Chapter title |
Activities of Vacuolar Cysteine Proteases in Plant Senescence
|
---|---|
Chapter number | 22 |
Book title |
Plant Senescence
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7672-0_22 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7670-6, 978-1-4939-7672-0
|
Authors |
Dana E. Martínez, Lorenza Costa, Juan José Guiamét |
Abstract |
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 predominantly locate 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 of vacuolar compartments may facilitate Cys-proteases purification and their concentration for further analysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivo examination of proteolytic activity by fluorescence microscopy using specific substrates which become fluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversibly with the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamide gels copolymerized with a substrate for proteases. Here we describe the three methods mentioned above for detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs. |
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