Improving sample preparation to investigate lignin intensity of xylem at the cellular level by confocal Raman microspectroscopy of Populus tomentosa

Confocal Raman microspectroscopy(CRM)is an important tool for analyzing the compositional distribution of cell walls in situ.In this study,we improved the sample preparation method using paraffin-embedded sections combined with hexane dewaxing to obtain high resolution Raman images.We determined that the cell wall components of fiber cells were different from those of ray cells and vessel cells in the xylem of Populus tomentosa.Acetyl bromide and CRM methods produced similar trends when the difference in lignin intensity in the xylem region was compared between transgenic PtrLac4 and wild-type P.tomentosa.However,CRM proved more useful to analyze the lignin distribution in each cell type and distinguished the detailed difference in lignin intensity at the cellular level.Thus,CRM proved to be a useful in situ method to rapidly analyze the spatial variation of lignin content in the xylem of woody plants.     

木质素单体生物合成途径及其修订

对上世纪90年代以来木质素单体生物合成途径的发展进行了综述，对木质素的组成、木质素生物合成途径中的步骤及其涉及到的酶类、近年来对合成途径的修订以及我国在木质素方面的研究现状进行了介绍。提出今后我国木质素研究将主要集中在改良木材的材性和改善草类的消化性方面，通过调节木质素合成中关键酶基因的表达，以改变木质素的含量或单体组成，以满足工业不同用材的需要及畜牧用草的需要。     

Distribution of lignin and lignin precursors in differentiating xylem of Japanese cypress and poplar

Lignin is an integral component of the cell wall of vascular plants. The mechanism of supply of lignin precursors from the cytosol into the cell wall of differentiating xylem has not yet been elucidated. The present study showed that a certain amount of coniferyl alcohol glucoside (coniferin) occurred in the differentiating xylem of Japanese cypress (Chamaecyparis obtusa), as previously reported in gymnosperms. Coniferin content peaked in the early stages of secondary wall formation and decreased during lignification. In contrast to gymnosperms, coniferin content was limited in the differentiating xylem of poplar (Populus sieboldii × Populus grandidentata). Moreover, coniferyl alcohol was not detected in all specimens. In the differentiating xylem of poplar, a higher amount of sinapyl alcohol occurred than glucoside (syringin). However, the phloem contained syringin and not sinapyl alcohol. The sinapyl alcohol content in the xylem peaked in the cells with ceasing cell wall formation, and decreased gradually towards the boundary of the annual ring, where the lignin content kept increasing. Sinapyl alcohol in the differentiating xylem of poplar may be used for the lignification of the xylem.     

通过RNAi技术抑制杨树c3h基因表达提高糖转化效率

利用克隆得到的毛白杨c3h1基因构建其RNAi抑制表达载体,通过根癌农杆菌介导的叶盘法转化银腺杨无性系84 K,Realtime PCR检测表明其转基因株系323、325和322中c3h1基因表达量较野生型植株分别下调89.04%、82.22%和68.38%;茎横切片组化染色和显微结构观察表明转基因植株木质部发育和木质素沉积方式发生了改变;木质素、纤维素含量测定及苯酚—硫酸法总糖含量与HPLC法可溶性总糖和单糖含量检测结果表明:转基因植株木质素含量平均降低23.00%,最高可达39.71%;酸前处理效率最高提高了41.39%;未经酸处理直接酶解的糖化效率是对照植株的2.34～2.72倍,322株系和323株系比对照植株经酸前处理后再酶解的糖化效率高出81.18%和375.53%。     

Lignin deposition during earlywood and latewood formation in Scots pine stems

Lignin deposition at consecutive secondary wall thickening stages of early and late xylem cells during annual ring wood formation in Scots pine (Pinus sylvestris L.) stems was studied. Lignin patterns, isolated by thioglycolic acid method, consisted of alcohol-soluble (LTGA-I) and alkali-soluble (LTGA-II) fractions. The sum of two fractions, being the total lignin content, gradually increased in the course of lignification. However, the increments of lignin amount at each development stage of early and late tracheids were different. The intensity of lignin deposition increased in the course of earlywood tracheid maturation and decreased toward the end of latewood cell differentiation. The deposition of two lignin fractions in each layer of forming wood also occurred oppositely. The increment of LTGA-I descended, whereas that of LTGA-II increased from the beginning to the end of early xylem lignification. In contrast, LTGA-I increment dropped, whereas LTGA-II rose during late xylem lignification. Gel permeation chromatography showed that the lignins, formed at the beginning of lignification, were more homogeneous and had higher molecular weight compared with the lignins at the end of cell differentiation. Besides, the content of cellulose, estimated as the residue after lignin isolation, and of cell wall substances, presented as cell wall cross-section areas, at consecutive maturation stages of early and late xylem cells have been found to be different. The data show that lignin deposition occurred in different conditions and with opposite dynamics during early and late xylem formation.     

杨树木质素合成酶hct基因的克隆及核苷酸序列分析

根据毛果杨全序列（AC185363.2）中唯一具有转移酶功能的保守区设计引物,以正在分化的2年生欧美杨107次生木质部总RNA为模板经RT-PCR扩增出hct基因片段,与pMD20-T载体连接,重组质粒经特异引物扩增、限制性内切酶酶切和测序鉴定。结果表明,扩增片段长度为709bp,包含一个708bp的开放阅读框,与毛果杨全序列及HCT2（EU603314）的同源性均为98%,编码的氨基酸序列与毛果杨HCT2氨基酸（ACC63883.1）的同源性为98%,断定我们克隆的cDNA为hct,属于转移酶超家族,GenBank登录号为FM202091,该重组质粒命名为pMD20-T-hct。     

Immunolocalization of an anionic peroxidase in differentiating poplar xylem

Peroxidases are the major candidate enzymes involved in dehydrogenative polymerization of monolignols. Peroxidases have the signal sequence at their N-terminus and this suggests that they are transported to extracellular spaces or developing cell walls. In this study, we focused on an anionic peroxidase isozyme encoded by prxA3a, which seems to be related to lignification. To investigate the localization of peroxidase in differentiating xylem cells of poplar (Populus sieboldii × Populus grandidentata), anti-PRX3 antibody was raised against the anionic peroxidase. Western blotting and peroxidase activity inhibition assay showed specificity of the antibody. Labeling by anti-PRX3 antibody was localized in vessels and fibers during the secondary wall formation and was observed along the plasma membrane beside the microtubules. The labeling was not seen in the cell wall, where localization of peroxidases was expected during lignification. The peroxidase isozyme, which is suggested to be involved in monolignol polymerization, is localized on the plasma membrane and its localization might be regulated by microtubules.     

昆虫取食和挥发物诱导的杨树叶片中LOX和PAL活性变化

为了探索昆虫取食诱导的木本植物体内所产生的防御反应,以合作杨（Populus simonii×P.pyramidalis,‘Opera8277’）扦插苗为实验材料,经杨扇舟蛾（Clostera anachoreta）幼虫取食后,检测叶片中茉莉酸（jasmonate,JA）途径中的关键酶——脂氧合酶（lipoxygenase,LOX）及苯基丙酸类合成途径中的限速酶——苯丙氨酸解氨酶（phenylalanine ammonia lyase,PAL）的活性变化。结果显示,LOX和PAL的活性不仅在虫咬叶片中出现增加,在虫咬叶片上部的系统叶片中也有显著升高,表明茉莉酸途径和苯基丙酸类合成途径被激活,而且防御反应被系统性诱导。并且,与虫咬植株邻近的健康杨树叶片中LOX和PAL的增加表明,杨树间存在由昆虫取食诱导挥发物介导的信息传递。熏蒸实验也证明,茉莉酸甲酯（methyl jasmonate,MeJA）能够作为气体信号诱导合作杨植株产生防御反应。     

Occurrence and significance of bacteria in living trees of Populus nigra L.

On the occurrence and significance of bacteria in living trees of Populus nigra L. Eleven strains of bacteria were isolated from sapwood and heartwood of living poplar trees (Populus nigra L.) and identified mostly as Erwinia, Xanthomonas, Agrobacterium and Acinetobacter. Most of them were able to attack milled wood and the wood components pectin, hemicelluloses and holocellulose; α-cellulose and lignin were not consumed. The capillary liquid in the xylem of poplar served as a nutrient for the isolated bacteria. The significance of these bacteria for wetwood formation is discussed.     

Molecular Cloning of a Cellulose Synthase Gene PtoCesA1 from Populus tomentosa and Its Genetic Transformation in Tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P tomentosa. Four anti-expression vectors with different fragments of PtoCesAl, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, ＂dwarf＂ phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems.     

木质素生物合成及其基因调控的研究进展

木质素是地球上数量仅次于纤维素的有机物, 在植物生长发育中具有重要的生物学功能, 也是生物质能源的来源之一, 但在制浆造纸过程中, 将木材原料中木质素与纤维素分离, 不仅能耗高, 成本高, 而且废弃物还污染环境.林木木质素改良对于提高纸浆得率和质量、降低造纸经济成本以及环境保护, 都具有重要意义.文中介绍了木质素生物合成途径及其基因调控的研究进展, 此外, 还介绍了木质素生物合成基因调控的研究趋势, 主要是木质素特异性启动子、双价和多基因结构的共抑制以及转录因子的调控.     

Genotypic variation in drought tolerance of poplar in relation to abscisic acid

We investigated effects of water stress and external abscisic acid (ABA) supply on shoot growth, stomatal conductance and water status in 1-year-old cuttings of a drought-sensitive poplar genotype Populus x euramericana cv. I-214 (Italica) and a drought-tolerant genotype P. 'popularis 35-44' (popularis). Populus popularis was more productive and maintained higher leaf water potentials throughout the drought treatment than cv. Italica. Supply of ABA to the xylem sap caused a greater decline in growth and more leaf abscission in shoots of cv. Italica than in shoots of P. popularis. Immediately after initiation of the drought treatment in P. popularis, the ABA concentration ([ABA]) of the xylem increased rapidly and stomatal conductance declined; however, stomatal conductance had returned to control values by the third day of the drought treatment, coincident with a gradual decline in xylem [ABA]. In contrast, xylem [ABA] of cv. Italica initially increased more slowly than that of P. popularis in response to the drought treatment, but the increase continued for 3 days at which time a tenfold increase in xylem [ABA] was observed that was followed by abscission of more than 40% of the leaves. We conclude that sensitivity of poplar roots to variation in soil water content varies by clone and that a rapid short-term accumulation of ABA in shoots in response to water stress may contribute to drought tolerance.     

Lignification in cell cultures of Pinus radiata: activities of enzymes and lignin topochemistry

Enzymatic and topochemical aspects of lignification were studied in a Pinus radiata D. Don cell culture system that was induced to differentiate tracheary elements and sclereids with lignified secondary cell walls. The activities of the lignin-related enzymes phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) increased concomitantly with cell differentiation, indicating that the increase in enzyme activity was related to lignification of the cell walls and was not induced by stress. This result also indicates that PAL and CAD are suitable markers for tracheary element differentiation in coniferous gymnosperms. To further characterize lignification in this cell culture system, cellular UV-microspectrophotometry and thioacidolysis were employed. Typical UV-absorption spectra of lignin were obtained from the secondary cell walls of the tracheary elements and sclereids and from the compound middle lamella connecting differentiated cells, and the presence of lignin was confirmed by thioacidolysis. Certain aspects of lignin topochemistry in the cell walls of the tracheary elements were similar to cell walls of P. radiata wood, such as the high lignin concentration in the compound middle lamella connecting adjacent cells and the lower lignin concentration in the secondary cell walls. Therefore, the P. radiata cell culture system appears to be well suited to study the formation of lignified secondary cell walls in coniferous gymnosperms.     

Ultraviolet microspectrophotometric investigation of the distribution of lignin in Prunus jamasakura differentiated on a three-dimensional clinostat

To examine the effect of gravity on lignin content and deposition in plant cells, we used ultraviolet (UV) microspectrophotometry and chemical methods to investigate the secondary xylem of Prunus jamasakura grown on a three-dimensional (3D) clinostat, which simulates microgravity. The stem of the 3D-clinostat specimens elongated with bending and the width of their secondary phloem increased. The UV absorbance of the 3D-clinostat specimens at 278 nm was higher than that of the control specimens, which were grown on the ground, in the wood fiber cell corner middle lamella, compound middle lamella, and fiber secondary wall; the UV absorbance in the vessel secondary wall did not differ between the specimens. The lignin content in the stem, including the bark, of the 3D-clinostat specimens, as determined using an acetyl bromide method, was less than that of the control specimens. In the specimens that differentiated on a 3D clinostat, the amount of lignin in the wood fibers increased, while the proportion of the lignified xylem in the stem decreased relative to control values.     

查尔酮合酶与查尔酮异构酶基因特征及转基因应用

查尔酮合酶和查尔酮异构酶一起构成了黄酮类化合物生物合成的限速酶.底物先是由查尔酮合酶的作用形成袖皮素查尔酮化合物,然后再由查尔酮异构酶异构化成不同的黄酮类化合物.查尔酮合酶和查尔酮异构酶广泛存在于多种植物中,涉及了苯丙氨酸代谢途径中各种具有防御性产物的生物合成,在植物的抗菌机制、抗胁迫、细胞的发育和分化、色素的积累和外源基因的表达等方面起着重要的作用.就查尔酮合酶和查尔酮异构酶的结构特点、编码基因的克隆及序列特征、基因的表达调控以及转基因应用等方面进行了系统的综述,并对其今后进一步的研究提出了适宜的建议.     

Mechanical constraints on lignin deposition during lignification

Summary The ultrastructure of lignifying cell walls in Pinus radiata D.Don was investigated using potassium permanganate staining and transmission electron microscopy. Lignin deposition occurred at numerous discrete sites within various cell wall regions, suggesting the presence of some initiating agent at these sites. In the middle lamella region, lignin deposition occurred by addition of protolignin monomers to spherical particles of lignin. Lignification was completed by expansion of these spherical particles, initially forming irregular patterns of lignification followed by infilling of adjacent areas. In contrast, lignification in the secondary wall occurred by deposition of protolignin monomers onto the ends of expanding lignin lamellae between cellulose microfibrils leading to greatly elongated patches of lignin due to the greater rate of deposition along the microfibril axis compared to that across it. It is concluded that the cellulose matrix in which lignin deposition occurs, in the secondary wall, can exert a mechanical influence which limits the rate of lignin deposition in the direction perpendicular to the microfibril axis.     

Understanding the role of the cytoskeleton in wood formation in angiosperm trees: hybrid aspen (Populus tremula x P. tremuloides) as the model species

The involvement of microfilaments and microtubules in the development of the radial and axial components of secondary xylem (wood) in hybrid aspen (Populus tremula L. x P. tremuloides Michx.) was studied by indirect immunofluorescent localization techniques. In addition to cambial cells, the differentiated cell types considered were early- and late-wood vessel elements, axial parenchyma, normal-wood fibers and gelatinous fibers, and contact and isolation ray cells. Microfilaments were rare in ray cambial cells, but were abundant and axially arranged in their derivatives once cell elongation had begun, and persisted in that orientation in mature ray cells. Microfilaments were axially arranged in fusiform cambial cells and persisted in that orientation in all xylem derivatives of those cells. Microtubules were randomly oriented in ray and fusiform cells of the cambial zone. Dense arrays of parallel-aligned microtubules were oriented near axially in the developing gelatinous fibers, but at a wide range of angles in normal-wood fibers. Ellipses of microfilaments were associated with pit development in fiber cells and isolation ray cells. Rings of co-localized microtubules and microfilaments were associated with developing inter-vessel bordered pits and vessel-contact ray cell contact pits, and, in the case of bordered pits, these rings decreased in diameter as the over-arching pit border increased in size. Although only microtubules were seen at the periphery of the perforation plate of vessel elements, a prominent meshwork of microfilaments overlaid the perforation plate itself. A consensus view of the roles of the cytoskeleton during wood formation in angiosperm trees is presented.