Physiological, structural, and proteomic analysis of chloroplasts during natural senescence of Ginkgo leaves

Abstract

A proteomic analysis was conducted to study the changes in chloroplast proteins during leaf senescence in Ginkgo under natural conditions. We also examined the physiological changes in senescing chloroplasts, including changes in photosynthesis and alterations in chloroplast ultrastructure. Decreased photosynthetic activity was observed during leaf senescence, while chloroplast structures were damaged with grana thylakoid unstacking and large accumulation of osmiophilic granules. Chloroplast proteins were extracted from Ginkgo leaves collected at an early stage (June) and late stage (October) of leaf senescence, and separated by two-dimensional gel electrophoresis. More than 850 protein spots were reproducibly detected, including 27 that were up-regulated and 21 that were down-regulated during senescence. Mass spectrometry analysis and database searches identified 17 spots, which represented 15 different proteins. Fifteen of these spots were chloroplast proteins, and the other two spots were proteins of unknown localization in Ginkgo leaves. Five of the proteins were previously reported as senescence-related proteins, and 10 were novel; (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate synthase, Ycf4, putative 97B2-like cytochrome P450, limonene synthase, light-independent protochlorophyllide reductase subunit B, taxadiene synthase, geranylgeranyl diphosphate synthase, ribosomal protein L2, RNA polymerase alpha chain, and RNA polymerase IV second largest subunit. These proteins are involved in photosynthesis, transcription and translation, secondary metabolism, and reactive oxygen species scavenging. The results indicated that the decline of photosynthesis was mainly due to the decrease in stomatal conductance and the degradation of the photosynthetic apparatus during leaf senescence.