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

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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.