Silencing barley cystatins HvCPI-2 and HvCPI-4 specifically modifies leaf responses to drought stress

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy2 and Icy4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy2 and Icy4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular HvPap-1, HvPap-12 and HvPap-16 C1A proteases. Metabolomics analyses lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.