Functional MYB transcription factor encoding gene AN2 is associated with anthocyanin biosynthesis in Lycium ruthenicum Murray

Background Lycium ruthenicum Murray is an important economic plant in China and contains higher levels of anthocyanins in its fruits than other Lyciums. However, the genetic mechanism of anthocyanin production in this plant is unknown. Results Based on previous transcriptome analysis, LrAN2 and LbAN2, encoding MYB transcription factors, were isolated from L. ruthenicum and L. barbarum, respectively. Both genes contained two introns, encoded 257 amino acids with two-Aa difference, and carried the unabridged HTH-MYB, MYB-like DNA-binding, and SANT domains. In the phylogenetic trees, LrAN2 and LbAN2 were found to be closely related to NtAN2, which regulates anthocyanin biosynthesis in tobacco. Overexpression of LrAN2 and LbAN2 induced anthocyanin biosynthesis in all tissues of tobacco. The anthocyanin content in the leaves of transgenic lines with LbAN2 was lower than LrAN2. It indicated that the function of LbAN2 was weaker than LrAN2. The AN2 transcript could be detected only in the fruits of L. ruthenicum and increased during fruit development, accompanied by anthocyanin accumulation. In natural population, the alleles LrAN2 and LrAN2 were associated strictly with L. ruthenicum and L. barbarum, respectively. Moreover, an AN2 genetic diversity study suggested that Lyciums with yellow, white, purple, and jujube red fruits were derived from L. ruthenicum. Conclusions Two AN2 alleles, from L. ruthenicum and L. barbarum, were functional MYB transcriptor regulating anthocyanin biosynthesis. The functional diversity and high expression level of LrAN2 could be the reason for high anthocyanin content in the fruit of L. ruthenicum. Lyciums with yellow, white, purple, and jujube red fruits were derived from L. ruthenicum based on AN2 sequence diversity. The results may be advantageous in identifying new varieties and breeding new cultivars. Electronic supplementary material The online version of this article (10.1186/s12870-019-1752-8) contains supplementary material, which is available to authorized users.


Background
Lycium ruthenicum Murray is a traditional Chinese herb listed in the Tibetan medical classic "Jing Zhu Ben Cao". The fruit of L. ruthenicum has been used for the treatment of menopause, heart disease and abnormal menstruation for thousands of years in China [1]. The high anthocyanin content in the fruit of L. ruthenicum is the distinctness characteristically different to other Lyciums, which also has been thought to contributed to its special medicinal value [2][3][4][5]. Nowadays, the molecular mechanism of a high level anthocyanin synthesis remains unclear.
Our previous research had identified the genes related to anthocyanin biosynthesis in the fruits of L. ruthenicum and L. barbarum based on transcriptome analysis. Compared with L. barbarum, 733,070 genes were upregulated while 25,779 genes appeared downregulated in the fruits of L. ruthenicum. All structural genes related to anthocyanin biosynthesis exhibited higher levels of expression in L. ruthenicum than L. barbarum, which implied the transcription factor was responsible for high anthocyanin content in the fruit of L. ruthenicum. The transcript level of bHLH genes in L. ruthenicum has no significant difference to L. barbarum, while the transcript level of the MYB transcription factor in L. ruthenicum was 35.66 times of L. barbarum. It could be induced that the MYB transcription factor played an important role in the black fruit formation of L. ruthenicum. Total four unigenes were homologous to the MYB transcription factor in the assembly sequence database, and these unigenes were thought to be derived from the same gene AN2 after further sequence alignment.
In this manuscript, the MYB transcription factor gene AN2s were isolated from L. ruthenium and L. barbarum to evaluate its role in the black fruit development of L. ruthenium. It will lay a good molecular foundation for the selection of superior resources and breeding of new varieties of L. ruthenicum.

Molecules characteristics
Previous experiments have shown that the expression of MYB transcription factor AN2 occurs at a higher level in fruits of L. ruthenicum than L. barbarum. LrAN2 and LbAN2 were isolated from L. ruthenicum and L. barbarum based on RNA-sequence data. Both open reading frames (ORFs) of LrAN2 and LbAN2 are 774 bp in length and encode 257 amino acids. Although five nucleotide differences exist in the ORFs of LrAN2 and LbAN2, only two amino acids differences have been discovered in translated sequences (Additional file 1: Figure S1). The nucleotide sequences of LrAN2 and LbAN2 contain 1383 bp and 1395 bp, respectively. Both contain two introns (Additional file 1: Figure S1), but 32 single nucleotide polymorphisms and one indel of 15 nucleotides in the second intron distinguish LrAN2 from LbAN2 (Additional file 1: Figure S1).
In order to determine the evolutionary relationship between Lycium AN2 and MYB transcription factors in other plants that regulate anthocyanin biosynthesis, amino acid sequences were downloaded from the NCBI database to construct a phylogenetic tree. LrAn2 and LbAN2 were the closest to NtAN2, AtMYB113, AtMYB114, AtPAP1 and AtPAP2 (Fig. 1). AN2 is clustered with other MYB transcription factors which were the main genes controlling anthocyanin synthesis in different tissues of other species. It includes many solanaceous plants, such as Solanum melongena (SmAN2), Solanum lycopersicum (SlAN2), Solanum tuberosum (StMTF2), Petunia x hybrida (PhAN2) and Capsicum annuum (CaAN2).
The amino acid sequences of CaAN2, SmAN2, PhAN2, SlAN2, and StMTF2 were downloaded to investigate the structural domains of LrAN2 and LbAN2. Both LrAN2 and LbAN2 contain the complete HTH_MYB, MYB-like DNA-binding, and SANT domains, which are important in regulating anthocyanin biosynthesis (Fig. 2). The two-Aa difference between LrAN2 and LbAN2, L > H existed in the HLH domain and R > Q was just outside of the N terminus of MYB R3 domain.

Transcription profile of LrAN2 and LbAN2
Semi quantitative PCR was used to compare the level of transcription for LrAN2 and LbAN2 in different tissues of L. ruthenicum and L. barbarum. The fruits of L. ruthenicum and L. barbarum take 36 days to develop. Fruit samples were collected at 9-day intervals for RT-PCR. The results showed that the LrAN2 transcript could be detected only in the fruit of L. ruthenicum and was absent from root, stem, leaf and fruit of L. barbarum (Fig. 3a). Correspondingly, only the fruit of L. ruthenicum accumulated anthocyanin. Interestingly, the expression of LrAN2 in L. ruthenicum increased with fruit development and was accompanied by anthocyanin accumulation (Fig. 3b, c).

Overexpression of AN2 induces anthocyanin biosynthesis in tobacco
The PJAM1502 constructs contained the double 35 s promoter which could drive the objective gene transcript in all tissues. Both transgenic lines, LrAN2 and LbAN2, could induce anthocyanin biosynthesis in root, stem, leaf, flower and seed of tobacco (Fig. 4a). The transgenic lines of LrAN2 showed the deeper purple leaf, while the transgenic lines of LbAN2 displayed the plaques purple leaf (Fig. 4a). The relative anthocyanin content of the LrAN2 transgenic lines was about 30 g − 1 fresh weight, while that of LbAN2 was 10 g − 1 fresh weight (Fig. 4b). All transgenic lines contained higher anthocyanin content  than the wild lines (Fig. 4b). These results show that both LrAN2 and LbAN2 can regulate anthocyanin biosynthesis by encoding MYB transcription factors.

Allelic variation of AN2 in natural populations of Lycium
Apart from red fruit (L. barbarum) and black fruit (L. ruthenicum) varieties, some mutated lines of the Lycium family, which are restricted to particular regions, carry yellow, jujube, purple, and white fruit. To explore the genetic relationship between L. ruthenicum, L. barbarum, and these mutated lines, the genomic sequences of homologous AN2 were isolated from the lines with yellow, jujube, purple, and white fruits. Only LbAN2 from red fruit (L. barbarum) carried the same insertion of 15 nucleotides (Fig. 5a, Additional file 1: Figure S1). The mutation lines were determined to be at the same phylogenetic branch with Black fruit, which indicates that they are derived from L. ruthenicum. A total of 54 L. ruthenicum and L. barbarum lines were collected from Qinghai, Xinjiang, Gansu and Ningxia, which are the main areas of distribution for L. ruthenicum and L. barbarum in China (Fig. 5b). Based on the indel in the second intron of the nucleotide sequence, a set of special primers was designed to distinguish LrAN2 from LbAN2. The special primers were effective in distinguishing L. barbarum from L. ruthenicum and other lines in China. All 24 L. ruthenicum lines were of the genotype LrAN2 and the 30 L. barbarum lines were of the genotype LbAN2 (Additional file 2: Table S1).

Discussion
In this study, we isolated two alleles LrAN2 and LbAN2 from L. ruthenicum and L. barbarum, respectively, and investigated the function of AN2 in regulating anthocyanin biosynthesis.
LrAN2 is a functional MYB transcription factor gene regulating anthocyanin biosynthesis. Firstly, LrAN2 contained the complete HTH_MYB, MYB-like DNA-binding, which was important for exercising regulating function as a MYB transcription factor. In phylogenetic tree, LrAN2 The phylogenetic tree of AN2 alleles in Lycium (a) and the geographical distribution of L. barbarum and L. ruthenicum (b). The tree was constructed using MEGA6, neighbor-joining phylogeny testing and 1000 boot strap replicates was the same branch of MYB transcription factors (NtAN2 and AtPAP1) [29,30]. NtAN2 gene encoded R2R3-MYB transcription factor and regulated anthocyanin synthesis in tobacco flowers [29]. While AtPAP1 encoded MYB75 transcription factor in Arabidopsis thaliana, which could induce purple anthocyanin production in most organs of Arabidopsis thaliana [30]. It implied LrAN2 was functional MYB transcriptor. Secondly, the transcript level of LrAN2 was substantially higher in black fruits relative to red fruits and other tissues with low anthocyanin content. The fruit development of L. ruthenicum was accompanied by the increase of anthocyanin content and the expression of LrAN2 gene. The transcript of LrAN2 was relative to the anthocyanin biosynthesis obviously. Thirdly, the direct evidence was that overexpression of LrAN2 promoted anthocyanin biosynthesis in tobacco, similarly to NtAN2 and AtPAP1.
Taken together, all results suggest that LrAN2 is a functional analog of NtAN2 encoding MYB transcription factors regulated to anthocyanin biosynthesis in plant cells [31][32][33][34]. Function diversity of AN2 is possibly related to the production of black-colored fruit in L. ruthenicum. Thirty-two single nucleotide polymorphisms and one indel of 15 nucleotides in the second intron could differentiate LrAN2 from LbAN2. In natural population, the alleles LrAN2 and LrAN2 were associated strictly with L. ruthenicum and L. barbarum, respectively. Five nucleotide differences in the ORFs of LrAN2 and LbAN2 produce two amino acids differences. The two-Aa difference L > H existed in the HLH domain and R > Q existed just outside of the N terminus of MYB R3 domain. Theoretically, they were crucial determinants of protein structure and possibly function. The heterologous expression of LrAN2 promoted anthocyanin biosynthesis in tobacco more effectively than LbAN2, which proved the function diversity exactly. Moreover, the transcript of LrAN2 couldn't been detect obviously in fruits of L. barbarum, which is consistent with our previous transcriptome experiment. Possible reason of high expression in L. ruthenicum could be the indels in their 2nd intron, because some indels could function in transcription regulation of anthocyanin biosynthesis. The promotor region could also contain some specific cis-recognition motif(s) to induce LrAN2 transcript in the fruit of L. ruthenicum. Considering LrAN2 was the only MYB transcription factor gene related to anthocyanin biosynthesis in black fruit of L. ruthenicum based on the transcriptome analysis, it could be inferred that both functional diversity and high expression level of LrAN2 could be the reason for high anthocyanin content in the fruit of L. ruthenicum. More works were being enforced to prove the role of LrAN2 in black fruit formation of L. ruthenicum.

Conclusion
In this study, two allelic genes LrAN2 and LbAN2 were isolated from L. ruthenicum and L. barbarum. They carried the function regulating anthocyanin biosynthesis as the MYB transcription factors. The functional diversity and high expression level of LrAN2 could be the reason for high anthocyanin content in the fruit of L. ruthenicum. Lyciums with yellow, white, purple, and jujube red fruits were derived from L. ruthenicum based on AN2 sequence diversity. The results may be advantageous in identifying new varieties and breeding new cultivars.

Plant material
The L. ruthenicum variety LMH1 and L. barbarum variety Ningqi 7 are planted widely in China and the two cultivars were chosen for this research as representatives of L. ruthenicum and L. barbarum. Twenty-four wild varieties of L. ruthenicum and 30 wild varieties of L. barbarum, which were collected from different provinces in Northwest China, including Xinjiang, Qinghai, Gansu and Ningxia, were used to study the relationship between fruit color and presence of the AN2 allele (Additional file 2: Table S1). Wolfberries in the colors purple, white, jujube red and yellow, from the genetically diverse area of the Qaidam basin, were used to study genetic variation of AN2. No permission was required in collecting the plants. Zenggen Liu is responsible for identifying and numbering these materials. All materials were preserved in the Northwest Plateau Institute of Biology, Chinese Academy of Sciences.

DNA and cDNA preparation
One g of stem tip leaves was selected to extract genomic DNA [35]. Root, stem, leaf, and fruit samples were collected from corresponding plants for RNA extraction. The Trizol total RNA extraction method was used for extracting Total RNA [36]. A reverse transcription kit (Thermo Fisher First Strand cDNA Synthesis Kit, Beijing, China) was used according to the instructions to synthesize a cDNA from RNA.

PCR and semi-quantitative PCR
Primers were synthesized by BGI Biological Technology Co., Ltd. The 50 μl reaction system included 10 μl 5×GC Buffer, 4 μl 10 mmol dNTP, 0.5 μl 20 pmol primer, and 0.5 μl (100 ng) cDNAs (Thermo Fisher Science, Beijing, China) were supplemented with ddH2O. The cycling conditions were as follows: 1 cycle at 98°C for 2 min, 35 cycles at 98°C for 10 s, 65°C for 30 s and 72°C for 2 min, followed by a cycle at 72°C for 10 min. All PCR was conducted in the GeneAmp PCR System 9700 (Thermo-Fisher Scientific, Shanghai, China). The high-fidelity Phushion DNA polymerase (Thermo-Fisher Scientific, Shanghai, China)were used in all PCR reactions. The PCR products were extracted with the Tiangen TIANgel Midi Purification Kit (Tiangen) from 1.0% agarose gels and were cloned into the pGEM-T Easy Vector plasmid (Promega Corporation, Madison, Wisconsin, USA). The recombinant plasmid was transformed into Escherichia coli DH5α cells, and six positive clones were sequenced in a commercial company (Huada Gene, Shenzheng, China).
The semi-quantitative RT-PCR experiments were conducted using previously published methods [37]. Selecting of different tissue parts, including root, stem, leaf and fruit. The amplification of tubulin gene transcripts was used to normalize the cDNA contents of various reverse transcription mixtures before PCR and to monitor the kinetics of thermo-amplification during PCR. The reproducibility of the transcriptional patterns revealed by semi-quantitative PCR was tested by at least three independent assays. Additional file 3: Table S2 contained all primers used in this study.

Overexpression of LrAN2 and LbAN2 in tobacco
The construct used for plant transformation was based on the binary vector PJAM1502, which contains a double 35 s promoter [38]. The construct PJAM1502:LrAN2 and PJAM1502:LbAN2 was based on the Gateway Cloning Kit (Invitrogen, USA). Binary vectors were electroporated into Agrobacterium tumefaciens strain GV3101. The leaf disc transformation method was used for tobacco transformation [39]. The selective medium of transgenic shoots contained 0.7% (w/v) agar, 3% (w/v) sucrose, 1.0 mg/L 1-Naphthaleneacetic acid (NAA), 1.0 mg/ L 6-benzylaminopurine (BAP), 150 mg/L kanamycin, and 300 mg/L Timentin (ticarcillin disodium and clavulanate potassium). The transgenic shoots grow up in the greenhouse with long-day lighting (16 h light/8 h dark) after 1 month. For further experiments, the T3 family lines carrying objective gene without the separation were used.

Relative anthocyanin content measurement
The fruits of 9, 18, 27 and 36 days after flowering were selected for estimating the anthocyanin content. The leaves of transgenic lines and wild lines were used for estimating the anthocyanin. Anthocyanin content was measured using the "Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines" method (AOAC Official Method 2005.02) and calculated using data sets from three independent experiments. Statistical analyses of the obtained data were performed using the software package SPSS for Windows 11.5 with a 95% confidence interval [40,41].
Genotyping the natural population of L. ruthenicum and L. barbarum with AN2sp To distinguish L. ruthenicum from L. barbarum, the polymorphic PCR marker, AN2sp was designed according to the nucleotide sequence difference between the second intron regions of LrAN2 and LbAN2. The primers of AN2sp are listed in Additional file 3: Table S2. The amplicons produced by AN2sp were 117 bp in length for LrAN2 and 132 bp in length for LbAN2 (Additional file 4: Figure S2). The geographical distribution map is made by ArcGIS 10.0 software and base map is derived from ArcMap version 10.2.