Formation of two chlorophyll-protein complexes during greening of etiolated bean leaves

BIOCHEMICAL

Vol. 45, No. 3, 1971

FORMATION DURING

J.H.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF TWO CHLOROPHYLL-PROTEIN

COMPLEXES

GREENING

LEAVES

OF ETIOLATED

Argyroudi-Akoyunoglou,

Department

of

Z.

Biology,

Nuclear

August

and

Research

Athens, Received

Feleki

BEAN

G.

Akoyunoglou,

Center

"Democritus",

Greece.

9, 1971

Summary. Etiolated bean leaves exposed to a series of light-dark cycles are devoid of Chl bl and contain agranal chloroplasts. lamellae of these chloropiasts are deficient in the chlorophyllprotein complex II, propably associated with PS II --in vivo. During

our

apparatus

we

greening (2

found

etiolated

min

white

that

very

ed.

The

Chl

studies

light high

by

tissue

of

disruption, bean

harvested

at the

dishes,

the

the

(1).

of

Chl

flashed I

(PS

4 to

I)

-a to

were

were Seven

grams

Chl may

form

Chl

-a between

on

moist

to

the

of

leaves

the

studying

the of

filter

chloroplast by

variety)

removal paper

intermittent

or withdrawn

the

sofubil-

kidney

1 Abbreviations: Chl: chlorophyll; SDS: sodium dodecyl 6-ALA: 6-aminolevulinic acid; DCIP: dichlorophenolindophenol. PS I: photosystem I; PS II: photosystem II. 606

only

obtained

the

were

so

establish-

by

(red

After

cycles (1)

-b are

examined

vulgaris

placed

exposed

period)

particles

age.

in

light-dark

complexes

Phaseolus of

of

was

formed

6 days

photosynthetic synthesized

dark

leaves

subchloroplast

plants

leaves and

98 min

300)

the

a system

chlorophyll-protein

between

Etiolated

cotyledon,

to

of

selectively

under

selectively

major

be

with

(50

the

SDS two

development

2 can

photosystem

digitonin

illumination

bean

that

or

petri

Chl

ratios

lamellae,

were

that

possibility

distribution

the

alternating

2 containing

ized

on

The

of in

one covered

continuous immediate-

sulfate;

Vol. 45, No. 3, 1971

ly

after

for

the

last

30 set

sucrose 0.01

in

of x g for

ml

water

of

in

chloroplasts

the

pellet in

0.1

x g for solubilized

Chl

in

min,

the

After

treated

and

Omni-mixer

at

at

pH The

the

7.0,

or

suspended

all

The was

the

for

between

lamellar

in

20

1 min.

x g fnr

30 min

homogenizer

with

1 ml

0.5%

SDS in

with

1 ml

was

hydroxylapatite molar 50

and

at of column

ratio

material

and

centrifuged complexes

by

electrophoresis.

through at

speed

suspension

separated

M

and

centrifuged

chlorophyll-protein

were

(0.5

filtration

full

36,000

speed

phosphate

was

a Potter-Elvejem

extracts

treatment

was

full

buffer

Following

obtained

and

at

sucrose

pellet

10 min

chloroplast

ml

the

pH 8.0.

gel

ground

homogenate

buffer

RESEARCH COMMUNICATIONS

M potassium

4'C.

centrifuged in

25

0.1

at

were

the

buffer

or

this

with

a Sorvall

lamellae

chromatography

and with

room

M phosphate

M Tris-HCl

36,000

pH 7.4

were was

light

cloth,

20

AND BIOPHYSICAL

blendor

a cold

using

The

1% SDS

at

cheece

6,000

of

a Waring

K EDTA),

0.1

flash

buffered

4 layers

the

BIOCHEMICAL

of

SDS/

100. entered

the

FLASHED

GREEN

ORIGIN

green

b YCIIOW green green yellow I=

ICOMPLEX I I COMPLEX II PIGMENTS

t Figure 1. plast lamellar bean leaves cycles.

Gel

electrophoresis chlorophyll-protein exposed to 72 hours

of

the solubilized complexes I continuous light 607

and

by SDS chloroII of etiolated or 46 light-dark

Vol. 45, No. 3, 1971

gels

BIOCHEMICAL

on electrophoresis.

tracts

of

Thornber sent.

green

--et

al

ed

bean

leaves

Rf

equal

to

(2).

that

only

one

complex

I

of

the

continuous

of

green

chlorophyll-protein

droxylapatite

by

to

similar

to

green

same

the

showed plant

was

Siegelman

(3).

--et

with

al

(4)

The

2 N KOH at

with

The ex-

two

studied

chloof by

phosphate

was

transformed

room

an

formation

Calcium and

flash-

their

the

further

pre-

I).

after

lamellae.

by

of

(Figure

clearly

ex-

were

extracts

plants,

the

obtained

band

tissue

bean

chromatography

titration

that

chlorophyll-protein of

of

complexes of

complexes

column according

very

illumination,

bands

hydroxylapatite prepared

was

pattern

electrophoresis

pattern

to

two

of

electrophoretic

chlorophyll-protein

that,

showed

rophyll-protein the

leaves Two

to

electrophoretic posure

The

bean

Contrary

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to

temperature

(b)

hyto

a

.

(a) 4

LD.C+

24 h C.L

1.1 )LD.C+24hC.L la -

06-’

(15i w-i a3as!0.1 -

Fraction

number

(lmllfmction)

Figure 2. Hydroxylapatite ed by SDS chloroplast etiolated bean leaves uous light (CL).

Fraction

column lamellar exposed

number

(lmllfractkm)

chromatography of the solubilizchlorophyll-protein complexes of to light-dark cycles (LDC) or contin

608

Vol. 45, No. 3, 1971

pH of about use with elution

BIOCHEMICAL

9.0.

The columns

5 ml 1% SDS in 0.1 was performed

ed by 0.35

with

M phosphate

eluted

sults

of such experiments

graphic

were recorded

pattern

of the

two chlorophyll-protein and 0.35

M (complex

contrary

the

flashed

phyll-protein ed after

peak. transfer

(1 x 5.5

0.1

pH 7.0.

were washed prior

buffer

M phosphate

pH 7.0.

buffer

The spectra

upon collection.

solubilized

phosphate

lamellae

bean leaves

eluted buffer

plants

2.

with

follow-

of the

frac-

the

to continuous

re-

The chromatoplants

shows

0.1 M (complex

respectively.

show only

Wavelength

Stepwise

pH 7.0

of green

The chlorophyll-protein of the

to

Representative

are shown in Figure

complexes, I)

cm)

M phosphate

buffer

tions

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

On

complex complex

IX)

the

I chloroII

is

form-

illumination.

(nm)

Figure 3. Absorption spectra of the chlorophyll-protein complexes eluted from hydroxylapatite columns with 0.35M (A,B,E) or O.lM (C,D) phosphate buffer pH 7.0. A: : tomato plants grown in a greenhouse. bean leaves after 24 hrs continuous light. B,C,D: etiolated E : etiolated bean leaves after 40 light-dark cycles. 609

BIOCHEMICAL

Vol. 45, No. 3, 1971

The

absorption

plexes

are

shown

eluted

with

0.1

tion

bands

the

complex

buffer,

of

at

670

the

of

Figure

3.

M phosphate 670

I

two

The

complex

652

nm in

the

Based can

on

Chl total

calculate

chlorophyll-protein the

II

greening

Chl

has

Light

26 LDC 40 LDC CL (24 hrs) LDC+CL (4 hrs) LDCtCL (24 hrs)

for

the bean

complexes

to

activities

PS I

of

between

attribute

Complexes I 28 30 33 36 28

the

lamellae

of by

by

bean 0.5%

can

studied

the

digitonin

not

chlorophyll-protein

and

to the

610

biochemical

measured

due

distribution

of

enriched

fragmentation exposed

exposure to a illumination the total based on absorp-

the be

particles

digitonin

leaves

two

Unfortunately,

complexes

subchloroplast

obtained

the

PS II.

We thus

system

disrupted

(2) and

these

inactivation.

solubilized by plants exposed

0 0 18 8 16

al

amount

total)

old etiolated bean leaves were used after of light-dark cycles (LDC) or continuous The values shown represent percentages of (CL). solubilized by SDS chloroplast lamellae, and are tion at 669 nm.

--et

the lamellae

5 days number

Thornber

on

I).

Chlorophyll-Protein (% Of II

Treatment

while

absorption

chloroplast

(Table

absorp-

applied

values

of

has

M phosphate

a strong units

approximate

and

0.35

Distribution of chlorophyll between chlorophyll-protein complexes in illumination conditions?

Table I. SDS lamellar to different

b

nm region,

with

absorption

process

chlorophyll-protein,

650-700

-a and

complexes

com-

chlorophyll-protein

eluted

predominantly

RESEARCH COMMUNICATIONS

contains

chlorophyll-protein

one

during

the

buffer,

nm and

nm.

columns,

formed

40 40

in

contains

band the

at

spectra

AND BlOPHYSlCAL

different 10,000

in (5). light x g (10

to

the

SDS

chlorophyll each

The

photochloroplast

conditions K)

and

were 144,000

x g

Vol. 45, No. 3, 1971

(144

K)

(5))

were

fractions

(active

separated.

fractions

is

phyll

of

and

14% in

given

leaves

144

of

22%

amount

in

the

lighter

are

46%

144

the

10

more

Sample

by

hrs

the

content digitonin (C.L.)

C.L.

of or

5dt29

a contamination and

This

by

Boardman and

when

the

are

10 days

is

expected,

older

plants

has

plants

(5)

illumination

was

10 K fraction, older

small

(6),

also

been

(7).

the 10 disruption intermittent

K and of

L.D.C.

lOdt24

144 K subchloroplast etiolated bean leaves (L.D.C.) illumination.

hrs

Anderson Boardman

C-L.

and (5)

Chl.

Chla -

Chl.

Chla -

Chl.

Chla -

Chl.

Chlb

%

Chlb -

"0

Chlb -

"0

Chlb -

"0

100 28.5 14.3

8.5 5.1 19.3

100 2 22

2.24

2.83

100

2. 2 1.8 2.9

in is that

in

the

the

chlorophyll

in

the

10 K fraction

continuous

K respectively.

by

2% of

the

that

chloroplast

amount

chloro-

Chla

Chloroplast Extract 10 K 144 K

propable

the

the

K fraction,

to

these

10 K fraction

Indeed,

exposed

in. higher

abundant

5d+24

chlorophyll

144

144

of

only

Anderson

leaves.

of

represented

Chlorophyll Table II. particles obtained exposed to continuous

Since

are

10.3%

present

PS II,

the

the

in

is

of

spinach

leaves

K and

b is

be

possible

for

hours,

to

is

11%

47% and

found

It

28%

in

present

the

mature

shown

is

values

of

is

respectively in

contrary,

The

10 K and

chlorophyll

is

On the

components.

24

the

it

reactions

of

24 hours

leaves

RESEARCH COMMUNICATIONS

PS I

10 K fraction

for

and

As

for

of

bean

Chl

II.

K fraction.

etiolated

since

Table

bean

old

in

distribution

and

the

representative

found

PS II

K fraction.

chlorophyll

for

AND BIOPHYSICAL

The

flashed

the

of

in

in

illuminated

the

chlorophyll and

BIOCHEMICAL

the

flashed

present the

missing

bean in

the

plants 10 K (PS

chlorophyll-protein

611

100

1.7 4.25

very II)

47 10.3

little,

2.27 5.34

if

fraction, from

any, it

these

46.2 11.7

of

seems plants

Vol. 45, No. 3,1971

is

the

one associated

selectively is

BIOCHEMICAL

synthesized

found

mainly

protein

in

isolated

solubilized

Electron

early

to

of

greening

continuous

the

complex,

formed

the

heavy

fraction, from

ever, hibits

seem

high

(ac-31) essentially

complex

the

of the

the

should

of grana

no stacking

lamellae for

PS II,

of

612

protein

account.

to

originate

stacks,while particles,

mutant

and when view-

grana

15). since

lacking

and thylakoids

Grana stacks, this

mutant

howex-

of Chlamydomonas reinhardii

to have normal lamellae

complex

stacks.

in PS II,

(14,

stroma

photosystems,

to PS II

of a barley

showed fewer

of

respect

into

seems

predominantly

A mutant

been found

be taken

separated

region

essential

activity.

In this

end membranes of grana

microscope

to be not

Chl b-rich

to PS I particles,

chloroplasts

single

the

protein

illumination. (13)

formation

that

Chl a-rich

amount of chlorophyll

more

has also

stacks

transfer

on the

suggest

French-press

the partition

PS II

showed

of the

of grana

after

results

the

corresponding

electron

granum but

only

may represent

and the

Chl &. had a lesser

per

the

continuous

of the

In addition,

the

of the SDS

chloroplasts

No indication

represent

predominantly lamellae

ed under

chlorophyll-

characteristic

complexes,

of Sane -et al.

stroma

originates

12).

with

structures

fraction

corresponding

however,

chlorophyll-protein

bean leaves

were formed

coupled

under

work

The light

and the

The

illumination.

may

grana

the recent

from

(11,

The latter

structures

while

the

thylakoids"

two chlorophyll-protein

lamellae

bean leaves,

fraction,

of flashed

"primary

These findings the

lamellae.

chromatography

may represent

the

was obtained; plants

flashed

of the

PS I --in vivo.

of

stage

RESEARCH COMMUNICATIONS

complex

hydroxylapatite

micrographs

presence

PS II

144 K (PS I)

after

with

the

Chl -a in the

the

lamellae

associated

the

with

AND BIOPHYSICAL

(16).

PS II On the

activity, contrary

but the

Vol. 45, No. 3, 1971

results

of

PS II

activity

Woo --et

al.

of

bundle

the

PS II

Homann

and

(18)

have sheath

and

a reduced

signal.

Bishop

in

chloroplasts,

these

sence

of in

between

the

reported (but

not

may

suggest

of

PS II

cedures

these two

we

a series

system

the

by by

an

the

of

be

Hence,

they

is

the

not

necessary

mutants agranal

of have

Hill

--et

brief

red

light

the

chlorophyll-protein by

in

that

leaves

undetected

-b

found

itself,

which

Chl

fluorescence

bean

of

-a to

only

De Greef

in

PS II

concluded PS II

(17).

deficient Chl

reduced

for

chloroplasts

are

altered

however,

fraction remains

are

absence

Recently, from

grana

plants

NADP + could

photosystems.

a small

that

(19),

chloroplasts

RESEARCH COMMUNICATIONS

tabacum

C4-cycle

and

ascorbate.

by

our

of

but

Chl

in

cells

coworkers

evolution

that

recently

-b-559,

that

Nicotiana

found

Cyt

oxygen b)

of

characterized

and

AND BIOPHYSICAL

indicate

are

and

DCIP

missing

Schmid

in a number

units,

ratio,

BIOCHEMICAL

activity the

pre-

what but

al.

accumulated

is

the

link

(20) Chl

illuminations.

the

analytical

a This

complex pro-

used.

Acknawledgements. research grant (No helpful discussions.

This 468).

research We wish

was to

supported thank Dr.

in G.

part by a NATO Papageorgiou for

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Argyroudi-Akoyunoglou, J.H., and Akoyunoglou, G., Plant Physiol., 46, 247 (1970). 'Ihornber, J.P., Gregory, R.P.E., Smith, C.A., and Bailey, J.L., Biochemistry, 5, 391 (1967). Thornber, J.P., (Personnal Communication). Anal. Siegelman, H.W., Wieczorek, G.A., and Turner, B.C., 13, 402 (1965). Biochem., Anderson, J.M., and Boardman, N.K., Biochim. Biophys. Acta, 112, 403 (1966). Akoyunoglou, G., and Argyroudi-Akoyunoglou, J.H., Physiol. Plantarum, 22, 288 (1969). Photosynthetica, 2, 285 (1969). Sestak, Z., Papageorgiou, G., Baloglou, A., Akoyunoglou, G., and Govindjee, (submitted for publication). Govindjee, Papageorgiou, G., and Rabinowich, E., Fluorescence: Instrumentation and Practice.G. Guibault Ed., Marcel Theory, Dekker,, N.Y., 511 (1967). 613

Vol. 45, No. 3, 1971

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 2c.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Dujardin, E., De Kouchkovsky, Y., and Sironval, C., Photosynthetica, 4, 233 (1970). Kirk, J.T.P., and Tinley-Basset, R.A.E., The Plastids, W.H. Freeman and Co., London, (1967). Sironval, C., Michel, J.M., Bronchart, R., and Englert-Dujardin, E Progress in Photosynthesis Research, I, 47 (1969). &IL ) P.V., Goodchild, D.J., and Park, R.B., Biochim. Biophys. Acta, 216, 162 (1970). Boardman, N.K., and Highkin, H.R., Biochim. Biophys. Acta, 126, 189 (1966). Goodchild, D.J., Highkin, H-R., and Boardman, N.K., Exptl. Cell Res., 43, 684 (1966). Goodenough, U.W., Armstrong, J.J., and Levine, R.P., Plant Physiol., 44, 1001 (1969). Homann, P.H., and Schmid, G.H., Plant Physiol., 42, 1619 (1967). Woo, K.C., Anderson, J.M., Boardman, N.K., Downton, W.J.S., Osmond, C.B., and Thorne, S.W., Proc. Natl. Acad. Sci., 67, 18 (1970). Bishop, D.G., Andersen, K.S., and Smillie, R.M., Biochim. Biophys. Res. Communs., 42, 74 (1971). De Greef, J., Butler, W.L., and Roth, T.F., Plant Physiol., 4J, 457 (1971).

614