Abstract
The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.
Highlights
Chloroplasts serve as the site of photosynthesis and are responsible for the production of many essential metabolites in higher plants
The Isolation of Three New Virescent Mutants—We have carried out large scale ethyl methanesulfonate (EMS) and activation tagging mutagenesis looking for genetic suppressors of the Arabidopsis variegation mutant yellow variegated [44]
The average FV/FM value of vir3-1 mature green tissues was 0.82. These results suggest that the quantum yield of PSII photochemistry was impaired in young vir3-1 leaves but was gradually recovered as vir3-1 leaves turned green (Fig. 1B)
Summary
F13N6#1F F13N6#1R F13N6#2F F13N6#2R F14G9#2F F14G9#2R T6H22#7F T6H22#7R F14J16#4F F14J16#4R 56180F 56180R 21960F 21960R 180CTPF 180CTPR VIR3GFPF VIR3GFPR VIR3-Flag-F VIR3-Flag-R VIR3-H235L-R VIR3-H235L-F HA-VIR3-F ACT2F ACT2R. In otp, the splicing of rpoC1 is compromised, which leads to abnormal plastidencoded plastid RNA polymerase activities [18]. In addition to these two categories of genes, virescent mutants have been observed at rather high frequency in our screens for yellow variegated (var2) suppressors [19]. We report the identification of three nonallelic virescent mutants, virescent (vir3-1), virescent (vir4-1) and virescent (vir5-1), following the existing naming system [25] These mutants exhibited yellow/white emerging leaves that gradually turned green as plants developed further. Our findings shed new light on the mechanisms of plant virescence and the regulation of chloroplast development
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