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SULPHUR UTILISATION EFFICIENCY IN OILSEED RAPE

F.J. Zhao1, M.M.A. Blake-Kalff 1, N. Riley1, M.J. Hawkesford2 and S.P. McGrath1

1 Soil Science Department, 2 Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, U.K.

ABSTRACT

Oilseed rape has a high demand for sulphur, requiring approximately 16 kg S to produce 1 t of seed. This has been attributed to the biosynthesis and accumulation of glucosinolates, which can account for up to 50% of the total S in seeds in single low varieties. However, glucosinolates usually account for less than 5% of the total S in the vegetative tissues of oilseed rape, and therefore, are not a major storage pool of S in the plant. Allocation and re-distribution of S in vegetative tissues were investigated using hydroponic, pot and field experiments. Under S-sufficient conditions, sulphate was the major storage pool, accounting for 40% of the total S in the young leaves and 70-90% in the middle and old leaves. When the external S supply was withdrawn, sulphate in the young leaves was utilised rapidly to fulfil cellular requirement for reduced S, whereas the remobilisation of the glucosinolate-S and glutathione-S pools was much less important. However, re-distribution of sulphate from the old leaves was found to be slow. There were considerable amounts of sulphate remaining in the old leaves when the young leaves started to show S deficiency symptoms. These results suggest that a large accumulation of sulphate in the mature leaves, coupled with a slow re-distribution of this S pool upon S-deficiency, are the main reasons for the high S demand in oilseed rape. The S requirement and the susceptibility to S deficiency of oilseed rape may be decreased if the re-distribution of sulphate from mature leaves can be enhanced.

KEYWORDS: glucosinolates, glutathione, S requirement, S uptake, sulphate, S utilisation.

INTRODUCTION

Brassica species are characterised by a high requirement for S. For example, winter oilseed rape grown under UK conditions requires approximately 16 kg S to produce 1 tonne of seed (McGrath et al. 1996). This compares markedly to a requirement of 2-3 kg S for the production of 1 tonne of cereal grain. In modern double-low oilseed rape varieties (low seed glucosinolates and low erucic acid), the S harvest index (S content in seed divided by total S in the whole crop) is typically only 0.25. This indicates that a large proportion of S taken up by the crop is retained in the vegetative tissues and pod walls. Because of the high S requirement, oilseed rape is among the most susceptible crops to S deficiency, and in recent years, S deficiency in this crop has increased substantially in the UK (McGrath et al. 1996).

Why does oilseed rape require so much S? A common, yet unproven, explanation has been the requirement of S for the biosynthesis of glucosinolates. Related to this explanation is a hypothesis that glucosinolates act as an important storage pool of S, which can be re-utilised upon shortage of S supply (Underhill 1980; Schnug and Haneklaus 1993). However, a survey of literature reveals quickly the fundamental weakness of both hypotheses, which is that glucosinolates are only a minor S pool in the vegetative tissues of oilseed rape for both single-low and double-low cultivars. Glucosinolates account typically for only <5% of the total S in oilseed rape leaves and stems (Porter et al. 1991; Fieldsend and Milford 1994; Griffiths et al. 1994; Marquard and Meuthen 1995). It is thus difficult to envisage a role of glucosinolates as a significant storage pool of S. In this study, we investigated the efficiency of S utilisation in oilseed rape by determining the allocation of S into different S compounds under S-sufficient or S-deficient conditions.

MATERIALS AND METHODS

Seedlings of oilseed rape (Brassica napus L. cv. Apex, a double-low cultivar) were grown hydroponically under controlled environmental conditions (Blake-Kalff et al. 1998). After pre-culture for 23 days with a full nutrient solution, plants were divided into two groups. One group continued to receive 1 mM SO42- and the other received no further S supply. Each treatment had three replicates. Plants were harvested on 0, 3, 6, 8 and 13 days after treatments started, and separated into young, middle and old leaves. Samples were frozen separately in liquid nitrogen and lyophilised for 72 hours, before being ground for analysis. Total S was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES), following digestion of leaf samples with HNO3/HClO4. Sulphate was extracted with deionised water at 90 oC for 2 hours, and determined by ion chromatography. Glutathione was extracted with a solution containing 5% (w/v) 5-sulfosalicylic acid and 6.3 mM diethylenetriaminepenta-acetic acid. Total glutathione was determined using the 5,5’-dithiobis(2-nitrobenzoic acid) recycling assay as described by Anderson (1985). Glucosinolates were extracted with methanol and determined by high performance liquid chromatography according to the protocols of Heaney et al. (1986). Insoluble S, representing mainly protein S, was calculated by subtracting the concentrations of sulphate, glutathione and glucosinolates from the concentration of total S.

RESULTS AND DISCUSSION

Concentrations of S in different pools are shown in Figure 1 for young and old leaves. It is clear that the pattern of S distribution was very different between the young and old leaves. Total S concentrations in the old leaves were almost double those in the young leaves. This difference was essentially due to a much larger sulphate concentration in the old leaves. Under S-sufficient conditions, sulphate accumulated in the old leaves and accounted for 70-90% of total S, whereas in the young leaves sulphate accounted for only 40% of total S. In contrast, the young leaves contained significantly more protein S, and far more glutathione and glucosinolates than the old leaves. Under S-sufficient conditions, glucosinolates accounted for 6.5% of the total S in the young leaves but only about 0.1% in the old leaves, indicating that they are the minor S compounds in oilseed rape leaves. Similarly, Porter et al. (1991) reported a rapid decrease in the concentration of glucosinolates in oilseed rape leaves after the leaves had fully expanded. This cannot be explained by a demand driven re-utilisation of glucosinolates, because there was an exceedingly large accumulation of sulphate in the old leaves. Although glutathione is undoubtedly essential for plant metabolism, it also represented a small fraction of leaf S, accounting for about 2 and 0.5% of the total S in the young and old leaves, respectively.

When the external S supply was interrupted, visual S deficiency symptoms were apparent in the young leaves first at day 6 and severe by day 13. All S pools, particularly sulphate, decreased rapidly in the young leaves from day 3 (Figure 1). The changes in the concentrations of different S pools occurred more slowly in the old leaves, and were evident only after day 6. By day 13, when the young leaves had developed severe S-deficiency symptoms, the old leaves still contained 50% of the total S as sulphate. There were only small changes in the concentrations of glutathione and glucosinolates in the old leaves in response to the withdrawal of external S supply.

Figure 1. Concentrations of sulphate, insoluble-S, glucosinolate-S and glutathione-S in young leaves (a) and old leaves (b) of oilseed rape.

Using S concentration and leaf growth data, it was possible to determine whether the young leaves received net import of S from other tissues after the external S supply was withheld. Essentially, import did not occur and the decrease in the concentration of total S in the young leaves was entirely due to growth dilution. However, there was internal re-distribution of S between different pools in the young leaves. By comparing leaf growth data and the changes in different pools of S in the young leaves, net gain or loss of S in these pools was also estimated (Table 1). The results showed that S in the sulphate, glutathione and glucosinolate pools was re-distributed to the insoluble S pool. The contribution of sulphate was by far the most important, being approximately 10 times greater than the contribution of glucosinolate S.

Table 1. Net gain or loss of S (Ámol g-1 DW) in different S pools in the young leaves of oilseed rape during the 13-day period when the external S supply was withheld

Insoluble S

Sulphate

Glutathione

Glucosinolates

+16.1

-21.3

-0.8

-2.2

CONCLUSIONS

This study showed that glucosinolates constitute a minor S pool in oilseed rape leaves, and do not play any significant role in storage of S that can be re-utilised upon S deficiency. The reason for a high S requirement in oilseed rape cannot be readily explained by glucosinolate biosynthesis either. In contrast, there is exceedingly large accumulation of sulphate, particularly in the old leaves. When the external S supply is limited, sulphate in the young leaves is utilised rapidly to maintain the insoluble (proteins) S pool. However, re-utilisation of sulphate that previously accumulated in the old leaves is slow, resulting in a low utilisation efficiency in oilseed rape. It is likely that sulphate in the old leaves is sequestrated predominantly in the vacuoles. Understanding tonoplast transport of sulphate would appear to be a key step for manipulation of S utilisation efficiency in oilseed rape.

ACKNOWLEDGEMENTS

This work was funded by the Home-Grown Cereals Authority of the United Kingdom. IACR-Rothamsted receives grant-aided funding from the Biotechnology and Biological Sciences Research Council.

REFERENCES

1. Anderson, M.E. 1985. Tissue glutathione. In: Handbook of Methods for Oxygen Radical Research, R.A. Greenwald, ed., CRC Press, Boca Raton, FL, pp 317-323.

2. Blake-Kalff, M.M.A., Harrison, K.R., Hawkesford, M.J., Zhao, F.J. and McGrath, S.P. 1998. Distribution of sulfur within oilseed rape leaves in response to sulfur deficiency during vegetative growth. Plant Physiology 118: 1337-1344.

3. Fieldsend, J. and Milford, G.F.J. 1994. Changes in glucosinolates during crop development in single- and double-low genotypes of winter oilseed rape (Brassica napus): I. Production and distribution in vegetative tissues and developing pods during development and potential role in the recycling of sulphur within the crop. Annals of Applied Biology 124: 531-542.

4. Griffiths D.W., MacFarlane-Smith, W.H. and Boag, B. 1994. The effect of cultivar, sample date and grazing on the concentration of S-methylcysteine sulphoxide in oilseed and forage rapes (Brassica napus). Journal of the Science of Food and Agriculture 64: 283-288.

5. Heaney, R.K., Spinks, R.K., Hanley, A.B. and Fenwick, G.R. 1986. Analysis of glucosinolates in rapeseed. Technical Bulletin, AFRC Food Research Institute, Norwich. 28 pp.

6. Marquard, R. and Meuthen, B. 1995. Investigation on the role of glucosinolates as a sulfur reserve for the rape plant. In: Rapeseed Today and Tomorrow, Proceedings of the 9th International Rapeseed Congress, Cambridge, Vol. 1. Pp. 274-276.

7. McGrath, S.P., Zhao, F.J. and Withers, P.J.A. 1996. Development of sulphur deficiency in crops and its treatment. Proceedings of the Fertiliser Society No. 379. The Fertiliser Society, Peterborough, UK.

8. Porter, A.J.R., Morton, A.M., Kiddle, G., Doughty, K.J. and Wallsgrove, R.M. 1991. Variation in the glucosinolate content of oilseed rape (Brassica napus L.) I. Effect of leaf age and position. Annals of Applied Biology 118: 461-467.

9. Schnug, E. and Haneklaus, S. 1993. Physiological backgrounds of different sulfur utilisation in Brassica napus varieties. Aspects of Applied Biology 34: 235-242.

10. Underhill, E.W. 1980. Glucosinolates. In: Encyclopedia of Plant Physiology Vol. 8 Secondary Plant Products. Ed. E.A. Bell and B.V. Charlwood B V. Springer-Verlag, Berlin. pp 493-511.

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