Characterization of WHIRLY1 functions in cereal crops

叶绿体是环境变化的主要传感器，特别是压力感知。植物生长在波动的环境中，需要协调调节光合作用，生长和防御。目前缺乏对叶绿体和核基因表达在非生物胁迫条件下如何共同调节的理解是作物改良的主要障碍。该项目涉及WHY1的功能表征，基于申请人实验室的最新工作，该工作证明该蛋白质在叶绿体和核生物学中具有重要作用，在这些细胞器之间具有信号传导作用。WHY1是在质体和细胞核中发现的单链DNA结合蛋白的WHIRLY家族的成员，非常适合BBSRC在“可持续地提高农业生产”领域的战略优先事项，特别是在“作物对非生物胁迫的更大适应力”的主题中。WHY 1作为病原体相关基因在细胞核中表达的转录激活因子，以及作为调节端粒长度的驱动蛋白样蛋白1的抑制因子。在叶绿体中，WHY 1是质体基因组稳定性和质体基因转录所必需的。然而，关于WHY1如何参与调节叶片发育和胁迫耐受性仍有许多不确定性。此外，WHY 1似乎是必不可少的质体核糖体生物合成在玉米，但不是在其他物种。该项目结合了Foyer实验室在WHIRLY蛋白方面的专业知识和Biogemma在谷物转化方面的专业知识，并创造了适合植物育种者的新性状。这些研究将提供有关WHY1在谷物中功能的新知识，并为最佳和胁迫条件下叶绿体到核的逆行信号机制提供新的见解。本研究的目的是：1）获得并鉴定缺失WHY 1的转基因RNAi小麦植株。（里程碑1：产生具有改变的WHY 1表达的转基因小麦品系）。2）产生并培育在叶肉或维管束鞘叶绿体中过表达WHY 1的转基因玉米植物。（里程碑2：具有改变的WHY 1表达的转基因玉米系的产生）3）为了确定修饰的WHY 1表达对植物对低氮可用性、干旱和强光胁迫的响应的影响，以将这些变化与叶绿体到核的信号传导联系起来（里程碑3：转基因系和野生型将与why1和why2 Mu转座子一起使用。诱导的玉米突变体，探讨WHY1蛋白在叶片发育过程中的叶绿体和叶肉细胞和维管束鞘细胞的核中的作用，在没有或存在胁迫（强光，干旱，低氮）。氧化还原调节的WHY1蛋白质在叶绿体的机制将被表征。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使用RNAseq和代谢物分析特定的品系。qPCR技术和林可霉素和其他抑制剂的筛选将用于抑制叶绿体到核的信号传导途径。还将在最佳和胁迫条件下生长的野生型和转基因品系中比较WHY1缺乏对根表型的影响。将在选定的品系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.