respect to the wild type in the later stages of growth. Photograph showing representative plants after 5 and 10 weeks growth.

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A Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc.11.81224 C B.5 Supplemental Figure 1.

SDH2 amino-acids sequences were aligned using the ClustalW alignment program (Higgins and Sharp, 1988).

l. (Solanum lycopersicum; SDH2-1, SGN-U563725; SDH2-2, SGN-U563726 ); S.t. (Solanum tuberosum; TC17348) ; N.b. (Nicotiana benthamiana; TC12172); A.

(Zea mays; AJ12374); S.o. (Saccharum officinarum; TC73886); C.r. (Chlamydomonas reinhardtii; XP_169629); S.c. (Saccharomyces cerevisiae ; J5487 ); H.

(B) Construction of a chimeric gene for the expression of tomato SDH antisense consisting of a 514 bp fragment encoding the CaMV 35S

1 A Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc C B.5 Supplemental Figure 1. Characterization and expression of tomato SDH2-2. (A) Dendogram of iron sulphur subunit of SDH. SDH2 amino-acids sequences were aligned using the ClustalW alignment program (Higgins and Sharp, 1988). The abbreviation and taxonomic names of the species and the accession numbers of the sequences used in the phylogenetic analysis are: S.l. (Solanum lycopersicum; SDH2-1, SGN-U563725; SDH2-2, SGN-U ); S.t. (Solanum tuberosum; TC17348) ; N.b. (Nicotiana benthamiana; TC12172); A.t. (Arabidopsis thaliana; AJ27891 (AtSDH2-1); AJ (AtSDH2-2); AJ (AtSDH2-3); O.s. (Oryza sativa; AB17428); Z.m. (Zea mays; AJ12374); S.o. (Saccharum officinarum; TC73886); C.r. (Chlamydomonas reinhardtii; XP_169629); S.c. (Saccharomyces cerevisiae ; J5487 ); H.s. (Homo sapiens ; U17248 ); D.m. (Drosophila melanogaster ; L2775 ); P.t. (Phaeodactylum tricornutum ; XM_ ). (B) Construction of a chimeric gene for the expression of tomato SDH antisense consisting of a 514 bp fragment encoding the CaMV 35S promoter and a 825- bp (antisense orientation) and the 2 bp ocs terminator; (C) Transgenic plants showed an enhanced aerial biomass with 1 respect to the wild type in the later stages of growth. Photograph showing representative plants after 5 and 1 weeks growth.

2 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc (A) SDH Relative values (B) SDH2-1 Relative values SDH 52 Supplemental Figure 2. Expression of tomato succinate dehydrogenase in epidermal fragments. (A) and (B) Relative transcript abundance of mitochondrial Complex II subunits (SDH2-2 and SDH2-1, respectively). The abundance of SDH mrnas was measured by quantitative RT-PCR and values are presented as mean ± SE of six individual plants per line. The expression of both isoforms was relatively low in lower epidermal fragments and unaltered in lower epidermal fragments of the transformants. Analyses were done in epidermal fragments of fully expanded leaves of 4-week-old plants. The lines used were:, black bars; SDH14, white bars;, dark grey bars; FL11, light grey bars, FL41, white bars., wild type. 2

3 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc SGN-U57313 SGN-U SGN-U SGN-U56331 SGN-U SGN-U56626 SGN-U58678 SGN-U FL 11 FL Relative expression Supplemental Figure 3. Relative expression of off target genes supposed to effect succinate dehydrogenase sequence in tomato. The transcripts analyzed here were: SGN-U579957, (glycolate oxidase, GOX); SGN-U58678, ribulose-phosphate 3-epimerase(R5P3E); SGN- U56626, similar to lipase (Arabidopsis thaliana); SGN-U584266, UDP-sulfoquinovose:DAG sulfoquinovosyltransferase / sulfolipid synthase (SQD2); SGN-U56331, CTD phosphatase-like protein 3 (CPL3); SGN-U591223, subtilase family protein; SGN-U595977, hypothetical protein; SGN-U57313, unnamed. Analyses were done in fully expanded leaves of 4-week-old plants. Values are presented as means ± SE of six individual plants. The lines used were:, black bars; SDH14, grey bars;, dark grey bars; FL11, light grey bars, FL41, white bars., wild type 3

4 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc Relative expression µmol DCPIP s -1 kg -1 protein SDH Plant age (weeks) Supplemental Figure 4. Analysis of succinate dehydrogenase expression and activity in tomato plants during normal plant development. (A) Relative transcript abundance of mitochondrial SDH2-2. The abundance of SDH mrnas was measured by quantitative RT-PCR and data were normalized to the mean response calculated for the 4-week-old leaves of the wild type; (B) The succinate-dependent dichlorophenolindophenol (DCPIP) reduction determined in enriched mitochondria from tomato leaves. The analyses were performed in four to nine week old plants, using fully developed source leaves. The youngest, fully expanded, outer leaves (third to fifth pair from the apex of plagiotropic branches) were harvested in the middle of the light period. Values are means ± SE of six independent samplings. Asterisk indicates values that were determined by the Students t-test to be significantly different (P <.5) from the wild-type within each week. The lines used were:, black bars; SDH14, white bars;, grey bars; SDH 52, dark grey bars. 4

5 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc [I] C1 C2 C3.4 C CO 2 evolution (as a % of 14 C applied) [II] [III] [IV] SDH Incubation time (h) Supplemental Figure 5. Carbon dioxide evolution from positionally labeled glucose in leaves of antisense SDH tomato plants. Evolution of 14 CO 2 from isolated leaf discs in the light. The leaf discs were taken from 4- week-old plants and were incubated in 1 mm MES-KOH solution, ph 6.5,.3 mm glucose supplemented with 2.32 kbq ml 1 of [1-14 C]-, [2-14C]-, [3,4-14 C]-, or [6-14 C]-glucose at an irradiance of 2 μmol m 2 s 1. The 14 CO 2 liberated was captured (at hourly intervals) in a KOH trap and the amount of radiolabel released was subsequently quantified by liquid scintillation counting. Values are presented as means ± SE of determinations on six individual plants per line. An asterisk indicates values that were determined by the 5Student s t test to be significantly different (P <.5) from the wild type.

6 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc Stomatal density (mm -2 ) A B Stomata index (%) C Stomatal aperture (µm) D Length of stomatal pore (µm) SDH 52 SDH 52 Supplemental Figure 6. Stomatal properties of 4-week-old transgenic and wildtype tomato plants. Stomata density (A), stomatal index (B), stomata aperture (width) (C), and length of stomatal pore (D). Stomatal properties were determined in five to eight different fields of.55 mm2 per leaflet and aperture measurements were determined in 9 to 12 guard cell pairs Data showed are mean ± SE obtained from six individual plants per line. The lines used were:, black bars; SDH14, light grey bars;, grey bars; SDH 52, dark grey bars. An asterisk indicates values that were determined by the Student s t-test to be significantly different (P <.5) from the wild type (). 6

7 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc Fresh weight loss (%) FL11 FL Time (min) Supplemental Figure 7. Fresh weight loss from detached leaves of wild type and transgenic tomato plants. Data show percentage of initial fresh weight lost from detached leaves incubated under greenhouse conditions. Results from one out of three representative experiments are shown. Analyses were done in fully expanded leaves of 4-week-old plants. Values are presented as means ± SD of six individual plants. The lines used were: wild type (), black triangles; SDH14, light grey triangle;, white square; FL11, dark grey circle, FL41, grey circle. 7

8 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc A specific leaf area Total leaf area (m 2 ) (m 2 kg -1 ) B SDH 52 Supplemental Figure 8. Growth variables of leaves of 4-week-old transgenic and wild-type tomato plants. Specific leaf area (A) and total leaf area (B). Data showed are mean ± SE obtained from six individual plants per line. The lines used were:, black bars; SDH14, white bars;, grey bars; SDH 52, dark grey bars. 8

9 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc O 2 uptake (nmol min -1 µg -1 Chl) A O 2 uptake (nmol min -1 µg -1 Chl) E O 2 evolution (nmol min -1 µg -1 Chl) B O 2 evolution (nmol min -1 µg -1 Chl) F 2 Fumarate (nmol µg -1 Chl) Malate (nmol µg -1 Chl) C D FL 11.. FL 41 Fumarate (nmol µg -1 Chl) Malate (nmol µg -1 Chl) G H FL 11 FL 41 Supplemental Figure 9. Respiratory and photosynthetic activities of GCP and MCP. GCP (guard cell protoplasts) and MCP (mesophyll cell protoplast) were isolated from leaves of wild type (), SDH and fumarase antisense lines. All measurements were performed in 4-5-week-old plants. Respiratoy O 2 uptake (A and E), photosynthetic O 2 evolution (B and F), malate (C and G) and fumarate (D and H) were analyzed either in GCP (A to D) or MCP (E to H). Data showed are mean ± SE of at least 4 biological replicates obtained from 4 independent experiments with comparable results. The lines used were:, black bars; SDH14, grey bars;, dark grey bars; FL11, light grey bars, FL41, white bars. An asterisk indicates values that were determined by the Student s t test to be significantly different (P <.5) from the. 9

10 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc Relative Stomatal Aperture Control ABA Malate Fumarate CsCl ABA+ CsCl Malate+ CsCl Fumarate+ CsCl Malate+ ABA Fumarate+ ABA FL 11 FL Supplemental Figure 1. Relative stomatal movement under malate, or fumarate and its combination with ABA and channel blocker. Effects of Malate and Fumarate and its combinations with ABA and CsCl. Detached leaves were floated on stomatal opening buffer (1 mm MES, 5 mm KCl, 5 μm CaCl 2, ph 6.15) under illumination for 2 h to induce stomatal opening, then treated with either 2 mm malate, 2 mm fumarate, 2 mm CsCl or 5 μm ABA and the combinations showed for additional 1. h. Thereafter stomatal apertures were measured. Data showed are mean ± SE of at least 12 stomata obtained from 3 independent experiments with comparable results. Data are normalized with respect to the mean response calculated for the wild type in the control treatment (to allow statistical assessment, individual plants from this set were normalized in the same way). The lines used were:, black bars; SDH14, dark grey bars;, grey bars; FL11, light grey bars, FL41, white bars. An asterisk indicates values that were determined by the Student s t test to be significantly different (P <.5) from the wild type or the corresponding control. 1

11 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc A B Malate (mmol kg -1 FW) µmol DCPIP s -1 kg -1 protein D Time (min) E C Fumarate (mmol kg -1 FW) MYB6:SDH-2 MYB6:SDH-3 MYB6:SDH-5 MYB6:SDH-8 MYB6:SDH-2 MYB6:SDH-3 MYB6:SDH-5 MYB6:SDH-8 Supplemental Figure 11. Physiological characterization of SDH2-2 lines under the control of a guard cell specific promoter MYB6. (A) The succinate-dependent dichlorophenolindophenol (DCPIP) reduction determined in enriched mitochondria from tomato leaves; malate (B) and fumarate (C) content were determined in leaves of 4-week-old transgenic and wild-type tomato plants; (D) Time taken for stomatal opening following a dark to light transition. (E) Time taken for stomatal closure following a light to dark transition. Note that there were no significant difference in final stomatal conductance in these experiments. Values are presented as mean ± SE of six individual plants per line. Values were determined by the Student s t- test to be not significantly different from wild type (P <.5). 11

12 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc Supplemental Table 1. Pigment contents in antisense succinate dehydrogenase tomato plants. Pigments were determined in 4-week-old fully expanded source leaves harvested 6 h into the photoperiod. Values presented are means ± SE of six individual plants per line. Values in bold were determined by the Student s t-test to be significantly different (P <.5) from the wild type (). SDH14 SDH43 SDH52 µmol kg -1 FW Neoxanthin 19.7 ± ± ± ± 5.4 Violaxanthin ± ± ± ± 5.7 Antheraxanthin 13.3 ± ± ± ±.5 Zeaxanthin 22.2 ± ± ± ± 1 β-carotene 11.1 ± ± ± ± 3.6 Lutein ± ± ± ± 18.6 Chlorophyll a ± ± ± ± Chlorophyll b ± ± ± ± 31.9 Chlorophyll a/b 3.1 ± ± ± ±.1 12

13 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc Supplemental Table 2. Relative metabolite content of the fully expanded leaves from 4-weekold-plants of the antisense succinate dehydrogenase plants. Leaves were harvested 6 h into the photoperiod. Data are normalized with respect to the mean response calculated for the wild type () (to allow statistical assessment, individual plants from this set were normalized in the same way). Values presented are the mean of relative values ± SE of measurements from six individual plants per genotype. Values in bold were determined by the Student s t-test to be significantly different (P <.5) from the. Amino acids SDH 52 Acetyl-Ala 1. ± ± ± ±.15 Acetyl-Gly 1. ± ±.9.99 ± ±.11 Acetyl-Ser 1. ± ± ±.5.37 ±.6 Alanine 1. ±.7.82 ± ± ±.9 Asparagine 1. ± ± ± ±.8 Aspartate 1. ±.6.64 ± ±.8.76 ±.13 Cysteine 1. ± ± ± ±.15 Glutamate 1. ± ± ± ±.6 Glutamine 1. ± ± ± ±.14 Glycine 1. ± ±.5.47 ±.6.49 ±.3 Homoserine 1. ± ± ± ±.13 Isoleucine 1. ±.9.72 ±.6.78 ±.4.77 ±.3 Norleucine 1. ±.9.58 ±.8.64 ± ±.7 Ornithine 1. ± ±.7.54 ±.9.5 ±.6 Phenylalanine 1. ± ±.5.59 ±.9.77 ±.1 Serine 1. ±.7 1. ± ± ±.1 Threonine 1. ±.1.88 ±.7.78 ±.7.8 ±.14 Organic acids 2-oxogluconate 1. ± ± ± ±.14 2-oxoglutarate 1. ±.13.8 ± ±.2.55 ±.4 6-amino caproic acid 1. ± ±.7.54 ±.1.69 ±.11 Citrate 1. ±.7.71 ± ± ±.6 Dehydroascorbate 1. ±.9.93 ±.1.96 ± ±.15 Glutarate 1. ± ± ±.6.51 ±.11 Glycerate 1. ±.6.71 ±.9.53 ± ±.4 Isocitrate 1. ±.6.54 ±.6.55 ±.4.71 ±.2 Maleate 1. ±.2.67 ± ±.6.22 ±.4 Oxaloacetate 1. ± ± ± ±.19 Phenylpyruvate 1. ± ± ±.9.96 ±.12 Pyroglutamate 1. ± ±.9.59 ±.8.68 ±.9 Pyruvate 1. ±.9.36 ±.5.31 ±.6.38 ±.7 Succinate 1. ± ± ± ±.18 Sugars Fructose 1. ± ± ± ±.12 Glucose 1. ±.7 2. ± ± ±.12 Maltose 1. ± ± ± ±.13 Ribose 1. ± ± ± ±.39 Sucrose 1. ± ± ± ±.13 Sugar alcohols Galactinol 1. ± ± ± ±.14 Glycerol 1. ± ± ± ±.5 Inositol 1. ± ± ± ±.12 Mannitol 1. ± ± ± ±.14 Sorbitol 1. ± ± ± ±.14 Other metabolites Putrescine 1. ± ± ± ±.32 Shikimate 1. ± ±.7.35 ±.9.46 ±.7 Spermine 1. ± ± ± ±.12 13

14 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc Supplemental Table 3. Gas exchange parameters derived by fitting A/C i data. Maximum carboxylation velocity of Rubisco (V cmax ), maximum electron transport rate (J max ), triose phosphate use (TPU) and J max /V cmax ratio variables were computed from the A/C i curve using the A/C i curve fitting model developed by Sharkey et al. (27). Values presented are means ± SE of six individual plants per line. Values in bold were determined by the Student s t-test to be significantly different (P <.5) from the wild type (). SDH14 SDH43 SDH52 V cmax (µmol m -2 s -1 ) 9.33 ± ± ± ± 2.74 J max (µmol m -2 s -1 ) ± ± ± ± 1.49 TPU (µmol m -2 s -1 ) 8.78 ± ± ± ±.19 J max /V cmax 1.25 ± ± ± ±.5 14

15 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc Supplemental Table 4. Primers used in the RT-PCR analyses performed in this study. Target Sequence ABCB 14 R 5 -ACAGTGTTCGCGCTCTTGACTG -3 ABCB14 F 5 -TCCCTGATGCAAGGTAGAACCG -3 ABI2 F 5 - AGTGAAACAGACGGGATTGAGC -3 ABI2 R 5 - AGCTGTAGATCCAACTGTCTCAGG -3 AHA1 F 5 - AGCTGGTCCTTCGTTGAACGTC -3 AHA1 R 5 - TGGCAACCAATTGGGCAATCAAG -3 CaSF 5 - ACCAATTGCCACATCCACAGTTG -3 CaSR 5 - ATAATGTGCCACCAGCCACTGC -3 CCR2 F 5 - CCAATCGGCCGAATTCCCATTG -3 CCR2 R 5 - CGACGAAGCACCTGTATTCAACG -3 CHX2 R 5 - ACTGCTGCCAATGTACCACACAC -3 CHX2 F 5 - TGGCTAAACGTTGTTCGGATGGG -3 CIPK15 F 5 - TCGCGACTACAATGTCAGCAAG -3 CIPK15 R 5 - GTTGTCTTTGCTACCTCCTCAAGC -3 CPK6 F 5 - TGTGGTGGCGGTGAATTGTTTG -3 CPK6 R 5 - TCAAATCGGCAGCCTTTCTCTCG -3 GPX3 F 5 - TGTAACCAGTTCCTGTGGCAAG -3 GPX3 R 5 - CTGGTGCATACAGTTTGCTGAATC -3 GTG1 F 5 - GGTTCAGGCCTTAGAGGAGCTATC -3 GTG1 R 5 - AGCAGCAGCTTCCTTAGCTTGG -3 KAT2 F 5 - TCAGCAGCAAACCAACCTGACTC -3 KAT2 R 5 - ACTTCTTGGTTCTGTCCGCTTGC -3 NIA2 F 5 - TGGCAAACACATATTCCTCTGTGC -3 NIA2 R 5 - TCGACTGTGCTAGTAGGCGTGTAG -3 OST1 F 5 - TGGATGGAAGTCCTGCACCAAG -3 OST1 R 5 - GTTGTGAATGCAACACCGAGGAC -3 PHOT1 F 5 - AGGATCTCGTGAAGGAGCAAACG -3 PHOT1 R 5 - CAATTCACACCGCGGAAGAACG -3 PHOT2 F 5 - TGCGATCGATTGGTGGGCTTTAG -3 PHOT2 R 5 - TGGTGTACGCCCATAGAGCATC -3 PLDALPHA1 F 5 - TTCTGAGAGCCCAGAGCCTGATTC -3 PLDALPHA1 R 5 - TCATGAAACGTCGTGCCTCCTG -3 RbcS F 5 - CGTTAGATGCATGCAGGTGT -3 RbcS R 5 - CATCGGAGAAGTCAGGAAGG -3 RBOHB F 5 - ACCAGTGTGAGGACACATTTCGC -3 RBOHB R 5 - TGCATGCTTGGTTAAGGTCTTGG -3 SDH2-1 R 5 - GGTTATCTGGATTCCATCGG-3 SDH2-1 F 5 - GCAGGTCCAACCTCAAGTC-3 SDH2-2 R 5 - GTTGTCTGGAGTCCATCGG-3 SDH2-2 F 5 - AGCACAGAAGGTAGAATCTAAG-3 STP1F 5 -GGGCCCACTTGGATGGTTAATTCC-3 STP1R 5 -ACCAGCTGAACGAGTTTCCAAAGG-3 tdt F 5 -ACGACGAAATTCCGAACAACGG -3 tdt R 5 -CGATGCCGGTAACTTCCATGTC -3 TIP4;1 F 5 - AAGTGGAATCTGGACTGACCATTG -3 TIP4;1 R 5 - CAAGCCCACCACCAATGAAAGG -3 UBI3 F 5 -AGGTTGATGACACTGGAAAGGTT-3 UBI3 R 5 -ATCGCCTCCAGCCTTGTTGTA-3 SGN-U579957_F 5 -ATTGTCATCCTGGGCCACTTCC-3 15

16 Supplemental Data. Araújo et al. (211). Plant Cell 1.115/tpc SGN-U579957_R SGN-U58678_F SGN-U58678_R SGN-U56626_F SGN-U56626_R SGN-U584266_F SGN-U584266_R SGN-U56331_F SGN-U56331_R SGN-U591223_F SGN-U591223_R SGN-U595977_F SGN-U595977_R SGN-U57313_F SGN-U57313_R 5 -CATAAAGCTGGAAGAAACGGATGC-3 5 -TGCGCCCTATCACAGATCTTCC-3 5 -AGGTACTCTCTGGTCAGGTTCGAC-3 5 -TGATTCAGCTGGGAGCAACACG-3 5 -GAAGGCATCCGAGAGGGTAAATGC-3 5 -ACCTCTCTCACTTGCTCTCAGC-3 5 -TCAGGCTTGAACTGTGCTACCTC-3 5 -TCTGGCAGGTTGCCGTATTGTC-3 5 -TGCCACAGAGGATGCAAATGGG-3 5 -CCAAGTGATTGACCCAACGTTCC-3 5 -GCGAACTCTGGTGTTCAAGGAG-3 5 -GGCAGTGGTTTGAGTTCTCTGC-3 5 -TCCCTTTAACCTCCGTCAACGC-3 5 -TTGTTCGGATCCTCGGGTCTTG-3 5 -AACCTCCACGCCACATCACATTC-3 16

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm, Germany b

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm, Germany b The Plant Cell, Vol. 23: 600 627, February 2011, www.plantcell.org ã 2011 American Society of Plant Biologists Antisense Inhibition of the Iron-Sulphur Subunit of Succinate Dehydrogenase Enhances Photosynthesis