Deg1 is a Ser protease peripherally attached to the lumenal side of the thylakoid membrane. Its physiological function is unknown, but its localization makes it a suitable candidate for participation in photoinhibition repair by degradation of the photosystem II reaction center protein D1. We transformed Arabidopsis thaliana with an RNA interference construct and obtained plants with reduced levels of Deg1. These plants were smaller than wild-type plants, flowered earlier, were more sensitive to photoinhibition, and accumulated more of the D1 protein, probably in an inactive form. Two C-terminal degradation products of the D1 protein, of 16 and 5.2 kD, accumulated at lower levels compared with the wild type. Moreover, addition of recombinant Deg1 to inside-out thylakoid membranes isolated from the mutant could induce the formation of the 5.2-kD D1 C-terminal fragment, whereas the unrelated proteases trypsin and thermolysin could not. Immunoblot analysis revealed that mutants containing less Deg1 also contain less FtsH protease, and FtsH mutants contain less Deg1. These results suggest that Deg1 cooperates with the stroma-exposed proteases FtsH and Deg2 in degrading D1 protein during repair from photoinhibition by cleaving lumen-exposed regions of the protein. In addition, they suggest that accumulation of Deg1 and FtsH proteases may be coordinated.

Deg1 is a chloroplastic protease involved in maintaining the photosynthetic machinery. Structural and biochemical analyses reveal that the inactive Deg1 monomer is transformed into the proteolytically active hexamer at acidic pH. The change in pH is sensed by His244, which upon protonation, repositions a specific helix to trigger oligomerization. This system ensures selective activation of Deg1 during daylight, when acidification of the thylakoid lumen occurs and photosynthetic proteins are damaged.

The proteases involved in proteolytic degradation in the thylakoid lumen are largely unknown. Western analysis with an antibody against the Escherichia coli periplasmic serine protease DegP suggested that pea chloroplasts contain a homologue of this protease. This homologue was peripherally bound to the luminal side of the thylakoid membrane and could only be removed by a combination of high salt and non-ionic detergent. Its level increased almost 2-fold in pea seedlings exposed to elevated temperature for 4 h, suggesting this protease's role in the chloroplast's heat response. Isolated thylakoid membranes containing the chloroplastic homologue of DegP degraded ␤-casein, an in vitro substrate of the bacterial protease. This activity was partially inhibited by a serine protease inhibitor, suggesting that at least part of the casein-degrading activity in the thylakoid membrane is attributable to DegP. The existence of chloroplastic DegP was further supported by isolating a full-length Arabidopsis cDNA (designated AtDegP) encoding a protein that is 37% identical and 60% similar to the E. coli protease. The amino terminus of the deduced amino acid sequence contained a bipartite transit peptide, typical of proteins targeted to the thylakoid lumen, and the mature portion of the protein contained the highly conserved serine protease catalytic triad His-Asp-Ser. The possible physiological roles of chloroplastic DegP protease are discussed.The chloroplast, the photosynthetic organelle of eukaryotic cells, is composed of six compartments: three different membranes and the three aqueous compartments delimited by them. The chloroplast envelope, composed of an outer membrane, an inner membrane, and an intramembrane space, surrounds the stroma, the soluble compartment where most carbon metabolism reactions take place. The third membrane is the extensive network of thylakoid membranes, harboring the photosynthetic antennae, the photosynthetic electron transport system, and the ATP synthesis machinery. The sixth compartment is the thylakoid lumen, into which protons are pumped to form a proton gradient across the thylakoid membrane. This gradient is the driving force for ATP synthesis. Proteins found within the lumen, either soluble or bound to the inner side of the thylakoid membrane, include the oxygenevolving complex of photosystem II, components of the photosynthetic electron transport system, and many as yet unidentified proteins. As in other biological systems, maintenance of the lumen is expected to require protein degradation to remove damaged or otherwise nonfunctional proteins. However, the proteolytic machinery in this compartment has never been detailed.Many examples of protein degradation in the chloroplast have been documented (for review, see Ref. 1). Specific degradation of luminal proteins has also been demonstrated, with plastocyanin being the best characterized example. When Chlamydomonas cells are grown in a Cu 2ϩ -deficient medium, apoplastocyanin is synthesized, imported into the chloroplast, and transloca...

The earliest visual changes of leaf senescence occur in the chloroplast as chlorophyll is degraded and photosynthesis declines. Yet, a comprehensive understanding of the sequence of catabolic events occurring in chloroplasts during natural leaf senescence is still missing. Here, we combined confocal and electron microscopy together with proteomics and biochemistry to follow structural and molecular changes during Arabidopsis leaf senescence. We observed that initiation of chlorophyll catabolism precedes other breakdown processes. Chloroplast size, stacking of thylakoids, and efficiency of PSII remain stable until late stages of senescence, whereas the number and size of plastoglobules increase. Unlike catabolic enzymes, whose level increase, the level of most proteins decreases during senescence, and chloroplast proteins are overrepresented among these. However, the rate of their disappearance is variable, mostly uncoordinated and independent of their inherent stability during earlier developmental stages. Unexpectedly, degradation of chlorophyll‐binding proteins lags behind chlorophyll catabolism. Autophagy and vacuole proteins are retained at relatively high levels, highlighting the role of extra‐plastidic degradation processes especially in late stages of senescence. The observation that chlorophyll catabolism precedes all other catabolic events may suggest that this process enables or signals further catabolic processes in chloroplasts.

During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b 6 f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state.