The Mechanism Of L2 And SLCHP16 Genes Regulating Fruit Ripening And Flower Organs Development In Tomato

Flowering and fruit ripening are the key to plant reproductive growth and population reproduction,as well as the important basis for crop yield with seeds or fruits as product organs.Tomato(Solanum lycopersicum.L)is an important cash crop and a model crop for studying flower and fruit development,the fruit is its main product organ,which provides rich nutrients for the human diet.Tomato is a climacteric fruit,and ethylene plays an important role in the regulation of fruit ripening.However,ethylene synthesis during fruit ripening is autocatalytic in the respiratory climacteric fruit,and the regulatory network is still unclear.Flower is the prerequisite for fruit production,so flower development has been the focus of research.There are many researches on the stage from flowering induction to flower organ formation,but the regulatory network from flower bud formation to flower full opening is still not well understood.In this paper,two research projects were carried out.First,we found a metalloproteinase L2,whose mutation causes fruit to turn white and delay ripening.Through this study,we found that L2 contribute to autocatalytic ethylene biosynthesis during fruit ripening in tomato.Secondly,we discovered a plant C1 domain protein Sl CHP16.Overexpression Sl CHP16 caused tomato buds to stop developing and unable to bear fruit.This study explored the molecular mechanism that affects flower growth and development.The main research contents are as follows:1.white fruit(wf)mutant was a naturally occurring mutant that was white at immaturity and significantly delayed ripening compared to normal fruit.Through the amplification analysis of the L2 locus in the wf mutant,we found that a 200 kb inversion occurred on chromosome 10 of the wf mutant,and one end of the inversion was located in the L2 locus that disrupts the L2 gene.L2 encodes a metalloproteinase,we using CRISPR-Cas9 technology knocked out L2 in wild type tomato and found that the l2-cr mutants produced fruit that were very similar to l2.In the l2-cr fruit,chloroplast development was impaired and the accumulation of carotenoids and lycopene occurred more slowly than in wild type.During fruit ripening in l2-cr mutants,the peak of ethylene release was delayed,less ethylene was produced and the expression of ACO genes was significantly suppressed.These data indicate that the L2 gene might affect the oxidation of ACC that produces ethylene.The results of yeast one-hybrid,EMSA and GUS staining showed that L2 was regulated by ethylene response factor ERF,and in particular,ERF.B3 could positively regulate L2 expression.In addition,ethephon treatment showed that L2 and ERF.B3 could be induced by exogenous ethylene,while L2 mutation reduced the sensitivity of fruits to exogenous ethylene.Thus,the expression of L2 is regulated by exogenous ethylene.Taken together,In the ethylene autonomous catalytic synthesis pathway,ethylene regulates the expression of L2 gene through ERF transcription factors,and L2 affects the activity and function of ACC oxidase by regulating the development of chloroplast thylakoid membrane,thereby affecting the synthesis of ethylene during tomato fruit ripening.2.By analyzing the C1 domain protein in tomato,we found that there are 21 C1 domain proteins in tomato,which are distributed on 9 chromosomes of tomato.Phylogenetic analysis showed that 21 C1 domain proteins were divided into 3subfamilies,most of which were expressed in roots.Sl CHP16 was expressed at a high level in fruits but low in flowers.Overexpression Sl CHP16 inhibited the development of tomato flower buds and prevented them from flowering and bearing.Interfering with the expression of Sl CHP16 can make tomato produce larger flowers.The flower primordia of Sl CHP16 over-expressed materials were observed by scanning electron microscopy,and it was found that the formation time of flower primordia and the formation of floral organs in the flower primordia of Sl CHP16 over-expressed materials were not affected.However,the growth and development of Sl CHP16 overexpressed materials stagnated after the formation of flower organs at the flower bud stage,and the number and size of cells in the flower did not change significantly with the growth of flower age.Transcriptome analysis showed that a large number of genes related to cell division and development were down-regulated in Sl CHP16 overexpressed materials.A 14-3-3 protein TFT12 was found to interact with Sl CHP16 by IP-MS/MS technique.TFT12 is specifically expressed in flowers,and the changes of its expression in flowers are synchronized with the development of flowers.Overexpression of TFT12 in tomato resulted in larger flowers.Co-expression experiments in tobacco leaves showed that TFT12 functions as a dimer,and Sl CHP16 affects the stability and dimer formation of TFT12 protein.Therefore,the overexpression of Sl CHP16 may affect the signal pathways related to flower development regulated by 14-3-3 protein in tomato flowers,causing flower development to stagnate.In summary,this study analyzed the wf mutant and found that a 200 kb inversion of chromosome 10 occurred in the wf mutant,which caused the L2 gene mutation.Analysis of the interaction proteins and upstream regulatory factors of L2 gene revealed that L2 gene is regulated by ERF transcription factors,and regulates fruit maturity by affecting the response and synthesis of ethylene.Through the analysis of the C1 domain protein in tomato,it was found that Sl CHP16 negatively regulates the development of tomato flowers,and interacts with the 14-3-3 protein TFT12 to coordinate the size of flower organs.