Mechanism Of SlAN2-like Promoter Variation Contributes To Anthocyanin Biosynthesis In Tomato Fruit

The antioxidative and anti-inflammatory effects of dietary anthocyanins make a major contribution to the quality of fruits.Although tomato(Solanum lycopersicum)fruits do not usually contain anthocyanins,the Aft(Anthocyanin fruit)tomato accession LA 1996 accumulates anthocyanin in its peel,which is controlled by high expression of a functional R2R3 MYB transcription factor SlAN2-like.Splicing mutations lead to a nonfunctional truncated SlAN2-like and therefore fail to produce anthocyanins in the wild-type tomato.However,the regulatory mechanism that governs SlAN2-like expression in wild natural tomato accessions is still unknown.In this study,Aft tomatoes with anthocyanin pigmentation and moneymaker(MM)tomatoes without anthocyanin pigmentation were used as research materials.The genetics,molecular biology,and epigenetics methods were used in combination with transgenic and gene editing technologies to illustrated the specific mechanism for the transcriptional regulating SlAN2-like expression in tomato fruits.The main results were presented as follows:1.SlAN2-like gene exhibits fruit specific high-evel expression and promotes anthocyanin accumulation.We checked the expression levels of four MYB gene from different tissus in LA 1996(Aft)and moneymaker(MM)using RT-qPCR and found a high correlation between SlAN2-like expression and anthocyanin accumulation in Aft fruit.The positive correlation of SlAN2-like expression level and anthocyanin contents was also confirmed using a F2[Aft×MM]population.The results showed that anthocyanin phenotype,SlAN2-like genotype,SlAN2-like gene expression and SIDFR gene expression in fruit were co-segregated.By using CRISPR/Cas9 to knock out the SlAN2-like gene of Aft tomato,it was found that the fruits of mutant didn't accumulated anthocyanins and reduced the expression levels of SlAN2-like and SIDFR genes significantly.Therefore,the anthocyanin biosynthesis in tomato fruits is regulated by the expression level of SlAN2-like gene.2.Variations at the SlAN2-like promoter affected the expression level of SlAN2-like gene and anthocyanin accumulation in tomato fruits.We sequenced a 2.4 kb fragment at the 5' end(upstream)of the translation start site of SlAN2-like gene from Aft and MM.Sequence alignments revealed that the region upstream of SlAN2-like is extremely polymorphic,containing a high density of SNPs and InDels from 1.2 kb to 1.8 kb from the translation start site.According the to the polymorphisms,we design an INDEL marker to genotyped all the collections of 25 accessions,uncovered two types of haplotypes:The Aft-type with anthocyanin pigmentation,and MM-type without anthocyanin pigmentation.Further investigation revealed that the anthocyanin contents in Aft-type cultivars were significantly higher than in the MM-type.Similar results were also obtained for the expression of SlAN2-like between the Aft-type and MM-type accessions,indicating that variation in the SlAN2-like promoter may result in the different levels of SlAN2-like expression and anthocyanin content in tomato fruit.To confirm the effect of sequence variations in the SlAN2-like promoter on anthocyanin biosynthesis,we generated four types of transgenic lines,MM-pSlAN2-likeAft::SlAN2-likeAft(MM-pA:A),Aft-pSlAN2-likeAft::SlAN2-likeAft(Aft-pA:A),MM-pSlAN2-likeMM::SlAN2-likeAft(MM-pM:A),MM-pSlAN2-likeAft::SlAN2-likeMM(MM-pA:M),by combination of the native promoter and gDNA of gene body region of SlAN2-like from Aft and MM.While MM-pA:A and Aft-pA:A exhibited increased anthocyanin contents in the fruit peel,the MM-pM:A line had a low level of SlAN2-like expression and slight anthocyanin accumulation.Although the MM-pA:M plants had a high level SlAN2-like expression,it failed to accumulate anthocyanin in the fruit peel,consistent with previous reports about the loss function of SlAN2-like due to alternative splicing transcript in MM accessions.Thus,a variation in the SlAN2-like promoter in the Aft-type is responsible for the enhanced expression of SlAN2-like,and the elevated SlAN2-like expression promoted anthocyanin pigmentation in the fruit peel of Aft-type accessions.3.The H3K27me3 level of SlAN2-like promoter is negatively associated with the expression level of SlAN2-like and anthocyanin accumulation.We noticed that stems,leaves,flowers,and fruit endocarps of MM-pA:A and Aft-pA:A plants accumulated anthocyanin,and correspondingly SlAN2-like was ectopically expressed in each.However,in nontransgenic Aft and MM plants,SlAN2-like was highly expressed in the exocarp of Aft fruit and barely detectable in any other tissues in Aft and none the tissues of MM plants,indicating a loss of fruit specificity of SlAN2-like expression in MM-pA:A and Aft-pA:A transgenic plants.It implied that the chromatin state of SlAN2-like gene may influence the tissue specific expression patterns of SlAN2-like.To investigate whether the fruit-specific expression of SlAN2-like is associated with epigenetic modification,we carry out McrBC-PCR and ChIP-qPCR to detect the DNA methylation and histone modification levels in different tissues of Aft and MM.The ChIP-qPCR results showed that the H3K27me3 level of SlAN2-like promoter in fruit peels of Aft was significantly lower than in other tissues.Meanwhile,the MM maintained a hyper-H3K27me3 state in all the tissues,which was correlated with low expression of SlAN2-like.To determine whether hypo-H3K27me3 of SIAN2-like promoter is a heritable epigenetic regulator of anthocyanin accumulation in Aft fruit peel,we selected individual F2 plants from reciprocal crosses between Aft and MM to test whether SIAN2-like expression and anthocyanin content cosegregate with a particular H3K27me3 status in SlAN2-like promoter.Consistently,the F2 plants with homozygous Aft-type SlAN2-like promoter alleles had significantly lower H3K27me3 levels,compared to those in F2 plants with homozygous MM-type SlAN2-like promoter allele.We also selected two representative accessions with different fruit colors from each type of SlAN2-like promoter variation to examine H3K27me3 levels in fruit peels.Opposite from the expression levels of SlAN2-like,the Aft-type accessions had lower H3K27me3 levels than in the MM-type accessions.Thus,the H3K27me3 level of SlAN2-like promoter is negatively associated with the expression of SlAN2-like and anthocyanin accumulation.These results suggest that three factors,Hypo-H3K27me3 of SlAN2-like promoter,expression level of SlAN2-like,and anthocyanin pigmentation are tightly coupled and appear to be stably inherited.Our results support the hypothesis that the fruit-specific expression of SlAN2-like in anthocyanin-pigmented accessions is due to hypo-H3K27me3 in its promoter and is responsible for anthocyanin accumulation.