Molecular Mechanisms Of GOLDEN2-LIKE Transcription Factors Regulating Adventitious Root Development In Arabidopsis

Adventitious roots,growing from non-root tissues,enhance tolerance of plants to various stress conditions and is a key limiting factor for vegetative reproduction.The formation of adventitious roots is regulated by external environmental and internal genetic factors.Auxin can promote plant root development and induce adventitious root formation.Previous studies have shown that the seedlings form long hypocotyls when cultured in the dark,and adventitious roots develop in hypocotyls when moved to light in Arabidopsis.At the same time,light also regulate adventitious root formation by affecting the expression of auxin-responsive genes.Chloroplast development is one of the important characteristics when Arabidopsis grows in light.The Arabidopsis GOLDEN2-LIKE(GLK)transcription factor promotes the chloroplast biogenesis by positively regulating the expression of photosynthesis-related genes.It also participates in retrograde signal transduction from chloroplast to nucleus,which can regulate the formation of adventitious roots by regulating chloroplast retrograde signal-related genes.The formation of adventitious roots is a complex process,and the molecular mechanisms that regulate them are rarely studied.This project mainly analyzed the adventitious root phenotype of seedlings under different culture conditions and corresponding mutants and overexpression materials,and analyzed the role of light signal,auxin signal,and chloroplasts development in the process of adventitious roots development.Our experiment showed that when Arabidopsis seedlings grow in the light,light can inhibit the induction of adventitious roots by NAA.DR5:GUS and DR5:GFP are auxin reporter genes.GUS staining and GFP fluorescence observation of DR5:GUS and DR5:GFP seedlings under different culture conditions showed that the auxin NAA response of seedlings grown in the light was weakened,light inhibited the response of seedlings to NAA.NAA can induce the expression of auxin response genes GH3.3,GH3.5,GH3.6,LBD16,LBD18,and LBD29.However,the ability of NAA-induced expression of auxin response genes is lower in the light than in the dark.In conclusion,our results indicated that the light inhibited the auxin-induced adventitious root development.Norflurazon,an inhibitor of chloroplast development enhances auxin-induced adventitious roots significantly and increases the auxin response of seedlings in light.At the same time,NF increases the induction fold of auxin response gene expression.The glk1 glk2,chloroplast development-related mutant,has more adventitious roots than the wild type and enhanced auxin response when grown in the medium with or without NAA.GLK-overexpression plants develop less adventitious roots than wild type and have weakened auxin response when grown in the medium with or without NAA.The glk1 glk2 double mutant was crossed with the auxin signal transduction mutant arf7.The hybrid plant harboring glk1 glk2 arf7 was able to restore the increased adventitious roots of glk1 glk2,and the glk1 glk2 arf7 mutant had similar phenotypes to arf7.This result suggested that the adventitious root phenotype of the glk1 glk2 mutant was related to auxin and that ARF7 located downstream of the GLK gene to regulate adventitious root development.GUN5 participates in chloroplast retrograde signals,and gun5 mutant has more adventitious roots than wild type with or without NAA treatment.NAA has a stronger ability to induce adventitious roots in gun5 mutant.The results of staining and fluorescence observation of the hybrid plants showed an enhanced response to auxin.q RT-PCR results showed that GLK gene could regulate the expression of GUN5.The results indicate that GLK-upregulated GUN5 is involved in auxin signaling.RNA-seq data showed that GLK and GUN5 were able to inhibit HSP90 gene expression.HSP can affect the nuclear localization of TIR1,which is an auxin receptor.Whether GLK and GUN5 regulate adventitious root development by affecting auxin signal transduction through HSP needs to be further studied.The innovative points of this work:Based on the research of the primary root and lateral roots,the study focus on Arabidopsis adventitious root formation.The work breaks through the limitations of anatomical morphology,physiological and biochemical levels.The molecular biology methods,such as q RT-PCR,RNA-seq,and genetic hybridization were used to analyze the molecular mechanisms of light signals,auxin signals,and chloroplast development in regulating adventitious root development and their mutual regulatory effects.