| Potato is one of the important food crops worldwide.Potato tubers are important food sources,asexual reproductive organs,and good materials for studying morphogenesis and development.Therefore,the research on tuber development has important theoretical significance and potential application value for understanding the formation and control mechanism of potato yield.As an important plant hormone,auxin plays a regulatory role in potato tuber development.Thus,functional study of its related response genes will help to reveal the regulatory mechanism of auxin during potato tuber development.In order to clarify functions of auxin-responsive genes during tuber development,molecular biology analysis was used to identify transcription factor family involved in auxin response in potato,and two family members that were abundantly expressed in stolons and tubers obtained through high-throughput expression data and quantitative expression validation.The biological functions of related genes during tuber development and functions of auxin transport genes were further explored by means of gene expression and subcellular localization,gene silencing and gene overexpression,and protein interaction.The main findings are as follows:(1)Some potato St LAX,StPIN and St ABCB auxin transporter genes were involved in the response to auxin and abiotic stress.Five AUX/LAX and 22 ABCB proteins related to auxin transport were identified from the potato genome by bioinformatics.The expression levels of some St LAX,StPIN and St ABCB genes changed in response to auxin,auxin transport inhibitors and abiotic stress.St LAX,StPIN and St ABCB gene promoter regions contain auxin-responsive and stress-related cis-acting elements targeted by specific transcription factors in response to different exogenous stimuli.The three typical auxin transporters St LAX2,StPIN2 and St ABCB4 are all membrane-localized proteins,impling their roles in regulating flow of auxin towards cell membrane.(2)StARF2 and StARF5 were involved in tuber development as transcription factors of auxin signaling.Nineteen ARF proteins were identified from the potato genome based on name and local BLAST sequence similarity searches.Combining RNA-seq expression data and q RT-PCR analysis of various tissues found that StARF2 was highly expressed in the whole process of tuber development,including stolons,young tubers and mature tubers,while StARF5 was abundantly expressed in stolons,indicating that StARF2 and StARF5 might be involved in tuber development.And the proteins encoded by StARF2 and StARF5 genes were both nuclear-localized proteins.Both StARF2 and StARF5 genes were up-regulated by auxin,indicating that they might be involved in auxin signaling pathway.(3)Both StARF2 and StARF5 genes were involved in the regulation of potato tuber development.StARF2 gene silencing resulted in delayed tuber formation in transgenic potato plants and three key quality indicators,including ascorbic acid,soluble sugar and soluble protein content were significantly reduced in transgenic potato tubers.Deletion of this gene affected normal expressions of marker genes that promote tuber formation(St BEL5,St CDF1,St SP6 A and St SWEET11)and auxin,gibberellin,abscisic acid-related genes(StPIN1,St GA2ox1,and St ABA).Overexpression of StARF5 increased stolon and tuber numbers,which resulted in increased tuber weight per plant.And overexpression of StARF5 promoted the accumulation of starch,soluble sugar and protein in transgenic potato tubers.Overexpression of this gene induced expressions of tuber formation positive regulators(St BEL5,St SP6 A and St CDF1),sucrose transport-related genes(St SWEET11 and St SUT1),auxin transporter gene StPIN1 and gibberellin synthesis-related gene St GA2ox1.The results showed that both StARF2 and StARF5 genes were involved in the regulation of potato tuber development.(4)Potato StARF2 could form an interaction complex with St IAA20 protein.Preliminary analysis of molecular structures and interaction network of potato ARF proteins showed that Aux/IAA was a potential interaction protein of ARF.Therefore,the interaction between StARF2 and St IAA20 proteins in yeast was preliminarily verified by yeast two-hybridization.Subcellular localization analysis showed that St IAA20 was also localized in the nucleus.Bimolecular fluorescence complementation(Bi FC)experiments again verified that StARF2 and St IAA20 proteins interacted in plant nucleus and performed transcriptional regulatory functions.In summary,StARF2 and StARF5 genes,as transcription factors of auxin signaling,were both involved in potato tuber development,and StARF2 could also form an interaction complex with St IAA20 protein and participate in the regulation of tuber development.In addition,auxin transporters in potato,as cell membrane-localized proteins,might play a role in mediating auxin homeostasis and redistribution between cells and regulating specific adaptive mechanisms tolerance to environmental stimuli. |
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