However, despite the importance of this process, the proteases responsible have not all been identified and characterized. These pathways involve autophagy, senescence-associated vacuoles (SAVs), chloroplast vesiculation, and selective chloroplast destruction via a 13-lipoxygenase. Four pathways of chloroplast breakdown have been identified in Arabidopsis. Protein degradation during senescence involves the disassembly and degradation of the photosystem and metabolic pathways and all other proteins of the living cell until no proteins remain for recycling. Leaf senescence is characterized by extensive protein degradation that enables remobilisation of nutrients, especially nitrogen, for use in other parts of the plant, such as newly developing organs, seeds or storage tissues. Senescence is the final stage in the development of cells, tissues, and organs and in the case of monocarpic species, entire plants. Major Cys protease activities are not essential for dark-induced and developmental senescence and only a knock out line lacking AALP shows a slight but significant delay in plant senescence.
However, a significant delay in progression of whole plant senescence was observed in aalp-1 and rd21A-1/aalp-1 mutants, visible in the reduced number of senescent leaves. Unexpectedly, despite the absence of dominating PLCP activities in these plants, the rubisco and chlorophyll decline in individually darkened leaves and the onset of whole plant senescence were unaltered. Eight protease knock-out lines and two protease over expressing lines were subjected to senescence phenotype analysis to determine the importance of individual protease activities to senescence. By contrast, despite increased VPE transcript levels, active VPE decreased in individually darkened leaves. ResultsĪBPP and mass spectrometry revealed an increased activity of several PLCPs, particularly RD21A and AALP. Using activity-based protein profiling (ABPP), a method that enables detection of active enzymes within a complex sample using chemical probes, the activities of PLCPs and VPEs were investigated in individually darkened leaves of Arabidopsis, and their role in senescence was tested in null mutants. Papain-like Cys Proteases (PLCPs) and Vacuolar Processing Enzymes (VPEs) are amongst the most highly expressed proteases during leaf senescence in Arabidopsis.