Protein Changes During Senescence in White Clover Leaves

Abstract

A proteomics approach has been used to study changes in the pattern of protein expression during leaf senescence in white clover. Senescence stages were characterised according to loss of chlorophyll and protein relative to mature green leaves. Cell ultrastructure in leaves at different stages of senescence was examined using transmission electron microscopy. The most obvious changes during senescence occurred in chloroplasts, with progressive loss of thylakoid membrane integrity and build up of osmiophilic globules in the stroma. In a quantitative analysis of 590 protein spots separated by two-dimensional electrophoresis, approximately 7% of proteins increased and 4% decreased in the early stages of senescence. These proteins may have roles in the catabolic processes that characterise senescence. Nearly half of the proteins analysed showed significant senescence-related changes in relative abundance, particularly those in the chloroplast fraction. Approximately a third of the protein spots present in mature green leaves were also visible in two-dimensional electrophoresis of isolated chloroplasts, and these spots represented a major proportion of the proteins showing senescence related declines in abundance. Several chloroplast proteins were tentatively identified by MALDI-TOF mass fingerprinting as rubisco large subunit, two rubisco subunit isoforms, a rubisco activase and the 33 kDa protein of the photosystem II oxygen-evolving complex. These proteins all showed significant declines late in senescence, indicating that the photosynthetic apparatus was being degraded and nitrogen remobilised. A putative glutamine synthetase in the chloroplast fraction also declined in abundance during senescence. Northern blot analysis indicated that expression of the mRNA for CP1, a putative cysteine protease, more than doubled late in senescence relative to earlier stages in development. CP1 may have a role in the large-scale degradation of leaf proteins during senescence. This study further emphasises the importance of proteolysis and nitrogen remobilisation in senescence. In particular, the breakdown of chloroplasts and degradation of chloroplast proteins with roles in photosynthesis was a primary feature of white clover leaf senescence. Further analysis of protein changes during senescence using this proteomics approach would allow the enzymes and regulatory factors involved in the process to be identified.