Functional Analysis Of A YUCCA Family Gene ToFZY4 In Tomato

The abscission of plant organs is a universal physiological phenomenon,which exists in the whole life cycle of a plants.In agriculture,abscission is closely related to the crop yield and the farmers income,and hence have attracted extensive attention from the biologists and the agronomists.The abscission of plant organs is a highly coordinated process,in which many endogenous plant hormones are involved in the molecular level.Thereinto,auxin,as an important plant hormone and a key signal molecule,plays an important role in the process of organ abscission.However,to date,whether the endogenous auxin biosynthesis is involved in the abscission process is still unknown.Indole-3-acetic acid(IAA)is the main form of auxin in plants,the biosynthesis of IAA can be conducted through two main pathways,namely the tryptophan(Trp)-dependent way and the Trp-independent way.The Trp-dependent way is the main biosynthesis pathway of IAA,among which,the indole-3-pyruvic acid(IPy A)pathway is the most important way for the IAA biosynthesis in higher plant.YUCCA protein is a kind of key enzyme that catalyzes the last step of the IAA biosynthesis in the IPy A pathway.Genes from the YUCCA family always have diverse expression patterns,which affect the local auxin biosynthesis and play important roles in both plant development and their responses to the environmental changes.The YUCCA proteins are the earliest discovered flavin-containing monooxygenases(FMOs)in plants,containing the highly conserved FAD-binding and NADPH-binding domains,which are critical for the function of a YUCCA protein.Six genes(ToFZY1 to ToFZY6)encoding the YUCCA-like FMOs have been reported in tomato.In the previous study of our laboratory,we found that the expression level changes of the ToFZY4 gene was closely related to the process of pedicel abscission.So we hypothesize that ToFZY4 is involved in the process of abscission in tomato.Based on these,in this work,we used the molecular,genetics,plant physiological and bioinformatical means to explore the biological function of ToFZY4,and the main results are as follows:1.Through the multiple alignment of protein sequences,the locations of the important domains for the protein encoded by the ToFZY4 gene was determined,and on this basis,the g RNAs targeting sites of ToFZY4 were designed.The gene editing vector of ToFZY4 was constructed based on the CRISPR/Cas9 technology,and the gene-editing mutant of ToFZY4gene(tofzy4)was obtained through the agrobacterium-mediated transformation of tomato.Through phenotypic observation,we accidentally found that ToFZY4 was involved in the process of tomato fruit abscission.When the ToFZY4 gene was mutated,the tomato fruit obtained the ability to abscise naturally under normal growth conditions.Gene expression level analyses of the factors related to the abscission process indicated that the bioactive auxin level decreased in the fruit pedicel abscission zone(AZ),and the local auxin transport was also inhibited.Meanwhile,the expression level of genes related to the cell wall degradation increased,and the abscission process of tofzy4 mutant initiated as early as in the breaker stage.Further gene expression pattern analysis showed that ToFZY4 was expressed in the placenta,columella and seeds,which may be related to the auxin transport between fruit and pedicel.And the expesssion level of ToFZY4 increased gradually with the ripening of the fruit in these tissues.At the same time,the expression level of ToFZY4 also increased in the fruit pedicel AZ at later stage of fruit development,but the expression level was relatively low.2.The overexpression vector of ToFZY4 was constructed,and the ToFZY4 overexpression plant(ToFZY4-OE)was obtained by agrobacterium-mediated transformation of tomato.The transgenic plants were identified at both the DNA and the RNA level.At present,6 ToFZY4-OE plants with significantly increased transcription of ToFZY4 have been obtained.3.3000 bp sequence before the ATG of ToFZY4 gene was selected for the promoter prediction of ToFZY4,and four core promoter regions were found.Two promoter sequences were amplified and obtained.The length of ToFZY4-P2 is 2591 bp,including four core promoter regions,while the length of ToFZY4-P1 is 2104 bp,with three core promoter regions.Then the p ToFZY4-1::GUS and p ToFZY4-2::GUS recombinants were constructed,and the respective transformed tomato plants were obtained through the agrobacteriummediated transformation.In summary,in this work,the mutant of ToFZY4 gene(tofzy4)was obtained through the gene editing technology based on CRISPR/Cas9.The phenotypic analysis showed that ToFZY4 was involved in the process of tomato fruit abscission,and the preliminary analysis of the gene expression level changes indicated that the declined bioactive auxin level in the tofzy4 mutants might be responsible for the abscising phenotype of the tofzy4 fruits.The creation of the ToFZY4 overexpression plants and promoter analysis transformants laid a material foundation for the subsequent study of the fruit abscission mechanism regulated by ToFZY4.The results of this study can aid the further mechanism study of the process of the plant organ abscission,and be beneficial for the development of plant organ abscission regulators in future.