| Target of rapamycin(TOR),a Ser/Thr protein kinase,has emerged as a central regulator in cell proliferation and growth as well as metabolism,and modulates downstream transcription,translation,autophagy and metabolism process by integrating nutrient,energy,and growth factors in all eukaryotic species.Though,TOR acts as a central coordinator of nutrient,energy,and stress signaling networks and plays an evolutionarily conserved role in anabolic and catabolic processes in eukaryotes.But little is known about the functions of TOR in photosynthesis and phytohormone signaling,which are unique features of plants in comparing to heterotrophic organisms from yeast to humans.In this study,combining genetics with and biochemical approaches as well as RNA-Seq analyses,we exploited molecular functions and dynamic regulation of TOR kinase in the transition from heterotrophic to photoautotrophic growth and photosynthetic growth in Arabidopsis.Finally,we got some new findings and presented some new insights in plant TOR signaling pathway.(1)In this study,we firstly screened different drugs which target to active-site of TOR(asTORis)for the physiological assays and compared the inhibitory effects on plants growth between these asTORis like the AZD8055,Torin1 and KU0063794.Based on the observation from these assays,we found that these asTORis not only could repress efficiently the growth of plants in a dose-dependent manner,but also caused deficient in photoautotrophic creation and failure to photoautotrophic growth at a high concentration of AZD8055 or Torin1 treatments.In addition,repression of TOR by AZD8055 also accelerated leaves senescence and reduced RNA levels in Arabidopsis seedlings.These results indicate that TOR signaling plays an important role in regulation of the transition from heterotrophic to photoautotrophic growth and photosynthetic growth in Arabidopsis.(2)Gene expression profiles were evaluated using RNA-Seq after treating Arabidopsis seedlings with AZD8055.Between the AZD8055 and DMSO control treatments,2780 DEGs were found out and most of them were assigned to one or more of three categories base on the GO assignments.Further categorization resulted in the identification of 404 different GO terms and their enrichment(corrected P < 0.05).A total of 96 KEGG pathways were detected and 19 were enriched.Most of these GO terms and KEEGs involved in ribosome biogenesis and amino acid,sugar,lipid metabolism process,suggesting that TOR conserved function in regulation od plants growth,and also reflect the high efficiency and specificity of AZD8055 in targeting to TOR in plants.(3)Importantly,the suppression of TOR by AZD8055 resulted in remodeling of the expression profile of the photosynthesis,and most of these DEGs enriched in chloroplast related GO terns and photosynthesis pathway.In addition,expression profile of various phytohormones(Auxin,CK,GA,Ethylene,BR,JA and SA)pathway were also reprogrammed.This data indicate that TOR plays a crucial role in modulating photosynthesis and phytohormone signaling in Arabidopsis.(4)Using engineered rapamycin-sensitive BP12-2 plants,the present study show that combined treatment with rapamycin and active-site TOR inhibitors(asTORis)results in additive or synergistic inhibition of TOR activity and plant growth in Arabidopsis.Combination index(CI)simulations displays that RAP + asTORis have a very low CI value(CI < 1)suggesting strong synergistic effects generated by RAP + asTORis on BP12-2 plants growth.These results may offer an efficient and economic method for TOR signaling studies,and may contribute to advancing knowledge and potential applications in other systems.(5)Additionally,RAP + asTORis could efficiently arrest the growth of BP12-2 plants and lead to that of growth arresting phenotype at their IC50 concentration.The growth arresting seedlings failed to photoautotrophic creation and growth.However,the etiolated seedlings could restore growth and differentiate into green true leaves and roots system after released from TOR inhibition implying that TOR may regulate cell differentiation in the process of the transition from heterotrophic to photoautotrophic growth.Intriguing,TEM observation showed a quantity of amyloplast and starch grain instead of chloroplast,or the proplastid and etioplast in the cotyledon of etiolated seedlings due to TOR severe inhibition.Similar results of starch grain accumulation companying with chloroplast degeneration phenotype were observed in chloroplast in the true leaves of seedlings after treated with TOR inhibitors.In addition,qRT-PCR result showed that the mark gens of photosynthesis and chlorophyll synthesis displayed obviously down-regulated when TOR inhibition.These results indicate that TOR may modulate chloroplast biogenesis or starch metabolism in the process of the transition from heterotrophic to photoautotrophic growth.(6)Based on high selectivity system of BP12-2,S6K2(Ribosomal protein S6 kinase 2)was identified as a direct downstream target of TOR,and the growth of TOR-suppressed plants could be rescued by up-regulating expression of S6K2 rather than S6K1.Western blot showed that the signals of At S6K2 phosphorylation were significantly reduced in response to various treatments with TOR inhibitors or their combination.These results suggest that TOR regulates photoautotrophic growth through the TOR-S6K2 signaling.(7)Systems,genetics and biochemical analyses including Y2 H scanning and CO-IP assays revealed that BIN2(Brassinosteriod Insensitive 2)as a novel downstream effector of S6K2 could be directly phosphorylated by S6K2,and the phosphorylation of BIN2 depends on TOR-S6K2 signaling in Arabidopsis.Genetic assays revealed BIN2 as the negative effector in photoautotrophic growth.These results suggest that TOR regulates the photoautotrophic establishment and growth partially depending on downstream S6K2-BIN2 signaling in Arabidopsis.In conclusion,we presented a TOR-S6K2-BIN2 signaling pathway which plays an important roles in regulating the photoautotrophic growth in Arabidopsis. |
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