JXB Advance Access originally published online on October 30, 2006
Journal of Experimental Botany 2006 57(15):3979-3988; doi:10.1093/jxb/erl169
RESEARCH PAPER |
The immunolocation of a xyloglucan endotransglucosylase/hydrolase specific to elongating tissues in Cicer arietinum suggests a role in the elongation of vascular cells
Departamento de Fisiología Vegetal, Facultad de Biología, Universidad de Salamanca, Centro Hispano-Luso de Investigaciones Agrarias, Pza Doctores de la Reina s/n, Salamanca 37007, Spain
* To whom correspondence should be addressed. E-mail: bdr{at}usal.es
Received 26 May 2006; Accepted 22 August 2006
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Abstract |
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In a previous work, a Cicer arietinum cDNA clone (CaXTH1) encoding a xyloglucan endotransglucosylase/hydrolase (XTH1) protein was isolated and characterized. CaXTH1 showed an expression pattern specific to growing tissue: mostly epicotyls and the upper growing internodes of adult stems. CaXTH1 mRNA was not detected in any other organs of either seedlings or adult plants, suggesting an involvement of the putative XTH encoded by CaXTH1 in the chickpea cell expansion process. After the generation of polyclonal antibodies by using the XTH1 recombinant protein and the analysis of the specificity of the antibodies for XTH proteins, here the specific location of the chickpea XTH1-cross-reacting protein in cell walls of epicotyls, radicles, and stems is reported, evaluated by western blot and immunocytochemical studies. The results indicate a function for this protein in the elongation of parenchyma cells of epicotyls and also in developing vascular tissue, suggesting a role in the elongation of vascular cells.
Key words: Cell wall, Cicer arietinum, elongation, epicotyl, vascular tissues, xyloglucan endotransglucosylase/hydrolase
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Introduction |
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The cell wall is the main factor that determines cell shape, and cell wall reconstruction enables its modification during cell elongation. The primary cell wall consists of three co-extensive polymer networks: the cellulose–xyloglucan framework, pectins, and structural protein. It is considered that the structural changes in these networks are regulated by enzymatic modification, and hence wall-modifying enzymes would be expected to play an important role in regulating the plasticity of the cell wall.
Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes that cut xyloglucan chains and transfer the fragment with the new reducing end either to another xyloglucan (called xyloglucan endotransglucosylase or XET activity) or to water (called xyloglucan endohydrolase or XEH activity). XTHs are thought to play an important role in the expansion and/or assembly of plant cell walls (Fry, 1995; Xu et al., 1996; Nishitani, 1997; Thompson et al., 1998) both by cutting and restructuring the existing wall-bound xyloglucan, and in some cases by rejoining newly secreted xyloglucan chains (Fry et al., 1992; Nishitani and Tominaga, 1992; Thompson et al., 1998; Thompson and Fry, 2001; Rose et al., 2002).
XET activity has indeed often been correlated with growth rate (Fry et al., 1992; Potter and Fry, 1994; Xu et al., 1995; Palmer and Davies, 1996; Catala et al., 1997), although not always, and it has also been detected in vegetative tissues that have ceased to elongate (Smith et al., 1996; Barrachina and Lorences, 1998), in ripening fruit (Redgwell and Fry, 1993; Maclachlan and Brady, 1994; Ishimaru and Kobayashi, 2002), and even during secondary cell wall formation (Bourquin et al., 2002). Currently, the role of XTHs as wall-modifying enzymes seems to be clear, but the existence of many different isoenzymes means that this activity could be related to processes other than growth.
In recent years, the expression of XTH genes has been found in particular in elongating tissues (Ma et al., 2001; Ji et al., 2003; Romo et al., 2005). Multiple isoforms of XTHs are expressed in different organs of different plant species in response to many hormonal, environmental, and developmental stimuli (Xu et al., 1995; Akamatsu et al., 1999; Yokoyama and Nishitani, 2001). Thus, the characterization of individual XTH genes and their corresponding proteins within a single species is essential if we are to understand their specific role.
The study of XTH mRNA expression patterns may suggest the physiological processes and/or tissue specificity in which one particular XTH participates. However, besides expression analysis of the different XTH genes, the localization of the protein could greatly increase current insights into the precise function of these enzymes.
The characterization of a cDNA clone from chickpea has previously been reported that encodes a putative XTH, whose expression occurs with high specificity in elongating tissues, suggesting that the encoded enzyme might function in cell extension (Romo et al., 2005). In the present work, using specific antibodies raised against the protein encoded by this clone, the highly specific localization of the chickpea XTH1-cross-reacting protein in elongating organs is reported, thus supporting its role in elongation and its possible involvement in elongation of vascular cells during the xylogenesis process.
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Materials and methods |
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Plant material and growth conditions
Chickpea seeds (Cicer arietinum L. cv. Pedrosillano) previously sterilized in 0.1% sodium hypochlorite were germinated in water, in the darkness at 25 °C and 80% relative humidity on glass plates covered with filter paper. The growth period studied ranged from 3 d to 8 d, after which the seedlings were harvested and epicotyls were collected. Hooks, epicotyls, mesocotyls (root–epicotyl junction zone), cotyledons, and roots from 4-d-old etiolated seedlings were also collected for subsequent studies.
Chickpea plants were grown in vermiculite, at 25 °C and 80% relative humidity, during 11 d (when the fifth internode appears), after which the stems and roots were harvested. Stems were divided into five internodes, numbered 1–5 from base to apex. The internode length was measured in plants from the sixth to the 12th day.
Expression and purification of the XTH1 fusion protein
The coding sequence of CaXTH1 (minus the signal sequence) was polymerase chain reaction (PCR)-amplified from the CaXTH1 plasmid clone. The two oligonucleotide primers used were: 5'-GAATTCGCTGCTCCAAGGAAACCAG-3' and 5'-CTCGAGTTAAATGTCACGGTCTCTTGTA-3', adding an EcoRI restriction site at the 5' end and an XhoI at the 3' end, respectively (underlined in the sequences). The PCR product was subcloned into the EcoRI–XhoI restriction site of the pET-28a(+) (Novagen, USA) expression vector and moved into the Escherichia coli strain BL21 (DE3) (Novagen, USA). As a control of expression, the plasmid pET-28a(+) was used without any insert.
The cells were first cultured overnight in 10 ml of Luria–Bertani (LB) medium (50 µg ml–1 kanamycin) at 37 °C. A 1 ml aliquot of the overnight culture was transferred to 200 ml of LB medium and allowed to continue to grow until the A600 reached 0.4–0.6. Isopropyl-ß-D-thiogalactopyranoside (IPTG) was added at 1 mM and allowed to induce for 3 h. Inclusion bodies were separated from the soluble fraction using the BugBuster Protein Extraction Reagent (Novagen, USA), and the subcellular location of the recombinant protein was determined by SDS–PAGE. The fusion protein was not soluble; several attempts to obtain the protein in the soluble fraction by modifying the culture temperature (27, 30, and 37 ºC) and the IPTG concentration (0.4 and 1 mM) were unsuccessful and therefore the inclusion bodies were harvested.
Purification of the recombinant XTH1 protein from the inclusion bodies was carried out by direct elution from the acrylamide gels, using the ProtoPrep II and the InsiteTM kits from National Diagnostics (USA), following the manufacturer's protocol.
Antibody production
For polyclonal antibody production, two white New Zealand female rabbits were immunized by multiple subcutaneous injections with recombinant XTH1 (50 µg each injection) emulsified with adjuvant at 1:3 dilution. Freund's complete adjuvant (Sigma, USA) was used for the first and incomplete adjuvant for the subsequent immunizations, at 2 week intervals. Pre-immune and immune sera were collected and IgGs were purified using an affinity column (HiTrap protein A HP, Amersham Biosciences, USA).
Cell wall protein extraction and western blotting
Cell walls were prepared as described in Dopico et al. (1989). Protein was extracted from freshly isolated cell walls with 1 M NaCl in Na-citrate/phosphate (10 mM, pH 5.5) at 4 °C for 48 h. The wall suspension was filtered through Miracloth (Calbiochem, USA), and the protein extract was dialysed against Na-acetate (20 mM, pH 5.0). The dialysed protein was centrifuged for 25 min at 6500 g and concentrated using an Amicon PM3 ultrafiltration cell (Amicon Corporation, USA). The protein was evaluated by Protein Assay (Bio-Rad, Baltimore, MD, USA).
For western blotting, proteins were separated by SDS–PAGE (Laemmli, 1970) and electro-transferred onto PVDF membranes (Amersham Biosciences, USA). Immunoblots were prepared essentially according to the procedure of Harlow and Lane (1988), using the anti-XTH1 IgGs at 1:10 000 dilution and a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, Bio-Rad, Germany) at 1:100 000. Blots were developed by chemiluminescence using the ECL Advanced Western Blotting Detection Kit (Amersham Biosciences, USA).
Two-dimensional electrophoresis (isoelectric focusing and SDS–PAGE)
For first dimension isoelectric focusing (IEF), 7 cm immobilized dry strips with pH gradients 6–11 (GE Healthcare) were rehydrated with 200 µg of protein sample in rehydration buffer [7 M urea, 2 M thiourea, 4% CHAPS, 0.015 µg µl–1 dithiothreitol (DTT), and 0.5% IPG buffer]. IEF was performed at 20 ºC using the IPGphor system (Amersham Pharmacia Biotech Biosciences, USA) with voltages from 100 V to 8000 V at maximum. For 2D SDS–PAGE, IPG strips were fixed vertically onto SDS–10% polyacrylamide gels. SDS–PAGE was carried out for 3 h at 25 mA. Two gels were made for each experiment. One of the gels was electro-transferred onto PVDF membranes (Amersham Biosciences, USA) and immunoblotted with anti-XTH antibodies as described above, the second gel was subsequently Commassie stained overnight using 0.1% Coomassie brilliant blue G250 (Bio-Rad, Richmond, CA, USA).
Trypsin digestion and peptide mass fingerprinting/mass spectrometry
The protein spot of interest was automatically excised from the gel using a Proteineer spII robotic spot scission station (Proteineersp, Bruker, Bremen, Germany) and digested by trypsin according to the protocol of Schevchenko et al. (1996). Gel plugs were treated in three successive washing steps with 250 µl of acetonitrile:ammonium bicarbonate [50% (v/v):25 mM], 250 µl of DTT:ammonium bicarbonate (20 mM:25 mM), and 250 µl of iodoacetamide:ammonium bicarbonate (100 mM:25 mM). After washing procedures, gel plugs were dried and digested for 16 h at 37 ºC using porcine trypsin (sequencing grade modified trypsin, Promega Corporation, Madison, WI, USA). The samples containing the trypsin-digested protein were mixed with a solution of 50% acetonitrile and 0.1% trifluoroacetic acid (TFA).
Mass spectra measurements of the peptides were obtained with a Bruker Ultraflex IV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker-Franzen Analytic GMBH, Bremen, Germany). All measurements were done under the following conditions: external calibration against a peptide mixture (Bruker, Bremen, Germany); and internal calibration for each spectrum against peaks arising from trypsin autoproteolysis to reach a typical mass measurement accuracy of ±30 ppm. The search for peptide mass fingerprint (PMF) matches was performed in different databases using the MascotTM software (Matriz Science, London, UK).
Immunocytochemical labelling
Epicotyls and radicles from 4-d-old seedlings and the five internodes from 11-d-old plants were cut in a drop of 4% (w/v) paraformaldehyde, 0.25% (v/v) glutaraldehyde, 100 mM sodium phosphate buffer (pH 6.8) and were immediately transferred to fixation solution and vacuum infiltrated. The tissues were subsequently dehydrated and embedded in Paraplast embedding medium at 60 °C. The embedded tissues were sliced into 12 µm sections using a microtome (Leica Instruments GMBH, Germany) and affixed to slides precoated with high molecular weight poly-L-lysine. Samples were deparaffinized with xylene and rehydrated through a graded ethanol series. Sections were then incubated for 5 min in 10 mM citrate buffer, pH 6.0, at 100 °C to inactivate endogenous alkaline phosphatase activity, since an alkaline phosphatase-conjugated secondary antibody is used to develop the reaction. Samples were washed twice in TRIS-buffered saline (TBS; 0.1 M TRIS, 0.1 M NaCl, pH 7.4). Free binding sites were blocked for 45 min with 5% bovine serum albumin (BSA) and 3% normal swine serum in TBS. Anti-XTH1 IgGs (1:100 dilution in TBS with 3% BSA) were applied to sections for 2 h at room temperature. Excess antibody was removed with extensive washing in 0.5% Tween-20, 1% BSA in TBS. After a second blocking, secondary antibody (goat anti-rabbit IgG conjugated with alkaline phosphatase, at 1:300 dilution in TBS with 3% BSA) was applied and then extensively washed as above. The colour reaction to visualize the antigen–antibody complexes was performed in TBS supplemented with 50 mM MgCl2, pH 9.5, containing 5-bromo-4-chloro-3-indolyl-phosphate and 4-nitroblue tetrazolium chloride. Sections were dehydrated in a graded ethanol series, dipped in xylene, and mounted in Entellan (Merck, USA). Photographs were taken using a microscope (Optiphot-2, Nikon, Tokyo) equipped with epifluorescence optics.
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Results |
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Heterologous expression of the recombinant protein XTH1 in Escherichia coli
CaXTH1 encodes a putative xyloglucan endotranglucosylase, as reported previously (Romo et al., 2005). In order to clarify the function of the protein encoded by CaXTH1 in the elongation processes, it was decided to carry out the expression of CaXTH1 in a heterologous system of E. coli in order to obtain the recombinant protein XTH1, which allowed polyclonal antibodies to be raised for immunolocation studies.
The expression of CaXTH1, except the signal peptide, in the pET28a/BL21 system led to the presence of a protein band of 36 kDa in the transformed E. coli cellular extracts, which was absent in the control (Fig. 1A). This molecular weight agrees with that estimated for the recombinant XTH1 protein. The protein appeared only 1 h after IPTG induction, increased up to 3 h after induction, and thereafter remained more or less constant (Fig. 1A). Study of the subcellular location of the recombinant protein XTH1 obtained in the BL21(DE3) strain 3 h after induction indicated that the protein appeared as inclusion bodies (Fig. 1B).
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Although the fusion protein XTH1 carried a six-histidine tail at the N-terminus in order to facilitate its purification, the recombinant protein was not retained by affinity chromatography in nickel columns. Its complete purification was therefore carried out by direct elution from the acrylamide gels, as described in the Materials and methods (Fig. 1C).
The purified recombinant XTH1 protein was used to raise antibodies in rabbits. By enzyme-linked immunosorbent assay (ELISA) assay it was proven that anti-XTH1 IgGs recognized the recombinant XTH1 protein (data not shown). In order to verify the specificity of anti-XTH1 IgGs to XTH proteins, 2D electrophoresis, western blot and MALDI-TOF analyses were carried out. Thus, total cell wall protein extract of epicotyls from 4-d-old chickpea seedlings were separated by 2D gel electrophoresis (Fig. 2A). In immunoblot analysis (Fig. 2B) using anti-XTH1 IgGs and the 2D separated proteins, the stronger signal corresponded to a 32 kDa polypeptide with a pI of 8.5, matching with the predicted molecular mass and the pI of XTH1 protein. This protein spot, indicated with number 1 in Fig. 2A, was excised from the gel and analysed by MALDI-TOF mass spectrometry after trypsin digestion. Comparison of the obtained PMFs (Fig. 2C) was performed with predicted PMFs of translated sequences of the SwissProt and NCBI databases. The result of these searches indicated that the chickpea peptides showed strong identity with XTHs isolated from Arabidopsis thaliana.
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Western blot immunodetection of the XTH1-cross-reacting protein in cell wall protein extracts
The anti-XTH1 IgGs were used for western blot analysis in different chickpea seedling and plant organs and developmental stages.
When total cell wall protein extract of seedling epicotyls was separated by SDS–PAGE, the anti-XTH1 IgGs recognized only a 32 kDa polypeptide, coinciding with the molecular weight of mature XTH1, and no other protein band was detected in the total cell wall protein extract. The chickpea XTH1-cross-reacting protein was detected during epicotyl growth, from day 3 to day 8. Figure 3A shows that the protein decreased with the age of epicotyls, according to the trend of CaXTH1 transcripts (Romo et al., 2005). The purified IgGs from the pre-immune serum did not recognize any cell wall protein.
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When the western blot was carried out with cell wall protein extracts from several parts of seedlings and plants, it was observed that the XTH1-cross-reacting protein was only detected in elongating organs such as epicotyls in seedlings (Fig. 3B) and stem internodes in 11-d-old plants (Fig. 3C). The maximum recognition was observed in apical internodes (fourth and fifth internodes), the highest being in the fourth internode which presents at that moment a very active growth, as shown in Fig. 4. No protein was detected in the more basal internodes, where there is no growth on the 11th day (Fig. 4). Again, these results agree with the CaXTH1 gene expression pattern (Romo et al., 2005) and confirm the presence of this protein mainly in elongating tissues.
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Immunolocation of the XTH1-cross-reacting protein
In order to determine the tissue and cellular location of the XTH1-cross-reacting protein in epicotyls and radicles from 4-d-old seedlings and in the stem internodes from 11-d-old plants, immunocytochemical studies were conducted, using the anti-XTH1antibodies, as described in the Materials and methods.
Since the aim was to establish a putative relationship between the detection of protein and growth, and it is known that chickpea epicotyls show a growth gradient that decreases from the apical to the basal zone (Seara et al., 1988), three 2 mm sections with different growth activity in the apical, central, and basal zone of 4-d-old epicotyls were separated as indicated in Fig. 5 and used for immunolocation studies.
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When slices from apical, central, and basal sections of epicotyls were immunolabelled with anti-XTH1 IgGs (Fig. 6), the XTH1-cross-reacting protein appeared clearly bound to the cell walls of the cortex and pith cells in the apical and central sections, detection being more pronounced in apical sections (Fig. 6a, c). Labelling decreased along the epicotyl axis from the apical growing zone to the basal zone (Fig. 6c, f, i). In the basal epicotyl section, the recognition was restricted to the cortex cell layer next to the vascular system, and a similar detection was observed in pith cells (Fig. 6g, h, i). Along the epicotyl, the XTH1-cross-reacting protein was also located in a specific way in the vascular tissue, in both the primary xylem and phloem cells (Fig. 6b, e, h). When slices from epicotyls were treated with the preimmune serum, no protein was recognized (Fig. 6j, k, l).
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Although the western blot results did not indicate the presence of XTH1-cross-reacting protein in radicles, bearing in mind that immunocytochemistry studies are more sensitive that western blot, immunolocation studies were also conducted in radicles. After removing the first 5 mm of the radicle tip where no growth was present, three 2 mm sections were separated as indicated in Fig. 5: an apical section (5–7 mm from the radicle tip) with a high growth activity; a central section, and a basal section (the 2 mm of the radicle closest to the cotyledon insertion) corresponding to the hypocotyl–radicle axis which does not present any growth activity (data not shown). Slices from these sections were used for immunocytochemical studies.
The immunolocation of XTH1-cross-reacting protein in chickpea radicles was, as expected, less pronounced than in epicotyls. In the apical zone (Fig. 7a, b), XTH1-cross-reacting protein was located in the cell walls of the endodermis layer, pericycle, and vascular tissues, the distribution being fairly uniform within the stele. A similar but much lower presence of the protein was seen in the basal zone of the radicle (Fig. 7c, d). In the zone of the hypocotyl–radicle axis, almost no protein was found (Fig. 7e, f). No protein was recognized in sections treated with preimmune serum (Fig. 7g, h, i).
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The location of XTH1 in chickpea stems was determined by studying its immunolocation in the five internodes of an 11-d-old plant (Fig. 8). After immunolabelling of internode transversal sections, no presence of XTH1-cross-reacting protein could be seen in the basal first, second, and third internodes, and detection was only evident in the apical fourth and fifth internodes, which exhibited active growth (Fig. 4). Higher magnification images of the fourth and fifth internode sections showed that the collenchyme tissue in the cortex exhibited strong incorporation of label. XTH1-cross-reacting protein was also detected in primary xylem and phloem cells. Almost all of the phloem cells appeared to be labelled in the fifth apical internode, (Fig. 8a, b), whereas in the fourth internode it could be seen that differentiated phloem fibres were not labelled (Fig. 8c, d).
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Discussion |
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The precise role of XTHs in cell growth is still under debate. It is known that Arabidopsis has 33 different XTH genes (Yokoyama and Nishitani, 2001). It can thus be expected that the different gene products would be active in different aspects of cell wall metabolism.
In an earlier work (Romo et al., 2005), an important involvement of the putative XTH encoded by CaXTH1 in the chickpea elongating process was suggested, based on several observations regarding its pattern of expression such as the high CaXTH1 expression in elongating organs, its up-regulation by growth-promoting hormones, or its down-regulation by inhibitory growth treatment. As in chickpea, in several species, genes encoding XTHs have been reported to show a specific expression in elongating organs and to be up-regulated by growth induced-hormones (Oh et al., 1998; Akamatsu et al., 1999; Nakamura et al., 2003).
Although gene expression studies are useful for checking the involvement of a specific protein in a specific process, the location of the corresponding protein encoded by a given gene could provide more information about the actual role of the protein in the process. In this sense, Vissenberg et al. (2005) recently reported the visualization of XET action at the subcellular level in single cells. As another approach to gain more insight into the role of XTHs, by using polyclonal antibodies raised against the E. coli-expressed recombinant protein encoded by CaXTH1, here the location of the XTH1-cross-reacting protein in seedlings and plants of C. arietinum and its visualization in specific cells of elongating organs are reported. The specificity of antibodies to XTH proteins was previously checked by MALDI-TOF analysis (Fig. 2).
In chickpea, the specific location of the XTH1-cross-reacting protein was detected in the cell walls of seedling epicotyls (Fig. 3B) and in stems from adult plants (Fig. 3C), all of them elongating organs, while it could not be detected in other organs such as meristematic hooks, cotyledons, or even adult roots, supporting its involvement in the cell elongation process. It is interesting that throughout epicotyl growth, the XTH1-cross-reacting protein level in cell walls decreased as the epicotyls aged and consequently decreased their growth rate (Fig. 3A). In the same way, in stems, the protein was only immunodetected in the cell walls of the more apical internodes (Figs 3, 8), mostly in the fourth one, with a very active growth (Fig. 4). These results are consistent with the CaXTH1 expression analysis carried out previously (Romo et al., 2005), and they support the direct involvement of the XTH1-cross-reacting protein in epicotyl and stem elongation. Further support comes from the fact that this protein could be immunodetected intensively in the cortex cells of apical zones of the epicotyls (Fig. 6), whereas detection was progressively less intense in the central and basal zones of the epicotyls, in agreement with the different growth capacities in the epicotyl zones (Fig. 6). A similar grading could be seen in the different radicle sections used for immunocytochemical studies (Fig. 7), indicating that this XTH1-cross-reacting protein could also be involved in radicle elongation, although the protein was not detected by western blot in this organ, which confirms the higher efficiency of immunocytochemistry as compared with immunodetection by western blot. However, protein detection in radicles was much lower than in epicotyls, indicating a greater involvement of an XTH1-cross-reacting protein in epicotyls than in radicle growth.
In addition to the involvement of chickpea XTH1 in cell wall extension in epicotyls and young stem internodes, this protein could be involved in other processes during development, as suggested by the specific location of the XTH1-cross-reacting protein in vascular tissues, specifically in primary xylem and phloem cells in both epicotyls and radicles (Figs 6, 7), and apical internodes (Fig. 8). The involvement of XTH in vascular differentiation has been indicated recently, and to date it has been reported that several XTH genes are broadly expressed throughout the primary vascular tissues (Xu et al., 1995; Antosiewicz et al., 1997; Oh et al., 1998; Nakamura et al., 2003). XET activity has also been observed in the plant vasculature (Vissenberg et al., 2000, 2004; Bourquin et al., 2002). This suggests that some members of the XTH family would be responsible for controlling the extensibility and properties of the cell wall in the different types of vascular cells.
In chickpea, it has previously been reported that mRNA CaXTH1 was observed in vascular tissues by in situ hybridization (Romo et al., 2005). The present results show that, as well as the CaXTH1 gene being preferentially expressed, the XTH1-cross-reacting protein is mainly located in immature growing vascular elements in epicotyls and radicles, where secondary wall thickening occurs (Figs 6 and 7). These results suggest that an XTH protein similar to XTH1 could play an essential role in the process of xyloglucan degradation in the growing xylem and phloem cells. The presence of XTH in vascular elements suggests that an increase in the degradation of xyloglucans in the cell wall would be taking place during the differentiation of vascular tissue. Several observations reported for different plants support this notion. Thus, in poplar vascular tissue, Bourquin et al. (2002) confirmed that xyloglucan molecules, which are present in the primary cell walls of immature tracheary elements, disappeared before the tracheary elements had matured. In soybean hypocotyls, xyloglucan molecules were not detected in the primary cell walls of protoxylem elements, but were present in the cell walls of surrounding cells (Ryser, 2003). Furthermore, in the Zinnia mesophyll cell transdifferentiation system, xyloglucans were degraded by hydrolytic enzymes that were secreted from tracheary elements before visible secondary wall thickening occurred (Stacey et al., 1995). Recently, it has been described that the protein encoded by the AtXTH27 gene plays an essential role in the disassembly of the primary cell wall architecture during the differentiation of tracheary elements that takes place during leaf development (Matsui et al., 2005). Regarding phloem cells, in Vigna angularis it has been reported that VaXTH1 and VaXTH2, both sequences similar to CaXTH1, are also expressed in vascular tissue, mainly in the protophloem cells of radicles (Nakamura et al., 2003).
In support of the involvement of a protein similar to XTH1 in early chickpea vascular differentiation, it is worth noting that in the hypocotyl–radicle axis (Fig. 7e, f), characterized by its loss of primary growth, no detection of protein was observed either in xylem or in phloem cells since in this zone mature vascular bundles were present. Also, in stem internodes (Fig. 8), XTH1-cross-reacting protein was found in xylem and phloem cells, but only in the early stages of differentiation. The label was associated specifically with the thickening of the secondary layer and disappeared in phloem fibres when the secondary wall was completed (Fig. 8c, d). A similar involvement of XTH in early phloem differentiation in poplar stems was reported by Bourquin et al. (2002) by evaluating XET activity.
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Acknowledgements |
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This work was supported by grants from the Dirección General de Investigación Científica y Técnica (DGICYT), Spain (BOS2002-01900) and the Junta de Castilla y León (SA124/04). The MALDI-TOF analysis was carried out in the Centro de Investigación del Cancer, Universidad de Salamanca-CSIC, www.proteored.org.
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Footnotes |
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The nucleotide sequence of CaXTH1 reported in this paper has been submitted to EMBL/GenBank database under accession number AJ004917.
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Abbreviations |
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IEF, isoelectric focusing; IPTG, isopropyl-ß-D-thiogalactopyranoside; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; PMF, peptide mass fragment; TBS, TRIS-buffered saline; XTH, xyloglucan endotransglucosylase/hydrolase.
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References |
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