Comparative study on antioxidative system in normal and vitrified shoots of Populus suaveolens in tissue culture

To explore the physiological and biochemical mechanism of the occurrence of vitrified shoots of Populus suaveolens in tissue culture, the changes in water, chlorphyll, lignin, H₂O₂, phenylalanine ammonialyase (PAL), malonaldehyde (MDA), protective enzymatic systems, and some key enzymes involved in the ascorbate- glutathione cycle were comparatively studied in both normal and vitrified shoots of P. suaveolens. The results show that the lower activities of peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and PAL, and the less contents of chlorphyll, lignin, ascorbate (ASA) and reduced glutathione (GSH) as well as the lower ratios of ASA / DHA and GSH / GSSG are observed in vitrified shoots than in normal ones during the whole culture period. While in comparison with normal shoots, the higher activity of superoxide dismutase (SOD) and the more concentrations of water, H₂O₂, MDA, dehydroascorbate (DHA) and oxidized glutathione (GSSG) are found in vitrified shoots. Statistical analysis indicates that the enhanced activity of SOD and the decreased activities of CAT and POD as well as some enzymes involved in the ascorbate-glutathione cycle might be closely correlated to the accumulation of H₂O₂. The less regeneration of ASA and GSH and the lower capacity of the ascorbate-glutathione cycle observed in vitrified shoots might be due to a significant decrease in APX, MDAR, DHAR and GR activities and a decline in redox status of ASA and GSH. The decreases in chlorphyll content might result in a decline in photosynthesis. The lower activities of POD and PAL could result in the decrease of lignin synthesis and cell wall ligination, which might be the key factor leading to the increase in water content. It is concluded that the deficiency of detoxification capacity caused by the lower capacity of the ascorbate-glutathione pathway and the decreased activity of protective enzymatic system might lead to the large accumulation of H₂O₂ and the enhancement of membrane lipid peroxidation, which might be the main cause leading to the occurrence of vitrifying shoots of P. suaveolens in tissue culture. [Supported by National Natural Science Foundation of China (Grant No. 30271093) and the Foundation of State-designated Base for Biology Researching and Teaching in Beijing Forestry University]     

The role of antioxidant system in freezing acclimation-induced freezing resistance of Populus suaveolens cuttings

We investigated the changes in the contents of H2O2, malonaldehyde (MDA) and endogenous antioxidants, the activities of protective enzymes and some critical enzymes involved in the ascorbate-glutathione (ASA-GSH) cycle as well as freezing resistance (expressed as LT50) and correlations mentioned above, in detail using Populus suaveolens cuttings. The purpose was to explore the physiological mechanism of the enhancement of freezing resistance induced by freezing acclimation at –20°C, and to elucidate the physiological mechanisms by which trees adapt to freezing. The results showed that freezing acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), monodehydroascorbate reductase (MDAR), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). And it increased the contents of reduced ascorbate (ASA), reduced glutathione (GSH), dehydroascorbate (DHA) and oxidized glutathione (GSSG). However, H2O2 and MDA contents and LT50 of cuttings were decreased. LT50 in cuttings was found to be closely correlated to the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, H2O2, MDA, ASA, GSH, DHA and GSSG during freezing acclimation. This suggested that the enhancement of freezing resistance of cuttings induced by freezing acclimation may relate to the distinct increase for the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, ASA, GSH, DHA, and GSSG. In addition, the observed levels of APX, DHAR, MDAR, GR, ASA, DHA, GSH and GSSG were higher than those of SOD, POD and CAT during freezing acclimation. It indicated that a higher capacity of the ASA-GSH cycle is required for H2O2 detoxification, and growth and development of cuttings. Based on the obtained results, it can be concluded that the ASA-GSH cycle plays an important role in enhancement of freezing resistance of P. suaveolens cuttings during freezing acclimation.     

保鲜处理对高节竹笋采后生理的影响

采用壳聚糖的3种保鲜处理方法对高节竹笋进行了试验比较,测定了高节竹笋采收后在贮藏过程中与竹笋老化密切相关的苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)的活性,以及纤维素和木质素含量.结果表明:竹笋采收后,随着贮藏时间的增加,PAL和POD活性在短时间内快速地增加,经过一定时间后减少,纤维素和木质素含量随竹笋的老化过程而增加.以壳聚糖涂膜和BCY保鲜剂共同作用的保鲜处理在7 d内各指标的增加速度最慢,保鲜效果最佳.     

甜龙竹笋采后不同时间及温度储藏下酶活性的变化

以甜龙竹笋为研究材料,采用植物ELISA检测试剂盒测定不同储藏时间(0、12、24、36、48、60、72 h)和不同储藏温度(0℃、4℃、常温对照)下PAL(苯丙氨酸解氨酶)、PPO(多酚氧化酶)、CAT(过氧化氢酶)、POD(过氧化物酶)、CAD(肉桂醇脱氢酶)的变化特征。结果表明:在常温(20~25℃)下,PAL活性呈现先上升后下降的趋势,POD、PPO、CAT、CAD呈现波动变化趋势;其次,4℃以下对PAL、POD、PPO、CAD活性存在抑制作用,对CAT活性影响不大。0℃、4℃储藏对采后甜龙竹笋酶活性有降低作用,可通过低温结合其他方法对甜龙竹笋进行保鲜处理,达到延缓甜龙竹笋老化的目的。     

Responses of antioxidant enzymes in the needles of hinoki cypress (Chamaecyparis obtusa) seedlings to nutrient solutions containing various calcium/aluminum ratios

An attempt was made to examine the possible connection between the various ratios of calcium/aluminum (Ca/Al) in the nutrient solution of plant cultures and the active oxygen scavenging system of hinoki cypress (Chamaecyparis obtusa) seedlings. The hinoki cypress seedlings were transferred to nutrient solutions containing 5 mM AlCl₃ together with various concentrations of Ca(NO₃)₂ in pots containing glass beads and Teflon tips. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) in the needles were estimated at several stages during the 7-day treatment. The samples treated with the lower Ca/Al solutions show the highest SOD activities. The activities of APX and GR, both of them involved in the ascorbate-glutathione cycle, showed the same tendency with decrease to higher Ca/Al ratio. These results indicate that rhizospheric Ca might compete with Al and ameliorate Al toxicity on and in the roots, the Al stress is not transformed to the needles after a few days, and the ascorbate-glutathione cycle in the hinoki cypress needles might fluctuate and be suppressed by the rhizospheric Al stress during the 7 days. This work was supported in part by funding from the Japan Science & Technology Corporation, the CREST program 1996–2001, and the Center for Forest Decline Studies.     

Water Chemistry Profiles under Four Tree Species at Gisburn, NW England

A study was made of the changes and variation in the chemistryof rainwater passing through the different strata of separateecosystems of four tree species (oak, alder, spruce and pine)on the same site at Gisburn (Bowland Forest), north-west England.Waters were sampled as rain, throughfall, forest-floor leachateand soil waters from both the A and B/C horizons; and analysedfor NH₄-N, NO₃-N, PO₄-P, K, Ca, Mg, Na, Al, SO₄-S, Cl, totalorganic carbon and pH. Species differences — often markedones — appear to exist in the concentrations of most solutesin most strata. The between-species differences in throughfallchemistry provide little guide to the chemistry of waters lowerdown the profile: the forest floor is a particularly importantsource of further species differences, e.g. variation in NO₃production and the resulting effects on acidity and other ions. The chemistry of the soil waters provides some indication ofpossible drainage losses, although the question of which horizonacts as the source of drainage waters on this site remains unanswered.The levels of H and Al, in particular, are very different betweenthe upper mineral soil (A horizon) and the lower B/C horizon.In general, and based on these concentration data only, oakis associated with smallest potential solute losses, pine thegreatest. Received 13 February 1990.     

Shading Reduces both Visible and Invisible Frost Damage to Norway Spruce Seedlings in the Field

Two-year-old cuttings of Norway spruce were subjected to nightfrosts in spring on an exposed site in southern Sweden. Shadingwas used to assess the influence of sunlight on the extent ofdamage resulting from night frost. Chlorophyll fluorescencewas measured in needles in flushing shoots, and in shoots atthe stage of bud burst. The F_v:F_m ratio was significantly lowerfor plants exposed to light, compared with shaded plants onthe days following the night frost (minimum temperature –6°C).The effect was similar both in 1-year-old and current year needles.The low F_v:F_m ratios indicate damage to photosystem H, causedby an interaction between sub freezing temperatures and highlight intensity. Shading also increased the survival of flushingshoots. It is suggested that regeneration of Norway spruce onsites exposed to frost should be carried out in partial shade,for example under a shelterwood.     

侧柏抗病单株对叶枯病抗性机制研究

本文比较测定了对叶枯病具有高度抗性的侧柏单株叶片中木质素和纤维素含量、苯丙氨酸解氨酶（ＰＡＬ）和超氧化物歧化酶（ＳＯＤ）、以及细胞含水量和肉质化程度。结果表明,抗病单株叶片细胞中木质素和纤维素的含量显著地高于普通的感病侧柏;抗病单株的ＰＡＬ活性和ＳＯＤ活性也明显高于感病株。并且发现ＰＡＬ活性和木质素含量之间具有高度相关性。抗病单株的细胞含水量和肉质化程度则显著地低于感病株。综上指标说明,抗病单株在生理生化和遗传上,明显地区别于普通感病株。     

Agrobacterium tumefaciens-mediated GUS gene transformation of Robinia pseudoacacia ‘Idaho’

Based on the plant regeneration system, a GUS gene transformation system to Idaho locust (Robinia pseudoacacia ‘Idaho’) mediated by Agrobacterium tumefaciens was established. The successful transformation was confirmed by regenerating the shoots from the infected leaves in the presence of hygromysin; by histochemical X-gluc assays of β-glucuronidase (GUS) and by PCR and PCR-Southern blotting analysis. The ratio of positive transgenic plants is 5.8% (5 out of 86 plants). With this system, the target gene DREB was introduced into the leaves of Idaho locust. The transgenic plants regenerated, which was verified by PCR-Southern blot-ting. It is suggested that the transformation system could be a new, simple, reliable and practical route to gene transformation of R. pseudoacacia ‘Idaho’ mediated with A. tumefaciens.     

渗透胁迫和钙处理对杉木幼苗膜脂过氧化及保护酶活性的影响

杉木幼苗用ＰＥＧ６０００渗透胁迫（－１．５ＭＰａ,－２．５ＭＰａ）,在４８ｈ内其叶片中Ｈ２Ｏ２含量和ＭＤＡ含量明显增加,Ｈ２Ｏ２含量从３５．５μｍｏｌ．ｇ＾－１分别增加到４３．０μｍｏｌ．ｇ＾－１和４８．０μｍｏｌ．ｇ＾－１;ＭＤＡ含量从１７０．５μｍｏｌ．ｍｇ＾－１分别增加到３４９．５μｍｏｌ．ｍｇ＾－１和３６５．２μｍｏｌ．ｍｇ＾－１,其变化与胁迫强度成正比;４８ｈ后Ｈ２Ｏ２含量下降,但ＭＤＡ     

Look back over the studies of lignin biochemistry

The role of the cinnamate pathway in monolignol biosynthesis based on feeding experiments with lignifying plant stems and characterization of the enzymes in the pathway, O-methyltransferase (OMT), cinnamyl alcohol dehydrogenase (CAD), etc. is discussed. Monolignol biosynthesis via metabolic grids according to newly characterized enzymes in the pathway is also reviewed and discussed. The cleavage mechanisms of side chains and aromatic rings by lignin peroxidase and laccase elucidated by using ¹⁸O, ²H, and ¹³C labeled lignin substructure dimers and DHP with ¹⁸O₂ and H₂ ¹⁸O are reviewed. Finally, the prospects of lignin biochemistry in the wood and paper industries are discussed according to the recent progress on gene technology on wood formation and microbial degradation of lignin.     

Cold tolerance of Acacia karroo and A. nilotica, two tree species from South Africa, in Beijing

Fynbos plant communities developed under geographic and climatic conditions unique to South Africa. Acacia karroo is the most widespread tree species, well known for its drought tolerance. A. nilotica exudes a good quality gum which is edible and suitable for confectionery. The two species have been introduced to China but little is known about their adaptability under the new environmental conditions. For our present study, the growth potential and physiological and biochemical parameters of the two species in response to cold stress were investigated. The results, measured as seed qualities (purity, thousand seed weights, germination, etc.) show differences between A. karroo and A. nilotica. The seed germination rate was between 10% and 60%. Seedlings were established in fields, pots and plugs. Those in the field grew well during the growing season but completely died in the winter. Those in pots or plugs were moved to and managed in the greenhouse. The plants were green in winter. The electrical conductivity of cells, the contents of water soluble sugars, proteins, malondialdehyde (MDA) increased during eight hours of low temperature treatment at 4℃. The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) decreased markedly during the eight hours of 4℃treatment. We conclude that A. karroo and A. nilotica have little adaptability to the cold winter weather and cannot survive in the outdoors in the Beijing area, but could grow well indoors as ornamentals.     

Differences in biosyntheses of guaiacyl and syringyl lignins in woods

Summary Metabolic differences in the formation of guaiacyl and syringyl lignins were explained in terms of the different functions of O-methyltransferases and reducing enzymes which participate in methylation and reduction of the hydroxycinnamic acid intermediates in the biosynthetic pathway of these two types of lignins. Sinapyl alcohol was dehydrogenated with peroxidase and H₂O₂ under various reaction conditions. Chemical properties of the dehydrogenation polymers (DHPs) formed were characterized, and the possible occurrence of syringyl lignin in hardwood was discussed. DHP and dimers of p-coumaryl alcohol were also characterized and discussed in relation to the formation of grass lignin which contains p-hydroxyphenyl propane as an additional lignin monomer.The authors are indebted to Messrs. Y. Nakamura and H. Kuroda in this Division and Mr. T. Yamasaki at Kagawa University for their cooperation in the course of these investigations     

Reaction behavior of lignin in supercritical methanol as studied with lignin model compounds

 The reaction behavior and kinetics of lignin model compounds were studied in supercritical methanol with a batch-type supercritical biomass conversion system. Guaiacol, veratrole, 2,6-dimethoxyphenol, and 1,2,3-trimethoxybenzene were used as model compounds for aromatic rings in lignin. In addition, 5-5, β-1, β-O-4, and α-O-4 types of dimeric lignin model compounds were used as representatives of linkages in lignin. As a result, aromatic rings and 5-5 (biphenyl)-type structures were stable in supercritical methanol, and the β-1 linkage was not cleaved in the β-1-type structure but converted rapidly to stilbene. On the other hand, β-ether and α-ether linkages of β-O-4 and α-O-4 lignin model compounds were cleaved rapidly, and these compounds decomposed to some monomeric compounds. Phenolic compounds were found to be more reactive than nonphenolic compounds. These results indicate that cleavages of ether linkages mainly contribute to the depolymerization of lignin, whereas condensed linkages such as the 5-5 and β-1 types are not cleaved in supercritical methanol. Therefore, it is suggested that the supercritical methanol treatment effectively depolymerizes lignin into the lower-molecular-weight products as a methanol-soluble portion mainly by cleavage of the β-ether structure, which is the dominant linkage in lignin. Received: December 19, 2001 / Accepted: April 30, 2002 Acknowledgments This research has been done under the research program for the development of technologies for establishing an ecosystem based on recycling in rural villages for the twenty-first century from the Ministry of Agriculture, Forestry and Fisheries, Japan; by a Grant-in-Aid for Scientific Research (B)(2) (no.12460144, 2001.4–2003.3) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and under the research program from Kansai Research Foundation for Technology Promotion, Japan. The authors thank them for their financial support. This study was presented in part at the 45th Lignin Symposium, Ehime, Japan, October 2000 and the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, Japan, April 2002 Correspondence to:S. Saka     

Microbial population in rotten living body ofSalix matsudana

The microbial population in rotten living body ofSalix matsudana caused byTrametes suaveolens (L.) was researched. 11 bacteria species (1 species ofBacillus, 2 species ofClostridium and 8 species of non-brood-cell bacteria), 1 species ofActinomyces that belongs to Lavendulac, 8 species of fungi and 6 species ofTrichoderma were isolated from rotten trunk. The hyphae ofTrametes suaveolens mainly existed between rotten sections and discoloration sections. In over-rotten section and healthy section the fungi (Trametes suaveolens) were not isolated. The microbes that lived in the discoloration section were the most in kinds and number and they were the pioneer microbes of wood rotting. Only after they dwelled in wood and eliminated its rot-resistance, could wood-rotting fungi invade wood and caused wood-rotting. Responsible editor: Zhu Hong     

Effect of sulfur-dioxide on protein-SH in needles of Picea abies1

The effect of SO₂ on the SH-groups of proteins in needles of Picea abies is studied. The content of total protein SH and glutathione is estimated with 5.5′-Dithio-bis-nitrobenzoic-acid (Ellman reagent). In addition structural protein SH is measured microspectrophotometrically in sections stained with the SH reagent, mercurochrome. The results indicate that GSH and also total protein SH and structural protein SH are increased in needles of Picea abies growing in SO₂ polluted areas. Compared to needles of trees in relatively unpolluted areas average increases of 2. 33 (GSH), 1.22 (total protein SH) and 1.55 (structural protein SH) are found.     

Lignin biochemistry: Biosynthesis and biodegradation

Summary Lignin biosynthesis via shikimate-cinnamate pathways in plants, and the biosynthetic differences of guaiacyl-and syringyl lignins between gymnosperms and angiosperms have been elucidated by tracer experiments using ¹⁴C labeled precursors and the following enzyme reactions. The formation of guaiacyl lignin but not syringyl lignin in gymnosperms was attributed to the following factors; absence of ferulate-5-hydroxylase, poor affinity of O-methyltransferase toward 5-hydroxyferulate, and lack of activation and/or reduction of sinapatc. A mechanism of lignin-carbohydrate complexes formation in wood cell walls was elucidated based on the reaction of the quinone methide of guaiacylglycerol--guaiacyl ether with sugars, and the analysis of DHP-polysaccharide complexes.The main cleavage mechanisms of side chains and aromatic rings of lignin model compounds and synthetic lignin (DHP) by white-rot fungi and their enzymes, lignin peroxidase and laccase have been elucidated using ²H, ¹³C and ¹⁸O-labeled lignin substructure dimcrs with ¹⁸O₂ and H₂ ¹⁸O. Side chains and aromatic rings of these substrates were cleaved via aryl cation radical and phenoxy radical intermediates, in reaction mediated only by lignin peroxidase/H₂O₂ and laccase/O₂.Academy Lecture presented at the Cellucon 88 in Japan, International Symposium on New Functionalisation Developments in Cellulosics and Wood, held in Kyoto, Japan, November 28 to December 1, 1988This paper is a conclusion of our investigations on the biosynthesis and microbial degradation of lignin for 30 years in the Research Section of Lignin Chemistry, Wood Research Institute, Kyoto University. The author is greatly indebted to Drs. M. Shimada, F. Nakatsubo, T. Yamasaki, H. Ohashi, M. Tanahashi, Y. Nakamura, H. Kuroda, H. Kutsuki, T. Katayama, Y. Kamaya, T. Umezawa and Messrs. H. Fushiki, M. Ohta, A. Noguchi, H. Namba, T. Habe, S. Kawai, S. Yokota, and T. Hattori for their kind cooperation in these investigations. These invstigations were supported in part by Grant-in-Aid Nos. 548047, 57480058, 59760124, 60760130, 60440015, 61760142, 61560193, 62790250 for Scientific Research, and 1980 Grant-in-Aid for Environmental Science (R-33-8), No. 403064 from the Ministry of Education, Science and Culture of Japan, and a 1980 Weyerhaeuser grant