水稻颖花开放前花器官茉莉酸水平变化及浆片茉莉酸信号基因表达分析

doi:10.3864/j.issn.0578-1752.2015.06.17

摘要/Abstract

摘要： 【目的】水稻颖花开放是由浆片膨大所启动的，并与花丝伸长和花药开裂同步发生。脂类衍生的茉莉酸（JA）激素在植物生殖发育中发挥重要调控作用。本文分析水稻颖花开放过程中花器官JA水平及浆片JA信号基因表达的动态变化，以进一步揭示内源JA信号在水稻颖花开放中的作用。【方法】以粳稻中花11颖花自然开放前18 h（18 h BF）和临近开放时0 h（0 h BF）的颖花器官（小穗梗、内外稃、浆片、雄蕊、雌蕊）为试验材料。100 mg液氮研磨的样品经甲醇﹕水﹕乙酸（80﹕19﹕1，体积比）混合液提取、氮气浓缩和0.22 μm滤膜过滤后，直接应用超高速液相色谱-电喷雾-质谱系统（UFLC-ESI-MS）测定JA含量。Trizol试剂提取水稻总RNA，DNaseⅠ消化残存的基因组DNA，M-MLV逆转录酶合成第一链cDNAs。JA生物合成和信号转导相关基因的表达分析采用SYBR Green实时定量 PCR技术，以水稻GAPDH作内参。试验设置2次生物学重复，差异显著性分析采用t测验。【结果】水稻颖花开放前18 h，花器官中小穗梗JA水平（16.0 ng·g^-1 FW）最高，内外稃次之（6.1 ng·g^-1 FW），雄蕊、浆片和雌蕊极低（小于4.0 ng·g^-1 FW）。颖花开放时，小穗梗JA水平下降了69%，内外稃升高了71%，雌蕊、雄蕊和浆片JA水平分别上升至19.0、77.2和52.4 ng·g^-1 FW。颖花开放时雄蕊和浆片的JA水平显著高于其他花器官，而小穗梗JA含量最低。与JA水平变化相一致，颖花开放时JA生物合成相关基因OsDAD1、OsAOS1、OsAOC及OsOPR7在雄蕊和浆片中的表达大幅上升，在内外稃中略有上升，而在小穗梗中的表达明显下调。但是，颖花开放时这些JA生物合成相关基因在雌蕊中的表达不上调，与JA水平变化不一致。OsLOX2和OsAOS2在小穗梗和内外稃中的表达水平较高，而在浆片、雄蕊、雌蕊中的表达水平极低。伴随颖花开放时浆片JA水平的大幅上升，浆片JA信号转导途径相关基因OsJAR1、OsCOI1b以及13个OsJAZs的表达也明显上调。其中，OsJAZ11的表达水平上升幅度最大，升高了近520倍。然而， OsJAR2、OsCOI1a、OsCOI2、OsJAZ5和OsJAZ15在浆片中的表达没有上调。【结论】内源JA在水稻颖花器官中的分布具有组织和发育时期特异性。结合外源JA对水稻颖花开放的灵敏诱导，颖花开放时浆片JA的积累以及JA生物合成和信号转导途径相关基因的差异性激活，强烈表明内源JA信号介导水稻浆片膨大的调控。

关键词: 水稻, 颖花开放, 颖花器官, 茉莉酸, 浆片膨大, 基因表达

Abstract: 【Objective】 The rice floret opening is driven by swelling of lodicules, and synchronized with filament elongation and anther dehiscence. The lipid-derived jasmonic acid (JA) is an important phytohormone in regulating plant reproductive development. In this study, the dynamic changes of endogenous jasmonic acid (JA) levels in floral organs and expression of JA signaling genes in lodicules were performed to investigate the roles of JA in rice floret opening. 【Method】 Floral organs (pedicels, lemmas and paleas, stamens, lodicules and pistils) of japonica rice Zhonghua 11 were harvested at 18 h and 0 h before natural floret opening. To quantify JA contents, 100 mg samples were ground in liquid nitrogen, extracted with mixed solution (methanol﹕water﹕acetic acid, 80﹕19﹕1, v/v/v), concentrated with nitrogen gas and filtered through 0.22 μm-pore filters, then measured using ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry (UFLC-ESI-MS). The rice total RNA was isolated with Trizol reagent. First-strand cDNAs were synthesized from DNaseI-treated total RNA using M-MLV reverse transcriptase. The expression patterns of genes related JA biosynthesis and signaling pathway were analyzed by real-time PCR using SYBR Green as dye. The rice GAPDH gene was used as endogenous control. The experiments were carried out with two biological replications. The statistical significance of differences between variants was estimated by t test.【Result】 At 18 h before floret opening, the highest JA level was found in pedicels (16.0 ng·g^-1 FW), followed by lemmas and paleas (6.1 ng·g^-1 FW), and with a very low accumulation in stamens, lodicules and pistils (less than 4.0 ng·g^-1 FW). During floret opening, JA levels increased by 71% in lemmas and paleas, and sharply increased in pistils, stamens and lodicules (reaching 19.0, 77.2 and 52.4 ng·g^-1 FW, respectively), contrasting with a 69% falling in pedicels. The JA levels in stamens and lodicules were significantly higher than that in other floral organ at floret opening, whereas the lowest JA level was found in pedicels. Consistent with the changes of JA levels, the expressionof OsDAD1, OsAOS1, OsAOC and OsOPR7 involved in JA biosynthesis pathway was sharply up-regulated in stamens and lodicules, and slightly increased in lemmas and paleas, but drastically down-regulated in pedicels during floret opening. Inconsistent with an increased JA level, no obvious expression changes of these JA biosynthetic genes were observed in pistils during floret opening. OsLOX2 and OsAOS2 had a high expression level in pedicels, lemmas and paleas, but a very low expression level in pistils, stamens and lodicules. With the rising of JA level, the expression of OsJAR1, OsCOI1b and 13 OsJAZs involved in JA signaling transduction was obviously up-regulated in lodicules during floret opening. Among them, the expression level of OsJAZ11 increased the most, and exhibited a 520-fold up-regulation. However, the expression level of OsJAR2, OsCOI1a, OsCOI1b, OsJAZ5 and OsJAZ15 was not increased in lodicules.【Conclusion】 The results of study showed that the occurrence of endogenous JA signature was tissue- and developmental stage- specific in rice floret. On the basis of the opening of rice florets induced by application of exogenous JA, the accumulation of JA and the differential activation of key genes in JA biosynthesis and signaling pathway in lodicules during floret opening suggested that endogenous JA was involved in regulation of lodicule swelling.

Key words: rice (Oryza sativa L.), floret opening, floral organ, jasmonic acid (JA), lodicule swelling, gene expression

黄俊宝，何永明，曾晓春，向妙莲，付永琦. 水稻颖花开放前花器官茉莉酸水平变化及浆片茉莉酸信号基因表达分析[J]. 中国农业科学, 2015, 48(6): 1219-1227.

HUANG Jun-bao, HE Yong-ming, ZENG Xiao-chun, XIANG Miao-lian, FU Yong-qi. Changes of JA Levels in Floral Organs and Expression Analysis of JA Signaling Genes in Lodicules Before Floret Opening in Rice[J]. Scientia Agricultura Sinica, 2015, 48(6): 1219-1227.

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参考文献

[1] Heslop-Harrison Y, Heslop-Harrison J S. Lodicule function and filament extension in the grasses: Potassiumion movement and tissue specialization. Annals of Botany, 1996, 77(6): 573-582.

[2] 何永明, 曾晓春, 向妙莲, 黄俊宝, 付永琦. 水稻花时调控研究进展. 湖北农业科学, 2014, 53(7): 1489-1492.

He Y M, Zeng X C, Xiang M L, Huang J B, Fu Y Q. Advances on floret opening time of rice. Hubei Agricultural Sciences, 2014, 53(7): 1489-1492. (in Chinese)

[3] 袁隆平. 杂交水稻学. 北京: 中国农业出版社, 2002: 246-279.

Yuan L P. Hybrid Rice Sciences. Beijing: China Agricultural Press, 2002: 246-279. (in Chinese)

[4] Ishimaru T, Hirabayashi H, Ida M, Takai T, San-Oh Y A, Yoshinaga S, Ando I, Ogawa T, Kondo M. A genetic resource for early-morning flowering trait of wild rice Oryza officinalis to mitigate high temperature-induced spikelet sterility at anthesis. Annals of Botany, 2010, 106(3): 515-520.

[5] Wasternack C. Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development. Annals of Botany, 2007, 100(4): 681-697.

[6] Chini A, Fonseca S, Fernández G, Adie B, Chico J M, Lorenzo O, García-Casado G, López-Vidriero I, Lozano F M, Ponce M R, Micol J L, Solano R. The JAZ family of repressors is the missing link in jasmonate signalling. Nature, 2007, 448(7154): 666-671.

[7] Wasternack C, Hause B. Jasmonates: Biosynthesis, perception, signal transduction and action in plant stress response, growth and development. Annals of Botany, 2013, 111(6): 1021-1058.

[8] Zeng X C, Zhou X, Zhang W, Murofushi N, Kitahara T, Kamuro Y. Opening of rice floret in rapid response to methyl jasmonate. Journal of Plant Growth Regulation, 1999, 18(4): 153-158.

[9] 闫芝芬, 周燮, 马春红, 崔四平, 魏建昆. 冠毒素和茉莉酸甲酯对诱导小麦、黑麦和高羊茅草颖花开放的效应. 中国农业科学, 2001, 34(3): 334-337.

Yan Z F, Zhou X, Ma C H, Cui S P, Wei J K. Inducing effect of coronatine and methyl jasmonate on the opening of spikelets in wheat, rye and mildew. Scientia Agricultura Sinica, 2001, 34(3): 334-337. (in Chinese)

[10] 宋平, 夏凯, 吴传万, 包冬萍, 陈丽莉, 周燮, 曹显祖. 雄性不育和可育水稻开颖对茉莉酸甲酯响应的差异. 植物学报, 2001, 43(5): 480-485.

Song P, Xia K, Wu C W, Bao D P, Chen L L, Zhou X, Cao X Z. Differential response of floret opening in male-sterile and male-fertile rices to methyl jasmonte. Acta Botanica Sinica, 2001, 43(5): 480-485. (in Chinese)

[11] 林俊城, 田小海, 殷桂香, 汤吉洪, 杨志刚. 人工调节籼型杂交水稻不育系花时的研究. 中国农业科学, 2008, 41(8): 2474-2479.

Lin J C, Tian X H, Yin G X, Tang J H, Yang Z G. Artificial regulation of the flowering time of CMS lines in Indica hybrid rice seed production. Scientia Agricultura Sinica, 2008, 41(8): 2474-2479. (in Chinese)

[12] 何永明, 林拥军, 曾晓春. 水稻颖花自然开放过程中茉莉酸(JA)生物合成的变化. 作物学报, 2012, 38(10): 1891-1899.

He Y M, Lin Y J, Zeng X C. Dynamic changes of jasmonic acid biosynthesis in rice florets during natural anthesis. Acta Agronomica Sinica, 2012, 38(10): 1891-1899. (in Chinese)

[13] 丁颖. 中国水稻栽培学. 北京: 农业出版社, 1961: 87-89.

Ding Y. Rice Cultivation in China. Beijing: Agriculture Press, 1961: 87-89. (in Chinese)

[14] Liu H, Li X, Xiao J, Wang S. A convenient method for simultaneous quantification of multiple phytohormones and metabolites: Application in study of rice-bacterium interaction. Plant Methods, 2012, 8: 2.

[15] 何永明, 曾晓春. 开花期水稻颖花实时定量R-PCR分析中内参基因的选择. 江西农业大学学报, 2012, 34(6): 1086-1092.

He Y M, Zeng X C. Reference gene selection for quantitative real-time PCR normalization in rice florets during anthesis. Acta Agriculturae Universitatis Jiangxiensis, 2012, 34(6): 1086-1092. (in Chinese)

[16] Ishiguro S, Kawai-Oda A, Ueda J, Nishida I, Okada K. The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. The Plant Cell, 2001, 13(10): 2191-2209.

[17] Du H, Liu H, Xiong L. Endogenous auxin and jasmonic acid levels are differentially modulated by abiotic stresses in rice. Frontiers in Plant Science, 2013, 4(1): 397.

[18] Peng Y L, Shirano Y, Ohta H, Hibino T, Tanaka K, Shibata D. A novel lipoxygenase from rice. Primary structure and specific expression upon incompatible infection with rice blast fungus. The Journal of Biological Chemistry, 1994, 269(5): 3755-3761.

[19] Chehab E W, Perea J V, Gopalan B, Theg S, Dehesh K. Oxylipin pathway in rice and Arabidopsis. Journal of Integrative Plant Biology, 2007, 49(1): 43-51.

[20] Agrawal G K, Tamogami S, Han O, Iwahashi H, Rakwal R. Rice octadecanoid pathway. Biochemical and Biophysical Research Communications, 2004, 317(1): 1-15.

[21] Tani T, Sobajima H, Okada K, Chujo T, Arimura S, Tsutsumi N, Nishimura M, Seto H, Nojiri H, Yamane H. Identification of the OsOPR7 gene encoding 12-oxophytodienoate reductase involved in the biosynthesis of jasmonic acid in rice. Planta, 2008, 227(3): 517-526.

[22] Mei C, Qi M, Sheng G, Yang Y. Inducible overexpression of a rice allene oxide synthase gene increases the endogenous jasmonic acid PR gene expression, and host resistance to fungal infection. Molecular Plant-Microbe Interactions, 2006, 19(10): 1127-1137.

[23] Staswick P E, Tiryaki I. The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis. The Plant Cell, 2004, 16(8): 2117-2127.

[24] Fonseca S, Chini A, Hamberg M, Adie B, Porzel A, Kramell R, Miersch O, Wasternack C, Solano R. (+)-7-iso-Jasmonoyl-_L-isoleucine is the endogenous bioactive jasmonate. Nature Chemical Biology, 2009, 5(5): 344-350.

[25] Wakuta S, Suzuki E, Saburi W, Matsuura H, Nabeta K, Imai R, Matsui H. OsJAR1 and OsJAR2 are jasmonyl-_L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling. Biochemical and Biophysical Research Communications, 2011, 409(4): 634-639.

[26] Lee H Y, Seo J S, Cho J H, Jung H, Kim J K, Lee J S, Rhee S, Do Choi Y. Oryza sativa COI homologues restore jasmonate signal transduction in Arabidopsis coi1-1 mutants. PLoS ONE, 2013, 8(1): e52802.

[27] Ye H, Du H, Tang N, Li X, Xiong L. Identification and expression profiling analysis of TIFY family genes involved in stress and phytohormone responses in rice. Plant Molecular Biology, 2009, 71(3): 291-305.

[28] Hause B, Stenzel I, Miersch O, Maucher H, Kramell R, Ziegler J, Wasternack C. Tissue-specific oxylipin signature of tomato flowers: Allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles. The Plant Journal, 2000, 24(1): 113-126.

[29] 甘立军, 夏凯, 周燮. 小麦颖花开放过程中花器不同部位茉莉酸类的动态变化. 南京农业大学学报, 2005, 28(2): 26-29.

Gan L J, Xia K, Zhou X. Fluctuations of jasmonates in all floret parts during anthesis process in wheat. Journal of Nanjing Agricultural University, 2005, 28(2): 26-29. (in Chinese)

[30] Browse J. Jasmonate passes muster: A receptor and targets for the defense hormone. Annual Review of Plant Biology, 2009, 60: 183-205.

[31] Xiao Y G, Chen Y, Charnikhova T, Mulder P P J, Heijmans J, Hoogenboom A, Agalou A, Michel C, Morel J B, Dreni L, Kater M M, Bouwmeester H, Wang M, Zhu Z, Ouwerkerk P B F. OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice. Plant Molecular Biology, 2014, 86(1): 19-33.

[32] Yamada S, Kano A, Tamaoki D, Miyamoto A, Shishido H, Miyoshi S, Taniguchi S, Akimitsu K, Gomi K. Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice. Plant and Cell Physiology, 2012, 53(12): 2060-2072.

[33] Cai Q, Yuan Z, Chen M J, Yin C S, Luo Z J, Zhao X X, Liang W Q, Hu J P, Zhang D B. Jasmonic acid regulates spikelet development in rice. Nature Communications, 2014, 5: 3476.

[34] Chini A, Fonseca S, Chico J M, Fernández-Calvo P, Solano R. The ZIM domain mediates homo- and heteromeric interactions between Arabidopsis JAZ proteins. The Plant Journal, 2009, 59(1): 77-87.

[35] Pauwels L, Goossens A. The JAZ proteins: A crucial interface in the jasmonate signaling cascade. The Plant Cell, 2011, 23(9): 3089-3100.

[36] 王忠, 顾蕴洁, 高煜珠. 水稻开颖机理的探讨: Ⅲ. 浆片的结构及其在开颖过程中内含物的变化. 作物学报, 1991, 17(2): 96-101.

Wang Z, Gu Y J, Gao Y Z. Studies on the mechanism of the anthesis of rice: III. Structure of the lodicule and changes of its content during flowering. Acta Agronomica Sinica, 1991, 17(2): 96-101. (in Chinese)

[37] Song S, Qi T, Huang H, Ren Q, Wu D, Chang C, Peng W, Liu Y, Peng J, Xie D. The jasmonate-ZIM domain proteins interact with the R2R3-MYB transcription factors MYB21 and MYB24 to affect jasmonate-regulated stamen development in Arabidopsis. The Plant Cell, 2011, 23(3): 1000-1013.

[38] Zhang H, Liang W, Yang X, Luo X, Jiang N, Ma H, Zhang D. Carbon starved anther encodes a MYB domain protein that regulates sugar partitioning required for rice pollen development. The Plant Cell, 2010, 22(3): 672-689.