Screening of FtUFGT genes in tartary buckwheat
To further study the UFGTs involved in flavonoid synthesis in tartary buckwheat, we used UDP-glucose: flavonoid 3-O-glucosyltransferase as a probe to screen the transcriptome of tartary buckwheat [19]. We obtained 41 UFGT Unigenes by scanning the transcriptome database. For further analysis of the function of these genes, we selected 34 Arabidopsis thaliana UGT genes that were previously used to construct a phylogenetic tree [20] (Additional file 1: Figure S1). As observed in a phylogenetic tree, UFGT in buckwheat was divided into 12 subfamilies (A, B, C, D, E, F, G, H, J, L, M, and P) and had different biological functions. On this basis, we selected seven unrevealed UFGT genes that were related to flavonoid synthesis and had a relatively high level of expression in the transcriptome for further study. All seven full-length UFGT genes were named, FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, FtUFGT15, FtUFGT40, and FtUFGT41, and were submitted to GenBank with accession numbers MG267387-MG267393. The genomic structure of these seven genes was analyzed by comparing their gDNA and cDNA sequences. There are three forms of intron-exon structures in these seven FtUFGT genes: type I contained three exons and two introns (FtUFGT41), type II contained two exons and one intron (FtUFGT15), and type III contained only one exon (FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, and FtUFGT40) (Additional file 2: Figure S2).
Sequence analyses of FtUFGTs
Multiple sequence analysis showed that the 7 UFGTs shared a conserved domain with the plant secondary product glycosyltransferase (PSPG) motif (Fig. 1) near their C-terminal domain, and the highly conserved amino acids were in positions 1 (W), 4 (Q), 8 (L), 10 (H), 12 (A/S), 14 (G), 16 (F/C), 19–24 (HC/SGW/FN/GS), 27 (E), 32 (G/N), 39 (P), 43 (E/D), and 44 (Q). This is consistent with the glycosyltransferases that are known to function in the biosynthesis of plant secondary metabolites [20]. The final glutamine (Q) residue within the PSPG motif is thought to confer specificity for UDP-glucose as the sugar donor [21]. Notably, all 7 UFGTs possess this Q, suggesting they may all use UDP-glucose as a sugar donor. The phylogenetic tree of putative FtUFGTs and Arabidopsis UDP glycosyltransferases indicated four clusters, which appear to be characterized by the specificity of the flavonoid glycosyltransferase activities (Fig. 2). Clusters I, II and III are characterized by flavonoid 3-O-glycosyltransferases, flavonoid 5-O-glycosyltransferases and flavonoid 7-O-glycosyltransferases, respectively. The results showed that six FtUFGT genes, including FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, FtUFGT40, and FtUFGT41, were clustered into the UF7GT cluster, and FtUFGT15 belonged to the UF5GT cluster. Additionally, P. frutescens and P. hybrida UF3GT did not cluster with P. frutescens and P. hybrida UF5GT, although they were derived from the same species. These results implied that the seven UDP glycosyltransferase clusters diverged before the speciation of monocot and dicot plants as reported by Imayama et al. [22] To further understand the relationship between these genes, we examined the location of the seven genes on the tartary buckwheat chromosomes. FtUFGT8 and FtUFGT15 are on chromosome Ft3, and the remainder is on different chromosomes. FtUFGT6 is on chromosome Ft7, FtUFGT7 is on chromosome Ft6, FtUFGT40 is on chromosome Ft5, and FtUFGT41 is on chromosome Ft2. However, we could not locate FtUFGT9 on the tartary buckwheat genome, possibly because of the differences between different species.
Expression of FtUFGT genes in different tissues
Tissue-specific expression of genes is often associated with specific developmental and physiological functions. Therefore, we detected the expression levels of these UFGT genes in three tissues (root, stem, and leaf) at different growth stages (seedling stage, cotyledon stage, true leaf stage, full-leaf stage, and full-bloom stage) of tartary buckwheat by real-time quantitative PCR (qRT-PCR). Most genes showed different expression patterns in tartary buckwheat, whereas FtUFGT15 and FtUFGT41 presented a similar expression pattern (Additional file 3: Figure S3). After the seedling stage, the expression levels of these two genes were highest in stems, followed by leaves, and lowest in roots, implying that they may participate in similar biological pathways. By contrast, both FtUFGT6, FtUFGT7, and FtUFGT40 have the highest expression in roots at different growth stages. Particularly, FtUFGT7 was almost undetectable in the leaves and stems after the seedling stage, suggesting that FtUFGT7 may be a root-specific gene. Additionally, the expression levels of FtUFGT8 and FtUFGT9 in the leaves were always at a relatively low level but showed opposite trends in roots and stems. At the same time, transcriptome data analysis and qRT-PCR were performed to determine the expression levels of these genes in flowering tartary buckwheat (Fig. 3a). The results indicated that gene expression did not change markedly compared with the early stage and remained consistent, maintaining almost the same tissue specificity in different growth stages of tartary buckwheat (Additional file 3: Figure S3).
Isolation and sequence analysis of the pFtUFGTs
To further reveal the response of the FtUFGT genes to the external environment and predict the regulatory pathways they may be involved in, we cloned the promoters starting between 1567 and 1594 bp upstream of the ATG start codon (Additional file 4: Figure S4). Analysis of the cis-regulatory elements in the promoter showed that these elements were classified into two groups based on their responsive functions: stress-responsive and hormone-responsive elements (Additional file 1: Table S2). The stress-responsive elements mainly included light-responsive elements (e.g., Box 4, ATTAAT), low-temperature (e.g., LTR, CCGAAA), drought (e.g., MBS, CAACTG), and high-temperature responsive elements (e.g., HSE, AGAAAATTCG). The hormone-responsive elements included abscisic acid (ABA)-responsive elements (ABRE, ACGTG), an auxin-responsive element (e.g., TGA-element, AACGAC), and the MeJA-responsive element (e.g., CGTCA-motif, CGTCA). Furthermore, several other types of cis-acting elements were found in these promoter sequences, including many TATA boxes, CAAT boxes, and MYB binding sites. The results of the analysis showed that these promoters contain numerous photoresponsive elements, and we also found that pFtUFGT8/15/41 contains more low-temperature response components than the other four promoters (Additional file 1: Table S1). It is well known that illumination directly affects the secondary metabolism of plants, and low temperature can induce the accumulation of anthocyanins by activating the expression of anthocyanin synthesis-related genes [23, 24]. Therefore, we speculate that FtUFGT8/15/41 may be involved in the production of anthocyanin.
FtUFGT8/15 gene expression is correlated with anthocyanin accumulation after cold treatment
Based on the analysis result of pFtUFGTs, we carried out low-temperature treatment on the tartary buckwheat seedlings and explored the effects of low temperature on the synthesis of anthocyanins and expression of FtUFGT genes. We found that, compared with the control group, the anthocyanin content of tartary buckwheat increased significantly after cold stress, and there was a significant difference after 2 h (P < 0.01) (Additional file 5: Figure S5). The difference was greatest after 3 h, which was 1.72 fold that of the control group. Similar results have been reported in previous literature [18, 24].
To analyze the relationship between these FtUFGT genes and anthocyanin accumulation, the expression profiles of FtUFGTs in tartary buckwheat under cold treatment were analyzed by qRT-PCR. Overall, the seven FtUFGT genes showed different expression patterns under cold stress (Additional file 5: Figure S5). The expression of the five FtUFGT genes, FtUFGT8, FtUFGT9, FtUFGT15, FtUFGT40, and FtUFGT41, were clearly enhanced. Among them, the response of FtUFGT9 and FtUFGT41 was the most rapid, increasing significantly after 0.5 h of stress and remaining at a relatively high level thereafter. FtUFGT8 and FtUFGT15 expression did not change much in the early stage of stress, and they rose rapidly after 6 h and reached the maximum at 16 h, 10.88-fold and 24.36-fold of the control, respectively. However, the FtUFGT7 gene showed downregulated expression. It remained unchanged within 0–2 h, significantly decreased after 3 h, and reached a minimum at 16 h, which was 0.27-fold that of the control.
Expression of FtUFGTs and flavonoid accumulation in tartary buckwheat sprouts after light treatment
Light is one of the most important environmental factors affecting flavonoid biosynthesis in plants [25]. From the results of the UFGT promoter structure analysis, it was found that the promoter portion of these genes contained numerous photoresponsive elements. Hence, we analyzed the trend between the expression of FtUFGT genes and accumulation of flavonoids in tartary buckwheat under light conditions. The results showed that the accumulation of four flavonols under light conditions indicated different trends (Additional file 6: Figure S6). Among them, the content of rutin was not significantly different from the control within 3 h after treatment, significant differences occurred after 6 h of treatment, lasting 16 h. Additionally, the change in quercetin and kaempferol indicated similar trends. The trend of treatment for 3 h was similar to that of rutin, but the accumulation under light conditions was significantly lower than that under dark conditions after 6 h of treatment. However, the content of myricetin was higher under the dark conditions than in the light throughout the treatment.
Subsequently, the expression profiles of FtUFGTs in tartary buckwheat under light treatment were analyzed by qRT-PCR. Overall, the seven FtUFGT genes indicated different expression patterns under light stress (Additional file 7: Figure S7). The expression of the three FtUFGT genes, FtUFGT6, FtUFGT15, and FtUFGT40, was not significantly different before 3 h of treatment, but there was a significant change after 6 h, and light conditions obviously inhibited the expression of these three genes. Additionally, the expression level of FtUFGT8 was decreased sharply after 0.5 h of treatment and reached the minimum value after 2 h, 0.504 times that of the control. Then, it rose rapidly and reached the maximum value after treatment for 16 h, 3.938 times that of the control group. FtUFGT41 and FtUFGT9 showed a trend of increasing first and then decreasing during the whole process. Overall, these seven genes all responded to light conditions, but the trends were somewhat different. It is speculated that they may play different roles in the flavonoid synthesis pathway.
FtUFGT8, FtUFGT15, and FtUFGT41 increase the anthocyanin content of transgenic plants
To further clarify the function of the seven selected FtUFGT genes in plants, transgenic Arabidopsis thaliana overexpressing FtUFGT genes were obtained by the floral dipping method. Eight resistant strains were selected in each plate and were found to be positive by RT-PCR. The results showed that the FtUFGT genes were expressed in all resistant seedlings but were not detected in wild type (WT) plants (Additional file 8: Figure S8). Thereafter, we selected three transgenic lines with higher FtUFGT gene expression levels among the T1 lines, and T3 homozygous plants were obtained for follow-up experiments.
When the transgenic and WT plants were grown on 1/2MS medium, the transgenic oxFtUFGT8, oxFtUFGT15, and oxFtUFGT41 seedlings developed a slight purple color, indicative of anthocyanin accumulation, which was not present in the four other transgenic lines (Fig. 4a). Therefore, we speculated that FtUFGT8, FtUFGT15, and FtUFGT41 may be involved in the synthesis of anthocyanins. After the plants grew to the flowering stage, their anthocyanin content was determined. Overexpression of the three genes FtUFGT8, FtUFGT15, and FtUFGT41 significantly increased the anthocyanin content of the transgenic plants, which were 2.50-, 1.78-, and 1.66-fold the content of the control group, respectively (P < 0.01) (Fig. 4b).
FtUFGTs affect the accumulation of major flavonols in transgenic Arabidopsis
To clarify the effect of FtUFGT genes on flavonoid biosynthesis in transgenic plants, we tested the three main flavonols (rutin, quercetin, and myricetin) by high performance liquid chromatography (HPLC) (Fig. 5, Additional file 9: Figure S9). For all genes, except for FtUFGT6, overexpression resulted in a significant decrease in rutin content in the transgenic plants (P < 0.05). Among them, FtUFGT8 transgenic plants showed the most significant reduction, at 0.35 fold that of wild type plants (P < 0.01). However, the effect of overexpression of genes on quercetin and rutin showed the opposite trend. Except for FtUFGT6 and FtUFGT9, the overexpression of other genes, including FtUFGT7, FtUFGT8, FtUFGT15, FtUFGT40, and FtUFGT41, significantly increased the content of quercetin in the transgenic plants, by 2.05-, 1.94-, 3.89-, 1.83-, and 2.05-fold of the WT, respectively (P < 0.01).
FtUFGT6 and FtUFGT15 affect the growth and development of transgenic plants
Unexpectedly, the overexpression of two of the FtUFGT genes affected the growth and development of transgenic plants. Leaf size of oxFtUFGT6 and oxFtUFGT15 plants showed greater differences than the WT and other transgenic plants when the same batch of transgenic plants were grown to approximately 40 days (Fig. 6a and b). The whole rosettes of the oxFtUFGT6 plants were significantly larger than those of WT plants, whereas oxFtUFGT15 showed the opposite trend (Fig. 7a). Divergent phenotypes were observed at different stages of development (Fig. 7b). At 47 days, oxFtUFGT6 plants bolted ahead of WT and oxFtUFGT15 plants. By day 51, the stems of oxFtUFGT6 plants and WT plants had grown 27 and 10 cm, respectively. The stems of oxFtUFGT15 plants grew 3 cm on day 57, at which time the plant heights of the other two plants reached 40 cm and 27 cm, respectively. Additionally, overexpression of FtUFGT6 also increased the number of tillers and time for seed maturation of transgenic plants. The number of tillers overexpressing FtUFGT6 reached 4 at 57 days, while the WT and oxFtUFGT15 plants had only one until the end. By the 64th day, the seeds of plants that had overexpressed FtUFGT6 had partially matured, while the other two were still dark green. Because AUXIN RESPONSE FACTORs ARF10 and ARF16 are the major auxin response factors in plants, the relative gene expression levels of ARF10 and ARF16 in these two transgenic plants were measured (Additional file 10: Figure S10). ARF10 and ARF16 expression levels increased in oxFtUFGT6 plants, and the ARF16 change was the most significant, reaching 4.01 times that of the control group. However, there was a different trend in FtUFGT15 transgenic plants. FtUFGT15 did not affect the gene expression of ARF10 but markedly suppressed ARF16 expression.
Taken together, overexpression of FtUFGT6/15 significantly affected the growth and development of transgenic plants. To investigate whether this effect also exists early in plant growth, we measured the developmental speed and root length of transgenic and WT seedlings on 1/2 MS medium. Overexpression of FtUFGT6 significantly increased the early developmental speed of transgenic plants (Fig. 8a). When wild-type plants still have only two cotyledons, most of the transgenic plants of FtUFGT6 have grown true leaves. Moreover, oxFtUFGT6 plant root growth was increased (Fig. 8b and c). On the contrary, oxFtUFGT15 seedling leaf and root growth did not significantly differ from wild type.