Purification and characterization of an anionic peroxidase from sycamore maple (Acer pseudoplatanus) cell suspension cultures

A 42 kDa anionic peroxidase (EC 1.11.1.7) having a pl of 3.6 was purified from suspension cultures of cells of sycamore maple ( Acer pseudoplatanus L.) grown in the dark by a combination of lectin-affinity, anion-exchange and gel permeation chromatography. The enzyme had an amino acid composition similar to that found for other anionic plant peroxidases, but the protein was blocked to amino-terminal protein sequencing. Commercially available antibodies against horseradish peroxidase were shown to cross-react with the sycamore maple enzyme on immunoblots. The purified peroxidase displayed differences in its affinity for each of the three monolignols, and these differences were compared to those found for a commercial preparation of horseradish peroxidase, as well as a laccase ( p -diphenol:O₂ oxidoreductase: EC 1.10.3.1) purified from sycamore maple cell suspension cultures. These results are discussed with respect to the role played by peroxidases in lignin deposition and host-pathogen response.     

Turnover of coniferin in pine seedlings

Coniferin (coniferyl alcohol-β-d-glucoside) was not detected in pine seeds (Picea abies) but it accumulated in the stems and roots of pine seedlings. Pulse labeling experiments with l-phenylalanine-[U-¹⁴C] and 100-day-old pine seedlings in hydroponic solution showed a turnover of coniferin with a half life of about 60 hr. Pulse labeling with ¹⁴CO₂ and seedlings kept in soil gave a half life of about 120 hr for coniferin. The results indicate that coniferin could be an intermediate of lignin biosynthesis in pine seedlings.     

Effect of ‘elicitors’ on extracellular peroxidase activity in suspension-cultured slash pine (Pinus elliottii Engelm.)

Peroxidase activity in the growth medium of suspension-cultured slash pine was observed over time and eliciting chitosan concentrations from non-damaging to lethal were followed. First significant differences in peroxidase activity were observed within 12h exposure to chitosan. Extracellular peroxidase activity decreased as chitosan increased until considerable cell death occurred and medium peroxidase activity increased. Chitin and fusiform rust mycelium elicited decreased peroxidase activity with no significant difference in viability. The highest levels of elicited lignification corresponded to the lowest extracellular peroxidase activity. Controls and the greatest chitosan concentration (which elicited 100% cell death) showed the highest peroxidase activity and no lignification.     

低氧气调包装对去壳雷笋褐变和木质化的影响

研究了在厚度为0.04 mm的聚乙烯(PE)袋内充气成分为2%O2、5%CO2和93%N2于10℃下贮藏时气调袋装去壳雷笋褐变和木质化进程.与对照相比,低氧气调包装(MAP)抑制了去壳雷笋贮藏前期丙二醛(MDA)的生成,显著抑制了过氧化物酶(POD)(P＜0.05)和苯丙氨酸解氨酶(PAL)活性(P＜0.01),最终显著抑制了笋肉的褐变(P＜0.01),显著抑制了木质素(P＜0.05)和纤维素的合成,从而延长了保鲜期.去壳雷笋的褐变可能由POD和PAL活性作用引起.POD和PAL活性的增加也可能诱导了木质素的合成.     

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.     

Effect of Inhibitors and Stimulators of Ethylene Production on Gall Development in Meloidogyne javanica-Infected Tomato Roots

Excised tomato roots infected with Meloidogyne javanica produced ethylene at 3-6 times the rate of noninfected roots. This increase in ethylene production started 5 days after inoculation. Gall growth and ethylene production in infected roots were accelerated by 1-aminocyclopropane-1-carboxylic acid (ACC), indole acetic acid (IAA), and ethrel known as ethylene production stimulators. When inhibitors of ethylene production, like aminoethoxyvinylglycine (AVG) or aminoxyacetic acid (AOA), or inhibitors of ethylene action like silver thiosulfate (STS), were applied, gall growth and ethylene production were inhibited. Enhanced expansion of parenchymatous cells was observed in sections from nematode-induced galls and ethylene-treated roots. Lignification of xylem elements and fibers in the vascular cylinder was markedly inhibited in the gall, compared with noninfected root tissue. Because ethylene is known to induce cell expansion and to inhibit lignification, it is suggested that this plant hormone plays a major role in the development of M. javanica-induced galls. Ethylene affects gall size by enhancing parenchymatous tissue development and allows expansion of giant cells and the nematode body by reducing tissue lignification.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

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.     

Anatomy and lignin distribution in reaction phloem fibres of several Japanese hardwoods

Background and Aims Although tension wood formation and the structure of gelatinous fibres (G-fibres) have been widely investigated, studies of the influence of the reaction phenomenon on phloem fibres have been few and incomplete in comparison with those of xylem wood fibres. This study was undertaken to clarify the influence of stem inclination on phloem fibres using several Japanese hardwood species that produce different G-fibre types in tension wood. Methods Eight hardwood species were inclined at 30-45° at the beginning of April. Specimens were collected in July and December. The cell-wall structure and lignin distribution of phloem fibres on both the tension and opposite sides were compared by light microscopy, ultraviolet microscopy, confocal laser scanning microscopy after staining with acriflavine, and transmission electron microscopy after staining with potassium permanganate. Key Results Three types of changes were found in tension-side phloem fibres: (1) increases in the proportion of the syringyl unit in lignin in the S(1) and S(2) layers and compound middle lamella (Cercidiphyllum japonicum), (2) formation of unlignified gelatinous layers (Melia azedarach and Acer rufinerve) and (3) increases in the number of layers (n) in the multi-layered structure of S(1) + S(2) + n (G + L) (Mallotus japonicus). Other species showed no obvious change in cell-wall structure or lignin distribution. Conclusions Phloem fibres of the tree species examined in our study showed three types of changes in lignin distribution and cell-wall structure. The reaction phenomenon may vary with tree species and may not be closely related to G-fibre type in tension wood.