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  • br Conflict of Interest br Statement of Authorship br Introd


    Conflict of Interest
    Statement of Authorship
    Introduction Plant endopeptidases are involved in many important biological processes. Recent developments in molecular biological techniques have revealed trace amounts of endopeptidases play essential roles in plant development, turnover of photosynthetic proteins and signal transduction during pathogenesis [1], [2], [3]. However, the physiological roles of many foliar endopeptidases remain unknown, because the identification and characterization of plant endopeptidases is hindered by the presence of multiple isozymes and the low activities of these enzymes. To overcome this problem, we developed an activity staining method, which is capable of high resolution isoelectric focusing (IEF) while also capable of detection of low proteolytic activities, because electrophoressis is carried out under mild conditions [4]. By using this method, many endopeptidases involved in degradation of storage proteins in geminating cucumber cotyledons were detected and their physiological roles were elucidated [5]. In addition to our previous study, zymogram analysis of endopeptidase has been used in several studies for germinating seedlings [6], [7], [8], however, there are few reports with senescing leaves. In this paper, we applied this method of analysis to endopeptidases in mature cucumber Natural Product Library and discussed their physiological roles.
    Materials and methods
    Results and discussion In this study, we used cotyledons as model leaves due to their distinct morphological changes during ontogenesis. Cucumber seeds germinated on the second day after sowing and the cotyledons started to expand on the fifth day. Endopeptidases appearing during this period were synthesized de novo and involved in the degradation of storage proteins [5]. The cotyledons continued to expand visibly until the 12th day when they had matured. After the 32th day mature cotyledons started to yellow and had dried up by the 46th day. Protein and Rubisco contents increased as the cotyledons expanded (7–12th day), and reached a maximum level on the 12th day (Fig. 1). Then their contents drastically decreased just after the cotyledons had fully expanded on the 12th day, and nearly reached the minimum level when cotyledons started showing signs of visible senescence. Makino et al. defined the beginning of senescence as the time when the total leaf total N and Rubisco contents begin to decrease [12]. According to their definition, we also defined the period (12–32nd day) as the early senescing stage and the period after cotyledons started yellowing (after 32nd day) as late senescing stage. The period when the cotyledons were expanding (7–12th day) was defined as the expanding stage. Changes in cucumber endopeptidase (CEP) activities in cotyledons at different ontogenic stages were analyzed by an activity staining method developed previously [4]. In cotyledons, three major CEPs with pIs of 4.3, 4.5, and 5.0 showed different patterns of changes in their activities (Fig. 2A). A CEP with a pI of 4.5 (CEP 4.5) appeared at the expanding stage and reached maximum activity on the 12th day. At early senescing stages, it was the most active endopeptidase. A CEP with a pI of 4.3 (CEP 4.3) was absent at the expanding stage, and appeared after 12th day. It was the most active of the three major endopeptidases in yellowish cotyledons during the late senescing stage. A CEP with a pI of 5.0 (CEP 5.0) showed a relatively constant activity throughout the all stages. We analyzed the substrate specificities of these CEPs using 12 artificial substrates as listed in Table 1. CEP 4.5 cleaved carbobenzoxy (CBZ)-Leu-Leu-Glu-NA and CEP 5.0 cleaved Benzoyl-Arg-NA, CBZ-Arg-Arg-NA, and CBZ-Ala-Ala-Lys-4OMe-NA. These results correspond to our previous studies of the purification of these two endopeptidases [13], [14]. Unfortunately, the substrate specificity of CEP 4.3 was not revealed in this experiment. In various plants, rapid degradation of Rubisco and photosynthetic activity is observed in mature leaves at the early senescing stage [12], [15], [16], [17], [18]. We also observed a rapid degradation of Rubisco in cucumber cotyledons (Fig. 1), and the activity of CEP 4.5 correlates with this degradation (Fig. 2A). This evidence leads us to suggest that CEP 4.5 may be involved in the active catabolism of proteins at an early senescing stage. Moreover, it may provide a source of additional amino acids for newly emerging leaves because the primary leaf had emerged after the 12th day. Previously, we purified and characterized CEP 4.5 as a cucumber glutamyl endopeptidase [13]. Amino acid sequencing showed that homologs are likely to be widely distributed among plant species. Therefore, CEP 4.5-like proteins might function commonly in fundamental intracellular processes in the leaves of various plants at the early senescing stage.