Transgenic plants having altered DAHP synthase activity
09909135 ยท 2018-03-06
Assignee
Inventors
- Gad Galili (Rehovot, IL)
- Asaph AHARONI (Rehovot, IL)
- Vered Tzin (Rehovot, IL)
- Sergey Malitsky (Rehovot, IL)
- Ilana Rogachev (Rehovot, IL)
Cpc classification
C12Y205/01054
CHEMISTRY; METALLURGY
C12N15/8243
CHEMISTRY; METALLURGY
C12N15/8251
CHEMISTRY; METALLURGY
International classification
C12N15/82
CHEMISTRY; METALLURGY
Abstract
The present invention provides transgenic plants comprising 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate (DAHP) Synthase that is insensitive to feedback inhibition, particularly to feedback inhibition by phenylalanine, producing increased amounts of the Shikimate pathway primary and secondary metabolites, including aromatic amino acids compared to corresponding non-transgenic plants.
Claims
1. A transgenic dicot plant comprising at least one plant cell comprising an exogenous polynucleotide encoding E. Coli AroG 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 and having at least a 7-fold increase in phenylalanine or prephenate as compared to the level of said phenylalanine or of said prephenate in a transgenic dicot plant expressing wild type E. coli AroG DAHPS set forth by SEQ ID NO:1, or as compared to a wild type control dicot plant of the same type.
2. The transgenic dicot plant of claim 1, wherein the polynucleotide encoding SEQ ID NO:2 comprises the nucleic acid sequence set forth in SEQ ID NO:3.
3. The transgenic dicot plant of claim 1, wherein the polynucleotide encoding SEQ ID NO:4 comprises the nucleic acid sequence set forth in SEQ ID NO:5.
4. A plant seed produced by the transgenic dicot plant of claim 1, wherein the seed is used for breeding a transgenic dicot plant having increased levels of phenylalanine or prephenate as compared to the level of said phenylalanine or of said prephenate in a transgenic dicot plant expressing wild type E. coli AroG DAHPS or as compared to a wild type control dicot plant of the same type.
5. A tissue culture comprising at least one transgenic cell of the dicot plant of claim 1 or a protoplast derived therefrom, wherein the tissue culture regenerates a transgenic dicot plant having at least a 7-fold increase in phenylalanine or prephenate as compared to the level of said phenylalanine or of said prephenate in a transgenic dicot plant expressing wild type E. coli AroG DAHPS or as compared to a wild type control dicot plant of the same type.
6. A method of modifying a flavor of fruit of a dicot plant, comprising (a) transforming a dicot plant cell with an exogenous polynucleotide encoding E. Coli AroG DAHPS having the amino acid sequence set forth in SEQ ID NO:4, and (b) regenerating the transformed cell into a transgenic dicot plant, wherein the transformed dicot plant has at least a 7-fold increase in phenylalanine or prephenate as compared to the level of said phenylalanine or of said prephenate in a transgenic dicot plant expressing wild type E. coli AroG DAHPS set forth by SEQ ID NO:1, or as compared to a wild type control dicot plant of the same type, and wherein said amino acid sequence modifies a flavor of a fruit as compared to said flavor of said fruit of a wild type control dicot plant of the same type.
7. The method of claim 6, wherein the polynucleotide encoding SEQ ID NO:4 comprises the nucleic acid sequence set forth in SEQ ID NO:5.
8. A method of growing a transgenic dicot plant having a increased levels of -phenylalanine or prephenate, the method comprising growing a seed of the transgenic dicot plant of claim 1, thereby growing the transgenic dicot plant.
9. The method of claim 6, wherein said dicot plant is a crop plant.
10. A transgenic dicot crop plant comprising at least one plant cell comprising an exogenous polynucleotide encoding E. Coli AroG 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) having the amino acid sequence set forth in SEQ ID NO: 4, wherein the transformed dicot crop plant has at least a 7-fold increase in phenylalanine or prephenate as compared to the level of said phenylalanine or of said prephenate in a transgenic dicot crop plant expressing wild type E. coli AroG DAHPS set forth by SEQ ID NO:1, or as compared to a wild type control dicot crop plant of the same type, and wherein said amino acid sequence modifies a flavor of a fruit as compared to said flavor of said fruit of a wild type control dicot crop plant of the same type.
11. The method of claim 1, wherein said amino acid sequence set forth by SEQ ID NO: 2 or 4 increases the level of phenylalanine by at least 7-fold in the transgenic dicot plant as compared to the level of said phenylalanine in a transgenic dicot plant of the same type expressing wild type E. coli AroG DAHPS.
12. A method of increasing production of a benzeniod phenylpropanoid volatile compound in a crop plant, comprising: (a) transforming a dicot plant cell with an exogenous polynucleotide encoding E. Coli AroG DAHPS having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 wherein said amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:4 increases production of said benzeniod phenylpropanoid volatile compound as compared to said production of said benzeniod phenylpropanoid volatile compound in a wild type control dicot plant of the same type, and (b) regenerating the transformed cell into a transgenic crop plant; wherein the transgenic dicot crop plant has at least a 7-fold increase in phenylalanine or prephenate as compared to the level of said phenylalanine or of said prephenate in a transgenic dicot crop plant expressing wild type E. coli AroG DAHPS set forth by SEQ ID NO: 1, or as compared to a wild type control dicot crop plant of the same type.
13. The method of claim 12, wherein said increased production of said benzeniod phenylpropanoid volatile compound is in a fruit or a flower of said crop plant.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(12) The present invention discloses transgenic plants transformed with exogenous nucleic acid encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) having a reduced sensitivity to feedback inhibition by a product of its activity, the aromatic amino acid phenylalanine, compared to a wild type DAHPS. The present invention shows for the first time that releasing this feedback inhibition in a plant cell results in increased accumulation of shikimate and the aromatic amino acids phenylalanine, tyrosine and tryptophan. Furthermore, the present invention now shows that expression of the feedback-insensitive DAHPS triggers the expression of genes associated with biotic stress, including pathogenesis-related genes, as well as genes associated with cell wall metabolism, secondary metabolism, biotic-stress related hormone metabolism (ethylene as well as salicylic acid that is produced via the Shikimate and Phenylalanine biosynthesis pathways), redox state regulation, transcription factors and signaling genes.
(13) The present invention now shows that transgenic plants expressing the feedback-insensitive DAHPS gene produce increased amount of phenylalanine compared to corresponding non transgenic plants. Moreover, the present invention now shows that the expression of a bacterial DAHPS polypeptide having reduced sensitivity to feedback inhibition in transgenic plants, particularly within the plastid of the plant cell leads to over production of secondary metabolites, which require phenylalanine and/or tyrosine and/or tryptophan and/or intermediate compounds produced through the phenylalanine pathway for their biosynthesis. Particularly, the second metabolites include phenylpropanoids selected from the group consisting of lignin, which is an essential component of the cell wall; chlorogenic acid (3-Caffeoylquinate), an antioxidant and inhibitor of the tumor promoting activity of phorbol esters; and flavonoids, which are plant pigment contributing to plant coloring and UV protection. The alteration of the Shikimate pathway may also lead to the production of phenylpropanoids class of benzeniods, which are volatile compounds essential for sweet, floral and fruity flavors.
(14) The present invention also provides a method of producing transgenic plants having increased amounts of at least one of shikimate, chorismate and an aromatic amino acid selected from the group consisting of phenylalanine, tryptophan and tyrosine as compared to a corresponding non transgenic plant. Also provided by the present invention are plant cells, comprising exogenous nucleic acids encoding DAHPS that is insensitive to feedback inhibition, particularly to feedback inhibition by phenylalanine, and plant seeds and progenies obtained from the transgenic plants.
(15) The present invention makes a significant contribution to the art by providing new strategies to engineer plants having the capability to modify the production of secondary metabolites. The present invention utilizes primary enzymes in the Shikimate pathway, not previously shown to be manipulated in plants by the release of feedback inhibition, for overproduction of shikimate and aromatic amino acids as well as secondary metabolites derived therefrom.
(16) The plants of the present invention are capable of overproducing secondary metabolites that have beneficial effects on the plant characteristics, for example fruit taste and aroma. Furthermore, the plants of the invention overproduce secondary metabolites required for their beneficial characterizations, which are naturally produced by the plant in insufficient amounts to be used commercially.
(17) Definitions
(18) As used herein, the terms having reduced sensitivity to feedback inhibition, insensitive to feedback inhibition or feedback insensitive with regard to DAHPS activity refer to complete or essential relieve of feedback inhibition at 1 mM of Phe, while showing similar specific enzymatic activity as the wild type (Hu et al. 2003, supra).
(19) The term plant is used herein in its broadest sense. It includes, but is not limited to, any species of woody, herbaceous, perennial or annual plant. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a root, stem, shoot, leaf, flower, petal, fruit, etc.
(20) As used herein, the term feedback inhibition refers to a cellular control mechanism of enzyme activity, in which an enzyme that catalyzes the production of a particular substance in the cell is inhibited when that substance has accumulated to a certain level.
(21) The term phenylalanine catabolic product(s) refers to classes of plant-derived organic compounds that are biosynthesized from the amino acid phenylalanine, particularly phenylpropanoids. The phenylpropanoids have a wide variety of functions in the plant, including defense against herbivores, microbial attack, or other sources of injury; as structural components of cell walls; as protection from ultraviolet light; as pigments; and as signaling molecules.
(22) The term 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS) as used herein refers to a protein having the enzymatic activity of converting Phosphoenolpyruvate (PEP) and Erythrose 4-Phosphate (E-4P) into 3-Deoxy-d-Arabino-Heptulosonate 7-Phosphate (DAHP) (
(23) The term gene refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of RNA or a polypeptide. A polypeptide can be encoded by a full-length coding sequence or by any part thereof. The term parts thereof when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, a nucleic acid sequence comprising at least a part of a gene may comprise fragments of the gene or the entire gene.
(24) The term gene also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5 and 3 ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5 of the coding region and which are present on the mRNA are referred to as 5 non-translated sequences. The sequences which are located 3 or downstream of the coding region and which are present on the mRNA are referred to as 3 non-translated sequences.
(25) The terms polynucleotide, polynucleotide sequence, nucleic acid sequence, and isolated polynucleotide are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA or hybrid thereof, that is single- or double-stranded, linear or branched, and that optionally contains synthetic, non-natural or altered nucleotide bases. The terms also encompass RNA/DNA hybrids.
(26) An isolated nucleic acid molecule is one that is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid (i.e., sequences encoding other polypeptides). Preferably, an isolated nucleic acid is free of some of the sequences which naturally flank the nucleic acid (i.e., sequences located at the 5 and 3 ends of the nucleic acid) in its naturally occurring replicon. For example, a cloned nucleic acid is considered isolated. A nucleic acid is also considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by agroinfection. Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
(27) The term recombinant refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
(28) The term construct as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest, a marker gene which in some cases can also be the gene of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.
(29) The term operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5 to the target mRNA, or 3 to the target mRNA, or within the target mRNA, or a first complementary region is 5 and its complement is 3 to the target mRNA.
(30) The terms promoter element, promoter, or promoter sequence as used herein, refer to a DNA sequence that is located at the 5 end (i.e. precedes) the protein coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as constitutive promoters. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in Okamuro J K and Goldberg R B (1989) Biochemistry of Plants 15:1-82.
(31) As used herein, the term an enhancer refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
(32) The term expression, as used herein, refers to the production of a functional end-product e.g., an mRNA or a protein.
(33) The term transgenic when used in reference to a plant or seed (i.e., a transgenic plant or a transgenic seed) refers to a plant or seed that contains at least one heterologous transcribeable polynucleotide in one or more of its cells. The term transgenic plant material refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous polynucleotide in at least one of its cells. A transgenic plant and a corresponding non transgenic plant as used herein refer to a plant comprising at least one cell comprising a heterologous transcribeable polynucleotide and to a plant of the same type lacking said heterologous transcribeable.
(34) The terms transformants or transformed cells include the primary transformed cell and cultures derived from that cell regardless to the number of transfers. All progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants.
(35) Transformation of a cell may be stable or transient. The term transient transformation or transiently transformed refers to the introduction of one or more exogenous polynucleotides into a cell in the absence of integration of the exogenous polynucleotide into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA), which detects the presence of a polypeptide encoded by one or more of the exogenous polynucleotides. Alternatively, transient transformation may be detected by detecting the activity of the protein (e.g. -glucuronidase) encoded by the exogenous polynucleotide.
(36) The term transient transformant refers to a cell which has transiently incorporated one or more exogenous polynucleotides. In contrast, the term stable transformation or stably transformed refers to the introduction and integration of one or more exogenous polynucleotides into the genome of a cell. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the exogenous polynucleotides. Alternatively, stable transformation of a cell may also be detected by enzyme activity of an integrated gene in growing tissue or by the polymerase chain reaction of genomic DNA of the cell to amplify exogenous polynucleotide sequences.
(37) The term stable transformant refers to a cell which has stably integrated one or more exogenous polynucleotides into the genomic or organellar DNA. It is to be understood that a plant or a plant cell transformed with the nucleic acids, constructs and/or vectors of the present invention can be transiently as well as stably transformed. The terms polypeptide, peptide and protein are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
(38) According to one aspect, the present invention provides a transgenic plant comprising at least one plant cell comprising an exogenous polynucleotide encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) having reduced sensitivity to feedback inhibition, wherein the transgenic plant comprises an increased amount of at least one of an aromatic amino acid, shikimate, chorismate or a catabolic product thereof compared to a corresponding non transgenic plant.
(39) According to certain embodiments, the polynucleotide encodes a bacterial DAHPS. According to certain typical embodiments, the polynucleotide encodes feedback insensitive E. coli DAHPS isoenzyme. According to further currently typical embodiments, the polynucleotide comprises E. coli AroG gene encoding a DAHPS having a reduced sensitivity to feedback inhibition.
(40) According to certain embodiments, the E. coli AroG gene encoding the feedback insensitive DAHPS has at least one point mutation at a position selected from the group consisting of position 150, 175, 179 and 209 of the wild type E. coli AroG DAHPS, having the amino acid sequence set forth in SEQ ID NO:1. According to one embodiment, proline at position 150 is replaced by leucine (P150L). According to other embodiments, the leucine at position 175 is replaced by glutamine (L175Q). According to further embodiments, leucine at position 179 is replaced by aspartic acid (L 179D). According to yet additional embodiments the phenylalanine at position 209 is replaced by alanine (F209A).
(41) According to certain currently preferred embodiments, the AroG gene encodes a DAHPS that shows reduced sensitivity to feedback inhibition by phenylalanine. According to these embodiments, the polynucleotide encodes DAHPS having the mutation L175Q (designated hereinafter AroG.sub.175). According to one embodiment, the feedback insensitive DAHPS has an amino acid sequence as set forth in SEQ ID NO:2.
(42) According to other currently preferred embodiments, the AroG gene encodes a DAHPS that is insensitive to feedback inhibition by phenylalanine. According to these embodiments, the polynucleotide encodes DAHPS having the mutation F209A (designated hereinafter AroG.sub.209). According to one embodiment, the feedback insensitive DAHPS has an amino acid sequence as set forth in SEQ ID NO:4.
(43) According to other currently typical embodiments, the AroG polynucleotide comprises the nucleic acid sequence set forth in any one of SEQ ID NO:3 and SEQ ID NO:5.
(44) Despite the major importance of the aromatic amino acid metabolism in plant primary and secondary production, still very little is known about the regulation of the conversion of primary carbon and nitrogen metabolites via the Shikimate pathway into the biosynthesis of the three aromatic amino acids phenylalanine, tyrosine and tryptophan.
(45) The present invention now shows that Arabidopsis and tomato plants expressing the AroG.sub.175 or AroG.sub.209 have enhanced levels of the three aromatic amino acids phenylalanine, tyrosine and tryptophan. In Arabidopsis, the levels of phenylalanine and tryptophan were found to be elevated more significantly, while in tomato the increase in the levels of phenylalanine and tyrosine was more prominent. This finding implies that DAHPS is a limiting enzyme that its activity regulates the conversion of primary carbon metabolites via the conversion of chorismate to aromatic amino acids as well as to metabolites that are derived directly from chorismate. Interestingly, among the two aromatic amino acids, phenylalanine accumulation was stimulated to a much higher degree than tryptophan in the AroGj.sub.75 expressing Arabidopsis plants (
(46) According to certain embodiments, the transgenic plant comprises an increased amount of at least one aromatic amino acid selected from the group consisting of phenylalanine, tyrosine, tryptophan or a combination thereof compared to a corresponding non transgenic plant. It is to be explicitly understood that each possibility represents a separate embodiment of the present invention.
(47) According to other embodiments, the transgenic plant comprises an increased amount of at least one catabolic product of phenylalanine compared to the corresponding non transgenic plant. According to certain embodiments, the catabolic product of phenylalanine is selected from several classes of metabolites including, but not limited to: terpenoids; glycoalkalodies (dehydrolycoperoside G, F or A); carotenoids (phytoene, phytofluene or lycopene); phenylpropanoids; lignin (caffeate, ferulate); coumarate and caffeoylquinate; flavonoids (Kaempferol, Quercetin derivatives and anthocyanins); phenylalanine derived glucosinolate (2-phenylethyl glucosinolate and phenylacetonitrile); and salicylate derivatives (hydroxybenzoate hexose and dihydroxybenzoate hexose). According to one embodiment, the catabolic product of tyrosine is homogentisate. According to one embodiment, the catabolic product of tryptophan is selected from several classes of metabolites selected from, but not limited to: tryptophan derived glucosinolates (1-(1H-indole-3-carboxylate) glucopyranose, 4-hydroxy-indolyl-3-methyl glucosinolate, 4-methoxyindole glucosinolate, 1-methoxyindole glucosinolate, 4-O-(Indole-3-acetyl)-dihexose); Auxin conjugates (4-O-(Indole-3-acetyl)-dihexose). The present invention further shows that the amount of additional classes of metabolites is elevated in the transgenic plant compared to a corresponding non-transgenic plant, including, but not limited to: monosaccharide (mannose, fructose, guanosine, fructose-6-phosphate); oligosaccharide (maltose, raffinose, trehalose and cellobiose); additional amino acids (glycine, glutamine, threonine, aspargine); polyamines (Putrecine); jasmonate conjugates (12-hydroxy jasmonate-hexose); methionine derived glucosinolates (3-benzoyloxypropyl glucosinolate, 8-methylthiooctyl glucosinolate, 7-methylthioheptyl glucosinolate and methylsulfonyloctyl glucosinolate); and combinations thereof.
(48) The present invention further shows that AroG.sub.175 expression triggers a significant increase in the accumulation of shikimate as well as of prephenate, the precursor of phenylalanine biosynthesis (
(49) In Arabidopsis, AroG.sub.175 expression increased the levels of a number of phenylalanine-derived secondary metabolites, including lignin precursors and their derivatives, anthocyanins, flavonoids, phenylalanine-derived glucosinolates, tryptophan derived glucosinolate, Methionine derived glucosinolates, Auxin, jasmonate and salicylate conjugates (
(50) In tomato fruit, AroG.sub.209 expression increased the levels of a number of phenylalanine-derived secondary metabolites including anthocyanins, flavonoids, coumarate and caffeoylquinate derivatives as well as monosaccharides and oligosaccharides (
(51) Taken together, these results imply the presence of a regulatory cross interaction between the fluxes of the shikimate and aromatic amino acid biosynthesis pathways and their further metabolism into various secondary metabolites. The results also indicate that DAHPS functions as an important regulatory enzyme in the conversion of primary to secondary metabolism in plants. Manipulating the expression of combinations of genes, particularly chorismate mutase, (CM), prephenate dehydratase (PDT) and 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS), having reduced sensitivity to feedback inhibition by phenylalanine, may thus result in synergistic effect on the accumulation the aromatic amino acids phenylalanine and tyrosine and metabolites derived thereof.
(52) As is shown in the Examples section hereinbelow, modulation of the DAHPS gene triggers significant expression changes in a moderate number of genes (109 genes). It appears that a considerable subset of these genes could be associated with biotic stresses, including those involved in hormone metabolism (cytokinin, absicisic acid, salicylate and jasmonate), several classes of transcription factors (TF) (MYBs, WRKYs and APETALA2/Ethylene-responsive element binding proteins), transportes, genes involved in signaling (calcium and cytokinin AAR-genes), genes encoding pathogenesis-related (PR) proteins including disease resistance proteins (TIR-NB proteins), and genes involved in redox regulation. These results are concomitant with the results of previous reports showing that pathogen attack and physical wounding stimulate the expression of the endogenous DAHPS genes in various plants, including Arabidopsis (Keith B. et al., 1991. Proceedings of the National Academy of Sciences 88(19):8821-8825), tomato (Gorlach J. et al., 1993. Plant Molecular Biology 23(4): 707-716) and potato (Muday G. K. and K. M. Herrmann, 1992. Plant Physiology 98(2): 496-500; Jones J. D. et al., 1995. Plant Physiology 108(4): 1413-1421). Additional studies also suggested that antisense inhibition of potato DAHPS transcript reduced the lignin content in the cell wall (Jones et al. 1995, supra).
(53) The present invention shows that in the AroG.sub.175 genotype, the change in the expression level of genes associated with synthesis of phenylalanine-derived secondary metabolites, including phenylpropanoids and metabolites associated with cell wall and salicylate metabolism correlated with changes in the levels of these classes of metabolites.
(54) Particularly, the levels of indole-glucosinolates, aliphatic-glucosinolates and phenylalanine-glucosinolates were significantly increased in the AroG.sub.175 lines in correlation to the increase in the mRNA levels of the following genes: (i) the gene encoding Flavin-Monooxygenase (FMO like glucosinolate s-oxygenase (AT1G65860; 2.1 fold increased) which catalyzes the conversion of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates and (ii) the gene encoding CYP81F2, a cytochrome P450 enzyme (AT5G57220; 2.7 fold increased), involved in indole glucosinolate metabolism. Without wishing to be bound by any specific theory or mechanism of action, the altered transcriptom and metabolome in AroG.sub.175 transgenic plants supports a regulatory link between glucosinolate metabolism and the Shikimate pathway.
(55) The expression level of 16 genes, categorized as genes associated with the circadian clock, was also induced in the AroG.sub.175-2 transcriptome. These circadian clock-related genes included those encoding MYB transcription factors (At1G01060 and At2G46830) and Zinc Finger DNA-binding proteins that regulate gene expression (At2G31380 and At5G15850). Clusters of circadian-regulated genes were previously found to connect important signaling networks that coordinate plant growth with rhythmic changes in the environment, including auxin, phenylpropanoid biosynthesis and starch metabolism. In addition, by over expression of the PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) transcription factor, which regulates the expression of several genes encoding key flavonoid enzymes, it has been shown that these target genes are regulated by the circadian clock. The co-regulation of PAP1 with the phenylpropanoids biosynthesis genes has also suggested that PAP1 acts as a master regulator of clock-controlled transcription of these genes (Harmer S. L. et al., 2000. Science 290(5499): 2110-2113). Hence, the findings of the present invention support the presence of a novel regulatory link between the expression of genes controlling physiological processes that are associated with the circadian clock and the conversion of primary to secondary metabolites.
(56) Another enriched gene category displaying increased expression was associated with nitrate assimilation, as it included genes encoding a nitrate transporter (At3g21670; NTP3) and two Nitrate Reductases (At1g77760; NIA1 and At1g37130; NIA2). The regulation of nitrogen metabolism is strongly associated with the regulation of glycolysis and the pentose phosphate pathway that serve as the donors of PEP and E-4P, the two substrates of DAHPS (Stitt M., 1999. Current Opinion in Plant Biology 2(3):178-186). In addition, nitrogen metabolism and translocation in plant cells has a major regulatory role in the competition between pathogens and their host plants for nitrogen availability. In the AroG.sub.175 transcriptome, genes associated with nitrate assimilation and disease resistance, including genes encoding PR proteins were induced.
(57) Without wishing to be bound by any specific theory or mechanism of action, the results of the present invention suggest that DAHPS activity, and perhaps the activity of other enzymes of the Shikimate pathway, influence the network connecting nitrogen metabolism with pathogen response in Arabidopsis plants.
(58) Several genes putatively encoding transcription factors were down regulated in the AroG.sub.175-2 line, including for example Zinc Finger B-box type proteins with a CCT domain (At3G07650, AT5G48250, At1G28050, At3G20810, At 1G07050). The expression of three other genes, encoding proteins associated with the circadian clock cascade, were decreased, including TOC1 (Timing of CAB expression 1; At5G61380), Pseudo-response regulator (At5G60100) and ELF4 (Early flowering 4; At2G40080).
(59) Producing the Transgenic Plants
(60) Cloning of a polynucleotide encoding the AroG DAHPS can be performed by any method as is known to a person skilled in the art. Various DNA constructs may be used to express the AroG DAHPS in a desired plant.
(61) The present invention provides a DNA construct or an expression vector comprising a polynucleotide encoding AroG DAHPS, which may further comprise regulatory elements, including, but not limited to, a promoter, an enhancer, and a termination signal.
(62) Among the most commonly used promoters are the nopaline synthase (NOS) promoter (Ebert et al., 1987 Proc. Natl. Acad. Sci. U.S.A. 84:5745-5749), the octapine synthase (OCS) promoter, caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987 Plant Mol. Biol. 9:315-324), the CaMV 35S promoter (Odell et al., 1985 Nature 313:810-812), and the figwort mosaic virus 35S promoter, the light inducible promoter from the small subunit of rubisco, the Adh promoter (Walker et al., 1987 Proc Natl Aca. Sci U.S.A. 84:6624-66280, the sucrose synthase promoter (Yang et al., 1990 Proc. Natl. Acad. Sci. U.S.A. 87:4144-4148), the R gene complex promoter (Chandler et al., 1989. Plant Cell 1:1175-1183), the chlorophyll a/b binding protein gene promoter, etc. Other commonly used promoters are, the promoters for the potato tuber ADPGPP genes, the sucrose synthase promoter, the granule bound starch synthase promoter, the glutelin gene promoter, the maize waxy promoter, Brittle gene promoter, and Shrunken 2 promoter, the acid chitinase gene promoter, and the zein gene promoters (15 kD, 16 kD, 19 kD, 22 kD, and 27 kD; Perdersen et al. 1982 Cell 29:1015-1026). A plethora of promoters is described in International Patent Application Publication No. WO 00/18963. According to certain currently preferred embodiments, the construct of the present invention comprises the constitutive CaMV 35S promoter or the E8 promoter (Deikman J. et al., Plant Physiol 100:2013-2017)
(63) The 3 non-coding sequences refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3 end of the mRNA precursor. The use of different 3non-coding sequences is exemplified by Ingelbrecht 1 L et al. (1989. Plant Cell 1:671-680).
(64) In particular embodiments of the present invention, the following elements were used to assemble the DNA constructs of the present invention: 1. A DNA sequence containing a cauliflower mosaic virus (CaMV) 35S promoter plus a CaMV omega translation enhancer upstream the translational initiation ATG codon, containing restriction enzyme sequences, termed 35S:PRO- (Shaul O. and G. Galili, 1993. Plant Mol Biol 23:759-768). 2. A DNA sequence containing E8, an ethylene biosynthesis-related gene which is induced by ethylene and activated to express at the beginning of fruit ripening. Expression of the E8 gene is spatially and temporally regulated in mature tomato fruit (Zhao et al., 2009 J Biosci 34:71-83). 3. A DNA sequence containing the 3 transcription termination and polyadenylation signals from the octopine synthase gene of Agrobacterium tumefacience, termed OCS-TER, with restriction enzyme sequences (Shaul and Galili 1993, supra).
(65) The above described sequences are used as regulatory elements that enable the expression of the encoding nucleic acid sequence within a plant cell. 4. A DNA sequence encoding a pea rbcS3 plastid transit peptide (SEQ ID NO:6), including the necessary restriction enzyme sequences (Shaul and Galili 1993, supra). The encoded peptide, when linked to the AroG DAHPS polypeptide, caused the migration of the later into the plastid. 5. A DNA sequence of the E. coli AroG.sub.WT gene (SEQ ID NO:16) which encode the E. coli AroG.sub.WT DAHPS enzyme (Having SEQ ID NO:1), or a DNA sequence of a mutated E. coli AroG.sub.175 (SEQ ID NO:3) encoding the mutated enzyme AroG.sub.175 DAHPS (SEQ ID NO:2) that is essentially not sensitive to feedback inhibition by phenylalanine or a DNA sequence of a mutated E. coli AroG.sub.209 (SEQ ID NO:5) encoding the mutated enzyme AroG.sub.209 DAHPS (SEQ ID NO:4) that is essentially not sensitive to feedback inhibition by phenylalanine. 6. A DNA sequence encoding three copies of a hemagglutinin (HA) epitope tag (SEQ ID NO:7). This epitope allows the detection of the recombinant AroG DAHPS polypeptide by immunoblots with antibodies for the HA epitope tag (Shevtsova et al., 2006. Eur J Neurosci 23:1961-1969).
(66) Those skilled in the art will appreciate that the various components of the nucleic acid sequences and the transformation vectors described in the present invention are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the constructs and vectors of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.
(67) According to yet another aspect, the present invention provides a method of inducing the synthesis of at least one of shikimate, chorismate and an aromatic amino acid in a plant, comprising (a) transforming a plant cell with an exogenous polynucleotide encoding DAHPS feedback insensitive and (b) regenerating the transformed cell into a transgenic plant comprising at least one cell having an altered content of at least one of shikimate, chorismate and an aromatic acid compared to a corresponding cell of a non transgenic plant.
(68) Methods for transforming a plant cell with nucleic acids sequences according to the present invention are known in the art. As used herein the term transformation or transforming describes a process by which a foreign DNA, such as a DNA construct, enters and changes a recipient cell into a transformed, genetically modified or transgenic cell. Transformation may be stable, wherein the nucleic acid sequence is integrated into the plant genome and as such represents a stable and inherited trait, or transient, wherein the nucleic acid sequence is expressed by the cell transformed but is not integrated into the genome, and as such represents a transient trait. According to preferred embodiments the nucleic acid sequence of the present invention is stably transformed into a plant cell.
(69) There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (for example, Potrykus I. 1991. Annu Rev Plant Physiol Plant Mol Biol 42:205-225; Shimamoto K. et al., 1989. Nature 338:274-276).
(70) The principal methods of the stable integration of exogenous DNA into plant genomic DNA includes two main approaches:
(71) Agrobacterium-mediated gene transfer: The Agrobacterium-mediated system includes the use of plasmid vectors that contain defined DNA segments which integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf-disc procedure, which can be performed with any tissue explant that provides a good source for initiation of whole-plant differentiation (Horsch et al., 1988. Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht). A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially useful in the generation of transgenic dicotyledenous plants.
(72) Direct DNA uptake: There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field, opening up mini-pores to allow DNA to enter. In microinjection, the DNA is mechanically injected directly into the cells using micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
(73) According to certain embodiments, transformation of the DNA constructs of the present invention into a plant cell is performed using Agrobacterium system.
(74) The transgenic plant is then grown under conditions suitable for the expression of the recombinant DNA construct or constructs. Expression of the recombinant DNA construct or constructs alters the quantity of at least one of shikimate, chorismate and an aromatic acid of the transgenic plant compared to their quantity in a non transgenic plant.
(75) The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In.: Methods for Plant Molecular Biology, (Eds.), 1988 Academic Press, Inc., San Diego, Calif.). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
(76) Selection of transgenic plants transformed with a nucleic acid sequence of the present invention as to provide transgenic plants having altered amount of shikimate, chorsimate, aromatic amino acids and secondary metabolites derived therefrom is performed employing standard methods of molecular genetic, known to a person of ordinary skill in the art. According to certain embodiments, the nucleic acid sequence further comprises a nucleic acid sequence encoding a product conferring resistance to antibiotic, and thus transgenic plants are selected according to their resistance to the antibiotic. According to other embodiments, the antibiotic serving as a selectable marker is one of the aminoglycoside group consisting of paromomycin and kanamycin. According to additional embodiments, the nucleic acid sequence further comprises a nucleic acid sequence encoding a product conferring resistance to an herbicide, including, but not limited to, resistant to Glufosinate ammonium. According to yet further embodiments, the nucleic acid sequence further comprises a polynucleotide encoding at least one copy of the hemagglutinin (HA) epitope tag, operably linked to the polynucleotide encoding AroG DAHPS. According to certain currently preferred embodiments, the nucleic acid sequence comprises a polynucleotide encoding three copies of the hemagglutinin (HA) epitope. According to these embodiments, proteins are then extracted and transgenic plants are selected according to the protein extracts reacting with HA-epitope antibodies.
(77) Extraction and detection of the metabolites synthesized by the transgenic plant cells can be performed by standard methods as are known to a person skilled in the art. According to certain embodiments, the metabolites of the present invention are extracted and analyzed by GC-MS as described by Mintz-Oron et al., 2008 (Plant Physiol 147(2):823-51), LC-MS and HPLC as described by Fraser et al. 2000 (Plant J 24(4):551-558) and as described in the Example section hereinbelow.
(78) The development or regeneration of plants containing the foreign, exogenous gene that encodes a protein of interest is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines, or pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one of skill in the art.
(79) There is a variety of methods in the art for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated.
(80) Also within the scope of this invention are seeds or plant parts obtained from the transgenic plants. Plant parts include differentiated and undifferentiated tissues, including but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture such as single cells, protoplasts, embryos, and callus tissue. The plant tissue may be in plant or in organ, tissue or cell culture.
(81) The following non-limiting examples hereinbelow describe the means and methods for producing the transgenic plants of the present invention. Unless stated otherwise in the Examples, all recombinant DNA and RNA techniques, as well as horticultural methods, are carried out according to standard protocols as known to a person with an ordinary skill in the art.
EXAMPLES
(82) Materials and Methods
(83) Plasmid Construction and Plant Transformation
(84) The coding DNA sequence of the E. coli AroG gene, encoding the DAHPS, was amplified by PCR with the following oligonucleotides: forward 5 CATGCATGCTGATGAATTATCAGAACGACGA-3 (SEQ ID NO:10) that introduces a SphI restriction site (underlined); and reverse 5-GGAATTCCCCGCGACGCGCTTTTACTG-3 (SEQ ID NO:11) that introduces an EcoRI restriction site (underlined). Two kinds of recombinant genes were constructed: AroGWT (the original sequence), encoding the feedback sensitive enzyme, and AroG.sub.175, having a point mutation at the 524 bp (leading to the replacement Leu175Glu), encoding the feedback insensitive enzyme. AroG175 was constructed using PCR with the following oligonucleotides: forward 5-GTGCACCGCGAACAGGCATCAGGGCTT-3 (SEQ ID NO:12) and reverse 5-AAGCCCTGATGCCTGTTCGCGGTGCAC-3 (SEQ ID NO:13). The AroG.sub.209, having a point mutation at the 625-626 bp (leading to the replacement Phe209Ala), encoding the feedback insensitive enzyme. AroG209 was constructed using PCR with the following oligonucleotides: forward 5-GCGCCGCACTGCGCCCTGTCCGTAACG-3 (SEQ ID NO:14) and reverse 5-CGTTACGGACAGGGCGCAGTGCGGCGC-3 (SEQ ID NO:15). The RuBisCO small subunit-3A plastid transit peptide (Shaul, O. and G. Galili. Plant Mol Biol, 1993. 23: p. 759-768) was fused in frame to the 5 end of the AroG open reading frame. The AroG 3 was fused to three copies of an HA epitope tag fused to an octopine synthase terminator. The chimeric gene and the vector were digested with the restriction enzymes NotI (BioLab) respectively and combined by T4 DNA ligase (BioLabs) and the entire fragment was sub-cloned into the Ti plasmid pBART, a derivative of pART27 (Gleave, A. P., Plant Mol Biol 1992. 20(6):1203-7). For sequence alignment, the NCBI database was used (http://www.ncbi.nlm.nih.gov). Agrobacterium tumefacies (A. tumefacies) strain EHA-105, was transformed with the plasmids by electrophoresis using gene pulser apparatus (Bio Rad).
(85) Wild type (WT) Arabidopsis thaliana, ecotype Colombia plants were inoculated by submersing inflorescences in the transformed A. tumefacies culture as previously described (Clough, S. J. and A. F. Bent, 1998. Plant J 16(6):735-43).
(86) Wild type tomato, ecotype M82 plants were inoculated by submersing cotyledon in the transformed A. tumefacies culture as previously described (McCormic S., Plant Tissue Manual. 1991. B6:109; Fillati, J. J. et al., 1987. Bio/Technology 5:726-730).
(87) Plant Material and Growth Condition
(88) Arabidopsis seeds were collected dried, and sowed on soil. Young seedlings were selected for transgenic plants selection by spraying with Basta (Glufosinate ammonium; Bayer CropScience). Tomato seeds sterilized and sowed on Petri dishes containing Nitsch complete medium pH 6 (Duchefa, Haarlem, Netherlands) supplemented with 1% sucrose and 1% plant agar. For transgenic tomato plants selection, 50 g/ml kanamycine was added to the growth medium. The seeds were imbibed for 48 h at 4 C. and transferred to a climate-controlled growth room with a regime of 16 h light/8 h dark (long day conditions) on 22 C.
(89) T2 generation plants were measured for progeny test and lines with a single gene insertion were selected based on 3:1 genetic segregation. 5MT (Sigma/Aldrich) Trp analog resistance test was performed as previously described (Li J. and R. L. Last, 1996. Plant Physiol 110(1):51-59; Tzin V. et al., 2009. Plant Journal 60(1):156-167).
(90) The resistant seedlings were removed to soil and grown in the greenhouse at 22 C. under long day conditions. For testing the response of plant growth to 5MT (Sigma/Aldrich), plant seeds homozygous for AroG.sub.175 and control plants were germinated on Nitsch medium as described above supplemented with 75, 100 or 150 M of 5MT (Li and Last 1996, supra)
(91) Selection of Transformed Lines
(92) Western blot analysis was performed using anti-HA tag antibodies in order to identify the mutated plants which translated the chemeric gene (Stepansky, A. and G. Galili. Plant Physiol, 2003. 133(3): p. 1407-15). Additionally, T.sub.2 generation plants were measured for progeny test and lines with a single insertion gene were selected based on 3:1 genetic segregation.
(93) Metabolomics Analysis
(94) Arabidopsis Transgenic Lines Samples Preparation
(95) Metabolic analysis was performed with aerial tissues of Arabidopsis seedlings (100 mg frozen powder) expressing the AroG.sub.WT and AroG.sub.175 and control lines (n=5-6). For UPLCqTOF-MS analysis, Arabidopsis samples were extracted in 450 l of methanol 80%. Sample preparation and injection conditions were as previously described (Mintz-Oron, S. et al., Plant Physiol, 2008. 147(2):823-51). For GC-MS analysis, the seedlings were harvested, frozen, ground and extracted in 450 l of methanol, 750 l methanol, 750 l water and 375 l chloroform as previously described (Malitsky, S. et al., Plant Physiol, 2008. 148(4):2021-2049). To assess whether the different lines in the analysis vary in their composition of metabolites, Student's t-test (JMP software) was performed. The resulting P-values were controlled for multiple hypotheses testing using a 5% FDR cutoff (Hochberg Y. and Y. Benjamini 1990. Statistics in Medicine 9(7):811-818). The PCA plots were generated using the TMEV4 software (Saeed, A. I. et al., Biotechniques, 2003. 34(2):374-378; Scholz, M. et al., 2004. Bioinformatics, 20(15):2447-2454).
(96) Tomato Transgenic Lines Samples Preparation
(97) Non-targeted metabolic analysis was performed with three developing stages of tomato fruits (500 mg frozen powder) expressing the AroG.sub.175 and AroG.sub.209 and WT (n=5), extracted in 1.5 ml of methanol 100%. Sample preparation and injection conditions were performed as previously described (Mintz-Oron, S., et al. Plant Physiol, 2008. 147(2): p. 823-51).
(98) UPLC-qTOF-MS and GC-MS Analyses
(99) The analysis of the raw UPLC-qTOF-MS data was performed using the XCMS software that performs chromatogram alignment, mass signal detection and peak integration (Smith, C. et al., Anal Chem, 2006. 78:779-787) from the Bioconductor package (v. 2.1) for the R statistical language (v. 2.6.1). XCMS was used with the following parameters: fwhm=10.8, step=0.05, steps=4, mzdiff=0.07, snthresh=8, max=1000. Injections of samples in the positive and negative ionization modes were performed in separate injection sets and pre-processing was done for each ionization mode independently. Differential mass ions were determined using a Student's 1-test (JMP software). The GC-MS analysis was performed as previously described (Malitsky, S. et al., Plant Physiol, 2008. 148(4):2021-2049), (n=5-6) The Xcalibur software v.1.4 (Thermo Finnigan; http://www.thermo.com/) was used for data analysis and compounds were identified by comparison of their retention index (R1) and mass spectrum to those generated for authentic standards analyzed on the same instrument. In cases when standards were not available, compounds were putatively identified by comparison of their R1 and mass spectrum to those present in the mass spectra library of Max-Planck-Institute for Plant Physiology, Golm, Germany (Q_MSRI_ID, http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/msri/gmd_msri.html) and the commercial mass spectra library NIST05 (www.nist.gov). The response values for metabolites resulting from the Xcalibur processing method were normalized to the ribitol internal standard. A Student's t-test analysis was done on metabolites level with significant changes in the two transformed AroG.sub.175 genotypes by using the JMP software. Then, the PCA plots were generated using the TMEV4 software (Saeed et al. 2003, supra; Scholz, et al. 2004, supra).
(100) RNA Extraction and Microarray Analysis
(101) All experiments analyzing RNA expression levels were carried out using two replicates of aerial tissues of 10 days old Arabidopsis seedlings from plants grown under controlled environment conditions. Total RNA was extracted as previously described (Chomczynski P., 1993. Biotechniques 15(3):532-537) and treated with DNAase RQ-1 (Promega). RNA was then amplified using 1-cycle Affimetrix labeling using the standard Affymetrix protocol. Hybridization, labeling, scanning and data extraction were performed according to the standard Affymetrix protocols. Transcriptome analysis was carried out using Partek Genome Suite software (www.partek.com). Pre-processing was carried out using the Robust Microarray Averaging (RMA) algorithm (Irizarry R. A. et al., 2003. Biostatistics 4(2):249-264). One way ANOVA analysis was performed, fold change between lines was calculated (two fold) and FDR was applied to correct form multiple comparisons (Hochberg and Benjamini 1990, supra). Over-representation analysis was performed by PageMan tool (http://mapman.mpimp-golm.mpg.de/general/ora/ora.shtml) (Usadel B. et al., 2006. BMC Bioinformatics 7:535). Visualization of metabolic pathways and other functional categories was preformed with the MapMan software tool (Usadel B. et al., 2005. Plant Physiol 138(3):1195-1204.). Common locus number (AGI) was used from TAIR nomenclature (http://www.arabidopsis.org). For a two-step quantitative RT-PCR, a Platinum SYBR SuperMix (Invitrogen) RT-PCRs were tracked on an ABI 7300 instrument (Applied Biosystems). Each sample was amplified by PCR using the same amount of cDNA template in triplicate reactions as previously describe (Mintz-Oron et al. 2008, supra).
Example 1
Generation of Transgenic Arabidopsis Plants Expressing a Bacterial Feedback Insensitive Dahps Enzyme and Initial Metabolic Analyses
(102) To study the importance of DAHPS in regulating fluxes bridging primary and secondary metabolism in plants, Arabidopsis plants were transformed with either a chimeric AroG.sub.WT or a chimeric AroG.sub.175 or AroG.sub.209 genes (
(103) A global view of the effect of the expression of the AroG.sub.WT, AroG.sub.175 and AroG.sub.209 transgenes on plant metabolism was obtained by performing gas chromatograph-mass spectrometry (GC-MS) analysis of polar compounds of derivatized extracts. 20 days-old plants from four independently transformed plants of each genotypes (2-3 independent analyses from each genotype; each derived from extracts made from 7 plants) as well as WT plants (five independent analyses) were taken for the analysis. The data set was first analyzed by a Principal Component Analysis (PCA). In this analysis, the metabolic profiles of the control and AroG.sub.WT genotypes grouped together, and were completely separated from the AroG.sub.175 and AroG.sub.209 genotypes (
(104) To test further whether AroG.sub.WT, AroG.sub.175 and AroG.sub.209 expression alters the levels of metabolites associated with the shikimate and aromatic amino acid biosynthesis pathways (
Example 2
Metabolic Analysis of Arabidopsis Plants Expressing the AroG175 Gene
(105) The effect of AroG.sub.175 expression on a wide range of primary and secondary metabolites was examined in two independently transformed homozygous AroG.sub.175 line, AroG.sub.175-2 and AroG.sub.175-21. These lines expressed either medium (AroG.sub.175-2) or relatively high (AroG.sub.175-21) level of the AroG.sub.175 polypeptide (
(106) In the LC-MS analysis, the AroG.sub.175-2 line generally displayed milder changes in the levels of the different metabolites compared to the AroG.sub.175-21 line (data not shown). Thus, the non-targeted LC-MS analysis focused only on metabolites that their level was significantly different between the AroG.sub.175-21 line and the control, but also showed similar direction of change in the AroG.sub.175-2 line. The non-targeted LC-MS analysis of AroG.sub.175-21 line revealed a total of 4473 mass signals, of which the levels of 2414 were significantly altered (up or down regulated) as compared to the control. Assuming an average of five mass signals per single metabolite (Malitsky S. et al., 2008. Plant Physiology 148(4):2021-2049), AroG.sub.175 expression in the AroG.sub.175-21 line resulted in altered levels of 500 metabolites. In regard to the two other aromatic amino acids, this analysis showed that the level of tryptophan was 2.6-fold higher in the AroG.sub.175-21 line, compared to the control, while its level in the AroG.sub.175-2 line was not significantly different from the control (
(107) The LC-MS analysis also showed that the level of a number of phenylalanine-derived phenylpropanoids secondary metabolites is significantly higher in AroG.sub.175-21 compared to the control plants (
(108) Since tryptophan level was higher in the AroG.sub.175-21, but not in the AroG.sub.175-2, as compared to the control lines (
Example 3
Generation of Transgenic Tomato Plants Expressing a Bacterial Feedback Insensitive Dahps Enzyme and Initial Metabolic Analyses
(109) To study the importance of DAHPS in regulating fluxes bridging primary and secondary metabolism in plants, tomato plants were transformed with either a chimeric AroG.sub.175 or AroG.sub.209 genes (
(110) At the first stage of studying the effect of expression of the AroG.sub.175 and AroG.sub.209 transgenes on plant metabolism, LC-MS analysis was performed for ripe tomato fruit from five independently transformed genotypes for each transgenic plants (5-6 independent analyses from each genotype; each derived from extracts made from 10 plants) as well as WT plants (five independent analyses). To get a global view on the metabolic effects of the AroG.sub.175 and AroG.sub.209 genotypes, the data set was first analyzed by a PCA plot. In this analysis, the metabolic profile of the control was completely separated from the AroG.sub.175 and AroG.sub.209 genotypes (
(111) TABLE-US-00001 TABLE 1 Accumulation of metabolites in developing tomato fruit obtained from plants expressing AroG.sub.209-9 gene and in fruit obtained wild-type (WT) tomato plants. Peel Flesh Metabolite MG Br Ripe MG Br Ripe Amino acids Phe 1.0 4.7 89.0 0.9 2.4 22.1 Tyr 1.6 2.4 171.9 0.9 2.7 15.3 Trp 0.6 2.2 4.1 0.5 1.4 1.4 Asn 0.4 0.5 0.5 0.6 0.9 0.5 Gln 0.1 0.1 0.4 0.3 0.7 0.4 Gly 0.5 1.3 1.7 1.0 1.2 1.5 Ile 0.7 1.2 2.2 1.0 1.1 2.0 N-acetyl-glutamate 0.1 0.2 0.4 0.7 0.8 0.4 Thr 0.5 1.0 1.1 0.7 1.0 1.2 Organic acid Fumaric acid 1.5 1.8 2.5 1.0 1.2 1.2 Prephenic acid 7.6 354.3 604.8 3.4 276.3 235.9 Shikimic acid 2.3 2.5 17.4 1.1 2.5 62.9 Sugars Cellobiose 0.9 1.3 8.0 1.0 1.2 22.0 Fructose 1.7 1.0 1.0 1.2 1.0 1.1 Fructose-6-phosphate 0.5 0.8 0.9 0.8 0.7 0.6 Maltose 1.3 1.2 1.4 1.2 0.9 1.5 Mannose 1.7 1.2 1.2 1.1 1.0 1.1 Raffinose 1.2 2.0 103.1 0.4 2.8 92.3 Trehalose 0.5 0.7 73.6 1.8 0.9 5.3 Polyamine Putrescine 1.1 0.6 0.3 0.8 0.8 0.2 Nucleosides Guanosine 0.8 0.9 1.9 1.0 0.7 1.6 Phenylpropanoids 3-Caffeoylquinic acid 0.9 1.3 2.9 1.9 1.1 2.2 4-Caffeoylquinic acid 0.7 1.0 2.1 1.3 0.8 1.4 Coumaric acid 0.7 2.7 164.8 0.4 2.6 613.6 Coumaric acid hexoside N.D N.D N.D 1.1 1.8 114.5 or deriv I Coumaric acid-hexose I 0.8 0.6 15.3 0.4 0.4 1.1 Kaempferol-glucose- 1.0 0.9 0.6 1.1 0.9 108.7 rhamnose Naringenin 0.9 0.4 0.6 0.5 10.1 1.8 Naringenin chalcone- 0.8 0.7 5.2 N.D N.D N.D hexose IV Quercetin 0.4 0.7 2.6 0.2 0.7 1.1 Quercetin-hexose- 1.0 1.1 3.9 N.D N.D N.D deoxyhexose-pentose- p-coumaric acid Tricaffeoylquinic acid 0.9 0.7 2.3 0.6 1.2 2.0 Steroidal Alkaloids Dehydrolycoperoside 2.8 1.1 2.6 1.5 1.5 2.9 G, F, A Cartenoids Phytoene N.D N.D 0.2 N.D N.D ~0.001 Phytofluene N.D N.D 0.1 N.D N.D ~0.001 Lycopene like N.D N.D 0.3 N.D N.D 0.55
(112) Metabolite levels are shown as fold change SE from five (UPLC-qTOF-MS analysis) or six (GC-MS analysis) or four (UPLC) independent samples in comparison with the WT. The metabolite levels are presented as fold change (AroG.sub.209-2/WT) in each developing stage. The three developing stages are: mature green (MG; 42 days post anthesis (DPA)), breaker (Br; 44 DPA) and red (red; 48 DPA) and separated to peel and flesh. P-values are shown as <0.05 (FDR). Terms in bold indicate a statistically significant increase as analyzed by two-way ANOVA and Student t-test. N.D. not detected.
Example 4
Metabolic Analysis of Tomato Fruits Expressing the AroG209 Gene
(113) The effect of AroG.sub.209 expression of T2 generation on a wide range of primary and secondary metabolites was examined on selected transformed homozygous AroG.sub.209-9 genotypes. This genotype contained a single insertion, based on PCR and antibiotic resistance. In order to study the effects of AroG.sub.209 expression on tomato fruit metabolism, tomato fruit in three developing stages were subjected to Lc-MA analysis: mature green (about 42 days post anthesis (DPA); breaker (about 44 DPA); and red (about 48 DPA). 5-6 independent analyses from AroG.sub.209 and WT genotypes at each developing stages were examined. Each analysis was derived from extracts made from three fruit collected from 10 plants. To get a global view on the metabolic effects of the AroG.sub.209 expression in tomato peel tissue, a PCA was performed of datasets obtained from 2,725 mass signals in negative ion mode. Interestingly, the mature green stage of both AroG.sub.209-9 and WT combine, a weak separation occurs in the breaker stage and a completely separation occurs in the red stage (
Example 5
Effects of AroG175 Expression on Gene-Expression Networks
(114) The effect of AroG.sub.175 expression on the global transcriptome of Arabidopsis was studied. A microarray analysis was performed with samples extracted from aerial tissues of 10 days old control and AroG.sub.175-2 seedlings, using the Affymetrix AtH1 GeneChip. The AroG.sub.175-2 line that was shown to have a moderate change in the metabolite was selected, to reduce the chance of pleiotropic effects. ANOVA analysis of the microarray results followed multiple hypotheses testing using a 5% FDR cutoff, indicated that expression of a relatively small number of genes (109 genes) showed a significant and at least 2-fold change between the control and AroG.sub.175-2 line, indicating a relatively moderate effect of AroG expression on the Arabidopsis transcriptome.
(115) Genes that were consistently up-regulated (77 genes) and down-regulated (32 genes) were classified by over-representation analysis using the PageMan and MapMan software tools (http://mapman.mpimpgolm.mpg.de/general/ora/ora.shtml; (Usadel et al. 2006, supra). The major effects of the AroG.sub.125-2 gene included a stimulation of the expression of 37 genes associated with biotic stress (Tables 2 and 3).
(116) TABLE-US-00002 TABLE 2 Over represented categories of up-regulated gene (minimum two fold increase) BIN (category number) Main number of P-value Page Man category Sub category (1) Sub category (2) probe sets (FDR) 10 Cell wall 3 1.2E08 10.8 Pectinesterases 2 5.4E06 12 Nitrate 3 5.4E06 metabolism 12.1.1 Nitrate reductase 2 5.4E06 16 Secondary 7 2.9E19 metabolism 16.2 Phenylpropanoids 2 5.4E06 16.4 N misc 3 1.2E08 16.4.1 Alkaloid-like 3 1.2E08 17 Hormone 4 2.8E11 metabolism 17.4 Cytokinin 2 5.4E06 17.4.2 Cytokinin- 2 5.4E06 signal transduction 20 Stress 10 2.7E27 20.1 Biotic stress 9 1.3E24 21 Redox 3 1.2E08 regulation 27 RNA 12 1.1E32 27.3 Regulation of 12 1.1E32 transcription 27.3.26 MYB - 2 5.4E06 transcription factor family 27.3.3 AP2/EREBP, 2 5.4E06 APETALA2/ Ethylene- responsive element binding protein family 27.3.5 ARR - 2 5.4E06 transcription factor family 27.3.7 C2C2(Zn) 2 5.4E06 Constans-like zinc finger family (circadian clock) 29 Protein 2 5.4E06 29.4 postranslational 2 5.4E06 modification 30 Signalling 7 2.9E19 30.2 Receptor 3 1.2E08 kinases 30.3 Calcium 4 2.8E11 33 Development 3 1.2E08 34 Transport 5 6.2E14
(117) TABLE-US-00003 TABLE 3 Over represented categories of down-regulated gene (minimum two fold decrease) BIN (category number) Main number of P-value Page Man category Sub category (1) Sub category (2) probsets (FDR) 11 Lipid 2 9.2E07 metabolism 27.3 RNA Regulation of 8 3.1E25 transcription 27.3.66 Psudo ARR 2 9.2E07 transcription factor family 27.3.7 C2C2(Zn) 3 8.4E10 Constans-like zinc finger family (circadian clock) 29 Protein 2 9.2E07 29.5 Protein 2 9.2E07 degradation
(118) The biotic stress category included genes associated with hormone metabolism (cytokinin, absicisic acid, salicylate and jasmonate), transcription factors (MYBs, WRKYs and APETALA2/Ethylene-responsive element binding proteins), signaling (calcium and cytokinin AAR-genes), pathogenesis (PR proteins), disease resistance (TIR-NB proteins), transporters and redox regulation. In addition, genes that putatively encode proteins associated with secondary metabolism were induced, including phenylprpanoid metabolism, Met-aliphatic-glucosinolates and a gene associated with Trp-derived indole-glucosinoltes. Another enriched category displaying increased expression was associated with nitrate assimilation. Notably, the expression level of 16 genes, categorized as genes associated with the circadian clock, was also induced in the AroG.sub.175-2 transcriptome. These circadian clock-related genes included those encoding MYB transcription factors, Zinc Finger B-box type proteins with a CCT DNA-binding domain and three other genes, encoding proteins associated with the circadian clock cascade including TOC1, Pseudo-response regulator and ELF4 (Early flowering 4). To corroborate the microarray results, a quantitative RT-PCR analysis was also performed on a representative sample of genes whose mRNA level was higher in the AroG.sub.175-2 than in the control line in the microarray analysis. This analysis, which was performed on both AroG.sub.175-2 and AroG.sub.175-21 lines, confirmed the gene expression results obtained by the microarray analysis.
Example 6
Aromatic Profile of Fruit from AroG209-9 Expressing Tomato Plants
(119) A panel of 10 expert trained flavorists evaluated the aroma of fruit samples obtained from tomato plants expressing the AroG.sub.209-9 gene. Preliminary tests were carried out to improve the ability of the assessors to recognize odour defects and consistently quantify sensory properties. The panelists had previously been trained in the quantitative description of tomato attributes according to selection trials based on French norms (ISO8586-1, AFNOR V09-003). For each fruit sample, flavorists evaluated whole fruits for peel profiling and cut fruitsfor the evaluation of flesh. Aroma evaluation was carried out by sniffing the samples by all panel members. Several attributes were chosen: acidic floral, fresh, green, metallic musty, ripe, spicy, and sweet in addition to global aroma intensity. Scores values were between 0 (none) to 5 (very strong). When completed, panel members discussed their scores and agreed on the final summarizing score of each aroma group. The results are summarized in
(120) The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.