Roles of C1A peptidases during barley leaf senescence mediated by abiotic stresses

Abstract

Abstract Protein breakdown and mobilization from old or stressed tissues, such as leaves, to growing and sink organs, such as grains or tubers, are some of the metabolic features associated with leaf senescence, essential for nutrient recycling. Senescence may be naturally activated by endogenous signals and/or modified by the prevalence of abiotic/biotic stresses, as a survival strategy. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and a dynamic protein traffic to ensure transformation of high molecular weight proteins into transportable and useful hydrolyzed products. C1A cysteine proteases are the most abundant key players responsible for the proteolytic activity during leaf senescence. Besides, cystatins, as specific modulators of C1A protease activities, exert a regulatory role along the process. In barley (Hordeum vulgare), the whole gene family members of C1A cysteine proteases and cystatins have been identified. Elucidating the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. Darkness and nitrogen starvation treatments were used to induce leaf senescence in barley. Both abiotic stresses strongly induced the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Morphological changes presuming chloroplast dismantling designated darkness as an ideal stressor for inducing and analyzing senescence. Differences in biochemical parameters and C1A gene expression and protein accumulation among wild-type and transgenic barley plants over-expressing or silencing this gene were detected under the stress. Besides, a lifespan-delayed phenotype of HvPap-1 silenced lines was evidenced, indicating a functional role for this protease along the senescence process. Proteolysis is likewise essential throughout the mobilization of storage proteins in barley grains during germination. Manipulation of the proteolytic machinery could enhance grain yield and quality through alterations along these stages. Transgenic barley plants silencing or over-expressing HvPap-1 showed differential accumulation of starch, proteins, and free amino acids in the grain. The phenotype displayed by silencing HvPap-1 lines, showing a drastic delay in germination, was particularly striking. Alterations in the proteolytic activities associated with changes in the expression levels of several C1A proteases were also detected. Similarly, down-regulating Icy-2, encoding one of the proteinaceous inhibitors of the studied cathepsin F-like protease, also brought about important effects on grain filling. The cooperative role of cystatins and their functional relationship with cysteine proteases have been highlighted in the current study by the enhanced/reduced tolerance of plants silencing phytocystatins towards drought. Two barley phytocystatins, HvCPI-2 and HvCPI-4, were induced by this stress. Alterations in the proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of target proteases. As a result, accelerated or delayed leaf senescence, depending on the silenced cystatin, was exhibited. Results support the potential use of these plants to modulate plant responses facing abiotic stress and, at the same time, to maintain or even increase crop yields under the evidenced climate change framework According to data reported in this thesis, manipulation of C1A proteases-cystatins interactions in barley has the potential to modulate sensitivity towards specific abiotic stresses through modifications over established developmental leaf senescence programs. In addition, the in vivo implication of this proteolytic network during remobilization of stored compounds along barley grain germination is demonstrated. As a general remark, caution should be taken when designing related biotechnological tools since the plant tries to compensate the genetic modifications by modulating the expression of some other proteases or inhibitors.