Proteome profiling highlights mechanisms underlying pigment and tocopherol accumulation in red and black rice seeds.

Rice is a major component of the human diet and feeds more than 50 million people across the globe. We previously developed two pigmented rice cultivars, Super-hongmi (red seeds) and Super-jami (black seeds), that are highly rich in antioxidants and exhibit high levels of radical scavenging activities. However, the molecular mechanism underlying the accumulation of pigments and different antioxidants in these rice cultivars remains largely elusive. Here, we report the proteome profiles of mature Super-hongmi and Super-jami seeds, and compared them with the Hopum (white seeds) using a label-free quantitative proteomics approach. This approach led to the identification of 5127 rice seed proteins of which 1628 showed significant changes in the pigmented rice cultivar(s). The list of significantly modulated proteins included a phytoene desaturase (PDS3) which suggested accumulation of ζ-carotene in red seeds while the black seeds seem to accumulate more of anthocyanins because of the higher abundance of dihydroflavonol 4-reductase. Moreover, proteins associated with lignin and tocopherol biosynthesis were highly increased in both red and black cultivars. Taken together, these data report the seed proteome of three different colored rice seeds and identify novel components associated with pigment accumulation in rice.

A new SNP in cyOsPPDK gene is associated with floury endosperm in Suweon 542.

Pyruvate orthophosphate dikinase (PPDK) is a component of glycolysis to mediate endosperm energy charge by adjusting the ratio of ATP to ADP and AMP that proposed to balance the flow of carbon into starch, protein, fatty acid and amino acid biosynthesis. However, these were inconsistent with the first report of a T-DNA insertional knockout mutant of the rice PPDK gene (flo4) showed that rice with inactivated PPDK gene failed to produce a opaque seeds. Therefore, the PPDK might have multifaceted functions in grain filling stage, which in some ways might depend on the direction of the reversible catalysis. Suweon 542 is a rice (Oryza sativa L.) mutant developed from Oryza sativa ssp. japonica cv. Namil. Suweon 542 has a milky-white floury endosperm suitable for dry filling, with low starch damage, low grain hardness, and fine flour particle size. The mutant locus on chromosome 5 controls the floury endosperm phenotype of Suweon 542. Fine mapping of this locus is required for efficient breeding of rice germplasm suitable for dry milling. In this study, whole genome of Suweon 542 and Milyang 23 were re-sequenced using Illumina HiSeq 2500. Co-segregation analysis of F3:4 family populations derived from Suweon 542/Milyang 23 was performed using eight CAPS markers and phenotypic evaluation of the endosperm. The target region was mapped to a 33 kb region and identified to encode cytosolic pyruvate orthophosphate dikinase protein (cyOsPPDK). A G→A SNP in exon 8 of cyOsPPDK resulting in a missense mutation from Gly to Asp at amino acid position 404 was responsible for the floury endosperm of Suweon 542. qRT-PCR experiments revealed that FLO4-4 was expressed to a considerably higher level in Suweon 542 than in Namil during the grain filling stage. Overall, fine mapping of FLO4-4 and candidate gene analysis provided further insight into the floury endosperm of rice, and reveal a novel SNP in cyOsPPDK gene can affect the floury endosperm phenotype through active PPDK gene during grain filling stage.

Teosinte Branched 1 modulates tillering in rice plants.

Tillering is an important trait of cereal crops that optimizes plant architecture for maximum yield. Teosinte Branched 1 (TB1) is a negative regulator of lateral branching and an inducer of female inflorescence formation in Zea mays (maize). Recent studies indicate that TB1 homologs in Oryza sativa (rice), Sorghum bicolor and Arabidopsis thaliana act downstream of the auxin and MORE AUXILIARY GROWTH (MAX) pathways. However, the molecular mechanism by which rice produces tillers remains unknown. In this study, transgenic rice plants were produced that overexpress the maize TB1 (mTB1) or rice TB1 (OsTB1) genes and silence the OsTB1 gene through RNAi-mediated knockdown. Because lateral branching in rice is affected by the environmental conditions, the phenotypes of transgenic plants were observed in both the greenhouse and the paddy field. Compared to wild-type plants, the number of tillers and panicles was reduced and increased in overexpressed and RNAi-mediated knockdown OsTB1 rice plants, respectively, under both environmental conditions. However, the effect was small for plants grown in paddy fields. These results demonstrate that both mTB1 and OsTB1 moderately regulate the tiller development in rice.

Single nucleotide polymorphisms and haplotype diversity in rice sucrose synthase 3.

Rice sucrose synthase 3 (RSUS3) is expressed predominantly in rice seed endosperm and is thought to play an important role in starch filling during the milky stage of rice seed ripening. Because the genetic diversity of this locus is not known yet, the full sequence of RSUS3 from 43 rice varieties was amplified to examine the distribution of DNA polymorphisms. A total of 254 sequence variants, including SNPs and insertion/deletions, were successfully identified in the 7733 bp sequence that comprises the promoter, exons and introns, and 3' downstream nontranscribed region (NTR). Eleven haplotypes were distinguished among the 43 rice varieties based on nucleotide variation in the 3 defined regions (5' NTR, transcript, and 3' NTR). The promoter region showed evidence of a base change on a cis-element that might influence the functional role of the motif in seed-specific expression. The genetic diversity of the RSUS3 gene sequences in the rice germplasm used in this study appears to be the result of nonrandom processes. Analysis of polymorphism sites indicated that at least 11 recombinations have occurred, primarily in the transcribed region. This finding provides insight into the development of a cladistic approach for establishing future genetic association studies of the RSUS3 locus.

Genome-Wide Association Study Reveals the Genetic Basis of Chilling Tolerance in Rice at the Reproductive Stage.

A genome-wide association study (GWAS) was used to investigate the genetic basis of chilling tolerance in a collection of 117 rice accessions, including 26 Korean landraces and 29 weedy rices, at the reproductive stage. To assess chilling tolerance at the early young microspore stage, plants were treated at 12 °C for 5 days, and tolerance was evaluated using seed set fertility. GWAS, together with principal component analysis and kinship matrix analysis, revealed five quantitative trait loci (QTLs) associated with chilling tolerance on chromosomes 3, 6, and 7. The percentage of phenotypic variation explained by the QTLs was 11-19%. The genomic region underlying the QTL on chromosome 3 overlapped with a previously reported QTL associated with spikelet fertility. Subsequent bioinformatic and haplotype analyses suggested three candidate chilling-tolerance genes within the QTL linkage disequilibrium block: Os03g0305700, encoding a protein similar to peptide chain release factor 2; Os06g0495700, encoding a beta tubulin, autoregulation binding-site-domain-containing protein; and Os07g0137800, encoding a protein kinase, core-domain-containing protein. Further analysis of the detected QTLs and the candidate chilling-tolerance genes will facilitate strategies for developing chilling-tolerant rice cultivars in breeding programs.

Increasing Coverage of Proteome Identification of the Fruiting Body of Agaricus bisporus by Shotgun Proteomics.

To increase coverage of protein identification of an Agaricus bisporus fruiting body, we analyzed the crude protein fraction of the fruiting body by using a shotgun proteomics approach where 7 MudPIT (Multi-Protein identification Technology) runs were conducted and the MS/MS spectra from the 7 MudPIT runs were merged. Overall, 3093 non-redundant proteins were identified to support the expression of those genes annotated in the genome database of Agaricus bisporus. The physicochemical properties of the identified proteins, i.e., wide pI value range and molecular mass range, were indicative of unbiased protein identification. The relative quantification of the identified proteins revealed that K5XI50 (Aldedh domain-containing protein) and K5XEW1 (Ubiquitin-like domain-containing protein) were highly abundant in the fruiting body. Based on the information in the Uniprot (Universal Protein Resource) database for A. bisporus, only approximately 53% of the 3093 identified proteins have been functionally described and approximately 47% of the proteins remain uncharacterized. Gene Ontology analysis revealed that the majority of proteins were annotated with a biological process, and proteins associated with coiled-coil (12.8%) and nucleotide binding (8.21%) categories were dominant. The Kyoto Encyclopedia of Genes and Genome analysis revealed that proteins involved in biosynthesis of secondary metabolites and tyrosine metabolism were enriched in a fruiting body of Agaricus bisporus, suggesting that the proteins are associated with antioxidant metabolites.

Study of Quantitative Trait Loci (QTLs) Associated with Allelopathic Trait in Rice.

In rice there are few genetic studies reported for allelopathy traits, which signify the ability of plants to inhibit or stimulate growth of other plants in the environment, by exuding chemicals. QTL analysis for allelopathic traits were conducted with 98 F8 RILs developed from the cross between the high allelopathic parents of 'Sathi' and non-allelopathic parents of 'Nong-an'. The performance of allelopathic traits were evaluated with inhibition rate on root length, shoot length, total length, root weight, shoot weight, and total weight of lettuce as a receiver plant. With 785 polymorphic DNA markers, we constructed a linkage map showing a total of 2489.75 cM genetic length and 3.17 cM of average genetic distance between each adjacent marker. QTL analysis detected on QTL regions on chromosome 8 responsible for the inhibition of shoot length and inhibition of total length. The qISL-8 explained 20.38% of the phenotypic variation for the inhibition on the shoot length. The qITL-8 explained 14.93% of the phenotypic variation for the inhibition on total length. The physical distance of the detected QTL region was 194 Kbp where 31 genes are located.

A New SNP in Rice Gene Encoding Pyruvate Phosphate Dikinase (PPDK) Associated with Floury Endosperm.

Rice varieties with suitable flour-making qualities are required to promote the rice processed-food industry and to boost rice consumption. A rice mutation, Namil(SA)-flo1, produces grains with floury endosperm. Overall, grains with low grain hardness, low starch damage, and fine particle size are more suitable for use in flour processing grains with waxy, dull endosperm with normal grain hardness and a high amylose content. In this study, fine mapping found a C to T single nucleotide polymorphism (SNP) in exon 2 of the gene encoding cytosolic pyruvate phosphate dikinase (cyOsPPDK). The SNP resulted in a change of serine to phenylalanine acid at amino acid position 101. The gene was named FLOURY ENDOSPERM 4-5 (FLO4-5). Co-segregation analysis with the developed cleaved amplified polymorphic sequence (CAPS) markers revealed co-segregation between the floury phenotype and the flo4-5. This CAPS marker could be applied directly for marker-assisted selection. Real-time RT-PCR experiments revealed that PPDK was expressed at considerably higher levels in the flo4-5 mutant than in the wild type during the grain filling stage. Plastid ADP-glucose pyrophosphorylase small subunit (AGPS2a and AGPS2b) and soluble starch synthase (SSIIb and SSIIc) also exhibited enhanced expression in the flo4-5 mutant.

Analysis of complete chloroplast genome sequence of Korean landrace Cymbidium goeringii.

The complete chloroplast genome sequence of Korean Cymbidium goeringii acc. smg222 was analyzed. Based on a comparison with Chinese C. goeringii, losses of nine ndh subunits (ndhA, ndhB, ndhC, ndhD, ndhE, ndhF, ndhH, ndhJ, and ndhK), three protein-coding genes (ycf 1-like, ycf 15, and ycf 68), six transfer RNAs, and one conserved open reading frame (orf 42). In addition, 219 InDels (insertion or deletion) and 171 simple sequence repeats were observed. Twenty-Five of which InDel markers have been evaluated, that useful for distinguishing Korean and Chinese Cymbidium associations based on the polymorphisms of chloroplast genomes between Korean Cymbidium goeringii acc. smg222 and Chinese C. goeringii and evaluation of genetic diversity. Finally, the phylogenetic relationships of the 39 Korean and 22 Chinese species was constructed based on the five InDel markers of them and obtained high support, indicating that our data may be useful in resolving relationships in this genus. The information about chloroplast DNA structure and gene variants of C. goeringii acc. smg222 chloroplast genome will provide sufficient phylogenetic information for resolving evolutionary relationships. The molecular markers developed in here will contribute to further research of Cymbidium species and conservation of endemic Cymbidium species.

In Vitro and in Vivo Anti-Hyperglycemic Activities of Taxifolin and Its Derivatives Isolated from Pigmented Rice (Oryzae sativa L. cv. Superhongmi).

Superhongmi is a new rice variety, which was developed for the enrichment of bioactive compounds through cross-breeding three varieties of rice breeds in Korea. The high-performance liquid chromatography coupled with a photodiode array detector quadrupole and tandem time-of-flight mass spectrometry (HPLC/PDA/QTOF-MS) analysis has revealed that superhongmi bran extract contained four taxifolin derivatives as well as cyanidin 3-glucoside. The high-performance countercurrent chromatography (CCC) and reversed-phase HPLC led to the isolation of aforementioned five compounds, and spectroscopic analysis identified cyanidin 3-glucoside (1), along with (2R,3R)-taxifolin 3-O-ß-d-glucopyranoside (2), (2R,3R)-4'-O-methyltaxifolin 3-O-ß-d-glucopyranoside (a novel compound) (3), (2R,3R)-taxifolin (4), and (2R,3R)-4'-O-methyltaxifolin (5). Compound 2 had the highest rat small intestinal sucrase inhibitory activity (0.54 mM) relevant for potentially managing postprandial hyperglycemia, followed by compound 1 (0.97 mM) and compound 4 (1.74 mM, IC50). The anti-hyperglycemic effect of compound 4 (taxifolin), a main peak in HPLC analysis was investigated using a Sprague-Dawley (SD) rat model. Compared to a control, taxifolin treatment (p < 0.001) reduced significantly after sucrose loading the observed postprandial blood glucose and the maximum blood glucose (Cmax) by 15% (203.60 ± 15.86 to 172.30 ± 12.74). These results indicate that taxifolin derivatives that inhibit the activity of carbohydrate-hydrolyzing enzymes resulting in reduced dietary carbohydrate absorption can potentially be used as a strategy to manage diabetes.

Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T(1) seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.

Map-based cloning of the ERECT PANICLE 3 gene in rice.

Panicle architecture in rice can have a strong influence on yield. Using N-methyl-N-nitrosourea mutagenesis, we isolated an erect panicle mutant, Hep, from Hwasunchalbyeo, a glutinous japonica rice cultivar. Genetic analysis revealed that the erect panicle phenotype was controlled by a single recessive mutation designated erect panicle 3 (ep3). Genetic mapping revealed that the ep3 mutation was located on the short arm of chromosome 2 in a 0.1 cM region delimited by the STS markers STS5803-5 and STS5803-7. The ep3 locus corresponded to 46.8 kb region and contained six candidate genes. Comparison of the DNA sequences of the candidate genes from wild-type and erect panicle plants revealed a single base-pair change in the second exon of LOC_Os02g15950, which is predicted to result in a nonsense mutation. LOC_Os02g15950 encodes a putative F-box protein containing 515 amino acids and is expressed throughout the plant during all growth stages. A line carrying a T-DNA insertion in LOC_ Os02g15950 was obtained and shown to have the same phenotype as the ep3 mutant, thus confirming the identification of LOC_Os02g15950 as the ERECT PANICLE 3 (EP3) gene. The ep3 mutation causes a significant increase in the number of small vascular bundles as well as the thickness of parenchyma in the peduncle, which results in the erect panicle phenotype.

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta.

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F(2) population derived from a cross between an introgression line, 'IR71033-121-15', from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, 'Junambyeo'. Resistance to BPH (biotype 1) was evaluated using 190 F(3) families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.

Effect of Germinated Pigmented Rice "Superjami" on the Glucose Level, Antioxidant Defense System, and Bone Metabolism in Menopausal Rat Model.

Women experience physical, mental, and social changes during menopause. It is important to maintain a healthy diet for effective menopause management. The effect of germinated Superjami, a deep violet colored rice cultivar, on the body weight, glucose level, antioxidant defense system, and bone metabolism in a menopausal rat model was investigated. The animals were randomly divided into three groups and fed with a normal diet (ND), a control diet supplemented with 20% (w/w) non-germinated Superjami flour (NGSF), or germinated Superjami flour (GSF) for eight weeks. The NGSF and GSF groups exhibited significantly lower body weight and fat, glucose and insulin contents, adipokine concentrations, and bone resorption biomarker levels, and higher antioxidant enzyme activities and 17-ß-estradiol content than the ND group (p < 0.05). The GSF group showed greater glucose homeostasis, antioxidative, and bone metabolism-improving effects compared with the NGSF group. These findings demonstrate that germination could further improve the health-promoting properties of Superjami and that this germinated pigmented rice cultivar could be useful in the treatment and management of menopause-induced hyperglycemia, oxidative stress, and bone turnover imbalance.

Next-generation sequencing yields the complete chloroplast genome of C. goeringii acc. smg222 and phylogenetic analysis.

Spring orchid (Cymbidium goeringii) is one of the most important species belonging to Orchidaceae owing to its aesthetic appeal, fragrant flowers and ideal characteristics for using as a houseplant. In this study, the complete chloroplast genome of Korean C. goeringii acc. smg222 was determined by Illumina sequencing. The circular double-stranded DNA of 148,441 bp consisted of two inverted repeat regions of 25,610 bp each, a large single copy region of 83,311 bp, and a small single copy region of 13,910 bp. The genome contained 122 genes, of which 104 were unique and 18 were duplicated within the IRs. The 104 unique genes included 70 protein-coding genes, 30 distinct tRNA genes, and four rRNA genes. Phylogenetic tree analysis revealed that C. goeringii acc. smg222 was clustered with Cymbidium kanran, a cymbidium species native to Korea.

Genome-wide association study reveals candidate genes related to low temperature tolerance in rice (Oryza sativa) during germination.

In this study, relative germination percentage (RGP) and delayed mean germination time (DMGT) were measured in various rice accessions at the germination stage and carried out association analysis to identify candidate genes related to low temperature germination (LTG) using a natural population comprising 137 rice cultivars and inbred lines selected from the Korean rice core set. Genome-wide association study using ~ 1.44 million high-quality SNPs, which were identified by re-sequencing all rice collections, revealed 48 candidate genes on chromosome 10 and 55 candidate genes on chromosome 11 in the high peak SNP sites of associated loci for RGP and DMGT, respectively. By detecting highly associated variations located inside genic regions and performing functional annotation of the genes, we detected 23 candidate genes for RGP and 18 genes for DMGT for LTG. In addition, the haplotype and sequence analysis of the candidate gene (Os10g0371100) with RGP trait and the candidate gene (Os11t0104240-00) with DMGT revealed correlation between sequences of functional variations and phenotypes. Several novel LTG-related candidate genes previously were known for the function during rice germination and uncovered their substantial natural variations. These candidate genes represent valuable resources for molecular breeding and genetic improvement of cold tolerance during rice germination.

Fine mapping and candidate gene analysis of the floury endosperm gene, FLO(a), in rice.

In addition to its role as an energy source for plants, animals and humans, starch is also an environmentally friendly alternative to fossil fuels. In rice, the eating and cooking quality of the grain is determined by its starch properties. The floury endosperm of rice has been explored as an agronomical trait in breeding and genetics studies. In the present study, we characterized a floury endosperm mutant, flo(a), derived from treatment of Oryza sativa ssp. japonica cultivar Hwacheong with MNU. The innermost endosperm of the flo(a) mutant exhibited floury characteristics while the outer layer of the endosperm appeared normal. Starch granules in the flo(a) mutant formed a loosely-packed crystalline structure and X-ray diffraction revealed that the overall crystallinity of the starch was decreased compared to wild-type. The FLO(a) gene was isolated via a map-based cloning approach and predicted to encode the tetratricopeptide repeat domain-containing protein, OsTPR. Three mutant alleles contain a nucleotide substitution that generated one stop codon or one splice site, respectively, which presumably disrupts the interaction of the functionally conserved TPR motifs. Taken together, our map-based cloning approach pinpointed an OsTPR as a strong candidate of FLO(a), and the proteins that contain TPR motifs might play a significant role in rice starch biosynthetic pathways.

PCR marker-based evaluation of the eating quality of japonica rice ( Oryza sativa L.).

Evaluation of eating quality in early breeding generations of rice is critical to developing varieties with better palatability. This paper reports DNA markers associated with eating quality of temperate japonica rice and an evaluation method aided by multiple regression analysis. A total of 30 markers comprising STSs, SNPs, and SSRs were tested for their association with palatability using 22 temperate japonica varieties with different palatability values. Eating quality-related traits of the 22 varieties were also measured. Of the 30 markers, 18 were found to be significantly associated with palatability and, consequently, a model regression equation with an R(2) value of 0.99 was formulated to estimate the palatability by the marker data set. Validation of the model equation using selected breeding lines indicated that the marker set and the equation are highly applicable to evaluation of the palatability of cooked rice in temperate japonica varieties.