| A short root mutant Osssr-1 (ssbm short root -1) was isolated from an EMS library of a rice variety SSBM. The size of elongation zone of the Osssr-1 is smaller and the maturation zone cell length of it is 1/3 of wild type (WT). Cell cycle marker-CYCB1,1:GUS showed less signal in Osssr-1.OsSSR-1 was mapped on short arm of chromosome 4, encoding anβ-1,4-endoglucanase (OsGLU3). The OsGLU3 was predicted to hydrolyze (3-1,4-glycosidic bond in CAZY (http://www.cazy.org/GH9_eukaryota.html). Sequence analysis revealed that a T to A point mutation was occurred at 2948bp.The point mutation leads to a Leu451 to His451 transition in the predict protein. The introduction of OsGLU3 genomic fragment into Osssr-1 fully complemented the mutant's defect, which indicates that OsGLU3 is responsible for the root elongation. A short root phenotype was also found in a OsGLU3 T-DNA insertion mutant Osglu3-2, which further corroborates that the short root defect of Osssr-1 is due to the mutation in OsGLU3.To examine the expression pattern of OsGLU3,αproOsGLU3::OsGLU3-GUS fusion construct was transferred into the Osglu3 mutant, which could completely rescue the mutant phenotype. The fusion protein presented in various organs including root tip, lateral root, leaf vein, stem-base and glume. Strong expression of OsGLU3-GUS was detected in the elongation zone and maturation zone of the root system, and in the root cap of adventitious root primordia.To determine the subcellular localization of OsGLU3, a fully functional OsGLU3-GFP fusion was also introducted into the Osglu3 mutant. The fusion protein OsGLU3-GFP distributed in both the cytoplasm compartment and plasma membrane in the cells of the root meristem and elongation zone and exclusively localized in plasma membrane in the cells of the root maturation zone.When treated by flucorescent styryl dye FM4-64 for 30 min, which used tracer for the clathin-dependented endocytic pathway, OsGLU3-GFP partly co-localized with FM4-64-labeled compartments, suggesting that OsGLU3 involved in an endocytic pathway. Added BFA after treated by FM4-64 for 30 min, OsGLU3-GFP completely co-localized with FM4-64-labeled compartments suggested that OsGLU3 involved in vesicle traffick.Osglu3 is also temperature sensitive. The Osssr-1 mutant displays a distinct defect in root elongation under 34℃, while it is almost normal under 26℃. But T-DNA insert mutant Osglu3-2 did not show temperature sensitivity. It suggested that the point mution in OsGLU3 resulted in the temperature sensitivity of mutated protein.The cellulose of root cell wall in Osglu3 was less than in wild type. However, there was no obvionsly differention in glucose concentration between mutant and wild type.The root elongation of Osglu3 under Pi starved was longer than under control Pi level, but it did not occur in Osglu3-2. It suggested that OsGLU3 had a role in the pathway of the root elongated in Pi starve.The mutants phenotype were recovered by 3% exogenous glucose, cellose, sucrose, fructose. The phenotype of T-DNA insert mutant Osglu3-2 were recovered partly by 3% exogenous glucose. So other genes involve in this process.In summary, the research revealed that OsGLU3 had a important role in sythesis of cellulose. Function loss of OsGLU3 leads to a defect in root cell elongation and cell division, consequently root elongation defect in rice.
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