Structural and functional diversity within the cystatin gene family of Hordeum vulgare

Phytocystatins are inhibitors of cysteine proteinases from plants putatively involved in defence and as endogenous regulators of protein turnover. Seven genes encoding cystatins (HvCPI-1 to HvCPI-7), identi-fied from EST collections and from an endosperm cDNA library, have been characterized. The intron–exon structure of their corresponding ORFs has been de-termined and the predicted three-dimensional models for the seven barley cystatins have been established, based on the known crystal structure of oryzacystatin I from rice. Only one out of the seven deduced proteins, HvCPI-7, had sequence variations affecting the three conserved motifs implicated in the enzyme–inhibitor interaction. In three cases, HvCPI-5, HvCPI-6, and HvCPI-7, amino acid differences lead to the prediction of important structural changes in their three-dimensional structures. Northern blot analysis indicated that the seven genes have different expression patterns in barley tissues. The recombinant proteins expressed in Escherichia coli showed distinct inhibitory properties in vitro, with differentKi values, against the three cysteine proteinases tested: papain, cathepsin B, and cathepsin H. Moreover, these recombinant proteins presented dif-ferential fungicidal characteristics inhibiting the growth of phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum in vitro. The resulting implications for the structural and functional diversity of the seven barley cystatins studied are discussed. Key words: Antifungal activity, barley cystatin genes, cysteine proteinase inhibitor, intron–exon structure, three-dimensional structure prediction