Functional Studies Of SlAS2 And SlTKN3 Regulating Tomato Fruit Development

Tomato is an important vegetable crop and a model plant for studying the development of berry fruit.The size and shape of tomato fruit not only determines its yield but also directly relates to its quality traits.The regulatory network of tomato fruit development is very complex,and transcription factors play a very important role in it.In the past,our laboratory was aimed at uncover the regulation network of tomato fruit development,transcriptome sequencing of early fruit ovary and ovules from multiple periods,we have found that multiple transcription factors are specifically expressed in the early fruit ovary or ovule.In this study,the mutant of the early fruit ovary-specific transcription factor Solyc11g008830(SlAS2)was obtained using CRISPR/Cas9 technology.The fruit of the mutant became smaller,the aspect ratio increased,the number of layers of the pericarp cells decreased,and the thickness of the peel became thinner than that of the wild type Moneymaker(MM),this indicates that SlAS2 is involved in the regulation of tomato fruit development.Further studies found that the number of cell layers in the fruit peel of the mutant was significantly lower than that of the wild type at 5 DPA and green ripening period,indicating that the change in the morphology of the mutant fruit may be due to the inhibition of the cell division in the tomato peel by the mutation of SlAS2.Moreover,the petals of the SlAS2 mutant were significantly smaller,the leaf polarity of the SlAS2 mutant was changed,and the leaves of the SlAS2 mutant were cup-shaped and needle-like.Therefore,SlAS2 has a similar function to the homologous gene in Arabidopsis and plays an important role in leaf polarity development and apical meristem maintenance.Solyc05g005090(SlTKN3)is a homologous gene of the AS2 target genes KNAT2 and KNAT6 in Arabidopsis thaliana and is also specifically expressed in the early fruit ovary wall.We have demonstrated that S1AS2 in tomato can inhibit the transcription of SlTKN3 in vitro by using dual luciferase assay.And the mutant of the early fruit ovary-specific transcription factor SlTKN3 was obtained using CRISPR/Cas9 technology,the fruit of the SlTKN3 mutant became smaller compared to the wild type MM,but the fruit aspect ratio was decreased as opposed to the SlAS2 mutant.The number of layers of pericarp cells in the SlTKN3 mutant was significantly increased,but the thickness of the pericarp was also thinned.In addition,SlTKN3 is also involved in the development of floral organs,leaves and shoot tips,and the flowers and leaves of the mutants are significantly larger.Therefore,it is shown that SlTKN3 acts as an inhibitor of cell division activity and promotes cell division after mutation.After overexpressing the plant SlTKN3 in tomato,the floral organ and leaf phenotype of the transgenic plants were similar to those of the SlAS2 mutant.In tomato,the floral organ and leaf phenotype of the SlTKN3 overexpressing transgenic plants were similar to those of the SlAS2 mutant.Therefore,we speculate that SlAS2 may participate in tomato fruit,flower and leaf development by regulating the transcription of SlTKN3.Further searching for genes that may be regulated by SlAS2 by transcriptome sequencing technology,it was found that there were differences in the expression levels of the 2282 gene in MM and slas2-cr 0 DPA fruits,of which 85 genes were specifically expressed in the ovary wall at the early stage of fruit development.GO clustering analysis showed that 85 ovary-specific genes play an important role in many biological processes.Taken together,our study demonstrates that early ovary-specific genes in the fruit play important roles in tomato fruit development.