Supplementary Figure 1 Chloroplast ultrastructure of mesophyll cells in the wild-type and er-ant1 mutant. The

seedlings were grown in the field for 12 or 15 DAG. All transmission electron microscopy samples were obtained

from the second leaf blade of both WT and er-ant1 mutant seedlings.

Supplementary Figure 2 Alignment of the deduced amino acid sequence of OsER-ANT1 with its homologs.

Identical amino acids in the proteins are shown as white letters on a black background. The predicted N-terminal

mitochondrial transit peptides of AAC1 to AAC3 are indicated by black squares. Asterisk (*) indicate amino acid

residues conserved in AACs with RRRMML motif, but not conserved in other AACs. Blue squares indicate

conserved amino acid residues L within RRRMML motif, which is only possessed by ER-located AACs. Sources

of AAC sequences are as follows: AtER-ANT1, AtAAC1 to AtAAC3, Arabidopsis thaliana (At5g17400,

At3g08580, At5g13490, At4g28390); OsER-ANT1, Oryza sativa L (Os11g0661300); M. truncatula, Medicago

truncatula (ABE81429); V. vinifera, Vitis vinifera (GSVIVG00000009001); B. taurus, Bos taurus (P02722).

Supplementary Figure 3 Activation of SPS and cyFBPase in the er-ant1 mutant. Data are expressed as means ±

SE. of three biological replicates.

To investigate the possible factors causing sucrose reduction in er-ant1 plants, we performed the assays of

cyFBPase and SPS activities of leaf tissues from 10 and 14 day-old rice seedlings. Enzymatic analysis shows that

cyFBPase and SPS are almost no difference between WT and er-ant1 seedlings at 10 DAG, in spite of their

activities in er-ant1 are lower than that in WT plants at 14 DAG (Figure S2), suggesting that the functional defects

in OsER-ANT1 have no direct effects on cyFBPase and SPS. cyFBPase was determined according to Holaday et

al (1992).SPS was assayed as described by Pavlinova, et al (2002).

References

Holaday AS, Martindale W, Alred R, Brooks AL, Leegood RC (1992) Changes in activities of enzymes of carbon

metabolism in leaves during exposure of plants to low temperature. Plant Physiology 98: 1105-1114

Pavlinova OA, Balakhontsev EN, Prasolova MF, Turkina MV (2002) Sucrose-phosphate synthase, sucrose

synthase, and invertase in sugar beet leaves. Russian Journal Plant Physiology 49: 68-73

Supplementary Figure 4 ATP contents of leaves in wild-type and er-ant1 plants. Seedlings of the wild type (WT)

and the er-ant1 mutant were grown under natural light conditions for 9, 12 or 15 days after germination (DAG).

Data are expressed as means ± SD. of three biological replicates. Asterisks indicate statistically significant

differences (Student’s t-test; ** P < 0.01).

The boiling water method was used for ATP extraction as descripted by Yang et al (2002). Leaf samples of

approximately 100 mg were collected and ground in liquid N2. Samples then were heated at 100 °C for 10 min in

1000 µl deionized water. The supernatant was stored for ATP determination. The concentration of ATP in each of

sample was determined by luciferin/luciferase luminescence assay as previously described by Song et al (2006).

References

Song CJ, Steinebrunner I, Wang X, Stout SC, Roux SJ. (2006). Extracellular ATP Induces the Accumulation of

Superoxide via NADPH Oxidases in Arabidopsis. Plant Physiol 140: 1222-1232

Yang N, Ho W, Chen Y, Hu M. (2002). A Convenient One-Step Extraction of Cellular ATP Using Boiling Water for

the Luciferin–Luciferase Assay of ATP. Anal Biochem 306: 323-327

Supplementary Table 1 Cosegregation of Ds insertion with the pale-green phenotype of the mutants

Population Phenotype Number of plants Genotype of Ds insertion locus χ2(3︰1)

++ Ds + DsDs

WT×d6-26 Normal 209 72 137 0 0.18

Pale-green 65 0 0 65

WT×d6-63 Normal 168 53 115 0 0.07

Pale-green 59 0 0 59

WT×d6-18 Normal 110 34 76 0 0.77

Pale-green 30 0 0 30

Total 641 159 328 154 0.28

χ20.05, 1 = 3.84

The mapping populations were constructed between the heterozygous Ds insertion plants (Ds/+) and wild-type plants (+/+). All F1

plant displayed wild-type phenotype, and their F2 progenies from the Ds/+ plants showed a segregation ratio of 3 : 1 (normal: pale-

green =487 : 154; χ2＝0.28<χ20.05,1=3.84), while the F2 progenies from the +/+ plants were of normal color (date not shown). All the

resulting plants showing the mutant phenotype were homozygotes (Ds/Ds) with Basta-resistant.

Supplementary Table 2 List of primers used in this study.Primers Gene Locus Sequence (5′→ 3′)

Os02g0115900-1F BiP1 Os02g0115900 GTCTTCTCGGCAAGTTCGAC

Os02g0115900-1R Os02g0115900 CGCTCCTTCACCTTCTTGTC

Os05g0428600F BiP2 Os05g0428600 GGGAGGAGAAGGAGAAGGTG

Os05g0428600R Os05g0428600 AAAGCTCATCGTGGTCATCC

Os06g0212900F BiP3 Os06g0212900 CACTGAGCGAGCAACAAGAG

Os06g0212900R Os06g0212900 ACAAGAACCTCGTCCACCAC

Os06g0716700F GRP94 Os06g0716700 AGCGAGAGCTTGAAGCAGAC

Os06g0716700R Os06g0716700 CCTCGACCTCCTCTTCCTCT

Os09g0451500F PDI Os09g0451500 CTGCTGCCATTTGCTTTGTA

Os09g0451500R Os09g0451500 AACCACCAACTCCAACTTGC

Os09g0347700F SEC61 Os09g0347700 CGATTGGTTCAGGAACTGGT

Os09g0347700R Os09g0347700 TTCAAATAAGTCGGGCAACC

Os04g0402100F CNX Os04g0402100 GAGAGAAGCAGCCCAACATC

Os04g0402100R Os04g0402100 TGCTTCGGACTCGGTAGACT

Os07g0246200F CRT Os07g0246200 TTCGAAGATGGATGGGAAAG

Os07g0246200R Os07g0246200 CTGCAGCACCAGGGTTTTAT

Os04g0675500F STT3 Os04g0675500 TTTTTGATTGCTGAGCATGG

Os04g0675500R Os04g0675500 CTACTGCGATTGCCACAAGA

Os05g0301500F Ribophorin I Os05g0301500 CTACTGGCGTTTCCCAATGT

Os05g0301500R Os05g0301500 TCTTCCCCTTCTCCAAAGGT

Os01g0911200F Ribophorin II Os01g0911200 TGTACAAGTGCTGGGCTCTG

Os01g0911200R Os01g0911200 CTGTGACAAAGATCGCCTCA

Os04g0397000F DAD1 Os04g0397000 ACATGGGAATAGTGGGGTCA

Os04g0397000R Os04g0397000 GCACAGAACGAAATCTGCAA

Os09g0553200F UGPase Os09g0553200 CGGACAACTTGGGTGCTATT

Os09g0553200R Os09g0553200 GGGACTTGAGCAATCTCCAA

Os05g0438600F FBPase Os05g0438600 CACTGGATGGCTCCTCAAAT

Os05g0438600R Os05g0438600 TTCCAGTGCTCAACACAAGC

OsSPS1F SPS1 Os01g0919400 GGGATGGATTTCAGCAGTGT

OsSPS1R Os01g0919400 GCTTTGACAAGGGTGGTGAT

Os03g0401300F SUS1 Os03g0401300 TGACTGGTCTGGTTGAGCTG

Os03g0401300R Os03g0401300 ACAAAAGCACCCTTGGTGTC

Os11g0113700F CIPK15 Os11g0113700 CTTGGTGTGAGACGCAAGAA

Os11g0113700R Os11g0113700 CCTGAGATTTAGCGCCTTTG

Os02g0178000F SnRK1 Os02g0178000 CCAGGAGCAGAAAACAAAGC

Os02g0178000R Os02g0178000 GACGCAGTGAGCTGGTGTTA

Os04g0629300F SnRK2 Os04g0629300 GCTTCTGCTGGGACGATAAG

Os04g0629300R Os04g0629300 AGTCTCCTTCCAGCTGTCCA

bZIP39F bZIP39 Os05g0411300 GATTCCACTCACCGGGAAGA

bZIP39R bZIP39 Os05g0411300 GAAGCGTGCAGGAGTAAGTG

bZIP60F bZIP60 Os07g0644100 TGTTCCGTCACATCATGGGA

bZIP60R bZIP60 Os07g0644100 GGCTTTCTCACTCGCAACAA

bZIP50F bZIP50 Os06g0622700 TCTAGAGGCCGAGTGTCGTC

bZIP50R bZIP50 Os06g0622700 GAGTAGGCACACGATGCTCA

OsIRE1F OsIRE1 Os07g0471000 CAATGCTGATAGCGGTGAGA

OsIRE1R OsIRE1 Os07g0471000 GTGGCCATACCTCGCATAGT

25SR-F 25S rRNA 　 AAGGCCGAAGAGGAGAAAGGT

25SR-R1 25S rRNA 　 TTGGCGGGCCGTTAAGCAGAAAAGA

AACP-1301F OsER-ANT1-GUS AAGCTTAAGAAGTTCTTGAGGTATAC

AACP-1301R OsER-ANT1-GUS CCATGGCGTCGACGGCGGATTCGGAG

RTL- AAC-15F OsER-ANT1-GFP AGATCTAATCCGCCGTCGACGATGCCA

RTL-AAC-990R OsER-ANT1-GFP TCTAGATCATTTCAATGCCCCTTTCATCTTG

Ds5′-1a Ds ACGGTCGGGAAACTAGCTCTAC

OsAAC-DsL partial OsER-ANT1 CCAATGTCATCCGATACTTCC

OsAAC-DsR partial OsER-ANT1 GCCATACAGATAACAAGGGTTC

Dsyb partial OsER-ANT1 ATTGGTGTGAGGCCAACATT

Dsyc partial OsER-ANT1 AGCGGGAGTACAACCACAAC

Ds3′-1a Ds GGTTCCCGTCCGATTTCGACT

Ds5′a Ds CTACCGTTTCCGTTTCCGTTTACC

Ds5′b Ds CCCGT TTCCGTTCCGTTTTCG

Ds5′c Ds GATAACGGTCGGTACGGGATTTTC

AD1 AD primer (AGCT) TCGA (GC) T (AT) T (GC) G (AT) GTT

AD3 AD primer(AT) GTG (AGCT) AG (AT) A (AGCT) CA (AGCT)

AGA

P6 Ac GGGGATCCTTCAACAATCTCCGAA

P7 Ac GGGGATCCGATGAAGTGGTTAGCC

5RT2-3 F partial OsER-ANT1 AGGGGCTACAACCTCATCCT

3RT2-3 R partial OsER-ANT1 　 TCTGTTTTGCTGCATGGAAG

AD Primer is an arbitrary degenerate (AD) primer having a lower melting temperature.