Functional Analysis Of OsEXPB2in Rice(Oryza Sativa)

Root system is essential for plants to adapt to hostile terrestrial environment inevolutionary process. The appearance of root system is a symbol of plant evolution.Roots play an important role in communication with environment, including absorptionof water and nutrient, anchorage to the soil and establishment of biotic interactions inrhizosphere. Although the morphology and structure of root looks plain, the molecularmechanism is a complicated biological progress with various factors involved. Recently,many genes had been identified which involved in plant root growth and development,among which are typical expansins. Expansins are cell wall-loosening proteins that playvital roles during plant growth. They play a distinctive role in a series of cell wallmodifications. To deeply elucidate the functions of expansins could provide newinsights into it. Here, we cloned a rice expansin gene, OsEXPB2(Accession No:AK061068) and characterized its biological functions. Furthermore, we confirmed itsinteracting proteins and signal transduction pathway in our research. The results are asfollows:Bioinformatics analysis of OsEXPB2indicated that it’s1259bp in length,encoding261amino acids. It predominantly consists of β-strands and coils. Theanalysis of OsEXPB2protein showed that it was a hydrophilic protein and contained aninside-out transmembrane structure. Phylogenetic analysis and multiple sequencealignment of OsEXPB2demonstrated that it possessed several conserved structuredomains and fell into a branch with OsEXPB3, TaEXPB23, TaEXPB11and ZmEXPB1.Subcellular localization of OsEXPB2gene revealed that it’s a secretory protein andlocated in extracellular with a high score. Functional analysis of OsEXPB2displayedthat it may participate in the process of protein transport and binding.Functional analysis of OsEXPB2demonstrated that it played a vital role inregulating root hair initiation and development. Cryo-SEM results showed that thesurface of RNAi roots was fairly smooth, indicating that OsEXPB2was associated withroot hair initiation and development. The anatomy of root structure indicated that thedevelopment of sclerenchyma was impaired. The morphology of the cells had changed.Therefore, the formation of root hairs was severe influenced. Additionally, acomparison between wild-types and RNAi lines revealed that there were significantdifferences in size and arrangement mode of endodermis cells. The results of Fourier transform infrared spectroscopy showed that there were no significant changes in cellwalls in RNAi lines.Transcriptional regulation of OsEXPB2indicated that the accumulation of mRNAwas influenced by many factors. Analysis of OsEXPB2promoter demonstrated that itpossessed many basic cis-elements, including CAAT box and TATA box, and amountof phytohormone response elements (such as AuxRR, GARE and ABRE), tissuespecific elements (RHERPATEXPA7, ROOTMOTIFTAPOX1, OSE1ROOTNODULEand OSE2ROOTNODULE) and abiotic stress response elements (DRE1, LTRE andMBS). Previous studies have demonstrated that RHEs are key regulators, which areimportant for those expansin genes. The spatial and temporal expression patterns ofOsEXPB2gene indicated that there were significant differences in different growthperiods and tissues. OsEXPB2specifically expressed in roots, especially in seedling root,and scarcely expressed in heading leaf blades, inflorescence, ripening leaf blades, stemand spike. When treated with phosphorus deficiency or water stress, OsEXPB2expression was increased. However, when treated with exogenous phytohormones, suchas ABA, GA3and IAA, OsEXPB2expression can be suppressed.Yeast two-hybrid and BiFC confirmed that OsEXPB2interacted with OsGPB-LR.OsGPB-LR is a guanine nucleotide-binding protein beta subunit-like protein, which ismade up of seven repetitive segments of Trp-Asp40-aa repeats. It’s reported to beinvolved in protein phosphorylation in ABA signaling pathway and drought stress.In conclusion, OsEXPB2is a typical cell wall-loosening protein that playsimportant roles in root hair initiation and development. In the present study, we usedtransgenic technology to study the functions of OsEXPB2and the molecular mechanism.Our study helps better understand the molecular regulation of plant root developmentand provides impetus for efforts to develop novel strategies that improve rice breeding.