Negative Regulator Of Anthocyanin Biosynthesis Research Articles

MsMYB62-like as a negative regulator of anthocyanin biosynthesis in Malus spectabilis

ABSTRACT Crabapple is a valuable tree species in gardens due to its captivating array of flower and leaf colors, rendering it a favored choice in landscaping. The economic and ornamental values of Malus crabapple are closely associated with the biosynthesis of anthocyanin, a pigment responsible for its vibrant hues. The intricate regulation of anthocyanin biosynthesis involves the concerted activity of various genes. However, the specific mechanism governing this process in crabapple warrants in-depth exploration. In this study, we explored the inhibitory role of MsMYB62-like in anthocyanin biosynthesis. We identified MsDFR and MsANS as two downstream target genes of MsMYB62-like. These genes encode enzymes integral to the anthocyanin biosynthetic pathway. The findings demonstrate that MsMYB62-like directly binds to the promoters of MsDFR and MsANS, resulting in the downregulation of their expression levels. Additionally, our observations indicate that the plant hormone cytokinins exert a suppressive effect on the expression levels of MsMYB62-like, while concurrently upregulating MsDFR and MsANS. This study reveals that the MsMYB62-like-MsDFR/MsANS module plays an important role in governing anthocyanin levels in Malus crabapple. Notably, the regulatory interplay is modulated by the plant hormone cytokinins.

CstPIF4 Integrates Temperature andCircadian Signals andInteracts with CstMYB16 to Repress Anthocyanins inCrocus.

Crocus sativus has emerged as an important crop because it is the only commercial source of saffron that contains unique apocarotenoids. Saffron is composed of dried stigmas of Crocus flower and constitutes the most priced spice of the world. Crocus floral organs are dominated by different classes of metabolites. While stigmas are characterized by the presence of apocarotenoids, tepals are rich in flavonoids and anthocyanins. Therefore, an intricate regulatory network might play a role in allowing different compounds to dominate in different organs. Work so far done on Crocus is focussed on apocarotenoid metabolism and its regulation. There are no reports describing the regulation of flavonoids and anthocyanins in Crocus tepals. In this context, we identified an R2R3 transcription factor, CstMYB16, which resembles subgroup 4 (SG4) repressors of Arabidopsis. CstMYB16 is nuclear localized and acts as a repressor. Overexpression of CstMYB16 in Crocus downregulated anthocyanin biosynthesis. The C2/EAR motif was responsible for the repressor activity of CstMYB16. CstMYB16 binds to the promoter of the anthocyanin biosynthetic pathway gene (LDOX) and reduces its expression. CstMYB16 also physically interacts with CstPIF4, which in turn is regulated by temperature and circadian clock. Thus, CstPIF4 integrates these signals and forms a repressor complex with CstMYB16, which is involved in the negative regulation of anthocyanin biosynthesis in Crocus. Independent of CstPIF4, CstMYB16 also represses CstPAP1 expression, which is a component of the MYB-bHLH-WD40 (MBW) complex and positively controls anthocyanin biosynthesis. This is the first report on identifying and describing regulators of anthocyanin biosynthesis in Crocus.

E3 ubiquitin ligases SINA4 and SINA11 regulate anthocyanin biosynthesis by targeting the IAA29-ARF5-1-ERF3 module in apple.

Auxin/indole-3-acetic acid (AUX/IAA) and auxin response factor (ARF) proteins are important components of the auxin signalling pathway, but their ubiquitination modification and the mechanism of auxin-mediated anthocyanin biosynthesis remain elusive. Here, the ARF MdARF5-1 was identified as a negative regulator of anthocyanin biosynthesis in apple, and it integrates auxin and ethylene signals by inhibiting the expression of the ethylene response factor MdERF3. The auxin repressor MdIAA29 decreased the inhibitory effect of MdARF5-1 on anthocyanin biosynthesis by attenuating the transcriptional inhibition of MdERF3 by MdARF5-1. In addition, the E3 ubiquitin ligases MdSINA4 and MdSINA11 played negative and positive regulatory roles in anthocyanin biosynthesis by targeting MdIAA29 and MdARF5-1 for ubiquitination degradation, respectively. MdSINA4 destabilized MdSINA11 to regulate anthocyanin accumulation in response to auxin signalling. In sum, our data revealed the crosstalk between auxin and ethylene signals mediated by the IAA29-ARF5-1-ERF3 module and provide new insights into the ubiquitination modification of the auxin signalling pathway.

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.

PlgMYBR1, an R2R3-MYB transcription factor, plays as a negative regulator of anthocyanin biosynthesis in Platycodon grandiflorus.

The online version contains supplementary material available at 10.1007/s13205-023-03490-6.

The R3-Type MYB Transcription Factor BrMYBL2.1 Negatively Regulates Anthocyanin Biosynthesis in Chinese Cabbage (Brassica rapa L.) by Repressing MYB–bHLH–WD40 Complex Activity

Chinese cabbage (Brassica rapa L.) leaves are purple in color due to anthocyanin accumulation and have nutritional and aesthetic value, as well as antioxidant properties. Here, we identified the R3 MYB transcription factor BrMYBL2.1 as a key negative regulator of anthocyanin biosynthesis. A Chinese cabbage cultivar with green leaves harbored a functional BrMYBL2.1 protein, designated BrMYBL2.1-G, with transcriptional repressor activity of anthocyanin biosynthetic genes. By contrast, BrMYBL2.1 from a Chinese cabbage cultivar with purple leaves carried a poly(A) insertion in the third exon of the gene, resulting in the insertion of multiple lysine residues in the predicted protein, designated BrMYBL2.1-P. Although both BrMYBL2.1 variants localized to the nucleus, only BrMYBL2.1-G interacted with its cognate partner BrTT8. Transient infiltration assays in tobacco leaves revealed that BrMYBL2.1-G, but not BrMYBL2.1-P, actively represses pigment accumulation by inhibiting the transcription of anthocyanin biosynthetic genes. Transient promoter activation assay in Arabidopsis protoplasts verified that BrMYBL2.1-G, but not BrMYBL2.1-P, can repress transcriptional activation of BrCHS and BrDFR, which was activated by co-expression with BrPAP1 and BrTT8. We determined that BrMYBL2.1-P may be more prone to degradation than BrMYBL2.1-G via ubiquitination. Taken together, these results demonstrate that BrMYBL2.1-G blocks the activity of the MBW complex and thus represses anthocyanin biosynthesis, whereas the variant BrMYBL2.1-P from purple Chinese cabbage cannot, thus leading to higher anthocyanin accumulation.

CRISPR/Cas9-mediated mutagenesis of VvbZIP36 promotes anthocyanin accumulation in grapevine (Vitis vinifera).

Anthocyanins are plant secondary metabolites that have a variety of biological functions, including pigmentation. The accumulation of anthocyanins is regulated by both transcriptional activators and repressors. Studies have shown that bZIP family members act primarily as positive regulators of anthocyanin biosynthesis, but there are few reports of negative regulation. Here, we report that a grapevine (Vitis vinifera) bZIP gene from group K, VvbZIP36, acts as a negative regulator of anthocyanin biosynthesis. Knocking out one allele of VvbZIP36 in grapevine with CRISPR/Cas9 promoted anthocyanin accumulation. Correlation analysis of transcriptome and metabolome data showed that a range of anthocyanin biosynthesis genes were activated in VvbZIP36 mutant plants relative to the wild type, resulting in the accumulation of related metabolites, including naringenin chalcone, naringenin, dihydroflavonols, and cyanidin-3-O-glucoside. The synthesis of stilbenes (α-viniferin), lignans, and some flavonols (including quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, and kaempferol-7-O-rhamnoside) was significantly inhibited, and several genes linked to their metabolism were downregulated in the mutant plants. In summary, our results demonstrate that VvbZIP36 is a negative regulator of anthocyanin biosynthesis that plays a role in balancing the synthesis of stilbenes (α-viniferin), lignans, flavonols, and anthocyanins.

The Basic Helix-Loop-Helix Transcription Factor SmbHLH1 Represses Anthocyanin Biosynthesis in Eggplant.

Many basic helix-loop-helix transcription factors (TFs) have been reported to promote anthocyanin biosynthesis in numerous plant species, but little is known about bHLH TFs that inhibit anthocyanin accumulation. In this study, SmbHLH1 from Solanum melongena was identified as a negative regulator of anthocyanin biosynthesis. However, SmbHLH1 showed high identity with SmTT8, which acts as a SmMYB113-dependent positive regulator of anthocyanin-biosynthesis in plants. Overexpression of SmbHLH1 in eggplant caused a dramatic decrease in anthocyanin accumulation. Only the amino acid sequences at the N and C termini of SmbHLH1 differed from the SmTT8 sequence. Expression analysis revealed that the expression pattern of SmbHLH1 was opposite to that of anthocyanin accumulation. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SmbHLH1 could not interact with SmMYB113. Dual-luciferase assay demonstrated that SmbHLH1 directly repressed the expression of SmDFR and SmANS. Our results demonstrate that the biological function of bHLHs in anthocyanin biosynthesis may have evolved and provide new insight into the molecular functions of orthologous genes from different plant species.

The transcription factor AtGLK1 acts upstream of MYBL2 to genetically regulate sucrose-induced anthocyanin biosynthesis in Arabidopsis

BackgroundThe regulation of anthocyanin biosynthesis by various factors including sugars, light and abiotic stresses is mediated by numerous regulatory factors acting at the transcriptional level. Here experimental evidence was provided in order to demonstrate that the nuclear GARP transcription factor AtGLK1 plays an important role in regulating sucrose-induced anthocyanin biosynthesis in Arabidopsis.ResultsThe results obtained using real-time quantitative PCR and GUS staining assays revealed that AtGLK1 was mainly expressed in the green tissues of Arabidopsis seedlings and could be induced by sucrose. The loss-of-function glk1 glk2 double mutant has lower anthocyanin levels than the glk2 single mutant, although it has been determined that loss of AtGLK1 alone does not affect anthocyanin accumulation. Overexpression of AtGLK1 enhances the accumulation of anthocyanin in transgenic Arabidopsis seedlings accompanied by increased expression of anthocyanin biosynthetic and regulatory genes. Moreover, we found that AtGLK1 also participates in plastid-signaling mediated anthocyanin accumulations. Genetic, physiological, and molecular biological approaches demonstrated that AtGLK1 acts upstream of MYBL2, which is a key negative regulator of anthocyanin biosynthesis, to genetically regulate sucrose-induced anthocyanin biosynthesis.ConclusionOur results indicated that AtGLK1 positively regulates sucrose-induced anthocyanin biosynthesis in Arabidopsis via MYBL2.

A Novel R2R3-MYB Transcription Factor PqMYB4 Inhibited Anthocyanin Biosynthesis in Paeonia qiui.

Paeonia qiui is a wild tree peony native to China. Its leaves show a clear purple-red color from the germination to the flowering stage, and it has high leaf-viewing value. A MYB transcription factor gene, designated as PqMYB4, was isolated from leaves of P. qiui based on transcriptome datas. The full-length cDNA of PqMYB4 was 693 bp, encoding 230 amino acids. Sequence alignment and phylogenetic analysis revealed that PqMYB4 was a R2R3-MYB transcription factor clustered with AtMYB4 in Arabidopsis thaliana. Moreover, it contained a C1 motif, an EAR repression motif and a TLLLFR motif in the C-terminal domains, which were unique in transcription repression MYB. Subcellular location analysis showed that PqMYB4 was located in the cell nucleus. PqMYB4 was highly expressed in the late stage of leaf development, and was negatively correlated with the anthocyanin content. The petiole of wild-type Arabidopsis seedlings was deeper in color than the transgenic lines of PqMYB4 and showed a little purple-red color. The seed coat color of Arabidopsis seeds that overexpressed PqMYB4 gene was significantly lighter than that of wild-type seeds. In transgenic Arabidopsis, the expression level of AtCHS, AtCHI, AtDFR and AtANS were down-regulated significantly. These results showed that PqMYB4 was involved in the negative regulation of anthocyanin biosynthesis in tree peony leaves, which can control the anthocyanin pathway genes. Together, these findings provide a valuable resource with which to further study the regulatory mechanism of anthocyanin biosynthesis in the leaf of P. qiui. They also benefit the molecular breeding of tree peony cultivars with colored leaf.

MicroRNA858 negatively regulates anthocyanin biosynthesis by repressing AaMYBC1 expression in kiwifruit (Actinidia arguta)

The anthocyanin biosynthetic pathway regulated by exogenous and endogenous factors through sophisticated networks has been extensively studied in kiwifruit (Actinidia arguta). However, the role of micro RNAs (miRNAs) as regulatory factor in this process is largely unclear. Here, we demonstrate that miR858 is a negative regulator of anthocyanin biosynthesis by repressing the target gene AaMYBC1 in red-colored kiwifruit. Transient co-transformation in Nicotiana benthamiana confirmed that miR858 could target AaMYBC1, which was identified to be an R2R3-type tanscription factor (TF). Subcellular localization showed that AaMYBC1 was located in the nucleus, indicating AaMYBC1 protein could act as a transcriptional regulator in plant cells. Functional protein association network analysis and the yeast two hybrid (Y2H) assay revealed that AaMYBC1 and AabHLH42 interact with each other. Silencing of AaMYBC1 using the virus-induced gene silencing method in the core of A. arguta ‘HB’ (‘Hongbaoshixing’, a kind of red-fleshed A. arguta cultivar) fruits reduced the accumulation of anthocyanin and decreased the expression of late biosynthetic genes. miR858 overexpression played a stronger role than AaMYBC1 silencing in the inhibition of coloration. With overexpression of miR858, A. arguta did not present coloration, and anthocyanin was hardly detected. Together, these results clarify the negative regulatory role of miR858 in mediating anthocyanin biosynthesis and accumulation in A. arguta, providing novel insights into the molecular mechanism of anthocyanin biosynthesis.

QTL-Seq and Sequence Assembly Rapidly Mapped the Gene BrMYBL2.1 for the Purple Trait in Brassica rapa

Anthocyanins have strong antioxidant activity and are believed to be healthy for human beings. The Brassica rapa L. ssp. chinensis var. purpurea “Zicaitai” is rich in anthocyanins. We constructed an F2 population of Zicaitai and “Caixin” (Brassica rapa ssp. parachinensis) and it shows clear segregation of the purple phenotype (i.e., variation in anthocyanin enrichment). Here, quantitative trait locus (QTL)-Seq was performed with two sample groups from the F2 population: one exhibiting an intense purple phenotype and the other showed a completely green phenotype. The results showed that the QTL-Seq and linkage analysis located different major loci. This indicates that there are two major genetic factors that plays different roles in regulating anthocyanin enrichment in Zicaitai. This was further supported by the data simulation of an in silico F2 population that QTL-Seq and linkage analysis can locate different major loci. Furthermore, the draft genomes of the two parents (Zicaitai and Caixin) were assembled and utilized to search for mutations in candidate genes. A ~100-bp insertion was found in the third exon of gene BrMYBL2.1 in Zicaitai. BrMYBL2.1 is a negative regulator of anthocyanin biosynthesis, while BrEGL3.2—previously located by linkage mapping—is a positive regulator. For these populations with multiple genes contributing large effects to a trait, a strategy of low depth re-sequencing of F2 individuals followed by QTL-Seq analysis with the free combination of sample groups is proposed. Furthermore, draft-sequence assembly of parental genomes together with QTL mapping is suggested as an efficient means for fine-mapping genes rapidly in segregating populations.

CmMYB#7, an R3 MYB transcription factor, acts as a negative regulator of anthocyanin biosynthesis in chrysanthemum.

'Jimba', a well-known white flowered chrysanthemum cultivar, occasionally and spontaneously produces red colored petals under natural cultivation, but there is little information about the molecular regulatory mechanism underlying this process. We analysed the expression patterns of 91 MYB transcription factors in 'Jimba' and 'Turning red Jimba' and identified an R3 MYB, CmMYB#7, whose expression was significantly decreased in 'Turning red Jimba' compared with 'Jimba', and confirmed it is a passive repressor of anthocyanin biosynthesis. CmMYB#7 competed with CmMYB6, which together with CmbHLH2 is an essential component of the anthocyanin activation complex, for interaction with CmbHLH2 through the bHLH binding site in the R3 MYB domain. This reduced binding of the CmMYB6-CmbHLH2 complex and inhibited its ability to activate CmDFR and CmUFGT promoters. Moreover, using transient expression assays we demonstrated that changes in the expression of CmMYB#7 accounted for alterations in anthocyanin content. Taken together, our findings illustrate that CmMYB#7 is a negative regulator of anthocyanin biosynthesis in chrysanthemum.

MdMYBDL1 employed by MdHY5 increases anthocyanin accumulation via repression of MdMYB16/308 in apple

Light is an important environmental factor affecting plant growth and development. Additionally, HY5 is a central factor that coordinates light signal transduction and regulates the expression of flower color-related genes. However, there are few reports describing the co-regulation of apple fruit coloration by MdHY5 and MYB transcription factors. In this study, we detected a light-inducible gene, MdMYBDL1, which encodes a MYB-like domain and is homologous to AtMYBD in Arabidopsis thaliana. Moreover, we observed that MdHY5 binds to the G-box element of the MdMYBDL1 promoter to upregulate expression. The overexpression of MdMYBDL1 enhanced anthocyanin accumulation in apple calli and inhibited the expression of MdMYB16 and its homolog, MdMYB308. Furthermore, MdMYB16 can form a dimer with MdMYB308 and functions as a negative regulator of anthocyanin biosynthesis. Interestingly, MdMYB16 and MdMYB308 promoter activities were inhibited by MdMYBDL1 and MdHY5. These findings imply that MdHY5 responds to light signals and functions upstream of different types of MYB transcription factors, ultimately regulating anthocyanin accumulation in apples.

PbCOP1.1 Contributes to the Negative Regulation of Anthocyanin Biosynthesis in Pear.

The synthesis of anthocyanin in pear (Pyrus bretschneideri) fruit is regulated by light. However, little is known about the molecular mechanisms of pear fruit coloring mediated by upstream light-signaling regulators. Here, the photoresponse factors CONSTITUTIVE PHOTOMORPHOGENIC (COP) 1.1 and 1.2 were cloned from ‘Red Zaosu’ peel to study their functions in pear fruit coloring. The overexpression vectors pBI121-PbCOP1.1 and pBI121-PbCOP1.2 were constructed to analyze their effects on anthocyanin synthesis in pear fruit. A protein sequence alignment and phylogenetic tree analysis revealed that PbCOP1 proteins are highly homologous with those of other species. An analysis of tissue differential expression showed that the greatest expression levels of PbCOP1s occurred in the leaves. Their expression levels increased in the leaves during development, when the leaves changed from red to green. The overexpression of PbCOP1s in the peel resulted in reduced anthocyanin synthesis at the injection sites. A quantitative PCR analysis of the injection sites showed that PbCOP1.1 significantly inhibited the expression of the anthocyanin synthesis-related genes CHI, DFR, UFGT2, bHLH3, HY5 and GST. Based on the above results, we hypothesize that PbCOP1.1 is an anthocyanin synthetic inhibitory factor of pear coloration.

A R2R3-MYB Transcription Factor, VvMYBC2L2, Functions as a Transcriptional Repressor of Anthocyanin Biosynthesis in Grapevine (Vitis vinifera L.).

In grapevine, the MYB transcription factors play an important role in the flavonoid pathway. Here, a R2R3-MYB transcription factor, VvMYBC2L2, isolated from Vitis vinifera cultivar Yatomi Rose, may be involved in anthocyanin biosynthesis as a transcriptional repressor. VvMYBC2L2 was shown to be a nuclear protein. The gene was shown to be strongly expressed in root, flower and seed tissue, but weakly expressed during the fruit development in grapevine. Overexpressing the VvMYBC2L2 gene in tobacco resulted in a very marked decrease in petal anthocyanin concentration. Expression analysis of flavonoid biosynthesis structural genes revealed that chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductase (LAR) and UDP glucose flavonoid 3-O-glucosyl transferase (UFGT) were strongly down-regulated in the VvMYBC2L2-overexpressed tobacco. In addition, transcription of the regulatory genes AN1a and AN1b was completely suppressed in transgenic plants. These results suggested that VvMYBC2L2 plays a role as a negative regulator of anthocyanin biosynthesis.

Mitogen-activated protein kinase kinase 6 negatively regulates anthocyanin induction in Arabidopsis

ABSTRACTArabidopsis mitogen-activated protein (MAP) kinase kinase 6 (MKK6) has previously been shown to play important roles in plant immunity and cytokinesis. Here we report that it is also involved in the negative regulation of anthocyanin biosynthesis in Arabidopsis. Elevated anthocyanin levels are observed in mkk6 knock out mutant plants. RNA-sequencing analysis revealed that the expression level of MYB75 (Myb transcription factor 75), which encodes a key positive regulator of anthocyanin biosynthesis, is up-regulated in mkk6 mutant plants, suggesting that increased MYB75 expression contributes to the elevated anthocyanin levels. In addition, suppression of high sucrose-induced anthocyanin production by pathogen associated molecular pattern-triggered immunity is also dependent on MKK6, further supporting that MKK6 is a key negative regulator of anthocyanin biosynthesis in Arabidopsis.

The Arabidopsis Transcription Factor ANAC032 Represses Anthocyanin Biosynthesis in Response to High Sucrose and Oxidative and Abiotic Stresses.

Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous sucrose as well as high light (HL) stress. Using biochemical, molecular and transgenic approaches, we show that ANAC032 represses anthocyanin biosynthesis in response to sucrose treatment, HL and oxidative stress. ANAC032 was found to negatively affect anthocyanin accumulation and the expression of anthocyanin biosynthesis (DFR, ANS/LDOX) and positive regulatory (TT8) genes as demonstrated in overexpression line (35S:ANAC032) compared to wild-type under HL stress. The chimeric repressor line (35S:ANAC032-SRDX) exhibited the opposite expression patterns for these genes. The negative impact of ANAC032 on the expression of DFR, ANS/LDOX and TT8 was found to be correlated with the altered expression of negative regulators of anthocyanin biosynthesis, AtMYBL2 and SPL9. In addition to this, ANAC032 also repressed the MeJA- and ABA-induced anthocyanin biosynthesis. As a result, transgenic lines overexpressing ANAC032 (35S:ANAC032) produced drastically reduced levels of anthocyanin pigment compared to wild-type when challenged with salinity stress. However, transgenic chimeric repressor lines (35S:ANAC032-SRDX) exhibited the opposite phenotype. Our results suggest that ANAC032 functions as a negative regulator of anthocyanin biosynthesis in Arabidopsis thaliana during stress conditions.

Repression of MYBL2 by Both microRNA858a and HY5 Leads to the Activation of Anthocyanin Biosynthetic Pathway in Arabidopsis

Extensive studies in various plants show that the anthocyanin biosynthetic process is affected by environmental factors and regulated by many transcription factors through sophisticated regulatory networks. However, it remains largely unclear about the roles of microRNA in this process. Here, we demonstrate that miR858a is a positive regulator of anthocyanin biosynthesis in Arabidopsis seedlings. Overexpression of miR858a enhances the accumulation of anthocyanins, whereas the reduced miR858a activity results in low levels of anthocyanins in STTM858 transgenic plants. We found that miR858a inhibits the expression of MYBL2, a key negative regulator of anthocyanin biosynthesis, by translational repression. In addition, ELONGATED HYPOCOTYL 5 (HY5) was shown to directly bind the MYBL2 promoter and represses its expression via specific histone modifications. Interestingly, we found that miR858a exhibits light-responsive expression in an HY5-dependent manner. Together, these results delineate the HY5-MIR858a-MYBL2 loop as a cellular mechanism for modulating anthocyanin biosynthesis, suggesting that integration of transcriptional and posttranscriptional regulation is critical for governing proper anthocyanin accumulation in response to light and other environmental factors.

Characterization of an activation-tagged mutant uncovers a role of GLABRA2 in anthocyanin biosynthesis in Arabidopsis.

In Arabidopsis, anthocyanin biosynthesis is controlled by a MYB-bHLH-WD40 (MBW) transcriptional activator complex. The MBW complex activates the transcription of late biosynthesis genes in the flavonoid pathway, leading to the production of anthocyanins. A similar MBW complex regulates epidermal cell fate by activating the transcription of GLABRA2 (GL2), a homeodomain transcription factor required for trichome formation in shoots and non-hair cell formation in roots. Here we provide experimental evidence to show that GL2 also plays a role in regulating anthocyanin biosynthesis in Arabidopsis. From an activation-tagged mutagenized population of Arabidopsis plants, we isolated a dominant, gain-of-function mutant with reduced anthocyanins. Molecular cloning revealed that this phenotype is caused by an elevated expression of GL2, thus the mutant was named gl2-1D. Consistent with the view that GL2 acts as a negative regulator of anthocyanin biosynthesis, gl2-1D seedlings accumulated less whereas gl2-3 seedlings accumulated more anthocyanins in response to sucrose. Gene expression analysis indicated that expression of late, but not early, biosynthesis genes in the flavonoid pathway was dramatically reduced in gl2-1D but elevated in gl2-3 mutants. Further analysis showed that expression of some MBW component genes involved in the regulation of late biosynthesis genes was reduced in gl2-1D but elevated in gl2-3 mutants, and chromatin immunoprecipitation results indicated that some MBW component genes are targets of GL2. We also showed that GL2 functions as a transcriptional repressor. Taken together, these results indicate that GL2 negatively regulates anthocyanin biosynthesis in Arabidopsis by directly repressing the expression of some MBW component genes.