A genome-wide analysis of the flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) dirigent protein family: from gene identification and evolution to differential regulation

Key message

Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress.

Abstract

Dirigent proteins (DIRs) were discovered during 8-8′ lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (?)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset of genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8′ linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (?)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense.

     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Effect of 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene on <Emphasis Type="Italic">cellulose synthase A</Emphasis> (<Emphasis Type="Italic">CesA</Emphasis>) genes in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L. ‘Nike’) seedlings

Introduction

Cellulose microfibril is a major cell wall polymer that plays an important role in the growth and development of plants. The gene cellulose synthase A (CesA), encoding cellulose synthases, is involved in the synthesis of cellulose microfibrils. However, the regulatory mechanism of CesA gene expression is not well understood, especially during the early developmental stages.

Objective

To identify factor(s) that regulate the expression of CesA genes and ultimately control seedling growth and development.

Methods

The presence of cis-elements in the promoter region of the eight CesA genes identified in flax (Linum usitatissimum L. ‘Nike’) seedlings was verified, and three kinds of ethylene-responsive cis-elements were identified in the promoters. Therefore, the effect of ethylene on the expression of four selected CesA genes classified into Clades 1 and 6 after treatment with 10^?4 and 10^?3 M 1-aminocyclopropane-1-carboxylic acid (ACC) was examined in the hypocotyl of 4–6-day-old flax seedlings.

Results

ACC-induced ethylene either up- or down-regulated the expression of the CesA genes depending on the clade to which these genes belonged, age of seedlings, part of the hypocotyl, and concentration of ACC.

Conclusion

Ethylene might be one of the factors regulating the expression of CesA genes in flax seedlings.

     

New Approach to Identification of Genes Controlling Cell Wall Biogenesis in the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

MCD4 codes for a protein presumably adding the phosphoethanolamine moiety to the first mannose residue of glycosylphosphatidylinositol (GPI) precursors in the yeast Saccharomyces cerevisiae. The role of this modification is still unclear. The phenotypic effects of some MCD4 mutations are probably unrelated to defects in GPI synthesis, suggesting additional functions for Mcd4p. To study the Mcd4p functions in more detail, a search for the genes whose mutations are lethal or semilethal in combination with the ssu21 mutation of MCD4 was performed. Six such mutations were isolated, including some mutations causing sensitivity to SDS and/or calcofluor white. Genes complementing two out of the six mutations were cloned and identified as MNN9, which is involved in the formation of outer chains of N-linked glycans of secreted proteins, and GWT1, which codes for an endoplasmic reticulum protein involved in GPI biosynthesis. In both cases, growth inhibition was probably caused by defective biogenesis of the cell wall and a misfolding of secreted proteins. The proposed approach is suitable for seeking new genes controlling cell wall biogenesis.     

Overexpression of UDP-glucose dehydrogenase from <Emphasis Type="Italic">Larix gmelinii</Emphasis> enhances growth and cold tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine diphosphate glucose dehydrogenase (UGDH) plays an important role in biosynthesis of hemicellulose by catalyzing oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in biosynthesis of the plant cell wall. In this study, a UGDH ortholog referred to as LgUGDH was isolated from Larix gmelinii using PCR and rapid amplification of cDNA ends techniques. Real-time PCR shows that the LgUGDH gene was expressed primarily in larch stems in addition to its roots and leaves, and Southern blot analysis indicates that UGDH is encoded by two paralogous genes in L. gmelinii. Overexpression of LgUGDH increased the content of soluble sugars and hemicelluloses and enhanced vegetative growth and cold tolerance in transgenic Arabidopsis thaliana. These results reveal that L. gmelinii UGDH participates in sucrose/polysaccharide metabolism and cell wall biosynthesis and may be a good candidate gene for enhancing plant growth, cold tolerance, and hemicellulose content.     

Effect of amikacin on cell wall glycopeptidolipid synthesis in <Emphasis Type="Italic">Mycobacterium abscessus</Emphasis>

Cultivation of the smooth colony Mycobacterium abscessus at the sub-minimum inhibitory concentration (MIC) of amikacin changed its growth pattern including its colony morphology (smooth to rough) and cell arrangement (dispersed to cord formation). In addition, reduced sliding motility and biofilm formation were observed. The amount of glycogpetidolipid (GPL) and mRNA expression of key genes involved in GPL synthesis were decreased in the amikacin-treated M. abscessus strain. An in vitro infection assay revealed that the amikacin-treated smooth M. abscessus strain induced more pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) than that of the smooth strain in murine macrophage cells. These results suggest that long-term exposure to a low concentration of amikacin causes a physical change in the cell wall which may increase its virulence.     

A long term evaluation of differential potassium fertilization of a creeping bentgrass putting green

Aims

Buckwheat (Fagopyrum esculentum) is highly tolerant to Al stress, but the molecular mechanisms remain largely unknown. This study aims to investigate a half-type ABC transporter gene (FeSTAR1) with respect to Al tolerance.

Methods

The expression of FeSTAR1 was examined and complementation test in atstar1 mutant was conducted. Furthermore, Al distribution and cell wall polysaccharides content were analyzed.

Results

FeSTAR1 is an ABC transporter protein with nucleotide binding domain, but lack of transmembrane domain. Consistently, FeSTAR1 is a soluble protein, localizing to both cytoplasm and nucleus. Al rapidly and specifically induced FeSTAR1 expression. Heterologous expression of FeSTAR1 in atstar1 rescued its Al tolerance, and exogenous applied UDP-glucose could alleviate Al sensitivity of atstar1 mutant, suggesting the connection between FeSTAR1 and UDP-glucose in terms of Al tolerance. Furthermore, FeSTAR1 complemented lines accumulated less Al in root cell wall than atstar1 mutant. Further cell wall fraction analysis showed that Al was largely confined to cell wall hemicellulose1, at which Al content was significantly lower in complemented lines. Consistent with Al distribution in different cell wall polysaccharides, complemented lines had lower hemicellulose1 content.

Conclusion

Our results indicate that FeSTAR1 is involved in Al resistance via possibly cell wall matrix polysaccharides metabolism in buckwheat.

     

Antisense expression of Gossypium barbadense UGD6 in Arabidopsis thaliana significantly alters cell wall composition

Uridine diphosphate-glucose dehydrogenase (UGD, EC1.1.1.22 oxidizes UDP-Glc (UDP-D-glucose) to UDP-GlcA (UDP-Dglucuronate), a critical precursor of cell wall polysaccharides. GbUGD6 from Gossypium barbadense is more highly expressed late in the elongation of cotton fibers (15 d post-anthesis (DPA)) and during the stage of secondary cell wall thickening (30 DPA). Subcellular localization analysis in onion epidermis revealed that fluorescently labeled GbUGD6 protein was distributed throughout the cell membrane, as well as the nucleus and vacuoles. Examination of UGD function in Arabidopsis revealed that the antisense GbUGD6 lines had shorter roots, deferred blossoming, compared to wild-type plants. Activities of associated enzymes were also affected by UGD reduction, and biochemical analysis of cell wall samples showed an increase in cellulose levels and a decrease in UGP-GlcA contents. The results of the present study as well as previous studies on UGD support the conclusion that UGD plays a major role in synthesizing polysaccharides synthesis in the cell wall.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of the new lysostaphin family endopeptidase catalytic domain from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Lysostaphin family endopeptidases, produced by Staphylococcus genus, are zinc-dependent enzymes that cleave pentaglycine bridges of cell wall peptidoglycan. They act as autolysins to maintain cell wall metabolism or as toxins and weapons against competing strains. Consequently, these enzymes are compelling targets for new drugs as well as are potential antimicrobial agents themselves against Staphylococcus pathogens, which depend on cell wall to retain their immunity against antibiotics. The rapid spread of methicillin and vancomycin-resistant Staphylococcus aureus strains draws demand for new therapeutic approaches. S. aureus gene sa0205 was found to be implicated in resistance to vancomycin and synthesis of the bacteria cell wall. The gene encodes for a catalytic domain of a lysostaphin-type endopeptidase. We aim to obtain the structure of the Sa0205 catalytic domain, the first solution structure of the catalytic domain of the lysostaphin family enzymes. In addition, we are to investigate the apparent binding of the second zinc ion, which has not been previously reported for the enzyme group. Herein, we present the backbone and side chain resonance assignments of Sa0205 endopeptidase catalytic domain in its one and two zinc-bound forms.     

The analysis of mutant phenotypes and tissue expression reveals a role of SNAREs VAMP721 and VAMP722 in seedling growth

Membrane traffic mediated by a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex contributes to plant growth and development. However, the functional significance of SNAREs involved in cell wall deposition and seedling development has not been sufficiently explored. In this study, we explored the roles of R-SNAREs VAMP721 (At1g04750) and VAMP722 (At2g33120) in seedling growth of Arabidopsis thaliana by histochemical staining, fluorescence labeling, and analyzing mutant phenotypes. Our results show a massive intracellular accumulation of cellulose and callose, and an abnormal deposition of callose at the expanding cell plate in vamp721vamp722 root cells compared with the wild type. Particularly, ectopic lignin accumulation was also observed in vamp721vamp722 root cells. The alteration of cell wall components was confirmed using Fourier transform infrared analysis. Plasma membrane integrity and cell viability were disturbed in the vamp721vamp722 seedling. Morphological observation shows that vamp721vamp722 mutations impaired development of roots, hypocotyl, cotyledon, and true leaf, and inhibited lateral root formation. Confocal images reveal that green fluorescent protein-tagged VAMP721 and VAMP722 showed a similar expression pattern and were expressed throughout all cells and tissues examined, including root and shoot apical meristems and cells of hypocotyls, cotyledons, and true leaves. Taken together, our results suggest that membrane traffic mediated by VAMP721 and VAMP722 is involved in seedling growth in A. thaliana.     

Increased expression of damage-associated molecular patterns (DAMPs) in osteoarthritis of human knee joint compared to hip joint

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of β-naphthoflavone (BNF), a potent AhR ligand, on the expression of AhR-dependent genes were examined by microarray and qPCR analysis in both, differentiated and undifferentiated HepaRG cell lines. To prove that BNF-induced changes of investigated genes were indeed AhR-dependent, we knock down the expression of AhR by stable transfection of HepaRG cells with shRNA. Regardless of genetical identity, our results clearly demonstrate different expression profiles of AhR-dependent genes between differentiated and undifferentiated HepaRG cells. Genes involved in metabolism of xenobiotics constitute only minute fraction of all genes regulated by AhR in HepaRG cells. Participation of AhR in induction of expression of genes associated with regulation of apoptosis or involved in cell proliferation as well as AhR-dependent inhibition of genes connected to cell adhesion could support suggestion of involvement of AhR not only in initiation but also in progression of carcinogenesis. Among the AhR-dependent genes known to be involved in metabolism of xenobiotics, cytochromes P4501A1 and 1B1 belong to the most inducible by BNF. On the contrary, expression of GSTA1 and GSTA2 was significantly inhibited after BNF treatment of HepaRG cells. Among the AhR-dependent genes that are not involved in metabolism of xenobiotics SERPINB2, STC2, ARL4C, and TIPARP belong to the most inducible by BNF. Our results imply involvement of Ah receptor in regulation of CYP19A1, the gene-encoding aromatase, and an enzyme responsible for a key step in the biosynthesis of estrogens.