Patent Application
20160068860
The invention relates to transgenic plants with improved phenotypic traits, including enhanced stress resistance. The improved traits are conferred by enhanced ABA receptor signalling. Also within the scope of the invention are related methods, uses, isolated nucleic acids and vector constructs.
The ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards increasing the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Alternatively methods can be used to modify or change, or “edit”, the existing genetic material in a targeted manner, altering just one or a few amino acids of the encoded protein, for example using mutagenesis or CRISPR technology. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits. Traits of particular economic interest are growth and stress resistance, as these are determinants of eventual crop yield.
Plants adapt to changing environmental conditions by modifying their growth. Plant growth and development is a complex process involves the integration of many environmental and endogenous signals that, together with the intrinsic genetic program, determine plant form. Factors that are involved in this process include several growth regulators collectively called the plant hormones or phytohormones. This group includes auxin, cytokinin, the gibberellins (GAs), abscisic acid (ABA), ethylene, the brassinosteroids (BRs), and jasmonic acid (JA), each of which acts at low concentrations to regulate many aspects of plant growth and development. Abiotic and biotic stress can negatively impact on plant growth leading to significant losses in agriculture. Even moderate stress can have significant negative impact on plant growth and thus reduce the yield of agriculturally important crop plants. In any given season or location, crops very commonly experience periods of moderate stress or one kind or another, which restricts the productivity of that crop. Therefore, finding a way to improve growth, in particular under stress conditions, is of great economic interest.
ABA plays a critical role both for plant biotic and abiotic stress response (Cutler et al., 2010). Since ABA is recognized as the critical hormonal regulator of plant response to water stress, both the ABA biosynthetic and signalling pathways can be considered as potential targets to improve plant performance under drought. Thus, it has been demonstrated that transgenic plants producing high levels of ABA display improved growth under drought stress compared to wild type (Iuchi et al. 2001; Qin & Zeevaart 2002). Priming of ABA biosynthesis can be obtained by direct over-expression of 9-cisepoxycarotenoid dioxygenase, a key enzyme in the biosynthetic pathway (Iuchi et al. 2001; Qin & Zeevaart 2002), or through the use of chemicals that accelerate ABA accumulation (Jakab et al., 2005). On the other hand, some examples are also known of Arabidopsis mutants (era1, abh1, pp2c combined mutants) affected in ABA signal transduction that show both an enhanced ABA response and drought resistant phenotypes (Pei et al., 1998; Hugouvieux et al., 2001; Saez et al., 2006). For instance, enhancement of abscisic acid sensitivity and reduction of water consumption has been achieved in Arabidopsis by combined inactivation of the protein phosphatases type 2C (PP2Cs) ABI1 and HAB1, leading to drought resistant plants (Saez et al., 2006).
Enhancing ABA signaling through the recently discovered PYR/PYL ABA receptors is another approach to improve plant drought resistance, for instance through over-expression of the receptors or generation of constitutively active versions (Santiago et al., 2009; Xaavedra et al., 2009; Mosquna et al., 2011, WO 2013/006263). However, pleiotropic effects due to sustained effects of high ABA levels or active ABA signalling might negatively affect plant growth, since abiotic stress responses divert resources required for normal growth. In Mosquna et al., 2011 and WO 2013/006263, constitutively active ABA mutant PYR1, PYL2 and PYL9 polypeptides are disclosed which inhibit PP2C in the absence of ABA. It was shown that only the combination of several specific amino acid substitutions was sufficient to convey the desired effects and produce variants with activation levels nearly indistinguishable from ABA saturation wild type PYR1.
Recent studies reveal at least two subclasses of PYR/PYL receptors, including monomeric and dimeric PYLs (Dupeux et al., 2011a; Hao et al., 2011). The dimeric receptors show a higher Kd for ABA (>50 μM, lower affinity) than monomeric ones (˜1 μM); however, in the presence of certain clade A protein phosphatases 2C (PP2Cs), both groups of receptors form ternary complexes with high affinity for ABA (Kd 30-60 nM) (Ma et al., 2009; Santiago et al., 2009a, b). A third subclass appears when we consider the trans-dimeric PYL3 receptor, which suffers a cis- to trans-dimer transition upon ligand binding to facilitate the posterior dissociation to monomer (Zhang et al., 2012). Dimeric receptors occlude their surface of interaction with the PP2C in the dimer, so they are strongly ABA-dependent for dissociation and adoption of a PP2C binding conformation (Dupeux et al., 2011). In vitro, monomeric ABA receptors are able to interact in the absence of ABA to some extent with the catalytic core of PP2Cs, although less stable complexes are formed compared to ternary complexes with ABA (Dupeux et al., 2011; Hao et al., 2011). In planta, tandem affinity purification (TAP) and mass spectrometrical analysis of PYL8-interacting partners was largely dependent on ABA to recover PYL8-PP2C complexes (Antoni et al., 2013).
Yeast two hybrid (Y2H) assays reveal both ABA-independent and ABA-dependent interactions among PYR/PYLs and PP2Cs. Y2H interactions of PYR/PYLs and PP2Cs that are dependent on exogenous ABA offer the possibility to set up screenings involving the generation of allele libraries and growth tests aimed to identify mutations that render ABA-independent interactions. Such mutations might lead in the plant cell to either receptors that interfere with PP2C function by enhancing association kinetics, steric hindrance or constitutively active receptors (not dependent on ABA-induced conformational changes) that inhibit PP2Cs in the absence of ABA. The interaction in Y2H assays of PYL4 and PP2CA, two representative members of the PYR/PYL and clade A PP2Cs families, respectively, was shown to be ABA-dependent (Lackmann et al., 2011). PYL4 shows high expression levels in different tissues and its inactivation is required to generate strongly ABA-insensitive combined pyr/pyl mutants (Gonzalez-Guzman et al., 2012). PP2CA plays a critical role to regulate both seed and vegetative responses to ABA, and regulates stomatal aperture through interaction with the anion channel SLAC1 and the kinase SnRK2.6/OST1 (Kuhn et al., 2006; Yoshida et al., 2006; Lee et al., 2009). In the absence of ABA, PP2C phosphatases interact with SnRK2 kinases to inhibit their autophosphorylation and activation. In the presence of ABA, inhibition of PP2C phosphatases by the ABA-receptor complex results in phosphorylation and activation of SnRK2 kinases, which in turn phosphorylate transcription factors that promote transcription of ABA-responsive genes.
Therefore, PP2CA is a physiologically relevant target to design PYR/PYL receptors that show a constitutive interaction with the phosphatase, affecting ABA signalling and plant stress response. Through the generation of PYL4 (At2g38310) allele libraries and Y2H assays, we identified several PYL4 mutations enabling ABA-independent interaction with PP2CA in yeast. Interestingly, upon over-expression of some PYL4 mutant receptors in Arabidopsis, we obtained enhanced sensitivity to ABA compared to wild-type PYL4 both in seed and vegetative tissues. Moreover, 35S:PYL4A194T and 5S:PYL4H82rV97A transgenic plants showed enhanced drought resistance compared to wt or 35S:PYL4 plants.
Thus, we describe a way of enhancing plant stress resistance, for example drought resistance, in a plant through the introduction of mutagenized versions of PYR/PYL, specifically PYL4, receptors. These carry single or multiple mutations and show effects in vegetative and non-vegetative tissue.
The inventors have shown that specific modifications in a PYL/PYR polypeptide can change the properties of the protein leading to improvements of agronomically important plant traits. In particular, the inventors have shown that certain modifications enable ABA independent interaction of the mutant protein with PP2C and lead to enhanced inhibition of PP2C compared to wt receptors. The ABA-independent interaction did not lead to major inhibition of the PP2C in the absence of ABA; however it improved ABA-dependent inhibition of the PP2C, for instance at low ABA levels. In other words, the modifications result in constitutive interaction of the receptor protein with PP2C, generating additional contact points between receptor and PP2C, which leads to improved ABA-dependent inhibition of the PP2C. The inventors have also shown that when the mutant protein is expressed in transgenic plants, Arabidopsis and barley, the plants show improved stress resistance, in particular to drought stress even in the absence of a stress inducible promoter. The invention therefore relates to PYL and PYR mutant polypeptides comprising one or more amino acid mutations or modifications, for example substitutions, compared to the wild type sequence and which confer ABA-independent interaction of the PYL/PYR receptor with PP2C and enhance ABA-dependent inhibition of the PP2C as well as their use in methods for conferring stress resistance to a plant. Mutations are exemplified herein with reference to the AtPYL4 wild type polypeptide (SEQ ID NO:3). However, mutant homolog/orthologs of AtPYL4 are also within the scope of the various aspects of the invention and these have the mutations as defined herein at corresponding/equivalent positions with reference to SEQ ID NO:3.
Specifically, the invention relates to an isolated mutant nucleic acid or a nucleic acid construct comprising a mutant nucleic acid wherein said nucleic acid encodes a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
Thus, said mutant nucleic acid comprises SEQ ID NO:1, 2 or 4 but has one or more modifications of said sequence resulting in said nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution. Examples of polypeptides according to the invention with specific mutations according to the invention are shown in SEQ ID NOs: 60-65.
In another aspect, the invention relates to a vector comprising an isolated mutant nucleic acid or a nucleic acid construct comprising a mutant nucleic acid wherein said nucleic acid encodes a mutant PYL or PYR polypeptide comprising an amino acid modification, preferably a substitution, at/corresponding to
In another aspect, the invention relates to a host cell comprising a vector comprising an isolated nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
In a further aspect, the invention relates to a transgenic plant expressing an isolated nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
In yet a further aspect, the invention relates to a method for increasing stress resistance in a transgenic plant comprising, introducing and expressing a nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitutions corresponding to
In yet a further aspect, the invention relates to a method for prolonging seed dormancy/preventing early germination/inducing hyperdormancy in a transgenic plant comprising, introducing and expressing a nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
In another aspect, the invention relates to a method for constitutive activation of the ABA signalling pathway comprising, introducing and expressing a nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
The invention also relates to a method for inhibiting the activity of a PP2C in a transgenic plant comprising, introducing and expressing a nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
The invention relates to a method for producing a transgenic plant with increased stress resistance comprising introducing and expressing a nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
The invention relates to a plant obtained or obtainable by a method of the invention ro described herein. Such methods include methods for malign transgenic plants as well as methods using targeted gene editing or mutagenesis.
In a final aspect, the invention relates to the use of a nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
In one embodiment of the various aspects above, the mutant PYL or PYR polypeptide is AtPYL4. In another embodiment, the mutant PYL or PYR polypeptide is a homolog/ortholog or functional variant of AtPYL4.
The invention is further described in the following non-limiting figures.
D, PYL4A194T prevents better than PYL4 the PP2CA-mediated dephosphorylation of OST1, ABF2 (1-173), ABI5 (1-200), and SLAC1 (1-186). Value 1 expresses protection of each substrate in the absence of ABA, and the normalized ratio expresses the fold number that either PYL4A194T or PYL4 enhanced protection of the substrate at the indicated concentration of ABA. A 1:1 phosphatase:receptor stoichiometry was used in this assay.
The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature. As used herein, the words “nucleic acid”, “nucleic acid sequence”, “nucleotide”, “nucleic acid molecule” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term “gene” or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences. In one embodiment, the isolated nucleic acid and the isolated nucleic acid used in the various methods and plants according to the invention is PYL/PYR cDNA. Examples of such sequences are given herein.
The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
For the purposes of the invention, “transgenic”, “transgene” or “recombinant” means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
(a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or
(b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
(c) a) and b)
are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above—becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic (“artificial”) methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815 both incorporated by reference.
A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the different embodiments of the invention are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. According to the invention, the transgene is integrated into the plant in a stable manner and preferably the plant is homozygous for the transgene.
The aspects of the invention involve recombination DNA technology, mutagenesis or genome editing and exclude embodiments that are solely based on generating plants by traditional breeding methods.
The invention relates to isolated plant PYL and PYR mutant nucleic acid and isolated mutant plant PYL and PYR polypeptides encoded by said mutant nucleic acid wherein said mutant polypeptides comprise one or more amino acid mutations or modifications, for example substitutions or deletions, compared to the wild type sequence, which promote ABA-independent interaction with PP2C and enhanced ABA-dependent inhibition of the PP2C, for instance at low ABA levels As explained herein, certain nucleic acids are modified in the mutant nucleic acids so that the resulting mutant protein is different from the wild type protein. The sequences shown herein show the wild type sequences and mutations in the mutant proteins with reference to positions in these sequences are set out herein. The invention also relates to methods for making transgenic plants with improved traits expressing said mutant polypeptides. Preferably, these mutations are located in the protein domain that interacts with a PP2C, for example PP2CA, and/or the domain that interacts with ABA (residues K59, E94, Y120, S122 and E141 in AtPYL4 as shown in
In one embodiment, the polypeptide of the various aspects of the invention has one or more mutation at one or more of the positions defined herein, but does not have mutations at one or more of the following positions with reference to SEQ ID NO: 3: H82, V105, V106, L109, A111, D177, F178, V181, C185 and/or S189. Also excluded are specific activating mutations as disclosed in WO 2013/006263, including mutations in PYL/PYR polypeptides corresponding to the following positions in PYR1: V83, 184, L87, A89, M158, F159, T162, L166, K170 (incorporated by reference). In one embodiment of the various aspects of the invention, the polypeptide of the invention does not have any additional mutations other than one or more of those mutations described herein. In one embodiment, the polypeptide of the invention does not have any additional activating mutations, that is mutations that affect ABA signalling.
Thus, in a first aspect, the invention relates to an isolated mutant nucleic acid encoding a mutant PYL or PYR polypeptide comprising one or more amino acid substitutions corresponding to one or more of position A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO: 3 and shown in
Thus, in one embodiment, the substitution is at A194 with reference to AtPLY4 as set forth in SEQ ID NO: 3 (see also SEQ ID NO: 55 and 60) or a position corresponding thereto. In another embodiment, the substitution is at V97 with reference to AtPLY4 as set forth in SEQ ID NO: 3 (see also SEQ ID NO:61) or a position corresponding thereto. In another embodiment, the substitution is at F130 with reference to AtPLY4 as set forth in SEQ ID NO: 3 (see also SEQ ID NO:64) or a position corresponding thereto. In another embodiment, the substitution is at C176 with reference to AtPLY4 as set forth in SEQ ID NO: 3 (see also SEQ ID NO:65) or a position corresponding thereto. In another embodiment, one or more of the residues at positions corresponding to A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO: 3 is deleted.
The invention also relates to an isolated nucleic acid encoding a mutant PYL or PYR polypeptide comprising two amino acid substitutions corresponding to/equivalent to positions H82 and V97 as set forth in SEQ ID NO:3 and shown in
Thus, the polypeptide preferably comprises one, two, three, four or more mutations as described above. Any combination of the substitutions or deletions specifically set out above is within the scope of the invention. For example, an amino acid substitution at position A194 or a position corresponding thereto may be combined with an amino acid substitution at V97 and/or F130 and/or 0176 or a position corresponding thereto. In one embodiment, other combinations of the mutations at positions A194, V97 and/or F130 and/or 0176 are also possible, for example with H82 and/or mutations in other residues in the domains that interact with ABA or a PP2C.
In one embodiment, an amino acid substitution at position V97 or a position corresponding thereto may be combined with an amino acid substitution at F130, A194 and/or 0176 or a position corresponding thereto. In one embodiment, an amino acid substitution at position F130 may be combined with an amino acid substitution at A194, V97 and/or 0176 or a position corresponding thereto. In one embodiment, an amino acid substitution at position 0176 may be combined with an amino acid substitution at A194, V97 and/or F130 or a position corresponding thereto.
A PP2C may be selected from HAB1 (Homology to AB11), ABI1 (Absciscic acid insensitive 1), ABI2 (Absciscic acid insensitive 2) or PP2CA. Preferably, the PP2C is PP2CA.
The amino acid substitutions at the positions set out above are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art. Alternatively, insertions can be made to render the site non-functional.
The mutations in the PYL/PYR polypeptides described herein are shown with reference to the amino acid positions as shown in SEQ NO:3 which designates the AtPLY4 wild type polypeptide sequence encoded by a nucleic acid shown in SEQ ID NOs:1, 2 or 4. Thus, in one embodiment, the mutant PYL or PYR polypeptide is encoded by a nucleic acid comprising, consisting or substantially consisting of a sequence substantially identical to SEQ ID NOs: 1, 2 or 4, a functional variant, ortholog or homolog thereof, but which has modifications so that transcription of the mutant nucleic acid results in a mutant protein comprising one or more of the mutations at the positions listed above. In other words, the mutant PYL or PYR polypeptide is encoded by a nucleic acid comprising, consisting or substantially consisting of a sequence substantially identical to SEQ ID NOs: 1, 2 or 4, a functional variant, ortholog or homolog thereof, but which comprises modifications in one or more the codons encoding the one or more residues listed above. These codons are 82, 97, 130, 176 and/or 194 in AtPYL4. As explained below, other PYL/PYR polypeptides share homology with AtPYL4 and residues for targeted manipulation that correspond to one or more of positions A194, V97, F130, H82 and 0176 in AtPYL4 can be identified by sequence comparison and alignment as described herein.
A PYL/PYR nucleic acid as used herein and according to the various aspects of the invention comprises SEQ ID NOs: 1, 2 or 4 or a functional variant or homolog/ortholog thereof, but wherein said nucleic acid is not the wild type nucleic acid shown in these sequences, but is a mutant nucleic acid that has a mutation in one or more codon which results in one or more mutation in the encoded polypeptide. Said mutation in the polypeptide is an amino acid substitution corresponding to
The term functional variant or homolog/ortholog of SEQ ID NOs: 1, 2 or 4 is described below and specific examples of such nucleic acids are also given below.
The sequence below shows the open reading frame of AtPYL4 (SEQ ID NO:2)
The one or more codons which are mutated according to the various aspects of the invention to obtain the mutations in the polypeptides of the invention are in bold and underlined. This is also shown below.
Examples of polypeptides according to the invention with specific mutations according to the invention are shown in SEQ ID NOs: 60-65. Functional variants or homolog/orthologs thereof with mutations at positions corresponding to the mutated positions in AtPYL4 are also within the scope of the invention.
In one embodiment, the mutant polypeptide according to the invention comprises an amino acid substitution corresponding to position A194 with reference to SEQ ID NO:3. Thus, the invention relates to an isolated mutant nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to position A194 with reference to SEQ ID NO:3 (see SEQ ID NO. 55 and 60). A194 is located in the C-terminal part of the C-terminal helix. The inventors have demonstrated that this mutation alone without the presence of additional mutations can confer a drought resistant phenotype to plants compared to a control wild type plant in the absence of other activating mutations in the domains that interact with ABA or a PP2C. The effect is observed in seeds and in vegetative tissue. In one embodiment, the mutant polypeptide does therefore not comprise any additional activating mutations. The A residue at position 194 or a position corresponding thereto in an AtPYL4 homolog/ortholog may be substituted with T, V, L, M, I or S. In a preferred embodiment, the substitution is with T, for example A194T in SEQ ID NO:3. In another embodiment, the mutant polypeptide according to the various aspects of the invention does not comprise any additional mutations.
Thus, in one embodiment, the polypeptide has an amino acid substitution selected from A194T and no other activating mutations in other residues in the domains that interact with ABA or a PP2C are present. In one embodiment, no other mutations are present. In one embodiment, the polypeptide does not have mutations at one or more of the following positions with reference to SEQ ID NO:3: H82, V105, V106, L109, A111, D177, F178, V181, C185 and/or S189. Also excluded are specific activating mutations as disclosed in WO 2013/006263 (incorporated by reference). In one embodiment, the mutant polypeptide with a mutation at A194 with reference to SEQ ID NO:3 is PYL4, a functional variant, homolog or ortholog thereof as described herein.
In another embodiment of the various aspects of the invention, the mutant polypeptide comprises an amino acid substitution corresponding to position V97 with reference to SEQ ID NO:3. In one embodiment, the polypeptide does not comprise any additional activating mutations. In another embodiment, the polypeptide does not comprise any additional mutations. In one embodiment, the polypeptide does not have mutations at the following positions with reference to SEQ ID NO:3: H82, V105, V106, L109, A111, D177, F178, V181, C185 and/or S189 or at a position corresponding thereto. Also excluded are specific activating mutations as disclosed in WO 2013/006263. In one embodiment, the mutant polypeptide is PYL4, a functional variant, homolog or ortholog thereof as described herein.
According to the various embodiment, the V residue at position 97 or a position corresponding thereto may be substituted with L, M, I, S or T. In a preferred embodiment, the substitution is 97A. In one embodiment, the mutant polypeptide is PYL4, a functional variant, homolog or ortholog thereof as described herein.
In another embodiment, the mutant polypeptide comprises an amino acid substitution corresponding to positions F130 and/or 0176 with reference to SEQ ID NO:3 or a position corresponding thereto. In one embodiment, the polypeptide does not comprise any further activating additional mutations. In another embodiment, the polypeptide does not comprise any additional mutations. The F residue at position 130 may be substituted with W. The C residue at position 176 may be substituted with K or H.
In one embodiment, the polypeptide does not have mutations at the following positions with reference to SEQ ID NO:3: H82, V105, V106, L109, A111, D177, F178, V181, C185 and/or S189 or at a position corresponding thereto. Also excluded are specific activating mutations as disclosed in WO 2013/006263. In a preferred embodiment, the substitution is F130Y and/or C176R. In one embodiment, the mutant polypeptide is PYL4, a functional variant, homolog or ortholog thereof as described herein. In one embodiment, no other mutations are present.
In another embodiment, the mutant polypeptide comprises an amino acid substitution corresponding to position H82 and an amino acid substitution corresponding to position V97 with reference to SEQ ID NO:3. Thus, at least two mutations are present in the polypeptide. Preferably, the polypeptide does not comprise any additional activating mutations. In another embodiment, the polypeptide does not comprise any additional mutations. In one embodiment, the polypeptide does not have mutations at the following positions with reference to SEQ ID NO:3: V105, V106, L109, A111, D177, F178, V181, C185 and/or S189 or at a position corresponding thereto. Also excluded are specific activating mutations as disclosed in WO 2013/006263. The H residue at position 82 may be substituted with K, N, Q, F, Y, W or P. In one embodiment, the residue is not P. In a preferred embodiment, the substitution is H82R. The V residue at position 97 may be substituted with L, M, I, S, T In one embodiment, the mutant polypeptide is PYL4, a functional variant, homolog or ortholog thereof as described herein.
The term “functional variant of a nucleic acid or peptide sequence” as used herein with reference to a mutant of SEQ ID NOs: 1, 2, 3 or 4 or homologs thereof as described herein refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full mutant sequence, for example confers increased stress resistance/yield and ABA-independent interaction with a PP2C when expressed in a transgenic plant. A functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, to the wild type sequences but which includes the target mutations as shown herein and is biologically active. Variants have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the wild type sequence. A variant may for example have restriction sites introduced in the coding sequence to facilitate cloning (see examples).
Thus, it is understood, as those skilled in the art will appreciate, that the aspects of the invention, including the methods and uses, encompass not only a mutant nucleic acid sequence comprising, consisting essentially or consisting of SEQ ID NOs: 1, 2 or 4 or a mutant polypeptide comprising, consisting essentially or consisting or SEQ ID NO: 3, which have the mutations described herein but are otherwise shown as in the referenced sequences, but also functional variants of the mutant sequences of SEQ ID NO: 1 to 4 or homologs thereof that do not affect the biological activity and function of the resulting mutant protein. In other words, the additional variations present in the variants do not affect ABA interaction or other biological functions and the phenotype of the transgenic plant expressing the variant is that of the transgenic plant expressing the mutant peptide as described above. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do however not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Alterations of the variants are not activating mutations.
Also, the various aspects of the invention the aspects of the invention, including the methods and uses, encompass not only a PYL, but also a fragment thereof. By “fragment” is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence encode protein fragments that retain the biological activity of the native protein and hence act to modulate responses to ABA.
In one embodiment according to the various aspects of the invention, the PYL/PYR mutant polypeptide is a mutant PYL4 polypeptide of AtPYL4 as shown in SEQ ID NO:3. The mutant has a modification, preferably a substitution at one or more of position A194, V97, C176 and/or F130 in PYL4 as set forth in SEQ ID NO:3 or at positions H82 and V97 in PYL4 as set forth in SEQ ID NO:3. Examples are shown in SEQ ID NOs: 60-65.
However, the invention also extends to functional homologs/orthologs of AtPYL4 with mutations in corresponding/equivalent positions when compared to the AtPYL4 sequence. A functional variant or homolog of AtPYL4 as shown in SEQ ID NO:3 is a PYL4 peptide which is biologically active in the same way as SEQ ID NO:3, in other words, for example it confers increased stress resistance, preferably against drought. The term functional homolog includes AtPYL4 orthologs in other plant species. In a preferred embodiment of the various aspects of the invention, the invention relates specifically to AtPYL4 or orthologs of AtPYL4 in other plants. Non-limiting examples of these are shown in
a) one or more of position A194, V97, C176 and/or F130 in PYL4 as set forth in SEQ ID NO:3 or
b) positions H82 and V97 in PYL4 as set forth in SEQ ID NO:3.
Corresponding wild type nucleic acid sequences are also shown herein as SEQ ID NOs: 6, 8, 10, 11, 13, 15, 17, 18, 20, 22, 24, 25, 27, 29, 30, 32, 33, 37, 38, 40 and 41. These have mutations in codons equivalent to the mutated codons in AtPYL4 as explained herein. Variants of homologous protein/nucleic acid sequences that retain the biological activity of the mutant sequence and which have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the sequence listed above are also included. This list is non-limiting and other homologous sequences of plants that are described herein, for example the AtPYL4 homolog/ortholog according to the various aspects of the invention is from other preferred plants, such as from crop plants. In a preferred embodiment, the AtPYL4 homolog/ortholog is from maize, rice, wheat, oilseed rape, sorghum, soybean, potato, tomato, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar are also included within the scope of the invention. In one embodiment, the AtPYL4 protein homolog/ortholog is as shown in SEQ ID NOs: 43-54.
Thus, the invention also relates to isolated mutant nucleic acids encoding functional homologs/orthologs of the AtPYL4 polypeptide with one or more mutation in corresponding positions when compared to the AtPYL4 mutant sequence of the invention and to isolated functional homologs/orthologs of the AtPYL4 mutant polypeptide with one or more mutation in corresponding positions when compared to the AtPYL4 sequence. It also extends to transgenic plants that express functional homologs/orthologs of the AtPYL4 polypeptide with one or more mutation at a position corresponding to A194, V97, F130, H82 and/or C176 or H82 and V97 when compared to the AtPYL4 sequence.
The homologue of a AtPYL4 polypeptide according to the various aspects of the invention has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the wild type amino acid represented by SEQ ID NO: 3. Preferably, overall sequence identity is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In another embodiment, the homolog of a AtPYL4 nucleic acid sequence has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the nucleic acid represented by SEQ ID NOs: 1, 2 or 4. Preferably, overall sequence identity is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The overall sequence identity is determined using a global alignment algorithm known in the art, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys).
Preferably, the AtPYL4 homolog/ortholog according to the various aspects of the invention comprises a conserved gate motif (residues 107-111 in AtPYL4: SGLPA; SEQ ID NO:57) and/or latch loops (residues 135-139 in AtPYL4: GDHRL; SEQ ID NO:58) and/or a conserved C-terminal alpha helix (residues 173-198 in AtPYL4: EETCDFVDVIVRCNLQSLAKIAENTA; SEQ ID NO:59) as shown in
Suitable homologues or orthologues can be identified by sequence comparisons and identifications of conserved domains using databases such as NCBI and Paint ensemble and alignment programmes known to the skilled person. The function of the homologue or orthologue can be identified as described herein and a skilled person will thus be able to confirm the function when expressed in a plant. Thus, one of skill in the art will recognize that analogous amino acid substitutions listed above with reference to SEQ ID NO:3 can be made in PYL4 receptors from other plants by aligning the PYL4 receptor polypeptide sequence to be mutated with the AtPYL4 receptor polypeptide sequence as set forth in SEQ ID NO: 3.
Thus, the nucleotide sequences of the invention and described herein can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly cereals. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen plant. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the ABA-associated sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2 d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
As a non-limiting example, an amino acid substitution in PYL4 that is analogous/equivalent to the amino acid substitution A194 in AtPYL4 as set forth in SEQ ID NO: 3 can be determined by aligning the amino acid sequences of AtPYL4 (SEQ ID NO:3) and a PYL4 amino acid sequence from another plant species and identifying the position corresponding to A194 in the PYL4 from another plant species as aligning with amino acid position A194 of AtPYL4. This is shown in
For example, according to the various aspects of the invention, a nucleic acid encoding a mutant PYL/PYR polypeptide, for example PYL4 which is a mutant version of the endogenous wt a mutant PYL/PYR polypeptide, for example PYL4 peptide in a plant may be expressed in said plant by recombinant methods. In another embodiment, a mutant a mutant PYL/PYR polypeptide, for example PYL4, which is a mutant version of a mutant PYL/PYR polypeptide, for example PYL4 peptide in a plant may be expressed in any plant of a second species as defined herein by recombinant methods.
For example, a mutant AtPYL4 or a homolog thereof according to the invention may be expressed in a crop plant. For example, a mutant AtPYL4 may be expressed in barley.
In one particular embodiment of the various aspects of the invention, the mutant nucleic acid is substantially identical to AtPYL4 as shown in SEQ ID No. 1, 2 or 4, a functional variant, homolog or otholog thereof, but has one or more modification of a codon as described herein so that it encodes a mutant polypeptide comprising an amino acid substitution corresponding to position A194 with reference to SEQ ID NO:3 (see also SEQ ID NO:55 and 60) or a position corresponding thereto.
In another embodiment, the various aspects of the invention relate to another member of the PYL/PYR receptor family wherein said PYL/PYR polypeptide is a mutant polypeptide comprising one or more amino acid modifications, selected from one or more amino acid substitutions corresponding to one or more of position A194, V97, F130 and/or C176 in PYL4 as set forth in SEQ ID NO:3 or amino acid substitutions corresponding to H82 and V97 in PLY4 as set forth in SEQ ID NO:3 or at a position corresponding thereto. In one embodiment, the mutant polypeptide comprises an amino acid substitutions corresponding to one or more of position A194, for example A194T. This may be present without the presence of other modifications or may be combined with other mutations in the amino acid sequence.
PYL/PYR bind PP2C via a PP2C binding interface which is characterised by conserved residues, including H82 in AtPYL4. A common motif of the PYL/PYR receptor family is also the conserved C-terminal helix which includes A194 in AtPYL4. A nucleic acid encoding a PYR/PYL polypeptide or a PYR/PYL polypeptide has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the nucleic acid represented by SEQ ID NO: 1, 2 or 4 or to the polypeptide represented by SEQ ID NO: 3 or a homolog thereof. Preferably, overall sequence identity is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Thus, term “PYR/PYL receptor polypeptide” refers to a protein characterized in part by the presence of one or more or all of a polyketide cyclase domain 2 (PF 10604), a polyketide cyclase domain 1 (PF03364), and a Bet V I domain (PF03364), which in wild-type form mediates abscisic acid (ABA) and ABA analog signaling. A wide variety of PYR/PYL receptor polypeptide sequences are known in the art. In some embodiments, a PYR/PYL receptor polypeptide comprises a polypeptide that is substantially identical to AtPYL4 (SEQ ID NO:3), AtPYL1 (SEQ ID NO:43), AtPYL2 (SEQ ID NO:44), AtPYL3 (SEQ ID NO:45), AtPYL5 (SEQ ID NO:46), AtPYL6 (SEQ ID NO:347), AtPYL7 (SEQ ID NO:48), AtPYL8 (SEQ ID NO:49), AtPYL9 (SEQ ID NO: 50), AtPYL10 (SEQ ID NO: 51), AtPYL11 (SEQ ID NO: 52), AtPYL12 (SEQ ID NO: 53), or AtPYL13 (SEQ ID NO: 4) or homologs thereof, but has one or more mutation at the positions corresponding to the targets in AtPYL4 as set out herein. In some embodiments, a PYR/PYL receptor polypeptide comprises a PYR polypeptide. In some embodiments, the PYR/PYL receptor polypeptide is as shown in
Orthologs of PYR/PYL receptor polypeptides in other plant species, for example as shown in
In another aspect, the invention relates to a nucleic acid construct or vector comprising an isolated nucleic acid as described herein. Thus, the vector comprises an isolated nucleic acid encoding a mutant PYL/PYR polypeptide, for example PYL4, comprising one or more amino acid substitutions corresponding to one or more of position A194, H82, V97, F130 or C176 in PLY4 as set forth in SEQ ID NO:3 or comprising amino acid substitutions corresponding to positions H82, and V97 as set forth in SEQ ID NO:3. For example, the substitution may be at position A194, such as A194T. As explained above, in one embodiment, other activating mutations are not present. Preferably, the vector further comprises a regulatory sequence which directs expression of the nucleic acid. In one embodiment, no other mutations are present.
The terms “regulatory element”, “regulatory sequence”, “control sequence” and “promoter” are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term “promoter” typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences. The term “regulatory element” also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
A “plant promoter” comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The “plant promoter” can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other “plant” regulatory signals, such as “plant” terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3′-regulatory region such as terminators or other 3′ regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern. The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
A “constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters include but are not limited to actin, HMGP, CaMV19S, GOS2, rice cyclophilin, maize H3 histone, alfalfa H3 histone, 34S FMV, rubisco small subunit, OCS, SAD1, SAD2, nos, V-ATPase, super promoter, G-box proteins and synthetic promoters.
A “strong promoter” refers to a promoter that leads to increased or overexpression of the gene. Examples of strong promoters include, but are not limited to, CaMV-35S, CaMV-35Somega, Arabidopsis ubiquitin UBQ1, rice ubiquitin, actin, or Maize alcohol dehydrogenase 1 promoter (Adh-1). The term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the control, for example wild-type, expression level. In one embodiment, the promoter is CaMV-35S.
In a one embodiment, the promoter is a constitutive or strong promoter. In a preferred embodiment, the regulatory sequence is an inducible promoter, a stress inducible promoter or a tissue specific promoter. The stress inducible promoter is selected from the following non limiting list: the HaHB1 promoter, RD29A (which drives drought inducible expression of DREB1A), the maize rabl7 drought-inducible promoter, P5CS1 (which drives drought inducible expression of the proline biosynthetic enzyme P5CS1), ABA- and drought-inducible promoters of Arabidopsis clade A PP2Cs (ABI1, ABI2, HAB1, PP2CA, HAI1, HAI2 and HAI3) or their corresponding crop orthologs.
In one embodiment, the promoter is not a stress inducible promoter. The promoter may also be tissue-specific.
Other regulatory sequences, such as terminator sequences may also be included.
The invention also relates to an isolated host cell transformed with a nucleic acid or vector as described above. The host cell may be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell. The invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described above.
The mutant nucleic acid or vector described above is used to generate transgenic plants using transformation methods known in the art. Thus, according to the various aspects of the invention, a nucleic acid comprising a sequence encoding for a mutant PYL/PYR polypeptide as described herein, for example a mutant PYL4 with reference to the wild type nucleic acid sequence as shown in SEQ ID No. 1, 2 or 4 is introduced into a plant and expressed as a transgene. The nucleic acid sequence is introduced into said plant through a process called transformation. The terms “introduction” or “transformation” as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plants is now a routine technique in many species. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.
To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
Thus, the invention relates to a transgenic plant comprising and expressing a mutant nucleic acid, nucleic acid construct comprising a mutant nucleic acid or a vector comprising a mutant nucleic acid wherein said mutant nucleic acid is a nucleic acid of the invention encoding a polypeptide of the invention as described herein. In one embodiment, the mutant nucleic acid encodes a mutant PYL/PYR, for example PYL4, polypeptide comprising a sequence as shown in SEQ ID NO:3, a functional variant or homolog thereof but which comprises an amino acid substitution at a position
a) corresponding to one or more of position A194, V97, C176 and/or F130 in PYL4 as set forth in SEQ ID NO:3 or
b) corresponding to positions H82 and V97 in PYL4 as set forth in SEQ ID NO:3.
Thus, the transgenic plant expresses a mutant version of SEQ ID NO: 3 with one or more mutation as described above (for example any of SEQ ID NOs:60-65) or expresses a mutant which is a homolog/ortholog of AtPYL4.
In one embodiment, the transgenic plant comprises and expresses a mutant nucleic acid encoding a mutant PYL/PYR, for example PYL4, polypeptide comprising a sequence as shown in SEQ ID. NO:3, a functional variant or homolog thereof but which comprises an amino acid substitution at a position A194. In one embodiment, the transgenic plant comprises and expresses a mutant nucleic acid encoding a mutant PYL4 polypeptide comprising a sequence as shown in SEQ ID NO:3, a functional variant or homolog thereof but which comprises an amino acid substitution at a position V97 or a position corresponding thereto. In one embodiment, the transgenic plant comprises and expresses a mutant nucleic acid encoding a mutant PYL4 polypeptide comprising a sequence as shown in SEQ ID NO:3, a functional variant or homolog thereof but which comprises an amino acid substitution at position C176 or a position corresponding thereto. In one embodiment, the transgenic plant comprises and expresses a mutant nucleic acid encoding a mutant PYL4 polypeptide comprising a sequence as shown in SEQ ID. NO:3, a functional variant or homolog thereof but which comprises an amino acid substitution at position F130 or a position corresponding thereto. In one embodiment, the transgenic plant comprises and expresses a mutant nucleic acid encoding a mutant PYL4 polypeptide comprising a sequence as shown in SEQ ID NO:3, a functional variant or homolog thereof but which comprises an amino acid substitution at positions H82 and V97 or at positions corresponding thereto. As explained above with reference to nucleic acids of the invention, any combinations of mutations at positions A194, V97, C176 and/or F130 in PYL4 as set forth in SEQ ID NO:3 are within the scope of the invention.
Thus, the sequence of the AtPYL4 mutant polypeptide is substantially identical to SEQ ID NO:3, but comprises an amino acid substitution at one or more of the positions above. As explained elsewhere, transgenic plants comprising a mutant nucleic acid, nucleic acid construct comprising a mutant nucleic acid or vector comprising a mutant nucleic acid wherein said nucleic acid encodes an ortholog of AtPYL4 with one or more mutation at one or more or corresponding position with reference to SEQ ID NO:3, are also within the scope of the invention. In one embodiment, the transgenic plant comprises and expresses a mutant nucleic acid which encodes a polypeptide that comprises an amino acid substitution corresponding to position A194 with reference to SEQ ID NO:3. In one embodiment, the polypeptide does therefore not comprise any additional activating mutations. In one embodiment, the polypeptide does not have mutations at the following positions with reference to SEQ ID NO:3: H82, V105, V106, L109, A111, D177, F178, V181, C185 and/or S189. Also excluded are specific activating mutations as disclosed in WO 2013/006263. In another embodiment, the polypeptide does not comprise any additional mutations. The A residue at position 194 may be substituted with V, L, M, I or S. In a preferred embodiment, the substitution is A194T. In one embodiment, the mutant polypeptide is PYL4, a functional variant, homolog or ortholog thereof, for example as shown in
In another embodiment, the transgenic plant comprises and expresses a mutant nucleic acid which comprises an amino acid substitution corresponding to position H82 and an amino acid substitution corresponding to position V97 with reference to SEQ ID NO:3. Preferably, the polypeptide does not comprise any additional activating mutations. In one embodiment, the polypeptide does not have mutations at the following positions with reference to SEQ ID NO:3: V105, V106, L109, A111, D177, F178, V181, C185 and/or S189. Also excluded are specific activating mutations as disclosed in WO 2013/006263. In another embodiment, the polypeptide does not comprise any additional mutations. The H residue at position 82 may be substituted with K, N, Q, F, Y, W or P. In a preferred embodiment, the substitution is H82R. The V residue at position 97 may be substituted with L, M, I, S, T or A. In one embodiment, the mutant polypeptide is PYL4, a functional variant, homolog or ortholog thereof, for example as shown in
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
Thus, the invention relates to a method for producing a transgenic plant as described above with improved stress resistance comprising incorporating a nucleic acid encoding for a mutant PYL/PYR polypeptide as defined in SEQ ID NO:3, a functional variant, homolog or ortholog thereof but which comprises a substitution of one or more amino acid corresponding to A194, V97, F130 and/or 0176 in PLY4 as set forth in SEQ ID NO:3 or a substitution of an amino acid corresponding to H82 and of an amino acid corresponding to V97 in PLY4 as set forth in SEQ ID NO:3 into a plant cell by means of transformation, and regenerating the plant from one or more transformed cells. Mutant polypeptides for use in this method are described elsewhere herein. In one embodiment, the PYL/PYR polypeptide is a PLY4 polypeptide as shown in
Another aspect of the invention provides a plant produced by a method described herein which displays improved stress resistance compared to control plant.
Control plants as defined herein are plants that do not express the nucleic acid or construct described above, for example wild type plants or 35S::PYL4 plants.
The plant of the various aspects of the invention is characterised in that it shows increased stress resistance, in particular to drought.
The invention also relates to a method for improving stress resistance or tolerance of a plant, for example drought resistance, comprising incorporating a nucleic acid encoding for a mutant PYL/PYR polypeptide as defined in SEQ ID NO:3, 43-54, a functional variant, homolog or ortholog of any of these sequences but which comprises a substitution of one or more amino acid corresponding to A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO:3 or a substitution of an amino acid corresponding to H82 and a substitution of an amino acid corresponding to V97 in PLY4 as set forth in SEQ ID NO:3 into a plant cell by means of transformation, and; regenerating the plant from one or more transformed cells. Mutant polypeptides for use in this method are described elsewhere herein. In one embodiment, the PYL/PYR polypeptide is a PLY4 polypeptide, for example as shown in
The stress is preferably abiotic stress and may be selected from drought, salinity, freezing (caused by temperatures below 0° C.), chilling (caused by low temperatures over 0° C.) and heat stress (caused by high temperatures). Preferably, the stress is drought.
The stress may be severe or preferably moderate stress. In Arabidopsis research, stress is often assessed under severe conditions that are lethal to wild type plants. For example, drought tolerance is assessed predominantly under quite severe conditions in which plant survival is scored after a prolonged period of soil drying. However, in temperate climates, limited water availability rarely causes plant death, but restricts biomass and seed yield. Moderate water stress, that is suboptimal availability of water for growth can occur during intermittent intervals of days or weeks between irrigation events and may limit leaf growth, light interception, photosynthesis and hence yield potential. Leaf growth inhibition by water stress is particularly undesirable during early establishment. There is a need for methods for making plants with increased yield under moderate stress conditions. In other words, whilst plant research in making stress tolerant plants is often directed at identifying plants that show increased stress tolerance under severe conditions that will lead to death of a wild type plant, these plants do not perform well under moderate stress conditions and often show growth reduction which leads to unnecessary yield loss. Thus, in one embodiment of the methods of the invention, yield is improved under moderate stress conditions. The transgenic plants according to the various aspects of the invention show enhanced tolerance to these types of stresses compared to a control plant and are able to mitigate any loss in yield/growth. The tolerance can therefore be measured as an increase in yield as shown in the examples. The terms moderate or mild stress/stress conditions are used interchangeably and refer to non-severe stress. In other words, moderate stress, unlike severe stress, does not lead to plant death. Under moderate, that is non-lethal, stress conditions, wild type plants are able to survive, but show a decrease in growth and seed production and prolonged moderate stress can also result in developmental arrest. The decrease can be at least 5%-50% or more. Tolerance to severe stress is measured as a percentage of survival, whereas moderate stress does not affect survival, but growth rates. The precise conditions that define moderate stress vary from plant to plant and also between climate zones, but ultimately, these moderate conditions do not cause the plant to die. With regard to high salinity for example, most plants can tolerate and survive about 4 to 8 dS/m. Specifically, in rice, soil salinity beyond ECe˜4 dS/m is considered moderate salinity while more than 8 dS/m becomes high. Similarly, pH 8.8-9.2 is considered as non-stress while 9.3-9.7 as moderate stress and equal or greater than 9.8 as higher stress.
Drought stress can be measured through leaf water potentials. Generally speaking, moderate drought stress is defined by a water potential of between −1 and −2 Mpa. Moderate temperatures vary from plant to plant and specially between species. Normal temperature growth conditions for Arabidopsis are defined at 22-24° C. For example, at 28° C., Arabidopsis plants grow and survive, but show severe penalties because of “high” temperature stress associated with prolonged exposure to this temperature. However, the same temperature of 28° C. is optimal for sunflower, a species for which 22° C. or 38° C. causes mild, but not lethal stress. In other words, for each species and genotype, an optimal temperature range can be defined as well as a temperature range that induces mild stress or severe stress which leads to lethality. Drought tolerance can be measured using methods known in the art, for example assessing survival of the transgenic plant compared to a control plant, or by determining turgor pressure, rosette radius, water loss in leaves, growth or yield. Regulation of stomatal aperture by ABA is a key adaptive response to cope with drought stress. Thus, drought resistance can also be measured by assessing stomatal conductance (Gst) and transpiration in whole plants under basal conditions (see
According to the invention, a transgenic plant has enhanced drought tolerance if the survival rates are at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold higher than those of the control plant after exposure to drought and/or after exposure to drought and re-watering. Also according to the invention, a transgenic plant has enhanced drought tolerance if the rosette radius is at least 10, 20, 30, 40, 50% larger than that of the control plant after exposure to drought and/or after exposure to drought and re-watering. The plant may be deprived of water for 10-30, for example 20 days and the re-watered. Also according to the invention, a transgenic plant has enhanced drought tolerance if stomatal conductance (Gst) and transpiration are lower than in the control plant, for example at least 10, 20, 30, 40, 50% lower.
Thus in one embodiment, the methods of the invention relate to increasing resistance to moderate (non-lethal) stress or severe stress. In the former embodiment, transgenic plants according to the invention show increased resistance to stress and therefore, the plant yield is not or less affected by the stress compared to wild type yields which are reduced upon exposure to stress. In other words, an improve in yield under moderate stress conditions can be observed.
In one embodiment, the method relates to improving drought tolerance of plant vegetative tissue.
The terms “increase”, “improve” or “enhance” are interchangeable. Yield for example is increased by at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40% or 50% or more in comparison to a control plant. The term “yield” in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters. The term “yield” of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant. Thus, according to the invention, yield comprises one or more of and can be measured by assessing: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased number of seed capsules/pods, increased seed size, increased growth or increased branching, for example inflorescences with more branches. Preferably, increased yield comprises an increased number of seed capsules/pods and/or increased branching. Yield is increased relative to control plants.
Thus, the invention also relates to improving yield under stress conditions, preferably moderate stress conditions, comprising incorporating a nucleic acid encoding for a mutant PYL/PYR polypeptide as defined in SEQ ID NO:3, a functional variant, homolog or ortholog thereof but which comprises a substitution of one or more amino acid corresponding to A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO:3 or a substitution of an amino acid corresponding to H82 and a substitution of an amino acid corresponding to V97 in PLY4 as set forth in SEQ ID NO:3 into a plant cell by means of transformation, and; regenerating the plant from one or more transformed cells. Mutant polypeptides for use in this method are described elsewhere herein. In one embodiment, the PYL/PYR polypeptide is a PLY4 polypeptide as shown in
The various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any of the methods described herein, and to all plant parts and propagules thereof unless otherwise specified. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
The invention also extends to harvestable parts of a plant of the invention as described above such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. The invention also relates to food products and food supplements comprising the plant of the invention or parts thereof.
The transgenic plant according to the various aspects of the invention described herein may be a moncot or a dicot plant. The plant PYL/PYR nucleic acid/polypeptide is a monocot or dicot PYL/PYR nucleic acid/polypeptide. Non-limiting examples of moncot or a dicot plants are given below.
A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (eg Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine or citrus species. In one embodiment, the plant is oilseed rape.
Also included are biofuel and bioenergy crops such as rape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).
A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana.
Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.
Most preferred plants are maize, rice, wheat, oilseed rape, sorghum, soybean, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
Sequences for a non-limiting list of preferred PYL4 orthologs are shown as SEQ ID NOs: 7, 9, 12, 14, 16, 19, 21, 23, 26, 28, 31, 34, 37, 39 or 42, but when used according to the invention, these have mutations at positions corresponding to those as set out for SEQ ID NO:3 herein. For example, a nucleic acid encoding SEQ ID No. 28 with one or more corresponding mutation may be introduced and expressed in rice, a nucleic acid encoding SEQ ID NO:19 with one or more corresponding mutation may be introduced and expressed in soybean, a nucleic acid encoding SEQ ID NO:14 with one or more corresponding mutation may be introduced and expressed in tobacco, a nucleic acid encoding SEQ ID NO:34 with one or more corresponding mutation may be introduced and expressed in maize or a nucleic acid encoding SEQ ID NO:31 with one or more corresponding mutation may be introduced and expressed in barley. Alternatively, the plant is any of the plants defined herein, preferably a crop plant such as maize, wheat, oilseed rape, sorghum, soybean, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar and the sequence expressed is a nucleic acid sequence encoding a mutant of SEQ ID NO:3 as defined herein.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
The invention also relates to the use of an isolated nucleic acid, nucleic acid construct or vector as described herein in increasing stress resistance, for example to drought, and/or yield of a plant. The invention also relates to the use of an isolated mutant nucleic acid, nucleic acid construct or vector as described herein in reducing stomatal conductance. The invention also relates to the use of an isolated nucleic acid, nucleic acid construct or vector as described herein in increasing water use efficiency. The term water use efficiency as used herein relates to the plants ability of using a water supply efficiently under normal or water deficit conditions. The invention also relates to corresponding methods, that is methods for increasing stress resistance, reducing stomatal conductance, increasing water use efficiency in a transgenic plant comprising introducing and expressing a nucleic acid encoding for a mutant PYL/PYR polypeptide as defined in SEQ ID NO:3, a functional variant, homolog or ortholog thereof but which comprises a substitution of one or more of the amino acid corresponding to A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO:3 or a position corresponding thereto or a substitution of an amino acid corresponding to H82 and a substitution of an amino acid corresponding to V97 in PYL4 as set forth in SEQ ID NO:3 or a position corresponding thereto into a plant. Preferably, the method is carried out at low ABA levels.
The invention also relates to a method for prolonging seed dormancy/preventing early germination/induce hyperdormancy in a transgenic plant comprising introducing and expressing a nucleic acid encoding for a mutant PYL/PYR polypeptide as defined in SEQ ID NO:3, a functional variant, homolog or ortholog thereof but which comprises a substitution of one or more of the amino acid corresponding to A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO:3 or a substitution of an amino acid corresponding to H82 and a substitution of an amino acid corresponding to V97 in PLY4 as set forth in SEQ ID NO:3 into a plant. Mutant polypeptides for use in this method are described elsewhere herein. In one embodiment, the PYL/PYR polypeptide is a PLY4 polypeptide as shown in
The invention also relates to a method for constitutive activation of the ABA signalling pathway comprising PLY4 receptor comprising introducing and expressing a nucleic acid encoding for a mutant PYL/PYR polypeptide as defined in SEQ ID NO:3, a functional variant, homolog or ortholog thereof but which comprises a substitution of one or more amino acid corresponding to A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO:3 or a substitution of an amino acid corresponding to H82 and a substitution of an amino acid corresponding to V97 in PLY4 as set forth in SEQ ID NO:3 into a plant. Mutant polypeptides for use in this method are described elsewhere herein. In one embodiment, the PYL/PYR polypeptide is a PLY4 polypeptide as shown in
The invention also relates to a method for inhibiting the activity of PP2C, preferably PP2CA, in a transgenic plant comprising introducing and expressing a nucleic acid encoding for a mutant PYL/PYR polypeptide as defined in SEQ ID NO:3, a functional variant, homolog or ortholog thereof but which comprises a substitution of an amino acid corresponding to one or more of A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO:3 or a substitution of an amino acid corresponding to H82 and a substitution of an amino acid corresponding to V97 in PLY4 as set forth in SEQ ID NO:3 into a plant. Mutant polypeptides for use in this method are described elsewhere herein. In one embodiment, the PYL/PYR polypeptide is a PLY4 polypeptide as shown in
The invention also relates to a method for improving ABA-dependent inhibition of PP2C, preferably PP2CA, in a transgenic plant comprising, introducing and expressing a mutant nucleic acid encoding for a mutant PYL/PYR polypeptide as defined in SEQ ID NO:3, a functional variant, homolog or ortholog thereof but which comprises a substitution of an amino acid corresponding to one or more of A194, V97, F130 and/or C176 in PLY4 as set forth in SEQ ID NO:3 or a substitution of an amino acid corresponding to H82 and a substitution of an amino acid corresponding to V97 in PLY4 as set forth in SEQ ID NO:3 into a plant. Preferably, inhibition is improved at low ABA levels.
The invention also relates to a method for identifying a mutation in a PYL/PYR polypeptide that confers drought resistance to vegetative tissue compressing mutagenising a plant population, regenerating progeny plants, exposing plants to drought conditions and comparing the phenotype to control plants and plants expressing AtPYL4 with a mutation at position A194. Plants with a phenotype similar to that of plants expressing AtPYL4 are identified and the sequences of PYL/PYR polypeptides are analysed.
In a further aspect, the invention relates to a method for producing a mutant plant expressing a PYR/PYL variant and which is characterised by one of the phenotypes described herein wherein said method uses mutagenesis and Targeting Induced Local Lesions in Genomes (TILLING) to target the gene expressing a PYR/PYL polypeptide. According to this method, lines that carry a specific mutation are produced that has a known phenotypic effect. For example, mutagenesis is carried out using TILLING where traditional chemical mutagenesis is flowed by high-throughput screening for point mutations. This approach does thus not involve creating transgenic plants. The plants are screened for one of the phenotypes described herein, for example a plant that shows increased stress resistance. A PYR/PYL locus is then analysed to identify a specific a PYR/PYL mutation responsible for the phenotype observed. Plants can be bred to obtain stable lines with the desired phenotype and carrying a mutation in a PYR/PYL locus.
Another technique that can be used for targeted DNA editing is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) (U.S. Pat. No. 8,697,359, Ran et al incorporated by reference). The CRISPR system can be used to introduce specific nucleotide modifications at the target sequence. Originally discovered in bacteria, where several different CRISPR cascades function as innate immune systems and natural defence mechanisms, the engineered CRISPR-Cas9 system can be programmed to target specific stretches of genetic code and to make cuts at precise locations. Over the past few years, those capabilities have been harnessed and used as genome editing tools, enabling researchers to permanently modify genes in mammalian and plant cells.
Thus, the invention relates to a method for generating a PYL/PYR mutant nucleic acid encoding a mutant PYL or PYR polypeptide comprising an amino acid substitution corresponding to
The method involves targeting of Cas9 to the specific genomic locus, in this case PYL/PYR, via a 20 nt guide sequence of the single-guide RNA. An online CRISPR Design Tool can identify suitable target sites (http://tools.genome-engineering.org, Ren et al).
Plants obtained through such methods are also within the scope of the invention. Thus, the invention relates to a non-transgenic plant obtained by mutagenesis or genome editing comprising and expressing a PYL/PYR nucleic acid which encodes a PYL/PYR mutant polypeptide that has a different sequence compared to the wild type sequence. The mutant polypeptide comprises an amino acid substitutions corresponding to
a) one or more of position A194, V97, C176 and/or F130 in PYL4 as set forth in SEQ ID NO:3 or
b) positions H82 and V97 in PYL4 as set forth in SEQ ID NO:3.
While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
All documents mentioned in this specification, including all reference to SEQ ID NOs in gene and protein databases are incorporated herein by reference in their entirety. Sequence versions are version 1 unless otherwise specified.
“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
The invention is further described in the following non-limiting examples.
PYL4 interacts in an ABA-dependent manner with PP2CA in Y2H assays (Lackman et al., 2011;
Y2H assays reveal both ABA-independent and -dependent interactions among PYR/PYLs and PP2Cs; however, PYR/PYL receptors inhibit the activity of clade A PP2Cs mostly in an ABA-dependent manner (Park et al., 2009; Ma et al., 2009; Santiago et al., 2009; Fujii et al., 2009). Thus, an ABA-independent interaction in Y2H assay does not necessarily imply capacity to inhibit phosphatase activity in the absence of ABA. Indeed, although most of the monomeric PYR/PYL receptors show ABA-independent interaction with different PP2Cs in Y2H assay, effective phosphatase inhibition requires ABA, and for instance, the in vivo binding of PYL8 to five clade A PP2Cs was largely dependent on ABA (Park et al., 2009; Ma et al., 2009; Santiago et al., 2009; Antoni et al., 2013). Therefore, we tested whether these mutations affected actually the activity of two clade A PP2Cs, i.e. PP2CA and HAB1. Using p-nitrophenyl phosphate (pNPP) as a substrate, we could detect a small inhibitory effect (20%) of PYL4A194T on the activity of PP2CA in the absence of ABA with respect to PYL4 (
Although phosphatase activity is usually measured using small phosphorylated molecules such as pNPP or phosphopeptides, in vivo phosphatase activity is addressed against phosphorylated proteins and therefore could involve substrate-dependent effects. Therefore, we also performed in vitro reconstitution of the ABA signaling cascade and measured the capacity of PYL4A194T or PYL4 to inhibit the dephosphorylation of several PP2CA targets, i.e. OST1/SnRK2.6, ΔC-ABF2 (residues 1-173) and ΔC-ABI5 (residues 1-200) transcription factors or N-terminal fragment (residues 1-186) of the anion channel SLAC1 (
The phenotype described below for 35S: PYL4A194T plants (see
Next we performed in vitro protein-protein interaction assays. Non-His tagged PYL4A194T could be co-purified with 6His-ΔNPP2CA using Ni-affinity chromatography in the absence of ABA, in contrast to PYL4 (
In order to study the putative effect of PYL4 mutations on ABA signaling in vivo, we generated transgenic plants that over-expressed hemmaglutinin (HA)-tagged versions of PYL4 or the mutant versions PYL4V97A, PYL4A194T, PYL4C176R F130Y and PYL4H82R V97A. Expression of the proteins in vegetative tissue was detected by immunoblot analysis and transgenic lines that expressed similar levels of PYL4 and mutant PYL4 proteins were selected for further analysis; however, PYL4H82R V97A lines consistently showed lower expression of the transgene compared to PYL4 or other mutant proteins (
We concentrated further analysis on PYL4A194T transgenic lines, where expression of the transgene remained stable in T4 lines. Seed germination and seedling establishment analyses of PYL4A194T OE lines confirmed the enhanced sensitivity to ABA observed in T3 seeds. Moreover, root and shoot growth analyses also revealed enhanced sensitivity to ABA in vegetative tissues (
Regulation of stomatal aperture by ABA is a key adaptive response to cope with drought stress. In order to probe stomatal function in non-transformed Col, PYL4 and PYL4A194T OE plants, we performed analysis of stomatal conductance (Gst) and transpiration in whole plants under basal conditions (
The lower Gst values of PYL4 and PYL4A194T OE plants suggest that under steady-state conditions, the stomata of PYL4 and PYL4A194T OE plants have reduced aperture compared to non-transformed Col plants. Indeed, direct measurements of stomatal aperture using whole leaf imaging revealed that stomata of both PYL4 and particularly PYL4A194T OE plants were more closed than those of non-transformed Col plants (
Finally, we performed drought resistance experiments under greenhouse conditions (
Transgenic Barley Plants Overexpressing Either PYL4A194T or PYL4H82R V97A show Enhanced Drought Tolerance at the Vegetative Stage
In order to demonstrate the efficacy of the mutant receptors in crop plants, we generated barley (Hordeum vulgare) transgenic plants that over-express mutant versions of Arabidopsis PYL4 receptors (encoded by the SEQ ID NO: 55 and 56). The demonstration of PYL4 technology in barley, in addition to the intrinsic value itself, would be invaluable in pointing the way for other cereal crops of huge agricultural value, such as maize, wheat and rice. Transgenic barley plants were generated (see methods) and they were subjected to drought stress (see methods). As a result, we found that after a 12-d period of drought, transgenic plants overexpressing either PYL4A194T or PYL4H82R V97A showed enhanced drought tolerance compared to non-transformed Golden promise wt plants (
Under non-stress conditions, endogenous levels of ABA play a critical role to regulate stomatal aperture, as revealed by the open stomata phenotype of multiple pyr/pyl mutants, and basal ABA signaling is also required for proper plant growth and development (Barrero et al., 2005; Gonzalez-Guzman et al., 2012; Antoni et al., 2013). On the other hand, plant response to drought is largely dependent on enhanced ABA biosynthesis and signaling in order to regulate both stomatal aperture and gene expression under water stress conditions. Thus, some mutants or transgenic plants showing enhanced response to ABA also display enhanced drought resistance and reduced water consumption (Pei et al., 1998; Hugouvieux et al., 2001; Saez et al., 2006). In this work we describe a novel approach to boost the interaction of PYL4 and PP2CA and to confer drought resistance through genetic engineering of mutated ABA receptors. We generated a PYL4 allele library by error-prone PCR mutagenesis and selected a mutation that enabled ABA-independent interaction of PYL4 with PP2CA. Y2H, in vitro protein-protein interaction and BiFC assays revealed that PYL4A194T showed a distinct pattern of interaction with PP2CA with respect to PYL4 (
Activation of ABA receptors by mutational stabilization of the agonist-bound conformation led to ABA-independent inhibition of HAB1, ABI1 and ABI2 (Mosquna et al., 2011). Triple and quadruple mutant combinations were constructed to generate constitutively active (CA) PYR1, PYL2 and PYL9 receptors, which efficiently blocked phosphatase activity in the absence of ABA. As a result, expression of a 35S:GFP-PYL2CA transgene in Arabidopsis seeds activated ABA signaling. However, the existence of a post-transcriptional mechanism that abolished expression of PYL2CA in vegetative tissue precluded further analysis (Mosquna et al., 2011). In contrast, expression of PYL4A194T could be detected in vegetative tissues of 35S:PYL4A194T transgenic plants, which showed hypersensitivity with respect to seed and vegetative responses to ABA. Moreover, 35S:PYL4A194T exhibited enhanced drought resistance compared to non-transformed or 35S:PYL4 OE plants. Particularly interesting features were the partial de-repression of ABA responsive genes, reduced stomatal aperture and transpiration of these lines under basal conditions, which likely contributes to the enhanced drought resistance observed in these plants. Indeed, these plants showed reduced Gst and transpiration under basal conditions as well as reduced water loss when submitted to drought stress (
The effect of PYL4A194T appeared to be specific for PP2CA with respect to HAB1, since it did not show a differential effect on HAB1 compared to PYL4. However, at this stage, we cannot exclude that other clade A phosphatases (for instance other members of the PP2CA branch) might also be differentially affected by PYL4A194T. Alignment of clade A PP2Cs reveals two subgroups (the ABM and PP2CA branches) and subtle differences in some regions of the proteins that could affect the interaction with PYR/PYLs (Santiago et al., 2012). Indeed, previous results revealed a certain specificity in the multiple interactions of the 9 clade A PP2Cs and 14 PYR/PYLs (Santiago et al., 2009; Szostkiewicz et al., 2009) and a differential inhibition of PP2CA by PYR/PYLs was recently reported (Antoni et al., 2012). Structural evidence for the PYL4A194T-PP2CA complex is currently not available; however, taking as model other complexes can be observed a clear difference in the length of the α2β4 loop of clade A PP2Cs, which is close to the receptor-phosphatase binding interface. Additionally, the A194 residue is located at the C-terminal helix of PYL4, close to the receptor-phosphatase binding interface. Therefore, the A194T mutation might also indirectly influence the interaction of the C-terminal helix of PYL4 with PP2CA.
In summary, taking into account the phenotype described here for PYL4A194T, the introduction of mutations in PYR/PYL genes that promote ABA-independent interactions with certain PP2Cs might serve as a new tool to ameliorate drought stress. Expression driven by a strong constitutive promoter might lead to some pleiotropic effects that negatively affect growth or yield of crop plants. Such a drawback could be bypassed by introducing stress-inducible or tissue-specific promoters that would drive the expression of the receptor only under stress conditions or in certain tissues. However, expression of either PYL4A194T or PYL4H82R V97A under control of the ubiquitin promoter in transgenic barley plants did not impair vegetative growth under non-stress conditions and on the other hand, it enhanced drought tolerance under stress conditions (
Arabidopsis thaliana plants were routinely grown under greenhouse conditions (40-50% relative humidity) in pots containing a 1:3 vermiculite-soil mixture. For plants grown under growth chamber conditions, seeds were surface sterilized by treatment with 70% ethanol for 20 min, followed by commercial bleach (2.5% sodium hypochlorite) containing 0.05% Triton X-100 for 10 min, and finally, four washes with sterile distilled water. Stratification of the seeds was conducted in the dark at 4° C. for 3 days. Seeds were sowed on Murashige-Skoog (MS) plates composed of MS basal salts, 0.1% 2-[N-morpholino]ethanesulfonic acid, 1% sucrose and 1% agar. The pH was adjusted to 5.7 with KOH before autoclaving. Plates were sealed and incubated in a controlled environment growth chamber at 22° C. under a 16 h light, 8 h dark photoperiod at 80-100 μE m-2 sec-1.
Construction of a PYL4 Mutant Library and Analysis of Yeast Two Hybrid Interaction with PP2CA
We conducted error-prone PCR mutagenesis by amplification of the PYL4 open reading frame using the following primers: FPYL4Ncol, 5″-GCAGCAGCCATGGTTGCCG TTCACCGTCCTTCT and RPYL4EcoRIstop: CGCACGAATTCACAGAGACA TCT TCTTCTT, and the following conditions: 2 mM dGTP, dCTP and dTTP, 0.5 mM dATP, 12 mM MgCl2 and Taq polymerase. The PCR product was Ncol-EcoRI doubly digested, cloned into the pGBKT7 vector and DH10B cells were transformed by electroporation. Thus, we generated an allele library in E. coli of approximately 10,000 PYL4 mutant clones. The sequencing of 50 clones revealed on average 1.7 non-silent mutations per clone in the PYL4 sequence (207 amino acids). The library was shuttled to yeast AH109 by co-transformation with pGAD7-PP2CA. Yeast transformants were pooled and clones able to grow in the absence of exogenous ABA in medium lacking histidine and adenine were selected. Yeast plasmids were extracted, sequenced and retransformed in yeast cells to recapitulate the phenotype. Protocols for yeast two hybrid assays were similar to those described previously (Saez et al., 2008).
BiFC Assay in N. benthamiana
Experiments were performed basically as described by Voinnet et al., (2003). The different binary vectors described above where introduced into Agrobacterium tumefaciens C58C1 (pGV2260) (Deblaere et al., 1985) by electroporation and transformed cells were selected in LB plates supplemented with kanamycin (50 μg/ml). Then, they were grown in liquid LB medium to late exponential phase and cells were harvested by centrifugation and resuspended in 10 mM morpholinoethanesulphonic (MES) acid-KOH pH 5.6 containing 10 mM MgCl2 and 150 mM acetosyringone to an OD600 nm of 1. These cells were mixed with an equal volume of Agrobacterium C58C1 (pCH32 35S:p19) expressing the silencing suppressor p19 of tomato bushy stunt virus (Voinnet et al., 2003) so that the final density of Agrobacterium solution was about 1. Bacteria were incubated for 3 h at room temperature and then injected into young fully expanded leaves of 4-week-old N. benthamiana plants. Leaves were examined after 3-4 days under a Leica TCS-SL confocal microscope and laser scanning confocal imaging system. Quantification of fluorescent protein signal was done as described (Gampala et al., 2007) using the National Institutes of Health (NIH) Image software ImageJ v1.37.
Constructs were done in pSPYNE-35S (Walter et al., 2004) as well as gateway vector pYFPC43 (a derivative of pMDC43 where GFP is replaced by YFPC, Belda-Palazon et al., 2012). The coding sequence of At2g38310 (PYL4) was cloned into the pENTR223.1-Sfi entry vector, kindly provided by ABRC (clone G12806). The coding sequence of PYL4A194T was PCR amplified, cloned into the pCR8/GW/TOPO and verified by sequencing. Next, constructs containing PYL4 and PYL4A194T were recombined by LR reaction into pYFPC43 destination vector. The coding sequence of HAB1 and PP2CA was excised from a pCR8/GW/TOPO construct using a double digestion BamHI-StuI and subcloned into BamHI-SmaI doubly digested pSPYNE-35S.
For small scale protein purifications, E. coli BL21 (DE3) cells transformed with the corresponding constructs were grown in 100 ml of LB medium to an OD600 of 0.6-0.8. At this point 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added and the cells were harvested after overnight incubation at 20° C. Pellets were resuspended in lysis buffer (50 mM Tris pH 7.5, 250 mM KCl, 10% Glycerol, 1 mM β-mercaptoethanol) and lysed by sonication with a Branson Sonifier 250. The clear lysate obained after centrifugation was purified by Ni-affinity. A washing step was performed using 50 mM Tris, 250 mM KCl, 20% Glycerol, 30 mM imidazole and 1 mM 3-mercaptoethanol washing buffer, and finally the protein was eluted using 50 mM Tris, 250 mM KCl, 20% Glycerol, 250 mM imidazole and 1 mM 3-mercaptoethanol elution buffer For protein-protein interaction experiments, the pET28a_ΔNPP2CA, pETM11_PYL4 wt and pETM11_PYL4A194T plasmids were transformed into E. coli BL21 (DE3). A total of 8 ml of an overnight culture were sub-cultured into 800 ml fresh 2TY broth (16 g Bacto tryptone, 10 g yeast extract, 5 g NaCl per litre of solution) plus kanamycin (50 μg ml-1). Protein expression was induced with 0.3 mM IPTG and the cells were harvested after overnight incubation at 20° C. Pellets were resuspended in 25 mM TrisHCl pH 8.0, 50 mM NaCl, 50 mM imidazole, 5 mM 3-mercaptoethanol and disrupted by sonication. After centrifugation (40 min, 40000 g) at 277 K, the clear supernatant was filtered (pore diameter 0.45 mm; Millipore Corporation, Bedford, Mass., USA). The 6His-tagged proteins were purified using Ni-NTA Agarose (Qiagen) according to the manufacturer's instructions. The filtered supernatant was mixed with the previously equilibrated beads. After incubation, a washing step with ten volumes of 25 mM TrisHCl pH 8.0, 50 mM NaCl, 20 mM imidazole, 5 mM 3-mercaptoethanol buffer was performed followed by the elution from the Ni2+ resin in a buffer with 500 mM imidazole. Imidazole was removed using a PD-10 column (GE Healthcare) and the His-tag was cleaved using TEV protease.
6His-ΔNPP2CA pellets were resuspended in 25 mM TrisHCl pH 8.0, 150 mM NaCl, 50 mM imidazole, 5 mM 3-mercaptoethanol, 5 mM Mg2+, mixed with 8 mg of either pure non-tagged (through TEV cleavage) PYL4 or PYL4A194T and disrupted by sonication. The crude extracts were treated as described above using His-Trap HP columns from GE Healthcare to the capture step according to the manufacturer's instructions. In all cases, the purified proteins were subjected to a size exclusion chromatography using a Superdex200 10/300 (Amersham Biosciences Limited, UK) to analyze the behavior in a gel filtration of each protein and to isolate the complex. In order to perform pull-down assays, 6His-ΔNPP2CA was purified, next immobilized on Ni-NTA agarose beads (Qiagen) and incubated with either pure non-tagged PYL4 or PYL4A194T. The mix was swirled 30 min at 4° C. and incubated in the absence or presence of 100 μM ABA. After three washes, proteins were eluted by adding 500 mM imidazol and analyzed by SDS-PAGE.
Phosphatase activity was measured using as a substrate either pNPP or phosphorylated ΔC-ABF2, ΔC-ABI5 and SLAC11-186 proteins. For the pNPP substrate, assays were performed in a 100 μl solution containing 25 mM Tris-HCl pH 7.5, 2 mM MnCl2 and 5 mM pNPP. Assays contained 2 μM phosphatase (PP2CA or HAB1), 4 μM receptor and the indicated concentrations of ABA. Phosphatase activity was recorded with a ViktorX5 reader at 405 nm every 60 seconds over 30 minutes and the activity obtained after 30 minutes is indicated in the graphics. In order to obtain phosphorylated ΔCABF2, ΔC-ABI5 and SLAC11-186 proteins, OST1 phosphorylation assays were done basically as described previously (Dupeux et al., 2011 b). ΔC-ABF2 and SLAC11-186 N-terminal fragments were prepared as described (Antoni et al., 2012; Vahisalu et al., 2010). ΔC-ABI5 recombinant protein (amino acid residues 1-200, containing the C1, C2 and C3 target sites of ABA-activated SnRK2s) was expressed in the pETM11 vector as described above. The reaction mixture containing the OST1 kinase and either ΔCABF2, ΔC-ABI5 or SLAC11-186 recombinant proteins were incubated for 50 min at room temperature in 30 μl of kinase buffer: 20 mM Tris-HCl pH 7.8, 20 mM MgCl2, 2 mM MnCl2, and 3.5 μCi of γ-32ATP (3000 Ci/mmol). Thus, OST1 was autophosphorylated and in turn it phosphorylated ΔC-ABF2, ΔC-ABI5 and SLAC11-186 proteins. Next, they were used as substrates of PP2CA that was preincubated (or not) for 10 with PYL4 or PYL4A194T (1:10 phosphatase:receptor ratio) either in the absence or presence of 30 μM ABA. The reaction was stopped by adding Laemmli buffer and the proteins were separated by SDS-PAGE using an 8% acrylamide gel and transferred to an Immobilon-P membrane (Millipore). Radioactivity was detected and quantified using a Phosphorimage system (FLA5100, Fujifilm). After scanning, the same membrane was used for Ponceau staining. The data presented are averages of at least three independent experiments.
PYL4 or PYL4 mutants were cloned into pCR8/GW/TOPO entry vector (Invitrogen) and recombined by LR reaction into the gateway compatible ALLIGATOR2 vector (Bensmihen et al., 2004). This construct drives expression of PYL4 under control of the 35S CaMV promoter and introduces a triple HA epitope at the N-terminus of the protein. Selection of transgenic lines is based on the visualization of GFP in seeds, whose expression is driven by the specific seed promoter At2S3. The ALLIGATOR2-35S:3HA-PYL4 or mutant constructs were transferred to Agrobacterium tumefaciens C58C1 (pGV2260) (Deblaere et al., 1985) by electroporation and used to transform Columbia wild type plants by the floral dip method. T1 transgenic seeds were selected based on GFP visualization and sowed in soil to obtain the T2 generation. At least three independent transgenic lines were generated for each construct. Homozygous T3 progeny was used for further studies and expression of HA-tagged protein in 21-d-old seedlings was verified by immunoblot analysis using anti-HA-peroxidase (Roche).
After surface sterilization of the seeds, stratification was conducted in the dark at 4° C. for 3 d. Approximately 100 seeds of each genotype were sowed on MS plates supplemented with different ABA concentrations per experiment. To score seed germination, radical emergence was analyzed at 72 h after sowing. Seedling establishment was scored as the percentage of seeds that developed green expanded cotyledons and the first pair of true leaves at 7-d.
Seedlings were grown on vertically oriented MS plates for 4 to 5 days. Afterwards, 20 plants were transferred to new MS plates lacking or supplemented with the indicated concentrations of ABA. The plates were scanned on a flatbed scanner after 10-d to produce image files suitable for quantitative analysis of root growth using the NIH software ImageJ v1.37. As an indicator of shoot growth, the maximum rosette radius was measured.
ABA treatment, RNA extraction and quantitative RT-PCR amplifications were performed as previously described (Saez et al., 2004).
The Arabidopsis whole-rosette gas exchange measurement device, plant growth practice and custom written program to calculate transpiration and Gst for water vapour have been described previously (Kollist et al. 2007; Vahisalu et al., 2008). For gas-exchange experiments, 25-28-d-old plants (rosette area 6-18 cm2) were used. Until measurements, plants were grown in growth chambers (AR-66LX and AR-22L, Percival Scientific, IA, USA) at 12/12 photoperiod, 23/18° C. temperature, air relative humidity of 70-80% and 150 μmol m-2 s-1 light. During gas exchange measurements, temperature, air relatively humidity, photoperiod and light in the cuvettes were kept as similar as possible to the
values in growth chambers. Photographs of plants were taken before and after the experiment and rosette leaf area was calculated using the NIH software ImageJ 1.37v. Leaf area values for the intermediary experimental period were calculated using linear regression between starting and final leaf area.
2-3 weeks-old seedlings grown in MS plates were used for water-loss assays. Four seedlings per genotype with similar growth, three independent experiments, were submitted to the drying atmosphere of a flow laminar hood. Kinetic analysis of waterloss was performed and represented as the percentage of initial fresh weight loss at each scored time point. Stomatal aperture measurements were done in leaves of 5-week-old plants grown under greenhouse conditions using whole leaf imaging (Chitrakar and Melotto, 2010). Staining of whole leaves with propidium iodide was conducted and the aperture of 30-40 stomata (ratio width/length, two independent experiments) was measured using a Leica TCS-SL confocal microscope.
Plants grown under greenhouse conditions (10 individuals per experiment, three independent experiments) were grown under normal watering conditions for 15 days and then subjected to drought stress by stopping irrigation during 20 days. Next, watering was resumed and survival rate was calculated after 3 days by counting the percentage of plants that had more than four green leaves. Photographs were taken at the start of the experiment (day 0), after 16 and 19 days of drought, and 3 days after rewatering. Shoot-growth and water-loss were measured as follows. Quantification of shoot-growth was performed at 2, 5, 7 and 9 d after stopping irrigation (day 0) by measuring the maximum rosette radius of the plants. Water-loss measurements were done in two leaves from each plant that were detached 11 d after the start of the experiment, next weighted, incubated in de-mineralized water for 3 h and weighed again. The difference in weight was considered as water loss and related to the initial fresh weight (μl H2O/g FW).
2-weeks-old seedlings grown in MS plates were used for these experiments. Twenty seedlings per genotype (two independent experiments) were submitted to the drying atmosphere of a flow laminar hood for 12 hours (25° C.±1° C., 25%±2% relative humidity), then rehydrated with 25 ml of water. Survival percentage was scored 3 days after rehydration by counting the percentage of plants that had at least four green leaves.
The Arabidopsis Genome Initiative locus identifiers for PYL4 and PP2CA are At2g38310 and At3g11410, respectively.
Barley Plant Material. Construction of the Vector and Transgenic Lines.
In order to demonstrate the efficacy of the mutant receptors in crop plants, we generated barley (Hordeum vulgare) transgenic plants that over-express mutant versions of Arabidopsis PYL4 receptors (encoded by the SEQ ID NO: 55 and 56). Transgenic barley (cv. Golden Promise) expressing either Arabidopsis PYL4A194T or PYL4H82R V97A open reading frame driven by the Ubiquitin promoter from the pBract214 vector was generated via Agrobacterium-mediated transformation (Bartlett et al. 2008).
The nucleotide sequence encoding either PYL4A194T or PYL4H82R V97A open reading frame was recombined by LR reaction from pCR8/GW/TOPO entry vector into the Gateway compatible pBract214 destination vector. The sequence introduced in barley is the Arabidopsis open reading frame carrying the indicated mutations. Also, the second codon (of the sequence as shown in SEQ ID NO; 2) was modified to GTT to get a Ncol site to facilitate cloning).
Immature embryos were inoculated with Agrobacterium strain AGL1 containing the over-expression vector pBract214 into which the genes of interest had been cloned. pBract214 also contains the hygromycin resistance gene to allow selection of transgenic tissues and plants. Following co-cultivation for 3 days, immature embryos were transferred to selective callus induction medium containing hygromycin to allow the selection of transformed tissue and timentin to remove Agrobacterium. After a total of 6 weeks callus induction, callus were moved to a transition medium in low light and then 2 weeks later to regeneration medium under full light. Regenerated plants were transferred to rooting tubes when shoots reached 2-3 cm in length. Plants with strong roots in hygromycin containing medium were then established in soil and grown to maturity under controlled environment conditions in order to obtain T1 seed progeny.
Barley plants (cv. Golden Promise) were routinely grown under greenhouse conditions (40-50% relative humidity, 23-24° C.) in pots containing a 1:3 vermiculite-soil mixture. Pots were grouped in trays where water was maintained approximately 0.2-1 cm above the bottom of the tray. For drought (D) stress experiments, four-week-old plants were watered with tap water for 12-d (minus D, water maintained 0.2-1 cm above the bottom of the tray) or were submitted to drought for 12-d (plus D, water withdrawal). One flag leaf per plant (10 individual plants for each genetic background, minus and plus D treatment) was weighed in order to obtain its fresh weight and dried for 16 h at 70° C. and weighed again to obtain its dry weight. After the 12 day drought period, plants that had been submitted to drought were re-watered (RW, water 1 cm above the bottom of the tray for 2-d, then water withdrawal) and weight per plant (destructive measurement to obtain above ground biomass) was obtained after 5-d (plus D, plus RW). The results obtained are described in
| Number | Date | Country | Kind |
|---|---|---|---|
| 13382177.7 | May 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/059772 | 5/13/2014 | WO | 00 |
