Department of Bioscience, School of Science and Technology, Kwansei Gakuin University,Sanda, Hyogo, Japan.
Photosynth Res. 2011 Sep;109(1-3):205-21. doi: 10.1007/s11120-011-9634-4. Epub 2011 Mar 2.
It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO(2) conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO(2) and 5% CO(2). However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO(2). Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm. These results suggest that CAs are constitutively present in the four chloroplastic membrane systems in P. tricornutum and that CO(2) responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.
人们认为细胞内碳酸酐酶（CA）是微藻碳浓缩机制的重要组成部分。在这项研究中，从海洋硅藻三角褐指藻和拟南芥的基因组序列中鉴定出了推定的 CA 编码基因。随后，确定了编码蛋白的亚细胞定位。在三角褐指藻和拟南芥的基因组中分别发现了 9 个和 13 个 CA 序列。三角褐指藻中的两个β-CA 基因与先前鉴定的 ptca1 和 ptca2 相对应。在三角褐指藻细胞中表达的 PtCA1:YFP 融合的免疫染色透射电子显微镜清楚地显示，PtCA1 定位于叶绿体中淀粉核结构的中心部分。除了这两个β-CA 基因外，三角褐指藻可能还含有五个α-CA 和两个γ-CA 基因，而拟南芥则含有三个α-CA、五个γ-CA、四个δ-CA 和一个 ζ-CA 基因。在光照和 CO2 条件不断变化的情况下，从两种硅藻生长的 mRNA 中进行的半定量反转录 PCR 显示，在空气中 CO2 水平和 5% CO2 下生长的细胞中，六个推定的α-CA 和γ-CA mRNA 的水平没有变化。然而，与光照相比，三角褐指藻中一个推定的α-CA 基因 CA-VII 的 mRNA 水平在黑暗中降低。在拟南芥中，分析了推定的α-CA（CA-1）、ζ-CA（CA-3）和δ-CA（CA-7）的 mRNA 积累水平，当细胞在 0.16% CO2 中生长时，所有水平都发现明显降低。通过在三角褐指藻和拟南芥中表达 GFP 融合，分析了八个推定 CA 的细胞内定位。在三角褐指藻中，CA-I 和 II 定位于周质腔，CA-III、VI 和 VII 位于叶绿体内质网，CA-VIII 定位于线粒体。另一方面，拟南芥 CA-1 定位于基质中，CA-3 定位于周质腔。这些结果表明，CA 在三角褐指藻的四个叶绿体膜系统中持续存在，并且 CO2 响应的 CA 存在于三角褐指藻的淀粉核以及拟南芥的基质和周质腔中。
