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，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。由于其在叶绿体和细胞核中的双重定位，RNA/DNA结合蛋白是感受叶绿体胁迫和将可塑性胁迫信号传递到细胞核的理想候选者。对改变了WHIRLY1水平的转基因大麦的研究表明，WHIRLY1参与了植物对非生物胁迫的响应，并提高了对白粉病的抗性。对所选基因的表达分析表明，在利用CRISPR RNA/Cas9内切酶技术产生的大麦hvwhy1基因敲除突变体中，WHIRLY1是大麦已知逆境基因的调节基因。对玉米和大麦突变体的研究进一步表明，WHIRLY1是叶绿体发育所必需的。在该项目的框架内，将确定WHIRLY1蛋白对应激反应重要的特定基序。此外，还将研究WHIRLY1蛋白在叶绿体发育中的功能是否可以从植物的逆境反应功能中分离出来。在细胞核中，WHIRLY1与NPR1一起参与水杨酸依赖的PR基因的激活。NPR1是水杨酸信号转导的中心成分，通常以寡聚体的形式存在于细胞质中。同样，叶绿体中的WHIRLY1形成更高阶的寡聚体。在与氧化还原变化相关的应激依赖的SA产生过程中，NPR1被单体化，然后迁移到细胞核中。如果WHIRLY1在叶绿体中的逆境感受类似于NPR1在细胞质中的逆境感受，那么可以假设，依赖于氧化还原的旋转复合体的单体对于逆境依赖的从叶绿体到细胞核的转移是必要的。为了测试寡聚化所需的旋转区中保守的半胱氨酸和赖氨酸对大麦WHIRLY1依赖的胁迫反应是否重要，我们将用突变的HvWHIRLY1序列和未突变的HvWHIRLY1序列来补充hvwhy1 KO突变体。转基因植物的特征将是它们对各种非生物胁迫因素以及白粉病与非生物胁迫因素相结合的反应能力。根据这些结果，将选择叶片材料，使用RNAseq进行全球基因表达分析。这些研究的目的是确定WHIRLY1的目标基因，这些基因在交叉耐受情况下被激活或抑制(白粉病感染与非生物胁迫相结合)。克劳斯·亨贝克教授在MLU Halle的研究小组将研究WHIRLY1是否通过表观遗传机制影响WHIRLY1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.