4.1 The neofunctionalization of NtMYB12a in tobacco
The AtMYB12 has been well characterized as a flavonol-specific regulator in Arabidopsis (Mehrtens et al., 2005). AtMYB12, AtMYB11 and AtMYB111 share high sequence identities in the MYB motifs, and all of these three MYBs retain the function of regulating flavonol biosynthesis in Arabidopsis, but their regulatory activities are tissue-specific (Stracke et al., 2007). The AtMYB12 mainly promotes the biosynthesis of flavonol in the roots, while the AtMYB111 works mainly in the cotyledons (Stracke et al., 2007). Tobacco is an allotetraploid specie with many duplicated genes presented in its genome (Edwards et al., 2017), and the functional evolution of homologous genes in tobacco is more alternative. NtMYB11, NtMYB12, and NtMYB111 were all encoded by two close homologous genes (Figure 1a), which shared high sequence identities in the R2R3 MYB motifs (Figure 1b). The tissue-specific expression patterns ofNtMYB11 , NtMYB12 , and NtMYB111 genes (Figure 2a) suggested that their regulatory functions in flavonol biosynthesis might also be differentiated to tissue-specific, which is similar to that occurred to the AtMYB11, AtMYB12, and AtMYB111 in Arabidopsis (Stracke et al., 2007). It’s noteworthy that the transcription of NtMYB11b and NtMYB111a genes could hardly be detected in all the tissues examined, but both theNtMYB12a and NtMYB12b showed high expression levels in many tissues (Figure 2a), indicating that NtMYB12a and NtMYB12b might have further functional differentiation.
The hypothesis about the functional differentiation of NtMYB12a and NtMYB12b was verified by generating RNAi plants of these two genes. The NtMYB12b was shown to be a flavonol-specific regulator, while the NtMYB12a was involved in the biosynthesis of both flavonol and anthocyanin (Figure 3c & 3d). RNA-seq was then performed to fully characterize the function of NtMYB12a, and found that the transcription levels of several lipase and LOX genes were significantly up-regulated in the NtMYB12a overexpression lines (Table 1, Figure 5a & 5b). The FA contents in the leaves and seeds ofNtMYB12a overexpression plants also significantly decreased (Figure 5c, 6d). The function of MYB12 in regulating FA metabolism has not been described in any other plant species so far, but it’s interestingly to see that several transcription factors involved in regulating flavonoids biosynthesis are also proved to affect the accumulation of FA or the embryo development in plants, including TT2 (Z. Wang et al., 2014), TT8 (Chen et al., 2014), NAC03(Dalman et al., 2017). The NtMYB12a might be a new transcription factor involved in the metabolism of both flavonoids and FA in tobacco.
The neofunctionalization of NtMYB12a in tobacco might be caused by two reasons. One is the sequence variation occurred to the C terminal of NtMYB12a protein. The amino acid sequence of NtMYB12a shares 87.91% identity with that of NtMYB12b (Supplementary Table S2). In detail, the amino acids of the R2R3 motifs within the two proteins were almost the same (only one substitution of Alanine to Glycine at 94th position), while the identity between the rest amino acid sequences of the two proteins was 83.97% (Figure 1b). Considering that both the NtMYB12a and NtMYB12b genes show strong transcriptional activities, and the variation of amino acids in the C terminals of the two homologous proteins, it is quite possible for NtMYB12a to have new functional differentiation. The other reason is that the function of MYB12 varies with different plant species. The AtMYB12 is a flavonol specific regulator in Arabidopsis (Mehrtens et al., 2005), but AtMYB12 could induce the expression of genes in both CQA (caffeoyl quinic acids) and flavonol biosynthetic pathways in tomato, and affect the contents of both flavonol and anthocyanin in tobacco (Luo et al., 2008). When overexpressed the citrus CsMYBF1 gene belonging to the flavonol-specific MYB subgroup in tomato, the contents of 5 sugars and 2-ketoglutaric acid significantly reduced in the transgenic fruits, while the contents of isocitric acid and glucuronic acid significantly increased (C. Liu et al., 2016). Therefore, theNtMYB12a might be a valuable candidate gene, which can increase flux to flavonoids metabolites and significantly affect the FA metabolism in tobacco.