Search

Seed quality is affected by different constituents of the seed. In general, seed lots are considered to be of high quality when they exhibit fast and homogeneous germination. When seeds are stored, they undergo different degrees of damage that have detrimental effects on their quality. Therefore, accurate prediction of the seed quality and viability levels of a seed lot is of high importance in the seed-producing industry. Here, we describe the use of activity-based protein profiling of proteases to evaluate the quality of artificially and naturally aged seeds of Arabidopsis thaliana. Using this approach, we have identified two protease activities with opposite behaviours in aged seeds of Arabidopsis that correlate with the quality status of the seeds. We show that vacuolar processing enzymes (VPEs) become more active during the ageing process, in both artificial and natural ageing treatments. Secondly, we demonstrate that serine hydrolases are active at the beginning of our artificial ageing treatment, but their labelling decreases along with seed viability. We present a list of candidate hydrolases active during seed germination and propose that these protease activities can be used in combination with VPEs to develop novel markers of seed quality.

Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. The secreted effector protein Pit2 is an inhibitor of papain-like cysteine proteases (PLCPs) essential for virulence. Pit2 inhibitory function relies on a conserved 14 amino acids motif (PID14). Here we show that synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. Mass spectrometry shows processing of Pit2 by maize PLCPs, which releases an inhibitory core motif from the PID14 sequence. Mutational analysis demonstrates that two conserved residues are essential for Pit2 function. We propose that the Pit2 effector functions as a substrate mimicking molecule: Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. Remarkably, the PID14 core motif is present in several plant associated fungi and bacteria, indicating the existence of a conserved microbial inhibitor of proteases (cMIP).

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.