METHODS OF AFFECTING NITROGEN ASSIMILATION IN PLANTS

Abstract

Provided herein are compositions and methods for producing transgenic plants. In specific embodiments, transgenic plants comprise a construct comprising a polynucleotide encoding CCA1, GLK1 or bZIP1, operably linked to a plant-specific promote, wherein the CCA1, GLK1 or bZIP1 is ectopically overexpressed in the transgenic plants, and wherein the promoter is optionally a constitutive or inducible promoter. In other embodiments, transgenic plants in which express a lower level of CCA1, GLK1 or bZIP1 are provided. Also provided herein are commercial products (e.g., pulp, paper, paper products, or lumber) derived from the transgenic plants (e.g., transgenic trees) produced using the methods provided herein.

Claims

1. A method for improving nitrogen assimilation and usage in a plant in which more nitrogen is available for biosynthesis, said method comprising fa) overexpressing GLK1 in the plant or (b) underexpressing bZIP1 in the plant or (c) overexpressing GLK1 and underexpressing bZIP1 in the plant.

2.-11. (canceled)

12. A method for altering nitrogen accumulation and storage in a plant, said method comprising (a) overexpressing bZIP1 in the plant or (b) underexpressing CCA1 in the plant or (c) overexpressing bZIP1 in the plant and underexpressing GLK1 in the plant.

13.-20. (canceled)

21. A transgenic plant comprising (i) a polynucleotide encoding GLK1 operatively linked to a promoter with activity in plants, wherein the promoter is optionally (a) a constitutive or inducible promoter or (b) associated with a constitutive or inducible regulatory element.sup.. or (ii) a polynucleotide encoding GLK1 operatively linked to a promoter with activity in plants and comprising a polynucleotide encoding CCA1 operatively linked to a second promoter with activity in plants, wherein the first and second promoter are optionally (a) a constitutive or inducible promoter or (b) associated with an constitutive or inducible regulatory element, and wherein the first and second promoter can be the same or different or (iii) a mutation in the coding sequence of bZIP1 or a mutation in the transcriptional or translational control sequences of bZIP1, such that the levels of bZIP1 produced in the plant are less compared to a non-transgenic control plant or (iv) a polynucleotide encoding bZIP1 operatively linked to a promoter with activity in plants, wherein the promoter is optionally (a) a constitutive or inducible promoter or (b) associated with a constitutive or inducible regulatory element or (v) a mutation in the coding sequence of CCA1 or a mutation in the transcriptional or translational control sequences of CCA1, such that the levels of CCA1 produced in the plant are less compared to a non-transgenic control plant or (vi) a polynucleotide encoding bZIP1 operatively linked to a promoter with activity in plants and comprising a mutation in the coding sequence of CCA1 or a mutation in the transcriptional or translational control sequences of CCA1, such that the levels of CCA1 produced in the plant are less compared to a non-transgenic control plant, wherein the promoter is optionally (a) a constitutive or inducible promoter or (b) associated with an constitutive or inducible regulatory element.

22.-47. (canceled)

48. A transgenic plant that exhibits increased nitrogen-assimilation capacity, increased nitrogen storage capacity, or both, as compared to a wild-type plant, the transgenic plant comprising a first gene construct comprising a polynucleotide encoding Basic Leucine Zipper 1 (bZIP1) operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding Myb Domain Protein 73 (MYB73) operatively linked to a second promoter with activity in plants, a third gene construct comprising a polynucleotide encoding Circadian Clock Associated 1 (CCA1) operatively linked to a third promoter with activity in plants, a fourth gene construct comprising a polynucleotide encoding Golden 2-like 1 (GLK1) operatively linked to a fourth promoter with activity in plants, a fifth gene construct comprising a polynucleotide encoding C3HC4 RING-type Zinc Finger (C3HC4) operatively linked to a fifth promoter with activity in plants, and a sixth gene construct comprising a polynucleotide encoding Basic Leucine Zipper 9 (bZIP9) operatively linked to a sixth promoter with activity in plants, wherein the first, second, third, fourth, fifth, and sixth promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first, second, third, fourth, fifth, and sixth promoters can be the same or different.

49. A transgenic plant that exhibits increased nitrogen-assimilation capacity as compared to a wild-type plant, the transgenic plant comprising (i) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding GLK1 operatively linked to a second promoter with activity in plants, a third gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a third promoter with activity in plants, and a fourth gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a fourth promoter with activity in plants, wherein the first, second, third, and fourth promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first, second, third, and fourth promoters can be the same or different; or (ii) a first gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a first promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, and a second gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression of MYB73, wherein the first and second promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first and second promoters can be the same or different; or (iii) (A) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding GLK1 operatively linked to a second promoter with activity in plants, or (B) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a second promoter with activity in plants, or (C) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, or (D) a first gene construct comprising a polynucleotide encoding GLK 1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a second promoter with activity in plants, or (E) a first gene construct comprising a polynucleotide encoding GLK 1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, or (F) a first gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, wherein the first and second promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first and second promoters can be the same or different; or (iv) (A) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression of MYB73 , or (B) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (C) a first gene construct comprising a polynucleotide encoding GLK1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form ofMYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression ofMYB73, or (D) a first gene construct comprising a polynucleotide encoding GLK1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (E) a first gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form ofMYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression ofMYB73, or (F) a first gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (G) a first gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form ofMYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression ofMYB73, or (H) a first gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, wherein the first and second promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first and second promoters can be the same or different; or (v) (A) a first gene construct comprising a polynucleotide encoding CCA1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding GLK1 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a third promoter with activity in plants, or (B) a first gene construct comprising a polynucleotide encoding CCA1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding GLK1 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a third promoter with activity in plants, or (C) a first gene construct comprising a polynucleotide encoding CCA1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a third promoter with activity in plants, or (D) a first gene construct comprising a polynucleotide encoding GLK1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a third promoter with activity in plants, or (E) a first gene construct comprising a polynucleotide encoding CCA1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression of MYB73, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (F) a first gene construct comprising a polynucleotide encoding GLK1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression of MYB73, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (G) a first gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression of MYB73, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (H) a first gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a second promoter with activity in plants, wherein the mutant form of MYB73 results in decreased expression of MYB73, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (I) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding GLK1 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a third promoter with activity in plants, wherein the mutant form ofMYB73 results in decreased expression ofMYB73, or (J) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a third promoter with activity in plants, wherein the mutant form ofMYB73 results in decreased expression ofMYB73, or (K) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a third promoter with activity in plants, wherein the mutant form ofMYB73 results in decreased expression ofMYB73, or (L) a first gene construct comprising a polynucleotide encoding GLK1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a third promoter with activity in plants, wherein the mutant form ofMYB73 results in decreased expression ofMYB73, or (M) a first gene construct comprising a polynucleotide encoding GLK1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a third promoter with activity in plants, wherein the mutant form ofMYB73 results in decreased expression ofMYB73, or (N) a first gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of MYB73 operatively linked to a third promoter with activity in plants, wherein the mutant form ofMYB73 results in decreased expression ofMYB73, or (O) a first gene construct comprising a polynucleotide encoding CCA 1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding GLK1 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP 1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (P) a first gene construct comprising a polynucleotide encoding CCA1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (Q) a first gene construct comprising a polynucleotide encoding CCA1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (R) a first gene construct comprising a polynucleotide encoding GLK1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (S) a first gene construct comprising a polynucleotide encoding GLK1 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, or (T) a first gene construct comprising a polynucleotide encoding C3HC4 operatively linked to a first promoter with activity in plants, a second gene construct comprising a polynucleotide encoding bZIP9 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP1 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP1 results in decreased expression of bZIP1, wherein the first, second, and third promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first, second and third promoters can be the same or different.

50. A transgenic plant that exhibits increased nitrogen-storage capacity as compared to a wild-type plant, the transgenic plant comprising (i) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a second promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, a third gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a third promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, and a fourth gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a fourth promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, wherein the first, second, third, and fourth promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first, second, third, and fourth promoters can be the same or different; or (ii) a first gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding MYB73 operatively linked to a second promoter with activity in plants, wherein the first and second promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first and second promoters can be the same or different; or (iii) (A) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, and a second gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a second promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, or (B) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, and a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, or (C) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, or (D) a first gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a first promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, and a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, or (E) a first gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a first promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, or (F) a first gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a first promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, wherein the first and second promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first and second promoters can be the same or different; or (iv) (A) a first gene construct comprising a polynucleotide encoding MYB73 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a second promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, or (B) a first gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a second promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA 1, or (C) a first gene construct comprising a polynucleotide encoding MYB73 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a second promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, or (D) a first gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a second promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, or (E) a first gene construct comprising a polynucleotide encoding MYB73 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, or (F) a first gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, or (G)a first gene construct comprising a polynucleotide encoding MYB73 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, or (H) a first gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a first promoter with activity in plants, and a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, wherein the first and second promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first and second promoters can be the same or different; or (v) (A) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a second promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, and a third gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a third promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, or (B) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a second promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, or (C) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, or (D) a first gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a first promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, and a third gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a third promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, or (E) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (F) a first gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a first promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, a second gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (G) a first gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a first promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, a second gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (H) a first gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a first promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, a second gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a second promoter with activity in plants, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (I) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA 1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a second promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (J) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA 1, a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (K) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA 1, a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (L) a first gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a first promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (M) a first gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a first promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (N) a first gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a first promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, and a third gene construct comprising a polynucleotide encoding MYB73 operatively linked to a third promoter with activity in plants, or (O) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a second promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, and a third gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a third promoter with activity in plants, or (P) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, and a third gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a third promoter with activity in plants, or (Q) a first gene construct comprising a polynucleotide encoding a mutant form of CCA1 operatively linked to a first promoter with activity in plants, wherein the mutant form of CCA1 results in decreased expression of CCA1, a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, and a third gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a third promoter with activity in plants, or (R) a first gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a first promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, a second gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a second promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, and a third gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a third promoter with activity in plants, or (S) a first gene construct comprising a polynucleotide encoding a mutant form of GLK1 operatively linked to a first promoter with activity in plants, wherein the mutant form of GLK1 results in decreased expression of GLK1, a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, and a third gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a third promoter with activity in plants, or (T) a first gene construct comprising a polynucleotide encoding a mutant form of C3HC4 operatively linked to a first promoter with activity in plants, wherein the mutant form of C3HC4 results in decreased expression of C3HC4, a second gene construct comprising a polynucleotide encoding a mutant form of bZIP9 operatively linked to a second promoter with activity in plants, wherein the mutant form of bZIP9 results in decreased expression of bZIP9, and a third gene construct comprising a polynucleotide encoding bZIP1 operatively linked to a third promoter with activity in plants, wherein the first, second, and third promoters are optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element, and wherein the first, second and third promoters can be the same or different.

51. A transgenic plant that exhibits increased nitrogen-assimilation or increased nitrogen storage capacity, as compared to a wild-type plant, the transgenic plant comprising a gene construct comprising a polynucleotide encoding MYB73 or C3HC4 operatively linked to a promoter with activity in plants, wherein the promoter is optionally (a) a constitutive, tissue-specific, or inducible promoter or (b) associated with a constitutive or inducible regulatory element.

52. The transgenic plant of claim 48, wherein bZIP 1,MYB73,CCA1,GLK1,C3HC4, and bZIP9 are ectopically overexpressed.

53. The transgenic plant of claim 48, wherein the first and second promoters are seed-specific promoters and the third, fourth, fifth, and sixth promoters are leaf-specific promoters.

54. The transgenic plant of claim 48, wherein the plant is a species of the genus Arabidopsis.

55. The transgenic plant of claim 48, wherein the plant is a species of woody, ornamental, decorative, crop, cereal, fruit, or vegetable.

56. The transgenic plant of claim 48, wherein the plant is a species of one of the following genuses: Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium, Arachis, Beta, Betula, Brassica, Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca, Linum, Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus, Populus, Prunus, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia.

57. A method of producing a plant-derived commercial product using the transgenic plant of claim 48, wherein optionally (a) said transgenic plant is a tree, and said commercial product is pulp, paper, a paper product, or lumber; or (b) said transgenic plant is tobacco, and said commercial product is a cigarette, cigar, or chewing tobacco; or (c) said transgenic plant is a crop, and said commercial product is a fruit or vegetable; or (d) said transgenic plant is a grain, and said commercial product is bread, flour, cereal, oat meal, or rice.

58. A method of producing a plant-derived commercial product using the transgenic plant of claim 49, wherein optionally (a) said transgenic plant is a tree, and said commercial product is pulp, paper, a paper product, or lumber; or (b) said transgenic plant is tobacco, and said commercial product is a cigarette, cigar, or chewing tobacco; or (c) said transgenic plant is a crop, and said commercial product is a fruit or vegetable; or (d) said transgenic plant is a grain, and said commercial product is bread, flour, cereal, oat meal, or rice.

59. A method of producing a plant-derived commercial product using the transgenic plant of claim 50, wherein optionally (a) said transgenic plant is a tree, and said commercial product is pulp, paper, a paper product, or lumber; or (b) said transgenic plant is tobacco, and said commercial product is a cigarette, cigar, or chewing tobacco; or (c) said transgenic plant is a crop, and said commercial product is a fruit or vegetable; or (d) said transgenic plant is a grain, and said commercial product is bread, flour, cereal, oat meal, or rice.

60. A method of producing a plant-derived commercial product using the transgenic plant of claim 51, wherein optionally (a) said transgenic plant is a tree, and said commercial product is pulp, paper, a paper product, or lumber; or (b) said transgenic plant is tobacco, and said commercial product is a cigarette, cigar, or chewing tobacco; or (c) said transgenic plant is a crop, and said commercial product is a fruit or vegetable; or (d) said transgenic plant is a grain, and said commercial product is bread, flour, cereal, oat meal, or rice.

Description

5. DESCRIPTION OF THE FIGURES

[0085] FIG. 1. Schematic diagram of the nitrogen assimilation pathway.

[0086] FIG. 2. Schematic diagram of the effects of transgenic overexpression or underexpression of master regulators CCA1 IGLK11bZIP 1 on nitrogen assimilation, usage and storage.

[0087] FIG. 3. Network analysis of genes regulated by organic N suggests CCA1 control N-assimilation in plants. In this Cytoscape-generated network, metabolic genes are drawn as triangles (transcription factors), hexagons (metabolic genes) whereas metabolites are shown as white circles. Arrows, diamonds or lines at the end of an edge indicate directionality of the interaction. To simplify, some of the genes connecting to GLK1 and CCA1 are grouped and summarized based on their associated functions (number in parenthesis indicate the number of genes in the group).

[0088] FIGS. 4A-B. Altered mRNA levels of target genes and binding of CCA1 protein to target gene promoter regions validate predicted regulation by CCA1. FIG. 4A: RT-qPCR was performed on CCA1-ox, glkl knockout, and wild-type plants to determine mRNA levels for ASN1, GLN1.3, and GDH1. Three biological and two technical replicates were carried out for each sample. mRNA levels were normalized to clathrin (At4g24550). The mean +/− standard error of the mean is shown. FIG. 4B: ChIP assays to show binding of CCA1 to GLN1.3, GDH1 and bZIP1 gene promoter regions. Control: input DNA control (no IP), -Ab: IP without antibody, CCA1: IP with the CCA1 antibody.

[0089] FIGS. 5A-D. Exposure of seedlings to pulses of inorganic and organic N shifts the phase of the circadian clock. FIGS. 5A-C: Plot of the phase shift of CCA1::LUC expression in response to 4-h pulse of inorganic N (20 mM KNO.sub.3/20 mM NH.sub.4NO.sub.3), 10 mM Glu, or 10 mM Gln against the time at which the pulse was administered to wild-type seedlings. Pulses were administered at 3-hr intervals spanning one complete circadian cycle and data were collected over the next 6 cycles. Phase shifts are double-plotted to emphasize the circadian pattern of the response. Phase advances (the peak in expression occurring earlier) are plotted as positive values and delays are plotted as negative values. FIG. 5D shows the CCA1::LUC expression of control (untreated) seedlings. In all panels, the entraining photocycle (16:8) is indicated by the vertical white (light) and gray (dark) bars. The mean +/− standard error of the mean is shown.

[0090] FIG. 6. Proposed model of the interaction between the Arabidopsis circadian clock and N-assimilatory pathway. Arrows indicate influences that affect the function of the two processes. Black arrow: Clock function would affect N-assimilation. This influence is at least partly due to the direct regulatory role of CCA1 on N-assimilation. Grey arrow: N-assimilation would influence clock function through downstream metabolites such as Glu, Gln and possibly other N-metabolites.

[0091] FIG. 7. Signaling by inorganic vs. organic nitrogen can be distinguished by using MSX and Glu treatments. (a) Treatments include N±MSX±Glu. (b) A simplified diagram of the N-metabolic pathway from inorganic nitrate (NO.sub.3) to organic Glu, and the block by MSX. Below this pathway are the predicted effects of the given treatments on nitrogen metabolism. Arrows indicate progression through the pathway. Line breaks, represented with a short perpendicular line, indicate the step in the pathway blocked by MSX. (c and d). The expected transcript levels for genes induced by inorganic nitrogen (c) vs. genes regulated by Glu or a Glu-derived metabolite (d).

[0092] FIGS. 8A-B. Analysis of the expression of asparagine synthetase genes. Shown is a comparison of ASN1 (FIG. 8A) and ASN2 (FIG. 8B) mRNA levels in control seedlings (transferred to 1 mM NO.sub.3) along with MSX control (treated with NO.sub.3 and 1 mM MSX) compared to seedlings treated with a stepwise combination of Nms, MSX, and Glu/Gln. (FIG. 8A) mRNA levels of ASN1 are increased in Nms, are sensitive to MSX treatment, and can be recovered with exogenous application of Glu or Gln. (FIG. 8B) mRNA levels of ASN2 are increased in Nms. However, this expression is insensitive to MSX treatment and is slightly repressed with exogenous application of Glu or Gln. mRNA levels were normalized to EIF4A (At3g13920).

[0093] FIGS. 9A-B. RT-qPCR confirmation of the regulation for two transcription factors TAZ and bZIP1. Shown is confirmation of TAZ (FIG. 9A) and bZIP1 (FIG. 9B) mRNA levels in control seedlings along with the MSX control and compared to seedlings treated with a stepwise combination of Nms, Nms+MSX, and Nms+MSX+Glu (or Gln). In both cases, although increased expression in the presence of N is blocked in the presence of MSX, this suppression can be overcome by exogenous application of Glu or Gln. Plants transferred to control media do not show mRNA levels different from treatments without MSX. Primers used for RT-qPCR are as follows: TAZ forward, 5′-TCCTCGTCTCGGTCTT-3′ (SEQ ID NO:1); reverse, 5′-CAACCACCAGGGATTC-3′ (SEQ ID NO:2); bZIP forward, 5′-TCAGGTTCCGACATAGATG-3′ (SEQ ID NO:3); reverse, 5′-CCACGGTGTACGTCTACA-3′ (SEQ ID NO:4).

[0094] FIG. 10. Analysis of the expression of bZIP1 in the CCA1-ox. To test some of the predictions of our network CCA1-ox and Col-0 plants were collected 3 h after dawn; three biological replicates were taken at each time point. RNA was extracted from whole seedlings (as described in Materials and Methods), and RT-qPCR was performed to measure mRNA levels for bZIP1 (At5g49450). Two technical replicates were carried out for each sample. mRNA levels were normalized to clathrin (At4g24550).

[0095] FIG. 11. Circadian regulation of the response of clock gene (CCA1) expression to N-assimilation inhibitors and inorganic and organic N. Mean±SEM luciferase activity of CCA1::LUC in response to exogenous inorganic N, Glu, or Gln is presented. Seedlings were entrained for 8 days in a 16-h white light/8-h dark photoperiod on MS medium containing 1 mM KNO.sub.3 before being transferred to continuous light and exposed for 4-h pulses of inorganic N (20 mM KNO.sub.3/20 mM NH.sub.4NO.sub.3), 10 mM Glu, or 10 mM Gln presented at 3-h intervals over one circadian cycle before return to MS medium containing 1 mM KNO.sub.3 in continuous light for luciferase measurements for 6 days. Luciferase activity values were normalized by the mean expression value for the treatment. The entraining photocycle is indicated by the vertical white (light) and gray (dark) bars.

[0096] FIG. 12. Schematic diagram of how CCA1 IGLK1 IbZIP 1 transcription factors coordinate the nitrogen regulation of genes in the nitrogen assimilation pathway.

6. DETAILED DESCRIPTION

[0097] Master control genes (CCA1, GLK1, and bZIP11) that control N-assimilation in response to Glu sensing have been identified in the present invention. As these genes are transcription factor hubs, they coordinate the N-regulation of the N-assimilatory gene network, with genome-wide responses associated with growth and development in plants. Thus, effecting genome-wide changes in N-assimilation, plant growth and development, by the transgenic manipulation of these master control genes in plants effects nitrogen use efficiency in vegetative tissues (leaves & roots) and also in seed. Changes in levels of N-assimilated into Gln effect changes in growth of vegetative tissues, while changes in levels of Asn affect seed development.

[0098] Thus, the present invention relates to the transgenic manipulation of these N-responsive master regulatory genes (CCA1, GLK1, and bZIP11) that control N-assimilation, and other related processes in response to N treatments, so as to increase the overall N-assimilation capacity, whether for increased N usage or N storage. The overexpression of these master control genes (e.g., uncoupled from Glu repression) effectively releases N-assimilation from the feedback repression loop by Glu- leading to increased N-assimilation and usage. As these regulatory genes serve to respond to Glu levels by reciprocally regulating the amount of N-assimilated into Gln versus the amount of Gln metabolized to Asn (for N-storage and transport), the manipulation of these genes in transgenic plants can be used to optimize N-assimilation into Gln versus Asn (FIG. 6). Increased N-assimilation is advantageous in all crops. Additionally, in seed crops, the increased synthesis of Asn increases N-transported and stored in seed.

[0099] Thus, in one embodiment, the present invention is directed to a method for improving nitrogen assimilation and usage in a plant in which more nitrogen is available for biosynthesis, said method comprising overexpressing GLK1 in the plant. In another embodiment, the present invention is directed to a method for improving nitrogen assimilation and usage in a plant in which more nitrogen is available for biosynthesis, said method comprising overexpressing CCA1 in the plant. In yet another embodiment, the present invention is directed to a method for improving nitrogen assimilation and usage in a plant in which more nitrogen is available for biosynthesis, said method comprising underexpressing bZIP1 in the plant.

[0100] In another embodiment, the method for improving nitrogen assimilation and usage in a plant in which more nitrogen is available for biosynthesis comprises overexpressing CCA1 and GLK1 in the plant. In another embodiment, the method for improving nitrogen assimilation and usage in a plant in which more nitrogen is available for biosynthesis comprises overexpressing CCA1 and underexpressing bZIP1 in the plant, or overexpressing GLK1 and underexpressing bZIP1 in the plant. In yet another embodiment, the method for improving nitrogen assimilation and usage in a plant in which more nitrogen is available for biosynthesis comprises overexpressing CCA1, overexpressing GLK1 and underexpressing bZIP1 in the plant.

[0101] The present invention is also directed to methods for altering nitrogen assimilation and storage, e.g., increasing nitrogen storage, in a plant. In one embodiment, the method comprises overexpressing bZIP1 in the plant. In another embodiment, the method comprises underexpressing CCA1 in the plant and/or underexpressing GLK1 in the plant. In another embodiment, the method overexpressing bZIP1 and underexpressing CCA1 and/or GLK1 in the plant. In yet another embodiment, the method comprises overexpressing bZIP1, and underexpressing CCA1 and underexpressing GLK1 in the plant.

[0102] In certain embodiments, the plant is species of woody, ornamental, decorative, crop, cereal, fruit, or vegetable. In other embodiments, the plant is a species of one of the following genuses: Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium, Arabidopsis, Arachis, Beta, Betula, Brassica, Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca, Linum, Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus, Populus, Prunus, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia.

[0103] The overexpression of a particular gene can be accomplished by any method known in the art, for example, by transforming a plant cell with a nucleic acid vector comprising the coding sequences of the desired gene operably linked to a promoter active in a plant cell such that the desired gene is expressed at levels higher than normal, i.e., levels found in a control/nontransgenic plant. Such promoters can be constitutively active in all or some plant tissues or can be inducible.

[0104] The underexpression of a desired gene can be accomplished by any method known in the art, such as knocking out the gene or mutating the gene transgenically such that lower than normal levels of the gene product is produced in the transgenic cells or plant. For example, such mutations include frame-shift mutations or mutations resulting in a stop codon in the wild-type coding sequence, thus preventing expression of the gene product. Another exemplary mutation would be the removal of the transcribed sequences from the plant genome, for example, by homologous recombination. Another method for underexpressing a gene is transgenically introducing an insertion or deletion into the transcribed sequence or an insertion or deletion upstream or downstream of the transcribed sequence such that expression of the gene product is decreased as compared to wild-type or appropriate control. Additionally, microRNA (native or artificial) can be used to target a particular encoding mRNA for degradation, thus reducing the level of the expressed gene product in the transgenic plant cell.

[0105] The present invention is also directed to a transgenic plant produced by any of the foregoing methods.

[0106] The present invention is also directed to compositions for modulating gene expression in plants. The compositions comprise constructs for the expression of CCA1, GLK1 or bZIP1. In certain embodiments, a construct of the invention comprises a promoter, such as a tissue specific promoter, which is expressed in a plant cell, such as a leaf cell, and promotes the expression of CCA1, GLK1 or bZIP1.

[0107] Any of a variety of promoters can be utilized in the constructs of the invention depending on the desired outcome. Tissue-specific or tissue-preferred promoters, inducible promoters, developmental promoters, constitutive promoters and/or chimeric promoters can be used to direct expression of the gene product in specific cells or organs the plant, when fused to the appropriate cell or organ specific promoter.

[0108] Chimeric constructs expressing CCA1, GLK1 or bZIP1 in transgenic plants (using constitutive or inducible promoters) can be used in the compositions and methods provided herein to enhance nitrogen assimilation and usage or increase nitrogen storage.

[0109] The present invention is also directed to a transgenic plant-derived commercial product. In one embodiment, the transgenic plant is a tree, and said commercial product is pulp, paper, a paper product, or lumber. In another embodiment, the transgenic plant is tobacco, and said commercial product is a cigarette, cigar, or chewing tobacco. In yet another embodiment, the transgenic plant is a crop, and said commercial product is a fruit or vegetable. In yet another embodiment, the transgenic plant is a grain, and said commercial product is bread, flour, cereal, oat meal, or rice. In another embodiment, the product is a biofuel or a plant oil.

6.1 Master Regulators

[0110] CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene encodes a MYB-related transcription factor involved in the phytochrome induction of a light-harvesting chlorophyll a/b-protein gene. The nucleotide and amino acid sequences of CCA1 from Arabidopsis are known, see Accession No. At2g46830. Further, orthologous CCA1 genes from other organisms are also known. For example, the CCA1 gene sequences from poplar can be found under Accession Nos. Poptrl#552368 or Poptrl#731468. The use of CCA1 in the present invention refers not only to the Arabidopsis gene but also the orthologous CCA1 gene from other species. Thus, in one embodiment, plant species-specific CCA1 genes can be used in plants of the same species, e.g., tobacco CCA1 can be overexpressed in tobacco. Additionally, such orthologous sequences can be identified and isolated using methods known in the art, such as hybridization methods and then testing the isolated sequences for CCA1 activity, as demonstrated infra. Other methods, such as alignment methods described supra can also be used to identify and isolate orthologous CCA1 sequences.

[0111] Golden 2-like genes (GLK) are members of the GARP superfamily of transcription factors. GLK genes are known to be involved in the regulation of chloroplast development in diverse plant species (Fritter et al., 2002, The Plant Journal 31:713-727). The nucleotide and amino acid sequences of GLK1 from Arabidopsis are known, see Accession No. At2g20570. Further, orthologous GLK1 genes from other organisms are also known. For example, the GLK1 gene sequences from poplar and rice can be found under Accession Nos. Poptr1#654401 and 0506g24070, respectively. The use of GLK1 in the present invention refers not only to the Arabidopsis gene but also the orthologous GLK1 gene from other species. Thus, in one embodiment, plant species-specific GLK1 genes can be used in plants of the same species, e.g., tobacco GLK1 can be overexpressed in tobacco. Additionally, such orthologous sequences can be identified and isolated using methods known in the art, such as hybridization methods and then testing the isolated sequences for GLK1 activity, such as DNA binding activity. Other methods, such as alignment methods described supra can also be used to identify and isolate orthologous GLK1 sequences.

[0112] bZIP1 is a transcription factor that belongs to the largest bZIP group in Arabadopsis, Group S (Jakoby et al., 2002, Trends Plant Sci 7:106-111). It is thought that Group S bZIP genes are involved in balancing carbohydrate demand and supply (Rook et al., 1998, Plant J 15:253-263). The nucleotide and amino acid sequences of bZIP1 from Arabidopsis are known, see Accession No. At5g49450. The use of bZIP1 in the present invention refers not only to the Arabidopsis gene but also the orthologous bZIP1 gene from other species. Thus, in one embodiment, plant species-specific bZIP1 genes can be used in plants of the same species, e.g., tobacco bZIP1 can be overexpressed in tobacco. Additionally, such orthologous sequences can be identified and isolated using methods known in the art, such as hybridization methods and then testing the isolated sequences for bZIP1 activity, such as DNA binding activity. Other methods, such as alignment methods described supra can also be used to identify and isolate orthologous bZIP1 sequences.

6.2 Modulation of Gene Expression

[0113] The methods of the invention involve modulation of the expression of one, two, three or more target nucleotide sequences in a plant, optionally in specific tissues such as vegetative tissues or leaves or seeds. That is, the expression of a target nucleotide sequence of interest may be increased or decreased. In specific embodiments, the target nucleot-ide sequences are CCA1, GLK1 or bZIP1, which can be increased or decreased.

[0114] The target nucleotide sequences may be endogenous or exogenous in origin. By “modulate expression of a target gene” is intended that the expression of the target gene is increased or decreased relative to the expression level in a plant that has not been altered by the methods described herein.

[0115] By “increased or over expression” is intended that expression of the target nucleotide sequence is increased over expression observed in conventional transgenic lines for heterologous genes and over endogenous levels of expression for homologous genes. Heterologous or exogenous genes comprise genes that do not occur in the plant of interest in its native state. Homologous or endogenous genes are those that are natively present in the plant genome. Generally, expression of the target sequence is substantially increased. That is expression is increased at least about 25%-50%, preferably about 50%-100%, more preferably about 100%, 200% and greater.

[0116] By “decreased expression” or “underexpression” it is intended that expression of the target nucleotide sequence is decreased below expression observed in conventional transgenic lines for heterologous genes and below endogenous levels of expression for homologous genes. Generally, expression of the target nucleotide sequence of interest is substantially decreased. That is expression is decreased at least about 25%-50%, preferably about 50%-100%, more preferably about 100%, 200% and greater.

[0117] Expression levels may be assessed by determining the level of a gene product by any method known in the art including, but not limited to determining the levels of the RNA and protein encoded by a particular target gene. For genes that encode proteins, expression levels may determined, for example, by quantifying the amount of the protein present in plant cells, or in a plant or any portion thereof. Alternatively, it desired target gene encodes a protein that has a known measurable activity, then activity levels may be measured to assess expression levels.

6.3 Transformation/Transfection

[0118] Any method or delivery system may be used for the delivery and/or transfection of the nucleic acid vectors encoding any of the master regulators of the present invention in the cell. The vectors may be delivered to the plant cell either alone, or in combination with other agents.

[0119] Transfection may be accomplished by a wide variety of means, as is known to those of ordinary skill in the art. Such methods include, but are not limited to, Agrobacterium-mediated transformation (e.g., Komari et al., 1998, Curr. Opin. Plant Biol., 1:161), particle bombardment mediated transformation (e.g., Finer et al., 1999, Curr. Top. Microbiol. Immunol., 240:59), protoplast electroporation (e.g., Bates, 1999, Methods Mol. Biol., 111:359), viral infection (e.g., Porta and Lomonossoff, 1996, Mol. Biotechnol. 5:209), microinjection, and liposome injection. Other exemplary delivery systems that can be used to facilitate uptake by a cell of the nucleic acid include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, and homologous recombination compositions (e.g., for integrating a gene into a preselected location within the chromosome of the cell). Alternative methods may involve, for example, the use of liposomes, electroporation, or chemicals that increase free (or “naked”) DNA uptake, transformation using viruses or pollen and the use of microprojection. Standard molecular biology techniques are common in the art (e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York). For example, in one embodiment of the present invention, Arabidopsis or another plant species is transformed with a gene encoding CCA1, GLK1 or bZIP1 using Agrobacterium.

[0120] One of skill in the art will be able to select an appropriate vector for introducing the encoding nucleic acid sequence in a relatively intact state. Thus, any vector which will produce a plant carrying the introduced encoding nucleic acid should be sufficient. The selection of the vector, or whether to use a vector, is typically guided by the method of transformation selected.

[0121] The transformation of plants in accordance with the invention may be carried out in essentially any of the various ways known to those skilled in the art of plant molecular biology. (See, for example, Methods of Enzymology, Vol. 153, 1987, Wu and Grossman, Eds., Academic Press, incorporated herein by reference).

[0122] Plant cells and plants can comprise two or more nucleotide sequence constructs. Any means for producing a plant comprising the nucleotide sequence constructs described herein are encompassed by the present invention. For example, a nucleotide sequence encoding the modulator can be used to transform a plant at the same time as the nucleotide sequence encoding the precursor RNA. The nucleotide sequence encoding the precursor mRNA can be introduced into a plant that has already been transformed with the modulator nucleotide sequence. Alternatively, transformed plants, one expressing the modulator and one expressing the RNA precursor, can be crossed to bring the genes together in the same plant. Likewise, viral vectors may be used to express gene products by various methods generally known in the art. Suitable plant viral vectors for expressing genes should be self-replicating, capable of systemic infection in a host, and stable. Additionally, the viruses should be capable of containing the nucleic acid sequences that are foreign to the native virus forming the vector. Transient expression systems may also be used.

[0123] Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (Toriyarna et al., 1988, Bio/Technology 6:1072-1074; Zhang et al., 1988, Plant Cell Rep. 7:379-384; Zhang et al., 1988, Theor. Appl. Genet. 76:835-840; Shimamoto et al., 1989, Nature 338:274-276; Datta et al., 1990, Bio/Technology 8: 736-740; Christou et al., 1991, Bio/Technology 9:957-962; Peng et al., 1991, International Rice Research Institute, Manila, Philippines, pp. 563-574; Cao et al., 1992, Plant Cell Rep. 11:585-591; Li et al., 1993, Plant Cell Rep. 12:250-255; Rathore et al., 1993, Plant Mol. Biol. 21:871-884; Fromm et al., 1990, Bio/Technology 8:833-839; Tomes et al., 1995, “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); D′Halluin et al., 1992, Plant Cell 4:1495-1505; Walters et al., 1992, Plant Mol. Biol. 18:189-200; Koziel et al., 1993, Biotechnology 11: 194-200; Vasil, I. K., 1994, Plant Mol. Biol. 25:925-937; Weeks et al., 1993, Plant Physiol. 102:1077-1084; Somers et al., 1992, Bio/Technology 10: 1589-1594; WO 92/14828). In particular, Agrobacterium mediated transformation is now emerging also as an highly efficient transformation method in monocots (Hiei et al., 1994, The Plant Journal 6:271-282). See also, Shimamoto, K., 1994, Current Opinion in Biotechnology 5:158-162; Vasil et al., 1992, Bio/Technology 10:667-674; Vain et al., 1995, Biotechnology Advances 13(4):653-671; Vasil et al., 1996, Nature Biotechnology 14:702).

[0124] The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practicing the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

6.3.1 Agrobacterium

[0125] A CCA1, GLK1 or bZIP1-encoding nucleic acid sequences or a nucleic acid designed to disrupt expression of CCA1, GLK1 or bZIP1 utilized in the present invention can be introduced into plant cells using Ti plasmids of Agrobacterium tumefaciens (A. tumefaciens), root-inducing (Ri) plasmids of Agrobacterium rhizogenes (A. rhizogenes), and plant virus vectors. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9, and Horsch et al., 1985, Science, 227:1229.

[0126] In using an A. tumefaciens culture as a transformation vehicle, it is most advantageous to use a non-oncogenic strain of Agrobacterium as the vector carrier so that normal non-oncogenic differentiation of the transformed tissues is possible. It is also preferred that the Agrobacterium harbor a binary Ti plasmid system. Such a binary system comprises 1) a first Ti plasmid having a virulence region essential for the introduction of transfer DNA (T-DNA) into plants, and 2) a chimeric plasmid. The chimeric plasmid contains at least one border region of the T-DNA region of a wild-type Ti plasmid flanking the nucleic acid to be transferred. Binary Ti plasmid systems have been shown effective in the transformation of plant cells (De Framond, Biotechnology, 1983, 1:262; Hoekema et al., 1983, Nature, 303:179). Such a binary system is preferred because it does not require integration into the Ti plasmid of A. tumefaciens, which is an older methodology.

[0127] In some embodiments, a disarmed Ti-plasmid vector carried by Agrobacterium exploits its natural gene transferability (EP-A-270355, EP-A-01 16718, Townsend et al., 1984, NAR, 12:8711, U.S. Pat. No. 5,563,055).

[0128] Methods involving the use of Agrobacterium in transformation according to the present invention include, but are not limited to: 1) co-cultivation of Agrobacterium with cultured isolated protoplasts; 2) transformation of plant cells or tissues with Agrobacterium; or 3) transformation of seeds, apices or meristems with Agrobacterium.

[0129] In addition, gene transfer can be accomplished by in planta transformation by Agrobacterium, as described by Bechtold et al., (C.R. Acad. Sci. Paris, 1993, 316:1194). This approach is based on the vacuum infiltration of a suspension of Agrobacterium cells.

[0130] In certain embodiments, a CCA1, GLK1, bZIP1-encoding nucleic acid or mutant thereof is introduced into plant cells by infecting such plant cells, an explant, a meristem or a seed, with transformed A. tumefaciens as described above. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots, roots, and develop further into plants.

[0131] Other methods described herein, such as microprojectile bombardment, electroporation and direct DNA uptake can be used where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g., bombardment with Agrobacterium-coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).

6.3.2 CaMV

[0132] In some embodiments, cauliflower mosaic virus (CaMV) is used as a vector for introducing a desired nucleic acid into plant cells (U.S. Pat. No. 4,407,956). CaMV viral DNA genome can be inserted into a parent bacterial plasmid creating a recombinant DNA molecule which can be propagated in bacteria. After cloning, the recombinant plasmid again can be cloned and further modified by introduction of the desired nucleic acid sequence. The modified viral portion of the recombinant plasmid can then be excised from the parent bacterial plasmid, and used to inoculate the plant cells or plants.

6.3.3 Mechanical and Chemical Means

[0133] In some embodiments, a CCA1, GLK1 or bZIP1-encoding nucleic acid or a nucleic acid designed to disrupt expression of CCA1, GLK1 or bZIP1 is introduced into a plant cell using mechanical or chemical means. Exemplary mechanical and chemical means are probided below.

[0134] As used herein, the term “contacting” refers to any means of introducing a a CCA1, GLK1 or bZIP1-encoding nucleic acid or a nucleic acid designed to disrupt expression of CCA1, GLK1 or bZIP1 into a plant cell, including chemical and physical means as described above. Preferably, contacting refers to introducing the nucleic acid or vector containing the nucleic acid into plant cells (including an explant, a meristem or a seed), via A. tumefaciens transformed with the, e.g., GLK1-encoding nucleic acid as described above.

6.3.3.1 Microinjection

[0135] In one embodiment, the CCA1, GLK1 or bZIP1-encoding nucleic acid or the nucleic acid designed to disrupt expression of CCA1, GLK1 or bZIP1 can be mechanically transferred into the plant cell by microinjection using a micropipette. See, e.g., WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al., 1987, Plant Tissue and Cell Culture, Academic Press, Crossway et al., 1986, Biotechniques 4:320-334.

6.3.3.2 PEG

[0136] In other embodiment, the nucleic acid can also be transferred into the plant cell by using polyethylene glycol (PEG)which forms a precipitation complex with genetic material that is taken up by the cell.

6.3.3.3 Electroporation

[0137] Electroporation can be used, in another set of embodiments, to deliver a nucleic acid to the cell, e.g., precursor miRNA, or a nucleotide sequence able to be transcribed to produce CCA1, GLK1 or bZIP1 protein (see, e.g., Fromm et al., 1985, PNA5, 82:5824). “Electroporation,” as used herein, is the application of electricity to a cell, such as a plant protoplast, in such a way as to cause delivery of a nucleic acid into the cell without killing the cell. Typically, electroporation includes the application of one or more electrical voltage “pulses” having relatively short durations (usually less than 1 second, and often on the scale of milliseconds or microseconds) to a media containing the cells. The electrical pulses typically facilitate the non-lethal transport of extracellular nucleic acids into the cells. The exact electroporation protocols (such as the number of pulses, duration of pulses, pulse waveforms, etc.), will depend on factors such as the cell type, the cell media, the number of cells, the substance(s) to be delivered, etc., and can be determined by those of ordinary skill in the art. Electroporation is discussed in greater detail in, e.g., EP 290395, WO 8706614, Riggs et al., 1986, Proc. Natl. Acad. Sci. USA 83:5602-5606; D'Halluin et al., 1992, Plant Cell 4:1495-1505). Other forms of direct DNA uptake can also be used in the methods provided herein, such as those discussed in, e.g., DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611, Paszkowski et al., 1984, EMBO J. 3:2717-2722.

6.3.3.4 Ballistic and Particle Bombardment

[0138] Another method for introducing a CCA1, GLK1 or bZIP1-encoding nucleic acid or a nucleic acid designed to disrupt expression of CCA1, GLK1 or bZIP1 into a plant cell is high velocity ballistic penetration by small particles with the nucleic acid to be introduced contained either within the matrix of such particles, or on the surface thereof (Klein et al., 1987, Nature 327:70). Genetic material can be introduced into a cell using particle gun (“gene gun”) technology, also called microprojectile or microparticle bombardment. In this method, small, high-density particles (microprojectiles) are accelerated to high velocity in conjunction with a larger, powder-fired macroprojectile in a particle gun apparatus. The microprojectiles have sufficient momentum to penetrate cell walls and membranes, and can carry RNA or other nucleic acids into the interiors of bombarded cells. It has been demonstrated that such microprojectiles can enter cells without causing death of the cells, and that they can effectively deliver foreign genetic material into intact tissue. Bombardment transformation methods are also described in Sanford et al. (Techniques 3:3-16, 1991) and Klein et al. (Bio/Techniques 10:286, 1992). Although, typically only a single introduction of a new nucleic acid sequence(s) is required, this method particularly provides for multiple introductions.

[0139] Particle or microprojectile bombardment are discussed in greater detail in, e.g., the following references: U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616; Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., 1995, “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al., 1988, Biotechnology 6:923-926.

6.3.3.5 Colloidal Dispersion

[0140] In other embodiments, a colloidal dispersion system may be used to facilitate delivery of a nucleic acid into the cell, for example, GLK1, or a nucleotide sequence able to disrupt expression of GLK1. As used herein, a “colloidal dispersion system” refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering to and releasing the nucleic acid to the cell. Colloidal dispersion systems include, but are not limited to, macromolecular complexes, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. One example of a colloidal dispersion system is a liposome. Liposomes are artificial membrane vessels. It has been shown that large unilamellar vessels (“LUV”), which-range in size from 0.2 to 4.0 microns, can encapsulate large macromolecules within the aqueous interior and these macromolecules can be delivered to cells in a biologically active form (e.g., Fraley et al., 1981, Trends Biochem. Sci., 6:77).

6.3.3.6 Lipids

[0141] Lipid formulations for the transfection and/or intracellular delivery of nucleic acids are commercially available, for instance, from QIAGEN, for example as EFFECTENE® (a non-liposomal lipid with a special DNA condensing enhancer) and SUPER-FECT® (a novel acting dendrimeric technology) as well as Gibco BRL, for example, as LIPOFECTIN® and LIPOFECTACE®, which are formed of cationic lipids such as N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (“DOTMA”) and dimethyl dioctadecylammonium bromide (“DDAB”). Liposomes are well known in the art and have been widely described in the literature, for example, in Gregoriadis, G., 1985, Trends in Biotechnology 3:235-241; Freeman et al., 1984, Plant Cell Physiol. 29:1353).

6.3.3.7 Other Methods

[0142] In addition to the above, other physical methods for the transformation of plant cells are reviewed in the following and can be used in the methods provided herein. Oard , 1991, Biotech. Adv. 9:1-11. See generally, Weissinger et al., 1988, sAnn. Rev. Genet. 22:421-477; Sanford et al., 1987, Particulate Science and Technology 5:27-37; Christou et al., 1988, Plant Physiol. 87:671-674; McCabe et al., 1988, Bio/Technology 6:923-926; Finer and McMullen, 1991, In vitro Cell Dev. Biol. 27P:175-182; Singh et al., 1998, Theor. Appl. Genet. 96:319-324; Datta et al., 1990, Biotechnology 8:736-740; Klein et al., 1988, Proc. Natl. Acad. Sci. USA 85:4305-4309; Klein et al., 1988, Biotechnology 6:559-563; Tomes, U.S. Pat. No. 5,240,855; Buising et al.,U.S. Pat. Nos. 5,322,783 and 5,324,646; Klein et al., 1988, Plant Physiol. 91:440-444; Fromm et al., 1990, Biotechnology 8:833-839; Hooykaas-Van Slogteren et al., 1984, Nature (London) 311:763-764; Bytebier et al., 1987, Proc. Natl. Acad. Sci. USA 84:5345-5349; De Wet et al., 1985, The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209; Kaeppler et al., 1990, Plant Cell Reports 9:415-418 and Kaeppler et al., 1992, Theor. Appl. Genet. 84:560-566; Li et al., 1993, Plant Cell Reports 12:250-255 and Christou and Ford, 1995, Annals of Botany 75:407-413; Osjoda et al., 1996, Nature Biotechnology 14:745-750; all of which are herein incorporated by reference.

6.4 Nucleic Acid Constructs

[0143] The CCA1, GLK1, bZIP 1 sequences of the invention may be provided in nucleotide sequence constructs or expression cassettes for expression in the plant of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to an encoding nucleotide sequence of the invention.

[0144] The expression cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.

[0145] In certain embodiments, an expression cassette can be used with a plurality of restriction sites for insertion of the sequences of the invention to be under the transcriptional regulation of the regulatory regions. The expression cassette can additionally contain selectable marker genes (see below).

[0146] The expression cassette will generally include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of the invention, e.g., GLK1 or a sequence designed to disrupt expression of GLK1, and a transcriptional and translational termination region functional in plants. The transcriptional initiation region, the promoter, may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0147] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al., 1991, Mol. Gen. Genet. 262:141-144; Proudfoot, 1991, Cell 64:671-674; Sanfacon et al., 1991, Genes Dev. 5:141-149; Mogen et al., 1990, Plant Cell 2:1261- 1272; Munroe et al., 1990, Gene 91:151-158; Ballas et al., 1989, Nucleic Acids Res. 17:7891-7903; and Joshi et al., 1987, Nucleic Acid Res. 15:9627-9639.

[0148] In some embodiments, a nucleic acid (e.g., encoding GLK1 or bZIP1) can be delivered to the cell in a vector. As used herein, a “vector” is any vehicle capable of facilitating the transfer of the nucleic acid to the cell such that the nucleic acid can be processed and/or expressed in the cell. The vector may transport the nucleic acid to the cells with reduced degradation, relative to the extent of degradation that would result in the absence of the vector. The vector optionally includes gene expression sequences or other components (such as promoters and other regulatory elements) able to enhance expression of the nucleic acid within the cell. The invention also encompasses the cells transfected with these vectors, including those cells previously described. In certain embodiments, the cells are transfected or transformed with a vector that specifically (or preferably) overexpresses CCA1 and/or GLK1 in the vegetative tissues of the plant, but not in the majority of other cell types of the plant.

[0149] To commence a transformation process in certain embodiments, it is first necessary to construct a suitable vector and properly introduce it into the plant cell. Vector(s) employed in the present invention for transformation of a plant cell include an encoding nucleic acid sequence operably associated with a promoter, such as a leaf-specifc promoter. Details of the construction of vectors utilized herein are known to those skilled in the art of plant genetic engineering.

[0150] In general, vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the nucleotide sequences (or precursor nucleotide sequences) of the invention. Viral vectors useful in certain embodiments include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses; adenovirus, or other adeno-associated viruses; mosaic viruses such as tobamoviruses; potyviruses, nepoviruses, and RNA viruses such as retroviruses. One can readily employ other vectors not named but known to the art. Some viral vectors can be based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the nucleotide sequence of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.

[0151] Genetically altered retroviral expression vectors can have general utility for the high-efficiency transduction of nucleic acids. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the cells with viral particles) are well known to those of ordinary skill in the art. Examples of standard protocols can be found in Kriegler, M., 1990, Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York, or Murry, E. J. Ed., 1991, Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J.

[0152] Another-example of a virus for certain applications is the adeno-associated virus, which is a double-stranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of-cell types and species. The adeno-associated virus further has advantages, such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages; and/or lack of superinfection inhibition, which may allow multiple series of transductions.

[0153] Another vector suitable for use with the method provided herein is a plasmid vector. Plasmid vectors, have been extensively described in the art and are well-known to those of skill in the art. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press. These plasmids may have a promoter compatible with the host cell, and the plasmids can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom-designed, for example, using restriction enzymes and ligation reactions, to remove and add specific fragments of DNA or other nucleic acids, as necessary. The present invention also includes vectors for producing nucleic acids or precursor nucleic acids containing a desired nucleotide sequence (which can, for instance, then be cleaved or otherwise processed within the cell to produce a precursor miRNA). These vectors may include a sequence encoding a nucleic acid and an in vivo expression element, as further described below. In some cases, the in vivo expression element includes at least one promoter.

[0154] Where appropriate, the gene(s) for enhanced expression may be optimized for expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons corresponding to the plant of interest. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al., 1989, Nucleic Acids Res. 17:477-498.

[0155] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When desired, the sequence is modified to avoid predicted hairpin secondary mRNA structures. However, it is recognized that in the case of nucleotide sequences encoding the miRNA precursors, one or more hairpin and other secondary structures may be desired for proper processing of the precursor into an mature miRNA and/or for the functional activity of the miRNA in gene silencing.

[0156] The expression cassettes can additionally contain 5′ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al., 1989, PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al., 1991, Nature 353:90-94); untranslated leader from the coat protein miRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al., 1987, Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al., 1989, Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al., 1991, Virology 81:382-385). See also, Della-Cioppa et al., 1987, Plant Physiol. 84:965-968.

[0157] In preparing the expression cassette, the various DNA fragments can be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers can be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0158] An illustrative vector enoding CCA1 for overexpression in plants is described infra. Wang et al., 1998, Cell 93:1207-1217 also describes a vector for overexpressing CCA1 in plants. An illustrative vector enoding GLK1 for overexpression in plants is described infra. Further, Fitter et al., 2002, The Plant Journal 31:713-727 describe an insertion mutant in the GLK1 gene such that expression of GLK1 is disrupted.

6.5 Promoters and Other Regulatory Sequences

[0159] In the broad method of the invention, at least one nucleic acid sequence encoding CCA1, GLK1 or bZIP1 or a nucleic acid designed to disrupt expression of same is operably linked with a promoter, such as a leaf-preferred or leaf-specific promoter. It may be desirable to introduce more than one copy of a polynucleotide into a plant for enhanced expression. For example, multiple copies of a GLK1 polynucleotide would have the effect of increasing production of GLK1 even further in the plant. In specific embodiments, the GLK1 polynucleotide is expressed primarily or entirely in vegetative cells of the plant.

[0160] In general, promoters are found positioned 5′ (upstream) of the genes that they control. Thus, in the construction of promoter gene combinations, the promoter is preferably positioned upstream of the gene and at a distance from the transcription start site that approximates the distance between the promoter and the gene it controls in the natural setting. As is known in the art, some variation in this distance can be tolerated without loss of promoter function. Similarly, the preferred positioning of a regulatory element, such as an enhancer, with respect to a heterologous gene placed under its control reflects its natural position relative to the structural gene it naturally regulates. In certain specific embodiments, bZIP1 is under the control of a seed-specific promoter, and may optionally comprise other regulatory elements that result in constitutive or inducible expression of bZIP1.

[0161] Thus, the nucleic acid, in one embodiment, is operably linked to a gene expression sequence, which directs the expression of the nucleic acid within the cell. A “gene expression sequence,” as used herein, is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the nucleotide sequence to which it is operably linked. The gene expression sequence may, for example, be a eukaryotic promoter or a viral promoter, such as a constitutive or inducible promoter. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription, for instance, as discussed in Maniatis et al., 1987, Science 236:1237. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). In some embodiments, the nucleic acid is linked to a gene expression sequence which permits expression of the nucleic acid in a plant cell. A sequence which permits expression of the nucleic acid in a plant cell is one which is selectively active in the particular plant cell and thereby causes the expression of the nucleic acid in these cells. Those of ordinary skill in the art will be able to easily identify promoters that are capable of expressing a nucleic acid in a cell based on the type of plant cell.

[0162] A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. Generally, the nucleotide sequence and the modulator sequences can be combined with promoters of choice to alter gene expression if the target sequences in the tissue or organ of choice. Thus, the nucleotide sequence or modulator nucleotide sequence can be combined with constitutive, tissue-preferred, inducible, developmental, or other promoters for expression in plants depending upon the desired outcome.

[0163] The selection of a particular promoter and enhancer depends on what cell type is to be used and the mode of delivery. For example, a wide variety of promoters have been isolated from plants and animals, which are functional not only in the cellular source of the promoter, but also in numerous other plant species. There are also other promoters (e.g., viral and Ti-plasmid) which can be used. For example, these promoters include promoters from the Ti-plasmid, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter, and promoters from other open reading frames in the T-DNA, such as ORF7, etc. Promoters isolated from plant viruses include the 35S promoter from cauliflower mosaic virus. Promoters that have been isolated and reported for use in plants include ribulose-1,3-biphosphate carboxylase small subunit promoter, phaseolin promoter, etc. Thus, a variety of promoters and regulatory elements may be used in the expression vectors of the present invention.

[0164] Promoters useful in the compositions and methods provided herein include both natural constitutive and inducible promoters as well as engineered promoters. The CaMV promoters are examples of constitutive promoters. Other constitutive mammalian promoters include, but are not limited to, polymerase promoters as well as the promoters for the following genes: hypoxanthine phosphoribosyl transferase (“HPTR”), adenosine deaminase, pyruvate kinase, and alpha-actin.

[0165] Promoters useful as expression elements of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, a metallothionein promoter can be induced to promote transcription in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art. The in vivo expression element can include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with the initiation of transcription, and can optionally include enhancer sequences or upstream activator sequences.

[0166] For example, in some embodiments an inducible promoter is used to allow control of nucleic acid expression through the presentation of external stimuli (e.g., environmentally inducible promoters), as discussed below. Thus, the timing and amount of nucleic acid expression can be controlled in some cases. Non-limiting examples of expression systems, promoters, inducible promoters, environmentally inducible promoters, and enhancers are well known to those of ordinary skill in the art. Examples include those described in International Patent Application Publications WO 00/12714, WO 00/11175, WO 00/12713, WO 00/03012, WO 00/03017, WO 00/01832, WO 99/50428, WO 99/46976 and U.S. Pat. Nos. 6,028,250, 5,959,176, 5,907,086, 5,898,096, 5,824,857, 5,744,334, 5,689,044, and 5,612,472. A general descriptions of plant expression vectors and reporter genes can also be found in Gruber et al., 1993, “Vectors for Plant Transformation,” in Methods in Plant Molecular Biology & Biotechnology, Glich et al., Eds., p. 89-119, CRC Press.

[0167] For plant expression vectors, viral promoters that can be used in certain embodiments include the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., Nature, 1984, 310:511; Odell et al., Nature, 1985, 313:810); the full-length transcript promoter from Figwort Mosaic Virus (FMV) (Gowda et al., 1989, J. Cell Biochem., 13D: 301) and the coat protein promoter to TMV (Takamatsu et al., 1987, EMBO J. 6:307). Alternatively, plant promoters such as the light-inducible promoter from the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al., 1984, EMBO J., 3:1671; Broglie et al., 1984, Science, 224:838); mannopine synthase promoter (Velten et al., 1984, EMBO J., 3:2723) nopaline synthase (NOS) and octopine synthase (OCS) promoters (carried on tumor-inducing plasmids of Agrobacterium tumefaciens) or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol., 6:559; Severin et al., 1990, Plant Mol. Biol., 15:827) may be used. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus, Rous sarcoma virus, cytomegalovirus, the long terminal repeats of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art.

[0168] To be most useful, an inducible promoter should 1) provide low expression in the absence of the inducer; 2) provide high expression in the presence of the inducer; 3) use an induction scheme that does not interfere with the normal physiology of the plant; and 4) have no effect on the expression of other genes. Examples of inducible promoters useful in plants include those induced by chemical means, such as the yeast metallothionein promoter which is activated by copper ions (Mett et al., Proc. Natl. Acad. Sci., U.S.A., 90:4567, 1993); In2-1 and In2-2 regulator sequences which are activated by substituted benzenesulfonamides, e.g., herbicide safeners (Hershey et al., Plant Mol. Biol., 17:679, 1991); and the GRE regulatory sequences which are induced by glucocorticoids (Schena et al., Proc. Natl. Acad Sci., U.S.A., 88:10421, 1991). Other promoters, both constitutive and inducible will be known to those of skill in the art.

[0169] A number of inducible promoters are known in the art. For resistance genes, a pathogen-inducible promoter can be utilized. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al., 1983, Neth. J. Plant Pathol. 89:245-254; Uknes et al., 1992, Plant Cell 4:645-656; and Van Loon, 1985, Plant Mol. Virol. 4:111-116. Of particular interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al., 1987, Plant Mol. Biol. 9:335-342; Matton et al., 1989, Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al., 1986, Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al., 1988, Mol. Gen. Genet. 2:93-98; and Yang, 1996, Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al., 1996, Plant J. 10:955-966; Zhang et al., 1994, Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al., 1993, Plant J. 3:191-201; Siebertz et al., 1989, Plant Cell 1:961-968; U.S. Pat. No. 5,750,386; Cordero et al., 1992, Physiol. Mol. Plant Path. 41:189-200; and the references cited therein.

[0170] Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter may be used in the DNA constructs of the invention. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan, 1990, Ann. Rev. Phytopath. 28:425-449; Duan et al., 1996, Nature Biotechnology 14:494-498); wunl and wun2, U.S. Pat. No. 5,428,148; winl and win2 (Stanford et al., 1989, Mol. Gen. Genet. 215:200-208); systemin (McGurl et al., 1992, Science 225:1570-1573); WIPI (Rohmeier et al., 1993, Plant Mol. Biol. 22:783-792; Eckelkamp et al., 1993, FEBS Letters 323:73-76); MPI gene (Corderok et al., 1994, Plant J. 6(2):141-150); and the like. Such references are herein incorporated by reference.

[0171] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al., 1991, Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al., 1998, Plant J. 14(2):247-257) and tetramiR167e-inducible and tetramiR167e-repressible promoters (see, for example, Gatz et al., 1991, Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

[0172] Where enhanced expression in particular tissues is desired, tissue-preferred promoters can be utilized. Tissue-preferred promoters include those described by Yamamoto et al., 1997, Plant J. 12(2):255-265; Kawamata et al., 1997, Plant Cell Physiol. 38(7):792-803; Hansen et al., 1997, Mol. Gen Genet. 254(3):337-343; Russell et al., 1997, Transgenic Res. 6(2):157-168; Rinehart et al., 1996, Plant Physiol. 112(3):1331-1341; Van Camp et al., 1996, Plant Physiol. 112(2):525-535; Canevascini et al., 1996, Plant Physiol. 12(2):513-524; Yamamoto et al., 1994, Plant Cell Physiol. 35(5):773-778; Lam, 1994, Results Probl. Cell Differ. 20:181-196; Orozco et al., 1993, Plant Mol. Biol. 23(6): 1129-1138; Matsuoka et al., 1993, Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al., 1993, Plant J 4(3):495-505.

[0173] The particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of structural gene product in the transgenic plant, e.g., GLK1 to cause upregulation of genes such as GLN1.3 and increased nitrogen assimilation, biomass, overall plant growth or yield, and/or other phenotypes described herein, as compared to wild type. The promoters used in the vector constructs of the present invention may be modified, if desired, to affect their control characteristics. In certain embodiments, chimeric promoters can be used.

[0174] There are promoters known which limit expression to particular plant parts or in response to particular stimuli. One skilled in the art will know of many such plant part-specific promoters which would be useful in the present invention. In certain embodiments, to provide pericycle-specific expression, any of a number of promoters from genes in Arabidopsis can be used. In some embodiments, the promoter from one (or more) of the following genes may be used: (i) At1g11080, (ii) At3g60160, (iii) At1g24575, (iv) At3g45160, or (v) At1g23130. In specific embodiments, we will also use (vi) promoter elements from the GFP-marker line used in Gifford et al. (in preparation) (see also, Bonke et al., 2003, Nature 426, 181-6; Tian et al., 2004, Plant Physiol 135, 25-38). Several of the predicted genes have a number of potential orthologs in rice and poplar and thus are predicted that they will be applicable for use in crop species; (i) Os04g44410, Os10g39560, Os06g51370, Os02g42310, Os01g22980, Os05g06660, and Poptr1#568263, Poptr1#555534, Poptr1#365170; (ii) Os04g49900, Os04g49890, Os01g67580, and Poptr1#87573, Poptr1#80582, Poptr1#565079, Poptr1#99223.

[0175] Promoters used in the nucleic acid constructs of the present invention can be modified, if desired, to affect their control characteristics. For example, the CaMV 35S promoter may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of light, to create a promoter which is active in leaves but not in roots. The resulting chimeric promoter may be used as described herein. For purposes of this description, the phrase “CaMV 35S” promoter thus includes variations of CaMV 35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the promoters may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression.

[0176] An efficient plant promoter that may be used in specific embodiments is an “overproducing” or “overexpressing” plant promoter. Overexpressing plant promoters that can be used in the compositions and methods provided herein include the promoter of the small sub-unit (“ss”) of the ribulose-1,5-biphosphate carboxylase from soybean (e.g., Berry-Lowe et al., 1982, J. Molecular & App. Genet., 1:483), and the promoter of the chorophyll a-b binding protein. These two promoters are known to be light-induced in eukaryotic plant cells. For example, see Cashmore, Genetic Engineering of plants: An Agricultural Perspective, p. 29-38; Coruzzi et al., 1983, J. Biol. Chem., 258:1399; and Dunsmuir et al., 1983, J. Molecular & App. Genet., 2:285.

[0177] The promoters and control elements of, e.g., SUCS (root nodules; broadbean; Kuster et al., 1993, Mol Plant Microbe Interact 6:507-14) for roots can be used in compositions and methods provided herein to confer tissue specificity.

[0178] In certain embodiment, two promoter elements can be used in combination, such as, for example, (i) an inducible element responsive to a treatment that can be provided to the plant prior to N-fertilizer treatment, and (ii) a plant tissue-specific expression element to drive expression in the specific tissue alone.

[0179] Any promoter of other expression element described herein or known in the art may be used either alone or in combination with any other promoter or other expression element described herein or known in the art. For example, promoter elements that confer tissue specific expression of a gene can be used with other promoter elements conferring constitutive or inducible expression.

6.6 Isolating Related Promoter Sequences

[0180] Promoter and promoter control elements that are related to those described in herein can also be used in the compositions and methods provided herein. Such related sequence can be isolated utilizing (a) nucleotide sequence identity; (b) coding sequence identity of related, orthologous genes; or (c) common function or gene products.

[0181] Relatives can include both naturally occurring promoters and non-natural promoter sequences. Non-natural related promoters include nucleotide substitutions, insertions or deletions of naturally-occurring promoter sequences that do not substantially affect transcription modulation activity. For example, the binding of relevant DNA binding proteins can still occur with the non-natural promoter sequences and promoter control elements of the present invention.

[0182] According to current knowledge, promoter sequences and promoter control elements exist as functionally important regions, such as protein binding sites, and spacer regions. These spacer regions are apparently required for proper positioning of the protein binding sites. Thus, nucleotide substitutions, insertions and deletions can be tolerated in these spacer regions to a certain degree without loss of function.

[0183] In contrast, less variation is permissible in the functionally important regions, since changes in the sequence can interfere with protein binding. Nonetheless, some variation in the functionally important regions is permissible so long as function is conserved.

[0184] The effects of substitutions, insertions and deletions to the promoter sequences or promoter control elements may be to increase or decrease the binding of relevant DNA binding proteins to modulate transcript levels of a polynucleotide to be transcribed. Effects may include tissue-specific or condition-specific modulation of transcript levels of the polypeptide to be transcribed. Polynucleotides representing changes to the nucleotide sequence of the DNA-protein contact region by insertion of additional nucleotides, changes to identity of relevant nucleotides, including use of chemically-modified bases, or deletion of one or more nucleotides are considered encompassed by the present invention.

[0185] Typically, related promoters exhibit at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, even more preferably, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. Such sequence identity can be calculated by the algorithms and computers programs described above.

[0186] Usually, such sequence identity is exhibited in an alignment region that is at least 75% of the length of a sequence or corresponding full-length sequence of a promoter described herein; more usually at least 80%; more usually, at least 85%, more usually at least 90%, and most usually at least 95%, even more usually, at least 96%, at least 97%, at least 98% or at least 99% of the length of a sequence of a promoter described herein.

[0187] The percentage of the alignment length is calculated by counting the number of residues of the sequence in region of strongest alignment, e.g., a continuous region of the sequence that contains the greatest number of residues that are identical to the residues between two sequences that are being aligned. The number of residues in the region of strongest alignment is divided by the total residue length of a sequence of a promoter described herein. These related promoters may exhibit similar preferential transcription as those promoters described herein.

[0188] In certain embodiments, a promoter, such as a leaf-preferred or leaf-specific promoter, can be identified by sequence homology or sequence identity to any root specific promoter identified herein. In other embodiments, orthologous genes identified herein as leaf-specific genes (e.g., the same gene or different gene that if functionally equivalent) for a given species can be identified and the associated promoter can also be used in the compositions and methods provided herein. For example, using high, medium or low stringency conditions, standard promoter rules can be used to identify other useful promoters from orthologous genes for use in the compositions and methods provided herein. In specific embodiments, the orthologous gene is a gene expressed only or primarily in the root, such as pericycle cells.

[0189] Polynucleotides can be tested for activity by cloning the sequence into an appropriate vector, transforming plants with the construct and assaying for marker gene expression. Recombinant DNA constructs can be prepared, which comprise the polynucleotide sequences of the invention inserted into a vector suitable for transformation of plant cells. The construct can be made using standard recombinant DNA techniques (Sambrook et al., 1989) and can be introduced to the species of interest by Agrobacterium-mediated transformation or by other means of transformation as referenced below.

[0190] The vector backbone can be any of those typical in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs and PACs and vectors of the sort described by (a) BAC: Shizuya et al., 1992, Proc. Natl. Acad. Sci. USA 89: 8794-8797; Hamilton et al., 1996, Proc. Natl. Acad. Sci. USA 93: 9975-9979; (b) YAC: Burke et al., 1987, Science 236:806-812; (c) PAC: Stemberg N. et al., 1990, Proc Natl Acad Sci USA. January; 87(1):103-7; (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., 1995, Nucl Acids Res 23: 4850-4856; (e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et al., 1983, J. Mol. Biol. 170: 827-842; or Insertion vector, e.g., Huynh et al., 1985, In: Glover N M (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press; T-DNA gene fusion vectors: Walden et al., 1990, Mol Cell Biol 1: 175-194; and (g) Plasmid vectors: Sambrook et al., infra.

[0191] Typically, the construct comprises a vector containing a sequence of the present invention operationally linked to any marker gene. The polynucleotide was identified as a promoter by the expression of the marker gene. Although many marker genes can be used, Green Fluorescent Protein (GFP) is preferred. The vector may also comprise a marker gene that confers a selectable phenotype on plant cells. The marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or phosphinotricin (see below). Vectors can also include origins of replication, scaffold attachment regions (SARs), markers, homologous sequences, introns, etc.

6.7 Tissue or Cell-Type Preferential Transcription

[0192] The invention also provides a method of providing increased transcription of a nucleic acid sequence in a selected tissue, such as vegetative tissues, leaves, seeds, fruit, etc. The method comprises growing a plant having integrated in its genome a nucleic acid construct comprising, an exogeneous gene encoding CCA1, GLK1 or bZIP 1, said gene operably associated with a tissue specific promoter, whereby transcription of said gene is increased (or decreased) in said selected tissue.

[0193] Specific promoters may be used in the compositions and methods provided herein. As used herein, “specific promoters” refers to a subset of promoters that have a high preference for modulating transcript levels in a specific tissue or organ or cell and/or at a specific time during development of an organism. By “high preference” is meant at least 3-fold, preferably 5-fold, more preferably at least 10-fold still more preferably at least 20-fold, 50-fold or 100-fold increase in transcript levels under the specific condition over the transcription under any other reference condition considered. Typical examples of temporal and/or tissue or organ specific promoters of plant origin that can be used in the compositions and methods of the present invention, inlcude RCc2 and RCc3, promoters that direct root-specific gene transcription in rice (Xu et al., 1995, Plant Mol. Biol. 27:237 and TobRB27, a root-specific promoter from tobacco (Yamamoto et al., 1991, Plant Cell 3:371). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues or organs, such as roots

[0194] “Preferential transcription” is defined as transcription that occurs in a particular pattern of cell types or developmental times or in response to specific stimuli or combination thereof. Non-limitative examples of preferential transcription include: high transcript levels of a desired sequence in root tissues; detectable transcript levels of a desired sequence in certain cell types during embryogenesis; and low transcript levels of a desired sequence under drought conditions. Such preferential transcription can be determined by measuring initiation, rate, and/or levels of transcription.

[0195] Promoters and control elements providing preferential transcription in a root can modulate growth, metabolism, development, nutrient uptake, nitrogen fixation, or modulate energy and nutrient utilization in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or in a leaf, is useful (1) to modulate root size, shape, and development; (2) to modulate the number of roots, or root hairs; (3) to modulate mineral, fertilizer, or water uptake; (4) to modulate transport of nutrients; or (4) to modulate energy or nutrient usage in relation to other organs and tissues. Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit nutrient usage in a root to be directed to the leaf instead, for instance.

[0196] Typically, promoter or control elements, which provide preferential transcription in cells, tissues, or organs of a root, produce transcript levels that are statistically significant as compared to other cells, organs or tissues. For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.

[0197] Root-preferred promoters are known and can be selected from the many available from the literature. See, for example, Hire et al., 1992, Plant Mol. Biol. 20(2): 207-218 (soybean root-preferred glutamine synthetase gene); Keller and Baumgartner, 1991, Plant Cell 3(10):1051-1061 (root-preferred control element in the GRP 1.8 gene of French bean); Sanger et al., 1990, Plant Mol. Biol. 14(3):433-443 (root-preferred promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); Miao et al., 1991, Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). Bogusz et al., 1990, Plant Cell 2(7):633-641 (root-preferred promoters from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa). Leach and Aoyagi, 1991, Plant Science (Limerick) 79(1):69-76 (ro1C and ro1D root-inducing genes of Agrobacterium rhizogenes); Teeri et al., 1989, EMBO J. 8(2):343-350) (octopine synthase and TR2′ gene); (VfENOD-GRP3 gene promoter); Kuster et al., 1995, Plant Mol. Biol. 29(4):759-772 and Capana et al., 1994, Plant Mol. Biol. 25(4):681-691 ro1B promoter. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179, root-specific glutamine synthetase (see Tingey et al., 1987, EMBO J., 6:1-9; Edwards et al., 1990, PNAS, 87:3439-3463). In addition, promoters of the above-listed orthologous genes in other plant species can be identified and used in the compositions and methods provided herein.

[0198] In specific embodiments, the compositions and methods provided herein use leaf-specific promoters operably associated to a nucleotide encoding bZIP1. In certain embodiments, the promoter is a constitutive or inducible promoter. In another specific embodiment, the compositions and methods provided herein use vegetative tissue-specific promoters operably associated to a nucleotide encoding CCA1 and/or GLK1. In certain embodiments, the promoter is a constitutive or inducible promoter.

6.8 Selectable Markers

[0199] Using any gene transfer technique, such as the above-listed techniques, an expression vector harboring the nucleic acid may be transformed into a cell to achieve temporary or prolonged expression. Any suitable expression system may be used, so long as it is capable of undergoing transformation and expressing of the precursor nucleic acid in the cell. In one embodiment, a pET vector (Novagen, Madison, Wis.), or a pBI vector (Clontech, Palo Alto, Calif.) is used as the expression vector. In some embodiments an expression vector further encoding a green fluorescent protein (“GFP”) is used to allow simple selection of transfected cells and to monitor expression levels. Non-limiting examples of such vectors include Clontech's “Living Colors Vectors” pEYFP and pEYFP-C.

[0200] The recombinant construct of the present invention may include a selectable marker for propagation of the construct. For example, a construct to be propagated in bacteria preferably contains an antibiotic resistance gene, such as one that confers resistance to kanamycin, tetracycline, streptomycin, or chloramphenicol. Suitable vectors for propagating the construct include plasmids, cosmids, bacteriophages or viruses, to name but a few.

[0201] In addition, the recombinant constructs may include plant-expressible selectable or screenable marker genes for isolating, identifying or tracking of plant cells transformed by these constructs. Selectable markers include, but are not limited to, genes that confer antibiotic resistances (e.g., resistance to kanamycin or hygromycin) or herbicide resistance (e.g., resistance to sulfonylurea, phosphinothricin, or glyphosate). Screenable markers include, but are not limited to, the genes encoding .beta.-glucuronidase (Jefferson, 1987, Plant Molec Biol. Rep 5:387-405), luciferase (Ow et al., 1986, Science 234:856-859), B and C1 gene products that regulate anthocyanin pigment production (Goff et al., 1990, EMBO J 9:2517-2522).

[0202] In some cases, a selectable marker may be included with the nucleic acid being delivered to the cell. As used herein, the term “selectable marker” refers to the use of a gene that encodes an enzymatic or other detectable activity (e.g., luminescence or fluorescence) that confers the ability to grow in medium lacking what would otherwise be an essential nutrient. A selectable marker may also confer resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be “dominant” in some cases; a dominant selectable marker encodes an enzymatic or other activity (e.g., luminescence or fluorescence) that can be detected in any cell or cell line.

[0203] Optionally, a selectable marker may be associated with the CCA1-, GLK1 or bZIP1-encoding nucleic acid. Preferably, the marker gene is an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Examples of suitable selectable markers include adenosine deaminase, dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidine kinase, xanthine-guanine phospho-ribosyltransferase and amino-glycoside 3′-O-phosphotransferase II. Other suitable markers will be known to those of skill in the art.

6.9 Selection and Identification of Transformed Plants and Plant Cells

[0204] According to the present invention, desired plants may be obtained by engineering the disclosed gene constructs into a variety of plant cell types, including but not limited to, protoplasts, tissue culture cells, tissue and organ explants, pollens, embryos as well as whole plants. In specific embodiments, the gene constructs are engineered into leaves, preferably with the use of a leaf-specific promoter.

[0205] In an embodiment of the present invention, the engineered plant material is selected or screened for transformants (those that have incorporated or integrated the introduced gene construct(s)) following the approaches and methods described below. An isolated transformant may then be regenerated into a plant. Alternatively, the engineered plant material may be regenerated into a plant or plantlet before subjecting the derived plant or plantlet to selection or screening for the marker gene traits. Procedures for regenerating plants from plant cells, tissues or organs, either before or after selecting or screening for marker gene(s), are well known to those skilled in the art.

[0206] A transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the β-glucuronidase, luciferase, B or C1 genes) that may be present on the recombinant nucleic acid constructs of the present invention. Such selection and screening methodologies are well known to those skilled in the art.

[0207] Physical and biochemical methods also may be also to identify plant or plant cell transformants containing the gene constructs of the present invention. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S1 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art.

6.10 Screening of Transformed Plants for Those With Improved Agronomic Traits

[0208] According to the present invention, to obtain plants with improved agronomic characteristics, the transformed plants may be screened for those exhibiting the desired physiological alteration. Alternatively, the transformed plants may be directly screened for those exhibiting the desired agronomic changes. A plant with the desired improvement can be isolated by screening the engineered plants for altered expression pattern or level of CCA1, GLK1 and/or bZIP1, or downstream gene products such as GLN1.3 or ASN1. A plant can also be screened for nutrient uptake, overall increased plant growth rate, enhanced vegetative yield, improved reproductive yields, increased levels of glutamine or asparagine, or increased nitrogen usage or storage. The screening of the engineered plants can involve Southern analysis to confirm the presence and number of transgene insertions; Northern analysis, RNase protection, primer extension, reverse transcriptase/PCR and the like to measure mRNA levels; measuring the amino acid composition, free amino acid pool or total nitrogen content of various plant tissues; measuring growth rates in terms of fresh weight gains over time; or measuring plant yield in terms of total dry weight and/or total seed weight, or a combination of any of the above methods. The procedures and methods for examining these parameters are well known to those skilled in the art.

[0209] In other embodiments, the screening of the transformed plants may be for improved agronomic characteristics (e.g., faster growth, greater vegetative or reproductive yields, or improved protein contents, etc.), as compared to unengineered progenitor plants, when cultivated under growth conditions (i.e., cultivated using soils or media containing or receiving sufficient amounts of nitrogen nutrients to sustain healthy plant growth).

[0210] Plants exhibiting increased growth and/or yield as compared with wild-type plants can be selected by visual observation, methods provided in the Examples, or other methods known in the art.

[0211] A “plant capable of increased yield” refers to a plant that can be induced to express its endogenous CCA1, GLK1 and/or bZIP1 gene to achieve increased yield. The term “promoter inducing amount” refers to that amount of an agent necessary to elevate such gene expression above such expression in a plant cell not contacted with the agent, by stimulating the endogenous promoter. For example, a transcription factor or a chemical agent may be used to elevate gene expression from native or chimeric CCA1, GLK1 and/or bZIP1 promoter, thus inducing the promoter and gene expression.

6.11 Cells

[0212] Optionally, germ line cells may be used in the methods described herein rather than, or in addition to, somatic cells. The term “germ line cells” refers to cells in the plant organism which can trace their eventual cell lineage to either the male or female reproductive cell of the plant. Other cells, referred to as “somatic cells” are cells which give rise to leaves, roots and vascular elements which, although important to the plant, do not directly give rise to gamete cells. Somatic cells, however, also may be used. With regard to callus and suspension cells which have somatic embryogenesis, many or most of the cells in the culture have the potential capacity to give rise to an adult plant. If the plant originates from single cells or a small number of cells from the embryogenic callus or suspension culture, the cells in the callus and suspension can therefore be referred to as germ cells. In the case of immature embryos which are prepared for treatment by the methods described herein, certain cells in the apical meristem region of the plant have been shown to produce a cell lineage which eventually gives rise to the female and male reproductive organs. With many or most species, the apical meristem is generally regarded as giving rise to the lineage that eventually will give rise to the gamete cells. An example of a non-gamete cell in an embryo would be the first leaf primordia in corn which is destined to give rise only to the first leaf and none of the reproductive structures.

6.12 Plant Regeneration

[0213] Following transformation, a plant may be regenerated, e.g., from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues, and organs of the plant. Available techniques are reviewed in Vasil et al., 1984, in Cell Culture and Somatic Cell Genetics of Plants, Vols. I, II, and III, Laboratory Procedures and Their Applications (Academic Press); and Weissbach et al., 1989, Methods For Plant Mol. Biol.

[0214] The transformed plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.

[0215] Normally, a plant cell is regenerated to obtain a whole plant from the transformation process. The term “growing” or “regeneration” as used herein means growing a whole plant from a plant cell, a group of plant cells, a plant part (including seeds), or a plant piece (e.g., from a protoplast, callus, or tissue part).

[0216] Regeneration from protoplasts varies from species to species of plants, but generally a suspension of protoplasts is first made. In certain species, embryo formation can then be induced from the protoplast suspension. The culture media will generally contain various amino acids and hormones, necessary for growth and regeneration. Examples of hormones utilized include auxins and cytokinins. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these variables are controlled, regeneration is reproducible.

[0217] Regeneration also occurs from plant callus, explants, organs or parts. Transformation can be performed in the context of organ or plant part regeneration (see Methods in Enzymology, Vol. 118 and Klee et al., Annual Review of Plant Physiology, 38:467, 1987). Utilizing the leaf disk-transformation-regeneration method of Horsch et al., Science, 227:1229, 1985, disks are cultured on selective media, followed by shoot formation in about 2-4 weeks. Shoots that develop are excised from calli and transplanted to appropriate root-inducing selective medium. Rooted plantlets are transplanted to soil as soon as possible after roots appear. The plantlets can be repotted as required, until reaching maturity.

[0218] In vegetatively propagated crops, the mature transgenic plants are propagated by utilizing cuttings or tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use.

[0219] In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The resulting inbred plant produces seed containing the newly introduced foreign gene(s). These seeds can be grown to produce plants that would produce the selected phenotype, e.g., increased lateral root growth, uptake of nutrients, overall plant growth and/or vegetative or reproductive yields.

[0220] Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences. Transgenic plants expressing the selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.

[0221] A preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-transgenic). Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

[0222] Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium. For transformation and regeneration of maize see, Gordon-Kamm et al., 1990, The Plant Cell, 2:603-618.

[0223] Plants cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., 1983, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp. 124-176; and Binding, Regeneration of Plants, Plant Protoplasts, 1985, CRC Press, Boca Raton, pp. 21-73.

[0224] The regeneration of plants containing the foreign gene introduced by Agrobacterium from leaf explants can be achieved as described by Horsch et al., 1985, Science, 227:1229-1231. In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al., 1983, Proc. Natl. Acad. Sci. (U.S.A.), 80:4803. This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.

[0225] The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., 1988, Academic Press, Inc., San Diego, Calif. This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., 1994, Springer, New York 1994; Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., 1988, American Society of Agronomy, Madison, Wis.

6.13 Plants and Plant Cells

[0226] Also provided herein are a plant cell having the nucleotide sequence constructs of the invention. A further aspect of the present invention provides a method of making such a plant cell involving introduction of a vector including the construct into a plant cell. For integration of the construct into the plant genome, such introduction will be followed by recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome. RNA encoded by the introduced nucleic acid construct may then be transcribed in the cell and descendants thereof, including cells in plants regenerated from transformed material. A gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, so such descendants should show the desired phenotype.

[0227] In certain embodiments, a plant cell comprises a GLK1 nucleotide sequence operably associated with a vegetative tissue specific promoter, which is optionally a constitutive or inducible promoter. In other embodiments, a plant cell comprises multiple copies of a GLK1 operably associated with a vegetative tissue specific promoter. In specific embodiments provided herein are plants (and plant cells thereof) that overexpress, contitutionally express and/or inducibly express GLK1 in the vegetative tissues of the plant, as compared to other tissues in the plant and/or as compared to a wild type plant.

[0228] The present invention also provides a plant comprising a plant cell as disclosed. Transformed seeds and plant parts are also encompassed.

[0229] In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, off-spring, clone or descendant. Plant extracts and derivatives are also provided.

[0230] Any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and algae (e.g., Chlamydomonas reinhardtii) may be used in the compositions and methods provided herein. Non-limiting examples of plants include plants from the genus Arabidopsis or the genus Oryza. Other examples include plants from the genuses Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium, Arachis, Beta, Betula, Brassica, Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca, Linum, Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus, Populus, Prunus, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia.

[0231] Plants included in the invention are any plants amenable to transformation techniques, including gymnosperms and angiosperms, both monocotyledons and dicotyledons.

[0232] Examples of monocotyledonous angiosperms include, but are not limited to, asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oats and other cereal grains.

[0233] Examples of dicotyledonous angiosperms include, but are not limited to tomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops or Brassica oleracea (e.g., cabbage, broccoli, cauliflower, brussel sprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers and various ornamentals.

[0234] Examples of woody species include poplar, pine, sequoia, cedar, oak, etc.

[0235] Still other examples of plants include, but are not limited to, wheat, cauliflower, tomato, tobacco, corn, petunia, trees, etc.

[0236] In certain embodiments, plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassaya, barley, pea, and other root, tuber, or seed crops. Exemplary cereal crops used in the compositions and methods of the invention include, but are not limited to, any species of grass, or grain plant (e.g., barley, corn, oats, rice, wild rice, rye, wheat, millet, sorghum, triticale, etc.), non-grass plants (e.g., buckwheat flax, legumes or soybeans, etc.). Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Other seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Other important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, and sorghum. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

[0237] Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations and geraniums. The present invention may also be applied to tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus, and pine.

[0238] The present invention may be used for transformation of other plant species, including, but not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum, Nicotiana benthamiana), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, Arabidopsis spp., vegetables, ornamentals, and conifers.

6.14 Cultivation

[0239] Methods of cultivation of plants are well known in the art. For example, for the cultivation of wheat see Alcoz et al., 1993, Agronomy Journal 85:1198-1203; Rao and Dao, 1992, J. Am. Soc. Agronomy 84:1028-1032; Howard and Lessman, 1991, Agronomy Journal 83:208-211; for the cultivation of corn see Tollenear et al., 1993, Agronomy Journal 85:251-255; Straw et al., Tennessee Farm and Home Science: Progress Report, Spring 1993, 166:20-24; Miles, S. R., 1934, J. Am. Soc. Agronomy 26:129-137; Dara et al., 1992, J. Am. Soc. Agronomy 84:1006-1010; Binford et al., 1992, Agronomy Journal 84:53-59; for the cultivation of soybean see Chen et al., 1992, Canadian Journal of Plant Science 72:1049-1056; Wallace et al., 1990, Journal of Plant Nutrition 13:1523-1537; for the cultivation of rice see Oritani and Yoshida, 1984, Japanese Journal of Crop Science 53:204-212; for the cultivation of linseed see Diepenbrock and Porksen, 1992, Industrial Crops and Products 1:165-173; for the cultivation of tomato see Grubinger et al., 1993, Journal of the American Society for Horticultural Science 118:212-216; Cerne, M., 1990, Acta Horticulture 277:179-182; for the cultivation of pineapple see Magistad et al., 1932, J. Am. Soc. Agronomy 24:610-622; Asoegwu, S. N., 1988, Fertilizer Research 15:203-210; Asoegwu, S. N., 1987, Fruits 42:505-509; for the cultivation of lettuce see Richardson and Hardgrave, 1992, Journal of the Science of Food and Agriculture 59:345-349; for the cultivation of mint see Munsi, P. S., 1992, Acta Horticulturae 306:436-443; for the cultivation of camomile see Letchamo, W., 1992, Acta Horticulturae 306:375-384; for the cultivation of tobacco see Sisson et al., 1991, Crop Science 31:1615-1620; for the cultivation of potato see Porter and Sisson, 1991, American Potato Journal, 68:493-505; for the cultivation of brassica crops see Rahn et al., 1992, Conference “Proceedings, second congress of the European Society for Agronomy”Warwick Univ., p.424-425; for the cultivation of banana see Hegde and Srinivas, 1991, Tropical Agriculture 68:331-334; Langenegger and Smith, 1988, Fruits 43:639-643; for the cultivation of strawberries see Human and Kotze, 1990, Communications in Soil Science and Plant Analysis 21:771-782; for the cultivation of songhum see Mahalle and Seth, 1989, Indian Journal of Agricultural Sciences 59:395-397; for the cultivation of plantain see Anjorin and Obigbesan, 1985, Conference “International Cooperation for Effective Plantain and Banana Research” Proceedings of the third meeting. Abidjan, Ivory Coast, p. 115-117; for the cultivation of sugar cane see Yadav, R. L., 1986, Fertiliser News 31:17-22; Yadav and Sharma, 1983, Indian Journal of Agricultural Sciences 53:38-43; for the cultivation of sugar beet see Draycott et al., 1983, Conference “Symposium Nitrogen and Sugar Beet” International Institute for Sugar Beet Research--Brussels Belgium, p. 293-303. See also Goh and Haynes, 1986, “Nitrogen and Agronomic Practice” in Mineral Nitrogen in the Plant-Soil System, Academic Press, Inc., Orlando, Fla., p. 379-468; Engelstad, 0. P., 1985, Fertilizer Technology and Use, Third Edition, Soil Science Society of America, p.633; Yadav and Sharmna, 1983, Indian Journal of Agricultural Sciences, 53:3-43.

6.15 Products of Transgenic Plants

[0240] Engineered plants exhibiting the desired physiological and/or agronomic changes can be used directly in agricultural production.

[0241] Thus, provided herein are products derived from the transgenic plants or methods of producing transgenic plants provided herein. In certain embodiments, the products are commercial products. Some non-limiting example include genetically engineered trees for e.g., the production of pulp, paper, paper products or lumber; tobacco, e.g., for the production of cigarettes, cigars, or chewing tobacco; crops, e.g., for the production of fruits, vegetables and other food, including grains, e.g., for the production of wheat, bread, flour, rice, corn; and canola, sunflower, e.g., for the production of oils or biofuels.

[0242] In certain embodiments, commercial products are derived from a genetically engineered (e.g., comprising overexpression of GLK1 in the vegetative tissues of the plant) species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and algae (e.g., Chlamydomonas reinhardtii), which may be used in the compositions and methods provided herein. Non-limiting examples of plants include plants from the genus Arabidopsis or the genus Oryza. Other examples include plants from the genuses Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium, Arachis, Beta, Betula, Brassica, Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca, Linum, Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus, Populus, Prunus, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia.

[0243] In some embodiments, commercial products are derived from a genetically engineered gymnosperms and angiosperms, both monocotyledons and dicotyledons. Examples of monocotyledonous angiosperms include, but are not limited to, asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oats and other cereal grains. Examples of dicotyledonous angiosperms include, but are not limited to tomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops or Brassica oleracea (e.g., cabbage, broccoli, cauliflower, brussel sprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers and various ornamentals.

[0244] In certain embodiments, commercial products are derived from a genetically engineered (e.g., comprising overexpression of bZIP1 in the leaves or seeds of the plant) woody species, such as poplar, pine, sequoia, cedar, oak, etc.

[0245] In other embodiments, commercial products are derived from a genetically engineered (e.g., comprising overexpression of CCA1 and GLK1 in the vegetative tissues of the plant) plant including, but are not limited to, wheat, cauliflower, tomato, tobacco, corn, petunia, trees, etc.

[0246] In certain embodiments, commercial products are derived from a genetically engineered crop plants, for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassaya, barley, pea, and other root, tuber, or seed crops. In one embodiment, commercial products are derived from a genetically engineered (e.g., comprising overexpression of CCA1 and GLK1 and underexpression of bZIP1 in the vegetative tissues of the plant) cereal crops, including, but are not limited to, any species of grass, or grain plant (e.g., barley, corn, oats, rice, wild rice, rye, wheat, millet, sorghum, triticale, etc.), non-grass plants (e.g., buckwheat flax, legumes or soybeans, etc.). In another embodiments, commercial products are derived from a genetically engineered (e.g., comprising overexpression of bZIP1 and optionally underexpression of CCA1 and/or GLK1 in leaf or seed tissue of the plant) grain plants that provide seeds of interest, oil-seed plants and leguminous plants. In other embodiments, commercial products are derived from a genetically engineered grain seed plants, such as corn, wheat, barley, rice, sorghum, rye, etc. In yet other embodiments, commercial products are derived from a genetically engineered (e.g., comprising overexpression of bZIP1 and optionally underexpression of CCA1 and/or GLK1 in leaf or seed tissue of the plant) oil seed plants, such as cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. In certain embodiments, commercial products are derived from a genetically engineered oil-seed rape, sugar beet, maize, sunflower, soybean, or sorghum. In some embodiments, commercial products are derived from a genetically engineered leguminous plants, such as beans and peas (e.g., guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.)

[0247] In certain embodiments, commercial products are derived from a genetically engineered horticultural plant of the present invention, such as lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations and geraniums; tomato, tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus, and pine.

[0248] In still other embodiments, commercial products are derived from a genetically engineered corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum, Nicotiana benthamiana), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, Arabidopsis spp., vegetables, ornamentals, and conifers.

6.16 Kits

[0249] In one aspect, the present invention provides any of the above-mentioned compositions in kits, optionally including instructions for use of the composition e.g., for the overexpression or underexpression of CCA1, GLK1 or bZIP 1. The “kit” typically defines a package including one or more compositions of the invention and the instructions, and/or analogs, derivatives, or functionally equivalent compositions thereof. Thus, for example, the kit can include a description of use of the composition for participation in any technique associated in the overexpression or underexpression of genes. The kit can include a description of use of the compositions as discussed herein. Instructions also may be provided for use of the composition in any suitable technique as previously described. The instructions may be of any form provided in connection with the composition.

[0250] The kits described herein may also contain one or more containers, which may contain the inventive composition and other ingredients as previously described. The kits also may contain instructions for mixing, diluting, and/or administrating the compositions in some cases. The kits also can include other containers with one or more solvents, surfactants, preservative and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting and/or administrating the compositions.

[0251] The compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders. When the composition provided is a dry powder, the composition may be reconstituted by the addition of a suitable solvent, which may also be provided. In embodiments where liquid forms of the composition are used, the liquid form may be concentrated or ready to use. The solvent will depend on the active compound(s) within the composition. Suitable solvents are well known, for example as previously described, and are available in the literature.

[0252] The invention also involves, in another aspect, promotion of the overexpression of a master regulatory gene of the present invention, e.g., CCA1/GLK1/bZIP1, according to any of the systems or methods described herein. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with compositions of the invention. “Instructions” can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.

7. EXAMPLE

7.1 Introduction

[0253] Nitrogen (N) is an essential nutrient and a metabolic signal that is sensed and transduced resulting in the control of gene expression in plants. Studies using nitrate reductase (NR) mutant plants, have shown that nitrate can serve as a metabolic signal for inorganic N that regulates gene expression in Arabidopsis thaliana and other plant species (Wang et al., 2004, Plant Physiol 136:2512-2522; Wang et al., 2003, Plant Physiol 132:556-567; Forde, 2002, Ann. Rev. Plant Biology 53:203-224; Scheible, et al., 2004, Plant Physiol 136:2483-2499). There is also ample though less direct evidence that the assimilated forms of N such as Glu or Gln may also serve as signals that regulate gene expression in plants (Rawat et al., 1999, Plant J 19:143-152; Oliveira et al., 1999, Plant Physiol 121:301-310). The ability of plants to sense and respond to levels of inorganic and organic N metabolites provides a mechanism to balance the availability of organic N resources within the plant with the need for N uptake. Because nitrate uptake, reduction and its assimilation into organic form require energy, a mechanism that activates this N assimilatory pathway based on sensing levels of organic N available in the plant is an efficient way to control N-use efficiency (Forde, 2002, Ann. Rev. Plant Biology 53:203-224). In plants, the transcription of genes involved in the uptake and assimilation of inorganic N is induced when levels of organic N are low. Conversely, the uptake and reduction of inorganic N are shut off when levels of organic N are high (reviewed in Scheible, et al., 1997, Plant Cell 9:783-798).

[0254] Recent microarray studies have shown that nitrate can cause changes in the expression of a large number of genes in Arabidopsis (Wang et al., 2004, Plant Physiol 136:2512-2522; Wang et al., 2003, Plant Physiol 132:556-567). Treatment of Arabidopsis seedlings with low levels of nitrate has been shown to increase the levels of mRNA for hundreds of genes within minutes of exposure. The nitrate-responsive genes include nitrate transporters, NR and nitrite reductase, putative transcription factors, stress responses genes, as well as genes whose products play roles in glycolysis, iron metabolism, and sulfate uptake (Wang et al., 2004, Plant Physiol 136:2512-2522; Wang et al., 2003, Plant Physiol 132:556-567). In a related study, N-starved plants underwent a transcriptome/metabolome analysis 30 min and 3 h after nitrate treatment (Scheible, et al., 2004, Plant Physiol 136:2483-2499). The expression of nitrate transporters (at 30 min) preceded the induction of amino acid biosynthetic genes and the repression of amino acid breakdown genes (at 3 h). In addition, increases in amino acid levels were observed, consistent with the changes in expression of the cognate amino acid biosynthesis genes. Putative nitrate-responsive regulatory factors including transcription factors, protein kinases/phosphatases and trehalose and hormone metabolic genes were also identified in that study. Recently, using a NR-null mutant, it was shown that nitrate and not a product of nitrate reduction and assimilation, regulates the expression of genes involved in energy production, metabolism, glycolysis and gluconeogenesis (Wang et al., 2004, Plant Physiol 136:2512-2522).

[0255] Nitrogen metabolism genes can be regulated by negative feedback of the products of N assimilation including downstream organic N metabolites such as Glu or Gln. For example, the expression of the ammonium transporter gene ammonium transporter 1 (AMT1.1) is repressed in treatments with high levels of inorganic N. It has been shown that this repression is blocked by methionine sulfoximine (MSX), a non-metabolizable analog of Glu that irreversibly inhibits glutamine synthetase and hence blocks N assimilation into Gln (Rawat et al., 1999, Plant J 19:143-152). Thus, it appears that organic forms of N may regulate the uptake of N in plants. In addition, the genes encoding asparagine synthetase 1 (ASND) and 2 (ASN2), are differentially regulated by organic and inorganic N sources. Organic N treatments were shown to positively regulate levels ofASN1 mRNA (Oliveira et al., 1999, Plant Physiol 121:301-310), whereas ASN2 gene expression appears to be responsive to inorganic N sources and not a downstream metabolite (Wang et al., 2004, Plant Physiol 136:2512-2522). Together, these studies prompt a model in which both inorganic as well as organic N sources can each regulate plant gene expression affecting N uptake, reduction and assimilation.

[0256] In study presented below, a genomic approach was used to identify gene networks whose expression is regulated by Glu or Glu-derived metabolites (organic N) in plants. Plants were treated with inorganic nitrogen sources in the presence or absence of MSX, which served to inhibit the assimilation of ammonium into Glu/Gln by blocking glutamine synthetase. The rationale for this approach was that a subset of nitrogen-responsive genes responding specifically to an organic signal (e.g. Glu/Gln) would not respond to nitrogen treatment if the synthesis of Glu/Gln was blocked by transient MSX treatments. Network analysis of the genes that responded to organic N revealed that transcription control of gene expression is important for a subnetwork of metabolic genes involved in the synthesis and degradation of asparagine (Asn), an important nitrogen-transport/storage compound synthesized when levels of nitrogen are abundant and degraded when nitrogen reserves are mobilized. The metabolic gene network discovered in this analysis provides molecular evidence for regulation of N-use at the level of gene expression. Moreover, the transcription factors regulated by organic N associated with this network provide a mechanistic link between circadian clock function and N-assimilation in plants.

7.2 Materials and Methods

[0257] Plant growth conditions. Wild type Arabidopsis thaliana ecotype Columbia-0 strain was used in all experiments unless indicated otherwise. Seeds were surface sterilized with ethanol and bleach as previously described (Brenner et al., 2000, Plant Physiol. 124:1615-1624) and sowed onto basal MS salts (Sigma, St Louis, Mo.) with 0.5% (w/v) sucrose, 0.8% BactoAgar, and 1mM KNO.sub.3. After 14 days under long day (16 hours light: 8 hours dark) at 22° C., plants were transiently treated for 2h in the light at the start of their light cycle by transferring them to basal medium with 0.5% sucrose and a combination of inorganic nitrogen sources (20 mM KNO.sub.3 and 20 mM NH.sub.4NO.sub.3) with or without 1 mM MSX (Sigma M-5379), 10 mM glutamate (Sigma G-1501) and/or 10 mM glutamine (Sigma G-3126): N; N+MSX; N+MSX+Glu respectively.

[0258] RNA isolation and quantitative real time PCR. RNA was isolated from whole plants with the TRIzol reagent and according to the instructions of the manufacturer (InVitrogen, Carlsbad, Calif.). cDNA synthesis from whole mRNA extractions was carried out according to kit manufacturer instructions (Invitrogen, Catalog number 11146-024). Real time quantitative PCR was carried out with a LightCycler (Roche Diagnostics, Mannheim, Germany) as described previously (Thum, K. E., Shasha, D. E., Lejay, L. V. & Coruzzi, G. M. (2003) Plant Physiol 132, 440-52).

[0259] Microarray experiments and analysis. cDNA synthesis, array hybridization, and normalization of the signal intensities were performed according to the instructions provided by Affymetrix (Santa Clara, CA). All raw microarray data was processed with MASv5.0 software as follows. Each hybridization was normalized to a median intensity of 150. Each treatment replica was compared with the two baselines to generate 4 comparisons per treatment. Data points with absent/marginal calls (Affymetrix quality control) in both baseline and treatment were removed. Data points with absent call in one hybridization and present call in the other hybridization were eliminated if the probe called present had a signal intensity of <100. The response of each gene was summarized using the Affymetrix change calls “I” for induced, “D” for decreased and “NC” for not changed. Data points were considered only if the change calls were consistent in at least 3 out of the 4 comparisons. This stepwise filtering resulted in a set of 834 genes that were detected and responded consistently in our experiments. We used custom made S-PLUS and PERL functions to analyze and visualize groups of genes with similar expression patterns based on the Affymetrix change calls.

[0260] Network analysis. For network analysis, an existing network model of plant gene interactions was used (Gutierrez, R. A., Lejay, L. V., Dean, A., Chiaromonte, F., Shasha, D. E. & Coruzzi, G. M. (2007) Genome Biol. 8(1):R7). In addition, protein:DNA interactions were predicted based as follows: The consensus sequence for transcription binding sites from well curated databases DATF (Guo, A., He, K., Liu, D., Bai, S., Gu, X., Wei, L. & Luo, J. (2005) Bioinformatics 21, 2568-2569) and AGRIS (Davuluri, R., Sun, H., Palaniswamy, S., Matthews, N., Molina, C., Kurtz, M. & Grotewold, E. (2003) BMC Bioinformatics 4, 25) were searched in 1500 base pairs of upstream sequence using the DNA pattern search tool from the RSA tools server with default parameters (van Heiden, J. (2003) Nucleic Acids Res 31, 3593 -3596). The search was performed in both strands of DNA, the upstream region was not allowed to overlap with the coding region of the upstream gene, motif matches were not allowed to overlap. A motif was considered over-represented if it was present in an upstream sequence more than 3 times the standard deviation above the mean occurrence in all the upstream sequences in the genome. A protein:DNA interaction was predicted when the upstream sequence of the gene contained an over representation of the regulatory motif for that transcription factor and the expression of the transcription factor and putative target gene was highly (≧0.7 or ≦−0.7) and significantly (p≦0.01) correlated. Similar regulatory predictions for other microarray data sets can be generated with the VirtualPlant system (http://www.virtualplant.org) using the “Gene Networks” tool.

[0261] Chromatin immunoprecipitation assays (ChIP): Immunoprecipitations (IP) were performed as previously described (Gendrel, A., Lippman, Z., Martienssen, R. A. & Colot, V. (2005) Profiling histone modification patterns in plants using genomic tiling microarrays Nat Methods 2, 219-224). Briefly, two weeks old wild-type and CCA1 -ox plants were collected at the beginning of the light cycle and immediately fixed in 1% formaldehyde for 15min in a vacuum at room temperature. Crosslinking was stopped by the addition of glycine to a final concentration of 0.125 M. Nuclei were prepared for chromatin isolation. The isolated chromatin was sonicated ten times for 20s each at 100% power (Diagenode Bioruptor) in an ice water bath. A small aliquot of sheared chromatin was removed to serve as control. The diluted chromatin was used for IP with the CCA1 antibody and one control IP without antibody. The primer sequences used for amplification of the CCA1 binding sites in each of the genes tested are listed in Table 1.

TABLE-US-00001 TABLE 1 Gene PUB locus Primer1 Primer2 bZIP1 At5g49450 5′-GATCGAAAATAAGGAAAGTGGG-3′ 5′-ACTGGTCACCTATTAAGGAAC-3′ (SEQ ID NO: 5) (SEQ ID NO: 6) TOC1 At5g61380 5′-TGGACGGTGGAGATTAAGTC-3′ 5′-ACGAAACGAAGCCGAATCCT-3′ (SEQ ID NO: 7) (SEQ ID NO: 8) ZTL At5g57360 5′-AGTCGCCGGAGATTATGAAGACGG- 5′-GGTTTTATCTACTTGACCCGACAG-3′ 3′ (SEQ ID NO: 9) (SEQ ID NO: 10) GDH1 At5g18170 5′-TGTTTCAATAGCATTAGCCTCCA-3′ 5′-TGGGGAATGTGACACACATAATC-3′ (SEQ ID NO: 11) (SEQ ID NO: 12) GLN1.3 Aa3g17820 5′-TTGAATCCGAAGAGGGGAAAA-3′ 5′-AACAACTGCTACCAATTTCCTTG-3′ (SEQ ID NO: 13) (SEQ ID NO: 14)

[0262] PCR amplifications included 95° C. for 2min followed by 36 cycles of 95° C. for 15s, 58° C. (for bZIP1, TOC1 and GDH1) or 60° C. (for ZTL and GLN1.3) for 30s and 72° C. for 30s.

[0263] Circadian phase response curves: CCA1::LUC seedlings were entrained on MS basal medium plus 0.5% sucrose and 1 mM KNO.sub.3 for 8 d in 16/8 h light/dark (100-150 pmol m.sup.−2 s.sup.−1), after which seedlings were moved into continuous light. At 3-hr intervals, seedlings (n=16 per treatment) were transferred to fresh solid medium plus 2mL liquid Nms or medium containing 10 mM Glu or 10 mM Gln for 4 hr, then rinsed in liquid entrainment medium 3 times for a total of 30 min and transferred individually to the wells of 96-well microtiter plates containing fresh solid media for luciferase activity measurements which were determined with a Packard TopCount scintillation counter as described (Salome, P. A., et al. (2002) The out of phase 1 mutant defines a role for PHYB in circadian phase control in Arabidopsis Plant Physiol 129, 1674-85). The period and phase of rhythms after the pulses were determined by fast-Fourier transform nonlinear least-square analysis (Plautz, J. D., et al. (1997) Quantitative analysis of Drosophila period gene transcription in living animals J Biol Rhythms 12, 204-17). The phase shifts were calculated as described (Covington, M. F., et al. (2001) ELF3 modulates resetting of the circadian clock in Arabidopsis Plant Cell 13, 1305-15).

7.3 Results

[0264] Inorganic versus organic N responses. To uncouple gene responses to inorganic N from those elicited by downstream products of inorganic N assimilation, treatments of Arabidopsis seedlings with combinations of inorganic N (nitrate and ammonium), organic forms of N (e.g., Glu, Gln), and MSX, an inhibitor of glutamine synthetase were performed (King et al., 1993, Plant Pysiol. 102:1279-1286) (FIG. 7). Genes regulated by inorganic N signals should be unaffected by MSX treatment. By contrast, genes responding to a downstream organic N signal should fail to show induction by inorganic N treatments if Glu/Gln synthesis is blocked by MSX. This block of induction by MSX should be relieved by Glu treatment. Following this rationale, two-week-old Arabidopsis seedlings grown on low concentrations of N (1 mM NO.sub.3.sup.−) were transferred to media containing 40 mM NO.sub.3.sup.− and 20 mM NH.sub.4.sup.+ (referred to as “Nms”). Seedlings were then harvested after a 2 h treatment time. This treatment was carried out alone (Nms), in the presence of 1 mM MSX (Nms+MSX) or 1 mM MSX and 10 mM Glu (Nms+MSX+Glu). The Nms treatment consists of the same N source found in standard MS salts which is the established standard amount of N for plant growth (Murashige et al., 1962, Plant Physiol. 15:473-497). A concentration of 1 mM MSX has previously been established as effective in blocking the N repression of AMT1.1 in Arabidopsis seedlings and in decreasing levels of internal organic N (Rawat et al., 1999, Plant J 19:143-152). A concentration of 10 mM for Glu treatments was chosen because this has been shown to be effective in the regulation of N assimilatory genes while not being high enough to be detrimental to plant growth or development (Oliveria et al., 1999, Plant Physiol. 121-301-310). To evaluate the effect of MSX alone, plants were exposed to growth media that contained MSX. To control for the effect of the plant transfer to distinct media, plants were transferred onto media plates without any of the treatment factors. This latter control was used as the base line for the microarray experiments described below.

[0265] To evaluate the experimental design, the mRNA level of genes shown to be responsive to organic N (ASN1) or inorganic N (ASN2) was determined by reverse transcription followed by real time quantitative PCR (RT-qPCR). This analysis showed that the ASN1 mRNA level was induced 3.5-fold by the Nms treatment as compared to the control (FIGS. 8A-B, compare Nms versus the control). This induction of ASN1 mRNA was blocked when MSX was present (FIG. 8A, Nms+MSX versus Nms). Importantly, when exogenous Glu or Gln was added, ASN1 levels were induced regardless of the presence of MSX (FIG. 8A, compare Control to Nms+MSX+Glu and Nms+MSX+Gln). These results indicate that the induction ofASN1 is due to Glu or a downstream metabolite, as shown previously (Lam et al., 1998, Plant J 16:345-353; Oliveira et al., 2001, Braz J Med Biol Res 34:567-575). In addition, the control treatments showed that MSX alone does not induce expression ofASN1 or ASN2 (FIGS. 8A-B). The addition of Glu or Gln partially blocked the induction ofASN2 by the Nms treatment (FIG. 8B), consistent with the negative regulation by amino acids seen previously (Lam et al., 1998, Plant J 16:345-353). Conversely, the induction ofASN2 mRNA by Nms was insensitive to MSX addition (FIG. 8B), suggesting the induction was mediated by an inorganic N source. This finding was consistent with previous data which indicates that ASN2 gene expression correlates with ammonium levels (Wong et al., 2004, Plant Physiol 134:332-812).

[0266] Global genomic responses to organic and inorganic nitrogen signals. To investigate global gene expression changes that are mediated by Glu or a Glu-derived metabolite, the plant transcriptome was monitored using the ATH1 Affymetrix gene chip. Total RNA was extracted from plants treated with Nms, Nms+MSX or Nms+MSX+Glu as described above; two biological replicates per treatment were performed. The Nms+MSX+Gln treatments were not analyzed using microarrays because Gln and Glu responses were similar in our hands (FIGS. 8A-B). RNA was labeled and hybridized to the microarrays, the raw intensity values were normalized and the data filtered as described in Materials and Methods. A gene was kept in the data set only if its expression was reproducible and reliable across the 3 different treatments (Nms, Nms+MSX, Nms+MSX+Glu). A total of 5,904 genes were identified that passed these stringent quality control criteria. In order to verify the microarray results, we analyzed the mRNA levels of selected genes by RT-qPCR including the TAZ zinc binding (At4g37610) and bZIP (At5g49450) transcription factors with results similar to the microarray data (FIGS. 9A-B). As expected, the genomic experiments verified the previous observation that AMT1.1 and ASN1 are regulated by organic N. In addition, the results identified additional genes regulated by organic N as described below.

[0267] Genes were categorized based on their response to the treatments using the Affymetrix change calls: induced (I), no change (NC) or decreased (D). Each gene was assigned a three-part code (e.g. I-NC-I) which corresponds to the gene expression response in the Nms, Nms+MSX, and Nms+MSX+Glu treatments respectively. 21 unique patterns of response were found (See Annex) and Table 2 (in each column the gene listed on the left is predicted to control the expression of the gene on the right, i.e., At1g74840 is predicted to control the expression of At2g47060, At5g24800 is predicted to control the expression of At5g13930).

TABLE-US-00002 TABLE 2 At1g74840 reg0.9 At2g47060 At5g24800 reg0.7 At5g13930 At5g14540 reg0.9 At5g53370 At5g48655 reg0.8 At5g53370 At3g61150 reg0.7 At4g30810 At1g74840 reg0.8 At5g01820 At2g46830 reg0.8 At5g01820 At5g24800 reg0.8 At2g30040 At5g48655 reg0.7 At2g30040 At2g33710 reg0.8 At2g30040 At4g17490 reg0.8 At2g30040 At2g46830 reg0.7 At2g30040 At5g44190 reg0.8 At2g30040 At1g74840 reg0.7 At4g28100 At2g20570 reg0.8 At4g28100 At1g22070 reg0.7 At4g33300 At2g46830 reg0.7 At1g06000 At5g48655 reg0.9 At5g11790 At1g74840 reg0.8 At4g36640 At1g22070 reg0.8 At4g19810 At5g24800 reg0.9 At4g19810 At5g49450 reg0.9 At2g39980 At3g01560 reg0.7 At2g15970 At1g43160 reg0.9 At2g15970 At1g22070 reg0.7 At2g15970 At2g20570 reg0.7 At2g22240 At5g49450 reg0.8 At1g49500 At2g20570 reg0.8 At1g27730 At2g46830 reg0.9 At1g27730 At4g37260 reg0.7 At3g04070 At5g24800 reg0.8 At2g36290 At5g48655 reg0.7 At5g63790 At5g48655 reg0.7 At4g28250 At2g04880 reg0.9 At5g45340 At2g25000 reg0.7 At5g45340 At2g38470 reg1.0 At5g45340 At5g44190 reg0.8 At1g29670 At5g14540 reg0.7 At1g70330 At1g53910 reg0.7 At1g70330 At5g14540 reg0.8 At3g58560 At2g22430 reg0.8 At2g30870 At5g47230 reg0.7 At2g30870 At5g61890 reg0.7 At2g30870 At2g22430 reg0.8 At2g35930 At2g38470 reg0.7 At1g33590 At3g01560 reg0.7 At5g06320 At5g14540 reg0.7 At5g06320 At3g61150 reg0.7 At2g44210 At5g49450 reg1.0 At2g44080 At3g61890 reg0.7 At5g60850 At1g22070 reg0.7 At3g59220 At2g20570 reg0.7 At1g60780 At1g43160 reg0.7 At1g74840 At5g61890 reg0.8 At1g74840 At5g49450 reg0.9 At4g27410 At5g14540 reg0.8 At2g40270 At4g37260 reg0.7 At1g77510 At5g14540 reg0.8 At4g33400 At1g74840 reg0.7 At4g36250 At3g61890 reg0.9 At5g05600 At1g74840 reg0.9 At1g53310 At2g22430 reg0.7 At4g14960 At1g43160 reg0.9 At5g15960 At5g61890 reg0.9 At5g15960 At2g22430 reg0.8 At3g23750 At1g53910 reg0.8 At3g23750 At5g61890 reg0.8 At3g19680 At3g01560 reg0.8 At2g47180 At1g43160 reg0.7 At2g47180 At5g47230 reg0.8 At2g47180 At5g61890 reg0.7 At2g47180 At2g22430 reg0.7 At5g01540 At5g14540 reg0.7 At2g39530 At5g48655 reg0.8 At2g39530 At2g33710 reg0.8 At2g39530 At1g74840 reg0.9 At1g51680 At1g22070 reg0.8 At4g39330 At5g24800 reg0.7 At4g39330 At1g53910 reg0.9 At4g03260 At5g14540 reg0.8 At1g76670 At2g22430 reg0.7 At1g76670 At3g61150 reg0.7 At1g76670 At5g24800 reg0.9 At5g02270 At2g20570 reg0.8 At5g35735 At5g61890 reg0.8 At3g47960 At1g74840 reg0.8 At3g21230 At2g04880 reg0.8 At4g31500 At4g31800 reg0.7 At4g31500 At3g61150 reg0.8 At5g63850 At3g01560 reg0.7 At2g46600 At4g37260 reg0.9 At1g10760 At1g74840 reg0.8 At3g54640 At5g49450 reg0.7 At1g76590 At1g25560 reg0.7 At1g76590 At1g68840 reg0.8 At1g76590 At5g24800 reg0.7 At5g44190 At1g74840 reg0.9 At5g60920 At5g14540 reg0.8 At5g60920 At1g25560 reg0.8 At4g38470 At1g68840 reg0.7 At4g38470 At2g20570 reg0.8 At2g37430 At2g46830 reg0.9 At2g37430 At2g04880 reg0.8 At2g37430 At2g25000 reg0.8 At2g37430 At2g30250 reg0.7 At2g37430 At2g38470 reg0.9 At2g37430 At4g01250 reg0.7 At2g37430 At2g20570 reg0.9 At1g74460 At2g46830 reg0.8 At1g74460 At1g22070 reg0.7 At4g23630 At3g01560 reg0.9 At3g14280 At5g14540 reg0.7 At3g14280 At3g61150 reg0.7 At3g14280 At2g20570 reg0.7 At3g14280 At2g46830 reg0.8 At3g14280 At2g04880 reg0.8 At5g44070 At2g38470 reg0.9 At5g44070 At4g01250 reg0.7 At5g44070 At5g24800 reg0.7 At1g73080 At2g33710 reg0.9 At3g56710 At4g17490 reg0.9 At3g56710 At2g20570 reg0.8 At3g56710 At2g46830 reg0.8 At3g56710 At1g74840 reg0.8 At2g30490 At3g01560 reg0.7 At2g42540 At1g43160 reg0.9 At2g42540 At5g61890 reg0.8 At2g42540 At1g74840 reg0.7 At1g47128 At2g46830 reg0.7 At1g47128 At5g24800 reg0.7 At4g11280 At2g20570 reg0.7 At3g60030 At4g37260 reg0.7 At3g47620 At5g49450 reg0.8 At3g21870 At5g14540 reg0.8 At4g33050 At5g48655 reg0.7 At4g33050 At3g01560 reg0.7 At5g01600 At1g43160 reg0.8 At5g01600 At1g43160 reg0.8 At3g05890 At1g74840 reg0.7 At2g16630 At4g37260 reg0.9 At2g16630 At2g04880 reg0.9 At5g45340 At2g25000 reg0.7 At5g45340 At2g38470 reg1.0 At5g45340 At3g01560 reg0.7 At2g22880 At5g48655 reg0.7 At2g22880 At2g04880 reg0.8 At3g52400 At1g43160 reg0.9 At1g29395 At5g61890 reg0.7 At1g29395 At1g74840 reg0.8 At2g16430 At1g74840 reg0.8 At4g23210 At1g22070 reg0.7 At4g23210 At5g49450 reg0.8 At4g21150 At1g22070 reg0.9 At3g48610 At5g24800 reg0.7 At3g48610 At5g47230 reg0.7 At2g38700 At1g74840 reg0.7 At4g30280 At5g14540 reg0.7 At1g53500 At5g48655 reg0.8 At1g53500 At3g01560 reg0.7 At2g23120 At5g14540 reg0.8 At5g09440 At2g22430 reg0.8 At5g09440 At1g74840 reg0.8 At5g09440 At5g24800 reg0.7 At2g41630 At2g20570 reg0.8 At1g56150 At2g46830 reg0.8 At1g56150 At5g49450 reg0.7 At1g10070 At2g22430 reg0.8 At3g21240 At3g01560 reg0.8 At5g16010 At4g37260 reg0.9 At5g46710 At1g25560 reg0.7 At5g11420 At1g68840 reg0.7 At5g11420 At5g24800 reg0.7 At4g01250 At5g44190 reg0.7 At3g07790 At2g30250 reg0.7 At1g13110 At2g38470 reg0.8 At1g13110 At4g01250 reg0.9 At1g13110 At1g22070 reg0.8 At2g25000 At2g20570 reg0.9 At2g25000 At2g46830 reg0.8 At2g25000 At2g04880 reg0.8 At2g25000 At2g38470 reg0.7 At2g25000 At4g31800 reg0.8 At2g25000 At5g24800 reg0.7 At1g35780 At3g01560 reg0.8 At5g11110 At1g74840 reg0.9 At5g11110 At1g43160 reg0.8 At5g11110 At5g47230 reg0.7 At5g11110 At5g61890 reg0.7 At5g11110 At3g61890 reg0.8 At4g12490 At1g74840 reg0.7 At1g51700 At1g74840 reg0.8 At3g55970 At4g37260 reg0.7 At3g55970 At4g17500 reg0.8 At5g49910 At1g22070 reg0.7 At4g39800 At1g74840 reg0.8 At4g39800 At2g20570 reg0.7 At4g39800 At2g46830 reg0.8 At4g39800 At2g20570 reg0.8 At2g13790 At2g46830 reg0.8 At2g13790 At3g61890 reg1.0 At1g52400 At2g20570 reg0.8 At5g18470 At2g46830 reg0.7 At5g18470 At2g22430 reg0.9 At1g53910 At5g14540 reg0.9 At2g40140 At5g48655 reg0.9 At2g40140 At2g20570 reg0.8 At2g40890 At2g46830 reg0.8 At2g40890 At1g74840 reg0.7 At3g55070 At4g37260 reg0.7 At1g68520 At1g74840 reg0.9 At1g14780 At1g43160 reg0.8 At1g14780 At5g61890 reg0.9 At1g14780 At2g22430 reg0.9 At2g39210 At2g20570 reg0.9 At1g76600 At2g46830 reg0.9 At1g76600 At2g04880 reg0.9 At1g76600 At2g25000 reg0.8 At1g76600 At2g38470 reg0.8 At1g76600 At5g48655 reg0.8 At5g19240 At2g22430 reg0.7 At3g02910 At2g22430 reg0.9 At3g63010 At3g61890 reg0.9 At3g49120 At1g74840 reg0.7 At3g49120 At4g37260 reg0.8 At3g49120 At5g14540 reg0.7 At3g52450 At2g23320 reg0.7 At3g52450 At4g31800 reg0.9 At3g52450 At1g25560 reg0.7 At5g58710 At1g68840 reg0.7 At5g58710 At5g49450 reg0.7 At5g58710 At3g01560 reg0.8 At4g25650 At1g53910 reg0.7 At4g04020 At4g17490 reg0.7 At4g04020 At5g49450 reg0.9 At3g47340 At5g49450 reg0.8 At1g77120 At5g48655 reg0.7 At2g26190 At4g37260 reg0.7 At5g49450 At1g22070 reg0.9 At1g20440 At1g22070 reg0.7 At5g59820 At5g48655 reg0.8 At1g26250 At1g74840 reg0.8 At5g07010 At4g37260 reg0.7 At5g07010 At1g74840 reg0.7 At3g54690 At2g20570 reg0.7 At3g54690 At2g46830 reg0.8 At3g54690 At2g20570 reg0.8 At5g26920 At2g46830 reg0.7 At5g26920 At3g61890 reg0.8 At2g02990 At1g74840 reg0.8 At5g61890 At5g49450 reg0.7 At2g34500 At1g74840 reg0.8 At4g01700 At2g46830 reg0.7 At4g01700 At2g20570 reg0.9 At5g59730 At2g46830 reg0.9 At5g59730 At3g01560 reg0.8 At3g52470 At5g14540 reg0.8 At3g52470 At5g48655 reg0.7 At3g52470 At2g46830 reg0.8 At3g02800 At5g49450 reg0.8 At4g24800 At4g37260 reg0.7 At4g24800 At2g20570 reg0.8 At5g25630 At2g46830 reg0.7 At5g25630 At5g24800 reg0.7 At5g28900 At2g20570 reg0.7 At4g27280 At2g46830 reg0.8 At4g27280 At5g49450 reg0.9 At3g13450 At5g24800 reg0.9 At1g20510 At2g20570 reg0.7 At1g20510 At2g46830 reg0.8 At1g20510 At5g44190 reg0.7 At1g20510 At2g46830 reg0.7 At1g14730 At5g61890 reg0.7 At1g43160 At3g01560 reg0.8 At5g06700 At5g14540 reg0.8 At5g06700 At1g22070 reg0.7 At5g06700 1g74840 reg0.8 At5g06700 At2g20570 reg0.7 At5g06700 5g24800 reg0.7 At4g34450 At1g43160 reg1.0 At2g28900 At1g74840 reg0.7 At2g28900 At4g37260 reg0.7 At2g28900 2g20570 reg0.7 At5g42310 At3g01560 reg0.8 At5g42310 At5g14540 reg0.8 At5g42310 At3g01560 reg0.7 At2g43620 At3g61890 reg0.8 At2g43620 At1g74840 reg0.7 At3g23810 At2g20570 reg0.8 At3g17820 At2g46830 reg0.7 At3g17820 At3g01560 reg0.8 At3g23820 At5g14540 reg0.7 At3g23820 At1g74840 reg0.8 At3g23820 At1g43160 reg0.7 At3g23820 At5g47230 reg0.8 At3g23820 At5g61890 reg0.8 At3g23820 At5g49450 reg0.9 At3g57520 At3g61890 reg0.9 At4g22212 At2g30250 reg0.7 At2g46225 At4g01250 reg0.7 At2g46225 At4g23810 reg0.8 At2g46225 At5g49450 reg0.7 At4g37260 At5g24800 reg0.7 At2g24940 At1g25560 reg0.9 At3g19390 At1g68840 reg0.9 At3g19390 At3g01560 reg0.8 At1g21790 At1g74840 reg0.8 At1g21790 At4g37260 reg0.8 At1g21790 At4g37260 reg0.7 At1g12780 At2g20570 reg0.7 At5g54490 At2g46830 reg0.9 At5g54490 At1g22070 reg0.9 At3g11670 At5g24800 reg0.7 At3g11670 At5g49450 reg−0.7 At2g30040 At1g25560 reg−0.7 At2g30040 At1g68840 reg−0.7 At2g30040 At2g20570 reg−0.9 At1g80180 At2g46830 reg−0.8 At1g80180 At1g22070 reg−0.7 At1g06760 At5g24800 reg−0.8 At5g61790 At5g48655 reg−0.7 At1g11545 At2g20570 reg−0.8 At1g07040 At2g46830 reg−0.8 At1g07040 At4g17500 reg−0.7 At2g15970 At2g22430 reg−0.9 At1g49500 At5g44190 reg−0.7 At3g04070 At4g37260 reg−0.8 At4g22710 At5g49450 reg−0.8 At5g63790 At5g14540 reg−0.7 At3g60320 At5g48655 reg−0.9 At3g60320 At2g22430 reg−0.7 At3g60320 At3g01560 reg−0.7 At4g37450 At4g37260 reg−0.7 At1g77450 At1g22070 reg−0.7 At1g42480 At2g46830 reg−0.7 At5g64570 At2g20570 reg−0.8 At5g39610 At2g22430 reg−0.8 At5g39610 At3g61890 reg−0.7 At2g34640 At5g24800 reg−0.8 At4g16660 At2g20570 reg−0.7 At5g60680 At2g46830 reg−0.7 At5g60680 At4g17500 reg−0.7 At2g47180 At1g22070 reg−0.9 At1g09210 At2g04880 reg−0.7 At1g68840 At2g38470 reg−0.8 At1g68840 At2g22430 reg−0.7 At1g49860 At5g44190 reg−0.8 At1g09240 At1g74840 reg−0.7 At1g76690 At4g37260 reg−0.9 At1g76690 At1g43160 reg−0.9 At4g31130 At5g61890 reg−0.7 At4g31130 At1g74840 reg−0.7 At4g31130 At4g37260 reg−0.8 At4g31130 At5g49450 reg−0.9 At2g38470 At5g44190 reg−0.7 At4g39980 At5g14540 reg−0.9 At4g12600 At4g37260 reg−0.8 At3g25230 At1g74840 reg−0.8 At1g32170 At2g20570 reg−0.7 At1g32170 At2g46830 reg−0.7 At1g32170 At1g43160 reg−0.7 At2g29490 At5g47230 reg−0.7 At2g29490 At5g61890 reg−0.8 At2g29490 At5g14540 reg−0.8 At5g05440 At5g48655 reg−0.8 At5g05440 At2g33710 reg−0.8 At4g39675 At2g20570 reg−0.7 At2g04280 At2g46830 reg−0.7 At2g04280 At1g74840 reg−0.7 At5g49480 At2g22430 reg−0.7 At5g49480 At3g61150 reg−0.7 At5g49480 At5g48655 reg−0.7 At1g76590 At4g37260 reg−0.7 At1g32920 At2g33710 reg−0.9 At4g38470 At4g17490 reg−0.8 At4g38470 At2g20570 reg−0.7 At4g38470 At2g46830 reg−0.8 At4g38470 At5g44190 reg−0.7 At5g15410 At2g20570 reg−0.7 At1g67910 At2g46830 reg−0.8 At1g67910 At3g01560 reg−0.7 At5g25460 At5g14540 reg−0.9 At5g25460 At5g48655 reg−0.8 At5g25460 At3g61150 reg−0.7 At3g48990 At1g22070 reg−0.8 At3g48990 At5g24800 reg−0.8 At3g48990 At5g49450 reg−0.7 At3g62960 At1g25560 reg−0.8 At3g56710 At1g68840 reg−0.7 At3g56710 At5g44190 reg−0.7 At3g51550 At1g74840 reg−0.8 At3g62120 At4g17500 reg−0.7 At2g42540 At5g49450 reg−0.8 At4g11280 At1g22070 reg−0.8 At3g15950 At3g01560 reg−0.9 At3g19130 At1g74840 reg−0.7 At4g21620 At4g37260 reg−0.8 At4g21620 At5g14540 reg−0.7 At3g21870 At5g48655 reg−0.7 At3g21870 At2g20570 reg−0.8 At3g15450 At2g46830 reg−0.8 At3g15450 At4g17500 reg−0.7 At5g01600 At4g17500 reg−0.7 At3g05890 At2g46830 reg−0.8 At1g22570 At5g48655 reg−0.9 At3g14310 At3g61150 reg−0.8 At5g65390 At2g20570 reg−0.8 At1g76160 At2g46830 reg−0.7 At1g76160 At4g17500 reg−0.7 At1g29395 At5g49450 reg−0.7 At5g66510 At1g74840 reg−0.7 At1g62480 At5g44190 reg−0.8 At3g07390 At2g20570 reg−0.8 At2g33830 At2g46830 reg−0.8 At2g33830 At3g61890 reg−0.7 At3g06680 At4g37260 reg−0.8 At5g23020 At5g24800 reg−0.7 At1g10070 At5g48655 reg−0.8 At4g36670 At2g38470 reg−0.9 At4g36670 At1g22070 reg−0.7 At2g44310 At5g24800 reg−0.7 At2g44310 At2g22430 reg−0.7 At2g44310 At3g61150 reg−0.7 At2g44310 At3g01560 reg−0.7 At4g12880 At5g14540 reg−0.8 At4g12880 At5g48655 reg−0.9 At4g12880 At1g74840 reg−0.8 At4g13770 At4g37260 reg−0.7 At4g13770 At3g61150 reg−0.7 At3g54400 At5g44190 reg−0.7 At5g06530 At5g48655 reg−0.9 At5g11420 At2g33710 reg−0.9 At5g11420 At4g17490 reg−0.8 At5g11420 At1g74840 reg−0.7 At1g19870 At4g37260 reg−0.7 At1g19870 At1g22070 reg−0.8 At1g19870 At4g17500 reg−0.7 At1g35780 At4g17500 reg−0.7 At5g11110 At5g49450 reg−0.9 At1g10960 At5g44190 reg−0.7 At5g06530 At5g44190 reg−0.7 At3g55970 At4g37260 reg−0.8 At3g11700 At3g01560 reg−0.7 At5g49910 At3g61890 reg−0.8 At4g23180 At1g74840 reg−0.9 At4g23180 At4g37260 reg−0.7 At4g23180 At1g74840 reg−0.8 At4g10480 At2g46830 reg−0.7 At4g10480 At2g22430 reg−0.7 At5g43970 At3g61150 reg−0.7 At5g43970 At3g61890 reg−0.7 At5g11670 At1g22070 reg−0.7 At1g56330 At5g48655 reg−0.7 At1g68520 At5g44190 reg−0.7 At1g68520 At3g61150 reg−0.7 At1g31420 At5g44190 reg−1.0 At2g23810 At2g20570 reg−0.9 At3g15630 At2g46830 reg−0.8 At3g15630 At2g20570 reg−0.9 At2g39570 At2g46830 reg−0.8 At2g39570 At5g49450 reg−0.8 At1g76600 At1g74840 reg−0.9 At2g06850 At5g14540 reg−0.7 At2g37640 At5g48655 reg−0.7 At2g37640 At2g20570 reg−0.8 At2g37640 At2g46830 reg−0.9 At2g37640 At1g74840 reg−0.7 At1g02930 At4g37260 reg−0.8 At1g02930 At2g33710 reg−0.8 At5g58710 At3g61890 reg−0.7 At1g64640 At1g74840 reg−0.8 At1g64640 At2g20570 reg−0.8 At3g49780 At2g46830 reg−0.9 At3g49780 At5g24800 reg−0.8 At3g49780 At5g24800 reg−0.7 At3g47340 At5g44190 reg−0.8 At1g09330 At5g48655 reg−0.7 At5g49450 At2g20570 reg−0.8 At5g49450 At2g46830 reg−0.7 At5g49450 At5g44190 reg−0.8 At5g07010 At5g49450 reg−0.7 At2g29550 At1g22070 reg−0.7 At2g34500 At1g74840 reg−0.8 At3g61440 At5g14540 reg−0.8 At2g24500 At5g24800 reg−0.7 At5g11520 At4g37260 reg−0.7 At1g62380 At5g48655 reg−0.7 At1g80070 At2g22430 reg−0.8 At3g13450 At5g14540 reg−0.8 At1g24530 At1g22070 reg−0.7 At1g24530 At5g49450 reg−0.7 At1g20510 At5g14540 reg−0.7 At1g73120 At4g17500 reg−0.7 At1g43160 At1g74840 reg−0.9 At1g53240 At2g20570 reg−0.8 At5g18170 At2g46830 reg−0.8 At5g18170 At3g61890 reg−0.8 At2g30010 At1g74840 reg−0.7 At2g30010 At4g37260 reg−0.9 At2g30010 At5g49450 reg−0.7 At2g30010 At5g24800 reg−0.9 At5g49700 At4g17500 reg−0.8 At2g28900 At5g24800 reg−0.8 At1g15690 At5g44190 reg−0.8 At4g12480 At2g04880 reg−0.9 At5g22920 At2g25000 reg−0.7 At5g22920 At2g38470 reg−1.0 At5g22920 At5g44190 reg−0.8 At3g23810 At5g48655 reg−0.8 At3g57520 At2g20570 reg−0.7 At3g57520 At2g46830 reg−0.8 At3g57520 At5g24800 reg−0.7 At3g57520 At1g53910 reg−0.8 At5g04340 At5g24800 reg−0.8 At2g05380 At2g33710 reg−0.7 At3g19390 At4g17490 reg−0.8 At3g19390 At4g37260 reg−0.8 At3g12740 At2g20570 reg−0.7 At1g12780 At3g61890 reg−0.8 At3g16530

[0268] The largest pattern was NC-NC-NC, representing 5,070 genes not affected by the treatments. The remainder 20 patterns (834 genes) were grouped into six classes that summarize the types of N-responses observed: (A) inorganic N, (B) inorganic N with Glu feedback, (C) organic N with no exogenous Glu rescue, (D) exogenous Glu, (E) exogenous and endogenous Glu and (F) exogenous and endogenous Glu with opposite effects (Table 3).

TABLE-US-00003 TABLE 3 Treatment Genes Nms + Nms + per Response Nms MSX MSX + Glu Genes class A) Inorganic nitrogen D D D 100 159 I I I 59 B) Inorganic nitrogen D D NC 30 48 and Glu I I NC 15 D D I 3 C) Internal Glu D NC NC 194 334 I NC NC 56 NC I I 49 NC D D 33 D I I 2 D) External Glu NC NC D 89 126 NC NC I 37 E) Internal/External NC I NC 55 164 Glu NC D NC 36 D NC D 47 I NC I 22 I D I 3 NC D I 1 F) Opposite D NC I 2 3 Internal/External Glu I NC D 1 834 Nitrogen responses. Patterns of expression based on their response to the Nms, Nms + MSX and Nms + MSX + Glu treatments. D = decreased; I = increased; NC = not changed. Genes were categorized into six classes based on these patterns.

[0269] The genes regulated in the experiments were compared to published results (Wang et al., 2004, Plant Physiol 136:2512-2522). This previous study identified 595 genes that responded similarly to nitrate treatment in both a NR-null mutant and wild-type plants. Because the mutant plants cannot assimilate nitrate, the responses observed were attributed to the action of nitrate as a signal and not a downstream metabolite. 80 out of these 595 genes showed consistent and reliable responses in the current experiments. Surprisingly, only 17 of these genes were found regulated by inorganic N signals in both studies (Table 4). Among these, we found nitrite reductase and several high affinity nitrate transporters. The majority, 58 of these 80 genes (73%), belonged to the C, D or E classes in the present studies suggesting that many previously described nitrate-responsive genes may respond to organic N signals (Table 4).

TABLE-US-00004 TABLE 4 Regulated by PUB LOCUS Gene Name Inorganic N At4g19170 9-cis-epoxycarotenoid dioxygenase, putative/neoxanthin cleavage enzyme, putative/carotenoid cleavage dioxygenase, putative similar to 9-cis- epoxycarotenoid dioxygenase [Phaseolus vulgaris][GI: 6715257]; neoxanthin cleavage enzyme, Lycopersicon esculentum, PATX: E325797 (68417.m02829) Inorganic N At3g61820 aspartyl protease family protein contains Pfam domain, PF00026: eukaryotic aspartyl protease (68416.m06939) Inorganic N At4g30190 ATPase 2, plasma membrane-type, putative/proton pump 2, putative/proton- exporting ATPase, putative strong similarity to SP Inorganic N At4g31500 cytochrome P450 83B1 (CYP83B1) Identical to Cytochrome P450 (SP: O65782)[Arabidopsis thaliana] (68417.m04474) Inorganic N At1g05340 expressed protein (68414.m00541) Inorganic N At1g19020 expressed protein (68414.m02367) Inorganic N At1g32920 expressed protein (68414.m04055) Inorganic N At2g41730 expressed protein (68415.m05158) Inorganic N At1g14870 expressed protein similar to PGPS/D12 [Petunia × hybrida] GI: 4105794; contains Pfam profile PF04749: Protein of unknown function, DUF614 (68414.m01778) Inorganic N At5g25350 F-box family protein contains Pfam PF00646: F-box domain and Pfam PF00560: Leucine Rich Repeat (6 copies); similar to F-box protein FBL6 (GI: 4432860) [Homo sapiens] (68418.m03007) Inorganic N At2g15620 ferredoxin--nitrite reductase, putative strong similarity to ferredoxin--nitrite reductase [Nicotiana tabacum] GI: 19893; contains Pfam profiles PF03460: Nitrite/Sulfite reductase ferredoxin-like half domain, PF01077: Nitrite and sulphite reductase 4Fe—4S domain (68415.m01789) GDSL-motif Inorganic N At5g55050 lipase/hydrolase family protein similar to family II lipases EXL3 GI: 15054386, EXL1 GI: 15054382, EXL2 GI: 15054384 from [Arabidopsis thaliana]; contains Pfam profile PF00657: GDSL-like Lipase/Acylhydrolase (68418.m06861) Inorganic N At5g18600 glutaredoxin family protein contains glutaredoxin domain, INTERPRO: IPR002109 (68418.m02201) Inorganic N At1g49860 glutathione S-transferase, putative similar to GI: 860955 from [Hyoscyamus muticus] (Plant Physiol. 109 (1), 253-260 (1995)) (68414.m05590) Inorganic N At1g08090 high-affinity nitrate transporter (ACH1) identical to trans-membrane nitrate transporter protein AtNRT2: 1 [Arabidopsis thaliana] GI: 3747058, high-affinity nitrate transporter ACH1 [Arabidopsis thaliana] GI: 3608362 (68414.m00885) Inorganic N At1g12940 high-affinity nitrate transporter, putative similar to trans-membrane nitrate transporter protein AtNRT2: 1 [Arabidopsis thaliana] GI: 3747058, high-affinity nitrate transporter ACH1 [Arabidopsis thaliana] GI: 3608362 (68414.m01503) Inorganic N At1g12110 nitrate/chlorate transporter (NRT1.1) (CHL1) identical to nitrate/chlorate transporter SP: Q05085 from [Arabidopsis thaliana]; contains Pfam profile: PF00854 POT family (68414.m01402) Organic and At1g55920 serine O-acetyltransferase, putative identical to GI: 608677 from [Arabidopsis Inorganic N thaliana] (68414.m06414) Organic and At2g16660 nodulin family protein similar to nodulin-like protein [Arabidopsis thaliana] Inorganic N GI: 3329368, nodule-specific protein Nlj70 [Lotus japonicus] GI: 3329366 (68415.m01912) Organic and At3g45140 lipoxygenase (LOX2) identical to SP Inorganic N Organic and At5g64410 oligopeptide transporter OPT family protein similar to SP Inorganic N Organic and At5g65010 asparagine synthetase 2 (ASN2) identical to asparagine synthetase (ASN2) Inorganic N [Arabidopsis thaliana] GI: 3859536 (68418.m08178) Organic N At1g14780 expressed protein (68414.m01767) Organic N At1g22160 senescence-associated protein-related similar to senescence-associated protein SAG102 (GI: 22331931) [Arabidopsis thaliana] (68414.m02770) Organic N At1g31770 ABC transporter family protein contains Pfam profile: PF00005: ABC transporter (68414.m03899) Organic N At1g32450 proton-dependent oligopeptide transport (POT) family protein contains Pfam profile: PF00854 POT family (68414.m04005) Organic N At1g47128 cysteine proteinase (RD21A)/thiol protease identical to SP Organic N At1g49500 expressed protein (68414.m05548) Organic N At1g56150 auxin-responsive family protein similar to SP: P33082 Auxin-induced protein X15. [Soybean] {Glycine max} (68414.m06450) Organic N At1g67910 expressed protein (68414.m07755) Organic N At1g74090 sulfotransferase family protein similar to SP Organic N At1g74710 isochorismate synthase 1 (ICS1)/isochorismate mutase identical to GI: 17223087 and GB: AF078080; contains Pfam profile PF00425: chorismate binding enzyme; contains TIGRfam profile TIGR00543: isochorismate synthases; identical to cDNA isochorismate synthase 1 precursor (ICS1) nuclear gene for plastid product GI: 17223086 (68414.m08655) Organic N At1g77760 nitrate reductase 1 (NR1) identical to SP Organic N At1g78000 sulfate transporter (Sultr1; 2) identical to sulfate transporter Sultr1; 2 [Arabidopsis thaliana] GI: 7768660; contaisn Pfam profiles PF00916: Sulfate transporter family and PF01740: STAS domain; contains TIGRfam profile TIGR00815: sulfate permease (68414.m09090) Organic N At2g15970 cold-acclimation protein, putative (FL3-5A3) similar to cold acclimation Organic N At2g27830 WCOR413-like protein gamma form [Hordeum vulgare] gi expressed protein (68415.m03374) Organic N At2g28550 AP2 domain-containing transcription factor RAP2.7 (RAP2.7) nearly identical to AP2 domain transcription factor RAP2.7 (GI: 2281639) [Arabidopsis thaliana] (68415.m03469) Organic N At2g30040 protein kinase family protein contains protein kinase domain, Pfam: PF00069 (68415.m03653) Organic N At2g31790 UDP-glucoronosyl/UDP-glucosyl transferase family protein contains Pfam profile: PF00201 UDP-glucoronosyl and UDP-glucosyl transferase (68415.m03881) Organic N At2g33710 AP2 domain-containing transcription factor family protein similar to RAP2.6 (GI: 17065542) {Arabidopsis thaliana} (68415.m04132) Organic N At2g33830 dormancy/auxin associated family protein contains Pfam profile: PF05564 dormancy/auxin associated protein (68415.m04151) Organic N At2g35930 U-box domain-containing protein similar to immediate-early fungal elicitor protein CMPG1 [Petroselinum crispum] GI: 14582200; contains Pfam profile PF04564: U-box domain (68415.m04410) Organic N At2g39200 seven transmembrane MLO family protein/MLO-like protein 12 (MLO12) identical to SP Organic N At2g39570 ACT domain-containing protein contains Pfam ACT domain PF01842 (68415.m04854) Organic N At2g40140 zinc finger (CCCH-type) family protein contains Pfam domain, PF00642: Zinc finger C-x8-C-x5-C-x3-H type (and similar) and Pfam domain, PF00023: Ankyrin repeat (68415.m04937) Organic N At2g43100 aconitase C-terminal domain-containing protein contains Pfam profile PF00694: Aconitase C-terminal domain (68415.m05350) Organic N At3g02910 expressed protein contains Pfam domain PF03674: Uncharacterised protein family (UPF0131) (68416.m00286) Organic N At3g05200 zinc finger (C3HC4-type RING finger) family protein (ATL6) contains Pfam profile: PF00097: Zinc finger, C3HC4 type (RING finger) (68416.m00567) Organic N At3g10520 non-symbiotic hemoglobin 2 (HB2) (GLB2) identical to SP Organic N At3g13930 dihydrolipoamide S-acetyltransferase, putative similar to dihydrolipoamide S- acetyltransferase [Zea mays] GI: 5669871; contains Pfam profiles PF00198: 2-oxo acid dehydrogenases acyltransferase (catalytic domain), PF00364: Biotin- requiring enzyme, PF02817: e3 binding domain (68416.m01759) Organic N At3g14940 phosphoenolpyruvate carboxylase, putative/PEP carboxylase, putative strong similarity to SP Organic N At3g15630 expressed protein (68416.m01982) Organic N At3g47520 malate dehydrogenase [NAD], chloroplast (MDH) identical to chloroplast NAD- malate dehydrogenase [Arabidopsis thaliana] GI: 3256066; contains InterPro entry IPR001236: Lactate/malate dehydrogenase; contains Pfam profiles PF00056: lactate/malate dehydrogenase, NAD binding domain and PF02866: lactate/malate dehydrogenase, alpha/beta C- terminal domain (68416.m05168) Organic N At3g48740 nodulin MtN3 family protein similar to MtN3 GI: 1619602 (root nodule development) from [Medicago truncatula] (68416.m05322) Organic N At3g48990 AMP-dependent synthetase and ligase family protein similar to peroxisomal- coenzyme A synthetase (FAT2) [gi: 586339] from Saccharomyces cerevisiae; contains Pfam AMP-binding enzyme domain PF00501; identical to cDNA; identical to cDNA adenosine monophosphate binding protein 3 AMPBP3 (AMPBP3)GI: 20799714 (68416.m05351) Organic N At3g49940 LOB domain protein 38/lateral organ boundaries domain protein 38 (LBD38) identical to SP Organic N At3g58990 aconitase C-terminal domain-containing protein contains Pfam profile PF00694: Aconitase C-terminal domain (68416.m06575) Organic N At3g60750 transketolase, putative strong similarity to transketolase 1 [Capsicum annuum] GI: 3559814; contains Pfam profiles PF02779: Transketolase, pyridine binding domain, PF02780: Transketolase, C-terminal domain, PF00456: Transketolase, thiamine diphosphate binding domain (68416.m06796) Organic N At3g61190 BON1-associated protein 1 (BAP1) identical to BON1-associated protein 1 [Arabidopsis thaliana] GI: 15487384; contains Pfam profile PF00168: C2 domain; supporting cDNA gi Organic N At3g61890 homeobox-leucine zipper protein 12 (HB-12)/HD-ZIP transcription factor 12 identical to homeobox-leucine zipper protein ATHB-12 (GI: 6899887) [Arabidopsis thaliana] (68416.m06951) Organic N At4g12280 copper amine oxidase family protein contains Pfam domain, PF01179: Copper amine oxidase, enzyme domain (68417.m01946) Organic N At4g13510 ammonium transporter 1, member 1 (AMT1.1) identical to SP Organic N At4g13770 cytochrome P450 family protein (68417.m02136) Organic N At4g24620 glucose-6-phosphate isomerase, putative similar to glucose-6-phosphate isomerase [Spinacia oleracea] GI: 3413511; contains Pfam profile PF00342: glucose-6-phosphate isomerase (68417.m03526) Organic N At4g30470 cinnamoyl-CoA reductase-related similar to cinnamoyl-CoA reductase from Pinus taeda [GI: 17978649], Saccharum officinarum [GI: 3341511] (68417.m04326) Organic N At4g36670 mannitol transporter, putative similar to mannitol transporter [Apium graveolens var. dulce] GI: 12004316; contains Pfam profile PF00083: major facilitator superfamily protein (68417.m05203) Organic N At4g37540 LOB domain protein 39/lateral organ boundaries domain protein 39 (LBD39) identical to SP Organic N At4g37610 TAZ zinc finger family protein/BTB/POZ domain-containing protein contains Pfam PF00651: BTB/POZ domain; contains Pfam PF02135: TAZ zinc finger; similar to Speckle-type POZ protein (SP: O43791) [Homo sapiens] (68417.m05321) Organic N At4g38470 protein kinase family protein similar to protein kinase [gi: 170047] from Glycine max; contains Pfam protein kinase domain PF00069 (68417.m05436) Organic N At4g39800 inositol-3-phosphate synthase isozyme 1/myo-inositol-1-phosphate synthase 1/ MI-1-P synthase 1/IPS 1 identical to SP Organic N At5g04950 nicotianamine synthase, putative similar to nicotianamine synthase [Lycopersicon esculentum][GI: 4753801], nicotianamine synthase 2 [Hordeum vulgare][GI: 4894912] (68418.m00524) Organic N At5g40850 urophorphyrin III methylase (UPM1) identical to urophorphyrin III methylase (GI: 1146165) [Arabidopsis thaliana]; similar to s-adenosyl-L-methionine- dependent uroporphyrinogen III methyltransferase (GI: 1490606) [Arabidopsis thaliana]; similar to Diphthine synthase (Diphtamide biosynthesis methyltransferase) (DPH5) (SP: P32469) [Saccharomyces cerevisiae]; contains Pfam PF00590: Tetrapyrrole (Corrin/Porphyrin) Methylases domain; contains TIGRFAM PF00590: Tetrapyrrole (Corrin/Porphyrin) Methylases (68418.m04960) Organic N At5g41670 6-phosphogluconate dehydrogenase family protein contains Pfam profiles: PF00393 6-phosphogluconate dehydrogenase C-terminal domain, PF03446 NAD binding domain of 6-phosphogluconate (68418.m05063) Organic N At5g45340 cytochrome P450 family protein similar to SP Organic N At5g46050 proton-dependent oligopeptide transport (POT) family protein contains Pfam profile: PF00854 POT family (68418.m05663) Organic N At5g48370 thioesterase family protein similar to SP Organic N At5g49480 sodium-inducible calcium-binding protein (ACP1)/sodium-responsive calcium- binding protein (ACP1) identical to NaCl-inducible Ca2+-binding protein GI: 2352828 from [Arabidopsis thaliana] (68418.m06123) Organic N At5g51830 pfkB-type carbohydrate kinase family protein contains Pfam profile: PF00294 pfkB family carbohydrate kinase (68418.m06426) Organic N At5g54170 expressed protein weak similarity to SP Organic N At5g58140 protein kinase family protein/non phototropic hypocotyl 1-like protein (NPL1) contains Pfam domains, PF00069: Protein kinase domain and PF00785: PAC motif; similar to SP: O48963 Nonphototropic hypocotyl protein 1 (Phototropin) [Mouse-ear cress] {Arabidopsis thaliana}; identical to cDNA non phototropic hypocotyl 1-like (NPL1) GI: 5391441 (68418.m07277) This table contains a list of genes that were previously identified as regulated by inorganic N (Wang et al., 2004 Plant Physiol 136: 2512-2522) and that were found regulated in the current study. The first column summarizes the regulatory pattern observed in this study.

[0270] Nitrogen signals control amino acid metabolism in Arabidopsis seedlings. To evaluate the biological significance of the observed patterns of response to the treatments, the distribution of functional categories in the six classes defined in Table 3 were analyzed using the BioMaps program (Gutierrez et al., 2007, Genome Biol. 8:R7). To focus on the most prominent biological processes affected, over-represented functional terms (p<0.01) with 5 or more genes (Table 5) were analyzed. This analysis indicated that inorganic N represses amino acid biosynthesis, and in particular a subset of genes related to the metabolism of S-containing amino acids. In addition, increased levels of internal Glu appears to induce the expression of genes involved in cell wall biosynthesis, especially genes in the xyloglucan:xyloglucosyl transferase family. Internal Glu also appears to repress genes involved in several aspects of metabolism, most prominently amino acid and carbohydrate metabolism. This analysis also showed that genes involved in secondary metabolism are repressed by both internal and external organic N sources. These results indicate that the balance between organic and inorganic N controls the expression of genes involved in N-reduction, N-assimilation and amino acid metabolism in Arabidopsis plants and coordinates N-assimilation with cellular processes including for example, cell wall biosynthesis.

TABLE-US-00005 TABLE 5 Response MIPS Functional Term p-value Genes Inorganic nitrogen repression amino acid biosynthesis (12) 0.00322 At3g54640, At4g13890, At3g01120, At5g37600, At3g23810, (100) At2g36880, At4g39980, At4g15560, At5g23020, At3g17390, At5g16570, At3g03780 Internal Glu induction (89) cell wall (7) 0.00933 At5g64570, At1g03870, At1g32170, At2g01850, At1g11545, At3g23730, At3g14310 Internal Glu repression (246) METABOLISM (98) 6.95E−05 At3g61190, At5g65620, At5g64440, At5g37990, At3g02360, At1g08920, At5g49720, At1g75680, At2g44160, At2g30490, At5g35170, At1g77760, At4g00370, At1g51680, At1g15130, At3g19420, At4g33580, At1g17840, At3g60750, At2g30870, At3g54690, At1g15950, At2g47180, At5g01820, At5g20070, At4g24620, At2g46830, At5g05730, At4g21850, At1g06640, At5g20980, At5g48370, At4g36640, At4g34050, At2g38010, At4g19810, At3g48690, At2g27860, At5g03555, At1g22610, At2g36690, At3g63010, At3g21240, At4g29900, At2g39210, At1g37130, At4g33680, At1g79380, At4g39800, At4g36250, At5g63850, At1g02400, At1g53500, At1g66900, At5g01800, At4g14440, At1g11840, At3g44720, At2g20360, At2g22240, At5g54960, At4g12280, At5g55910, At1g03590, At2g38700, At5g54160, At1g65960, At2g16430, At5g11110, At2g30040, At4g11570, At3g21230, At5g58140, At4g39640, At5g37510, At4g00360, At3g01560, At4g30440, At2g40890, At2g47880, At3g48560, At5g49630, At1g76670, At5g43370, At4g30470, At4g25300, At2g29450, At2g40140, At4g39330, At1g07890, At4g30280, At1g12000, At2g36290, At5g53370, At1g74710, At3g45640, At3g23820, At3g17820 amino acid metabolism (28) 0.00017 At3g61190, At5g64440, At5g63850, At4g39640, At5g05730, At4g21850, At1g06640, At5g20980, At1g02400, At2g44160, At3g48560, At5g49630, At2g30490, At1g11840, At1g77760, At3g44720, At1g22610, At4g25300, At5g54960, At3g60750, At2g36690, At1g07890, At3g54690, At1g37130, At1g65960, At1g74710, At4g33680, At3g17820 complex cofactor binding (13) 0.00235 At4g36250, At3g60750, At5g37510, At4g36640, At1g15950, At2g44160, At3g48560, At1g53500, At2g30490, At2g27860, At4g33680, At5g54960, At4g12280 C-compound and carbohydrate 0.00673 At4g36250, At3g02360, At5g49720, At1g75680, At2g44160, utilization (36) At1g53500, At1g11840, At2g20360, At2g22240, At5g54960, At4g33580, At3g60750, At3g54690, At1g15950, At5g11110, At2g47180, At4g11570, At4g24620, At3g21230, At5g37510, At5g20980, At4g30440, At3g48560, At3g48690, At2g27860, At1g76670, At4g30470, At4g39330, At4g30280, At3g21240, At3g63010, At2g36290, At1g12000, At5g53370, At3g23820, At4g39800 Internal/External Glu secondary metabolism (19) 0.0036 At3g16150, At1g13110, At4g15390, At3g58990, At1g06000, repression (102) At4g39950, At4g22710, At4g30210, At1g20510, At2g05710, At5g48010, At5g26030, At1g02500, At5g40850, At1g10360, At1g05010, At2g34460, At5g47990, At5g45340

[0271] Network analysis reveals a metabolic gene network connected to regulatory transcription factors regulated by organic N. To uncover the mechanism underlying gene regulation in response to sensing Glu or a Glu-derived product, network analysis was used to identify the subnetwork of genes regulated by organic N (FIG. 3). The subnetwork of N-regulated genes using an Arabidopsis multinetwork was generated as described previously (Gutierrez et al., 2007, Genome Biol 8:R7). Cytoscape was used to visualize the resulting subnetworks wherein genes were represented as nodes connected by edges that represented distinct interactions (e.g., metabolic reactions, regulatory interactions). In addition to the interactions described previously (Gutierrez et al., 2007, Genome Biol 8:R7), regulatory connections were predicted between genes and associated transcription factors (see, Materials and Methods). In order to identify putative “master regulators” that control the expression of genes regulated by organic N, the transcription factors regulated in these experiments were ranked based on the number of regulatory connections in the subnetwork (Table 6). At the top of the list, were a Myb family transcription factor (At1g74840), the central clock gene CCA1 (At2g46830) and a golden 2-related transcription factor (GLK1; At2g20570). Interestingly, both CCA1 and GLK1 were predicted to positively affect the expression of a gene for glutamine synthetase (GLN1. 3) (which uses Glu in a biosynthetic reaction), and to negatively affect the expression of a glutamate dehydrogenase gene (GDH1) (which catabolizes Glu) (FIG. 3). Moreover, the analysis suggests that both CCA1 and GLK1 block the expression of a bZIP transcription factor (bZIP1) which is predicted to induce the expression of the Gln-dependent ASN1 gene. Thus, Glu regulation of the GLK1 and CCA1 transcription factors appears to coordinate the expression of genes involved in making Gln (GLN1.3) vs. those involved in metabolizing Gln into Glu (ASN1, GDH1). In addition, another gene in this gene subnetwork encodes a putative asparaginase gene (ANS) that controls the degradation of Asn (to Asp and Glu) (FIG. 3). The ASN1 (Asn biosynthesis) and ANS (Asn degradation) genes had inverse expression patterns (correlation =−0.51), suggesting that Glu coordinates the reciprocal regulation of Asn synthesis and degradation by coordinating an antiregulation of the cognate.

TABLE-US-00006 TABLE 6 Number of PUB connections LOCUS Annotation 51 At1g74840 myb family transcription factor. 47 At2g46830 myb-related transcription factor (CCA1) 46 At2g20570 golden2-like transcription factor (GLK1) 31 At4g37260 myb family transcription factor (MYB73) 30 At5g24800 bZIP1 transcription factor family protein contains 30 At5g49450 bZIP1 family transcription factor 29 At5g14540 proline-rich family protein contains proline rich extensin domains. 29 At5g48655 zinc finger (C3HC4-type RING finger) family protein 24 At1g22070 bZIP1 family transcription factor (TGA3) 23 At3g01560 proline-rich family protein contains proline rich extensin domains. 20 At2g22430 homeobox-leucine zipper protein 6 (HB-6) 19 At5g44190 myb family transcription factor (GLK2) 16 At1g43160 AP2 domain-containing protein RAP2.6 (RAP2.6) 15 At3g61890 homeobox-leucine zipper protein 12 (HB-12) 14 At5g61890 AP2 domain-containing transcription factor family protein similar to RAP2.6 12 At3g61150 homeobox-leucine zipper family protein 11 At2g38470 WRKY family transcription factor 11 At4g17500 ethylene-responsive element-binding protein 1 (ERF1) 10 At2g25000 WRKY family transcription factor

[0272] Validation of network model predictions highlights the regulatory role of CCA1 in the N-assimilatory pathway. The model in FIG. 3 predicts that CCA1 and/or GLK1 genes are important regulators of genes involved in N-assimilation and over-expression of either one of these genes would repress the expression ofASN1 and GDH1 and induce the expression of the GLN1.3 gene. Conversely, a knockout of the CCA1 or GLK1 gene should increase ASN1 and GDH1 expression levels and diminish GLN1.3 mRNA levels. To test these hypotheses, we used previously characterized CCA1 overexpressor (CCA1-ox) (Wang et al., 1998, Cell 93:1207-1217) and GLK1 gene knockout (glk1) (Fitter et al., 2002, Plant J 31:713-727) lines. A stronger phenotype for the overexpressor lines was anticipated as compared to the knockout, as the model predicts redundancy in the function of CCA1 and GLK1 in regulating ASN1, GDH1 and GLN1.3 gene expression. CCA1 -ox, glk1 and wild-type plants were grown for two weeks as above, and samples were collected in the morning (3 h after dawn). Total RNA was extracted from whole seedlings and RT-qPCR was performed to determine mRNA levels for ASN1, GLN1.3 and GDH1 in the three genotypes. As shown in FIGS. 4A-B, all three genes tested showed altered expression patterns in the mutant lines utilized (as determined by analysis of variance, p<0.05) which were consistent with the predicted network model shown in FIG. 1. In addition, bZIP1 mRNA level was also repressed in CCA1-ox (FIG. 10). ASN1, GDH1 and GLN1.3 mRNA levels were not altered in the glkl line, with the exception of a small increase in GDH1 mRNA levels. This is probably due to the redundant function of GLK1 and CCA1 in regulating the expression of the tested genes. In contrast, and as predicted by the model shown in FIG. 3, ASN1 and GDH1 levels were decreased in the CCA1 -ox line. Also consistent with the predictions of the model, GLN1.3 mRNA levels were increased as compared to wild-type in the CCA1-ox.

[0273] The network model predicts that the effect of CCA1 on the expression of the target genes will be direct. To test this hypothesis, ChIP assays were used using a CCA1 antibody (FIG. 4B). As controls, it was demonstrated that the ChIP assays could detect binding of CCA1 protein to a region of the TOC1 promoter, a known target of CCA1, but was not able to detect the ZTL promoter which has no circadian oscillation at the mRNA level. Consistent with the model for CCA1, ChIP assays in both wild-type and CCA1-ox lines were able to confirm binding of CCA1 to the promoter regions of GLN1.3,GDH1 and bZIP1 promoters. These results support the model, and indicate that CCA1 regulates expression of bZIP1, GDH1 and GLN1.3 genes directly, and indirectly for ASN1 through bZIP1.

[0274] N-nutrient signals act as input to the Arabidopsis circadian clock. CCA1 is a key component of a negative feedback loop at the center of the Arabidopsis circadian clock (McClung, 2006, Plant Cell 18:792-803; Millar, 2004, J. Exp. Bot. 55:277-283). Because the results showed that N-treatments affected CCA1 expression, it was hypothesized that N might serve as an input capable of affecting the circadian clock function. To test this hypothesis, pulses of inorganic or organic N were provided at intervals spanning a circadian cycle and determined the effects on the phase of the oscillation in CCA1::LUC expression. Each treatment resulted in stable phase shifts indicating that N status serves as an input to the circadian clock (FIGS. 5A-D and FIG. 11). Inorganic N and 10 mM Glu treatments conferred slight phase advances whereas 10 mM Gln conferred only delays. The Nms and Glu pulses did not affect the period but the Gln pulse shortened the period as determined by one-way analysis of variance and Dunn's multiple comparison tests. Thus, the clock regulates a number of steps in N metabolism, such as NR expression and activity (Pilgrim et al., 1993, Plant Mol Biol 23:349-64) and ASN1 expression as demonstrated herein (see also Harmer et al., 2000, Science 290:2110-2113). In turn, N status feeds back to the clock, at least in part through its effect on CCA1 expression.

7.4 Discussion

[0275] In the present study, genomic and pharmacological approaches were used to distinguish organic from inorganic N responses in Arabidopsis seedlings. The majority of the genes regulated by the N-treatments used in this study (81%) are responding to organic N signals. Among the genes regulated by organic N, we distinguished two classes of genes: (i) genes that responded only to external Glu application and (ii) genes that responded to internal and external sources of Glu (Table 3). The difference in these two expression patterns raises the possibility that there are different mechanisms for sensing internally produced cellular Glu vs. extra cellular Glu that is transported between cells. The other possibility is that the differences in internal vs. external Glu responses observed in this study reflect distinct threshold responses to Glu levels. There is precedence for internal and external Glu sensing mechanisms in other organisms. Bacteria regulate ammonium assimilation via a mechanism involving PII, a sensor that measures levels of a-ketoglutarate and Glu (Arcondeguy et al., 2001, Microbiol Mol Biol Rev 65:80-105). Plants contain a PII protein that is localized to chloroplasts (Hsieh et al., 1998, Proc Natl Acad Sci USA 95:13965-13970), a potential sensor of internal levels of Glu. By contrast, extracellular Glu is sensed by Glu receptors in animal brains (Sykova, 2004, Neuroscience 129:861-876). The presence of Glu receptor genes in plants (Lacombe et al., 2001, Science 292:1486-1487) raises the possibility that Glu receptors in plants may serve to sense levels of external apoplastic transported Glu.

[0276] Analysis of the genes regulated by N, identified a gene network with transcription factors that appear to regulate the expression of N-assimilatory genes. New to this study is the finding that the NR genes (NIA1, NIA2) are repressed by organic N, as is GLN1.3, which is involved in Gln biosynthesis. Within this N-regulated network we also found genes involved in N uptake and metabolism including an ammonium transporter (AMT1.1), genes involved in assimilating N into and out of Asn (ASN1, ANS), as well as amino acid transporter genes. Organic N negatively regulated AMT1.1 and ANS, but induced the ASN1 gene. It was therefore hypothesized that in the presence of Glu, or a Glu-derived metabolite, Asn production is optimized and regulated at the level of transcription by increasing levels of ASN1 and decreasing levels of ANS transcripts. These results are consistent with Asn serving as a major N storage compound (Lam et al., 1994, Plant Physiol 106:1347-1357) controlled by the ASN1 gene, and suggest a mechanism to maximize Asn production, degradation and distribution depending on levels of internal sources of organic N.

[0277] The network analysis proposed a mechanism for transcriptional regulation of N-assimilation. ASN1 was a predicted target of the transcription factor bZIP1; GDH1, GLN1.3 and bZIP1 were predicted targets of GLK1 and CCA1. Because bZIP1 is also regulated by carbon (Gutierrez et al., 2007, Genome Biol 8:R7), this gene may be an integrator of C and N signaling for regulation of N-assimilation in Arabidopsis. This our network model was validated by measuring mRNA levels of the target genes in CCA1-ox and glk1 knockout lines. As predicted, ASN1 and GDH1 mRNA levels were down regulated and GLN1.3 mRNA was elevated in the CCA1-ox line. In contrast, mRNA levels for these three genes were not affected in the glk1 knockout line. The lack of a molecular phenotype in the glk1 knockout may be explained by the fact that CCA1 and GLK1 are predicted to have the same regulatory function in the subnetwork. The predictions that CCA1 directly targets a number of genes in the network was validated using CCA1 antibodies in ChIP experiments. Because CCA1 is one of the central components of the circadian clock in Arabidopsis, regulation of CCA1 expression in response to organic N suggests that the circadian clock may receive N nutritional inputs in plants. Thus, in addition to light and temperature (Millar, 2004, J. Exp. Bot. 55:277-283; McClung, 2001, Ann. Rev. Plant Physiol Plant Mol.Biol. 52:139-162), nutrients -such as N- may act as input for the clock. The phase response curve analysis results presented herein are consistent with weak (type 1) resetting similar to those observed in response to light pulses in Lemna gibba (Kondo, 1983, Plant Cell Physiol. 24:659-665), KC1 or ethanol pulses in Phaseolus coccineus (Bunning and Moser, 1973, Proc Natl Acad Sci USA 70:3387-3389) and cAMP or imidazole pulses in Trifolium repens (Bollig et al., 1978, Planta 141:225-230), strengthening the hypothesis that N status feeds back to the clock, at least in part through its effect on CCA1 expression. In Arabidopsis, light pulses evoke strong delays (-8 h) in the early night and strong advances (5-10 h) later in the night (Covington et al., 2001, Plant Cell 13:1305-1315). Although the molecular basis of these phase shifts is not definitively established, they may involve induction of CCA1 by light (Wang et al., 1998, Cell 93:1207-1217). In the above-experiments, N treatment would decrease CCA1 mRNA abundance. That this elicits only small phase shifts suggests that posttranscriptional regulation buffers against CCA1 activity changes from reduced mRNA, at least over the time frames tested with our 4-h N pulses. Alternatively, N treatment may also modulate other clock components either at the mRNA, protein abundance or protein activity level in ways that reduce the magnitude of the phase shifts in response to CCA1 mRNA decrease. The emerging view of the circadian clock is as a key integrator of multiple metabolic and physiologic processes (Lam et al., 1994, Plant Physiol 106:1347-1357; Kondo, 1983, Plant Cell Physiol. 24:659-665). As such it receives input not only from environmental stimuli but also from multiple metabolic pathways, many of which are subject to circadian regulation. Thus, the clock regulates a number of steps in N metabolism, such as NR expression and activity (Bunning and Moser, 1973, Proc Natl Acad Sci USA 70:3387-3389) and ASN1 expression. In turn, N status feeds back to the clock, at least in part through its effect on CCA1 expression. This feedback is more subtle than the effects of saturating light pulses and our results are consistent with N status fine tuning clock function rather than conferring large changes such as those observed in response to light (FIG. 6).

[0278] Oscillations in the mRNA of genes that code for metabolic enzymes could have an impact on metabolite levels, as recently shown (Gibon et al., 2006, Genome Biology 7:R76). Predicting time of food availability is key for the survival in most animals (Stephan, 2002, J Biol Rhythms 17:284-292). The data presented herein suggest that this may also be the case in Arabidopsis, e.g., anticipating the availability of carbon skeletons produced by photosynthesis to assimilate inorganic N into amino acids. Moreover, this data provides a plausible molecular mechanism for how this could happen in plants via CCA1 . The present study thus provides evidence that plant nutrition, like in animals, is tightly linked to circadian functions as previously hypothesized (Harmer et al., 2000, Science 290:2110-2113). Recently, it was shown that the central clock gene Pert is necessary for food anticipation in mice (Feillet et al., 2006, Curr Biology 16:2016-2022). The present data indicates that the central clock gene CCA1 plays a role in circadian regulation of N-assimilation in plants (FIG. 12). This data is consistent with a model in which the N-assimilatory pathway is a downstream target of the clock with CCA1 being the direct regulatory factor. Moreover, Glu or other Glu-derived signal act as input to the circadian clock providing a link between plant N-nutrition and circadian rhythms.

8. EQUIVALENTS

[0279] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0280] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties.