Molecular Mechanism Of ARF19-WOX9/13 Mediated Exogenous Auxin Inducing The Embryogenic Cell Formation In Lilium

Somatic embryogenesis(SE) is a powerful evidence of plant cells totipotency and is also the most promising regeneration pathway for efficient propagation,germplasm conservation and genetic improvement in Lilium.The formation of embryonic cells in the early stage of SE is of great significance to the induction cycle and proliferation rate.Reasonable regulation of the formation of embryonic cells is conducive to the industrialized application of SE reproduction in lily.We first defined the origin of somatic embryos in lily and confirmed that auxin is necessary for the initiation of embryogenic program.In this study,WOX gene,a key regulator of stem cell development,was used as the entry point to verify the function in embryogenic cell formation.By exploring the interaction between ARF protein(the core response factor of auxin signaling pathway)and WOX,the molecular mechanism of auxin regulating WOX expression via ARF and inducing embryogenic cell formation was clarified.The results will provide a theoretical basis for the production and germplasm improvement of lily,and hopefully provide a possible way to analyze the important scientific topic of plant cell totipotency.The main results are as follows:1.Based on transcriptome analysis in the early stage of somatic embryo induction,combined with q RT-PCR and in situ hybridization,the key regulatory factors of embryogenic cell formation LpWOX9 and LpWOX13 were excavated.Subsequently,the CDS and promoter sequences of LpWOX9 and LpWOX13 were cloned.Cis-acting element analysis showed that both the promoters contained ARF binding sites.By observing the initial stage of somatic embryo induction in Lilium pumilum DC.Fisch.,it was clear that the critical time of embryogenic cell formation was the 9 th day.The content of endogenous auxin in 0-9 d showed that there was no significant difference of endogenous IAA,ME-IAA and ICA,indicating that the content of endogenous auxin was not enough to be used as a marker of the emergence of plant embryogenic cells.2.The high expression of LpWOX9 and LpWOX13 significantly promoted the formation of embryogenic cells and somatic embryogenesis in lilium.The embryogenic cell formation period of LpWOX9 and LpWOX13 overexpression lines was 6 d,the time required for the somatic embryo induction rate to reach 100% was 20 d,and the somatic embryo proliferation coefficients reached 2.52 and 2.08 respectively.Compared with the empty line,the time of embryogenic cell formation of LpWOX9 and LpWOX13 overexpression lines was advanced by 3 d.The cycle of somatic embryo induction was significantly shortened by 8 d.And the proliferation rate of somatic embryo was increased by 68% and 38.7%,respectively.LpWOX9 and LpWOX13 overexpression plants could not form somatic embryos lack of exogenous auxin,indicating that the promoting effects of LpWOX9 and LpWOX13 on somatic embryos cannot exist independently of auxin.3.The interaction between all differentially expressed Lp ARFs,LpWOX9 and LpWOX13 in the early stage of somatic embryo induction was verified.Lp ARF19 can inhibit the transcription of LpWOX13,and LpWOX13 can promote the transcription of LpWOX9.Exogenous auxin induced low-level expression of Lp ARF19,which promoted the expression of LpWOX13 and LpWOX9.Meanwhile,LpWOX9 weakens the inhibition of LpWOX13 transcription by binding to Lp ARF19 protein,then promotes the expression of LpWOX13 and LpWOX9.LpWOX9 promotes its own expression level by binding to LpWOX13 protein.In conclusion,this study confirmed the direct regulation of auxin signaling pathway on WOX by analyzing the interaction between LpWOX9,LpWOX13 and Lp ARF19,to reveal the molecular mechanism of somatic embryogenesis in lilium.The results will provide theoretical basis and practical reference for the propagation and genetic improvement of lily bulbs based on somatic embryogenesis,and provide ideas for the interpretation of totipotency in plants.