A BHLH Transcription Factor,SlJAF13,Promotes Anthocyanin Biosynthesis In Aft Tomato Fruits

Anthocyanins are natural pigments in plants that play essential roles in multiple biological processes,such as attracting pollinators and dispersers as well as protecting plants from biotic and abiotic stresses.Unlike modern tomato（Solanum lycopersicum）cultivars,LA1996 harbors the dominant Aft allele,which is associated with anthocyanin accumulation in tomato fruit peel.Anthocyanin biosynthetic enzymes are transcriptionally regulated by the MYB–bHLH–WD40（MBW）complex.However,although the roles of WD40 and MYB proteins in anthocyanin biosynthesis in the tomato are well understood,those of bHLH factors remain unknown.Here,we isolated an anthocyanin-deficient tomato mutant following the ethyl methanesulfonate treatment of Aft and identified SlJAF13,encoding a class IIIf basic helix-loop-helix（bHLH）transcription factor（TF）,as the gene responsible for its mutant phenotype.The molecular mechanism of SlJAF13 regulating anthocyanin synthesis in Aft tomato was investigated by transcriptome analysis,function complementation,protein-protein interaction and transcriptional activation experiments.The main results are as follows:1.Screening and mapping of anthocyanin-deficient at4 mutant（1）Among mutants treated by EMS（ethyl methanesulfonate）,one mutant,named at4,lacking anthocyanins in the peel during fruit ripening was selected for further analysis.The phenotypic segregation ratio showed that the anthocyanin deficiency trait of the at4 mutant is controlled by a single recessive gene.（2）The candidate gene SlJAF13 was identified by Mutmap and CAPS molecular markers with a single SNP（C3166T）,which was predicted to cause an amino acid substitution from proline to leucine（P405L）.The P⁴⁰⁵ residue of SlJAF13 was highly conserved among the IIIf subgroup bHLH TFs.The transgenic SlJAF13-Com complementation lines showed anthocyanin accumulation in the fruit peel.2.SlJAF13 involved in the transcriptional activation of SlAN1 for anthocyanin biosynthesis（1）RNA-seq analysis of the peels of Aft（WT）and sljaf13 mutant fruits at the mature green stage.The most highly enriched gene ontology（GO）term for the downregulated genes was‘flavonoid biosynthetic process’in sljaf13 mutant,including bHLH TF SlAN1 gene.Mutations in SlAN1 using the CRISPR/Cas9 system in the Aft background largely impaired anthocyanin biosynthesis and SlDFR expression in the fruit peel but did not affect the expression of SlJAF13.（2）Transcriptional activation experiments,such as Ch IP-q PCR,EMSA and double luciferase reporter assay,as well as protein interaction experiments of Y2H,LCI and pull-down,confirmed that SlJAF13 forms an MBW complex with SlAN2-like and SlAN11,which is involved in the transcriptional activation of SlAN1 for anthocyanin biosynthesis.（3）The P450L substitution did not affect the DNA-binding ability of SlJAF13.SlJAF13^P450L failed to interact with the WD40 protein SlAN11,but not with the MYB protein SlAN2-like,suggesting that the P450L substitution may affect formation of the SlJAF13–SlAN11–SlAN2-like complex.3.SlJAF13 involved in the transcriptional repression of SlJAZs for anthocyanin biosynthesis（1）Gene ontology analysis showed that SlJAF13 is involved in inhibiting the expression level of core suppressor SlJAZs gene in jasmonic acid metabolic pathway.Transient transfection,transcriptional activation and protein interaction experiments verified that SlJAZ proteins（eg.SlJAZ2）interact with bHLH and WD40 TFs to attenuate the transcriptional activation function of the MBW complexes,resulting in the inactivation of downstream genes.（2）Upon the perception of Me JA,the JAZ repressor is reported ubiquitinated and degraded,which increased in SlAN1 expression and restored anthocyanin accumulation in the sljaf13 mutant,but the increased level of SlJAF13 protein cannot compensate for the slan1mutant phenotype.（3）Transcriptional activation experiments,such as Ch IP-q PCR,EMSA and double luciferase reporter assay,as well as protein interaction experiments of Y2H,LCI,Co IP and pull-down,confirmed that SlMYC2 dramatically activated p SlJAZ2,but the heterodimerization of SlMYC2 with SlJAF13 interferes with its ability to bind and activate to p SlJAZ2.In conclusion,we report an unexpected mechanism,whereby SlJAF13 promotes the production of anthocyanins by performing dual functions:（1）acting as an upstream regulator to activate the transcription of SlAN1 and SlAN2-like,which encode members of the MBW complex;and（2）suppressing the transcription of SlJAZs,which encode JA signaling repressors,thus interfering with the MBW complex formation.Further mechanistic investigation revealed for the first time that SlJAF13 interacts with SlMYC2 to inhibit SlMYC2-dependent activation of SlJAZ2 transcription.Hence,we propose that SlJAF13 activates the expression of anthocyanin biosynthesis genes by promoting the activity of the MBW complex via two signaling pathways in the Aft tomato fruit.This study revealed the regulatory network of anthocyanin synthesis mediated by bHLH family genes,and provided a new insight into the regulatory mechanism of anthocyanin biosynthesis in tomato fruits.