[go: up one dir, main page]

WO2025238216A1 - Procédés d'amélioration de la tolérance au stress - Google Patents

Procédés d'amélioration de la tolérance au stress

Info

Publication number
WO2025238216A1
WO2025238216A1 PCT/EP2025/063553 EP2025063553W WO2025238216A1 WO 2025238216 A1 WO2025238216 A1 WO 2025238216A1 EP 2025063553 W EP2025063553 W EP 2025063553W WO 2025238216 A1 WO2025238216 A1 WO 2025238216A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
mutation
cngc
acid sequence
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/063553
Other languages
English (en)
Inventor
Myriam CHARPENTIER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
JOHN INNES CENTRE
Innes John Institute
Original Assignee
JOHN INNES CENTRE
Innes John Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by JOHN INNES CENTRE, Innes John Institute filed Critical JOHN INNES CENTRE
Publication of WO2025238216A1 publication Critical patent/WO2025238216A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to methods of improving stress tolerance, and specifically, maintaining the fertility of plants under stress, and in particular heat stress.
  • Such methods comprise introducing one or more mutations into at least one nuclear localized cyclic nucleotide-gated ion channel (CNGC) gene.
  • CNGC nuclear localized cyclic nucleotide-gated ion channel
  • Heat stress both chronic and short-term, is known to have a detrimental effect on the health and seed production of plants.
  • Heat stress-induced plant phenotypes include, growth retardation, wilted plant architecture, low pollen viability, abnormal fertilization, and poor grain filling and seed setting.
  • Heat stress has been demonstrated to negatively affect the female reproductive structure by causing irreversible anatomical and physiological changes, leading to a reduction in the number of ovules.
  • the reduction in the number of ovules has been found to be related to disruption in the normal development of the ovary (Shi et al., 2022).
  • male reproductive development is sensitive to spikes in temperature, with direct consequences on fertility and seed productivity, likely due to metabolic and physiological changes triggered by heat stress.
  • a method of improving abiotic stress tolerance in a plant comprising introducing at least one mutation in a plant, part thereof or plant cell, wherein the at least one mutation is in at least one gene encoding a nuclear localized cyclic nucleotide-gated ion channel (CNGC), wherein the at least one mutation is in at least one conserved motif, wherein the motif comprises the residues XDPX, and wherein the abiotic stress tolerance is heat stress.
  • CNGC nuclear localized cyclic nucleotide-gated ion channel
  • abiotic stress tolerance - or heat stress tolerance - is improved during fertilisation.
  • a method of maintaining plant fertility under stress comprising introducing at least one mutation in a plant, part thereof or plant cell, wherein the at least one mutation is in at least one gene encoding a nuclear localized cyclic nucleotide-gated ion channel (CNGC), wherein the at least one mutation is in at least one conserved motif, wherein the motif comprises the residues XDPX.
  • CNGC nuclear localized cyclic nucleotide-gated ion channel
  • the cyclic nucleotide-gated ion channel may be CNGC15.
  • the CNCG gene encodes an amino acid sequence as defined in any one of SEQ ID NOs 1 to 42, 232 to 236, 247, 248 or 253 or a functional variant or homologue thereof.
  • the at least one mutation is in at least one conserved motif, wherein the motif comprises the residues XDPX, preferably XDPL, more preferably VDPL.
  • the at least one mutation is at least one substitution at one more positions in VDPL, wherein preferably the substitution is a P for L or S and/or a L for F.
  • the mutation may be a gain of function mutation - and is referred to herein as cngc15 GoF .
  • a method of improving abiotic stress tolerance in a plant comprising introducing and expressing, in a plant, a nucleic acid construct, wherein said nucleic acid construct comprises a nucleic acid sequence encoding a nuclear-localised cyclic nucleotide-gated ion channel (CNGC) amino acid sequence, wherein the CNGC amino acid sequence comprises at least one mutation in at least one conserved motif, wherein the motif comprises the residues XDPX, preferably XDPL, more preferably VDPL; and wherein the abiotic stress is heat stress.
  • abiotic stress tolerance - or heat stress tolerance - is improved during fertilisation.
  • a method of maintaining plant fertility under stress comprising introducing and expressing, in a plant, a nucleic acid construct, wherein said nucleic acid construct comprises a nucleic acid sequence encoding a nuclear-localised cyclic nucleotide-gated ion channel (CNGC) amino acid sequence, wherein the CNGC amino acid sequence comprises at least one mutation in at least one conserved motif, wherein the motif comprises the residues XDPX, preferably XDPL, more preferably VDPL.
  • CNGC nuclear-localised cyclic nucleotide-gated ion channel
  • the cyclic nucleotide-gated ion channel may be CNGC15.
  • the nucleic acid sequence encodes a CNGC polypeptide as defined in one of SEQ ID NOs 85 to 231, 242 to 246, 251, 252, 255 or a functional variant thereof.
  • the method may also maintain plant fertility when the plant is grown under low fertiliser conditions or in the absence of fertiliser.
  • a method for identifying and/or selecting a plant that has improved abiotic stress tolerance in a plant comprising screening a population of plants, parts thereof or plant cells and detecting in the plant or plant germplasm at least one polymorphism in at least one conserved domain of a cyclic nucleotide-gated ion channel (CNGC) gene, preferably a polymorphism in a VDPL motif; and selecting said plant.
  • CNGC cyclic nucleotide-gated ion channel
  • a method for identifying and/or selecting a plant that will maintain its fertility under stress conditions comprising screening a population of plants, parts thereof or plant cells and detecting in the plant or plant germplasm at least one polymorphism in at least one conserved domain of a cyclic nucleotide-gated ion channel (CNGC) gene, preferably a polymorphism in a VDPL motif; and selecting said plant.
  • CNGC cyclic nucleotide-gated ion channel
  • the plant is selected from wheat, rice, maize, soybean, tomato, barley, sugar cane, sorghum, sunflower, sugar beet, rye, cotton, potato, peanut, flax (common flax or linseed), strawberry, oilseed rape and any leguminous plant.
  • the stress is heat stress.
  • Figure 1A shows a picture of representative siliques on A.thaliana inflorescence grown under heat stress.
  • Figure 1B shows representative pictures of the silique in figure 1A dissected to observe seed set. Only the cngc15KO/CNGC15 GoF lines present developed seeds. The scale bar represents 1cm.
  • FIG. 1 Representative pictures of the inflorescence with developed siliques under heat stress conditions. The grey bar represents 1cm.
  • nucleic acid 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.
  • 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.
  • polypeptide and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
  • Cyclic nucleotide-gated ion channels or CNGCs are calcium permeable cation transport channels. Plant CNGCs are tetrameric and have six transmembrane domains, with a cytosolic N-terminal (NT) and C-terminal (CT) region per subunit. Members of the CNGC15 family are localized to the nuclear envelope, where they participate in nuclear Ca 2+ oscillations, which are crucial for root growth and symbiosis establishment. In one embodiment, the CNGC is selected from one or more of the CNGC15 sub-family/sub-type.
  • a “genetically altered plant” is a plant that has been genetically altered compared to the naturally occurring wild-type (WT) plant.
  • a genetically altered plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as targeted genome modification or genome editing.
  • the plant genome has been altered compared to the wild-type using a mutagenesis method.
  • Such plants have an altered phenotype as described herein, such as maintained plant fertility under heat stress. Therefore, in this example, these phenotypes are conferred by the presence of an altered plant genome, for example the mutation of at least one gene encoding a CNCG15 gene.
  • the aspects of the invention involve recombination DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods.
  • the invention relates to methods of improving or enhancing abiotic stress tolerance in plants.
  • 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).
  • the stress is heat stress.
  • the stress may be severe or preferably moderate stress.
  • the plant may show improved or enhanced stress tolerance during or following fertilisation.
  • the terms “improving” or “enhancing” are used interchangeably.
  • heat stress refers to exposure to temperatures exceeding the optimal temperature range for growth of a plant. It is understood that the optimal range of temperatures varies between plants and not all plants have the same optimal temperature range. In other words, the specific temperature threshold for heat stress varies between plants species. Heat stress can disrupt essential physiological processes and can lead to stunted growth, reduced yields, and reduced or abolished fertility.
  • the optimum temperature of plants is between 21 and 29°C, more preferably between 22 and 26°C.
  • an increase in temperature of between 10 and 15°C above the optimal temperature for a given plant leads to heat stress.
  • the plant is exposed to above optimum daytime temperatures. In another embodiment, the plant is exposed to above optimum nighttime temperatures. In another embodiment, the plant is exposed to above optimum daytime and night-time temperatures. Daytime may be a period of between 10 to 20 hours, preferably around 16 hours. Night-time may be a period of between 12 to 4 hours, preferably around 8 hours.
  • the plant may be exposed to temperatures of at least 22°C, or at least 23°C, or at least 24°C, or at least 25°C, or at least 26°C, or at least 27°C, or at least 28°C or at least 29°C, or at least 30°C, or at least 31 °C, or at least 32°C, or at least 33°C, or at least 34°C, or at least 35°C, or at least 36°C or more.
  • the plants may be exposed to a temperature of at least 32°C or more. Preferably these are daytime temperatures.
  • the plant may be exposed to temperatures of at least 16°C, or at least 17°C, or at least 18°C, or at least 19°C, or at least 20°C, or at least 21 °C, or at least 22°C, or at least 23°C, or at least 24°C, or at least 25°C, or at least 26°C, or at least 27°C, or at least 28°C or at least 29°C, or at least 30°C or more.
  • the plants may be exposed to a temperature of at least 26°C or more. Preferably these are night-time temperatures.
  • the plant may be exposed to a daytime temperature of at least 32°C or more and a night-time temperature of at least 26°C or more.
  • the temperatures described above are all temperatures that can lead to heat stress in a plant.
  • the plant may be exposed to the above temperatures for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days or more. In practice there may be no minimum or maximum duration.
  • thermotolerance or thermoresistance
  • thermotolerance and thermoresistance may be used interchangeably herein and refer to the ability of a plant to grow and/or survive at high temperatures (i.e. above optimum temperature).
  • a plant’s natural tolerance of heat is their basal thermotolerance.
  • acquired thermotolerance can be considered to be an enhanced level of thermotolerance after exposure to a heat stress.
  • CNGC15 GoF a gain of function mutation
  • CNGC15 GoF a gain of function mutation
  • CNGC15 confers heat stress resistance (or tolerance; such terms may be used interchangeably) during the fertilisation period in plants.
  • the inventors have found that introducing a gain of function mutation into a CNGC in plants maintains fertility - compared to wildtype or control plants that lack the mutation - under heat stress.
  • a plant has improved or enhanced stress tolerance, and in particular, heat stress tolerance during fertilisation if the plant remains (or maintains fertility) or has improved fertility compared to a wild-type or control plant that has not been exposed to any stress, in particular heat stress.
  • the invention therefore also provides a method of maintaining or improving plant fertility under stress, wherein said method comprises introducing at least one mutation in a plant, part thereof or plant cell, wherein the at least one mutation is in at least one gene encoding a nuclear localized cyclic nucleotide-gated ion channel (CNGC).
  • CNGC nuclear localized cyclic nucleotide-gated ion channel
  • the at least one mutation is in at least one conserved motif.
  • the invention provides a method of maintaining plant fertility under stress, or being fertile under stress conditions, wherein said method comprises introducing and expressing, in a plant, a nucleic acid construct, wherein said nucleic acid construct comprises a nucleic acid sequence encoding a nuclear-localised cyclic nucleotide- gated ion channel (CNGC) amino acid sequence, wherein the CNGC amino acid sequence comprises at least one mutation.
  • CNGC nuclear-localised cyclic nucleotide- gated ion channel
  • the at least one mutation is in at least one conserved motif.
  • maintaining fertility means that the fertility of the plant is the same as the fertility of a control or wild-type plant that has not been exposed to heat stress. That is, the fertility of the plant has not decreased or significantly decreased compared to the fertility of (an equivalent) plant that has been exposed to heat stress.
  • Seed development can be used as a measure of the fertility of a plant.
  • the fertility of a plant can be determined by measuring seed development - for example, measuring seed setting, seed size and/or seed number.
  • the development and/or size (length and/or width and/or number) of one or more silique (siliquae) - the seed capsule - can be used to determine the fertility of a plant.
  • the size or number of the silique (or siliquae) and/or the development of seeds is the same (or not significantly different) as that in a wild-type or control plant that has not been exposed to stress, particularly heat stress, it can be considered that the fertility of the plant is maintained.
  • the size or number of the silique (or siliquae) and/or the development of seeds is improved or better or increased than the size or number of silique (or siliquae) and/or the development of seeds in a wild-type or control plant that has been exposed to stress, particularly heat stress, it can also be considered that the fertility of the plant is improved.
  • An increase may be at least an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more compared in the size or number of siliquae and/or in the development of seeds (e.g. seed size, number of seeds) in a wild-type or control plant that has been exposed to stress. Any of the above can be measured using standard techniques in the art.
  • maintaining fertility may mean maintaining ovule and/or pollen viability.
  • maintaining ovule and/or pollen viability under heat stress means that at least one of ovule viability or pollen viability is not significantly reduced under heat stress compared to a wild-type or control plant that has not been exposed to heat stress.
  • Ovule viability may refer to the capacity of an ovule to develop into a mature seed following fertilisation.
  • a viable ovule is a healthy cell within the plant ovary that has the potential to form a seed after successful fertilisation.
  • Pollen viability may refer to the ability of pollen to mature and fertile.
  • Viable pollen cells are able to germinate, form a pollen tube and fertilise the ovule leading to seed development.
  • the viability of pollen may be measured by determining one or more of (i) seed set in a fertilised plant (e.g. number of fertilised ovules per ovary) and/or (ii) cytochemical staining of granules and/or (iii) analysing germination of pollen in vitro or on styles.
  • Heat stress can also cause meiosis to stop in plants, consequently reducing or abolishing the fertility of said plants. Without being bound by theory, it may be that mutating CNGC and specifically CNGC15, protects meiotic pathways from any impact from heat stress.
  • the invention also provides a method of maintaining meiosis under stress, wherein said method comprises
  • CNGC nuclear localized cyclic nucleotide-gated ion channel
  • nucleic acid construct comprises a nucleic acid sequence encoding a CNGC amino acid sequence, wherein the CNGC amino acid sequence comprises at least one mutation, and wherein preferably the at least one mutation is in at least one conserved motif.
  • at least one mutation in at least one gene is meant that where the gene of a CNGC15 subtype is present as more than one copy or homologue (with the same or slightly different sequence) there is at least one mutation in at least one (endogenous) gene.
  • all genes are mutated.
  • the mutation in the gene sequences leads to a mutation in the amino acid sequence of the CNGC.
  • the CNGC15 family comprises MtCNGC15a, b and c.
  • the method comprises introducing at least one mutation in MtCNGC15a or at least one mutation in MtCNGC15b or at least one mutation in MtCNGC15c.
  • the method comprises introducing at least one mutation in MtCNGC15a and at least one mutation in MtCNGC15c or homologues thereof.
  • the method comprises introducing at least one mutation in MtCNGC15a and at least one mutation in MtCNGC15b or homologues thereof.
  • the method comprises introducing at least one mutation in MtCNGC15b and at least one mutation in MtCNGC15c or homologues thereof.
  • the method comprises introducing at least one mutation in MtCNGC15a and at least one mutation in MtCNGC15b and at least one mutation in MtCNGC15c or homologues thereof.
  • the MtCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 12 or a functional variant or homologue thereof
  • MtCNGC15b comprises or consists of SEQ ID NO: 13 or a functional variant or homologue thereof
  • MtCNGC15c comprises or consists of SEQ ID NO: 14 or a functional variant or homologue thereof.
  • the MtCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 54 or a functional variant or homologue thereof
  • MtCNGC15b comprises or consists of SEQ ID NO: 55 or a functional variant or homologue thereof
  • MtCNGC15c comprises or consists of SEQ ID NO: 56 or a functional variant or homologue thereof.
  • the AtCNGC15 family comprises one member: AtCNGC15.
  • the method comprises introducing at least one mutation in at least one gene encoding AtCNGC15.
  • the AtCNGC15 amino acid sequence comprises or consists of SEQ ID NO: 1 or a functional variant or homologue thereof.
  • the AtCNGC15 nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 43 or a functional variant or homologue thereof.
  • the GmCNGC15 family comprises five members, GmCNGC15 a, b, c, d and e.
  • the method comprises introducing at least one mutation (in at least one gene of) at least one, two, three or four of GmCNGC15 a, b, c, d and e. In an alternative embodiment, the method comprises introducing at least one mutation (in at least one gene of) all of GmCNGC15 a, b, c, d and e.
  • the GmCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 2 or a functional variant or homologue thereof
  • GmCNGC15b comprises or consists of SEQ ID NO: 3 or a functional variant or homologue thereof
  • GmCNGC15c comprises or consists of SEQ ID NO: 4 or a functional variant or homologue thereof
  • GmCNGC15d comprises or consists of SEQ ID NO: 5 or a functional variant or homologue thereof
  • GmCNGC15e comprises or consists of SEQ ID NO: 6 or a functional variant or homologue thereof.
  • the GmCNGC15a nucleic acid sequence comprises or consists of SEQ ID NO: 44 or a functional variant or homologue thereof
  • GmCNGC15b comprises or consists of SEQ ID NO: 45 or a functional variant or homologue thereof
  • GmCNGC15c comprises or consists of SEQ ID NO: 46 or a functional variant or homologue thereof
  • GmCNGC15d comprises or consists of SEQ ID NO: 47 or a functional variant or homologue thereof
  • GmCNGC15e comprises or consists of SEQ ID NO: 48 or a functional variant or homologue thereof.
  • the SICNGC15 family comprises three members; SICNGC15a, SICNGC15b and SICNGC15c.
  • the method comprises introducing at least one mutation in (in at least one gene of) SICNGC15a, SICNGC15b and SICNGC15c.
  • the method comprises introducing at least one mutation in (in at least one gene of) SICNGC15a and SICNGC15b.
  • the method comprises introducing at least one mutation in (in at least one gene of) SICNGC15a and SICNGC15c.
  • the method comprises introducing at least one mutation in (in at least one gene of) SICNGC15b and SICNGC15c.
  • the method comprises introducing at least one mutation in (in at least one gene of) SICNGC15a and SICNGC15b and SICNGC15c.
  • the SICNGC15a amino acid sequence comprises or consists of SEQ ID NO: 29 or a functional variant or homologue thereof
  • SICNGC15b comprises or consists of SEQ ID NO: 30 or a functional variant or homologue thereof
  • SICNGC15c comprises or consists of SEQ ID NO: 31 or a functional variant or homologue thereof.
  • the SICNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 71 or a functional variant or homologue thereof
  • SICNGC15b comprises or consists of SEQ ID NO: 72 or a functional variant or homologue thereof
  • SICNGC15c comprises or consists of SEQ ID NO: 73 or a functional variant or homologue thereof.
  • the CNGC15 family comprises one member: ZmCNGC15.
  • the method comprises introducing at least one mutation in at least one gene encoding ZmCNGC15.
  • the ZmCNGC15 amino acid sequence comprises or consists of SEQ ID NO: 32 or a functional variant or homologue thereof.
  • the ZmCNGC15 nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 74 or a functional variant or homologue thereof.
  • the CNGC15 family comprises seven members, TrCNGC15 a, b, c, d, e, f and g.
  • the method comprises introducing at least one mutation in (in at least one gene of) at least one, two, three, four, five or six of TrCNGC15.
  • the method comprises introducing at least one mutation in (in at least one gene of) all of TrCNGC15 a, b, c, d, e, f and g.
  • the TrCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 33 or a functional variant or homologue thereof
  • TrCNGC15b comprises or consists of SEQ ID NO: 34 or a functional variant or homologue thereof
  • TrCNGC15c comprises or consists of SEQ ID NO: 35 or a functional variant or homologue thereof
  • TrCNGC15d comprises or consists of SEQ ID NO: 36 or a functional variant or homologue thereof
  • TrCNGC15e comprises or consists of SEQ ID NO: 37 or a functional variant or homologue thereof
  • TrCNGC15f comprises or consists of SEQ ID NO: 38 or a functional variant or homologue thereof
  • TrCNGC15g comprises or consists of SEQ ID NO: 39 or a functional variant or homologue thereof.
  • the method comprises introducing at least one mutation in TrCNGC15a and at least one mutation in TrCNGC15c.
  • the TrCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 75 or a functional variant or homologue thereof
  • TrCNGC15b comprises or consists of SEQ ID NO: 76 or a functional variant or homologue thereof
  • TrCNGC15c comprises or consists of SEQ ID NO: 77 or a functional variant or homologue thereof
  • TrCNGC15d comprises or consists of SEQ ID NO: 78 or a functional variant or homologue thereof
  • TrCNGC15e comprises or consists of SEQ ID NO: 79 or a functional variant or homologue thereof
  • TrCNGC15f comprises or consists of SEQ ID NO: 80 or a functional variant or homologue thereof
  • TrCNGC15g comprises or consists of SEQ ID NO: 81 or a functional variant or homologue thereof.
  • the genetically altered plant comprises at least one mutation in TrCNGC15a and at least
  • the CNGC15 family comprises one member: OsCNGC15.
  • the method comprises introducing at least one mutation in at least one gene encoding OsCNGC15.
  • the OsCNGC15 amino acid sequence comprises or consists of SEQ ID NO: 28 or a functional variant or homologue thereof.
  • the OsCNGC15 nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 70 or a functional variant or homologue thereof.
  • the CNGC15 family comprises HvCNGC15a, b and c.
  • the method comprises introducing at least one mutation in HvCNGC15a or at least one mutation in HvCNGC15b or a homologue thereof or at least one mutation in HvCNGC15c or a homologue thereof.
  • the genetically altered plant comprises at least one mutation in HvCNGC15a and at least one mutation in HvCNGC15c or homologues thereof.
  • the genetically altered plant comprises at least one mutation in HvCNGC15a and at least one mutation in HvCNGC15b or homologues thereof.
  • the genetically altered plant comprises at least one mutation in HvCNGC15b and at least one mutation in HvCNGC15c or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in HvCNGC15a and at least one mutation in HvCNGC15b and at least one mutation in HvCNGC15c or homologues thereof. In one embodiment, the HvCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 40 or a functional variant or homologue thereof, HvCNGC15b comprises or consists of SEQ ID NO: 41 or a functional variant or homologue thereof and HvCNGC15c comprises or consists of SEQ ID NO: 42 or a functional variant or homologue thereof.
  • the HvCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 82 or a functional variant or homologue thereof
  • HvCNGC15b comprises or consists of SEQ ID NO: 83 or a functional variant or homologue thereof
  • HvCNGC15c comprises or consists of SEQ ID NO: 84 or a functional variant or homologue thereof.
  • the CNGC15 family comprises AhCNGC15a, b and c.
  • the method comprises introducing at least one mutation in AhCNGC15a or at least one mutation in AhCNGC15b or a homologue thereof or at least one mutation in AhCNGC15c or a homologue thereof.
  • the method comprises introducing at least one mutation in AhCNGC15a and at least one mutation in AhCNGC15c or homologues thereof.
  • the method comprises introducing at least one mutation in AhCNGC15a and at least one mutation in AhCNGC15b or homologues thereof.
  • the method comprises introducing at least one mutation in AhCNGC15b and at least one mutation in AhCNGC15c or homologues thereof. In another embodiment, the method comprises introducing at least one mutation in AhCNGC15a and at least one mutation in AhCNGC15b and at least one mutation in AhCNGC15c or homologues thereof.
  • the AhCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 232 or a functional variant or homologue thereof
  • AhCNGC15b comprises or consists of SEQ ID NO: 234 or a functional variant or homologue thereof
  • AhCNGC15c comprises or consists of SEQ ID NO: 233 or a functional variant or homologue thereof.
  • the AhCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 237 or a functional variant or homologue thereof
  • AhCNGC15b comprises or consists of SEQ ID NO: 239 or a functional variant or homologue thereof
  • AhCNGC15c comprises or consists of SEQ ID NO: 238 or a functional variant or homologue thereof.
  • the CNGC family comprises LuCNGCa and c.
  • the method comprises introducing at least one mutation in LuCNGCa or at least one mutation in LuCNGCc or a homologue thereof.
  • the method comprises introducing at least one mutation in LuCNGCa and at least one mutation in LuCNGCc or homologues thereof.
  • the LuCNGCa amino acid sequence comprises or consists of SEQ ID NO: 235 or a functional variant or homologue thereof
  • LuCNGCc comprises or consists of SEQ ID NO: 236 or a functional variant or homologue thereof.
  • the LuCNGCa nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 240 or a functional variant or homologue thereof and LuCNGCc comprises or consists of SEQ ID NO: 241 or a functional variant or homologue thereof.
  • the CNGC family comprises BnCNGC15a.
  • the method comprises introducing at least one mutation in BnCNGC15a or a homologue thereof.
  • the BnCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 247, 248 or a functional variant or homologue thereof.
  • the BnCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 249, 250 or a functional variant or homologue thereof.
  • the CNGC family comprises F.vCNGC15a.
  • the method comprises introducing at least one mutation in F.vCNGC15a or a homologue thereof.
  • the F.vCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 253 or a functional variant or homologue thereof.
  • the F.vCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 254 or a functional variant or homologue thereof.
  • an ‘endogenous’ nucleic acid or gene may refer to the native or natural sequence in the plant genome - for example, one of the abovereferenced nucleic acid or amino acid sequences (SEQ ID NO: 43 to 84, 237 to 241, 249, 250, 254 and SEQ ID NO: 1 to 42, 232 to 236, 247, 248 and 253).
  • variant or “functional variant” as used herein with reference to any of the sequences defined herein refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant (e.g. wild-type) sequence.
  • a functional variant is one that retains the wild-type function - i.e. acts as cyclic nucleotide gated channel and can generate nuclear calcium oscillations.
  • 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.
  • a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active.
  • Alterations in a nucleic acid sequence that results in the production of a different amino acid at a given site that does not affect the functional properties of the encoded polypeptide are well known in the art.
  • 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.
  • a “variant” or a “functional variant” has 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%,
  • homolog also designates a CNGC, specifically a CNGC15 gene orthologue from other plant species. Suitable homologues can be identified by sequence comparisons and identifications of conserved domains as described above. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example when overexpressed in a plant. A homolog may also have, in increasing order of preference, at least 50%, 51 %, 52%,
  • the homolog is Arabidopsis
  • the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 1 or a variant thereof.
  • the homolog is soybean
  • the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 2, 3, 4, 5 or 6 or a variant thereof.
  • the homolog is Medicago
  • the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 7, 8, 9, 10, 11 ,12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 or a variant thereof.
  • the homolog is tomato
  • the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 29, 30 or 31 or a variant thereof.
  • the homolog is maize, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 32 or a variant thereof.
  • the homolog is wheat, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 33, 34, 35, 36, 37, 38 or 39 or a variant thereof.
  • the homolog is rice, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 28 or a variant thereof.
  • the homolog is barley, and the CNGC15 comprises or consists of SEQ ID NO: 40, 41 , or 42 or a variant thereof.
  • the homolog is peanut, and the CNGC15 comprises or consists of SEQ ID NO: 232, 233, 234 or a variant thereof.
  • the homolog is flax (common flax or linseed), and the CNGC comprises or consists of SEQ ID NO: 235, 236 or a variant thereof.
  • the homolog is strawberry, and the CNGC comprises or consists of SEQ ID NO: 253 or variant thereof.
  • the homolog is oilseed rape
  • the CNGC comprises or consists of SEQ ID NO: 247, 248 or a variant thereof.
  • CNGC15 or the CNGC15 subtype comprises at least one highly conserved motif.
  • the methods of the invention comprise introducing at least one mutation in at least one of conserved motifs.
  • the motif comprises the sequence XDPX, wherein X is any amino acid. More preferably, the motif comprises the sequence XDPL. Even more preferably, the conserved motif comprises the sequence VDPL.
  • the mutation is a mutation at one or more positions in the XDPX motif.
  • the mutation is a point mutation or a substitution mutation. That is, a mutation that exchanges one nucleotide base for another and that leads to a change in the codon causing the nucleic acid sequence to encode a different amino acid at that position.
  • the mutation is selected from a substitution of P for another amino acid, preferably a substitution of P for S.
  • the mutation is selected from one or more of the following mutations in VDPL: a substitution of V for another amino acid; and/or a substitution of D for another amino acid; and/or a substitution of P for another amino acid; preferably L or S and/or a substitution of L for another amino acid, preferably F.
  • the mutation in the VDPL is a substitution of P for L.
  • the mutation in the VDPL is a substitution of P for S.
  • the mutation in the VDPL is a substitution of L for F.
  • VDPL is mutated to VDLL.
  • VDPL is mutated to VDSL.
  • VDPL is mutated to VDPF.
  • VDPL is mutated to VDSF.
  • VDPL is mutated to VDLF.
  • the mutation is selected from a substitution of P for another amino acid, preferably a substitution of P for S.
  • the mutation is selected from one or more of the following mutations in IDPL: a substitution of I for another amino acid; and/or a substitution of D for another amino acid; and/or a substitution of P for another amino acid; preferably S; and/or a substitution of L for another amino acid, preferably F or M.
  • the mutation in the IDPL is a substitution of P for S.
  • the mutation in the IDPL is a substitution of L for F.
  • the mutation is selected from one or more of the following mutations in IDPM: a substitution of I for another amino acid; and/or a substitution of D for another amino acid; and/or a substitution of P for another amino acid; preferably S; and/or a substitution of M for another amino acid.
  • the mutation in the IDPM is a substitution of P for S.
  • IDPL is mutated to IDSL.
  • IDPM is mutated to IDSM.
  • IDPL is mutated to IDSF.
  • the mutation is at the following positions is selected from one of the following substitutions in Table 1 :
  • the method comprises introducing at least one mutation into a CNGC15 nucleic acid sequence, wherein the CNGC15 nucleic acid sequence comprises or consists of: a. a nucleic acid sequence encoding a polypeptide comprising at least one XDPX, preferably a XDPL motif, and more preferably a VDPL motif or a variant thereof; b. a nucleic acid sequence encoding a polypeptide as defined in one of SEQ ID Nos 1 to 42, 232 to 236, 247, 248, 253; or c. a nucleic acid sequence as defined in one of SEQ ID Nos 43 to 84, 237 to 241 , 249, 250, 254; or d.
  • nucleic acid sequence with at least 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 at least 99% overall sequence identity to either (a) or (c); or e. a nucleic acid sequence encoding a CNGC15 polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to the nucleic acid sequence of any of (a) to (d).
  • Hybridization of such sequences may be carried out under stringent conditions.
  • 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 hours.
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • the mutation is introduced using targeted genome editing. That is, in one embodiment, the invention relates to a method and plant that has been generated by genetic engineering methods as described above, and does not encompass naturally occurring varieties or generating plants by traditional breeding methods.
  • Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events.
  • the genome editing method that is used according to the various aspects of the invention is CRISPR.
  • sgRNAs can be used with a modified Cas9 protein, such as nickase Cas9 or nCas9 or a “dead” Cas9 (dCas9) fused to a “Base Editor” - such as an enzyme, for example a deaminase such as cytidine deaminase, or TadA (tRNA adenosine deaminase) or ADAR or APOBEC. These enzymes are able to substitute one base for another. As a result no DNA is deleted, but a single substitution is made (Kim et al., 2017; Gaudelli et al. 2017).
  • the method may use sgRNA together with a template or donor DNA constructs, to introduce a targeted SNP or mutation, in particular one of the substitutions described herein, into a CNGC gene.
  • introduction of a template DNA strand following a sgRNA-mediated snip in the double-stranded DNA, can be used to produce a specific targeted mutation (i.e. a SNP) in the gene using homology directed repair.
  • prime editing can be used to introduce the specific mutation (Anzalone et al., 2019).
  • a catalytically impaired Cas9 endonuclease is fused to an engineered reverse transcriptase programmed with a prime editing guide RNA (pegRNA) that is both specific to the target site and encodes the desired edit.
  • pegRNA prime editing guide RNA
  • a method of producing a genetically altered plant, part thereof or plant cell that has increased tolerance to heat stress compared to wild-type or control plants comprising introducing at least one mutation in at least one CNGC gene, preferably a CNGC15 subtype.
  • the mutation is in a nucleic acid sequence encoding a CNGC15 subtype selected from one of the sequences defined in SEQ ID NO: 43 to 84, 237 to 241, 249, 250, and 254, as described in detail above.
  • Plants obtained or obtainable and seeds obtained or obtainable from such plants by such method which carry a functional mutation or dominant mutation in at least one endogenous CNGC15 gene are also within the scope of the invention.
  • the progeny plant is stably transformed with the CRISPR constructs, and comprises the exogenous polynucleotide which is heritably maintained in the plant cell.
  • the method may include steps to verify that the construct is stably integrated.
  • the method may also comprise the additional step of collecting seeds from the selected progeny plant.
  • the method may comprise obtaining a DNA sample from a transformed plant and carrying out DNA amplification to detect the at least one mutation in the CNGC gene, and preferably a substitution in the VDPL motif.
  • the method may further comprise at least one or more of the steps of assessing the phenotype of the genetically altered plant, and measuring fertility (e.g. seed production, seed size, seed setting or silique size). In other words, the method may involve the step of screening the plants for the desired phenotype.
  • mutagenesis methods can be used to introduce at least one mutation into at least one CNGC gene. These methods include both physical and chemical mutagenesis. A skilled person will know further approaches can be used to generate such mutants, and methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • mutagenesis is physical mutagenesis, such as application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons or protons.
  • the targeted population can then be screened to identify a substitution mutation in a CNGC gene.
  • the method comprises mutagenizing a plant population with a mutagen.
  • the mutagen may be a fast neutron irradiation or a chemical mutagen, for example selected from the following non-limiting list: ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N- nitrosurea (ENU), triethylmelamine (1'EM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N'-nitro- Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethyl- benz(a)anthracene (DMBA), ethylene oxide, hexamethyl
  • EMS ethy
  • the method used to create and analyse mutations is targeting induced local lesions in genomes (TILLING), reviewed in Henikoff et al, 2004.
  • TILLING induced local lesions in genomes
  • seeds are mutagenised with a chemical mutagen, for example EMS.
  • the resulting M1 plants are self-fertilised and the M2 generation of individuals is used to prepare DNA samples for mutational screening.
  • DNA samples are pooled and arrayed on microtiter plates and subjected to gene specific PCR.
  • the PCR amplification products may be screened for mutations in the CNGC15 gene using any method that identifies heteroduplexes between wild type and mutant genes.
  • dHPLC denaturing high pressure liquid chromatography
  • DCE constant denaturant capillary electrophoresis
  • TGCE temperature gradient capillary electrophoresis
  • the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences.
  • Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program.
  • Any primer specific to a CNGC nucleic acid sequence may be utilized to amplify the CNGC nucleic acid sequence within the pooled DNA sample.
  • the primer is designed to amplify the regions of a CNGC gene where useful mutations are most likely to arise e.g. in the XDPL motif that is highly conserved as explained elsewhere.
  • the PCR primer may be labelled using any conventional labelling method.
  • the method used to create and analyse mutations is EcoTILLING.
  • EcoTILLING is molecular technique that is similar to TILLING, except that its objective is to uncover natural variation in a given population as opposed to induced mutations. The first publication of the EcoTILLING method was described in Comai et al. (2004).
  • the method may comprise introducing and expressing a nucleic acid construct wherein said nucleic acid construct comprises a nucleic acid sequence encoding a CNGC amino acid sequence, wherein the CNGC amino acid sequence comprises at least one mutation, and wherein preferably the at least one mutation is in at least one conserved motif.
  • the method comprises introducing a nucleic acid construct comprising a nucleic acid sequence that encodes a CNGC polypeptide from the same plant - for example, a wheat CNGC polypeptide is expressed in wheat.
  • the nucleic acid construct comprises a nucleic acid sequence encoding a CNGC polypeptide from a different plant - for example, a wheat CNGC polypeptide is expressed in rice.
  • the nucleic acid construct may be stably incorporated into the plant genome.
  • the nucleic acid sequence encodes a CNGC polypeptide as defined in any one of SEQ ID Nos: 85 to 231, 242 to 246, 251 , 252, 255 or a functional variant thereof.
  • the nucleic acid sequence encoding a CNGC polypeptide is also preferably operably linked to a regulatory sequence.
  • the regulatory sequence is a promoter.
  • operably linked 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 “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.
  • the nucleic acid molecule 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.
  • operably linked 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.
  • the promoter is the endogenous or native CNGC, specifically CNGC15 promoter.
  • the promoter is a constitutive promoter.
  • 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.
  • the promoter is a tissue-specific promoter.
  • Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development.
  • the tissuespecific promoter is a root-specific promoter.
  • the root-specific promoter is the P1534 promoter (as described in Li et al. 2019).
  • a method of making a genetically altered plant comprising introducing and expressing a nucleic acid construct as described herein in a plant.
  • the method comprises a. selecting a part of the plant; b. transfecting at least one cell of the part of the plant of paragraph (a) with at least one nucleic acid construct as described above; c. regenerating at least one plant derived from the transfected cell or cells; d. selecting one or more plants obtained according to paragraph (c) that express the CNGC polypeptide, for example a CNGC polypeptide as defined in SEQ ID NO: 85 to 231 , 242 to 246, 251 , 252, 255 or a functional variant thereof.
  • Transformation methods as used herein for generating a genetically altered plant of the invention are known in the art.
  • a CRISPR or nucleic acid construct as described herein is introduced into a plant and expressed as a transgene.
  • the construct is introduced into said plant through a process called transformation.
  • transformation encompasses 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 therefrom.
  • 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 CRISPR or nucleic acid construct 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.
  • Transformation of plants is now a routine technique in many species.
  • any of several transformation methods may be used to introduce a CRISPR or nucleic acid construct 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 microinjection. 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.
  • the plant material obtained in the transformation is subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • 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.
  • the transformed plants are screened for the presence of a selectable marker.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • 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 nontransformed 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).
  • the method may further comprise regenerating a genetically altered plant from the transformed plant or plant cell, and obtaining a progeny plant derived from the transgenic plant, wherein said progeny exhibits at least one mutation in a CNGC15 gene as described and shows an increase in nodulation and/or AM.
  • a genetically altered plant of the present invention may also be obtained by transference of any of the sequences of the invention by crossing, e.g., using pollen of the genetically altered plant described herein to pollinate a wild-type or control plant, or pollinating the gynoecia of plants described herein with other pollen that is not transformed or genetically altered as described herein.
  • the methods for obtaining the plant of the invention are not exclusively limited to those described in this paragraph; for example, genetic transformation of germ cells from the ear of wheat could also be carried out as mentioned, but without having to regenerate a plant afterwards.
  • a plant obtained or obtainable by the above-described methods In a further aspect, there is provided a seed obtained or obtainable from the plant. Also included in the scope of the invention is progeny plants obtained from the seed and as well as seed obtained from the progeny plants.
  • the use of the genetically altered plant, part thereof or plant cell of the invention to improve or increase stress tolerance, as described above.
  • the use of the genetically altered plant of the invention to maintain or be fertile under stress conditions, and in particular under heat stress conditions. That is, there is provided the use of the genetically altered plant of the invention in breeding when the plant is grown under stress, and in particular heat stress conditions.
  • the genetically altered plant, part thereof or plant cell of the invention may
  • (i) have at least one mutation, wherein the at least one mutation is in at least one gene encoding a nuclear localized cyclic nucleotide-gated ion channel (CNGC), preferably in at least one conserved motif, as described above; or
  • CNGC nuclear localized cyclic nucleotide-gated ion channel
  • nucleic acid construct comprises a nucleic acid sequence encoding a CNGC amino acid sequence, wherein the CNGC amino acid sequence comprises at least one mutation, and wherein preferably the at least one mutation is in at least one conserved motif.
  • a method for identifying and/or selecting a plant that has improved abiotic stress tolerance in a plant comprising screening a population of plants, parts thereof or plant cells and detecting in the plant or plant germplasm at least one polymorphism in at least one conserved domain of a cyclic nucleotide-gated ion channel (CNGC) gene, preferably a polymorphism in a VDPL motif; and selecting said plant.
  • CNGC cyclic nucleotide-gated ion channel
  • a method for identifying and/or selecting a plant that will maintain its fertility under stress conditions comprising screening a population of plants and detecting in the plant or plant germplasm at least one polymorphism in at least conserved domain of CNGC, preferably a conserved domain of a CNGC15, preferably a polymorphism in the VDPL motif, as described above, compared to a control plant or a plant from the same or different plant population, and selecting said plant.
  • Suitable tests for assessing the presence of a polymorphism would be well known to the skilled person, and include but are not limited to, Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs-which are also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNPs).
  • RFLPs Restriction Fragment Length Polymorphisms
  • RAPDs Randomly Amplified Polymorphic DNAs
  • AP-PCR Arbitrarily Primed Polymerase Chain Reaction
  • DAF Sequence Characterized Amplified Regions
  • AFLPs Am
  • the method comprises a) obtaining a nucleic acid sample from a plant and b) carrying out nucleic acid amplification of CNGC alleles using one or more primer pairs.
  • the method may further comprise introgressing the chromosomal region comprising at least one of said CNGC polymorphisms as described above into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.
  • a plant according to all aspects of the invention described herein may be a monocot or a dicot plant.
  • the plant is a crop plant.
  • crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.
  • the plant is Arabidopsis or Medicago truncatula.
  • the plant may be a dicot or a monocot.
  • the plant is selected from wheat, rice, potatoes, maize, soybean, tomato, barley, sugar cane, sorghum, sunflower, sugar beet, rye, cotton, peanut, flax (common flax or linseed), strawberry, oilseed rape and any leguminous plant.
  • plant encompasses whole plants and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned expresses the nucleic acid construct of the invention.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores.
  • the invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.
  • the plant part or harvestable product is a seed or grain. Therefore, in a further aspect of the invention, there is provided a seed or grain produced from the methods described herein. Accordingly, in one aspect of the invention there is provided seed, wherein the seed comprises at least one mutation in CNGC15 as described herein or expresses the nucleic acid construct or CRISPR construct of the invention. Also provided is a progeny plant obtained from the seed as well as seed obtained from that progeny.
  • a control plant as used herein according to all of the aspects of the invention is a plant, which has not been modified according to the methods of the invention. Accordingly, in one embodiment the control plant does not express a nucleic acid construct of the invention or a CRSIPR construct or alternatively the plant does not have one or more mutations in the CNCG15 polypeptide as described herein. In one embodiment, the control plant is a wild type plant. Alternatively, a control plant is a plant that has not been exposed to heat stress. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.
  • Heat stress is known to have a dramatic and detrimental effect on the seed production of crops as well as model plant species, such as Arabidopsis. Heat stress affects the size of the siligue and the maturation of the seeds in wild-type Arabidopsis plants, this is shown in Figures 1 and 2. However, the inventors have found that introducing a gain of function mutation into CNGC15 rescues the seed setting and seed size defects observed in wild-type plants. This is shown in Figure 1 where it can be seen that only cngc15KO/CNGC15 GoF plants developed seeds. The mutant complemented with the CNGC15 GoF was the only plant the presented normal seed development, demonstrating that CNGC15 GoF can confer heat stress resistance during fertilization period in Arabidopsis thaliana.
  • Arabidopsis thaliana ColO wild type, mutant knock out CNGC15 (cngc15 KO), and the cngc15 KO expressing either CNGC15 or CNGC15 GoF driven by its native promoter were grown until the first flowering at 22°C (16 hours day, 8 hours night) and then transferred to heat stress conditions (16h day at 32°C, 8h night at 26°C). After 20 days, seed set was analysed.
  • VDPL conserved domain
  • Mutant Polypeptide Sequences in Glycine Max Mutations are in Bold and Underlined Mutant Polypeptide Sequences in Medicago truncatula; Mutations are in Bold and Underlined Mutant Polypeptide Sequences in Oryza sativa var.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne des procédés d'amélioration de la tolérance au stress abiotique, et plus particulièrement, le maintien de la fertilité de plantes soumises à des conditions de stress, et en particulier de stress thermique. Lesdits procédés comprennent l'introduction d'une ou de plusieurs mutations dans au moins un gène de canal ionique déclenché par des nucléotides cycliques (CNGC) localisé nucléaire.
PCT/EP2025/063553 2024-05-17 2025-05-16 Procédés d'amélioration de la tolérance au stress Pending WO2025238216A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
LU507268 2024-05-17
LULU507268 2024-05-17

Publications (1)

Publication Number Publication Date
WO2025238216A1 true WO2025238216A1 (fr) 2025-11-20

Family

ID=91186826

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/063553 Pending WO2025238216A1 (fr) 2024-05-17 2025-05-16 Procédés d'amélioration de la tolérance au stress

Country Status (1)

Country Link
WO (1) WO2025238216A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4873192A (en) 1987-02-17 1989-10-10 The United States Of America As Represented By The Department Of Health And Human Services Process for site specific mutagenesis without phenotypic selection
WO2023073224A1 (fr) * 2021-10-29 2023-05-04 John Innes Centre Procédés d'augmentation de l'endosymbiose racinaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4873192A (en) 1987-02-17 1989-10-10 The United States Of America As Represented By The Department Of Health And Human Services Process for site specific mutagenesis without phenotypic selection
WO2023073224A1 (fr) * 2021-10-29 2023-05-04 John Innes Centre Procédés d'augmentation de l'endosymbiose racinaire

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"Techniques in Molecular Biology", 1983, MACMILLAN PUBLISHING COMPANY
CUI YONGMEI ET AL: "CYCLIC NUCLEOTIDE-GATED ION CHANNELs 14 and 16 Promote Tolerance to Heat and Chilling in Rice", PLANT PHYSIOLOGY, vol. 183, no. 4, 11 June 2020 (2020-06-11), Rockville, Md, USA, pages 1794 - 1808, XP093221832, ISSN: 0032-0889, DOI: 10.1104/pp.20.00591 *
DUSZYN MARIA ET AL: "Cyclic nucleotide gated channels (CNGCs) in plant signalling-Current knowledge and perspectives", JOURNAL OF PLANT PHYSIOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 241, 27 August 2019 (2019-08-27), XP085872770, ISSN: 0176-1617, [retrieved on 20190827], DOI: 10.1016/J.JPLPH.2019.153035 *
GHOSH SOMA ET AL: "Calcium signaling and transport machinery: Potential for development of stress tolerance in plants", vol. 29, 1 January 2022 (2022-01-01), pages 100235, XP093182549, ISSN: 2214-6628, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S2214662822000019> DOI: 10.1016/j.cpb.2022.100235 *
KUNKEL ET AL., METHODS IN ENZYMOL., vol. 154, 1987, pages 367 - 382
KUNKEL, PROC. NATL. ACAD. SCI., vol. 82, 1985, pages 488 - 492
PENG XUAN ET AL: "Cyclic nucleotide-gated ion channel 6 mediates thermotolerance in Arabidopsis seedlings by regulating nitric oxide production via cytosolic calcium ions", vol. 19, no. 1, 20 August 2019 (2019-08-20), GB, XP093221838, ISSN: 1471-2229, Retrieved from the Internet <URL:http://link.springer.com/article/10.1186/s12870-019-1974-9/fulltext.html> DOI: 10.1186/s12870-019-1974-9 *
RESENTINI FRANCESCA ET AL: "The impact of heat stress in plant reproduction", FRONTIERS IN PLANT SCIENCE, vol. 14, 7 December 2023 (2023-12-07), CH, XP093221840, ISSN: 1664-462X, DOI: 10.3389/fpls.2023.1271644 *
TUNC-OZDEMIR MERAL ET AL: "A Cyclic Nucleotide-Gated Channel (CNGC16) in Pollen Is Critical for Stress Tolerance in Pollen Reproductive Development", vol. 161, no. 2, 12 December 2012 (2012-12-12), pages 1010 - 1020, XP093221842, ISSN: 1532-2548, Retrieved from the Internet <URL:http://academic.oup.com/plphys/article-pdf/161/2/1010/37168433/plphys_v161_2_1010.pdf> DOI: 10.1104/pp.112.206888 *
ZHU ET AL.: "Measuring spatial and temporal Ca2+ signals in Arabidopsis plants", J VIS EXP., vol. 91, 2 September 2014 (2014-09-02)

Similar Documents

Publication Publication Date Title
US12234467B2 (en) Diplospory gene
US11873499B2 (en) Methods of increasing nutrient use efficiency
US11725214B2 (en) Methods for increasing grain productivity
WO2019038417A1 (fr) Méthodes pour augmenter le rendement en grain
US10793868B2 (en) Plants with increased seed size
CN113874388B (zh) 孤雌生殖基因
US20200255846A1 (en) Methods for increasing grain yield
US20230081195A1 (en) Methods of controlling grain size and weight
CN110386967B (zh) 与植物雄性育性相关的蛋白SiMS1及其编码基因与应用
US20230323384A1 (en) Plants having a modified lazy protein
CN110407921A (zh) 来源于谷子的植物籽粒发育相关蛋白sgdw1及其编码基因和应用
CN104168760A (zh) 编码fasciated ear3(fea3)的核苷酸序列及其使用方法
WO2023073224A1 (fr) Procédés d&#39;augmentation de l&#39;endosymbiose racinaire
JPWO2008120410A1 (ja) エンドリデュプリケーション促進活性を有する遺伝子
CN104592370A (zh) OsPYL9蛋白及其编码基因和应用
WO2025238216A1 (fr) Procédés d&#39;amélioration de la tolérance au stress
US20220251590A1 (en) Increasing water use efficiency in plants
EA043050B1 (ru) Способы повышения урожая зерна