Inhibits Anthocyanin Biosynthesis Research Articles

Enhancing Red Table Grape Coloration Using Tsikoudia: A Novel and Sustainable Approach.

Achieving optimal coloration in red table grapes, especially in warm-climate regions, presents significant challenges due to high temperatures that inhibit anthocyanin biosynthesis. Conventional methods to enhance grape coloration, including the use of abscisic acid (ABA), ethephon, foliar nutrient supplementation, and viticultural practices like cluster trimming and girdling, have limitations related to cost, regulatory restrictions, and potential adverse effects on grapes quality. This study proposes the application of tsikoudia, a traditional Greek alcoholic beverage, as a novel, sustainable, and cost-effective alternative to conventional practices. Tsikoudia, applied during the veraison stage, significantly improved the coloration of 'Crimson Seedless' and 'Red Globe' grapes by enhancing anthocyanin accumulation and altering color parameters. Specifically, lightness (L*), chroma (C*), and hue angle (h), measured using the CIE-Lab color system, were reduced, while the Color Index for Red Grapes (CIRG) was increased. Additionally, total anthocyanin content, determined through spectrophotometric analysis, also showed an increase. These changes indicate a more intense red coloration. This research highlights the effectiveness of tsikoudia in improving grape coloration and contributes to the development of more sustainable viticultural practices.

Anthocyanin Accumulation in Grape Berry Skin Promoted by Endophytic Microbacterium sp. che218 Isolated from Wine Grape Shoot Xylem.

Grape berry skin coloration is a key determinant of the commercial value of red wines. Global warming caused by climate change has inhibited anthocyanin biosynthesis in berry skins, leading to poor coloration. Through two-year field experiments, the endophyte che218 isolated from grape shoot xylem promoted anthocyanin accumulation in berry skins. The che218 enhanced anthocyanin biosynthesis in grapevine cultured cells. In the 2022 growing season, applying che218 to grape bunches enhanced anthocyanin accumulation in berry skins on day 20 post-treatment. However, the anthocyanin accumulation enhancing effect of che218 became negligible at harvest. In the 2023 growing season, che218 enhanced anthocyanin accumulation in berry skins on day 15 post-treatment and at harvest (day 30 post-treatment) and also upregulated the transcription of mybA1 and UFGT, two genes that regulate anthocyanin biosynthesis in berry skins. Whole genome sequencing demonstrated that che218 is an unidentified Microbacterium species. However, it remains unknown how che218 is involved in the biosynthesis of anthocyanin in berry skins. This study provides insights into the development of an eco-friendly endophyte-mediated technique for improving grape berry skin coloration, thereby mitigating the effects of global warming on berry skin coloration.

An ‘activator-repressor’ loop controls the anthocyanin biosynthesis in red-skinned pear

The color of red-skinned pear (Pyrus spp.) is primarily attributed to accumulation of anthocyanins, which provide nutritional benefits for human health and are closely associated with the commercial value of fruits. Here, we reported the functional characterization of a R2R3-MYB repressor PyMYB107, which forms an ‘activator-repressor’ loop to control anthocyanin accumulation in the red-skinned pear. PyMYB107 overexpression inhibited anthocyanin biosynthesis in both pear calli and fruits, while virus-induced gene silencing of PyMYB107 increased anthocyanin accumulation in pear fruits. Furthermore, ectopic expression of PyMYB107 decreased anthocyanin accumulation in tomato, strawberry and tobacco. PyMYB107 can competitively bind to PybHLH3 with PyMYB10/MYB114, thereby suppressing the transcriptional activation of key anthocyanin biosynthesis genes, PyANS and PyUFGT. Site-directed mutagenesis showed that mutations within the R3 domain and EAR motif of PyMYB107 eliminated its repressive activity. Additionally, PyMYB107 exhibited a comparable expression pattern to PyMYB10/MYB114 and was transcriptionally activated by them. Our finding advanced comprehension of the repression mechanism underlying anthocyanin accumulation, providing valuable molecular insights into improving quality of pear fruits.

The SMXL8-AGL9 module mediates crosstalk between strigolactone and gibberellin to regulate strigolactone-induced anthocyanin biosynthesis in apple.

Although the strigolactone (SL) signaling pathway and SL-mediated anthocyanin biosynthesis have been reported, the molecular association between SL signaling and anthocyanin biosynthesis remains unclear. In this study, we identified the SL signal transduction pathway associated with anthocyanin biosynthesis and the crosstalk between gibberellin (GA) and SL signaling in apple (Malus × domestica). ELONGATED HYPOCOTYL5 (HY5) acts as a key node integrating SL signaling and anthocyanin biosynthesis, and the SL-response factor AGAMOUS-LIKE MADS-BOX9 (AGL9) promotes anthocyanin biosynthesis by activating HY5 transcription. The SL signaling repressor SUPPRESSOR OF MAX2 1-LIKE8 (SMXL8) interacts with AGL9 to form a complex that inhibits anthocyanin biosynthesis by downregulating HY5 expression. Moreover, the E3 ubiquitin ligase PROTEOLYSIS1 (PRT1) mediates the ubiquitination-mediated degradation of SMXL8, which is a key part of the SL signal transduction pathway associated with anthocyanin biosynthesis. In addition, the GA signaling repressor REPRESSOR-of-ga1-3-LIKE2a (RGL2a) mediates the crosstalk between GA and SL by disrupting the SMXL8-AGL9 interaction that represses HY5 transcription. Taken together, our study reveals the regulatory mechanism of SL-mediated anthocyanin biosynthesis and uncovers the role of SL-GA crosstalk in regulating anthocyanin biosynthesis in apple.

Transcriptomic and metabolomic analyses revealCmMYB308 as a key regulator in the pink flower color variation of 'Dante Purple' chrysanthemum.

CmMYB308 was identified as a key regulator in chrysanthemum flower color variation from purple to pink by conducting transcriptome and metabolome analysis. CmMYB308 can inhibit anthocyanin biosynthesis by suppressing the expression of CmPAL, CmC4H, and Cm4CL. Flower color variation is a widespread natural occurrence that plays a significant role in floral breeding. We discovered a variation in the flower of the chrysanthemum cultivar 'Dante Purple' (abbreviated as 'DP'), where the flower color shifted from purple to pink. We successfully propagated these pink flowers through tissue culture and designated them as DPM. By conducting transcriptome and metabolome analysis, we identified a reduction in the expression of critical genes involved in anthocyanin biosynthesis-CmPAL, CmC4H, and Cm4CL-in the DPM. This downregulation led to an accumulation of phenylalanine and cinnamic acid within the general phenylpropanoid pathway (GPP), which prevented their conversion into cyanidin and cyanidin 3-glucoside. As a result, the flowers turned pink. Additional transformation and biochemical experiments confirmed that the upregulation of CmMYB308 gene expression in the DPM directly suppressed CmPAL-1 and CmC4H genes, which indirectly affected Cm4CL-3 expression and ultimately inhibited anthocyanin biosynthesis in the DPM. This study offers a preliminary insight into the molecular mechanism underlying chrysanthemum flower color mutation, paving the way for genetic improvements in chrysanthemum flower color breeding.

Tartary Buckwheat (Fagopyrum tataricum) FtTT8 Inhibits Anthocyanin Biosynthesis and Promotes Proanthocyanidin Biosynthesis.

Tartary buckwheat (Fagopyrum tataricum) is an important plant, utilized for both medicine and food. It has become a current research hotspot due to its rich content of flavonoids, which are beneficial for human health. Anthocyanins (ATs) and proanthocyanidins (PAs) are the two main kinds of flavonoid compounds in Tartary buckwheat, which participate in the pigmentation of some tissue as well as rendering resistance to many biotic and abiotic stresses. Additionally, Tartary buckwheat anthocyanins and PAs have many health benefits for humans and the plant itself. However, little is known about the regulation mechanism of the biosynthesis of anthocyanin and PA in Tartary buckwheat. In the present study, a bHLH transcription factor (TF) FtTT8 was characterized to be homologous with AtTT8 and phylogenetically close to bHLH proteins from other plant species. Subcellular location and yeast two-hybrid assays suggested that FtTT8 locates in the nucleus and plays a role as a transcription factor. Complementation analysis in Arabidopsis tt8 mutant showed that FtTT8 could not recover anthocyanin deficiency but could promote PAs accumulation. Overexpression of FtTT8 in red-flowering tobacco showed that FtTT8 inhibits anthocyanin biosynthesis and accelerates proanthocyanidin biosynthesis. QRT-PCR and yeast one-hybrid assay revealed that FtTT8 might bind to the promoter of NtUFGT and suppress its expression, while binding to the promoter of NtLAR and upregulating its expression in K326 tobacco. This displayed the bidirectional regulating function of FtTT8 that negatively regulates anthocyanin biosynthesis and positively regulates proanthocyanidin biosynthesis. The results provide new insights on TT8 in Tartary buckwheat, which is inconsistent with TT8 from other plant species, and FtTT8 might be a high-quality gene resource for Tartary buckwheat breeding.

SnRK1 inhibits anthocyanin biosynthesis through both transcriptional regulation and direct phosphorylation and dissociation of the MYB/bHLH/TTG1 MBW complex.

Plants have evolved an extensive specialized secondary metabolism. The colorful flavonoid anthocyanins, for example, not only stimulate flower pollination and seed dispersal but also protect different tissues against high light, UV- and oxidative stress. Their biosynthesis is highly regulated by environmental and developmental cues and induced by high sucrose levels. Expression of the biosynthetic enzymes involved is controlled by a transcriptional MBW complex, comprising (R2R3) MYB- and bHLH-type transcription factors (TF) and the WD40 repeat protein TTG1. Anthocyanin biosynthesis is obviously useful but also carbon- and energy-intensive and non-vital. Consistently, the SnRK1 protein kinase, a metabolic sensor activated in carbon- and energy-depleting stress conditions, represses anthocyanin biosynthesis. Here we show that Arabidopsis SnRK1 represses MBW complex activity both at the transcriptional and post-translational level. In addition to repressing expression of the key transcription factor MYB75/PAP1, SnRK1 activity triggers MBW complex dissociation, associated with loss of target promoter binding, MYB75 protein degradation and nuclear export of TTG1. We also provide evidence for direct interaction with and phosphorylation of multiple MBW complex proteins. These results indicate that repression of expensive anthocyanin biosynthesis is an important strategy to save energy and redirect carbon flow to more essential processes for survival in metabolic stress conditions.

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.

The ethylene-responsive transcription factor PpERF9 represses PpRAP2.4 and PpMYB114 via histone deacetylation to inhibit anthocyanin biosynthesis in pear.

Ethylene induces anthocyanin biosynthesis in most fruits, including apple (Malus domestica) and plum (Prunus spp.). By contrast, ethylene inhibits anthocyanin biosynthesis in pear (Pyrus spp.), but the underlying molecular mechanism remains unclear. In this study, we identified and characterized an ethylene-induced ETHYLENE RESPONSE FACTOR (ERF) transcription factor, PpETHYLENE RESPONSE FACTOR9 (PpERF9), which functions as a transcriptional repressor. Our analyses indicated PpERF9 can directly inhibit expression of the MYB transcription factor gene PpMYB114 by binding to its promoter. Additionally, PpERF9 inhibits the expression of the transcription factor gene PpRELATED TO APETALA2.4 (PpRAP2.4), which activates PpMYB114 expression, by binding to its promoter, thus forming a PpERF9-PpRAP2.4-PpMYB114 regulatory circuit. Furthermore, PpERF9 interacts with the co-repressor PpTOPLESS1 (PpTPL1) via EAR motifs to form a complex that removes the acetyl group on histone H3 and maintains low levels of acetylated H3 in the PpMYB114 and PpRAP2.4 promoter regions. The resulting suppressed expression of these 2 genes leads to decreased anthocyanin biosynthesis in pear. Collectively, these results indicate that ethylene inhibits anthocyanin biosynthesis by a mechanism that involves PpERF9-PpTPL1 complex-mediated histone deacetylation of PpMYB114 and PpRAP2.4. The data presented herein will be useful for clarifying the relationship between chromatin status and hormone signaling, with implications for plant biology research.

Persulfidation of transcription factor MYB10 inhibits anthocyanin synthesis in red-skinned pear.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that delays color change during fruit ripening. Whether H2S affects anthocyanin biosynthesis in red-skinned pears (Pyrus L.) remains unclear. Here, we found that H2S substantially inhibits anthocyanin accumulation in red-skinned pears and the expression of several genes encoding transcription factors is affected in response to H2S signaling. For example, PyMYB10 and PyMYB73 were down-regulated, whereas PyMYB114 and PyMYB6 were up-regulated. Bioinformatics analysis showed that PyMYB73 and PyMYB6, each containing an EAR motif, may negatively regulate anthocyanin accumulation. Transient expression analysis showed that PyMYB73 substantially promotes anthocyanin biosynthesis by co-transforming with PyMYB10/PyMYB114 + PybHLH3; however, PyMYB6 inhibited anthocyanin biosynthesis in strawberry (Fragaria vesca) receptacles and pear fruits, and PyMYB73 interacted with PyMYB10 and PyMYB6 but not PyMYB114 or PybHLH3. Further investigation showed that Cys194 and Cys218 of PyMYB10 were modified by persulfidation and that PyMYB10Cys218Ala substantially increased anthocyanin accumulation by a transient transformation system. Co-transformation of PyMYB10Cys218Ala + PyMYB73/PyMYB6 also promoted anthocyanin accumulation in pear fruits. Yeast two-hybrid assays showed that the mutation of PyMYB10 did not affect the interaction between PyMYB10 and PyMYB73, but it inhibited interaction with PyMYB6. Moreover, H2S weakened the interaction between PyMYB10 and PyMYB73 but enhanced the interaction with PyMYB6. Thus, we provided a model in which PyMYB10 undergoes persulfidation at Cys218, enhancing the interaction with PyMYB6 and reducing the interaction with PyMYB73. These subsequently results in lower expression of the anthocyanin biosynthesis-related genes Pyrus dihydroflavonol 4-reductase (PyDFR), Pyrus anthocyanidin synthase (PyANS), Pyrus UDP-glucose: flavonoid 3-glucosyl transferase (PyUFGT) and Pyrus glutathione S-transferase (PyGST), thereby inhibiting anthocyanin accumulation in red-skinned pears. Our findings provided a molecular mechanism for H2S-mediated anthocyanin biosynthesis in red-skinned pears.

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.

PpZAT5 suppresses the expression of a B-box gene PpBBX18 to inhibit anthocyanin biosynthesis in the fruit peel of red pear.

BBX (B-box) proteins play a vital role in light-induced anthocyanin biosynthesis. PpBBX18 was an indispensable regulator for the induction of anthocyanin biosynthesis in the peel of red pear fruit (Pyrus pyrifolia Nakai.). However, the upstream regulation of BBX genes has not been well characterized. In this study, PpZAT5, a cysteine2/histidine2-type transcription factor, was discovered as the upstream negative regulator of PpBBX18. The results showed that PpZAT5 functions as a transcriptional repressor and directly binds to the CAAT motif of PpBBX18 and inhibits its expression. PpZAT5 expression was inhibited by light, which is converse to the expression pattern of anthocyanin-related structural genes. In addition, less anthocyanin accumulated in the PpZAT5-overexpressing pear calli than in the wild-type pear calli; on the contrary, more anthocyanin accumulated in PpZAT5-RNAi pear calli. Moreover, the crucial genes involved in light-induced anthocyanin biosynthesis were markedly down-regulated in the transcriptome of PpZAT5 overexpression pear calli compared to wild-type. In conclusion, our study indicates that PpBBX18 is negatively regulated by a C2H2-type transcriptional repressor, PpZAT5, which reduces anthocyanin content in pear. The present results demonstrate an upstream molecular mechanism of PpBBX18 and provide insights into light-induced anthocyanin biosynthesis.

Ethylene Inhibits Anthocyanin Biosynthesis by Repressing the R2R3-MYB Regulator SlAN2-like in Tomato.

Fruit ripening is usually accompanied by anthocyanin accumulation. Ethylene is key in ripening-induced anthocyanin production in many fruits. However, the effects of fruit ripening and ethylene on anthocyanin biosynthesis in purple tomato fruits are unclear. This study shows that bagged fruits of the purple tomato cultivar ‘Indigo Rose’ failed to produce anthocyanins at the red ripening stage after bag removal. In contrast, the bagged immature fruits accumulated a significant amount of anthocyanins after removing the bags. The transcriptomic analyses between immature and red ripening fruit before and after bag removal revealed that anthocyanin-related genes, including the key positive R2R3-MYB regulator SlAN2-like, were repressed in the red ripening fruit. The 86 identified transcription factors, including 13 AP2/ERF, 7 bZIP, 8 bHLH and 6 MYB, showed significantly different expressions between immature and red ripening fruits. Moreover, subjecting bagged immature fruits to exogenous ethylene treatment significantly inhibited anthocyanin accumulation and the expression of anthocyanin-related genes, including the anthocyanin structure genes and SlAN2-like. Thus, ethylene inhibits anthocyanin biosynthesis by repressing the transcription of SlAN2-like and other anthocyanin-related genes. These findings provide new insights into anthocyanin regulation in purple tomato fruit.

A novel transcription factor CmMYB012 inhibits flavone and anthocyanin biosynthesis in response to high temperatures in chrysanthemum

Flavones are among the major colorless pigments synthesized through branches of the flavonoid pathway in plants. However, due to the absence of a gene encoding flavone synthase (FNS) in the model plant Arabidopsis thaliana species, the regulatory mechanism of FNS-catalyzed flavone biosynthesis has rarely been studied in plants. Here, it was found that flavones play a predominant role in the elimination of excess reactive oxygen species (ROS) at high temperatures in colorless plant organs. A novel atypical subgroup 7 (SG7) R2R3-MYB transcription factor, CmMYB012, was found to be induced in response to prolonged high temperatures and to inhibit flavone biosynthesis by directly regulating CmFNS. Moreover, CmMYB012 was also found to inhibit anthocyanin biosynthesis by suppressing the expression of CmCHS, CmDFR, CmANS, and CmUFGT. CmMYB012 overexpression exerted a negative influence on plant fitness and pink flower color formation, while CmMYB012 suppression had the opposite effect in response to high temperatures. Our findings provide new insights into the mechanisms by which high temperatures regulate the metabolism of flavones and anthocyanins to affect plant fitness and flower color formation.

The apple MdCOP1-interacting protein 1 negatively regulates hypocotyl elongation and anthocyanin biosynthesis

BackgroundIn plants, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is a key negative regulator in photoperiod response. However, the biological function of COP1-interacting protein 1 (CIP1) and the regulatory mechanism of the CIP1-COP1 interaction are not fully understood.ResultsHere, we identified the apple MdCIP1 gene based on the Arabidopsis AtCIP1 gene. Expression pattern analysis showed that MdCIP1 was constitutively expressed in various tissues of apple, and responded to stress and hormone signals at the transcriptional level. Ectopic expression of MdCIP1 complemented the phenotypes of the Arabidopsis cip1 mutant, and MdCIP1 inhibited anthocyanin biosynthesis in apple calli. In addition, the biochemical assay demonstrated that MdCIP1 could interact with MdCOP1 protein by their coiled-coil domain, and MdCIP1-OX/cop1–4 had a similar phenotype in photomorphogenesis with the cop1–4 mutant, suggesting that COP1 is epistatic to CIP1. Furthermore, the transient transformation assay indicated that MdCIP1 repressed anthocyanin biosynthesis in an MdCOP1-mediated pathway.ConclusionTake together, this study finds that MdCIP1 acts as a repressor in regulating hypocotyl elongation and anthocyanin biosynthesis through MdCOP1 in apple.

Ethylene-activated PpERF105 induces the expression of the repressor-type R2R3-MYB gene PpMYB140 to inhibit anthocyanin biosynthesis in red pear fruit.

Ethylene induces anthocyanin biosynthesis in most fruits, including apple (Malus domestica), strawberry (Fragaria × ananassa), and plum (Prunus spp.). However, ethylene inhibits anthocyanin biosynthesis in pear (Pyrus spp.), but the underlying molecular mechanism has not been characterized. In this study, ethylene induced the expression of PpERF105, which encodes a transcription factor. PpERF105 functioned as a transcriptional activator, but it inhibited anthocyanin biosynthesis in pear. A transcriptome analysis revealed that PpERF105 activated the expression of PpMYB140, which encodes an R2R3-MYB transcriptional repressor. Moreover, PpMYB140 directly inhibited the expression of anthocyanin-related structural genes. It also competed with PpMYB114 for the binding to bHLH3, ultimately resulting in the formation of the MYB140-bHLH-WDR complex rather than the conventional MBW complex, thereby further inhibiting anthocyanin biosynthesis. Furthermore, PpMYB140 prevented the overaccumulation of anthocyanins in the absence of ethylene. Collectively, our study data indicate that ethylene-induced PpERF105 inhibits anthocyanin biosynthesis by upregulating PpMYB140 expression. Our findings may be useful for elucidating the molecular basis of the ethylene-mediated inhibition of anthocyanin biosynthesis in fruit.

High temperature at veraison inhibits anthocyanin biosynthesis in berry skins during ripening in ‘Kyoho’ grapevines

We examined the effects of high temperature (HT) at veraison (the onset of ripening) on coloration and anthocyanin biosynthesis in berry skins of ‘Kyoho’ grapevines (Vitis labruscana L.). The vines were subjected to control, HT (6 °C higher than the control for 10 days), and intermittent HT (IHT; 6 °C higher than the control for 4 days followed by control temperature for 3 days and then 6 °C higher than the control for another 3 days) conditions from 50 to 60 days after full bloom (DAFB) in temperature-controlled rooms. Under control conditions, berry skins were tinted purple from 55 DAFB and turned to reddish-purple thereafter until 80 DAFB, concurrently with the anthocyanin accumulation. The HT and IHT treatments greatly inhibited the coloration and anthocyanin accumulation, with greater inhibition by the HT treatment. The HT and IHT treatments significantly inhibited the expressions of early (EBGs) and late anthocyanin biosynthetic genes (LBGs), and the transcription factor gene VlMYBA2. Abscisic acid (ABA) contents in the control berry skins increased from 50 DAFB, peaked at 55 DAFB, and decreased thereafter. The HT and IHT treatments reduced the increase in ABA contents, with no significant difference between HT- and IHT-treated vines. Gibberellin (GA) contents decreased during veraison in the berry skins of control and IHT-treated vines, but remained unchanged in those of HT-treated vines. These results suggest that the coloration and anthocyanin biosynthesis in berry skins are associated with changes in the ABA/GA ratio.

LbNR-Derived Nitric Oxide Delays Lycium Fruit Coloration by Transcriptionally Modifying Flavonoid Biosynthetic Pathway.

Anthocyanin-derived fleshy fruit pigmentation has become an excellent system for studying the regulatory network underlying fruit ripening and quality. The transcriptional control of anthocyanin biosynthesis by MYB–bHLH–WDR complexes has been well established, but the intermediate signals through which the environmental or developmental cues regulate these transcription factors remain poorly understood. Here we found that nitric oxide (NO) production during Lycium fruit ripening decreased progressively presenting a negative relationship with anthocyanins. After cloning of the nitric reductase (NR) gene from Lycium barbarum (LbNR) plants, we demonstrated that LbNR-derived NO partially inhibited anthocyanin biosynthesis but enhanced proanthocyanidin (PA) accumulation, and delayed fruit coloration. Application of the NO donor, sodium nitroprusside (SNP), produced a similar effect. The endogenous or exogenous NO downregulated the transcripts both of the regulatory genes and the structural genes that related to anthocyanin biosynthesis, while upregulated both of those genes that related to PA biosynthesis. Given there is a significant negative relationship between the levels of anthocyanins and PAs during Lycium fruit ripening, NO not only inhibited anthocyanin de novo biosynthesis but redirected the flavonoid biosynthetic pathway from anthocyanins to PA production. Two types of LrMYB transcription factors of opposite nature, namely anthocyanin-specific and PA-specific, which belong to the R2R3-MYB subfamily and 1R-MYB subfamily, respectively, were identified from L. ruthenicum fruits. It was further found that NO acts by antagonizing the ABA signaling, a phytohormone we have previously shown playing a positive role in Lycium fruit coloration. Our results provided particularly novel information about NO–ABA–anthocyanin interplay during Lycium fruit development and ripening, which may fill a gap between the developmental cues and the transcriptional regulation of anthocyanin biosynthesis.

MdMYB6 regulates anthocyanin formation in apple both through direct inhibition of the biosynthesis pathway and through substrate removal

Anthocyanin biosynthesis and sugar metabolism are important processes during plant growth, but the molecular interactions underlying these pathways are still unclear. In this work, we analyzed the anthocyanin and soluble sugar contents, as well as the transcript levels of transcription factors that are known to be related to the biosynthesis of anthocyanin in ‘Hongcui 1’ apple flesh during fruit development. Overexpression of MdMYB6 in red-fleshed calli was found to reduce anthocyanin content and result in downregulated expression of the MdANS and MdGSTF12 proteins. Yeast one-hybrid and electrophoretic mobility shift analyses showed that MdMYB6 could directly bind to the promoters of MdANS and MdGSTF12, indicating that MdMYB6 could inhibit anthocyanin biosynthesis by regulating MdANS and MdGSTF12. Overexpression of MdTMT1 in the Arabidopsis tmt1 mutant restored the glucose and fructose contents to the wild-type levels, while overexpression of MdTMT1 in red-fleshed calli increased the contents of glucose and fructose but reduced the contents of UDP-glucose, UDP-galactose, and anthocyanin. Using a GUS reporter system, yeast one-hybrid, chromatin immunoprecipitation-PCR and electrophoretic mobility shift analyses, we found that MdMYB6 could bind to the promoter of MdTMT1, resulting in increased promoter activity. Overexpression of MdMYB6 in calli overexpressing MdTMT1 increased the expression of MdTMT1, which led to reduced contents of UDP-glucose and UDP-galactose and decreased anthocyanin content compared to those of the calli that overexpressed MdTMT1. This finding suggested that MdMYB6 could also inhibit anthocyanin biosynthesis by regulating MdTMT1 to decrease the contents of UDP-glucose and UDP-galactose. Taken together, these results showed that MdMYB6 and MdTMT1 play key roles in both anthocyanin biosynthesis and sugar transport.

Transposon-induced methylation of the RsMYB1 promoter disturbs anthocyanin accumulation in red-fleshed radish.

Red-fleshed radish (Raphanus sativus L.) is a unique cultivar whose taproot is rich in anthocyanins beneficial to human health. However, the frequent occurrence of white-fleshed mutants affects the purity of commercially produced radish and the underlying mechanism has puzzled breeders for many years. In this study, we combined quantitative trait location by genome resequencing and transcriptome analyses to identify a candidate gene (RsMYB1) responsible for anthocyanin accumulation in red-fleshed radish. However, no sequence variation was found in the coding and regulatory regions of the RsMYB1 genes of red-fleshed (MTH01) and white-fleshed (JC01) lines, and a 7372 bp CACTA transposon in the RsMYB1 promoter region occurred in both lines. A subsequent analysis suggested that the white-fleshed mutant was the result of altered DNA methylation in the RsMYB1 promoter. This heritable epigenetic change was due to the hypermethylated CACTA transposon, which induced the spreading of DNA methylation to the promoter region of RsMYB1. Thus, RsMYB1 expression was considerably down-regulated, which inhibited anthocyanin biosynthesis in the white-fleshed mutant. An examination of transgenic radish calli and the results of a virus-induced gene silencing experiment confirmed that RsMYB1 is responsible for anthocyanin accumulation. Moreover, the mutant phenotype was partially eliminated by treatment with a demethylating agent. This study explains the molecular mechanism regulating the appearance of white-fleshed mutants of red-fleshed radish.