Characterization of IbMYB and IbNAC anthocyanin-related genes in sweet potato
In previous reports, TFs AtMYB75 (AtPAP1) in Arabidopsis thaliana and MdMYB10 (DQ267897) and PyMYB114 (ASY06612.1) in nonmodel species were found to be R2R3-type MYBs, which may work together with other TFs in regulating anthocyanin biosynthesis [11, 12, 15]. Here, we found that the Itf12g05820.t1 gene was highly homologous with AtPAP1 (AT1G56650) by BLAST alignment against the sweet potato genome database. It was named IbMYB340 after multiple sequence alignment revealed its similarity with other typical R2R3-MYB TFs in other species. In addition, IbMYB44 was identified from FaMYB44.1 by homologous sequence alignment; FaMYB44 is a transcriptional repressor that negatively regulates sucrose accumulation in strawberry receptacles through interplay with FaMYB10 . Phylogenetic analysis indicated that IbMYB340 belongs to the activator clade, together with other known MYB transcription factors with the R2R3 domain (Fig. 1b). By contrast, TF IbMYB44, which harbors the transcriptional repressor domain LxLxL (Fig. 1c), was phylogenetically related to FaMYB44.1 (Fig. 1b), which acts as a transcriptional repressor in strawberry . Thus, we speculated that these two MYB TFs may regulate anthocyanin biosynthesis in sweet potato.
We retrieved sequence information for 98 NAC TFs containing a NAC domain in the N-terminal region from the sweet potato genome database. Amino acid sequences of NACs in sweet potato and peach PpBL (ALK27819.1), which can promote the activity of PpMYB10.1 by acting as a heterodimer with PpNAC1, resulting in anthocyanin accumulation in tobacco leaves , were used to construct a phylogenetic tree (Fig. 1a). Three NAC genes, IbNAC56a, IbNAC56b, and IbNAC25, were candidates according to the phylogenetic analysis results.
Then, we quantified the expression abundance of candidate genes in tuberous roots of different sweet potato cultivars. As shown in Fig. 2a, the IbNAC56a and IbNAC56b expression levels were significantly higher in purple-fleshed sweet potato cultivars ‘Xuzi No. 8’ and ‘Zhezi No. 3’ than in yellow-fleshed sweet potatoes ‘Sushu No. 8’ and ‘Guangshu No. 87’ or in white-fleshed sweet potatoes ‘Lizixiang’ and ‘Xushu No. 18’, whereas the expression patterns of IbNAC25 were the opposite. Therefore, IbNAC56a and IbNAC56b were selected for subsequent experiments.
Moreover, the transcript abundance of IbMYB340 and IbbHLH2 was significantly higher in ‘Xuzi No. 8’ and ‘Zhezi No. 3’ than in the other cultivars, while the expression of IbMYB44 was maintained at lower levels in purple-fleshed sweet potatoes than in yellow-/white-fleshed sweet potatoes. Furthermore, ‘Lizixiang’ and ‘Xushu No. 18’ presented higher transcript levels of IbMYB44 than did ‘Sushu No. 8’ and ‘Guangshu No. 87’. The expression level of IbANS was also higher in the purple-fleshed cultivars except for ‘Hanzi’, while the yellow-/white-fleshed cultivars showed relatively low gene expression. However, the expression of IbUFGT and IbDFR showed no obvious difference.
Further, we analyzed the correlation among anthocyanin biosynthesis-related gene expression levels in the different sweet potato cultivars. As shown in Fig. 2b, the expression of IbNAC56a, IbNAC56b, IbbHLH2 and IbANS displayed noticeably positive correlations with IbMYB340 expression, and the correlation coefficients ranged from 0.77 to 0.98. However, the expression of IbMYB44 had negative correlations with the expression of IbMYB340, IbNAC56a, IbNAC56b, IbbHLH2, IbANS and IbUFGT, suggesting that there might be an opposite effect of IbMYB340 and IbMYB44. In addition, IbNAC25 was negatively correlated with other factors (except IbMYB44, IbDFR and IbUFGT).
Anthocyanin was induced by cotransforming IbMYB340, IbbHLH2 and IbNAC56a or IbNAC56b in tobacco leaves
The results of a functional analysis of IbMYB340, IbbHLH2 and IbNAC56a or IbNAC56b using a transient expression assay in tobacco leaves are shown in Fig. 3a. No anthocyanin accumulation was detected at the injection areas of tobacco leaves when transformed with IbNAC56a, IbNAC56b or IbbHLH2 alone, while slight pigmentation appeared with IbMYB340 injected alone. However, an enhanced color was visible at injection regions 7 day after transformation with both IbMYB340 and IbNAC56a/IbNAC56b or IbbHLH2. When IbNAC56a or IbNAC56b was cotransformed with IbMYB340 and IbbHLH2, obvious intense pigmentation was detected. The quantification of total anthocyanin contents indicated that anthocyanin biosynthesis was induced by cotransformation of three TFs, which was more than that from two TFs or IbMYB340 alone, whereas tobacco leaves did not show any visible anthocyanin accumulation after transformation with the empty vector or with IbNAC56a or IbNAC56b alone (Fig. 3b). We next analyzed the color of the tobacco leaves. The L* values apparently declined when the pigmentation appeared; by contrast, the a*/b* ratio significantly increased (Fig. 3c-d).
Heterologous overexpression of IbMYB340, IbbHLH2 and IbNAC56a or IbNAC56b induces anthocyanin biosynthesis in strawberry receptacles
To further confirm the roles of IbMYB340 and IbNAC56a or IbNAC56b in anthocyanin biosynthesis, we cotransformed IbMYB340 and IbNAC56a or IbNAC56b in strawberry receptacles via agroinfiltration, and diploid strawberry (Fragaria vesca) ‘Yellow Wonder’ 5AF7 was used for the transient transformation experiment. The transient expression assays in strawberry receptacles showed that coinfiltration of IbMYB340 with any of the other candidate TFs resulted in apparent pigmentation accumulation 7 day after transformation, while the coinfiltration of three TFs caused deeper red pigmentation than did two TFs or IbMYB340 alone. However, no pigmentation was observed when the empty vector was infiltrated; IbNAC56a or IbNAC56b were infiltrated separately; or IbNAC56a and IbNAC56b were coinfiltrated. In addition, pigmentation was also visible in the injection region of IbMYB340 alone (Fig. 4a). Measurements of the induced anthocyanin are shown in Fig. 4b. The total anthocyanin contents of the three TFs cotransformed were significantly higher than those when the two TFs or one TF alone were cotransformed. The L* and a*/b* values of the injection regions of the strawberry receptacles declined or increased sharply when pigmentation appeared and stayed at significantly relatively low or high levels, respectively (Fig. 4c-d). Heterologous overexpression systems in strawberry receptacles presented phenotypic changes similar to those presented in the tobacco leaves in this study. We observed great anthocyanin pigmentation when IbMYB340 was cotransformed with IbbHLH2 and IbNAC56a or IbNAC56b together.
Expression patterns of anthocyanin-related genes in strawberry receptacles
To explore the regulatory models of the TFs IbMYB340, IbbHLH2 and IbNAC56a or IbNAC56b, several critical anthocyanin-related genes, FvPAL, FvF3H, FvANS, FvDFR, FvUFGT and FvRAP were analyzed in induced-color strawberry receptacles by RT-qPCR. FvANS expression was greatly increased when IbMYB340, IbbHLH2 and IbNAC56a or IbNAC56b were cotransformed (P < 0.01, Fig. 5a). Interestingly, the transformation of IbMYB340 alone showed a higher FvANS expression level than did cotransformation of IbMYB340 and of IbbHLH2 and IbNAC56a or IbNAC56b (P < 0.01 or P < 0.05, respectively). For FvDFR, transformation of TFs, including IbNAC56b or IbMYB340 alone, resulted in noticeably gene expression levels that were higher than those from the other TF combinations (P < 0.01), while the additional IbbHLH2 with IbMYB340 and IbNAC56a cotransformation caused an obvious increase in FvDFR expression levels (P < 0.01, Fig. 5b). Figure 5d-e illustrates similar changes in the expression levels of FvRAP and FvF3H; these two genes were highly expressed in the cotransformation with different TFs or IbMYB340 alone, except for IbMYB340 + IbNAC56a (P < 0.01). In addition to IbMYB340 + IbNAC56a, IbMYB340 + IbbHLH2 also maintained a relatively low expression level of FvRAP and FvF3H compared with that of other cotransformation types, excluding the empty vector pSAK277 (P < 0.01). From Fig. 5c, we can see that FvUFGT was significantly expressed in response to the cotransformation of IbMYB340 + IbNAC56b or IbMYB340 alone (P < 0.01), whereas other cotransformation types seemed not to activate the transcription of FvUFGT. In addition, it seemed that FvPAL could not be activated by different cotransformation types except for the cotransformation of IbMYB340 and IbNAC56b (Fig. 5f). As mentioned above, almost all the genes involved in the anthocyanin biosynthetic pathway and vacuolar transport were highly expressed in the abovementioned cotransformed strawberries, especially when IbMYB340, IbbHLH2, and IbNAC56a or IbNAC56b were cotransformed. This result suggested that the IbMYB340, IbbHLH2 and IbNAC56a or IbNAC56b genes might regulate or coregulate anthocyanin synthesis by forming a regulatory complex.
The regulatory complex MYB340-bHLH2-NAC56 promotes anthocyanin biosynthesis by binding to the IbANS promoter
We speculated that the products of the IbMYB340, IbbHLH2, IbNAC56a and IbNAC56b genes might form regulatory complexes to regulate anthocyanin synthesis. As such, the possible interactions of IbMYB340 with IbbHLH2, IbNAC56a and IbNAC56b were studied via Y2H assays. First, we found that cotransformed yeast cells harboring pGADT7 and IbMYB340 failed to grow on SD − Trp/−Leu/−His/−Ade media, indicating that IbMYB340 could not activate downstream gene expression in yeast by itself. As a result, strong growth on SD-Trp-Leu-His-Ade + AbA media was observed when we cotransformed the complete amino acid sequence of IbMYB340 with IbbHLH2, IbNAC56a or IbNAC56b. However, the Y2H assays showed no interaction between IbbHLH2 and IbNAC56a or IbNAC56b (Fig. 6a). These results suggest that IbMYB340 can interact in yeast with IbbHLH2, IbNAC56a and IbNAC56b.
Additionally, we further validated the results obtained in yeast via firefly luciferase complementation assays in tobacco. Coexpression of NLuc-IbMYB340 and CLuc-IbbHLH2 or IbNAC56a or IbNAC56b vectors reversed the intense luciferase enzyme activity. In contrast, we detected no apparent luciferase enzyme activity in any of the control groups, including the groups with Nluc-IbMYB340 with CLuc and CLuc-IbbHLH2/-IbNAC56a/-IbNAC56b with NLuc (Fig. 6b-d). Taken together, these results indicated that IbMYB340 can interact with IbNAC56a and IbNAC56b in tobacco leaves. These results were in agreement with the results of the yeast two-hybrid assay.
We next tested the transactivation activity of the candidate TFs with the IbANS promoter via dual-luciferase reporter assays (Fig. 6f). Cotransformation of IbMYB340, IbbHLH2 and IbNAC56a or IbNAC56b showed more transactivation on the IbANS promoter than did IbMYB340 cotransformation with IbbHLH2 and IbNAC56a or IbNAC56b. However, it seemed that cotransformation of IbMYB340 and any other TFs or IbMYB340 alone made no difference, while the transformation of IbbHLH2 or IbNAC56a slightly promoted the activity (Fig. 6f). Furthermore, Y1H assays were performed to demonstrate whether the IbANS promoter region is bound directly by IbMYB340 and IbNAC56a or IbNAC56b. Promoter structure analysis revealed multiple cis-regulatory elements, including a MYB motif (T/CAACCA) and a NAC-binding site (CACG) (Fig. 6e). In this assay, we transformed pGADT7-IbMYB340 /IbNAC56a/IbNAC56b prey vectors into Y1H Gold cells harboring pAbAi-IbANS1/2/3 bait vectors and tested them on SD/−Ura/−Leu/AbA plates. The transformants coexpressing the prey vectors pGADT7-IbMYB340 and pAbAi-IbANS1/3 were grown on SD/−Ura/−Leu/AbA400 plates, while the pAbAi-IbANS1/2/3 bait vectors could not grow on SD/−Ura/AbA400 plates, suggesting that IbMYB340 was capable of binding to the 1st (-956 bp to -755 bp) and 3rd (-310 bp to -105 bp) IbANS promoter fragments rather than the 2nd IbANS promoter fragment (Fig. 6g). However, we found no direct association between IbNAC56a or IbNAC56b and the promoter of IbANS, although several NAC-binding sites were located in different individual promoter regions (Fig. 6g). Taken together, these results indicated that the different regulatory complexes (MYB340-bHLH2-NAC56a and MYB340-bHLH2-NAC56b) directly activated the expression of IbANS by binding to the MYB motif element.
IbMYB44 suppresses anthocyanin accumulation by competitively inhibiting the regulatory complex formation of IbMYB340-IbbHLH2-IbNAC56a or IbNAC56b
To study the role of IbMYB44 in anthocyanin biosynthesis, we cotransformed IbMYB340 and IbMYB44 at different ratios into the abaxial side of tobacco leaves to test the transcriptional repression effect of IbMYB44. As shown in Fig. 7a, the pigmentation in tobacco leaves gradually diminished with an increasing proportion of IbMYB44. Using a dual-luciferase reporter assay, we then investigated the transactivation activity of the IbANS promoter when IbMYB44 and IbMYB340 were cotransformed in tobacco leaves. The results showed that steadily declining IbANS promoter activity occurred when the IbMYB340:IbMYB44 ratio decreased from 1:0 to 1:4. However, it seemed that there were no changes between several ratios of IbMYB340:IbMYB44: 1:0.5, 1:0.67, 1:1 and 1:1.5 (Fig. 7b). The total anthocyanin contents and a*/b* values declined significantly at different ratios, their changes were consistent with the phenotypes of the tobacco leaves mentioned above, and the L* values apparently increased (Fig. 7c-e). Thus, when cotransformed with IbMYB340, IbMYB44 could decrease anthocyanin biosynthesis.
We further verified the interaction of IbMYB44 with IbMYB340 and IbNAC56a or IbNAC56b. Y2H analysis showed that the cells cotransformed with IbMYB44 with IbMYB340 and IbNAC56a or IbNAC56b could grow on SD-Trp-Leu-His-Ade + AbA plates (Fig. 7f). Moreover, the marked luciferase enzyme activity was rescued by the control infiltrated with IbMYB340 + IbMYB44, IbMYB44 + IbNAC56a or IbMYB44 + IbNAC56b; by contrast, Nluc-IbMYB340/IbMYB44 coinfiltrated with CLuc or CLuc-IbMYB44/IbNAC56a/IbNAC56b coinfiltrated with Nluc did not result in a sufficient level of luciferase enzyme activity (Fig. 7g-i). Overall, IbMYB44 could interact with IbMYB340, IbNAC56a or IbNAC56b, suggesting that IbMYB44 suppressed anthocyanin accumulation probably through competitive inhibition of IbMYB340, IbNAC56a or IbNAC56b.