OST1 is Limiting in ABA Responses of Arabidopsis Guard Cells Biswa R. Acharya 1, Byeong Wook Jeon 1,...

OST1 is Limiting in ABA Responses of Arabidopsis Guard Cells Biswa R. Acharya1, Byeong Wook Jeon1, Wei Zhang1, 2* and Sarah M. Assmann1, *

*Corresponding author (WZ):

Tel: (86)-531-88364332e-mail: [email protected]

*Corresponding author (SMA):

Tel.: (814)863-9579FAX: (814) 360-1089e-mail: [email protected]

Supporting Information

mailto:[email protected]

Fig. S1. Identification of C-terminal FLAG peptide-tagged transgenic OST1

overexpressing Arabidopsis thaliana lines.

Expression status of indicated transgenic lines was determined by RT-PCR analysis.

Forward primer specific to OST1 (N-terminus region) and reverse primer specific to the

region encoding for C-terminal FLAG peptide-tag of OST1-FLAG were used. Actin

gene specific primers were used as internal control. Empty vector transgenic plants

(EV) and Ler were used as negative controls

Actin

Ler

Ler

OV

1O

V2

OV

3O

V4

OV

5O

V6

OV

7O

V8

OV

9EV

1EV

3

OST1-FLAG

0 1 µM 10 µM 50 µM

closure

Ape

rtur

e (µ

m)

0 1 µM 10 µM 50 µM

Ape

rtur

e (µ

m)

openinga

b

Fig. S2. ABA inhibition of light induced stomatal opening and ABA promotion of

stomatal closure in Arabidopsis thaliana wild type (Ler) and empty vector lines (EV1 and

EV3).

Data are means ± SE of three independent replicates. In each replicate, > 130 stomata

were measured for each genotype per treatment. The means of the stomatal apertures of

EV lines are not significantly different from those of wild type (Ler), as assessed by two-

sample t-test (P > 0.05).

(a) ABA inhibition of stomatal opening in wild type and EV control lines.

(b) ABA promotion of stomatal closure in wild type and EV control lines.

0

2

4

6

Ler EV1 EV3

0

2

4

6

Ler EV1 EV3

Ape

rtur

e (µ

m)

Ape

rtur

e (µ

m)

Time (h)

Time (h)

a

b

0 1 2 3 4 51

2

3

4

5

6

7 Ler EV1 EV3 OV1 OV4 ost1-1 ost1-2

0 1 2 32

3

4

5

6

7 Ler (EtOH) EV1 (EtOH) EV3 (EtOH) OV1 (EtOH) OV4 (EtOH) ost1-1 (EtOH) ost1-2 (EtOH) Ler (ABA) EV1 (ABA) EV3 (ABA) OV1 (ABA) OV4 (ABA) ost1-1 (ABA) ost1-2 (ABA)

Fig S3. Time courses of stomatal aperture changes induced by 10 µM ABA in

Arabidopsis thaliana wild type (Ler), two empty vector control lines (EV1 and EV3),

two OST1 overexpressing lines (OV1 and OV4) and two ost1 mutants (ost1-1 and ost1-

2).

For opening experiments, the intact leaves of each genotypes were floated on opening

solution and kept in darkness for 2.5 h. After 2.5 h, leaves were illuminated with white

light at 450 µmol m-2 sec-1. Two leaves each were taken at indicated time points for

measurement of stomatal aperture. For closure experiments, the intact leaves of each

genotypes were floated on closing solution and illuminated for 2.5 h to induce stomatal

opening. After 2.5 h illumination, leaves were treated with 10 µM ABA. Two leaves

each were taken at indicated time points for measurement of stomatal aperture. Data are

means ± SE of three independent replicates. In each replicate, > 180 stomata were

measured for each genotype per treatment.

(a) ABA inhibition of light-induced stomatal opening

(b) ABA promotion of stomatal closure. At 0.5 h (↑), the means of the stomatal

apertures of OV1 and OV4 are statistically different (two-sample t-test) from those of

wild-type (Ler) (P < 0.05), but the means of the stomatal apertures of EV lines are not

significantly different from those of wild-type (P > 0.05).

a b

Fig. S4. ABA inhibition of whole cell K+in currents in guard cells of Arabidopsis thaliana

wild type (Ler) and empty vector lines (EV1 and EV3).

(a) Typical whole-cell recordings of K+ currents with or without 50 µM ABA treatment.

(b) I/V curves (mean ± SE) of time-activated whole-cell K+ currents indicated in (a).

Numbers of guard cells analyzed were: Ler (8), Ler + ABA (7), EV1 (7), EV1 + ABA (6),

EV3 (6), EV3 + ABA (7).

Fig. S5. Arabidopsis thaliana KAT1 does not interact with AtrbohD.

Protein-protein interaction between KAT1 and AtrbohD was performed by BiFC assays in epidermal

cells of Nicotiana benthamiana. KAT1-VenusN was co-expressed with VenusC-AtrbohD (by

cotransformation method). A representative confocal image does not show fluorescence from BiFC,

which indicates that KAT1 does not interact with AtrbohD. Similar results were obtained in more

than three replicates.

(BiFC: BiFC signal; Chl: chlorophyll autofluorescence)

BiFC Chl

BiFC Chl

a b

Fig. S6. ABA activation of whole cell anion currents in guard cells of Arabidopsis thaliana

wild type (Ler) and empty vector lines (EV1 and EV3).

(a) Typical whole-cell recordings of anion currents (Ler, EV1 and EV3) with or without 50

µM ABA treatment.

(b) I/V curves (mean ± SE) of time-activated anion currents as indicated in (a). Numbers of

guard cells analyzed were: Ler (7), Ler + ABA (8), EV1 (6), EV1 + ABA (7), EV3 (7), EV3

+ ABA (8).

50 p

A

20 s

Control 50 M ABA

Ler

ost1-1

ost1-2

Voltage (mV)

-150 -100 -50 0 50

Ani

on c

urre

nt (

pA

)

-60

-40

-20

0

20

Ler Ler+ABA ost1-1 ost1-1+ABA ost1-2 ost1-2+ABA

a b

Fig. S7. ABA activation of guard cell anion currents is impaired in Arabidopsis thaliana ost1

mutants under weak pH buffered pipette solution condition.

(a) Typical whole-cell anion currents in wild type and ost1 mutant guard cells with or without 50

µM ABA. Time and current scales as shown in top left panel apply to all traces.

(b) I/V curves (mean ± SE) of time-activated anion currents as recorded in (A). Numbers of

guard cells analyzed were: Ler (7), Ler + ABA (9), ost1-1 (7), ost1-1 + ABA (9), ost1-2 (9),

ost1-2 + ABA (10).

Fig. S8. Arabidopsis thaliana SLAC1 does not interact with AtrbohF.

Protein-protein interaction between SLAC1 and AtrbohF was performed by BiFC assay in epidermal

cells of Nicotiana benthamiana. SLAC1-VenusN was coexpressed with VenusC-AtrbohF (by


which indicates that SLAC1 does not interact with AtrbohF. Similar negative results were obtained in

more than three replicates.


BiFC Chl

BiFC

Fig. S9. ABA activation of whole cell Ba2+ currents through Ca2+-permeable channels in guard

cells of Arabidopsis thaliana wild type (Ler) and empty vector lines (EV1 and EV3).

(a-c) Typical whole-cell recordings of Ba2+ currents (Ler, EV1 and EV3) with or without 50 µM

ABA treatment.

(d) I/V curves (mean ± SE) of time-activated Ba2+ currents as indicated in (A-C). Numbers of

guard cells analyzed were: Ler (9), Ler + ABA (9), EV1 (8), EV1 + ABA (7), EV3 (9), EV3 +

ABA (7).

a

b

c

d

Fig. S10. ABA-induced ROS production in guard cells of Arabidopsis thaliana wild type (Ler)

and empty vector lines (EV1 and EV3).

(a) Representative images of ROS production in response to 50 µM ABA in indicated genotypes.

(b) Kinetics of ROS production in guard cells after treatment with 50 µM ABA (final

concentration) or with ethanol (solvent control).

Results are the averages ± SE (n > 6) from at least 3 independent experiments; Ler + ABA

(n=10), EV1 + ABA (n=11), EV3 + ABA (n=12), Ler (n=13), EV1 (n=12), and EV3 (n=10).

EV3EV1Ler

0

10 min

30 min

a

Flu

ores

cenc

e (%

of

init

ial)

Time (min)

LerLer + ABAEV1EV1 + ABAEV3EV3 + ABA

100

120

140

160

0 5 10 15 20 25 30

b

Fig. S11. Arabidopsis thaliana KAT1 does not interact with AtrbohF.

Protein-protein interaction between KAT1 and AtrbohF was performed by BiFC assay in epidermal

cells of Nicotiana benthamiana. KAT1-VenusN coexpressed with VenusC-AtrbohF (by


which indicates that KAT1 does not interact with AtrbohF. Similar negative results were obtained in

more than three replicates.


BiFC Chl

KAT1-Cub KAT2-Cub

OST1-NubG _ _

NubG-OST1 _ _

Nubwt-OST1

(positive control)

+++ ++

(NubG Vector)

(Negative Control)

_ _

Table S1. Interaction analyses between Arabidopsis thaliana OST1 and K+ channel proteins,

KAT1 and KAT2, in yeast (mbSUS assay).

Protein-protein interaction analysis between Nub-fusion proteins of OST1 (OST1-NubG and

NubG-OST1) and Cub-fusion protein of K+ channel proteins. Growth phenotypes of yeast on

interaction-selective media (minimal media containing 150 µM methionine). -: No yeast

growth (no interaction); ++: Moderate yeast growth (moderate positive interaction); +++:

Heavy yeast growth (heavy positive interaction). Note that Cub fusions are always on the C-

terminus of the protein, while two different NubG constructs: C-terminal (X-NubG) and N-

terminal (NubG-X) are used.

Video S1. ROS imaging in a guard cell pair of Arabidopsis thaliana wild type (Ler) in response

to 50 μM ABA.

For all videos, reactive oxygen species production in Arabidopsis guard cells was monitored

using the dye 2′,7′-dichlorofluorescin diacetate (H2DCF-DA, Invitrogen , USA) (Murata et al.,

2001) and analyzed by image J software (NIH, Bethesda, MD, USA) as earlier (Zhang et al.,

2011). Leaves from Arabidopsis plants (indicated genotype) were incubated for 3 h in 30 mM

KCl and 10 mM MES-KOH, pH 6.5 under white light (150 µmol m-2 sec-1). After incubation,

leaves were cut into slices (less than 3 mm2). Sliced leaves were then incubated with H2DCF-DA

at a final concentration of 30 µM for 20 min under dark. After 20 min, excess dye was removed

by three washes with distilled water. Leaf slices were then mounted on to an incubation chamber

with medical adhesive (Hollister, Libertyville, IL). Samples were then incubated for the

indicated time with 50 µM ABA or with an equal volume of ethanol added as a solvent control.

Guard cells were then observed with a laser scanning confocal microscope (LSM 510 Meta; Carl

Zeiss, Thornwoood, NY, USA) using a C-Apochromat 40×/1.2 W corr water immersion

objective. H2DCF-DA was excited by the 488-nm line of the argon laser at 4.9 A tube current

and 99% attenuation (equal to 1% laser intensity). The fluorescence of H2DCF was detected by a

bandpass emission filter (500-550 nm). Time-lapse imaging of guard cell pairs was performed at

30 sec interval for 30 min. Movie files were generated using Image J software.

Video S2. ROS imaging in a guard cell pair of Arabidopsis thaliana ost1-1 mutant in

response to 50 μM ABA.


response to 50 μM ABA.

Video S4. ROS imaging in a guard cell pair of Arabidopsis thaliana empty vector line EV1

in response to 50 μM ABA.


in response to 50 μM ABA.

Video S6. ROS imaging in a guard cell pair of Arabidopsis thaliana OST1 overexpressing

line OV1 in response to 50 μM ABA.


line OV4 in response to 50 μM ABA.

Video S8. ROS imaging in a guard cell pair of Arabidopsis thaliana wild type (Ler) in

response to ethanol.


response to ethanol.


in response to ethanol.


line OV1 in response to ethanol.

OST1 is Limiting in ABA Responses of Arabidopsis Guard Cells Biswa R. Acharya 1, Byeong Wook Jeon 1,...

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