4.2 NtMYB12a acts downstream of sucrose signaling to stimulate FA degradation in tobacco
The major FA of tobacco leaves are linolenic acid (18:3) and linoleic acid (18:2), which comprise about 60% and 15% of total FA, respectively. The contents of FA increase rapidly during tobacco seed development, and linoleic acid comprises 75% of the tobacco seed oil (Chu & Tso, 1968). The linolenic acid and linoleic acid are the major PUFAs, which are preferred substrates of LOX enzymes for generating a diverse class of phyto-oxylipins, including jasmonates, divinylethers and green leaf volatiles (GLVs) (Nalam et al., 2013; ul Hassan, Zainal, & Ismail, 2015). In the present study, we show that NtMYB12a directly bind to the regulatory regions of NtLOX5 , NtLOX6 , NtSFAR4 , andNtGDSL2 genes (Figure 7), and thus promote the transcription levels of these four genes (Figure 5a & 5b). The percentage of PUFAs significantly decreased in the leaves of NtMYB12a-OE lines, but significantly increased in the leaves of NtMYB12a-RNAi and mutant lines (Figure 5d). We further showed that over expression ofNtLOX5 and NtLOX6 genes lead to the reduction of FA content and percentage of PUFAs in tobacco seeds. The NtSFAR4 is a close homologous protein of the Arabidopsis AtSFAR4, which plays an important role in FA degradation during post‑germination and seedling development in Arabidopsis, thus reducing the FA content (Huang et al., 2015). Several GDSL esterase/lipase have been proved to be involved in FA degradation in Arabidopsis seeds (Chen, Du, et al., 2012), and the BnGLIP gene of Brassica napus expressed inN. benthamiana also shows lipase activity (Tan et al., 2014). Therefore, the inhibition of FA accumulation by NtMYB12a in tobacco is achieved by directly promoting the transcription of NtLOX5 , NtLOX6 ,NtSFAR4 , and NtGDSL2 genes.
The transcripts of CHS , CHI , F3H , F3’H ,FLS , DFR , and LDOX genes were evidently induced by sucrose in Arabidopsis seedlings (Solfanelli, Poggi, Loreti, Alpi, & Perata, 2006). The expression level of NtMYB12a gene was also significantly induced by sucrose treatment (Figure 9), suggesting the regulation of sucrose on the EBGs might be mediated by MYB12 in tobacco. We further showed that sucrose treatment significantly reduced the FA content in the WT seedlings (Figure 10c). The content of FA inntmyb12a seedlings under sucrose treatment was higher than that in the WT seedlings under sucrose treatment, but lower than that in the WT seedlings under normal condition (Figure 10c), suggesting that loss function of NtMYB12a partially suppressed the decrease of FA content in tobacco seedlings caused by sucrose treatment.
The expression levels of NtMYB12a , NtLOX6 , andNtGDSL2 genes were all significantly elevated by sucrose treatment, and knock-out of NtMYB12a repressed the induction of sucrose on the expression of NtLOX6 and NtGDSL2 (Figure 10b), indicating that NtMYB12a mediates the promotion of sucrose on the transcription of NtLOX6 and NtGDSL2 . It’s noteworthy that the transcription of NtLOX5 and NtSFAR4 genes was not affected in the WT seedlings under sucrose treatment, but sucrose could significantly reduce the transcription of these two genes in the absence of NtMYB12a function (Figure 10b), suggesting that sucrose and NtMYB12a might play opposite roles in regulating the expression of NtLOX5and NtSFAR4 genes. It has been proved that sucrose can block GA (gibberellin acid) mediated degradation of DELLA proteins, which further induces the biosynthesis of anthocyanin (Y. Li, Van den Ende, & Rolland, 2014), and prevents the degradation of FA by repressing the SFAR genes in Arabidopsis (Chen, Du, et al., 2012). But meanwhile sucrose can also promote the accumulation of auxin in plant by degrading the auxin conjugates (Meir, Philosoph-Hadas, Epstein, & Aharoni, 1985; Meir, Riov, Philosoph-Hadas, & Aharoni, 1989) and inducing the transcription of auxin biosynthesis gene YUCCA (Le, Schmelz, & Chourey, 2010; Lilley, Gee, Sairanen, Ljung, & Nemhauser, 2012). High concentration of auxin could inhibit the accumulation of FA in microalgae Scenedesmus(Dao et al., 2018). Therefore, sucrose can enhance the activity of NtMYB12a, which further suppresses the accumulation of FA in tobacco by targeting important lipoxygenase and lipase genes. Meanwhile, sucrose might also participate in regulating FA metabolism by balancing GA and auxin signals, which remains to be further studied.
In conclusion, we summarize the functions of NtMYB12a via a schematic that illustrates a regulatory network in which NtMYB12a promotes the biosynthesis of flavonoids and the degradation of FA in tobacco (Figure 11). We found that NtMYB12a can directly bind to the regulatory regions of NtLOX5 , NtLOX6 , NtSFAR4 , and NtGDSL2genes, and promote the transcription of these four genes. These genes encode the lipoxygenase or lipase enzymes, which have been proved to reduce the FA content in plants by catalyzing the degradation of FA. Sucrose significantly induces the transcription of NtMYB12a gene, as well as the NtLOX6 and NtGDSL2 genes, and loss function of NtMYB12a interrupts the induction of sucrose on the expression ofNtLOX6 and NtGDSL2 , indicating NtMYB12a mediates the promotion of sucrose on the transcription of NtLOX6 andNtGDSL2 . The regulation of NtMYB12a on the transcription ofNtLOX5 and NtSFAR4 genes is independent of sucrose.