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.