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.     

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.     

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.     

Purification and properties of the three o-diphenol-O-methyltransferases of tobacco leaves

Three o-diphenol-O-methyltransferases (OMTs I, II and III) which catalyse the monomethylation of various o-diphenols using S-adenosyl-L-methionine as methyl donor were isolated and purified about 210-, 70-, and 70-fold, respectively, from leaves of Nicotiana tabacum cv Samsun NN. They had slightly different MWs (93 000, 90 000 and 100000 for OMTs 1, 11 and Ill respectively) and slightly different pls (5.21, 4.80 and 4.74). The activities of all three enzymes were very stable when stored at 0° but they had different sensitivities to ultrafiltration and to heat treatment (45°). In none of the enzymes was there any change in reaction rate when Mg²⁺ ions or EDTA were added. The three enzymes exhibited very high and similar affinities towards the substrate S-adenosylmethionine and the reaction product S-adenosylhomocysteine, but they differed markedly in specificities towards the various o-diphenolic substrates. Relative methylation efficiencies were estimated from the calculation of the V/K_m ratios that led to the following decreasing order of best substrates: 5-hydroxyferulic acid > caffeic acid > homo-catechol > esculetin > protocatechuic aldehyde > catechol > hydrocaffeic acid > chlorogenic acid, for OMT I, and: homocatechol > catechol > protocatechuic aldehyde > esculetin ≈ cafreic acid > 5-hydroxyferulic acid, for both OMTsIIandIII. Most of the o-diphenols assayed were methylated exclusively in the meta position, but all three tobacco OMTs showed both para and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and escultin. Since K_m values towards the two position of methylation were always found to be identical, we conclude that each enzyme bears only one catalytic site.     

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.     

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.     

Significance of caffeic acid-O-methyltransferase in lignification of cultured tobacco cells

Caffeic acid-O-methyltransferase, activity was assayed in the callus and suspension cultured cells of tobacco. Lignification of cells was observed only in a kinetin (10^?5 M) culture and not in an auxin (10^?6 M 2,4-D or 10^?5 M IBA) culture. Enzyme activity in the kinetin cultured cells was much higher than in the stock culture and the rise in enzyme activity coincided with the onset of lignification.