Xylem sap collection and extraction methodologies to determine in vivo concentrations of ABA and its bound forms by gas chromatography-mass spectrometry (GC-MS)

Andrew G Netting,Ian C Dodd,Julian C Theobald

doi:10.1186/1746-4811-8-11

Abstract

BackgroundAccurate quantification of xylem sap ABA concentrations is important to underpin models of root-to-shoot ABA signalling to predict the physiological effects of soil drying. Growing tomato plants in a whole plant pressure chamber allowed sequential xylem sap collection from a detached leaf, the petiole stub of an otherwise intact plant and finally the de-topped root system of the same plant, to determine the impact of xylem sap sampling methodology on xylem ABA concentration. Since xylem sap can contain bound forms of ABA, a novel gas chromatography-mass spectrometry (GC-MS) procedure was developed to chemically separate free ABA from two in planta bound ABA forms known as Adducts I and II and ABA-glucose-ester (ABA-GE).ResultsXylem sap ABA concentrations were highly dependent on the sampling methodology used: the highest concentrations were detected in sap collected by applying an overpressure to detached leaves following the measurement of leaf water potential. Irrespective of xylem sap source, the wild-type cultivars Ailsa Craig and Rheinlands Ruhm had higher free ABA concentrations than a range of ABA-deficient mutants (notabilis, flacca and sitiens). However, in the mutants, concentrations of bound forms of ABA were similar to wild-type plants, and similar to free ABA concentrations.ConclusionsAlthough xylem concentrations of these bound ABA forms and ABA-GE suggest they have a limited physiological impact on ABA homeostasis in tomato, the methods developed here will allow a more complete understanding of ABA biochemistry and root-to-shoot signalling in species known to have higher concentrations of these compounds.

Highlights

Accurate quantification of xylem sap abscisic acid (ABA) concentrations is important to underpin models of root-toshoot ABA signalling to predict the physiological effects of soil drying
Whole plant transpiration rate (E) apparently decreased, and leaf water potential (Ψleaf) increased as whole plant leaf area increased (Figure 3). Despite this developmental confounding and considerable inter-plant variation, it was apparent that E was lower and Ψleaf higher in the wild-type Ailsa Craig (AC) and Rheinlands Ruhm (RR) plants (Figure 3 insets)
In contrast to previous work where whole plant transpiration rate generally increased with increasing severity of ABA deficiency [17], there were no significant differences between the ABA deficient mutants (Figure 3a inset)

Summary

Introduction

Accurate quantification of xylem sap ABA concentrations is important to underpin models of root-toshoot ABA signalling to predict the physiological effects of soil drying. Reciprocal grafting studies of rootstock, it is possible that water stress could induce synthesis of bound forms of ABA known as Adducts I and II [6,7] in the roots. These compounds could be transported in the xylem to the shoots where they would release ABA in the xylem sap of the leaves, as the soil dries. The widely accepted pathway for ABA biosynthesis [8] commences with the oxygenation of 9’cis-neoxanthin or 9-cis-violaxanthin to give the C-15 aldehyde, xanthoxin. Since the role of adduct biochemistry in modulating ABA concentrations in these mutants remains obscure, adduct concentrations in xylem sap collected from these mutants was determined

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