3.6 Sucrose enhances the effect of NtMYB12a on FA metabolism
The transcriptome analysis showed that many DEGs in theNtMYB12a-OE plants were involved in the starch and sucrose metabolism (Supplemental Figure S2c). To further test whether sucrose affects the activity of NtMYB12a, the expression pattern ofNtMYB12a gene under various sucrose concentration was firstly analyzed with the ProNtMYB12a:GUS seedlings. The GUS signal was mainly detected in the shoot apical meristem (SAM) of the seedlings on the normal MS medium, but strong GUS activity was also detected in the fresh leaves of the seedlings under 150 mM and 200 mM sucrose treatment (Figure 9a). Meanwhile, no GUS signal was detected in the leaves of seedlings under 0 mM and 50 mM sucrose treatment (Figure 9a). qRT-PCR analysis also showed that the expression level ofNtMYB12a was highest in the seedlings under 150 mM sucrose treatment, while the transcription level of this gene in the seedlings under 200 mM sucrose treatment was also significantly higher than that in the control seedlings (Figure 9b).
RNA-seq analysis showed that the expression levels of 1367 genes were up-regulated under 150 mM sucrose treatment, while those of 2266 genes were down-regulated (Supplemental Figure S5a & 5b). Gene annotation showed that these DEGs were mainly involved in plant hormone signal transduction, phenylpropanoid biosynthesis, response to tress, and so on (Supplemental Figure S5c).Venn analysis showed that the transcription levels of 29 genes were up-regulated by both the NtMYB12a OE and sucrose treatment (Figure 9c). Among the 29 genes, there were three genes encoding NtGDSL2, NtGDSL3, and NtLOX6 (Table 2), respectively. When germinated on MS medium containing 150 mM sucrose, theNtMYB12a-OE seedlings were obviously smaller than the WT, RNAi, and mutant plants (Figure 10a). The expression levels of NtLOX6and NtGDSL2 genes in the WT seedlings increased by about 4 folds under 150 mM sucrose treatment (Figure 10b). However, the expression levels of these two genes in the NtMYB12a-RNAi and mutant seedlings under 150 mM sucrose treatment showed no significant difference with those in the RNAi and mutant seedlings under normal condition (Figure 10b). By contrast, the transcription ofNtLOX5 and NtSFAR4 genes were not affected in the WT seedlings treated with sucrose, but sucrose significantly decreased the expression levels of these two genes in the NtMYB12a-RNAi and mutant seedlings (Figure 10b).
When grown on the normal MS medium, the FA content in theNtMYB12a-OE seedlings was about 33% less than that in the WT seedlings (Figure 10c). Sucrose treatment reduced FA content in the WT seedlings by about 44%, and the FA content in the OE lines under sucrose treatment reduced by 60% compared to that in the normal WT seedlings, suggesting that sucrose treatment enhanced the effect of NtMYB12a on FA degradation in tobacco seedlings. TheNtMYB12a-RNAi and mutant seedlings under normal condition contained 64% and 56% more FA than the normal WT seedlings, respectively. The FA content of the ntmyb12a mutant seedlings under sucrose treatment was 21% less than that in the normal WT seedlings, but was significantly higher than that in the WT seedlings under sucrose treatment (Figure 10c), indicating that loss function of NtMYB12a partially suppressed the decrease of FA content in tobacco seedlings caused by sucrose treatment.