Activity-Based Profiling of Papain-like Cysteine Proteases During Late-Stage Leaf Senescence in Barley

Leaf senescence is a developmental process that allows nutrients to be remobilized and transported to sink organs. Previously, papain-like cysteine proteases (PLCPs) have been found to be highly expressed during leaf senescence in different plant species. In this study, we analyzed active PLCPs in barley (<i>Hordeum vulgare</i> L.) leaves during the terminal stage of natural senescence. Anion exchange chromatography of protein extracts from barley leaves, harvested six weeks after anthesis, followed by activity assays using the substrates Z-FR-AMC and Z-RR-AMC, revealed a single prominent peak corresponding to active PLCPs. This hydrolytic activity was completely inhibited by E-64, a potent and irreversible inhibitor of cysteine proteases. Fractions enriched for PLCP activity were affinity-labeled with DCG-04 and subjected to SDS-PAGE fractionation, separating two major bands at 43 and 38 kDa. These bands were analyzed using tandem mass spectrometry, allowing the identification of eleven PLCPs. Identified enzymes belong to eight PLCP subfamilies, including CTB/cathepsin B-like (<i>Hv</i>Pap-19 and -20), RD19/cathepsin F-like (<i>Hv</i>Pap-1), ALP/cathepsin H-like (<i>Hv</i>Pap-12 or aleurain), SAG12/cathepsin L-like A (<i>Hv</i>Pap-17), CEP/cathepsin L-like B (<i>Hv</i>Pap-14), RD21/cathepsin L-like D (<i>Hv</i>Pap-6 and -7), cathepsin L-like E (<i>Hv</i>Pap-13 and -16), and XBCP3 (<i>Hv</i>Pap-8). Among the identified PLCPs, <i>Hv</i>Pap-6 was the most abundant. Peptides corresponding to <i>Hv</i>Pap-6 were identified in both the 43 kDa and 38 kDa bands in approximately the same quantity based on total spectral count. Thus, our results indicate that two active <i>Hv</i>Pap-6 isoforms can be isolated from barley leaves at late senescence.