Importance Of Less Conserved Domain LRRs In Signaling Of Receptor BRI1

Brassinosteroids(BRs)are essential plant steroid hormone perceived by a membrane-bound receptor called BRI1,on the cell surface.This triggers a signaling cascade that activates and inactivates different sets of BR responsive genes to regulate plant growth and development.BRI1 comprises of LRRs(Leucine Rich Repeats),embedded between the signaling peptide and the transmembrane domain followed by an intracellular kinase domain.Many mutant alleles have been identified in Arabidopsis but the mutation sites of these mutants were most present on the BR binding domain and cytoplasmic domains and demonstrated that both these domains are crucial for the normal functions of BRI1.Conversely much fewer mutations were found in the LRR region especially for the first 10 LRRs.It may be due to the fact that this domain was not critical for biological activities of BRI1 or may be neglected by researchers for lacking of observable phenotype.Therefore this study was conducted with the objective to explore the importance of this domain especially in the less conserved region of the first 10 LRRs in order to comprehensively understand the function of the LRR domain in the normal functioning of the BRI1.In the present investigation the sequences of BRI1 and its homologues BRL1,BRL2 and BRL3 were used to compute phylogenetic tree in which three clades were formed.It showed that BRL2 appeared earlier than BRL1 and BRL3,whereas,BRI1 appeared later during the evolution.Sequences of Oryza sativa,Brachypodium distachyon,Setaria italic,Sorghum bicolor,Zea mays,Arabidopsis thaliana,Solanum lycopersicum,Solanum tuberosum,Populus trichocarpa,Glycine max,Phaseolus vulgaris,Amborella trichopoda)were compared and found that LRR1-10 of BRI1 was less conserved in these species.We obtained bri1-235,a weak allele of bri1.This mutant showed a weak morphological phenotype compared with the WT wild type(WT)i.e.round leaves and short petioles,prolonged life span,reduced rosette size and smaller plant height that is about half of the WT.Upon amplification of the genomic region of BRI1 using PCR and then DNA sequencing in bri1-235,a single base alteration from C to T resulted in an amino acid substitution from serine(Ser)to phenylalanine(Phe)at the 156th amino acid position of LRR domain was identified.The subcellular localization of bri1-235 was checked by using endoglycosidase H and it showed that bri1-235 protein was completely retained in the endoplasmic reticulum which was further confirmed by using confocal laser scanning microscopy on bri1-235-GFP.To check the sensitivity of this mutant to BR and BRZ,WT and bril-235 were grown under different concentration of 24-epibrassinolide and BRZ.Roots of bri1-235 were decreased with the increasing concentration of eBL while hypocotyl length was increased but relatively much slower than that of WT.Under BRZ treatments both roots and hypocotyls lengths of bri1-235 decreased faster that WT in a concentration-dependent manner.These results suggest that bril-235 is a BR insensitive mutant.To confirm that I have got the right gene,I cloned the BRI1 gene with BRI1:BRI1-GFP promoter to compliment bri1-235 mutants.The transgenic plants were then obtained by Agrobacterium tumefaciens-mediated floral dip method.The transgenic plants of pBRI1:BRI1-GFP/bri1-235 produced longer petiole length,elongated leaves and total plant stature,similar to WT.I also got sbilbri1-235 plants by cross and it partially rescued bri1-235 phenotypes.Since SBI1 is a BRI1 inhibitor,these results suggest that bri1-235 is a functional receptor.Furthermore,I found that PCZ(a specific BR biosynthetic inhibitor)greatly reduced the hypocotyls and roots of both pBRI1:bri1-235:GFP/bril-235 and bril-235sbil as compared to bri1-235.We conclude that bri1-235 is a partial loss-of-function mutant.BR homeostasis is subjected to feedback regulation in which DWF4 and CPD involved in BR biosynthesis are downregulated when BR signaling is activated.Thus high transcript levels indicate low BR signaling and vice versa.BAS1 is involved in BR inactivation which is positively regulated by activated BR signaling.In bril-235 the DWF4 and CPD showed higher transcript levels and remained unchanged upon exogenous application of eBL which demonstrated lower BR signaling in this mutant,while it was decreased in WT and pBRI1:BRI1-GFP/bri1-235.that exhibited the high BR signaling Consistently the expression of BAS 1 was decreased in bri1-235 compared to WT and pBRI1:BRI1:GFP/bri1-235,confiriming lower BR signaling in bri1-235.Several studies showed that exogenous application of BR increased the stress resistance in plants.No study was conducted by using genetic mutants of BR to elucidate the role of of BRs.On the stress response.Therefore I conducted experiments by using this new mutant of BR signaling,bril-235 under different stresses.For this,bri1-235 and WT plants were grown under abiotic stress(control,100Mm of NaCl and 100Mm of NaCl+1?M of BR)and biotic stress(M.phaseolina inoculation+1?M of BR and control).Both stresses reduced the shoot and root fresh and dry weight and also the lengths of shoot and roots.The relative water contents and chlorophyll contents were more dramatically decreased in bri1-235 than in WT.The Sugar contents,proline and protein contents were increased under stresses.Salinity stress increased the antioxidants i.e.SOD,H2O2,POD,MDA while CAT activity was decreased in both WT and bri1-235 plant,however the magnitude of rising and fall was different.The application of exogenous 24-epibressinolide ameliorated the stress condition and increased the overall growth of WT and bri1-235 plants as compared to the control.bril-235 was more sensitive than WT under stressed conditions.So BR signaling is crucial to cope with stressed environment.The finding of this new mutant bri1-235 provides an insight to elucidate detailed functions of less conserved LRRs of BRI1 during growth,development and stress response.