A Study Of BR Signaling In European Spruce

Brassinosteroids(BRs)are a class of steroidal phytohormones that are widely found in plants.In angiosperms,brassinolide,the most active BR and castasterone,the second most active BR,are synthesised by CYP85A2 and CYP85A/A1,respectively.In Arabidopsis,BRs activate their downstream targets via the essential receptor BRI1(BR Insensitive 1).In addition,BRI1-likes 1,3(BRL1/3)can but BRL2 cannot bind BRs.Blast search shows that CYP85A and BRL1/3 first appeared in gymnosperms.However,whether active BR can be synthesized in gymnosperms and how BRs function in gymnosperms are unknown.Based on the components of BR signals in many extant plants and through phylogenetic analysis and functional identification of related genes,this study explored the functions of BR and its receptor in gymnosperms,so as to provide a basis for further studying the molecular mechanism of adaptive function evolution in terrestrial plants.Currently,forward genetics study is difficult in gymnosperms.Thus,molecular complementation of the respective genetic mutants of angiosperms essentially becomes the best alternative to study the biological functions of gymnosperm genes.The main results are as follows:1.Phylogenetic analysis confirmed that PaCYP85A shared a common ancestor with OsCYP85A,which shared a common ancestor with AtCYP85A1 and AtCYP85A2.PaCYP85A under the 35S promoter rescued AtCyp85a2 mutants better than that of AtCYP85A1 but slightly and clearly worse than that of OsCYP85A and AtCYP85A2,respectively,confirming that P.abies may only synthesize the second most active CS but not the most active BL.2.In this study,petri dish with filter paper was used to investigate the effects of brassinosteroids on seed germination under normal and drought stress conditions.The results showed that on 7,14 and 21 days,eBL could promote the germination of Picea abies seeds,regardless of treatments by normal or drought stress condition.This effect was BR concentration-dependent.It had the best effect when BL concentration was 500nmol.At the same time,BL treated seeds reduced the effect of drought stress on their germination.3.The homologous analysis suggests that BRI1 is angiosperm-specific while BRL1 is seed plant-specific.There were two copies of BRL1,PaBRL1-1 and PaBRL1-2,in Picea.By sequence similarity analysis,there are only BRL1 orthologues(PaBRL1-1 and PaBRL1-2)but no BRI1 orthologue in P.abies.4.This study found that PaBRL1-1 and PaBRL1-2 were broadly expressed in root,stem and leaf of Picea,and PaBRL1-1 had higher expression than PaBRL1-2.Consistently,the promoters of PaBRL1-1::GUS fusion was expressed more intensely than that of PaBRL1-2::GUS fusion in Arabidopsis.The expression of PaBRL1-1 and PaBRL1-2 under the AtBRI1 promoter could not complement the statures of bri1-301 mutants.However,neither BR treatment of Picea seedlings nor expression of PaBRL1-1 and PaBRL1-2 in Atbril promoted plant heights,yet the BR responsive genes were activated.Meanwhile,dephosphorylation of BES1 occurred in transgenic plants when treated with BRs,indicating that there are active BR signals in transgenic plants with the expression of PaBRL1-1 and PaBRL1-2.5.This study showed that expressing chimeric constructs AtBRI1:PaBRL1-1 and PaBRL1-1:AtBRI1 under the AtBRI1 promoter completely rescued Atbri1-301 statures.However,AtBRI1:PaBRL1-2 could only partially while PaBRL1-2:AtBRI1 could not,rescue the statures of Atbri1 mutants.Nevertheless,they all had physiological and molecular BR responses,suggesting that all the transgenic plants can express functional chimeric receptors.Together,these results further confirm that PaBRL1 is likely a weak BR receptor in P.abies.Furthermore,PaBRL1-1 has a stronger receptor function than PaBRL1-2.6.Expressing chimeric constructs AtBRI1 with the BD1-3 of PaBRL1,we found drastically functional enhancement in AtBRI1 with the BD3 of PaBRL1-1 or PaBRL1-2 such that they completely rescued bri1 phenotypes.AtBRI1 with the BD3 of PaBRL1-1 had stronger function than that of PaBRL1-2,which was further confirmed by swapping the BD3 of PaBRL1-1:AtBRI1 with that of PaBRL1-2:AtBRI1.Altogether,these data confirm that PaBRLl has weaker BR receptor function than that of AtBRI1 and AtBRL1.To assure the function of PaBRL1 is BR specific,a couple of conserved residues were mutated to reduce BR-binding.Expression of PaBRL1-1Y627A and AtBRL1Y699A rescued neither bril-301 statures nor bril-301 molecular functions for BR signaling.Further,AtBRL1F584L and PaBRL1-1:AtBRI1F656L resulted in an almost complete loss of their ability to rescue bril-301 statures,yet the molecular functions for BR signaling were most retained.As the kinase activity of PaBRL1 was lower than that of AtBRI1,although PaBRL1-1 is a bona fide BR receptor,it has weaker receptor function than AtBRI1 and AtBRL1 receptors.This study confirmed for the first time the active and weak BR signals in gymnosperm through studies of BR synthesis and BR receptors,suggesting gradual co-evolution of BR receptor and ligand in plant evolution.Together with no BRL1 in seedless plants,these results reveal the possible emergence of BR function in seed plants,filling a gap between angiosperm-derived BRI1 and seedless plant-originated EMS1 and BRL2.Therefore,this study can provide a new venue to further investigate the functional divergence of BRI1/EMS1 signaling during evolution.