Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy–immunogold labeling

The lignification process in mature Norway spruce [Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)–immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.     

Structure of lignins in developing xylem of Norway spruce

The developing xylem in a Norway spruce (Picea abies) clone was investigated during a growth season and compared to lignin from sapwood of the same tree clone. Klason and acid-soluble lignin contents were determined as well as the carbohydrate monomer distribution and protein content. By analyzing lignin thioacidolysis products, it was shown that only guaiacyl units could be detected in the materials, and the relative amount of beta-O-4' bonds was assessed. Monomeric and selected dimeric lignin products were identified by mass spectrometry. The specimens were embedded and thin sections examined by microscopy to determine the state of cell differentiation in the samples. In the spring and early summer, growth was very rapid and the intention was to collect tissue in which exclusively the middle lamella/primary cell wall had begun to lignify. Combining data regarding Klason lignin, protein content and carbohydrate monomer distribution with microscopy, it was found that the developing xylem sample from mid-June contained lignin from exclusively middle lamella/primary wall. The Klason lignin content in the developing xylem during the growth season was 20%, 5% and 10% in April, June and August, respectively. Thioacidolysis showed that the lignin had more condensed structures than lignin from the reference Norway spruce clone wood. Mass spectrometry showed that the developing xylem specimens from June and August contained more lignin structures with end-groups than the reference sample. These results suggest that lignification in the cambial layer and early developing xylem may take place more in a bulk fashion during the summer.     

Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies)

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.     

Peroxidase and not laccase is the enzyme responsible for cell wall lignification in the secondary thickening of xylem vessels inLupinus

Summary The post-exponential growth phase of lupin (Lupinus albus cv. Multolupa) hypocotyls is characterized by a strong deposition of lignins in the primary and secondary walls of the xylem vessels. Coinciding with this phenomenon, there is a clearly peroxidatic activity in both the primary cell walls and the outer-most layers of the secondary thickening of the xylem vessels, as demonstrated by 3,3-diaminobenzidine cytochemistry. This activity was completely inhibited by KCN and the removal of H₂O₂ and was not due to laccase since this enzyme shows an almost total inability to oxidize 3,3-diaminobenzidine both in the presence and in the absence of H₂O₂. The absence of laccase-like activities in cell walls of vascular cells was supported by the fact that cell wall proteins from vascular cells were only capable of oxidizing 3,3-diaminobenzidine and coniferyl alcohol in the presence of H₂O₂. These results support the idea of an exclusive role of peroxidase (and exclude any role for laccase) in lignin formation in the secondary thickening of xylem vessels inLupinus.     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis     

Detection in situ and characterization of lignin in the<Emphasis Type="Italic"> G</Emphasis>-layer of tension wood fibres of<Emphasis Type="Italic"> Populus deltoides</Emphasis>

The occurrence of lignin in the additional gelatinous (G-) layer that differentiates in the secondary wall of hardwoods during tension wood formation has long been debated. In the present work, the ultrastructural distribution of lignin in the cell walls of normal and tension wood fibres from poplar (Populus deltoides Bartr. ex Marshall) was investigated by transmission electron microscopy using cryo-fixation–freeze-substitution in association with immunogold probes directed against typical structural motifs of lignin. The specificity of the immunological probes for condensed and non-condensed guaiacyl and syringyl interunit linkages of lignin, and their high sensitivity, allowed detection of lignin epitopes of definite chemical structures in the G-layer of tension wood fibres. Semi-quantitative distribution of the corresponding epitopes revealed the abundance of syringyl units in the G-layer. Predominating non-condensed lignin sub-structures appeared to be embedded in the crystalline cellulose matrix prevailing in the G-layer. The endwise mode of polymerization that is known to lead to these types of lignin structures appears consistent with such an organized cellulose environment. Immunochemical labelling provides the first visualization in planta of lignin structures within the G-layer of tension wood. The patterns of distribution of syringyl epitopes indicate that syringyl lignin is deposited more intensely in the later phase of fibre secondary wall assembly. The data also illustrate that syringyl lignin synthesis in tension wood fibres is under specific spatial and temporal regulation targeted differentially throughout cell wall layers.Abbreviations G-layer Gelatinous layer - G Guaiacyl monomeric unit - PATAg Periodic acid–thiocarbohydrazide–silver proteinate - S Syringyl monomeric unit     

巴西橡胶树HbMYB52基因的克隆及其在拟南芥中的表达

为揭示Hb MYB52在巴西橡胶树(Hevea brasiliensis)木材发育过程中的功能,从其转录组中分离克隆到1个MYB转录因子G21亚组成员基因,命名为Hb MYB52,开放阅读框为726 bp,编码242个氨基酸的蛋白,在木质部中高度表达。在拟南芥(Arabidopsis thaliana)中过表达Hb MYB52,虽未改变转基因植株株型,但植株维管束间纤维细胞壁明显增厚,同时抑制了木质纤维、导管次生壁形成。转基因拟南芥株系3和株系6中纤维素和木质素含量减少,相应各组分合成的关键酶基因的表达量也不同程度下降;株系8产生了木质素异位沉积,且木质素合成关键酶基因表达活跃。因此,推测Hb MYB52参与了植物次生壁形成调控,在拟南芥次生壁形成中可能发挥了双重功能:一方面负调控维管束次生壁形成以及各组分的生物合成,另一方面具有促进束间纤维次生壁增厚的作用。     

Secretion of vascular coating components by xylem parenchyma cells of tomatoes infected withVerticillium albo-atrum

Summary Massive infusion of conidia ofVerticillium albo-atrum into the xylem of tomato induces a cell wall coating response in resistant and susceptible near-isolines. In the early stages two types of coating material develop in the xylem vessels. The first, designated type A, is formed in association with xylem parenchyma cells that lack secondary walls; the localized accumulation of type A coating in the in the adjacent intercellular spaces, primary walls (i.e., pit membranes) and vessels occurs in conjunction with localized development of apposition wall layers within the parenchyma cells. Type B coating is initially formed in association with xylem parenchyma cells with secondary walls; the localized accumulation of typeB coating in the adjacent intercellular spaces, primary walls (i.e., pit membranes) and vessels occurs in conjunction with development of protective layers within the parenchyma cells. Most vessels are surrounded by a number of parenchyma cells including both cell types; therefore, in most vessels the coatings are mixed in later stages of development (i.e.,> 48 hours). The formation of both types of coating is stopped by the application of L--aminooxy--phenylpropionate, a specific inhibitor of phenylpropanoid synthesis. Histochemically, type A coating resembles lignin and type B, suberin. The data suggest that the coating response is due, wholly or in part to hypersecretion and/or chemical modification of normal cell wall components, induced by the pathogen.     

Cell-wall-bound oxidases from tobacco (Nicotiana tabacum) xylem participate in lignin formation

A band of cells closest to the cambium in the xylem of tobacco (Nicotiana tabacum L. cv. Samsun) stems oxidized 2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS), o-dianisidine and syringaldazine in the absence of exogenously added hydrogen peroxide. The oxidation was not prevented by catalase which suggests that the oxidation is not dependent on the production and utilisation of endogenous hydrogen peroxide by cell-wall peroxidases. Cell walls, isolated from tobacco xylem, also oxidized these substrates in the absence of added hydrogen peroxide. The cell walls consumed molecular oxygen whilst oxidizing a range of compounds including coniferyl alcohol. The substrate preference and sensitivity to inhibitors suggest the presence of laccasetype polyphenol oxidases (p-diphenol:O₂ oxidoreductase EC 1.14.18.1) which are covalently bound to the wall. The oxidation of coniferyl alcohol by the xylem cell walls was confirmed by assays based on the disappearance of coniferyl alcohol and was not affected by the presence of 500 units·mi^-1 catalase or Superoxide dismutase. Prolonged incubation of cell walls with coniferyl alcohol led to the production of a yellow-orange water-insoluble material that precipitated with the cell walls. Although a proportion of this material was soluble in methanol, the majority was tightly associated with the cell walls. These coloured cell walls had elevated lignin contents when assayed by the acetyl-bromide method. Fourier transforminfrared spectroscopic analysis of the coloured cell walls indicated that the increased lignin content is due to the deposition of guaiacyl-type lignin. Digestion of the xylem cell walls with Driselase, a mixture of fungal glycases, produced a wall residue that had a dramatically reduced ability to oxidize ABTS in the absence of added H₂O₂. However, oxidase activity could not be detected in the Driselase-solubilized extract, although small amounts of oxidase activity could be recovered from the Driselaseresistant wall residue by extraction in 3 M CaCl₂.Abbreviations ABTS 2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) - dl-DOPA 3-(3,4-dihydroxyphenyl)-alanine - FTIR Fourier transform infra-red - o-D o-dianisidine - o-pD o-phenylenediamine - SYR syringaldazine The authors acknowledge funding from the Scottish Office Agriculture and Food Department. They would like to thank Professor J.R. Hillman for his support, Dr. G.D. Lyon for his help and advice with the oxygen electrode and Mrs F. Carr for lignin determinations.     

Cellular characteristics of a traumatic frost ring in the secondary xylem of <Emphasis Type="Italic">Pinus radiata</Emphasis>

The effect of a short-term low temperature on cellular characteristics of a frost ring in radiata pine (Pinus radiata) secondary xylem was investigated using various microscopic techniques. Cell walls in the frost ring, that formed in the earlywood due to an abrupt drop in the temperature one night in the Spring, were poorly developed, lacking in the proper thickness and the proportion of wall constituents. In majority of the cases, the cell walls were highly convoluted and the secondary walls developed poorly and incompletely. Judging by irregular deposition of lignin, it appears that the control mechanism ensuring an orderly deposition of monolignols failed to function properly. The highly porous texture of some secondary walls indicated that cellulosic and hemicellulosic framework was affected, which would explain irregular lignification of cell walls. Thus the frost ring constitutes a serious defect in the timber, being a zone of weakness along which the wood is likely to split during processing, such as drying.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Wound-induced lignin and suberin deposition in a woody angiosperm (Eucalyptus gunnii Hook.): Histochemistry of early changes in young plants

Summary A simple system was developed to investigate the deposition of lignin and synthesis occurring in response to mechanical wounding in the woody angiospermEucalyptus gunnii Hook. The spatiotemporal deposition of these phenolic polymers was histochemically characterized in stem tissue through a combination of fluorescent microscopy and specific stains. Lignin and suberin deposition was detectable 24 h post wounding in the xylem wound zone and by 3 days post wounding in the bark wound zone where a welldeveloped necrophylactic (wound) periderm could be observed by 7 days post wounding. Close examination suggests that the spatial reinforcement of cell walls with lignin and/or suberin is carefully orchestrated so as to rapidly produce an effective protective barrier. Specific lignin colour reactions indicate that the lignin formed in response to wounding in both the bark and xylem wound zones is relatively poor in syringyl monomers as compared to that of developmental xylem lignin.Abbreviations P-HCl phloroglucinol-HCl - RH relative humidity - NP necrophylactic periderm     

Studies onArtemisia afra Jacq.: The phloem in stem and leaf

Summary The structure of the phloem was studied in stem and leaf ofArtemisia afra Jacq., with particular attention being given to the sieve element walls. Both primary and secondary sieve elements of stem and midvein have nacreous walls, which persist in mature cells. Histochemical tests indicated that the sieve element wall layers contained some pectin. Sieve element wall layers lack lignin. Sieve elements of the minor veins (secondary and tertiary veins) lack nacreous thickening, although their walls may be relatively thick. These walls and those of contiguous transfer cells are rich in pectic substances. Transfer cell wall ingrowths are more highly developed in tertiary than in secondary veins.     

海岛棉GbHCT13基因的功能验证

该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉GbHCT13基因(GenBank 登录号MW048849)在纤维发育中的功能。结果显示：(1)成功构建重组载体pCAMBIA3301 GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转GbHCT13基因拟南芥T₃代植株4株;qRT PCR检测表明,转基因植株中GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中CAD、CCoAOMT、PAL和4CL与GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转GbHCT13基因棉花植株3株;转GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的CAD、CCoAOMT、CCR、PAL 4个基因的表达均呈降低趋势,说明抑制GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明,GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与GbHCT13基因在模式植物拟南芥中的基本一致。     

Chemical composition of Casparian strips isolated from Clivia miniata Reg. roots: evidence for lignin

Endodermal cell walls and xylem vessels were isolated enzymatically from Clivia miniata Reg. roots. Transmission-electron-microscopic investigation of cross-sections of intact C. miniata roots and scanning-electron-microscopic investigation of isolated endodermal cell walls indicated that the root endodermis of C. miniata is essentially in its primary state of development. Isolated Casparian strips and xylem vessels were subjected to two different degradation methods usually applied to prove the existence of lignin, namely, cupric oxide oxidation and thioacidolysis. The reaction products obtained were typical aromatic derivatives of the natural lignin precursors coniferyl and sinapyl alcohols, and, in traces, of p-coumaryl alcohol, indicating the occurrence of lignin in the polymers from both Casparian strips and xylem vessels. The qualitative chemical compositions of the polymers from the two sources were similar, whereas the quantitative compositions were different, indicating that the molecular structure of the lignin polymer in the Casparian strips was different from that in the xylem vessels. Thus, for the first time, direct chemical evidence has been obtained that Casparian strips of C. miniata roots contain lignin as a major cell wall polymer.The author is indebted to Prof. Dr. G. Krohne (Zentrale Abteilung für Elektronenmikroskopie, Universität Würzburg, Germany) and to Prof. Dr. R. Guggenheim (Labor für Rasterelektronenmikroskopie, Universität Basel, Schweiz) for offering the opportunity for transmission-electron-microscopic and low-temperature scanning-electron-microscopic investigations, respectively. Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.     

Gene expression profiles in different stem internodes reveal the genetic regulation of primary and secondary stem development in <Emphasis Type="Italic">Betula platyphylla</Emphasis>

Secondary growth of stems is an important process for the radial increase of trees. To gain an insight into the molecular mechanisms underlying stem development from primary to secondary growth and to provide information for molecular research and breeding in Betula platyphylla (birch), the gene expression profiles of material from the first, third, and fifth internodes (IN) of 3-month-old seedlings were analyzed. Compared with the first IN, 177 genes were up-regulated and 157 genes down-regulated in the third IN; in the fifth IN, 180 genes were up-regulated and 275 genes were down-regulated. The expressions of 24 genes were up-regulated and 6 genes were down-regulated in the fifth IN relative to the third IN. The differentially expressed genes were annotated as having roles in cambium, xylem, and phloem development and formation; including cell wall expansion, cellulose biosynthesis, lignin biosynthesis and deposition, xylem extension, cell wall modification, and growth hormone responses. The expressions of genes related to cell wall expansion and cellulose biosynthesis in the primary cell wall were down-regulated in the third and fifth IN relative to the first IN. Genes involved in lignin biosynthesis, xylem extension, and cellulose synthesis in the secondary cell wall were up-regulated in the third and fifth IN relative to the first IN. These results described the patterns of gene expression during stem development in birch and provided candidate genes for further functional characterization.     

Lignin composition in cambial tissues of poplar

The cambial tissues of a Populus balsamifera, Balsam poplar clone were studied during a growth season. The Klason and acid-soluble lignin contents were determined as well as the carbohydrate monomer distribution and the protein content. Both the phloem and the xylem sides of the cambial region were examined. The samples were analyzed by thioacidolysis and structures of dimeric products were determined by mass spectrometry after desulphuration. Chemical analysis of samples during the growth season was combined with microscopy of embedded specimens that showed the state of cell differentiation at the time of sampling. In spring and early summer, growth is very rapid and the intention was to collect tissue in which exclusively the middle lamella/primary cell wall had begun to lignify. The Klason lignin, protein content and carbohydrate monomer distribution showed that all the specimens from the cambial tissues sampled during a growth season contained predominantly middle lamella and primary walls; except for the developing xylem sampled in August where the carbohydrate composition showed that secondary walls were present. Thioacidolysis showed that the lignin from the cambial tissues had more condensed structures than the lignin from the reference balsam poplar clone wood. More guaiacyl than syringyl units were detected and mass spectrometry showed that the cambial tissues contained more lignin structures with end-groups than the reference sample. These results suggest that lignification in the cambial layer and early developing xylem may take place predominantly in a bulk fashion during the summer.     

Characterization of basic <Emphasis Type="Italic">p</Emphasis>-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming <Emphasis Type="Italic">Picea abies</Emphasis> suspension culture

A Norway spruce (Picea abies) tissue culture line that produces extracellular lignin into the culture medium has been used as a model system to study the enzymes involved in lignin polymerization. We report here the purification of two highly basic culture medium peroxidases, PAPX4 and PAPX5, and isolation of the corresponding cDNAs. Both isoforms had high affinity to monolignols with apparent K_m values in μM range. PAPX4 favoured coniferyl alcohol with a six-fold higher catalytic efficiency (V_max/K_m) and PAPX5 p-coumaryl alcohol with a two-fold higher catalytic efficiency as compared to the other monolignol. Thus coniferyl and p-coumaryl alcohol could be preferentially oxidized by different peroxidase isoforms in this suspension culture, which may reflect a control mechanism for the incorporation of different monolignols into the cell wall. Dehydrogenation polymers produced by the isoforms were structurally similar. All differed from the released suspension culture lignin and milled wood lignin, in accordance with previous observations on the major effects that e.g. cell wall context, rate of monolignol feeding and other proteins have on polymerisation. Amino acid residues shown to be involved in monolignol binding in the lignification-related Arabidopsis ATPA2 peroxidase were nearly identical in PAPX4 and PAPX5. This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.     

Lignification in the flax stem: evidence for an unusual lignin in bast fibers

In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maüle reagents and KMnO₄-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl–syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical woody plant lignin, thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.     

An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine

Arabinogalactan proteins (AGPs) are abundant plant proteoglycans implicated in plant growth and development. Here, we report the genetic characterization, partial purification and immunolocalization of a classical AGP (PtaAGP6, accession number AF101785) in loblolly pine (Pinus taeda L.). A PtaAGP6 full-length cDNA clone was expressed in bacteria. PtaAGP6 resembles tomato LeAGP-1 and Arabidopsis AtAGP17-19 in that they all possess a subdomain composed of basic amino acids. The accessibility of this domain in the glycoprotein makes it possible to label the PtaAGP6 epitopes on the cell surface or in the cell wall with polyclonal antibodies raised against this subdomain. The antibodies recognize the peptide of the basic subdomain and bind to the intact protein molecule. A soluble protein-containing fraction was purified from the differentiating xylem of pine trees by using -glucosyl Yariv reagent (-glcY) and was recognized by antibodies against the basic subdomain. Immunolocalization studies showed that the PtaAGP6 epitopes are restricted to a file of cells that just precede secondary cell wall thickening, suggesting roles in xylem differentiation and wood formation. The location of apparent labeling of the PtaAGP6 epitopes is separated from the location of lignin deposition. Multiple single nucleotide polymorphisms (SNPs) were detected in EST variants. Denaturing HPLC analysis of PCR products suggests that PtaAGP6 is encoded by a single gene. Mobility variation in denaturing gel electrophoresis was used to map PtaAGP6 SNPs to a site on linkage group 5.