Seeds of two independent ethanol-inducible TOR RNAi lines (5.2 and 6.3, described in Deprost et al., 2007) as well as an ethanol-inducible GUS overexpressing line (as a control) (Deprost et al., 2007) were grown in vitro under long day conditions (16 h light/8 h night) for 7 days on solid 1/5 Murashige and Skoog medium supplemented with sucrose 0.3% (w/v) at a constant temperature of 25°C and a light intensity of 75 μE.m^-2.s^-1. The plants were subsequently treated with ethanol vapor for either 3 or 10 days. Whole plantlets from two independent biological replicates of each condition were then harvested in the middle of the light period and directly snap frozen in liquid nitrogen, grinded and subjected immediately to the ribosome enrichment protocol.

Ribosomal subunits (40S and 60S), monoribosomes (80S) and polyribosomes were isolated from the plantlet powder according to Bailey-Serres and Freeling (1990) with minor modifications. Freshly harvested and grinded plantlets were homogenized at a final concentration of 10% (w/v) in the ice-cold extraction buffer (0.2 M Tris-HCl [pH 9], 0.4 M KCl, 0.025 M EGTA, 0.035 M MgCl₂, 0.2 M sucrose) supplemented with 2% (v/v) Triton X-100, 2% (v/v) Tween 20, 2% (v/v) NP-40 and 1% (w/v) sodium deoxycholate. The extracts were incubated on ice for 10 min to solubilize membrane-bound ribosomes and centrifuged at 2880 × g for 15 min at 4°C. The supernatants were layered over a sucrose cushion (0.04 M Tris-HCl [pH 9], 0.2 M KCl, 0.005 M EGTA, 0.03 M MgCl₂, 1.75 M sucrose) and ultracentrifuged at 225 000 × g for 14 h. The ribosome enriched pellet was resuspended in 300 μl of Laemmli buffer (Laemmli, 1970) and denatured at 100°C for 10 min.

For the proteomic characterization, ribosome enriched fractions were first submitted to a short migration through the stacking gel of a SDS-PAGE, in order to remove the rRNA and the possible chemical contaminant, including detergents. After a Coomassie staining, the unique band of proteins, for each sample, was cut and divided into five pieces that were submitted, in gel, to the tryptic digestion, reduction and alkylation. Peptide containing fractions were then analyzed by nano LC-MS/MS as previously described (Boex-Fontvieille et al., 2013). Briefly on-line liquid chromatography was performed on a NanoLC-Ultra system (Eksigent). Eluted peptides were analyzed with a Q-Exactive mass spectrometer (Thermo Electron) using a nano-electrospray interface (non-coated capillary probe, 10 μ i.d; New Objective). Peptides and the corresponding proteins were identified and grouped with X!TandemPipeline using the X!Tandem Piledriver (2015.04.01) release (Craig and Beavis, 2004) and the TAIR10 protein library with the phosphorylation of serine, threonine and tyrosine as a potential peptide modification. Precursor mass tolerance was 10 ppm and fragment mass tolerance was 0.02 Th. Identified proteins were filtered and grouped using the X!TandemPipeline v3.3.4¹. Data filtering was achieved according to a peptide E-value lower than 0.01. The false discovery rate (FDR) was estimated to 0.92%. Relative quantification was performed using the MassChroQ software (Valot et al., 2011) by peak area integration on extracted ion chromatograms (XICs) within a 10 ppm window, after LC-MS/MS chromatogram alignment and spike filtering.

Arabidopsis seedlings grown on MS agar plates in standard 16/8 h and 21/17°C day/night conditions were transferred to liquid MS media supplemented with 10 μM NAA (Sigma-Aldrich). Total protein extracts were precipitated with 0.1 M ammonium acetate in 100% methanol, reduced, alkylated and digested overnight with trypsin (Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate. Resulting peptides were vacuum-dried and re-suspended in 250 mM acetic acid with 30% acetonitrile for phosphopeptide enrichment with Phos-Select Iron Affinity Gel (Sigma-Aldrich) according to the protocol from Thingholm et al. (2008). Eluted phosphopeptides were desalted and analyzed by nano LC-MS/MS on a TripleTOF 5600 (Sciex, Canada) coupled a NanoLC-2DPlus system with nanoFlex ChiP module (Eksigent, Sciex).

Transcriptomic and translatomic analyses were performed on two biological replicates using 7-day-old plantlets from the two independent TOR RNAi and GUS control lines grown in vitro and treated with ethanol for 24 h. Transcriptome analyses using CATMA arrays were performed on total RNA preparations as previously described (Moreau et al., 2012). For translatomic analyses total RNA was extracted and polysomal fractions were purified on sucrose gradients after ultracentrifugation as previously described (Deprost et al., 2007; Sormani et al., 2011). Polysome-bound RNAs were extracted using guanidinium hydroxychloride and precipitated by isopropanol and linear acrylamide as a carrier. Subsequently, RNAs were reverse transcribed and hybridized on CATMA arrays as described above for the determination of differentially translated mRNAs (Sormani et al., 2011). Statistical analysis of each comparison was based on two dye swaps and followed by the analysis described by Gagnot et al. (2008) and Moreau et al. (2012). Briefly, an array-by-array normalization was performed to remove systematic biases. To determine differentially expressed genes, we performed a paired t-test on the log ratios averaged on the dye swap. The raw p-values were adjusted by the Bonferroni method, which controls the family-wise error rate to keep a strong control of the false positives in a multiple-comparison context. We considered as being differentially expressed the probes with a Bonferroni P-value ≤0.05, as described by Gagnot et al. (2008). The results are available online in the CatDB database².

Antibodies directed against phosphorylated RPS6A were obtained by conjugating to keyhole limpet hemocyanin the SRLpSSAAAKPSVTA (phosphoSer240) peptide (produced by Proteogenix, Schiltigheim, France) and injecting two New Zealand White female rabbits (performed by Proteogenix). Seven injections were performed over a period of 56 days. Then a preliminary ELISA test was performed at day 63 to evaluate the titer of the antibodies and the rabbits were bled at day 70. The obtained antisera were first depleted against immobilized non-phosphorylated peptide then specific antibodies were purified using the phosphorylated SRLpSSAAAKPSVTA peptide. The specificity of the purified antibodies was evaluated using an ELISA test with the phosphorylated and non-phosphorylated peptides (produced by Proteogenix) (Supplementary Figure S3).

Primary antibodies used in this study are directed against mammalian RPS6 (Cell Signaling Technology #2317S), rapeseed RPL13 (Sáez-Vásquez et al., 2000) and Arabidopsis RPS14 (Agrisera AS09 477).

For detection of phosphorylated RPS6, total proteins were extracted from either wild-type (Col-0), TOR RNAi or control Arabidopsis lines using the Laemmli buffer and blotted with our RPS6 phospho-specific antibody. Bradford assay (Bio-Rad) was performed to quantify total protein concentrations. Ten micrograms of proteins were separated by SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (PVDF, Bio-Rad) by electroblotting. Membranes were probed with either antiphospho-RPS6 (P-RPS6) rabbit polyclonal antiserum (dilution 1:5000) or with anti-RPS6 mouse monoclonal IgG (dilution 1:1000). Goat anti-rabbit IgG-HRP (horseradish peroxidase 1:2000, Santa Cruz Biotechnologies) and Goat anti-mouse IgG-HRP (1:2000, Santa Cruz Biotechnologies) were used as secondary antibodies. Immunodetection was performed by using enhanced chemiluminescent (ECL) substrates for HRP as recommended by the manufacturer (Clarity Western ECL blotting substrate Bio-Rad). Transferred proteins on PVDF membranes were visualized by Ponceau S staining.

The 5′ UTR sequences of the ribosomal proteins mRNAs were obtained from the TAIR10 database³. The identification of the motifs was performed with the online MEME software⁴ (Bailey et al., 2009) with a motif recognition size comprised between 6 and 50 nt. Only the representative isoforms of the genes were used.

Arabidopsis RPS6A: At4g31700 and RPS6B: At5g10360. Proteomic raw data are available in the Protic database under the following accession name: tor_inactivation⁵.

The aim of this study was to identify, by an untargeted proteomic analysis, modifications in the Arabidopsis ribosome fraction in response to TOR inactivation. First, we evaluated the suitability of our ribosome extraction method for LC-MS/MS analysis. To do so, 7-day-old seedlings of Arabidopsis (Col-0) were harvested and, to prevent protease, phosphatase, or kinase activities, immediately submitted to the ribosome purification protocol (see, Materials and Methods). Finally, high molecular weight particles, including polysomes, were pelleted by ultracentrifugation through a sucrose cushion.

We then submitted the ultracentrifugated fraction to SDS-PAGE and resolved proteins were stained using silver nitrate (Figure 1A). The obtained protein profile is typical of purified plant ribosomal fractions (Carroll et al., 2008). The presence of ribosomal proteins in this fraction was confirmed by Western blot analysis which allowed to normalize the protein fractions by diluting the samples according to Western blot quantifications (Figure 1B).

To inactivate TOR, we used two independent ethanol-inducible TOR RNAi lines (based on the AlcR/AlcA operon) that we previously obtained and characterized (Deprost et al., 2007) and compared them to a control line expressing an ethanol-inducible GUS gene (GUS control, Deprost et al., 2007; Dobrenel et al., 2011).

Seedlings of two independent RNAi lines and of the GUS control were grown for 7 days in vitro and then treated with ethanol to induce TOR inactivation. We repeated the same experiment twice and then analyzed the six samples together. In order to remove eventual contaminating rRNA as well as the chemicals, the samples were first submitted to a short migration through a SDS-PAGE stacking gel. Proteomic as well as phosphoproteomic analyses by LC-MS/MS identified a total of 5936 spectra, corresponding to 1508 unique peptide sequences (raw data are available in the Protic database). Peptides were matched to protein sequences from TAIR10 and grouped according to sequence homology using the X!TandemPipeline with the phosphorylation of serine, threonine and tyrosine as a potential peptide modification. By this method, 361 different proteins were potentially identified by at least two peptide sequences, belonging to 217 groups (corresponding presumably to protein families, based on sequence homologies). Among these 361 proteins, 210 were identified by the presence of at least two proteotypic (i.e., specific of a given protein) peptides. Based on the previous annotations of the ribosomal proteins (Sormani et al., 2011; Tiller and Bock, 2014; Hummel et al., 2015), we found that more than half of the 361 proteins were ribosomal proteins (147 correspond to cytosolic ribosomes and 46 to organelle ribosomes) (Figure 2A). Using an in silico analysis, Sormani et al. (2011) refined the annotation of ribosomal proteins (including plastidic and mitochondrial ones). We used this list to identify the proteins (and therefore the peptides) corresponding to the mitochondrial and plastidic ribosomes. All identified organellar peptides corresponded to plastidic ribosomes.

Target of Rapamycin inactivation in the RNAi lines did not largely affect the number of detected peptides originating from the cytosolic ribosomes and thus most peptides were found both in the RNAi and in the control lines (between 419 and 449 peptides for RNAi2 and GUS lines, respectively; Figure 2B). Conversely the pool of peptides coming from pRPs was specifically depleted in the TOR RNAi lines with only 116 and 111 peptides detected for the TOR RNAi1 and RNAi2 lines, respectively, compared to 201 peptides in the control GUS line (Figure 2C). One third of the pRPs peptides were only present in the control GUS line (74 out of 218) whereas a much smaller number of peptides (17) were specifically found in the RNAi lines.

In order to exclude biases that could be caused by potential technical issues, like the mass spectrometer being occupied by some abundant peptides eluting near the peptides of interest, we quantified the traces of the peaks corresponding to the identified peptides of the plastidic and cytoplasmic RPs. These peak areas were then compared between the RNAi and GUS control lines for each peptide (Figure 2D). Such a quantitative approach confirmed that most of peptides resulting from the fragmentation of the pRPs are indeed less abundant in the TOR RNAi samples. This also confirmed that TOR inactivation did not have any strong effect on the accumulation of the cRPs peptides (Figure 2D). Thus these results suggest a global decrease in the abundance of pRPs resulting in a lower number and amount of peptides detected in the LC-MS/MS analysis.

We then used the number of spectra (or peptide hits) per protein to estimate the protein abundance for the pRPs and cRPs. Indeed it has been shown that there is a proportional relationship between these two values (Allet et al., 2004; Liu et al., 2004). We exclusively used the spectra corresponding to proteotypic peptides in order to avoid a bias that would be caused by the very high sequence homology within ribosomal protein families. By this method, we showed a coordinated down-regulation of the pRPs while the cRPs have a much less coordinated profile and are globally only slightly affected by the TOR inactivation (Figure 3). To better understand the role of TOR in regulating plant gene expression, we performed transcriptomic and translatomic analyses after TOR inactivation, in which we monitored the mRNA levels on total and polysome-bound RNA samples. The variations in the abundance of polysome-bound mRNA were determined after purification of polysomes on a sucrose gradient, extraction of RNA and microarray hybridization. To better identify the primary TOR-regulated mRNA targets, we decided to shorten the time of ethanol-mediated RNAi induction. Two biological repetitions were performed using each time the two independent RNAi lines. The resulting four transcriptomic and translatomic experiments were submitted to a statistical analysis to identify common differentially expressed genes when compared to the GUS control lines (see Materials and Methods). When focusing on the ribosomal proteins, we found a large difference in the expression profiles of the corresponding nuclear genes depending on whether the gene product is part of the cytoplasmic or the plastidic ribosome (Figure 3; Supplementary Table S1). First, almost all detected nuclear-encoded mRNA coding for pRPs, whether located in the small or in the large subunit, showed either no change in abundance or were down-regulated after TOR inactivation when compared to the GUS control line treated with ethanol (Figure 3A). On the opposite mRNAs coding for cRPs were mostly up-regulated (Figures 3B,C) except for RPL18a-1 which was the only transcript showing a reproducible decrease in abundance. The translatomic analysis mostly mirrored the transcriptomic variations but for some genes coding for cytosolic ribosomes, like RPL40 and RPL41, the total mRNA abundance did not vary significantly whereas they were more engaged in polysomes compared to the GUS control, suggesting a higher translation of these genes (Figure 3C). Taken together, these data suggest that there is a coordinated down regulation of the nuclear genes coding for the pRPs at the transcriptional, translational (polysomal loading), and protein levels in response to TOR inactivation (Figure 3A).

Next we examined whether this co-regulation involves the recognition of a conserved motif in the 5′ UTR sequences of the nuclear-encoded mRNA coding for pRPs. These sequences were analyzed for enriched motifs using the MEME software (Bailey and Elkan, 1994). A strongly significant motif composed of a stretch of pyrimidines was identified in this set of 5′ UTRs (Figure 4A). This sequence is reminiscent of the animal TOP motif found within the 5′UTR of mammalian ribosomal protein encoding mRNAs, which confers translational regulation by TOR (Meyuhas and Kahan, 2015). The MEME analysis also revealed the presence of a second A/G-enriched motif (Figure 4A). On the contrary, the 5′ UTRs of the mRNAs coding for cRPs were significantly enriched for a TTTAGGGTTT motif (Figure 4B), which is similar to the telo-box consensus sequence already identified in the promoters of these genes (Figure 4B) (McIntosh et al., 2011; Wang et al., 2011). Next we analyzed the 5′ UTRs of the nuclear genes coding for pRPs, which were less engaged in polysomes after TOR inactivation (Figure 3A). MEME analysis identified a shorter pyrimidine-rich motif that is more similar to canonical TOP motifs, but even more to the pyrimidine-rich translational element (PRTE), a motif identified in animal genes which translation is controlled by TOR (Figure 4C) (Hsieh et al., 2012). This motif is found in the majority of animal TOR targets and, unlike conventional TOP motifs, does not reside at the start of the mRNA sequence (Hsieh et al., 2012). Consistently, the motif we identified is also rarely present at the start of the analyzed mRNAs (Figure 4D; Supplementary Figure S1) but is significantly enriched in pRP transcripts translation of which is affected by TOR inactivation. A purine-rich motif was also identified in this subset of genes and was often found 3′ to the PRTE-like motif (Figure 4D).

We then mined the public Genevestigator transcriptome database (Hruz et al., 2008) for information about the expression profile of plastidic ribosomal genes encoded by the nuclear genome. Interestingly these genes were found to be down-regulated in estradiol-inducible TOR RNAi lines (Xiong et al., 2013; Supplementary Figure S2) which suggests that TOR inactivation reproducibly reduces their expression. Nuclear genes coding for pRPs are also strongly induced during germination or after light treatment, which is consistent with their role in the formation of the photosynthetic machinery. Conversely they were repressed in response to various stresses such as pathogen infection, drought, hypoxia, or increased temperature as well as in response to extended night. Finally these genes were slightly induced in one experiment of sucrose feeding but globally repressed in several experiments of nitrogen starvation (Supplementary Figure S2).

Even if most of the cRPs were not affected by TOR inactivation (Figure 2D) we found 30 peptides which were significantly down regulated (t-test p-value < 0.05; Supplementary Table S2). In most of the case, these peptides are not proteotypic, thus making the conclusions more complicated. The two cytosolic RPS6 paralogs were identified with three different proteotypic peptides that are significantly down-regulated in a quantitative manner following TOR inactivation (Figure 3B). In total, 15 peptides were detected for this protein family. Among these 15 peptides, we identified a phosphorylated peptide corresponding to the RPS6B C-terminal extremity. The X!Tandem analysis predicted a phosphorylation site on Ser240 (SRLpSSAPAKPVAA: Figure 5; Supplementary Figure S3). This serine seems to be conserved in the eukaryotic RPS6 proteins, including plants, and was previously identified as being phosphorylated following TOR activation in yeast and animals (Pende, 2006; Meyuhas, 2008; Yerlikaya et al., 2016) (Figure 6). In order to clarify the actual position of the phosphorylated residue in this peptide, the phosphoproteomic results were submitted to a Phoscalc statistical analysis (MacLean et al., 2008). This analysis failed to clearly identify the phosphorylated site between Ser237, 240 or 241 (Supplementary Figures S3C,D). Nevertheless it showed that this RPS6B C-terminal peptide carries only one phosphate group. An independent phosphoproteomic analysis of Arabidopsis seedlings treated with auxin was also performed (see Materials and Methods). Indeed previous studies suggested that auxin mediates TOR, and thus S6K1, activation in Arabidopsis as shown by phosphorylation of TOR at Ser2424 and S6K1 at Thr449 (Schepetilnikov et al., 2013). Since S6K1 is directly involved in the phosphorylation of RPS6, we asked whether this ribosomal protein is phosphorylated at Ser240 in Arabidopsis extracts treated by auxin. After purification of the phosphopeptides by immobilized metal affinity chromatography (IMAC), Ser240 was again identified as a potential phosphorylation site both in RPS6A and B (Supplementary Table S3; Supplementary Figures S3E,F). In some cases RPS6A Ser240 was identified together with Ser237 (pSRLpSSAPAKPVAA) but again the discrimination between phosphorylated Ser240 and Ser241 was not always possible (Supplementary Table S3).

Even if the precise localization of the phosphorylated serine residue in the RPS6B protein C-terminus could not be determined without ambiguities, we compared the abundance of the C-terminal monophospho-SRLSSAPAKPVAA peptide between the TOR RNAi lines and the GUS controls. After 72 h of exposure to ethanol we observed a modest decrease in the RPS6B C-terminal phosphorylated peptide (Figure 5A). This decrease in phosphorylation was only observed in one of two experiments. Since the abundance of the phosphorylated peptide was already low in the control line for the second experiment, it could be that the decrease in phosphorylation level was less obvious in this case. The corresponding RPS6A peptide was not found in this analysis. Thus to confirm this TOR-dependent decrease in RPS6 C-terminus phosphorylation, we performed a longer silencing induction by ethanol for 10 days. As for the 72 h treatment, biological duplicates have been used. The abundance of the monophospho C-terminal peptides was compared for both RPS6A and RPS6B proteins between the RNAi and the control GUS lines (Figures 5B,C). The amount of phosphorylated C-terminal peptide was lower (with a mean of 40% decrease) for both the RPS6A and the RPS6B proteins in the TOR RNAi lines when treated with ethanol for 10 days.

To confirm this phosphoproteomic analysis, a phospho-specific antibody was raised against a synthetic SRLpSSAAAKPSVTA peptide in which only Ser240 was phosphorylated. The obtained polyclonal antibody was purified against this peptide and the eluted antibody fraction detected a single band corresponding in size to RPS6 proteins (Supplementary Figure S4A; Figure 7). Furthermore this antibody was found to be highly specific for the phosphorylated SRLpSSAAAKPSVTA peptide, showing no reaction with the control non-phosphorylated peptide in an ELISA test (Supplementary Figure S4B).

This antibody was used in an optimized Western blot assay and the obtained signal was very strong even when the antibody was diluted 1/5000. This band co-migrated with the band decorated by a specific monoclonal antibody against mammalian RPS6 (Figure 7) which was subsequently used to quantify the amount of total RPS6 in protein extracts. Both treating Arabidopsis seedlings with AZD-8055, a strong and specific second generation TOR inhibitor (Montané and Menand, 2013), or silencing the expression of TOR by a 7-day ethanol treatment, resulted in a significant and dose-dependent decrease in the signal obtained with the RPS6 phospho-specific antibody, whereas the total amount of RPS6 protein only decreased slightly (Figures 7A,C). Since this antibody is highly specific for the phosphorylated RPS6 protein, this confirms that there is a reproducible decrease in Ser240 phosphorylation level following TOR inactivation. Accordingly it was previously shown in animals that AZD inhibits TOR, and as a consequence S6K activity and RPS6 phosphorylation (Chresta et al., 2010). TOR and S6K were previously shown to be activated by sugars like sucrose and glucose (Xiong et al., 2013). Consistently RPS6 phosphorylation was augmented by the addition of sucrose when supplied to sugar-starved Arabidopsis seedlings (Figure 7B). Next we examined the kinetic of changes in RPS6 phosphorylation after either AZD-8055 or sucrose treatments (Figure 8). As soon as 1 h after AZD addition, a significant decrease in Ser240 phosphorylation was observed (Figure 8A). For sucrose, an increase in phosphorylation was detected 2 h after supply. However it is difficult at this stage to discriminate between a direct signaling effect of sucrose and an indirect consequence of sugar metabolism (Figure 8B). Altogether these data suggest a strong positive correlation between the RPS6 phosphorylation level, as detected by this specific polyclonal antibody, and TOR activity. Therefore, it seems that in plants, like in animals and yeast, RPS6 phosphorylation can be used as a robust and sensitive readout for TOR activity.