Phylogenetically distant barley legumains have a role in both seed and vegetative tissues

Abstract

Legumains or vacuolar processing enzymes are cysteine peptidases (C13 family, clan CD) with increasingly recognized physiological significance in plants. They have previously been classified as seed and vegetative legumains. In this work, the entire barley legumain family is described. The eight members of this family belong to the two phylogenetic clades in which the angiosperm legumains are distributed. An in-depth molecular and functional characterization of a barley legumain from each group, HvLeg-2 and HvLeg-4, was performed. Both legumains contained a signal peptide and were located in the endoplasmic reticulum, were expressed in seeds and vegetative tissues, and when expressed as recombinant proteins showed legumain and caspase proteolytic activities. However, the role of each protein seemed to be different in their target tissues. HvLeg-2 responded in leaves to biotic and abiotic stimuli, such as salicylic acid, jasmonic acid, nitric oxide, abscisic acid, and aphid infestation, and was induced by gibberellic acid in seeds, where the protein is able to degrade storage globulins. HvLeg-4 responded in leaves to wounding, nitric oxide, and abscisic acid treatments, and had an unknown role in the germinating seed. From these results, a multifunctional role was assumed for these two phylogenetically distant legumains, achieving different physiological functions in both seed and vegetative tissues.

Vacuolar processing enzyme 4 contributes to maternal control of grain size in barley by executing programmed cell death in the pericarp

“…a). These results corresponds to the activity optima found for barley VPE2a (HvLeg‐2) and VPE3 (HvLeg‐4; Julian et al ., ). Further, we measured the activity of the recombinant VPE4 against caspase‐1, caspase‐3, caspase‐4, caspase‐6 and caspase‐8 substrates at pH 5 (Fig.…”

“…VPE or legumain (Julian et al ., ) is the first identified enzyme with caspase‐like activity in plants and exhibits activity against caspase‐1 substrate (Hatsugai et al ., , ). An Arabidopsis mutant with all four VPE genes knocked out lacks any caspase‐1‐like activity, which indicates that VPE is the major contributor to this activity (Kuroyanagi et al ., ).…”

Digestive proteases in bodies and faeces of the two-spotted spider mite, Tetranychus urticae

“…This inhibition profile contrasts with those obtained for cathepsin B-and L-like activities in T. urticae, which were inhibited by both IAA and E-64 or only by E-64, respectively, indicating that the substrate used to measure legumain-like activity was not hydrolysed by these other cysteine proteases. In both plants and animals, legumains are enzymes with high specificity for peptide hydrolysis on the carboxyl side of asparagine residues that act as lysosomal/-vacuolar processing enzymes of biologically relevant protein substrates (Chen et al, 2000;Müntz and Shutov, 2002;Julián et al, 2013). In addition, legumains have been shown to be involved in the digestion of dietary proteins in haematophagous ticks (Sojka et al, 2007(Sojka et al, , 2013 and platyhelminth parasites (Delcroix et al, 2006), being in both cases compartmentalised in acidic pH endosomal/lysosomal vesicles in the gut cells.…”

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

“…The βVPE , γVPE , and δVPE ORFs were inserted in the expression vector pPICZαA, and the recombinant plasmid were transformed into Pichia pastoris GS115 using lithium acetate/single-stranded carrier DNA/PEG (LiAc/SS-DNA/PEG) method. The positive colonies were identified and cultivated using methods described previously [32]. To induce expression, we added methanol every 24 h to maintain a final concentration of 0.75%.…”