BLOTING TRANSITION SPECIFIC GENETIC & BIOCHEMICAL EVENTS IN ARABIDOPSIS THALIANA

印迹转变特异性基因

• 批准号: 6481228
• 负责人: ROBERT L VELLANOWETH
• 金额: $ 7.33万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6481228
• 关键词: Arabidopsis; ascorbate; cell senescence; differential display technique; electron spin resonance spectroscopy; enzyme activity; free radical oxygen; gene expression; gene induction /repression; genetic transcription; high performance liquid chromatography; leaf; lipoxygenase; molecular cloning; northern blottings; nucleic acid sequence; peroxidases; plant genetics; plant growth /development; polymerase chain reaction; posttranscriptional RNA processing

The goal of this proposal is to expand on our current studies showing that significant changes in reactive oxygen species (ROS) metabolism occur in leaf tissue as Arabidopsis plants undergo the fundamental developmental transition from a vegetative state to a reproductive state. This transition, referred to in this proposal as the bolting transition, involves a conversion of the principal shoot apical meristem from one giving rise to leave to one forming a floral meristem which produces a long bolt with terminal flowers. We have demonstrated that the bolting transition is associated with a decline in measured ascorbate peroxidase (APX) activity and with a concomitant increase in lipid peroxidation in leaf tissue. The APX decline and subsequent peroxidation are hypothesized to provide intracellular signals initiating the ordered cascade of gene expressing leading to senescence. In this proposal, experiments are described that will test particular mechanisms of the bolting transition-specific alterations in leaf APX and lipoxygenase activities. In addition, experiments are proposed to exploit the molecular genetic advantages of the Arabidopsis system to investigate the genetic cascades from bolting to senescence by isolating bolting transition-induced genes and screening for late senescing mutant phenotypes. The following specific aims are proposed: 1) Analyze the bolting transition specific down-regulation of leaf ascorbate peroxidase at the transcriptional and post-transcriptional levels; 2) Determine the means of induction of lipoxygenase activity following APX decline; 3) Isolate, sequence, and characterize cDNA clones or genes isolated by differential display whose level of expression changes in leaf tissue immediately following the bolting transition; and 4) Screen mutagenized populations of the ethylene-insensitive Arabidopsis mutant etr1-1 for mutants exhibiting delayed following in response to darkness-induced senescence. Our long term objective is to determine the overall scheme of senescence induction in gene expression program induced as early as the floral transition. Our observation that floral initiation alters ROS metabolism in leaf tissue provides an opportunity to investigate communication between two distinct organs in the plant. We expect that the investigation of these early events will allow the elucidating of the initial signals and the signaling signals and the signaling mechanism response for the life-ending gene expression program known as senescence.

本提案的目的是扩大我们目前的研究表明，在拟南芥植物经历从营养状态到生殖状态的基本发育转变过程中，叶组织中活性氧（ROS）代谢发生了显著变化。这种转变，在本提案中被称为抽苔转变，涉及到主枝顶端分生组织从一个产生叶片到一个形成花的分生组织的转化，这个分生组织产生一个带顶生花的长抽苔。我们已经证明，抽苔过渡期与测定的抗坏血酸过氧化物酶（APX）活性下降有关，并伴随着叶组织中脂质过氧化的增加。据推测，APX的下降和随后的过氧化作用提供了细胞内信号，启动了导致衰老的有序级联基因表达。在本提案中，实验描述了将测试抽苔过渡特异性改变叶片APX和脂氧合酶活性的特定机制。此外，还提出利用拟南芥系统的分子遗传优势，通过分离抽苔过渡诱导基因和筛选晚衰老突变表型，研究抽苔到衰老的遗传级联反应。提出以下具体目标：1)从转录和转录后水平分析抽苔转代叶片抗坏血酸过氧化物酶特异性下调；2)确定APX下降后脂氧合酶活性的诱导方法；3)分离、测序和鉴定通过差异展示分离的表达水平在抽苔转化后立即发生变化的cDNA克隆或基因；4)筛选拟南芥乙烯不敏感突变体etr1-1的突变群体，寻找对黑暗诱导的衰老表现出延迟跟随反应的突变体。我们的长期目标是确定早在花转化时诱导的基因表达程序诱导衰老的总体方案。我们观察到花的形成会改变叶片组织中的ROS代谢，这为研究植物中两个不同器官之间的交流提供了机会。我们期望对这些早期事件的研究将有助于阐明生命终止基因表达程序衰老的初始信号和信号传导信号以及信号传导机制。