Functional Dissection Of SlARFs In Regulating Tomato Leaf Shape Development

Leaves are the main photosynthetic organs and serve as the major mediator with the environment above the soil surface.Leave morphogenesis and differentiation is a highly flexible process,which leads to the diversity of leaf shape.Variation is one of the most conspicuous aspects of plant diversity in leaf shape.As one of the most important horticultural crop in the world,tomato(Solanum lycopersium)is the typical model plant for plant growth and development research.The Aux/IAA genes family involves in various aspects of plant growth and development and Sl IAA9 gene belongs one o f them.Research indicated that Sl IAA9-RNAi transgenic tomato lines presented fruit parthenocarpy and simple leaves.A single-base C deletion in the coding region of Sl IAA9 gene results in tomato entire mutant,which also establishes simple leaves.Previous research demonstrates that the interaction between Aux/IAAs and ARFs refer to unique biological functions.In this study,candidate Sl ARF proteins were screened as the interactor of Sl IAA9 protein by yeast two-hybrid system.In order to validate the function of Sl ARF genes,an important direction for further work might be to study using TRV-mediated virus-induced gene silencing(VIGS)to down-regulate candidate Sl ARF genes expression for functional characterization in tomato entire mutation leaves.The main results were as follows:1.Y2H(Yeast Two-Hybrid),Bi FC and pull-down results indicated that Sl IAA9 protein could interacts with Sl ARF6 A,Sl ARF8 A,Sl ARF8 B and Sl ARF24 protein.2.The expression of Sl ARFs gene family in AC(compound leaves material)and entire mutant(simple leaves material)leaves were analysed.The results presented entire mutation alters the expression patterns of multiple Sl ARFs.3.entire mutant infected with o ne,double or triple combinations of p TRV2-Sl ARFs were used to tested their function using VIGS technology,respectively.Ultimately,the VIGS results suggested that simultaneous silencing of these four genes converted the leaf shape from simple to compound in entire mutant plants.These results suggested that simultaneous silencing of these four genes regulates the leaf shape from simple to compound in entire mutant plants.In addition,IAA content in these materials was significant increased than that in entire mutant.4.Furthermore,we specifically simultaneous designed four guide RNAs(g RNAs)to target with Sl ARF8 B,Sl ARF8 A,Sl ARF6 A and Sl ARF24 sites in tomato entire mutant materials using CRISPR/Cas9 technology,and there are 2 T0 plants(CR4-3 and CR4-4)presented compound leaves in some degree compared with entire mutation simple leaves.There are different mutated alleles in the two plants,no mutation was detected in the site T3(Sl ARF6A),various indels was detected in the site T1(Sl ARF8B),site T2(Sl ARF8A)and site T4(Sl ARF24).These results signified that Sl ARF8 A,Sl ARF8 B and Sl ARF24 gene were simultaneous knocked out in CR4-3 and CR4-4 lines,which could make them functional recovery in a certain degree.5.CR4-3 and CR4-4 plants presented not only compound leaves,but also increased fruit parthenocarpy.IAA content in these two lines was significant increased than that in entire mutant.These results established that the functional recovery in entire mutant was regulated by auxin.6.Y1H(Yeast O ne-Hybrid)and LUC(dual luciferase)assay indicated that Sl ARF6 A and Sl ARF24 bind to the Sl PIN1 promoter.We propose that Sl ARFs may regulate leaf growth and development through binding to the Sl PIN1 promoter.