JXB Advance Access originally published online on June 27, 2005
Journal of Experimental Botany 2005 56(418):2173-2181; doi:10.1093/jxb/eri217
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RESEARCH PAPER |
The modulating effect of the perisperm–endosperm envelope on ABA-inhibition of seed germination in cucumber
1Institute of Environment Management and Plant Sciences, Vikram University, Ujjain (MP) 456 010, India
2Center for Research on Wild Plants, Utsunomiya University, 350 Mine-machi, Utsunomiya 321-8505, Japan
* To whom correspondence should be addressed. Fax: +91 0734 2511226. E-mail: dilipamr{at}sancharnet.in; dilipamritphale{at}yahoo.co.in
Received 2 January 2005; Accepted 2 May 2005
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Abstract |
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Abscisic acid (ABA) markedly reduced the germination of developing seeds at much lower concentrations (ABA50=0.1 mM) compared with that of mature seeds (ABA50=1.6 mM) in cucumber (Cucumis sativus L. cv. Green long). The perisperm–endosperm (PE) envelope in developing seeds showed partly differentiated lipid and callose layers, considerable ABA biosynthetic activity in endosperm cells, and appreciable permeability to applied ABA. The decrease in the sensitivity of seeds to applied ABA was coincident with the complete development of lipid and callose layers, diminished ABA biosynthetic activity in endosperm cells in imbibed mature seeds, and moderate permeability of the PE envelope to applied ABA. Decoated seeds pretreated with chloroform showed decreased germination (ABA50=0.4 mM) in response to applied ABA and increased ABA permeation through the PE envelope. ABA thus allowed to permeate into embryonic tissues substantially reduced the pregerminative activity of ß-glucanase in the radicles. The structure and biophysical/biochemical properties of the PE envelope seem to modulate the effect of ABA on the germination of developing and mature cucumber seeds.
Key words: Abscisic acid, callose, chloroform treatment, cucumber, Cucumis sativus, perisperm–endosperm envelope, permeability, seed germination, wall hydrolases
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Abscisic acid (ABA) is known as a positive regulator of dormancy and a negative regulator of seed germination (Bewley, 1997
; Koornneef et al., 2002). A link between ABA content and germinability was found in soybean, tomato and wheat, but not in castor bean and sunflower (King, 1976; Ackerson, 1984; Kermode et al., 1989; Bianco et al., 1994; Hilhorst, 1995). In addition to ABA content, sensitivity to ABA may also influence seed dormancy and germination (Romagosa et al., 2001). A decrease in germination sensitivity to applied ABA during seed maturation is known in castor bean, muskmelon, and wheat (Walker-Simmons, 1987; Kermode et al., 1989; Welbaum et al., 1990). Kermode et al. (1989) suggested that altered ABA sensitivity in castor bean seeds could be a consequence of tissue dehydration. On the other hand, Welbaum et al. (1990, 2000) suggested that a drop in endogenous ABA content in muskmelon seeds during maturation could have resulted in an apparent decrease in ABA response in mature seeds. In cucumber, muskmelon, and other cucurbitaceous seeds, a thin perisperm–endosperm envelope completely encloses the embryo (Singh, 1953; Sreenivasulu and Amritphale, 1999). The envelope consists of a single layer of endosperm cells, covered by a thick, non-cellular layer of callose-rich material and a thin lipid-rich outer layer in cucumber and muskmelon seeds (Yim and Bradford, 1998; D Amritphale, unpublished work). Further, Yim and Bradford (1998) showed that the envelope in muskmelon displayed apoplastic semipermeability and also that it was acquired only at later stages of seed development. Preliminary studies showed that applied ABA effectively reduced the germination of developing cucumber seeds, but not that of mature seeds. The present work was therefore undertaken to determine whether the observed change in germination response of cucumber (Cucumis sativus L. cv. Green long) seeds to applied ABA was because of its reduced permeation into the embryo due to altered structural and biophysical/biochemical properties of the perisperm–endosperm (PE) envelope.
In addition to serving as a semipermeable layer, the envelope in cucumber, muskmelon, and other cucurbitaceous seeds is known to act as the primary barrier to radicle emergence (Welbaum et al., 1995; Sreenivasulu and Amritphale, 1999). Since Ikuma and Thimann (1963) first suggested that endosperm weakening is the consequence of enzymatic action, a number of studies have provided evidence for the collaborative/successive action of several cell wall hydrolases (Leubner-Metzger, 2003a, and references therein). ABA is suggested to inhibit germination in tobacco, tomato, and several other seeds by way of affecting the activity of wall hydrolases. The developing consensus is that mannanases and ß-glucanases might be involved in the weakening of endosperm in a number of seeds. Noticeably, in addition to the presence of a callose-rich layer, the cell walls of the envelope in muskmelon, a species closely related to cucumber, are also rich in mannan and galactomannan polymers (Welbaum et al., 1998). Therefore, in order to undedrstand the reduced sensitivity of germination in mature cucumber seeds to applied ABA, its effect on the activity of endo-ß-mannanase and ß-glucanase was also studied.
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Materials and methods |
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Seed material
Cucumber (Cucumis sativus L. cv. Green long) seeds were purchased from Mahyco Ltd., India. After testing the seeds for germination in the laboratory (90–95% germination at 25 °C in the dark), they were stored in air-tight plastic containers at –10 °C until used. Cucumber plants were also grown in the experimental field of the Institute by following the cultural practices suggested by Mahyco Ltd., Jalna, India. Female flowers were tagged at anthesis and fruits were harvested at 35 days post-anthesis (DPA). Developing seeds were extracted from freshly harvested fruits. After removing the mucilaginous endocarp, the seeds were used for the experiments with ABA or fixed in a formaldehyde–glacial acetic acid–50% ethanol (FAA) solution (1:1:18, by vol.) for seed anatomy.
Treatment with organic solvents and autoclaving
Chloroform or ethanol treatment was administered by dipping mature, whole or decoated cucumber seeds in the solvent (assay >99%) for 10 min at 25 °C with gentle stirring followed by air-drying for 24 h. For autoclaving, decoated seeds were imbibed in distilled water for 3 h at 25 °C and then autoclaved at 120 °C for 20 min.
Effect of ABA on seed germination
To study the effect of ABA on germination, mature seeds (whole or decoated) with or without chloroform or ethanol pretreatment and developing seeds at 35 DPA were treated with various concentrations of ABA in four replicates of 25 seeds each. The pH of the aqueous stock solution of ABA was adjusted to 7.0. Seeds were placed in 10 cm Petri dishes on two circles of Whatman No. 1 filter paper moistened with an adequate amount of distilled water or ABA solution as per the recommendation of Ellis et al. (1985). The Petri dishes containing seeds were wrapped with plastic film to reduce evaporation and kept at 25 °C in the dark. Seeds were scored daily for radicle emergence through the testa or PE envelope. On day 5, ungerminated seeds were transferred to fresh Petri dishes containing water or ABA and germination was recorded for an additional period of 4 d. Data for cumulative germination percentage are presented.
Seed anatomy
Twenty micrometre thick sections of dry, mature, decoated seeds were cut with a Plant Microtome Model MTH-1 (Nippon Medical and Chemical Instruments Co., Ltd., Japan). Developing seeds, fixed in FAA, were cut similarly after washing with distilled water. The sections were stained with 0.05% aniline blue in 0.1 M phosphate buffer (pH 8.2) or a saturated solution of Sudan III in 70% ethanol or both in accordance with Yim and Bradford (1998) except that toluidine blue O and Sudan IV were not used. The sections were viewed on a DAS Microskop Leica DMLB (Leica Microsystems, Germany), and images were captured using a Canon PowerShot S40 and downloaded for archiving to ZoomBrowser EX software (Canon, USA).
ABA quantification
Samples were transferred into a 3 ml vial containing 1 ml of 80% aqueous acetone solution of [13C2]ABA (5 ng ml–1). The vials were sealed and sonicated in an ultrasonic cleaner below 6 °C for 10 min. These vials were stored in a dark cold room at 4 °C for at least 7 d. The resulting solutions were filtered (Disk filter; Merck, Tokyo, Japan), concentrated, and the residues were dissolved in 100 µl methanol. Aliquots of 10 µl were injected to a high performance liquid chromatography (HPLC)-connected tandem mass spectrometer. HPLC separation was performed using a JASCO U980 HPLC instrument (JASCO, Tokyo, Japan) equipped with an ODS (C18) column (Mightysil RP-18, 2x250 mm, 5 µm; Kanto Chemicals Co. Ltd, Tokyo, Japan). The mobile phase was 40% methanol in water containing 0.25% acetic acid (v/v) and the methanol content was linearly increased to 100% in 30 min. The column was then washed with 100% methanol for 20 min to elute all the injected materials. The flow rate was 0.2 ml min–1. Column temperature was set to 40 °C. Mass spectrometry was performed on a Quattro LC mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray source. For the analysis of ABA, the MS was operated in electrospray ionization negative mode (ESI-negative). The drying gas as well as the nebulizing gas was nitrogen generated from pressurized air in an N2G nitrogen generator (Parker-Hanifin Japan, Tokyo, Japan). The nebulizer gas flow was set to approximately 100 l h–1 and the desolvation gas flow to 500 l h–1. The interface temperature was set to 400 °C and the source temperature to 150 °C. The capillary and cone voltages were 2.80 kV and 20 V, respectively. MS/MS experiments were performed using argon as the collision gas, and the collision energy was set to 13 V. The collision gas pressure was 1.5x10–3 mbar. Data acquisition and analysis were performed using the software MASSLynx (version 3.2, Micromass, Manchester, UK) running under Windows NT (version 4.0) on a Pentium PC. For the quantification of ABA, multiple reaction monitoring (MRM) was employed with monitoring transitions of m/z 263.1>153.1 for ABA and 265.1>153.1 for the internal standard, [13C2]ABA. The quantification limit for ABA was approximately 0.5 pg.
ABA permeation into 2-mm-diameter agarose gel discs
Gel solution containing agarose (0.6% w/v) and 0.1 mM ABA was prepared in a 0.05 M phosphate buffer (pH 6.8). The gel solution (0.25 ml) was added to a 1.5-cm-diameter well in a 10-welled plastic strip in triplicate and allowed to set. PE envelopes were isolated from developing seeds at 35 dpa and split longitudinally into halves. Mature, decoated seeds with or without chloroform pretreatment were imbibed in distilled water for 3 h and their PE envelopes were similarly isolated and split longitudinally. The halves were placed either with their inner or outer surface on the agarose gel containing ABA (Fig. 1). A 0.6% (w/v) 1.0 mm-thick agarose gel in 0.05 M phosphate buffer (pH 6.8) was separately cast and discs were punched with a 2 mm cork borer. There was little difference in the results of preliminary experiments whether the agarose gel disc was placed in the centre of the half of PE envelope or a little away from the centre. Furthermore, the amount of ABA permeating through the PE envelope into an agarose gel disc during a given permeation period was at least 20–25% lower than the ABA content of a disc placed directly on the ABA-containing agarose gel. Hence, (i) one agarose gel disc was placed in the centre of each half of the PE envelope, and (ii) permeation of ABA was allowed to occur for 6 h or 12 h at 25 °C in the darkness in water vapour-saturated, self-sealing polythene bags. After the permeation period, the discs were freeze-dried and their ABA content was determined as described above. Because the amount of ABA accumulated into the gel discs during the permeation was several orders of magnitude higher than the amount of ABA secreted into the gel discs, corrections for the latter were not made.
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ABA diffusion into 2-cm-diameter agarose gel discs
Gel solution containing agarose (0.6% w/v) with or without fluridone was prepared in a 0.05 M phosphate buffer (pH 6.8). The stock solution of fluridone was prepared with acetone and the final concentration of acetone in the gel solution was <0.1% (v/v). The gel solution was poured between two glass plates to obtain a 1.0 mm thick gel. Discs were punched with a 2-cm cork borer. In one set, PE envelopes (n=10, in triplicate) were isolated from 3-h water-imbibed mature seeds or from developing seeds at 35 DPA following Welbaum et al. (2000)
and their ABA content was determined as described above. In the other set, PE envelopes (n=10, in triplicate per treatment) were split longitudinally into halves. Each of the halves was placed with its inner surface on a 2-cm-diameter agarose gel disc and incubated at 25 °C in the dark in water-vapour saturated self-sealing polythene bags. After a 12-h incubation period, the halves were removed and the ABA content of the gel discs and the halves was determined as described above.
Enzyme extraction
Decoated cucumber seeds, with or without chloroform pretreatment, were imbibed for 3 h or 15 h in distilled water or ABA solution as described above in the germination experiments and their PE envelopes or embryos were isolated. Caps (n=25) were excised from the radicle end of the PE envelopes, whereas radicles (n=25) were excised from the embryos. For endo-ß-mannanase activity, the caps or radicles were extracted in triplicate in 0.4 ml each of 0.1 M citric acid–0.2 M disodium phosphate buffer (pH 6.2) with a pestle and mortar, whereas for ß-glucanase activity, caps and radicles were extracted in triplicate in 0.4 ml of 15 mM Na-acetate buffer (pH 5.5). The pH of buffer solutions was kept 6.2 and 5.5 for endo-ß-mannanase and ß-glucanase activity, respectively, on the basis of preliminary experiments. The extract in each case was centrifuged at 10 000 g for 10 min at 4 °C. The supernatant was pipetted into the Ultrafree-MC filter unit PL-5 and the retentate, after dilution to 0.1 ml with an appropriate buffer, was used for the assay of the respective enzyme.
Enzyme assays
(i) Activity of endo-ß-mannanase (EC 3.2.1.78):
Gels (5x3x0.5 cm) containing 7.5% (w/v) polyacrylamide and 0.1% (w/v) locust bean gum were prepared in 0.1 M citric acid–0.2 M disodium phosphate buffer (pH 6.2). Hollow plastic cylinders (diameter 3 mm; height 2 mm) were gently pressed on the polyacrylamide gel. A 10-µl aliquot of the diluted retentate was loaded into a plastic cylinder on the gel. The enzyme was allowed to act for 24 h at 25 °C in darkness after which the cylinders were removed and the gels were stained immediately in accordance with Toorop et al. (1996) with a little modification as follows. The gels were washed after incubation with distilled water for 20 min and stained with a 0.4% (w/v) aqueous solution of Congo Red for 30 min at about 25 °C. To prevent folding in 95% (v/v) ethanol, the gels were first washed in 50% (v/v) ethanol for 5 min and then transferred to 95% (v/v) ethanol for 10 min. This was followed by a 5-min rewash in 50% (v/v) ethanol after which the gels were allowed to destain overnight in 1 M KCl solution at 10 °C and photographed. The diameters of the hydrolysed areas were measured in two directions to the nearest 0.1 mm with calipers and averaged. The enzyme activity in nkatals was calculated according to a standard curve for Aspergillus mannanase corrected, if necessary, for the diameters of the cleared areas of the 0.075 and 0.00075 nkat Aspergillus mannanase positive controls for each plate.
(ii) Activity of ß-glucanase [ß(1
3)-glucanohydrolase, EC 3.2.1.6]:
ß-glucanase activity was assayed using Laminaria digitata laminarin as the substrate following Salyers et al. (1977) and Morohashi and Matsushima (2000) with a slight modification as follows. The assay mixture contained 0.5 mg laminarin, 50 µmol Na-acetate buffer (pH 5.5), and 0.1 ml enzyme solution in a total volume of 0.4 ml. Incubation was at 37 °C for 30 min and the reaction was stopped by boiling for 5 min. A boiled enzyme control was run in parallel. No increase in glucose was observed in the boiled enzyme control. The enzyme activity was evaluated by measuring glucose formation with GOD-POD method using the kit and the procedure given by E Merck (India) Ltd., Mumbai, India.
Chemicals
S-Abscisic acid (S-ABA) was a generous gift from Dr Yasuo Kamuro (BAL Planning Co., Ltd., Japan). Congo Red and Locust Bean Gum (Fluka, Switzerland), commercially prepared endo-ß-mannanase from Aspergillus niger (Megazyme, Intl., Ireland), fluridone (Aldrich, USA) and laminarin (Sigma, USA) were used. [13C2-1,2]ABA was kindly provided by Dr Tadao Asami (RIKEN, Japan). Other chemicals used in this study were from Bengal Chemical and Pharmaceutical Ltd., HiMedia Laboratories, and Sisco Research Laboratories, India.
Statistical analysis
All the experiments were repeated at least twice and were found to be reproducible. The results presented are a typical set of three replicates (ABA quantification and enzyme activity) or four replicates (germination experiments) for each treatment. Germination percentages were arcsin 
% transformed to normalize the variances of binomial data before subjecting them to analysis of variance. ABA50 values were calculated from a polynomial equation fitted to the plot of ABA concentrations versus germination percentages.
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Results |
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Cumulative germination percentages of developing and mature seeds at increasing concentrations of exogenously applied ABA are shown in Fig. 2. The response curves show that applied ABA markedly reduced the germination of developing seeds at much lower concentrations (ABA50=0.1 mM) compared with that of mature seeds (ABA50=1.6 mM). In an earlier study, Sreenivasulu and Amritphale (1999)
showed PE envelope to be the primary barrier to radicle emergence in cucumber. Transection of a mature, decoated seed showed that the PE envelope in cucumber consisted of a single layer of live endosperm cells (EN) covered by a thick aniline blue-staining layer (CL, callose layer), which in turn was covered by a relatively thin Sudan-staining layer (LL, lipid layer) (Fig. 3A). Notably, developing seeds at 35 DPA had only partly differentiated lipid (LL) and callose (CL) layers (Fig. 3B). In a related study, the PE envelope in developing cucumber seeds was found to be highly permeable to 2,3,5-triphenyltetrazolium chloride (TTC) compared with that in mature seeds (D Amritphale, unpublished work). It therefore seemed possible that the greater inhibition of germination in developing seeds in response to applied ABA could be partly due to the greater permeability of their PE envelopes to ABA. In order to check this possibility, PE envelopes isolated from developing seeds at 35 DPA as well as from 3-h water-imbibed mature seeds were tested for their permeability to ABA using 2-mm-diameter agarose gel discs. The amount of ABA accumulated into the agarose gel discs through the halves of the PE envelopes in developing seeds during a 6-h incubation period was significantly greater than that in mature seeds regardless of the surface (Table 1). There was little difference in the amount of ABA accumulated into the agarose gel discs across the two surfaces of the PE envelope in developing seeds. By contrast, a considerably greater amount of ABA accumulated in the discs when allowed to enter through the inner surface of the PE envelope in mature seeds than when allowed to enter through the outer one.
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While conducting the permeability experiments described above, ABA was detected in 2-mm-diameter agarose gel discs placed as a control on the inner surface of the halves of the PE envelopes that were incubated on agarose gels without ABA. To check whether the ABA diffused into gel discs could be ascribed to de novo biosynthesis in the endosperm cells of the PE envelopes, in one set, the envelopes from developing seeds as well as mature seeds were analysed for their ABA content immediately after isolation. In the other set, halves of the isolated PE envelopes were incubated with their inner surface on 2-cm-diameter agarose gel discs. After 12 h, the halves and discs were both analysed separately for ABA content. Notably, the amount of ABA diffused into agarose gel discs per PE envelope was 45% and 26% greater in developing and mature seeds, respectively, than the amount of ABA originally present per PE envelope, i.e. prior to incubation (Table 2). In addition, there was still a substantial amount of ABA present in the PE envelopes after the incubation. A significant fraction of the ABA that diffused into the discs thus appeared to be synthesized de novo. In order to verify this, halves of the PE envelopes were allowed to incubate with their inner surface on 2-cm-diameter agarose gel discs containing fluridone, an ABA biosynthesis inhibitor, and the discs were analysed for ABA. Notably, fluridone at 10 µM caused a 35–40% reduction in the amount of ABA that diffused into the agarose gel discs in developing and mature seeds (Fig. 4). An increase in fluridone concentration to 100 µM reduced ABA diffusion by 70–75%.
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It is evident from the above findings that the decrease in germination response of mature cucumber seeds to ABA was coincident, if not correlated, with decreased ABA permeability and reduced ABA biosynthetic potential of the PE envelope. Khan (1977)
quoted several references where the treatment of seeds with organic solvents was shown to increase the permeation of several compounds including ABA. Pretreatment of decoated seeds with ethanol had little effect on their germination response to ABA. On the other hand, germination in chloroform-pretreated decoated seeds was reduced to about 40% and 70% of the control at 0.1 mM ABA and 1.0 mM ABA, respectively (Fig. 5). The ABA50 value for germination also decreased from 1.8 mM in control to 0.4 mM in chloroform-pretreated seeds. Chloroform treatment appeared to have little or no effect by itself on the rate of germination and cumulative germination percentage of seeds because, in both chloroform-untreated and -treated seeds, the initial phase of water uptake lasted up to 9 h, initiation of radicle emergence occurred at 16 h, and germination was nearly complete by 22 h (data not given).
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Yim and Bradford (1998)
showed chloroform treatment to remove the lipid layer of the endosperm envelope in muskmelon seeds. Notably, TTC could permeate through the PE envelope of chloroform-treated decoated cucumber seeds as evident by formazan deposition into the cells of the endosperm layer, but not through the PE envelope of ethanol-treated seeds (Fig. 6A, B). It was thus possible that the increased ABA-inhibition of germination in chloroform-treated seeds was partly on account of the greater ABA permeation through the PE envelope. In order to check this possibility, ABA was allowed to accumulate into 2-mm-diameter agarose gel discs through the outer surface of the halves of the PE envelopes isolated from seeds with or without chloroform pretreatment. Notably, the accumulation of ABA into the agarose gel discs was significantly greater through the halves of the PE envelope isolated from chloroform-treated seeds than those from chloroform-untreated seeds (Fig. 7). In a related experiment, seeds were autoclaved in order to kill the cells of the endosperm layer of the PE envelope. ABA accumulated into agarose gel discs to a greater extent when allowed to permeate through the halves of the PE envelopes from autoclaved seeds than from those of viable seeds. Furthermore, ABA permeation through the PE envelope was considerably greater in chloroform-pretreated autoclaved seeds than in autoclaved seeds without chloroform treatment (Fig. 7; t-test for paired comparison in all the treatments significant at P=0.05).
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For germination in cucumber seeds, the radicle is required to protrude through the layer of endosperm cells and the non-cellular layer of the PE envelope. As mentioned earlier, endosperm cell walls in muskmelon, a species closely related to cucumber, are known to be composed primarily of mannan or galactomannan polymers (Welbaum et al., 1998
). As shown in Fig. 3A and B, the non-cellular layer in the cucumber PE envelope comprised a thin Sudan-staining (lipid-rich) layer and a thick aniline blue-staining (callose-rich) layer. Therefore, it was of interest to know whether ABA inhibited germination in cucumber seeds by affecting the hydrolytic degradation of the PE envelope. For this, decoated seeds pretreated with chloroform were allowed to imbibe in ABA-containing medium and the pre-germinative activities of endo-ß-mannanase and ß-glucanase were determined. Endo-ß-mannanase activity was present in the caps of the PE envelopes from the beginning of imbibition of the seeds (3 h) to the initiation of the radicle (15 h), but was not detected in the radicles (Figs 8, 9A). By contrast, ß-glucanase activity appeared just prior to emergence in the radicles, but was not present in the caps of the PE envelopes (Fig. 9B). There was little reduction in endo-ß-mannanase activity at 0.1 mM ABA. Even 1.0 mM ABA, which reduced germination in chloroform-pretreated seeds by 70% of the control, could inhibit the activity of endo-ß-mannanase by not more than 20% (Table 3). On the other hand, a substantial reduction in ß-glucanase activity was recorded with ABA not only at 1.0 mM, but also at 0.1 mM.
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Discussion |
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Despite questions regarding the biological relevance of the exogenous ABA inhibition of seed germination, it is a convenient quantitative assay for ABA sensitivity and has been used to dissect ABA response mechanism(s) (Finkelstein et al., 2002
). The germination of developing seeds in cucumber was markedly reduced at relatively lower concentrations of applied ABA, whereas that of mature seeds was reduced only marginally even at relatively higher ABA concentrations (Fig. 2). A decrease in germination response to applied ABA during seed maturation is known in castor bean, muskmelon, and wheat (Walker-Simmons, 1987; Kermode et al., 1989; Welbaum et al., 1990). Welbaum et al. (1990, 2000) suggested that a drop in endogenous ABA content in muskmelon seeds during maturation could have resulted in an apparent decrease in ABA sensitivity in mature seeds. Higher levels of ABA were also found in the PE envelopes (Table 2) and embryos (data not given) in cucumber seeds at 35 DPA than in imbibed mature seeds. However, in our view, reduced germination sensitivity of mature cucumber seeds to applied ABA in the latter might also be explained in terms of decreased permeability of their PE envelopes. This is because (i) the amount of ABA accumulated into agarose gel discs through the halves of PE envelopes during a 6-h incubation period was significantly lower in mature seeds than in developing seeds (Table 1), (ii) the lipid and callose layers of the PE envelope were only partly differentiated in developing seeds showing significantly higher germination response to applied ABA (Figs 2, 3B), (iii) reduction in the sensitivity of seeds to applied ABA coincided with complete development of the lipid and callose layers in the PE envelope (Figs 2, 3A), and (iv) treatment of mature, decoated seeds with chloroform not only increased ABA accumulation into gel discs (Fig. 7), but also caused a reduction in seed germination in response to applied ABA (Fig. 5). Indeed, Debeaujon and Koornneef (2000) attributed increased sensitivity to applied gibberellins to differences in uptake of various compounds by testa mutants in Arabidopsis. As mentioned earlier, Yim and Bradford (1998) showed that chloroform treatment had no apparent effect on the callose layer in muskmelon seeds, whereas the lipid layer was removed. Interestingly, relatively greater permeation of ABA through the PE envelope was observed in cucumber, not only in chloroform-pretreated viable seeds having a layer of live endosperm cells but also in chloroform-pretreated autoclaved seeds with a layer of dead endosperm cells compared with their respective controls (Fig. 7). Therefore, in contrast to Yim and Bradford (1998), who attributed the apoplastic semipermeability of the envelope in muskmelon seeds to the callose layer only, these data suggest that the lipid layer might also contribute, at least partly, to the semipermeability of the PE envelope in cucumber.
The free ABA content is high in developing seeds and is generally low or even undetectable in mature seeds (Black, 1991; Romagosa et al., 2001). As mentioned earlier, Welbaum et al. (2000), who carried out a detailed analysis of the content and compartmentation of ABA in different parts of muskmelon seeds at various developmental stages, also found that the ABA content declined during seed maturation. While the above study showed that the envelope in muskmelon seeds at various developmental stages contained ABA in considerable amounts, it did not attempt to examine whether it was derived from adjacent maternal/embryonic tissues or was synthesized in the cells of endosperm. Although the present data cannot resolve the issue, they are indicative of the de novo biosynthesis of ABA in the endosperm cells of the PE envelope in developing seeds as well as in imbibing mature seeds (Table 2). Moreover, the inhibiting effect of fluridone (Fig. 4) also suggested de novo ABA biosynthesis in the endosperm cells in both developing and imbibing mature cucumber seeds. As to the relevance of de novo ABA synthesis in the PE envelopes in developing seeds, it may possibly have a role in preventing precocious germination in cucumber where germinable seeds, similar to that in other fleshy fruits, are held at relatively high water content for extended periods during fruit development. At present, it is not possible to assign any definite role to ABA biosynthesis in the PE envelopes in mature cucumber seeds. However, it may be important under environmental conditions that are non-conducive to germination.
Welbaum et al. (1990) showed that the inhibitory effect of ABA on muskmelon seed germination was primarily due to an increase in the apparent minimum turgor threshold required for germination, which can be operationally defined as the yield threshold for radicle emergence. Because the strength of the seed coverings is known to contribute to the yield threshold that must be exceeded for radicle emergence to occur, it is quite likely that the inhibitory effect of ABA was realized, at least partially, due to its inhibitory effect on the activity of wall hydrolases involved in the weakening of these layers. The composition of endosperm cell walls is not known in cucumber; however, as mentioned earlier, endosperm cell walls happen to be rich in mannans in muskmelon, a species closely related to cucumber. Nearly complete inhibition of germination in muskmelon with 100 µM ABA was accompanied by only a 30% reduction in endo-ß-mannanase activity (Welbaum et al., 2000). It was also found that 1.0 mM ABA, which caused 70% inhibition of germination in chloroform-pretreated cucumber seeds (Fig. 5), reduced endo-ß-mannanase activity by about 20% only (Table 3). Since ABA did not inhibit endo-ß-mannanase activity, this suggested that some other wall hydrolase(s) might be involved in the ABA-inhibition of seed germination in cucumber. The other way round, it could also mean that such a hydrolase(s) that is ABA-regulated might be involved in the weakening of the PE envelope in cucumber. Wu et al. (2000) proposed endosperm weakening in tomato involving ABA-sensitive ß-1,3-glucanase, but there was no evidence for a ß-1,3-glucan substrate in the walls of tomato endosperm cells. Similarly, an ABA-inhibited ß-1,3-glucanase was shown to be causally involved in the endosperm rupture in tobacco seed germination, but a ß-1,3-glucan substrate has also not been reported (Leubner-Metzger, 2003a, b). Notably, a thick callose (ß-1,3-glucan) layer is present in the PE envelopes in cucumber (Fig. 3A) and muskmelon (Yim and Bradford, 1998). However, Witmer et al. (2003) did not find ß-1,3-glucanase activity in mature muskmelon seeds. Although it was not possible to distinguish between endo- and exo-glucanase activities with the assay employed, nevertheless, a measurable ß-1,3-glucan (laminarin)-hydrolysing activity was observed in the radicles in mature cucumber seeds just before the splitting of the PE envelope (Fig. 9B). Interestingly, ABA concentrations, which significantly reduced the germination of chloroform-pretreated seeds in cucumber (Fig. 5), also caused a substantial decrease in the ß-glucanase activity in radicles (Table 3). However, further experiments are necessary in order to draw any conclusion regarding the correlation between ABA-inhibition of seed germination and ß-glucanase activity in cucumber.
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Acknowledgements |
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We are grateful to the University Grants Commission, New Delhi for partial financial support in the form of SAP-DRS Research Project and to Megazyme Intl., Wicklow, Ireland for their generous gift of Aspergillus niger endo-ß-mannanase.
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References |
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