(a) (b)

(c) (d)

(e) (f)

(g)

Figure S1.

Figure S1. Comparison of OsPCR1-6 and GW2 transcript levels in the grains of devel-

oping gw2 and wild-type isogenic lines. OsPCR1-6 (a-f) and GW2 (g) expression in gw2

and the corresponding WT seedlings (HS vs. CR413, HY vs. gw2 k/o). RNA was ex-

tracted from developing grains (3 days before pollination (DBP) to 12 DAP, from

Hwaseong (HS), gw2-NIL (CR413), Hwayeong (HY), and GW2 knockout (gw2 k/o)

plants grown in rice paddy soil). Expression analysis was performed using qRT-PCR

and a gene-specific primer set. The values were normalized to actin transcript levels

and indicate the average and standard error (n=3).

OsPCR1 sense lines

OsPCR1 anti-sense lines

Figure S2. Analysis of correlation between grain weight and OsPCR1 expression

level in OsPCR1 transgenic plants. The OsPcr1 mRNA levels were analyzed by

qRT-PCR and compared to the grain weight shown in figure. qRT-PCR was per-

formed with the total RNAs using the OsPCR1RT-F and OsPCR1RT-R primers and

actin primers for an internal standard. The graph was generated by GraPad Prism,

and correlation analysis between phenotype and OsPCR1 expression level was

performed using nonlinear regression (curve fit) parameter with polynomial fourth

order in GraPad Prism program.

OsPCR1-YFP

FM4-64 Merged

Back/white

Figure S3. Tissue-specific expression of OsPCR1 and subcellular localization of Os-

PCR1-YFP. in root cells of transgenic rice. (a) OsPCR1 expression patterns in differ-

ent tissues of 2-week-old seedlings and plants of 5 day-after-pollination stage (5

DAP). R: root, S: shoot, N II: node II, N I: node I, LS I: leaf sheath I, FL: flag leaf, I II:

internode II, I I: internode I, Ra: rachis, Sp: spikelet. (b) Subcellular localization of Os-

PCR1-YFP in root cells of transgenic rice. Roots were treated with FM4-64 for 30 min

to label the plasma membrane. Bar = 10 µm.

Seedlings Plants of 5 DAP

R S N II N I LS I FL I II I I Ra Sp

OsPCR1

OsActin1

(a)

(b)

O.sativa Kaluheenati O.sativa Rambhog

O.rufipogon(IRGC105491) O.sativa Kalodhan

O.sativa ARC7047 O.sativa Shoni O.sativa ARC7291

O.sativa Kasalath O.rufipogon(W1944)

O.grandiglumis O.sativa Rexmont

O.sativa Milyang23 O.sativa IR58 O.sativa Khautanchiem

O.sativa Nipponbare O.sativa Ilpum O.sativa Hwayeong

O.sativa Hwaseong

87

100

90

75

66

42

44

67

45

48

46

88

0.01

Figure S4. Sequence divergence of rice PCR1 genes in different Oryza. The phylogenetic

tree was constructed using the neighbor-joining method implemented in MEGA version

4.0 and was based on multiple alignments of PCR1 genomic DNA sequences from differ-

ent rice accessions. The bootstrap values (percentage) of 1000 replicates are shown at

the branching points. The scale bars indicate unit of branch length.

H S G R2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 3 43 5 3 6 3 7 3 8 3 9 4 04 1 4 2 4 3 4 4

H SG R2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 3 43 5 3 6 3 7 3 8 3 9 4 04 1 4 2 4 3 4 4Figure S5. Genotype analysis of OsPCR1 in 25 Korean rice accessions. (a) A 320-

bp fragment was detected in japonica-type using the HS-PCR1 marker. (b) A 290-bp

fragment was amplified from indica-type rice using the primer set of GR-PCR1 . The

sample numbers are the same as in Table S1. The products were divided into japon-

ica- or indica-type PCR1 by DNA band analysis using Perkin Elmer LabChip GX

(Caliper Life Sciences, Inc., Hopkinton, MA, USA).

(a)

(b)

Ch55 Os

Os Ch55

SalI

PCR1 CO:

PCR1 OC:

½ SG½ SG, 30 µM Cd

PCR1

EV Os Ch55 OC CO

PCR1

EV Os Ch55 OC CO

(a)

(b)

Figure S6.

Ch55 PCR1 1 M Y P P D P S K S G E S S A A A E V P P L A P A P E P V T G I P V G M F Y P A P P M E R V V S C R MO. sativa Hwaseong PCR1 1 M Y P P D P S K S G E S S A A - - - - - - - - A P E P V T G I P V G M F Y P A P P M E R V V S C R M

Ch55 PCR1 51 A P A A G G A W T T A L C D C S D D C N T C C M A C W C P C I P V G Q I A E I V D R G S S S C A L NO. sativa Hwaseong PCR1 43 A P A A G G A W T T A L C D C A D D C N T C C M A C W C P C I P V G Q I A E I V D R G S S S C A L N

Ch55 PCR1 101 A V L Y C L V F H V S A G M C QW V Y S C A Y R A R L R A A Y D L P E T P C S D C L V T F C C Q T CO. sativa Hwaseong PCR1 93 A V L Y C L V F H V S A G M C QW V Y S C A Y R A R L R A A Y D L P E T P C S D C L V T F C C Q T C

Ch55 PCR1 151 S I A Q M H R E L K N R G H D L T L - - - - - - - - - - - - - - - - - - - - - - - - - -O. sativa Hwaseong PCR1 143 S I A Q M H R E L K N R G H D P N L GW E V N S R R T M M T P P Q H Q A M E G M T T P S

Figure S6. Comparison of Cd resistance in DTY 167 yeast strains expressing the PCR1

genes from Hwaseong and the weedy rice Ch55. (a) Comparison of PCR1 amino acid se-

quences of O. sativa cv. Hwaseong and Ch55, which exhibits lead resistance (Yang et al.

2000). (b) Phenotype analysis of DTY 167 yeast cells transformed with OsPCR1, Ch55 PCR1,

a swapping mutant of japonica-type (N’) and Ch55 (C’) PCR1 (OCPCR1; white and blue bar),

and a swapping mutant of indica- (N’) and japonica- (C’) type PCR1 (COPCR1; blue and white

bar). Yeast cells were grown in the absence of uracil on synthetic glucose (SDura-) liquid

medium, spotted on uracil-free synthetic galactose (SGura-) agar plates in the absence or pres-

ence of CdCl2 and cultured at 30ºC for 3-5 days.