| Abstract |
The appearance and stabilization of the nuclear-coded LHCII protein (light-harvesting pigment-protein complex serving PS II) during thylakoid biogenesis is under control of phytochrome-regulated Lhcb transcription, the endogenous circadian clock, and the amount of Chl accumulated. LHCII is barely immunodetected in thylakoids of dark-grown leaves, and contrary to the situation found in leaves exposed to continuous light (CL) (where it comprises up to 60% of the thylakoid protein), its level in leaves exposed to intermittent light (ImL) is very low, despite the presence of in vitro translatable Lhcb mRNA. Furthermore, these plants, when transferred to CL following prolonged preexposure to ImL, do not accumulate a high amount of additional Chl or LHCII, even though they possess in vitro translatable Lhcb mRNA. The level of LHCII in ImL plants however, depends on the duration of the dark phase in light-dark cycles, increasing as the duration is decreased, suggesting that the rate of Chl accumulation, relative to that of the other thylakoid components, is an important factor in LHCII stabilization. Indeed, in leaves exposed to CL for limited time, and then transferred to conditions where Chl synthesis is completely stopped but synthesis of reaction center (RC) proteins and new photosynthetic units continues, the preaccumulated LHCII is degraded (either in the dark or in light in the presence of Chl-synthesis inhibitors). Based on these results it was earlier proposed that the rate of Chl accumulation relative to that of the RC and the LHCII proteins regulates the LHCII stabilization during thylakoid biogenesis. RC and LHCII apoproteins may compete for the limited amount of Chl, which is required to anchor the proteins in the thylakoid membrane, rescuing them from degradation. It was proposed that the RC proteins, possibly having higher affinity for Chl, are the first to be stabilized under limited Chl accumulation, while the LHCs, in the absence of Chl binding are degraded. However, in view of the recent finding that thylakoids possess thylakoid-bound proteolytic activity against the universal substrate azocoll and the endogenous or/and exogenously added LHCII apoprotein, also under phytochrome and circadian control and activated under conditions inhibiting Chl accumulation, the question was raised as to whether the inability of the ImL plant to stabilize LHCII is rather due to the presence or activation of such a protease. The aim of this work was to study the nature of the thylakoidal protease against the LHCII apoprotein, its regulation during thylakoid biogenesis as well as its role on the function of the photosynthetic membrane in bean leaves (Phaseolus vulgaris, var. Red kidney). This study has shown that the proteolytic activity depends on the developmental stage of the plant. PLBs of 6-day old etiolated plants were found to possess low activity that increased parallel to leaf tissue age. Similarly, intact etioplasts of bean plants exhibit proteolytic activity against the exogenously added LHCII apoprotein that increases as etiolation is prolonged. The activity increases in the membrane fraction and not in the stroma, where it remains low and constant, suggesting that the gradual appearance of the proteolytic activity in thylakoids during development most probably reflects the synthesis of the proteolytic system and not changes in the association of the protease to prothylakoids. The proteolytic activity increased by a pulse of white light applied to etiolated plants; the increase was similar to that observed following a number of light-dark cycles, or following brief exposure to CL (up to 25 h), suggesting that the protease activity is induced by light. However, exposure of etiolated plants to prolonged CL resulted in reduced proteolytic activity mainly vs endogenous LHCII, in a manner inversely proportional to Chl accumulation. This reduction in activity was not observed in TX-100-solubilized thylakoids, suggesting that it probably does not reflect a direct effect of Chl on the activity of the protease. Although, TX-100-solubilization greatly increased the activity of thylakoids vs endogenous LHCII in plants exposed to prolonged CL, it did not affect the activity in PLBs or primary thylakoids vs exogenous LHCII apoprotein. Furthermore, addition of TX-100-solubilized Chl-rich mature green thylakoids to Chl-deficient primary thylakoids did not reduce the activity of the latter. Chl, therefore seems to act by shielding of the LHCII apoprotein, rescuing the protein from proteolytic attack. Our results further showed that in ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i. e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast and is responsible for the LHCII stabilization, and give a clue as to why plants in ImL with short dark-intervals contain LHCII (more developed leaves, enhanced proteolytic activity), whereas those with long dark-intervals possess only photosystem-unit cores and lack LHCII (less developed leaves, low proteolytic activity). In the present study the involvement of a membrane-bound, but only peripherally attached, protease in the degradation of LHCII apoprotein has been established. This protease is partially or completely removed by low-salt or NaBr-washing, respectively, the activity being gained in the wash. In etioplasts, as well as in chloroplasts, the proteolytic activity is mainly detected in the membrane fraction while in the stroma it remains low. Under the conditions studied, low proteolytic activity was also found in the envelope fraction of chloroplasts, while no activity was detected in the thylakoid lumen. In the present study it was also observed that the association of the protease with thylakoids may be under cation control. The protease was found to act mainly against POR and LHCII, suggesting that it constitutes a regulatory mechanism, the main target of which are Chl-binding proteins. Other characteristics of the proteolytic activity is its heat and SDS-stability. Using plastidic (CAP) or cytoplasmic (CHI) protein synthesis inhibitors, it was found that they both inhibit the proteolytic activity, pointing to a protease complex, the subunits of which are synthesized in both compartments, as in the case of the clp protease. Preexposure of TX-100-solubilized thylakoids to light increases their proteolytic activity, depending on the light intensity as well as on the duration of the exposure. Mg-ATP addition to the incubation medium, further enhances the proteolytic activity, which reaches its previous level when NaF is added concominant whith Mg-ATP. This effect is not detected in this extend, when stroma or NaBr-wash samples are used, suggesting that possibly a thylakoidal factor is responsible for the in vitro high-light and Mg-ATP activation of the proteolytic activity. Addition of Mg2+ or Ca2+ in to the incubation medium enhances the proteolytic activity, while Ni2+ has no effect and Zn2+ or Cd2+ both inhibit the thylakoidal proteolytic activity. In an attempt to explain how Cd2+ inhibits and LHCII accumulation, we used plants exposed to ImL. We found that under the conditions studied, both Chl and LHCII accumulation were drastically reduced, although the LDS-solubilized total leaf protein remained unaffected. However, on the basis of total Chl present, the amount of stabilized LHCII was similar in both Cd-treated and non-treated samples. Additionally, the reduction of LHCII was not due to the level of proteolysis, since the thylakoid-bound protease was drastically inhibited. Northen hybridization analysis indicated that Cd affects LHCII accumulation by reducing drastically the steady-state level of Lhcb transcripts. In the present work, the involvement of the proteolytic activity in the grana unstacking was also studied. We found that benzamidine, a serine-type protease inhibitor, which inhibited both thylakoidal and stromal proteolytic activity, inhibited also the low-salt-induced grana unstacking. Nevertheless, NaBr-washing of thylakoids, known to completely remove the proteolytic activity from the thylakoids, had a partial effect on the low-salt-induced grana unstacking, indicating that this proteolytic activity may be only partly responsible for grana unstacking. In an attempt to separate the protease we analyzed several samples by SDS-PAGE and we found that the proteolytic activity against exogenous LHCII apoprotein is detected at the origin of the resolving gel, suggesting, that the protease is either of high molecular weight, or a supramolecular complex that could not be further resolved under the conditions studied.
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