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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.