Plants resilient to environmental stresses

Abstract

Plants, seeds, or parts thereof with increased or enhanced tolerance or resilience to abiotic or environmental stresses via stably integrated tardigrade CAHS transgenes conferring such resilience. CAHS sequences, vectors, and methods thereof for increasing or enhancing tolerance or resilience to abiotic or environmental stresses as compared to untreated controls.

Description

[0001] PLANTS RESILIENT TO ENVIRONMENTAL STRESSES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the priority benefit of U. S. Provisional Patent Application Serial No. 63 / 753,708, filed February 4, 2025, entitled WHEAT RESILIENT TO ENVIRONMENTAL STRESSES, incorporated by reference in its entirety herein.

[0004] SEQUENCE LISTING

[0005] The following application contains a sequence listing submitted electronically as a Standard ST.26 compliant XML file entitled "61657SequenceListing.xml," created on February 4, 2026, as 33,631 bytes in size, the contents of which are incorporated herein.

[0006] BACKGROUND

[0007] Technical Field

[0008] The present disclosure relates to increasing tolerances to a number of environmental extremes (e.g., abiotic stressors) by expressing tardigrade-specific Cytosolic- Abundant Heat Soluble (CAHS) proteins in plants resulting in more resilient plants to these extremes.

[0009] Description of Related Art

[0010] Climate change is an increasing concern worldwide, particularly for agriculture. Extreme weather events, including excessively hot or cold temperatures and droughts, are becoming more frequent and significant, negatively impacting crop production. These abiotic stress adversely affects the growth and development of plants and limits agricultural productivity, often causing losses of billions of dollars per year. Molecularly transforming plants by inserting specific DNA sequences into their genomes is one way to create new varieties with desired traits, such as resistance to abiotic stress. One approach involves transferring the target molecular function from one species with a desirable trait to another species that needs that trait.

[0011] Tardigrades (also known as water bears) are a phylum of eight-legged segmented microinvertebrates and are among the most resilient animals known, with individual species able to survive extreme conditions - such as exposure to extreme temperatures, extreme pressures (both high and low), air deprivation, radiation, dehydration / desiccation, freezing, and starvation - that would quickly kill most other forms of life. Tardigrades have even survived exposure to outer space radiation conditions. Various species of tardigrades are known, with Hypsibius exemplaris being the currentcorrect name for the specific species widely used in scientific research today, although prior to 2018 this species of research tardigrade was incorrectly identified as Hypsibius dujardini. While both names are in literature, in 2018, researchers conducted a detailed morphological and genetic analysis of the specific “Z151” culture used in labs worldwide and found it was quite different compared to the wild-type species, H. dujardini, found in Europe populations (see Gqsiorek, P., Stec, D., Morek, W., & Michalczyk, L. (2018). An integrative redescription of Hypsibius dujardini (Doyere, 1840), the nominal taxon for Hypsibioidea (Tardigrada: Eutardigrada). Zootaxa, 4415(1), 45-75). The “laboratory” tardigrade (77. exemplaris) was the species that was fully sequenced first and from which all the genomic data sequences were derived. Therefore, it is worth noting that before 2018, almost every paper mentioning 77. dujardini in a laboratory or research context is likely actually referring to the animal that we now know as 77. exemplaris.

[0012] One reason for a tardigrade’s impressive ability to withstand harsh environmental stresses is special proteins that help it tolerate extremes. These proteins act as a shield for water bears. One class of proteins, Cytosolic-abundant heat soluble (CAHS) proteins, have been shown to provide cellular protection to environmental extremes in transgenic yeast, bacteria, cyanobacteria, and human cells.

[0013] CAHS proteins are defined by a repetitive, short sequence motif that confers intrinsic disorder and unique biophysical properties essential for cellular protection during extreme dehydration. Typically, CAHS proteins have a -18-20 amino-acid repetitive motif that occurs multiple times in tandem within each protein. This motif is compositionally biased, containing an alternating pattern of hydrophobic residues (e.g., alanine, leucine, isoleucine, valine) and charged or polar residues (e.g., lysine, arginine, glutamate, aspartate, glutamine). As a result, CAHS proteins remain intrinsically disordered under hydrated conditions.

[0014] Upon desiccation, the CAHS motifs drive a sol-gel-glass transition, in which the disordered proteins assemble into an amorphous, non-crystalline vitrified state. This glassy matrix immobilizes cellular macromolecules, suppresses aggregation, and stabilizes proteins and membranes during water loss. Experimental work has shown that disruption of this vitrification behavior abolishes protection, underscoring the functional importance of the motif architecture rather than specific amino-acid sequences.

[0015] SUMMARY

[0016] The present disclosure pertains to genetically-modified plants, seeds, and plant parts with improved tolerance or resilience to abiotic or environmental stresses. By stably integrating tardigrade cytosolic abundant heat-soluble (CAHS) transgenes into the plant genome, the described technologyoffers methods, sequences, and plant materials that enable enhanced resistance to stress conditions such as heat, cold, drought, and salinity, as compared to untreated controls.

[0017] In one aspect, a method of producing a genetically-modified plant having enhanced tolerance to an abiotic stress as compared to a control plant is provided. The method comprises transforming a plant with a tardigrade CAHS transgene encoding a protein that is stably integrated into the plant genome to yield a genetically-modified plant, such as a plant selected from the group consisting of wheat, oat, barley, rice, maize, millet, rye, sorghum, triticale, buckwheat, quinoa, soybean, bean, pea, alfalfa, potato, sweet potato, cassava, yam, tomato, pepper, tobacco, Arabidopsis, and cotton.

[0018] The tardigrade transgene may comprise a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3, a sequence having at least about 75% identity thereto, a sequence encoding a CAHS protein of SEQ ID NO:1 or SEQ ID NO:5-11, or a nucleotide sequence encoding a CAHS protein having at least about 50% amino acid identity to SEQ ID NO:1 and being a functional analog thereof. Transformation may be carried out on plant tissue cultured on media by Agrobacterium-mediated transformation, PEG-mediated uptake, electroporation, particle bombardment, or microinjection. In one aspect, the method further includes regenerating plants from transformed tissue by inducing callus formation, shoot regeneration, and rooting; and may include self-pollinating to produce genetically-modified seed or crossing with a second plant to produce progeny exhibiting enhanced tolerance to heat stress.

[0019] Under non-stress conditions, the genetically-modified plants exhibit a phenotype substantially similar to control plants; however, under environmental stress conditions such as heat, cold, drought, salinity, or combinations thereof, they exhibit improved performance, increased survival rates, and enhanced biomass accumulation.

[0020] In a further aspect, a genetically-modified plant having enhanced tolerance to an abiotic stress as compared to a control plant is provided. The plant comprises a tardigrade transgene stably integrated into the plant’s genome and encoding a CAHS protein, wherein the transgene is a nucleotide sequence selected from SEQ ID NO:2 or SEQ ID NO:3, embodiments having at least about 75% sequence identity thereto, sequences encoding CAHS proteins of SEQ ID NO:1 or SEQ ID NO:5-11, or functional analogs comprising CAHS proteins with at least about 50% amino acid identity to SEQ ID NO:1. Accordingly, the genetically-modified plant exhibits enhanced tolerance to abiotic stresses without compromising growth under favorable conditions.

[0021] In a further aspect, one or more genetically-modified seeds produced by the method described herein are provided. Each seed comprises the heterologous tardigrade transgene encoding the CAHSprotein that confers enhanced tolerance to abiotic stress. These seeds may be used to establish crop populations exhibiting increased resilience to environmental challenges.

[0022] In yet another aspect, a cereal plant, seed, or part thereof, such as those selected from wheat or maize, comprising or expressing a transgene encoding tardigrade CAHS6 protein (SEQ ID NO:1; GenBank Accession No. P0CU48) is provided. The presence of the CAHS6 transgene in such plant material confers resilience to heat, drought, cold, salinity, or combinations thereof, thereby improving agricultural productivity in suboptimal environments.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure (FIG.) 1A is a plasmid map pAHC17-CAHS6 of the gene of interest after cloning and confirmed by sequencing.

[0025] FIG. IB plasmid map of pAHC20-BlpR that confers resistance to glufosinate and is used as a selective marker of plant transformations.

[0026] FIG. 1C is a plasmid map of pMDC-GmUbi-CAHS6 used in the Agrobacterium-mediated transformations of Arabidopsis thaliana and Nicotiana benthamiana.

[0027] FIG. 2A is a sequence alignment of core domain of CAHS Proteins. Multiple sequence alignment of five CAHS protein variants. The alignment identifies the conserved amphiphilic alphahelical domain. Asterisks (*) denote 100% residue conservation; colons (:) denote strong physicochemical similarity; periods (.) denote weak similarity.

[0028] FIG. 2B shows a consensus motif for functional analogs in the CAHS family with variable and invariable amino acid residues represented by the codes.

[0029] FIG. 3 is a graph showing days to germination (DTG) of three wheat genotypes under PEG-induced osmotic stress. Bars show estimated marginal means ± SE from a two-way ANOVA (Genotype, PEG treatment, and GxT), for BW, 7949D 2 and 7975B 6 at 0, -0.3 and -0.5 MPa from PEG.

[0030] FIG. 4 is a graph showing seedling length (SeL) of three wheat genotypes under PEG-induced osmotic stress. Bars show estimated marginal means ± SE from a two-way ANOVA (Genotype, PEG treatment, and their interaction) for BW, 7949D_2 and 7975B_6 at 0, -0.3 and -0.5 MPa.

[0031] FIG. 5 is a graph showing seedling biomass (SB) of three wheat genotypes (BW, 7949D_2, and 7975B 6) grown under control (0 MPa) and PEG-induced osmotic stress (-0.3 and -0.5 MPa). Bars represent estimated marginal means from a two-way ANOVA (Genotype, Treatment, and their interaction), with error bars showing standard errors.FIG. 6 is a graph showing seedling vigor index (SVI) of three wheat genotypes (BW, 7949D 2, and 7975B 6) under PEG-induced osmotic stress. Bars represent estimated marginal means from a two-way ANOVA (Genotype, Treatment, and their interaction) and error bars indicate ± standard error.

[0032] FIG. 7 is a graph showing the percentage of green leaves per seedling for four Arabidopsis genotypes (BW, AA001, AA019, AA021) after exposure to control, heat, or freeze treatments. Reduction in color is indicative of cell damage due to treatments. Bars show mean % green leaves per seedling, and error bars indicate the standard error of the mean for each genotype x temperature combination.

[0033] DETAILED DESCRIPTION

[0034] Our invention provides tolerances to a number of environmental extremes and abiotic stresses by expressing tardigrade-specific Cytosolic-Abundant Heat Soluble (CAHS) proteins in plants to yield plants resilient to these extremes. Upon dehydration, CAHS proteins transform from a soluble state into a fibrous network (gelation). This network fills the plant cell, preventing collapse and acting as a physical, water-interacting shield. CAHS proteins contain long helical domains with flexible tails, allowing them to form stable, stress-dependent dimers and networks. CAHS proteins possess a highly conserved motif (specifically "motif 1") that is essential for maintaining solubility and protecting enzyme activity during freeze-thaw or dehydration treatments. Specific variants include CAHS6 and CAHS8, which form these protective hydrogels, as seen in Hypsibius exemplaris. as well as other members of the protein family described hereinafter and analogs thereof, e.g., CAHS1, CAHS2, CAHS3, CAHS4, CAHS5, RvCAHSl, and / or PrCAHS 1.

[0035] CAHS6 (P0CU48.1) or CAHS8 (P0CU50) - Hypsibius exemplaris MSGRNVESHMERNEKVVVNNSGHADVKKQQQQVEHTEFTHTEVKAPLIHPAPPIISTGAA GLAEEIVGQGFTASAARISGGTAEVHLQPSAAMTEEARRDQERYRQEQESIAKQQEREMEK KTEAYRKTAEAEAEKIRKELEKQHARDVEFRKDLIESTIDRQKREVDLEAKMAKRELDREG QLAKEALERSRLATNVEVNFDSAAGHTVSGGTTVSTSDKMEIKRN (SEQ IDNO:1).

[0036] CAHS1 (P0CU45) - Hypsibius exemplaris MSGRNVESHMERNEKVVVNNSGHADVKKHQQQVEHTEFTHTEVKAPLIHPAPPIISTGAA GLAEEIVGQGFTASAARISGGTAEVHLQPSAAMTEEARRDQERYRQEQESIAKQQEREMEKKTEAYRKTAEAEAEKIRKELEKQHARDVEFRKDLIESTIDRQKREVDLEAKMAKRELDREG QLAKEALERSRLATNVEVNFDSAAGHTVSGGTTISSSDKMEIKRN (SEQ ID NO:5)

[0037] CAHS2 (P0CU46) - Hypsibius exemplaris MSQQYEKKVERTEVVYGGDRRVEGSASASAEKTTNYTHTEIRAPMVNPLPPIISTGAAGLA QEIVGEGFTASATRISGAAATTQVLESQASREQAFKDQEKYSREQASIARAHDKDLEKKTE EYRKTAEAEAEKIRKELEKQHARDVEFRKDLVESAIDRQKREVDLEAKYAKKELEHEREL AMNALEQSKMATNVQVQMDTAAGTTVSGGTTVSEHTEVHDGKEKKSLGEKIKSLF (SEQ ID NO: 6)

[0038] CAHS3 (P0CU43) - Hypsibius exemplaris MSNYQQESSYQSDRSNNGQQQEQQKKEVEHSSYTHTDVKVNMPNLIAPFISSSAGLAQEL VGEGFQASVSRITGASGELTVIDEAETEEARRDMEAKAREQELLSRQFEKELERKTEAYRK QQEVETEKIRKELEKQHLRDVEFRKELMEQTIENQKRQIDLEARYAKKELERERNKVKVRL ERSKFHTDIQVNMEAAAGSTHSGSSS VAVSESEKFQTNN (SEQ ID NO: 7)

[0039] CAHS4 (P0CU44) - Hypsibius exemplaris MSNYQQESSYQSDRSNNGQQQEQQKKEVEHSSYTHTDVKMNPNLIAPFISSSAGLAQELV GEGFQASVSRITGASGELTVIDEAETEEARRDLEAKAREQELLSRQFEKELERKTEAYRKQQ EVETEKIRKELEKQHLRDVEFRKELMEQTIENQKRQIDLEARYAKKELERERNKVKVRLER SKFHTDIQVNMEAAAGSTHSGSSS VAVSESEKFQTNN (SEQ ID NO: 8)

[0040] CAHS5 (P0CU47) - Hypsibius exemplaris MATKESKYERVEKVNVDADGATLVKNIGEDRGPDMGNYQDKRPANLVPGAPAGVIPNRI ESLPTDRGQRLREHLSESERLGRS STS SKS S SFVEPSLKYRGEIGPIGKNGEF VAS SNRQNS SS NVS S SDNSERASPASRNSNPGMNNGMTTQRTTVITESS VQGLGAQRTVPIQPHQQREDHEV ITHESHARAPETVVVIPTTRFESAQSLESRRDGRTYTEDKELTIPAPVVVAPQIHAHQQVNM SGGTSATIHATTDLHLASEAQINDMGPEEYRYRAKVEALARIHEDETSRKAAAYRNAVEA D AELIRQTLERQHMRDIEFRKDL VE S S VDRQQQEIRLEAEYAMRALEQERVNARAALDQ A MASTNIDVNIDSAIGTTHSQGRVTTTSESRTSQARGPATAAVI (SEQ ID NO:9)RvCAHSl (BAM37958.1) - Ramazzottius varieornatus MPYEKHVEQTVVEKTEQPGHSSTHHAPAQRTVAREQEEVVHKEFTHTDIRVPHIDAPPPIIA ASAVGLAEEIVSHGFQASAARISGASTEVDMRPSPKLAEEARRDAERYQKEHEMINRQAEA TLQKKAEEYRHQTEAEAEKIRRELEKQHERDIQFRKDLIDQTIEKQKREVDLEAKMAKREL DREAQLAKEALERSRMATNVEVTLDTAAGHTVSGGTTVSSVDKVETVRERKHH (SEQ ID NO: 10)

[0041] PrCAHSl (P0CU51) - Paramacrobiotus richtersi MSRYEKPVEVPEKEHVEQTIVEKTEQPGHGATHHAAHAAPAQRTVAREQEEVVHKEFTHT DIRVPHIDAPPPIIAASAVGLAEEIVSHGFQASAARISGASTEVDMRPSPKLAEEARRDAERY QKEHEMINRQAEATLQKKAEEYRHQTEAEAEKIRRELEKQHERDIQFRKDLIDQTIEKQKR EVDLEAKMAKRELDREAQLAKEALERSRMATNVEVTLDTAAGH (SEQ ID NO:11).

[0042] CAHS proteins have three functional domains: 1) a highly disordered N-terminal Domain that promotes protein-protein interactions and keeps the protein soluble until gelling is triggered, 2) a central helical region that forms the structural core which is amphipathic and 3) a C-terminal domain the charged and works in tandem with the NTD to ensure the network remains flexible. Within these domains there are key functional motifs. The central helical region contains 90-150 amino acid residues so that hydrophobic amino acids such as Leucine, Isoleucine and Valine are on one side of the helix and amino acids such as Lysine and Glutamic acid are on the opposite side of the helix. The N-terminus and C-terminus of the protein contains amino acids such as Tyrosine, glutamine and glycine. The C-terminus also contain 19-mer repeat motif common to most CAHS proteins, which is characterized by a heptad repeat pattern denoted as (a-b-c-d-e-f-g)n. Within this framework, positions “a” and “d” comprise the hydrophobic core of the helix, while positions “e” and “g” comprise charged or polar residues. In a preferred embodiment, the hydrophobic seam at positions a and d is populated by residues selected from the group consisting of Leucine (L), Valine (V), Isoleucine (I), Alanine (A), and Phenylalanine (F). The stability of the resulting filament is further enhanced by electrostatic interactions between oppositely charged residues at positions “e” and “g” (e.g., Lysine-Glutamic Acid salt bridges), which facilitate the inter-helical packing observed during desiccation-induced phase transition. The present invention relates to a family of Cytosolic Abundant Heat Soluble (CAHS) proteins derived from the tardigrade Hypsibius exemplaris. As illustrated in FIG. 2A, these proteins (P0CU48.1, P0CU45, P0CU44, P0CU43, P0CU46, and P0CU47) are characterized by a highly conserved central-to-C-terminal domain. This domain is predicted to form an amphipathic alpha-helix, a structural motif where hydrophobic and hydrophilic residues are segregated on opposite faces of the helical cylinder. Under conditions of cellular dehydration (desiccation), the increased concentration of these helical domains facilitates a rapid, reversible phase transition. The proteins self-assemble into a network of interconnected filaments, forming a “hydrogel” that physically stabilizes sensitive macromolecules, such as enzymes and membranes, preventing their denaturation or mechanical collapse, as mentioned above.

[0043] With reference to FIG. 2A, the Multiple Sequence Alignment (MSA) reveals a core 19-mer consensus motif. While the N-terminal "tail" regions of these proteins exhibit significant intrinsic disorder and length variability, the helical core maintains a rigid conservation of key residues. Particularly, the selected motive is invariant across all disclosed variants. The phenylalanine (F) residue provides a hydrophobic anchor, while the adjacent charged residues (E, R, and K) facilitate the electrostatic interactions necessary for inter-molecular cross-linking during filament assembly.

[0044] Looking at the pairwise identities of the conserved core region (Table A), some variants such as P0CU48.1 and P0CU46 share 94% identity. More divergent variants share at least 50% and up to 64% identity, yet they retain the essential heptad repeat, described herein, required for retention of the functional activity to be considered a functional analog, e.g., biological activity such as heat, cold, and drought protection.

[0045] Table B identifies the coordinates of the conserved CAHS motifs within the parent polypeptides P0CU48.1, P0CU45, P0CU44, P0CU43, P0CU46, and P0CU47. These boundaries define the minimum functional unit required for desiccation-induced filamentation and subsequent molecular stabilization. The invention expressly contemplates fragments consisting of, or comprising, these identified residue ranges.

[0046] Table A. Pairwise sequence identity matrix of the conserved helical core domain (50 amino acids). Identity is calculated as the percentage of identical residues over the alignment length. This matrix defines the structural relationship between the claimed variants.

[0047] P0CU48.1 P0CU45 P0CU44 P0CU43 P0CU46 P0CU47 P0CU48.1 100% 100% 72% 72% 94% 60%

[0048] P0CU45 100% 100% 72% 72% 94% 60%

[0049] P0CU44 72% 72% 100% 100% 72% 50%

[0050] P0CU43 72% 72% 100% 100% 72% 50%

[0051] P0CU46 94% 94% 72% 72% 100% 64%

[0052] P0CU47 60% 60% 50% 50% 64% 100%Table B. Motif Boundaries for Sequence Listing. The table identifies the coordinates of the conserved CAHS motifs within the parent polypeptides P0CU48.1, P0CU45, P0CU44, P0CU43, P0CU46, and P0CU47. These boundaries define the minimum functional unit required for desiccation-induced fllamentation and subsequent molecular stabilization. The invention expressly contemplates fragments consisting of, or comprising, these identified residue ranges.

[0053] Protein ID / Start End Sequence Segment (Motif Core)

[0054] Accession Residue Residue

[0055] P0CU48.1 119 168 YRI< TAEAEAEKIRI< ELEKQHARDVEFRI< DLIE 94205 STIDRQKREVDLEAKMAK (SEQ ID NO: 13) P0CU45 119 168 YRKTAEAEAEKIRKELEKQHARDVEFRKDLIE 94205 iso STIDRQKREVDLEAKMAK (SEQ ID NO:13) P0CU44 116 165 YRKQQEVETEKIRKELEKQHLRDVEFRKELME 77611 QTIENQKRQIDLEARYAK (SEQ ID NO:14) P0CU43 118 167 YRKQQEVETEKIRKELEKQHLRDVEFRKELME 77580 QTIENQKRQIDLEARYAK (SEQ ID NO: 14) P0CU46 112 161 YRKTAEAEAEKIRKELEKQHARDVEFRKDLVE 86272 S AIDRQKREVDLEAK YAK (SEQ ID NO: 15) P0CU47 324 373 YRNAVEADAELIRQTLERQHMRDIEFRKDLVE 89226 SSVDRQQQEIRLEAEYAM (SEQ ID NO: 16)

[0056] Other tardigrade species have CAHS proteins, which have highly similar 19-mer motifs that protect those species from environmental extremes. For example, Ramazzottius varieornatus and Paramacrobiotus richtersi both contain similar structural motifs (Table C). A pairwise identity matrix containing suggests these sequences represent a specific conserved functional sub-class within the CAHS family across different species (Table D). For example, P0CU48.1 shares 92% identity with PrCAHSl, and P0CU47 shares 98% identity with RvCAHSl. Based on the alignment of these 8 sequences shown, the consensus motif is shown in FIG. 2B.

[0057] Table C. Multiple Sequence Alignment of Cross-Species CAHS Core Domains. Tardigrade species containing CAHS proteins with similar structural motifs to H. exemplaris CAHS proteins. Multiple sequence alignment (MSA) of CAHS helical core domains across three distinct tardigrade genera. The alignment highlights the conservation of the 19-mer motif architecture. Asterisks (*)denote 100% conservation across all nine sequences; colons (:) denote high physicochemical conservation, particularly at the hydrophobic a and d heptad positions.

[0058] Species Protein ID Sequence Alignment (Conserved Helical Core Domain) H. exemplaris P0CU48.1 YRKTAEAEAEKIRKELEKQHARDVEFRKDLIESTID RQI< REVDLEAI< AI< (SEQ ID NO: 13)

[0059] H. exemplaris P0CU45 YRKTAEAEAEKIRKELEKQHARDVEFRKDLIESTID RQKREVDLEAKM AK (SEQ ID NO: 13)

[0060] H. exemplaris P0CU44 YRKQQEVETEKIRKELEKQHLRDVEFRKELMEQTIE NQKRQIDLEARYAK (SEQ ID NO: 14)

[0061] H. exemplaris P0CU43 YRKQQEVETEKIRKELEKQHLRDVEFRKELMEQTIE NQKRQIDLEARYAK (SEQ ID NO: 14)

[0062] H. exemplaris P0CU46 YRKTAEAEAEKIRKELEKQHARDVEFRKDLVESAID RQKREVDLEAKYAK (SEQ ID NO:17)

[0063] H. exemplaris P0CU47 YRNAVEADAELIRQTLERQHMRDIEFRKDLVESSVD RQQQEIRLEAEYAM (SEQ ID NO:18)

[0064] R. varieornatus RvCAHSl YREKAEADADKVRKELEKQHMRDIEFRKDLVESSV DRQQQEIRLEAEYAM (SEQ ID NO: 19)

[0065] P. richtersi PrCAHSl YRKTAEAEAEKIRKELEKQHLRDIEFRKDLIESTIDR QKREVDLEARYAK (SEQ ID NO:20)

[0066] Consensus —

[0067]

[0068] Table D. Pairwise Sequence Identity Matrix (Phylum-Wide). Pairwise identity matrix for representative CAHS core domains from H. exemplaris (He), R. varieornatus (Rv), and P. richtersi (Pr). Note the high identity (98%) between HeP0CU47 and RvCAHS1, suggesting these sequences represent a specific conserved functional sub-class within the CAHS family across different species.

[0069] P0CU48.1 P0CU44 P0CU47 RvCAHSl PrCAHSl P0CU48.1 (He) 100% 72% 60% 60% 92%

[0070] P0CU44 (He) 72% 100% 50% 50% 74%

[0071] P0CU47 (He) 60% 50% 100% 98% 62%

[0072] RvCAHSl (Rv) 60% 50% 98% 100% 62%

[0073] PrCAHSl (Pr) 92% 74% 62% 62% 100%Table E. Motif Boundaries for Cross-Species Sequence Listing. Coordinates and residue ranges for the primary functional helical domains of CAHS proteins from diverse tardigrade species. These sequences define the "core fragments" claimed in the present invention as being sufficient to confer desiccation tolerance to biological materials.

[0074] Species Protein ID Start End Sequence Segment (Functional Unit)

[0075] H. exemplar is P0CU48.1 119 168 YRKTAEAEAEKIRKELEKQHARDVEFRKD LIESTIDRQKREVDLEAKMAK (SEQ ID NO:13)

[0076] H. exemplaris P0CU47 324 373 YRNAVEADAELIRQTLERQHMRDIEFRKD LVESSVDRQQQEIRLEAEYAM (SEQ ID NO: 16)

[0077] R. varieornatus RvCAHSl 132 181 YREKAEADADKVRKELEKQHMRDIEFRK DLVESSVDRQQQE1RLEAEYAM (SEQ ID NO: 19)

[0078] P. richtersi PrCAHSl 124 173 YRKTAEAEAEKIRKELEKQHLRDIEFRKDL IESTIDRQKREVDLEARYAK (SEQ ID NO: 20)

[0079] Enhanced plant lines are generated by inserting an extreme-environment stable protein (EESP), aka the CAHS protein sequence, into the plant genome such that the CAHS transgene is stably integrated and heritable in progeny. Upon expression of the transgene, the CAHS protein enhances the plant’s ability to tolerate and withstand against environmental stresses. Some applications could be heat and drought tolerances on adult plants and freeze (winter kill) tolerances on seedlings. The technology could be applicable in a variety of cereal crops and other plants.

[0080] Thus, the disclosure concerns a method for enhancing plant resilience to environmental extremes, such as drought and temperature fluctuations (extreme heat and freezing), by expressing EESPs in plants and other crops. In one aspect, the invention involves the molecular transformation of plants to express tardigrade CAHS proteins. These proteins function as “shields” that protect cellular components during desiccation and other abiotic stresses through a “sol-gel-glass” transition. In this state, the proteins form a non-crystalline matrix that stabilizes membranes and prevents the aggregation of vital macromolecules during water loss.

[0081] In more detail, the present invention is concerned with plants having stable integration of a transgene conferring increased tolerance or resistance to abiotic stresses relative to a control plant,and in particular tardigrade-specific CAHS6 transgene, e.g., a transgene encoding for SEQ ID NO:1, or analogs thereof.

[0082] Unless otherwise indicated by the context, references herein to a “plant” or “plants” include tissues, organs, or parts thereof (e.g., leaves, stems, tubers), fruit, or cells thereof. The invention is also concerned with various methods of increasing plant tolerance or resistance to abiotic stresses by stable integration and expression of a CAHS transgene in a plant. Methods of creating such genetically-modified plants are also provided, along with nucleic acid constructs, vectors, and fusion proteins useful in such methods. The invention is suitable for use with various plants, including both monocotyledons (i.e., plants having one cotyledon (seed-leaf), aka “monocots”) and dicotyledons (i.e., plants having two cotyledons, aka “dicots”). Non-limiting examples of plants suitable for the disclosed embodiments include grains (e.g., wheat, oat, barley, rice, maize, millet, rye, sorghum, triticale, buckwheat, quinoa), legumes (e g., soybeans, beans, peas, alfalfa), tubers (e.g., potatoes, sweet potatoes, cassava, yam), nightshades (e.g., tomatoes, peppers, tobacco), other cash crops (e.g., cotton), as well as research lines, e.g., Arabidopsis, and the like. While SEQ ID NO: 2 can be used to generate transgenic monocots or dicots, a further sequence was optimized for dicot expression, as SEQ ID N0:3:

[0083] Modified DNA sequence of CAHS6 protein for dicot expression.

[0084] |P0CU48.1 |CAHS6_HYPEX_dicot_optimized ATGAGTCAGGATCGTAACGTTGAGAGCCACATGGAAAAGGAATGAGAAAAGTAGTCG TCAACAGCAGTGGTCACGCCGACGTCAAGAAACAGCAACAGCAGGTAGAGCACACCG AACTCACCCACACAGAAGTGAAGGCGCCGCTCACCCATCCTGCTCCACCGATAATCTC CACTGGCGCCGCCGGCCTTGCCGAAGAAATTGTTGGCCAAGGGTTCACGGCCTCTGCT GCACGCATAAGTGGAGGTACTGCAGAGGTCCATCTGCAGCCCTCGGCGGCCATGACGG AGGAGGCACGGCGGGACCAGGAAAAGATATCGACAAGAGCAGGAGAGCATTGCAAAG CAACAAGAACGCGAGATGGAGAAGAAGACTGAAGCATACCGCAAAACAGCTGAAGCT GAGGCCGAGAAGATCAGGAAAGAGCTGGAAAAGCAGCATGCTAGAGATGTTGAGTTC AGGAAAGATTTGATTGAGTCCACCATCGACAGACAGAAGCGGGAAGTTGATCTAGAGG CTAAAATGGCGAAGAGGGAGCTTGACCGTGAGGGGCAGCTGGCCAAGGAGGCGCTCG AGAGGTCGCGCCTCGCCACCAACGTGGAGGTGAACTTTGATTCTGCGGCGGGGCATAC AGTAAGCGGCGGCACCACGGTGTCCACAAGCGACAAGATGGAGATCAAGAGAAATTG

[0085] A (SEQ ID NO:3).The inventive methods can be used to produce plants with enhanced tolerance to a variety of environmental or abiotic stresses. The term “abiotic” stress is used herein to refer to non-living chemical and / or physical factors in the environment that affect plant growth and / or development. Examples include extreme temperatures (heat or cold), water availability (e.g., drought), salinity (e.g., salt), and the like. Such abiotic factors are considered “stressors” when they influence the environment beyond its normal range of variation to adversely affect plant growth and / or development. For example, heat or cold stress may occur when the plant is subjected to temperatures at least about 10-20°C higher or lower than the normal plant growing temperatures. “Normal plant growing temperatures” refers to temperature ranges suggested for optimal growth and yield, which for most species are known in the art. For example, many plants prefer a daytime temperature of between about 24°C and about 28°C, and a nighttime temperature of between about 19°C and about 21 °C. In general, preferred nighttime temperatures are about 5°C to 7°C lower than daytime temperatures. The tolerance of the transgenic plant is considered to be “enhanced” when the transgenic plant’s growth or development is superior to the growth and development of a control plant under the same conditions or stressors, even if the transgenic plant is not completely resistant to or unaffected by the stressor.

[0086] As noted above, the transgenic plants have stable expression of integrated transgene. The term “stable” integration or expression is used herein to refer to a nucleic acid sequence gene that originates from a source outside of a particular plant species and is stably integrated into the genome of another (host) plant species to create the transgenic plant with the transgene being incorporated into germline as a heritable element. For example, the term as it is used in reference to expression of an encoding nucleic acid, refers to introduction of the encoding nucleic acid in an expressible form into the host plant, but more specifically integration of the expressible nucleic acid into the genome of the plant, which can be passed to progeny. Transformation techniques for plants are well known in the art and include particle bombardment-mediated delivery, and / or microinjection, Agrobacterium-mediated techniques, PEG- or electroporation-mediated uptake, or any other techniques involving the insertion, introduction or uptake of exogenous genetic material by the plant.

[0087] In some embodiments, increased or enhanced tolerance to an abiotic stressor in the plant is enhanced by expressing in the plant a CAHS transgene which comprises (or consists of) a sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO:2 or 3; (b) a nucleotide sequence having at least about 75% sequence identity (preferably at least about 80% sequence identity, and more preferably at least about 90% sequence identity) to SEQ ID NO:2 or 3 (i.e., conservatively modified variants or functional analogs thereof); (c) a nucleotide sequenceencoding a CAHS protein comprising SEQ ID NO: 1, 5, 6, 7, 8, 9, 10, or 11; (d) a nucleotide sequence encoding a CAHS protein having at least about 50% amino acid identity (preferably at least about 75% amino acid identity, more preferably at least about 85% amino acid identity, and even more preferably at least 90-98% amino acid identity) to SEQ ID NO: 1 and functional analogs thereof (i.e., those sequence analogs retaining the functional characteristics thereof, e.g., SEQ ID NO:4, 5, 6, 7, 8, 9, 10, or 11). The “functional characteristics” of the abiotic stress tolerance proteins refers to the ability of the expressed CAHS protein to undergo the sol-gel transition under abiotic stress and act as a protective hydrogel in the plant cells. Thus, “conservatively modified variants” or “functional analogs” of the disclosed nucleic acid and amino acid sequences are contemplated herein, as long as the resulting proteins retain the ability described, and generally fall within the category of the CAHS family of proteins.

[0088] In one or more embodiments, the method of enhancing the tolerance of a plant to abiotic stress comprises introducing and expressing in a plant cell a nucleic acid construct encoding a tardigrade-specific CAHS, and preferably CAHS6 (SEQ ID NO: 1) or a functional analog thereof. A recombinant plant cell comprising the nucleic acid construct, preferably stably incorporated into its genome, is also provided herein. The nucleic acid construct can comprise a nucleic acid coding sequence which is operably linked to a promoter that drives expression in the plant cell. Suitable promoters include the Glycine max polyubiquitin promoter (GmUbi), cauliflower mosaic virus promoter (CaMV35S), the rice actin promoter, the maize ubiquitin promoter, tissue specific promoter, or other stressinducible promoters that can be selected by those of ordinary skill in the art. A “stress-inducible promoter” is one that is activated in response to an abiotic stressor. A “tissue specific promoter” induces the expression of the transgene in specific tissues, organs, or developmental stages.

[0089] More preferably, the transgenic plant is prepared by introducing into a plant cell or tissue a vector or plasmid comprising the nucleic acid construct encoding for the CAHS protein. Thus, in one or more embodiments, a plant cell transformed with an expression vector or plasmid described herein is also provided. In further embodiments, a vector or plasmid is provided for preparing a transgenic plant having enhanced resistance to abiotic stress. The vector or plasmid comprises an expression cassette comprising a nucleic acid construct, which encodes an abiotic stress tolerance gene, operably linked a suitable promoter for driving expression of the nucleic acid in the plant cell.

[0090] In some embodiments, there is provided an isolated nucleotide CAHS transgene sequence which comprises (or consists of) a sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO:2 or 3; (b) a nucleotide sequence having at least about 75% sequence identity (preferably at least about 80% sequence identity, and more preferably at least about90% sequence identity) to SEQ ID NO:2 or 3 (i.e., conservatively modified variants or functional analogs thereof); (c) a nucleotide sequence encoding a CAHS protein comprising SEQ ID NO:1, 5, 6, 7, 8, 9, 10, or 11; (d) a nucleotide sequence encoding a CAHS protein having at least about 50% amino acid identity (preferably at least about 75% amino acid identity, more preferably at least about 85% amino acid identity, and even more preferably at least 90-98% amino acid identity) to SEQ ID NO:1 and functional analogs thereof (i.e., those retaining the functional characteristics thereof, e.g., SEQ ID NO:4, 5, 6, 7, 8, 9, 10, or 11). The sequence can be linked to reporter amino acid sequences by one or more peptide bonds. In some embodiments, the reporter is a fluorescent protein, such as green fluorescent protein (GFP). Other suitable reporters include beta-glucuronidase (GUS), luciferase, dsRed (red fluorescent protein), and the like.

[0091] Methods of the invention include, culturing plant tissue (e.g., leaf, cotyledon, or hypocotyl explants) on a suitable media (e.g., Murashige and Skoog (MS), or Chu (N6)), followed by introduction of the CAHS transgene into the tissue using suitable techniques, such as those described above and in the working examples. Expression of the transgene results in transformed or modified tissue. Reporter genes or other selection criteria can be used to verify transformation (which can be on the same or different expression vectors). The transformed tissue can then be used to regenerate transgenic whole plants having enhanced tolerance to abiotic stressors. Transgenic plants can be regenerated using various techniques depending upon the plant species involved. In one or more embodiments, regeneration comprises inducing callus formation from the transformed tissue, and regeneration of shoots, followed by rooting of the shoots in soil or other appropriate rooting media to generate the whole plant. In other embodiments, Agrobacterium-mediated floral dip can be used to generate transgenic lines without the requirement for intermediate tissue culture steps.

[0092] The resulting transgenic plants can be crossed to prepare progeny, and preferably homozygous progeny or seeds. Thus, abiotic stress-tolerant plants can also be produced indirectly by breeding parent transgenic plants having enhanced abiotic stress tolerance with other abiotic stress-tolerant plants, or even with other cultivars having additional desired characteristics (e.g., pest or herbicide resistance, geographic adaptation, stalk strength, etc.). The resulting progeny can then be screened to identify abiotic stress-tolerant progeny.

[0093] In one or more embodiments, the invention is also concerned with a process of producing (transgenic) seed. In some embodiments, the method comprises self-pollination of a transgenic plant as described herein. In some embodiments, the method comprises crossing a first plant with a second plant, wherein at least one of the first or second plants is a transgenic plant having enhanced abiotic stress tolerance, as described herein. In some embodiments, the first and second plants are bothtransgenic plants as described herein. In one or more embodiments, the first and second plants can be crossed via cross-pollination using insects (e.g., flies in cloth cages), manual (hand) pollination, and the like.

[0094] Advantageously, transgenic plants according to the various embodiments of the invention have enhanced tolerance to abiotic stress. However, unlike many other transgenic plants with similar improvements in tolerance to one or more specific stresses, transgenic plants according to the invention have a phenotype / morphology that is otherwise substantially similar to, and in some cases, nearly identical to wild type or non-transgenic plants of the same species (when such wild type plants are grown under non-stress conditions). In other words, the seedling biomass, seedling length, seedling vigor, or shape, size, and / or abundance of foliage and / or fruit / vegetable is substantially similar between the transgenic plants and wild type plants. Plants are considered to be “substantially similar” herein if those skilled in the art have difficulty visually distinguishing between the genetically-modified plant (grown under stress) and a control plant when grown under normal growing conditions. In contrast, when grown under stress, transgenic plants according to the various embodiments of the invention, have significantly improved morphologies as compared to control plants grown under the same stress conditions. For example, the transgenic plant may have one or more of the following improved characteristics: seedling biomass, seedling length, seedling vigor, vigorous plant growth, abundant foliage, longer primary roots, yield, height, and / or shoot water potential, when grown in the presence of one or more abiotic stressors. Similarly, the transgenic plants have significantly improved recovery after stress.

[0095] Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and / or integrations of the specific embodiments described herein.

[0096] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0097] A “control” plant, as used in the present invention, refers to a plant used to compare againsttransgenic or genetically modified plants according to the invention for the purpose of identifying changes in the transgenic or genetically modified plant. The control plant is of the same species as the transgenic plant. In some cases, the control plant may be a wild type (native) plant, although cultivars and genetically altered plants that otherwise have normal abiotic stress tolerance can also be used as a reference for comparison. A “wild type” plant is a plant that has not been genetically modified or treated in an experimental sense. A “wild-type” gene is one that has the characteristics of a gene isolated from a naturally occurring source. A “wild-type” gene product is one that has the characteristics of a gene product isolated from a naturally occurring source, whereas “modified” genes or gene products are those having modifications in sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Likewise, “genetically-modified” cells, tissues, seeds, plants etc. are those that have been altered to include a transgene and / or to change the expression, activity, or function of the target genes or gene products, as opposed to nonmodified cells, tissues, etc. The term is synonymous with “genetically-engineered.” References herein to “genes” encompass both the partial (fragment) or complete coding sequence of a gene or transgene (including its cDNA sequence), its complement, and its 5' or 3' untranslated regions.

[0098] The term “operably linked” refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced

[0099] The term “vector” refers to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term includes recombinant DNA molecules containing a desired coding sequence(s) and appropriate nucleic acid sequences (e.g., promoters) necessary for the expression of the operably linked coding sequence in a particular host organism.

[0100] The term “transform” is used herein to refer to the introduction of foreign DNA into cells. Transformation may be accomplished by a variety of means known to the art and described herein.

[0101] The term “isolated” when used in relation to a nucleic acid, refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural environment. That is, an isolated nucleic acid is one that is present in a form or setting that is different from that in which it is found in nature.

[0102] The terms “sequence identity” or “amino acid identity” are used herein to describe the sequence relationships between two or more nucleic acid or amino acid sequences when aligned for maximum correspondence over a specified comparison window. The percentage of “identity” isdetermined by comparing two optimally aligned sequences over the comparison window. For “optimal alignment” of the two sequences, it will be appreciated that the portion of the sequence in the comparison window may include gaps (e.g., deletions or additions) as compared to the reference sequence, which does not contain additions or deletions. After alignment, the number of matched positions (i.e., positions where the identical nucleic acid base or amino acid residue occurs in both sequences) is determined and then divided by the total number of positions in the comparison window. This result is then multiplied by 100 to calculate the percentage of sequence or amino acid identity. It will be appreciated that a sequence having a certain percentage of sequence identity to a reference sequence does not necessarily have to have the same total number of nucleotides or amino acids (see e.g., microRNAs discussed above). Thus, a sequence having a certain level of “identity” includes sequences that correspond to only a portion (i.e., 5' non-coding regions, 3' non-coding regions, coding regions, etc.) of the reference sequence.

[0103] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

[0104] EXAMPLES

[0105] The following examples set forth methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.

[0106] EXAMPLE 1

[0107] Identification of Intrinsically Disordered Proteins, reverse translation, codon optimization and vector construction

[0108] This study’s objective is to develop synthetic wheat (Triticum aestivum L.) lines by inserting an extreme-environment stable protein (EESP) sequence into the wheat genome. Our invention describes the use of this protein in wheat to enhance the plant’s ability to tolerate and withstand against environmental stresses. Tardigrade Cytosolic-Abundant Heat Soluble (CAHS) proteins are characterized by a conserved biophysical motif comprising intrinsically disordered, low-complexityregions with periodic charge patterning that enables reversible formation of amphipathic a-helices under dehydration or crowding stress. This motif drives stress-induced self-assembly into filamentous or gel-like networks that physically stabilize cellular components, thereby conferring tolerance to desiccation and extreme environmental conditions.

[0109] We retrieved the protein sequence from Genbank for CAHS6 protein (aka CAHS 94205; GenBank Accession # P0CU48) below, and in silica, reverse translated the sequence into a wheat, codon optimized sequence and further cloned the sequence into the pACH17 vector. This plasmid contains the maize ubiquitin promoter which constitutively drives expression of the transgene. The protein sequences identified and characterized by others as Intrinsically Disordered Proteins (which assist tardigrades with survival during desiccation) were used as a starting point for the work. The protein sequence identified as Genomic ID CAHS 94205 (UniProt ID P0CU48.1 aka CAHS6 (SEQ ID NO:1)* was used to reverse translate a codon optimized DNA sequence (SEQ ID NO:2).

[0110] The CAHS6 was in silica optimized for wheat transcription and translation by retrieving the protein sequence from NCBI (Accession # P0CU48) and codon optimized for wheat using the “Triticum aestivumIThinopyrum intermedium alien addition line [gbpln]: 2” table to yield SEQ ID NO:2.

[0111] Reverse translation of sp|P0CU48.1|CAHS6_HYPEX codon optimized for wheat.

[0112] ATGTCAGGACGTAATGTTGAATCACACATGGAAAGGAATGAGAAAGTAGTCGTCAACA ACAGTGGTCACGCCGACGTCAAGAAACAGCAACAGCAGGTGGAGCACACCGAATTCA CCCACACAGAAGTGAAGGCGCCGCTCATCCATCCTGCTCCACCGATAATCTCCACTGGC GCCGCCGGCCTTGCCGAAGAAATTGTTGGCCAAGGGTTCACGGCCTCTGCTGCACGCA TAAGTGGAGGTACTGCAGAGGTCCATCTGCAGCCCTCGGCGGCCATGACGGAGGAGGC ACGGCGGGACCAGGAAAGATATCGACAAGAGCAGGAGAGCATTGCAAAGCAACAAGA ACGCGAGATGGAGAAGAAGACTGAAGCATACCGCAAAACAGCTGAAGCTGAGGCCGA GAAGATCAGGAAAGAGCTGGAAAAGCAGCATGCTAGAGATGTTGAGTTCAGGAAAGA TTTGATTGAGTCCACCATCGACAGACAGAAGCGGGAAGTTGATCTAGAGGCTAAAATG GCGAAGAGGGAGCTTGACCGTGAGGGGCAGCTGGCCAAGGAGGCGCTCGAGAGGTCG CGCCTCGCCACCAACGTGGAGGTGAACTTTGATTCTGCGGCGGGGCATACAGTAAGCG GCGGCACCACGGTGTCCACAAGCGACAAGATGGAGATCAAGAGAAATTGA (SEQ ID NO:2), (TGA added as stop codon).The gene of interest (GOI) was then cloned into pAHC17 plasmid backbone and confirmed through Oxford Nanopore (ONT) long-read nanopore sequencing (FIG. 1A), see SEQ ID NO: 12, with BamHi site at nts 2924-2929 and 3620-3625 and the CAHS6 gene at nts 2936-3619. The selective marker plasmid contains the BlpR gene, also driven by ZmUbi, conferring resistance to glufosinate (FIG. IB).

[0113] *CAHS D is the biological name used in many research papers, while P0CU48 (aka CAHS6) and P0CU50 (aka CAHS8) are the protein database ID numbers for identical amino acids derived from two different gene sequences. For clarity, P0CU48, P0CU50, and CAHS D all share the exact same 227 amino acid sequence for the protein sequence (SEQ ID NO: 1).

[0114] >sp|P0CU50.1|CAHS8_HYPEX Cytosolic-abundant heat soluble protein 94063 O =Hypsibius exemplaris OX=2072580 GN=CAHS 94063 PE=1 SV=1 (227AA) MSGRNVESHMERNEKVVVNNSGHADVKKQQQQVEHTEFTHTEVKAPLIHPAPPIISTGAA GLAEEIVGQGFTASAARISGGTAEVHLQPSAAMTEEARRDQERYRQEQESIAKQQEREMEK KTEAYRKTAEAEAEKIRKELEKQHARDVEFRKDLIESTIDRQKREVDLEAKMAKRELDREG QLAKEALERSRLATNVEVNFDSAAGHTVSGGTTVSTSDKMEIKRN (SEQ ID NO:1).

[0115] Two different accession numbers for this same protein can be found in UNIPROT because tardigrades have multiple copies of the same gene in their genome. P0CU48 refers to the gene locus 94205, and P0CU50 refers to gene locus 94063. Although the gene sequences differ, they both produce the same “CAHS D” protein having SEQ ID NO: 1.

[0116] EXAMPLE 2

[0117] Generation of Transgenic Wheat lines expressing CAHS6

[0118] The plasmid constructions (FIG. 1A and IB) were co-bombarded in ten independent biolistic experiments using approximately 1500 independent wheat embryos. Embry ogenic calli was transferred into glufosinate selection and regeneration cycles, where morphological differentiation occurred normally as described in the published literature. Ninety-eight plants were generated under glufosinate selection, and transferred to soil. PCR analyses were performed on DNA samples of these plants for the presence of the CAHS6 gene. RT-PCR analysis of these lines confirmed that 18 were expressing the CAHS6 gene. Lines were self-fertilized and tested in the T2 generation to confirm expression and to identify homozygous lines. The majority of the lines derived from single seed descent contained the GOI and were expressing the transgene. Seed from the T2 generation werebulked for environmental stress tests. We also had the protein tested (in silico) for allergenicity and found it has no (low) potential to produce allergens.

[0119] EXAMPLE 3

[0120] Screening of seedlings using PEG stress to simulate drought stress Drought is one limiting factor in agriculture and can greatly impact crops such as wheat, soybean and com in the past as well as the future. For example, in Kansas wheat yield typically fluctuates between 40 and 52 bu / ac however drought can affect wheat yields significantly. In 2002 and 2006 wheat yields were in the low 30s bu / ac, in 2014 wheat yields were 33.2 bu / ac and in 2023 where yields were just 35 bu / ac and 29% of the crop was abandoned. Soybean is also affected by drought. In 2012, both US soybean and corn production dropped by 22% and 16%, respectively due to drought, and in 2024 Kansas and Ohio both reported soybean lowered by about 10%. By 2036 portions of the Midwest could see yield reduced by 25 % decline in corn and 43 % for soybeans due to climate change.

[0121] Different methods can be used to simulate drought. One method is by the use of polyethylene glycol (PEG) which simulates drought by lowering the osmotic potential of the surrounding medium, a process that mimics the reduced water availability in drying soil. High-molecular- weight PEG (.g. PEG-6000) cannot cross the cell membranes of plant roots. Instead, it remains in the external medium, where it binds with water molecules to increase the solution’s osmotic pressure. This creates a water deficit similar to soil-based drought. This example demonstrates having wheat expressing the CAHS6 protein it has better resiliency to drought than control wheat plants as demonstrated by decreased germination time, and greater growth rates shown below.

[0122] Methodology

[0123] PEG-induced osmotic stress treatments

[0124] After transformation, 10 PCR-positive To plants were selected for planting and advancing to Ti. These second-generation lines were again tested for the presence of GDI, and those lines from the same event that showed a close to 3:1 Mendelian segregation were selected for advancing to the T2 generation. They were tested again for the GOI presence, and all plants that were PCR-positives were considered putative homozygous lines and selected for the in vitro screening with Polyethylene Glycol (PEG) induced drought stress. A total of two putative homozygous lines were selected, 7949D_2 and 7975B_6, in addition to the wild-type Bobwhite (BW), for the screening.

[0125] To simulate drought stress in in vitro cultivation media, PEG 6000 was used. Three different PEG osmotic stress levels were used: 0 MPa (no PEG added), -0.3 MPa (143 g / L), and -0.5 MPa (192g / L). PEG concentrations were calculated following published protocols considering an incubation temperature of 20°C. For seedling germination, the media used was ½ MS with 1 g / L MES buffer, 10 g / L sucrose, 0.2% PPM, and 2 g / L of Gelrite. Media were prepared and autoclaved to half the final volume, and the PEG was diluted in water and autoclaved to the final volume for the other half. They were combined immediately after autoclaving, then 13 to 15 mL of media with PEG was dispensed into each glass culture tube. Seeds were incubated in a 20% bleach solution containing one drop of Tween 20 at room temperature in a rotary shaker for 20 minutes. After the incubation time, seeds were rinsed four to five times with autoclaved, double-distilled water. After rinsing, the seeds were soaked in water for 10 min, then transferred to the media. Seeds were incubated for three days in the dark at 20 °C and then moved to the light with a 16h day and an 8h dark cycle until completion, 10 days after sowing.

[0126] Plant Material and Genotypes

[0127] Transgenic material was generated by plant transformation using the bombardment method using Triticum aestivum cv Bobwhite immature embryo calli following published protocols. Two plasmids were used for the transformations, one for the gene of interest (GO I) and a second one for the selective marker gene. The GOI plasmid has the CAHS6 gene from the tardigrade Hypsibius exemplaris driven by the ZmUbi promoter. The CAHS6 was in silico optimized for wheat transcription and translation by retrieving the protein sequence from NCBI (Accession # P0CU48), reverse translated, and codon optimized and then cloned into pAHC17 plasmid backbone and confirmed through Sanger sequencing as described in Example 1.

[0128] Experimental design

[0129] The experiment followed a complete randomized factorial design with three genotypes (wildtype, 7949D_2, and 7975B_6), and three PEG treatments (0 MPa, -0.3 MPa, -0.5 MPa; Table 1). For each combination of genotype x PEG levels, 15 seeds were sown, one seed per tube, giving a total of 135 experimental units. In a spreadsheet, each tube was assigned a number from 1 to 135, and then randomly assigned to a spot on the tray.

[0130] Table 1 - Summary of the experimental design and sample size

[0131] Seeds for Seeds for Genotype Treatment Seeds sowed germination traits analysis measurement O MPa 15 11 5

[0132] BW (wildtype) -0.3 MPa 15 15 5

[0133] -0.5 MPa 15 14 5 7949D 2 O MPa 15 15 5-0.3 MPa 15 15 5

[0134] -0.5 MPa 15 15 5 O MPa 15 15 5 7975B_6 -0.3 MPa 15 14 5

[0135] -0.5 MPa 15 15 5

[0136] Germination and seedlings measurements

[0137] The traits measured and calculated as a stress response difference among the genotypes to the in vitro induced drought stress were: days to germinate (DTG), seed germination percentage (SGP), coleoptile length (CL), shoot length (ShL), root length (RL), seedling length (SeL), root fresh weight (RFW), shoot fresh weight (SFW), root dry weight (RDW), shoot dry weight (SDW), seedling biomass (SB), seedling vigor index (SVI), and coefficient of relative inhibition (CRI). The stress tolerance index (STI) was calculated using the mean data for biomass trait under PEG-induced stress.

[0138] All the seeds were monitored daily to detect how many days it took to germinate after sowing. The DTG was recorded when the radicle emerged was approximately >0.3 mm. With this information, SGP was calculated per genotype per treatment.

[0139] After 10 days, five random tubes for each treatment and each genotype were selected for the measurements. Any residual media from the roots was washed out with tap water, and the germinated seeds were measured. Using a ruler in centimeters, the coleoptile, shoot, and root length were measured. The seedling length is the sum of the shoot and root lengths. All length measurements were recorded in millimeters.

[0140] Once the seedling lengths were recorded, their shoot and root fresh weights were logged, and they were placed in an oven set to 65°C for 2 days. After drying, the root and shoot dry weights were measured. The seedling biomass is the sum of both root and shoot dry weights. Seeds that didn’t germinate were classified as “no germination,” and visibly contaminated tubes were recorded as “contaminated” and excluded from the final data.

[0141] The SVI, CRI, and STI were calculated as shown below:

[0142] SVI = SGP × SeLgenotype mean

[0143] SBMcontroiS B Mstress

[0144] CRI SBMCOntrol

[0145] _ Traitcontroigenotype X Traitsf-ressgenotype

[0146] ‘ ‘trait, str ess ~ “2

[0147]

[0148] V raitcontrol, all genotypes )Statistical analysis

[0149] All statistical analyses and graphs were performed and built in RStudio. The data were analyzed with Genotype, PEG treatment, and their interaction as fixed effects, and three levels of PEG. Five random tubes were selected for the measurements. Fresh and dry weights below the scale’s limit of detection, but with germination detected, were considered zero for the statistical analysis. Contaminated tubes were removed from the analysis.

[0150] Results

[0151] Overall, across all traits analyzed, both genotype and PEG treatments had significant effects. Osmotic stress induced by PEG, simulating in vitro drought stress, consistently reduced germination and seedling growth. From the three genotypes, both transgenes performed differently compared to the wildtype BW. On the other hand, the interaction between genotypes and treatment was overall not significant. This means that the difference among the genotypes consistently changes to adapt to each treatment level, although genotypes significantly differ and the PEG stress is strong (Table 2).

[0152] Table 2 - Summary of ANOVA (continuous traits) and GLM (germination probability) for wheat seedlings under PEG-induced osmotic stress. Effects of genotype (G), PEG treatment (T), and their interaction (G×T) on each trait. F = F-statistic from ANOVA; χ² = likelihood ratio chi-square from GLM. Significance codes: ns = p > 0.05;. = p < 0.10; * = p < 0.05; ** = p < 0.01; *** = p < 0.001

[0153] Model

[0154] Trait Effect df Test Value p-value Sig.

[0155] type

[0156] DTG (days to germinate) ANOVA Genotype (G) 2 F 35.51 < 0.001 ***

[0157] Treatment (T) 2 F 98.72 < 0.001 *** G T 4 F 3.83 0.0058 ** Germination (SGP, GLM Genotype (G) 2 χ29.66 0.008 ** GLM) (logit) Treatment (T) 2 χ28.61 0.0135 * (response: Germinated) G × T 4 χ20 1 ns CL (coleoptile length) ANOVA Genotype (G) 2 F 45.39 < 0.001 ***

[0158] Treatment (T) 2 F 12.34 < 0.001 *** G x T 4 F 1.5 0.2234 ns ShL (shoot length) ANOVA Genotype (G) 2 F 33.97 < 0.001 ***

[0159] Treatment (T) 2 F 63.39 < 0.001 *** G x T 4 F 0.11 0.9774 nsRL (root length) ANOVA Genotype (G) 2 F 19.5 < 0.001 ***

[0160] Treatment (T) 2 F 57.73 < 0.001 *** G x T 4 F 2.07 0.1052 ns SeL (seedling length) ANOVA Genotype (G) 2 F 34.2 < 0.001 ***

[0161] Treatment (T) 2 F 75.56 < 0.001 *** G x T 4 F 0.73 0.5782 ns SFW (shoot fresh ANOVA Genotype (G) 2 F 16.81 < 0.001 *** weight) Treatment (T) 2 F 83.99 < 0.001 ***

[0162] G x T 4 F 0.22 0.9246 ns

[0163] RFW (root fresh weight) ANOVA Genotype (G) 2 F 14.08 < 0.001 ***

[0164] Treatment (T) 2 F 95.07 < 0.001 *** G x T 4 F 2.53 0.0571

[0165] SDW (shoot dry weight) ANOVA Genotype (G) 2 F 4.92 0.014 *

[0166] Treatment (T) 2 F 26.75 < 0.001 *** G x T 4 F 0.23 0.9222 ns RDW (root dry weight) ANOVA Genotype (G) 2 F 8.39 0.0012 **

[0167] Treatment (T) 2 F 23.06 < 0.001 *** G x T 4 F 0.29 0.8818 ns SB (seedling biomass) ANOVA Genotype (G) 2 F 22.48 < 0.001 ***

[0168] Treatment (T) 2 F 38.74 < 0.001 *** G x T 4 F 0.91 0.4664 ns SVI (seedling vigor ANOVA Genotype (G) 2 F 43.99 < 0.001 *** index) Treatment (T) 2 F 89.61 < 0.001 ***

[0169] G x T 4 F 1.06 0.39 ns

[0170] Germination

[0171] The seed germination percentage was overall consistently 100%, with the exception of genotype control BW in moderate osmotic stress (-0.3MPa), which was 73.3% (Table 3). Considering the overall results, this deviation could be caused by other factors that are not related to osmotic stress itself. For that, more tests should be performed. However, in the big picture, we can say that the osmotic stress hasn’t affected the germination percentage. Yet, the days it took to germinate weresignificantly impacted by the PEG levels. The stress caused the BW germination delay compared to both transgenic lines 7949D 2 and 7975B 6 that had faster germination. Even in the control media with 0 MPa, transgenic lines germinated in one day on average, compared to two days for the BW. The moderate stress -0.3 MPa increased the days to germination for all genotypes, with an average of three days for the transgenes and almost four days for the BW. And for the higher stress (-0.5 MPa), the germination of the transgenes increased for three days on average and more than five days for BW (FIG. 3).

[0172] Table 3 - Estimated marginal means (± SE) of days to germination (DTG) and seed germination percentage (SGP) for three wheat genotypes under PEG-induced osmotic stress. Within each PEG level, DTG means followed by different lowercase letters differ significantly at a = 0.05 (Tukey’s test).

[0173] Treatment (MPa) Genotype DTG (days)1Group (DTG) SGP (%)2O MPa BW 2.00 ± 0.26 b 100

[0174] O MPa 7949D_2 1.07 ± 0.22 a 100

[0175] O MPa 7975B_6 1.00 ± 0.22 a 100

[0176] -0.3 MPa BW 3.73 ± 0.26 a 73.3

[0177] -0.3 MPa 7949D_2 3.07 ± 0.22 a 100

[0178] -0.3 MPa 7975B_6 2.93 ± 0.23 a 100

[0179] -0.5 MPa BW 5.43 ± 0.23 b 100

[0180] -0.5 MPa 7949D_2 3.13 ± 0.22 a 100

[0181] -0.5 MPa 7975B_6 3.33 ± 0.22 a 100

[0182] 1DTG (days to germination) estimated marginal means ± standard error from a two-way ANOVA model (Genotype, Treatment, and their interaction). Within each PEG treatment, means followed by different lowercase letters (group DTG) differ significantly according to Tukey’s test (a = 0.05).

[0183] 2SGP values are derived from a binomial GLM (germinated vs. not germinated) and expressed as percentages (probabilityx100). For this experiment, SGP was 100% for all genotypextreatment combinations except BW at -0.3 MPa (73.3%).

[0184] Seedling length

[0185] The seedling length is strongly affected by the genotype and treatment level (FIG. 4).

[0186] Following a similar pattern from DTG, all genotypes performed better under control conditions (0MPa). Additionally, 7975B_6 (297.6 mm) and 7949D_2 (324.2 mm) lines were longer than BW (228.8 mm), showing that the transgene lines, on average, even under control conditions, seem to perform better than the wildtype. When the osmotic stress was introduced, the lengths of all genotypes decreased. However, transgene seedlings still performed better, being longer compared with the control genotype, which had a more pronounced reduction. For the -0.3 MPa BW was 66 mm, while 7975B_6 was 177.4 mm and 7949D_2 was 189.2 mm, on average. While for the -0.5 MPa BW was only 7.4 mm, when the 7975B_6 was 138.6 mm and 7949D_2 was 147.8 mm, on average. These data shows that the reduction in length of the BW seedlings was more than 3 OX comparing the 0 MPa with the highest PEG stress. While the CAHS6 genotypes 7975B 6 and 7949D 2 were reduced by about 2X (Table 4)

[0187] Table 4 - Estimated marginal means (± SE) of seedling length (SeL) for three wheat genotypes under PEG-induced osmotic stress. Within each PEG level, means followed by different lowercase letters differ significantly at a = 0.05 (Tukey’s test).

[0188] Treatment (MPa) Genotype SeL (mm)1Group O MPa BW 228.8 ± 19.3 a

[0189] O MPa 7975B_6 297.6 ± 19.3 b

[0190] O MPa 7949D 2 324.2 ± 19.3 b

[0191] -0.3 MPa BW 66.0 ± 19.3 a

[0192] -0.3 MPa 7975B_6 177.4 ± 19.3 b

[0193] -0.3 MPa 7949D_2 189.2 ± 19.3 b

[0194] -0.5 MPa BW 7.4 ± 19.3 a

[0195] -0.5 MPa 7975B_6 138.6 ± 19.3 b

[0196] -0.5 MPa 7949D_2 147.8 ± 19.3 b

[0197] 1SeL (seedling length) estimated marginal means ± standard error from a two-way ANOVA model (Genotype, Treatment, and their interaction). Within each PEG treatment, means followed by different lowercase letters differ significantly according to Tukey’s test (a = 0.05).

[0198] Seedling biomass

[0199] Following the pattern of the other traits, seedling biomass also showed better performance for the CAHS6 lines across all PEG levels, including in the control treatment (FIG. 5). All genotypes had a greater biomass when under non-stress conditions. BW presented 20.368 mg, 7975B_626.215 mg, and 7949D_229.512 mg of biomass. When under osmotic stress, a pronounced reduction of thebiomass was observed in the -0.5 MPa. BW presented 0.354 mg, while 7975B_6 13.938 mg, and 7949D 2 16.012 mg of biomass. This represents an approximate 57X reduction in BW and less than 2X in both transgene lines. Overall, both CAHS6 lines were able to sustain seedlings biomass better than the non-transgenic BW under -0.3 MPa and -0.5 MPa (Table 5).

[0200] Table 5 - Estimated marginal means (± SE) of seedling biomass (SB) for three wheat genotypes under PEG-induced osmotic stress. Within each PEG level, means followed by different lowercase letters differ significantly at a = 0.05 (Tukey’s test).

[0201] Treatment (MPa) Genotype SB (mg)1Group O MPa BW 20.368 ± 2.27 a

[0202] O MPa 7975B_6 26.215 ± 2.27 ab

[0203] O MPa 7949D_2 29.512 ± 2.27 b

[0204] -0.3 MPa BW 6.408 ± 2.27 a

[0205] -0.3 MPa 7975B_6 15.278 ± 2.27 b

[0206] -0.3 MPa 7949D_2 16.712 ± 2.27 b

[0207] -0.5 MPa BW 0.354 ± 2.27 a

[0208] -0.5 MPa 7975B_6 13.938 ± 2.27 b

[0209] -0.5 MPa 7949D_2 16.012 ± 2.27 b

[0210] 1SB (seedling biomass) estimated marginal means ± standard error from a two-way ANOVA model (Genotype, Treatment, and their interaction). Within each PEG treatment, means followed by different lowercase letters differ significantly according to Tukey’s test (a = 0.05).

[0211] Seedling vigor index

[0212] SVI is an index that measures how well the seedling performed. For that, it combines the seed germination percentage with the seedling length. So, when SVI is high, it means that it had a good performance in the germination and or the growth, indicating a better establishment in the condition tested. Here we observed the same consistent pattern as before. Besides all genotypes being vigorous in the absence of PEG, the CAHS6 genotypes still performed better overall in all PEG levels (FIG.

[0213] 6). Additionally, the increase in stress decreased the BW SVI more sharply compared to the other two genotypes (Table 6).Table 6 - Estimated marginal means (± SE) of the seedling vigor index (SVI = SGP x SeL) for three wheat genotypes under PEG-induced osmotic stress. Within each PEG level, means followed by different lowercase letters differ significantly at a = 0.05 (Tukey’s test).

[0214] Treatment (MPa) Genotype SVI1Group

[0215] 0 MPa BW 22880 ± 1790 a

[0216] O MPa 7975B_6 29760 ± 1790 b

[0217] O MPa 7949D_2 32420 ± 1790 b

[0218] -0.3 MPa BW 4818 ± 1790 a

[0219] -0.3 MPa 7975B_6 17740 ± 1790 b

[0220] -0.3 MPa 7949D_2 18920 ± 1790 b

[0221] -0.5 MPa BW 740 ± 1790 a

[0222] -0.5 MPa 7975B_6 13860 ± 1790 b

[0223] -0.5 MPa 7949D_2 14780 ± 1790 b

[0224] 1SVI (seedling vigor index) calculated as SGP x SeL, where SGP is the seed germination percentage and SeL is seedling length (mm). Values are estimated marginal means ± standard error from a two-way ANOVA model. Within each PEG treatment, means followed by different lowercase letters differ significantly according to Tukey’s test (a = 0.05).

[0225] Coefficient of relative inhibition (CRI) and stress tolerance index

[0226] CRI takes into consideration the seedling biomass mean of each genotype in each treatment and compares it with the seedling biomass of the control. With that, a value closer to zero means low or no inhibition, while a value closer to one means a very strong inhibition of the treatment. Looking to the Table 7, we can see that both transgene genotypes showed a medium inhibition close to 0.5. On the other hand, BW increased the CRI when the stress level increased, being closer to one in the highest PEG level tested.

[0227] STI combines the biomass of the control and of the treatments to show how productive a genotype is without stress and how much biomass it keeps under stress. Here, in contrast with CRI, a higher STI means a better performance under stress. When observing the results in the Table 7, we notice that the genotype 7949D 2 performed better, maintaining a STI over 0.7 for both moderate and higher level of PEG, while the genotype 7975B 6 had a good performance of approximately 0.6 STI in the moderate stress and reduced to approximately 0.5 in the higher stress. However, BW had the worst performance with 0.2 STI in the moderate stress and close to zero in the higher stress, as was observed with the CRI.Table 7 - Mean seedling biomass (SB), coefficient of relative inhibition (CRI), and stress tolerance index (STI) for three wheat genotypes under PEG-induced osmotic stress.

[0228] Genotype Treatment (MPa) SB mean (mg)1CRI2STI SB37949D_2 OMPa 29.5

[0229] 7949D_2 -0.3 MPa 16.7 0.434 0.767 7949D_2 -0.5 MPa 16 0.457 0.734 7975B 6 OMPa 26.2

[0230] 7975B_6 -0.3 MPa 15.3 0.417 0.622 7975B_6 -0.5 MPa 13.9 0.468 0.568

[0231] BW 0 MPa 20.4

[0232] BW -0.3 MPa 6.41 0.685 0.203

[0233] BW -0.5 MPa 0.354 0.983 0.011

[0234] 1SB mean is the mean seedling biomass per genotype * treatment, based on five randomly sampled seedlings per cell.

[0235] 2CRI (coefficient of relative inhibition) was calculated as CRI = (SB control - SB treatment) / SB control, where SB control is the mean biomass at 0 MPa for each genotype. Higher CRI indicates stronger inhibition under stress.

[0236] 3STI SB (stress tolerance index for seedling biomass) was calculated as STI SB = (SB_ctrl_genotype x SB_mean_genotype, treatment) / (SB_ctrl_all2), where SB_ctrl_all is the overall mean biomass of all genotypes at 0 MPa. STI SB is reported only for stress treatments (0 MPa values shown as “-”).

[0237] EXAMPLE 4

[0238] Expression of CAHS6 gene in Arabidopsis thaliana

[0239] This Example describes a method for the stable in planta transformation of Arabidopsis thaliana with genetic constructs encoding the CAHS6 (aka CAHS D) protein (a Cytosolic Abundant Heat Soluble tardigrade-specific intrinsically disordered protein). This protocol utilizes an Agrobacterium-mediated floral dip procedure to generate transgenic lines without the requirement for intermediate tissue culture steps and could be used for any CAHS proteins.

[0240] Agrobacterium tumefaciens strain GV3101, harboring a binary vector comprising a sequence encoding the CAHS6 protein under the regulatory control of the constitutive Glycine max polyubiquitin promoter (GmUbi), is cultured in liquid LB medium supplemented with appropriate selection antibiotics. The 5 mL pre culture is incubated at 28 °C with orbital shaking at 200 rpmovernight from a single colony. On the next day 150 mL of inoculum culture is grown from 5 mL pre culture until an O. D. 600nm of 1.5 is achieved. The bacterial cells are harvested by centrifugation at 4,000 x g for 10 minutes and resuspended in an infiltration medium to a final O. D. 600nm of approximately 0.8. The infiltration medium comprises 5.0% (w / v) sucrose and 0.05% (v / v) of a silicone surfactant (e.g., Silwet L-77).

[0241] Arabidopsis thaliana (ecotype Col-0) plants are grown under long-day conditions (16 h light / 8 h dark) at 22 °C. The primary inflorescence is clipped approximately one week prior to transformation to induce the growth of multiple secondary bolts. The transformation is performed by inverting the plants and submerging the aerial inflorescences into the Agrobacterium suspension for 5 minutes with short and gentle agitation to disperse air bubbles. Following submersion, the plants are placed in a horizontal position within a tray and covered with a plastic dome overnight, kept in the dark to maintain high humidity (>90% RH), thereby optimizing bacterial infection of the germline.

[0242] Seeds (T₁ generation) are harvested from the treated plants upon reaching maturity and desiccation. Transgenic individuals (T₁ and T₂ generations) are identified by surface-sterilizing the seeds and germinating them on Murashige and Skoog (MS) medium supplemented with 12 mg / L Glufosinate).

[0243] Survivors exhibiting normal root development and green primary leaves are transplanted to soil. The presence and stable integration of the CAHS6 transgene are confirmed via PCR amplification of the CAHS6 sequence from genomic DNA. Expression levels of the CAHS6 protein are further characterized in T₁ and subsequent T₂ generations using RT-PCR.

[0244] EXAMPLE 5

[0245] Expression of CAHS6 gene in Nicotiana benthamiana

[0246] The transformation of Nicotiana benthamiana used the same genetic construct with the CAHS6 protein encoded from the Arabidopsis transformation of Example 4. The leaf disc Agrobacterium-mediated stable transformation protocol from the literature was followed. The strains used were GV3101 and LAB4404. For the leaf discs, two newer fully expanded leaves were disinfected with 10% bleach solution with one drop of Tween-20 pr 50 mL. Leaves were soaked in this solution for 10 minutes and then rinsed with sterile double distilled water. Leaf discs or pieces of no more than five millimeters were transferred to the preculture medium with the adaxial side up. Leaf pieces were incubated for 24 hours at 25 °C under 18 hour light cycle. A 5 mL bacteria pre culture in YEP supplemented with antibiotics was carried out overnight at 28 °C with orbital shaking at 200 rpm from a single colony. On the next day 2 mL of the pre culture was transferred to 50 mL ofYEP medium with antibiotics and incubated with shaking again for 6 to 8 hours. Cells were harvested by centrifugation at 4000 × g for 10 minutes, and the pellet was resuspended to a final OD600 of 0.5- 1.0 in liquid cultivation medium (MS-based media supplemented with BAP and NAA). Pre cultured explants were inoculated for 30 minutes followed by blotting on sterile filter paper and transferred to a solidified cocultivation medium with the adaxial side up. They were incubated for 3 days at 25 °C under an 18-hour light cycle. After the transformation step, explants were transferred to regeneration media every two weeks. After two weeks, shoots become visible and are individually transferred once they get longer than 3 mm for the rooting medium, avoiding clones by avoiding shoots from the same callus cluster. Once roots were stablished they were transferred to the soil. PCR of the CAHS6 detected 12 positive transformed T₀ plants. Seeds were harvested and then screened in ½ MS media supplemented with 12 g / L of glufosinate. These seedlings with good root and leaf development were selected for further testing.

[0247] EXAMPLE 6

[0248] Cold tolerance of A. thaliana

[0249] Methodology

[0250] Seeds from floral dip transformation were screened for the gene of interest by germinating them in ½ MS media supplemented with 1% sucrose, 0.5 g / L MES, and 0.2% PPM, and 12 mg / L of glufosinate. A total of 12 T₁ plants tested positive for CAHS6 by PCR. Three of them were selected for the temperature stress tolerance test: AA001, AA019, and AA021. A wildtype (WT) was also used in the tests. T₂ seeds were soaked with 70% ethanol for 1 minute, then after removing the ethanol, they were soaked in 20% bleach with 1 drop of Tween 20 for 10 minutes. Seeds were then washed with autoclaved, double-distilled water at least four times. Right after, the transgene seeds were spread on ½ MS media + glufosinate, and the WT on media with no selective agent. Plates were kept at 4 °C for 2 days and then moved to 20 °C with a 16-hour photoperiod. Twelve seedlings with 7-day old were moved to a new plate and exposed to the cold temperature. Plates were moved to the -20 °C in the dark for 60 minutes, and then transferred to the 4 °C for 16 hours in the dark. Then they were transferred to a new plate and moved to 20 °C with a 16-hour photoperiod for recovery. After at least 3 days, seedlings were evaluated for the number of green / healthy leaves and bleached leaves. The percentage of green leaves was calculated to estimate cold tolerance.

[0251] Green leaves percentage under cold stress

[0252] In the cold treatment, plants were frozen for a few minutes, and after recovery, all WT seedlings exhibited bleached leaves, with no leaves remaining green (FIG. 7). In contrast, transgenelines AA001, AA019, and AA021 showed a few green leaves, with similar responses among them, having approximately 26 - 32 % of their leaves green (Table 8). These results highlight the potential of the CAHS6 expression in protecting plants from cold / freeze stresses.

[0253] Table 8 - Mean (± SE) percentage of green leaves (undamaged) per seedling for four A thaliana genotypes under control, heat, and freeze treatments.

[0254] Genotype Control (% green) Heat (% green) Freeze (% green) WT 100.0 ± 0.0 66.7 ± 7.8 0.0 ± 0.0

[0255] AA001 100.0 ± 0.0 86.5 ± 4.9 29.6 ± 6.5

[0256] AA019 100.0 ± 0.0 60.7 ± 6.7 26.4 ± 10.2

[0257] AA021 100.0 ± 0.0 25.8 ± 7.6 32.4 ± 8.9

[0258] Values represent mean ± standard error of the percentage of green leaves per seedling for each genotype x temperature treatment. Each mean is based on 12 seedlings per genotype and treatment.

[0259] EXAMPLE 7

[0260] Heat tolerance of A. thaliana

[0261] Methodology

[0262] The model plant A. thaliana (At), wild type and transformed ones with CAHS6, were tested for heat tolerance. At T2 seedlings were used in this assay. Seeds were cleaned by soaking in 70% ethanol for 1 minute, then in 20% bleach with one drop of Tween20 for 10 minutes, and rinsed with autoclaved double-distilled water at least four times. Decontaminated seeds were then transferred to ½ MS media supplemented with 1% sucrose, 0.5 g / L MES, and 0.2% PPM for germination. Transgene seeds were sown in media supplemented with 12g / L of glufosinate. Seeds were incubated for two days at 4°C in the dark before being moved to the chamber at 20°C with a 16h light and 8h dark cycle. Seedlings that were seven days old were treated with heat stress at 45°C for 60 minutes in an incubator in the dark and then moved back to the chamber. Seedlings were left to recover for five days and then scored for survival. Seedlings that were heat-treated were evaluated for the number of green and bleached leaves. The percentage of green leaves was calculated after leaf counting. Green leaves percentage under cold stress

[0263] For the heat treatment, transgene line AA001 showed the best response to the stress (FIG. 7) with the highest proportional number of green leaves with a mean of approximately 86% of green leaves compared to approximately 66% of green leaves for the WT (see Table 8, above).EXAMPLE 8

[0264] Production of Transgenic Maize Plants Expressing CAHS

[0265] This Example describes a method for the genetic transformation, selection, and recovery of transgenic maize plants containing sequences encoding tardigrade-specific CAHS protein. The techniques for transformation, selection, and recovery can follow published protocols.

[0266] Briefly, maize ears are harvested approximately ten to fourteen days after anthesis. The harvested ears are surface sterilized using a solution of 20% commercial bleach and 0.05% Tween 20 for 20 minutes, followed by three rinses with distilled water. Immature embryos, measuring between 0.5 and 1.5 mm in length, are excised and cultured with the scutellum side facing up on maize initiation medium (M10+). These embryos are maintained for 1-2 days in low light at 25 °C prior to transformation.

[0267] Before the introduction of the CAHS transgene, immature embryos are placed on M10+ medium supplemented with 0.2 M mannitol and 0.2 M sorbitol for four hours. The embryos are then transformed using a particle inflow gun to deliver the genetic construct. Twelve to sixteen hours following genetic transformation, the maize embryos are transferred to M10+ medium containing 5 mg / L glufosinate, which serves as a selection agent for bar gene expression. The cultures are maintained at 25 °C and transferred to fresh selection medium every two weeks.

[0268] Approximately six to eight weeks post-transformation, cultures exhibiting defined embryogenic calli are moved to a maturation medium containing 5 mg / L glufosinate for a period of two weeks. Once shoots have formed, the cultures are transferred to a regeneration medium (also containing 5 mg / L glufosinate) and cultured for two to three weeks under light conditions. After root formation occurs, the regenerated plantlets are transferred to soil and grown to maturity. To confirm stable integration and expression, DNA and RNA are isolated from leaf tissue to perform Southern-blot, RT-PCR analyses, respectively. Finally, T₁ and T₂ progeny are grown to assess the long-term stability of the promoter and the CAHS gene.

Claims

CLAIMS:

1. A method of producing a genetically-modified plant having enhanced tolerance to an abiotic stress as compared to a control plant, said method comprising:transforming a plant with a tardigrade transgene to yield a genetically-modified plant, wherein said transgene is stably integrated into the genome of said genetically-modified plant and encodes a tardigrade CAHS protein, thereby enhancing the tolerance of said genetically-modified plant to abiotic stress, andwherein said genetically-modified plant is selected from the group consisting of wheat, oat, barley, rice, maize, millet, rye, sorghum, triticale, buckwheat, quinoa, soybeans, beans, peas, alfalfa, potatoes, sweet potatoes, cassava, yam, tomatoes, peppers, tobacco, Arabidopsis, and cotton.

2. The method of claim 1, wherein said CAHS transgene comprises a sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO:2 or 3; (b) a nucleotide sequence having at least about 75% sequence identity to SEQ ID NO:2 or 3; (c) a nucleotide sequence encoding a CAHS protein comprising SEQ ID NO:1, 5, 6, 7, 8, 9, 10, or 11; (d) a nucleotide sequence encoding a CAHS protein having at least about 50% amino acid identity to SEQ ID NO: 1 and being a functional analog thereof.

3. The method of claim 2, wherein said functional analogs comprise a sequence encoding a CAHS protein comprising SEQ ID NO:5, 6, 7, 8, 9, 10, or 11.

4. The method of claim 1, wherein said plant comprises a plant tissue, and said transforming comprising:culturing said tissue on media; andintroducing said transgene into the cells of said tissue to yield transformed tissue.

5. The method of claim 4, further comprising:regenerating whole plants from said tissue, wherein said regenerated plants have enhanced tolerance to an abiotic stress.

6. The method of claim 5, wherein said regenerating comprises:inducing callus formation from said transformed tissue;regenerating shoots; androoting of said shoots in rooting media to regenerate said whole plant.

7. The method of claim 4, wherein said introducing is selected from the group consisting of: Agrobacterium-mediated transformation, PEG-mediated uptake, electroporation-mediated uptake, particle bombardment-mediated delivery, and microinjection.

8. The method of claim 1, said method further comprising: crossing said genetically-modified plant with a second plant to thereby produce progeny having enhanced tolerance to heat stress.

9. The method of claim 1, said method further comprising self-pollinating said genetically-modified plant to thereby produce genetically-modified seed, wherein said seed comprises said heterologous, abiotic stress tolerance gene.

10. The method of claim 1, wherein said genetically-modified plant comprises a phenotype that is substantially similar to a phenotype of said control plant when said plants are grown under non-stress conditions.

11. The method of claim 1, wherein said genetically-modified plant comprises an improved phenotype as compared to said control plant, when said plants are grown under abiotic stress.

12. The method of claim 1, wherein said abiotic stress is selected from the group consisting of: heat, cold, drought, salinity, and combinations thereof.

13. A genetically-modified seed produced according to the method of claim 1.

14. A genetically-modified plant having enhanced tolerance to an abiotic stress as compared to a control plant, said genetically-modified plant having a tardigrade transgene stably integrated into the genome of said genetically-modified plant, wherein said tardigrade encodes a tardigrade CAHS protein, thereby enhancing the tolerance of said genetically-modified plant to abiotic stress.

15. The genetically-modified plant of claim 14, said plant expressing a CAHS protein comprising SEQ ID NO:1 or a CAHS protein having at least about 50% amino acid identity to SEQ ID NO:1 and being a functional analog thereof.

16. The genetically-modified plant of claim 15, wherein said functional analogs comprise a sequence encoding a CAHS protein comprising SEQ ID NO:5, 6, 7, 8, 9, 10, or 11.

17. A method of producing genetically-modified plants having enhanced tolerance to an abiotic stress, said method comprising: crossing a first plant with a second plant to thereby produce progeny, wherein at least one of said first or second plants is a genetically-modified plant according to claim 14, 15, or 16, said progeny having enhanced tolerance to an abiotic stressor.

18. A cereal plant, seed, or part thereof, preferably wheat or corn, resilient to environmental stresses, said cereal plant, seed, or part thereof comprising or expressing a transgene encoding for tardigrade CAHS6 protein (aka CAHS 94205) (GenBank Accession # P0CU48):msgrnveshm ernekvvvnn sghadvkkqq qqvehtefth tevkaplihp appiistgaaglaeeivgqg ftasaarisg gtaevhlqps aamteearrd qeryrqeqes iakqqeremekkteayrkta eaeaekirke lekqhardve frkdliesti drqkrevdle akmakreldregqlakeale rsrlatnvev nfdsaaghtv sggttvstsd kmeikrn (SEQ ID NO: 1).

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