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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (12): 1802-1808.doi: 10.3724/SP.J.1006.2018.01802

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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 Online:2018-12-12 Published:2018-08-20
  • Contact: Xiao ZHANG E-mail:xzhang@henu.edu.cn
  • 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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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

Fig. 1

Hypocotyl phototropic curvature in wild type (WT) and mutant seedlings in response to unilateral blue light at the indicated fluence rates A and B: phototropic phenotype of WT, rpt2-2, phot1, phot1 rpt2-2, phot2 rpt2-2, and phot1 phot2 to 100 μmol m-2 s-1 (A) or 0.01 μmol m-2 s-1 (B) unilateral blue light for 12 h. C and D: bar graph of the phototropic curvature of seedlings from A (C) or B (D). Each column represents an average of three experiments (15-20 measurements each) ±SD."

Fig. 2

Screening of proteins interacted with RPT2 by yeast two hybrid A: construction of recombinant yeast two hybrid vector; B: analysis of toxicity of recombinant yeast two-hybrid vector; C: validation of autonomously activate of bait gene; D: the color analysis of vectors of Yeast library screening."

Table 1

List of RPT2-interacting proteins identified in a yeast two-hybrid screen"

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.

Fig. 3

RPT2 interacted with PHOT1, JAC1, RIP1, and RIP6 proteins A: RPT2 interacted with PHOT1; B: RPT2 interacted with RIP1; C: RPT2 interacted with JAC1; D: RPT2 interacted with RIP6."

Fig. 4

Hypocotyl phototropism induced by blue light in rip1 mutant of Arabidopsis A and B: phototropic phenotype of WT, rpt2-2, rip1-1, and rip1-2 in response to blue light (100 μmol m-2 s-1 for A and 0.01 μmol m-2 s-1 for B); C: measurement of phototropic curvature for A and B. The values are the average of three independent experiments (15-20 measurements each) ±SD."

Fig. 5

Arabidopsis double mutant rpt2-2 rip1 recovered hypocotyl phototropisim induced by high blue light A: phototropic phenotype of WT, rpt2-2, rpt2-2 rip1-1, and rpt2-2 rip1-2 in response to 100 μmol m-2 s-1 blue light; B: measurement of phototropic curvature for A. The values are the average of three independent experiments (15-20 measurements each) ±SD."

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