Distribution of isoflavones in lupin hypocotyls. Possible control of cell wall peroxidase activity involved in lignification

The distribution of 4 key isoflavones (luteone, genistein, 2'-hydroxygenistein and wighteone) in lupin ( Lupinus albus L. cv. multolupa) hypocotyls shows a gradient that diminishes from young to old tissues. A spatial gradient occurs within the hypocotyl, and a temporal gradient in both the outermost vascular and epidermal tissues. Not only does a gradient exist in respect to the quantity of isoflavones, but there is also a gradient in respect to the type of isoflavone. Thus, wighteone is mainly associated with the non-meristematic zones of the lupin hypocotyl. A close relationship was found between the distribution and the localization in the walls of phloem cells of both peroxidase (EC 1.11.1.7) and isoflavones. This observation suggests an in vivo peroxidase-isoflavone interconnection. In fact, lupin isoflavones are able to inhibit the peroxidase-catalyzed oxidation of the lignin precursor coniferyl alcohol, probably due to the co-oxidation of isoflavones in the reaction media. The results are discussed on the basis of a possible role for isoflavones in controlling cell wallperoxidase activity involved in the lignification of phloem cells.     

Purification and properties of cinnamyl alcohol dehydrogenase from higher plants involved in lignin biosynthesis

The purification and characterization of an NADP(H) specific cinnamyl alcohol oxidoreductase is reported. The enzyme, which has been purified 600 ×, shows an absolute specificity for the cinnamyl moiety. The reaction is readily reversible but is strongly inhibited by aldehyde substrates. The enzyme belongs to class A of NAD(P) specific oxidoreductases. The distribution of this activity throughout a wide variety of taxonomically different plant groups as well as plant parts has revealed a possible correlation with lignin biosynthesis.     

Peroxidase involvement in lignification in water-impermeable seed coats of weedy leguminous and malvaceous species

Abstract. The development of water impermeable seed coats of two members each of the leguminoseae family [ Crotalaria spectabilis Roth, Sesbania exaltata (Raf) Cory] and the malvaceae family [Anoda cristata (L.) Schlecht, Abutilon theophrasti Medic.] was investigated. Highest peroxidase (POD) activity of Anoda and Abutilon seed coat extracts was highly correlated with the developmental stages when soluble phenolics were maximally converted into lignin. Although extensive lignification occurred during seed coat development in both legumes, the patterns of POD activity, soluble phenolic levels and time of lignification were different from those of the malvaceous species. POD activity levels in developing coats of the malvaceous seeds increased as phenolics decreased. Both POD activity and phenolic levels decreased during seed coat development of the legumes. POD was immunocytochemically and immunochemically detected in seed coats of all four species; however, results for polyphenol oxidase were negative. The results confirmed POD involvement in lignification of leguminous and malvaecous species and support and extend our earlier view that POD is involved in lignin formation during development of impermeable seed coats.     

Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway. The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors. The structure of ATP A2 was solved to 1.45 Å resolution at 100 K. Docking of p-coumaryl, coniferyl and sinapyl alcohol in the substrate binding site of ATP A2 were analysed on the basis of the crystal structure of a horseradish peroxidase C-CN-ferulic acid complex. The analysis indicates that the precursors p-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant cell wall.     

Physiological and biochemical events leading to vitrification of plants cultured in vitro

Water content, peroxidase activity and isoperoxidases, phenylalanine ammonia-lyase activity and phenolic content were comparatively analyzed in tissues of normal and vitreous plants cultured in vitro. The release of ethylene in flask atmospheres by normal and vitrifying plants was also measured. On the basis of the results, it is hypothesized that vitrification results from a burst of ethylene controlled by the peroxidase-IAA-oxidase system. An initiating stress (e.g. excess of cytokinins or of NH₄⁺ ions) would mediate the enhancement of the activity of soluble and membrane-bound peroxidases through a rapid modification of the phenolic level. The excess of ethylene in the atmosphere of stressed plants would retroinhibit its own biosynthesis and as a consequence decrease the activities of PAL and acidic peroxidases, thus hindering lignification processes. A parallel decrease in cellulose synthesis due to a diverted conversion of sugars to amino acids is expected (from data in the literature). Deficiency of both cellulose and lignin would allow more water uptake due to reduced wall pressure and bring about the hyperhydric malformations.     

Distribution of cell-wall xylans in bryophytes and tracheophytes: new insights into basal interrelationships of land plants

Xylans are known to be major cellulose-linking polysaccharides in secondary cell walls in higher plants. We used two monoclonal antibodies (LM10 and LM11) for a comparative immunocytochemical analysis of tissue and cell distribution of xylans in a number of taxa representative of all major tracheophyte and bryophyte lineages. The results show that xylans containing the epitopes recognized by LM10 and LM11 are ubiquitous components of secondary cell walls in vascular and mechanical tissues in all present-living tracheophytes. In contrast, among the three bryophyte lineages, LM11 binding was detected in specific cell-wall layers in pseudoelaters and spores in the sporophyte of hornworts, while no binding was observed with either antibody in the gametophyte or sporophyte of liverworts and mosses. The ubiquitous occurrence of xylans containing LM10 and LM11 epitopes in tracheophytes suggests that the appearance of these polysaccharides has been a pivotal event for the evolution of highly efficient vascular and mechanical tissues. LM11 binding in the sporophyte of hornworts, indicating the presence of relatively highly substituted xylans (possibly arabinoxylans), separates these from the other bryophytes and is consistent with recent molecular data indicating a sister relationship of the hornworts with tracheophytes.     

Major transitions in the evolution of early land plants: a bryological perspective

Background Molecular phylogeny has resolved the liverworts as the earliest-divergent clade of land plants and mosses as the sister group to hornworts plus tracheophytes, with alternative topologies resolving the hornworts as sister to mosses plus tracheophytes less well supported. The tracheophytes plus fossil plants putatively lacking lignified vascular tissue form the polysporangiophyte clade. Scope This paper reviews phylogenetic, developmental, anatomical, genetic and paleontological data with the aim of reconstructing the succession of events that shaped major land plant lineages. Conclusions Fundamental land plant characters primarily evolved in the bryophyte grade, and hence the key to a better understanding of the early evolution of land plants is in bryophytes. The last common ancestor of land plants was probably a leafless axial gametophyte bearing simple unisporangiate sporophytes. Water-conducting tissue, if present, was restricted to the gametophyte and presumably consisted of perforate cells similar to those in the early-divergent bryophytes Haplomitrium and Takakia. Stomata were a sporophyte innovation with the possible ancestral functions of producing a transpiration-driven flow of water and solutes from the parental gametophyte and facilitating spore separation before release. Stomata in mosses, hornworts and polysporangiophytes are viewed as homologous, and hence these three lineages are collectively referred to as the 'stomatophytes'. An indeterminate sporophyte body (the sporophyte shoot) developing from an apical meristem was the key innovation in polysporangiophytes. Poikilohydry is the ancestral condition in land plants; homoiohydry evolved in the sporophyte of polysporangiophytes. Fungal symbiotic associations ancestral to modern arbuscular mycorrhizas evolved in the gametophytic generation before the separation of major present-living lineages. Hydroids are imperforate water-conducting cells specific to advanced mosses. Xylem vascular cells in polysporangiophytes arose either from perforate cells or de novo. Food-conducting cells were a very early innovation in land plant evolution. The inferences presented here await testing by molecular genetics.     

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.     

A cladistic evaluation of the lower and higher green plants (Viridiplantae)

This paper presents a cladistic analysis and classification of the green plants (Viridiplantae). It is intended to be more balanced than previous treatments because more attention is paid to the lower green plants, or green algae. A major dichotomy is reflected by the structure of the flagellar root system of motile cells. Taxonomically, this justifies recognition of two divisions of green plants, viz.Chlorophyta sensu stricto, andAnthocerotophyta. The latter embraces a portion of the green algae plus all embryophytes. From a cladistic viewpoint it is doubtful whether land plants are strictly monophyletic.     

The mediation of veratryl alcohol in oxidations promoted by lignin peroxidase: the lifetime of veratryl alcohol radical cation

The kinetics of decay of veratryl alcohol radical cation, generated by cerium(IV) ammonium nitrate induced oxidation of veratryl alcohol, have been followed spectrophotometrically in a stopped-flow apparatus. In acidic aqueous acetonitrile the radical cation was found to decay by a first-order process, due to deprotonation from the alpha-carbon leading to an alpha-hydroxybenzyl radical with the rate constant of 17.1+/-0.5 s(-1). This value is in full agreement with those obtained by pulse radiolysis studies but much lower than the value (1.2x10(3) s(-1)) indirectly determined by EPR experiments. The implications of these results with respect to the possible role of veratryl alcohol as a mediator in the oxidative biodegradation of lignin catalysed by lignin peroxidase are discussed.     

Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L

Adventitious rooting of Ebenus cretica cuttings was studied in order to examine a) the rooting ability of different genotypes in relation to electrophoretic patterns of peroxidases. b) the activity and electrophoretic patterns of soluble and wall ionically bound peroxidases, the lignin content and anatomical changes in the control and IBA treated cuttings of and genotypes in the course of adventitious root formation. In addition, a fraction of soluble cationic peroxidases was separated by gel filtration chromatography from the total soluble peroxidases of a genotype. No rooting occurred in cuttings without IBA-treatment. In both genotypes, electrophoretic patterns of soluble anionic peroxidases revealed two common peroxidase isoforms, while a fast-migrating anionic peroxidase isoform (A3) appeared only in genotypes. Both genotypes showed similar patterns of soluble, as well as wall ionically bound cationic peroxidase isoforms. The number of isoforms was unchanged during the rooting process (induction, initiation and expression phase) but an increase in peroxidase activity (initiation phase) followed by decrease has been found in IBA-treated cuttings. During initiation phase the lignin content was almost similar to that on day 0 in genotype while it was reduced at by about 50% in genotype at the respective time. Microscopic observations revealed anatomical differences between genotypes. According to this study, the and genotypes display differences in anatomy, lignin content, activity of soluble peroxidases and the electrophoretic patterns of soluble anionic peroxidase isoforms. The A3-anionic peroxidase isoform could be used as biochemical marker to distinguish and genotypes of E. cretica and seems to be correlated to lignin synthesis in rooting process.     

Peroxidase activity, ethylene production, lignification and growth limitation in shoots of a nonrooting mutant of tobacco

The rooting recalcitrant rac Nicotiana tabacum cv Xanthi mutant has been multiplied in vitro under the form of shoots in parallel to wild-type. rac Shoots grew at a lower rate and did not root whatever the treatments when compared to those of wild-type shoots. They were characterized by a higher lignin level, a higher total specific peroxidase activity with higher activity of both acidic and basic isoperoxidases (although missing and supernumerary isoenzymes were observed), and higher ethylene production. These observations might be causally related to growth inhibitions as similar incidences have been noted in different stress-induced growth limitation, through cell wall rigidification and auxin catabolism. The relationship between these aspects and rooting recalcitrance remains to be explored.     

Distribution of free and bound forms of cis-trans and trans-trans abscisic acid in broad-bean plants in relation to apical dominance

Levels of endogenous abscisic acid (ABA; free and bound forms) have been determined by gas chromatography in stems and buds of broad-bean plants ( Vicia faba L. cv. Aguadulce) in relation to apical dominance. A downward gradient of free cis-trans ABA occurred along the stem, from the apical bud to the roots. Except for the actively growing apical bud the levels of free cis-trans ABA were higher in the buds than in the corresponding nodes. An inverse correlation can be set up between levels of free cis-trans ABA and growth of buds, except for the cotyledonary ones. High levels of bound ABA ( cis-trans form) are correlated with the growth of the apical bud and that of the axillary bud ax1. The hormonal regulation of the growth of the cotyledonary buds, which contained high levels of trans-trans ABA in bound forms, is apparently different from that of the other buds.     

Investigation of hydrogen peroxide formation in plants

A convenient and simple electrophoretic procedure was used to study the NAD(P)H-dependent generation of the hydrogen peroxide needed for the polymerization of coniferyl alcohol by peroxidases from the wood of Ailanthus glandulosa. The results showed that an NAD(P)H-dependent generation of hydrogen peroxide could be brought about by either: a FMN or riboflavin-dependent system; or a Mn²⁺ -dependent system. The most active system was the one incorporating Mn²⁺, followed closely by that incorporating riboflavin. In nature it appears that the method of hydrogen peroxide formation is determined by the amounts of cofactors present in the lignifying tissue. Because no quantitative data are available in the literature, further studies of the concentrations of these cofactors in the plant cell-wall are needed.     

Oxidation of hydroxycinnamic acid and hydroxycinnamyl alcohol derivatives by laccase and peroxidase. Interactions among p-hydroxyphenyl,guaiacyl and syringyl groups during the oxidation reactions

Fungal laccase oxidized derivatives of hydroxycinnamic acid. The rates decreased in the order sinapic acid > ferulic acid ≥p-coumaric acid. The laccase oxidized sinapyl alcohol faster than coniferyl alcohol. The rates of oxidation of the hydroxycinnamic acid derivatives by an isoenzyme of peroxidase from horseradish decreased in the order p-coumaric acid > ferulic acid ≥ sinapic acid. The peroxidase oxidized coniferyl alcohol much faster than sinapyl alcohol. The laccase and the peroxidase predominantly oxidized (a) ferulic acid in a reaction mixture that contained p-coumaric acid and ferulic acid, (b) sinapic acid in a mixture of p-coumaric acid plus sinapic acid, and (c) sinapic acid in a mixture of ferulic acid plus sinapic acid. In a reaction mixture that contained both coniferyl and sinapyl alcohols, both fungal laccase and horseradish peroxidase predominantly oxidized sinapyl alcohol. From these results, it is concluded (1) that the p-hydroxyphenyl radical can oxidize guaiacyl and syringyl groups and produce their radicals and (2) that the guaiacyl radical can oxidize the syringyl group under formation of its radical; and that (3) in both cases the reverse reactions are very slow.     

Bioelectrocatalytic properties of lignin peroxidase from Phanerochaete chrysosporium in reactions with phenols,catechols and lignin-model compounds

Bioelectrocatalytic reduction of H₂O₂ catalysed by lignin peroxidase from Phanerochaete chrysosporium (LiP) was studied with LiP-modified graphite electrodes to elucidate the ability of LiP to electro-enzymatically oxidise phenols, catechols, as well as veratryl alcohol (VA) and some other high-redox-potential lignin model compounds (LMC). Flow-through amperometric experiments performed at +0.1 V vs. Ag|AgCl demonstrated that LiP displayed significant bioelectrocatalytic activity for the reduction of H₂O₂ both directly (i.e., in direct electron transfer (ET) reaction between LiP and the electrode) and using most of studied compounds acting as redox mediators in the LiP bioelectrocatalytic cycle, with a pH optimum of 3.0. The bioelectrocatalytic reduction of H₂O₂ mediated by VA and effects of VA on the efficiency of bioelectrocatalytic oxidation of other co-substrates acting as mediators were investigated. The bioelectrocatalytic oxidation of phenol- and catechol derivatives and 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulphonate) by LiP was independent of the presence of VA, whereas the efficiency of the LiP bioelectrocatalysis with the majority of other LMC acting as mediators increased upon addition of VA. Special cases were phenol and 4-methoxymandelic acid (4-MMA). Both phenol and 4-MMA suppressed the bioelectrocatalytic activity of LiP below the direct ET level, which was, however, restored and increased in the presence of VA mediating the ET between LiP and these two compounds. The obtained results suggest different mechanisms for the bioelectrocatalysis of LiP depending on the chemical nature of the mediators and are of a special interest both for fundamental science and for application of LiP in biotechnological processes as solid-phase bio(electro)catalyst for decomposition/detection of recalcitrant aromatic compounds.     

Fourier-transform infrared and Raman spectroscopic evidence for the incorporation of cinnamaldehydes into the lignin of transgenic tobacco (Nicotiana tabacum L.) plants with reduced expression of cinnamyl alcohol dehydrogenase

Xylem from stems of genetically manipulated tobacco plants which had had cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity down-regulated to a greater or lesser degree (clones 37 and 49, respectively) by the insertion of antisense CAD cDNA had similar, or slightly higher, lignin contents than xylem from wild-type plants. Fourier-transform infrared (FT-IR) microspectroscopy indicated that down-regulation of CAD had resulted in the incorporation of moieties with conjugated carbonyl groups into lignin and that the overall extent of cross-linking, particularly of guaiacyl (4-hydroxy-3-methoxyphenyl) rings, in the lignin had altered. The FT-Raman spectra of manipulated xylem exhibited maxima consistent with the presence of elevated levels of aldehydic groups conjugated to a carbon-carbon double bond and a guaiacyl ring. These maxima were particularly intense in the spectra of xylem from clone 37, the xylem of which exhibits a uniform red coloration, and their absolute frequencies matched those of coniferaldehyde. Furthermore, xylem from clone 37 was found to have a higher content of carbonyl groups than that of clone 49 or the wild-type (clone 37: clone 49: wild-type; 2.4:1.6:1.0) as measured by a degradative chemical method. This is the first report of the combined use of FT-IR and FT-Raman spectroscopies to study lignin structure in situ. These analyses provide strong evidence for the incorporation of cinnamaldehyde groups into the lignin of transgenic plants with down-regulated CAD expression. In addition, these non-destructive analyses also suggest that the plants transformed with antisense CAD, in particular clone 37, may contain lignin that is less condensed (cross-linked) than that of the wild-type. Received: 27 May 1996 / Accepted: 30 July 1996