- Email: [email protected]
Effect of Sulphur and Iron-EDDHA on Chlorophyll Synthesis, Iron Enzymes and Crop Yield of Sugarcane
Biochem. Physiol. Pflanzen (BPP), Bd. 164, S. 509-513 (1973) University of Udaipur, Rajasthan College of Agriculture, Department of Agronomy
Effect of Sulphur and Iron-EDDHA on Chlorophyll Synthesis, Iron Enzymes and Crop Yield of Sugarcane By P. N. MATHUR, H. S. DUNGARWAL, H. G. SINGH and M. S. SAROHA (Received February 14, 1973)
Summary A field experiment was conducted to study the effect of sulphur and iron on iron chlorosis of sugarcane (Saccharum officinarum L.) grown on calcareous soil. Sulphur and iron treatments increased the chlorophyll content, and catalase and peroxidase activity several times that of chlorotic leaves while the aconitase activity remained unaffected. While both treatments reduced the phosphorus content only sulphur treatment decreased the iron content significantly. Chlorosis appears to be due to a reduced physiological availability of iron due either to a reduced permeability of intracellular membranes or competition with some other element for the same reactive site.
Introduction
Rice, sugarcane, peanut, peas and certain other legumes when grown on calcareous soils develop severe symptoms of chlorosis resembling iron deficiency. SINGH (1966) and SINGH and SINGH (1966) reported partial prevention of chlorosis by repeated application of iron chelates. Subsequently it was shown (SINGH and GUPTA 1968) that application of elemental sulphur to soil was a much more effective means of preventing chlorosis and that the chlorotic leaves contained much more iron than green leaves (SINGH 1970 and SINGH 1971). Recently ZECH (1970), SAGLIO (1969) and WALLACE (1971) also reported that chlorotic leaves from plants grown on calcareous soils contained more iron than green leaves. SINGH (1970) proposed that the chlorosis in these soils could be due to a physiological unavailability of iron in the plants. In this paper evidence has been presented in favour of this hypothesis. Material and methods The experimental material for this study forms part of a replicated trial comparing efficacy of different sources of sulphur and iron in sugarcane (Saccharum officinarum L. Var. Co 419). Three treatments one each for control, 500 kg Sjha (applied 21 days before planting) and 2 kgjha sequestrene 138 iron chelate, which showed definite gradations of chlorosis were selected from all
510
P. N. MATHUR, H. S. DUNGARWAL, H. G. SINGH and M. S. SAROHA
the three replications. The crop was planted in March, 1971. Young upper leaves of four month old plants were used for analysis. The leaves from control plots at this stage showed severe symptoms of chlorosis and were pale yellow in appearance, though necrosis had not begun. Nitrogen content was estimated by the method of MITOHELL (1972), phosphorus by Vanadatemolybdate yellow colour method (USDA hand book No. 60), sulphur by the turbidimetric method (TABATABAI and BRE:VIMER 1970), iron by the orthophenanthroline method (CHAPMAN and PRATT 1961) and free amino acids by the ninhydrin method. Chlorophyll was determined by the method of AROXON (1944), peroxidase activity by o-dianisidine method of SHANNON et al. (1971) and catalase activity by the method of VON EULER and JOSEPHSON (1927). Protein was estimated by the method of LOWRY et al. (1951).
Results
When compared with control, an application of sulphur increased cane yield by 24.7% whereas that of Fe-EDDHA increased it only by 10.8%. The Fe-EDDHA application reduced phosphorus content by 33 % as compared to control but did not affect nitrogen, sulplmr, iron and free amino acids. The application of sulphur had no effect on nitrogen, phosphorus and free amino acid content of leaves but reduced iron content by 60% and increased sulphur content by 23% (table 1). Table 1. Effect of sulphur and Fe-EDDHA application on cane yield, elemental composition and free amino acids of sugarcane leaves Fe (ppm)
Free amino acid (%)
Treatments
Yield (qj50 sq m)
N (%)
P (%)
S (%)
Control
4.4
1.88
0.18
0.48
779
1.56 1.52 1.52
Sulphur
5.53
1.90
0.15
0.59
314
Fe-EDDHA
4.97
1.86
0.12
0.49
719
±
0.036 0.124
0.029
0.010
0.01
34.5
0.071
NS
0.040
0.04
67.7
XS
SEm
LSD at 5% NS
=
F test not significant
Chlorophyll content of leaves from plants that received sulphur or iron tleatment was 11 to 12 times of that in chlorotic leaves. The green leaves contained 10 times more c!ilorophyll a and If) times more chlorophyll b as compared to chlorotic leaves. The chlorophyll content of sulphur and iron treated plants did not differ significantly (table 2). The catalase and peroxidase activity was increased by 76 and 71 per cent respectively by sulphur application and 90 and 100 per cent with Fe-EDDHA application. The aconitase activity was not altered significantly by either of the treatments (table 2).
511
Effect of Sulphur and Iron-EDDHA on Chlorophyll Synthesis usw.
Table 2. Effect of sulphur and Fe- EDDHA application on chlorophyll content, and enzymatic activities of sugarcane leaves Treatments
Chlorophyll
Total b (mg/g (mg/g (mg/g fresh weight) fresh weight) fresh weight)
Catalase tug H 2 0 2 / min/mg protein)
Peroxidase (.dOD 460/ min/mg protein)
Aconitase (.dOD 240/ min/mg protein)
0.10 0.92 1.06 0.12 0.48
2.57 4.52 4.89 0.17 0.66
6.00 10.28 12.03 0.20 0.77
2.48 2.96 2.78 0.38 NS
11
Control Sulphur Fe-EDDIIA SEm ± CD at 5 % Nfl
=
0.04 0.56 0.55 0.08 0.30
0.14 1.48 1.61 0.20 0.77
F test not significant
Discussion
Chlorophyll and the haeme portion of enzymes catalase and peroxidase are derived from the same tetrapyrrole biosynthetic chain upto the protoprophyrin stage. }iARSH et al. (1963a) showed that haeme content and the activity of catalase was reduced concomittant with a decreased chlorophyll, in iron deficiency chlorosis of cowpea. In the present study the activity of haematin enzymes catalase and peroxidase was increased together with an increase in the chlorophyll content by both sulphur and iron treatments. Since iron and sulphur treatments did not differ in the magnitude of their effect on chlorophyll and haeme synthesis it appears that iron is the element which is limiting the synthesis of both metalloporphyrins. However, as sulphur treatment reduces the iron content drastically, it is apparent that the iron content per se is not the determining factor in the normal synthesis of prophyrins. A reduced availability of this element at the site of prophyrin biosynthesis may therefore be the cause of chlorosis. The yield of sugarcane is increased much more by sulphur than chelated iron showing that the former may potentiate other physiological processes besides chlorophyll and prophyrin synthesis that contribute to the overall grcwth of the plants. MARSH et al. (1963 b) showed that iron deficiency did not effect the rate of synthesis of chlorophyll from C-14 labelled b-amino levulinic acid, though the incorporation of labelled succinate and
512
P. N. MATHUR, H. S. DUNGARWAL, H. G. SINGH and M. S. SAROHA
ILJIN (1951) reported that protein synthesis in chlorotic leaves of cowpea plants was reduced as evidenced by an increase in its free amino acid content. However, we did not observe any difference in the free amino acid content of chlorotic and non chlorotic leaves. The exact mechanism by which the physiological availability of iron for porphyrin synthesis is reduced, is not yet clear. It appears that excess of some other element (Ca ?) either reduces the intracellular permeability of this element or competes with it for the same reactive site, an effect that is counteracted by excess sulphur.
Acknowledgements Sequestrene 138 Fe was generously supplied by Ciba-Geigy, Easle, Switzerland.
References ARNON, D. I., Copper enzymes in isolated chloroplasts-polyphenol oxidase in B. vulgaris. Plant Physiol. 24, 1-15 (1949). CHAPMAN, H. D., and PRATT, P. F., Methods of analysis for soils, plants and waters. University of California, Division of Agricultural Sciences. 1961. lLJIN, W. S., Metabolism of plants affected with lime induced chlorosis (calciose) I. Nitrogen metabolism. Plant and Soil 3, 239-256 (1951). LOWRY, D. H., ROSENBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with folin phenol reagent. J. BioI. Chern. 193, 265 (1951). MARSH, H. V. Jr., EVANS, H. J., and MALTRONE, G., (a) Investigation of the role of iron in chlorophyll metabolism I. Effect of iron deficiency on chlorophyll and haeme content and on the activity of certain enzymes in leaves. Plant Physiol. 38, 632-638 (1963). - - - (b) Investigation of the role of iron in chlorophyll metabolism. II. Effect of iron deficiency on chlorophyll synthesis. Plant Physiol. 39, 638-642 (1963). MITCHELL, H. L., Micro determination of nitrogen in plant tissue. Jour. A.O.A.C. 00, 1-3 (1972). SAGLIO, P., Iron nutrition of grapes, I. Test for inducing iron chlorosis with a combination of bicarbonate and orthosphate in two strains: one susceptible and other resistant. Ann. Physiol. Veg. 11, 27-31 (French) (1969). cf. Chern. Abstr. 73, 87000-R, (1970). SHANNON, L. M., URITANI, L., and IMASEKI, De novo synthesis of peroxidase isoenzymes in sweet potato slices. Plant Physiol. 47, 493-498 (1971). SINGH, H. G., How to control chlorosis in peas. Indian Fmg. 16, 17-18 (1966). - SINGH, R. M., Role of iron in preventing the death of rice seedlings in nursery. Indian J. Agron. 11, 310-311 (1966). - GUPTA, P. C., Nature and control of chlorosis in paddy seedlings on calcareous soil. Indian J. Agric. Sci. 38, 714-719 (1968). SINGH, H. G., Effect of sulphur in preventing the occurrence of chlorosis in peas. Agron. J. 62, 708-711 (1970). - Sulphur application prevents chlorosis and insures large crop yield on alkaline calcareous soils. Indian Fmg. 20, 21-23 (1971).
Effect of Sulphur and Iron-EDDHA on Chlorophyll Synthesis usw.
513
TARATABAI, ~1. A., and BREMNER, J. M., A turbidimetric method of determining sulphur in plant materials. Agron. J. 62,806-808 (1970). VON EULER, and JOSEPHSON, K., Described by Chance, B. and Maehly. Catalase. In methods in Enzymology Vol. II by COLOWICK and KAPLAN (ed.) 1927 779. WALLACE, A., Do iron chlorotic leaves contain more iron than green leaves? In "Regulation of the micronutrient stutus of plants by chelating agents and other factors" by WALLACE, A. (ed.). University of California, Los Angeles, California 1971 a, 194-196. Organic acids and activities of some enzymes in leaves and roots of iron deficient and iron sufficient corn plants. In "Regulation of the micronutrients status of plants by chelating agents and other factors", by WALLACE, A. (ed.). University of California, Los Angeles, California 1971 b, 106-116. ZECH, W., Study of lime induced chlorosis in Pinus sylvestris by analysis of needles. Z. Pflanzenerhaehr. Bodenk. 125, 1-16 (German) d. Chern. Abstr. 73, 55178b (1970). Authors' address: Dr. P. N. MATHUR, H. S. DUNGRAWAL, H. G. SINGH and M. S. SAROHA, University of Udaipur, Rajasthan College of Agriculture, Department of Agronomy, Udaipur (India).
Effect of Sulphur and Iron-EDDHA on Chlorophyll Synthesis, Iron Enzymes and Crop Yield of Sugarcane By P. N. MATHUR, H. S. DUNGARWAL, H. G. SINGH and M. S. SAROHA (Received February 14, 1973)
Summary A field experiment was conducted to study the effect of sulphur and iron on iron chlorosis of sugarcane (Saccharum officinarum L.) grown on calcareous soil. Sulphur and iron treatments increased the chlorophyll content, and catalase and peroxidase activity several times that of chlorotic leaves while the aconitase activity remained unaffected. While both treatments reduced the phosphorus content only sulphur treatment decreased the iron content significantly. Chlorosis appears to be due to a reduced physiological availability of iron due either to a reduced permeability of intracellular membranes or competition with some other element for the same reactive site.
Introduction
Rice, sugarcane, peanut, peas and certain other legumes when grown on calcareous soils develop severe symptoms of chlorosis resembling iron deficiency. SINGH (1966) and SINGH and SINGH (1966) reported partial prevention of chlorosis by repeated application of iron chelates. Subsequently it was shown (SINGH and GUPTA 1968) that application of elemental sulphur to soil was a much more effective means of preventing chlorosis and that the chlorotic leaves contained much more iron than green leaves (SINGH 1970 and SINGH 1971). Recently ZECH (1970), SAGLIO (1969) and WALLACE (1971) also reported that chlorotic leaves from plants grown on calcareous soils contained more iron than green leaves. SINGH (1970) proposed that the chlorosis in these soils could be due to a physiological unavailability of iron in the plants. In this paper evidence has been presented in favour of this hypothesis. Material and methods The experimental material for this study forms part of a replicated trial comparing efficacy of different sources of sulphur and iron in sugarcane (Saccharum officinarum L. Var. Co 419). Three treatments one each for control, 500 kg Sjha (applied 21 days before planting) and 2 kgjha sequestrene 138 iron chelate, which showed definite gradations of chlorosis were selected from all
510
P. N. MATHUR, H. S. DUNGARWAL, H. G. SINGH and M. S. SAROHA
the three replications. The crop was planted in March, 1971. Young upper leaves of four month old plants were used for analysis. The leaves from control plots at this stage showed severe symptoms of chlorosis and were pale yellow in appearance, though necrosis had not begun. Nitrogen content was estimated by the method of MITOHELL (1972), phosphorus by Vanadatemolybdate yellow colour method (USDA hand book No. 60), sulphur by the turbidimetric method (TABATABAI and BRE:VIMER 1970), iron by the orthophenanthroline method (CHAPMAN and PRATT 1961) and free amino acids by the ninhydrin method. Chlorophyll was determined by the method of AROXON (1944), peroxidase activity by o-dianisidine method of SHANNON et al. (1971) and catalase activity by the method of VON EULER and JOSEPHSON (1927). Protein was estimated by the method of LOWRY et al. (1951).
Results
When compared with control, an application of sulphur increased cane yield by 24.7% whereas that of Fe-EDDHA increased it only by 10.8%. The Fe-EDDHA application reduced phosphorus content by 33 % as compared to control but did not affect nitrogen, sulplmr, iron and free amino acids. The application of sulphur had no effect on nitrogen, phosphorus and free amino acid content of leaves but reduced iron content by 60% and increased sulphur content by 23% (table 1). Table 1. Effect of sulphur and Fe-EDDHA application on cane yield, elemental composition and free amino acids of sugarcane leaves Fe (ppm)
Free amino acid (%)
Treatments
Yield (qj50 sq m)
N (%)
P (%)
S (%)
Control
4.4
1.88
0.18
0.48
779
1.56 1.52 1.52
Sulphur
5.53
1.90
0.15
0.59
314
Fe-EDDHA
4.97
1.86
0.12
0.49
719
±
0.036 0.124
0.029
0.010
0.01
34.5
0.071
NS
0.040
0.04
67.7
XS
SEm
LSD at 5% NS
=
F test not significant
Chlorophyll content of leaves from plants that received sulphur or iron tleatment was 11 to 12 times of that in chlorotic leaves. The green leaves contained 10 times more c!ilorophyll a and If) times more chlorophyll b as compared to chlorotic leaves. The chlorophyll content of sulphur and iron treated plants did not differ significantly (table 2). The catalase and peroxidase activity was increased by 76 and 71 per cent respectively by sulphur application and 90 and 100 per cent with Fe-EDDHA application. The aconitase activity was not altered significantly by either of the treatments (table 2).
511
Effect of Sulphur and Iron-EDDHA on Chlorophyll Synthesis usw.
Table 2. Effect of sulphur and Fe- EDDHA application on chlorophyll content, and enzymatic activities of sugarcane leaves Treatments
Chlorophyll
Total b (mg/g (mg/g (mg/g fresh weight) fresh weight) fresh weight)
Catalase tug H 2 0 2 / min/mg protein)
Peroxidase (.dOD 460/ min/mg protein)
Aconitase (.dOD 240/ min/mg protein)
0.10 0.92 1.06 0.12 0.48
2.57 4.52 4.89 0.17 0.66
6.00 10.28 12.03 0.20 0.77
2.48 2.96 2.78 0.38 NS
11
Control Sulphur Fe-EDDIIA SEm ± CD at 5 % Nfl
=
0.04 0.56 0.55 0.08 0.30
0.14 1.48 1.61 0.20 0.77
F test not significant
Discussion
Chlorophyll and the haeme portion of enzymes catalase and peroxidase are derived from the same tetrapyrrole biosynthetic chain upto the protoprophyrin stage. }iARSH et al. (1963a) showed that haeme content and the activity of catalase was reduced concomittant with a decreased chlorophyll, in iron deficiency chlorosis of cowpea. In the present study the activity of haematin enzymes catalase and peroxidase was increased together with an increase in the chlorophyll content by both sulphur and iron treatments. Since iron and sulphur treatments did not differ in the magnitude of their effect on chlorophyll and haeme synthesis it appears that iron is the element which is limiting the synthesis of both metalloporphyrins. However, as sulphur treatment reduces the iron content drastically, it is apparent that the iron content per se is not the determining factor in the normal synthesis of prophyrins. A reduced availability of this element at the site of prophyrin biosynthesis may therefore be the cause of chlorosis. The yield of sugarcane is increased much more by sulphur than chelated iron showing that the former may potentiate other physiological processes besides chlorophyll and prophyrin synthesis that contribute to the overall grcwth of the plants. MARSH et al. (1963 b) showed that iron deficiency did not effect the rate of synthesis of chlorophyll from C-14 labelled b-amino levulinic acid, though the incorporation of labelled succinate and
512
P. N. MATHUR, H. S. DUNGARWAL, H. G. SINGH and M. S. SAROHA
ILJIN (1951) reported that protein synthesis in chlorotic leaves of cowpea plants was reduced as evidenced by an increase in its free amino acid content. However, we did not observe any difference in the free amino acid content of chlorotic and non chlorotic leaves. The exact mechanism by which the physiological availability of iron for porphyrin synthesis is reduced, is not yet clear. It appears that excess of some other element (Ca ?) either reduces the intracellular permeability of this element or competes with it for the same reactive site, an effect that is counteracted by excess sulphur.
Acknowledgements Sequestrene 138 Fe was generously supplied by Ciba-Geigy, Easle, Switzerland.
References ARNON, D. I., Copper enzymes in isolated chloroplasts-polyphenol oxidase in B. vulgaris. Plant Physiol. 24, 1-15 (1949). CHAPMAN, H. D., and PRATT, P. F., Methods of analysis for soils, plants and waters. University of California, Division of Agricultural Sciences. 1961. lLJIN, W. S., Metabolism of plants affected with lime induced chlorosis (calciose) I. Nitrogen metabolism. Plant and Soil 3, 239-256 (1951). LOWRY, D. H., ROSENBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with folin phenol reagent. J. BioI. Chern. 193, 265 (1951). MARSH, H. V. Jr., EVANS, H. J., and MALTRONE, G., (a) Investigation of the role of iron in chlorophyll metabolism I. Effect of iron deficiency on chlorophyll and haeme content and on the activity of certain enzymes in leaves. Plant Physiol. 38, 632-638 (1963). - - - (b) Investigation of the role of iron in chlorophyll metabolism. II. Effect of iron deficiency on chlorophyll synthesis. Plant Physiol. 39, 638-642 (1963). MITCHELL, H. L., Micro determination of nitrogen in plant tissue. Jour. A.O.A.C. 00, 1-3 (1972). SAGLIO, P., Iron nutrition of grapes, I. Test for inducing iron chlorosis with a combination of bicarbonate and orthosphate in two strains: one susceptible and other resistant. Ann. Physiol. Veg. 11, 27-31 (French) (1969). cf. Chern. Abstr. 73, 87000-R, (1970). SHANNON, L. M., URITANI, L., and IMASEKI, De novo synthesis of peroxidase isoenzymes in sweet potato slices. Plant Physiol. 47, 493-498 (1971). SINGH, H. G., How to control chlorosis in peas. Indian Fmg. 16, 17-18 (1966). - SINGH, R. M., Role of iron in preventing the death of rice seedlings in nursery. Indian J. Agron. 11, 310-311 (1966). - GUPTA, P. C., Nature and control of chlorosis in paddy seedlings on calcareous soil. Indian J. Agric. Sci. 38, 714-719 (1968). SINGH, H. G., Effect of sulphur in preventing the occurrence of chlorosis in peas. Agron. J. 62, 708-711 (1970). - Sulphur application prevents chlorosis and insures large crop yield on alkaline calcareous soils. Indian Fmg. 20, 21-23 (1971).
Effect of Sulphur and Iron-EDDHA on Chlorophyll Synthesis usw.
513
TARATABAI, ~1. A., and BREMNER, J. M., A turbidimetric method of determining sulphur in plant materials. Agron. J. 62,806-808 (1970). VON EULER, and JOSEPHSON, K., Described by Chance, B. and Maehly. Catalase. In methods in Enzymology Vol. II by COLOWICK and KAPLAN (ed.) 1927 779. WALLACE, A., Do iron chlorotic leaves contain more iron than green leaves? In "Regulation of the micronutrient stutus of plants by chelating agents and other factors" by WALLACE, A. (ed.). University of California, Los Angeles, California 1971 a, 194-196. Organic acids and activities of some enzymes in leaves and roots of iron deficient and iron sufficient corn plants. In "Regulation of the micronutrients status of plants by chelating agents and other factors", by WALLACE, A. (ed.). University of California, Los Angeles, California 1971 b, 106-116. ZECH, W., Study of lime induced chlorosis in Pinus sylvestris by analysis of needles. Z. Pflanzenerhaehr. Bodenk. 125, 1-16 (German) d. Chern. Abstr. 73, 55178b (1970). Authors' address: Dr. P. N. MATHUR, H. S. DUNGRAWAL, H. G. SINGH and M. S. SAROHA, University of Udaipur, Rajasthan College of Agriculture, Department of Agronomy, Udaipur (India).