Identification And Characterization Of New Bri1 TILLING Mutants In Arabidopsis Thaliana

Brassinosteroids(BRs)are a class of steroidal phytohormones playing essential roles during normal plant growth and development.In Arabidopsis,BRs are perceived by their major receptor BRI1 and co-receptor BAK1,both of which are single-pass transmembrane leucine-rich repeat receptor-like protein kinases(LRR-RLKs).In the absence of BRs,the kinase activity of BRI1 is inhibited by its associated inhibitory protein BKI1.When BRs are present,they can directly interact with the extracellular domains of BRI1 and BAK1,causing dissociation of BKI1 and association of BRI1 kinase domain with BAK1 kinase domain.The BRI1-BL-BAK1 complex can then initiate early BR signaling events and activate its downstream signaling cascade.Identification and characterization of a mutational spectrum for a specific protein can help to elucidate its detailed cellular functions.During the past two decades,over 20 unique bri1 alleles have been isolated,which helped to determine the significance of each domain within BRI1.Some weak alleles,such as bri1-119,bri1-5,and bri1-9,on the other hand,are excellent genetic tools used for characterizing the entire BR signaling pathway via extragenic modifier screens.Identification of many key regulatory components by using weak bri1 mutants has contributed significantly to our current knowledge of the BR signaling pathway,from BR perception to downstream responsive gene regulation.In order to deeply understand the molecular mechanisms of BRI1 in regulating the BR signaling pathway,we analyzed all BRI1 point mutations generated from a TILLING project.All 87 BRI1 TILLING mutations were obtained from the Arabidopsis Biological Resource Center(ABRC)and 83 mutations were confirmed by a dCAPs method.These mutations were generated in Columbia(Col-0)background containing an er mutation.A high dose of EMS could introduce mutations in other genes that could result in BRI1-unrelated phenotypes.In order to remove those additional mutations,we backcrossed the mutants with Col-0 for at least three generations.Offspring plants containing BRI1 mutations were selected by using the dCAPs approach.After backcrossing three times,most mutations were found to exhibit no morphological differences from wild-type plants.Only 9 mutants consistently exhibit an allelic series of typical bri1 phenotypes,from subtle to severe morphological alterations,including 4 subtle(bri1-705,bri1-706,bri1-710,and bri1-711),1 weak(bri1-702),and 4 strong(bri1-703,bri1-704,bri1-708,and bri1-709)alleles.We also carried out biochemical analyses to investigate possible mechanisms of these mutations in affecting BR signaling.For example,bri1-702,the first weak allele in the activation loop of BRI1 identified so far,exhibited not only reduced autophosphorylation activity of BRI1 in vitro but also reduced sensitivity of BAK1 phosphorylation response to exogenously applied BL in vivo.bri1-705 is a new subtle allele containing a point mutation in the extracellular portion.Overexpression of mBAK1 failed to generate a dominant negative phenotype in bri1-705,implying the mutation in bri1-705 may interfere with the interaction between bri1-705 and BAK1.Molecular dynamic simulation of structural analysis suggests that this mutation likely disrupts the formation of hydrogen bonds among BRI1,brassinolide(BL),and BAK1.Surprisingly,bri1-706,a subtle morphological mutant with a mutation in the extracellular portion of BRI1,showed insensitive root growth inhibition to exogenously applied BL which is similar to the null bri1 mutants.To our knowledge,this is the first bri1 mutant showing that phenotypic severity is not positively correlated with the degree of insensitivity to exogenous BL application.In this study,we also found bri1-301 possess kinase activity in vivo,clarified a previous report arguing that kinase activity may not be necessary for the function of BRI1.These data provide additional insights into our understanding of the early events in the BR signaling pathway.The new mutants identified from these studies can be used as genetic tools for further dissecting the functions of BRI1,as well as utilizing as references for studying the biological functions of other RLKs in plants.