Collaborative Research: Investigations of the plant and bacterial family of LOV-domain flavoproteins

智能优点：植物光感受器识别光信号，检测光方向、光强度、光颜色和光持续时间。它们利用这些信号来调控植物发育的几乎每一个阶段，从种子萌发到开花、果实发育和衰老。已知的植物光感受器有四类：能够感知红光和近红外光的光敏色素，以及隐色素、光敏素和与光敏剂有亲缘关系的三个光感受器家族，它们都可以感知蓝光和近紫外光。向光素是光感受器，它将植物生长引向光源(向光性)；调整叶片角度以最大限度地捕捉光合作用所需的光线；调整进行光合作用的小细胞器(叶绿体)的位置以展开它们并最大限度地减少自身遮蔽，以便在暗光下最大限度地捕捉光线(或移动到最大限度地遮蔽自身以避免因过多光线造成损害的位置)；以及诱导叶表面的小气孔(称为气孔)打开，以促进光合作用所需的二氧化碳的吸收。趋光素含有两个连续的氨基酸片段，它们形成了一个口袋，包围着核黄素衍生物的分子，核黄素衍生物是一种黄色色素，负责吸收蓝光。这些片段被指定为LOV结构域，因为它们与感知光、氧或电压的结构域相似。在吸收蓝光时，光激活的色素改变蛋白质的形状，启动导致最终生物反应的步骤--无论是向光性、叶片定位、叶绿体运动或气孔开放。这种光吸收蛋白质口袋(LOV结构域)也在低等植物、许多不同种类的真菌和大量细菌的重要蛋白质中发现。布鲁氏菌是一种有毒的动物病原体，在这种细菌中，光吸收已被证明可以诱导细菌毒力增加10倍。这种反应是由一个含有LOV结构域的蛋白介导的。本项目有两个重点：第一，继续研究LOV结构域的形状变化是如何传递到诱导几种生物反应之一的信号中，使用生化技术来鉴定在光激发下与包含LOV结构域的蛋白相互作用的其他蛋白质。其目的是确定从光吸收到反应的完整的事件链。其次，它将利用生物物理和生化方法研究根瘤菌的光反应，根瘤菌与豆类一起是固氮所必需的。与布鲁氏菌病病原体一样，根瘤菌也有一种含有LOV结构域的信号蛋白，该蛋白会影响其在光照下的行为。这项研究将使用生物物理和生化方法来追踪从光吸收到细菌行为改变的步骤，来研究光在这种农业上重要的细菌中所扮演的先前未被描述的角色。该项目旨在阐明一个小的含有色素的蛋白质口袋一方面可以提高细菌的致病能力，另一方面可以在蓝光激活时叶片上的气孔打开的共同机制。更广泛的影响：将研究和教育结合起来，培养博士后研究员和研究生，并指导本科生(加州大学洛杉矶分校需要一篇研究论文)和暑期实习生，本科生和高中都是该项目的重要组成部分。许多学生是由少数族裔项目赞助的-MARC(少数族裔研究职业准入)、MBR(NIH少数民族生物医学研究支持)、暑期社区大学学生在ACCESS计划下工作(ACCESS：通向未来的文凭桥梁，NIH桥梁计划的一部分)和CAMP(加州少数族裔参与联盟)。温斯洛·布里格斯在当地一个州立公园向公众发表了解释性演讲，部分是基于他的研究。将继续以暑期实习的形式向高中生、教师和大学本科生进行宣传。最后，预计这项研究本身将最终影响农业和卫生问题。

Intellectual Merit: Plant photoreceptors recognize light signals that detect light direction, light intensity, light color, and light duration. They use these signals to regulate almost every phase of plant development from seed germination through flowering, fruit development, and senescence. There are four known groups of plant photoreceptors: the phytochromes that can sense red and near-infrared light, and the cryptochromes, phototropins, and a family of three photoreceptor relatives of the phototropins, all of which can sense both blue and near-ultraviolet light. The phototropins are the photoreceptors that direct plant growth toward a light source (phototropism); adjust the leaf angle to maximize light capture for photosynthesis; adjust the positions of the small organelles that carry out photosynthesis (chloroplasts) to spread them out and minimize self-shading in order to maximize light capture in dim light (or to move into positions maximizing self-shading to avoid damage caused by too much light); and to induce the opening of small pores in the leaf surface (called stomata) to facilitate the uptake of carbon dioxide needed for photosynthesis. The phototropins contain two sequential segments of amino acids that form a pocket surrounding a molecule of a riboflavin derivative, a yellow pigment that is responsible for absorbing blue light. The segments are designated LOV domains for their similarity to domains that sense light, oxygen, or voltage. On absorption of blue light, the light-activated pigment alters the shape of the protein to initiate the steps leading to the eventual biological response - be it phototropism, leaf positioning, chloroplast movement, or stomatal opening. This light-absorbing protein pocket (the LOV domain) is also found in important proteins in lower plants, many different kinds of fungi, and a large number of bacteria. In the bacterium Brucella, a virulent animal pathogen, light absorption has been shown to induce a ten-fold increase in bacterial virulence. This response is mediated by a LOV-domain-containing protein. The present project has two foci: First, to continue to investigate how the change in shape of the LOV domain is transmitted into a signal that induces one of the several biological responses, using biochemical techniques to identify other proteins which interact with the LOV-domain-containing proteins on light excitation. The objective is to determine the complete chain of events from light absorption to response. Second, it will investigate, using biophysical and biochemical methods, the light responses of the bacterium Rhizobium, essential together with legumes for nitrogen fixation. Like the brucellosis pathogen, Rhizobium has a LOV-domain-containing signaling protein that affects its behavior on exposure to light. The research will investigate the previously un-described role of light in this agriculturally important bacterium using biophysical and biochemical methods to trace the steps from light absorption to alteration in the bacterial behavior. The project aims to elucidate the common mechanism by which a single small pigment-containing protein pocket can elevate pathogenesis in a bacterium on the one hand, and the opening of stomatal pores on a leaf on the other when activated by blue light. Broader Impact: Integration of research and education in the training of post-doctoral fellows and graduate students and mentoring of undergraduate students (a research thesis is required at UCSC) and summer interns, both undergraduate and high school is an essential component of this project. Many of the students are sponsored by minority programs - MARC (Minority Access to Research Careers), MBRS (NIH Minority Biomedical Research Support), Summer Community College Students working under the ACCESS program (ACCESS: Baccalaureate Bridges to the future, part of NIH Bridges program), and CAMP (California Alliance for Minority Participation). Winslow Briggs gives interpretive talks to the public at a local state park based partially on his research. Outreach to high-school students, teachers and college undergraduates will continue in the form of summer internships. Finally, it is anticipated that the research itself will ultimately impact both agriculture and health issues.