Re-identification Of Tomato AP2/ERF Transcription Factor Superfamily And Functional Analysis Of SlERF.D.3

Tomato(Solanum lycopersicum)is popular among people because of its rich nutritional value and unique flavor.It is one of the vegetables that is widely grown in the world.The ERF transcription factor family belongs to the AP2/ERF superfamily.Studies have shown that it is involved in regulating hormone signal transduction,stress responses and plant growth and development.Based on the completion of tomato genome sequencing,some scholars have identified the AP2/ERF superfamily of tomato.However,with the update of tomato genome database,the more comprehensive identification of AP2/ERF members in tomato is of great significance for predicting the function of unknown genes.In this study,we systematically updated tomato AP2/ERF superfamily members and performed the analysis of ERF family genes,including their chromosomal localization,conserved motifs,gene structure,promoter cis-acting elements and responses to Botrytis cinerea and auxin treatments.The function of SlERF.D.3 gene was further identified by using tomato transgenic material.The specific results are as follows:Through bioinformatics analysis,a total of 176 members of the AP2/ERF superfamily were identified in this paper including 141 genes of the ERF family and 42 newly identified members of the ERF subfamily.These new members were distributed on all chromosomes except chromosome 7 and there were 19 genes only containing exons.By using the evolutionary relationship and the promoter cis-acting element prediction,19 genes that may be related to stress responses were screened out from the ERF family genes,and gene expression levels were detected under the treatments of Botrytis cinerea and IAA.Among them 9 genes responded to Botrytis cinerea treatment and 12 genes responded to IAA treatment.As a member of the D subclass of the ERF transcription factor family,the SlERF.D.3 gene can quickly respond to IAA treatment and its function is unknown.Subcellular localization prediction result suggested that SlERF.D.3 protein may be located in the nucleus.Construction of the pGBKT7-SLRERF.D.3 expression vector proved that this transcription factor had transcriptional activation activity in yeast.Real-time quantitative PCR analysis showed that both ACC and MeJA treatments could induce the expression of SlERF.D.3 gene,and it increased rapidly within 1 h after treatment.ABA treatment always suppressed the expression of this gene.Tissue expression-specific analysis showed that the SlERF.D.3 gene was expressed in different organs and tissues of wild-type tomatoes especially in the ovary,which indicated that SlERF.D.3 may play a role in fruit development.In order to clarify the function of SlERF.D.3,its coding region was cloned from tomato variety "Ailsa Craig" and constructed into the overexpression vector.Three transgenic tomato lines overexpressing SlERF.D.3 were obtained through agrobacterium-mediated transformation.Phenotypic identification results showed that the overexpressed lines had more slender and downward curling leaves compared with the wild type.While no fruit-set was observed in WT unpollinated pistils,around 22% of unfertilized transgenic ovaries showed spontaneous parthenocarpic growth with the development of a normal locular tissue.The different expression of tomato EXP5 and ACO4 in wild-type and transgenic ovary suggested that overexpression of SlERF.D.3 can affect the expression of genes related to fruit development.This indicated that SlERF.D.3 was a parthenocarpy-related gene.Overexpression of SlERF.D.3 significantly increased the auxin content and affected the expression of auxin-related genes compared with wild-type tomato.Auxin-induced parthenocarpy and fruit development are partially dependent on GA.In order to investigate whether SlERF.D.3 played a role in regulating the expression of GA biosynthetic genes,this study examined the expression levels of GA20-oxidase gene family members in the ovary of wild-type and transgeneic plants at different developmental stages.The results showed that overexpression of SlERF.D.3 affected the transcription levels of SlGA20ox1?SlGA20ox2 and SlGA20ox3.In order to investigate whether overexpression of SlERF.D.3 affects fruit quality,this study measured some fruit quality indexes.The results showed that the content of vitamin C and free amino acids in transgenic tomato fruits were increased and the content of soluble protein were decreased compared with wild-type fruits,there was no significant difference in soluble sugar and soluble solids.