Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety.

Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) from wheat affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions of specific proteins. The objective of this study was to link LMW-GS genes and haplotypes characterized using well known Glu-A3, Glu-B3, and Glu-D3 gene-specific primers to their protein products in a single wheat variety. A total of 36 LMW-GS genes and pseudogenes were amplified from the Korean cultivar Keumkang. These include 11 Glu-3 gene haplotypes, two from the Glu-A3 locus, two from the Glu-B3 locus, and seven from the Glu-D3 locus. To establish relationships between gene haplotypes and their protein products, a glutenin protein fraction was separated by two-dimensional gel electrophoresis (2-DGE) and 17 protein spots were analyzed by N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS). LMW-GS proteins were identified that corresponded to all Glu-3 gene haplotypes except the pseudogenes. This is the first report of the comprehensive characterization of LMW-GS genes and their corresponding proteins in a single wheat cultivar. Our approach will be useful to understand the contributions of individual LMW-GS to the end-use quality of flour.

Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress.

KEY MESSAGE: Overexpression of OsGS gene modulates oxidative stress response in rice after exposure to cadmium stress. Our results describe the features of transformants with enhanced tolerance to Cd and abiotic stresses. Glutamine synthetase (GS) (EC 6.3.1.2) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Exposure of plants to cadmium (Cd) has been reported to decrease GS activity in maize, pea, bean, and rice. To better understand the function of the GS gene under Cd stress in rice, we constructed a recombinant pART vector carrying the GS gene under the control of the CaMV 35S promoter and OCS terminator and transformed using Agrobacterium tumefaciens. We then investigated GS overexpressing rice lines at the physiological and molecular levels under Cd toxicity and abiotic stress conditions. We observed a decrease in GS enzyme activity and mRNA expression among transgenic and wild-type plants subjected to Cd stress. The decrease, however, was significantly lower in the wild type than in the transgenic plants. This was further validated by the high GS mRNA expression and enzyme activity in most of the transgenic lines. Moreover, after 10 days of exposure to Cd stress, increase in the glutamine reductase activity and low or no malondialdehyde contents were observed. These results showed that overexpression of the GS gene in rice modulated the expression of enzymes responsible for membrane peroxidation that may result in plant death.

Effects of reduced prolamin on seed storage protein composition and the nutritional quality of rice.

Rice seed storage proteins accumulate in two types of protein body (PB-I and PB-II) that are nutrient sources for animals. PB-I is indigestible and negatively affects rice protein quality. To improve the nutritional value of rice seeds we are aiming to engineer the composition and accumulation of endogenous seed storage proteins. In this study we generated transgenic rice plants in which 13 kD prolamin genes were suppressed by RNA interference (13 kD pro-RNAi). Analysis based on qRT-PCR confirmed that the targeted 13 kD prolamins were markedly suppressed, and were compensated for by an increase in other storage proteins including 10 kD prolamin, glutelins, and chaperone proteins. The storage protein profiles further revealed that the levels of 13 kD prolamins were significantly reduced, while that of the glutelin precursor was slightly increased and the remaining storage proteins did not change. Amino acid analysis showed that the reduction of 13 kD prolamins resulted in a 28% increase in the lysine content relative to the wild type, indicating that the 13 kD pro-RNAi rice seeds are more nutritious. Furthermore, a reduction in the levels of 13 kD prolamins resulted in abnormal formation of PB-I, which was small and had no lamellar structure. These results suggest that alteration of prolamins can contribute to improving the nutritional quality of rice.

Inactivation of the CTD phosphatase-like gene OsCPL1 enhances the development of the abscission layer and seed shattering in rice.

Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h, from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F(2) plants and five F(3) lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3' splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 (OsCPL1). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development.

Overexpression of IbFAD8 Enhances the Low-Temperature Storage Ability and Alpha-Linolenic Acid Content of Sweetpotato Tuberous Roots.

Sweetpotato is an emerging food crop that ensures food and nutrition security in the face of climate change. Alpha-linoleic acid (ALA) is one of the key factors affecting plant stress tolerance and is also an essential nutrient in humans. In plants, fatty acid desaturase 8 (FAD8) synthesizes ALA from linoleic acid (LA). Previously, we identified the cold-induced IbFAD8 gene from RNA-seq of sweetpotato tuberous roots stored at low-temperature. In this study, we investigated the effect of IbFAD8 on the low-temperature storage ability and ALA content of the tuberous roots of sweetpotato. Transgenic sweetpotato plants overexpressing IbFAD8 (TF plants) exhibited increased cold and drought stress tolerance and enhanced heat stress susceptibility compared with non-transgenic (NT) plants. The ALA content of the tuberous roots of TF plants (0.19 g/100 g DW) was ca. 3.8-fold higher than that of NT plants (0.05 g/100 g DW), resulting in 8-9-fold increase in the ALA/LA ratio in TF plants. Furthermore, tuberous roots of TF plants showed better low-temperature storage ability compared with NT plants. These results indicate that IbFAD8 is a valuable candidate gene for increasing the ALA content, environmental stress tolerance, and low-temperature storage ability of sweetpotato tuberous roots via molecular breeding.

Plant growth promotion and Penicillium citrinum.

BACKGROUND: Endophytic fungi are known plant symbionts. They produce a variety of beneficial metabolites for plant growth and survival, as well as defend their hosts from attack of certain pathogens. Coastal dunes are nutrient deficient and offer harsh, saline environment for the existing flora and fauna. Endophytic fungi may play an important role in plant survival by enhancing nutrient uptake and producing growth-promoting metabolites such as gibberellins and auxins. We screened roots of Ixeris repenes (L.) A. Gray, a common dune plant, for the isolation of gibberellin secreting endophytic fungi. RESULTS: We isolated 15 endophytic fungi from the roots of Ixeris repenes and screened them for growth promoting secondary metabolites. The fungal isolate IR-3-3 gave maximum plant growth when applied to waito-c rice and Atriplex gemelinii seedlings. Analysis of the culture filtrate of IR-3-3 showed the presence of physiologically active gibberellins, GA1, GA3, GA4 and GA7 (1.95 ng/ml, 3.83 ng/ml, 6.03 ng/ml and 2.35 ng/ml, respectively) along with other physiologically inactive GA5, GA9, GA12, GA15, GA19, GA20 and, GA24. The plant growth promotion and gibberellin producing capacity of IR-3-3 was much higher than the wild type Gibberella fujikuroi, which was taken as control during present study. GA5, a precursor of bioactive GA3 was reported for the first time in fungi. The fungal isolate IR-3-3 was identified as a new strain of Penicillium citrinum (named as P. citrinum KACC43900) through phylogenetic analysis of 18S rDNA sequence. CONCLUSION: Isolation of new strain of Penicillium citrinum from the sand dune flora is interesting as information on the presence of Pencillium species in coastal sand dunes is limited. The plant growth promoting ability of this fungal strain may help in conservation and revegetation of the rapidly eroding sand dune flora. Penicillium citrinum is already known for producing mycotoxin citrinin and cellulose digesting enzymes like cellulase and endoglucanase, as well as xylulase. Gibberellins producing ability of this fungus and the discovery about the presence of GA5 will open new aspects of research and investigations.

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice.

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.

Immunomodulatory activity of Ulmus davidiana Planch (Ulmaceae) water and ethanolic extracts on bone cells: Stimulation of proliferation, alkaline phosphatase activity and type I collagen synthesis.

Ulmus davidiana Planch (Ulmaceae) (UD) frequently appears as the main ingredient in prescriptions for bone injuries, however, the action mechanism is unclear. In the present study, (i) the effect of the aqueous extract of UD on bone cells was investigated in vitro and (ii) the immunomodulatory activity of UD was investigated with regard to cellular and humoral immunity. The osteoprecursor cells (OPC) were incubated in the medium with different concentrations of the UD and the cell proliferation was studied. When the concentration of UD was <100µg/ml, the proliferation of OPC was enhanced. However, the proliferation of OPC was inhibited by UD with the concentrations >180µg/ml. Under most treatments, the cells presented low expression for cyclooxygenase-2 (Cox-2) protein. On the other hand, oral administration of the ethanolic and water extracts of UD, at the doses of 20, 50, 100 and 200mg/kg in mice, dose-dependently potentiated the delayed-type hypersensitivity reaction induced both by sheep red blood cells (SRBC) and oxazolone. It significantly enhanced the production of circulating antibody titers in mice in response to SRBC. UD had no any effect on macrophage phagocytosis. Chronic administration of UD significantly ameliorated the total white blood cell counts and also restored the myelosuppressive effects induced by cyclophosphamide. From the results, it was concluded that UD directly stimulates the proliferation, alkaline phosphatase activity, protein secretion and particularly type I collagen synthesis of OPC in a dose-dependent manner, and that UD possesses immunomodulatory activity.

The complete chloroplast genome sequence of Perilla citriodora (Makino) Nakai.

Perilla citriodora is a diploid and an aromatic herb species belonging to the family Lamiaceae and widely distributed in East Asia. The complete chloroplast genome sequence of P. citriodora was generated by de novo assembly using whole genome next-generation sequences. The assembled chloroplast genome of P. citriodora was 152 588 bp in length and structurally divided into four distinct regions; large single copy region (83 699 bp), small single copy region (17 537 bp), and a pair of inverted repeat regions (25 676 bp). A total of 115 genes were annotated including 81 protein-coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic relationship with previously reported chloroplast genomes revealed that P. citriodora is most closely related to P. frutescens, a tetraploid Perilla species.

A high-density SNP genetic map consisting of a complete set of homologous groups in autohexaploid sweetpotato (Ipomoea batatas).

Sweetpotato (Ipomoea batatas) is an autohexaploid species with 90 chromosomes (2n = 6x = 90) and a basic chromosome number of 15, and is therefore regarded as one of the most challenging species for high-density genetic map construction. Here, we used single nucleotide polymorphisms (SNPs) identified by double-digest restriction site-associated DNA sequencing based on next-generation sequencing technology to construct a map for sweetpotato. We then aligned the sequence reads onto the reference genome sequence of I. trifida, a likely diploid ancestor of sweetpotato, to detect SNPs. In addition, to simplify analysis of the complex genetic mode of autohexaploidy, we used an S1 mapping population derived from self-pollination of a single parent. As a result, 28,087 double-simplex SNPs showing a Mendelian segregation ratio in the S1 progeny could be mapped onto 96 linkage groups (LGs), covering a total distance of 33,020.4 cM. Based on the positions of the SNPs on the I. trifida genome, the LGs were classified into 15 groups, each with roughly six LGs and six small extra groups. The molecular genetic techniques used in this study are applicable to high-density mapping of other polyploid plant species, including important crops.

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.

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest.

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars.

AllergenPro: an integrated database for allergenicity analysis and prediction.

UNLABELLED: The National Agricultural Biotechnology Information Center (NABIC) reconstructed an AllergenPro database for allergenic proteins analysis and allergenicity prediction. The AllergenPro is an integrated web-based system providing information about allergen in foods, microorganisms, animals and plants. The allergen database has the three main features namely, (1) allergen list with epitopes, (2) searching of allergen using keyword, and (3) methods for allergenicity prediction. This updated AllergenPro outputs the search based allergen information through a user-friendly web interface, and users can run tools for allergenicity prediction using three different methods namely, (1) FAO/WHO, (2) motif-based and (3) epitope-based methods. AVAILABILITY: The database is available for free at http://nabic.rda.go.kr/allergen/

SNP-based analysis of genetic diversity in anther-derived rice by whole genome sequencing.

BACKGROUND: Anther culture has advantage to obtain a homozygous progeny by induced doubling of haploid chromosomes and to improve selection efficiency for invaluable agronomical traits. Therefore, anther culturing is widely utilized to breed new varieties and to induce genetic variations in several crops including rice. Genome sequencing technologies allow the detection of a massive number of DNA polymorphism such as SNPs and Indels between closely related cultivars. These DNA polymorphisms permit the rapid identification of genetic diversity among cultivars and genomic locations of heritable traits. To estimate sequence diversity derived from anther culturing, we performed whole-genome resequencing of five Korean rice accessions, including three anther culture lines (BLB, HY-04 and HY-08), their progenitor cultivar (Hwayeong), and an additional japonica cultivar (Dongjin). RESULTS: A total of 1,165 × 106 raw reads were generated with over 58× coverage that detected 1,154,063 DNA polymorphisms between the Korean rice accessions and Nipponbare. We observed that in Hwayeong and its progenies, 0.64 SNP was found per one kb of Nipponbare genome, while Dongjin, bred by a conventional breeding method, had a lower number of SNPs (0.45 SNP/kb). Among 1,154,063 DNA polymorphisms, 29,269 non-synonymous SNPs located on 30,013 genes and these genes were functionally classified based on gene ontology (GO). We also analyzed line-specific SNPs which were estimated 1 ~ 3% of the total SNPs. The frequency of non-synonymous SNPs in each accession ranged from 26 SNPs in Hwayeong to 214 SNPs in HY-04. CONCLUSIONS: The genetic difference we detected between the progenies derived from anther culture and their mother cultivar is due to somaclonal variation during tissue culture process, such as karyotype change, chromosome rearrangement, gene amplification and deletion, transposable element, and DNA methylation. Detection of genome-wide DNA polymorphisms by high-throughput sequencer enabled to identify sequence diversity derived from anther culturing and genomic locations of heritable traits. Furthermore, it will provide an invaluable resource to identify molecular markers and genes associated with diverse traits of agronomical importance.

NABIC marker database: A molecular markers information network of agricultural crops.

UNLABELLED: In 2013, National Agricultural Biotechnology Information Center (NABIC) reconstructs a molecular marker database for useful genetic resources. The web-based marker database consists of three major functional categories: map viewer, RSN marker and gene annotation. It provides 7250 marker locations, 3301 RSN marker property, 3280 molecular marker annotation information in agricultural plants. The individual molecular marker provides information such as marker name, expressed sequence tag number, gene definition and general marker information. This updated marker-based database provides useful information through a user-friendly web interface that assisted in tracing any new structures of the chromosomes and gene positional functions using specific molecular markers. AVAILABILITY: The database is available for free at http://nabic.rda.go.kr/gere/rice/molecularMarkers/

FSTVAL: a new web tool to validate bulk flanking sequence tags.

BACKGROUND: Information about a transgene locus is one of the major concerns in transgenic research because expression of the transgene or a gene interrupted by the integration event could be affected. Thus, the flanking sequences obtained from transgenic plants need to be analyzed in terms of genomic context, such as genic and intergenic regions. This process may consist of several steps: 1) elimination of a vector sequence from the flanking sequence, 2) finding the locations in the target genome, and 3) statistics of the integration sites. These steps could be automated for flanking sequences from several dozens of transgenic plants generated in an ordinary targeted gene expression strategy. It would be indispensable in a genome-wide mutagenesis screen using T-DNA or transposons because these projects often generate several thousands of transgenic lines and just as many loci of the transgene among the transgenic plants. RESULTS: We present an open access web tool, flanking sequence tags validator (FSTVAL), to manage bulk flanking sequence tags (FSTs). FSTVAL automatically evaluates the FSTs and finds the best mapping positions of the FST against a known genome sequence. The statistics, in terms of genic and intergenic regions, are presented as a table, a distribution map, and a frequency graph along the chromosomes. Currently, 17 plant genome sequences, including Arabidopsis thaliana, Oryza sativa, and Glycine max, are available as reference genomes. We evaluated the utility and accuracy of the tool with 5,144 rice FSTs. The whole process, from uploading the sequences to generating tables of insertions, required a few minutes, with less than 4 clicks in the web environment. CONCLUSIONS: Run for 1 year and tested over 1,000 times, we have confirmed FSTVAL efficiently handles bulk FSTs. FSTVAL is freely available without login at http://bioinfo.mju.ac.kr/fstval/.

RiceArrayNet: a database for correlating gene expression from transcriptome profiling, and its application to the analysis of coexpressed genes in rice.

Microarray data can be used to derive understanding of the relationships between the genes involved in various biological systems of an organism, given the availability of databases of gene expression measurements from the complete spectrum of experimental conditions and materials. However, there have been no reports, to date, of such a database being constructed for rice (Oryza sativa). Here, we describe the construction of such a database, called RiceArrayNet (RAN; http://www.ggbio.com/arraynet/), which provides information on coexpression between genes in terms of correlation coefficients (r values). The average number of coexpressed genes is 214, with sd of 440 at r >or= 0.5. Given the correlation between genes in a gene pair, the degrees of closeness between genes can be visualized in a relational tree and a relational network. The distribution of correlated genes according to degree of stringency shows how each gene is related to other genes. As an application of RAN, the 16-member L7Ae ribosomal protein family was explored for coexpressed genes and gene expression values within and between rice and Arabidopsis (Arabidopsis thaliana), and common and unique features in coexpression partners and expression patterns were observed for these family members. We observed a correlation pattern between Os01g0968800, a drought-responsive element-binding transcription factor, Os02g0790500, a trehalose-6-phosphate synthase, and Os06g0219500, a small heat shock factor, reflecting the fact that genes responding to the same biological stresses are regulated together. The RAN database can be used as a tool to gain insight into a particular gene by examining its coexpression partners.