Study On Phytohormone Regulated Potato Tuber Formation And The Function Of Related WRKY TFs

Potato（Solanum tuberosum L.）is one of the most important crops in the world being considered as a staple food in many developing countries.As a complex biological phenomenon,tuberization is regulated by many signals,such as environmental factors,plant hormones and genetic background.Among various phytohormones,ABA is a positive modulator,while GA is an inhibitor of the process.However,ABA-GA signaling crosstalk and molecular mechanism during tuberization are still unclear.Understanding the regulation of tuber induction is essential to devise strategies to improve tuber yield and quality.As one of high-throughput functional genomics techniques,RNA-Seq technology is powerful for screening functional genes.Identification of key genes responsive to tuberization is an essential for the elucidation of the molecular mechanisms,and thereby the breeding or genetic engineering of high-yielding varieties.The exact levels of various hormones in leaves and stolons were quantified at four consecutive stages.The contents of ABA and JA in leaves and stolon/tuber and accumulation of JA in stolon/tuber increased throughout the whole tuber development,while JA amounts increased from tuberization induction to tuber initiation and decreased from tuber grow to ripe stage.The levels of GA3 decreased after tuberization induction both in leaves and in stolons.IAA amounts showed an increase-decrease tendency with a peak at tuber initiation stage.The results showed IAA and GA3 are essential for elongation of stolon.In addition,ABA and JA are positive regulatory factors for tuberization,while GA is a dominant negative regulator that inhibits tuber formation.The roles of exogenous ABA and GA₃ application in tuber formation in vitro were studied.The results showed that exogenous GA₃ inhibited tuber formation,whereas exogenous ABA stimulated tuberization.GA3 inhibited tuber formation at a very low concentration of 0.01μM,tuber formation was completely inhibited at a GA₃concentration of 0.5μM.It’s clear that GA acted as a dominant negative regulator,and ABA was unable to counteract the inhibition caused by high concentration of GA3.ABA significantly promoted the accumulation of starch suggesting ABA may induce the expression of starch synthesis genes.A total of 261 DEGs respones to ABA were identified,including 168 up-regulated genes and 93 down-regulated genes.Four tuberization mark genes,Patatin,GA2ox1,SP6A and BEL5 were up-expressed,which is consistent with the promotion effects of ABA on tuber formation.However,ABA may not directly regulating the expression of these mark genes.Pathway enrichment analysis showed that phytohormone signalings were markedly enriched in environmental information processing,including ABA,ethylene,JA and GA.Several classes of stress-related genes including WRKY,AP2/ERF,HSP,MYB were specifically up-regulated or down-regulated in respone to ABA,indicating that these genes may induced or negative feedback by ABA or genes in ABA signaling.There was a high correlation coefficient between the qRT-PCR and transcriptome results confirming that the transcriptomic results were acceptable.Differentially expressed WRKY genes were chosen to study their roles in tuberization ABA-GA crosstalk.All differentially expressed WRKY genes were cloned,and bioinformatic analysis were performed.Further,prokaryotic expression of WRKY57 protein was used to confirm bioinformatics analysis.StWRKY57 gene was over expressed in potato and Arabidopsis thaliana,the overexpressor lines will be helpful in future studies to determine the ABA/GA levels and tuber formation in wild-type and transgenic line,and propose model for the regulation of tuberization in WRKY transgenic potato plants.Upstream sequence（up to 1500 nt from ATG）of ABA/GA metabolism and signaling pathway genes（StGA2ox1,StNECD2,StABF,StABI）was extracted from the genome and known cis-elements were mapped onto the promoters using Plant Cis-acting Regulatory DNA Elements（PLACE）.The presence of W-box elements indicated that WRKY transcription factor may regulate ABA-GA crosstalk by directly regulating transcription of ABA/GA metabolism and signaling pathway genes.