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.     

Carry-over of thigmomorphogenetic characteristics in calli derived fromBryonia dioica internodes

Calli have been initiated in vitro from young internodes (control and rubbed) ofBryonia dioica, where previously it had been shown, using intact plants, that rubbing induced limited growth through enhanced lignification. Calli derived from rubbed internodes were somewhat more compact and showed biochemical changes, i.e. enhanced activity of total peroxidase and isoperoxidases, enhanced production of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene, enhanced tissue capacity to convert ACC into ethylene, enhanced activity of phenylalanine ammonia-lyase (PAL) and higher content of lignin, which characterized rubbed internodes. Differences in ethylene metabolism between the two types of calli tended to fade from the third week onwards of initial culture, whereas lignin content, peroxidase activity and peroxidase isoenzyme pattern appeared to be more persistant rubbing-induced markers for several subcultures. The results point to the persistance of environmentally induced changes in gene expression.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

Gibberellic acid and dwarfism effects on the growth dynamics of B73 maize (Zea mays L.) leaf blades: a transient increase in apoplastic peroxidase activity precedes cessation of cell elongation

The relationship between apoplastic peroxidase (EC 1.11.1.7) activity and cessation of growth in maize (Zea mays L.) leaf blades was investigated by altering elongation zone length. Apoplastic peroxidase activity in the elongation and secondary cell wall deposition zones of elongating leaf blades of the maize inbred line B73 was used as a control and compared to leaves of the dwarf mutant D8-81127, a near-isogenic line of B73 unresponsive to gibberellins, and to leaves of B73 plants to which gibberellic acid (GA(3)) had been applied via root uptake. Elongation zone length was increased by treatment with GA(3) through an increase in cell number as well as increased final cell length. The shorter elongation zone of dwarf leaves occurred primarily through reduced final cell length. Although elongation zone length differed among dwarf, control, and GA(3)-treated leaf blades, in all three treatments a transient increase in apoplastic peroxidase activity preceded a reduction in the segmental elongation rate in leaves. A peroxidase isoenzyme with pI 7.0 occurred in the leaf elongation zone during growth deceleration in all three treatments, and its activity decreased as growth displaced tissue into the region of secondary cell wall deposition. Growth cessation for all treatments coincided with the first appearance of peroxidase isozymes with pIs of 5.6 and 5.7. Based on the activity of particular isozymes relative to growth and differentiation, the pI 7.0 isoenzyme is most likely to be involved in cessation of cell elongation, while isozymes with pIs 5.6 and 5.7 are likely to be active in lignification.     

INDOLE ACETIC ACID OXIDASE AND PEROXIDASE ACTIVITIES IN NORMAL AND CROWN GALL TISSUE CULTURES OF PARTHENOCISSUS TRICUSPIDATA

Lipetz , Jacques , and Arthur W. Galston . (Yale U., New Haven.) Indole acetic acid oxidase and peroxidase activities in normal and crown gall tissue cultures of Parthenocissus tricuspidata. Amer. Jour. Bot. 46(3) : 193-196. Illus. 1959.—Normal and crown gall cells of P. tricuspidata grown in pure culture were examined for IAA oxidase and peroxidase activities. No IAA oxidase activity could be demonstrated in dialyzed or undialyzed homogenates of either tissue; however, crown gall tissue, but not normal tissue, was found to produce an extracellular IAA oxidase which required Mn⁺⁺ and DCP as co-factors. Normal tissue, but not crown gall tissue was found to contain high levels of substances which spared IAA from destruction by a pea IAA oxidase preparation. Peroxidase activity was found to be higher in normal than in crown gall homogenates, but crown gall tissue released considerably more peroxidase into the external medium. The differences in the auxin requirements and growth rate between normal and crown gall cells appear not to be easily explicable in terms of differential auxin destruction.     

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     

Rapid cellular responses to auxin and the regulation of growth

Abstract The cellular responses rapidly evoked by auxin are reviewed, and related to a consideration of how growth rate is regulated in excised segments and in whole dicotyledonous plants. Two processes, synthesis of proteins and of cell wall components, are both promoted by auxin and essential for auxin-stimulated growth, whereas other processes show little promotion by auxin or do not appear essential for growth. Current models for the cellular regulation of growth by auxin are briefly discussed, and a new model presented. Auxin is suggested to act by bringing about a transient increase in cytosolic Ca²⁺ levels, which through the stimulation of protein kinases converts a cytoplasmic protein factor to an active state capable of binding auxin. The protein-auxin complex induces mRNA synthesis, which effects the increased synthesis of cell wall components and their incorporation into the wall, resulting in wall loosening and growth. It is proposed that the factor limiting growth in floating excised segments may initially be cell wall pH, but that this is not the case in whole plants and growth is instead mediated by increased protein and matrix cell wall synthesis. Differences are noted between monocotyledonous coleoptiles and dicotyledonous stems in some metabolic processes possibly involved in auxin growth responses, and it is cautioned that observations made on one tissue may not necessarily be applicable to the other. Care should also be taken in applying conclusions drawn from studies on excised tissue to the interpretation of growth regulation in the whole plant.     

Production of peroxidases by hairy roots ofBrassica napus

Hairy roots cultures derived from leaf explants ofBrassica napus L. produced and secreted peroxidases. The enzyme activity in the medium increased with growth but it remained nearly constant in the tissue. The changes in extracellular peroxidase activity seemed to be correlated with the increase in a basic peroxidase of pI: 9.6. Four isoenzymes with pI in the range 8.5–9.6 and a neutral peroxidase of pI 6.3 were the most important peroxidases detected in cell extracts. Ca²⁺ addition at the beginning of the culture stimulated both the excretion of peroxidase to the medium and the enzyme activity in hairy roots but the isoenzyme profiles did not show qualitative changes during the growth cycle for both culture conditions.     

Peroxidase isoenzymes of the Avena coleoptile

The bulk of the peroxidases of Avena coleoptile sections exist in soluble and salt-extractable, wall-associated fractions with lesser amounts in membranous and wall-bound fractions. In the presence of auxin the peroxidase levels remain nearly constant while in the absence of auxin the peroxidase of each fraction increases 2-to 6-fold in 22 hr. There are qualitative and quantitative changes in the isoenzyme patterns with time, but these changes are independent of auxin. It is concluded that the peroxidase changes are induced by isolation of the tissues from the coleoptile and are unrelated to the growth rate.     

Cadmium interferes with auxin physiology and lignification in poplar

Cadmium (Cd) is a phytotoxic heavy metal that causes rapid growth reduction. To investigate if Cd interferes with the metabolism of auxin, a major growth hormone in plants, poplars (Populus × canescens) expressing a heterologous GH3::GUS reporter gene were exposed to 50 μM Cd in hydroponic solutions. Growth, photosynthetic performance, lignification, peroxidase activity, auxin concentration, and GUS staining were determined in order to record the activities of GH3 enzymes in the stem apex, the elongation zone, wood in the zone of radial growth, and in roots. Cd-induced growth reductions were tissue-specific decreasing in the order: roots>wood>shoot elongation and leaf initiation, whereas Cd concentrations increased in the order: leaves相似文献   

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.     

Phenolics, lignin content and peroxidase activity in Picea omorika lines

We analyzed low molecular mass phenolics, lignin content and both soluble and cell wall bound peroxidase activity in the needles of three Picea omorika (Pancic) Purkyne lines grown in the generative seed orchard. The highest values of the total phenol content as well as of catechine, caffeic acid, coniferyl alcohol, isoferulic acid and lignin concentration were detected in B5 line (“semidichotomy” line). The soluble guaiacol peroxidase activity was the highest in A3 line (line “borealis”). The highest activity of cell wall bound peroxidases was measured in B5 line, and it was in correlation with lignin content.     

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.     

Influence of Mechanical Stress on Auxin-Stimulated Growth of Excised Pea Stem Sections

The auxin to cytokinin ratios are described for promoting growth in the in vitro cultures of soybean (Glycine max (L.) Merr. cv. Bragg) and perennial clover (Trifolium repens L. cv. Regal Ladinc). Callus growth was induced on somatic tissue with 50:1 auxin to cytokinin (w/w) ratio. A 5:1 ratio served for initiation of cell suspensions from callus and for subsequent growth of callus from cells in suspension. A 1:2 ratio served for regeneration of buds and plantlets from the callus grown from cells. Although (2,4-dichlorophenoxy) acetic acid was the auxin for suspension and regenerative cultures, (2,4,5-trichlorophenoxy)acetic acid was the more effective auxin for initiation of callus on somatic tissue. All cultures were grown with 6-furfurylaminopurine as the cytokinin. The phytohormones strongly influenced the rates of culture growth, but determination of culture type was augmented by dl-alpha tocopherol acetate and iron. Tocopherol and a relatively high complement of iron promoted growth of juvenile cultures, whereas low level of iron and absence of tocopherol favored growth to comparatively more differentiated cultures. Without tocopherol, no callus formed on somatic tissue during the allotted period of incubation. Tocopherol plus a complement of low iron enabled growth of callus on rapidly growing somatic tissue. A high level of iron enabled comparatively more callus growth but suppressed growth of somatic tissue. In suspension cultures tocopherol and a high iron level enhanced dispersion of cells. A low iron complement in the absence of tocopherol induced growth of callus from cells and subsequent regeneration of buds and plantlets from the callus.     

Rapid Effects of IAA on Cell Surface Proteins from Intact Carrot Suspension Culture Cells

Suspension cultures of carrot (Daucus carrota L.) which had an absolute requirement for exogenously supplied auxin were grown in medium containing indoleacetic acid (IAA) as the sole auxin source. Putative cell surface proteins were extracted from the intact cells. Resupply of IAA to cultures partially depleted of auxin resulted in rapidly increased activities of three enzyme activities subsequently extracted. Two of the enzyme activities which increased, peroxidase and pectinesterase, have been implicated in the literature as important to cell wall development, structure, and growth. The other enzyme activity which was increased, IAA oxidase, may be involved in the degradation of IAA In vivo. Polypeptides in the extracts were found to increase equally as rapidly as the enzymes in response to IAA as determined with sodium dodecyl sulfate-polyacrylamide electrophoretic gels stained with silver. It is not known whether the changes in enzyme and polypeptide levels in the protein extracts were due to auxin effects on protein synthesis, transport, or extractability.     

Promotion of peroxidase activity in the cell wall of Nicotiana

Peroxidase catalyzes the oxidation of indole-3-acetic acid. The primary products of this reaction stimulate growth in plants. Therefore, our concept is that an increase in peroxidase activity will increase the effect of indole-3-acetic acid as a growth hormone. Our objective was to study the effect of 2,3,5-triiodobenzoic acid, a growth regulator, on isoperoxidases in the cell wall and cytoplasm of Nicotiana. Isoperoxidases from the cell wall and cytoplasmic fractions were separated by acrylamide gel electrophoresis. We found that 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase peroxidase activity in the cell wall. Since both 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase the activity of the same isoperoxidase, we conclude that 2,3,5-triiodobenzoic acid synergizes rather than antagonizes auxin action, and we suggest that this increase in indole-3-acetic acid oxidase activity sensitizes plant tissues to auxin.     

Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.