Wilt‐induced ABA biosynthesis, gene expression and down‐regulation of rbcS mRNA levels in Arabidopsis thaliana

Abstract

The kinetics of wilt‐induced abscisic acid (ABA) biosynthesis were investigated in shoots of Arabidopsis thaliana (L.) Heynh Landsberg erecta. ABA concentrations were measured using a radioimmunoassay (RIA) based on the monoclonal antibody MAC 252, and the RIA validated by comparison with combined gas chromatography‐mass spectrometry using a [2H3] labelled internal standard. The basal ABA content of Arabidopsis shoots was ca 10 ng g−1 fresh weight; the concentrations had increased ca 4‐fold within 30 min of the initiation of wilting, increased ca 8‐fold after 4 h and 11‐fold after 8 h. This stress‐induced ABA production was dependent on de novo gene expression; pre‐treatment of leaves and shoots with the metabolic inhibitors cordycepin and cycloheximide reduced the rate of subsequent stress‐induced ABA biosynthesis from 12.5 ng g−1 h−1 to 1 ng g−1 h−1 and 0 ng g−1 h−1, respectively. In vitro translation of mRNA isolated from shoots subjected to wilting or ABA treatment followed by one‐dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed only minor changes. The effects of wilting and ABA on the content of total ribulose 1,5‐bisphosphate carboxylase/oxygenase small sub‐unit (rbcS) mRNA were also determined. Both wilting and exogenous ABA resulted in a substantial reduction in the amount of rbcS mRNA, an effect readily reversed by rehydration of wilted shoots. However, the effects of wilting were not mediated solely by newly‐synthesised endogenous ABA, as wilting also reduced rbcS mRNA levels in the ABA‐deficient aba‐1 mutant, which did not produce ABA in response to loss of turgor. The amount of rbcS mRNA was higher in aba‐1 shoots, suggesting that cellular rbcS mRNA levels are normally down‐regulated by ABA. Cold treatment induced ABA production in wild type shoots only, but resulted in an increased rbcS mRNA content of both wild type and aba‐1 shoots.