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. 2019 Jun 18;19(1):263.
doi: 10.1186/s12870-019-1876-x.

FtMYB8 from Tartary buckwheat inhibits both anthocyanin/Proanthocyanidin accumulation and marginal Trichome initiation

Yunji Huang  1 , Qi Wu  1 , Shuang Wang  1 , Jiaqi Shi  1 , Qixin Dong  1 , Panfeng Yao  1 , Guannan Shi  1 , Shuangxiu Xu  1 , Renyu Deng  1 , Chenglei Li  1 , Hui Chen  1 , Haixia Zhao  2
Affiliations

FtMYB8 from Tartary buckwheat inhibits both anthocyanin/Proanthocyanidin accumulation and marginal Trichome initiation

Yunji Huang et al. BMC Plant Biol. .

Abstract

Background: Because flavonoids and trichomes play crucial roles in plant defence, their formation requires fine transcriptional control by multiple transcription factor families. However, little is known regarding the mechanism of the R2R3-MYB transcription factors that regulate both flavonoid metabolism and trichome development.

Results: Here, we identified a unique SG4-like-MYB TF from Tartary buckwheat, FtMYB8, which harbours the C2 repression motif and an additional TLLLFR repression motif. The expression profiles of FtMYB8 combined with the transcriptional activity of PFtMYB8 promoter showed that FtMYB8 mRNA mainly accumulated in roots during the true leaf stage and flowering stage and in bud trichomes and flowers, and the expression of this gene was markedly induced by MeJA, ABA and UV-B treatments but repressed by dark treatment. Overexpression of FtMYB8 in Arabidopsis reduces the accumulation of anthocyanin/proanthocyanidin by specifically inhibiting TT12 expression, which may depend on the interaction between FtMYB8 and TT8. Interestingly, this interaction may also negatively regulate the marginal trichome initiation in Arabidopsis leaves.

Conclusions: Taken together, our results suggest that FtMYB8 may fine-tune the accumulation of anthocyanin/proanthocyanidin in the roots and flowers of Tartary buckwheat by balancing the inductive effects of transcriptional activators, and probably regulate trichome distribution in the buds of Tartary buckwheat.

Keywords: Anthocyanin; Arabidopsis; FtMYB8; Proanthocyanidin; Tartary buckwheat; Trichome.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of SG4-like-MYB expression and anthocyanin/PA content at the flowering stage. a Heat map of SG4-like-MYBs. Each column represents one tissue, and each line represents one gene that is displayed on the right. The colour intensity of the blue and red rectangles reflect low and high Z-scores for mRNA accumulation. b Expression pattern of SG4-like-MYBs. FtH3 was used as a reference gene. The accumulation of each SG4-like-MYB mRNA in the stems was defined as “1” for each developmental stage. Means were calculated from three repeats, and error bars reflect ±SDs. c Anthocyanin/PA content in different tissues
Fig. 2
Fig. 2
Molecular identification of FtMYB8. a Multiple sequence alignment of FtMYB8. The R2 and R3 SANT domains are underlined. The bHLH interaction motif and three conserved motifs are indicated with black boxes. b Phylogenetic relationships of FtMYB8. The GenBank accession numbers are displayed to the right of the protein names. FtMYB8 is highlighted with a black square
Fig. 3
Fig. 3
Histochemical localization of GUS expression under PFtMYB8 promoter control in transgenic plants. a-g Histochemical staining of 4-, 6-, 11-, 18-, 25-, 32- and 42-day-old PFtMYB8:GUS seedlings. a-g Histochemical GUS staining of whole seedlings. (a-1) Cotyledons and hypocotyl of a 4-day-old plant. (a-2) Root of a 4-day-old plant. (b-1) Bud of a 6-day-old plant. (b-2)-(b-3) Roots of a 6-day-old plant. (c-1) Bud of an 11-day-old plant. (c-2) Root of an 11-day-old plant. (d-1) Bud of an 18-day-old plant. (d-2) Root of an 18-day-old plant. (e-1) Bud of a 25-day-old plant. (e-2) Root of a 25-day-old plant. (f-1) Stem of a 32-day-old plant. (f-2) Root primordia of a 32-day-old plant. (g-1)-(g-3) Root primordia of a 42-day-old plant. (g-4) Crowded bud of a 42-day-old plant. (g-5) Stem of a 42-day-old plant. (g-6) Leaf of a 42-day-old plant. (g-7) Flower of a 42-day-old plant. (g-8) Silique of a 42-day-old plant
Fig. 4
Fig. 4
Analyses of the response of the PFtMYB8 promoter to environmental factors and hormone signals. a-f Histochemical GUS staining of PFtMYB8:GUS seedlings under normal conditions (a, b) or subjected for 5 h to 50 μM MeJA (C), 10 μM ABA (D), UV-B (e) or dark (f). (a-f) Histochemical GUS staining of whole seedlings. (n-1)-(n-3) Histochemical GUS staining of the buds (n-1), roots (n-2) or roots (n-3), where n represents a-f. g Abundance analyses of GUS mRNA in WT and transgenic Arabidopsis whole seedlings. Total RNA was extracted after 5 h of treatment, and each sample contained at least 30 seedlings. Relative expression of the GUS gene was evaluated by the 2-ΔΔCT method, and the accumulation of GUS mRNA in whole transgenic seedlings under normal conditions was defined as “1”. Arabidopsis Atactin2 was used as a reference gene. Means were calculated from three repeats, and error bars reflect ±SDs. ** and * represent extremely significant differences and significant differences, respectively (*P < 0.05, **P < 0.01)
Fig. 5
Fig. 5
Overexpression of FtMYB8 reduces the anthocyanin/PA content in transgenic tobacco plants. a Changes in the floral phenotype in tobacco plants overexpressing FtMYB8. b, c Anthocyanins/PA content in transgenic tobacco leaves. d Detection of flavonoid biosynthetic gene transcriptional levels in FtMYB8 transgenic tobacco leaves. mRNA accumulation of six genes was monitored by qRT-PCR in the WT and transgenic lines. Relative expression was evaluated by the 2−ΔΔCT method, and gene expression levels were defined as “1” in WT tobacco. Tobacco Ntβ-actin was used as a reference gene. Means were calculated from three repeats, and error bars reflect ±SDs. *P < 0.05
Fig. 6
Fig. 6
Effect of FtMYB8 on the accumulation of anthocyanin/PA, gene expression and development of marginal trichomes. a Photographs of WT and transgenic seedlings grown in MS medium with 3% sucrose. (B-1) Mature Arabidopsis seeds. (B-2) PA accumulation in mature seeds stained by vanillin-HCl. c,d Anthocyanin/PA content in transgenic lines and WT plants. e Each sample used for total RNA extraction contained at least 50 seedlings. The mRNA abundances of ten genes were monitored by qRT-PCR in the WT and transgenic lines. The relative expression was evaluated by the 2−ΔΔCT method, and gene expression levels were defined as “1” in WT Arabidopsis seedlings. Arabidopsis Atactin2 was used as a reference gene. f Comparison of trichome distribution between transgenic plants and WT plants at the true leaf stage by stereomicroscopy. g Total number of trichomes at the true leaf stage in the WT and transgenic lines. Each sample used to count the total number of trichomes contained at least 15 leaves. h Percentage of marginal trichomes in true leaves in the WT and transgenic lines. Each sample used to calculate the percentage of marginal trichomes contained at least 15 leaves. Means were calculated from three repeats and error bars reflect ±SDs. *P < 0.05, **P < 0.01
Fig. 7
Fig. 7
Molecular interaction identification of FtMY8 transcription factor. a FtMYB8 interacts with AtTT8/FtTT8/FtGL3 in yeast. Image showing the growth of AH109 [pGADT7-FtMYB8 × pGBKT7-AtTT8/−AtGL3/−AtEGL3/−AtTTG1/−FtTT8/−FtGL3/−FtEGL3/−FtTTG1] yeast on SD/−Leu-Trp plates and SD/−Leu-Trp-His-Ade plates. b Transient expression assays displaying that FtMYB8 inhibits AtTT12 expression. (a, c and e) Representative images of Nicotiana benthamiana leaves 48 h after infiltration are shown. (b, d and f) Quantitative analysis of luminescence intensity represent a, c and e, respectively. Means were calculated from three repeats and error bars reflect ±SDs. *P < 0.05
Fig. 8
Fig. 8
Potential working model for the function of FtMYB8. FtMYB8 mRNA mainly accumulates in the roots during the true leaf stage and flowering stage and in bud trichomes and flowers, and the expression of this gene is enhanced by ABA, MeJA and UV-B signals and suppressed by dark treatment. FtMYB8 could interact with TT8/GL3, and this abnormal interaction may disrupt the normal formation of the MYBs-TT8/GL3-TTG1 complex. FtMYB8 may form MBW complex with TT8/GL3 and TTG1, and then, these MBW complexes may reduce the accumulation of anthocyanin/PA by downregulating TT12 expression. FtMYB8 may also inhibit the initiation of marginal trichomes by forming MBW complexes and/or decreasing the levels of active TT8/GL3 proteins
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