Tracing Cationic Nutrients from Xylem into Stem Tissue of French Bean by Stable Isotope Tracers and Cryo-Secondary Ion Mass Spectrometry

Fluxes of mineral nutrients in the xylem are strongly influenced by interactions with the surrounding stem tissues and are probably regulated by them. Toward a mechanistic understanding of these interactions, we applied stable isotope tracers of magnesium, potassium, and calcium continuously to the transpiration stream of cut bean (Phaseolus vulgaris) shoots to study their radial exchange at the cell and tissue level with stem tissues between pith and phloem. For isotope localization, we combined sample preparation with secondary ion mass spectrometry in a completely cryogenic workflow. After 20 min of application, tracers were readily detectable to various degrees in all tissues. The xylem parenchyma near the vessels exchanged freely with the vessels, its nutrient elements reaching a steady state of strong exchange with elements in the vessels within 20 min, mainly via apoplastic pathways. A slow exchange between vessels and cambium and phloem suggested that they are separated from the xylem, parenchyma, and pith, possibly by an apoplastic barrier to diffusion for nutrients (as for carbohydrates). There was little difference in these distributions when tracers were applied directly to intact xylem via a microcapillary, suggesting that xylem tension had little effect on radial exchange of these nutrients and that their movement was mainly diffusive.Long-distance transport of nutrients in stems is strongly influenced by the interaction of the moving xylem sap with the surrounding tissues (e.g. phloem; Stout and Hoagland, 1939; Biddulph and Markle, 1944). The importance of this radial exchange was highlighted in studies on budgets of carbon/nitrogen and mineral nutrients (Pate et al., 1979; Jeschke et al., 1985, 1991; Wolf et al., 1991). The composition of a solution is changed during perfusion of stem pieces (Gilmer and Schurr, 2007), suggesting that xylem sap composition is regulated. Thus, the fluxes of nutrients in the xylem could be regulated through the ionic concentration and also from the influence of nutrient concentration (e.g. potassium) on hydraulic properties (Thompson and Zwieniecki, 2005). The transport of these nutrients in stems, therefore, does not occur in a simple pipeline connecting roots with leaves but in pathways that involve many tissues in the stem, in the same way that photoassimilate transport is not confined to sieve tubes (van Bel, 2003). However, perfusion experiments with stem pieces may be inappropriate for elucidating these interactions (van Ieperen, 2007), since lateral flow may be promoted by the unnatural pressure regime. This reservation also applies when the root pressure chamber is used to extract sap, for example, in experiments that showed strong interactions between xylem and adjacent tissues (Siebrecht et al., 2003; Gilmer and Schurr, 2007). Therefore, studies of nutrient and water movement in the xylem should use techniques that minimize any perturbation of the water status of all stem tissues.Isotope tracers are ideal for studies toward a mechanistic understanding of nutrient exchange between the transpiration stream and different stem tissues, because they are chemically identical to the traced elements. Enriched stable isotopes are available for most nutrients and can be detected at subcellular spatial resolution with imaging mass spectrometric techniques such as secondary ion mass spectrometry (SIMS), provided that the distribution of diffusible tracers can be preserved until completion of the analysis. Strict cryogenic sample preparation followed by analysis with SIMS below −130°C (cryo-SIMS) has been shown to satisfy this criterion (Metzner et al., 2008), with scanning electron microscopy of the frozen samples (cryo-SEM) for quality control and detailed anatomical information of the individual tissues.Here, we used this cryogenic protocol to examine the exchange between xylem vessels and stem tissue of French bean (Phaseolus vulgaris ‘Fardenlosa Shiny’), with stable isotope tracers for potassium, calcium, and magnesium applied to the transpiration stream of a cut shoot. Based on earlier microanalytical studies on the diffusion kinetics of cationic nutrients moving into roots (Kuhn et al., 2000; Horst et al., 2007), we selected two different periods of tracer application, namely 20 min to show any potential diffusion barriers and 240 min to show distribution patterns after reaching a steady state in nutrient exchange between xylem and surrounding tissue. We evaluated our standard method of tracer application, via the cut stem, in which stem water status was disturbed, in an ancillary experiment where the solution entered via microcapillary directly into xylem under tension.