Regulation of the development of the first leaf of oats (Avena sativa L.) : characterization and subcellular localization of proteases

The loss of chlorophyll during the senescence of leaves is preceded by a decrease in protein content. Proteases responsible for the degradation of the proteins have been implicated in the regulation of the senescence process. The first leaf of the seedling of oats ( <em>Avena sativa</em> L.) demonstrates the typical pattern of leaf senescence in cereals and was chosen to study the properties and subcellular localization of proteases throughout leaf development.In Chapter 1, a general introduction is given that indicates the significance of the breakdown of proteins in leaves for seed yield and quality in cereals. Also in this chapter, protease classification is documented, and the possible role of changing protease activities in the regulation of protein degradation is discussed.Oat leaves contain two major proteases with pH optima at pH 4.5 ("acidic protease") and 7.5 ("neutral protease"). During natural development of greenhouse-grown plants both types of enzymes showed highest activities in young and fully-grown leaves, and decreased throughout the course of senescence. Also in detached leaves incubated in the dark and, therefore, subject to accelerated ageing, loss of protein was not accompanied by increases in protease activities. In the light, protease activities increased, but the rate of protein loss was greatly reduced. Thus, protein breakdown appears to be independent of the amount of protease present and additional synthesis of the major proteases is not required for protein loss during senescence (Chapter 1).This apparent paradox could be resolved if proteases and their substrates were spatially separated and brought into contact by a controlled decompartmentalization. Therefore, the distribution and subcellular localization of the major proteases were determined. Protoplasts were prepared as a first step in the isolation of vacuoles. However, the cell wall-degrading enzyme mixtures used for protoplast isolation were seriously contaminated by proteases interfering with the determination of the endogenous protease activity in the isolated protoplasts. These extra proteases could be inactivated by heating the cell well-degrading enzyme mixture at 50°C for 10 min at pH 6.5. This treatment did not impair the cell walldegrading activity. Protoplasts isolated with heated enzymes showed similar protease activities as washed protoplasts, isolated with untreated enzymes. This proved that contaminating proteases were effectively removed during protoplast washing, and that the protease activity measured in isolated protoplasts was derived from the protoplasts themselves. (Chapter 3).Vacuoles were isolated by osmotically lysing the protoplasts in the presence of K _{<font size="-1">2</font>} HPO _{<font size="-1">4</font>} . The maximum concentration of phosphate by which lysis occurred decreased progressively with increasing leaf age. By using the appropriate phosphate concentrations, it became possible to isolate clean vacuoles from leaves up to an advanced stage of senescence, when the leaves had lost more than 50% of their protein. From leaves older than 17 days, only vacuoplasts (vacuoles with adhering cytoplasm, within a resealed plasma membrane) could be obtained. The integrity of both the plasmalemma and the tonoplast decreased in these older leaves and this phenomenon might be linked with increased decompartmentalization at a late stage of senescence (Chapter 4).When the distribution of the proteases was determined in different subcellular fractions, on an average 16% of the acidic protease activity was washed out of the intercellular space of the leaves. The major part of the acidic activity was located within the vacuole. The neutral protease was absent from both these compartments and must, therefore, be cytoplasmic. During the course of leaf development, all of the acidic protease activity present in protoplasts was recovered in the vacuoles, as long as clean vacuoles could be isolated (i.e. up to 17 days). It seems most likely that protein degradation is controlled by import of protein substrates into the vacuole (Chapter 5).The acidic and neutral proteases were partly purified by gel filtration and anion-exchange chromatography (Chapter 6). The enzymes hardly separated, indicating that they have similar molecular weights and charges. The neutral activity was stabilized by merceptoethanol and inhibited by inhibitors of metallopeptidases, whereas the acidic one was not. Both activities were inhibited to varying extents by sulphydryl- and serinetype inhibitors. Both enzymes were endopeptideses. The instability of, in particular, the neutral protease seriously hampers its further purification and characterization.Furthermore, by activity staining after electrophoretic separation and using aminoacyl-2-naphthyl am ides as substrates, five aminopeptidases and one trypsin-like endopeptidase were identified (Chapter 7). The main aminopeptidase was largely unspecific. Two aminopeptidases showed preference for arginine and lysine. An iminopeptidase acted on proline. The fifth aminopeptidase was most active with methionine. All enzymes were active at pH 4.5, 6.0 and 7.5, but showed highest activities at low rather then at neutral pH. The trypsine-like endopeptidase was active at pH 4.5 and 7.5, was inhibited by <u>o</u> -phenanthroline and was not identical with either the acidic or the neutral protease described previously. Its activity decreased during leaf development. During storage of protein extracts in the cold this arginine-specific endopeptidase associated with ribulosebisphophate carboxylase, concomitant with a loss of this protein band. As to how far this enzyme has a function in protein breakdown <u>in</u><u>vivo</u> has to be further elucidated.The results of this study show that the levels of protease activities are not correlated with the rate of over all protein degradation. The acidic protease has no entry to the substrates to be degraded. In contrast, the neutral protease appears to be located together with protein substrates and organelles in the cytoplasm, but seems to selectively degrade only a few proteins. Whereas the mechanism of normal protein turnover remains to be elucidated, the slow, steady decline of protein during natural development may be controlled by, on the one hand, compartmentalization of the acidic protease in the vacuole and, on the other hand, by maintaining a pH removed from the optimum for exopeptidese action in the cytoplasm.