Identification And Characterization Of Anthocyanin Biosynthetic Genes In Mulberry Plants

Anthocyanins are the most important water-soluble plant pigments, which are widely distributed in a variety of plant and are the main pigments assigned to different colors of flowers and fruits. Anthocyanins not only play important roles in physiological and biochemical activities of plants, but also exhibit lots of biological activities in antioxidant, anti-inflammatory, anti-carcinogenic, and anti-cardiovascular disease.Anthocyanins belong to flavonoid according to its chemical structure. The biosynthesis of anthocyanins is part of flavonoid bosynthesis. This is a complex biosynthetic pathway involved many structure genes and regulated genes. This pathway has been clarified in a lot of model plants.Mulberry is a perennial woody plant, besides its traditional usage as the forage for silkworm, also has other important applications, such as ecological protection, Chinese medicine resources, nutrition and health, and so on. Mulberry fruits are also popular because of their delicious taste and considerable nutritional value. Anthocyanins are the most abundant nutrient components in mulberry fruits, which also have important biological activities. Over11anthocyanins have been identified in mulberry and the main components are cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside. Characterization of anthocyanin biosynthetic genes will facilitate the genetic improvement of anthocyanins in mulberry.In this research, we identified anthocyanin biosynthetic genes in the mulberry genome using bioinformatic methods. The qRT-PCR was conducted to study the transcriptional levels of the identified genes during fruit development in two mulberry cultivars with different fruit colors. For these key genes, Western blotting was used to confirm the protein expression patterns. UPLC was used to identify and quantify the anthocyanins in mulberry during fruit ripening. The relationship between the expression of anthocyanin biosynthetic genes and anthocyanin contents was analyzed. Then we characterized gene functions and promoter activities by plant transgenic technology. The main results are as follows:1. Identification and bioinformatic analysis of mulberry anthocyanin biosynthetic genes.We identified nine putative anthocyanin biosynthetic genes in Morus notabilis by bioinformatic methods, including two CHS genes, two F3H genes, two F3’H genes, and one gene each of CHI, DFR, and ANS. Eight of these genes were cloned except for ANS (We cloned ANS in another mulberry species M. atropurputea Roxb.). A F3’5’H gene was not found in the mulberry genome. Gene structure analysis revealed the presence of introns in all identified genes. The number and position of gene introns were generally conserved when compared with those of other plants.The deduced amino acid sequences of the nine predicted mulberry anthocyanin biosynthetic genes were blast searched against the SwissProt database. Most genes had quite high sequence identities ranged from65to93%except for MnF3H2, which showed less than40%similarity. The results of domain prediction and multiple sequence alignment indicated that the catalytic domains and active sites were conserved in these mulberry proteins.The cis-acting regulatory elements in promoters of mulberry anthocyanin biosynthetic genes were predicted in plantCARE and the results were cataloged into three types. The light-responsive elements were in type I, which was the most abundant ones including Box I, G-Box, ATCC-motif, and so on. Hormone-responsive elements were in type II, such as abscisic acid responsiveness element, ethylene-responsive element, CGTCA-motif, and TGACG-motif involved in the MeJA-responsiveness. Elements in type III were stress-responsive elements, such as fungal elicitor responsive element Box W1, HSE element involved in heat stress responsiveness, LTR element involved in low-temperature responsiveness. In addition, MBSI and MBSII elements were also predicted in the promoters of MnF3’H1, MnDFR and MnANS, which were MYB binding sites involved in flavonoid biosynthetic genes regulation. This result suggested that mulberry anthocyanin biosynthesis pathway was also regulated by the MYB type transcription factors.2. Expression of mulberry anthocyanin biosynthetic genes during fruit development in two mulberry cultivarsExperiments were carried out to confirm the expression levels of the anthocyanin biosynthetic genes in six tissues of M. notabilis using qRT-PCR. The results revealed that the expression pattern of these genes were quite different and genes in the same family were also different. For example, the MnCHS1gene was expressed exclusively in male flowers and its transcriptional level was much higher than that of MnCHS2, which was mainly expressed in roots and female flowers. In the cases of MnF3H genes, MnF3Hl was expressed mainly in roots and male flowers, while MnF3H2was highly expressed in shoots, leaves, and female flowers. For two MnF3’H genes, the expression of MnF3’H2was restricted to roots, while MnF3’H1was broadly expressed in several tissues. Differences in the expression of members of the same gene subfamilies might be the cause of functional differences and specialization of anthocyanin biosynthesis. No MnANS transcripts were detected in any of the six types of tissues and this was consistent with that no anthocyanin was measured in M. notabilis.The copy numbers of the anthocyanin biosynthetic genes in M. notabilis and two mulberry cultivars with different fruit colors (Da10with purple fruit and Zhenzhubai with white fruit) were investigated by Southern blotting. Generally, there were less gene copies in M. notabilis than in DalO or Zhenzhubai and the hybridization patterns of Da10were similar to those of Zhenzhubai. The expression patterns of the nine mulberry genes associated with anthocyanin biosynthesis were assessed in the fruits of these two mulberry cultivars. Based on these expression patterns, we divided the genes with similar pattern into four types. It is worth noting that genes in type I included CHS1, CHI, F3H1, F3’H1, and ANS, which expressed increasingly during fruit ripening in the Da10cultivar, but only trace expression of these genes was observed in the Zhenzhubai cultivar. For the two key genes (DFR and ANS) in the late steps of anthocyanin biosynthesis, we used the polyclonal antibodies to detect their protein expression levels. Western blotting results indicated the protein expression of ANS was similar with its transcrptional expression, but DFR exhibited a different pattern. The transcrptional levels of DFR were decreasing during fruit ripening in the Da10cultivar but protein levels were increasing. We speculated there was a negative feedback regulation in DFR transcriptional regulation. The types and concentrations of anthocyanins in two mulberry cultivars with differently colored fruit were determined using UPLC. The results indicated that the two compounds present in the fruits of Da10were cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside. The concentrations of the two anthocyanins were increased significantly during fruit ripening and the degree of cyanidin-3-O-glucoside increasing was much higher than that of cyanidin-3-O-rutinoside. No anthocyanin was detected in Zhenzhubai fruits at any stage. This result was consistent with the expression patterns of mulberry anthocyanin biosynthesis genes.Previous studies indicated that the main anthocyanins in mulberry were cyanidin derivatives and few pelargonidin derivatives. Anthocyanins identified in our results were also cyanidin derivatives. The anthocyanin biosynthesis pathway indicated that different anthocyanin types were synthesized from the same precursor naringenin. Here, the F3’5’H gene was not found in the mulberry genome, which is the key gene for the formation of delphinidin. We then speculated that the absence of the F3’5’H gene in mulberry may account for the composition of anthocyanins in mulberry plants. On the other hand, Sequence analysis indicated that MnDFR belongs to the Asp-type DFRs, which are unable to convert dihydrokaempferol to leucopelargonidin. This would explain why cyanidin derivatives are the most abundant anthocyanins in mulberry plants.3. Functional research of anthocyanin biosynthetic genes in mulberry plantsThe promoter activity of MnDFR was examined using GUS as a reporter gene in transgenic Arabidopsis. GUS staining indicated that MnDFR promoter only exhibited activity in root and the activity was decreased when treated in dark. When treated in high temperature, the MnDFR promoter activity was induced in leaf. This result suggested that MnDFR may be involved in reactions that induced by high temperature stress.We alao transformed MnDFR and MnANS into Arabidopsis to observe their effect on anthocyanin accumulation in Arabidopsis. Compared with the wild type, the basal part of transgenic Arabidopsis stem exhibited apparent red color. During the growth of plants, the whole stem exhibited light red color and this phenomenon was much more apparent in transgenic plants with MnANS than MnDFR. The reason for this difference may be that ANS is the downstream enzyme in the anthocyanin biosynthesis pathway than DFR, so the effect of ANS may be more effective.We further transformed eight of these anthocyanin biosynthetic genes except for ANS into tobacco to analyze the heterologous expression of these genes in tobacco. The results revealed that no significant change have been observed in transgenic plants but the anthocyanin contents of tobacco flower. Different genes exhibited different effects. The content of anthocyanins in transgenic tobacco with MnCHSl, MnCHS2, MnCHI, and MnDFR increased, with MnF3Hl and MnF3H2decreased, and no apparent changes were observed in those transgenic tobacco with MnF3’HI and MnF3’H2.In the present study, nine putative genes involved in anthocyanin biosynthesis in mulberry plants were identified and cloned. Bioinformatic analysis indicated that these genes were conserved with their counterparts of other plants in sequence similarity, gene structures, catalytic domains and active sites. qRT-PCR, Western blotting and UPLC experiments were carried out in developmental fruits of mulberry cultivars with different fruit colors. The results indicated that anthocyanin concentrations correlated with the expression levels of genes associated with most anthocyanin biosynthetic steps during the fruit ripening process. Transgenic experiments revealed that heterologous expression of these genes affected the anthocyanin accumulation in tobacco and Arabidopsis, particular in Arabidopsis. For the first time, our studies comprehensively studied the mulberry anthocyanin biosynthetic genes on the molecular level, clarified the relationship between composition of anthocyanins and its biosynthetic genes in mulberry. This study may facilitate genetic engineering for improvement of the anthocyanin content in mulberry fruit.