Transcription Factor HY5 Regulates Adventitious Root And Callus Formation And Shoot Regeneration In Arabidopsis Thaliana

Photomorphogenesis is a process in which light regulates the formation of plant tissues and organs by activating a series of signal molecules.As a positive regulator of photomorphogenesis,the transcription factor LONG HYPOCOTYL 5(HY5)is involved in many plant growth and development processes,such as cell elongation and proliferation,chloroplast development,pigment accumulation,and nutrient assimilation.HY5 also plays an important role in other signaling pathways such as hormone,nutrition,terpene synthesis,defense,and temperature response.De novo organogenesis commonly includes the formation of adventitious root and shoot regeneration,all of which are based on the pluripotency and totipotency of plant cells.The main factors controlling organogenesis include wound.light,stress,hormone,transcriptional and epigenetic factors.It will lay a theoretical foundation for high-efficient propagation of plants on the basis of revealling the interaction of different factors,stem cell specification,cell fate determination and cellular reprogramming.In previous studies,we found that HY5 mediates root-autonomous lateral roots development and root system architecture in Arabidopsis.The adventitious root,calli,and adventitious shoot,similar to lateral root formation,possibly all derived from the regeneration potential cells with root primordial properties.Light is an important environmental factor for adventitious shoot induction.Altogether,we speculate that HY5 may play an important role in the de novo organogenesis.Our study attempts to reveal HY5 function and potential molecular mechanisms in the regulation of de novo organogenesis through plant physiological,genetic and molecular approaches using genetic materilas.The main conclusions are as follows.1.The experimental procedure of the adventitious root formation,calli formation and 'two-step' shoot regeneration were established for phenotypic analysis.The number of adventitious roots in hy5 mutants and the number of adventitious roots formation in a single hy5 mutant seedling are significantly stronger than that of wild-type plants;the calli formation ability of hy5 mutant is significantly stronger than that of wild-type plants(using the whole seedling as explants);the shoot regenerative capacity of hy5 mutants is much weaker than that of wild-type plants(using excised Arabidopsis hypocotyls as explants).Our data suggested that HY5 is implicated in regulating adventitious root,calli formation,and shoot regeneration in Arabidopsis;2.HY5 is widely expressed during different stages of adventitious root and calli formation,and begins to express 48 hours and onwards on the SIM(shoot induction medium).Therefore,HY5 is suggested to play a significant role in adventitious root,calli formation,and shoot regeneration in Arabidopsis;3.The loss-of-function of HY5 homolog protein HYH and a downstream transcription factor PIF4 result in no alteration in adventitious root and calli formation,and shoot regeneration.Additionally,HYH and PIF4 are hardly not expressed during adventitious root and calli formation,and shoot regeneration.It is suggested that HYH and PIF4 are not involved in HYS-mediated adventitious root,calli formation,and shoot regeneration in Arabidopsis;4.The expression time,intensity or range of DR5:GUS hy5-2 in adventitious root and calli formation are earlier,stronger than DR5:GUS as revealed by auxin response reporter DR5:GUS,indicating that it is possible that HY5 modulate the adventitious root and calli formation by affecting auxin distribution and/or accumulation;5.With the increase of concentration,there was no significant change in the number of shoot regeneration of hy5 mutant,and explants were not greenish.It is suggested that elevated cytokinin could not resue the defect in shoot regeneration of hy5 mutants,HY5 is a key gene in cytokinin-induced shoot regeneration;6.The reporters that essential during shoot regeneration including PIN1:GUS,ARR5:GUS,WUS:GUS-GFP,are not altered in the hy5 mutant,implying that HY5-regulated shoot regeneration is not accomplished through affecting ARR5-mediated cytokinin signaling and/or acquisition of regenerative competence.In summary,We found that HY5,a positive regulator of photomorphogenesis,is implicated in adventitious roots and calli formation.Particularly,the loss-of-function of HY5 leads to a severe defect in shoot regeneration.Moreover,the hy5 mutant is insensitive to cytokinin,and increased exogenous cytokinin could not resue the defective phenotype of hy5 mutants in shoot regeneration,suggesting that the loss of HY5 function lead to heavy impairment in cytokinin-induced shoot regeneration.Combined with previous studies and our results,we suggest that HY5 regulates the balance between auxin and cytokinin in adventitious roots and calli formation.In addition,as a regulator of photomorphogenesis,HY5 may integrate light and cytokinin signaling in modulating shoot regeneration.