Gas chromatographic analysis of low molecular weight organic acids in roots and shoots of durum wheat plants

Abstract

Low molecular weight organic acids (LMWOAs) in plant tissue have been shown to play a role in plant response to metals. Therefore, there is a need for effective analytical methods capable of quantitating LMWOAs in plants. Several solvents were examined for the efficiency of the LMWOA extraction from plant tissue of durum wheat (Triticum turgidum var. durum) and for the suitability of the resulting extracts for the gas chromatographic (GC) analysis. Water, 1 N HCl, 0.5 N KCl, 0.5 N HCl in MeOH at room temperature, and water and 1 N HCl at 100°C were tested. Increased temperature and use of 1 N HCl improved the recovery of most of the LMWOAs as compared to water or KCl extractions at room temperature. However, recovery of fumaric and t-aconitic acids was lowered by the addition of HCl and/or increased temperature of extraction probably because of the instability of the double bond. The highest efficiency for oxalic acid was obtained with HCl/MeOH extraction; however, the chromatograms contained many peaks interfering with those of other LMWOAs, rendering this extractant unsuitable for the GC analysis. Thus the extraction with 1 N HCl at room temperature was found to be the most suitable; 1 N HCl removed the majority of the acids studied without artifacts appearing in the chromatograms. Non-volatile LMWOAs were analyzed by GC after sample trimethylsilylation (TMS) and after sample methylation, while volatile LMWOAs were injected directly into the GC without sample derivatization. The combination of the three chromatographic separations allowed for the determination of oxalic, malonic, succinic, fumaric, malic, t-aconitic, citric, acetic, and propionic acids in roots and shoots of wheat plants. This method was applied to two durum wheat cultivars, i.e., Kyle and Arcola grown hydroponically for two weeks. LMWOA concentrations were greater in shoots than in roots for both cultivars. Individual and total LMWOA concentrations in roots and shoots did not differ significantly between cultivars. Further studies are needed to identify the factors controlling LMWOA production in plant tissue and its significance in processes occurring in plants and at the root/soil interface.