JXB Advance Access originally published online on July 7, 2006
Journal of Experimental Botany 2006 57(11):2639-2650; doi:10.1093/jxb/erl027
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RESEARCH PAPER |
The role of arabinogalactan proteins binding to Yariv reagents in the initiation, cell developmental fate, and maintenance of microspore embryogenesis in Brassica napus L. cv. Topas
Key Laboratory of the MOE for the Development Biology, College of Life Science, Wuhan University, Wuhan 430072 China
*To whom correspondence should be addressed. E-mail: mxsun{at}whu.edu.cn
Received 18 October 2005; Accepted 13 April 2006
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Abstract |
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Arabinogalactan proteins (AGPs) are extracellular proteoglycans involved in plant growth and development. The addition of ß-D-glucosyl Yariv reagent (ßGlcY), a synthetic phenylglycoside that specifically reacts with AGPs, to the culture medium notably disturbed microspore embryogenesis in a concentration-dependent manner. The initiation of microspore embryogenesis was clearly inhibited by 30 µM ßGlcY and completely inhibited by 50 µM ßGlcY. The transfer of microspore-derived embryos at different developmental stages into NLN6 medium containing 50 µM ßGlcY prohibited their normal development, as
21.24, 43.99, and 59.73%, respectively, of the treated globular-, heart-, and torpedo-stage embryos exhibited numerous root hair-like structures. Both heart-stage and torpedo-stage embryos showed a rapid growth of roots with a large number of clustered root hairs. Some root hair-like structures were also observed on the apical portions of embryos. Microscopy of the treated embryos revealed that the basic patterns of cells at both the radial and apical–basal axes were greatly altered, such that the cells lost their ability to carry out programmed embryogenesis. These results show that the ßGlcY–AGP interaction modulates the developmental fate of embryonic cells, especially epidermal cells, and thereby strongly affects root generation and development. Immunofluorescence microscopy revealed that both JIM8 and JIM13 binding to AGP co-localize with ßGlcY-binding sites. Thus, AGPs binding to ßGlcY, co-localized with Jim8- and Jim13-binding protein, appear to play a crucial role in the initiation of Brassica microspore embryogenesis and the maintenance of cell differentiation during embryonic development. In addition, these proteins may also be involved in the regulation of root generation. Key words: Arabinogalactan protein, Brassica napus L. cv. Topas, cell fate, development expansion, microspore embryogenesis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Arabinogalactan proteins (AGPs) are a large family of plant proteoglycans that belong to the hydroxyproline-rich glycoprotein superfamily (Nothnagel, 1997; Majewska-Sawka and Nothnagel, 2000). Studies using antibody probes, in addition to the biochemical isolation and characterization of these proteins, have shown that AGPs are widely distributed throughout the plant kingdom. At the organ level, AGPs are present in leaves, stems, roots, flowers, and seeds (Fincher et al., 1983; Basile and Basile, 1987). At the subcellular level, AGPs are located on the plasma membrane (Pennell et al., 1991; Zhu et al., 1993; Serpe and Nothnagel, 1996) and in the cell wall (Li et al., 1992; Serpe and Nothnagel, 1994). In addition to their predominant localization at the plant cell surface, AGPs have been detected in the extracellular space (Samson et al., 1984) and in several organelles, including the Golgi apparatus (Pennell et al., 1991) and multivesicular bodies (Herman and Lamb, 1992). It has been demonstrated that the expression of AGPs is temporally and spatially regulated during plant development.
Yariv phenylgycosides (Yariv et al., 1962), such as ß-glucosyl Yariv reagent (ßGlcY), are synthetic probes that bind to and aggregate AGPs. Although the mechanisms by which ßGlcY selectively and non-covalently binds to AGPs are not yet fully understood (van Holst and Clarke, 1985), Yariv reagents are frequently used as colorimetric histochemical probes to localize specifically the distribution of AGPs. Previous work has shown that the addition of ßGlcY to living cells disturbs AGP function. ß-D-Mannosyl Yariv (ßManY) reagents differ from ßGlcY reagents only in the isomerization of the hydroxyl group at carbon atom 2 of the sugar, which prevents their binding to AGPs. ßManY thus provides an excellent control in studies of AGPs using ßGlcY (Yariv et al., 1967; Nothnagel, 1997). In the present study, ßGlcY and ßManY reagents were used to examine the function of AGPs, yielding insight into the possible roles of these proteins in plant development.
Although their biological function has not been precisely identified, there is evidence that AGPs are involved in processes of plant growth and development, such as cell proliferation (Serpe and Nothnagel, 1994), cell expansion (Willats and Knox, 1996; Shi et al., 2003), cell differentiation (Pennell and Roberts, 1990; Knox et al., 1991), cell elongation (Park et al., 2003), the regulation of somatic embryogenesis (Egertsdotter and Von Arnold, 1995; Kreuger and Van Holst, 1995; Thompson and Knox, 1998; Chapman et al., 2000), pollen tube growth (Cheung et al., 1995; Roy et al., 1998; Wu et al., 2000), and programmed cell death (Gao and Showalter, 1999; Chavesa et al., 2002). Immunocytochemistry experiments have demonstrated the developmental regulation of some AGP epitopes in organs and in somatic embryogenesis (Stacey et al., 1990; for a review, see Knox, 1997).
Guha and Maheshwari (1964) discovered that pollen grains from isolated anthers of Datura can switch from their normal gametophytic developmental mode to an embryogenic pathway and develop into haploid embryos. In vitro embryogenesis from haploid male gametophytes has subsequently been demonstrated in anthers and in both isolated microspore and pollen culture in many species of flowering plants (Dunwell, 1986; Srivastava and Johri, 1988). Haploid embryo formation in Brassica napus L. offers an attractive model system that has been frequently used for various developmental studies in vitro (Zaki and Dickinson, 1991; Telmer et al., 1995; Touraev, 1997; Maraschin et al., 2005). The high frequency of embryogenesis and the availability of growth regulator-free medium make this species an ideal system for studying both the effects of ßGlcY and the function of ßGlcY-reactive AGPs in androgenesis. In the present study, ßGlcY was used to perturb AGPs in living Brassica microspores and microspore-derived embryos. The results revealed that ßGlcY treatment disturbs both the normal process of embryogenesis and the basic structural pattern of the embryos. Furthermore, the altered developmental fate of embryonic epidermal cells indicates that AGPs binding to ßGlcY play a crucial role in microspore embryogenesis.
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Materials and methods |
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Plant material
Plants of Brassica napus cv. Topas were grown under greenhouse conditions at the College of Life Sciences of Wuhan University. The temperature was maintained between 18 °C and 23 °C under a 16 h light/8 h dark cycle.
Isolation and culture of microspores
Microspores at the late unicellular stage of development and pollen at the early bicellular stage were obtained from flower buds with a length ranging from 2.8 mm to 3.2 mm. The developmental stages of freshly isolated microspores and pollen were determined by fluorescence microscopy after staining with 4 µg of 4',6-diamidino-2-phenylindole (DAPI) ml–1. The microspores were isolated in B5 medium, cultured in NLN13 medium according to the method of Hause et al. (1994), and then cultured in the dark at 32 °C.
Treatment of cultured microspores and microspore-derived embryos with ßGlcY
Microspores were cultured following the standard embryo induction procedure using NLN13 medium supplemented with 0 (control), 10, 30, or 50 µM ßGlcY. Additional control cultures were grown in medium containing 10 or 50 µM ßManY, which does not react with AGPs. Untreated microspore-derived embryos were transferred in low numbers to diluted medium (NLN6) containing 50 µM ßGlcY at 7–8 d (globular stage and heart stage) or 14–16 d (torpedo stage and plantlets) from the start of embryo induction. As a second control, the effect of sucrose starvation on the plantlets was examined using NLN medium containing 0, 3, or 6 g l–1 sucrose.
Determination of microspore viability
At the end of the culture period, microspore viability was determined using fluorescein diacetate (FDA) at a concentration of 2.5 µg ml–1. Aliquots of microspores were stained with FDA and then observed under a fluorescence microscope. Approximately 1000 microspores were scored in each treatment group by examining 10 different microscopic fields of view on multiple slides.
Immunofluorescent labelling with monoclonal antibodies JIM4, JIM8, and JIM13
The distribution of AGPs was investigated with the rat monoclonal antibodies JIM8, JIM13, and JIM4.
Samples were fixed overnight in 4% paraformaldehyde and 0.1 M phosphate-buffered saline (PBS), pH 7.2. After washing twice for 5 min each time in PBS buffer, the samples were blocked in 5% bovine serum albumin (BSA) in culture medium for 1 h at room temperature. Subsequently, they were incubated with monoclonal antibodies JIM8 (diluted 1:20 with 0.1 M PBS containing 0.1% BSA), JIM4, and JIM13 (diluted 1:10 with 0.1 M PBS containing 0.1% BSA) at room temperature for 2 h. After rinsing in PBS three times (5 min each), the samples were then incubated with fluorescein isothiocyanate (FITC)-labelled goat anti-rat IgG antiserum (Sigma) diluted 1:20 in the same buffer at room temperature for 2 h. After a final rinse series in PBS, the samples were mounted on slides with 5% n-propyl gallate in glycerol for observation. The controls were prepared following the same procedure but omitting the primary antibody incubation.
Tissue fixation and microtomy
The cultures were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 6.8) and then embedded in Epon 812 resin. Sections were cut to a thickness of 5 µm, dried onto polylysine-coated slides, and stained with toluidine blue.
Microscopy and image collection
The samples were examined under either an inverted microscope (Leica DM IRB, Germany) or a stereo microscope (Olympus SZX12). A cooled CCD (MicroMAX Princeton Instruments Inc., Coherent Life Sciences, Australia) and a confocal microscope (Leica TCS 4D LCSM) were used to view and record the images.
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Results |
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Initiation of microspore embryogenesis is inhibited by ßGlcY
Haploid embryos were obtained at high frequencies from single isolated microspores cultured in liquid NLN13 medium under the conditions described above. In order to evaluate whether ßGlcY interferes with the initiation of microspore embryogenesis, microspore cultures were grown under the same conditions used for the induction of Brassica microspore embryos, except that the medium was supplemented with 10, 30, or 50 µM ßGlcY. Microspore division was altered in cultures grown in ßGlcY-containing medium for 48 h. Only
19.41% and 12.92% of microspores treated with 10 µM and 30 µM ßGlcY, respectively, could divide, while microspore division was completely inhibited by 50 µM ßGlcY (Figs 1, 3C). In contrast, division was not inhibited in untreated microspores (Fig. 1) or in microspores treated with ßManY (Fig. 1), which does not bind to AGPs and hence serves as an important control.
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Microspores were cultured for a week in ßGlcY-free medium or ßManY-containing medium (Fig. 3D), resulting in a large number of embryos at different stages of development. In contrast, only a few proembryos were present in medium containing 10 µM (Fig. 3A) or 30 µM (Fig. 3B) ßGlcY, while globular- and/or heart-stage embryos occurred even less frequently.
To determine whether inhibition of microspore division by ßGlcY affects viability, the microspores were stained with FDA. After 24 h of treatment with 50 µM ßGlcY, microspore viability was markedly reduced to 47%; after 48 h, the percentage of dead cells was 93% (Fig. 2). By contrast, the percentage of dead microspores remained nearly constant at 21% and 23% after 24 h and 48 h under control conditions and in microspores treated with ßManY, respectively, indicating that the presence of the reagent itself in the medium had no toxic effect on microspore viability.
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ßGlcY-binding site distribution in living microspore-derived embryos
When ßGlcY binds to its specific target, AGPs, in microspore-derived embryos, the ßGlcY–AGP binding sites show a red coloration, clearly indicating the distribution of AGPs. In globular-stage embryos, ßGlcY-binding sites were present in the entire embryo proper (Fig. 4A), especially in the epidermal layer and in adjacent cell layers. In the later globular stage, ßGlcY-binding sites were most apparent where the radicle eventually forms. Thus, the orientation of the apical–basal axis of the embryo can be predicted according to the prevalence of ßGlcY-binding sites. This was confirmed when the globular embryo started to elongate and its root pole became morphologically observable. At this stage, ßGlcY-binding sites were predominantly located in the basal part of the embryo (Fig. 4B). In heart-stage embryos, the red coloration was concentrated in both the shoot apex and the root pole (Fig. 4C). In torpedo-stage embryos, staining was observed in the entire embryo proper, mainly in the cotyledon, the basal part of the radicle, and in provascular tissues (the future stele) where cells undergo rapid multiplication. In addition, at this stage, the apical portion accumulated more ßGlcY-binding sites than those observed in other parts during younger stages (Fig. 4D).
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Immunofluorescent labelling of AGPs containing JIM4, JIM8, and JIM13 epitopes
Three selected monoclonal antibodies, JIM4, JIM8, and JIM13, were used to localize their binding proteins and compared with ßGlcY-binding sites. In globular-stage embryos, the distribution of the JIM8 epitopes was conspicuously punctuated on the surface of proembryos (Fig. 5A1, A2). During the transition period of the embryos, JIM8 epitopes were mainly located on the basal part of the embryos (Fig. 5B1, B2). As the embryos differentiated, JIM8 epitopes were predominantly located in both the shoot apex and the basal part of the embryos (Fig. 5C1, C2). Confocal microscopy further confirmed that JIM8 epitopes were evenly distributed on proembryos, distributed in a polar fashion on transition embryos, and concentrated on differentiated embryos (Fig. 5D–F). The fluorescent intensity of JIM13 epitopes was much less than that of JIM8 epitopes on proembryos (Fig. 5G1, G2). Once the embryos started to differentiate, the fluorescent intensity of JIM13 epitopes became stronger and was also mainly located on the basal part of the embryos (Fig. 5H1, H2). Similar to JIM8 epitopes, JIM13 epitopes were also distributed on both the shoot apex and root pole of heart-stage embryos (Fig. 5I1, I2). However, in contrast to JIM8, JIM13 epitopes were notably located on the suspensor (Fig. 5H, 5I). The distribution of JIM4 epitopes was hardly observed on the proembryos, and a weak signal could be detected on the suspensors (data no shown). Clearly, in the proembryo stage, ßGlcY-binding sites were co-localized with JIM8 epitopes, while in the differentiated embryo stage they co-localized with both JIM13 and JIM8 epitopes.
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ßGlcY inhibits the development of microspore-derived embryos
In order to determine the effects of ßGlcY on the development of microspore-derived embryos, three stages of the embryos, globular, heart-shaped, and torpedo embryos, were selected and cultured in the sucrose-diluted NLN6 medium containing 50 µM ßGlcY.
After globular embryos were grown for 7 d in medium containing ßGlcY, only 2% (n=253) developed into the next stage, as determined by the presence of morphologically observable changes, while the remaining embryos persisted in the globular stage but increased slightly in size. Similarly, 2.5% (n=266) of the heart-shaped embryos and 4% (n=270) of the torpedo embryos cultured in 50 µM ßGlcY developed into the next stage, whereas the remaining embryos showed various morphological changes. As in earlier experiments, control embryos at the same stages that were cultured in normal NLN6 medium or ßManY-containing NLN6 medium developed into normal mature embryos.
ßGlcY alters the cell developmental fate and structural pattern of embryos
Although the different stage embryos treated with 50 µM ßGlcY did not develop into the next stage, they showed a surprising range of morphological changes. In 21.24% (n=253) of the globular embryos, the epidermal cells of the embryo proper appeared to bulge over it after 7 d of culture, and root hair-like structures were generated from these bulged cells (Fig. 6A, B). In 30.27% of the globular embryos, no obvious root hair-like structures were seen on the embryo proper; instead, the epidermal cells appeared loose and expanded radially. In 43.99% (n=266) of the heart-shaped and 59.73% (n=270) of the torpedo embryos, clustered root hair-like structures were observed in the future root pole (Fig. 6C), and in some embryos root hair-like structures had even arisen from cotyledons (Fig. 6D). On these cotyledons, large protruding epidermal cells were also seen among the root hair-like structures (Fig. 6E).
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A similar phenomenon was observed in cultures of mature embryos in that all of the mature embryos transferred into ßGlcY-containing medium failed to grow normally. The epidermal cells at the hypocotyl appeared greatly loosened after 24 h, and root hair-like structures were subsequently produced (Fig. 6F, G). However, the responses of the mature embryos to ßGlcY differed from those of early-stage embryos. ßGlcY did not appear to react with cells of all the other regions except the junction of the hypocotyl and radicle (Fig. 6F, G), nor did cells in those regions differ morphologically from those of untreated mature embryos or from those treated with ßManY.
When heart-shaped and torpedo embryos were kept in culture medium containing 50 µM ßGlcY for a few more days, both showed rapid growth of roots with a large number of clustered root hairs (Fig. 7A). In such rapidly elongated roots, new lateral roots were soon produced (Fig. 7B) and developed into root branches (Fig. 7C), similar to what is observed during seed germination. However, the apical part of the embryos could not develop into normal cotyledons (Fig. 7D). It therefore seems that the binding of ßGlcY mainly accelerates root generation and growth, but also interferes with apically oriented development. Embryos at the same stages as those cultured as controls in ßGlcY-free medium or with ßManY grew into mature embryos and exhibited normal cotyledons and radicles (Fig. 7E) but did not germinate in the medium.
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The sectioning of the ßGlcY-treated embryos revealed that their basic structural pattern along both the radial and apical–basal axes was greatly altered. Typically, in late globular embryos that had started to elongate when the apical–basal axis was appearing, the epidermal cells underwent extensive expansion, becoming irregularly arranged and varying in shape (Fig. 8A). Most of the embryonic cells in the apical region were converted into typical parenchymal tissue, with notable gaps between cells and large vesicles in the cells. Only a few cells were dividing, indicating that the embryos had lost their programmed ability to undergo embryogenesis. Microscopy sections also showed that the root hair-like structures were derived from epidermal cells of the apical region (Fig. 8B). However, in the basal part of the embryos, although some of the cells were parenchyma-like, large groups of vigorously dividing cells were still visible (Fig. 8C), which might explain the root generation. In contrast, untreated embryos or embryos treated with ßManY at the same developmental stages showed normal structures and cell types (Fig. 8D–F).
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Reduction of the sucrose concentration does not influence embryo development
Kreuger et al. (1995) reported that embryogenic cyclamen cell suspension cultures could be induced to produce roots rather than embryos when grown in medium containing a low concentration of sucrose. To clarify whether epidermal cell expansion and root formation by Brassica microspore-derived embryos were related to a reduction of the sucrose content in the culture medium, from 13 to 6 g l–1, the four above-mentioned stages of embryos were transferred into the same medium as used in the previous experiment but containing 0, 3, or 6 g sucrose l–1 without ßGlcY. The results showed that no root hair-like structures or roots were produced by early embryos grown in sucrose-containing media. All of the early embryos developed normally, and the morphological expansion of the hypocotyls of mature embryos was not observed.
This result demonstrated that the alteration of the developmental fate of the cell was not caused by the reduction of sucrose but was induced by ßGlcY treatment.
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Discussion |
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Previous studies have indicated that distinct classes of AGPs are involved in the complex interactions between different cell types in suspension cultures and that they act indirectly on somatic embryo development (Kreuger and van Holst, 1995; Toonen et al., 1997; Thompson and Knox, 1998; Chapman et al., 2000). In the present work, a concentration-dependent inhibitory effect of ßGlcY on the initiation of microspore embryogenesis was shown. The occurrence of the first round of division in embryogenesis was reduced to 19.41% and 12.92% when microspores were treated with 10 µM and 30 µM ßGlcY, respectively, and division was completely inhibited by 50 µM ßGlcY. Further analysis showed that the complete inhibition by 50 µM ßGlcY resulted in the loss of microspore viability. Microspore division was not inhibited and there was no effect on viability in untreated cultures or in cultures treated with ßManY, indicating that the effect of ßGlcY treatment was AGP dependent. AGPs are thought to play a role in several cellular and developmental processes that are regulated by phytohormones. For example, van Hengel (2001) demonstrated that the addition of AGPs to carrot protoplasts increased cell division in a manner similar to that of auxin, whereas the binding of AGPs by ßGlcY in carrot suspension cells had the complementary effect of suppressing cell division (Thompson and Knox, 1998). Similarly, the inhibition of microspore embryogenesis initiation may also have been due to the suppression of cell division. However, normal microspore embryogenesis does not require the presence of auxin in the medium; thus, whether the initiation of microspore embryogenesis involves endocellular auxin regulation is unclear.
The present work also showed that AGPs binding to ßGlcY are widely involved in the development of microspore-derived embryos and that the ßGlcY–AGP interaction modulates the developmental fate of the early embryo, particularly during the transition from the globular to the heart stage. The shift in embryo symmetry from radial at the globular stage to bilateral at the heart stage represents the initiation of embryo differentiation. Normally, the establishment of the root and shoot meristems is a key event in embryo development after the apical–basal axis is established. The present results show that ßGlcY treatment blocks all of these developmental processes. Furthermore, epidermal cell expansion and the generation of root hair-like structures were observed in all embryos at different stages. The shoot meristem did not appear; instead, embryonic cells developed into mature tissues, i.e. parenchymal cells, indicating that the programmed cell developmental fate and the normal structural pattern of the embryo along both the radicle and apical–basal axes were greatly altered. These data support the reasoning that ßGlcY-reactive AGPs maintain developmental balance and bipolar growth (Thompson and Knox, 1998) and further demonstrate the functional significance of ßGlcY-binding AGPs in early pattern formation, cell developmental fate, and the maintenance of normal embryogenesis.
There is some evidence indicating that AGPs are also involved in cell expansion (Willats and Knox, 1996; Ding and Zhu, 1997). Low levels of AGPs in tobacco cells adapted to NaCl resulted in less extensibility of their walls, as compared with unadapted cells (Zhu et al., 1993). Recently, Vissenberg et al. (2001a) provided direct evidence that ßGlcY inhibits cellulose deposition on the protoplasts of cultured tobacco cells and prevents the elongation of these cells. The orientation of cellulose microfibrils controls the orientation of cell expansion. In the present work, cell radial expansion caused by ßGlcY was restricted to epidermal cells, suggesting that ßGlcY directly interacts with surface AGPs of epidermal cells and that this interaction affects cellulose synthesis, which is critical for cell expansion in a controlled manner.
The specific binding of AGPs to ßGlcY has long been known. Recently, >20 gene sequences encodings core proteins of AGPs in Arabidopsis and a large number of hybrid proteins have been identified in a range of species in which AGP-like sequences occur in the same molecule as other protein motifs such as lipid transfer protein, phytocyanin, and fasciclin (Schultz et al., 2000, 2002; Borner et al, 2002; Johnson et al., 2003). The evidence has demonstrated that some proteins will confer binding by Yariv reagents (Mashiguchi et al., 2004; Motose et al., 2004). Indeed, AGPs comprise a highly complex family of proteins. AGPs are now divided into three subclasses: AGPs, chimeric AGP, and hybrid AGP, based on their structural features (Schultz et al., 2002). Early nodulin-like proteins (ENODLs) and lipid transfer protein-like proteins (LTPLs) interacting with ßGLcY should also be classified as chimeric AGPs according to recent investigations (Mashiguchi et al., 2004; Motose et al., 2004). It is widely accepted that JIM8, JIM13, and JIM4 antibodies recognize certain carbohydrate epitopes of AGPs. Therefore, these three monoclonal antibodies were selected and used. The results showed that the distributions of JIM8- and Yariv reagent-binding sites co-incided with each other during proembryo development. As the embryos started to differentiate, both JIM8 and JIM13 epitopes were located predominantly in the shoot apex and the basal part of the embryos, which also coincided with Yariv reagent-binding sites in this period. JIM13 and JIM4 have high affinity for a ß-D-GlcpA-(1
3)-
-GlapA-(12)-L-Rha oligosaccharide (Yates et al., 1996), but recognize distinct epitopes, clearly demonstrated by their recognition of different patterns of cells, and JIM4 appeared to be relatively species specific. JIM13 binds to cells in the region of the developing xylem, whereas JIM4 binds only to pericycle cells at the carrot root apex (Knox et al., 1989, 1991). The JIM8 antibody recognizes epitopes containing large amounts of galactosyl, arabinosyl, and rhamnogalacturonan residues. The epitopes are mainly located in the embryo proper, suspensor cells, and sperm (Pennell et al., 1991). In the present work, the co-localization of JIM13- and JIM8-binding proteins with ßGlcY-binding sites suggests that JIM8-binding AGP might play a principal role in proembryo stages, whereas both JIM8- and JIM13-binding AGPs could be involved in embryo differentiation.
Immunofluorescence microscopy experiments have shown that the JIM8 AGP epitope is distributed on the plasma membrane of Brassica microspores (Pennell et al., 1991). Other investigators have proposed that the ßGlcY-induced aggregation of plasma membrane AGPs generates physical stresses that directly tear the membrane open, causing gradual cell death (Guan and Nothnagel, 2004). The mechanisms may involve the binding of ßGlcY to plasma membrane AGPs, thereby trapping other, nearby membrane proteins and activating signal transduction pathways that direct the fate of the cell (Gao and Showalter, 1999; Guan and Nothnagel, 2004). This proposal may well explain the phenomenon observed in the current experiments. However, the binding of ßGlcY indeed altered the developmental fate of cells, especially epidermal cells, but how AGPs binding to ßGlcY are involved in directing cell developmental fate and the nature of their role in signal transduction are not yet known.
Recently it was reported that the knockout of each single AGP core polypeptide gene has not shown an obvious phenotype, which was thought probably to be because of genetic redundancy (Gaspar et al., 2001). The results emphasized the complexity of such a multigene-controlled mechanism of AGP-related cell fate determination. The present results also suggested that several classes of AGPs might contribute to the same process of embryogenesis, each of them playing a unique role and acting together in regulating the same process. Although it is not yet clear how AGPs binding to ßGlcY are involved in directing cell developmental fate, ßGLcY binding experiments showed the dramatic effect and clear phenotype in each stage of embryogenesis. Therefore, it is a useful tool to combine with the genetic analysis assay and offers a valuable clue with the specific phenotype in seeking the mechanism of action of AGPs in cell differentiation during embryogenesis.
In normal embryonic development, roots are not generated during the heart and torpedo stages. However, in the present work, the ßGlcY–AGP interaction promoted rapid root generation during both stages. It is interesting that this response to ßGlcY paralleled the effects of shoot apex ablation in the carrot somatic embryo (Schiavone and Racusen, 1990), which also promoted root development. Schiavone (1988) suggested that the shoot apex controls root elongation through the production of auxin and polar auxin transport in the carrot somatic embryo. Recently, phytohormones were shown to regulate both root growth and the development of lateral roots (Beaudoin et al., 2000; Brady et al., 2003; De Smet et al., 2003). Root hair formation can also be modulated by hormones and inhibitors (Le et al., 2001; Vissenberg et al., 2001b). In the experiments reported here, microscopy clearly showed that ßGlcY binding totally altered the developmental fate of the cell at the apical region of the embryos and thus promoted root growth, similar to the effect of apical tissue ablation. A reasonable explanation is that the binding of ßGlcY disturbs AGPs function and alters the developmental fate of apical cells. Thus, the apical cells lose their normal function of controlling root generation and development, resulting in overgrown roots and aborted shoots in ßGlcY-treated embryos. The data also suggest that ßGlcY–AGP interactions change the expression level and/or transport of endogenous hormones, which provides additional support for the hypothesis that the shoot pole is the site of action of ßGlcY (Thompson and Knox, 1998). Finally, it could be deduced that, during microspore embryogenesis, AGPs are involved in bipolar development processes that are co-operatively regulated by phytohormones.
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Acknowledgements |
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We thank Dr JP Knox (Centre for Plant Science, University of Leeds, Leeds, UK) for the gifts of Yariv reagents and JIM8 antibody, and for his helpful comments during the preparation of this manuscript. We also thank Dr K Roberts (John Innes Institute, UK) for providing JIM4 and JIM13 antibodies. The project was supported by the National Outstanding Youth Science Fund (30225066), the National Natural Science Fund of China (30370743, 90408002, 30521004), and ‘PCSIRT’.
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Abbreviations |
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AGP, arabinogalactan protein; ßGlcY, ß-D-glucosyl Yariv reagent; ßManY, ß-D-mannosyl Yariv reagent; BSA, bovine seum albumin; FDA, fluorescence diacetate; PBS, phosphate-buffered saline.
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References |
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