FtBPM3 modulates the orchestration of FtMYB11‐mediated flavonoids biosynthesis in Tartary buckwheat

Tartary buckwheat (Fagopyrum tataricum, TB) is rich in bioactive flavonoids, which have a variety of biological activities (Ghorbani, 2017). Jasmonates (JAs) are essential phytohormones, which play key roles in regulating the formation of numerous secondary metabolites, including flavonoids rutin (Li et al., 2019; Zhou and Memelink, 2016). It has been reported that JAs could induce flavonoids accumulation and identified a class of JAs‐responsive R2R3‐MYB TFs, such as FtMYB11, a repressor of rutin biosynthesis in TB (Zhang et al., 2018; Zhou et al., 2017). JAs lead to FtMYBs degradation by 26S proteasome pathway (Zhang et al., 2018). However, the posttranslational regulation of these FtMYBs has not been reported.

Recently, we reported genome re‐sequencing data of 510 TB accessions and genome‐wide association study (GWAS) of three main flavanols, among which the kaempferol‐3‐O‐rutinoside (KC) is stable and less affected by environmental factors compared with quercetin and rutin (Zhang et al., 2021). Four associated loci passing the threshold P < 1 × 10⁻⁵ were identified, and three associated loci repetitively in two years (Figure 1a). However, no known flavonoids metabolism genes were identified and no genes were significantly induced by JA (data not shown). Interestingly, FtBPM3 (FtPinG0808645400), a homolog of Arabidopsis BTB‐POZ/MATH (BPM) E3 ligase, which functions as an important regulator of JA‐responsive TFs activity and stability (Chico et al., 2020; Li et al., 2021) was associated with Ft8:31558491. Further analysis showed KC content (Figure 1b) and FtBPM3 expression (Figure 1c) was higher in the A/A genotype than the A/C genotype, suggesting FtBPM3 was a candidate gene controlling flavonoids biosynthesis in TB.

Figure 1.

FtBPM3 modulates the orchestration of FtMYB11‐mediated flavonoids biosynthesis in Tartary buckwheat. (a) Local Manhattan plot of association loci with the KC contents. (b) Box plots show KC contents in two haplotypes. (c) The expression levels of FtBPM3 in A/A and A/C type. (d‐e) FtCHS and FtBPM3 expression patterns (d) and flavonoids concentration (e) in different tissues. DW, Dry weight. (f‐g) Flavonoids content (f) and the expression levels of FtC4H, FtCHS and FtFLS (g) in different types of TB hairy roots in response to 50 μΜ MeJA for 4 h. (h) Immunoblot analysis of FtBPM3‐HA protein levels in FtBPM3OE lines. (i‐l) Y2H (i‐j), BIFC (k) and Pull‐down assay (l) of FtMYB11 and FtBPM3 or its derivatives. (m) FtMYB11 SBC‐like motif and derivatives. (n‐o) Y2H and BIFC assay of FtBPM3 and FtMYB11^P156A. (p) Immunoblot analysis of FtMYB11‐HA protein levels in Arabidopsis suspension protoplasts expressing 35S::FtBPM3. (q) Immunoblot analysis of FtBPM3‐GFP, FtMYB11‐HA and FtMYB11^AAA‐HA protein levels in WT or Coil‐1 Arabidopsis suspension protoplasts treated with 50 μm MeJA. (r) Trans‐activation assays of FtPAL promoter by FtMYB11, FtBPM3 or FtBPM3ΔN. (s‐t) Trans‐activation assays of FtBPM3pro‐GUS or FtBPM3mpro‐GUS by FtMYB11 and/or FtBPM3. (u) Transcriptional activity assays of FtMYB11 binding to the FtBPM3pro or FtBPM3mpro fragments in yeast. (v) EMSA of a wild type or mutated probe of FtBPM3pro with FtMYB11. (w‐y) Model of the fine‐tuned ‘ping‐pong’ regulatory mechanism between FtMYB11 and FtBPM3 activity in JAs signalling pathway. JA‐pathway activation stabilizes FtBPM3 (w). Without JA, the ubiquitination of FtBPM3 is degraded by the 26S proteasome (x), FtBPM3 targets FtMYB11 for degradation (y). Data in (b), (c), (g) and (u) are means ± SE of three biological repeats (Student’s t‐test, *P < 0.05, **P < 0.01).

To test whether the expression pattern of FtBPM3 is consistent with TB flavonoids biosynthesis, the expression patterns of FtBPM3 and CHS (chalcone synthase), a key gene in TB flavonoids biosynthesis and the flavonoids content in different tissues TB plant were studied. The expression of FtBPM3 appeared closely associated with CHS (Figure 1d) and flavonoids content (Figure 1e), indicating the expression of FtBPM3 is probably involved in flavonoids accumulation. The flavonoids levels (Figure 1f) and the expression of three genes (FtC4H, FtCHS and FtFLS) (Figure 1g) in the FtBPM3 overexpressing hairy roots lines were significantly higher than those in control. MeJA (methyl jasmonate) treatment drastically increased the accumulation of flavonoids in FtBPM3 overexpressing transgenic lines (Figure 1f), indicating that FtBPM3 protein is stable in response to MeJA. Immunoblot analysis of FtBPM3‐HA protein levels in FtBPM3 overexpressing lines revealed that both MeJA and MG132 drastically increased FtBPM3‐HA accumulation (Figure 1h), indicating FtBPM3 protein is subject to 26S proteasome‐mediated degradation. Taken together, these results demonstrated FtBPM3 protein accumulation promotes JA‐induced flavonoids biosynthesis.

Yeast two‐hybrid (Y2H) assays were used to examine whether FtBPM3 assembles with JA‐responsive subgroup 4 MYB repressors and found FtBPM3 interacts strongly with FtMYB11, while FtMYB13 and FtMYB15 did not (Figure 1i). And this interaction requires the N‐terminal MATH domain of FtBPM3 (Figure 1j). However, no YFP signal was observed by bimolecular fluorescence complementation (BiFC) assay in the combination of full‐length FtBPM3 and FtMYB11, but FtBPM3ΔC lacking a BTB‐POZ domain does (Figure 1k), indicating that FtBPM3 probably targets FtMYB11 for protein degradation. Pull down assay found direct physical interaction between HA‐FtBPM3 and His‐FtMYB11 (Figure 1l). These results consistently support the direct interaction between FtBPM3 and FtMYB11.

It has been reported that Arabidopsis BPM proteins interact with their target proteins through the speckle‐type POZ protein (SPOP)‐binding consensus (SBC)‐like motif ϕ‐π‐S‐X‐S/T (ϕ, nonpolar; π, polar; X, any amino acid) (Morimoto et al., 2017). To test whether SBC‐like motif in FtMYB11 was responsible for the recognition of FtMYB11 by FtBPM3, the interaction between FtBPM3 and FtMYB11 derivatives (Figure 1m), was tested by Y2H assays. FtMYB11^S155A, FtMYB11^S157A and FtMYB11^AAA lost interaction with FtBPM3, while FtMYB11^P156A did not affect the interaction (Figure 1n). BiFC assay showed a YFP signal only observed in the nucleus of Arabidopsis protoplasts upon co‐expression of FtBPM3ΔC‐cYFP with nYFP‐FtMYB11^P156A (Figure 1o). These results indicate that the SBC‐like motif of FtMYB11 is hence sufficient for the interaction with FtBPM3.

The interaction between FtBPM3 and FtMYB11 suggested that FtMYB11 could be targets of CUL3^FtBPM3 E3 ubiquitin ligases. Thus, we sought to examine whether increment of FtBPM3 expression affects FtMYB11 stability using a transient expression assay in Arabidopsis protoplasts. Total proteins were extracted from protoplasts co‐transformed with FtMYB11‐HA, with or without FtBPM3‐His and subjected to immunoblot analysis. The addition of FtBPM3 led to FtMYB11 degradation, whereas the internal control was not significantly affected (Figure 1p). Since both FtBPM3 and FtMYB11 are responsive to JA at protein levels, we then tested whether their stability was associated with the 26S proteasome in a JA‐dependent manner in wild type (WT) and Coil‐1 protoplasts. JA treatment significantly decreased FtMYB11‐HA protein level, but not for FtMYB11^AAA‐HA, and drastically increased the accumulation of FtBPM3‐His with time in WT background (Figure 1q). However, the level of two proteins was not changed under JA treatment in Coil‐1 background, indicating FtBPM3 targets FtMYB11 for degradation depending on JA signalling.

Our previous results showed that FtMYB11 directly represses the FtPAL gene expression (Zhou et al., 2017). To further elucidate the effect of FtBPM3 on the activity of FtMYB11, Arabidopsis protoplast trans‐activation assays were performed. The co‐transformation of FtPALpro‐GUS reporter and 35S::FtMYB11 effector resulted in strong repression (Figure 1r). The addition of the 35S::FtBPM3 effector resulted in the repression of FtMYB11 activity, but 35S::FtBPM3ΔN has no effect. These results support the above protein interaction and FtBPM3 could regulate FtMYB11 activity.

Sequence analysis showed the promoter of FtBPM3 contains one type II element, which was recognized by FtMYB11 (Zhou et al., 2017). Arabidopsis trans‐activation assays showed FtMYB11 repressed the FtBPM3pro‐GUS reporter and had no effect on FtBPM3mpro‐GUS (Figure 1s,t), indicating this type II element is important for transcriptional repression. The addition of FtBPM3 released the repression activity of FtMYB11 (Figure 1t), indicating FtMYB11 may directly repress FtBPM3 gene expression via binding to the type II element. Yeast one‐hybrid (Y1H) assays identified the direct interactions between FtMYB11 and FtBPM3 promoter fragment, but not with its derivative (Figure 1u). Electrophoretic mobility shift assay (EMSA) showed incubation of the wild type with His‐FtMYB11 produced shifts, while the mutant 80‐bp probes did not result in shifts (Figure 1v). These results illustrated that FtMYB11 could directly repress the FtBPM3 gene expression.

In summary, we provide a new framework to understand the fine‐tuned ‘ping‐pong’ regulatory mechanism between FtMYB11 and FtBPM3 activity. We uncovered a negative feedback regulatory loop of FtMYB11 protein levels mediated by the E3 ligase CUL3^FtBPM3 that facilitates termination of FtBPM3 mRNA accumulation to avoid the overaccumulation of FtBPM3 (Figure 1w‐y). This ‘ping‐pong’ mechanism is necessary for resetting JA signalling and to avoid harmful runaway responses, which optimize plant fitness and provide a theoretical basis for the cultivation of TB with high flavonoid content.

Conflict of interest

The authors declare no conflicts of interest.

Author contributions

M.Z., M.I.G. and S.U.P conceived and supervised the research. M.D., K.Z., Q.Z. and Y.H conducted the experiments. H.Z. and M.H analysed the data. M.D., M.I.G., S.U.P., M.Z. and Y.H wrote the paper.

Ding, M. , Zhang, K. , He, Y. , Zuo, Q. , Zhao, H. , He, M. , Georgiev, M. I. , Park, S. U. and Zhou, M. (2021) FtBPM3 modulates the orchestration of FtMYB11‐mediated flavonoids biosynthesis in Tartary buckwheat. Plant Biotechnol J, 10.1111/pbi.13587