Identification of the Eutrema Salsugineum EsMYB90 gene important for anthocyanin biosynthesis
Anthocyanins contribute to coloration and antioxidation effects in different plant tissues. MYB transcription factors have been demonstrated to be a key regulator for anthocyanin synthesis in many plants. However, little information was available about the MYB genes in the halophyte species Eutrema salsugineum . Result: Here we report the identification of an important anthocyanin biosynthesis regulator Es MYB90 from Eutrema salsugineum , which is a halophyte tolerant to multiple abiotic
... iple abiotic stresses. Our phylogenetic and localization analyses supported that Es MYB90 is an R2R3 type of MYB transcription factor. Ectopic expression of EsMYB90 in tobacco and Arabidopsis enhanced pigmentation and anthocyanin accumulation in various organs. The transcriptome analysis revealed that 42 genes upregulated by Es MYB90 in 35S : EsMYB90 tobacco transgenic plants are required for anthocyanin biosynthesis. Moreover, our qRT-PCR results showed that Es MYB90 promoted expression of early ( PAL , CHS , and CHI ) and late ( DFR , ANS , and UFGT ) anthocyanin biosynthesis genes in stems, leaves, and flowers of 35S : EsMYB90 tobacco transgenic plants. Conclusions: Our results indicated that Es MYB90 is a MYB transcription factor, which regulates anthocyanin biosynthesis genes to control anthocyanin biosynthesis. Our work provides a new tool to enhance anthocyanin production in various plants. Keywords : Anthocyanin, flavonoid, Eutrema salsugineum , R2R3 MYB transcription factor, Es MYB90, transcriptional regulation, anthocyanin biosynthesis genes. Background Flavonoids which are derivatives of the phenylpropanoid/flavonoid pathway mainly contain proanthocyanidins (PAs), anthocyanins and flavonols [1-3]. As important pigments, anthocyanins are responsible for red, purple, violet and blue colors in flowers, fruits, and leaves, which determine economic traits of crops and ornamental plants . Anthocyanins are the end products of a specific branch in the phenylpropanoid/flavonoid biosynthesis pathway. Enzymes involved in anthocyanin biosynthesis have been extensively studied in many plant species . Catalyzed by phenylalanine ammonia-lyase (PAL), the initial step of the flavonoid pathway is the conversion of phenylalanine into trans-cinnamic acid , while chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthesis to form naringenin chalcone. Chalcone isomerase (CHI) cyclizes chalcone to 4 form naringenin . The naringenin is then converted into dihydrokaempferol (DHK) by flavanone 3 βhydroxylase (F3H). DHK is further hydroxylated to dihydroquercetin (DHQ) by flavonoid 3 'hydroxylase (F3'H), or to dihydromyricetin (DHM) by flavonoid 3',5'-hydroxylase (F3'5'H). Dihydroflavonol 4-reductase (DFR) converts DHQ into leucocyanidin, which is further converted into anthocyanidins by anthocyanidin synthase (ANS). Finally, UDP-glucose: flavonoid 3-Oglucosyltranferase (UFGT) catalyzes glycosylation of anthocyanidins to form anthocyanins [8,10-12]. MYB transcription factors play a central role in regulating expression of genes encoding major enzymes for anthocyanin biosynthesis via forming the transcriptional complex containing MYB-bHLH-WD40 (MBW) [1,13-15]. Expression of early biosynthesis genes (EBGs) such as CHS and CHI, is regulated by MYB11, MYB12 and MYB111, whereas PAP1 to PAP4 (AtMyb75, AtMyb90, AtMyb113, and AtMyb114) control expression of late biosynthesis genes (LBGs) including DFR, ANS, and UFGT in Arabidopsis[16,17]. In Arabidopsis, up-regulation of one of MYB75, MYB90, MYB113 and MYB114 genes is sufficient to increase anthocyanin accumulation in young leaves [15,17]. For example, the well-known Arabidopsis AtMYB75 (PAP1) gene directs anthocyanin production in leaves, roots, flowers, and fruits [18,19]. Overexpression of AtMyb75 in Arabidopsis and tobacco results in upregulation of PAL, CHS and DFR genes [19,20]. Similarly, AtMYB75 induces anthocyanin production in tomato (Solanum lycopersicum L.) via promoting the DFR expression . Furthermore, the sequence variation of AtMYB90 (PAP2) is causal for natural variation in anthocyanin accumulation [17,21]. AtMYB90 may act together with TTG1 (a WD40 protein) and different bHLH partners including TT8, GL3 or EGL3 [15,18,22]. Moreover, in Arabidopsis the ternary complexes formed by R2R3-MYB, bHLH and the WD repeat protein activate the biosynthetic genes required for proanthocyanidin accumulation in the innermost cell layer of the seed coat . The R2R3 MYB protein TT2 (MYB123) is also a key regulator of proanthocyanidin accumulation in developing seeds . MYB transcription factors are involved in regulation of anthocyanin synthesis in many plants, such as Arabidopsis [15,17,24,25], cauliflower (Brassica oleracea var botrytis)  , bok choy (Brassica rapa var. chinensis) , apple (Malus × domestica) [11,28-30], peach(Prunus persica)[14,31], pear (Pyrus pyrifolia) [13,32,33],strawberry(Fragaria x ananassa), snapdragon (Antirrhinum majus) , Chrysanthemum , grape hyacinth (Muscari armeniacum) , grapevine (Vitis vinifera) [37,38], chinese bayberry(Myrica rubra) [6,39], Epimedium sagittatum [40,41], poplar (Populus spp)  and potato (Solanum tuberosum L) . In addition, some MYB genes are up-regulated under various stress conditions [15,17]. However, the transcriptional regulation of anthocyanin synthesis by MYB in stress-tolerant plants is not well studied. Eutrema salsugineum (salt cress), a stress-tolerant model halophyte, is highly tolerant to cold, salt, drought, oxidative stress, and nitrogen deficiency. In Eutrema salsugineum, many stress-tolerant related genes, such as SOS1, HKT1, and nsLTP4, have been identified[44-49]. By analyzing the differentially expressed regulatory genes between Arabidopsis and E.salsugineum, it was found that the regulatory functions of 307 transcription factors in 50 different families were significantly different. Another study found that EsMYB96/WAX1 from E.salsugineum under the RD29A promoter improved drought tolerance with increased accumulation of cuticular wax and ascorbic acid in transgenic Arabidopsis . So far, there is no research report on the anthocyanin synthesis of EsMYBs in E.salsugineum. Here, we reported our functional analysis of the MYB transcription factor EsMYB90 in anthocyanin synthesis. Our phylogenetic and localization analyses suggest that EsMYB90 is an R2R3 type of MYB transcriptional factor. Ectopic expression of EsMYB90 in tobacco and Arabidopsis led to significantly increased pigmentation and production of anthocyanins in leaves, stems, and flowers. Our further RNA-seq and qRT-PCR analyses showed that EsMYB90 promoted expression of anthocyanin early biosynthesis genes (EBGs: NtCHS, NtCHI, and NtF3H) and late biosynthesis genes (LBGs: NtDFR, NtANS, and NtUFGT) in 35S:EsMYB90 tobacco transgenic plants. Our study identified a MYB transcription factor, which plays an important role in plant anthocyanin biosynthesis. Results Database mining identifies EsMYB90, a candidate regulator for anthocyanin synthesis Eutrema Salsugineum is a stress-tolerance halophyte, which produces purple flower buds after vernalization . Since MYB genes are required for anthocyanin synthesis [13,15,36] we identified which MYB controls this purple phenotype in E. salsugineum. After comparing MYB genes obtained from the transcriptome of E. salsugineum based on our previously published results, with 72 MYB 6 genes known acting as proanthocyanin (PA) and anthocyanin regulators in other plants, we found one candidate MYB gene, named as EsMYB90. To determine the relationship of EsMYB90 to characterized flavonoid and PA MYBs, we performed similarity analysis at the protein level. Our results showed that EsMYB90 has 80.5%, 78.9%, 78.4%, 74.4%, 69.4%, 65.9%, 50% identities respectively to 7 MYB proteins, i.e. BoMYB1, AtMYB90, BrMYB114, AtMYB75, CrMYB114, AtMYB113, and AtMYB114 (Fig. 1A) . In addition, similarities between EsMYB90 and other 10 MYB proteins range from 44.1% to 39.0% (Fig. 1A) . Those MYB proteins with high similarities to EsMYB90 belong to the class of R2R3-MYB which have a conserved DNA-binding domain (R2 and R3 repeats) in the N-terminal and a variable C-terminal region [41,53,54]. The ANDV motif (marked by red A box in Fig.1A), a characteristic identifier for anthocyanin-promoting MYBs in dicots , existed in EsMYB90, AtMYB90, AtMYB75, AtMYB113, AtMYB114, AmROSEA1, StMYB113, EsMYBA1(AGT39060), VvMYBA1, MrMYB1, and FaMYB10, while the C-terminal-conserved motif KPRPR [S/T]F for Arabidopsis anthocyanin-promoting MYBs [25,36] (marked by blue B box in Fig.1A), was only found in EsMYB90, AtMYB75, AtMYB90, AtMYB113, and MrMYB1. Moreover, EsMyb90 has a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif (marked by black arrows in Fig.1A), which is required for interaction with R/B-like bHLH proteins [10,16]. To further identify the relationship of EsMYB90 to other MYB proteins, we generated a phylogenetic tree with 29 MYB proteins involved in anthocyanin synthesis in 16 plants. Our results demonstrated that EsMYB90 was clustered in the clade I (Fig. 1B) , which consists of AtMYB75, AtMYB90, AtMYB113, AtMYB114, BoMYB1 and BrMYB114 that are important for anthocyanin accumulation [17,19,25,26,27,55]. EsMYB90 has a relatively farer phylogenetic relationship to MYB proteins in clades II, III, and IV, although those MYBs promote biosynthesis of PA and anthocyanin, except for EsMYB5(XP_006407201) have no research reports yet. In summary, our results suggest that EsMYB90 is a R2R3-MYB, which may function in proanthocyanin and anthocyanin synthesis. Expression pattern of EsMYB90 in E.salsugineum and subcellular localization of the protein MYB transcriptional factors. For instance, at least four MBW complexes assembled with various MYBs are involved in the PA accumulation in the innermost cell layer of Arabidopsis seed coat . Thus, our results suggest that different from other plants less MYBs regulate anthocyanin biosynthesis in E. salsugineum and EsMYB90 is a major player. The regulatory mechanisms for flavonoid biosynthesis are conserved in higher plants . The MYB proteins usually determine the involvement of MBW complexes in specific pathways [1,60]. In the MBW complex, MYBs have the highest binding specificity compared with bHLH and WD40. MYB and bHLH bind to at least one of three conserved cis elements, i.e. MYB-core, AC-rich, and E/G-box, in promoters of four LBGs (late biosynthesis genes: DFR, TT19, TT12, and AHA10), which specify their expression in the seed coat of Arabidopsis [23,60]. Expression of LBGs are regulated by MYB75, MYB90, MYB113 and MYB114 in Arabidopsis [15,16]. Our phylogenetic analysis showed that EsMYB90 was in the same clade with Arabidopsis MYB75, MYB90, MYB113, and MYB114, suggesting that EsMYB90 may play a more important role in regulation of LBGs (DFR, ANS/LDOX and UFGT) expressions, which is in accordance with our research results. In addition, our results show that EsMYB90 promotes expression of early biosynthesis genes, such as PAL, CHS, CHI, and F3H which agrees with findings in other plants [1,10,36]. Furthermore, it is possible that EsMYB90 upregulates expressions of NtMYB and NtbHLH genes involved in anthocyanin biosynthesis, because expressions of were significantly increased in 35S:EsMYB90 tobacco transgenic plants (Additional file 2). Overall, our results showed that ectopic expression of the novel EsMYB90 gene can strongly induce the anthocyanin biosynthesis by promoting expression of anthocyanin biosynthesis pathway genes, particularly functioning in the LBGs. Our study also paved the way for the application of EsMYB90 to genetically engineering crops and ornamental plants. Conclusions 13 EsMYB90, a R2R3 MYB transcription factor, is localized in nucleus. 35S:EsMYB90 transgenic tobacco and Arabidopsis showed purple-red, purple-black phenotypes, and accumulated more anthocyanin in the leaves, stems and flowers compared with wild type. The results showed that ectopic expression of EsMYB90 in stems, leaves and flowers of transgenic tobacco could significantly enhanced the expression of anthocyanin biosynthetic genes including EBGs (PAL, CHS, and CHI) and LBGs (DFR, ANS, and UFGT), particularly in LBGs. The study suggested that EsMYB90 plays a key role in regulating anthocyanin biosynthesis, and it provide new clues to increase the content of anthocyanin in transgenic plants. Methods Plant materials and growth conditions The seeds of Eutrema Salsugineum (Shandong ecotype), Arabidopsis thaliana (Landsberg ecotype) and tobacco (Nicotiana tabacum cv SR1) are preserved and presented by Shandong Provincial Key Laboratory of Plant Stress Research, College of Life Science, Shandong Normal University. Wild-type and transgenic plants of Nicotiana tabacum and Arabidopsis thaliana (Columbia-0) were grown in a mixture of vermiculite, perlite and peat moss (1:1:1) in a greenhouse with 25°C, a photoperiod of 16 h light /8 h dark. Eutrema Salsugineum were grown in a growth chamber with 22°C, a photoperiod of 16 h light /8 h dark and 70% relative humidity. T3 generation homozygous transgenic and wild-type tobacco plants were used for analyses. After 8 weeks growth of tobacco plants at the period of 8-9 leaves, the sixth leaves of wild-type and transgenic plants were collected for transcriptome and qRT-PCR identification of the transcriptome. Three biological repeats for transcriptome and qRT-PCR were performed. In tobacco, stems, young leaves (YL), mature leaves (ML), flowers, and fruit pods at the flowering stage, and mature seeds were used to determine the content of anthocyanins, meanwhile the stems, young leaves (YL), and flowers were used to examine expression of anthocyanin biosynthesis genes. Similarly, Arabidopsis roots, stems, leaves, flowers, and fruit pods from plants growing 4 weeks, and mature seeds were used to determine the content of anthocyanins. In all cases, samples were frozen immediately in liquid nitrogen and stored at -80°C. Three repeats of all tests were conducted. Construction generation and plant transformation The whole coding sequence of EsMYB90 was PCR-amplified from the Eutrema Salsugineum cDNA using the forward primer 5′-CCGGAATTCTTTAGAATACTTATTGGTCC-3′ and the reverse primer 5′-CGCGGATCCATCAGAGACAGATATTAGTTGG-3′ with EcoR I and BamH I restriction enzyme sites at the 5' and 3', respectively (Additional file 9). The resulting EsMYB90 fragment was cloned into the pMD18-T vector (Takara, USA). After sequencing confirmation, the EsMYB90 fragment was subcloned into the EcoRI -BamHI sites of the pCAMBIA3301H vector, where EsMYB90 was under the control of the CaMV 35S promoter. The expression vector (35S:EsMYB90-pCAMBIA3301H) was finally introduced into the Agrobacterium tumefaciens strain GV3101. Transformation of N. tabacum was performed using the leaf disc method essentially as reported by Horsch et al. . Transgenic tobacco seedlings were selected on the MS medium containing 6 mg / L of bastar and 300 mg / L of cefalexin. Transformation of A. thaliana Columbia-0 was performed using the floral-dipping method  and transformants were screened by spraying 0.1% of bastar herbicide. The presence of the transgene was further confirmed by PCR using specific primers for EsMYB90. The homozygous transgenic N. tabacum and A. thaliana were used for subsequent phenotypic and functional analysis. Anthocyanin analysis Stems, young leaves (YL), mature leaves (ML), flowers, and fruit pods of tobacco growing about 8 weeks at the flowering stage, and the mature seeds were sampled, respectively. Similarly, stems, leaves, flowers, fruit pods, and roots of Arabidopsis growing about 4 weeks at the bolting period, as well as the mature seeds were also collected. All materials were frozen immediately in liquid nitrogen and ground to powders. The anthocyanin content was determined using an improved method described by Neff and Chory . The measurements of A 530 and A 657 were conducted with a spectrophotometer (UV-1800, Shimadzu). The results were calculated by the equation (A530-0.25*A657)/fresh weight. Three replicates were performed for each sample. QZ conceived and designed the experiments. QZ wrote the manuscript with contributions from DZ, CM, XW, SG, and CG. CG, YQ, SG,CL1, CZ, and CL2 carried out most of the experiments. CL1 and CZ took care of the plants. QZ,YQ,CG and CL2 analyzed the data. All authors read and approved the final manuscript. metabolite analysis reveals the role of delphinidin metabolism in flower colour in Grape hyacinth.