ANALYTICAL
BIOCHEMISTRY
200,235-238
(1992)
A Modified Calorimetric Method for the Determination of Orthophosphate in the Presence of High ATP Concentrations’ Pilar Gonzblez-Romo, Sobeida SBnchez-Nieto, and Marina Gavilanes-Ruiz Departamento de Bioquimica, DEPg, Facultad de Quimica, Universidad National Authma de M&co, Ciudad Universitaria, Mexico City 04510, Mexico
Received
June
3,199l
We describe a modified calorimetric method that quantitates inorganic phosphate linearly up to 60 nmol, with high stability of the developed color and with a low interference by ATP concentration (up to 30 mu). This method is very suitable for use in ATPase enzymatic assays, especially with enzymes that have low specific activities and (or) high K, values for ATP. o 1992 Academic
Press,
Inc.
Enzymatic ATP hydrolysis has been measured with several methods; these include determination of ADP by coupling enzymes (1,2), by ““Pi release from [y32P]ATP hydrolysis (3,4), or by calorimetric reactions (5-9). Most of the calorimetric reactions are based on the formation of a phosphomolybdate complex in an acid medium followed by a reduction or complexation with basic dyes that yield colored complexes (5,9). An inconvenience of these methods is that spontaneous ATP splitting occurs during the color developing phase, not only in the incubation phase of the enzyme with ATP. Many improvements to these methods, including reagents and different experimental conditions, have been introduced with the aim of increasing sensitivity and color stability and decreasing the nonenzymatic ATP hydrolysis. Chifflet et al. (10) have reported a colorimetric method for Pi determination in samples with high protein content and low ATPase activity. We have introduced some modifications to the Chifflet et al. procedure in order to measure ATP hydrolysis at high ATP 1 Supported by the DGAPA, UNAM, Mexico, under ment IN206691 and by the International Foundation Sweden, under Grant Agreement c/1647-1. 0003-2697/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Grant Agreefor Science,
concentrations; this is of value in studies with plant ATPases, which in some cases have high K, values for ATP (11-14). The method described here allows the measurement of ATP hydrolysis at concentrations of ATP as high as 30 mM; it is linear up to 60 nmol Pi and the developed color is stable up to 5 h. MATERIALS
SDS was obtained from Sigma Chemical Co. (L-4509) and Extran from Merck. The rest of the chemicals used were of analytical grade. The following solutions were made with double-distilled water and in glassware previously washed with Extran and soaked in concentrated H,SO, for at least 24 h: Solution A is 12% SDS.’ Solution B is 12% ascorbic acid in 1 N HCl. Solution C is 2% ammonium molybdate in HCl; B and C solutions stay stable for 2 months at 4°C. Solution D is prepared by mixing 1 ml B with 1 ml C; the mixture turns yellow with time and thus it must be used within 24 h. Solution E is 2% sodium citrate and 2% sodium metaarsenite in 2% acetic acid in H,O. METHODS
Preparation of a standard curve. K,HPO, was previously dried and a 100 InM stock solution was prepared; this was diluted to 1 mM. Aliquots equivalent to 2 to 100 nmol Pi were taken to a final volume of 150 ~1 with double-distilled water. Then 150 ~1 of solution A was added and mixed. This was followed by the addition of
’ Abbreviations nylmethylsulfonyl
used: PEP, phospho(enol)pyruvate; fluoride; SDS, sodium dodecyl
PMSF,
phe-
sulfate. 235
Inc. reserved.
236
GONZALEZ-ROMO,
40
80 nmol
SANCHEZ-NIETO,
120
Pi
FIG. 1. Comparison
of linear ranges of absorbance and nanomoles of Pi between the original (0) [Ref. (lo)] and the present procedure (w). The standard curves were experimentally built as described under Methods.
300 ~1 of solution D. After 3 min, but before 7 min, 450 ~1 of solution E was added and mixed. Samples were incubated for 20 min at room temperature, and readings were made at 850 nm. The color developed was stable up to 5 h. ATP hydrolysis assays. Protein (10 to 100 pg) from microsomal membranes from 6-day-old maize roots was added to a medium that contained 250 mM sucrose, 10 mM Tris-HCl, pH 7.0,7 PM carbonyl cyanide m-chlorophenylhydrazone, and ATP/Mg in equimolar ratios from 1 to 30 mM. ATP hydrolysis was allowed to proceed for 3 to 60 min at 3O’C. The reaction was stopped by the addition of 150 ~1 of solution A (if necessary, the SDS concentration of solution A may be increased as indicated below). The color reagents were added as described for the standard curve. Blanks were made with samples of membranes added after the SDS solution (solution A) to the ATP hydrolysis medium at equivalent times of incubation at 30°C. For measurements of ATP hydrolysis in the presence of an ATP regenerating system, 5 to 7 units of pyruvate kinase in 2.2 M (NH&SO, and 5 mM phospho(enol)pyruvate were added to the ATP hydrolysis medium. Blanks were included in all assays. RESULTS
AND
GAVILANES-RUiZ
tive solutions. With these modifications the assay of Pi could be increased from 20 to 60 nmol Pi (Fig. 1). The reproducibility of our protocol is high in triplicates (variation coefficient of 2%), as well as in determinations of the same samples at different days. ATP interference. The effect of ATP concentration present in a typical ATP hydrolysis mixture incubated for 30 min at 30°C, but without any enzyme included, was evaluated in the experiment depicted in Fig. 2, showing that the nonenzymatic ATP hydrolysis in a wide range of ATP concentrations results in very low absorbance values at 850 nm at the end of the colorimetric assay. As an example, 15 mM ATP in the medium contributed to only 8.7 nmol Pi (0.2 ODU), which represents 15% of the Pi potentially estimated in the linear range (60 nmol or 1.4 ODU). This is an advantage with some preparations, such as plant membrane ATPases, which do not have high specific activities and some of which exhibit high Km values for ATP (11-14); thus in assays of maximal rates, a lo-fold excess of substrate must be used. Another advantage of our procedure is that the blue color developed by the samples remains stable for 5 h even in the presence of 30 mM ATP in the medium (Fig. 2), which means that there is no additional spontaneous ATP splitting during color development. The present method is also satisfactory for assay of Pi using coupled ATPase systems. Table 1 shows that Pi from PEP and ATP yields only about 11 nmol Pi in the blanks in our incubation time of 30 min at 3O’C. Therefore there is
0 t
t .
B
DISCUSSION
range. The modifications to the method (10) included an increase in the volume tion to 1050 ~1 and the doubling of the ascorbic ammonium molybdate concentrations in their Linear
AND
original of reacacid and respec-
FIG. 2.
Stability and tolerance of the modified procedure to ATP concentrations. Indicated ATP concentrations were added to 160 pl of water and incubated at 3O’Y! for 30 min. Color was developed as described under Methods. The tubes of the experiment were read at times of 20 min (0) and 1 (0), 2 (A), 3 (A), 4 (m), and 5 h (0).
COLORIMETRIC
METHOD
OF
ORTOPHOSPHATE
sufficient margin for detection of Pi derived from enzymic hydrolysis. Tolerance to protein concentrations and other chemicals. The procedure detects Pi linearly in the presence of up to 0.7 mg of microsomal protein per milliliter. However, soluble. proteins such as bovine serum albumin can be present even at concentrations of 33.3 mgfml without interference with the assay. An additional advantage of the method with the latter proteins is that centrifugation is not necessary. Other compounds that did not interfere with the assay were 2 M (NH&SO,, 2 M sucrose, 200 IIIM KCl, 200 InM NaCl, 200 mM Mg,SO,, 200 mM KNO,, 200 mM 2-(N-morpho1ine)ethane sulfonic acid, 200 mM 1,3-bis(tris[hydroxymethyl]methylamino)propane, 50 mM Tris, 40 mM /3mercaptoethanol, 10 mM PMSF, 2 mM NaN,, 2 mM Na,MoO,, 300 pM Na,VO,, and 24% SDS. However, the following compounds caused precipitation or changes in color: 250 mM mannitol, 50 mM Hepes, 50 mM Na citrate, 50 mM CaCl,, 100 mM K,SO,, 20 mM EDTA, 10 rn’,* EGTA, and 2% Na deoxycholate. ATP hydrolysis from radicle microsomal fraction. Figure 3 shows a time course of ATP hydrolysis by microsomal fractions from maize radicles in the absence and presence of an ATP regenerating system measured with the modified version of Pi determination. Only a slight increase in the ATP hydrolysis in the presence of the regenerating system was found, which is probably due to the large excess of substrate in the incubation media and the small concentration of accumulated product in the control. Thus it seems clear that the method can provide accurate readings in a broad time span of ATP hydrolysis with ATPases of low specific activity. In conclusion, the present procedure has the following advantages over that previously reported by Chifflet et al. (10): (1) the use of high ATP concentrations up to 30 mM; (2) the tolerance to high concentrations of many chemicals, including some that are very common in the TABLE
Pi Detection
in the
ATP
1
Regenerating
System
Mixture Absorbance
Components 250 mM sucrose, 5 mM ATP 10 mM ATP 5 units PK” 5 units PK + 2 5 units PK + 2 5 units PK + 2
10
mM mM mM
mM
PEP PEP PEP
850 nm Tris-HCl,
pH 7.0
+ 5 mM ATP + 10 mM ATP
o PK was in a 2.2 M (NH&SO, suspension. Note. The components indicated were present ing of color as described under Methods.
0.026 0.070 0.142 0.033 0.133 0.183 0.263
nmol 1.061 2.66 5.81 1.37 5.26 7.47 10.73
Pi
237
DETERMINATION
00
30 Hydrolysis
ttme (mm I
FIG. 3.
ATP hydrolysis of maize root microsomal fractions. One hundred micrograms of microsomal protein was added to a medium with (0) and without (0) an ATP regenerating system. ATP hydrolysis was measured as described under Methods.
determination of ATP hydrolysis from plant membranes (PMSF, NaN, , Na,MoO,, Na,VO, ,and KNO,); (3) the stability of the developed color (at least 5 h) and the fact that the solutions can be prepared in advance without changes in color due to Pi contamination; and (4) the range of protein that can be used in the assay was up to 0.7 mg/ml of microsomal protein, but soluble proteins such as albumin can be added to the Pi assay in concentrations as high as 35 mg/ml without interference. We consider the described method very useful in cases where high ATP concentrations and/or low-specific-activity ATPases are measured. ACKNOWLEDGMENTS The authors are Gomez-Puyou and of the manuscript, Vidal-Guerrero for technical assistance.
grateful to Dr. M. Tuena de Gbmez-Puyou, Dr. A. Dr. D. Gonzalez-Halphen for their careful revision to Miss Rocio M. Santos-Osnaya and Miss Maria typing this work, and to Mr. Sergio Villegas for his
REFERENCES prior
to the develop-
1. Pullman, M. E., Penefsky, H. S., Datta, J. Biol. Chm. 235, 3322-3329.
A., and Racker,
E. (1960)
238
GONZALEZ-ROMO,
SANCHEZ-NIETO,
2. Gomez-Puyou, A., Tuena de Gomez-Puyou, M., and De Meis, L. (1980) Eur. J. Biochem. 159,133-140. 3. Sugino, Y., and Miyoshi, Y. (1964) J. Bid. Chem. 239,2360-3364. 4. Martins, 0. B., and De Meis, L. (1985) J. Biol. Chem. 260,67766781. 5. Fiske, C. H., and Subbarow, Y. (1925) J. Bid. Chem. 66,375-400. 6. Ames, B. N. (1966) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 8, pp. 115-118, Academic Press, New York. 7. Van Belle, H. (1970) Anal. Biochem. 33, 132-142. 8. Itaya, K., and Vi, M. (1966) Clin. Quim. Actu 14, 361-366.
AND
GAVILANES-RUiZ
9. Debruyne, 10. Chifflet,
Biochem. 11. DuPont,
I. (1983) S., Torriglia, 16&l-4.
Anal. Biochem. 130,454-460. A., Chiesa,
F. M., Burke,
S. (1988)
And.
R. M. (1981)
Plant
R., and Tolosa,
L. L., and Spanswick,
Physiol. 67, 59-63. 12. De Michelis, 81,542-547. 13. Kawata, 1410.
M. I., and Spanswick,
T., and Yoshida,
14. St. Marty-Fleurence, P. (1988) Plant
S. (1988)
F., Bourdil,
sci. 54,177-184.
R. M.
Plant Physiol.
(1986)
Plant Cell Physiol. 29,1399I., Rossignol,
M.,
and Blein,
J.
BIOCHEMISTRY
200,235-238
(1992)
A Modified Calorimetric Method for the Determination of Orthophosphate in the Presence of High ATP Concentrations’ Pilar Gonzblez-Romo, Sobeida SBnchez-Nieto, and Marina Gavilanes-Ruiz Departamento de Bioquimica, DEPg, Facultad de Quimica, Universidad National Authma de M&co, Ciudad Universitaria, Mexico City 04510, Mexico
Received
June
3,199l
We describe a modified calorimetric method that quantitates inorganic phosphate linearly up to 60 nmol, with high stability of the developed color and with a low interference by ATP concentration (up to 30 mu). This method is very suitable for use in ATPase enzymatic assays, especially with enzymes that have low specific activities and (or) high K, values for ATP. o 1992 Academic
Press,
Inc.
Enzymatic ATP hydrolysis has been measured with several methods; these include determination of ADP by coupling enzymes (1,2), by ““Pi release from [y32P]ATP hydrolysis (3,4), or by calorimetric reactions (5-9). Most of the calorimetric reactions are based on the formation of a phosphomolybdate complex in an acid medium followed by a reduction or complexation with basic dyes that yield colored complexes (5,9). An inconvenience of these methods is that spontaneous ATP splitting occurs during the color developing phase, not only in the incubation phase of the enzyme with ATP. Many improvements to these methods, including reagents and different experimental conditions, have been introduced with the aim of increasing sensitivity and color stability and decreasing the nonenzymatic ATP hydrolysis. Chifflet et al. (10) have reported a colorimetric method for Pi determination in samples with high protein content and low ATPase activity. We have introduced some modifications to the Chifflet et al. procedure in order to measure ATP hydrolysis at high ATP 1 Supported by the DGAPA, UNAM, Mexico, under ment IN206691 and by the International Foundation Sweden, under Grant Agreement c/1647-1. 0003-2697/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Grant Agreefor Science,
concentrations; this is of value in studies with plant ATPases, which in some cases have high K, values for ATP (11-14). The method described here allows the measurement of ATP hydrolysis at concentrations of ATP as high as 30 mM; it is linear up to 60 nmol Pi and the developed color is stable up to 5 h. MATERIALS
SDS was obtained from Sigma Chemical Co. (L-4509) and Extran from Merck. The rest of the chemicals used were of analytical grade. The following solutions were made with double-distilled water and in glassware previously washed with Extran and soaked in concentrated H,SO, for at least 24 h: Solution A is 12% SDS.’ Solution B is 12% ascorbic acid in 1 N HCl. Solution C is 2% ammonium molybdate in HCl; B and C solutions stay stable for 2 months at 4°C. Solution D is prepared by mixing 1 ml B with 1 ml C; the mixture turns yellow with time and thus it must be used within 24 h. Solution E is 2% sodium citrate and 2% sodium metaarsenite in 2% acetic acid in H,O. METHODS
Preparation of a standard curve. K,HPO, was previously dried and a 100 InM stock solution was prepared; this was diluted to 1 mM. Aliquots equivalent to 2 to 100 nmol Pi were taken to a final volume of 150 ~1 with double-distilled water. Then 150 ~1 of solution A was added and mixed. This was followed by the addition of
’ Abbreviations nylmethylsulfonyl
used: PEP, phospho(enol)pyruvate; fluoride; SDS, sodium dodecyl
PMSF,
phe-
sulfate. 235
Inc. reserved.
236
GONZALEZ-ROMO,
40
80 nmol
SANCHEZ-NIETO,
120
Pi
FIG. 1. Comparison
of linear ranges of absorbance and nanomoles of Pi between the original (0) [Ref. (lo)] and the present procedure (w). The standard curves were experimentally built as described under Methods.
300 ~1 of solution D. After 3 min, but before 7 min, 450 ~1 of solution E was added and mixed. Samples were incubated for 20 min at room temperature, and readings were made at 850 nm. The color developed was stable up to 5 h. ATP hydrolysis assays. Protein (10 to 100 pg) from microsomal membranes from 6-day-old maize roots was added to a medium that contained 250 mM sucrose, 10 mM Tris-HCl, pH 7.0,7 PM carbonyl cyanide m-chlorophenylhydrazone, and ATP/Mg in equimolar ratios from 1 to 30 mM. ATP hydrolysis was allowed to proceed for 3 to 60 min at 3O’C. The reaction was stopped by the addition of 150 ~1 of solution A (if necessary, the SDS concentration of solution A may be increased as indicated below). The color reagents were added as described for the standard curve. Blanks were made with samples of membranes added after the SDS solution (solution A) to the ATP hydrolysis medium at equivalent times of incubation at 30°C. For measurements of ATP hydrolysis in the presence of an ATP regenerating system, 5 to 7 units of pyruvate kinase in 2.2 M (NH&SO, and 5 mM phospho(enol)pyruvate were added to the ATP hydrolysis medium. Blanks were included in all assays. RESULTS
AND
GAVILANES-RUiZ
tive solutions. With these modifications the assay of Pi could be increased from 20 to 60 nmol Pi (Fig. 1). The reproducibility of our protocol is high in triplicates (variation coefficient of 2%), as well as in determinations of the same samples at different days. ATP interference. The effect of ATP concentration present in a typical ATP hydrolysis mixture incubated for 30 min at 30°C, but without any enzyme included, was evaluated in the experiment depicted in Fig. 2, showing that the nonenzymatic ATP hydrolysis in a wide range of ATP concentrations results in very low absorbance values at 850 nm at the end of the colorimetric assay. As an example, 15 mM ATP in the medium contributed to only 8.7 nmol Pi (0.2 ODU), which represents 15% of the Pi potentially estimated in the linear range (60 nmol or 1.4 ODU). This is an advantage with some preparations, such as plant membrane ATPases, which do not have high specific activities and some of which exhibit high Km values for ATP (11-14); thus in assays of maximal rates, a lo-fold excess of substrate must be used. Another advantage of our procedure is that the blue color developed by the samples remains stable for 5 h even in the presence of 30 mM ATP in the medium (Fig. 2), which means that there is no additional spontaneous ATP splitting during color development. The present method is also satisfactory for assay of Pi using coupled ATPase systems. Table 1 shows that Pi from PEP and ATP yields only about 11 nmol Pi in the blanks in our incubation time of 30 min at 3O’C. Therefore there is
0 t
t .
B
DISCUSSION
range. The modifications to the method (10) included an increase in the volume tion to 1050 ~1 and the doubling of the ascorbic ammonium molybdate concentrations in their Linear
AND
original of reacacid and respec-
FIG. 2.
Stability and tolerance of the modified procedure to ATP concentrations. Indicated ATP concentrations were added to 160 pl of water and incubated at 3O’Y! for 30 min. Color was developed as described under Methods. The tubes of the experiment were read at times of 20 min (0) and 1 (0), 2 (A), 3 (A), 4 (m), and 5 h (0).
COLORIMETRIC
METHOD
OF
ORTOPHOSPHATE
sufficient margin for detection of Pi derived from enzymic hydrolysis. Tolerance to protein concentrations and other chemicals. The procedure detects Pi linearly in the presence of up to 0.7 mg of microsomal protein per milliliter. However, soluble. proteins such as bovine serum albumin can be present even at concentrations of 33.3 mgfml without interference with the assay. An additional advantage of the method with the latter proteins is that centrifugation is not necessary. Other compounds that did not interfere with the assay were 2 M (NH&SO,, 2 M sucrose, 200 IIIM KCl, 200 InM NaCl, 200 mM Mg,SO,, 200 mM KNO,, 200 mM 2-(N-morpho1ine)ethane sulfonic acid, 200 mM 1,3-bis(tris[hydroxymethyl]methylamino)propane, 50 mM Tris, 40 mM /3mercaptoethanol, 10 mM PMSF, 2 mM NaN,, 2 mM Na,MoO,, 300 pM Na,VO,, and 24% SDS. However, the following compounds caused precipitation or changes in color: 250 mM mannitol, 50 mM Hepes, 50 mM Na citrate, 50 mM CaCl,, 100 mM K,SO,, 20 mM EDTA, 10 rn’,* EGTA, and 2% Na deoxycholate. ATP hydrolysis from radicle microsomal fraction. Figure 3 shows a time course of ATP hydrolysis by microsomal fractions from maize radicles in the absence and presence of an ATP regenerating system measured with the modified version of Pi determination. Only a slight increase in the ATP hydrolysis in the presence of the regenerating system was found, which is probably due to the large excess of substrate in the incubation media and the small concentration of accumulated product in the control. Thus it seems clear that the method can provide accurate readings in a broad time span of ATP hydrolysis with ATPases of low specific activity. In conclusion, the present procedure has the following advantages over that previously reported by Chifflet et al. (10): (1) the use of high ATP concentrations up to 30 mM; (2) the tolerance to high concentrations of many chemicals, including some that are very common in the TABLE
Pi Detection
in the
ATP
1
Regenerating
System
Mixture Absorbance
Components 250 mM sucrose, 5 mM ATP 10 mM ATP 5 units PK” 5 units PK + 2 5 units PK + 2 5 units PK + 2
10
mM mM mM
mM
PEP PEP PEP
850 nm Tris-HCl,
pH 7.0
+ 5 mM ATP + 10 mM ATP
o PK was in a 2.2 M (NH&SO, suspension. Note. The components indicated were present ing of color as described under Methods.
0.026 0.070 0.142 0.033 0.133 0.183 0.263
nmol 1.061 2.66 5.81 1.37 5.26 7.47 10.73
Pi
237
DETERMINATION
00
30 Hydrolysis
ttme (mm I
FIG. 3.
ATP hydrolysis of maize root microsomal fractions. One hundred micrograms of microsomal protein was added to a medium with (0) and without (0) an ATP regenerating system. ATP hydrolysis was measured as described under Methods.
determination of ATP hydrolysis from plant membranes (PMSF, NaN, , Na,MoO,, Na,VO, ,and KNO,); (3) the stability of the developed color (at least 5 h) and the fact that the solutions can be prepared in advance without changes in color due to Pi contamination; and (4) the range of protein that can be used in the assay was up to 0.7 mg/ml of microsomal protein, but soluble proteins such as albumin can be added to the Pi assay in concentrations as high as 35 mg/ml without interference. We consider the described method very useful in cases where high ATP concentrations and/or low-specific-activity ATPases are measured. ACKNOWLEDGMENTS The authors are Gomez-Puyou and of the manuscript, Vidal-Guerrero for technical assistance.
grateful to Dr. M. Tuena de Gbmez-Puyou, Dr. A. Dr. D. Gonzalez-Halphen for their careful revision to Miss Rocio M. Santos-Osnaya and Miss Maria typing this work, and to Mr. Sergio Villegas for his
REFERENCES prior
to the develop-
1. Pullman, M. E., Penefsky, H. S., Datta, J. Biol. Chm. 235, 3322-3329.
A., and Racker,
E. (1960)
238
GONZALEZ-ROMO,
SANCHEZ-NIETO,
2. Gomez-Puyou, A., Tuena de Gomez-Puyou, M., and De Meis, L. (1980) Eur. J. Biochem. 159,133-140. 3. Sugino, Y., and Miyoshi, Y. (1964) J. Bid. Chem. 239,2360-3364. 4. Martins, 0. B., and De Meis, L. (1985) J. Biol. Chem. 260,67766781. 5. Fiske, C. H., and Subbarow, Y. (1925) J. Bid. Chem. 66,375-400. 6. Ames, B. N. (1966) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 8, pp. 115-118, Academic Press, New York. 7. Van Belle, H. (1970) Anal. Biochem. 33, 132-142. 8. Itaya, K., and Vi, M. (1966) Clin. Quim. Actu 14, 361-366.
AND
GAVILANES-RUiZ
9. Debruyne, 10. Chifflet,
Biochem. 11. DuPont,
I. (1983) S., Torriglia, 16&l-4.
Anal. Biochem. 130,454-460. A., Chiesa,
F. M., Burke,
S. (1988)
And.
R. M. (1981)
Plant
R., and Tolosa,
L. L., and Spanswick,
Physiol. 67, 59-63. 12. De Michelis, 81,542-547. 13. Kawata, 1410.
M. I., and Spanswick,
T., and Yoshida,
14. St. Marty-Fleurence, P. (1988) Plant
S. (1988)
F., Bourdil,
sci. 54,177-184.
R. M.
Plant Physiol.
(1986)
Plant Cell Physiol. 29,1399I., Rossignol,
M.,
and Blein,
J.
