Lim et al, Supplemental Figure S1. OsRING-H2 type : 5 OsRING-HC type : 1 OsRING-v type : 1 OsRING-H2...
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Transcript of Lim et al, Supplemental Figure S1. OsRING-H2 type : 5 OsRING-HC type : 1 OsRING-v type : 1 OsRING-H2...
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.