Control Col-0 arr2-5 arr2-6 A 3 Supplemental Data. Arnaud ... · 0.010 0.015 0.020 Relative expression ... Heat map showing levels of ex pression of AHK and type-B ARR genes (log2

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Supplemental Figure 1. ARR2 participates in t-Z-mediated stomatal closure. (Supports Figure 1.)(A) Kinetic of stomatal closure in response to 1 µM t-zeatin (t-Z) or Control solution.(B) Stomatal apertures in Col-0 WT, arr2-5 and arr2-6 after 2 h of incubation with Control solution or 1 µM t-Z. Values are means ± SE (n ≥ 100). Different letters indicate significant differences at P < 0.001 based on a Tukey’s HSD test (See ANOVA table; Supplemental Dataset 1).(C) T-DNA insertional mutation sites in arr2-4, arr2-5 and arr2-6 mutant lines (upper panel). Black and gray filled boxes represents exon and 5' and 3' untranslated regions respectively. Inverted triangles indicate the T-DNA insertions and arrows denote the positions of the primers used in RT-qPCR experiments. RT-qPCR analysis of ARR2 transcript in WT (Col-0) and arr2-4, arr2-5 and arr2-6 mutants (lower panel). Transcript levels were normalized to UBQ1. Relative ARR2/UBQ1 expression ratios are shown. Error bars indicate SE (n=4). (D) Heat map showing levels of ex pression of AHK and type-B ARR genes (log2 intensities) in guard cells (GC) and leaves (upper panel; Pandey et al., 2010) and in guard cells (GC) and mesophyll cells (MC) treated (+) or not (-) with the transcription inhibitors actinomycin and cordycepin (lower panel; Yang et al., 2008).

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Supplemental Data. Arnaud et al. (2017). Plant Cell 10.1105/tpc.16.00583

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Supplemental Figure 2. Cytokinin inhibits the COR-dependent reopening of stomata. (Supports Figure 2.)(A) Stomatal apertures in Col-0 WT, arr2-5 and arr2-6 after 2 h of incubation with Control solution or 5 µM flg22.(B) Stomatal apertures in Col-0 WT, IPT1 and IPT3 overexpressing (OE) lines exposed to Mock control or 108 cfu/ml Pst DC3000 (Pst) for 1 and 4 h. In (A) and (B), values are means ± SE (n ≥ 100). Different letters indicate significant differences at P < 0.001 based on a Tukey’s HSD test (See ANOVA table; Supplemental Dataset 1).(C) Bacterial growth in Col-0 WT at 3 days after spray-inoculation with Pst DC3000 at 108 cfu/ml together with 1 µM t-Z or Control solution.(D) and (E) Bacterial growth at 3 days after spray-inoculation with Pst DC3000 at 108 cfu/ml in Col-0 WT, ARR2 OE line (D), IPT1 OE and IPT3 OE lines (E). In (C) to (E), values are the means ± SE (n = 4). Asterisks ind icate significant d ifferences between Control and t-Z treatment (C) and between WT and ARR2 OE lines (D) based on a two-tailed Stud ent's t-test (*P<0.05). In (E), different letters indicate significant differences at P < 0.05 based on a Tukey’s HSD test (See ANOVA table; Supplemental Dataset 1).(F) and (G) COR-med iated inhibition of flg22-triggered stomatal closure is impaired by t-Z or ARR2 overexpression. Stomatal aperture in Col-0 and ARR2 OE line (F), or in Col-0 WT treated with Control solution or 1 µM t-Z (G), exposed to Control solution, 1.6 µM COR, 5 µM flg22, or 5 µM flg22 and 1.6 µM COR together (flg22+COR) for 2.5 h. Values are means ± SE (n ≥ 100). Different letters indicate significant differences at P < 0.001 based on a Tukey’s HSD test (See ANOVA table; Supplemental Dataset 1).

ACol-0 arr2-5 arr2-6

a a a a aa aa

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Supplemental Figure 3. CK-mediated stomatal closure is dependent on apoplastic peroxidases. (Supports Figure 3.)(A) ROS production detected by H

2DCFDA fluorescence in guard cells of Col-0 WT, ARR2 and IPT3

overexpression (OE) lines 30 min after treatments with Control solution or 5 µM flg22. Values are means ± SE (n ≥ 60). (B) Stomatal aperture in Col-0 WT and IPT3 overexpressing lines (IPT3 OE) exposed to Control solution, 1 mM ascorbate (ASC), 20 µM DPI, 2 mM SHAM or 1 µM sodium azide for 3 h. Values are means ± SE (n ≥ 100). In (A) and (B), different letters indicate significant differences at P < 0.001 based on a Tukey’s HSD test (See ANOVA table; Supplemental Dataset 1). (C) RT-qPCR analysis of RBOHD and RBOHF transcript levels in guard cell protoplasts isolated from leaves of 5-week-old Col-0 WT and ARR2 overexpressing line (ARR2 OE). Transcript levels were normalized to UBQ1. Error bars indicate SE of three independent experiments (n = 3). Asterisks indicate statistically significant differences between Col-0 and ARR2 OE based on a two-tailed Student's t-test (**P<0.01).

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PRX12PRX63PRX66PRX67PRX42PRX31PRX19PRX25PRX73PRX41PRX9PRX46PRX10PRX48PRX65PRX35PRX24PRX57PRX28PRX7PRX68PRX1/2PRX6PRX3PRX27PRX43PRX61PRX40PRX55PRX64PRX39PRX44PRX56PRX36PRX26PRX60PRX13PRX16PRX45PRX18PRX20PRX8PRX72PRX11PRX17PRX30PRX14/15PRX38PRX54PRX5PRX22/23PRX59PRX53PRX70PRX47PRX21PRX50/51PRX32/33/34PRX58PRX37PRX49PRX69PRX71PRX62PRX52PRX4A

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Supplemental Figure 4. The expression of a set of PRX genes is induced by bacteria or PAMPs in leaves. (Supports Figure 4.)(A) Expression profile of PRX genes in response to elicitor treatments and bacteria infection. Fold change (log

2; treatment versus control) are color-coded (red and green for relatively higher

or lower expression, respectively) as indicated in the color bar. White indicates no change between conditions. Data are from AtGenExpress (F. Brunner and T. Nürnberger, unpublished). Note that each couple of tandem-duplicated genes PRX4/5, PRX33/34, PRX37/38, PRX50/51 and PRX69/70/71 show high sequence homology (Valério et al., 2004) indicating possible cross-hybridization, and the PRX1/2 pair of genes as well as PRX17/18, PRX22/23, PRX32/33/34 and PRX50/51 share the same microarray probe set.(B) Expression profile of PRX genes in guard cells and mesophyll cells. Heat map showing levels of gene expression (log

2 intensities) in guard cells (GC) and leaves (upper panel; Pandey

et al., 2010) and in guard cells and mesophyll cells (MC) treated or not with the transcription inhibitors actinomycin and cordycepin (lower panel; Yang et al., 2008). Genes early-induced or repressed by elicitors or bacteria are respectively highlighted in red and green, and genes late-induced by avirulent bacteria are indicated in orange.


5

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→ ←

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→ ←

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←→

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←A B

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Supplemental Figure 5. Molecular characterization of prx mutants. (Supports Figure 5.)(A) T-DNA insertional mutation sites in prx mutant lines. Black and gray filled boxes represent exons and 5' and 3' untranslated regions respectively. Inverted triangles indicate the T-DNA insertions and arrows denote the positions of the primers used in the RT-PCR and RT-qPCR experiments in (B and C). Numbers indicate positions of T-DNA insertions relative to ATG initiation codon. (B) and (C) RT-PCR (B) and RT-qPCR (C) analyses of PRX transcripts in leaves of 5-week-old Col-0 WT and prx mutant lines. EF1 was used as a control for cDNA quantity. Numbers in parentheses indicate the number of PCR cycles used to amplify the PRX genes. Transcript levels determined by RT-qPCR were normalized to UBQ1 as an internal control. Relative gene/UBQ1 expression ratios are shown. Error bars indicate SE (n = 4).

→


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Supplemental Figure 6. PRX33 and PRX34 expression in ARR2 OE/prx33-3 and ARR2 OE/prx34-2 lines. (Supports Figure 6.)(A) and (B) RT-qPCR and (A) and RT-PCR (B) analyses of PRX33 and PRX34 transcripts in leaves of 5-week-old Col-0 WT, ARR2 OE, ARR2 OE/prx33-3 and ARR2 OE/prx34-2 lines, and prx33-3 and prx34-2 mutants. Transcript levels determined by RT-qPCR were normalized to UBQ1. Error bars indicate SE of three independent experiments, each consisting of three biological replicates (n = 9). The changes in transcript levels are relative to Col-0 WT. Different letters indicate significant differences at P < 0.05 based on a Tukey’s HSD test (See ANOVA table; Supplemental Dataset 1). For RT-PCR, numbers in parentheses indicate the number of PCR cycles used to amplify the PRX genes. EF1 was used as a control for cDNA quantity.

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Supplemental Figure 7. CK functions with SA in guard cells independently of the ABA pathway. (Supports Figure 8.)(A) and (B) ROS production detected by H

2DCFDA fluorescence in guard cells of Col-0 WT,

npr1-1 and sid2-2 mutants (A) and ARR2 and IPT3 overexpression (OE) lines (B) 30 min after treatment with Control solutions, 5 µM flg22, 1 µM t-Z or 10 µM SA.(C) Stomatal apertures in Col-0 WT and tga3-3 mutant after 2 h of incubation with Control solution, 1 µM t-Z or 5 µM flg22. (D) Basal stomatal apertures in Col-0 WT, tga3-3 mutant, ARR2 OE and tga3-3/ARR2 OE lines. (E) ROS production detected by H

2DCFDA fluorescence in guard cells of Col-0 WT and ost1-3

mutant 30 min after treatments with Control solution or 1 µM t-Z.For (A), (B) and (E), results are shown as the mean of ≥60 stomata ± SE. For (C) and (D), results are shown as the mean of ≥100 stomata measurements ± SE. Different letters indicate significant differences at P < 0.001 based on a Tukey’s HSD test (See ANOVA table; Supplemental Dataset 1).

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8

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Supplemental Figure 8. ARR2 overexpression enhances plant growth.(A) Phenotype of representative 5-week-old Col-0 WT and ARR2 overexpressing (ARR2 OE) plants.(B) Shoot fresh weight measurements of 5 week-old plants. Error bars indicate SE (n = 10). Asterisks indicate significant differences between the WT and the ARR2 OE line based on a two-tailed Student's t-test (**P<0.01).

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Supplemental Figure 9. RBOHD and RBOHF expression is not affected in prx33-3 and prx34-2 mutants. RBOHD and RBOHF transcript levels were determined by RT-qPCR and normalized to UBQ1 as an internal control. Relative gene/UBQ1 expression ratios are shown. Error bars indicate SE (n = 4).


10

Supplemental Table 1. Cytokinin concentrations in Col-0 (WT) leaves at 1 h and 3 h after

inoculation with Pst DC3000 bacteria.

pmole/gDW Mock 1 h Pst DC3000 1 h Mock 3 h Pst DC3000 3 h

tZ 14.28 ± 1.37 13.40 ± 1.49 13.56 ± 0.85 10.83 ± 1.41

tZR 75.38 ± 7.63 73.23 ± 2.92 75.43 ± 7.48 57.04 ± 6.67

tZRPs 161.49 ± 15.81 129.78 ± 3.74 64.19 ± 3.07 45.84 ± 6.11*

cZ 2.94 ± 0.44 8.2 ± 1.32* 7.9 ± 1.49 7.76 ± 0.89

cZR 41.14 ± 9.92 94.05 ± 23.52 240.2 ± 72.56 198.65 ± 33.53

cZRPs 23.95 ± 3.62 35.52 ± 8.31 60.99 ± 4.92 84.58 ± 3.44**

DZ ND ND ND ND

DZR ND ND ND ND

DZRPs ND ND ND ND

iP 0.86 ± 0.03 1.45 ± 0.08*** 1.06 ± 0.24 1.01 ± 0.16

iPR 17.67 ± 0.98 31.17 ± 4.88* 46.21 ± 14.94 31.23 ± 5.32

iPRPs 131.53 ± 16.14 154.59 ± 13.79 54.64 ± 3.35 53.6 ± 0.69

tZ7G 497.16 ± 44.82 487.84 ± 14.65 433.0 ± 15.97 434.09 ± 27.68

tZ9G 383.82 ± 32.91 399.23 ± 13.76 402.49 ± 13.92 364.64 ± 32.53

tZOG 41.94 ± 1.68 37.34 ± 1.78 35.04 ± 0.74 36.74 ± 2.25

cZOG 7.65 ± 0.28 7.8 ± 0.68 5.64 ± 0.47 7.12 ± 0.77

tZROG 9.9 ± 0.90 9.08 ± 0.70 8.08 ± 0.62 7.48 ± 0.95

cZROG 17.3 ± 2.16 17.26 ± 0.60 19.45 ± 2.77 19.47 ± 1.25

tZRPsOG ND ND ND ND

cZRPsOG ND ND ND ND

DZ9G 1.72 ± 0.04 1.61 ± 0.14 1.77 ± 0.09 1.79 ± 0.17

iP7G 111.86 ± 2.54 117.09 ± 3.12 107.25 ± 4.28 108.06 ± 4.02

iP9G 23.73 ± 1.04 25.04 ± 1.45 21.74 ± 0.83 21.60 ± 0.68

Arabidopsis Col-0 plants were grown on soil for 4 to 5 weeks before harvest. Leaves were

syringe-infiltrated with Mock control (10 mM MgCl2) or 108 cfu/ml Pst DC3000 bacteria and

collected 1 h and 3 h after infiltration for the measurement of CK concentrations. Data are

means ± SE of three independent experiments (n = 3). Asterisks indicate statistically

significant differences between Mock control and Pst DC3000 bacteria treatments based on a

two-tailed Student's t-test (*P<0.05; **P<0.01; ***P<0.01). gDW, gram dry weight; tZ, trans-

zeatin; tZR, tZ riboside; tZRPs, tZ ribotides; cZ, cis-zeatin; cZR, cZ riboside; cZRPs, cZ

ribotides; DZ, dihydrozeation; DZR, DZ riboside; DZRPs, DZ ribotide; iP, N6-(Δ2-

isopentenyl)adenine; iPR, iP riboside; iPRPs, iP ribotides; tZ7G, tZ-7-N-glucoside; tZ9G, tZ-9-

N-glucoside; tZOG, tZ-O-glucoside; cZOG, cZ-O-glucoside; tZROG, tZR-O-glucoside;

cZROG, cZR-O-glucoside; tZRPsOG, tZR 5'-phosphate-O-glucoside; cZRPsOG, cZR 5'-


11

phosphate-O-glucoside; DZ9G, DZ-9-N-glucoside; iP7G, iP-7-N-glucoside; iP9G, iP-9-N-

glucoside; ND, not detected.

Supplemental Table 2. Expression levels of PRX genes in guard cell protoplasts of Col-0

(WT) and the ARR2 overexpressing line (ARR2 OE), and in Col-0 after flg22 treatment.

Gene Col-0 ARR2 OE Control flg22

PRX4 1 ± 0.01 0.21 ± 0.02*** 1 ± 0.04 5.41 ± 1.10**

PRX5 1 ± 0.03 1.10 ± 0.23 1 ± 0.06 11.49 ± 4.41

PRX22 ND ND ND ND

PRX23 ND ND ND ND

PRX32 1 ± 0.05 0.92 ± 0.11 1 ± 0.19 2.30 ± 0.77

PRX33 1 ± 0.09 2.06 ± 0.24** 1 ± 0.13 8.74 ± 1.34***

PRX34 1 ± 0.01 0.89 ± 0.05 1 ± 0.02 2.07 ± 0.15***

PRX37 1 ± 0.10 0.91 ± 0.11 1 ± 0.11 30.33 ± 5.26***

PRX38 ND ND 1 ± 0.60 13.09 ± 4.05*

PRX50 1 ± 0.18 2.82 ± 0.45** 1 ± 0.09 4.53 ± 1.26*

PRX51 1 ± 0.02 0.78 ± 0.17 1 ± 0.03 2.90 ± 0.68*

PRX62 1 ± 0.15 0.62 ± 0.13 1 ± 0.10 21.90 ± 5.05**

PRX69 1 ± 0.13 1.23 ± 0.20 1 ± 0.40 1.75 ± 0.44

PRX70 1 ± 0.22 1.30 ± 0.07 1 ± 0.80 11.70 ± 2.52***

PRX71 1 ± 0.10 1.22 ± 0.26 1 ± 0.03 19.18 ± 2.70***

ARR5 1 ± 0.18 1.94 ± 0.16** 1 ± 0.09 0.90 ± 0.16

ARR6 1 ± 0.11 5.13 ± 0.76** 1 ± 0.12 0.94 ± 0.32

Plants were grown on soil for 4 to 5 weeks before harvest. For flg22 treatment, about 50

leaves of Col-0 (WT) were incubated for 2 h in stomatal buffer without (Control) or with 1 µM

flg22. Guard cell protoplasts were isolated in the presence of transcriptional inhibitors.

Transcript levels were determined by RT-qPCR and normalized to both UBQ1 and EF1. Data

are means ± SE of three independent experiments (n = 3). The changes in transcript levels

are relative to Col-0 WT or Control treatment (expression value = 1). ND, not detected.

Asterisks indicate statistically significant differences between WT and ARR2 OE lines or

between Control and flg22 treatments based on a two-tailed Student's t-test (*P<0.05;**P<0.01; ***P<0.01).


12

Supplemental Table 3. Expression levels of PRX genes in whole leaves of Col-0 (WT),

ARR2 and IPT3 overexpressing lines (ARR2 OE and IPT3 OE), and in Col-0 after Control or

flg22 treatments.

Gene Col-0 ARR2 OE Col-0 IPT3 OE Control flg22

PRX4 1 ± 0.11 1.01 ± 0.20 1 ± 0.11 1.35 ± 0.20 1 ± 0.11 5.02 ± 1.27*

PRX5 ND ND ND ND 1 ± 0.22 49.80 ± 12.04**

PRX22 1 ± 0.24 0.87 ± 0.46 1 ± 0.38 0.37 ± 0.22 1 ± 0.34 0.21 ± 0.08*

PRX23 1 ± 0.43 1.12 ± 0.54 1 ± 0.28 0.37 ± 0.14 1 ± 0.57 0.07 ± 0.07*

PRX32 1 ± 0.10 0.90 ± 0.15 1 ± 0.09 0.98 ± 0.07 1 ± 0.20 0.68 ± 0.15

PRX33 1 ± 0.22 6.25 ± 2.04* 1 ± 0.09 2.35 ± 0.44** 1 ± 0.15 9.59 ± 2.37**

PRX34 1 ± 0.12 2.46 ± 0.55* 1 ± 0.04 1.52 ± 0.25 1 ± 0.10 3.75 ± 0.94*

PRX37 1 ± 0.11 2.61 ± 0.53* 1 ± 0.06 1.38 ± 0.22 1 ± 0.24 23.39 ± 6.26**

PRX38 1 ± 0.27 0.64 ± 0.11 1 ± 0.16 0.91 ± 0.24 1 ± 0.21 57.84 ± 12.22**

PRX50 1 ± 0.08 0.92 ± 0.16 1 ± 0.06 0.90 ± 0.11 1 ± 0.07 3.31 ± 0.54**

PRX51 1 ± 0.08 0.79 ± 0.09 1 ± 0.11 0.95 ± 0.13 1 ± 0.08 5.11 ± 1.17**

PRX62 1 ± 0.54 1.07 ± 0.58 1 ± 0.65 4.94 ± 2.55 1 ± 0.19 2.49 ± 0.88

PRX69 1 ± 0.24 1.39 ± 0.33 1 ± 0.23 1.01 ± 0.35 1 ± 0.08 14.19 ± 2.46**

PRX70 1 ± 0.08 2.61 ± 0.56* 1 ± 0.08 1.26 ± 0.19 1 ± 0.05 35.14 ± 4.47**

PRX71 1 ± 0.12 1.95 ± 0.56 1 ± 0.05 1.47 ± 0.20* 1 ± 0.11 4.28 ± 0.43**

PR1 1 ± 0.19 36.44 ± 16.36** 1 ± 0.21 2.25 ± 0.95 1 ± 0.21 2.75 ± 1.13

Plants were grown on soil for 4 to 5 weeks before harvest. For flg22 treatment, Col-0 (WT)

leaves were syringe-infiltrated with 10 mM MgSO4 (Control) or 1 µM flg22 and collected after

2 h. Transcript levels were determined by RT-qPCR and normalized to both UBQ1 and EF1.

Data are means ± standard error of three independent experiments, each consisting of three

biological replicates (n = 9). The changes in transcript levels are relative to Col-0 WT or

Control treatment (expression value = 1). ND, not detected. Asterisks indicate statistically

significant differences between WT and OE lines or between Control and flg22 treatments

based on a two-tailed Student's t-test (*P<0.05; **P<0.01).


13

Supplemental Table 4. Positions of putative B-type ARR binding sites (AGATT) in PRX4, PRX33, PRX34 and PRX71 promoters.

Gene Position from transcription start site Strand orientation Q-PCR primers

PRX4 -2016 (-)-1800 (+)-1741 (+) pPRX4a-1396 (+)-936 (+)-594 (+) pPRX4b-480 (-)-327 (+) pPRX4c-307 (+) pPRX4c-234 (+) pPRX4c

PRX33 -2032 (+)-1871 (+) pPRX33a-1679 (-) pPRX33a-1303 (-) pPRX33b-1216 (+) pPRX33b-583 (+)-364 (+) pPRX33c-296 (+) pPRX33c

PRX34 -1912 (+)-1661 (+) pPRX34f-1591 (+) pPRX34f-1578 (-) pPRX34f-1513 (-) pPRX34f-1091 (-) pPRX34g-879 (+) pPRX34g-685 (-)-366 (+) pPRX34h-309 (-) pPRX34h-88 (+)

PRX71 -1975 (+)-1424 (-)-1428 (-)-1159 (-) pPRX71a-963 (-) pPRX71a-651 (-)-599 (+)-133 (-) pPRX71b-44 (-) pPRX71b


14

Supplemental Table 5. Description of the mutant lines used in the study.

Gene name TAIR locus Genotype Mutation References

AHK2 AT5G35750 ahk2-2tk Higuchi et al., 2004

AHK3 AT1G27320 ahk3-3 SALK_069269 Higuchi et al., 2004

AHK4/CRE1 AT2G01830 cre1-12 SALK_048970 Higuchi et al., 2004

ARR1 AT3G16857 arr1-4 SALK_042196 Mason et al., 2005


arr2-5 GK-269G01 Hill et al., 2013

arr2-6 SALK_142235C This study

ARR10 AT4G31920 arr10-5 SALK_098604 Yokoyama et al., 2007



ARR14 AT2G01760 arr14-1 GK-147B02 Ishida et al., 2008


OST1/SNRK2.6/SNRK2E AT4G33950 ost1-3 SALK_008068 Yoshida et al., 2002

ABA1 AT5G67030 aba1-3 CS3100 Rock et al., 1991

NPR1 AT1G64280 npr1-1 CS3726 Cao et al., 1994

SID2/ICS1 AT1G74710 sid2-2 CS16438 Wildermuth et al., 2001

TGA3 AT1G22070 tga3-3 Salk_088114C Choi et al., 2010

RBOHD AT5G47910 rbohD CS9555 Torres et al., 2002

RBOHF AT1G64060 rbohF CS9557 Torres et al., 2002

PRX4 AT1G14540 prx4-1 SALK_110617C This study

prx4-2 SALK_044730C This study


prx33-3 GK-014E05 This study

PRX34 AT3G49120 prx34-1 SALK_051769 This study

prx34-2 GK-728F08 This study

PRX37 AT4G08770 prx37-1 GK-158H12 This study




PRX62 AT5G39580 prx62-1 GK-287E07 This study

PRX69 AT5G64100 prx69-1 SAIL_691_G12 This study



15

Supplemental Table 6. PCR primers used for genotyping the mutant lines.

Genotype Forward primers 5' to 3' Reverse primers 5' to 3'

ahk2-2tk GAGGGAGACTTTGGCAAACATG GGTAGGCTCGTGTCATAGAC

ahk3-3 CCTTGTGATTGCGTTACTTGTTGCAC CTCTCAAATCAAACCGCACCTCCT

cre1-12 AGCCTTCACCGGTTAGGGATGAG CTCCACATCAAACGCACCACCAAG

arr1-4 GCATGTTGGTCTAGAGATGGACTTAC TTAAGATTGCCTTGGTGCTGCGATAC

arr2-4 AGCTCGAGGAAGCGGAAGGAAG TTCCGCTGCTCTGAACATTCTGTC

arr2-5 GCCATGCCCTTCATAAATCTTTC ACAATGCGCTCTCTGCTCTGT

arr2-6 AAGGACATCTCTACACCAACGTATATAC AGACAATGCGCTCTCTGCTCTGT

arr10-5 GCATGAGCTGCACAATAAATTCCTAG AGCGGCAAACCCTGAAGTATGTTTC

arr11-3 GCTGGTCCCAAGAAGATATTGGATC GCCAAGTCAAGTCTAAGTCAGTGTC

arr12-1 CAACGACGAACCAAGCACAAAAGG CTTGCTGATTAGCCACACCACTGA

arr14-1 ATCAACGATCAGTTTCCTAGTGG TCACTAACCGAAACAACAACAGTC

arr18-2 TGGATAAAGTTAAGACTGTTGGAGACTCA TTGTCATCCACAGCAAGAACCCTC

ost1-3 CAGCAGTGAGTGGTCCAATGGAT ACAGTTGCTTCTGCAATGATCTGC

tga3-3 GGCGCACGATAAGGTCAACTTTTG GGACTCTCCCAACCAGATAACTGT

rbohD CATGGGTTATTGCGTTTGTGTCGCCAA GGATACTGATCATAGGCGTGGCTCCA

rbohF ACTTCCGATATCCTTCAACCAACTCTTTG CTCTCGTCGTTGATTTGTGACCAATACT

prx4-1 GTCAACGTAGGCCAGCTTCAACA AAGCCTACCTTTGAACGTGAGG

prx4-2 GCGTTTAGGGCTATCGCAGAC TCACCAGAACATCTCCACACGG

prx33-2 CAACTTACCCCTACTTTTTACGACACT TCGGGCTGAATTTGCATTTCCAAGT

prx33-3 TGGAAATGCAAATTCAGCCCGA CAGATCGAAATCCACTAAGACG

prx34-1 TCCGTTAGCGTTCCACATTAGTTGT TTGAGCAGCGGACAAAGATGCATG

prx34-2 CACCCCTACCTTCTACGATAG CCATTTGTTCCTCTGAAGCAAG

prx37-1 CAGGTATCATTGTCTCATGCTCAA TCCGTCCTAAACGATGTGGTGT

prx38-1 GTGGCGTAGTATAGTAGCGCAT GTCGGGTAAACCGGTTTCACC

prx50-1 CCCTCATATAGATAGATCATCCTAGT CGAAGAGTGAAGTGAGTTTATTGAC

prx51-1 CGGCACACACAGCACAAAGCA AAGTGCATTGAGTTGGTTGAGATCA

prx62-1 CGAAGCCAGCTCATGACCAA GAGTTAGGTCCCGATAAAAGCAC

prx69-1 CAAGGGAACCGTGGCTCAAACA ACTTCCCTCGTCTAACTCCACT

prx71-1 CGCCGTGAATGCTGGTTTCAG CCCACGGCTTAGGTTGTTGTAG

Salk T-DNA TGGTTCACGTAGTGGGCCATCG

SAIL T-DNA CCCAAATTACCAATACATTACACTAGC

GABI-Kat T-DNA ATATTGACCATCATACTCATTGC

DsLox T-DNA GCTCTTGCTAAGCTCCTCGAGTT


16

Supplemental Table 7. Primers used for RT-PCR and quantitative RT-PCR.

Gene Forward primers 5' to 3' Reverse primers 5' to 3'

RT-qPCR

AHK3 CCTTGTGATTGCGTTACTTGTTGCAC CTCTCAAATCAAACCGCACCTCCT

ARR2 CCTTCTCTGATCGTTCGTTTTCTG ATCAACGACAAGAACTCGAAGATTC

GAATCAGCTAGGCGTTGACAAAG GCAAGGCTCTGAGGAGGGAG

ARR5 GCGTCCCGAGATGTTAGATATCT CTCGATGAACTTCCGATCAACCATA

ARR6 CCGAGGAAGATGGAGATTCTCAA CCGACTGATTTTTCCTCAACATCC

RBOHD GACGATGAGTACGTGGAGATCA GGAGGTGGTGTTGTTGAGGCT

RBOHF CATCTATCGCTCCGATTTCGCT CTCTCGTCGTTGATTTGTGACCA

PRX4 GCGTTTAGGGCTATCGCAGAC AAGCCTACCTTTGAACGTGAGG

PRX5 AGCATCATCCTCACGTCATCGA CTAACGGAGGATCTGATCTTCGAA

PRX33 ATCGTCCTTCTGATCTTGTTGCG GCAGATCGAAATCCACTAAGACG

PRX34 ATCGTCCTTCTGATCTCGTTGCT GATCAAAATCTACCAAGGCACTTC

PRX22 GCACTTCCGATCTAGTTGCTCTT ACACAATCGACGGAGTTCGACA

PRX23 CCTTCGGATTTAGTCGCCCTC ACACAATCGACGGAGTTCAACG

PRX32 CCAGTGCCAATTTATTATGGATAGAC GACGGTCTGGTTACCATTGCG

PRX37 CCTTCCAGCTCCATTCTTTACC GGTCGGGTAAACCAGTGTTACT

PRX38 CCTTCCAGGTCCATCTTCTACA GTCGGGTAAACCGGTTTCACC

PRX50 AGGCTAGATGGACTATCATCAACA CGAAGAGTGAAGTGAGTTTATTGAC

PRX51 GATGGACTTTCGTCGTCGGCTT AAGTGCATTGAGTTGGTTGAGATCA

PRX62 TCGGACCACTGTGGCATCTCA GAGTTAGGTCCCGATAAAAGCAC

PRX69 CTGCTGGCTGCGGTCTAGTAA ACTTCCCTCGTCTAACTCCACT

PRX70 GGGACAGATTCTTCAACTACGACA CACTTCCCGTGTCCAGCACTA

PRX71 CGCCGTGAATGCTGGTTTCAG GGTGTTAGCTCCGGATATAAGGA

EF1α TGAGCACGCTCTTCTTGCTTTCA GTGGTGGCATCCATCTTGTTACA

UBQ1 GCTTGCTCGTAAGTACAATCAGG GGCCTCAACTGGTTGCTGTGA

PR1 AAAACTTAGCCTGGGGTAGCGG CCACCATTGTTACACCTCACTTTG

RT-PCR

PRX33 ACTTACCCCTACTTTTTACGACACT GCAGATCGAAATCCACTAAGACG

PRX34 TCACCCCTACCTTCTACGATAG GATCAAAATCTACCAAGGCACTTC

PRX37 CAGGTATCATTGTCTCATGCTCAA GGTCGGGTAAACCAGTGTTACT

PRX38 TCCGCAAGTCTTTGATATTGTAACCAA GTCGGGTAAACCGGTTTCACC

PRX50 AGGCTAGATGGACTATCATCAACA TGAGACCCATGACCCATCCTAT

PRX51 ATTCTCTCTCTTTTTCTCGCCATCA AAGTGCATTGAGTTGGTTGAGATCA

PRX62 CACACTTGCGGTCTTATCTAAAACAC GAGTTAGGTCCCGATAAAAGCAC

PRX69 CAAGGGAACCGTGGCTCAAACA ACTTCCCTCGTCTAACTCCACT

PRX71 CGCCGTGAATGCTGGTTTCAG CCCACGGCTTAGGTTGTTGTAG

EF1α GAGGCTGGTATCTCTAAGGATGGT CTGTGGGAGCAAAGGTAACAACCA


17

Supplemental Table 8. Primers used for ChIP qPCR.

Name Forward primers 5' to 3' Reverse primers 5' to 3'

pEF1 CCGTCACCTTTTCGATCATCAAG CCTTACGCGTTTTGGGCCTTTC

pARR5 CACTCACCATTACTTGACTATTCTC GTGGGATTTTGTCTGTCTGATAGC

pPRX4a AGGGGTGTCATTTTCATCTAGCTCT TCCATACATGCACTGATCTAGTAACA

pPRX4b GGCTAGTGGTTACGGTATGGACA CTGGCCTACGTTGACCAAACCA

pPRX4c ACCCCGAACAAACACTCTTTTGTTG GTTTCTGCGATGATTATTGAACTCTCA

pPRX33a GTCATGGGTTACTATGCACGCA GTAGGGTGTCAACTAAAGCTTAAGT

pPRX33b GTTGTGTGGAAATAAGCGGTTTTCT ACCCAATAAATTCAAACAATGGAAACCA

pPRX33c TGGCCCGTTTCTTGGTTTTTGC CACCGCACATCAAAGAAGACTAG

PRX33d ACTTACCCCTACTTTTTACGACACT GGGTCCGATCTTAGCTCGTTG

PRX34e GTGGCCTTGCATTGATTTATGATAC ACCCTATGAATCGCTTCTCGTTC

pPRX34f TTGAGAGATTTTTTCAGAAGCATATTGG GACGTATAGCGACTTAATATCCTCA

pPRX34g AGGGCACCGTTTAGTAGTATAGG CGACGACCTTTCAAAATCTCTTGG

pPRX34h AGCGTTCCACATTAGTTGTAAAGAG GATGTCAAAATTCGACCATAAAATATAACC

pPRX71a AGCTAGGGACCTTGGTTTGTAGA CACCAGACCACATTTTTTCAATGTG

pPRX71b ACGCGCTTTTGAATCTCCCACAA GAGGAGATTGATTAGTCAAAGAATGA


18

Supplemental Table 9. Gene sequences used in this article and the corresponding

accession numbers.

Genes Accession numbers (AGI) Genes Accession numbers (AGI)

AHK2 AT5G35750 PRX4 AT1G14540

AHK3 AT1G27320 PRX5 AT1G14550

AHK4/CRE1 AT2G01830 PRX33 AT3G49110

ARR1 AT3G16857 PRX34 AT3G49120


ARR10 AT4G31920 PRX23 AT2G38390

ARR11 AT1G67710 PRX32 AT3G32980

ARR12 AT2G25180 PRX37 AT4G08770

ARR14 AT2G01760 PRX38 AT4G08780

ARR18 AT5G58080 PRX50 AT4G37520



OST1/SNRK2.6/SNRK2E AT4G33950 PRX69 AT5G64100

ABA1 AT5G67030 PRX70 AT5G64110

NPR1 AT1G64280 PRX71 AT5G64120

SID2/ICS1 AT1G74710 PR1 AT2G14610

TGA3 AT1G22070 EF1α AT5G60390

RBOHD AT5G47910 UBQ1 AT3G52590

RBOHF AT1G64060

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Documents

Control Col-0 arr2-5 arr2-6 A 3 Supplemental Data. Arnaud ... · 0.010 0.015 0.020 Relative expression ... Heat map showing levels of ex pression of AHK and type-B ARR genes (log2