RUI: The role of the AtcDPPIV Dipeptidyl Aminopeptidase in Photoacclimation

拟南芥AtcDPPIV基因编码一种叶绿体定位蛋白，该蛋白与动物蛋白酶具有同源性，该蛋白酶从具有脯氨酸作为第二氨基酸的底物的氨基端切割前两个氨基酸。植物在弱光条件下生长时，AtcDPPIV mRNA含量丰富，但在亚饱和中光条件下生长两天后，AtcDPPIV mRNA含量大幅减少。当cch1突变体从低光照转移到中等光照时，未观察到AtcDPPIV mRNA水平的下降。cch1突变体在中等光照下具有较小的光收获复合体，因此在中等光照下收获的光比WT少。这些观察结果表明，AtcDPPIV基因表达与光收获有关，而不仅仅是光强度。为了了解AtcDPPIV的功能，研究人员分析了AtcDPPIV基因中两个独立T-DNA插入的突变体。这些品系显示出微妙的光驯化表型，表明AtcDPPIV二肽基氨基肽酶可能在调节植物适应不同光强方面发挥作用。为了验证这一假设，我们提出了六个目标：1)证明两个插入系的光驯化表型是由于AtcDPPIV的互补而导致的AtcDPPIV的缺失。2)过表达AtcDPPIV和AtcDPPIV::GFP，以确定是否过表达导致光驯化表型。3)过表达AtcDPPIV::GFP以确认预测的叶绿体定位，并产生AtcDPPIV抗体用于亚细胞器定位。4)确定重组AtcDPPIV蛋白酶的底物特异性。5)利用酵母双杂交系统鉴定与AtcDPPIV相互作用的蛋白。6)更详细地描述光驯化过程中观察到的延长和紧凑的生长习性，并确定AtcDPPIV插入系是否发生了微妙的影响。人们对光驯化过程中的信号传导知之甚少，AtcDPPIV二肽基氨基肽酶可能是一个尚未被描述的重要信号级联的一部分。AtcDPPIV蛋白在双子体、单子体和许多环境细菌中高度保守，这表明它具有重要的保守功能。更广泛的影响该提案的更广泛的影响包括来自南加州少数民族校园的本科生和硕士生的培训。在研究事业中增加少数族裔人数的最好方法是在他们学习的早期阶段为他们提供一个有趣的、具有挑战性的、可行的项目。还包括培养一名有效教学和卓越研究的博士后研究员。该实验室的许多项目已被纳入生物科学专业的本科课程中，受到了大量学生的影响。此外，PI还协调一项三年级科学教学项目，该项目目前为长滩公立学校系统的160名三年级学生提供为期八周的动手科学课程。

The Arabidopsis AtcDPPIV gene encodes a chloroplast-localized protein that has homology to animal proteases that cleave off the first two amino acids from the amino terminus of substrates that have a proline as the second amino acid. AtcDPPIV mRNA is abundant when plants are grown in low light, but is greatly reduced after two days at sub-saturating moderate light. The decrease in AtcDPPIV mRNA levels is not observed when the cch1 mutant is transferred from low to moderate light. The cch1 mutant has a smaller light harvesting complex under moderate light, and is therefore harvesting less light than WT under moderate light. These observations suggest that AtcDPPIV gene expression correlates with light harvesting, not just light intensity. To understand the function of AtcDPPIV, two mutants with independent T-DNA insertions into the AtcDPPIV gene have been analyzed. These lines show subtle photoacclimation phenotypes suggesting that the AtcDPPIV dipeptidyl aminopeptidase may play a role in adjusting plants to different light intensities. To test this hypothesis, six objectives are proposed: 1) Demonstrate that the photoacclimation phenotypes in the two insertion lines are due to loss of AtcDPPIV by complementation with AtcDPPIV.2) Overexpress AtcDPPIV and AtcDPPIV::GFP to determine if overexpression results in a photoacclimation phenotype.3) Overexpress AtcDPPIV::GFP to confirm predicted chloroplast localization and produce an AtcDPPIV antibody for suborganelle localization.4) Determine the substrate specificity of recombinant AtcDPPIV protease.5) Use the yeast two-hybrid system to identify proteins that interact with AtcDPPIV.6) Characterize the extended and compact growth habits observed during photoacclimation in more detail, and determine whether subtle effects are occurring in the AtcDPPIV insertion lines.Little is known about signaling during photoacclimation, and the AtcDPPIV dipeptidyl aminopeptidase could be part of an important signaling cascade that has not yet been described. The AtcDPPIV protein is highly conserved in dicots, monocots and in numerous environmental bacteria, suggesting that it has an important, conserved function.Broader ImpactsThe broader impacts of this proposal include the training of undergraduate and Masters students from a minority campus in Southern California. The best way to increase the numbers of underrepresented minorities in research careers is to offer them an interesting, challenging, feasible project at an early stage in their studies. Also included is the training of a postdoctoral researcher in effective teaching as well as excellence in research. Many of the projects in the laboratory have been included in the undergraduate curriculum in Biological Sciences in both lower and upper division courses, and thus a large number of students have been impacted by this work. In addition, the PI coordinates a third grade science teaching project that currently provides an eight-week hands-on science program to 160 third grade students in the Long Beach public school system.