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，而硅藻被认为承担了大约一半的海洋光合作用。参与光合作用捕获光能的分子机制在进化上是古老而保守的。然而，在光合作用过程中，提供固定的无机碳（即与碳水化合物结合）的途径是相当多样化的。这些途径是为了响应全球二氧化碳浓度的下降而进化而来的，它们会提高初级碳固定酶RubisCO周围的二氧化碳浓度，而RubisCO既低效又位于叶绿体内。本研究的目的是鉴定和表征三角藻（Phaeodactylum tricornutum）和假海藻（thalassisira pseudonana）这两种海洋硅藻的二氧化碳浓缩机制的分子成分，这两种硅藻的全基因组序列和基因操作系统都是可用的。研究重点将是碳酸氢盐转运体和碳酸酐酶，它们是二氧化碳浓缩机制的重要组成部分。虽然这些已经在三角棘球绦虫和伪棘球绦虫基因组中使用计算方法进行了鉴定，但对计算预测的细胞学或功能验证很少。包括显微镜、免疫定位、天然蛋白-荧光蛋白融合物的过表达、基于RNA干扰的表达敲除和膜入口质谱在内的各种方法将被用于将细胞学和功能信息与基因组实体联系起来。当基因组或转录组变得可用时，这些信息将有助于扩展到其他硅藻。这些数据将用于检验和改进二氧化碳浓缩机制功能的概念模型。更广泛的影响研究生和本科生将接受浮游植物生理学和遗传学交叉的培训。通过与东南海洋科学卓越教育中心的合作，更广泛的社区将参与其中，调查人员将与K-12教师和外联专家合作，开发用于K-12教室的海洋酸化材料。

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.