Characterization of Arabidopsis mutants defective in the regulation of chlorophyllide a oxygenase.
Chlorophyll b is one of the major light-harvesting pigments produced by land plants, green algae and several cyanobacterial species. It is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which in higher plants consists of three domains, namely, A, B, and C. We previously demonstrated that the C domain exhibits a catalytic function, whereas the A domain destabilizes the CAO protein in the presence of chlorophyll b, thus regulating the cellular level of CAO. In a previous study, we also presented genetic evidence demonstrating the involvement of Clp protease in the destabilization of CAO. In this study, in order to gain further insight into the regulatory mechanism of CAO, we screened for mutants defective in the control of CAO accumulation. Seeds from an Arabidopsis transgenic plant overexpressing a chimeric protein consisting of the A and B domains of CAO and green fluorescent protein (GFP) were mutagenized by ethyl methane sulfonate. We screened the progenies of the transgenic plants by laser-scanning confocal microscopy, and isolated a total of 66 mutants exhibiting significant GFP fluorescence. By immunoblotting analysis, we confirmed that these mutants accumulated the fusion protein of the N-terminal domains of CAO and GFP at a high level. We further divided these mutants into seven groups by distribution patterns of the fusion protein, and characterized them by pigment and immunoblotting analyses. Based on these analyses, we proposed a model to describe the regulatory mechanism of CAO.
Kanematsu
Satoshi
S
Institute of Low Temperature Science, Hokkaido University, N19 W8, Kita-ku, Sapporo, 060-0819, Japan.
Sakuraba
Yasuhito
Y
Tanaka
Ayumi
A
Tanaka
Ryouichi
R
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D002734:Chlorophyll / Q000096:biosynthesis; D000077194:Chlorophyll A; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D018613:Microscopy, Confocal; D009154:Mutation / Q000235:genetics*; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002734:Chlorophyll; D049452:Green Fluorescent Proteins; D010105:Oxygenases; C115272:chlorophyll a oxygenase; D000077194:Chlorophyll A