Map-Based Cloning And Function Analysis Of Hoffman’s Anthocyaninless In Tomato (Solanum Lycopersicum)

As an important kind of secondary metabolite, Anthocyanin pigments play many roles in plants, including attractting insects that pollinate and help disperse seeds and providing protection against biotic and abiotic stresses. Til now, many of the transcription factors(TFs) that mediate anthocyanin accumulation have been identified through studies of Arabidopsis, maize and petunia, however, much less works has been conducted on anthocyanin biosynthesis in tomato. In this study, Hoffman’s Anthocyaninless(AH) was identified through map-based cloning, which encoded a b HLH TF controlling anthocyanin biosynthesis in tomato. Combining with genetic transformation assay, genes expression analysis under difference developmental stages and low temperature stress, and RNA-seq analyses, we revealed that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses. The main contents and results are as follows:1. The green phenotype of FMTT271 is controlled by a single recessive locus. Allele test was performed and indicated that this single recessive locus is the allele of ah, so we named it ah. Using the BC1 and BC4S1 mapping population, ah was fine mapped in a 130-kb interval on chromosome 9 between the CAPS markers, CAPS2 and CAPS4. A total of 5 candidate genes were predicted to be present in this region based on the ITAG2.4 annotation of the reference tomato genome.2. Based on the results of homologous analysis and q RT-PCR analysis, Solyc09g065100 was suggested to be the best candidate gene for the ah locus. Sequencing of the predicted full-length c DNA of Solyc09g065100, amplified by reverse transcription(RT)-PCR of the AH/AH and ah/ah genotypes, revealed a single G to T substitution at base 550 of the c DNA clone(exon 6 in the genomic clone) in the ah mutant. This substitution is predicted to result in the conversion of glycine(Gly) to a stop codon, truncating the predicted protein by 501 amino acids. The full-length Solyc09g065100 c DNA was expressed under the control of the constitutive cauliflower mosaic virus 35 S promoter in the ah mutant, FMTT271. Plants from all T1 generation transgenic lines can complement the phenotypes of ah mutant, indicating that Solyc09g065100 is the AH. We further inferred that AH may control anthocyanin accumulation by up-regulating the transcript levels of anthocyanin late biosynthetic genes based on the results of expression analysis of anthocyanin biosynthesis genes and AH transactivation assay.3. AH expression is developmentally regulated and that its expression in turn may regulate the expression of anthocyanin biosynthetic genes, resulting a decrease of anthoycnain accumulation during seedlings development. In addition, low temperature stress can induce expression of AH in tomato leaves, and results in a high anthocyanin content, suggested that AH functions as a regulator of anthocyanin biosynthesis in response to low temperature stress. Moreover, DAB staining and trypan blue staining results suggested that AH regulates anthocyanin biosynthesis, thereby playing a protective role to low temperature stress.4. RNA-seq analyses of hypocotyls from NILs revealed that 551 DEGs comprising 285 that were up- regulated and 266 that were down-regulated in PH relative to GH. GO terms assigned to genes upregulated in PH relative to GH were significantly enriched in ‘response to abiotic(heat, light stimulus, temperature stimulus, oxidative stress) stimulus’, ‘response to hormone(jasmonic acid, gibberellin, and ethylene) stimulus’ and ‘flavonoid biosynthetic process’, while the down-regulated genes were enriched in ‘response to abiotic stimulus’, ‘oxidation reduction’ and ‘rhythmic process’. RNA-seq analyses of leaves from NILs revealed 39 DEGs may be influenced by AH expression under low temperature stress. Among the 39 DEGs, 10 genes were up-regulated in 16-PL relative to 16-GL, including the anthocyanin biosynthetic genes F3’5’H and DFR. The others showed a downregulation. GO enrichment analysis showed that the up-regulated genes were significantly enriched in the terms ‘phenylpropanoid biosynthetic process’, ‘cellular amino acid derivative biosynthetic process’ and ‘aromatic compound biosynthetic process’, while the down regulated genes were enriched in the ‘response to carbohydrate stimulus’ category.5. Based on the expression pattern of AH and Sl AN2, Sl ANT1, Sl ANT1-like, Sl AN2-like, we suggested that AH interact with Sl AN2 or Sl ANT1-like to control anthocyanin biosynthesis in tomato. Since the expression of F3’5’H and DFR are always co-expressed with AH, we hypothesized that they are directly regulated by AH. Indeed, Numerous E-box sequences(CANNTG) were discovered in each of the upstream sequences(2000bp for F3’5’H and 1500 bp for DFR) of these genes.