Patent Application
20130326734
This invention generally relates to plant molecular and cellular biology. In alternative embodiments, the invention provides compositions and methods for manipulating the exchange of water and/or carbon dioxide (CO2) through plant stomata by combining the control of expression of CO2 sensor genes with the control of expression of OST1 (Open Stomata 1) protein kinase and related protein kinases SnRK2.2 and SnRK2.3, and their genes. In alternative embodiments, the invention provides plants, plant tissues and cells, having increased water use efficiency, and drought-resistant plants, plant tissues and cells; and methods for engineering of water transpiration and water use efficiency in plants, and engineering plants with increased water use efficiency and drought-resistant plants, plant tissues and cells.
Stomatal pores in the epidermis of plant leaves enable the control of plant water loss and the influx of CO2 into plants from the atmosphere. Carbon dioxide is taken up for photosynthetic carbon fixation and water is lost through the process of transpiration through the stomatal pores. Each stomate is made up of a specialized pair of cells named guard cells, which can modify the size of the stomatal pore by controlling guard cell turgor status.
An important trait in agriculture, in biotechnological applications and the production of biofuels is the water use efficiency of plants. The water use efficiency defines how well a plant can balance the loss of water through stomata with the net CO2 uptake into leaves for photosynthesis and hence its biomass accumulation. Several biotic and abiotic factors influence the state of stomatal opening thereby optimizing the water use efficiency of a plant in a given condition.
The concentration of CO2 regulates stomatal movements, where high levels of CO2 will lead to stomatal closing and low levels of CO2 will induce stomatal opening. Thus CO2 regulates CO2 influx into plants and plant water loss on a global scale.
In alternative embodiments, the invention provides methods for increasing the water use efficiency of a guard cell, a plant, plant leaf, plant organ or plant part; or increasing the rate of growth or biomass production in a plant, plant leaf, plant organ or plant part (e.g., under conditions of drought or increased atmospheric carbon dioxide); or enhancing the carbon dioxide (CO2) sensitivity of a plant, plant leaf, plant organ or plant part; or down-regulating or decreasing carbon dioxide (CO2) and/or water exchange in a guard cell of a plant, plant leaf, plant organ or plant part; comprising:
(a) in a cell of the plant, plant leaf, plant organ or plant part, or in a plant guard cell, increasing the expression and/or activity of:
(b) the method of (a), wherein the increasing of expression and/or activity of the OST1, SnRK2.2- or SnRK2.3 protein kinase is by: (1) providing a heterologous OST1-, SnRK2.2- or SnRK2.3-expressing nucleic acid (e.g., a gene or message) and expressing the gene, message and/or protein in the guard cell, plant, plant leaf, plant organ or plant part; (2) increasing of expression and/or activity of a homologous OST1 -, SnRK2.2- or SnRK2.3-expressing nucleic acid (e.g., a gene or message); or, (3) a combination of (1) and (2);
(b) the method of (a), further comprising in the cell of the plant, plant leaf, plant organ or plant part, or in the plant guard cell, increasing the expression and/or activity of a CO2 a sensor protein or a carbonic anhydrase by: (1) providing a heterologous CO2 sensor protein-expressing nucleic acid (e.g., a gene or message), or a carbonic anhydrase-expressing nucleic acid (e.g., a gene or message) and expressing the gene, message and/or protein in the guard cell, plant, plant leaf, plant organ or plant part; (2) increasing of expression and/or activity of a homologous CO2 sensor protein-expressing nucleic acid (e.g., a gene or message), or a homologous CO2 sensor protein-expressing nucleic acid (e.g., a gene or message), or a homologous OST1 carbonic anhydrase-expressing nucleic acid (e.g., a gene or message); or, (3) a combination of (1) and (2); or
(c) the method of (b), wherein the carbonic anhydrase is a β-carbonic anhydrase;
thereby increasing the water use efficiency of the guard cell, plant, plant leaf, plant organ or plant part; or increasing the rate of growth or biomass production in the plant, plant leaf, plant organ or plant part; or enhancing the carbon dioxide (CO2) sensitivity of the plant, plant leaf, plant organ or plant part; or down-regulating or decreasing carbon dioxide (CO2) and/or water exchange in the guard cell of the plant, plant leaf, plant organ or plant part.
In alternative embodiments, the invention provides methods for up-regulating or increasing carbon dioxide (CO2) and/or water exchange in a guard cell, a plant, plant leaf, plant organ or plant part; decreasing the water use efficiency of a guard cell, a plant, plant leaf, plant organ or plant part; or decreasing (desensitizing) the carbon dioxide (CO2) sensitivity of a plant, plant leaf, plant organ or plant part; or upregulating or increasing carbon dioxide (CO2) and/or water exchange in a guard cell of a plant, plant leaf, plant organ or plant part; comprising:
(a) in a cell of the plant, plant leaf, plant organ or plant part, or in a plant guard cell, decreasing the expression and/or activity of:
(b) the method of (a), wherein the decreasing of expression and/or activity of the OST1, SnRK2.2 or SnRK2.3 protein kinase is by: (1) providing a heterologous antisense or iRNA OST1, SnRK2.2 or SnRK2.3 protein kinase nucleic acid (e.g., to decrease the expression or activity of a gene or message), or any nucleic acid inhibitory to the expression or the OST1, SnRK2.6 or SnRK2.6 protein kinase; and, expressing the inhibitory nucleic acid, the antisense or the iRNA in the guard cell, plant, plant leaf, plant organ or plant part; (2) decreasing of expression and/or activity of a homologous OST1 , SnRK2.2- or SnRK2.3 kinase-expressing nucleic acid (e.g., a gene or message); or, (3) a combination of (1) and (2);
(b) the method of (a), further comprising in the cell of the plant, plant leaf, plant organ or plant part, or in the plant guard cell, decreasing the expression and/or activity of a CO2 a sensor protein or a carbonic anhydrase by: (1) providing a heterologous antisense or iRNA to a CO2 sensor protein- or a carbonic anhydrase-expressing nucleic acid (e.g., a gene or message), or any nucleic acid inhibitory to the expression of the CO2 sensor protein or the carbonic anhydrase, and expressing the inhibitory nucleic acid, the antisense or the iRNA in the guard cell, plant, plant leaf, plant organ or plant part; (2) decreasing of expression and/or activity of a homologous CO2 sensor protein-expressing nucleic acid (e.g., a gene or message) or a homologous carbonic anhydrase-expressing nucleic acid (e.g., a gene or message); or, (3) a combination of (1) and (2); or
(c) the method of (b), wherein the carbonic anhydrase is a β-carbonic anhydrase;
thereby up-regulating or increasing carbon dioxide (CO2) and/or water exchange in the guard cell, plant, plant leaf, plant organ or plant part; decreasing the water use efficiency of the guar cell, plant, plant leaf, plant organ or plant part; or increasing the rate of growth or biomass production in the plant, plant leaf, plant organ or plant part; or decreasing (desensitizing) the carbon dioxide (CO2) sensitivity of the plant, plant leaf, plant organ or plant part; or up-regulating or increasing carbon dioxide (CO2) and/or water exchange in the guard cell of the plant, plant leaf, plant organ or plant part.
In alternative embodiments of the methods, the polypeptide having carbonic anhydrase activity comprises an amino acid sequence having between about 75% to 100% sequence identity with an amino acid sequence of (comprising) SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46.
In alternative embodiments of the methods, the polypeptide having carbonic anhydrase activity is encoded by a nucleotide sequence of (comprising) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45. In alternative embodiments of the methods, the polypeptide having OST1 protein kinase activity comprises an amino acid sequence having between 75% to 100% sequence identity with an amino acid sequence of (comprising) SEQ ID NO:12 or SEQ ID NO:14; or the polypeptide having OST1 protein kinase activity is encoded by a nucleotide sequence of (comprising) SEQ ID NO:11 or SEQ ID NO:13.
In alternative embodiments of the methods, the plant is characterized by controlled CO2 exchange under ambient 365 ppm CO2, elevated ppm CO2 or reduced ppm CO2, or the plant is characterized by controlled water exchange under ambient 365 ppm CO2, elevated ppm CO2 or reduced ppm CO2.
In alternative embodiments of the methods, the CO2 sensor protein-expressing nucleic acid or gene, carbonic anhydrase-expressing nucleic acid, message or gene, and/or the protein kinase-expressing nucleic acid, message or gene, is oeprably 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.
In alternative embodiments of the methods, the up-regulating or increasing carbon dioxide (CO2) and/or water exchange in a guard cell of a plant, plant cell, plant leaf, plant organ or plant part; decreasing the water use efficiency of a guard cell, a plant, plant leaf, plant organ or plant part; or decreasing (desensitizing) the carbon dioxide (CO2) sensitivity of a plant, plant leaf, plant organ or plant part; or upregulating or increasing carbon dioxide (CO2) and/or water exchange in a guard cell or a plant, plant leaf, plant organ or plant part; comprises:
(a) providing (i) a nucleic acid inhibitory to the expression of a CO2 sensor protein-expressing nucleic acid or a CO2 sensor gene or transcript (mRNA), each encoding a polypeptide having a carbonic anhydrase (CA) activity or a β-carbonic anhydrase activity; and/or (ii) a nucleic acid inhibitory (e.g., antisense, iRNA) to the expression an and OST1, SnRK2.2- or SnRK2.3 protein kinase-expressing nucleic acid or an OST1 , SnRK2.2- or SnRK2.3 protein kinase gene or transcript;
(b) expressing the nucleic acid inhibitory to the expression of the CO2 sensor protein-expressing nucleic acid, gene or transcript (e.g., expressing an antisense, iRNA or inhibitory nucleic acid) in a guard cell; and/or, expressing a nucleic acid inhibitory to the expression of the protein kinase-expressing nucleic acid, gene or transcript,
thereby up-regulating or increasing carbon dioxide (CO2) and/or water exchange in a guard cell; decreasing the water use efficiency of a guard cell, a plant, plant leaf, plant organ or plant part; or decreasing (desensitizing) the carbon dioxide (CO2) sensitivity of a plant, plant leaf, plant organ or plant part; or upregulating or increasing carbon dioxide (CO2) and/or water exchange in a guard cell or a plant, plant leaf, plant organ or plant part.
In alternative embodiments of the methods, the nucleic acid inhibitory to the expression of a CO2 sensor protein-expressing nucleic acid comprises:
(a) a nucleotide sequence of at least about 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with a nucleotide sequence encoding a polypeptide having carbonic anhydrase activity,
the polypeptide optionally comprising an amino acid sequence having between about 75% and 100% sequence identity with an amino acid sequence of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, or
(b) a partial or complete complementary sequence of the nucleotide sequence (a).
In alternative embodiments of the methods, the nucleic acid inhibitory to the expression of a CO2 sensor protein-expressing nucleic acid comprises:
(a) a nucleotide sequence of at least about 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with a nucleotide sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45; or
(b) a partial or complete complementary sequence of the nucleotide sequence (a).
In alternative embodiments of the methods, the nucleic acid inhibitory to the expression of the polypeptide having OST1 protein kinase activity comprises:
(a) a nucleotide sequence of at least about 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with a nucleotide sequence encoding an amino acid sequence having between 75% and 100% sequence identity with amino acid sequence of SEQ ID NO:12 or SEQ ID NO: 14; or
(b) a partial or complete complementary sequence of the nucleotide sequence (a).
In alternative embodiments of the methods, the nucleic acid inhibitory to the expression of the polypeptide having OST1 protein kinase activity comprises:
(a) a nucleotide sequence of at least about 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with a nucleotide sequence of SEQ ID No.11 or SEQ ID NO:13; or
(b) a partial or complete complementary sequence of the nucleotide sequence (a).
In alternative embodiments of the methods, the nucleic acid inhibitory to the expression of a CO2 sensor protein-expressing nucleic acid comprises the nucleotide sequence of at least about 11, 12, 13, 14, 15, 16, 17, 18 or 19 or more nucleotides and a complementary sequence to the nucleotide sequence of at least about 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides.
In alternative embodiments of the methods, the nucleotide sequence comprising the at least about 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides is a nucleotide sequence comprising at least 50 or 100 or 300 nucleotides having between 75 to 100% sequence identity to the nucleotide sequence encoding a polypeptide having carbonic anhydrase activity and/or nucleotide sequence encoding a polypeptide having OST1 protein kinase activity.
In alternative embodiments of the methods, the plant is characterized by controlled CO2 exchange under ambient 365 ppm CO2, elevated ppm CO2 or reduced ppm CO2, or the plant is characterized by controlled water exchange under ambient 365 ppm CO2, elevated ppm CO2 or reduced ppm CO2.
In alternative embodiments of the methods, the CO2 sensor protein-inhibitory nucleic acid an/or the OST1 protein kinase-inhibitory nucleic acid is 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.
In alternative embodiments, the invention provides methods for regulating water exchange in a cell of a plant, plant leaf, plant organ or plant part comprising:
(a) expressing or increasing the expression of a CO2 sensor protein-encoding or a carbonic anhydrase-encoding gene or transcript, and an OST1, SnRK2.2- or SnRK2.3 protein kinase-encoding gene or transcript, by providing a CO2 sensor protein expressing and an OST1, SnRK2.2- or SnRK2.3 protein kinase nucleic acid, gene or transcript, as set forth in a composition or method of this invention, in the plant, guard cell, plant cell, plant leaf, plant organ or plant part; or
(b) decreasing the expression of a CO2 sensor protein encoding gene or transcript or a carbonic anhydrase gene or transcript and an OST1, SnRK2.2- or SnRK2.3 protein kinase-encoding gene or transcript in the plant, guard cell, plant cell, plant leaf, plant organ or plant part, by expressing a nucleic acid inhibitory to the expression of the CO2 sensor protein-expressing or carbonic anhydrase-expressing nucleic acid, gene or transcript and the OST1, SnRK2.2- or SnRK2.3 protein kinase-expressing nucleic acid, gene or transcript, as set forth in a method of the invention, in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part;
thereby regulating water exchange, wherein down-regulating or decreasing water exchange is achieved by expression or increased expression of the carbonic anhydrase or CO2 sensor protein and the protein kinase and wherein up-regulating or increasing water exchange is achieved by reduction of expression of the carbonic anhydrase or CO2 sensor protein and the protein kinase in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part.
In alternative embodiments of the methods, the increasing or decreasing of the expression is in the plant guard cell.
In alternative embodiments, the invention provides methods for regulating water uptake or water loss in a plant, plant cell, plant leaf, plant organ or plant part comprising:
(b) decreasing the expression of a CO2 sensor protein encoding gene or transcript or a carbonic anhydrase gene or transcript and an OST1, SnRK2.2- or SnRK2.3 protein kinase-encoding gene or transcript in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part, by expressing a nucleic acid inhibitory to the expression of the CO2 sensor protein-expressing or carbonic anhydrase-expressing nucleic acid, gene or transcript and the OST1, SnRK2.2- or SnRK2.3 protein kinase-expressing nucleic acid, gene or transcript, as set forth in a method of this invention, in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part;
thereby regulating water uptake or water loss, wherein down-regulating water uptake or causing water conservation is achieved by expression or increased expression of the carbonic anhydrase or CO2 sensor protein and the OST1, SnRK2.2- or SnRK2.3 protein kinase and wherein up-regulating water exchange or increasing water loss is achieved by reduction of expression of the carbonic anhydrase or CO2 sensor protein and the OST1, SnRK2.2- or SnRK2.3 protein kinase in the plant, plant cell, plant leaf, plant organ, or plant part. The increasing or decreasing of the expression can occur in the plant guard cell.
In alternative embodiments, the invention provides methods for making a plant with enhanced water use efficiency (WUE), or drought-resistant plant, plant cell, plant leaf, plant organ or plant part, comprising:
expressing or increasing the expression of a CO2 sensor protein-encoding or a carbonic anhydrase-encoding gene or transcript, and an OST1, SnRK2.2- or SnRK2.3 protein kinase-encoding gene or transcript, by providing a CO2 sensor protein expressing and an OST1, SnRK2.2- or SnRK2.3 protein kinase nucleic acid, gene or transcript, as set forth in a composition or method of this invention, in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part
thereby regulating water uptake or water loss and increasing the WUE in the plant, plant cell, plant leaf, plant organ, or plant part.
The increasing of the expression can occur in the plant guard cell.
In alternative embodiments, the invention provides methods for making a heat-resistant plant, guard cell, plant cell, plant leaf, plant organ, or plant part, comprising:
decreasing the expression of a CO2 sensor protein encoding gene or transcript or a carbonic anhydrase gene or transcript and an OST1, SnRK2.2- or SnRK2.3 protein kinase-encoding gene or transcript in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part, by expressing a nucleic acid inhibitory to the expression of the CO2 sensor protein-expressing or carbonic anhydrase-expressing nucleic acid, gene or transcript and the OST1, SnRK2.2- or SnRK2.3 protein kinase-expressing nucleic acid, gene or transcript, as set forth in a method of the invention, in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part,
thereby making a heat-resistant plant, guard cell, plant cell, plant leaf, plant organ, or plant part.
The decreasing of the expression can occur in the plant guard cell.
In alternative embodiments, the invention provides methods for opening a stomatal pore in a guard cell, plant, plant part, a plant organ, a plant leaf, or a plant cell, comprising:
decreasing the expression of a CO2 sensor protein encoding gene or transcript or a carbonic anhydrase gene or transcript and an OST1, SnRK2.2- or SnRK2.3 protein kinase-encoding gene or transcript in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part, by expressing a nucleic acid inhibitory to the expression of the CO2 sensor protein-expressing or carbonic anhydrase-expressing nucleic acid, gene or transcript and the OST1, SnRK2.2- or SnRK2.3 protein kinase-expressing nucleic acid, gene or transcript, as set forth in a method of the invention, in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part,
thereby opening a stomatal pore in the guard cell, plant, plant part, plant organ, plant leaf, or plant cell.
The decreasing of the expression can occur in the plant guard cell.
In alternative embodiments, the invention provides methods for closing a stomatal pore on a guard cell in the epidermis or a plant, a plant leaf, plant organ or a plant cell, comprising:
expressing or increasing the expression of a CO2 sensor protein-encoding or a carbonic anhydrase-encoding gene or transcript, and an OST1, SnRK2.2- or SnRK2.3 protein kinase-encoding gene or transcript, by providing a CO2 sensor protein expressing and an OST1, SnRK2.2- or SnRK2.3 protein kinase nucleic acid, gene or transcript, as set forth in a composition or method of this invention, in the plant, guard cell, plant cell, plant leaf, plant organ, or plant part
thereby closing a stomatal pore on the guard cell in the epidermis of the plant, plant leaf, plant organ or plant cell.
The expression or increase in expression can occur in the plant guard cell.
In alternative embodiments, the invention provides methods for enhancing or optimizing biomass accumulation in a plant, a plant leaf, a plant organ, a plant part, a plant cell, or seed by balancing the loss of water through stomata with the net CO2 uptake for photosynthesis, and hence enhancing or optimizing biomass accumulation in the plant, plant leaf, plant part, plant organ, plant cell, or seed, comprising opening or closing stomatal pores using a method of the invention.
In alternative embodiments, the invention provides methods for reducing leaf temperature and enhancing transpiration in a plant, a plant leave, or a plant cell, comprising opening a stomatal pore a cell or cells of the plant using a method of the invention.
In alternative embodiments, the plant is, or the guard cell, plant cell, plant part or plant organ, is isolated and/or derived from: (i) a dicotyledonous or monocotyledonous plant; (ii) wheat, oat, rye, barley, rice sorghum, maize (corn), tobacco, a legume, a lupins, potato, sugar beet, pea, bean, soybean (soy), a cruciferous plant, a cauliflower, rape (or rapa or canola), cane (sugarcane), flax, cotton, palm, sugar beet, peanut, a tree, a poplar, a lupin, a silk cotton tree, desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle, jute, or sisal abaca; or, (c) a species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Curcurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Hellanthus, Heterocallis, Hordeum, Hyascyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Man[iota]hot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, or Zea.
In alternative embodiments, the invention provides transgenic guard cells, plants, plant cells, plant tissues, plant seeds or fruits, plant parts or plant organs, comprising:
(a) (1) a heterologus OST1 protein kinase-expressing nucleic acid or an OST1 protein kinase gene or mRNA (message) encoding a polypeptide with OST1 protein kinase activity; or
(2) a heterologous protein kinase SnRK2.2- or SnRK2.3-expressing nucleic acid or an SnRK2.2- or SnRK2.2 protein kinase gene or mRNA (message) encoding a polypeptide with SnRK2.2- or SnRK2.2 protein kinase activity; or
(b) the transgenic plant cell, plant, plant part or plant organ of (a), further comprising a heterologous nucleic acid, gene or transcript encoding a protein having a carbonic anhydrase (CA) activity of a β-carbonic anhydrase activity, or encoding a CO2 sensor protein,
wherein optionally the nucleic acid, gene or transcript is 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;
and optionally the nucleic acid, gene or transcript is stably integrated into the genome of the guard cell, plant, plant cell, plant tissue, plant seed or fruit, plant part or plant organ, or is contained in an episomal vector in the guard cell, plant, plant cell, plant tissue, plant seed or fruit, plant part or plant organ.
In alternative embodiments, the invention provides transgenic guard cells, plants, plant cells, plat tissues, plant seeds or fruits, plant parts or plant organs, comprising:
(a)(1) a heterologus nucleic acid that is inhibitory to an OST1 protein kinase-expressing nucleic acid or an OST1 protein kinase gene or mRNA (message) encoding a polypeptide with OST1 protein kinase activity, or is inhibitory to the activity or the kinase; or
(2) a heterologus nucleic acid that is inhibitory to a protein kinase SnRK2.2 - or SnRK2.3-expressing nucleic acid or an SnKR2.2- or SnRK2.3 protein kinase gene or mRNA (message) encoding a polypeptide with SnRK2.2 - or SnRK2.3 protein kinase activity, or is inhibitory to the activity or the kinase; or
(b) the transgenic plant cell, plant, plant part or plant organ of (a), further comprising a heterologous nucleic acid that is inhibitory to a gene or transcript encoding a protein having a carbonic anhydrase (CA) activity or a β-carbonic anhydrase activity, or is inhibitory to a gene or transcript encoding a CO2 sensor protein,
wherein optionally the inhibitory nucleic acid is 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;
and optionally the inhibitory nucleic acid is stably integrated into the genome of the guard cell, plant, plant cell, plant tissue, plant seed or fruit, plant part or plant organ, or is contained in an episomal vector in the guard cell, plant, plant cell, plant tissue, plant seed or fruit, plant part or plant organ,
and optionally the inhibitory nucleic acid comprises an antisense RNA or an iRNA.
In alternative embodiments, the invention provides transgenic guard cells, plants, plant cells, plant tissues, plant seeds or fruits, plant parts or plant organs, comprising:
(a) a first and second recombinant gene, wherein the first recombinant gene comprises an expression-increasing recombinant first gene or an expression-inhibiting first recombinant gene, and wherein the second recombinant gene comprises an expression-increasing second recombinant gene or an expression-inhibiting second recombinant gene;
wherein the expression increasing first recombinant gene comprises:
optionally further comprising a transcription termination and polyadenylation signal;
wherein the expression-inhibiting first recombinant gene comprises the following operably linked DNA fragments:
optionally further comprising a transcription termination and polyadenylation signal;
wherein the expression-increasing second recombinant gene comprises:
optionally further comprising a transcription termination and polyadenylation signal;
wherein the expression inhibiting second recombinant gene:
optionally further comprising a transcription termination and polyadenylation signal.
In alternative embodiments, the nucleic acid (e.g., a DNA fragment) encoding a polypeptide having a carbonic anhydrase (CA) activity or a β-carbonic anhydrase activity encodes a polypeptide comprising an amino acid sequence having between 75% and 100% sequence identity with an amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46. In alternative embodiments, the polypeptide having carbonic anhydrase activity is encoded by a nucleotide sequence of (comprising) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45. In alternative embodiments, nucleic acid (e.g., DNA fragment) encoding the polypeptide with OST1, SnRK2.2- or SnRK2.3 protein kinase activity encodes a polypeptide comprising an amino acid sequence having between 75% and 100% sequence identity with an amino acid sequence of (comprising) SEQ ID NO:12 or SEQ ID NO:14. In alternative embodiments, the polypeptide having OST1 protein kinase activity is encoded by a nucleotide sequence selected from the nucleotide sequence of (comprising) SEQ ID NO:11 or SEQ ID NO:13.
In alternative embodiments, the nucleic acid (e.g., DNA fragment), which when transcribed yield an inhibitory nucleic acid (e.g., an inhibitory ribonucleic acid) to the expression of a CO2 sensor protein-expressing nucleic acid comprises a nucleotide sequence of at least 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucelotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with a nucleotide sequence encoding a polypeptide having carbonic anhydrase activity comprising an amino acid sequence having between 75% and 100% sequence identity with an amino acid sequence selected from the amino acid sequence of (comprising) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, or a complete or partial complement thereof.
In alternative embodiments, the nucleic acid (e.g., DNA fragment), which when transcribed yield a ribonucleic acid inhibitory to the expression of a CO2 sensor protein-expressing nucleic acid comprises a nucleotide sequence of at least 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides having at least 94% sequence identity with a nucleotide sequence selected from the nucleotide sequence of (comprising) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, or a complete or partial complement thereof.
In alternative embodiments, the ribonucleic acid inhibitory to the expression of a CO2 sensor protein-expressing nucleic acid comprises the nucleotide sequence at least 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucelotides and a complementary sequence to the nucleotide sequence at least 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucelotides.
In alternative embodiments, the nucleic acid (e.g., DNA fragment), which when transcribed yield a ribonucleic acid inhibitory to the expression of a OST1 kinase protein-expressing nucleic acid comprises a nucleotide sequence of at least 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with a nucleotide sequence encoding a polypeptide having OST1 protein kinase activity comprising an amino acid sequence having between 75% and 100% sequence identity with an amino acid sequence selected from the amino acid sequence of (comprising) SEQ ID NO:12 or SEQ ID NO:14, or a complete or partial complement thereof.
In alternative embodiments, the nucleic acid (e.g., DNA fragment), which when transcribed yield a ribonucleic acid inhibitory to the expression of a OST1 protein kinase encoding nucleic acid comprises a nucleotide sequence of at least 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity with a nucleotide sequence selected from the nucleotide sequence of (comprising) SEQ ID NO:11 or SEQ ID NO:13, or a complete or partial complement thereof.
In alternative embodiments, the ribonucleic acid inhibitory to the expression of a CO2 sensor protein-expressing nucleic acid comprises the nucleotide sequence of at least 19 nucleotides and a complementary sequence to the nucleotide sequence of at least 19 nucleotides.
In alternative embodiments, the first recombinant gene is an expression increasing first recombinant gene, and the second recombinant gene is an expression increasing second recombinant gene. The first recombinant gene can be an expression inhibiting first recombinant gene, and the second recombinant gene is an expression inhibiting second recombinant gene. The first recombinant gene can be an expression increasing first recombinant gene, and the second recombinant gene is an expression inhibiting second recombinant gene. The first recombinant gene can be an expression inhibiting first recombinant gene, and the second recombinant gene is an expression increasing second recombinant gene.
In alternative embodiments, the plant is or the guard cell, plant, plant cell, plant tissue, plant seed or fruit, plant part or plant organ is isolated and/or derived from: (i) a dicotyledonous or monocotyledonous plant; (ii) wheat, oat, rye, barley, rice, sorghum, maize (corn), tobacco, a legume, a lupins, potato, sugar beet, pea, bean, soybean (soy), a cruciferous plant, a cauliflower, rape (or rapa or canola), cane (sugarcane), flax, cotton, palm, sugar beet, peanut, a tree, a poplar, a lupin, a silk cotton tree, desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle, jute, or sisal abaca; or, (c) a species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Curcurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Hellanthus, Heterocallis, Hordeum, Hyascyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Man[iota]hot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, or Zea.
In alternative embodiments, the invention provides methods for altering the opening or closing of stomatal cells in a plant, plant part or plant organ, comprising providing cells of a guard cell, plant, plant cell, plant tissue, plant seed or fruit, plant part or plant organ with a first and second recombinant gene, wherein the first recombinant gene is selected from an expression increasing recombinant first gene or an expression inhibiting first recombinant gene, and wherein the second recombinant gene is selected from an expression increasing second recombinant gene or an expression inhibiting second recombinant gene as set forth in a composition or method of this invention, for
In alternative embodiments, the first recombinant gene is an expression increasing first recombinant gene, and the second recombinant gene is an expression increasing second recombinant gene. The first recombinant gene can be an expression inhibiting first recombinant gene, and the second recombinant gene is an expression inhibiting second recombinant gene. The first recombinant gene can be an expression increasing first recombinant gene, and the second recombinant gene is an expression inhibiting second recombinant gene.
In alternative embodiments, the invention provides kits comprising a compound or compounds used to practice the methods of the invention, and optionally instructions to practice a method invention.
The details of one or more 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 embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figures are described in detail herein.
Like reference symbols in the various drawings indicate like elements.
In alternative embodiments, the invention provides compositions and methods for manipulating the exchange of water and carbon dioxide (CO2) through plant stomata by controlling both CO2 sensor genes, which can be designated “CO2 Sen genes” and OST1 (Open Stomata 1, also known as SnRK2.6), SnRK2.2 or SnRK2.3 protein kinase genes (SnRK2 genes are SNF1 Related Protein Kinase Subfamily 2 genes) SNF1 is “Sucrose non-fermenting 1”). The invention provides compositions and methods for over or under-expressing CO2 sensor nucleic acids and CO2 sensor polypeptides and OST1, SnRK2.2 or SnRK2.3 protein kinase genes. The invention provides compositions and methods for over-expressing CO2 sensor nucleic acids and CO2 sensor polypeptides and OST1, SnRK2.2 or SnRK2.3 protein kinase genes, to engineer and improved CO2 response in a plant, plant part, plant organ, a leaf, and the like.
While the invention is not based on any particular mechanism of action, embodiments of the invention are based on the elucidation of the mechanism for CO2 control of gas exchange in plants. The inventors demonstrated that bicarbonate, but not elevated CO2, acts as intracellular signaling molecule to activate SLAC1-mediated anion channels. Elevated bicarbonate enhances (primes) the [Ca2+]i sensitivity of SLAC1 channel activation. The ht1-2 kinase mutant is found to enhance the HCO3
The inventors' analysis of OST1 on CO2 regulation of stomatal movements and anion channels demonstrate that the OST1 protein kinase is a major regulator of CO2-induced stomatal closing and CO2 activation of anion channels in guard cells, leading to a new model for CO2 control of gas exchange in plants and further possibilities to modulate the exchange of water and/or carbon dioxide (CO2) through plant stomata.
Over-expression of one or several CO2 sensor genes, including the CO2 sensor nucleic acids (e.g., as genes or messages or transcripts), or CO2 sensor polypeptides, and overexpression of OST1 protein kinase encoding nucleic acids (such as genes, messages or transcripts) evokes an improved CO2 response. Thus, overexpression of both CO2 sensor proteins and OST1, SnRK2.2 or SnRK2.3 protein kinase enhances WUE and produces a more efficient and drought resistant plant, particularly in light of the continuously rising atmospheric CO2 concentrations.
In alternative embodiments, the invention provides transgenic plants (including crop plants, such as a field row plants), cells, plant tissues, seeds and organs, and the like, (which in alternative embodiments express one or more recombinant nucleic acids encoding all or one of the CO2Sen proteins, and all or one of the OST1, SnRK2.2- or SnRK2.3 protein kinases) which can close their stomata to a greater extent that wild-type plants, thereby preserving their water usage. Because water use efficiency defines how well a plant can balance the loss of water through stomata with the net CO2 uptake for photosynthesis, and hence its biomass accumulation, the compositions and methods of the invention can also be used to increase a plant's biomass, and thus the compositions and methods of the invention have applications in the biofuels/alternative energy area.
In alternative embodiments, the invention also provides compositions and methods for inhibiting the expression of CO2Sens genes, transcripts and CO2Sensor proteins and of OST1, SnRK2.2- or SnRK2.3 protein kinase genes, transcripts and CO2Sensor proteins using e.g. inhibitory RNA mediated repression (including antisense RNA, co-suppression RNA, siRNA, microRNA, double-stranded RNA, hairpin RNA and/or RNAi) of the expression of CO2 sensors and OST1, SnRK2.2- or SnRK2.3 protein kinase in cells, such as guard cells, in any plant including agricultural crops.
In alternative embodiments, the invention provides transgenic plants which have a lower expression of CO2sens proteins and OST1, SnRK2.2- or SnRK2.3 protein kinases (CO2sensor and OST1, SnRK2.2- or SnRK2.3-under-expressing plants) and can open their stomata to a greater extent than wild-type plants.
In alternative embodiments, the invention provides plants, plant cells, plant organs and the like, e.g., agriculture crops, that can withstand increased temperatures—thus preventing a “breakdown” of metabolism, photosynthesis and growth. Thus, compositions and methods of this invention, by inhibiting both the expression of CO2Sensor nucleic acids and/or CO2Sens proteins as well as expression of OST1, SnRK2.2- or SnRK2.3 protein kinase, help crops that otherwise would be sensitive to elevated temperatures to cope with the increased atmospheric CO2 concentrations, also reducing or ameliorating an accelerated increase in leaf temperatures.
In alternative embodiments, the invention provides compositions and methods comprising inhibitory RNA (including antisense and RNAi) for repression of CO2 sensors and OST1, SnRK2.2- or SnRK2.3 protein kinase expression in guard cells to reduce leaf temperature though enhancing transpiration in these crops and also to maximize crop yields.
In alternative embodiments, the invention provides compositions and methods for down-regulating/decreasing or alternatively increasing carbon dioxide (CO2) and/or water exchange in a plant, e.g., through the guard cell of a plant, plant cell, plant leaf, plant organ or plant part comprising inter alia use of a polypeptide having carbonic anhydrase, and an OST1, SnRK2.2- or SnRK2.3 protein kinase.
While the invention is not based on any particular mechanism of action, embodiments of compositions and methods of the invention are based on regulation of the opening or closing of stomata, including regulation of the efficiency of the exchange of water and CO2 through stomata can further be modulate or balanced in a more controlled way by controlling CO2 sensor and OST1, SnRK2.2- or SnRK2.3 protein kinase genes and/or transcripts thereby expressing or increasing the expression of CO2 sensor genes and/or transcripts and simultaneously decreasing the expression of OST1, SnRK2.2- or SnRK2.3 protein kinase genes and/or transcripts or inversely by decreasing the expression of CO2 sensor genes and/or transcripts and simultaneously expressing or increasing the expression of OST1, SnRK2.2- or SnRK2.3 protein kinase genes and/or transcripts.
In alternative embodiments, the invention provides methods for down-regulating or decreasing carbon dioxide (CO2) and/or water exchange in a guard cell of a plant, plant cell, plant leaf, plant organ or plant part comprising expressing in a cell a polypeptide having a carbonic anhydrase (carbonate dehydratase) activity, or a β-carbonic anhydrase activity in combination with a polypeptide having OST1, SnRK2.2- or SnRK2.3 protein kinase activity.
In alternative embodiments, any carbonic anhydrase (carbonate dehydratase) can be used, e.g., including plant or bacterial carbonic anhydrase (carbonate dehydratase) enzymes. Exemplary carbonic anhydrase (carbonate dehydratase) enzymes that can be used to practice this invention include carbonic anhydrase (carbonate dehydratase) enzymes isolated or derived from:
Oryza sativa (japonica cultivar-group) Osl2g0153500 (Osl2g0153500) mRNA, complete
Oryza sativa (japonica cultivar-group) Os1lgO153200 (Os1lgO153200) mRNA,
Oryza sativa (japonica cultivar-group) Os09g0464000 (Os09g0464000) mRNA, complete
Oryza sativa (japonica cultivar-group) Os09g0454000 (Os09g0454500) mRNA, complete
Oryza sativa (japonica cultivar-group) Os08g0470200 (Os08g0470200) mRNA, complete
Oryza sativa (japonica cultivar-group) Os08g0423500 (Os08g0423500) mRNA, complete
Oryza sativa (japonica cultivar-group) Os06g0610100 (Os06g0610100) mRNA, complete
Oryza sativa (japonica cultivar-group) Os02g0533300 (Os02g0533300) mRNA, complete
Oryza sativa (japonica cultivar-group) Os01g0640000 (Os01g0640000) mRNA, complete
Oryza sativa (japonica cultivar-group) Os01g0639900 (OsO1g0639900) mRNA, partial
Oryza sativa (indica cultivar-group) clone OSS-385-480-G10 carbonic anhydrase mRNA,
Oryza sativa carbonic anhydrase 3 mRNA, complete cds
Oryza sativa chloroplast carbonic anhydrase mRNA, complete cds
Zea mays carbonic anhydrase (LOC542302), mRNA
Zea mays Golden Bantam carbonic anhydrase mRNA, complete cds
Zea mays carbonic anhydrase mRNA, complete cds
Zea mays putative carbonic anhydrase homolog mRNA, partial cds gi|168561|
Glycine max mRNA for carbonic anhydrase
Lycopersicon esculentum mRNA for chloroplast carbonic anhydrase (ca2 gene)
Lycopersicon esculentum mRNA for carbonic anhydrase (ca1 gene)
Nicotiana langsdorffu × Nicotiana sanderae neclarin III (NEC3) mRNA,
Nicotiana tabacum beta-carbonic anhydrase (CA) mRNA, complete cds; nuclear gene for
Nicotiana tabacum mRNA for carbonic anhydrase, partial cds
Nicotiana paniculata mRNA for NPCA1, complete cds
Nicotiana tabacum chloroplastic carbonic anhydrase mRNA, 3′ end
Nicotiana tabacum chloroplast carbonic anhydrase gene, complete cds
Nicotiana benthamiana clone 30F62 chloroplast carbonic anhydrase mRNA, partial cds;
Nicotiana benthamiana clone 30C84 chloroplast carbonic anhydrase mRNA, partial cds;
Nicotiana benthamiana clone 3 OB 10 chloroplast carbonic anhydrase mRNA, partial cds;
Hordeum vulgare carbonic anhydrase mRNA, complete cds
Gossypium hirsutum carbonic anhydrase isoform 2 (CA2)
Gossypium hirsutum carbonic anhydrase isoform 1 (CA1) mRNA, partial cds; nuclear
Populus tremula × Populus tremuloides carbonic anhydrase (CA1a) mRNA, nuclear gene
Populus tremula × Populus tremuloides carbonic anhydrase (CA1b) mRNA, nuclear gene
Cucumis
Cucumis sativus clone CU8F3 carbonic anhydrase mRNA, partial cds
Medicago
M. sativa mRNA for carbonic anhydrase
Phaseolus
Phaseolus vulgaris partial mRNA for carbonic anhydrase (ca gene)
Pisum
P. sativum carbonic anhydrase mRNA, complete cds
Pyrus
Pyrus pyrifolia strain Whangkeumbae carbonic anhydrase isoform 1 (Cal1)
Prunus
Prunus dulcis clone Pdbes-E45 putative carbonic anhydrase mRNA, partial cds
Vigna
Vigna radiata carbonic anhydrase (CipCal) mRNA, complete cds; nuclear gene for
In alternative embodiments, carbonic anhydrase encoding nucleic acids from any carbonic anhydrase gene, e.g., including plant and bacterial genes, can be used to practice this invention; for example, a nucleic acid from any carbonic anhydrase gene of any plant can be used, including any carbonic anhydrase-encoding nucleic acid sequence from any gene family of Arabidopsis, e.g., any carbonic anhydrase-encoding nucleic acid sequence from an Arabidopsis family, e.g., from Arabidopsis thaliana, can be used to practice the compositions and methods of this invention, such as the nucleic acid sequences encoding a polypeptide having the amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46. Such nucleotide sequences include the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.
In alternative embodiments, carbonic anhydrases encoding nucleic acids may be used having between 75% and 100% sequence identity to any of the nucleotide sequences above, which include those having at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % sequence identity to a nucleotide sequence encoding an amino acid sequence of any of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, such as a nucleotide sequence having 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.
In alternative embodiments, OST1, SnRK2.2- or SnRK2.3 protein kinase encoding genes include genes encoding a polypeptide with OST1 protein kinase activity having between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 12 or SEQ ID 14 including those having 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:14. such nucleotide sequences may have 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of SEQ ID 11 or 13.
In alternative embodiments, compositions and methods of the invention comprise combinations, wherein the carbonic anhydrase can be either a β carbonic anhydrase 4 or a β carbonic anhydrase 1. In alternative embodiments, alternative (exemplary) combinations are:
i) Expressing, increasing the expression, upregulating a polypeptide with β carbonic anhydrase activity having an amino acid sequence sharing between 75% and 100% sequence identity to an amino acid of SEQ ID 8 (CA1) and expressing, increasing the expression or upregulating a polypeptide with OST1 protein kinase activity sharing between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 12 (OST1.1)
ii) Expressing, increasing the expression, upregulating a polypeptide with β carbonic anhydrase activity having an amino acid sequence sharing between 75% and 100% sequence identity to an amino acid of SEQ ID 8 (CA1) and expressing, increasing the expression or upregulating a polypeptide with OST1 protein kinase activity sharing between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 14 (OST1.2)
iii) Expressing, increasing the expression, upregulating a polypeptide with β carbonic anhydrase activity having an amino acid sequence sharing between 75% and 100% sequence identity to an amino acid of SEQ ID 3 (CA4) and expressing, increasing the expression or upregulating a polypeptide with OST1 protein kinase activity sharing between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 12 (OST1.1)
iv) Expressing, increasing the expression, upregulating a polypeptide with β carbonic anhydrase activity having an amino acid sequence sharing between 75% and 100% sequence identity to an amino acid of SEQ ID 3 (CA4) and expressing, increasing the expression or upregulating a polypeptide with OST1 protein kinase activity sharing between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 14 (OST1.1)
v) Expressing, increasing the expression, upregulating the expression of CA1 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 7 (CA1) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 11 (OST1.1)
vi) Expressing, increasing the expression, upregulating the expression of CA1 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 7 (CA1) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 13 (OST1.2)
vii) Expressing, increasing the expression, upregulating the expression of CA4 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 1 (CA4) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 11 (OST1.1)
viii) Expressing, increasing the expression, upregulating the expression of CA4 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 1 (CA4) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 13 (OST1.2)
ix) Expressing, increasing the expression, upregulating the expression of CA4 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 2 (CA4) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 11 (OST1.1)
x) Expressing, increasing the expression, upregulating the expression of CA4 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 2 (CA4) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 13 (OST1.2)
xi) Reducing or downregulating the expression of a polypeptide with β carbonic anhydrase activity having an amino acid sequence sharing between 75% and 100% sequence identity to an amino acid of SEQ ID 8 (CA1) and expressing, increasing the expression or upregulating a polypeptide with OST1 protein kinase activity sharing between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 12 (OST1.1)
xii) Reducing or downregulating the expression of a polypeptide with β carbonic anhydrase activity having an amino acid sequence sharing between 75% and 100% sequence identity to an amino acid of SEQ ID 8 (CA1) and expressing, increasing the expression or upregulating a polypeptide with OST1 protein kinase activity sharing between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 14 (OST1.2)
xiii) Reducing or downregulating the expression of a polypeptide with β carbonic anhydrase activity having an amino acid sequence sharing between 75% and 100% sequence identity to an amino acid of SEQ ID 3 (CA4) and expressing, increasing the expression or upregulating a polypeptide with OST1 protein kinase activity sharing between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 12 (OST1.1)
xiv) Reducing or downregulating the expression of a polypeptide with β carbonic anhydrase activity having an amino acid sequence sharing between 75% and 100% sequence identity to an amino acid of SEQ ID 3 (CA4) and expressing, increasing the expression or upregulating a polypeptide with OST1 protein kinase activity sharing between 75% and 100% sequence identity to the amino acid sequence of SEQ ID 14 (OST1.2)
xv) Reducing or downregulating the expression of a CA1 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 7 (CA1) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 11 (OST1.1)
xvi) Reducing or downregulating the expression of a CA1 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 7 (CA1) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 13 (OST1.2)
xvii) Reducing or downregulating the expression of a CA4 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 1 (CA4) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 11 (OST1.1)
xviii) Reducing or downregulating the expression of a CA4 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 1 (CA4) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 13 (OST1.2)
xix) Reducing or downregulating the expression of a CA4 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 2 (CA4) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 11 (OST1.1)
xx) Reducing or downregulating the expression of a CA4 nucleotide sequence having between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 2 (CA4) and expressing, increasing the expression or upregulating the expression of OST1 protein kinase nucleotide sequence sharing between 75% and 100% sequence identity to an nucleotide sequence of SEQ ID 13 (OST1.2)
In alternative embodiments, the invention provides combinations between upregulating one protein and downregulating the expression of another protein, e.g., as set forth in the above paragraphs i) to xx), which can be made as described herein.
In alternative embodiments, expression or upregulating of the expression of a protein can be achieved by introduction (e.g., through transformation or crossing with a transgenic plant) or a recombinant gene comprising one, several or all of the following operably linked fragments
In alternative embodiments, nucleic acids, protein coding sequences or genes used to practice the invention is oeprably 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 the invention 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, nucleic acids, 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 sequences in accordance with the invention 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 the invention, e.g., in a trangenic 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 2A1 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 Mol. 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 PTA13 (e.g., see U.S. Pat. No. 5,792,929), promoters active in vascular tissue (e.g., see Ringli and Keller (1998) Plant Mol. Biol. 37:977-988), flower-specific (e.g., see Kaiser et al. (1995) Plant Mol. Biol. 28:231-243), pollen (e.g., see Baerson et al. (1994) Plant Mol. Biol. 26:1947-1959), carpels (e.g., see Ohl et al. (1990) Plant Cell 2:, pollen and ovules (e.g., see Baerson et al. (1993) Plant Mol. Biol. 22:255-267), auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant Mol. Biol. 39: 979-990 or Baumann et al., (1999) Plant Cell 11:323-334), cytokinin-inducible promoter (e.g., see Guevara-Garcia (1998) Plant Mol. Biol. 38:743-753), promoters responsive to gibberellin (e.g., see Shi et al. (1998) Plant Mol. 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 this invention are those that elicit expression in response to heat (e.g., see Ainley et al. (1993) Plant Mol. 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 (1989) Plant Cell 1:961-968); pathogens (such as the PR-I promoter described in Buchel et al. (1999) Plant Mol. Biol. 40: 387-396, and the PDF 1.2 promoter described in Manners et al. (1998) Plant Mol. Biol. 38: 1071-1080), and chemicals such as methyl jasmonate or salicylic acid (e.g., see Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. 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 AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristem promoter elF4. Tissue specific promoters which are active throughout the life cycle of a particular tissue can be used. In one aspect, the nucleic acids of the invention are operably linked to a promoter active primarily only in cotton fiber cells, in one aspect, the nucleic acids of the invention 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 nucleic acids can be operably linked to the Fb12A gene promoter to be preferentially expressed in cotton fiber cells (Ibid). See also, John (1997) Proc. Natl. Acad. Sci. USA 89:5769-5775; 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 the nucleic acids of the invention. 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 the nucleic acids of the invention 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) Mol. 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 Mol. 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 which are inducible upon exposure to plant hormones, such as auxims, are used to express the nucleic acids used to practice the invention. For example, the invention can use the auxin-response elements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxim-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) Mol. 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, nucleic acids used to practice the invention 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 of the invention can be induced at a particular stage of development of the plant.
In alternative embodiments, the invention also provides for transgenic plants containing an inducible gene encoding for polypeptides used to practice the invention 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, downregulation of CO2sensor genes or OST1, SnRK2.2 or SnRK2.3 genes or transcripts can be achieved by introduction of a recombinant gene expressing inhibitory RNA targeted towards CO2sensor genes or OST1, either separately or together.
In alternative embodiments, the invention provides an antisense inhibitory molecules comprising a sequence used to practice this invention (which include both sense and antisense strands), e.g., which target CO2sensor genes or OST1, SnRK2.2 or SnRK2.3 genes or transcripts. Naturally occurring or synthetic nucleic acids can be used as antisense oligonucleotides. The antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening. The antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl)glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J. 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholino carbamate nucleic acids, as described above.
In one aspect, the invention provides an RNA inhibitory molecule, a so-called “RNAi” molecule, comprising a sequence used to practice this invention. In alternative embodiments, the RNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAi molecule can comprise a double-stranded RNA (dsRNA) molecule, e.g., siRNA, miRNA (microRNA) and/or short hairpin RNA (shRNA)molecules. The RNAi molecule, e.g., siRNA (small inhibitory RNA) can inhibit expression of a CO2Sen genes or OST1 genes, and/or miRNA (micro RNA) to inhibit translation of a CO2Sen genes or OST1 genes.
In alternative aspects, the RNAi is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. While the invention is not limited by any particular mechanism of action, the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). A possible basic mechanism behind RNAi, e.g., siRNA for inhibiting transcription and/or miRNA to inhibit translation, is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In one aspect, the RNAi's of the invention are used in gene-silencing therapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one aspect, the invention provides methods to selectively degrade RNA using the RNAi's of the invention. The process may be practiced in vitro, ex vivo or in vivo. In one aspect, the RNAi molecules of the invention can be used to generate a loss-of-function mutation in a cell, an plant tissue or organ or seed, or a plant.
In alternative embodiments, intracellular introduction of the RNAi (e.g., miRNA or siRNA) is by internalization of a target cell specific ligand bonded to an RNA binding protein comprising and RNAi (e.g., microRNA) is adsorbed. The ligand is specific to a unique target cell surface antigen. The ligand can be spontaneously internalized after binding to the cell surface antigen. If the unique cell surface antigen is not naturally internalized after binding to its ligand, internalization can be promoted by the incorporation of an arginine-rich peptide, or other membrane permeable peptide, into the structure of the ligand or RNA binding protein or attachment of such a peptide to the ligand or RNA binding protein. See, e.g., U.S. Patent App. Pub. Nos. 20060030003; 20060025361; 20060019286; 20060019258. In one aspect, the invention provides lipid-based formulations for delivering, e.g., introducing nucleic acids of the invention as nucleic acid-lipid particles comprising and RNAi molecule to a cell, see e.g., U.S. Patent App. Pub. No. 20060008910.
In alternative embodiments, methods for making and using RNAi molecules, e.g., siRNA and/or miRNA, for selectively degrade RNA include, e.g., U.S. Pat. No. 6,506,559; 6,511,824; 6,515,109; 6,489,127.
In alternative embodiments, known and routine methods for making expression constructs, e.g., vectors or plasmids, from which an inhibitory polynucleotide (e.g., a duplex siRNA of the invention) is transcribed are used. A regulatory region (e.g., promoter, enhancer, silencer, splice donor, acceptor, etc.) can be used to transcribe an RNA strand or RNA strands of an inhibitory polynucleotide from an expression construct. When making a duplex siRNA (e.g., to a CO2Sen gene, or OST1, SnRK2.2 or SnRK2.3 gene) inhibitory molecule, the sense and antisense strands of the targeted portion of the targeted IRES can be transcribed as two separate RNA strands that will anneal together, or as a single RNA strand that will form a hairpin loop and anneal with itself.
For example, in alternative embodiments, a construct targeting a portion of a CO2Sen gene or OST1, SnRK2.2 or SnRK2.3 gene is inserted between two promoters (e.g., two plant, viral, bacteriophage T7 or other promoters) such that transcription occurs bidirectionally and will result in complementary RNA strands that may subsequently anneal to form an inhibitory siRNA of the invention. Alternatively, a targeted portion of a CO2Sen gene or OST1, SnRK2.2 or SnRK2.3 can be designed as a first and second coding region together on a single expression vector, wherein the first coding region of the targeted gene is in sense orientation relative to its controlling promoter, and wherein the second coding region of the gene is in antisense orientation relative to its controlling promoter. If transcription of the sense and antisense coding regions of the targeted portion of the targeted gene occurs from two separate promoters, the result may be two separate RNA strands that may subsequently anneal to form a gene or inhibitory siRNA, e.g., a CO2Sen gene-or OST1, SnRK2.2 or SnRK2.3 gene inhibitory siRNA used to practice the invention.
In alternative embodiments, transcription of the sense and antisense targeted portion of the targeted nucleic acid, e.g., a CO2Sen gene, or OST1, SnRK2.2 or SnRK2.3 gene, is controlled by a single promoter, and the resulting transcript will be a single hairpin RNA strand that is self-complementary, e.g., forms a duplex by folding back on itself to create a (e.g., CO2Sen gene, or OST1, SnRK2.2 or SnRK2.3 gene)-inhibitory siRNA molecule. In this configuration, a spacer, e.g., of nucleotides, between the sense and antisense coding regions of the targeted portion of the targeted (e.g., CO2Sen gene-or OST1, SnRK2.2 or SnRK2.3) gene can improve the ability of the single strand RNA to form a hairpin loop, wherein the hairpin loop comprises the spacer. In one embodiment, the spacer comprises a length of nucleotides of between about 5 to 50 nucleotides. In one aspect, the sense and antisense coding regions of the siRNA can each be on a separate expression vector and under the control of its own promoter.
In alternative embodiments, the invention provides ribozymes capable of binding CO2 sensor and/or OST1, SnRK2.2 or SnRK2.3 coding sequence, gene or message. These ribozymes can inhibit gene activity by e.g., targeting mRNA.
Strategies for designing ribozymes and selecting the gene specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the reagents and sequences used to practice this invention.
Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it can be released from that RNA to bind and cleave new targets repeatedly
In alternative embodiments, the invention provides transgenic plants, plant parts, plant organs or tissue, and seeds comprising nucleic acids, polypeptides, expression cassettes or vectors or a transfected or transformed cell of the invention. The invention also provides plant products, e.g., seeds, leaves, extracts and the like, comprising a nucleic acid and/or a polypeptide according to the invention. In alternative embodiments, the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). The invention also provides methods of making and using these transgenic plants and seeds. The transgenic plant or plant cell expressing a polypeptide of the present invention 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 the invention 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 CO2Sen protein production is regulated by endogenous transcriptional or translational control elements, or by a heterologous promoter, e.g., a promoter of this invention. The invention also provides “knockout plants” where insertion of gene sequence by, e.g., homologous recombination, has disrupted the expression of the endogenous gene. Means to generate “knockout” plants are well-known in the art.
The nucleic acids and polypeptides used to practice the invention can be expressed in or inserted in any plant, plant part, plant cell or seed. Transgenic plants of the invention, or a plant or plant cell comprising a nucleic acid used to practice this invention (e.g., a transfected, infected or transformed cell) can be dicotyledonous or monocotyledonous. Examples of monocots comprising a nucleic acid of this invention, e.g., as monocot transgenic plants of the invention, 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 a nucleic acid of this invention, e.g., as dicot transgenic plants of the invention, 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 a nucleic acid of this invention, including the transgenic plants and seeds of the invention, 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, Curcurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Hellanthus, Heterocallis, Hordeum, Hyascyamus, 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, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, or Zea.
The nucleic acids and polypeptides used to practice this invention 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.
In alternative embodiments, the invention provides transgenic plants, plant cells, organs, seeds or tissues, comprising and expressing the nucleic acids used to practice this invention, e.g., CO2Sen gene and proteins and OST1, SnRK2.2 or SnRK2.3 genes; for example, the invention provides plants, e.g., transgenic plants, plant cells, organs, seeds or tissues that show improved growth under limiting water conditions; thus, the invention provides drought-tolerant plants, plant cells, organs, seeds or tissues (e.g., crops).
A transgenic plant of this invention can also include the machinery necessary for expressing or altering the activity of a 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) incorporating the polynucleotides of the invention and/or expressing the polypeptides of the invention 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, of the invention, standard techniques can be used to introduce the 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 dictoyledonous 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, the invention uses Agrobacterium tumefaciens mediated transformation. Transformation means introducing 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; 5,589615; 5,750871; 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 are identified. The modified trait can by 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 of the invention 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, the invention also provides “knockout plants” where insertion of gene sequence by, e.g., homologous recombination, has disrupted the expression of the endogenous gene. 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 the invention 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, e.g., Christou (1997) Plant Mol. biol. 35:197-203; Pawlowski (1996) Mol. 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 acids, 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 the nucleic acids of the invention leads to phenotypic changes, plants comprising the recombinant nucleic acids of the invention can be sexually crossed with a second plant to obtain a final product. Thus, the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a plant of the invention and another plant. The desired effects (e.g., expression of the polypeptides of the invention to produce a plant in which flowering behavior is altered) can be enhanced when both parental plants express the polypeptides, e.g., a CO2 sensor and OST1, SnRK2.2 or SnRK2.3 gene of the invention. The desired effects can be passed to future plant generations by standard propagation means.
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.
The following non-limiting Example demonstrates that genes and proteins of a CO2 signaling pathway and the use of CO2 sensor genes and OST1, SnRK2.2 or SnRK2.3 protein kinase genes can modulate stomatal movement.
Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols 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 et al. (2000) PCR—Basics: From Background to Bench, First Edition, Spring Verlag, Germany.
Throughout the description and Examples, reference is made to the following sequences:
SEQ ID NO:1: nucleotide sequence of β carbonic anhydrase 4 (CA4) from Arabidopsis thaliana (At1g70410)
SEQ ID NO:2: nucleotide sequence of β carbonic anhydrase 4 (CA4) from Arabidopsis thaliana—coding sequence.
SEQ ID NO:3: nucleotide sequence of β carbonic anhydrase 4 (CA4) from Arabidopsis thaliana.
SEQ ID NO:4: nucleotide sequence of β carbonic anhydrase 6 (CA6) from Arabidopsis thaliana (At1g58180)
SEQ ID NO:5: nucleotide sequence of β carbonic anhydrase 6 (CA6) from Arabidopsis thaliana—coding sequence.
SEQ ID NO:6: nucleotide sequence of β carbonic anhydrase 6 (CA6) from Arabidopsis thaliana.
SEQ ID NO:7: nucleotide sequence of β carbonic anhydrase 1 (CA1) from Arabidopsis thaliana—variant 1
SEQ ID NO:8: nucleotide sequence of β carbonic anhydrase 1 (CA1) from Arabidopsis thaliana—variant 1
SEQ ID NO:9: nucleotide sequence of β carbonic anhydrase 1 (CA1) from Arabidopsis thaliana—variant 2
SEQ ID NO:10: nucleotide sequence of β carbonic anhydrase 1 (CA1) from Arabidopsis thaliana—variant 2
SEQ ID NO:11: nucleotide sequence of OST1 protein kinase cDNA from Arabidopsis thaliana —variant 1
SEQ ID NO:12: amino acid sequence of OST1 protein kinase cDNA from Arabidopsis thaliana —variant 1
SEQ ID NO:13: nucleotide sequence of OST1 protein kinase cDNA from Arabidopsis thaliana —variant 2
SEQ ID NO:14: amino acid sequence of OST1 protein kinase cDNA from Arabidopsis thaliana —variant 2
SEQ ID NO:15: nucleotide sequence of A. thaliana β carbonic anhydrase 2 (CA2) cDNA (At5g14740)
SEQ ID NO:16: amino acid sequence of A. thaliana β carbonic anhydrase 2 (CA2) cDNA (At5g14740)
SEQ ID NO:17: nucleotide sequence of A. thaliana α carbonic anhydrase 1 (CA1) cDNA (At3g52720)
SEQ ID NO:18: nucleotide sequence of A. thaliana α carbonic anhydrase 1 (CA1) cDNA (At3g52720)
SEQ ID NO:19: nucleotide sequence of A. thaliana α carbonic anhydrase 2 (CA2) cDNA (At2g28210)
SEQ ID NO:20: amino acid sequence of A. thaliana α carbonic anhydrase 1 (CA1) cDNA (At3g52720)
SEQ ID NO:21: nucleotide sequence of A. thaliana α carbonic anhydrase 3 (CA3) cDNA (At5g04180)
SEQ ID NO:22: amino acid sequence of A. thaliana α carbonic anhydrase 3 (CA3) cDNA (At5g04180)
SEQ ID NO:23: nucleotide sequence of A. thaliana α carbonic anhydrase 4 (CA4) cDNA (At4g20990)
SEQ ID NO:24: amino acid sequence of A. thaliana α carbonic anhydrase 2 (CA4) cDNA (At4g20990)
SEQ ID NO:25: nucleotide sequence of A. thaliana α carbonic anhydrase 5 (CA5) cDNA (At1g08065)
SEQ ID NO:26: amino acid sequence of A. thaliana α carbonic anhydrase 5 (CA5) cDNA (At1g08065)
SEQ ID NO:27: nucleotide sequence of A. thaliana α carbonic anhydrase 6 (CA6) cDNA (At4g21000)
SEQ ID NO:28: amino acid sequence of A. thaliana α carbonic anhydrase 6 (CA6) cDNA (At4g21000)
SEQ ID NO:29: nucleotide sequence of A. thaliana α carbonic anhydrase 7 (CA7) cDNA (At1g08080)
SEQ ID NO:30: amino acid sequence of A. thaliana α carbonic anhydrase 7 (CA7) cDNA (At1g08080)
SEQ ID NO:31: nucleotide sequence of A. thaliana α carbonic anhydrase 8 (CA8) cDNA (At5g56330)
SEQ ID NO:32: amino acid sequence of A. thaliana α carbonic anhydrase 8 (CA8) cDNA (At5g56330)
SEQ ID NO:33: nucleotide sequence of A. thaliana β carbonic anhydrase 3 (CA3) cDNA (At1g23730)
SEQ ID NO:34: amino acid sequence of A. thaliana β carbonic anhydrase 3 (CA3) cDNA (At1g23730)
SEQ ID NO:35: nucleotide sequence of A. thaliana β carbonic anhydrase 5 (CA5) cDNA (At4g33580)
SEQ ID NO:36: amino acid sequence of A. thaliana β carbonic anhydrase 5 (CA5) cDNA (At4g33580)
SEQ ID NO:37: nucleotide sequence of A. thaliana γ carbonic anhydrase 1 (CA1) cDNA (At1g19580)
SEQ ID NO:38: amino acid sequence of A. thaliana γ carbonic anhydrase 1 (CA1) cDNA (At1g19580)
SEQ ID NO:39: nucleotide sequence of A. thaliana γ carbonic anhydrase 2 (CA2) cDNA (At1g47260)
SEQ ID NO:40: amino acid sequence of A. thaliana γ carbonic anhydrase 2 (CA2) cDNA (At1g47260)
SEQ ID NO:41: nucleotide sequence of A. thaliana γ carbonic anhydrase 3 (CA3) cDNA (At5g66510)
SEQ ID NO:42: amino acid sequence of A. thaliana γ carbonic anhydrase 3 (CA3) cDNA (At5g66510)
SEQ ID NO:43: nucleotide sequence of A. thaliana γ carbonic anhydrase like 1 (CAL1) cDNA (At5g63510)
SEQ ID NO:44: amino acid sequence of A. thaliana γ carbonic anhydrase like 1 (CAL1) cDNA (At5g63510)
SEQ ID NO:45: nucleotide sequence of A. thaliana γ carbonic anhydrase2 (CAL2) cDNA (At3g48680)
SEQ ID NO:46: amino acid sequence of A. thaliana γ carbonic anhydrase 2 (CAL2) (At3g48680)
The Arabidopsis mutant lines analyzed in this study were ca1;ca4 (Hu et al, 2010), Slac1-1, slac1-3 (Vahisalu et al, 2008), ht1-2 (Hashimoto et al, 2006), ost1-1, ost1-2 (Mustilli et al, 2002), ost1-3 (Yoshida et al, 2002), abi1-1, abi2-1 and pyr1;pyl1; pyl2;pyl4 in the backcrossed Columbia background (Nishimura et al, 2010), Plants were grown in a plant growth chamber at 21° C. temperature, 65%-85% humidity, except that abi1-1 and abi2-1 were grown constantly at 75-85% humidity and a 16-h-light/8-h-dark photoperiod regime at ˜75 μmol m−2s−1.
Arabidopsis guard cell protoplasts were isolated as described previously (Siegel et al, 2009). Whole-cell patch-clamp experiments were performed as described previously (Pei et al, 1997). During recordings of S-type anion currents, the membrane voltage was stepped to potentials starting at±35 mV to −145 mV for 7 s with −30 mV decrements and the holding potential was +30 mV. The interpulse period was 5 s. Liquid junction potentials (LJP) were determined using Clampex 10.0. No leak subtraction was applied for all current-voltage curves. Steady-state currents were the average currents during the last 500 ms of pulses. Detail contents of solutions are discussed, below (see “supplementary data”). Bicarbonate (CsHCO3) was freshly dissolved in the pipette solution before patch clamp experiments and pH was adjusted to the indicated values. The pipette solution was stored using air-tight precision glass syringes during patch clamp experiments to slow CO2 equilibration with the surrounding air and was not stored overnight. The concentrations of free CO2 and bicarbonate in solutions were calculated using the Henderson-Hasselbalch equation (pH=pK1+log [HCO3−]/[CO2]) (Hauser et al, 1995). [HCO3−] represents the free bicarbonate concentration; [CO2] represents the free CO2 concentration. A value, pK1=6.352, was used for calculations (Speight, 2005). To independently measure CO2 concentrations in the solutions at different pH values, an InPro 5000 CO2 sensor (Mettler Tolego 400, Mettler-Toledo Inc, USA) was used for dissolved CO2. The InPro 5000 sensor employs a gas permeable silicone membrane. The significance of differences between data sets was assessed by noncoupled double-tailed Student's t-test analysis. Values of P<0.05 were considered statistically significant.
The Pt-GFP cDNA was amplified with the primers PGF (5′-AACCATGGCGCAGACCTTCCTCTAT-3′, with NcoI site) and PGR (5′-AACTGCAGAGGCGTCTCGCATATCTC-′, with PstI site) from the construct pART7-PrGFP (Schulte et al, 2006), kindly provided by Dr. Christoph Plieth. The sequenced PCR product was digested with NcoI and PstI and then subcloned into the binary expression vector pGreenII 0179-pGCP(D1)-terminator under the control of guard cell specific promoter pGC1 (Yang et al, 2008). The construct pGC1::PtGFP was transformed to the Agrobacterium strain GV3101 containing helper plasmid pSOUP and then was introduced into Arabidopsis (Col-0) by the floral dip method (Clough & Bent, 1998).
Fluorescence imaging was performed with a TE300 inverted microscope using a TE-FM Epi-Fluorescence attachment (Nikon) as previously described (Allen et al, 2000). Fluorescence images at excitation wavelengths of 470 nm and 440 nm were taken every 2 s using light from a 75-Watt xenon short arc lamp (Osram, Germany). 32° neutral density filters were used to reduce bleaching of fluorescent reporter. Metafluor software (MDS, Inc.) was used to control filter wheels, shutter and COOLSNAP™ (CoolSNAP) CCD camera from Photomerics when recording and also processing raw data. The fluorescence ratio F470/F440 of Pt-GFP was analyzed as a detection of pH shifts (Schulte et al, 2006). Intact epidermes from pGC1::PtGFP expressing leaves were prepared and affixed to glass coverslips using medical adhesive (Hollister Incorporated Libertyville, Ill. USA) and then adhered to a glass slide with a hole in the middle generating a well, as described (Hu et al, 2010; Siegel et al, 2009; Young et al, 2006).
For recording intracellular Pt-GFP fluorescence in response to changes in extracellular pH incubation buffers, the pH of incubation buffers containing 10 mM MES, 10 mM KCl and 50 μM CaCl2 at 5.0 and 7.5 was adjusted by adding Tris-HCl. The well was perfused with incubation buffer at pH 5.0 for 15 min to obtain a background value and subsequently perfused with buffer at pH 7.5 for 15 min and returned to pH 5.0 again. For recording intracellular Pt-GFP fluorescence in response to constant extracellular pH and added weak acid, the perfusion buffers contained 10 mM MES, 10 mM KCl and 50 μM CaCl2, pH 5.6 supplemented with the indicated concentrations of sodium butyrate. For recording the Pt-GFP fluorescence of guard cells in response to CO2 changes, the incubation buffer (10 mM MES, 10 mM KCl and 50μM CaCl2, pH 6.15) was continually bubbled with 800 ppm CO2 or bubbled with air through soda lime, which was considered as nominal 0 ppm CO2 inside the buffer. Note that the final CO2 concentrations to which leaf epidermes were exposed were as reported previously using the same experimental set up and conditions (Young et al, 2006). The well was perfused with buffers shifting from 800 ppm to 0 ppm CO2 via a peristaltic pump and teflon tubing. Background fluorescence intensities at 470 nm were measured in regions lacking guard cells and are also shown for the corresponding experiments.
Bicarbonate Activates S-Type Anion Currents in ca1;ca4 Double Mutant Guard Cell Protoplasts
The βCA1 and βCA4 carbonic anhydrases act as upstream regulators in CO2-induced stomatal movements in guard cells (Hu et al, 2010). Elevated CO2 together with bicarbonate concentrations activate S-type anion channel currents in wild type Arabidopsis guard cells. Previous studies of CO2 regulation of anion channels have only analyzed wild type guard cells (Brearley et al, 1997; Hu et al, 2010; Raschke et al, 2003). Therefore, we investigated whether elevated bicarbonate and intracellular CO2 can by-pass the ca1;ca4 mutant and activate S-type anion currents in ca1;ca4 mutant guard cells. The addition of 13.5 mM total bicarbonate to the pipette solution (equivalent to 11.5 mM free bicarbonate ([HCO3−]i)/2 mM free [CO2] at pH 7.1) activated anion currents in patch clamped ca1;ca4 guard cell (
Bicarbonate Activated S-Type Anion Currents are Greatly Impaired in slac1 Mutant Guard Cell Protoplasts
The reversal potential of CO2+HCO3− activated whole-cell currents was +24.0±3.6 mV (n=8), which was close to the imposed chloride equilibrium potential of +31.1 mV, supports the hypothesis that CO2+HCO3− activate guard cell anion channels. The bicarbonate and CO2 concentrations used for anion current activation were very high (
Next, we analyzed whether these anion currents show a clear HCO3− permeability in wild type guard cells. The total bicarbonate was elevated to 50 mM in the pipette solution at pH 7.1 (corresponds to 43.4 mM free [HCO3−]i and 6.6 mM free [CO2]). Under this high [HCO3−] condition, the reversal potential of whole-cell currents was +26.0±0.9 mV (
Carbonic anhydrases reversibly catalyze the conversion of CO2 into bicarbonate ions and free protons (Chandrashekar et al, 2009; Supuran, 2008). Whether high [CO2], [HCO3−], [H+] or a combination of these mediates activation of S-type anion channels in Arabidopsis guard cells remains to be investigated (Hu et al, 2010). We investigated whether intracellular acidification is capable of activating S-type anion currents in wild type guard cell protoplasts. Intracellular acidification at pH 6.1 alone did not significantly activate S-type anion channel currents compared with control recordings at pH 7.1 (
Previous research has shown no intracellular pH shift in Vicia faba guard cells in response to [CO2] shifts (Brearly et al, 1997). To further investigate whether cytosolic pH is affected in Arabidopsis guard cells in response to [CO2] shifts, a ratiometric pH indicator Pt-GFP (Schulte et al, 2006) under the control of a strong guard cell preferential promoter pGC1 (Yang et al, 2008) was transformed into Arabidopsis guard cells (
To see whether elevated intracellular [HCO3−] is sufficient to activate anion currents at low [H−] and low [CO2], 13.5 mM total CsHCO3 was added to the pipette solution and the free [HCO3−] was calculated as 13.04 mM with 0.46 mM free [CO2] at pH 7.8. These analyses clearly showed that compared with the control recordings (
Extracellular bicarbonate was next tested on activation of S-type anion currents in wild type guard cells. After obtaining whole-cell recordings in wild type guard cells, the bath solution (200 μl) was perfused for 2 min at 1 ml min−1 with a solution that contained 11.5 mM free [HCO3−]i and 2 mM [CO2] at pH 7.1; see
The above analyses of activation of S-type anion currents were all conducted at 2 μM cytosolic free Ca2+ ([Ca2+]i) (
A summary of cytosolic free Ca2+ and HCO3− activation of S-type anion channels are shown in Table I. These data demonstrate a requirement for an elevated [Ca2+]i in HCO−-mediated activation of guard cell anion channels and provide direct and mechanistic evidence for the model that CO2-induced stomatal closing enhances the ability of [Ca2+]i to activate stomatal closing mechanisms (Young et al, 2006).
a-f Current values from FIG. 4G for comparision. Data are mean ± s.e.
Lower [Bicarbonate] is Sufficient for Activation of S-Type Anion Channel Currents in ht1-2 Guard Cells
The Arabidopsis HT1 protein kinase functions as a negative regulator of CO2-induced stomatal closing (Hashimoto et al, 2006). To test whether HT1 functions in the CO2/HCO3− SLAC1 signaling pathway (
The OST1 protein kinase was previously demonstrated to mediate ABA-induced stomatal closing. Recessive ost1 mutants disrupt ABA-induced stomatal closure as well as ABA inhibition of light-induced stomatal opening, but low CO2 induction of stomatal opening remained unaffected in the ost1-2 mutant, indicating that OST1 doesn't participate in CO2 signaling (Mustilli et al, 23002; Yushida et al, 2002). Here, the effect of OST1 on bicarbonate activation of S-type anion channels was investigated. Using the same recording solutions in
Elevated CO2-induced stomatal closure was also impaired in ost1-3 mutant leaf epidermes compared to wild type controls in genotype-blind assays (
ABA Receptor pyr1;pyl1;pyl2;pyl4 Quadruple Mutant and Type 2C Protein Phosphatases abi1-1 and abi2-1 Mutants Maintain Functional CO2 Response
The PYR/RCAR ABA receptor family was recently identified in Arabidopsis as major ABA receptors (Ma et al, 2009; Park et al, 2009). Since these ABA receptors tightly regulate and form complexes with SnRK2 kinases including OST1 (Fujii et al, 2009; Ma et al, 2009; Nishimura et al, 2010; Park et al, 2009), CO2 regulation of gas exchange in intact pyr1;pyl1;pyl2;pyl4 leaves was analyzed to see the requirement of ABA receptors for this CO2 response. Intact leaves of the pyr1;pyl1;pyl2;pyl4 quadruple mutant showed clear CO2 responses upon [CO2] changes; see
ABI1 and ABI2 encode type 2C protein phosphatases (PP2Cs) (Leung et al, 1994; Leung et al, 1997, Meyer et al, 194; Rodriguez et al, 1998). The dominant mutants abi1-1 and abi2-1 exhibit ABA insensitivity in seed germination, root growth responses and guard cells signaling (Koornneef et al, 1984; Pei et al, 1997). ABI1, PYR1 and OST1 interact with each other in ABA signaling (Nishimura et all, 2010; Park et al, 2009), thereafter CO2 regulation of gas exchange in abi1-1 and abi2-1 intact leaves were analyzed as well. Note that abi1-1 and abi2-1 leaves can wilt easily and therefore all gas exchange experiments were conducted on well-watered plants at ˜75-85% humidity, abi1-1 and abi2-1 mutants showed slightly impaired responses to changes of [CO2] compared with wild type Co1-0 plants (
Elevated [CO2] in leaf intercellular spaces (Ci) and elevated atmosphere [CO2] cause closing of stomatal pores (Medlyn et al, 2001). Carbonic anhydrases have been identified that function early in CO2 signal transduction (Hu et al, 2010). However, major questions in CO2 signal transduction have arisen. Whether CO2 or bicarbonate ion or a combination of these function in CO2 signal transduction in guard cells remained unclear. The presented findings demonstrate that bicarbonate acts as an intracellular signaling molecule in CO2 signal transduction, by activating SLAC1-mediated S-type anion channels in guard cells. We further found a synergistic action of intracellular HCO3− with cytosolic Ca2+, that requires both of these small molecules of CO2 signaling to proceed. We also report the characterization of the cellular functions and relative positions within the CO2 signal transduction cascade of mutants that strongly affect CO2 control of stomatal movements, including ca1;ca4, slac1 and ht1, ht1-2 mutant guard cells show hypersensitivity to intracellularly applied HCO3−, but continue to require cytosolic CA2+ for activation of SLAC1-dependent anion currents. In addition, we have unexpectedly found that loss-of-function mutations in the OST1 protein kinase cause a strong CO2 insensitivity of stomatal regulation by analyses of S-type anion channel regulation, stomatal movements and gas exchange in intact leaves and in whole plants, which leads to a new model for early CO2 signal transduction in guard cells.
Previous stomatal movement assays indicated that the OST1 protein kinase may not function in CO2 inhibition of stomatal opening (Mustilli et al, 2002). Unexpectedly, we have found here in ost1 mutant guard cells in both Columbia and Landsberg accessions show a dramatic impairment in CO2 regulation of stomatal conductance in intact leaves. Recent studies have shown that the OST1 kinase activates SLAC1 channels via phosphorylation (Geiger et al, 2009; Lee et al 2009; Vahisalu et al, 2010). Together our findings of impairment in bicarbonate activation of S-type anion currents in ost1-2 and ost1-3 mutant guard cells (
The PYR/RCAR abscisic acid receptors form a linear signal transduction module together with type 2C protein phosphatases and the OST1 protein kinase (Fujii et al, 2009; Ma et al, 2009; Nishimura et al, 2010; Park et al, 2009; Santiago et al, 2009; Umezawa et al, 2009). A quadruple mutant in four highly-expressed guard cell ABA receptors pyr1;pyl1;pyl2;pyl4 shows a strong impairment in ABA-induced stomatal closing (Nishimura et al, 2010). In contrast CO2 regulation remained functional in intact leaves (
The dominant protein phosphatase 2C (PP2C) mutants, abi1-1 and abi2-1, have been reported to conditionally affect CO2 signaling in guard cells (Leymarie et al 1998a; Leymarie et al, 1998b; Webb & Hetherington, 1997). ABI1 interacts with the OST1 protein kinase (Belin et al, 2006; Nishimura et al 2010; Umezawa et al. 2009; Vlad et al, 2009; Yoshida et al, 2006). The present study on CO2 signaling and research indicating ABA-independent activation of the OST1 protein kinase (Yoshida et al, 2006; Zheng et al, 2010) indicates that the early ABA signaling module consisting of ABA receptors, PP2Cs and OST1/SnRK2 kinases (Ma et al, 2009; Park et al, 2009) may be more complex than present models (Fujii et al, 2009).
Elevated bicarbonate activation of S-type anion currents in ca1;ca4 double mutant guard cells (
The intra cellular concentrations of bicarbonate and CO2 used in patch clamp experiments in the present study for S-type anion channel activation were higher than physiological concentrations in planta. Note that patch clamping of guard cells includes dialysis of the cytoplasm (Hamill et al, 1981) and it is possible that additional diluted small molecules or proteins are required for full sensitivity of this HCO3− response. Furthermore, typically high CO2 and HCO3− concentrations are used in electrophysiological studies, up to 72 mM HCO3− (Chandrashekar et al, 2009; Hu et al, 2010: Loriol et al, 2008; Yarmolinsky et al, 2009), although these experiments were conducted in different systems. The close correlation of high HCO3− regulation of S-type anion channels in the present study and the impaired CO2 response phenotypes in intact leaves of the Arabidopsis cal1;cal4, slac1, ht1-2 and ost1 mutants (
Intracellular acidification activates slow anion channel currents in the plasma membrane of Arabidopsis hypocotyl cells (Colcombet et al, 2005). However, intracellular acidification did not activate S-type anion currents in Arabidopsis guard cells, even in the presence of elevated 2 μM free [Ca2+]i (
Calcium is a second messenger that transduces diverse stimuli in plants (Blatt, 2000; Hetherington & Brownlee, 2004; Kim et al, 2010; Kudla et al, 2010; Sanders et al, 1999). Elevated CO2 caused an increase in [Ca2+]i in Commelina Communis guard cells (Webb et al, 1996). Furthermore, elevated CO2 caused a dampening of spontaneous repetitive [Ca2+]i, transients whereas low CO2 caused rapid [Ca2+]i transients in Arabidopsis guard cells (Young et al, 2006), which can be attributed to CO2-induced depolarization of guard cells (Grabov & Blatt, 1998; Klusener et al, 2002; Staxen et al, 1999). In both plant species abolishment of [Ca2+]i elevations abolished CO2-induced stomatal closing (Schwartz, 1985; Webb et al, 1996; Young et al. 2006). Time-resolved [Ca2+]i, imaging experiments led to the Ca2+ sensitivity priming hypothesis, in which CO2 was hypothesized to enhance (prime) the Ca2+ sensitivity of signaling mechanisms that relay CO2-induced stomatal closure (Young et al, 2006). However, additional and direct evidence for this CO2 signaling hypothesis has been lacking. Recent studies showed that ABA enhances (primes) the [Ca2+]i, sensitivity of S-type anion channel and Kin+ channel regulation, strongly supporting the hypothesis that ABA primes [Ca2+]i signal transduction (Siegel et al, 2009).
ABA increases cytosolic Ca2+ concentration by activating plasma membrane Ca2+ channels in Vicia faba and Arabidopsis guard cells (Grabov & Blatt, 1998; Hamilton et al, 2000; Murata et al, 2001; Pei et al., 2000; Schroeder & Hagiwara, 1990). Cytosolic [Ca2+]i interacts with other signaling molecules including nitric oxide (NO) (Garcia-Mata et al, 2003) and cytosolic pHi (Grabov & Blatt, 1997) in ion channels regulation in guard cells. Recently, Chen et al (2010) showed that cytosolic free [Ca2+]i interacts with protein phosphorylation events during slow anion currents activation.
The present study shows that elevated bicarbonate enhances the [Ca2+]i sensitivity in S-type anion channels activation (
The HT1 protein kinase functions as a negative regulator of CO2 signaling (Hashimoto et al, 2006) and our recent study showed that HT1 is epistatic to βCA1 and βCA4 in CO2 responses pathway (Hu et al, 2010). However, the role of HT1 within the guard cell signaling network had not been further analyzed. The ht1-2 mutant exhibits a hypersensitive response in bicarbonate activation of S-type anion currents, demonstrating that the HT1 kinase functions as a negative regulator and affects CO2 signaling downstream of HCO3− production and upstream of anion channel activation (
In conclusion, the present study identifies the OST1 protein kinase and the synergistic roles of the intercellular small molecules HCO3− and Ca2+ in guard cell CO2 signal transduction and anion channel regulation. Furthermore, characterization of the positions and roles of OST1, the HT1 protein kinase, the βCA1 and βCA4 carbonic anhydrases, PYR/RCAR ABA receptors, ABI1 and ABI2 PP2Cs and SLAC1 in CO2 regulation of S-type anion channels, leads to a revised model for CO2 signal transduction (
For analyses of S-type anion currents, the pipette solution contained 150 mM CsCl, 2 mM MgCl2, 6.7 mM EGTA, 2.61 mM CaCl2 (150 mM [Ca2+]i), 4.84 mM CaCl2 (0.6 μM [Ca2+]i), or 6.03 mM CaCl2 (2 μM [Ca2+]i), 5 mM Mg-ATP, 5 mM Tris-GTP, 1 mM HEPES/Tris, pH 7.1. For experiments analyzing effects of protons on S-type anion currents, the pipette solution contained 150 mM CsCl, 2 mM MgCl2, 6.7 mM EGTA, 0.6 mM CaCl2 (2 μM [Ca2+]i), 5 mM Mg-ATP, 5 mM Tris-GTP, 1 mM Mes/Tris, pH 6.1. For experiments with pipette solution at pH 7.8, the pipette medium contained 150 mM CsCl, 2 mM MgCl2, 2 μM free [Ca2+]i, 5 mM Mg-ATP, 5 mM Tris-GTP, 1 mM HEPES/Tris. Calcium affinities of EGTA and free Ca2+ concentrations were calculated using the WEBMAXC tool (http://www.stanford.edu/-epatton/webmaxc/webmaxcE.htm), which considers pH, [ATP] and ionic conditions. The bath solution contained 30 mM CsCl, 2 mM MgCl2, 5 mM CaCl2 and 10 mM Mes/Tris, pH 5.6. Osmolalities of all solutions were adjusted to 485 mmol1·kg−1 for bath solutions and 500 mmol·kg−1 for pipette solutions by addition of D-sorbitol.
Stomatal conductance measurements of 5-week-old plants in response to the imposed [CO2] at a light (PAR) fluence rate of 150 μmol m−2s−1 were conducted with a Li-6400 gas exchange analyzer with a fluorometer chamber (Li-Cor Inc.) as described-previously (Hu at al, 2010). To reduce the wilting of abi1-1 and abi1-2 mutant leaves, all plants ware analyzed with a humidifier that humidified the air surrounding plants to ˜75-85%. Relative stomatal conductance values of intact leaves were calculated by normalization relative to 365 or 400 ppm just before transition to 800 ppm [CO2]. Data shown are mean±s.e. of at least 3 leaves per genotype in the same experimental set.
For whole-plant gas-exchange experiments, 24 to 26-day-old plants were used. Plants were grown in pots as described previously (Kollist et al, 2007). For monitoring CO2-induced changes in whole-plant stomatal conductance, a custom made device for Arabidopsis whole-plant gas-exchange measurements was used (Kollist et al, 2007). Before application of different CO2 treatments, plants were acclimated in the measuring cuvettes for at least 1 h (Vahisalu et al, 2008). Experiments were performed at photosynthetic photon flux density of 150±3 μmol m−2s−1, relative humidity of 60-70% (vapor pressure deficit=0.9-1.2 kPa) and air temperature of 24-25° C. Photographs of plants were taken before the experiment and rosette leaf area was calculated using ImageJ 1.37v (National Institutes of Health, USA). Stomatal conductance for water vapor was calculated us described previously (Kollist et al, 2007; Vahisalu et al. 2008). Data were normalized relative to the stomatal conductance at 400 ppm [CO2] just before the transition to 100 ppm [CO2].
Three to 4-week-old plants grown in a plant-growth chamber were used for analyses of stomatal movements in response to ambient and elevated [CO2]. Intact leaf epidermal layers with no mesophyll cells in the vicinity and ambient or high [CO2] (800 ppm) incubation buffers were prepared as described (Hu et al., 2010; Young et al, 2006). Leaf epidermal layers were pre-incubated for 1.5 h in a buffer containing 10 mM MES, 10 mM KCl, 50 μM CaCl2 at pH 6.15 and then perfused with incubation buffers continually bubbled with ambient air or 800 ppm CO2 for 30 min. Stomatal apertures were measured using ImageJ software and analyzed. Data shown are from genotype blind analyses (n=3 experiments, 40 stomata per experiment and condition).
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.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/022331 | 1/24/2012 | WO | 00 | 8/21/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61438618 | Feb 2011 | US |
