Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

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 in Zinnia mesophyll cell cultures

Mechanically isolated mesophyll cells of Zinnia elegans differentiate into tracheary elements (TEs) when cultured in a medium containing adequate auxin and cytokinin. Differentiation in this culture system is relatively synchronous, rapid (occuring within 3 days of cell isolation) and efficient (with up to 65% of the mesophyll cells differentiating into TEs), and does not require prior mitosis. The Zinnia system has been used to investigate (a) cytological and ultrastructural changes occurring during TE differentiation, such as the reorganization of microtubules controlling secondary wall deposition, (b) the influences of calcium and of various plant hormones and antihormones on TE differentiation, and (c) biochemical changes during differentiation, including those occurring during secondary wall deposition, lignification and autolysis. This review summarizes experiments in which the Zinnia system has served as a model for the study of TE differentiation.     

Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to transdifferentiate semi-synchronously into tracheary elements (TEs). This system has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for in cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with the transition from primary to secondary cell wall formation. At very early stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the culture medium were found. Changes in the secretion of pectic polysaccharides, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recognize these three classes of cell-wall molecule, respectively. Twenty-four hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thickenings of mature TEs but not in their surrounding primary walls. Methyl-esterified pectic polysaccharides are present in all cell walls and are secreted into the culture medium throughout the time course of differentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively unbranched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amount of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control medium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from inductive medium 6 h before any secondary thickenings are visible in the cells. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, although methylesterified pectin is present. All three epitopes are expressed in the vascular bundles, and the AGP is specifically localized in the xylem cells. Fucosylated xyloglucan is also present in the epidermal tissue, and the AGP is present in guard cells. The dynamic behaviour of these specific cell-wall molecules is tightly correlated with differentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate determination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.     

Characterization of basic <Emphasis Type="Italic">p</Emphasis>-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming <Emphasis Type="Italic">Picea abies</Emphasis> suspension culture

A Norway spruce (Picea abies) tissue culture line that produces extracellular lignin into the culture medium has been used as a model system to study the enzymes involved in lignin polymerization. We report here the purification of two highly basic culture medium peroxidases, PAPX4 and PAPX5, and isolation of the corresponding cDNAs. Both isoforms had high affinity to monolignols with apparent K_m values in μM range. PAPX4 favoured coniferyl alcohol with a six-fold higher catalytic efficiency (V_max/K_m) and PAPX5 p-coumaryl alcohol with a two-fold higher catalytic efficiency as compared to the other monolignol. Thus coniferyl and p-coumaryl alcohol could be preferentially oxidized by different peroxidase isoforms in this suspension culture, which may reflect a control mechanism for the incorporation of different monolignols into the cell wall. Dehydrogenation polymers produced by the isoforms were structurally similar. All differed from the released suspension culture lignin and milled wood lignin, in accordance with previous observations on the major effects that e.g. cell wall context, rate of monolignol feeding and other proteins have on polymerisation. Amino acid residues shown to be involved in monolignol binding in the lignification-related Arabidopsis ATPA2 peroxidase were nearly identical in PAPX4 and PAPX5. This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.     

The heterochromatin as a marker for protoplast differentiation of <Emphasis Type="Italic">Cucumis sativus</Emphasis>

The protoplast cultures of Cucumis sativus in two culture systems were used to study heterochromatin reassembly during dedifferentiation of isolated protoplasts and their subsequent differentiation into calli and proembryos. Here we show that dedifferentiation of the cucumber mesophyll cells is accompanied by a dramatic reduction in size and numbers of nuclear chromocenters. Although chromocenters were newly established during protoplast culture, the measured relative heterochromatin content differed according to the culture system used. Protoplast culture leading to proembryo formation displayed a lower level of relative heterochromatin content than cultures resulting in calli and the relative heterochromatin content reached values close to those estimated for somatic embryos.     

Chitosan and a fungal elicitor inhibit tracheary element differentiation and promote accumulation of stress lignin-like substance in Zinnia elegans xylogenic culture

We investigated the effect of elicitors on xylem differentiation and lignification using a Zinnia elegans xylogenic culture system. Water-soluble chitosan and a fungal elicitor derived from Botrytis cinerea were used as elicitors. Elicitor addition at the start of culturing inhibited tracheary element (TE) differentiation in a concentration-dependent manner, and 30 μg mL^?1 of chitosan or 16.7 μg mL^?1 of the fungal elicitor strikingly inhibited TE differentiation and lignification. Addition of chitosan (at 50 μg mL^?1) or the fungal elicitor (at 16.7 μg mL^?1) during the culturing period also inhibited TE differentiation without inhibiting cell division, except for immature TEs undergoing secondary wall thickening. Elicitor addition after immature TE appearance also caused the accumulation of an extracellular lignin-like substance. It appears that elicitor addition at the start of culturing inhibits the process by which dedifferentiated cells differentiate into xylem cell precursors. Elicitor addition during culturing also appears to inhibit the transition from xylem cell precursors to immature TEs, and induces xylem cell precursors or xylem parenchyma cells to produce an extracellular stress lignin-like substance.     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.     

Extracellular peroxidases of suspension culture cells of spruce (Picea abies): fungal elicitor-induced inactivation

Extracellular peroxidases of suspension cultures of spruce (Picea abies) (L.) (Karst) become inactivated when the cell suspension is elicited with a cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii. In contrast, cellular peroxidases are induced under these conditions. Both changes of activity are reflected in the isoenzyme profiles.Inactivation of the extracellular peroxidases is caused by an effector, arising from the cells after contact with the elicitor. Formation of the effector is limited to the beginning of elicitation, showing maximal activity at this period of time. Subsequently it becomes increasingly ineffective, probably due to inactivation. The effector is able to also inactivate commercial (horseradish) peroxidase. Inactivation was not the result of the action of a protease present in the medium.The elicitor exerts two different effects on the spruce cell suspension culture. It induces synthesis of enzymes correlated with lignin synthesis and an accumulation of lignin-like material. It also induces secretion of the negative effector which inactivates extracellular peroxidases.The elicitor-induced inactivation is not specific for peroxidases. Other extracellular enzymes, -glucosidase and acid phosphatase (secreted by the cells into the medium) and -amylase and pectinase (from Aspergillus strains) are also inactivated.     

Acidic phiorogludnol test positive reaction in Cultures of isolated mesophyll protoplasts ofBrassica napus L. and with secreted brown exudates

Cultures of isolated mesophyll protoplasts ofBrassica napus L.have shown a positive reaction with the acidic phloroglucinol test for lignin (Wiesner reagent) 30 h after the beginning of the culture period. The reaction was observed in protoplasts and in a very early stage connected with divisions, in which acidic phloroglucinol positive material was primarily deposited like a girdle surrounding the division plane. Brown exudates, which are a serious problem even in young cultures derived from isolated protoplasts, have been secreted into the medium by cells, and also showed a positive reaction. The same staining pattern has been found with toluidine blue 0.     

Possible involvement of DNA-repair events in the transdifferentiation of mesophyll cells ofZinnia elegans into tracheary elements

In cultures of isolated mesophyll cells ofZinnia elegans, transdifferentiation into tracheary elements is induced by a combination of auxin and cytokinin and is blocked by inhibitors of DNA synthesis and poly (ADP-ribose) synthesis. During transdifferentiation, a very low level of synthesis of nuclear DNA was found in some cultured cells by microautoradiography after pulse-labeling with [³H]thymidine. Density profiles of nuclear DNA that had been double-labeledin vivo with bromodeoxyuridine (BrdU) and [³H]thymidine indicated that this DNA synthesis was repair-type synthesis. The sedimentation velocity of nucleoids increased during the culture of isolated mesophyll cells and the increase was dependent on phytohormones. This phenomenon may reflect the rejoining of DNA strand breaks after repair-type DNA synthesis during transdifferentiation. Treatment of cells with inhibitors of DNA synthesis or of poly(ADP-ribose) synthesis prevented the increase in the sedimentation velocity of nucleoids. The data suggest the involvement of DNA-repair events in the transdifferentiation of mesophyll cells into tracheary elements.     

Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori

We found that a whole cell suspension of Alexandrium taylori, which is toxic to Artemia, causes species-specific hemolysis against mammalian erythrocytes. Among the erythrocytes tested, rabbit and guinea-pig erythrocytes were highly sensitive, but human, sheep, and cattle erythrocytes were insensitive. The cell-free culture supernatant also showed potent hemolytic activity toward rabbit erythrocytes as seen in whole cell suspension. The hemolytic activity in the culture medium gradually increased with increase in cell number during exponential growth phase, and relatively high activity was maintained even after reaching the death phase. These results suggest that the hemolytic substance is actively released into the medium from A. taylori cells rather than simple leakage from ruptured or dead cells, and a part of them are steadily accumulated in the medium during the algal growth. Chemical characterization with ultrafiltration and trypsin-treatment suggested that the hemolytic substance released into the medium is protein-like compound with molecular weight more than 10,000 Da. The ammonium sulfate precipitated fraction obtained from the cell-free supernatant of A. taylori showed cytotoxic effect on HeLa cells as well as the hemolytic activity in a similar concentration range on a protein content basis. Our results suggest that A. taylori produces a novel proteinaceous hemolytic exotoxin.     

L-α-Aminooxy-β-phenylpropionic acid inhibits lignification but not the differentiation to tracheary elements of isolated mesophyll cells of Zinnia elegans

The influence of L-α-aminooxy-β-phenylpropionic acid (AOPP), an inhibitor of L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), and thus of lignin formation, on the differentiation of tracheary elements from isolated mesophyll cells of Zinnia elegans L. cv. Canary Bird was investigated. At low concentrations of AOPP (5–25 μ,M) lignification of differentiating cells was almost completely prevented whereas number of differentiating cells, viability of the cells, fresh and dry weights, and the packed cell volume were higher than corresponding parameters in control cultures. At higher concentrations of AOPP (50–75 μ M ) the formation of tracheary elements was inhibited but division, elongation and expansion of cells were still observed. Cells cultured for 96 h in the presence of 100 μ M AOPP were morphologically similar to cells at 12 h of culture, the time at which AOPP was added. At concentrations of AOPP that did not inhibit differentiation, AOPP caused an increase in the amounts of uronic acid and total carbohydrate (per unit volume of cell suspension) in the extracellular polysaccharide fraction and in the total cell wall fraction, although these parameters were not significantly different from control values when expressed on a dry weight basis. AOPP caused the release of polysaccharides which contained xylose into the medium when added before the onset of visible differentiation and the release of polysaccharides which contained glucose when added at the time when the formation of the secondary cell wall thickenings took place. The results indicate that AOPP at low concentrations specifically inhibits the formation of lignin without adversely affecting the synthesis of cell wall polysaccharides, although the proper integration of these compounds into the wall may be disturbed. O-Benzylhydroxyla-mine, on the other hand, did not prove to be a useful agent to affect lignin synthesis in differentiating Zinnia cells.     

Effect of Ultraviolet (UV-B) Radiation on the Formation and Localization of Phenolic Compounds in Tea Plant Callus Cultures

The effect of ultraviolet (UV-B) radiation on the accumulation and tissue localization of phenolic compounds in two strains of callus cultures of tea plant (Camellia sinensisL.) were investigated. The strains differed in their morphological and physiological characteristics and biosynthetic capacity. UV-B radiation hampered culture growth, decreased the size of callus-forming cells and promoted the accumulation of soluble and, to a lesser extent, polymeric forms of phenolic compounds, such as lignin. This accumulation was accompanied by an increase in the phenolic compound deposition in cell walls and intercellular space and by deposition of a lignin-like material on the surface of callus cultures. The strain characterized by an increased formation of phenolic compounds was more resistant to UV-B radiation as compared to that with lower phenolic productivity.     

Inhibition of phenylalanine ammonia-lyase activity causes the depression of lignin accumulation of secondary wall thickening in isolatedZinnia mesophyll cells

Summary Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 M AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.Abbreviations AIP 2-aminoindan-2-phosphonic acid - PAL phenylalanine ammonia-lyase     

An efficient in vitro plantlet regeneration of <Emphasis Type="Italic">Cryptocoryne wendtii</Emphasis> and <Emphasis Type="Italic">Cryptocoryne becketti</Emphasis> through shoot tip culture

An efficient micropropagation protocol was established for Cryptocoryne wendtii and Cryptocoryne becketti using shoot tips explants. Multiple shoots were induced from shoot tip explants of both species cultured on agar-gelled as well as liquid MS medium supplemented with 0.5 mg/L BA and 0.2 mg/L IBA (proliferation medium). The multiple shoots of both the species formed on agar-gelled as well as liquid medium were vigorously growing with well-developed roots and leaves after 4 weeks of culture. Highest number of multiple shoots was obtained from shoot tip explants of both the species cultured in liquid proliferation medium after 4 weeks of culture. The shoot tip explants of C. wendtii and C. becketti, that were cultured in liquid proliferation medium (2 weeks) followed by culturing on agar-gelled proliferation medium (2 weeks) also produced the multiple shoots. Shoot tips cultured on agar-gelled medium produced the least number of multiple shoots after 4 weeks of culture. Histological study did not show any abnormalities in the leaves of in vitro plantlets of both the species, cultured in agar-gelled and liquid proliferation medium. The leaves of the in vitro plantlets formed normal mesophyll cells and vascular bundles. More than 95% of the acclimatized plantlets grew vigorously without any morphological abnormalities.     

Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells by Phytophthora capsici

Cell suspension cultures of three varieties of Capsicum annuum L., each with a different degree of sensitivity to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate with a hypersensitive reaction. They showed the synthesis or accumulation of PR-proteins with peroxidase (EC 1.11.1.7) activity and the accumulation of lignin-like polymer (as measured by derivatization with thioglycolic acid). The cultivation medium was optimised for both plant and fungus growth in order to avoid any stress during their combination. The resistant pepper variety, Smith-5, showed a more intense response to the elicitor preparations than the sensitive varieties, Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate. After elicitation, the cell walls thickened through the accumulation of lignin, as can be observed by staining microscope preparations with methylene blue. Elicitation also reduced the level of total peroxidase activity in the susceptible varieties, while such activity increased in resistant varieties, and was accompanied by de novo expression of acidic peroxidase isoenzymes in the extracellular and cell wall fractions. Of note was the PR protein of pI 5.7 showing peroxidase activity, which was induced by both elicitor types in the elicited cell suspensions of the resistant variety alone, making it a marker of resistance. The increases in the activity of these peroxidases in the resistant variety are in concordance with the accumulation of lignin observed 24 h after inoculation by both elicitors from the fungus. The possible role of these isoenzymes in lignin biosynthesis, used to reinforce the cell walls against fungal penetration of the cells, is discussed. These results are in accordance with those previously observed in plant stem sections.     

Involvement of local intercellular communication in the differentiation of zinnia mesophyll cells into tracheary elements

The transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans L.) into tracheary elements (TEs) has been well studied as a model of plant cell differentiation. In order to investigate intercellular communication in this phenomenon, two types of culture method were developed, in which mesophyll cells were embedded in a thin sheet of agarose gel and cultured on solid medium, or embedded in microbeads of agarose gel and cultured in liquid medium. A statistical analysis of the two-dimensional distribution of TEs in the thin-sheet cultures demonstrated their aggregation. In the microbead cultures, the frequency of TE differentiation was shown to depend on the local cell density (the cell density in each microbead): TE differentiation required local cell densities of more than 10⁵ cells ml⁻¹. These results suggest that TE differentiation involves cell-cell communication mediated by a locally acting diffusible factor. This presumptive factor was characterized by applying a modified version of the sheet culture, which used two sheets of different cell densities, a low-density sheet and a high-density sheet. Differentiation of TEs in the former could be induced only by bringing it into contact with the latter. Insertion of a 25-kDa-cutoff membrane between the high-density and low-density sheets severely suppressed such induction of TEs in the low-density sheet while a 300-kDa-cutoff membrane suppressed induction only slightly. Insertion of agarose sheets containing immobilized pronase E or trypsin also interfered with the induction of TEs in the low-density sheets. Thus, a proteinaceous macromolecule of 25–300 kDa in molecular weight was assumed to mediate the local intercellular communication required for TE differentiation. This substance was designated “xylogen” with reference to its xylogenic activity. The time of requirement for xylogen during TE differentiation was assessed by experiments in which cells in the low-density sheet were separated from xylogen produced in the high-density sheet at various times by insertion of a 25-kDa-cutoff membrane between the two sheets, and was estimated to be from the 36th hour to the 60th hour of culture (12–36 h before visible thickening of secondary cell walls of TEs). Received: 13 July 2000 / Accepted: 4 October 2000     

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.