Foliar application of elemental sulphur on metabolism of sulphur and nitrogen compounds in leaves of sulphur-deficient wheat

1991

Phytochemistry, Vol. 30, No. 3, pp. 729-732, Printed in Great Britain.

0031-9422/91 33.00+0.00 Pergamon Press plc

FOLIAR APPLICATION OF ELEMENTAL SULPHUR ON METABOLISM OF SULPHUR AND NITROGEN COMPOUNDS LEAVES OF SULPHUR-DEFICIENT WHEAT JACQUES LANDRY,

STEPHANIE

LEGRIS-DELAPORTE

and

FRANCOISE

FERRON

Laboratoire de Chimie Biologique, INRA, Centre de Biotechnologie Agro-Industrielle, 78850 Thiverval-Grignon, (Received in reoisedform

Key Word Index--Triticum deficiency.

IN

France

10 July 1990)

aestiuum; Gramineae; wheat leaf uptake; metabolism; elemental sulphur; sulphur

Abstract-Wheat seedlings grown in the presence and absence of sulphur, given as sulphate, were compared for their ability to take up elemental S applied to the third leaf and to incorporate it as it was or as metabolites (sulphate, oxidized and reduced glutathione, cysteine + cystine, methionine) or proteins. Sulphur application on S-deficient plants enhanced the extent and duration of S uptake, and induced some changes in metabolism of sulphur and nitrogen fractions to reduce drift effects caused by the deficiency.

INTRODUCTION

A small proportion (between 1 and 2%) of elemental sulphur, when applied in the micronized and “S-1abelled state on the third leaf of wheat, grown under normal conditions, was found to be absorbed and metabolized into sulphate, cysteine + cystine, methionine, glutathione and proteins [ 1,2]. The contribution to this phenomenon of microorganisms converting S into sulphate may be excluded. Bacteria use S mainly under anaerobic conditions [3]. They were not detected at the surface of treated leave when examined by scanning electron microscopy [l, 21. Moreover, the kinetic curve of S-incorporation, as determined from 4 hr after leaf treatment, displayed no lag, the occurrence of which would suggest S conversion into sulphate by bacteria followed by excretion of the latter and its uptake by host leaf [Z]. The preponderance of sulphate among 35S-labelled metabolites suggested a link between the concentration of this ion and the metabolism of elemental S, which prompted us to study the effects of sulphate deficiency. We compared the uptake and the fate of elemental S in the foliar system of wheat grown in the presence and absence of sulphate. The compositions of the free amino acid pool and amino acids incorporated into protein were also examined because they are claimed to be perturbed in leaf as well as in seed by S starvation [4-91. A preliminary report of these data has been presented in poster form

cm RESULTS AND DISCUSSION

The sulphate-deprived plants, as used in the present experiment, exhibited a physiological time lag from three to four days, as assessed by the emergence of third and fourth leaves, pale green foliage and pronounced decrease in leaf size (fr. wt lowered by 20-70%). In addition to these visual symptons, the S content of any leaf was found to be reduced in comparison with control plants. Thus,

contents (pg S per mg fr. wt) in leaves nos l-3 amounted to 0.39,0.23 and 0.62, respectively, for S-deprived plants, and 0.46, 0.44 and 0.80 for control plants. Changes pertaining to the 35S radioactivity incorporated, as a whole and as metabolites and proteins, by the third, fourth and first plus second leaves, originating from wheat plants grown in the presence or absence of sulphate after S application are shown in Table 1. Lipid S is not taken into consideration since it was found only in treated leaves with a percentage ranging from 0.2 to 3.1% of total recovered radioactivity. Foliar application of elemental 35S on the third leaf of S-control plants resulted in the appearance of radioactivity in leaves whether treated or not. The magnitude and variations of labelling during the course of the experiment were dependent on the leaf range and the basis used for expressing data. For the third leaf the radioactivity, either total or incorporated as protein into fresh matter or entire leaf, passed through a maximum at two days after application (DAA) while labelled metabolites showed a stability for the first two DAA then a decline. For the non-treated leaves the radioactivity, either total or detected as metabolites and proteins, increased with time. However, for the fourth leaf labelled metabolites per mg fr. wt declined between two and seven DAA. On the other hand, the rate of S-incorporation, defined as the percentage of radioactivity applied to the third leaf and recovered in lipid, metabolites and protein present in the bulk of foliar system, increased then remained stable from two DAA. The radioactivity decrease recorded for the third leaf between two and seven DAA reflects cessation of elemental 35S uptake (as shown by the stability of Sincorporation rate for the same period), associated with the translocation of S-metabolites throughout the nontreated leaves, and with the protein turnover (as revealed by the decline of labelled proteins). The dilution effect seems unlikely as leaf weight increased slightly from two DAA. In contrast, the decline in the content of labelled 729

J.

730

Table 1. Incorporation

et al.

LANDRY

of radioactivity from 35S into fresh matter (Bq mg-‘) and into leaves (kBq)* of wheat Control

1DAA

2DAA

S-deficient 7DAA

1DAA

7DAA

2DAA

a. Whole leaf 3 4 ll2.t

15.50 1.42 0.37

4.65 0.17 0.10

17.80 2.04 0.97

7.12 0.43 0.32

13.90 1.73 1.42

5.76 0.71 0.64

17.70 0.95 0.26

4.95 0.04 0.02

20.70 3.12 0.45

6.23 0.13 0.09

24.70 3.25 1.94

7.41 0.91 0.70

b. Metabolites 3 4 l/2

10.80 0.75 0.20

3.25 0.09 0.06

10.60 1.00 0.42

4.25 0.18 0.14

7.88 0.52 0.43

3.23 0.22 0.39

12.50 0.51 0.15

3.00 0.02 0.02

12.80 1.50 0.25

3.85 0.06 0.05

15.80 0.79 0.57

4.75 0.23 0.20

4.30 0.67 0.17

1.30 0.08 0.04

6.95 1.05 0.55

2.76 0.22 0.18

5.73 1.17 0.98

2.35 0.49 0.45

4.87 0.51 0.11

1.17 0.02 0.02

7.37 1.70 0.20

2.21 0.07 0.04

8.8) 2.33 1.37

2.66 0.68 0.50

c. Proteins 3 4 l/2 d. Incorporation rate %

1.16

1.42

1.45

1.49

1.51

2.87

*Values are the average of two experiments. tAverage value for first and second leaves.

Table 2. Effect of foliar application of elemental S and S deficiency on composition metabolites identified from alcohol-soluble fraction from third leaf of wheat* S-deficient

Control Metabolites

1DAA

2DAA

7DAA

1DAA

2DAA

7DAA

Elemental S so:Methionine Cystine + cysteine Glutathione Non-migrating compoundst Total

3.1 37.0 1.3 25.0 7.5 16.0 89.9

4.0 36.0 1.9 21.0 12.5 11.0 86.4

3.7 59.0 3.5 13.0 10.5 9.0 98.7

3.1 24.0 1.5 19.0 12.0 23.0 82.6

4.4 28.0 1.3 19.0 12.0 24.5 89.2

4.8 49.0 2.6 15.0 10.3 11.0 92.9

*Values are the average of two experiments. tcompounds at origin after chromatography

metabolites in the fresh matter of the fourth leaf is related to the emergence of the fifth leaf and hence, to the appearance of an additional sink, and to the dilution effect since leaf weight doubled between two and seven DAA. From a quantitative point of view the radioactivity, when expressed on a mg fr. wt basis or on a leaf basis, was higher in the third than in fourth leaf, the latter being more radioactive than the average of the first and second leaves. Therefore, the magnitude of S metabolism in nontreated leaves was dependent on their physiological age. Similar changes were observed in plants grown in the absence of S and treated with S. The only difference was concerned with the third leaf which continued to take up elemental 35S at seven DAA although the leaf weight remained fairly constant from two DAA. Comparison of S-control and S-deficient plants for analogous leaves at the same stage after S-application showed that the radioactivity incorporated into the fresh matter by Sdeficient plant was higher for the third leaf at any stage,

of

and electrophoresis (probably small proteins).

for the fourth leaf from two DAA and for first plus second leaves at seven DAA. This confirms the influence of leaf age on S-metabolism and the physiological lag of Sdeficient plants, also revealed by the rate of S-incorporation, which began to increase after two DAA reaching at seven DAA a value double that recorded for S-control plants. The distribution of S-metabolites for the third leaf as identified and evaluated from alcohol soluble material is given in Table 2. Oxidized and reduced glutathione, cysteine + cystine, immobile compounds and sulphate were by far the major metabolites, the preponderance of sulphate increasing with the time of leaf contact with S. Methionine and elemental S represented small percentages. Elemental S probably arose from its incomplete removal from leaf washing with chloroform and its solution by aqueous alcohol. It is noteworthy that Joyard et al. [ll] have detected the most stable form (S,) of elemental S in spinach chloroplasts when incubated with

Effects of foliage-applied S Table 3. Effect of foliar application of elemental S and S deficiency on composition of protein amino acids assayed from the free amino acid pool of the third leaf of wheat (nmol g- ’ fr. wt). Control

S-deficient

Amino acid

OS

+s

OS

+s

Asp Glu Asn Ser Gln Gly His Arg Thr Ala Pro Tyr Val Met CYS Be Leu Phe LYS Total

480 2080 3190 2260 1530 320 110 50 375 700 70 90 130 tr* 70 70 70 tr tr 11620

590 2420 5760 2190 3230 760 170 65 400 775 70 130 170 40 200 90 75 tr tr 17 175

890 5990 30 580 3570 4700 2200 270 80 610 800 75 140 200 tr tr 135 105 tr tr 51250

880 2800 11130 2540 2615 505 120 50 430 890 70 90 120 40 70 70 80 tr tr 22 530

*tr: Traces.

35SO:-. Sulphur starvation did not markedly alter the distribution of metabolites which underwent the same variation as for S-control plants from two DAA. At seven DAA, sulphate which remained the most abundant metabolite had a percentage lower, but a level per mg fr. wt per leaf higher than that found for S-control leaves. The same was observed by Friedrich and Schrader following S deprivation in maize seedlings [12]. The composition of protein amino acids quantified from the free amino acid pool of the third leaf also reflected the effects of S-application and S-starvation at 1DAA (Table 3). About 78% of the total free amino acids of S-control plants was found as glutamic acid, asparagine, glutamine and serine. Sulphur application did not alter this percentage, but increased the total free amino acid concentration of fresh matter by doubling the content of glycine, by trebling that of cystine + cysteine and by allowing one to quantify the methionine level. Sulphur starvation amplified the preponderance of the four major amino acids (86%) and promoted a large accumulation of all other amino acids, except methionine, cystine + cysteine, phenylalanine and lysine which were at non quantitatively detectable levels. Accordingly the contents of aspartic and glutamic acids, and serine had increased two-fold, asparagine by lo-fold, glutamine by three-fold and glycine by seven-fold. Sulphur-application reduced the starvation drift effect markedly as the content of most amino acids were close to those recorded for treated control leaves. However, the asparagine content remained very high reaching ca four-fold as much as that of the controls.

731

The large accumulation of free asparagine in organs of S-deprived plants has been noted, among others, by Mertz et al. [4] for lucerne leaf, by Coic et al. [S] for barley leaf and grain and by Macnicol [8] for pea cotyledons. Therefore, the significant increase of free asparagine in control leaves after application of elemental S indicates stress. This, suggested by the deviations of photorespiratory metabolism recorded with samples analysed 4 hr after application [2], could result from the large amount (ca 10 mg) of S applied on the leaf. Bagging, used to reduce S sublimation, is thought to have a very limited influence on leaf metabolism as the period between application and harvesting for amino acid analysis was relatively short (1 day). In contrast to the alteration in distribution of free amino acids, the amino acid composition of proteins isolated as alcohol-insoluble material was unaffected by S-application or S-starvation or both (data not shown). This has been also reported in the case of S-application and wheat grain proteins [13]. It differs from the observations of Colic et al. [S] with barley. These authors reported an enrichment of foliar proteins from S-deprived plants in aspartic acid + asparagine representing the third of the protein mass. Such a divergence may be explained by protein contamination with free asparagine incompletely extracted by aqueous ethanol at -10”. With seeds it has been found that S-starvation induces not only a large accumulation of free amino acids, mainly asparagine, but also changes in the distribution of the main protein fractions without perturbing their amino acid composition [8, 91. Modification of amino acid composition of the seed proteins from plants grown without S reflected changes associated with preferential synthesis of storage proteins poor in S-containing amino acids to compensate the deficiency [9, 14, 151. The fact that the amino acids of leaf proteins was insensitive to the S status of the plant suggests that differences in S supply induces only discrete changes, if any, in leaf proteins. The data presented in this paper therefore show that foliar application of elemental S to S-deficient wheat plants promoted a greater efficiency and duration of S uptake. It induced some changes in the metabolism of S and N compounds present in leaves, reducing the stress effects caused by the deficiency. EXPERIMENTAL

Wheat (T. aestivum) cv. Champlein was grown in Vermiculite supplied with a nutrient soln containing 1.5 mequiv of SOi- 1-i or devoid of this anion [16], a 15 hr photoperiod, a quantum flux of 200 pm01 m-’ see-’ and 70/90% relative humidity for 3 weeks. 35S-labelled elemental S was obtained by stirring a colloidal dispersion of radioactive S with 10 times as much micronized S, (Thiovit from Sandoz) free ofadjuvantaccording to ref. [2]. The mixt., resuspended in adjuvant, was applied to the 3rd leaf with a brush (35&600 KBq). Treated leaves were enclosed in a plastic bag to reduce S losses by sublimation. Individual leaves were washed with H,O, if treated with CHCI,, and frozen in liquid N,. Samples were ground in the presence of finely powdered sand, NaF and KBH, and extracted with 50% EtOH. The supernatant contained S-containing metabolites. The residue, after washing with CHCl, for isolation of lipid-sol. metabolites, was constituted of proteins mainly. 3SS-labelled metabolites from the EtOH-sol. fr. were analysed by ZD-TLC fractionation on cellulose [lq. Thirteen labelled

J. LANDRY etal.

132

compounds, 7 of which were identified, were isolated [Z]. EtOH-sol. material and hydrolysates (6 M HCl, 110”, 24 hr without and with performic oxidation) of the EtOH-insol. residue were analysed for amino acids by HPLC of their phenylthiocarbamyl derivatives [18]. Sepns were performed using two coupled columns [13]. Acknowledgement-The

authors wish to thank Mrs S. Delhaye

for amino acid analyses.

6. Eppendorfer, W. (1968) Pkznt Soil 26, 129. 7. Byers, M. and Bolton, J. (1979) J. Sci. Food Agric. 30, 251. 8. Macnicol, P. K. (1983) FEES Letters 156, 55. 9. Baudet, J., Huet, J. C., Jolivet, E., Lesaint, C., Moss&, J. and Pemollet, J. C. (1986) Physiol. Plantarum 68, 608. 10. Legris-Delaporte, S., Ferron, F., Landry, J. and Costes, C. (1987) in Elemental Sulphur in Agriculture Vol. 1 (Coleno, A., ed.), p. 365. Syndicat FranCais du Soufre, Marseille, France. 11. Joyard, J., Forest, E., Blee, E. and Deuce, R. (1988) Plant Physiol. 88, 961. 12. Friedrich, J. W. and Larry, L. E. (1978) Plant Physiol. 61,900. 13. Leg&-Delaporte, S. and Landry, J. (1987) J. Cereal Sci. 6,

REFERENCES

1. Legris-Delaporte, S., Ferron, F., Landry, J. and Costes, C. (1987) in Elemental Sulphur in Agriculture Vol. 2 (Colbno, A., ed.), p. 681. Syndicat Franwis du Soufre, Marseille, France. 2. Legris-Delaporte, S., Ferron, F., Landry, J. and Costes, C. (1987) PIant Physiol. 85, 1026. 3. Brune, D. C. (1989) Biochim. Eiophys. Acta 975, 189. 4. Mertz, E. T., Singleton, V. L. and Garey, C. L. (1952) Arch.

119. 14. Randall, P. J., Thomson, J. A. and Schroeder, H. E. (1979) Aust. .I. PIant Physiol. 6, 11. 15. Shewry, P. R., Franklin, J., Parmar, S., Smith, S. J. and Miflin, B. J. (1983) .J. Cereal Sci. 1, 21. 16. Coic, Y. and Lesaint, C. (1973) Rev. Hortic. 2316, 29. 17. Ferron, F., Coudret, A. and Gaudillere, J. P. (1978) Bull. Sot.

Biochem. Biophys. 38, 139. 5. Cok, Y., Fauconneau, G., Pion, R., Lesaint, Godefroy, S. (1962) Ann. Physiol. Vbg. 4, 295.

Bot. Fr. Actual. Bat. 125, 189. 18. Bidlingmeyer, B. A., Cohen, S. A. and Tarvin, T. L. (1984) J. Chromatogr. 336,93.

C. and