Sun-Hwa Ha

Overexpression of the OsERF71 transcription factor Alters Rice Root Structure and Drought Resistance

OsERF71 alters root structure to enhance drought resistance. Plant responses to drought stress require the regulation of transcriptional networks via drought-responsive transcription factors, which mediate a range of morphological and physiological changes. AP2/ERF transcription factors are known to act as key regulators of drought resistance transcriptional networks; however, little is known about the associated molecular mechanisms that give rise to specific morphological and physiological adaptations. In this study, we functionally characterized the rice (Oryza sativa) drought-responsive AP2/ERF transcription factor OsERF71, which is expressed predominantly in the root meristem, pericycle, and endodermis. Overexpression of OsERF71, either throughout the entire plant or specifically in roots, resulted in a drought resistance phenotype at the vegetative growth stage, indicating that overexpression in roots was sufficient to confer drought resistance. The root-specific overexpression was more effective in conferring drought resistance at the reproductive stage, such that grain yield was increased by 23% to 42% over wild-type plants or whole-body overexpressing transgenic lines under drought conditions. OsERF71 overexpression in roots elevated the expression levels of genes related to cell wall loosening and lignin biosynthetic genes, which correlated with changes in root structure, the formation of enlarged aerenchyma, and high lignification levels. Furthermore, OsERF71 was found to directly bind to the promoter of OsCINNAMOYL-COENZYME A REDUCTASE1, a key gene in lignin biosynthesis. These results indicate that the OsERF71-mediated drought resistance pathway recruits factors involved in cell wall modification to enable root morphological adaptations, thereby providing a mechanism for enhancing drought resistance.

Unraveling the Light-Specific Metabolic and Regulatory Signatures of Rice through Combined in Silico Modeling and Multiomics Analysis

Combined in silico modeling and multiomics data analysis elucidate the transcriptional control of rice cellular metabolism upon light signaling. Light quality is an important signaling component upon which plants orchestrate various morphological processes, including seed germination and seedling photomorphogenesis. However, it is still unclear how plants, especially food crops, sense various light qualities and modulate their cellular growth and other developmental processes. Therefore, in this work, we initially profiled the transcripts of a model crop, rice (Oryza sativa), under four different light treatments (blue, green, red, and white) as well as in the dark. Concurrently, we reconstructed a fully compartmentalized genome-scale metabolic model of rice cells, iOS2164, containing 2,164 unique genes, 2,283 reactions, and 1,999 metabolites. We then combined the model with transcriptome profiles to elucidate the light-specific transcriptional signatures of rice metabolism. Clearly, light signals mediated rice gene expressions, differentially regulating numerous metabolic pathways: photosynthesis and secondary metabolism were up-regulated in blue light, whereas reserve carbohydrates degradation was pronounced in the dark. The topological analysis of gene expression data with the rice genome-scale metabolic model further uncovered that phytohormones, such as abscisate, ethylene, gibberellin, and jasmonate, are the key biomarkers of light-mediated regulation, and subsequent analysis of the associated genes’ promoter regions identified several light-specific transcription factors. Finally, the transcriptional control of rice metabolism by red and blue light signals was assessed by integrating the transcriptome and metabolome data with constraint-based modeling. The biological insights gained from this integrative systems biology approach offer several potential applications, such as improving the agronomic traits of food crops and designing light-specific synthetic gene circuits in microbial and mammalian systems.

Marker development for the identification of rice seed color

Pigmented traits in rice seeds are regarded as important breeding goals for crop improvement. Marker-assisted selection is very helpful when screening for target seed color traits in the early stages of plant development. Among the genes involved in the biosynthesis of anthocyanins and proanthocyanins (PAs) that are candidates for marker development, we examined the expression of five genes encoding CHS, CHI, F3H, DFR, and ANS in the seeds of non-pigmented white and pigmented black and red rice cultivars. The transcript levels of all these genes except for CHI are higher in pigmented rice than in non-pigmented rice. Sequence variations in these biosynthetic genes revealed that the DFR gene harbors a single nucleotide substitution that generates a premature stop codon in white rice. Additional sequence variations in two regulatory genes, OSB1 and Rc, were also compared among the same cultivars. The sequence of the OSB1 gene in black rice was found to differ from that in red and white rice. The sequence of the Rc gene in red rice also differed from that in white and black rice. Based on these variations, we developed two CAPS markers for DFR and OSB1 genes and an Indel marker for the Rc gene. The combined use of these three markers could discriminate rice seeds harboring white, black and red color. We validated the usefulness of these markers in 34 rice cultivars. Hence, the combined application of our three new markers may have utility to screen the seed color prior to seed setting in rice breeding programs.

Identification and quantification of carotenoids in paprika fruits and cabbage, kale, and lettuce leaves

Twelve carotenoids were identified in Korean leafy vegetables and paprikas. by high-performance liquid chromatography, Carotenoid contents varied greatly, with red paprika having a higher antheraxanthin and capsanthin contents than other paprikas. Orange paprika had higher levels of zeaxanthin, β-cryptoxanthin, lutein, and α-carotene compared to those of other paprikas. The results of Pearson’s correlation analysis using quantitative data of carotenoids revealed that significant positive relationships were apparent between capsanthin and antheraxanthin (r=0.9870, p <0.0001), zeaxanthin and α-cryptoxanthin (r=0.9951, p <0.0001), as well as lutein and α-carotene (r=0.9612, p <0.0001). Because the correlations between carotenoids levels have provided valuable information regarding metabolic associations, this technique will contribute to identifying metabolic links for carotenoid biosynthesis.

Metabolic and transcriptional regulatory mechanisms underlying the anoxic adaptation of rice coleoptile

Rice is a unique crop plant since it can survive under anoxia condition by flooding and also germinate and grow up to coleoptile through its distinctive adaptations. Despite several decades of research on this topic, the current knowledge on the molecular machinery of rice under anoxia is very limited. Therefore, we unraveled the possible regulatory mechanisms by resorting to systems biology approach which combines the metabolic modeling and transcriptome analysis. Such integrative analysis highlight the critical role of MYB, bZIP, ERF and ZnF transcription factors in up-regulating the fermentation and sucrose metabolism genes to generate sufficient energy for cellular growth.

Molecular and Biochemical Analysis of Two Rice Flavonoid 3'-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain.

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.

Light-specific transcriptional regulation of the accumulation of carotenoids and phenolic compounds in rice leaves

ABSTRACT Carotenoids and phenolic compounds are important subgroups of secondary metabolites having an array of functional roles in the growth and development of plants. They are also major sources for health and pharmaceutical benefits, and industrially relevant biochemicals. The control of the biosynthesis of these compounds depends mainly on the quality and quantity of different light sources. Thus, to unravel their light-specific transcriptional regulation in rice leaves, we performed promoter analysis of genes upregulated in response to blue and red lights. The analysis results suggested a crosstalk between different phytohormones and the involvement of key transcription factors such as bHLH, bZIP, MYB, WRKY, ZnF and ERF [jasmonic acid inducible], in the regulation of higher accumulation of carotenoids and phenolic compounds upon blue light. Overall, the current analysis could improve our understanding of the light-specific regulatory mechanism involved in the biosynthesis of secondary metabolites via possible critical links between different TFs in rice leaves.

RNAi-mediated suppression of dihydroflavonol 4-reductase in tobacco allows fine-tuning of flower color and flux through the flavonoid biosynthetic pathway.

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.

Improved quantification of γ-aminobutyric acid in rice using stable isotope dilution gas chromatography–mass spectrometry

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 GABA‑d6 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.

A Radish Basic Helix-Loop-Helix Transcription Factor, RsTT8 Acts a Positive Regulator for Anthocyanin Biosynthesis

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.