Cell Wall Lignin is Polymerised by Class Ⅲ Secretable Plant Peroxidases in Norway Spruce

Class Ⅲ secretable plant peroxidases occur as a large family of genes in plants with many functions and probable redundancy. In this review we are concentrating on the evidence we have on the catalysis of lignin polymerization by class Ⅲ plant peroxidases present in the apoplastic space in the xylem of trees. Some evidence exists on the specificity of peroxidase isozymes in lignin polymerization through substrate specificity studies, from antisense mutants in tobacco and poplar and from tissue and cell cult...     

Norway spruce(Picea abies) laccases:Characterization of a laccase in a lignin-forming tissue culture

Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell wal s water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce(Picea abies(L.)Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences(genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five full-length laccase c DNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture.This laccase has an acidic p H optimum(p H 3.8–4.2) for coniferyl alcohol oxidation. It has a high af finity to coniferyl alcohol with an apparent K m value of 3.5 m M; however, the laccase has a lowe catalytic ef ficiency(V max/K m) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases o coniferous plants.     

TRANSGENIC PLANTS EXPRESSING BACILLUS THURINGIENSIS DELTA-ENDOTOXINS

Commercial varieties of transgenic Bacillus thuringiensis (Bt) plants have been developed in many countries to control target pests. Initially, the expression of native Bt genes in plants was low due to mRNA instability, improper splicing, and post-translation modifications. Subsequently, modifications of the native Bt genes greatly enhanced expression levels. This is a review of the developments that made modem high-expression transgenic Bt plants possible, with an emphasis on the reasons for the low-level expression of native Bt genes in plant systems, and the techniques that have been used to improve plant expression of Bt toxin genes.     

Gene expression profiles of the one-carbon metabolism pathway

One-carbon metabolism plays a critical role in both DNA methylation and DNA synthesis. Accumulating evidence has shown that interruptions of this pathway are associated with many disease outcomes including cardiovascular diseases and cancers. Mechanistic studies have been performed on genetic polymorphisms involved in one-carbon metabolism. However, expression profiles of these inter-related genes are not well-known. In this study, we examined the gene expression profiles of 11 one-carbon metabolizing genes by quantifying the mRNA level of the lymphocyte among 54 healthy individuals and explored the correlations of these genes. We found these genes were expressed in lymphocytes at moderate levels and showed significant inter-person variations, We also applied principle component analysis to explore potential patterns of expression. The components identified by the program agreed with existing knowledge about one-carbon metabolism. This study helps us better understand the biological functions of one-carbon metabolism.     

SDG714 Regulates Specific Gene Expression and Consequently Affects Plant Growth via H3K9 Dimethylation

Histone lysine methylation is known to be involved in the epigenetic regulation of gene expression in all eukaryotes including plants. Here we show that the rice SDG714 is primarily responsible for dimethylation but not trimethylation on histone H3K9 in vivo. Overexpression of YFP-SDG714 in Arabidopsis significantly inhibits plant growth and this inhibition is associated with an enhanced level of H3K9 dimethylation. Our microarray results show that many genes essential for the plant growth and development were downregulated in transgenic Arabidopsis plants overexpressing YFP-SDG714. By chromatin immunoprecipitation analysis, we show that YFP-SDG714 is targeted to specific chromatin regions and dimethylate the H3K9, which is linked with heterochromatinization and the downregulation of genes. Most interestingly, when YFP-SDG714 production is stopped, the inhibited plants can partially restore their growth, suggesting that the perturbation of gene expression caused by YFP-SDG714 is revertible. Taken together, our results point to an important role of SDG714 in H3K9 dimethylation, suppression of gene expression and plant growth, and provide a potential method to regulate gene expression and plant development by an on-off switch of SDG714 expression.     

Abiotic and Biotic Stresses and Changes in the Lignin Content and Composition in Plants

Lignin is a polymer of phenylpropanoid compounds formed through a complex biosynthesis route,represented by a metabolic grid for which most of the genes involved have been sequenced in several plants,mainly in the model-plants Arabidopsis thaliana and Populus.Plants are exposed to different stresses,which may change lignin content and composition.In many cases,particularly for plant-microbe interactions,this has been suggested as defence responses of plants to the stress.Thus,understanding how a stressor modulates expression of the genes related with lignin biosynthesis may allow us to develop study-models to increase our knowledge on the metabolic control of lignin deposition in the cell wall.This review focuses on recent literature reporting on the main types of abiotic and biotic stresses that alter the biosynthesis of lignin in plants.     

An Integrative Analysis of the Effects of Auxin on Jasmonic Acid Biosynthesis in Arabidopsis thaliana

Auxin and jasmonic acid （JA） are two plant phytohormones that both participate in the regulation of many developmental processes. Jasmonic acid also plays important roles in plant stress response reactions. Although extensive investigations have been undertaken to study the biological functions of auxin and JA, little attention has been paid to the cross-talk between their regulated pathways. In the few available reports examining the effects of auxin on the expression of JA or JA-responsive genes, both synergetic and antagonistic results have been found. To further investigate the relationship between auxin and JA, we adopted an integrative method that combines microarray expression data with pathway information to study the behavior of the JA biosynthesis pathway under auxin treatment. Our results showed an overall downregulation of genes involved in JA biosynthesis, providing the first report of a relationship between auxin and the JA synthesis pathway in Arabidopsis seedlings.     

Genome-wide analysis and expression profiling of the phospholipase D gene family in Gossypium arboreum

The plant phospholipase D(PLD)plays versatile functions in multiple aspects of plant growth,development,and stress responses.However,until now,our knowledge concerning the PLD gene family members and their expression patterns in cotton has been limited.In this study,we performed for the first time the genome-wide analysis and expression profiling of PLD gene family in Gossypium arboretum,and finally,a total of 19 non-redundant PLD genes(GaPLDs)were identified.Based on the phylogenetic analysis,they were divided into six well-supported clades(α,β/γ,δ,ε,ζ and φ).Most of the GaPLD genes within the same clade showed the similar exon-intron organization and highly conserved motif structures.Additionally,the chromosomal distribution pattern revealed that GaPLD genes were unevenly distributed across 10 of the 13 cotton chromosomes.Segmental duplication is the major contributor to the expansion of Ga PLD gene family and estimated to have occurred from19.61 to 20.44 million years ago when a recent large-scale genome duplication occurred in cotton.Moreover,the expression profiling provides the functional divergence of GaPLD genes in cotton and provides some new light on the molecular mechanisms of GaPLDα1 and GaPLDδ2 in fiber development.     

Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.)

The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles.     

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.     

Simultaneously disrupting AtPrx2,AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem

Plant class III heme peroxidases catalyze lignin polymerization. Previous reports have shown that at least three Arabidopsis thaliana peroxidases, At Prx2, At Prx25 and At Prx71,are involved in stem lignification using T-DNA insertion mutants,atprx2, atprx25, and atprx71. Here, we generated three double mutants, atprx2/atprx25, atprx2/atprx71, and atprx25/atprx71,and investigated the impact of the simultaneous de ficiency of these peroxidases on lignins and plant growth. Stem tissue analysis using the acetyl bromide method and derivatization followed by reductive cleavage revealed improved lignin characteristics, such as lowered lignin content and increased arylglycerolb-aryl(b-O-4) linkage type, especially b-O-4 linked syringyl units, in lignin, supporting the roles of these genes in lignin polymerization. In addition, none of the double mutants oexhibited severe growth defects, such as shorter plant stature, dwar fing, or sterility, and their stems had improved cell wall degradability. This study will contribute to progress in lignin bioengineering to improve lignocellulosic biomass.     

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.     

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.     

Development and diversity of lignin patterns

Different patterns of lignified cell walls are associated with diverse functions in a variety of plant tissues. These functions rely on the stiffness and hydrophobicity that lignin polymers impart to the cell wall. The precise pattern of subcellular lignin deposition is critical for the structure–function relationship in each lignified cell type. Here, we describe the role of xylem vessels as water pipes, Casparian strips as apoplastic barriers, and the role of asymmetrically lignified endocarp b cells in exploding seed pods. We highlight similarities and differences in the genetic mechanisms underpinning local lignin deposition in these diverse cell types. By bringing together examples from different developmental contexts and different plant species, we propose that comparative approaches can benefit our understanding of lignin patterning mechanisms.

Diverse lignin patterns underpin distinct functions in different plant tissues.     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

Terpenoids are transported in the xylem sap of Norway spruce

Norway spruce is a conifer storing large amounts of terpenoids in resin ducts of various tissues. Parts of the terpenoids stored in needles can be emitted together with de novo synthesized terpenoids. Since previous studies provided hints on xylem transported terpenoids as a third emission source, we tested if terpenoids are transported in xylem sap of Norway spruce. We further aimed at understanding if they might contribute to terpenoid emission from needles. We determined terpenoid content and composition in xylem sap, needles, bark, wood and roots of field grown trees, as well as terpenoid emissions from needles. We found considerable amounts of terpenoids—mainly oxygenated compounds—in xylem sap. The terpenoid concentration in xylem sap was relatively low compared with the content in other tissues, where terpenoids are stored in resin ducts. Importantly, the terpenoid composition in the xylem sap greatly differed from the composition in wood, bark or roots, suggesting that an internal transport of terpenoids takes place at the sites of xylem loading. Four terpenoids were identified in xylem sap and emissions, but not within needle tissue, suggesting that these compounds are likely derived from xylem sap. Our work gives hints that plant internal transport of terpenoids exists within conifers; studies on their functions should be a focus of future research.