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To characterize the mechanisms of amino acid accumulation under sulphur (S)-deficiency and its physiological significance in Brassica napus, stable isotopes 15N and 34S were employed. The plants were exposed for 9 days to S-deficient conditions (0.05 mM vs 1.5 mM sulphate). After 9 days of S-deficiency, leaf-osmotic potential and total chlorophyll content significantly decreased. S uptake decreased by 94%, whereas N uptake and biomass were not significantly changed. Using 15N and 34S labelling, de novo synthesis of amino acids and proteins derived from newly absorbed NO3 and SO42 and the content of N and S in the previously synthesized amino acids and proteins were quantified. At the whole plant level, S-deficiency increased the pool of amino acids but resulted in strong decrease of incorporation of newly absorbed NO3 and SO42 into amino acids by 22.2 and 76.6%, respectively, compared to the controls. Total amount of N and S incorporated into proteins also decreased by 28.8 and 62.1%, respectively. The levels of 14N- and 32S-proteins (previously synthesized proteins) strongly decreased, mainly in mature leaves. The data thus indicate that amino acid accumulation under short-term S-deficiency results from the degradation of previously synthesized proteins rather than from de novo synthesis.