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Apoplastic iron (Fe) in roots represents an essential Fe storage pool. Reallocation of apoplastic Fe is of great importance to plants experiencing Fe deprivation, but how this reallocation process is regulated remains elusive, likely because of the highly complex cell wall structure and the limited knowledge about cell wall biosynthesis and modulation. Here, we present genetic and biochemical evidence to demonstrate that the Cdi-mediated galactosylation of rhamnogalacturonan-II (RG-II) is required for apoplastic Fe reallocation. Cdi is expressed in roots and up-regulated in response to Fe deficiency. It encodes a putative glycosyltransferase localized to the Golgi apparatus. Biochemical and mass spectrometry assays showed that Cdi catalyzes the transfer of GDP-L-galactose to the terminus of side chain A on RG-II. Disruption of Cdi essentially decreased RG-II dimerization and hence disrupted cell wall formation, as well as the reallocation of apoplastic Fe from roots to shoots. Further transcriptomic, Fourier transform infrared spectroscopy, and Fe desorption kinetic analyses coincidently suggested that Cdi mediates apoplastic Fe reallocation through extensive modulation of cell wall components and consequently the Fe adsorption capacity of the cell wall. Our study provides direct evidence demonstrating a link between cell wall biosynthesis and apoplastic Fe reallocation, thus indicating that the structure of the cell wall is important for efficient usage of the cell wall Fe pool., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Taking into account that the industrial processing of passion fruit generates significant amounts of waste (only the peels represent 51% of the total mass of the fruit), in the present study an economic analysis was conducted to evaluate industrial line viability for pectin extraction from passion fruit peels. Knowing that absolute ethanol (99.50% purity), used in the precipitation and washing steps, has a higher cost, a simulation of extractive distillation was performed using solvents ethylene glycol and glycerol, in the software Aspen Plus v.11, being possible to recover 99.63% of ethanol for both solvents. The results of the economic evaluation showed that the process using ethylene glycol has an advantage, mainly due to its higher profitability (1.13 times higher), lower production cost (94.86% of the price using glycerol), and a lower breakeven point (around 3% smaller). The financial indicators showed profitability and attractiveness for the implementation of this processing line.

Appropriate pectin deposition in cell walls is important for cell growth in plants. Rhamnogalacturonan II (RG-II) is a portion of pectic polysaccharides; its borate crosslinking is essential for maintenance of pectic networks. However, the overall process of RG-II synthesis is not fully understood. To identify a novel factor for RG-II deposition or dimerization in cell walls, we screened Arabidopsis mutants with altered boron (B)-dependent growth. The mutants exhibited alleviated disorders of primary root and stem elongation, and fertility under low B, but reduced primary root lengths under sufficient B conditions. Altered primary root elongation was associated with cell elongation changes caused by loss of function in AtTMN1 (Transmembrane Nine 1)/EMP12, which encodes a Golgi-localized membrane protein of unknown function that is conserved among eukaryotes. Mutant leaf and root dry weights were lower than those of wild-type plants, regardless of B conditions. In cell walls, AtTMN1 mutations reduced concentrations of B, RG-II specific 2-keto-3-deoxy monosaccharides, and rhamnose largely derived from rhamnogalacturonan I (RG-I), suggesting reduced RG-II and RG-I. Together, our findings demonstrate that AtTMN1 is required for the deposition of RG-II and RG-I for cell growth and suggest that pectin modulates plant growth under low B conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis ( Arabidopsis thaliana ) QUA2 , qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development., (© 2020 American Society of Plant Biologists. All rights reserved.)

Virus-induced gene silencing (VIGS) is a transient based reverse genetic tool used to elucidate the function of novel gene in N. benthamiana. In current study, 14 UDP-D-glucuronate 4-epimerase (GAE) family members were identified and their gene structure, phylogeny and expression pattern were analyzed. VIGS system was optimized for the functional characterization of NbGAE6 homologous genes in N. benthamiana. Whilst the GAE family is well-known for the interconversion of UDP-D-GlcA and UDP-D-GalA during pectin synthesis. Our results revealed that the downregulation of these genes significantly reduced the amount of GalA in the homogalacturunan which is the major component of pectin found in primary cell wall. Biphenyl assay and high performance liquid chromatography analysis (HPLC) depicted that the level of 'GalA' monosaccharide reduced to 40-51% in VIGS plants as compared to the wild type plants. Moreover, qRT-PCR also confirmed the downregulation of the NbGAE6 mRNA in VIGS plants. In all, this is the first comprehensive study of the optimization of VIGS system for the provision of rapid silencing of GAE family members in N. benthamiana, eliminating the need of stable transformants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Arabidopsis thaliana MYB43 (AtMYB43) is suggested to be involved in cell wall lignification. PtrMYB152, the Populus orthologue of AtMYB43 , is a transcriptional activator of lignin biosynthesis and vessel wall deposition. In this research, MYB43 genes from Brassica napus (rapeseed) and its parental species B. rapa and B. oleracea were molecularly characterized, which were dominantly expressed in stem and other vascular organs and showed responsiveness to Sclerotinia sclerotiorum infection. The BnMYB43 family was silenced by RNAi, and the transgenic rapeseed lines showed retardation in growth and development with smaller organs, reduced lodging resistance, fewer silique number and lower yield potential. The thickness of the xylem layer decreased by 28%; the numbers of sclerenchymatous cells, vessels, interfascicular fibers, sieve tubes and pith cells in the whole cross section of the stem decreased by 28%, 59%, 48%, 34% and 21% in these lines, respectively. The contents of cellulose and lignin decreased by 17.49% and 16.21% respectively, while the pectin content increased by 71.92% in stems of RNAi lines. When inoculated with S. sclerotiorum , the lesion length was drastically decreased by 52.10% in the stems of transgenic plants compared with WT, implying great increase in disease resistance. Correspondingly, changes in the gene expression patterns of lignin biosynthesis, cellulose biosynthesis, pectin biosynthesis, cell cycle, SA- and JA-signals, and defensive pathways were in accordance with above phenotypic modifications. These results show that BnMYB43, being a growth-defense trade-off participant, positively regulates vascular lignification, plant morphology and yield potential, but negatively affects resistance to S. sclerotiorum . Moreover, this lignification activator influences cell biogenesis of both lignified and non-lignified tissues of the whole vascular organ.

Plant polysaccharides (cellulose, hemicellulose, pectin, starch) are either direct (i.e. leaf starch) or indirect products of photosynthesis, and they belong to the most abundant organic compounds in nature. Although each of these polymers is made by a specific enzymatic machinery, frequently in different cell locations, details of their synthesis share certain common features. Thus, the production of these polysaccharides is preceded by the formation of nucleotide sugars catalyzed by fully reversible reactions of various enzymes, mostly pyrophosphorylases. These 'buffering' enzymes are, generally, quite active and operate close to equilibrium. The nucleotide sugars are then used as substrates for irreversible reactions of various polysaccharide-synthesizing glycosyltransferases ('engine' enzymes), e.g. plastidial starch synthases, or plasma membrane-bound cellulose synthase and callose synthase, or ER/Golgi-located variety of glycosyltransferases forming hemicellulose and pectin backbones. Alternatively, the irreversible step might also be provided by a carrier transporting a given immediate precursor across a membrane. Here, we argue that local equilibria, established within metabolic pathways and cycles resulting in polysaccharide production, bring stability to the system via the arrangement of a flexible supply of nucleotide sugars. This metabolic system is itself under control of adenylate kinase and nucleoside-diphosphate kinase, which determine the availability of nucleotides (adenylates, uridylates, guanylates and cytidylates) and Mg2+, the latter serving as a feedback signal from the nucleotide metabolome. Under these conditions, the supply of nucleotide sugars to engine enzymes is stable and constant, and the metabolic process becomes optimized in its load and consumption, making the system steady and self-regulated., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Pectin is a major polysaccharide component that promotes plant growth and fiber elongation in cotton. In previous studies, the galacturonosyltransferase-like (GATL) gene family has been shown to be involved in pectin synthesis. However, few studies have been performed on cotton GATL genes. Here, a total of 33, 17, and 16 GATL genes were respectively identified in Gossypium hirsutum , Gossypium raimondii , and Gossypium arboreum . In multiple plant species, phylogenetic analysis divided GATL genes into five groups named GATL-a to GATL-e, and the number of groups was found to gradually change over evolution. Whole genome duplication (WGD) and segmental duplication played a significant role in the expansion of the GATL gene family in G. hirsutum . Selection pressure analyses revealed that GATL-a and GATL-b groups underwent a great positive selection pressure during evolution. Moreover, the expression patterns revealed that most of highly expressed GhGATL genes belong to GATL-a and GATL-b groups, which have more segmental duplications and larger positive selection value, suggesting that these genes may play an important role in the evolution of cotton plants. We overexpressed GhGATL2 , GhGATL9 , GhGATL12, and GhGATL15 in Arabidopsis and silenced the GhGATL15 gene in cotton through a virus induced gene silencing assay (VIGS). The transgenic and VIGS lines showed significant differences in stem diameter, epidermal hair length, stamen length, seed size, and fiber length than the control plant. In addition, the pectin content test proved that the pectin was significantly increased in the transgenic lines and reduced in VIGS plants, demonstrating that GhGATL genes have similar functions and act on the pectin synthesis to regulate plant growth and fiber elongation. In summary, we performed a comprehensive analysis of GhGATL genes in G. hirsutum including evolution, structure and function, in order to better understand GhGATL genes in cotton for further studies.

Apple pomace, an abundant accessible source of carbohydrate platform chemicals, is refractory to cellulase degradation because of the main barrier problem of pectin constitute. A rapid and portable method for the coproduction of pectin and fermentable sugars was developed using the pretreatment of acetic acid, followed by enzymatic hydrolysis. Compared with pectinase, acetic acid pretreatment provided the highest pectin yield of 19.1% and the highest enzymatic hydrolysis yield from apple pomace. The acidic pretreated apple pomace cellulose was easily and completely hydrolyzed into fermentable sugars. More than 98.2% conversion of cellulose was achieved in a batch hydrolysis using a cellulase loading of 25 FPU/g cellulose and 10% total solids without any special strategies. A mass balance analysis showed that 95.5 g pectin and 110.2 g fermentable sugars were produced from 500-g oven-dried apple pomace. The integrated process is suggestive of environment-friendly and recyclable methods for the industrial utilization of apple pomace.

Key Message: A possible transcription factor TLP2 was identified to be involved in the regulation of HG biosynthesis in Arabidopsis seed mucilage. TLP2 can translocate into nucleus from plasma membrane by interacting with NF-YC3. The discovery of TLP2 gene function can further fulfill the regulatory network of pectin biosynthesis in Arabidopsis thaliana. Arabidopsis seed coat mucilage is an excellent model system to study the biosynthesis, function and regulation of pectin. Rhamnogalacturonan I (RG-I) and homogalacturonan (HG) are the major polysaccharides constituent of the Arabidopsis seed coat mucilage. Here, we identified a Tubby-like gene, Tubby-like protein 2 (TLP2), which was up-regulated in developing siliques when mucilage began to be produced. Ruthenium red (RR) staining of the seeds showed defective mucilage of tlp2-1 mutant after vigorous shaking compared to wild type (WT). Monosaccharide composition analysis revealed that the amount of total sugars and galacturonic acid (GalA) decreased significantly in the adherent mucilage (AM) of tlp2-1 mutant. Immunolabelling and dot immunoblotting analysis showed that unesterified HG decreased in the tlp2-1 mutant. Furthermore, TLP2 can translocate into nucleus by interacting with Nuclear Factor Y subunit C3 (NF-YC3) to function as a transcription factor. RNA-sequence and transactivation assays revealed that TLP2 could activate UDP-glucose 4-epimerase 1 (UGE1). In all, it is concluded that TLP2 could regulate the biosynthesis of HG possibly through the positive activation of UGE1.

Knockdown (KD) expression of GAlactUronosylTransferase 4 ( GAUT4 ) in switchgrass improves sugar yield and ethanol production from the biomass. The reduced recalcitrance of GAUT4 -KD transgenic biomass is associated with reduced cell wall pectic homogalacturonan and rhamnogalacturonan II content and cross-linking, and the associated increases in accessibility of cellulose to enzymatic deconstruction. To further probe the molecular basis for the reduced recalcitrance of GAUT4 -KD biomass, potential recalcitrance-related factors including the physicochemical properties of lignin and hemicellulose are investigated. We show that the transgenic switchgrass have a lower abundance of ferulate and lignin-carbohydrate complex cross-linkages, reduced amounts of residual arabinan and xylan in lignin-enriched fractions after enzymatic hydrolysis, and greater coalescence and migration of lignin after hydrothermal pretreatment in comparison to the wild-type switchgrass control. The results reveal the roles of both decreased lignin-polymer and pectin cross-links in the reduction of recalcitrance in PvGAUT4 -KD switchgrass., Competing Interests: The authors declare no competing interests.

Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase ( GAUT ) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana Here, we established a heterologous GAUT expression system in HEK293 cells and show that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed >45-fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high-molecular-weight polymeric HG (>100 kDa) in a substrate concentration-dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate- and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high-molecular-weight products., (© 2018 Amos et al.)

Sugar beet pulp pectin is an attractive source for the production of pectic oligosaccharides, an emerging class of potential prebiotics. The main aim of the present work was to investigate a new process allowing to produce pectic oligosaccharides in a continuous way by means of a cross flow enzyme membrane reactor while using a low-cost crude enzyme mixture (viscozyme). Preliminary experiments in batch and semi-continuous setups allowed to identify suitable enzyme concentrations and assessing filtration suitability. Then, in continuous experiments in the enzyme membrane reactor, residence time and substrate loading were further optimized. The composition of the obtained oligosaccharide mixtures was assessed at the molecular level for the most promising conditions and was shown to be dominated by condition-specific arabinans, rhamnogalacturonans, and galacturonans. A continuous and stable production was performed for 28.5 h at the optimized conditions, obtaining an average pectic oligosaccharide yield of 82.9 ± 9.9% (w/w), a volumetric productivity of 17.5 ± 2.1 g/L/h, and a specific productivity of 8.0 ± 1.0 g/g E/h. This work demonstrated for the first time the continuous and stable production of oligosaccharide mixtures from sugar beet pulp using enzyme membrane reactor technology in a setup suitable for upscaling.

During somatic embryogenesis (SE), explant cells undergo changes in the direction of their differentiation, which lead to diverse cell phenotypes. Although the genetic bases of the SE have been extensively studied in Arabidopsis thaliana, little is known about the chemical characteristics of the wall of the explant cells, which undergo changes in the direction of differentiation. Thus, we examined the occurrence of selected pectic and AGP epitopes in explant cells that display different phenotypes during SE. Explants examinations have been supplemented with an analysis of the ultrastructure. The deposition of selected pectic and AGP epitopes in somatic embryos was determined. Compared to an explant at the initial stage, a/embryogenic/totipotent and meristematic/pluripotent cells were characterized by a decrease in the presence of AGP epitopes, b/the presence of AGP epitopes in differentiated cells was similar, and c/an increase of analyzed epitopes was detected in the callus cells. Totipotent cells could be distinguished from pluripotent cells by: 1/the presence of the LM2 epitope in the latest one, 2/the appearance of the JIM16 epitope in totipotent cells, and 3/the more abundant presence of the JIM7 epitope in the totipotent cells. The LM5 epitope characterized the wall of the cells that were localized within the mass of embryogenic domain. The JIM8, JIM13 and JIM16 AGP epitopes appeared to be the most specific for the callus cells. The results indicate a relationship between the developmental state of the explant cells and the chemical composition of the cell walls., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Pectins are plant cell wall natural heteropolysaccharides composed mainly of α-1-4 d-galacturonic acid units, which may or may not be methyl esterified, possesses neutral sugars branching that harbor functional moieties. Physicochemical features as pH, temperature, ions concentration, and cosolute presence, affect directly the extraction yield and gelling capacity of pectins. The chemical and structural features of this polysaccharide enables its interaction with a wide range of molecules, a property that scientists profit from to form new composite matrices for target/controlled delivery of therapeutic molecules, genes or cells. Considered a prebiotic dietary fiber, pectins meetmany regulations easily, regarding health applications within the pharmaceutical industry as a raw material and as an agent for the prevention of cancer. Thus, this review lists many emergent pectin-based composite materials which will probably palliate the impact of obesity, diabetes and heart disease, aid to forestall actual epidemics, expand the ken of food additives and food products design.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Plant cell walls are composed of an intricate network of polysaccharides and proteins that varies during the developmental stages of the cell. This makes it very challenging to address the functions of individual wall components in cells, especially for highly complex glycans. Fortunately, structurally defined oligosaccharides can be used as models for the glycans, to study processes such as cell wall biosynthesis, polysaccharide deposition, protein-carbohydrate interactions, and cell-cell adhesion. Synthetic chemists have focused on preparing such model compounds, as they can be produced in good quantities and with high purity. This Review contains an overview of those plant and algal polysaccharides that have been elucidated to date. The majority of the content is devoted to detailed summaries of the chemical syntheses of oligosaccharide fragments of cellulose, hemicellulose, pectin, and arabinogalactans, as well as glycans unique to algae. Representative synthetic routes within each class are discussed in detail, and the progress in carbohydrate chemistry over recent decades is highlighted.

Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 μM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The pectin in plant cell walls consists of three domains: homogalacturonan, rhamnogalacturonan (RG)-I, and RG-II. It is predicted that around 50 different glycosyltransferases are required for their biosynthesis. Among these, the activities of only a few glycosyltransferases have been detected because pectic oligosaccharides are not readily available for use as substrates. In this study, fluorogenic pyridylaminated RG-I-backbone oligosaccharides (PA-RGs) with 3-14 degrees of polymerization (DP) were prepared. Using these oligosaccharides, the activity of RG-I:rhamnosyltransferase (RRT), involved in the biosynthesis of the RG-I backbone diglycosyl repeating units (-4GalUAα1-2Rhaα1-), was detected from the microsomes of azuki bean epicotyls. RRT was found to prefer longer acceptor substrates, PA-RGs with a DP > 7, and it does not require any metal ions for its activity. RRT is located in the Golgi and endoplasmic reticulum. The activity of RRT coincided with epicotyl growth, suggesting that RG-I biosynthesis is involved in plant growth., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The study was focused on assessing the presence of arabinogalactan proteins (AGPs) and pectins within the cell walls as well as prenyl lipids, sodium and chlorine content in leaves of Tilia x euchlora trees. The leaves that were analyzed were collected from trees with and without signs of damage that were all growing in the same salt stress conditions. The reason for undertaking these investigations was the observations over many years that indicated that there are trees that present a healthy appearance and trees that have visible symptoms of decay in the same habitat. Leaf samples were collected from trees growing in the median strip between roadways that have been intensively salted during the winter season for many years. The sodium content was determined using atomic spectrophotometry, chloride using potentiometric titration and poly-isoprenoids using HPLC/UV. AGPs and pectins were determined using immunohistochemistry methods. The immunohistochemical analysis showed that rhamnogalacturonans I (RG-I) and homogalacturonans were differentially distributed in leaves from healthy trees in contrast to leaves from injured trees. In the case of AGPs, the most visible difference was the presence of the JIM16 epitope. Chemical analyses of sodium and chloride showed that in the leaves from injured trees, the level of these ions was higher than in the leaves from healthy trees. Based on chromatographic analysis, four poly-isoprenoid alcohols were identified in the leaves of T. x euchlora. The levels of these lipids were higher in the leaves from healthy trees. The results suggest that the differences that were detected in the apoplast and symplasm may be part of the defensive strategy of T. x euchlora trees to salt stress, which rely on changes in the chemical composition of the cell wall with respect to the pectic and AGP epitopes and an increased synthesis of prenyl lipids.

Background: Processing of fresh produce exposes cut surfaces of plant cell walls that then become vulnerable to human foodborne pathogen attachment and contamination, particularly by Salmonella enterica. Plant cell walls are mainly composed of the polysaccharides cellulose, pectin and hemicelluloses (predominantly xyloglucan). Our previous work used bacterial cellulose-based plant cell wall models to study the interaction between Salmonella and the various plant cell wall components. We demonstrated that Salmonella attachment was favoured in the presence of pectin while xyloglucan had no effect on its attachment. Xyloglucan significantly increased the attachment of Salmonella cells to the plant cell wall model only when it was in association with pectin. In this study, we investigate whether the plant cell wall polysaccharides mediate Salmonella attachment to the bacterial cellulose-based plant cell wall models through specific carbohydrate interactions or through the effects of carbohydrates on the physical characteristics of the attachment surface., Results: We found that none of the monosaccharides that make up the plant cell wall polysaccharides specifically inhibit Salmonella attachment to the bacterial cellulose-based plant cell wall models. Confocal laser scanning microscopy showed that Salmonella cells can penetrate and attach within the tightly arranged bacterial cellulose network. Analysis of images obtained from atomic force microscopy revealed that the bacterial cellulose-pectin-xyloglucan composite with 0.3 % (w/v) xyloglucan, previously shown to have the highest number of Salmonella cells attached to it, had significantly thicker cellulose fibrils compared to other composites. Scanning electron microscopy images also showed that the bacterial cellulose and bacterial cellulose-xyloglucan composites were more porous when compared to the other composites containing pectin., Conclusions: Our study found that the attachment of Salmonella cells to cut plant cell walls was not mediated by specific carbohydrate interactions. This suggests that the attachment of Salmonella strains to the plant cell wall models were more dependent on the structural characteristics of the attachment surface. Pectin reduces the porosity and space between cellulose fibrils, which then forms a matrix that is able to retain Salmonella cells within the bacterial cellulose network. When present with pectin, xyloglucan provides a greater surface for Salmonella cells to attach through the thickening of cellulose fibrils.

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.

Background: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth., Results: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content., Conclusions: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

Pectins are complex polysaccharides that contain acidic sugars and are major determinants of the cohesion, adhesion, extensibility, porosity and electrostatic potential of plant cell walls. Recent evidence has solidified their positions as key regulators of cellular growth and tissue morphogenesis, although important details of how they achieve this regulation are still missing. Pectins are also hypothesized to function as ligands for wall integrity sensors that enable plant cells to respond to intrinsic defects in wall biomechanics and to wall degradation by attacking pathogens. This update highlights recent advances in our understanding of the biosynthesis of pectins, how they are delivered to the cell surface and become incorporated into the cell wall matrix and how pectins are modified over time in the apoplast. It also poses unanswered questions for further research into this enigmatic but essential class of carbohydrate polymers., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Figures of persimmons for the world's top ten persimmon producing countries are about 4000,000 tons in 2011 and are increasing every year according to FAO statistics. However, there is not any report on pectin production by microbial with persimmon peel as the source. Optimization of fermentation conditions of pectin production from Aspergillus terreus in submerged culture and partial characterization of pectin were carried out in the work. An optimum fermentation condition for pectin production was obtained through a central composite rotatable design in response surface methodology as follows: fermentation time, 30.09 h, temperature, 25.00 °C and the initial pH in the fermentation medium, 6.90, respectively and the pectin yield reached the maximal value 0.449 g/g. Persimmon peel pectin had highly methoxylated (62.51%), high galacturonic acid content (82.28%) than citrus pectin, and was classified as the highly methoxylated pectin, the results indicated that persimmon peel had potential good resources for pectin production. The investigation can make it available to utilize persimmon peel to produce high methoxyl pectin for food industry, pharmacy and cosmetic manufacture., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Based on the fungus Penicillium verruculosum, we created strains with a complex of extracellular enzymes that contains both cellulolytic enzymes of the fungus and heterologous pectin lyase A from P. canescens and endo- 1,4-α-polygalacturonase from Aspergillus niger. The endopolygalacturonase and pectin lyase activities of enzyme preparations obtained from culture media of the producer strains reached 46-53 U/mg of protein and 1.3-2.3 U/mg of protein, respectively. The optimal temperature and pH values for recombinant pectin lyase and endopolygalacturonase corresponded to those described in the literature for these enzymes. The content of heterologous endopolygalacturonase and pectin lyase in the studied enzyme preparations was 4-5% and 23% of the total protein content, respectively. The yield of reducing sugars upon the hydrolysis of sugar beet and apple processing wastes with the most efficient preparation was 41 and 71 g/L, respectively, which corresponded to a polysaccharide conversion of 49% and 65%. Glucose was the main product of the hydrolysis of sugar beet and apple processing wastes.

Background: Cold storage induces chilling injury (CI) disorders in peach fruit (woolliness/mealiness, flesh browning and reddening/bleeding) manifested when ripened at shelf life. To gain insight into the mechanisms underlying CI, we analyzed the transcriptome of 'Oded' (high tolerant) and 'Hermoza' (relatively tolerant to woolliness, but sensitive to browning and bleeding) peach cultivars at pre-symptomatic stages. The expression profiles were compared and validated with two previously analyzed pools (high and low sensitive to woolliness) from the Pop-DG population. The four fruit types cover a wide range of sensitivity to CI. The four fruit types were also investigated with the ROSMETER that provides information on the specificity of the transcriptomic response to oxidative stress., Results: We identified quantitative differences in a subset of core cold responsive genes that correlated with sensitivity or tolerance to CI at harvest and during cold storage, and also subsets of genes correlating specifically with high sensitivity to woolliness and browning. Functional analysis indicated that elevated levels, at harvest and during cold storage, of genes related to antioxidant systems and the biosynthesis of metabolites with antioxidant activity correlates with tolerance. Consistent with these results, ROSMETER analysis revealed oxidative stress in 'Hermoza' and the progeny pools, but not in the cold resistant 'Oded'. By contrast, cold storage induced, in sensitivity to woolliness dependant manner, a gene expression program involving the biosynthesis of secondary cell wall and pectins. Furthermore, our results indicated that while ethylene is related to CI tolerance, differential auxin subcellular accumulation and signaling may play a role in determining chilling sensitivity/tolerance. In addition, sugar partitioning and demand during cold storage may also play a role in the tolerance/sensitive mechanism. The analysis also indicates that vesicle trafficking, membrane dynamics and cytoskeleton organization could have a role in the tolerance/sensitive mechanism. In the case of browning, our results suggest that elevated acetaldehyde related genes together with the core cold responses may increase sensitivity to browning in shelf life., Conclusions: Our data suggest that in sensitive fruit a cold response program is activated and regulated by auxin distribution and ethylene and these hormones have a role in sensitivity to CI even before fruit are cold stored.

Background: Since tomato is an important food component, it is imperative to enhance its yield against the activities of many devastating fungal pathogens such as Alternaria alternata. The exploitation of plant innate resistance by cultivation of resistant varieties is an effective measure in this regard. In the present study, 28 tomato varieties were tested against 32 A. alternata isolates, and representative varieties were further evaluated to determine the extent and basis of their antifungal resistance., Results: A significant increase (104.7%) in polyphenols was recorded in the resistant variety Dinaar compared with the susceptible variety Red Tara. Dinaar also exhibited 100% enhancement of alkaloids and terpenoids along with a 30.7% increase in cell wall hemicellulose content. Significant differences were found in physical barriers (cellulose, lignin and pectin) of the representative varieties when stained tissue sections were subjected to colorimetric analysis. Similarly, polyphenol oxidase, peroxidase and phenylalanine ammonia lyase showed increases of 78.37, 114.67 and 125.11% respectively in the resistant variety. Higher expression of glucanase genes was evident from native gel analysis, in which not only the number of isozymes but also the quantity of individual isozymes was significantly increased., Conclusion: The resistant variety Dinaar had strong antifungal resistance and can therefore be recommended as suitable for cultivation in the agricultural system of Pakistan., (© 2013 Society of Chemical Industry.)

The expression of the rolB gene was found to increase the pectic yield in Rubia cordifolia cells, while the rolC gene inhibited the pectin production, which correlated with its expression level. The expression of the rolA, rolB, and rolC genes led to an increase in the content of arabinogalactan (AG) in cells. The increase in the expression of the rolB and rolC genes resulted in a more significant reduction in the content of arabinose residues in pectin, which was accompanied by an increased activity of alpha-L-arabinofuranosidase in cells. Moreover, the amount of galactose residues in pectin increased with the enhancement of the rolB expression due to a decrease in the activity of beta-galactosidase in cells. The content of galacturonic acid residues in pectin from transgenic cultures increased in the following order: rolC < rolB < rolA. The amount of arabinose residues in AG decreased independently of the gene type. The amount of arabinose residues in AG was found to be considerably reduced when the rolB expression level was increased.

Cell wall biosynthesis is indispensable for pollen tube growth. Despite its importance to sexual reproduction, the molecular mechanisms of pollen tube wall biosynthesis remain poorly understood. Here, we report functional characterization of two putative Arabidopsis galacturonosyltransferase genes, GAUT13 and GAUT14, which are essential for pollen tube growth. GAUT13 and GAUT14 encode the proteins that share a high amino acid sequence identity and are located in the Golgi apparatus. The T-DNA insertion mutants, gaut13 and gaut14, did not exhibit any observable defects, but the gaut13 gaut14 double mutants were defective in pollen tube growth; 35.2-37.3% pollen tubes in the heterozygous double mutants were swollen and defective in elongation. The outer layer of the cell wall did not appear distinctly fibrillar in the double mutant pollen tubes. Furthermore, distribution of homogalacturonan labeled with JIM5 and JIM7 in the double mutant pollen tube wall was significantly altered compared to wild-type. Our results suggest that GAUT13 and GAUT14 function redundantly in pollen tube growth, possibly through participation in pectin biosynthesis of the pollen tube wall.

Recent progress in the identification and characterization of pectin biosynthetic proteins and the discovery of pectin domain-containing proteoglycans are changing our view of how pectin, the most complex family of plant cell wall polysaccharides, is synthesized. The functional confirmation of four types of pectin biosynthetic glycosyltransferases, the identification of multiple putative pectin glycosyl- and methyltransferases, and the characteristics of the GAUT1:GAUT7 homogalacturonan biosynthetic complex with its novel mechanism for retaining catalytic subunits in the Golgi apparatus and its 12 putative interacting proteins are beginning to provide a framework for the pectin biosynthetic process. We propose two partially overlapping hypothetical and testable models for pectin synthesis: the consecutive glycosyltransferase model and the domain synthesis model.

The present study deals with the production of cold active polygalacturonase (PGase) by submerged fermentation using Thalassospira frigidphilosprofundus, a novel species isolated from deep waters of Bay of Bengal. Nonlinear models were applied to optimize the medium components for enhanced production of PGase. Taguchi orthogonal array design was adopted to evaluate the factors influencing the yield of PGase, followed by the central composite design (CCD) of response surface methodology (RSM) to identify the optimum concentrations of the key factors responsible for PGase production. Data obtained from the above mentioned statistical experimental design was used for final optimization study by linking the artificial neural network and genetic algorithm (ANN-GA). Using ANN-GA hybrid model, the maximum PGase activity (32.54 U/mL) was achieved at the optimized concentrations of medium components. In a comparison between the optimal output of RSM and ANN-GA hybrid, the latter favored the production of PGase. In addition, the study also focused on the determination of factors responsible for pectin hydrolysis by crude pectinase extracted from T. frigidphilosprofundus through the central composite design. Results indicated 80% degradation of pectin in banana fiber at 20 °C in 120 min, suggesting the scope of cold active PGase usage in the treatment of raw banana fibers.

Arabinogalactan proteins (AGPs), are a group of highly glycosylated proteins that are found throughout the plant kingdom. To date, glycosyltransferases that glycosylate AGP backbone have remained largely unknown. In this study, a gene (GhGalT1) encoding a putative β-1,3-galactosyltransferase (GalT) was identified in cotton. GhGalT1, belonging to CAZy GT31 family, is the type II membrane protein that contains an N-terminal transmembrane domain and a C-terminal galactosyltransferase functional domain. A subcellular localization assay demonstrated that GhGalT1 was localized in the Golgi apparatus. RT-PCR analysis revealed that GhGalT1 was expressed at relatively high levels in hypocotyls, roots, fibers and ovules. Overexpression of GhGalT1 in Arabidopsis promoted plant growth and metabolism. The transgenic seedlings had much longer primary roots, higher chlorophyll content, higher photosynthetic efficiency, the increased biomass, and the enhanced tolerance to exogenous D-arabinose and D-galactose. In addition, gas chromatography (GC) analysis of monosaccharide composition of cell wall fractions showed that pectin was changed in the transgenic plants, compared with that of wild type. Three genes (GAUT8, GAUT9 and xgd1) involved in pectin biosynthesis were dramatically up-regulated in the transgenic lines. These data suggested that GhGalT1 may be involved in regulation of pectin biosynthesis required for plant development.

β-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, β-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but β-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased β-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher β-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto β-1,4-galactopentaose. GALS1 specifically formed β-1,4-galactosyl linkages and could add successive β-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as β-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.

Whole genome transcript correlation-based approaches have been shown to be enormously useful for candidate gene detection. Consequently, simple Pearson correlation has been widely applied in several web based tools. That said, several more sophisticated methods based on e.g. mutual information or Bayesian network inference have been developed and have been shown to be theoretically superior but are not yet commonly applied. Here, we propose the application of a recently developed statistical regression technique, the LASSO, to detect novel candidates from high throughput transcriptomic datasets. We apply the LASSO to a tissue specific dataset in the model plant Arabidopsis thaliana to identify novel players in Arabidopsis thaliana seed coat mucilage synthesis. We built LASSO models based on a list of genes known to be involved in a sub-pathway of Arabidopsis mucilage synthesis. After identifying a putative transcription factor, we verified its involvement in mucilage synthesis by obtaining knock-out mutants for this gene. We show that a loss of function of this putative transcription factor leads to a significant decrease in mucilage pectin.

Polygalacturonase is an enzyme that hydrolyzes external and internal α (1-4) glycosidic bonds of pectin to decrease the viscosity of fruits juices and vegetable purees. Several bacterial strains were isolated from soil and rotten vegetables and screened for polygalacturonase production. The strain which produced maximum polygalacturonase was identified Bacillus licheniformis on the basis of taxonomic studies and 16S rDNA analysis. The isolated bacterial strain produced maximum polygalacturonase at 37 °C after 48 h of fermentation. Among various carbon sources apple pectin (1.0%) showed maximum enzyme production. Different agro industrial wastes were also used as substrate in batch fermentation and it was found that wheat bran is capable of producing high yield of enzyme. Maximum polygalacturonase production was obtained by using yeast extract (0.3%) as a nitrogen source. It was observed that B. licheniformis KIBGE IB-21 is capable of producing 1015 U/mg of polygalacturonase at neutral pH., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Two Chinese jujube (Zizyphus jujuba) cultivars ('Huanghua' and 'Zhanhua') at unripe and ripe stages were investigated. During ripening, the weight, pH, hardness, and chewiness of both cultivars decreased while titratable acidity, total soluble solid content, and pectin contents increased. More than 75% pectins of the fruits were water-soluble pectin (WSP). Pectins shared the common major compositions of Gal, Rha and GalUA. For both cultivars, most of the chain widths were between 47 and 70 nm for unripe while less than 40 nm for ripe jujubes. Compared to unripe fruits, all pectins of ripe fruits had less percentage of wide and long pectin chains. All the pectin contents of cultivar 'Huanghua' changed to a degree greater than that of cultivar 'Zhanhua' during ripening. Changes of pectin nanostructure and neutral sugar composition might be responsible for the major physicochemical properties of jujubes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise, in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.

Ethylene-responsive transcription factor (ERF) family genes, which are involved in regulation of metabolic pathways and/or are useful for metabolic engineering, were investigated in the cultured cells of Arabidopsis thaliana. The pectin content in the gelatinous precipitates after the ethanol precipitation of extracts derived from calli of a transgenic cell line, A17, overexpressing an ERF gene (At1g44830), increased in comparison with the control. Expression of genes involved in pectin biosynthesis was up-regulated in the A17 calli. Overexpression of the ERF gene coordinately activates the pectin biosynthetic pathway genes and increases the content of pectin. These results therefore will be useful as a genetic resource for engineering pectin biosynthesis in plants.

Background: Like sweet orange (Citrus sinensis), tangerine (Citrus reticulata) is another citrus crop grown widely throughout the world. However, whether it shares a common mechanism with sweet orange in forming a given mastication trait is still unclear. In this study, three 'Nanfeng' tangerine cultivars, 'Yangxiao-26' ('YX-26') with inferior mastication trait, elite 'YX-26' with moderate mastication trait and 'Miguang' ('MG') with superior mastication trait, were selected to investigate the formation mechanism of mastication trait., Results: 'MG' had the lowest contents of total pectin, protopectin and lignin and the highest gene expression levels of citrus polygalacturonase (PG) and pectin methylesterase (PME) at the end of fruit ripening, whereas 'YX-26' had the lowest water-soluble pectin (WSP) content, the highest lignin content and the lowest PG and PME expression levels. The contents of cellulose and hemicellulose were similar among the three tangerines., Conclusion: The fruit mastication trait of C. reticulata was determined by the proportions of WSP and protopectin as well as lignin content, not by cellulose and hemicellulose contents. Pectin content could be a major contribution to the feeling of mastication trait, while PG and PME exhibited an important role in forming a given mastication trait according to the present results as well as previous results for C. sinensis., (Copyright © 2011 Society of Chemical Industry.)

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

Efficient and cost-effective conversion of plant biomass to usable forms of energy requires a thorough understanding of cell wall biosynthesis, modification and degradation. To elucidate these processes, we assessed the expression dynamics during enzymatic removal and regeneration of rice cell walls in suspension cells over time. In total, 928 genes exhibited significant up-regulation during cell wall removal, whereas, 79 genes were up-regulated during cell wall regeneration. Both gene sets are enriched for kinases, transcription factors and genes predicted to be involved in cell wall-related functions. Integration of the gene expression datasets with a catalog of known and/or predicted biochemical pathways from rice, revealed metabolic and hormonal pathways involved in cell wall degradation and regeneration. Rice lines carrying Tos17 mutations in genes up-regulated during cell wall removal exhibit dwarf phenotypes. Many of the genes up-regulated during cell wall development are also up-regulated in response to infection and environmental perturbations indicating a coordinated response to diverse types of stress.

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In plant cells, boron (B) occurs predominantly as a borate ester associated with rhamnogalacturonan II (RG-II), but the function of this B-RG-II complex has yet to be investigated. 3-Deoxy-D-manno-2-octulosonic acid (KDO) is a specific component monosaccharide of RG-II. Mutant plants defective in KDO biosynthesis are expected to have altered RG-II structure, and would be useful for studying the physiological function of the B-RG-II complex. Here, we characterized Arabidopsis CTP:KDO cytidylyltransferase (CMP-KDO synthetase; CKS), the enzyme activating KDO as a nucleotide sugar prior to its incorporation into RG-II. Our analyses localized the Arabidopsis CKS protein to mitochondria. The Arabidopsis CKS gene occurs as a single-copy gene in the genome, and we could not obtain cks null mutants from T-DNA insertion lines. Analysis using +/cks heterozygotes in the quartet1 background demonstrated that the cks mutation rendered pollen infertile through the inhibition of pollen tube elongation. These results suggest that KDO is an indispensable component of RG-II, and that the complete B-RG-II complex is essential for the cell wall integrity of rapidly growing tissues.

A novel biotechnological process for the production of high-quality flax fibers was developed. In this process, decorticated fibers from green flax were washed with 0.5% soda solution and treated with the pectinolytic strain Geobacillus thermoglucosidasius PB94A. Before drying the fibers, they were treated with the textile softener Adulcinol BUN. If the fibers contained contaminant shives, a bleaching step with hydrogen peroxide was performed before the softener treatment. In experiments where fibers were treated by the new process, and in which the bacterial solutions were reused seven times, the fiber quality was similar in all batches. The resolution of the treated fibers was 2.7 ± 0.4 and the fineness was 11.1 ± 1.1 dtex, while the starting material had a resolution of 7.3 and a fineness of 37 dtex. The new biotechnological treatment eliminates the weather-associated risks of the traditional fiber retting completely and produces consistently high-quality fibers that can be used to produce fine linen yarns.

Pectins are complex polysaccharides that are essential components of the plant cell wall. In this study, a novel putative Arabidopsis S-adenosyl-L-methionine (SAM)-dependent methyltransferase, termed QUASIMODO 3 (QUA3, At4g00740), has been characterized and it was demonstrated that it is a Golgi-localized, type II integral membrane protein that functions in methylesterification of the pectin homogalacturonan (HG). Although transgenic Arabidopsis seedlings with overexpression, or knock-down, of QUA3 do not show altered phenotypes or changes in pectin methylation, this enzyme is highly expressed and abundant in Arabidopsis suspension-cultured cells. In contrast, in cells subjected to QUA3 RNA interference (RNAi) knock-down there is less pectin methylation as well as altered composition and assembly of cell wall polysaccharides. Taken together, these observations point to a Golgi-localized QUA3 playing an essential role in controlling pectin methylation and cell wall biosynthesis in Arabidopsis suspension cell cultures.

L-galactose (L-Gal), a monosaccharide involved in L-ascorbate and rhamnogalacturonan II (RG-II) biosynthesis in plants, is produced in the cytosol by a GDP-D-mannose 3,5-epimerase (GME). It has been recently reported that the partial inactivation of GME induced growth defects affecting both cell division and cell expansion (Gilbert, L., Alhagdow, M., Nunes-Nesi, A., Quemener, B., Guillon, F., Bouchet, B., Faurobert, M., Gouble, B., Page, D., Garcia, V., Petit, J., Stevens, R., Causse, M., Fernie, A. R., Lahaye, M., Rothan, C., and Baldet, P. (2009) Plant J. 60, 499-508). In the present study, we show that the silencing of the two GME genes in tomato leaves resulted in approximately a 60% decrease in terminal L-Gal content in the side chain A of RG-II as well as in a lower capacity of RG-II to perform in muro cross-linking. In addition, we show that unlike supplementation with L-Gal or ascorbate, supplementation of GME-silenced lines with boric acid was able to restore both the wild-type growth phenotype of tomato seedlings and an efficient in muro boron-mediated cross-linking of RG-II. Our findings suggest that developmental phenotypes in GME-deficient lines are due to the structural alteration of RG-II and further underline the crucial role of the cross-linking of RG-II in the formation of the pectic network required for normal plant growth and development.

Plant cell wall polysaccharides are amongst the most complex, heterogeneous and abundant bio-molecules on earth. This makes the biosynthetic enzymes, namely the glycosyltransferases and polysaccharide synthases, important research targets in plant science and biotechnology. As an initial step to characterize At4g01220, a putative Arabidopsis thaliana encoding glycosyltransferases in CAZy GT-family-77 that is similar to three known xylosyltransferases involved in the biosynthesis of the pectic polysaccharide, rhamnogalacturonan II, we conducted an expression analysis. In transgenic Arabidopsis thaliana plants containing a fusion between the At4g01220 promoter and the gusA reporter gene we found the expression to be spatially and developmentally regulated. Analysis of Nicotiana benthamiana transfected with the At2g01220::YFP fusion protein revealed that the fusion protein resided in a Brefeldin A-sensitive compartment consistent with a sub-cellular location in the Golgi apparatus. In addition, in silico expression analysis from the Genevestigator database revealed that At4g01220 was up-regulated upon treatment with isoxaben, an inhibitor of cellulose synthesis, which, together with a co-expression analysis that identified a number of plant cell wall co-related biosynthetic genes, suggests involvement in cell wall biosynthesis with pectin being a prime candidate. The data presented provide insights into the expression, sub-cellular location and regulation of At4g01220 under various conditions and may help elucidate its specific function., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

In flowering plants, the growth of pollen tubes is essential for the delivery of sperm to the egg cells. Although many factors (including cell-wall properties) are involved in this process, little is known about the underlying molecular mechanisms that regulate the growth of pollen tubes. We report here the characterization of an Arabidopsis mutant male gametophyte defective 4 (mgp4) that is severely defective in pollen tube growth. The mgp4 mutation also impairs root growth of pollen-rescued mgp4 mutant plants generated by expressing MGP4 cDNA under the control of a pollen grain/tube-specific promoter. The MGP4 gene encodes a putative xylosyltransferase and is expressed in many organs/tissues, including pollen tubes and roots. MGP4 protein expressed in Pichia pastoris exhibited xylosyltransferase activity and transferred d-xylose onto l-fucose. The pectic polysaccharide rhamnogalacturonan II (RG-II), isolated from 7-day-old pollen-rescued mutant seedlings, exhibited a 30% reduction in 2-O-methyl d-xylose residues. Furthermore, an exogenous supply of boric acid enhanced RG-II dimer formation and partially restored the root growth of the pollen-rescued mutant seedlings. Taken together, these results suggest that MGP4 plays important roles in pollen tube and root growth by acting as a xylosyltransferase involved in the biosynthesis of pectic RG-II., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)