Mg-dechelatase Encoded by Chlamydomonas Stay-Green is Involved in the Formation of Photosystem II but not in Chlorophyll Degradation

Pheophytin (Pheo) a is an indispensable molecule in photosystem (PS) II, and is a product in the first step of Chlorophyll (Chl) degradation as well. So, it is seemingly suggested that the STAY-GREEN (SGR) gene, which encodes an Mg-dechelatase that catalyzes the conversion of Chl a to Pheo a, is involved in both the formation of PSII and the degradation of Chl. In this study, a series of experiments were carried out to investigate the physiological functions of Chlamydomonas SGR. Two Chlamydomonas sgr null mutants were prepared by screening an insertionmutant library. The Fv/Fm ratios of wild type (WT), sgr mutants, and complementations under several light conditions were observed. The lower Fv/Fm ratios of sgr mutants suggested a lower PSII activity. High-performance liquid chromatography (HPLC) analysis showed reduced Pheo a/Chl levels of sgr mutants, implying a reduced PSII levels. The growth rates in the presence (TAP medium) and absence (HSM medium) of a carbon source were examined. The sgr mutants exhibited reduced photomixotrophical and photoautotrophic growth rate. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) and immunoblotting analysis showed the PSII levels were reduced in sgr mutants, with PSI and LHCII levels unchanged. The reduced PSII levels were verified by low-temperature fluorescence spectroscopy of whole cells. These results indicate that Chlamydomonas SGR is involved in PSII formation. In Nitrogen (N) starvation under both photomixotrophic and photoautotrophic conditions, Chl degradation proceeded in the sgr mutants as in WT. The qRT-PCR results presented that the expression level of SGR kept unchanged during N starvation. These results indicate that SGR is not required for Chl degradation. Compare with Arabidopsis WT, sgr 1 2 l triple mutant, a mutant with the complete absence of SGR activity, showed similar growth speed and Fv/Fm ratio at the developmental stage. Immunoblotting analysis showed that the proteins in PSs and LHCII of sgr triple mutants were normally synthesized at the developmental stage. HPLC analysis showed similar pigments levels in Arabidopsis WT and sgr triple mutant at the developmental stage. BN-PAGE results demonstrated PS SC of sgr triple mutants were normally assembled at the developmental stage but less disassembled at senescence stage. At senescence stage, sgr triple mutants showed stay green phenotype and reduced Fv/Fm ratios. These results indicate Arabidopsis SGR participates in Chl degradation but not in PSII formation. According to the above results, Chlamydomonas SGR is involved in PSII formation but not in Chl degradation.

(主査) 特任教授 田中 歩, 教授 藤田 知道, 助教 伊藤 寿

生命科学院（生命科学専攻）