调控拟南芥ROP10介导ABA信号转导的RopGEFs基因筛选与初步分析
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摘要
小G蛋白ROPs(Rho-related GTPase from plants)是植物生长发育过程中重要的“分子开关”,参与调控了多种信号转导过程。ROP10蛋白是ABA应答的特异性负调控因子,但其信号转导机理尚未阐明。ROP鸟苷酸交换因子RopGEFs (guanine nucleotide exchange factor)为ROPs的上游调控子,使非活性ROPs转变为活性形式。拟南芥基因组编码14个植物特有的RopGEFs,但是他们在ABA信号转导途径中的作用尚有待确定。本文筛选并初步分析作用于ROP10上游以调控ABA应答的RopGEFs,为更详尽认识ROP10介导的ABA信号传导通路及作用机制奠定基础。
1)本文确定了RopGEFs的组织表达特异性。在此基础上,比较了拟南芥野生型幼苗材料中RopGEFs及ROP10基因在外源ABA不同浓度和不同时间处理下的表达情况。不同的RopGEFs显示出不同的ABA应答水平,其中RopGEF1,2,3,5,11和14可能参与了ABA信号转导过程；
2)以RopGEFs T-DNA插入缺失突变体为材料,研究外源ABA不同浓度处理下突变体中ROP10基因的表达情况。结果显示ROP10基因的转录水平在不同突变体中显示不同的表达变化模式。其中ROP10基因在RopGEF1 T-DNA插入缺失突变体中的表达基本不受ABA处理浓度的影响,推测RopGEF1可能在ROP10介导的ABA信号转导途经中起调控作用。另外,RopGEF2,5,11和14也可能通过其他信号途径参与了ABA应答；
3)对ROP10, RopGEF1,2,11和14 T-DNA插入缺失突变体在外源ABA处理后的萌发率、根长、气孔开度及持水性等表型研究表明,突变体的生理表型相对野生型没有明显的变化。这可能与RopGEFs基因家族的功能冗余有关。
本研究初步筛选出可能参与ROP10介导的ABA传导途径的RopGEFs基因,其功能仍需通过突变体构建、分析和蛋白质学方法进一步研究验证。
Small G protein ROPs (Rho-related GTPase from plants) plays pivotal roles in plant growth, development and signal transduction by acting as "molecular switches" ROP10 participents specifically in the negative regulation of ABA signaling, but its signaling mechanism remains poorly understood. RopGEFs (guanine nucleotide exchange factor) acted as an up-regulator to catalyse the ROPs cycling between actived sate and inactived sate. It have been cloned 14 RopGEFs in Arabidopsis. The screening of the RopGEFs as a regulator in ROP10-dependent ABA signaling pathway may help us to understand the ABA transduction pathway and its mechanism.
1) The tissue-specific expressions of RopGEFs in Arabidopsis thaliana was confirmed. Then the expression profiles of ROP10 and RopGEFs seedlings in response to ABA with different concentration and time-course were detected and different RopGEFs showed verious expression characters. These results suggest that RopGEF1,2,3,5,11 and 14 may participant in ABA signal transdution.
2) The expression of ROP10 in RopGEFs T-DNA insert mutants was measured in response to ABA. The results show that expression levels of ROP 10 appeared differently in these mutants under ABA treatment. The RopGEF1 may play a role in ROP10-dependent ABA signaling pathway as its expression was almost not affected by ABA concentration. The RopGEF 2,5,11 and 14 may also invole in ABA signal transdution by other regulation pahtway.
3) The phenotypes such as seed germination rate, root length, stomatal aperture and water loss between the mutants and wild-type under ABA treatment were observed and there was no obvious distinction could be found between the mutants and the wide type. This may be explained by the gene redundancy. More detailed functional study for the preliminary screened RopGEFs in this work will be followed in the future.
1)本文确定了RopGEFs的组织表达特异性。在此基础上,比较了拟南芥野生型幼苗材料中RopGEFs及ROP10基因在外源ABA不同浓度和不同时间处理下的表达情况。不同的RopGEFs显示出不同的ABA应答水平,其中RopGEF1,2,3,5,11和14可能参与了ABA信号转导过程；
2)以RopGEFs T-DNA插入缺失突变体为材料,研究外源ABA不同浓度处理下突变体中ROP10基因的表达情况。结果显示ROP10基因的转录水平在不同突变体中显示不同的表达变化模式。其中ROP10基因在RopGEF1 T-DNA插入缺失突变体中的表达基本不受ABA处理浓度的影响,推测RopGEF1可能在ROP10介导的ABA信号转导途经中起调控作用。另外,RopGEF2,5,11和14也可能通过其他信号途径参与了ABA应答；
3)对ROP10, RopGEF1,2,11和14 T-DNA插入缺失突变体在外源ABA处理后的萌发率、根长、气孔开度及持水性等表型研究表明,突变体的生理表型相对野生型没有明显的变化。这可能与RopGEFs基因家族的功能冗余有关。
本研究初步筛选出可能参与ROP10介导的ABA传导途径的RopGEFs基因,其功能仍需通过突变体构建、分析和蛋白质学方法进一步研究验证。
Small G protein ROPs (Rho-related GTPase from plants) plays pivotal roles in plant growth, development and signal transduction by acting as "molecular switches" ROP10 participents specifically in the negative regulation of ABA signaling, but its signaling mechanism remains poorly understood. RopGEFs (guanine nucleotide exchange factor) acted as an up-regulator to catalyse the ROPs cycling between actived sate and inactived sate. It have been cloned 14 RopGEFs in Arabidopsis. The screening of the RopGEFs as a regulator in ROP10-dependent ABA signaling pathway may help us to understand the ABA transduction pathway and its mechanism.
1) The tissue-specific expressions of RopGEFs in Arabidopsis thaliana was confirmed. Then the expression profiles of ROP10 and RopGEFs seedlings in response to ABA with different concentration and time-course were detected and different RopGEFs showed verious expression characters. These results suggest that RopGEF1,2,3,5,11 and 14 may participant in ABA signal transdution.
2) The expression of ROP10 in RopGEFs T-DNA insert mutants was measured in response to ABA. The results show that expression levels of ROP 10 appeared differently in these mutants under ABA treatment. The RopGEF1 may play a role in ROP10-dependent ABA signaling pathway as its expression was almost not affected by ABA concentration. The RopGEF 2,5,11 and 14 may also invole in ABA signal transdution by other regulation pahtway.
3) The phenotypes such as seed germination rate, root length, stomatal aperture and water loss between the mutants and wild-type under ABA treatment were observed and there was no obvious distinction could be found between the mutants and the wide type. This may be explained by the gene redundancy. More detailed functional study for the preliminary screened RopGEFs in this work will be followed in the future.
引文
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[3]Cultler S, Ghassemian M, Bonetta D, et al. A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis. Science,1996, 273(5279):1239-1241
[4]Himmelbach A, Hoffmann T, Leube M, et al. Homeodomain protein ATHB6 is a target of the protein phosphatase ABI1 and regulates hormone responses in Arabidopsis. Embo Journal,2002,21(12):3029-3038
[5]Xiong L, Lee Bh, Ishitani M, et al. FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Development,2001,15(15):1971-1984
[6]Lu C, Fedoroff N. A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin. Plant Cell,2000,12(12):2351-2366
[7]Finkelstein R R, Gampala S S, Rock C D. Abscisic acid signaling in seeds and seedlings. Plant Cell,2002,14(Suppl):S15-S45
[8]王绍明,李学禹.中国拟南芥属植物种质资源及其地理分布.石河子大学学报(自然科学版),2001,5(2)：102-108
[9]Meinke D W, Cherry J M, Dean C, et al. Arabidopsis thaliana:A model plant for genome analysis. Science,1998,2(5389):678-682
[10]Eagles C F, Wareing P E. The role of growth substances in the regulation of onion bud dormancy. Physiologia Plantarum,1964,17(3):697-709
[11]童超.ABA生理功能与信号转导相关综述.科技资讯,2008,10(2)：44-45
[12]Pastor A, Lopez-Carbonell M, Alegre L. Abscisic acid immunolocalization and ultrastructural changes in water-stressed lavender (Lavandula stoechas L) plants. Plant Physiology,1999,105(2):272-279
[13]Rensburg L V, Kruger H. Immunogold localization and quantification of cellular and subcellular abscisic acid, prior to and during drought stress. Biotech Histochem,1996,71(1):38-43
[14]Wilkinson S, Corlett J E, Oger L, et al. Effects of xylem pH on transpiration from wild-type and flacca tomato leaves. Plant Physiology,1998, 117(2):703-709
[15]Zhang J, Davies W J. Abscisic acid produced in dehydrating roots may enable the plant to measure the water status of the soil[J]. Plant Cell and Environment, 1998,13(1):277-285
[16]胡学博,宋凤鹏,郑重.高等植物中蛋白磷酸酶2C的结构与功能.细胞生物学杂志,2005,27(1)：29-34
[17]Xiong L, Zhu J K. Cell signaling during cold, drought and salt stress[J]. Plant Physiology,2003,133(1):29-36
[18]Zhu, J K. Salt and drought stress signal transduction in plants. Annual Review of Plant Biology,2002,53:247-73
[19]Anderson B E, Ward J M, Schroeder J I. Evidence for extracellular recption site for abscisic acid in commelina guard cells. Plant Physiology,1994, 104(3):1177-1182
[20]Pandey G K, Cheong Y H, Kim K N, et al. The calcium sensor calcineurin B-Like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis. Plant Cell,2004,16(3):1912-1924
[21]Gosti F, Beaudoin N, Serizet C, et al. ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling. Plant Cell,1999,11 (4):1897-1910
[22]Rodriguez P L. Protein phosphatase 2C (PP2C) function in higher plants. Plant Molecular Biology,1998,38(3):919-927
[23]Merlot, S., et al. The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. Plant J,2001. 25(3):295-303
[24]Welin B V, Olson A, Nylander M, et al. Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana. Plant Molecular Biology,1994,10(26):131-144
[25]Abe H, Urao T, Ito T, et al. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell,2003, 15(1):63-78
[26]Ghassemian M, Nambara E, Cutler S, et al. Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis. The Plant Cell,2000, 12:1117-1126
[27]Assmann S M. Signal transduction in guard cells. Annual Review of Cell Biology,1993,9:345-375
[28]Blatt M R, Grabov A. Signaling gates in abscisic acid-mediated control of guard cell ion channels. Physiol Plant,1997,100(3):481-490
[29]Li J, Wang X Q, Watson M B, et al. Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase. Science,2000, 287(5451):300-303
[30]张骁,上官周平,高俊凤,等.保卫细胞的ABA信号转导.2001,27(5)：361-368
[31]Dure L S, Crouch M, Harada J, et al. Common amino acid sequence domain among the LEA proteins of hughen plants. Plant Molecular Biology,1989, 12(2):475-486
[32]Yanaguchi-Shunnzaki K, Koizum M, Urao S, et al. Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana:sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant and Cell Physiology,1992, 33(3):217-224
[33]Schultz T F, Quatrano R S. Evidence for surface perception of abscisic acid by rice suspension cells as assayed by Em gene expression. Plant Science,1997, 130(1):63-71
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