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Blackwell Science, LtdOxford, UK PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2001 25 747 Xylem transport of abscisic acid conjugates A. Sauter et al. Original ArticleBEES SGML Plant, Cell and Environment (2002) 25, 223–228 A possible stress physiological role of abscisic acid conjugates in root-to-shoot signalling A. SAUTER,1 K.-J. DIETZ2 & W. HARTUNG1 1Julius-von-Sachs-Institut der Universität Würzburg, Lehrstuhl Botanik I, Würzburg, Germany and 2Lehrstuhl für Stoffwechselphysiologie und Biochemie der Pflanzen, Universität Bielefeld, Universitätsstraße 25, D-33501 Bielefeld, Germany ABSTRACT Abscisic acid (ABA) conjugates, predominantly their glucose esters, have recently been shown to occur in the xylem sap of different plants. Under stress conditions, their concentration can rise substantially to levels that are higher than the concentration of free ABA. External ABA conjugates cannot penetrate apoplastic barriers in the root. They have to be hydrolysed by apoplastic enzymes in the root cortex. Liberated free ABA can then be redistributed to the root symplast and dragged directly across the endodermis to the stele. Endogenous ABA conjugates are formed in the cytosol of root cells, transported symplastically to the xylem parenchyma cells and released to the xylem vessels. The mechanism of release is unknown; it may include the action of ABC-transporters. Because of its extremely hydrophilic properties, ABA-GE is translocated in the xylem of the stem without any loss to the surrounding parenchyma. After arrival in the leaf apoplast, transporters for ABA-GE in the plasmalemma have to be postulated to redistribute the conjugates to the mesophyll cells. Additionally, apoplastic esterases can cleave the conjugate and release free ABA to the target cells and tissues. The activity of these esterases is increased when barley plants are subjected to salt stress. Key-words: ABA-GE; apoplast; leaf; stem; xylem transport. Abbreviations: ABA, abscisic acid; ABAxyl, ABA in the sylem sap; ABA-GE, abscisic acid glucose ester; ABAGExyl, abscisic acid glucose ester in the sylem; IWF, intercellular washing fluid. stantially high concentrations. In vacuoles they are trapped because of their hydrophilic properties and withdrawn from further metabolism. ABA glucose ester and other non-hydrolysable unidentified conjugates were detected by Kaiser, Weiler & Hartung (1985) in vacuoles of barley mesophyll cells. Hormone conjugates, however, can also serve as longdistance transport forms in the xylem fluid. The major transport form of cytokinins in the xylem is the zeatin riboside (Fußeder et al. 1992; Bano et al. 1993, 1994; Kamboj, Blake & Baker 1998). Gibberellins are conjugated predominantly with glucose. They have been detected in the bleeding sap of trees by Dathe et al. (1982). Auxin conjugates have never been reported to be translocated in the xylem. Recently, much attention has been paid to ABA conjugates in the xylem sap of various plants. Munns & King (1988) and Munns et al. (1993) reported that an unidentified ABA conjugate, the ‘adduct’, is more important as a longdistance stress signal than free ABA. After arrival in the leaf tissue, this complex form is believed to be physiologically more active than ABA. Bano et al. (1993, 1994) were the first to identify ABA glucose ester (ABA-GE) in the xylem sap of rice and sunflower plants. Other examples for xylem-transported ABA conjugates are cited and discussed below. Nothing is known about the origin of root-borne ABA conjugates, their radial transport in roots to the xylem elements, their transport in the xylem vessels of the stem, as well as their fate in the leaf apoplast. Figure 1 presents a schematic plant, numbering several factors that influence transport and formation of ABA-GE. This article discusses the present knowledge and open questions of this potentially important group of hormonal long-distance stress signals. INTRODUCTION Plant hormone homeostasis is largely maintained by their biosynthesis and metabolism. ABA can be degraded oxidatively to phaseic acid (PA) and dihydrophaseic acid (DPA) and auxin to 3-methyleneoxindole. Gibberellins are converted to less active or inactive gibberellin metabolites. An alternative mechanism of biotransformation of hormones is their conjugation with sugars, amino acids and peptides. These conjugated forms are usually physiologically inactive and deposited in plant vacuoles where they can reach subCorrespondence: W. Hartung. Fax: + 49 931 888 6158; e-mail: [email protected] © 2002 Blackwell Science Ltd CONJUGATED ABA IN TRANSPORT FLUIDS Over the last decade ABA conjugates have been analysed in the xylem sap of various plants (Table 1). Bano et al. (1993) first reported a rise in concentration of conjugated ABA in the xylem sap of non-irrigated sunflower and rice. After rewatering the concentration of free and bound ABA decreased again. The conjugates of the major ABA metabolites PA and DPA were also present in sunflower xylem sap. Salt stress has also been shown to increase ABA-GExyl in barley and maize plants (Dietz et al. 2000; Sauter, unpub223 224 A. Sauter et al. C leaf cc st tr 1 B stem phloem xylem A root palisade parenchyma 2 stoma 2 ABA-GE ABA ß-D-glucosidase ABC-transporter ? xylem phloem 1 1 2 4 3 exodermis rhizodermis cortex 5 endodermis xylem pericycle Figure 1. A schematic presentation of the origin, the transport and the fate of ABA-GE in a vegetative plant. The arrows indicate the flows of ABA-GE (red) and free ABA (blue) in roots (a), stems (b) and leaves (c). The numbering refers to the text. Extracellular enzyme activity is coloured light green, the putative transporter of the xylem parenchyma plasma membrane light blue. The cell wall (grey), the thickenings of the endodermis and the Casparian band of the exo- and endodermis are yellow. In the leaf (c) a schematic vascular bundle, the crosssectioned palisade parenchyma cells and a stoma are presented. The colouring is as in (a). cc, companion cell; st, sieve tube; tr, tracheid. Table 1. Content of free and bound ABA in xylem sap of different plants coping with various stress conditions Species Rice a Sunflowerb Barleyc Maized Anastatica hierochunticae Maizef Betula pendulag Treatment Free ABA (nM) Well watered Drought-stressed Rewatered Well watered Drought-stressed 24 h Rewatered 2–3 h Control 50 mM NaCl 100 mM NaCl Control 100 mM NaCl Control 250 mM NaCl +40 mM CaCl2 Control Single-rooted plants Control Dry 7·2 92 52 10 252 32 1·8 2·2 3·8 2·8 5·5 3·7 42·9 30·9 4·1 31 829 ± ± ± ± ± ± ± ± ± ± ± 1 3 12 0·6 7·5 0·5 0·6 0·3 0·2 0·5 0·4 ± ± ± ± 6·1 0·4 17 267 Bound ABA (nM) 73 499 87 120 546 145 0·3 0·3 0·9 1·5 2·5 0·2 33·9 0·4 1·0 15 263 ± ± ± ± ± ± ± ± ± ± ± 7·5 75 5·7 0·6 104 2·2 0·1 0·2 0·2 0·3 0·2 ± ± ± ± 0·1 0·2 16 63 Ratio ABA : ABA-conjugate 0·1 0·2 0·6 0·1 0·5 0·2 5·8 7·3 4·4 1·8 2·2 16·1 1·3 77 4 2·1 3·2 a Bano et al. 1993; bBano et al. 1994; cDietz et al. 2000; dSauter, unpublished results; eHartung & Jeschke (1999); fJeschke et al. 1997b; gFort et al. 1998 © 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 223–228 Xylem transport of abscisic acid conjugates 225 Xylem Table 2. Concentration of ABA and ABA- Phloem Treatment Free ABA (nM) Bound ABA (nM) Free ABA (nM) Bound ABA (nM) Control Low P 180 ± 40 1100 ± 800 1·4 ± 0·3 4·4 ± 0·3 1800 ± 100 8300 ± 2300 90 ± 30 160 ± 30 lished results). A substantial rise of ABA-GExyl was observed by Grimmer (1993; cited by Hartung & Jeschke 1999) in the winter annual desert plant Anastatica hiërochuntica that had been treated with a mixture of NaCl and CaCl2 in similar concentrations to those that have been found in the solution of the soils of the original desert habitat. Here the ratio of free ABA : conjugated ABA decreased from 16·1 : 1 in control plants to 1·3 : 1 in saltstressed plants. ABA-GE was also present in the xylem sap of maize plants that were supplied by their seminal roots only (Jeschke et al. 1997a). These plants suffer from drought stress in the leaves although the roots have been watered sufficiently. Single-rooted plants transported ABA-GE in a higher concentration and the ratio of free ABA : conjugated ABA was reduced from 77 : 1 in control plants to 4 : 1. Fort et al. (1998) investigated seedlings of Betula pendula cultivated in a split root system. As the soil of both compartments started to dry ABA-GExyl increased as well as free ABA. Jeschke et al. (1997a) have investigated both xylem and phloem sap of phosphate-deficient Ricinus communis L. (Table 2). Under these condition xylem sap concentration of ABA-GE rose 3·1-fold. Phloem sap concentration of control plants was higher by a factor of 64 in comparison with the xylem, the ABA-GE concentration in the phloem of phosphorus-deficient plants was enhanced 1·8-fold. From these data it was concluded that leaves may also be a source for ABA-GExyl (Fig. 1c). Conjugated ABA in the xylem was also increased in castor beans when plants were supplied with N by foliar application only (Hartung, Peuke & Davies 1999). Ammonium spraying proved to be more effective than nitrate spraying (Peuke & Hartung unpublished results). It can be concluded from this compilation that ABA conjugates in the xylem are widely or even ubiquitously distributed among plants and that their fraction of the total ABA pool consisting of bound and free ABA frequently increases under stressful growth conditions. NATURE OF THE ABA CONJUGATE Munns et al. (1993) postulated that root-borne ABA might be travelling in the xylem in a complexed form (the ‘adduct’) and that this compound exhibits a stronger inhibitory activity on stomatal conductance than free ABA. Under conditions of storage this substance changed from a low molecular weight precursor into a highly polymerized © 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 223–238 GE in stem base xylem sap and phloem sap of Ricinus communis L. grown under control and phosphorus deficient conditions (after Jeschke et al. 1997b) compound. However, the exact chemical structure of this substance remains unknown. Hansen & Dörffling (1999) investigated the content of conjugated ABA in the xylem sap of drought-stressed sunflower plants by high-performance liquid chromqtography. They detected five conjugates in the xylem sap of wellwatered plants. The sap of drought-stressed plants contained six ABA conjugates. Some conjugates were alkaline stable and could only be cleaved by the enzyme β-glucosidase. Thus the enzymatic test characterized these ABA conjugates as ABA glucose esters with β-glucosidic linkages. Baier, Gimmler & Hartung (1990) have shown that the permeability coefficient of guard cell membranes of Valerianella locusta is extremely low for ABA-GE (PABA-GE ∼ 10−8 ms−1). Bearing in mind that the basic permeability of the guard cell plasmalemma seems to be higher than that of the mesophyll and the root cortex by a factor of 10 and 100, respectively (Baier et al. 1990; Daeter, Slovik & Hartung 1993) biomembranes can be regarded as impermeable for ABA-GE. Therefore, the question arises by which mechanism the ABA-GE that is synthesized in the cytosol of the root cortex, is released to the stelar apoplast and the xylem. During xylem transport in the stem no loss to the surrounding tissues is to be expected as may happen in the case of free ABA. Having arrived in the leaf two mechanisms could be involved in the avoidance of extremely high apoplastic concentrations of ABA-GE. First, the transporters for ABA-GE, located in the mesophyll plasmalemma could redistribute the conjugates to the mesophyll and second, apoplastic glucosidases could cleave the physiologically inactive conjugates and release free ABA to their targets, the stomata or the growing cells of young leaves. RADIAL TRANSPORT OF ABA GLUCOSE ESTER IN ROOTS ABA glucose esters in the xylem can originate from external or internal sources. The soil solution under different crops contains ABA-GE in concentrations up to 35 nM. Sauter & Hartung (2000) have shown that external ABAGE is not transported laterally to the stele in significant amounts even when roots lack an exodermis such as roots from hydroponically cultivated maize seedlings (Fig. 1a, 2). When lateral water flow in the root systems was increased by applying vacuum to the mesocotyl of decapitated seedlings in the presence of 100 nM ABA-GE in the external medium, its concentration in the xylem decreased. Simultaneously, however, ABAxyl increased, suggesting cleavage 226 A. Sauter et al. Tissue Table 3. ABA-GE content of isolated Medium Treatment Cortex (pmol g−1 FG−1) Stele (pmol g−1 FG−1) Cortex (pmol m−2) Stele (pmol m−2) 1 2 3 7·8 ± 3·1 6·5 ± 3·7 16·7 ± 7·2 15·6 ± 3·7 27·7 ± 8·8 47·0 ± 13·8 0·4 ± 0·2 0·4 ± 0·2 0·8 ± 0·3 1·0 ± 0·3 2·1 ± 0·9 4·3 ± 1·5 of the conjugate by an apoplastic esterase in the cortex (Fig. 1a, 1). Indeed the intracellular washing fluid of maize roots contained a β-glucosidase which could be inhibited by ABA-GE. The same type of experiment was performed with root systems of aeroponically cultivated maize seedlings. Free ABA did not increase in the xylem sap. It was concluded that the Casparian bands formed in the hypodermis of aeroponically cultivated maize plants (Freundl, Steudle & Hartung 1998; 2000) prevent the uptake and radial transport of ABA-GE in roots (Fig. 1a, 3). These results show that for the extremely hydrophilic ABA-GE both the endodermis and the exodermis are perfect transport barriers. Applying the flux equation of radial ABA-flow in roots (Freundl et al. 1998) the reflection coefficient σ of ABA-GE for maize roots is 1·0. Therefore ABAGE formed in the cytosol of cortical cells must be translocated symplastically to the stele (Fig. 1a, 4). Having arrived in the xylem parenchyma cells ABA-GE must be released across the plasma membrane to the stelar apoplast. Stelar tissues of maize release ABA-GE with rates that can be three to five times higher than from cortical tissues (Table 3). Efflux of ABA-GE from isolated stelar tissues of maize is particularly high under conditions when ABA degradation is inhibited. In experiments with intact plants, salt stress (100 nM) stimulated ABA-GEefflux to the xylem three-fold. The mechanism of the membrane transport of ABA-GE remains unknown. Sauter & Hartung (2000) speculated that transmembrane transport of ABA conjugates could be mediated by ABC-transporters (Fig. 1a, 5). Sidler et al. (1998) provided evidence that an AtPGP1 transporter is localized in the plasmalemma of Arabidopsis thaliana seedlings in both root and shoot and perhaps involved in hormone-regulated developmental processes. According to preliminary data of Enrico Martinoia (Neuchatel, CH, personal communication) membrane transport of another hormone conjugate, the indolyl-acetyl-aspartate, seems to be mediated by an ABC-transporter. FATE OF ABA GLUCOSE ESTER IN THE STEM Free ABA that is translocated over long distances in the xylem through stems may be redistributed to the surrounding parenchyma, especially when the xylem sap is acid (Fig. cortical and stelar tissue segments after preincubation of intact root segments for 24 h with (1) 5 nM ABA, (2) 50 nM ABA and (3) 50 nM ABA plus tetcyclacis. The tissue ABA-GE concentrations were determined immediately after preloading, the medium was analysed after preloaded segments were kept for 120 min in a hormone-free medium (after Sauter & Hartung 2000) 1b, 2). This could result in a significant ABA loss during xylem transport. Indeed, Jokhan, Harink & Jackson (1999) and Jeschke & Hartung (2000) were able to show that during transport in the stem the ABA signal can be weakened significantly. The ABA-GE-molecule, however, because of its physico-chemical properties must be expected to stay in the xylem unchanged until its arrival in the leaves, as long as there is no glucose esterase activity in the xylem sap (Fig. 1b, 1). Indeed, xylem saps, both from stressed and unstressed plants were absolutely free of any enzymatic activity that could release ABA from its glucose esters. Sauter and Hartung (unpublished results) have perfused 10 cm lengths of isolated second internode segments of Phaseolus coccineus with buffers that contain ABA-GE, in concentrations that might be expected under stress conditions, by application of vacuum to the apical cut surface. ABA-GE passes the bean internodes unchanged without being taken up by the stem tissues (Fig. 1b, 1). ABA-GE proved to be perfectly suited for efficient long-distance transport. FATE OF ABA GLUCOSE ESTER IN THE LEAF As discussed above ABA-GE can only play a role as a hormonal stress signal when ABA is released from the physiologically inactive conjugate by apoplastic enzymes (Fig. 1c, 1,2). The action of enzymes that cleave hormone conjugates in the leaf apoplast becomes evident by comparing the ratios of free ABA : conjugated ABA in the xylem sap (4– 7) of barley and intracellular washing fluids of barley leaves (22–32; Table 4). Intercellular washing fluids (IWF) are Table 4. Eleven-day-old barley seedlings were grown at varying salt concentrations. Xylem sap of excised roots as wells an intercellular washing fluid of the primary leaves were analysed for free and bound ABA (after Sauter & Hartung 2000) ABA-GE (nM) Ratio of ABA : ABA-GE NaCl (mM) Xylem sap IWF Xylem sap IWF 0 50 100 0·32 ± 0·09 0·50 ± 0·26 1·31 ± 0·30 0·07 ± 0·01 0·09 ± 0·02 0·16 ± 0·03 5·8 7·3 4·4 22·3 32·7 29·4 © 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 223–228 Xylem transport of abscisic acid conjugates 227 PERSPECTIVES AND CONCLUDING REMARKS 1·0 0·9 This review shows that ABA conjugates must be discussed as important and effective hormonal stress signals that intensify the ABA long-distance signal. The following problems should be investigated in the future: (1) the chemical structure of all ABA conjugates of the xylem sap needs to be elucidated; (2) the transport of ABA conjugates across root and mesophyll plasma membranes should be investigated under control and stress conditions, including a possible involvement of ABC-transporters; and (3) apoplastic β-glucosidases that hydrolyse ABA-glucose esters need to be purified and characterized. Extinction (405 nm) 0·8 0·7 0·6 0·5 0·4 0·3 0·2 0·1 0·0 3 4 5 6 7 8 9 10 pH Figure 2. Isoelectric focusing of extracellular β-glucosidases of barley primary leaves. Intercellular washing fluid (IWF) was prepared as described previously (Dietz et al. 2000). The protein of 300 µL IWF protein was separated by isoelectric focusing in immobilized pH gradients (Immobilin strips 3–10, Pharmacia, Uppsala, Sweden). The strips were cut in sections and the activity was determined using the substrate p-nitrophenol-β-Dglucopyranoside. The data represent means of three replicates ±SD. ACKNOWLEDGMENTS We are grateful to Deutsche Forschungsgemenchaft (SFB 261, TP A3) for financial support and W. J. Davies (Lancaster) and Dr E. Hose for their interest and stimulating discussions and to Miss B. Dierich for technical help. REFERENCES known to contain a large set of glycosidases. For example, Holden & Rohringer (1985) detected 10 spots in twodimensional electropherograms of barley with activity towards p-nitrophenol β-glucoside substrate. Figure 2 demonstrates the occurrence of at least three isoforms of βglucoside hydrolases in intercellular washing fluid of barley leaves with isoelectric points between 6·4 and 7·4. The average apparent molecular mass was 50 kDa. A quick search in the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000) allows the identification of more than 30 genes assigned to β-glucosidases with targeting addresses to the secretory pathway, and even more genes coding for putative β-glucosidases. Thus, there exists a large family of βglucoside hydrolases in plants with mostly unknown substrate specificity. ABA glucose esters could either be cleaved by hydrolases with high selectivity towards hormone conjugates or by β-glucosidases with rather low substrate selectivity. Indeed, Dietz et al. (2000) detected an extracellular β-glucosidase activity in barley leaves that is able to release free ABA from its conjugate. This enzyme activity increased seven-fold when plants were salt stressed. An increase of apoplastic pH as it occurs in stressed leaves (Wilkinson & Davies 1997) also enhances the activity of this enzyme. 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