The Components of the CO2 Concentrating Mechanism of Chlamydomonas reinhardtii

水生光合生物几乎占地球二氧化碳固定和氧气进化的50%。由于CO2在水中扩散缓慢，藻类发展了促进CO2和HCO3-吸收的方法来增强光合作用。包括莱茵衣藻（Chlamydomonas reinhardtii）在内的单细胞藻类具有内部浓缩二氧化碳的能力，使这些生物能够在非常低的外部二氧化碳浓度下进行光自养生长。这种CO2浓缩机制是可诱导的；它只存在于在限制二氧化碳条件下生长的细胞中。已经提出了一个模型来解释二氧化碳是如何在真核单细胞藻类中集中的。在这个模型中，存在一种机制，即碳酸氢盐积累、Rubisco的包装以及在Rubisco的位置产生CO2。这个模型成功地解决了藻类如何浓缩二氧化碳的问题，二氧化碳是一种很容易通过生物膜扩散的分子。从本质上讲，不容易穿过膜的HCO3-首先在叶绿体中积累，二氧化碳在固定二氧化碳的酶，核酮糖- 1,5 -二磷酸羧化酶/加氧酶（Rubisco）处或附近产生。这使得Rubisco能够在CO2从细胞中泄漏之前催化羧基化反应。在过去的三年中，已经证明Rubisco特异性地位于叶绿体类pyrenoid。碳酸酐酶使HCO3-脱水，在叶绿体中产生二氧化碳。此外，还发现了低CO2环境下生长诱导的膜蛋白。这些膜蛋白的功能仍有待证实，但它们可能具有HCO3-转运体的功能。在这个建议中，已经概述了三种方法，将确定这些膜蛋白的生理作用。在第一种方法中，通过插入诱变产生一系列突变。然后，转化体将被筛选为不能在低二氧化碳水平下生长和在二氧化碳浓缩机制中有缺陷的细胞。在插入位于基因内的情况下，这种方法将允许克隆在二氧化碳浓缩机制中起作用的基因，促进该基因的鉴定。第二种方法是组成性地表达通常只在低二氧化碳下表达的特定膜蛋白，以观察它们是否能增强二氧化碳的吸收和固定。在这种情况下，感兴趣的基因将与组成启动子融合。第三种方法是敲除特定的基因，看看它们是否对二氧化碳浓缩机制的运作是必需的。这种方法依赖于罕见的同源重组事件，这些事件已被证明发生在莱茵哈特氏菌中。已经概述了一种方法，该方法采用阳性选择标记和链接的阴性选择标记，这将使我们能够识别罕见的同源重组。这种方法在其他真核生物中已经取得了成功。二氧化碳浓缩机制的蛋白质特征将有助于我们对藻类如何提高其二氧化碳固定效率的理解。对这一过程的了解也将有助于科学家预测海洋生物对全球二氧化碳浓度增加的反应。

Aquatic photosynthetic organisms account for almost 50% of the Earth's CO2 fixation and O2 evolution. Due to the slow diffusion of CO2 in water, algae have developed methods of facilitating CO2 and HCO3- uptake to enhance photosynthesis. Unicellular algae, including Chlamydomonas reinhardtii, have the capacity to concentrate CO2 internally, allowing these organisms to grow photoautotrophically at very low external CO2 concentrations. This CO2 concentrating mechanism is inducible; it is present only in cells that have been grown with limiting CO2 conditions. A model has been proposed to explain how CO2 is concentrated in eukaryotic unicellular algae. In this model there is a mechanism for bicarbonate accumulation, a packaging of Rubisco and a generation of CO2 at the location of Rubisco. This model successfully addresses the question of how algae can concentrate CO2, a molecule that can readily diffuse through biological membranes. Essentially, HCO3- which does not cross membranes readily is first accumulated in the chloroplast and CO2 is generated at or near the enzyme that fixes CO2, ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco). This allows Rubisco to catalyze the carboxylation reaction before the CO2 can leak from the cell. In the past three years it has been demonstrated that Rubisco is specifically located in the chloroplast pyrenoid. The carbonic anhydrase that dehydrates HCO3-, generating CO2 in the chloroplast has also been identified. In addition, membrane proteins induced by growth on low CO2 have been discovered. The function of these membrane proteins remains to be proven but they might function as HCO3- transporters. In this proposal three approaches have been outlined that will identify the physiological role of these membrane proteins. In the first approach, a series of mutations will be generated by insertional mutagenesis. The transformants will then be screened for cells that cannot grow on low CO2 levels and are defective in the CO2 concentrating mechanism. In cases where the insertion is located within the gene this approach will allow for the cloning of a gene that plays a part in the CO2 concentrating mechanism, facilitating the identification of that gene. The second approach will be to constitutively express specific membrane proteins that are normally only expressed under low CO2 to see if they enhance CO2 uptake and fixation. In this case, the gene of interest will be fused with a constitutive promoter. The third approach is to knock out specific genes to see if they are required for the operation of the CO2 concentrating mechanism. This approach relies upon rare homologous recombination events which have been demonstrated to occur in C. reinhardtii. An approach has been outlined that employs a positive selectable marker and a linked negative selectable marker that will allow us to identify rare homologous recombinations. This approach has been successful with other eukaryotic organisms. The characterization of the proteins of the CO2 concentrating mechanism will add to our understanding of how algae have increased their efficiency of CO2 fixation. An understanding of this process will also help scientists predict how marine organism might respond to increasing global CO2 concentrations.