Function Analysis Of Strigolactone Receptor Gene DAD2 In Tomato

Strigolactones(SLs)are a new type of plant hormones that participate in many aspects of plant growth and development,including root development,leaf shape,leaf senescence,stem secondary growth,and response to stress.SLs synthesis and signal transduction pathways have been extensively studied in Arabidopsis and rice,but the function of SLs receptor DWARF14(D14)/DECREASED APICAL DOMINANCE 2(DAD2)in tomato has not been studied yet.Tomato is one of the most important vegetable crops in the world,and has been widely used as a model plant for studying fruit development and maturation in basic theoretical research.To date,three SLs biosynthesis genes Sl CCD7,Sl CCD8,and Sl MAX1 have been identified in tomato,which can regulate axillary bud outgrowth in tomato.However,SL signaling genes,and particularly the sole SL receptor gene,remain to be identified.In the present study,the receptor gene of SLs was identified in tomato by bioinformatic analysis,named Sl DAD2,and then sldad2 mutant was obtained using CRISPR/Cas9 gene editing system to study the role of Sl DAD2 gene in tomato growth and development.The possible reasons for the phenotypic changes of the sldad2 mutant were further explored by RNA-seq,q PCR verification and other methods.The main findings are as follows:(1)Identification of tomato SLs receptor gene Sl DAD2.We used the amino acid sequences of the identified functional SLs receptor proteins Os D14,At D14 and Ph DAD2 in rice,Arabidopsis and petunia to compare them in the protein database and construct a phylogenetic tree.The phylogenetic tree was divided into four branches.Among them,the branch with Os D14,At D14 and Ph DAD2 is called the D14 family.The only homologous sequence XP＿004238093.1 in tomato is classified into this branch.Therefore,we speculated that it may be the receptor for SLs in tomato,named Sl DAD2.Motif analysis results showed that motif 5 might be important for D14 protein and KAI2 protein as SL and KAR receptors.The conserved amino acids in D14 protein and KAI2 protein was further studied by multiple sequence alignment,and the result showed that the seven specificity determined positions(SDP)of Sl DAD2/D14 protein and KAI2 protein were different.The q PCR analysis showed that Sl DAD2 was most highly expressed in leaves,followed by flowers and fruits.(2)The sldad2 mutant was generated by the CRISPR/Cas9 gene editing system and the phenotype was observed.Two types of homozygous mutants were screened out.The mutants showed obvious dwarfing,with significantly increased branches and branch lengths.The shape of the leaves was also changed,the width of the leaves did not change significantly,but the length of the leaves decreased,and the biomass of the mutant roots decreased.The mutant fruit became smaller,and the stem and flowers were almost unchanged.The expression of SLs synthesis and signal genes was detected by q PCR.The expression of SLs biosynthesis genes Sl D27 and Sl CCD8 increased in roots and stems.The transcription level of signal genes Sl MAX2 A and Sl MAX2 B in stems decreased,but the hypothetical Sl D53 increased.The expression of Sl BRC1 in the 4th leaf bud of the mutant decreased.In addition,the expression of the cytokinin biosynthesis gene Sl IPT3 in the stem of mutant increased significantly,while the expression of Sl IPT2 and Sl IPT4 decreased slightly.The expression of the auxin biosynthesis genes Sl FZY,Sl TAR1 and the transfer genes Sl PIN1,Sl PIN3,Sl PIN4,Sl PIN7 and Sl PIN9 in the 4th stem node of the mutant increased.The expression of Sl PIN1,Sl PIN3,Sl PIN4,Sl PIN7 and Sl PIN9 in the roots of mutant all increased.(3)The leaf color of the sldad2 mutant became lighter,and the content of chlorophyll and carotenoids was reduced,and the number of chloroplasts was reduced,and the arrangement of granum lamella in the chloroplast was loose.According to the RNA-seq data,the expression of key rate-limiting enzyme genes in the chlorophyll synthesis pathway,HEMA(Solyc04g076870.4)and CHLH were down-regulated.Besides,the expressions of POR,FC(Solyc05g018650.4),HO1,CRD1,DVR and CBR were also down-regulated.In the carotenoid synthesis pathway,the key rate-limiting enzyme genes PDS(Solyc02g083410.4)and CRTISO(Solyc05g010180.4)were down-regulated,and the accuracy of RNA-seq was verified by q PCR.(4)Through GO enrichment analysis,we found that among the more significant types of enrichment,except for toxin catabolism and toxin metabolism,the other 6 categories are closely related to photosynthesis.The most significant enrichment of KEGG pathway is the enrichment of photosynthetic antenna protein.The expression of Lhca and Lhcb families encoding photosynthetic antenna proteins is down-regulated,Lhca1,Lhca3,and Lhca4 in PSI are down-regulated,Lhcb1,Lhcb2,Lhcb3,Lhcb5,and Lhcb6 are down-regulated in PSII,and the Fv/Fm ratio of mutants is decreased,indicating that the Sl DAD2 gene mainly plays a role in the regulation of photosynthesis.In summary,the only receptor for SLs was found in tomato and named Sl DAD2 in this study,and the gene was knocked out by CRISPR/Cas9 system.The influence of SLs on tomato plant growth was studied through the mutant sldad2,and RNA-seq revealed that the lighter leaf color of the mutant may be related to the down-regulation of the expression of the rate-limiting enzyme for photosynthetic pigments,and the down-regulation of the LHC family were down-regulated in the mutants,and the Fv/Fm ratio of the mutants was also found to be reduced.These provide references for further research on the molecular mechanism of tomato SLs.