The Molecular Mechanism Of Flower Color Difference Between Nelumbo Nucifera And N. Lutea

Lotus (Nelumbonaceae:Nelumbo Adans.) is an aquatic perennial plant with ornamental, medical and edible values. The genus Nelumbo contains two species: Nelumbo nucifera Gaertn. (sacred lotus) and N. lutea Pers. (American lotus).These two species disjunctly distributed in North America-Eastern Asia and North of Australia, but there is no reproductive isolation between two species and their hybrids are fertile. Among external morphologies, petal color is the most obvious difference that distinguishes the two species of Nelumbo. The petal colors of the wild sacred lotus are red, pink, white, red/white pied, while the American lotus is yellow. So far, the molecular mechanism of flower coloration in lotus has remaind poor understood. The flower color difference in red and white cultivars has been stuided, but the flower color difference between two speceis is still not studied. What lead to the striking difference in flower color between closely related two species in Nelumbo? To unveil this mystery, the wild sacred lotus with red flowers obtained from Heilongjiang province, China and wild American lotus with yellow flowers obtained from Florida, USA were used in this study. Firstly, anthocyanin content of red and yellow flowers was measured using pectrophotometry. Secondly, through gene cloning, local blast and phylogenetic analysis, we identified structural genes and regulatory genes involved in anthocyanin synthesis and transport. Thirdly, the comparison of nucleotide sequence and the expression level among above genes revealed potential key genes involved in flower color differences of two species. Finally, combined with yeast two-hybrid experiments and transgenic methods, the function of candidate gene was tested. The main results are as following:(1) A high content of anthocyanin was detected in wild sacred lotus with red flowers, while almost no anthocyanin was detected in wild American lotus with yellow flowers. The result indicated that the anthocyanin biosynthetic pathway may be blocked in American lotus. A high absorption at 350 nm in both extracts suggested that the existence of flavones and flavonols in both red and yellow petals. The flavones and flavonols were synthesized in the intermediate steps of anthocyanin biosynthetic pathway, therefore we inferred that the upstream genes located in this pathway were active and the block may be located in the downstream of this pathway.(2) Through degenerated PCR technology, DFR1 and ANSI genes were obtained from two species. Due to the public of lotus genome, we constructed the local blast. Through blasting local data and constructing phylogenetic tree, four UFGT genes (UGT78A1-A4) and two GST genes (GSTF11 and GSTF12) involved in anthocyanin synthesis were identified from lotus genome. The transcripts of four UFGT genes and one GST (GSTF11) gene were detected in the cDNAs of red flowers. A comparison of nucleotide sequences and deduced amino acids among above structural genes showed a high homology and almost no amino acid differences in conserved regions and sites in two species. The qRT-PCR results showed that DFR1, ANSI, and UFGT2 had a higher expression level in yellow petals than in red petals. The expression levels of three UFGT genes (UGT1, UGT3 and UGT4) in yellow petals of N. lutea were lower than that in red petals of N. nucifera. The sqRT-PCR result showed that the transcripts of GSTs gene was only detected in red flowers of N. nucifera and were no detected in yellow flowers of N. lutea. The lack of transcripts of GST genes may be associated with the loss of anthocyanin in yellow flowers of N. lutea.(3) A total of 95 MYBs,10 bHLHs and 17 WD40s were obtained in the genome database of N. nucifera. Through aligning with anthocyanin-related regulatory genes from other species and constructing phylogenetic tree, nine MYB genes, two bHLH genes and one WD40 gene in lotus were considered as candidate genes regulating anthocyanin synthesis. Only one MYB gene (named NnMYBS) and one bHLH gene (named NnbHLHl) involved in anthocyanin synthesis were isolated from cDNA of red flowers. Sequence analysis of regulatory genes among two species showed that the deduced amino acid of TTG1 was the identical in two species, and for bHLH protein, only one amino acid differences occurred in the bHLH domain. A striking difference is that two amino acid substitutions led to a premature stop codon in MYB5 gene. Thirteen types and fourteen types of cis-acting regulatory elements were identified in the promoter region of NnMYB5 gene and NIMYB5 gene, respectively. Among these elements, many light-responsive elements were identified in the promoters of MYB5 from two species.(4) Population analysis allowed the identification of three variants at the MYB5 locus:(i) the functional allele, encoding a 291 amino acid polypeptide present in the homozygous state in red flowers of N. nucifera, (ii) two nucleotide substitutions (GAA/TAG) in the second exon leading to a premature stop codon, (iii) a single-base deletion in the second exon causing a premature stop codon. Remarkably, these two putatively inactive forms of the gene were recognized only in yellow flowers of N. lutea. Inactive form with nonsense mutation exists in all eight populations, whereas inactive form with frameshift mutation exists in six populations of eight populations (NLP1 and NLP8). All inactive form with frameshift mutation coexists with inactive form with nonsense mutation as a heterozygous genotype. Inactive form with nonsense mutation could produce a homozygous genotype. However, not homozygous individuals with framshift mutation were detected.(5)Yeast two-hybrid experiments showed that NnMYB5 can interact with NnbHLH and NnTTG1 proteins and also interact with NlbHLH1. In addition, NnTTG1 interacts with NnbHLH 1 and N1bHLH1. Above results indicated that MYB5, bHLH1 and TTG1 can be composed of MBW complex. The interaction of NnMYB5 and NnbHLH1 was stronger than that of NnMYB5 and N1bHLH1, indicating that the amino acid differences between N1bHLH1 and NnbHLH1 may affect the interaction with NnMYB5.(6) Over-expression of NnMYB5 gene in Arabidopsis led to anthocyanin accumulation in immature seeds and flower stalks, suggesting that NnMYB5 is an transcription activator of anthocyanin synthesis. The up-regulation of AtGSTF12 gene in NnMYB5 over-expression plants indicated that NnMYB5 can regulate the expression of AtGSTFl2.Based on above results, we can make conclusions as follows:the differences of petal coloration between red lotus and yellow American lotus might be caused by the differences in the coding region of MYB5 in two species, which might affected its regulation of structural genes and further influenced the expression level of structural genes and the accumulation of anthocyanin.