光合作用信号途径调控陆地棉矮化的机理研究
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摘要
【目的】了解光合作用信号途径基因调控陆地棉矮化的机理。【方法】以现蕾期陆地棉矮化突变体LA-1及其近等基因系LH-1茎尖为材料,通过同位素标记相对和绝对定量(Isobaric tags for relative and absolute quantitation, i TRAQ)结合液相质谱串联(LC-MS/MS)的定量蛋白质组学技术及定量反转录-聚合酶链式反应(Quantitative reverse-polymerase chain reaction, qRT-PCR)技术,分析两种材料中蛋白水平及m RNA水平基因表达情况,并通过生理生化方法检测两种材料光合能力差异。【结果】光合作用信号途径差异表达蛋白PsbO、PsaE、PsaH、PetF-1、PetF-2在LA-1中表达量下调,PetC和delta表达量上调。m RNA水平,除delta基因在两种材料中表达量无显著差别外,其它基因与蛋白水平具有同样的表达趋势。现蕾后,LA-1的净光合速率(P_n)、气孔导度(Cond)显著小于LH-1,蒸腾速率(Tr)极显著小于LH-1,而胞间CO2浓度(Ci)无显著差异。与LH-1相比,LA-1的实际光化学效率(Y_Ⅱ)、光系统Ⅱ(PS Ⅱ)的潜在活性(F_v/F_0)显著减少,叶绿素荧光非光化学猝灭(NPQ)显著增大。【结论】陆地棉矮化突变体LA-1的矮化与光合作用信号途径存在相关性,为进一步研究LA-1矮化的分子机理及棉花的矮化育种提供基础。
[Objective] Here, we examined the mechanism of the photosynthetic pathway in dwarfed upland cotton(Gossypium hirsutum L.). [Methods] The upland cotton dwarf line LA-1 and the near-isogenic line LH-1 were used as research materials.We screened for dwarf-related differentially expressed genes at protein and m RNA levels by applying the isobaric tags for relative and absolute quantitation method in combination with LC-MS/MS quantitative proteomic technology and quantitative reverse-polymerase chain reaction. The difference in the photosynthetic abilities between the two materials were detected using physiological and biochemical methods. [Results] An analysis on the differential expression of proteins encoded by photosynthetic system elements revealed that PsbO, PsaE, PsaH, PetF-1 and PetF-2 were down-regulated, while PetC and delta were up-regulated in LA-1. The expression trends of the m RNA levels were the same as at the protein levels, except for those of the delta gene. The net photosynthetic rate, stomatal conductance and transpiration rate in LA-1 were lower than those LH-1, but the intercellular CO2 concentration was not significantly different after budding. This indicated that the non-stomatal factor led to a decreased net photosynthetic rate in LA-1. Chlorophyll fluorescence detection revealed that the actual photosynthetic efficiency and potential photochemical activity of photosystem II in LA-1 were significantly decreased, while non-photochemicalquenching was significantly increased. [Conclusion] The dwarfism of LA-1 is related to the photosynthetic pathway, and the results lay a foundation for exploring key dwarf-related genes and the molecular basis for dwarfism in upland cotton.
[Objective] Here, we examined the mechanism of the photosynthetic pathway in dwarfed upland cotton(Gossypium hirsutum L.). [Methods] The upland cotton dwarf line LA-1 and the near-isogenic line LH-1 were used as research materials.We screened for dwarf-related differentially expressed genes at protein and m RNA levels by applying the isobaric tags for relative and absolute quantitation method in combination with LC-MS/MS quantitative proteomic technology and quantitative reverse-polymerase chain reaction. The difference in the photosynthetic abilities between the two materials were detected using physiological and biochemical methods. [Results] An analysis on the differential expression of proteins encoded by photosynthetic system elements revealed that PsbO, PsaE, PsaH, PetF-1 and PetF-2 were down-regulated, while PetC and delta were up-regulated in LA-1. The expression trends of the m RNA levels were the same as at the protein levels, except for those of the delta gene. The net photosynthetic rate, stomatal conductance and transpiration rate in LA-1 were lower than those LH-1, but the intercellular CO2 concentration was not significantly different after budding. This indicated that the non-stomatal factor led to a decreased net photosynthetic rate in LA-1. Chlorophyll fluorescence detection revealed that the actual photosynthetic efficiency and potential photochemical activity of photosystem II in LA-1 were significantly decreased, while non-photochemicalquenching was significantly increased. [Conclusion] The dwarfism of LA-1 is related to the photosynthetic pathway, and the results lay a foundation for exploring key dwarf-related genes and the molecular basis for dwarfism in upland cotton.
引文
[1]李合生.现代植物生理学[M].北京:高等教育出版社, 2006,45-46.Li Hesheng. Modern plant physiology[M]. Beijing:Higher Education Press, 2006, 45-46.
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[3] Li Beibei, Xu Wenzhong, Xu Yunyuan, et al. Integrative study on proteomics, molecular physiology, and genetics reveals an accumulation of cyclophilin-like protein, Ta CYP20-2, leading to an increase of Rht protein and dwarf in a novel GA-insensitive mutant(gaid)in wheat[J]. Journal of Proteome Research, 2010, 9:4242-4253. http://doi.org/10.1021/pr100560v
[4] Khush G S. Green revolution:preparing for the 21stcentury[J].Genome, 1999, 42(4):646-655. http://doi.org/10.1139/gen-42-4-646
[5] Hirano I K, Ordonio R L, Matsuoka M. Engineering the lodging resistance mechanism of post-green revolution rice to meet future demands[J]. Proceeding of the Japan Academy, Series B Physical and Biology Science, 2017, 93(4):220-233. http://doi.org/10.2183/pjab.93.014
[6]季高翔,何守朴,潘兆娥,等.基于重测序开发的In Del标记定位陆地棉矮化突变体[J].棉花学报, 2018, 30(6):29-35. http://doi.org/10.11963/1002-7807.jgxdxm.20181121Ji Gaoxiang, He Shoupu, Pan Zhao'e, et al. Localization of a dwarfing mutation in upland cotton using inDel markers based on genome re-sequencing data[J]. Cotton Science, 2018, 30(06):29-35.
[7] Rojas-González J A, Soto-Súarez M, García-Díazá, et al. Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana[J]. Journal of Experimental Botany, 2015,66(9):2673-2689. http://doi.org/10.1093/jxb/erv062
[8] Chen Su, Yuan Hongmei, Liu Guifeng, et al. A label-free differential quantitative proteomics analysis of a Ta LEA-introduced transgenic Populus simonii×Populus nigra dwarf mutant[J].Molecular Biology Reports, 2012, 39:7657-7664.
[9] Hu Wenjun, Chen Lin, Qiu Xiaoyun, et al. Morphological, physiological and proteomic analyses provide insights into the improvement of castor bean productivity of a dwarf variety in comparing with a high-stalk variety[J]. Frontiers in Plant Science,2016, 7:1473-1484. http://doi.org/10.3389/fpls.2016.01473
[10] Johnson M P. Photosynthesis[J]. Essays in Biochemistry, 2016,60(3):255–273. http://doi.org/10.1042/EBC20160016
[11]黄华宏,陈奋学,童再康,等.矮生杉木光合特性及叶绿素荧光参数研究[J].北京林业大学学报, 2009, 31(2):69-73. http://doi.org/10.13332/j.1000-1522.2009.02.017Huang Huahong, Chen Fenxue, Tong Zaikang, et al. Photosynthetic properties and chlorophyll florescence parameters of dwarf Chinese fir[J]. Journal of Beijing Forestry University,2009, 31(2):69-73.
[12] Wu Chuntai, Zhou Baoliang, Zhang Tianzhen. Isolation and characterization of a sterile-dwarf mutant in Asian cotton(Gossypium arboretum L.)[J]. Journal of Genetics and Genomics, 2009, 36(6):343-353. http://doi.org/10.1016/S1673-8527(08)60123-x
[13]王静静.杂交兰矮化突变体形态及相关生理生化特性分析[D].南京:南京农业大学, 2014:51-52.Wang Jingjing. Studies on the morphology and physiological and biochemical characteristics of a dwarf mutant of cymbidium hybrids[D]. Nanjing:Nanjing Agricultural University, 2014:51-52.
[14] Tu Xiaoju, Li Juan, Wang Qiming, et al. Quantitative proteomic analysis of upland cotton stem terminal buds reveals phytohormone-related pathways associated with dwarfism[J]. Biologia Plantarum, 2016, 61(1):106-114. http://doi.org/10.1007/s10535-016-0644-0
[15] Ata-Ul-Karim S T, Cao Q, Zhu Y, et al. Non-destructive assessment of plant nitrogen parameters using leaf chlorophyll measurements in rice[J]. Frontiers in Plant Science, 2016, 7:1829.http://doi.org/10.3389/fpls.2016.01829
[16] Baena-Gonzalez E, Aro E M. Biogenesis, assembly and turnover of photosystem II units[J]. Philosophical Transactions of the Royal Society of London, Series B, 2002, 357(1426):1451-1460. http://doi.org/10.1098/rstb.2002.1141
[17] Barber J. Photosystem II:its function, structure, and implications for artificial photosynthesis[J]. Biochemistry, 2014, 79(3):185-196. http://doi.org/10.1134/S0006297914030031
[18] Popelkova H, Yocum C F. PsbO, the manganese-stabilizing protein:analysis of the structure-function relations that provide insights into its role in photosystemⅡ[J]. Journal of Photochemistry and Photobiology B:Biology, 2011, 104(1-2):179-190. http://doi.org/10.1016/j.jphotobiol.2011.01.015
[19] Murakami R, Ifuku K, Takabayashi A, et al. Functional dissection of two Arabidopsis PsbO proteins:PsbO1 and Psb O2[J].The Febs Journal, 2005, 272(9):2165-2175. http://doi.org/10.1111/j.1742-4658.2005.04636x
[20] Busch A, Hippler M. The structure and function of eukaryotic photosystem I[J]. Biochimica et Biophysica Acta(BBA)-Bioenergetics, 2011, 1807(8):864-877. http://doi.org/10.1016/j.bbabio.2010.09.009
[21] Caspy I, Nelson N. Structure of the plant photosystem I[J]. Biochemical Society Transactions. 2018, 46(2):285-294. http://doi.org/10.1042/BST20170299
[22] Jeanjean R, Latifi A, Matthijs H C, et al. The Psa E subunit of photosystem I prevents light-induced formation of reduced oxygen species in the cyanobacterium Synechocystis sp. PCC 6803[J]. Biochimica et Biophysica Acta(BBA)-Bioenergetics, 2008,1777(3):308-316. http://doi.org/10.1016/j.bbabio.2017.11.009
[23] Varotto C, Pesaresi P, Meurer J, et al. Disruption of the Arabidopsis photosystem I gene psaE1 affects photosynthesis and impairs growth[J]. The Plant Journal, 2000, 22(2):115-124.http://doi.org/10.1046/j.1365-313x.2000.00717.x
[24] Naver H, Haldrup A, Scheller H V. Cosuppression of photosystem I subunit PSI-H in Arabidopsis thaliana. Efficient electron transfer and stability of photosystem I is dependent upon the PSI-H subunit[J]. Journal of Biological Chemistry, 1999, 274(16):10784-10789. http://doi.org/10.1074/jbc.274.16.10784
[25] Schneider D, Berry S, Volkmer T, et al. PetC1 is the major Rieske iron-sulfur protein in the cy tochrome b6f complex of Synechocystis sp. PCC 6803[J]. Journal of Biological Chemistry, 2004, 279(38):39383-39388. http://doi.org/10.1074/jbc.M406288200
[26] Gelsler D A, Papke C, Obata T, et al. Downregulation of theδ-subunit reduces mitochondrial ATP synthase levels, alters respiration, and restricts growth and gametophyte development in Arabidopsis[J]. The Plant Cell, 2012, 24(7):2792-2811. http://doi.org/10.1105/tpc.112.099424
[27]张超,孙君灵,贾银华,等.棉花极端矮化突变体AS98性状与分子标记分析[J].核农学报, 2014, 28(2):186-192. http://doi.org/10.11869/j.issn.100-8551.2014.02.0186Zhang Chao, Sun Junling, Jia Yinhua, et al. Traits analysis and molecular mapping of a cotton supper-dwarf mutant AS98[J].Agriculturae Nucleatae Sinica, 2014, 28(2):186-192.
[2]沈允钢,程建峰.光合作用与农业生产[J].植物生理学报, 2010,46(6):513-516. http://doi.org/10.13592/j.cnki.ppj.2010.06.004Shen Yungang, Cheng Jianfeng. Photosynthesis and agricultural production[J]. Plant Physiology Journal, 2010, 46(6):513-516.
[3] Li Beibei, Xu Wenzhong, Xu Yunyuan, et al. Integrative study on proteomics, molecular physiology, and genetics reveals an accumulation of cyclophilin-like protein, Ta CYP20-2, leading to an increase of Rht protein and dwarf in a novel GA-insensitive mutant(gaid)in wheat[J]. Journal of Proteome Research, 2010, 9:4242-4253. http://doi.org/10.1021/pr100560v
[4] Khush G S. Green revolution:preparing for the 21stcentury[J].Genome, 1999, 42(4):646-655. http://doi.org/10.1139/gen-42-4-646
[5] Hirano I K, Ordonio R L, Matsuoka M. Engineering the lodging resistance mechanism of post-green revolution rice to meet future demands[J]. Proceeding of the Japan Academy, Series B Physical and Biology Science, 2017, 93(4):220-233. http://doi.org/10.2183/pjab.93.014
[6]季高翔,何守朴,潘兆娥,等.基于重测序开发的In Del标记定位陆地棉矮化突变体[J].棉花学报, 2018, 30(6):29-35. http://doi.org/10.11963/1002-7807.jgxdxm.20181121Ji Gaoxiang, He Shoupu, Pan Zhao'e, et al. Localization of a dwarfing mutation in upland cotton using inDel markers based on genome re-sequencing data[J]. Cotton Science, 2018, 30(06):29-35.
[7] Rojas-González J A, Soto-Súarez M, García-Díazá, et al. Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana[J]. Journal of Experimental Botany, 2015,66(9):2673-2689. http://doi.org/10.1093/jxb/erv062
[8] Chen Su, Yuan Hongmei, Liu Guifeng, et al. A label-free differential quantitative proteomics analysis of a Ta LEA-introduced transgenic Populus simonii×Populus nigra dwarf mutant[J].Molecular Biology Reports, 2012, 39:7657-7664.
[9] Hu Wenjun, Chen Lin, Qiu Xiaoyun, et al. Morphological, physiological and proteomic analyses provide insights into the improvement of castor bean productivity of a dwarf variety in comparing with a high-stalk variety[J]. Frontiers in Plant Science,2016, 7:1473-1484. http://doi.org/10.3389/fpls.2016.01473
[10] Johnson M P. Photosynthesis[J]. Essays in Biochemistry, 2016,60(3):255–273. http://doi.org/10.1042/EBC20160016
[11]黄华宏,陈奋学,童再康,等.矮生杉木光合特性及叶绿素荧光参数研究[J].北京林业大学学报, 2009, 31(2):69-73. http://doi.org/10.13332/j.1000-1522.2009.02.017Huang Huahong, Chen Fenxue, Tong Zaikang, et al. Photosynthetic properties and chlorophyll florescence parameters of dwarf Chinese fir[J]. Journal of Beijing Forestry University,2009, 31(2):69-73.
[12] Wu Chuntai, Zhou Baoliang, Zhang Tianzhen. Isolation and characterization of a sterile-dwarf mutant in Asian cotton(Gossypium arboretum L.)[J]. Journal of Genetics and Genomics, 2009, 36(6):343-353. http://doi.org/10.1016/S1673-8527(08)60123-x
[13]王静静.杂交兰矮化突变体形态及相关生理生化特性分析[D].南京:南京农业大学, 2014:51-52.Wang Jingjing. Studies on the morphology and physiological and biochemical characteristics of a dwarf mutant of cymbidium hybrids[D]. Nanjing:Nanjing Agricultural University, 2014:51-52.
[14] Tu Xiaoju, Li Juan, Wang Qiming, et al. Quantitative proteomic analysis of upland cotton stem terminal buds reveals phytohormone-related pathways associated with dwarfism[J]. Biologia Plantarum, 2016, 61(1):106-114. http://doi.org/10.1007/s10535-016-0644-0
[15] Ata-Ul-Karim S T, Cao Q, Zhu Y, et al. Non-destructive assessment of plant nitrogen parameters using leaf chlorophyll measurements in rice[J]. Frontiers in Plant Science, 2016, 7:1829.http://doi.org/10.3389/fpls.2016.01829
[16] Baena-Gonzalez E, Aro E M. Biogenesis, assembly and turnover of photosystem II units[J]. Philosophical Transactions of the Royal Society of London, Series B, 2002, 357(1426):1451-1460. http://doi.org/10.1098/rstb.2002.1141
[17] Barber J. Photosystem II:its function, structure, and implications for artificial photosynthesis[J]. Biochemistry, 2014, 79(3):185-196. http://doi.org/10.1134/S0006297914030031
[18] Popelkova H, Yocum C F. PsbO, the manganese-stabilizing protein:analysis of the structure-function relations that provide insights into its role in photosystemⅡ[J]. Journal of Photochemistry and Photobiology B:Biology, 2011, 104(1-2):179-190. http://doi.org/10.1016/j.jphotobiol.2011.01.015
[19] Murakami R, Ifuku K, Takabayashi A, et al. Functional dissection of two Arabidopsis PsbO proteins:PsbO1 and Psb O2[J].The Febs Journal, 2005, 272(9):2165-2175. http://doi.org/10.1111/j.1742-4658.2005.04636x
[20] Busch A, Hippler M. The structure and function of eukaryotic photosystem I[J]. Biochimica et Biophysica Acta(BBA)-Bioenergetics, 2011, 1807(8):864-877. http://doi.org/10.1016/j.bbabio.2010.09.009
[21] Caspy I, Nelson N. Structure of the plant photosystem I[J]. Biochemical Society Transactions. 2018, 46(2):285-294. http://doi.org/10.1042/BST20170299
[22] Jeanjean R, Latifi A, Matthijs H C, et al. The Psa E subunit of photosystem I prevents light-induced formation of reduced oxygen species in the cyanobacterium Synechocystis sp. PCC 6803[J]. Biochimica et Biophysica Acta(BBA)-Bioenergetics, 2008,1777(3):308-316. http://doi.org/10.1016/j.bbabio.2017.11.009
[23] Varotto C, Pesaresi P, Meurer J, et al. Disruption of the Arabidopsis photosystem I gene psaE1 affects photosynthesis and impairs growth[J]. The Plant Journal, 2000, 22(2):115-124.http://doi.org/10.1046/j.1365-313x.2000.00717.x
[24] Naver H, Haldrup A, Scheller H V. Cosuppression of photosystem I subunit PSI-H in Arabidopsis thaliana. Efficient electron transfer and stability of photosystem I is dependent upon the PSI-H subunit[J]. Journal of Biological Chemistry, 1999, 274(16):10784-10789. http://doi.org/10.1074/jbc.274.16.10784
[25] Schneider D, Berry S, Volkmer T, et al. PetC1 is the major Rieske iron-sulfur protein in the cy tochrome b6f complex of Synechocystis sp. PCC 6803[J]. Journal of Biological Chemistry, 2004, 279(38):39383-39388. http://doi.org/10.1074/jbc.M406288200
[26] Gelsler D A, Papke C, Obata T, et al. Downregulation of theδ-subunit reduces mitochondrial ATP synthase levels, alters respiration, and restricts growth and gametophyte development in Arabidopsis[J]. The Plant Cell, 2012, 24(7):2792-2811. http://doi.org/10.1105/tpc.112.099424
[27]张超,孙君灵,贾银华,等.棉花极端矮化突变体AS98性状与分子标记分析[J].核农学报, 2014, 28(2):186-192. http://doi.org/10.11869/j.issn.100-8551.2014.02.0186Zhang Chao, Sun Junling, Jia Yinhua, et al. Traits analysis and molecular mapping of a cotton supper-dwarf mutant AS98[J].Agriculturae Nucleatae Sinica, 2014, 28(2):186-192.