ABSTRACT

We aim to gain a better understanding of the biochemical and molecular processes that determine the allocation of sulphur in pods and seeds of oilseed rape (Brassica napus). The crop has a high demand for S, supporting the production of glucosinolates (S-containing amino acid derivatives) as well as seed storage proteins with a relatively high S content. Yet the plant appears to use S rather inefficiently: when sufficient S for optimal growth is supplied, much remains in the mature crop as sulphate in the pod walls. We are studying the biochemical pathways of sulphate assimilation and the biosynthesis of S amino acids and glucosinolates, and the distribution of these pathways between the pod walls and developing seeds of cultivars with high and low seed glucosinolate content. The study entails measuring the flux through the pathways, the content of intermediates, the activities of enzymes and the expression of the corresponding genes.

INTRODUCTION - THE DEMAND FOR SULPHUR

The aim of this project is to gain a better understanding of the biochemical and molecular processes that determine the allocation of sulphur in pods and seeds of oilseed rape (Brassica napus). Brassica crops such as rape have a high requirement for S. Storage proteins in the seeds have a relatively high S content and Brassicas produce substantial concentrations of S-containing secondary metabolites, the glucosinolates, which have important roles in plant defence metabolism and as flavour components (Wallsgrove et al., 1999). Glucosinolates are derived from amino acids and contain both a thio-glucose and a sulphate residue (Fig 1), which are introduced into the molecule from cysteine and the sulphate donor compound, PAPS. One of the main classes of glucosinolates in Brassicas are the alkenyl glucosinolates derived from methionine. Hence the synthesis of glucosinolates has direct consequences for S-amino acid metabolism and the demand for S.

Fig 1. Structure of Glucosinolates

SULPHATE ACCUMULATION IN THE PODS

Despite the high requirement for S, the rape plant appears to use it rather inefficiently. When sufficient S for optimal growth is supplied, much remains in the mature crop as inorganic sulphate in the pod walls. This is even more evident in cultivars with low seed glucosinolate content: the double-low cultivar Cobra accumulated a higher proportion of S as sulphate in the pod walls than did the single-low cultivar Bienvenu (Zhao et al., 1993b).

S-metabolism in the rest of the plant appears to be very similar in the two cultivars: overall S uptake and total S content did not differ (Zhao et al., 1993a). Furthermore, the leaves of the two cultivars showed similar developmental profiles of glucosinolate content (Porter et al., 1991). S-allocation seems to differ greatly only in the pods and seeds of the two cultivars.

We have confirmed a similar pattern under glasshouse conditions to that seen in the field, but with an even more pronounced difference between the cultivars. During the period of pod and seed development there was a large increase in the sulphate content of the pod walls of Cobra (as was seen in the field-grown crop), but a fall in sulphate in the pod walls in Bienvenu (Fig 2). The sulphate content of the seeds was quite low and fell slightly in both cultivars.

Fig 2. Sulphate content of pods and seeds during development

Pod walls and seeds of glasshouse-grown rape were harvested at growth stages 6,1-6,9 (fully mature), freeze-dried then extracted in hot water. Inorganic sulphate was determined by ion chromatography.

THE FLOW OF SULPHUR

We can consider this system in a simplified scheme for the flow of S in the developing pods and seeds (Fig 3). Sulphate is assimilated into S-amino acids, which are the substrates for synthesis of both storage proteins and glucosinolates - the major sinks of S in the seeds. The protein content of the cultivars is about the same, and the total S content of the seeds closely reflects the glucosinolate content (Zhao et al., 1993b). The lower seed glucosinolate content in Cobra is associated with a larger accumulation of free sulphate in the pod walls, compared to Bienvenu.

POSSIBLE ORIGINS OF DIFFERENCES

We are investigating this system to gain a more detailed understanding of the biochemical and molecular mechanisms controlling the partitioning of S. We can envisage two major possibilities that could give rise to the differences in S-allocation:

(1) A restriction in glucosinolate biosynthesis could lead to reduced amino acid consumption and accumulation of the initial substrate, sulphate.

(2) Alternatively, there may be some limitation in the assimilation of sulphate, again leading to accumulation of the substrate. Under conditions where the supply of amino acids is restricted, production of the storage proteins may be maintained at the expense of glucosinolate synthesis.

Fig 3. Scheme for flux of S in pods and seeds

APPROACHES

We are using a range of techniques to investigate these questions:

• radio-labelling experiments allow us to describe the flux of S through the biochemical pathways and the distribution of these pathways between the pod walls and developing seeds;

• measurements of the pool sizes of intermediates in the pathways, in conjunction with information from the labelling experiments, should identify sites in the pathways where the cultivars differ;

• measurements of the activity of enzymes at key points in the pathways, and the expression of the corresponding genes (where suitable probes are available) should reveal how differences in gene expression give rise to the altered partitioning of S in the two cultivars.

Conclusion

The developing pods and seeds of oilseed rape present a useful system to study the processes of S-allocation. By a combined approach using techniques of biochemistry and molecular biology we are investigating the regulation of these processes.

Acknowledgements

This work is funded by a RASP grant from BBSRC. IACR receives grant-in-aid from BBSRC.

References

1. Porter et al. (1991) Ann. App. Biol. 118: 461-467

2. Wallsgrove et al. (1999) Glucosinolates. In: Plant Amino Acids: Biochemistry and Biotechnology (ed. B.K.Singh), Marcel Dekker Inc., New York. pp 523-561.

3. Zhao et al. (1993a) Plant and Soil 150: 69-76

4. Zhao et al. (1993b) J Sci Food Agric 62: 111-119