5 STUDIES ON THE SULPHUR METABOLISM OF PLANTS II. THE EFFECT OF NITROGEN SUPPLY ON THE AMOUNTS OF PROTEIN SULPHUR, SULPHATE SULPHUR AND ON THE VALUE OF THE RATIO OF PROTEIN NITROGEN TO PROTEIN SULPHUR IN LEAVES AT DIFFERENT STAGES DURING THE LIFE CYCLE OF THE PLANT BY B. S. BARRIENi & J. G. WOOD From the Department of Botany, University of Adelaide, South Australia (With figures in the text) INTRODUCTION IN a previous paper (Wood & BarHen, 199) an experiment was described wherein the amounts of sulphur fractions were determined in plants subjected to increasing doses of ammonium salts. In this experiment the ratio of protein nitrogen to protein sulphur showed a tendency to increase with increased ammonium treatment; but the amount of variation in both quantities was so small that, with the method of analysis employed, it could not be said with certainty that such change in the ratio was significant. Therefore we decided to determine the value of this ratio at stages during the life cycle of a plant and to employ a more accurate method for determining protein sulphur. We are greatly indebted to Dr A. H. K. Petrie of the Waite Research Institute who placed at our disposal dried material of leaves of Sudan grass {Andropogon sudanensis, Leppan and Bosman) which had been used in an experiment concerned with the relation of ontogeny to nutrition (Ballard & Petrie, 196; Petrie, 19; Williams, 196, 198). EXPERIMENTAL The leaf material was obtained from plants subjected to three different nitrogen treatments; seven harvests from each treatment were taken during the life cycle of the plant. Details of the experimental procedure have been described by Ballard & Petrie (196). In brief, the three treatments were: treatment I, 0-5 g. NaNOg per pot; treatment II, -5 g, NaN0 per pot; treatment III, -50 g. per pot. Other nutrients, including sulphate, were supplied at constant initial concentration. The first harvest * Research Assistant under Federal Governnnent Research Grant to Austrahan Universities,
58 B. S. BARRIEN and J. G. WOOD took place 9 days after planting and before the nutrients were applied; subsequent harvests took place 1,, 0, 55, 6, and 10 days after planting respectively. The analytical procedure was as follows: A weighed amount, not exceeding g. of the carefully dried and finely ground leaf material was extracted with 100 ml. distilled water at 60 C. for i hr., after which it was boiled for 5 min. The mixture was cooled to 0 C. and 0 ml. of a % tannin solution in 0-5 % HCl added. The mixture was allowed to stand for 15 min. and then filtered and the residue washed with I % tannin solution. Sulphate sulphur was determined in the filtrate by precipitation with BaClj after acidifying with HCl. The residue was dried and, including the filter paper, pressed into a compact pellet which was then ignited in oxygen at 00 lb. pressure in an Emerson calorimeter bomb. After cooling, the bomb was thoroughly washed out and the resulting solution evaporated, after addition of bromine water, to a volume of about 100 ml. After filtration, appropriate dilution and acidification, the sulphate formed during the oxidation was precipitated with BaCla. All weighings were carried out on materials and vessels allowed to equilibrate with air in the balance case. Duplicate analyses of the leaf material all agreed within i %. RESULTS The results of analysis have been expressed as absolute values (grams of substance per plant) and as relative values (percentage of dry matter). These are given in Tables I and II and are expressed graphically in Figs, i and. In addition, relevant figures for protein nitrogen and amount of dry matter from Petrie's data (19) are given in Tables I and II. Table I. Absolute amounts* of dry-matter protein sulphur, protein nitrogen, sulphate sulphur afid water in leaves Treatment I Mean dry wt. per plant SOiS 6 0-00 0-051 0-88 0-1-6 1-65 1-9 0-066 0-96 -95 1-01 16-6 10-96 -6 0-0068 0-06 O-55S 1-0-0010 O-OI 1 0-11 0- t O-OI 1 0-19 1-0 -i8-19 -65 0-89 Treatment II Mean dry wt. per plant SOS 1 0-00 0-0 0-9 I-000-5 - - 0-066 0-9 9-5 9-80 50-1- 1-8 0-0068 0-05 0-60 1-9 -66-1-88 0-0010 00108 0-1 0-0 0-0 0-61 0-65 O-OI 1 0-119 1-6 -1 8-01 5- -6
Studies on the sulphur metabolism of plants 59 Mean dry wt. per plant 0-00 0-0199 0-58 0-98 -8-61 -0 Table I {continued) 0-066 0-0 8-6 8-9 66-8 61- -6 Treatment III 0-0068 0-0 0-59 i-8i -5-95 -0 SOS 0-0010 0-0085 0-08 0-60 0-55 0-0-6 * Amounts of water in grams, of other fractions in milligrams, per plant, -f Insufficient material for analysis. 0-011 0-099 1-6 -15 8-9- -9 Table II. Percentage amounts of protein sulphur, protein nitrogen, sulphate sulphur and water* in leaves Treatment I SOS Ratio -90-8 -6-0 i-io 0-66 O- 0-160 0-8 019 0-158 0-05 t 0-05 0-05 0-0 0-0 t 91 51 90 06 5 160 6 18-1 15-5 1-1-9 1-5 Treatment II SOS Ratio -90-99 -19-98 1-65 1-0-6 0-160 0-0-06 0-19 0-10 0-108 0-069 0-05 0-06 0-0 0-0 0-0 0-018 0-0 91 509 98 1 68 05 9 18-1 16-15-5 15-1- 11-9-5-90 -8-8 -0-1-1 1-1 0-160 0-18 0-05 0-195 0-158 0-1 0-09 Treatment III SOS 0-05 0-0 009 0-08 0-019 0-0 0-0 91 96 90 99 19 * The amounts for water are the amounts associated with 100 g. dry weight, f Insufficient material for analysis. Ratio 18-1 1-16-0 15-15-0 1-5 1-
6o B. S. BARRIEN and J. G. WOOD Absolute drifts Protein sulphur (Fig. i). Protein sulphur in eacb treatment rises to a maximum value and thereafter declines. The most striking feature of the curves for amount of protein sulphur plotted against time is that they follow the trend of the curve for amount of dry matter. Time in days Fig. 1. Absolute amounts of sulphate sulphur, dry matter, protein sulphur, and protein nitrogen of total leaves per plant. From data of Table I. E indicates times of exsertion of inflorescences and F time of flowering. Although the form of the curve for amount of protein sulphur resembles in a general way that for protein nitrogen, yet the positions of the maxima are not coincident except in the case of treatment II. In treatments I and III the maximum for protein sulphur is later than for protein nitrogen.
Studies on the sulphur metabolism of plants 61 Inorganic sulphate sulphur (Fig. i). The curves for amount of sulphate sulphur plotted against time follow in each case the trends of the curves for amounts of dry matter, water and protein sulphur. Relative drifts Protein sulphur (Fig. ). The curves for amount of protein sulphur resemble those for protein nitrogen; the amount of protein sulphur reaches a maximum at Time in days Time in days Fig.. Amounts of sulphate sulphur, protein nitrogen, protein sulphur, and water in grams per 100 grams dry matter, from data of Table II. E indicates times of exsertion of inflorescences and F time of flowering. harvest and thereafter declines. The percentage decrease in protein sulphur with time is less than in the case of protein nitrogen. The close connexion between synthesis of protein sulphur and nitrogen supply is also seen at harvest of the highest nitrogen treatment: protein sulphur, like protein nitrogen and also dry matter, is lower in amount than in the other treatments owing to a temporary reduction in growth. New Phyt. XXXVIII, '
6 B. S. BARRIEN and J. G. WOOD Inorganic sulphate sulphur (Fig. ). In treatments I and II the percentage of sulphate sulphur shows little change in harvests 1-, thereafter it decreases. In treatment III there is a slow fall in value between harvests 1- and thereafter a more rapid fall. Protein nitrogen/protein sulphur ratios. These are illustrated in Fig.. The values for this ratio show two definite trends. First, the value of the ratio increases with treatment and second, the value of the ratio decreases with time in each treatment. Time in davs c o 0 ISEATMENT 1 c c o Fig.. Values of the ratio of protein nitrogen to protein sulphur plotted against time. From data of Table II. DISCUSSION The relation of protein sulphur to protein nitrogen and to the amounts of dry matter and water The increase in amount of protein sulphur at each harvest with increased nitrogen treatment (Figs, i, ) when the initial amount of sulphate supplied remains unchanged confirms the results already described by Wood & Barrien (199). In explanation of the increase in value of the protein nitrogen/protein sulphur ratio with treatment, which is evident at each harvest, we are confronted with two alternatives. Either there is only one protein which varies in constitution or there is more than one protein, each with a different sulphur content, and the relative proportions of these proteins change with treatment. The latter alternative appears the more likely, for Foreman (198) and Bawden & Pirie (198) have demonstrated the existence of several proteins with distinct physical properties in leaves of normal plants. The explanation we advance is that with increased nitrogen treatment one or more proteins, relatively rich in sulphur, increase in amount, but at the same time are diluted with one or more other proteins containing relatively less suphur.
Studies on the sulphur metabolism of plants 6 For the present this explanation applies only to the special case of Sudan grass. It is possible that in some species the ratio of protein nitrogen to protein sulphur may decrease in value with treatment owing to dilution of a protein with another containing relatively more sulphur. It is also possible, as in the case of vicilin and legumelin, reserve proteins of the pea seed, that the dilutant may contain only a very small amount of sulphur in the molecule. A further possibility is that both diluting and diluted proteins may have the same sulphur content. From this viewpoint the decrease in value with time of the protein nitrogen/ protein sulphur ratio in Sudan grass could be ascribed to the more rapid decrease in the sulphur-poor protein compared with the sulphur-rich protein. It is tempting in the present case to consider the sulphur-rich protein as the more stable cytoplasmic protein and the sulphur-poor protein as a reserve protein. This idea receives some support from the data for the absolute amounts of protein sulphur and protein nitrogen and their relation to the amount of dry matter. The trends of the curves for protein sulphur follow those for amounts of dry matter and the positions of the maxima are coincident, whereas the maximum value for protein nitrogen is attained earlier than that of protein sulphur. If protein sulphur is regarded as a measure of the cytoplasmic protein, then increase in dry matter might be expected to be proportional to increase in protein sulphur. It is obvious, however, that all that can be said with certainty is that amount of protein sulphur is more highly correlated with amount of dry matter than is protein nitrogen. Of the factors contributing to decrease in amount of protein sulphur the more important appear to be formation of the inflorescences {vide Fig. i), decrease in water content (Petrie & Wood, 198) and, possibly, depletion of the external supply of nitrogen: it is not probable that the supply of sulphate was depleted to such an extent that it became limiting, since at harvest the amount of sulphate on a dryweight basis had fallen to only one-half of the amount originally present. Inorganic sidphate sitlphur Rate of growth of the leaves is probably the greatest single factor determining the amount of sulphate sulphur present in the leaves. The absolute amount of sulphate sulphur is greater the greater the dry weight of the leaves. In absence of data for stems and roots no certainty can be reached, but the decline in percentage content of sulphate sulphur is probably due to a partial depletion of the external supply which would bring about a decline in the rate of import of sulphate and consequently a decline in the percentage sulphate content. SUMMARY This paper describes the changes in amounts of protein sulphur and of sulphate sulphur during the life cycle of grass plants {Andropogon sttdanensis Leppan & Bosman) which received three different initial supplies of nitrogen, the initial supply of sulphate remaining the same. Increased nitrogen supply caused an increase in the amount of protein sulphur. As in the case of protein nitrogen, the highest nitrogen treatment caused at first a 1-
6 B. S. BARRIEN and J. G. WOOD depression in amount of protein sulphur due to an effect of treatment on growth rate. The absolute content of protein sulphur rises to a maximum and thereafter decreases. The position of this maximum is coincident with the maxima for amounts of dry matter and of water, but is not coincident with the maximum for protein nitrogen which is attained earlier. On a relative basis, after an initial rise, the amount of protein sulphur decreases throughout the life cycle, although the rate of decrease is less rapid than that of protein nitrogen. The latter effect is seen in the value of the ratio of protein nitrogen to protein sulphur. This ratio decreases in amount throughout the life cycle of the plant, and at any harvest is higher the higher the nitrogen treatment. It is suggested that with increased nitrogen treatment, protein sulphur increases in amount but at the same time is diluted with a protein containing relatively less sulphur; and also that the latter protein is utilized more rapidly within the plant than is the sulphur-rich protein. Various factors contributing to the decrease in amount of protein sulphur are discussed. REFERENCES BALLARD, L. A. T. & PETRIE, A. H. K. (196). Aust. J. exp. Biol. med. Sci. 1. BAWDEN, F. C. & PiRiE, N. W. (198). J. exp. Path. 19. FOREMAN, F. W. {198). J. agric. Sci. 8. PETRIE, A. H. K. (19). Aust. J. exp. Biol. med. Sci. 15. PETRIE, A. H. K. & WOOD, J. G. (198). Ann. Bot., Lond., N.S.. WILLIAMS, R. F. (196). Aust. J. exp. Biol. med. Sci. 1. (198). Aust. J. exp. Biol. med. Sci. 16. WOOD, J. G. & BARRIEN, B. S. (199). Neiv Phytol. 8.