Progress 03/01/13 to 02/28/18
Outputs
Progress Report Objectives (from AD-416): The likelihood of global climate change increases the need for strategies for protecting crop productivity from environmental extremes, including heat, drought, and cold, all of which ultimately impact yield. Pollination and fertilization, in particular, are sensitive to environmental extremes. Pollination and fertilization require an extensive dialog between the tissues of the pistil and the pollen tube. Pollination and fertilization are also model processes for analyzing cell- cell recognition, cell-cell communication and coordination at the molecular, cellular and tissue levels. The long term goal of this project is to identify and characterize the genes and cellular processes that regulate pollen development, pollination, and fertilization, in order to identify genes that can be manipulated to better regulate pollen development, pollination and fertilization. This knowledge will better enable breeders to develop new strategies for genetically hardening these sensitive processes for the field. Genes that are necessary for pollen and sperm function(s) are often evolutionarily conserved and thus identifiable using comparative genomic strategies; such conserved genes are candidates for manipulations across a wide range of plant species. Objective 1: Identify and analyze, at genetic, molecular and protein levels, interactions that occur in pollen development, in pollen-pistil interactions or in sperm-egg interactions that determine the outcomes of these processes. Objective 2: Identify and characterize evolutionarily conserved regions of the genome whose function is to mediate pollen development and/or pollen-pistil interactions. Objective 3: Determine new means to regulate pollen development and pollen-pistil interactions. Approach (from AD-416): Objective 1: We hypothesize that protein complexes composed of receptor kinases and other interacting proteins will mediate cell signaling during pollen tube growth and pollen-pistil interactions. We further hypothesize that the components of the complexes will vary at different stages of pollen tube growth. To test this hypothesis we will use biochemical approaches such as co-immunoprecipitation and yeast two hybrid interactions, and in vivo imaging techniques, such as BiMolecular Fluorescence Complementation, to determine what proteins are present in the complexes at different stages of pollen tube growth, and how and when individual proteins participate in these complexes. These approaches have been successful in our previous studies. It is possible that particular interactions will fail to be confirmed biochemically, either because a third partner is necessary, or because the interaction is weak or transitory. If so, a genetic approach will be used to determine the functions of candidate proteins. Objective 2: We hypothesize that genes that encode pollen-specific proteins whose amino acid sequences are highly conserved across angiosperms will play important roles during pollen function. Conversely, we hypothesize that genes encoding pollen-expressed proteins that exhibit enhanced amino acid variation across angiosperms, or that are family- specific, might contribute to speciation. To test these hypotheses, we will identify candidate genes from RNA-seq analyses and use comparative genomics and multi-sequence alignments to identify conserved (or conversely, highly variable) protein domains. The bioinformatic approach is well-established and robust, but for particular genes it might not prove fruitful. However, since there are many such candidate genes, we anticipate at least partial success. Similarly, demonstrating a critical reproductive function for genes will follow well-established protocols (such as gene knock-outs and phenotypic analyses), but it is likely that some candidates will be excluded because no phenotype will be seen. Objective 3: We hypothesize that manipulating gene expression of key genes will improve stress tolerance during reproduction. To test this hypothesis, we will first establish robust methods for applying transient stresses to growing pollen tubes, then identify genes whose expression changes upon a stress treatment, such as temperature. Genes up-regulated upon stress treatment are candidates for stress tolerance, i.e. overexpressing the gene to higher levels might improve stress tolerance under those stress conditions, while down-regulating such genes (for example, via a gene knockout) is predicted to increase stress sensitivity and would validate the role of the gene in response to stress. Again, as in objective 2, some candidates might not fulfill this goal, but as there are many candidate genes, it is likely that the approach will prove fruitful. This is the final report for project 2030-21000-037-00D, which expired February 2018 and was replaced by bridging project 2030-21000-047-00D. Although the scientist in charge of this project retired in 2016, considerable progress was made on the objectives prior to departure. Towards Objective 1, the ARS scientist, in collaboration with researchers at the Shanghai Institute of Plant Physiology and Ecology, identified a protein that interacted with both a receptor kinase and the actin cytoskeleton. They discovered that these interactions were critical for pollen tube growth. Progress was also made on Objective 2, to select candidate genes for analyses. They showed that a gene encoding a protein phosphatase, called AHG3, is expressed in the vegetative cell of the pollen grain, but surprisingly, a fusion of the AHG3 protein with green fluorescent protein was localized in nuclei of the sperm cells. This finding supports the idea that transcripts move from the vegetative cell to the sperm cells. This work exposed an unknown communication between the cell that grows the pollen tube and the gametic cells inside the pollen grain. Another gene selected for analysis was expressed at high levels in pollen. In collaboration with researchers at Uppsala University in Sweden, they discovered that this �pollen� gene also has a function in the female, as a mutation in this gene causes a defect in integument formation, resulting in altered seed shape and reduced seed set. Progress was also made on Objective 3, towards manipulating key genes to enhance tolerance to stress in reproductive tissues. Various stress conditions were tested for effects on pollen tube growth in vitro. Plants carrying a mutation in a pollen-specific gene encoding an unknown protein were found to exhibit variable seed set, depending on the growth conditions of the plant. Genes encoding unknown proteins or previously uncharacterized transcription factors were analyzed for seed set defects, and the metabolome of a mutant in methionine biosynthesis was analyzed, in order to pinpoint the reason for seed set defects in this mutant. Many cereal crops, such as wheat and rice, have tillers that increase cereal yield. During the domestication of maize from its ancestor, teosinte, the tillers were suppressed in exchange for the large maize ear. The major gene responsible for the domestication from teosinte to maize is the transcription factor, TEOSINTE BRANCHED1 (TB1). The TB1 mutant reverts maize plants to teosinte-like plants in regards to architecture. TB1 mutants have many tillers and the tillers end in male flowers instead of ears. Progress was made in determining the genes that are regulated by TB1. These genes also have mutant phenotypes and when they are combined, they resemble the TB1 mutant.
Impacts
(N/A)
Publications
- Qin, P., Loraine, A.E., McCormick, S.M. 2018. Cell-specific cis-natural antisense transcripts (cis-NATS) in the sperm and the pollen vegetative cells of Arabidopsis thaliana. F1000Research. 7:93.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): The likelihood of global climate change increases the need for strategies for protecting crop productivity from environmental extremes, including heat, drought, and cold, all of which ultimately impact yield. Pollination and fertilization, in particular, are sensitive to environmental extremes. Pollination and fertilization require an extensive dialog between the tissues of the pistil and the pollen tube. Pollination and fertilization are also model processes for analyzing cell- cell recognition, cell-cell communication and coordination at the molecular, cellular and tissue levels. The long term goal of this project is to identify and characterize the genes and cellular processes that regulate pollen development, pollination, and fertilization, in order to identify genes that can be manipulated to better regulate pollen development, pollination and fertilization. This knowledge will better enable breeders to develop new strategies for genetically hardening these sensitive processes for the field. Genes that are necessary for pollen and sperm function(s) are often evolutionarily conserved and thus identifiable using comparative genomic strategies; such conserved genes are candidates for manipulations across a wide range of plant species. Objective 1: Identify and analyze, at genetic, molecular and protein levels, interactions that occur in pollen development, in pollen-pistil interactions or in sperm-egg interactions that determine the outcomes of these processes. Objective 2: Identify and characterize evolutionarily conserved regions of the genome whose function is to mediate pollen development and/or pollen-pistil interactions. Objective 3: Determine new means to regulate pollen development and pollen-pistil interactions. Approach (from AD-416): Objective 1: We hypothesize that protein complexes composed of receptor kinases and other interacting proteins will mediate cell signaling during pollen tube growth and pollen-pistil interactions. We further hypothesize that the components of the complexes will vary at different stages of pollen tube growth. To test this hypothesis we will use biochemical approaches such as co-immunoprecipitation and yeast two hybrid interactions, and in vivo imaging techniques, such as BiMolecular Fluorescence Complementation, to determine what proteins are present in the complexes at different stages of pollen tube growth, and how and when individual proteins participate in these complexes. These approaches have been successful in our previous studies. It is possible that particular interactions will fail to be confirmed biochemically, either because a third partner is necessary, or because the interaction is weak or transitory. If so, a genetic approach will be used to determine the functions of candidate proteins. Objective 2: We hypothesize that genes that encode pollen-specific proteins whose amino acid sequences are highly conserved across angiosperms will play important roles during pollen function. Conversely, we hypothesize that genes encoding pollen-expressed proteins that exhibit enhanced amino acid variation across angiosperms, or that are family- specific, might contribute to speciation. To test these hypotheses, we will identify candidate genes from RNA-seq analyses and use comparative genomics and multi-sequence alignments to identify conserved (or conversely, highly variable) protein domains. The bioinformatic approach is well-established and robust, but for particular genes it might not prove fruitful. However, since there are many such candidate genes, we anticipate at least partial success. Similarly, demonstrating a critical reproductive function for genes will follow well-established protocols (such as gene knock-outs and phenotypic analyses), but it is likely that some candidates will be excluded because no phenotype will be seen. Objective 3: We hypothesize that manipulating gene expression of key genes will improve stress tolerance during reproduction. To test this hypothesis, we will first establish robust methods for applying transient stresses to growing pollen tubes, then identify genes whose expression changes upon a stress treatment, such as temperature. Genes up-regulated upon stress treatment are candidates for stress tolerance, i.e. overexpressing the gene to higher levels might improve stress tolerance under those stress conditions, while down-regulating such genes (for example, via a gene knockout) is predicted to increase stress sensitivity and would validate the role of the gene in response to stress. Again, as in objective 2, some candidates might not fulfill this goal, but as there are many candidate genes, it is likely that the approach will prove fruitful. Progress in FY 2017 entailed research on plant architecture, specifically tillering. Many cereal crops, such as wheat and rice, have tillers that increase cereal yield. During the domestication of maize from its ancestor, teosinte, the tillers were suppressed in exchange for the large maize ear. The major gene responsible for the domestication from teosinte to maize is the transcription factor, TEOSINTE BRANCHED1 (TB1). The TB1 mutant reverts maize plants to teosinte-like plants in regards to architecture. TB1 mutants have many tillers and the tillers end in male flowers instead of ears. Progress was made in determining the genes that are bound and regulated by TB1. These genes also have mutant phenotypes and when they are combined, they resemble the TB1 mutant.
Impacts
(N/A)
Publications
- Chen, Y., Zou, T., McCormick, S. 2016. S-Adenosylmethionine synthetase 3 is important for pollen tube growth. Plant Physiology. 172(1):244-253. doi:10.1104//.16.00774.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): The likelihood of global climate change increases the need for strategies for protecting crop productivity from environmental extremes, including heat, drought, and cold, all of which ultimately impact yield. Pollination and fertilization, in particular, are sensitive to environmental extremes. Pollination and fertilization require an extensive dialog between the tissues of the pistil and the pollen tube. Pollination and fertilization are also model processes for analyzing cell- cell recognition, cell-cell communication and coordination at the molecular, cellular and tissue levels. The long term goal of this project is to identify and characterize the genes and cellular processes that regulate pollen development, pollination, and fertilization, in order to identify genes that can be manipulated to better regulate pollen development, pollination and fertilization. This knowledge will better enable breeders to develop new strategies for genetically hardening these sensitive processes for the field. Genes that are necessary for pollen and sperm function(s) are often evolutionarily conserved and thus identifiable using comparative genomic strategies; such conserved genes are candidates for manipulations across a wide range of plant species. Objective 1: Identify and analyze, at genetic, molecular and protein levels, interactions that occur in pollen development, in pollen-pistil interactions or in sperm-egg interactions that determine the outcomes of these processes. Objective 2: Identify and characterize evolutionarily conserved regions of the genome whose function is to mediate pollen development and/or pollen-pistil interactions. Objective 3: Determine new means to regulate pollen development and pollen-pistil interactions. Approach (from AD-416): Objective 1: We hypothesize that protein complexes composed of receptor kinases and other interacting proteins will mediate cell signaling during pollen tube growth and pollen-pistil interactions. We further hypothesize that the components of the complexes will vary at different stages of pollen tube growth. To test this hypothesis we will use biochemical approaches such as co-immunoprecipitation and yeast two hybrid interactions, and in vivo imaging techniques, such as BiMolecular Fluorescence Complementation, to determine what proteins are present in the complexes at different stages of pollen tube growth, and how and when individual proteins participate in these complexes. These approaches have been successful in our previous studies. It is possible that particular interactions will fail to be confirmed biochemically, either because a third partner is necessary, or because the interaction is weak or transitory. If so, a genetic approach will be used to determine the functions of candidate proteins. Objective 2: We hypothesize that genes that encode pollen-specific proteins whose amino acid sequences are highly conserved across angiosperms will play important roles during pollen function. Conversely, we hypothesize that genes encoding pollen-expressed proteins that exhibit enhanced amino acid variation across angiosperms, or that are family- specific, might contribute to speciation. To test these hypotheses, we will identify candidate genes from RNA-seq analyses and use comparative genomics and multi-sequence alignments to identify conserved (or conversely, highly variable) protein domains. The bioinformatic approach is well-established and robust, but for particular genes it might not prove fruitful. However, since there are many such candidate genes, we anticipate at least partial success. Similarly, demonstrating a critical reproductive function for genes will follow well-established protocols (such as gene knock-outs and phenotypic analyses), but it is likely that some candidates will be excluded because no phenotype will be seen. Objective 3: We hypothesize that manipulating gene expression of key genes will improve stress tolerance during reproduction. To test this hypothesis, we will first establish robust methods for applying transient stresses to growing pollen tubes, then identify genes whose expression changes upon a stress treatment, such as temperature. Genes up-regulated upon stress treatment are candidates for stress tolerance, i.e. overexpressing the gene to higher levels might improve stress tolerance under those stress conditions, while down-regulating such genes (for example, via a gene knockout) is predicted to increase stress sensitivity and would validate the role of the gene in response to stress. Again, as in objective 2, some candidates might not fulfill this goal, but as there are many candidate genes, it is likely that the approach will prove fruitful. Progress was made toward Objective 2. Progress towards other objectives was limited due to retirement of the lead scientist. Using salary lapse funds, a postdoc was funded who worked on reproductive biology. Arabidopsis has four enzymes in the methionine metabolic pathway. According to RNA-seq data, MAT3 has the highest expression in pollen. We obtained a T-DNA insertion line and determined that gene expression levels were reduced. mat3 mutants have shorter siliques and reduced seed set. Self-pollinated heterozygous plants (+/mat3) exhibited a distorted segregation ratio that differed significantly from the expected Mendelian ratio of 1:2:1. To test for potential defects in male or female gametophyte transmission, we performed reciprocal crosses using +/mat3 heterozygotes as either the male or female parent. Male transmission was more affected than female transmission. We examined pollen germination in vitro and in vivo. For in vitro pollen germination, we measured germination percentage and pollen tube lengths. mat3 pollen showed reduced pollen germination and had shorter pollen tubes than WT. For in vivo pollination, we used WT pollen on WT stigmas or mat3 pollen on mat3 stigmas, and then observed pollen tubes 12 hours after pollination. Pollen tubes of WT had penetrated through the style and reached close to the end of the transmitting tract, while in mat3, pollen tubes had just begun to penetrate into the style. We confirmed that the defect was due to mat3 by complimenting the mutation using a transgene. The construct also allowed us to localize the MAT3 protein to the cytoplasm and nucleus. We hypothesized that the pollen phenotype might be caused by increased amounts of methionine. Ethionine is a toxic analog of methionine, and cells that over accumulate free methionine exhibit increased resistance to ethionine. We added ethionine to pollen germination medium and measured pollen tube lengths in WT and mat3. Inhibition was dose- dependent and was more obvious in WT than in mat3, suggesting that free methionine accumulates in mat3 pollen. To verify if methionine over- accumulation indeed affected pollen tube growth, exogenous methionine was added to in vitro pollen germination medium. The pollen tube lengths were shorter in WT than in mat3 and the effect was again dose-dependent. These data support the idea that methionine can inhibit pollen tube growth and that the short mat3 pollen tubes might be caused by methionine over- accumulation. To determine whether there was any overlap of these changes in mat3 pollen and pollen tubes, we performed metabolomics analysis on WT and mat3/mat3 mature pollen and pollen tubes after 3 hours of germination, using GC-TOF/MS. Accomplishments 01 S-adenosylmethionine synthetase 3 is required during pollen germination and pollen tube growth in Arabidopsis. SAM (S-adenosylmethionine) is widely used in a variety of biological reactions and participates in the Met (methionine) metabolic pathway. In Arabidopsis, one of the 4 S- adenosyl methionine synthetase (SAMS) genes, MAT3, is highly expressed in pollen. ARS scientists at Albany, California showed that mat3 mutants have impaired pollen tube growth and reduced seed set. Metabolomics analyses confirmed that mat3 pollen and pollen tubes over- accumulate methionine and that mat3 pollen has several metabolite profiles, for example, those of polyamine biosynthesis, which are different from those of wild type. Additionally, they showed that disruption of methionine metabolism in mat3 pollen affected tRNA and histone methylation levels. These results suggest a connection between metabolism and epigenetics and reveal a crucial role for regulating methionine levels in plants in order to ensure reproductive fitness.
Impacts
(N/A)
Publications
- Zheng, B., He, H., Zheng, Y., Wu, W., Mccormick, S.M. 2014. An ARID domain- containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana. PLoS Genetics. 10(7):e1004421. doi: 10. 1371/journal.pgen.1004421.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): The likelihood of global climate change increases the need for strategies for protecting crop productivity from environmental extremes, including heat, drought, and cold, all of which ultimately impact yield. Pollination and fertilization, in particular, are sensitive to environmental extremes. Pollination and fertilization require an extensive dialog between the tissues of the pistil and the pollen tube. Pollination and fertilization are also model processes for analyzing cell- cell recognition, cell-cell communication and coordination at the molecular, cellular and tissue levels. The long term goal of this project is to identify and characterize the genes and cellular processes that regulate pollen development, pollination, and fertilization, in order to identify genes that can be manipulated to better regulate pollen development, pollination and fertilization. This knowledge will better enable breeders to develop new strategies for genetically hardening these sensitive processes for the field. Genes that are necessary for pollen and sperm function(s) are often evolutionarily conserved and thus identifiable using comparative genomic strategies; such conserved genes are candidates for manipulations across a wide range of plant species. Objective 1: Identify and analyze, at genetic, molecular and protein levels, interactions that occur in pollen development, in pollen-pistil interactions or in sperm-egg interactions that determine the outcomes of these processes. Objective 2: Identify and characterize evolutionarily conserved regions of the genome whose function is to mediate pollen development and/or pollen-pistil interactions. Objective 3: Determine new means to regulate pollen development and pollen-pistil interactions. Approach (from AD-416): Objective 1: We hypothesize that protein complexes composed of receptor kinases and other interacting proteins will mediate cell signaling during pollen tube growth and pollen-pistil interactions. We further hypothesize that the components of the complexes will vary at different stages of pollen tube growth. To test this hypothesis we will use biochemical approaches such as co-immunoprecipitation and yeast two hybrid interactions, and in vivo imaging techniques, such as BiMolecular Fluorescence Complementation, to determine what proteins are present in the complexes at different stages of pollen tube growth, and how and when individual proteins participate in these complexes. These approaches have been successful in our previous studies. It is possible that particular interactions will fail to be confirmed biochemically, either because a third partner is necessary, or because the interaction is weak or transitory. If so, a genetic approach will be used to determine the functions of candidate proteins. Objective 2: We hypothesize that genes that encode pollen-specific proteins whose amino acid sequences are highly conserved across angiosperms will play important roles during pollen function. Conversely, we hypothesize that genes encoding pollen-expressed proteins that exhibit enhanced amino acid variation across angiosperms, or that are family- specific, might contribute to speciation. To test these hypotheses, we will identify candidate genes from RNA-seq analyses and use comparative genomics and multi-sequence alignments to identify conserved (or conversely, highly variable) protein domains. The bioinformatic approach is well-established and robust, but for particular genes it might not prove fruitful. However, since there are many such candidate genes, we anticipate at least partial success. Similarly, demonstrating a critical reproductive function for genes will follow well-established protocols (such as gene knock-outs and phenotypic analyses), but it is likely that some candidates will be excluded because no phenotype will be seen. Objective 3: We hypothesize that manipulating gene expression of key genes will improve stress tolerance during reproduction. To test this hypothesis, we will first establish robust methods for applying transient stresses to growing pollen tubes, then identify genes whose expression changes upon a stress treatment, such as temperature. Genes up-regulated upon stress treatment are candidates for stress tolerance, i.e. overexpressing the gene to higher levels might improve stress tolerance under those stress conditions, while down-regulating such genes (for example, via a gene knockout) is predicted to increase stress sensitivity and would validate the role of the gene in response to stress. Again, as in objective 2, some candidates might not fulfill this goal, but as there are many candidate genes, it is likely that the approach will prove fruitful. Progress was made on objective 1, to elucidate the molecular mechanisms that occur during pollen development, pollen-pistil interactions and sperm-egg interactions. In collaboration with researchers at Uppsala University, Sweden, we showed that messenger RNA from the vegetative cell of pollen grains trafficks to the sperm cells within the pollen grain. This previously undocumented intercellular communication has implications for plant reproduction. This work was submitted to Proceedings of the National Academy of Science and was accepted pending revision, titled, "Intercellular communication in pollen: AHG3 transcripts move from the vegetative cell to sperm in Arabidopsis thaliana". Progress was made on objective 2, to select candidate genes for analyses. We found that a gene selected for analysis because of its expression level in pollen surprisingly has a function in the female, as a mutation in this gene causes a defect in integument formation, resulting in altered seed shape and reduced seed set. Progress was made on objective 3, towards manipulating key genes to enhance tolerance to stress in reproductive tissues. Genes encoding unknown proteins or previously uncharacterized transcription factors were analyzed for seed set defects, and the metabolome of a mutant in methionine biosynthesis was analyzed, in order to pinpoint the reason for seed set defects in this mutant. Accomplishments 01 Genes that function in plant reproduction. An ARS scientist at the Plant Gene Expression Center in Albany, California, in collaboration with researchers at Shanghai Institute of Plant Physiology and Ecology, showed that overexpression of a pollen receptor kinase, LePRK1, caused pollen tubes to change their growth mode to a blebbing mode. In blebbing mode, the tube expands in the middle, instead of the normal tip growth. This blebbing phenotype therefore indicates that the expression level of LePRK1 is crucial for normal pollen tube growth. The work leads to a better understanding of plant reproductive biology, critical for flowering plants in agriculture.
Impacts
(N/A)
Publications
- Gui, C., Dong, X., Liu, H., Huang, W., Barberini, M.L., Gao, X., Muschietti, J., Mccormick, S.M., Tang, W. 2014. Overexpression of the tomato pollen receptor kinase LePRK1 rewires pollen tube growth to a blebbling mode. The Plant Cell. 26:3538-3555.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): The likelihood of global climate change increases the need for strategies for protecting crop productivity from environmental extremes, including heat, drought, and cold, all of which ultimately impact yield. Pollination and fertilization, in particular, are sensitive to environmental extremes. Pollination and fertilization require an extensive dialog between the tissues of the pistil and the pollen tube. Pollination and fertilization are also model processes for analyzing cell- cell recognition, cell-cell communication and coordination at the molecular, cellular and tissue levels. The long term goal of this project is to identify and characterize the genes and cellular processes that regulate pollen development, pollination, and fertilization, in order to identify genes that can be manipulated to better regulate pollen development, pollination and fertilization. This knowledge will better enable breeders to develop new strategies for genetically hardening these sensitive processes for the field. Genes that are necessary for pollen and sperm function(s) are often evolutionarily conserved and thus identifiable using comparative genomic strategies; such conserved genes are candidates for manipulations across a wide range of plant species. Objective 1: Identify and analyze, at genetic, molecular and protein levels, interactions that occur in pollen development, in pollen-pistil interactions or in sperm-egg interactions that determine the outcomes of these processes. Objective 2: Identify and characterize evolutionarily conserved regions of the genome whose function is to mediate pollen development and/or pollen-pistil interactions. Objective 3: Determine new means to regulate pollen development and pollen-pistil interactions. Approach (from AD-416): Objective 1: We hypothesize that protein complexes composed of receptor kinases and other interacting proteins will mediate cell signaling during pollen tube growth and pollen-pistil interactions. We further hypothesize that the components of the complexes will vary at different stages of pollen tube growth. To test this hypothesis we will use biochemical approaches such as co-immunoprecipitation and yeast two hybrid interactions, and in vivo imaging techniques, such as BiMolecular Fluorescence Complementation, to determine what proteins are present in the complexes at different stages of pollen tube growth, and how and when individual proteins participate in these complexes. These approaches have been successful in our previous studies. It is possible that particular interactions will fail to be confirmed biochemically, either because a third partner is necessary, or because the interaction is weak or transitory. If so, a genetic approach will be used to determine the functions of candidate proteins. Objective 2: We hypothesize that genes that encode pollen-specific proteins whose amino acid sequences are highly conserved across angiosperms will play important roles during pollen function. Conversely, we hypothesize that genes encoding pollen-expressed proteins that exhibit enhanced amino acid variation across angiosperms, or that are family- specific, might contribute to speciation. To test these hypotheses, we will identify candidate genes from RNA-seq analyses and use comparative genomics and multi-sequence alignments to identify conserved (or conversely, highly variable) protein domains. The bioinformatic approach is well-established and robust, but for particular genes it might not prove fruitful. However, since there are many such candidate genes, we anticipate at least partial success. Similarly, demonstrating a critical reproductive function for genes will follow well-established protocols (such as gene knock-outs and phenotypic analyses), but it is likely that some candidates will be excluded because no phenotype will be seen. Objective 3: We hypothesize that manipulating gene expression of key genes will improve stress tolerance during reproduction. To test this hypothesis, we will first establish robust methods for applying transient stresses to growing pollen tubes, then identify genes whose expression changes upon a stress treatment, such as temperature. Genes up-regulated upon stress treatment are candidates for stress tolerance, i.e. overexpressing the gene to higher levels might improve stress tolerance under those stress conditions, while down-regulating such genes (for example, via a gene knockout) is predicted to increase stress sensitivity and would validate the role of the gene in response to stress. Again, as in objective 2, some candidates might not fulfill this goal, but as there are many candidate genes, it is likely that the approach will prove fruitful. Progress was made on objective 1, to elucidate the molecular mechanisms that occur during pollen development, pollen-pistil interactions and sperm-egg interactions. In collaboration with with researchers at the Shanghai Institute of Plant Physiology and Ecology, we found that a protein called KPP (short for kinase partner protein) interacted with both a receptor kinase and the actin cytoskeleton and that these interactions were critical for pollen tube growth. Progress was made on objective 2, to select candidate genes for analyses. We showed that a gene encoding a protein phosphatase, called AHG3, is expressed in the vegetative cell of the pollen grain, but surprisingly, a fusion of the AHG3 protein with green fluorescent protein was localized in nuclear speckles in the nuclei of the sperm cells. This finding supports the idea that transcripts move from the vegetative cell to the sperm cells. This work exposed an unknown communication between the cell that grows the pollen tube and the gametic cells inside the pollen grain. Progress was made on objective 3, towards manipulating keys to enhance tolerance to stress in reproductive tissues. Various stress conditions were tested for effects on pollen tube growth in vitro. Plants carrying a mutation in a pollen-specific gene encoding an unknown protein were found to exhibit variable seed set, depending on the growth conditions of the plant. Accomplishments 01 Genes that function in plant reproduction. An ARS scientist at the Plant Gene Expression Center in Albany, California, in collaboration with researchers at Shanghai Institute of Plant Physiology and Ecology, showed that a female-expressed protein, STIG1, interacted with both a pollen receptor kinase and with lipids called phosphoinositides. We used mutational analyses to pinpoint the precise amino acids required for these protein interactions. These dual interactions are important for pollen tube growth. In collaboration with researchers at the Gulbenkian Institute in Portugal, an ARS scientist in Albany, California, showed that two sperm-specific tetraspanin proteins are localized at the sperm-sperm interface, suggesting that they might be required for the association of the sperm with the vegetative cell nucleus, an association that is critical for sperm delivery to the female. The work leads to advances in reproduction, critical for flowering plants in agriculture.
Impacts
(N/A)
Publications
- Boavida, L.C., Qin, P., Broz, M., Becker, J.D., McCormick, S.M. 2013. Arabidopsis tetraspanins are confined to discrete expression domains and cell types in reproductive tissues and form homo-and heterodimers when expressed in yeast. Plant Physiology. 163:696-712.
- Huang, W., Liu, H., McCormick, S.M., Tang, W. 2014. Tomato pistil factor STIG1 promotes in vivo pollen tube growth by binding to phosphatidylinositol 3-phosphate and the extracellular domain of the pollen receptor kinase LePRK2. The Plant Cell. DOI: 10.1105/tpc.114.123281.
- McCormick, S.M. 2013. Pollen (quick guide). Current Biology. 23:R988-990.
- Zhao, X., Wang, Q., Li, S., Ge, F., Zhou, L., McCormick, S.M., Zhang, Y. 2013. The Juxtamembrane and carboxy-terminal domains of Arabidopsis PRK2 are critical for ROP-induced growth in pollen tubes. Journal of Experimental Botany. 64:5599-5610.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): The likelihood of global climate change increases the need for strategies for protecting crop productivity from environmental extremes, including heat, drought, and cold, all of which ultimately impact yield. Pollination and fertilization, in particular, are sensitive to environmental extremes. Pollination and fertilization require an extensive dialog between the tissues of the pistil and the pollen tube. Pollination and fertilization are also model processes for analyzing cell- cell recognition, cell-cell communication and coordination at the molecular, cellular and tissue levels. The long term goal of this project is to identify and characterize the genes and cellular processes that regulate pollen development, pollination, and fertilization, in order to identify genes that can be manipulated to better regulate pollen development, pollination and fertilization. This knowledge will better enable breeders to develop new strategies for genetically hardening these sensitive processes for the field. Genes that are necessary for pollen and sperm function(s) are often evolutionarily conserved and thus identifiable using comparative genomic strategies; such conserved genes are candidates for manipulations across a wide range of plant species. Objective 1: Identify and analyze, at genetic, molecular and protein levels, interactions that occur in pollen development, in pollen-pistil interactions or in sperm-egg interactions that determine the outcomes of these processes. Objective 2: Identify and characterize evolutionarily conserved regions of the genome whose function is to mediate pollen development and/or pollen-pistil interactions. Objective 3: Determine new means to regulate pollen development and pollen-pistil interactions. Approach (from AD-416): Objective 1: We hypothesize that protein complexes composed of receptor kinases and other interacting proteins will mediate cell signaling during pollen tube growth and pollen-pistil interactions. We further hypothesize that the components of the complexes will vary at different stages of pollen tube growth. To test this hypothesis we will use biochemical approaches such as co-immunoprecipitation and yeast two hybrid interactions, and in vivo imaging techniques, such as BiMolecular Fluorescence Complementation, to determine what proteins are present in the complexes at different stages of pollen tube growth, and how and when individual proteins participate in these complexes. These approaches have been successful in our previous studies. It is possible that particular interactions will fail to be confirmed biochemically, either because a third partner is necessary, or because the interaction is weak or transitory. If so, a genetic approach will be used to determine the functions of candidate proteins. Objective 2: We hypothesize that genes that encode pollen-specific proteins whose amino acid sequences are highly conserved across angiosperms will play important roles during pollen function. Conversely, we hypothesize that genes encoding pollen-expressed proteins that exhibit enhanced amino acid variation across angiosperms, or that are family- specific, might contribute to speciation. To test these hypotheses, we will identify candidate genes from RNA-seq analyses and use comparative genomics and multi-sequence alignments to identify conserved (or conversely, highly variable) protein domains. The bioinformatic approach is well-established and robust, but for particular genes it might not prove fruitful. However, since there are many such candidate genes, we anticipate at least partial success. Similarly, demonstrating a critical reproductive function for genes will follow well-established protocols (such as gene knock-outs and phenotypic analyses), but it is likely that some candidates will be excluded because no phenotype will be seen. Objective 3: We hypothesize that manipulating gene expression of key genes will improve stress tolerance during reproduction. To test this hypothesis, we will first establish robust methods for applying transient stresses to growing pollen tubes, then identify genes whose expression changes upon a stress treatment, such as temperature. Genes up-regulated upon stress treatment are candidates for stress tolerance, i.e. overexpressing the gene to higher levels might improve stress tolerance under those stress conditions, while down-regulating such genes (for example, via a gene knockout) is predicted to increase stress sensitivity and would validate the role of the gene in response to stress. Again, as in objective 2, some candidates might not fulfill this goal, but as there are many candidate genes, it is likely that the approach will prove fruitful. This report documents progress for project number 5335-21000-037-00D, which started in March 2013 and continues research from Project Number 5335-21000-036-00D, entitled "Molecular Developmental Genetics of Pollen and Pollen-Pistil Interactions in Crop Plants." Progress was made on objective 1, to elucidate the molecular mechanisms that occur during pollen development, pollen-pistil interactions and sperm-egg interactions. In collaboration with researchers at the Shanghai Institute of Plant Physiology and Ecology, we found that overexpression of the receptor kinase LePRK1 in tomato changed the pollen tube growth mode from tubular to a blebbing mode, and showed that this phenotype was due to interactions with the actin cytoskeleton. Progress was made on objective 2, to select candidate genes for analyses. We showed that the tetraspanin family, of which there are 17 members in Arabidopsis thaliana, exhibit cell-type specific expression patterns. Most interestingly, we showed that two sperm-specific tetrapanins are localized at the sperm-sperm interface, suggesting that they might be required for the association of the sperm cells themselves and for the association of the sperm cells with the vegetative cell nucleus. Accomplishments 01 Genes that function in pollen. In collaboration with researchers at University of North Carolina-Charlotte, we used deep sequencing technology (so-called RNA-Seq) to discover that Arabidopsis pollen expresses about 1000 more genes than previously known, that it expresses some genes that are not in the current genome annotation or that are incorrectly annotated, and that transcripts from some pollen- expressed genes are spliced differently than the known splice variants in other cell types. We have and continue to use the RNA-Seq dataset to select candidate genes for functional analyses. In, addition, we showed that a type II ROP GTPase is localized to the plasma membrane that surrounds the two sperm cells inside the pollen grain. We swapped domains of a type I ROP and a type II ROP to show that localization surrounding the sperm cells is due to the C-terminal part of the type II ROP, specifically to some cysteines in that part of the protein. The work leads to advances in reporduction, critical cor flowering plants in agriculture.
Impacts
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Publications
- Loraine, A., Mccormick, S.M., Estrada, A., Patel, K., Qin, P. 2013. RNA- Seq of Aradopsis pollen uncovers novel transcription and alternative splicing. Plant Physiology. 162: 1092-1109.
- Scarpin, R., Sigaut, L., Pietrasanta, L., Zheng, B., Mccormick, S.M., Muschietti, J. 2013. Cajal bodies are developmentally regulated during pollen development abd pollen tube growth in Arabidopsis thaliana. Molecular Plant. 6: 1355-1357.
- Li, S., Zhou, L., Feng, Q., Mccormick, S.M., Zhang, Y. 2013. The C- terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane. Molecular Plant. 6:1362-1364.
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