Light dependent formation of δ-aminolevulinic acid in etiolated leaves of higher plants

Vol. 49, No. 2,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LIGHT DEPENDENT FORMATION OF 6-AMINOLEVULLNIC

ACID IN ETLOLATED LEAVES

OF HIGHER PLANTS

E. HABBLand S. KLEIN

Department of Received

Botany, The Hebrew University,

August

JemrsaZem,Israel

24,1972

SUMMARY Levulinic acid inhibited the synthesis of chlorophyll and caused accumulation of &sminolevulinic acid in greening etiolated leaves of corn and bean seedlings. This is the first direct evidence for the No stoichiometric synthesis of 6-aminolevulinic acid in higher plants. relationship was found between the inhibion of cholorophyll synthesis and d-aminolevulinic acid accumulation. The formation of 8-aminolevulinic acid was light dependent and the rate of its formation increased after an initial lag period of 2-4 hours. Aminolevulinic

acid synthetase participates

phyll in photosynthetic succinyl-CoA and glycine

bacteria by catalyzing 111.

in the synthesis of chlorothe formation of ALA from

It has been suggested that in algae and higher

plants also, ALA is an intermediate

in the biosynthesis of chlorophyll

The production of ALA in algae has only recently Beale 143 showed that light of LA, a competitive

f2,3].

been demonstrated directly.

grown ChloreZZa cells excrete ALA in the presence

inhibitor

of ALA dehydratase [S].

A similar

situation

exists in Eughna, where ALA accumulates in greening cells in the presence of LA [6].

So far,

no direct

evidence is available

in higher plants as part of the biosynthetic

for the synthesis of ALA

pathway leading to chlorophyll.

In this communication we shall show that in higher plants LA inhibits

chloro-

phyll synthesis as in Ch~orella and Euglena, and that ALA accumulates under these conditions.

Abbreviations:

ALA - 6- aminolevulinic 364

Copyright 0 1972 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

acid; LA - levulinic

acid.

BIOCHEMICAL

Vol. 49, No. 2,1972

AND BIOPHYSICAL RESEARCH COMMUNtCATIONS

MATERIALS AND METHODS

Corn

(Zea

bulgarian)

seeds

The primary green

leaves

flasks

were

the

the leaves

were

at 10,000

1 M Na acetate

For (Sigma),

g for

15 min.

concentrated

according

paper

[9]

acid

and on silica

acetylacetone,

gel

from

leaves

with

Levulinic

acid

(grade

I,

80% acetone Sigma)

corn

various

and pestle,

of 4% and the extract

extracts

centri-

to pH 4.6 with and Granick

or commercially

[8].

obtained

with

[7].

obtained

ethylacetate

The ALA-pyrrole

and (2-methyl-

was chromatographed Germany)

and determined

was purified

After

in small

After

a mortar

to Mauzerall

(M. Voelm,

a

of white

of solutions.

extracted

thus

at 22'.

under

cut bases

was brought

and Elliot

plates

80 ft.c.

with

var.

at pH 6.0.

their

water

the leaf

pyrrole)

extracted

uptake

according

to Irving

3-acetyl-4-propionic

with

The supernatant

ALA from with

stood

collected

from 8-9 day old

concentration

and ALA determined

was condensed

Leaves

in ice-cold

to a final

were

with

vuzga.ris, in the dark

solutions

the dark. but

ground

chromatography,

in vermiculite

illuminated

to accelerate

CClsCOOH was added

fuged

were in

(Phaseozus

bean seedlings

similarly,

of a fan

and bean

on the appropriate

leaves

treated

170)

and grown

day old

or remained

in front

periods

germinated

and floated

light

seedlings

Neve Yaar

of 9-10

in the dark

fluorescent

cold

were

safe-light

2 hours

var.

mugs,

[8].

on

Chlorophyll

was

spectrophotometrically.

as described

by Beale

[4].

RESULTS AND DISCUSSION

Levulinic

acid

of ALA when added In the dark known

only

to enhance

inhibited to etiolated

traces

chlorophyll corn

synthesis

and bean leaves

of ALA accumulated.

chlorophyll

synthesis

exposed

In the presence

in the bean

365

and caused

[lo],

accumulation

to light

(Table

of sucrose,

more ALA accumulated

1).

Vol. 49, No. 2,1972

BIOCHEMICAL

The effect acid (ALA)

TABLE 1.

of levulinic in greening

AND BIOPHYSICAL RESEARCH COMMJNKATIONS

acid (LA) on the formation of &aminolevulinic leaves of corn and bean seedlings

Corn Treatment

Water,

light

LA, light Sucrose, Sucrose Water,

light + LA, light dark

LA, dark Sucrose, Sucrose

Bean

ALA nmolelg

dark + LA, dark

Chlorophyll IJPiP

ALA wAef g

Chlorophyll !-dg

0.04

zk 0.03

318 k 35

0.44

f 0.17

60 f 11

7.62

f 0.22

184 f 19

5.40

f 1.00

32+

7

0.08

f 0.05

239 f 25

1.08

f 0.20

187 t 22

4.66

f 0.55

163 f 24

8.78

f 1.50

76 + 12

0.13

f 0.03

14*+

3

0.25

?: 0.24

16*f

0.15

-i 0.04

7+

2

0.50

f 0.50

0.12

f 0.05

0.36

f 0.01

20* 9k

4 3

0.00 0.00

1

*Protochlorophyll(ide) Duration levulinic Results

than

corn - 22 hours; bean - 18 hours. Concentration of of experiments: acid: 20 mM for corn; 15 mM for bean. Sucrose concentration 0.2 M. are given per g fresh weight as mean and standard error of 4-6 experiments.

in its

In corn,

absence.

and less

ALA was formed

compared

with

Figure tion

leaves

chromatograms

-were

from commercial

in

leaf

The Rf values

the leaf

leaves given

for

However, extracts.

and a decrease

was identified

with

no indications

with

synthesis

LA and sucrose

as

of LA on the forma-

the concentration

the pyrrole

can interfere

treated

chlorophyll

concentrations

Increasing

identical

This

were

of various

extracts

inhibited

LA alone.

those

by thin

obtained

to values with were

layer

layer for

reported

found

for

synthesis.

chromato-

and 0.2 on paper prepared

in the literature determination

the presence

by ion-exchange

50 mM

and paper

the pyrrole

the calorimetric

was established

366

of LA up-to in chlorophyll

- 0.5 on thin

ALA and corresponded

Aminoketones

ALA [7].

were

slightly

in ALA accumulation

The ALA in the

[8,91.

which

and ALA.

an increase

graphy -

when the

1 shows the effect

of chlorophyll

caused

sucrose

of

of aminoketones chromatography

191,

Vol. 49, No. 2,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. The effect of various concentrations of levulinic acid on the formation of 6-aminolevulinic acid and chlorophyll in greening corn leaves. Etiolated 8 day old leaves were treated with levulinic acid for 2 hours in the dark and a following period of 22 hours in light.

thin

layer

and paper

of Granick

[ill.

chromatography

Evidently,

due to the presence

Small which that

amounts

the leaves found

synthesized

in either

bean

and corn

leaves

difference

ring

were

that

(Table

matched

or corn.

8 molecules

to that

detected

method

the Ehrlich

reagent

in the LA containing

media

on

to lo-15%

of

143 reported

that

the difference

and absence this

5-10

of LA.

between

was

of ALA are required

than

in the

the amount

of chlorophyll

the conditions

relationship

higher

treated

in ChloreZZa

Under

of ALA found

times

amounted

in amounts

stoichiometric

The amount

was always

course

with

The ALA in the media Beale

experiments

1, Fig.

also

floated.

in chlorophyll

The time similar

of ALA were

exactly

our

formed

calorimetric

of ALA-pyrrole.

in the presence

ing during

account

the product

in the extracts.

of ALA excreted

and by the modified

prevail-

does not extracts

of both

that

expected

from

and untreated

leaves,

taking

for

the formation

occur bean

the into

of a tetrapyrrole

1). of ALA accumulation

of chlorophyll

accumulation

361

in corn

leaves

in untreated

treated leaves

with

LA is

(Fig.

2).

Vol. 49, No. 2, 1972

BIOCHEMICAL

Fig. 2. The effect chlorophyll in greening o light, water X dark, water

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of levulinic acid on the accumulation corn leaves. s light, 20 mM levulinic acid + dark, 20 mM levulinic acid

of ALA and

6-

4 4 10

hours

Fig. 3. The accumulation of ALA in corn leaves in the dark after illumination for 5 hours. 0 control: leaves treated with 20 mM levulinic acid in continuous light. s same treatment but leaves were returned to darkness after 5 hours in light.

366

BIOCHEMICAL

Vol. 49, No. 2, 1972

TABLE 2.

The effect of succinate and glycine greening corn leaves in the presence

levulinic

acid

0.05

M glycine

0.05

M glycine

0.05

M succinate

0.05

M succinate

(LA) + LA + LA

*

Mean and standard

When leaves

were

accumulation

error

returned

ceased

within

ALA synthetase

0.03

* 0.02

7.35

t 0.23

0.04

0.02

0.00

1.72

4.95

11.05

0.00

0.00

0.00

1.88

6.10

10.94

2-3 hours

that

has yet

no proof

The direct plants formation phyll is this

light

dependent

suggestion.

on ALA

that

formation

This

f 0.48

in tissues comprise

process

(Table

the that

is

from

and related The parallel indicates

continuous

first

direct

higher

plant

to light.

plants

and

observation tissues

produce

by ALA synthetase.

of ALA accumulation

that

in corn

[13,14].

[2,12]

The finding

to a decrease

369

in higher

ALA increases

and accelerates that

synthesis

on chlorophyll

two processes

the chloro-

ALA accumulation

in chlorophyll

of sucrose these

of chlorophyll

exogenous

in the dark

that

cessation

leaves

of higher

catalyzed

the

ALA

2).

observations

effect

to light,

resembles

too requires

The finding this

exposure

of illuminated

of ALA in the synthesis

in the light

also

3).

by the enhancement

and glycine

involvement

here

of protochlorophyll(ide)

production

(Fig.

upon transfer

reported

suggested

has been deduced

5 hours

to be detected

ALA is

by succinate

after

ALA accumulation

in such tissues.

leaves

f 0.98

of 3 experiments

of ALA synthesis

is

light

0.03

the data

however,

22 hrs

4.51

to our knowledge

in itself

weighi

1.36

synthesis

and indicates

of ALA in acid.

0.00

to the dark

of protochlorophyll(ide)

This,

ALA/ g fresh 13 hrs light

5 hrs light

Water

RESEARCH COMMUNICATIONS

on the accumulation of 20 mM levulinic

umole

Treatment

darkness

AND BlOPHYSlCAt

supports

synthesis

are related.

and The

Vol. 49, No. 2, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS L

lack

of stoichiometry

synthesis system

between

in the presence from a tightly

the low intensity chlorophyll

ALA formation

of chlorophyll

of LA may be due to the release

coupled

of light

accumulation

and inhibition

control

mechanism,

used in our

It

experiments

and the formation

of the ALA producing

may also

to avoid

of ALA might

be related

bleaching

to since

have different

light

requirements.

It

has been

the metabolic a light

phyll

control

dependent

approach

suggested

the

of chlorophyll

formation

more directly

that

of AU

problems

formation

of ALA plays

biosynthesis in higher

concerning

[2,3].

plants the

a central

in

The demonstration

makes it

control

role

possible

mechanisms

to

of chloro-

synthesis.

ACKNOWLEDGMENTS for

We wish technical

to thank Mrs. assistance.

E. Neeman, Mrs.

H. Barenholz

and Mr.

M. Guy

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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