Anthocyanins are antioxidants used as natural colorants and are beneficial to human health. Anthocyanins contribute to reactive oxygen species detoxification and sustain plant growth and development under different environmental stresses. They are phenolic compounds that are broadly distributed in nature and are responsible for a wide range of attractive coloration in many plant organs. Anthocyanins are found in various parts of plants such as flowers, leaves, stems, shoots, and grains. Considering their nutritional and health attributes, anthocyanin-enriched rice or pigmented rice cultivars are a possible alternative to reduce malnutrition around the globe. Anthocyanin biosynthesis and storage in rice are complex processes in which several structural and regulatory genes are involved. In recent years, significant progress has been achieved in the molecular and genetic mechanism of anthocyanins, and their synthesis is of great interest to researchers and the scientific community. However, limited studies have reported anthocyanin synthesis, transportation, and environmental conditions that can hinder anthocyanin production in rice. Rice is a staple food around the globe, and further research on anthocyanin in rice warrants more attention. In this review, metabolic and pre-biotic activities, the underlying transportation, and storage mechanisms of anthocyanins in rice are discussed in detail. This review provides potential information for the food industry and clues for rice breeding and genetic engineering of rice.

Anthocyanins belong to the group of flavonoid compounds broadly distributed in plant species responsible for attractive colors. In black rice (Oryza sativa L.), they are present in the stems, leaves, stigmas, and caryopsis. However, there is still no scientific evidence supporting the existence of compartmentalization and trafficking of anthocyanin inside the cells. In the current study, we took advantage of autofluorescence with anthocyanin’s unique excitation/emission properties to elucidate the subcellular localization of anthocyanin and report on the in planta characterization of anthocyanin prevacuolar vesicles (APV) and anthocyanic vacuolar inclusion (AVI) structure. Protoplasts were isolated from the stigma of black and brown rice and imaging using a confocal microscope. Our result showed the fluorescence displaying magenta color in purple stigma and no fluorescence in white stigma when excitation was provided by a helium–neon 552 nm and emission long pass 610–670 nm laser. The fluorescence was distributed throughout the cell, mainly in the central vacuole. Fluorescent images revealed two pools of anthocyanin inside the cells. The diffuse pools were largely found inside the vacuole lumen, while the body structures could be observed mostly inside the cytoplasm (APV) and slightly inside the vacuole (AVI) with different shapes, sizes, and color intensity. Based on their sizes, AVI could be grouped into small (Ф < 0.5 um), middle (Ф between 0.5 and 1 um), and large size (Ф > 1 um). Together, these results provided evidence about the sequestration and trafficking of anthocyanin from the cytoplasm to the central vacuole and the existence of different transport mechanisms of anthocyanin. Our results suggest that stigma cells are an excellent system for in vivo studying of anthocyanin in rice and provide a good foundation for understanding anthocyanin metabolism in plants, sequestration, and trafficking in black rice.

Anthocyanin is a flavonoid compound with potential antioxidant properties beneficial to human health and sustains plant growth and development under different environmental stresses. In black rice, anthocyanin can be found in the stems, leaves, stigmas, and caryopsis. Although the anthocyanin biosynthesis in rice has been extensively studied, limited knowledge underlying the storage mechanism and transporters is available. This study undertook the complementation of computational and transcriptome analysis to decipher a potential multidrug and toxic compound extrusion (MATE) gene candidate for anthocyanin transportation in black rice caryopsis. The phylogenetic analysis showed that OsMATE34 has the same evolutionary history and high similarities with VvAM1, VvAM3, MtMATE2, SlMATE/MTP77, RsMATE8, AtFFT, and AtTT12 involved in anthocyanin transportation. RNA sequencing analysis in black caryopsis (Bc; Bc11, Bc18, Bc25) and white caryopsis (Wc; Wc11, Wc18, Wc25), respectively, at 11 days after flowering (DAF), 18 DAF, and 25 DAF revealed a total of 36,079 expressed genes, including 33,157 known genes and 2922 new genes. The differentially expressed genes (DEGs) showed 15,573 genes commonly expressed, with 1804 and 1412 genes uniquely expressed in Bc and Wc, respectively. Pairwise comparisons showed 821 uniquely expressed genes out of 15,272 DEGs for Wc11 vs. Bc11, 201 uniquely expressed genes out of 16,240 DEGs for Wc18 vs. Bc18, and 2263 uniquely expressed genes out of 16,240 DEGs for Wc25 vs. Bc25. Along with anthocyanin biosynthesis genes (OsPAL, OsCHS, OsCHI, OsF3H, OsDFR, OsANS, and OsUFGT/Os3GT), OsMATE34 expression was significantly upregulated in all Bc but not in Wc. OsMATE34 expression was similar to OsGSTU34, a transporter of anthocyanin in rice leaves. Taken together, our results highlighted OsMATE34 (Os08g0562800) as a candidate anthocyanin transporter in rice caryopsis. This study provides a new finding and a clue to enhance the accumulation of anthocyanin in rice caryopsis.

Rice grain yield is a complex and highly variable quantitative trait consisting of several key components, including the grain weight, the effective panicles per unit area, and the grain number per panicle (GNPP). The GNPP is a significant contributor to grain yield controlled by multiple genes (QTL) and is crucial for improvement. Attempts have been made to find genes for this trait, which has always been a challenging and arduous task through conventional methods. We combined a BSA analysis, RNA profiling, and a metabolome analysis in the present study to identify new candidate genes involved in the GNPP. The F2 population from crossing R4233 (high GNPP) and Ce679 (low GNPP) revealed a frequency distribution fitting two segregated genes. Three pools, including low, middle, and high GNPP, were constructed and a BSA analysis revealed six candidate regions spanning 5.38 Mb, containing 739 annotated genes. Further, a conjunctive analysis of BSA-Seq and RNA-Seq showed 31 differentially expressed genes (DEGs) in the candidate intervals. Subsequently, a metabolome analysis showed 1024 metabolites, with 71 significantly enriched, including 44 up and 27 downregulated in Ce679 vs. R4233. A KEGG enrichment analysis of these 31 DEGs and 71 differentially enriched metabolites (DEMs) showed two genes, Os12g0102100 and Os01g0580500, significantly enriched in the metabolic pathways’ biosynthesis of secondary metabolites, cysteine and methionine metabolism, and fatty acid biosynthesis. Os12g0102100, which encodes for the alcohol dehydrogenase superfamily and a zinc-containing protein, is a novel gene whose contribution to the GNPP is not yet elucidated. This gene coding for mitochondrial trans-2-enoyl-CoA reductase is involved in the biosynthesis of myristic acid, also known as tetradecanoic acid. The Os01g0580500 coding for the enzyme 1-aminoclopropane-1-carboxylate oxidase (OsACO7) is responsible for the final step of the ethylene biosynthesis pathway through the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene. Unlike Os12g0102100, this gene was significantly upregulated in R4233, downregulated in Ce679, and significantly enriched in two of the three metabolite pathways. This result pointed out that these two genes are responsible for the difference in the GNPP in the two cultivars, which has never been identified. Further validation studies may disclose the physiological mechanisms through which they regulate the GNPP in rice.

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