Plant & Cell Physiol. 21(8): 1505-1513 (1980)
Accum.ulation Or sugars in cucum.ber leaves during calcium. starvation Hideaki Matsumoto! and Koh Teraoka
(Received September 4, 1980)
To determine the metabolic role of calcium we grew cucumber plants without calcium. Symptoms of leaf injury such as chlorosis and marginal curling appeared a few days after the removal of calcium. The level of the soluble sugars in calcium deficient leaves increased to more than l O-fold that in the control at the late stage of treatment. In contrast, the level of the soluble sugars in the root decreased because of the calcium deficiency. The contents of soluble and bound calcium, in contrast to soluble sugars, decreased only in the young leaves of calcium deficient plants. The content of each soluble sugar measured by liquid chromatography was stable in the control leaves during treatment. Changes in sucrose, fructose, glucose, maltose and galactose in calcium deficient leaves were similar to the change in the total soluble sugars. The increases in stachyose and the mixture of raffinose and cellobiose took place only at the late stage of calcium starvation. The starch content in calcium deficient leaves was somewhat higher than that of the control, except for the remarkable decrease at the late stage. a-Amylase activities were not altered much in either the control or the calcium deficient plant during 5 days of treatment, but a clear increase took place at the late stage of calcium starvation. The reason for the distinct increase in the soluble sugars in calcium deficient leaves could be explained by the decline in transport due to the calcium deficiency. Key words: Accumulation - Calcium deficiency - Cucumber leaf - Sugar.
Calcium (Ca 2 +) is an essential element in higher plants, but its physiological function has not been understood fully. It is generally accepted that Ca 2 + is involved in various fundamental physiological functions of plants that comprise the structure of the cell wall, the membrane, chromatin and enzyme activities (2, 6, 7). Most studies of the physiological functions of Ca 2 + in plants have concentrated on the interrelationship between Ca 2 + and ion transport (1, 11). In a previous report on metabolic disorders in the cucumber during Ca 2+ starvation, we reported that the induction of nitrate reductase in leaves was repressed strongly (10). Our results indicated that the main reason for the repression of nitrate reductase was damage to the root, which caused inhibition of the absorption and transport of nitrate. However, excised Ca 2 + deficient leaves could not induce nitrate reductase at the maximum rate even in the presence of sufficient nitrate, evidence that some metabolic disorders other than the shortage of nitrate may be 1
Correspondence for this paper should be addressed to H. M. 1505
Downloaded from http://pcp.oxfordjournals.org/ at University of California, Davis on February 10, 2015
Institute for Agricultural and Biological Sciences, Okayama University, Chuo 2-20-1, Kurashiki, Okayama 710, Japan
1506
H. Matsumoto and K. Teraoka
involved in the decreased level of nitrate reductase in Ca 2+ deficient leaves. Studies of the metabolic disorders due to a Ca 2+ deficiency have been limited. Thus, we decided to investigate the role of Ca 2+ in metabolism with particular emphasis on changes in the sugar level associated with a Ca 2+ deficiency because, as far as we know, there have been no published reports on this relationship.
Materials and m.ethods
Cucumber (Cucumis sativus cv. Seiriki No.2) was used. The culture solution and conditions were the same as reported previously (10). The culture solution of approximately 30 liter for 36 plants was changed weekly. When the 4th or 5th leaf appeared, the samples were treated to produce a Ca 2+ deficiency. The culture solution was changed every other day during treatment. Deionized water was used in the treatment, and samples were aerated throughout the experiment. Three young leaves and the whole root of each plant were used for the measurements.
Extraction and determination
ofsoluble sugars and starch
The samples were weighed then homogenized with 80% ethanol in Waring blendor for 3 min. The homogenate was centrifuged for 15 min at 4,000 rpm. This extraction of soluble sugars from the precipitate was repeated. The combined alcohol extract was evaporated to remove ethanol, then it was centrifuged at 15,000 X g for 15 min. The supernatant was passed through columns (1 X 12 cm) packed with Amberlite IR 120 (H+) and IRA 45 (OH-). The eluate was evaporated to dryness under a vacuum at 40°C. Soluble sugars were dissolved in 5 ml of distilled water. A portion of the soluble sugars was analyzed by the anthrone method with glucose as the standard to determine the total content of soluble sugars. Starch in the residue of the alcohol extract was solubilized in 52 % perchloric acid then
precipitated with the iodine-potassium iodine reagent. Iodine was eliminated from the precipitated starch-iodine mixture with alcoholic NaOH (5). Finally, the starch was solubilized in 26% perchloric acid and its content determined with anthrone reagent.
Fractionation
ofsoluble sugars
A portion of the soluble sugars was filtered through a membrane filter (Toyo TM-2, 13 m/m, 0.45/-lm) then injected into the column (0.8 X 13 cm) of a Nihon Denshi model ]LC 6-AUH liquid chromatograph. The column was packed with LC-R-3A quartenary ammonium ion exchange resin. Operating conditions were as follows: the flow speed 0.51 mljmin, temperature 52°C and a buffer system of 56 ml of 0.13 M sodium borate (pH 7.5), 46 ml of 0.25 M sodium borate (pH 9.0) and 92 ml of 0.35 M sodium borate (pH 9.6). Identification of each sugar was made by comparing the retention time of the sample and the authentic standard. Furthermore, the identification of each soluble sugar was verified by paperchromatography. The sample was spotted on Toyo filter paper No. 51A, after which the paper was developed three times by the ascending method with a solvent system of n-butanolacetic acid-water (4 : 1 : 2). After drying, the sugars were detected by the silver
Downloaded from http://pcp.oxfordjournals.org/ at University of California, Davis on February 10, 2015
Plant materials
Sugar accumulation and Ca 2 + deficiency
1507
nitrate dip method (14). Raffinose and cellobiose could not be separated by these methods, so their total amount is expressed as raffinose.
a-Amylase assay
Measurement
ofsoluble and bound Ca2+
Each leaf of 5-leaved plants cultured in complete medium or in medium lacking Ca 2 + was excised, then homogenized in distilled water and centrifuged at 10,000 xg for 15 min. The residue was re-extracted and centrifuged. The supernatants were combined, and the final residue was decomposed in concentrated H 2 S0 4 by heating it. Ca 2 + in the combined water extract and the sample digested in H 2 S0 4 was analyzed with a Hitachi atomic absorption spectrophotometer, model 207. The Ca 2 + in the whole root also was measured by the method used for the leaf sample. Results
Symptoms of a Ca2 + deficiency in the cucumber After the removal of Ca 2 + from the culture solution, cucumber growth was almost completely inhibited and the appearance of the root became dull, after which the top withered 4~6 days (10). Typical injury symptoms in leaves, which showed marginal curling and chrolosis, appeared after 3-5 days and progressed rapidly (Fig. 1). Plants nearly had died after approximately 10 days of treatment.
Level of total soluble sugars As shown in Fig. 2, the soluble sugar content in the leaves increased distinctly after the removal of Ca 2+ to more than 10-fold the control level at the late stage. The increasing pattern seemed to be biphasic; a slow increase in the early half stage up to 4 days and a rapid one in the latter half stage. However, the level of free sugars in the root was opposite to that in the leaves. A stable and severe decrease was observed after 2 days of treatment. In the following experiment we investi-
Downloaded from http://pcp.oxfordjournals.org/ at University of California, Davis on February 10, 2015
The sample was homogenized with 3 times its volume of 0.05 M potassium phosphate buffer (pH 7.0) containing 10 mM 2-mercaptoethanol in a Waring blendor at maximum speed for 2 min. The homogenate was passed through 4 layers of cheese cloth then centrifuged at 15,000 xg for 20 min. The supernatant was used as the enzyme. The reaction mixture consisted of 2.0 ml of 0.20/0 amylose (approximate molecular weight 16,000) adjusted to pH 7.5, 1.0 ml of 0.5 M sodium acetate buffer (pH 5.5) and an appropriate volume of enzyme. The final volume was adjusted to 4.0 ml with distilled water and the reaction took place at 30aC for 30 min. After incubation, the reaction was stopped by the addition of 5 ml of 1 M acetic acid. Then a 1.0 ml sample was taken and combined with 10 ml of 0.005% 12 containing 0.05% KI. The color was read at 680 nm. Enzyme was omitted in the blank test. a-Amylase activity was expressed by the blue value. Protein was determined by the method of Lowry et al. using bovine serum albumin as the standard (8).
1508
H. Matsumoto and K. Teraoka
Fig. 1. Cucumber leaf cultured in complete medium (left) and one lacking Ca2 + (right) for 5 days. Marginal curling and chlorosis spots near the petiole are present in the Ca 2 + deficient leaf.
gated whether the increase in soluble sugars in the leaves was linked with the loss of Ca 2 +.
Levels
of soluble
and bound Ca2 +
In view of the low physiological mobility of Ca-! (3), the Ca 2 + content in both
Leaf
'-
1.2 Root
~1.0 ='~
Ul
•
~.: O.
...A
~
Accum.ulation Or sugars in cucum.ber leaves during calcium. starvation Hideaki Matsumoto! and Koh Teraoka
(Received September 4, 1980)
To determine the metabolic role of calcium we grew cucumber plants without calcium. Symptoms of leaf injury such as chlorosis and marginal curling appeared a few days after the removal of calcium. The level of the soluble sugars in calcium deficient leaves increased to more than l O-fold that in the control at the late stage of treatment. In contrast, the level of the soluble sugars in the root decreased because of the calcium deficiency. The contents of soluble and bound calcium, in contrast to soluble sugars, decreased only in the young leaves of calcium deficient plants. The content of each soluble sugar measured by liquid chromatography was stable in the control leaves during treatment. Changes in sucrose, fructose, glucose, maltose and galactose in calcium deficient leaves were similar to the change in the total soluble sugars. The increases in stachyose and the mixture of raffinose and cellobiose took place only at the late stage of calcium starvation. The starch content in calcium deficient leaves was somewhat higher than that of the control, except for the remarkable decrease at the late stage. a-Amylase activities were not altered much in either the control or the calcium deficient plant during 5 days of treatment, but a clear increase took place at the late stage of calcium starvation. The reason for the distinct increase in the soluble sugars in calcium deficient leaves could be explained by the decline in transport due to the calcium deficiency. Key words: Accumulation - Calcium deficiency - Cucumber leaf - Sugar.
Calcium (Ca 2 +) is an essential element in higher plants, but its physiological function has not been understood fully. It is generally accepted that Ca 2 + is involved in various fundamental physiological functions of plants that comprise the structure of the cell wall, the membrane, chromatin and enzyme activities (2, 6, 7). Most studies of the physiological functions of Ca 2 + in plants have concentrated on the interrelationship between Ca 2 + and ion transport (1, 11). In a previous report on metabolic disorders in the cucumber during Ca 2+ starvation, we reported that the induction of nitrate reductase in leaves was repressed strongly (10). Our results indicated that the main reason for the repression of nitrate reductase was damage to the root, which caused inhibition of the absorption and transport of nitrate. However, excised Ca 2 + deficient leaves could not induce nitrate reductase at the maximum rate even in the presence of sufficient nitrate, evidence that some metabolic disorders other than the shortage of nitrate may be 1
Correspondence for this paper should be addressed to H. M. 1505
Downloaded from http://pcp.oxfordjournals.org/ at University of California, Davis on February 10, 2015
Institute for Agricultural and Biological Sciences, Okayama University, Chuo 2-20-1, Kurashiki, Okayama 710, Japan
1506
H. Matsumoto and K. Teraoka
involved in the decreased level of nitrate reductase in Ca 2+ deficient leaves. Studies of the metabolic disorders due to a Ca 2+ deficiency have been limited. Thus, we decided to investigate the role of Ca 2+ in metabolism with particular emphasis on changes in the sugar level associated with a Ca 2+ deficiency because, as far as we know, there have been no published reports on this relationship.
Materials and m.ethods
Cucumber (Cucumis sativus cv. Seiriki No.2) was used. The culture solution and conditions were the same as reported previously (10). The culture solution of approximately 30 liter for 36 plants was changed weekly. When the 4th or 5th leaf appeared, the samples were treated to produce a Ca 2+ deficiency. The culture solution was changed every other day during treatment. Deionized water was used in the treatment, and samples were aerated throughout the experiment. Three young leaves and the whole root of each plant were used for the measurements.
Extraction and determination
ofsoluble sugars and starch
The samples were weighed then homogenized with 80% ethanol in Waring blendor for 3 min. The homogenate was centrifuged for 15 min at 4,000 rpm. This extraction of soluble sugars from the precipitate was repeated. The combined alcohol extract was evaporated to remove ethanol, then it was centrifuged at 15,000 X g for 15 min. The supernatant was passed through columns (1 X 12 cm) packed with Amberlite IR 120 (H+) and IRA 45 (OH-). The eluate was evaporated to dryness under a vacuum at 40°C. Soluble sugars were dissolved in 5 ml of distilled water. A portion of the soluble sugars was analyzed by the anthrone method with glucose as the standard to determine the total content of soluble sugars. Starch in the residue of the alcohol extract was solubilized in 52 % perchloric acid then
precipitated with the iodine-potassium iodine reagent. Iodine was eliminated from the precipitated starch-iodine mixture with alcoholic NaOH (5). Finally, the starch was solubilized in 26% perchloric acid and its content determined with anthrone reagent.
Fractionation
ofsoluble sugars
A portion of the soluble sugars was filtered through a membrane filter (Toyo TM-2, 13 m/m, 0.45/-lm) then injected into the column (0.8 X 13 cm) of a Nihon Denshi model ]LC 6-AUH liquid chromatograph. The column was packed with LC-R-3A quartenary ammonium ion exchange resin. Operating conditions were as follows: the flow speed 0.51 mljmin, temperature 52°C and a buffer system of 56 ml of 0.13 M sodium borate (pH 7.5), 46 ml of 0.25 M sodium borate (pH 9.0) and 92 ml of 0.35 M sodium borate (pH 9.6). Identification of each sugar was made by comparing the retention time of the sample and the authentic standard. Furthermore, the identification of each soluble sugar was verified by paperchromatography. The sample was spotted on Toyo filter paper No. 51A, after which the paper was developed three times by the ascending method with a solvent system of n-butanolacetic acid-water (4 : 1 : 2). After drying, the sugars were detected by the silver
Downloaded from http://pcp.oxfordjournals.org/ at University of California, Davis on February 10, 2015
Plant materials
Sugar accumulation and Ca 2 + deficiency
1507
nitrate dip method (14). Raffinose and cellobiose could not be separated by these methods, so their total amount is expressed as raffinose.
a-Amylase assay
Measurement
ofsoluble and bound Ca2+
Each leaf of 5-leaved plants cultured in complete medium or in medium lacking Ca 2 + was excised, then homogenized in distilled water and centrifuged at 10,000 xg for 15 min. The residue was re-extracted and centrifuged. The supernatants were combined, and the final residue was decomposed in concentrated H 2 S0 4 by heating it. Ca 2 + in the combined water extract and the sample digested in H 2 S0 4 was analyzed with a Hitachi atomic absorption spectrophotometer, model 207. The Ca 2 + in the whole root also was measured by the method used for the leaf sample. Results
Symptoms of a Ca2 + deficiency in the cucumber After the removal of Ca 2 + from the culture solution, cucumber growth was almost completely inhibited and the appearance of the root became dull, after which the top withered 4~6 days (10). Typical injury symptoms in leaves, which showed marginal curling and chrolosis, appeared after 3-5 days and progressed rapidly (Fig. 1). Plants nearly had died after approximately 10 days of treatment.
Level of total soluble sugars As shown in Fig. 2, the soluble sugar content in the leaves increased distinctly after the removal of Ca 2+ to more than 10-fold the control level at the late stage. The increasing pattern seemed to be biphasic; a slow increase in the early half stage up to 4 days and a rapid one in the latter half stage. However, the level of free sugars in the root was opposite to that in the leaves. A stable and severe decrease was observed after 2 days of treatment. In the following experiment we investi-
Downloaded from http://pcp.oxfordjournals.org/ at University of California, Davis on February 10, 2015
The sample was homogenized with 3 times its volume of 0.05 M potassium phosphate buffer (pH 7.0) containing 10 mM 2-mercaptoethanol in a Waring blendor at maximum speed for 2 min. The homogenate was passed through 4 layers of cheese cloth then centrifuged at 15,000 xg for 20 min. The supernatant was used as the enzyme. The reaction mixture consisted of 2.0 ml of 0.20/0 amylose (approximate molecular weight 16,000) adjusted to pH 7.5, 1.0 ml of 0.5 M sodium acetate buffer (pH 5.5) and an appropriate volume of enzyme. The final volume was adjusted to 4.0 ml with distilled water and the reaction took place at 30aC for 30 min. After incubation, the reaction was stopped by the addition of 5 ml of 1 M acetic acid. Then a 1.0 ml sample was taken and combined with 10 ml of 0.005% 12 containing 0.05% KI. The color was read at 680 nm. Enzyme was omitted in the blank test. a-Amylase activity was expressed by the blue value. Protein was determined by the method of Lowry et al. using bovine serum albumin as the standard (8).
1508
H. Matsumoto and K. Teraoka
Fig. 1. Cucumber leaf cultured in complete medium (left) and one lacking Ca2 + (right) for 5 days. Marginal curling and chlorosis spots near the petiole are present in the Ca 2 + deficient leaf.
gated whether the increase in soluble sugars in the leaves was linked with the loss of Ca 2 +.
Levels
of soluble
and bound Ca2 +
In view of the low physiological mobility of Ca-! (3), the Ca 2 + content in both
Leaf
'-
1.2 Root
~1.0 ='~
Ul
•
~.: O.
...A
~
