The role of WHIRLY1 in chloroplast-based perception of stress and in chloroplast-to-nucleus signaling

植物的发育和生产力需要光合作用装置对环境进行有效的适应，并受到胁迫因素的限制。胁迫诱导的光合作用装置的干扰在叶绿体中被感知，并被传递到细胞核以调节基因表达，从而导致植物的驯化。叶绿体在胁迫过程中产生的信号包括光合作用电子链中的活性氧(ROS)和氧化还原变化。到目前为止，从叶绿体到细胞核的信号传递机制还不是很清楚。本项目将研究位于同一细胞的叶绿体和细胞核中的RNA/DNA结合蛋白WHIRLY1是否参与了基于叶绿体的胁迫感知和胁迫诱导的叶绿体到核的信号转导。初步研究表明，WHIRLY1不仅位于叶绿体类核中，而且位于类囊体膜上，是感知光合作用信号和调节叶绿体基因表达的理想候选适配子。因此，在高光胁迫期间，转基因大麦的叶绿体发育受到干扰，WHIRLY1基因的数量减少。WHIRLY1可以形成低聚物，也可以作为单体存在。这种单体形式可能是对胁迫下植物叶绿体氧化还原变化的响应。将对暴露在不同胁迫条件下的大麦植株进行生化分析，以确定齐聚物与单体的比例。另外还将对分离的叶绿体进行研究，在叶绿体中，光合作用抑制剂的应用将改变氧化还原状态和/或ROS的产生。通过改变WHIRLY1的转基因大麦品系来研究WHIRLY1对适应不同胁迫条件的影响，即干旱和高光冷。光合作用和核基因表达的测量是设想的。WHIRLY1在大麦叶绿体和细胞核之间的分布将被免疫学分析。为了研究逆境诱导WHIRLY1亚细胞分布变化的机制，可以利用过量表达WHIRLY1突变序列的转基因拟南芥。除了负责蛋白质齐聚的赖氨酸残基K91外，可能参与WHIRLY1构象氧化还原相关变化的保守半胱氨酸残基C159已经被丙氨酸取代。此外，已经制备了表达叶绿体内突变序列的转体烟草植株。WHIRLY1亚细胞分布的可视化将通过积累荧光融合蛋白(WHIRLY1：ILOV)的拟南芥植物来完成。

Plant development and productivity require an efficient adaptation of the photosynthetic apparatus to the environment and are limited by stress factors. Stress-induced disturbances in the photosynthetic apparatus are sensed in chloroplasts and are transmitted to the nucleus to adjust gene expression leading to an acclimation of the plants. Among the well-studied signals produced during stress in chloroplasts are reactive oxygen species (ROS) and redox changes in the photosynthetic electron chain. So far, the mechanisms of signal transference from chloroplasts to the nucleus are not well understood.In this project it will be investigated whether the RNA/DNA binding protein WHIRLY1, being dually located in chloroplasts and the nucIeus of the same cell is involved in chloroplast-based perception of stress and in stress-induced chloroplast-to-nucleus signaling. Preliminary studies showed that WHIRLY1 is located in chloroplast nucleoids as well as at the thylakoid membranes, making it an ideal candidate adaptor perceiving signals from photosynthesis and adjusting plastid gene expression. Accordingly, during high light stress chloroplast development is disturbed in transgenic barley plants with reduced amount of WHIRLY1. WHIRLY1 can form oligomers or can be present as a monomer. The monomeric form might be formed in response to redox changes in chloroplasts of stress exposed plants. The ratio of oligomers to monomers will be analysed biochemically with barley plants exposed to different stress conditions. Additional investigations will be done with isolated chloroplasts, in which the redox state and/or the production of ROS will be altered by application of inhibitors of photosynthesis. Transgenic barley lines with altered amounts of WHIRLY1 will be used to investigate the impact of WHIRLY1 on acclimation towards various stress conditions, i.e. drought and high irradiance in combination with cold. Measurements of photosynthesis and nuclear gene expression are envisaged. The distribution of WHIRLY1 between chloroplasts and nucleus will be immunologically analysed in barley. For investigations on the mechanisms of putative stress-induced changes in the subcellular distribution of WHIRLY1, transgenic Arabidopsis plants overexpressing mutated sequences of WHIRLY1 are available. Besides the lysine residue K91 responsible for oligomerization of the protein the conserved cysteine residue C159 possibly involved in redox dependent changes of the WHIRLY1 conformation has been replaced by alanine. Additionally, transplastomic tobacco plants expressing the mutated sequences inside the plastids have been prepared. Visualization of the subcellular distribution of WHIRLY1 will be done with Arabidopsis plants accumulating fluorescing fusion proteins (WHIRLY1:iLOV).