Cloning And Identification Of Fruit Shape Development And Seed Dormancy Genes In Tomato

As an important vegetable crop,tomato is very popular among consumers and is one of the largest consumption vegetables in the world.Seeds symbolize the beginning of life,which is crucial for the plants life circle.Seed dormancy refers to the phenomenon that mature seeds with viability still do not germinate under suitable conditions.Seeds release dormancy by sensing changes in the external environment,thereby starting the process of life and helping plants avoid adverse environmental conditions.Once seed germination process initiating,the seeds development into seedlings and transform into vegetative growth stage,and will confronting with diverse environment conditions.Adverse environment conditions will cause yield loss,in severe cases will lead to plants death.Tomato fruits as edible part possess high nutritional values.With the development of society,requirements of consumers for the appearance quality of tomato fruits are becoming much more diverse.Simultaneously,normal fruit development and mature process will guarantee the fertility of next generation.Therefore,it is of great significance to perform the research about seed dormancy,fruit shape development and stress response of tomato.In this study,the key genes controlling seed dormancy MAPK11,the fruit developmental gene PT and stress response gene ATL78L were cloned and identified,and the functions analysis of these two genes were performed using molecular biology,biochemistry and cytology methods.The main results are as follows:Ⅰ.The mechanism of MAPK11 regulating seed dormancy in tomato1.Using 255 accessions natural population with GWAS,we identified 8 loci that were significantly associated with seed dormancy.Among of these loci,AAO3 and ABI5 were previously reported to regulate seed germination in Arabidopsis.A novel locus with the highest SNP value(ch07＿62133707,P-value<5.28×10^-17)on chromosome 7 attracted our attention.We named this locus TOMATO SEED DORMANCY ON CHROMOSOME 7（TSD7）for further analysis.2.To test for variation in TSD7,we found that the SNP with the highest value is located in MAPK11.We then sequenced the promoter region of MAPK11 and detected an interesting variation in dormant and non-dormant tomatoes,which changed the HSE in MAPK11 promoter.Moreover,we found that MAPK11 was expressed at especially high levels in dormant tomato seeds.So we concluded that MAPK11 was the best candidate gene for the TSD7 locus.3.We performed a genetic complementation test and found that the seed germination rates of the OE lines were lower than the TS-9.In contrast,the seeds of the RNAi lines germinated at higher rates than TS-34.Based on these data,we conclude that MAPK11can regulate seed dormancy and that the MAPK11 gene is TSD7.4.We found that the germination of the OE lines was hypersensitive to ABA than TS-9,whereas,ABA inhibitor promotes the germination of OE lines using ABA treatment assay.To further analysis using q RT-PCR and ABA content measurement,we found that MAPK11 positively regulates ABA signaling and that MAPK11 affects seed dormancy by potentially acting upstream of ABA biosynthesis at the level of transcription.Additionly,we found that MAPK11 promotes seed dormancy by regulating transcription and by acting downstream of KAO and upstream of GA20OX2 and GA2OX.5.We found that MAPK11 affects ABA signaling and promotes seed dormancy by influencing the phosphorylation of Sn RK2.2 on Ser-154 and Thr-301 in vivo using phosphoproteomics.6.MAPK11 may inhibit the activation of Sn RK1 by binding and phosphorylating Sn RK1 on Ser-29.Sn RK1 binds ABI5 and downregulates the expression of ABI5,which contributes to ABA signaling and controls seed germination.In addition,we found that Sn RK1 inhibits seed dormancy in tomato using transgenic germination experiment.Ⅱ.Cloning and regulatory mechanism of tomato pointed tip fruit mature gene PT1.Using F2 population of AC and LA4053 hybridization for BSA analysis,four sites controlling the fruit pointed tip were identified.Further fine mapping of the site on chromosome 5,we cloned the key gene PT that controls the fruit pointed tip formation.2.Analysis of PT revealed that a single base mutation in the exon resulted in a change in amino acids,generating two different alleles of PT^H and PT^R.It is further predicted that the variation in the spatial structure of the two alleles may contribute to the regulation difference of downstream genes.3.Overexpression of PT^R promoted the formation of fruit pointed tip,and rin assisted PT^R to form a more remarkable pointed tip.In addition,PT^R positively regulates the transcription of ACO1 by binding to the promoter of ACO1 and promotes fruits ripening,which is dependent on rin.4.Overexpression of PT^H forms pear-shaped fruit without pointed tip.PT^H negatively regulates the transcription of FUL2 by binding to the promoter of FUL2,knock out PT^Hcan remove the inhibition on FUL2,thus promoted the formation of fruit pointed tip.Ⅲ.The mechanism of ATL78L regulating abiotic stress in tomato1.A wild tomato（Solanum habrochaites）cold-induced RING-H2 finger gene,ShATL78L,was isolated,which has been identified as an abiotic stress responsive gene in tomato.The results showed that ShATL78L was constitutively expressed in various tissues such as root,lea petiole,stem,flower,and fruit.Cold stress up-regulated ShATL78L in the cold-tolerant S.habrochaites compared to the susceptible cultivated tomato（S.lycopersicum）.Furthermore,ShATL78L expression was also regulated under different stresses such as drought,salt,heat,wound,osmotic stress,and exogenous hormones.2.Functional characterization showed that cultivated tomato overexpressing ShATL78L had improved tolerance to cold,drought and oxidative stresses compared to the wild-type and the knockdown lines.To understand the underlying molecular mechanism of ShATL78L regulating abiotic stress responses,we performed yeast one-hybrid and two-hybrid assays and found that RAV2 could bind to the promoter of ShATL78L and alter its transcription,and CSN5B could interact with ShATL78L to regulate abiotic stress responses.