The effects of LsMYB3 disruption were tissue-specific and varied among genetic backgrounds, resulting in increased anthocyanin accumulation in wild lettuce spines and cultivated lettuce leaves, suggesting its involvement in the lettuce MBW regulatory network.
Abstract
Anthocyanins contribute to pigmentation, nutritional quality, and stress responses in plants. Although red spines are a characteristic trait of wild lettuce (Lactuca serriola), the genetic basis underlying spine pigmentation remains unclear. Here, we identified LsMYB3, a gene controlling spine color variation in wild lettuce. LsMYB3 encodes an R2R3-MYB transcription factor lacking a canonical repression motif but functioning as a negative regulator of anthocyanin biosynthesis. CRISPR/Cas9-mediated knockout of LsMYB3 enhanced anthocyanin accumulation, whereas overexpression suppressed pigmentation and converted red spines to green. Sequence analysis revealed that the natural Lsmyb3 allele carries a conserved Cys-to-Ser substitution within the R2 domain and a 1-bp deletion causing premature protein truncation, indicating loss of function. Yeast one-hybrid and dual-luciferase assays demonstrated that LsMYB3 directly represses LsDFR, a key anthocyanin biosynthetic gene. Notably, the effects of LsMYB3 disruption were tissue-specific and varied among genetic backgrounds, resulting in increased anthocyanin accumulation in wild lettuce spines and cultivated lettuce leaves. In addition, LsMYB3 physically interacted with the bHLH regulator RLL1, suggesting its involvement in the lettuce MBW regulatory network. Together, our findings identify LsMYB3 as a key negative regulator of anthocyanin biosynthesis and provide new insights into the evolution and diversification of pigmentation traits in lettuce.
This study uncovers a rare case in which a deletion of just two amino acids is sufficient to generate a potent dominant-negative regulator, designated SmMYB1alf-D, which enables reliable prediction of fruit color and provides a breeding strategy to precisely manipulate anthocyanin metabolism.
Yan Li, Yiwen Tian, Wanyue Li et al.· Plant Communications· 0 citations
LrMYB113 drives anthocyanin biosynthesis in Lycium ruthenicum by forming an MBW complex and directly activating LrDFR and LrANS promoters, providing a genetic target for enhancing flavonoid production. Lycium ruthenicum Murray (black goji berry), a Solanaceae medicinal plant, is valued for its high flavonoid content. However, the transcriptional regulation of flavonoid biosynthesis in L. ruthenicum remains unclear, hindering its pharmaceutical development. Here, we identified and characterized LrMYB113, an R2R3-MYB transcription factor, as a key regulator of anthocyanin biosynthesis in L. ruthenicum. Phylogenetic analysis grouped LrMYB113 into the anthocyanin-associated S6 subgroup of MYBs. Heterologous expression of LrMYB113 in tobacco induced pigment accumulation and upregulated anthocyanin pathway genes. LrMYB113 overexpression in L. ruthenicum hairy roots enhanced accumulation of four acylated anthocyanins and activated anthocyanin pathway genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed LrMYB113 interacts with bHLHs (LrJAF13/LrAN1b) and WD40 (LrAN11) to form an MBW complex. Promoter binding and transactivation assays demonstrated LrMYB113 directly binds to and activates LrDFR and LrANS promoters. Dual-luciferase assays showed LrMYB113 alone strongly activates LrDFR and LrANS promoters; MBW complexes enhanced activity compared to individual bHLH/WD40 but not to LrMYB113 alone. Our findings identify LrMYB113 as a critical regulator of anthocyanin biosynthesis in L. ruthenicum, shedding light on flavonoid molecular mechanisms and supporting genetic improvement for pharmaceutical use.
Tingting Li, Zihan Zhang, Jingjin Wang et al.· Plant Cell Reports· 0 citations
Anthocyanin biosynthesis is a key process determining flower color in lotus. In this study, 145 MYB transcription factors were identified from the lotus genome, of which 124 belong to the R2R3-MYB subfamily. Based on phylogenetic analysis and short-term light-induced transcriptome data, NnMYB38 and NnMYB114 were selected for further characterization. Expression of NnMYB38 and NnMYB114 was significantly higher in red-flowered lotus, with NnMYB38 showing a positive correlation with anthocyanin content. Transient overexpression in Nicotiana benthamiana revealed that NnMYB38 induced significant anthocyanin accumulation by upregulating multiple structural genes. In transgenic Arabidopsis, both MYB TFs promoted anthocyanin accumulation in mature seeds, and the seedlings of NnMYB114-OE line exhibited red pigmentation. Dual-luciferase assays confirmed that both transcription factors significantly activated the promoters of NnDFR and NnANS, indicating their crucial regulatory roles in anthocyanin biosynthesis. In this study, we screened and identified the regulatory functions of two MYB TFs in lotus anthocyanin biosynthesis, providing new insights into the molecular mechanisms underlying lotus flower coloration.
Lonicera japonica Thunb. is a traditional medicinal plant rich in bioactive flavonoids, but the transcriptional regulation of flavonoid biosynthesis remains unclear. In this study, we identified LjMYB106, an R2R3-MYB transcription factor associated with flavonoid accumulation, from floral developmental transcriptomes. Sequence and phylogenetic analyses showed that LjMYB106 contains conserved R2 and R3 MYB domains and is closely related to homologous MYB proteins. Heterologous overexpression of LjMYB106 in Arabidopsis thaliana and Nicotiana benthamiana increased total flavonoid and p-coumaric acid contents. Integrated transcriptomic and widely targeted metabolomic analyses in A. thaliana showed that LjMYB106 overexpression was associated with changes in phenylpropanoid and flavonoid biosynthetic pathways. qRT-PCR validation further showed that PAL, C4H, 4CL, CHS, DFR, and ANS homologs were up-regulated, whereas CHI, F3H, and FLS homologs were down-regulated in LjMYB106-overexpressing plants. Subcellular localization and yeast transactivation assays indicated that LjMYB106 is a nuclear protein with transcriptional activation activity. These results suggest that LjMYB106 is a candidate regulator of phenylpropanoid and flavonoid metabolism and provide a basis for further studies of MYB-mediated flavonoid regulation in L. japonica.
Jiangxin Yang, Jingjie Zhang, Xing-Yu Nie et al.· Frontiers in Plant Science· 0 citations
Apple rust, caused by the fungal pathogen Gymnosporangium yamadae, leads to substantial yield losses and significant economic damage. In the rust-resistant cultivar Malus ‘Profusion’, rust infection triggers anthocyanin synthesis at infection sites as a defense mechanism to restrict fungal proliferation. Although small noncoding RNAs (miRNAs) play important roles in regulating anthocyanin biosynthesis, their specific functions under rust stress remain poorly characterized. In this study, small RNA sequencing revealed that miR166a is a key rust-responsive regulator. Its direct targeting and negative regulation of MpATHB8 were confirmed through luciferase assays, GUS staining, and gene expression analyses. Functional validation via transient and stable transformation in Malus demonstrated that suppressing miR166a expression using short tandem target mimics or overexpressing MpATHB8 promoted anthocyanin accumulation and enhanced resistance to rust. In contrast, overexpressing miR166a or silencing MpATHB8 suppressed anthocyanin synthesis and increased susceptibility to the pathogen. Further evidence indicates that the MpATHB8 protein activates anthocyanin biosynthesis by binding to and inducing the promoter of MpMYB10b. These findings reveal a miR166a-MpATHB8-MpMYB10b regulatory module that enhances rust resistance through anthocyanin metabolism in M. ‘Profusion’. Our findings provide novel insights into the miRNA-mediated regulation of anthocyanin metabolism and facilitate the breeding of rust-resistant and anthocyanin-enriched Malus cultivars.
