Lim et al, Supplemental Figure S1. OsRING-H2 type : 5 OsRING-HC type : 1 OsRING-v type : 1 OsRING-H2 type : 9 OsRING-HC type : 8 OsRING-v type : 2 OsRING-H2 type : 9 OsRING-HC type : 8 OsRING-v type : 2 OsRING-H2 type : 4 OsRING-HC type : 6 OsRING-v type : 2 Supplemental Figure S1. Subcellular distribution of 47 OsRFP proteins. Subcellular localization of OsRFPs was predicted using the WoLF PSORT program (

A Lim et al, Supplemental Figure S2.

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

C

D

E F

G

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 (

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#3 35S: 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 T 3 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. Germination rate A BC D E FG H Supplemental Figure S4. Germination rate of 7 overexpressed OsRFP genes in Arabidopsis. Three independent T 3 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 length (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 length (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 length (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. H 2 O 2 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 H 2 O 2 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) Survival rate (%) 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.