Patent Application
20220154204
This invention generally relates to agriculture and molecular biology. In alternative embodiments, provided are methods for: enhancing drought tolerance of crop plants and trees, enhancing salinity of tolerance of plants such as crop plants, enhancing early monitoring of drought, salinity and cold stress by plants such as crop plants and trees, enhancing stress resistance in plants such as crop plants and trees, by increasing the expression of or the activity of a Raf-like mitogen-activated protein (MAP) kinase kinase (MAPKK) kinase δ B3 family enzyme (or a Raf-like MAPKK kinase δ B3 family enzyme) (an M3K δ B3 family enzyme) in a plant or a tree cell or a plant or a tree.
The response of plants to reduced water availability is controlled by a complex osmotic stress and abscisic acid (ABA)-dependent signal transduction network. The core ABA signaling components are snf1-related protein kinase2s (SnRK2s) which are activated by ABA-dependent inhibition of type 2C protein phosphatases and by an unknown ABA-independent osmotic stress signaling pathway.
Limited water availability is one of the key factors that negatively impacts crop yields. The plant hormone abscisic acid (ABA) and the signal transduction network it activates, enhance plant drought tolerance through stomatal closure, and inhibition of seed germination and growth (Finkelstein, 2013). As plants are constantly exposed to changing water conditions, reversibility and robustness of the ABA signal transduction cascade is important for plants to balance growth and drought stress resistance. Core ABA signaling components have been established (Ma et al., 2009; Park et al., 2009): the ABA receptors PYRABACTIN RESISTANCE (PYR/PYL) or REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) inhibit type 2C protein phosphatases (PP2Cs) (Ma et al., 2009; Park et al., 2009), resulting in the activation of the SnRK2 protein kinases SnRK2.2, 2.3 and OST1/SnRK2.6 (Umezawa et al., 2009; Vlad et al., 2009). The SnRK2 kinases phosphorylate and thus regulate the activity of downstream components such as ion channels and transcription factors (Fujii et al., 2009; Geiger et al., 2009; Lee et al., 2009; Takahashi et al., 2013), which leads to stomatal closure and changes in gene expression. Activation of SnRK2 protein kinases requires phosphorylation of the SnRK2 kinases themselves, and in vitro experiments using purified recombinant OST1/SnRK2.6 suggest that phosphorylation of the activation-loop is an important step (Belin et al., 2006). However, it has remained unclear whether direct auto-phosphorylation or trans-phosphorylation by unknown protein kinases re-activates these SnRK2 protein kinase in response to stress.
Previous studies showed that ABA-dependent phosphorylation of substrate proteins of SnRK2 could be reconstituted in vitro using only recombinant PYR/RCAR ABA receptors, PP2Cs and SnRK2 proteins. (Fujii et al., 2009; Brandt et al., 2012; Takahashi et al., 2017). Recombinant SnRK2 proteins used in these studies, unlike SnRK2s in plant cells, had high intrinsic kinase activities even before ABA treatment. Therefore it is not clear whether autophosphorylation accounts for the ABA-dependent SnRK2 reactivation after PP2C-dependent inhibition.
The Arabidopsis genome encodes ten SnRK2 kinases, and at least nine of them are activated in response to osmotic stress (Boudsocq et al., 2004). Interestingly, osmotic stress-induced activation of SnRK2 protein kinases can occur independently of ABA signaling (Yoshida et al., 2006). The osmotic stress sensing mechanism and upstream signal transduction mechanisms leading to SnRK2 activation remain largely unknown in plants.
In alternative embodiments, provided are methods for
the method comprising increasing the expression and/or activity of a Raf-like mitogen-activated protein (MAP) kinase kinase (MAPKK) kinase δ B3 family enzyme (an M3K δ B3 family enzyme) in a plant or a tree, or a plant cell or a tree cell by: inserting in the a plant or a tree, or a plant cell or a tree cell, a heterologous M3K δ B3 family enzyme-expressing nucleic acid, wherein the nucleic acid is operatively linked to a transcriptional regulatory element that is capable of expressing the M3K δ B3 family enzyme in the plant or tree or plant cell or tree cell, resulting in increasing the amount of M3K δ B3 family enzyme expression or M3K δ B3 family enzyme activity in the plant or tree or plant cell or tree cell.
The method of claim 1, wherein the transcriptional regulatory element comprises a promoter, and optionally the promoter comprises an inducible promoter, a constitutive promoter, a guard cell specific promoter, a drought-inducible promoter, a stress-inducible promoter or a guard cell active promoter, and optionally the promoter comprises: a pRAB18 drought and ABA-induced promoter; a pGC1 guard cell promoter; a constitutive CAMV 35 promoter; a constitutive pUbi10 promoter.
In alternative embodiments, of methods as provided herein:
In alternative embodiments, provided are transgenic guard cells, plants, plant cells, plant tissues, plant seeds or fruits, or plant parts or plant organs that expresses a heterologous M3K δ B3 family enzyme, comprising: a heterologous M3K δ B3 family enzyme-expressing nucleic acid operatively linked to transcriptional regulatory element, and optionally the transcriptional regulatory element comprises a promoter, and optionally the promoter comprises an inducible promoter, a constitutive promoter, a guard cell specific promoter, a drought-inducible promoter, a stress-inducible promoter or a guard cell active promoter, and optionally the promoter comprises: a pRAB18 drought and ABA-induced promoter; a pGC1 guard cell promoter; a constitutive CAMV 35 promoter; a constitutive pUbi10 promoter.
In alternative embodiments, provided are uses of transgenic guard cells, plants, plant cells, plant tissues, plant seeds or fruits, or plant parts or plant organs as provided herein for: enhancing or creating drought tolerance of crop plants and trees, enhancing or creating salinity of tolerance of a plant, wherein optionally the plant is a crop plant, early monitoring of drought, salinity and cold stress by a plant or a tree, wherein optionally the plant is a crop plant, or, enhancing or creating stress resistance in a plant or a tree, wherein optionally the plant is a crop plant.
In alternative embodiments, provided are transgenic guard cells, plants, plant cells, plant tissues, plant seeds or fruits, or plant parts or plant organs as provided herein for use in: enhancing or creating drought tolerance of crop plants and trees, enhancing or creating salinity of tolerance of a plant, wherein optionally the plant is a crop plant, early monitoring of drought, salinity and cold stress by a plant or a tree, wherein optionally the plant is a crop plant, or, enhancing or creating stress resistance in a plant or a tree, wherein optionally the plant is a crop plant.
The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.
In alternative embodiments, provided are methods for: enhancing drought tolerance of crop plants and trees, enhancing salinity of tolerance of plants such as crop plants, enhancing early monitoring of drought, salinity and cold stress by plants such as crop plants and trees, enhancing stress resistance in plants such as crop plants and trees, by increasing the expression of or the activity of a Raf-like mitogen-activated protein (MAP) kinase kinase (MAPKK) kinase δ B3 family enzyme (or a Raf-like MAPKK kinase δ B3 family enzyme) (an M3K δ B3 family enzyme) in a plant or a tree cell or a plant or a tree.
In alternative embodiments, provided are methods for enhancing plant and tree drought and salinity stress resistance and protect yields of plants such as crop plants and trees exposed to drought stress. In alternative embodiments, provided are methods for enhancing drought and salinity tolerance of plants such as crop plants and trees. In alternative embodiments, provided are methods for enhancing early monitoring of drought and salinity-linked osmotic stress in plants such as crop plants and trees, which can boost early mounting of stress resistance in the plants and trees.
Through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified key functionally-redundant Raf-like MAPKK kinases (M3Ks) that activate OST1/SnRK2 kinases. Reactivation of dephosphorylated SnRK2 requires these M3Ks, and ABA-induced OST1/SnRK2.6 activation and S-type anion channel activation requires the presence of M3Ks. M3K knock-out plants show not only reduced sensitivity to ABA but also strongly impaired osmotic stress-induced SnRK2 activation. Our results demonstrate that these Raf-like M3Ks are required for ABA- and osmotic stress-activation of SnRK2 kinases, ensuring robust ABA and osmotic stress signal transduction and indicate that increased resistance to these stresses can be engineered through targeted over-expression and enhancement of these protein activities.
This newly recognized mechanism can be used to boost both drought and salinity osmotic stress sensing as well abscisic acid drought and temperature resistance responses in plants.
The described advances can be used via over-expression of the identified mechanisms using promoters and/or genome editing using several genome editing platforms, including non-restricted technologies, to: enhance drought tolerance of crop plants and trees; enhance salinity of tolerance of crop plants; enhance early monitoring of drought, salinity and cold stress by crop plants and trees; provide for early mounting of stress resistance in crop plants and trees.
Genome editing can be accomplished using transcription activator-like effector nuclease (TALEN) gene editing, see e.g., Zhang et al Plant Physiology (2013) vol 161(1):pg 20-27; Haun et al (2014) Plant Biotechnology Journal, Vol 12(7): 934-40; or unrestricted TALEN™ (ThermoFisher) technology.
In alternative embodiments, promoters that can be used to drive the over-expression of an M3K δ B3 family enzyme in a plant or a tree cell or a plant or a tree for practicing exemplary methods as provided herein, including enhancing drought and salinity tolerance, comprise: a pRAB18 drought and ABA-induced promoter; a pGC1 guard cell promoter; a constitutive CAMV 35 promoter; a constitutive pUbi10 promoter.
In alternative embodiments, M3K nucleic acids and M3K-protein coding sequences or genes used to practice methods as provided herein are operably linked to a plant expressible promoter, an inducible promoter, a constitutive promoter, a guard cell specific promoter, a drought-inducible promoter, a stress-inducible promoter or a guard cell active promoter. Promoters used to practice methods as provided herein include a strong promoter, particularly in plant guard cells, and in some embodiments is guard cell specific, e.g., the promoters described in WO2008/134571.
In alternative embodiments, M3K nucleic acids and M3K-protein coding sequences or genes also can be operatively linked to any constitutive and/or plant specific, or plant cell specific promoter, e.g., a cauliflower mosaic virus (CaMV) 35S promoter, a mannopine synthase (MAS) promoter, a 1′ or 2′ promoter derived from T-DNA of Agrobacterium tumefaciens, a figwort mosaic virus 34S promoter, an actin promoter, a rice actin promoter, a ubiquitin promoter, e.g., a maize ubiquitin-1 promoter, and the like.
Examples of constitutive plant promoters which can be useful for expressing the M3K-encoding sequences in accordance with methods as provided herein include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odell et al. (1985) Nature 313: 810-812); the nopaline synthase promoter (An et al. (1988) Plant Physiol. 88: 547-552); and the octopine synthase promoter (Fromm et al. (1989) Plant Cell 1: 977-984).
A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a sequence in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like.
Numerous known promoters have been characterized and can be employed to promote expression of a polynucleotide used to practice methods as provided herein, e.g., in a transgenic plant or cell of interest. For example, tissue specific promoters include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2Al 1 promoter (e.g., see U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (e.g., see Bird et al. (1988) Plant MoI. Biol. 11: 651-662), root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such as PTA29, PTA26 and PTAl 3 (e.g., see U.S. Pat. No. 5,792,929), promoters active in vascular tissue (e.g., see Ringli and Keller (1998) Plant MoI. Biol. 37: 977-988), flower-specific (e.g., see Kaiser et al. (1995) Plant MoI. Biol. 28: 231-243), pollen (e.g., see Baerson et al. (1994) Plant MoI. Biol. 26: 1947-1959), carpels (e.g., see OhI et al. (1990) Plant Cell 2: pollen and ovules (e.g., see Baerson et al. (1993) Plant MoI. Biol. 22: 255-267), auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant MoI. Biol. 39: 979-990 or Baumann et al., (1999) Plant Cell 11: 323-334), cytokinin-inducible promoter (e.g., see Guevara-Garcia (1998) Plant MoI. Biol. 38: 743-753), promoters responsive to gibberellin (e.g., see Shi et al. (1998) Plant MoI. Biol. 38: 1053-1060, Willmott et al. (1998) Plant Molec. Biol. 38: 817-825) and the like.
Additional promoters that can be used to practice methods as provided herein are those that elicit expression in response to heat (e.g., see Ainley et al. (1993) Plant MoI. Biol. 22: 13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al. (1989) Plant Cell 1: 471-478, and the maize rbcS promoter, Schaffher and Sheen (1991) Plant Cell 3: 997-1012); wounding (e.g., wunl, Siebertz et al. (1989) Plant Cell 1: 961-968); pathogens (such as the PR-I promoter described in Buchel et al. (1999) Plant MoI. Biol. 40: 387-396, and the PDF 1.2 promoter described in Manners et al. (1998) Plant MoI. Biol. 38: 1071-1080), and chemicals such as methyl jasmonate or salicylic acid (e.g., see Gatz (1997) Annu. Rev. Plant Physiol. Plant MoI. Biol. 48: 89-108). In addition, the timing of the expression can be controlled by using promoters such as those acting at senescence (e.g., see Gan and Amasino (1995) Science 270: 1986-1988); or late seed development (e.g., see Odell et al. (1994) Plant Physiol. 106: 447-458).
In alternative embodiments, tissue-specific and/or developmental stage-specific promoters are used, e.g., promoter that can promote transcription only within a certain time frame of developmental stage within that tissue. See, e.g., Blazquez (1998) Plant Cell 10:791-800, characterizing the Arabidopsis LEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77, describing the transcription factor SPL3, which recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene API; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristem promoter eIF4. Tissue specific promoters which are active throughout the life cycle of a particular tissue can be used. In one aspect, M3K-encoding nucleic acids used to practice methods as provided herein are operably linked to a promoter active primarily only in cotton fiber cells, hi one aspect, M3K-encoding nucleic acids used to practice methods as provided herein are operably linked to a promoter active primarily during the stages of cotton fiber cell elongation, e.g., as described by Rinehart (1996) supra. The M3K-encoding nucleic acids used to practice methods as provided herein can be operably linked to the Fbl2A gene promoter to be preferentially expressed in cotton fiber cells (Ibid). See also, John (1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat. Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promoters and methods for the construction of transgenic cotton plants. Root-specific promoters may also be used to express M3K-encoding nucleic acids used to practice methods as provided herein. Examples of root-specific promoters include the promoter from the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol. 123:39-60). Other promoters that can be used to express M3K-encoding nucleic acids used in methods as provided herein include, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific promoters, or some combination thereof, a leaf-specific promoter (see, e.g., Busk (1997) Plant J. 11:1285 1295, describing a leaf-specific promoter in maize); the ORF 13 promoter from Agrobacterium rhizogenes (which exhibits high activity in roots, see, e.g., Hansen (1997) supra); a maize pollen specific promoter (see, e.g., Guerrero (1990) MoI. Gen. Genet. 224:161 168); a tomato promoter active during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers can be used (see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specific promoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant MoI. Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa making it a useful tool to target the expression of foreign genes to the epidermal layer of actively growing shoots or fibers; the ovule-specific BEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No. U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583, describing a plant promoter region is capable of conferring high levels of transcription in meristematic tissue and/or rapidly dividing cells.
In alternative embodiments, plant promoters used in methods as provided herein can be inducible upon exposure to plant hormones, such as auxins; these promoters can be used to express M3K nucleic acids used in methods as provided herein. For example, exemplary methods can use the auxin-response elements El promoter fragment (AuxREs) in the soybean {Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) MoI. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900-1902).
In alternative embodiments, M3K-encoding nucleic acids used in methods as provided herein can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324). Using chemically- {e.g., hormone- or pesticide-) induced promoters, i.e., promoter responsive to a chemical which can be applied to the transgenic plant in the field, expression of a polypeptide can be induced at a particular stage of development of the plant.
In alternative embodiments, provided are transgenic plants containing an inducible gene encoding for polypeptides used to practice methods as provided herein whose host range is limited to target plant species, such as corn, rice, barley, wheat, potato or other crops, inducible at any stage of development of the crop.
In alternative embodiments, a tissue-specific plant promoter may drive expression of operably linked sequences in tissues other than the target tissue. In alternative embodiments, a tissue-specific promoter that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well, is used.
In alternative embodiments, proper polypeptide expression may require polyadenylation region at the 3′-end of the coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant (or animal or other) genes, or from genes in the Agrobacterial T-DNA.
In alternative embodiments, provided are transgenic plants, plant parts, plant organs or tissue, and seeds comprising a nucleic acid that encodes an M3K δ B3 family enzyme, and expression cassettes or vectors, or a transfected or transformed cell, or transgenic plant comprising or having contained therein an M3K δ B3 family enzyme-encoding nucleic acid. Also provided are plant products, e.g., seeds, leaves, extracts and the like, comprising an M3K δ B3 family enzyme-encoding nucleic acid.
In alternative embodiments, the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Also provided are methods of making and using these transgenic plants and seeds. The engineered transgenic plant or plant cell over-expressing an M3K δ B3 family polypeptide may be constructed in accordance with any method known in the art. See, for example, U.S. Pat. No. 6,309,872.
Nucleic acids and expression constructs used to practice methods as provided herein can be introduced into a plant cell by any means. For example, nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or, the nucleic acids or expression constructs can be episomes. Introduction into the genome of a desired plant can be such that the host's a CO2Sen protein production is regulated by endogenous transcriptional or translational control elements, or by a heterologous promoter, e.g., a promoter used to drive expression of an M3K δ B3 family enzyme-expressing nucleic acid.
Also provided are engineered plants where insertion of gene sequence into the genome by, e.g., homologous recombination, inserts an M3K δ B3 family polypeptide-encoding nucleic acid sequence.
The nucleic acids practice methods as provided herein can be expressed in or inserted in any plant, plant part, plant cell or seed.
Transgenic plants or a plant or plant cell comprising a nucleic acid used to practice methods as provided herein (e.g., a transfected, infected or transformed cell) can be dicotyledonous or monocotyledonous. Examples of monocots comprising an M3K δ B3 family enzyme-expressing nucleic acid, e.g., as monocot transgenic plants as provided herein, are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn). Examples of dicots comprising an M3K δ B3 family enzyme-expressing nucleic acid, e.g., as dicot transgenic plants as provided herein, are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana. Thus, plant or plant cell comprising an M3K δ B3 family enzyme-expressing nucleic acid, including the transgenic plants and seeds as provided herein, include a broad range of plants, including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Cojfea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solarium, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.
The nucleic acids used to practice methods as provided herein can be expressed in or inserted in any plant cell, organ, seed or tissue, including differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, cotyledons, epicotyl, hypocotyl, leaves, pollen, seeds, tumor tissue and various forms of cells in culture such as single cells, protoplast, embryos, and callus tissue. The plant tissue may be in plants or in organ, tissue or cell culture.
Transgenic plants In alternative embodiments, provided are transgenic plants, plant cells, organs, seeds or tissues, comprising and expressing the nucleic acids used to practice methods as provided herein, including M3K δ B3 family-expressing genes; for example, provided are plants, e.g., transgenic plants, plant cells, organs, seeds or tissues that show improved growth under limiting water conditions; thus, provided are drought-tolerant plants, plant cells, organs, seeds or tissues (e.g., crops).
A transgenic plant as provided herein can also include the machinery necessary for increasing the expression or activity of an M3K δ B3 family polypeptide encoded by an endogenous gene, for example, by altering the phosphorylation state of the polypeptide to maintain it in an activated state.
Transgenic plants (or plant cells, or plant explants, or plant tissues) over-expressing M3K δ B3 family polypeptide can be produced by a variety of well-established techniques as described above.
Following construction of a vector, most typically an expression cassette, including a polynucleotide, e.g., encoding a transcription factor or transcription factor homolog, standard techniques can be used to introduce the M3K δ B3 family polynucleotide into a plant, a plant cell, a plant explant or a plant tissue of interest. In one aspect the plant cell, explant or tissue can be regenerated to produce a transgenic plant.
The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledonous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al., eds., (1984) Handbook of Plant Cell Culture—Crop Species, Macmillan Publ. Co., New York, N. Y.; Shimamoto et al. (1989) Nature 338: 274-276; Fromm et al. (1990) Bio/Technol. 8: 833-839; and Vasil et al. (1990) Bio/Technol. 8: 429-434.
Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and
In alternative embodiments, an Agrobacterium tumefaciens mediated transformation is used. Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence.
Successful examples of the modification of plant characteristics by transformation with cloned sequences which serve to illustrate the current knowledge in this field of technology, and include for example: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,619,042.
In alternative embodiments, following transformation, plants are selected using a dominant selectable marker incorporated into the transformation vector. Such a marker can confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.
In alternative embodiments, after transformed plants are selected and grown to maturity, those plants showing a modified trait (e.g., overexpression of M3K δ B3 family enzyme) are identified. The modified trait can be any of those traits described above. In alternative embodiments, to confirm that the modified trait is due to changes in expression levels or activity of the transgenic polypeptide or polynucleotide can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.
Nucleic acids and expression constructs can be introduced into a plant cell by any means. For example, nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or, the nucleic acids or expression constructs can be episomes. Introduction into the genome of a desired plant can be such that the host's CO2 sensor production is regulated by endogenous transcriptional or translational control elements.
In alternative embodiments, provided are “knockout plants” where insertion of a gene sequence by, e.g., homologous recombination, can result in M3K over-expression. Means to generate “knockout” plants are well-known in the art, see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao (1995) Plant J 7:359-365. See discussion on transgenic plants, below.
In alternative embodiments, making transgenic plants or seeds comprises incorporating sequences used to practice methods as provided herein and, in one aspect (optionally), marker genes into a target expression construct (e.g., a plasmid), along with positioning of the promoter and the terminator sequences. This can involve transferring the modified gene into the plant through a suitable method. For example, a construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. For example, see, e.g., Christou (1997) Plant MoI. Biol. 35:197-203; Pawlowski (1996) MoI. Biotechnol. 6:17-30; Klein (1987) Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use of particle bombardment to introduce transgenes into wheat; and Adam (1997) supra, for use of particle bombardment to introduce YACs into plant cells. For example, Rinehart (1997) supra, used particle bombardment to generate transgenic cotton plants. Apparatus for accelerating particles is described U.S. Pat. No. 5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000 particle acceleration instrument; see also, John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describing particle-mediated transformation of gymnosperms.
In alternative embodiments, protoplasts can be immobilized and injected with a nucleic acid, e.g., an expression construct. Although plant regeneration from protoplasts is not easy with cereals, plant regeneration is possible in legumes using somatic embryogenesis from protoplast derived callus. Organized tissues can be transformed with naked DNA using gene gun technique, where DNA is coated on tungsten microprojectiles, shot 1/100th the size of cells, which carry the DNA deep into cells and organelles. Transformed tissue is then induced to regenerate, usually by somatic embryogenesis. This technique has been successful in several cereal species including maize and rice.
In alternative embodiments, a third step can involve selection and regeneration of whole plants capable of transmitting the incorporated target gene to the next generation. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants from transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins.
In alternative embodiments, after the expression cassette is stably incorporated in transgenic plants, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Since transgenic expression of an M3K δ B3 family enzyme-expressing nucleic acid leads to phenotypic changes, plants comprising the recombinant nucleic acids comprising an M3K δ B3 family enzyme-expressing nucleic acid can be sexually crossed with a second plant to obtain a final product. Thus, a seed containing an M3K δ B3 family enzyme-expressing nucleic acid can be derived from a cross between two transgenic plants as provided herein, or a cross between a plant comprising an M3K δ B3 family enzyme-expressing nucleic acid and another plant. The desired effects (e.g., over-expression of an M3K δ B3 family enzyme) can be enhanced when both parental plants express the polypeptides, e.g., an M3K δ B3 family enzyme. The desired effects can be passed to future plant generations by standard propagation means.
Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary and/or Detailed Description sections.
As used in this Specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR—Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
This example demonstrates that methods as provided herein by increasing Raf-like mitogen-activated protein (MAP) kinase (MAPKK) kinase (M3K) activity (i.e., the expression of an M3K δ B3 family enzyme) in a plant or a tree cell can: enhance drought tolerance of crop plants and trees, enhance salinity of tolerance of plants such as crop plants, enhance early monitoring of drought, salinity and cold stress by plants such as crop plants and trees, enhance stress resistance in plants such as crop plants and trees.
Through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant Raf-like MAPKK kinases (M3Ks) that activate OST1/SnRK2 kinases. Reactivation of dephosphorylated SnRK2 requires these M3Ks, and ABA-induced OST1/SnRK2.6 activation and S-type anion channel activation requires the presence of M3Ks. M3K knock-out plants show not only reduced sensitivity to ABA but also strongly impaired osmotic stress-induced SnRK2 activation. Our results demonstrate that these Raf-like M3Ks are required for ABA- and osmotic stress-activation of SnRK2 kinases, ensuring robust ABA and osmotic stress signal transduction.
Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.
Data provided herein identifies a family of MAP kinase kinase kinases (M3Ks) that are essential for reactivation of SnRK2 protein kinases after PP2C dephosphorylation. We show that the OST1/SnRK2.6 protein kinase cannot reactivate itself after dephosphorylation. Three independent reconstitution assays and in planta analyses show the function of these M3Ks in SnRK2 kinase reactivation and ABA signalling. Moreover interestingly, triple M3K knockout mutant analyses show that the identified M3Ks are required for the rapid osmotic stress activation of SnRK2 kinases, in a less-well understood, previously proposed, pathway parallel to ABA signalling.
Isolation of ABA-Insensitive MAPKK-Kinase amiRNA Mutants
By unbiased forward genetic screening of seeds from over 1,500 independent T2 artificial microRNA (amiRNA)-expressing lines in pools (approximately 45,000 seeds screened) for ABA-insensitive seed germination, we isolated up to ˜290 putative mutants. In secondary screening of the surviving putative mutants in the next (T3) generation, progeny from 25 of the putative mutant plants continued to show a clearly reduced ABA sensitivity, including seeds propagated from three amiR-ax1117-expressing plants (
ST1/SnRK2.6 Re-Activation after Dephosphorylation
We investigated phosphorylation of purified recombinant GST-tagged OST1/SnRK2.6 protein kinase after dephosphorylation in vitro. To test whether OST1/SnRK2.6 could be re-activated by autophosphorylation, after dephosphorylation, the GST-OST1/SnRK2.6 protein bound on glutathione sepharose 4B resin was incubated with calf intestinal alkaline phosphatase (CIAP), and [γ-32P]-ATP was added to the reaction after wash out of CIAP. Surprisingly, we found that OST1/SnRK2.6 showed very low autophosphorylation activity even after the protein phosphatase had been removed (
We investigated whether the amiRNA-targeted M3Ks may directly activate OST1/SnRK2.6. In-gel kinase assays were carried out in vitro after incubation of the dephosphorylated His-OST1/SnRK2.6 with GST-tagged recombinant M3K kinase domains and His-tagged full-length M3Ks in the presence of ATP. Notably, three M3Ks from the subgroup B3, named M3Kδ1, 66 and 67, were found to strongly activate OST1/SnRK2.6, whereas the other M3Ks targeted by the corresponding amiRNA did not clearly activate OST1/SnRK2.6 under the imposed conditions in vitro (
M3Kδ1 Phosphorylates a Critical Ser171 for OST1 activation
Mass spectrometry analyses revealed that M3Kδ1 phosphorylated the OST1/SnRK2.6 residues Ser171, Ser175 and Thr176 in the OST1 activation loop (
We created transgenic Arabidopsis plants stably expressing OST1-HF (S171A) in the ost1-3 background29,30. Expression of OST1-HF (S171A) did not rescue the ABA-insensitive stomatal conductance response and the low leaf temperature phenotype of the ost1-3 mutant in two independent lines (
Patch-clamp analyses of the ost1-3 complementation lines showed the essential role of Ser171 for ABA-induced S-type anion channel activation in Arabidopsis guard cells (
Reconstitution of early ABA signalling with MAPKK-kinases Previous studies have reconstituted ABA-dependent phosphorylation of OST1/SnRK2.6 substrates in vitro using recombinant proteins14,18. Recombinant OST1/SnRK2.6 has many phosphorylated sites and a significant protein kinase activity in vitro16. However, we find that prior dephosphorylated OST1/SnRK2.6 could unexpectedly not be re-activated by itself (
OST1/SnRK2.6-mediates activation of the S-type anion channel SLAC1 in Xenopus oocytes12,13, and ABA-induced SLAC1 activation was reconstituted in oocytes17. These results strongly depended on artificial BiFC tags that force interaction of the SLAC1 channel with OST1/SnRK2.6 proteins12,17, indicating that the BiFC tag might cause an unknown artificial effect. When expressing SLAC1 and OST1/SnRK2.6 proteins without any tag in oocyte experiments in the present study, SLAC1 was not significantly activated (
In additional experiments, we co-injected cRNA for the ABA receptor PYL9/RCAR1, together with the ABI1 PP2C, OST1 SnRK2.6, SLAC1 and M3Ks into oocytes, to test whether ABA-dependent SLAC1 anion channel activation could be reconstituted with these components. ABA could activate SLAC1 in oocytes only in the presence of low concentrations of either M3Kδ1, M3Kδ6 or M3Kδ7 mRNAs (
The reduced steady-state stomatal conductance in the m3k amiRNA line indicates additional effects of this artificial microRNA and/or compensatory effects of impaired stomatal closing response mutants31,32. Higher order mutant combinations will be required to investigate this hypothesis. Based on the lower steady-state stomatal conductance, the impaired response to ABA (
We isolated T-DNA insertion mutants (m3kδ1 (SALK_048985), m3kδ6-1 (SALK_004541), m3kδ6-2 (SALK_001982) and m3kδ7 (SALK_082710)) (
We confirmed knock-out of full-length expression of M3Kδ1 and M3Kδ7 in the T-DNA lines, while there was partial expression of the kinase domain of M3Kδ6 in the m3kδ6-1 line (
To further test the function of these M3Ks, we created amiRNA lines predicted to target only the triple combination of M3Kδ1, M3Kδ6 and M3Kδ7 and found that three independent amiRNA lines showed ABA-insensitivities in seed germination (
In-gel kinase assays showed that ABA-induced activation of SnRK2 kinase in the m3kδ1crispr m3kδ6-2 m3kδ7crispr triple was slightly less strong than in wild type plants (
In-gel kinase assays suggest that the M3Ks have a major role in osmotic stress signalling in Arabidopsis (
Co-immunoprecipitation of M3Kδ6- and OST1/SnRK2.6-expressed in mesophyll cell protoplasts did not show a clear interaction (
In the present study, a combination of genetic screening for functional redundancy in abscisic acid responsiveness and multiple biochemical and signal transduction analyses in vitro and in planta have identified and characterized members of the Raf-like MAPKK-kinase δ B3 family that are required for full activation of SnRK2 protein kinases in abscisic acid signal transduction in vitro (
Dephosphorylation of the OST1/SnRK2.6 kinase was unexpectedly found not to result in OST1/SnRK2.6 re-activation by SnRK2 auto-phosphorylation alone. The identified M3Kδs, but not other analyzed CPK and MPK12 protein kinases that function in ABA signalling24-27, were found to be required for re-activation of OST1/SnRK2.6. Moreover, the M3Kδ1 kinase greatly enhances the activities of other ABA signalling protein kinases SnRK2.2 and SnRK2.3 (
A previous proof-of-concept screen using artificial microRNAs that target multiple homologous genes isolated a plant predicted to target seven M3Ks of the B-family21. A Physcomitrella single gene encoding a M3K, ARK, was also identified which functions in SnRK2 activation28. Recent studies show that ARK kinase is required for Physcomitrella ABA and drought stress responses including phosphorylation of transcription factors through SnRK2 kinases41,42. Here, in forward genetic screening we have isolated amiRNA expressing lines that target M3K members of the B family (
Osmotic stress is known to rapidly activate SnRK2 protein kinases20,34,43. Rapid osmotic stress signalling includes a prominent ABA-independent pathway that leads to activation of transcription factors44,45. However, the upstream osmotic stress signalling mechanisms remain incompletely understood. Recent studies suggest that PP2Cs involved in ABA signalling dephosphorylate SnRK2.446-48. The M3K ARK is required for osmotic stress tolerance in Physcomitrella28,42. Interestingly, the identified M3Kδs play a critical role in the rapid osmotic stress activation of SnRK2 protein kinases (
Osmotic and salt stresses induce a rapid cytosolic Ca2+ increase51-54. An ABA-independent osmotic stress signalling pathway has been characterized that triggers rapid gene expression44,55. Recent research shows that the Arabidopsis NGATHA1 transcription factor mediates the ensuing drought stress-induced ABA accumulation through enhanced expression of the ABA biosynthesis NINE-CIS-EPOXYCAROTENOID DIOXYGENASE, NCED356. Gel shift assays indicate that osmotic stress causes a rapid post-translational modification of M3Kδ6 (
Using amiRNA libraries21, we screened amiRNA lines for ABA-insensitive seed germination phenotypes using ½ MS plate supplemented with 2 μM ABA57. The underlying amiRNA sequences were identified from genomic DNA by PCR and sequencing (m3k amiRNA: 5′-TTGGAGCCATCCATTCAGCCG-3′ (SEQ ID NO:1), amiR-ax1117: 5′-TCCAAAATCGCAAACCTTCAC-3′) (SEQ ID NO:2). We used an amiRNA line targeting human myosin 2 gene (HsMYO2) as a control.
10 μg GST-OST1/SnRK2.6 proteins were bound to glutathione sepharose 4B beads and incubated with 30 U CIAP for 2 hr at room temperature. The beads were washed with T-TBS (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.05% Tween-20) three times, and GST-OST1 protein was eluted with 30 μL elution buffer (50 mM Tris-HCl pH 8.0, 10 mM reduced glutathione). 5 μL GST-OST1 solution was added in phosphorylation buffer [50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 μM free Ca2+ buffered by 1 mM EGTA and CaCl2 (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaMgATPEGTA-NIST.htm), 0.1% Triton X-100, and 1 mM DTT] with or without 1 μg of the protein kinases CPKδ, CPK23, MPK12 or 0.1 μg of the indicated MAPKK kinases (M3Ks). The phosphorylation reactions were started by addition of 200 μM ATP and 1 ρCi [γ-32P]ATP. After 60 min incubation at room temperature, these reactions were stopped by addition of SDS-PAGE loading buffer. Note that the mobilities of recombinant and transgenic proteins in the present study depend on the linked tags. For example, the OST1/SnRK2.6 6×His-tag also includes sequences including thrombin and enterokinase cleavage sites and restriction enzyme sites in the pET-30a(+) vector used for E. coli expression of OST1/SnRK2.6 in
15-20 Arabidopsis seedlings (7-9-day-old) grown on ½ MS plates were treated with 10 μM ABA or 0.3 M mannitol for 15 min at room temperature and grinded with a pestle and mortar in 400 μL extraction buffer (50 mM MOPS-KOH pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 10 mM NaF, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μM leupeptin) on ice. After 10 min centrifugation at 13,000 g, the supernatants were transferred to new tubes, and proteins were precipitated by acetone precipitation. Proteins were dissolved in SDS-PAGE loading buffer and separated in 9% acrylamide gels. In-gel kinase assays were performed as described previously58. In brief, gels were incubated in washing buffer (25 mM Tris-HCl pH 8.0, 0.5 mM DTT, 0.1 mM Na3VO4, 5 mM NaF, 0.5 mg ml−1 BSA, and 0.1% Triton X-100) for 30 min three times and in renaturation buffer (25 mM Tris-HCl pH 8.0, 1 mM DTT, 0.1 mM Na3VO4, and 5 mM NaF) for 30 min once. Gels were further incubated in renaturation buffer at 4° C. overnight followed by further incubation in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mM DTT, and 1 mM EGTA) for 30 min. Phosphorylation reactions were carried out in reaction buffer with 50 μCi [γ-32P]-ATP for 60 min at room temperature. Gels were washed in 5% trichloroacetic acid and 1% phosphoric acid four times for 30 min each. Storage phosphor screens or X-ray films were used for detection.
0.43 μmol His-OST1/SnRK2.6, 0.17 μmol His-PYR1/RCAR11 and 0.06 μmol GST-M3Kδ6 kinase domain were incubated in 200 μL phosphorylation buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1% Triton X-100, and 1 mM DTT) with 200 μM ATP for 10 min, and 20 μL solution was transferred to a new tube and 10 μL 3×SDS-PAGE loading buffer was added to stop the reaction. Then, 0.01 μmol His-HAB1 was added to the reaction solution, and 20 μL solution were transferred to a new tube to stop the reaction by addition of 10 μL 3×SDS-PAGE loading buffer after 10 min incubation. 50 μM ABA was added to the reaction and 20 μL reactions were transferred to new tubes to stop the reaction after 5, 10 or 30 min incubation. Proteins were separated by SDS-PAGE, and OST1/SnRK2.6 activity was detected by in-gel kinase assays.
30 μg GST-OST1/SnRK2.6(D140A) and 2.5 μg GST-M3Kδ1 kinase domain were incubated in phosphorylation buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1% Triton X-100, and 1 mM DTT) with 1 mM ATP for 2 hr at room temperature. Proteins were precipitated by acetone precipitation and dissolved in SDS-PAGE loading buffer. After SDS-PAGE and CBB staining, protein bands of GST-OST1/SnRK2.6(D140A) were excised and analyzed by LC-MS/MS17. For in vivo Ser-171 phosphorylation, OST1/SnRK2.6-GFP was transiently expressed in Arabidopsis mesophyll cell protoplasts. The protoplasts were incubated with or without 20 μM ABA for 15 min, and OST1/SnRK2.6 proteins were purified by immunoprecipitation using anti-GFP antibodies. After SDS-PAGE and CBB staining, OST1/SnRK2.6-GFP bands were excised and analyzed by LC-MS/MS17
Infrared-based gas exchange analyzer systems were used including an integrated Multiphase Flash Fluorometer (Li-6800-01A or Li-6400; Li-Cor Inc.) for gas exchange analyses. Plants were grown on soil in Percival growth cabinets at a 12/12 h, 21° C./21° C. day/night cycle, a photosynthetic photon flux density of ˜90 mmol m-2 s-1, and 70 to 80% relative humidity for 6 to 7 weeks. Mature rosette leaves were detached at the basal part of petiole by a razor blade, and re-cut twice under distilled and deionized water. The petioles of the leaves were then immersed in ddH2O for gas exchange analysis. The detached leaves were clamped and the environment of the leaf chamber was controlled at 400 ppm ambient CO2, 23-24° C., ˜65% relative air humidity, 150 μmol m−2 s−1 photon flux density, and 500 μmol s−1 flow rate until stomatal conductance stabilized. One or 2 μM±-ABA was applied to the petiole for kinetic stomatal conductance response analyses as described59.
Guard cell protoplasts from 4 to 6 week-old Arabidopsis plants were prepared24,33 ABA-activated S-type anion channel current recordings were carried out by using an Axon 200A amplifier (Axon instruments) and a Digidata 1440A low-noise data acquisition system. Epidermal tissues were isolated from one or two rosette leaves and collected using a nylon mesh (100-μm pore size). Subsequently the epidermal tissues were incubated in 10-ml protoplast isolation solution containing 500 mM D-mannitol, 1% cellulase R-10 (Yakult Pharmaceutical Industry), 0.5% macerozyme R-10 (Yakult Pharmaceutical Industry), 0.5% bovine serum albumin, 0.1% kanamycin sulfate, 0.1 mM CaCl2), 0.1 mM KCl, and 10 mM ascorbic acid for 16 hr at 25° C. on a circular shaker at 50 rpm. Guard cell protoplasts were collected through a nylon mesh (10-μm pore size) and then washed two times with protoplast suspension solution containing 500 mM D-sorbitol, 0.1 mM CaCl2), and 0.1 mM KCl (pH 5.6 with KOH) by centrifugation (200 g for 5 min at room temperature). Isolated guard cell protoplasts were stored on ice before use.
S-type anion currents in guard cell protoplasts were recorded using the whole-cell patch-clamp technique24,33. The pipette solution was composed of 150 mM CsCl, 2 mM MgCl2, 5.86 mM CaCl2), 6.7 mM EGTA, and 10 mM Hepes-Tris (pH 7.1). 5 mM Mg-ATP was added to the pipette solution freshly before use. The bath solution was composed of 30 mM CsCl, 2 mM MgCl2, 1 mM CaCl2), and 10 mM MES-Tris (pH 5.6). Osmolalities of the pipette solution and the bath solution were adjusted to 500 mosmol kg−1 and 485 mosmol kg−1 using D-sorbitol, respectively. In
The PCR amplified cDNA fragments of OST1, SLAC1, PYL9/RCAR1, ABI1, M3Kδ1, M3Kδ6 and M3Kδ7 were cloned into the oocyte expression vector pNB1 by using an advanced uracil-excision based cloning strategy as previously described60. The mutant isoforms OST1-S171A, M3Kδ6-K775W and M3Kδ7-K740W were generated using the Quikchange Site-Directed Mutagenesis kit (Agilent Technologies). Linearized plasmids were used to generate cRNAs via the mMESSAGE mMACHINE® T7 kit (Thermo Fisher Scientific, Catalog number: AM1344). Surgically extracted ovaries of Xenopus laevis were ordered from Nasco (Fort Atkinson, Wis., product number: LM00935) and Ecocyte Bio Science US (Austin, Tex.) and oocytes were isolated as previously described61. 5 ng cRNA of each construct OST1, OST1-S171A, SLAC1, PYL9/RCAR1, ABI1 and 0.5 ng cRNAs of each construct M3Kδ1, M3Kδ6, M3Kδ7, M3Kδ6-K775W, M3Kδ7-K740W were co-injected into isolated oocytes in the indicated combinations. Oocytes were then incubated at 16° C. for 2 days in ND96 buffer (1 mM CaCl2), 1 mM MgCl2, 96 mM NaCl, 10 mM MES/Tris, pH=7.5). Osmolarity was adjusted to 220 mosmol kg−1 by D-sorbitol. Using a Cornerstone (Dagan) TEV-200 amplifier and a Digidata 1440A low-noise data acquisition system with pClamp software (Molecular Devices), two-electrode voltage clamp recordings were performed in a bath solution containing 1 mM CaCl2), 2 mM KCl, 24 mM NaCl, 70 mM Na-gluconate, 10 mM MES/Tris, pH 7.4, Osmolarity was adjusted to 220 mosmol kg−1 by D-sorbitol. ABA was injected into oocytes to achieve a final concentration of 50 μM for analyses of ABA activation of SLAC1 currents. Steady state currents were recorded with 3 second voltage pulses ranging from +40 mV to −120 mV in −20 mV decrements, followed by a “tail” voltage of −120 mV and the holding potential was kept at 0 mV.
SLAC1-mediated currents in oocytes vary showing either time-dependent relaxation or more instantaneous currents when using a chloride bath solution61,62. Furthermore, ion channel activities display different magnitudes from one oocyte batch to another due to protein expression level variation among batches of oocytes. To avoid time-of-measurement and inter-batch dependence in the data, H2O-injected control and other indicated controls were included in each batch of oocytes and control experiments were recorded intermittently with the investigated conditions. Data from one representative oocyte batch are shown from the same batch in each figure panel and at least three independent batches of oocytes were investigated and showed consistent findings.
Mesophyll cell protoplasts were isolated as described previously63 from 3-4-week old Arabidopsis leaves. 10-20 μg of pUC18 plasmids carrying 35S:OST1/SnRK2.6-GFP:nosT or 35S:M3Kδ6-FLAG:nosT and 30 μg protoplasts were used for 20% PEG-mediated transient expression. After overnight incubation in incubation buffer (10 mM MES-KOH pH 6.0, 0.4 M mannitol, 20 mM KCl, 1 mM CaCl2), protoplasts were incubated in 10 μM ABA or 0.8 M mannitol or in control buffer for 15 min and harvested by centrifugation at 13,000 g for 1 min. After the supernatants were removed, 20 μL SDS-PAGE loading buffer was added and incubated at 95° C. for 3 min.
Plants grown 4-5 weeks on soil were sprayed with 20 μM ABA dissolved in water. After 3 hr under white light in the growth room, images were captured using an infrared thermal imaging camera (T650sc; FLIR, Wilsonville, Oreg.). Leaf temperatures were determined as average temperatures of each whole leaf area by using Fiji software (ImageJ version: 2.0.0-rc-59/1.51n).
The m3kδ1 and m3kδ7 CRISPR/Cas9 deletion knock-out mutants were generated using CRISPR/Cas9 gene editing technology64-66 in the m3kδ6-2 mutant background. We used two guide RNAs to generate a large deletion in each target gene. The target sequences in M3Kδ1 were TACGGAAGCTCCACATCGGCGG (SEQ ID NO:3) and GATGCAAGTCGTTGGAGCTGTGG (SEQ ID NO:4) (PAM sites are underlined). Targets for M3Kδ7 were GACGGAGTTCCAGATCTCCGGG (SEQ ID NO:5) and CCAGAGAGCAGCAGTTCCCAGT (SEQ ID NO:6).
The designed m3kδ1crispr mutants were genotyped with the primer pair Delta1-GT1 and Delta1-GT2, which would generate a fragment of about 750 bp when the designed deletion took place. The primer pair could not amplify WT genomic DNA due to the large size of the fragment. To determine zygosity of m3kδ1crispr mutants, we used the primer set Delta1-GT1+Delta1-GT3, which amplifies a 777 bp fragment from WT DNA, but could not amplify a band in a homozygous mutant.
For m3kδ7crispr mutants, we used Delta7-GT1 and Delta7-GT4, which would generate a fragment of about 1390 bp if mutant DNA is used as PCR template. The primer pair could not amplify WT DNA because of the large fragment size. The Delta7-GT1 and Delta7-GT3 primer pair was able to generate a fragment of 1125 bp when WT DNA was used as PCR template. The Delta7-GT1/GT3 was used to differentiate homozygous m3kδ7crispr mutants from heterozygous m3kδ7crispr mutants. After isolating homozygous m3kδ1crispr m3kδ6-2 and m3kδ7crispr m3kδ6-2 mutants, these lines were crossed and homozygous triple mutants were recovered in the T2 generation. Primers for genotyping:
Creating amiRNA Knock-Downs Targeting M3Kδ1, δ6 and δ7
The amiRNA sequence was designed using the WMD3 (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi) and PHANTOM database (http://phantomdb.ucsd.edu). The amiRNA containing the target sequence (5′-TACGACTTGCATCGGGTTCAA-3′) (SEQ ID NO:13) for M3Kδ1, M3Kδ6 and M3Kδ7 was amplified by PCR using primers
and inserted into the vector pFH003221. Arabidopsis (Col-0) plants were used for floral-dip transformation. Three independent homozygous T3 seeds were used in the seed germination assays.
Constructs for BiFC analyses were generated by ligation of coding sequences of ABI1, RopGEF1, OST1/SnRK2.6, SnRK2.2, SnRK2.4, SnRK2.10, M3Kδ6, and M3Kδ7 into pSPYCE(M) or pSPYNE173 using the USER Cloning technology (see
Cotyledon greening assays were analyzed by two-way ANOVA followed by Tukey's tests. Leaf temperatures were analyzed by one-way ANOVA followed by Tukey's tests.
a, Seeds of amiR-HsMYO wild-type (control line) or amiR-ax1117 mutant were sowed on ½ MS medium containing 2 μM ABA, or 0.02% EtOH as control, for germination assays. Representative images showing seed germination after 6 days. b, The percentage of seedlings showing green cotyledons was analyzed. Data represent mean±s.d. n=4 experiments. Each experiment included 64 seeds for each genotype. Letters at the top of columns are grouped based on two-way ANOVA and Tukey's test, P<0.05. c, Identification of the amiRNA sequence in amiR-ax1117 plants. Black box labels the sequence of amiR-ax1117. The amiR-ax1117 is predicted to include Raf-like protein kinase genes M3Kδ5, M3Kδ7, M3Kδ1, M3Kδ6 and M3KS-CTR1 kinase (see
a, The inactive M3Kδ6 kinase domain mutant (K775W) did not re-activate His-OST1/SnRK2.6 in vitro. b, Inactive GST-OST1/SnRK2.6-D140A kinase protein was incubated with M3Kδ6 kinase domain, and in vitro phosphorylation assays were performed with 32P-ATP. c, Recombinant inactive OST1(D140A) and M3Kδ1 kinase domains were incubated with ATP. A mass spectrum of phosphorylated OST1 peptide (SSVLHpSQPK) is shown. pS indicates phosphorylated Ser171 of OST1(D140A). d, Phosphorylation at Ser171 was not detectable after in vitro auto-phosphorylation of OST1/SnRK2.6, but was consistently phosphorylated in the presence of M3Kδ1. e, OST1(S171A)-GFP was transiently expressed in Arabidopsis mesophyll cell protoplasts. Protoplasts were incubated with 10 μM ABA or control buffer for 15 min, and OST1/SnRK2.6 activities were analyzed by in-gel kinase assays.
a and b, In vitro reconstitution of ABA-induced OST1/SnRK2 activation without M3Kδ6 (a) or with M3Kδ6 (b). The recombinant proteins His-PYR1/RCAR11, His-OST1/SnRK2.6 without (a) or with (b) GST-M3Kδ6 kinase domain were mixed. After addition of His-HAB1, protein solutions were incubated for 10 min. Then, 50 μM ABA was added to the protein solution. Reactions were stopped at the indicated times. OST1/SnRK2.6 kinase activities were detected by in-gel kinase assays. c, Recombinant His-PYR1/RCAR11, His-HAB1, His-OST1/SnRK2.6, His-AKS1 and GST-M3Kδ6 kinase domain were mixed as indicated above the gel. 50 μM ABA was added before (lane 5) or after (lanes 6 and 8) 10 min incubation at room temperature. Then, 100 μM ATP was added (lanes 2 to 8) to trigger phosphorylation reactions for 10 min. Note that M3Kδ6 is required for ABA-induced AKS1 phosphorylation when ABA is added 10 min after exposure to HAB1-PP2C-including mix (compare lanes 6 and 8). Reactions were stopped by addition of SDS-PAGE loading buffer. Phosphorylation of AKS1 is detected by binding of 14-3-3Phi (At1g35160) to the phosphorylated AKS1 protein15. AKS1 phosphorylation is shown by protein-blot (top), and protein amount is monitored by immuno-blot (bottom). d-f, Reconstitution of ABA-activation of SLAC1 channels in Xenopus oocytes, in the presence or absence of M3Ks. (d) Representative whole cell chloride current recordings of oocytes co-expressing the indicated proteins, without (control) or with injection of 50 μM ABA (+ABA). Currents were recorded in response to voltage pulses ranging from +40 mV to −120 mV in −20 steps with a holding potential at 0 mV and a final tail potential of −120 mV. (e) Mean current-voltage curves of oocytes co-expressing the indicated proteins, with or without injection of ABA. The symbols of H2O control, OST1+SLAC1, PYL9/RCAR1+ABI1+OST1+SLAC1, PYL9/RCAR1+ABI1+OST1+SLAC1+ABA and PYL9/RCAR1+ABI1+OST1+SLAC1+M3Ks overlapped. Single symbols are shown for some data points for better viewing. (f) Average SLAC1-mediated currents at −100 mV, co-expressing the indicated proteins, in the presence or absence of 50 μM ABA.
Data from 3 independent batches of oocytes showed similar results. One representative batch of oocytes is shown, with the number of oocytes in that batch indicated in parentheses. H2O, OST1+SLAC1, PYL9/RCAR1+ABI1+OST1+SLAC1 and PYL9/RCAR1+ABI1+OST1+SLAC1+ABA controls are the same data in both panels in (e) as the data are from the same oocyte batch. Error bars denote mean±s.e.m. Means with letters (a, b, c and d) are grouped based on one-way ANOVA and Tukey's multiple comparisons test, P<0.05.
a, Genome structures and T-DNA insertion sites of M3K genes are shown. b, Genomic regions of CRISPR/Cas9-mediated M3Kδ1 and M3Kδ7 gene deletions are shown. These deletions were introduced in the m3kδ6-2 T-DNA knock out mutant as a background. c, RT-PCR assays show transcripts of kinase domains of M3Ks in the m3kδ1crispr m3kδ6-2 m3kδ7crispr triple mutant. d, m3kδ1crispr m3kδ6-2 m3kδ7crispr triple mutant seedlings were grown on ½ MS plates supplemented with 2 μM ABA or ethanol (control) for 16 days. e, Seedlings showing green cotyledons as in (d) were counted. n=3 (EtOH) and n=4 (ABA) experiments, means+/−s.d., 45 seeds per genotype were used in each experiment. f, RT-PCR shows M3Kδ1, δ6 and δ7 expression in the indicated m3k T-DNA insertion mutants. δ6(KD) refers to primers that amplify the M3Kδ6 kinase domain in the m3kδ6-1 T-DNA line. g, m3kδ1 m3kδ6-1 m3kδ7 T-DNA triple mutant plants were grown on ½MS plates supplemented with 0.8 μM ABA for 9 days. h, Seedlings showing green cotyledons as in (g) were counted. n=3, means+/−s.d., 60-88 seeds were used per genotype in each assay. i, Three amiRNA lines targeting M3Kδ1, 66 and 67 were grown on ½MS plates supplemented with EtOH (control) or 2 μM ABA for 9 days. As a control line, the amiRNA-HsMYO line21 was used. j, Seedlings showing green cotyledons as in (i) were counted. n=3 (EtOH) and 4 (ABA) experiments, means+/−s.d., 81 seeds per genotype were analyzed in each experiment. (e, h and i) Letters at the top of columns are grouped based on two-way ANOVA and Tukey's test, P<0.05.
a, m3kδ1crispr m3kδ6-2 m3kδ7crispr triple mutant seedlings were incubated in 10 μM ABA or 0.3 M mannitol (Osmo) for 15 min. SnRK2 activities were tested by in-gel kinase assays. Arrowhead shows SnRK2 activity23. b, Normalized band intensities as shown in (a) were measured by using ImageJ. n=4, means+/−s.e.m. c, m3kδ1 m3kδ6-1 m3kδ7 T-DNA triple mutant seedlings were incubated in 10 μM ABA or 0.3 M mannitol (Osmo) for 15 min. SnRK2 activities were analyzed by in-gel kinase assays. d, Normalized band intensities as shown in (c) were measured by using ImageJ. n=4 experiments, means+/−s.e.m. e, Recombinant GST-tagged Arabidopsis SnRK2 protein kinases were incubated with M3Kδ1 kinase domain. SnRK2 kinase activities were analyzed by in-gel kinase assays. f, M3Kδ6-FLAG was transiently expressed in Arabidopsis mesophyll cell protoplasts. Protoplasts were incubated in 0.8 M mannitol (Osmo) for 15 min. M3Kδ6 proteins were detected by immuno-blot using anti-FLAG antibody. In the Osmo lane, the M3Kδ6 band showed a slight mobility shift as indicated by an asterisk.
All M3Ks in subgroup B and selected M3Ks in subgroup C1-7 are shown (see e.g., MAPK Group, Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7, 301-308 (2002)) # and * indicates genes targeted by the amiR-ax1117, and the m3k amiRNA, respectively.
Recombinant kinase inactive GST-OST1/SnRK2.6 (D140A) protein was incubated with M3Kδ1, M3Kδ6 or M3Kδ7 kinase domains, and in vitro phosphorylation assays were performed with 32P-ATP.
a, Recombinant GST-OST1/SnRK2.6 proteins carrying S171A, S175A or T176A mutation were used for in vitro autophosphorylation. OST1/SnRK2.6 (S175A) has no kinase activity (Belin, C. et al. Plant Physiol 141, 1316-1327 (2006)). b, OST1/SnRK2.6-GFP variants (WT, S171A, S175A or T176A) were transiently expressed in Arabidopsis mesophyll cell protoplasts. Protoplasts were incubated in 10 μM ABA for 15 min. OST1/SnRK2.6 protein kinase activity was detected by in-gel kinase assays (top panel). OST1/SnRK2.6-GFP proteins were detected by immuno-blot using GFP antibody (bottom panel). c, OST1-GFP (S171E) kinase activity was tested as shown in (b).
a, The sequence of identified phosphorylated peptide by mass spectrometry. OST1/SnRK2.6-GFP was expressed in Arabidopsis mesophyll cell protoplasts and purified by immunoprecipitation with GFP antibodies before or after 20 μM ABA treatment for 15 min. S(+79.97) indicates phosphorylated serine residues. Values (Control and ABA) indicate normalized peak areas for phosphorylated peptides. The phosphorylated peptide was not detected in the control sample. In contrast the peptide was clearly phosphorylated in response to ABA. b, An annotated mass spectrum of the phosphorylated peptide in the presence of ABA is shown.
a, Stomatal conductances were analyzed in detached leaves of stable transgenic Arabidopsis [pUBQ10:OST1-HF ost1-3 (OST1_comp1) or pUBQ10:OST1(S171A)-HF ost1-3 (S171A_comp1)]. 2 μM ABA was applied at 0 min. Different independent transgenic lines from those shown in
a, Representative whole cell chloride current recordings of oocytes injected with the indicated cRNAs. Currents were recorded in response to voltage pulses ranging from +40 mV to −120 mV in −20 mV steps with a holding potential at 0 mV and a final tail potential of −120 mV. b-d, Mean current-voltage curves of oocytes co-expressing OST1 and SLAC1, in the presence or absence of the indicated M3K proteins. In panel (b) and (d), the symbols of H2O, OST1+SLAC1 and SLAC1+M3Ks overlapped. One symbol is shown for some data points for better viewing. (e) Average SLAC1 mediated currents at −100 mV, co-expressing OST1, in the presence or absence of the indicated M3K proteins. M3K6 and OST1 and/or SLAC1 cRNA were injected at a concentration ratio of 1 to 10 to 10 (see main text). Data from one representative batch of oocytes are shown, with the number of oocytes in that batch indicated in parentheses. Control H2O and OST1+SLAC1 data are the same data in (b), (c) and (d), as these data are from the same batch of oocytes (see Methods). Four independent batches of oocytes showed similar results. Error bars denote mean±s.e.m. f, Mean current-voltage curves of Xenopus oocytes injected with the indicated cRNAs. g, Average SLAC1-mediated currents from panel (f) at −120 mV, co-expressing OST1 isoforms, in the presence of M3Kδ1. The injected cRNA ratio of M3Kδ1 and OST1 isoforms was 1 to 10 (see main text). Letters on the bottom of columns are grouped based on one-way ANOVA with Tukey's test, P<0.01.
a-d, Mean current-voltage curves of Xenopus oocytes injected with the indicated cRNAs. The symbols of OST1-S171A+SLAC1+M3Kδ6 are not visible because the symbols overlap. e, Average SLAC1-mediated currents from panels (a) to (d) at −100 mV, co-expressing OST1 or the kinase inactive OST1-S171A mutant isoform, in the presence of the indicated M3Ks or the kinase-inactive M3Kδ6-K775W and M3Kδ7-K740W isoforms. When M3K6 and OST1 cRNAs were co-injected the ratio of M3K6 and OST1 cRNA was 1 to 10 (see main text). Data from one representative oocyte batch are shown. Results from 3 independent batches of oocytes showed similar results. H2O, SLAC1 and OST1+SLAC1 controls are the same in panels (a), (b), (c) and (d), as these were recorded in the same batch of oocytes. Error bars denote mean±s.e.m.
a, Leaves from m3k amiRNAi and the control amiRNA-HsMYO line (expressing an amiRNA targeting human myosin 2), which has no target gene in Arabidopsis plants were analyzed in time-resolved stomatal conductance analyses in which 1 μM ABA was added to the transpiration stream via the petiole (as described in e.g., Ceciliato, P. et al. Plant Methods 15, doi:10.1186/s13007-019-0423-y (2019)) as indicted by the red arrowhead (n=3 leaves from 3 independent plants per genotype, +/−s.d.). b, Normalized relative stomatal conductance to the first data point shown in (a). c-h, ABA-activated S-type anion channel currents were investigated by patch-clamp analyses using guard cell protoplasts from the wildtype parent Col-0 (c, d), the HsMYO amiRNA control line (e, f), and the m3k amiRNA line (g, h). Representative current traces (c, e, g) and average current voltage relationships (d, f, h) of S-type anion channel currents are shown. Data presented are means+/−s.e.m.
a, m3k double (m3kδ6-2/δ7) and triple (m3kδ1/δ6-1/δ7) mutants were grown on ½ MS plates supplemented with 2 μM ABA or EtOH for 9 days. Seedlings showing green cotyledons were counted. n=3 experiments, means+/−s.d., 45-48 seeds were used per genotype and condition in each experiment. b, m3k double (m3kδ1/δ7) and triple (m3kδ1 crispr δ6-2/δ7crispr) mutants were grown on ½ MS plates supplemented with 2 μM ABA or EtOH for 16 days. Seedlings showing green cotyledons were counted. n=3 experiments (EtOH) and n=4 experiments (ABA), means+/−s.d., 45 seeds were used per genotype and condition in each experiment. The crispr and Col results are the same as
Replicate of in-gel kinase assay using m3k9J δ6-1/δ7 triple mutant is shown. See
a, m3kδ1/δ7 double mutant and m3kδ1crispr δ6-2/δ7crispr triple mutant seedlings were grown on ½ MS plates supplemented with 0.4 M mannitol for 3 days. Green cotyledons were counted. n=4 experiments, means+/−s.d., 64 seeds per genotype were used in each experiment. b, Three amiRNA lines targeting M3Kδ1, 66 and 67 were grown on ½ MS plate supplemented with 0.4 M mannitol for 3 days. As a control line, HsMYO was used. n=4 experiments, means+/−s.d., 64 seeds per genotype were used in each experiment. Letters at the top of columns are grouped based on two-way ANOVA and Tukey's test, P<0.05.
a, GST-SnRK2.3 protein was incubated with the kinase domains of M3Kδ1, M3Kδ6 or M3Kδ7 and in-gel kinase assays were conducted. b, SnRK2.2-GFP (WT or S180A) and SnRK2.3-GFP (WT or S172A) were expressed in Arabidopsis mesophyll cell protoplasts and purified by immunoprecipitation with GFP antibodies. The isolated proteins bound on magnetic immunoprecipitation beads were used for in vitro phosphorylation assays using histone as an artificial substrate. Phosphorylation reactions were started by the addition of 32P-ATP. After 30 min, reactions were stopped by the addition of 3×SDS-PAGE sample buffer.
a, Co-immunoprecipitation experiments using transiently expressed M3Kδ6 and OST1/SnRK2.6 in Arabidopsis mesophyll cell protoplasts. OST1/SnRK2.6-GFP or GFP control co-expressed with M3Kδ6-FLAG were immunoprecipitated with GFP antibodies. Precipitated proteins were analyzed by immunoblots using GFP or FLAG antibody. b and c, BiFC analyses of nYFP-M3Kδ6 (b) or nYFP-M3Kδ7 (c) with OST1/SnRK2.6-cYFP, SnRK2.2-cYFP, 2.4-cYFP and 2.10-cYFP infiltrated in 6-week-old Nicotiana benthamiana leaves. nYFP-GEF1/cYFP-ABI1 combination was used as a positive control. All images are at the same scale. Scale bars=50 μm. d and e, BiFC quantifications measured from maximal projections of z-stacks and normalized over an infiltration control expressing p19 only. BiFC quantifications were analyzed by one-way ANOVA followed by Tukey's tests. Confocal images were acquired using identical settings for each BiFC experiment. Means±s.e.m. (n=45).
a, m3k triple (m3kδ1/δ6-1/δ7) and m3k quadruple (m3kδ1/δ5/δ6-1/δ7) mutant plants were grown on ½ MS plates supplemented with 0.8 μM ABA for 6 days (m3kδ5=SALK_025685). Green emerging cotyledons were counted. n=6 experiments, means+/−s.d., 81 seeds were used per genotype and per condition in each experiment.
Letters at the top of columns are grouped based on two-way ANOVA and Tukey's test, P<0.05. b, Gene expression levels of B3 subgroup M3K genes and three SnRK2 genes in guard cells and mesophyll cells. Data were obtained from the public microarray database eFP Browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi), see Yang et al. Plant Methods 4, 6, doi:10.1186/1746-4811-4-6 (2008).
A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This U.S. Utility Patent Application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/816,492, Mar. 11, 2019. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.
This invention was made with government support under ES010337 and GM060396 awarded by the National Institutes of Health and under MCB-1900567 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/022013 | 3/11/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62816492 | Mar 2019 | US |
