Anthocyanin Biosynthesis Genes Research Articles

Genotypic Variation of Purple Rice in Response to Shading in Yield, Anthocyanin Content, and Gene Expression.

Purple rice (Oryza sativa L.) contains anthocyanin, which acts as an antioxidant and functional food for humans. The levels of anthocyanin growth and production in rice are mainly controlled by the availability of light. However, shade can affect anthocyanin biosynthesis genes. Therefore, the objective of this study was to determine the yield and anthocyanin content among four purple rice varieties, which provide the difference in colors of purple and green leaves. This study also evaluated gene expression affected by shading treatment to understand the relation of grain anthocyanin and expression level. This research was conducted using a split plot design using four levels of shading (levels of shading from anthesis to maturity) with three replications, no shading, 30% shading, 50% shading, and 70% shading, as the main plots and purple rice varieties as subplots, KJ CMU-107, K2, K4, and KDK10, from anthesis to maturity. Shading significantly decreased yield and yield components, but increased grain anthocyanin content. Nonetheless, the response of yield and grain anthocyanin content to shading did not show a significant different between purple and green leaf varieties. In addition, the level of OsDFR gene expression was different depending on the shading level in four rice varieties. The OsDFR gene presented the highest expression at shading levels of 30% for K4 and 50% for KDK10, while the expression of the OsDFR gene was not detected in the purple rice varieties with green leaves (KJ CMU-107 and K2). The response of grain anthocyanin and gene expression of OsDFR to light treatment did not show significantly differences between the purple and green leaf varieties, suggesting that the appearance of anthocyanin in leaves might be not related to anthocyanin synthesis in the grain. Taken together, the results suggest that some purple rice varieties were more suitable for planting under low light intensity based on a lower level of grain yield loss, strong shade tolerance, and high anthocyanin content in leaf and grain pericarp. However, it is necessary to explore the effects of light intensity on genes and intermediates in the anthocyanin synthesis pathway for further study.

Three R2R3-MYB transcription factors from banana (Musa acuminata) activate structural anthocyanin biosynthesis genes as part of an MBW complex

ObjectiveBananas are one of the most popular fruits in the world, providing food security and employment opportunities in several developing countries. Increasing the anthocyanin content of banana fruit could improve the health-promoting properties. Anthocyanin biosynthesis is largely regulated at the transcriptional level. However, relatively little is known about the transcriptional activation of anthocyanin biosynthesis in banana.ResultsWe analysed the regulatory activity of three Musa acuminata MYBs that were predicted by bioinformatic analysis to transcriptionally regulate anthocyanin biosynthesis in banana. MaMYBA1, MaMYBA2 and MaMYBPA2 did not complement the anthocyanin-deficient phenotype of the Arabidopsis thaliana pap1/pap2 mutant. However, co-transfection experiments in A. thaliana protoplasts showed that MaMYBA1, MaMYBA2 and MaMYBPA2 function as components of a transcription factor complex with a bHLH and WD40 protein, the so called MBW complex, resulting in the activation of the A. thaliana ANTHOCYANIDIN SYNTHASE and DIHYDROFLAVONOL 4-REDUCTASE promoters. The activation potential of MaMYBA1, MaMYBA2 and MaMYBPA2 was increased when combined with the monocot Zea mays bHLH ZmR instead of the dicot AtEGL3. This work paves the path towards decoding the MBW complex-mediated transcriptional activation of anthocyanin biosynthesis in banana. It will also facilitate research towards increased anthocyanin content in banana and other monocot crops.

The Influence of Melatonin Treatment in the Vinification of Feteasca Neagra and Cabernet Sauvignon Wines on the Profile of Polyphenolic Compounds and Antioxidant Activity.

Until recently, the main antioxidant role among wine constituents was attributed to polyphenolic compounds, but once the presence of melatonin in wines was confirmed, an interesting new field of research opened up due to its possible synergistic effects with other antioxidants in the winemaking process, which may lead to a change in the profile of polyphenolic compounds and antioxidant activity. In order to investigate the evolution of active principles from the phenylpropanoid metabolism associated with the synergistic effects of melatonin, for the first time, a melatonin treatment was performed in the pre-stage of the different winemaking processes of Feteasca Neagra and Cabernet Sauvignon wines with different melatonin concentrations. After comparing the acquired results for the evolution of the polyphenolic compound profile and antioxidant activity of treated wines, we ascertained an increase in the antioxidant compound concentrations, especially in resveratrol, quercetin, and cyanidin-3-glucoside, directly proportional to the used melatonin concentration; an intensification in activity of PAL and C4H enzymes; and the modification in the expression of specific anthocyanin biosynthesis genes, especially UDP-D-glucose-flavonoid-3-O-glycosyltransferase. It was also shown that the application of melatonin in the pre-stage of the winemaking process can be successfully used to obtain red wines with increased antioxidant activity (almost 14%).

Sulfo-peptide CLEL6 inhibits anthocyanin biosynthesis in Arabidopsis thaliana.

Post-translationally modified peptides are now recognized as important regulators of plant stress responses. Here, we identified the small sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in etiolated and in light-stressed Arabidopsis (Arabidopsis thaliana) seedlings. CLEL6 function depends on proteolytic processing of the CLEL6 precursor by subtilisin-like serine proteinase 6.1 (SBT6.1) and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST). Loss-of-function mutants of either sbt6.1 or tpst showed significantly higher anthocyanin accumulation than the wild type upon light stress. The anthocyanin overaccumulation phenotype of sbt6.1 and tpst was suppressed by application of mature CLEL6. Overexpression and external application of CLEL6 inhibited the expression of anthocyanin biosynthesis genes in etiolated and light-stressed seedlings, confirming the role of CLEL6 as an inhibitor of anthocyanin biosynthesis. Small post-translationally modified peptides are perceived by leucine-rich-repeat receptor like kinases. Using a quintuple mutant of ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors, we showed the essential function of the RGI receptor family in CLEL6 signaling. Our data indicate that overexpression or application of CLEL6 inhibit anthocyanin biosynthesis through RGI receptors. We propose that CLEL6 inhibits anthocyanin biosynthesis in etiolated seedlings, and that anthocyanin biosynthesis is derepressed when CLEL6 expression is down-regulated upon light exposure. Hyperaccumulation of anthocyanins in light-stressed tpst and sbt6.1 mutant seedlings suggests that CLEL6, or related sulfopeptides, continue to act as negative regulators to limit pigment accumulation in the light.

High light triggers flavonoid and polysaccharide synthesis through DoHY5-dependent signaling in Dendrobium officinale.

Dendrobium officinale is edible and has medicinal and ornamental functions. Polysaccharides and flavonoids, including anthocyanins, are important components of D. officinale that largely determine the nutritional quality and consumer appeal. There is a need to study the molecular mechanisms regulating anthocyanin and polysaccharide biosynthesis to enhance D. officinale quality and its market value. Here, we report that high light (HL) induced the accumulation of polysaccharides, particularly mannose, as well as anthocyanin accumulation, resulting in red stems. Metabolome and transcriptome analyses revealed that most of the flavonoids showed large changes in abundance, and flavonoid and polysaccharide biosynthesis was significantly activated under HL treatment. Interestingly, DoHY5 expression was also highly induced. Biochemical analyses demonstrated that DoHY5 directly binds to the promoters of DoF3H1 (involved in anthocyanin biosynthesis), DoGMPP2, and DoPMT28 (involved in polysaccharide biosynthesis) to activate their expression, thereby promoting anthocyanin and polysaccharide accumulation in D. officinale stems. DoHY5 silencing decreased flavonoid- and polysaccharide-related gene expression and reduced anthocyanin and polysaccharide accumulation, whereas DoHY5 overexpression had the opposite effects. Notably, naturally occurring red-stemmed D. officinale plants similarly have high levels of anthocyanin and polysaccharide accumulation and biosynthesis gene expression. Our results reveal a previously undiscovered role of DoHY5 in co-regulating anthocyanin and polysaccharide biosynthesis under HL conditions, improving our understanding of the mechanisms regulating stem color and determining nutritional quality in D. officinale. Collectively, our results propose a robust and simple strategy for significantly increasing anthocyanin and polysaccharide levels and subsequently improving the nutritional quality of D. officinale.

Metabolome and transcriptome analyses reveal the molecular mechanisms of LcMYB1 regulating anthocyanin accumulation in litchi hairy roots

Agrobacterium rhizogenes-mediated hairy root culture offer a promising approach for gene function analysis and production of plant secondary metabolites. Here, we obtained red litchi hairy roots using A. rhizogenes-mediated LcMYB1 transformation. Using high performance liquid chromatography, the main anthocyanins in the red hairy roots were determined to be cyanidin 3-rutinoside and cyanidin 3-glucoside. A total of 164 metabolites were significantly upregulated or downregulated in the red hairy roots, which were mostly involved in flavone and flavonol pathway, and flavonoid pathway. The transcriptome analysis revealed 472 differentially expressed genes (DEGs). Up-regulated genes were considerably enriched in anthocyanin, flavone and flavonol biosynthesis. Integrative metabolite profiling and transcriptome analyses showed that LcF3′H, LcUFGT1, and LcGST4 were key structural genes in anthocyanin biosynthesis. However, the expression of Cinnamyl-alcohol dehydrogenase (CAD) and Peroxidase (POD) leading to the production of lignin were significantly down-regulated, suggesting flavonoids and lignin compete with each other in the phenylpropanoid pathway. A total of 52 DEGs were identified as transcription factors. Correlation analysis showed that 8 transcription factors were positively correlated with LcUFGT1, and LcGST4, involving in anthocyanin biosynthesis. These findings clarify the molecular mechanisms of LcMYB1 regulating anthocyanin accumulation in litchi hairy roots.

Metabolomics and transcriptomics provide insights into the molecular mechanisms of anthocyanin accumulation in the seed coat of differently colored mung bean (Vigna radiata L.)

Black mung bean is rich in anthocyanin, however, the accumulation and the molecular mechanism of anthocyanin synthesis in black mung bean are unclear. In this study, anthocyanin metabolomics and transcriptomics on the seed coats of two different colors of mung bean were performed to clarify the composition of anthocyanins, and identify transcription factors involved in regulating anthocyanin biosynthesis. In the mature stage, 23 kinds of anthocyanin compounds were identified. All anthocyanin components contents were significantly higher in seed coat of black mung bean compare with green mung bean. Transcriptome analysis suggested that most of the structural genes for anthocyanin biosynthesis and some potential regulatory genes were significantly differentially expressed. WGCNA suggested VrMYB90 was an important regulatory gene in anthocyanin biosynthesis. Arabidopsis thaliana overexpressing VrMYB90 showed significant accumulation of anthocyanins. PAL, 4CL, DFR, F3′5′H, LDOX, F3′H and UFGT were up-regulated in 35S:VrMYB90 Arabidopsis thaliana. These findings provide valuable information for understanding the synthesis mechanism of anthocyanins in black mung bean seed coats.

Genome-wide identification and bioinformatics analysis of the WRKY transcription factors and screening of candidate genes for anthocyanin biosynthesis in azalea (Rhododendron simsii).

WRKY transcription factors have been demonstrated to influence the anthocyanin biosynthesis in many plant species. However, there is limited knowledge about the structure and function of WRKY genes in the major ornamental plant azalea (Rhododendron simsii). In this study, we identified 57 RsWRKY genes in the R. simsii genome and classified them into three main groups and several subgroups based on their structural and phylogenetic characteristics. Comparative genomic analysis suggested WRKY gene family has significantly expanded during plant evolution from lower to higher species. Gene duplication analysis indicated that the expansion of the RsWRKY gene family was primarily due to whole-genome duplication (WGD). Additionally, selective pressure analysis (Ka/Ks) suggested that all RsWRKY duplication gene pairs underwent purifying selection. Synteny analysis indicated that 63 and 24 pairs of RsWRKY genes were orthologous to Arabidopsis thaliana and Oryza sativa, respectively. Furthermore, RNA-seq data was used to investigate the expression patterns of RsWRKYs, revealing that 17 and 9 candidate genes may be associated with anthocyanin synthesis at the bud and full bloom stages, respectively. These findings provide valuable insights into the molecular mechanisms underlying anthocyanin biosynthesis in Rhododendron species and lay the foundation for future functional studies of WRKY genes.

NataMYB4, a flower specific gene, regulates the flavonoid biosynthesis in Chinese Narcissus

R2R3 MYBs play a crucial role in flower development and its associated flavonoid biosynthesis, but their regulatory network in Chinese Narcissus is not fully understood. Anthocyanin, flavonol and proanthocyanin are very important secondary metabolites with various biological functions and their biosynthesis is controlled by MYB transcriptional factors. NataMYB4, as a member of the R2R3-MYB gene family, isolated from Chinese Narcissus, was found mainly expressed in flowers, particularly in petals and corona. In addition to the R2-R3 region, NataMYB4 displays a conserved motif “NF/YWSV/MEDF/LW” at the end of the C-terminal. Transient expression in tobacco leaves showed that NataMYB4 significantly decreased the red pigmentation that was induced by an anthocyanin activator StMYB. Ectopic overexpression of NataMYB4 in tobacco altered the flower color by reducing the pigmentation and anthocyanin contents in transgenic flowers. However, the contents of total phenolic acid and total flavonol were increased in the transgenic tobacco lines. qPCR analysis indicated that transcript levels of anthocyanin biosynthesis genes were down-regulated in transgenic flowers carrying the NataMYB4 gene, while the flavonol biosynthetic pathway key gene FLS showed higher expression level in transgenic tobacco flowers. Dual luciferase assays showed that co-infiltration of NataMYB4 and NatabHLH1 significantly activated the promoter of the Chinese Narcissus FLS gene. Furthermore, a yeast two-hybrid assay confirmed that NataMYB4 interacts with NatabHLH1. The outcomes of our effort in this study provide novel insights into the regulation of flavonoid biosynthetic pathways in Chinese Narcissus.

Coexpression of Structural and Regulatory Genes of the Flavonoid Pathway Reveals the Characteristics of Anthocyanin Biosynthesis in Eggplant Organs (<i>Solanum melongena</i> L.)

Eggplant (Solanum melongena L.) is an economically important vegetable crop whose purple-colored fruits are enriched with anthocyanidins. In this work in the eggplant genome, homologues of the main known structural (CHS1, CHS2, CHI, F3H, F35H, DFR, ANS, and UFGT) and regulatory (TT8, GL3, bHLH137, bHLH143, MYB1, MYB2, and MYB75) anthocyanin biosynthesis genes, as well as anthocyanidin transporter gene (GSTF12), were identified. The expression of these genes was characterized in comparisonwith the content of the total anthocyanins and the color of the leaf, flower petals, and fruit peel. It was shown that the gene expression pattern corresponds to the color and the presence of anthocyanins in the tissue, and also indicates the presence of organ-specific characteristics of the regulation of transcription of genes encoding transcription factors of the MBW complex. The results of correlation analysis confirm the involvement of SmbHLH137, SmTT8, SmMYB2, and SmMYB75 genes in the regulation of the expression of structural genes in flower petals and SmGL3, SmTT8, and SmMYB1 in fruit peel.

Effects of Different Light Wavelengths on Fruit Quality and Gene Expression of Anthocyanin Biosynthesis in Blueberry (Vaccinium corymbosm).

Different light wavelengths display diverse effects on fruit quality formation and anthocyanin biosynthesis. Blueberry is a kind of fruit rich in anthocyanin with important economic and nutritional values. This study explored the effects of different light wavelengths (white (W), red (R), blue (B) and yellow (Y)) on fruit quality and gene expression of anthocyanin biosynthesis in blueberry. We found that the B and W treatments attained the maximum values of fruit width, fruit height and fruit weight in blueberry fruits. The R treatment attained the maximum activities of superoxide dismutase (SOD) and peroxidase (POD), and the Y treatment displayed the maximum contents of ascorbic acid (AsA), glutathione (GSH) and total phenol in fruits, thus improving blueberry-fruit antioxidant capacity. Interestingly, there were differences in the solidity-acid ratio of fruit under different light-wavelength treatments. Moreover, blue light could significantly improve the expression levels of anthocyanin biosynthesis genes and anthocyanin content in fruits. Correlation and principal component analysis showed that total acid content and antioxidant enzymes were significantly negatively correlated with anthocyanin content in blueberry fruits. These results provide new insights for the application of light wavelength to improve blueberry fruit quality and anthocyanin content.

The MYB transcription factor RcMYB1 plays a central role in rose anthocyanin biosynthesis.

Rose (Rosa hybrida) is one of most famous ornamental plants in the world, and its commodity value largely depends on its flower color. However, the regulatory mechanism underlying rose flower color is still unclear. In this study, we found that a key R2R3-MYB transcription factor, RcMYB1, plays a central role in rose anthocyanin biosynthesis. Overexpression of RcMYB1 significantly promoted anthocyanin accumulation in both white rose petals and tobacco leaves. In 35S:RcMYB1 transgenic lines, a significant accumulation of anthocyanins occurred in leaves and petioles. We further identified two MBW complexes (RcMYB1-RcBHLH42-RcTTG1; RcMYB1-RcEGL1-RcTTG1) associated with anthocyanin accumulation. Yeast one-hybrid and luciferase assays showed that RcMYB1 could active its own gene promoter and those of other EBGs (early anthocyanin biosynthesis genes) and LBGs (late anthocyanin biosynthesis genes). In addition, both of the MBW complexes enhanced the transcriptional activity of RcMYB1 and LBGs. Interestingly, our results also indicate that RcMYB1 is involved in the metabolic regulation of carotenoids and volatile aroma. In summary, we found that RcMYB1 widely participates in the transcriptional regulation of ABGs (anthocyanin biosynthesis genes), indicative of its central role in the regulation of anthocyanin accumulation in rose. Our results provide a theoretical basis for the further improvement of the flower color trait in rose by breeding or genetic modification.

Genome-wide identification of MATE, functional analysis and molecular dynamics of DcMATE21 involved in anthocyanin accumulation in Daucus carota

Anthocyanins are a subclass of flavonoids that are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Multidrug and toxic compound extrusion transporters (MATE) is a family of membrane transporters that transport ions and secondary metabolites, such as anthocyanins, in plants. Although various studies on MATE transporters have been carried out on different plant species, this is the first comprehensive report to mine the Daucus carota genome to identify the MATE gene family. Our study identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from the genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. In addition, we analyzed RNA-seq data obtained from the European Nucleotide Archive to screen for the expression of DcMATEs involved in anthocyanin biosynthesis. Among the identified DcMATEs, DcMATE21 correlated with anthocyanin content in the different D. carota varieties. In addition, the expression of DcMATE21 and anthocyanin biosynthesis genes was correlated under abscisic acid, methyl jasmonate, sodium nitroprusside, salicylic acid, and phenylalanine treatments, which were substantiated by anthocyanin accumulation in the in vitro cultures. Further molecular membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket, showing extensive H-bond interactions with 10 crucial amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. The current investigation, using RNA-seq, in vitro cultures, and molecular dynamics studies revealed the involvement of DcMATE21 in anthocyanin accumulation in vitro cultures of D. carota.

Proteomic and metabolomic analyses showing the differentially accumulation of NnUFGT2 is involved in the petal red-white bicolor pigmentation in lotus (Nelumbo nucifera)

Bicolor flower lotus is rare with high ornamental value. During the long history of breeding and artificial selection, a very famous lotus cultivar ‘Da Sajin’ with red and white picotee bicolor petals were obtained. In order to reveal the mechanism underlying the formation of its picotee bicolor pattern in the petal, an integrative metabolomics and proteomics analyses were conducted between red and white parts of its petals. The results showed that the defect of anthocyanidin 3-O-glucosyltransferases (UFGTs) accumulation resulted in the failure of the glycosylation of anthocyanidin, the last step of anthocyanin biosynthesis in white part of the petals. And proteomic data and biochemical analysis showed that the defect of UFGTs accumulation is not related to their transcription, but because of their degradation. Function of one differentially accumulated NnUFGT were proven being involved in anthocyanin biosynthesis through both in-vitro enzyme assay and in-vivo transgenic analyses. This regulation on the protein accumulation of structural genes in anthocyanin biosynthesis was not explored in any other plants, and hence supposed to be a novel mechanism for the formation of picotee bicolor pattern flower. The results not only provide some new insights into the understanding of lotus flower coloration, but also might assist the breeding of flower lotus.

The long noncoding RNA LINC15957 regulates anthocyanin accumulation in radish.

Radish (Raphanus sativus L.) is an important root vegetable crop belonging to the Brassicaceae family. Anthocyanin rich radish varieties are popular among consumers because of their bright color and high nutritional value. However, the underlying molecular mechanism responsible for skin and flesh induce anthocyanin biosynthesis in transient overexpression, gene silencing and transcriptome sequencing were used to verify its function in radish anthocyanin accumulation, radish remains unclear. Here, we identified a long noncoding RNA LINC15957, overexpression of LINC15957 was significantly increased anthocyanin accumulation in radish leaves, and the expression levels of structural genes related to anthocyanin biosynthesis were also significantly increased. Anthocyanin accumulation and expression levels of anthocyanin biosynthesis genes were significantly reduced in silenced LINC15957 flesh when compared with control. By the transcriptome sequencing of the overexpressed LINC15957 plants and the control, 5,772 differentially expressed genes were identified. A total of 3,849 differentially expressed transcription factors were identified, of which MYB, bHLH, WD40, bZIP, ERF, WRKY and MATE were detected and differentially expressed in the overexpressed LINC15957 plants. KEGG enrichment analysis revealed the genes were significant enriched in tyrosine, L-Phenylalanine, tryptophan, phenylpropanol, and flavonoid biosynthesis. RT-qPCR analysis showed that 8 differentially expressed genes (DEGs) were differentially expressed in LINC15957-overexpressed plants. These results suggested that LINC15957 involved in regulate anthocyanin accumulation and provide abundant data to investigate the genes regulate anthocyanin biosynthesis in radish.

Sucrose non-fermenting1-related protein kinase VcSnRK2.3 promotes anthocyanin biosynthesis in association with VcMYB1 in blueberry.

Sucrose non-fermenting1-related protein kinase-2 (SnRK2) is a plant-specific protein kinase family and an important component of the abscisic acid (ABA) signaling pathway. However, there is a lack of relevant studies in blueberry (Vaccinium corymbosum). In this study, we identified six SnRK2 family members (from VcSnRK2.1 to VcSnRK2.6) in blueberries for the first time. In addition, we found that VcSnRK2.3 expression was not only positively correlated with fruit ripening but was also induced by ABA signaling. Transient expression in blueberry fruits also proved that VcSnRK2.3 promoted anthocyanin accumulation and the expression of anthocyanin synthesis-related genes such as VcF3H, VcDFR, VcANS, and VcUFGT. Transgenic Arabidopsis thaliana seeds and seedlings overexpressing VcSnRK2.3 showed anthocyanin pigmentation. Yeast two-hybrid assays (Y2H) and Bimolecular fluorescence complementation assays (BiFC) demonstrated that VcSnRK2.3 could interact with the anthocyanin positive regulator VcMYB1. Finally, VcSnRK2.3 was able to enhance the binding of VcMYB1 to the VcDFR promoter. Via regulation transcription of anthocyanin biosynthesis genes, VcSnRK2.3 promoted anthocyanin accumulation in blueberry. The above results suggest that VcSnRK2.3 plays an important role in blueberry anthocyanin synthesis, is induced by ABA, and can interact with VcMYB1 to promote anthocyanin biosynthesis in blueberry.

Magnesium improved fruit quality by regulating photosynthetic nitrogen use efficiency, carbon-nitrogen metabolism, and anthocyanin biosynthesis in 'Red Fuji' apple.

Both nitrogen (N) and magnesium (Mg) play important roles in biochemical and physiological processes in plants. However, the application of excessive N and insufficient Mg may be the factor leading to low nitrogen utilization rate (NUE) and fruit quality degradation in apple production. In this study, we analyzed the effects of different application rates of Mg (0, 50, 100, 150, 200 kg/ha) on the photosynthetic nitrogen use efficiency (PNUE), the accumulation and distribution of carbon (C), N metabolism, anthocyanin biosynthesis and fruit quality of the 'Red Fuji' apple in 2018 and 2019. The results showed that the application of Mg significantly increased the 15NUE and increased the allocation rate of 15N in the leaves whereas the 15N allocation rate in the perennial organs and fruits was decreased. With the increase in Mg supply, the activities of N metabolism enzymes (NiR, GS, and GOGAT) were significantly promoted and the content of intermediate products in N metabolism ( , , and free amino acid) was significantly decreased. Furthermore, an appropriate rate of Mg significantly promoted the net photosynthetic rate (Pn) and photosynthetic nitrogen use efficiency (PNUE), enhanced the enzyme activities of C metabolism (SS, SPS, S6PDH), and increased the contents of sorbitol and sucrose in leaves. In addition, Mg upregulated the gene expression of sugar transporters (MdSOT1, MdSOT3, MdSUT1, and MdSUT4) in fruit stalk and fruit fresh; 13C isotope tracer technology also showed that Mg significantly increased the 13C allocation in the fruits. Mg also significantly increased the expression of anthocyanin biosynthesis genes (MdCHS and MdF3H) and transcription factors (MdMYB1 and MdbZIP44) and the content of anthocyanin in apple peel. The comprehensive analysis showed that the appropriate application of Mg (150 kg/ha) promoted PNUE, C-N metabolism, and anthocyanin biosynthesis in apple trees.

High temperature inhibited the accumulation of anthocyanin by promoting ABA catabolism in sweet cherry fruits.

Color is an essential appearance characteristic of sweet cherry (Prunus avium L.) fruits and mainly determined by anthocyanin. Temperature plays an important role in the regulation of anthocyanin accumulation. In this research, anthocyanin, sugar, plant hormone and related gene expression were analyzed using physiological and transcriptomic methods in order to reveal the effects of high temperature on fruit coloring and the related mechanism. The results showed that high temperature severely inhibited anthocyanin accumulation in fruit peel and slowed the coloring process. The total anthocyanin content in fruit peel increasedby 455% and 84% after 4 days of normal temperature treatment (NT, 24°C day/14°C night) and high temperature treatment (HT, 34°C day/24°C night), respectively. Similarly, the contents of 8 anthocyanin monomers were significantly higher in NT than in HT. HT also affected the levels of sugars and plant hormones. The total soluble sugar content increased by 29.49% and 16.81% in NT and HT, respectively, after 4 days of treatment. The levels of ABA, IAA and GA20 also increased in both the two treatments but more slowly in HT. Conversely, the contents of cZ, cZR and JA decreased more rapidly in HT than in NT. The results of the correlation analysis showed that the ABA and GA20 contents were significantly correlated with the total anthocyanin contents. Further transcriptome analysis showed that HT inhibited the activation of structural genes in anthocyanin biosynthesis as well as the repression of CYP707A and AOG, which dominated the catabolism and inactivation of ABA. These results indicate that ABA may be a key regulator in the high-temperature-inhibited fruit coloring of sweet cherry. High temperature induces higher ABA catabolism and inactivation, leading to lower ABA levels and finally resulting in slow coloring.

ETHYLENE RESPONSE FACTORS 4.1/4.2 with an EAR motif repress anthocyanin biosynthesis in red-skinned pears.

Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of 'Starkrimson' pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3-PyMYB114-PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.

Selenium treatment promotes anthocyanin accumulation in radish sprouts (Raphanus sativus L.) by its regulation of photosynthesis and sucrose transport

Red radish sprout become a popular dietary vegetable because of its unique flavor, abundant nutrients and short production cycle. As a cruciferous plant, it has strong ability to absorb and assimilate Se which can promote the content of anthocyanin in plants. However, the mechanisms of Se on anthocyanin accumulation are still unclear. In this study, we explored that appropriate Se promoted growth, antioxidant system and nutrients in radish sprouts. The enhancement of photosynthesis by Se treatment resulted in more sucrose synthesis in radish sprouts. And the transport of sucrose from cotyledon to hypocotyl promoted by Se through up-regulating the gene expression of sucrose transporters, and more sucrose increased the expression of anthocyanin biosynthesis genes to promote anthocyanin accumulation in hypocotyl. These results reveal the beneficial effect of Se on radish sprouts quality, and provide a new insight into the function of Se on sucrose-induced anthocyanin accumulation in radish sprouts.