Lim et al, Supplemental Figure S1.

OsRING-H2 type : 5 OsRING-HC type : 1OsRING-v type : 1

OsRING-H2 type : 9OsRING-HC type : 8OsRING-v type : 2

OsRING-H2 type : 9OsRING-HC type : 8OsRING-v type : 2

OsRING-H2 type : 4OsRING-HC type : 6OsRING-v type : 2

Supplemental Figure S1. Subcellular distribution of 47 OsRFP proteins. Subcellular localization of OsRFPs was predicted using

the WoLF PSORT program (http://psort.hgc.jp/form.html).

OsRFPH2-3 QDO/A α-gal activity P.L. TIGR annotation (Rice Genome Annotation Resource) TIGR Locus

2 + + Cytosol ubiquitin family protein Os02g06640

9 + - Cytosol expressed protein Os02g22130

15 + +++ Chloroplast peptidyl_prolyl cis_trans isomerase, FKBP_type Os02g52290

16 + ++ Chloroplast chlorophyll A_B binding protein Os07g37240

17 + +++ Plasma membrane aquaporin protein Os02g44630

28 + ++ Cytosol jacalin-like lectin domain containing protein Os01g24710

29 + + Cytosol expressed protein Os10g21190

30 + + Extracellular expressed protein Os02g26294

33 + + Mitochondria ufm1_conjugating enzyme 1 Os10g13800

34 + + Mitochondria PRLI-interacting factor A Os02g58570

35 + +++ Extracellular WIP5 _ Wound_induced protein precursor Os11g37970

37 + + Chloroplast calvin cycle protein CP12 Os03g19380

39 + + Nucleus expressed protein Os05g02070

40 + +++ Chloroplast cysteine-rich repeat secretory protein 55 precursor Os03g16950

42 + + Vacuole LTPL129 _ Protease inhibitor/seed storage/LTP family protein precursor Os06g43600

45 + + Chloroplast expressed protein Os06g38210

46 + ++ Cytosol RING finger and CHY zinc finger domain containing protein 1 Os01g52110

47 + +++ Nucleus coiled_coil domain containing 49 Os08g39090

50 + +++ Nucleus expressed protein Os05g02070

53 + + Chloroplast universal stress protein domain containing protein Os05g28740

54 + + Chloroplast 40S ribosomal protein S26 Os05g39960

55 + + Chloroplast ribulose bisphosphate carboxylase small chain, chloroplast precursor Os02g05830

57 + + Mitochondria transferase family protein Os04g42250

60 + +++ Extracellular DUF581 domain containing protein Os04g49670

A

Lim et al, Supplemental Figure S2.

OsRFPH2-14 QDO/X/A α-gal activity P.L TIGR annotation (Rice Genome Annotation Resource) TIGR Locus

3 + + Cytosol naringenin,2_oxoglutarate 3_dioxygenase Os04g49194

4 + + Cytoskeleton aminotransferase Os04g52450

5 + +++ Chloroplast expressed protein Os09g00999

7 + + Cytosol aminotransferase Os01g18640

11 + +++ Nucleus expressed protein Os05g02070

14 + +++ Chloroplast copper methylamine oxidase precursor Os04g40040

19 + +++ Nucleus expressed protein Os09g01000

24 + + Nucleus RNA recognition motif containing protein Os03g46770

25 + + Chloroplast expressed protein Os02g33070

28 + ++ Cytosol pathogen-related protein Os01g14590

29 + ++ Chloroplast expressed protein Os01g68300

31 + +++ Cytosol jacalin_like lectin domain containing protein Os01g24710

34 + + Nucleus metallothionein Os01g74300

35 + + Nucleus thioredoxin Os05g07690

36 + ++ Nucleus expressed protein Os06g36360

37 + ++ Nucleus expressed protein Os01g48830

45 + ++ Chloroplast photosystem I reaction center subunit Os09g30340

46 + +++ Cytosol profilin domain containing protein Os06g05880

52 + ++ Cytosol jacalin-like lectin domain containing protein Os01g24710

60 + + Chloroplast mTERF family protein Os06g12040

61 + + Chloroplast OsGH3.3 _ Probable indole_3_acetic acid_amido synthetase Os01g12160

63 + ++ Extracellular polygalacturonase inhibitor 1 precursor Os07g38130

64 + +++ Chloroplast universal stress protein domain containing protein Os05g28740

66 + ++ Chloroplast inorganic H+ pyrophosphatase Os06g43660

67 + ++ Chloroplast expressed protein Os07g17184

69 + ++ Chloroplast heparanase_like protein precursor Os03g11320

70 + + Nucleus AMSH_like protease Os01g47262

71 + + Cytosol ubiquitin family protein Os02g06640

72 + ++ Cytosol 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase protein Os03g06620

74 + +++ Mitochondria expressed protein Os01g15270

B

Lim et al, Supplemental Figure S2. (continued from previous page.)

OsRFPH2-16 QDO/X/A α-gal activity P.L TIGR annotation (Rice Genome Annotation Resource) TIGR Locus

4 + +++ Chloroplast glycosyl hydrolase Os05g15770

11 + +++ Mitochondria expressed protein Os01g15270

16 + ++ Cytosol vacuolar ATP synthase subunit E Os01g46980

18 + ++ Extracellular OsFBX27 _ F_box domain containing protein Os01g59180

19 + +++ Nucleus expressed protein Os08g07280

21 + + Nucleus zinc finger, RING-type Os04g58320

28 + ++ Nucleus expressed protein Os09g01000

30 + ++ Chloroplast expressed protein Os01g12200

31 + ++ Nucleus expressed protein Os05g02070

34 + ++ Chloroplast WIP4 _ Wound_induced protein precursor Os11g37960

35 + ++ Cytosol ubiquitin family protein Os02g06640

36 + ++ Cytosol jacalin_like lectin domain containing protein Os01g24710

37 + ++ Chloroplast OsSub25 - Putative Subtilisin homologue Os03g02750

40 + ++ Chloroplast expressed protein Os09g00999

49 + ++ Chloroplast endoribonuclease Os07g33240

50 + ++ Nucleus hypothetical protein Os06g27510

52 + + Cytosol jacalin-like lectin domain containing protein Os01g24710

57 + ++ Vacuole inorganic H+ pyrophosphatase Os02g09150

64 + +++ Nucleus OsFBT7 _ F_box and tubby domain containing protein Os05g36190

65 + + Cytosol kinase, pfkB family Os01g01620

68 + ++ Nucleus mps one binder kinase activator_like 1A Os10g25674

69 + ++ Plasma membrane glucan endo-1,3-beta-glucosidase precursor Os07g32600

71 + ++ Cytosol ubiquitin family protein Os02g06640

76 + + Nucleus serine/arginine repetitive matrix protein 1 Os05g01540

78 + + Cytosol expressed protein Os10g21190

94 + + Chloroplast expressed protein Os04g57760

96 + + Chloroplast universal stress protein domain containing protein Os05g28740

97 + + Chloroplast phytochrome A Os03g51030

C

Lim et al, Supplemental Figure S2. (continued from previous page.)

OsRFPH2-23 QDO/X/A α-gal activity P.L TIGR annotation (Rice Genome Annotation Resource) TIGR Locus

3 + + Cytosol expressed protein Os10g21190

4 + ++ Cytosol CBS domain_containing protein Os01g69090

5 + +++ Nucleus RNA recognition motif containing protein Os11g40510

6 + +++ Chloroplast cysteine_rich repeat secretory protein 55 precursor Os03g16950

7 + +++ Chloroplast glycosyltransferase Os02g32750

11 + + Cytosol isocitrate lyase Os07g34520

13 + + Vacuole LTPL129 - Protease inhibitor/seed storage/LTP family Os06g43600

15 + ++ Cytosol expressed protein Os10g21190

18 + + Extracellular ML domain protein Os07g06590

19 + + Cytoskeleton actin Os03g50885

20 + + Extracellular glycosyl hydrolases family 17 Os01g51570

21 + +++ Cytosol jacalin-like lectin domain containing protein Os01g24710

22 + ++ Chloroplast aldehyde dehydrogenase Os02g49720

23 + ++ Cytosol expressed protein Os11g01060

24 + ++ Cytosol DIP1 Os05g34070

25 + ++ Cytosol RPA1B - Putative single-stranded DNA binding complex Os03g11540

26 + ++ Chloroplast expressed protein Os09g00999

29 + ++ E.R. expressed protein Os11g16330

30 + ++ Cytosol jacalin_like lectin domain containing protein Os01g24710

31 + ++ Chloroplast universal stress protein domain containing protein Os07g36600

32 + + Chloroplast expansin precursor Os07g29290

33 + +++ Chloroplast cysteine-rich repeat secretory protein 55 precursor Os03g16950

34 + + Cytosol ubiquitin family protein Os02g06640

35 + ++ Nucleus trehalose-6-phosphate synthase Os08g31980

36 + + Extracellular CHIT5 _ Chitinase family protein precursor Os05g33140

37 + + Mitochondria peptidase Os06g10770

D

Lim et al, Supplemental Figure S2. (continued from previous page.)

OsRFPHC-2 QDO/X/A α-gal activity P.L TIGR annotation (Rice Genome Annotation Resource) TIGR Locus

1 + + Cytosol expressed protein Os02g22130

4 + + Chloroplast PB1 domain containing protein Os11g25780

5 + + Chloroplast PsbP Os07g04840

6 + + Cytosol ubiquitin family protein Os02g06640

11 + + Nucleus serine/arginine repetitive matrix protein 1 Os05g01540

21 + + Cytosol fructose-bisphospate aldolase isozyme Os01g67860

22 + ++ Extracellular ML domain protein Os07g06590

55 + + Nucleus expressed protein Os09g01000

OsRFPHC-13QDO/X/A α-gal activity P.L TIGR annotation (Rice Genome Annotation Resource) TIGR Locus

2 + + Mitochondria expressed protein Os02g01500

37 + + Chloroplast putrescine-binding periplasmic protein-related Os04g51310

38 + +++ Chloroplast peroxidase precursor Os04g51300

39 + + Plasma membrane lectin protein kinase family protein Os06g35870

41 + + Cytosol expressed protein Os02g22130

42 + + Chloroplast expressed protein Os08g25420

43 + + Nucleus ubiquitin-conjugating enzyme Os03g57790

E

F

Lim et al, Supplemental Figure S2. (continued from previous page.)

OsRFPv-6 QDO/X/A α-gal activity P.L TIGR annotation (Rice Genome Annotation Resource) TIGR Locus

2 + ++ Cytosol DUF538 domain containing protein Os01g11240

3 + + Mitochondria transposon protein Os12g19680

4 + + Cytosol UDP-glucoronosyl and UDP-glucosyl transferase domain containing proteinOs03g55010

6 + + Cytosol ribosomal protein L5 Os05g11710

7 + + Chloroplast expressed protein Os04g46100

10 + + Plasma membrane Plant PDR ABC transporter associated domain containing protein Os08g43120

11 + + Nucleus WRKY53 Os05g27730

12 + + Nucleus serine/arginine repetitive matrix protein 1 Os05g01540

13 + + Chloroplast RCLEA5 - Root cap and Late embryogenesis related family protein precursorOs01g73730

14 + + Nucleus transcription factor Os02g07800

15 + +++ Nucleus ELF3 protein Os01g38530

16 + +++ Chloroplast indole-3-glycerol phosphate synthase, chloroplast precursor Os08g23150

17 + + Chloroplast aconitate hydratase protein Os08g09200

21 + + Cytosol phytoene synthase, chloroplast precursor Os06g51290

22 + + Cytosol transferase family protein Os06g06180

25 + + Nucleus expressed protein Os09g01000

28 + ++ Chloroplast AP2 domain containing protein Os02g52670

29 + +++ Nucleus expressed protein Os01g57968

30 + + Extracellular periplasmic beta-glucosidase precursor Os03g53860

31 + + Chloroplast cytochrome P450 71E1 Os03g63310

34 + ++ Nucleus BEE 1 Os08g42470

42 + + Chloroplast expressed protein Os02g29390

45 + +++ Plasma membrane ABC transporter, ATP-binding protein Os03g08380

50 + + Chloroplast expressed protein Os09g00999

52 + ++ Nucleus no apical meristem protein Os07g48550

54 + + Chloroplast ZOS6-07 - C2H2 zinc finger protein Os06g46910

55 + + Plasma membrane ABC transporter family protein Os04g49900

61 + +++ Nucleus Myb-like DNA-binding domain containing protein Os02g04640

63 + ++ Chloroplast expressed protein Os07g41390

70 + +++ Chloroplast SAM-dependent methyltransferase Os03g36910

73 + +++ Plasma membrane aquaporin protein Os07g26690

G

Lim et al, Supplemental Figure S2. (continued from previous page.)

Lim et al, Supplemental Figure S2. (continued from previous page.)

Supplemental Figure S2. Proteins that positively interacted with OsRFPH2-3 (A), OsRFPH2-14 (B), OsRFPH2-16 (C), OsRFPH2-

23 (D), OsRFPHC-2 (E), OsRFPHC-13 (F), or OsRFPv-6 (G) from yeast 2-hybrid screening and their predicted subcellular

distributions. ‘+’ indicates cell growth on QDO/X/A (synthetic defined medium lacking Ade, His, Leu, and Trp with 40 μg/mL X-α-

Gal and 70 ng/mL aureobasidin) or density of α-GAL activity (‘-’: no, ‘+’: weak, ‘++’: strong, ‘+++’: very strong). Subcellular

localizations of positive interacting proteins with OsRFPs were predicted using the WoLF PSORT program

(http://psort.hgc.jp/form.html).

OsRFPv-6

AtUBC10

OsRFPHC-13

AtUBC10

OsRFPH2-23

AtUBC10

OsRFPH2-16

AtUBC10

OsRFPH2-14

AtUBC10

OsRFPHC-3

AtUBC10

OsRFPH2-3

AtUBC10

OsRFPHC-2

AtUBC10

#1 #2 #335S:EGFP

35S:EGFP #1 #2 #3

Lim et al, Supplemental Figure S3.

Supplemental Figure S3. RT-PCR analysis of 3 independent col-0/35S:OsRFP-EGFPs T3 transgenic plants

(#1, #2, and #3) and the control plant (35S:EGFP). Total RNA was extracted from 14-d-old Arabidopsis

seedlings, and first-strand cDNA was synthesized using a cDNA synthesis kit (Takara-Bio, Ohtsu, Japan).

Semi-quantitative RT-PCR was conducted using each gene specific primer set. The AtUBC10 (At5g25760)

gene was used as an internal PCR control.

Lim et al, Supplemental Figure S4.

Germ

inati

on r

ate

Germ

inati

on r

ate

A B C D

E F G H

Supplemental Figure S4. Germination rate of 7 overexpressed OsRFP genes in Arabidopsis. Three independent T3 seeds

from 35S:OsRFPH2-3-EGFP (A), 35S:OsRFPH2-14-EGFP (B), 35S:OsRFPH2-16-EGFP (C), 35S:OsRFPH2-23-EGFP (D),

35S:OsRFPHC-2-EGFP (E), 35S:OsRFPHC-3-EGFP (F), 35S:OsRFPHC-13-EGFP (G), and 35S:OsRFPv-6-EGFP (H) with control

plants (35S:EGFP) were plated on 1/2 MS. Germination percentages for each experiment were scored and calculated at 1-d

intervals for 7 d. Data represent means ± SDs of 3 independent experiments (n = 3).

Root

leng

th (

Cm

)

35S:EGFP

35S:OsRFPH2-3#1 #2 #3

35S:EGFP

35S:OsRFPH2-14#1 #2 #3

35S:EGFP

35S:OsRFPH2-16#1 #2 #3

35S:EGFP

35S:OsRFPH2-23#1 #2 #3

35S:EGFP

35S:OsRFPHC-2#1 #2 #3

35S:EGFP

35S:OsRFPHC-3#1 #2 #3

35S:EGFP

35S:OsRFPHC-13#1 #2 #3

35S:EGFP

35S:OsRFPv-6#1 #2 #3

******

*****

Mock

100 mM NaCl

Root

leng

th (

Cm

)

A

B

*

******

****

******

*******

********

**** **

35S:EGFP

35S:OsRFPH2-3#1 #2 #3

35S:EGFP

35S:OsRFPH2-14#1 #2 #3

35S:EGFP

35S:OsRFPH2-16#1 #2 #3

35S:EGFP

35S:OsRFPH2-23#1 #2 #3

35S:EGFP

35S:OsRFPHC-2#1 #2 #3

35S:EGFP

35S:OsRFPHC-3#1 #2 #3

35S:EGFP

35S:OsRFPHC-13#1 #2 #3

35S:EGFP

35S:OsRFPv-6#1 #2 #3

Lim et al, Supplemental Figure S5.

-0.5 MPa PEG

Root

leng

th (

Cm

)

C

35S:EGFP

35S:OsRFPH2-3#1 #2 #3

35S:EGFP

35S:OsRFPH2-14#1 #2 #3

35S:EGFP

35S:OsRFPH2-16#1 #2 #3

35S:EGFP

35S:OsRFPH2-23#1 #2 #3

35S:EGFP

35S:OsRFPHC-2#1 #2 #3

35S:EGFP

35S:OsRFPHC-3#1 #2 #3

35S:EGFP

35S:OsRFPHC-13#1 #2 #3

35S:EGFP

35S:OsRFPv-6#1 #2 #3

*** ******** **

**

**

******

******

******

******

Lim et al, Supplemental Figure S5. (Continued from previous page.)

Supplemental Figure S5. Root length assay for 8 overexpressed OsRFP genes in Arabidopsis under 100 mM NaCl and -0.5 MPa

PEG treatments. Three independent seeds from 35S:OsRFPH2-3-EGFP, 35S:OsRFPH2-14-EGFP, 35S:OsRFPH2-16-EGFP,

35S:OsRFPH2-23-EGFP, 35S:OsRFPHC-2-EGFP, 35S:OsRFPHC-3-EGFP, 35S:OsRFPHC-13-EGFP, and 35S:OsRFPv-6-EGFP with control

plants (35S:EGFP) were plated on 1/2 MS, and germinated seeds were transferred 1/2 MS (A) or 1/2 MS containing 100 mM NaCl (B)

or -0.5 MPa PEG (C). The root lengths of each transgenic plant and control plant were photographed after 7 d and analyzed using

Image J software. Data represent means ± SDs of 3 independent experiments (n = 30). ‘*’ and ‘**’ indicate significant differences

of overexpressing transgenic lines in comparison to the control at p < 0.05 and p < 0.01, respectively (Student’s t -test).

35S:EGFP

35S:OsRFPH2-3

#1 #2 #3

35S:EGFP

35S:OsRFPHC-2

#1 #2 #3

35S:EGFP

35S:OsRFPH2-14

#1 #2 #3

35S:EGFP

35S:OsRFPHC-3

#1 #2 #3

35S:EGFP

35S:OsRFPH2-16

#1 #2 #3

35S:EGFP

35S:OsRFPHC-13

#1 #2 #3

35S:EGFP

35S:OsRFPH2-23

#1 #2 #3

35S:EGFP

35S:OsRFPv-6

#1 #2 #3

Lim et al, Supplemental Figure S6.

Supplemental Figure S6. H2O2 production in response to dehydration stress. Detached rosette leaves from

2-week-old control (35S:EGFP) and transgenic lines (35:OsRFP-EGFPs) were treated with dehydration for 90

min. Indicated leaf samples were incubated with 1 mg/mL DAB solution for 4 h, after which the solution was

replaced with bleaching solution. Induced H2O2 production was visualized as a dark brown color.

35S:EGFP

35S:OsRFPH2-3 #2

35S:EGFP

35S:OsRFPH2-14 #3

35S:EGFP

35S:OsRFPH2-16 #1

35S:EGFP

35S:OsRFPH2-23 #1

14 days with water

10 days without water

5 days after rewatering

14 days with water

10 days without water

5 days after rewatering

35S:EGFP

35S:OsRFPHC-3 #2

35S:EGFP

35S:OsRFPHC-2 #1

35S:EGFP

35S:OsRFPHC-13 #1

35S:EGFP

35S:OsRFPv-6 #3

4%(3/75)

67.6%(48/71)

7%(5/71)

49.3%(37/75)

10.2%(8/78)

35.8%(28/78)

6.7%(5/74)

47.9%(35/73)

2.6%(2/75)

23%(18/78)

8.0%(6/75)

30.6%(23/75)

7.5%(6/79)

58.9%(43/73)

4.2%(2/74)

16%(12/75)

Su

rviv

al ra

te (

%)

Su

rviv

al ra

te (

%)

Lim et al, Supplemental Figure S7.

Supplemental Figure S7. Increased water tolerance of overexpressed OsRFP genes in Arabidopsis. Overexpression of OsRFPH2-3-

EGFP, OsRFPH2-14-EGFP, OsRFPH2-16-EGFP, OsRFPH2-23-EGFP, OsRFPHC-2-EGFP, OsRFPHC-3-EGFP, OsRFPHC-13-EGFP, and OsRFPv-

6-EGFP in Arabidopsis conferred tolerance to drought stress. Fourteen-day-old control (35S:EGFP) and transgenic lines were grown for

10 days without irrigation. Survival rates were measured at 5 days after rewatering.