Abstract
We have characterized a nuclear mutant of barley, viridis-115, lacking photosystem II (PSII) activity and compared it to wild-type seedlings during light-induced chloroplast development. Chloroplasts isolated from wild-type and viridis-115 seedlings illuminated for 1 h synthesized similar polypeptides and had similar protein composition. After 16 h of illumination, however, mutant plastids exhibited reduced ability to radiolabel D1, CP47, and several low Mr membrane polypeptides, and by 72 h, synthesis of these proteins was undetectable. Immunoblot analysis showed that plastids of dark-grown wild-type barley lacked several PSII proteins (D1, D2, CP47, and CP43) and that 16 h of illumination resulted in the accumulation of these polypeptides. In contrast, these polypeptides did not accumulate in illuminated viridis-115 seedlings, although mutant plastids accumulated two PSII proteins that participate in oxygen evolution, oxygen-evolving enhancers 1 and 3. Northern analysis showed that the levels of psbA and psbB mRNA in mutant plastids were equal to or greater than levels in wild-type plastids throughout the developmental period examined here. These results indicate that the nuclear mutation present in viridis-115 affects the translation and stability of the chloroplast-encoded D1 and CP47 polypeptides and that its influence is expressed after the onset of light-induced chloroplast development.
Highlights
In order to gain more insight on how nuclear gene products regulate chloroplast gene expression, we have characterized a nuclear mutant of barley, viridis-115, which is deficient in PSII activity.' We show that the nuclear mutation results in inhibition of translation and decreased stability of the chloroplast-encoded D l and CP47 polypeptides in mature chloroplasts despite the presence of transcripts for these proteins
Description of Viridis-115-Viridis-115 is anuclear mutant of barley deficient in PSII activity.' The molecular basis for the absence of PSII activity in the vir-115 mutant is unknown; initial characterization of mutant seedlings revealed the presence of plastids with giant grana that exhibit a reduction in the number of PSII complexes relative to the other electron transport complexes in mutant seedlings.' Immunoblot analysis demonstrated that the vir-115 mutant contains normal amounts of the 23-kDa polypeptide (OEE2) involved in oxygen evolution [34]
Plastids were isolated from wild-type and mutant seedlings grown 4.5-5.5 days in the dark and illuminated for an additional 1, 16,or 72 h.Intact plastids were fractionated into membrane and soluble polypeptides, and the samples were electrophoresed on Na
Summary
Plastids were isolated from wild-type (lanes I, 3, 5, 7, 9,and 11) and mutant (lanes 2, 4, 6, 8, 10, and 12) seedlings grown 4.5-5.5 days in the dark and illuminated for an additional 1 (lanes 1, 2,7, and 8), 16 (lanes3, 4, 9,and IO),or 72 h (lanes 5, 6, 11, and12).Intact plastids were fractionated into membrane and soluble polypeptides, and the samples were electrophoresed on Na- Thylakoid membranes were probed with antibodies to the nuclear-encoded OEEl and OEE3 proteins of the oxygenevolving complexto quantify the accumulation of these PSII polypeptides in wild-type and vir-115 seedlings (Fig. 2, lanes 11-15).
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