Studies On The Molecular Regulation Of The Anthocyanin Biosynthesis Related To Leaf Color Of Solenostemon Scutellarioides

Solenostemon scutellarioides (Coleus blumei), one of perennial herbs of the genus Solenostemon (Lamiaceae), is one kind of important and famous foliage plant with multicolor leaves, which has an extensive application and attracts great attentions. However, the molecular mechanism of color emerging in colored-leaf plant are poorly understood. Furthermore, the cloning and expression of genes involved in coloration of leaf in S. scutellarioides are not reported up to now. The limited understanding about the mechanism of color emerging in colored-leaf delayed the development of molecular breeding and leaf color engeneering of foliage plant with S. scutellarioide as representative. Because anthocyanins play a key role in forming colorful leaf, in this dissertation, we report the study on molecular regulation of anthocyanin biosynthesis in S. scutellarioide. Four key enzyme genes in this pathway and two Myb transcription factors related with phenylpropanoid metabolism were cloned from red leaves of S. scutellarioide.cv. C4 cultivar. Their molecular characterization, expression files and functional activity were analyzed, in detail, by using bioinformatic method, southern blot, RT-PCR and expression in transgenic tobacco. We got the main results listed below:1. Cloning of the S. scutellarioides CHS gene (SsCHS) and its expression in transgenic tobaccoChalcone synthase (CHS) is a key enzyme and catalyzes the first step in flavonoid and anthocyanin biosynthesis. The full-length cDNA of CHS gene from S. scutellarioides (SsCHS), and its genomic DNA sequence were obtained by using RACE (rapid amplification of cDNA ends) method. The GenBank accession No. of SsCHS is EF522149 and EF522150 for mRNA and genomic sequence, respectively. The comparison between cDNA and genomic DNA sequences revealed that SsCHS is composed of two exons and one intron, one of the typical features of CHS genes. Its deduced amino acid sequence was found to have extensive homology with other plant CHS proteins, especially to Perilla. frutescens (Lamiaceae) 2 CHSs, via multiple alignments in NCBI blast and Vector NT I Suite 10.0 software. The comparative modeling revealed that the 3D model of putative SsCHS protein closely similar with that of Medicago sativa CHS2 protein (MsCHS2), the crystallogram and function of which has been elucidated, and all active sites in SsCHS protein are the same as in MsCHS2. Southern blot analysis indicated that SsCHS belongs to a multigene family, which has 5-8 memebers.Semi-quantitative RT-PCR revealed that SsCHS in red S. scutellarioides cultivar has expression in stem, leaf and flower, especially highly expressed in leaf, while no transcripts could be detected in root. In red leaf cultivar, the transcript levels of SsCHS could be induced by white light or drought stress.The sense, antisense and RNAi plant expression vectors of SsCHS were constructed and transformed into tobacco W38 by Agrobacterium tumefaciens. In 45 lines of transgenic plants, the content of total flavonoids in 71% sense transgenic tobacco leaves increases significantly, the highest is nearly up to 3 folds, which indicate that the synthesis of total flavonoids are promoted by overexpression of SsCHS. However, the content of total flavonoids in 11 % sense transgenic tobacco leaves decrease remarkably, which may be caused by co-supresssion between SsCHS and endogenous NtCHS. At the same time, the relative content of anthocyanins of 22 lines are improved significantly, and 6 lines are no significant changes, and 4 lines decrease in transgenic tobacco leaves of 32 lines that total flavonoids increased significantly. The results indicate that possibly there are some factors that regulate distribution of different products from branch pathways of flavonoid.2. Cloning and expression characterization of the S. scutellarioides F3H gene family (SsF3H)Flavanone 3-hydroxylase (F3H, or FHT) is the third key enzyme in the flavonoid and anthocyanin biosynthetic pathway, located in the early-stage of biosynthesis of anthocyanin, providing a branching point for the biosynthesis of different flavonoids. Two full-length flavanone 3-hydroxylase genes, SsF3Hl (GenBank accession No. EF522151and EF522152 for mRNA and gene) and SsF3H2 (EF522153 and EF522154 for mRNA and gene), were cloned from red leaves of S. scutellarioides by RACE technology for the first time. Both of them contain 2 introns with canonical GT…AG splicing sites. The genomic and full-length cDNA sequences of SsF3H1 are 1454 base pair (bp) and 1101 bp, respectively. SsF3Hl has a 1092 bp open reading frame (ORF) encoding a 363-amino-acid polypeptide. While in SsF3H2, genomic sequence is 1495 bp and cDNA is 1175bp, ORF is 1095bp encoding a 364-amino-acid polypeptide. NCBI blastn and NCBI blastp indicated that SsF3Hl and SsF3H2 share high homologies to plant F3H genes, especially to P. frutescens F3H. NCBI Conserved Domain Search found that they both contain 2 conserved domains, one is 2OG-FeII Oxy (pfam03171, 2OG-Fe(II) oxygenase super family) and another is PcbC (COG3491, Isopenicillin N synthase and related dioxygenases). The invariable conserved amino acid residues, H, D and H, for ligating ferrous iron and the RXS motif for participating in 2-oxogutarate binding site all present in SsF3H1 and SsF3H2 at the similar positions like other F3Hs, which indicates that the two deduced SsF3Hs belong to 2-oxoglutarate dependent dioxygenase requiring 2-oxoglutarate, Fe²⁺, and molecular oxygen for activity. Their predicted tertiary structures, by homology-based modeling, are similar to the F3Hs structure reported and conform to typical characters. Southern blot analysis indicated that in S. scutellarioides there are about 2-3 F3H copies.As semi-quantitative RT-PCR showed, the overall of SsF3H gene family was observed in stem, leaf and flower, while no expression in root. Expression patterns of individual SsF3H member are consensus with those of SsF3H gene family. In leaf of red cultivar, under white light or drought stress conditions, the expression of SsF3H increased.The sense and antisense plant expression vectors of SsF3H2 gene were constructed and transformed into tobacco W38, respectively, and the 76-sense and 64-antisense transformed lines with Km resistance were obtained.3. Cloning of the S. scutellarioides DFR gene (SsDFR) and its expression in transgenic tobaccoDihydroflavonol 4-reductase (DFR) is the first critical enzyme in the later part of anthocyanins biosynthesis. The full-length cDNA and genomic DNA sequences of DFR gene were isolated from S. scutellarioides, named as SsDFR, using RACE ampliation. The GenBank accession No. of SsDFR is EF522155 and EF522156 for mRNA and gene, respectively. The SsDFR gene has 1792 bp and contains 5 introns with canonical GT…AG splicing sites, while the 1322-bp cDNA has an ORF of 1101 bp, encoding a polypeptide of 366 amino acids. The nucleotide and amino acid sequences of SsDFR share extensive homologies to plant DFR genes, especially high similarities to P. frutescens DFR gene by NCBI blastn and NCBI blastp. NCBI Conserved Domain Search found that the deduced SsDFR protein contain a typic 3Beta_HSD (3-beta hydroxysteroid dehydrogenase/ isomerase family) domain, belonging to a 3-beta hydroxysteroid dehydrogenase_superfamily. The high-conserved amino acid residues, V10-Y33, for binding NADPH and the 26 aa region, T134-K159, for determining the substrate specificity of DFR all present in SsDFR corresponding with other DFRs. The homology modeling revealed that the predicted tertiary structure of SsDFR protein has almost the same 3D structure to Vitis vinifereat DFR protein (VvDFR) that the crystallogram and function have been elucidated. Southern blot analysis indicated that SsDFR seems only have 2 copies.The results of RT-PCR indicated that the high expression level of SsDFR was observed in stem, leaf and flower, and expression in leaf is comparative higher than those in stem and flower, while no expression detected in root. In addition, the expression levels of SsDFR in leaf of red cultivar could be induced by white light or drought stress.The sense and antisense plant expression vectors of SsDFR were constructed and transformed into tobacco W38, respectively. In 35 lines of overexpression of SsDFR, the content of total flavonoids in transgenic tobacco leaves all are improved to different extent from 0.019% to 49.78%, in which 28 lines increase significantly (P< 0.01 or 0.05). Further analysis in transgenic tobacco leaves showed that all sense transgenic lines accumulate significantly higher levels of anthocyanins than wild-type control, increment from 2.88% to 282.81% (P< 0.01 or 0.05). The results indicate that the overexpression of SsDFR is able to increase the accumulation of anthocyanins, significantly.4. Cloning of the S. scutellarioides ANS gene family (SsANS) and its expression in transgenic tobaccoAnthocyanidin synthase (ANS), the second critical enzyme in the later part of anthocyanins biosynthesis, catalyzes the penultimate step in the biosynthesis of the anthocyanin class of flavonoids. The full-length cDNA and genomic DNA sequences of two ANS genes were isolated from S. scutellarioides, named as SsANS1 and SsANS2 using RACE method. The GenBank accession No. for mRNA and gene of SsANS1 and SsANS2 are EF522157 and EF5221528, EF522159 and EF522160, respectively. Both of them contain 1 intron with canonical GT-AG splicing sites. The genomic and full-length cDNA sequences of SsANS1 are 1655 bp and 1184 bp, while 1627 bp and 1193 bp for SsANS2, respectively. They both have an 1101-bp ORF encoding a polypeptide of 366 amino acid residues. NCBI blastn and NCBI blastp indicated that SsANS1 and SsANS2 share high homologies to plant ANS genes, especially to P. frutescens ANS. NCBI Conserved Domain Search found that the deduced two SsANS proteins both contain 2 conserved domains, one is 2OG-FeII_Oxy (pfam03171, 2OG-Fe(II) oxygenase super family) and another is PcbC (COG3491. Isopenicillin N synthase and related dioxygenases).The conserved amino acids ligating ferrous iron and residues participating in 2-oxoglutarate binding (R-X-S) were identified in two SsANSs consistent with other ANSs, which indicates that the SsANSs belong to the class of 2-oxoglutarate dependent dioxygenases (2-ODDs), and share consistent homology with the other 2-ODDs of the flavonoid pathway, F3H. ANSs and F3Hs both depend on 2-oxoglutarate, Fe²⁺, and molecular oxygen for activity. The homology modeling revealed that the 3D models of two SsANSs protein closely resembles the Arabidopsis thaliana ANS protein (AtANS), whose crystallogram and function have been elucidated. Southern blot analysis indicated that SsANS has 2 copies, corresponding to the two SsANS genes.Semi-quantitative RT-PCR revealed that strong expression of SsANS could be observed in stem, leaf and flower of red cultivar, while no expression in root. Expression patterns of individual SsANS member are similar to those of SsANS family, and expression of SsANS1 in leaf is higher than SsANS2, while the reverse case could be observed in flower. In leaf of red cultivar, the transcript levels of SsANS could be induced by white light or drought stress. Expression of SsCHS, SsF3H, SsDFR and SsANS from 5. scutellarioides was all induced by white light or drought stress, possibly there might be co-expression among of them.The sense and antisense plant expression vectors of SsANS1 were constructed and transformed into tobacco W38, respectively. And the sense transgenic tobacco plants were analyzed. In 22 lines of SsANS1 overexpression, the content of total flavonoids in transgenic tobacco leaves all are improved to different extent, increment from 0.12% to 48.25%, of which 13 lines increase significantly (P< 0.01 or 0.05). The content of total flavonoids of SsANS1 overexpression is equal to that of overexpression of SsDFR. The relative content of transgenic tobacco leaves anthocyanins were analyzed, and the result shows that they all are improved in varied degree, increment from 2.88% to 246.58%, of which 18 lines are significant differences (P<0.01 or 0.05). The results indicate that the overexpression of SsANS1 is able to increase the accumulation of anthocyanins significantly.5. Cloning of the S. scutellarioides MYB R2R3 transcription factors (SsMYB2 and SsMYB3) and their expression in transgenic tobaccoThe full-length cDNA and genomic DNA sequences of two different MYB R2R3 transcription factors were isolated from S. scutellarioides, named SMYB2 and SsMYB3 using RACE method. The GenBank accession No. of mRNA and gene of SMYB2 and SsMYB3 are EF522160 and EF522161, EF522163 and EF522164, respectively. They both contain 1 intron with canonical GT-AG splicing sites. The genomic and full-length cDNA sequences of SsMYB2 are 1108 bp and 994 bp, with a 732-bp ORF encoding a polypeptide of 243 amino acids, while 931 bp and 826 bp, with a 747-bp ORF encoding a polypeptide of 248 amino acids for SsMYB3, respectively. NCBI Conserved Domain Search indicated that the deduced proteins of SsMYB2 and SsANS3 both have two typical conserved R2R3-Myb DNA-binding domains in N-terminus. The SsMYB2 protein sequence showed greatest homology to VvMYB4, a grape MYB R2R3 transcription factor (GenBank accession no. ABL61515), with 75.0% overall identity and 98.1% identity in the R2R3 DNA-binding domain. The R3 repeat region of SsMYB2 in N-terminus contains the bHLH (basic helix-loop-helix) motif [D/E]Lx2[R/K]x3Lx6Lx3R for interaction with bHLH proteins, and in non-conserved C-terminal region there are three conserved motifs, C1 motif ( LlsrGIDPxT/SH RxI/l ) , C2 motif (pdLNLD/ElxiG/S) and Zinc-finger motif (CX_1-2CX_7-12CX_1-2C) relating to the special function, in which the C2 motif forms part of the region involved in the repression of transcription. Phylogenetic analysis reveals that SsMYB2 and AmMYB308 from Antirrhinum majus form a closely related subgroup, which further groups with AtMYB4 and FaMYB1 (strawberry) and all belong to subgroup 4. The putative function of SsMYB2 is relevant to suppress expression of genes encoding enzymes that are part of lignin biosynthetic pathway. The SsMYB3 protein sequence showed close homology to VvMYBPA1, a grapevine MYB R2R3 transcription factor regulating proanthocyanidin synthesis (GenBank accession no. CAJ90831), with 82.9% identity within the MYB R2R3 domain, 67% overall identity). The R3 repeat region of SsMYB3 in N-terminus contains the conserved bHLH motif for interaction with bHLH proteins, but in non-conserved C-terminal region there is a lack of conserved motif. Phylogenetic analysis reveals that SsMYB3 and VvMYBPA1 form a closely related subgroup, which further groups with ZmP and AtMYB12. The putative function of SsMYB3 is relevant to regulate branch pathways of flavonoid metabolism, such as flavonols or proanthocyanidins.Semi-quantitative RT-PCR indicated that SsMYB2 has expression in root, stem, leaf and flower in red cultivar, and high expression could be detected in leaf and flower, while weak expression in root and stem. Under white light conditions, transcripts of SsMYB2 is up-regulated, and even weak expression could be detected before weak light treatment. Expression of SsMYB2 has down-regulation under drought stress, and about 1/3 expression decreased than control. The transcripts of SsMYB3 could be dramatically induced after 1-24 h white light treatment. Similar to SsMYB2, expression of SsMYB1 also down-regulated under drought stress conditions.The sense plant expression vectors of both SsMYB2 and SsMYB3 were constructed and transformed into tobacco W38. Overexpression of SsMYB2 got the same phenotype in transgenic tobacco that had been observed for AmMYB308 and AtMYB4: plants that grows slowly and with pale leaves showed premature white lesions on the mature leaves indicative of reduced levels of hydroxycinnamic acid derivatives by repressing the expression of the gene encoding the key enzyme in lignins biosynthesis. In 13 lines of overexpression of SsMYB2, the content of total flavonoids in transgenic tobacco leaves all are improved in varied degree, increment from 0.12% to 48.25%, of which 10 lines increased significantly (P< 0.01 or 0.05), and the maximum is 3 fold of control. The transgenic tobacco plants of overexpression of SsMYB3 appeared normal in morphology, growth characteristics and leaf color, compared with that of control. In transgenic tobacco leaves of 17 lines of overexpression of SsMYB3, there are large difference of the content of total flavonoids. 12 lines increase significantly (P< 0.01 or 0.05) and the maximum is 3.2 fold of control. However 3 lines don't have significant change (P>0.05), and 2 lines decreased significantly (P<0.05). The results suggest that the overexpression of either SsMYB2 or SsMYB3 can efficiently regulate the different pathways of phenylpropanoid metabolism in transgenic tobacco.6 Formation of color in stem and leaf of green cultivar are related to the expression levels of SsDFR and SsANSThe transcript levels of SsCHS, SsF3H, SsDFR and SsANS in root, stem, leaf and flower of green cultivar (S. scutellarioide.cv. C3) were detected using Semi-quantitative RT-PCR. The results showed that strong expression of 4 genes could be observed in flower, stem, and leaf with different expression patterns, but no expression could be observed in root. SsCHS displayed high expression in leaf and flower, and weak expression in stem. The highest overall of SsF3H family could be detected in flower, followed by stem, while very weak expression was also observed in stem. Two SsF3H members had strong expression in flower and weak expression in leaf, and expression of SsF3H2 could not be founded in stem. The high expression of SsDFR in flower could be detected, while weak expression in stem and no expression in leaf. The overall expression of SsANS family highly expressed in flower and weakly in root, while none of them could be detected in stem. The transcript level of SsANS1 in leaf was higher than SsANS2, while the reverse case displayed in flower. After drought stress in green cultivar, leaf and stem appeared notable accumulation of anthocyanin and became red. RT-PCR indicated that all the 4 genes were up-regulated in induced leaves, and expression of SsDFR and SsANS obviously increased, indicating that drought stress could activate transcript of SsDFR and SsANS.From above evidences, the possible implication is that the difference of color in leaf and stem between green and red S. scutellarioides cultivar could not caused by the differential expression of SsCHS gene and SsF3H gene family. It is possible that transcriptional inactivation of SsDFR and down-regulated expression of SsANS family in leaf, and transcriptional inactivation of SsANS in stem in green cultivar leaded to non-accumulation of anthocyanin in leaf and stem, and displayed green color. We speculate that the phenomenon may be related to mutant inactivation of different transcription factors, which regulated SsDFR and SsANS genes in stem and leaf of green cultivar, respectively.