Journal of Experimental Botany, Vol. 55, No. 396, pp. 423-431, February 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
Zinnia elegans uses the same peroxidase isoenzyme complement for cell wall lignification in both single-cell tracheary elements and xylem vessels
Received 23 June 2003; Accepted 16 October 2003
Department of Plant Biology (Plant Physiology), University of Murcia, E-30100 Murcia, Spain
* To whom correspondence should be addressed. Fax: +34 968 363 963. E-mail: rosbarce{at}um.es
Abbreviations: IWF, intercellular washing fluid; NEIEF, non-equilibrium isoelectric focusing.
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Abstract |
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The nature of the peroxidase isoenzyme complement responsible for cell wall lignification in both Zinnia elegans seedlings and Z. elegans tracheary single-cell cultures have been studied. Results showed that both hypocotyls and stems from lignifying Z. elegans seedlings express a cell wall-located basic peroxidase of pI
10.2, which was purified to homogeneity. Molecular mass determination under non-denaturing conditions showed an Mr of about 43 000, similar to that of other plant peroxidases. The purified Z. elegans peroxidase showed absorption maxima at 403 (Soret band), and at 496–501 and 632–635 (
and ß absorption bands), indicating that this enzyme is a high spin ferric haem protein, belonging to the plant peroxidase superfamily, the prosthetic group being ferric protoporphyrin IX. The N-terminal amino acid sequence of this Z. elegans basic peroxidase was KVAVSPLS (peptide motif in bold), which shows strong homologies with the N-amino acid terminus of other strongly basic plant peroxidases. Isoenzyme and western blot analyses showed that this peroxidase isoenzyme is also expressed in trans-differentiating Z. elegans tracheary single-cell cultures. The results also showed that Z. elegans tracheary single-cell cultures not only express the same peroxidase isoenzyme as the Z. elegans lignifying xylem, but that this peroxidase isoenzyme acts as a marker of tracheary element differentiation in Z. elegans mesophyll single-cell cultures. From these results, it may be concluded that Z. elegans uses a single programme, i.e. an identical peroxidase isoenzyme complement, for lignification of the xylem, regardless of the existence of different ontogenesis pathways from either mesophyll cells (in the case of tracheary elements) or cambial derivatives (in the case of xylem vessels). Key words: Lignifying xylem, peroxidase isoenzyme, tracheary single cell cultures, Zinnia elegans.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Lignins are three-dimensional phenolic heteropolymers covalently associated with polysaccharides in plant cell walls (Anterola and Lewis, 2002). They are mainly localized in the impermeable water-transport conduits of the xylem and other supporting tissues, such as phloem fibres, and result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction that can be mediated by both peroxidases and laccases (Ros Barceló, 1997; Liu et al., 1994) leading to an optically inactive hydrophobic heteropolymer (Ralph et al., 1999). The process of sealing plant cell walls through lignin deposition is known as lignification, and provides mechanical strength to the stems, protecting cellulose fibres from chemical and biological degradation (Grabber et al., 1988). In this context, plant cell wall lignification is one of the main restrictive factors in the use and recycling of plant biomass (Anterola and Lewis, 2002).
Zinnia elegans is a annual-cycle flowering plant belonging to the Asteraceae family frequently used as a model for lignification studies (Fukuda, 1997). The cell wall of lignifying Z. elegans hypocotyls and stems contain a basic peroxidase (EC 1.11.1.7
[EC]
) of pI 10.2, which shows coniferyl alcohol oxidase activity (Ros Barceló and Aznar-Asensio, 1999; Pomar et al., 2002), i.e. an oxidase activity in the absence of H2O2. This basic peroxidase is capable of oxidizing both hydroxycinnamyl alcohols and aldehydes with Km values in the µM range, and shows some specificity for syringyl-type phenols (Ros Barceló and Pomar, 2001). It is worth noting that the affinity of this strongly basic peroxidase for hydroxycinnamyl alcohols and aldehydes is similar (Ros Barceló and Pomar, 2001) to that shown by the preceding enzymes in the lignin biosynthetic pathway (microsomal 5-hydroxylases and cinnamyl alcohol dehydrogenase), which also use hydroxycinnamyl alcohols and aldehydes as substrates. These catalytic properties indicate that the one-way highway of lignin macromolecule construction has no metabolic ‘potholes’ in which the lignin building blocks might accumulate. All these constraints strongly suggest that this peroxidase is one of the enzymes responsible for cell wall lignification, a consideration which is supported by its localization in lignifying xylem vessels (Ros Barceló et al., 2000, 2002).
Z. elegans mesophyll cells capable of undergoing tracheary element differentiation have been considered as a suitable model for studying the sequence of events which takes place during xylem differentiation, including secondary cell wall synthesis (Fukuda, 1997; Roberts and McCann, 2000). In fact, they have been used as a model for monitoring the expression of enzymes from the lignin biosynthetic pathway, especially the route segment that is concerned with the phenylpropanoid backbones (Fukuda and Komamine, 1982; Demura et al., 2002; Milloni et al., 2002). Differentiation of mesophyll cells into tracheary elements is synchronously induced in a large number of cells in a relatively homogeneous cell population system, making it possible to study the biochemistry and physiology of xylogenesis free from the complexity which heterogeneous plant tissues impose (Roberts and McCann, 2000; Milloni et al., 2002). However, to the authors’ knowledge, there are no available data confirming that tracheary single-cell cultures may be regarded as a full reliable model of what occurs in the lignifying xylem, especially during the last step of the lignification, i.e. the assembly process. This uncertainty is extrapolated to the architecture of the major surface on which lignins are largely deposited, the cell wall secondary thickening.
In vascular plants, the pattern of secondary cell wall formation is species-specific and is of taxonomic importance, since this pattern is used in phylogenetic studies (Kenrick and Crane, 1997). In Z. elegans tracheary single-cell cultures, the patterns of secondary cell wall formation are multivariate, and lead simultaneously to tracheids with annular, spiral, reticulate, scaliform, or pitted secondary thickenings (Falconer and Seagull, 1985). However, it may be expected that such variability in the secondary thickenings shown by Z. elegans tracheary single-cell cultures will be unlike that which occurs in the Z. elegans lignifying xylem.
In the case of peroxidase, one of the enzymes responsible for the polymerization of hydroxycinnamyl alcohols into lignins, several studies describe its expression in Z. elegans tracheary single-cell cultures (Masuda et al., 1983; Sato et al., 1993, 1995a, b; Church and Galston, 1988), but no direct comparison has been made between peroxidase isoenzyme expression in both systems. This led to a search for homologies and differences at the peroxidase isoenzyme level between the Z. elegans lignifying xylem and the Z. elegans tracheary single-cell culture model using isoenzyme and western blot analyses.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material
Seedlings of Zinnia elegans (cv. Envy, Chiltern Seeds, Cumbria, England) were grown (Ros Barceló et al., 2000) for 26 d in a greenhouse under daylight conditions at 25 °C on Humus KingTM (type 3) (Impra SL, El Ejido, Almería, Spain), containing 30% organic C, 0.5% organic N, and 52% total organic material, pH 5.5–6.0. Fertilizers present in the humus were 120–160 mg l–1 N, 100–130 mg l–1 P2O5 and 150–200 mg l–1 K2O. Plants containing only the first internode were used in these studies, and they are characterized by presenting fully lignifying hypocotyls and weakly lignifying stems.
Z. elegans tracheary single-cell cultures
First true leaves of 14-d-old seedlings were removed, surface-sterilized in 5% (v/v) commercial NaOCl, and rinsed in sterile distilled water. Single cells were isolated and cultured for 6 d in a differentiating medium as described by Fukuda and Komamine (1982). Controls were performed with mesophyll cells cultured in a non-inductive medium (Fukuda and Komamine, 1982). Light micrographs were taken of cell suspensions in situ viewed in bright field with a Leica DMRB microscope. Staining with Calcofluor white was performed as described by Falconer and Seagull (1985). Lignin autofluorescence was photographed by excitation at 350 nm using a barrier filter with a transmission cut-off at 420 nm.
Isolation and fractionation of Z. elegans tracheary single-cell cultures and assay of enzymatic activities
Cells were separated from the culture medium by centrifugation at 100 g for 1 min at 4 °C. The supernatant constituted the apoplastic protein fraction. Sedimented cells were washed by resuspension in culture medium, and broken by a freezing/thawing cycle. Cell debris were extracted for 30 min at 4 °C with 50 mM Na-acetate buffer pH 5.0 containing 1.0 M KCl, and the protease inhibitors, 1.0 mM phenylmethylsulphonylfluoride and 1.0 mM benzamidine. The homogenate was centrifuged at 1300 g for 1 min at 4 °C, and the supernatant constituted the cellular protein fraction. Both the apoplastic and cellular protein fractions were desalted by chromatography on PD-10 Sephadex G-25 (Pharmacia Biotech.) columns equilibrated in 50 mM Na-acetate buffer pH 5.0 containing the protease inhibitors, 1.0 mM phenylmethylsulphonylfluoride and 1.0 mM benzamidine, and concentrated using Ultrafree-0.5 (Millipore). The assay of peroxidase activity using 3,3',5,5'-tetramethylbenzidine, coniferyl alcohol, and sinapyl alcohol was performed as described by Ros Barceló and Pomar (2001).
Transmission electron microscope studies
Mesophyll cells cultured for 4 d in a non-inductive medium were fixed, stained for peroxidase using 3,3'-diaminobenzidine, and processed for electron microscope studies as described by Ros Barceló et al. (1991).
Scanning electron microscopy
Both unfixed and uncryoprotected tissues were directly frozen in liquid nitrogen. Fractured 0.5 cm thick sections were dehydrated in a graded ethanol series, substituted in 2-methyl-2-propanol, and lyophilized in the critical state. Specimens were mounted on specimen carrier stubs, coated with gold, and observed under a scanning electron microscope (JEOL 6100) operated at 10 kV.
Total protein extraction, purification of the basic peroxidase from Z. elegans and microsequencing
Total protein was extracted from hypocotyls, leaves and stems by homogenizing in 0.2 M Na-acetate buffer, pH 5.0, containing 1.0 M KCl and 0.1 M CaCl2 at 4 °C, and centrifugation at 30 000 g for 30 min. Desalting and protein concentration was performed as above. Protein was determined according to Bradford (1976).
The Z. elegans basic peroxidase was purified from lignifying hypocotyls as described by Ros Barceló and Aznar-Asensio (1999). The protein was characterized as pure by SDS-PAGE (Ros Barceló and Aznar-Asensio, 1999). The visible spectrum of the protein was recorded in 50 mM Na-acetate buffer, pH 5.0. The N-terminal amino acid sequence of the Z. elegans basic peroxidase was determined by transferring the 43 kDa SDS-PAGE purified band onto a PVDF membrane, and deblocking the N terminus with pyrrolidone carboxyl peptidase (EC 3.4.19.3 [EC] ), which removes amino-terminal L-pyroglutamic acid from peptides and proteins (Hirano et al., 1993). The N-terminal sequence was determined by automated Edman degradation at the CIB (CSIC, Madrid Spain)
Intercellular washing fluids
To recover the intercellular washing fluids (IWF), either hypocotyls or stem sections (5.0 g FW), measuring less than 5 mm, were soaked in deionized water and subsequently vacuum-infiltrated for 5 min at 1.0 kPa and 4 °C with 50 mM TRIS-acetate buffer pH 5.0 containing 1.0 M KCl and 50 mM CaCl2. The sections were quickly dried and centrifuged in a 25 ml syringe barrel placed within a centrifuge tube at 900 g for 5 min at 4 °C. Desalting and concentration of the apoplastic protein was performed as above.
Electrophoretic analysis and western blot
Isoelectric focusing in non-equilibrium conditions (NEIEF) and cationic PAGE was performed as described by Ros Barceló et al. (2002). Protein migration was followed at 4 °C using cytochrome c as a migration marker. Peroxidase isoenzymes were stained with either 4-methoxy--naphthol or 3,3'-diaminobenzidine (Ros Barceló et al., 2002). SDS-PAGE was performed on 10% (w/v) polyacrylamide gels using a MiniProtean II electrophoresis kit and a pH 8.8 electrophoresis buffer composed of 192 mM glycine and 25 mM TRIS containing 0.1% SDS. SDS-PAGE was performed at 100 V for 20 min followed by 150 V for 70 min at 4 °C. Proteins were stained by the ammoniacal silver method (Oakley et al., 1980).
Western blot analyses of NEIEF gels were performed using anti-horseradish peroxidase (anti-HRP) rabbit IgGs as primary antibodies and HRP-conjugated goat anti-rabbit IgGs as secondary antibodies (Pomar et al., 2002). Endogenous peroxidase electroblotted onto Immun-Blot PVDF membranes (0.2 µm) was inactivated by heating (100 °C for 5 min). Peroxidase activity was stained with 4-methoxy--naphthol as above. Immunodot-blots were performed using the Bio-Dot SF (Bio-Rad) kit according to the manufacturer. Controls were performed in the absence of either anti-HRP rabbit IgGs or HRP-conjugated goat anti-rabbit IgGs.
Chemicals
Coniferyl alcohol, sinapyl alcohol, Calcofluor white, anti-horseradish peroxidase rabbit IgGs (P7899), HRP-conjugated goat anti-rabbit IgGs (A9169), and pyroglutamate aminopeptidase (L-pyrrolidine carboxyl peptidase, EC 3.4.19.3
[EC]
) were purchased from Sigma (Madrid, Spain). The rest of the chemicals were obtained from various suppliers and were of the highest purity available.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The Z. elegans basic peroxidase is constitutively expressed in leaves, stems and hypocotyls
The lignifying xylem is a common tissue present in young leaves, stems, and hypocotyls, its contribution to total tissues (vascularization degree) being lower in leaves and greater in hypocotyls. Protein fingerprints, as determined by SDS-PAGE (Fig. 1A), revealed that, among the major proteins, none was totally specific for a given organ, the observed differences being more quantitative (Fig. 1A, arrowheads) than qualitative. NEIEF of total peroxidase activity revealed the major presence of a strongly basic peroxidase in these three organs (Fig. 1B, arrowhead), and these results were confirmed by cationic PAGE (Fig. 1C, arrowhead).
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The Z. elegans basic peroxidase is located in the cell wall
To study the subcellular (apoplastic) localization of this Z. elegans basic peroxidase, intercellular washing fluids (IWFs) were obtained from both hypocotyls and stems. IWFs were free of any noticeable degree of contamination by the symplastic marker, glucose-6-phosphate dehydrogenase, which never exceeded 0.1% of that localized in the tissue fractions from which IWFs have been removed Ros Barceló and Aznar-Asensio, 1999). Further confirmation of the absence of noticeable symplastic contamination in this apoplastic fraction was obtained by protein fingerprint analysis. SDS-PAGE analyses of the major proteins in the symplastic fractions of both hypocotyls (Fig. 2A, lane hs) and stems (Fig. 2A, lane ss) showed the presence of specific proteins (arrowheads), which were almost totally absent from the respective apoplastic fractions (Fig. 2A, lanes ha and sa). Likewise, the yields in the recovery of IWFs were extraordinarily high since certain apoplastic proteins (Fig. 2A, lane sa, arrow) were absent from symplastic (IWF-extracted tissue) fractions. NEIEF analysis of the apoplast protein isolated from hypocotyls (Fig. 2B, lane ha) and stems (Fig. 2B, lane sa) confirmed the presence of this strongly basic peroxidase, supporting a cell wall (apoplastic) localization for the protein.
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Molecular characterization of Z. elegans basic peroxidase
The Z. elegans peroxidase was purified from lignifying hypocotyls. The protein was pure according to SDS-PAGE and showed an isoelectric point close to 10.2. NEIEF of the purified protein made it possible to compare the protein purity (Fig. 3A, lane p) with the assay of its catalytic activity in gel (Fig. 3A, lane ca). Molecular mass determination under non-denaturing conditions showed an Mr of about 43 000, which is similar to that of other plant peroxidases. The purified Z. elegans peroxidase showed absorption maxima at 403 (Soret band), and at 496–501 and 632–635 (
and ß absorption bands), indicating that this enzyme is a high spin ferric haem protein, belonging to the plant peroxidase superfamily, the prosthetic group therefore being ferric protoporphyrin IX. Finally, the Z. elegans basic peroxidase was shown to possess antigenic epitopes, which were recognized by antibodies directed against horseradish peroxidase, when antigenic properties were tested by immunodot-blot (Fig. 3B).
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The N-terminal amino acid sequence of the Z. elegans basic peroxidase was determined by transferring the SDS-PAGE purified band onto a PVDF membrane. Microsequencing was then carried out by deblocking the N terminus with pyrrolidone carboxyl peptidase, which removes amino-terminal L-pyroglutamic acid from peptides and proteins, and subsequent Edman degradation. The sequence obtained, KVAVSPLS (peptide motif in bold), was also contained in the N-amino acid terminus of other peroxidases (Fig. 4).
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Tracheary element differentiation in Z. elegans single-cell cultures is accompanied by a plethora of secondary cell wall thickening architectures which contrast with the single secondary cell wall thickening architecture of lignifying Z. elegans vessels
From the final architecture of the respective secondary thickenings, it is easy to appreciate that the mechanism governing tracheary element differentiation in Z. elegans mesophyll single-cell cultures is, at least in part, different from the mechanism governing xylem (vessel) differentiation from cambial derivatives in Z. elegans stems and hypocotyls. Thus, Z. elegans tracheary single-cell cultures showed a multivariate pattern of secondary thickenings which includes reticulate (Fig. 5A), pitted (Fig. 5B), and spiral (Fig. 5C) architectures, which appear to be lignified as revealed by lignin auto-fluorescence (Fig. 5D). Even in the same tracheary element, reticulated (Fig. 5B, arrows) and pitted (Fig. 5B, arrowheads) architectures may be present simultaneously, leading to aberrant or ‘chimeric’ differentiation patterns.
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This multi-determination of the final architecture of the secondary thickenings shown by Z. elegans tracheary single-cell cultures contrasts with the uniformity of the determination pattern of the secondary cell wall differentiation programme in Z. elegans protoxylem vessels (Fig. 6A), where only spiral architectures are developed both in stems (Fig. 6B, arrows) and in hypocotyls (data not shown). These differences suggest that the differentiation of tracheary elements from mesophyll cells does not exactly involve the same mechanisms or the same cellular signalling as differentiation of the xylem from cambial derivatives. In fact, leaf wounding appears to be critical (Roberts and McCann, 2000) for mesophyll cells to acquire competence for trans-differentiation in the Z. elegans tracheary single-cell culture model.
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From these results, it may be concluded that secondary cell wall formation in Z. elegans tracheary single-cell cultures is probably directed by a network of signals which differs from that which guides the differentiation of xylem vessels from cambial derivatives. If the pattern of secondary cell wall formation is different in Z. elegans tracheary single-cell cultures from the pattern found in Z. elegans xylem vessels, the authors speculated to what extent these ontogenic differences could also affect the expression of the peroxidase isoenzymes responsible for secondary cell wall lignification. To answer this, peroxidase isoenzyme expression was monitored by isoelectric focusing during tracheary element differentiation in Z. elegans single-cell cultures. Isoelectric focusing was selected as a tool for isoenzyme analysis since the isoelectric point property is determined by the balance of acid and basic amino acids of the protein, and thus reflects similarities in the primary structure of the isoenzymes.
Z. elegans single-cell cultures undergoing tracheary element differentiation express a basic peroxidase isoenzyme
The differentiation of Z. elegans mesophyll single-cells into tracheids is concomitant with the expression of a basic peroxidase isoenzyme (Fig. 7A). The increase in activity of this basic peroxidase isoenzyme during the trans-differentiation of mesophyll cell into tracheary elements (Fig. 7A, arrowhead) was related to the amount of the isoenzyme in the culture media when this was determined by western blot (Fig. 7B, arrowhead). It is worth noting that neither acidic nor neutral peroxidase isoenzymes were expressed during the trans-differentiation of mesophyll cells (Fig. 7A), despite the fact that, in some plant species, acidic peroxidase isoenzymes have been associated with lignifying vascular bundles (Carpin et al., 1999).
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Since it is known that coniferyl and sinapyl alcohols are utilized by Z. elegans for lignification (Pomar et al., 2002), and that Z. elegans basic peroxidase is capable of oxidizing both lignin precursors (Ros Barceló and Pomar, 2001), the activity of this peroxidase isoenzyme during trans-differentiation of mesophyll cells was followed both in the cellular and in the apoplast (culture medium) protein fraction using coniferyl (Fig. 8B) and sinapyl acohol (Fig. 8C). 3,3',5,5'-Tetramethylbenzidine was also used as a substrate (Fig. 8A) for comparative purposes. Enzymatic assays were performed at pH 5.0, the pH at which tracheary trans-differentiation takes place (Roberts and Haigler, 1994). Results showed that the increases in peroxidase activities during trans-differentiation of mesophyll cells (Fig. 8) was clearly related to the increase in the basic peroxidase isoenzyme in cells (Fig. 7A), and to the increase in the amount of apoplastic isoenzyme as determined by western blot (Fig. 7B).
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The basic peroxidase isoenzyme acts as a marker of tracheary element differentiation
From the results of isoenzyme analysis (Fig. 7) it may be expected that peroxidase isoenzyme expression is concomitant with tracheary element differentiation (Fig. 8) when the latter was monitored using the light microscope. Thus, peroxidase isoenzyme expression begins to take place 24 h before secondary cell wall formation starts, and this time-course is logical since lignification of the primary walls occurs before the lignification of the secondary walls (Terashima and Fukushima, 1988), and secondary cell wall lignification cannot take place until secondary cell wall thickenings are in progress.
Furthermore, the time-course of the expression of this basic peroxidase isoenzyme during the differentiation of Z. elegans tracheary elements (Fig. 7) is related to the expression of certain proteins (Fig. 9) which act as molecular markers of tracheary element differentiation in Z. elegans cell cultures. Of special mention was a 43 kDa protein which was specifically expressed after 72 h in the cells (Fig. 9A, arrow) but not in the culture medium (Fig. 9B). This protein has been identified as an RNase and has been considered a marker of tracheary differentiation in single-cell cultures (Fukuda, 1997). Protein bands at both 45 kDa and 40 kDa (Fig. 9, arrowheads) have also been considered markers of the trans-differentiation process (Stacey et al., 1995), and were expressed with a time-course pattern similar to that of the basic peroxidase (Fig. 7).
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The time-course of the expression of this basic peroxidase isoenzyme during the differentiation of Z. elegans tracheary elements as monitored by NEIEF (Fig. 7) also correlated well with the expression profiles for a cDNA coding for a putative peroxidase during trans-differentiation of Z. elegans mesophyll cells (Demura et al., 2002), in which microarray analysis of the gene expression of more than 8000 cDNA clones showed the presence of one sole putative peroxidase (Demura et al., 2002), despite the fact that molecular weight-based electrophoretic techniques resolved several isoforms of the enzyme (Masuda et al., 1983; Church and Galston, 1988; Sato et al., 1993).
That the expression of this peroxidase isoenzyme forms part of the trans-differentiation programme of mesophyll cells into tracheary elements was confirmed by the absence of its expression in mesophyll cells. Z. elegans mesophyll cell cultures showed no peroxidase when this was assayed in vitro, and no peroxidatic activities (electron dark deposits) could be detected cytochemically using the 3,3'-diaminobenzidine/H2O2 probe (Fig. 10A). Furthermore, western blot analyses were unable to detect this peroxidase isoenzyme in the culture medium of mesophyll cells (Fig. 10B, lane ni), but it was easily detectable in the culture medium of mesophyll cells undergoing tracheary element differentiation (Fig. 10B, lane i). Taken together, these results showed that the expression of this basic peroxidase isoenzyme forms part of the programme of trans-differentiation in Z. elegans single-cell cultures.
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The basic peroxidase isoenzyme expressed by tracheary single-cell cultures is the same isoenzyme that is expressed by the lignifying xylem
Finally, peroxidase isoenzyme patterns from tracheary elements were compared with the peroxidase isoenzyme patterns of stems and hypocotyls. Again, isoelectric focusing was selected as a tool for isoenzyme analysis since the isoelectric point property is determined by the balance of acid and basic amino acids of the protein, and thus reflects similarities in the primary structure of the isoenzymes. Results showed that the basic peroxidase isoenzyme expressed in Z. elegans tracheary elements (Fig. 11, lane t) was the same isoenzyme that was expressed by the stems (Fig. 11, lane s) and the hypocotyls (Fig. 11, lane h).
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From these results, it may be concluded that Z. elegans uses a single programme, i.e. one sole peroxidase isoenzyme complement, for xylem lignification, regardless of the existence of different ontogenesis pathways from either mesophyll cells (in the case of tracheary elements) or cambial derivatives (in the case of xylem vessels). The results also showed that Z. elegans tracheary single-cell cultures not only express the same peroxidase isoenzyme as the Z. elegans lignifying xylem, but that this peroxidase isoenzyme acts as a marker of tracheary element differentiation in Z. elegans mesophyll single-cell cultures. These results are not surprising since peroxidase isoenzymes with an isolectric point homologous to this Z. elegans peroxidase isoenzyme have been described in lignifying tissues of several vascular plants (Ros Barceló et al., 1998). Likewise, the presence of an isoelectric point-homologous peroxidase isoenzyme in ancestral vascular plant species, such as cycads (the nearest living relatives of pteridosperms) (LV Gómez Ros, MA Pedreño, and A. Ros Barceló, unpublished results), also suggests that this isoenzyme represents a highly conserved evolutionary gateway for lignin monomer polymerization, as its special catalytic properties strongly suggest (Ros Barceló and Pomar, 2001).
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Acknowledgements |
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This work was supported by grants from the Fundación Séneca (project no. PI-70/00615/FS/01) and MCYT (BMC2001-0271 and BOS2002-03550). MLS holds a fellowship from the Fundación Séneca.
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References |
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