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1.
J Agric Food Chem ; 67(26): 7399-7409, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31244203

RESUMO

Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily. We isolated OsFLS from the rice ( Oryza sativa) cultivar "Ilmi" OsFLS includes highly conserved 2-ODD-specific motifs and FLS-specific regions. Recombinant OsFLS exhibited both FLS and flavanone 3ß-hydroxylase (F3H) activities, converting dihydroflavonols into flavonols and flavanones into dihydroflavonols, respectively, and more efficiently used dihydrokaempferol than dihydroquercetin as a substrate. OsFLS was expressed in both nonpigmented and pigmented rice seeds and was developmentally regulated during seed maturation. Transgenic tobacco ( Nicotiana tabacum) plants expressing OsFLS produced pale pink or white flowers with significantly increased levels of kaempferol-3- O-rutinoside and dramatically reduced levels of anthocyanin in their petals. Additionally, pod size and weight were reduced compared to the wild type. Several early and late biosynthetic genes of flavonoid were downregulated in the transgenic flowers. We demonstrated that OsFLS is a bifunctional 2-ODD enzyme and functions in flavonol production in planta.


Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Oryza/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Antocianinas/biossíntese , Cor , Flavonóis/biossíntese , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Cetoglutáricos/metabolismo , Oryza/genética , Tabaco/genética , Tabaco/metabolismo
2.
Metab Eng ; 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30503392

RESUMO

Carotenoid pigments are valuable components of the human diet. A notable example is ß-carotene, or provitamin A, which is converted into the derivatives astaxanthin and capsanthin, via the common intermediate zeaxanthin. To generate rice varieties producing diverse carotenoids beyond ß-carotene, we specifically used a Capsicum ß-carotene hydroxylase gene, B (CaBch) and a codon optimized version of the same gene, stB (stBch) to increase zeaxanthin synthesis. We also used a recombinant BAK gene (CaBch-2A-HpBkt), consisting of the CaBch sequence and a Haematococcus ß-carotene ketolase gene (HpBkt) linked by a bicistronic 2A sequence, as well as a codon optimized recombinant stBAK gene (stBch-2A-stBkt) to create astaxanthin synthesis. The four cassettes to seed-specifically express the B, stB, BAK and stBAK genes were individually combined with a PAC gene (CaPsy-2A-PaCrtI) cassette to previously impart ß-carotene-enriched trait in rice endosperm. The single T-DNA vectors of B-PAC, stB-PAC, BAK-PAC and stBAK-PAC resulted in the accumulation of zeaxanthin and astaxanthin in the endosperm of the transgenic rice seeds. In addition, an extended version on the carotenoid pathway was introduced into rice to allow the production of capsanthin, by intercrossing a B-PAC rice line with a Ccs rice line, which harbors a Capsicum capsanthin-capsorubin synthase gene. Ultimately, we developed three functional rice varieties: B-PAC (0.8µg/g zeaxanthin, deep yellow), stBAK-PAC (1.4µg/g ketocarotenoids, including astaxanthin, pinkish red) and B-PAC x Ccs (0.4µg/g of ketoxanthophylls, including capsanthin, orange-red) with the similar levels of total carotenoids to PAC rice, suggesting the capacity was dependent on ß-carotene levels. Collectively, a combination of genetic engineering and conventional breeding is effective for multi-step metabolic engineering and biochemical pathway extension.

3.
Food Chem ; 266: 375-380, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30381200

RESUMO

An accurate method for the analysis of γ-aminobutyric acid (GABA) in rice grain was developed using trimethylsilyl (TMS) derivatization and stable isotope dilution gas chromatography-mass spectrometry. When this method was used with GABA­d6 as an internal standard (IS), the observed GABA concentration was maintained at 100% of the initial concentration with increasing storage time of the vial in the autosampler. In contrast, when using ribitol as an IS and multiple injections from one vial or single injections from different vials, the observed GABA concentration was 85 and 113% of the initial concentration upon increased storage time, respectively. The improved method recoveries at two different spike levels were between 93.3 and 97.8%, with relative standard deviations of less than 3.3%. The GABA content of resveratrol-enriched transgenic rice was compared with that of its non-transgenic counterpart from two field sites, and statistically non-significant differences were observed between the two grains.

4.
BMC Plant Biol ; 18(1): 291, 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30463509

RESUMO

BACKGROUND: Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour. RESULTS: A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes. CONCLUSIONS: While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

5.
Front Plant Sci ; 9: 1388, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30294338

RESUMO

The RsMYB1 transcription factor (TF) controls the regulation of anthocyanin in radishes (Raphanus sativus), and its overexpression in tobacco and petunias strongly enhances anthocyanin production. However, there are no data on the involvement of RsMYB1 in the mechanisms underlying abiotic stress tolerance, despite strong sequence similarity with other MYBs that confer such tolerance. In this study, we used the anthocyanin-enriched transgenic petunia lines PM6 and PM2, which overexpress RsMYB1. The tolerance of these lines to heavy metal stress was investigated by examining several physiological and biochemical factors, and the transcript levels of genes related to metal detoxification and antioxidant activity were quantified. Under normal conditions (control conditions), transgenic petunia plants (T2-PM6 and T2-PM2) expressing RsMYB1, as well as wild-type (WT) plants, were able to thrive by producing well-developed broad leaves and regular roots. In contrast, a reduction in plant growth was observed when these plants were exposed to heavy metals (CuSO4, ZnSO4, MnSO4, or K2Cr2O7). However, T2-PM6 and T2-PM2 were found to be more stress tolerant than the WT plants, as indicated by superior results in all analyzed parameters. In addition, RsMYB1 overexpression enhanced the expression of genes related to metal detoxification [glutathione S-transferase (GST) and phytochelatin synthase (PCS)] and antioxidant activity [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX)]. These results suggest that enhanced expression levels of the above genes can improve metal detoxification activities and antioxidant activity, which are the main components of defense mechanism included in abiotic stress tolerance of petunia. Our findings demonstrate that RsMYB1 has potential as a dual-function gene that can have an impact on the improvement of anthocyanin production and heavy metal stress tolerance in horticultural crops.

6.
J Exp Bot ; 69(21): 5105-5116, 2018 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-30124964

RESUMO

Carotenoids of staple food crops have a high nutritional value as provitamin A components in the daily diet. To increase the levels of carotenoids, inhibition of carotenoid-cleavage dioxygenases (CCDs), which degrade carotenoids, has been considered as a promising target in crop biotechnology. In this study, suppression of the OsCCD1, OsCCD4a, and OsCCD4b genes using RNAi was verified in transgenic rice plants by quantitative RT-PCR and small RNA detection. Leaf carotenoids were significantly increased overall in OsCCD4a-RNAi lines of the T1 generation, and the highest accumulation of 1.3-fold relative to non-transgenic plants was found in a line of the T2 generation. The effects on seed carotenoids were determined via cross-fertilization between ß-carotene-producing transgenic rice and one of two independent homozygous lines of OsCCD1-RNAi, OsCCD4a-RNAi, or OsCCD4b-RNAi. This showed that carotenoids were increased to a maximum of 1.4- and 1.6-fold in OsCCD1-RNAi and OsCCD4a-RNAi, respectively, with a different preference toward α-ring and ß-ring carotenoids; levels could not be established in OsCCD4b-RNAi. In addition, the contents of four carotenoids decreased when OsCCD1, OsCCD4a, and OsCCD4b were overexpressed in E. coli strains accumulating phytoene, lycopene, ß-carotene, and zeaxanthin. OsCCD1 and OsCCD4a had a similar high carotenoid degrading activity, followed by OsCCD4b without substrate specificity. Overall, our results suggest that suppresing OsCCD4a activity may have potential as a tool for enhancing the carotenoid content of seed endosperms and leaves in rice.

7.
Int J Mol Sci ; 19(8)2018 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-30060460

RESUMO

Anthocyanins are responsible pigments for giving attractive colors of plant organs and nutraceutical benefits of grains. Anthocyanin biosynthesis is known to be regulated by transcription factors and other regulatory proteins. In rice (Oryza sativa), the R2R3 MYB transcription factor (TF) OsC1 and a bHLH TF, OsB2, were previously reported to control anthocyanin biosynthesis in vegetative tissues and seeds, respectively; however, the regulatory mechanisms of the anthocyanin biosynthesis by TFs remain largely unknown. In this study, we identified OsBBX14, a homolog of Arabidopsis thaliana B-box domain protein 22 (AtBBX22), and investigated its function. The transcript level of OsBBX14 was high in pigmented rice seeds and gradually increased as the seeds matured. The ectopic expression of OsBBX14 in Arabidopsis resulted in a dramatic increase in anthocyanin accumulation in its seedlings. Using a steroid receptor-based inducible activation system, OsBBX14 and OsHY5 were found to directly activate OsC1 or OsB2 in an independent or collaborative manner. Yeast two hybrid revealed that the second B-box domain of OsBBX14 physically interacts with the bZIP domain of OsHY5. These results suggest that the anthocyanin biosynthesis in rice is induced and finely tuned by OsBBX14 in collaboration with OsHY5.


Assuntos
Antocianinas/biossíntese , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plântula/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Clorofila/biossíntese , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oryza/genética , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Plântula/crescimento & desenvolvimento , Ativação Transcricional
8.
Front Plant Sci ; 9: 818, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29971078

RESUMO

Wheat gliadins are a complex group of proteins that contribute to the functional properties of wheat flour doughs and contain epitopes that are relevant for celiac disease (CD) and wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, we extracted ethanol-soluble gliadin fractions from flour of the Korean bread wheat cultivar Keumkang. Proteins were separated by 2-dimensional gel electrophoresis (2-DE) using a pI range of 6-11 in the first dimension and subjected to tandem mass spectrometry. α-, γ-, and ω-gliadins were identified as the predominant proteins in 31, 28, and one 2-DE spot, respectively. An additional six ω-gliadins were identified in a separate experiment in which a pI range of 3-11 was used for protein separation. We analyzed the composition of CD- and WDEIA-relevant epitopes in the gliadin sequences from Keumkang flour, demonstrating the immunogenic potential of this cultivar. Detailed knowledge about the complement of gliadins accumulated in Keumkang flour provides the background necessary to devise either breeding or biotechnology strategies to improve the functional properties and reduce the adverse health effects of the flour.

9.
Int J Mol Sci ; 19(1)2018 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-29361688

RESUMO

Flower color is a main target for flower breeding. A transgenic approach for flower color modification requires a transgene and a flower-specific promoter. Here, we expressed the B-peru gene encoding a basic helix loop helix (bHLH) transcription factor (TF) together with the mPAP1 gene encoding an R2R3 MYB TF to enhance flower color in tobacco (Nicotiana tabacum L.), using the tobacco anthocyanidin synthase (ANS) promoter (PANS) to drive flower-specific expression. The transgenic tobacco plants grew normally and produced either dark pink (PANSBP_DP) or dark red (PANSBP_DR) flowers. Quantitative real time polymerase chain reaction (qPCR) revealed that the expression of five structural genes in the flavonoid biosynthetic pathway increased significantly in both PANSBP_DP and PANSBP_DR lines, compared with the non-transformed (NT) control. Interestingly, the expression of two regulatory genes constituting the active MYB-bHLH-WD40 repeat (WDR) (MBW) complex decreased significantly in the PANSBP_DR plants but not in the PANSBP_DP plants. Total flavonol and anthocyanin abundance correlated with flower color, with an increase of 1.6-43.2 fold in the PANSBP_DP plants and 2.0-124.2 fold in the PANSBP_DR plants. Our results indicate that combinatorial expression of B-peru and mPAP1 genes under control of the ANS promoter can be a useful strategy for intensifying flower color without growth retardation.


Assuntos
Flores/genética , Regulação da Expressão Gênica de Plantas , Fenótipo , Pigmentação/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Especificidade de Órgãos/genética , Plantas Geneticamente Modificadas , Tabaco/genética , Tabaco/metabolismo
10.
3 Biotech ; 8(1): 56, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29354367

RESUMO

Although many studies on low-molecular-weight glutenin subunit (LMW-GS) function have been reported, a comprehensive comparison between specific genes and their protein product is still lacking. This study aimed to link the 43 genes isolated from the Korean wheat variety "Jokyoung" in the authors' previous study to their protein products. Proteins were separated using two-dimensional gel electrophoresis (2-DGE) and identified by tandem mass spectrometry (MS/MS) at the gene haplotype level. Using MS/MS analysis of 17 protein spots, two spots were identified in the Glu-A3 locus and the corresponding haplotype was GluA3-13(Glu-A3c). Six spots were identified in the Glu-B3 locus and the corresponding haplotypes were GluB3-33 and GluB3-43 (Glu-B3h). Eight spots were identified in the Glu-D3 locus and the corresponding haplotypes were GluD3-11, GluD3-21, GluD3-31, GluD3-5, and GluD3-6 (Glu-D3a), and one spot was contaminated with gamma gliadin. Phylogenetic analysis and alignment of nucleotide and amino acid sequences assigned 35 of the 43 genes to seven haplotypes: GluA3-13, GluB3-43, GluD3-11, GluD3-21, GluD3-31, GluD3-42, and GluD3-5. Taken together, except for GluB3-33 and GluD3-6, which were not isolated, linking of each gene to the corresponding protein products at the gene haplotype level was accomplished using proteomic tools and phylogenetic analysis.

11.
Breed Sci ; 67(4): 398-407, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29085250

RESUMO

Two-dimensional gel electrophoresis (2-DGE) was used as a complement to SDS-PAGE to determine the allelic compositions of LMW-GS in 32 Korean wheat cultivars. Protein patterns generated by 2-DGE from each cultivar were compared to patterns from standard wheat cultivars for each allele. At the Glu-A3 locus, thirteen c, twelve d, three e (null), two g and two new alleles were identified. At the Glu-B3 locus, one b, nineteen d, four h, one i and five ad alleles were identified. At the Glu-D3 locus, twenty-three a, four b, four c and one l alleles were identified. When compared to results obtained previously using SDS-PAGE, there were discrepancies in the allelic designations of 10 of 32 cultivars (31%). While SDS-PAGE is a rapid and relatively simple method for assessing LMW-GS composition, the similar mobilities of the proteins makes it difficult to discriminate certain alleles. 2-DGE is a more complicated technique, but provides a more accurate picture of the complement of the LMW-GS in a given cultivar. In addition to providing essential information for wheat breeders, the 2-DGE reference maps generated in this study will make it possible to study the contributions of individual LMW-GS to flour quality.

12.
Int J Mol Sci ; 18(11)2017 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-29156580

RESUMO

Rice (Oryza sativa L.) is a primary global food cereal. However, when compared to wheat, rice has poor food processing qualities. Dough that is made from rice flour has low viscoelasticity because rice seed lacks storage proteins that are comparable to gluten protein from wheat. Thus, current research efforts aim to improve rice flour processing qualities through the transgenic expression of viscoelastic proteins in rice seeds. In this study, we characterized the transgenic expression of wheat glutenin subunits in rice seeds. The two genes 1Dx5_KK and 1Dy10_JK, which both encode wheat high-molecular-weight glutenin subunits that confer high dough elasticity, were cloned from Korean wheat cultivars KeumKang and JoKyung, respectively. These genes were inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter and were expressed in the high-amylose Korean rice cultivar Koami (Oryza sativa L.). Individual expression of both glutenin subunits was confirmed by SDS-PAGE and immunoblot analyses performed using T3 generation of transgenic rice seeds. The subcellular localization of 1Dx5_KK and 1Dy10_JK in the rice seed endosperm was confirmed by immunofluorescence analysis, indicating that the wheat glutenin subunits accumulate in protein body-II and novel protein body types in the rice seed. These results contribute to our understanding of engineered seed storage proteins in rice.


Assuntos
Endosperma/metabolismo , Glutens/genética , Glutens/metabolismo , Oryza/genética , Triticum/metabolismo , Clonagem Molecular , Peso Molecular , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Engenharia de Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Análise de Sequência de Proteína , Triticum/genética
13.
Front Plant Sci ; 8: 1917, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29167678

RESUMO

The MYB-bHLH-WDR (MBW) complex activates anthocyanin biosynthesis through the transcriptional regulation. RsMYB1 has been identified as a key player in anthocyanin biosynthesis in red radish (Raphanus sativus L.), but its partner bHLH transcription factor (TF) remains to be determined. In this study, we isolated a bHLH TF gene from red radish. Phylogenetic analysis indicated that this gene belongs to the TT8 clade of the IIIF subgroup of bHLH TFs, and we thus designated this gene RsTT8. Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of Arabidopsis thaliana protoplasts harboring the RsTT8-sGFP construct. We evaluated anthocyanin biosynthesis and RsTT8 expression levels in three radish varieties (N, C, and D) that display different red phenotypes in the leaves, root flesh, and root skins. The root flesh of the C variety and the leaves and skins of the D variety exhibit intense red pigmentation; in these tissues, RsTT8 expression showed totally positive association with the expression of RsMYB1 TF and of five of eight tested anthocyanin biosynthesis genes (i.e., RsCHS, RsCHI, RsF3H, RsDFR, and RsANS). Heterologous co-expression of both RsTT8 and RsMYB1 in tobacco leaves dramatically increased the expression of endogenous anthocyanin biosynthesis genes and anthocyanin accumulation. Furthermore, a yeast two-hybrid assay showed that RsTT8 interacts with RsMYB1 at the MYB-interacting region (MIR), and a transient transactivation assay indicated that RsTT8 activates the RsCHS and RsDFR promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, with RsTT8 restored red pigmentation, and resulted in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively. Together, these results show that RsTT8 functions as a regulatory partner with RsMYB1 during anthocyanin biosynthesis.

14.
Molecules ; 22(7)2017 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-28672820

RESUMO

The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differences in retention times (RTs) for subunits corresponding to HMW-GS alleles were determined using 16 standard wheat cultivars with known HMW-GS compositions. Subunits that were not identified unambiguously by RP-HPLC were distinguished by SDS-PAGE or inferred from association with linked subunits. The method was used to verify the allelic compositions of 32 Korean wheat cultivars previously determined using SDS-PAGE and to assess the compositions of six new Korean cultivars. Three cultivars contained subunits that were identified incorrectly in the earlier analysis. The improved RP-HPLC method combined with conventional SDS-PAGE provides for accurate, efficient and reliable identification of HMW-GS and will contribute to efforts to improve wheat end-use quality.


Assuntos
Glutens/química , Glutens/isolamento & purificação , Triticum/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Melhoramento Vegetal , Triticum/classificação
15.
J Agric Food Chem ; 65(26): 5287-5298, 2017 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-28537403

RESUMO

We isolated cDNAs encoding flavonol synthase (FLS) from the red onion "H6" (AcFLS-H6) and the yellow onion "Hwangryongball" (AcFLS-HRB). We found three amino acid variations between the two sequences. Kinetic analysis with recombinant proteins revealed that AcFLS-HRB exhibited approximately 2-fold higher catalytic efficiencies than AcFLS-H6 for dihydroflavonol substrates and that both proteins preferred dihydroquercetin to dihydrokaempferol. The expression patterns of flavonoid biosynthesis genes corresponded to the accumulation patterns of flavonoid aglycones in both onions. Whereas the other flavonoid biosynthesis genes were weakly expressed in the HRB sheath compared to that of H6, the expression of FLS was similar in both onions. This relatively enhanced FLS expression, along with the higher activity of AcFLS-HRB, could increase the quercetin production in the HRB sheath. The quercetin content was approximately 12-fold higher than the cyanidin content in the H6 sheath, suggesting that FLS has priority in the competition between FLS and dihydroflavonol 4-reductase (DFR) for their substrate dihydroquercetin.


Assuntos
Flavonoides/biossíntese , Cebolas/enzimologia , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Cor , Flavonoides/química , Regulação da Expressão Gênica de Plantas , Cinética , Estrutura Molecular , Cebolas/química , Cebolas/genética , Cebolas/metabolismo , Oxirredutases/química , Oxirredutases/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética
16.
Plant Physiol Biochem ; 109: 482-490, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27842297

RESUMO

To examine flux regulation in the flavonoid pathway of tobacco flowers, we suppressed two genes for dihydroflavonol 4-reductase (NtDFR 1 and 2) by RNA interference (Ri)-mediated post transcriptional gene silencing in pink-flowered tobacco. Two phenotypes were observed, pale pink (DFR-Ri_PP)- and white (DFR-Ri_W)-flowered lines. The relative mRNA levels of NtDFR genes in DFR-Ri_PP and DFR-Ri_W lines were reduced by 79%-95% relative to non-transformed (NT) plants. DFR-Ri_W lines had five-fold higher levels of small interference RNAs compared to DFR-Ri_PP lines. Expression of eight structural genes in the flavonoid pathway was significantly increased in DFR-Ri_W lines but not in DFR-Ri_PP lines based on quantitative RT-PCR. Anthocyanin contents correlated with flower color, with a reduction of 72%-97% in DFR-Ri_PP and DFR-Ri_W lines. Decreases in anthocyanin in flower were proportional with reductions of proanthocyanidin content in seeds. Two pale pink lines, DFR-Ri_PP 17 and 20, with anthocyanin decreases and the lowest level of DFR gene silencing, had higher (dihydro) flavonol production than a white flowered line, DFR-Ri_W 67. This finding suggests that suppression of DFR can increase the total levels of flavonoids due to (dihydro) flavonol biosynthesis. Our observations that higher suppression of DFR had a greater influence on the expression of flavonoid biosynthetic genes demonstrates the key role of DFR in the pathway and allows selection among DFR-Ri lines for plants with specific gene expression profiles to fine-tune flux through the pathway.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Flavonoides/biossíntese , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Tabaco/genética , Tabaco/metabolismo , Vias Biossintéticas/genética , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Pigmentação/genética , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo
17.
Front Plant Sci ; 7: 1624, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27843443

RESUMO

Seed storage proteins (SSPs) such as glutelin, prolamin, and globulin are abundant components in some of the most widely consumed food cereals in the world. Synthesized in the rough endoplasmic reticulum (ER), SSPs are translocated to the protein bodies. Prolamins are located at the spherical protein body I derived from the ER, whereas, glutelins and globulin are accumulated in the irregularly shaped protein bodies derived from vacuoles. Our previous studies have shown that the individual suppression of glutelins, 13-kDa prolamins and globulin caused the compensative accumulation of other SSPs. Herein, to investigate the phenotypic and molecular features of SSP deficiency transgenic rice plants suppressing all glutelins, prolamins, and globulin were generated using RNA interference (RNAi). The results revealed that glutelin A, cysteine-rich 13-kDa prolamin and globulin proteins were less accumulated but that glutelin B and ER chaperones, such as binding protein 1 (BiP1) and protein disulfide isomerase-like 1-1 (PDIL1-1), were highly accumulated at the transcript and protein levels in seeds of the transformants compared to those in the wild-type seeds. Further, the transcription of starch synthesis-related genes was reduced in immature seeds at 2 weeks after flowering, and the starch granules were loosely packaged with various sphere sizes in seed endosperms of the transformants, resulting in a floury phenotype. Interestingly, the rates of sprouting and reducing sugar accumulation during germination were found to be delayed in the transformants compared to the wild-type. In all, our results provide new insight into the role of SSPs in the formation of intracellular organelles and in germination.

18.
Plant Sci ; 252: 144-150, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27717450

RESUMO

The effects of three different sucrose concentrations on plant growth and anthocyanin accumulation were examined in non-transgenic (NT) and transgenic (T2) specimens of the Petunia hybrida cultivar 'Mirage rose' that carried the anthocyanin regulatory transcription factors B-Peru+mPAP1 or RsMYB1. Anthocyanin accumulation was not observed in NT plants in any treatments, whereas a range of anthocyanin accumulation was observed in transgenic plants. The anthocyanin content detected in transgenic plants expressing the anthocyanin regulatory transcription factors (B-Peru+mPAP1 or RsMYB1) was higher than that in NT plants. In addition, increasing sucrose concentration strongly enhanced anthocyanin content as shown by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, wherein increased concentrations of sucrose enhanced transcript levels of the transcription factors that are responsible for the induction of biosynthetic genes involved in anthocyanin synthesis; this pattern was not observed in NT plants. In addition, sucrose affected plant growth, although the effects were different between NT and transgenic plants. Taken together, the application of sucrose could enhance anthocyanin production in vegetative tissue of transgenic Petunia carrying anthocyanin regulatory transcription factors, and this study provides insights about interactive effects of sucrose and transcription factors in anthocyanin biosynthesis in the transgenic plant.


Assuntos
Antocianinas/genética , Petunia/genética , Proteínas de Plantas/fisiologia , Sacarose/farmacologia , Fatores de Transcrição/fisiologia , Antocianinas/biossíntese , Antocianinas/metabolismo , Regulação da Expressão Gênica de Plantas , Petunia/efeitos dos fármacos , Petunia/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Int J Mol Sci ; 17(9)2016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27649148

RESUMO

Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3',4'-dihydroxylated leucocyanidin. 3'-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3'H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3'H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/biossíntese , Oryza/enzimologia , Substituição de Aminoácidos , Vias Biossintéticas , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hidroxilação , Oryza/classificação , Oryza/genética , Oryza/metabolismo , Folhas de Planta/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Análise de Sequência de DNA , Especificidade por Substrato
20.
J Agric Food Chem ; 64(21): 4426-34, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-27172980

RESUMO

A total of 38 bioactive compounds, including glucosinolates, carotenoids, tocopherols, sterols, and policosanols, were characterized from nine varieties of Chinese cabbage (Brassica rapa L. subsp. pekinensis) to determine their phytochemical diversity and analyze their abundance relationships. The metabolite profiles were evaluated with principal component analysis (PCA), Pearson correlation analysis, and hierarchical clustering analysis (HCA). PCA and HCA identified two distinct varieties of Chinese cabbage (Cheonsangcheonha and Waldongcheonha) with higher levels of glucosinolates and carotenoids. Pairwise comparisons of the 38 metabolites were calculated using Pearson correlation coefficients. The HCA, which used the correlation coefficients, clustered metabolites that are derived from closely related biochemical pathways. Significant correlations were discovered between chlorophyll and carotenoids. Additionally, aliphatic glucosinolate and carotenoid levels were positively correlated. The Cheonsangcheonha and Waldongcheonha varieties appear to be good candidates for breeding because they have high glucosinolate and carotenoid levels.


Assuntos
Brassica rapa/química , Carotenoides/análise , Glucosinolatos/análise , Brassica rapa/classificação , Brassica rapa/genética , Brassica rapa/metabolismo , Cruzamento , Carotenoides/metabolismo , Glucosinolatos/metabolismo , Metabolômica
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