Abstract
Jasmonates are plant hormones that induce the accumulation of many secondary metabolites, such as rutin in buckwheat, via regulation of jasmonate-responsive transcription factors. Here, we report on the identification of a clade of jasmonate-responsive subgroup 4 MYB transcription factors, FtMYB13, FtMYB14, FtMYB15, and FtMYB16, which directly repress rutin biosynthesis in Fagopyrum tataricum. Immunoblot analysis showed that FtMYB13, FtMYB14, and FtMYB15 could be degraded via the 26S proteasome in the COI1-dependent jasmonate signaling pathway, and that this degradation is due to the SID motif in their C-terminus. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that FtMYB13, FtMYB14, and FtMYB15 interact with the importin protein Sensitive to ABA and Drought 2 (FtSAD2) in stem and inflorescence. Furthermore, the key repressor of jasmonate signaling FtJAZ1 specifically interacts with FtMYB13. Point mutation analysis showed that the conserved Asp residue of the SID domain contributes to mediating protein-protein interaction. Protoplast transient activation assays demonstrated that FtMYB13, FtMYB14, and FtMYB15 directly repress phenylalanine ammonia lyase (FtPAL) gene expression, and FtSAD2 and FtJAZ1 significantly promote the repressing activity of FtMYBs. These findings may ultimately be promising for further engineering of plant secondary metabolism.
Highlights
Buckwheat is a type of pseudocereal of the genus Fagopyrum, within the Polygonaceae family
FtMYB13D281N-green fluorescent protein (GFP), FtMYB14D266N-GFP, and FtMYB15D258NGFP protein were found only in the cytoplasm (Supplementary Fig.S6).These results suggest that the conservedAsp residue of the SAD2 Interaction Domain (SID) domains of FtMYB13, FtMYB14, and FtMYB15 are necessary for their transport into the nucleus mediated by FtSAD2.As shown in Fig. 1, FtSAD2 transcription was induced by JA and transcripts accumulated in stem, leaves and flowers, similar to the pattern of transcript accumulation of FtMYB13, FtMYB14, and FtMYB15.This indicates that FtSAD2 probably directly regulates the transcriptional activity of these FtMYBs
The above data show that FtMYB13, FtMYB14, FtMYB15, and FtMYB16 repress the expression of FtMYB15 directly repress phenylalanine ammonia lyase (FtPAL), suggesting that these factors probably regulate rutin biosynthesis.To functionally test the roles of these MYB transcription factor (TF) and their derivatives in planta, we investigated the accumulation of rutin in F. tataricum hairy roots overexpressing FtMYB13-HA, FtMYB13D281N-HA, FtMYB13D283N-HA, FtMYB13D285N-HA, FtMYB14-HA, FtMYB14D266N-HA, FtMYB15-HA, FtMYB15D258N-HA, FtMYB16-HA, FtSAD2-HA, or FtJAZ1-HA
Summary
Buckwheat is a type of pseudocereal of the genus Fagopyrum, within the Polygonaceae family. Tartary buckwheat (TB; Fagopyrum tataricum), common buckwheat (CB; Fagopyrum esculentum), and golden buckwheat (Fagopyrum cymosum), are widely cultivated in Asia and Europe (Zhang et al, 2012). TB research interest has increased due to its higher content of bioactive flavonoids (e.g. rutin, orientin, vitexin, quercetin, isovitexin, and isoorientin) compared with CB, and its potential to contribute to diverse health benefits. The concentration of rutin, a major flavonoid, can reach 81 mg g–1 in the groats of TB compared with the concentration of 0.2 mg g–1 reported in CB (Wijngaard and Arendt, 2006). TB flour accumulates ~10-fold more total flavonoids than CB (Qin et al, 2010).buckwheat was considered to be a model plant for studying flavonoid biosynthesis due toits high accumulation of rutin, especially in the early days of research
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