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.