作物学报 ›› 2013, Vol. 39 ›› Issue (11): 1952-1961.doi: 10.3724/SP.J.1006.2013.01952
张小红1,许鹏博1,2,郭萌萌1,2,徐兆师2,李连城2,陈明2,*,马有志2
ZHANG Xiao-Hong1,XU Peng-Bo1,2,GUO Meng-Meng1,2,XU Zhao-Shi2,LI Lian-Cheng2,CHEN Ming2,*,MA You-Zhi2
摘要:
拟南芥G蛋白复合体(异源三聚体包括α、β、γ亚基)参与植物多个信号转导途径,G蛋白复合体通过膜上的G蛋白偶联受体(GPCR)接受胞外信号后通过3个亚基将信号传递给下游效应器。目前,有关植物G蛋白复合体的效应器及其信号传递途径的报道较少,寻找新的G蛋白的效应器有助于阐明G蛋白复合体相关的信号传导途径。本研究以拟南芥G蛋白α亚基GPA1为诱饵蛋白,利用泛素分离系统筛选拟南芥cDNA文库,获得一个与GPA1互作的铜离子结合蛋白AtBCB。荧光双分子杂交(BiFC)试验证明,GPA1与AtBCB的互作发生在细胞膜上。基因表达特性分析结果显示,GPA1和AtBCB受金属铝胁迫的诱导表达。进一步以野生型拟南芥(WT)、GPA1拟南芥突变体gpa1-4和AtBCB拟南芥突变体bcb为材料,研究该基因对植物耐金属铝胁迫的功能,结果显示,在无胁迫情况下,2个突变体和WT根部的丙二醛含量无显著差异;在100 µmol L–1 Al3+处理下,gpa1-4突变体根部丙二醛含量显著(P<0.05)低于WT低;bcb根部丙二醛含量极显著(P<0.01)高于WT。对3个铝胁迫响应基因(苹果酸转运体基因AtALMT1、半类型ABC转运蛋白基因ALS1和ABC转运蛋白基因ALS3)的表达进行Real-time PCR分析,比较它们在突变体和野生型之间的表达差异,发现在有铝和无铝处理情况下,ALS1和ALS3的表达水平在突变体和WT间均无显著差异;在铝处理下,gpa1-4中AtALMT1的表达量极显著高于WT;在bcb中的表达量显著低于WT。以上结果表明,植物通过细胞膜上的G蛋白α亚基GPA1和铜离子结合蛋白AtBCB的相互作用调控下游基因AtALMT1的表达,参与植物对铝胁迫的响应,其中GPA1对铝胁迫耐受起负向作用,AtBCB对铝胁迫耐受起正向作用。
| [1]Chakravorty D, Trusov Y, Zhang W, Acharya B R, Sheahan M B, McCurdy D W, Assmann S M, Botella J R. An atypical heterotrimeric G-protein gamma-subunit is involved in guard cell K+-channel regulation and morphological development in Arabidopsis thaliana. Plant J, 2011, 67: 840–851[2]Warpeha K M, Upadhyay S, Yeh J, Adamiak J, Hawkins S I, Lapik Y R, Anderson M B, Kaufman L S. The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis. Plant Physiol, 2007, 143: 1590–1600[3]Botto J F, Ibarra S, Jones A M. The heterotrimeric G-protein complex modulates light sensitivity in Arabidopsis thaliana seed germination. Photochem Photobiol, 2009, 85: 949–954[4]Fox A R, Soto G C, Jones A M, Casal J J, Muschietti J P, Mazzella M A. Cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin synthesis in Arabidopsis. Plant Mol Biol, 2012, 80: 315–324[5]Wang X Q, Ullah H, Jones A M, Assmann S M. G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science, 2001, 292: 2070–2072[6]Mudgil Y, Uhrig J F, Zhou J, Temple B, Jiang K, Jones A M. Arabidopsis N-MYC downregulated-like 1, a positive regulator of auxin transport in a G protein-mediated pathway. Plant Cell, 2009, 21: 3591–3609[7]Huang J, Taylor J P, Chen J G, Uhrig J F, Schnell D J, Nakagawa T, Korth K L, Jones A M. The plastid protein thylakoid formation 1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis. Plant Cell, 2006, 18: 1226–1238[8]Assmann S M, Fan L M, Zhang W, Chen J G, Taylor J P, Jones A M. Abscisic acid regulation of guard-cell K+ and anion channels in G beta- and RGS-deficient Arabidopsis lines. Proc Natl Acad Sci USA, 2008, 105: 8476–8481[9]Llorente F, Blanco C A, Rodriguez C S, Jorda L, Molina A. ERECTA receptor-like kinase and heterotrimeric G protein from Arabidopsis are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina. Plant J, 2005, 43: 165–180[10]Lapik Y R, Kaufman L S. The Arabidopsis cupin domain protein AtPirin1 interacts with the G protein alpha subunit GPA1 and regulates seed germination and early seedling development. Sci STKE, 2003, 15: 1578–1590[11]Assmann S M, Pandey S. The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling. Plant Cell, 2004, 16: 1616–1632[12]Zhao J, Wang X. Arabidopsis phospholipase D alpha 1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. Sci Signal, 2004, 279: 1794–1800[13]Broder Y C, Katz S, Aronheim A. The ras recruitment system, a novel approach to the study of protein–protein interactions. Curr Biol, 1998, 8: 1121–1130[14]Hubsman M, Yudkovsky G, Aronheim A. A novel approach for the identification of protein–protein interaction with integral membrane proteins. Nucl Acids Res, 2001, 29: e18[15]Yuan G-L (苑国良). Characterizing Arabidopsis G Protein Interactors by Reverse Ras Recruitment System. PhD Disseratation of Shandong Agricultural University, 2009 (in Chinese with English abstract)[16]Kaufman L S, Warpeha K M, Gibbons J, Carol A, Slusser J, Tree R, Durham W. Adequate phenylalanine synthesis mediated by G protein is critical for protection from UV radiation damage in young etiolated Arabidopsis thaliana seedlings. Plant Cell Environ, 2008, 31: 1756–1770[17]Friedman E J, Wang H X, Jiang K, Perovic I, Deshpande A, Pochapsky T C, Temple B R S, Hicks S N, Harden T K, Jones A M. Aci-reduction dioxygenase1 (ARD1) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis. 2011, 286: 30107–30118[18]Tsugama D, Liu H, Liu S, Takano T. Arabidopsis heterotrimeric G protein β subunit interacts with a plasma membrane 2C-type protein phosphatase, PP2C52. Biochimt Biophysica Acta (BBA)-Mol Cell Res, 2012: 2254–2260[19]Stagljar I, Korostensky C, Johnsson N, te Heesen S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci USA, 1998, 95: 5187–5192[20]Takumi S, Otani M, Shimada T. Effect of six promoter-intron combinations on transient reporter gene expression in einkorn, emmer and common wheat cells by particle bombardment. Plant Sci, 1994, 103: 161–166[21]Li M(李敏), Yang S(杨双), Ruan Y-Y(阮燕晔), Fan J-J(樊金娟), Zhang L-J(张立军). PCR identification Arabidopsis T-DNA mutant of atsuc3. Plant Physiol Commun (植物生理学通讯), 2006, 42(1): 91–94 (in Chinese)[22]Murphy A, Taiz L. A new vertical mesh transfer technique for metal-tolerance studies in Arabidopsis–ecotypic variation and copper-sensitive mutants. Plant Physiol, 1995, 108: 29–38[23]Ono K, Yamamoto Y, Hachiya A, Matsumoto H. Synergistic inhibition of growth by aluminum and iron of tobacco (Nicotiana tabacum L.) cells in suspension culture. Plant Cell Physiol, 1995, 36: 115–125[24]Van Gysel A, Van Montagu M, Inzé D. A negatively light-regulated gene from Arabidopsis thaliana encodes a protein showing high similarity to blue copper-binding proteins. Gene, 1993, 136: 79–85[25]Richards K D, Schott E J, Sharma Y K, Davis K R, Gardner R C. Aluminum induces oxidative stress genes in Arabidopsis thaliana. Plant Physiol, 1998, 116: 409–418[26]Ezaki B, Sasaki K, Matsumoto H, Nakashima S. Functions of two genes in aluminium (Al) stress resistance: repression of oxidative damage by the AtBCB gene and promotion of efflux of Al ions by the NtGDI1gene. J Exp Bot, 2005, 56: 2661–2671[27]Kochian L V. Cellular Mechanisms of Aluminum Toxicity and Resistance in Plants. Annu Rev Plant Physiol Plant Mol Biol, 1995, 46: 237–260[28]Ezaki B, Gardner R C, Ezaki Y, Matsumoto H. Expression of aluminum-induced genes in transgenic Arabidopsis plants can ameliorate aluminum stress and/or oxidative stress. Plant Physiol, 2000, 122: 657–666[29]Ezaki B, Katsuhara M, Kawamura M, Matsumoto H. Different mechanisms of four aluminum (Al)-resistant transgenes for Al toxicity in Arabidopsis. Plant Physiol, 2001, 127: 918–927[30]Hoekenga O A, Maron L G, Piñeros M A, Cançado G M, Shaff J, Kobayashi Y, Ryan P R, Dong B, Delhaize E, Sasaki T. AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. Proc Natl Acad Sci USA, 2006, 103: 9738–9743[31]Larsen P B, Geisler M J, Jones C A, Williams K M, Cancel J D. ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis. Plant J, 2004, 41: 353–363[32]Larsen P B, Cancel J, Rounds M, Ochoa V. Arabidopsis ALS1 encodes a root tip and stele localized half type ABC transporter required for root growth in an aluminum toxic environment. Planta, 2007, 225: 1447–1458[33]Hamm H E. The many faces of G protein signaling. J Biol Chem, 1998, 273: 669–672[34]Adman E T. Structure and function of copper-containing proteins. Curr Opin Struct Biol, 1991, 1: 895–904[35]Joo J H, Wang S, Chen J, Jones A, Fedoroff N V. Different signaling and cell death roles of heterotrimeric G protein α and β subunits in the Arabidopsis oxidative stress response to ozone. Plant Cell Onl, 2005, 17: 957–970[36]Delhaize E, Ryan P R, Hebb D M, Yamamoto Y, Sasaki T, Matsumoto H. Engineering high-level aluminum tolerance in barley with the ALMT1 gene. Proc Natl Acad Sci USA, 2004, 101: 15249–15254[37]Larsen P B, Degenhardt J, Tai C Y, Stenzler L M, Howell S H, Kochian L V. Aluminum-resistant Arabidopsis mutants that exhibit altered patterns of aluminum accumulation and organic acid release from roots. Plant Physiol, 1998, 117: 9–17[38]Iuchi S, Koyama H, Iuchi A, Kobayashi Y, Kitabayashi S, Kobayashi Y, Ikka T, Hirayama T, Shinozaki K, Kobayashi M. Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance. Proc Natl Acad Sci USA, 2007, 104: 9900–9905[39]Xia J, Yamaji N, Kasai T, Ma J F. Plasma membrane-localized transporter for aluminum in rice. Proc Natl Acad Sci USA, 2010, 107: 18381–18385 |
| [1] | 崔致远, 秦晨展, 刘星雨, 张海, 曾康, 黄国强, 徐景升. 甘蔗类四跨膜蛋白ScTSPAN18与6K2互作应答SCMV侵染研究[J]. 作物学报, 2026, 52(6): 1618-1630. |
| [2] | 刘晓宁, 张颖, 蔡曼蕾, 马昊, 苗智博, 曹宁, 连荣芳, 徐全乐. LsSAT2与LsAAE3互作调控山黧豆ODAP水平[J]. 作物学报, 2025, 51(8): 2220-2227. |
| [3] | 周恩强, 缪亚梅, 周瑶, 姚梦楠, 赵娜, 王永强, 朱宇翔, 薛冬, 李宗迪, 石宇欣, 李波, 汪凯华, 顾春燕, 王学军, 魏利斌. 基于种子发育转录组的豌豆bZIP基因家族分析及种子发育候选基因的鉴定[J]. 作物学报, 2025, 51(4): 914-931. |
| [4] | 玉泉馨, 杨宗桃, 张海, 程光远, 焦文迪, 曾康, 罗廷绪, 黄国强, 王璐, 徐景升. 甘蔗类钙调素ScCML13与SCMV运动蛋白P3N-PIPO的互作研究[J]. 作物学报, 2024, 50(7): 1855-1866. |
| [5] | 李万, 李成, 程敏, 吴芳. 磷转运蛋白StPHO1.2提高马铃薯耐热性[J]. 作物学报, 2024, 50(2): 394-402. |
| [6] | 王连南, 李远超, 余乃通, 麦伟涛, 李亚军, 陈新. MeTCP3a转录因子在木薯叶片发育中的功能鉴定[J]. 作物学报, 2024, 50(11): 2720-2730. |
| [7] | 玉泉馨, 杨宗桃, 张海, 程光远, 周营栓, 焦文迪, 曾康, 罗廷绪, 黄国强, 张木清, 徐景升. 甘蔗VAMP相关蛋白ScPVA12与甘蔗花叶病毒P3N-PIPO的互作研究[J]. 作物学报, 2023, 49(9): 2472-2484. |
| [8] | 柏成成, 姚小尧, 王雨璐, 王赛玉, 李金莹, 蒋有为, 靳舒荣, 陈春杰, 刘渔, 魏星玥, 徐新福, 李加纳, 倪郁. 甘蓝型油菜长链烷烃合成相关基因的克隆及其与BnCER1-2的互作[J]. 作物学报, 2023, 49(4): 1016-1027. |
| [9] | 杜鹃, 彭晓君, 侯娟, 刘腾飞, 刘增, 宋波涛. 马铃薯淀粉酶StBAM9互作蛋白的鉴定及其互作机制分析[J]. 作物学报, 2023, 49(10): 2643-2653. |
| [10] | 杨宗桃, 焦文迪, 张海, 张克闽, 程光远, 罗廷绪, 曾康, 周营栓, 徐景升. 甘蔗谷胱甘肽硫转移酶ScGSTF1与P3N-PIPO互作应答甘蔗花叶病毒侵染的研究[J]. 作物学报, 2023, 49(10): 2665-2676. |
| [11] | 刘淑娴, 杨宗桃, 程光远, 张海, 周营栓, 商贺阳, 黄国强, 徐景升. 甘蔗易化子家族蛋白ScZIFL1与6K2互作应答SCMV侵染[J]. 作物学报, 2022, 48(12): 3080-3090. |
| [12] | 许彬, 曹绍玉, 苏甜, 彭梦玲, 吕霞, 李振林, 张国平, 许俊强. 结球甘蓝类钙调蛋白CMLs与花粉萌发NPG1及NPGRs相互作用研究[J]. 作物学报, 2022, 48(11): 2934-2944. |
| [13] | 张海, 程光远, 杨宗桃, 刘淑娴, 商贺阳, 黄国强, 徐景升. 甘蔗PsbR亚基应答SCMV侵染及其与SCMV-6K2的互作[J]. 作物学报, 2021, 47(8): 1522-1530. |
| [14] | 李兰兰, 母丹, 严雪, 杨陆可, 林文雄, 方长旬. OsPAL2;3对水稻化感抑制稗草能力的调控作用[J]. 作物学报, 2021, 47(2): 197-209. |
| [15] | 孟钰玉, 魏春茹, 范润侨, 于秀梅, 王逍冬, 赵伟全, 魏新燕, 康振生, 刘大群. 小麦TaPP2-A13基因的表达响应逆境胁迫并与SCF复合体接头蛋白TaSKP1相互作用[J]. 作物学报, 2021, 47(2): 224-236. |
|
||