EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

Vol.

92, No.

January

1, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

15, 1980

EGTA,

Pages

A CALCIUM OF

CHELATOR, INHIBITS SPINACH CHLDROPLASTS

Rita

Sarr,

Karen

S.

ELECTRON TRANSPORT AT TWO DIFFERENT

Troxel

and

of Biological Purdue University West Lafayette IN

Frederick

Department

Received

November

IN PHOTOSYSTEM SITES L.

206-212

II

Crane

Sciences 47907

29,1979

Summary A fifteen Ca2+ chelator, through both indophenol, are inhibited

minute incubation of spinach chloroplasts with the divalent EGTA, in concentrations 50-250 pM, inhibits electron transport photosystems. All photosystem II partial reactions, including ferricyanide and the DCMU-insensitive silicomolybdate reduction from 70-1004. The photosystem II donor reaction, diphenyl . . . indicating that the inhibition ~4~~"~~~~s'a:~~~p~~~'~~2~'s~~~' ~~~'~~~~d~he first Ca2+ effect noted (site II) is not on the water ixidation enzyme, as is commonly assumed, but The other photosystem II between the Mn2+ site and plastoquinone A pool. site I), occurs in the region between plastoquinone A effect of EGTA (Cazt and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD +methyl viologen is given by incubation with 200 UM EGTA for 15 min. Ca2+ site II activity can be restored Ca2+ site I responds to Ca2+ and plastocyanin added with 20 mM CaC12. jointly. More than 90% activity in the ascorbate + TMPD + methylviologen Various ways in which Ca2+ ions could affect reaction can be restored. Since EGTA is more likely chloroplast structure and function are discussed. to penetrate chloroplast membranes than EDTA, which is known to remove CFl, the coupling factor, from chloroplast membranes, and since Mg2+ ions it is concluded that Ca2+ may function are ineffective in restoring activity, in the electron transport chain of chloroplasts in a hitherto unsuspected manner. Introduction Fredricks Nat

or

Kt

Piccioni increased Phormidium

and salts

and

Jagendorf

inhibited Mauzerall

Hill

Hill

found

rates Since

found

that

reaction

(2,3,4)

reaction

luridum.

(1)

rates that

twentyfold they

had

Ca2+

the in

used

high

in

salts

stimulated,

Anacystis

addition cell-free concentrations

but

nidulans. of

Ca2+

ions

extracts

from of

EGTA

Abbreviations: DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DPC - diphenylcarbazide; DCIP - 2,&dichloroindophenol; DBMIB - 2,5-dibromo-3-methylEGTA - ethyleneglycol bis ( a - aminoethyl 6-isopropyl-p-benzoquinone; ether)-N,Nl-tetraacetic acid; FeCN - potassium ferricyanide; MV - methylII; SM - silicomolybdic viologen; PS I - phot system I; PS II - photosystem diamine. acid; TMPD - N,N,N 1P,N - tetramethyl-p-phenylene 0006-291X/80/010206-07$01.00/0 Cop.vrighi 0 1980 hv Academic Press, Inc. All rights of reproducrion in any form reserved.

206

Vol.

92, No.

(1

BIOCHEMICAL

1, 1980

mM)

in

the

occurred

to

original us

breaking

that,

maybe,

of

with

concentrations

various

inhibited

50%

partial by

Piccioni

II were

Ca*+

found

may

be

Materials

be

required

and

study

not

French an

that

by

means

further

along

the

electron

inhibited

by

their

of

activity

EGTA --in

was

chloroplast

site

postulated requiring

transport

treatment,

depleting

transport

as

the

it

chloroplasts

various

was

of

spinach

itself,

(5)

the

way

electron

oxidation Brand

COMMUNICATIONS

treatment,

efficient

found

or

for

press

washing

and

water

RESEARCH

tried

studies

(2,3,4)

sites to

by

devised

EGTA

that

Mauzerall

2 other

this

BIOPHYSICAL

cells had

Further

showed

and

they

of

more.

reactions

Ca2+ , but PS

Cazt.

or

of

We in

chloroplasts

AND

chain implying

in that

vitro.

Methods.

Sucrose (0.4 M)NaCl(O.05 M) chloroplasts (SN chloroplasts) were prepared by grinding 20 g deveined spinach leaves, obtained from the local market, for 15 sec. in 80 ml of SN grinding solution in a Waring blendor. The slurry was filtered through 8 layers of cheesecloth, then through a layer of Miracloth and centrifuged in 2 centrifuge tubes at 200 xg for 2 min. The supernatant was transferred to clean centrifuge tubes and recentrifuged at 600 xg for 10 min. to obtain a chloroplast pellet, which was gently suspended with a brush in 5 ml SN. Chlorophyll was determined according to Arnon (6). 02 evolution or uptake on chloroplast partial reactions was measured with a Clark-type oxygen electrode, as reported previously (7). Reaction rates were recorded with a Sargent-Welch SRG recorder. Electron donation to PS II with the diphenyl carbazide/DCIP system was measured spectrophotometrically at 600 nm, using 21.2 as the extinction coefficient for DCIP (8). The EGTA treatment of chloroplasts consisted of incubating chloroplasts (2.5 mg chlorophyll/40 ml EGTA solution) with loo-250 PM EGTA solutions for 15 min. at 4OC. Chloroplasts were pelleted by centrifugation at 600 xg for 10 min. Control chloroplasts were incubated with distilled water in place of EGTA solutions, but were treated alike otherwise. For rewashing treated and control chloroplasts,SN was used where indicated. EGTA was purchased from the S igma Chemical Co. and recrystallized from a 50% solution of isopropanol. Results

and

Discussion.

Studies

of

DCMU-insensit

showed,

that

moderate

reaction

at

pH 6 and

hibited

it

at

removal

of

Ca*+

reactions.

As

similar from Fig.

ive

silicomolybdate

concentrations at

pH 8 (Fig.

of l),

concentrations. chloroplasts 2 shows,

a 15

reduction

CaC12

(=

while This

20 mM)

Mg *+, lead

would

affect

min.

incubation

Sr2+ us

various of

to

by stimulated and

I

Photosystem

Ba2+

this ions

in-

investigate,

how

chloroplast

partial

chloroplasts

with

Vol.

92, No.

BIOCHEMICAL

1, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

+25

0 MgCI,,pH6 l A A 0 n

Mgcl,. PI-l8 Cdl,. pH6 cm,, pH8 sq,.pH6 SrCI..pH8 v BoCI,,pH6 t BaCl*.pH8

0 MV(+mde), pH7 0 DCIP,pH7 A osc. +TMPD -WvlV o osc.+DAD+MV v 0s~. t DCIP-wMV

;-25 a0 % 8

-75

I 20

40

I 60

60

1 100

IONS ADDED (mM)

Fig.

1 150

I 200

, 250

EGTA (vM1

The

2. The Effect of Various Concentrations of EGTA on Electron Transport in Spinach Chloroplasts after a Fifteen Minute Treatment. The control rate for the reaction H20 -t MV (t azide) was 838 pequivalents/mg chl . hr; for the H20 + DCIP reaction - 481; for the ascorbate + TMPD for the ascorbate t DCIP + MV - 981. The reaction + MV - 1124; mixture for the H20 -+ MV ( t azide) reaction contained chloroplasts (50 ug chlorophyll), 25 mM Tris-Mes, pH 7, 0.5 mM MV, 0.5 mM Na azide and 5 mM NH4Cl; for the H20 -+ DCIP reaction chloroplasts, buffer and NH4Cl as above plus 0.5 mM DCIP; for the reaction ascorbate + TMPD -f MV, chloroplasts, 25 mM Tris-Mes, pH 8, 5 pM DCMU, 0.5 mM Na azide, 0.5 mM MV, 50 pM TMPD, and 1 mM Na ascorbate; for the ascorbate + DCIP -+ MV reaction everything as for the TMPD reaction except 0.5 mM DCIP t indicates stimutation, - inhibition of rate in in place of TMPD. relation to control.

200

pM

The

only

before

02

1 100

Other Ions on the DCMU-Insensitive 1. The Effect of Ca*' and Various Silicomolybdate Reduction by Photosystem II of Spinach Chloroplasts. control rate at pH 8 was 152 uequiv./mg chl . hr, at pH 6 - 188. The reaction mixture contained chloroplasts (50 pg chlorophyll), 25 mM Tris-Mes buffer, pH 6 or 8, 5 pM DCMU, silicomolybdic acid, 85 PM at pH 6, 250 pM at pH 8, and various ions in concentrations indicated; + indicates stimulation, - inhibition of rate in relation to control.

Fig.

plus

1 50

EGTA

inhibited

PS

I donor

DCIP

electron reaction

+ MV reaction,

P700,

the

reaction

transport

in

unaffected indicating center

by that

chlorophyll

208

the

both the

photosystems treatment

EGTA complex

from was

inhibition in

the sites

PS I.

50-90%. ascorbate occur

Vol.

92, No.

BIOCHEMICAL

1, 1980

Localization chain

was

partial

of

attempted reactions.

reduction Ca2+

in ions '

shown

both

at

the

knowing, evolved/mg

of

45 mM CaC12.

II +

chloroplasts. of

(data

not

was

present.

for

Ca2+

restore

Ca2+

is

DCIP,

offer

proof,

site,

where

diphenyl

pool,

but

not

there

is

due data

and

to

EGTA

on

Mauzerall

presented

chloroplasts

in is

strongly

in

indicating

II

in

EGTA-treated

rate

of

pH 6,

but

with

data

the

the

showed

Ca*+

effect

at

oxidation effect

of or to

study. suggested

be

pH 8,

when

3D,

where

Ca 2+

ions

Brand

past

Specificity by

209

on (5)

the

for restoration

is

water

water

joint

by >50%

the

restoration 3C):

Ca*+

dibromothymoquinone restoration

between

of

but

It as

the

may

be,

assumed

Ca 2+

site, the

2+

plastoquinone

assumed.

algae,

studies

Mn

the

oxidation,

in

-f

the

between

oxidation Ca2+

the

diphenylcarbazide

commonly

in

of

EGTA-treated

(Fig.

and is

presence

of

+ MV pathway

II

site

pequiv

component

in

reaction,

as

by

another

in

Fig.

10

reduction

Only

H20

electrons,

itself,

was in

the

affected

at

in

donor

donates

CaC12

chloroplasts

II

PS

but

differences

not

without

chloroplasts

that

the

CaC12

be misleading

plastocyanin.

restored

by

75 pequivalents

was

exogenous

control

stimulation

without

3B),

be

of

in

EGTA-treated

well,

rates

appear this

II

PS

(2,3,4)

treatment

PS

II

PS

addition

reaction 750%

to

as

I).

carbazide

a separate

by

may

on

water

increased

component

shown that

It

(Fig.

site

the

3A may

CaC12

CaCl2

various

silicomolybdate

reduction

pathway

along

The Fig.

transport

data,

same

of

and

transport

of

to

50%.

by

the

These by

Piccioni

respond

transport

activity

that

addition

reduction

electron could

indophenol

electron

activity

the

in

. hr.

plastocyanin shown

while

than

COMMUNICATIONS

DCMU-insensitive

silicomolybdate

missing

Ferricyanide

ions

of

not

The

pH 8,

RESEARCH

the

responded

more

I electron

PS

the

chloroplasts

the

did

addition

at

in

the

3A shows,

changed

of to

restore

chloroplasts

rate

reduction

methylviologen PS

Fig.

BIOPHYSICAL

lesions

to

chlorophyll

responded

the

As

not

the

02

also

trying

EGTA-treated

that

The

by

pH 6 and

was

in

EGTA-created

EGTA-treated

chloroplasts

of

the

AND

by

sites

as

shown

by

Ca 2+

site

II

EGTA-treated of

the

Vol.

92, No.

1, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

+ 100

0 SM ItDCMUI,

pH 6, CONTROL

0 SM PDCMU), pH 6, EGTA A SM WDCMIJ), pH 8. CONTROL A SM PDCMU),

= e E

pH 6, EGTA

g

+75

a MV kwide), CONTROL A MV i+azideI, EGTA

-2500051 60

CaC12

-25

ImM)

0

15

60

. 0 CONTROL 0 SHOCKED . EGTA

200

D

0 F&N, CONTROL, pH 6 . FeCN, EGTA, PH 6 A FeCN ~tDBMIB), CONTROL, A FeCN

ItDEMIE),

BEFORE EGTA CONTROC

pH 8

EGTA, pH 6

c

0

60

15

Fig.

3. Restoration Photosystem in the reaction

II

75

of Electron Transport in Various and I by Caz+ Ions in EGTA-Treated at pH 6 and Hz0 + SM (+OCMU),

210

8;

Partial Reactions Chloroplasts. AB- in the reactions

of

75

Vol.

92, No.

BIOCHEMICAL

1, 1980

Table

I.

The

Effect

Diphenylcarbazide

of

AND

and

Ca*+

+ Indophenol,

BIOPHYSICAL

Other

in

Ions

on the

Spinach

CONC. hM)

Transport

CONTROL DCIP REDUCED

None KC1

7.5

MgCl*

15

CaC12

II

Reaction,

Rate') EGTA-TREATED DCIP REDUCED

y*) o

100 15

Photosystem

COMMUNICATIONS

Chloroplasts

Electron ION ADDED

RESEARCH

100

%

50

-50

200

t100

60

-40

200

+lOO

55

-45

175

+ 75

105

+5

f 75

25

-75

MnC12

7.5

175

cuc12

7.5

75

- 25

40

-60

FeC12

7.5

85

- 15

45

-55

ZnC12

1.5

110

f 10

48

-52

1) pmoles 2)+

DCIP

reduced/mg

indicates

stimulation

Reaction mixture (50 ug chlorophyll), and 0.5 mM DCIP.

with

other

DCIP

reduction

chl

ions in

, - inhibition

for

(Table

of

the reaction, 25 mM Tris-Mes,

I).

the

. hr.

It

can

be

rate

in

relation

to

control.

DPC + DCIP contained chloroplasts pH 7.5, 5 mM NH4C1, 0.5 mM DPC

seen

that

-f

DCIP

diphenylcarbazide

only

Ca*+

pathway

could

in

fully

restore

EGTA-treated

chloro-

plasts. Data cyanin

for

the

region,

Ca2+

on

that

full

the

is

Ca2+

site

presented

ascorbate

I,

which

in

detail

+ TMPD

restoration

of

has

been

elsewhere.

+ MV reaction

activity

narrowed

required

down

to

Restoration

in

plasto-

studies

EGTA-treated

the

the

with

chloroplasts

addition

of

both

Ca

showed 2+

and

plastocyanin. The chloroplasts

exact

role is

in

unknown

which at

Ca

2t

present.

. influences Previous

electron studies

transport by

Gross

in and

spinach associates

Fig. 3 (continued) Hz0 * DCIP and H20 + MV (+ azide); C- in the reaction H20 + FeCN, pH 6 and H20 + FeCN (+DBMIB), pH 8. D- in the donor reaction DPC + DCIP. Various concentration of Ca*+ added as indicated. The control rate in A of H20 + SM (+DCMU), pH 6, was 248, in EGTA-treated chloroplasts 23; control at pH 8 - 164, EGTA - 10; in B the control rate for the reaction H20 h DCIP was 817, for EGTA - 158; the control rate of H20 + MV (+ azide) was 1139, in EGTA - 158; in C the control rate for H20 -f FeCN, pH 6 was 344, in EGTA, pH 6 it was 62; control H20 + FeCN (+DBMIB), pH 8 - 152, EGTA 0; in 0 the control rate of DPC + DCIP was 130 in shocked control chloroplasts, in EGTA chloroplasts - 43. All rates are expressed as uequiv./mg chl . hr. + indicates stimulation, - inhibition of rate in relation to control.

211

Vol.

92, No.

1, 1980

(9,10,11) in

the

have

emphasized

distribution

of

"spillover". untreated

in

"spillover". restoration

the

at

2 sites

II

reaction

PS

present

in

was

the

site

in

the

plastocyanin

Ca2+

ions '

at

in

which

the make

way, it

this

in

is

which

Ca

2+

2+ II

Bose

and of

has

totally

unknown.

is

functions

in

a more

may

be

Arntzen

the

2+

to

(12)

found

may

the

be

electron

a role

along

with

in

the Ca2+

before. that

distinguish transport

Ca2+

electron

organization

that The

chloroplast to

the

sites

plays

role

related

described

termed

binding

Ca

direct

cations.

It

underway

that

2+

chloroplasts,

been

to

Ca

data

divalent

not

attached are

their

cation

photosystems

2 specific

plays

site

absence

studies

2+

Ca

COMMUNICATIONS

divalent

two

EGTA-treated

region,

plastocyanin Further

in

Ca since

inactive

site

function.

alternatives

II.

the

found from

with that

center,

center

(9)

RESEARCH

a non-specific

between

deduced

study

PS

as

Hess

implies

reaction

of

and They

studies,

BIOPHYSICAL

energy

Gross

The

AND

participation

excitation

chloroplasts.

transport of

its

However,

in

the

BIOCHEMICAL

I,

site The

role

organizes

membranes

to

between

various

chain

of

chloroplasts. Acknowledgements.

wish for

This study was supported to thank Dr. E. Ullrich his gift of plastocyanin.

by from

N.S.F. the

Grant Chemistry

PCM-7820458. Department,

The authors Purdue University,

References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Fredricks A.T. (1964) Arch. Biochem. Biophys. 104, 39-49. , W.W. and Jagendorf, Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605-609. Piccioni, R.G. and Mauzerall, D.C. (1978) Biochim. Biophys. Acta 504, 384-397. Piccioni, R.G. and Mauzerall, D.C. (1978) Biochim. Biophys. Acta 504, 398-405. Brand, J. (1979) FEBS Lett. 103, 114-117. Arnon, D.I. (1949) Plant Physiol. 24, 1-15. Sireci, J.E., Plotner, A., Barr, R. and Crane, F.L. (1978) Biochem. Biophys. Res. Communs. 85, 976-982. Armstrong, J. McD. (1964) Biochim. Biophys. Acta 86, 194-197. Gross, E.L. and Hess, S.C. (1974) Biochim. Biophys. Acta 339, 334-346. Gross, E.L., Zimmermann, R.J. and Hormats, G.F. (1976) Biochim. Biophys. Acta 440, 59-67. E.L. and Arntzen, C.J. (1976) Arch. Davis, D.J., Armond, P.H., Gross, Biochem. Biophys. 175, 64-70. Bose, S. and Arntzen, C.J. (1978) Arch. Biochem. Biophys. 185, 567-575.

212