# Patent

## Title
Transgenic plant with increased stress tolerance and yield

## Abstract

Polynucleotides are disclosed which are capable of enhancing growth, yield under water-limited conditions, and/or increased tolerance to an environmental stress of a plant transformed to contain such polynucleotides. Also provided are methods of using such polynucleotides and transgenic plants and agricultural products, including seeds, containing such polynucleotides as transgenes.

## Background
This application is a national stage application under 35 U.S.C. §371 of PCT/EP2008/053382, filed Mar. 20, 2008, which claims benefit of U.S. provisional application Nos. 60/896,505, filed Mar. 23, 2007. The entire contents of each of the above-identified applications are incorporated herein by reference.
Please add the abstract as set forth in the attached supplemental sheet as a separate page.


FIELD OF THE INVENTION
This invention relates generally to transgenic plants which overexpress nucleic acid sequences encoding polypeptides capable of conferring increased stress tolerance and consequently, increased plant growth and crop yield, under normal or abiotic stress conditions. Additionally, the invention relates to novel isolated nucleic acid sequences encoding polypeptides that confer upon a plant increased tolerance under abiotic stress conditions, and/or increased plant growth and/or increased yield under normal or abiotic stress conditions.
BACKGROUND OF THE INVENTION
Abiotic environmental stresses, such as drought, salinity, heat, and cold, are major limiting factors of plant growth and crop yield. Crop yield is defined herein as the number of bushels of relevant agricultural product (such as grain, forage, or seed) harvested per acre. Crop losses and crop yield losses of major crops such as soybean, rice, maize (corn), cotton, and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many underdeveloped countries.
Water availability is an important aspect of the abiotic stresses and their effects on plant growth. Continuous exposure to drought conditions causes major alterations in the plant metabolism which ultimately lead to cell death and consequently to yield losses. Because high salt content in some soils results in less water being available for cell intake, high salt concentration has an effect on plants similar to the effect of drought on plants. Additionally, under freezing temperatures, plant cells lose water as a result of ice formation within the plant. Accordingly, crop damage from drought, heat, salinity, and cold stress, is predominantly due to dehydration.
Because plants are typically exposed to conditions of reduced water availability during their life cycle, most plants have evolved protective mechanisms against dessication caused by abiotic stresses. However, if the severity and duration of dessication conditions are too great, the effects on development, growth, plant size, and yield of most crop plants are profound. Developing plants efficient in water use is therefore a strategy that has the potential to significantly improve human life on a worldwide scale.
Traditional plant breeding strategies are relatively slow and require abiotic stress-tolerant founder lines for crossing with other germplasm to develop new abiotic stress-resistant lines. Limited germplasm resources for such founder lines and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Breeding for tolerance has been largely unsuccessful.
Many agricultural biotechnology companies have attempted to identify genes that could confer tolerance to abiotic stress responses, in an effort to develop transgenic abiotic stress-tolerant crop plants. Although some genes that are involved in stress responses or water use efficiency in plants have been characterized, the characterization and cloning of plant genes that confer stress tolerance and/or water use efficiency remains largely incomplete and fragmented. To date, success at developing transgenic abiotic stress-tolerant crop plants has been limited, and no such plants have been commercialized.
In order to develop transgenic abiotic stress-tolerant crop plants, it is necessary to assay a number of parameters in model plant systems, greenhouse studies of crop plants, and in field trials. For example, water use efficiency (WUE), is a parameter often correlated with drought tolerance. Studies of a plant's response to desiccation, osmotic shock, and temperature extremes are also employed to determine the plant's tolerance or resistance to abiotic stresses. When testing for the impact of the presence of a transgene on a plant's stress tolerance, the ability to standardize soil properties, temperature, water and nutrient availability and light intensity is an intrinsic advantage of greenhouse or plant growth chamber environments compared to the field.
WUE has been defined and measured in multiple ways. One approach is to calculate the ratio of whole plant dry weight, to the weight of water consumed by the plant throughout its life. Another variation is to use a shorter time interval when biomass accumulation and water use are measured. Yet another approach is to use measurements from restricted parts of the plant, for example, measuring only aerial growth and water use. WUE also has been defined as the ratio of CO2 uptake to water vapor loss from a leaf or portion of a leaf, often measured over a very short time period (e.g. seconds/minutes). The ratio of 13C/12C fixed in plant tissue, and measured with an isotope ratio mass-spectrometer, also has been used to estimate WUE in plants using C3 photosynthesis.
An increase in WUE is informative about the relatively improved efficiency of growth and water consumption, but this information taken alone does not indicate whether one of these two processes has changed or both have changed. In selecting traits for improving crops, an increase in WUE due to a decrease in water use, without a change in growth would have particular merit in an irrigated agricultural system where the water input costs were high. An increase in WUE driven mainly by an increase in growth without a corresponding jump in water use would have applicability to all agricultural systems. In many agricultural systems where water supply is not limiting, an increase in growth, even if it came at the expense of an increase in water use (i.e. no change in WUE), could also increase yield. Therefore, new methods to increase both WUE and biomass accumulation are required to improve agricultural productivity.
Concomitant with measurements of parameters that correlate with abiotic stress tolerance are measurements of parameters that indicate the potential impact of a transgene on crop yield. For forage crops like alfalfa, silage corn, and hay, the plant biomass correlates with the total yield. For grain crops, however, other parameters have been used to estimate yield, such as plant size, as measured by total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, and leaf number. Plant size at an early developmental stage will typically correlate with plant size later in development. A larger plant with a greater leaf area can typically absorb more light and carbon dioxide than a smaller plant and therefore will likely gain a greater weight during the same period. This is in addition to the potential continuation of the micro-environmental or genetic advantage that the plant had to achieve the larger size initially. There is a strong genetic component to plant size and growth rate, and so for a range of diverse genotypes plant size under one environmental condition is likely to correlate with size under another. In this way a standard environment is used to approximate the diverse and dynamic environments encountered at different locations and times by crops in the field.
Harvest index, the ratio of seed yield to above-ground dry weight, is relatively stable under many environmental conditions and so a robust correlation between plant size and grain yield is possible. Plant size and grain yield are intrinsically linked, because the majority of grain biomass is dependent on current or stored photosynthetic productivity by the leaves and stem of the plant. Therefore, selecting for plant size, even at early stages of development, has been used as to screen for plants that may demonstrate increased yield when exposed to field testing. As with abiotic stress tolerance, measurements of plant size in early development, under standardized conditions in a growth chamber or greenhouse, are standard practices to measure potential yield advantages conferred by the presence of a transgene.
There is a need, therefore, to identify additional genes expressed in stress tolerant plants and/or plants that are efficient in water use that have the capacity to confer stress tolerance and/or increased water use efficiency to the host plant and to other plant species. Newly generated stress tolerant plants and/or plants with increased water use efficiency will have many advantages, such as an increased range in which the crop plants can be cultivated, by for example, decreasing the water requirements of a plant species. Other desirable advantages include increased resistance to lodging, the bending of shoots or stems in response to wind, rain, pests, or disease.
SUMMARY OF THE INVENTION
The present inventors have discovered that transforming a plant with certain polynucleotides results in enhancement of the plant's growth and/or response to environmental stress, and accordingly the yield of the agricultural products of the plant is increased, when the polynucleotides are present in the plant as transgenes. The polynucleotides capable of mediating such enhancements have been isolated from Physcomitrella patens, Brassica napus, Zea mays, Linum usitatissimum, Oryza satvia, Glycine max, or Triticum aestivum and are listed in Table 1, and the sequences thereof are set forth in the Sequence Listing as indicated in Table 1.











TABLE 1








Polynucleotide
Amino acid


Gene Name
Gene ID
Organism
SEQ ID NO
SEQ ID NO



















PpTPT-1
EST 214
P. patens
1
2


PpCDC2-1
EST 280
P. patens
3
4


PpLRP-1
EST 298a
P. patens
5
6



EST 298b
P. patens
55
56



EST 298c
P. patens
57
58


PpRBP-1
EST 300
P. patens
7
8


PpPD-1
EST 362
P. patens
9
10


PpMSC-1
EST 378
P. patens
11
12


PpMBP-1
EST 398
P. patens
13
14


PpAK-1
EST 407
P. patens
15
16


PpZF-6
EST 458
P. patens
17
18


PpCDK-1
EST 479
P. patens
19
20


PpZF-7
EST 520
P. patens
21
22


PpMFP-1
EST 544
P. patens
23
24


PpLRP-2
EST 545
P. patens
25
26


PpPPK-1
EST 549
P. patens
27
28


PpSRP-1
EST 554
P. patens
29
30


PpCBL-1
EST 321
P. patens
31
32


PpCBL-2
EST 416
P. patens
33
34


PpHD-1
EST 468
P. patens
35
36


BnHD-1
BN51361834
B. napus
37
38


BnHD-2
BN50000854
B. napus
39
40


ZmHD-1
ZM59324542
Z. mays
41
42


LuHD-1
LU61552369
L. usitatissimum
43
44


OsHD-1
OS34631911
O. sativa
45
46


GmHD-1
GM59700314
G. max
47
48


GmHD-2
GM49753757
G. max
49
50


GmHD-3
GM50270592
G. max
51
52


TaHD-1
TA60089198
T. aestivum
53
54









In one embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a tRNA 2′-phosphotransferase.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a cell division control protein kinase.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a leucine-rich repeat protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a Ran-binding protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a plastid division protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a mitochondrial substrate carrier protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a MADS-box protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polunucleotide encoding an adenosine kinase-1 protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a zinc finger-6 protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a cyclin-dependent kinase regulatory subunit protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a zinc finger-7 protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a MAR-binding protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a leucine rich repeat receptor protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a phytochrome protein kinase protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a synaptobrevin protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a calcineurin B protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a caleosin protein.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a histone deacetylase protein.
In a further embodiment, the invention concerns a seed produced by the transgenic plant of the invention, wherein the seed is true breeding for a transgene comprising the polynucleotide described above. Plants derived from the seed of the invention demonstrate increased tolerance to an environmental stress, and/or increased plant growth, and/or increased yield, under normal or stress conditions as compared to a wild type variety of the plant.
In a still another aspect, the invention concerns products produced by or from the transgenic plants of the invention, their plant parts, or their seeds, such as a foodstuff, feedstuff, food supplement, feed supplement, cosmetic or pharmaceutical.
The invention further provides the isolated polynucleotides identified in Table 1 or in Table 2 below, and polypeptides identified in Table 1. The invention is also embodied in recombinant vector comprising an isolated polynucleotide of the invention.
In yet another embodiment, the invention concerns a method of producing the aforesaid transgenic plant, wherein the method comprises transforming a plant cell with an expression vector comprising an isolated polynucleotide of the invention, and generating from the plant cell a transgenic plant that expresses the polypeptide encoded by thepolynucleotide. Expression of the polypeptide in the plant results in increased tolerance to an environmental stress, and/or growth, and/or yield under normal and/or stress conditions as compared to a wild type variety of the plant.
In still another embodiment, the invention provides a method of increasing a plant's tolerance to an environmental stress, and/or growth, and/or yield. The method comprises the steps of transforming a plant cell with an expression cassette comprising an isolated polynucleotide of the invention, and generating a transgenic plant from the plant cell, wherein the transgenic plant comprises the polynucleotide.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating the phylogenetic relationship among the disclosed PpHD-1 (SEQ ID NO:36), BnHD-1 (SEQ ID NO:38), BnHD-2 (SEQ ID NO:40), ZmHD-1 (SEQ ID NO:42), LuHD-1 (SEQ ID NO:44), OsHD-1 (SEQ ID NO:46), GmHD-1 (SEQ ID NO:48), GmHD-2 (SEQ ID NO:50), GmHD-3 (SEQ ID NO:52), and TaHD-1 (SEQ ID NO:54) amino acid sequences. The diagram was generated using Align X of Vector NTI.
FIG. 2 shows an alignment of the disclosed amino acids sequences: PpHD-1 (SEQ ID NO:36), BnHD-1 (SEQ ID NO:38), BnHD-2 (SEQ ID NO:40), ZmHD-1 (SEQ ID NO:42), LuHD-1 (SEQ ID NO:44), OsHD-1 (SEQ ID NO:46), GmHD-1 (SEQ ID NO:48), GmHD-2 (SEQ ID NO:50), GmHD-3 (SEQ ID NO:52), and TaHD-1 (SEQ ID NO:54). The alignment was generated using Align X of Vector NTI.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. As used herein, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be used.
In one embodiment, the invention provides a transgenic plant that overexpresses an isolated polynucleotide identified in Table 1, or a homolog thereof. The transgenic plant of the invention demonstrates an increased tolerance to an environmental stress as compared to a wild type variety of the plant. The overexpression of such isolated nucleic acids in the plant may optionally result in an increase in plant growth or in yield of associated agricultural products, under normal or stress conditions, as compared to a wild type variety of the plant. Without wishing to be bound by any theory, the increased tolerance to an environmental stress, increased growth, and/or increased yield of a transgenic plant of the invention is believed to result from an increase in water use efficiency of the plant.
As defined herein, a “transgenic plant” is a plant that has been altered using recombinant DNA technology to contain an isolated nucleic acid which would otherwise not be present in the plant. As used herein, the term “plant” includes a whole plant, plant cells, and plant parts. Plant parts include, but are not limited to, stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores, and the like. The transgenic plant of the invention may be male sterile or male fertile, and may further include transgenes other than those that comprise the isolated polynucleotides described herein.
As used herein, the term “variety” refers to a group of plants within a species that share constant characteristics that separate them from the typical form and from other possible varieties within that species. While possessing at least one distinctive trait, a variety is also characterized by some variation between individuals within the variety, based primarily on the Mendelian segregation of traits among the progeny of succeeding generations. A variety is considered “true breeding” for a particular trait if it is genetically homozygous for that trait to the extent that, when the true-breeding variety is self-pollinated, a significant amount of independent segregation of the trait among the progeny is not observed. In the present invention, the trait arises from the transgenic expression of one or more isolated polynucleotides introduced into a plant variety. As also used herein, the term “wild type variety” refers to a group of plants that are analyzed for comparative purposes as a control plant, wherein the wild type variety plant is identical to the transgenic plant (plant transformed with an isolated polynucleotide in accordance with the invention) with the exception that the wild type variety plant has not been transformed to contain an isolated polynucleotide of the invention.
As defined herein, the term “nucleic acid” and “polynucleotide” are interchangeable and refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. An “isolated” nucleic acid molecule is one that is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid (i.e., sequences encoding other polypeptides). For example, a cloned nucleic acid is considered isolated. A nucleic acid is also considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by transformation. Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. While it may optionally encompass untranslated sequence located at both the 3′ and 5′ ends of the coding region of a gene, it may be preferable to remove the sequences which naturally flank the coding region in its naturally occurring replicon.
As used herein, the term “environmental stress” refers to a sub-optimal condition associated with salinity, drought, nitrogen, temperature, metal, chemical, pathogenic, or oxidative stresses, or any combination thereof. The terms “water use efficiency” and “WUE” refer to the amount of organic matter produced by a plant divided by the amount of water used by the plant in producing it, i.e., the dry weight of a plant in relation to the plant's water use. As used herein, the term “dry weight” refers to everything in the plant other than water, and includes, for example, carbohydrates, proteins, oils, and mineral nutrients.
Any plant species may be transformed to create a transgenic plant in accordance with the invention. The transgenic plant of the invention may be a dicotyledonous plant or a monocotyledonous plant. For example and without limitation, transgenic plants of the invention may be derived from any of the following diclotyledonous plant families: Leguminosae, including plants such as pea, alfalfa and soybean; Umbelliferae, including plants such as carrot and celery; Solanaceae, including the plants such as tomato, potato, aubergine, tobacco, and pepper; Cruciferae, particularly the genus Brassica, which includes plant such as oilseed rape, beet, cabbage, cauliflower and broccoli); and Arabidopsis thaliana; Compositae, which includes plants such as lettuce; Malvaceae, which includes cotton; Fabaceae, which includes plants such as peanut, and the like. Transgenic plants of the invention may be derived from monocotyledonous plants, such as, for example, wheat, barley, sorghum, millet, rye, triticale, maize, rice, oats, switchgrass, miscanthus, and sugarcane. Transgenic plants of the invention are also embodied as trees such as apple, pear, quince, plum, cherry, peach, nectarine, apricot, papaya, mango, and other woody species including coniferous and deciduous trees such as poplar, pine, sequoia, cedar, oak, willow, and the like. Especially preferred are Arabidopsis thaliana, Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat, linseed, potato and tagetes.
As shown in Table 1, one embodiment of the invention is a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a tRNA 2′-phosphotransferase polypeptide. In yeast, the RNA 2′-phosphotransferase Tpt1 protein is an essential protein that catalyzes the final step of tRNA splicing. Although this family of proteins is conserved in eukaryotes, bacteria, and archaea, its function has only been well characterized in yeast. tRNA splicing is conserved in all three major kingdoms, but the mechanisms and enzymes involved differ. These differences leave the exact function of RNA 2′-phoshotransferase proteins in plants unclear, although the enzymatic activity has been demonstrated in tobacco nuclear extracts. All of the RNA 2′phosphotransferase family members contain a conserved core domain, exemplified by amino acids 98 to 287 of SEQ ID NO:2, and members from Escherichia coli, Arabidopsis thaliana, Schizosaccharomyces pombe, and Homo sapiens are capable of complementing the Saccharomyces cerevisiae tpt1 mutant, indicating similarity of function.
The transgenic plant of this embodiment may comprise any polynucleotide encoding a tRNA 2′-phosphotransferase. Preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a tRNA 2′-phosphotransferase having a sequence comprising amino acids 98 to 287 of SEQ ID NO:2. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a tRNA 2′-phosphotransferase having a sequence comprising amino acids 1 to 323 of SEQ ID NO:2.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a cell division control 2 (CDC2) protein kinase. The CDC2 proteins belong to a specific family of cyclin-dependent kinases (CDKs) in plants commonly referred to as the CDKA family. All of the CDKA proteins contain a highly conserved core kinase domain with a PSTAIRE motif that is the principle site for cyclin interaction to form active CDK-cyclin complexes. An exemplary PSTAIRE motif is represented as amino acids 4 to 287 of SEQ ID NO:4. The CDKA proteins are also subject to posttranslational modification. Phosphorylation of the conserved threonine 14 and tyrosine 15 positions inactivates the CDKA, and phosphorylation of the conserved threonine 161 position activates the CDKA. In yeast these CDKs are involved specifically in G1/S and G2/M controls. In plants, CDKA's are proposed to function in both S and M phase progression and to be involved in cell proliferation and maintenance of cell division competence in differentiating tissues. In Arabidopsis thaliana for example, a mutation of the CDKA1 gene leads to male gametophytic lethality and impairs seed development by reducing seed size.
The transgenic plant of this embodiment may comprise any polynucleotide encoding a CDC2 protein kinase. Preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a CDKA protein having a sequence comprising amino acids 4 to 287 of SEQ ID NO:4. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a CDKA protein having a sequence comprising amino acids 1 to 294 of SEQ ID NO:4.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a leucine-rich repeat (LRR) protein. LRRs are typically found in proteins as 20 to 29 amino acids repeats, each containing an 11 amino acid conserved region with the consensus sequence of LXXLXLXXN/CXL with X as any amino acid and L as valine, leucine, or phenylalanine. The LRR protein of the present invention contains an LRR represented by amino acids 422 to 441 of SEQ ID NO:6. The generally accepted major function of LLRs is to provide a structural scaffold for the formation of protein-protein interactions. LLR-containing proteins are known to be involved in hormone-receptor interactions, enzyme inhibition, cell adhesion, celluar trafficking, plant disease resistance, and bacterial virulence.
The transgenic plant of this embodiment may comprise any polynucleotide encoding an LRR protein having a sequence comprising amino acids 422 to 441 of SEQ ID NO:6. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a LRR protein having a sequence comprising amino acids 1 to 646 of SEQ ID NO:6.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a Ran-binding protein. Ran GTPase (RanGTP) proteins belong to a subfamily of small GTP-binding proteins that are involved in nucleocytoplasmic transport, and are involved in controlling nuclear functions throughout the cell cycle. The Ran binding proteins 1 (RanBP1s) are cytoplasmic proteins that form a complex with the GTP form of RanGTP. The binding domain of RanBP1 s that interacts with RanGTP has been identified and is represented by amino acids 51 to 172 of SEQ ID NO:8. The formation of this RanGTP-RanBP1 complex is key to promoting the initial dissociation of RanGTP from transport factors that are exported from the nucleus to the cytoplasm.
The transgenic plant of this embodiment may comprise any polynucleotide encoding RanBP1 protein having a sequence comprising amino acids 51 to 172 of SEQ ID NO:8. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a RanBP1 protein having a sequence comprising amino acids 1 to 213 of SEQ ID NO:8.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a plastid division protein. The FtsZ plastid division proteins are characterized by domains represented by amino acids 139 to 332 of SEQ ID NO:10. The transgenic plant of this embodiment may comprise any polynucleotide encoding a plastid division protein having a sequence comprising amino acids 139 to 332 of SEQ ID NO:10. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a plastid division protein having a sequence comprising amino acids 1 to 490 of SEQ ID NO:10.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a mitochondrial substrate carrier protein. The mitochondrial substrate carrier proteins are characterized by domains represented by amino acids 1 to 98 of SEQ ID NO:12. The transgenic plant of this embodiment may comprise any polynucleotide encoding a mitochondrial substrate carrier protein having a sequence comprising amino acids 1 to 98 of SEQ ID NO:12. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a mitochondrial substrate carrier protein having a sequence comprising amino acids 1 to 297 of SEQ ID NO:12.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a MADS-box protein. The DNA binding and dimerization domains of SRF-type transcription factors comprise MADS-box domains represented by amino acids 9 to 59 of SEQ ID NO:14. The transgenic plant of this embodiment may comprise any polynucleotide encoding an SRF-type transcription factor protein comprising a MADS-box domain having a sequence comprising amino acids 9 to 59 of SEQ ID NO:14. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a MADS-box protein having a sequence comprising amino acids 1 to 187 of SEQ ID NO:14.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding an adenosine kinase-1 (ADK-1) protein. The pfkB family of carbohydrate kinases designated ADK-1 comprise domains represented by amino acids 23 to 339 of SEQ ID NO:16. The transgenic plant of this embodiment may comprise any polynucleotide encoding an ADK-1 protein comprising a domain having a sequence comprising amino acids 23 to 339 of SEQ ID NO:16. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding an ADK-1 protein having a sequence comprising amino acids 1 to 343 of SEQ ID NO:16.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a zinc finger-6 (ZF-6) protein. These proteins comprise an IBR domain represented by amino acids 210 to 272 of SEQ ID NO:18. The transgenic plant of this embodiment may comprise any polynucleotide encoding a ZF-6 protein comprising a domain having a sequence comprising amino acids 210 to 272 of SEQ ID NO:18. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a ZF-6 protein having a sequence comprising amino acids 1 to 594 of SEQ ID NO:18.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a cyclin-dependent kinase regulatory subunit (CDK) protein. These proteins comprise a domain represented by amino acids 1 to 72 of SEQ ID NO:20. The transgenic plant of this embodiment may comprise any polynucleotide encoding a CDK protein comprising a domain having a sequence comprising amino acids 1 to 72 of SEQ ID NO:20. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a CDK protein having a sequence comprising amino acids 1 to 91 of SEQ ID NO:20.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a zinc finger-7 (ZF-7) protein. These proteins comprise a C3HC4-type domain represented by amino acids 20 to 60 of SEQ ID NO:22. The transgenic plant of this embodiment may comprise any polynucleotide encoding a ZF-7 protein comprising a domain having a sequence comprising amino acids 20 to 60 of SEQ ID NO:22. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a ZF-7 protein having a sequence comprising amino acids 1 to 347 of SEQ ID NO:22.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a MAR-binding protein. The transgenic plant of this embodiment comprises a polynucleotide encoding a MAR-binding protein having a sequence comprising amino acids 1 to 814 of SEQ ID NO:24.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a leucine rich repeat receptor protein kinase. The LRP-2 protein of the present invention contains several LRRs, represented by amino acids 111 to 133 of SEQ ID NO:26, amino acids 135 to 158 of SEQ ID NO:26, amino acids 160 to 182 of SEQ ID NO:26, and amino acids 184 to 207 of SEQ ID NO:26. The transgenic plant of this embodiment may comprise any polynucleotide encoding an LRP-2 protein having a sequence comprising amino acids 111 to 133 of SEQ ID NO:26, amino acids 135 to 158 of SEQ ID NO:26, amino acids 160 to 182 of SEQ ID NO:26, and amino acids 184 to 207 of SEQ ID NO:26. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a LRR protein having a sequence comprising amino acids 1 to 251 of SEQ ID NO:26.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a phytochrome protein kinase protein. The transgenic plant of this embodiment comprises a polynucleotide encoding a phytochrome protein kinase protein having a sequence comprising amino acids 1 to 689 of SEQ ID NO:28.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a synaptobrevin-related protein.
These proteins comprise a synaptobrevin domain represented by amino acids 127 to 215 of SEQ ID NO:30. The transgenic plant of this embodiment may comprise any polynucleotide encoding a synaptobrevin-related protein comprising a domain having a sequence comprising amino acids 127 to 215 of SEQ ID NO:30. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a synaptobrevin-related protein having a sequence comprising amino acids 1 to 222 of SEQ ID NO:30.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a calcineurin B protein. In plants, a family of proteins has been found that are calcium sensor proteins with similarity to both the regulatory B subunit of calcineurin and neuronal calcium sensors of animals. These proteins have been termed calcineurin B-like proteins (CBL). These CBL proteins contain EF hand motifs that are structurally important for calcium binding and interact specifically with a group of Ser/Thr protein kinases, designated as CBL-interacting protein kinases (CIPK). CIPKs most likely represent targets of calcium sensed and transduced by CBL proteins.
Each EF hand consists of a loop of 12 amino acids flanked by two alpha helices, which binds a single calcium ion via the loop domain. These proteins have also been found to bind magnesium ions. Proteins with four EF hand motifs usually have two structural domains, each formed by a pair of EF hand motifs separated by a flexible linker. Binding of the metal ion to the EF hand protein leads to a conformational change that exposes a hydrophobic surface, which binds to a target sequence. Many EF hand containing proteins also contain a myristoylation site at the N-terminus, with consensus sequence of MGXXXS/T, with X representing any amino acid. Myristoylation at this site promotes protein-protein or protein membrane interaction. This myristoylation site is not present in the EST321 (SEQ ID NO:32) sequence, potentially indicating that the EST321 protein could belong to a different class of EF hand domain containing proteins.
The calcineurin B subunit protein of the present invention contains several EF hand motifs, represented by amino acids 37 to 65 of SEQ ID NO:32, amino acids 106 to 134 of SEQ ID NO:32, and amino acids 142 to 170 of SEQ ID NO:32. The transgenic plant of this embodiment may comprise any polynucleotide encoding a calcineurin B subunit protein having a sequence comprising amino acids 37 to 65 of SEQ ID NO:32, amino acids 106 to 134 of SEQ ID NO:32, and amino acids 142 to 170 of SEQ ID NO:32. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a calcineurin B subunit protein having a sequence comprising amino acids 1 to 182 of SEQ ID NO:32.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a caleosin-related protein. Caleosins are a family of proteins are presumably modulated by calcium-binding and phosphorylation state and are thought to be involved in fusion of membranes and oil bodies. These proteins contain several domains, an N-terminal region with a single calcium ion binding EF-hand motif, a central hydrophobic region with a potential membrane anchor, and a C-terminal region with conserved protein kinase phosphorylation sites. The presence of only a single EF hand motif is unusual for most EF hand containing proteins. It has been postulated that this single EF hand domain may interact with the membrane surface or another protein in order to form the coordinated double EF hand domain interaction found in most other EF hand proteins.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a caleosin-related protein. These proteins comprise a caleosin domain represented by amino acids 26 to 229 of SEQ ID NO:34. The transgenic plant of this embodiment may comprise any polynucleotide encoding a caleosin-related protein comprising a domain having a sequence comprising amino acids 26 to 229 of SEQ ID NO:34. More preferably, the transgenic plant of this embodiment comprises a polynu-cleotide encoding a caleosin-related protein having a sequence comprising amino acids 1 to 239 of SEQ ID NO:34.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a histone deacetylase protein. Nucleosomes consist of histones and DNA, which are essential for packaging DNA into chromosomes. Lysine at the N-terminal ends of core histones are the predominant sites for acetylation and methylation, and histone deacetylases catalyze the removal of the acetyl group from these lysine side chains. Active genes are preferentially associated with highly acetylated histones and inactive genes are associated with hypoacetylated histones. Acetylation results in charge neutralization of histones and weakens histone/DNA contacts. In plants, histone hyperacetylation is correlated with gene activity.
Histones are found to be associated with large multisubunit complexes. Three distinct families of histone deacytelases are found in plants, the RPD3/HDA family, SIR2 family, and the plant specific HD2 family. The RPD3/HDA1 family is found in all eukaryotic organisms, and members possess a complete histone deacetylase domain. Some histone deacetylase proteins possess unique regions outside the histone deacetylase domain that may be important for function and/or specificity of these proteins.
The histone deacetylases of the present invention are characterized by the following domains: from amino acids 6 to 318 of SEQ ID NO:36; from amino acids 6 to 318 of SEQ ID NO:38; from amino acids 20 to 332 of SEQ ID NO:40; from amino acids 8 to 322 of SEQ ID NO:42; from amino acids 6 to 318 of SEQ ID NO:44; from amino acids 23 to 333 of SEQ ID NO:46; from amino acids 8 to 321 of SEQ ID NO:48; from amino acids 6 to 318 of SEQ ID NO:50; from amino acids 56 to 382 of SEQ ID NO:52; and from amino acids 23 to 333 of SEQ ID NO:54.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an polynucleotide encoding a histone deacetylase protein. The transgenic plant of this embodiment may comprise any polynucleotide encoding a histone deacetylase protein comprising a domain having a sequence selected from the group consisting of amino acids 6 to 318 of SEQ ID NO:36; amino acids 6 to 318 of SEQ ID NO:38; amino acids 20 to 332 of SEQ ID NO:40; amino acids 8 to 322 of SEQ ID NO:42; amino acids 6 to 318 of SEQ ID NO:44; amino acids 23 to 333 of SEQ ID NO:46; amino acids 8 to 321 of SEQ ID NO:48; amino acids 6 to 318 of SEQ ID NO:50; amino acids 56 to 382 of SEQ ID NO:52; and amino acids 23 to 333 of SEQ ID NO:54. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a histone deacteylase protein selected from the group consisting of a protein having a sequence comprising amino acids 1 to 431 of SEQ ID NO:36; a protein having a sequence comprising amino acids 1 to 426 of SEQ ID NO:38; a protein having a sequence comprising amino acids 1 to 470 of SEQ ID NO:40; a protein having a sequence comprising amino acids 1 to 363 of SEQ ID NO:42; a protein having a sequence comprising amino acids 1 to 429 of SEQ ID NO:44; a protein having a sequence comprising amino acids 1 to 518 of SEQ ID NO:46; a protein having a sequence comprising amino acids 1 to 334 of SEQ ID NO:48; a protein having a sequence comprising amino acids 1 to 429 of SEQ ID NO:50; a protein having a sequence comprising amino acids 1 to 417 of SEQ ID NO:52; and a protein having a sequence comprising amino acids 1 to 519 of SEQ ID NO:54.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a leucine-rich repeat (LRR) protein. LRRs are typically found in proteins as 20 to 29 amino acids repeats, each containing an 11 amino acid conserved region with the consensus sequence of LXXLXLXXN/CXL with X as any amino acid and L as valine, leucine, or phenylalanine. The LRR protein of the present invention contains an LRR represented by amino acids 422 to 441 of SEQ ID NO:56. The generally accepted major function of LLRs is to provide a structural scaffold for the formation of protein-protein interactions. LLR-containing proteins are known to be involved in hormone-receptor interactions, enzyme inhibition, cell adhesion, celluar trafficking, plant disease resistance, and bacterial virulence.
The transgenic plant of this embodiment may comprise any polynucleotide encoding an LRR protein having a sequence comprising amino acids 422 to 441 of SEQ ID NO:56. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a LRR protein having a sequence comprising amino acids 1 to 698 of SEQ ID NO:56.
In another embodiment, the invention provides a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a leucine-rich repeat (LRR) protein. LRRs are typically found in proteins as 20 to 29 amino acids repeats, each containing an 11 amino acid conserved region with the consensus sequence of LXXLXLXXN/CXL with X as any amino acid and L as valine, leucine, or phenylalanine. The LRR protein of the present invention contains an LRR represented by amino acids 422 to 441 of SEQ ID NO:58. The generally accepted major function of LLRs is to provide a structural scaffold for the formation of protein-protein interactions. LLR-containing proteins are known to be involved in hormone-receptor interactions, enzyme inhibition, cell adhesion, celluar trafficking, plant disease resistance, and bacterial virulence.
The transgenic plant of this embodiment may comprise any polynucleotide encoding an LRR protein having a sequence comprising amino acids 422 to 441 of SEQ ID NO:58. More preferably, the transgenic plant of this embodiment comprises a polynucleotide encoding a LRR protein having a sequence comprising amino acids 1 to 665 of SEQ ID NO:58.
The invention further provides a seed produced by a transgenic plant expressing polynucleotide listed in Table 1, wherein the seed contains the polynucleotide, and wherein the plant is true breeding for increased growth and/or yield under normal and/or stress conditions and/or increased tolerance to an environmental stress as compared to a wild type variety of the plant. The invention also provides a product produced by or from the transgenic plants expressing the polynucleotide, their plant parts, or their seeds. The product can be obtained using various methods well known in the art. As used herein, the word “product” includes, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, cosmetic or pharmaceutical. Foodstuffs are regarded as compositions used for nutrition or for supplementing nutrition. Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs. The invention further provides an agricultural product produced by any of the transgenic plants, plant parts, and plant seeds. Agricultural products include, but are not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
In a preferred embodiment, an isolated polynucleotide of the invention comprises a polynucleotide having a sequence selected from the group consisting of the polynucleotide sequences listed in Table 1. These polynucleotides may comprise sequences of the coding region, as well as 5′ untranslated sequences and 3′ untranslated sequences. Table 2 describes potential start and end positions of the coding regions of the P. patens polynucleotides of the invention, and alternative open reading frames that may be present in the sense or antisense strands of these polynucleotides. Alternatively, the polynucleotides of the invention can comprise only the coding region of the nucleotide sequences listed in Table 1, as indicated in Table 2, or the polynucleotides can contain whole genomic fragments isolated from genomic DNA. Thus the invention is also embodied as an isolated polynucleotide having a sequence selected from the group consisting of the sequences listed in Table 1 or Table 2.














TABLE 2





Gene Name
GENE ID
SEQ ID NO
ORFs
Orf Number
Strand
Start Position
End Position






















PpTPT-1
EST 214
1
1
1
sense
113
1104


PpCDC2-1
EST 280
3
2
1
sense
37
921


PpCDC2-1
EST 280
3
2
2
antisense
380
42


PpLRP-1
EST 298a
5
1
1
sense
144
2084



EST 298b
55
1
1
sense
143
2236



EST 298c
57
1
1
sense
1
1998


PpRBP-1
EST 300
7
1
1
sense
55
696


PpPD-1
EST 362
9
2
1
sense
47
1519


PpPD-1
EST 362
9
2
2
antisense
1197
604


PpMSC-1
EST 378
11
2
1
sense
453
1346


PpMSC-1
EST 378
11
2
2
antisense
1314
1027


PpMBP-1
EST 398
13
1
1
sense
33
878


PpAK-1
EST 407
15
3
1
sense
25
1056


PpAK-1
EST 407
15
3
2
sense
381
632


PpAK-1
EST 407
15
3
3
antisense
506
270


PpZF-6
EST 458
17
1
1
sense
126
1910


PpCDK-1
EST 479
19
2
1
sense
248
523


PpCDK-1
EST 479
19
2
2
antisense
304
104


PpZF-7
EST 520
21
2
1
sense
276
1319


PpZF-7
EST 520
21
2
2
sense
583
813


PpMFP-1
EST 544
23
1
1
sense
127
2571


PpLRP-2
EST 545
25
3
1
sense
225
980


PpLRP-2
EST 545
25
3
2
sense
416
694


PpLRP-2
EST 545
25
3
3
antisense
469
167


PpPPK-1
EST 549
27
1
1
sense
145
2214


PpSRP-1
EST 554
29
1
1
sense
20
688


PpCBL-1
EST 321
31
2
1
sense
43
591


PpCBL-1
EST 321
31
2
2
sense
803
1171


PpCBL-2
EST 416
33
1
1
sense
16
735


PpHD-1
EST 468
35
2
1
sense
166
1461


PpHD-1
EST 468
35
2
2
antisense
420
175









A polynucleotide of the invention can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, P. patens cDNAs of the invention were isolated from a P. patens library using a portion of the sequence disclosed herein. Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon the nucleotide sequence shown in Table 1. A nucleic acid molecule of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecule so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to the nucleotide sequences listed in Table 1 can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
“Homologs” are defined herein as two nucleic acids or polypeptides that have similar, or substantially identical, nucleotide or amino acid sequences, respectively. Homologs include allelic variants, analogs, and orthologs, as defined below. As used herein, the term “analogs” refers to two nucleic acids that have the same or similar function, but that have evolved separately in unrelated organisms. As used herein, the term “orthologs” refers to two nucleic acids from different species, but that have evolved from a common ancestral gene by speciation. The term homolog further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in Table 1 due to degeneracy of the genetic code and thus encode the same polypeptide. As used herein, a “naturally occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural polypeptide).
To determine the percent sequence identity of two amino acid sequences (e.g., one of the polypeptide sequences of Table 1 and a homolog thereof), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide or nucleic acid). The amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence then the molecules are identical at that position. The same type of comparison can be made between two nucleic acid sequences.
Preferably, the isolated amino acid homologs, analogs, and orthologs of the polypeptides of the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more identical to an entire amino acid sequence identified in Table 1. In another preferred embodiment, an isolated nucleic acid homolog of the invention comprises a nucleotide sequence which is at least about 40-60%, preferably at least about 60-70%, more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, or more identical to a nucleotide sequence shown in Table 1 or Table 2.
For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences is determined using the Vector NTI 9.0 (PC) software package (Invitrogen, 1600 Faraday Ave., Carlsbad, Calif. 92008). A gap opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings. For purposes of a multiple alignment (Clustal W algorithm), the gap opening penalty is 10, and the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide is equivalent to a uracil nucleotide.
Nucleic acid molecules corresponding to homologs, analogs, and orthologs of the polypeptides listed in Table 1 can be isolated based on their identity to said polypeptides, using the polynucleotides encoding the respective polypeptides or primers based thereon, as hybridization probes according to standard hybridization techniques under stringent hybridization conditions. As used herein with regard to hybridization for DNA to a DNA blot, the term “stringent conditions” refers to hybridization overnight at 60° C. in 10× Denhart's solution, 6×SSC, 0.5% SDS, and 100 μg/ml denatured salmon sperm DNA. Blots are washed sequentially at 62° C. for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS, and finally 0.1×SSC/0.1% SDS. As also used herein, in a preferred embodiment, the phrase “stringent conditions” refers to hybridization in a 6×SSC solution at 65° C. In another embodiment, “highly stringent conditions” refers to hybridization overnight at 65° C. in 10× Denhart's solution, 6×SSC, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA. Blots are washed sequentially at 65° C. for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS, and finally 0.1×SSC/0.1% SDS. Methods for nucleic acid hybridizations are described in Meinkoth and Wahl, 1984, Anal. Biochem. 138:267-284; well known in the art (see, for example, Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York, 1995; and Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, New York, 1993). Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent or highly stringent conditions to a nucleotide sequence listed in Table 1 corresponds to a naturally occurring nucleic acid molecule.
There are a variety of methods that can be used to produce libraries of potential homologs from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene is then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (See, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983, Nucleic Acid Res. 11:477).
Additionally, optimized nucleic acids can be created. Preferably, an optimized nucleic acid encodes a polypeptide that has a function similar to those of the polypeptides listed in Table 1 and/or modulates a plant's growth and/or yield under normal and/or water-limited conditions and/or tolerance to an environmental stress, and more preferably increases a plant's growth and/or yield under normal and/or water-limited conditions and/or tolerance to an environmental stress upon its overexpression in the plant. As used herein, “optimized” refers to a nucleic acid that is genetically engineered to increase its expression in a given plant or animal. To provide plant optimized nucleic acids, the DNA sequence of the gene can be modified to: 1) comprise codons preferred by highly expressed plant genes; 2) comprise an A+T content in nucleotide base composition to that substantially found in plants; 3) form a plant initiation sequence; 4) to eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites; or 5) elimination of antisense open reading frames. Increased expression of nucleic acids in plants can be achieved by utilizing the distribution frequency of codon usage in plants in general or in a particular plant. Methods for optimizing nucleic acid expression in plants can be found in EPA 0359472; EPA 0385962; PCT Application No. WO 91/16432; U.S. Pat. No. 5,380,831; U.S. Pat. No. 5,436,391; Perlack et al., 1991, Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al., 1989, Nucleic Acids Res. 17:477-498.
An isolated polynucleotide of the invention can be optimized such that its distribution frequency of codon usage deviates, preferably, no more than 25% from that of highly expressed plant genes and, more preferably, no more than about 10%. In addition, consideration is given to the percentage G+C content of the degenerate third base (monocotyledons appear to favor G+C in this position, whereas dicotyledons do not). It is also recognized that the XCG (where X is A, T, C, or G) nucleotide is the least preferred codon in dicots, whereas the XTA codon is avoided in both monocots and dicots. Optimized nucleic acids of this invention also preferably have CG and TA doublet avoidance indices closely approximating those of the chosen host plant. More preferably, these indices deviate from that of the host by no more than about 10-15%.
The invention further provides an isolated recombinant expression vector comprising a polynucleotide as described above, wherein expression of the vector in a host cell results in the plant's increased growth and/or yield under normal or water-limited conditions and/or increased tolerance to environmental stress as compared to a wild type variety of the host cell. The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. As used herein with respect to a recombinant expression vector, “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in a bacterial or plant host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides encoded by nucleic acids as described herein.
Plant gene expression should be operatively linked to an appropriate promoter conferring gene expression in a timely, cell specific, or tissue specific manner. Promoters useful in the expression cassettes of the invention include any promoter that is capable of initiating transcription in a plant cell. Such promoters include, but are not limited to, those that can be obtained from plants, plant viruses, and bacteria that contain genes that are expressed in plants, such as Agrobacterium and Rhizobium.
The promoter may be constitutive, inducible, developmental stage-preferred, cell type-preferred, tissue-preferred, or organ-preferred. Constitutive promoters are active under most conditions. Examples of constitutive promoters include the CaMV 19S and 35S promoters (Odell et al., 1985, Nature 313:810-812), the sX CaMV 35S promoter (Kay et al., 1987, Science 236:1299-1302) the Sep1 promoter, the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171), the Arabidopsis actin promoter, the ubiquitan promoter (Christensen et al., 1989, Plant Molec. Biol. 18:675-689), pEmu (Last et al., 1991, Theor. Appl. Genet. 81:581-588), the figwort mosaic virus 35S promoter, the Smas promoter (Velten et al., 1984, EMBO J 3:2723-2730), the super promoter (U.S. Pat. No. 5,955,646), the GRP1-8 promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), promoters from the T-DNA of Agrobacterium, such as mannopine synthase, nopaline synthase, and octopine synthase, the small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter, and the like.
Inducible promoters are preferentially active under certain environmental conditions, such as the presence or absence of a nutrient or metabolite, heat or cold, light, pathogen attack, anaerobic conditions, and the like. For example, the hsp80 promoter from Brassica is induced by heat shock; the PPDK promoter is induced by light; the PR-1 promoters from tobacco, Arabi-dopsis, and maize are inducible by infection with a pathogen; and the Adh1 promoter is induced by hypoxia and cold stress. Plant gene expression can also be facilitated via an inducible pro-moter (For a review, see Gatz, 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters are especially suitable if gene expression is wanted to occur in a time specific manner. Examples of such promoters are a salicylic acid inducible promoter (PCT Application No. WO 95/19443), a tetracycline inducible promoter (Gatz et al., 1992, Plant J. 2: 397-404), and an ethanol inducible promoter (PCT Application No. WO 93/21334).
In one preferred embodiment of the present invention, the inducible promoter is a stress-inducible promoter. For the purposes of the invention, stress-inducible promoters are preferentially active under one or more of the following stresses: sub-optimal conditions associated with salinity, drought, nitrogen, temperature, metal, chemical, pathogenic, and oxidative stresses. Stress inducible promoters include, but are not limited to, Cor78 (Chak et al., 2000, Planta 210:875-883; Hovath et al., 1993, Plant Physiol. 103:1047-1053), Cor15a (Artus et al., 1996, PNAS 93(23):13404-09), Rci2A (Medina et al., 2001, Plant Physiol. 125:1655-66; Nylander et al., 2001, Plant Mol. Biol. 45:341-52; Navarre and Goffeau, 2000, EMBO J. 19:2515-24; Capel et al., 1997, Plant Physiol. 115:569-76), Rd22 (Xiong et al., 2001, Plant Cell 13:2063-83; Abe et al., 1997, Plant Cell 9:1859-68; Iwasaki et al., 1995, Mol. Gen. Genet. 247:391-8), cDet6 (Lang and Palve, 1992, Plant Mol. Biol. 20:951-62), ADH1 (Hoeren et al., 1998, Genetics 149:479-90), KAT1 (Nakamura et al., 1995, Plant Physiol. 109:371-4), KST1 (Müller-Röber et al., 1995, EMBO 14:2409-16), Rha1 (Terryn et al., 1993, Plant Cell 5:1761-9; Terryn et al., 1992, FEBS Lett. 299(3):287-90), ARSK1 (Atkinson et al., 1997, GenBank Accession #L22302, and PCT Application No. WO 97/20057), PtxA (Plesch et al., GenBank Accession #X67427), SbHRGP3 (Ahn et al., 1996, Plant Cell 8:1477-90), GH3 (Liu et al., 1994, Plant Cell 6:645-57), the pathogen inducible PRP1-gene promoter (Ward et al., 1993, Plant. Mol. Biol. 22:361-366), the heat inducible hsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold inducible alpha-amylase promoter from potato (PCT Application No. WO 96/12814), or the wound-inducible pinII-promoter (European Patent No. 375091). For other examples of drought, cold, and salt-inducible promoters, such as the RD29A promoter, see Yamaguchi-Shinozalei et al., 1993, Mol. Gen. Genet. 236:331-340.
Developmental stage-preferred promoters are preferentially expressed at certain stages of development. Tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem. Examples of tissue-preferred and organ-preferred promoters include, but are not limited to fruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred, integument-preferred, tuber-preferred, stalk-preferred, pericarp-preferred, leaf-preferred, stigma-preferred, pollen-preferred, anther-preferred, petal-preferred, sepal-preferred, pedicel-preferred, silique-preferred, stem-preferred, root-preferred promoters, and the like. Seed-preferred promoters are preferentially expressed during seed development and/or germination. For example, seed-preferred promoters can be embryo-preferred, endosperm-preferred, and seed coat-preferred (See Thompson et al., 1989, BioEssays 10:108). Examples of seed-preferred promoters include, but are not limited to, cellulose synthase (celA), Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1), and the like.
Other suitable tissue-preferred or organ-preferred promoters include the napin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Bae-umlein et al., 1991, Mol. Gen. Genet. 225(3): 459-67), the oleosin-promoter from Arabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoter from Brassica (PCT Application No. WO 91/13980), or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2): 233-9), as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice, etc. Suitable promoters to note are the Ipt2 or Ipt1-gene promoter from barley (PCT Application No. WO 95/15389 and PCT Application No. WO 95/23230) or those described in PCT Application No. WO 99/16890 (promoters from the barley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene, and rye secalin gene).
Other promoters useful in the expression cassettes of the invention include, but are not limited to, the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the β-conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15 kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2, and bronze promoters, the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonase promoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546), and the SGB6 promoter (U.S. Pat. No. 5,470,359), as well as synthetic or other natural promoters.
Additional flexibility in controlling heterologous gene expression in plants may be obtained by using DNA binding domains and response elements from heterologous sources (i.e., DNA binding domains from non-plant sources). An example of such a heterologous DNA binding domain is the LexA DNA binding domain (Brent and Ptashne, 1985, Cell 43:729-736).
In a preferred embodiment of the present invention, the polynucleotides listed in Table 1 are expressed in plant cells from higher plants (e.g., the spermatophytes, such as crop plants). A polynucleotide may be “introduced” into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection, and the like. Suitable methods for transforming or transfecting plant cells are disclosed, for example, using particle bombardment as set forth in U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,302,523; 5,464,765; 5,120,657; 6,084,154; and the like. More preferably, the transgenic corn seed of the invention may be made using Agrobacterium transformation, as described in U.S. Pat. Nos. 5,591,616; 5,731,179; 5,981,840; 5,990,387; 6,162,965; 6,420,630, U.S. patent application publication number 2002/0104132, and the like. Transformation of soybean can be performed using for example a technique described in European Patent No. EP 0424047, U.S. Pat. No. 5,322,783, European Patent No. EP 0397 687, U.S. Pat. No. 5,376,543, or U.S. Pat. No. 5,169,770. A specific example of wheat transformation can be found in PCT Application No. WO 93/07256. Cotton may be transformed using methods disclosed in U.S. Pat. Nos. 5,004,863; 5,159,135; 5,846,797, and the like. Rice may be transformed using methods disclosed in U.S. Pat. Nos. 4,666,844; 5,350,688; 6,153,813; 6,333,449; 6,288,312; 6,365,807; 6,329,571, and the like. Other plant transformation methods are disclosed, for example, in U.S. Pat. Nos. 5,932,782; 6,153,811; 6,140,553; 5,969,213; 6,020,539, and the like. Any plant transformation method suitable for inserting a transgene into a particular plant may be used in accordance with the invention.
According to the present invention, the introduced polynucleotide may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes. Alternatively, the introduced polynucleotide may be present on an extra-chromosomal non-replicating vector and may be transiently expressed or transiently active.
Another aspect of the invention pertains to an isolated polypeptide having a sequence selected from the group consisting of the polypeptide sequences listed in Table 1. An “isolated” or “purified” polypeptide is free of some of the cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of a polypeptide in which the polypeptide is separated from some of the cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of a polypeptide of the invention having less than about 30% (by dry weight) of contaminating polypeptides, more preferably less than about 20% of contaminating polypeptides, still more preferably less than about 10% of contaminating polypeptides, and most preferably less than about 5% contaminating polypeptides.
The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one skilled in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities are abundant and well known to one skilled in the art.
The invention is also embodied in a method of producing a transgenic plant comprising at least one polynucleotide listed in Table 1 or Table 2, wherein expression of the polynucleotide in the plant results in the plant's increased growth and/or yield under normal and/or water-limited conditions and/or increased tolerance to an environmental stress as compared to a wild type variety of the plant comprising the steps of: (a) introducing into a plant cell an expression vector comprising at least one polynucleotide listed in Table 1 or Table 2, and (b) generating from the plant cell a transgenic plant that expresses the polynucleotide, wherein expression of the polynucleotide in the transgenic plant results in the plant's increased growth and/or yield under normal or water-limited conditions and/or increased tolerance to environmental stress as compared to a wild type variety of the plant. The plant cell may be, but is not limited to, a protoplast, gamete producing cell, and a cell that regenerates into a whole plant. As used herein, the term “transgenic” refers to any plant, plant cell, callus, plant tissue, or plant part, that contains at least one recombinant polynucleotide listed in Table 1 or Table 2. In many cases, the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.
The present invention also provides a method of increasing a plant's growth and/or yield under normal and/or water-limited conditions and/or increasing a plant's tolerance to an environmental stress comprising the steps of increasing the expression of at least one polynucleotide listed in Table 1 or Table 2 in the plant. Expression of a polynucleotide listed in Table 1 or Table 2 can be increased by any method known to those of skill in the art.
The effect of the genetic modification on plant growth and/or yield and/or stress tolerance can be assessed by growing the modified plant under normal and/or less than suitable conditions and then analyzing the growth characteristics and/or metabolism of the plant. Such analysis techniques are well known to one skilled in the art, and include dry weight, wet weight, polypeptide synthesis, carbohydrate synthesis, lipid synthesis, evapotranspiration rates, general plant and/or crop yield, flowering, reproduction, seed setting, root growth, respiration rates, photosynthesis rates, metabolite composition, etc., using methods known to those of skill in bio-technology.
The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof.
EXAMPLE 1
Identification of P. patens Open Reading Frames
cDNA libraries made from plants of the species P. patens (Hedw.) B.S.G. from the collection of the genetic studies section of the University of Hamburg were sequences using standard methods. The plants originated from the strain 16/14 collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire (England), which was subcultured from a spore by Engel (1968, Am. J. Bot. 55:438-446).
Sequences were processed and annotated using the software package EST-MAX commercially provided by Bio-Max (Munich, Germany). The program incorporates practically all bioinformatics methods important for functional and structural characterization of protein sequences. For reference, see the website at pedant.mips.biochem.mpg.de. The most important algorithms incorporated in EST-MAX are: FASTA (Very sensitive sequence database searches with estimates of statistical significance; Pearson, 1990, Methods Enzymol. 183:63-98); BLAST (Very sensitive sequence database searches with estimates of statistical significance; Altschul et al., 1990, Journal of Molecular Biology 215:403-10); PREDATOR (High-accuracy secondary structure prediction from single and multiple sequences; Frishman and Argos, 1997, Proteins 27:329-335); CLUSTALW (Multiple sequence alignment; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680); TMAP (Transmembrane region prediction from multiply aligned sequences; Persson and Argos, 1994, J. Mol. Biol. 237:182-192); ALOM2 (Transmembrane region prediction from single sequences; Klein et al., 1984, Biochim. Biophys. Acta 787:221-226. Version 2 by Dr. K. Nakai); PROSEARCH (Detection of PROSITE protein sequence patterns; Kolakowski et al., 1992, Biotechniques 13, 919-921); BLIMPS (Similarity searches against a database of ungapped blocks, Wallace and Henikoff, 1992, Comput Appl Biosci. 8(3):249-54); PATMAT (a searching and extraction program for sequence, pattern and block queries and databases, CABIOS 8:249-254. Written by Bill Alford).
P. patens partial cDNAs (ESTs) were identified in the P. patens EST sequencing program using the program EST-MAX through BLAST analysis. The full-length nucleotide cDNA sequences were determined using known methods. The identity and similarity of the amino acid sequences of the disclosed polypeptide sequences to known protein sequences are shown in Tables 2-19 (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum 62).







TABLE 3







Comparison of PpTPT-1 (SEQ ID NO: 2)


to known RNA 2′ phosphotransferases










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)













XP_550615
O. sativa
45.4
56.2


NP_197750
A. thaliana
34.1
43.0


NP_182058
A. thaliana
39.6
48.9


NP_594515
Schizosaccharomyces
21.3
29.1



pombe


NP_788477
Drosophila
25.5
34.7



melanogaster
















TABLE 4







Comparison of PpCDC2-1 (SEQ ID NO: 4) to


known Cell Division Control Protein 2










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





S42049
Picea abies
92.9
96.3


Q40790
Pinus contorta
92.5
96.3


CDC2_CHERU
Chenopodium
91.5
95.9



rubrum


Q9AUH4
Populus tremula ×
90.5
95.9



P. tremuloides


Q8W2D3
Helianthus annuus
89.5
95.2
















TABLE 5







Comparison of PpLRP-1 (SEQ ID NO: 6) to


known Leucine Rich Repeat Family Proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





NP_912570
O. sativa
27.6
42.1


NP_921136
O. sativa
27.2
40.3


AAF71805
A. thaliana
24.4
33.6


NP_177947
A. thaliana
28.1
38.5


G96811
A. thaliana
28.8
40.3
















TABLE 6







Comparison of PpRBP-1 (SEQ ID NO:


8) to known Ran binding protein 1










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





Q94K24
Lycopersicon
50.7
63.3



esculentum


Q9LUZ8
A. thaliana
39.4
51.9


NP_200667
A. thaliana
44.3
58.3


O04149
A. thaliana
44.1
55.1


NP_172194
A thaliana
44.4
55.1
















TABLE 7







Comparison of PpPD-1 (SEQ ID NO: 10) to


known Plastid division ftsZ proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)













Q70ZZ6
P. patens
100
100


Q75ZR3
Nannochloris
42.6
53.0



bacillaris


ZP_00177632
Crocosphaera watsonii
41.3
51.8


NP_440816
Synechocystis sp.
41.0
52.1


T51092
Synechocystis sp.
40.0
51.5
















TABLE 8







Comparison of PpMSC-1 (SEQ ID NO: 12) to known


Mitochondiral substrate carrier proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





NP_194188
A. thaliana
56.6
70.0


T05577
A. thaliana
56.7
69.8


Q66PX4
Saccharum
55.4
69.9



officinarum


NP_179836
A. thaliana
54.3
71.4


D84613
A. thaliana
54.5
71.2
















TABLE 9







Comparison of PpMBP-1 (SEQ ID NO:


14) to known MADS-box proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





Q8LPA5
P. patens
63.0
63.0


Q6QAF0
P. patens
62.6
62.6


Q9FE71
P. patens
53.4
55.8


Q9FE89
P. patens
49.1
53.7


Q8LLC8
Lycopodium
46.1
59.5



annotinum
















TABLE 10







Comparison of PpAK-1 (SEQ ID NO: 16)


to known Adenosine kinase proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)













ADK_PHYPA
P. patens
100
100


NP_195950
A. thaliana
66.3
77.8


XP_466836
O. sativa
68.2
77.6


Q84P58
O. sativa
62.9
71.5


NP_187593
A. thaliana
64.7
76.6
















TABLE 11







Comparison of PpZF-6 (SEQ ID NO:


18) to known zinc finger proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





NP_180709
A. thaliana
61.1
71.0


XP_472997
O. sativa
63.4
72.1


NP_176722
A. thaliana
61.8
72.4


T02366
A. thaliana
54.7
64.1


NP_172080
A. thaliana
58.9
69.0
















TABLE 12







Comparison of PpCDK-1 (SEQ ID NO: 20) to known


Cyclin-dependent kinase regulatory subunits










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





Q6JJ57
Ipomoea
76.9
83.5



trifida


Q6T300
G. max
79.1
82.4


NP_180364
A. thaliana
74.7
82.4


XP_470214
O. sativa
80.2
84.6


Q8GZU5
Populus tremula ×
75.8
80.2



P. tremuloides
















TABLE 13







Comparison of PpZF-7 (SEQ ID NO: 22)


to known RING zinc finger proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





Q6F3A0
O. sativa
34.7
47.3


Q852N7
O. sativa
35.5
48.5


Q7XJB5
O. sativa
33.1
45.9


NP_851050
A. thaliana
35.3
46.2


NP_851051
A. thaliana
35.3
46.2
















TABLE 14







Comparison of PpMFP-1 (SEQ ID NO: 24) to


known MAR binding filament-like protein 1










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





MFP1_TOBAC
Nicotiana tabacum
18.0
30.5


NP_914440
O. sativa
16.8
30.1


MFP1_ARATH
A. thaliana
19.2
30.3


NP_188221
A. thaliana
20.1
31.1


T07111
Lycopersicon
19.6
31.6



esculentum
















TABLE 15







Comparison of PpLRP-2 (SEQ ID NO: 26) to known Leucine


rich repeat receptor-like protein kinases










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)













Q9XGG1
Sorghum bicolor
9.3
16.1


NP_189183
A. thaliana
9.2
14.6


Q708X5
Cicer arietinum
18.0
28.6


XP_474976
O. sativa
5.9
10.3


Q9LSU7
A. thaliana
9.2
14.0
















TABLE 16







Comparison of PpPPK-1 (SEQ ID NO: 28)


to known light-sensor protein kinases










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)













S27396
Ceratodon purpureus
5.7
11.4


P93098
Ceratodon purpureus
5.8
11.5


PHY1_CERPU
Ceratodon purpureus
5.7
11.4


NP_564829
A. thaliana
19.0
32.1


H96666
A. thaliana
19.5
31.6
















TABLE 17







Comparison of PpSRP-1 (SEQ ID NO: 30)


to Synaptobrevin-related proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





NP_180871
A. thaliana
73.0
84.2


Q7X9C5
Pyrus pyrifolia
64.9
75.2


NP_180826
A. thaliana
55.4
63.8


Q681H0
A. thaliana
71.2
83.8
















TABLE 18







Comparison of PpCBL-1 (SEQ ID NO:


32) to known Calcineurin B proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





XP_3133573
Anopheles gambiae
19.1
36.7


NP_505885
Caenorhabditis
16.6
34.8



elegans


Q95P81
Bombyx mori
18.7
36.4


NP_524874
D. melanogaster
17.1
35.3
















TABLE 19







Comparison of PpCBL-2 (SEQ ID NO: 34)


to known caleosin-related proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





Q9SQ57
Sesamum indicum
64.9
78.4


T07092
G. max
67.5
74.6


NP_194404
A. thaliana
61.2
74.7


NP_200335
A. thaliana
62.1
72.8


XP_473140
O. sativa
60.2
73.0
















TABLE 20







Comparison of PpHD-1 (SEQ ID NO: 36)


to known histone deacetylase proteins










Public Database

Sequence
Sequence


Accession #
Species
Identity (%)
Similarity (%)





Q8W508
Zea mays
81.9
88.6


NP_190054
A. thaliana
78.7
88.2


T47443
A. thaliana
76.1
85.8


Q6JJ24
Ipomoea trifida
68.2
75.6


Q7XLX3
Oryza sativa
70.1
77.5









EXAMPLE 2
Cloning of Full-Length cDNAs from Other Plants
Canola, soybean, rice, maize, linseed, and wheat plants were grown under a variety of conditions and treatments, and different tissues were harvested at various developmental stages. Plant growth and harvesting were done in a strategic manner such that the probability of harvesting all expressable genes in at least one or more of the resulting libraries is maximized. The mRNA was isolated from each of the collected samples, and cDNA libraries were constructed. No amplification steps were used in the library production process in order to minimize redundancy of genes within the sample and to retain expression information. All libraries were 3′ generated from mRNA purified on oligo dT columns. Colonies from the transformation of the cDNA library into E. coli were randomly picked and placed into microtiter plates.
Plasmid DNA was isolated from the E. coli colonies and then spotted on membranes. A battery of 288 33P radiolabeled 7-mer oligonucleotides were sequentially hybridized to these membranes. To increase throughput, duplicate membranes were processed. After each hybridization, a blot image was captured during a phosphorimage scan to generate a hybridization profile for each oligonucleotide. This raw data image was automatically transferred to a computer. Absolute identity was maintained by barcoding for the image cassette, filter, and orientation within the cassette. The filters were then treated using relatively mild conditions to strip the bound probes and returned to the hybridization chambers for another round of hybridization. The hybridization and imaging cycle was repeated until the set of 288 oligomers was completed.
After completion of the hybridizations, a profile was generated for each spot (representing a cDNA insert), as to which of the 288 33P radiolabeled 7-mer oligonucleotides bound to that particular spot (cDNA insert), and to what degree. This profile is defined as the signature generated from that clone. Each clone's signature was compared with all other signatures generated from the same organism to identify clusters of related signatures. This process “sorts” all of the clones from an organism into clusters before sequencing.
The clones were sorted into various clusters based on their having identical or similar hybridization signatures. A cluster should be indicative of the expression of an individual gene or gene family. A by-product of this analysis is an expression profile for the abundance of each gene in a particular library. One-path sequencing from the 5′ end was used to predict the function of the particular clones by similarity and motif searches in sequence databases.
The full-length DNA sequence of the P. patens PpHD-1 (SEQ ID NO:35) was blasted against proprietary databases of canola, soybean, rice, maize, linseed, and wheat cDNAS at an e value of e−10 (Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). All the contig hits were analyzed for the putative full length sequences, and the longest clones representing the putative full length contigs were fully sequenced. Two homologs from canola (BnHD-1, SEQ ID NO:38 and BnHD-2, SEQ ID NO:40), one homolog from maize (ZmHD-1, SEQ ID NO:42), one homolog from linseed (LuHD-1, SEQ ID NO:44), one sequence from rice (OsHD-1, SEQ ID NO:46) three sequences from soybean (GmHD-1, SEQ ID NO:48, GmHD-2, SEQ ID NO:50, and GmHD-3, SEQ ID NO:52) and one sequence from wheat (TaHD-1, SEQ ID NO:54) were identified. The degree of amino acid identity and similarity of these sequences to the closest known public sequence is indicated in Table 21 (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62).







TABLE 21







Degree of Amino Acid Identity and Similarity of Histone Deacetylases











Gene Name
Public Database

Sequence
Sequence


(SEQ ID NO)
Accession #
Species
Identity (%)
Similarity (%)





BnHD-1
NP_190054
A. thaliana
  96%
98.1%


(SEQ ID NO: 38)


BnHD-2
NP_201116
A. thaliana
92.6%
95.3%


(SEQ ID NO: 40)


ZmHD-1
NP_563817
A. thaliana
66.4%
77.2%


(SEQ ID NO: 42)


LuHD-1
NP_190054
A. thaliana
87.4%
94.4%


(SEQ ID NO: 44)


OsHD-1
Q7Y0Y8
O. sativa
 100%
 100%


(SEQ ID NO: 46)


GmHD-1
NP_563817
A. thaliana
63.1%
  74%


(SEQ ID NO: 48)


GmHD-2
NP_190054
A. thaliana
85.6%
92.1%


(SEQ ID NO: 50)


GmHD-2
NP_567921
A. thaliana
67.2%
77.4%


(SEQ ID NO: 52)


TaHD-1
Q7Y0Y8
O. sativa
89.4%
93.8%


(SEQ ID NO: 54)









EXAMPLE 3
Stress-Tolerant Arabidopsis Plants
A fragment containing the P. patens polynucleotide was ligated into a binary vector containing a selectable marker gene. The resulting recombinant vector contained the corresponding polynucleotide listed in Table 1 in the sense orientation under the constitutive super promoter. The recombinant vectors were transformed into Agrobacterium tumefaciens C58C1 and PMP90 plants according to standard conditions. A. thaliana ecotype C24 plants were grown and transformed according to standard conditions. T1 plants were screened for resistance to the selection agent conferred by the selectable marker gene, and T1 seeds were collected.
The P. patens polynucleotides were overexpressed in A. thaliana under the control of a constitutive promoter. T2 and/or T3 seeds were screened for resistance to the selection agent conferred by the selectable marker gene on plates, and positive plants were transplanted into soil and grown in a growth chamber for 3 weeks. Soil moisture was maintained throughout this time at approximately 50% of the maximum water-holding capacity of soil.
The total water lost (transpiration) by the plant during this time was measured. After 3 weeks, the entire above-ground plant material was collected, dried at 65° C. for 2 days and weighed. The ratio of above-ground plant dry weight (DW) to plant water use is water use efficiency (WUE). Tables 22-41 present WUE and DW for independent transformation events (lines) of transgenic plants overexpressing the P. patens polynucleotides. Least square means (LSM), standard errors, and significant value (P) of a line compared to wild-type controls from an Analysis of Variance are presented. The percent improvement of each P. patens polynucleotides line as compared to wild-type control plants for WUE and DW is also presented.







TABLE 22







A. thaliana lines overexpressing PpTPT-1 (SEQ ID NO: 2)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P
















DW
Wild-type

0.136
0.011
—
—



PpTPT-1
8
0.217
0.025
60
0.0033



(SEQ ID
6
0.222
0.028
64
0.0047



NO: 2)
5
0.226
0.032
67
0.0085




10
0.228
0.025
68
0.0009




2
0.230
0.032
70
0.0063


WUE
Wild-type

2.270
0.085
—
—



PpTPT-1
8
2.274
0.190
0
0.9822



(SEQ ID
6
2.308
0.212
2
0.8656



NO: 2)
2
2.426
0.245
7
0.5465




5
2.675
0.245
18
0.1207
















TABLE 23







A. thaliana lines overexpressing PpCDC2-1 (SEQ ID NO: 4)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.088
0.009
—
—



PpCDC2-
4
0.117
0.016
33
0.1147



1
1
0.125
0.016
42
0.0473



(SEQ ID



NO: 4)


WUE
Wild-type

1.446
0.097
—
—



PpCDC2-
4
1.890
0.186
31
0.036 



1
1
1.947
0.186
35
0.0183



(SEQ ID



NO: 4)
















TABLE 24







A. thaliana lines overexpressing PpLRP-1 (SEQ ID NO: 6)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.109
0.033
—
—



PpLRP-1
10
0.161
0.034
48
0.2835



(SEQ ID



NO: 6)


WUE
Wild-type

1.782
0.119
—
—



PpLRP-1
10
2.205
0.169
24
0.0529



(SEQ ID



NO: 6)
















TABLE 25







A. thaliana lines overexpressing PpRBP-1 (SEQ ID NO: 8)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.088
0.008
—
—



PpRBP-1
2
0.130
0.017
48
0.0276



(SEQ ID



NO: 8)


WUE
Wild-type

1.446
0.102
—
—



PpRBP-1
2
2.301
0.214
59
0.0004



(SEQ ID



NO: 8)
















TABLE 26







A. thaliana lines overexpressing PpPD-1 (SEQ ID NO: 10)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P
















DW
Wild-type

0.114
0.006
—
—



PpPD-1
3
0.171
0.019
50
0.0045



(SEQ ID
1
0.171
0.017
50
0.0019



NO: 10)
8
0.174
0.019
53
0.0026




2
0.182
0.019
60
0.0007




11
0.191
0.017
67
<.0001




5
0.201
0.019
76
<.0001




9
0.204
0.017
79
<.0001


WUE
Wild-type

1.958
0.058
—
—



PpPD-1
11
2.328
0.165
19
0.0353



(SEQ ID
1
2.343
0.165
20
0.0286



NO: 10)
8
2.354
0.180
20
0.0383




2
2.450
0.180
25
0.0102




3
2.517
0.180
29
0.0036




5
2.552
0.180
30
0.002 




9
2.572
0.165
31
0.0006
















TABLE 27







A. thaliana lines overexpressing PpMSC-1 (SEQ ID NO: 12)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.136
0.011
—
—



PpMSC-1
3
0.227
0.027
68
0.0026



(SEQ ID
2
0.240
0.025
77
0.0002



NO: 12)
1
0.247
0.025
82
<.0001




4
0.271
0.022
100 
<.0001


WUE
Wild-type

2.270
0.085
—
—



PpMSC-1
2
2.343
0.191
 3
0.7268



(SEQ ID
4
2.631
0.174
16
0.0654



NO: 12)
1
2.820
0.191
24
0.0097
















TABLE 28







A. thaliana lines overexpressing PpMBP-1 (SEQ ID NO: 14)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.110
0.005
—
—



PpMBP-1
1
0.154
0.017
39
0.0146



(SEQ ID



NO: 14)


WUE
Wild-type

1.620
0.066
—
—



PpMBP-1
1
2.144
0.209
32
0.0182



(SEQ ID



NO: 14)
















TABLE 29







A. thaliana lines overexpressing PpAK-1 (SEQ ID NO: 16)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.136
0.011
—
—



PpAK-1
1
0.185
0.022
36
0.049 



(SEQ ID
5
0.216
0.022
59
0.0014



NO: 16)
3
0.217
0.027
60
0.0063




7
0.217
0.024
60
0.0026




8
0.227
0.024
68
0.0008




4
0.230
0.024
69
0.0006


WUE
Wild-type

2.270
0.084
—
—



PpAK-1
8
2.285
0.188
1
0.9394



(SEQ ID
7
2.358
0.188
4
0.6683



NO: 16)
5
2.374
0.172
5
0.5851




3
2.377
0.211
5
0.6369




4
2.403
0.188
6
0.5191
















TABLE 30







A. thaliana lines overexpressing PpZF-6 (SEQ ID NO: 18)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.107
0.015
—
—



PpZF-6
4
0.131
0.018
22
0.3091



(SEQ ID



NO: 18)


WUE
Wild-type

1.897
0.316
—
—



PpZF-6
4
2.026
0.371
 7
0.7946



(SEQ ID



NO: 18)
















TABLE 31







A. thaliana lines overexpressing PpCDK-1 (SEQ ID NO: 20)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.088
0.009
—
—



PpCDK-1
7
0.148
0.017
69
0.0029



(SEQ ID



NO: 20)


WUE
Wild-type

1.446
0.102
—
—



PpCDK-1
7
1.963
0.195
36
0.0207



(SEQ ID



NO: 20)
















TABLE 32







A. thaliana lines overexpressing PpZF-7 (SEQ ID NO: 22)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P
















DW
Wild-type

0.114
0.006
—
—



PpZF-7
1
0.151
0.019
32
0.0617



(SEQ ID
10
0.153
0.019
35
0.0456



NO: 22)
2
0.159
0.019
39
0.0226




7
0.160
0.019
40
0.0198




3
0.163
0.019
43
0.0139




6
0.175
0.019
54
0.0021




9
0.176
0.019
55
0.0018




5
0.177
0.019
56
0.0014




8
0.196
0.019
72
<.0001




4
0.217
0.019
90
<.0001


WUE
Wild-type

1.958
0.057
—
—



PpZF-7
2
2.185
0.176
12
0.2224



(SEQ ID
10
2.237
0.176
14
0.1331



NO: 22)
7
2.242
0.176
15
0.1262




9
2.327
0.176
19
0.0479




3
2.359
0.176
20
0.0318




6
2.378
0.176
21
0.0245




1
2.435
0.176
24
0.0108




5
2.490
0.176
27
0.0045




8
2.537
0.176
30
0.002 




4
2.707
0.176
38
<.0001
















TABLE 33







A. thaliana lines overexpressing PpMFP-1 (SEQ ID NO: 24)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P
















DW
Wild-type

0.110
0.005
—
—



PpMFP-1
4
0.128
0.016
16
0.3008



(SEQ ID
2
0.130
0.016
18
0.25 



NO: 24)
10
0.145
0.016
31
0.0465




3
0.146
0.016
32
0.0417




6
0.159
0.016
44
0.0048




7
0.164
0.016
48
0.0022




5
0.166
0.016
50
0.0015




1
0.168
0.016
52
0.0011




8
0.172
0.016
56
0.0004


WUE
Wild-type

1.620
0.064
—
—



PpMFP-1
3
1.979
0.203
22
0.0929



(SEQ ID
8
2.049
0.203
26
0.0451



NO: 24)
7
2.049
0.203
26
0.0449




4
2.095
0.203
29
0.0267




1
2.113
0.203
30
0.0215




6
2.178
0.203
34
0.0094




5
2.217
0.203
37
0.0055




10
2.324
0.203
43
0.0011




2
2.345
0.203
45
0.0008
















TABLE 34







A. thaliana lines overexpressing PpLRP-2 (SEQ ID NO: 26)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.136
0.011
—
—



PpLRP-2
4
0.199
0.029
47
0.042 



(SEQ ID
2
0.206
0.023
52
0.0078



NO: 26)
3
0.224
0.029
65
0.0049




1
0.227
0.023
67
0.0007




5
0.235
0.026
74
0.0006




8
0.266
0.040
96
0.0026


WUE
Wild-type

2.270
0.090
—
—



PpLRP-2
4
2.360
0.224
4
0.7073



(SEQ ID
5
2.402
0.200
6
0.5481



NO: 26)
1
2.404
0.183
6
0.5095




8
2.471
0.317
9
0.5426
















TABLE 35







A. thaliana lines overexpressing PpPPK-1 (SEQ ID NO: 28)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P
















DW
Wild-type

0.108
0.007
—
—



PpPPK-1
4
0.157
0.020
45
0.023 



(SEQ ID
2
0.159
0.018
47
0.0097



NO: 28)
10
0.175
0.020
62
0.0018




9
0.177
0.022
64
0.0037


WUE
Wild-type

1.951
0.078
—
—



PpPPK-1
4
2.043
0.219
 5
0.6913



(SEQ ID
9
2.158
0.245
11
0.4225



NO: 28)
2
2.177
0.200
12
0.2948




10
2.523
0.219
29
0.0149
















TABLE 36







A. thaliana lines overexpressing PpSRP-1 (SEQ ID NO: 30)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P
















DW
Wild-type

0.114
0.006
—
—



PpSRP-1
7
0.152
0.018
33
0.0495



(SEQ ID
6
0.159
0.018
39
0.0196



NO: 30)
1
0.162
0.020
42
0.026 




10
0.164
0.017
44
0.0054




9
0.167
0.015
46
0.0015




8
0.174
0.018
53
0.0019




2
0.179
0.018
57
0.0008


WUE
Wild-type

1.958
0.057
—
—



PpSRP-1
10
2.109
0.161
 8
0.3776



(SEQ ID
8
2.197
0.177
12
0.1991



NO: 30)
9
2.239
0.149
14
0.0802




2
2.302
0.177
18
0.0659




6
2.405
0.177
23
0.017 




1
2.450
0.197
25
0.0178
















TABLE 37







A. thaliana lines overexpressing PpCBL-1 (SEQ ID NO: 32)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.114
0.006
—
—



PpCBL-1
5
0.156
0.019
37
0.034 



(SEQ ID
8
0.163
0.019
43
0.0153



NO: 32)
4
0.179
0.019
57
0.0012




3
0.180
0.019
58
0.0011




6
0.181
0.017
59
0.0003




1
0.182
0.017
59
0.0003




9
0.214
0.017
87
<.0001


WUE
Wild-type

1.958
0.057
—
—



PpCBL-1
3
2.109
0.177
 8
0.4181



(SEQ ID
5
2.152
0.177
10
0.2991



NO: 32)
8
2.158
0.177
10
0.2836




1
2.298
0.162
17
0.0489




6
2.306
0.162
18
0.0438




9
2.319
0.162
18
0.0367




4
2.440
0.177
25
0.0105
















TABLE 38







A. thaliana lines overexpressing PpCBL-2 (SEQ ID NO: 34)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P
















DW
Wild-type

0.114
0.006
—
—



PpCBL-2
9
0.156
0.017
37
0.0226



(SEQ ID
10
0.170
0.017
49
0.0027



NO: 34)
1
0.179
0.017
57
0.0005




4
0.188
0.017
65
<.0001




7
0.188
0.017
65
<.0001




3
0.192
0.017
68
<.0001




8
0.194
0.017
70
<.0001




2
0.203
0.017
78
<.0001


WUE
Wild-type

1.958
0.054
—
—



PpCBL-2
9
1.944
0.168
−1
0.9357



(SEQ ID
2
2.314
0.168
18
0.0451



NO: 34)
10
2.322
0.168
19
0.0405




8
2.448
0.168
25
0.0061




3
2.545
0.168
30
0.0011




4
2.569
0.168
31
0.0007




1
2.617
0.168
34
0.0003




7
2.771
0.168
42
<.0001
















TABLE 39







A. thaliana lines overexpressinq PpHD-1 (SEQ ID NO: 36)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

1.620
0.065
—
—



PpHD-1
7
1.976
0.207
22
0.1027



(SEQ ID
3
1.985
0.207
23
0.0944



NO: 36)
6
2.144
0.207
32
0.0169




2
2.374
0.207
47
0.0007




8
2.444
0.207
51
0.0002


WUE
Wild-type

0.110
0.005
—
—



PpHD-1
2
0.126
0.016
14
0.3655



(SEQ ID
8
0.143
0.016
30
0.0566



NO: 36)
6
0.149
0.016
35
0.0246




3
0.152
0.016
37
0.0177




7
0.191
0.016
73
<.0001
















TABLE 40







A. thaliana lines overexpressing PpLRP-1 (SEQ ID NO: 56)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.109
0.033
—
—



PpLRP-1
10
0.161
0.034
48
0.2835



(SEQ ID



NO: 56)


WUE
Wild-type

1.782
0.119
—
—



PpLRP-1
10
2.205
0.169
24
0.0529



(SEQ ID



NO: 56)
















TABLE 41







A. thaliana lines overexpressing PpLRP-1 (SEQ ID NO: 58)













Measure-



Standard
%



ment
Genotype
Line
LSM
Error
Improvement
P





DW
Wild-type

0.109
0.033
—
—



PpLRP-1
10
0.161
0.034
48
0.2835



(SEQ ID



NO: 58)


WUE
Wild-type

1.782
0.119
—
—



PpLRP-1
10
2.205
0.169
24
0.0529



(SEQ ID



NO: 58)









EXAMPLE 4
Stress-Tolerant Rapeseed/Canola Plants
Canola cotyledonary petioles of 4 day-old young seedlings are used as explants for tissue culture and transformed according to EP1566443. The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can be used. A. tumefaciens GV3101:pMP90RK containing a binary vector is used for canola transformation. The standard binary vector used for transformation is pSUN (WO 02/00900), but many different binary vector systems have been described for plant transformation (e.g. An, G. in Agrobacterium Protocols, Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, N.J.). A plant gene expression cassette comprising a selection marker gene and a plant promoter regulating the transcription of the cDNA encoding the polynucleotide is employed. Various selection marker genes can be used including the mutated acetohydroxy acid synthase (AHAS) gene disclosed in U.S. Pat. Nos. 5,767,366 and 6,225,105. A suitable promoter is used to regulate the trait gene to provide constitutive, developmental, tissue or environmental regulation of gene transcription.
Canola seeds are surface-sterilized in 70% ethanol for 2 min, incubated for 15 min in 55° C. warm tap water and then in 1.5% sodium hypochlorite for 10 minutes, followed by three rinses with sterilized distilled water. Seeds are then placed on MS medium without hormones, containing Gamborg B5 vitamins, 3% sucrose, and 0.8% Oxoidagar. Seeds are germinated at 24° C. for 4 days in low light (<50 μMol/m2s, 16 hours light). The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 3 days on MS medium including vitamins containing 3.75 mg/I BAP, 3% sucrose, 0.5 g/I MES, pH 5.2, 0.5 mg/l GA3, 0.8% Oxoidagar at 24° C., 16 hours of light. After three days of co-cultivation with Agrobacterium, the petiole explants are transferred to regeneration medium containing 3.75 mg/l BAP, 0.5 mg/l GA3, 0.5 g/l MES, pH 5.2, 300 mg/l timentin and selection agent until shoot regeneration. As soon as explants start to develop shoots, they are transferred to shoot elongation medium (A6, containing full strength MS medium including vitamins, 2% sucrose, 0.5% Oxoidagar, 100 mg/l myo-inositol, 40 mg/l adenine sulfate, 0.5 g/l MES, pH 5.8, 0.0025 mg/l BAP, 0.1 mg/l IBA, 300 mg/l timentin and selection agent).
Samples from both in vitro and greenhouse material of the primary transgenic plants (TO) are analyzed by qPCR using TaqMan probes to confirm the presence of T-DNA and to determine the number of T-DNA integrations.
Seed is produced from the primary transgenic plants by self-pollination. The second-generation plants are grown in greenhouse conditions and self-pollinated. The plants are analyzed by qPCR using TaqMan probes to confirm the presence of T-DNA and to determine the number of T-DNA integrations. Homozygous transgenic, heterozygous transgenic and azygous (null transgenic) plants are compared for their stress tolerance, for example, in the assays described in Example 3, and for yield, both in the greenhouse and in field studies.
EXAMPLE 5
Screening for Stress-Tolerant Rice Plants
Transgenic rice plants comprising a polynucleotide of the invention are generated using known methods. Approximately 15 to 20 independent transformants (TO) are generated. The primary transformants are transferred from tissue culture chambers to a greenhouse for growing and harvest of T1 seeds. Five events of the T1 progeny segregated 3:1 for presence/absence of the transgene are retained. For each of these events, 10 T1 seedlings containing the transgene (hetero- and homozygotes), and 10 T1 seedlings lacking the transgene (nullizygotes) are selected by visual marker screening. The selected T1 plants are transferred to a greenhouse. Each plant receives a unique barcode label to link unambiguously the phenotyping data to the corresponding plant. The selected T1 plants are grown on soil in 10 cm diameter pots under the following environmental settings: photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytime temperature=28° C. or higher, night time temperature=22° C., relative humidity=60-70%. Transgenic plants and the corresponding nullizygotes are grown side-by-side at random positions. From the stage of sowing until the stage of maturity, the plants are passed several times through a digital imaging cabinet. At each time point digital, images (2048×1536 pixels, 16 million colours) of each plant are taken from at least 6 different angles.
The data obtained in the first experiment with T1 plants are confirmed in a second experiment with T2 plants. Lines that have the correct expression pattern are selected for further analysis. Seed batches from the positive plants (both hetero- and homozygotes) in T1 are screened by monitoring marker expression. For each chosen event, the heterozygote seed batches are then retained for T2 evaluation. Within each seed batch, an equal number of positive and negative plants are grown in the greenhouse for evaluation.
Transgenic plants are screened for their improved growth and/or yield and/or stress tolerance, for example, using the assays described in Example 3, and for yield, both in the greenhouse and in field studies.
EXAMPLE 6
Stress-Tolerant Soybean Plants
The polynucleotides of Tables 1 and 2 are transformed into soybean using the methods described in commonly owned copending international application number WO 2005/121345, the contents of which are incorporated herein by reference. The transgenic plants are then screened for their improved growth under water-limited conditions and/or drought, salt, and/or cold tolerance, for example, using the assays described in Example 3, and for yield, both in the greenhouse and in field studies.
EXAMPLE 7
Stress-Tolerant Wheat Plants
Transformation of wheat is performed with the method described by Ishida et al., 1996, Nature Biotech. 14745-50. Immature embryos are co-cultivated with Agrobacterium tumefaciens that carry “super binary” vectors, and transgenic plants are recovered through organogenesis. This procedure provides a transformation efficiency between 2.5% and 20%. The transgenic plants are then screened for their improved growth and/or yield under water-limited conditions and/or stress tolerance, for example, is the assays described in Example 3, and for yield, both in the greenhouse and in field studies.
EXAMPLE 8
Stress-Tolerant Corn Plants
Agrobacterium cells harboring the genes and the maize ahas gene on the same plasmid are grown in YP medium supplemented with appropriate antibiotics for 1-3 days. A loop of Agrobacterium cells is collected and suspended in 1.5 ml M-LS-002 medium (LS-inf) and the tube containing Agrobacterium cells is kept on a shaker for 1-4 hours at 1,000 rpm.
Corncobs [genotype J553×(HIIIAxA188)] are harvested at 7-12 days after pollination. The cobs are sterilized in 20% Clorox solution for 15 minutes followed by thorough rinse with sterile water. Immature embryos with size 0.8-2.0 mm are dissected into the tube containing Agrobacterium cells in LS-inf solution.
Agro-infection is carried out by keeping the tube horizontally in the laminar hood at room temperature for 30 minutes. Mixture of the agro infection is poured on to a plate containing the co-cultivation medium (M-LS-011). After the liquid agro-solution is piped out, the embryos transferred to the surface of a filter paper that is placed on the agar co-cultivation medium. The excess bacterial solution is removed with a pipette. The embryos are placed on the co-cultivation medium with scutellum side up and cultured in the dark at 22° C. for 2-4 days.
Embryos are transferred to M-MS-101 medium without selection. Seven to ten days later, embryos are transferred to M-LS-401 medium containing 0.50 μM imazethapyr and grown for 4 weeks (two 2-week transfers) to select for transformed callus cells. Plant regeneration is initiated by transferring resistant calli to M-LS-504 medium supplemented with 0.75 μM imazethapyr and grown under light at 25-27° C. for two to three weeks. Regenerated shoots are then transferred to rooting box with M-MS-618 medium (0.5 μM imazethapyr). Plantlets with roots are transferred to potting mixture in small pots in the greenhouse and after acclimatization are then transplanted to larger pots and maintained in greenhouse till maturity.
The copy number of the transgene in each plantlet is assayed using Taqman analysis of genomic DNA, and transgene expression is assayed using qRT-PCR of total RNA isolated from leaf samples.
Using assays such as the assay described in Example 3, each of these plants is uniquely labeled, sampled and analyzed for transgene copy number. Transgene positive and negative plants are marked and paired with similar sizes for transplanting together to large pots. This provides a uniform and competitive environment for the transgene positive and negative plants. The large pots are watered to a certain percentage of the field water capacity of the soil depending the severity of water-stress desired. The soil water level is maintained by watering every other day. Plant growth and physiology traits such as height, stem diameter, leaf rolling, plant wilting, leaf extension rate, leaf water status, chlorophyll content and photosynthesis rate are measured during the growth period. After a period of growth, the above ground portion of the plants is harvested, and the fresh weight and dry weight of each plant are taken. A comparison of the drought tolerance phenotype between the transgene positive and negative plants is then made.
Using assays such as the assay described in Example 3, the pots are covered with caps that permit the seedlings to grow through but minimize water loss. Each pot is weighed periodically and water added to maintain the initial water content. At the end of the experiment, the fresh and dry weight of each plant is measured, the water consumed by each plant is calculated and WUE of each plant is computed. Plant growth and physiology traits such as WUE, height, stem diameter, leaf rolling, plant wilting, leaf extension rate, leaf water status, chlorophyll content and photosynthesis rate are measured during the experiment. A comparison of WUE phenotype between the transgene positive and negative plants is then made.
Using assays such as the assay described in Example 3, these pots are kept in an area in the greenhouse that has uniform environmental conditions, and cultivated optimally. Each of these plants is uniquely labeled, sampled and analyzed for transgene copy number.
The plants are allowed to grow under theses conditions until they reach a predefined growth stage. Water is then withheld. Plant growth and physiology traits such as height, stem diameter, leaf rolling, plant wilting, leaf extension rate, leaf water status, chlorophyll content and photosynthesis rate are measured as stress intensity increases. A comparison of the dessication tolerance phenotype between transgene positive and negative plants is then made.
Segregating transgenic corn seeds for a transformation event are planted in small pots for testing in a cycling drought assay. These pots are kept in an area in the greenhouse that has uniform environmental conditions, and cultivated optimally. Each of these plants is uniquely labeled, sampled and analyzed for transgene copy number. The plants are allowed to grow under theses conditions until they reach a predefined growth stage. Plants are then repeatedly watered to saturation at a fixed interval of time. This water/drought cycle is repeated for the duration of the experiment. Plant growth and physiology traits such as height, stem diameter, leaf rolling, leaf extension rate, leaf water status, chlorophyll content and photosynthesis rate are measured during the growth period. At the end of the experiment, the plants are harvested for above-ground fresh and dry weight. A comparison of the cycling drought tolerance phenotype between transgene positive and negative plants is then made.
In order to test segregating transgenic corn for drought tolerance under rain-free conditions, managed-drought stress at a single location or multiple locations is used. Crop water availability is controlled by drip tape or overhead irrigation at a location which has less than 10 cm rainfall and minimum temperatures greater than 5° C. expected during an average 5 month season, or a location with expected in-season precipitation intercepted by an automated “rain-out shelter” which retracts to provide open field conditions when not required. Standard agronomic nomic practices in the area are followed for soil preparation, planting, fertilization and pest con-trol. Each plot is sown with seed segregating for the presence of a single transgenic insertion event. A Taqman transgene copy number assay is used on leaf samples to differentiate the transgenics from null-segregant control plants. Plants that have been genotyped in this manner are also scored for a range of phenotypes related to drought-tolerance, growth and yield. These phenotypes include plant height, grain weight per plant, grain number per plant, ear number per plant, above ground dry-weight, leaf conductance to water vapor, leaf CO2 uptake, leaf chlorophyll content, photosynthesis-related chlorophyll fluorescence parameters, water use efficiency,leaf water potential, leaf relative water content, stem sap flow rate, stem hydraulic conductivity, leaf temperature, leaf reflectance, leaf light absorptance, leaf area, days to flowering, anthesis-silking interval, duration of grain fill, osmotic potential, osmotic adjustment, root size, leaf extension rate, leaf angle, leaf rolling and survival. All measurements are made with commercially available instrumentation for field physiology, using the standard protocols provided by the manufacturers. Individual plants are used as the replicate unit per event.
In order to test non-segregating transgenic corn for drought tolerance under rain-free conditions, managed-drought stress at a single location or multiple locations is used. Crop water availability is controlled by drip tape or overhead irrigation at a location which has less than 10 cm rainfall and minimum temperatures greater than 5° C. expected during an average 5 month season, or a location with expected in-season precipitation intercepted by an automated “rain-out shelter” which retracts to provide open field conditions when not required. Standard agronomic practices in the area are followed for soil preparation, planting, fertilization and pest control. Trial layout is designed to pair a plot containing a non-segregating transgenic event with an adjacent plot of null-segregant controls. A null segregant is progeny (or lines derived from the progeny) of a transgenic plant that does not contain the transgene due to Mendelian segregation. Additional replicated paired plots for a particular event are distributed around the trial. A range of phenotypes related to drought-tolerance, growth and yield are scored in the paired plots and estimated at the plot level. When the measurement technique could only be applied to individual plants, these are selected at random each time from within the plot. These pheno-types include plant height, grain weight per plant, grain number per plant, ear number per plant, above ground dry-weight, leaf conductance to water vapor, leaf CO2 uptake, leaf chlorophyll content, photosynthesis-related chlorophyll fluorescence parameters, water use efficiency, leaf water potential, leaf relative water content, stem sap flow rate, stem hydraulic conductivity, leaf temperature, leaf reflectance, leaf light absorptance, leaf area, days to flowering, anthesis-silking interval, duration of grain fill, osmotic potential, osmotic adjustment, root size, leaf extension rate, leaf angle, leaf rolling and survival. All measurements are made with commercially available instrumentation for field physiology, using the standard protocols provided by the manufacturers. Individual plots are used as the replicate unit per event.
To perform multi-location testing of transgenic corn for drought tolerance and yield, five to twenty locations encompassing major corn growing regions are selected. These are widely distributed to provide a range of expected crop water availabilities based on average temperature, humidity, precipitation and soil type. Crop water availability is not modified beyond standard agronomic practices. Trial layout is designed to pair a plot containing a non-segregating transgenic event with an adjacent plot of null-segregant controls. A range of pheno-types related to drought-tolerance, growth and yield are scored in the paired plots and estimated at the plot level. When the measurement technique could only be applied to individual plants, these are selected at random each time from within the plot. These phenotypes included plant height, grain weight per plant, grain number per plant, ear number per plant, above ground dry-weight, leaf conductance to water vapor, leaf CO2 uptake, leaf chlorophyll content, photo-synthesis-related chlorophyll fluorescence parameters, water use efficiency, leaf water potential, leaf relative water content, stem sap flow rate, stem hydraulic conductivity, leaf temperature, leaf reflectance, leaf light absorptance, leaf area, days to flowering, anthesis-silking interval, duration of grain fill, osmotic potential, osmotic adjustment, root size, leaf extension rate, leaf angle, leaf rolling and survival. All measurements are made with commercially available instru-mentation for field physiology, using the standard protocols provided by the manufacturers. Individual plots are used as the replicate unit per event.

## Claims

1. A transgenic plant cell or a transgenic plant transformed with an expression cassette comprising an isolated polynucleotide encoding a polypeptide having a sequence comprising amino acids 1 to 91 of SEQ ID NO:20.

2. An isolated polynucleotide having a sequence selected from the group consisting of
(a) a polynucleotide having a sequence comprising nucleotides 248 to 520 of SEQ ID NO:19; and
(b) a polynucleotide which encodes a polypeptide having a sequence comprising amino acids 1 to 91 of SEQ ID NO:20.

3. An isolated polypeptide having a sequence comprising amino acids 1 to 91 of SEQ ID NO:20.

4. A method of producing a transgenic plant comprising the steps of:
(a) introducing into a plant cell an expression vector comprising at least one polynucleotide having a sequence selected from the group consisting of (i) a polynucleotide having a sequence comprising nucleotides 248 to 520 of SEQ ID NO:19 and (ii) a polynucleotide which encodes a polypeptide having a sequence comprising amino acids 1 to 91 of SEQ ID NO:20; and
(b) generating from the plant cell a transgenic plant that expresses the polynucleotide.

5. A method of increasing a plant's growth or yield under normal or water-limited conditions or increasing a plant's tolerance to an environmental stress comprising the steps of: (a) inserting into an expression vector a polynucleotide having a sequence selected from the group consisting of (i) a polynucleotide having a sequence comprising nucleotides 248 to 520 of SEQ ID NO:19 and (ii) a polynucleotide which encodes a polypeptide having a sequence comprising amino acids 1 to 91 of SEQ ID NO:20; and (b) introducing the expression vector into the plant.