Target of rapamycin (TOR) acts as a master regulator to control cell growth by integrating nutrient, energy, and growth factors in all eukaryotic species. TOR plays an evolutionarily conserved role in regulating the transcription of genes associated with anabolic and catabolic processes in Arabidopsis, but little is known about the functions of TOR in photosynthesis and phytohormone signaling, which are unique features of plants. In this study, AZD8055 (AZD) was screened as the strongest active-site TOR inhibitor (asTORi) in Arabidopsis compared with TORIN1 and KU63794 (KU). Gene expression profiles were evaluated using RNA-seq after treating Arabidopsis seedlings with AZD. More than three-fold differentially expressed genes (DEGs) were identified in AZD-treated plants relative to rapamycin-treated plants in previous studies. Most of the DEGs and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways involved in cell wall elongation, ribosome biogenesis, and cell autophagy were common to both AZD- and rapamycin-treated samples, but AZD displayed much broader and more efficient inhibition of TOR compared with rapamycin. Importantly, the suppression of TOR by AZD resulted in remodeling of the expression profile of the genes associated with photosynthesis and various phytohormones, indicating that TOR plays a crucial role in modulating photosynthesis and phytohormone signaling in Arabidopsis. These newly identified DEGs expand the understanding of TOR signaling in plants. This study elucidates the novel functions of TOR in photosynthesis and phytohormone signaling and provides a platform to study the downstream targets of TOR in Arabidopsis.
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Target of rapamycin (TOR), a master sensor for growth factors and nutrition availability in eukaryotic species, is a specific target protein of rapamycin. Rapamycin inhibits TOR kinase activity viaFK506 binding protein 12 kDa (FKBP12) in all examined heterotrophic eukaryotic organisms. In Arabidopsis, several independent studies have shown that AtFKBP12 is non-functional under aerobic condition, but one study suggests that AtFKBP12 is functional during anaerobic growth. However, the functions of AtFKBP12 have never been examined in parallel under aerobic and anaerobic growth conditions so far. To this end, we cloned the FKBP12 gene of humans, yeast, and Arabidopsis, respectively. Transgenic plants were generated, and pharmacological examinations were performed in parallel with Arabidopsis under aerobic and anaerobic conditions. ScFKBP12 conferred plants with the strongest sensitivity to rapamycin, followed by HsFKBP12, whereas AtFKBP12 failed to generate rapamycin sensitivity under aerobic condition. Upon submergence, yeast and human FKBP12 can significantly block cotyledon greening while Arabidopsis FKBP12 only retards plant growth in the presence of rapamycin, suggesting that hypoxia stress could partially restore the functions of AtFKBP12 to bridge the interaction between rapamycin and TOR. To further determine if communication between TOR and auxin signaling exists in plants, yeast FKBP12 was introduced into DR5::GUS homozygous plants. The transgenic plants DR5/BP12 were then treated with rapamycin or KU63794 (a new inhibitor of TOR). GUS staining showed that the auxin content of root tips decreased compared to the control. DR5/BP12 plants lost sensitivity to auxin after treatment with rapamycin. Auxin-defective phenotypes, including short primary roots, fewer lateral roots, and loss of gravitropism, occurred in DR5/BP12 plants when seedlings were treated with rapamycin+KU63794. This indicated that the combination of rapamycin and KU63794 can significantly inhibit TOR and auxin signaling in DR5/BP12 plants. These studies demonstrate that TOR is essential for auxin signaling transduction in Arabidopsis.
Target of Rapamycin (TOR) is an eukaryotic protein kinase and evolutionally conserved from the last eukaryotic common ancestor (LECA) to humans. The growing evidences have shown that TOR signaling acts as a central controller of cell growth and development. The downstream effectors of TOR have been well-identified in yeast and animals by using the immunosuppression agent rapamycin. However, less is known about TOR in plants. This is largely due to the fact that plants are insensitive to rapamycin. In this study, AZD8055 (AZD), the novel ATP-competitive inhibitor of TOR, was employed to decipher the downstream effectors of TOR in Arabidopsis. One AZD insensitive mutant, TOR-inhibitor insensitive-1 (trin1), was screened from 10,000 EMS-induced mutation seeds. The cotyledons of trin1 can turn green when its seeds were germinated on ½ MS medium supplemented with 2 μM AZD, whereas the cotyledons greening of wild-type (WT) can be completely blocked at this concentration. Through genetic mapping, TRIN1 was mapped onto the long arm of chromosome 2, between markers SGCSNP26 and MI277. Positional cloning revealed that TRIN1 was an allele of ABI4, which encoded an ABA-regulated AP2 domain transcription factor. Plants containing P35S::TRIN1 or P35S::TRIN1-GUS were hypersensitive to AZD treatment and displayed the opposite phenotype observed in trin1. Importantly, GUS signaling was significantly enhanced in P35S::TRIN1-GUS transgenic plants in response to AZD treatment, indicating that suppression of TOR resulted in the accumulation of TRIN1. These observations revealed that TOR controlled seed-to-seedling transition by negatively regulating the stability of TRIN1 in Arabidopsis. For the first time, TRIN1, the downstream effector of TOR signaling, was identified through a chemical genetics approach.
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