Journal of Experimental Botany, Vol. 51, No. 346, pp. 937-944,
May 2000
© 2000 Oxford University Press
Extracellular ß-glucosidase activity in barley involved in the hydrolysis of ABA glucose conjugate in leaves
1 Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
2 Lehrstuhl für Stoffwechselphysiologie und Biochemie der Pflanzen, Universität Bielefeld, Universitätsstraße 25, D-33501 Bielefeld, Germany
Received 20 August 1999; Accepted 31 January 2000
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Abstract |
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Abscisic acid conjugate concentrations increased in barley xylem sap under salinity, whereas it remained at a low level in the intercellular washing fluid (IWF) of barley primary leaves (Hordeum vulgare cv. Gerbel). Here it is shown that IWF contains ß-glucosidase activity which releases abscisic acid (ABA) from the physiologically inactive ABA-glucose conjugate pool in the leaf apoplast. The following data support this conclusion and give the first biochemical and physiological characterization of the extracellular glucosidase activity in barley. Free ABA was released by the incubation of ABA glucose ester with IWF. The product exhibited the retention time of authentic ABA upon separation by thin layer chromatography and was identified by ABA-ELISA. p-Nitrophenol-ß-D-glucopyranoside (pNPG) was used as the substrate for ß-glucosidases. The KM(pNPG) was 1.8 mmol l-1. The activity was affected by ABA glucopyranoside in a competitive type of inhibition with a KI of 400 µmol l-1. Various hormone conjugates were compared with respect to their inhibitory effect on beta;-glucosidase activity. Inhibition was highest for the ABA glucopyranoside and the zeatin riboside, but insignificant for ABA methyl ester and zeatin-9-ß-D- glucoside. The specific activity of the ß-glucosidase was 16-fold greater in IWF as compared to crude leaf extracts confirming its extracellular compartmentation. The activity of ß-glucosidase was strongly increased after growth in hydroponic medium supplemented with NaCl. The data support the hypothesis that the glucose conjugate is a long-distance transport form of ABA.
Key words: Abscisic acid, abscisic acid glucose ester, apoplast, ß-glucosidase, barley, salt stress, root : shoot signalling.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Abscisic acid (ABA) functions as a stress hormone in higher and lower plants. The kinetics of the tissue response following the exogenous addition or endogenous increase of ABA may be fast on a time scale of less than 1 min. For example, ABA induces rapid stomatal closure as a short-term response to water deficit (Raschke, 1975
; Blatt, 1990). On the other hand, ABA is a mediator of the long-term adaptation of plants by altering gene expression and inducing drought and frost hardiness (Bartels et al., 1988; Reany et al., 1989; Hollenbach et al., 1997). Various factors determine the ABA concentration at the site of action, particularly the rate of synthesis, long-distance transport, tissue susceptibility, compartmentation, conjugation, and oxidative degradation of ABA (Hetherington and Quatrano, 1991; Hartung and Slovik, 1991).
Conjugation to glucosyl- and methyl-groups metabolizes ABA to a physiologically inactive form (Boyer and Zeevaart, 1982). ABA conjugates accumulate in plant tissues with age and during stress treatments. The conjugates are mainly compartmentalized into the vacuole (Kaiser et al., 1985) where they constitute a rather inert pool of ABA which was even suggested to serve as a direct indicator of the previous stress history of the plant. Conjugated ABA gradually increased with age in the leaves of 4-month-old Hyoscyamus niger plants and represented almost 95% of the total ABA pool in the oldest senescent leaf (Weiler, 1980). A similarly high content of conjugated ABA was detected in the primary leaves of 27-d-old bean plants.
However, ABA conjugates are not only associated with the vacuole, but are also found in the xylem sap of stressed plants (Bano et al., 1993, 1994; Jeschke et al., 1997; Hartung and Jeschke, 1999; Sauter and Hartung, 2000). The function of the extracellular pool of ABA conjugates is unknown. Different forms of conjugates occur in the xylem sap, ABA glucose ester being by far the most dominant form under drought (Hansen and Dörffling, 1999). Three possible fates of this extracellular ABA glucopyranoside must be discussed. (1) The ABA conjugate may accumulate in the aqueous phase of the cell wall. Consequently, the concentration of ABA conjugate should increase in the apoplast with age and stress phases similar to the vacuolar pool. However, such an accumulation is not observed. (2) The ABA conjugate may be imported into the cells. Until now, no such transport system has been identified in the plasma membrane of leaf cells. Conversely, transport of the ABA glucose ester is considered to be insignificant across plasma membranes because of their extremely low permeability for ABA-GE (Baier et al., 1988). (3) The ABA conjugates may be cleaved enzymatically once they reach the apoplasmic space of the leaves by the transpiration stream. The liberated ABA may then be taken up into the cells and induce metabolic changes or may act on guard cells from outside. The apoplasmic space is an extraplasmic compartment with typical hydrolytic activities (Boller and Kende, 1979; Dietz, 1996). Examples of such apoplasmic enzyme activities are the 
-mannosidase, - and ß-galactosidase and phosphodiesterase. The present work aimed at identifying an apoplasmic enzyme activity possibly involved in the metabolism of ABA glucopyranoside by catalysing the hydrolysis of ABA glucose conjugates. Such an enzyme would then be likely to play a role in the ABA-dependent stress adaptation of plants.
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Material and methods |
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Plant growth
Barley was grown in soil culture in a growth cabinet at a 16/8 h light/dark cycle. If not stated otherwise the analyses were performed with leaf extracts of 10-d-old seedlings. In order to investigate the effect of elevated salt concentrations, plants were cultured in hydroponic medium as described previously (Brune et al., 1994
). The basal medium which contained about 0.46 mM Na+ from added trace element salts was supplemented with NaCl as indicated.
Isolation of intercellular washing fluid and xylem sap
Barley primary leaves were infiltrated with a solution containing 1 mmol l-1 CaCl2 at about 10 Pa for 10 min. Following the release of the vacuum, the surface of the infiltrated leaves was dried carefully with tissue. The leaves were placed into tubes with perforated bottoms which were inserted into a larger centrifugation tube. The leaves were spun at 1000 g for 15 min. The intercellular washing fluid was collected from the bottom of the larger centrifugation tube and either immediately used for the experiments or frozen at -80 °C until further use. Xylem sap was isolated from the cut surface of the mesocotyl of 11-d-old plants using a suction technique at -0.045 MPa as described previously (Freundl et al., 1998).
Determination of enzyme activity
The standard enzyme assay contained 400 µl of a 100 mmol l-1 citrate buffer adjusted to pH 4.8 with KOH. p-Nitrophenol-ß-D-glucopyranoside was dissolved in dimethylformamide at a stock concentration of 200 mg ml-1. 10 µl substrate stock solution and usually 12.5 µl IWF-sample were added to the buffer. After 60 min of incubation at 37 °C, the reaction was terminated by addition of 1 ml of 200 mmol l-1 Na2CO3 solution. The amount of liberated p-nitrophenol was quantified spectrophotometrically at 405 nm using the molar extinction coefficient 
=18 300 
Absxlx(mol cm)-1 (Del Campillo and Shannon, 1982).
Quantification of ABA and ABA-conjugate by ELISA
Water was added to the incubation mixture to a final volume of 1 ml; the pH was adjusted to pH 2.5 with HCl. Lipophilic compounds were extracted three times with 1 ml ethylacetate and the combined fractions dried in a vacuum concentrator. The samples were taken up in TBS buffer (TRIS-buffered saline, 50 mmol l-1 TRIS, 150 mmol l-1 NaCl, 1 mmol l-1 MgCl2, pH 7.8), as described earlier (Weiler, 1986). The aqueous fractions which contain ABA-conjugate were hydrolysed with NaOH (1 M) for 1 h, adjusted to pH 3.0 with HCl and partitioned against ethyl acetate as described above. ABA, released from ABA conjugates was determined by ELISA.
Synthesis of radiolabeled ABA-ß-D-glucopyranoside and the analysis of ABA and ABA-metabolites by thin layer chromatography
ABA-ß-D-glucopyranoside was synthesized from 0.4 µmol [14C[ABA in 200 µl acetone by the addition of 0.4 µmol diethanolamine and 0.4 µmol -acetobromoglucose at room temperature for 24 h according to the method of Lehmann and Schütte (Lehmann and Schütte, 1977). The reaction was stopped by the addition of 50 mmol l-1 NaHCO3, followed by extraction with ethylacetate. Acetyl groups were cleaved with the esterase extract of Helianthus annuus. ABA glucose ester was purified by thin layer chromatography (silica gel 60, F254, Merck, Darmstadt, Germany, toluol : ethylacetate : methanol : acetic acid, 50 : 30 : 7 : 4, by vol.) and purified as described previously (Daeter and Hartung, 1995). 800 Bq of purified [14C]ABA glucose ester were incubated with 300 µl IWF of barley primary leaves. Following an incubation at 22 °C for 5 h and 22 h, released free ABA was analysed by TLC as described previously (Daeter and Hartung, 1995).
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Results |
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ABA and ABA conjugate contents of xylem sap and intercellular washing fluid of barley seedlings
When root systems of 11-d-old barley seedlings were stressed with 50 and 100 mM NaCl, both ABA and ABA-conjugates of the xylem sap increased substantially. Free ABA ranged from 1.8 nM (control) up to 5.8 nM (100 mM NaCl) and conjugated ABA ranged from 0.3 nM (control) to 1.3 nM (100 mM NaCl), with a ratio of ABA/ABA-conjugate of 4–7. It is known from the investigations of Hansen and Dörffling that ABA glucose ester constitutes the main fraction of the ABA conjugate pool (Hansen and Dörffling, 1999
).
The apoplastic fluid harvested from the leaves of the same plants exhibited similar ABA concentrations as the xylem sap. The extracellular concentration of ABA-conjugate, however, was below 0.2 nM resulting in ABA/ABA-conjugate ratios of 22–32 (Fig. 1). It should be noted that barley cv. Gerbel is moderately salt-tolerant and showed no major growth inhibition in the presence of 100 mM NaCl.
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Barley leaves contain ß-D-glucosidase in the apoplast
Intercellular washing fluid of 10-d-old barley seedlings was assayed for ß-D-glucosidase activity using p-nitrophenol-ß-D-glucopyranoside as a substrate. In the mean of eight independent determinations, with three replicates each, the activity was 1.01±0.33 pmol p-nitrophenol being released µl-1 IWF s-1 (mean ±SD). The ß-D-glucosidase activity was characterized in respect to affinity, pH-optimum and age dependence (Fig. 2). The rate of hydrolysis increased with substrate concentration yielding half saturation at 2.4 mmol l-1 p-nitrophenol-ß-D-glucopyranoside. The pH-dependency revealed maximum activity at pH 5.5. A pronounced shoulder was seen at pH 5. This indicates the existence of isoforms. IWF was isolated from barley primary leaves between 8 and 15 d after sowing. The activity of ß-D-glucosidase declined with leaf age. In primary leaves of 2-week-old barley seedlings the activity was only about half of that in IWF of 1-week-old plants. Younger seedlings were not investigated since IWF in sufficient quantities could not be isolated from primary leaves at that stage of development due to their small size. The specific activity of ß-D-glucosidase was determined in IWF and crude leaf extracts using p-nitrophenol-ß-D-glucopyranoside as a substrate. In IWF, the activity was 5.77±0.73 nmol mg-1 protein s-1 (mean±SE, n=5) and 0.36±0.02 nmol mg-1 protein s-1 (mean±SE, n=5) in crude extracts of the same leaves. Thus, the specific activity was enriched 16-fold in IWF as compared to crude extract.
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Competitive inhibition of barley apoplast ß-D-glucosidase by ABA glucose ester
The question of a possible function of the apoplasmic ß-D-glucosidase in ABA conjugate metabolism was first addressed in competition experiments (Fig. 3). The ß-D-glucosidase assay with p-nitrophenol-substrate was supplemented with increasing concentrations of ABA glucose ester. Two concentrations of p-nitrophenol substrate were tested, 0.1 and 0.2 mg ml-1 corresponding to 0.33 and 0.66 mmol l-1 p-nitrophenol-ß-D-glucopyranoside. ABA glucose conjugate was added at concentrations of 1 to 5 mM. The activity of the p-nitrophenol-ß-D-glucopyranoside-dependent ß-D-glucosidase decreased with increasing ABA conjugate concentration. However, the ß-D-glucosidase activity appeared not to approach zero for high (infinite) ABA glucopyranoside concentrations. As far as can be deduced from the two concentration dependences, the ABA glucopyranoside-insensitive ß-D-glucosidase-activity in the IWF was independent of the substrate concentration at the two concentrations used in the assay and amounted to about 0.08 pmol µl-1 s-1. The ABA glucopyranoside-insensitive ß-D-glucosidase, therefore, seemed to have a higher affinity towards p-nitrophenol-ß-D-glucopyranoside than the ABA glucopyranoside-sensitive ß-D-glucosidase. Its activity was close to maximum both at 0.33 and 0.66 mmol l-1 p-nitrophenol-ß-D-glucopyranoside. The assumed rate of the ABA glucopyranoside-insensitive ß-D-glucosidase-activity was subtracted from the total rate to estimate the ABA glucopyranoside-sensitive activity
of the ß-D-glucosidase. The data were plotted as 1/ versus the ABA glucopyranoside concentration (Fig. 3B). From the intersection of the two regression lines, the Ki of the ß-D-glucosidase for ABA glucopyranoside was calculated to be 400 µmol l-1.
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Other hormone conjugates were compared with the ABA glucopyranoside with regard to their inhibitory effect on the ß-D-glucosidase (Fig. 4
). The inhibition decreased in the order of ABA glucopyranoside, zeatin riboside, 6-benzyladenine glucopyranoside and was insignificant for ABA methyl ester and zeatin glucopyranoside. The distinct pattern of inhibitory activity of the various conjugates was indicative of a role of the ß-D-glucosidase in ABA glucopyranoside metabolism.
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Hydrolysis of ABA from ABA conjugate by apoplast ß-D-glucosidase
Finally, experiments were performed to demonstrate immediate hydrolysis of ABA glucopyranoside by IWF proteins. ABA glucopyranoside was added at a concentration of 10-6 mol l-1 to IWF and incubated at the physiological pH of 6.5 for up to 1 h and 4 h. The reaction mix was analysed by ELISA. The monoclonal antibody used in the ELISA assay did not react with ABA glucopyranoside at this low concentration. As can be seen from Fig. 5A, ABA was released from the conjugate. The amount of ABA released increased with incubation time, however, not linearly, since after 4 h of incubation only the 2.5-fold amount of ABA was released as compared to 1 h. In addition to the identification of free ABA liberated from ABA-GE by apoplastic ß-glucosidase in the ELISA, an independent experiment was performed with radiolabelled ABA glucopyranoside. The products were analysed by chromatography on a thin layer of silica gel where a scanner for ß-particle emission was used (Fig. 5B). The sample loaded without incubation exclusively revealed the peak of [14C]-ABA glucopyranoside with the corresponding high retention factor (lane 1). After 5 h of incubation, 59% of the radiolabel was associated with a product separating with a lower retention factor than ABA glucopyranoside (lane 2). The RF value of the hydrolysis product was identical with the RF-value of authentic free [14C]-ABA (lane 4).
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Increase in apoplast ß-D-glucosidase activity under salinity
ABA glucopyranoside concentrations in xylem exudates are known to increase when plants are grown at elevated salt concentrations (Hartung and Jeschke, 1999). Under the assumption of a physiological role of the ABA glucopyranoside in signalling the stress situation from the roots to the shoot there is an argument for an increased requirement of hydrolysis of ABA glucopyranoside in the shoot apoplast under salinity. Barley seedlings were grown for 8 d in hydroponic medium containing NaCl at concentrations of up to 200 mM. IWF was extracted and analysed for ß-glucosidase using p-nitrophenol-ß-D-glucopyranoside as substrate (Fig. 6). The apoplasmic ß-glucosidase activity of NaCl-less grown barley was normalized to 100%. The activity decreased slightly when NaCl was added to the growth medium at the low concentration of 25 mM. At higher salt concentrations in the growth medium, the ß-glucosidase activity rose considerably with increasing salinity and reached the maximum with about 700% of the control at 150 mM. No significant stimulation of ß-glucosidase activity was seen upon increasing the NaCl concentration from 150 mmol l-1 up to 200 mM.
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Discussion |
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Conjugation is an important mechanism to inactivate plant hormones. The main portion of ABA glucopyranoside is compartmented into the vacuole where it is believed to be stored permanently as a terminal metabolite of ABA. Another portion of ABA glucopyranoside is synthesized in the roots and transported in the xylem to the shoot (Hansen and Dörffling, 1999
; Fig. 1
). Until now it was not known whether and how this ABA metabolite participates in inter-organ signalling. The data presented in this communication provide evidence that ß-glucosidase activity is expressed in barley leaves which hydrolyses ABA glucopyranoside in the apoplast thereby releasing the physiologically active free ABA. The liberated ABA may be taken up into the cells and trigger responses involved in the adaptation of the plant to the prevailing stress condition, in addition to free ABA. Two main questions need to be discussed in that context. First, what is the advantage of transport of ABA conjugate under stress as compared to transport of free ABA? Formation of glucosylconjugates requires two equivalents of ATP. One mol of ATP is needed per mol glucose to synthesize glucose-6-phosphate which then, in turn, is activated by UTP to yield UDPglucose under release of pyrophyophate. UDPglucose is the substrate of the glycosylation reaction. The extra energy consumption for ABA glucopyranoside synthesis should be balanced by physiological advantages of ABA glucopyranoside transport. The second question is addressed to the proposed role of the apoplasmic ß-glucosidase in ABA metabolism. How do the enzymic properties relate to this function? Answers to both questions will be discussed in the following two paragraphs.
The role of ABA glucopyranoside in plants
In animal sciences, the definition of hormones was originally based on a distinction of the site of synthesis and release from the, usually distant, site of action. Plant growth regulators such as abscisic acid are not hormones in that strict sense since most plant cells respond to the plant hormones, for example to abscisic acid, with a general or specific programme of physiological changes (Hetherington and Quatrano, 1991). Glycosidic conjugation of abscisic acid at the site of synthesis and liberation by glucosidases at the target site, however, would essentially fit to the definition of homones known from animal sciences. Following its synthesis in the root cells, the addition of the glucosyl residue to the free form inactivates the messenger abscisic acid, so that the ABA glucopyranoside does not induce genetic responses in the roots. A second advantage of conjugate synthesis may be related to the transport process of ABA. The distribution of free ABA across biomembranes is mainly dependent on pH gradients. The protonated form of ABA is highly hydrophobic and easily permeates biomembranes. Conversely, biomembranes are highly impermeable for the ABA anion. As a consequence, ABA is trapped in alkaline compartments of the cells. ABA redistribution in light/dark-transients and under stress may essentially be understood on the basis of this simple model of ABA diffusion (Hartung and Slovik, 1991; Slovik and Hartung, 1992). Until now, a transporter for abscisic acid has only been identified in the plasma membrane of the leaf epidermis (Daeter and Hartung, 1993) and extreme root tips (Astle and Rubery, 1980). This transporter facilitates ABA uptake at a slightly alkaline pH of the surrounding medium (Daeter and Hartung, 1993). Since the cytoplasmic pH usually is more alkaline than the apoplast (Pfanz and Dietz, 1987), the proton gradient from the apoplast to the cytoplasm favours ABA accumulation in the cytoplasm. The pH-value of the IWF isolated and used in this study was about 6.5. ABA is only released from the cells along the gradient. Glycosylation of ABA may be a mechanism to allow for the export of ABA from the cells independent of the prevailing cytoplasmic proton concentration and transmembrane proton gradients. Various glycosylated metabolites are exported from the cytoplasm into the extraplasmic compartments. For example, phenolic glycosides are transported into the vacuole and apoplast where they are stored and function as UV protectants or phytoalexins (Keller et al., 1997; Schnitzler et al., 1996; Dietz et al., 1994). Transporters with a general substrate specificity for glycosylated compounds are likely to be involved in catalysing the export of ABA glucopyranoside from the cytoplasm of the parenchyma cells of the central cylinder in the roots into the xylem.
The enzymic properties of the ß-glucosidase
The apoplasmic ß-D-glucosidase has an acidic pH optimum with low residual activity above pH 7. The IWF of barley leaves has a pH of about 6.2 to 6.5 which is suboptimal for the ß-D-glucosidase. Further alkalization as reported to occur under drought stress (Hartung and Slovik, 1991) will decrease the remaining ß-D-glucosidase activity. Therefore, it was important to investigate whether hydrolysis of ABA glucopyranoside can occur after incubation with unbuffered IWF. The result presented in Fig. 5 reveals a capacity of the apoplast to hydrolyse ABA glucopyranoside even at low (10-6 M) substrate concentrations, despite the apparently high dissociation constant of the ABA-glucopyranoside/ß-glucosidase complex of about 400 µM.
Another important question to be discussed relates to the specificity of the apoplasmic esterases. The competition experiments of Fig. 4 show that, within the hormone conjugates tested, ABA-GE is most active. With the exception of zeatine riboside all the other conjugates exhibited a substantially lower competitive effect than ABA-GE. Despite this observation, there is no evidence available which would allow the argument for an ABA-GE-specific ß-glucosidase. Conversely, it appears more likely that ß-glucosidase(s) with a broad substrate specificity also hydrolyse ABA-GE.
The specific activity of the ß-glucosidase was increased 16-fold in IWF compared to the leaf extract. The contamination of IWF with cytosolic material is below 0.5% (Mimura et al., 1990). A comparison of both figures prove that (i) the ß-glucosidase activity in the IWF is not a contamination by symplastic material but, nevertheless, that (ii) ß-glucosidase activity is also contained in the symplast. Preliminary experiments show three peaks of ß-glucosidase activity following separation of IWF proteins in native isoelectric focusing gels, whereas the predominant activity in crude extracts focuses as one peak at a more acidic pH (data not shown).
Hartung and Jeschke showed that salt stress especially, applied in combinations of NaCl and CaCl2, increased the concentration of ABA-GE in the xylem sap of the desert plant Anastatica hiërochuntica dramatically (Hartung and Jeschke, 1999). A similar result was obtained in salt-stressed maize plants (Sauter and Hartung, 2000). In this paper it is shown that there exists a radial transport pathway for ABA-GE from the root cortex to the root central cylinder. The finding that salt treatment increased ABA-glucose-esterase activity in the barley leaf IWF may be important in this context, because this could be an additional mechanism to increase the ABA concentration at the primary site of action. In this context it was also an important observation that ABA-conjugate concentrations of IWF are lower by a factor of 5–8 compared to the xylem sap. This is in accord with an efficient cleavage of ABA-GE in the leaf apoplast after import from the xylem.
In conclusion, the results presented here are in full support of the hypothesis that the extracellular ß-glucosidase of barley leaves serves a specific role in ABA-dependent root:shoot signalling in plants, supplementing the signalling pathway of free ABA.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereiche 251 TP A2 of the University of Würzburg and SFB 549 TP A6 of the University of Bielefeld) and the Graduiertenkolleg. Excellent technical assistance by Ms Barbara Dierich and Ms Martina Holt is gratefully acknowledged.
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Notes |
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3 To whom correspondence should be addressed at the University of Bielefeld. Fax: +49 521 106 6039. E-mail:karl-josef.dietz{at}biologie.uni-bielefeld.de
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References |
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F. Jiang and W. Hartung Long-distance signalling of abscisic acid (ABA): the factors regulating the intensity of the ABA signal J. Exp. Bot., January 1, 2008; 59(1): 37 - 43. [Abstract] [Full Text] [PDF]
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M. A. Else, J. M. Taylor, and C. J. Atkinson Anti-transpirant activity in xylem sap from flooded tomato (Lycopersicon esculentum Mill.) plants is not due to pH-mediated redistributions of root- or shoot-sourced ABA J. Exp. Bot., September 1, 2006; 57(12): 3349 - 3357. [Abstract] [Full Text] [PDF]
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N. M. Holbrook, V.R. Shashidhar, R. A. James, and R. Munns Stomatal control in tomato with ABA-deficient roots: response of grafted plants to soil drying J. Exp. Bot., June 1, 2002; 53(373): 1503 - 1514. [Abstract] [Full Text] [PDF]
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A. Sauter and W. Hartung Radial transport of abscisic acid conjugates in maize roots: its implication for long distance stress signals J. Exp. Bot., May 1, 2000; 51(346): 929 - 935. [Abstract] [Full Text] [PDF]
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