Cell Wall Monosaccharide Composition Research Articles

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

OsVTC1-1 RNAi Mutant with Reduction of Ascorbic Acid Synthesis Alters Cell Wall Sugar Composition and Cell Wall-Associated Proteins

Ascorbic acid (AsA) or Vitamin C is an antioxidant molecule and plays an important role in many biological processes in plants. GDP-D-mannose pyrophosphorylase (GMP or VTC1) catalyzes the synthesis of GDP-D-mannose, which is a precursor for AsA production and is used for cell wall polysaccharide and glycoprotein synthesis. In rice, the OsVTC1 gene consists of three homologs, including OsVTC1-1, OsVTC1-3 and OsVTC1-8. In this study, we characterized wild type (WT) and OsVTC1-1 RNAi lines (RI1-2 and RI1-3) and showed that the transcript levels of most genes in the AsA synthesis pathway, AsA content and leaf anatomical parameters in RNAi lines were reduced, revealing that OsVTC1-1 is involved in AsA synthesis. To further study the role of OsVTC1-1 gene, cell wall monosaccharide composition, transcriptome and proteome were compared, with specific attention paid to their wild type and OsVTC1-1 RNAi lines. Mannose and galactose composition (mole%) were decreased in OsVTC1-1 RNAi lines. Additionally, reduction of cell wall-associated proteins, such as kinesin, expansin, beta-galactosidase and cellulose synthase were observed in OsVTC1-1 RNAi lines. Our results suggest that OsVTC1-1 gene plays an important role in AsA synthesis and in cell wall-related processes.

Phylogenomic Analyses of Nucleotide-Sugar Biosynthetic and Interconverting Enzymes Illuminate Cell Wall Composition in Fungi.

The fungi are an enormously successful eukaryotic lineage that has colonized every aerobic habitat on Earth. This spectacular expansion is reflected in the dynamism and diversity of the fungal cell wall, a matrix of polysaccharides and glycoproteins pivotal to fungal life history strategies and a major target in the development of antifungal compounds. Cell wall polysaccharides are typically synthesized by Leloir glycosyltransferases, enzymes that are notoriously difficult to characterize, but their nucleotide-sugar substrates are well known and provide the opportunity to inspect the monosaccharides available for incorporation into cell wall polysaccharides and glycoproteins. In this work, we have used phylogenomic analyses of the enzymatic pathways that synthesize and interconvert nucleotide-sugars to predict potential cell wall monosaccharide composition across 491 fungal taxa. The results show a complex evolutionary history of these cell wall enzyme pathways and, by association, of the fungal cell wall. In particular, we see a significant reduction in monosaccharide diversity during fungal evolution, most notably in the colonization of terrestrial habitats. However, monosaccharide distribution is also shown to be varied across later-diverging fungal lineages.IMPORTANCE This study provides new insights into the complex evolutionary history of the fungal cell wall. We analyzed fungal enzymes that convert sugars acquired from the environment into the diverse sugars that make up the fundamental building blocks of the cell wall. Species-specific profiles of these nucleotide-sugar interconverting (NSI) enzymes for 491 fungi demonstrated multiple losses and gains of NSI proteins, revealing the rich diversity of cell wall architecture across the kingdom. Pragmatically, because cell walls are essential to fungi, our observations of variation in sugar diversity have important implications for the development of antifungal compounds that target the sugar profiles of specific pathogens.

Abrogation of glucosidase I–mediated glycoprotein deglucosylation results in a sick phenotype in fission yeasts: Model for the human MOGS-CDG disorder

Glucosidase I (GI) removes the outermost glucose from protein-linked Glc3Man9GlcNAc2 (G3M9) in the endoplasmic reticulum (ER). Individuals with congenital disorders of glycosylation MOGS-CDG bear mutations in the GI-encoding gene (gls1). Although GI absence has been reported to produce lethality in Schizosaccharomyces pombe yeasts, here we obtained two viable Δgls1 mutants, one with a very sick but not lethal phenotype (Δgls1-S) and the other with a healthier one (Δgls1-H). The sick strain displayed only G3M9 as an ER protein-linked oligosaccharide, whereas the healthier strain had both G3M9 and Man9GlcNAc2 The lipid-linked oligosaccharide patterns of the two strains revealed that the most abundantly formed glycans were G3M9 in Δgls1-S and Glc2Man9GlcNAc2 in Δgls1-H, suggesting reduced Alg10p glucosyltransferase activity in the Δgls1-H strain. A mutation in the alg10+ gene was indeed observed in this strain. Our results indicated that abrogated G3M9 deglucosylation was responsible for the severe defects observed in Δgls1-S cells. Further studies disclosed that the defects could not be ascribed to disruption of glycoprotein entrance into calnexin-folding cycles, inhibition of the oligosaccharyltransferase by transfer reaction products, or reduced proteasomal degradation of misfolded glycoproteins. Lack of triglucosylated glycoprotein deglucosylation neither significantly prevented glycan elongation in the Golgi nor modified the overall cell wall monosaccharide composition. Nevertheless, it resulted in a distorted cell wall and in the absence of underlying ER membranes. Furthermore, Golgi expression of human endomannosidase partially restored normal growth in Δgls1-S cells. We propose that accumulation of G3M9-bearing glycoproteins is toxic and at least partially responsible for defects observed in MOGS-CDG.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants.

Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance.

Developing xylem-preferential expression of PdGA20ox1, a gibberellin 20-oxidase 1 from Pinus densiflora, improves woody biomass production in a hybrid poplar.

Woody biomass has gained popularity as an environmentally friendly, renewable and sustainable resource for liquid fuel production. Here, we demonstrate biotechnological improvement of the quantity and quality of woody biomass by employing developing xylem (DX)-preferential production of gibberellin (GA), a phytohormone that positively regulates stem growth. First, for the proof of concept experiment, we produced transgenic Arabidopsis plants expressing GA20-oxidase, a key enzyme in the production of bioactive GAs, from Pinus densiflora (PdGA20ox1) under the control of either a constitutive 35S promoter, designated 35S::PdGA20ox1, or a DX-specific promoter (originated from poplar), designated DX15::PdGA20ox1. As we hypothesized, both transgenic Arabidopsis plants (35S::PdGA20ox1 and DX15::PdGA20ox1) exhibited an accelerated stem growth that resulted in a large increase of biomass, up to 300% compared to wild-type control plants, together with increased secondary wall thickening and elongation of fibre cells. Next, we applied our concept to the production of transgenic poplar trees. Both transgenic poplar trees (35S::PdGA20ox1 and DX15::PdGA20ox1) showed dramatic increases in biomass, up to 300%, with accelerated stem growth and xylem differentiation. Cell wall monosaccharide composition analysis revealed that in both Arabidopsis and poplar, glucose and xylose contents were significantly increased. However, undesirable phenotypes of 35S::PdGA20ox1 poplar, including poor root growth and leaf development, were found. Interestingly, DX15::PdGA20ox1 poplar resulted in a reduction of undesirable phenotypes. Our results indicate that the controlled production of GAs through a tissue-specific promoter can be utilized as an efficient biotechnological tool for producing enhanced plant biomass, minimizing unwanted effects.

Overexpression of gibberellin 20-oxidase1 from Pinus densiflora results in enhanced wood formation with gelatinous fiber development in a transgenic hybrid poplar.

Gibberellins (GAs) are important regulators of plant shoot biomass growth, and GA 20-oxidase (GA20ox) is one of the major regulatory enzymes in the GA biosynthetic pathway. Previously, we showed that the expression levels of a putative GA20ox1 (i.e., PdGA20ox1) in stem tissue of 3-month-old seedlings of 12 families of Pinus densiflora were positively correlated with stem diameter growth across those same families growing in an even-aged 32-year-old pine forest (Park EJ, Lee WY, Kurepin LV, Zhang R, Janzen L, Pharis RP (2015) Plant hormone-assisted early family selection in Pinus densiflora via a retrospective approach. Tree Physiol 35:86-94). To further investigate the molecular function of this gene in the stem wood growth of forest trees, we produced transgenic poplar lines expressing PdGA20ox1 under the control of the 35S promoter (designated as 35S::PdGA20ox1). By age 3 months, most of the 35S::PdGA20ox1 poplar trees were showing an exceptional enhancement of stem wood growth, i.e., up to fourfold increases in stem dry weight, compared with the nontransformed control poplar plants. Significant increases in endogenous GA1, its immediate precursor (GA20) and its catabolite (GA8) in elongating internode tissue accompanied the increased stem growth in the transgenic lines. Additionally, the development of gelatinous fibers occurred in vertically grown stems of the 35S::PdGA20ox1 poplars. An analysis of the cell wall monosaccharide composition of the 35S::PdGA20ox1 poplars showed significant increases in xylose and glucose contents, indicating a qualitative increase in secondary wall depositions. Microarray analyses led us to find a total of 276 probe sets that were upregulated (using threefold as a threshold) in the stem tissues of 35S::PdGA20ox1 poplars relative to the controls. 'Cell organization or biogenesis'- and 'cell wall'-related genes were overrepresented, including many of genes that are involved in cell wall modification. Several transcriptional regulators, which positively regulate cell elongation through GA signaling, were also upregulated. In contrast, genes involved in defense signaling were appreciably downregulated in the 35S::PdGA20ox1 stem tissues, suggesting a growth versus defense trade-off. Taken together, our results suggest that PdGA20ox1 functions to promote stem growth and wood formation in poplar, probably by activating GA signaling while coincidentally depressing defense signaling.

Variations in cell wall monosaccharide composition during seed development in Coffea arabica L. Comparison between Coffea arabica var. Bourbon and Coffea arabica var. Laurina

Cell wall polysaccharide composition changed over seed development. Differences between B and BP only concerned the fruit growth period. Coffea arabica var. Laurina, also known as ‘Bourbon Pointu’ (BP), is a natural mutant of Coffea arabica var. Bourbon (B). BP is known for its high organoleptic quality, which is today compared with the well-known ‘Blue Mountain’. Cell wall composition of seeds could explain the better cup quality of BP. To test this hypothesis, we investigated possible impacts of the laurina mutation on the cell wall composition of seeds over a time course, and more precisely on polysaccharides. The identification of cell wall polysaccharides (CWP) was deduced from permethylation analysis, whereas cell wall monosaccharide (CWM) composition was estimated using trifluoroacetic acid (TFA) and H2SO4. The observation of CWM over time allowed defining three phases. The φ 1 phase, from the 8th to 14th week after flowering (WAF), was characterized by the predominance of arabinogalactans, arabinoxylans and arabinans. The φ 2 phase, from the 14th to 20th WAF, was mainly characterized by the increased importance of highly branched galactomannans at the expense of arabinoxylans. Lastly, galactomannans constituted the main CWP present in the third phase (from the 20th to the 32nd WAF), associated with arabinogalactans and arabinans. Variations in CWP composition were connected to fruit and seed development. The end of the φ 1 phase coincides with the end of the fruit growth, i.e., with the end of the endosperm development. During the φ 2 phase, endosperm becomes milky and then hard due to the galactomannan deposition. Moreover, the φ 3 phase corresponded to the fruit maturation stage in which CWM composition did not change over time. Galactomannans were less substituted and constitute the main seed CWP. Lastly, the evidence of laurina mutation impact on cell wall polysaccharides of seeds was only observed during the fruit growth period. Consequently, the difference of cup quality between B and BP would not be due to CWP composition at the end of the φ 3 phase, i.e., when coffee beans are harvested.

AtC3H14, a plant‐specific tandem CCCH zinc‐finger protein, binds to its target mRNAs in a sequence‐specific manner and affects cell elongation in Arabidopsis thaliana

AtC3H14 (At1 g66810) is a plant-specific tandem CCCH zinc-finger (TZF) protein that belongs to the 68-member CCCH family in Arabidopsis thaliana. In animals, TZFs have been shown to bind and recruit target mRNAs to the cytoplasmic foci where mRNA decay enzymes are active. However, it is not known whether plant TZF proteins such as AtC3H14 function. So far, no mRNA targets of plant TZFs have been identified. We have obtained several lines of experimental evidence in support of our hypothesis that AtC3H14 is involved in post-transcriptional regulation of its target genes. Nucleic acid binding assays using [(35) S]-labeled AtC3H14 protein showed that AtC3H14 could bind to ssDNA, dsDNA, and ribohomopolymers, suggesting its RNA-binding activity. RNA immunoprecipitation (RIP) assay identified several putative target RNAs of AtC3H14, including a polygalacturonase, a well-known cell wall modifying gene. RNA electrophoretic mobility shift assays (RNA-EMSA) were used to confirm the RIP results and demonstrate that the TZF domain of AtC3H14 is required for the target RNA binding. Microarray analysis of 35S::AtC3H14 plants revealed that many of the cell wall elongation and/or modification-associated genes were differentially expressed, which is consistent with the cell elongation defect phenotype and the changes in the cell wall monosaccharide composition. In addition, yeast activation assay showed that AtC3H14 also function as a transcriptional activator, which is consistent with the previous finding that AtC3H14 activate the secondary wall biosynthesis genes. Taken together, we conclude that AtC3H14 may play a key role in both transcriptional and post-transcriptional regulation.

Two poplar cellulose synthase-like D genes, PdCSLD5 and PdCSLD6, are functionally conserved with Arabidopsis CSLD3

Root hairs are tip-growing long tubular outgrowths of specialized epidermal cells, and are important for nutrient and water uptake and interaction with the soil microflora. Here we characterized two poplar cellulose synthase-like D (CSLD) genes, PdCSLD5 and PdCSLD6, the most probable orthologs to the Arabidopsis AtCSLD3/KOJAK gene. Both PdCSLD5 and PdCSLD6 are strongly expressed in roots, including in the root hairs. Subcellular localization experiments showed that these two proteins are located not only in the polarized plasma membrane of root hair tips, but also in Golgi apparatus of the root hair and non-hair-forming cells. Overexpression of these two poplar genes in the atcsld3 mutant was able to rescue most of the defects caused by disruption of AtCSLD3, including root hair morphological changes, altered cell wall monosaccharide composition, increased non-crystalline β-1,4-glucan and decreased crystalline cellulose contents. Taken together, our results provide evidence indicating that PdCSLD5 and PdCSLD6 are functionally conserved with AtCSLD3 and support a role for PdCSLD5 and PdCSL6 specifically in crystalline cellulose production in poplar root hair tips. The results presented here also suggest that at least part of the mechanism of root hair formation is conserved between herbaceous and woody plants.

Two Poplar Glycosyltransferase Genes, PdGATL1.1 and PdGATL1.2, Are Functional Orthologs to PARVUS/AtGATL1 in Arabidopsis

Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However, the biosynthesis of xylan in woody plants, where this polysaccharide is a major component of wood, is poorly understood. Here, we characterize two Populus genes, PdGATL1.1 and PdGATL1.2, the closest orthologs to the Arabidopsis PARVUS/GATL1 gene, with respect to their gene expression in poplar, their sub-cellular localization, and their ability to complement the parvus mutation in Arabidopsis. Overexpression of the two poplar genes in the parvus mutant rescued most of the defects caused by the parvus mutation, including morphological changes, collapsed xylem, and altered cell wall monosaccharide composition. Quantitative RT-PCR showed that PdGATL1.1 is expressed most strongly in developing xylem of poplar. In contrast, PdGATL1.2 is expressed much more uniformly in leaf, shoot tip, cortex, phloem, and xylem, and the transcript level of PdGATL1.2 is much lower than that of PdGATL1.1 in all tissues examined. Sub-cellular localization experiments showed that these two proteins are localized to both ER and Golgi in comparison with marker proteins resident to these sub-cellular compartments. Our data indicate that PdGATL1.1 and PdGATL1.2 are functional orthologs of PARVUS/GATL1 and can play a role in xylan synthesis, but may also have role(s) in the synthesis of other wall polymers.

Yeasts associated with termites: a phenotypic and genotypic characterization and use of coevolution for dating evolutionary radiations in asco- and basidiomycetes

Thirtynine yeast isolates or dimorphic fungi were obtained from the hindgut of the lower termites Mastotermis darwiniensis (Mastotermitidae), Zootermopsis angusticollis, Z. nevadensis (Hodotermitidae), Neotermes jouteli (Kalotermitidae), Reticulitermes santonensis, Heterotermes indicola (Rhinotermitidae) and the roach Cryptocercus punctulatus. Using RAPD-PCR the 39 yeast isolates were assigned to 13 different species. Commonly yeast species were specific to the termite species isolates from. There were only two yeast species which were found in different species of lower termites. Based on phenotypic characters Debaryomyces hansenii showed a high score in four species. The qualitative and quantitative yeast cell wall monosaccharide composition, the ubiquinone system, partial sequencing of 18S ribosomal DNA (bases 1273 to 948; numbering according to the gene of Saccharomyces cerevisiae), and the ultrastructure of septal pores indicate that 11 yeast species belong to the Endomycetales. Although ascospores were lacking, two of these species were identified to belong to the genus Debaryomyces. One remaining yeast isolate was identified as a Sporothrix anamorph representative for the filamentous Ascomycetes (Ophiostomataceae s. str.); the second species showed affinities to the Basidiomycetes in particular to the genus Trichosporon. Comparing an additional 18S rDNA fragment (bases 595 to 993) and RAPD-PCR data using different species type strains of the genus Sporothrix, the filamentous ascomycete was genotypically identified as Sporothrix albicans. Sporothrix. albicans, although phylogenetically closely related to S. schenckii var. schenckii and Ophiostoma stenoceras remains genotypically distinct. An emended species description of S. albicans is presented. Evidence is provided that the yeasts isolated from the hindgut can be considered symbionts.

Firmness and Cell Wall Characteristics of Pasteurized Jalapeño Pepper Rings Affected by Calcium Chloride and Acetic Acid

ABSTRACTPepper rings packed in brine containing CaCl2 were firmer, had higher bound calcium, chelator soluble pectin and pectin DE, and less water‐soluble pectin (WSP) than peppers packed in brine containing no CaCl2. Pepper rings packed in low acid brines (1% and 1.2% acetic acid) were firmer and had less WSP than those packed in high acid brine. Those samples (4% acetic acid) resulted in softening and pectin solubilization, but CaCl2 resulted in less softening. Monosaccharide composition of cell walls was not affected by CaCl2 or acetic acid. Firmness retention in CaCl2 treated samples was probably due to greater association between calcium ions and pectic substances, which resisted acid hydrolysis.