Endopeptidase activity and nitrogen mobilization in senescing leaves of Nicotiana rustica in light and dark

Abstract

The time course of endopeptidase activity (digestion of azocasein at pH 4.6) in leaves of intact plants of Nicotiana rustica L. was studied and related to changes in the contents of chlorophyll, total nitrogen and soluble and insoluble protein nitrogen. Endopeptidase activity increased several fold during senescence. However, the course of protein degradation did not reflect the steep slope of azocaseolytic activity. When single mature leaves were darkened, senescence proceeded faster than in illuminated leaves but the amount of nitrogen mobilized and translocated did not differ greatly between darkened and illuminated leaves. However, in contrast to leaves in light, azocaseolytic activity did not increase. Gelatin zymograms obtained using isoelectric focusing of extracts of mature leaves showed several bands in the pH 4.0 to 6.5 region of the gels. During senescence in both light and dark the position and number of bands remained largely unchanged. In leaves in light, the activity of endopeptidases focusing in the range pH 4.1 to 5.0 increased greatly. In leaves in dark, however, no major changes in activity could be detected. The results suggest that in tobacco leaves endopeptidase activity normally increases considerably during senescence but this increase is not a prerequisite for an effective protein degradation. Separation and analysis of free amino acids showed that during senescence in light the levels of all amino acids decreased considerably. In leaves senescing in the dark there were large increases in the levels of glutamine and asparagine, concomitant decreases in glutamate and aspartate, and considerable increases in all other amino acids.

Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

“…Protease activity was measured by the method of Weckenmann and Martin, 47 using azocasein as substrate. Absorbance of the released-azo-dye-was measured at 340 nm and one unit of activity was defined as the activity producing an increase of 0.01 units of absorbance during 1 h incubation.…”

Seasonal protease activity in storage tissue of the deciduous grass Molinia caerulea

“…The leaves of several plant species, either prior to or at an early stage of senescence, have also been shown to exhibit maximum azocaseinase activity around pH 5 (Weckenmann & Martin, 1984 ;Fro$ hlich & Feller, 1992 ;Morris et al, 1996). At later stages of leaf senescence, increases in the relative azocaseinase activity at higher pH values have been reported (Fro$ hlich & Feller, 1992 ;Stieger & Feller, 1994), though this has not been universally observed (Weckenmann & Martin, 1984 ;Morris et al, 1996). The relative azocaesinase activity at higher pH values in Lolium temulentum leaves increased in detached leaves but not in leaves still attached to the plant (Morris et al, 1996).…”

“…The first 3n5 cm$ of solution eluted after sample application was used to assay for protease activity. The protease activity on azocasein was then estimated as described by Weckenmann & Martin (1984), except that 4 h incubation was used (preliminary tests confirmed the reaction was linear over this time period), and the reaction was stopped using 12% (w\v) trichloroacetic acid. Incubations were performed at pH 5n0 and also at the values indicated in Fig.…”

“…1. Enzyme activity was calculated using the definition of Weckenmann & Martin (1984), 1 unit of enzyme activity being that amount which caused an increase of 1 absorbance unit at 340 nm during 1 h of incubation. In crude enzyme extracts, the formation of chromophore products from azoprotein substrates indicates general protease activity, although such formation is considered to be slower by exopeptidases compared with endopeptidases (Storey, 1986).…”

“…The extraction and estimation of protease activity were adapted from procedures described by Weckenmann & Martin (1984). Fresh tissue (approx.…”