The role of chlorophyll a oxygenase in the regulation of chlorophyll a/chlorophyll b ratio

叶绿素b是陆地植物、绿藻和原生藻类中普遍存在的一种辅助色素。它的生物合成在适应各种光环境中起着关键作用。我们通过插入诱变的方法分离了6个叶绿素b缺失突变体，并分离到了一个与甲基单加氧酶序列相似的负责叶绿素b合成的基因，该基因含有一个可能与[2Fe-2S]Rieske中心和一个单核铁结合的结构域。这些结果表明，叶绿素a加氧酶(CAO)参与了叶绿素b的形成。为了了解CAO的反应机理，我们在大肠杆菌中表达了CAO，并对其进行了酶学研究。在大肠杆菌中产生的重组酶催化了一个不寻常的两步加氧酶反应，并将叶绿素A转化为叶绿素B。光系统的捕光效率在很大程度上取决于其光合作用天线的大小。为了验证这一假说，我们在35S花椰菜花叶病毒启动子的控制下，将叶绿素b生物合成酶(CAO)的基因转化到拟南芥中，实现了CAO基因的高表达。在绿化期，当内源CAO的表达很低时，叶绿素a/b的比值从28急剧下降到7.3，表明成功地促进了叶绿素b的生物合成。我们还发现，在转基因植株的全绿莲座状叶片中，叶绿素a/b的比值从2.85降至2.65，外围捕光复合体与核心天线复合体的比值增加了20。这些结果表明，转基因植株的天线尺寸增加，这表明CAO通过控制叶绿素b的合成来调节天线尺寸。

Chlorophyll b is an ubiquitous accessory pigment in land plants, green algae and prochlorophytes. Its biosynthesis plays a key role in the adaptation to various light environments. We isolated 6 chlorophyll b-less mutants by insertional mutagenesis and isolated a gene responsible for chlorophyll b synthesis whose sequence is similar to those of methyl monooxygenases, and contains putative binding domains for a [2Fe-2S] Rieske center and for a mononuclear iron. These results demonstrate that chlorophyll a oxygenase (CAO) is involved in the chlorophyll b formation. In order to know the reaction mechanisms, we expressed the CAO in E.coli and carried out the enzymatic studies. The recombinant enzyme produced in E.coli catalyses an unusual two-step oxygenase reaction and converted chlorophyllide a to chlorophyllide b.The light-harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis (CAO) in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early greening phase, when the intrinsic CAO expression is very low, the chlorophyll a-to-b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We also found with full-green rosette leaves of transgenic plants that the chlorophyll a-to-b ratio was reduced from 2.85 to 2.65 and that the ratio of the peripheral light-harvesting complexes to the core antenna complex increased by 20. These results indicated that the antenna size increased in transgenic plants, suggesting that CAO regulates antenna size by controlling chlorophyll b synthesis.