Overexpression of R2R3-MYB IbMYB1a induces anthocyanin pigmentation in soybean cotyledon.

KEY MESSAGE: This is the first report showing anthocyanin accumulation in the soybean cotyledon via genetic transformation of a single gene. Soybean [Glycine max (L.) Merrill] contains valuable components, including anthocyanins. To enhance anthocyanin production in Korean soybean Kwangankong, we utilized the R2R3-type MYB gene (IbMYB1a), known for inducing anthocyanin pigmentation in Arabidopsis. This gene was incorporated into constructs using two promoters: the CaMV 35S promoter (P35S) and the ß-conglycinin promoter (Pß-con). Kwangankong was transformed using Agrobacterium, and the presence of IbMYB1a and Bar transgenes in T0 plants was confirmed through polymerase chain reaction (PCR), followed by gene expression validation. Visual inspection revealed that one P35S:IbMYB1a and three Pß-con:IbMYB1a lines displayed seed color change. Pß-con:IbMYB1a T1 seeds accumulated anthocyanins in cotyledon outer layers, whereas P35S:IbMYB1a and non-transgenic black soybean (Cheongja 5 and Seum) accumulated anthocyanins in the seed coat. During the germination and growth phase, T1 seedlings from Pß-con:IbMYB1a lines exhibited anthocyanin pigmentation in cotyledons for up to 1 month without growth aberrations. High-performance liquid chromatography confirmed cyanidin-3-O-glucoside as the major anthocyanin in the Pß-con:IbMYB1a line (#3). We analyzed the expression patterns of anthocyanin biosynthesis genes, chalcone synthase 7,8, chalcone isomerase 1A, flavanone 3-hydroxylase, flavanone 3'-hydroxylase, dihydroflavanol reductase 1, dihydroflavanol reductase 2, anthocyanidin synthase 2, anthocyanidin synthase 3, and UDP glucose flavonoid 3-O-glucosyltransferase in transgenic and control Kwangankong and black soybean (Cheongja 5 and Seum) seeds using quantitative real-time PCR. We conclude that the induction of gene expression in transgenic plants in comparison with Kwangankong was attributable to IbMYB1a transformation. Notably, flavanone 3-hydroxylase, flavanone 3'-hydroxylase, and dihydroflavanol reductase 1 were abundantly expressed in black soybean seed coat, distinguishing them from transgenic cotyledons.

Uncovering transcriptional reprogramming during callus development in soybean: insights and implications.

Callus, a valuable tool in plant genetic engineering, originates from dedifferentiated cells. While transcriptional reprogramming during callus formation has been extensively studied in Arabidopsis thaliana, our knowledge of this process in other species, such as Glycine max, remains limited. To bridge this gap, our study focused on conducting a time-series transcriptome analysis of soybean callus cultured for various durations (0, 1, 7, 14, 28, and 42 days) on a callus induction medium following wounding with the attempt of identifying genes that play key roles during callus formation. As the result, we detected a total of 27,639 alterations in gene expression during callus formation, which could be categorized into eight distinct clusters. Gene ontology analysis revealed that genes associated with hormones, cell wall modification, and cell cycle underwent transcriptional reprogramming throughout callus formation. Furthermore, by scrutinizing the expression patterns of genes related to hormones, cell cycle, cell wall, and transcription factors, we discovered that auxin, cytokinin, and brassinosteroid signaling pathways activate genes involved in both root and shoot meristem development during callus formation. In summary, our transcriptome analysis provides significant insights into the molecular mechanisms governing callus formation in soybean. The information obtained from this study contributes to a deeper understanding of this intricate process and paves the way for further investigation in the field.

Mutation of GmIPK1 Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds.

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP5) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T0 generation at editing frequencies of 0.1-84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1-9 nucleotides and insertions of 1 nucleotide in several soybean lines (T0). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T1 plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T2 seeds but did not show any defects in plant growth and seed development.

Dissecting seed pigmentation-associated genomic loci and genes by employing dual approaches of reference-based and k-mer-based GWAS with 438 Glycine accessions.

The soybean is agro-economically the most important among all cultivated legume crops, and its seed color is considered one of the most attractive factors in the selection-by-breeders. Thus, genome-wide identification of genes and loci associated with seed colors is critical for the precision breeding of crop soybeans. To dissect seed pigmentation-associated genomic loci and genes, we employed dual approaches by combining reference-based genome-wide association study (rbGWAS) and k-mer-based reference-free GWAS (rfGWAS) with 438 Glycine accessions. The dual analytical strategy allowed us to identify four major genomic loci (designated as SP1-SP4 in this study) associated with the seed colors of soybeans. The k-mer analysis enabled us to find an important recombination event that occurred between subtilisin and I-cluster B in the soybean genome, which could describe a special structural feature of ii allele within the I locus (SP3). Importantly, mapping analyses of both mRNAs and small RNAs allowed us to reveal that the subtilisin-CHS1/CHS3 chimeric transcripts generate and act as an initiator towards 'mirtron (i.e., intron-harboring miRNA precursor)'-triggered silencing of chalcone synthase (CHS) genes. Consequently, the results led us to propose a working model of 'mirtron-triggered gene silencing (MTGS)' to elucidate a long-standing puzzle in the genome-wide CHS gene silencing mechanism. In summary, our study reports four major genomic loci, lists of key genes and genome-wide variations that are associated with seed pigmentation in soybeans. In addition, we propose that the MTGS mechanism plays a crucial role in the genome-wide silencing of CHS genes, thereby suggesting a clue to currently predominant soybean cultivars with the yellow seed coat. Finally, this study will provide a broad insight into the interactions and correlations among seed color-associated genes and loci within the context of anthocyanin biosynthetic pathways.

Comparative transcriptome analysis reveals higher expression of stress and defense responsive genes in dwarf soybeans obtained from the crossing of G. max and G. soja.

BACKGROUND: Plant height is an important component of plant architecture and significantly affects crop breeding practices and yield. Dwarfism in plants prevents lodging and therefore it's a desired trait in crops. OBJECTIVE: To find differentially expressed genes to classify and understand the regulation of genes related to plant growth in mutant dwarf soybeans, which appeared in the F5 generation. METHODS: We obtained a few segregated dwarf soybeans in the populations derived from the crossing of Glycine max var. Peking and Glycine soja var. IT182936 in an F5 RIL population. These dwarf soybeans may be useful genetic resources for plant breeders, geneticists and biologists. Using the Illumina high-throughput platform, transcriptomes were generated and compared among normal and dwarf soybeans in triplicate. CONCLUSION: We found complex relationship of the expressed genes to plant growth. There were highly significantly up-/downregulated genes according to the comparison of gene expression in normal and dwarf soybeans. The genes related to disease and stress responses were found to be upregulated in dwarf soybeans. Such over-expression of disease resistance and other immune response genes can be targeted to understand how the immune genes regulate the response of plant growth. In addition, photosynthesis-related genes showed very low expression in dwarf lines. The transcriptome expression and genes classified as related to plant growth may be useful resources to researchers studying plant growth.

Translational genomics and multi-omics integrated approaches as a useful strategy for crop breeding.

Recent next generation sequencing-driven mass production of genomic data and multi-omics-integrated approaches have significantly contributed to broadening and deepening our knowledge on the molecular system of living organisms. Accordingly, translational genomics (TG) approach can play a pivotal role in creating an informational bridge between model systems and relatively less studied plants. This review focuses mainly on addressing recent advancement in omics-related technologies, a diverse array of bioinformatic resources and potential applications of TG for the crop breeding. To accomplish above objectives, information on omics data production, various DBs and high throughput technologies was collected, integrated, and used to analyze current status and future perspectives towards omics-assisted crop breeding. Various omics data and resources have been organized and integrated into the databases and/or bioinformatic infrastructures, and thereby serve as the ome's information center for cross-genome translation of biological data. Although the size of accumulated omics data and availability of reference genomes are different among plant families, translational approaches have been actively progressing to access particular biological characteristics. When multi-layered omics data are integrated in a synthetic manner, it will allow providing a stereoscopic view of dynamic molecular behavior and interacting networks of genes occurring in plants. Consequently, TG approach will lead us to broader and deeper insights into target traits for the plant breeding. Furthermore, such systems approach will renovate conventional breeding programs and accelerate precision crop breeding in the future.

Reconstruction of a composite comparative map composed of ten legume genomes.

The Fabaceae (legume family) is the third largest and the second of agricultural importance among flowering plant groups. In this study, we report the reconstruction of a composite comparative map composed of ten legume genomes, including seven species from the galegoid clade (Medicago truncatula, Medicago sativa, Lens culinaris, Pisum sativum, Lotus japonicus, Cicer arietinum, Vicia faba) and three species from the phaseoloid clade (Vigna radiata, Phaseolus vulgaris, Glycine max). To accomplish this comparison, a total of 209 cross-species gene-derived markers were employed. The comparative analysis resulted in a single extensive genetic/genomic network composed of 93 chromosomes or linkage groups, from which 110 synteny blocks and other evolutionary events (e.g., 13 inversions) were identified. This comparative map also allowed us to deduce several large scale evolutionary events, such as chromosome fusion/fission, with which might explain differences in chromosome numbers among compared species or between the two clades. As a result, useful properties of cross-species genic markers were re-verified as an efficient tool for cross-species translation of genomic information, and similar approaches, combined with a high throughput bioinformatic marker design program, should be effective for applying the knowledge of trait-associated genes to other important crop species for breeding purposes. Here, we provide a basic comparative framework for the ten legume species, and expect to be usefully applied towards the crop improvement in legume breeding.

Functional classification of rice flanking sequence tagged genes using MapMan terms and global understanding on metabolic and regulatory pathways affected by dxr mutant having defects in light response.

BACKGROUND: Rice is one of the most important food crops for humans. To improve the agronomical traits of rice, the functions of more than 1,000 rice genes have been recently characterized and summarized. The completed, map-based sequence of the rice genome has significantly accelerated the functional characterization of rice genes, but progress remains limited in assigning functions to all predicted non-transposable element (non-TE) genes, estimated to number 37,000-41,000. RESULTS: The International Rice Functional Genomics Consortium (IRFGC) has generated a huge number of gene-indexed mutants by using mutagens such as T-DNA, Tos17 and Ds/dSpm. These mutants have been identified by 246,566 flanking sequence tags (FSTs) and cover 65 % (25,275 of 38,869) of the non-TE genes in rice, while the mutation ratio of TE genes is 25.7 %. In addition, almost 80 % of highly expressed non-TE genes have insertion mutations, indicating that highly expressed genes in rice chromosomes are more likely to have mutations by mutagens such as T-DNA, Ds, dSpm and Tos17. The functions of around 2.5 % of rice genes have been characterized, and studies have mainly focused on transcriptional and post-transcriptional regulation. Slow progress in characterizing the function of rice genes is mainly due to a lack of clues to guide functional studies or functional redundancy. These limitations can be partially solved by a well-categorized functional classification of FST genes. To create this classification, we used the diverse overviews installed in the MapMan toolkit. Gene Ontology (GO) assignment to FST genes supplemented the limitation of MapMan overviews. CONCLUSION: The functions of 863 of 1,022 known genes can be evaluated by current FST lines, indicating that FST genes are useful resources for functional genomic studies. We assigned 16,169 out of 29,624 FST genes to 34 MapMan classes, including major three categories such as DNA, RNA and protein. To demonstrate the MapMan application on FST genes, transcriptome analysis was done from a rice mutant of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene with FST. Mapping of 756 down-regulated genes in dxr mutants and their annotation in terms of various MapMan overviews revealed candidate genes downstream of DXR-mediating light signaling pathway in diverse functional classes such as the methyl-D-erythritol 4-phosphatepathway (MEP) pathway overview, photosynthesis, secondary metabolism and regulatory overview. This report provides a useful guide for systematic phenomics and further applications to enhance the key agronomic traits of rice.

CSGM Designer: a platform for designing cross-species intron-spanning genic markers linked with genome information of legumes.

BACKGROUND: Genetic markers are tools that can facilitate molecular breeding, even in species lacking genomic resources. An important class of genetic markers is those based on orthologous genes, because they can guide hypotheses about conserved gene function, a situation that is well documented for a number of agronomic traits. For under-studied species a key bottleneck in gene-based marker development is the need to develop molecular tools (e.g., oligonucleotide primers) that reliably access genes with orthology to the genomes of well-characterized reference species. RESULTS: Here we report an efficient platform for the design of cross-species gene-derived markers in legumes. The automated platform, named CSGM Designer (URL: http://tgil.donga.ac.kr/CSGMdesigner), facilitates rapid and systematic design of cross-species genic markers. The underlying database is composed of genome data from five legume species whose genomes are substantially characterized. Use of CSGM is enhanced by graphical displays of query results, which we describe as "circular viewer" and "search-within-results" functions. CSGM provides a virtual PCR representation (eHT-PCR) that predicts the specificity of each primer pair simultaneously in multiple genomes. CSGM Designer output was experimentally validated for the amplification of orthologous genes using 16 genotypes representing 12 crop and model legume species, distributed among the galegoid and phaseoloid clades. Successful cross-species amplification was obtained for 85.3% of PCR primer combinations. CONCLUSION: CSGM Designer spans the divide between well-characterized crop and model legume species and their less well-characterized relatives. The outcome is PCR primers that target highly conserved genes for polymorphism discovery, enabling functional inferences and ultimately facilitating trait-associated molecular breeding.

An Acidic PATHOGENESIS-RELATED1 Gene of Oryza grandiglumis is Involved in Disease Resistance Response Against Bacterial Infection.

Wild rice, Oryza grandiglumis shows hyper-resistance response to pathogen infection. In order to identify genes necessary for defense response in plants, we have carried out a subtractive hybridization coupled with a cDNA macroarray. An acidic PATHOGENESIS-RELATED1 (PR1) gene of the wild rice is highly identical to the acidic PR1 genes of different plant species. The OgPR1a cDNA has an apparent single open reading frame with a predicted molecular mass 40,621 Da and an isoelectic point of 5.14. Both in silico analysis and a transient expression assay in onion epidermal cells revealed that the OgPR1a protein could be localized in intercellular space in plants. The OgPR1a mRNA was strongly transcribed by the exogenous treatment with ethylene and jasmonic acid as well as protein phosphatase inhibitors. Additionally, ectopic expression of the OgPR1a conferred disease resistance on Arabidopsis to the bacterial and fungal infections.

Cross-family translational genomics of abiotic stress-responsive genes between Arabidopsis and Medicago truncatula.

Cross-species translation of genomic information may play a pivotal role in applying biological knowledge gained from relatively simple model system to other less studied, but related, genomes. The information of abiotic stress (ABS)-responsive genes in Arabidopsis was identified and translated into the legume model system, Medicago truncatula. Various data resources, such as TAIR/AtGI DB, expression profiles and literatures, were used to build a genome-wide list of ABS genes. tBlastX/BlastP similarity search tools and manual inspection of alignments were used to identify orthologous genes between the two genomes. A total of 1,377 genes were finally collected and classified into 18 functional criteria of gene ontology (GO). The data analysis according to the expression cues showed that there was substantial level of interaction among three major types (i.e., drought, salinity and cold stress) of abiotic stresses. In an attempt to translate the ABS genes between these two species, genomic locations for each gene were mapped using an in-house-developed comparative analysis platform. The comparative analysis revealed that fragmental colinearity, represented by only 37 synteny blocks, existed between Arabidopsis and M. truncatula. Based on the combination of E-value and alignment remarks, estimated translation rate was 60.2% for this cross-family translation. As a prelude of the functional comparative genomic approaches, in-silico gene network/interactome analyses were conducted to predict key components in the ABS responses, and one of the sub-networks was integrated with corresponding comparative map. The results demonstrated that core members of the sub-network were well aligned with previously reported ABS regulatory networks. Taken together, the results indicate that network-based integrative approaches of comparative and functional genomics are important to interpret and translate genomic information for complex traits such as abiotic stresses.

Population structure and domestication revealed by high-depth resequencing of Korean cultivated and wild soybean genomes.

Despite the importance of soybean as a major crop, genome-wide variation and evolution of cultivated soybeans are largely unknown. Here, we catalogued genome variation in an annual soybean population by high-depth resequencing of 10 cultivated and 6 wild accessions and obtained 3.87 million high-quality single-nucleotide polymorphisms (SNPs) after excluding the sites with missing data in any accession. Nuclear genome phylogeny supported a single origin for the cultivated soybeans. We identified 10-fold longer linkage disequilibrium (LD) in the wild soybean relative to wild maize and rice. Despite the small population size, the long LD and large SNP data allowed us to identify 206 candidate domestication regions with significantly lower diversity in the cultivated, but not in the wild, soybeans. Some of the genes in these candidate regions were associated with soybean homologues of canonical domestication genes. However, several examples, which are likely specific to soybean or eudicot crop plants, were also observed. Consequently, the variation data identified in this study should be valuable for breeding and for identifying agronomically important genes in soybeans. However, the long LD of wild soybeans may hinder pinpointing causal gene(s) in the candidate regions.

Genome-wide identification and analysis of Japonica and Indica cultivar-preferred transcripts in rice using 983 Affymetrix array data.

BACKGROUND: Accumulation of genome-wide transcriptome data provides new insight on a genomic scale which cannot be gained by analyses of individual data. The majority of rice (O. sativa) species are japonica and indica cultivars. Genome-wide identification of genes differentially expressed between japonica and indica cultivars will be very useful in understanding the domestication and evolution of rice species. RESULTS: In this study, we analyzed 983 of the 1866 entries in the Affymetrix array data in the public database: 595 generated from indica and 388 from japonica rice cultivars. To discover differentially expressed genes in each cultivar, we performed significance analysis of microarrays for normalized data, and identified 490 genes preferentially expressed in japonica and 104 genes in indica. Gene Ontology analyses revealed that defense response-related genes are significantly enriched in both cultivars, indicating that japonica and indica might be under strong selection pressure for these traits during domestication. In addition, 36 (34.6%) of 104 genes preferentially expressed in indica and 256 (52.2%) of 490 genes preferentially expressed in japonica were annotated as genes of unknown function. Biotic stress overview in the MapMan toolkit revealed key elements of the signaling pathway for defense response in japonica or indica eQTLs. CONCLUSIONS: The percentage of screened genes preferentially expressed in indica was 4-fold higher (34.6%) and that in japonica was 5-fold (52.2%) higher than expected (11.1%), suggesting that genes of unknown function are responsible for the novel traits that distinguish japonica and indica cultivars. The identification of 10 functionally characterized genes expressed preferentially in either japonica or indica highlights the significance of our candidate genes during the domestication of rice species. Functional analysis of the roles of individual components of stress-mediated signaling pathways will shed light on potential molecular mechanisms to improve disease resistance in rice.

Inflammatory responses in the muscle coat of stomach and small bowel in the postoperative ileus model of guinea pig.

PURPOSE: Small intestinal function returns first after surgery, and then the function of the stomach returns to normal after postoperative ileus (POI). The aim of this study was to investigate inflammatory responses in the muscle coat of stomach and small intestine in guinea pig POI model. MATERIALS AND METHODS: The distance of charcoal migration from pylorus to the distal intestine was measured. Hematoxylin and eosin (H&E) and immunohistochemical stain for calprotectin were done from the histologic sections of stomach, jejunum and ileum obtained at 3 and 6 hour after operation. Data were compared between sham operation and POI groups. RESULTS: The distance of charcoal migration was significantly reduced in the 3 and 6 hour POI groups compared with sham operated groups (p<0.05). On H&E staining, the degree of inflammation was significantly higher in the stomach of 3 hour POI groups compared with jejunum and ileum of POI groups or sham operated groups (p<0.05). Calprotectin positive cells were significantly increased in the muscle coat of stomach of 3 hour POI groups compared with jejunum and ileum of POI groups or sham operated groups (p<0.05). There was strong association between the degree of inflammation and calprotectin positive cells in stomach. CONCLUSION: Postoperative ileus induced by cecal manipulation significantly increased the degree of inflammation and calprotectin positive cells in the muscle coat of stomach as a remote organ. The relevance of degree of inflammation and the recovery time of ileus should be pursued in the future research.

Water deficit modulates the response of Vitis vinifera to the Pierce's disease pathogen Xylella fastidiosa.

Pierce's disease, caused by the bacterium Xylella fastidiosa, is one of the most devastating diseases of cultivated grape, currently restricted to the Americas. To test the long-standing hypothesis that Pierce's disease results from pathogen-induced drought stress, we used the Affymetrix Vitis GeneChip to compare the transcriptional response of Vitis vinifera to Xylella infection, water deficit, or a combination of the two stresses. The results reveal a redirection of gene transcription involving 822 genes with a minimum twofold change (P < 0.05), including the upregulation of transcripts for phenylpropanoid and flavonoid biosynthesis, pathogenesis-related proteins, abscisic acid- and jasmonic acid-responsive biosynthesis, and downregulation of transcripts related to photosynthesis, growth, and nutrition. Although the transcriptional response of plants to Xylella infection was largely distinct from the response of healthy plants to water stress, we find that 138 of the pathogen-induced genes exhibited a significantly stronger transcriptional response when plants were simultaneously exposed to infection and drought stress, suggesting a strong interaction between disease and water deficit. This interaction between drought stress and disease was mirrored in planta at the physiological level for aspects of water relations and photosynthesis and in terms of the severity of disease symptoms and the extent of pathogen colonization, providing a molecular correlate of the classical concept of the disease triangle in which environment impacts disease severity.

Genetic modification of the soybean to enhance the ß-carotene content through seed-specific expression.

The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the ß-conglycinin (ß) or CaMV-35S (35S) promoter to generate ß-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for ß-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for ß-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the ß-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T(2) generation plants revealed that the best line among the ß-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was ß-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high ß-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.

Complete genome sequence of the metabolically versatile plant growth-promoting endophyte Variovorax paradoxus S110.

Variovorax paradoxus is a microorganism of special interest due to its diverse metabolic capabilities, including the biodegradation of both biogenic compounds and anthropogenic contaminants. V. paradoxus also engages in mutually beneficial interactions with both bacteria and plants. The complete genome sequence of V. paradoxus S110 is composed of 6,754,997 bp with 6,279 predicted protein-coding sequences within two circular chromosomes. Genomic analysis has revealed multiple metabolic features for autotrophic and heterotrophic lifestyles. These metabolic diversities enable independent survival, as well as a symbiotic lifestyle. Consequently, S110 appears to have evolved into a superbly adaptable microorganism that is able to survive in ever-changing environmental conditions. Based on our findings, we suggest V. paradoxus S110 as a potential candidate for agrobiotechnological applications, such as biofertilizer and biopesticide. Because it has many associations with other biota, it is also suited to serve as an additional model system for studies of microbe-plant and microbe-microbe interactions.

A new esterase EstD2 isolated from plant rhizosphere soil metagenome.

Soil metagenome constitutes a reservoir for discovering novel enzymes from the unculturable microbial diversity. From three plant rhizosphere metagenomic libraries comprising a total of 142,900 members of recombinant plasmids, we obtained 14 recombinant fosmids that exhibited lipolytic activity. A selected recombinant plasmid, pFLP-2, which showed maximum lipolytic activity, was further analyzed. DNA sequence analysis of the subclone in pUC119, pELP-2, revealed an open reading frame of 1,191 bp encoding a 397-amino-acid protein. Purified EstD2 exhibited maximum enzymatic activity towards p-nitrophenyl butyrate, indicating that it is an esterase. Purified EstD2 showed optimal activity at 35 °C and at pH 8.0. The K(m) and K(cat) values were determined to be 79.4 µM and 120.5/s, respectively. The esterase exhibited an increase in enzymatic activity in the presence of 15% butanol and 15% methanol. Phylogenetic analysis revealed that the lipolytic protein EstD2 may be a member of a novel family of lipolytic enzymes. Several hypothetical protein homologs of EstD2 were found in the database. A hypothetical protein from Phenylobacterium zucineum HLK1, a close homolog of EstD2, displayed lipolytic activity when the corresponding gene was expressed in Escherichia coli. Our results suggest that the other hypothetical protein homologs of EstD2 might also be members of this novel family.

Integration of novel SSR and gene-based SNP marker loci in the chickpea genetic map and establishment of new anchor points with Medicago truncatula genome.

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 x C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM-ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2-21 alleles and polymorphic information content value 0.04-0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago.

Molecular characterization of OsPAP2: transgenic expression of a purple acid phosphatase up-regulated in phosphate-deprived rice suspension cells.

A phosphate starvation-induced, purple, acid phosphatase cDNA was cloned from rice, Oryza sativa. The cDNA encoding the phosphatase (OsPAP2) has 1,893 bp with an open reading frame of 630 amino acid residues. The deduced amino acid sequence of OsPAP2 shows identities of 60-63% with other plant purple acid phosphatases and appears to have five conserved motifs containing the residues involved in metal binding. OsPAP2 expression is up-regulated in the rice plant and in cell cultures in the absence of phosphate (P( i )). The induced expression of OsPAP2 is a specific response to P( i ) starvation, and is not affected by the deprivation of other nutrients. OsPAP2 expression was responsive to the level of P( i )-supply, and transcripts of OsPAP2 were abundant in P( i )-deprived roots. The OsPAP2 cDNA was expressed as a 69 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsPAP2 gene was introduced into Arabidopsis via an Agrobacterium-mediated transformation. Functional expression of the OsPAP2 gene in the transgenic Arabidopsis line was confirmed by northern and western blot analyses, as well as by phosphatase activity assays. These results suggest that the OsPAP2 gene can be used to develop new transgenic dicotyledonous plants that are able to adapt to P( i )-deficient conditions.