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Supplemental Materials: Padilla-Chacon et al. Running title: Heterologous expression of Hxt2 in Arabidopsis thaliana Supplemental Methods Data treatment and conversion to a Mapman compatible format The data column with the expression ratio value (WT versus transgenic) was used as primary data. The two sampling time points (6h and 12h after addition 3% glucose) were statistically treated as biological replicates and mean values and standard errors were calculated accordingly. Using the AGI locus number in one column, we generated an additional ID column with the corresponding identifier number from Affymetrix microarrays. This was done with a javascript macro and using the excel lookup functions. Since the microarray chip from Arizona university contains various amounts of duplicated spots, and since different AGI codes can be converted to the same Affymetrix at_ID_code, we had to filter the data table in order to contain only unique at-ID_codes. For that purpose, for the duplicated IDs we estimated average transcript levels using the apply function of the statistical program (R). Subsequently, a filter was used to mark as non significant all those genes for which the standard error was larger than the mean expression value of the genes. Values were also marked as non-significant when the difference between the two contrasting samples (WT versus transgenic) was smaller than the sum of the two standard errors. Visualization of Microarray results with Mapman The visualization tool MapMan (Thimm et al., 2004; http://gabi.rzpd.de/projects/MapMan/) was used to analyze the microarray data. The mapping files structure the Arabidopsis genes into main BINS and sub BINS for display on the schematic maps of metabolism and cellular processes. The overview figures (Figs S2-4) were prepared using mapping file version Ath_AFFY_ATH1_TAIR8. The mean ratio values between wild type and transgenic line L2 were converted to log2 scale, and imported into MapMan, which converts the data values to a false color scale. Transcripts called not present are shown as gray, transcripts that change by less than a given threshold are white, transcripts that increase are blue, and transcripts that decrease are red. In the scale used for the present figures, a 2-fold change was required to produce a visible coloration, and the scale saturated at an 8-fold change (= 3 on a log2 scale). Supplemental Results The metabolic overview figures (see MapMan figures below) suggest a coordinated reprogramming of gene expression when comparing WT and L2 seedlings exposed to high glucose. There were significant changes for many genes directly involved in metabolism, transport and assimilation. For example, a MATE family transporter protein (Fig S1) and the sucrose transporter SUT4 gene were induced (Fig S4). Raffinose and minor sugar metabolism metabolism was also affected (Fig S1 and S2). The transcript levels of some key glycolytic enzymes were also induced including pyruvate kinase and glyceralheyde-3P deshydrogenase. The fact that not only glucose and fructose phosphorylating activities increased (see Fig 6), but also some other genes in glycolysis is additional evidence that glycolytic flux was indeed increased in the transgenic lines gown at high glucose. Sucrose synthase was weakly induced and cytosolic invertase was repressed (Fig S3). A 1 invertase inhibitor was also induced (Fig S1). This change presumably reflects a coordinated switch. The starch synthesis the small subunit of ADP-glucose pyrophosphorylase was repressed. There were relatively few changes in the expression of genes involved in cell wall polysaccharide synthesis. Interestingly, a subset of genes for cell wall-modifying enzymes was also induced, including some members of the expansin family. Few genes involved in fatty acid metabolism were altered, for example, diacylglicerol kinase was repressed. In the nitrogen metabolism the gene of glutamate deshydrogenase was strongly induced. Our results suggest that there is a reprogramming of metabolism by heterologous expression of the transporter Hxt2, something that would not to be expected if Hxt2 would function only as a sugar transporter per se. Therefore, we postulate that Hxt2 alters sugar signaling and perception, and that this effect is not only reflected as a phenotype of less glucose sensitivity (see Fig 4), but also observed at the level of gene expression. Some of the changes in the Mapman figures are not apparent from raw inspection of lists of affected genes because many of the changes are relatively small. Some minor changes become only visible when genes are systematically collected in BIN groups or functional categories. Supplemental Discussion on Yeast Sugar Transporters and Signalling Yeast have developed a system to sense extracellular concentrations of sugars that ensures a coordination between the supply of carbohydrates from the environment and the enzymatic machinery of the cells (Lalonde et al., 1999; Lalonde et al., 2004). External glucose is sensed by SNF3 and RGT2. Despite their high homology with glucose permeases, these transmembrane proteins do not mediate significant glucose transport (Ozcan et al., 1996). However, both have a large intracellular domain, and from functional analyses, it appears that the sensing function requires two interacting domains: the membrane-spanning domain necessary for glucose recognition and the C-terminal extensions necessary for intracellular reporting. Both are required for the transmission of the glucose signal (Ozcan et al., 1998). SNF3 is required for repression of the high-affinity transporters Hxt2, Hxt6 and Hxt7 at high glucose concentrations (Liang and Gaber, 1996; Vagnoli et al., 1998). The C-terminal domains of SNF3 and RGT2 mediate the signaling function of proteins. The C-terminal extension of SNF3 contains two nearly identical repeats of 25 amino acids. Deletions of these extensions leads to a loss of sensor function (Ozcan et al., 1998; Vagnoli et al., 1998). 2 Supplemental Figures Metabolism Log2FC p-value -2.15 0.003 -2.12 0.008 -2.04 0.002 -1.91 0.005 -1.75 0.005 -1.70 0.003 -1.68 0.003 -1.64 0.004 -1.50 0.007 -1.49 0.006 -1.49 0.004 -1.42 0.005 -1.42 0.005 -1.40 0.010 -1.37 0.006 -1.36 0.005 -1.36 0.005 -1.34 0.006 -1.31 0.006 -1.29 0.009 -1.27 0.007 -1.24 0.006 -1.17 0.009 -1.17 0.009 -1.16 0.010 -1.14 0.009 -1.14 0.009 1.13 0.009 1.19 0.008 1.23 0.007 At3g55680 At5g20250 At5g55370 At5g07440 At1g02880 At3g26445 At5g02230 At2g29300 At1g71120 At5g57590 At5g19690 At4g39800 At5g27410 At1g31490 At1g54630 At1g21840 At4g28395 At1g55810 At1g77420 At4g31790 At4g11640 At4g36530 At5g37170 At5g07000 At5g04490 At2g22250 At4g21860 At1g17745 At4g26860 At5g01210 Membrane Log2FC p-value Transcription Log2FC p-value Unknown Log2FC p-value invertase/pectin methylesterase inhibitor family protein profile PF04043: Plant invertase/pectin inhibitor -1.92 TIGR00797: 0.001 At1g27000 bZIP family Pfam transcription factor -2.13 contains 0.009 Pfam At1g66760 MATE efflux family protein methylesterase contains TIGRfam profile: MATE efflux family protein, profile PF01554: Uncharacterized membrane protein family -1.98 0.003 At1g69760 raffinose synthase family protein / seed imbibition-1.84 protein,0.004 putative At1g79450 (din10) similar toLEM3 seed (ligand-effect imbibition protein GB:AAA32975 from [Hordeum vulgare]; contains nonconsensus ATContains donor splice site at intron domain. 1; contains Pfam profile PF05691: Raffinose 0.004 At1g21760 F-box familyGI:4585976; protein PF|00646 F-box ESTs gb|Z37267, gb|R90412, gb|Z37268 and gb|T88189 come from -1.87 0.003 At1g10150 modulator 3) family GI:167100 protein /-1.90 CDC50 family protein similar to GI:4966357; GI:11994416; GI:9757735 from [Arabidopsis thaliana] long-chain-alcohol O-fatty-acyltransferase family-1.80 protein /0.005 wax synthase family protein contains similarity to transporter wax synthase similarity to wax synthase wax - Simmondsia PID:g5020219 similar to wax synthase from Simmondsiariboside-insensitive c -1.77 0.002 At5g36670 PHD fingerchinensis, family protein -1.84 [gi:5020219] 0.002 At3g12300 At1g70330 equilibrative nucleoside family protein contains similarity tosynthase SWISS-PROT:Q14542 equilibrative nucleoside transporter 2 (Equilibrative nitrobenzylmercaptopurine nucleoside tra glutamate dehydrogenase 2 (GDH2) identical to -1.68 glutamate dehydrogenase 2 (GDHauxin 2) [Arabidopsis thaliana] SWISS-PROT:Q38946 -1.59 0.003 At5g39810 family protein contains similarity to to hypothetical of [Arabidopsis thaliana] thaliana] gi|4322486|gb|AAD16 -1.83 proteins 0.002 At5g07490 0.006 At5g57090 transport protein (EIR1) identical to auxin transport protein EIR1 [ArabidopsisMADS-box thaliana] gi|3377507|gb|AAC39513; identical root gravitropism control protein [Arabidopsis thiamin pyrophosphokinase, putative similar to thiamin [Mus musculus] gi|6468206|dbj|BAA87040 At5g65210 bZIP family GI:4585976; transcription GI:11994416; factor (TGA1) GI:9757735 identical to transcription factor thaliana] (TGA1) GI:16550 from [Arabidopsis thaliana] -1.76 0.002 At5g17210 -1.67 pyrophosphokinase 0.003 At1g79450 LEM3 (ligand-effect modulator 3) family protein /-1.57 CDC50 0.004 family protein similar to GI:4966357; from [Arabidopsis glycosyl transferase family 17 protein contains Pfam PF04724: Glycosyltransferase family 17 oligopeptide transport (POT) family -1.49 protein 0.003contains At5g64950 mitochondrial transcription termination factor-related / -1.70 mTERF-related Pfam profile PF02536: mTERF 0.003 contains At4g19645 -1.57profile 0.003 At5g62730 proton-dependent Pfam profile: PF00854 POT family haloacid dehalogenase-like hydrolase family protein contains accession IPR005834: Haloacid dehalogenase-like -1.47 NSF 0.007 At1g32430 F-box VTI1a family protein domain Pfam:PF00646 -1.69 0.006 At4g23670 -1.52 0.003InterPro At5g39510 vesicle transport v-SNARE 11 (VTI11) /hydrolase vesicle soluble attachment protein receptor (VTI1A)contains identicalF-box to SP|Q9SEL6 Vesicle transport v-SNARE 11 (AtVTI11) (Vesicle transport v-SNARE protein V tropinone reductase, putative / tropine dehydrogenase, to tropinonesulfate reductase SP:P50165 fromto[Datura 0.004 thaliana] At5g10960 CCR4-NOT transcription complex, subu -1.64to SWISS-PROT:Q9UFF9 0.003 At5g07570 -1.43 putative 0.006 similar At1g77990 transporter identical sulfatestramonium] transporter-1.43 [Arabidopsis GI:1498120CCR4-NOT transcription complex protein, putative similar GDSL-motif lipase/hydrolase family protein similar to family II lipases EXL3 GI:15054386 [Arabidopsisfamily thaliana]; contains Pfam profile PF00657: GDSL-like Lipase/Acylhydrolase 0.009 At4g31720 transcription transporter, initiation factor IID II(TFIID) 23-30kDa subunit familyAt1g32400 protein contains Pfam profile: PF03540 transcriptio -1.63(TAF2H) 0.003 -1.34 0.008 At5g02180 amino from acid transporter protein belongs to -1.41 INTERPRO:IPR002422 amino acid/polyamine family aminotransferase class III family protein low similarity acidankyrin aminotransferase BioA [Mesorhizobium loti] GI:12044306; contains Pfam profile PF00202: class III family protein contains -1.36 Pfam:PF00023 0.008 At5g15790 zinc fingeraminotransferase, (C3HC4-type RING finger) profile:At1g04030 PF00097 zinc finger, C3HC4 type (RING finger) -1.60 Pfam 0.003 -1.31 to 7,8-diaminopelargonic 0.005 At4g03500 repeat family protein contains ankyrin repeats, oligosaccharyl transferase STT3 subunit family protein Oligosaccharyl transferase STT3 subunit {Saccharomyces contains Pfam profile PF02516: Oligosaccharyl transferase STT3Pfam subunit -1.28cerevisiae}; 0.007 At3g18960 transcriptional factor B3 family protein contains profile B3At1g24370 DNA binding domain -1.56 PF02362: 0.003 -1.31 similar 0.006to SP|P39007 At1g30540 ATPase, BadF/BadG/BcrA/BcrD-type family contains Pfam PF01869: BadF/BadG/BcrA/BcrD ATPase family; similar to Transcriptional regulators of NagC/XylR (ROK) family, sugar kinase (GI:15023013) [Clo inositol-3-phosphate synthase isozyme 1 / myo-inositol-1-phosphate synthase 1 / MI-1-P synthase 1 / IPS 1 identical Inositol-3-phosphate synthase isozyme (EC 5.5.1.4) (Myo-inositol-1phosphate synthase 1) (MI-1-P 1) (IPS 1) {Arabi -1.15 0.008nodule At5g07610 F-box family protein similar to unknown protein (emb|CAB85517.1) -1.54 0.003synthase At1g36050 -1.31 0.006 At5g50790 nodulin MtN3 family protein similartotoSP|P42801 MtN3 GI:1619602 (root development) from 1[Medicago truncatula] aminotransferase class IV family protein contains-1.31 Pfam profile: aminotransferase class IV -1.15 zinc-binding family protein similar to zinc-binding protein [Pisum0.003 sativum] At3g12940 GI:16117799; contains Pfam profile PF04640 : Prot -1.54 0.008 PF01063 At4g35080 high-affinity nickel-transport family protein contains Pfam0.009 domain, At1g32700 PF03824: High-affinity nickel-transport protein transferase family protein contains similarity to anthranilate N-hydroxycinnamoyl GI:3288180, from cancer (Dianthus caryophyllus); Pfam profile PF02458 transferase family 1.22 resistance 0.007 At4g28890 zinc finger RING finger) family protein contains profile:At5g50900 PF00097 zinc finger, C3HC4 type (RING finger) -1.51 Pfam 0.008 -1.26 0.007 At1g71960benzoyltransferase ABC transporter family protein GI:2239091 similar to breast proteincontains GB:AAC97367 from(C3HC4-type [Homo sapiens] acyl carrier protein 3, chloroplast (ACP-3) nearly -1.18 identical0.008 to SP|P25702 Acyl carrierS1 protein 3, chloroplast protein-related precursor (ACP) {Arabidopsis thaliana} 1.22similarity 0.007to S1 At4g25470 DRE-binding protein (DREB1C) / CRT/DRE-binding factor to DREB1C GI:3738228 from [Arabidopsis thali -1.512 (CBF2) 0.006identical At3g61710 At1g11765 self-incompatibility contains weak self-incompatibility protein [Papaver rhoeas] gi|452430|emb|CAA52380 expressed protein weak similarity to SP|Q07402 -1.16 Urease accessory protein ureF [strain TB-90] {Bacillus sp.}(AGP21) 0.010 At1g55330 arabinogalactan-protein 1.31 0.007 At3g08020 PHD finger protein-related contains low similarity to PHD-finger domain proteins -1.50 0.008 At5g15990 lipid transfer protein, putative identical to anther-specific [gi:2746339]; contains Pfam protease family0.006 domain 1.34 At4g34370 IBR domain-containing protein similar to SP|Q94981 Ariadne-1 protein (Ari-1) {Drosophila melanogaster}; contains Pfam prof -1.50 At1g53560 -1.16 gene 0.010ATA7 At4g18190 purine permease family inhibitor/seed protein similarstorage/LTP to purine permease [Arabidopsis thaliana] GI:7620007; contains Pfam profile PF03151: Domain of unknown0.005 function, DUF250 uracil phosphoribosyltransferase, putative / UMP-1.15 pyrophosphorylase, putative / UPRTase, putative similar MLO to SP|O65583 Uracil/ MLO-like phosphoribosyltransferase (EC 2.4.2.9) (UMP pyrophosphorylase) (UPRTase) {Arabidopsis thaliana}; contains Pfam profile PF00485: PhSWISS-PROT:P93766, NC 1.92 protein 0.0014 (MLO4) At3g04250 family protein-related contains TIGRFAM TIGR01640: F-box protein interaction domain -1.41 0.004 At5g46560 0.009 At1g11000 seven transmembrane family protein identical to F-box membrane protein Mlo4 [Arabidopsis thaliana] gi|14091578|gb|AAK53797; similar to MLO protein hydrolase, alpha/beta fold family protein similar to monoglyceride from [Homo sapiens] GI:14594904, musculus] GI:2632162; profile PF00561: hydrolase, alpha/beta fold family 1.93 contains 0.001 Pfam agamous-like box protein AGL1 / shatterproof 1-1.40 (AGL1) (Keratinocyte (SHP1) to SP|P29381 Agamous-like MADS box prot 0.005 identical At4g01915 -1.13 0.010 lipase At4g18905 transducin family protein /[Mus WD-40 repeat family protein contains 5 At3g58780 (4 significant) WD-40 repeats; MADS similar to periodic tryptophan protein 1 homolog protein IEF SSP 9502) (PWP1)(SP:Q13610) (P diphthine synthase, putative (DPH5) similar to Diphthine (Diphtamide biosynthesis methyltransferase) (DPH5) [Saccharomyces cerevisiae]; similar to CGI-30 DPH5, Saccharo -1.38 to methyltransferase 0.005 At1g76185 1.19 synthase 0.008 At1g74240 mitochondrial substrate carrier family(SP:P32469) protein contains Pfam profile: PF00153 mitochondrial carrierprotein protein(GI:4680699) [Homo sapiens]; similar At5g36690 At2g35520 At5g25470 sulfotransferase family protein similar to steroid sulfotransferase 3 [Brassica napus] GI:3420008, steroid sulfotransferase 1 [Brassica napus] GI:3420004; contains Pfam profile PF00685: Sulfotransferase domain -1.30 0.005 At1g14530 phosphatidate cytidylyltransferase family protein contains Pfam profile: PF01148 phosphatidate cytidylyltransferase -1.30 0.005 At5g55240 aminotransferase class I and II family protein similar to aspartate aminotransferase from Bacillus stearothermophilus SP|Q59228, Thermus aquaticus SP|O33822; contains Pfam profile PF00155 aminotransferase, classes I and II 0.006 -1.29 At2g47760 methionine sulfoxide reductase domain-containing protein / SeIR domain-containing protein low similarity to pilin-like transcription factor [Homo sapiens] GI:5059062, SP|P14930 Peptide methionine sulfoxide reductase msrA/msrB (EC 1.8.4.6) {Neisseria gonor -1.28 0.008 At1g79970 D-3-phosphoglycerate dehydrogenase / 3-PGDH identical to SP|O04130 -1.28 0.006 At3g25930 Log2FC p-value Log2FC p-value alanine racemase family protein contains Pfam domain, PF01168: Alanine racemase, N-terminal domain -1.24 0.007 At1g80550 -1.65 0.004 At5g18600 glutaredoxin family protein contains glutaredoxin-1.28 domain,0.007 INTERPRO:IPR002109 At3g61110 40S ribosomal protein S27 (ARS27A) identical to cDNA-1.22 ribosomal protein S27 (ARS27A) GI:4193381 transferase family protein contains Pfam profile PF02458 transferase family 0.007 At3g58080 -1.55 0.005 At5g51630 disease resistance protein (TIR-NBS-LRR class),-1.21 putative0.008 domain At1g01100 signature TIR-NBS-LRR exists, suggestive a disease resistance 60S acidic ribosomal proteinofP1 (RPP1A) similar to protein. 60S ACIDIC RIBOSOMAL PROTEIN P1 GB:O23095 from [Arabidopsis t -1.20 0.007 At5g53905 -1.43 0.004 At1g50950 thioredoxin-related contains weak hit to Pfam PF00085: Thioredoxin; contains 2 predicted transmembrane domains -1.18 0.008 At3g09100 mRNA capping enzyme family protein similar to mRNA-1.19 capping enzyme [Xenopus laevis] GI:7239232; contains Pfam profiles 0.008 At2g01100 -1.38 0.004 At4g21380 S-locus protein kinase, putative (ARK3) identical-1.17 to PIR|T05180|T05180 S-receptor29 kinase ARK3 precursor - [Arabidopsis thaliana]/ RNA-binding protein cp29, putative similar to 29 kDa ribonucleoprotein chlor 0.008 At1g60000 kDa ribonucleoprotein, chloroplast, putative -1.18 0.008 At3g12590 -1.36 0.006 At2g23080 casein kinase II alpha chain, putative identical to-1.17 probable0.010 casein kinase II, alpha chain [Arabidopsis thaliana] SWISS-PROT:O64817; similar to casein kinase II, alpha chainfactor, 1 [Arabidopsis thaliana] SWISS-PR At5g42820 U2 snRNP auxiliary factor small subunit, putative strong similarity to U2 snRNP auxiliary small subunit [Oryza sativa] G -1.14 0.009 At1g67570 -1.34 0.009 At1g16920 Ras-related GTP-binding protein, putative similar 1.14 to GTP0.009 binding protein GI:218228DNA-directed from [Vicia faba]; to cDNA small GTP-binding proteinto(Rab11) GI:451859 At2g24120 RNA identical polymerase, chloroplast (RPOPT) identical SP|O24600 DNA-directed RNA polymerase, chloroplast pre -1.14 0.009 At1g80860 -1.33 0.006 At1g22280 protein phosphatase 2C, putative / PP2C, putative1.14 similar0.010 to protein phosphatase type 2C GI:4336436 family from [Lotus japonicus] At2g47220 3' exoribonuclease domain 1 protein-related similar to polynucleotide phosphorylase [Pisum sativum] GI:2286200, poly -1.14 0.009 At4g09260 -1.30 0.006 At5g63140 calcineurin-like phosphoesterase family protein contains Pfam profile: PF00149 calcineurin-like phosphoesterase 1.24 0.006 At5g04210 RNA recognition motif (RRM)-containing protein contains InterPro entry IPR000504: RNA-binding region RNP-1 (RNA recogn -1.12 0.010 At5g16800 -1.30 0.007 At5g64620 invertase/pectin methylesterase inhibitor family protein similarity to SP|P83326 mitochondrial Pectinesterasetranscription inhibitor (Pectin methylesterase inhibitor) (PMEI) {Actinidia chinensis}; contains Pfam profile PF04043: Plant 1.56 low 0.005 At2g44020 termination factor-related / mTERF-related contains Pfam profile PF02536: mTERF -1.11 0.010 At3g08030 meprin and TRAF homology domain-containing protein / MATH domain-containing protein weak similarity to ubiquitin-specific protease 12 [Arabidopsis thaliana] GI:11993471; contains weak hit to Pfam PF00917: MATH domain -1.25 0.008 At1g74520 ABA-responsive protein (HVA22a) identical to AtHVA22a [Arabidopsis thaliana] GI:4884932 1.13 0.009 At3g03280 aspartyl protease family protein contains Pfam profile PF00026: Eukaryotic aspartyl protease; similar to CND41, chloroplast nucleoid DNA binding protein (GI:2541876) {Nicotiana tabacum} -1.22 0.009 At1g07960 thioredoxin family protein low similarity to protein disulfide isomerase 4 [Giardia intestinalis] GI:13489047; contains Pfam profile PF00085: Thioredoxin 1.22 0.007 At2g34220 ubiquitin-protein ligase, putative similar to SP|P39940 Ubiquitin--protein ligase RSP5 (EC 6.3.2.-) {Saccharomyces cerevisiae}; contains Pfam profiles PF00240: Ubiquitin family, PF00632: HECT-domain (ubiquitin-transferase) -1.20 0.007 At5g64813 GTP-binding protein-related contains weak similarity to Ras-related protein Rab-27A (Rab-27) (GTP-binding protein Ram) (Swiss-Prot:P51159) [Homo sapiens] 1.26 0.010 At3g02950 Der1-like family protein / degradation in the ER-like family protein contains Pfam profile: PF04511 Der1-like family -1.13 0.009 At4g25780 pathogenesis-related protein, putative similar to gene PR-1 protein - Medicago truncatula, SP|Q40374; contains Pfam profile PF00188: SCP-like extracellular protein 1.28 0.006 At3g07660 protease inhibitor/seed storage/lipid transfer protein (LTP) family protein similar to lipid transfer protein - Hordeum vulgare, EMBL:AF109195; contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 1.14 0.010 At5g26751 shaggy-related protein kinase alpha / ASK-alpha (ASK1) identical to shaggy-related protein kinase alpha SP:P43288 GI:460832 from [Arabidopsis thaliana] 1.29 0.006 At2g39705 Ulp1 protease family protein contains Pfam profile PF02902: Ulp1 protease family, C-terminal catalytic domain; similar to EST gb|N96456 1.17 0.008 At2g14440 leucine-rich repeat protein kinase, putative similar to light repressible receptor protein kinase [Arabidopsis thaliana] gi|1321686|emb|CAA66376; contains leucine rich repeat (LRR) domains, Pfam:PF00560; co 1.29 0.009 At5g37470 protease inhibitor/seed storage/lipid transfer protein (LTP) family protein contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 1.42 0.005 At2g37290 RabGAP/TBC domain-containing protein low similarity to Rab6 GTPase activating protein, GAPCenA [Homo sapiens] GI:12188746; contains Pfam profile PF00566: TBC domain 1.35 0.006 At3g10770 1.47 0.008 At1g62820 calmodulin, putative similar to calmodulin SP:P04465 from [Trypanosoma brucei gambiense]; contains INTERPRO:IPR002048 calcium-binding EF-hand domain 1.49 0.007 At5g47870 1.53 0.005 At3g23860 GTP-binding protein-related low similarity to SP|Q99ME9 Nucleolar GTP-binding protein 1 (Chronic renal failure gene protein) {Mus musculus} 1.58 0.003 At3g14730 1.64 0.004 At1g22920 COP9 signalosome subunit 5B / CSN subunit 5B (CSN5B) / c-JUN coactivator protein AJH1, putative (AJH1) COP9 complex subunit CSN5-1; identical to Arabidopsis homologs of a c-Jun coactivator AJH1 G 1.61 0.002 At1g30320 1.65 0.004 At4g21865 1.68 0.009 At2g25800 1.80 0.002 At1g19500 serine racemase, putative similar to serine racemase musculus] GI:6448865; extracellular contains Pfam profile PF00291: Pyridoxal-phosphate dependent -1.36 0.007 1.37 [Mus 0.005 At5g19110 dermal glycoprotein-related / EDGP-related similar toenzyme extracellular dermal glycoprotein EDGP precursor [Daucus carota] GI:285741 hydrolase, alpha/beta fold family protein low similarity to hydrolase [Sphingomonas sp.] GI:3426124; contains Pfam profile PF00561: hydrolase, alpha/beta fold family -1.35 0.005 O-methyltransferase family 2 protein similar to caffeic acid 3-O-methyltransferase [Populus tremuloides][SP|Q00763] -1.31 0.006 Signaling RNA related Proteases Log2FC p-value -1.52 0.004 -1.39 0.007 -1.32 0.006 -1.30 0.008 -1.18 0.008 1.40 0.009 1.42 0.004 At3g58430 At1g08210 At4g12570 At4g04860 At5g09370 At1g10570 At4g08530 Figure S1. Transcript levels measured by microarray (log2 fold change between WT and transgenic). Plants were cultivated in 1% sucrose for 12 days and then transferred to a media without sugars for 48 h in dark before being induced with 3% glucose for 6 and 12 h. Total RNA was extracted and hybridized to the 22k (ATv3.4.2.Z) Arabidopsis microarray (Arizona University). Statistical analysis was performed with both R and excel using stringent selection criteria (p<0.01, log2 fold change >1, and net intensity >30). Significantly regulated genes were grouped into functional categories by manual curation of the currently available TAIR annotations. Within each functional category, genes are listed by the magnitude of the log2 fold change (log2FC). Blue color indicates up-regulation, whereas red color down-regulation. 3 light reactions raffinose metabolism cell wall modification lipids metabolism glycolisis N-metabolism Figure S2. MapMan overview of metabolism Expression of genes involved in metabolism after addition of 3% glucose for 6 and 12h. The experiment was carried out as described in "Materials and Methods." The results are the mean of two biological replicates. The results are displayed using the MapMan software (Thimm et al., 2004). The results are shown as fold changes between wild type to transgenic seedlings after 6 and 12 h of having being transferred to 3% glucose. All results are shown on a log2 scale coded to red or blue color. 4 glucose-6P isomerase aldolase glycealdihyde-3P-deshydrogenase piruvate kinase Figure S3 MapMan overview of glycolysis. See also legend of Fig S1 sucrose synthase small subunit of AGPase neutral Invertase sucrose transporter SUT4 ß-amylase glucan phosphorilase Figure S4 MapMan overview of sucrose synthesis and degradation, and starch metabolism. See also legend of Fig S1. 5