| During the terminal phase of leaf development (i.e., senescence), the photosynthetic membranes of chloroplasts (thylakoids) are broken down and the photosynthetic activity of leaves drops to negligible levels. The main objective of this work was to analyze the mechanism(s) of the breakdown of thylakoids during senescence of soybean (Glycine max L. Merr.) leaves. To this end, a combination of biochemical, physiological and molecular approaches were used in conjunction with microscopic observations of senescing cells. Also, a cytoplasmically-inherited mutant of soybean (cytG) that retains chlorophyll during senescence was characterized in detail and used as a probe of this breakdown process.;Among the thylakoid protein complexes, cytochrome b6/f was the first to decline, followed by photosystems I and II, and their respective light-harvesting antennae. The cytG mutation partially inhibited the loss of chlorophyll, specially chlorophyll b, and of the light-harvesting complex of photosystem II. The retention of light-harvesting complex II in cytG resulted in light-accelerated inactivation and breakdown of photosystem II reaction center.;In senescing chloroplasts, there was a marked accumulation of lipid globules (plastoglobuli) containing chlorophyll and thylakoid proteins. Some materials present in plastoglobuli were secreted out of senescing chloroplasts. The secreted materials formed a cytosolic globule ("bleb") squeezing through the outer chloroplast envelope membrane. Among the thylakoid components secreted were chlorophyll, lipids and at least one photosystem II protein (CP47). These observations suggest that part of the breakdown of thylakoid components may occur outside the chloroplast, probably in the cytosol or vacuole.;Treatment with a highly specific inhibitor of thiol proteases indicated that members of this class of proteases are involved in the degradation of several thylakoid proteins (e.g., ATP synthase, photosystem II proteins) but not others (e.g., light-harvesting complex II). One thiol protease was expressed early during senescence, and it appeared to be regulated at the translational/post-translational level.;These findings indicate that secretion of thylakoid components out of the chloroplast, and translational/post-translational regulation of the expression of thiol protease(s) may be important control points in the senescence process. |
