Characterization of WHIRLY1 functions in cereal crops

谷类作物中 WHIRLY1 功能的表征

• 批准号: 1824370
• 金额: --
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1824370
• 关键词: Characterization; WHIRLY1; functions; cereal; crops

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.

叶绿体是环境变化的主要传感器，特别是压力感知。在波动的环境中生长的植物需要光合作用、生长和防御的协调调节。目前对叶绿体和核基因表达如何在非生物胁迫条件下共同调节缺乏了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是叶绿体和核生物学中的重要参与者，在这些细胞器之间的信号传导中发挥作用。 WHY1 是单链 DNA 结合蛋白 WHIRLY 家族的成员，在质体和细胞核中都有发现，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“增强作物对非生物胁迫的抵抗力”这一主题。 WHY1 充当细胞核中病原体相关基因表达的转录激活子，并充当调节端粒长度的驱动蛋白样蛋白 1 的阻遏物。在叶绿体中，WHY1 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 如何参与叶片发育和胁迫耐受性的调节仍存在很多不确定性。此外，WHY1 似乎对于玉米质体核糖体生物发生至关重要，但在其他物种中则不然。该项目结合了 Foyer 实验室在 WHIRLY 蛋白方面的专业知识与 Biogemma 在谷物转化和创造适合植物育种者的新性状方面的专业知识。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细胞核逆行信号机制提供新的线索。该提案的目标是：1) 生产和表征缺乏 WHY1 的转基因 RNAi 小麦植物。 （里程碑 1：产生 WHY1 表达改变的转基因小麦品系）。2) 产生并表征在叶肉或束鞘叶绿体中过度表达 WHY1 的转基因玉米植物。 （里程碑 2：产生 WHY1 表达改变的转基因玉米品系）3) 确定修改后的 WHY1 表达对植物对低氮利用率、干旱和高光胁迫的反应的影响，将这些变化与叶绿体和细胞核信号传导联系起来（里程碑 3：生成转录物和代谢物概况数据）。转基因品系和野生型将与 Why1 和 Why2 Mu 转座子诱导的玉米一起使用 突变体以探索 WHY1 蛋白在叶绿体以及叶肉细胞核和束鞘细胞中在缺乏或存在胁迫（强光、干旱、低氮）的情况下叶片发育过程中的作用。将描述叶绿体中 WHY1 蛋白的氧化还原调节机制。除了使用生理学、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，学生还将在选定条件下对特定品系使用 RNAseq 和代谢物分析。 qPCR 技术以及林可霉素和其他抑制剂的筛选将用于表征叶绿体到细胞核的信号传导途径。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 缺陷对根表型的影响。将评估选定品系的田间行为和产量影响。