Studies Of Regulatory Mechanism Of Auxin Receptor SlTIR1/AFBs Family Genes During Tomato Growth And Developmental Process

Auxin is one kind of important plant hormone. Auxin participates in a variety of physiological and development processes and responses in plant. Auxin promotes the binding of the Aux/IAA proteins to auxin receptor TIR1/AFBs. This binding leads to the degradation of Aux/IAA and to the subsequent release of ARF. Auxin signaling control different physiological processes in the plant. Responses of plants to auxin is an extremely complex process, which involved in a strict regulation mechanism. Therefore, studies on the action mechanism of auxin is significant to realize the growth and developmental progress of plant.Tomato(Solanum lycopersicum) is generally considered as an important cash crop. It displays developmental traits not found in Arabidopsis such as sympodial, compound leaves, glandular trichomes, inflorescence and fruit growth, repining. TIR1/AFB as auxin receptor, is the key point in auxin signaling pathways. It is very important for addressing the functions of TIR1/AFB in plant. However, little is known about auxin receptor regulated-auxin signaling invovling compound leaves, floral organs, fruit development and repining. In this study, we focoused on expression patterns, proteinprotein interaction and developmental regulation of SlTIR1/AFBs auxin receptor homologues to reveal regulatory mechanism of auxin signaling involvling in tomao growth and developmental process. The main results are as follows:(1)A total of 4 TIR1/AFB auxin receptor homologues were indentified in tomato genome. Structural analysis of the four Sl TIR1/AFB genes showed that they displayed similar numbers of introns and exons. The open reading frames(ORF) of SlTIR1/AFBs were predicated to encode TIR1/AFB-like proteins containing canonical domains(Fbox and LRR). Phylogenetic analysis indicated that tomato SlTIR1/AFBs family genes belonged to TIR1, AFB4 and AFB6 sub-family.(2)qPCR analysis showed that SlTIR1/AFB gene family members have different expression patterns during tomato growth and developmental stage. Meanwhile, GUS histochemical staining of proSlTIR1/AFBs-GUS transgenic lines indicated that SlTIR1/AFB genes are expressed differently in various tissues, but largely overlapping domains. Indeed, SlTIR1 A mainly expressed in sepal, anther, ovary and early fruit placenta. SlTIR1 B strongly expressed in petiole, leaf vein, ovule, placenta and pericarp. SlAFB4 mainly expressed lateral root, sepal, stigma and early fruit placenta. SlAFB6 showed prominent expression in petiole, sepal anther and early fruit pericarp. In addition, qPCR analysis and GUS histochemical staining of proSlTIR1/AFBs-GUS transgenic lines indicated that auxin negatively regulates SlTIR1/AFB genes expression.(3)To address the subcellular localization of Sl-TIR1/AFBs proteins, we generated Sl-TIR1/AFBs:GFP fusion protein constructs and transiently expressed in BY2 tobacco cells. Microscopy analysis showed that the GFP signal for the Sl-TIR1A:GFP, SlTIR1B:GFP,Sl-AFB4:GFP and Sl-AFB6:GFP fusion protein were exclusively localized to the nucleus. To determine whether Sl-TIR1/AFBs exhibit the characteristics of auxin receptors, we performed Yeast two-hybrid and BiFC assays. Our results showed that SlTIR1 A, SlTIR1 B, SlAFB4 and SlAFB6 interacted with ASK1 and presumably form an SCF complex. In addition, F-box and LRR domain can affect the binding ability of Sl-TIR1/AFBs and Aux/IAA.(4)Yeast two-hybrid and BiFC assays indicated that Sl-TIR1/AFBs interacted with Sl-Aux/IAAs in an auxin-dependent manner. Moreover, these results established the framework of TIR1/AFB-Aux/IAA protein-protein interactions. qPCR analysis of tomato Aux/IAA and ARF genes indicated that SlTIR1/AFBs influenced the expression of these genes.(5)qPCR analysis indicated that there were the increased expression of each target genes in overexpressing transgenic lines and reduced expression in RNAi transgenic lines. The effect of exogenous IAA or NAA on root growth indicated that modification of SlTIR1/AFB expression may result in differential auxin response. SlTIR1A/B makes major contribution to plant auxin response.(6)SlTIR1A-OX lines presented alteration of floral development and fertilizationindependent fruit set. It was found that SlTIR1 A modulates the expression of auxin synthesis, transport and signaling and cell division and expansion genes during fruit-set. SlTIR1B-overexpression(SlTIR1B-OX) lines have a pleiotropic phenotype including reduced apical dominance associated with modified pattern of axillary bud development, dark green leaves, increased leaf number, altered fruit development and decreased fruit set. Sl-AFB4 is involved in latral root development.(7)Overexpression of SlmiR393 resulted in decrease mRNA levels of SlTIR1 A, SlTIR1 B and SlAFB6. Four mutations were introduced into the SlmiR393 binding site in the coding sequence and generated a SlmiR393-resistant form of SlAFB6(mSlAFB6). Over-expression of mSlAFB6 affected the formation of lateral organ boundaries. RNAseq analysis identified 27 differential expression genes(DEGs) belonging to 6 TF families, such as NAC, LOB, YABBY, TCP, zf-HD and HB-KNOX. 22 DEGs were involved in the metabolism and signaling transduction of auxin. These results indicated that the important roles of the TFs and hormones in regulation of lateral organ boundaries.