INSTITUTO DE FISIOLOGIA VEGETAL
Unidad Ejecutora - UE
capítulos de libros
Activities of vacuolar cysteine proteases in plant senescence
GUIAMET J.J.; MARTÍNEZ D.E; COSTA M.L.
Plant Senescence: Methods and Protocols,
Lugar: New York; Año: 2018; p. 283 - 297
Plant senescence is accompanied by a marked increase in proteolytic activities, and cysteine proteases(Cys-protease) represent the prevailing class among the responsible proteases. Cys-proteases predominantlylocate to lytic compartments, i.e., to the central vacuole (CV) and to senescence-associated vacuoles(SAVs), the latter being specific to the photosynthetic cells of senescing leaves. Cellular fractionation ofvacuolar compartments may facilitate Cys-proteases purification and their concentration for furtheranalysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivoexamination of proteolytic activity by fluorescence microscopy using specific substrates which becomefluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversiblywith the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamidegels copolymerized with a substrate for proteases. Here we describe the three methods mentioned abovefor detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs.