Pollen exine contains complex biopolymers of aliphatic lipids and phenolics.Abnormal development of pollen exine often leads to plant sterility.Molecular mechanisms regulating exine formation have been studied extensi...Pollen exine contains complex biopolymers of aliphatic lipids and phenolics.Abnormal development of pollen exine often leads to plant sterility.Molecular mechanisms regulating exine formation have been studied extensively but remain ambiguous.Here we report the analyses of three GDSL esterase/lipase protein genes,OsGELP34,OsGELP110,and OsGELP115,for rice exine for-mation.OsGELP34 was identified by cloning of a male sterile mutant gene.OsGELP34 encodes an endoplasmic reticulum protein and was mainly expressed in anthers during pollen exine formation.osgelp34 mutant displayed abnormal exine and altered expression of a number of key genes required for pollen development.OsGELP110 was previously identified as a gene differentially expressed in meiotic anthers.OsGELP110 was most homologous to OsGELP115,and the two genes showed similar gene expression patterns.Both OsGELP110 and OsGELP115 proteins were localized in peroxisomes.Individual knockout of OsGELP110 and OsGELP115 did not affect the plant fertility,but double knockout of both genes altered the exine structure and rendered the plant male sterile.OsGELP34 is distant from OsGELP110 and OsGELP115 in sequence,and osgelp34 and osgelp110/osgelp115 mutants were different in anther morphology despite both were male sterile.These results suggested that OsGELP34 and OsGELP110/OsGELP115 catalyze different compounds for pollen exine development.展开更多
Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination.Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective ape...Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination.Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture.The mutant gene encodes a novel plasma membrane-localized legume-lectin receptor kinase that we named OsLecRK-S.7.OsLecRK-S.7 was expressed at different levels in all tested tissues and throughout anther development.In vitro kinase assay showed OsLecRK-S.7 capable of autophosporylation.Mutation in s13283(E560K)and mutation of the conserved ATP binding site(K418E)both knocked out the kinase activity.Mass spectrometry showed Thr376,Ser378,Thr386,Thr403,and Thr657 to be the autophosphorylation sites.Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees,but did not abolish the gene function in fertility complementation.oslecrk-s.7 mutant plant overexpressing OsLecRK-S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers,and these phenotypes were abolished by E560K or K418E mutation.The results indicated that OsLecRK-S.7 was a key regulator of pollen development.展开更多
Large-scale production of male sterile seeds can be achieved by introducing a fertility-restoration gene linked with a pollen-killer gene into a recessive male sterile mutant.We attempted to construct this system in r...Large-scale production of male sterile seeds can be achieved by introducing a fertility-restoration gene linked with a pollen-killer gene into a recessive male sterile mutant.We attempted to construct this system in rice by using a late-stage pollen-specific(LSP)promoter driving the expression of maizeα-amylase gene ZM-AA1.To obtain such promoters in rice,we conducted comparative RNA-seq analysis of mature pollen with meiosis anther,and compared this with the transcriptomic data of various tissues in the Rice Expression Database,resulting in 269 candidate LSP genes.Initial test of nine LSP genes showed that only the most active OsLSP3 promoter could drive ZM-AA1 to disrupt pollen.We then analyzed an additional 22 LSP genes and found 12 genes stronger than OsLSP3 in late-stage anthers.The promoters of OsLSP5 and OsLSP6 showing higher expression than OsLSP3 at stages 11 and 12 could drive ZM-AA1 to inactivate pollen,while the promoter of OsLSP4 showing higher expression at stage 12 only could not drive ZM-AA1 to disrupt pollen,suggesting that strong promoter activity at stage 11 was critical for pollen inactivation.The strong pollen-specific promoters identified in this study provided valuable tools for genetic engineering of rice male sterile system for hybrid rice production.展开更多
Calcium-dependent protein kinases(CPKs)are serine/threonine protein kinases that function in plant stress responses. Although CPKs are recognized as key messengers in signal transduction, the specific roles of CPKs ...Calcium-dependent protein kinases(CPKs)are serine/threonine protein kinases that function in plant stress responses. Although CPKs are recognized as key messengers in signal transduction, the specific roles of CPKs and the molecular mechanisms underlying their activity remain largely unknown. Here, we characterized the function of Os CPK_(24), a cytosol-localized calciumdependent protein kinase in rice. Os CPK_(24) was universally and highly expressed in rice plants and was induced by cold treatment. Whereas Os CPK_(24) knockdown plants exhibited increased sensitivity to cold compared to wild type(WT), Os CPK_(24)-overexpressing plants exhibited increased cold tolerance. Plants overexpressing Os CPK_(24) exhibited increased accumulation of proline(an osmoprotectant) and glutathione(an antioxidant) and maintained a higher GSH/GSSG(reduced glutathione to oxidized glutathione) ratio during cold stress compared to WT. In addition to these effects in response to cold stress, we observed the kinase activity of Os CPK_(24) varied under different calcium concentrations. Further,Os CPK_(24) phosphorylated Os Grx_(10), a glutathionedependent thioltransferase, at rates modulated by changes in calcium concentration. Together, our results support the hypothesis that Os CPK_(24) functions as a positive regulator of cold stress tolerance in rice, a process mediated by calcium signaling and involving phosphorylation and the inhibition of Os Grx_(10) to sustain higher glutathione levels.展开更多
基金We thank the Microscope Center in Life Science Schoolof Sun Yat-sen University for using their facilities formicroscopic analysis,and Yao-Guang Liu for the CRISPR/Cas9 systemThis work was supported by grants from Major Program of Guangdong Basic and Applied Re-search(grant number 2019B030302006)+3 种基金National Nat-ural Science Foundation of China(grant numbersU1901203 and 31801344)Natural Science Foundation ofGuangdong Province(grant number 2018B030308008)Shenzhen Commission on Innovation and Technology Programs(grant number JCYJ20180507181837997)Guangzhou Science and Technology lnnovation Com-mission(grant number 201804010034).
文摘Pollen exine contains complex biopolymers of aliphatic lipids and phenolics.Abnormal development of pollen exine often leads to plant sterility.Molecular mechanisms regulating exine formation have been studied extensively but remain ambiguous.Here we report the analyses of three GDSL esterase/lipase protein genes,OsGELP34,OsGELP110,and OsGELP115,for rice exine for-mation.OsGELP34 was identified by cloning of a male sterile mutant gene.OsGELP34 encodes an endoplasmic reticulum protein and was mainly expressed in anthers during pollen exine formation.osgelp34 mutant displayed abnormal exine and altered expression of a number of key genes required for pollen development.OsGELP110 was previously identified as a gene differentially expressed in meiotic anthers.OsGELP110 was most homologous to OsGELP115,and the two genes showed similar gene expression patterns.Both OsGELP110 and OsGELP115 proteins were localized in peroxisomes.Individual knockout of OsGELP110 and OsGELP115 did not affect the plant fertility,but double knockout of both genes altered the exine structure and rendered the plant male sterile.OsGELP34 is distant from OsGELP110 and OsGELP115 in sequence,and osgelp34 and osgelp110/osgelp115 mutants were different in anther morphology despite both were male sterile.These results suggested that OsGELP34 and OsGELP110/OsGELP115 catalyze different compounds for pollen exine development.
基金This work was supported by the Major Program of Guangdong Basic and Applied Research(2019B030302006)National Natural Science Foundation of China(U1901203,31801344,andU1704232)+3 种基金Natural Science Foundation of Guangdong Province(2018B030308008,2017A030313104,and 2018A0303130270)Shenzhen Commission on Innovation and Technology Programs(JCYJ20180507181837997)China Postdoctoral Science Foundation(2019M652938)the Innovation Project of Graduate School of South China Normal Uni-versity(2017LKXM011).
文摘Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination.Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture.The mutant gene encodes a novel plasma membrane-localized legume-lectin receptor kinase that we named OsLecRK-S.7.OsLecRK-S.7 was expressed at different levels in all tested tissues and throughout anther development.In vitro kinase assay showed OsLecRK-S.7 capable of autophosporylation.Mutation in s13283(E560K)and mutation of the conserved ATP binding site(K418E)both knocked out the kinase activity.Mass spectrometry showed Thr376,Ser378,Thr386,Thr403,and Thr657 to be the autophosphorylation sites.Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees,but did not abolish the gene function in fertility complementation.oslecrk-s.7 mutant plant overexpressing OsLecRK-S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers,and these phenotypes were abolished by E560K or K418E mutation.The results indicated that OsLecRK-S.7 was a key regulator of pollen development.
基金This work was supp orted by the Nationa l Key Research and Development Plan Program(2016YFD0100406 and 2016YFD0101801)National Natural Science Foundation of China(U1901203,31901532,and U1704232)+3 种基金Natural Science Foundation of Guangdong Province(2018B030308008)Shenzhen Commission on Innovation and Technology Programs(JCYJ20180507181837997)China Postdoctoral Science Foundation(2019M652938)Natural Science Foundation of Fujian Province(2017J01427)。
文摘Large-scale production of male sterile seeds can be achieved by introducing a fertility-restoration gene linked with a pollen-killer gene into a recessive male sterile mutant.We attempted to construct this system in rice by using a late-stage pollen-specific(LSP)promoter driving the expression of maizeα-amylase gene ZM-AA1.To obtain such promoters in rice,we conducted comparative RNA-seq analysis of mature pollen with meiosis anther,and compared this with the transcriptomic data of various tissues in the Rice Expression Database,resulting in 269 candidate LSP genes.Initial test of nine LSP genes showed that only the most active OsLSP3 promoter could drive ZM-AA1 to disrupt pollen.We then analyzed an additional 22 LSP genes and found 12 genes stronger than OsLSP3 in late-stage anthers.The promoters of OsLSP5 and OsLSP6 showing higher expression than OsLSP3 at stages 11 and 12 could drive ZM-AA1 to inactivate pollen,while the promoter of OsLSP4 showing higher expression at stage 12 only could not drive ZM-AA1 to disrupt pollen,suggesting that strong promoter activity at stage 11 was critical for pollen inactivation.The strong pollen-specific promoters identified in this study provided valuable tools for genetic engineering of rice male sterile system for hybrid rice production.
基金funded by the National Natural Science Foundation of China (31571753)the National High Technology Research and Development Program of China
文摘Calcium-dependent protein kinases(CPKs)are serine/threonine protein kinases that function in plant stress responses. Although CPKs are recognized as key messengers in signal transduction, the specific roles of CPKs and the molecular mechanisms underlying their activity remain largely unknown. Here, we characterized the function of Os CPK_(24), a cytosol-localized calciumdependent protein kinase in rice. Os CPK_(24) was universally and highly expressed in rice plants and was induced by cold treatment. Whereas Os CPK_(24) knockdown plants exhibited increased sensitivity to cold compared to wild type(WT), Os CPK_(24)-overexpressing plants exhibited increased cold tolerance. Plants overexpressing Os CPK_(24) exhibited increased accumulation of proline(an osmoprotectant) and glutathione(an antioxidant) and maintained a higher GSH/GSSG(reduced glutathione to oxidized glutathione) ratio during cold stress compared to WT. In addition to these effects in response to cold stress, we observed the kinase activity of Os CPK_(24) varied under different calcium concentrations. Further,Os CPK_(24) phosphorylated Os Grx_(10), a glutathionedependent thioltransferase, at rates modulated by changes in calcium concentration. Together, our results support the hypothesis that Os CPK_(24) functions as a positive regulator of cold stress tolerance in rice, a process mediated by calcium signaling and involving phosphorylation and the inhibition of Os Grx_(10) to sustain higher glutathione levels.