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Tomato fruit ripening is a unique process of nutritional and energy metabolism. Target of rapamycin (TOR), a conserved serine/threonine protein kinase in eukaryotes, controls cell growth and metabolism by integrating nutrient, energy, and hormone signals. However, it remains unclear whether TOR participates in the modulation of tomato fruit ripening. Here, we showed that the manipulation of SlTOR by chemical or genetic methods greatly alters the process of tomato fruit maturation. Expression pattern analysis revealed that the transcripts of SlTOR declined as fruit ripening progressed. Moreover, suppression of SlTOR by TOR inhibitor AZD8055 or knock down of its transcripts by inducible RNA interference, accelerated fruit ripening, and led to overall effects on fruit maturity, including changes in colour and metabolism, fruit softening, and expression of ripening-related genes. Genome-wide transcription analysis indicated that silencing SlTOR reprogrammed the transcript profile associated with ripening, including cell wall and phytohormone pathways, elevated the expression of ethylene biosynthetic genes, and further promoted ethylene production. In contrast, the ethylene action inhibitor 1-MCP efficiently blocked fruit maturation, even following SlTOR inhibition. These results suggest that accelerated fruit ripening caused by SlTOR inhibition depends on ethylene, and that SlTOR may function as a regulator in ethylene metabolism.

Target of rapamycin (TOR) operates as a hub of the signal transduction that integrates nutrient and energy signaling to promote cell proliferation and growth through mediating the transcriptional and post- transcriptional regulator networks in all eukaryotic species. MicroRNAs (miRNAs) are widespread classes of small, single-stranded, non-coding endogenous RNAs and are widely found in eukaryotes, which play a vital role in regulating gene expression by degrading targeted mRNAs or translational repression at post-transcriptional level. Recent studies found that there were necessarily close connections between miRNA and TOR pathways in mammals. However, there is little information about the interplay between the miRNA and TOR in plants. Thus, the aim of this study was to identify potential TOR-miRNA-mRNA regulatory networks in TOR signaling through global mRNA and microRNA expression profiling in potato. Based on the previous high-throughput transcriptome sequencing and filtering, a total of 2,899 genes were significantly differentially expressed in potato under TOR inhibitors treatment. Pathway analysis revealed that these genes were significantly enriched in multiple metabolic processes. Similarly, in the present study, suppression of TOR resulted in 41 miRNAs up-regulated and 45 down-regulated, revealing that TOR plays a crucial role in the regulation of miRNA regulatory network. Furthermore, integrated mRNA and miRNA expression profiling uncovered that these miRNAs participated in large-scale metabolic process in the TOR signal pathway in potato, such as regulation of autophagy and ubiquitination, and biosynthesis of secondary metabolites. Overall, the results shed new insight into TOR related post-transcriptional gene regulatory networks in potato and suggesting TOR-miRNA-targeting genes relevant networks as a potential genetic resource for potato improvement.

Potassium (K) is an essential inorganic nutrient needed by plants for their growth and development. The conserved target of rapamycin (TOR) kinase, a well-known nutrition signaling integrator, has crucial roles in regulating growth and development in all eukaryotes. Emerging evidence suggests that TOR is a core regulator of nutrient absorption and utilization in plants. However, it is still unclear whether there is a causative link between the TOR pathway and potassium absorption. Here, we show that the expression of some potassium transporters and channels was regulated by TOR, and the suppression of TOR activity significantly affected potassium uptake in Arabidopsis and potato. Furthermore, we discovered that a Type 2A phosphatase-associated protein of 46 kDa (TAP46), a direct TOR downstream effector, could interact with CBL-interacting protein kinase 23 (CIPK23) in Arabidopsis and potato. In Arabidopsis, the K+ channel AKT1 conducting K+ uptake was significantly regulated by Calcineurin B-like Calcium Sensor Protein 1/9 (CBL1/9)-CIPK23 modules. We found that the cbl1cbl9, cipk23 (lks1-2 and lks1-3), and akt1 mutants were more hyposensitive to the TOR inhibitor than the wild-type, and the TOR inhibitor induced the downregulation of K+ uptake rate in the wild-type more than in these mutants. In addition, the overexpression of CIPK23 could effectively restore the defects in growth and potassium uptake induced by the TOR inhibitors. Thus, our work reveals a link between TOR signaling and CIPK23 and provides new insight into the regulation of potassium uptake in plants.

Target of rapamycin (TOR) acts as a master regulator in coordination of cell growth with energy and nutrient availability. Despite the increased appreciation of the essential role of the TOR complex in interaction with phytohormone signaling, little is known about its function on ethylene signaling. Here, through expression analysis, genetic and biochemical approaches, we reveal that TOR functions in the regulation of ethylene signals. Transcriptional analysis indicates that TOR inhibition by AZD8055 upregulated senescence- and ethylene-related genes expression. Furthermore, ethylene insensitive mutants like etr1-1, ein2-5 and ein3 eil1, showed more hyposensitivity to AZD8055 than that of WT in hypocotyl growth inhibition. Similarly, blocking ethylene signals by ethylene action inhibitor Ag+ or biosynthesis inhibitor aminoethoxyvinylglycine (AVG) largely rescued hypocotyl growth even in presence of AZD8055. In addition, we also demonstrated that Type 2A phosphatase-associated protein of 46 kDa (TAP46), a downstream component of TOR signaling, physically interacts with 1-aminocy-clopropane-1-carboxylate (ACC) synthase ACS2 and ACS6. Arabidopsis overexpressing ACS2 or ACS6 showed more hypersensitivity to AZD8055 than WT in hypocotyl growth inhibition. Moreover, ACS2/ACS6 protein was accumulated under TOR suppression, implying TOR modulates ACC synthase protein levels. Taken together, our results indicate that TOR participates in negatively modulating ethylene signals and the molecular mechanism is likely involved in the regulation of ethylene biosynthesis by affecting ACSs in transcription and protein levels.

Target of rapamycin (TOR) acts as a master regulator in coordination of cell growth with energy and nutrient availability. Despite the increased appreciation of the essential role of the TOR complex in interaction with phytohormone signaling, little is known about its function on ethylene signaling. Here, through expression analysis, genetic and biochemical approaches, we reveal that TOR functions in the regulation of ethylene signals. Transcriptional analysis indicates that TOR inhibition by AZD8055 upregulated senescence- and ethylene-related genes expression. Furthermore, ethylene insensitive mutants like etr1-1, ein2-5 and ein3 eil1, showed more hyposensitivity to AZD8055 than that of WT in hypocotyl growth inhibition. Similarly, blocking ethylene signals by ethylene action inhibitor Ag+ or biosynthesis inhibitor aminoethoxyvinylglycine (AVG) largely rescued hypocotyl growth even in presence of AZD8055. In addition, we also demonstrated that Type 2A phosphatase-associated protein of 46 kDa (TAP46), a downstream component of TOR signaling, physically interacts with 1-aminocy-clopropane-1-carboxylate (ACC) synthase ACS2 and ACS6. Arabidopsis overexpressing ACS2 or ACS6 showed more hypersensitivity to AZD8055 than WT in hypocotyl growth inhibition. Moreover, ACS2/ACS6 protein was accumulated under TOR suppression, implying TOR modulates ACC synthase protein levels. Taken together, our results indicate that TOR participates in negatively modulating ethylene signals and the molecular mechanism is likely involved in the regulation of ethylene biosynthesis by affecting ACSs in transcription and protein levels.

Brassinosteroid (BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3s are the main negative regulator kinases. BRs have been shown to play vital roles in cotton fiber elongation. However, the underlying mechanism is still elusive. In this study, fibers of a BR-defective mutant Pagoda 1 (pag1), and its corresponding wild-type (ZM24) were selected for a comparative global phosphoproteome analysis at critical developmental time points: fast-growing stage (10 days after pollination (DPA)) and secondary cell wall synthesis stage (20 DPA). Based on the substrate characteristics of GSK3, 900 potential substrates were identified. Their GO and KEGG annotation results suggest that BR functions in fiber development by regulating GhSKs (GSK3s of Gossypium hirsutum L.) involved microtubule cytoskeleton organization, and pathways of glucose, sucrose and lipid metabolism. Further experimental results revealed that among the GhSK members identified, GhSK13 not only plays a role in BR signaling pathway, but also functions in developing fiber by respectively interacting with an AP2-like ethylene-responsive factor GhAP2L, a nuclear transcription factor Gh_DNF_YB19, and a homeodomain zipper member GhHDZ5. Overall, our phosphoproteomic research advances the understanding of fiber development controlled by BR signal pathways especially through GhSKs, and also offers numbers of target proteins for improving cotton fiber quality.

Summary Botrytis cinerea is the causal agent of grey mould for more than 200 plant species, including economically important vegetables, fruits and crops, which leads to economic losses worldwide. Target of rapamycin (TOR) acts a master regulator to control cell growth and proliferation by integrating nutrient, energy and growth factors in eukaryotic species, but little is known about whether TOR can function as a practicable target in the control of plant fungal pathogens. Here, we characterize TOR signalling of B. cinerea in the regulation of growth and pathogenicity as well as its potential value in genetic engineering for crop protection by bioinformatics analysis, pharmacological assays, biochemistry and genetics approaches. The results show that conserved TOR signalling occurs, and a functional FK506‐binding protein 12 kD (FKBP12) mediates the interaction between rapamycin and B. cinerea TOR (BcTOR). RNA sequencing (RNA‐Seq) analysis revealed that BcTOR displayed conserved functions, particularly in controlling growth and metabolism. Furthermore, pathogenicity assay showed that BcTOR inhibition efficiently reduces the infection of B. cinerea in plant leaves of Arabidopsis and potato or tomato fruits. Additionally, transgenic plants expressing double‐stranded RNA of BcTOR through the host‐induced gene silencing method could produce abundant small RNAs targeting BcTOR, and significantly block the occurrence of grey mould in potato and tomato. Taken together, our results suggest that BcTOR is an efficient target for genetic engineering in control of grey mould, and also a potential and promising target applied in the biocontrol of plant fungal pathogens.

Target of rapamycin (TOR) operates as a hub of the signal transduction that integrates nutrient and energy signaling to promote cell proliferation and growth through mediating the transcriptional and post- transcriptional regulator networks in all eukaryotic species. MicroRNAs (miRNAs) are widespread classes of small, single-stranded, non-coding endogenous RNAs and are widely found in eukaryotes, which play a vital role in regulating gene expression by degrading targeted mRNAs or translational repression at post-transcriptional level. Recent studies found that there were necessarily close connections between miRNA and TOR pathways in mammals. However, there is little information about the interplay between the miRNA and TOR in plants. Thus, the aim of this study was to identify potential TOR-miRNA-mRNA regulatory networks in TOR signaling through global mRNA and microRNA expression profiling in potato. Based on the previous high-throughput transcriptome sequencing and filtering, a total of 2,899 genes were significantly differentially expressed in potato under TOR inhibitors treatment. Pathway analysis revealed that these genes were significantly enriched in multiple metabolic processes. Similarly, in the present study, suppression of TOR resulted in 41 miRNAs up-regulated and 45 down-regulated, revealing that TOR plays a crucial role in the regulation of miRNA regulatory network. Furthermore, integrated mRNA and miRNA expression profiling uncovered that these miRNAs participated in large-scale metabolic process in the TOR signal pathway in potato, such as regulation of autophagy and ubiquitination, and biosynthesis of secondary metabolites. Overall, the results shed new insight into TOR related post-transcriptional gene regulatory networks in potato and suggesting TOR-miRNA-targeting genes relevant networks as a potential genetic resource for potato improvement.

•The components of the target of rapamycin (TOR) signaling pathway have been well characterized in heterotrophic organisms from yeast to humans. However, because of rapamycin insensitivity, embryonic lethality in tor null mutants and a lack of reliable ways of detecting TOR protein kinase in higher plants, the key players upstream and downstream of TOR remain largely unknown in plants. •Using engineered rapamycin-sensitive Binding Protein 12-2 (BP12-2) plants, the present study showed that combined treatment with rapamycin and active-site TOR inhibitors (asTORis) results in synergistic inhibition of TOR activity and plant growth in Arabidopsis. •Based on this system, we revealed that TOR signaling plays a crucial role in modulating the transition from heterotrophic to photoautotrophic growth in Arabidopsis. Ribosomal protein S6 kinase 2 (S6K2) was identified as a direct downstream target of TOR, and the growth of TOR-suppressed plants could be rescued by up-regulating S6K2. Systems, genetic, and biochemical analyses revealed that Brassinosteriod Insensitive 2 (BIN2) acts as a novel downstream effector of S6K2, and the phosphorylation of BIN2 depends on TOR-S6K2 signaling in Arabidopsis. •By combining pharmacological with genetic and biochemical approaches, we determined that the TOR-S6K2-BIN2 signaling pathway plays important roles in regulating the photoautotrophic growth of Arabidopsis.

In the agriculture industry, adventitious root formation is a core issue of plants asexual propagation. However, the underlying molecular mechanism of adventitious root formation is far beyond understanding. In present study we found that target of rapamycin (TOR) signaling plays a key role in adventitious root formation in potato and Arabidopsis. The core components of TOR complex including TOR, RAPTOR, and LST8 are highly conserved in potato, but the seedlings of potato are insensitive to rapamycin, implying FK506 Binding Protein 12 KD (FKBP12) lost the function to bridge the interaction of rapamycin and TOR in potato. To dissect TOR signaling in potato, the rapamycin hypersensitive potato plants (BP12-OE) were engineered by introducing yeast FKBP12 (ScFKBP12) into potato. We found that rapamycin can significantly attenuate the capability of adventitious root formation in BP12-OE potatoes. KU63794 (KU, an active-site TOR inhibitor) combined with rapamycin can more significantly suppress adventitious root formation of BP12-OE potato than the single treatments, such as KU63794 or rapamycin, indicating its synergistic inhibitory effects on potato adventitious root formation. Furthermore, RNA-seq data showed that many genes associated with auxin signaling pathway were altered when BP12-OE potato seedlings were treated with rapamycin + KU, suggesting that TOR may play a major role in adventitious root formation via auxin signaling. The auxin receptor mutant tir1 was sensitive to TOR inhibitors and the double and quadruple mutants including tir1afb2, tir1afb3, and tir1afb1afb2afb3 displayed more sensitive to asTORis than single mutant tir1. Consistently, overexpression of AtTIR1 in Arabidopsis and potato can partially overcome the inhibitory effect of asTORis and promote adventitious root formation under asTORis treatments. These observations suggest that TOR signaling regulates adventitious root formation by mediating auxin signaling in Arabidopsis and potato.

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 via FK506 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.