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Anthocyanin regulatory and structural genes associated with violet flower color of Matthiola incana

Abstract

Main conclusion

MiMYB1 and MibHLH2 play key roles in anthocyanin biosynthesis in Matthiola incana flowers. We established a transient expression system using Turnip mosaic virus vector in M. incana.

Abstract

Garden stock (Matthiola incana (L.) R. Br.) is a popular flowering plant observed from winter to spring in Japan. Here we observed that anthocyanin accumulation in ‘Vintage Lavender’ increased with flower development, whereas flavonol accumulation remained constant throughout flower development. We obtained five transcription factor genes, MiMYB1, MibHLH1, MibHLH2, MiWDR1, and MiWDR2, from M. incana floral cDNA contigs. Yeast two-hybrid analyses revealed that MiMYB1 interacted with MibHLH1, MibHLH2, and MiWDR1, but MiWDR2 did not interact with any transcription factor. Expression levels of MiMYB1 and MibHLH2 increased in petals during floral bud development. Their expression profiles correlated well with the temporal profiles of MiF3ʹH, MiDFR, MiANS, and Mi3GT transcripts and anthocyanin accumulation profile. On the other hand, MibHLH1 was expressed weakly in all organs of ‘Vintage Lavender’. However, high expression levels of MibHLH1 were detected in petals of other cultivars with higher levels of anthocyanin accumulation than ‘Vintage Lavender’. MiWDR1 and MiWDR2 maintained constant expression levels in petals during flower development and vegetative organs. Transient MiMYB1 expression in 1-month-old M. incana seedlings using a Turnip mosaic virus vector activated transcription of the endogenous anthocyanin biosynthetic genes MiF3ʹH, MiDFR, and MiANS and induced ectopic anthocyanin accumulation in leaves. Therefore, MiMYB1 possibly interacts with MibHLH2 and MiWDR1, and this trimeric protein complex activates the transcription of anthocyanin biosynthetic genes in M. incana flowers. Moreover, MibHLH1 acts as an enhancer of anthocyanin biosynthesis with the MiMYB1–MibHLH2–MiWDR1 complex. This study revealed the molecular mechanism involved in the regulation of anthocyanin accumulation levels in M. incana flowers.

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Acknowledgements

We thank Ms. Nobue Nakamura, Shizuoka University, for technical support. This work was supported by JSPS KAKENHI Grant Number 17H03765 and Ministerio de Ciencia, Innovación y Universidades (Spain; co-financed European Union FEDER funds) Grant BIO2017-83184-R.

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Correspondence to Takashi Nakatsuka.

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Supplementary file1 Supplementary Fig. S1 Chromatographs of anthocyanins, flavonols, and hydrolytic aglycones extracted from Matthiola incana ‘Vintage Lavender’. Flavonoids extracted from petals at stage S4 were separated using HPLC and monitored at 520 nm (a) and 360 nm (b). Two major peaks detected at 520 nm were annotated as (1) cyanidin 3-[2-(2-(sinapoyl)-xylosyl)-6-(p-coumaroyl)-glucoside]-5-[6-(malonyl)-glucoside] and (2) cyanidin 3-[2-(2-(sinapoyl)-xylosyl)-6-(feruloyl)-glucoside]-5-[6-(malonyl)-glucoside]. Three major peaks detected at 360 nm were annotated as (3) 1-(sinapoyl)-glucopyranoside, (4) kaempferol 3-(2-rhamnopyranosyl)-β-glucopyranoside-7-rhamnopyranosid, and (5) kaempferol 3--(2-rhamnopyranosyl)-arabinopyranoside-7-rhamnopyranoside. The hydrolyzed extracts collected from ‘Vintage Lavender’ petals were monitored at 530 nm (c) and 360 nm (d), respectively. (e and f) Chromatographs of standards, cyanidin (Cy), delphinidin (Dp), pelargonidin (Pg), kaempferol (K), quercetin (Q), and myricetin (M). (PDF 1649 kb)

Supplementary file2 Supplementary Fig. S2 Phylogenetic tree inferred from the amino acid sequences of the R2R3 region of MiMYBs with R2R3-MYB from other species. This phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrapping data sets. R2R3-MYB name and GenBank accession numbers are as follows: Arabidopsis thaliana MYB11 (NP_191820), MYB12 (O22264), MYB111 (NP_199744), MYB113 (NP_176811), MYB114 (NP_176812), PAP1 (Q9FE25), PAP2 (Q9ZTC3), and TT2 (Q9FJA2); cauliflower (Brassica oleracea var. botrytis) BoMYB2 (ADP76651); garden stock (Matthiola incana) MiMYB1 (present study, red closed circle); grape (Vitis vinifera) MYBA1-1 (Q8L5P3); Japanese gentian (Gentiana triflora) MYB3 (A9ZMI4); petunia (Petunia hybrida) AN2 (Q9M72); maize (Zea mays) C1 (P10290) and P1 (P27898); snapdragon (Antirrhinum majus) MIXTA (Q38739), ROSEA1 (ABB83826), ROSEA2 (ABB8327), and VENOSA (ABB83828); and radish (Raphanus sativus) RsMYB1 (AKM95888). Scale bar represents 0.1 substitutions per site. (PDF 59 kb)

Supplementary file3 Supplementary Fig. S3 Phylogenetic tree inferred from the amino acid sequences of bHLH derived from Matthiola incana and other species. This phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrapping data sets. bHLH name and GenBank accession numbers are as follows: snapdragon (Antirrhinum majus) delila (Q38736); Arabidopsis thaliana MYC1 (Q8W2F1), EGL3 (Q9CAD0), GL3 (Q9FN69), and TT8 (Q9FT81); cauliflower (Brassica oleracea var. botrytis) BoTT8 (ADP76654); garden stock (M. incana) MibHLH1 and MibHLH2 (present study, red closed circles); Japanese gentian (Gentiana triflora) bHLH1 (B7XEI1), gerbera (Gerbera hybrida) GMYC1 (O82686); maize (Zea mays) In1 (Q41875) and Lc (P13526); morning glory (Ipomoea nil) bHLH1 (Q1JV08), bHLH2 (Q1JV07), and bHLH3 (Q1JV06); rice (Oryza sativa) Ra (Q40643) and Rc (Q2I7J3); perilla (Perilla frutescens) F3G1 (Q852P3) and MYC-RP (Q9ZQS4); petunia (Petunia hybrida) AN1 (Q9FEA1) and JAF13 (O64908); and radish (Raphanus sativus) RsTT8 (ASF79354). Scale bar represents 0.1 substitutions per site. (PDF 60 kb)

Supplementary file4 Supplementary Fig. S4 Phylogenetic tree inferred from the amino acid sequences of WDR derived from Matthiola incana and other species. This phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrapping data sets. WDR name and GenBank accession number are as follows: Arabidopsis thaliana AtAN11 (AAC18912.1) and AtTTG1 (NP_001318637); garden stock (M. incana) MiWDR1 and MiWDR2 (present study, red closed circles); morning glory (Ipomoea nil) InTTG1 (XP_019200425) and InWDR1 (BAE94407); perilla (Perilla frutescen) PFWD (BAB58883); petunia (Petunia hybrida) AN11 (AAC18914); and maize (Zea may) MP1 (NP_001169326) and PAC1 (NP_001310302). (PDF 57 kb)

Supplementary file5 Supplementary Fig. S5 Transient expression of MiMYB1 in Nicotiana benthamiana. a) The expression levels of transgenes were measured by RT-qPCR. Total RNA was isolated from the inoculated leaves of 7 days after inoculation. The expression levels of TuMVUK1, GFP, and MiMYB1 were normalized using that of NtUBQ-C. Values and error bars are expressed as mean ± standard error (n = 5). b) Anthocyanin amounts in TuMVUK1:GFP- and TuMVUK1:MiMYB1-infected N. benthamiana leaves. Anthocyanins were extracted from inoculated leaves at 7 days per inoculation (n = 10). N.D. indicates not detected. ns indicates no significant difference between TuMV:GFP and TuMV:MYB1 using Student’s t-test (P < 0.05). (c-d) N. benthamiana leaves at 15 days after inoculation with TuMVUK1:MiMYB1/GV3101 (c) and TuMVUK1:GFP/GV3101 (d). e) GFP fluorescence was observed in TuMVUK1:GFP-infected leaves under blue light. Bar = 1 cm. (PDF 103 kb)

Supplementary file6 Supplementary Fig. S6 Anthocyanin analysis of TuMV-infected Matthiola incana plants. Anthocyanins extracted from the upper leaves at 15 days after sap inoculation were monitored at a wavelength of 520 nm. The chromatographs of extracts from TuMVUK1:MiMYB1- and TuMVUK1:GFP-infected plants indicated as red and black lines, respectively. (PDF 169 kb)

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Nuraini, L., Ando, Y., Kawai, K. et al. Anthocyanin regulatory and structural genes associated with violet flower color of Matthiola incana. Planta 251, 61 (2020). https://doi.org/10.1007/s00425-020-03351-z

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Keywords

  • Basic helix–loop–helix
  • Garden stock
  • R2R3MYB
  • Turnip mosaic virus
  • WD40 repeat