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Salicornia europaea is among the most salt-tolerant of plants, and is widely distributed in non-tropical regions. Here, we investigated whether maternal habitats can influence different responses in physiology and anatomy depending on environmental conditions. We studied the influence of maternal habitat on S. europaea cell anatomy, pectin content, biochemical and enzymatic modifications under six different salinity treatments of a natural-high-saline habitat (~ 1000 mM) (Ciechocinek [Cie]) and an anthropogenic-lower-saline habitat (~ 550 mM) (Inowrocław [Inw]). The Inw population showed the highest cell area and roundness of stem water storing cells at high salinity and had the maximum proline, carotenoid, protein, catalase activity within salt treatments, and a maximum high and low methyl esterified homogalacturonan content. The Cie population had the highest hydrogen peroxide and peroxidase activity along with the salinity gradient. Gene expression analysis of SeSOS1 and SeNHX1 evidenced the differences between the studied populations and suggested the important role of Na + sequestration into the vacuoles. Our results suggest that the higher salt tolerance of Inw may be derived from a less stressed maternal salinity that provides a better adaptive plasticity of S. europaea. Thus, the influence of the maternal environment may provide physiological and anatomical modifications of local populations., (© 2022. The Author(s).)

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal d-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume d-GalUA with the highest reported maximum specific rate of 0.23 g g CDW -1 h -1 in synthetic minimal medium when glycerol was added. By means of a 13 C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two 'respiratory' carbon sources to ethanol and demonstrates a fast and complete consumption of d-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Floral patterning is a complex task. Various organs and tissues must be formed to fulfill reproductive functions. Flower development has been studied, mainly looking for master regulators. However, downstream changes such as the cell wall composition are relevant since they allow cells to divide, differentiate, and grow. In this review, we focus on the main components of the primary cell wall-cellulose, hemicellulose, and pectins-to describe how enzymes involved in the biosynthesis, modifications, and degradation of cell wall components are related to the formation of the floral organs. Additionally, internal and external stimuli participate in the genetic regulation that modulates the activity of cell wall remodeling proteins.

Dissecting the genetic mechanisms underlying dioecy (i.e., separate female and male individuals) is critical for understanding the evolution of this pervasive reproductive strategy. Nonetheless, the genetic basis of sex determination remains unclear in many cases, especially in systems where dioecy has arisen recently. Within the economically important plant genus Solanum (∼2,000 species), dioecy is thought to have evolved independently at least 4 times across roughly 20 species. Here, we generate the first genome sequence of a dioecious Solanum and use it to ascertain the genetic basis of sex determination in this species. We de novo assembled and annotated the genome of Solanum appendiculatum (assembly size: ∼750 Mb scaffold N50: 0.92 Mb; ∼35,000 genes), identified sex-specific sequences and their locations in the genome, and inferred that males in this species are the heterogametic sex. We also analyzed gene expression patterns in floral tissues of males and females, finding approximately 100 genes that are differentially expressed between the sexes. These analyses, together with observed patterns of gene-family evolution specific to S. appendiculatum, consistently implicate a suite of genes from the regulatory network controlling pectin degradation and modification in the expression of sex. Furthermore, the genome of a species with a relatively young sex-determination system provides the foundational resources for future studies on the independent evolution of dioecy in this clade., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Angiosperm cell adhesion is dependent on interactions between pectin polysaccharides which make up a significant portion of the plant cell wall. Cell adhesion in Arabidopsis may also be regulated through a pectin-related signaling cascade mediated by a putative O-fucosyltransferase ESMERALDA1 (ESMD1), and the Epidermal Growth Factor (EGF) domains of the pectin binding Wall associated Kinases (WAKs) are a primary candidate substrate for ESMD1 activity. Genetic interactions between WAKs and ESMD1 were examined using a dominant hyperactive allele of WAK2, WAK2cTAP, and a mutant of the putative O-fucosyltransferase ESMD1. WAK2cTAP expression results in a dwarf phenotype and activation of the stress response and reactive oxygen species (ROS) production, while esmd1 is a suppressor of a pectin deficiency induced loss of adhesion. Here we find that esmd1 suppresses the WAK2cTAP dwarf and stress response phenotype, including ROS accumulation and gene expression. Additional analysis suggests that mutations of the potential WAK EGF O-fucosylation site also abate the WAK2cTAP phenotype, yet only evidence for an N-linked but not O-linked sugar addition can be found. Moreover, a WAK locus deletion allele has no effect on the ability of esmd1 to suppress an adhesion deficiency, indicating WAKs and their modification are not a required component of the potential ESMD1 signaling mechanism involved in the control of cell adhesion. The WAK locus deletion does however affect the induction of ROS but not the transcriptional response induced by the elicitors Flagellin, Chitin and oligogalacturonides (OGs)., Competing Interests: The authors have declared that no competing interests exist.

Temperature is one of the critical factors affecting gene expression in bacteria. Despite the general interest in the link between bacterial phenotypes and environmental temperature, little is known about temperature-dependent gene expression in plant pathogenic Pectobacterium atrosepticum , a causative agent of potato blackleg and tuber soft rot worldwide. In this study, twenty-nine P. atrosepticum SCRI1043 thermoregulated genes were identified using Tn5-based transposon mutagenesis coupled with an inducible promotorless gusA gene as a reporter. From the pool of 29 genes, 14 were up-regulated at 18 °C, whereas 15 other genes were up-regulated at 28 °C. Among the thermoregulated loci, genes involved in primary bacterial metabolism, membrane-related proteins, fitness-corresponding factors, and several hypothetical proteins were found. The Tn5 mutants were tested for their pathogenicity in planta and for features that are likely to remain important for the pathogen to succeed in the (plant) environment. Five Tn5 mutants expressed visible phenotypes differentiating these mutants from the phenotype of the SCRI1043 wild-type strain. The gene disruptions in the Tn5 transposon mutants caused alterations in bacterial generation time, ability to form a biofilm, production of lipopolysaccharides, and virulence on potato tuber slices. The consequences of environmental temperature on the ability of P. atrosepticum to cause disease symptoms in potato are discussed.

A 91 kDa heteropolysaccharide (F2) was isolated from Mangifera indica fruit via extraction with H 2 O, purification by C 2 H 5 OH, starch removal and ion exchange chromatography. This polymer was made up mostly of Ara, Gal, Glc, Rha, Xyl, and GalA in a 37: 29: 9:3:2:19 molar proportion. It inherited a small backbone containing GalpA and Rhap units substituted with very large side chains containing differently linked Ara and Gal units plus esterified gallic acid (GA) residue. Several enzymes generated oligosaccharides including (i) Ara 2-10 Ac 6 - 22 , (ii) Gal 1-8 Ac 5 - 26 and (iii) GA 1 Gal 1 Ac 7 were characterized. This polysaccharide, which showed dose dependent antioxidant activity, exhibited synergism with gallic acid, and formed a complex (K = 1.2 × 10 6  M -1 ) with β-lactoglobulin. Accordingly, H 2 O treatment produces a polysaccharide with desired biochemical properties; this could be effective in designing innovative functional food with flexible makeup., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.)

Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato ( Solanum lycopersicum ), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein., (© 2020 American Society of Plant Biologists. All rights reserved.)

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

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.)

Poplar is a superior forestation species with high adaptability. The woody tissue of poplar is mainly derived from cell wall. Cell wall formation determines cell shape and woody growth. Pectin is rich in primary cell wall, but it is also involved in the regulation of wood formation. In our study, we cloned a gene from poplar (Populus tomentos), designed as PtoPME35, which encodes a putative pectin methylesterase. PtoPME35 has higher sequence similarity with Arabidopsis AtPME35. Gene expression analysis shows that PtoPME35 has a constitutive expression pattern in multiple tissues, with the highest expression in stem. Subcellular localization result indicates that PtoPME35 is localized to the cell wall. To elucidate the biological function of PtoPME35 in vivo, we generated overexpression plants in poplar and Arabidopsis. The degree of pectin methylesterification is decreased in PtoPME35-overexpressing transgenic poplar, although no obvious phenotypes were displayed. In PtoPME35-overexpressing Arabidopsis plants, stomatal opening is inhibited and water loss rate is decreased under the drought condition. Moreover, the expression levels of drought-stress responsive genes were higher with mannitol treatment in PtoPME35-overexpressing Arabidopsis plants than in wild type controls. Accordingly, these results suggest that PtoPME35 may regulate osmotic stress responses by modulating stomatal functions., Competing Interests: Declaration of competing interest We declare that we have no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

On imbibition, Arabidopsis ( Arabidopsis thaliana ) seeds release polysaccharides from their epidermal cells that form a two-layered hydrogel, termed mucilage. Analysis of a publicly available data set of outer seed mucilage traits of over 300 accessions showed little natural variation in composition. This mucilage is almost exclusively made up of rhamnogalacturonan I (RGI), highlighting the importance of this pectin for outer mucilage function. In a genome-wide association study, observed variations in polymer amount and macromolecular characteristics were linked to several genome polymorphisms, indicating the complexity of their genetic regulation. Natural variants with high molar mass were associated with a gene encoding a putative glycosyltransferase called MUCILAGE-RELATED70 (MUCI70). muci70 insertion mutants produced many short RGI polymers that were highly substituted with xylan, confirming that polymorphism in this gene can affect RGI polymer size. A second gene encoding a putative copper amine oxidase of clade 1a (CuAOα1) was associated with natural variation in the amount of RGI present in the outer mucilage layer; cuaoα1 mutants validated its role in pectin production. As the mutant phenotype is unique, with RGI production only impaired for outer mucilage, this indicates that CuAOα1 contributes to a further mechanism controlling mucilage synthesis., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Chrysanthemum ( Chrysanthemum morifolium (Ramat.) Kitamura) plants have great ornamental value, but their flowers can also be a source of pollen contamination. Previously, morphological and cytological studies have shown that anthers of some chrysanthemum cultivars such as 'Qx-115' fail to dehisce, although the underlying mechanism is largely unknown. In this study, we investigated the molecular basis of anther indehiscence in chrysanthemum via transcriptome analysis of a dehiscent cultivar ('Qx-097') and an indehiscent cultivar ('Qx-115'). We also measured related physiological indicators during and preceding the period of anther dehiscence. Our results showed a difference in pectinase accumulation and activity between the two cultivars during dehiscence. Detection of de-esterified pectin and highly esterified pectin in anthers during the period preceding anther dehiscence using LM19 and LM20 monoclonal antibodies showed that both forms of pectin were absent in the stomium region of 'Qx-097' anthers but were abundant in that of 'Qx-115' anthers. Analysis of transcriptome data revealed a significant difference in the expression levels of two transcription factor-encoding genes, CmLOB27 and CmERF72 , between 'Qx-097' and 'Qx-115' during anther development. Transient overexpression of CmLOB27 and CmERF72 separately in tobacco leaves promoted pectinase biosynthesis. We conclude that CmLOB27 and CmERF72 are involved in the synthesis of pectinase, which promotes the degradation of pectin. Our results lay a foundation for further investigation of the role of CmLOB27 and CmERF72 transcription factors in the process of anther dehiscence in chrysanthemum.

Stomatal pores are vital for the diffusion of gasses into and out of land plants and are, therefore, gatekeepers for photosynthesis and transpiration. Although much published literature has described the intercellular signaling and transcriptional regulators involved in early stomatal development, little is known about the cellular details of the local separation between sister guard cells that give rise to the stomatal pore or how formation of this pore is achieved. Using three-dimensional (3D) time-lapse imaging, we found that stomatal pore formation in Arabidopsis ( Arabidopsis thaliana ) is a highly dynamic process involving pore initiation and enlargement and traverses a set of morphological milestones in 3D. Confocal imaging data revealed an enrichment of exocytic machinery, de-methyl-esterified pectic homogalacturonan (HG), and an HG-degrading enzyme at future pore sites, suggesting that both localized HG deposition and degradation might function in pore formation. By manipulating HG modification via enzymatic, chemical, and genetic perturbations in seedling cotyledons, we found that augmenting HG modification promotes pore formation, whereas preventing HG de-methyl-esterification delays pore initiation and inhibits pore enlargement. Through mechanical modeling and experimentation, we tested whether pore formation is an outcome of sister guard cells being pulled away from each other upon turgor increase. Osmotic treatment to reduce turgor pressure did not prevent pore initiation but did lessen pore enlargement. Together, these data provide evidence that HG delivery and modification, and guard cell pressurization, make functional contributions to stomatal pore initiation and enlargement., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

l-Arabinose and d-galactose are the principal constituents of l-arabinogalactan, and also co-occur in other hemicelluloses and pectins. In this work we hypothesized that similar to the induction of relevant glycoside hydrolases by monomers liberated from these plant heteropolymers, their respective catabolisms in saprophytic and phytopathogenic fungi may respond to the presence of the other sugar to promote synergistic use of the complex growth substrate. We showed that these two sugars are indeed consumed simultaneously by Aspergillus nidulans, while l-arabinose is utilised faster in the presence than in the absence of d-galactose. Furthermore, the first two genes of the Leloir pathway for d-galactose catabolism - encoding d-galactose 1-epimerase and galactokinase - are induced more rapidly by l-arabinose than by d-galactose eventhough deletion mutants thereof grow as well as a wild type strain on the pentose. d-Galactose 1-epimerase is hyperinduced by l-arabinose, d-xylose and l-arabitol but not by xylitol. The results suggest that in A. nidulans, l-arabinose and d-xylose - both requiring NADPH for their catabolisation - actively promote the enzyme infrastructure necessary to convert β-d-galactopyranose via the Leloir pathway with its α-anomer specific enzymes, into β-d-glucose-6-phosphate (the starting substrate of the oxidative part of the pentose phosphate pathway) even in the absence of d-galactose., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tomato ( Solanum lycopersicum ) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and β-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL , PG2a , and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to β-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Nephrotoxicity is a major toxic effect in chemotherapy, which constitutes up to 60% of hospitalized acute kidney injury (AKI). Very few treatment options exist to slow the transition from AKI to subsequent chronic kidney diseases (CKD). Here, we demonstrate that galectin-3 (Gal-3), a β-galactoside binding lectin that plays an important role in kidney fibrosis and renal failure, is one of the key factors for renal injury progression. Ectopic overexpression of Gal-3 significantly decreased the viability of HEK293, simultaneously inducing of cell cycle arrest and apoptosis. However, inhibition of Gal-3, mediated by modified citrus pectin (MCP), predominantly antagonized the pro-apoptotic effects. Mice were pre-treated with normal or 1% MCP-supplemented drinking water 1 week before cisplatin injection. Analyses of serum creatinine and renal tissue damage indicated that MCP-treated mice demonstrated increased renal function and attenuated renal fibrosis after cisplatin-induced injury. MCP-treated mice also demonstrated decreased renal fibrosis and apoptosis, as revealed by masson trichrome staining and Western blot analysis of cleaved caspase-3. Additionally, the protective role of Gal-3 inhibition in the kidney injury was shown to be mediated by protein kinase C α (PKC-α), which promoted cell apoptosis and collagen I synthesis in HEK293 cells. These results demonstrated the potential Gal-3 and PKC-α as therapeutic targets for the treatment of AKI and CKD., (© 2018 The Author(s).)

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under- or over-express a xylem-abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under- and over-expressing aspen lines developed xylem with lower-specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties., (© 2018 Scandinavian Plant Physiology Society.)

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.)

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.

Etiolated growth in darkness or the irreversible transition to photomorphogenesis in the light engages alternative developmental programs operating across all organs of a plant seedling. Dark-grown Arabidopsis de-etiolated by zinc (dez) mutants exhibit morphological, cellular, metabolic, and transcriptional characteristics of light-grown seedlings. We identify the causal mutation in TRICHOME BIREFRINGENCE encoding a putative acyl transferase. Pectin acetylation is decreased in dez, as previously found in the reduced wall acetylation2-3 mutant, shown here to phenocopy dez. Moreover, pectin of dez is excessively methylesterified. The addition of very short fragments of homogalacturonan, tri-galacturonate, and tetra-galacturonate, restores skotomorphogenesis in dark-grown dez and similar mutants, suggesting that the mutants are unable to generate these de-methylesterified pectin fragments. In combination with genetic data, we propose a model of spatiotemporally separated photoreceptive and signal-responsive cell types, which contain overlapping subsets of the regulatory network of light-dependent seedling development and communicate via a pectin-derived dark signal., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although pectin-derived polysaccharides can antagonize galectin function in various pathological disorders, the nature of their binding interactions needs to be better defined for developing them as drugs. Moreover, given their relatively large size and complexity, pectin-derived polysaccharides are also useful as model systems to assess inter-polysaccharide and protein-polysaccharide interactions. Here, we investigated interactions between galectin-3 (Gal-3) and pectin-derived polysaccharides: a rhamnogalacturonan (RG) and two homogalacturonans (HGs). BioLayer Interferometry and fluorescence-linked immunosorbent assays indicate that these polysaccharides bind Gal-3 with macroscopic or apparent K D values of 49 nM, 46 µM, and 138 µM, respectively. 15 N- 1 H heteronuclear single quantum coherence (HSQC) NMR studies reveal that these polysaccharides interact primarily with the F-face of the Gal-3 carbohydrate recognition domain. Even though their binding to Gal-3 does not inhibit Gal-3-mediated T-cell apoptosis and only weakly attenuates hemagglutination, their combination in specific proportions increases activity synergistically along with avidity for Gal-3. This suggests that RG and HG polysaccharides act in concert, a proposal supported by polysaccharide particle size measurements and 13 C- 1 H HSQC data. Our model has HG interacting with RG to promote increased avidity of RG for Gal-3, likely by exposing additional lectin-binding sites on the RG. Overall, the present study contributes to our understanding of how complex HG and RG polysaccharides interact with Gal-3., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Boron (B) is an essential micronutrient for the growth and development of plants. Oilseed rape (Brassica napus L.) is a staple oleaginous crop, which is greatly susceptible to B deficiency. Significant differences in tolerance of low-B stresses are observed in rapeseed genotypes, but the underlying mechanism remains unclear, particularly at the single-cell level. Here we provide novel insights into pectin-mediated cell wall (CW) mechanical properties implicated in the differential tolerance of low B in rapeseed genotypes. Under B deficiency, suspension cells of the low-B-sensitive genotype 'W10' showed more severely deformed morphology, lower viabilities and a more easily ruptured CW than those of the low-B-tolerant genotype 'QY10'. Cell rupture was attributed to the weakened CW mechanical strength detected by atomic force microscopy; the CW mechanical strength of 'QY10' was reduced by 13.6 and 17.4%, whereas that of 'W10' was reduced by 29.0 and 30.4% under 0.25 and 0.10 μM B conditions, respectively. The mechanical strength differences between 'QY10' and 'W10' were diminished after the removal of pectin. Further, 'W10' exhibited significantly higher pectin concentrations with much more rhamnogalacturonan II (RG-II) monomer, and also presented obviously higher mRNA abundances of pectin biosynthesis-related genes than 'QY10' under B deficiency. CW regeneration was more difficult for protoplasts of 'W10' than for those of 'QY10'. Taking the results together, we conclude that the variations in pectin-endowed CW mechanical properties play key roles in modulating the differential genotypic tolerance of rapeseed to low-B stresses at both the single-cell and the plant level, and this can potentially be used as a selection trait for low-B-tolerant rapeseed breeding., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In this study, we analyze 31 transgenic lines and their respective untransformed background lines to determine the transgene effects on targeted structures including the pectin components rhamnogalacturonan I (RG-I) and homogalacturonan (HG), neutral side chains (galactan/arabinangalactan), acetylation of pectin, and cellulose level. Modification arising from the pectin backbone- or pectin side chain transgenic lines either increased or decreased the HG:RG-I ratio, side chain length, and methyl esterification of pectin in the tuber cell wall. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level-targeting transgenic lines yielded an unexpectedly high HG:RG-I ratio and longer pectic side chains. These results clearly demonstrate that in effects of a transgene are not restricted to the direct activity of the targeted enzyme but have consequences for the structure of the cell wall matrix. Analysis of whole cell wall structure is therefore necessary to assess the complete effect, direct and indirect, of a transgene., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo., Competing Interests: The authors have declared that no competing interests exist.

Due to its organoleptic and nutraceutical qualities, strawberry fruit (Fragaria x ananassa, Duch) is a worldwide important commodity. The role of ethylene in the regulation of strawberry cell wall metabolism was studied in fruit from Toyonoka cultivar harvested at white stage, when most changes associated with fruit ripening have begun. Fruit were treated with ethephon, an ethylene-releasing reagent, or with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action, maintaining a set of non-treated fruit as controls for each condition. Ethephon treated-fruit showed higher contents of hemicelluloses, cellulose and neutral sugars regarding controls, while 1-MCP-treated fruit showed a lower amount of those fractions. On the other hand, ethephon-treated fruit presented a lower quantity of galacturonic acid from ionically and covalently bound pectins regarding controls, while 1-MCP-treated fruit showed higher contents of those components. We also explored the ethylene effect over the mRNA accumulation of genes related to pectins and hemicelluloses metabolism, and a relationship between gene expression patterns and cell wall polysaccharides contents was shown. Moreover, we detected that strawberry necrotrophic pathogens growth more easily on plates containing cell walls from ethephon-treated fruit regarding controls, while a lower growth rate was observed when cell walls from 1-MCP treated fruit were used as the only carbon source, suggesting an effect of ethylene on cell wall structure. Around 60% of strawberry cell wall is made up of pectins, which in turns is 70% made by homogalacturonans. Our findings support the idea of a central role for pectins on strawberry fruit softening and a participation of ethylene in the regulation of this process., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Cell walls are a distinguishing characteristic of plants essential to their survival. The pectin content of primary cell walls in grasses and dicots is distinctly different. Polygalacturonases (PGs) can degrade pectins and participate in multiple developmental processes of plants. This study comprehensively compared the evolution, expression, and cis-regulatory element of PGs in grasses and dicots. A total of 577 PGs identified from five grasses and five dicots fell into seven clades. Evolutionary analysis demonstrated the distinct differences between grasses and dicots in patterns of gene duplication and loss, and evolutionary rates. Grasses generally contained much fewer clade C and F members than dicots. We found that this disparity was the result of less duplication and more gene losses in grasses. More duplications occurred in clades D and E, and expression analysis showed that most of clade E members were expressed ubiquitously at a high overall level and clade D members were closely related to male reproduction in both grasses and dicots, suggesting their biological functions were highly conserved across species. In addition to the general role in reproductive development, PGs of clades C and F specifically played roles in root development in dicots, shedding light on organ differentiation between the two groups of plants. A regulatory element analysis of clade C and F members implied that possible functions of PGs in specific biological responses contributed to their expansion and preservation. This work can improve the knowledge of PGs in plants generally and in grasses specifically and is beneficial to functional studies.

Main Conclusion: A combined approach, using a carbohydrate microarray as a support for genomic data, has revealed subtle plant cell-wall remodelling during Tuber melanosporum and Corylus avellana interaction. Cell walls are involved, to a great extent, in mediating plant-microbe interactions. An important feature of these interactions concerns changes in the cell-wall composition during interaction with other organisms. In ectomycorrhizae, plant and fungal cell walls come into direct contact, and represent the interface between the two partners. However, very little information is available on the re-arrangement that could occur within the plant and fungal cell walls during ectomycorrhizal symbiosis. Taking advantage of the Comprehensive Microarray Polymer Profiling (CoMPP) technology, the current study has had the aim of monitoring the changes that take place in the plant cell wall in Corylus avellana roots during colonization by the ascomycetous ectomycorrhizal fungus T. melanosporum. Additionally, genes encoding putative plant cell-wall degrading enzymes (PCWDEs) have been identified in the T. melanosporum genome, and RT-qPCRs have been performed to verify the expression of selected genes in fully developed C. avellana/T. melanosporum ectomycorrhizae. A localized degradation of pectin seems to occur during fungal colonization, in agreement with the growth of the ectomycorrhizal fungus through the middle lamella and with the fungal gene expression of genes acting on these polysaccharides.

ERF transcription factors play critical roles in plant immune responses. Here, we report the function of AtERF014, a nucleus-localized transcriptional activator, in Arabidopsis immunity. Expression of AtERF014 was induced by Pseudomonas syringae pv. tomato (Pst) and Botrytis cinerea (Bc). AtERF014-overexpressing (OE) plants displayed increased Pst resistance but decreased Bc resistance, whereas AtERF014-RNAi plants exhibited decreased Pst resistance but increased Bc resistance. After Pst infection, expression of salicylic acid (SA)-responsive genes AtPR1 and AtPR5 in AtERF014-OE plants and of a jasmonic acid/ethylene-responsive gene AtPDF1.2 in AtERF014-RNAi plants was intensified but expression of AtPDF1.2 in AtERF014-OE plants and of AtPR1 and AtPR5 in AtERF014-RNAi plants was weakened. After Bc infection, expression of AtPR1 and AtPR5 in AtERF014-OE plants was attenuated but expression of AtPR1, AtPR5 and AtPDF1.2 in AtERF014-RNAi plants was strengthened. Pathogen- and flg22-induced ROS burst, expression of PTI genes and SA-induced defense were partially suppressed in AtERF014-RNAi plants, whereas pathogen-induced ROS and flg22-induced immune response were strengthened in AtER014-OE plants. Altered expression of AtERR014 affected expression of pectin biosynthetic genes and pectin content in AtERF014-RNAi plants was decreased. These data demonstrate that AtERF014 acts as a dual regulator that differentially modulates immunity against Pst and Bc in Arabidopsis.

AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The root cap covers the tip of the root and functions to protect the root from environmental stress. Cells in the last layer of the root cap are known as border cells, or border-like cells (BLCs) in Arabidopsis (Arabidopsis thaliana). These cells separate from the rest of the root cap and are released from its edge as a layer of living cells. BLC release is developmentally regulated, but the mechanism is largely unknown. Here, we show that the transcription factor NIN-LIKE PROTEIN7 (NLP7) is required for the proper release of BLCs in Arabidopsis. Mutations in NLP7 lead to BLCs that are released as single cells instead of an entire layer. NLP7 is highly expressed in BLCs and is activated by exposure to low pH, a condition that causes BLCs to be released as single cells. Mutations in NLP7 lead to decreased levels of cellulose and pectin. Cell wall-loosening enzymes such as CELLULASE5 (CEL5) and a pectin lyase-like gene, as well as the root cap regulators SOMBRERO and BEARSKIN1/2, are activated in nlp7-1 seedlings. Double mutant analysis revealed that the nlp7-1 phenotype depends on the expression level of CEL5 Mutations in NLP7 lead to an increase in susceptibility to a root-infecting fungal pathogen. Together, these data suggest that NLP7 controls the release of BLCs by acting through the cell wall-loosening enzyme CEL5., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pectin, which is enriched in primary cell walls and middle lamellae, is an essential polysaccharide in all higher plants. Homogalacturonans (HGA), a major form of pectin, are synthesized and methylesterified by enzymes localized in the Golgi apparatus and transported into the cell wall. Depending on cell type, the degree and pattern of pectin methylesterification are strictly regulated by cell wall-localized pectin methylesterases (PMEs). Despite its importance in plant development and growth, little is known about the physiological functions of pectin in rice, which contains 43 different types of PME. The presence of pectin in rice cell walls has been substantiated by uronic acid quantification and immunodetection of JIM7 monoclonal antibodies. We performed PME activity assays with cell wall proteins isolated from different rice tissues. In accordance with data from Arabidopsis, the highest activity was observed in germinating tissues, young culm, and spikelets, where cells are actively elongating. Transcriptional profiling of OsPMEs by real-time PCR and meta-analysis indicates that PMEs exhibit spatial- and stress-specific expression patterns during rice development. Based on in silico analysis, we identified subcellular compartments, isoelectric point, and cleavage sites of OsPMEs. Our findings provide an important tool for further studies seeking to unravel the functional importance of pectin modification during plant growth and abiotic and biotic responses of grass plants., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background and Aims: Quercus suber (cork oak) is a dominant tree of the Fagaceae in forests of the south-west Iberian Peninsula. It is monoecious with a long progamic phase that provides a comprehensive system for comparative studies in development and sexual reproduction. In this study the distribution of arabinogalactan protein (AGPs) and pectin epitopes in anthers of Q. suber was assessed to map these hydroxyproline-rich glycoproteins and the galacturonate-rich acidic polysaccharides during pollen development. Methods Immunolocalization in male flowers was performed with a set of monoclonal antibodies directed against the carbohydrate moiety that recognizes AGPs and pectins. To identify AGP genes involved in cork oak male flower development, a search was conducted for annotated AGP genes in the available transcriptome data of the Cork Oak EST Consortium database (www.corkoakdb.org)., Key Results: Ubiquitous labelling in all cell types was obtained with anti-homogalacturan antibodies for methyl-esterified pectins. In contrast, the antibody that labelled non-methyl-esterified homogalacturans had a preferential presence in microsporocyte cells walls at the beginning of pollen development. Intense labelling was obtained with anti-AGP antibodies both in the tapetum and in the intine wall near the pollen apertures and later in the generative cell wall and vegetative cell. Evaluation of the putative AGPs highly expressed in the male gametophyte was achieved by quantitative RT-PCR analysis in male and female cork oak flowers., Conclusions: Four putative AGP genes were identified that are preferentially expressed in the male flower compared with the female flower. The putative Arabidopsis thaliana orthologues of these genes are associated with preferential expression in pollen, suggesting that the AGPs probably play a significant role in cork oak reproduction., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Plant cells are surrounded by a carbohydrate-rich extracellular matrix known as the cell wall. Primary cell walls are laid down around dividing and elongating cells and consist largely of the polysaccharides cellulose, hemicelluloses, and pectin along with approximately 10% protein. Specific cells such as xylem vessels and fibers lay down a secondary wall rich in cellulose, hemicellulose, and lignin, with lesser amounts of pectin. Most of the models depict the plant cell wall as a matrix of separate polysaccharides. However, the recent identification of a proteoglycan that contains covalently attached pectin and xylan indicates that at least some of these wall glycans exist as domains within a single glycopolymer and that current models of the wall need to be revised. Furthermore, several cell wall biosynthesis mutants, including the secondary cell wall mutant irregular xylem (irx) 8, are affected in multiple cell wall polymers making it challenging to define the biochemical function of the mutated gene. The goal of this review is to provide a background for studying genes which encode secondary cell wall biosynthetic proteins whose mutation affects multiple wall polymers including xylan and lignin. We first review the phenotypes of the irx mutants and then summarize the current understanding of the structure and synthesis of xylan and lignin along with a review of transcription factors known to affect secondary wall synthesis. This review is intended to serve as a resource for those studying genes that encode proteins involved in the synthesis of plant secondary wall lignin and xylan.

Background: Pectins are acidic sugar-containing polysaccharides that are universally conserved components of the primary cell walls of plants and modulate both tip and diffuse cell growth. However, many of their specific functions and the evolution of the genes responsible for producing and modifying them are incompletely understood. The moss Physcomitrella patens is emerging as a powerful model system for the study of plant cell walls. To identify deeply conserved pectin-related genes in Physcomitrella, we generated phylogenetic trees for 16 pectin-related gene families using sequences from ten plant genomes and analyzed the evolutionary relationships within these families., Results: Contrary to our initial hypothesis that a single ancestral gene was present for each pectin-related gene family in the common ancestor of land plants, five of the 16 gene families, including homogalacturonan galacturonosyltransferases, polygalacturonases, pectin methylesterases, homogalacturonan methyltransferases, and pectate lyase-like proteins, show evidence of multiple members in the early land plant that gave rise to the mosses and vascular plants. Seven of the gene families, the UDP-rhamnose synthases, UDP-glucuronic acid epimerases, homogalacturonan galacturonosyltransferase-like proteins, β-1,4-galactan β-1,4-galactosyltransferases, rhamnogalacturonan II xylosyltransferases, and pectin acetylesterases appear to have had a single member in the common ancestor of land plants. We detected no Physcomitrella members in the xylogalacturonan xylosyltransferase, rhamnogalacturonan I arabinosyltransferase, pectin methylesterase inhibitor, or polygalacturonase inhibitor protein families., Conclusions: Several gene families related to the production and modification of pectins in plants appear to have multiple members that are conserved as far back as the common ancestor of mosses and vascular plants. The presence of multiple members of these families even before the divergence of other important cell wall-related genes, such as cellulose synthases, suggests a more complex role than previously suspected for pectins in the evolution of land plants. The presence of relatively small pectin-related gene families in Physcomitrella as compared to Arabidopsis makes it an attractive target for analysis of the functions of pectins in cell walls. In contrast, the absence of genes in Physcomitrella for some families suggests that certain pectin modifications, such as homogalacturonan xylosylation, arose later during land plant evolution.

A paradigm regarding rhamnogalacturonans II (RGII) is their strictly conserved structure within a given plant. We developed and employed a fast structural characterization method based on chromatography and mass spectrometry, allowing analysis of RGII side chains from microgram amounts of cell wall. We found that RGII structures are much more diverse than so far described. In chain A of wild-type plants, up to 45% of the l-fucose is substituted by l-galactose, a state that is seemingly uncorrelated with RGII dimerization capacity. This led us to completely reinvestigate RGII structures of the Arabidopsis thaliana fucose-deficient mutant mur1, which provided insights into RGII chain A biosynthesis, and suggested that chain A truncation, rather than l-fucose to l-galactose substitution, is responsible for the mur1 dwarf phenotype. Mass spectrometry data for chain A coupled with NMR analysis revealed a high degree of methyl esterification of its glucuronic acid, providing a plausible explanation for the puzzling RGII antibody recognition. The β-galacturonic acid of chain A exhibits up to two methyl etherifications in an organ-specific manner. Combined with variation in the length of side chain B, this gives rise to a family of RGII structures instead of the unique structure described up to now. These findings pave the way for studies on the physiological roles of modulation of RGII composition., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Pollen grains are surrounded by a sculpted wall, which protects male gametophytes from various environmental stresses and microbial attacks, and also facilitates pollination. Pollen wall development requires lipid and polysaccharide metabolism, and some key genes and proteins that participate in these processes have recently been identified. Here, we summarise the genes and describe their functions during pollen wall development via several metabolic pathways. A working model involving substances and catalytic enzyme reactions that occur during pollen development is also presented. This model provides information on the complete process of pollen wall development with respect to metabolic pathways., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Rice (Oryza sativa L.) is a typical Si-accumulating plant and is able to accumulate Si up to >10 % of shoot dry weight. The cell wall has been reported to become thicker under Si-deficient condition. To clarify the relationship between Si accumulation and cell wall components, the physical properties of, and macromolecular components and Si content in, the pectic, hemicellulosic, and cellulosic fractions prepared from rice seedlings grown in hydroponics with or without 1.5 mM silicic acid were analyzed. In the absence of Si (the -Si condition), leaf blades drooped, but physical properties were enhanced. Sugar content in the cellulosic fraction and lignin content in the total cell wall increased under -Si condition. After histochemical staining, there was an increase in cellulose deposition in short cells and the cell layer just beneath the epidermis in the -Si condition, but no significant change in the pattern of lignin deposition. Expression of the genes involved in secondary cell wall synthesis, OsCesA4, OsCesA7, OsPAL, OsCCR1 and OsCAD6 was up-regulated under -Si condition, but expression of OsCesA1, involved in primary cell wall synthesis, did not increase. These results suggest that an increase in secondary cell wall components occurs in rice leaves to compensate for Si deficiency.

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.

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.

Although processed potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase (PME) activity as a potential factor impacting on textural properties, and the expression of a gene encoding an isoform of PME (PEST1) was associated with cooked tuber textural properties. In this study, a transgenic approach was undertaken to investigate further the impact of the PEST1 gene. Antisense and over-expressing potato lines were generated. In over-expressing lines, tuber PME activity was enhanced by up to 2.3-fold; whereas in antisense lines, PME activity was decreased by up to 62%. PME isoform analysis indicated that the PEST1 gene encoded one isoform of PME. Analysis of cell walls from tubers from the over-expressing lines indicated that the changes in PME activity resulted in a decrease in pectin methylation. Analysis of processed tuber texture demonstrated that the reduced level of pectin methylation in the over-expressing transgenic lines was associated with a firmer processed texture. Thus, there is a clear link between PME activity, pectin methylation and processed tuber textural properties., (© 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Wood properties are ultimately related to the morphology and biophysical properties of the xylem cell wall. Although the cellulose and lignin biosynthetic pathways have been extensively studied, modifications of other wall matrix components during secondary growth have attracted relatively less attention. In this work, thirty-eight new Eucalyptus cDNAs encoding cell wall-modifying proteins from nine candidate families that act on the cellulose-hemicellulose and pectin networks were cloned and their gene expression was investigated throughout the developing stem. Semi-quantitative RT-PCR revealed distinct, gene-specific transcription patterns for each clone, allowing the identification of genes up-regulated in xylem or phloem of stem regions undergoing secondary growth. Some genes, namely an endo-1,4-beta-glucanase, one mannan-hydrolase and three pectin methylesterases showed transcription in juvenile and also in mature stages of wood development. The patterns of gene expression using samples from tension and opposite wood disclosed a general trend for up-regulation in tension wood and/or down-regulation in opposite wood. Localised gene expression of two selected representative clones, EGl-XTH1 and EGl-XTH4, obtained through in situ hybridization confirms the RT-PCR results and association with secondary xylem formation. Likewise, immunolocalisation studies with the anti-pectin antibody (JIM5) also supported the idea that the development of tissue-specific pectin characteristics is important during secondary growth. These results emphasize an involvement of hemicellulose and pectin biochemistry in wood formation, suggesting that the controlled and localised modification of these polysaccharides may define cell properties and architecture and thus, contribute to determining different biophysical characteristics of Eucalyptus wood., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

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.

A dynamic mathematical model has been developed and validated to describe the synthesis of pectate lyases (Pels), the major virulence factors in Dickeya dadantii. This work focuses on the simultaneous modeling of the metabolic degradation of pectin by Pel enzymes and the genetic regulation of pel genes by 2-keto-3-deoxygluconate (KDG), a catabolite product of pectin that inactivates KdgR, one of the main repressors of pel genes. This modeling scheme takes into account the fact that the system is composed of two time-varying compartments: the extracellular medium, where Pel enzymes cleave pectin into oligomers, and the bacterial cytoplasm where, after internalization, oligomers are converted to KDG. Using the quasi-stationary state approximations, the model consists of some nonlinear differential equations for which most of the parameters could be estimated from the literature or from independent experiments. The few remaining unknown parameters were obtained by fitting the model equations against a set of Pel activity data. Model predictions were verified by measuring the time courses of bacterial growth, Pel production, pel mRNA accumulation, and pectin consumption under various growth conditions. This work reveals that pectin is almost totally consumed before the burst of Pel production. This paradoxical behavior can be interpreted as an evolutionary strategy to control the diffusion process so that as soon as a small amount of pectin is detected by the bacteria in its surroundings, it anticipates more pectin to come. The model also predicts the possibility of bistable steady states in the presence of constant pectin compounds.

The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50-60 degrees C, with some enzyme activity retained up to 80 degrees C. Its activation energy was 5.352calmol(-1). PGase I showed a higher affinity towards PGA than citric pectin (Km=0.55+/-0.02 and 0.72+/-0.02mgml(-1), respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on mur1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants (Col-0) and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels (approximately tensile strength) of the hypocotyls of the mutants with pectin alterations (mur1, qua2) were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for mur1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.