Nicotiana tabacum
1 INTRODUCTION
Flavonoids are important polyphenolic secondary metabolites in plants,
and the biosynthetic process of flavonoids has been well studied in
Arabidopsis by using the series of transparent testa (tt )
mutants (Buer, Imin, & Djordjevic, 2010).
The content and distribution of flavonoids in Arabidopsis are precisely
regulated by multiple transcription factors, especially the well-known
MBW (MYB-bHLH-WD40) protein complex (Baudry
et al., 2004; Gonzalez, Zhao, Leavitt, &
Lloyd, 2008; Xu, Dubos, & Lepiniec,
2015; Xu et al., 2014). Interestingly,
several transcription factors involved in flavonoids biosynthesis also
participate in regulating FA metabolism. TRANSPARENT TESTA2 (TT2) is
proved to regulate embryonic FA biosynthesis by targeting FUSCA3during the early developmental stage of Arabidopsis seeds
(Z. Wang et al., 2014). TT8 inhibits seed
FA accumulation by targeting several seed development regulators in
Arabidopsis (Chen et al., 2014).
Overexpression of the SiTTG1 gene from Setaria italica can
induce the transcription of genes involved in accumulation of seed FA in
developing seeds of Arabidopsis ttg1-13 plants
(K. Liu et al., 2017). Many ttmutants in Arabidopsis are deficient in flavonoids biosynthesis, but
show higher contents of FA than WT plants
(Z. Wang et al., 2014), indicating a
close relationship between these two metabolites.
FA and FA-derived complex lipids are the main energy reserves in many
higher plant seeds. The biosynthesis of FA is a complex physiological
and biochemical process, involving the co-expression of many enzymes,
and the participation of several cell structures, such as plastid,
cytoplasm and endoplasmic reticulum. Seed oil is mainly stored in the
form of triacylglycerol (TAG), which is formed by three FA molecules
connected to the skeleton of one glycerol molecule
(Baud & Lepiniec, 2009). The degradation
of seed oil is initiated with the breaking of TAG into FA and glycerin,
which might be realized by three ways, including lipase hydrolysis, acyl
CoA diesterylglycerol acyltransferase (DAGAT) pathway
(Zou et al., 1999), and lipoxygenase
pathway (Bannenberg, Martinez, Hamberg, &
Castresana, 2009). Several GDSL type lipases have been shown to
stimulate FA degradation in Arabidopsis
(Chen, Du, et al., 2012;
Huang, Lai, Chen, Chan, & Shaw, 2015).
The free polyunsaturated fatty acids (PUFAs) including linolenic acid
and linoleic acid are the preferred substrate for LOX enzymes to
generate oxidation products like aldehydes, alcohols, ketones, acids
(B. Li et al., 2016;
Sjovall, Virtalaine, Lapvetelainen, &
Kallio, 2000). Wheat germ (WG) contains large amount of unsaturated
lipids, which makes them sensitive to rancidity during storage due to
the presence of lipase (LA) and LOX (Kumar
& Krishna, 2015; B. Li et al., 2016).
About 95% of the total FA esters in tobacco are of chain length 16
carbons or 18 carbons, including palmitate, stearate, oleate, linoleate,
and linolenate (16:0, 18:0, 18:1, 18:2, and 18:3, respectively)
(Chu & Tso, 1968). The PUFA 18:2 and
18:3 are the major FA in tobacco. For instance, the relative amount of
linolenic acid (18:3) in tobacco leaves is about 30% at early
developing stages, but increases to 60 % at maturity stage, while the
percentage of other FAs (18:2, 18:1, 18:0, and 16:0) decrease
progressively with leaf development. There is a rapid increase of FA
content in tobacco flowers developed into seedpods, and the linoleic
acid (18:2) comprises 75 % of tobacco seed oil
(Chu & Tso, 1968).
MYB12 is a flavonol-specific regulator, which in parallel activates the
transcriptions of several EBGs, including CHS (Chalcone
synthase ), CHI (Chalcone isomerase ), F3H(Flavanone 3-hydroxylase ), and FLS (Flavonol
synthase ) in Arabidopsis (Mehrtens,
Kranz, Bednarek, & Weisshaar, 2005). MYB12 does not need a bHLH or a
WD protein as partner, but shares significant structural and functional
similarity with MYB11 and MYB111 (Stracke
et al., 2007). In Arabidopsis, MYB12, MYB11, and MYB111 form the
subgroup 7 of the R2R3-MYB family, but show differential spatial
activity. For instance, flavonol biosynthesis in the roots is mainly
controlled by MYB12, while in cotyledons the flavonol biosynthesis is
primarily controlled by MYB111 (Stracke et
al., 2007). MYB12 gene has been cloned in many plant species,
including grape (Czemmel et al., 2009),
tomato (Ballester et al., 2010), apple
(N. Wang et al., 2017), buckwheat
(Matsui et al., 2018), and pear
(Zhai et al., 2019). Interestingly,
AtMYB12 activates the biosynthesis of both flavonol and caffeoyl quinic
acid in tomato fruit (Luo et al., 2008),
though it has been well characterized as a flavonol-specific regulator
in Arabidopsis. Moreover, when overexpressed in tobacco, AtMYB12 not
only promotes the accumulation of flavonol, but also regulates the
transcription of genes involved in many pathways, such as amino acid
metabolism, carbohydrate and lipid metabolism, auxin response, and
defense response (Misra et al., 2010).
Subsequent research confirms that over-expressed AtMYB12 enhances
the tolerance of transgenic Arabidopsis plants to the salt and drought
stresses (F. Wang et al., 2016). However,
the exact role of MYB12 in other pathways still needs to be studied.
Tobacco is often used as a model plant to study the function of MYB12
from other plant species. One NtMYB12 gene has been identified
from tobacco so far, and was shown to positively regulate flavonol
biosynthesis, as well as to enhance plant tolerance to low Pi stress
(Song et al., 2019). However, whether
there are duplicated NtMYB12 genes in allotetraploid tobacco
genome, and whether the duplicated NtMYB12 genes have functional
differentiation in tobacco have not been reported so far. Thus, we
identified two duplicated NtMYB12 genes from tobacco genome, and
verified their regulation on flavonoids biosynthesis. We further found
that sucrose induced the transcription of NtMYB12a gene, which
directly targets NtSFAR4 , NtLOX5 , NtLOX6 , andNtGDSL2 genes to stimulate the FA degradation in tobacco.
Consequently, our results provide evidence for revealing the multiple
roles of MYB12 in plants.