Functional Analysis of Rice Target of Rapamycin OsTOR in Regulating Root Elongation

To clone the target of rapamycin gene OsTOR in rice, and investigate its regulatory mechanism in root elongation, elucidate its biological function in rice development, and provide a molecular basis for improving rice traits and increasing its yield.

In this research, the japonica rice variety Dongjin was used as material for OsTOR expression pattern analysis via qRT- PCR. OsTOR was cloned by RT-PCR, and the overexpression vector 35S:OsTOR-GFP was constructed. The overexpression lines OsTOR-OX were generated by Agrobacterium-mediated rice genetic transformation to examine the subcellular localization of OsTOR. Rice seedlings were treated with TOR inhibitor AZD-8055, and the phenotype was observed. The root tips of inhibitor-treated and OsTOR-OX plants were cleared and the cell morphology in the meristematic zone was then visualized by CLSM. Transcriptome sequencing was performed on 7-day-after-germination (DAG) seedlings with or without AZD-8055 treatment. The expression of cell cycle related genes was further analyzed by qRT-PCR.

OsTOR was evolutionally closely related to barley and wheat TORs. It contains HEAT repeat domain, FAT, FRB, PIKKc kinase domain and FATc domain. OsTOR was localized in cytoplasm and cell membrane, and expressed in various tissues: leaves, leaf sheaths and roots of 7 DAG seedlings, as well as flag leaf blades, flag leaf sheaths, inflorescences, mature anthers, and unpollinated pistil of mature plant. AZD-8055 inhibited seedling growth and root elongation, accompanied by reduced cell proliferation in the root meristem. In contrast, OsTOR overexpression promoted root elongation. Transcriptome analysis revealed 225 genes were upregulated and 121 genes were downregulated upon AZD-8055 treatment. Among the upregulated genes, approximately 28% were associated with cell process, 18.6% with response to stimulus, and some others were involved in degradation. Downregulated genes were mainly involved in biosynthetic processes, protein metabolism, and cell cycle regulation. qRT-PCR results further confirmed that reduced OsTOR activity led to decreased expression of cell cycle-related genes.

OsTOR was localized in cytoplasm and cell membrane, and was ubiquitously expressed in various tissues at different developmental stages of rice. Reduced OsTOR activity restrained seedling growth and root elongation, whereas OsTOR overexpression promoted root elongation. This effect was primarily attributed to altered expression of cell cycle-related genes, which subsequently affected cell proliferation in root meristem.