Supplemental Data. Takahashi et al. Plant Cell (2014) /tpc

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Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc.4.363 Fructose Glucose Sucrose Gentiobiose Gentianose Fructose STDs +GLU +INV Gentianose Supplemental Figure.

1 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc Fructose Glucose Sucrose Gentiobiose Gentianose Fructose STDs +GLU +INV Gentianose Supplemental Figure. TLC analysis of sugars hydrolyzed from gentianose. Gentianose (GF) were hydrolyzed by β-glucosidase obtained from Ustilago esculenta (GLU; Nakajima et al., ) and purified recombinant protein of INV. After hydrolysis, sugars were separated by silica gel TLC with ethyl acetate-acetic acid-water (3::) as the mobile phase and stained thymol-sufuric acid reagent. Fructose was stained pink color, but glucose was stained violet color.

2 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc cv. Maciry cv. SpB nmol mg - DW 5 5 G 3 G nmol mg - DW GF.... nmol mg - DW 5 5 GF Relative expression.... nmo mg - DW Relative expression INV INV Supplemental Figure. Variety comparison of changes in gentiooligosaccharide concentrations and INV expression in OWBs. The concentration of gentiobiose (G) and gentianose (GF), as well as the expression level of INV in OWBs of cv. Maciry and cv. SpB were compared. Each value represents the mean ± SD calculated from three independent experiments.

3 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc A [kda] [kda] M INV M INV CBB anti-myc antibody B GF hydrolytic activity (%) ph Temperature ( o C) Supplemental Figure 3. Characterization of recombinant INV protein. (A) SDS-PAGE and western-blot analysis of recombinant INV. Purified INV protein produced in P. pastoris was detected by Coomassie brilliant blue (CBB) staining or westernblotting using the anti-myc antibody. (B) The effect of ph and temperature on gentianose (GF) hydrolytic activity of INV. Each value represents the mean ± SD calculated from triplicate measurements.

Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc.4.363 GFP BF Merge At-GRP5 INV Control Supplemental Figure 4. Subcellular localization of INV-sGFP.

4 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc GFP BF Merge At-GRP5 INV Control Supplemental Figure 4. Subcellular localization of INV-sGFP. Transiently expressed GFP (control, upper-row) and GFP-fusion protein (INV, middle row) in Nicotiana benthamiana leaves. GFP channel (left), bright field (BF, center) and merged image (right) are shown. Bars indicate 5 µm. The subcellular localization of the GFP fusion protein was examined 3 days after incubation under constitutive dark conditions. The At-GFP5 protein was used as a vacuolar protein marker (Mangeon et al, 9).

Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc.4.363 T B G-AP (.

IOWBs were cultured on solid MS medium containing.5% G-AP at o C with a 6/8 h light/dark photoperiod.

5 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc T B G-AP (.5 mg/ml) T B T B T B IOWB IOWB IOWB 3 Supplemental Figure 5. Uptake of G-AP into IOWBs. IOWBs were cultured on solid MS medium containing.5% G-AP at o C with a 6/8 h light/dark photoperiod. After 4 h, IOWBs (n=3) were divided in half, and metabolites in the top (T) and bottom (B) halves of IOWBs were extracted as described in METHODS. G-AP was separated by TLC and the fluorescence of G-AP was detected.

6 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc Relative expression 4 3 PDH nmol mg - DW G+GF Supplemental Figure 6. Changes in PDH expression and the sum concentrations of gentiobiose and gentianose in field grown OWBs. The expression level of PDH and the sum of gentiobiose (G) and gentianose (GF) concentrations in OWBs of cv. SpB was measured. Each value represents the mean ± SD calculated from three independent experiments.

7 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc SiR.5 OASL.5 OASL 3 OASL3.5 GCL Relative expressions GSHS. EGS. CBL 4 MeS 6 MeS SAMS 8 SAMS 4 SAMDC.4 ACO 3 SAHH SAHH 5 5 DHAR DHAR 5 5 GR 5 5 GR Supplemental Figure 7. Changes in gentiobiose-affected gene expression in field-grown OWBs before and during budbreak. Expression levels of genes that gentiobiose induced in IOWBs were compared in field grown OWBs. Each value represents the mean ± SD calculated from three independent experiments.

Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc.4.363 Control week 4 µm ethephon week 5 mm ACC week Control 3 weeks 4 µm ethephon 3 weeks 5 mm ACC 3 weeks Supplemental Figure 8.

8 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc Control week 4 µm ethephon week 5 mm ACC week Control 3 weeks 4 µm ethephon 3 weeks 5 mm ACC 3 weeks Supplemental Figure 8. Effect of treatment of ethylene precursors on budbreak of gentian OWBs. Field-grown endodormant OWBs (G. triflora cv. SpB) harvested in September were treated with water (Control), 4 µm ethephon, or 5 mm ACC. After incubation at oc for 3 weeks, budbreak of OWBs was measured. Scale bars indicate cm.

Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc.4.363 SF ( day) % G ( day) G (nmol mg - DW) 6 4 4 36 Hours Met (nmol mg - DW).5.5 4 36 Hours SF ( days) SF (3 days) % G ( days) % G (3 days) SAM (nmol mg - DW) Glu-cys (nmol mg - DW).

9 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc SF ( day) % G ( day) G (nmol mg - DW) Hours Met (nmol mg - DW) Hours SF ( days) SF (3 days) % G ( days) % G (3 days) SAM (nmol mg - DW) Glu-cys (nmol mg - DW) Hours 4 36 Hours Cys (nmol mg - DW) GSH (nmol mg - DW) Hours 4 36 Hours Supplemental Figure 9. Effect of gentiobiose treatment on germination of Arabidopsis seeds. Arabidopsis seeds were placed on sugar-free (SF) or % gentiobiose (G)-containing MS agar medium and incubated at 4 o C for 4 days before transferring to a growth chamber at o C under continuous light (6 µmol m - s - ). Metabolites in the seeds were extracted and quantified as described in METHODS. Each value represents the mean ± SD calculated from three independent experiments.

10 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc Supplemental Table. Metabolite profiles in OWBs (G. triflora x G. scabra (5-543)) harvested in October, uary, and ch Concentrations (nmol g - DW) PC loadings October uary ch PC PC Gly 44. ± ± ± Ala 4.8 ± ± ± Asn ± ± ± Asp ± ± ± Lys 3.53 ± ± ± Gln ± ± ± Glu ± ± ± His ± ± ± Arg ± ± ± Ser 3.8 ± ± ± Pro ± ± ± Val ± ± ± Thr 495. ± ± ± Leu/Ile 4.4 ± ± ± Met.34 ± ± ± Phe 6.7 ± ± ± Tyr. ± ± ± Trp ± ± ± Putrescine ± ± ± GABA ± ± ± Spermidine ± ± ± Citrulline 6.7 ± ± ± Spermine 9.68 ± ± ± Arg-suc 4.3 ± ±..38 ± SAM 5.44 ± ± ± Glucose 583. ± ± ± Sucrose ± ± ± Gentiobiose ± ± ± Gentianose ± ± ± Arabinose 8. ± ± ± Galactose 6.67 ± ± ± AMP 4.75 ± ± ± ADP 79.4 ± ± ± ATP 83.4 ± ± ± GMP 8.9 ± ±.64.4 ± GDP 3. ± ± ± GTP 36.7 ± ± ± UDPG ± ± ± NAD ± ± ± NADP 5.35 ± ± ± NADPH ± ± ± Pyruvate ± ± ± Fumarate ± ± ± Succinate 5.78 ± ±.79.4 ± OG ± ± ± PEP.55 ± ± ± DHAP 3.74 ± ± ± Aconitate 6.66 ± ± ± Shikimate.9 ± ± ± PGA 69.9 ± ± ± Ru5P/R5P 366. ± ± ± G6P ± ± ± PG.3 ± ± ± RuBP 3.33 ± ± ± FBP 4.7 ± ± ± Isocitrate ± ± ± Citrate ± ± ± Malate ± ± ± Data presented are means ± SD of five independent experiments. DW, dry weight; GABA, γ-amino butyrate; SAM, S- adenosyl methionine; UDPG, UDP glucose; 3PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate; DHAP, dihydroxyacetone phosphate; Ru5P, ribulose 5-phosphate; R5P, Rib 5-phosphate; G6P, Glc 6-phosphate; 6PG, 6- phosphogluconate; RuBP, ribulose,5-bisphosphate; FBP, Fru,6-bisphosphate.

11 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc Supplemental Table. Metabolite concentrations in IOWBs (G. triflora (cv. Ihatovo)) cultured with sugar free (SF) or with % gentiobiose containing MS medium (% G). Metabolites AVR (nmol mg - DW) SD Ratio F-test T-test SF % G SF % G G/SF Alanine Arginine Asparagine Aspartate Citrulline Cysteine E-4 GABA Glu-Cys Glutamate Glutamine Glycine Histidine Homocysteine Isoleucine Leucine Lysine Methionine Ornithine Phenylalanine Proline SAM Serine Spermidine Spermine Threonine Tryptophan Tyrosine Valine ACC GSH E-5 GSSG OG PGA PG Aconitate Ascorbate E-6 DHA Citrate DHAP Fumarate G6P Malate NAD NADH NADP NADPH PEP Shikimate Succinate Data presented are calculated from four independent experiments. DW, dry weight; GABA, γ-amino butyrate; Glu-Cys, γ-glutamyl cysteine; SAM, S-adenosyl methionine; ACC, -Aminocyclopropane-- carboxylic acid; OG, -oxoglutarate; 3PGA, 3-phosphoglycerate; 6PG, 6-phosphogluconate; DHAP, dihydroxyacetone phosphate; G6P, Glc 6-phosphate; PEP, phosphoenolpyruvate.

12 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc Supplementary Table 3. List of primers used in this study Forward (5 3 ) Reverse (5 3 ) Cloning INV ATGCCATCCAAAAATTCAAAATCT ATAGGATCTAATGTCGGCGGAGTT HXK ATGGGGAAGGTAGCTGTGACGGCGG AGACTCCTCTAAACCAATGTACTG PGM ATGACATTCTGTACC TGTTATCACTGTTGGCTTCTC UGP ATGGAAACTCCTGCAA TATGTCTTGAGGACTGCTAAC SPS ATGGCGGGAAATGATTGGATAAACA GAGAACTTGTAGCTTCTCTAACGATG SUS ATGGCGCCTAAGTTAGAGAAAATA ATGCTCATCATCCACAGCAAG SiR ATGGCGACGTCGTATGGAGCAGCAGC CTTCTCAGCAAAACGGGAAGAGTGAG OASL ATGGCACAGGAAAAGATTGGTATT GGGATCAAAGGTCATGTTCTCTGC OASL ATGGCATCATCTTCTTCTTCTTC CAATCAGGGCACTGGTACCATCTT OASL3 ATGGCTGCTGCTGCTTTAGCAAATCT ATCAACTGACACAGGCTGCATATTC ECS ATGGCACTTATGTCACAAGCGGGATC GTATAGTAGCTCTTCGAAAACAGGT GSHS ATGGGTTCTGCATATTCTTCTTCTGA CCAACCGTAACAGCTGCATTTGCAC CBL ATGGCGTCTTCCTTGTTCCGTCAA AGCCGGTGCAGTTCTTAGTGCGTAAT CGS ATGGCCGTCTCCAGCTGCGCTAGGGT ATGGTCTCCAAAGCCTGCAGGATA MeS ATGGCATCACACATCGTTGGATACCC TTTGGCACTTGAGAGCTGTGTGCGG MeS ATGGCATCCCACATCGTTGGATATC TTTAGCACTGGCAAGCTGTGTGCGG SAMS ATGGACACTTTTCTATTCACCTCA AGCTTTGGGCTTGAGGATCTTGACA SAMS ATGGATACCTTCTTGTTCACTTCTG TGGCTTGTCGCTTATGAGGGGCTTG SAMDC ATGTCGGAAATGCCGGTCTCTGCGA CTCCTTTTCTTCATCTTCATCCTCCT ACO ATGGCATTCCCAATAGTAAACATGGAGAAG TCAAACGGGGGCAATGGGTCCCAAATTAATG SAHH ATGTCTCTCTCCGTCGAGAAGACCA ATACCTATAGGCCAAAGGCTTGTAT SAHH ATGTCTTTGCTCGTGGACAAAACCA ATACCTATAGTGAAGAGGCTTATAA DHAR ATGTCGACCGCCAAGTTAACATCA ACCACCCATCACCTTCGGCCGCCA DHAR ATGGCTCCGCCCGTTGAAGTAGCT TGCCTCAACCTTTGGCTTCCATCC GR ATGTCGGGTTTGGGAAGGAAGATG AAGGTTTGTTTTCGGTTTAACCGC GR ATGGCGACGTCTCTTGCTACGCCG GACCCCAGCAGTGGCTTTGATCTC AtGRP5 ATGGCTTCCAAGTCACTCTTTCTTGT ATGATGTCCACCACCGAAACCACCT Real-time PCR INV GTGGGTCTCGGGTTGAGATA AATCCAGCCCCATAAAATCC HXK TGTGATATTGGGAACGGGAACTA ATCTTGCCACTTGGGAATCG PGM TTGGGTGGTGATGGTCGAT ACCATTGCCTGCGGAAATT UGP TGAGTTGGGACCCGAGTTTAAG CACGATACTGGGAATGGATTTGA SPS CCTTTGGAGAATGTCGACCTTT CTCATCGATATTATCCCGGTTACC SUS CTGGAGTTTATGGCTTCTGGAAAT GAACATCTCAAGGTAACGCCTTGT SiR CCACTTTGTCCTTTGGCAATC CCCTCTTAAGCACATCCGGTAT OASTL AAAACCTGGCCCGCATAAA CAAAACACCAGGAATGAATCCA OASTL TTTCCTGGCCATCAGATTTGT AAGGTAGCCAACCCAATGTCA OASTL3 TCTTCCCTAGTCACTCCATTTGC ATTCGAACTGTGGGTCCAGAA ECS AACCCAACGGTTATCTCAGCAT TCCGATCTCCATCTGTGTCAAG GSHS GCTTCCCTTCCTTCGAATCTG CTGCCTTCTCGGTCTCCATTT CBL TTGGAAGCGTAAAGTCCCTCAT GGTATGCTCGCGTGTGACA CGS GGGCCTAAAAAGTCATCCTGAA CACCACCAAAACCAGTCATCTG MeS GCAATCTTATGGTTCTCGTTGTGT GCTTGGGTCGACTCACATCA MeS GGGTCAACCCGGATTGTG CAGAGCCGGCTTGACTTCTC SAMS TTCGGGATACCTGCAGAGAGA TCAGCATCCAGGCCAACA SAMS TGCCTCTGAGCCATGTCCTT TCTTGCGGACTTCGGTCAA SAMDC AATTATACACGTGGGAGCTTCATCT GAAGCTACGATGCGGAAACG ACO GAAGCTGTTGAGGGTGAAGTCA TCAGGGATTTCAGAGATGTTGGA SAHH TCGACATGCTCGGGTTAGAGA GGTTTGTGGCTTGATCGTGAT SAHH GAATTCAAAACGTCTGGGACAAT ACGATCTGGAACTCAGCATTATCA DHAR GGGAAGCCGGCGAGTAA CGCCGACTTCGCCATTAAT DHAR AGATCCCAATGATGGCTCAGA GATGCTCATCCAATGCTTTCAA GR GCCTGTTGCCTTGATGGAA TCGGCTGCTCACCAAAAAC GR CCTTCGAAGCCCAAAAAGATT CCAGCAAACTCAACAGCGATAT UBQ GGAAGCAGTTGGAAGATGGC GGCCAAAGTTCGTCCGTCCTC Vector construction for protein expression CCGCTCGAGAAAAGAATGCCATCCAAAAATT INV CAAAATCTC TGCTCTAGAAGATAGGATCTAATGTCGGCGGA GTT

13 Supplemental Data. Takahashi et al. Plant Cell (4).5/tpc Supplemental references Nakajima, M., Yamashita, T., Takahashi, M., Nakano, Y., Takeda, T. (). Identification, cloning, and characterization of β-glucosidase from Ustilago esculenta. Appl. Microbiol. Biotechnol. 93: Mangeon, A., Magioli, C., Menezes-Salgueiro, A.D., Cardeal, V., de Oliveira, C., Galvao, V.C., gis, R., Engler, G., and Sachetto-tins, G. (9). AtGRP5, a vacuole-located glycine-rich protein involved in cell elongation. Planta 3:

Amino Acids. Amino Acids. Fundamentals. While their name implies that amino acids are compounds that contain an NH. 3 and CO NH 3

Amino Acids. Amino Acids. Fundamentals. While their name implies that amino acids are compounds that contain an NH. 3 and CO NH 3 Fundamentals While their name implies that amino acids are compounds that contain an 2 group and a 2 group, these groups are actually present as 3 and 2 respectively. They are classified as α, β, γ, etc..