The activity of the 20S proteasome is maintained in detached wheat leaves during senescence in darkness

Abstract

In the present paper, we studied the participation of the 20S proteasome, the proteolytic component of the ubiquitin-proteasome pathway, in the remobilization of bulk proteins in senescing wheat leaves. The detached leaves of 15-d-old plants were incubated in darkness for several days, and various proteolytic activities were analysed in soluble extracts prepared at 0, 48 and 96 h after detachment. The endoproteolytic activity, measured at pH 7.5 and 5.4, increased more than 10-fold and the total peptidasic activity increased up to 5-fold after 96 h of incubation in the dark, when expressed as specific activity. In the same period, the leaf-protein content decreased to less than 50% of that present at the initial time. The 20S proteasome chymotrypsin-like activity remained constant when it was expressed as activity per leaf fresh weight and resulted 2-fold higher in terms of specific activity. The western blot analysis showed that the amount of 20S proteasome protein and ubiquitin-protein conjugates also remained constant until 4 d of incubation in darkness. These results indicate that the ubiquitin-proteasome pathway remains functional until the late phases of senescence suggesting that it may participate in the regulatory aspects of the process rather than in the massive protein breakdown.