supplementary information - nature c6 c4 c5 c7 a groups at2g31010 at3g58640 ... plants were...

Supplementary Information A Raf-like protein kinase BHP mediates blue light-dependent stomatal opening Maki Hayashi1, Shin-ichiro Inoue1, Yoshihisa Ueno1,3, Toshinori Kinoshita1,2,*

1Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan 2Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602, Japan 3Current address: Department of Agriculture, Ryukoku University, Seta Oe-cho, Otsu, Shiga 520-2194, Japan *Corresponding author: Toshinori Kinoshita Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602, Japan Tel/ Fax: +81-52-789-4778 E-mail: [email protected]

Exp

ress

ion

leve

l in

guar

d ce

lls

HT1

b

B1

B2

B3

B4

C1

C2

C3

C6

C4

C5

C7

a G

roup

s A

t2g3

1010

At3

g586

40 A

t2g4

2640

At4

g230

50 A

t1g6

7890

At5

g494

70 A

t3g0

6620

At3

g066

30 A

t3g0

6640

At1

g087

20 A

t5g1

1850

At1

g181

60 A

t1g7

3660

At4

g244

80 A

t5g0

3730

At1

g162

70 A

t1g7

9570

At2

g350

50 A

t1g0

4700

At3

g247

15 A

t3g4

6920

At5

g576

10 A

t1g1

4000

At3

g598

30 A

t2g4

3850

At2

g318

00 A

t4g1

8950

At3

g587

60 A

t2g1

7700

At4

g357

80 A

t4g3

8470

At3

g275

60 A

t5g4

0540

At5

g501

80 A

t5g0

1850

At3

g507

20 A

t3g5

0730

At5

g667

10 A

t2g2

4360

At4

g311

70 A

t1g6

2400

At3

g469

30 A

t5g5

8950

At3

g014

90 A

t5g5

0000

At3

g227

50 A

t4g1

4780

At3

g632

60 A

t3g0

7980

At3

g135

30 A

t4g0

8500

At4

g084

80 A

t4g0

8470

At4

g120

20 A

t1g5

3570

At1

g637

00 A

t5g6

6850

At3

g060

30 A

t1g0

9000

At1

g549

60

0

500

1000

1500

2000

2500

3000

At2

g310

10

At3

g586

40

At2

g426

40

At4

g230

50

At1

g678

90

At5

g494

70

At3

g066

20

At3

g066

30

At3

g066

40

At1

g087

20

At5

g118

50

At1

g181

60

At1

g736

60

At4

g244

80

At5

g037

30

At1

g162

70

At1

g795

70

At2

g350

50

At1

g047

00

At3

g247

15

At3

g469

20

At5

g576

10

At1

g140

00

At3

g598

30

At2

g438

50

At2

g318

00

At4

g189

50

At3

g587

60

At2

g177

00

At4

g357

80

At4

g384

70

At3

g275

60

At5

g405

40

At5

g501

80

At5

g018

50

At3

g507

20

At3

g507

30

At5

g667

10

At3

g469

30

At5

g589

50

At1

g624

00

At2

g243

60

At4

g311

70

At3

g014

90

At5

g500

00

At3

g227

50

At4

g147

80

At3

g632

60

B1 B2 B3 B4 C1 C2 C3 C4 C5 C6 C7

Supplementary Figure 1. Expression levels of Arabidopsis Raf-like kinase genes in guard cells. (a) Phylogenetic relationship in the Arabidopsis Raf-like kinases. The tree was similarly created as described in Fig. 4b. Amino acid sequences of the kinase domains from the members were used. The Raf-like kinases subfamily was classified as B1-B4 and C1-C7 groups, as reported previously1. (b) Gene expression levels of Raf-like kinases in guard cells. Data of the expression levels in guard cells were obtained from a public microarray da tabase eFP b rowse r ( h t t p : / / ba r . u to ron to . ca /e fp / cg i - b i n /e fpWeb .cg i?dataSource=Guard_Cell). White columns indicate the high expression genes in guard cells over 500 in the database.

B1

B4

B3 SALK_ 061250

SALK_ 005607 WT

At3g58640

TUB2

SALK_ 025685 WT

At4g24480

TUB2

At3g24715

TUB2

At2g35050

TUB2

SALK_ 205151 WT

SALK_ 107170 WT

C1 C2

At1g14000

TUB2

SALK_ 002267 WT

At4g35780

TUB2

SALK_ 139573 WT

C6

At2g24360

TUB2

SALK_ 105195 WT C3

At5g50180

TUB2

SALK_ 119787 WT

Groups

C7

At3g01490

At5g50000

SALK_ 005187 WT

SAIL_ 63F05

TUB2

Supplementary Figure 2. Confirmation of gene knockouts in the T-DNA insertion mutants in this study. RT-PCR was performed using total RNAs from the rosette leaves in wild-type (WT) and T-DNA insertion mutants. All genes were amplified by PCR using the specific primer sets (see Supplementary Table 2). TUB2 was amplified as an internal control.

0 1 2 3 4 5

Dark

Light

B1

B4

B3

0

1

2

3

4

Dark

Light

0 1 2 3 4 5

Dark

Light

0

1

2

3

4

Dark Light

C1

0

1

2

3

4

Dark Light

Sto

mat

al a

pertu

re

(µm

)

Sto

mat

al a

pertu

re

(µm

)

Sto

mat

al a

pertu

re

(µm

)

Sto

mat

al a

pertu

re

(µm

)

Sto

mat

al a

pertu

re

(µm

)

C2

Sto

mat

al a

pertu

re

(µm

)

0

1

2

3

4

Dark

Light

C6 C3

0 1 2 3 4 5

Dark

Light

Sto

mat

al a

pertu

re

(µm

)

0 1 2 3 4 5

Dark

Light

Sto

mat

al a

pertu

re

(µm

)

C7

Sto

mat

al a

pertu

re

(µm

)

0

1

2

3

Dark Light

WT

SALK_061250

SALK_005607 WT SALK_025685

WT SALK_205151 WT SALK_107170



SALK_005187

(At3g01490) SAIL_63F05

(At5g50000)

Groups

At4g24480 At3g58640

At2g35050 At3g24715

At5g50180

At1g14000 At4g35780

At2g24360

*

Supplementary Figure 3. Measurements of the blue light-dependent stomatal opening in the T-DNA insertion mutants. Values represent means±s.d. (n=3); measurement of 30 stomata in each experiment. * indicates values that statistically differ from WT blue light sample (Student’s t test; *p<0.01).

a

BHP

TUB2

bhp-2 WT

0

2

4

Col #3 #6

Dark

Light

#2

Sto

mat

al

aper

ture

(µm

)

WT

Anti-PKSO

Anti- H+-ATPase

#1

WT proGC1: PKSO

#1 #2

I

proGC1: PKSO

**

**

0

0.5

1

1.5

Col #3 #6

R B

Red Red+Blue

Sig

nal i

nten

sity

(R

elat

ive

valu

e)

WT #1 #2

WT #1 #2

proGC1: PKSO

proGC1: PKSO

Red

Red+Blue

b

c

G

bhp-2 (WiscDsLox345_348B17)

ATG TGA

0

1

2

3

Col gabi wisc

Dark Light

WT bhp-1 bhp-2

Sto

mat

al

aper

ture

(µm

)

* *

Supplementary Figure 4. Blue light-dependent stomatal opening in T-DNA insertion alleles of BHP. (a) Schematic representation of the BHP gene (At4g18950) and position of the T-DNA insertion in the bhp-2 mutant. Boxes and lines indicate exons and introns, respectively. (b) Knockout of the BHP gene in the bhp-2 mutant. (c) Blue light-dependent stomatal opening in leaves from WT, bhp-1, and bhp-2. The experiment was performed as described in Fig. 2d. Values represent means±s.d. (n=4); measurement of 30 stomata in each experiment. * indicates values that statistically differ from the corresponding WT (Student’s t test; *p<0.01).

b

0

0.5

1

1.5

2

2.5

Red

Blue

FC S

igna

l int

ensi

ty

(Rel

ativ

e va

lue)

WT SALK_002267

0

0.5

1

1.5

Sig

nal i

nten

sity

(R

elat

ive

valu

e)

WT SALK_002267

c

a

0

0.5

1

1.5

2

2.5

3

3.5

0 1 10

WT SALK_002267 S

tom

atal

ap

ertu

re (µ

m)

FC (µM) 0 1 10

Anti-H+-ATPase

Anti-pThr

* **

Supplementary Figure 5. Immunohistochemical detection of guard cell H+-ATPase and fusicoccin (FC) induced-stomatal opening in the At1g14000 knockout mutant. (a) Phosphorylation of the guard cell H+-ATPase in SALK_002267 in response to blue light and fusicoccin (FC). The immunohistochemical experiments using anti-pThr antibody were performed and are shown in Fig. 2h. Values represent means±s.d. (n=3); measurement of 30 stomata in each experiment. (b) Immunohistochemical detection of the amount of guard cell H+-ATPase in WT and SALK_002267 using anti-H+-ATPase antibody. The relative signal intensity was expressed as the ratio of the signal intensity from the SALK_002267 mutant to that from WT. Values indicate means±s.d. (n=3), and measurements of 30 stomata in each experiment. (c) Stomatal opening in response to FC in WT and SALK_002267 epidermis. Epidermal fragments from dark-adapted plants were incubated in reaction buffer 2 (see Methods) containing FC at the indicated concentrations for 3 h in the dark. Data represent means±s.d. (n=4). Thirty stomata were measured in each experiment. * and ** indicate values that statistically differ from the corresponding WT (Student’s t test; **p<0.01, *p<0.05).

-50

0

50

100

150

WT bhp-1 phot1 phot2

WT

bhp-1

phot1 phot2 WT

bhp-1

phot1 phot2

a

b

c

WT bhp-1

phot1 phot2

WT bhp-1

phot1 phot2

Blue light

Pho

totro

pic

cu

rvat

ure

(deg

ree)

Supplementary Figure 6. Phot-mediated blue light responses in bhp-1 mutant. (a) Phototropism in WT, bhp-1, and phot1-5 phot2-1 (phot1 phot2). Etiolated seedlings were irradiated with unilateral blue light (0.5 µmol m-2 s-1) for 14 h. Values represent means±s.d. (n=18-19). (b) Slit band assays for the chloroplast movement in WT, bhp-1, and phot1 phot2. Left and right panels indicate the chloroplast avoidance and accumulation responses, respectively. Detached leaves were irradiated with blue light at 100 µmol m-2 s-1 or 5 µmol m-2 s-1 for 30 min through a slit to induce the avoidance and accumulation responses, respectively. Arrowheads indicate the irradiated areas. (c) Leaf flattening. Rosette leaves were detached from 5-week-old plants, and photos were taken from the adaxial (left panel) and abaxial (right panel) sides.

D BL

Input GST- 14-3-3

GST- BHP

Anti-GST

Anti-phot1

Pulldown

GST-BHP

GST-14-3-3

D BL D BL

a

+� + +� +

Immunoblot (Anti-FLAG)�

GST-BHP�

FLAG-PP1 �Input �

GST

Pulldown�

- � + - � +

GST

Immunoblot (Anti-GST) �

GST-BHP�

+� - +� -

b

Input GST-

14-3-3 GST-BHP

Anti-GST

Pulldown

GST-BHP

GST-14-3-3

Anti- H+-ATPase

c

Supplementary Figure 7. In vitro pull-down assays for interaction of BHP with phot1, H+-ATPase, and PP1. (a,b) The recombinant GST-14-3-3 protein or GST-BHP was purified from E. coli cells using glutathione-Sepharose 4B beads, and the proteins were reacted with microsomal proteins from Arabidopsis etiolated seedlings. The boundary matrices were subjected to SDS-PAGE and then immunoblot using anti-GST, anti-phot1, and anti-H+-ATPase antibodies. (a) Pull-down of assay of phot1 and BHP. (b) Pull-down of assay of H+-ATPase and BHP. The GST-14-3-3 was used as a positive control for the binding to phot1 and the H+-ATPase. (c) In vitro pull-down assay for interaction of BHP with PP1. Both proteins were expressed in E. coli and used. Extract from E. coli cells expressing GST or GST-BHP was mixed with that expressing FLAG-PP1 and reacted with glutathione-Sepharose 4B beads. Proteins on the beads were subjected to SDS-PAGE and then immunoblotting using anti-GST and anti-FLAG antibodies.

BLUS1-YFPC AHA1-YFPC AHA2-YFPC phot1-YFPC phot2-YFPC YFPC

D

a

b

BHP -YFPN

AHA1 -YFPC

AHA2 -YFPC

phot1 -YFPC

phot2 -YFPC

AHA1-YFPN BHP-YFPN AHA2-YFPN BHP-YFPN

phot1-YFPN BHP-YFPN phot2-YFPN BHP-YFPN

AHA1 AHA2

phot1 phot2

Supplementary Figure 8. In vivo interactions of BHP with signaling components in blue light-dependent stomatal opening by BiFC assay. (a) Fluorescence images of the interactions between BHP and BLUS1, AHA1, AHA2, phot1, and phot2 in BiFC assays. Experiments were performed as shown in Fig. 5b. For negative control, the C-terminal half of YFP (YFPC) only was co-expressed with BHP-YFPN. Scale bar is 150 µm. (b) Comparison of BHP-H+-ATPase or -phototropin interactions with the positive interaction controls in BiFC assays. Because H+-ATPase and phototropins form multimer complexes2,3, co-expression of AHA1-YFPN/AHA1-YFPC, AHA2-YFPN/AHA2-YFPC, phot1-YFPN/phot1-YFPC, and phot2-YFPN/phot2-YFPC were used as positive controlsScale bar represents 150 µm.

Inhibitor Mammaliantarget

Putative targets inArabidopsis

Identity (%)

PKC

c-Raf

EGFRK

32

33

Sphingosine

GW5074

BML-265

Raf-like kinaseAt4g38470


32

38

41

39

38

38

37

32

S6 kinae-2At3g08720

Putative proteinkinase At1g48490





Tyrphostin 9 PDGFRK Raf-like kinaseAt4g31170

39


37


S6 kinase-1At3g08730

Tyrphostin 9, Sphingosine, GW5074 and BML-265 were screened from the Inhibitor library (Enzo; BML-2832) as inhibitors that suppress effectively the blue light-dependent H+-ATPase phosphorylation and stomatal opening. Detection of the guard cell H+-ATPase was performed by the immunohistochemical method (Fig. 1a). The targets of the inhibitors are known in the mammalian cells. Putative targets in Arabidopsis were assumed by BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the full-length amino acid sequences, and three proteins with high similarity are shown in the list.

Supplementary Table 1. Putative targets of the selected inhibitors in Arabidopsis.

Gene Experiments

F: GAGGGTACCGCTCCCATGAGCACAGCAACAAGGCCTTCCR: CTTTTGCTCCATCCCGTCAACGGCTTTAGAATCATTAGAAG

Primer sequence

F: GAGGGTACCGCTCCCATGTCAGGTCTCGAAGATATCAAGR: CTTTTGCTCCATCCCCACAGTGTAGTGATGTCCTGC

F: GAGGGTACCGCTCCCATGTCAGGTCTCGAAGATATCAAGR: GTATGGGTACATCCCCACAGTGTAGTGATGTCCTGC

F: GAGGGTACCGCTCCCATGTCGAGTCTCGAAGATATCAAGR: CTTTTGCTCCATCCCCACAGTGTAGTGACTGGGAGTTTC

F: GAGGGTACCGCTCCCATGTCGAGTCTCGAAGATATCAAGR: GTATGGGTACATCCCCACAGTGTAGTGACTGGGAGTTTC

F: GAGGGTACCGCTCCCATGGCGACGACGACGACGR: GTATGGGTACATCCCAATCTTTGTGGACATCATGAACTTG

F: GAGGGTACCGCTCCCATGGCTCGGAACAAGCTCGAGTTCR: GTATGGGTACATCCCACCCAAAACACTATCTTTATCAGC

F: GAGGGTACCGCTCCCATGGAACCAACAGAAAAACCATCGR: CTTTTGCTCCATCCCAAAAACATTTGTTTGCAGATCTTCTAGC

F: GAGGGTACCGCTCCCATGGAACCAACAGAAAAACCATCGR: GTATGGGTACATCCCAAAAACATTTGTTTGCAGATCTTCTAGC

F: GAGGGTACCGCTCCCATGGAGAGGCCAAGAGCCCCTR: CTTTTGCTCCATCCCGAAGAGGTCAATGTCCAAGTCCG

F: GAGGGTACCGCTCCCATGGAGAGGCCAAGAGCCCCTR: GTATGGGTACATCCCGAAGAGGTCAATGTCCAAGTCCG

F: AATCTTGCAATGGATTCTTTGACTGR: CAAGGTTAATAACATTGATTGAAGCAG

F: AGTGAATCTTAGATTGGGAAGATGCR: TTCTCGCTGTCGTCATTATCTCTG

F: AAATGAAGGAGAAGGCGGAGAGTGR: ATCCCAAAGCTATACACATCGCAC

F: ATGAAAGAAGGAAAGGATGGGTTTGR: TTAAGGACCACGTTTCCTTCGG

F: GAGGGTACCGCTCCCATGGAAGAGGATTATCAACAGCR: CTTTTGCTCCATCCCCAAATGTGAACCGGATGATG

F: ATGGATCAAGCAAAAGGTTATGAACATGR: TTACTTGTGGATTTGGTGGTTGACAG

F: ATGTCAGATAGATGGGCTCGACR: AGGAAATTGGCAATGGGAGAG

F: CAATGGTGATCTGATGGTAGR: TCTGAATCGGTTTTGAGATG

F: ATGGAAGAGGATTATCAACAGCR: TCACAAATGTGAACCGGATG

F: ATGGCGATCAAAGAGGAGACR: TAGGCCTGAAAAGTAACCAC

for RT-PCR

for RT-PCRAt3g58640 B1

F: ATGGCGATCAAAGAGGAGACR:TAGGCCTGAAAAGTAACCAC

At4g24480 B3

F: TCAGAGTGAGATTTTAACGAAATGCR:CTATAGTATGGGAGCTGATTTAGTTGG

At2g35050 B4

At3g24715 B4

VIK At1g14000 C1

BHP At4g18950C1

At4g35780 C2

At5g50180C3

At2g24360 C6

At3g01490 C7

At5g50000C7

BHPBHP-YFPN

BLUS1BLUS1-YFPC

PHOT1 PHOT1-YFPN

PHOT1-YFPC

PHOT2 PHOT2-YFPN

PHOT2-YFPC

AHA1 AHA1-YFPN

AHA1-YFPC

AHA2AHA2-YFPN

AHA2-YFPC

PP1 TOPP4-YFPC

INHIBITOR3 INHIBITOR3-YFPN for BiFC

for BiFC

for BiFC

for BiFC

for BiFC

for BiFC

for BiFC

for BiFC

for BiFC

for BiFC

for BiFC

for BiFC

for RT-PCR

for RT-PCR

for RT-PCR

for RT-PCR

for RT-PCR

for RT-PCR

for RT-PCR

for RT-PCR

for RT-PCR

Supplementary Table 2. Primers used in RT-PCR and BiFC assay.

1.  Ichimura, K. et al. Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7, 301-308 (2002).

2.  Kanczewska, J. et al. Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer. Proc Natl Acad Sci USA 102, 11675-11680 (2005).

3.  Katsura, H., Zikihara, K., Okajima, K., Yoshihara, S. & Tokutomi, S. Oligomeric structure of LOV domains in Arabidopsis phototropin. FEBS Lett 583, 526-530 (2009).

Supplementary References

supplementary information - nature c6 c4 c5 c7 a groups at2g31010 at3g58640 ... plants were...

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