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.