Cytological And Molecular Basis Of Brassinosteroid Regulating Seedling Roots Development In Wheat

Wheat(Triticum aestivum L.)is one of the world's most important crops.The root system is the key organ for controlling the fate of wheat plants through its anchoring efficiency to the soil as well as water and nutrient absorption.The root traits therefore are the critical targets to modify for grain yield and drought tolerance improvement.Brassinosteroid(BR),a major plant hormone,plays critical roles in regulating plant growth and development and responding to various biotic and abiotic stresses.The regulatory role of BR on root development has been well studied in Arabidopsis,but it remains largely unknown in hexaploid wheat,preventing BR utilization in this important crop.In this study,bread wheat(hexaploidy wheat)of the ‘Chinese Spring' variety was used.To evaluate the roles of BR in wheat root development,the exogenous BR(EpiBL)and the BR synthesis inhibitor brassinozole(BRZ)were applied to wheat seedlings.The function of BR in wheat root development were comprehensively investigated by cytological observation.Combined with wheat root transcriptome analysis and genetic transformation,this study identified the characteristics and verified the functions of wheat BR pathway genes,and initially explored the corresponding regulation of BR pathway in wheat.Our results layed a foundation for understanding the mechanism of BR regulating wheat root development and the application of BR for wheat root improvement.It also provides a reference for studying BR pathways in other polyploid plants.This study showed the following results and conclusions:The root length,root diameter and lateral root(LR)number was regulated by BR in a concentration-dependent manner.Cytological observation confirmed that BR regulates the root diameter by controlling both the cell number and the cell diameter of the root,but confers different effects in different cell types on the horizontal plane.For LR,BR regulates the LR numbers by enhancing lateral root primordium(LRP)initiation and accelerated the LR emergence process at the same time.Cytological observation suggested that BR regulating the root diameter and LR emergence were two novel roles of BR in root development.Transcriptome analysis of BR and BRZ treated wheat roots showed that BR synthesis and some sugar metabolism pathways were significantly enriched,indicating that BR could regulate the sugar metabolism pathway in wheat roots.Meanwhile,many auxin signaling pathway genes(ARF7,ARF19,IAA14,IAA28),which are involved in root development,were not only respond to BR,but also their expression patterns are consistent with root phenotypic changes,indicating that BR regulate root development by interacted with auxin signaling pathway.Analyses of BR homologous genes demonstrated that the genetic framework of the wheat BR pathway was close to that of rice,but contained highly redundant homologous copies of genes from the subgenome A,B and D,which also called triplets.In the BR-treated root transcriptome,these triplet copies exhibited different expression level but the same response to BR,especially the BR feedback genes,TaDWF4,TaCPD and TaBR6 OX.Expression of wheat TaDWF4 copies in Real-time PCR experiment also confirmed that the functions among the copy genes were similar,indicating that the wheat BR pathway gene has functional redundancy.TaDWF4s transgenic results confirmed that the wheat TaDWF4 s could inhibit the defective phenotypes of dwf4-44 mutant with short roots and few LRs,while its overexpression in wildtype could significantly increase LR density.In addition,the wheat TaSK1-m(mutant)s overexpressing lines showed phenotypes with short roots but increased lateral root density,which is similar to the root phenotypes of the bin2-1 gain-of-function mutant in Arabidopsis.In addition,the expression of TaDWF4 s and TaSKs genes in Arabidopsis roots confirmed that multiple homologous copies maintained their conserved function in regulating root development.Besides,genetic experiments confirmed that point mutant TaSK transgenes dominant negatively affected the function of AtBIN2 and its multiple homologs in Arabidopsis,indicating that this site-mutation pattern may also operate in wheat to inhibit the many homologs in the wheat genome,thus providing a promising approach to dissect wheat BR signaling and manipulate BR signaling by genetic modification.