Phenylboronic Acid-Functionalized Ratiometric Surface-Enhanced Raman Scattering Nanoprobe for Selective Tracking of Hg2+ and CH3Hg+ in Aqueous Media and Living Cells.

The development of appropriate molecular tools to monitor different mercury speciation, especially CH3Hg+, in living organisms is attractive because its persistent accumulation and toxicity are very harmful to human health. Herein, we develop a novel activity-based ratiometric SERS nanoprobe to selectively monitor Hg2+ and CH3Hg+ in aqueous media and in vivo. In this nanoprobe, a new bifunctional Raman probe bis-s-s'-[(s)-(4-(ethylcarbamoyl)phenyl)boronic acid] (b-(s)-EPBA) was synthesized and immobilized on the surface of gold nanoparticles via a Au-S bond, in which the phenylboronic acid group was employed as the recognition unit for Hg2+ and CH3Hg+ based on the Hg-promoted transmetalation reaction. In the presence of Hg2+ and CH3Hg+, a new surface-enhanced Raman scattering (SERS) peak aroused from of C-Hg appeared at 1080 cm-1, and the SERS intensity at 1002 cm-1 belonged to the B-O symmetric stretching decreased simultaneously. The quantitative tracking of Hg2+ and CH3Hg+ was realized based on the SERS intensity ratio (I1080/I1303) with rapid response (∼4 min) and high sensitivity, with detection limits of 10.05 and 25.13 nM, respectively. Moreover, the SERS sensor was used for the quantitative detection of Hg2+ and CH3Hg+ in four actual water samples with a high accuracy and excellent recovery. More importantly, cell imaging experiments showed that AuNPs@b-(s)-EPBA could quantitatively detect intracellular CH3Hg+ and had a good concentration dependence in ratiometric SERS imaging. Meanwhile, we demonstrated that AuNPs@b-(s)-EPBA could detect and image CH3Hg+ in zebrafish. We anticipate that AuNPs@b-(s)-EPBA could potentially be used to study the physiological functions related to CH3Hg+ in the future.

Promoting sustainable development: Revisiting digital economy agglomeration and inclusive green growth through two-tier stochastic frontier model.

In the global wave of digitization, digital economic agglomeration, as an emerging model, profoundly impacts the economy, environment, and society. Countries worldwide are formulating strategies and policies to promote the development of digital economic agglomeration, yet they also face challenges of widening digital divide and environmental sustainability. Existing research primarily focuses on the positive effects of the digital economy, with limited assessment of the dual effects of digital economic agglomeration on sustainable development. This study utilizes panel data from 282 Chinese cities between 2011 and 2021, employing a two-tier stochastic frontier model. It reexamines the dual impacts and intrinsic mechanisms of digital economic agglomeration, attempting to capture regional and temporal variations in the dual effects to address this research gap. The study shows that: (1) The positive effect of digital economy agglomeration is much more than the negative effect, resulting in a positive net effect that shows an overall increasing trend with significant regional disparities. (2) Digital economic agglomeration has a significant negative spatial spillover effect, promoting local inclusive green growth while inhibiting inclusive green growth in neighboring cities. (3) Regarding the mediating mechanisms, industrial structure, technological innovation and resource allocation efficiency have positive indirect effects on inclusive green growth, while environmental regulation intensity has a negative indirect effect, and it has a nonlinear effect under the threshold constraint of the mediating mechanisms. This study provides policy insights for promoting inclusive green growth, emphasizing the need to consider regional differences in resource distribution, ecological environment, and social demands. It advocates for the organic integration of the digital economy across different regions, reducing polarization effects, and enhancing diffusion effects.

Analysis and experimental research of transient temperature rise characteristics of high-speed cylindrical roller bearing.

In this paper, on one hand, the time-varying characteristics of the heat source and thermal boundary conditions of the high-speed spindle system were analyzed considering the thermal-structural coupling effect. And a transient bearing temperature field prediction method combining the thermal network method and finite element method was proposed. Furthermore, the relationships between time step, calculation efficiency and calculation results were analyzed. On the other hand, a online real-time monitoring system of the transient temperature of the cylindrical roller bearing inner ring for maximum speed of 13,000 r/min was designed and implemented using fibre optic sensing technology. Comparing with the conventional static thermal analysis results, it is verified that the simulation method proposed in this paper has higher accuracy. This paper provides a new approach for analysing and testing the thermal characteristics of high-speed spindle system.

OsGSTU34, a Bz2-like anthocyanin-related glutathione transferase transporter, is essential for rice (Oryza sativa L.) organs coloration.

Anthocyanins are a flavonoid compound known as one of the most important chromogenic substances. They play several functions, including health promotion and sustaining plants during adverse conditions. They are synthesized at the endoplasmic reticulum and sequestered in the vacuole. In this work, we generated knock-out lines of OsGSTU34, a glutathione transporter's tau gene family, with no transgene line and off-target through CRISPR/Cas9 mutagenesis and highlighted the loss of pigmentation in rice flowers, leaves, stems, shoots, and caryopsis. The anthocyanin quantification in the wild-type BLWT and mutant line BLG34-8 caryopsis showed that cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside (P3G) were almost undetectable in the mutant line. A tandem mass tag (TMT) labeling proteomic analysis was conducted to elucidate the proteomic changes in the BLWT and BLG34-8. The result revealed that 1175 proteins were altered, including 408 that were down-regulated and 767 that were upregulated. The accumulation of the OsGSTU34-related protein (Q8L576), along with several anthocyanin-related proteins, was down-regulated. The enrichment analysis showed that the down-regulated proteins were enriched in different pathways, among which the phenylpropanoid biosynthesis pathway, flavonoid biosynthesis metabolites, and anthocyanin biosynthesis pathway. Protein interaction network prediction revealed that glutathione-S-transferase (Q8L576) was connected to the proteins involved in the flavonoid and anthocyanin biosynthesis pathways, such as flavanone 3-dioxygenase 1 (Q7XM21), leucoanthocyanidin dioxygenase 1 (Q93VC3), 4-coumarate-CoA ligase 2 (Q42982), phenylalanine ammonia-lyase (P14717), chalcone synthase 1 (Q2R3A1), and 4-coumarate-CoA ligase 5 (Q6ZAC1). However, the expression of the most important anthocyanin biosynthesis gene was not altered, suggesting that only the transport mechanism was affected. Our findings highlight new insight into the anthocyanin pigmentation in black rice and provide new perspectives for future research.

Zn-coordination polymers for fluorescence sensing various contaminants in water.

Luminescent coordination polymers (LCPs) have garnered significant attention from researchers as promising materials for detecting contaminants. In this paper, three new LCPs ([Zn(tib)(opda)]n⋅H2O (1), [Zn3(tib)2(mpda)3]n⋅5H2O (2), [Zn (tib)(ppda)]n⋅H2O (3)) with different structures (LCP 1-3: 1D, 2D, 1D) using phenylenediacetic acid isomers and 1,3,5-tris (1-imidazolyl) benzene (tib) are synthesized. The specific surface areas (BET) of LCP 1-3 are 4 m2/g, 19 m2/g, and 13 m2/g respectively. LCP 1-3 exhibit excellent fluorescence properties and can serve as fluorescent probe for the detection of inorganic contaminants and organic contaminants. Due to the large BET of LCP 2, the detection limits for trace analytes surpass those of LCP 1 and 3. The detection limits of LCP 2 for Fe3+, nitrobenzene (NB), chloramphenicol (CAP), and pyrimethanil (PTH) are 8.3 nM, 0.016 µM, 0.19 µM, and 0.032 µM, respectively, and the fluorescence quenching rates are 98.6 %, 98.8 %, 92.3 %, and 98.8 %, respectively. These values outperform most reported in the literature. The quantum yields of LCP 1-3 are 11.84 %, 25.22 %, 22.00 % respectively. Real sample testing of LCP 1-3 reveals favorable performance, where spiked recoveries of LCP 2 for the detection of pyrimethanil in grape skins ranged from 99.62 % to 119.3 % with a relative standard deviation (RSD) of 0.627 % to 4.56 % (n = 3). The fluorescence quenching mechanism was attributed to a combination of photoelectron transfer (PET), resonance energy transfer (RET), and competitive absorption (CA). This study advances the application of LCPs in luminescence sensing and contributes to the expansion of novel materials for detecting environmental pollutants.

A Subtle Change in the Flexible Achiral Spacer Does Matter in Supramolecular Chirality: Two-Fold Odd-Even Effect in Polymer Assemblies.

Precise control over the chirality and morphologies of polymer assemblies, a remaining challenge for both chemists and materials scientists, is receiving ever-increasing attention in the recent years. Herein, we report the subtle manipulation of the achiral spacers from the chiral stereocenter to the azobenzene (Azo) unit, of which the chiroptical consistency or chiroptical inversion of self-assemblies could be successfully controlled and present "two-fold" odd-even effect. Furthermore, morphological transitions from 0D spherical micelles, 1D worms, and nanowires to 3D vesicles, spindle- and dumbbell-shaped vesicles were also unexpectedly found to exhibit odd-even correlations. These observations were collectively elucidated by mesomorphic properties, stacking modes, chiroptical dynamics, and stimuli-responsive behaviors. Negligible modifications to the spacer structures can enable remarkable modulation of supramolecular chirality and anisotropic topologies in polymer assemblies, which is of great significance for the design of complex chiral functional polymers.

Integrative HPLC profiling and transcriptome analysis revealed insights into anthocyanin accumulation and key genes at three developmental stages of black rice (Oryza sativa. L) caryopsis.

Introduction: Anthocyanins are plants' secondary metabolites belonging to the flavonoid class with potential health-promoting properties. They are greatly employed in the food industry as natural alternative food colorants for dairy and ready-to-eat desserts and pH indicators. These tremendous advantages make them economically important with increasing market trends. Black rice is a rich source of anthocyanin that can be used to ensure food and nutritional security around the world. However, research on anthocyanin accumulation and gene expression during rice caryopsis development is lacking. Methods: In this study, we combined high-performance liquid chromatography (HPLC) and transcriptome analysis to profile the changes in anthocyanin content and gene expression dynamics at three developmental stages (milky, doughy, and mature). Results: Our results showed that anthocyanin accumulation started to be visible seven days after flowering (DAF), increased rapidly from milky (11 DAF) to dough stage, then started decreasing after the peak was attained at 18 DAF. RNA-seq showed that 519 out of 14889, 477 out of 17914, and 1614 out of 18810 genes were uniquely expressed in the milky, doughy, and mature stages, respectively. We performed three pairwise comparisons: milky vs. dough, milky vs. mature, and dough vs. mature, and identified 6753, 9540, and 2531 DEGs, respectively. The DEGs' abundance was higher in milky vs. mature, with 5527 up-regulated genes and 4013 down-regulated genes, while it was smaller in dough vs. mature, with 1419 up-regulated genes and 1112 down-regulated DEGs. This result was consistent with the changes in anthocyanin profiling, and the expression of structural, regulatory, and transporter genes involved in anthocyanin biosynthesis showed their highest expression at the dough stage. Through the gene expression profile and protein interaction network, we deciphered six main contributors of the anthocyanin peak observed at dough stage, including OsANS, OsDFR, OsGSTU34, OsMYB3, OsbHLH015, and OsWD40-50. Discussion: This study is the first to report the investigation of anthocyanin and gene expression at three developmental stages of black rice caryopsis. The findings of this study could aid in predicting the best harvesting time to achieve maximum anthocyanin content and the best time to collect samples for various gene expression analysis, laying the groundwork for future research into the molecular mechanisms underlying rice caryopsis coloration.

Development and application of CRISPR/Cas9 to improve anthocyanin pigmentation in plants: Opportunities and perspectives.

Since its discovery in 2012, the novel technology of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has greatly contributed to revolutionizing molecular biology. It has been demonstrated to be an effective approach for identifying gene function and improving some important traits. Anthocyanins are secondary metabolites responsible for a wide spectrum of aesthetic coloration in various plant organs and are beneficial for health. As such, increasing anthocyanin content in plants, especially the edible tissue and organs, is always a main goal for plant breeding. Recently, CRISPR/Cas9 technology has been highly desired to enhance the amount of anthocyanin in vegetables, fruits, cereals, and other attractive plants with more precision. Here we reviewed the recent knowledge concerning CRISPR/Cas9-mediated anthocyanin enhancement in plants. In addition, we addressed the future avenues of promising potential target genes that could be helpful for achieving the same goal using CRISPR/Cas9 in several plants. Thus, molecular biologists, genetic engineers, agricultural scientists, plant geneticists, and physiologists may benefit from CRISPR technology to boost the biosynthesis and accumulation of anthocyanins in fresh fruits, vegetables, grains, roots, and ornamental plants.

On-line pre-column FRAP-based antioxidant reaction coupled with HPLC-DAD-TOF/MS for rapid screening of natural antioxidants from different parts of Polygonum viviparum.

Polygonum viviparum L. (PV) is a widely used resource plant with high medicinal, feeding and ecological values. Our studies show that PV has strong antioxidant activity. However, up to date, the antioxidant activity and components in other parts were not fully elucidated. In the present study, a new online pre-column ferric ion reducing antioxidant power (FRAP)-based antioxidant reaction coupled with high performance liquid chromatography-diode array detector-quadrupole-time-of-flight mass spectrometry (HPLC-DAD-TOF/MS) was developed for rapid and high-throughput screening of natural antioxidants from three different parts of PV including stems and leaves, fruits and rhizomes. In this procedure, it was assumed that the peak areas of compounds with potential antioxidant activity in HPLC chromatograms would be greatly diminished or vanish after incubating with the FRAP. The online incubation conditions including mixed ratios of sample and FRAP solution and reaction times were firstly optimized with six standards. Then, the repeatability of the screening system was evaluated by analysis of the samples of stems and leaves of PV. As a result, a total of 21 compounds mainly including flavonoids and phenolic acids were screened from the three parts of PV. In conclusion, the present study provided a simple and effective strategy to rapidly screen antioxidants in natural products.

Unexpected chirality transition and inversion mediated by dissolution-aggregation and the odd-even effect.

The evolution of hierarchical chirality at macromolecular and supramolecular levels in biological systems is ubiquitous; however, achieving precise control over transitions between them in polymer systems is still challenging. Here, we reported multiple chiroptical transitions and inversion phenomena in side-chain azobenzene (Azo) polymers, PAzo-l/d-m (m = 3, 6, 7, 8, 9, and 10, where m is the total number of atoms from the chiral stereocenter to the Azo unit), with different distances from the chiral stereocenter to the Azo unit. In the case of m = 3, an unexpected macromolecular-to-supramolecular chirality transition and inversion occurred in situ when the Azo-polymer underwent from a macromolecular-dissolved state to a supramolecular-aggregated state. To our surprise, an exciton-coupling induced multiple chiroptical inversion was observed upon the heating-assisted reassembly treatment, which was demonstrated to be driven by H- to J-aggregation transition. Furthermore, the odd-even effect was first established to regulate the supramolecular helical orientations (left- or right-handedness) in side-chain Azo-polymer assemblies.

Morphology engineering and photothermal effect derived from perylene diimide based derivative for boosting photocatalytic hydrogen evolution of ZnIn2S4.

The construction of excellent photocatalysts for splitting water into hydrogen is highly desirable to realize carbon neutralization. In this work, an innovative and well-designed S-scheme photocatalyst composed of ultrathin ZnIn2S4 (ZIS) nanosheets uniformly anchored on the surface of organic semiconductor PDIIM is successfully fabricated. Within the heterojunction, perylene diimide with an imidazole group (PDIIM) is strategically applied as a structure template, which plays a crucial role in optimizing the morphology, increasing the active sites of sulfur vacancies, providing the additional photothermal effect, and promoting photogenerated charge separation of the catalyst. The photocatalytic H2 generation rate of the ZIS/PDIIM heterojunction with an optimized mass ratio reaches up to 13.04 mmol/g/h, which is 2.64 times and 14.02 times higher than that of pristine ZIS and PDIIM, respectively. The outstanding photocatalytic activity is attributed to the synergistic effect of the above advantages. Importantly, the photothermal effect induced by PDIIM belonging to the perylene diimide-based derivative was discovered to accelerate photocatalytic H2 generation for the first time. This work provides valuable insight into the utilization of perylene diimide-based derivatives in the construction of multi-effect enhancement photocatalysts and their application in photothermal-assisted photocatalytic hydrogen evolution.

Combined Analysis of BSA-Seq Based Mapping, RNA-Seq, and Metabolomic Unraveled Candidate Genes Associated with Panicle Grain Number in Rice (Oryza sativa L.).

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.

Increased precipitation and nitrogen addition accelerate the temporal increase in soil respiration during 8-year old-field grassland succession.

Ecological succession after disturbance plays a vital role in influencing ecosystem structure and functioning. However, how global change factors regulate ecosystem carbon (C) cycling in successional plant communities remains largely elusive. As part of an 8-year (2012-2019) manipulative experiment, this study was designed to examine the responses of soil respiration and its heterotrophic component to simulated increases in precipitation and atmospheric nitrogen (N) deposition in an old-field grassland undergoing secondary succession. Over the 8-year experimental period, increased precipitation stimulated soil respiration by 11.6%, but did not affect soil heterotrophic respiration. Nitrogen addition increased both soil respiration (5.1%) and heterotrophic respiration (6.2%). Soil respiration and heterotrophic respiration linearly increased with time in the control plots, resulting from changes in soil moisture and shifts of plant community composition from grass-forb codominance to grass dominance in this old-field grassland. Compared to the control, increased precipitation significantly strengthened the temporal increase in soil respiration through stimulating belowground net primary productivity. By contrast, N addition accelerated temporal increases in both soil respiration and its heterotrophic component by driving plant community shifts and thus stimulating soil organic C. Our findings indicate that increases in water and N availabilities may accelerate soil C release during old-field grassland succession and reduce their potential positive impacts on soil C accumulation under future climate change scenarios.

Chiral Expression and Morphology Control in Polymer Dispersion Systems.

The chiral self-assembly of polymers in dispersions plays an important role in chiral chemistry and self-assembly. In general, both polymerization-induced self-assembly (PISA) and post-polymerization self-assembly can be used to prepare polymer nanoassemblies. In chirality induction or transfer processes, the self-assembly manner of the polymers greatly affects the chiral expression of the nanostructure and cannot be ignored in the chiral fields. Moreover, unique chiral expression and morphological transition of polymer assemblies in dispersions enable the preparation of advanced functional chiroptical materials. Herein, this Review discusses recent advances in chiral expression and morphology control in polymer dispersion systems, particularly the comparison between traditional post-polymerization self-assembly and in situ PISA strategies, to predict and advance chirality control in polymers.

The Acetylation Modification of SP1 Regulates the Protein Stability in Silkworm.

Acetylation is a highly conservative and reversible post-translational modification. Acetylation modification can regulate gene expression by altering protein function and is widely identified in an increasing number of species. Previously, the acetylated proteome of silkworm was identified by combining acetylated polypeptide enrichment with nano-HPLC/MS/MS; the identification revealed that the SP proteins (SPs) were high acetylated. In this study, the acetylation of SP1, one of the SPs, was further confirmed using immunoprecipitation (IP) and Western blotting. Then, we found the acetylation could upregulate SP1 protein expression by enhancing the protein stability. Further research found that the acetylation of SP1 protein can competitively inhibit its ubiquitination and thus improve the stability and cell accumulation of SP1 protein by inhibiting the ubiquitin-mediated proteasome degradation pathway. This result provides a basis for acetylation to regulate the nutrient storage and utilization of silkworm.

Accumulation of 22 kDa α-zein-mediated nonzein protein in protein body of maize endosperm.

Protein bodies (PBs), the major protein storage organelle in maize (Zea mays) endosperm, comprise zeins and numerous nonzein proteins (NZPs). Unlike zeins, how NZPs accumulate in PBs remains unclear. We characterized a maize miniature kernel mutant, mn*, that produces small kernels and is embryo-lethal. After cloning the Mn* locus, we determined that it encodes the mitochondrial 50S ribosomal protein L10 (mRPL10). MN* localized to mitochondria and PBs as an NZP; therefore, we renamed MN* Non-zein Protein 1 (NZP1). Like other mutations affecting mitochondrial proteins, mn* impaired mitochondrial function and morphology. To investigate its accumulation mechanism to PBs, we performed protein interaction assays between major zein proteins and NZP1, and found that NZP1 interacts with 22 kDa α-zein. Levels of NZP1 and 22 kDa α-zein in various opaque mutants were correlated. Furthermore, NZP1 accumulation in induced PBs depended on its interaction with 22 kDa α-zein. Comparative proteomic analysis of PBs between wild-type and opaque2 revealed additional NZPs. A new NZP with plastidial localization was also found to accumulate in induced PBs via interaction with 22 kDa α-zein. This study thus reveals a mechanism for accumulation of NZPs in PBs and suggests a potential application for the accumulation of foreign proteins in maize PBs.

Controlling the Multiple Chiroptical Inversion in Biphasic Liquid-Crystalline Polymers.

While controlling the chirality and modulating the helicity is a challenging task, it attracts great research interest for gaining a better understanding of the origin of chirality in nature. Herein, structurally similar azobenzene (Azo) vinyl monomers were designed in which the alkyl chains comprised the chiral stereocenter with different achiral tail lengths. Combining the synchronous polymerization, supramolecular stacking and self-assembly, the multiple chiroptical inversion of the Azo-polymer supramolecular assemblies can be modulated by the tail length and DP of Azo blocks during in situ polymerization. The DP-, UV light-, temperature-, aging time-dependent chiroptical properties and liquid-crystalline (LC) characterization indicated that the amorphous-to-LC phase transition and biphasic LC interconversion allow the transcription of intra-chain π-π stacking, inter-chain H- and J-aggregation, thereby controlling the dynamic multiple reversal of supramolecular chirality.

Computational and Transcriptomic Analysis Unraveled OsMATE34 as a Putative Anthocyanin Transporter in Black Rice (Oryza sativa L.) Caryopsis.

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.

Subcellular Localization and Vesicular Structures of Anthocyanin Pigmentation by Fluorescence Imaging of Black Rice (Oryza sativa L.) Stigma Protoplast.

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.

Recent Insights into Anthocyanin Pigmentation, Synthesis, Trafficking, and Regulatory Mechanisms in Rice (Oryza sativa L.) Caryopsis.

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.