# Patent

## Title
Compositions and methods for the improvement of plants

## Abstract

The invention provides compositions and methods useful for producing plant cells or plants with altered tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 60/853,194, filed Oct. 20, 2006. The priority application is incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled Sequence-Listing-DAIRY94005AUS.txt, created Oct. 20, 2007, which is 443 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to compositions and methods for producing plants with improved stress tolerance.
2. Description of the Related Art
Environmental abiotic stresses, including drought stress, cold stress, freezing stress, heat stress and salinity stress are major factors limiting plant growth and productivity. Crop losses and reduction in yield of major crops including maize, wheat and rice caused by such stresses represent significant economic issues and also lead to food shortages in several underdeveloped countries.
The development of stress tolerant plants has the potential to reduce or solve at least some of these problems. The use of traditional plant breeding strategies to produce new lines of plants that exhibit tolerance to these types of stresses has been slow. Lack of sufficient germplasm resources and incompatibility between distantly related plant species, present significant problems in conventional breeding. Further, the cellular processes leading to tolerance to such stresses are complex and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways. This limits the success of both traditional breeding and that of genetic engineering approaches to development of stress tolerant plants. It would be beneficial to identify genes and proteins involved in controlling the complex processes leading to stress tolerance.
Regulators of gene expression, such as transcription factors, involved in controlling stress tolerance may be particularly useful in genetic engineering of plants, as a single gene may control a whole cascade of genes leading to the tolerance phenotype. Furthermore, there is sometimes commonality in many aspects of the different types of stress tolerant responses referred above. For example, genes that increase tolerance to cold or salt may also improve drought stress tolerance. This has been demonstrated in the case of the transcription factor AtCBF/DREB 1 (Kasuga et al., 1999 Nature Biotech 17: 287-91) and the vacuolar pyrophosphatase, AVP1 (Gaxiola et al., 2001 PNAS 98:11444-19).
Whilst some potentially useful genes have been identified, the identification and cloning of plant genes that confer tolerance to stress remains fragmented and incomplete. Although it is assumed that stress induced proteins may have a role in stress tolerance, evidence is still lacking and the function of many such stress responsive genes is unknown.
The hot and dry weather conditions in New Zealand and other countries in the summer period can have significant effect upon the yield of ryegrass. This is invariably during the dairy milking season and therefore has real effects on cost of dairy production through either reduced milk yield or the use of supplementary feeds and/or irrigation systems.
It would be beneficial to identify genes, which have the capacity to confer stress tolerance in stress susceptible plant species. The development of stress tolerant crops will provide many advantages such as increasing yield and producing plants that may be cultivated in previously unsuitable environmental conditions. Thus, there exists a need for compositions and methods for producing plants with improved stress tolerance relative to their wild-type counterparts.
It is an object of the invention to provide improved compositions and methods for developing plant varieties with improved tolerance of at least one of the following stresses; drought, cold, freezing, heat and salinity, or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION
In a first aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:1 or a variant thereof, wherein the variant encodes a polypeptide which modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:2 or a variant thereof, wherein the variant encodes a polypeptide which modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the environmental stress is drought stress.
Preferably the variants of SEQ ID NO:1 and 2 encode polypeptides comprising the amino acid sequence:







(SEQ ID NO: 248)


RKRKX1X2X3RGX4RX5RPWGKWAAEIRDPRX6GX7RX8WLGTX9X10


 


X11X12EX13AAX14AYX15X16X17X18RRIRX19X20KAX21VNFP






wherein X1=N, S or T, X2=Q or R, X3=Y or F, X4=I or V, X5=Q or R, X6=K, E or Q, X7=V, S, I or A, X8=V, E or I, X9=F or Y, X10=N, S, D, K or G, X11=T or S, X12=A or P, X13=E or K, X14=R, M or K, X15=D, X or G, X16=A, V, S, I or T, X17=E or A, X18=A or T, X19=G or X, X20=K, S, T or N and X21=K or E.

Exemplary polynucleotide variants of SEQ ID NO:1 and SEQ ID NO:2 are disclosed herein and identified as SEQ ID NOs:13-25 of the sequence listing.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:1.
In a further aspect the invention provides an isolated polynucleotide consisting of the sequence of SEQ ID NO:1.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:2.
In a further aspect the invention provides an isolated polynucleotide consisting of the sequence of SEQ ID NO:2.
In a further aspect the invention provides polynucleotides comprising fragments of SEQ ID NO:1. Polynucleotides comprising fragments of the polynucleotide variants also form part of the invention.
In a further aspect the invention provides polynucleotides comprising fragments of SEQ ID NO:2. Polynucleotides comprising fragments of the polynucleotide variants also form part of the invention.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:4 or a variant thereof, wherein the variant encodes a polypeptide which modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:5 or a variant thereof, wherein the variant encodes a polypeptide which modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the environmental stress is drought stress.
Preferably the variants of SEQ ID NO:4 and SEQ ID NO:5 encode polypeptides comprising the amino acid sequence:







(SEQ ID NO: 249)









WRX1IX2RX3X4VX5X6X7TPTQVASHIAQKX8X9X10R






wherein X1=G or N, X2=A or S, X3=H, K, N, S or T, X4=F or Y, X5=A, F, I, K, M, N, P, T or V, X6=S or T, X7=K or R, X8=F or Y, X9=F or Y and X10=I, L, M or S.

Exemplary polynucleotide variants of SEQ ID NO:4 and SEQ ID NO:5 are disclosed herein and identified as SEQ ID NOs:39-66 of the sequence listing.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:4.
In a further aspect the invention provides an isolated polynucleotide consisting of the sequence of SEQ ID NO:4.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:5.
In a further aspect the invention provides an isolated polynucleotide consisting of the sequence of SEQ ID NO:5.
In a further aspect the invention provides polynucleotides comprising fragments of SEQ ID NO:4. Polynucleotides comprising fragments of the polynucleotide variants also form part of the invention.
In a further aspect the invention provides polynucleotides comprising fragments of SEQ ID NO:5. Polynucleotides comprising fragments of the polynucleotide variants also form part of the invention.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:7 or a variant thereof, wherein the variant encodes a polypeptide which modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:8 or a variant thereof, wherein the variant encodes a polypeptide which modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the environmental stress is drought stress.
Preferably the variants of SEQ ID NO:7 and SEQ ID NO:8 encode polypeptides comprising the amino acid sequence:







(SEQ ID NO: 250)


IPX1X2X3X4SWRKYGQKPIKGSX5X6PRGYYKCSX7X8X9X10X11X12


 


X13X14X15HVERX16X17X18X19X20X21MLX22VTYEX23X24HX25


 


H






wherein X1=A, G, P, Q or S, X2=D or G, X3=D or E, X4=F or Y, X5=K or P, X6=F, H or Y, X7=S or T, X8=I, L, M or V, X9=R or T, X10=G or S, X11=C or Y, X12=L or P, X13=A or L, X14=F, K or R, X15=K or N, X16=A, C, D or S, X17=I, L, M, P or V, X18=A, D, E or S, X19=D or E, X20=A, P, S or T, X21=A, S or T, X22=I, M or V, X23=D or G, X24=D or E, and X25=C, H, N or R.

Exemplary polynucleotide variants of SEQ ID NO:7 and SEQ ID NO:8 are disclosed herein and identified as SEQ ID NOs: 95-126 of the sequence listing.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:7.
In a further aspect the invention provides an isolated polynucleotide consisting of the sequence of SEQ ID NO:7.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:8.
In a further aspect the invention provides an isolated polynucleotide consisting of the sequence of SEQ ID NO:8.
In a further aspect the invention provides polynucleotides comprising fragments of SEQ ID NO:7. Polynucleotides comprising fragments of the polynucleotide variants also form part of the invention.
In a further aspect the invention provides polynucleotides comprising fragments of SEQ ID NO:8. Polynucleotides comprising fragments of the polynucleotide variants also form part of the invention.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:10 or a variant thereof, wherein the variant encodes a polypeptide which modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:11 or a variant thereof, wherein the variant encodes a polypeptide which modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the environmental stress is drought stress.
Preferably the variants of SEQ ID NO:10 and SEQ ID NO:11 encode polypeptides comprising the amino acid sequence:







(SEQ ID NO: 251)


X1X2X3X4AIX5X6X7X8X9GVX10X11X12X13X14X15X16ZEFX17


 


IX18X19X20LTX21X22X23X24X25PGX26X27YA






wherein X1=D or E, X2=I, L or V, X3=F, I, L, M or V, X4=C, F, I or L, X5=F, I or L, X6=I or L, X7=H or P, X8=P or Q, X9=L or V, X10=F or C, X11=F or L, X12=K or R, X13=F, K, R, V or Y, X14=A, G or K, X15=C, I or L, X16=A, C, G, K or Q, X17=C, F, L, M or W, X18=A, C, D or S, X19=L or V, X20=I, L, P or V, X21=C, F, I, L or V, X22=F or L, X23=A or G, X24=F, W or Y, X25=F, I, L or V, X26=I, L or V, X27=I, L or V and wherein Z is a sequence of 1-27 amino acids.

Exemplary polynucleotide variants of SEQ ID NO:10 and SEQ ID NO:11 are disclosed herein and identified as SEQ ID NOs:159-200 of the sequence listing.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:10.
In a further aspect the invention provides an isolated polynucleotide consisting of the sequence of SEQ ID NO:10.
In a further aspect the invention provides an isolated polynucleotide comprising the sequence of SEQ ID NO:11.
In a further aspect the invention provides an isolated polynucleotide consisting of the sequence of SEQ ID NO:11.
In a further aspect the invention provides polynucleotides comprising fragments of SEQ ID NO:10. Polynucleotides comprising fragments of the polynucleotide variants also form part of the invention.
In a further aspect the invention provides polynucleotides comprising fragments of SEQ ID NO:11. Polynucleotides comprising fragments of the polynucleotide variants also form part of the invention.
The polynucleotides of the invention may be derived from any plant species.
In one embodiment the polynucleotide is derived from a gymnosperm species.
In a further embodiment the polynucleotide is derived from an angiosperm species.
In a preferred embodiment the polynucleotide of the invention is derived from a forage plant species. Preferably the species is selected from those of the following genera: Lolium, Festuca, Dactylis, Bromus, Trifolium, Medicago, Phleum, Phalaris, Holcus, Lotus, Plantago and Cichorium. 
In a more preferred embodiment the polynucleotide of the invention is derived from the genera Lolium or Trifolium. Particularly preferred are the species Lolium perenne and Trifolium repens. Most preferably the polynucleotide is derived from Lolium perenne. 
The isolated polynucleotides of the invention are useful in methods for modulating in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
The isolated polynucleotides of the invention are also useful in methods for selecting plants tolerant to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
In a further aspect the invention provides an isolated polypeptide comprising the sequence of SEQ ID NO:3 or a variant thereof, wherein the variant modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the environmental stress is drought stress.
Preferably the variants of SEQ ID NO:3 comprise the amino acid sequence:







(SEQ ID NO: 248)


RKRKX1X2X3RGX4RX5RPWGKWAAEIRDPRX6GX7RX8WLGTX9X10


 


X11X12EX13AAX14AYX15X16X17X18RRIRX19X20KAX21VNFP






wherein X1=N, S or T, X2=Q or R, X3=Y or F, X4=I or V, X5=Q or R, X6=K, E or Q, X7=V, S, I or A, X8=V, E or I, X9=F or Y, X10=N, S, D, K or G, X11=T or S, X12=A or P, X13=E or K, X14=R, M or K, X15=D, X or G, X16=A, V, S, I or T, X17=E or A, X18=A or T, X19=G or X, X20=K, S, T or N and X21=K or E.

Exemplary polypeptide variants of SEQ ID NO:3 are disclosed herein and identified as SEQ ID NOs:26-38 of the sequence listing.
In a further aspect the invention provides an isolated polypeptide comprising the sequence of SEQ ID NO:3.
In a further aspect the invention provides an isolated polypeptide consisting of the sequence of SEQ ID NO:3.
In a further aspect the invention provides polypeptides comprising fragments of SEQ ID NO:3. Polypeptides comprising fragments of variants, also form part of the invention.
In a further aspect the invention provides an isolated polypeptide comprising the sequence of SEQ ID NO:6 or a variant thereof, wherein the variant modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the environmental stress is drought stress.
Preferably the variants of SEQ ID NO:6 comprise the amino acid sequence:







(SEQ ID NO: 249)









WRX1IX2RX3X4VX5X6X7TPTQVASHIAQKX8X9X10R






wherein X1=G or N, X2=A or S, X3=H, K, N, S or T, X4=F or Y, X5=A, F, I, K, M, N, P, T or V, X6=S or T, X7=K or R, X8=F or Y, X9=F or Y and X10=1, L, M or S.

Exemplary polypeptide variants of SEQ ID NO:6 are disclosed herein and identified as SEQ ID NOs:67-94 of the sequence listing.
In a further aspect the invention provides an isolated polypeptide comprising the sequence of SEQ ID NO:6.
In a further aspect the invention provides an isolated polypeptide consisting of the sequence of SEQ ID NO:6.
In a further aspect the invention provides polypeptides comprising fragments of SEQ ID NO:6. Polypeptides comprising fragments of variants, also form part of the invention.
In a further aspect the invention provides an isolated polypeptide comprising the sequence of SEQ ID NO:9 or a variant thereof, wherein the variant modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the environmental stress is drought stress.
Preferably the variants of SEQ ID NO:9 comprise the amino acid sequence:







(SEQ ID NO: 250)


IPX1X2X3X4SWRKYGQKPIKGSX5X6PRGYYKCSX7X8X9X10X11X12


 


X13X14X15HVERX16X17X18X19X20X21MLX22VTYEX23X24HX25


 


H






wherein X1=A, G, P, Q or S, X2=D or G, X3=D or E, X4=F or Y, X5=K or P, X6=F, H or Y, X7=S or T, X8=I, L, M or V, X9=R or T, X10=G or S, X11=C or Y, X12=L or P, X13=A or L, X14=F, K or R, X15=K or N, X16=A, C, D or S, X17=I, L, M, P or V, X18=A, D, E or S, X19=D or E, X20=A, P, S or T, X21=A, S or T, X22=I, M or V, X23=D or G, X24=D or E, and X25=C, H, N or R.

Exemplary polypeptide variants of SEQ ID NO:9 are disclosed herein and identified as SEQ ID NOs:127-158 of the sequence listing.
In a further aspect the invention provides an isolated polypeptide comprising the sequence of SEQ ID NO:9.
In a further aspect the invention provides an isolated polypeptide consisting of the sequence of SEQ ID NO:9.
In a further aspect the invention provides polypeptides comprising fragments of SEQ ID NO:9. Polypeptides comprising fragments of variants, also form part of the invention.
In a further aspect the invention provides an isolated polypeptide comprising the sequence of SEQ ID NO:12 or a variant thereof, wherein the variant modulates in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the environmental stress is drought stress.
Preferably the variants of SEQ ID NO:12 comprise the amino acid sequence:







(SEQ ID NO: 251)


X1X2X3X4AIX5X6X7X8X9GVX10X11X12X13X14X15X16ZEFX17


 


IX18X19X20LTX21X22X23X24X25PGX26X27YA






wherein X1=D or E, X2=I, L or V, X3=F, I, L, M or V, X4=C, F, I or L, X5=F, I or L, X6=I or L, X7=H or P, X8=P or Q, X9=L or V, X10=F or C, X11=F or L, X12=K or R, X13=F, K, R, V or Y, X14=A, G or K, X15=C, I or L, X16=A, C, G, K or Q, X17=C, F, L, M or W, X18=A, C, D or S, X19=L or V, X20=I, L, P or V, X21=C, F, I, L or V, X22=F or L, X23=A or G, X24=F, W or Y, X25=F, I, L or V, X26=I, L or V, X27=I, L or V and wherein Z is a sequence of 1-27 amino acids.

Exemplary polypeptide variants of SEQ ID NO:12 are disclosed herein and identified as SEQ ID NOs:201-242 of the sequence listing.
In a further aspect the invention provides an isolated polypeptide comprising the sequence of SEQ ID NO:12.
In a further aspect the invention provides an isolated polypeptide consisting of the sequence of SEQ ID NO:12.
In a further aspect the invention provides polypeptides comprising fragments of SEQ ID NO:12. Polypeptides comprising fragments of variants, also form part of the invention.
In one embodiment the polypeptide is derived from a gymnosperm species.
In a further embodiment the polypeptide is derived from an angiosperm species.
In a preferred embodiment the polypeptide of the invention is derived from a forage plant species. Preferably the species is selected from those of the following genera: Lolium, Festuca, Dactylis, Bromus, Trifolium, Medicago, Phleum, Phalaris, Holcus, Lotus, Plantago and Cichorium. 
In a more preferred embodiment the polypeptide of the invention is derived from the genera Lolium or Trifolium. Particularly preferred are the species Lolium perenne and Trifolium repens. Most preferably the polypeptide of the invention is derived from Lolium perenne. 
In a further aspect the invention provides a polynucleotide encoding a polypeptide of the invention.
In a further aspect the invention provides a genetic construct which comprises a polynucleotide of the invention.
In a further aspect the invention provides a genetic construct which comprises a polynucleotide encoding a polypeptide of the invention.
In a further aspect the invention provides a genetic construct which comprises a polynucleotide of any one of SEQ ID NO:1, SEQ ID NO:2 or a variant or fragment thereof.
In a further aspect the invention provides a genetic construct which comprises the polynucleotide of SEQ ID NO:1.
In a further aspect the invention provides a genetic construct which comprises the polynucleotide of SEQ ID NO:2.
In a further aspect the invention provides a genetic construct which comprises a polynucleotide of any one of SEQ ID NO:4, SEQ ID NO:5 or a variant or fragment thereof.
In a further aspect the invention provides a genetic construct which comprises the polynucleotide of SEQ ID NO:4.
In a further aspect the invention provides a genetic construct which comprises the polynucleotide of SEQ ID NO:5.
In a further aspect the invention provides a genetic construct which comprises a polynucleotide of any one of SEQ ID NO:7, SEQ ID NO:8 or a variant or fragment thereof.
In a further aspect the invention provides a genetic construct which comprises the polynucleotide of SEQ ID NO:7.
In a further aspect the invention provides a genetic construct which comprises the polynucleotide of SEQ ID NO:8.
In a further aspect the invention provides a genetic construct which comprises a polynucleotide of any one of SEQ ID NO:10, SEQ ID NO:11 or a variant or fragment thereof.
In a further aspect the invention provides a genetic construct which comprises the polynucleotide of SEQ ID NO:10.
In a further aspect the invention provides a genetic construct which comprises the polynucleotide of SEQ ID NO:11.
Preferably the constructs of the invention are expression constructs as herein defined. Preferably expression constructs of the invention include an environmental stress responsive promoter operably linked polynucleotide sequence. Preferably the environmental stress responsive promoter is responsive to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
Preferably the expression construct includes a promoter comprising the sequence of SEQ ID NO:243 or a fragment, region, cis-element or variant of the sequence capable of regulating transcription of an operably linked polynucleotide sequence.
In a further aspect the invention provides a vector which comprises a genetic construct of the invention.
In a further aspect the invention provides a host cell which comprises a genetic construct of the invention.
In a further aspect the invention provides methods for the recombinant production of polypeptide of the invention comprising the steps of:

    
    
        a) culturing a host cell comprising a genetic construct of the invention, such as an expression construct as defined herein, capable of expressing a polypeptide of the invention, and
        b) separating the expressed polypeptide.
    
    


In a further aspect the invention provides a plant cell which comprises one or more of the genetic constructs of the invention. In a preferred embodiment the genetic construct comprises the polynucleotide sequence of SEQ ID NO:1 or a variant or fragment thereof.
In a further aspect the invention provides a plant cell which comprises one or more of the genetic constructs of the invention. In a preferred embodiment the genetic construct comprises the polynucleotide sequence of SEQ ID NO:2 or a variant or fragment thereof.
In a further aspect the invention provides a plant cell which comprises one or more of the genetic constructs of the invention. In a preferred embodiment the genetic construct comprises the polynucleotide sequence of SEQ ID NO:4 or a variant or fragment thereof.
In a further aspect the invention provides a plant cell which comprises one or more of the genetic constructs of the invention. In a preferred embodiment the genetic construct comprises the polynucleotide sequence of SEQ ID NO:5 or a variant or fragment thereof.
In a further aspect the invention provides a plant cell which comprises one or more of the genetic constructs of the invention. In a preferred embodiment the genetic construct comprises the polynucleotide sequence of SEQ ID NO:7 or a variant or fragment thereof.
In a further aspect the invention provides a plant cell which comprises one or more of the genetic constructs of the invention. In a preferred embodiment the genetic construct comprises the polynucleotide sequence of SEQ ID NO:8 or a variant or fragment thereof.
In a further aspect the invention provides a plant cell which comprises one or more of the genetic constructs of the invention. In a preferred embodiment the genetic construct comprises the polynucleotide sequence of SEQ ID NO:10 or a variant or fragment thereof.
In a further aspect the invention provides a plant cell which comprises one or more of the genetic constructs of the invention. In a preferred embodiment the genetic construct comprises the polynucleotide sequence of SEQ ID NO:11 or a variant or fragment thereof.
In a further aspect the invention provides a plant cell with altered expression of a polynucleotide or polypeptide of the invention.
In a further aspect the invention provides a plant cell genetically modified to alter expression of a polynucleotide or polypeptide of the invention.
In a further aspect the invention provides a plant which comprises a plant cell of the invention.
In a further aspect the invention provides methods for altering in a plant, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity, the method comprising transformation of a plant cell, or plant with a genetic construct of the invention capable of altering expression of a polynucleotide/polypeptide of the invention.
In a further aspect the invention provides methods for altering tolerance to drought stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide/polypeptide of the invention.
In a further aspect the invention provides methods for altering tolerance to cold stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide/polypeptide of the invention.
In a further aspect the invention provides methods for altering tolerance to freezing stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide/polypeptide of the invention.
In a further aspect the invention provides methods for altering tolerance to heat stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide/polypeptide of the invention.
In a further aspect the invention provides methods for altering tolerance to salinity stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide/polypeptide of the invention.
In a further aspect the invention provides methods for altering tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide involved in modulation in a plant of tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
In a further aspect the invention provides methods for altering tolerance to drought stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide involved in modulation tolerance to drought stress in a plant.
In a further aspect the invention provides methods for altering tolerance to cold stress in a plant the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide involved in modulation of tolerance to cold stress in a plant.
In a further aspect the invention provides methods for altering tolerance to freezing stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide involved in modulation of tolerance to freezing stress in a plant.
In a further aspect the invention provides methods for altering tolerance to heat stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide involved in modulation of tolerance to heat stress in a plant.
In a further aspect the invention provides methods for altering tolerance to salinity stress in a plant, the method comprising transformation of a plant with a genetic construct of the invention capable of altering expression of a polynucleotide involved in modulation of tolerance to salinity stress in a plant.
It will be understood by those skilled in the art that transformation of a plant may involve transforming a plant cell(s) and regenerating a transformed plant from the transformed plant cell(s).
In a further aspect the invention provides a method for selecting a plant with increased tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to drought stress, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to cold stress, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to freezing stress, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to heat stress, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to salinity stress, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to drought stress, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to cold stress, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to freezing stress, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to heat stress, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a method for selecting a plant with increased tolerance to salinity stress, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a plant cell or plant produced by a method of the invention.
In a further aspect the invention provides a plant cell selected by a method of the invention.
In a further aspect the invention provides a population or group of plants selected by a method of the invention.
The plant cells and plants of the invention may be from any species.
In one embodiment the plant cells and plants of the invention are from gymnosperm species.
In a further embodiment the plant cells and plants of the invention are from angiosperm species.
In a preferred embodiment the plant cells and plants of the invention are derived from forage plant species. Preferably the forage species is selected from those of the following genera: Lolium, Festuca, Dactylis, Bromus, Trifolium, Medicago, Phleum, Phalaris, Holcus, Lotus, Plantago and Cichorium. 
In a more preferred embodiment of the invention the plant cells and plants are from the genera Lolium or Trifolium. Particularly preferred are the species Lolium perenne and Trifolium repens. Most preferably the plant cells and plants of the invention are derived from Lolium perenne. 



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be better understood with reference to the accompanying drawings in which:
FIG. 1 shows a map of a vector, for plant transformation, comprising ORF24 (SEQ ID NO:2).
FIG. 2 shows the sequence (SEQ ID NO:244) of a vector, for plant transformation, comprising ORF24 and corresponding to the map in FIG. 1. Sequence in bold corresponds to the CaMV35S promoter. Sequence in italics corresponds to ORF24. Sequence underlined corresponds to 3′terminator sequence from CaMV35S gene. Sequence in regular font corresponds to vector sequence.
FIG. 3 shows a map of another vector, for plant transformation, comprising ORF24 (SEQ ID NO:2).
FIG. 4 shows the sequence (SEQ ID NO:252) of another vector, for plant transformation, comprising ORF24 and corresponding to the map in FIG. 3. Sequence in bold corresponds to the Lolium perenne promoter (SEQ ID NO:243). Sequence in italics corresponds to ORF24. Sequence underlined corresponds to 3′terminator sequence from CaMV35S gene. Sequence in regular font corresponds to vector sequence.
FIG. 5 shows an alignment of polypeptides of the invention, including ORF24 (SEQ ID NO:3 labeled as “translated”) and sequences which are variants of SEQ ID NO:3 from several species (AF071893.P=SEQ ID NO:15; AY572463GD=SEQ ID NO:18; AJ606475.F=SEQ ID NO:16; AY044235.L=SEQ ID NO:17; AY246274.0=SEQ ID NO:20; AY286010.N SEQ ID NO:19; AJ515026=SEQ ID NO:21; AF537220=SEQ ID NO:22; NM 104269=SEQ ID NO:23; translated=SEQ ID NO:3; predicted=SEQ ID NO:3; AF542184.T==SEQ ID NO:13; BQ802250.T=SEQ ID NO:25; AF190770.0=SEQ ID NO:14; and AY103951.Z=SEQ ID NO:24) and illustrates a consensus region (shown within bold box) identified by the applicants which is present in all of the such sequences.
FIG. 6 shows a map of a vector, for plant transformation, comprising ORF68 (SEQ ID NO:5).
FIG. 7 shows the sequence (SEQ ID NO:245) of a vector, for plant transformation, comprising ORF68 and corresponding to the map in FIG. 6. Sequence in bold corresponds to the CaMV35S promoter. Sequence in italics corresponds to ORF68. Sequence underlined corresponds to 3′terminator sequence from CaMV35S gene. Sequence in regular font corresponds to vector sequence.
FIG. 8 shows a map of another vector, for plant transformation, comprising ORF68 (SEQ ID NO:5).
FIG. 9 shows the sequence (SEQ ID NO:253) of a vector, for plant transformation, comprising ORF68 and corresponding to the map in FIG. 8. Sequence in bold corresponds to the Lolium perenne promoter (SEQ ID NO:243). Sequence in italics corresponds to ORF68. Sequence underlined corresponds to 3′terminator sequence from CaMV35S gene. Sequence in regular font corresponds to vector sequence.
FIG. 10 shows an alignment of polypeptides of the invention, including ORF68 (SEQ ID NO:6) and sequences which are variants of SEQ ID NO:6 from several species (AAU06309.1=SEQ ID NO:91; CAI30890.1=SEQ ID NO:93; AAL78742.1=SEQ ID NO:92; AAK91894.1=SEQ ID NO:94; CB289054.1=SEQ ID NO:84; AA045179.1=SEQ ID NO:89; CF207250.1=SEQ ID NO:79; CF838179.1=SEQ ID NO:81; CK281819.1=SEQ ID NO:82; AAS09982.1=SEQ ID NO:52; CD822965.1=SEQ ID NO:57; CF436607.1 =SEQ ID NO:59; BE130418.1=SEQ ID NO:60; AA047339.1=SEQ ID NO:90; CF667220.1 =SEQ ID NO:58; ORF68 =SEQ ID NO:6; BJ259031.1=SEQ ID NO:67; BAD72233.1=SEQ ID NO:68; CD225976.1=SEQ ID NO:70; CA132547.1=SEQ ID NO:75; AJ615010.1=SEQ ID NO:71; CAC24845.1=SEQ ID NO:73; BG127541.1=SEQ ID NO:76; BQ120809.2=SEQ ID NO:77; BM520331.1=SEQ ID NO: 72; AW570097.1=SEQ ID NO:83; CO077451.1=SEQ ID NO:69; AJ502953.1=SEQ ID NO:74; and BP950240.1=SEQ ID NO:78 and illustrates a consensus region (shown within bold box) identified by the applicants which is present in all of the such sequences.
FIG. 11 shows a map of a vector, for plant transformation, comprising ORF69 (SEQ ID NO:8).
FIG. 12 shows the sequence (SEQ ID NO:246) of a vector, for plant transformation, comprising ORF69 and corresponding to the map in FIG. 11. Sequence in bold corresponds to the CaMV35S promoter. Sequence in italics corresponds to ORF69. Sequence underlined corresponds to 3′terminator sequence from CaMV35S gene. Sequence in regular font corresponds to vector sequence.
FIG. 13 shows a map of another vector, for plant transformation, comprising ORF69 (SEQ ID NO:8).
FIG. 14 shows the sequence (SEQ ID NO:254) of another vector, for plant transformation, comprising ORF69 and corresponding to the map in FIG. 13. Sequence in bold corresponds to the Lolium perenne promoter (SEQ ID NO:243). Sequence in italics corresponds to ORF69. Sequence underlined corresponds to 3′terminator sequence from CaMV35S gene. Sequence in regular font corresponds to vector sequence.
FIG. 15 shows an alignment of polypeptides of the invention, including ORF69 (SEQ ID NO:9) and sequences which are variants of SEQ ID NO:9 from several species (CO092212.1=SEQ ID NO:135; CO903744.1=SEQ ID NO:137; BU835049.1=SEQ ID NO:136; CO979572.1=SEQ ID NO:134; CA921208.1=SEQ ID NO:139; BG351755.1=SEQ ID NO:138; CD484064.1=SEQ ID NO:132; BJ576569.1=SEQ ID NO:140; BX831747.1=SEQ ID NO:133; AY011121.1=SEQ ID NO:131; CB879962.1=SEQ ID NO:141; BE361091.1=SEQ ID NO:144; AY077758.1=SEQ ID NO:145; CO210220.1=SEQ ID NO:147., CF205668.1=SEQ ID NO:146; CV291964.1=SEQ ID NO:152; AAD32676.1=SEQ ID NO:155; DAA05115.1=SEQ ID NO: 157; AB020023.1 SEQ ID NO:148; CK295284.1SEQ ID NO:150; BT014501.1=SEQ ID NO:158; CV707436.1=SEQ ID NO:153; AAS66778.1=SEQ ID NO:156; U56834.1=SEQ ID NO:151; BP083458.1=SEQ ID NO:154; BM336249.1=SEQ ID NO:143; BF291316.1=SEQ ID NO:128; CA141214.1=SEQ ID NO: 130; ORF69=SEQ ID NO:9; CV000669.1=SEQ ID NO:149; BM956319.1=SEQ ID NO:142; AK070648.1=SEQ ID NO:127; and CV782162.1=SEQ ID NO:129) and illustrates a consensus region (shown within bold box) identified by the applicants which is present in all of the such sequences.
FIG. 16 shows a map of a vector, for plant transformation, comprising ORF9 (SEQ ID NO:11).
FIG. 17 shows the sequence of a vector (SEQ ID NO:247), for plant transformation, comprising ORF9 and corresponding to the map in FIG. 16. Sequence in bold corresponds to the CaMV35S promoter. Sequence in italics corresponds to ORF9. Sequence underlined corresponds to 3′terminator sequence from CaMV35S gene. Sequence in regular font corresponds to vector sequence.
FIG. 18 shows a map of another vector, for plant transformation, comprising ORF9 (SEQ ID NO:11).
FIG. 19 shows the sequence of another vector (SEQ ID NO:255), for plant transformation, comprising ORF9 and corresponding to the map in FIG. 18. Sequence in bold corresponds to the Lolium perenne promoter (SEQ ID NO:243). Sequence in italics corresponds to ORF9. Sequence underlined corresponds to 3′terminator sequence from CaMV35S gene. Sequence in regular font corresponds to vector sequence
FIG. 20 shows an alignment of polypeptides of the invention, including ORF9 (SEQ ID NO: 12) and sequences which are variants of SEQ ID NO: 12 from several species (CD913648=SEQ ID NO:219; BI780134=SEQ ID NO:220; ORF9=SEQ ID NO:12; AY107179.1=SEQ ID NO:204; CF042363=SEQ ID NO:221; CD229091=SEQ ID NO:218; AAS72368=SEQ ID NO:202; AC093089=SEQ ID NO:206; CF319949=SEQ ID NO:222; AY060504=SEQ ID NO:230; AC005770=SEQ ID NO:234; CN 183349=SEQ ID NO:239; CA836518=SEQ ID NO:238; BU870658=SEQ ID NO:240; Q9ARD5=SEQ ID NO:203; AJ310995=SEQ ID NO:212; U00966.1=SEQ ID NO:213; AJ310994=SEQ ID NO:217; Z25537=SEQ ID NO:224; AK070872=SEQ ID NO:205; NM 104551=SEQ ID NO:236; AY103848=SEQ ID NO:209; AY607689=SEQ ID NO:214; AY105302=SEQ ID NO:216; NM 183567=SEQ ID NO:223; AY554051=SEQ ID NO:211; NM 184595=SEQ ID NO:215; AY084701=SEQ ID NO:235; AB061265=SEQ ID NO:237; AY316308=SEQ ID NO:233; AAR87655=SEQ ID NO:241; BX842170=SEQ ID NO:231; BX825999=SEQ ID NO:232; AY496072=SEQ ID NO:242; AB030211=SEQ ID NO:207; AK062410=SEQ ID NO:208; AY108684=SEQ ID NO:210; AAN06944=SEQ ID NO:201; NP 194795=SEQ ID NO:184; NP 179982=SEQ ID NO:229; NP 974629=SEQ ID NO:225; NP 194794=SEQ ID NO:227; and AAT11798=SEQ ID NO:228) and illustrates a consensus region (shown within bold box) identified by the applicants which is present in all of the such sequences.
FIG. 21 shows four hydroponic tanks, representing eight randomized blocks with 15 plants in each block (10 transgenic lines and 5 wild type clones), totaling 120 plants (80 transgenic lines and 40 wild type clones).
FIG. 22 shows the Electron Transfer Rate (ETR) (a) and yield (b) of photosystem II after 24 hours recovery from 24 hours stress as measured with PAM2000.
FIG. 23 shows the Electron Transfer Rate (ETR) (a) and yield (b) of photosystem II after 24 hours recovery from 42 hours stress as measured with PAM2000.
FIG. 24 shows fresh (a) and dry weight (b) of leaves from transgenic and wildtype ryegrass plants and the difference between fresh and dry weights (c) in grams after nine days of recovery from three cycles of drought-stress.
FIG. 25 shows dry weight of roots after nine days of recovery from three cycles of drought-stress.
FIG. 26 shows the visual appearance of plants after the third drought-stress lasting for 42 hours; a) Wild type (left) transgenic line D24-145 (right); b) Wild type (left) transgenic line D9-144 (right); c) Wild type (left) transgenic line D9-150 (right).
FIG. 27 shows (a) Transgenic ryegrass growing in bins filled with soil during progressive drought stress; (b) Transgenic ryegrass over-expressing ORF 24 in comparison to wildtype at 2.3% soil VWC.
FIG. 28 shows the aboveground biomass (dry weight) produced before (a; d; g) and after drought-stress (b; e; h) and the ability to grow during drought-stress is shown (c, f and i).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The term “comprising” as used in this specification and claims means “consisting at least in part of”; that is to say when interpreting statements in this specification and claims which include “comprising”, the features prefaced by this term in each statement all need to be present but other features can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.
The term “plant” is intended to include a whole plant, any part of a plant, propagules and progeny of a plant.
The term ‘propagule’ means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.
The term “tolerance or tolerant to drought stress” is intended to describe a plant or plants which perform more favourably in any aspect of their growth and development under sub-optimal hydration conditions than do suitable control plants in the same conditions.
The term “tolerance or tolerant to drought stress” is also intended to describe a plant or plants which perform more favourably in any aspect of their growth and development after having been subjected to sub-optimal hydration conditions. That is, plants that show improved recovery after a period of sub-optimal hydration.
The term “tolerance or tolerant to cold stress” is intended to describe a plant or plants which perform more favourably in any aspect of their growth and development under sub-optimal-reduced reduced temperature conditions than do suitable control plants in the same conditions.
The term “tolerance or tolerant to freezing stress” is intended to describe a plant or plants which perform more favourably in any aspect of their growth and development under temperature conditions of less than or equal to 0° C., than do suitable control panels in the same conditions.
The term “tolerance or tolerant to heat stress” is intended to describe a plant or plants which perform more favourably in any aspect of their growth and development under sub-optimal elevated temperature conditions than do suitable control plants in the same conditions.
The term “tolerance or tolerant to salinity” is intended to describe a plant or plants which perform more favourably in any aspect of their growth and development under sub-optimal elevated salinity conditions than do suitable control plants in the same conditions.
With reference to the selection methods of the invention, a plant with increased tolerance to environmental stress refers to a plant, selected from a population of plants, which performs more favourably in any aspect of growth and development under stress conditions than does an average member of the population under the same conditions.
The term “polynucleotide(s),” as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, genetic constructs, vectors and modified polynucleotides.
A “fragment” of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, e.g., a sequence that is at least 15 nucleotides in length. The fragments of the invention comprise 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.
The term “primer” refers to a short polynucleotide, usually having a free 3′OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.
The term “probe” refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay. The probe may consist of a “fragment” of a polynucleotide as defined herein.
The term “polypeptide”, as used herein, encompasses amino acid chains of any length, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.
A “fragment” of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity of the polypeptide. Polypeptide fragments are at least 5 amino acids, preferably at least 10 amino acids, more preferably at least 20 amino acids, more preferably at least 30 amino acids in length, more preferably 40 amino acids in length, more preferably 50 amino acids in length.
The term “isolated” as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.
The term “recombinant” refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and/or is recombined with sequences that are not present in its natural context.
A “recombinant” polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.
As used herein, the term “variant” refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polynucleotides possess biological activities that are the same or similar to those of the inventive polypeptides or polynucleotides. The term “variant” with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.
Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% identity to any one of SEQ ID NO:1, 2, 4, 5, 7, 8, 10 and 11. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the invention.
Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN from the BLAST suite of programs, version 2.2.5 [Nov. 2002]) in b12seq (Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI. The default parameters of b12seq are utilized except that filtering of low complexity parts should be turned off.
The identity of polynucleotide sequences may be examined using the following UNIX command line parameters:
bl2seq-i nucleotideseq1-j nucleotideseq2-F F-p blastn
The parameter-F F turns off filtering of low complexity sections. The parameter-p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line “Identities=”.
Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No. 6. pp. 276-277. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences.
Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
Use of BLASTN as described above is preferred for use in the determination of sequence identity for polynucleotide variants according to the present invention.
Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polynucleotides may be determined using the publicly available b12seq program from the BLAST suite of programs (version 2.2.5 [Nov. 2002]) from NCBI.
The similarity of polynucleotide sequences may be examined using the following UNIX command line parameters:
bl2seq-i nucleotideseq1-j nucleotideseq2-F F-p tblastx
The parameter-F F turns off filtering of low complexity sections. The parameter-p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an “E value” which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.
Variant polynucleotide sequences preferably exhibit an E value of less than 1×10−5 more preferably less than 1×10−6 more preferably less than 1×10−9, more preferably less than 1×10−12, more preferably less than 1×10−15, more preferably less than 1×10−18 and most preferably less than 1×10−21 when compared with any one of the specifically identified sequences.
Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in any one of SEQ ID NO:1, 2, 4, 5, 7, 8, 10 and 11, or complements thereof under stringent conditions.
The term “hybridize under stringent conditions”, and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C. (for example, 10° C.) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm=81.5+0.41% (G+C−log(Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C. below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)° C.
With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec. 6; 254(5037):1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov. 1; 26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C. below the Tm.
Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a “silent variation”. Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.
Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available b12seq program from the BLAST suite of programs (version 2.2.5 [Nov. 2002]) from NCBI via the tblastx algorithm as previously described.
The term “variant” with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% identity to a sequences any one of SEQ ID NO:3, 6, 9 and 12. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.
Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov. 2002]) in b12seq, which is publicly available from NCBI. The default parameters of b12seq are utilized except that filtering of low complexity regions should be turned off.
Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.
Use of BLASTP as described above is preferred for use in the determination of polypeptide variants according to the present invention.
Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available b12seq program from the BLAST suite of programs (version 2.2.5 [Nov. 2002]) from NCBI. The similarity of polypeptide sequences may be examined using the following UNIX command line parameters:
bl2seq-i peptideseq1-j peptideseq2-F F-p blastp
Variant polypeptide sequences preferably exhibit an E value of less than 1×10−5, more preferably less than 1×10−6, more preferably less than 1×10−9, more preferably less than 1×10−12, more preferably less than 1×10−15, more preferably less than 1×10−18 and most preferably less than 1×10−21 when compared with any one of the specifically identified sequences.
The parameter-F F turns off filtering of low complexity sections. The parameter-p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an “E value” which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
The term “genetic construct” refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.
The term “vector” refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli. 
The term “expression construct” refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5′ to 3′ direction:

    
    
        a) a promoter functional in the host cell into which the construct will be transformed,
        b) the polynucleotide to be expressed, and
        c) a terminator functional in the host cell into which the construct will be transformed.
    
    


The term “coding region” or “open reading frame” (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5′ translation start codon and a 3′ translation stop codon. When inserted into a genetic construct, a “coding sequence” is capable of being expressed when it is operably linked to promoter and terminator sequences.
“Operably-linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
The term “noncoding region” refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5′ UTR and the 3′ UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.
Terminators are sequences, which terminate transcription, and are found in the 3′ untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
The term “promoter” refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
A “transgene” is a polynucleotide that is taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
A “transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.
An “inverted repeat” is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,









(5′)GATCTA.......TAGATC(3′)


 



(3′)CTAGAT.......ATCTAG(5′)






Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
The terms “to alter expression of” and “altered expression” of a polynucleotide or polypeptide of the invention, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the invention is modified thus leading to altered expression of a polynucleotide or polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The “altered expression” can be related to an increase or decrease in the amount of messenger RNA and/or polypeptide produced and may also result in altered activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.
The applicants have identified polynucleotides from ryegrass (SEQ ID NO:1 and SEQ ID NO:2) which encode a polypeptide (SEQ ID NO:3) which modulates in plants, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity. The applicants have also identified polynucleotide variants of SEQ ID NO:1 and SEQ ID NO:2 (SEQ ID NOs:13-25) encoding polypeptide variants of SEQ ID NOs:3 (SEQ ID NOs:26-38) which modulate in plants, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
The applicants have identified polynucleotides from ryegrass (SEQ ID NO:4 and SEQ ID NO:5) which encode a polypeptide (SEQ ID NO:6) which modulates in plants, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity. The applicants have also identified polynucleotide variants of SEQ ID NO:4 and SEQ ID NO:5 (SEQ ID NOs:39-66) encoding polypeptide variants of SEQ ID NO:6 (SEQ ID NOs:67-94) which modulate in plants, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
The applicants have identified polynucleotides from ryegrass (SEQ ID NO:7 and SEQ ID NO:8) which encode a polypeptide (SEQ ID NO:9) which modulates in plants, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity. The applicants have also identified polynucleotide variants of SEQ ID NO:7 and SEQ ID NO:8 (SEQ ID NOs:95-126) encoding polypeptide variants of SEQ ID NOs:9 (SEQ ID NOs:127-158) which modulate in plants, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
The applicants have identified polynucleotides from ryegrass (SEQ ID NO:10 and SEQ ID NO:11) which encode a polypeptide (SEQ ID NO:12) which modulates in plants, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity. The applicants have also identified polynucleotide variants of SEQ ID NO:10 and SEQ ID NO:1 (SEQ BD NOs:159-200) encoding polypeptide variants of SEQ ID NOs:12 (SEQ ID NOs:201-242) which modulate in plants, tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity
The invention provides plants altered relative to suitable control plants in tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity. The invention provides both plants with both increased tolerance to the above and plants with decreased tolerance to above characteristic stresses. The invention also provides methods for the production or selection of such plants.
The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polynucleotides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polynucleotides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
Further methods for isolating polynucleotides of the invention include use of all, or portions of, the any on of the polynucleotides having the sequence set forth in SEQ ID NOs:1, 2, 4, 5, 7, 8, 10, 11 13-25, 39-66, 95-126 and 159-242 as hybridization probes. The technique of hybridizing labeled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65° C. in 5.0 X SSC, 0.5% sodium dodecyl sulfate, 1×Denhardt's solution; washing (three washes of twenty minutes each at 55° C.) in 1.0×SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0.5×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C. An optional further wash (for twenty minutes) can be conducted under conditions of 0.1×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C.
The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion and oligonucleotide synthesis.
A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods would include PCR-based methods, 5′RACE (Frohman M A, 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer/database-based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. Additionally when down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species. Variants (including orthologues) may be identified by the methods described.
Variant polynucleotides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer, useful to amplify variants of polynucleotide molecules of the invention by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.
Further methods for identifying variant polynucleotides of the invention include use of all, or portions of, the polynucleotides having the sequence set forth in SEQ ID NOs:1, 2, 4, 5, 7, 8, 10 and 11 as hybridization probes to screen a plant genomic or cDNA libraries as described above. Typically probes based on a sequence encoding a conserved region of the corresponding amino acid sequence may be used. Hybridisation conditions may also be less stringent than those used when screening for sequences identical to the probe.
The variant sequences of the invention, including both polynucleotide and polypeptide variants, may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.
An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov. 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from the National Center for Biotechnology Information (NCBI), National Library of Medicine. Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
The “hits” to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce “Expect” values for alignments. The Expect value (E) indicates the number of hits one can “expect” to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680, or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).
Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego.
PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (www.expasy.org/prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.
Proteins can be classified according to their sequence relatedness to other proteins in the same genome (paralogues) or a different genome (orthologues). Orthologous genes are genes that evolved by specification from a common ancestral gene and normally retain the same function as they evolve. Paralogous genes are genes that are duplicated within a genome and genes may acquire new specificities or modified functions which may be related to the original one. Phylogenetic analysis methods are reviewed in Tatusov et al., 1997, Science 278, 631-637,).
In addition to the computer/database methods described above, polypeptide variants of the invention may be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.
The function of a variant polynucleotide of the invention in modulating tolerance to environmental stress plant may be assessed by altering expression of the polynucleotide in a plant by methods known in the art and/or described herein, and, analyzing performance of the transformed plant in comparison to a control plant, under conditions of environmental stress. Further plant transformation protocols for several species are known to those skilled in the art. A list of such protocols is provided herein.
The polypeptides of the invention, including variant polypeptides, may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco Calif., or automated synthesis, for example using an Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.). Mutated forms of the polypeptides may also be produced during such syntheses.
The polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification,).
Alternatively the polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.
Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).
The invention provides a host cell which comprises a genetic construct or vector of the invention. Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.
Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
Host cells of the invention may also be useful in methods for production of an enzymatic product generated by an expressed polypeptide of the invention. Such methods may involve culturing the host cells of the invention in a medium suitable for expression of a recombinant polypeptide of the invention, optionally in the presence of additional enzymatic substrate for the expressed polypeptide of the invention. The enzymatic product produced may then be separated from the host cells or medium by a variety of art standard methods.
The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention. Plants comprising such cells also form an aspect of the invention.
Tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity in a plant, may also be altered through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide which modulates for example, tolerance to drought stress, in such plant cells and plants. Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polynucleotides or polypeptides which modulate for example, tolerance to drought stress in such plant cells and plants.
Methods for transforming plant cells, plants and portions thereof with polynucleotides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenberg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.
A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297). For example, strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.
Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.
Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detest presence of the genetic construct in the transformed plant.
The promoters suitable for use in the constructs of this invention are functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is herein incorporated by reference.
Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.
Selectable markers commonly used in plant transformation include the neomycin phosphotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene (hpt) for hygromycin resistance.
Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.
Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. “Regulatory elements” is used here in the widest possible sense and includes other genes which interact with the gene of interest.
Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide of the invention may include an antisense copy of a polynucleotide of the invention. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.
An “antisense” polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,









5′GATCTA 3′ (coding strand)
3′CTAGAT 5′ (antisense strand)



 


3′CUAGAU 5′ mRNA
5′GAUCUA 3′ antisense RNA






Genetic constructs designed for gene silencing may also include an inverted repeat as herein defined. The preferred approach to achieve this is via RNA-interference strategies using genetic constructs encoding self-complementary “hairpin” RNA (Wesley et al., 2001, Plant Journal, 27: 581-590).
The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually a spacer of at least 3-5 bp between the repeated regions is required to allow hairpin formation.
Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.
The term genetic construct as used herein also includes small antisense RNAs and other such polynucleotides effecting gene silencing.
Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5′ or 3′ untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al., 2002, Plant Physiol. 128(3): 844-53; Jones et al., 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a polynucleotide of the invention is also contemplated.
The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5′ or 3′ UTR sequence, or the corresponding gene.
Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (McIntyre, 1996, Transgenic Res, 5, 257)
Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.
The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); maize (U.S. Pat. Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9,: 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073 and 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci. 104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); and cereals (U.S. Pat. No. 6,074,877).
Several further methods known in the art may be employed to alter expression of a nucleotide and/or polypeptide of the invention. Such methods include but are not limited to Tilling (Till et al., 2003, Methods Mol Biol, 2%, 205), so called “Deletagene” technology (Li et al., 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors. (e.g. Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al., 2003, Nat. Biotechnol., 21(1), 35). Transposon tagging approaches may also be applied. Additionally peptides interacting with a polypeptide of the invention may be identified through technologies such as phage-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a polypeptide of the invention. Use of each of the above approaches in alteration of expression of a nucleotide and/or polypeptide of the invention is specifically contemplated.
Methods are also provided for selecting plants altered tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity. Such methods involve testing of plants for altered for the expression of a polynucleotide or polypeptide of the invention. Such methods may be applied at a young age or early developmental stage to accelerate breeding programs directed toward at least one of the characteristics described which may not be easily assessed until a later age or developmental stage.
The expression of a polynucleotide, such as a messenger RNA, is often used as an indicator of expression of a corresponding polypeptide. Exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). Polynucleotides or portions of the polynucleotides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with altered tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity. For example an altered level in a plant, of a polypeptide involved in modulating tolerance to drought stress may be used as an indicator of eventual tolerance to drought stress in such a plant. The polynucleotides of the invention may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.
Alternatively antibodies may be raised against polypeptides of the invention. Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998). Such antibodies may be used in methods to detect altered expression of polypeptides which modulate flower size in plants. Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).
These approaches for analysis of polynucleotide or polypeptide expression and the selection of plants with altered expression are useful in conventional breeding programs designed to produce varieties with altered in tolerance to at least one environmental stress selected from drought, cold, freezing, heat and salinity.
The plants of the invention may be grown and either selfed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.
EXAMPLES
The invention will now be illustrated with reference to the following non-limiting examples.
Example 1
Identification of Polynucleotides which Modulate Tolerance to Environmental Stresses
Introduction:
Perennial ryegrass (Lolium perenne L.) is a cool temperate pasture plant from the family Gramineae and the tribe Festucaceae. To generate a profile of relative gene expression patterns in ryegrass, RNA was extracted from samples obtained from ambient temperature growth, cold grown, hydrated, dehydrated and rehydrated or dehydration pre- and post-grazed plants during autumn, summer, spring and winter, and used for constructing a SAGE (serial analysis of gene expression) (Velculescu et al. 1995, Science 270: 484-487) library.
Materials and Methods:
Perennial ryegrass (Lolium perenne L.) cv. Bronsyn was used throughout this study. Field grown samples were collected from active paddocks at Dexcel, Hamilton, New Zealand during the peak of each season. Grass samples were collected from pre-grazed (15 days post grazing) and post-grazed (1 day post grazing) ryegrass swards. Tufts of grass samples were harvested from 3-6 randomly chosen sites and stored in dry-ice after snap-freezing with liquid nitrogen. During spring, immature spike and floral initials were also harvested. For stress-treatment, the following conditions were used on lab-grown ryegrass: Mature lab-grown perennial ryegrass that was grown in growth chamber for 15 months at 85% RH, 20° C./18° C. and 16 h/8 h day/night regime; Hydrated control grown for 55 days at 85% RH, 20° C./18° C. and 16 h/8 h day/night regime; 6 days at 70% RH, 22° C./16° C. and 16 h/8 h day/night regime, seedlings were kept watered throughout their life; Dehydrated sample watered only for 55 days at 85% RH, 20° C./18° C. and 16 h/8 h day/night regime; 3 days at 70% RH, 28° C./20° C. and 16 h/8 h day/night regime; 3 days at 50% RH, 28° C./20° C. and 16 h/8 h day/night regime; Rehydrated samples were from dehydrated plants that was watered for 24 hours and grown at 70% RH, 22° C./16° C. and 16 h/8 h day/night regime; Cold-stressed plants were grown for 55 days at 85% RH, 20° C./18° C. and 16 h/8 h day/night regime; 7 days at 70% RH, 22° C./16° C. and 16 h/8 h day/night regime; 7 days at 70% RH, 6° C./2° C. and 16 h/8 h day/night regime, seedlings were kept watered throughout their life.
Construction of SAGE Libraries
RNA was extracted using TRIZOL® reagent (Invitrogen, Calif., USA) and by the protocol described by the manufacturer from tissue that was ground in liquid nitrogen. For each SAGE library 100 μg of total RNA was used and the libraries were created using I-SAGETM or I-SAGETM Long kit (Invitrogen, Calif., USA) according to manufacturer's protocol. From each library 960-1,920 clones were sequenced (Australian Genome Research Facility, Brisbane, Australia) and the tags extracted using the SAGE2000 software.
SAGE Bioinformatics:
The relational database was designed to hold tags, libraries and expression counts of the SAGE experiments. Each tag sequence (including enzyme sequence) was searched against the whole Ryegrass non-overlapping Gene thresher and the EST sets. The search was carried out in both direction and used exact match only. Results were loaded to the relational database using General Feature Format (GFF) approach.
All Ryegrass Gene thresher and the EST sequences were annotated using homology searches against some or all the following public and propriety databases:

    
    
        AGI TIGR Gene Indices, Arabidopsis, release 11, Jan. 2004
        OGI TIGR Gene Indices, Rice, release 14-1, Jan. 2004
        GENESEQN Derwent patent DNA sequences 2002 Dec. 7
        GENESEQP Derwent patent amino acid sequences 2002 Dec. 7
        Os_unigene Oryza sativa Unigene unique sequences 2004-03-18
        est_others Other EST sequences (mammal, fungi, prokaryote) 2003 Mar. 11
        est_plant Viridiplantae subset of Non-redundant Database of GenBank+EMBL+DDBJ EST Divisions 2004-03-15
        nr All non-redundant GenBank CDS translations+PDB+SwissProt+PIR 2003 Mar. 11
        nr_pant Plant subset of HS subset of BT subset of All non-redundant GenBank CDS translations+PDB+SwissProt+PIR 203 Aug. 8
        nt All Non-redundant GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or HTGS -sequences) Mar. 11, 2003
        nt_monocots Monocot subset of All Non-redundant GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or HTGS sequences) Mar. 11, 2003
        swissprot The last major release of the SWISS-PROT protein sequence database (no updates) 2003 Mar. 28
    
    


A cutoff of E value less than E-05 was used and maximum of 10 targets per database were stored in the relational database.
Tags Annotation:
Tags with hits to the Ryegrass sets were annotated by creating a summary of all the annotations of the involved sequences. The summary was generated using an algorithm to calculate the frequency of the occurrence of each word in the annotations and rank them in descending order based on the number off occurrences. The summary was limited to 10 words and a void word list was used to filter out insignificant information. The resulting summary line was used as an indication of what the tags were likely to be. Actual numbers are displayed; giving additional information that could be used to evaluate the significance of each of the words in the summary. This method of automatic annotation using keyword counts is similar to the Automatic comment that is used by the ProDom database to annotate the automatically generated protein domain families.
Detailed annotation based on the top hits of the involved sequences was displayed when viewing tags data.
Four polynucleotide sequences of particular interest were identified in the above analysis. These are ORF24 (corresponds to SEQ ID NO:1 and 2) and ORF68 (corresponding to SEQ ID NO:4 and 5), ORF69 (corresponding to SEQ ID NO:7 and 8) and ORF9 (corresponding to SEQ ID NO:10 and 11).
ORF24 appears to encode a heat shock transcription factor. The transcript accumulates in dehydrated and winter tissues. The full transcript profile is shown in table 1.












TABLE 1





SAGE_TAG
CCCATTTCTG
tpm*
SAGE_TAG
CCCATTTCTGCCGATTT
tpm




















Winter Pre-grazed
1
36
Winter Pre-grazed
1
36


Winter Post-grazed
2
75
Winter Post-grazed
2
75


Winter roots
1
61
Winter roots
1
61


Spring Pre-grazed
0
0
Spring Pre-grazed
0
0


Spring Post-grazed
0
0
Spring Post-grazed
0
0


Inflorescence
1
41
Inflorescence
1
41


Summer Post-grazed
0
0
Summer Post-grazed
0
0


Autumn Pre-grazed
0
0
Total
5
30


Autumn Post-grazed
0
0


Mature
1
80


Cold-stressed
0
0


Hydrated
1
64


Dehydrated
3
176


Rehydrated
1
33


Total
11
40





*tpm = Tag counts per million tags






ORF68 appears to encode an ethylene responsive element binding protein (EREBP) transcription factor and the transcript accumulates in winter tissues. The full transcript profile is shown in table 2 below.












TABLE 2





SAGE_TAG
TCGCTGAAGA
tpm*
SAGE_TAG
TCGCTGAAGATCTTGGC
tpm




















Winter Pre-grazed
1
36
Winter Pre-grazed
1
36


Winter Post-grazed
1
37
Winter Post-grazed
1
37


Winter roots
2
121
Winter roots
2
121


Spring Pre-grazed
0
0
Spring Pre-grazed
0
0


Spring Post-grazed
0
0
Spring Post-grazed
0
0


Inflorescence
0
0
Inflorescence
0
0


Summer Post-grazed
0
0
Summer Post-grazed
0
0


Autumn Pre-grazed
0
0
Total
4
28


Autumn Post-grazed
0
0


Mature
0
0


Cold-stressed
0
0


Hydrated
0
0


Dehydrated
0
0


Rehydrated
0
0


Total
4
14





*tpm = Tag counts per million tags






ORF69 appears to be a MYB-like transcription factor and the transcript accumulates in cold-stressed, dehydrated and rehydrating tissues. The full transcript profile is shown in table 3 below.












TABLE 3





SAGE_TAG
TATGTAAAGG
tpm*
SAGE_TAG
TATGTAAAGGTTGCGAA
tpm




















Winter Pre-grazed
0
0
Winter Pre-grazed
0
0


Winter Post-grazed
0
0
Winter Post-grazed
0
0


Roots (winter)
0
0
Roots (winter)
0
0


Spring Pre-grazed
0
0
Spring Pre-grazed
0
0


Spring Post-grazed
0
0
Spring Post-grazed
0
0


Inflorescence
1
41
Inflorescence
1
41


Summer Post-grazed
0
0
Summer Post-grazed
0
0


Autumn Pre-grazed
0
0
Total
1
6


Autumn Post-grazed
0
0


Mature
1
80


Cold-stressed
1
54


Hydrated
0
0


Dehydrated
1
59


Rehydrated
1
33


Total
5
19





*tpm = Tag counts per million tags






ORF9 appears to be an extra-cellular stress-tolerance peptide and the transcript accumulates in cold-stressed and rehydrating tissues. The full transcript profile is shown in table 4 below.











TABLE 4







SAGE_TAG
TTTTTGTGAA
tpm*




















Winter Pre-grazed
0
0



Winter Post-grazed
1
37



Winter roots
0
0



Spring Pre-grazed
0
0



Spring Post-grazed
1
61



Inflorescence
0
0



Summer Post-grazed
1
51



Autumn Pre-grazed
1
35



Autumn Post-grazed
3
108



Mature
0
0



Cold-stressed
3
163



Hydrated
0
0



Dehydrated
0
0



Rehydrated
3
99



Total
13
40







*tpm = Tag counts per million tags






Example 2
Identification Variants of ORFs 24, 68, 69 and 9
The polypeptide sequence encoded by the ORFs 24, 68, 69 and 9 were used as seed sequences to perform BLASTP search against NR_PLANT database (release date 30-07-04). Besides BLASTP, a TBLASTN search was also performed against EST_PLANT database (release date Jul. 15, 2004) and NT_PLANT database (release date Jul. 15, 2004). To identify the variants cut-off e value was generally set at greater than 1e-05, which was determined based upon the associated score value.
Selected variant sequences were aligned using the EMBOSS tool EMMA (Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994, CABIOS, 10, 19-29.), which is an interface to the popular multiple alignment program ClustalW. Aligned sequences were visualised using another EMBOSS tool called prettyplot, which displays aligned sequences with colouring and boxing.
Example 3
Preparation of Vectors Comprising Polynucleotides of the Invention for Plant Transformation
Vectors Comprising ORF24
Two vectors for over-expressing ORF24 were produced by standard molecular biology techniques. A map of the first vector (pCORF24) is shown in FIG. 1. The sequence of the first vector is shown in FIG. 2 and SEQ ID NO:244. A map of the second vector (pDORF24) is shown in FIG. 3. The sequence of the second vector is shown in FIG. 4 and SEQ ID NO:252.
Vector Comprising ORF68
Two vectors for over-expressing ORF68 were produced by standard molecular biology techniques. A map of the first vector (pCORF68) is shown in FIG. 6. The sequence of the first vector is shown in FIG. 7 and SEQ ID NO:245. A map of the second vector (pDORF68) is shown in FIG. 8. The sequence of the second vector is shown in FIG. 9 and SEQ ID NO:253.
Vector Comprising ORF69
Two vectors for over-expressing ORF69 were produced by standard molecular biology techniques. A map of the first vector (pCORF69) is shown in FIG. 11. The sequence of the first vector is shown in FIG. 12 and SEQ ID NO:246. A map of the second vector (pDORF69) is shown in FIG. 13. The sequence of this vector is shown in FIG. 14 and SEQ ID NO:254.
Vector Comprising ORF9
Two vectors for over-expressing ORF9 were produced by standard molecular biology techniques. A map of the first vector (pCORF9) is shown in FIG. 16. The sequence of the first vector is shown in FIG. 17 and SEQ ID NO:247. A map of the second vector (pDORF9) is shown in FIG. 18. The sequence and features of this vector is shown in FIG. 19 (SEQ ID NO:255).
Example 4
Transformation of Plants within the Polynucleotides of the Invention
Donor Plant Production to Obtain Tissue Culture Explants
Seeds to establish contamination free in-vitro cultures were surface sterilized for 3 minutes with 70% (v/v) ethanol; followed by 60 minutes with sodium hypochlorite solution (2.4% active Chlorine) supplemented with a surfactant (0.1% (w/v) of Tween-20 and five rinses with autoclaved distilled water. Plantlets of perennial ryegrass (Lolium perenne L.) cultivar ‘Limes’ (DSV Lippstadt/Germany) were clonally propagated in a 90 mm petridish, containing Murashige and Skoog basal medium supplemented with 0.1 mg/l Benzylaminopurine; pH 5.8 and was solidified with 3.0 g/l phytagel (Sigma), at 16° C. during night and 20° C. during the day with 14/12 h light/dark cycle. Light intensity of at least 360 μEm−2s−1 at plant height was maintained with sodium vapor lights (SON-T AGRO 400, Phillips). Axillary buds approximately 4-10 mm in size were excised and placed on callus induction medium. 12 explants were cultured per 90 mm petri-dish at 20 μEm−2s−1 and 25° C. for 28 to 56 days and calli sub-cultured to fresh medium every 14 days.
Biolistic Gene Transfer, Selection and Regeneration of Transgenic Plants
Calli were bombarded with DNA-coated particles six to ten weeks after culture of explants. Four to six hours prior to biolistic gene transfer calli were sub-cultured on medium with additional 64 g.l−1 mannitol and retransferred to mannitol free callus subculture medium after the particle bombardment. Regeneration medium differed from the callus induction medium in the phytohormone composition (no 2,4-D and BAP) and the carbohydrate source and concentration (20 g-l−1 sucrose). Calli were cultured in low light at 20 μE.m−2.s−1 and 24° C. and regenerated initially at 50 μE.m−2.s−1 with a 16 h day, 8 h night cycle at 24° C. Two weeks after transfer to regeneration media light intensity was increased to 130 μE.m−2.s−1 with fluorescent lamps (Philips TL-D 58 W/840R).
The plasmid pJFnpt contains the selectable nptII gene, encoding the enzyme neomycin phosphotransferase II under control of the maize ubiquitin promoter and first intron (Christensen and Quail 1996, Transgenic Res., 5, 213-218). The nptII expression cassette from pJFnpt was inserted into the pPZP 111 vector [Hajdukiewicz et al., 1994, Plant Mol Biol 25 (6) 989-94]. The plasmids pPZP 111, pDORF24, pDORF68, pDORF69, and pDORF9 were isolated as supercoiled DNA using a commercially available DNA Maxiprep Kit (QIAGEN). Vector backbone was removed from both the selectable marker gene expression cassette as well as from the target gene expression cassette by restriction digest, gel electrophoresis and gel purification prior gene transfer. This produced minimal expression cassettes comprising promoter-ORF-terminator for each of the four ORF sequences, and promoter-nptII-terminator from the selectable marker pPZP 111 plasmid. Genetic transformation of perennial ryegrass was essentially carried out as described previously (Altpeter, F., Xu, J. and Ahmed S. 2000, Molecular Breeding, 6, 519-528). In brief, minimal transgene expression cassettes without vector backbone were precipitated on gold particles and delivered to target tissue in a 2:1 molar ratio (target gene expression cassette: selectable marker gene expression cassette) using a DuPont PDS-1000/He (BioRad, USA) device and 1100 psi rupture disks [Sanford et al., 1991 Journal of Methods in Cell and Molecular Biology 3, 3-16]. Particle density was adjusted by the final volume of ethanol in the gold-DNA suspension to 50 μg per bombardment. Five μl of the DNA coated particles were spread on the surface of the microcarrier. Thirty to 35 callus pieces were put in the center of a petridish per bombardment six to ten weeks after callus initiation.
Selection was initiated five to seven days after biolistic gene transfer into calli. Two to three biweekly callus subcultures on CIM medium with 50 mg.l−1 paromomycin were followed by two to three biweekly subcultures on 50 mg 1-1 paromomycin containing SRM medium. Four to eight weeks after transfer of selected calli to light, rooted transgenic plants were screened by performing an ELISA for nptII expression using leaf protein extracts. nptII positive plants were further screened by performing a genomic PCR involving ORF24 or ORF68 specific primers as appropriate. Positive primary transformants were transferred to soil under controlled environment conditions and kept at 15° C./12° C. day/night with a 12 hour photoperiod and 400 μE.m−2.s−1. Illumination was provided by sodium vapor lamps (Philips SON-T AGRO 400) and vegetatively propagated to produce clones of uniform size and growth. RT-PCR was carried out using standard methodology on regenerated plants to determine the transgene expression levels and lines for drought screening were selected based on the transgene expression level.
Transgenic lines transformed with the ryegrass promoter DHN-driven ORF9 cassette excised from pDORF9, used in further experiments included D9-144 and D9-150.
Transgenic lines transformed with the ryegrass promoter DHN-driven ORF24 cassette excised from pDORF24, used in further experiments included D24-106, D24-107, D24-145 and D24-266.
Transgenic lines transformed with the ryegrass promoter DHN-driven ORF68 cassette excised from pDORF68, used in further experiments included D68-135, D68-137, D68-230 and D68-239.
Transgenic lines transformed with the ryegrass promoter DHN-driven ORF69 cassette excised from pDORF69, used in further experiments included D69-270; D69-143 and D69-201.
Example 5
Alteration in Tolerance to Environmental Stress in Plants Transformed with Polynucleotides of the Invention
Drought Screening in Growth Chamber Based Hydroponics System.
Clones of selected lines and a non-transgenic control line were established in a hydroponics system that was set up in a growth chamber. The experimental setup involved four hydroponic tanks, representing eight randomized blocks with 15 plants in each block (10 transgenic lines and 5 wild type clones), totaling 120 plants (80 transgenic lines and 40 wild type clones) (FIG. 21). The transgenic lines were D24-145; D24-266, D9-144; D9-150, D68-135; D68-137; D68-230, D69-270; D69-143 and D69-201. After establishment, the plants were exposed to three rounds of drought-stress (plants lifted up from the hydroponic system) comprising of 19 h drought followed by 11 days of recovery in the first cycle and then by 24 hours of drought and 24 h recovery in the second cycle. The third drought stress was performed after 18 days of recovery from the second drought stress and the plants were drought stressed for 42 hours. Biometric parameters such as Quantum yield of Photosystem II (yield) and Electron Transfer Rate (ETR) were measured using a Pulse Modulated Fluorometer (PAM2000) before the drought stress, at the end of the drought cycle, after 24 hours of recovery and at the end of the recovery period. Each data point in the figure represents the average of 12 measurements (three measurements per plant and four plants per line).
After 19-hours first round of drought-stress, ETR and yield was measured before returning the plants to the hydroponics system for recovery. Line D69-270, line D9-144 and line D68-135 had a significant higher yield and ETR than wildtype. All other transgenic lines were not significantly different than wildtype. Following 24-hours recovery from the 19-hour drought-stress, ETR and yield were measured again. Line D68-135 which performed well during stress did not recover well and performed significantly worse than the wildtype. Line D24-145 recovered significantly better than the wildtype. All other lines, including D69-270 and D9-144 did not show differences from wildtype. After 11 days of recovery, Line D68-135 and line D24-266 did not recover well after the first drought stress and performed significantly worse than the wildtype, while line D68-230 and line D9-150 showed significantly better yields and ETR's than the wildtype, and recovered even to pre-stress values. All other lines did not show significant differences to the wildtype (data not shown).
After the 24-hour second cycle of drought-stress, photosystemII measurements identified lines D24-266 and D69-143 to be performing significantly worse while four lines (D24-145, D68-137, D9-150 and D69-270) were found to perform significantly better than the wildtype. Line D24-266 which already performed badly under stress could not recover well even after 24 hours recovery from 24-hour stress, while line D69-201 performed significantly worse than the wildtype. Lines D68-137, D68-230, D9-144, D9-150 and D69-270 recovered significantly better than the wildtype (FIGS. 22a and 22b).
Yield and ETR were measured after a 24 hours recovery period following the 42-hour third cycle of drought-stress carried out 18 days after the 24-hour drought-stress. Lines D9-144, D9-150, D24-145, D68-230 and D69-270 out performed the wildtype and showed significantly higher yield and ETR than the wildtype while three transgenic lines, D69-143; D68-135 and D24-266, performed poorly (FIGS. 23a and 23b). Following nine days of recovery from the 42-hour drought stress, fresh and dry weights (after 48 h at 80 degrees C.) were evaluated separately for roots and shoots. The five above-mentioned transgenic lines (D9-144, D9-150, D24-145, D68-230 and D69-270) had a significantly higher fresh weight (FIG. 24a) and dry weight (FIG. 24b) than the wildtype. The difference between fresh and dry weight (FIG. 24c) indicates that transgenic lines D9-144, D9-150, D24-145, D68-230 and D69-270 had significantly more hydrated leaves at the time of harvest then the wildtypes. The root dry weight (FIG. 25), for seven (including the five above-mentioned lines) of the ten transgenic lines were significantly higher than the wildtype.
FIG. 26 indicates how viable the wild type plants and transgenic lines D24-145, D9-144 and D9-150 are after three cycles of drought stress as they appeared just after the end of the third drought stress, which lasted for 42 hours.
Drought Screening in SUN-Lit Chambers
Transgenic lines over-expressing ORF 9, ORF24, ORF68 or ORF69 were selected for a detailed physiological analysis in SUN-LIT chambers following their performance in hydroponic culture. Transgenic ryegrass and a wildtype ryegrass (WT) were vegetatively propagated in the greenhouse before transplanting to the SPAR chamber, i.e., D9-144; D9-150; D9-187; D24-106; D24-107; D24-145; D68-137; D68-230; D68-239; D68-296; D69-198; D69-258; D69-306, non-transgenic WT. These lines were randomized in a block design of 4 to 8 replications and grown in three bins (FIG. 27a).
Soil Moisture Monitoring
The soil moisture (VWC, volumetric water content) was recorded with a TDR300 at an interval of two to three days. Measurements were taken in each row between each of the plants at 20 cm depth (there were 28 positions for monitoring soil water status in each chamber). Following the establishment period subsurface irrigation was cut off. Soil moisture content declined and reached volumetric water content (VWC) below 2.3%. A period of no-irrigation was followed by a re-growth period.
Above-Ground Biomass
Leaf clipping dry weight was determined before (>4.3% VWC) and after drought stress (<2.3% VWC). All leaves were cut at 2.5 cm clipping height. The fresh weights (FW) of leaves were measured immediately, then leaves were dried at 80° C. for 48 h and the dry weight (DW) was measured. The difference between fresh weight and dry weight was used as an indicator of early recovery from drought stress. The ability to grow under drought-stress is calculated as percentage of inverse mass loss, which is calculated as the difference of dry weight and dry weight over fresh weight, i.e. (1-[{Fresh weight—Dry weight}/Fresh weight]) %. None of the transgenic lines produced significantly more biomass then the wildtype during the first weeks after establishment (data not shown). However with the onset of drought-stress, line D24-145 (FIG. 27b and FIG. 28c) and lines D68-137 and D68-239 produced significantly more biomass than wildtype. The aboveground biomass produced before drought-stress is shown in FIGS. 28a; 28d; 28g, after drought-stress is shown in FIGS. 28b; 28e; 28h and the ability to grow during drought-stress is shown in FIGS. 28c, 28f and 28i. 
The above examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention.
SUMMARY OF SEQUENCES













SEQ ID NO:
TYPE
SPECIES
REFERENCE


















1
Polynucleotide
Lolium perenne
ORF24 cDNA


2
Polynucleotide
Lolium perenne
ORF24 genomic


3
Polypeptide
Lolium perenne
ORF24


4
Polynucleotide
Lolium perenne
ORF68 cDNA


5
Polynucleotide
Lolium perenne
ORF68 genomic


6
Polypeptide
Lolium perenne
ORF68


7
Polynucleotide
Lolium perenne
ORF69 cDNA


8
Polynucleotide
Lolium perenne
ORF69 genomic


9
Polypeptide
Lolium perenne
ORF69


10
Polynucleotide
Lolium perenne
ORF9 cDNA


11
Polynucleotide
Lolium perenne
ORF9 genomic


12
Polypeptide
Lolium perenne
ORF9


13
Polynucleotide
Triticum aestivum
AF542184


14
Polynucleotide
Oryza sativa
AF190770


15
Polynucleotide
Prunus armeniaca
AF071893


16
Polynucleotide
Fagus sylvatica
AJ606475


17
Polynucleotide
Lycopersicon esculentum
AY044235


18
Polynucleotide
Gossypium barbade
AY572463


19
Polynucleotide
Nicotiana tabacum
AY286010


20
Polynucleotide
Capsicum annuum
AY246274


21
Polynucleotide
Cicer arietinum
AJ515026


22
Polynucleotide
Glycine max
AF537220


23
Polynucleotide
Arabidopsis thaliana
NM 104269


24
Polynucleotide
Zea mays
AY103951


25
Polynucleotide
Triticum monococcum
BQ802250


26
Polypeptide
Triticum aestivum
AF542184


27
Polypeptide
Oryza sativa
AF190770


28
Polypeptide
Prunus armeniaca
AF071893


29
Polypeptide
Fagus sylvatica
AJ606475


30
Polypeptide
Lycopersicon esculentum
AY044235


31
Polypeptide
Gossypium barbade
AY572463


32
Polypeptide
Nicotiana tabacum
AY286010


33
Polypeptide
Capsicum annuum
AY246274


34
Polypeptide
Cicer arietinum
AJ515026


35
Polypeptide
Glycine max
AF537220


36
Polypeptide
Arabidopsis thaliana
NM 104269


37
Polypeptide
Zea mays
AY103951


38
Polypeptide
Triticum monococcum
BQ802250


39
Polynucleotide
Triticum aestivum
dbj BJ259031.1


40
Polynucleotide
Oryza sativa
AP002746


41
Polynucleotide
Gossypium raimondii
gb CO077451.1


42
Polynucleotide
Sorghum bicolor
gb CD225976.1


43
Polynucleotide
Triticum turgidum
emb AJ615010.1


44
Polynucleotide
Glycine soja
gb BM520331.1


45
Polynucleotide
Hordeum vulgare
gb AJ303355


46
Polynucleotide
Medicago truncatula
emb AJ502953.1


47
Polynucleotide
Saccharum officinarum
gb CA132547.1


48
Polynucleotide
Lycopersicon esculentum
gb BG127541.1


49
Polynucleotide
Solanum tuberosum
gb BQ120809.2


50
Polynucleotide
Bruguiera gymnorrhiza
dbj BP950240.1


51
Polynucleotide
Vitis vinifera
gb CF207250.1


52
Polynucleotide
Arabidopsis thaliana
gb AAS09982.1


53
Polynucleotide
Citrus sinensis
gb CF838179.1


54
Polynucleotide
Nicotiana benthamiana
gb CK281819.1


55
Polynucleotide
Glycine max
gb AW570097.1


56
Polynucleotide
Vitis aestivalis
gb CB289054.1


57
Polynucleotide
Brassica napus
gb CD822965.1


58
Polynucleotide
Pinus taeda
gb CF667220.1


59
Polynucleotide
Allium cepa
gb CF436607.1


60
Polynucleotide
Mesembryanthemum crystallinum
gb BE130418.1


61
Polynucleotide
Malus xiaojinensis
gb AY196776


62
Polynucleotide
Zea mays
AY207047


63
Polynucleotide
Hevea brasiliensis
AY712938


64
Polynucleotide
Antirrhinum majus
AY077454


65
Polynucleotide
Cucumis sativus
AJ870304


66
Polynucleotide
Solanum demissum
AC091627


67
Polypeptide
Triticum aestivum
dbj BJ259031.1


68
Polypeptide
Oryza sativa
dbj BAD72233.1


69
Polypeptide
Gossypium raimondii
gb CO077451.1


70
Polypeptide
Sorghum bicolor
gb CD225976.1


71
Polypeptide
Triticum turgidum
emb AJ615010.1


72
Polypeptide
Glycine soja
gb BM520331.1


73
Polypeptide
Hordeum vulgare
emb CAC24845.1


74
Polypeptide
Medicago truncatula
emb AJ502953.1


75
Polypeptide
Saccharum officinarum
gb CA132547.1


76
Polypeptide
Lycopersicon esculentum
gb BG127541.1


77
Polypeptide
Solanum tuberosum
gb BQ120809.2


78
Polypeptide
Bruguiera gymnorrhiza
dbj BP950240.1


79
Polypeptide
Vitis vinifera
gb CF207250.1


80
Polypeptide
Arabidopsis thaliana
gb AAS09982.1


81
Polypeptide
Citrus sinensis
gb CF838179.1


82
Polypeptide
Nicotiana benthamiana
gb CK281819.1


83
Polypeptide
Glycine max
gb AW570097.1


84
Polypeptide
Vitis aestivalis
gb CB289054.1


85
Polypeptide
Brassica napus
gb CD822965.1


86
Polypeptide
Pinus taeda
gb CF667220.1


87
Polypeptide
Allium cepa
gb CF436607.1


88
Polypeptide
Mesembryanthemum crystallinum
gb BE130418.1


89
Polypeptide
Malus xiaojinensis
gb AAO45179.1


90
Polypeptide
Zea mays
gb AAO47339.1


91
Polypeptide
Hevea brasiliensis
gb AAU06309.1


92
Polypeptide
Antirrhinum majus
gb AAL78742.1


93
Polypeptide
Cucumis sativus
emb CAI30890.1


94
Polypeptide
Solanum demissum
gb AAK91894.1


95
Polynucleotide
Oryza sativa
dbj AK070648.1


96
Polynucleotide
Aegilops speltoides
gb BF291316.1


97
Polynucleotide
Triticum aestivum
gb CV782162.1


98
Polynucleotide
Saccharum officinarum
gb CA141214.1


99
Polynucleotide
Dactylis glomerata
gb AY011121.1


100
Polynucleotide
Amborella trichopoda
gb CD484064.1


101
Polynucleotide
Arabidopsis thaliana
emb BX831747.1





CNS0A16X


102
Polynucleotide
Glycine max
gb CO979572.1


103
Polynucleotide
Gossypium raimondii
gb CO092212.1


104
Polynucleotide
Populus tremula x Populus tremuloides
gb BU835049.1


105
Polynucleotide
Malux x domestica
gb CO903744.1


106
Polynucleotide
Solanum tuberosum
gb BG351755.1


107
Polynucleotide
Medicago truncatula
gb CA921208.1


108
Polynucleotide
Ipomoea nil
dbj BJ576569.1


109
Polynucleotide
Hordeum vulgare
gb CB879962.1


110
Polynucleotide
Crocus sativus
gb BM956319.1


111
Polynucleotide
Zea mays
gb BM336249.1


112
Polynucleotide
Sorghum bicolor
gb BE361091.1


113
Polynucleotide
Physcomitrella patens
gb AY077758.1


114
Polynucleotide
Vitis sp
gb CF205668.1


115
Polynucleotide
Picea engelmannii x Picea
gb CO210220.1




sitchensis


116
Polynucleotide
Nicotiana tabacum
dbj AB020023.1


117
Polynucleotide
Liriodendron tulipifera
gb CV000669.1


118
Polynucleotide
Nicotiana benthamiana
gb CK295284.1


119
Polynucleotide
Petroselinum crispum
gb U56834.1





PCU56834


120
Polynucleotide
Asparagus officinalis
gb CV291964.1


121
Polynucleotide
Poncirus trifoliata
gb CV707436.1


122
Polynucleotide
Lotus corniculatus var. japonicus
dbj BP083458.1


123
Polynucleotide
Avena sativa
gb AAD32676.1


124
Polynucleotide
Capsella rubella
gb AAS66778.1


125
Polynucleotide
Oryza sativa subsp. indica
tpg DAA05115.1


126
Polynucleotide
Lycopersicon esculentum
gb BT014501.1


127
Polypeptide
Genus species
dbj AK070648.1


128
Polypeptide
Aegilops speltoides
gb BF291316.1


129
Polypeptide
Triticum aestivum
gb CV782162.1


130
Polypeptide
Saccharum officinarum
gb CA141214.1


131
Polypeptide
Dactylis glomerata
gb AY011121.1


132
Polypeptide
Amborella trichopoda
gb CD484064.1


133
Polypeptide
Arabidopsis thaliana
emb BX831747.1





CNS0A16X


134
Polypeptide
Glycine max
gb CO979572.1


135
Polypeptide
Gossypium raimondii
gb CO092212.1


136
Polypeptide
Populus tremula x Populus
gb BU835049.1




tremuloides


137
Polypeptide
Malux x domestica
gb CO903744.1


138
Polypeptide
Solanum tuberosum
gb BG351755.1


139
Polypeptide
Medicago truncatula
gb CA921208.1


140
Polypeptide
Ipomoea nil
dbj BJ576569.1


141
Polypeptide
Hordeum vulgare
gb CB879962.1


142
Polypeptide
Crocus sativus
gb BM956319.1


143
Polypeptide
Zea mays
gb BM336249.1


144
Polypeptide
Sorghum bicolor
gb BE361091.1


145
Polypeptide
Physcomitrella patens
gb AY077758.1


146
Polypeptide
Vitis sp
gb CF205668.1


147
Polypeptide
Picea engelmannii x Picea
gb CO210220.1




sitchensis


148
Polypeptide
Nicotiana tabacum
dbj AB020023.1


149
Polypeptide
Liriodendron tulipifera
gb CV000669.1


150
Polypeptide
Nicotiana benthamiana
gb CK295284.1


151
Polypeptide
Petroselinum crispum
gb U56834.1





PCU56834


152
Polypeptide
Asparagus officinalis
gb CV291964.1


153
Polypeptide
Poncirus trifoliate
gb CV707436.1


154
Polypeptide
Lotus corniculatus var. japonicus
dbj BP083458.1


155
Polypeptide
Avena sativa
gb AAD32676.1


156
Polypeptide
Capsella rubella
gb AAS66778.1


157
polypeptide
Oryza sativa subsp. indica
tpg DAA05115.1


158
Polypeptide
Lycopersicon esculentum
gb BT014501.1


159
Polynucleotide
Triticum aestivum
gb AAN06944.1


160
Polynucleotide
Oryza sativa
gb AAS72368.1


161
Polynucleotide
Hordeum vulgare
sp Q9ARD5





LT02 HORVU


162
Polynucleotide
Zea mays
gb AY107179.1


163
Polynucleotide
Oryza sativa
dbj AK070872.1


164
Polynucleotide
Oryza sativa
gb AC093089.1


165
Polynucleotide
Oryza sativa
dbj AB030211.1


166
Polynucleotide
Oryza sativa
dbj AK062410.1


167
Polynucleotide
Zea mays
gb AY103848.1


168
Polynucleotide
Zea mays
gb AY108684.1


169
Polynucleotide
Oryza sativa
gb AY554051.1


170
Polynuclotide
Hordeum vulgare
emb AJ310995.1





HVU310995


171
Polynucleotide
Lophopyrum elongatum
gb U00966.1





U00966


172
Polynucleotide
Oryza sativa
gb AY607689.1


173
Polynucleotide
Oryza sativa
ref NM 184595.1


174
Polynucleotide
Zea mays
gb AY105302.1


175
Polynucleotide
Hordeum vulgare
emb AJ310994.1





HVU310994


176
Polynucleotide
Sorghum bicolor
gb CD229091.1





CD229091


177
Polynucleotide
Triticum aestivum
gb CD913648.1





CD913648


178
Polynucleotide
Hordeum vulgare
gb BI780134.2





BI780134


179
Polynucleotide
Zea mays
gb CF042363.1





CF042363


180
Polynucleotide
Oryza sativa
gb CF319949.1





CF319949


181
Polynucleotide
Oryza sativa
ref NM 183567.1


182
Polynucleotide
Hordeum vulgare
emb Z25537.1





HVBLT101


183
Polynucleotide
Arabidopsis thaliana
ref NP 974629.1


184
Polynucleotide
Arabidopsis thaliana
ref NP 194795.1


185
Polynucleotide
Arabidopsis thaliana
ref NP 194794.1


186
Polynucleotide
Brassica rapa
gb AAT11798.1


187
Polynucleotide
Arabidopsis thaliana
ref NP 179982.1


188
Polynucleotide
Arabidopsis thaliana
gb AY060504.1


189
Polynucleotide
Arabidopsis thaliana
emb BX842170.1





CNS09YE9


190
Polynucleotide
Arabidopsis thaliana
emb BX825999.1





CNS0A6CK


191
Polynucleotide
Poncirus trifoliate
gb AY316308.1


192
Polynucleotide
Arabidopsis thaliana
gb AC005770.3


193
Polynucleotide
Arabidopsis thaliana
gb AY084701.1


194
Polynucleotide
Arabidopsis thaliana
ref NM 104551.1


195
Polynucleotide
Solanum tuberosum
dbj AB061265.1


196
Polynucleotide
Mesembryanthemum crystallinum
gb CA836518.1





CA836518


197
Polynucleotide
Citrus sinensis
gb CN183349.1





CN183349


198
Polynucleotide
Populus balsamifera
gb BU870658.1





BU870658


199
Polynucleotide
Physcomitrella patens
gb AAR87655.1


200
Polynucleotide
Physcomitrella patens
gb AY496072.1


201
Polypeptide
Triticum aestivum
gb AAN06944.1


202
Polypeptide
Oryza sativa
gb AAS72368.1


203
Polypeptide
Hordeum vulgare
sp Q9ARD5





LT02 HORVU


204
Polypeptide
Zea mays
gb AY107179.1


205
Polypeptide
Oryza sativa
dbj AK070872.1


206
Polypeptide
Oryza sativa
gb AC093089.1


207
Polypeptide
Oryza sativa
dbj AB030211.1


208
Polypeptide
Oryza sativa
dbj AK062410.1


209
Polypeptide
Zea mays
gb AY103848.1


210
Polypeptide
Zea mays
gb AY108684.1


211
Polypeptide
Oryza sativa
gb AY554051.1


212
Polypeptide
Hordeum vulgare
emb AJ310995.1





HVU310995


213
Polypeptide
Lophopyrum elongatum
gb U00966.1





U00966


214
Polypeptide
Oryza sativa
gb AY607689.1


215
Polypeptide
Oryza sativa
ref NM 184595.1


216
Polypeptide
Zea mays
gb AY105302.1


217
Polypeptide
Hordeum vulgare
emb AJ310994.1





HVU310994


218
Polypeptide
Sorghum bicolor
gb CD229091.1





CD229091


219
Polypeptide
Triticum aestivum
gb CD913648.1





CD913648


220
Polypeptide
Hordeum vulgare
gb BI780134.2





BI780134


221
Polypeptide
Zea mays
gb CF042363.1





CF042363


222
Polypeptide
Oryza sativa
gb CF319949.1





CF319949


223
Polypeptide
Oryza sativa
ref NM 183567.1


224
Polypeptide
Hordeum vulgare
emb Z25537.1





HVBLT101


225
Polypeptide
Arabidopsis thaliana
ref NP 974629.1


226
Polypeptide
Arabidopsis thaliana
ref NP 194795.1


227
Polypeptide
Arabidopsis thaliana
ref NP 194794.1


228
Polypeptide
Brassica rapa
gb AAT11798.1


229
Polypeptide
Arabidopsis thaliana
ref NP 179982.1


230
Polypeptide
Arabidopsis thaliana
gb AY060504.1


231
Polypeptide
Arabidopsis thaliana
emb BX842170.1





CNS09YE9


232
Polypeptide
Arabidopsis thaliana
emb BX825999.1





CNS0A6CK


233
Polypeptide
Poncirus trifoliata
gb AY316308.1


234
Polypeptide
Arabidopsis thaliana
gb AC005770.3


235
Polypeptide
Arabidopsis thaliana
gb AY084701.1


236
Polypeptide
Arabidopsis thaliana
ref NM 104551.1


237
Polypeptide
Solanum tuberosum
dbj AB061265.1


238
Polypeptide
Mesembryanthemum crystallinum
gb CA836518.1





CA836518


239
Polypeptide
Citrus sinensis
gb CN183349.1





CN183349


240
Polypeptide
Populus balsamifera
gb BU870658.1





BU870658


241
Polypeptide
Physcomitrella patens
gb AAR87655.1


242
Polypeptide
Physcomitrella patens
gb AY496072.1


243
Polynucleotide
Lolium perenne
promoter


244
Polynucleotide
vector
ORF24


245
Polynucleotide
vector
ORF68


246
Polynucleotide
vector
ORF69


247
Polynucleotide
vector
ORF9


248
Polypeptide
consensus
ORF24


249
Polypeptide
consensus
ORF68


250
Polypeptide
consensus
ORF69


251
Polypeptide
consensus
ORF9


252
Polynucleotide
vector
ORF24


253
Polynucleotide
vector
ORF68


254
Polynucleotide
vector
ORF69


255
Polynucleotide
vector
ORF9

## Claims

1. An isolated polynucleotide comprising:
a) a sequence encoding a polypeptide with at least 95% identity to the amino acid sequence of SEQ ID NO: 6; or
b) the complement of the sequence of a).

2. The isolated polynucleotide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 249.

3. The isolated polynucleotide of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO:6.

4. The isolated polynucleotide of claim 1, wherein the polypeptide-encoding sequence in a) has at least 95% identity to the sequence of SEQ ID NO: 4 or 5.

5. The isolated polynucleotide of claim 1, wherein the polypeptide-encoding sequence in a) has at least 95% identity to the coding sequence of SEQ ID NO: 4 or 5.

6. The isolated polynucleotide of claim 1, wherein the polypeptide-encoding sequence in a) has the sequence of SEQ ID NO: 4 or 5.

7. The isolated polynucleotide of claim 1, wherein the polypeptide-encoding sequence in a) has the coding sequence of SEQ ID NO: 4 or 5.

8. An isolated polynucleotide comprising:
a) a sequence with at least 95% identity to the nucleotide sequence of SEQ ID NO: 4 or 5; or
b) the complement of the sequence of a).

9. A genetic construct comprising the polynucleotide of claim 1.

10. A host cell comprising the genetic construct of claim 9.

11. A host cell genetically modified to comprise the polynucleotide of claim 1.

12. A plant cell or plant comprising the genetic construct of claim 9.

13. A plant cell or plant genetically modified to comprise the polynucleotide of claim 1.

14. A plant part, propagule, progeny or seed of the plant of claim 13, wherein the plant part, propagule, progeny or seed is genetically modified to comprise a polynucleotide comprising:
a) a sequence encoding a polypeptide with at least 95% identity to the amino acid sequence of SEQ ID NO: 6,; or
b) the complement of the sequence of a).

15. A method of producing a plant cell or plant the method comprising the step of transforming a plant cell or plant with
at least one polynucleotide of claim 1.

16. A plant produced by the method of claim 15.

17. A plant part, propagule, progeny or seed of the plant of claim 16, wherein the plant part, propagule, progeny or seed comprises the at least one polynucleotide of claim 1 used to transform the plant cell or plant.