RETRACTED ARTICLE: Sulfur availability regulates plant growth via glucose-TOR signaling

Growth of eukaryotic cells is regulated by the target of rapamycin (TOR). The strongest activator of TOR in metazoa is amino acid availability. The established transducers of amino acid sensing to TOR in metazoa are absent in plants. Hence, a fundamental question is how amino acid sensing is achieved in photo-autotrophic organisms. Here we demonstrate that the plant Arabidopsis does not sense the sulfur-containing amino acid cysteine itself, but its biosynthetic precursors. We identify the kinase GCN2 as a sensor of the carbon/nitrogen precursor availability, whereas limitation of the sulfur precursor is transduced to TOR by downregulation of glucose metabolism. The downregulated TOR activity caused decreased translation, lowered meristematic activity, and elevated autophagy. Our results uncover a plant-specific adaptation of TOR function. In concert with GCN2, TOR allows photo-autotrophic eukaryotes to coordinate the fluxes of carbon, nitrogen, and sulfur for efficient cysteine biosynthesis under varying external nutrient supply.

Specific transcriptional down-regulation of ribosome proteins in sir1-1. The entire protein biosynthesis pathway is presented and modified based on Mapman TM . The compared sets of genes contained transcripts which were identified by microarray analysis to be significantly up-or down regulated by more than 1.5-fold in sir1-1 or serat tko in comparison to the wild type (n=3, p<0.05). A clear enrichment of down-regulated genes encoding for ribosome proteins and proteins involved in RNA processing is evident in the root of sir1-1. An increase of down-regulated genes is also observed in the category of RNA processing in sir1-1 root. Please note that there are only significantly altered transcripts shown and hence no white squares appear in the graphic. However, light blue signals appear as white signal due to automatic settings of Mapman TM (http://mapman.gabipd.org/). Bar diagrams represents the frequency distribution of down-regulated genes in indicated process. Striped lines in the diagrams indicate +1 and -1 log ratio.
Supplementary Fig. 4 Transcript levels of 25s rRNA in cysteine synthesis depleted mutants. Total RNA was extracted from shoots and roots of 7-week old wild type (black), serat tko (grey) and sir1-1 (white) grown hydroponically on ½ Hoagland medium. Relative transcript levels of 25s rRNA were determined according to Ren, Qiu 3 . Primers used for specific amplification of 25s rRNA are listed in Table S4. (n=3, mean±s.e.m., one way ANOVA, *, p<0.05) Supplementary Fig. 5 Supplementary Fig. 5 Limitation of SiR activity decreases TOR activity. (a) Shoot phenotype and (b) fresh weight of 5-week old wild type (black), sir1-3 (white) and sir1-4 (grey) grown on soil under short day conditions 4 (n=3, mean±s.e.m., one way ANOVA, *, p<0.05) . (c) SiR protein level in wild type, sir1-3 and sir1-4 was determined by immunological detection with a specific antiserum shown in d (n=3, mean±s.e.m., one way ANOVA, *, p<0.05). (d) Immunological detection of S6K-p (52 kDa), S6K (52 kDa) and SiR (72 kDa) with specific antisera. Coomassie stained protein served as loading control (LC). TOR activity was determined by antibodies against S6K-p (52 kDa) and S6K (52 kDa) and (e) The ratio of S6K-p/S6K was calculated as a measure for in vivo TOR activity in the different mutants and displayed as x-fold of wild type level for easier comparison (n=3, mean±s.e.m., one way ANOVA, *, p<0.05).    Determination of SERAT Enzyme Activity SERAT activity in leaves was determined as described in Heeg et al., 2008, with the only difference that 2 units purified recombinant OAS-TL 11 was used to achieve the abundance of OAS-TL activity. The assay was performed in a total volume of 100 µl containing 58 µl protein extract from 100 mg plant material, 50 mM Hepes/KOH pH 7.5, 10 mM Na2S, 5 mM DTT, 10 mM serine and 2 unites OASTL. The reaction mix was incubated at 25°C for 5 min. Then 1 mM acetyl-coA was added and incubated at 25 °C for 30 min. The reaction was stopped by addition of 50 µl 20% TCA. After centrifugation, the supernatant was transferred quantitatively to 200 µl ninhydrin and 100 µl acetic acid and boiled at 100°C for 10 min. Then samples were cooled down and mixed with 550 µl 100% ethonal. The derivatives of ninhydrincysteine was measured at 560 nm with the photometer UvikonXL.
Total RNA extraction RNA was extracted from 50 mg of plant materials using the peqGOLD total RNA kit (peqLAB, Erlangen). The manufacturer's protocol was slightly modified to optimize the extraction. Briefly plant material was homogenized in 400 µl lysis buffer T. DNase digestion was performed on the RNA binding column for 15 min at room temperature. RNA was eluted by 50 µl RNase free water. RNA concentration was determined by Nanodrop.
cDNA synthesis RNA was converted to cDNA using cDNA synthesis kit (Fermentas), according to the manufacturer's protocol with 0.5 µg RNA and 0.5 µl oligo dT primers (for SULTR1;1, APR2 and SDI1 detection) or 25s RNA_RT primer (Supplementary  Table 4, for 25s rRNA detection). RNA was incubated with oligo dT at 65°C for 5 min. Then cDNA synthesis was carried out at 42°C for 1 hour. The synthesized cDNA for mRNA detection was diluted 1:5 for qPCR. The synthesized cDNA for 25s rRNA detection was diluted 1:10000 for qPCR. 25s rRNA was measured according to Ren  Metabolites measurement Thiol metabolites were extracted with 0.1 M HCL and separated on a LiChroCART 125x4 mm LiChrospher 60 RP-select column and quantified with the fluorescent dye monobromobimane (Synchem) described by 12 . Soluble sugars were extracted by 80% ethanol and separated on Dionex ICS-3000 system with CarboPac PA1 with CarboPac PA1-Guard column at 25°C.

Pinciple Component Analysis
The PCA of metabolites data was performed by MetaboAnalyst 3.0. Data was normalized to WT and log-transformed. Data scale was auto-scaled 14 .
Cell size measurement Cell wall was stained by 0.1 % SR 2200 solution (0.1% SR 2200, 1% DMSO, 0.05% Triton-X100, 5 % glycerol, 4 % paraformaldehyde) and immediately analyzed under a confocal scanhead LSM 510 META mounted on an Axiovert 200M microscope. Cell size was measured from the cell wall staining along single epidermal cell files. The meristematic zone was defined as the region of isodiametric cells from the QC up to the cell that was twice the length of the immediately preceding cell 7 . Cell size was measured by the online-software CellSet (CPIB) 8 .