Enhancement of polysaccharides accumulation in <Emphasis Type="Italic">Dendrobium officinale</Emphasis> by exogenously applied methyl jasmonate

The accumulation of polysaccharides, activities of sucrose metabolism enzymes, and the expression of sucrose biosynthetic genes in Dendrobium officinale were significantly affected by exogenous methyl jasmonate (MeJA). Application of MeJA increased the content of polysaccharides and the highest polysaccharide production occurred in the samples treated with 200 μM MeJa. The MeJA application influenced polysaccharide biosynthesis rather than degradation because the activities of sucrose metabolism enzymes and the expressions of sucrose biosynthetic genes were upregulated by MeJA. Interestingly, low MeJA concentrations promoted accumulation of Dendrobium polysaccharides, while high MeJA amounts played an inhibitory role. The content of major constituent of polysaccharides, glucose and mannose, also increased after MeJa treatment.     

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.     

Acaricidal and repellent effects of <Emphasis Type="Italic">Cnidium officinale</Emphasis>-derived material against <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis> (Acari: Dermanyssidae)

The acaricidal activity of a methanolic extract and fractions from the rhizome of Cnidium officinale against Dermanyssus gallinae adults was investigated. The C. officinale methanolic extract exhibited 100% acaricidal activity after 48 h of treatment at a dose of 4000 ppm. The acaricidal constituents of the plant were sequentially partitioned with several solvents and then purified using silica gel column chromatography and high-performance liquid chromatography. Gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy revealed (Z)-ligustilide as a constituent of C. officinale. Acaricidal activity was examined in three experimental tests (spray, fumigation and contact), with the spraying method being the most effective. The methanolic extract of C. officinale showed both contact and fumigant activities, though only fumigant activity was observed with (Z)-ligustilide. The fumigant effects of the methanolic extract and (Z)-ligustilide caused 86.5 and 62.6% mortality, respectively, of D. gallinae adults at 48 h. Among (Z)-ligustilide, acaricides (bifenthrin, cypermethrin and spinosad) and butylidenephthalide, bifenthrin displayed the highest acaricidal activity, and the activity of butylidenephthalide was 2.3-fold higher than that of (Z)-ligustilide. These results suggest that C. officinale-derived material can be used for the development of a control agent for D. gallinae.     

Effects of chilling stress on the accumulation of soluble sugars and their key enzymes in <Emphasis Type="Italic">Jatropha curcas</Emphasis> seedlings

As osmolytes and signaling molecules, soluble sugars participate in the response and adaptation of plants to environmental stresses. In the present study, we measured the effect of chilling (12 °C) stress on the contents of eight soluble sugars in the leaves, cotyledons, stems, and roots of Jatropha curcas seedlings, as well as on the activities of eight rate-limiting enzymes that are critical to the metabolism of those soluble sugars. Chilling stress promoted both starch hydrolysis and soluble sugar accumulation. The soluble sugar contents of the leaves and cotyledons were affected more than that of the stems and roots. Meanwhile, the activities of the corresponding metabolic enzymes (e.g., β-amylase, uridine diphosphate glucose phosphorylase, and sucrose phosphate synthase) also increased in some organs. The gradual increase of soluble neutral alkaline invertase activity in the four studied organs suggested that sucrose catabolic production, such as glucose and fructose, was especially important in determining resistance to chilling stress and hexose signal transduction pathway. In addition, the substantial accumulation of raffinose family oligosaccharides and increase in corresponding metabolic enzyme activity suggested that galactinol and raffinose play an important role in determining the chilling resistance of J. curcas. Together, these findings establish a foundation for determining the relationship between the chilling resistance and soluble sugar accumulation of J. curcas and for investigating the mechanisms underlying sugar signaling transduction and stress responses.     

Molecular cloning and functional analysis of <Emphasis Type="Italic">DoUGE</Emphasis> related to water-soluble polysaccharides from <Emphasis Type="Italic">Dendrobium officinale</Emphasis> with enhanced abiotic stress tolerance

UDP glucose 4-epimerase (UGE), an enzyme with significant impacts on sugar metabolism, catalyzes the reversible inter-conversion between UDP-glucose and UDP-galactose. However, very little is known about whether UGE plays a critical role in the accumulation of water-soluble polysaccharide (WSP) and its relationship to abiotic stress tolerance. Here, DoUGE from D. officinale, encoding UGE localized in the cytoplasm, was initially cloned and analyzed. DoUGE exhibited highly tissue-specific expression patterns. The highest expression was in the stems of seedlings and adult plants. The content of WSPs ranged from 168.43 to 416.12 mg g^?1 DW from developmental stages S1 to S4, the highest value being in S3. DoUGE was expressed throughout S1 to S4, with a maximum in S3. This trend was similar in three cultivated varieties (T10, T32-5 and T636). There was a positive correlation between DoUGE expression and the content of WSPs (R ² ?=?0.94; p?<?0.01). Furthermore, promoter analysis showed its possible role in responses to abiotic stresses. Transgenic Arabidopsis thaliana seedlings overexpressing DoUGE accumulated 34.84–44.78% more WSPs, showed 26.24–32.79% more UGE activity, and had a 1.19–1.31-fold higher chlorophyll content than the wild type. Transgenic plants also showed a 50.84 and 34.33% increase in the average content of glucose and galactose, respectively. Transgenic lines growing in half-strength Murashige and Skoog medium containing 150 mM NaCl or 200 mM mannitol displayed enhanced root length and fresh weight, as well as lower proline and malondialdehyde accumulation under salt and osmotic stresses, indicating that the DoUGE gene could be used to improve tolerance to abiotic stress in crops and medicinal or ornamental plants. Our results provide genetic evidence for the involvement of DoUGE in the regulation of WSP content during plant development in D. officinale, as well as in enhanced tolerance to salt and osmotic stresses.     

Improvement of uridine production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by metabolic engineering

Objectives

To construct a Bacillus subtilis strain for improved uridine production.

Results

The AAG_2846–2848 fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation.

Conclusion

This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.

     

Identification,characterization, and expression of the <Emphasis Type="Italic">SWEET</Emphasis> gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis> and <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

Sugars are important molecules that function not only as primary metabolites, but also as nutrients and signal molecules in plants. The sugar transport protein genes family SWEET has been recently identified. The availability of the Dendrobium officinale and Phalaenopsis equestris genome sequences offered the opportunity to study the SWEET gene family in this two orchid species. We identified 22 and 16 putative SWEET genes, respectively, in the genomes of D. officinale and P. equestris using comprehensive bioinformatics analysis. Based on phylogenetic comparisons with SWEET proteins from Arabidopsis and rice, the DoSWEET and PeSWEET proteins could be divided into four clades; among these, clade II specifically lacked PeSWEETs and clade IV specifically lacked DoSWEETs, and there were orthologs present between D. officinale and P. equestris. Protein sequence alignments suggest that there is a predicted serine phosphorylation site in each of the highly conserved MtN3/saliva domain regions. Gene expression analysis in four tissues showed that three PeSWEET genes were most highly expressed in the flower, leaf, stem, and root, suggesting that these genes might play important roles in growth and development in P. equestris. Analysis of gene expression in different floral organs showed that five PeSWEET genes were highly expressed in the column (gynostemium), implying their possible involvement in reproductive development in this species. The expression patterns of seven PeSWEETs in response to different abiotic stresses showed that three genes were upregulated significantly in response to high temperature and two genes were differently expressed at low temperature. The results of this study lay the foundation for further functional analysis of SWEET genes in orchids.     

Characteristic of Polysaccharide Complexes from <Emphasis Type="Italic">Centaurea scabiosa</Emphasis> L. and <Emphasis Type="Italic">Centaurea pseudomaculosa</Emphasis> Dobrocz.

We characterized polysaccharide complexes from Centaurea scabiosa L. and Centaurea pseudomaculosа Dobrocz. We proposed the technique of sequential selection of water-soluble polysaccharides and pectin substances from the aerial parts of studied objects. We have discovered that the content of water-soluble polysaccharides in the aerial parts of C. scabiosa was 2.8 times higher (2.7 ± 0.3%, n = 3) than in C. pseudomaculosа (0.97 ± 0.50%, n = 3). The content of pectin substances in the aerial parts of C. scabiosa was 2 times higher (7.6 ± 0.4%, n = 3) than in C. pseudomaculosа (3.9 ± 0.3%, n = 3). The residues of D-galacturonic acid, L-rhamnose, D-xylose, D-mannose, D-glucose, and D-galactose are the monomeric units of polysaccharide complexes from C. scabiosa and C. pseudomaculosa. Using ion-exchange chromatography, three polysaccharide fractions (molecular weights 667, 722, and 1027 kDa), whose monomer units are D-galacturonic acid, L-rhamnose, D-galactose, D-xylose, and D-glucose were isolated from the water-soluble polysaccharides of C. scabiosa.     

Cross regulation between mTOR signaling and <Emphasis Type="Italic">O</Emphasis>-GlcNAcylation

The hexosamine biosynthetic pathway (HBP) integrates glucose, amino acids, fatty acids and nucleotides metabolisms for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is the nucleotide sugar donor for O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) processes. O-GlcNAc transferase (OGT) is the enzyme which transfers the N-acetylglucosamine (O-GlcNAc) residue onto target proteins. Several studies previously showed that glucose metabolism dysregulations associated with obesity, diabetes or cancer correlated with an increase of OGT expression and global O-GlcNAcylation levels. Moreover, these diseases present an increased activation of the nutrient sensing mammalian target of rapamycin (mTOR) pathway. Other works demonstrate that mTOR regulates protein O-GlcNAcylation in cancer cells through stabilization of OGT. In this context, we studied the cross-talk between these two metabolic sensors in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells. We report that levels of OGT and O-GlcNAcylation are increased in obese mice colon tissues and colon cancer cells and are associated with a higher activation of mTOR signaling. In parallel, treatments with mTOR regulators modulate OGT and O-GlcNAcylation levels in both normal and colon cancer cells. However, deregulation of O-GlcNAcylation affects mTOR signaling activation only in cancer cells. Thus, a crosstalk exists between O-GlcNAcylation and mTOR signaling in contexts of metabolism dysregulation associated to obesity or cancer.     

Characterization of <Emphasis Type="Italic">SWEET</Emphasis> family members from loquat and their responses to exogenous induction

The plant SWEET family is a sugar transporter family that plays a significant role in plant development. Here, seven loquat SWEET family members were identified by RNA-seq. These were designated as EjSWEET1, EjSWEET2a, EjSWEET2b, EjSWEET2c, EjSWEET4, EjSWEET15, and EjSWEET17. Phylogenetic and predictive functional annotation analyses suggest that the loquat SWEETs are classified as having sucrose, glucose and fructose transportation features. The in vivo responses of loquat SWEETs to exogenous sugar or NaCl was investigated by applying high concentrations of sugar or salt to 7-month-old loquat seedlings cultured in a nutrient medium. The results showed that most loquat SWEET genes can respond to exogenous applications of sucrose, glucose, fructose and salt. The response of EjSWEET1 to exogenous fructose was faster than the others, indicating that EjSWEET1 is more sensitive to exogenous fructose compared with other loquat SWEETs. EjSWEET15 can be induced by sucrose, but is suppressed by glucose. This indicates its possible role in sucrose transporting. The response of loquat SWEETs to NaCl showed broadly similar patterns compared to sugars. However, after a longer time of NaCl treatment, most loquat SWEETs are upregulated, especially EjSWEET15. This indicates its long-term response to high salinity.     

Carrageenans from <Emphasis Type="Italic">Sarcothalia crispata</Emphasis> and <Emphasis Type="Italic">Gigartina skottsbergii</Emphasis>: Structural Analysis and Interpolyelectrolyte Complex Formation for Drug Controlled Release

The aims of the present study were to characterize for the first time the carrageenan extracted from cystocarpic stage of S. crispata collected in the Patagonian coast of Argentina, and to prepare interpolyelectrolytic complexes (IPECs) between the polysaccharide extracted from cystocarpic stage of Sarcothalia crispata and Gigartina skottsbergii thalli, and basic butylated methacrylate copolymer (Eudragit E), in order to test their potential for the controlled release of ibuprofen as model drug. The structural determination revealed that the polysaccharides extracted from S. crispata and G. skottsbergii were mainly constituted by κ-carrageenan, particularly in the case of G. skottsbergii; however, significant amounts of ι- and ν-carrageenan were also detected in both polygalactans. The differences in diad composition and possibly in their distribution along the polysaccharide chain of both carrageenans would favor a different arrangement in the resulting IPEC structure. The smaller pores observed by scanning electron microscopy in the IPEC of S. crispata suggest that the kinks in the polysaccharide backbone are evenly distributed, resulting in a slower ibuprofen release compared to the IPEC of G. skottsbergii.     

A highly branched storage polyglucan in the thermoacidophilic red microalga <Emphasis Type="Italic">Galdieria maxima</Emphasis> cells

The thermoacidophilic red alga Galdieria maxima is capable of heterotrophic growth. The content of carbohydrates in G. maxima grown heterotrophically increases by a factor of 4, reaching as much as 60% of cell dry weight. The increase in the level of carbohydrates in cells is due to accumulation of a storage α-glycan. According to a specific cleavage to glucose catalyzed by amyloglucosidase and the high positive specific optical rotation characteristic of polyglucans, this polysaccharide can be classified as a floridean starch. The data of ¹H NMR spectroscopy and the results of methylation showed that the average length of the unbranched regions of the polysaccharide molecule is six to seven glucose residues. The degree of branching of the starch molecule of G. maxima is greater than that of storage polysaccharides of other red algae, glycogens of yeast, and phytoglycogens of cyanobacteria.     

Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of non-stoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, theposition of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by ¹³C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner.

Open image in new window

where L-Rhap3Rlac2Ac is 2-O-acetyl-3-O-[(R-1-carboxyethyl]-L-rhamnose

     

Metabolic reconstruction and experimental verification of glucose utilization in <Emphasis Type="Italic">Desulfurococcus amylolyticus</Emphasis> DSM 16532

Desulfurococcus amylolyticus DSM 16532 is an anaerobic and hyperthermophilic crenarchaeon known to grow on a variety of different carbon sources, including monosaccharides and polysaccharides. Furthermore, D. amylolyticus is one of the few archaea that are known to be able to grow on cellulose. Here, we present the metabolic reconstruction of D. amylolyticus’ central carbon metabolism. Based on the published genome, the metabolic reconstruction was completed by integrating complementary information available from the KEGG, BRENDA, UniProt, NCBI, and PFAM databases, as well as from available literature. The genomic analysis of D. amylolyticus revealed genes for both the classical and the archaeal version of the Embden-Meyerhof pathway. The metabolic reconstruction highlighted gaps in carbon dioxide-fixation pathways. No complete carbon dioxide-fixation pathway such as the reductive citrate cycle or the dicarboxylate-4-hydroxybutyrate cycle could be identified. However, the metabolic reconstruction indicated that D. amylolyticus harbors all genes necessary for glucose metabolization. Closed batch experimental verification of glucose utilization by D. amylolyticus was performed in chemically defined medium. The findings from in silico analyses and from growth experiments are discussed with respect to physiological features of hyperthermophilic organisms.     

Sucrose dependent mineral phosphate solubilization in <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> PSI3 by heterologous overexpression of periplasmic invertases

Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.     

Polysaccharide degradation systems of the saprophytic bacterium <Emphasis Type="Italic">Cellvibrio japonicus</Emphasis>

Study of recalcitrant polysaccharide degradation by bacterial systems is critical for understanding biological processes such as global carbon cycling, nutritional contributions of the human gut microbiome, and the production of renewable fuels and chemicals. One bacterium that has a robust ability to degrade polysaccharides is the Gram-negative saprophyte Cellvibrio japonicus. A bacterium with a circuitous history, C. japonicus underwent several taxonomy changes from an initially described Pseudomonas sp. Most of the enzymes described in the pre-genomics era have also been renamed. This review aims to consolidate the biochemical, structural, and genetic data published on C. japonicus and its remarkable ability to degrade cellulose, xylan, and pectin substrates. Initially, C. japonicus carbohydrate-active enzymes were studied biochemically and structurally for their novel polysaccharide binding and degradation characteristics, while more recent systems biology approaches have begun to unravel the complex regulation required for lignocellulose degradation in an environmental context. Also included is a discussion for the future of C. japonicus as a model system, with emphasis on current areas unexplored in terms of polysaccharide degradation and emerging directions for C. japonicus in both environmental and biotechnological applications.     

Mining and characterization of EST-SSR markers for <Emphasis Type="Italic">Zingiber officinale</Emphasis> Roscoe with transferability to other species of Zingiberaceae

Zingiber officinale is a model spice herb, well known for its medicinal value. It is primarily a vegetatively propagated commercial crop. However, considerable diversity in its morphology, fiber content and chemoprofiles has been reported. The present study explores the utility of EST-derived markers in studying genetic diversity in different accessions of Z. officinale and their cross transferability within the Zingiberaceae family. A total of 38,115 ESTs sequences were assembled to generate 7850 contigs and 10,762 singletons. SSRs were searched in the unigenes and 515 SSR-containing ESTs were identified with a frequency of 1 SSR per 25.21 kb of the genome. These ESTs were also annotated using BLAST2GO. Primers were designed for 349 EST-SSRs and 25 primer pairs were randomly picked for EST SSR study. Out of these, 16 primer pairs could be optimized for amplification in different accessions of Z. officinale as well as other species belonging to Zingiberaceae. GES454, GES466, GES480 and GES486 markers were found to exhibit 100% cross-transferability among different members of Zingiberaceae.