The induction of two subtilisin-like proteases (P1 and P2) associated with stress-induced senescence in young plants was investigated in adult wheat plants during the grain-filling period. Western blot analysis of flag leaf extracts showed that P1 was induced very late in the life cycle of the plants (9 days post-anthesis) and that 7 days later it reached a 2.5-fold increase with respect to the initial value at anthesis. On the other hand, the P2 signal was already detected previous to anthesis and increased soon after anthesis, reaching a fourfold increase by the end of the grain-filling period. The induction of P1 and P2 temporally correlates with the degradation of the Rubisco small and large subunits in the flag leaf, as well as with nitrogen (N) accumulation in the ears. At the same time, a decrease in the endogenous concentration of the cytokinins isopentenyladenine and isopentenyladenosine (iP + iPA) in the leaves was observed. In detached leaves senescing in the dark, the levels of both proteases were affected by 6-benzylaminopurine application: the induction of P1 was completely prevented, whereas the induction of P2 was reduced. Our findings demonstrate that both P1 and P2 are expressed in leaves of adult plants and are induced during natural senescence. These results enable us to postulate their participation in N remobilization to developing grains during monocarpic senescence and their regulation by a cytokinin-mediated mechanism.
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