The R2R3 MYB Ruby1 is activated by two cold responsive ethylene response factors, via the retrotransposon in its promoter, to positively regulate anthocyanin biosynthesis in citrus.

Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in animal and human nutrition. Low temperature (LT) promotes anthocyanin biosynthesis in many species including blood orange. A retrotransposon in the promoter of Ruby1, which encodes an R2R3 MYB transcription factor, controls cold-induced anthocyanin accumulation in blood orange flesh. However, the specific mechanism remains unclear. In this study, we characterized two LT-induced ETHYLENE RESPONSE FACTORS (CsERF054 and CsERF061). Both CsERF054 and CsERF061 can activate the expression of CsRuby1 by directly binding to a DRE/CRT cis-element within the retrotransposon in the promoter of CsRuby1, thereby positively regulating anthocyanin biosynthesis. Further investigation indicated that CsERF061 also forms a protein complex with CsRuby1 to co-activate the expression of anthocyanin biosynthetic genes, providing a dual mechanism for the upregulation of the anthocyanin pathway. These results provide insights into how LT mediates anthocyanin biosynthesis and increase the understanding of the regulatory network of anthocyanin biosynthesis in blood orange.

The introgression of BjMYB113 from Brassica juncea leads to purple leaf trait in Brassica napus.

The purple leaves of Brassica napus are abundant in anthocyanins, which are renowned for their role in conferring distinct colors, stress tolerance, and health benefits, however the genetic basis of this trait in B. napus remains largely unelucidated. Herein, the purple leaf B. napus (PL) exhibited purple pigments in the upper epidermis and a substantial increase in anthocyanin accumulation, particularly of cyanidin, compared to green leaf B. napus (GL). The genetic control of the purple leaf trait was attributed to a semi-dominant gene, pl, which was mapped to the end of chromosome A03. However, sequencing of the fragments amplified by the markers linked to pl indicated that they were all mapped to chromosome B05 from B. juncea. Within this B05 chromosomal segment, the BjMYB113 gene-specific marker showed perfect co-segregation with the purple leaf trait in the F2 population, suggesting that the BjMYB113 introgression from B. juncea was the candidate gene for the purple leaf trait in B. napus. To further verify the function of candidate gene, CRISPR/Cas9 was performed to knock out the BjMYB113 gene in PL. The three myb113 mutants exhibited evident green leaf phenotype, absence of purple pigments in the adaxial epidermis, and a significantly reduced accumulation of anthocyanin compared to PL. Additionally, the genes involved in positive regulatory (TT8), late anthocyanin biosynthesis (DFR, ANS, UFGT), as well as transport genes (TT19) were significantly suppressed in the myb113 mutants, further confirming that BjMYB113 was response for the anthocyanin accumulation in purple leaf B. napus. This study contributes to an advanced understanding of the regulation mechanism of anthocyanin accumulation in B. napus.

Physiological and molecular responses of 'Hamlin' sweet orange trees expressing the VvmybA1 gene under cold stress conditions.

MAIN CONCLUSION: Overexpression of VvmybA1 transcription factor in 'Hamlin' citrus enhances cold tolerance by increasing anthocyanin accumulation. This results in improved ROS scavenging, altered gene expression, and stomatal regulation, highlighting anthocyanins' essential role in citrus cold acclimation. Cold stress is a significant threat to citrus cultivation, impacting tree health and productivity. Anthocyanins are known for their role as pigments and have emerged as key mediators of plant defense mechanisms against environmental stressors. This study investigated the potential of anthocyanin overexpression regulated by grape (Vitis vinifera) VvmybA1 transcription factor to enhance cold stress tolerance in citrus trees. Transgenic 'Hamlin' citrus trees overexpressing VvmybA1 were exposed to a 30-day cold stress period at 4 °C along with the control wild-type trees. Our findings reveal that anthocyanin accumulation significantly influences chlorophyll content and their fluorescence parameters, affecting leaf responses to cold stress. Additionally, we recorded enhanced ROS scavenging capacity and distinct expression patterns of key transcription factors and antioxidant-related genes in the transgenic leaves. Furthermore, VvmybA1 overexpression affected stomatal aperture regulation by moderating ABA biosynthesis, resulting in differential responses in a stomatal opening between transgenic and wild-type trees under cold stress. Transgenic trees exhibited reduced hydrogen peroxide levels, enhanced flavonoids, radical scavenging activity, and altered phytohormonal profiles. These findings highlighted the role of VvmybA1-mediated anthocyanin accumulation in enhancing cold tolerance. The current study also underlines the potential of anthocyanin overexpression as a critical regulator of the cold acclimation process by scavenging ROS in plant tissues.

Overexpression of oilseed rape trehalose-6-phosphate synthesis gene BnaC02.TPS8 confers sensitivity to low nitrogen and high sucrose-induced anthocyanin accumulation in Arabidopsis.

MAIN CONCLUSION: Overexpression of BnaC02.TPS8 increased low N and high sucrose-induced anthocyanin accumulation. Anthocyanin plays a crucial role in safeguarding photosynthetic tissues against high light, UV radiation, and oxidative stress. Their accumulation is triggered by low nitrogen (N) stress and elevated sucrose levels in Arabidopsis. Trehalose-6-phosphate (T6P) serves as a pivotal signaling molecule, sensing sucrose availability, and carbon (C) metabolism. However, the mechanisms governing the regulation of T6P synthase (TPS) genes responsible for anthocyanin accumulation under conditions of low N and high sucrose remain elusive. In a previous study, we demonstrated the positive impact of a cytoplasm-localized class II TPS protein 'BnaC02.TPS8' on photosynthesis and seed yield improvement in Brassica napus. The present research delves into the biological role of BnaC02.TPS8 in response to low N and high sucrose. Ectopic overexpression of BnaC02.TPS8 in Arabidopsis seedlings resulted in elevated shoot T6P levels under N-sufficient conditions, as well as an increased carbon-to-nitrogen (C/N) ratio, sucrose accumulation, and starch storage under low N conditions. Overexpression of BnaC02.TPS8 in Arabidopsis heightened sensitivity to low N stress and high sucrose levels, accompanied by increased anthocyanin accumulation and upregulation of genes involved in flavonoid biosynthesis and regulation. Metabolic profiling revealed increased levels of intermediate products of carbon metabolism, as well as anthocyanin and flavonoid derivatives in BnaC02.TPS8-overexpressing Arabidopsis plants under low N conditions. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses demonstrated that BnaC02.TPS8 interacts with both BnaC08.TPS9 and BnaA01.TPS10. These findings contribute to our understanding of how TPS8-mediated anthocyanin accumulation is modulated under low N and high sucrose conditions.

A novel R2R3-MYB transcription factor PbMYB1L of Pyrus bretschneideri regulates cold tolerance and anthocyanin accumulation.

KEY MESSAGE: PbMYB1L enhances the cold tolerance and anthocyanin accumulation of transgenic Arabidopsis by regulating the expression of genes related to the cold-responsive genes pathway and anthocyanin synthesis pathway. MYB transcription factors (TFs) have been demonstrated to play diverse roles in plant growth and development. In the present study, we identified a novel R2R3-MYB transcription factor, PbMYB1L, from the peel of 'Red Zaosu' pear (Pyrus bretschneideri), which was induced by cold stress and acted as a positive regulator in anthocyanin biosynthesis. Notably, the transgenic Arabidopsis lines exhibited enhanced tolerance to cold stress. Compared to the Arabidopsis wild-type plants, the transgenic lines displayed longer primary roots and reduced reactive oxygen species (ROS) levels including O2-, hydrogen peroxide (H2O2), and malondialdehyde (MDA). Furthermore, significant upregulation of key cold-responsive genes AtCBF1, AtCBF2, AtCBF3, AtCBF4, and AtKIN1 was observed in the transgenic plants under cold stress conditions compared to wild type. Arabidopsis plants overexpressing PbMYB1L had significant anthocyanin accumulation in leaves after cold treatment with quantitative results indicating higher expression of anthocyanin structural genes compared to wild type. These findings suggest that PbMYB1L not only plays a vital role in conferring cold tolerance but also acts as a crucial regulator of anthocyanin biosynthesis.

Transcriptome Analysis Reveals Coexpression Networks and Hub Genes Involved in Papillae Development in Lilium auratum.

Lilium is a genus of important ornamental plants with many colouring pattern variations. Lilium auratum is the parent of Oriental hybrid lilies. A typical feature of L. auratum is the presence of red-orange special raised spots named papillae on the interior tepals. Unlike the usual raised spots, the papillae are slightly rounded or connected into sheets and usually have hairy tips. To elucidate the potential genes regulating papillae development in L. auratum, we performed high-throughput sequencing of its tepals at different stages. Genes involved in the flavonoid biosynthesis pathway were significantly enriched during the colouration of the papillae, and CHS, F3H, F3'H, FLS, DFR, ANS, and UFGT were significantly upregulated. To identify the key genes involved in the papillae development of L. auratum, we performed weighted gene coexpression network analysis (WGCNA) and further analysed four modules. In total, 51, 24, 1, and 6 hub genes were identified in four WGCNA modules, MEbrown, MEyellow, MEpurple, and MEred, respectively. Then, the coexpression networks were constructed, and important genes involved in trichome development and coexpressed with anthocyanin biosynthesis genes, such as TT8, TTG1, and GEM, were identified. These results indicated that the papillae are essentially trichomes that accumulate anthocyanins. Finally, we randomly selected 12 hub genes for qRT-PCR analysis to verify the accuracy of our RNA-Seq analysis. Our results provide new insights into the papillae development in L. auratum flowers.

UVR8-TCP4-LOX2 module regulates UV-B tolerance in Arabidopsis.

The phytohormone jasmonate (JA) coordinates stress and growth responses to increase plant survival in unfavorable environments. Although JA can enhance plant UV-B stress tolerance, the mechanisms underlying the interaction of UV-B and JA in this response remain unknown. In this study, we demonstrate that the UV RESISTANCE LOCUS 8 - TEOSINTE BRANCHED1, Cycloidea and PCF 4 - LIPOXYGENASE2 (UVR8-TCP4-LOX2) module regulates UV-B tolerance dependent on JA signaling pathway in Arabidopsis thaliana. We show that the nucleus-localized UVR8 physically interacts with TCP4 to increase the DNA-binding activity of TCP4 and upregulate the JA biosynthesis gene LOX2. Furthermore, UVR8 activates the expression of LOX2 in a TCP4-dependent manner. Our genetic analysis also provides evidence that TCP4 acts downstream of UVR8 and upstream of LOX2 to mediate plant responses to UV-B stress. Our results illustrate that the UV-B-dependent interaction of UVR8 and TCP4 serves as an important UVR8-TCP4-LOX2 module, which integrates UV-B radiation and JA signaling and represents a new UVR8 signaling mechanism in plants.

The Synergistic Effects of Environmental and Genetic Factors on the Regulation of Anthocyanin Accumulation in Plant Tissues.

Anthocyanin accumulation is responsible for the coloration of apple fruit, and their accumulation depends on the expression of anthocyanin biosynthesis-related genes. Light is an environmental stimulus that induces fruit color by regulating genes involved in the anthocyanin biosynthesis pathway. In this study, the roles of light and genetic factors on fruit coloration and anthocyanin accumulation in apple fruit were investigated. Three genes in the anthocyanin biosynthesis pathway, MdCHS, MdANS, and MdUFGT1, were synthesized and cloned into a viral-based expression vector system for transient expression in 'Ruby S' apple fruits. Apple fruits were agroinfiltrated with expression vectors harboring MdCHS, MdANS, and MdUFGT1. Agroinfiltrated apple fruits were then either kept in the dark (bagged fruits) or exposed to light (exposed fruits). The agroinfiltrated fruits showed significantly different coloration patterns, transcript expression levels, and anthocyanin accumulation compared to the control fruits. Moreover, these parameters were higher in exposed fruits than in bagged fruits. For stable expression, MdCHS was introduced into a binary vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The ectopic overexpression of MdCHS in transgenic rice calli showed a high accumulation of anthocyanin content. Taken together, our findings suggest that light, together with the overexpression of anthocyanin biosynthesis genes, induced the coloration and accumulation of anthocyanin content in apple fruits by upregulating the expression of the genes involved in the anthocyanin biosynthesis pathway.

Genome-Wide Identification and Comparative Transcriptome Methods Reveal FaMDHAR50 Regulating Ascorbic Acid Regeneration and Quality Formation of Strawberry Fruits.

Ascorbic acid (AsA) is a crucial water-soluble antioxidant in strawberry fruit, but limited research is currently available on the identification and functional validation of key genes involved in AsA metabolism in strawberries. This study analyzed the FaMDHAR gene family identification, which includes 168 genes. Most of the products of these genes are predicted to exist in the chloroplast and cytoplasm. The promoter region is rich in cis-acting elements related to plant growth and development, stress and light response. Meanwhile, the key gene FaMDHAR50 that positively regulates AsA regeneration was identified through comparative transcriptome analysis of 'Benihoppe' strawberry (WT) and its natural mutant (MT) with high AsA content (83 mg/100 g FW). The transient overexpression experiment further showed that overexpression of FaMDHAR50 significantly enhanced the AsA content by 38% in strawberry fruit, with the upregulated expression of structural genes involved in AsA biosynthesis (FaGalUR and FaGalLDH) and recycling and degradation (FaAPX, FaAO and FaDHAR) compared with that of the control. Moreover, increased sugar (sucrose, glucose and fructose) contents and decreased firmness and citric acid contents were observed in the overexpressed fruit, which were accompanied by the upregulation of FaSNS, FaSPS, FaCEL1 and FaACL, as well as the downregulation of FaCS. Additionally, the content of pelargonidin 3-glucoside markedly decreased, while cyanidin chloride increased significantly. In summary, FaMDHAR50 is a key positive regulatory gene involved in AsA regeneration in strawberry fruit, which also plays an important role in the formation of fruit flavor, apperance and texture during strawberry fruit ripening.

Combination analysis of single-molecule long-read and Illumina sequencing provides insights into the anthocyanin accumulation mechanism in an ornamental grass, Pennisetum setaceum cv. Rubrum.

KEY MESSAGE: Combination analysis of single-molecule long-read and Illumina sequencing provide full-length transcriptome information and shed new light on the anthocyanin accumulation mechanism of Pennisetum setaceum cv. 'Rubrum'. Pennisetum setaceum cv. 'Rubrum' is an ornamental grass with purple leaves widely used in landscaping. However, the current next-generation sequencing (NGS) transcriptome information of this species is not satisfactory due to the difficulties in obtaining full-length transcripts. Furthermore, the molecular mechanisms of anthocyanin accumulation in P. setaceum have not been thoroughly studied. In this study, we used PacBio full-length transcriptome sequencing (SMRT) combined with NGS technology to build and improve the transcriptomic datasets and reveal the molecular mechanism of anthocyanin accumulation in P. setaceum cv. 'Rubrum'. Therefore, 280,413 full-length non-chimeric reads sequences were obtained using the SMRT technology. We obtained 97,450 high-quality non-redundant transcripts and identified 5352 alternative splicing events. In addition, 93,066 open reading frames (ORFs), including 57,457 full ORFs and 2910 long non-coding RNA (lncRNAs) were screened out. Furthermore, 10,795 differentially expressed genes were identified using NGS. We also explored key genes, synthesis pathways, and detected lncRNA involved in anthocyanin accumulation, providing new insights into anthocyanin accumulation in P. setaceum cv. 'Rubrum'. To our best knowledge, we provided the full-length transcriptome information of P. setaceum cv. 'Rubrum' for the first time. The results of this study will provide baseline information for gene function studies and pave the way for future P. setaceum cv. 'Rubrum' breeding projects.

Metabolome and transcriptome profiling unveil the mechanisms of light-induced anthocyanin synthesis in rabbiteye blueberry (vaccinium ashei: Reade).

BACKGROUND: Blueberry is one of the most important fruit crops worldwide. Anthocyanin is an important secondary metabolites that affects the appearance and nutritive quality of blueberries. However, few studies have focused on the molecular mechanism underlying anthocyanin accumulation induced by light intensity in blueberries. RESULTS: The metabolic analysis revealed that there were 134 significantly changed metabolites in the natural light compared to the control, and flavone, flavonol, and anthocyanins were the most significantly increased. Transcriptome analysis found 6 candidate genes for the anthocyanin synthesis pathway. Quantitative reverse transcription PCR (qRT-PCR) results confirmed changes in the expression levels of genes encoding metabolites involved in the flavonoid synthesis pathways. The flavonoid metabolic flux in the light intensity-treatment increased the accumulation of delphinidin-3-O-arabinoside compared to under the shading-treatment. Furthermore, we performed qRT-PCR analysis of anthocyanin biosynthesis genes and predicted that the gene of VcF3'5'H4 may be a candidate gene for anthocyanin accumulation and is highly expressed in light intensity-treated fruit. Through the co-expression analysis of transcription factors and anthocyanin synthesis pathway genes, we found that the VcbHLH004 gene may regulate VcF3'5'H4, and then we transformed VcbHLH004 heterologously into tomato to verify its function. CONCLUSION: These results provide novel insights into light intensity regulation of blueberry anthocyanin accumulation and represent a valuable data set to guide future functional studies and blueberry breeding.

Comparative Transcriptome Analysis between Two Potato Cultivars in Tuber Induction to Reveal Associated Genes with Anthocyanin Accumulation.

Anthocyanins are generally accumulated within a few layers, including the epidermal cells of leaves and stems in plants. Solanum tuberosum cv. 'Jayoung' (hereafter, JY) is known to accumulate anthocyanin both in inner tissues and skins. We discovered that anthocyanin accumulation in the inner tissues of JY was almost diminished (more than 95% was decreased) in tuber induction condition. To investigate the transcriptomic mechanism of anthocyanin accumulation in JY flesh, which can be modulated by growth condition, we performed mRNA sequencing with white-colored flesh tissue of Solanum tuberosum cv. 'Atlantic' (hereafter, 'Daeseo', DS) grown under canonical growth conditions, a JY flesh sample grown under canonical growth conditions, and a JY flesh sample grown under tuber induction conditions. We could identify 36 common DEGs (differentially expressed genes) in JY flesh from canonical growth conditions that showed JY-specifically increased or decreased expression level. These genes were enriched with flavonoid biosynthetic process terms in GO analysis, as well as gene set enrichment analysis (GSEA) analysis. Further in silico analysis on expression levels of anthocyanin biosynthetic genes including rate-limiting genes such as StCHS and StCHI followed by RT-PCR and qRT-PCR analysis showed a strong positive correlation with the observed phenotypes. Finally, we identified StWRKY44 from 36 common DEGs as a possible regulator of anthocyanin accumulation, which was further supported by network analysis. In conclusion, we identified StWRKY44 as a putative regulator of tuber-induction-dependent anthocyanin accumulation.

B-Box Transcription Factor FaBBX22 Promotes Light-Induced Anthocyanin Accumulation in Strawberry (Fragaria × ananassa).

B-box transcription factors (TFs) play a vital role in light-induced anthocyanin accumulation. Here, the FaBBX22 gene encoding 287 amino acids B-box TF was isolated from the cultivated strawberry variety 'Benihoppe' and characterized functionally. The expression analysis showed that FaBBX22 was expressed in the roots, stems, leaves, flowers and fruits, and its transcription level was upregulated under the red- or blue-light irradiation. FaBBX22 was localized in the nucleus and showed trans-acting activity in yeast cells. Ectopic overexpression of FaBBX22 in Arabidopsis enhanced the accumulation of anthocyanin. Additionally, we obtained transgenic strawberry calli that overexpressed the FaBBX22 gene, and strawberry calli coloration assays showed that FaBBX22 increased anthocyanin accumulation by upregulating the expression of anthocyanin biosynthetic genes (FaPAL, FaANS, FaF3'H, FaUFGT1) and transport gene FaRAP in a light-dependent manner. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays indicated that FaBBX22 interacted with FaHY5. Furthermore, mutation of the 70th Asp residue in FaBBX22 protein to an Ala residue disrupted the interaction between FaBBX22 and FaHY5. Further, a transient expression assay demonstrated that the co-expression of FaBBX22 and FaHY5 could strongly promote anthocyanin accumulation in strawberry fruits. Collectively, these results revealed the positive regulatory role of FaBBX22 in light-induced anthocyanin accumulation.

Bagging Strategy and Identification of Coloring Mode of 'Xinqihong' Pear.

'Xinqihong' is a recently selected and well-colored red pear (Pyrus bretschneideri Rehd.) cultivar that is popular in the marketplace owing to the bright red color and high quality of the fruit. The red pigmentation is strongly associated with the light signal. However, its responses to bagging treatment and to light exposure after shading are unknown. In this study, the fruit were treated with three types of fruit bags. 'Xinqihong' fruit colored rapidly in response to light stimulation. A white fruit bag was optimal for bagging of 'Xinqihong' fruit. To ensure satisfactory red pigmentation, the fruit required exposure to 30 days of light after bag removal. A transcriptome analysis was conducted to screen light-signal-related genes and identify their possible functions. PbCRY1 activated the promoter of PbHY5.2 and enhanced its expression. PbHY5.2 activated the promoter activity of PbUFGT and induced anthocyanin synthesis, and also showed self-activation characteristics. Both PbCRY2 and PbPHY1 induced anthocyanin accumulation. Thus, blue-light receptors played an important role in anthocyanin synthesis. This study provides a theoretical basis for the bagging cultivation of new varieties of 'Xinqihong', and lays a foundation for the study of the mechanisms of red pear fruit coloring in response to light signals.

Effects of ethylene on berry ripening and anthocyanin accumulation of 'Fujiminori' grape in protected cultivation.

BACKGROUND: Although the grape berries are deliberated as a non-climacteric fruit, ethylene seems to be involved in grape berry ripening. However, the precise role of ethylene in regulating the ripening of non-climacteric fruits is poorly understood. RESULTS: Exogenous ethephon (ETH) can stimulate the concentration of internal ethylene and accelerate the accumulation of anthocyanins in berries of 'Fujiminori', including malvidin-, delphinidin-, and petunidin-derivatives (3',4',5'-trihydroxylated anthocyanins) and cyanidin-derivatives (3',4'-dihydroxylated anthocyanins). The content of 3',4',5'-trihydroxylated anthocyanins was extremely higher than 3',4'-dihydroxylated anthocyanins, and ethylene did not affect the composition of anthocyanins in grape. Furthermore, we observed the expression of anthocyanin structural and regulatory genes as well as ethylene biosynthesis and response genes in response to ETH treatment. The anthocyanins accumulation is significantly associated with increased expression of anthocyanin structural (VvPAL, Vv4CH, VvCHS, VvCHI, VvF3H, and VvUFGT) and regulatory genes (VvMYBA1, VvMYBA2, and VvMYBA3), which persisted over the 12 days. In addition, exogenous ETH affected the endogenous ethylene biosynthesis (VvACO2 and VvACO4) and the downstream ethylene regulatory network (VvERS1, VvETR2, VvCTR1, and VvERF005). CONCLUSIONS: These findings bring new insights into the physiological and molecular function of ethylene during berry development and ripening in grapes. © 2021 Society of Chemical Industry.

Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit.

Light is indispensable for the accumulation of anthocyanin in the peel of red pear fruit (Pyrus pyrifolia Nakai). ELONGATED HYPOCOTYL 5 (HY5) is considered to be a critical regulator for induction of anthocyanin biosynthesis, but detailed characterization of its regulatory mechanism is needed. In this study, multiple genetic and biochemical approaches were applied to identify the roles of P. pyrifolia HY5 (PpHY5) and two B-box (BBX) proteins, PpBBX18 and PpBBX21, in the transcriptional regulation of PpMYB10. The functions of the two BBX proteins were analyzed in overexpression lines using pear calli-based approaches. On its own PpHY5 was unable to activate downstream genes. The two BBX proteins, PpBBX18 and PpBBX21, physically interacted with PpHY5 and antagonistically regulated anthocyanin biosynthesis in Arabidopsis and pear. PpBBX18 formed a heterodimer with PpHY5 via two B-box domains, in which PpHY5 bound to the G-box motif of PpMYB10 and PpBBX18 provided the trans-acting activity, thus inducing transcription of PpMYB10. PpBBX21 interacted with PpHY5 and PpBBX18 and hampered formation of the PpHY5-PpBBX18 active transcription activator complex, and subsequently repressed anthocyanin biosynthesis. The present results demonstrate the fine-tuned regulation of anthocyanin biosynthesis via transcriptional regulation of PpMYB10 by PpHY5-associated proteins and provide insights into light-induced anthocyanin biosynthesis.

Auxin regulates anthocyanin biosynthesis through the auxin repressor protein MdIAA26.

Auxin plays an important role in plant growth and development; for example, it regulates the elongation and division of plant cells, the formation of plantlet's geotropism and phototropism, and the growth of main lateral roots and hypocotyl. IAA gene is associated with auxin and can response to biotic and abiotic stress in plants. However, the regulatory effect of auxin on anthocyanin accumulation has been rarely reported. In this study, we show that auxin inhibites the accumulation of anthocyanin and decreases the expression of genes related to anthocyanin synthesis in calli, leaves, and seedlings of apple. The expression levels of MdIAA family genes were determined, and we found that MdIAA26 significantly responded to auxin, which also induced MdIAA26 degradation. Functional analysis of MdIAA26 showed that overexpressing MdIAA26 in apple calli and Arabidopsis could promote the accumulation of anthocyanin and up-regulate the genes related to anthocyanin synthesis. Furthermore, the MdIAA26-overexpressing Arabidopsis could counteract auxin-induced inhibition on anthocyanin accumulation, which indicates that auxin inhibits the accumulation of anthocyanin in apple by degrading MdIAA26 protein.

An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation.

MYB transcription factors (TFs) have been demonstrated to play diverse roles in plant growth and development through interaction with basic helix-loop-helix (bHLH) TFs. MdbHLH33, an apple bHLH TF, has been identified as a positive regulator in cold tolerance and anthocyanin accumulation by activating the expressions of MdCBF2 and MdDFR. In the present study, a MYB TF MdMYB308L was found to also positively regulate cold tolerance and anthocyanin accumulation in apple. We found that MdMYB308L interacted with MdbHLH33 and enhanced its binding to the promoters of MdCBF2 and MdDFR. In addition, an apple RING E3 ubiquitin ligase MYB30-INTERACTING E3 LIGASE 1 (MdMIEL1) was identified to be an MdMYB308L-interacting protein and promoted the ubiquitination degradation of MdMYB308L, thus negatively regulated cold tolerance and anthocyanin accumulation in apple. These results suggest that MdMYB308L acts as a positive regulator in cold tolerance and anthocyanin accumulation in apple by interacting with MdbHLH33 and undergoes MdMIEL1-mediated protein degradation. The dynamic change in MYB-bHLH protein complex seems to play a key role in the regulation of plant growth and development.

MiR399d and epigenetic modification comodulate anthocyanin accumulation in Malus leaves suffering from phosphorus deficiency.

Inorganic phosphorus (Pi) deficiency induces anthocyanin accumulation in the leaves of some plant species; however, the molecular mechanisms underlying this phenomenon have not been well characterized. Here, we showed that microRNA399d (miR399d), high-affinity Pi transporter McPHT1;4, and McMYB10 are strongly induced in Malus leaves suffering from Pi deficiency. By culturing explants of transiently transformed plants in MS medium under conditions of Pi sufficiency and Pi deficiency, miR399d and McPHT1;4 were shown to play essential roles in the response to Pi deficiency and to play positive roles in the regulation of anthocyanin biosynthesis. Silencing of McHDA6 expression and treatment with the inhibitor trichostatin A suggested that the low expression of McHDA6 simultaneously reduced the transcription of McMET1 and decreased the methylation level of the McMYB10 promoter; however, the expression of McMYB10 and anthocyanin content were increased. Bimolecular fluorescence complementation and yeast two-hybrid assays revealed that McHDA6 binds directly to McMET1 through its BAH2 and DNMT1-RFD domains. Based on the results of our study, we propose a mechanism for the molecular regulation of anthocyanin biosynthesis, namely, the miR399d and epigenetic modification comodulation model, to explain the phenomenon in which leaves turn red under conditions of Pi deficiency.

Dynamic regulation of anthocyanin biosynthesis at different light intensities by the BT2-TCP46-MYB1 module in apple.

Teosinte branched1/cycloidea/proliferating (TCP) transcription factors play a broad role in plant growth and development, but their involvement in the regulation of anthocyanin biosynthesis is currently unclear. In this study, anthocyanin biosynthesis induced by different light intensities in apple (Malus domestica) was found to be largely dependent on the functions of the MdMYB1 and MdTCP46 transcription factors. The expression of MdTCP46 was responsive to high light intensity, and under these conditions it promoted anthocyanin biosynthesis by direct interactions with MdMYB1 that enhanced the binding of the latter to its target genes. MdTCP46 also interacted with a bric-a-brac/tramtrack/broad (BTB) protein, MdBT2, that is responsive to high light intensity, which ubiquitinated MdTCP46 and mediated its degradation via the 26S proteasome pathway. Our results demonstrate that the dynamic regulatory module MdBT2-MdTCP46-MdMYB1 plays a key role in modulating anthocyanin biosynthesis at different light intensities in apple, and provides new insights into the post-transcriptional regulation of TCP proteins.