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Effect of Salinity on Ribonuclease Activity of Vigna unguiculata Cotyledons during Germination
J. PlantPhysiol. Vol.J32. pp. 307-311 (1988)
Effect of Salinity on Ribonuclease Activity of Vigna unguiculata Cotyledons during Germination E.
GOMES FILHO
and L
SODEK
Departamento de Fisiologia Vegetal, Universidade Estadual de Campinas, 13081 Campinas, SP, Brasil Received June 12, 1987 . Accepted October 18, 1987
Summary The presence of iso-osmotic solutions (-4.6 bar) of NaCl (0.1 M) and mannitol (0.19 m) during the germination of Vigna unguiculata (L.) Walp inhibited the growth of seedlings to a similar degree, suggesting that the salt effect is mainly osmotic. In the presence of salt, RNase activity of the cotyledons rose more slowly than the control during the initial stages of germination, but thereafter rose sharply to values about twice those of the control. The slower initial rise in RNase was apparently an osmotic effect, since an identical curve was obtained with mannitol. The later sharp rise in activity was not seen in mannitoltreated seedlings, suggesting that some specific salt effect was responsible. Cycloheximide, used under conditions that did not inhibit growth, inhibited the increase in RNase. Although polyacrylamide-gel electrophoresis revealed the presence of six RNase isoenzymes in the extracts, essentially all the activity was due to a single isoenzyme. The main RNase isoenzyme hydrolysed RNA producing all four cyclic (2',3') nucleotides, but no 3'-nucleotides, and had no activity towards DNA.
Key words: Vigna unguiculata, germination, RNase, salinity.
Introduction Salinity inhibits seed germination for two reasons. First, the water potential gradient between the seed and the external medium is reduced (osmotic effect), which leads to a diminished influx of water (Uhvits, 1946; Prisco and O'Leary, 1970). Second, the metabolism of the seed is affected, causing the inhibition of seed reserve mobilization (Prisco and Vieira, 1976; Gomes Filho and Prisco, 1978; Prisco et al., 1981; Gomes Filho et aI., 1983). The metabolic effects may be due, in part, to an accumulation of ions to toxic levels (Uhvits, 1946; Prisco and O'Leary, 1970). Studies of the effects of salinity on enzymes involved in seed reserve mobilization have shown that, in general, those enzymes already present at high levels in the quiescent seed are little affected by salt during germination (Prisco and Vieira, 1976; Gomes Filho and Prisco, 1978). On the other hand, those enzymes present at low levels of activity are strongly affected by salinity (Prisco et al., 1981; Gomes Filho et al., 1983). RNase is an example of the latter category. In a previous study (Gomes Filho et al., 1983), it was reported that salinity inhibits this enzyme during the first few days of germina© 1988 by Gustav Fischer Verlag, Stuttgart
tion, together with an inhibition of the mobilization of RNA in the reserve organs. In view of the central role of RNA in cell metabolism, it is possible that impaired RNA metabolism could be the primary effect of salinity on germination, but at present there is no evidence that the change in RNA metabolism in the cause or consequence of the NaCI effect. In this study we investigated the nature of the salt effect on RNase activity during germination of cowpea. Attempts were made to separate the osmotic and ionic components of the salt effect, as ;well as to determine the nature of the increase in enzyme' activity and the possible involvement of Isoenzymes.
Materials and Methods Seed germination and growth measurements Seeds of Vigna unguiculata (L.) Walp cv. pitiuba were sterilized for 30 min in a commercial bleach (2.5 % active chlorine) diluted 20 times with water. The seeds were germinated in rolls of paper, as de-
308
E. GOMES FILHO and L. SODEK
scribed previously (Gomes Filho and Prisco, 1978). Seedling growth was measured by dry and fresh weights of different parts of the seedling on the seventh day after germination. Dry weight was determined after drying at 100°C for 24 h.
Osmotic potential Sodium chloride was used in the germination medium at 0.1 M, which, according to Lang (1967), has an osmotic potential of -4.6 bar at 25°C. When mannitol was used, this was prepared at 0.19 m, calculated from the Van't Hoff equation to have the same osmotic potential as 0.1 M NaC!.
Ribonuclease activity Cotyledons were homogenized in 0.1 M potassium phosphate buffer, pH 5.7, with a cold mortar and pestle. After centrifugation at 3000g for 10 min, the supernatant was adjusted to pH5.1 with HCl, and left in ice for 16 h. The supernatant obtained after a further centrifugation was used in the enzyme assay. The RNase assay was based on that of Tuve and Anfinsen (1960) with minor modifications. The assay mixture contained 50 mM sodium citrate buffer, pH 5.2 (3.0 ml), 1 % yeast RNA (Nutritional Biochemical Company, USA; purified by precipitation from alcohol as the sodium salt) (1.0 ml), and extract (1.0 ml). The assay mixture was incubated at 40°C, and after intervals of 5 and 25 min, 1 ml aliquots were taken for analysis. The aliquots were mixed immediately with 0.2 ml of 0.75 % uranyl acetate in 25 % HCI04 , and left in ice for at least 30 min. After removal of the RNA precipitate by centrifugation at 1000 g for 5 min, the supernatant was diluted 25 times with water and read in a spectrophotometer at 260 m. Activities were expressed as units pere cotyledon per hour, where one unit corresponds to an absorbance increase of 0.01.
Deoxyribonuclease activity DNase was assayed according to Wilson (1968), using heat-denatured calf thymus DNA as substrate, at pH 5.8.
Hydrolysis product analysis A routine assay mixture was used, except that the incubation period continued for up to 24 h. Aliquots (10 to 40 ILl) were applied to cellulose TLC plates and analysed using the two solvent systems described by Walters and Loring (1966). A second aliquot was treated with 0.1 N HCl for 30 min at 30°C prior to chromatography. This treatment transforms any cyclic (2', 3')-nucleotides to 2'- and 3'-nucleotides, thereby differentiating between cyclic nucleotides and the nucleosides, which have similar mobilities in these solvents.
Statistical analysis The data were subjected to an analysis of variance, and when F was significant (P<0.05), the Tukey test was used to compare means (Steel and Torrie, 1980).
Results and Discussion
Seedling growth The fresh and dry weights of the seedlings 7 days after imbibition (Table 1) show that growth was strongly inhibited by NaCI and mannitol. Apparently, the presence of NaCI or mannitol in the germination medium reduces the uptake of water by the seedlings (dry weights less affected than fresh weights) and inhibits the mobilization of the cotyledon reserves to the growing embryonic axis. These data are in agreement with others involving studies of germination in the presence of NaCI or non-ionic osmotic solution such as polyethylene glycol or mannitol (Uhvits, 1946; Prisco and O'Leary, 1970; Prisco and Vieira, 1976; Machado et al., 1976; Sheoran and Garg, 1978; Gomes Filho et al., 1983; Kawasaki et aI., 1983). Since the inhibition imposed by NaCI was no more severe than that of mannitol, it would appear that the water stress effect (one of the salinity stress components) was the principal factor related to growth inhibition.
Electrophoresis Polyacrylamide disc-gel electrophoresis was carried out as described by Davis (1964), with the modifications introduced by Wilson (1978). The spacer gel contained 3 % acrylamide, 0.75 % bisacrylamide, and 62 mM tris adjusted to pH 6.7 with citric acid. The separating gel contained 7 % acrylamide, 0.148 % bis-acrylamide and 0.37M tris-HCl, pH 8.9. The electrode buffer was 10mM tris with 76 mM glycine, pH 8.3. Samples (150 ILl) containing 1 mg of protein together with added sucrose (10 %) were layered onto the gel surface. Electrophoresis was carried out at room temperature, using a current of 2 rnA per gel tube for 20 min followed by 4 rnA for 90 to 120min. RNase isoenzymes were detected by incubating the gels with RNA at Img/ml in sodium citrate buffer (50mM, pH5.2) for 20 min, followed by staining for 1 min in toluidine blue, as described by Wilson (1978).
Experiments with Cycloheximide Cycloheximide (20 ILg! ml) was applied to the germinating seeds either by soaking the seeds with the inhibitor for 24h (quiescent seeds) or by brushing 25 ILl of this solution onto each cotyledon (germinated seeds). On average, the cotyledons absorbed the applied inhibitor solution within 1 h, after which the seedlings were returned to the original germination conditions. For each treatment, a control was set up where water was used instead of the inhibitor.
Table 1: Fresh and dry weights of Vigna unguiculata seedlings 7 days after imbibition in water and iso-osmotic solutions (-4.6 bar) of NaCI and mannitol. Each value represents the mean of 12 replicates, each replicate corresponding to the average measurement of 10 seedlings. dry weIght (mg) fresh weight (mg) roots shoots roots shoots cots. cots. Control 8.5 a 54.3 a 5.6 b 272 a 1640 a 36 a NaCl 7.9 ab 43.0 b 26.8 a 176 b 608 b 84 b 136 c 496 c 82 b Mannitol 7.5 b 39.2 b 30.5 a Values with the same letters (withm columns) are not significantly different (P<0.05) by the T ukey test.
RNase activity In the control seedlings, RNase activity of the cotyledons, initially very low, increased markedly during germination, reaching a maxium near the fifth day after germination (Fig. 1). The activity pattern appears to be inversely related to the RNA content of the tissue, which is high in the quiescent seed and decreases during germination (Beevers and Guernsey, 1966; Palmiano and Juliano, 1972; Gomes Filho et aI., 1983).
Salinity and RNase in Vigna
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Fig. 1: Changes in RNase activity of Vigna unguiculata cotyledons during germination in water (0) and iso-osmotic solutions (-4.6 bar) of NaCI (.) and mannitol (1':.). Each value represents the mean of 3 replicate extracts. Vertical bars represent the LSD value.
In the saline treatment, RNase activity also increased with germination, reaching its maximum value on the seventh day. Although activity in the control was higher than that of the saline treatment up to the third day after germination, thereafter the situation inverted and the activity in the saline treatment increased to almost twice the maximum attained by the control. In the case of mannitol, the activity increased slowly throughout the experiment. The activity curve was identical to that of the NaCI treatment up to the third day, but thereafter increased much more slowly, reaching its highest value on day nine. The effect of NaCI on RNase was similar to that reported previously (Gomes Filho et al., 1983). The activity curve obtained with mannitol was clearly different from that of NaCI and could well be a simple retardation of the control curve, for two reasons. First, the highest activity recorded with mannitol, on day nine, is close to the maximum activity of the control, obtained on day five. Second, the growth parameters (seedling fresh weight and cotyledon dry weight) recorded on day nine for the mannitol treatment were close to those of the control on day five (data not shown), suggesting that the treated plants at day nine reached a similar stage of development as the control plants at day five. Since the activity curves for both mannitol and NaCI-treated plants were identical up to day three, it would appear that initially the effect of NaCI is purely osmotic. The subsequent high RNase activity of the cotyledons in the presence of NaCl must be a specific salt effect, since this increase was not accompanied by the mannitol-treated seedlings. Nevertheless, this specific salt effect on RNase activity does not apparently lead to any change in seedling growth, in view of the similar dry weights obtained with NaCI and mannitol (Table 1). This would
309
suggest that RNase may only playa secondary role in the germination process.
Polyacrylamide-gel electrophoresis When the cotyledon extracts were submitted to polyacrylamide-gel electrophoresis, at least six bands of apparent RNase activity could be detected (Fig.2), with Rms corresponding to 0.17 (band 1), 0.28 (band2), 0.37 (band3), 0.41 (band4), 0.57 (bandS), and 0.80 (band6). Band6 (designated «main band») was practically nonexistent in the quiescent seed, but increased tremendously in intensity during germination, while the others «
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Fig. 2: RNase isoenzymes after electrophoresis of cotyledon extracts of Vigna unquiculata at different stages of germination in water and O.IM NaCl.
310
E. GOMES FILHO and L. SODEK (Bl Nael
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Table 2: Densitometry readings (peak areas) of RNase isoenzymes after electrophoresis of cotyledon extracts of Vigna unguiculata treated with water and cycloheximide (CHI) for 24 h prior to analysis. A = 3-day-old seedlings germinated in water; B = 6-day-old seedlings germinated in NaCI. Isoenzyme band numbering as for Fig. 2.
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Fig. 3: Changes in RNase activity over a 24 hour period in Vigna unquiculata cotyledons treated with water (0) and 20 /lg/ml cycloheximide (~) during germination in water (A) and NaCl (B). The change in activity was determined for days 0 to 1 (a), 1 to 2 (b), 2 to 3 (c), 3 to 4 (d), 4 to 5 (e), 5 to 6 (1) and 6 to 7 (g). Three replicate extracts were used for each determination.
Studies with cycloheximide When cotyledons at different ages were treated with cycloheximide and assayed for RNase activity 24 h later, the activities at all ages were substantially lower than the corresponding controls (Fig. 3). This was true for both NaCI treated and control seedlings, suggesting that most, if not all, the increase in activity is dependent on protein synthesis. Others (Bryant et al., 1976a, 1976 b; Kapoor and Sachar, 1976) have reported an increase in RNase totally or partially dependent on protein synthesis, using cycloheximide, but in these experiments the seeds were left to germinate in the presence of the inhibitor for an extended period. The inconvenience of this resides in the strong reduction of growth which puts in doubt whether the observed inhibition is not simply a consequence of the delay in germination. In our own case, the inhibitor was studied for 24 h periods only, during which growth inhibition was not observed (data not shown). Although stronger inhibitions of enzyme activity were possible either by extending the period of treatment or increasing the concentration of the inhibitor, this leads to inhibition of growth. These data are in contrast with those of Barker et al. (1974) who found that the increase in RNase activity during germination of Pisum arvense was independent of protein synthesis. When cycloheximide was applied at ages when RNase activity was decreasing, e.g. days 4 to 5 of the control, the inhibitor caused an even greater decrease in RNase. This suggests that RNase undergoes turnover such that when degradation exceeds synthesis the inhibition of synthesis will lead to an apparent increase in degradation. Polyacrylamide-gel electrophoresis of the extracts showed that cycloheximide reduced the intensity of the main band only (Table 2), consistent with the conclusion that virtually all the increase in activity is due to this isoenzyme. A similar
effect of this inhibitor on one of two isoenzymes of RNase in the cotyledons of germinating Pisum sativum was reported by Bryant et al. (1976 b).
Characteristics of the enzyme Some characteristics of the cotyledon RNase were determined in an attempt to classify the enzyme according to Wilson's (1975) scheme for plant RNases. Using extracts of the NaCI treatment 6 days after germination, when virtually all the activity was due to the main band isoenzyme, no DNase activity could be detected. This suggests that the enzyme is a RNase and not a nuclease. The RNase activity was also virtually insensitive to EDTA, a further property of non-nuclease-type RNases. The products of RNA hydrolysis were examined with the partially purified enzyme (extracted from gel slices taken from the centre of the main band) and this revealed the formation of all four cyclic {2',3')-nucleotides, but no 3'-nucleotides. According to the classification scheme of Wilson (1975), this RNase could be of the I or II type in that it does not hydrolyse DNA and produces cyclic nucleotides. However, in contrast to RNase I or II there was no evidence for the further hydrolysis of any of the cyclic nucleotides to 3'nucleotides. Furthermore, the enzyme showed a broad pH optimum between pH 5 and 6, unaffected by salt, which also differs from both the RNase I and II types.
Conclusions The effect of NaCI on RNase activity of Vigna cotyledons during germination presents two distinct phases. First, a delayed increase in RNase activity, due to the osmotic component and apparently associated with the slower growth of the seedling, and second, a subsequent sharp rise in RNase activity due to a specific salt effect. The specific salt effect does not appear to be associated with any notable change in seedling growth, suggesting that the increase in RNase is not a primary event in the germination process. Acknowledgements EGF is grateful to the Coordenadoria de Aperfeic;oamento de Pessoal de Ensino Superior (CAPES) for a grant.
Salinity and RNase in Vigna
References BARKER, G. R., C. M. BRAY, and T.J. WALTER: The development of ribonuclease and acid phosphatase during germination of Pisum arvense. Biochem. J. 142, 211-219 (1974). BEEVERS, L. and F. S. GUERNSEY: Changes in some nitrogenous components during the germination of pea seeds. Plant Physiol. 41, 1455-1458 (1966). BRYANT, J. A., S. C. GREENWAY, and G. A. WEST: Development of nuclease activity in cotyledons of Plsum sativum L. Planta 130, 137-140 (1976 a). - - - Iso-enzymes of acid ribonuclease in cotyledons of Pisum sativum L. Planta 130,141-144 (1976 b). CHAPPEL, J., W. VAN DER WILDEN, and M. J. CHRISPEELS: The biosynthesis of RNase and its accumulation in protein bodies in the cotyledons of mung bean Vigna radlata seedlings. Dev. BioI. 76, 115-125 (1980). DAVIS, B. J.: Disc electrophoresis. II. Method and application to human serum protein. Ann. N. Y. Acad. Sci. 121, 404-427 (1964). GOMES FILHO, E. andJ. T. PRISCO: Effects of NaCI salinity In vivo and In vitro on the proteolytic activity of Vigna sinensis (L.) Savi cotyledons during germination. Rev. Brasil. Bot. 1, 83-88 (1978). GOMES FILHO, E., J. T. PRISCO, F. A. P. CAMPOS, and J. ENEAS FILHO: Effects of NaCI salinity in VIVO and In vitro on ribonuclease activity of Vigna unguiculata cotyledons during germination. Physiol. Plant. 59, 183 -188 (1983). JACOBSEN, H. J.: Analysis of RNase isozymes in germinating pea cotyledons by polyacrylamide-gel electrophoresis. Plant Cell Physiol. 21, 659-665 (1980). KApOOR, H. C. and R. C. SACHAR: Stimulation of ribonuclease activity and its isoenzymes in germinating seeds of cowpea (Vigna si· nensis) by gibberellic acid and adenosine-Y-,5 '-cyclic monophosphate. Experientia 32,558-560 (1976). KAWAZAKI, T., T. AKIBA, and M. MORITSUGU: Effects of high concentrations of sodium chloride and polyethylene glycol on the growth and ion absorbtion in plants. Plant Soil 75, 75-85 (1983). LANG, A. R. G.: Osmotic coefficients and water potentials of sodium chloride solutions from 0 to 40°C. Austral. J. Chern. 20, 2017 -2023 (1967). MACHADO, R. C. R., A. B. RENA, and C. VIEIRA: Efeito da desidratas;ao osmotica na germinac;:ao de sementes de vinte cultivares de feijao (Phaseolus vulgariS) L. Rev. Ceres 128, 310-320 (1976).
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PALMIANO, E. P. and B. O. JULIANO: Biochemical changes in the rice grain during germination. Plant Physiol. 49, 751-756 (1972). PIETRZAK, M., H. CUDNEY, and M. MALUSZYNSKI: Purification and properties of two ribonucleases and a nuclease from barley seeds. Biochim. Biophys. Acta 614, 102-112 (1980). PRISCO, J. T. and W. O'LEARY: Osmotic and «toxic» effects of salinity on germination of Phaseolus vulgaris L. seeds. Turrialba 20, 177 -184 (1970). PRISCO, J. T. and G. H. F. VIEIRA: Effects of NaCI salinity on nitrogenous compounds and proteases during germination of Vigna sinensis seeds. Physiol. Plant. 36, 317 -320 (1976). PRISCO, J. T., J. ENEAS FILHO, and E. GOMES FILHO: Effect of NaCI salinity on cotyledon starch mobilization during germination of Vigna unguiculata (L.) Walp seeds. Rev. Brasil. Bot. 4, 63-71 (1981). SHEORAN, I. S. and O. P. GARG: Effect of salinity on the activities of RNase, DNase and protease during germination and early seedling growth of mung bean. Physiol. Plant. 44, 171-174 (1978). STEEL, R. G. D. and J. H. TORRIE: Principles and Procedures of Statistics: A Biometrical Approach, 2nd Edn. McGraw-Hill, New York, 1980. TUVE, T. W. and C. B. ANFINSEN: Preparation and properties of spinach ribonuclease. J. BioI. Chern. 235, 3437 -3441 (1960). UHVITS, R.: Effect of osmotic pressure on water absorption and germination of alfalfa seeds. Amer. J. Bot. 33, 278 - 285 (1946). WALTERS, T. L. and H. S. LORING: Enzymes of nucleic acid metabolism from mung bean sprouts. I. Fractionation and concentration of phosphomonoesterase, ribonuclease M1 and M2, 3'-nucleotidase, and deoxyribonuclease. J. BioI. Chern. 241, 2870-2875 (1966). WILSON, C. M.: Plant nucleases 1. Separation and purification of two ribonucleases and one nuclease from corn. Plant Physiol. 43, 1332-1338 (1968). - A rapid staining technique for detection of RNase after polyacrylamide gel electrophoresis. Analyt. Biochem. 31, 506 - 511 (1969). - Plant nucleases. III. Polyacrylamide gel electrophoresis of corn ribonuclease isoenzymes. Plant Physiol. 48, 64-68 (1971). - Plant nucleases. Ann. Rev. Plant Physiol. 26, 187 -208 (1975). - Plant nucleases. V. Survey of corn ribonuclease II isoenzymes. Plant Physiol. 61, 861-863 (1978).
Effect of Salinity on Ribonuclease Activity of Vigna unguiculata Cotyledons during Germination E.
GOMES FILHO
and L
SODEK
Departamento de Fisiologia Vegetal, Universidade Estadual de Campinas, 13081 Campinas, SP, Brasil Received June 12, 1987 . Accepted October 18, 1987
Summary The presence of iso-osmotic solutions (-4.6 bar) of NaCl (0.1 M) and mannitol (0.19 m) during the germination of Vigna unguiculata (L.) Walp inhibited the growth of seedlings to a similar degree, suggesting that the salt effect is mainly osmotic. In the presence of salt, RNase activity of the cotyledons rose more slowly than the control during the initial stages of germination, but thereafter rose sharply to values about twice those of the control. The slower initial rise in RNase was apparently an osmotic effect, since an identical curve was obtained with mannitol. The later sharp rise in activity was not seen in mannitoltreated seedlings, suggesting that some specific salt effect was responsible. Cycloheximide, used under conditions that did not inhibit growth, inhibited the increase in RNase. Although polyacrylamide-gel electrophoresis revealed the presence of six RNase isoenzymes in the extracts, essentially all the activity was due to a single isoenzyme. The main RNase isoenzyme hydrolysed RNA producing all four cyclic (2',3') nucleotides, but no 3'-nucleotides, and had no activity towards DNA.
Key words: Vigna unguiculata, germination, RNase, salinity.
Introduction Salinity inhibits seed germination for two reasons. First, the water potential gradient between the seed and the external medium is reduced (osmotic effect), which leads to a diminished influx of water (Uhvits, 1946; Prisco and O'Leary, 1970). Second, the metabolism of the seed is affected, causing the inhibition of seed reserve mobilization (Prisco and Vieira, 1976; Gomes Filho and Prisco, 1978; Prisco et al., 1981; Gomes Filho et aI., 1983). The metabolic effects may be due, in part, to an accumulation of ions to toxic levels (Uhvits, 1946; Prisco and O'Leary, 1970). Studies of the effects of salinity on enzymes involved in seed reserve mobilization have shown that, in general, those enzymes already present at high levels in the quiescent seed are little affected by salt during germination (Prisco and Vieira, 1976; Gomes Filho and Prisco, 1978). On the other hand, those enzymes present at low levels of activity are strongly affected by salinity (Prisco et al., 1981; Gomes Filho et al., 1983). RNase is an example of the latter category. In a previous study (Gomes Filho et al., 1983), it was reported that salinity inhibits this enzyme during the first few days of germina© 1988 by Gustav Fischer Verlag, Stuttgart
tion, together with an inhibition of the mobilization of RNA in the reserve organs. In view of the central role of RNA in cell metabolism, it is possible that impaired RNA metabolism could be the primary effect of salinity on germination, but at present there is no evidence that the change in RNA metabolism in the cause or consequence of the NaCI effect. In this study we investigated the nature of the salt effect on RNase activity during germination of cowpea. Attempts were made to separate the osmotic and ionic components of the salt effect, as ;well as to determine the nature of the increase in enzyme' activity and the possible involvement of Isoenzymes.
Materials and Methods Seed germination and growth measurements Seeds of Vigna unguiculata (L.) Walp cv. pitiuba were sterilized for 30 min in a commercial bleach (2.5 % active chlorine) diluted 20 times with water. The seeds were germinated in rolls of paper, as de-
308
E. GOMES FILHO and L. SODEK
scribed previously (Gomes Filho and Prisco, 1978). Seedling growth was measured by dry and fresh weights of different parts of the seedling on the seventh day after germination. Dry weight was determined after drying at 100°C for 24 h.
Osmotic potential Sodium chloride was used in the germination medium at 0.1 M, which, according to Lang (1967), has an osmotic potential of -4.6 bar at 25°C. When mannitol was used, this was prepared at 0.19 m, calculated from the Van't Hoff equation to have the same osmotic potential as 0.1 M NaC!.
Ribonuclease activity Cotyledons were homogenized in 0.1 M potassium phosphate buffer, pH 5.7, with a cold mortar and pestle. After centrifugation at 3000g for 10 min, the supernatant was adjusted to pH5.1 with HCl, and left in ice for 16 h. The supernatant obtained after a further centrifugation was used in the enzyme assay. The RNase assay was based on that of Tuve and Anfinsen (1960) with minor modifications. The assay mixture contained 50 mM sodium citrate buffer, pH 5.2 (3.0 ml), 1 % yeast RNA (Nutritional Biochemical Company, USA; purified by precipitation from alcohol as the sodium salt) (1.0 ml), and extract (1.0 ml). The assay mixture was incubated at 40°C, and after intervals of 5 and 25 min, 1 ml aliquots were taken for analysis. The aliquots were mixed immediately with 0.2 ml of 0.75 % uranyl acetate in 25 % HCI04 , and left in ice for at least 30 min. After removal of the RNA precipitate by centrifugation at 1000 g for 5 min, the supernatant was diluted 25 times with water and read in a spectrophotometer at 260 m. Activities were expressed as units pere cotyledon per hour, where one unit corresponds to an absorbance increase of 0.01.
Deoxyribonuclease activity DNase was assayed according to Wilson (1968), using heat-denatured calf thymus DNA as substrate, at pH 5.8.
Hydrolysis product analysis A routine assay mixture was used, except that the incubation period continued for up to 24 h. Aliquots (10 to 40 ILl) were applied to cellulose TLC plates and analysed using the two solvent systems described by Walters and Loring (1966). A second aliquot was treated with 0.1 N HCl for 30 min at 30°C prior to chromatography. This treatment transforms any cyclic (2', 3')-nucleotides to 2'- and 3'-nucleotides, thereby differentiating between cyclic nucleotides and the nucleosides, which have similar mobilities in these solvents.
Statistical analysis The data were subjected to an analysis of variance, and when F was significant (P<0.05), the Tukey test was used to compare means (Steel and Torrie, 1980).
Results and Discussion
Seedling growth The fresh and dry weights of the seedlings 7 days after imbibition (Table 1) show that growth was strongly inhibited by NaCI and mannitol. Apparently, the presence of NaCI or mannitol in the germination medium reduces the uptake of water by the seedlings (dry weights less affected than fresh weights) and inhibits the mobilization of the cotyledon reserves to the growing embryonic axis. These data are in agreement with others involving studies of germination in the presence of NaCI or non-ionic osmotic solution such as polyethylene glycol or mannitol (Uhvits, 1946; Prisco and O'Leary, 1970; Prisco and Vieira, 1976; Machado et al., 1976; Sheoran and Garg, 1978; Gomes Filho et al., 1983; Kawasaki et aI., 1983). Since the inhibition imposed by NaCI was no more severe than that of mannitol, it would appear that the water stress effect (one of the salinity stress components) was the principal factor related to growth inhibition.
Electrophoresis Polyacrylamide disc-gel electrophoresis was carried out as described by Davis (1964), with the modifications introduced by Wilson (1978). The spacer gel contained 3 % acrylamide, 0.75 % bisacrylamide, and 62 mM tris adjusted to pH 6.7 with citric acid. The separating gel contained 7 % acrylamide, 0.148 % bis-acrylamide and 0.37M tris-HCl, pH 8.9. The electrode buffer was 10mM tris with 76 mM glycine, pH 8.3. Samples (150 ILl) containing 1 mg of protein together with added sucrose (10 %) were layered onto the gel surface. Electrophoresis was carried out at room temperature, using a current of 2 rnA per gel tube for 20 min followed by 4 rnA for 90 to 120min. RNase isoenzymes were detected by incubating the gels with RNA at Img/ml in sodium citrate buffer (50mM, pH5.2) for 20 min, followed by staining for 1 min in toluidine blue, as described by Wilson (1978).
Experiments with Cycloheximide Cycloheximide (20 ILg! ml) was applied to the germinating seeds either by soaking the seeds with the inhibitor for 24h (quiescent seeds) or by brushing 25 ILl of this solution onto each cotyledon (germinated seeds). On average, the cotyledons absorbed the applied inhibitor solution within 1 h, after which the seedlings were returned to the original germination conditions. For each treatment, a control was set up where water was used instead of the inhibitor.
Table 1: Fresh and dry weights of Vigna unguiculata seedlings 7 days after imbibition in water and iso-osmotic solutions (-4.6 bar) of NaCI and mannitol. Each value represents the mean of 12 replicates, each replicate corresponding to the average measurement of 10 seedlings. dry weIght (mg) fresh weight (mg) roots shoots roots shoots cots. cots. Control 8.5 a 54.3 a 5.6 b 272 a 1640 a 36 a NaCl 7.9 ab 43.0 b 26.8 a 176 b 608 b 84 b 136 c 496 c 82 b Mannitol 7.5 b 39.2 b 30.5 a Values with the same letters (withm columns) are not significantly different (P<0.05) by the T ukey test.
RNase activity In the control seedlings, RNase activity of the cotyledons, initially very low, increased markedly during germination, reaching a maxium near the fifth day after germination (Fig. 1). The activity pattern appears to be inversely related to the RNA content of the tissue, which is high in the quiescent seed and decreases during germination (Beevers and Guernsey, 1966; Palmiano and Juliano, 1972; Gomes Filho et aI., 1983).
Salinity and RNase in Vigna
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I
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Fig. 1: Changes in RNase activity of Vigna unguiculata cotyledons during germination in water (0) and iso-osmotic solutions (-4.6 bar) of NaCI (.) and mannitol (1':.). Each value represents the mean of 3 replicate extracts. Vertical bars represent the LSD value.
In the saline treatment, RNase activity also increased with germination, reaching its maximum value on the seventh day. Although activity in the control was higher than that of the saline treatment up to the third day after germination, thereafter the situation inverted and the activity in the saline treatment increased to almost twice the maximum attained by the control. In the case of mannitol, the activity increased slowly throughout the experiment. The activity curve was identical to that of the NaCI treatment up to the third day, but thereafter increased much more slowly, reaching its highest value on day nine. The effect of NaCI on RNase was similar to that reported previously (Gomes Filho et al., 1983). The activity curve obtained with mannitol was clearly different from that of NaCI and could well be a simple retardation of the control curve, for two reasons. First, the highest activity recorded with mannitol, on day nine, is close to the maximum activity of the control, obtained on day five. Second, the growth parameters (seedling fresh weight and cotyledon dry weight) recorded on day nine for the mannitol treatment were close to those of the control on day five (data not shown), suggesting that the treated plants at day nine reached a similar stage of development as the control plants at day five. Since the activity curves for both mannitol and NaCI-treated plants were identical up to day three, it would appear that initially the effect of NaCI is purely osmotic. The subsequent high RNase activity of the cotyledons in the presence of NaCl must be a specific salt effect, since this increase was not accompanied by the mannitol-treated seedlings. Nevertheless, this specific salt effect on RNase activity does not apparently lead to any change in seedling growth, in view of the similar dry weights obtained with NaCI and mannitol (Table 1). This would
309
suggest that RNase may only playa secondary role in the germination process.
Polyacrylamide-gel electrophoresis When the cotyledon extracts were submitted to polyacrylamide-gel electrophoresis, at least six bands of apparent RNase activity could be detected (Fig.2), with Rms corresponding to 0.17 (band 1), 0.28 (band2), 0.37 (band3), 0.41 (band4), 0.57 (bandS), and 0.80 (band6). Band6 (designated «main band») was practically nonexistent in the quiescent seed, but increased tremendously in intensity during germination, while the others «
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Fig. 2: RNase isoenzymes after electrophoresis of cotyledon extracts of Vigna unquiculata at different stages of germination in water and O.IM NaCl.
310
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Table 2: Densitometry readings (peak areas) of RNase isoenzymes after electrophoresis of cotyledon extracts of Vigna unguiculata treated with water and cycloheximide (CHI) for 24 h prior to analysis. A = 3-day-old seedlings germinated in water; B = 6-day-old seedlings germinated in NaCI. Isoenzyme band numbering as for Fig. 2.
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Fig. 3: Changes in RNase activity over a 24 hour period in Vigna unquiculata cotyledons treated with water (0) and 20 /lg/ml cycloheximide (~) during germination in water (A) and NaCl (B). The change in activity was determined for days 0 to 1 (a), 1 to 2 (b), 2 to 3 (c), 3 to 4 (d), 4 to 5 (e), 5 to 6 (1) and 6 to 7 (g). Three replicate extracts were used for each determination.
Studies with cycloheximide When cotyledons at different ages were treated with cycloheximide and assayed for RNase activity 24 h later, the activities at all ages were substantially lower than the corresponding controls (Fig. 3). This was true for both NaCI treated and control seedlings, suggesting that most, if not all, the increase in activity is dependent on protein synthesis. Others (Bryant et al., 1976a, 1976 b; Kapoor and Sachar, 1976) have reported an increase in RNase totally or partially dependent on protein synthesis, using cycloheximide, but in these experiments the seeds were left to germinate in the presence of the inhibitor for an extended period. The inconvenience of this resides in the strong reduction of growth which puts in doubt whether the observed inhibition is not simply a consequence of the delay in germination. In our own case, the inhibitor was studied for 24 h periods only, during which growth inhibition was not observed (data not shown). Although stronger inhibitions of enzyme activity were possible either by extending the period of treatment or increasing the concentration of the inhibitor, this leads to inhibition of growth. These data are in contrast with those of Barker et al. (1974) who found that the increase in RNase activity during germination of Pisum arvense was independent of protein synthesis. When cycloheximide was applied at ages when RNase activity was decreasing, e.g. days 4 to 5 of the control, the inhibitor caused an even greater decrease in RNase. This suggests that RNase undergoes turnover such that when degradation exceeds synthesis the inhibition of synthesis will lead to an apparent increase in degradation. Polyacrylamide-gel electrophoresis of the extracts showed that cycloheximide reduced the intensity of the main band only (Table 2), consistent with the conclusion that virtually all the increase in activity is due to this isoenzyme. A similar
effect of this inhibitor on one of two isoenzymes of RNase in the cotyledons of germinating Pisum sativum was reported by Bryant et al. (1976 b).
Characteristics of the enzyme Some characteristics of the cotyledon RNase were determined in an attempt to classify the enzyme according to Wilson's (1975) scheme for plant RNases. Using extracts of the NaCI treatment 6 days after germination, when virtually all the activity was due to the main band isoenzyme, no DNase activity could be detected. This suggests that the enzyme is a RNase and not a nuclease. The RNase activity was also virtually insensitive to EDTA, a further property of non-nuclease-type RNases. The products of RNA hydrolysis were examined with the partially purified enzyme (extracted from gel slices taken from the centre of the main band) and this revealed the formation of all four cyclic {2',3')-nucleotides, but no 3'-nucleotides. According to the classification scheme of Wilson (1975), this RNase could be of the I or II type in that it does not hydrolyse DNA and produces cyclic nucleotides. However, in contrast to RNase I or II there was no evidence for the further hydrolysis of any of the cyclic nucleotides to 3'nucleotides. Furthermore, the enzyme showed a broad pH optimum between pH 5 and 6, unaffected by salt, which also differs from both the RNase I and II types.
Conclusions The effect of NaCI on RNase activity of Vigna cotyledons during germination presents two distinct phases. First, a delayed increase in RNase activity, due to the osmotic component and apparently associated with the slower growth of the seedling, and second, a subsequent sharp rise in RNase activity due to a specific salt effect. The specific salt effect does not appear to be associated with any notable change in seedling growth, suggesting that the increase in RNase is not a primary event in the germination process. Acknowledgements EGF is grateful to the Coordenadoria de Aperfeic;oamento de Pessoal de Ensino Superior (CAPES) for a grant.
Salinity and RNase in Vigna
References BARKER, G. R., C. M. BRAY, and T.J. WALTER: The development of ribonuclease and acid phosphatase during germination of Pisum arvense. Biochem. J. 142, 211-219 (1974). BEEVERS, L. and F. S. GUERNSEY: Changes in some nitrogenous components during the germination of pea seeds. Plant Physiol. 41, 1455-1458 (1966). BRYANT, J. A., S. C. GREENWAY, and G. A. WEST: Development of nuclease activity in cotyledons of Plsum sativum L. Planta 130, 137-140 (1976 a). - - - Iso-enzymes of acid ribonuclease in cotyledons of Pisum sativum L. Planta 130,141-144 (1976 b). CHAPPEL, J., W. VAN DER WILDEN, and M. J. CHRISPEELS: The biosynthesis of RNase and its accumulation in protein bodies in the cotyledons of mung bean Vigna radlata seedlings. Dev. BioI. 76, 115-125 (1980). DAVIS, B. J.: Disc electrophoresis. II. Method and application to human serum protein. Ann. N. Y. Acad. Sci. 121, 404-427 (1964). GOMES FILHO, E. andJ. T. PRISCO: Effects of NaCI salinity In vivo and In vitro on the proteolytic activity of Vigna sinensis (L.) Savi cotyledons during germination. Rev. Brasil. Bot. 1, 83-88 (1978). GOMES FILHO, E., J. T. PRISCO, F. A. P. CAMPOS, and J. ENEAS FILHO: Effects of NaCI salinity in VIVO and In vitro on ribonuclease activity of Vigna unguiculata cotyledons during germination. Physiol. Plant. 59, 183 -188 (1983). JACOBSEN, H. J.: Analysis of RNase isozymes in germinating pea cotyledons by polyacrylamide-gel electrophoresis. Plant Cell Physiol. 21, 659-665 (1980). KApOOR, H. C. and R. C. SACHAR: Stimulation of ribonuclease activity and its isoenzymes in germinating seeds of cowpea (Vigna si· nensis) by gibberellic acid and adenosine-Y-,5 '-cyclic monophosphate. Experientia 32,558-560 (1976). KAWAZAKI, T., T. AKIBA, and M. MORITSUGU: Effects of high concentrations of sodium chloride and polyethylene glycol on the growth and ion absorbtion in plants. Plant Soil 75, 75-85 (1983). LANG, A. R. G.: Osmotic coefficients and water potentials of sodium chloride solutions from 0 to 40°C. Austral. J. Chern. 20, 2017 -2023 (1967). MACHADO, R. C. R., A. B. RENA, and C. VIEIRA: Efeito da desidratas;ao osmotica na germinac;:ao de sementes de vinte cultivares de feijao (Phaseolus vulgariS) L. Rev. Ceres 128, 310-320 (1976).
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PALMIANO, E. P. and B. O. JULIANO: Biochemical changes in the rice grain during germination. Plant Physiol. 49, 751-756 (1972). PIETRZAK, M., H. CUDNEY, and M. MALUSZYNSKI: Purification and properties of two ribonucleases and a nuclease from barley seeds. Biochim. Biophys. Acta 614, 102-112 (1980). PRISCO, J. T. and W. O'LEARY: Osmotic and «toxic» effects of salinity on germination of Phaseolus vulgaris L. seeds. Turrialba 20, 177 -184 (1970). PRISCO, J. T. and G. H. F. VIEIRA: Effects of NaCI salinity on nitrogenous compounds and proteases during germination of Vigna sinensis seeds. Physiol. Plant. 36, 317 -320 (1976). PRISCO, J. T., J. ENEAS FILHO, and E. GOMES FILHO: Effect of NaCI salinity on cotyledon starch mobilization during germination of Vigna unguiculata (L.) Walp seeds. Rev. Brasil. Bot. 4, 63-71 (1981). SHEORAN, I. S. and O. P. GARG: Effect of salinity on the activities of RNase, DNase and protease during germination and early seedling growth of mung bean. Physiol. Plant. 44, 171-174 (1978). STEEL, R. G. D. and J. H. TORRIE: Principles and Procedures of Statistics: A Biometrical Approach, 2nd Edn. McGraw-Hill, New York, 1980. TUVE, T. W. and C. B. ANFINSEN: Preparation and properties of spinach ribonuclease. J. BioI. Chern. 235, 3437 -3441 (1960). UHVITS, R.: Effect of osmotic pressure on water absorption and germination of alfalfa seeds. Amer. J. Bot. 33, 278 - 285 (1946). WALTERS, T. L. and H. S. LORING: Enzymes of nucleic acid metabolism from mung bean sprouts. I. Fractionation and concentration of phosphomonoesterase, ribonuclease M1 and M2, 3'-nucleotidase, and deoxyribonuclease. J. BioI. Chern. 241, 2870-2875 (1966). WILSON, C. M.: Plant nucleases 1. Separation and purification of two ribonucleases and one nuclease from corn. Plant Physiol. 43, 1332-1338 (1968). - A rapid staining technique for detection of RNase after polyacrylamide gel electrophoresis. Analyt. Biochem. 31, 506 - 511 (1969). - Plant nucleases. III. Polyacrylamide gel electrophoresis of corn ribonuclease isoenzymes. Plant Physiol. 48, 64-68 (1971). - Plant nucleases. Ann. Rev. Plant Physiol. 26, 187 -208 (1975). - Plant nucleases. V. Survey of corn ribonuclease II isoenzymes. Plant Physiol. 61, 861-863 (1978).