The chloroplast-nucleus located WHIRLY1 of barley as master regulator of cross-tolerance towards abiotic and biotic stresses

为了确保产量的稳定性，作物需要对环境的变化做出快速反应，并有效地适应经常同时发生的多种胁迫因素。关于其在叶绿体和细胞核中的双重定位，RNA/DNA结合蛋白WHIRLY是叶绿体中感受胁迫和将质体胁迫信号传递到细胞核的理想候选者。对具有改变的WHIRLY 1水平的转基因大麦系的研究表明，WHIRLY 1参与植物对非生物胁迫的响应并促进对白粉病的抗性。对所选基因表达的分析显示，在通过CRISPR RNA/Cas9内切核酸酶技术产生的大麦hvwhy 1敲除突变体中，WHIRLY 1是大麦中众所周知的胁迫基因的调节因子。对玉米和大麦突变体的研究进一步表明，WHIRLY 1是叶绿体发育所必需的。在该项目的框架下，将鉴定对应激反应重要的WHIRLY 1蛋白的特定基序。此外，还将研究WHIRLY 1蛋白在叶绿体发育中的功能性是否可以与植物胁迫反应中的功能性分开。在细胞核中，WHIRLY 1与NPR 1一起参与PR基因的水杨酸依赖性激活。NPR 1是水杨酸信号传导的核心成分，通常以寡聚体的形式位于细胞质中。同样，叶绿体中的WHIRLY 1形成更高级的寡聚体。在与氧化还原变化有关的SA的应激依赖性产生期间，NPR 1被单体化，然后迁移到细胞核中。如果叶绿体中的WHIRLY 1的应激感知与细胞质中的NPR 1相似，则可以假设WHIRLY复合物的氧化还原依赖性单体化对于从叶绿体到细胞核的应激依赖性转移是必需的。为了测试寡聚化所需的WHIRLY结构域中的保守半胱氨酸和赖氨酸是否对大麦的WHIRLY 1依赖性应激反应重要，将用突变的HvWHIRLY 1序列和HvWHIRLY 1的非突变序列补充hvwhy 1 KO突变体。将表征转基因植物对各种非生物胁迫因子以及与非生物胁迫因子组合的白粉病的反应能力。基于结果，将选择叶材料用于使用RNAseq的全局基因表达分析。这些研究的目的是确定WHIRLY 1的靶基因，这些基因在交叉耐受性情况下（白粉病感染与非生物胁迫相结合）被激活或抑制。Klaus Humbeck教授在MLU Halle的研究小组将研究WHIRLY 1是否通过表观遗传机制影响WHIRLY 1靶基因的转录。

In order to ensure yield stability, crop plants need to react rapidly to changes in the environment and to acclimate efficiently to multiple stress factors which often occur simultaneously. With regard to its dual localization in chloroplasts and nucleus, the RNA/DNA binding protein WHIRLY is an ideal candidate for perception of stress in the chloroplast and for transfer of plastidic stress-signals to the nucleus. Investigations with transgenic barley lines having altered levels of WHIRLY1 revealed that WHIRLY1 is involved in responsiveness of plants towards abiotic stresses and promotes the resistance towards powdery mildew. Analyses of the expression of selected genes revealed that in a hvwhy1 knockout mutant of barley that has been produced by CRISPR RNA/Cas9 endonuclease technology, revealed that WHIRLY1 is a regulator of well-known stress genes in barley. Studies on mutants of maize and barley furthermore showed that WHIRLY1 is necessary for the development of chloroplasts. In the framework of this project specific motifs of the WHIRLY1 protein being important for stress responses are to be identified. Furthermore, it will be investigated whether the functionality of the WHIRLY1 protein in chloroplast development can be separated from the functionality in stress responses of plants. In the nucleus, WHIRLY1, together with NPR1, is involved in the salicylic acid-dependent activation of PR genes. NPR1 which is a central component of salicylic acid signaling, normally is located in the cytoplasm in form of oligomers. As well WHIRLY1 in chloroplasts forms higher order oligomers. During stress dependent production of SA in connection with redox changes, NPR1 is monomerized and then migrates into the cell nucleus. If the stress perception by WHIRLY1 in the chloroplasts is similar to that by NPR1 in the cytoplasm, it can be assumed that a redox-dependent monomerization of the WHIRLY complex is necessary for the stress dependent transfer from the chloroplasts into the cell nucleus. To test whether the conserved cysteine and a lysine in the WHIRLY domain required for oligomerization are important for WHIRLY1 dependent stress responses of barley, the hvwhy1 KO mutant will be complemented with mutated HvWHIRLY1 sequences and the nonmutated sequence of HvWHIRLY1. The transgenic plants will be characterized with regard to their ability to react to various abiotic stress factors as well as powdery mildew in combination with abiotic stress factors. Based on the results, leaf material will be selected for global gene expression analysis using RNAseq. The aim of these investigations is to identify target genes of WHIRLY1 that are either activated or repressed in a cross-tolerance situation (powdery mildew infection in combination with abiotic stress). Prof. Klaus Humbeck's research group at MLU Halle will investigate whether WHIRLY1 influences the transcription of WHIRLY1 target genes via epigenetic mechanisms.