Journal of Experimental Botany, Vol. 52, No. 364, pp. 2097-2103,
November 1, 2001
© 2001 Oxford University Press
Original Papers |
Abscisic acid-specific binding sites in the flesh of developing apple fruit
Laboratory of Molecular Developmental Biology of Fruit Trees, Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing 100094, PR China
Received 26 February 2001; Accepted 2 July 2001
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Abstract |
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Abscisic acid (ABA) specific-binding sites localized in the cytosol were identified and characterized in the flesh of developing apple (Malus pumila L. cv. Starkrimon) fruit. ABA binding activity was scarcely detectable in the microsomes but high ABA binding activity in the cytosolic fraction was detected. The ABA-binding sites possessed a protein nature with both active serine residues and thiol-groups of cysteine residues in their functional binding sites. ABA binding was shown to be saturable, reversible and of high affinity. A Scatchard plot provided evidence for two different ABA binding proteins, one with higher affinity (Kd=2.3 nM) and the other with lower affinity (Kd=58.8 nM). Phaseic acid, trans-ABA and (-)-ABA had essentially no affinity for the binding proteins, indicating their stereo-specificity to bind physiologically active cis-(+)-ABA. The time-course, pH- and temperature-dependence of the ABA-binding proteins were determined. It is hypothesized that the detected ABA-binding proteins may be putative ABA-receptors that mediate ABA signals during fruit development.
Key words: Abscisic acid, ABA-binding protein, apple fruit.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Abscisic acid (ABA) plays a major role in various aspects of plant growth and development, including seed maturation and germination, adaptation to environmental stresses, as well as fruit development (Coombe, 1976
, 1992; Davies and Zhang, 1991; Rock and Quatrano, 1995). It is well known that the ripening of climacteric fruits including apple is triggered by ethylene, but many studies have demonstrated that a sharp increase of ABA in fruits was followed by a dramatic increase of ethylene, and externally applied ABA significantly stimulated ethylene biosynthesis and hastened fruit ripening (Lieberman et al., 1977; Vendrell and Buesa, 1989; Buta and Spaulding, 1994; Martfnze-Madrid et al., 1996; Chen and Zhang, 2000). Thus, it is considered that ABA plays an important role in the regulation of fruit ripening. Another important effect of ABA on fruit physiology is to stimulate sugar accumulation in fruits, thus improving fruit yield and quality (Beruter, 1983; Yamaki and Asakura, 1991; Ofosu-Anim et al., 1996; Lu et al., 1999; Xia et al., 2000). However, although the conventional physiology and biochemistry of ABA action on fruit development has been well studied, the cellular and molecular mechanisms of ABA action remain unknown.
Recent advances in hormone signal transduction have considerably extended current knowledge of the processes of hormone action. As a plant hormonal signal, ABA should be firstly perceived by cells through binding to an extracellular or intracellular specific site or receptor (McAinsh et al., 1991; Michael and Valeria, 1993; Giraudat et al., 1994; Leung and Giraudat, 1998), and then the ABA-receptor complex can activate some intracellular messengers that transduce the ABA signal to target enzymes or target genes, resulting in short-term or long-term physiological responses (McAinsh et al., 1991; Giraudat et al., 1994; Cutler et al., 1996; Merlot and Giraudat, 1997; Leung and Giraudat, 1998; Li et al., 2000). In this ABA signal transduction cascade, the molecular perception of the ABA signal by cells is considered a key step (McAinsh et al., 1991; Michael and Valeria, 1993; Giraudat et al., 1994; Leung and Giraudat, 1998). A number of approaches have contributed to the detection and examination of the ABA-binding sites that are considered putative ABA receptors responsible for perceiving the ABA signal. ABA-binding sites have been identified in microsomes of leaves (Hocking et al., 1978), guard cell protoplasts of Vicia faba (Hornberg and Weiler, 1984), the cytosolic fraction of wheat (Veliev, 1991), microsomes of rice seedling (Chen et al., 1992), maize roots (Chen and Zhu, 1996), Arabidopsis thaliana leaves (Pedron et al., 1998) and grape berries (Zhang et al., 1999). In the present research ABA-binding sites were identified and characterized in the flesh of developing apple fruit.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material
Apple fruits (Malus pumila L. cv. Starkrimon) were harvested 60–90 d after full bloom from 5-year-old trees grown in a field located in the western suburbs of Beijing.
Preparation of microsomes and cytosolic fraction
Microsomes and cytosolic fractions were prepared following the method of Zhang et al. (Zhang et al., 1999) with modifications. The freshly harvested fruits were frozen with liquid N2 and then crushed. The crushed flesh was homogenized with cold medium in a volume ratio of 1:3 (flesh:medium). This medium contained 200 mM sucrose, 1 mM EDTA (ethylenediamine tetra-acetic acid, Sigma, St Louis, USA), 0.5% (w/v) casein, 2 mM DTT (dithiothreitol, Sigma), 0.1% soluble PVP (polyvinylpyrrolidone, Sigma), 5 mM ascorbic acid, 10 mM MgCl2, and 0.1 M TRIS-NaOH [TRIS=tris(hydroxymethyl)-amino methane, Sigma], pH 7.8. After filtration of the homogenate through four layers of 600 µm nylon cloth, the obtained filtrate was centrifuged at 5000 g for 15 min, and the supernatant obtained after the centrifugation was further centrifuged at 50 000 g for 1 h. The supernatant obtained after this last centrifugation was dialysed and concentrated in PEG (polyethylene glycol compound with molecular weight of 15 000
20 000, from Sigma), giving the cytosolic fraction in which the protein concentration was approximately 2 mg ml-1. The microsomal pellet obtained after the centrifugation at 50 000 g was resuspended in a suspension buffer containing 250 mM mannitol, 1 mM EDTA, 2 mM DTT, 2 mM MgCl2, and 0.05 M MES-NaOH (MES=2-(N-morpholino)ethane sulphonic acid, Boehringer Mannheim), pH 5.0. The suspended microsomes and the concentrated cytosolic fraction were either kept at 0 °C for immediate use or frozen with liquid N2 and stored at -70 °C. All steps were performed at 0–4 °C.
Protein and enzyme analysis
Protein concentrations in the microsomes or cytosolic fraction were determined by the method using bovine serum albumin as the standard (Bradford, 1976). The membrane composition in the microsomal fraction was evaluated by measuring the activity of the marker enzymes. Following enzymes were selected as the membrane markers: P-type ATPase for plasma membranes, V-type ATPase for tonoplast, F-type ATPase for chloroplast/ mitochondrial membranes, and latent-UDPase for membranes of Golgi apparatus. ATPase activity was determined by the method described previously (Hodges and Leonard, 1974). NADPH-cytochrome reductase (NADPH-CytC reductase) was taken as a marker for the ER membrane and was assayed by the method of Leonard (Leonard, 1973).
Assay of 3H-ABA binding to subcellular fractions
The 3H-ABA binding to the subcellular fractions was analysed by the method of Zhang et al. (Zhang et al., 1999) with modifications. The incubation medium of the microsomes contained 250 mM sorbitol, 1 mM MgCl2, 1 mM EDTA, 20 µM NADPH (ß-nicotinamide adenine dinucleotide 3'-phosphate, Sigma), 2 mM DTT, 0.05 M MES-NaOH (pH 5.5), 40 nM 3H-(±)-ABA (2.37x1012 Bq mmol-1, purity 98.4%, Amersham Pharmacia Biotech Ltd., Buckinghamshire, UK). The total incubation volume was 100 µl. The microsomes were incubated in vitro at 30 °C for 50 min in an amount equivalent to 100 µg protein. After the in vitro incubation, the mixtures were quickly centrifuged through nitrocellulose filters (diameter 0.22 µm, Amersham Pharmacia) to remove free 3H-ABA, and the filters were immediately washed with the incubation buffer diluted five times. After this wash, the filters were placed in vials containing 5 ml scintillation cocktail (0.5 g l-1 PPO and 0.01 g l-1 POPOP in toluene/Triton X-100=3:1, v/v), and the vials were oscillated overnight. The radioactivity retained on the filters was counted in a liquid scintillometer (Beckman LS-5801). The specific binding was determined by the difference between the radioactivity bound to the microsomes incubated only with 3H-ABA (total binding) and the radioactivity bound in the presence of 1000-fold molar excess (40 µM) of unlabelled (±)ABA (Sigma; unspecific binding). The unlabelled (±)ABA was added into the incubation medium at the same time with 3H-ABA.
The 3H-ABA binding to the cytosolic fraction was assayed in a similar way. The in vitro incubation medium of the cytosolic fraction contained 40 nM 3H-(±)ABA, 100 µg protein of cytosolic fractions, 1 mM MgCl2, 20 µM NADPH, 2 mM DTT, 0.05 M MES-NaOH (pH 5.5, except when analysing ABA binding at different pH), and the total volume of each assay was 200 µl. Unlabelled (±)ABA (40 µM; Sigma) was added to the incubation medium to determine unspecific binding. For analysing ABA-binding kinetics, 3H-ABA was added to the binding medium at a concentration gradient from 0.8 to 133 nM while unlabelled ABA was added in concentrations as high as 1000 times those of the 3H-ABA. The Scatchard plot was used for kinetic analysis (Scatchard, 1949). The cytosolic fraction was incubated in vitro at 30 °C for 50 min (except when analysing the time-course or temperature dependence of ABA binding), and then the incubation mixture was quickly placed in ice. Following the addition of 100 µl DCC (Dextran T70-coated charcoal, Sigma), the mixtures were maintained in ice for 10 min, and then centrifuged for removing DCC. The supernatant (100 µl) was used for the radioactivity measurement. A preliminary experiment for validating the methods of ABA binding assay showed that the Dextran–charcoal absorption method mentioned above gave substantially the same results as those by the equilibrium dialysis technique (Venis, 1985), but the latter was not used, mainly because of the long time needed to attain binding equilibrium (about 2–3 h).
The stereospecificity of the 3H-ABA binding sites in the cytosolic fraction was determined using three ABA analogues competing possibly for the same binding sites: phaseic acid, trans-ABA (2-trans-4-trans-ABA) and (-)-ABA (Sigma). These three substances are structurally similar to physiologically active (+)-ABA but are shown to be functionally inactive from several experiments (Walton, 1983; Zeevaart et al., 1991). The three ABA analogues and (+)-ABA (Sigma) were assayed in concentrations ranging from 10 nM to 105 nM. The conditions of incubation were the same as those described above (the incubation buffer containing 40 nM 3H-ABA).
Trypsin treatment of cytosolic fraction
The cytosolic fraction was incubated with either trypsin (Amersham Pharmacia) or inactivated trypsin at various concentrations of 50, 100, and 200 mg l-1 at 25 °C for 10 min, and the ABA binding assay was performed with the treated cytosolic fraction thereafter. The inactivated trypsin was obtained by a treatment in boiling water for 5 min.
Assay of ABA-binding inhibition and recovery in the cytosolic fraction
The cytosolic fraction was preincubated at 4 °C for 50 min in a MES-buffer containing 250 mM sorbitol, 1 mM MgCl2, 1 mM EDTA, 5 mM ascorbic acid, 2 µM NADPH, 1 mM DTT, and 0.05 M MES-NaOH (pH 5.5) (buffer A). This preincubation buffer also contained 0, 5, 10, 50, 100 or 200 µM PMSF (phenylmethylsulphonyl fluoride, Sigma) or IAAM (iodoacetamide, Sigma), or 0.2 mM PMSF+2 mM serine. After the preincubation, the mixture was assayed for 3H-ABA binding. The other assays were as follows: (1) The cytosolic fraction was pre-incubated at 4 °C for 50 min in buffer A without 1 mM DTT but containing 0.05 mM p-CMPS (p-chloromercuriphenylsulphonic acid, Sigma). (2) After the same treatment as in (1), DTT (1 mM) was added into the medium in which the cytosolic fraction was incubated at 4 °C again for 50 min. (3) The cytosolic fraction was pre-incubated at 4 °C for 50 min in buffer A without 1 mM DTT but containing 0.2 mM IAAM. (4) After the same treatment as in (3), cysteine (1 mM), or DTT (1 mM) was added into the medium in which the cytosolic fraction was incubated at 4 °C again for 50 min. After treatments (1), (2), (3) or (4), the mixtures were immediately assayed for 3H-ABA binding.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The ABA-binding sites are localized in the cytosol but not on plasmalemma nor on endomembranes
The microsomes and the cytosolic fraction were isolated from the flesh of developing apple fruit. The results of the marker enzyme assays (Table 1
) showed a dominant presence of P-type ATPase activity and a detectable activity of V- and F-type ATPase, latent-UDPase and NADPH-cytochrome reductase in the microsomes, suggesting that the isolated microsomes, enriched with plasma membranes and also containing endo-membranes, were biochemically active. The assays of 3H-ABA binding via in vitro incubation of the subcelluar fractions (Table 2) showed that, in the microsomes possessing high activity for their marker enzymes, ABA binding activity was barely detectable, but the ABA binding activity was predominantly present in the cytosolic fraction, indicating that ABA-binding sites are subcellularly localized in the cytosol but essentially not on the plasmalemma nor on endomembranes in the flesh of developing apple fruits. This result is different from that obtained with grape berries that have ABA-binding sites predominantly on endomembranes (Zhang et al., 1999).
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) sensitive ATPase; V-ATPase, nitrate (NO3-) sensitive ATPase; F-ATPase, sodium azide (N3Na) sensitive ATPase; NADPH-CytC, NADPH-cytochrome reductase. Values represent the means±SD (n=5).
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ABA binding to the cytosolic fraction is pH-, temperature- and time-dependent
ABA binding to the cytosolic fraction was shown to be highly sensitive to the pH of the incubation medium (Fig. 1). The binding was optimal at pH 5.5, and sharply decreased below or above pH 5.5 (Fig. 1). Previous studies reported that the optimum pH for ABA-binding sites was from 4.0 to 8.0 according to different plants or organs (Hornberg and Weiler, 1984; Chen et al., 1992; Chen and Zhu, 1996; Zhang et al., 1999). These differences in optimum pH may be associated with the different physiological significance of the ABA-binding sites. The remarkable pH-dependent ABA binding in the present experiment suggests that there may exist, at the binding domain of the ABA-binding sites, some amino acid residues with positive charges, since the carboxyl group of ABA is almost completely ionized at pH 5.5 (Parry and Horgan, 1991). This means a possible involvement of pH in regulating the sensitivities of the fruit cells to the ABA signal.
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The ABA binding to the cytosolic fraction was also highly temperature-dependent (Fig. 2
). The binding was optimum at 30 °C, below or above which the binding was sharply decreased (Fig. 2). This optimum temperature is higher than that of the ABA-binding sites reported previously (Chen et al., 1992; Chen and Zhu, 1996; Zhang et al., 1999).
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Figure 3
illustrates the time-course of the ABA binding to the cytosolic fraction. The binding attained 50% of the maximum after approximately 10 min of incubation, and approached the maximum after 50 min, at which time a binding plateau was found (Fig. 3).
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Inhibition and recovery assays of ABA binding proved both the protein nature of the ABA-binding sites and the existence of –SH and active serine residues in their functional domain
Figure 4 shows that a short treatment of the cytosolic fraction with trypsin led to a marked decrease of their subsequent ability to bind ABA. As compared with the control (untreated microsomes), treatment with 50 mg l-1 and 100 mg l-1 trypsin reduced the binding by about 70% and by more than 80%, respectively; and the ABA-binding sites were almost completely abolished by 200 mg l-1 trypsin, whereas the binding activity was not influenced by the treatment of 200 mg l-1 inactivated trypsin. This indicates that hydrolytic domains sensitive to proteinase may exist at the ABA binding sites.
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The ABA binding to the cytosolic fraction was remarkably reduced by PMSF or IAAM treatments (Fig. 5
). PMSF is a specific modifier of active serine residues in many enzymes, and IAAM is an alkylating reagent for the thiol-group (–SH) of cysteine in peptides (Price and Stevens, 1989). The ID50 (median inhibition dose) of the two inhibitors was similarly around 25–30 µM, but the maximum inhibition of IAAM (near 100%) was higher than that of PMSF (63%) (Fig. 5; Table 3). The inhibiting effect of PMSF was partly overcome by the addition of serine (Table 3), which provided further proof of the importance of active serine residues for the ABA binding to the cytosolic fraction. The inhibiting effect of IAAM was partly overcome (Table 3) by a second incubation containing either cysteine or DTT, a protective agent for thiol-groups, verifying that the ABA binding to the cytosolic fraction needs thiol-groups of cysteine residues in a reduced state. In the assay with p-CMPS, which is a thiol inhibitor, its inhibiting effect on ABA binding to the cytosolic fraction was nearly completely overcome by an incubation containing DTT (Table 3), strongly supporting the suggestion that the reduced thiol-group plays an important role in ABA binding to ABA-binding sites. These results not only revealed the existence of active serine residues and thiol-groups of cysteine residues in functional ABA binding sites, but also, together with the results of the trypsin treatments mentioned above, proved the protein nature of the detected ABA binding sites. The thiol group-dependent ABA-binding activity in the flesh of apple fruits is completely different from grape berries of which the ABA-binding sites are highly disulphide bond-dependent (Zhang et al., 1999).
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) or IAAM (•) on 3H-ABA-specific binding activity to the cytosolic fraction of the flesh tissue. The cytosolic fraction was preincubated in buffer containing PMSF or IAAM, and then immediately incubated in the binding medium containing 40 nM 3H-ABA at pH 5.5 and 30 °C for 50 min. A preincubation without adding PMSF or IAAM was taken as the control according to which the percentages of binding inhibition by PMSF or IAAM were calculated. Points indicate the means±SD (n=5).
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Kinetics and stereospecificity of ABA binding revealed two ABA highly specific-binding proteins of high affinity
The 3H-ABA binding to the cytosolic fraction was shown to be saturable with increasing 3H-ABA concentrations (Fig. 6), and this saturation curve could be divided into two curves (Fig. 6A, B). The unspecific binding was lower than 10% and was linear (Fig. 6). In Fig. 7, the Scatchard plotting according to the data presented in the saturation curves (Fig. 6) indicates two linear functions that give evidence of two different ABA binding proteins. One possesses higher affinity to bind ABA with a low dissociation constant (Kd=2.3 nM) and maximum binding (Bmax=39.9 pmol g-1 protein) than the other, which has a Kd of 58.8 nM and Bmax of 229.6 pmol g-1 protein (Fig. 7). Compared with the ABA-binding proteins reported previously that had a Kd of 3–4 nM in Vicia faba guard cell protoplasts (Hornberg and Weiler, 1984), and of 266 nM in rice leaf microsomes (Chen et al., 1992) and of 6.3–50 nM in microsomes of grape berries (Zhang et al., 1999), the affinity of these two ABA-binding proteins can be considered as relatively high. It is also necessary to note that the ABA-binding protein in apple fruit is different from that in grape berry not only in its subcellular localization but also in its number of classes: there is only one class of the binding protein found in grape berry throughout the fruit developmental processes (Zhang et al., 1999).
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In Fig. 8
, phaseic acid (PA), trans-ABA and (-)-ABA are shown to have very poor competition for 3H-ABA binding to the cytosolic fractions. In the incubation buffer containing only 40 nM 3H-(±)-ABA, the binding was not significantly reduced even by 105 nM trans-ABA or (-)-ABA (2500 times as much as 3H-ABA), and nor by 103 nM PA. PA at 104–105 nM displaced from the ABA-binding protein only 20–30% of the bound 3H-ABA, whereas the 3H-ABA binding was nearly completely displaced by 103 nM unlabelled (+)-ABA (Fig. 8). This competition assay demonstrates that the ABA-binding protein possesses a high stereospecificity to bind the physiologically active cis-(+)-ABA, suggesting its specificity for natural ABA. The efficient displacement effect of bound 3H-ABA by unlabelled ABA (Fig. 8) also indicates the reversibility of the ABA binding. All the kinetic properties shown in the present research, of saturability, reversibility, high affinity, and especially stereospecificity of the ABA binding proteins to bind cis-(+)-ABA closely meet the expected primary criteria of a hormone receptor (Venis, 1985).
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), trans-ABA (
). Incubation was performed at pH 5.5 and 30 °C for 50 min in the binding medium containing 40 nM 3H-ABA with unlabelled (+)-ABA or (-)-ABA or either of the other two ABA analogues. Points indicate the means±SD (n=5).Apple fruit is a climacteric fruit, different from non-climacteric grape berry (Coombe, 1976
). The differences between the two kinds of fruit in the properties of their ABA-binding proteins such as those mentioned above in temperature sensitivity, thiol-group- or disulphide-bond-dependence, number of the protein classes, and subcellular localization, may reflect the possible differences in ABA signal transduction pathway and final physiological significance. It is difficult, however, to explain these differences clearly according to the current research. This requires future research and is a challenging field to reveal the first event of ABA signal transduction (Leung and Giraudat, 1998).
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Acknowledgments |
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This research was supported by the National Natural Science Foundation of China (grant nos. 39730340, 39870487 and 30070532) and by the National Key Basic Research Project of China (grant no. G 1999011700).
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Notes |
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1 To whom correspondence should be addressed. Fax: +861062891899. E-mail: zhangdp{at}public2.east.cn.net
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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