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Calcium in Relation to Nitrogen Metabolism
Biochem. Physiol. Pflanzen (BPP), Bd. 164, S. 547-565 (1973) Botany Department, Allahabad University, Allahabad, India
Calcium in Relation to Nitrogen Metabolism II. Changes in Free Amino Acids and Amides in Peanut Plants
By R. N. PAL and M. M. LALORAYA With 12 figures (Received March 23, 1973)
Summary The effect of high as well as low levels of calcium on the free amino-acids and amides content in different plant parts of peanut, Arachis hypogaea was investigated. Low levels of calcium cause accumulation of amino acids and amides in peanut plants. However, the effect varies from harvest to harvest and in different parts of the plant. Thus while at first harvest in roots, ex-alanine and y-aminobutyric acid are the chief amino acids to accumulate but at second harvest asparagine accumulation is also observed. In stems, the major amino acids to accumulate at the first harvest are y-aminobutyric acid and glutamic acid but at second harvest ex-alanine and glutamine also accumulate. Arginine accnmulation is observed only in case of leaves. The accumulation of arginine, asparagine, ex-alanine and glutamic acid is observed even at the fourth harvest when most of the amino-acids show depletion. In general, amino acids were less at high levels of calcium as compared with control plants.
Introduction
Mineral deficiencies in plants frequently lead to marked increase in the concentration of free amino-acids and amides. STEINBERG et al. (1956) observed that deficiencies of calcium, magnesium or potassium caused increase in the concentration of free (X-amino nitrogen ranging from double to nearly seven fold in the leaves of tobacco and they have suggested that several of the symptoms of mineral disorders could be assigned to the accumulation of toxic concentrations of certain amino compounds of which L-hydroxyproline and L( +)-isoleucine were shown to be outstandingly toxic. L( +)-Isoleucine and hydroxyproline especially produced severe mottling, necrosis, strap shaped leaves and rosetting. PLESHKOV and FOWDEN (1959) found that asparagine and glutamine increased with calcium deficiency in barley leaves. FREIBERG and STEWARD (1960) reported that in banana plant amide glutamine accumulated in the younger calcium deficient leaves. Tso and McMURTREY (1960) reported that free amino-acids notably aspartic acid, glutamic acid and their amides; proline and serine accumulated in calcium deficient tobacco plants. It is 37
Biochem. Physiol. Pflanzen, Bd. 164
548
R. N. PAL and M. M. LALORAYA
evident that the effects seem to vary in different plants and subject to very important factors inherent in different experimental conditions and also differ in various deficiency conditions. In our previous communication (PAL and LALORAYA 1973), we have reported the effect of calcium levels on the protein and soluble-nitrogen content of peanut and linseed plants. In the present investigation an attempt has been made to study the effects of high as well as low levels of calcium on the free amino-acid composition of peanut plants. Material and Methods The plants of peanut (Arachis hypogaea) var. Big Japan were grown in sand culture as described elsewhere (PAL and L.UORA YA 1972). ARNON and HOAGLANDS (1940) nutrient solution was used in the present investigation. The plants were grown on following levels of calcium. Ca-1: Ca-2: Ca-3: Ca-4 (Control): Ca-5: Ca-6: Ca-7: Ca-8:
0.1 M 0.03 M 0.01 M 0.003 M 0.001 M 0.0003 M 0.0001 M Minus calcium
)
Calcium chloride in addition to Ca(N0 3 )2 which is present in the normal control solution Ca(N0 3 )2 Ca(N0 3 )2 + 0.004 M NaN0 3 Ca(N0 3 )2 + 0.0054 M NaN0 3 Ca(N0 3 )2 + 0.0058 M NaN0 3 + 0.006 M NaN0 3
The plants were harvested at four different growth intervals viz., 25, 40, 55 and 70 days of growth. The alcoholic extract of different plant parts of peanut was made and free amino-acids were analyzed by the two dimensional ascending paper chromatographic technique (PAL and LALORAYA 1967). Whatman No.1 filter paper was used in the present investigation. Phenol (80 % in double distilled water) saturated with 0.5 % ammonia solution was used as the first running solvent, whereas n-butanol, acetic acid and water (4: 1: 5) was the second solvent. The dried chromatograms were sprayed with 0.1 % ninhydrin and the various amino-acid spots were developed by heating the chromatograms in an oven at 80°C for 30 minutes. The amino acids were identified on the basis of their Rf values and colour reactions in different solvent systems. The quantitative estimation of individual amino acids was done by the colorimetric methods. The data are expressed in terms of glycine.
Results
The free amino acids present in different parts of the peanut plant and the changes in individual amino acids at different harvests are shown in figs. 1 to 12. A. First Harvest (25 days) The results are shown in figs. 1 to 3. In roots, a slight accumulation of free amino acids at low calcium levels is observed but the accumulation is most marked in case of IX-alanine and asparagine. However, there is slight decrease of amino acids at high calcium levels but ami des asparagine and y-methyleneglutamine show slight increase as compared to the control plants.
549
Calcium in Relation to Nitrogen Metabolism etc.
In stems, the amino-acids and amides show much more fluctuations at different levels of calcium. Leucines and phenylalanine show slight depletion at hig'h calcium levels, but it is more or less same at 10 IV calcium levels as compared to the control. Valine and y-aminobutyric acid show accumulation while arginine shows decrease both at high and low calcium levels. x Alanine does not show much change. Glutamic acid and aspartic acid show slight decrease at high calcium levels but its concentration is somewhat more in lower calcium levels. Asparagine shows depletion both at high and low calcium levels but at complete calcium deficiency, i. e., Ca-8 it shows some
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a.ccumulation, while the other amide glutamine shows accumulation both at high and low calcium levels. y-Methyleneglutamine, the third amide known to occur in this plant, shows accumulation at high calcium levels but decreases at low calcium levels. y-Methyleneglutamicacid shows some accumulation both at high and low calcium levels. In leaves, the amino-acids in general show accumulation at low calcium levels and depletion at high calcium levels, but iX-alanine, arginine, glycine and serine show slight accumulation at higher calcium levels too. Though the amino-acids valine, ~
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551
Calcium in Relation to Nitrogen Metabolism etc.
y-aminobutyric acid, tyrosine and aspartic acid show accumulation at low calcium levels, their concentration is less at minus calcium level, i. e., Ca-8 as compared to the control plants. y-Methyleneglutamine shows depletion both at high and low calcium levels. This is in contrast to the behaviour of the other two amides asparagine and glutamine, which show depletion at high calcium levels and accumulation at low calcium levels. B. Second Harvest (40 days) The results are shown in figs. 4 to 6. In roots, amino-acids in general show decrease at high calcium levels but show slight accumulation at low calcium levels. ~
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The accumulation at low calcium level is most marked in case of asparagine and y-aminobutyric acid. The concentration of arginine is less both at high and low calcium levels. It is interesting to note that asparagine, the only amino-acid amide present in root, shows accumulation both at high and low calcium levels. In stem also, free amino-acids in general are depleted at high calcium levels but accumulate at low calcium levels. The depletion at high calcium levels is most marked in case of ,x-alanine, glutamic acid and valine. The amino-acid arginine and the amide glutamine show accumulation both at high calcium levels as well as in low calcium
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Fig. 4. Changes in asparagine, glutamine, aspartic acid and glutamic acid at different levels of calcium in peanut plants at second harvest (40 days).
553
Calcium in Relation to Nitrogen Metabolism etc.
levels. y-Aminobutyric acid also shows some accumulation at high calcium levels but at the highest calcium level, i. e., Ca-l, it shows lesser amount as compared to the control set. In leaves too, similar pattern of changes in free amino-acids and amides is observed as in case of stems. The amino-acids in general decrease in high calcium levels but show increase in low calcium levels. However, all the amino-acids do not show similar increase in their content at low calcium levels. The accumulation is most marked in case of iX-alanine, glutamic acid, aspartic acid and the amide glutamine. ;;
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C. Third Harvest (55 days) The results are shown in figs. 7 to 9. In roots, there is in general a decrease of free amino-acids and amides both at high and low calcium levels, but valine, yaminobutyric acid and glycine & serine show slight accumulation at low calcium levels. The depletion at low as well as at high calcium levels is most marked in case of ami des asparagine and glutamine and amino-acids, glutamic acid, aspartic acid and arginine. In stems also, the same pattern of amino-acid changes is observed, i. C., the decrease both at high and low calcium levels. The decrease of amino-acids at high "0
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Calcium in Relation to Nitrogen Metabolism etc.
calcium levels is most marked in case of leucines & phenylalanine, valine, ex-alanine, glutamic acid, aspartic acid and asparagine, whereas the depletion at low calcium levels is pronounced in case of ex-alanine, arginine and the ami des asparagine and glutamine. In leaves, however, free amino-acids and almdes show decrease at high calcium levels but show slight accumulation at low calcium levels. The depletion of aminoacids at high calcium level is most marked only in case of ex-alanine, glutamic acid, aspartic acid and the amide asparagine, while the accumulation at low calcium level
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is marked in case of leucines & phenylalanine, valine and iX-alanine. In contrast to other amino-acids, which show some accumulation, glycine & serine show decrease in their content at low calcium levels. D. Fourth Harvest (70 days) The results are shown in figs. 10 to 12. In roots, there is a general decrease of free amino-acids and amides at high calcium levels while at low calcium levels marked fluctuations are observed. The decrease of amino-acids at low calcium levels is
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557
Calcium in Relation to Nitrogen Metabolism etc.
observed in case of leucines & phenylalanine, glutamic acid, aspartic acid and glycine & serine. Valine and y-aminobutyric acid show little or no change. iX-Alanine shows accumulation at low calcium levels but at complete calcium deficient set, i. e., Ca-8, it shows decrease in its content as compared to the control plants. Amides glutamine and asparagine show slight accumulation at low calcium levels. In the stems also, there is general depletion of free amino-acids and amides at high calcium levels. At low calcium levels amino-acids show fluctuations in their contents, whereas leucines & phenylalanine, y-aminobutyric acid, iX-alanine, arginine, and the amides asparagine and glutamine show decrease in their content; glutamic acid, aspartic acid and glycine & serine show increase in their content as compared to the control plants. However, it is interestmg to note that glutamic acid and aspartic acid show accnmulation both at high calcium levels as well as at low calcium levels. In the leaves, at the fourth harvest also, there is decrease of free amino-acids and ami des at high calcium levels except for valine, (x-alanine and aspartic acid, which show accumulation. At low calcium levels, there is general accumulation of amino-acids and amides but leucines & phenylalanine, and glycine & serine show decrease in their content. The accumulation is most marked in case of iX-alanine, glutamic acid, aspartic acid, arginine and amide asparagine. In pods and pegs at the fourth harvest, there is general accumulation of free amino-acids at high calcium levels, but the accumulation is most marked in
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558
R. N. PAL and M. M.
LALORAYA
case of glutamic acid, aspartic acid, tyrosine and IX-alanine. The amide, asparagine and glycine & serine, threonine and valine show little or no change at higher calcium levels. The accumulation of amino-acids at low calcium levels is due to y-aminobutyric acid, tyrosine, aspartic acid and the amides asparagine and glutamine, while the other amino-acids, e. g., leucines & phenylalanine, IX-alanine, glutamic acid, threonine, arginine and glycine & serine show decrease in their content as compared to the control plants. The analysis of free amino-acids and ami des could not be done at complete calcium deficiency, i. e., Ca-8 due to lack of sufficient pegs. It has been
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559
Calcium in Relation to Nitrogen Metabolism etc.
shown that pod formation is completely inhibited at complete calcium deficiency conditions (PAL 1970). Pegs do not develop into normal pods as in case of control, and plants of higher calcium levels.
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Fig. 12. Changes in arginine and glycine & serine at different levels of calcium in peanut plants at fourth harvest (70 days).
Discussion
In general, mineral deficiencies are known to cause accumulation of free amino acids and amides in plants and often of some specific amino acids. However, the effect seems to vary from plant to plant. A good deal of work has been done in the recent years on the effects of micro- as well as macro-nutrient deficiencies on the amino acid composition of a large number of plant species. The results of some of these studies are summarized in table 1. It is clear from these studies that both macro as well as micronutrient deficiencies cause an accumulation of free amino acids and amides in plants but a critical study of their data brings out the fact that in most cases the accumulation was mainly due to amino acid arginine and ami des asparagine or glutamine, or in certain cases both. Low levels of calcium cause accumula.tion of amino acids and amides in peanut plants. The effect varies from harvest to harvest however, and in different parts of the plant. Thus while at the first harvest in roots, ex-alanine and y-aminobutyric acid are the chief amino acids to accumulate with no indication of asparagine accumulation; at the second harvest asparagine accumulation is also observed. Similarly in stem, the major amino acids to accumulate at the first harvest are y-aminobutyric
Calcium in Relation to Nitrogen Metabolism etc.
561
acid and glutamic acid but at the second harvest iX-alanine and glutam'ne also accumulate. Arginine accumulation is observed only in case of leaves. At the first harvest along with arginine, iX-alanine and glutamic acid also show accumulation but at the second harvest a large number of amino acids and ami des accumu'ate in large quantities. Among these are the amides glutamine and asparagine. The accumulation of arginine, asparagine, iX-alanine and glutamic acid are observed even at the fourth harvest when most of the other amino acids show depletion. In most of the earlier reports only leaves have been analysed and only at the time after deficiency symptoms had appeared. The results with the leaves of peanut, is, therefore, comparable to the other reports in the literat.ure. The accumulation chiefly of amino acid arginine and amides asparagine and glutamine in most of the plants studied under different. mineral deficiencies suggest that accumulation of these compounds is not due to specific requirement of these minerals for their utilization but rather due to some general effect of the mineral deficiency. Working on the effect of phosphorus deficiency on the free amino acid content of certain leguminous plants, RANJAN et al. (1962) observed that arginine accumulation was detectable when the deficiency effects on growth, i. e., retardation of growth, was observed and that the degree of accumulation increased with the advancing age of the plant. It was suggested that accumulation of arginine was associated with restricted growth of the plant. rather than any effect of the mineral on its utilization and conversion. This is also supported by the present observation. Arginine is known to be a major component of the free amino acid pool in many legumes and FOWDEN (1954) has reported its presence in Arachis hypogaea seeds. But as the seeds germinate, the growing axes predominantly contain the amides which make bulk of soluble nitrogen pool, while arginine is not traceable in the growing axis. Depletion of any substance with growth may indicate their utilization, while accumulation is a manifestation of formation exceeding utilizat.ion of the compound. It cannot be said, however, that. synthesis or accumulation of amides is associated with the vigorous growth of the tissue, for in leaves undergoing senescence and breakdown of proteins, amides asparagine and glutamine are also known to accumulate in large quantities. Accumulation of amides asparagine and glutamine may, therefore, be due to the enhanced breakdown of protein in mineral deficient plants coupled with their poor ut.ilization in the growth of the plants. Indeed amides asparagine and glutamine and the amino acid arginine are recognized as store house of available nitrogen for the growth and metabolic activities of the growing plant (RANJAN et al. 1962). It will be observed that while the total soluble nitrogen declines under calcium deficiency (PAL and LALORAYA 1973), some amino acids show a tendency to accumulate. It must be noted, however, that all t.he amino acids do not accumulate,
L
562
R. N. PAL and M. M. LALORAYA
Table 1
Effects of mineral deficiencies on the accumulation of free amino-acids and amides in plants
Mineral deficiency/Plant
Amino-acids accumulated
Refereces
A. Macron u trien t
1. Calcium Banana Barley Mint Tobacco
Turnip
Glutamine and glutamic acid. FREIBERG and STEWARD (1960) Asparagine and glutamine. PLESHKOV and FOWDEN (1959) Asparagine in long days and STEWARD et al. (1959) glutamine in short days. Asparagine, Glutamine, aspartic Tso and McMURTREY (1960) acid, glutamic acid, proline and serine. Glutamine and glycine. THOMPSON and MORRIS (1956)
2. Magnesium Banana
Potato Tobacco
3. Phosphorus Alfalfa Banana Legumes Linseed Turnip
Glutamine in young leaves and asparagine & pipe colic acid in older leaves. Arginine, asparagine, threonine, leu cines & phenylalanine. Asparagine and proline
Arginine, asparagine and glutamine. Aspartic acid, glutamic acid & glutamine. Arginine and asparagine. Arginine Glutamine, isoleucine and proline
FREIBERG and STEWARD (1960)
MULDER and BAKEMA (1956) Tso and McMURTREY (1960)
GLEITER and PARKER (1957) FREIBERG and STEWARD (1960) RANJAN et al. (1962) RANJAN and MALAVIYA (1962) THOMPSON and MORRIS (1956)
4. Potassium Barley Potato Tobacco
Arginine, asparagine, glutamine, RICHARDS and BERNER (1954) leu cines and phenylalanine MULDER (1949) Free tyrosine Tso and McMURTREY (1960) Asparagine
5. Sulphur Alfalfa
Arginine and aspartic acid
Desmodium Flax Tomato White clover
Arginine, glycine and Serine Arginine Arginine Arginine
MERTZ et al. (1952) MERTZ and MATSUMOTO (1956) COLEMAN (1957) -do-do-do-
Calcium in Rebtion to Nitrogen Metabolism etc.
563
Fortsetzung Tabelle 1
B. Micronutrient 1. Boron
Tobacco
Asparagine and tyramine
STEINBERG (1956)
Arginine and asparagine Aspartic acid, glutamic acid, proline and ex-alanine
STEINBERG et al. (1956) POSSINGHAM (1956)
2. Copper Tobacco Tomato
3. :Manganese C,tuliflower
Tobacco Tomato
Arginine, asparagine, glutamine, HEWITT et al. (1949) aspartic acid, glutamic acid, ex-alanine & proline. STEINBERG (1956) Arginine and asparagine Aspartic acid POSSINGHAM (1956)
4. Molybdenum Tobacco
Lysine
STEINBERG et al. (1956)
Arginine Arginine and asparagine Asparagine Asparagine and glutamine Arginine, asparagine and glutamine. Asparagine and glutamine
HOLLEY and CAIN (1955) DEKoCK and MORRISON (1958) STEWARD et al. (1959) STEINBERG (1956) Tso and McMURTREY (1960)
Arginine, asparagine and glutamine. Arginine, asparagine and glutamine.
Tso and McMURTREY (1960)
5. Iron Blue berry 1iIustard 1iIint Tobacco Tobacco Tomato
POSSINGHAM (1956)
6. Zinc Tobacco Tomato
POSSINGHAM (1956)
several reduce ill quantities and this would explain the low values of soluble nitrogen in spite of accumulation of certain individual amino acids. Also, the changes in mtrogenous compounds other than amino acids, like urea which undergo changes during deficiency and toxicity conditions, may also contribute to this observed pattern. The effect of calcium ion on growth of the plant may be related also to its controlling effect on the uptake of water and other elements like potassium and nitrate, which are dependent on calcium. Removal of calcium would also restrict the uptake of potassium which is essential for maintaining the hydration state of cells and also 38 Biochem. Physiol. Pflanzen, Bd. 164
564
R. N. PAL and M. M. LALORAYA
has a role in protein synthesis. Likewise, excess of calcium would inhibit water uptake directly by altering the permeability of the cell, which is well known (Cf. STOCKING 1956). The restricted entry of water into the plant may thereby exert an overall inhibitory effect on growth. Even during water deficit, metabolic processes are altered in a fashion very similar to that observed in mineral deficient plants (BANERJI and LA LORA YA 1962). Breakdown of proteins and nucleic acids, and accumulation of free amino acids during water deficit eonditions are now well documented (GATES and BONNER 1960; CHEN et al. 1964). It is tller!'fore, likely that many of the effects of mineral deficiency and toxicity may be related to such general effects as the hydration state of the cells imposed by the ionic imbalance inside the root cells.
Acknowledgement This research was supported by the grant from U.S. PL-480 scheme No. FG-In. 232, which is thankfully acknowledged.
Literature ARNON, D. 1., and HOAGLAND, D. R., Crop production in soil with special reference to factors influencing yield and absorption of inorganic nutrients. Soil Sci. 50, 463 (1940). BANERJI, D., and LALORAYA, M. M., Amino-acid metabolism in Phaseolus radiatus plants under condition of reduced water uptake. Proc. 49th Ind. Sci. Congo Session, Cuttak (INDIA): Abst.1962. CHEN, D., KESSLER, B., and MONS ELISE, S. P., Studies on water regime and nitrogen metabolism of citrus seedlings grown under water stress. Plant Physiol. 39, 379 (1964). COLEMAN, R. G., The effect of sulphur deficiency on the free amino acids of some plants. Aust. J. BioI. Sci. 10, 50 (1957). DEKoCK, P. C., and MORRISON, R. 1., The metabolism of chlorotic leaves. I-Amino acids. Biochem. J. 70, 266 (1958). FOWDEN, L., The nitrogen metabolism of groundnut plants: The role of y-methyleneglutamine and y-methyleneglutamic acid. Ann. Bot. 18, 417 (1954). FREIBERG, S. R., and STEWARD, F. C., Physiological investigations on the banana plant. IIIFactors which affect the nitrogen compounds of the leaves. Ann. Bot. 24, 247(1960). GATES, C. T., and BONNER, J., Effect of water stress on RNA metabolism. Plant PhysioI. 34, 49 (1960). GLEITER, M. E., and PARKER, H. E., The effect of phosphorus deficiency on the amino acids of alfalfa. Arch. Biochem. Biophys. 71, 430 (1957). HEWITT, E. J., JONES, E. W., and WILLIAMS, A. H., Relation of molybdenum and manganese to the free amino acids of the cauliflower. Nature HlS, 681 (1949). HOLLEY, R. W., and CAIN, J. C., Accumulation of arginine in plants affected with iron deficiency type chlorosis. Science 121, 172 (1955). MERTZ, E. T., SINGLETON, V. L., and GAREY, C. L., The effects of sulphur deficiency on the aminoacids of alfalfa. Arch. Biochem. 38, 139 (1952). - MATSUMOTO, H., Further studies on the amino acids and proteins of sulphur deficient alfalfa. Arch. Biochem. Biophys. 63, 50 (1956).
Calcium in Relation to Nitrogen Metabolism etc.
565
MULDER, E. G., Mineral nutrition in relation to the biochemistry and physiology of potatoes. Plant and Soil 2, 59 (1949). - BAKEMA, K., The effect of the nitrogen, phosphorus, potassium and magnesium nutrition of potato plants on the content of free amino acids and on the amino-acid composition of the protein of the tubers. Plant and Soil 7, 135 (1956). PAL, R. N., Studies on effects of calcium on growth and metabolic activities of certain crop plants. PH. D. THESIS, Allahabad Univ., Allahabad, India 1970. - LALORAYA, M. M., Nitrogen metabolism of the Tamarindus indica: Changes in y-methyleneglutamine and y-methyleneglutamic acid during seedling growth. Physio!. Plant. 20, 789 (1967). - - Effect of calcium levels on chlorophyll synthesis in peanut and linseed plants. Biochem. Physio!. Pflanzen (BPP) HIS, 443 (1972). - - Calcium in relation to nitrogen metabolism. I-Changes in protein and soluble-nitrogen in peanut and linseed plants. Biochem. Physiol. Pflanzen (BPP) 164, 315 (1973). PLESHKOV, B. P., and FOWDEN, L., Amino acid composition of the proteins of barley leaves in relation to mineral nutrition and age of plants. Nature 183, 1445 (1959). POSSINGHAM, J. V., The effect of mineral nutrition on the content of free amino acids and amides in tomato plants. I - A comparison of effects of deficiencies of copper, zinc, manganese, iron and molybdenum. Aust. J. Bio!. Sci. 9, 539 (1956). RANJAN, S., and MALAVIYA, B., Effect of phosphorus deficiency on the free and protein bound amino acids of the linseed plant. Flora 152, 399 (1962). - PANDE, R. M., SRIVASTAVA, R. K., and LALORAYA, M. M., Effect of phosphorus deficiency on the metabolic changes in free amino-acids in certain leguminous crop plants. Nature 193, 997 (1962). RICHARDS, F. J., and BERNER, E., Physiological studies in plant nutrition. XVII. A general survey of the free amino acids of barley as affected by mineral nutrition with special reference to potassium supply. Ann. Bot. 18, 15 (1954). STEINBERG, R. A., Metabolism of inorganic nitrogen by plants. In "Inorganic Nitrogen Metabolism", Eds. McELROY, W. D., and GLASS, B., JOHN HOPKINS Maryland 1956, 153-158. - BOWLING, J. D., and McMURTREY, J. E. JR., Accumulation of amino acids as a chemical basis for physiological symptoms in tobacco manifesting frenching and mineral deficiency symptoms. Plant Physio!. 25, 279 (1956). STEWARD, F. C., CRANE, F., MILLER, K., ZACHARIUS, R. M., RABSON, R., and MARGOLIS, D., Nutritional and environmental effects on the nitrogen metabolism of plants. In "Utilization of nitrogen and its compounds by plants". Symposia Soc. Expt. Bio!. 13, 148 (1959). STOCKING, C. R., Hydration and Cell physiology. Encyclo. Plant Physio!. 2, 22 (1956). Ed. RUHLAND, W., Germany. THOMPSON, J. F., and MORRIS, C. J., The effect of potassium deficiency on the amino acid composition of turnips. Plant Physio!. 31, (Supp!.). IX. Tso, T. C., and McMURTREY, J. E. JR., Mineral deficiency and organic constituents in tobacco plants. II - Amino acids. Plant Physio!. 35, 865 (1960). Authors' address: Dr. R. N. PAL, Regional Fruit Research Station, Punjab Agricultural University, ABORAR 152116, and Dr. M. M. LALORAYA, Botany Department, University School of Sciences, Gujarat University, Ahmedabad-9 (INDIA).
38*
Calcium in Relation to Nitrogen Metabolism II. Changes in Free Amino Acids and Amides in Peanut Plants
By R. N. PAL and M. M. LALORAYA With 12 figures (Received March 23, 1973)
Summary The effect of high as well as low levels of calcium on the free amino-acids and amides content in different plant parts of peanut, Arachis hypogaea was investigated. Low levels of calcium cause accumulation of amino acids and amides in peanut plants. However, the effect varies from harvest to harvest and in different parts of the plant. Thus while at first harvest in roots, ex-alanine and y-aminobutyric acid are the chief amino acids to accumulate but at second harvest asparagine accumulation is also observed. In stems, the major amino acids to accumulate at the first harvest are y-aminobutyric acid and glutamic acid but at second harvest ex-alanine and glutamine also accumulate. Arginine accnmulation is observed only in case of leaves. The accumulation of arginine, asparagine, ex-alanine and glutamic acid is observed even at the fourth harvest when most of the amino-acids show depletion. In general, amino acids were less at high levels of calcium as compared with control plants.
Introduction
Mineral deficiencies in plants frequently lead to marked increase in the concentration of free amino-acids and amides. STEINBERG et al. (1956) observed that deficiencies of calcium, magnesium or potassium caused increase in the concentration of free (X-amino nitrogen ranging from double to nearly seven fold in the leaves of tobacco and they have suggested that several of the symptoms of mineral disorders could be assigned to the accumulation of toxic concentrations of certain amino compounds of which L-hydroxyproline and L( +)-isoleucine were shown to be outstandingly toxic. L( +)-Isoleucine and hydroxyproline especially produced severe mottling, necrosis, strap shaped leaves and rosetting. PLESHKOV and FOWDEN (1959) found that asparagine and glutamine increased with calcium deficiency in barley leaves. FREIBERG and STEWARD (1960) reported that in banana plant amide glutamine accumulated in the younger calcium deficient leaves. Tso and McMURTREY (1960) reported that free amino-acids notably aspartic acid, glutamic acid and their amides; proline and serine accumulated in calcium deficient tobacco plants. It is 37
Biochem. Physiol. Pflanzen, Bd. 164
548
R. N. PAL and M. M. LALORAYA
evident that the effects seem to vary in different plants and subject to very important factors inherent in different experimental conditions and also differ in various deficiency conditions. In our previous communication (PAL and LALORAYA 1973), we have reported the effect of calcium levels on the protein and soluble-nitrogen content of peanut and linseed plants. In the present investigation an attempt has been made to study the effects of high as well as low levels of calcium on the free amino-acid composition of peanut plants. Material and Methods The plants of peanut (Arachis hypogaea) var. Big Japan were grown in sand culture as described elsewhere (PAL and L.UORA YA 1972). ARNON and HOAGLANDS (1940) nutrient solution was used in the present investigation. The plants were grown on following levels of calcium. Ca-1: Ca-2: Ca-3: Ca-4 (Control): Ca-5: Ca-6: Ca-7: Ca-8:
0.1 M 0.03 M 0.01 M 0.003 M 0.001 M 0.0003 M 0.0001 M Minus calcium
)
Calcium chloride in addition to Ca(N0 3 )2 which is present in the normal control solution Ca(N0 3 )2 Ca(N0 3 )2 + 0.004 M NaN0 3 Ca(N0 3 )2 + 0.0054 M NaN0 3 Ca(N0 3 )2 + 0.0058 M NaN0 3 + 0.006 M NaN0 3
The plants were harvested at four different growth intervals viz., 25, 40, 55 and 70 days of growth. The alcoholic extract of different plant parts of peanut was made and free amino-acids were analyzed by the two dimensional ascending paper chromatographic technique (PAL and LALORAYA 1967). Whatman No.1 filter paper was used in the present investigation. Phenol (80 % in double distilled water) saturated with 0.5 % ammonia solution was used as the first running solvent, whereas n-butanol, acetic acid and water (4: 1: 5) was the second solvent. The dried chromatograms were sprayed with 0.1 % ninhydrin and the various amino-acid spots were developed by heating the chromatograms in an oven at 80°C for 30 minutes. The amino acids were identified on the basis of their Rf values and colour reactions in different solvent systems. The quantitative estimation of individual amino acids was done by the colorimetric methods. The data are expressed in terms of glycine.
Results
The free amino acids present in different parts of the peanut plant and the changes in individual amino acids at different harvests are shown in figs. 1 to 12. A. First Harvest (25 days) The results are shown in figs. 1 to 3. In roots, a slight accumulation of free amino acids at low calcium levels is observed but the accumulation is most marked in case of IX-alanine and asparagine. However, there is slight decrease of amino acids at high calcium levels but ami des asparagine and y-methyleneglutamine show slight increase as compared to the control plants.
549
Calcium in Relation to Nitrogen Metabolism etc.
In stems, the amino-acids and amides show much more fluctuations at different levels of calcium. Leucines and phenylalanine show slight depletion at hig'h calcium levels, but it is more or less same at 10 IV calcium levels as compared to the control. Valine and y-aminobutyric acid show accumulation while arginine shows decrease both at high and low calcium levels. x Alanine does not show much change. Glutamic acid and aspartic acid show slight decrease at high calcium levels but its concentration is somewhat more in lower calcium levels. Asparagine shows depletion both at high and low calcium levels but at complete calcium deficiency, i. e., Ca-8 it shows some
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550
R. N.
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a.ccumulation, while the other amide glutamine shows accumulation both at high and low calcium levels. y-Methyleneglutamine, the third amide known to occur in this plant, shows accumulation at high calcium levels but decreases at low calcium levels. y-Methyleneglutamicacid shows some accumulation both at high and low calcium levels. In leaves, the amino-acids in general show accumulation at low calcium levels and depletion at high calcium levels, but iX-alanine, arginine, glycine and serine show slight accumulation at higher calcium levels too. Though the amino-acids valine, ~
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551
Calcium in Relation to Nitrogen Metabolism etc.
y-aminobutyric acid, tyrosine and aspartic acid show accumulation at low calcium levels, their concentration is less at minus calcium level, i. e., Ca-8 as compared to the control plants. y-Methyleneglutamine shows depletion both at high and low calcium levels. This is in contrast to the behaviour of the other two amides asparagine and glutamine, which show depletion at high calcium levels and accumulation at low calcium levels. B. Second Harvest (40 days) The results are shown in figs. 4 to 6. In roots, amino-acids in general show decrease at high calcium levels but show slight accumulation at low calcium levels. ~
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The accumulation at low calcium level is most marked in case of asparagine and y-aminobutyric acid. The concentration of arginine is less both at high and low calcium levels. It is interesting to note that asparagine, the only amino-acid amide present in root, shows accumulation both at high and low calcium levels. In stem also, free amino-acids in general are depleted at high calcium levels but accumulate at low calcium levels. The depletion at high calcium levels is most marked in case of ,x-alanine, glutamic acid and valine. The amino-acid arginine and the amide glutamine show accumulation both at high calcium levels as well as in low calcium
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553
Calcium in Relation to Nitrogen Metabolism etc.
levels. y-Aminobutyric acid also shows some accumulation at high calcium levels but at the highest calcium level, i. e., Ca-l, it shows lesser amount as compared to the control set. In leaves too, similar pattern of changes in free amino-acids and amides is observed as in case of stems. The amino-acids in general decrease in high calcium levels but show increase in low calcium levels. However, all the amino-acids do not show similar increase in their content at low calcium levels. The accumulation is most marked in case of iX-alanine, glutamic acid, aspartic acid and the amide glutamine. ;;
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C. Third Harvest (55 days) The results are shown in figs. 7 to 9. In roots, there is in general a decrease of free amino-acids and amides both at high and low calcium levels, but valine, yaminobutyric acid and glycine & serine show slight accumulation at low calcium levels. The depletion at low as well as at high calcium levels is most marked in case of ami des asparagine and glutamine and amino-acids, glutamic acid, aspartic acid and arginine. In stems also, the same pattern of amino-acid changes is observed, i. C., the decrease both at high and low calcium levels. The decrease of amino-acids at high "0
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555
Calcium in Relation to Nitrogen Metabolism etc.
calcium levels is most marked in case of leucines & phenylalanine, valine, ex-alanine, glutamic acid, aspartic acid and asparagine, whereas the depletion at low calcium levels is pronounced in case of ex-alanine, arginine and the ami des asparagine and glutamine. In leaves, however, free amino-acids and almdes show decrease at high calcium levels but show slight accumulation at low calcium levels. The depletion of aminoacids at high calcium level is most marked only in case of ex-alanine, glutamic acid, aspartic acid and the amide asparagine, while the accumulation at low calcium level
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is marked in case of leucines & phenylalanine, valine and iX-alanine. In contrast to other amino-acids, which show some accumulation, glycine & serine show decrease in their content at low calcium levels. D. Fourth Harvest (70 days) The results are shown in figs. 10 to 12. In roots, there is a general decrease of free amino-acids and amides at high calcium levels while at low calcium levels marked fluctuations are observed. The decrease of amino-acids at low calcium levels is
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557
Calcium in Relation to Nitrogen Metabolism etc.
observed in case of leucines & phenylalanine, glutamic acid, aspartic acid and glycine & serine. Valine and y-aminobutyric acid show little or no change. iX-Alanine shows accumulation at low calcium levels but at complete calcium deficient set, i. e., Ca-8, it shows decrease in its content as compared to the control plants. Amides glutamine and asparagine show slight accumulation at low calcium levels. In the stems also, there is general depletion of free amino-acids and amides at high calcium levels. At low calcium levels amino-acids show fluctuations in their contents, whereas leucines & phenylalanine, y-aminobutyric acid, iX-alanine, arginine, and the amides asparagine and glutamine show decrease in their content; glutamic acid, aspartic acid and glycine & serine show increase in their content as compared to the control plants. However, it is interestmg to note that glutamic acid and aspartic acid show accnmulation both at high calcium levels as well as at low calcium levels. In the leaves, at the fourth harvest also, there is decrease of free amino-acids and ami des at high calcium levels except for valine, (x-alanine and aspartic acid, which show accumulation. At low calcium levels, there is general accumulation of amino-acids and amides but leucines & phenylalanine, and glycine & serine show decrease in their content. The accumulation is most marked in case of iX-alanine, glutamic acid, aspartic acid, arginine and amide asparagine. In pods and pegs at the fourth harvest, there is general accumulation of free amino-acids at high calcium levels, but the accumulation is most marked in
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558
R. N. PAL and M. M.
LALORAYA
case of glutamic acid, aspartic acid, tyrosine and IX-alanine. The amide, asparagine and glycine & serine, threonine and valine show little or no change at higher calcium levels. The accumulation of amino-acids at low calcium levels is due to y-aminobutyric acid, tyrosine, aspartic acid and the amides asparagine and glutamine, while the other amino-acids, e. g., leucines & phenylalanine, IX-alanine, glutamic acid, threonine, arginine and glycine & serine show decrease in their content as compared to the control plants. The analysis of free amino-acids and ami des could not be done at complete calcium deficiency, i. e., Ca-8 due to lack of sufficient pegs. It has been
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559
Calcium in Relation to Nitrogen Metabolism etc.
shown that pod formation is completely inhibited at complete calcium deficiency conditions (PAL 1970). Pegs do not develop into normal pods as in case of control, and plants of higher calcium levels.
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Discussion
In general, mineral deficiencies are known to cause accumulation of free amino acids and amides in plants and often of some specific amino acids. However, the effect seems to vary from plant to plant. A good deal of work has been done in the recent years on the effects of micro- as well as macro-nutrient deficiencies on the amino acid composition of a large number of plant species. The results of some of these studies are summarized in table 1. It is clear from these studies that both macro as well as micronutrient deficiencies cause an accumulation of free amino acids and amides in plants but a critical study of their data brings out the fact that in most cases the accumulation was mainly due to amino acid arginine and ami des asparagine or glutamine, or in certain cases both. Low levels of calcium cause accumula.tion of amino acids and amides in peanut plants. The effect varies from harvest to harvest however, and in different parts of the plant. Thus while at the first harvest in roots, ex-alanine and y-aminobutyric acid are the chief amino acids to accumulate with no indication of asparagine accumulation; at the second harvest asparagine accumulation is also observed. Similarly in stem, the major amino acids to accumulate at the first harvest are y-aminobutyric
Calcium in Relation to Nitrogen Metabolism etc.
561
acid and glutamic acid but at the second harvest iX-alanine and glutam'ne also accumulate. Arginine accumulation is observed only in case of leaves. At the first harvest along with arginine, iX-alanine and glutamic acid also show accumulation but at the second harvest a large number of amino acids and ami des accumu'ate in large quantities. Among these are the amides glutamine and asparagine. The accumulation of arginine, asparagine, iX-alanine and glutamic acid are observed even at the fourth harvest when most of the other amino acids show depletion. In most of the earlier reports only leaves have been analysed and only at the time after deficiency symptoms had appeared. The results with the leaves of peanut, is, therefore, comparable to the other reports in the literat.ure. The accumulation chiefly of amino acid arginine and amides asparagine and glutamine in most of the plants studied under different. mineral deficiencies suggest that accumulation of these compounds is not due to specific requirement of these minerals for their utilization but rather due to some general effect of the mineral deficiency. Working on the effect of phosphorus deficiency on the free amino acid content of certain leguminous plants, RANJAN et al. (1962) observed that arginine accumulation was detectable when the deficiency effects on growth, i. e., retardation of growth, was observed and that the degree of accumulation increased with the advancing age of the plant. It was suggested that accumulation of arginine was associated with restricted growth of the plant. rather than any effect of the mineral on its utilization and conversion. This is also supported by the present observation. Arginine is known to be a major component of the free amino acid pool in many legumes and FOWDEN (1954) has reported its presence in Arachis hypogaea seeds. But as the seeds germinate, the growing axes predominantly contain the amides which make bulk of soluble nitrogen pool, while arginine is not traceable in the growing axis. Depletion of any substance with growth may indicate their utilization, while accumulation is a manifestation of formation exceeding utilizat.ion of the compound. It cannot be said, however, that. synthesis or accumulation of amides is associated with the vigorous growth of the tissue, for in leaves undergoing senescence and breakdown of proteins, amides asparagine and glutamine are also known to accumulate in large quantities. Accumulation of amides asparagine and glutamine may, therefore, be due to the enhanced breakdown of protein in mineral deficient plants coupled with their poor ut.ilization in the growth of the plants. Indeed amides asparagine and glutamine and the amino acid arginine are recognized as store house of available nitrogen for the growth and metabolic activities of the growing plant (RANJAN et al. 1962). It will be observed that while the total soluble nitrogen declines under calcium deficiency (PAL and LALORAYA 1973), some amino acids show a tendency to accumulate. It must be noted, however, that all t.he amino acids do not accumulate,
L
562
R. N. PAL and M. M. LALORAYA
Table 1
Effects of mineral deficiencies on the accumulation of free amino-acids and amides in plants
Mineral deficiency/Plant
Amino-acids accumulated
Refereces
A. Macron u trien t
1. Calcium Banana Barley Mint Tobacco
Turnip
Glutamine and glutamic acid. FREIBERG and STEWARD (1960) Asparagine and glutamine. PLESHKOV and FOWDEN (1959) Asparagine in long days and STEWARD et al. (1959) glutamine in short days. Asparagine, Glutamine, aspartic Tso and McMURTREY (1960) acid, glutamic acid, proline and serine. Glutamine and glycine. THOMPSON and MORRIS (1956)
2. Magnesium Banana
Potato Tobacco
3. Phosphorus Alfalfa Banana Legumes Linseed Turnip
Glutamine in young leaves and asparagine & pipe colic acid in older leaves. Arginine, asparagine, threonine, leu cines & phenylalanine. Asparagine and proline
Arginine, asparagine and glutamine. Aspartic acid, glutamic acid & glutamine. Arginine and asparagine. Arginine Glutamine, isoleucine and proline
FREIBERG and STEWARD (1960)
MULDER and BAKEMA (1956) Tso and McMURTREY (1960)
GLEITER and PARKER (1957) FREIBERG and STEWARD (1960) RANJAN et al. (1962) RANJAN and MALAVIYA (1962) THOMPSON and MORRIS (1956)
4. Potassium Barley Potato Tobacco
Arginine, asparagine, glutamine, RICHARDS and BERNER (1954) leu cines and phenylalanine MULDER (1949) Free tyrosine Tso and McMURTREY (1960) Asparagine
5. Sulphur Alfalfa
Arginine and aspartic acid
Desmodium Flax Tomato White clover
Arginine, glycine and Serine Arginine Arginine Arginine
MERTZ et al. (1952) MERTZ and MATSUMOTO (1956) COLEMAN (1957) -do-do-do-
Calcium in Rebtion to Nitrogen Metabolism etc.
563
Fortsetzung Tabelle 1
B. Micronutrient 1. Boron
Tobacco
Asparagine and tyramine
STEINBERG (1956)
Arginine and asparagine Aspartic acid, glutamic acid, proline and ex-alanine
STEINBERG et al. (1956) POSSINGHAM (1956)
2. Copper Tobacco Tomato
3. :Manganese C,tuliflower
Tobacco Tomato
Arginine, asparagine, glutamine, HEWITT et al. (1949) aspartic acid, glutamic acid, ex-alanine & proline. STEINBERG (1956) Arginine and asparagine Aspartic acid POSSINGHAM (1956)
4. Molybdenum Tobacco
Lysine
STEINBERG et al. (1956)
Arginine Arginine and asparagine Asparagine Asparagine and glutamine Arginine, asparagine and glutamine. Asparagine and glutamine
HOLLEY and CAIN (1955) DEKoCK and MORRISON (1958) STEWARD et al. (1959) STEINBERG (1956) Tso and McMURTREY (1960)
Arginine, asparagine and glutamine. Arginine, asparagine and glutamine.
Tso and McMURTREY (1960)
5. Iron Blue berry 1iIustard 1iIint Tobacco Tobacco Tomato
POSSINGHAM (1956)
6. Zinc Tobacco Tomato
POSSINGHAM (1956)
several reduce ill quantities and this would explain the low values of soluble nitrogen in spite of accumulation of certain individual amino acids. Also, the changes in mtrogenous compounds other than amino acids, like urea which undergo changes during deficiency and toxicity conditions, may also contribute to this observed pattern. The effect of calcium ion on growth of the plant may be related also to its controlling effect on the uptake of water and other elements like potassium and nitrate, which are dependent on calcium. Removal of calcium would also restrict the uptake of potassium which is essential for maintaining the hydration state of cells and also 38 Biochem. Physiol. Pflanzen, Bd. 164
564
R. N. PAL and M. M. LALORAYA
has a role in protein synthesis. Likewise, excess of calcium would inhibit water uptake directly by altering the permeability of the cell, which is well known (Cf. STOCKING 1956). The restricted entry of water into the plant may thereby exert an overall inhibitory effect on growth. Even during water deficit, metabolic processes are altered in a fashion very similar to that observed in mineral deficient plants (BANERJI and LA LORA YA 1962). Breakdown of proteins and nucleic acids, and accumulation of free amino acids during water deficit eonditions are now well documented (GATES and BONNER 1960; CHEN et al. 1964). It is tller!'fore, likely that many of the effects of mineral deficiency and toxicity may be related to such general effects as the hydration state of the cells imposed by the ionic imbalance inside the root cells.
Acknowledgement This research was supported by the grant from U.S. PL-480 scheme No. FG-In. 232, which is thankfully acknowledged.
Literature ARNON, D. 1., and HOAGLAND, D. R., Crop production in soil with special reference to factors influencing yield and absorption of inorganic nutrients. Soil Sci. 50, 463 (1940). BANERJI, D., and LALORAYA, M. M., Amino-acid metabolism in Phaseolus radiatus plants under condition of reduced water uptake. Proc. 49th Ind. Sci. Congo Session, Cuttak (INDIA): Abst.1962. CHEN, D., KESSLER, B., and MONS ELISE, S. P., Studies on water regime and nitrogen metabolism of citrus seedlings grown under water stress. Plant Physiol. 39, 379 (1964). COLEMAN, R. G., The effect of sulphur deficiency on the free amino acids of some plants. Aust. J. BioI. Sci. 10, 50 (1957). DEKoCK, P. C., and MORRISON, R. 1., The metabolism of chlorotic leaves. I-Amino acids. Biochem. J. 70, 266 (1958). FOWDEN, L., The nitrogen metabolism of groundnut plants: The role of y-methyleneglutamine and y-methyleneglutamic acid. Ann. Bot. 18, 417 (1954). FREIBERG, S. R., and STEWARD, F. C., Physiological investigations on the banana plant. IIIFactors which affect the nitrogen compounds of the leaves. Ann. Bot. 24, 247(1960). GATES, C. T., and BONNER, J., Effect of water stress on RNA metabolism. Plant PhysioI. 34, 49 (1960). GLEITER, M. E., and PARKER, H. E., The effect of phosphorus deficiency on the amino acids of alfalfa. Arch. Biochem. Biophys. 71, 430 (1957). HEWITT, E. J., JONES, E. W., and WILLIAMS, A. H., Relation of molybdenum and manganese to the free amino acids of the cauliflower. Nature HlS, 681 (1949). HOLLEY, R. W., and CAIN, J. C., Accumulation of arginine in plants affected with iron deficiency type chlorosis. Science 121, 172 (1955). MERTZ, E. T., SINGLETON, V. L., and GAREY, C. L., The effects of sulphur deficiency on the aminoacids of alfalfa. Arch. Biochem. 38, 139 (1952). - MATSUMOTO, H., Further studies on the amino acids and proteins of sulphur deficient alfalfa. Arch. Biochem. Biophys. 63, 50 (1956).
Calcium in Relation to Nitrogen Metabolism etc.
565
MULDER, E. G., Mineral nutrition in relation to the biochemistry and physiology of potatoes. Plant and Soil 2, 59 (1949). - BAKEMA, K., The effect of the nitrogen, phosphorus, potassium and magnesium nutrition of potato plants on the content of free amino acids and on the amino-acid composition of the protein of the tubers. Plant and Soil 7, 135 (1956). PAL, R. N., Studies on effects of calcium on growth and metabolic activities of certain crop plants. PH. D. THESIS, Allahabad Univ., Allahabad, India 1970. - LALORAYA, M. M., Nitrogen metabolism of the Tamarindus indica: Changes in y-methyleneglutamine and y-methyleneglutamic acid during seedling growth. Physio!. Plant. 20, 789 (1967). - - Effect of calcium levels on chlorophyll synthesis in peanut and linseed plants. Biochem. Physio!. Pflanzen (BPP) HIS, 443 (1972). - - Calcium in relation to nitrogen metabolism. I-Changes in protein and soluble-nitrogen in peanut and linseed plants. Biochem. Physiol. Pflanzen (BPP) 164, 315 (1973). PLESHKOV, B. P., and FOWDEN, L., Amino acid composition of the proteins of barley leaves in relation to mineral nutrition and age of plants. Nature 183, 1445 (1959). POSSINGHAM, J. V., The effect of mineral nutrition on the content of free amino acids and amides in tomato plants. I - A comparison of effects of deficiencies of copper, zinc, manganese, iron and molybdenum. Aust. J. Bio!. Sci. 9, 539 (1956). RANJAN, S., and MALAVIYA, B., Effect of phosphorus deficiency on the free and protein bound amino acids of the linseed plant. Flora 152, 399 (1962). - PANDE, R. M., SRIVASTAVA, R. K., and LALORAYA, M. M., Effect of phosphorus deficiency on the metabolic changes in free amino-acids in certain leguminous crop plants. Nature 193, 997 (1962). RICHARDS, F. J., and BERNER, E., Physiological studies in plant nutrition. XVII. A general survey of the free amino acids of barley as affected by mineral nutrition with special reference to potassium supply. Ann. Bot. 18, 15 (1954). STEINBERG, R. A., Metabolism of inorganic nitrogen by plants. In "Inorganic Nitrogen Metabolism", Eds. McELROY, W. D., and GLASS, B., JOHN HOPKINS Maryland 1956, 153-158. - BOWLING, J. D., and McMURTREY, J. E. JR., Accumulation of amino acids as a chemical basis for physiological symptoms in tobacco manifesting frenching and mineral deficiency symptoms. Plant Physio!. 25, 279 (1956). STEWARD, F. C., CRANE, F., MILLER, K., ZACHARIUS, R. M., RABSON, R., and MARGOLIS, D., Nutritional and environmental effects on the nitrogen metabolism of plants. In "Utilization of nitrogen and its compounds by plants". Symposia Soc. Expt. Bio!. 13, 148 (1959). STOCKING, C. R., Hydration and Cell physiology. Encyclo. Plant Physio!. 2, 22 (1956). Ed. RUHLAND, W., Germany. THOMPSON, J. F., and MORRIS, C. J., The effect of potassium deficiency on the amino acid composition of turnips. Plant Physio!. 31, (Supp!.). IX. Tso, T. C., and McMURTREY, J. E. JR., Mineral deficiency and organic constituents in tobacco plants. II - Amino acids. Plant Physio!. 35, 865 (1960). Authors' address: Dr. R. N. PAL, Regional Fruit Research Station, Punjab Agricultural University, ABORAR 152116, and Dr. M. M. LALORAYA, Botany Department, University School of Sciences, Gujarat University, Ahmedabad-9 (INDIA).
38*