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Appearance of endopeptidases during the senescence of cucumber leaves
Plant Science 162 (2002) 615 /619 www.elsevier.com/locate/plantsci
Appearance of endopeptidases during the senescence of cucumber leaves Yasuo Yamauchi a,*, Toshio Sugimoto b, Kuni Sueyoshi b,1, Yoshikiyo Oji b, Kiyoshi Tanaka a a
Laboratory of Plant Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan b Faculty of Agriculture, Kobe University, Rokkodai 1-1, Nada-ku, Kobe 657-8501, Japan Received 5 September 2001; received in revised form 10 December 2001; accepted 14 December 2001
Abstract In cucumber (Cucumis sativus L.) leaves at different ontogenic stages, a differential appearance of three major endopeptidases was observed by employing activity staining using gelatin as a substrate. On the basis of this observation, we discussed their physiological roles in senescing leaves. The most active endopeptidase in young mature leaves was a glutamyl endopeptidase with a pI of 4.5. It might be involved in active protein catabolism in young leaves because its activity became maximal just after the leaf had fully expanded and when protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents rapidly decreased. An endopeptidase with a pI of 4.3 was not observed in young leaves, however, it was highly active in senescing leaves. Interestingly, its activity in cotyledons was eliminated when the upper metabolically active leaves were removed. This implies that the appearance of this enzyme is regulated by the presence of sink tissues, and it is involved in the degradation of protein in senescing leaves facilitating N transfer to upper developing leaves. A trypsin-like endopeptidase with a pI of 5.0 showed relatively constant activity during the whole period. This endopeptidase has been shown to be inhibited by arginine, guanidino compounds and Mg2 , therefore, it might exist constitutively and its activity might be regulated mainly at a post-translational level responding to nutrient and environmental conditions. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cucumis sativus ; Cucumber; Endopeptidase; Senescence; N transfer
1. 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 /3]. However, the physiological roles of many foliar endopeptidases remain unknown, because the identification and characAbbreviations: CBZ, carbobenzoxy; CEP, cucumber endopeptidase; IEF, isoelectric focusing; 2-ME, 2-mercaptoethanol; NA, naphthylamide; Rubisco, ribulose-1,5-bisphosphate carboxylase/ oxygenase; SCG, substrate containing gel; VSP, vegetative storage protein. * Corresponding author. Tel./fax: 81-0857-31-6702. E-mail address: [email protected] (Y. Yamauchi). 1 Present address: Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan.
terization 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 /8], however, there are few reports with senescing leaves. In this paper, we applied this method of analysis to endopeptidases in mature cucumber leaves and discussed their physiological roles.
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2. Materials and methods 2.1. Materials All of the naphthylamide (NA) derivatives and Fast Garnet GBC were purchased from Sigma (St. Louis, Mo, USA). All other reagents were purchased from Wako Pure Chemical Co Ltd (Osaka, Japan). Cucumber seeds (Cucumis sativus L. suyo) purchased from Takii Seed Co Ltd (Kyoto, Japan) were sown and grown hydrotopically with Hoagland solution in a green house at 25 8C. Plants grown under an N-starvation were supplied with a modified media with nitrogen removed. Harvested leaves were frozen in liquid N2 and stored at /20 8C until use. Leaf age used in all experiments was defined as days after sowing. 2.2. Activity staining of endopeptidases Solid phase detection of proteolytic activity was performed by using the substrate containing gel (SCG)-IEF method [4]. Leaves (1 g each) were homogenized with 10 volumes of 50 mM ammonium acetate, containing 5 mM 2-mercaptoethanol (2-ME). After centrifugation at 20 000 /g for 20 min, the supernatant was dialyzed in the same buffer and then freeze-dried. The proteins were dissolved in 20 ml of IEF-sample buffer solution, and then electrophoresed on an SCG containing 0.1% (w/v) gelatin as a substrate and Ampholine with a pH range from 3.5 to 10.0 (Amersham Pharmacia Biotech, Sweden), or SERVALYT, pH range 4.0 /6.0 (Serva Electrophoresis GmbH, Germany). After electrophoresis, the SCG was incubated in 100 mM sodium acetate buffer (pH 5.0) containing 2 mM 2-ME at 37 8C. The SCG was stained in 0.1% (w/v) Coomassie Brilliant Blue R-250 and destained with methanol (40%) and acetic acid (10%). Activity was recognized as clear band against a blue background. Activity staining using artificial substrates was performed by using the method of Mort and Leduc [9]. This technique, briefly summarized, includes subjecting a sample of the enzyme to the SCG-IEF method using an SCG containing SERVALYT with a pH range between 4.0 and 6.0. After which, strips of filter paper, moistened in 2 mM of various artificial substrates (stock was dissolved in dimethyl sulfoxide) in 50 mM sodiumacetate buffer (pH 5.0) or 50 mm Hepes-KOH (pH 7.0) are overlaid on the SCG. After incubation at 37 8C, activity was visualized by immersing the strips in 0.1 M sodium-acetate buffer (pH 4.0) containing 0.1% (w/v) Fast Garnet GBC. 2.3. Protein analysis Foliar proteins were extracted in 5 volumes of 50 mM potassium phosphate buffer (pH 7.0). After centrifuga-
tion, the soluble protein concentration was determined by the method of Lowry et al. [10]. SDS-PAGE and the subsequent staining and destaining of gel was performed by the method of Laemmli [11]. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content was determined by densitometric analysis after SDSPAGE. A known amount of purified Rubisco was run in to the same gel as a standard and stained with Coomassie Brilliant Blue R-250.
3. 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
Fig. 1. Temporal changes in total protein (A) and Rubisco (B) contents in N-supplied (k) and N-starved (m) cucumber cotyledons. Abbreviations used in the figure, exp, ear, and lat represent expanding, early senescing, and late senescing stages, respectively.
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Fig. 2. Temporal appearance of CEP activities at different ontogenic stages. Enzyme samples prepared from cotyledons of N-supplied (A) and N-starved plants (B) were subjected to the SCG-IEF method using the SCG containing wide range ampholyte (pH 3.5 /10.0). Abbreviations are same as Fig. 1.
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)-LeuLeu-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 Table 1 Substrate specificities of CEPs Substrate hydrolyzed by CEP 4.5, Carbobenzoxy (CBZ)-Leu-LeuGlua-naphthylamide (NA) Substrates hydrolyzed by CEP 5.0, CBZ-Arg-Arg-NA; Benzoyl-ArgNA; CBZ-Ala-Ala-Lys-4OMe-NA Substrates not hydrolyzed by any endopeptidases, CBZ-Phe-Arg-NA; Benzoyl-Arg-Gly-Phe-Phe-Leu-NA; CBZ-Gly-Gly-Leu-NA; BenzoylPhe-NA; Glutaryl-Gly-Gly-Phe-NA; Acetyl-Ala-Ala-Pro-Ala-NA; CBZ-Pro-Ala-Gly-Pro-NA; CBZ-Pro-His-Leu-Leu-Val-Tyr-Ser-NA a
The amino acid residue on the carboxyl-terminal side of cleavage bond is indicated with bold letters.
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the early senescing stage [12,15/18]. 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. The rate of protein degradation is often affected by Nstarvation [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 contrast 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 Nsupplied 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
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Fig. 3. Analysis of appearance of CEP 4.3 and 4.5 in intact plant versus plants with upper leaves removed. After enzyme solutions were prepared, endoproteolytic activities were detected by the SCG-IEF method. (A) Disappearance of CEP 4.3 in cotyledons with upper leaves removed. C, 1, and 2 represent cotyledon, primary, and secondary leaves, respectively. (B) Distribution of CEP 4.3 and 4.5 in each leaf position of an intact plant just after the fifth leaf had emerged. Leaves are numbered from lower to upper.
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 sink / source 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 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
Fig. 4. Temporal appearance of CEP activities in primary leaf. (A) Enzyme solutions were applied to the SCG-IEF method using the SCG containing narrow range ampholyte (pH 4.0 /6.0). (B) After electrophoresis of enzyme solution prepared from primary leaf on the 28th day, cleaving activities were detected using benzoyl-Arg-NA (lane 1), CBZ-Ala-Ala-Lys-4OMe-NA (lane 2) and CBZ-Leu-Leu-Glu-NA (lane 3) as the substrates. Abbreviations are same as Fig. 1.
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. In this study, advanced activity staining of endopeptidases provided deep insights about their physiological roles. The combination of results in this study with data about biochemical properties obtained from individual
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studies of CEPs gives further information. Moreover, it also shows that this method for separating and studying individual endopeptidases will allow us to readily characterize and purify them. In the future, molecular analysis of CEP 4.3 is needed for elucidation of its characteristic behavior.
References [1] S. Seo, M. Okamoto, T. Iwai, M. Iwano, K. Fukui, A. Isogai, N. Nakajima, Y. Ohashi, Reduced levels of chloroplast FtsH protein in tobacco mosaic virus infected tobacco leaves accelerate the hypersensitive reaction, Plant Cell 12 (2000) 917 /932. [2] K. Takechi, M. Sodmergen, M. Murata, F. Motoyoshi, W. Sakamoto, The YELLOW VARIEGATED (VAR2 ) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis , Plant Cell Physiol. 41 (2000) 1334 /1346. [3] Y. Yamamoto, N. Inagaki, K. Satoh, Overexpression and characterization of carboxyl-terminal processing protease for precursor D1 protein, J. Biol. Chem. 276 (2001) 7518 /7525. [4] Y. Yamauchi, T. Sugimoto, Y. Oji, A method for the separation and detection of proteases using substrate-containing gel isoelectric focusing electrophoresis, Biosci. Biotech. Biochem. 59 (1995) 767 /768. [5] Y. Yamauchi, T. Sugimoto, K. Sueyoshi, Y. Oji, Emergence of proteases in germinating cucumber cotyledons and their roles in the two-step degradation of storage protein, Plant Cell Physiol. 37 (1996) 279 /284. [6] W. Mitsuhashi, T. Minamikawa, Synthesis and posttranslational activation of sulfhydryl-endopeptidase in cotyledons of germinating Vigna mungo seeds, Plant Physiol. 89 (1989) 274 /279. [7] E.G. de Barros, B.A. Larkins, Purification and characterization of Zein-degrading proteases from endosperm of germinating maize seeds, Plant Physiol. 94 (1990) 297 /303. [8] R. Wrobel, B.L. Jones, Appearance of endoproteolytic enzymes during the germination of barley, Plant Physiol. 100 (1992) 1508 / 1516.
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[9] J.S. Mort, M. Leduc, A simple, economical method for staining gels for cathepsin B-like activity, Anal. Biochem. 119 (1982) 148 / 152. [10] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 /275. [11] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680 /685. [12] A. Makino, T. Mae, K. Ohira, Relation between nitrogen and ribulose-1,5-bisphosphate carboxylase in rice leaves from emergence through senescence, Plant Cell Physiol. 25 (1984) 429 /437. [13] Y. Yamauchi, Y. Ejiri, T. Sugimoto, K. Sueyoshi, Y. Oji, K. Tanaka, A high molecular weight glutamyl endopeptidase and its endogenous inhibitors from cucumber leaves, J. Biochem. 130 (2001) 257 /261. [14] Y. Yamauchi, T. Sugimoto, K. Sueyoshi, Y. Oji, K. Tanaka, A serine endopeptidase from cucumber leaves is inhibited by Larginine, guanidino compounds and divalent cations, Phytochemistry 58 (2001) 677 /682. [15] J.M. Hopkinson, Studies on the expansion of the leaf surface, J. Exp. Bot. 15 (1964) 125 /137. [16] M.B. Peoples, M.J. Dalling, Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves, Planta 138 (1978) 153 /160. [17] J.W. Friedrich, R.C. Huffaker, Photosynthesis, leaf resistances, and ribulose-1,5-bisphosphate carboxylase degradation in senescing barley leaves, Plant Physiol. 65 (1980) 1103 /1107. [18] K. Ono, H. Hashimoto, S. Katoh, Changes in the number and size of chloroplasts during senescence of primary leaves of wheat grown under different conditions, Plant Cell Physiol. 36 (1995) 9 / 17. [19] V.A. Wittenbach, Effect of pod removal on leaf photosynthesis and soluble protein composition of field-grown soybeans, Plant Physiol. 73 (1983) 121 /124. [20] D.B. DeWald, H.S. Mason, J.E. Mullet, The vegetative storage proteins VSPa and VSPb are acid phosphatases active on polyphosphates, J. Biol. Chem. 267 (1992) 15958 /15964.
Appearance of endopeptidases during the senescence of cucumber leaves Yasuo Yamauchi a,*, Toshio Sugimoto b, Kuni Sueyoshi b,1, Yoshikiyo Oji b, Kiyoshi Tanaka a a
Laboratory of Plant Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan b Faculty of Agriculture, Kobe University, Rokkodai 1-1, Nada-ku, Kobe 657-8501, Japan Received 5 September 2001; received in revised form 10 December 2001; accepted 14 December 2001
Abstract In cucumber (Cucumis sativus L.) leaves at different ontogenic stages, a differential appearance of three major endopeptidases was observed by employing activity staining using gelatin as a substrate. On the basis of this observation, we discussed their physiological roles in senescing leaves. The most active endopeptidase in young mature leaves was a glutamyl endopeptidase with a pI of 4.5. It might be involved in active protein catabolism in young leaves because its activity became maximal just after the leaf had fully expanded and when protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents rapidly decreased. An endopeptidase with a pI of 4.3 was not observed in young leaves, however, it was highly active in senescing leaves. Interestingly, its activity in cotyledons was eliminated when the upper metabolically active leaves were removed. This implies that the appearance of this enzyme is regulated by the presence of sink tissues, and it is involved in the degradation of protein in senescing leaves facilitating N transfer to upper developing leaves. A trypsin-like endopeptidase with a pI of 5.0 showed relatively constant activity during the whole period. This endopeptidase has been shown to be inhibited by arginine, guanidino compounds and Mg2 , therefore, it might exist constitutively and its activity might be regulated mainly at a post-translational level responding to nutrient and environmental conditions. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cucumis sativus ; Cucumber; Endopeptidase; Senescence; N transfer
1. 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 /3]. However, the physiological roles of many foliar endopeptidases remain unknown, because the identification and characAbbreviations: CBZ, carbobenzoxy; CEP, cucumber endopeptidase; IEF, isoelectric focusing; 2-ME, 2-mercaptoethanol; NA, naphthylamide; Rubisco, ribulose-1,5-bisphosphate carboxylase/ oxygenase; SCG, substrate containing gel; VSP, vegetative storage protein. * Corresponding author. Tel./fax: 81-0857-31-6702. E-mail address: [email protected] (Y. Yamauchi). 1 Present address: Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan.
terization 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 /8], however, there are few reports with senescing leaves. In this paper, we applied this method of analysis to endopeptidases in mature cucumber leaves and discussed their physiological roles.
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 6 0 7 - 0
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2. Materials and methods 2.1. Materials All of the naphthylamide (NA) derivatives and Fast Garnet GBC were purchased from Sigma (St. Louis, Mo, USA). All other reagents were purchased from Wako Pure Chemical Co Ltd (Osaka, Japan). Cucumber seeds (Cucumis sativus L. suyo) purchased from Takii Seed Co Ltd (Kyoto, Japan) were sown and grown hydrotopically with Hoagland solution in a green house at 25 8C. Plants grown under an N-starvation were supplied with a modified media with nitrogen removed. Harvested leaves were frozen in liquid N2 and stored at /20 8C until use. Leaf age used in all experiments was defined as days after sowing. 2.2. Activity staining of endopeptidases Solid phase detection of proteolytic activity was performed by using the substrate containing gel (SCG)-IEF method [4]. Leaves (1 g each) were homogenized with 10 volumes of 50 mM ammonium acetate, containing 5 mM 2-mercaptoethanol (2-ME). After centrifugation at 20 000 /g for 20 min, the supernatant was dialyzed in the same buffer and then freeze-dried. The proteins were dissolved in 20 ml of IEF-sample buffer solution, and then electrophoresed on an SCG containing 0.1% (w/v) gelatin as a substrate and Ampholine with a pH range from 3.5 to 10.0 (Amersham Pharmacia Biotech, Sweden), or SERVALYT, pH range 4.0 /6.0 (Serva Electrophoresis GmbH, Germany). After electrophoresis, the SCG was incubated in 100 mM sodium acetate buffer (pH 5.0) containing 2 mM 2-ME at 37 8C. The SCG was stained in 0.1% (w/v) Coomassie Brilliant Blue R-250 and destained with methanol (40%) and acetic acid (10%). Activity was recognized as clear band against a blue background. Activity staining using artificial substrates was performed by using the method of Mort and Leduc [9]. This technique, briefly summarized, includes subjecting a sample of the enzyme to the SCG-IEF method using an SCG containing SERVALYT with a pH range between 4.0 and 6.0. After which, strips of filter paper, moistened in 2 mM of various artificial substrates (stock was dissolved in dimethyl sulfoxide) in 50 mM sodiumacetate buffer (pH 5.0) or 50 mm Hepes-KOH (pH 7.0) are overlaid on the SCG. After incubation at 37 8C, activity was visualized by immersing the strips in 0.1 M sodium-acetate buffer (pH 4.0) containing 0.1% (w/v) Fast Garnet GBC. 2.3. Protein analysis Foliar proteins were extracted in 5 volumes of 50 mM potassium phosphate buffer (pH 7.0). After centrifuga-
tion, the soluble protein concentration was determined by the method of Lowry et al. [10]. SDS-PAGE and the subsequent staining and destaining of gel was performed by the method of Laemmli [11]. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content was determined by densitometric analysis after SDSPAGE. A known amount of purified Rubisco was run in to the same gel as a standard and stained with Coomassie Brilliant Blue R-250.
3. 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
Fig. 1. Temporal changes in total protein (A) and Rubisco (B) contents in N-supplied (k) and N-starved (m) cucumber cotyledons. Abbreviations used in the figure, exp, ear, and lat represent expanding, early senescing, and late senescing stages, respectively.
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Fig. 2. Temporal appearance of CEP activities at different ontogenic stages. Enzyme samples prepared from cotyledons of N-supplied (A) and N-starved plants (B) were subjected to the SCG-IEF method using the SCG containing wide range ampholyte (pH 3.5 /10.0). Abbreviations are same as Fig. 1.
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)-LeuLeu-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 Table 1 Substrate specificities of CEPs Substrate hydrolyzed by CEP 4.5, Carbobenzoxy (CBZ)-Leu-LeuGlua-naphthylamide (NA) Substrates hydrolyzed by CEP 5.0, CBZ-Arg-Arg-NA; Benzoyl-ArgNA; CBZ-Ala-Ala-Lys-4OMe-NA Substrates not hydrolyzed by any endopeptidases, CBZ-Phe-Arg-NA; Benzoyl-Arg-Gly-Phe-Phe-Leu-NA; CBZ-Gly-Gly-Leu-NA; BenzoylPhe-NA; Glutaryl-Gly-Gly-Phe-NA; Acetyl-Ala-Ala-Pro-Ala-NA; CBZ-Pro-Ala-Gly-Pro-NA; CBZ-Pro-His-Leu-Leu-Val-Tyr-Ser-NA a
The amino acid residue on the carboxyl-terminal side of cleavage bond is indicated with bold letters.
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the early senescing stage [12,15/18]. 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. The rate of protein degradation is often affected by Nstarvation [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 contrast 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 Nsupplied 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
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Fig. 3. Analysis of appearance of CEP 4.3 and 4.5 in intact plant versus plants with upper leaves removed. After enzyme solutions were prepared, endoproteolytic activities were detected by the SCG-IEF method. (A) Disappearance of CEP 4.3 in cotyledons with upper leaves removed. C, 1, and 2 represent cotyledon, primary, and secondary leaves, respectively. (B) Distribution of CEP 4.3 and 4.5 in each leaf position of an intact plant just after the fifth leaf had emerged. Leaves are numbered from lower to upper.
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 sink / source 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 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
Fig. 4. Temporal appearance of CEP activities in primary leaf. (A) Enzyme solutions were applied to the SCG-IEF method using the SCG containing narrow range ampholyte (pH 4.0 /6.0). (B) After electrophoresis of enzyme solution prepared from primary leaf on the 28th day, cleaving activities were detected using benzoyl-Arg-NA (lane 1), CBZ-Ala-Ala-Lys-4OMe-NA (lane 2) and CBZ-Leu-Leu-Glu-NA (lane 3) as the substrates. Abbreviations are same as Fig. 1.
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. In this study, advanced activity staining of endopeptidases provided deep insights about their physiological roles. The combination of results in this study with data about biochemical properties obtained from individual
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studies of CEPs gives further information. Moreover, it also shows that this method for separating and studying individual endopeptidases will allow us to readily characterize and purify them. In the future, molecular analysis of CEP 4.3 is needed for elucidation of its characteristic behavior.
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