Triton X-100 solubilized thylakoids, isolated from Phaseolus vulgaris chloroplasts, degrade endogenous or exogenously added LHC II. The degradation, as monitored by immunodetection of the remaining LHC II after incubation at 37°C, is activated by Mg(++) and inhibited by pCMB, EDTA, PMSF and benzamidine; the activity under high light conditions parallels chlorophyll photooxidation. The thylakoid-bound proteolytic activity is under phytochrome control. Etiolated plants pretreated by a white light pulse, and kept in the dark thereafter, show enhanced proteolytic activity, which follows rhythmical oscillations. On the other hand, chloramphenicol pretreatment of etiolated plants, prior to their transfer to continuous light, reduces the proteolytic activity against LHC II. The results suggest that the degradation involves a serine type protease, which depends on SH group(s), coded by the plastid genome; the protease action on LHC II is regulated by Mg(++), phytochrome, the biological clock and chlorophyll accumulation in the thylakoid. The stroma lamellar fraction, separated from French press disrupted chloroplasts, exhibits higher activity towards exogenous LHC II than the grana fraction. The stroma of intact chloroplasts exhibits also high proteolytic activity, which is drastically reduced when the lysis medium is supplemented with cations. This suggests that the protease is bound mainly on stroma lamellae and peripheral granal membranes, its association to the membranes being possibly under cation control.
The appearance of the light harvesting II (LHC II) protein in etiolated bean leaves, as monitored by immunodetection in LDS-solubilized leaf protein extracts, is under phytochrome control. A single red light pulse induces accumulation of the protein, in leaves kept in the dark thereafter, which follows circadian oscillations similar to those earlier found for Lhcb mRNA (Tavladoraki et al. (1989) Plant Physiol 90: 665-672). These oscillations are closely followed by oscillations in the capacity of the leaf to form Chlorophyll (Chl) in the light, suggesting that the synthesis of the LHC II protein and its chromophore are in close coordination. Experiments with levulinic acid showed that PChl(ide) resynthesis does not affect the LHC II level nor its oscillations, but new Chl a synthesis affects LHC II stabilization in thylakoids, implicating a proteolytic mechanism. A proteolytic activity against exogenously added LHC II was detected in thylakoids of etiolated bean leaves, which was enhanced by the light pulse. The activity, also under phytochrome control, was found to follow circadian oscillations in verse to those in the stabilization of LHC II protein in thylakoids. Such a proteolytic mechanism therefore, may account for the circadian changes observed in LHC II protein level, being implicated in pigment-protein complex assembly/stabilization during thylakoid biogenesis.
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