3.3 NtMYB12a prevents the accumulation of FA in tobacco
To explore the potential new functions of NtMYB12a, the transcriptomes of NtMYB12a-OE and WT plants were detected by RNA-seq (Supplemental Figure S2a). There were 1,152 DEGs between the OE and WT plants, among which the expression levels of 692 genes were up-regulated, and those of 453 genes were down-regulated in theNtMYB12a-OE plants (Supplemental Figure S2b). Gene annotation showed that these DEGs were mainly involved in plant-pathogen interaction, starch and sucrose metabolism, plant hormone signal transduction, flavonoid biosynthesis, lipid metabolism, and so on (Supplemental Figure S2c). It is noteworthy that the lipid-related DEGs mainly encoded the GDSL esterase lipase, lipooxygenase (LOX) and FAH1 (Table 1), which could catalyze the dehydration of FA as described in the previous studies (Chauvin, Caldelari, Wolfender, & Farmer, 2013; Nagano et al., 2012; Nalam, Keereetaweep, & Shah, 2013).
The relative expression levels of NtLOX6 , NtLOX5 ,NtFAH1 , NtSFAR4 , NtGDSL1 , NtGDSL2 ,NtGDSL3 , and NtGDSL4 genes significantly increased by more than 2 folds in the NtMYB12a-OE plants compared to those in the WT plants, but the transcripts of these genes significantly decreased in the RNAi and mutant plants (Figure 5a & 5b). When compared to that in the WT plants, the FA contents in the leaves of three independentNtMYB12a-OE lines decreased by 31%-48%, while those in the leaves of three independent RNAi lines increased by 2.3 to 3.2 fold, and those in the mutant plants also increase by 1.5 to 1.7 folds (Figure 5c). The percentage of PUFAs in the leaves of WT plants was about 71%. However, the percentage of PUFAs in the NtMYB12a-OE lines was about 43%-59%, and the percentage of PUFAs was more than 81% in all the RNAi and mutant plants (Figure 5d).
In the developing seedpods of WT plants, the transcripts ofNtSFAR4 , NtLOX5 , NtLOX6 , NtFAH1 , andNtGDSL1 genes could be detected at 10 DAF, then increased to higher levels at 20 DAF, but finally decreased to low levels at 30 DAF (Figure 6a). The NtGDSL2 , NtGDSL3 , and NtGDSL4genes showed low expression levels during the whole developmental process of WT seedpods. Generally, the expression levels of these eight genes were all up-regulated in the NtMYB12a-OE plants at each time point, especially at 30 DAF, when their expression levels were more than five folds of those in the WT plants (Figure 6a). By contrast, the expression levels of these eight genes in the developing seedpods ofNtMYB12a-RNAi and mutant plants were lower than those of the WT plants at each time point (Figure 6a). There were no significant changes occurred to the phenotype and weight of the mature seeds of theNtMYB12a transgenic and mutant plants (Figure 6b & 6c). However, when compared to those in the WT plants, the FA contents in the mature seeds of three independent NtMYB12a-OE plants significantly decreased by 29.6% to 42.3%, while the contents in the three RNAi lines increased by 55.9% to 78.1%, and the FA contents in the mutant lines even increased by 85.6% to 106.3% (Figure 6d). Taken together, our results indicate that NtMYB12a could induce the transcription ofLOX and lipase genes, and then prevent the FA accumulation in tobacco leaves and seeds.