Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2.

Plastoglobules (PGs) are plastidial lipid droplets enclosed by a polar monolayer born from the thylakoid membrane when plants require active lipid metabolism, including carotenogenesis, under the environmental stress and during plastid transition. Despite the fact that many proteins are reported to target PGs, their translocation mechanism has remained largely unexplored. To elucidate this process, we studied the influence of three hydrophobic regions (HR)-HR1 (1-45th aa), HR2 (46-80th aa), and HR3 (229-247th aa)-of rice phytoene synthase 2 (OsPSY2, 398 aa), which has previously shown to target PGs. As results, HR1 includes the crucial sequence (31-45th aa) for chloroplast import and the stromal cleavage occurs at a specific alanine site (64th aa) within HR2, verifying that a N-terminal 64-aa-region works as the transit peptide (Tp). HR2 has a weak PG-targeting signal by showing synchronous and asynchronous localization patterns in both PGs and stroma of chloroplasts. HR3 exhibited a strong PG-targeting role with the required positional specificity to prevent potential issues such as non-accumulation, aggregation, and folding errors in proteins. Herein, we characterized a Tp and two transmembrane domains in three HRs of OsPSY2 and propose a spontaneous pathway for its PG-translocation with a shape embedded in the PG-monolayer. Given this subplastidial localization, we suggest six sophisticated tactics for plant biotechnology applications, including metabolic engineering and molecular farming.

Differential Regulation of an OsIspH1, the Functional 4-Hydroxy-3-Methylbut-2-Enyl Diphosphate Reductase, for Photosynthetic Pigment Biosynthesis in Rice Leaves and Seeds.

The methylerythritol 4-phosphate (MEP) pathway is responsible for providing common precursors for the biosynthesis of diverse plastidial terpenoids, including chlorophylls, carotenoids, and phytohormones, in plants. In rice (Oryza sativa), the last-step genes encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase [HDR/isoprenoid synthesis H (IspH)] have been annotated in two genes (OsIspH1 and OsIspH2) in the rice genome. The spatial transcript levels indicated that OsIspH1 is highly expressed in all tissues at different developmental stages, whereas OsIspH2 is barely expressed due to an early stop in exon 1 caused by splicing error. OsIspH1 localized into plastids and osisph1, a T-DNA inserted knockout mutant, showed an albino phenotype, indicating that OsIspH1 is the only functional gene. To elucidate the role of OsIspH1 in the MEP pathway, we created two single (H145P and K407R) and double (H145P/K407R) mutations and performed complementation tests in two hdr mutants, including Escherichia coli DLYT1 strains and osisph1 rice plants. The results showed that every single mutation retained HDR function, but a double mutation lost it, proposing that the complementary relations of two residues might be important for enzyme activity but not each residue. When overexpressed in rice plants, the double-mutated gene, OsIspH1MUT , reduced chlorophyll and carotenoid biosynthesis in the leaves and seeds. It confirmed the crucial role of OsIspH1 in plastidic terpenoid biosynthesis, revealing organ-specific differential regulation of OsIspH1 in rice plants.

Tissue-specific enhancement of OsRNS1 with root-preferred expression is required for the increase of crop yield.

INTRODUCTION: Root development is a fundamental process that supports plant survival and crop productivity. One of the essential factors to consider when developing biotechnology crops is the selection of a promoter that can optimize the spatial-temporal expression of introduced genes. However, there are insufficient cases of suitable promoters in crop plants, including rice. OBJECTIVES: This study aimed to verify the usefulness of a new rice root-preferred promoter to optimize the function of a target gene with root-preferred expression in rice. METHODS: osrns1 mutant had defects in root development based on T-DNA insertional mutant screening and CRISPR technology. To optimize the function of OsRNS1, we generated OsRNS1-overexpression plants under two different promoters: a whole-plant expression promoter and a novel root-preferred expression promoter. Root growth, yield-related agronomic traits, RNA-seq, and reactive oxygen species (ROS) accumulation were analyzed for comparison. RESULTS: OsRNS1 was found to be involved in root development through T-DNA insertional mutant analysis and gene editing mutant analysis. To understand the gain of function of OsRNS1, pUbi1::OsRNS1 was generated for the whole-plant expression, and both root growth defects and overall growth defects were found. To overcome this problem, a root-preferential overexpression line using Os1-CysPrxB promoter (Per) was generated and showed an increase in root length, plant height, and grain yield compared to wild-type (WT). RNA-seq analysis revealed that the response to oxidative stress-related genes was significantly up-regulated in both overexpression lines but was more obvious in pPer::OsRNS1. Furthermore, ROS levels in the roots were drastically decreased in pPer::OsRNS1 but were increased in the osrns1 mutants compared to WT. CONCLUSION: The results demonstrated that the use of a root-preferred promoter effectively optimizes the function of OsRNS1 and is a useful strategy for improving root-related agronomic traits as well as ROS regulation.

Overexpression of OsMYBR22/OsRVE1 transcription factor simultaneously enhances chloroplast-dependent metabolites in rice grains.

The OsMYBR22 (same to OsRVE1), an R1type-MYB transcription factor belonging to the rice CCA1-like family, was upregulated under blue light condition, which enhanced the chlorophyll and carotenoid accumulation. The overexpression of OsMYBR22 in rice (Oryza sativa, L) led to everlasting green seeds and leaves of a darker green. Transgene expression patterns showed more concordance with chlorophyll than carotenoid profiles. The transcript levels of most genes related to chlorophyll biosynthesis and degradation examined were similarly repressed in the late maturing stages of seeds. It proposed that rice seeds have the feedback regulatory mechanism for chlorophyll biosynthesis and also implied that evergreen seed traits might be caused due to the inhibition of degradation rather than the promotion of biosynthesis for chlorophylls. Metabolomics revealed that OsMYBR22 overexpression largely and simultaneously enhanced the contents of nutritional and functional metabolites such as chlorophylls, carotenoids, amino acids including lysine and threonine, and amino acid derivatives including γ-aminobutyric acid, which are mostly biosynthesized in chloroplasts. Transmission electron microscopy anatomically demonstrated greener phenotypes with an increase in the number and thickness of chloroplasts in leaves and the structurally retentive chloroplasts in tubular and cross cells of the seed inner pericarp region. In conclusion, the molecular actions of OsMYBR22/OsRVE1 provided a new strategy for the biofortified rice variety, an "Evergreen Rice," with high accumulation of chloroplast-localized metabolites in rice grains.

An OsKala3, R2R3 MYB TF, Is a Common Key Player for Black Rice Pericarp as Main Partner of an OsKala4, bHLH TF.

Rice (Oryza sativa) pericarp exhibits various colors due to the accumulation of anthocyanins and/or proanthocyanidins. Previous work revealed that the two basic helix-loop-helix (bHLH) transcription factors OsKala4 and OsRc are key regulators for the black and red pericarp traits, respectively, and their inactivation results in rice with white pericarp. However, their pericarp-specific R2R3 MYB partner remained unknown. Here, we characterized the role of the R2R3 MYB gene OsKala3 in rice pericarp pigmentation through genetic and molecular approaches. A rice protoplast transfection assay showed that OsKala3 is a nuclear-localized protein. Furthermore, OsKala3 physically interacted with OsKala4 in a yeast two-hybrid analysis. Co-transfection assays in rice protoplasts revealed that OsKala3 and OsKala4 mediate the activation of anthocyanin biosynthetic genes. Notably, the OsKala3 promoter region exhibited an insertion polymorphism specifically in rice cultivars with black pericarp, creating two tandem repeats while red and white varieties harbor only one. The number of repeats within the OsKala3 promoter correlated with increased transactivation by OsKala3, thus providing a rationale for the black pericarp characteristic of cultivars with two repeats. These results thus provide evidence for the molecular basis of anthocyanin biosynthesis in rice pericarp and may facilitate the introduction of this beneficial trait to other rice cultivars through marker-assisted breeding.

Metabolomic Variability of Different Soybean Genotypes: ß-Carotene-Enhanced (Glycine max), Wild (Glycine soja), and Hybrid (Glycine max × Glycine soja) Soybeans.

We obtained a new hybrid soybean (Hybrid) by hybridizing ß-carotene-enhanced soybean (BCE; Glycine max L.) containing the phytoene synthase-2A-carotene desaturase gene and wild-type soybean (Wild; Glycine soja). To investigate metabolic changes between variants, we performed metabolic profiling of leaves (three growth stages) and seeds. Multivariate analyses revealed significant metabolic differences between genotypes in seeds and leaves, with seeds showing accumulation of phytosterols, tocopherols, and carotenoids (BCE only), indicating co-induction of the methylerythritol 4-phosphate and mevalonic acid pathways. Additionally, Hybrid produced intermediate levels of carotenoids and high levels of amino acids. Principal component analysis revealed metabolic discrimination between growth stages of soybean leaves and identified differences in leaf groups according to different genotypes at 8, 12, and 16 weeks, with Wild showing higher levels of environmental stress-related compounds relative to BCE and Hybrid leaves. The metabolic profiling approach could be a useful tool to identify metabolic links in various soybean cultivars.

Diversity of Plastid Types and Their Interconversions.

Plastids are pivotal subcellular organelles that have evolved to perform specialized functions in plant cells, including photosynthesis and the production and storage of metabolites. They come in a variety of forms with different characteristics, enabling them to function in a diverse array of organ/tissue/cell-specific developmental processes and with a variety of environmental signals. Here, we have comprehensively reviewed the distinctive roles of plastids and their transition statuses, according to their features. Furthermore, the most recent understanding of their regulatory mechanisms is highlighted at both transcriptional and post-translational levels, with a focus on the greening and non-greening phenotypes.

Metabolite Profiling Reveals Distinct Modulation of Complex Metabolic Networks in Non-Pigmented, Black, and Red Rice (Oryza sativa L.) Cultivars.

Comprehensive profiling of primary and secondary metabolites was performed to understand metabolic differences associated with color formation in pigmented rice (Oryza sativa L.). Overall, 110 metabolites from non-pigmented, black, and red rice cultivars were identified. Black and red rice contained high levels of flavonoids associated with plant color. Black rice also contained high levels of terpenoids (carotenoids, tocopherols, phytosterols, and monoterpenes). The non-pigmented rice contained relatively low levels of secondary metabolites. Multivariate and pathway analyses were performed to data-mine the metabolite profiles. Hierarchical clustering analysis of correlation coefficients revealed metabolite clusters based on nitrogen and carbon sources. These clusters suggested a negative correlation between nitrogen and carbon. Pathway analysis revealed that black rice was rich in carbon-based secondary metabolites, with relatively low levels of primary metabolites compared with other rice cultivars. These data highlight the complex interactions between nitrogen and carbon metabolism of primary and secondary metabolites in rice. For the first time, the relationships and metabolic differences in terpenoid content (monoterpenes, triterpenes, and tetraterpenes) of non-pigmented and pigmented rice cultivars were analyzed. These findings should greatly contribute to the understanding of pigmented rice metabolome and inform breeding programs for new rice cultivars.

2A-linked bi-, tri-, and quad-cistrons for the stepwise biosynthesis of ß-carotene, zeaxanthin, and ketocarotenoids in rice endosperm.

Foot-and-mouth disease virus (FMDV) 2A constructs have been successfully used for the production of "Golden Rice", a ß-carotene producing rice strain. However, to allay public fears and opposition to plants carrying a mammalian pathogenic viral sequence, 2A-like synthetic sequences from Thosea asigna virus and Infectious myonecrosis virus were used to coordinate the coexpression of carotenoid biosynthetic genes. Here, up to four carotenogenic genes encoding PSY, CRTI, BCH and BKT were concatenated and produced ß-carotene, zeaxanthin, and ketocarotenoids (astaxanthin and adonixanthin) in transgenic rice seeds displaying color variation due to the difference in carotenoid content and composition.

The Predicted Functional Compartmentation of Rice Terpenoid Metabolism by Trans-Prenyltransferase Structural Analysis, Expression and Localization.

Most terpenoids are derived from the basic terpene skeletons of geranyl pyrophosphate (GPP, C10), farnesyl-PP (FPP, C15) and geranylgeranyl-PP (GGPP, C20). The trans-prenyltransferases (PTs) mediate the sequential head-to-tail condensation of an isopentenyl-PP (C5) with allylic substrates. The in silico structural comparative analyses of rice trans-PTs with 136 plant trans-PT genes allowed twelve rice PTs to be identified as GGPS_LSU (OsGGPS1), homomeric G(G)PS (OsGPS) and GGPS_SSU-II (OsGRP) in Group I; two solanesyl-PP synthase (OsSPS2 and 3) and two polyprenyl-PP synthases (OsSPS1 and 4) in Group II; and five FPSs (OsFPS1, 2, 3, 4 and 5) in Group III. Additionally, several residues in "three floors" for the chain length and several essential domains for enzymatic activities specifically varied in rice, potentiating evolutionarily rice-specific biochemical functions of twelve trans-PTs. Moreover, expression profiling and localization patterns revealed their functional compartmentation in rice. Taken together, we propose the predicted topology-based working model of rice PTs with corresponding terpene metabolites: GPP/GGPPs mainly in plastoglobuli, SPPs in stroma, PPPs in cytosol, mitochondria and chloroplast and FPPs in cytosol. Our findings could be suitably applied to metabolic engineering for producing functional terpene metabolites in rice systems.

Improving Nutritional and Functional Quality by Genome Editing of Crops: Status and Perspectives.

Genome-editing tools including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats (CRISPR) system have been applied to improve the quality of staple, oilseed, and horticultural crops with great accuracy and efficiency compared to conventional breeding. In particular, the CRISPR method has proven to be a feasible, cost-effective and versatile tool allowing precise and efficient editing of plant genomes in recent years, showing great potential in crop improvement. Until now, various genome-edited crops with enhanced commercial value have been developed by not only global companies but also small laboratories in universities, suggesting low entry barriers with respect to manpower and capital. In this study, we review the current applications of genome editing technologies to improve the nutritional and functional quality and preferred traits of various crops. Combining this rapidly advancing genome-editing technology and conventional breeding will greatly extend the potential of genome-edited crops and their commercialization.

Metabolite Profiling and Chemometric Study for the Discrimination Analyses of Geographic Origin of Perilla (Perilla frutescens) and Sesame (Sesamum indicum) Seeds.

Perilla and sesame are traditional sources of edible oils in Asian and African countries. In addition, perilla and sesame seeds are rich sources of health-promoting compounds, such as fatty acids, tocopherols, phytosterols and policosanols. Thus, developing a method to determine the geographic origin of these seeds is important for ensuring authenticity, safety and traceability and to prevent cheating. We aimed to develop a discriminatory predictive model for determining the geographic origin of perilla and sesame seeds using comprehensive metabolite profiling coupled with chemometrics. The orthogonal partial least squares-discriminant analysis models were well established with good validation values (Q2 = 0.761 to 0.799). Perilla and sesame seed samples used in this study showed a clear separation between Korea and China as geographic origins in our predictive models. We found that glycolic acid could be a potential biomarker for perilla seeds and proline and glycine for sesame seeds. Our findings provide a comprehensive quality assessment of perilla and sesame seeds. We believe that our models can be used for regional authentication of perilla and sesame seeds cultivated in diverse geographic regions.

A high-throughput platform for interpretation of metabolite profile data from pepper (Capsicum) fruits of 13 phenotypes associated with different fruit maturity states.

Nowadays, novel tools have been developed for efficient analysis and visualization of large-scale metabolite profile data associated with metabolic pathways. A high-throughput platform using PathVisio 3 combined with multivariate analysis is proposed for the first time. Additionally, this is the first analysis of the relationships among terpenoids monoterpene, sesquiterpene, triterpene, and tetraterpene during pepper fruit ripening, and their changes. This platform was successfully applied to interpret large-scale data related to 131 metabolites from mature and immature fruits of 13 pepper phenotypes. The carotenoid-derived volatiles, such as dihydroactinidiolide and ß-ionone were closely correlated with carotenoids, indicating that the synthesis and degradation of carotenoids occurred in pepper fruit mature stage. Using PathVisio 3, the metabolic changes in pathway could be presented quickly, revealing the accumulation of stress-related metabolites, such as proline, capsaicin, and phenylalanine, in the mature stage. This approach could provide useful information about comprehensive biochemical regulation of fruit ripening.

Discrimination of Adzuki Bean (Vigna angularis) Geographical Origin by Targeted and Non-Targeted Metabolite Profiling with Gas Chromatography Time-of-Flight Mass Spectrometry.

As international food trade increases, consumers are becoming increasingly interested in food safety and authenticity, which are linked to geographical origin. Adzuki beans (Vigna angularis) are cultivated worldwide, but there are no tools for accurately discriminating their geographical origin. Thus, our study aims to develop a method for discriminating the geographical origin of adzuki beans through targeted and non-targeted metabolite profiling with gas chromatography time-of-flight mass spectrometry combined with multivariate analysis. Orthogonal partial least squares discriminant analysis showed clear discrimination between adzuki beans cultivated in Korea and China. Non-targeted metabolite profiling showed better separation than targeted profiling. Furthermore, citric acid and malic acid were the most notable metabolites for discriminating adzuki beans cultivated in Korea and China. The geographical discrimination method combining non-targeted metabolite profiling and pareto-scaling showed excellent predictability (Q2 = 0.812). Therefore, it is a suitable prediction tool for the discrimination of geographical origin and is expected to be applicable to the geographical authentication of adzuki beans.

Phytoene synthase 2 can compensate for the absence of PSY1 in the control of color in Capsicum fruit.

Phytoene synthase 1 (PSY1) and capsanthin-capsorubin synthase (CCS) are two major genes responsible for fruit color variation in pepper (Capsicum spp.). However, the role of PSY2 remains unknown. We used a systemic approach to examine the genetic factors responsible for the yellow fruit color of C. annuum 'MicroPep Yellow' (MY) and to determine the role of PSY2 in fruit color. We detected complete deletion of PSY1 and a retrotransposon insertion in CCS. Despite the loss of PSY1 and CCS function, both MY and mutant F2 plants from a cross between MY and the 'MicroPep Red' (MR) accumulated basal levels of carotenoids, indicating that other PSY genes may complement the loss of PSY1. qRT-PCR analysis indicated that PSY2 was constitutively expressed in both MR and MY fruits, and a color complementation assay using Escherichia coli revealed that PSY2 was capable of biosynthesizing a carotenoid. Virus-induced gene silencing of PSY2 in MY resulted in white fruits. These findings indicate that PSY2 can compensate for the absence of PSY1 in pepper fruit, resulting in the yellow color of MY fruits.

Phosphate-Starvation-Inducible S-Like RNase Genes in Rice Are Involved in Phosphate Source Recycling by RNA Decay.

The fine-tuning of inorganic phosphate (Pi) for enhanced use efficiency has long been a challenging subject in agriculture, particularly in regard to rice as a major crop plant. Among ribonucleases (RNases), the RNase T2 family is broadly distributed across kingdoms, but little has been known on its substrate specificity compared to RNase A and RNase T1 families. Class I and class II of the RNase T2 family are defined as the S-like RNase (RNS) family and have showed the connection to Pi recycling in Arabidopsis. In this study, we first carried out a phylogenetic analysis of eight rice and five Arabidopsis RNS genes and identified mono-specific class I and dicot-specific class I RNS genes, suggesting the possibility of functional diversity between class I RNS family members in monocot and dicot species through evolution. We then compared the in silico expression patterns of all RNS genes in rice and Arabidopsis under normal and Pi-deficient conditions and further confirmed the expression patterns of rice RNS genes via qRT-PCR analysis. Subsequently, we found that most of the OsRNS genes were differentially regulated under Pi-deficient treatment. Association of Pi recycling by RNase activity in rice was confirmed by measuring total RNA concentration and ribonuclease activity of shoot and root samples under Pi-sufficient or Pi-deficient treatment during 21 days. The total RNA concentrations were decreased by < 60% in shoots and < 80% in roots under Pi starvation, respectively, while ribonuclease activity increased correspondingly. We further elucidate the signaling pathway of Pi starvation through upregulation of the OsRNS genes. The 2-kb promoter region of all OsRNS genes with inducible expression patterns under Pi deficiency contains a high frequency of P1BS cis-acting regulatory element (CRE) known as the OsPHR2 binding site, suggesting that the OsRNS family is likely to be controlled by OsPHR2. Finally, the dynamic transcriptional regulation of OsRNS genes by overexpression of OsPHR2, ospho2 mutant, and overexpression of OsPT1 lines involved in Pi signaling pathway suggests the molecular basis of OsRNS family in Pi recycling via RNA decay under Pi starvation.

Stepwise pathway engineering to the biosynthesis of zeaxanthin, astaxanthin and capsanthin in rice endosperm.

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 2 A 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 (0.8 µg/g zeaxanthin, deep yellow), stBAK-PAC (1.4 µg/g ketocarotenoids, including astaxanthin, pinkish red) and B-PAC x Ccs (0.4 µg/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.

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 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.

A Rice B-Box Protein, OsBBX14, Finely Regulates Anthocyanin Biosynthesis in Rice.

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.

RNAi-mediated suppression of three carotenoid-cleavage dioxygenase genes, OsCCD1, 4a, and 4b, increases carotenoid content in rice.

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.