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

Xylem 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.Although 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.Roots in drying soil produce chemical signals such as abscisic acid (ABA) [1] that can be transported to the shoots to modify their physiology. While this remains an attractive hypothesis, extensive studies have demonstrated that this signal can be modified by synthesis and metabolism of ABA along the transport pathway [2], and the fundamental basis of root-sourced ABA signalling has been challenged. Reciprocal grafting studies of wild-type (WT) plants and ABA-deficient mutants [3-5] have generally concluded that stomatal closure is independent of root-synthesised ABA since stomatal conductance of WT scions decreased as the soil dried, independently of whether they were grafted on a WT or ABA-deficient rootstock [3]. This closure occurred in plants where shoot turgor was maintained (by root pressurisation) as the soil dried, seemingly excluding a role for local induction of ABA synthesis in the leaves due to decreased turgor [3]. Instead, this suggests that a chemical signal from the roots may increase the apoplastic ABA concentration in the leaves to cause stomatal closure. In WT sc