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  • MDL 28170 The rate of protein degradation is often affected


    The rate of protein degradation is often affected by N-starvation [12], therefore, we examined the influence of N-starvation against the appearance of CEPs. N-starved cucumber cotyledons contained less amounts of protein and Rubisco, but the temporal changes in protein and Rubisco contents were similar to those of N-supplied plants (Fig. 1). In N-starved plants, CEP 4.3 was almost eliminated in MDL 28170 to CEP 4.5 and 5.0 which showed a relatively similar pattern to the case of N-supplied plants (Fig. 2B). This result led to the hypothesis that CEP 4.3 might be regulated by the nitrogen status of the plant. However, N-starved cucumber plants almost cease development after the primary leaf had emerged on the 12th day, therefore, changes in the developing rate of the plants might also affect the appearance of CEP 4.3. The major role of a proteolytic system during the senescing stages is to supply N for metabolically active leaves by the degradation of proteins in senescing leaves. CEP 4.3 possibly plays such a role because its activity is increased in senescing cotyledons of N-supplied plants after the primary leaf had emerged. This led to another hypothesis that CEP 4.3 might be regulated by the presence of sink tissues. To determine which hypothesis can be supported, we examined whether or not CEP 4.3 appears in cotyledons after the upper leaves of plants with N were removed. In such plants, we expected that CEP 4.3 would be eliminated if it is regulated by the presence of a sink tissue. As shown in Fig. 3A, CEP 4.3 is eliminated in the cotyledons of plants grown in the presence of N in the medium when the upper leaves are removed. In addition, CEP 4.3 reappeared in cotyledons when the new leaves emerged following the removal of leaves (data not shown). This data suggests that CEP 4.3 is regulated by the existence of sink tissues but not nitrogen in the medium. Furthermore, CEP 4.3 was observed only in leaves of lower positions and its activity was higher the lower the leaf was positioned (Fig. 3B). These results support the hypothesis that the appearance of CEP 4.3 is determined by the existence of sink tissues, such as upper leaves, whereas there is likely to be little effect of N-states on the appearance of CEPs. Well-characterized foliar proteins whose amounts are affected by removal of sink tissue, are vegetative storage proteins (VSPs) [19]. VSPs in soybeans are acid phosphatases that accumulate after the removal of sink tissues [20], as opposed to the disappearance of CEP 4.3. This opposite behavior of CEP 4.3 and VSPs is logical because the VSPs are proteins of nitrogen source and CEP 4.3 is the enzyme catalyzing breakdown of proteins in source tissues such as senescing leaves. Therefore, regulation of expression of VSPs and CEP 4.3 might share the same mechanism involving a sinkÔÇôsource relationship. CEP 5.0 showed a relatively constant activity during the whole experimental period (Fig. 2). We previously purified CEP 5.0 and characterized it as a basic amino acid-specific serine-type endopeptidase possibly regulated by endogenous substances such as l-Arg and/or guanidino compounds, and divalent cations [14]. Apparently the unchanged activity of CEP 5.0 shown in this study implies that CEP 5.0 exists constitutively in cucumber leaves and that changes in endogenous substances influenced by environmental and nutrient conditions regulate its activity. Changes in activities of CEPs found in cotyledons appeared to be a common phenomena in cucumber leaves because MDL 28170 a similar pattern was also observed in the primary leaves (Fig. 4). According to definition in the case of cotyledons, the period when the primary leaf expanded (from 12th to 24th day) and following maturity (after 24th day) were defined as expanding and early senescing stages, respectively. Primary leaves at late senescing stage, i.e. yellowing leaves, were not observed in this experimental period. CEP 4.5 and 5.0 were active at the expanding and early senescing stages, whereas CEP 4.3 did not exist at the expanding stage and then appeared at early senescing stage when the second leaf was emerging. This result suggests that the physiological roles of CEP 4.3, 4.5 and 5.0 in primary leaves are possibly identical to those in cotyledons as discussed above. Besides these CEPs, several minor endopeptidases with pIs of 4.1 and 4.7 were observed at early senescing stage because a narrow range ampholyte was used for IEF. The appearance of CEP 4.1 and 4.7 at identical times as CEP 4.3 implies that these CEPs also might be involved in N-transfer of senescing leaves.