Characterization of senescence-associated protease activities involved in the efficient protein remobilization during leaf senescence of winter oilseed rape

Abstract

Oilseed rape (Brassica napus L.) is a crop plant characterized by a poor nitrogen (N) use efficiency that is mainly due to low N remobilization efficiency during the sequential leaf senescence of the vegetative stage. As a high leaf N remobilization efficiency was strongly linked to a high remobilization of proteins during leaf senescence of rapeseed, our objective was to identify senescence-associated protease activities implicated in the protein degradation. To reach this goal, leaf senescence processes and protease activities were investigated in a mature leaf becoming senescent in plants subjected to ample or low nitrate supply. The characterization of protease activities was performed by using in vitro analysis of RuBisCO degradation with or without inhibitors of specific protease classes followed by a protease activity profiling using activity-dependent probes. As expected, the mature leaf became senescent regardless of the nitrate treatment, and nitrate limitation enhanced the senescence processes associated with an enhanced degradation of soluble proteins. The characterization of protease activities revealed that: (i) aspartic proteases and the proteasome were active during senescence regardless of nitrate supply, and (ii) the activities of serine proteases and particularly cysteine proteases (Papain-like Cys proteases and vacuolar processing enzymes) increased when protein remobilization associated with senescence was accelerated by nitrate limitation. Short statement: Serine and particularly cysteine proteases (both PLCPs and VPEs) seem to play a crucial role in the efficient protein remobilization when leaf senescence of oilseed rape was accelerated by nitrate limitation.

Response to Plant Hormones of Senescence-related Genes for Cucumis sativus L. in Cotyledon Development

“…As discussed earlier section about SAG 3-5 for cysteine protease following cysteine synthesis by CsOAS-TL, a prolonged ethylene exposure revealed that gradual increase of the SAG 3-5 gene expression in this experiment (Fig. 4B), which could be agreed with the protein remobilization process during cotyledon senescence [25].…”

“…Furthermore, cysteine biosynthesis is part of the regulatory network that mediates between inorganic sulfur supply and the demand for reduced sulfur during plant growth and in response to environmental changes. In accordance with biosynthesis of cysteine, crucial role of senescence-associated serine and cysteine proteases was proposed in the efficient protein remobilization during leaf senescence of oilseed rapeseed [25].…”

The Plastid and Mitochondrial Peptidase Network in Arabidopsis thaliana: A Foundation for Testing Genetic Interactions and Functions in Organellar Proteostasis

“…Arabidopsis cysteine peptidases, such as the cathepsins and metacaspases, include many of the most abundant peptidases in plants with well-established functions in a variety of processes, including senescence and programmed cell death (Poret et al, 2016). Other cysteine peptidases are deubiquitinating enzymes (DUBs) and deSUMOylating enzymes (SUMO isopeptidases); neither ubiquitinylated nor SUMOylated proteins have been reported within the plastids or mitochondria, making it therefore less likely to find any of these DUBs or SUMO isopeptidases within the organelles.…”

Subunit‐selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections

“…So far we have introduced over 40 activity‐based probes into plant science to monitor, for example, Cys proteases, glycosidases, subtilases, acyltransferases and glutathione transfereases, and many of these probes are widely used in plant science (Morimoto and van der Hoorn, ). DCG‐04, for instance, is a probe for papain‐like Cys proteases (Greenbaum et al ., ; van der Hoorn et al ., ) that has been instrumental for the discovery of pathogen‐derived inhibitors (Rooney et al ., ; Tian et al ., ; Shabab et al ., ; Van Esse et al ., ; Song et al ., ; Kaschani et al ., ; Lozano‐Torres et al ., ; Mueller et al ., ), deciphering protease‐inhibitor arms‐races and effector adaptation upon a host jump (Hörger et al ., ; Dong et al ., ), and identifying senescence‐associated proteases (Martínez et al ., ; Carrión et al ., ; Poret et al ., ;). Likewise, proteasome probes have been used to describe post‐translational activation of the proteasome during SA signaling (Gu et al ., ); the selective suppression of the nuclear proteasome by bacterial phytotoxin SylA (Kolodziejek et al ., ; Misas‐Villamil et al ., ); and the regulation of the proteasome by NAC transcription factor RPX (Nguyen et al ., ), the validation and availability of next‐generation chemical probes will underpin exciting scientific discoveries.…”