Chemical composition analysis and transcriptomics reveal the R2R3-MYB genes and phenol oxidases regulating the melanin formation in black radish.

Melanins are dark-brown to black-colored biomacromolecules which have been thoroughly studied in animals and microorganisms. However, the biochemical and molecular basis of plant melanins are poorly understood. We first characterized melanin from the black radish (Raphanus sativus var. niger) 'HLB' through spectroscopic techniques. p-Coumaric acid was identified as the main precursor of radish melanin. Moreover, a joint analysis of transcriptome and coexpression network was performed for the two radish accessions with black and white cortexes, 'HLB' and '55'. A set of R2R3-type RsMYBs and enzyme-coding genes exhibited a coexpression pattern, and were strongly correlated with melanin formation in radish. Transient overexpression of two phenol oxidases RsLAC7 (laccase 7) or RsPOD22-1 (peroxidase 22-1) resulted in a deeper brown color around the infiltration sites and a significant increase in the total phenol content. Furthermore, co-injection of the transcriptional activator RsMYB48/RsMYB97 with RsLAC7 and/or RsPOD22-1, markedly increased the yield of black extracts. Spectroscopic analyses revealed that these extracts are similar to the melanin found in 'HLB'. Our findings advance the understanding of structural information and the transcriptional regulatory mechanism underlying melanin formation in radish.

Neofunctionalization of an OMT cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus.

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and O-methyltransferases (OMTs). However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.

The role of the transcriptional repressor CssR in Corynebacterium glutamicum in response to phenolic compounds.

The cssR gene (ncgl1578) of Corynebacterium glutamicum encodes a repressor of the TetR (tetracycline regulator) family. Its role in the stress response to antibiotics/heavy metals has been investigated, but how CssR functions in response to phenolic compounds in C. glutamicum has been rarely studied. In this study, we applied transcriptomic analysis, ß-galactosidase analysis, qRT-PCR, and EMSAs to analyze the target genes and functions of CssR in response to phenolic compounds. Consistent with the upregulation of genes involved in the degradation of phenolic compounds, the ΔcssR mutant was more resistant to various phenolic compounds than was the wild-type strain. Furthermore, the addition of phenolic compounds induced the expression of corresponding genes (ncgl0283, ncgl1032, ncgl1111, ncgl2920, ncgl2923, and ncgl2952) in vivo. However, the DNA binding activity of CssR to the promoter of phenolic compound-degrading genes was undetected in vitro. Additionally, we also found that CssR indirectly negatively regulates the expression of cell wall/membrane/envelope biogenesis-related genes, which may enhance resistance to stress caused by phenolic compounds. Together, our findings demonstrate that CssR is a key regulator that copes with stress conditions induced by phenolic compounds, thus greatly expanding our understanding of the functions of TetR family transcription factors.

Characterization of oxidative stress-induced cgahp, a gene coding for alkyl hydroperoxide reductase, from industrial importance Corynebacterium glutamicum.

Alkyl hydroperoxide reductase (Ahp), comprised of four different subunits AhpC, AhpD, AhpE, and AhpF, is a thiol-based antioxidative enzyme with the ability to protect bacteria against oxidative stress. Functionally, AhpC and AhpE considered as peroxidases directly detoxify peroxides, while AhpD and AhpF as oxidoreductases restore oxidized peroxidases to their reduced form. Corynebacterium glutamicum ncgl0877 encodes a putative Ahp with a unique Cys-Pro-Phe-Cys (C-P-G-C) active-site motif, similar with those of the thiol-disulfide oxidoreductases such as thioredoxin (Trx), mycoredoxin-1 (Mrx1) and AhpD. However, its physiological and biochemical functions remain unknown in C. glutamicum. Here, we report that NCgl0877, designated CgAhp, is involved in the protection against organic peroxide (OP) stress. The cgahp-deleted strain is notably more sensitive to OP stress. The cgahp expression is controlled by a MarR-type transcriptional repressor OasR (organic peroxide- and antibiotic-sensing regulator). The physiological role of CgAhp in resistance to OP stresses is corroborated by its induced expression under stresses. Although CgAhp has a weak peroxidase activity toward OP, it mainly supports the OP-scavenging activity of the thiol-dependent peroxidase preferentially linked to the dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide succinyltransferase (SucB)/NADH system. The C-P-G-C motif of CgAhp is essential to maintain the reductase activity. In conclusion, our study identifies CgAhp, behaving like AhpD, as a key disulfide oxidoreductase involved in the oxidative stress tolerance and the functional electron donor for peroxidase.

Dynamic profiling of intact glucosinolates in radish by combining UHPLC-HRMS/MS and UHPLC-QqQ-MS/MS.

Glucosinolates (GSLs) and their degradation products in radish confer plant defense, promote human health, and generate pungent flavor. However, the intact GSLs in radish have not been investigated comprehensively yet. Here, an accurate qualitative and quantitative analyses of 15 intact GSLs from radish, including four major GSLs of glucoraphasatin (GRH), glucoerucin (GER), glucoraphenin (GRE), and 4-methoxyglucobrassicin (4MGBS), were conducted using UHPLC-HRMS/MS in combination with UHPLC-QqQ-MS/MS. Simultaneously, three isomers of hexyl GSL, 3-methylpentyl GSL, and 4-methylpentyl GSL were identified in radish. The highest content of GSLs was up to 232.46 µmol/g DW at the 42 DAG stage in the 'SQY' taproot, with an approximately 184.49-fold increase compared to the lowest content in another sample. That the GSLs content in the taproots of two radishes fluctuated in a similar pattern throughout the five vegetative growth stages according to the metabolic profiling, whereas the GSLs content in the '55' leaf steadily decreased over the same period. Additionally, the proposed biosynthetic pathways of radish-specific GSLs were elucidated in this study. Our findings will provide an abundance of qualitative and quantitative data on intact GSLs, as well as a method for detecting GSLs, thus providing direction for the scientific progress and practical utilization of GSLs in radish.

A Siamese tracker with "dynamic-static" dual-template fusion and dynamic template adaptive update.

In recent years, visual tracking algorithms based on Siamese networks have attracted attention for their desirable balance between speed and accuracy. The performance of such tracking methods relies heavily on target templates. Static templates cannot cope with the adverse effects of target appearance change. The dynamic template method, with a template update mechanism, can adapt to the change in target appearance well, but it also causes new problems, which may lead the template to be polluted by noise. Based on the DaSiamRPN and UpdateNet template update networks, a Siamese tracker with "dynamic-static" dual-template fusion and dynamic template adaptive update is proposed in this paper. The new method combines a static template and a dynamic template that is updated in real time for object tracking. An adaptive update strategy was adopted when updating the dynamic template, which can not only help adjust to the changes in the object appearance, but also suppress the adverse effects of noise interference and contamination of the template. The experimental results showed that the robustness and EAO of the proposed method were 23% and 9.0% higher than those of the basic algorithm on the VOT2016 dataset, respectively, and that the precision and success were increased by 0.8 and 0.4% on the OTB100 dataset, respectively. The most comprehensive real-time tracking performance was obtained for the above two large public datasets.

Primary Bitter Taste of Citrus is Linked to a Functional Allele of the 1,2-Rhamnosyltransferase Gene Originating from Citrus grandis.

1,2-Rhamnosyltransferase (1,2RhaT) catalyzes the final step of production of flavanone neohesperidoside (FNH) that is responsible for the primary bitter taste of citrus fruits. In this study, species-specific flavonoid profiles were determined in 87 Citrus accessions by identifying eight main flavanone glycosides (FGs). Accumulation of FNHs was completely correlated to the presence of the 1,2RhaT gene in 87 citrus accessions analyzed using a novel 1,2RhaT-specific DNA marker. Pummelo (Citrus grandis) was identified as the genetic origin for a function allele of 1,2RhaT that underpinned FNH-bitterness in modern citrus cultivars. In addition, genes encoding six MYB and five bHLH transcription factors were shown to coexpress with 1,2RhaT and other flavonoid pathway genes related to FNH accumulation, indicating that these transcription factors may affect the fruit taste of citrus. This study provides a better understanding of bitterness formation in Citrus varieties and a genetic marker for the early selection of nonbitterness lines in citrus breeding programs.

Comparative profiling and natural variation of polymethoxylated flavones in various citrus germplasms.

Citrus fruits are the main dietary source of polymethoxylated flavones (PMFs) with significant effects on consumer health. In this study, eleven main PMFs were evaluated in the fruit flavedo or leaves of 116 citrus accessions via UPLC-DAD-ESI-QTOF-MS/MS combined with HPLC-DAD analysis, which revealed significant species-specific and spatiotemporal characteristics. All Citrus reticulata and their natural or artificial hybrids were found to have detectable PMFs, especially in the fruit flavedo of the wild or early-cultivated mandarins at early fruit development stages. However, PMFs were not detected in citrons, pummelos, kumquats, trifoliata oranges, papedas, Chinese box oranges and 'Mangshanyegan'. The results enlightened that PMF accumulation only in mandarins and mandarin hybrids is a phenotype inherited from mandarin ancestors. This study provides a comprehensive PMF profile in various citrus germplasms and will benefit future functional citrus breeding practices aimed at designing plants rich in total or specific PMFs for health benefits.

Integrated Transcriptomic and Metabolomic analysis reveals a transcriptional regulation network for the biosynthesis of carotenoids and flavonoids in 'Cara cara' navel Orange.

BACKGROUND: Carotenoids and flavonoids are important secondary metabolites in plants, which exert multiple bioactivities and benefits to human health. Although the genes that encode carotenogenesis and flavonoid biosynthetic enzymes are well characterized, the transcriptional regulatory mechanisms that are related to the pathway genes remain to be investigated. In this study, 'Cara cara' navel orange (CNO) fruit at four development stages were used to identify the key genes and TFs for carotenoids and flavonoids accumulation. RESULTS: In this study, CNO was used to investigate the profiles of carotenoids and flavonoids by a combination of metabolomic and transcriptomic analyses. The important stage for the accumulation of the major carotenoid, lycopene was found to be at 120 days after florescence (DAF). The transcripts of five carotenogenesis genes were highly correlated with lycopene contents, and 16, 40, 48, 24 and 18 transcription factors (TFs) were predicted to potentially bind 1-deoxy-D-xylulose-5-phosphate synthase (DXS1), deoxyxylulose 5-phosphate reductoisomerase (DXR), geranylgeranyl diphosphate synthase (GGPPS2), phytoene synthase (PSY1) and lycopene ß-cyclase (LCYB) promoters, respectively. Narirutin was the most abundant flavonoid in the flesh at the early stages, 60 DAF was the most important stage for the accumulation of flavonoids, and 17, 22, 14, 25, 24 and 16 TFs could potentially bind phenylalanine ammonia-lyase (PAL-1 and PAL-4), 4-Coumarate-CoA ligase (4CL-2 and 4CL-5), chalcone synthase (CHS-1) and chalcone isomerase (CHI) promoters, respectively. Furthermore, both sets of 15 candidate TFs might regulate at least three key genes and contribute to carotenoids/flavonoids accumulation in CNO fruit. Finally, a hierarchical model for the regulatory network among the pathway genes and TFs was proposed. CONCLUSIONS: Collectively, our results suggest that DXS1, DXR, GGPPS2, PSY1 and LCYB genes were the most important genes for carotenoids accumulation, while PAL-1, PAL-4, 4CL-2, 4CL-5, CHS-1 and CHI for flavonoids biosynthesis. A total of 24 TFs were postulated as co-regulators in both pathways directly, which might play important roles in carotenoids and flavonoids accumulation in CNO fruit.

Volatile Compounds in Fruit Peels as Novel Biomarkers for the Identification of Four Citrus Species.

The aroma quality of citrus fruit is determined by volatile compounds, which bring about different notes to allow discrimination among different citrus species. However, the volatiles with various aromatic traits specific to different citrus species have not been identified. In this study, volatile profiles in the fruit peels of four citrus species collected from our previous studies were subjected to various analyses to mine volatile biomarkers. Principal component analysis results indicated that different citrus species could almost completely be separated. Thirty volatiles were identified as potential biomarkers in discriminating loose-skin mandarin, sweet orange, pomelo, and lemon, while 17 were identified as effective biomarkers in discriminating clementine mandarins from the other loose-skin mandarins and sweet oranges. Finally, 30 citrus germplasms were used to verify the classification based on ß-elemene, valencene, nootkatone, and limettin as biomarkers. The accuracy values were 90.0%, 96.7%, 96.7%, and 100%, respectively. This research may provide a novel and effective alternative approach to identifying citrus genetic resources.

Cit1,2RhaT and two novel CitdGlcTs participate in flavor-related flavonoid metabolism during citrus fruit development.

Neohesperidosides are disaccharides that are present in some flavonoids and impart a bitter taste, which can significantly affect the commercial value of citrus fruits. In this study, we identified three flavonoid-7-O-di-glucosyltransferase (dGlcT) genes closely related to 1,2-rhamnosyltransferase (1,2RhaT) in citrus genomes. However, only 1,2RhaT was directly linked to the accumulation of neohesperidoside, as demonstrated by association analysis of 50 accessions and co-segregation analysis of an F1 population derived from Citrus reticulata × Poncirus trifoliata. In transgenic tobacco BY2 cells, over-expression of CitdGlcTs resulted in flavonoid-7-O-glucosides being catalysed into bitterless flavonoid-7-O-di-glucosides, whereas over-expression of Cit1,2RhaT converted the same substrate into bitter-tasting flavonoid-7-O-neohesperidoside. Unlike 1,2RhaT, during citrus fruit development the dGlcTs showed an opposite expression pattern to CHS and CHI, two genes encoding rate-limiting enzymes of flavonoid biosynthesis. An uncoupled availability of dGlcTs and substrates might result in trace accumulation of flavonoid-7-O-di-glucosides in the fruit of C. maxima (pummelo). Past human selection of the deletion and functional mutation of 1,2RhaT has led step-by-step to the evolution of the flavor-related metabolic network in citrus. Our research provides the basis for potentially improving the taste in citrus fruit through manipulation of the network by knocking-out 1,2RhaT or by enhancing the expression of dGlcT using genetic transformation.