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.