Genome Reduction and Secondary Metabolism of the Marine Sponge-Associated Cyanobacterium Leptothoe

Sponges form symbiotic relationships with diverse and abundant microbial communities. Cyanobacteria are among the most important members of the microbial communities that are associated with sponges. Here, we performed a genus-wide comparative genomic analysis of the newly described marine benthic cyanobacterial genus Leptothoe (Synechococcales). We obtained draft genomes from Le. kymatousa TAU-MAC 1615 and Le. spongobia TAU-MAC 1115, isolated from marine sponges. We identified five additional Leptothoe genomes, host-associated or free-living, using a phylogenomic approach, and the comparison of all genomes showed that the sponge-associated strains display features of a symbiotic lifestyle. Le. kymatousa and Le. spongobia have undergone genome reduction; they harbored considerably fewer genes encoding for (i) cofactors, vitamins, prosthetic groups, pigments, proteins, and amino acid biosynthesis; (ii) DNA repair; (iii) antioxidant enzymes; and (iv) biosynthesis of capsular and extracellular polysaccharides. They have also lost several genes related to chemotaxis and motility. Eukaryotic-like proteins, such as ankyrin repeats, playing important roles in sponge-symbiont interactions, were identified in sponge-associated Leptothoe genomes. The sponge-associated Leptothoe stains harbored biosynthetic gene clusters encoding novel natural products despite genome reduction. Comparisons of the biosynthetic capacities of Leptothoe with chemically rich cyanobacteria revealed that Leptothoe is another promising marine cyanobacterium for the biosynthesis of novel natural products.     

A Draft Genome Sequence Reveals the Helminthosporium solani Arsenal for Cell Wall Degradation

Helminthosporium solani is a slow-growing fungal pathogen belonging to the family Massarinaceae. It causes blemishes on potato tubers, affecting processing and fresh market trade. Despite its worldwide distribution, little is known about the biology of H. solani. Here we report the generation of a draft genome sequence of H. solani with an estimated genome size of ~35 megabases, for use in phylogenetic and pathogenicity studies of this fungal pathogen. This sequence is also the first reference genome within the family Massarinaceae. We identified a large suite of genes in the H. solani genome that encode putative cell wall degrading enzymes. Based on comparison with other genomes, we speculate that H. solani is a hemibiotroph or necrotroph. The presence of a large number of genes in the glycoside hydrolase (GH) 10 and 43 families, which aid in the hydrolysis of glucoronoarabinoxylan, also suggests that H. solani may be able to survive on grass hosts and indicates the need to re-examine the life cycle and host range of this pathogen.     

Comparative Analysis of Glycoside Hydrolases Activities from Phylogenetically Diverse Marine Bacteria of the Genus Arenibacter

A total of 16 marine strains belonging to the genus Arenibacter, recovered from diverse microbial communities associated with various marine habitats and collected from different locations, were evaluated in degradation of natural polysaccharides and chromogenic glycosides. Most strains were affiliated with five recognized species, and some presented three new species within the genus Arenibacter. No strains contained enzymes depolymerizing polysaccharides, but synthesized a wide spectrum of glycosidases. Highly active β-N-acetylglucosaminidases and α-N-acetylgalactosaminidases were the main glycosidases for all Arenibacter. The genes, encoding two new members of glycoside hydrolyses (GH) families, 20 and 109, were isolated and characterized from the genomes of Arenibacter latericius. Molecular genetic analysis using glycosidase-specific primers shows the absence of GH27 and GH36 genes. A sequence comparison with functionally-characterized GH20 and GH109 enzymes shows that both sequences are closest to the enzymes of chitinolytic bacteria Vibrio furnissii and Cellulomonas fimi of marine and terrestrial origin, as well as human pathogen Elisabethkingia meningoseptica and simbionts Akkermansia muciniphila, gut and non-gut Bacteroides, respectively. These results revealed that the genus Arenibacter is a highly taxonomic diverse group of microorganisms, which can participate in degradation of natural polymers in marine environments depending on their niche and habitat adaptations. They are new prospective candidates for biotechnological applications due to their production of unique glycosidases.     

A New,Quick, and Simple Protocol to Evaluate Microalgae Polysaccharide Composition

In this work, a new methodological approach, relying on the high specificity of enzymes in a complex mixture, was developed to estimate the composition of bioactive polysaccharides produced by microalgae, directly in algal cultures. The objective was to set up a protocol to target oligomers commonly known to be associated with exopolysaccharides’ (EPS) nutraceutical and pharmaceutical activities (i.e., rhamnose, fucose, acidic sugars, etc.) without the constraints classically associated with chromatographic methods, while maintaining a resolution sufficiently high to enable their monitoring in the culture system. Determination of the monosaccharide content required the application of acid hydrolysis (2 M trifluoroacetic acid) followed by NaOH (2 M) neutralization. Quantification was then carried out directly on the fresh hydrolysate using enzyme kits corresponding to the main monosaccharides in a pre-determined composition of the polysaccharides under analysis. Initial results showed that the enzymes were not sensitive to the presence of TFA and NaOH, so the methodology could be carried out on fresh hydrolysate. The limits of quantification of the method were estimated as being in the order of the log of nanograms of monosaccharides per well, thus positioning it among the chromatographic methods in terms of analytical performance. A comparative analysis of the results obtained by the enzymatic method with a reference method (high-performance anion-exchange chromatography) confirmed good recovery rates, thus validating the closeness of the protocol. Finally, analyses of raw culture media were carried out and compared to the results obtained in miliQ water; no differences were observed. The new approach is a quick, functional analysis method allowing routine monitoring of the quality of bioactive polysaccharides in algal cultures grown in photobioreactors.     

Marine polysaccharides: a source of bioactive molecules for cell therapy and tissue engineering

The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans, is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics. Advances in our understanding of the biosynthesis, structure and function of complex glycans from mammalian origin have shown the crucial role of this class of molecules to modulate disease processes and the importance of a deeper knowledge of structure-activity relationships. Marine environment offers a tremendous biodiversity and original polysaccharides have been discovered presenting a great chemical diversity that is largely species specific. The study of the biological properties of the polysaccharides from marine eukaryotes and marine prokaryotes revealed that the polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides such as glycosaminoglycans. Marine polysaccharides present a real potential for natural product drug discovery and for the delivery of new marine derived products for therapeutic applications.     

Seaweed polysaccharides and derived oligosaccharides stimulate defense responses and protection against pathogens in plants

Plants interact with the environment by sensing "non-self" molecules called elicitors derived from pathogens or other sources. These molecules bind to specific receptors located in the plasma membrane and trigger defense responses leading to protection against pathogens. In particular, it has been shown that cell wall and storage polysaccharides from green, brown and red seaweeds (marine macroalgae) corresponding to ulvans, alginates, fucans, laminarin and carrageenans can trigger defense responses in plants enhancing protection against pathogens. In addition, oligosaccharides obtained by depolymerization of seaweed polysaccharides also induce protection against viral, fungal and bacterial infections in plants. In particular, most seaweed polysaccharides and derived oligosaccharides trigger an initial oxidative burst at local level and the activation of salicylic (SA), jasmonic acid (JA) and/or ethylene signaling pathways at systemic level. The activation of these signaling pathways leads to an increased expression of genes encoding: (i) Pathogenesis-Related (PR) proteins with antifungal and antibacterial activities; (ii) defense enzymes such as pheylalanine ammonia lyase (PAL) and lipoxygenase (LOX) which determine accumulation of phenylpropanoid compounds (PPCs) and oxylipins with antiviral, antifugal and antibacterial activities and iii) enzymes involved in synthesis of terpenes, terpenoids and/or alkaloids having antimicrobial activities. Thus, seaweed polysaccharides and their derived oligosaccharides induced the accumulation of proteins and compounds with antimicrobial activities that determine, at least in part, the enhanced protection against pathogens in plants.     

CRISPR/Cas9: Development and Application in Rice Breeding

Rice(Oryza sativa L.) is an important staple food crop worldwide due to its adaptability to different environmental conditions. Because of its great economic and social importance, there is a constant requirement for new varieties with improved agronomic characteristics, such as tolerance to different biotic(such as bacterium, fungus, insect and virus) and abiotic stresses(such as salinity, drought and temperature), higher yield and better organoleptic and nutritional value. Among the new genome editing technologies, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)(CRISPR/Cas) system allows precise and specific edition in a targeted genome region. It is one of the most frequently used techniques for the study of the function of new genes and for the development of mutant lines with enhanced tolerance to biotic and abiotic stresses, herbicide resistance or improved yield. The wide varieties of applications for this technology include simple non-homologous end joining, homologous recombination, gene replacement, and base editing. In this review, we analyzed how some of these applications have been used in rice cultivars to obtain rice varieties better adapted to current environmental conditions and market requirements.     

转基因与常规杂交相结合改良水稻耐盐性

通过农杆菌介导法和基因枪法将CMO、BADH、mtlD、gutD和SAMDC基因以单价或双价的形式导入水稻常规品种中,再结合常规杂交育种,选育5价强耐盐性的转基因水稻植株。1~5价9类组合的水稻品系,经PCR分子检测,在转基因后代中多价目的基因聚合,遗传稳定,且分子检测与田间耐盐性的表现一致。转基因植株在盐碱地中能正常生长,拓展了水稻常规品种耐盐性。并已获得耐0.5%~1.0%NaCl的T秀水11——品3、品6和品7等9份优良株系或中间材料。     

Screen of Genes Linked to High-Sugar Content in Stems by Comparative Genomics

One of the great advantages of the fully sequenced rice genome is to serve as a reference for other cereal genomes in particular for identifying genes linked to unique traits. A trait of great interest is reduced lignocellulose in the stem of related species in favor of fermentable sugars as a source of biofuels. While sugarcane is one of the most efficient biofuel crops, little is known about the underlying gene repertoire involved in it. Here, we take advantage of the natural variation of sweet and grain sorghum to uncover genes that are conserved in rice, sorghum, and sugarcane but differently expressed in sweet versus grain sorghum by using a microarray platform and the syntenous alignment of rice and sorghum genomic regions containing these genes. Indeed, enzymes involved in carbohydrate accumulation and those that reduce lignocellulose can be identified.     

Laminarin,a Major Polysaccharide in Stramenopiles

During the processes of primary and secondary endosymbiosis, different microalgae evolved to synthesis different storage polysaccharides. In stramenopiles, the main storage polysaccharides are β-1,3-glucan, or laminarin, in vacuoles. Currently, laminarin is gaining considerable attention due to its application in the food, cosmetic and pharmaceuticals industries, and also its importance in global biogeochemical cycles (especially in the ocean carbon cycle). In this review, the structures, composition, contents, and bioactivity of laminarin were summarized in different algae. It was shown that the general features of laminarin are species-dependence. Furthermore, the proposed biosynthesis and catabolism pathways of laminarin, functions of key genes, and diel regulation of laminarin were also depicted and comprehensively discussed for the first time. However, the complete pathways, functions of genes, and diel regulatory mechanisms of laminarin require more biomolecular studies. This review provides more useful information and identifies the knowledge gap regarding the future studies of laminarin and its applications.     

Hemicellulase production in Chrysosporium lucknowense C1

Filamentous fungi are widely used for enzyme production for the biofuel industry. The ascomycetous fungus Chrysosporium lucknowense C1 was isolated as a natural producer of neutral cellulases. It is at present an attractive alternative to well known fungi like Aspergillus sp. and Trichoderma reesei for protein production on a commercial scale. Besides many cellulases, a large number of hemicellulases (particularly xylanases and arabinofuranosidases) and esterases (acetyl xylan esterases and ferulic acid esterases) encoding genes have also been identified in the C1 genome. Many of these extracellular enzymes have been selectively expressed in C1 and then purified and characterized. Four arabinofuranosidases, two acetyl xylan esterases, two ferulic acid esterases, an α-glucuronidase and four xylanases have been purified and characterized. All these enzymes were found to be active towards arabinoxylans, demonstrating the high potential of C1 as a producer of hemicellulolytic enzymes.     

巴西橡胶树乳管细胞中5个橡胶合成关键酶基因节律性表达研究

以巴西橡胶树无性系PR107、RRIM600、CATAS7-33-97和CATAS8-79的未开割树为材料,采用荧光定量PCR技术,分析乳管细胞中HMGR1、FDPS、SRPP、REF和HRT2等5个橡胶生物合成关键酶基因的昼夜表达模式。结果表明：5个基因的表达均具有节律性,而且不同基因在同一无性系中以及同一基因在不同无性系间的节律性表达模式都有一定程度的差异。结果为分析不同环境因素对橡胶生物合成相关酶基因节律性表达的影响具有一定参考价值。     

稻瘟病菌类LxAR家族基因的预测及表达分析

在侵染水稻过程中,稻瘟病菌分泌一系列效应因子来抑制寄主免疫系统。无毒效应因子AVR Pii和AvrPiz-t的氨基酸序列含有类LxAR基序,基于此,本研究利用生物信息学技术,从稻瘟病菌基因组中筛选获得了99个含有类LxAR基序的假定效应因子(MoLLEs)。该家族蛋白都为小分子蛋白(70~300 aa),富含半胱氨酸氨基,绝大多数为未知功能的蛋白。24个蛋白含已知的功能域,可能涉及了多种生命过程,其中有9个蛋白编码植物细胞壁降解酶。通过表达模式分析发现,MoLLEs家族基因成员的表达受到饥饿胁迫、附着胞生长发育以及侵染水稻等过程的诱导,此外一部分基因受到附着胞发育关键基因PMK1调控。基因表达交叉分析表明,部分基因同时在附着胞发育和致病过程中上调表达。在与水稻的相互作用中,MoLLEs在稻瘟病菌侵染不同阶段均有上调表达,并且部分基因受亲和或非亲和反应特异性诱导。      

大豆天冬氨酸代谢途径关键酶基因电子定位与结构分析

运用电子定位的方法,利用大豆基因组物理图和遗传图的整合图谱,完成了17个大豆天冬氨酸代谢途径中关键酶基因的定位以及结构分析。结果表明:这些酶基因分别定位在A1、B2、D1a、D1b、D2、F、G、H、J、L和N等11个连锁群上,并获得了相应连锁群区间两侧的标记;在基因结构分析上,AHAS、DHDPS、HK与TS没有内含子,DHAD基因的内含子数目最多,为13个,AK基因有12个内含子。通过定位获得的对应分子标记可以为基因分子辅助育种奠定基础,而基因结构信息能更好的实现基因功能分析。     

The Glycolytic Enzymes Activity in the Midgut of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) adult and their Seasonal Changes

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is an important pest of maize. The diet of the D. virgifera imago is rich in starch and other polysaccharides present in cereals such as maize. Therefore, knowledge about enzymes involved in digestion of such specific food of this pest seems to be important. The paper shows, for the first time, the activities of main glycolytic enzymes in the midgut of D. virgifera imago: endoglycosidases (α-amylase, cellulase, chitinase, licheninase, laminarinase); exoglycosidases (α- and β-glucosidases, α- and β-galactosidases) and disaccharidases (maltase, isomaltase, sucrase, trehalase, lactase, and cellobiase). Activities of α-amylase, α-glucosidase, and maltase were the highest among assayed endoglycosidases, exoglycosidases, and disaccharidases, respectively. This indicates that in the midgut of D. virgifera imago α-amylase, α-glucosidase and maltase are important enzymes in starch hydrolysis and products of its digestion. These results lead to conclusion that inhibition of most active glycolytic enzymes of D. virgifera imago may be another promising method for chemical control of this pest of maize.     

天然橡胶合成关键酶基因Hmg1启动子克隆与序列分析

在巴西橡胶树天然橡胶的生物合成途径中,3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-Hydroxy-3-MethylglutarylCoenzymeAReductase,HMGR)是关键酶之一,为了利用其基因的启动子和瞬时表达系统来筛选促进橡胶树生物合成的调节剂,利用GenomicWalking方法从高产树热研7-33-97的叶片基因组DNA中克隆了Hmg15′端调控序列。序列分析结果表明,巴西橡胶树Hmg1启动子转录起始位点的保守序列CTCATCA,与其他植物基因的非常一致;在5′端调控序列中发现几个典型的真核生物顺式调控元件TATA-motif、CAAT-motif、CGTCA-motif、ERE等。用克隆得到的Hmg15′端启动子序列分别替换了pCAMBIA1302中的CaMV35S启动子,构建了序列缺失pCAMBIA1302表达载体pCAMBIA1302H900、pCAMBIA1302H500和pCAMBIA1302H300。     

Characterization of the exopolysaccharide produced by Salipiger mucosus A3, a halophilic species belonging to the Alphaproteobacteria, isolated on the Spanish Mediterranean seaboard

We have studied the exopolysaccharide produced by the type strain of Salipiger mucosus, a species of halophilic, EPS-producing (exopolysaccharide-producing) bacterium belonging to the Alphaproteobacteria. The strain, isolated on the Mediterranean seaboard, produced a polysaccharide, mainly during its exponential growth phase but also to a lesser extent during the stationary phase. Culture parameters influenced bacterial growth and EPS production. Yield was always directly related to the quantity of biomass in the culture. The polymer is a heteropolysaccharide with a molecular mass of 250 kDa and its components are glucose (19.7%, w/w), mannose (34%, w/w), galactose (32.9%, w/w) and fucose (13.4%, w/w). Fucose and fucose-rich oligosaccharides have applications in the fields of medicine and cosmetics. The chemical or enzymatic hydrolysis of fucose-rich polysaccharides offers a new efficient way to process fucose. The exopolysaccharide in question produces a solution of very low viscosity that shows pseudoplastic behavior and emulsifying activity on several hydrophobic substrates. It also has a high capacity for binding cations and incorporating considerable quantities of sulfates, this latter feature being very unusual in bacterial polysaccharides.     

Regulatory Function of Histone Modifications in Controlling Rice Gene Expression and Plant Growth

Histone modifications play pivotal roles in chromatin remodeling and gene regulation. Rice genome possesses multiple genes encoding different classes of histone modification enzymes. Specific histone modification patterns in rice are associated with either heterochromatic or euchromatic regions or related to gene expression. Functional studies of several rice genes encoding histone deacetylases and histone methyltransferases and demethylases reveal specific regulators involved in transposon repression, development regulation, and responses to environmental conditions. Functional interplay between rice histone modification regulators in gene regulation and transposon silencing and their implication in rice epigenetic variation are discussed.