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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