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SUPPLEMENTAL FIGURES:
Supplemental Figure 1. The pigment contents in leaves from wild-type and wsp1
mutant plants at different developmental stages.
A–C. L3, leaves from three-leaf stage plants; L4, leaves from four-leaf stage plants;
FL, flag leaves from heading stage plants; P, young panicles from heading stage
plants. In A–C, the pigment contents were measured using the third leaves of
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seedlings. FW: fresh weight; Chla and Chlb: chlorophyll a and b, respectively. Bars
represent the SD of three measurements. Student’s t-test was performed using the raw
data; asterisks indicate statistically significant differences at P<0.01.
Supplemental Figure 2. Phenotypic characterization of the wsp1 mutant at a low
temperature (20°C).
(A) Phenotypes of wild-type (left) and wsp1 mutant (right) plants at the three-leaf
stage following growth at 20°C. (B) A comparison of the pigment content at the three-
leaf stage between wsp1 plants grown at 20°C (wsp1-lc) and wsp1 plants grown under
natural conditions (wsp1-nc).
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Supplemental Figure 3. Phylogeny of the WSP1 protein family.
A. Positions of the putative transit peptide region (CTP) and MORF in WSP1. B. A
phylogenetic tree representing an alignment of WSP1 proteins. The tree was
constructed using the neighbor-joining algorithm. Bootstrap values are shown at each
node. Bars indicate the genetic distance based on the branch length. C. A comparison
of MORF protein family sequences from rice and Arabidopsis. The red box indicates
the mutant position in wsp1.
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Supplemental Figure 4. Analyses of the tissue expression and sub-cellular
localization of WSP1.
A. Schematic illustration of a rice plant with a fully expanded third leaf. L1, L2, L3,
and L4 indicate the first, second, third, and fourth leaves, respectively; developmental
stages (P0–P6) are also indicated. SB (shoot base) corresponds to a 5-mm piece from
the bottom of the shoot and contains as yet unmerged leaves at stages P0–P3. B. qRT-
PCR analysis of WSP1 in the SB, L1, L2, L3, and L4 tissues of wild-type seedlings.
The SD was calculated from three independent experiments. C. qRT-PCR analysis of
WSP1 in the root, young leaf, old leaf culm, and panicle tissues of wild-type
seedlings. D–G. Rice protoplasts expressing the WSP1-GFP fusion protein. D. GFP
fluorescence. E. Chloroplast autofluorescence. F. Bright field image of GFP. G.
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Merged image of D and E. Bars=5 μm.
Supplemental Figure 5. A transcription analysis of chloroplast-encoded and nuclear-
encoded genes.
A. Some chloroplast-encoded genes were detected in wild-type and wsp1 plants. B.
Some nuclear-encoded genes were detected in wild-type and wsp1 plants. These genes
were mainly involved in chlorophyll biosynthesis, chloroplast development, and
photosynthesis.
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Supplemental Figure 6. Immunoblot analysis of photosynthetic protein abundance.
A. Total leaf proteins were analyzed by probing immunoblots with antiserum against
representative subunits of photosystem I PsaA(A1) and PsaB(A2), photosystem II (D1
and D2), and ATP synthase (AtpB). The same amount of protein was immunoblotted
with Hsp90 antibody (bottom). B. The gray values show a quantitative analysis of
proteins from wild-type and wsp1 mutant plants.
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Supplemental Figure 7. RNA editing analysis of the complementary line (CP). The
Rpob-467,545,560, rps14-80, ndhG(-11), and ndhD-878 sites were edited normally in
the complementary line (CP).
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Supplemental Figure 8. Splicing analysis of all rice chloroplast transcripts with
introns in wild-type plants and the white sectors of wsp1 plants. The genes
corresponding to the transcripts are shown on the left. Spliced (S) and unspliced (U)
transcripts are indicated on the right. RT-PCR was performed using RNA from the
leaves of three-leaf stage seedlings.
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Supplemental Figure 9. A yeast two-hybrid interaction assay between WSP1 and
MORF families in rice. WSP1 was fused to the pGBKT7 vector (WSP1-BD). Other
MORF protein were fused to the pGADT7 vector. –L T, control medium (SD –Leu/–
Trp); –L T H A, selective medium (SD –Leu/–Trp/–His/–Ade). In addition, WLP1
(AK099041, RPL13) was also used to examine the interaction with WSP1 protein.
The empty pGBKT7 and pGAD-T7 vectors served as negative controls.
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SUPPLEMENTAL TABLES:
Supplemental Table 1. Statistical analysis of major agronomic traits of wild-type and
wsp1 plants. Each value represents the mean ±SD from 20 plants. Statistical analyses
(Student’s t-test, P <0.05) did not detect any significant differences between the wild-
type and wsp1 mutant plants except the number of spikelets per panicle .
Geno-type
Plant
height
(cm)
Number of tillers per
plant
Panicle
length (cm)
Number of
spikelets
per panicle
Seed
setting rate
(%)
1000-grain
weight (g)
wild-type 105.4±3.2 25.6±2.8 21.1±1.2 145.9±7.5 95.6±2.5 25.8±1.8
wsp1 104.2±3.1 26.1±2.4 21.2±0.9 134.3±6.2* 93.7±3.6 25.7±2.1
Supplemental Table 2. Main photosynthetic parameters measurement of wild-type
and wsp1 plants during the flowering stage in the nature condition. Each value
represents the mean ±SD from at least 5 leaves. Statistical analyses (Student’s t-test, P
<0.01) detected a significant difference between the wild-type and wsp1 mutant
plants.
Genotype
photosynthetic efficiency
(umol/m2s)
CO2 conductance
(mol/m2s) Ci (μmol/mol)
Trmmol
(mmol/m2s)
wild-type 27.50±0.18 0.52±0.02 276.91±0.27 7.84±0.05wsp1 16.37±0.91** 0.19±0.09** 236.88±0.51** 4.15±0.04**
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Supplemental Table 3. RNA editing sites in the plastids of wild type and wsp1
mutant.
Gene Codon position
Edited codon
Amino acid change
Wta
wsp1
1 atpA 383 uCa S→L + +2 ndhA 158 uCa S→L + +3 357 uCc S→F + +4 ndhB 156 cCa P→L + +5 196 Cau H→Y + +6 204 uCa S→L + +7 235 uCc S→F + +8 246 cCa P→L + +9 277 uCa S→L + +10 279 uCa S→L + +11 494 cCa P→L + +12 ndhD 293 uCa S→L + -13 ndhF 21 uCa S→L + +14 ndhG 5’-
UTR-11 C→U + -
15 rpl2 1 aCg T→M + +16 rpoB 156 uCg S→L + -17 182 uCa S→L + -18 187 uCa S→L + -19 rps14 27 uCa S→L + -20 ycf3 62 aCg T→M + +21 rps8 61 uCa S→L + +
a:Abbreviations:+, editing; -, no editing.
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129130131132133134135136137138139140141142143
Supplemental Table 4. Co-expressed genes of WSP1 were calculated by CREP
(http://crep.ncpgr.cn) Module Gene Correlator analysis. Gene Correlator for
discovering genes co-expressed with target gene. Gene Correlator calculates all
Pearson correlations by selecting each gene against all genes in the microarray and the
corresponding gene pairs with correlation coefficients larger than 0.85 are listed.
Co-expressed gene Correlation Gene Prediction LOC_Os01g32350 0.92 ATP-dependent Clp protease proteolytic subunitLOC_Os03g43430 0.91 THA4LOC_Os04g52100 0.91 methionine aminopeptidase 1BLOC_Os12g37710 0.89 thylakoid lumenal 21.5 kDa proteinLOC_Os10g36860 0.88 YhbY domain containing proteinLOC_Os10g32300 0.88 TPR Domain containing proteinLOC_Os11g41910 0.88 GTP-binding protein engALOC_Os09g10760 0.87 plastid-specific 30S ribosomal protein 2LOC_Os03g58540 0.87 small GTP-binding protein domainLOC_Os02g08380 0.87 complex interacting protein 9LOC_Os11g37130 0.87 HCF106C precursor proteinLOC_Os03g48040 0.86 ferredoxin-6, chloroplast precursorLOC_Os07g09370 0.86 pentatricopeptide repeat protein LOC_Os12g37610 0.86 ribosomal protein S6 containing proteinLOC_Os07g38300 0.86 ribosome recycling factorLOC_Os11g05552 0.86 signal recognition particle 54 kDa proteinLOC_Os03g05806 0.85 ribosomal large subunit pseudouridine synthase
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Supplemental Table 5. Primers used for fine mapping, RT-PCR, and vector
construction, RNA splicing and RNA editing.
Fine mapping and complementary testMarker Forward sequence (5’-3’) Reverse sequence (5’-3’)
Indel1-1 CGTCACTCGTACGGTTAGCA AGACGGGACGAGAGCAAAC
Indel1-2 AGCCTAAATTCATGTGGATCA TCACCAGCGATAGCAAGGTA
3017 CGAACCCGGACTGAGATAAG ACCCGACAATTTTCCCTTTT
3020 CCACTACATATGCGGTTTTCA TTTCACACAATTTTGAACAGTCA
WSP`-Se F/R TGTCATGCGAACGGTACTTTG CATAGCGAGTTCGGTCACTG
WSP1-cDNA-F/RGATATCCTCTCCTCCCCTTCTCTT
CG
TCTAGATGCAGGAGCTGCTGTTCT
AA
WSP1-GFP-
F/R ACTAGTATGGCCACCGCCGC TCTAGACCGCTGGTACGCCTGAT
dCAPS1TTGACAGGGCTACCAGGTGTTCTA
TTTGAGC(AluI)CTCCGTTTCATATTATAAAACTTTC
QRT-PCRGene Forward sequence (5’-3’) Reverse sequence (5’-3’)WSP1 GATCGACTGCTACATCCAGACC ACAACTCAGCCCATAGTCCT
ACTIN TGCTATGTACGTCGCCATCCAG AATGAGTAACCACGCTCCGC
PsaA GCGAGCAAATAAAACACCTTTC GTACCAGCTTAACGTGGGGG
PsbA CCCTCATTAGCAGATTCGTTTT ATGATTGTATTCCAGGCAGAC
cab1R AGATGGGTTTAGTGCGACGAG TTTGGGATCGAGGGAGTATTT
Cab2R TGTTCTCCATGTTCGGCTTCT GCTACGGTCCCCACTTCACT
rbcS TCCGCTGAGTTTTGGCTATTT GGACTTGAGCCCTGGAAGG
rbcL CTTGGCAGCATTCCGAGTAA ACAACGGGCTCGATGTGATA
YGL1 AACCTTACCGTCCTATTCCTT CCATACATCTAACAGAGCACC
HEMA1 CGCTATTTCTGATGCTATGGGT TCTTGGGTGATGATTGTTTGG
CAO1 GATCCATACCCGATCGACAT CGAGAGACATCCGGTAGAGC
DVR CGAGCCCAGGTTCATCAAGGTGC CCTCCCGATCTTGCCGAACTC
RNRS ACGTGCTCGCCTTCTTCG CAGCGCATGG CCCAGT
RpoA CCATTCCCACAAGCAAAAAT TCTTACCGCCTTCCGTAGAA
RpoB AAGAATTCCTGACTATTGCATGGGAA GTTAGAGCGCCGATGGGTAAC
RpoT GTTGAACGTGAGTTCAAAGTCC GGGTCCCTAA CTTCACTTGGG
Rps12 AGCCGTTTGCTACCAATGG TGATCGGTACCAATGAATAGG
V2 GAGGAGTTCCTCACGATGAT AGCATCAATGATAGACTCC
Wlp1 CTAAGGGCAGACTGGGAAGA TTGATAGGCAGTGGAACAGG
ndhA GTCTATGGACTGATATGGATTCTACCGCTTAGTTCCATCTGCTATA
GCTT
RNA splicing test primerGene Forward Sequence(5’-3’) Reverse sequenceatpF TTTTAGCTCACTGGCCATCC TTCATCGCCCTTTGTTTTTC
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162
petB ATTCAGACCTCGCAACCAGA GTTGGCCTCGGTCATTATGT
ycf3-1 TGATAAGACCTTCTCAATTGTAGCC GTGTGTATAAGGCCTATGTTATAGAGT
ycf3-2 AGAGCATACAAAGGCTTTGGAAT TTCAACCAGTTCTGTGCTTCAATATA
rpl2 ATCGATTTTCGACGGAATCA GGACCTCCCCAGATGGTAAT
petD CCATGAAGAGGCTCCGTAAG ATCATTGACGGCTCGAGAAC
rpl16 TGCTTCGTATTGTCGAGATCC TCGTGTCATTGCTCTTCGTC
ndhA TCCTTTGGGATATCGCTTTG TCTTCCTCCGCTTCTGGTAA
ndhB TGGGGCAAGCTCTTCTATTC AGCAAGGAGATTCCCCAATA
rps16 TTCAAGGAAATAGAGAATAG AACATGTGGTAGAAAGCAA
trnK TATGGGTTGCCCGGGACTCG GGTTGCTAACTCAATGGTAG
D1 GCCGAATACACCAGCTACAC TGGGTATGCGTCCTTGGATT
RNA editing test primerGene Forward Sequence(5’-3’) Reverse sequencendhA-1 GGACCGTCTATAGCAGTCAT CTGACGCCAAAGATTCCATC
ndhA-2 GGTGGAATTTGTCTATTCCC TTTCTCTTGTTTGAGAGGAC
ndhB-1 GGCTATAACAGAGTTTCTGT CCAGAAGAAGATGCCATTCG
ndhB-2 CTTGGTTTCAATAGGACTCC TAAAAGAGGGTATCCTGAGC
ndhD GGTTACCAGATACCCATGGG AAAATAGCTCCATTGAGTCC
ndhF CAATATGCATGGGTAATCCC AACCAGGATTCCTACAGTAG
ndhG CCTAATCCCTTTTTTCTTCC TCAAGACATTTATAGCTCCC
rpl2 CCGGGTTATTCTATTCCACT TACGCATTTCGATTAGGGTC
rps8 GGAACTGTTCGGGTAGTATC GGAATTCCTTGATAGTTGGC
rps14 GTTTGATTCAGAGAGAGAGG TTCTCGAAGTATGTGTCCGG
rpoB GTCCTGGTATTTACTACCGC TCCCCACCTACACAAGCAAA
atpA GACTCAATCTGGAGACGTTT TAGTAGCTATCTGCTCTTCC
ycf3 TGTGGTAAGAAGGGGTTTCG GCGAATAATTCCGACAACCT
rpoC2 GGTCCTTGGGGATTCTTGAT TCTTGTTTTGTGGGTAACGG
RT-PCR analysis for ndhANdhA-F/R ATGATAATAGACAGGGTACAG GTGAAACAAGTTGGGAAGAAG
Yeast Two-Hybrid AnalysisGene Forward Sequence(5’-3’) Reverse sequence
WSP1-BD
CATGGAGGCCGAATTCATGGCCA
CCGCCGCAGCAG
GCAGGTCGACGGATCCTCACCG
CTGGTACGCCTGAT
LOC_Os06g02600-AD
GGAGGCCAGTGAATTCCTAGGGT
TTTACCCCGCGATGGCCG
CGAGCTCGATGGATCCCTTCTG
ATAATCAGATCACCGT
LOC_Os09g04670-AD GGAGGCCAGTGAATTCGCGATGG
TGTCGGCGTCGCGCT
CGAGCTCGATGGATCCTGTTGT
TCTACTGGTAATTCCT
LOC_Os09g33480-AD GGAGGCCAGTGAATTCATGGCGT
CGGCGTCGCGCTTC
CGAGCTCGATGGATCCTCTAGC
AGTGTTCTACTGGT
LOC_Os03g04490-AD GGAGGCCAGTGAATTCATGGCGG
CCGGAGCAGCAG
CGAGCTCGATGGATCCCTACTG
CTGCAGATTTGAAG
WLP1(RPL13) -AD GAATTCATGGCTACGGCCATCGCA GGATCCCTACTTCTCAGACTTCT
GTATTCTTTTATC
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