Stress-induced changes in protease composition are determined by nitrogen supply in non-nodulating white clover

An inbreeding line of white clover has been identified which remains non-nodulated under appropriate physiological conditions and so the nitrogen concentration of the plant can be manipulated by altering the nitrate supply to the roots. Non-nodulating plants were used to test the hypothesis that acclimation to nitrogen limitation in white clover involves changes in protease activity and composition. These results indicate that acclimation to nitrogen limitation involves the realignment of constituent proteases without necessarily incurring significant changes in total protease activity. Plants grown at 2.5, 5.0, 7.5, and 10 mM nitrate showed a positive correlation between nitrate supply and foliar protein concentration. Protein profiles, revealed by Coomassie-stained SDS-PAGE, were unchanged between treatments for a given amount of protein. Serine, aspartate/metalloprotease, and two cysteine proteases were identified in the leaves. Although total protease activity per gram fresh weight was unchanged between treatments, the relative contributions of these four proteases was determined by nitrate supply. When plants were stressed further by withholding nitrate there was an increase in cysteine protease activity, but a senescence-related aspartate/metalloprotease was not visible. Hence, while protease expression in white clover leaves responded to the current and past nitrogen status of the plant, the proteases involved in remobilization during nutrient limitation were distinct from those involved during the main senescence period. It is suggested that nitrogen limitation induced an early, reversible stage of senescence in which perturbations in protease activity facilitated the degradation of non-essential proteins in order to increase the chances of plant survival or seed set.