# Patent

## Title
Plants having increased desiccation tolerance, increased drought tolerance or increased water use efficiency

## Abstract

A transgenic plant engineered to have increased desiccation tolerance, increased drought tolerance or increased water use efficiency, the plant transformed with an artificial DNA construct comprising a transcribable nucleic acid molecule encoding a polypeptide requiring both ABA and ABI3 to influence desiccation tolerance. Also provided are DNA constructs and methods of producing a transgenic plant engineered to have increased desiccation tolerance, increased drought tolerance or increased water use efficiency.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Provisional Application Ser. No. 61/660,277 filed on 15 Jun. 2013, which is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable.
MATERIAL INCORPORATED-BY-REFERENCE
The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
To survive on land, the earliest land plants had to develop mechanisms to tolerate desiccation. Modern seed plants possess an array of morphological features to retain water (such as conductive tissues, waxy layers on leaves, and openings in leaves that can be control to prevent water loss) and have retained desiccation tolerance in only a few specialized structures (e.g., seeds). Present-day bryophytes (mosses), in contrast, lack water transport and retention tissues, presumably like early land plants. As a result, their normal state is at equilibrium with the surrounding air, creating a water-deficit condition that most seed plants could not tolerate.
The plant specific transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) functions in ABA signaling during seed maturation and germination. It has been reported that ABI3 is evolutionarily conserved in the non-seed plant Physcomitrella patens. Furthermore, both ABA and ABI3 are required for P. patens vegetative tissue to survive desiccation.
SUMMARY OF THE INVENTION
Among the various aspects of the present disclosure is the provision of a transgenic plant engineered to have increased desiccation tolerance, increased drought tolerance or increased water use efficiency. In some embodiments, the plant transformed with an artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription: a promoter that functions in a plant; a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a sequence at least 95% identical thereto; and a transcriptional termination sequence; wherein the transgenic plant has an increased desiccation tolerance compared to a plant without the DNA construct. In some embodiments, the transgenic plant has increased desiccation tolerance. In some embodiments, the transgenic plant has increased drought tolerance. In some embodiments, the transgenic plant has increased water use efficiency. In some embodiments, the transgenic plant has two or more of increased desiccation tolerance, increased drought tolerance or increased water use efficiency. In some embodiments, the transgenic plant has all of increased desiccation tolerance, increased drought tolerance and increased water use efficiency.
In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: 8, or a sequence at least 95% identical thereto.
In some embodiments, the DNA construct comprises at least two polynucleotides independently selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a sequence at least 95% identical thereto. In some embodiments, the plant comprises at least two DNA constructs, each DNA construct comprising a polynucleotide independently selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a sequence at least 95% identical thereto.
In some embodiments, the plant comprises SEQ ID NO: 8 and one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, the plant comprises SEQ ID NO: 8 and one or more of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5. In some embodiments, the plant comprises: SEQ ID NO: 8 and SEQ ID NO: 1; SEQ ID NO: 8 and SEQ ID NO: 2; SEQ ID NO: 8 and SEQ ID NO: 5; SEQ ID NO: 8, SEQ ID NO: 1, and SEQ ID NO: 2; SEQ ID NO: 8, SEQ ID NO: 1, and SEQ ID NO: 5; SEQ ID NO: 8, SEQ ID NO: 2, and SEQ ID NO: 5; or SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5.
In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a seed-specific promoter.
In some embodiments, the desiccation tolerance of the transgenic plant is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 250%, at least about 500%, at least about 750%, or at least about 1000%, as compared to a plant not comprising the DNA construct.
In some embodiments, the plant is maize, bean, soybean, wheat, barley, hay, rice, peanut, cotton, tomato, cucurbit, okra, eggplant, pepper, sugar beet, sugarcane, cassava, potatoes, palm, rapeseed, sunflower, coconut, olive, flax, safflower, sesame, apple, pear, grape, strawberry, blackcurrant, redcurrant, gooseberry, guava, lucuma, chili pepper, pomegranate, kiwifruit, cranberry, blueberry, blackberry, raspberry, boysenberry, banana, plum, cherry, peach, apricot, mango, orange, lime, lemon, grapefruit, pineapple, fig, mulberry, hedge apple, osage-orange, or breadfruit.
Another aspect of the present disclosure provides a plant part of a transgenic plant described above.
Another aspect of the present disclosure provides an artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription: a promoter that functions in a plant; a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a sequence at least 95% identical thereto; and a transcriptional termination sequence.
Another aspect of the present disclosure provides a method of increasing desiccation tolerance of a plant comprising: transforming a plant with an artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription: a promoter that functions in a plant; a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a sequence at least 95% identical thereto; and a transcriptional termination sequence; wherein the transgenic plant has an increased desiccation tolerance, increased drought tolerance or increased water use efficiency. compared to a plant without the DNA construct.
Other objects and features will be in part apparent and in part pointed out hereinafter.



DESCRIPTION OF THE DRAWINGS
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
FIG. 1 is a series of charts and bar graphs providing a graphical overview of quantitative proteomic analysis using iTRAQ.
FIG. 2 is a chart providing an overview of sample preparation for iTRAQ analysis.
FIG. 3 is a series of images depicting Northern blot analysis of mRNA expression for the following genes: Accession No. gi168047556, Phypa ID 169276; Accession No. gi167999540, Phypa ID 112415; Accession No. gi168049029, Phypa ID 194258; Accession No. gi168000434, Phypa ID 113212; Accession No. gi168038393, Phypa ID 190133; Accession No. gi168002106, Phypa ID 159190; Accession No. gi168015696, Phypa ID 122983; and Accession No. gi168052229, Phypa ID 27775.
FIG. 4 is a series of images showing over-expression lines of Accession No. gi168047556, Phypa ID 169276, to A abi3 and resulting desiccation tolerance with ABA. FIG. 4A is an image of Northernblot analysis of overexpression lines of Phypa—169276 to WT. FIG. 4B is a pair of images depicting results from a desiccation assay in which tissues from 6-day-old WT, Δabi3 and Phypa—169276 overexpression lines to Δabi3 were treated with ABA (10 μM) for 24 hours. Tissues were dried for 30 hours, rehydrated with sterile distilled water, and incubated for 1 week.
FIG. 5 is a series of images showing over-expression lines of Accession No. gi168047556, Phypa ID 169276, to WT and resulting stronger desiccation tolerance than WT with ABA. FIG. 5A is an image of Northernblot analysis of overexpression lines of Phypa—169276 to A abi3. FIG. 5B is a pair of images depicting results from a desiccation assay in which tissues from 6-day-old WT, Δabi3 and Phypa—169276 overexpression lines to Δabi3 were treated with ABA (10 μM) for 24 hours. Tissues were dried for 48 hours, rehydrated with sterile distilled water, and incubated for 1 week.



DETAILED DESCRIPTION OF THE INVENTION
The present disclosure is based, at least in part, on the discovery of reduced desiccation tolerance of a moss line having a knock-out gene encoding a protein controlled by ABA and ABI3 during the desiccation-rehydration process. Such genes were identified according to quantitative proteomics analysis using iTRAQ (Isobaric Tags for Relative and Absolute Quantification) to reveal the regulatory network controlled by ABA and ABI3 during the desiccation-rehydration process. As described herein, from the thousands of proteins identified, close to one hundred were shown to increase more than two point five-fold with ABA treatment, and eight were shown to require both ABI3 and ABA. These results suggest that the gene regulatory pathway that requires both ABA and ABI3 control a relatively few proteins, and that these proteins can be used to increase desiccation tolerance, increase drought tolerance or increase water use efficiency in plant tissues.
ABA and ABI3 are generally discussed in Marella et al. 2006 The Plant Journal 46, 1032-1044; Khandelwal et al. 2010 Science 327, 546; and Yotsui et al. 2013 New Phytologist 199, 101-109. Desiccation tolerance in angiosperm seeds and in vegetative tissues of the moss Physcomitrella patens use similar regulatory pathways. The phytohormone abscisic acid (ABA) is a key controlling molecule that allows seeds to survive desiccation by activating a set of genes whose products presumably protect seed tissue to water loss. ABA acts through regulatory (transcription) factors such as ABSCISIC ACID INSENSITIVE 3 (ABI3). ABA and homologs of ABI3 are both found in P. patens and using a promoter active in wheat seed and linked to a reporter genes, it was found that ABA can activate this reporter gene in moss cells. Furthermore, one of three homologs of ABI3 found in P. patens partially complements the Arabidopsis abi3-6mutant. Both of these latter results indicate that the ABA signaling pathway exists in moss and that the regulatory components are similar. Thus, gene regulatory pathways that include both ABA and ABI3 originally evolved for cellular protection from water deficits but independently have been used to provide desiccation tolerance in normal tissues of bryophytes and in angiosperm seeds. For at least the above reasons, proteins controlled by ABA and ABI3 during the desiccation-rehydration process in moss can be employed in similar fashion in higher plants not containing such native proteins or expressing the same or similar proteins at lower levels or under different regulatory control.
According to an approach described herein, a plant can be transformed so as to express or overexpress a polypeptide responsive to ABA and requiring ABI3 so as to provide or increase tolerance or resistance to desiccation. For example, a plant can be transformed to express or overexpress one or more polypeptides encoded by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof.
Proteins
As described herein, from the thousands of proteins identified in quantitative proteomics analysis using iTRAQ, eight were shown to require both ABI3 and ABA. Because the gene regulatory pathway that requires both ABA and ABI3 control a relatively few proteins, these proteins can be used to increase desiccation tolerance, increase drought tolerance or increase water use efficiency in plant tissues.
One aspect of the present disclosure provides a plant in which one or more proteins encoded by a transcribable nucleic acid molecule selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, are expressed or overexpressed in a plant so as to provide or increase desiccation tolerance, increase drought tolerance or increase water use efficiency. Such transcribable nucleic acid molecule(s) can be heterologous nucleotide sequences. A transcribable nucleic acid molecule of a heterologous construct can include a nucleic acid molecule that is already present in the host cell, a nucleic acid molecule from another organism, a nucleic acid molecule from a different organism, or a nucleic acid molecule generated externally, such as a nucleic acid molecule containing an antisense message of a gene, or a nucleic acid molecule encoding an artificial or modified version of a gene. A transcribable nucleic acid molecule can be any sequence encoding a polypeptide of interest. For example, a transcribable nucleic acid molecule can be a gene encoding a polypeptide having a particular activity of interest. A transcribable nucleic acid molecule can be gene selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof. Such transcribable nucleic acid molecule(s) can be incorporated into a construct for transformation of a plant according to conventional techniques.
For example, one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, can be expressed or overexpressed in a plant. As another example, two or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, can be expressed or overexpressed in a plant. As another example, three or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, can be expressed or overexpressed in a plant. As another example, four or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, can be expressed or overexpressed in a plant. As another example, five or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, can be expressed or overexpressed in a plant. As another example, six or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, can be expressed or overexpressed in a plant. As another example, seven or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, can be expressed or overexpressed in a plant. As another example, all of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a variant thereof, can be expressed or overexpressed in a plant.
A plant cell can be transformed with one or more heterologous nucleotide sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a nucleotide sequence having at least about 90% sequence identity thereto, or a complementary sequence to any of these sequences, such that expression or overexpression of the encoded polypeptide provides or increases tolerance of desiccation or desiccation resistance. For example, a plant can be transformed with a nucleotide sequence having at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, such that expression or overexpression of the encoded polypeptide provides or increases tolerance of desiccation or desiccation resistance. Such transcribable sequences can be heterologous nucleotide sequences.
A plant can be transformed with an isolated polynucleotide that hybridizes to any of the above discussed nucleic acid sequences under stringent conditions thereto over the entire length of said sequence; said stringent conditions comprising incubation at 65° C. in a solution comprising 6×SSC (0.9 M sodium chloride and 0.09 M sodium citrate); and which encodes a polypeptide that when expressed or overexpressed in the plant provides or increases tolerance of desiccation or desiccation resistance.
Sequences for nucleotides referred to by Accession No. and Phypa ID can be obtained at, for example, the website cosmoss.org.
A transcribable nucleic acid molecule can be Accession No. gi168047556, Phypa ID 169276 or SEQ ID NO: 1, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, such that expression or overexpression of the encoded polypeptide provides or increases desiccation tolerance.
A transcribable nucleic acid molecule can be Accession No. gi167999540, Phypa ID 112415 or SEQ ID NO: 2, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, such that expression or overexpression of the encoded polypeptide provides or increases desiccation tolerance.
A transcribable nucleic acid molecule can be Accession No. gi168052229, Phypa ID 27775 or SEQ ID NO: 3, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, such that expression or overexpression of the encoded polypeptide provides or increases desiccation tolerance.
A transcribable nucleic acid molecule can be Accession No. gi168049029, Phypa ID 194258 or SEQ ID NO: 4, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, such that expression or overexpression of the encoded polypeptide provides or increases desiccation tolerance.
A transcribable nucleic acid molecule can be Accession No. gi168000434, Phypa ID 113212 or SEQ ID NO: 5, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, such that expression or overexpression of the encoded polypeptide provides or increases desiccation tolerance.
A transcribable nucleic acid molecule can be Accession No. gi168038393, Phypa ID 190133 or SEQ ID NO: 6, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, such that expression or overexpression of the encoded polypeptide provides or increases desiccation tolerance.
A transcribable nucleic acid molecule can be Accession No. gi168002106, Phypa ID 159190 or SEQ ID NO: 7, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, such that expression or overexpression of the encoded polypeptide provides or increases desiccation tolerance.
A transcribable nucleic acid molecule can be Accession No. gi168015696, Phypa ID 122983 or SEQ ID NO: 8, or a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, such that expression or overexpression of the encoded polypeptide provides or increases desiccation tolerance.
Using targeted gene deletion techniques, deletion lines are developed for combinations of the four genes shown to require both ABI3 and ABA as follows: Accession No. gi168047556, Phypa ID 169276 (SEQ ID NO: 1); Accession No. gi167999540, Phypa ID 112415 (SEQ ID NO: 2); Accession No. gi168000434, Phypa ID 113212 (SEQ ID NO: 5); and Accession No. gi168015696, Phypa ID 122983 (SEQ ID NO: 8).
As shown herein, transcribable nucleic acid molecule having a sequence of Accession No. gi168015696, Phypa ID 122983 (SEQ ID NO: 8) was shown to be strongly associated with desiccation tolerance. As such, a transformed plant can be engineered with a transcribable nucleic acid molecule of SEQ ID NO: 8, or a variant thereof, so as to increase desiccation tolerance, increase drought tolerance or increase water use efficiency of the plant. Furthermore, in addition to SEQ ID NO: 8, or a variant thereof, a plant can be transformed with one or more additional transcribable nucleic acid molecules described herein. For example, a plant can be transformed with SEQ ID NO: 8, or a variant thereof, and at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a variant thereof. Exemplary combinations include, but are not limited to: SEQ ID NO: 8 and SEQ ID NO: 1; SEQ ID NO: 8 and SEQ ID NO: 2; SEQ ID NO: 8 and SEQ ID NO: 5; SEQ ID NO: 8, SEQ ID NO: 1, and SEQ ID NO: 2; SEQ ID NO: 8, SEQ ID NO: 1, and SEQ ID NO: 5; SEQ ID NO: 8, SEQ ID NO: 2, and SEQ ID NO: 5; SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5. For example, a plant can be transformed with SEQ ID NO: 8, or a variant thereof, and SEQ ID NO: 1, or a variant thereof. One of ordinary skill will understand that plants transformed with other combinations of transcribable nucleic acid molecules described herein can be generated.
Constructs
Any of the transcribable polynucleotide molecule sequences described above can be provided in a construct. Constructs of the present disclosure generally include a promoter functional in the host plant operably linked to a transcribable polynucleotide molecule, such as provided in SEQ ID NO: 1, 3, 5, or 7, and variants thereof as discussed herein.
Exemplary promoters are discussed above. One or more additional promoters may also be provided in the recombinant construct. These promoters can be operably linked to any of the transcribable polynucleotide molecule sequences described herein.
In addition, constructs can include, but are not limited to, additional polynucleotide molecules from an untranslated region of the gene of interest. These additional polynucleotide molecules can be derived from a source that is native or heterologous with respect to the other elements present in the construct.
Promoters
A transcribable nucleic acid molecule encoding a polypeptide responsive to ABA and requiring ABI3 so as to provide or increase tolerance or resistance to desiccation can be operably linked to a promoter for transformation of a plant cell. The promoter can be any promoter functional in a plant cell (see e.g., Weise et al. Applied Microbiology and Biotechnology 70(3), 337-345; Saidi et al. 2005 Plant Molecular Biology 59(5), 697-711; Horstmann et al. 2004 BMC Biotechnology 4; Holtorf et al. 2002 Plant Cell Reports 21(4), 341-346; Zeidler et al. 1996 Plant Molecular Biology 30(1), 199-205). The promoter can be an inducible promoter.
The promoter can be any promoter endogenously associated with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or analogs thereof in other species.
Examples of promoters than can be used in accord with methods and compositions described herein include, but are not limited to, ubiquitin promoter; factor EF1α gene promoter (see e.g., US App Pub No. 2008/0313776); rice tungro bacilliform virus (RTBV) gene promoter (see e.g., US App Pub No. 2008/0282431); cestrum yellow leaf curling virus (CmYLCV) promoter (see e.g., Stavolone et al. Plant Molecular Biology 53(5), 663-673); tCUP cryptic promoter system (see e.g., Malik et al. 2002 Theoretical and Applied Genetics 105(4), 505-514); T6P-3 promoter (see e.g., JP2002238564); S-adenosyl-L-methionine synthetase promoter (see e.g., WO/2000/037662); Raspberry E4 gene promoter (see e.g., U.S. Pat. No. 6,054,635); cauliflower mosaic virus 35S promoter (see e.g., Benfey et al. 1990 Science 250(4983), 959-966); figwort mosaic virus promoter (see e.g., U.S. Pat. No. 5,378,619); conditional heat-shock promoter (see e.g., Saidi et al. 2005 Plant Molecular Biology 59(5), 697-711); promoter subfragments of the sugar beet V-type H+-ATPase subunit c isoform (see e.g., Holtorf et al. 2002 Plant Cell Reports 21(4), 341-346); beta-tubulin promoter (see e.g., Jost et al. 2005 Current Genetics 47(2), 111-120); and bacterial quorum-sensing components (see e.g., You et al. 2006 Plant Physiology 140 (4), 1205-1212). For example, a promoter can be a heat-shock promoter. As another example, a promoter can be an inducible promoter providing for expression at germination or seedling stage.
A promoter can be a tissue specific promoter. For example, a transcribable nucleic acid molecule described herein can be operably linked to a pollen-, flower-, seed-, leaf-, or stem-specific promoter. As another example, a transcribable nucleic acid molecule described herein can be operably linked to a seed-specific promoter.
Inclusion of a termination region control sequence is optional, and if employed, then the choice is be primarily one of convenience, as the termination region is relatively interchangeable. The termination region may be native to the transcriptional initiation region (the promoter), may be native to the nucleic acid sequence of interest, or may be obtainable from another source.
A promoter of the present disclosure can be incorporated into a construct using marker genes as described and tested for an indication of gene expression in a stable host system. As used herein the term “marker gene” refers to any transcribable nucleic acid molecule whose expression can be screened for or scored in some way.
Plant
A plant or plant cell can be transformed with a construct including a transcribable nucleic acid molecule described herein operably linked to a promoter.
As used herein, the term “plant” can include plant cells, plant protoplasts, plant cells of tissue culture from which a plant can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants such as pollen, flowers, seeds, leaves, stems, and the like.
A host plant can be any plant in which it is desirable to increase desiccation tolerance, increase drought tolerance or increase water use efficiency. A host plant can be a flowering plant, conifer, fern or moss. A host plant can be angiosperm or a gymnosperm. A host plant can be monocot or a dicot. A host plant can be an agronomically important plant, such as maize (corn), bean, soybean, wheat, barley, hay (e.g., alfalfa and legume and grass mixtures), rice, peanut, cotton, tomato, cucurbit (e.g., squash, pumpkin, gourd, cucumber, melon, watermelon, zuchini), okra, eggplant, pepper, sugar beet, sugarcane, cassava, potatoes, palm, rapeseed (canola), sunflower, coconut, olive, flax, safflower, sesame, apple, pear, grape, strawberry, blackcurrant, redcurrant, gooseberry, guava, lucuma, chili pepper, pomegranate, kiwifruit, cranberry, blueberry, blackberry, raspberry, boysenberry, banana, plum, cherry, peach, apricot, mango, orange, lime, lemon, grapefruit, pineapple, fig, mulberry, hedge apple, osage-orange, or breadfruit.
A transformed plant or plant cell can be analyzed for the presence of a gene of interest and the expression level or profile conferred by the construct of the present disclosure. Those of skill in the art are aware of the numerous methods available for the analysis of transformed hosts. For example, methods for host analysis include, but are not limited to Southern blots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, and immunodiagnostic assays.
Tolerance
Desiccation tolerance is generally understood as an ability of an organism to withstand or endure extreme dryness, or drought-like conditions, or the ability to continue a plant function (i.e., reduce strain, i.e., observed biological changes that occur in response to stress, i.e., an environmental condition that is capable of causing a biologically injurious change) despite reductions in plant water potential (see generally, Salisbury, Units, Symbols, and Terminology for Plant Physiology 1996 Oxford University Press, New York, ISBN019509445X). Desiccation tolerance is generally understood As described herein, desiccation tolerance in a plant can be provided or increased by engineering a plant to express or overexpress one or more polypeptides encoded by a transcribable nucleic acid molecule described herein (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or variants thereof).
Processes to assess desiccation tolerance of a plant are well known (see e.g. Jenks and Wood 2007 Plant Desiccation Tolerance, 340 p., Wiley-Blackwell, ISBN 978-0-8138-1263-2; Luttge et al. 2011 Plant Desiccation Tolerance, Ecological Studies, Vol. 215, 386 p., Springer, ISBN 978-3-642-19105-3; Khandelwal et al. 2010 Science 327: 546. Except as otherwise noted herein, therefore, assessing desiccation tolerance of a plant can be carried out in accordance with such processes. For example, desiccation tolerance can be assessed according to survival or growth rate during or after dehydration/rehydration cycles (see e.g., Example 6, Example 7).
A plant transformed with one or more transcribable nucleic acid molecules described herein can exhibit increased desiccation tolerance. For example, a plant transformed with one or more transcribable nucleic acid molecules described herein can exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 250%, at least about 500%, at least about 750%, at least about 1000%, or more, increased desiccation tolerance as compared to a non-transformed control plant of the same or similar type.
Drought tolerance is understood to be an ability of a plant to survive or yield under drought conditions despite lack of available soil water to meet its maximal needs (see generally, Salisbury, Units, Symbols, and Terminology for Plant Physiology 1996 Oxford University Press, New York, ISBN019509445X). Drought conditions are understood to be a generally insufficient availability of soil water on a meso- or macro-environmental scale to supply the maximal requirements of otherwise well-adapted plants (see generally, Salisbury, Units, Symbols, and Terminology for Plant Physiology 1996 Oxford University Press, New York, ISBN019509445X). Processes to assess drought tolerance of a plant are well known (see generally, Werner et al. 1991 Planta 186, 99-103). Except as otherwise noted herein, therefore, assessing drought tolerance of a plant can be carried out in accordance with such processes.
A plant transformed with one or more transcribable nucleic acid molecules described herein can exhibit increased drought tolerance. For example, a plant transformed with one or more transcribable nucleic acid molecules described herein can exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 250%, at least about 500%, at least about 750%, at least about 1000%, or more, increased drought tolerance as compared to a non-transformed control plant of the same or similar type.
Water use efficiency is understood to be a comparative measure of plant productivity per unit water used (see generally, Salisbury, Units, Symbols, and Terminology for Plant Physiology 1996 Oxford University Press, New York, ISBN019509445X). Agricultural water use efficiency can be defined for a growing season either as yield per unit irrigation water applied or biomass produced per unit of transpiration. Instantaneous physiological water use efficiency can be defined as moles of carbon dioxide fixed by photosynthesis per mole of water transpired. Processes to assess water use efficiency (e.g., agricultural water use efficiency or instantaneous physiological water use efficiency) of a plant are well known (e.g., LI-6400XT Portable Photosynthesis System, LI-COR Biosciences). Except as otherwise noted herein, therefore, assessing water use efficiency of a plant can be carried out in accordance with such processes.
A plant transformed with one or more transcribable nucleic acid molecules described herein can exhibit increased water use efficiency (e.g., agricultural water use efficiency or instantaneous physiological water use efficiency). For example, a plant transformed with one or more transcribable nucleic acid molecules described herein can exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 250%, at least about 500%, at least about 750%, at least about 1000%, or more, increased water use efficiency (e.g., agricultural water use efficiency or instantaneous physiological water use efficiency) as compared to a non-transformed control plant of the same or similar type.
Molecular Engineering
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
The terms “heterologous DNA sequence”, “exogenous DNA segment” or “heterologous nucleic acid,” as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
A “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
The “transcription start site” or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative.
“Operably-linked” or “functionally linked” refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
A “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
A constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3′ transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3′-untranslated region (3′ UTR). Constructs can include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
“Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term “untransformed” refers to normal cells that have not been through the transformation process.
“Wild-type” refers to a virus or organism found in nature without any known mutation.
Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLASTTM, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr. Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
“Highly stringent hybridization conditions” are defined as hybridization at 65° C. in a 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65° C. in the salt conditions of a 6×SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65° C. in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm=81.5° C.+16.6(log10[Na+])+0.41(fraction G/C content)−0.63(% formamide)−(600/l). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1-1.5° C. for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).
Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, C., et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem. Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iTTM RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinoformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3′ overhangs.
Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.
Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
EXAMPLES
The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
Example 1
Mass Spectrometric Analysis
Nano LC-MS/MS analyses were performed on an LTQ-Orbitrap XL (Thermo Fisher Scientific, Waltham, Mass., USA) equipped with a nano-ESI source and coupled to a Paradigm MG4 pump (MichromBioresources, Auburn, Calif., USA) and autosampler (HTC PAL, CTC Analytics, Zwingen, Switzerland). Peptide mixtures were separated on MagicC18AQ column (100 mm×150 mm, 3.0 mm particle size, 300 Å, MichromBioresources, Auburn, Calif., USA) with a flow rate of 500 nl/min. Linear gradient of 5-35% buffer B in buffer A for 150 min, 30-95% buffer B in buffer A for 4 min, and 95% buffer B and 5% buffer A for 5 min and finally decreased to 5% buffer B in buffer A was employed (buffer A=0.1% formic acid in 2% acetonitrile, buffer B=0.1% formic acid in 90% acetonitrile). Up to three CID and HCD spectra were acquired in a data-dependent acquisition mode following each full scan (m/z, 400-1,500).
Example 2
iTRAQ
The following example describes quantitative proteomics analysis using iTRAQ (Isobaric Tags for Relative and Absolute Quantification) to reveal the regulatory network controlled by ABA and ABI3 during the desiccation-rehydration process. A graphical overview of the proteomic analysis using iTRAQ is provided in FIG. 1.
The frozen Physcomitrella protonemata samples were crushed to frozen powder with a Multi-Beads Shocker. The frozen powder was then treated with urea lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail). After centrifugation at 13,200 rpm for 15 min, the supernatant was recovered and stored at −85° C. until use. Protein concentrations were determined with the RC-DC Protein Assay kit, using BSA as the concentration standard. A graphical overview of sample preparation for iTRAQ analysis is provided in FIG. 2.
Extracted proteins were purified by using 2D-clean up kit. Subsequently, 100 μg of each protein were dissolved in 10 μl of 7 M urea, 2 M Thiourea, 4% CHAPS and 20 μl of 1M TEAB pH8.5, reduced, alkylated and digested with trypsin according to the manufacturer's protocol. Trypsin-digested peptides were concentrated to 30 μl by a centrifugal concentrator. Samples were labeled with the iTRAQ reagent according to the manufacturer's protocol. WT without ABA was labeled with iTRAQ reagent 113 and 114, WT with ABA and dry was labeled with 115 and 116, Δabi3 without ABA was labeled with 117 and 118, Δabi3 with ABA and dry was labeled with 119 and 121. The labeled peptide samples were then pooled and desalted with Sep-Pak Light C18 Cartridges and peptides were dried in a centrifugal prior to strong cation exchange (SCX) fractionation.
iTRAQ Data Analysis.
Protein identification and quantification for iTRAQ analysis was carried out using Proteome Discoverer software (v. 1.1) (Thermo Fisher Scientific) against NCBInr database. The MS/MS data were investigated only against the Physcomitrella patens. Search parameters for peptide and MS/MS mass tolerance were 10 ppm and 0.8 Da, respectively, with allowance for two missed cleavages made from the trypsin digest. Carbamidomethylation (Cys) and iTRAQ8plex (Lys, N-terminal) were specified as static modifications, whereas iTRAQ8plex (Tyr), and oxidation (Met) were specified as variable modifications in the database search. The false discovery rate of 1% was calculated by Proteome Discoverer based on a search against a corresponding randomized database. Relative protein abundances were calculated using the ratio of iTRAQ reporter ion in the MS/MS scan.
Example 3
Identification of ABA Responsible Proteins in WT and Δabi3
Protein identification and quantification for iTRAQ analysis was carried out using Proteome Discoverer software (ver.1.1) (Thermo Fisher Scientific) against NCBInr database. The MS/MS data were investigated only against the P. patens. Also compared were proteins identified by iTRAQ analysis with mRNAs identified by microarray analysis. Also confirmed was expression of mRNA codes proteins identified with a significant change both WT and Δabi3.
Results of iTRAQ analysis identified a total of 3582 proteins as being ABA responsive, with a false discovery rate (FDR) of less than 1%. A subset of 98 proteins from this group, including 19 LEA proteins, were increased more than two point five-fold with ABA treatment (see e.g., TABLE 1). The ABA responsible proteins could be sorted into nine categories according to their biological function as described by EMBL-EBI. These categories include seed maturation (19.39%), defense (9.18%), metabolism (16.33%), protein synthesis (2.04%), ion transport (2.04%), transcription (3.06%), unclassified (7.14%), unknown moss specific (21.43%) and unknown (19.39%).
Sequences for nucleotides referred to by Accession No. and Phypa ID can be obtained at, for example, the website cosmoss.org.
Eight proteins were identified as having a significant change between WT and Δabi3 with ABA and desiccation, which are encoded by the ORF regions of the following nucleotide sequences: Accession No. gi168047556, Phypa ID 169276 (SEQ ID NO: 1); Accession No. gi167999540, Phypa ID 112415 (SEQ ID NO: 2); Accession No. gi168052229, Phypa ID 27775 (SEQ ID NO: 3); Accession No. gi168049029, Phypa ID 194258 (SEQ ID NO: 4); Accession No. gi168000434, Phypa ID 113212 (SEQ ID NO: 5); Accession No. gi168038393, Phypa ID 190133 (SEQ ID NO: 6); Accession No. gi168002106, Phypa ID 159190 (SEQ ID NO: 7); and Accession No. gi168015696, Phypa ID 122983 (SEQ ID NO: 8).
Only four proteins required both ABI3 and ABA: Accession No. gi168047556, Phypa ID 169276 (SEQ ID NO: 1); Accession No. gi167999540, Phypa ID 112415 (SEQ ID NO: 2); Accession No. gi168000434, Phypa ID 113212 (SEQ ID NO: 5); and Accession No. gi168015696, Phypa ID 122983 (SEQ ID NO: 8).







TABLE 1







Protein identification and quantification for iTRAQ analysis was carried out using Proteome Discoverer


software (ver.1.1) (Thermo Fisher Scientific) against NCBInr database. The MS/MS data were investigated only against


the P. patens. Double lines show proteins identified with a significant change between WT and Δabi3 with ABA and


desiccation by iTRAQ analysis. Blue letters shows proteins were increased more than two point five-fold with ABA treatment.



















microarray







iTRAQ
Fold change




















Up-regulation folds with ABA and dry
WT with
[Δ0] vs
[Δ24] vs


















Accession
Phypa ID
annotation
Coverage
#peptides
[WT]1
[WT]2
[Δabi3]1
[Δabi3]2
ABA
[WT0]
[WT24]




















Seed maturation




































LATE EMBRYOGENESIS ABUNDANT RELATED
9.74
4
5.882
12.767 
1.304
1.140
1541.92
−1.93
−58.14


 










Small hydrophilic plant seed protein
45.98
6
2.543
3.445
1.042
0.703
38977.95
1.01
−4.11


 


gi167997373
110912
Late embryogenesis abundant protein (LEA) family protein
34.69
19
11.652 
20.860 
39.883 
8.981
125.78
−3.12
−2.47


gi168034736
166566
late embryogenesis abundant domain-containing protein/LEA domain-containing 
32.63
14
8.804
18.853 
30.765 
4.071
18.70
−42.82
−2.11




protein











gi168042734
140531
Seed maturation protein
30.54
25
7.144
11.457 
7.383
3.522
1013.14
1.63
−1.48


gi168020025
 34885
Late embryogenesis abundant protein (LEA) family protein
59.78
26
7.066
11.882 
20.671 
8.355
53.70
−1.26
−1.08


gi168025625
233250
late embryogenesis abundant domain-containing protein/LEA domain-containing 
39.25
60
6.363
11.633 
6.322
3.023
47.79
−27.74
−2.56




protein











gi168058121
 53626
late embryogenesis abundant domain-containing protein/LEA domain-containing 
51.32
12
8.133
10.262 
6.884
4.241
59.03
−2.37
−3.85




protein











gi168055759
223670
late embryogenesis abundant domain-containing protein/LEA domain-containing
48.22
19
8.105
8.266
6.569
3.388
99.49
−3.93
−4.98




protein











gi168033892
166392
late embryogenesis abundant domain-containing protein/LEA domain-containing 
35.59
67
6.048
8.486
4.787
2.683
139.48
−38.50
−3.69




protein











gi168066331
173331
Dehydrin family protein
36.21
25
5.745
8.388
11.005 
3.498
16.60
1.27
−1.29


gi168063969
153020
Phypa_459381 mRNA sequence for Late embryogenesis abundant protein, 
22.03
6
5.503
8.335
7.841
2.530
48.37
−1.53
−1.98




group 3 (LEA) [Zea mays]











gi168058168
171483
LATE EMBRYOGENESIS ABUNDANT (PLANTS) LEA-RELATED
14.47
13
5.063
8.277
12.739 
5.634
3.25
1.54
1.34


gi168065609
154342
late embryogenesis abundant protein, putative/LEA protein, putative
26.48
12
4.235
3.092
4.757
2.627
29.53
−2.92
−3.65


gi168063809
152915
late embryogenesis abundant domain-containing protein/LEA domain-containing 
59.71
23
3.897
6.431
5.241
2.437
566.05
−8.83
−4.09




protein











gi168005886
 25919
late embryogenesis abundant protein, putative/LEA protein, putative
24.32
11
3.898
6.474
4.586
2.232
41.62
−1.18
−1.98


gi38176433
221321
Dehydrin Hoe
47.29
45
3.229
4.896
4.857
2.642
3414.72
−9.57
−14.00


gi168063260
172612
late embryogenesis abundant protein
35.16
36
3.198
5.858
7.317
3.886
1.91
−1.68
1.23


gi167997371
 64056
Late embryogenesis abundant (LEA) protein
12.9
7
2.785
4.972
5.271
2.113
—
—
—

















Defense




































Cysteine-rich secretary protein Family
11.03
2
11.242 
16.993 
5.382
3.260
844.05
−2.05
−6.88


 










LANC-LIKE PROTEIN
1.52
1
7.648
13.508 
3.565
1.965
49.86
1.13
−2.35


 


gi165001128
 65913
Hsp20/alpha crystallin family/SMALL HEAT-SHOCK PROTEIN (HSP20) 
5.23
2
4.383
4.436
2.500
1.287
—
—
—




FAMILY











gi117557347
114091
CALCIUM-DEPENDENT LIPID-BINDING PROTEIN (CALB RELATED)
5.76
6
7.369
10.927 
8.478
5.718
48.53
−1.00
−1.53


gi168053997
147280
ASPARAGINE SYNTHETASE
6.43
3
2.348
10.608 
7.725
5.489
−1.05
−1.85
−1.99


gi168031730
166082
AWPM-19-like family
14.37
10
6.455
10.595 
8.875
4.396
42.12
1.28
1.06


gi168038266
217333
14-3-3 protein
31.13
40
2.993
4.898
6.656
3.678
14.41
1.18
1.26


gi169010337
161093
NITROGEN METABOLIC REGULATION PROTEIN NMR-RELATED
28.63
31
2.781
5.180
8.105
2.463
7.78
1.52
1.38


gi165033788
 83561
proton gradient regulation 5
31.08
4
2.749
5.233
4.524
2.894
−1.88
−1.49
−1.23


gi168020452
125936
Caleasin related protein
10.62
4
2.739
4.181
3.743
3.636
40756.19
−1.23
−17.78

















Metabolism




































SHORT-CHAIN DEHYDROGENASES/REDUCTASE
5.54
4
48.871 
66.814 
1.849
2.140
5731.04
−11.86
−600.71


 










ALDO/KETO REDUCTASE
18.59
9
6.070
8.439
1.407
0.962
1780.14
−4.26
−68.22


 


gi168023967
127465
ALCOHOL DEHYDROGENASE RELATED
6.15
10
9.977
16.928 
6.469
2.861
3671.93
−4.43
−21.87


gi168021556
210293
Phosphaenolpyruvate carboxykinase
9.31
11
3.656
5.632
2.738
0.960
−1.17
−2.03
−1.06


gi168002724
202843
C-terminal domain of 1-Cys peroxidedoxin/PEROXIREDOXIN
40.72
18
7.327
10.990 
4.973
1.661
250.33
−4.79
−17.88


gi168059949
150414
INORGANIC PYROPHOSPHATASE
11.59
6
6.851
10.058 
7.487
4.838
—
—
—


gi168018523
124664
ZINC FINGER FIVE DOMAIN CONTAINING PROTEIN
14.46
15
6.693
8.366
7.938
4.187
87.36
1.46
−1.47


gi167999436
158682
LysM domain contain protein
11.55
16
4.829
6.897
5.989
3.608
73.53
1.30
−1.33


gi168008176
205052
ALDO/KETO REDUCTASE
22.12
16
3.970
5.988
4.740
2.289
25.81
1.26
−1.34


gi168057984
197372
CATALASE
15.64
20
3.746
4.530
3.893
2.046
1107.08
−2.88
−2.04


gi168003088
 25822
GRAM domain family protein
11.6
7
3.538
6.055
5.397
1.956
3.09
−1.33
−1.28


gi168032507
214778
AMINOHYDROLASE
3.97
5
3.278
4.835
4.337
1.640
3.35
−1.35
−1.74


gi168016771
123644
ALCOHOL DEHYDROGENASE RELATED
2.4
3
3.132
4.553
2.938
1.046
4.73
1.01
−2.35


gi168015766
 49632
GLUTAREDOXIN
7.07
2
3.025
4.597
3.055
1.709
67.93
1.37
−1.44


gi168029940
213846
PHOSPHOLIPASE D
9.81
10
2.755
3.331
3.538
2.389
7.22
1.86
1.36


gi168006265
176782
CARBOXYLASE:PYRUVATE/ACETYL-COA/PROPIONYL-COA
5.42
5
2.705
3.899
4.289
1.089
1.41
−1.89
−1.84


gi168059614
150204
IRON-SULFUR DOMAIN CONTAINING PROTEIN
5.06
6
2.509
3.837
2.440
1.013
1.09
−1.36
−1.25


gi168046840
193394
SHORT-CHAIN DEHYDROGENASES/REDUCTASE
10.59
4
2.501
3.485
2.039
1.485
4.56
2.09
1.34

















Protein synthesis/fate




























gi168055678
 2950
ALPHA/BETA HYDROLASE RELATED
6.42
3
6.683
10.643 
7.782
5.336
22.62
2.16
−1.12


gi168029624
213747
Thioredoxin-like protein
12.87
9
5.509
11.244 
11.237 
4.390
13.29
−1.13
−1.58


gi168061135
198530
Ribosomal protein L14
41.04
10
3.323
5.528
7.645
3.892
1.03
1.10
1.12


gi168035968
136081
Ribosomal protein L6
16.09
9
3.037
5.487
6.332
3.307
—
—
—

















Transport




























gi168022840
 77363
MYOSIN-VII
1.32
8
10.266 
17.875 
13.708 
5.168
—
—
—


gi167998408
111412
Calcium-activated chloride channel
2.55
4
6.062
9.280
8.310
4.149
180.85
1.51
−1.46


gi168065723
110643
TRANSLOCASE OF INNER MITOCHONDRIAL MEMBRANE TIM22
42.86
11
3.329
2.934
4.831
2.851
19.87
1.45
1.23


gi168020639
163517
COPPER TRANSPORT PROTEIN ATOXI-RELATED
5.26
1
3.367
4.432
4.658
2.993
4.68
1.43
1.18


gi168045663
168786
NAD DEPENDENT EPIMERASE/DEHYDRATASE
18.46
31
2.539
4.638
3.623
2.435
9.54
−1.27
−1.46

















Signal transduction and












regulation of transcription




























gi168058456
171520
AP2 domain contain protein
2.42
1
3.175
2.725
1.409
0.734
—
—
—


gi168011320
178765
TRANSFORMING GROWTH FACTOR INDUCED PROTEIN
11.45
2
4.706
2.109
6.835
2.499
5260.38
−24.95
−20.38


gi168049682
144692
EF hand-containing protein
16.05
9
4.898
6.011
3.887
2.639
37.80
1.27
−1.33


gi168003523
159544
TRANSCRIPTIONAL REPRESSOR PROTEIN YY
3.14
10
3.366
2.668
2.686
3.787
1.16
−1.70
−1.68


gi168048824
 91567
SIN3B-RELATED
1.8
9
3.664
4.632
3.698
2.285
—
—
—

















Unclassified




























gi168004061
 67470
Viral A-type inclusion protein repeat
27.67
68
8.707
14.363 
3.385
2.826
1241.23
−8.83
−40.02


gi168002515
159259
EFSD domain-containing protein
3.61
10
7.558
11.857 
8.100
6.675
−1.43
−1.15
−1.18


gi168058093
171446
MITOCHONDRIAL CARRIER PROTEIN RELATED
14.19
16
5.253
3.537
8.016
5.396
7.14
1.05
−1.41


gi168068011
173681
SOUL heme-binding family protein
11.61
5
2.861
4.963
3.141
1.917
8.71
−1.09
−1.30


gi167999364
 24243
DNA repair ATPase-related
16.92
11
2.873
4.448
3.835
1.176
5.79
1.11
−1.20

















Unknown moss specific




































there are no functional annotations for this locus
21.36
8
17.953 
36.378 
2.598
1.611
1444.27
−13.06
−122.71


 


gi168051653
170187
there are no functional annotations for this locus
13.18
15
10.449 
22.863 
8.953
2.543
290.94
−85.68
−108.25


gi168008551
160757
there are no functional annotations for this locus
19.73
9
5.398
8.476
3.107
1.380
6145.67
−1.44
−54.04


gi168059789
 96882
there are no functional annotations for this locus
3.2
3
3.539
5.337
1.968
1.223
—
—
—


gi168007348
 69345
there are no functional annotations for this locus
21.02
13
14.460 
22.863 
9.253
5.506
2842.01
−2.79
−11.70


gi168057763
171387
there are no functional annotations for this locus
38.72
60
11.339 
22.695 
12.358 
4.991
522.28
1.18
−1.94


gi168012482
161636
there are no functional annotations for this locus
43.14
71
9.932
39.421 
18.834 
5.523
240.83
−43.67
−28.37


gi168010327
 70813
there are no functional annotations for this locus
13.74
2
9.891
19.845 
9.793
5.527
—
—
—


gi168057767
171388
there are no functional annotations for this locus
34.18
24
6.623
9.543
6.791
3.823
25.07
2.22
−1.32


gi168008419
160688
there are no functional annotations for this locus
22.9
10
6.769
33.766 
11.196 
3.456
8379.37
−4.08
−4.54


gi168024980
229196
there are no functional annotations for this locus
12.75
11
5.871
8.356
3.917
2.055
744.02
−2.72
−38.98


gi168039669
232091
there are no functional annotations for this locus
11.4
10
4.773
8.798
31.42
2.210
1357.51
−2.94
−4.95


gi168041604
 87717
there are no functional annotations for this locus
6.89
4
4.240
7.736
4.123
1.724
—
—
—


gi168033047
233882
there are no functional annotations for this locus
20.55
6
3.766
6.352
13.538 
4.122
204.00
−1.43
−1.33


gi167998520
 64336
there are no functional annotations for this locus
17.25
21
3.375
4.705
2.678
3.358
116.59
1.86
1.21


gi168053822
 99184
there are no functional annotations for this locus
6.96
1
3.297
5.506
4.099
1.866
—
—
—


gi168066383
173340
there are no functional annotations for this locus
43.15
99
3.367
4.891
5.914
2.746
67.40
−1.17
−1.39


gi168004453
159758
there are no functional annotations for this locus
6.44
9
2.943
4.315
4.528
2.315
3548.78
−1.90
−2.35


gi168052182
170304
there are no functional annotations for this locus
15.38
18
2.928
5.398
5.494
2.943
157.33
−1.11
−1.57


gi168019178
163180
there are no functional annotations for this locus
19.23
5
2.773
4.078
3.657
3.082
2.51
1.91
1.66


gi168041447
 87628
there are no functional annotations for this locus
2.09
4
2.659
4.285
3.595
3.712
—
—
—

















Unknown




































there are no functional annotations for this locus
30.41
18
15.344 
23.949 
1.718
1.459
304.25
−1.38
−7.69


 


gi167999378
 64955
unknown
4.91
3
6.898
8.994
2.643
5.668
—
—
—


gi168003483
159520
there are no functional annotations for this locus
35.48
34
8.882
11.253 
9.372
5.286
73.72
−5.87
−1.86


gi168044535
168559
Protein of unknown function (DUF1253)
2.53
4
5.140
8.104
3.588
1.886
1.09
1.11
1.16


gi168040629
167699
Protein of unknown function (DUF567)
21.43
15
4.223
5.449
8.505
3.811
9.15
1.35
1.01


gi168039405
107294
Protein of unknown function (DUF567)
24.24
16
4.024
4.405
3.956
2.356
196.52
−1.07
−1.29


gi167999195
 64697
there are no functional annotations for this locus
6.5
4
3.863
8.858
9.538
4.495
—
—
—


gi168052628
 93565
unknown
6.42
4
3.590
4.859
3.938
2.119
1.34
1.02
−1.02


gi168036875
189292
there are no functional annotations for this locus
11.36
6
3.588
5.008
5.917
3.670
161.37
1.61
−1.59


gi168064705
 99618
unknown
4.47
7
3.363
4.287
3.749
1.972
—
—
—


gi168046560
235391
unknown
24
24
2.781
3.119
4.376
2.128
3290.74
−4.37
−4.47









Example 4
Analysis OF mRNA Expression
Northern blot analysis of mRNA expression was conducted for the eight genes (Accession No. gi168047556, Phypa ID 169276; Accession No. gi167999540, Phypa ID 112415; Accession No. gi168049029, Phypa ID 194258; Accession No. gi168000434, Phypa ID 113212; Accession No. gi168038393, Phypa ID 190133; Accession No. gi168002106, Phypa ID 159190; Accession No. gi168015696, Phypa ID 122983; and Accession No. gi168052229, Phypa ID 27775) significantly changed between WT and A abi3 with ABA and desiccation treatment.
Exemplary results are depicted in FIG. 3.
Example 5
Transgenic Moss Methods
This example describes methods for isolation of transgenic moss specific to the used vector. Necessary molecular characterization (locus genotyping and copy number evaluation) of such transgenic plants is not part of the present protocol, but follow standard techniques in common use in plant molecular biology field. Similarly, vector cloning (specific to each project) as well DNA amplification methods and restriction cut needed prior any moss specific procedure are described here. Maps and sequences of both vectors used in this particular project are available.
Transformation methods are consistent with Cove et al. (2008) The Moss Physcomitrella patens. A Novel Model System for Plant Development and Genomic Studies. IN: Emerging Model Organisms, a Laboratory Manual, Volume I. (Eds: D. A. Crotty and A. Gann). Chapter 3, pp. 69-104. Cold Spring Harbor Laboratory Press, New York. Desiccation assay methods are consistent with Werner et al 1991 Planta 186, 99-103; Charron and Quatrano 2009 Molecular Plant 2, 478-486; Khandelwal et al. 2010 Science 327, 546; and Yotsui et al. 2013 New Phytologist 199, 101-109.
Gene deletion methods are consistent with Cove et al. (2008) The Moss Physcomitrella patens, A Novel Model System for Plant Development and Genomic Studies, IN: Emerging Model Organisms, a Laboratory Manual, Vol I, Eds Crotty and Gann, Chapter 3, pp. 69-104, Cold Spring Harbor Laboratory Press, New York; Perroud and Quatrano 2008 The Plant Cell 20, 411-422; and Marella et al. 2006 The Plant Journal 46, 1032-1044. Desiccation assay methods are consistent with Werner et al 1991 Planta 186, 99-103; Charron and Quatrano 2009 Molecular Plant 2, 478-486; Khandelwal et al. 2010 Science 327, 546; and Yotsui et al. 2013 New Phytologist 199, 101-109.
Methods are described in three different protocols: protoplast production, protoplast transformation, and protoplast regeneration and plant selection. Such procedure was shown to work similarly with both strains used in the study Gg-1 (female) and R40 (male).
Protocols described hereafter are performed at room temperature (<22° C.) if not mentioned otherwise and in a sterile environment, e.g. vertical or horizontal laminar flow hood. Standard in vitro plant precautions concerning sterility (e.g. surface cleaning, reagent filtering and autoclaving) are required.
Protoplast Production.
Tissue from four Petri dishes of six-days old moss protonemata grown on BCDA medium in a Petri dish are harvested and transferred rapidly into 15 ml of 8.5% mannitol without letting the tissue dry. Five ml of driselase 2% (for a final driselase concentration of 0.5%) is added and incubated 45 minutes at room temperature with occasional gentle plate swirling. The suspension should be green after 45 minutes with a rapidly sedimenting colored component, protoplasts. The suspension is filtered through 100 μm sieve and let incubate at room temperature for 15 minutes. The suspension is filtered to 30-35 μm sieve and the flow-though containing isolated protoplasts are poured into a tube that can withstand low speed centrifugation (e.g. 50 ml conical “Falcon” tube). The tube is centrifuge for 5 minutes at 250 g to sediment the protoplast.
At this stage, a dark green pellet should be visible and the supernatant should be brownish (driselase color), not green. The supernatant is discarded and the pellet resuspended into 20 ml of mannitol 8.5% supplemented with 10 mM CaCl2 (wash #1), then centrifuged 5 minutes at 250 g to sediment the protoplast. At this stage, a dark green pellet should be visible and the supernatant should be mostly clear. Resuspension of the pellet and centrifugation are repeated (wash #2).
The pellet is resuspended into 20 ml of mannitol 8.5% supplemented with 10 mM CaCl2 and the protoplast number is evaluated in suspension using a hemacytometer (Fisher Scientific, USA, Cat. #0267110).
Thus is provided a suspension with a known number of protoplast that can be used for transformation (see below). Alternatively, protoplast can be plated on PRMB for a regeneration test. Typical protoplast yield is one million protoplasts per Petri dish plate.
Protoplast Transformation.
In a 15 ml sterile tube, 15 μg of vector DNA was resuspended in sterile TE with a maximum of 30 μl volume. This DNA can be produced by standard PCR amplification or plasmid prep amplification. With plasmid amplified DNA, however, the vector must be cut on each side of the transformation vector producing a linear, open-ended DNA fragment for transfection. This fragment type improves DNA integration and reduces the number of episomal transient transformants.
Added to the TE plus DNA is 300 μl of protoplast resuspended in MMM buffer. Protoplast concentration in the MMM buffer can be anywhere between 1.2×106 to 1.6×106 without affecting transformation efficiency, with the volume ratio for each component presented in this protocol. 300 μl of PEG transformation solution is added and mixed gently but thoroughly, producing visually uniform solution. The tube is incubated for 5 minutes in a 45° C. water bath then allowed to stand at room temperature for 10 minutes.
The PEG transformation solution is toxic to the protoplasts. Using a serial dilution approach (one dilution per minute), the transformation mix is added to successively five times 300 μl, then five times 1 ml of mannitol 8.5% supplemented with 10 mM CaCl2. The tube is mixed gently by swirling after each dilution step. After dilution, the tube is allowed to stand at room temperature for 30 minutes. At this stage protoplasts have been transformed and are ready to be plated.
To concentrate, the protoplast suspension is centrifuged for 5 minutes at 250 g to sediment the protoplast. Supernatant is discarded and up to 2 ml of mannitol 8.5% supplemented with 10 mM CaCl2 is added. Three ml of 45° C. PRMT (previously melted in the microwave and cooled down to 45° C. in a water bath) is added and mixed well by pipetting. 1.25 ml of the mix is quickly poured per PRMB plate overlaid with sterile cellophane (4 plates per transformation tube). Plates are allowed to stand for 30 minutes to one hour in a laminar flow hood (covered and sealed) before transferring the plate to the an incubator set at 25° C. and with a long light cycle (16 hours light/8 hours dark).
Plant Regeneration and Selection.
Week 1: Protoplast Regeneration.
Protoplasts are grown for 6 to 7 days on the plates poured for the transformation. Plates are observed for: 1) the number of dividing protoplasts, since this is the actually number that count to evaluate transformation efficiency (it should be as high as possible), and 2) bacterial or fungal contamination. After a week of growth, there should be between 2 to 15 cells per plant.
Week 2: First Selection.
Cellophane containing the regenerating protoplasts is transferred onto a BDCA plate supplemented with the appropriate antibiotic (for which resistance is present in the transformation vector) (e.g., Hygromicin B at 25 μg/L). Plates are incubated for a week in standard growth conditions. After this step, there should be left about 50 to 250 growing plants.
Week 3-4: Release from First Selection.
Cellophane containing living transformants and dead non-transformants are transferred onto a BDCA plate. Most of the growing moss plants are still transient transformants. The two weeks of selection release allows strong growth of the plants leading to dilution of the episomal element so such plant will end up to be antibiotic sensitive and die in the next selection step.
Week 5: Second Selection.
Cellophane is transferred onto BDCA plate supplemented with the appropriate antibiotic (for which resistance is present in the transformation vector) (e.g., Hygromicin B at 25 μg/l). This step will kill up to 90% of the remaining growing plants, leaving stable (or chromosome integrated) transformant.
Picking Transformants.
After 6-8 days of second selection, a stereoscope is used to pick any plant still growing and transfer such plant onto BCDA medium for tissue amplification. Filament (the most sensitive cell of a transient transformant) growing at the edge of plants are examined as an indicator of stably transformed plants.
Example 6
Overexpression of PHYPA ID 169276
The following example describes overexpression of Phypa ID 169276. Methods are consistent with Example 5 except as indicated otherwise.
A protein corresponding to Accession No. gi168047556, Phypa ID 169276, was over-expressed in Δabi3 and WT. Resulting over-expressing lines were subjected to a desiccation assay, in which tissues from 6-day-old over-expressing lines were treated with ABA (10 μM) for 24 hours, tissues were dried for 30 hours, rehydrated with sterile distilled water, and incubated for 1 week.
Results showed that over-expression lines of protein Phypa—169276 in provided desiccation tolerance when treated with ABA, similar to WT (see e.g., FIG. 4).
Results also showed that over-expression lines of Phypa—169276 in WT acquired stronger desiccation tolerance with ABA than WT (see e.g., FIG. 5). These results support that Phypa—169276 plays a role in desiccation tolerance in P. patens vegetative tissue.
These results support that the gene regulatory pathway that requires both ABA and ABI3 control a relatively few proteins, and that these proteins can be used to increase desiccation tolerance, increase drought tolerance or increase water use efficiency in plant tissues.
Example 7
Knockout Studies
The following example describes knock out studies. Methods are consistent with Example 5 except as indicated otherwise.
Using targeted gene deletion techniques, deletion lines were developed for of the four genes shown to require both ABI3 and ABA as follows: Accession No. gi168047556, Phypa ID 169276 (SEQ ID NO: 1); Accession No. gi167999540, Phypa ID 112415 (SEQ ID NO: 2); Accession No. gi168000434, Phypa ID 113212 (SEQ ID NO: 5); and Accession No. gi168015696, Phypa ID 122983 (SEQ ID NO: 8).
Each knock-out line was subjected to a desiccation assay, in which tissues from 6-day-old knock-out lines were treated with ABA (10 μM) for 24 hours, tissues were dried for 30 hours, rehydrated with sterile distilled water, and incubated for 1 week.
Results showed that lines with Accession No. gi168047556, Phypa ID 169276 (SEQ ID NO: 1); Accession No. gi167999540, Phypa ID 112415 (SEQ ID NO: 2); or Accession No. gi168000434, Phypa ID 113212 (SEQ ID NO: 5) knocked out were approximately as tolerant to desiccation as controls.
But the line with Accession No. gi168015696, Phypa ID 122983 (SEQ ID NO: 8) knock out was clearly not as tolerant to desiccation as controls (or other knock outs) and exhibited reduced growth. Experiments with the SEQ ID NO: 8 knock out line were repeated with similar results.
Example 8
Multiple Knockout Studies
The following example describes prophetic multiple knock out studies. Methods are consistent with Example 5 except as indicated otherwise.
Using targeted gene deletion techniques, deletion lines are developed for combinations of the four genes shown to require both ABI3 and ABA as follows: Accession No. gi168047556, Phypa ID 169276 (SEQ ID NO: 1); Accession No. gi167999540, Phypa ID 112415 (SEQ ID NO: 2); Accession No. gi168000434, Phypa ID 113212 (SEQ ID NO: 5); and Accession No. gi168015696, Phypa ID 122983 (SEQ ID NO: 8).
Accession No. gi168015696, Phypa ID 122983 (SEQ ID NO: 8) knock out is tested in combination with each of the other three knock outs as well as in combinations of two of the three and all three. Exemplary knock out combinations are as follows:
SEQ ID NO: 8 and SEQ ID NO: 1
SEQ ID NO: 8 and SEQ ID NO: 2
SEQ ID NO: 8 and SEQ ID NO: 5
SEQ ID NO: 8, SEQ ID NO: 1, and SEQ ID NO: 2
SEQ ID NO: 8, SEQ ID NO: 1, and SEQ ID NO: 5
SEQ ID NO: 8, SEQ ID NO: 2, and SEQ ID NO: 5
SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5
Results are expected to show that lines having multiple knockouts of the four genes show poorer performance in the desiccation assay or do not survive the desiccation assay, especially those knockouts of SEQ ID NO: 8 in combination with one or more gene knock outs.










SEQUENCE LISTING















SEQ ID NO: 1


Coding region of Phypa_169276


ATGGCAACAGTGAACGGTACATCAATAATGCAGACTACCAACTTAGCAATCACACCCGTGGCTGCAAGCCA


TTTGGGCTCGCGCACCGTCCAGCTCGGAGTCACACGCGTACTCTACAACGCCACCCTTACCACCAGGTCTG


CAAGGAGGCTCGTCGTGGTCGCCGCCGGAAACCCGGAACGAATTGACAACTTCGTCGACGGAGCCAGGAAA


GACGCCAGCCAAAACGCGAAGAACTTCGGCAACCAGGTGGCGGACGCCTTCGGCAACGCCCAGGAGACCGC


GAAGGACGTGGGTCGGGACATGGGCGCCAAGGCACAGGAAGCCGTCGACCAAGGCAGTAAGAAGGTCGACG


AGGCAGGAGACAAGGCCAGGGATGTAGCCAAGGAAGTGAGGGGCTCCACCGAGGATACCTTGAATGCTGCC


AAAGCAAACAACGGCCAATCTGTTGCGGACAAGGCGCAAAATGCTGCATCCAACGTGGGTGACAATTTGAA


GCAGAACTTCGATTACGGAACTGGGGCCGTGCAGAACGCCGCCGATGACGCGAGCAAAAACGTGAAAGACG


CCACTAACCGCAACCTCTAG


 


SEQ ID NO: 2


Coding region of Phypa_112415


ATGTCGAGCCAGGAAGATCTTGACGCCAAGGCCGCCGCTGGAGAGACGGTGGTGCCCGGAGGCACTGGCGG


AAGGTCCTTTCAAGCTCAGAAGAACCTTGCAGAAGGCCGCAGCAAAGGAGGGCAAGCTCGTGCGGAGCAGC


TGGGTCATGAAGGATACGTAGAGATGGGCAAGAAGGGAGGCTCTGCTACAACTGACATGTCCAGTGGAGGC


GCTGCCGAGGCTGCCGGTCGCGGCATCGACGAGACCAAGTTCACCACCTAG


 


SEQ ID NO: 3


Coding region of Phypa_27775


ATGAAGATGACGGTGGTTGCGGTGATGGCCGTGGTTCTGATCCTCGGTAGCGAGCTGCCTCGCTACTCGAG


CGCAGTTTCGAGCGAGGCTGAGGAGTTTGTGTCAGCGCACAACGCAGCGAGGGCTGATGTGGACGTAGGGC


CTTTGGTTTGGAGCCACAAGCTGGAGGATTACGCGCGCAAGTACGGGGAGGAGCAGCGCGATCATCACAAT


TGCGCCATGGTGCACTCGCGGGGTCCGTATGGGGAGAACCTTTTCTGGGGCTACGGGAAGTCGTTTGCGCC


CGCGGATGCCGTGCGCTCGTGGGTGGATGAGAAACAGCACTACGACTACGACTCGAATTCCTGCGCGTCGG


GGAAGGTGTGCGGGCACTATACGCAGGTGGTGTGGGCGGACACAAAGGAGGTCGGGTGCGCTTCAATCACC


TGCCATGACAAGGCCACGTTCATCATCTGCAGCTATAATCCGCCTGGCAACTTCGTGGGCGAATGGCCATA


CAAGCGTGCCGGAACCAAGCATTCACATAGGCGATCTCATGAAAACGACGACTCTCGCACAAGCTCCCATA


AAACCACAGCCCAGACCACAGACGATGCCAACTCCGACCTCAAGCTCCGCTCAGTACATGAGTGTATGAGC


CAGTGTATGAATGATTGCTTCTCGATGTCTGGGTCCAGCCATCACCATCGCTCGAACTACTATCAGTAG


 


SEQ ID NO: 4


Coding region of Phypa_194258


ATGGCTAATCGGTACTTCCCCAACACTATGCCAGATTATTCATTGAGTTTGGAAGGCTCAGAAGCGGATTA


TTCAGTGATGGATCCTGACACGCTGCTGGAGGTCGCCATGTCCATCAAAGATGAGGTGATACAAGCAACAT


GGATACGAAAAGGCCGAAGGGTTTCTGACCCCATTCTCTACACAGGGGTGTTGGGAACAGCTTTTCTGTGC


TTTAAGGCATATCAAATTACAGGAAGCAAAGAGGATCTCACACTCTGCAGCGAGATCGTTGATAGTTGTAC


CGTTGCCGCTAAATCTTTGCACAAATACGTCACTTTTTTATGTGGGCAGCCAGGCATATATGCCTTAGGAG


CAGCAGCGGCAAAAAGCAGCGGGGATGAGCAAAGTCTTCATCCATATCTTCAGCTTTTCCATAAGGTTTCC


AAAAACCAAACTCTTGCTGTGGGAGCGGAAGAGGGTGGTATGGGAATGCCTTACGAACTTTTATATGGACG


TGCGGGGTTCTTATGGGCTGCTCTCTTTGTCAACAAGCATGTCGGCGAGGGGACCATTCCTTCGAGCACTA


CGGGTCCCATAGTTGATGCGATTTTAGCTGGCGGACGAGCTGGAGCTTCACAAACACAGTCTCCATTGATG


TATCAGTGGCACGGATCAAGATATTGGGGAGGAGCACATGGTCTGGCTGGTATCATGCACACTCTCATGCA


CTTTCCCTTGAACAAAAAAGATGAAGAGGATGTCAAGGGAACACTACGATACATGATCGCGCGCCGCTTTC


CTAGTGGCAACTACCCATCCAGCGAGGGCAATGCAACAGACCGGTTGGTGCATTGGTGCCACGGAGCACCT


GGTATTGCCATGACTTTGTGCAAGGCGTCTAAGGTATTTCCTGATGAAATGGAGTTCCAACATGCAGCAGT


TGAAGCAGGAGATGTCGTATGGAGTCGAGGTCTACTTCGGAAGTTGGGCCTCTGTCATGGGATCAGTGGAA


ATACCTACGTCTTTCTGTCTTTGTACCAATCAACAGGAGGGAAACAGCACCTCTTCAGAGCTCAACAATTC


GCCACTTTCCTTCACAAAAACGCCAGAACGTTGATCGAGTCAGGAGAGATGCACGGCGGTGACCATCCCTA


CTCTCTGTTTGAAGGCTTGGCAGGAACTGCATGCCTCTTTTTTGACTTGACAAAACCAGAGATGGCAAGAT


TCCCTGCTTATGAGCTTTAG


 


SEQ ID NO: 5


Coding region of Phypa_113212


ATGGCATCGTACAAGAGGACCTGTACAAGTGGCAACACGGCTTGCTGGGCACTGGACACATCAAATGGATT


GCATGACGCTCTCTGGGGCCTTGTCGTCATGCACCACCGCAATGGCCATTACCCGAGTCTGGAAGCATCGC


TAGCATTTCGATCCTCTCCCTCGCCTTCAAAACCTCCGGGGAACACTCCAACCATCGCAGTCGTCATTCAA


GTGAATTCCATTCTCGTATTGCACCGAACAAGAACTTTTCTGTTGACCAGTGATCTTGAATCTTTCGTAAA


TTTTAGCTGTAGTCGGATTTACGTTTTCGCAATGGCGGAATTCCGACCCGCACAAGAGCAGTCGCAGCACC


CTGGAGAGGAGCATTTGATGGATCCTGTGCCTCGTCACCACGGAACCAACTACAAGGCTGCTGGCAAGTTG


AAGGGAAAGATAGCTCTAGTGACAGGCGGTGACTCCGGCATAGGTCGTGCCGTGGGCGTGCTCTTCACACG


AGAAGGTGCCACAGTGGCCTTCACATATGTGAAAGGAGCGGAAGAAAAAGATGCGGTAGACACGATTAATT


TGCTGAAGCAGTACAAGGCAGAAGGTGGTGGCGAAGGAGAACCTCTTGCAATTCCCTGCGATCTAGGATTC


GACGAGCAGTGCAAGAAGGTTGTAGACAAAGTCGTTGAGAAGTATGGCCGAATCGACATCCTGGTCAACAA


TGCGGCAGAGCAGCACGTCGTGGAGAACATTGAGGATCTCCAGCCTGAGCAGCTGGAGCGGACGTTCCGCA


CCAACATCTTCTCCCAGTTTTACCTCGTAAGACATGCCTTGAAGCATATGAAAGAGGGTAGCTGTATTATT


AATACGACTTCCGTTAATGCATTCAAGGGCAACACCACACTTCTAGACTACACTTCCACAAAGGGCGCCAT


TCTTGCCTTCACTCGGGGACTGGCTCTTCAGCTTGTCAAGCGCGGAATTCGAGTAAATGCGGTAGCGCCAG


GCCCAATTTGGACGCCGCTCATTCCAGCATCAATGGGTCAAGGATCGCCTGAGAAGATGAAATCCTTCGGT


TCGCAATGCCCAATGGGCCGTGCTGGTGAGCCAGAGGAAATCGCAACGGCGTATGTTTTCCTTGCTTCAGA


GGATTCCTCTTACTTCACTGGCCAAACCCTGCACCCGAACGGTGGAATAGTTGTGAACGCATAA


 


SEQ ID NO: 6


Coding region of Phypa_190133


ATGGTGAATCAAGCATTGGAGGCTATGCCGAAGCTTAAGTTGAACACTGGCACATGTATTTCCGCCGTTGG


GCTAGGAACCTGGCAGGCGGATCCAGGCTTGGTTAAACAAGCTGTCAAGGAAGCTGTAAAAGTTGGGTACC


GCCACATCGACTGCGCCAAGGCTTACAAAAACGAGGACGAGGTGGGAGAAGCTTTGCAGGAGCTTTTCAAG


GAAGGCGTTGTTAAGCGTGAAGACTTGTGGATCACGTCGAAACTTTGGTGCACTGACCACAACCCTGCGGA


TGTGGAGCCGGCATTGGACGGGTCGATTGAGCGTTTGCAATGTGGCTACCTTGATCTGTACCTGATGCATT


GGCCGGTCGCCTTGAAGAAGGATGCTCAAGGCACAGGTCCAGATGACTTTGCTCCTCTCAATGTCGCGGCG


ACATGGGCAGCTATGGAAAAGTGCTACGAGAAGGGGAAAGCGAAGGCTATCGGAATCAGTAACTTCTCTGT


AGAGAAAACGAAGGACTTGTTGTCTAAATGCAAAGTACGACCTGCTGTGAACCAGGTGGAATGCCACCCTC


TTTGGCAGCAGAAGAAGTTGTGGCCATACTTAAAGTCCGAGGGCATTCACTTGACAGCTTACTCTCCAATC


GGATCCTCAAACAGTCCCTTCGCAACAATTAACGTCTTAGAGCTTCCCACAGTCACGAAATTGGCTGAGAA


ATACAAAAGGTCTCCATCACAGATAGTTCTCCGATGGAACATCCAACAAGGTCATAGCGTGCTGCCAAAGA


GCACTCACGCTGATCGGCTTGCGTCCAACATTGAGATCTTCGACTTCGAACTCAATGAAGAAGATCTCAAG


GAATTCGACAAAATTGAGCAGCACCGCCTTCTCCTTGGAGACGACATGTGGATAAATGACAAAACCAGTCC


ATACAAGACGGTGGAGGAGCTCTGGGACGGAGACATCTAA


 


SEQ ID NO: 7


Coding region of Phypa_159190


ATGGCGAAGTCCGCCGCCGCCGTGGTGATATGTGTGCTTTTCTTGGGGGTGTTCATGGGGACTCCTGCTGT


GGCGAGCAGGAAGATGTGCAAAGGAGCGGTTTCCCTCGCCATTCGTGCGCTTTGCATTCCTACCACAGAGT


CGTTGTTCATGAAGCACGATGGGAAGCACTCGTGCGCGTATAATTTGACTAACGGTGACAGAGCTGTTGGT


GTGGCGTATAACTTGGATGATGACGTAGAGTCTCGCCGGTCTGAGCTTACTGCTGTTTTTGCTGACTACGA


CAAGGTTTACGAAGGGAAAGACTGCCTGAACACAATCCAGATTAGTGCCCTGCTCACTTTAGATGCTAAAC


GTGCCCTTGATAGAGCTGCCAAAAGCGTTAAGTCCTTAGACGACCAGTGCTGCGACGTCATGGCCGTTTTT


GGCGACATCCAACACTCTGGAGGCAAGGACGTGTACCAGGAAAATGCGTTTGATGACTTCATTGAAGCTGT


GAGTGCCAAGAAATGGGAGAAGGGAGTCGAGTTGCTGGATAGAACCAGGTGGTGCAATGACAACAAAGATA


GATGTGATGACGATAAAAAGATCATCTCTGAAGGTTGCAAGGGTACATTCGCCACTGGAGTCATTGCTCAG


GTCACCGACGCTCTGAAAGCTTAG


 


SEQ ID NO: 8


Coding region of Phypa_122983


ATGATGGGTACTCGGAACGTGTTGGTTTTTGCTGTTGGGTTGTTTCTTGTGCTGAGCTTTGCTGCAGCTAG


CGGCAGTAAACATGACGACAACCATGAGCTGATGATCGGGGGGCTGGACATCGACTACAAGTGCCGGCCCG


GGAACTATACCACCACCAAGGACAAGGAGCTCACGCAGGTTGCTCTGAACTTGGAGTATTTCGAAGCGGAG


TATTTCTTGTGGGGAGCTTACGGCTATGGTTTGGACAAGATTGCTCCCTACTTGGTCGATGGCGGTCCCCC


CCCCATTGGTGCTCAGAAGGCCAATTTGGACGCTTACTATACCGATATCTACATTCAGATGGGGCTTCAGG


AAGTTGGACATCTCAGGGCGATCAAGAGAGCTTTGGGTGATCCTCCCAGGTGTGCATTCCCTCGCACTCAG


CTGGACATTAGCAAAAAGACCTGGGCCGATACGATGGACAAGGCGTTCCTGCAGACTTTCGGCGAGAAGCT


GAACCCTCCCTATGATCCTTATGAAGACAGCTTGAAGTACCTCATCTCCACGTACACCATTCCCTACGTTG


GGTTGACGGGATACGTCGGCGCTAACCCCGAGTTGAAGGGATACAATGCCAAGAAGCTTGTGGCTGGTTTG


CTGGGTGTGGAGTCGGGTCAAGACGCTATCATTCGCACCGAAATGTACCGCCAGAAGAACAAGAAGGTCAG


CCCGTACAAGTACACCGTGGCGGATTTCAGCAACGCCATTTCCAACTTGAGGAACAACTTGTCCCACGCTT


TCGTTGACGAAGGATTGGTGGTGCCAAATGAGCTGGGTGCTGAAATGATGGTGACTGGTAACATATTGTCT


GCTGACAACGACTCTCTCTCCTACCCTCGTACTGCCGAGCAGGTGTTCGAAACTGTTTACGGGACCGGAGA


TGCTTCCAAGCCCGGAGGCTTCTATCCCAAGGGCTGTCAGGGTGTCATCGCCGCGAGTTACCTTGATTAG

## Claims

1. A transgenic plant engineered to have increased desiccation tolerance, increased drought tolerance or increased water use efficiency, the plant transformed with an artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription:
a promoter that functions in a plant;
a polynucleotide consisting of SEQ ID NO: 8; and
a transcriptional termination sequence;
wherein the transgenic plant has an increased desiccation tolerance, increased drought tolerance or increased water use efficiency compared to a plant without the DNA construct.

2. The transgenic plant of claim 1, wherein
(a) the DNA construct further comprises at least one polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; or
(b) the plant comprises a second DNA construct comprising a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

3. The transgenic plant of claim 2, wherein the plant comprises SEQ ID NO: 8 and one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

4. The transgenic plant of claim 2, wherein the plant comprises SEQ ID NO: 8 and one or more of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5.

5. The transgenic plant of claim 4, wherein the plant comprises:
SEQ ID NO: 8 and SEQ ID NO: 1;
SEQ ID NO: 8 and SEQ ID NO: 2;
SEQ ID NO: 8 and SEQ ID NO: 5;
SEQ ID NO: 8, SEQ ID NO: 1, and SEQ ID NO: 2;
SEQ ID NO: 8, SEQ ID NO: 1, and SEQ ID NO: 5;
SEQ ID NO: 8, SEQ ID NO: 2, and SEQ ID NO: 5; or
SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5.

6. The transgenic plant of claim 1, wherein the promoter is an inducible promoter.

7. The transgenic plant of claim 1, wherein the promoter is a tissue-specific promoter.

8. The transgenic plant of claim 7, wherein the promoter is a seed-specific promoter.

9. The transgenic plant of claim 1, wherein the drought tolerance is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 250%, at least about 500%, at least about 750%, or at least about 1000%, as compared to a plant not comprising the DNA construct.

10. The transgenic plant of claim 1, wherein the water use efficiency is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 250%, at least about 500%, at least about 750%, or at least about 1000%, as compared to a plant not comprising the DNA construct.

11. The transgenic plant of claim 1, wherein the plant is selected from the group consisting of maize, bean, soybean, wheat, barley, hay, rice, peanut, cotton, tomato, cucurbit, okra, eggplant, pepper, sugar beet, sugarcane, cassava, potatoes, palm, rapeseed, sunflower, coconut, olive, flax, safflower, sesame, apple, pear, grape, strawberry, blackcurrant, redcurrant, gooseberry, guava, lucuma, chili pepper, pomegranate, kiwifruit, cranberry, blueberry, blackberry, raspberry, boysenberry, banana, plum, cherry, peach, apricot, mango, orange, lime, lemon, grapefruit, pineapple, fig, mulberry, hedge apple, osage-orange, and breadfruit.

12. A plant part of the transgenic plant of claim 1.

13. An artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription:
a promoter that functions in a plant;
a polynucleotide consisting of SEQ ID NO: 8; and
a transcriptional termination sequence;
wherein the promoter is heterologous to SEQ ID NO: 8.

14. A method of increasing desiccation tolerance, drought tolerance, or water use efficiency of a plant comprising:
transforming a plant with an artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription:
a promoter that functions in a plant;
a polynucleotide consisting of SEQ ID NO: 8; and
a transcriptional termination sequence;
wherein the transgenic plant has an increased desiccation tolerance, increased drought tolerance, or increased water use efficiency compared to a plant without the DNA construct.