In situ characterization of a NO-sensitive peroxidase in the lignifying xylem of Zinnia elegans

The lignifying xylem from Zinnia elegans stems gives an intense reaction with 3,3',5,5'-tetramethylbenzidine (TMB), a reagent previously reported to be specific for peroxidase/H2O2. However, the staining of lignifying xylem cells with TMB is apparently the result of two independent mechanisms: one, the catalase-sensitive (H2O2-dependent) peroxidase-mediated oxidation of TMB, and the other, the catalase-insensitive oxidation of TMB, probably mediated by xylem oxidases which are specific from lignifying tissues. The catalase-insensitive oxidation of TMB by the Z. elegans xylem was sensitive to sodium nitroprusside (SNP), a nitric oxide (NO)-releasing compound that, when used at 5.0 mM, is capable of sustaining NO concentrations of 6.1 &mgr;M in the aqueous phase. This effect of SNP was totally reversed by 150 &mgr;M 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO), an efficient NO scavenger in biological systems, so the above-mentioned effect must be ascribed to NO, and not to other nitrogen oxides. This response of the catalase-insensitive TMB-oxidase activity of the lignifying Z. elegans xylem was similar to that shown by a basic peroxidase isolated from the intercellular washing fluid, which showed TMB-oxidase activity, and which was also inhibited by 5 mM SNP, the effect of SNP also being reversed by 150 &mgr;M PTIO. These results suggest that peroxidase was the enzyme responsible for the NO-sensitive catalase-insensitive TMB-oxidase activity of the lignifying Z. elegans xylem. Further support for this statement was obtained from competitive inhibitor-dissected histochemistry, which showed that this stain responded to peroxidase-selective competitive inhibitors, such as ferulic acid and ferrocyanide, in a similar way to the Z. elegans basic peroxidase. From these results, we conclude that this NO-sensitive catalase-insensitive oxidation of TMB is apparently performed by the Z. elegans basic peroxidase, and that the regulation of this enzyme by NO may constitute an intrinsically programmed event during the differentiation and death of the xylem.     

Analysis of expression profiles of three peroxidase genes associated with lignification in Arabidopsis thaliana

We have investigated the mechanism of lignification during tracheary element (TE) differentiation using a Zinnia elegans xylogenic culture. In the process, we isolated ZPO-C , a peroxidase gene of Z. elegans that is expressed specifically in differentiating TEs. ZPO-C is suggested to be involved in lignification of Z. elegans TEs in vivo and in vitro. Furthermore, a peroxidase gene of Arabidopsis thaliana ( AtPrx66 ), which is homologous to ZPO-C , was identified. The expression profile and functions of the gene in planta remain to be investigated. In this study, we performed promoter :: β-glucuronidase (GUS) assays to investigate the expression profiles and functions of the ZPO-C -like peroxidases in A. thaliana . We generated transgenic A. thaliana lines carrying AtPrx66, AtPrx47 or AtPrx64 (peroxidases showing high sequence similarity to AtPrx66 ) promoter :: GUS reporter gene fusions. The GUS activities of AtPrx66, AtPrx47 and AtPrx64 promoter :: GUS lines were arranged concentrically from the center to the periphery in the roots of seedlings. Furthermore, histochemical GUS assays using inflorescence stems showed that AtPrx66, AtPrx47 and AtPrx64 promoter-driven GUS were mainly expressed in the differentiating vessels, xylem parenchyma and sclerenchyma, respectively. These results suggest that the gene expressions of these three peroxidases, which showed high sequence similarity to one another, are differentially regulated in various tissues and organs. In addition, our results suggest that while AtPrx66 and AtPrx47 are associated with lignification of vessels, AtPrx64 is associated with lignification of sclerenchyma.     

Effect of copper excess on superoxide dismutase,catalase, and peroxidase activities in sunflower seedlings (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

The changes in lipid peroxidation, antioxidative and lignifying enzyme activities were studied in leaves and stems of Cu-stressed sunflower seedlings. In both organs, membrane lipid peroxidation was enhanced by copper treatment. Additionally, catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) activities were modulated: The activity of superoxide dismutase was enhanced in both plant organs. Differently, catalase activity was not affected in leaves but significantly reduced in stems. Peroxidase (EC 1.11.1.7) activities were also changed. Guaiacol peroxidase activity was increased in leaves and stems. In the same way, electrophoretic analysis of the anionic isoperoxidases involved in lignification (syringaldazine peroxidase) revealed qualitative and quantitative changes on the isoenzyme patterns. These modifications were accompanied by the increase of the NADH-oxidase activity in ionically cell wall bound fraction. It appeared that the growth delay caused by copper excess could be related to the activation of lignifying peroxidases.     

Nitric oxide production by the differentiating xylem of Zinnia elegans

Nitric oxide (NO) is currently regarded as a signal molecule involved in plant cell differentiation and programmed cell death. Here, we investigated NO production in the differentiating xylem of Zinnia elegans by confocal laser scanning microscopy to answer the question of whether NO is produced during xylem differentiation. Results showed that NO production was mainly located in both phloem and xylem regardless of the cell differentiation status. However, there was evidence for a spatial NO gradient inversely related to the degree of xylem differentiation and a protoplastic NO burst was associated with the single cell layer of pro-differentiating thin-walled xylem cells. Confirmation of these results was obtained using trans-differentiating Z. elegans mesophyll cells. In this system, the scavenging of NO by means of 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO) inhibits tracheary element differentiation but increases cell viability. These results suggest that plant cells, which are just predetermined to irreversibly trans-differentiate in xylem elements, show a burst in NO production, this burst being sustained as long as secondary cell wall synthesis and cell autolysis are in progress.     

Association of caffeoyl coenzyme A 3-O-methyltransferase expression with lignifying tissues in several dicot plants.

Caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) was previously shown to be associated with lignification in both in vitro tracheary elements (TEs) and organs of zinnia (Zinnia elegans). However, it is not known whether this is a general pattern in dicot plants. To address this question, polyclonal antibodies against zinnia recombinant CCoAOMT fusion protein were raiseed and used for immunolocalization in several dicot plants. The antibodies predominantly recognized a protein band with a molecular mass of 28 kD on western analysis of tissue extracts from zinnia, forsythia (Forsythia suspensa), tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), and soybean (Glycine max). Western analyses showed that the accumulation of CCoAOMT protein was closely correlated with lignification in in vitro TEs of zinnia. Immunolocalization results showed that CCoAOMT was localized in developing TEs of young zinnia stems and in TEs, xylem fibers, and phloem fibers of old stems. CCoAOMT was also found to be specifically associated with all lignifying tissues, including TEs, xylem fibers, and phloem fibers in stems of forsythia, tobacco, alfalfa, soybean, and tomato (Lycopersicon esculentum). The presence of CCoAOMT was evident in xylem ray parenchyma cells of forsythia, tobacco, and tomato. In forsythia and alfalfa, pith parenchyma cells next to the vascular cylinder were lignified. Accordingly, marked accumulation of CCoAOMT in these cells was observed. Taken together, these results showed a close association of CCoAOMT expression with lignification in dicot plants. This supports the hypothesis that the CCoAOMT-mediated methylation branch is a general one in lignin biosynthesis during normal growth and development in dicot plants.     

An antigen expressed during plant vascular development crossreacts with antibodies towards KLH (keyhole limpet hemocyanin).

An antigen present in plant vascular tissue crossreacts with antibodies towards keyhole limpet hemocyanin (KLH). The antigen is present in xylem and vascular cambium, as evidenced by immunocytochemical staining of plant sections. This cell type assignment was confirmed by staining of mesophyll cell cultures from Zinnia elegans L. undergoing tracheary cell differentiation. The strongest staining both in sections and cell cultures occurred in cells and tissues during early stages of differentiation. Although the anti-KLH antibodies can easily be removed by affinity purification, our findings suggest that a certain caution is needed when KLH is used as an immunological carrier for studies in plants.     

Changes in the activity and mRNA of cinnamyl alcohol dehydrogenase during tracheary element differentiation in zinnia.

Changes in the enzymatic activity of cinnamyl alcohol dehydrogenase (CAD) and in the expression of a gene for CAD during tracheary element (TE) differentiation were investigated in cultures of single cells isolated from the mesophyll of zinnia (Zinnia elegans). In cultures in which TE differentiation was induced (TE-inductive cultures), CAD activity increased from h 36 after the start of culture (12 h before the start of thickening of the secondary cell wall) and peaked at h 72, when lignin was actively being deposited. In control cultures in which TE differentiation was not induced, CAD activity remained at a very low level for 5 d. Some isoforms of CAD were detected only in the TE-inductive cultures by native gel electrophoresis and subsequent staining for CAD activity. A cDNA clone for CAD, ZCAD1, was isolated from Z. elegans using a cDNA clone for CAD from Aralia cordata as the probe. RNA gel-blot analysis revealed that in the TE-inductive cultures the level of ZCAD1 mRNA increased from h 36 and peaked at h 48 to 60. No such increases were observed in control cultures. These results indicated that both the gene expression and the activity of CAD are strictly regulated, in association with lignification, during TE differentiation in Z. elegans.     

An alternative methylation pathway in lignin biosynthesis in Zinnia.

S-Adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is implicated in disease resistant response, but whether it is involved in lignin biosynthesis is not known. We isolated a cDNA clone for CCoAOMT in differentiating tracheary elements (TEs) induced from Zinnia-isolated mesophyll cells. RNA gel blot analysis showed that the expression of the CCoAOMT gene was markedly induced during TE differentiation from the isolated mesophyll cells. Tissue print hybridization showed that the expression of the CCoAOMT gene is temporally and spatially regulated and that it is associated with lignification in xylem and in phloem fibers in Zinnia organs. Both CCoAOMT and caffeic acid O-methyltransferase (COMT) activities increased when the isolated Zinnia mesophyll cells were cultured, whereas only CCoAOMT activity was markedly enhanced during lignification in the in vitro-differentiating TEs. The induction pattern of the OMT activity using 5-hydroxyferuloyl CoA as substrate during lignification was the same as that using caffeoyl CoA. Taken together, the results indicate that CCoAOMT is associated with lignification during xylogenesis both in vitro and in the plant, whereas COMT is only involved in a stress response in vitro. We propose that CCoAOMT is involved in an alternative methylation pathway in lignin biosynthesis. In Zinnia in vitro-differentiating TEs, the CCoAOMT mediated methylation pathway is dominant.     

Neighboring Parenchyma Cells Contribute to Arabidopsis Xylem Lignification,while Lignification of Interfascicular Fibers Is Cell Autonomous

Lignin is a critical structural component of plants, providing vascular integrity and mechanical strength. Lignin precursors (monolignols) must be exported to the extracellular matrix where random oxidative coupling produces a complex lignin polymer. The objectives of this study were twofold: to determine the timing of lignification with respect to programmed cell death and to test if nonlignifying xylary parenchyma cells can contribute to the lignification of tracheary elements and fibers. This study demonstrates that lignin deposition is not exclusively a postmortem event, but also occurs prior to programmed cell death. Radiolabeled monolignols were not detected in the cytoplasm or vacuoles of tracheary elements or neighbors. To experimentally define which cells in lignifying tissues contribute to lignification in intact plants, a microRNA against CINNAMOYL CoA-REDUCTASE1 driven by the promoter from CELLULOSE SYNTHASE7 (ProCESA7:miRNA CCR1) was used to silence monolignol biosynthesis specifically in cells developing lignified secondary cell walls. When monolignol biosynthesis in ProCESA7:miRNA CCR1 lines was silenced in the lignifying cells themselves, but not in the neighboring cells, lignin was still deposited in the xylem secondary cell walls. Surprisingly, a dramatic reduction in cell wall lignification of extraxylary fiber cells demonstrates that extraxylary fibers undergo cell autonomous lignification.     

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system

Summary. The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O₂⁻ and H₂O₂) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O₂⁻ production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl₃ assays, which monitor H₂O₂ production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl₃) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H₂O₂ is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H₂O₂ in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H₂O₂ was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H₂O₂ produced by this in vitro culture system is destined for use during lignin biosynthesis. Correspondence and reprints: Departamento de Biologia Vegetal, Facultad de Biologia, Universidad de Murcia, 30100 Murcia, Spain.     

Downregulation of the Basic Peroxidase Isoenzyme from Zinnia elegans by Gibberellic Acid

Hypocotyl formation during the epigeal germination of seedlings is under strict hormonal regulation. In a 3 d old Zinnia elegans seedling system, gibberellic acid (GA3) exerts an opposite effect to that exerted by light on hypocotyl photomorphogenesis because GA3 promotes an etiolated-like growth with an inhibition of radial (secondary) growth. For this reason, the effect of GA3 on the basic peroxidase isoenzyme from Z. elegans (ZePrx), an enzyme involved in hypocotyl lignin biosynthesis, was studied. The r...     

O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction

The nature and specificity of the Wiesner test (phloroglucinol-HCl reagent) for the aromatic aldehyde fraction contained in lignins is studied. Phloroglucinol reacted in ethanol-hydrochloric acid with coniferyl aldehyde, sinapyl aldehyde, vanillin, and syringaldehyde to yield either pink pigments (in the case of hydroxycinnamyl aldehydes) or red-brown pigments (in the case of hydroxybenzaldehydes). However, coniferyl alcohol, sinapyl alcohol, and highly condensed dehydrogenation polymers derived from these cinnamyl alcohols and aldehydes did not react with phloroglucinol in ethanol-hydrochloric acid. The differences in the reactivity of phloroglucinol with hydroxycinnamyl aldehydes and their dehydrogenation polymers may be explained by the fact that, in the latter, the unsubstituted (alpha,beta-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the beta carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuumvar. annuum, Populus albaL., and Pinus halepensisL. demonstrated the presence of 4- O-linked hydroxycinnamyl aldehyde end groups and 4- O-linked 4-hydroxy-3-methoxy-benzaldehyde (vanillin) end groups in lignins. However, given the relatively low abundance of 4- O-linked vanillin in lignifying cell walls and the low extinction coefficient of its red-brown phloroglucinol adduct, it is unlikely that vanillin contributes to a great extent to the phloroglucinol-positive stain reaction. These results suggest that the phloroglucinol-HCl pink stain of lignifying xylem cell walls actually reveals the 4- O-linked hydroxycinnamyl aldehyde structures contained in lignins. Histochemical studies showed that these aldehyde structures are assembled, as in the case of coniferyl aldehyde, during the early stages of xylem cell wall lignification.     

Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinnia

In a culture system in which single cells isolated from the mesophyll of Zinnia elegans L. differentiate to tracheary elements (TEs), two inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5), L-α-aminooxy-β-phenylpropionic acid (AOPP) at 10 μM inhibited lignification without reducing the number of TEs formed. These inhibitors caused intracellular changes in peroxidase (EC 1.11.1.7) activities. The inhibitors increased the activity of peroxidases bound to the cell walls and especially the activity of peroxidase bound ionically to the cell walls. In contrast, the activity of extracellular peroxidase decreased. There were five isoenzymes, P1-P5, in the ionically bound peroxidase of cultured Zinnia cells. Among the isoenzymes, P4 and P5 appeared to be specific for TE differentation. Treatment with AOPP and AIP resulted in increases in the activities of P2, P4 and P5 isoenzymes, with the most prominent increase in P5 activity. The addition of lignin precursors, including coniferyl alcohol, to the AOPP-treated cells restored lignification, and suppressed the alteration of peroxidase isoenzyme patterns caused by AOPP. The relationship between the wall-bound peroxidases and lignification during TE differentiation is discussed in the light of these results.     

Involvement of extracellular dilignols in lignification during tracheary element differentiation of isolated Zinnia mesophyll cells

During differentiation of isolated Zinnia mesophyll cells into tracheary elements (TEs), lignification on TEs progresses by supply of monolignols not only from TEs themselves but also from surrounding xylem parenchyma-like cells through the culture medium. However, how lignin polymerizes from the secreted monolignols has not been resolved. In this study, we analyzed phenol compounds in culture medium with reversed-phase HPLC, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry, and found 12 phenolic compounds including coniferyl alcohol and four dilignols, i.e. erythro-guaiacylglycerol-beta-coniferyl ether, threo-guaiacylglycerol-beta-coniferyl ether, dehydrodiconiferyl alcohol and pinoresinol, in the medium in which TEs were developing. Coniferyl alcohol applied to TE-inductive cultures during TE formation rapidly disappeared from the medium, and caused a sudden increase in dilignols. Addition of the dilignols promoted lignification of TEs in which monolignol biosynthesis was blocked by an inhibitor of phenylalanine anmmonia-lyase (PAL), L-alpha-aminooxy-beta-phenylpropionic acid (AOPP). These results suggested that dilignols can act as intermediates of lignin polymerization.     

Tracheary element differentiation is correlated with inhibition of cell expansion in xylogenic mesophyll suspension cultures.

To test the hypothesis that xylogenesis is coupled to cell growth suppression, cell expansion in Zinnia elegans L. var. Envy mesophyll suspension cultures was manipulated by varying the extracellular osmolarity and the effect on xylogenesis was examined. Cell expansion and tracheary element differentiation were inversely related along a gradient of extracellular osmolarity ranging from 200 to 400 mOsm, supporting the hypothesis that tracheary element differentiation is coupled to cessation of cell expansion. Above 300 mOsm, reduction in the number of cells that differentiated into tracheary elements coincided with an increase in the number of plasmolyzed cells as extracellular osmolarity was increased, indicating that plasmolysis inhibits tracheary element differentiation, although not specifically. Using the plasmolysis method we showed that cellular osmolarity within populations of isolated Zinnia mesophyll cells ranges from 250 to 600 mOsm with a mean of 425 mOsm. The broad range in cellular osmolarity within Zinnia mesophyll cell populations, coupled with inhibition of differentiation in the low range due to cell expansion and in the high range due to plasmolysis, may help explain why tracheary element differentiation in Zinnia suspension cultures is never complete nor perfectly synchronous and enable further optimization of this culture system.     

Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.)

The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles.     

Purification of cationic peroxidases bound ionically to the cell walls from the roots ofZinnia elegans

Cell wall-bound and tracheary element-specific peroxidase isoenzymes, designated P5A and P5B, were shown previously to be associated with lignification during the differentiation into tracheary elements of single cells isolated from the mesophyll ofZinnia elegans (Satoet al. Planta 189: 584–589, 1993; Planta 196: 141–147, 1995). Isoenzymes corresponding to P5 (RP5A and RP5B) were present at a relatively high level in the roots ofZinnia elegans. These isoenzymes were purified from theZinnia roots by several column-chromatographic steps. Both RP5A and RP5B had molecular masses of 35 kDa. Purified RP5A and RP5B were cleaved by CNBr and the partial amino acid sequences of these isoenzymes were determined.