Characterization of WHIRLY1 functions in cereal crops

叶绿体是环境变化的主要感受器，尤其是对胁迫的感知。生长在多变环境中的植物需要光合作用、生长和防御的协调调节。目前缺乏对非生物胁迫条件下叶绿体和核基因表达如何共同调控的了解，这是作物改良的主要障碍。该项目涉及WHY1的功能特征，是基于申请人实验室最近的工作，该工作证明了该蛋白质在叶绿体和核生物学中发挥重要作用，并在这些细胞器之间发挥信号作用。WHY1是单链DNA结合蛋白Whirly家族中的一员，该蛋白存在于叶绿体和细胞核中，非常符合BBSRC在“可持续提高农业生产”领域的战略重点，特别是在“提高作物对非生物胁迫的适应能力”这一主题上。WHY1在细胞核中作为病原体相关基因表达的转录激活因子，并作为调节端粒长度的动蛋白样蛋白1的抑制因子。在叶绿体中，WHY1对于叶绿体基因组的稳定和叶绿体基因的转录是必需的。然而，关于WHY1如何参与叶片发育和抗逆性的调节仍有许多不确定的地方。此外，WHY1似乎是玉米叶绿体核糖体生物发生所必需的，但在其他物种中不是。该项目结合了Foyer实验室在Whirly蛋白质方面的专业知识，以及Biogmea在谷物转化和创造适合植物育种者的新特征方面的专业知识。这些研究将为了解WHY1在谷物中的功能提供新的知识，并为在最佳和胁迫条件下叶绿体到细胞核的逆行信号机制提供新的线索。这项建议的目的是：1)培育和鉴定缺失WHY1的转基因RNAi小麦植株。(里程碑1：产生改变了WHY1表达的转基因小麦品系)2)产生并鉴定在叶肉或束鞘叶绿体中过度表达WHY1的转基因玉米植株。(里程碑2：获得WHY1表达改变的转基因玉米品系)3)确定修改的WHY1表达对植物对低氮供应、干旱和强光胁迫的反应的影响，以将这些变化与叶绿体和核信号联系起来(里程碑3：转录本和代谢物图谱数据的生成)。转基因品系和野生型将与Why1和Why2Mu转座子诱导的玉米突变体一起用于探索WHY1蛋白在没有或存在胁迫(高光、干旱、低氮)时叶片发育过程中叶肉和束鞘细胞叶绿体和细胞核中的作用。将描述叶绿体中WHY1蛋白的氧化还原调节机制。除了生理学、生物化学和细胞生物学方法来研究光合作用和叶片发育外，学生还将在选定的条件下对特定的品系进行RNAseq和代谢物分析。定量聚合酶链式反应技术和林可霉素和其他抑制剂的筛选将被用来描述叶绿体到细胞核的信号通路。还将比较WHY1缺乏对野生型和转基因品系在最佳和胁迫条件下生长的根表型的影响。将在选定的行中评估田间行为和产量影响

Chloroplasts are major sensors of environmental change, particularly stress perception. Plants growing in fluctuating environments require coordinated regulation of photosynthesis, growth and defence. The current lack of understanding of how chloroplast and nuclear gene expression are co-regulated under abiotic stress conditions is a major impediment to crop improvement. This project, which concerns the functional characterization of WHY1, is based on recent work in the applicant's laboratory that demonstrated this protein as an important player in chloroplast and nuclear biology, with roles in signalling between these organelles. WHY1 is a member of the WHIRLY family of single-stranded DNA-binding proteins that is found in both plastids and the nucleus, fits well into BBSRC's strategic priorities in the area of "sustainably enhancing agricultural production", particularly in the topic of "greater resilience of crops to abiotic stresses". WHY1 acts as a transcription activator for pathogen-related gene expression in the nucleus and as a repressor for the kinesin-like protein 1 that modulates telomere length. In chloroplasts, WHY1 is required for plastid genome stability and plastid gene transcription. However, much remains uncertain concerning how WHY1 participates in the regulation of leaf development and stress tolerance. Moreover, WHY1 appears to be essential for plastid ribosome biogenesis in maize but not in other species. This project combines the expertise of the Foyer lab on WHIRLY proteins with that of Biogemma in cereal transformation and the creation of new traits suitable for plant breeders. These studies will provide new knowledge concerning WHY1 functions in cereals and shed new light on chloroplast to nucleus retrograde signalling mechanisms under optimal and stress conditions. The objectives of this proposal are:1) To produce and characterise transgenic RNAi wheat plants lacking WHY1. (Milestone 1: generation of transgenic wheat lines with altered WHY1 expression).2) To generate and characterise transgenic maize plants over-expressing WHY1 in either the mesophyll or bundle sheath chloroplasts. (Milestone 2: generation of transgenic maize lines with altered WHY1 expression)3) To determine the effects of modified WHY1 expression on plant responses to low nitrogen availability, drought and high light stresses to link these changes to chloroplast to nucleus signalling (Milestone 3: Generation of transcript and metabolite profile data).The transgenic lines and wild types will be used together with why1 and why2 Mu transposon-induced maize mutants to explore the roles of the WHY1 protein in the chloroplasts and nuclei of mesophyll and bundle sheath cells during leaf development in absence or presence of stress (high light, drought, low nitrogen). The mechanisms of redox regulation of the WHY1 protein in chloroplasts will be characterised. In addition to physiology, biochemistry and cell biology approaches to study photosynthesis and leaf development, the student will use RNAseq and metabolite profiling on specific lines under selected conditions. qPCR techniques and screens with lincomycyin and other inhibitors will be used to characterise chloroplast to nucleus signalling pathways. The effects of WHY1 deficiency on root phenotypes will also be compared in wild type and transgenic lines grown under optimal and stress conditions. Field behaviour and yield impacts will be assessed in selected lines