膜结合转录因子NAC091调控植物内质网胁迫应答的分子机理研究

When plants are exposed to various stresses, such as heat, salinity and pathogen infection，unfolded and misfolded proteins are accumulated in endoplasmic reticulum (ER), which trigger a well-conserved pathway called the unfolded protein response (UPR) to mitigate the ER stress and promote cell survival. Two membrane-associated transcription factors (MTFs), AtbZIP28 and AtbZIP60, play important roles in transmitting ER stress signals from the ER to the nucleus in Arabidopsis. Recently, we reported the identification of two Arabidopsis membrane-associated transcription factors, AtNAC062 and AtNAC089, both of which play important roles in ER stress response in plants. AtNAC091 were found to be also be up-regulated by ER stress inducer tunicamycin (TM) , an N-glycosylation inhibitor commonly used to specifically induce ER stress. It was found that the truncated AtNAC091D protein formed both a homodimer, and also a heterodimer with AtNAC062D or AtNAC089D respectively in yeast and tobacco cells. In the current proposal, we are planning to characterize another MTF, AtNAC091. The biological function and underlying molecular mechanisms of AtNAC091 in plants, and its relationship with environmental stress tolerance will be substantially investigated, which should be important for understanding the ER stress signal transduction and provide more opportunities to explore new components involved in UPR

当植物细胞受到高温、盐害与病原菌侵染等逆境干扰时，错误折叠蛋白会大量积累在内质网，细胞启动内质网胁迫应答反应，通过蛋白酶切和mRNA非常规剪切活化膜结合转录因子AtbZIP28和AtbZIP60调节下游基因，增强蛋白折叠能力，促进细胞生存。申请人前期发现NAC家族两个膜结合转录因子AtNAC062和 AtNAC089 在内质网胁迫应答信号转导途径中发挥重要作用。此外，前期研究结果表明另一拟南芥膜结合转录因子AtNAC091受内质网胁迫诱导上调，所编码的蛋白AtNAC091与AtNAC062、AtNAC089都具有相互作用，但AtNAC091在内质网胁迫应答中的功能尚不清楚。本项目拟通过分子生物学和遗传学等技术研究AtNAC091在环境胁迫应答中的生物功能、作用机理及其与高温等逆境胁迫抗性的关系，为研究植物内质网胁迫信号途径提供新的切入点，对进一步认识和研究内质网胁迫信号转导奠定基础

蛋白质折叠对真核细胞进行正常生命活动具有非常重要的作用，分泌蛋白和大多数膜蛋白只有在内质网进行折叠加工后，最终才能发挥生物学功能。然而，复杂而精细的蛋白折叠过程很容易受到外界环境胁迫的干扰。植物遭受逆境会导致内质网中大量蛋白无法折叠或错误折叠，从而引起内质网胁迫，此时细胞会通过启动未折叠蛋白应答途径帮助蛋白折叠以缓解胁迫。我们利用实时荧光定量PCR发现NAC091受内质网胁迫诱导上调表达，且这种诱导表达依赖于bZIP60，同时发现NAC091启动子序列上有一段经典的ERSE-I元件，进一步说明NAC091参与内质网胁迫途径，且上游调控因子是bZIP60。通过亚细胞定位分析显示内质网胁迫下NAC091有一个从细胞膜迁移到细胞核的过程。通过转录激活活性分析表明去掉跨膜域的NAC091D具有转录激活活性，且酵母双杂交和双荧光分子实验显示NAC091D进入细胞核后能形成同源二聚体，也能与其他3个与内质网胁迫相关的NACs(NAC062D、NAC089D和NAC103）形成异源二聚体。此外，在zip28zip60双突变体背景下诱导表达NAC091能够提高植物对内质网胁迫的抗性，而通过CRISPR cas9构建的nac091突变体后，植物在内质网胁迫途径中与野生型并没有明显表型区别，说明可能还有其他因子发挥作用，功能冗余。采用RNA-seq技术研究NAC091调控的下游基因，发现NAC091上调的不是经典的UPR基因（例如分子伴侣等），而是一些转录因子(PIF4，NAC094和bHLH等）和酶等，说明在内质网胁迫途径中NAC091通过上调转录因子来促进细胞生存。同时通过非生物胁迫处理，发现NAC091还受NaCl、4℃、ABA等诱导上调，说明NAC091还参与其他生物和非生物胁迫途径。