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Influence of low temperature on phosphatase in roots of Verbascum thapsus, a biennial weed
CRYOl3IOLOGT
14, 121-123 ( 1977)
Influence
of Low Temperature Verbascum thapsus,
on Phosphatase in Roots of a Biennial Weed
JAhlES II. GLIER ASD JOHN L. Ct\RlJSO Depadmcnt
of Biological Cincinnati,
Sciences, Ohio
Low temperatures were reported to stimulate starch hydrolysis in roots of \‘erha.scum thapsus, a biennial weed which has a lowtemperature requirement for flowering (3). Only a few similar cases of starch degradation in other plant species have been associated with the activation of enzymes by the lowering of temperatures (1, 5). Lo~vtemperature activation of alpha-amylasc, beta-amylase, and glucan phosphorylase in S’erbascum roots was also observed, however, and this suggests a genetically controlled adaptive response to declining tcmpcratures (4). The present report describes the increased activities of two nonspecific enzymes, namely, acid and alkaline phosphatase, in Verbasczlm roots cxposcd to decreasing temperatures.
Procedures for growing rosettes of \‘erbascurn in the greenhouse (24-29”C), esposing the rosettes to decreasing tcmperaturcs ( 20-15-10-4” C ) under controlled conditions, and obtaining cstracts from roots for enzyme assay have already been described (4). Supernatants obtained from final ccntrifugations at 48,OOOg were analyzed for soluble protein (7) which n-as used in establishing specific activities of phosphatases. Determination of these activities was based on the production of p-nitro_-- --Receivd
September 26, 1975.
1977 by Academic Press, Inr. Copyright All rights o? reproduction in any form reserved.
Uniocrsity 45221
of Cincinnati,
phenol from p-nitrophenyl phosphate, as described elsewhere (2). One unit of activity of either enzyme was defined as that amount which caused the release of 1 pn~ of p-nitrophenol per min, at 25°C. The reaction mixture llsed in our assay for alkaline phosphatase was modified in that it contained 0.03 M magnesium chloride. The reaction mixture used in the determimtion of acid phosphatase contained 0.15 px sodium acetate at pH 5.0 with 0.01 nr EDT,4, and no magnesium chloride.
When temperatures were lowered from greenhouse conditions to lS“C, both acid and alkaline phosphatase reached their respective peaks in activjtics (Fig. 1). The maximum range in specific activities (units of activity per milligram protein) was 0.007 for acid phosphatase and 0.021 for alkaline phosphatase. While the activity of acid phosphatase virtually disappeared by the end of several weeks at 4”C, alkaline phosphatasc declined in activity much more slowly under this condition and eventually approsimated the activity of that seen in roscttcs growing in the greenhouse. A second peak in activities of both enzymes is seen in roots of \‘erbascurn rosettes which are returned to the greenhouse following their exposure to low temperatures (Fig. 2). The rise in activities of these enzymes occurs during the early stages of
ISSS 0011-2240
BRIEF COMMUNICATIONS
122
FIG. 1. Activities of acid and alkaline phosphatase in roots of Verbascum rosettes as influenced by declining temperatures. Two samples were used for each enzyme assay and two replicates were made from each sample.
stem elongation. Alkaline phosphatase had greater activities than acid phosphatase throughout the bolting period. DISCUSSION
The peaks in activities attained by acid and alkaline phosphatase at 15°C coincide with the peak in activity previously seen in alpha-amylase at this temperature. The peaks of activities in phosphatases 1 week following cold treatment coincides with the second peaks in activities of alpha-amylase, beta-amylase, and glucan phosphorylase (4). Whereas the starch degradative enzymes were presumed to furnish the plant with soluble sugars and were postulated to have a direct role in survival of the overwintering rosette, one cannot ascribe a direct role to the phosphatases at this time. The hydrolysis of organic monophosphate esters may furnish orthophosphate which is utilized in the phosphorylation of soluble carbohydrates which in turn are increasing in number due to the action of the starch degrading enzymes already mentioned. The controlling mechanism for the phosphatases in Verbascum is unknown. If future studies show that, for Verbascum, phosphate becomes limiting at low trmperatures, then a mechanism which is known for another plant may appear. Derepression of alkaline
phosphatase which had been previously known for phosphate-deficient bacteria has also been reported for Lemna, a higher plant, upon depletion of orthophosphate in the culture media (6). In studies of bacteria and plants, addition of phosphate to the culture media generally brings about a decrease in phosphatase activity (8, 9). Howcvcr, since no increase in enzyme synthesis has been demonstrated in Verbascum, a model of dercpression can only be suggested for future analysis. An alternative explanation is of course that greater enzyme activities are due to activation of preexisting enzymes, a situation which was strongly suggested for starch-degrading enzymes in cabbage at hardening temperatures (1). Nevertheless, the decrease in activities of phosphatases in Verbascum roots at lO”C, and even more dramatically so at 4”C, is similar to the decreasing activities of starch degrading enzymes at these temperatures. This decrease may prove to be reflective of an overall decrease in protein synthesis at these lower temperatures. The second increase in phosphatase activities in roots of rosettes which underwent the cold treatment and which display early signs of bolting might again reflect a necessity for greater levels of phosphate which, in this case, could be used by rapidly growing plants. A great amount of biosynthesis must
Fro. 2. Activities of acid and alkaline phosplrntase in roots of rosettes returned to the green11ouse following cold treatment. The peaks in activities occur during the early stages of the bolting process.
BRIEF
COhlhlUNIC.4TIOSS
occur during this period of rapid gro\\tll, not only for elongating stems of Vcrbasctm, but for bolting specimens of other coldrequiring biennial plant species as well.
The temperature peak ( 15°C ) of acid and alkaline phosphatase in this study coincides with a peak in alpha-amylase as seen in an earlier study of roots of Vedmscum thapsus. It is speculated that one of the results of higher phosphatasc activities ma) be increased amount of orthophosphatc which can be utilized in phosphorylation of soluble carbohydrates which in turn are in greater supply due to the higher activities of the starch-degrading enzymes. A second peak in activities of acid and alkaline phosphatase was seen in plants which were returned to the greenhouse following cold treatment. This incrcasc in enzymatic activities is also similar to increases in activities of three starch degrading enzymes studied earlier. Alkaline phosphatase showed grcatcr activities than did acid phosphatase at lower temperatures (10 and 4°C) and under greenhouse conditions following cold treatment.
.)‘) I -<)
1. ljc:tr, J, A rapid dcgrudatioll of starch at 11ard~‘11ing telnperaturt,s, ~T~yJbiO~OgrJ 10, 78-81 (1973). C. A fine-structure 2. Garen, A., and Levinlhal, gtwetic and chrn~ical study of the enzyme alkaline phosphatasc of E. coli I. Purification and characterization of alkaline phosphatase. Biocl~inl. Bioph!y~. Acts 38, 470-483 ( 1960). 3. Glier, J. II., and Caruso, J. L. Low-temperature induction of starch degradation in roots of a biennial weed. Cryobiology 10, 328-330
(1973). 4. Glier, J. II., alld Carmo, J. L. The iuflwnce of low temperatures on activities of starch degradntive enzymes in a cold-requiring plant. &ochen~. BiOlJhy.% Res. CorIfmfrn. 58, 573578 (1974). 5. Levitt, J. “Response of Plants to Environmental Stresses.” Academic Press, New York, 1972. 6. Liedtke, V. &I., and Ohmann, E. Synthese und Inaktivierung von alkalischer Phosphatase in Lemna minor. Flora 160, 378-390 (1969). ‘7. Lowry, 0. II., Rosel~rougl~, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagc’nt. J. Bid. C7wtn. 193, 26x-275 ( 19Fjl). 8. Price, C. A. Repression of acid phosphatase synthesis in Eulgenn gmcilk. Science 135, 46 (1962). 9. Vrki, K., and Sate, S. Effects of inorganic phosphate on the extracellular acid phosphatase activity of tobacco cells cultured in vitro. PhJsiol. Plunt. 24, 5OG-511 ( 1971 ).
14, 121-123 ( 1977)
Influence
of Low Temperature Verbascum thapsus,
on Phosphatase in Roots of a Biennial Weed
JAhlES II. GLIER ASD JOHN L. Ct\RlJSO Depadmcnt
of Biological Cincinnati,
Sciences, Ohio
Low temperatures were reported to stimulate starch hydrolysis in roots of \‘erha.scum thapsus, a biennial weed which has a lowtemperature requirement for flowering (3). Only a few similar cases of starch degradation in other plant species have been associated with the activation of enzymes by the lowering of temperatures (1, 5). Lo~vtemperature activation of alpha-amylasc, beta-amylase, and glucan phosphorylase in S’erbascum roots was also observed, however, and this suggests a genetically controlled adaptive response to declining tcmpcratures (4). The present report describes the increased activities of two nonspecific enzymes, namely, acid and alkaline phosphatase, in Verbasczlm roots cxposcd to decreasing temperatures.
Procedures for growing rosettes of \‘erbascurn in the greenhouse (24-29”C), esposing the rosettes to decreasing tcmperaturcs ( 20-15-10-4” C ) under controlled conditions, and obtaining cstracts from roots for enzyme assay have already been described (4). Supernatants obtained from final ccntrifugations at 48,OOOg were analyzed for soluble protein (7) which n-as used in establishing specific activities of phosphatases. Determination of these activities was based on the production of p-nitro_-- --Receivd
September 26, 1975.
1977 by Academic Press, Inr. Copyright All rights o? reproduction in any form reserved.
Uniocrsity 45221
of Cincinnati,
phenol from p-nitrophenyl phosphate, as described elsewhere (2). One unit of activity of either enzyme was defined as that amount which caused the release of 1 pn~ of p-nitrophenol per min, at 25°C. The reaction mixture llsed in our assay for alkaline phosphatase was modified in that it contained 0.03 M magnesium chloride. The reaction mixture used in the determimtion of acid phosphatase contained 0.15 px sodium acetate at pH 5.0 with 0.01 nr EDT,4, and no magnesium chloride.
When temperatures were lowered from greenhouse conditions to lS“C, both acid and alkaline phosphatase reached their respective peaks in activjtics (Fig. 1). The maximum range in specific activities (units of activity per milligram protein) was 0.007 for acid phosphatase and 0.021 for alkaline phosphatase. While the activity of acid phosphatase virtually disappeared by the end of several weeks at 4”C, alkaline phosphatasc declined in activity much more slowly under this condition and eventually approsimated the activity of that seen in roscttcs growing in the greenhouse. A second peak in activities of both enzymes is seen in roots of \‘erbascurn rosettes which are returned to the greenhouse following their exposure to low temperatures (Fig. 2). The rise in activities of these enzymes occurs during the early stages of
ISSS 0011-2240
BRIEF COMMUNICATIONS
122
FIG. 1. Activities of acid and alkaline phosphatase in roots of Verbascum rosettes as influenced by declining temperatures. Two samples were used for each enzyme assay and two replicates were made from each sample.
stem elongation. Alkaline phosphatase had greater activities than acid phosphatase throughout the bolting period. DISCUSSION
The peaks in activities attained by acid and alkaline phosphatase at 15°C coincide with the peak in activity previously seen in alpha-amylase at this temperature. The peaks of activities in phosphatases 1 week following cold treatment coincides with the second peaks in activities of alpha-amylase, beta-amylase, and glucan phosphorylase (4). Whereas the starch degradative enzymes were presumed to furnish the plant with soluble sugars and were postulated to have a direct role in survival of the overwintering rosette, one cannot ascribe a direct role to the phosphatases at this time. The hydrolysis of organic monophosphate esters may furnish orthophosphate which is utilized in the phosphorylation of soluble carbohydrates which in turn are increasing in number due to the action of the starch degrading enzymes already mentioned. The controlling mechanism for the phosphatases in Verbascum is unknown. If future studies show that, for Verbascum, phosphate becomes limiting at low trmperatures, then a mechanism which is known for another plant may appear. Derepression of alkaline
phosphatase which had been previously known for phosphate-deficient bacteria has also been reported for Lemna, a higher plant, upon depletion of orthophosphate in the culture media (6). In studies of bacteria and plants, addition of phosphate to the culture media generally brings about a decrease in phosphatase activity (8, 9). Howcvcr, since no increase in enzyme synthesis has been demonstrated in Verbascum, a model of dercpression can only be suggested for future analysis. An alternative explanation is of course that greater enzyme activities are due to activation of preexisting enzymes, a situation which was strongly suggested for starch-degrading enzymes in cabbage at hardening temperatures (1). Nevertheless, the decrease in activities of phosphatases in Verbascum roots at lO”C, and even more dramatically so at 4”C, is similar to the decreasing activities of starch degrading enzymes at these temperatures. This decrease may prove to be reflective of an overall decrease in protein synthesis at these lower temperatures. The second increase in phosphatase activities in roots of rosettes which underwent the cold treatment and which display early signs of bolting might again reflect a necessity for greater levels of phosphate which, in this case, could be used by rapidly growing plants. A great amount of biosynthesis must
Fro. 2. Activities of acid and alkaline phosplrntase in roots of rosettes returned to the green11ouse following cold treatment. The peaks in activities occur during the early stages of the bolting process.
BRIEF
COhlhlUNIC.4TIOSS
occur during this period of rapid gro\\tll, not only for elongating stems of Vcrbasctm, but for bolting specimens of other coldrequiring biennial plant species as well.
The temperature peak ( 15°C ) of acid and alkaline phosphatase in this study coincides with a peak in alpha-amylase as seen in an earlier study of roots of Vedmscum thapsus. It is speculated that one of the results of higher phosphatasc activities ma) be increased amount of orthophosphatc which can be utilized in phosphorylation of soluble carbohydrates which in turn are in greater supply due to the higher activities of the starch-degrading enzymes. A second peak in activities of acid and alkaline phosphatase was seen in plants which were returned to the greenhouse following cold treatment. This incrcasc in enzymatic activities is also similar to increases in activities of three starch degrading enzymes studied earlier. Alkaline phosphatase showed grcatcr activities than did acid phosphatase at lower temperatures (10 and 4°C) and under greenhouse conditions following cold treatment.
.)‘) I -<)
1. ljc:tr, J, A rapid dcgrudatioll of starch at 11ard~‘11ing telnperaturt,s, ~T~yJbiO~OgrJ 10, 78-81 (1973). C. A fine-structure 2. Garen, A., and Levinlhal, gtwetic and chrn~ical study of the enzyme alkaline phosphatasc of E. coli I. Purification and characterization of alkaline phosphatase. Biocl~inl. Bioph!y~. Acts 38, 470-483 ( 1960). 3. Glier, J. II., and Caruso, J. L. Low-temperature induction of starch degradation in roots of a biennial weed. Cryobiology 10, 328-330
(1973). 4. Glier, J. II., alld Carmo, J. L. The iuflwnce of low temperatures on activities of starch degradntive enzymes in a cold-requiring plant. &ochen~. BiOlJhy.% Res. CorIfmfrn. 58, 573578 (1974). 5. Levitt, J. “Response of Plants to Environmental Stresses.” Academic Press, New York, 1972. 6. Liedtke, V. &I., and Ohmann, E. Synthese und Inaktivierung von alkalischer Phosphatase in Lemna minor. Flora 160, 378-390 (1969). ‘7. Lowry, 0. II., Rosel~rougl~, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagc’nt. J. Bid. C7wtn. 193, 26x-275 ( 19Fjl). 8. Price, C. A. Repression of acid phosphatase synthesis in Eulgenn gmcilk. Science 135, 46 (1962). 9. Vrki, K., and Sate, S. Effects of inorganic phosphate on the extracellular acid phosphatase activity of tobacco cells cultured in vitro. PhJsiol. Plunt. 24, 5OG-511 ( 1971 ).