Chloroplast Function in Fatty Acid Mutants of Arabidopsis

本项目是基于分离一系列具有叶片脂肪酸组成特异性改变的拟南芥突变体。这些突变体促进了叶绿体甘油合成的生物化学以及膜组成在叶绿体结构和功能中的作用的研究。突变体表现出一系列非常惊人的表型，这些表型是由叶绿体脂肪酸组成的变化引起的。在fab1突变体中，饱和脂肪酸的增加导致光合作用在2℃下2-3周后崩溃，并导致叶绿体分解。fad3、fad7、fad8突变体缺乏三烯脂肪酸，但在22℃、120 ~ 150 μ mol量子/m2/s光照条件下，其生长和光合作用正常。最初在这个突变体中发现的表型是由于它们无法合成茉莉酸盐，一种从亚麻酸中提取的氧脂素。低温强光下fad3、fad7、fad8植株光合作用受到影响。在5℃下筛选生长减少的其他拟南芥突变体没有发现额外的脂质突变体，但确实导致了低温反应的其他遗传决定因素的分离和克隆。缺乏多不饱和脂肪酸的fad2 fad6突变体在蔗糖培养基上实现了强大的营养生长，但不能自养生长。当以叶绿素为基础表达时，脂质变化对低温下短期光合作用的影响是非常温和的。相反，突变系的特征表明膜组成对光系统的组装或长期维持具有重要意义。本提案中描述的实验的总体目标是了解这些表型的生化和生物物理基础。对突变体的光合作用和光抑制的测量将允许对突变体的表型进行详细的比较。已经进行了实验，以产生具有中间脂肪酸组成的额外突变系，并筛选分离抑制fab1低温表型的突变。抑制子的分离是识别导致突变表型的基因和过程的一种特别有效的方法，fad3、fad7、fad8和fad2、fad6系的抑制子筛选也将进行。在拟南芥中可用的基因组学工具将允许克隆抑制基因和表征它们所涉及的生物过程。对拟南芥突变体的观察和研究人员在其他系统中的研究结果表明，蛋白质进入叶绿体或转运到类囊体膜可能会因脂肪酸组成的变化而中断。将对野生型和每个突变体进行蛋白质运输试验，以验证这一假设，并确定几种蛋白质靶向机制中的哪一种受到突变体中所代表的膜组成的不同变化的影响。这项工作将提高对叶绿体膜结构和光合作用功能的理解，这是修改这些过程以提高植物生产力的重要先决条件。

This project is based on the isolation of a series of Arabidopsis mutants with specific alterations in leaf fatty acid composition. These mutants have facilitated investigations of the biochemistry of chloroplast glycerolipid synthesis and the roles of membrane composition in chloroplast structure and function. The mutants exhibit a series of very striking phenotypes that result from changes in the fatty acid composition of chloroplasts. In the fab1 mutant, increased saturated fatty acids result in the collapse of photosynthesis after 2-3 weeks at 2 deg. C and an associated breakdown of chloroplasts. The fad3 fad7 fad8 mutants lack trienoic fatty acids but their growth and photosynthesis are normal at 22 deg. C under 120-150 umol quanta/m2/s of light. The phenotypes initially discovered in this mutant result from their inability to synthesize jasmonate, an oxylipin that is derived from linolenic acid. Photosynthesis is affected in fad3 fad7 fad8 plants at low temperatures and under high light. A screen for additional Arabidopsis mutants with reduced growth at 5 deg. C did not identify additional lipid mutants but did lead to the isolation and cloning of other genetic determinants of low-temperature responses. A fad2 fad6 mutant that is deficient in polyunsaturated fatty acids achieves strong vegetative growth on sucrose media but is incapable of autotrophic growth. The effects of lipid changes on short-term photosynthesis at low temperature are very modest when expressed on a chlorophyll basis. Instead, characterization of the mutant lines points to the significance of membrane composition being on assembly or long-term maintenance of the photosystems. The overall goal of the experiments described in this proposal is to understand the biochemical and biophysical bases of these phenotypes. Measurements of photosynthesis and photoinhibition in the mutants will allow detailed comparisons of the mutant phenotypes. Experiments have been carried out to generate additional mutant lines with intermediate fatty acid compositions and to screens to isolate mutations that suppress the low-temperature phenotype of fab1. The isolation of suppressors is a particularly powerful approach for identifying genes and processes contributing to a mutant phenotype and suppressor screens with the fad3 fad7 fad8 and fad2 fad6 lines will also be performed. Genomics tools available in Arabidopsis will allow cloning of the suppressor genes and characterization of the biological processes in which they are involved. Observations on the Arabidopsis mutants and results from researchers working in other systems suggest that protein import into the chloroplast or transport into and through the thylakoid membrane may be disrupted by changes in fatty acid composition. Assays of protein trafficking in wild type and each mutant will be carried out to test this hypothesis and to determine which of the several protein-targeting mechanisms are affected by the different changes in membrane composition that are represented in the mutants. The improved understanding of chloroplast membrane structure and photosynthetic function that will result from this work is an important prerequisite to modifying these processes for increased plant productivity.