3.2 Functional divergence of NtMYB12 genes in regulating flavonoid biosynthesis
Both NtMYB12a and NtMYB12b showed high expression levels in tobacco leaves, buds, flowers, and capsules (Figure 2a). In the developing tobacco leaves, the expression levels of NtMYB12a andNtMYB12b genes decreased gradually from the seedling stage to the early senescence stage, but slightly increased in the late senescence stage (Figure 2b). NtMYB11a was mainly expressed in the roots, buds, and flowers, and NtMYB111b showed high expression levels in axillary buds, capsules, and seeds (Figure 2a). The transcription ofNtMYB11b and NtMYB111a genes could hardly be detected in all the tissues examined. Since both NtMYB12a and NtMYB12bgenes showed high transcriptional activity, their subcellular localization was then investigated in tobacco protoplast. As shown in Figure 2c, the location of NtMYB12a-GFP and NtMYB12b-GFP are complete match with that of the NLS marker protein, indicating NtMYB12a and NtMYB12b are nuclear proteins.
To explore the roles of NtMYB12a and NtMYB12b in regulating flavonoid biosynthesis, five independent NtMYB12a-RNAi andNtMYB12b-RNAi lines were generated with gene specific CDS fragments (Figure 3a), respectively. The NtMYB12a gene was specifically silenced in the NtMYB12a-RNAi lines, while theNtMYB12b gene was specifically silenced in theNtMYB12b-RNAi lines (Figure 3b). Silencing of NtMYB12areduced the flavonol content by about 31%-76% in the leaves of five RNAi lines, while silencing of NtMYB12b reduced the flavonol content by about 51%-86% (Figure 3c).The flavonol contents in threeNtMYB12a-RNAi lines were significant higher than those of the five NtMYB12b-RNAi lines (Figure 3c), indicating a weak role of NtMYB12a in regulating flavonol biosynthesis. The contents of anthocyanin in four NtMYB12b-RNAi lines showed no significant differences with that in the WT plants (Figure 3d). However, the anthocyanin contents of the five NtMYB12a-RNAi lines were all significant lower than that of the WT plants (Figure 3d). Moreover, the expression levels of NtCHS , NtCHI , NtF3H , andNtFLS genes were all significantly down-regulated in the fiveNtMYB12b-RNAi lines, but were not significantly affected in someNtMYB12a-RNAi lines (Supplemental Figure S1).
NtMYB12b seems to be a flavonol-specific regulator in tobacco, but NtMYB12a might also be involved in regulating anthocyanin biosynthesis. To fully characterize the role of NtMYB12a in regulating flavonoid biosynthesis, the NtMYB12a over-expression (OE) andntmyb12a mutant plants were generated, respectively. The flowers of NtMYB12a OE plants were dark red, while the color of the mutant plants turned light red (Figure 4a). In accordance with this, the anthocyanin contents in the flowers of two NtMYB12a OE lines were significant higher than that in WT flowers, and the flowers of the three mutant lines possessed higher anthocyanin contents than the WT plants (Figure 4b). The anthocyanin contents also significantly increased in the leaves of two NtMYB12a OE lines, but significantly decreased in the leaves of two ntmyb12a mutant lines (Figure 4c). Moreover, the flavonol contents of the flowers and leaves were significant higher in the NtMYB12a OE lines than those in the WT plants, but significant lower in the ntmyb12a mutant lines (Figure 4d). These results indicate NtMYB12a might possess new functions in tobacco besides promoting flavonol biosynthesis.