Analysis of major tomato xylem organic acids and PITC-derivatives of amino acids by RP-HPLC and UV detection

Abstract

Major amino acids and organic acids in xylem exudates of tomato plants were separated by reversed phase high performance liquid chromatography (RP-HPLC) and quantified by UV detection. Before separation, amino acids were converted into their phenylisothiocyanate (PITC) derivatives. In a single run, Asp, Glu, Ser, Gin, His, Thr, Ala, Tyr, Val, Met, Cys, lie, Leu, Phe, and Lys could be separated and detected down to the pmol level. Unresolved peaks were obtained for Asn and Gly and for Arg and Pro. For organic acid analysis, exudates were pre-treated by perfusion over a prepacked Adsorbex SCX cation exchange column, to eliminate exudate amino acids. Elution recoveries for organic acids were close to 100%. The exudate organic acids were separated by ion suppression RP-HPLC chromatography, and peaks could be resolved for L-malic acid, malonic acid, maleic acid, citric acid and fumaric acid, down to the pmol level. UV signals for exudate ascorbic acid, and succinic acid were below the limits of detection. Determination of oxalic acid and tartaric acid was impossible, due to the presence of the exudate salt peak in the chromatogram. The results indicate the potential of the methods applied, and show the applicability of RP-HPLC analysis for the determination of both amino acids and organic acids in xylem exudates.

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment, Stem Colonization, and Virulence

“…Nitrate is abundant and highly mobile in soil. Nitrate is also relatively abundant in xylem sap, but other accessible nitrogen sources such as amino acids are present as well, though not at comparable concentrations (40)(41)(42)(43). Our finding that nasA is dispensable for R. solanacearum growth and competitive fitness in tomato xylem suggests strongly that this environment offers the bacterium sufficient amounts of non-nitrate nitrogen sources.…”

Effects of cadmium on the behaviour of citric acid in isolated tomato xylem cell walls

“…Stem internodes were submerged in tap water at 20 °C for 40 d, after which the unaffected xylem cell wall parts of the internodes were isolated by rinsing in tap water (Senden et al, 1992A). Purification was carried out according to Ritchie and Larkum (1982) by soaking and washing the xylem material in 0.5% (v/v) Triton X-100, acetone and distilled water for 4 d. The xylem cell wall material was dried, powdered and sieved into particles with diameters ranging from 0.125 to 0.250 mm.…”

“…The uptake and synthesis of organic compounds in the roots (Collins and Reilly, 1968;Tonin el al, 1990), and the presence of amino acids and cation-organic complexes in xylem fluid have been reported to seriously affect the movement of metals in the xylem, both in longitudinal and lateral directions (Tiffin, 1966(Tiffin, 1970Bradfield, 1976;White et al, 1981a, b, c;Van de Geijn and Pikaar, 1982;McGrath and Robson, 1984;Senden and Wolterbeek, 1990). In this context, citric acid has received considerable attention, probably because it is generally a substantial fraction of the organic acids in the xylem (White et al, 1981a;Senden et al, 1992a), and because it is a relatively strong metal complexer (Sillen and Martell, 1964).…”

Analysis of free amino acids during fermentation by Bacillus subtilis using capillary electrophoresis

“…Conventional techniques for analysis of amino acids include ion exchange chromatography (IEC), using amino acid analyzer [9] and reverse-phase (RP)-HPLC [10,11], usually using a post-or pre-column derivatization with various kinds of chromophores [12] or fluorophores [13,14]. Amino acid analysis using an amino acid analyzer is expensive and time-consuming.…”