Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

BIOCHEMICAL

Vol. 53, No. 3, 1973

ABSORBANCE

AND BIOPHYSICAL

AND FLUORESCENCE

PROTOCHLOROPHYLLIDE

IN

Merrill

PROPERTIES

ETIOLATED

L.

Received

of Biological of Illinois Chicago, Illinois

June 7,

OF

BEAN

LEAVES

Gassman

Dept. University

RESEARCH COMMUNICATIONS

Sciences Chicago 60680

at

Circle

1973

SUMMARY Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm in vivo at -196' and a fluorescence emission peak at 639nm --invi=t room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with --in vivo absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of &-aminolevulinic acid to etiolated leaves causes the synthesis of nontransformable protochlorophyllide with an absorption peak also at 631nm in vivo at -196“ but with a fluorescence emission peak at 643nm -K viva at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous g-aminolevulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf. Etiolated three

species

species

can

-196"

in

species

of be

the

’ is

‘650

the

red

a lesser

extent

in

etiolated-leaf

etiolated

leaves 1

phyllide;

@I I973

of

the

PChld

in

to

It

is

also

conta

occurs

only

its

levels

increase

Abbreviations: PChld, ALA, &-aminolevulinic

in

protochlorophyllide; acid. 693

3).

The

first,

in

This P637,

The

second,

eaves

but

to

the

when

a greater

phototransformable to

Chld

concentrations ALA

at

vivo.

(4).

low

pigment vivo

phototransformable

Psz8, but

These --in

2,

ning

least

maxima

ho 1 ochrome Chld

at

PChld'.

(1,

etiolated

extracts

by Academic Press, Inc. in any form reserved.

of reproduction

spectrum

light

contain

absorption

the

extent

species,

their

of

by

(1).

seedlings precursor

by

region

to

third

chlorophyll

transformed

holochrome

angiosperm

identified

occurs

The

of

characteristic is

PChld

Copyright All rights

leaves

is Chld,

(1). in

adminischloro-

Vol. 53, No. 3,1973

tered to

to

the

Chld

by

heat,

properties

of (1,

The the

acid,

these

three

is

the

PChld

same

absorption

that

of

bility

must

be

considered

from

the

state

or or

therefore,

that

a

converted

by

The

fluorescence

-196"

treatment

have

been

in

in 22". a dim

spectrum

through

at

in with

maximum

emission differs

than

is that

in of

at

least

bean

All

manipulations

P637, treat-

22“

of

the

possi-

PChld

species dif-

heat-denatured

PChld

PChld types

the

significant

a physically

two

and,

of

nontrans-

leaves.

AND METHODS var.

darkness

-196'

two

Thus,

nontransformable be

at

but

photoinactive

that

and

at

a small

species.

treatment

total

in

Red

moist

Kidney)

plant

(10). of

leaf

Model

accessory.

694

356

A single

were

for

material The

primary

Perkin-Elmer

seeds

vermiculite

of

safe-light

a cryogenic

vivo

peak

to

the

vulgaris

a Hitachi

--in

these

may

layers

indicate

of

etiolated

two

results

a combination

ALA

green

characterize

by

that

there

to

P650

endogenous

(Phaseolus

germinated

equipped

at

as

and

of

MATERIALS

-196"

be

denaturation

environment

the

PChld

under

can

undertaken

ALA-derived

exogenous

holochrome

out

act

heat

species

from

about

to

11).

The

fluorescence

extent

at

10,

were

species.

or

PChld

Bean

'637

species

here

by

the

heat-generated

formable

and

9,

PChld

derived

However,

ferent

phototransformed

seems

'650

(2,

reported

administration,

derived

directly

freezing-and-thawing,

compounds

has

it

ments.

not

RESEARCH COMMUNICATIONS

3).

species

ALA

8).

of

studies

whether

is

circumstances,

7,

nontransformable

this

by

(6,

AND BIOPHYSICAL

P628

some

P65D

a variety

reported

(5).

under

of

P628

with

leaves

but,

precursor to

BIOCHEMICAL

in

7-9

were vivo

tissue

days carried

absorption was

measured

spectrophotometer thickness

of

BIOCHEMICAL

Vol.53,No.3,1973

filter

paper

scanned

served

at

as

a speed

60mm/min.

The

of

slit

single

layer

Hitachi

with The

mode. exit

surface. angles

to

wave

of

response

MPF-2A

excitation the

sample

and was

the

beam

and

passed

exciting

before

the

rate

50mm/min. of

exit of

The

either

445

and

the

14.5mW/cm2 was

through

the

a

"ratio" the was

leaf the

leaf

taken

at

right

a 600nm

emission

sample.

at

short-

monochromator.

set

at

2nm.

50nm/min

and

recorded

at

not

corrected

emission

a of

filter

was

were

on

nm with

slit

spectra

the

22"

in

cut-off

the

of

(0.5nm).

accessory

at

surface

entering

monochromator at

set

was

leaf

speed

operated

long-wave

light

a chart

spectrophotometer

monochromator

exciting

were

at

Fluorescence

A 500nm

Spectra

0.125mm

solid

photomultiplier

40nm.

filter

at

recorded

the

scanned

speed

the

at

were

from

emission

were

set

Fluorescence

cut-off

The

was

monochromator

the of

recorded

in

RESEARCH COMMUNICATIONS

(10).

and

spectra

Model

to

between intensity

356

tissue

a R446S

open

placed The

leaf

excitation

slit

the

emission of

blank

60mm/min

Perkin-Elmer

equipped

a reference

on

Fluorescence

AND BIOPHYSICAL

The a chart

for

monochromator

or

holochrome

was

spectra

the

the

photomulti-

plier. Phototransformation charging at

a xenon

22"

(10).

minutes

of arc

(60

PChld

W-sec)3

Fluorescence

after

the

cm from

spectra

actinic

the

were

achieved

surface

recorded

by

of

from

the

3 to

disleaf

5

flash. RESULTS

The bean

absorption

leaf

is

apparent

at

maximum with

spectrum

shown 650nm,

is

observed

a light

flash

seconds,

curve

e of

in

Figure

at

-196"

1A

(curve

characteristic at at

637nm. 22'

Figure

and

If then

1A is

of

a 7-day-old

a).

of

P

an

etiolated

650'

brought obtained.

An

etiolated

absorption

A smaller leaf to The

-196' peaks

peak absorption

is

irradiated

within at

a few 650nm

is

Vol. 53, No. 3, 1973

BIOCHEMICAL

and

637nm

have

disappeared

the

first

Chld

photoproduct

peak

at

628nm

548,

552,

data

agree

and

and

Figure

1A are

with

time

Curves

a and

b of

leaves

this

phenomenon

P650

is

under of

the

continuous

P628 d in

Figure which

lated

bean

leaf

Curve

c in

Figure

which

was

followed

infiltrated by

boiling

estimation

631nm

of

absorption

been

the

in

water

for

band

bands

in

at

these

two

hours,

for

of

this

loss

wheat

in

leaves

when

the

pool

(15).

six

for

30

gave

etio-

seconds. leaf

darkness curves In

631nm.

half-maximum

an

a similar in

Both at

of

of

hours

seconds.

curves

diminished.

description

inhibited

water

maximum

widths

of

has

P650

spectrum

30

spectra

spectrum

in

of

P628.

s ix

in

level

for

of

However,

is

1OmM ALA

ion

complete

absorption

absorption the

of

synthesis

boiled

1OmM ALA

increases

and

level

absorption

with

symmetrical

a single, an

is

the

These

respectively,

accumulat

three

a certain

1A is

18

c,

elsewhere.

exceeds

at

cytochromes.

absorption

for

Chld

peaks

631-632nm

A more

why

small

with

at

the

addition

3).

irradiation

has

a

to

the

ALA

treated

reason

In

the

clear.

low-level

ALA-generated Curve

be

appears.

b and

hour,

not

of

infiltrated

1B indicate

is

characteristic

due

absorbance

sixth

678nm,

also

(1,

are

with

will

probably

al

indicating

Figure

this

--et

RESEARCH COMMUNICATIONS

are

probably

The

infiltrated

for

14)

darkness,'curves

By the

reason

13,

leaves

infiltration,

respectively. The

Kahn

obtained.

of

similar

of

in

at

There

are

bean

hours

a peak

(12,

which

those

7-day-old

four

and

apparent.

556nm

with

When two

becomes

AND BIOPHYSICAL

exhibit addition,

height

of

the

a value

of

28nm

spectra

of

four

or

6 hours

* 2nm. Figure etiolated in

darkness.

of

PChld

2 presents leaves

fluoresence

infiltrated It

shifts

the

can from

be

seen

639nm

to

emission

with

1OmM ALA

that

the

643nm

696

for

0,

fluorescence as

nontransformable

2,

4,

emission

peak PChld

BIOCHEMICAL

Vol. 53, No. 3,1973

builds

up

the

curve

c-640nm, ducible

in

reported

leaf;

i.e.

d-643nm.

other to

results at

in

curve These

suggest

638nm

the

shorter

curve peaks

Heat-killed

at

that

RESEARCH COMMUNICATIONS

a-639nm,

emission

experiments.

fluoresce

a slightly

AND BIOPHYSICAL

at

were

barley

room

endogenous

b-639nm, quite

repro-

leaves

have

temperature

(16).

nontransformable

wavelength

than

the

curve

These

PChld

PChld

been

derived

emits

by

ALA

administration. To

test

whether

the

fluorescence

PChld

holochrome

was

or

endogenous

nontransformable

of

Figure

3 was

carried

curves

b and

d are

for

6 hours

in

boiled

in

Curves

a and

show in

ALA-treated

at

643nm

lengths

by

PChld

or

of

for

leaves

curve the

d was

639nm

emission

without

compared

to

band The

c).

in

is

at

639nm

(curves

peaks

of

PChld

heating,

is

642-643nm

in

b and

which

level

the

emission

d PChld

band wave-

heat-denatured that

leaves, c).

leaves,

of

shorter

indicate

a and

b and

large

to

were

spectra.

curves

with

untreated

ALA-treated (curves

while the

results

1OmM ALA

leaves the

in

leaves;

with

associated

PChld

shown

untreated

shifted

of

emission

of

appreciably

emission

of

controls,

peak

heating,

at

Because

PChld

experiment

recording

639nm

denatured

ALA-derived

d depict

at

heat

infiltrated

to

bands

not

(curve

c are

c and

643nm. as

a and

prior

of of

the

were

Curves

at

that

PChld,

which

emission

bands

either

Curves

30 seconds

c show

holochrome

rescence

out.

leaves

in

to

darkness.

water

emission

similar

emission

the

fluo-

either

with

However,

the

with

or

without

d).

DISCUSSION Evidence the

absorption,

endogenous are

has

different

been

presented

properties or

derived from

of from

those

that

the

nontransformable

heat-denaturation of

the

fluorescence,

nontransformable

697

PChld, of

PChld PChld

but

not

whether holochrome, derived

Vol. 53, No.3,

BIOCHEMICAL

1973

AND BIOPHYSICAL

600

RESEARCH COMMUNICATIONS

640 Wavelength

A

\ / i,

Wavelength

i

Inml 600

640 Wavelength

A.

B.

inm)

if\

!

Fig.

720

680

680

720 (nml

1. The _in vivo absorption spectra of etiolated bean leaves at -196'. a. (-) control; b. ce+v) treated with 1OmM ALA for 2 hours; c. G-V) treated with 1OmM ALA for 4 hours; d. (--) control, boiled in H20 for 30 sec.; e. c-e-) control, after flash of 81gyL) b: &a+.) Fti l!O')fir 2

treated treated reated 30

with with with sec.

1OmM ALA 1OmM ALA 1OmM ALA

for for for

3 hours; 6 hours; 6 hours

followed

by

boiling

Fig.

2. The fluorescence emission spectra of etiolated bean leaves at 22" following a flash of light. b. (a**) treated with 1OmM ALA for 2 hours; a. (-) control; c. k-3 treated with 1OmM ALA for 4 hours; d. (--) treated with 1OmM ALA for 6 hours. The electrical gain for curve d was l/9 that used in the other curves. 698

3

BIOCHEMICAL

Vol.53,No.3,1973

from

ALA

clear

administration.

but

may

pigments;

e.g.

polymeric

state

One to

The

reflect

a hydrophobic of

the

and

may

ALA

the

apparent

emission

ly,

however,

for

for

the

is

that

emit

peak

the

of

leaves;

ALA-treated

displaced

leaves

from

Another is

that

PChld.

If

pigments

the

tracted

into

ether,

and

benzene: red

ether Sundqvist Fig.

has as

is

PChld (15)

same which

the

this

in

shift

in

the

Rf and

extracted treating

are

considerably

Upon

exto gel

spot

this

properties

in

wheat

in

with

elution,

leaves.

etiolated

(1)

silica

1 green

untreated

endo-

transferred on

fluorescence

emission

are

ether,

= 0.54.

wave-

from

leaves

only

from

the

643nm

following:

chromatography

at

at

(3)

the

bean

magex-

band

species

petroleum

of

ALA-PChld

on

unlike-

ether

emission

porphyrins.

(4:1:1),

absorption

that

found

based

thin-layer

obtained,

reports

are

chemical

with

ethanol

is

traces

and

etiolated

is

only

other

unlikely

shifting

(1)

PChld

peak

synthesized

thereby

(425-465nm) of

be

explanation

symmetrical;

washed by

is

This

a different

also

acetate:

the

(2)

ALA-treated

purified

fluorescence

PChld.

bands

be

acetone,

ethyl

pigment

may

of

the

emission

may

640nm,

reasons:

for

This

not

the

and/or

ALA-PChld

porphyrins

fluorescence

explanation

genous

is of

a protein

the

near

quite

Soret

ALA-PChld

difference

environment

protoporphyrin

is

excite the

the

in

other

following

tracts

to

shift

of

and

used

this

of

fluorescence

protoporphyrin

lengths

in

interior

nesium

ALA-treated

for

pigments.

wavelengths

from

in

reason

a difference

explanation

longer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(2) leaves

3. The fluorescence leaves at 22O following

emission spectra of etiolated bean a flash of light. t reated with 1OmM ALA for 6 hours; boiled in H 0 for 30 sec.; with 1OmM ALi for 6 hours followed by boiling

treated H 0 for 30 sec. Tie electrical gain for 27x that of curve d.

curve

a was

699

9x

that

of

curves

b and

in c and

Vol.53,No.3,1973

with

BIOCHEMICAL

[14C]

ALA

[14C]

PChld;

which

forms

identical

etiolated

This

the

PChld

level

of

in

the

C678,

to

shorter

in

with

Chld

shorter

(12).

this

shown irradiated

The

in

mixed

emission

cannot

be

wavelength a PChld-Chld Kahn

maximum

--et

al

also

be

Chld

Fig.

3).

is

cor-

from

683

rapid

to

longer

of

ALA-PChld

rather

peak

of

can

wave-

than

emission

(17)

peak

even

in d,

be

to

ascribed

However,

PChld

to rein-

have

recently

form

in

Fig.

longer

to

the

the

longwave

heat-treated

weakly

3).

Thus,

this to

wavelengths formation shift

leaves,

due exclusively

(1,

3)

attribute

the

phototransformation

a secondary of

PChld

dimer

shifted

photoproduct,

amounts

Sauer

the

the

of in

when

ALA-

Chld

anomalous formation

of

dimer.

following to

may

cannot

and

is

The

than

with

in

682nm

(12).

concentrations

emission

(curve

shift

to

Chld

the

b,

up

leaves.

dimer.

produced

curve

maximum

large

mixed

PChld

occurs

peak

691

rather

of

Mathis

leaves

2;

first

Chld

protein.

builds

ALA-PChld

of

increasing

the

Fig.

PChld

chemically

emission

from

the

displacement

bean

ALA-treated

PChld

of

of

etiolated

probably

absorption

of

aggregation

PChld-Chld

shift

its

presence

at

d,

the

holochrome

Chld

(675nm),

a natural

a PChld-Chld

and

the

The

the

presence

explanation.

that

the

emission

band

in an

to

c and

wavelengths

to

is

Thus,

ALA-PChld

of

Chld

phototransformable

P65.,.

treatment

in

the

wavelengths

forces

vivo

the

absorption

(683nm),

ascribed

in

position

in

with

binds

(curves

22"

shift

was

ALA

a displacement

at

lengths

associated

RESEARCH COMMUNICATIONS

of

nontransformable

the

displacement

675nm

formation

which

wavelengths

with

the

exogenous

leaf,

related to

PChld

from to

lower

in

i.e.

As the

to

results

AND BIOPHYSICAL

the

peak endogenous

of

628nm to

Chld.

both

Therefore,

nontransformable

700

absorption

peak

-in

nontransformable the

true

PChld

may

PChld absorption lie

at

wave-

BIOCHEMICAL

Vol. 53, No. 3,1973

lengths the

longer

absorption

d of

Figure

(7,

628nm.

peaks

of

lA,

curve

here

of and

the

three

the The

cursors

using

state

of

effectiveness of

P650

of in

at

1B)

631nm

and

at

pigments

in

these

-196"

of

[curve at

22"

absorption PChld

described

spectra

one

method

of

the

intact

leaf.

different

position

633-636nm

fluorescence

than

the

at

identical

their

currently

by

nontransformable

more

of

is

supported

species

Figure

types

for

is

apparently

discrepancy

necessity

identify

these

the

the

This

c of

lO).Nevertheless,

spectra

the

than

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

forms

being

investigated.

supported

in

point

analysis

of

P628

out

to

as

pre-

ACKNOWLEDGMENT This Foundation Illinois,

investigation grant Chicago

was

GB 35627 Circle

and

by

a grant

Research

part

by

from

the

National University

Science of

Board.

REFERENCES 1. 2. 3.

4. 5. 6. 7. 8. 9. ::: 12. 13.

Kahn, A., Boardman, N. K. and Thorne, S. W. (197O),J. Mol. Biol. 48, 85-101. Dujardin, E., and Sironval, C. (1970). Photosynthetica 4, 129-138. Boardman, N. K., Anderson, J. M., Kahn, A., Thorne, S. W., and Treffry, T. E. (1970). In Autonomy and Biogenesis of Mitochondria and Chloroplasts N. K. Boardman, A. W. Linnane, North Holland, Amsterdam. and R. M. Smillie, eds. pp. 70-84, Shibata, K. (1957). J. Biochem. 44, 147-173. Granick, S. (1963). Proc. V Int. Cong. Biochem. 5, N. M. Sissakian, ed. pp. 176-185, Pergamon Press, Oxford. Granick, S., and Gassman, M. (1970). Plant Physiol. 45, 201-205. Sundqvist, C. (1970). Physiol. Plant. 23, 412-424. Murray, A. E., and Klein, A. 0. (1971). Plant Physiol. 48, 383-388. Butler, W. L., and Briggs, W. R. (1966). Biochim. Biophys. Acta l&, 45-53. Gassman, M. L. (1973). Plant Physiol. 51, 139-145. Henningsen, K. W. (1970). J. Cell. Sci.I, 587-621. Gassman, M., Granick, S., and Mauzerall, D. (1968). Biochem. Biophys. Res. Commun. 32, 295-300. Sironval, C., Brouers, M., Michel, J.-M., and Kuiper, Y. (1968). Photosynthetica 2, 268-287.

701

Vol. 53, No. 3,1973

14. ;i: 17.

Bonner, Sundqvist, Virgin, In Modern Tracey, Mathis,

BIOCHEMICAL

AND BIOPHYSICAL

B.

RESEARCH COMMUNICATIONS

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