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作物学报 ›› 2018, Vol. 44 ›› Issue (12): 1802-1808.doi: 10.3724/SP.J.1006.2018.01802

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

拟南芥RPT2与RIP1互作调节下胚轴向光弯曲的功能鉴定

赵翔,朱自亿,王潇楠,慕世超,张骁()   

  1. 河南大学生命科学学院/棉花生物学国家重点实验室/植物逆境生物学重点实验室, 河南开封475004
  • 收稿日期:2018-03-21 接受日期:2018-08-20 出版日期:2018-12-12 网络出版日期:2018-08-20
  • 通讯作者: 张骁
  • 基金资助:
    本研究由河南省高等学校青年骨干教师培养计划项目(2015GGJS-020);河南省高校科技创新人才支持计划项目(17HASTIT035);国家自然科学基金项目(31670289);国家自然科学基金项目(31570294);河南省基础与前沿技术研究计划项目资助(142300413225)

Functional Analysis of Hypocotyl Phototropism Modulated by RPT2-Interacting Protein RIP1 in Arabidopsis thaliana L.

Xiang ZHAO,Zi-Yi ZHU,Xiao-Nan WANG,Shi-Chao MU,Xiao ZHANG()   

  1. Key Laboratory of Plant Stress Biology / State Key Laboratory of Cotton Biology / College of Life Sciences, Henan University, Kaifeng 475004, Henan, China
  • Received:2018-03-21 Accepted:2018-08-20 Published:2018-12-12 Published online:2018-08-20
  • Contact: Xiao ZHANG
  • Supported by:
    This study was supported by the Training Plan for Young Backbone Teachers in Colleges and Universities in Henan(2015GGJS-020);the Sponsored by Program for Science and Technology Innovation Talents in Universities of Henan Province(17HASTIT035);the National Natural Science Foundation of China(31670289);the National Natural Science Foundation of China(31570294);the Basic and Advanced Technology Research Project of Henan (142300413225)(142300413225)

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摘要：

拟南芥突变体rpt2-2表型类似于蓝光受体双突变体phot1 phot2, 缺失强蓝光诱导的下胚轴向光弯曲, 不同于双突变体phot1 rpt2-2下胚轴向光性正常, 表明在RPT2上游存在受PHOT1抑制的旁路调节途径。本文以RPT2为诱饵蛋白进行酵母文库筛选, 成功筛选到包括JAC1 和PHOT1在内的6个与RPT2互作的蛋白(RIPs, RPT2 Interacting Proteins)。酵母互作验证显示, 其中有4个蛋白可以与RPT2相互作用。表型分析显示基因RIP1对应的2个突变体rip1-1和rip1-2, 发现rip1-1和rip1-2单突变体下胚轴向光弯曲正常, 而rpt2-2 rip1-1和rpt2-2 rip1-2双突变体表型类似于phot1 rpt2-2双突变体, 恢复拟南芥下胚轴向光弯曲反应, 暗示RIP1蛋白可能调节PHOT1介导强蓝光抑制反应, 分析鉴定蛋白RIP1的生物学功能将有助于揭示PHOT1介导强蓝光抑制反应的机制。

关键词: 拟南芥, 蓝光, 向光素, RPT2

Abstract:

The rpt2-2 single mutant lost phototropism, but phot1 rpt2-2 double mutant shows phototropic response, indicating that PHOT1 has a function in inhibiting hypocotyl phototropism, and RPT2 maybe play vital role in these prosses. Here, we used RPT2 as bait protein to screen yeast library and successfully obtained six proteins interacted with RPT2 including JAC1 and PHOT1. Yeast hybridization verified that four of these proteins could interact with RPT2. Phenotypic analysis of two mutants of gene RIP1 showed that the single mutant rip1-1 and rip1-2 had normal phototropism in response to high blue light, but rpt2-2 rip1-1 and rpt2-2 rip1-2 double mutant were defective in phototropism, similar with the phot1 rpt2-2 double mutant. These results suggested that this protein may modulate PHOT1-mediated high blue light inhibitory response. Functional analysis of this protein will be helpful to promote the discovery of the mechanism of phot1-mediated inhibitory response.

Key words: Arabidopsis thaliana, blue light, phototropins, RPT2

图1

单侧强弱蓝光差异诱导拟南芥野生型和突变体下胚轴向光弯曲 A和B: 拟南芥野生型和突变体rpt2-2、phot1、phot1 rpt2-2、phot2 rpt2-2和phot1 phot2响应单侧蓝光(A: 100 μmol m-2 s-1; B: 0.01 μmol m-2 s-1)的下胚轴向光弯曲表型。C和D: 蓝光(C: 100 μmol m-2 s-1; D: 0.01 μmol m-2 s-1)诱导下胚轴向光弯曲度测量统计结果。图中每个数据分别来自3次独立重复试验, 大约15~20颗苗的平均值±标准误。"

图2

拟南芥RPT2互作蛋白酵母库筛选体系建立 A: 重组酵母双杂交载体的构建; B: 表达蛋白的毒性检测; C: 酵母库筛选诱饵蛋白自激活验证; D: 酵母库筛选结果显色分析。"

表1

RPT2互作蛋白筛选目标蛋白列表"

目的基因
Bait gene		 筛选基因
Prey gene		 功能注释
Annotation
RIP4/PHOT1 蓝光受体PHOT1, 可调节强弱蓝光诱导的下胚轴向光反应, 调节弱光诱导的叶绿体聚光运动、向光性。
Blue light photoreceptor. Mediates blue light-induced phototropism, chloroplast accumulation, stomatal opening, and leaf flattening.
RIP2/KIX8 编码KIX8蛋白, 与PPD2相互作用, 调节植物拟分生组织分裂, 调控植物叶片大小发育。
Encodes KIX8. Interacts with PPD2. Regulates meristem division and leaf size.
RIP1 功能未知。Unknown protein.
RPT2 RIP3 功能未知。Unknown protein.
RIP6 核蛋白, 功能未知。Hypothetical nuclear protein.
RIP5/JAC1 JAC1蛋白, 定位在细胞质及叶绿体中, 参与叶绿体聚光运动但不参与叶绿体避光运动, 同时响应蓝光诱导。
Located in the chloroplast and cytoplasm. Involved in chloroplast accumulation, chloroplast avoidance movement, and cellular response to blue light.

图3

RPT2与PHOT1、JAC1、RIP1和RIP6蛋白相互作用 A: RPT2与PHOT1相互作用; B: RPT2与RIP1相互作用; C: RPT2与JAC1相互作用; D: RPT2与RIP6相互作用。"

图4

拟南芥rip1突变体下胚轴弯曲反应 A和B: 拟南芥野生型和突变体 rpt2-2、rip1-1和rip1-2响应单侧蓝光(A: 100 μmol m-2 s-1; B: 0.01 μmol m-2 s-1)的下胚轴向光弯曲表型; C: 弯曲度的测量统计结果。图中每个数据分别来自3次独立重复试验, 大约15~20颗苗的平均值±标准误。"

图5

拟南芥双突变体rpt2-2 rip1恢复下胚轴向光弯曲反应 A: 拟南芥野生型和突变体 rpt2-2、 rpt2-2 rip1-1和rpt2-2 rip1-2响应单侧蓝光(100 μmol m-2 s-1)的下胚轴向光弯曲表型; B: 弯曲度的测量统计结果。图中每个数据分别来自3次独立重复试验, 大约15~20颗苗的平均值±标准误。"

[1] Takemiya A, Inoue S, Doi M, Kinoshita T, Shimazaki K . Phototropins promote plant growth in response to blue light in low light environments. Plant Cell, 2005, 17: 1120-1127
doi: 10.1105/tpc.104.030049 pmid: 15749755
[2] de Carbonnel M, Davis P, Roelfsema M R G, Inoue S, Schepens I, Lariguet P, Geisler M, Shimazaki K, Hangarter R, Fankhauser C . The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning. Plant Physiol, 2010,152:1391-1405
doi: 10.1104/pp.109.150441
[3] Huala E, Oeller P W, Liscum E, Han I S, Larsen E, Briggs W R . Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science, 1997,278:2120-2123
doi: 10.1126/science.278.5346.2120 pmid: 9405347
[4] Demarsy E, Fankhauser C . Higher plants use LOV to perceive blue light. Curr Opin Plant Biol, 2009,12:69-74
doi: 10.1016/j.pbi.2008.09.002 pmid: 18930433
[5] Christie J M . Phototropin blue-light receptors. Annu Rev Plant Biol, 2007,58:21-45
doi: 10.1146/annurev.arplant.58.032806.103951
[6] Kagawa T, Sakai T, Suetsugu N, Oikawa K, Ishiguro S, Kato T, Tabata S, Okada K, Wada M . Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response. Science, 2001,291:2138-2141
[7] Shimazaki K, Doi M, Assmann S M, Kinoshita T . Light regulation of stomatal movement. Annu Rev Plant Biol, 2007, 58: 219-247
[8] Kinoshita T, Doi M, Suetsugu N, Kagawa T, Wada M, Shimazaki K . phot1 and phot2 mediate blue light regulation of stomatal opening. Nature, 2001, 414: 656-660
doi: 10.1038/414656a pmid: 11740564
[9] Harada A, Sakai T, Okada K . Phot1 and phot2 mediate blue light-induced transient increases in cytosolic Ca 2+ differently in Arabidopsis leaves. Proc Natl Acad Sci USA, 2003,100:8583-8588
doi: 10.1073/pnas.1336802100 pmid: 166272
[10] Zhao X, Wang Y L, Qiao X R, Wang J, Wang L D, Xu C S, Zhang X . Phototropins function in high-intensity blue light-induced hypocotyl phototropism in Arabidopsis by altering cytosolic calcium. Plant Physiol, 2013,162:1539-1551
doi: 10.1104/pp.113.216556 pmid: 23674105
[11] Sakai T, Wada T, Ishiguro S, Okada K . RPT2: a signal transducer of the phototropic response in Arabidopsis. Plant Cell, 2000,12:225-236
[12] Blakeslee J J, Peer W A, Murphy A S . Auxin transport. Curr Opin Plant Biol, 2005,8:494-500
doi: 10.1016/j.pbi.2005.07.014
[13] Whippo C W, Hangarter R P . Phototropism: bending towards enlightenment. Plant Cell, 2006,18:1110-1119
doi: 10.1105/tpc.105.039669
[14] Stone B B, Stowe-Evans E L, Harper R M, Celaya R B, Ljung K, Sandberg G, Liscum E . Disruptions in AUX1-dependent auxin influx alter hypocotyl phototropism in Arabidopsis. Mol Plant, 2008,1:129-144
doi: 10.1093/mp/ssm013 pmid: 20031920
[15] Briggs W R . Phototropism: some history, some puzzles, and a look ahead. Plant Physiol, 2014,164:13-23
doi: 10.1104/pp.113.230573
[16] Haga K, Mayama T, Yamada M, Sakaic T . Arabidopsis ROOT PHOTOTROPISM2 contributes to the adaptation to high-intensity light in phototropic responses. Plant Cell, 2015,27:1098-1112
doi: 10.1105/tpc.15.00178 pmid: 25873385
[17] Lariguet P, Schepens I, Hodgson D, Pedmale U V, Trevisan M, Kami C, de Carbonnel M, Alonso J M, Ecker J R, Liscum E . PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism. Proc Natl Acad Sci USA, 2006,103:10134-10139
doi: 10.1073/pnas.0603799103 pmid: 16777956
[18] Inada S, Ohgishi M, Mayama T, Okada K, Sakai T . RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana. Plant Cell, 2004,16:887-896
doi: 10.1105/tpc.019901 pmid: 15031408
[19] Inoue S, Kinoshita T, Takemiya A, Doi M, Shimazaki K . Leaf positioning of Arabidopsis in response to blue light. Mol Plant, 2008, 1: 15-26
doi: 10.1093/mp/ssm001 pmid: 20031912
[20] Motchoulski A, Liscum E . Arabidopsis NPH3: a NPH1 photoreceptor-interacting protein essential for phototropism. Science, 1999,286:961-964
doi: 10.1126/science.286.5441.961 pmid: 10542152
[21] 赵青平, 赵翔, 慕世超, 肖慧丽, 张骁 . 拟南芥下胚轴向光弯曲P2SA2基因的克隆与功能鉴定. 作物学报, 2015,41:585-592
doi: 10.3724/SP.J.1006.2015.00585
Zhao Q P, Zhao X, Mu S C, Xiao H L, Zhang X . Functional analysis and mapping of gene P2SA2 involved in hypocotyl phototropism of Arabidopsis thaliana. Acta Agron Sin, 2015,41:585-592 (in Chinese with English abstract)
doi: 10.3724/SP.J.1006.2015.00585
[22] Tsuchida-Mayama T, Sakai T, Hanada A, Uehara Y, Asami T, Yamaguchi S . Role of the phytochrome and cryptochrome signaling pathways in hypocotyl phototropism. Plant J, 2010,62:653-662
doi: 10.1111/j.1365-313X.2010.04180.x pmid: 20202166
[23] Zhao X, Zhao Q P, Xu C Y, Wang J, Zhu J D, Shang B S, Zhang X . Phot2-regulated relocation of NPH3 mediates phototropic response to high-intensity blue light in Arabidopsis thaliana. J Integr Plant Biol, 2018,60:562-577
doi: 10.1111/jipb.12639 pmid: 29393576
[24] 何乙坤, 钟敏, 胡同乐, 王树桐, 段豪, 丁丽, 王亚南, 曹克强 . 利用酵母双杂交筛选与苹果褪绿叶斑病毒CP互作的寄主因子. 中国农业科学, 2014,47:4821-4829
doi: 10.3864/j.issn.0578-1752.2014.24.005
He Y K, Zhong M, Hu T L, Wang S T, Duan H, Ding L, Wang Y N, Cao K Q . Screening of the host factors interacting with CP of apple chlorotic leaf spot virus by Yeast Two-Hybrid system. Sci Agric Sin, 2014,47:4821-4829 (in Chinese with English abstract)
doi: 10.3864/j.issn.0578-1752.2014.24.005
[25] 王怀琴, 郭晓荣, 杨新兵, 苏娇, 曹晓燕 . 利用酵母双杂交筛选与丹参R2R3-MYB类转录因子SmPAP1互作的蛋白. 基因组学与应用生物学, 2016,35:2819-2826
Wang H Q, Guo X R, Yang X B, Su J, Cao X Y . Screening of the proteins interacting with SmPAP1 of R2R3-MYB transcription factor from salvia miltiorrhiza bunge by Yeast Two-Hybrid system. Genom Appl Biol, 2016,35:2819-2826 (in Chinese with English abstract)
[26] Briggs W R, Beck C F, Cashmore A R, Christie J M, Hughes J, Jarillo J A, Kagawa T, Kanegae H, Liscum E, Nagatani A, Okada K, Salomon M, Rüdiger W, Sakai T, Takano M, Wada M, Watson J C . The phototropin family of photoreceptors. Plant Cell, 2001,13:993-997
doi: 10.1109/9.341804 pmid: 11424903
[27] Sakai T, Kagawa T, Kasahara M, Swartz T E, Christie J M, Briggs W R, Wada M, Okada K . Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proc Natl Acad Sci USA, 2001,98:6969-6974
doi: 10.1073/pnas.101137598 pmid: 11371609
[28] Briggs W R, Christie J M . Phototropins 1 and 2: versatile plant blue-light receptors. Trends Plant Sci, 2002,7:204-210
doi: 10.1016/S1360-1385(02)02245-8 pmid: 11992825
[29] Suetsugu N, Wada M . Evolution of the cp-actin-based motility system of chloroplasts in green plants. Front Plant Sci, 2016,7:561
doi: 10.3389/fpls.2016.00561
[30] Thakur J K, Agarwal P, Parida S, Bajaj D, Pasrija R . Sequence and expression analyses of KIX domain proteins suggest their importance in seed development and determination of seed size in rice, and genome stability in Arabidopsis. Mol Genet Genomics, 2013,288:329-346
doi: 10.1007/s00438-013-0753-9 pmid: 23756993
[31] Gonzalez N, Pauwels L, Baekelandt A， DeMilde L, Leene J V, Besbrugge N, Heyndrickx K S, Pérez A C, Durand A N, Clercq R D, Van De Slijke E, Vanden Bossche R, Eeckhout D, Gevaert K, Vandepoele K, De Jaeger G, Goossens A, Inzé D . A repressor protein complex regulates leaf growth in Arabidopsis. Plant Cell, 2015,27:2273-2287
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