MsCYS1, a developmentally-regulated cystatin from alfalfa

Several roles have been attributed to cystatins in plants, ranging from the regulation of host [endogenous] cysteine proteases to the inhibition of herbivorous pest [exogenous] proteases. We report here the cloning, expression and functional characterization of a novel cystatin from alfalfa, Medicago sativa L. The new sequence, isolated from a cDNA expression library prepared from young leaves, encodes a protein, MsCYS1, with the typical inhibitory motifs of cystatins, namely the central signature motif QxVxG, a GG doublet in the N-terminal trunk, and a W residue in the C-terminal region, about 30 amino acids distant from the central inhibitory motif. As shown by a protein-based phylogenetic reconstruction, MsCYS1 is a close relative of other cystatins from Fabaceae presumably involved in the regulation of endogenous proteases. This cystatin is developmentally regulated in stems and leaves, and not induced by stress signals including methyl jasmonate, known to activate cystatins involved in plant defense. A recombinant form of MsCYS1 expressed in Escherichia coli was shown to strongly inhibit alfalfa leaf cysteine proteases while showing weak affinity for the digestive cysteine proteases of different herbivorous pests. Overall, these observations suggest an endogenous protease regulatory role for MsCYS1, possibly associated with the early development of stems and leaves.