Abstract
Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PL) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PL and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid (SA) treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three SA activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves.
Highlights
Proteases determine a variety of biological processes ranging from organ maturation, senescence and programmed cell death (PCD) (van der Hoorn and Jones, 2004; van der Hoorn, 2008)
Protein fractions corresponding to the three major peaks observed in Figure 1B were pooled and active papain-like cysteine proteases (PLCP) were labeled using DCG-04, a probe that binds covalently and irreversible to the active site of PLCPs allowing us to monitor the availability of active sites rather than their abundance (Greenbaum et al, 2000; van der Hoorn et al, 2004)
CP1C was the only PLCP found in position A and B of peak 1 and it was found in position A of peak 2
Summary
Proteases determine a variety of biological processes ranging from organ maturation, senescence and programmed cell death (PCD) (van der Hoorn and Jones, 2004; van der Hoorn, 2008). PLCPs are known to be involved in growth related senescence (Noh and Amasino, 1999a; McLellan et al, 2009), PCD (Gilroy et al, 2007; Coll et al, 2011; Lampl et al, 2013), predicted to be important for resource acquisition (Adamczyk et al, 2010) and act as hubs in plant immunity, where they are involved in the perception of microbes, initiation of signaling cascades and activation of responses against pathogens (Misas-Villamil and van der Hoorn, 2008; Jashni et al, 2015; Misas-Villamil et al, 2016) Due to their crucial roles in the regulation of various cellular processes, PLCP activity is tightly controlled via autocatalytic posttranslational modifications, as well as by endogenous inhibitors such as cystatins and serpins (Martinez and Diaz, 2008; Ochieng and Chaudhuri, 2010; Martínez et al, 2012; van der Linde et al, 2012a; Lampl et al, 2013). Their general enzymatic activity involves a nucleophilic attack of the thiol-group at the substrate carboxylterminus where His acts as a proton acceptor (base) for the catalytic Cys and Asn plays an important role for the orientation of the His (Rawlings et al, 2018)
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