Reconstitution of the Jasmonate Signaling Pathway in Plant Protoplasts
Reconstitution of the Jasmonate Signaling Pathway in Plant Protoplasts
复制标题
植物原生质体中茉莉酸信号通路的重建
DOI：
10.3390/cells8121532
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发表时间：
2019-12-01
期刊：
影响因子：
6
通讯作者：
Gatz, Christiane
中科院分区：
文献类型：
--
作者：
Li, Ning;Uhrig, Joachim F.;Gatz, Christiane
植物激素茉莉酸（JA）在植物的各种发育过程和环境适应中起着重要作用。JA信号通路已经在参考植物拟南芥（Arabidopsis thaliana）中得到充分阐明。它始于F-box蛋白COI 1（E3连接酶SCFCOI 1的一部分）对活性JA衍生物茉莉酸-异亮氨酸（JA-Ile）的感知。JA-Ile的结合使得COI1和JAZ阻遏蛋白之间能够相互作用。JAZ蛋白的后续降解导致转录因子的激活，MYC 2.在这里，我们证明了该途径可以在瞬时转化的原生质体中重建。分析JAZ1-fLuc融合蛋白作为在coil原生质体中瞬时表达的COI 1的功能的稳定性允许JAZ和COI 1两者的结构功能分析。使用该系统，我们发现COI1中保守的半胱氨酸影响稳态COI1蛋白水平。使用JAZ1启动子控制下的荧光素酶报告基因能够解决MYC2抑制所需的JAZ1的那些特征。有趣的是，保守的TIFY基序先前描述的相互作用与NINJA招募辅阻遏物TOPLESS是不必要的镇压。这一结果支持另一种抑制模式，即抑制性JAZ和促进性MEDIATOR 25在MYC 2处直接竞争。最后，使用原生质体从aos coi1双突变体，这是缺乏JA的合成和感知，我们提供了一个系统，有可能研究不同的COI1变体在不同的配体的存在下的活动。
The phytohormone jasmonic acid (JA) plays an important role in various plant developmental processes and environmental adaptations. The JA signaling pathway has been well-elucidated in the reference plant Arabidopsis thaliana. It starts with the perception of the active JA derivative, jasmonoyl-isoleucine (JA-Ile), by the F-box protein COI1 which is part of the E3-ligase SCFCOI1. Binding of JA-Ile enables the interaction between COI1 and JAZ repressor proteins. Subsequent degradation of JAZ proteins leads to the activation of transcription factors like e.g., MYC2. Here we demonstrate that the pathway can be reconstituted in transiently transformed protoplasts. Analysis of the stability of a JAZ1-fLuc fusion protein as a function of COI1 transiently expressed in coi1 protoplasts allows structure function analysis of both JAZs and COI1. Using this system, we found that conserved cysteines in COI1 influence steady state COI1 protein levels. Using a luciferase reporter gene under the control of the JAZ1 promoter enable to address those features of JAZ1 that are required for MYC2 repression. Interestingly, the conserved TIFY-motif previously described to interact with NINJA to recruit the corepressor TOPLESS is not necessary for repression. This result is in favor of the alternative repression mode that proposes a direct competition between repressive JAZs and promotive MEDIATOR25 at MYC2. Finally, using protoplasts from the aos coi1 double mutant, which is deficient in JA synthesis and perception, we provide a system that has the potential to study the activity of different COI1 variants in the presence of different ligands.