Collaborative Research: Physiological and Genetic Characterization of C02 Concentrating Mechanisms in Marine Diatoms

地球上大约50%的光合作用发生在海洋中，而硅藻被认为负责大约一半的海洋光合作用。参与光合作用的光能捕获的分子机制在进化上是古老的，而且保存得很好。然而，在光合作用过程中提供固定的无机碳(即结合到碳水化合物中)的途径是相当多样化的。这些途径是随着全球二氧化碳浓度的下降而演变的，它们提高了初级固碳酶Rubisco周围的二氧化碳浓度，Rubisco是低效的，而且位于叶绿体内。这项拟议研究的目标是鉴定和表征三角褐指藻和假海链藻这两种海洋硅藻的二氧化碳浓缩机制的分子组成，这两种硅藻的全基因组序列和遗传操作系统是可用的。研究的重点将是碳酸氢盐转运体和碳酸氢酶，它们是已知的二氧化碳浓缩机制的基本组成部分。虽然已经用计算方法在三角锥虫和拟青霉基因组中确定了这些基因，但很少有细胞学或功能上的验证来验证计算预测。各种方法，包括显微镜，免疫定位，天然蛋白-荧光蛋白融合的过度表达，基于RNA干扰的表达下调，以及膜进口质谱仪将被用来连接细胞学和功能信息与基因组实体。这些信息将有助于随着基因组或转录本的出现而延伸到其他硅藻。这些数据将被用来测试和改进二氧化碳浓缩机制功能的概念模型。广泛影响一名研究生和本科生将在浮游植物生理学和遗传学的交叉点接受培训。更广泛的社区将通过与东南海洋科学教育卓越中心合作，其中调查人员将与K-12教师和外联专家合作，编写关于海洋酸化的材料，供K-12课堂使用。

Approximately 50% of the photosynthesis on Earth occurs in the oceans, and diatoms are thought to be responsible for around half of this marine photosynthesis. The molecular machinery involved in the capture of light energy for photosynthesis is evolutionarily ancient and well conserved. However, the pathways for supplying the inorganic carbon that is fixed (i.e. incorporated into carbohydrates) during photosynthesis are quite diverse. These pathways, which evolved in response to a decrease in global carbon dioxide concentrations, elevate the concentration of carbon dioxide around the primary carbon-fixing enzyme RubisCO, which is both inefficient and located within the chloroplast. The goal of the proposed research is to identify and characterize the molecular components of the carbon dioxide concentrating mechanisms in Phaeodactylum tricornutum and Thalassiosira pseudonana, two marine diatoms for which whole genome sequences and genetic manipulation systems are available. A research focus will be bicarbonate transporters and carbonic anhydrases, which are known to be essential components of the carbon dioxide concentrating mechanisms. While these have been identified in the P. tricornutum and T. pseudonana genomes using computational approaches, there is little cytological or functional verification of the computational predictions. A variety of methods including microscopy, immunolocalization, over-expression of native protein-fluorescent protein fusions, RNA interference based expression knockdown, and membrane inlet mass spectrometry will be used to connect cytological and functional information to genomic entities. Such information will facilitate an extension to other diatoms as genomes or transcriptomes become available. The data will be used to test and refine conceptual models of the function of the carbon dioxide concentrating mechanisms.Broader ImpactsA graduate student and undergraduate students will be trained at the intersection of phytoplankton physiology and genetics. The wider community will be engaged through a collaboration with the Centers for Ocean Sciences Education Excellence-South East in which the investigators will collaborate with K-12 teachers and outreach specialists to develop materials on ocean acidification for use in K-12 classrooms.