SUPPLEMENTAL DATA

Supplemental figures

Supplemental Figure 1. Gene structures of CBSX1 and sequence alignment between

CBSX1 and CBSX2.

(A) CBSX1 contained seven introns and eight exons. The closed boxes indicate exons and

the lines indicate introns.

(B) The N-terminal signal sequence for targeting to the chloroplast is also included for alignment,

and the predicted cleavage sites for CBSX1 and CBSX2 are indicated as black and blue arrows,

respectively (as predicted by ChloroP server, http://www.cbs.dtu.dk/services/ChloroP/). The

secondary structural elements at the top of each alignment correspond to those of CBSX2.

The boxes colored green, yellow and red indicate, respectively, the first CBS domain (CBS1),

the second CBS domain (CBS2), and an additional a-helical segment (a5) of corresponding

colored regions in Figure 2A. Invisible segments in the structure are marked as black dots.

Strictly conserved and conservatively substituted residues are boxed in red and in white,

respectively. Every 10th residue for CBSCX1 and CBSX2 is indicated at the top and bottom

of each alignment, respectively.

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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Supplemental Figure 2. Expression patterns and subcellular localization of CBSX1.

From 57 proCBSX1:GUS T2 transgenic lines, six individual lines were selected and examined

for their GUS expression. One of those six individual GUS lines was shown in Figure 1.

Two additional GUS-expressing lines are presented here from (A) to (H). (A)-(D) the second

GUS-expressing transgenic plant. (E)-(H) the third transgenic plant.

(A) and (E) Cotyledon.

(B) and (F) Inflorescence cluster.

(C) and (G) Rosette leaf.

(D) and (H) Cauline leaf.

(I) Subcellular localization of CBSX1:smGFP protein. The guard cells of

Pro35S:CBSX1:smGFP transgenic Arabidopsis seedlings (T2 generation) were observed

under a fluorescence microscope.

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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Supplemental Figure 3. Size exclusion chromatography (Superose 12 GL 10/300;

Amersham-Pharmacia) elution profile of the CBSX1 homodimer.

CBSX1 protein formed a homodimer. Red line represents gel filtration elution profile of

CBSX1. Dashed-gray colored lines represent molecular weight marker, 67kDa (BSA) and

45kDa (Ovalbumin), respectively.

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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Supplemental Figure 4. Ribbon diagram comparing the overall structure of CBS

domain proteins.

Plant (CBSX2 from Arabidopsis thaliana), bacterial (Bacillus subtilis and Escherichia coli),

archaeal (Methanococcus jannaschii), lower eukaryotic (Schizosaccharomyces pombe) and

higher eukaryotic (Homo sapiens) CBS domain proteins are compared in the same view to

Figure 2C (right) in the main text. PDB ID codes and Z-scores from DALI server for each

structure are provided. Two-fold molecular symmetry axes are also indicated.

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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Supplemental Figure 5. Enhancing the activities of Trxs and FTR in the presence of

CBSXs and adenosine-containing ligands.

(A) Assay for NADPH oxidation through the reduction of Trx m by FTR, coupled to insulin

reduction by Trx m. The consumption of NADPH was measured by absorbance at 340 nm;

values represent means ± SD (n=3).

(B) Assay for reduction activity of Trx m using insulin substrate measured by the turbidity at

650 nm. The values are normalized to the activity of Trx m only designated as 1.0. Error bar

was calculated based on three independent experiments. Values are means ± SD (n=3).

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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(C) Assay for the reducing activity of Trx f, m, x, and y using insulin protein and measured

by absorbance at 650 nm. The error bars were calculated based on three independent

experiments. The values are means SD (n=3).

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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Supplemental Figure 6. Dehiscence and senescence of wild-type and 35:CBSX1

transgenic plants.

(A) and (B) Inflorescences of wild-type and 35S:CBSX1. A, anther; Ov, ovary; Sg, stigma; Sy,

style; PG, pollen grain.

(C) A series of flower buds on an inflorescence of the wild type and the 35S:CBSX1 plants.

Developmental series of flowers from young to old within a single inflorescence were

photographed using a dissecting microscope.

(D) Delayed apical senescence phenotype of 35S:CBSX1 plants.

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Supplemental Figure 7. Images of scanning electron microscopy and transmission

electron microscopy.

(A) SEM images of anther. A very narrow crevice was formed in the stomium region.

(B) SEM images of stigma. There were no phenotypic alterations between the wild-type and

the transgenics.

(C) TEM images of the secondary cell wall of the anther.

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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Supplemental Figure 8. Expression levels of NST1, NST2 and MYB26 and expression level of CBSX1 under various stress condition and hormone treatment.

Legend on next page

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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Supplemental Figure 8, cont. Expression levels of NST1, NST2 and MYB26 and expression

level of CBSX1 under various stress condition and hormone treatment.

(A) Expression patterns of the NSTs and MYB26 gene by Real Time-PCR from 35S:CBSX1,

wild-type, and cbsx1 buds.

(B) Expression patterns of the CBSX1 gene by Real Time-PCR and RT-PCR under various

stress condition in seedling stage.

(C) Expression patterns of CBSX1 gene by Real Time-PCR and RT-PCR under plant

hormones treatment in seedling stage.

Supplemental Data. Yoo et al. (2011). Plant Cell 10.1105/tpc.111.089847

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Supplemental Tables

Supplemental Table 1. Proteins interacting with CBSX1.

No. Description Locus No.1 ATF1, TRXF1, Thioredoxin f-type 1 At3g027302 ATHM2, Thioredoxin m-type 2 At4g035203 ATX, ATHX, Thioredoxin x At1g503204 ATY1, TRX-Y1, Thioredoxin y1 At1g767605 BHLH101 At5g041506 S-adenosyl-L-methionine-dependent methyltransferases superfamily protein At3g173657 CKX7 At5g214828 Sec14p-like phosphatidylinositol transfer family protein At1g721609 Aconitase/3-isopropylmalate dehydratase protein At2g4309010 Pyruvate kinase family protein At5g5635011 Unknown protein At1g2214012 Translation initiation factor SUI1 family protein At4g2713013 HAP5A, NF-YC1, ATHAP5A At3g4859014 REF1, ALDH1A, Aldehyde dehydrogenase 1A At3g2450315 UDP-Glycosyltransferase superfamily protein At4g2756016 ATP-dependent protease La (LON) domain protein At1g1974017 PSBR, Photosystem II subunit R At1g7904018 Unknown protein At1g5012019 Acylphosphatase family At5g0337020 Ribulose bisphosphate carboxylase (small chain) family protein At5g3843021 FBA1, Fructose-bisphosphate aldolase 1 At2g2133022 P-loop containing nucleoside triphosphate hydrolases superfamily protein At3g1035023 Expressed protein At1g1176024 Unknown protein At2g4639025 PI4KBETA1, Phosphatidylinositol 4-OH kinase beta1 At5g6407026 Lactate/malate dehydrogenase family protein At1g5324027 HSK, Homoserine kinase, DMR1, Downy mildew resistant 1 At2g1726528 Rhodanese/Cell cycle control phosphatase superfamily protein At2g4076029 ATGRX2, Glutaredoxin, CXIP2, CAX-interacting protein 2 At2g38270

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Supplemental Table 2. Data collection and refinement statistics

MAD Native Peak Edge RemoteH RemoteL

Data Collection Space group P6522 Cell dimension A, b, c (Å ) 73.045, 73.045, 253.453

�, �, � (°) 90, 90, 120No. of molecules/asu 2 X-ray sources a PAL 6B PF AR-NW12A Resolution range (Å ) b 1.91 (1 .97-1.91) 2.30 (2.38-2.30) Wavelength 1.23985 0.97888 0.97928 0.95000 0 .98317 Rsym (%) c 5.7 (39.4) 5.3 (20.2) 4.6 (20.1) 4.8 (20.3) 4 .3 (46.7)Total reflections 797,712 413,069 410,408 411,024 413,300 Unique reflections 31,938 18,832 18,731 18,821 18,920 Completeness (%) b 99.4 (97.6) 99.3 (94.9) 99.3 (94.8) 98.9 (91.9) 99.4 (96.0) I/� (I) b 45.0 (2 .2) 55.9 (5.9) 55.32 (5.6) 53.9 (5.5) 55.0 (5.5)Redundancy b 25.0 (15.3) 21.9 (16.5) 21.9 (16.4) 21.8 (16.2) 21.8 (16.2) FOM d 0.571/0.690 (SOLVE/RESOLVE)

Refinement Resolution range (Å ) 35.13-1.91

No. of reflections 29,856 Rwork / Rfree (%) e 0.220/0.247 No. of atoms

Protein 2,174 Water 219 Ions 1 (glycerol), 7 (acetate)

R.M.S. deviations Bond lengths ( ) 0.007 Bond angles (°) 1.153

Ramachandran plot Most favored regions (%) 98.53 Additional allowed regions (%) 1.47 Disallowed regions (%) 0.00 PDB ID 3SL7

a PAL, Pohang Accelerator Laboratory, Korea; PF, Photon Factory, Japan

b Values in parentheses are for reflections in the highest resolution bin

c Rsym = �h�i�I(h,i)-<I(h)>�� �h�i I(h,i), where I(h,i) is the intensity of the ith measurement of h and < I(h)> is the corresponding average value for all I measurements. d Figure of merit = �� P(�)ei�� � P(�)�, where P(�) is the phase probability distribution and � is the phase. e Rwork and Rfree = ���FO�-�FC��� � �FO� for the working set and test set (10%) of reflections.

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