Mechanism Of Gibberellin Under Low Night Temperature Inducing The Increase Of Tomato Locule At Seedling Stage

Tomato is the vegetable crop with the largest facility production area in our country,but the low temperature adversity in early spring induces the formation of a large number of multi-locule malformed fruits,which severely reduces the commercial value of the fruits and becomes an important factor affecting the efficiency of facility tomatoes.The multi-locule malformation of tomato is due to the abnormal carpel formation during the flower bud differentiation period,which leads to the formation of multi-locule malformations fruit.Studies have shown that the formation of multi-locule malformations fruit was mainly caused by internal and external factors,internal factor is the genetic feature,and the external factor is the environment,nutrition and some exogenous substances,among which low temperature and gibberellin（GA）are the most important factors inducing the formation of tomato multi-locule malformation,but the mechanism of low temperature and gibberellin inducing the occurrence of tomato multi-locule malformation is still unknown.This study was based on the previous research that flower bud differentiation stage under low night temperature in tomato could significantly increase the activity of gibberellin in shoot apical and induce multi-locule malformation fruit,and exogenous gibberellin（GA₃）could also significantly induce multi-locule malformation fruit.Further transcriptome analysis was conducted to identify the key gibberellin types induced by low night temperature and the key regulatory genes of gibberellin content accumulation in tomato shoot apical.This study used CRISPR/cas9 technology to construct key gene knockout mutants,and further analyzed the changes in tomato floral organs and locule number of knockout mutant seedlings under normal temperature and low night temperature,as well as changes in the content of gibberellin in the shoot apical,to determine the cause of gibberellin accumulation in shoot apical under low night temperature.This study also found that tomato gibberellin signal suppressor SlDELLA function loss mutant pro showed changes in flower organs and locule number,and then,the interaction between SlDELLA and SlWUS was detected by yeast two-hybrid,bimolecular fluorescence complementation（BIFC）and pull-down tests,while yeast one-hybrid,dual luciferase and EMSA experiments were used to analyze its co-involvement with SlWUS in the regulation of SlCLV3 transcription,which clarified the molecular mechanism of SlDELLA regulating the occurrence of tomato multi-locule malformations.The main findings are as follows:1.In this study,we identified gibberellin hydrolase SlGA2oxs gene,a key gene involved in regulating the formation of tomato multi-locule malformations under low night temperature.After low night temperature treatment of tomato"MLK1"seedlings,shoot apical RNA-seq results showed that differentially expressed genes were mainly enriched in“plant hormone signal transduction”and“diterpenoid biosynthetic pathways”.Further analysis of the diterpenoid gibberellin-related synthesis,metabolism and response gene expression levels found that the expression of gibberellin hydrolase SlGA2oxs gene was down-regulated after low night temperature treatment.Then it was found that active GA₁ and GA₄ accumulated under low night temperature.After exogenous application of active GA₁,GA₃,GA₄ and GA₇at tomato“MLK1”early stage of flower bud differentiation,the locule number increased,and GA₄ had the most significant effect.These results indicated that low night temperature at tomato seedling stage reduced SlGA2oxs gene expression in shoot apical,which resulted in the accumulation of active GA₁ and GA₄,and then induced the increase of locule number.2.In this study,it was confirmed that gibberellin hydrolase genes SlGA2ox1 and SlGA2ox3 were involved in inducing tomato multi-locule fruit formation under low night temperature.We obtained the T2 generation tomato ga2ox1ga2ox3 knockout mutant by gene editing technology,and the mutation way is the deletion of large fragments of the target sequence.The changes of flower organs and fruit locule number of ga2ox1ga2ox3 knockout mutant T2 generation seedlings were further analyzed under normal temperature and low night temperature.The results showed that the number of flower organs and locule number of ga2ox1ga2ox3 knockout mutants were higher than those of the wild-type Micro-Tom at normal temperature,but did not reach a significant difference,while it increased significantly after low night temperature treatment.The GAs contents of tomato ga2ox1ga2ox3 knockout mutants at normal temperature and low night temperature were further detected.The results showed that the GA₁ and GA₄ contents in the shoot apical of the ga2ox1ga2ox3 mutant at normal temperature increased not significantly compared with the wild type,while it increased significantly under low night temperature conditions.In order to further analyze the reasons for the insignificant changes in floral organs and locule of the ga2ox1ga2ox3 mutant at normal temperature,this study detected the gene expression levels of other three C19-GA2oxs,SlGA2ox2,SlGA2ox4 and SlGA2ox5 of ga2ox1ga2ox3 mutant shoot apical under normal temperature and low night temperature conditions.It was found that the SlGA2ox4 and SlGA2ox5 genes were significantly up-regulated in the ga2ox1ga2ox3 mutant,indicating that the C19-SlGA2oxs family members have functional redundancy in the process of regulating tomato locule formation,which suggested that SlGA2ox1 and SlGA2ox3 are not the only ways to induce the formation of tomato multi-locule under low night temperature,but also induce the formation of multi-locule malformations through other ways.3.This study clarified the mechanism of gibberellin signal inhibitor SlDELLA regulating locule formation.Compared with the wild type,the floral organs and fruit locule of tomato SlDELLA loss-of-function pro mutants increased significantly,and the expression levels of SlWUS in tomato 35S::SlDELLA over-expression plants were significantly down-regulated.It also verified that SlDELLA directly interacts with the shoot apical specific transcription factor SlWUS based on yeast two-hybrid,bi-molecular fluorescence complementation（Bi FC）and pull-down test,and based on yeast one-hybrid,dual-luciferase and EMSA assays,the interaction of SlDELLA and SlWUS can inhibit SlWUS from combining with the promoter of SlCLV3 to participate in the regulation of SlCLV3 transcription,which clarified the molecular mechanism of SlDELLA regulating the occurrence of tomato multi-locule malformations.