Tartary Buckwheat (Fagopyrum tataricum) FtTT8 Inhibits Anthocyanin Biosynthesis and Promotes Proanthocyanidin Biosynthesis

Tartary buckwheat (Fagopyrum tataricum) is an important plant, utilized for both medicine and food. It has become a current research hotspot due to its rich content of flavonoids, which are beneficial for human health. Anthocyanins (ATs) and proanthocyanidins (PAs) are the two main kinds of flavonoid compounds in Tartary buckwheat, which participate in the pigmentation of some tissue as well as rendering resistance to many biotic and abiotic stresses. Additionally, Tartary buckwheat anthocyanins and PAs have many health benefits for humans and the plant itself. However, little is known about the regulation mechanism of the biosynthesis of anthocyanin and PA in Tartary buckwheat. In the present study, a bHLH transcription factor (TF) FtTT8 was characterized to be homologous with AtTT8 and phylogenetically close to bHLH proteins from other plant species. Subcellular location and yeast two-hybrid assays suggested that FtTT8 locates in the nucleus and plays a role as a transcription factor. Complementation analysis in Arabidopsis tt8 mutant showed that FtTT8 could not recover anthocyanin deficiency but could promote PAs accumulation. Overexpression of FtTT8 in red-flowering tobacco showed that FtTT8 inhibits anthocyanin biosynthesis and accelerates proanthocyanidin biosynthesis. QRT-PCR and yeast one-hybrid assay revealed that FtTT8 might bind to the promoter of NtUFGT and suppress its expression, while binding to the promoter of NtLAR and upregulating its expression in K326 tobacco. This displayed the bidirectional regulating function of FtTT8 that negatively regulates anthocyanin biosynthesis and positively regulates proanthocyanidin biosynthesis. The results provide new insights on TT8 in Tartary buckwheat, which is inconsistent with TT8 from other plant species, and FtTT8 might be a high-quality gene resource for Tartary buckwheat breeding.


Introduction
Tartary buckwheat (Fagopyrum tataricum), belonging to the genus Fagopyrum within the family Polygonaceae, is a kind of crop utilized for both food and medicine [1].As the country of origin and main producer of Tartary buckwheat, China owns abundant germplasm resources and has a long cultivation history [2,3].Moreover, Chinese people take full advantage of buckwheat to make a variety of food and health products, such as noodles, steamed bun, cakes, cookies, bread, pasta, tea, liquor, and pillows [4][5][6].Buckwheat products attract more and more consumers worldwide due to their rich nutrition and human-beneficial metabolites, especially abundant bioactive flavonoids, like rutin, quercetin, tannin, and proanthocyanidin, among others [7,8].Rutin has been reported to have numerous bioactive properties, including antioxidant, antihypertension, anti-inflammatory, antidiabetic, and gastric lesion protecting activities, as well as preventing cognitive impairments like Alzheimer's disease [9][10][11].
Tartary buckwheat seeds have been utilized as rice and flour.However, their sprouts are also rich in nutrients, and possess the same health benefits as the seeds.Therefore, sprouts have become increasingly popular as functional greens worldwide [12,13].Additionally, Tartary buckwheat sprouts contain no allergic proteins that exist in the seeds and contain anthocyanins that are absent in the seeds [14][15][16].Anthocyanin is a kind of water-soluble natural pigment that belongs to the flavonoid family.Except for pigmentation for the flowers, fruits, leaves, etc., of plants, anthocyanin also plays roles in resistance to biotic and abiotic stress as well as in seed pollination and dispersal [17][18][19].
The anthocyanin biosynthesis pathway is one branch of the flavonoid biosynthesis pathway, which includes chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), and anthocyanin 3-O-glucosyltransferase (UFGT).In this pathway, two substrates, leucoanthocyantin and anthocyanidin, are catalyzed by leucoanthocyanin reductase (LAR) and anthocyanidin reductase (ANR), respectively, which leads to the biosynthesis of proanthocyanidins (PAs), another kind of flavonoid and contributes to the pigmentation of the plant's seed coat [20][21][22].Nowadays, the biosynthesis pathway and regulation mechanism of anthocyanin and PA have been well established and deeply characterized in model plants Arabidopsis thaliana [23][24][25], Petunia hybrida [26,27], Zay maize [28,29], as well as other plant species.Notably, three transcription factors, (TFs) MYB, bHLH, and WD40, are the main transcriptional regulators for flavonoid biosynthesis [30].MYB and bHLH can play the role alone by binding to target gene promoters, while WD40 does not and it regulates flavonoid biosynthesis by forming a protein complex with MYB and bHLH (MBW) [30][31][32].At present, some MYB transcription factors have been reported to be involved in the regulation of anthocyanin and proanthocyanidin biosynthesis [33][34][35][36][37]. Ft-MYB15 [33] promotes anthocyanins accumulation.The overexpression of FtMYB1 and FtMYB2 significantly enhances PAs accumulation in Nicotiana tabacum [34].FtMYB3 [35], FtMYB8 [36], and FtMYB18 [37] act as negative regulators of anthocyanin and proanthocyanidin biosynthesis in Tartary buckwheat.However, the literature focuses on MYB, and the regulation functions of bHLH and WD40 remain under-researched.Here, a bHLH transcription factor gene FtTT8 was characterized and verified to possess bidirectional regulation functions that inhibit anthocyanin accumulation and enhance proanthocyanidin biosynthesis.This conclusion is distinct from that of other plant species and provides new insights into TT8's role in the regulation of flavonoid biosynthesis.

Cloning and Molecular Characterization of FtTT8
The full length of the CDS sequence of FtTT8 was obtained from Tartary buckwheat seedlings, which contained 2199 bp and encoded a protein of 732 amino acids.Sequence alignments with the NCBI database revealed FtTT8 to be a member of the bHLH transcription factor family.Phylogenetic analysis of FtTT8 and bHLH transcript factors from other species that have been reported to be involved in the regulation of anthocyanin and proanthocyanidin biosynthesis showed that the FtTT8 gene is closely related to LfTT8 and belongs to the IIIf subfamily (Figure 1).Most of these bHLH TFs contain 10 motifs (Figure 1).Since the LfTT8 TF was reported to regulate anthocyanin biosynthesis in Liquidambar formosana [38], we therefore speculated that the FtTT8 gene is a strong candidate to be responsible for anthocyanin pigmentation in Tartary buckwheat.bHLH TFs from nine species that were more closely related to FtTT8 were chosen to analyze the conserved domains.The results showed that they contain a specific conserved bHLH (basic helix1 loop helix2) domain, the N-terminal MIR (MYB-interacting region), and the C-terminal ACT-like domain (Figure 2).The MIR is critical for binding to MYB TFs [39], therefore, FtTT8 may play a role in regulating anthocyanin or proanthocyanidin biosynthesis by interacting with the MYB protein.
may play a role in regulating anthocyanin or proanthocyanidin biosynthesis by interacting with the MYB protein.

Analysis of Subcellular Location and Transcription Activation of FtTT8
Subcellular location analysis displayed that FtTT8-GFP merged with RFP (the nucleus location reference), while the GFP from the control vector was distributed throughout the cell (Figure 3A).This implied that FtTT8 is located in the nucleus and plays the role of a transcript factor.The result of the yeast two-hybrid assay suggests that the transcript factor FtTT8 possesses transcription activation activity (Figure 3B).

Complementation of the Arabidopsis tt8 Mutant by FtTT8
Complementation analysis in the Arabidopsis attt8 mutant with FtTT8 driven by the CaMV35S promoter was conducted to verify the function of FtTT8 in the regulation of anthocyanin and PA biosynthesis.FtTT8 transgenic Arabidopsis plants (three of each) were screened out and they showed a much higher expression level of FtTT8, whereas the wild-type (WT) and attt8 mutant had no expression of this gene (Figure 4A).Firstly, when observing the seedlings of these tree types, the color red could obviously be observed in the leaves and hypocotyl of WT seedlings, while no red was evident in the attt8 mutant and the FtTT8 over expression lines (Figure 4A), indicating that FtTT8 was unable to promote anthocyanin biosynthesis.Meanwhile, DMACA staining was performed for PA analysis.The results showed that the attt8 mutant contained less PA than the WT; however, the PA content of the attt8 mutant transformed by FtTT8 increased remarkably, nearly to the WT plant level (Figure 4B).Content measurement of the total anthocyanins and PAs showed that the WT contained higher anthocyanins, while the attt8 mutant and the FtTT8-transformed plants had similar anthocyanin content, and both were low (Figure 4C); the attt8 mutant had lower PA content than the WT, but PA content was improved in the FtTT8-transformed plants, and close to the WT (Figure 4D), which was consistent with phenotype investigation.

Analysis of Subcellular Location and Transcription Activation of FtTT8
Subcellular location analysis displayed that FtTT8-GFP merged with RFP (the nucleus location reference), while the GFP from the control vector was distributed throughout the cell (Figure 3A).This implied that FtTT8 is located in the nucleus and plays the role of a transcript factor.The result of the yeast two-hybrid assay suggests that the transcript factor FtTT8 possesses transcription activation activity (Figure 3B).To analyze the effect of FtTT8 on the expression of key genes in the anthocyanin and PA biosynthesis pathway, the expression levels of CHS, CHI, F3H, DFR, ANS, UFGT, and ANR in the leaves of the WT, attt8 mutant, and FtTT8-transformed plants were analyzed by qRT-PCR.In the WT plants, except for ANR, the expression levels of other genes were very low, in the FtTT8-overexpression plants, most genes were up regulated, especially CHS, CHI, F3H, and ANR, which had higher expressions than those in the WT plants (Figure 4E).Key genes shared in the anthocyanin and PA biosynthesis pathway DFR and ANS increased slightly, while UFGT expression remained very low, not overly different from that of the mutant, but the gene in the key and speed-limiting enzyme ANR, which leads to the PA biosynthesis pathway, was significantly upregulated in the FtTT8overexpression lines compared to the attt8 mutant, and even higher than in the WT plant (Figure 4E).

Complementation of the Arabidopsis tt8 Mutant by FtTT8
Complementation analysis in the Arabidopsis attt8 mutant with FtTT8 driven by the CaMV35S promoter was conducted to verify the function of FtTT8 in the regulation of anthocyanin and PA biosynthesis.FtTT8 transgenic Arabidopsis plants (three of each) were screened out and they showed a much higher expression level of FtTT8, whereas the wildtype (WT) and attt8 mutant had no expression of this gene (Figure 4A).Firstly, when observing the seedlings of these tree types, the color red could obviously be observed in the leaves and hypocotyl of WT seedlings, while no red was evident in the attt8 mutant and the FtTT8 over expression lines (Figure 4A), indicating that FtTT8 was unable to promote anthocyanin biosynthesis.Meanwhile, DMACA staining was performed for PA analysis.The results showed that the attt8 mutant contained less PA than the WT; however, the PA content of the attt8 mutant transformed by FtTT8 increased remarkably, nearly to the WT plant level (Figure 4B).Content measurement of the total anthocyanins and PAs showed that the WT contained higher anthocyanins, while the attt8 mutant and the FtTT8-transformed plants had similar anthocyanin content, and both were low (Figure 4C); the attt8 mutant had lower PA content than the WT, but PA content was improved in the FtTT8transformed plants, and close to the WT (Figure 4D), which was consistent with phenotype investigation.
To analyze the effect of FtTT8 on the expression of key genes in the anthocyanin and PA biosynthesis pathway, the expression levels of CHS, CHI, F3H, DFR, ANS, UFGT, and ANR in the leaves of the WT, attt8 mutant, and FtTT8-transformed plants were analyzed by qRT-PCR.In the WT plants, except for ANR, the expression levels of other genes were very low, in the FtTT8-overexpression plants, most genes were up regulated, especially CHS, CHI, F3H，and ANR, which had higher expressions than those in the WT plants (Figure 4E).Key genes shared in the anthocyanin and PA biosynthesis pathway DFR and ANS increased slightly, while UFGT expression remained very low, not overly different from that of the mutant, but the gene in the key and speed-limiting enzyme ANR, which leads to the PA biosynthesis pathway, was significantly upregulated in the FtTT8-overexpression lines compared to the attt8 mutant, and even higher than in the WT plant (Figure 4E).The color of the WT seed coat was brown, while that of the att8 mutant was light yellow.However, after the attt8 mutant was complemented by FtTT8, the seed coat color returned to brown (Figure 5A).Consistently, DMACA staining showed that attt8 mutant seed coats nearly had no PAs, while the seed coat color of the WT and transgenic plants changed to dark brown (Figure 5B), suggesting that they contained high levels of PAs content, and this was verified by the measurement of PA content (Figure 5C).Additionally, most common genes shared in the anthocyanin and PA biosynthesis pathway were upregulated in the FtTT8-transformed seeds (Figure 5D), whereas, UFGT was still lowly expressed and had no significant difference from that of the attt8 mutant; however, ANR expression increased to even higher levels than those of the WT plants (Figure 5D).All the The color of the WT seed coat was brown, while that of the att8 mutant was light yellow.However, after the attt8 mutant was complemented by FtTT8, the seed coat color returned to brown (Figure 5A).Consistently, DMACA staining showed that attt8 mutant seed coats nearly had no PAs, while the seed coat color of the WT and transgenic plants changed to dark brown (Figure 5B), suggesting that they contained high levels of PAs content, and this was verified by the measurement of PA content (Figure 5C).Additionally, most common genes shared in the anthocyanin and PA biosynthesis pathway were upregulated in the FtTT8-transformed seeds (Figure 5D), whereas, UFGT was still lowly expressed and had no significant difference from that of the attt8 mutant; however, ANR expression increased to even higher levels than those of the WT plants (Figure 5D).All the above results suggest that FtTT8 could not accelerate anthocyanin accumulation but promotes PA biosynthesis in Arabidopsis.

Overexpression of FtTT8 in K326 Tobacco
To further identify the function of FtTT8 in the regulation of anthocyanin and PA biosynthesis, overexpression of FtTT8 in K326 tobacco (with red flowers) was conducted.The flower of wild-type K326 tobacco had no expression of the FtTT8 gene, and its color was dark red; however, the FtTT8-transformed tobacco flower became light pink with high expression levels of FtTT8 (Figure 6A).After DMACA staining, the brown color had no obvious difference between the WT and transgenic plants, and brown spots were similarly detected under the microscope (Figure 6B).Consistent with the results, the total anthocyanin content of the transgenic tobacco flower was significantly lower than that in theWT flower (Figure 6C), and the PA content showed no obvious difference between them (Figure 6D).The effect of FtTT8 on the expression profiles of structural genes in the anthocyanin and PA biosynthesis pathway were analyzed by qRT-PCR.Except for F3H and DFR, which were more highly expressed in the transgenic tobacco flower, the other genes were downregulated in the transgenic tobacco flower (Figure 6E).
Interestingly, PAs accumulated more in the FtTT8-transformed tobacco leaves than in WT plants, but anthocyanin pigment was invisible to the naked eye (Figure 7A).Detecting the content of anthocyanins and PAs showed that total anthocyanins accumulated less while PAs accumulated more (about a three-fold difference) in transgenic tobacco leaves by FtTT8 than those in WT plants (Figure 7B,C).When analyzing the expression levels of structural genes in the anthocyanin and PA biosynthesis pathway, common genes, including CHS, CHI and F3H, were slightly upregulated in the FtTT8-transformed tobacco leaves, while DFR and ANS were greatly increased (Figure 7D).However, UFGT, which catalyzed anthocyanin production, was downregulated in the transgenic plants, which was consistent with the lower content of total anthocyanin.However, surprisingly, in the two key genes, ANR and LAR, that lead to the branch of PA synthesis, the former saw no change, while the latter showed a dramatic approximately 250-fold increase in the transgenic plants (Figure 7D), indicating that LAR plays the main role in the catalyzation of PA production.These gene expression profiles were consistent with the pigment accumulation.All of the results suggest that FtTT8 negatively regulates anthocyanin biosynthesis but positively regulates PA biosynthesis when transformed in K326 tobacco.above results suggest that FtTT8 could not accelerate anthocyanin accumulation but promotes PA biosynthesis in Arabidopsis.ilarly detected under the microscope (Figure 6B).Consistent with the results, the total anthocyanin content of the transgenic tobacco flower was significantly lower than that in theWT flower (Figure 6C), and the PA content showed no obvious difference between them (Figure 6D).The effect of FtTT8 on the expression profiles of structural genes in the anthocyanin and PA biosynthesis pathway were analyzed by qRT-PCR.Except for F3H and DFR, which were more highly expressed in the transgenic tobacco flower, the other genes were downregulated in the transgenic tobacco flower (Figure 6E).

FtTT8 Combined to the Promoters of NtUFGT and NtLAR
UFGT and LAR were the speed-limiting enzymes that led to the anthocyanin synthesis branch and PA synthesis branch, respectively, in K326 tobacco, and the results of the qRT-PCR (Figures 6E and 7D) show that NtUFGT displayed significantly different expression in the flowers and leaves of the WT and transgenic plants, and NtLAR expressed remarkable differently between the WT and transgenic tobacco leaves.Furthermore, both the 2mp promoters of NtUFGT and NtLAR contain G-box (CACGTG) (Figure 8A,B), which is reported to be a bHLH binding site [40].Therefore, we speculate that NtUFGT and NtLAR might be the downstream target genes of the transcription factor FtTT8.To verify this hypothesis, a yeast one-hybrid experiment was performed, and the results suggested that FtTT8 could combine with the promoters of NtUFGT (Figure 8C) and NtLAR (Figure 8D), respectively.

FtTT8 Combined to the Promoters of NtUFGT and NtLAR
UFGT and LAR were the speed-limiting enzymes that led to the anthocyanin synth sis branch and PA synthesis branch, respectively, in K326 tobacco, and the results of t qRT-PCR (Figures 6E and 7D) show that NtUFGT displayed significantly different expre

The Different Functions of FtTT8 in the Regulation Anthocyanin and Proanthocyanidin Biosynthesis
It has been well documented that AtTT8 positively regulates anthocyanin and PA biosynthesis in Arabidopsis [41], and that its homologs in other plant species, such as pear [42,43], lotus [44], strawberry [45], and Medicago truncatula [46], among others, have similar functions.For example, our previous study showed that in NnTT8-overexpression Arabidopsis, lotus NnTT8 can interact with AtMYB114 to promote anthocyanin and PA accumulation in leaves and seeds, which may be due to activating transcript levels of ANS and UFGT genes [44].However, some bHLH TFs, including BnbHLH92a in Brassica napus [47] and bHLH92 in sheepgrass [48], inhibited anthocyanin and PA accumulation.Hu et al. reported that BnbHLH92a binds directly to the ANS gene promoter and represses its expression.In addition, BnbHLH92a can interact with a WD40 protein (BnTTG1) and represses the biosynthesis of anthocyanins and PAs in rapeseed [47].Overexpression of LcbHLH92 in Arabidopsis significantly inhibited the expression levels of the DFR and ANS genes in leaves and seeds, and led to a decrease in anthocyanins and proanthocyanidins, respectively [48].Phylogenetic relationship analysis showed that FtTT8 was closely related to LfTT8 and PhAN1 (Figure 1), all of them belonging to the IIIf-1 subfamily of bHLH TFs.Furthermore, like other bHLH regulators of anthocyanin and PA synthesis, the FtTT8

The Different Functions of FtTT8 in the Regulation Anthocyanin and Proanthocyanidin Biosynthesis
It has been well documented that AtTT8 positively regulates anthocyanin and PA biosynthesis in Arabidopsis [41], and that its homologs in other plant species, such as pear [42,43], lotus [44], strawberry [45], and Medicago truncatula [46], among others, have similar functions.For example, our previous study showed that in NnTT8-overexpression Arabidopsis, lotus NnTT8 can interact with AtMYB114 to promote anthocyanin and PA accumulation in leaves and seeds, which may be due to activating transcript levels of ANS and UFGT genes [44].However, some bHLH TFs, including BnbHLH92a in Brassica napus [47] and bHLH92 in sheepgrass [48], inhibited anthocyanin and PA accumulation.Hu et al. reported that BnbHLH92a binds directly to the ANS gene promoter and represses its expression.In addition, BnbHLH92a can interact with a WD40 protein (BnTTG1) and represses the biosynthesis of anthocyanins and PAs in rapeseed [47].Overexpression of LcbHLH92 in Arabidopsis significantly inhibited the expression levels of the DFR and ANS genes in leaves and seeds, and led to a decrease in anthocyanins and proanthocyanidins, respectively [48].Phylogenetic relationship analysis showed that FtTT8 was closely related to LfTT8 and PhAN1 (Figure 1), all of them belonging to the IIIf-1 subfamily of bHLH TFs.Furthermore, like other bHLH regulators of anthocyanin and PA synthesis, the FtTT8 protein also contained a bHLH domain, a MYB-interacting region (MIR) and ACT-like domain (Figure 2).In addition, FtTT8 was located in the nucleus, and had transcriptional activation activity (Figure 3).These results indicate that FtTT8 was a transcription factor which may have a similar function to the other IIIf-1 subfamily of bHLHs in regulating anthocyanin and PA biosynthesis.
To verify this assumption, the complementation analysis of the Arabidopsis tt8 mutant with FtTT8 was conducted.The results were not consistent with the predicted results as there was no accumulation of anthocyanin pigments observed in the transgenic plant, where the color was just close to the colorless attt8 mutant (Figure 4A).Additionally, the low anthocyanin content and no significant change in the expression of the anthocyanin-related gene AtUFGT, which encodes the speed-limiting enzyme (Figure 4C,E), indicated that FtTT8 cannot enhance anthocyanin accumulation in Arabidopsis.Conversely, the seed coat color of the attt8 mutant was recovered by transformation with FtTTT8 from light yellow to brown (Figure 5A), and DMACA staining (Figures 4B and 5B), measurement of proanthocyanidin content (Figures 4D and 5C) of Arabidopsis leaves and seeds, as well as the expression level of the speed-limiting anthocyanin-related genes AtANR (Figures 4B and 5D), all suggested that FtTT8 was able to promote proanthocyanidin biosynthesis.
The transformation of K326 tobacco (with red flowers) with FtTT8 was conducted to further prove the functions of FtTT8.To our surprise, the flower color of the transgenic tobacco turned lighter than that of the WT (Figure 6A), and lower contents of anthocyanins in the flowers and leaves of the transgenic plant (Figures 6C and 7B), as well as the downregulated expression level of the speed-limiting gene of anthocyanin biosynthesis NtUFGT (Figures 6E and 7D), indicated that FtTT8 prevented anthocyanin biosynthesis in tobacco.Although PA accumulation in the FtTT8-transformed tobacco showed no significant change in the flowers compared with the WT (Figure 6B,D), deeper DMACA staining (Figure 7A), a large increase in PA content (Figure 7C), and remarkably upregulated expression of NtLAR (Figure 7D) in the leaves of transgenic tobacco, demonstrated that FtTT8 promoted PA accumulation in tobacco leaves.
Therefore, unlike other bHLH TFs, which either simultaneously improved anthocyanin and PA biosynthesis [44,46], or simultaneously suppressed anthocyanin and PA accumulation [47,48], FtTT8 possessed bidirectional regulating effects on anthocyanin and PA biosynthesis that negatively regulated anthocyanin biosynthesis and positively regulated PA biosynthesis.This finding is new and interesting; however, the mechanism of different roles of FtTT8 on regulation of anthocyanin and PA biosyntheis pathways is unknown, it may be due to several different amino acid residues in the conserved domains in FtTT8 or for other reasons.The mechanism of competitiveness between the synthesis of both secondary metabolites remains unclear.Furthermore, if FtTT8 regulates other flavonoid composition syntheses, such as flavones, or flavonols, what regulates FtTT8 as well as which MYB and WD40 TFs can combine with it to play this function, need to be studied.

FtTT8 Interacted with the Promoter of NtUFGT and NtLAR to Play Roles in the Regulation of Anthocyanin and Proanthocyanidin Biosynthesis
It has been reported that bHLH TFs play roles by interaction with G-box DNA element of their regulated gene in most plants [40,49].For example, AtbHLH106 confers salt tolerance on Arabidopsis by integrating the functions of multiple genes through its G-box [50].NtUFGT and NtLAR were the speed-limiting enzymes, which lead to the anthocyanin and proanthocyanidin synthesis branch, respectively in tobacco (Figure 9).Combined with the expression level obtained by qRT-PCR, that showed downregulation of NtUFGT (Figure 6E) and upregulation of NtLAR (Figure 7D), which was consistent with anthocyanin (Figure 6A,C) and PA accumulation (Figure 7A,C), both NtUFGT and NtLAR promoters contained G-box (Figure 8A,B), indicating that NtUFGT and NtLAR were probably the downstream target genes of FtTT8.Therefore, we speculate that FtTT8 interacts with the G-box of the NtUFGT promoter, and represses the expression of NtUFGT, resulting in less anthocyanin biosynthesis in transgenic tobacco flowers and leaves.Meanwhile, FtTT8 inter-acts with the G-box of the NtLAR promoter, and activates the expression of NtLAR, leading to improved proanthocyanidins accumulation in transgenic tobacco leaves (Figure 9).anthocyanin biosynthesis in transgenic tobacco flowers and leaves.Meanwhile, FtTT8 interacts with the G-box of the NtLAR promoter, and activates the expression of NtLAR, leading to improved proanthocyanidins accumulation in transgenic tobacco leaves (Figure 9).

Plant Materials
The seeds of Tartary buckwheat cultivar 'Jinqiao 2' from the Research Center of Buckwheat Industry Technology, Guizhou Normal University were planted based on the paper bed germination method with some modification [51], and placed in an artificial climate box at 25 °C, under a 16 h/8 h photoperiod and 80% humidity.The seven-day sprouts were collected and frozen in liquid N2 immediately and stored at −80 °C for further experiments.

Cloning and Characterization of FtTT8
The protein of Tartary buckwheat TT8 (FtTT8) was obtained by homologous alignment with Arabidopsis AtTT8 (AT4G09820) based on a Tartary buckwheat database (http://mbkbase.org/Pinku1/(accessed on 8 August 2023)), and full-length specific primers were designed using Primes 5 according to the ORF sequence of the FtTT8 gene.The primer sequences are listed in Table S1.
Extraction of the total RNA of the samples mentioned above was performed using an OminiPlant RNA kit (Kangwei, Beijing, China), and cDNA was synthesized using a Re-verTra Ace ® qPCR RT kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions.Using the cDNA as the template, and full-length specific primers obtained above, the CDS of FtTT8 was amplified by a PCR program using high-fidelity thermostable DNA polymerase (TaKaRa, Dalian, China).Then, the amplified PCR product was cloned to T vector (pMD19-T, TaKaRa, China), and transformed to E.coil strain DH5α and the detected positive clones were sent to a biological company (Sangon Biotech, Shanghai, China) for sequencing.Phylogenic analysis was achieved using MEGA 7.026 software [52], and motif prediction was analyzed by MEME (https://meme-suite.org/meme/tools/meme (accessed

Plant Materials
The seeds of Tartary buckwheat cultivar 'Jinqiao 2' from the Research Center of Buckwheat Industry Technology, Guizhou Normal University were planted based on the paper bed germination method with some modification [51], and placed in an artificial climate box at 25 • C, under a 16 h/8 h photoperiod and 80% humidity.The seven-day sprouts were collected and frozen in liquid N 2 immediately and stored at −80 • C for further experiments.

Cloning and Characterization of FtTT8
The protein of Tartary buckwheat TT8 (FtTT8) was obtained by homologous alignment with Arabidopsis AtTT8 (AT4G09820) based on a Tartary buckwheat database (http:// mbkbase.org/Pinku1/(accessed on 8 August 2023)), and full-length specific primers were designed using Primes 5 according to the ORF sequence of the FtTT8 gene.The primer sequences are listed in Table S1.
Extraction of the total RNA of the samples mentioned above was performed using an OminiPlant RNA kit (Kangwei, Beijing, China), and cDNA was synthesized using a ReverTra Ace ® qPCR RT kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions.Using the cDNA as the template, and full-length specific primers obtained above, the CDS of FtTT8 was amplified by a PCR program using high-fidelity thermostable DNA polymerase (TaKaRa, Dalian, China).Then, the amplified PCR product was cloned to T vector (pMD19-T, TaKaRa, China), and transformed to E.coil strain DH5α and the detected positive clones were sent to a biological company (Sangon Biotech, Shanghai, China) for sequencing.Phylogenic analysis was achieved using MEGA 7.026 software [52], and motif prediction was analyzed by MEME (https://meme-suite.org/meme/tools/meme (accessed on 12 August 2023)), then visualized using the TBtools program [53].bHLH transcription factors from other species involved in the regulation of anthocyanin and PA biosynthesis were obtained from the NCBI database by sequence alignments with FtTT8.Multi-alignment was performed using the online website for multiple sequence alignment by CLUSTALW (https://www.genome.jp/tools-bin/clustalw(accessed on 13 August 2023)).

Arabidopsis Protoplast Transient Expression Assay
The CDS of FtTT8 was amplified and cloned into the pC1300S-GFP vector using the homologous recombination method, and the specific primers are listed in Table S1.The recombined vector pC1300S-GFP-FtTT8 and reference vector Ghd7:RFP (located in the nucleus) were co-transformed into Arabidopsis protoplast following a previous protocol [54].While the empty vectors pC1300S-GFP and Ghd7:RFP were co-transformed as a control.The observation of GFP and RFP signals was performed on confocal laser scanning microscope (STELLARIS 8, Leica, Wetzlar, Germany).

Yeast Two-Hybrid Assay
The CDS of FtTT8 was amplified by specific primers (listed in Table S1) and recombined into a pGBKT7 (BD) vector, then the combination vector FtTT8-BD was co-transformed into the AH109 yeast strain with the PGADT7 (AD) vector using the LiAc-PEG-mediated transformation method.The combination of AD and BD was used as a negative control, while pGADT7-T and pGBKT7-p53 were used as positive controls.These yeast cells were cultured on a medium which lacked Leu and Trp (SD/-L/-T), followed by transferring several clones onto a medium lacking Leu, Trp, His, and Ade (SD/-L/-T/-H/-A), and SD/-L/-T/-H/-A with X-α-gal, respectively.

Transformation of Arabidopsis tt8 Mutant and Tobacco by FtTT8 Gene
The CDS of FtTT8 was amplified by PCR using specific primers (Table S1) and recombined into the overexpression vector PMDC83, the constructed 35S:FtTT8 vector was transformed into the Arabidopsis tt8 mutant using the Agrobacterium-mediated floral dip method [16,54].The T3 homozygous transgenic lines were used for further analysis.Meanwhile, the 35S:FtTT8 transformed into tobacco (K326 cultivar with red flowers) by Agrobacterium through leaf disc transformation and the pigmentation of the transgenic tobacco was analyzed.

Detection of Anthocyanins and Proanthocyanidins (PAs)
The total anthocyanins of the fresh leaves of the WT and transgenic Arabidopsis plants were extracted following the protocol described by Park et al. [55].Then, the extracted solutions were measured at absorption lengths of 530 nm and 657 nm using a UV9600 spectrophotometer (Beifen-Ruili, Beijing, China), and the total anthocyanin content was obtained by the formula Q Anthocyanin = (A 530 − 0.25 × A 657 )/M (Q represents the total anthocyanin content, M represents the sample weight).Each sample was processed with three biological replicates.
p-Dimethylaminocinnamaldehyde (DMACA) solution (0.1% w/v in methanol containing 1% (v/v) HCl) was used to visualize the PAs according to the method described by Abeynayake et al. [56].Briefly, fresh samples (Arabidopsis seeds, leaves, and tobacco leaves and flowers) were soaked in GAA solution (ethyl alcohol: glacial acetic acid = 1:1 (v/v)) for full de-coloration first, then stained with 0.1% DMACA for 20 min with slow shaking.After staining, the samples were washed with 70% ethyl alcohol until the color was stable and were examined under the microscope.Meanwhile, the content of PAs was measured.In brief, firstly, standard of procyanidin B2 was accurately weighted and dissolved in methanol, which was then diluted 2-fold to produce appropriate concentration ranges, which were 7.81~125 µg/mL, to establish the calibration curve in order to quantify the PAs, while methanol was used as blank control.Samples were ground into fine power in liquid N 2 , then weighed at 5 mg into a 10 mL tube, respectively, 5 mL methanol was added, and ultrasonic extraction was performed for 20 min.After centrifugation, the filter residue was washed with methanol 3 times, and the washing liquids were combined with filtrates into a 10 mL measuring flask and diluted with methanol to volume and mixed well.Then, 1 mL of standard series solution and sample solution were collected, respectively, and then mixed with 6 mL of HCl-n-butanol (5:95, v/v) and 0.2 mL of NH 4 FeSO 4 and heated in a boiling water bath for 40 min.They were then cooled to room temperature in ice water and the absorbance at the wavelength of 546 nm of the UV9600 spectrophotometer (Beifen-Ruili, Beijing, China) was measured.The PA content was obtained by calibration curve.

qRT-PCR Analysis
The expression levels of the structural genes on the pathways of anthocyanin and PA biosynthesis (AtCHS, AtCHI, AtF3H, AtDFR, AtANS, AtUFGT and AtANR) in wildtype (WT) and transgenic Arabidopsis thaliana seedlings and seeds, as well as NtCHS, NtCHI, NtF3H, NtDFR, NtANS, NtUFGT, NtLAR, and NtANR in WT and transgenic K326 tobacco were detected via qRT-PCR.QRT-PCR was performed using the C1000 TM thermal cycler coupled with a CFX96 TM detection module (Bio-Rad, Santa Clara, CA, USA) using the 2 × iQ TM SYBR Green Super mix (Bio-Rad, Santa Clara, CA, USA).AtActin [57] and NtActin [58] were used as the internal controls.The expression levels of the genes were obtained by the 2 −∆∆Ct method with three technical replicates.The primers of these genes are listed in Table S1.

Yeast One-Hybrid Analysis
A 2000 bp promoter sequence of AtUFGT and AtANR was amplified by PCR and cloned into the pHIS2 vector, respectively, and the CDS of FtTT8 was cloned into a pGADT7 (AD) vector.Then, the combination vector AD-FtTT8 was co-transformed into the AH109 yeast strain with vectors pHIS2-proAtUFGT and pHIS2-proAtANR, respectively.The combination of AD-p53 and pHIS2-p53 was used as a positive control, while the combinations of AD-p53 and pHIS2-proAtUFGT/pHIS2-proAtANR, AD-FtTT8, and pHIS2-p53 were negative controls.These yeast cells were cultured on a medium which lacks Leu and Trp (SD/-L/-T), then several clones were transferred to a medium lacking His, Leu, and Trp with 3AT (60 mM).The primers of these genes are listed in Table S1.

Conclusions
In the present study, FtTT8, a bHLH transcription factor, is located in the nucleus and exhibits transcriptional activation activity.Unlike TT8 from other plant species, which either promotes both anthocyanin and PA biosynthesis or inhibits the biosynthesis of these flavonoid components, FtTT8 suppresses anthocyanin biosynthesis but improves PA accumulation.In addition, FtTT8 may inhibit UFGT expression and activate LAR expression, respectively, by binding to G-boxes in the promoters of these two key genes that regulates anthocyanin and PA biosynthesis.This new insight enriches the roles of TT8 and sets the stage for further in-depth studies of the mechanism of TT8 regulation in flavonoid biosynthesis.

Figure 2 .
Figure 2. Protein sequence alignment of FtTT8 and other bHLH proteins that regulate anthocyanin and proanthocyanidin synthesis.Note: All nine species are the same as those listed in Figure 1.

Figure 2 .
Figure 2. Protein sequence alignment of FtTT8 and other bHLH proteins that regulate anthocyanin and proanthocyanidin synthesis.Note: All nine species are the same as those listed in Figure 1.

Figure 4 .
Figure 4. Complementation analysis of the Arabidopsis tt8 mutant seedlings with FtTT8.(A) Seedlings of the WT, attt8 mutant, and FtTT8-transformed plants and expression of FtTT8 in three types of Arabidopsis plants.(B) DMACA staining of leaves of the WT, attt8 mutant, and FtTT8 overexpressing Arabidopsis plants.(C) Total anthocyanin content.(D) PA content.(E) Expression changes of the anthocyanin and PA biosynthesis pathway genes by the overexpression of FtTT8 in Arabidopsis leaves (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4 .
Figure 4. Complementation analysis of the Arabidopsis tt8 mutant seedlings with FtTT8.(A) Seedlings of the WT, attt8 mutant, and FtTT8-transformed plants and expression of FtTT8 in three types of Arabidopsis plants.(B) DMACA staining of leaves of the WT, attt8 mutant, and FtTT8 overexpressing Arabidopsis plants.(C) Total anthocyanin content.(D) PA content.(E) Expression changes of the anthocyanin and PA biosynthesis pathway genes by the overexpression of FtTT8 in Arabidopsis leaves (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6 .Figure 6 .
Figure 6.Overexpression analysis of FtTT8 in the K326 tobacco flower.(A) Flowers of WT and FtTT8-transformed K326 tobacco (OE, overexpression).(B) DMACA staining of flowers of WT and FtTT8 overexpressing tobacco.(C) Total anthocyanin content and (D) PA content in the WT andFigure 6. Overexpression analysis of FtTT8 in the K326 tobacco flower.(A) Flowers of WT and FtTT8-transformed K326 tobacco (OE, overexpression).(B) DMACA staining of flowers of WT and FtTT8 overexpressing tobacco.(C) Total anthocyanin content and (D) PA content in the WT and FtTT8 overexpressing tobacco flowers.(E) Expression changes of the anthocyanin and PA biosynthesis pathway genes by the overexpression of FtTT8 in tobacco flowers (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7 .
Figure 7. Overexpression analysis of FtTT8 in K326 tobacco leaves.(A) DMACA staininf flowers of WT and FtTT8 overexpressing tobacco.(B) Total anthocyanins content and (C) PAs content in WT and FtTT8 overexpressing tobacco leaves.(D) Expression changes of anthocyanin and PA biosynthesis pathway genes by the overexpression of FtTT8 in tobacco leaves (** p < 0.01, *** p < 0.001).

Figure 8 .
Figure 8. Analysis of the promoters of NtUFGT and NtLAR and yeast one-hybrid assays.G-box in the promoter of NtUFGT (A) and NtLAR (B), yeast one-hybrid assays for the combination between the FtTT8 and NtUFGT promoters (C), and FtTT8 and NtLAR (D), respectively.

Figure 8 .
Figure 8. Analysis of the promoters of NtUFGT and NtLAR and yeast one-hybrid assays.G-box in the promoter of NtUFGT (A) and NtLAR (B), yeast one-hybrid assays for the combination between the FtTT8 and NtUFGT promoters (C), and FtTT8 and NtLAR (D), respectively.

Figure 9 .
Figure 9. Anthocyanin and PA biosynthesis pathways and FtTT8 regulation in K326 tobacco.Note: ↑ means positive regulation and ↓ means negative regulation.

Figure 9 .
Figure 9. Anthocyanin and PA biosynthesis pathways and FtTT8 regulation in K326 tobacco.Note: ↑ means positive regulation and ↓ means negative regulation.