Tomato (Solanum lycopersicum, cv. Micro Tom) seeds were sterilized with 70% ethanol for 2 min, followed by 10% sodium hypochlorite for 10 min, and then washed 5–6 times with distilled water. The surface-sterilized seeds were germinated on square Petri dishes (100 × 100 × 17 mm) with half-strength Murashige–Skoog (0.5 × MS) agar supplemented with different inhibitors (rapamycin, AZD8055, TORIN1, and KU63794, selleckchem) at increasing concentrations (0, 1, 5, and 10 μM). The seedlings were photographed and primary root length and fresh weight were measured at 7 days after germination (DAG), respectively. In addition, normal seedlings of 5DAG were transferred to 0.5 × MS medium on Petri dishes with 10 μM different inhibitors (rapamycin, AZD8055, TORIN1, and KU63794) for 5 more days' growth. Then, the fresh weight and root length were measured. All plants were grown in growth chambers at 25°C and a photoperiod of 16 h (100-mmol/m²/s fluorescence bulb light) followed by 8 h of dark unless indicated otherwise.

Seven days of tomato seedlings were transplanted to the 0.5 × MS medium with 10 μM rapamycin, 10 μM KU63794, and 0.1% DMSO as control and incubated for 24 h, the whole plant including shoot and root were harvested and ground in liquid nitrogen. Three independent biological replicates for each treatment (DMSO, rapamycin or KU63794) were produced. Three individual plants were chosen randomly to prepare one pooled RNA sample as one biological repetition. The RNA extraction, quality evaluation, and cDNA library construction were done as described in previous report (Dong et al., 2015). The libraries were then sequenced using the Illumina HiSeqTM 2000. Genes were defined as differentially expressed if they exhibited a two-fold or greater change between the treatment and control with a false discovery rate (FDR) of 5% or less. All the differentially expressed genes (DEGs) were annotated on the NCBI NR database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The resulting InterPro and BLAST annotations were performed into GO annotations and all the GO terms were mapped to the GO categories. The significantly functional GO enrichment was evaluated using the Fisher's exact test within Blast2GO (false discovery rate FDR <0.05; Conesa et al., 2005). Significantly enriched KEGG pathways were identified using a hypergeometric test and Benjamini–Hochberg FDR correction and performed using KOBAS2.0 software (http://kobas.cbi.pku.edu.cn/; Xie et al., 2011; Kanehisa et al., 2015). Comparisons of the DEGs got from rapamycin vs. DMSO (rapamycin) and KU63794 vs. DMSO (KU63794) were conducted based on the online Venny analysis (http://bioinfogp.cnb.csic.es/tools/venny_old/index.html; Oliveros, 2007). The heat map was generated from RNA-seq data with software of HemI (Heatmap Illustrator, version 1.0).

Total RNA of plants was extracted for qRT-PCR and Illumina RNA-seq by using the RNAprep Pure Plant Kit (TianGen Biotech). Total RNA (1 μg) was reverse transcribed by using the PrimeScript RT Reagent Kit (TAKARA). Gene expression was analyzed by quantitative RT-PCR using SYBR® Premix Ex Taq™II (TAKARA) on a Bio-Rad CFX96 Real-time PCR system (Bio-Rad). The primers of the candidate genes and the reference gene Actin (Solyc03g078400) are listed in Table S1. Expression was calculated with the comparative cycle threshold (Ct) method, which involves normalizing against the geometric mean of the housekeeping genes (Actin) for each treatment.

The coding sequences of tomato FKBP12 were amplified by PCR with the primer pairs (Forward: 5′-GCGGCCGCATGGGAGTGGAGAAGGAAGT-3′, Reverse: 5′-CCTGCAGGTTGTGCACCTAGGACTTCAA-3′, NotI and Sbf I sites underlined). PCR products were cloned into the T1-Simple cloning vector (TransGen Biotech) and sequenced. The full length CDS of TOR-like in tomato (XM_004230627.1) was amplified using PrimeSTAR® Max DNA Polymerase Kit (TAKARA) with the primer pairs (Forward: 5′-AACTAGTGCGGCCGCATGGCTGCCACCGTTCAGGCGA-3′, Reverse: 5′-CCCGGGACCTGCAGGCCAAAATGGACACCACCCAAC-3′) which contained 15 bp extensions homologous to entry vector p8GWN ends. And then, the purified PCR product was cloned on p8GWN (linearized by NotI and Sbf I) by Seamless cloning using In-Fusion® HD Cloning Kit (Clontech) following the user manual. The CDS sequence was verified by sequencing. Sequence analysis was conducted by using programs deposited in the NCBI network (https://www.ncbi.nlm.nih/gov). The deduced amino acid sequences were aligned using the CLUSTAL W 1.81 and the phylogenetic tree was constructed with the MEGA software.

Arabidopsis thaliana L. (Columbia ecotype) was used in this study. To generate gateway entry vector constructs, the SlFKBP12 fragment was inserted into the downstream of CaMV 35S promoter and fused HA-tag sequence carrying p8GWN Entry vector via digestion with NotI/Sbf I, the experimental protocols were described in previous report (Ren et al., 2011). Then the expression cassette was introduced pEarleyGate303 (pEG303) by using the LR Clonase II reaction (Life Technologies). The pEG303 is the destination vector of a Gateway system established by previous report (Earley et al., 2006). The resulting destination vectors were used for plant transformation. The floral dipping method was employed for generating transgenic plants (Zhang et al., 2006). The transformation and screening of primary transformants were performed according to Zhang's method (Zhang et al., 2006).

SlFKBP12-expressing lines of T3 generation were grown on the 0.5 × MS plates with differing concentrations of rapamycin (0, 0.5, 1, 5, and 10 μM) at 22°C and 16-h/8-h light/dark cycle. The fresh weight and root length were measured as described in previous report (Xiong et al., 2016). The hypocotyl lengths of SlFKBP12-expressing lines were measured after plants were grown on 0.5 × MS medium supplemented with 10 μM rapamycin in dark for 5 days. ScFKBP12-expressing plants were previously generated by Ren et al. were set as rapamycin sensitive controls (Ren et al., 2012). Root hair was photographed by using the OLYMPUS MVX10 stereoscopic microscopes (Olympus, Japan).

To extract the protein, 100 mg fresh plant sample were collected from wild type Arabidopsis, ScFKBP12,- and SlFKBP12-expressing lines, respectively, then ground in liquid nitrogen and quickly transferred into a 1.5 mL EP tube for further protein extraction. Four hundred microliters of RIPA lysis buffer (including 10 mM Tris, pH 8, 150 mM NaCl, 2% Triton, 1 mM PMSF, protease inhibitor, and 10 mM CaCl2, Sigma) were added to the above EP tubes and incubated together for 1 h at 4°C with constant shaking, and then centrifuged for 5 min at 12,000 rpm at 4°C to remove cell debris. Subsequently, 200 μL supernatant mixed with 40 μL 6 × protein loading buffer were added to the EP tubes, and boiled in boiling water for 5 min. After incubation, the samples were separated by SDS-PAGE, and the SlFKBP12 protein was detected by immunoblotting with antibodies (EarthOx) against HA-Tag. For the SlTOR detection, extracted protein from tomato seedlings was detected by immunoblotting using polyclonal antibody against Arabidopsis TOR(N) (Abiocode) based on high amino acid sequence identity between SlTOR with AtTOR. ECL Western Blotting Detection Reagents (Thermo) were used for detection.

For the protein gel shifting assay, 5DAG seedlings of ScFKBP12- or SlFKBP12-expressing Arabidopsis line were collected after treated with various concentration rapamycin (0, 1, 5, and 10 μM) for 24 h, and then divided into two groups. Then the total proteins were extracted by using the denaturing lysis buffer and native lysis buffer according to the protocols of Minute™Total Protein Extraction Kit for Plant Tissus (Invent Biotech). The extracted proteins after treated with denaturing lysis buffer were mixed with 6 × protein loading buffer and boiled for denaturation, then the proteins were separated in 10% SDS-PAGE and detected by immunoblotting. Another group of extracted proteins with native lysis buffer were mixed with 4 × native protein loading buffer before separated in 10% SDS-PAGE.

For TOR inhibitors combination assays, the surface-sterilized tomato seeds were treated with 10 μM rapamycin, 10 μM KU63794, or the two drugs combination. After 7 days of incubation, the fresh weight of seedlings was measured for evaluating inhibitory effects. As for transgenic Arabidopsis, sterilized seeds from SlFKBP12-expressing line 8, ScFKBP12-expressing line, and wild type were incubated on 0.5 × MS plate supplemented with 5 μM rapamycin, 5 μM KU63794, or their combination. Ten days after germinatioin, the seedlings were photographed and the fresh weight of seedlings was measured to assess the growth inhibition.

Tomato TOR gene (SlTOR) was found through questing for the homologs of Arabidopsis TOR coding region sequence (CDS) against tomato genome (Tomato Genome, 2012; https://solgenomics.net/) and NCBI (http://www.ncbi.nlm.nih.gov/), respectively. The results shows that a single TOR homolog gene (Solyc01g106770) locates on chromosome 1 of tomato (Figure 1A). However, 18 bases pair nucleotide acids of TOR-like are missing in the version of Sol Genomics Network compared with that of NCBI database (GenBank accession no. XM_010316867). To confirm the sequence of CDS of TOR in Micro TOM, the full length CDS of TOR has been amplified and cloned (Figure S1A). The sequencing results show that the CDS of TOR is identical to the version of NCBI rather than Sol Genomics Network. Based on this sequence, we find that full length genome of SlTOR spans about 35.3 kb and contains 57 exons and 56 introns (Figure 1A).

The tomato TOR protein sequence seems to consist of 2470 amino acid residues with a predicted molecular mass of 278 kDa and the western blotting result showed that the protein size of SlTOR is similar to that as of AtTOR (Figure S1B). Alignment of SlTOR with TOR protein sequences from other organisms shows consistent domain organization with a high identification and conservation of the FRB and kinase domains as well as in a C-terminal domain named FATC (Figure 1B), which is conserved in the phosphatidylinositol 3-kinase related protein kinase (Schmelzle and Hall, 2000; Fruman and Rommel, 2014). Highly conserved amino acids are also present throughout the N-term region (Figure 1B), which contains repeat motif sequence named HEAT repeats and have been reported to be in relation to proteins interactions (Schmelzle and Hall, 2000; Hara et al., 2002; Xiong and Sheen, 2014). Phylogenetic analysis shows a closer evolutionary relationship between SlTOR and AtTOR than with the other TORs (Figure 1C).

In yeast or animals, TOR protein participates in forming two structurally and functionally distinct multiprotein complexes, TORC1 and TORC2, and each complex recruits shared and distinct TOR interacting components (Helliwell et al., 1994; Xiong and Sheen, 2014). As Arabidopsis, in tomato, two genes encoding RAPTOR homologous on chromosomes 9 (Solyc09g014780) and 10 (Solyc10g076260), but one gene encoding LST8 (Solyc03g059310) homolog was found, which are the core components of TORC1. By contrast, there is no evidence of a plant TORC2 complex to date due to specific components of this complex, RICTOR, was not found in tomato (Table 1). The results indicate the existence of a conserved and functional TORC1 in tomato.

In order to understand TOR signaling in tomato, asTORis (KU63794, TORIN1, and AZD8055) was applied to treat tomato seedlings, meanwhile, rapamycin also was used to test rapamycin-sensitivity of tomato. It is surprising that the tomato seedling growth was observed obvious growth inhibition reflecting on reducing fresh weight and root length (Figures 2A,B) at 10 μM rapamycin compared to the control indicated that SlFKBP12 may be functional in bridging the interaction between rapamycin and tomato TOR (Figure 2). Similar to wild type Arabidopsis treated with different asTORis (Montané and Menand, 2013; Dong et al., 2015), the dosage-dependent effect of asTORis on fresh weight and primary root length were observed when tomato was treated with the asTORis (KU63794, TORIN1, and AZD8055; Figure 2). In addition, the 5 DAG (days after germination) tomato seedling were transferred to 0.5 × MS medium supplemented with different inhibitors (rapamycin, KU63794, TORIN1, and AZD8055) to further assess inhibitory effects on seedlings growth. After 5 days growing with the inhibitors, the tomato seedling growth was inhibited in all the inhibitors tested (Figure S2). Taken together, these results imply that a functional FKBP12 existing in tomato and a conserved SlTOR function on controlling the growth of tomato seedlings.

The rapamycin response in eukaryotes depends on the formation of the ternary complex of rapamycin-FKBP12-FRB domain of TOR. The conservation of SlTOR with other TORs and the interchangeable FKBP12 in plants drove us to mine information on SlFKBP12 to understand the growth inhibition of tomato caused by rapamycin (Xu et al., 1998; Menand et al., 2002; Crespo et al., 2005; Agredano-Moreno et al., 2007). The amino acid sequence of SlFKBP12 (Solyc01g105710, GenBank accession no. NP_001233825) was downloaded from the Sol genomics network (https://solgenomics.net/) and aligned to various FKBP12s from other eukaryotic organisms (Figure 3A). The identity between the SlFKBP12 protein and its homologs in other organisms ranges from 47 to 90% at the amino acid level. Phylogenetic development analysis showed S. lycopersicum had the closest relationship with Nicotiana sylvestris (Figure 3B). The drug-binding pocket of HsFKBP12 formed by several hydrophobic residues (Phe36, Phe46, Val55, Ile56, Trp59, Tyr82, Ile91, and Phe99) in quarternary structure is conserved in the SlFKBP12 sequence (Van Duyne et al., 1993). The glycine (Gly53) residues, which are involved in the formation of X-sheet, and loop-loop packing (Van Duyne et al., 1991), are also conserved (Figure 3A). These conserved features also suggest that SlFKBP12 proteins maintain a secondary structure with a five-stranded X-sheet, a 40's loop, and an 80's loop that are characteristic of the human FKBP12 (Clardy, 1995).

Early studies showed that the FKBP12 protein from yeast or humans could restore the rapamycin sensitivity in Arabidopsis (Mahfouz et al., 2006; Sormani et al., 2007; Ren et al., 2012). To further confirm the SlFKBP12 function on bridging the interaction between TOR and rapamycin, the coding region of SlFKBP12 was cloned and introduced into Arabidopsis. As shown in Figure 4A, no obvious morphological phenotypes appeared in any of these transgenic lines. However, the transgenic lines with SlFKBP12 displayed response to rapamycin as Arabidopsis expressing ScFKBP12 (Ren et al., 2012) on a solid cultured conditions. Results showed that the inhibitory effect of rapamycin on primary root length and plant fresh weight was most effective in the ScFKBP12 line, followed by SlFKBP12-8, 7, 5, and 6 (Figures 4A–F). At concentrations around 0.5 μM rapamycin, all the transgenic lines displayed smaller cotyledon and leaves reflecting in the decreasing fresh weight compared with WT (Figure 4C). With increasing concentrations of rapamycin, the transgenic lines were increasingly inhibited. In the dark, rapamycin could repress the hypocotyl elongatioin of all the SlFKBP12-expressing Arabidopsis lines significantly, similar to the ScFKBP12-expressing line, while there was no obvious difference observed between the treatment of rapamycin and DMSO in WT (Figure S3A). At 5 days after germination, hypocotyl elongation inhibition was about 50% in all the transgenic lines comparing rapamycin to DMSO (Figure S3B). These results suggest that SlFKBP12 can partly restore sensitivity to rapamycin in Arabidopsis.

To further confirm that SlFKBP12 are able to brigde the interaction between rapamycin and TOR. Protein gel shifting assay was performed to detect the band difference between the free monomer FKBP12 protein and FKBP-RAP-TOR complex in ScFKBP12 and SlFKBP12-expressing plants (SlFKBP12-8) after treated with various concentration of rapamycin (0, 1, 5, and 10 μM). As shown in Figure 5A, with increasing of rapamycin, SlFKBP12 protein level displayed a decline trend in native stage but not denaturing molecular stage (Figure 5A), indicating that there was an increasing FKBP-RAP-TOR complex formation with the increasing rapamycin concentrations. Similar results also were observed in ScFKBP12-expressing plants (Figure 5A). These results suggest that, like ScFKBP12, SlFKBP12 has the functions to bridge the interaction between TOR and RAP in plants. However, the FKBP-RAP-TOR complex was not detected in the gel shifting assay. The possible reason is that the molecular weights of TOR, RAPTOR, and LST8 protein are 280, 150, 35 KD, respectively. The total molecular weight of TOR complex (containing TOR, RAPTOR, LST8, FKBP12, rapamycin, and some unknown proteins) is more than 500 KD. Usually, it is very difficult to transfer a big protein from gel onto membrane if the protein molecular weight is more than 250 KD.

It was reported that rapamycin and asTORis combination could create significant addictive or synergistic inhibitory effects on rapamycin sensitive Arabidopsis plants due to rapamycin and asTORis binding to various target sites of TOR (Deng et al., 2016; Xiong et al., 2016). We therefore examine the effect of rapamycin combined with asTORis on tomato and SlFKBP12-expressing Arabidopsis. As shown in Figure 5B, comparing to rapamycin and KU63794 alone treatment (Figure 2), the combination of these two drugs showed more severe inhibitory effect on tomato seedlings growth after 7 days incubation. The fresh weight reduced to about 20% when treated with rapamycin and KU63794 combination compared with single drug treatment (Figure 5C). These results indicate that the additive or synergistic effects can be created by rapamycin and KU63794 combination in tomato. Similar results also were observed on SlFKBP12-expressing Arabidopsis when treated with rapamycin and KU63794 combination (Figure 5D) and the additive or synergistic inhibitory effects is identical to that ScFKBP12 plants after same treatment (Figures 5D,E). Taken together, our data indicate that SlFKBP12 could bridge the interaction between rapamycin and TOR.

To further seek the function of SlFKBP12 and TOR signaling on tomato seedling growth, whole gene expression profile analysis of tomato seedling growth under TOR inhibition was performed by RNA-Seq. Rapmycin and KU63794 are very specific TOR inhibitors and lack non-specific targets to other kinases in plants (Tables S2, S3). Therefore, rapamycin and KU63794 were selected to treat seedlings for investigating the early molecular landscape in response to TOR suppression. RNA Sequencing for global expression profiling changes were conducted in tomato after the treatment of 24 h 10 μM rapamycin, 10μM KU63794, and corresponding volume of DMSO as control, respectively. As shown in Figure 6B, 1791 DEG's (1108 down-regulated and 683 up-regulated) out of 34,191 were identified in rapamycin treatments (Tables S4A,C), and 1550 DEGs (959 down-regulated and 591 up-regulated) out of 34,723 genes identified from KU63794 (Tables S4B,D). All the DEGs were annotated in the NCBI NR database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the Sol Genomics Network website (https://solgenomics.net/; Tables S4C,D). The two treatments shared 1233 overlapping DEGs and nearly all of the overlapping genes (753 down-regulated and 479 up-regulated) showed the same tendency of up-regulated or down-regulated expression (Figures 6C–E, Table S4E). Some overlapping DEGs were selected randomly from the RNA-Seq data and the same trends were validated by real time-PCR (Figure S4). There were 5 genes in the top 15 down-regulated genes in both the treatment of rapamycin and KU63794, while 7 genes among the top 15 up-regulated genes, were observed simultaneously in both treatments (Table S5). Our data reflects that rapamycin and KU63794 likely have overlap on function as TOR inhibitors.

To further understand the function of these DEGs, GO assignments, KEGG pathways, and their enrichments were analyzed, respectively. Approximately 70.18% DEGs from the rapamycin data and 70.32% DEGs from the KU63794 data were assigned to one or more of three GO categories: cellular component (CC), molecular function (MF), and biological process (BP). A total of 282 and 281 GO terms were enriched in tomato under the TOR inhibition with rapamycin and KU63794, respectively (Tables S6A,B). Of the GO category biological process, the top 20 enriched GO terms are showed on Figures 7A,B. With respects to Gene Ontology, there were 15, 19, and 17 GO terms overlapped in the BP, CC, and MF categories, respectively, for both treatments (Figures 7C,D). In addition, 19 out of 183 KEGG pathways were enriched in the rapamycin data, and 18 out of 184 KEGG pathways were enriched in the KU63794 data (Tables S7A,B). High overlapping of enrichments in GO assignments and KEGG pathways suggest that rapamycin and KU63794 share a similar molecular mechanism in repressing TOR activity in tomato.

The overlapping enriched GOs and KEGG pathways include many TOR conserved functions identified in Arabidopsis, such as cell wall biogenesis, photosynthesis, transporters. In this study, total of 59 DEGs involved in photosynthesis were detected under the treatment with either rapamycin or KU63794 (Table S8). The “photosynthesis (light reaction)” KEGG pathway was one of the most enriched pathways in both treatments (Table S8). Out of 47 DEGs, there were 34 down-regulated, overlapping genes, indicating that light reaction associated genes were significantly repressed under the inhibition of TOR with rapamycin and KU63794. In the category of cell wall biogenesis, down-regulated genes included extensins, expansins, xyloglucan endotransglycosylase/hydrolase (XTE/XTH), and cellulose synthase (CESA), etc. (Figure 8, Table S9). For example, there were eight overlapping down-regulated genes encoding XTE. In cell wall restructuring, the expansins and extensins function in loosening the cell wall to slide the microfibril matrix network. The enzyme XTE/XTH is used for cutting the xyloglucans (XG). CESA is employed to connect monosaccharides to the end of polysaccharides to synthesize the cellulose chains. Transporters that are essential for anabolic processes in plants were also down-regulated both in rapamycin- and KU63794-treated tomato. The transporters included boron transporters, nitrate transporters, sulfate transporters, phosphate transporters, zinc transporters, etc., (Table S10). Taken together, these data indicates that TOR plays a vital role in regulating tomato seedling growth.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.