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Abstract
I have analyzed the dynamics of protein metabolism, protein modifications and subcellular localization of three specific proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), acyl carrier protein (ACP) and carboxypeptidase, during normal and induced senescence.
Marked qualitative and quantitative changes to proteins in spatially distinct cellular compartments were demonstrated during ripening and upon wounding stress of tomato (Lycopersicon esculentum cv. Pik-Red). The polypeptides identified include ACP, pathogenesis-related proteins, cellulase, peroxidase, large subunit of rubisco, sorbitol-6-phosphate dehydrogenase and carboxypeptidase. More proteins in the fruit at pink stage showed a higher turnover rate than those in green fruit, soluble proteins being more unstable relative to the membrane-associated proteins. Tomato proteins were modified by fucosylation or palmitoylation throughout ripening, even during advanced ripening when protein synthesis in vivo was limiting. The generality of differential regulation of protein synthesis versus posttranslational modifications of proteins during tomato senescence was supported by similar findings in the aquatic angiosperm, Spirodela oligorrhiza, induced to senesce under oxidative stress. Results on rubisco turnover in Spirodela, wheat (Triticum aestivum L.) and Chlamydomonas demonstrated high susceptibility of this protein to oxidative stress. This sensitivity was related to crosslinking of large subunits of rubisco via Cys-247, translocation of the holoenzyme to the membranes, and finally protein degradation. These results suggest that the redox state of plant cells may be closely intertwined with cellular switches shifting the leaf from growth and maturation to senescence and death.
The carboxypeptidase (CP) activity in tomato was found to increase with ripening and wounding stress. An antibody reacting specifically with non-denatured CP from fruit and leaf was characterized. Two major polypeptides of about 67-kD and 43-kD were found to constitute the major CP activity.
Immunocytochemical data confirmed a chloroplast location for rubisco, the general presence of ACP (in mitochondria, chloroplasts and cytoplasm) and a vacuolar location for CP. In the tomato leaf, CP was specifically localized to vacuoles of the vascular parenchymal cells, suggesting that it may be involved in the distribution of amino acids and in nitrogen metabolism. Chloroplast membranes were found to be more resistant to oxidative stress damage than the tonoplast and plasmalemma. (Abstract shortened by UMI.)
