Method for improving stress tolerance of plants

Abstract

The present invention relates to a method of improving stress tolerance and/or preventing growth reduction of a plant by introducing a polynucleotide encoding a Repetitive Proline-rich Protein (RePRP) into the plant.

Claims

1. A method of improving stress tolerance while preventing growth reduction of a plant, comprising: (a) transforming plant cells with a vector comprising a nucleic acid operably linked to a promoter to obtain recombinant plant cells expressing a Repetitive Proline-rich Protein (RePRP), wherein the nucleic acid encodes the RePRP protein, wherein the vector comprises SEQ ID NO: 12, 13 or 14; (b) growing the recombinant plant cells obtained in (a) to generate a plurality of transgenic plants; and (c) selecting a transgenic plant from the plurality of transgenic plants generated in (b) that exhibits an improved tolerance to stress and substantially no growth reduction, as compared with a non-transgenic plant counterpart growing under the same conditions.

2. The method of claim 1, wherein the transgenic plant exhibits less yield reduction as compared with a non-transgenic plant counterpart.

3. The method of claim 1, wherein the transgenic plant is a monocot plant.

4. The method of claim 3, wherein the monocot plant is rice, barley, wheat, rye, oat, corn, bamboo, sugarcane, onion, leek or ginger.

5. The method of claim 1, wherein, the transgenic plant is a dicot plant.

6. The method of claim 5, wherein, the transgenic plant is Arabidopsis, soybean, peanut, sunflower, safflower, cotton, tobacco, tomato, pea, chickpea, pigeon pea or potato.

7. The method of claim 1, wherein the stress is abiotic stress selected from the group consisting of osmotic stress, drought stress, salt stress, or a combination thereof.

8. The method of claim 1, wherein the vector comprises SEQ ID NO: 12.

9. The method of claim 1, wherein the vector comprises SEQ ID NO: 13.

10. The method of claim 1, wherein the vector comprises SEQ ID NO: 14.

11. A method of improving stress tolerance while preventing reduction in growth and/or productivity of a plant, comprising: (a) transforming plant cells with a vector comprising a nucleic acid operably linked to a promoter to obtain recombinant plant cells expressing a Repetitive Proline-rich Protein (RePRP), wherein the nucleic acid encodes the RePRP protein, wherein the vector comprises SEQ ID NO: 12, 13 or 14; (b) growing the recombinant plant cells obtained in (a) to generate a plurality of transgenic plants; and (c) selecting a transgenic plant from the plurality of transgenic plants generated in (b) that exhibits an improved tolerance to stress and substantially no reduction in growth and productivity, as compared with a non-transgenic plant counterpart growing under the same conditions.

12. The method of claim 11, wherein the vector comprises SEQ ID NO: 12.

13. The method of claim 11, wherein the vector comprises SEQ ID NO: 13.

14. The method of claim 1, wherein the RePRP protein is from rice, barley, wheat, maize and sorghum.

15. The method of claim 11, wherein the ReEPRP protein is from rice, barley, wheat, maize and sorghum.

16. The method of claim 11, wherein the vector comprises SEQ ID NO: 14.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

(2) In the drawings:

(3) FIG. 1 shows that OsRePRP2.1 over-expression enhanced salinity tolerance in rice. Three-leaf old seedlings were treated with 250 mM NaCl for 5 days, and then recovered to normal condition for 12 days. The survival rate of OsRePRP2.1OX transgenic plants was significantly higher than TNG67. Data are means ±SD from three experimental repeats.

(4) FIG. 2 shows that OsRePRP2.1 over-expression in rice enhanced drought tolerance. Three-leaf old seedlings were treated with 30% PEG for 20 hours, and then recovered to normal condition for 10 days. The survival rate of OsRePRP2.1OX transgenic plants was significantly higher than that of TNG67. Data are means ±SD from three experimental repeats.

(5) FIG. 3 shows that OsRePRP2.1 over-expression in rice enhanced the plant recovery after drought treatment. Watering of two-week old plants was withheld for 13 days, and watering was then reassumed for 12 days. Half of OsRePRP2.1OX plants survived and grew new leaves, while a few TNG67 plants survived.

(6) FIG. 4 shows that OsRePRP RNAi plants were sensitive to abiotic stress treatments. Three-leaf old seedlings were treated for 250 mM NaCl for 5 days or 30% PEG for 24 hours. The survival rate was recorded after 12 days of recovery. The average survival rate of RNAi lines was significantly lower than WT (TNG67) in both stress treatments, while OsRePRP2.1 over-expression lines showed up to 80% survival rate.

(7) FIGS. 5A-5B show that induced expression of OsRePRP2.1 transgenic rice plants were tolerant to PEG treatment. Two-week old plants were treated with 30% PEG for 18 hours, then recovered to normal condition for 10 days. FIG. 5A shows that the survival rate of ABRC321:OsRePRP2.1 transgenic plants (T2 generation) was significantly higher than that of TNG67. Data are means ±SD from two experimental repeats. FIG. 5B shows similar root image of 14-day old rice seedlings of wild type and ABRC321:OsRePRP2.1 plants.

(8) FIGS. 6A-6C show that OsRePRP2.1 over-expression in Arabidopsis plants grow normally, similar to non-transgenic Arabidopsis plants under normal conditions. FIG. 6A shows 50-day-old matured Arabidopsis plants grown under normal conditions. FIG. 6B shows root image of 10-day-old Arabidopsis seedlings grown on the normal agar plate. FIG. 6C shows the root length data expressed by the means ±SD from each lines.

DETAILED DESCRIPTION

(9) Unless defined otherwise, all technical and scientific terms used herein have the same meanings as is commonly understood by one of skill in the art to which this invention belongs.

(10) As used herein, the articles “a” and “an” refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

(11) The term “comprise” or “comprising” is generally used in the sense of include/including which means permitting the presence of one or more features, ingredients or components. The term “comprise” or “comprising” encompasses the term “consists” or “consisting of.”

(12) The present studies found that, unexpectedly, Repetitive Proline-rich Proteins (RePRPs) improved features of transgenic plants overly expressing such, for example growth properties and/or stress tolerance. Accordingly, provided herein are transgenic plants overly expressing a RePRP as described herein, vectors for expressing the RePRP, methods for making the transgenic plants, and methods for improving growth properties or stress tolerance of plants by over-expressing a RePRP protein.

(13) I. Repetitive Proline-Rich Proteins (RePRPs)

(14) Repetitive Proline-rich Proteins (RePRPs) are ABA induced glycoproteins in plants. These proteins include a signal peptide at the N-terminus followed by a Proline-rich domain occupying about 70% of the protein. The Proline-rich domain contains numerous repetitive PX.sub.1PX.sub.2 motifs (SEQ ID NO: 25) and constitutes the hydrophilic regions, wherein P is a proline residue and X.sub.1 and X.sub.2 are any amino acid residues other than proline and more particularly are highly glycosylated with arabinose and glucose on multiple hydroproline residues converted from proline. The motif can be repeated “n” times, represented by (PX.sub.1PX.sub.2)n, for which it is understood that X.sub.1 or X.sub.2 can be the same or different in each repeat and the identifies of X.sub.1 or X.sub.2 residue are not necessarily preserved throughout the “n” repeats of the residue. It is further understood that each of the repeats can be connected end to end (directly), or with one or more intervening amino acid(s) (indirectly). In some embodiments, n is an integer of 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, or 80 or more. In some embodiments, RePRPs have a content of proline e.g 20% or more, 30% or more, 40% or more, 50% or more based on the total number of the amino acid residues of this protein. In some embodiments, X.sub.1 or X.sub.2 is selected from the group consisting of lysine (Lys, K), glutamate (Glu, E), Asparagine (Asn, N), Aspartate (Asp, D), tyrosine (Tyr, Y), valine (Val, V), Histidine (His, H), isoleucine (Ile, I), glycine (Gly, G), threonine (Thr, T), glutamine (Gln, Q), serine (Ser, S). In some embodiments, RePRPs have 200 to 500 amino acids in length e.g. about 200, 250, 300 or 350 amino acid in length.

(15) According to the present disclosure, the terms “polypeptide,” “peptide” and “protein” as used herein refer to a polymer formed of amino acid residues, wherein one or more amino acid residues are naturally occurring amino acids or artificial chemical mimics.

(16) The RePRPs described herein can be a naturally occurring protein of any suitable species. Exemplary RePRPs may be from plants preferably from monocot plants, including, but not limited to, rice, barley, wheat, maize and sorghum. In rice, there are two subclasses of RePRPs, (1) RePRP1 including RePRP1.1 (SEQ ID NO: 1) and RePRP1.2 (SEQ ID NO: 2) and (2) RePRP2, including RePRP2.1 (SEQ ID NO: 3) and RePRP2.2 (SEQ ID NO: 4).

(17) In some embodiments, the RePRPs may comprise the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4. Alternatively, the RePRPs may be a naturally occurring protein that is highly homologous to SEQ ID NO: 1, 2, 3 or 4, for example, sharing at least 85% sequence identity in the entire length (e.g., at least 90%, at least 93%, at least 95%, or at least 97%). Such RePRPs can be readily identified from publically available gene database (e.g., GenBank) using SEQ ID NO: 1, 2, 3 or 4 as a query.

(18) It is understandable a polypeptide may have a limited number of changes or modifications that may be made within a certain portion of the polypeptide irrelevant to its activity or function and still result in a molecule with an acceptable level of equivalent biological activity or function. Modifications and changes may be made in the structure of such polypeptides and still obtain a molecule having similar or desirable characteristics. For example, certain amino acids may be substituted for other amino acids in the peptide/polypeptide structure (other than the conserved region) without appreciable loss of activity. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. For example, arginine (Arg), lysine (Lys), and histidine (His) are all positively charged residues; and alanine (Ala), glycine (Gly) and serine (Ser) are all in a similar size. Therefore, based upon these considerations, arginine (Arg), lysine (Lys) and histidine (His); and alanine (Ala), glycine (Gly) and serine (Ser) may be defined as biologically functional equivalents. One can readily design and prepare recombinant genes for microbial expression of polypeptides having equivalent amino acid residues.

(19) Therefore, in some embodiments, the RePRPs can be a functional variant of a naturally occurring RePRP. Such a functional variant may share a high sequence identity with the wild-type counterpart, for example, at least 85% (e.g., 90%, 95%, 96%, 97%, 98% or 99%) sequence identify to the amino acid sequence of the wild-type counterpart and possess substantially similar bioactivities as the wild-type counterpart.

(20) To determine the percent identity of two amino acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence). In calculating percent identity, typically exact matches are counted. The determination of percent homology or identity between two sequences can be accomplished using a mathematical algorithm known in the art, such as BLAST and Gapped BLAST programs, the NBLAST and XBLAST programs, or the ALIGN program.

(21) II. Vectors Encoding RePRPs

(22) In some aspects, the present invention provides vectors comprising a nucleic acid encoding any of the RePRPs described herein. The term “nucleic acid” or “polynucleotide” refers to a polymer composed of nucleotide units, including naturally occurring deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as any analogs thereof. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “nucleic acid” typically refers to relatively larger polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.” The term “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

(23) The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide (e.g., a gene, a cDNA, or an mRNA) to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Therefore, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. It is understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described there to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

(24) In some embodiments, a nucleic acid encoding a RePRP protein as described herein is SEQ ID NO: 5 (encoding OsRePRP1.1), SEQ ID NO: 6 (encoding OsRePRP1.2), SEQ ID NO: 7 (encoding OsRePRP2.1), or SEQ ID NO: 8 (encoding OsRePRP2.2).

(25) A “vector,” as used herein, can be a recombinant nucleic acid-based vehicle to artificially carry foreign genetic material into a host cell, in which the foreign genetic material can be replicated and/or expressed. The vector as described herein may be a cloning and/or an expression vector. The vector may be in linear or circular form. It may remain episomal or integrate into the host cell genome when introduced into a host cell. In some embodiments, the vector can be a viral vector or a non-viral vector (e.g., a plasmid). In particularly examples, the vectors may be plant vectors or Agrobacterium vectors.”

(26) In some embodiments, the nucleic acid encoding a RePRP protein can be operably linked to a promoter in the vector to drive expression of RePRP either in vitro or in vivo. As used herein, the term “operably linked” may mean that a polynucleotide is linked to an expression control sequence e.g. a promoter in such a manner to enable expression of the polynucleotide when a proper molecule (such as a transcriptional factor) is bound to the expression control sequence. As used herein, the term “expression control sequence” or “regulatory sequence” means a DNA sequence that regulates the expression of the operably linked nucleic acid sequence in a certain host cell.

(27) Examples of vectors include, but are not limited to, plasmids, cosmids, phages, YACs or PACs. Typically, in vectors, the given nucleotide sequence is operably linked to the regulatory sequence such that when the vectors are introduced into a host cell, the given nucleotide sequence can be expressed in the host cell under the control of the regulatory sequence. The regulatory sequence may comprises, for example and without limitation, a promoter sequence (e.g., the cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, T7 promoter, and alcohol oxidase gene (AOX1) promoter), a start codon, a replication origin, enhancers, an operator sequence, a secretion signal sequence (e.g., alpha-mating factor signal) and other control sequence (e.g., Shine-Dalgano sequences and termination sequences). Preferably, vectors may further contain a marker sequence (e.g., an antibiotic resistant marker sequence) for the subsequent screening/selection procedure.

(28) In some examples, the promoter described herein may be heterologous to the nucleic acid encoding the RePRP in the vector. As used herein, a promoter heterologous to a coding sequence (a gene) refers to a promoter that is not the natural promoter that controls (drives) expression of the gene in native state. For example, the vector of the present disclosure may comprise a promoter derived from a non-RePRP gene.

(29) In some instances, the promoter described herein can be constitutive, which initiates transcription independent of the influence of regulation. Exemplary constitutive promoters include, but are not limited to a maize ubiquitin (Ubi) promoter, a rice actin (Actl) promoter, and a cauliflower mosaic virus 35S (CaMV35S) promoter.

(30) In other instances, the promoter described herein can be inducible, which initiates transcription in a regulated manner, for example, in the presence or absence of a particular factor. Exemplary inducible promoters include an ethanol inducible promoter (e.g., a AlcR/AlcA promoter) or a β-estradiol inducible promoter (e.g., a XVE promoter, see Examples section below). Exemplary promoters inducible by biotic or abiotic stress (e.g., osmotic stress, drought stress, salt stress, high or low temperatures, hypoxia, anoxia, hydration, pH, chemicals, hormones or a combination thereof) include an Arabidopsis rd29A promoter, an Arabidopsis corl SA promoter, an Arabidopsis kinl promoter, an Arabidopsis heat-shock factor (HSF) promoter, an alpha-amylase promoter, and a synthetic ABRC321 promoter.

(31) In certain embodiments, a promoter sequence as used in the invention is a synthetic ABRC321 promoter, having SEQ ID NO: 9 (1xABRC321), SEQ ID NO: 10 (2xABRC321) or SEQ ID NO: 11 (3xABRC321), preferably SEQ ID NO: 11 (3xABRC321)

(32) In some embodiments, a vector comprising a nucleic acid encoding a RePRP protein operably linked to a promoter comprises a fused promoter/coding region fragment of SEQ ID NO: 12 (Ubi:OsRePRP2.1).

(33) In some embodiments, a vector comprising a nucleic acid encoding a RePRP protein operably linked to a promoter comprises a fused promoter/coding region fragment of SEQ ID NO: 13 (3xABRC321i:OsRePRP2.1).

(34) In some embodiments, a vector comprising a nucleic acid encoding a RePRP protein operably linked to a promoter comprises a fused promoter/coding resin fragment of SEQ ID NO: 14 (35S:OsRePRP2.1)

(35) Any of the vectors described herein may be prepared via conventional recombinant technology.

(36) III. Host Cells, Transgenic Plants, and Methods for Making Them

(37) Some aspects of the present invention provide host cells (e.g., an Agrobacterium cell or a plant cell) comprising any of the vectors as described herein. These host cells (or called recombinant cells) carry exogenous/foreign genetic materials (e.g., the vectors described herein), which can be introduced into the host cell via conventional practice. “Exogenous genetic materials” as used herein can mean that the genetic materials are not originally present in the cells and instead artificially introduced into the cells of a parent thereof. In some instances, the exogenous genetic material may be derived from a different species as the host cell. In some other instances, the exogenous genetic material may be derived from the same species as the host cell and introduced into the host cell such that the resultant recombinant cell comprises extra copies of the genetic material as compared with the wild-type counterpart. The term “transformation” or “transform” as used herein refers to the introduction of exogenous genetic materials into a host cell such as a plant cell.

(38) In certain embodiments, the host cell may be an Agrobacterium host cell. In certain embodiments, the host cell may be a plant cell, for example, a cell from a monocotyledonous plant or a dicotyledonous plant.

(39) Suitable conventional methods are available to make the recombinant cells described herein. Examples of such methods include electroporation, PEG operation, particle bombardment, micro injection of plant cell protoplasts or embryogenic callus or other plant tissue, or Agrobacterium-mediated transformation.

(40) RePRP expression (e.g., before and after transformation of a vector presented herein in a host cell) may be detected using methods known in the art. For example, reverse transcriptional polymerase chain reaction (RT-PCR) may be used to determine RePRP mRNA expression. Additional detection methods include western blot analysis and an enzyme-linked immunosorbent assay (ELISA) with an anti-RePRP antibody for protein detection.

(41) The present invention also provides a transgenic plant comprising an exogenous nucleic acid operably linked to a promoter, wherein the exogenous nucleic acid (a transgene) encodes any of the RePRPs as described herein.

(42) As used herein, plants may be a full plant or a part thereof, including a fruit, shoot, stem, root, leaf or seed, or various types of cells in culture (e.g., single cells, protoplasts, embryos, callus, protocorm-like bodies, and other types of cells). As described above, a plant of the present disclosure may be a monocot or a dicot.

(43) In some embodiments, the plants as described herein are monocotyledonous plants. Examples of monocots include, but are not limited to, rice, barley, wheat, rye, oat, corn, bamboo, sugarcane, onion, leek and ginger.

(44) In other embodiments, the plants described herein are dicotyledonous plants. Exemplary dicot plants include Arabidopsis, soybean, peanut, sunflower, safflower, cotton, tobacco, tomato, pea, chickpea, pigeon pea and potato.

(45) A variety of procedures that can be used to engineer a stable transgenic plant are available in this art. In one embodiment of the present invention, the transgenic plant is produced by transforming a tissue of a plant, such as a protoplast or leaf-disc of the plant, with a recombinant Agrobacterium cell comprising a nucleic acid encoding a desired protein (e.g. RePRPs) and generating a whole plant from the transformed plant tissue. In another embodiment, a nucleic acid encoding a desired protein can be introduced into a plant via gene gun technology, particularly if transformation with a recombinant Agrobacterium cell is not efficient in the plant.

(46) Specifically, a “transgenic plant” described herein can refer to a plant that comprises a transgene (such as an exogenous nucleic acid comprising a RePRP gene operably linked to a suitable promoter) allowing for expression of a RePRP gene in the transgenic plant.

(47) In some embodiments, the transgenic plants, described herein, overexpress RePRPs. As used herein, the term “overexpression” can refer to the production of a gene product (e.g. RePRPs) in transgenic plants that exceeds levels of production in non-transgenic (wild type) counterpart plants, including but not limited to constitutive or induced expression. For example, the level of the RePRPs in the transgenic plant may be at least 10% higher (e.g., 20% higher, 30% higher, 50% higher, 1-fold higher, 2-fold higher, 5-folder higher, 10-fold higher, or above) as compared with that in non-transgenic (wild type) counterpart plants. In some instances, the wild-type parent does not express the RePRPs.

(48) According to the present invention, a transgenic plant as disclosed herein may exhibit improved stress tolerance (e.g., biotic stress or abiotic stress). Biotic stress can be stress that occurs as a result of damage done to plants by other living organisms, such as pathogens e.g. bacteria, viruses, fungi, parasites, beneficial and harmful insects. Abiotic stress can be the negative impact of non-living factors on the living organisms in a specific environment. In some embodiments, the abiotic stress is osmotic stress, drought stress, salt stress, or a combination thereof.

(49) In some embodiments, improving the stress tolerance of a plant refers to increasing the ability of a plant to survive under stress. For example, the survival rate of a transgenic plant as disclosed herein may be at least 20% higher (e.g., 30% higher, 50% higher, 1-fold higher, 2-fold higher, 5-folder higher, 10-fold higher, 20-fold higher, 50-fold higher, 100-folder higher, or above) than the survival rate of its wild-type counterpart, under stress and/or during recovery from stress.

(50) In some embodiments, a transgenic plant as disclosed herein exhibits substantially no growth reduction compared to its wild-type counterpart under the same condition. In other words, the presence of the transgene can have no substantial detriment effects in plant growth. For example, plant height, panicle length, panicle numbers or root number/length of a transgenic plant as disclosed herein may be less than 15% (e.g. less than 10%, less than 5% or below) reduction or unaffected (maintained the same) than that of its wild-type counterpart under the same growth condition.

(51) In some embodiments, a transgenic plant as disclosed herein exhibits substantially no yield reduction under stress and/or during recovery from stress, compared to normal, non-stress conditions. For example, grain yield reduction of a transgenic plant as disclosed herein may be less than 15% (e.g. less than 10%, less than 5% or below) under stress and/or during recovery from stress, compared to normal, non-stress conditions. In particular, the yield reduction of the transgenic plant of the present invention, under stress and/or during recovery from stress versus normal, non-stress conditions, is less than that of its wild-type counterpart.

(52) In some embodiments, a transgenic plant as disclosed herein may exhibit improved ability to survive under salt stress. Salt stress may be mimicked by exposure to sodium chloride (NaCl) at 100 mM or higher (e.g. 150 mM or higher, 200 mM or higher, 250 mM or higher). In some embodiments, plants may be allowed to recover from salt stress.

(53) In some embodiments, a transgenic plant as disclosed herein may exhibit is improved ability to survive under osmotic stress. Osmotic stress may be mimicked by exposure to polyethylene glycol (PEG) at 20% or higher (e.g. 30% or higher). In some embodiments, osmotic stress is mimicked under 30% PEG6000. In some embodiments, plants may be allowed to recover from osmotic stress.

(54) In some embodiments, a transgenic plant as disclosed herein may exhibit improved ability to survive under drought stress. Drought stress may be mimicked by dehydration. In some embodiments, recovery from drought stress may be achieved through rehydration.

(55) Accordingly, the present invention also provides methods of producing the transgenic plants described herein. The method may comprise: (a) transforming a plant cell with a nucleic acid operably linked to a promoter to obtain a recombinant plant cell expressing a RePRP protein, wherein the nucleic acid encodes the RePRP protein; and (b) growing the recombinant plant cell obtained in (a) to generate the transgenic plant.

(56) The present invention further provides methods for improving growth (e.g., under stress and/or during recovery from stress) or stress tolerance of a plant. The method may comprise: (a) transforming plant cells with a vector comprising a nucleic acid operably linked to a promoter to obtain recombinant plant cells expressing a RePRP protein, wherein the nucleic acid encodes the RePRP protein; (b) growing the recombinant plant cells obtained in (a) to generate a plurality of transgenic plants; and (c) selecting a transgenic plant from the plurality of transgenic plants generated in (b) that exhibits an improved feature in respect of stress tolerance or growth as described herein, including a higher survival rate to abiotic stress, substantially no growth reduction, or a combination thereof, as compared with a non-transgenic plant counterpart growing under the same condition. In some embodiments, the transgenic plant as selected is that exhibits substantially no growth/yield reduction under stress, compared to normal, non-stress conditions. In some embodiments, the transgenic plant as selected is that exhibits similar growth features and/or less yield reduction under stress compared to its non-transgenic plant counterpart.

(57) Without further elaboration, it is believed that one skilled in the art can, based on the disclosure herein, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely descriptive, and not limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLES

(58) In this study, we generated OsRePRP2.1 over-expressing transgenic plants and found that OsRePRP2.1 over-expression makes plants more tolerant to drought and salinity conditions. In the field test with semi-drought conditions, OsRePRP2.1 over-expression lines also showed no substantial growth retardation and less reduction in grain yield than that in wild type plants. In order to further reduce yield penalty in the OsRePRP2.1 over-expression plants, stress-induced expression of OsRePRP2.1 controlled by the synthetic 3XABRC321 promoter are introduced into transgenic rice. These transgenic plants with ABA/stress induced over-expression of OsRePRP2.1 show significantly higher drought tolerance level and maintain normal plant growth under non-stress conditions.

(59) 1. Materials and Methods

(60) 1.1 Plant Materials and Growth Conditions

(61) Seeds of wild-type rice (Oryza sativa L., cv. Tainung 67; “TNG67”) were sterilized with 2% sodium hypochloride for 30 min. and washed thoroughly with distilled water. To obtain uniform germination, rice seeds were soaked in distilled water at 37° C. for 1 day in darkness and then germinated in 20 cm petri dishes containing distilled water at 37° C.

(62) Transgenic rice seeds were germinated in water containing hygromycin-B (30 μg/ml; Invitrogen) at 28° C. for 3 days to select for transgene containing seedlings. Uniformly germinated seeds were then selected and cultivated in a beaker containing half-strength Kimura B solution (Hsu et al., 2003). The hydroponically cultivated seedlings were grown at 28° C. at 90% relative humidity in 14 h light/10 h dark conditions with a light intensity of 100˜105 μmol photons m.sup.−2 sec.sup.−1).

(63) Arabidopsis seeds were sterilized with 0.6% sodium hydrochloride for 15 mins and washed thoroughly with distilled water. To select transgene containing plants, seeds were sown on half strength of Murashige and Skoog (MS) medium with 1% sucrose and 0.8% agar (pH5.7) with kanamycin (50 μg/ml; Invitrogena) at 4° C. in the darkness for 2 days, and then grew on plates at 22° C. in 16 hr light/8 hr dark cycle for 5 days. Homozygous transgenic plants and wild-type seeds grew on the normal half strength of MS medium with 1% sucrose and 0.8% agar (pH5.7) without antibiotics for 10 days and transferred to soil conditions for the phenotype observation.

(64) 1.2 Generation of Transgenic Plants

(65) OsRePRP2.1 over-expressing transgenic rice were generated as follows. The OsRePRP2.1 coding region amplified by genomic PCR was cloned behind the maize Ubi1 promoter with its first intron in the pPZP binary vector. This vector contains a combined promoter/coding region fragment of SEQ ID NO: 12 (Ubi:OsRePRP2.1). Transgenic rice lines were generated with this construct via Agrobacterium-mediated transformation essentially as described in Hong et al., 2004, in the rice transformation laboratory at the Institute of Molecular Biology, Academia Sinica, Taiwan.

(66) OsRePRP2.1 over-expressing transgenic Arabidopsis plants were generated as follows. The OsRePRP2.1 coding region was clones behind the 35S promoter in the pKGW vector. The vector contains a combined promoter/coding region fragment of SEQ ID: 14 (35S:OsRePRP2.1). Transgenic Arabidopsis plants were generated with this construct via Agrobacterium-mediated vacuum infiltration transformation performed in Transgenic Plant Laboratory, Academia Sinica, Taiwan

(67) OsRePRP-RNAi knockdown lines were generated as follows. The coding regions of the OsRePRP1.1 and 2.1 genes were amplified by genomic PCR and fused together in pCR8/GW/TOPO (Invitrogen). The combined fragment was excised and cloned into the pANDA binary vector described in Miki et al., 2004) via LR recombination (LR Clonase, Invitrogen). Transgenic lines were obtained from this construct as described in the preceding paragraph.

(68) A stress-inducible OsRePRP2.1 construct was obtained by fusing the OsRePRP2.1 coding region to the 3XABRC321 promoter (SEQ ID NO: 7) (Chen et al., 2015) in the pENTR vector (Invitrogen). The combined promoter/coding region fragment was cloned into the pZP200 binary vector by LR recombination. This vector contains a combined promoter/coding region fragment of SEQ ID NO: 13 (3XABRC321:OsRePRP2.1). Again, transgenic plants were generated via Agrobacterium-mediated transformation.

(69) 1.3 RT-PCR Analysis

(70) For the determination of OsRePRP transcript levels, total RNA was isolated from rice tissues using TRIzol reagent (Invitrogen) according to the supplier's recommendations. First-strand cDNA was synthesized using the SuperScript II first-strand synthesis system (Invitrogen). Gene specific primer sets used for RT-PCR to quantify OsRePRP gene expression are provided in Table 1 below. The rice OsActin gene was used as an internal control.

(71) TABLE-US-00001 TABLE 1 RT-PCR Primers RT-PCR analysis Gene Primer Sequence (5′ to 3′) OsRePRP1.1 Forward ACAAGCTCACAGTTCAGTTACGTACAAC (SEQ ID NO: 15) Reverse GCGCTCCTTCCTCGGGT (SEQ ID NO: 16) OsRePRP1.2 Forward GATCACAGAAGCTCACAGTTCAGTT (SEQ ID NO: 17) Reverse TGACTCGCTCGCTCCTCC (SEQ ID NO: 18) OsRePRP2.1 Forward ATGAGGAGATCAATCCTCTCACTG (SEQ ID NO: 19) Reverse TCAGTTCCCGGGCACAATTATAG (SEQ ID NO: 20) OsRePRP2.2 Forward AATGTTCCTGATCACATTGCCAAT (SEQ ID NO: 21) Reverse CATACCAAAACTATGCGGAATCAT (SEQ ID NO: 22) OsActin Forward CTGATGGACAGGTTATCACC (SEQ ID NO: 23) Reverse CAGGTAGCAATAGGTATTACAG (SEQ ID NO: 24)

(72) 1.4 Stress Treatments

(73) For hydroponic system growth, wild type (TNG67) and transgenic plants (10 seedlings for each line) were grown in the same pot for 2 weeks under normal half-strength Kimura B solution. Two-week old seedlings (three-leaf old) were exposed to (i) 30% PEG in water (PEG 6000; Merck) for 18-20 hours, or (ii) 250 mM NaCl in half-strength Kimura B solution for 5 days. Seedlings were recovered in normal half-strength Kimura B solution for 10-12 days, followed by an evaluation of plant survival rate.

(74) For growth in soil, seven 2-day germinated seeds per pot were grown in a soil mixture of 1:1 v/v clay and vermiculite for two weeks. Watering of two-week old seedlings (three-leaf old) was withheld for 12-14 days, and plants were then re-watered for 12 days before pictures were taken and survival rates were measured.

(75) 2. Results

(76) 2.1 OsRePRP2.1 Over-Expression Enhances Tolerance to High Salinity and Dehydration

(77) Multiple stably transformed lines were generated that overexpress the OsRePRP2.1 gene (“OsRePRP2.1OX”). Ectopic expression of this gene was controlled by the maize ubiquitin promoter. These stably transformed lines are already in T4 generation of homozygous plants. The transcript levels of several OsRePRP genes in roots from these lines were quantified by RT-PCR using the primers shown in Table 1 above.

(78) The results are shown in Table 2 below.

(79) TABLE-US-00002 TABLE 2 Relative mRNA expression of OsRePRP transformed rice lines TNG67 OsRePRP2.1OX endogenous normal salt treated #2 #10 #19 OsRePRP2.1 1 1.9 2.0 2.1 1.9 OsRePRP2.2 1 1.5 1.4 1.2 1.3 OsRePRP1.1 1 1.6 1.1 1.2 1.0 OsRePRP1.2 1 1.2 0.9 0.9 0.9

(80) OsRePRP2.1 transcript levels were significantly higher in over-expression lines, as compared to those in wild type TNG67 plants under normal condition. The expression level of OsRePRP2.1 induced by salt treatment in TNG67 plants was similar to that observed in untreated OsRePRP2.1 over-expression lines. The expression level of OsRePRP2.2, OsRePRP 1.1 and OsRePRP 1.2 was not significantly affected by overexpression of OsRePRP21.

(81) 2.2 OsRePRP2.1 Over-Expression Enhances Tolerance to High Salinity

(82) The transgenic rice plants described above were tested for their response to high salinity conditions in order to determine whether OsRePRP-mediated root architecture adjustments were beneficial to rice plants under stress. Ten individual plants of each transgenic line were grown side by side with and TNG67 plants in a hydroponic culture chamber. Three-leaf old plants were treated with 250 mM NaCl for five days, and then moved to normal culture medium for a 12 day recovery period. After the recovery period, the survival rate of each line was compared to that of TNG67. The results indicated that three independent OsRePRP2.1OX transgenic rice plants had a 25˜40% survival rate, as compared to less than 5% for TNG67. See FIG. 1. Clearly, OsRePRP2.1 over-expression enhances rice plant adaptation to salinity conditions.

(83) 2.3 OsRePRP2.1 Over-Expression Enhances Tolerance to Dehydration

(84) In addition to salinity, water deficit, i.e., dehydration, is another important environment stress inhibiting rice plant growth. Dehydration was mimicked by treating rice plants with 30% PEG for 20 h. More specifically, three-leaf old plants were treated with 30% PEG for 20 h and then switched to normal culture medium for recovery. Survival rate was determined after 10 days of recovery. The results showed that three independent OsRePRP2.1OX transgenic rice plants had survival rates as high as 75%, as compared to only 15% in TNG67 wild-type plants. See FIG. 2. Furthermore, when the PEG treatment time was reduced to 16 hours, the shrunken leaves of OsRePRP2.1OX transgenic rice plants were able to fully expand in just one day after recovery and survival rate went up to 95%.

(85) 2.4 OsRePRP2.1 Over-Expression Enhances Tolerance to Drought Conditions

(86) When grown in soil, the OsRePRP2.1OX transgenic rice plants also showed high recovery rates after 13 days of withholding water in compared to no WT plants recovered. See FIG. 3. According to these results of abiotic stress tests, the ABA/stress induced OsRePRP family not only modulated root growth but was also involved in the stress tolerance processes.

(87) 2.5 RNA Interference (“RNAi”)-Mediated Knockdown of OsRePRP Expression Reduces Stress Tolerance in Rice

(88) OsRePRP RNAi transgenic rice lines were generated to reduce the expression of all four OsRePRP genes. Rice OsRePRP family transcripts were determined by RT-PCR in salt treated roots of OsRePRP RNAi lines as described above. The results are shown in Table 3 below.

(89) TABLE-US-00003 TABLE 3 Relative RePRP Gene expression in RNAi knockdown lines TNG67 OsRePRP RNAi, salt treated endogenous normal salt treated #3 #5 #6 #7 OsRePRP1.1 1 1.56 0.69 0.04 0.08 0.18 OsRePRP1.2 1 1.18 0.76 0.21 0.47 0.54 OsRePRP2.1 1 3.26 1.97 0.08 0.14 0.80 OsRePRP2.2 1 1.42 0.71 0.02 0.09 0.33

(90) The four OsRePRP RNAi lines tested each showed significantly lower levels of RNA transcripts of the endogenous OsRePRP1.1, OsRePRP1.2, OsRePRP2.1, and OsRePRP2.2 genes, even after salt treatment.

(91) 2.6 OsRePRP Knock-Down Transgenic Rice Plants are Sensitive to Salinity and Drought Conditions

(92) OsRePRP RNAi transgenic rice plants were tested for their sensitivities to salt and to PEG as described above. In the salinity test (250 mM NaCl), the average survival rate of RNAi lines was 3.2%, less than half of the 9.1% displayed by wild-type TNG67 plants. Under the same conditions, OsRePRP2.1OX transgenic plants had a survival rate of as high as 80%.

(93) Turning to the drought test (30% PEG), the average survival rate of RNAi lines, i.e., 2.7%, was lower than the 4.2% survival rate of TNG67 plants. Under the same conditions, between 45% and 83% of OsRePRP2.1OX transgenic rice plants survived. See FIG. 4. According to these results, knocking down expression of OsRePRP genes in rice led to greater sensitivity to salinity and drought treatments.

(94) 2.7 OsRePRP2.1 Overexpressing Transgenic Rice Plants are Tolerant to Drought Conditions in the Field

(95) Field drought tests were performed to evaluate the tolerance level of TNG67 wild-type and OsRePRP2.1OX transgenic plants. Field tests were performed twice in the first and second growing seasons of 2015. In these two seasons, OsRePRP2.1OX lines were grown side by side with TNG67 plants, with 24 individuals for each line grown with a spacing of 25×25 cm between each plant in an irrigated filed and in a non-irrigation field at the same time. Tests were initiated by transplanting 25-day old transgenic seedlings to the genetically modified organism-certified field at National Chung Hsing University, Taiwan,

(96) Normal irrigated conditions were achieved by flooding the field with 1-5 cm of water until the end of the active tilling stage, i.e., 60-70 days after imbibition, at which time the water was drained. Soil was kept moist until the end of tillering stage. The field was then flooded again with 3-10 cm of water until the milky stage, and then water was again drained.

(97) In the non-irrigated field, soil was kept just moist instead of flooding during the entire planting period. Plant growth and grain yield were observed. The results are summarized in Table 4 below.

(98) TABLE-US-00004 TABLE 4 Results of plant growth and grain yield reduction of OsRePRP overexpressing plants. Plant growth Irrigated Non-irrigated Plant Panicle Panicle Plant Panicle Panicle Grain yield height(cm).sup.1 length(cm).sup.1 number.sup.1 height(cm).sup.1 length(cm).sup.1 number.sup.1 Loss %.sup.2 TNG67 101.6 ± 2.9 19.7 ± 2.1 11.3 ± 2.2 97.8 ± 3.3 19.5 ± 2.2 10.7 ± 2.2 15.0 OX#2 105.2 ± 3.4 19.9 ± 2.4 10.9 ± 1.4 94.9 ± 5.9 19.7 ± 2.3 10.7 ± 2.6 5.3 OX#10 103.4 ± 4.4 18.8 ± 2.3 10.6 ± 1.2 93.6 ± 5.7 17.8 ± 2.0 10.5 ± 1.9 4.2 OX#19 101.2 ± 3.2 19.3 ± 2.4 11.1 ± 1.3 94.0 ± 4.5 19.4 ± 2.3  9.2 ± 1.9 13.6 .sup.1Plant height, panicle length and panicle number were measured for each individual of each line for two seasons in 2015 and shown as means ± SD (n = 24). .sup.2Plant grain yield was measured for each individual of each line for two seasons in 2015. Grain yield loss (%) was shown as an average of two-season loss (%) of each line, wherein each season loss of each line was calculated as follows: (means of grain yield in irrigated condition − means of grain yield in non-irrigated condition)/means of grain yield in irrigated condition × 100 (%).

(99) The results showed that plant height, panicle length, and panicle numbers were similar in OsRePRP2.1OX and TNG67 plants under normal irrigation conditions. In addition, OsRePRP2.1 overexpressing transgenic Arabidopsis plants were produced and the growth was found similar to non-transgenic Arabidopsis plant under normal conditions. See FIGS. 6A-6C. It shows that overexpression of OsRePRP2.1 does not affect the plant growth including plant height, panicle length, panicle number and root length.

(100) On the other hand, TNG67 plants grown in non-irrigated filed caused grain yield loss of about 15% as compared to TNG67 plants grown in irrigated conditions. In contrast, OsRePRP2.1OX transgenic rice plants showed less than 15% reduction in rice yield when grown in non-irrigated filed as compared to OsRePRP2.1OX transgenic rice plants grown in irrigated conditions; among them, surprisingly, some lines (e.g. OX#2 and OX#10) exhibit very low grain loss, only about 5% loss. These observations suggest that over-expression of OsRePRP2.1 in rice helps plants survive and maintain near normal seed production under drought conditions.

(101) 2.8 Transgenic Rice Plants Having Stress-Induced Overexpression of OsRePRP2.1

(102) Although constitutive expression of OsRePRP2.1 in rice enhanced drought tolerance level, over-expression also resulted in a yield penalty under non-stress conditions.

(103) Rice plants were generated that contain a transgene containing OsRePRP2.1 controlled by a synthetic ABA/stress inducible promoter, 3XABRC321, which directs high level gene expression only under stress conditions (see Chen et al., 2015).

(104) T1 and T2 plants were tested for drought tolerance in the PEG stress model mentioned above. After PEG treatment in hydroponic culture systems, 3 independent T2 rice plant lines carrying an inducible ABRC321:OsRePRP2.1 construct showed the significant higher survival rate as compared to wild type plants.

(105) Furthermore, root growth inhibition in over-expression lines was not observed in induced expression lines. See FIGS. 5A-5B. Plant growth of ABRC321:OsRePRP2.1 transgenic plants also were not affected. Similar plant height, panicle length and panicle numbers of ABRC321:OsRePRP2.1 transgenic rice as those in wild type plants were observed under the normal condition. See Table 5.

(106) TABLE-US-00005 TABLE 5 Results of plant growth of ABRC321:OsRePRP2.1 transgenic plants. Plant height Panicle Panicle (cm) length (cm) numbers TNG67 95.9 ± 3.3 18.5 ± 2.7  8.3 ± 1.4 ABRC:P2.1#2 86.3 ± 3.3 16.6 ± 2.5 10.5 ± 2.0 ABRC:P2.1#10 90.7 ± 3.8 17.8 ± 2.5  7.5 ± 1.9 ABRC:P2.1#12 91.6 ± 4.8 17.5 ± 2.7 10.9 ± 1.6 1. Plant height, panicle length and panicle number were measured for each individual of each line in the first season of 2017 and shown as means ± SD (n = 15).

(107) These results indicated that ABRC321:OsRePRP2.1 inducible transgenic rice plants were drought tolerant by virtue of OsRePRP2.1 expression. Yet, these plants did not suffer any growth retardation effects attributed to overexpression of OsRePRP2.1.

(108) Given the above, the present invention provides technologies to improve stress tolerance and/or preventing growth reduction of a plant by introducing a polynucleotide encoding a Repetitive Proline-rich Protein (RePRP) into the plant. The invention helps plants not only to survive under stress but also maintain growth and productivity, which is beneficial to agricultural development.

(109) The following references can be used to better understand the background of the application: Boyer, J. S. (1982), Science 218, 443-448. Chen et al (2015), Plant Biotechnology J. 13, 105-116. Hong et al. (2004), Transgenic Research 13, 29-39. Hsu et al. (2003), Plant, Cell Environment 26, 867-874. Kar (2011), Plant Signaling Behavior 6, 1741-1745. Miki et al. (2004), Plant Cell Physiology 45, 490-495. Saab et al. (1990), Plant Physiology 93, 1329-1336. Sharp et al. (2004), J. Experimental Botany 55, 2343-2351. Tilman et al. (2002), Nature 418, 671-677. Tseng et al. (2013), Plant Physiology 163, 118-134. Xu et al. (2013), The New Phytologist 197, 139-150.

(110) The contents of the above references are hereby incorporated by reference in their entirety.

OTHER EMBODIMENTS

(111) All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

(112) From the above description, a person skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the present invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

(113) TABLE-US-00006 >OsRePRP1.1 (SEQ ID NO: 1) MARRSPCLTAAVLLLGALAVASALVDEAAAAGQGLGHGARFMSKQGRAMYEKPPELEPKPKPKPHPKHESK PEPKPEPKPEPKPYPEPKPETKPELKPEPKPNPEPKPEPKPEPKPEPKPYPEPKPKPKPEPKPEPKPEHKP EPKPEPEPKPYPKPKPEPKPGPKPEPKPEPKPHPEPKPEPKPKPVPHPEPKPEPKPEPKPHPEPKPEPKPE PKLHPKPEPKPHPEPEPKLKPEPKPEPKPEPEPKPEPKPEPKPEPKPYPKPKPEPKPVPKPKPIPHPGPKP KPKPDPKLEPKPHPEPKPHPMPEPEPKPKPEPKPEPKPYPEPKPKLKPEPKPGPKPIAPPNKHKPPHMPPA TNQ >OsRePRP1.2 (SEQ ID NO: 2) MARRSPCLAVAMLLLGALAVASAFIDEAAAAGRGLGHGARFMSKQGRVTYEKLPEPEPKPKPKPHPKPTPK PEPKPEPEPKPVPEPEPKPEPKPEPKPEPKPEPKPYPEPKPEPKPEPKPEPEPKPEPKPEPKPEPKPYPEP KPEPKPEPKPEPKPEPKPKPEPKPHPEPKPDPKPEPKPHPEPEPKPEPKPEPKPHPEPEPKPEPKPEPKPE PKPEPKPEPKPKPKPEPKPKPEPKPYPEPKPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPEPKPKPEPKP HPKPEPKPEPKPEPKPEPKPEPKPEPKPEPEPKPEPKPEPKPEPKPYPEPKPDPKPEPKPHPEPKPEPKPQ PEPKPEPKPEPKPEPKPEPKPEPKPYPEPKPEPKPKPKPEPKPEAPPKKHKPPHIPPATDQ >OsRePRP2.1 (SEQ ID NO: 3) MRSILSLCFHLALAIALAANVPDHIANGRVIEAKSDPKPADPNPKPDPIPKPQPETKPSPQPNPQPNPQPD PKPSPQPDPKPTPQPEPKQDPKPNPQPDPKPSPQPDPKPTPQPDPKQDPQPNPQPDPKPTPQPNPKQDPQP NPQPDPKPTPQPDPKQDPQPNPQPSPKADPKPNPKPKPQPEPSPNPKPEPKPEPKPEPSPNPKPNPNPKPE PQPDPKPEPKPQPEPSLPKPPPLSPAIAIIVPGN >OsRePRP2.2 (SEQ ID NO: 4) MRRSILSLCFHLALVIALAANVPDIANGRVIEAKSDPKPADPKPKPDPTPKPQPETKPSPQPNPQPNPQPD PKPSPQPDPKPTPQPEPKQDPQPNPQPDPKQSPQPDPKPTPQPNPKQDPQPNPQPDPKPTLQPNPKQDPQP NPQPNPKPTPQLDPKQDPQPNPQPSPKADPKPNPKPKPQPEPSPNPKPEPKPEPKPEPSPNPKPNPNPKPE PQPDPKPEPKPQPEPSQPKLPPLSPAIAIIVPGN >OsRePRP1.1 (SEQ ID NO: 5)    1 caacagcaga agtgagagag ggagaagaag ataagcgaag aggaggagct tagcttgcca   61 gccatggcta ggcgctctcc ttgcctcact gccgccgtgc tcctgcttgg ggcattggcg  121 gtggcgagcg ctttagttga tgaagcggcg gcagctggcc agggactcgg ccatggcgcc  181 cgcttcatga gcaagcaggg ccgtgcgatg tacgagaagc cgccagagct ggagccgaag  241 ccaaagccaa agcctcatcc taagcatgaa tcaaaaccgg agccaaagcc agaacctaag  301 ccggagccaa agccataccc agagccgaag ccagagacga aaccggagct aaagccagaa  361 ccaaaaccta atccagaacc taaacctgag cctaagcctg aaccaaaacc agaaccaaag  421 ccatacccag agccgaagcc aaagcccaaa ccggagccaa agccagaacc aaaacctgag  481 cataaacctg aaccaaaacc agaaccagaa ccaaagccat acccaaagcc aaagccagag  541 ccaaaaccgg ggcccaaacc cgagccgaag ccagagccta agccacaccc agaaccgaaa  601 ccggagccca aaccaaagcc agtgccacac cctgaaccaa aaccggaacc aaagccggag  661 cccaaaccac acccagaacc aaagcctgag ccgaaacccg agcctaagct acacccgaag  721 cctgagccaa agccacaccc agagcctgag cctaagctta aacctgaacc aaaaccagag  781 ccaaagccag agcctgaacc gaagcccgag ccaaagcctg aaccaaaacc agagcctaaa  841 ccatatccaa agccaaaacc ggaacctaaa ccggtgccga agccgaagcc cattccacac  901 ccaggaccaa aaccaaagcc taaacctgac ccaaagctag agcccaagcc acacccggag  961 ccaaaaccac atccgatgcc tgaacctgaa ccaaagccta agcccgaacc aaagccagag 1021 cctaaaccat acccagaacc aaagcctaaa ctgaaacctg aacctaagcc tggaccgaaa 1081 cctatagcac cgccgaacaa gcacaagccg ccgcacatgc caccagcgac aaaccagtga 1141 cggcgatcgc tggagaccga gcatttgctg gctgcacggt tgaggcaccg acgacattat 1201 ttcacccgag gaaggagcgc tagcgagtca ctacactgta ccgtttctgg aataaagtga 1261 tgagctagct ttctgcttgc cttttctttt cctctcttat tttcctttta tttcatgttg 1321 gtttttcgga tgtgccactg ctagctagtg taattaaatt atttattatg tgcctaccgt 1381 catttttatt accgtgtctg tgacattcta ttgtctattg gcattattct cattgtaaaa 1441 tcttttggta atattatttg tcatcatttt tacccagctt ctaaaaaaaa aaa >OsRePRP1.2 (SEQ ID NO: 6)    1 atggcgaggc gctctccttg cctcgccgtc gccatgctcc tgcttggggc gttggcggtg   61 gcgagcgcct tcattgatga agcggcggct gctggccggg ggctcggcca tggcgcccgc  121 ttcatgagca agcagggtcg tgtgacatac gagaagctgc cggagccgga gccgaagcca  181 aagccaaagc ctcatcctaa acccacgcca aaacctgagc ccaagccaga gccggagcca  241 aaaccagtac ctgagcctga gcctaaaccg gaaccaaagc cagaaccaaa acctgagcct  301 aagcctgaac ctaaaccata cccagagcca aaaccggagc cgaagccaga gccaaaacct  361 gagccggagc ctaaacctga gcctaagcca gaaccaaaac cagaaccaaa gccgtaccca  421 gagccgaagc cagagccaaa accggaaccg aagccggaac caaaaccgga gcccaaacca  481 aagccagagc ccaaaccaca cccagaacca aagcctgatc cgaaacctga gcctaagcca  541 cacccagagc ctgagcctaa gcctgaacct aagcctgagc ccaagccaca ccctgagcct  601 gaaccaaagc ctgagcctaa gcctgagcca aagccagaac caaagccgga gccaaaacct  661 gaaccaaaac caaagccaaa gccagagcca aagccaaagc ctgagcccaa gccataccct  721 gagcctaagc ctaagcctga accaaagcct gagcctaagc ctgagccaaa gccagaacca  781 aagccggagc caaaacctga accaaaacca gagccaaagc cagagccaaa gccaaagcct  841 gagcccaagc cacaccctaa gcctgagcct aagcctgagc ccaagccaga accaaagcca  901 gagccaaaac ctgaaccaaa accagagcca aaaccagagc ctgaaccgaa gcctgagcca  961 aagcctgaac caaaaccaga gcccaaacca tatccagagc ctaaaccgga tcccaaacca 1021 gaacccaaac cacacccaga accaaagcca gagcccaagc cacagccgga gccaaaacca 1081 gagccgaagc ctgaacctaa accagagcct aagcccgaac caaaaccgga gcctaaacca 1141 tacccagagc caaagcctga accgaaacct aagcctaagc ctgagccaaa acctgaagca 1201 cctccgaaga agcacaagcc gccgcacata ccgccagcga ccgaccagtg a OsRePRP2.1 (SEQ ID NO: 7)    1 aacacaccta actaccacag cttgtgaact atcaagagtg agtagtagag tttgcagtga   61 caacgagatg aggagatcaa tcctctcact gtgcttccat ttggcgcttg tcattgcatt  121 ggcagcaaat gttcctgaca ttgccaatgg acgcgtgatt gaagctaaat ctgatccaaa  181 gccagcagat cccaagccta aacctgaccc aacaccaaaa ccacaaccag agacaaagcc  241 cagtccacag cctaaccctc aacctaaccc acagccagat ccaaaaccat caccgcagcc  301 tgatccaaaa cctacaccac agcctgaacc aaaacaagat cctcaaccaa acccacagcc  361 ggatccaaaa caatcgccgc agcctgaccc aaaacctaca ccacagccta acccaaaaca  421 agatcctcaa ccgaacccac aacctgaccc aaaaccaacg ctgcaaccta acccaaaaca  481 agatcctcag ccgaacccac agcctaaccc gaaaccaacg ccacagcttg acccgaaaca  541 agatcctcaa ccgaacccac aacctagccc caaagctgac ccaaaaccaa atccaaagcc  601 taagccacaa ccggagccga gcccaaatcc taagccggag ccaaaacctg aacccaaacc  661 tgagccgagt cctaacccca agccaaatcc taatcccaag ccggagccac agcctgatcc  721 taagccagaa cccaagcctc agccagagcc gtctcaacca aagctgccac cactttcacc  781 agcaatagct ataattgtgc ccgggaactg agtagacttg gttgtttgct acgtatgatc  841 ccgcatactt ttggtatgta ctattgctct agtgactatt tgtgtgtttt tcgtgtgttg  901 ttcactagtg tgtccatgtg gctatctatg tgttttctta atgccgttgc atatgagcag  961 gcgtgcttct tataataaag catacataca tacatacata catacataca tacatatata 1021 tatacacgtg tgttatgtat gtgcgtacat accatcaata aaaagagcat gtatccctgt 1081 gtgtcaat >OsRePRP2.2 (SEQ ID NO: 8)    1 aacacaccta gctaccacag cttgtgtact gtcaagagtg agtagtagag tttgtagtga   61 caacgagatg agatcaatcc tctcactgtg cttccatttg gcgcttgcca ttgcattggc  121 ggcaaatgtt cctgatcaca ttgccaatgg acgcgtgatt gaagctaaat ctgatccaaa  181 gccagcagat cccaatccta aacctgaccc aacaccaaaa ccacaaccag agacaaagcc  241 cagtccacag cctaaccctc aacctaaccc acagccagat ccaaaaccat caccgcagcc  301 tgacccaaaa cctacaccac agcctgaacc aaaacaagat cctaaaccaa acccacaacc  361 ggatccaaaa ccatctccgc agcctgaccc gaaacctaca ccacagcctg acccaaaaca  421 agatcctcaa ccgaacccac aacctgaccc aaaaccaacg ccgcaaccta acccaaaaca  481 agatcctcag ccgaacccac agcctgaccc aaaaccaacg ccacagcctg acccgaaaca  541 agatcctcaa ccgaacccgc aacctagccc caaagctgac ccaaaaccaa atccaaagcc  601 taagccacaa ccggagccga gcccaaatcc taagccggag ccaaagcctg aacccaaacc  661 tgagccaagt cctaacccca agccaaatcc taatcctaag ccggagccac agcctgatcc  721 taagccagaa cccaagcctc agccagagcc atctctgcca aagccaccac ctctttcacc  781 agcaatagct ataattgtgc ccgggaactg agtagacttt ttgctacgta tgattccgca  841 tagttttggt atgtactatt gctctagtga ctatctatgt gtttgtcgtg tgttgttcac  901 tggtgtatgt gtccatgtgg ctatctatgt gttttcttaa tgctgttgca tctgagcagg  961 cgtgcttctt ataataaagc atatatatgc acgtgtgtta tgtatgtgcg tacatatata 1021 ccatgaataa aaagagcatg tatccctgtg tgtcact >Synthetic promoter 1XABRC321 (SEQ ID NO: 9)   1 ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg tctagagtcg  61 actgcagcaa ttccggcatg ccgcagcaca ctataaatac ctggccagac acacaagctg 121 aatgcatcag ttctccatcg tactcttcga gagcacagca agagag >Synthetic promoter 2XABRC321 (SEQ ID NO: 10)   1 ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa  61 cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg tctagagtcg actgcagcaa 121 ttccggcatg ccgcagcaca ctataaatac ctggccagac acacaagctg aatgcatcag 181 ttctccatcg tactcttcga gagcacagca agagag >Synthetic promoter 3XABRC321 (SEQ ID NO: 11)   1 ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa  61 cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa cgcgtgtcct 121 ccctacgtgg cggctcgaga ttgccaccgg tctagagtcg actgcagcaa ttccggcatg 181 ccgcagcaca ctataaatac ctggccagac acacaagctg aatgcatcag ttctccatcg 241 tactcttcga gagcacagca agagag >Ubi:OsRePRP2.1 (SEQ ID NO: 12)    1 ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta   61 agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta  121 tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa  181 tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga  241 gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt  301 ttttttgcaa atagcttcac ctatataata cttcatccat tttattagta catccattta  361 gggtttaggg ttaatggttt ttatagacta atttttttag tacatctatt ttattctatt  421 ttagcctcta aattaagaaa actaaaactc tattttagtt tttttattta ataatttaga  481 tataaaatag aataaaataa agtgactaaa aattaaacaa atacccttta agaaattaaa  541 aaaactaagg aaacattttt cttgtttcga gtagataatg ccagcctgtt aaacgccgtc  601 gacgcagtct aacggacacc aaccagcgaa ccagcagcgt cgcgtcgggc caagcgaagc  661 agacggcacg gcatctctgt cgctgcctct ggacccctct cgagagttcc gctccaccgt  721 tggacttcgt ccgctgtcgg catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg  781 cacggcaggc ggcctcctcc tcctctcacg gcaccggcag ctacggggga ttcctttccc  841 accgctcctt cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc  901 tctttcccca acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat  961 ccacccgtcg gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ctctctacct 1021 tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1081 tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1141 tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1201 atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1261 gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1321 tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1381 agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1441 gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1501 ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg cttttgttcg 1561 cttggttgtg atgatgtggt gtggttgggc ggtcgttcat tcgttctaga tcggagtaga 1621 atactgtttc aaactacctg gtgtatttat taattttgga actgtatgtg tgtgtcatac 1681 atcttcatag ttacgagttt aagatggatg gaaatatcga tctaggatag gtatacatgt 1741 tgatgtgggt tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc 1801 taaccttgag tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg 1861 atatacttgg atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca 1921 tacgctattt atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt 1981 tacttctgca gatgaggaga tcaatcctct cactgtgctt ccatttggcg cttgtcattg 2041 cattggcagc aaatgttcct gacattgcca atggacgcgt gattgaagct aaatctgatc 2101 caaagccagc agatcccaag cctaaacctg acccaacacc aaaaccacaa ccagagacaa 2161 agcccagtcc acagcctaac cctcaaccta acccacagcc agatccaaaa ccatcaccgc 2221 agcctgatcc aaaacctaca ccacagcctg aaccaaaaca agatcctcaa ccaaacccac 2281 agccggatcc aaaacaatcg ccgcagcctg acccaaaacc tacaccacag cctaacccaa 2341 aacaagatcc tcaaccgaac ccacaacctg acccaaaacc aacgctgcaa cctaacccaa 2401 aacaagatcc tcagccgaac ccacagccta acccgaaacc aacgccacag cttgacccga 2461 aacaagatcc tcaaccgaac ccacaaccta gccccaaagc tgacccaaaa ccaaatccaa 2521 agcctaagcc acaaccggag ccgagcccaa atcctaagcc ggagccaaaa cctgaaccca 2581 aacctgagcc gagtcctaac cccaagccaa atcctaatcc caagccggag ccacagcctg 2641 atcctaagcc agaacccaag cctcagccag agccgtctca accaaagctg ccaccacttt 2701 caccagcaat agctataatt gtgcccggga actga >3xABRC321i:OsRePRP2.1 (SEQ ID NO: 13)    1 ggtaccgcaa cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa   61 cgcgtgtcct ccctacgtgg cggctcgaga ttgccaccgg ggtaccgcaa cgcgtgtcct  121 ccctacgtgg cggctcgaga ttgccaccgg tctagagtcg acctgcagca attccggcat  181 gccgcagcac actataaata cctggccaga cacacaagct gaatgcatca gttctccatc  241 gtactcttcg agagcacagc aagagagtga tcatttcagg taagatctag agtcgacctg  301 caggcgaccg tatgtatatt accctatctc taccttgcaa atcgcgtgtg tacggatctt  361 ctccgtggtc gagccgagtg attgctgatc tgatatccta tctgctgctt cgtttccttg  421 cgcaggccaa gcatcacgct gctgtaccct ctgtaagttg atcagtcgct tgtggtactt  481 tttagtacgt ggggaagtaa tccttgtgct ggatgtgacc ctggcggatc tgtataatac  541 aggtatgcgg atcccccggg ctgcaggaat tcgatatcaa gctcaccatg aggagatcaa  601 tcctctcact gtgcttccat ttggcgcttg tcattgcatt ggcagcaaat gttcctgaca  661 ttgccaatgg acgcgtgatt gaagctaaat ctgatccaaa gccagcagat cccaagccta  721 aacctgaccc aacaccaaaa ccacaaccag agacaaagcc cagtccacag cctaaccctc  781 aacctaaccc acagccagat ccaaaaccat caccgcagcc tgatccaaaa cctacaccac  841 agcctgaacc aaaacaagat cctcaaccaa acccacagcc ggatccaaaa caatcgccgc  901 agcctgaccc aaaacctaca ccacagccta acccaaaaca agatcctcaa ccgaacccac  961 aacctgaccc aaaaccaacg ctgcaaccta acccaaaaca agatcctcag ccgaacccac 1021 agcctaaccc gaaaccaacg ccacagcttg acccgaaaca agatcctcaa ccgaacccac 1081 aacctagccc caaagctgac ccaaaaccaa atccaaagcc taagccacaa ccggagccga 1141 gcccaaatcc taagccggag ccaaaacctg aacccaaacc tgagccgagt cctaacccca 1201 agccaaatcc taatcccaag ccggagccac agcctgatcc taagccagaa cccaagcctc 1261 agccagagcc gtctcaacca aagctgccac cactttcacc agcaatagct ataattgtgc 1321 ccgggaactg a >35S:OsRePRP2.1 (SEQ ID NO: 14)    1 tcgagggatc cgtcccccgt gttctctcca aatgaaatga acttccttat atagaggaag   61 ggtcttgcga aggatagtgg gattgtgcgt catcccttac gtcagtggag attccagata  121 ggcctaacgc ttgtccaaga tctattcagg attccagata ggcctaacgc ttgtccaaga  181 tctattcagg atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt  241 ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc  301 ttcaacgatg gcctttcctt tatcgcaatg atggcatttg taggagccac cttccttttc  361 cactatcttc acaataaagt gacagatagc tgggcaatgg aatccgagga ggtttccgga  421 taatgaggag atcaatcctc tcactgtgct tccatttggc gcttgtcatt gcattggcag  481 caaatgttcc tgacattgcc aatggacgcg tgattgaagc taaatctgat ccaaagccag  541 cagatcccaa gcctaaacct gacccaacac caaaaccaca accagagaca aagcccagtc  601 cacagcctaa ccctcaacct aacccacagc cagatccaaa accatcaccg cagcctgatc  661 caaaacctac accacagcct gaaccaaaac aagatcctca accaaaccca cagccggatc  721 caaaacaatc gccgcagcct gacccaaaac ctacaccaca gcctaaccca aaacaagatc  781 ctcaaccgaa cccacaacct gacccaaaac caacgctgca acctaaccca aaacaagatc  841 ctcagccgaa cccacagcct aacccgaaac caacgccaca gcttgacccg aaacaagatc  901 ctcaaccgaa cccacaacct agccccaaag ctgacccaaa accaaatcca aagcctaagc  961 cacaaccgga gccgagccca aatcctaagc cggagccaaa acctgaaccc aaacctgagc 1021 cgagtcctaa ccccaagcca aatcctaatc ccaagccgga gccacagcct gatcctaagc 1081 cagaacccaa gcctcagcca gagccgtctc aaccaaagct gccaccactt tcaccagcaa 1141 tagctataat tgtgcccggg aactacccat acgatgttcc agattacgct tga