Proline enhances primary photochemical activities in isolated thylakoid membranes of Brassica juncea by arresting photoinhibitory damage

Vol. 181, No. 3, 1991 December 31, 1991

PROLINE

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1238-1244

ENElANCES PRIHARY PHOTOCEl3HICAL ACTIVITIES THYLAKOID MEMBRANES OF B-CA JUNCEA BY PIiOTOINKIBITORY DAMAGE

2*

ALIAl,

p. PARDHA SARADHIl AND PRASANNA MOHANTY

1 Centre for Biosciences, Jamia Millia New Delhi - 110 025, India 2

IN ISOLATED ARRESTING

Islamiar

School of Life Sciences, Jawaharlal Nehru New Delhi - 110 067, India

UniVerSityr

Received November 15, 1991 The presence of L-proline in tne reaction mixture enhances the photosystem II (H~O-SDCPIP) and.whole chain (H20+MV) catalysed electron isolated from the cotyledonary leaves transport activities of thylakoids Of seedlings raised in the absence and the presence Shabbica juncea of NaCl. The extent of stimulation in activities was higher in tne thylakoids of NaCl raised plants than the controls. The extent of proline mediated stimulation was seen even in the presence of uncoupler NH Cl suggesting that this stimulation is not due to uncoupling. However, pA otosystem I (DCPIPH2+lyv) catalysed photoreaction remained almost insensitive to proline. The presence of proline in the incubation medium brought about a significant reduction in the time dependent loss in photochemical activity suggesting that proline prevents of thylakoids exposed to strong light photoinhibitory loss in chloroplast activity. Also, proline brought about peroxidation linked a considerable reduction in the production of lipid We suggest that proline promaiondialdehyde auring strong tiumination. tects the components invoived in water oxidation capacity by reducing the production of free radicals and/or scavenging the free radicals and 0 1991Academic Press,Inc. thereby reducing thylakoid lipid peroxidation.

Proline from

bacteria

stresses tissues

(1,2).

against

to

gets accumulated higher Elevated

stress

by

plants level

in the

wide range of organisms right

when subjected of

regulating activities

proline

is

to

believed

osmotic potential of enzymes (5)

(4) I NAD/NADH ratio (l), scavenger of free radicals (6). Although accumulated in leaves in high quantities

various

proline

to

environmental protect

(2,3), and/or

plant

cytosolic pH sting as a

has been known to ge:

and tne chloroplasts

nave been

* Author for correspondence. At&eviatiis: Chl, Chlorophyll; DCPIP, 2,6-dichlorophenoi indophenoi: HEPES, 4-(2-Hydroxyethyl)-lpiperazine-ethane-sulphonic acid: MDA, malodialdehyde; MV, methyl viologen; PSI Photosystem; TBA, thiobarbimethyl) - aminomethane: WC, whole chain. turic acid; Tris, Tris-(hydroxy 0006-291X/91$1.50 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

1238

Vol.

181,

No.

BIOCHEMICAL

3, 1991

reported

to

be the

site

of

been made to see whether

AND

proline

BIOPHYSICAL

synthesis

RESEARCH

(7)

so

far

primary

photochemical

presence of proline in high Recently glycine-betaine reactions.

mulates

during

has

the

present

stress

the

absence

ennances and

shown

we report

Our results

pnotochemical

prevents

bringing

been

the

protect

effect

for

of proline

tne first

of PS Z

photoinhibitory

loss

in lipid

PS II

activity

in

(8).

on primary the

time,

in isolated

have

quantities aEects which also accu-

thylakoid membranes of seedlings raised in the show,

activities

about a reduction

to

the

in the isolated B/tasclica juncca

of NaCl.

no efforts

the

communication,

chemical reactions donary leaves of

COMMUNICATIONS

photocotyle-

presence

that

thylakoid

photochemical

In

or

tne proline membranes

activities

by

peroxidation.

MATERIAL

AND

METHODS

Seeds of 8ta66ica juncea cv. Dira 367 obtained Agricultural Research Institute (New Delhi, India), were grown H5 (9) media with or without NaCl (200mM) as described earlier

from Indian on modified (10).

Thylakoids were isolated from the cotyledonary leaves of &tlci,5sicajuncea seedlings 10 days after inoculation of the seeds as reported by Alia et al. (10). PS II mediated electron transport activities were measured both polarographically (11) and spectrophotometricaly (12). Whole chain electron transport was monitored with MV as electron acceptor and activity of PS I was measured with reduced DCPIP as electron donor and MV as electron acceptor as described earlier (10). All polarograp_hp measurements were carried out under rate saturating intensity (-480Wm ) at 25?1"C. by exposing

Thylakoid suspensions were subjected t photoinhibitory treatment them to strong white light (~650 Wm3 ) at 2551°C (13).

For measuring MDA content, isolated thylakoids were washed with 50 mM tris-HCl buffer (pH 8.0) containing 175 mM NaCl and resuspended in tne same buffer. The MDA content was determined by using thiobarbituric acid reaction (14). RESULTS

The stimulation tally was

of

proline

of DCPIP supported

as well obtained

affected with 'The lipophilic zoquinone forming

presence

could

AND

in

not

a deep

increase electron be used

coloured

the

reaction

PS II activity

as spectrophotometrically). with 1 M proline and further PS II

DISCUSSION

this

in the acceptors

(measured

The maximal stimulation

both

brought

about

polarographi-

extent of stimulation remained almost un-

concentration of proline (Fig. 1). like phenylenediamine and p-ben-

as they complex.

mixture

reacted Proline

instantaneously also

electron transport (H20+MV) reaction measured as (Fig. 2a). However, PS I mediated photochemical mained insensitive to proline (Fig. 2b) suggesting 1239

stimulated MV dependent

with

proline

whole

chain

02 uptake

reaction(wpIpH2+MVj rethe effect of proline

that

BIOCHEMICAL

Vol. 181, No. 3, 1991

= 0' 0 Fig.

0.5

1.

Effect

1.0 15 [Proline], M of

4 2.0

AND BIOPHYSICAL

1 I 1.0 1.5 [Proline], M

1 0.5

01 0

I 2.5

RESEARCH COMMUNICATIONS

2.5

2.0

varying

concentrations of proline on PS II (H 0-f electron transport activity in thylakhids isolated from cotyledonary leaves of &a66ica juncea seedlings raised in the absence (-o-) and the presence (-o-) of NaCl. (A) O2 evolution measured polarqrapnically and

DCPIP) mediated

(B) the DCPIP reduction monitored spectrophdometrically at 590 nm. The vertical lines represent standard deviation of the mean value of three independent experiments.

is

localized

plants

to

raised

PS II.

in the

Thylakoids

isolated

medium containing

from

cotyledonary

200 mM NaCl

leaves

showed higher

of

rates

of WC and PS II mediated photoreactions as compared to those from control plants (10). Proline could bring about stimulation of both WC and PS II mediated

electron

raised

plants

transport (Figs.

in these thylakoids 250

0 Fig.

2.

activities

in the thylakoids

1 and 2 ) and the extent

as compared to those obtained 800

A

0.5

1.0 1.5 [Proline], M

2.0

2.5

isolated

of stimulation from

from

control

plants.

B

5 [Proline], M

Effect of varying concentrations of proline on (A) whole chain (H O--MY) and (H) PS I (DCPIPH~JIYV) electron tranTne 0. uptake sport a c& vities. measured polarographically leaves of 3ha4bica in thylakoids isolated I+rom cotyledonary juncea seedlings raised in the absence (-o-) and tne lines represent standard pK@S@nCe (-0-l of NaCl. The vertical deviation of the mean value of three independent experiments. 1240

NaCl

was higher

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AND

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RESEARCH

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Table 1. Effect of proline (1N) without and with 5 mM NH Cl on the whole chain (H2OjNV) electron transport activity in thylakoi 4 s isolated seedlings raised in from cotyledonary leaves of ikauica juncea the absence (Control) and the presence of NaCl. Figures in parenthesis indicate the percentage stimulation to their respective controls. Electron transport rates were measured as mentioned in materials and methods. Mean value of the three independent experiments and their variation is given as f SD. Electron transport rate (p mole 02 consumed/mgChl/h) Source

Control NaCl

69.933 (lOti)

108.0?08 (155)

83.9209 (100)

135+17 ( 160)

123.099

171.5+10 (133)

184.5Kl.5 (100)

259.2+18 (140)

(ioo)

The enhancement in photochemical

WC (Table

activities

of uncoupler, NH4C1 in the photochemical phorylation

(5

1) and PS II

with

proline

mm),

suggesting that

activities

is

(data

not shown) mediated

was observed not

even in the presence

proline

related

to

induced enhancement uncoupling

of

phos

from electron transport.

membranes The exposure of thylakoid NaCl raised plants to strong white light resulted

from

both

control

in a reduction

and

of the

water oxidation capacity (H2O+i*1V). The extent bf loss in the activity increased with time of illumination (Fig. 3). The rate of loss in activity was faster

in thylakoids

stress is

NaCl

koids

to

siderably

koids

incubated

(13). under

(Fig. 3). The exposed to high Light low

in particular PS II The absorption characteristics subjected

photodestruction

the incubation

mixture

capacity exposed 30

to

The loss in the exactly

similar

water oxidation

conditions

in dark

of thylacapacity was con-

in photochemical activities in thylaintensity could be due to damage of photopigment protein complex (13, 15, 16, 17). of the thylakoids were marginally altered

to strong light

no significant

the susceptibility

plants.

reduction

systems

when

plants than the control

known to enhance substantially

photoinhibition

of thylakoids

from NaCl raised

for of

significantly

50 min (data not shown),

pigments. prevented

The presence of

suggesting proline

in

the loss in water oxidation

of the thylakoids (from both control and NaCl raised plants) to high light intensity (Fig. 3). The extent of protection was 35%. However, proline did not bring about any significant change

in the water oxidation

capacity

of thylakoids 1241

incubated in dark.

The reduc-

Vol.

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No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

120 6

10 20

0

30

Time,

Fig. 3.

40

50

60

0

+ Proline - Prokne

10

20

30

Time,

min

dark

light

* *

-D +

40

50

60

min

Proline induced suppression in the loss of whole chain electron transport activity of the thylakoids isolated from cotyledonary leaves of 3m~bica jwwea seedlings raised in (A) absence apd (R) presence of NaCl, exposed to strong light (650 tirn-&j. 'I'hylakoids equivaiem to 20 pM Chl were lncubated in medium containing 20 mM HEPES-KOH (pH 7.51, 100 ml"j sucrose, 10 mM NaCl and 2 mM MgC12 without and

with proline (1 PI) at 291°C. All values are expressed as percentage of the respective zero time readings. The data

represent

the

mean

of

values

of

three

independent

experiments.

tion in the photoinhibition by proline could venge free radicals (6) since free radical and uric protein

a

acid

are known to inhioit

lignt

due to its ability to scascavengers like propylgtiate

be

induced

degradation

of 32 kDa

(17).

The exposure of thylakoids to high light intensity brought significant enhancement in the production of MDA. However, the

of

MDA produced

was

considerably

thylakoids

by

higher

tent in the control ever,

only a little

raised

when compared to controls.

tnylakoids

(over the respective

of seedlings

initial

was values)

2.3 fold after

about amount

in presence of The rise

as against

50 min of light

NaCl

in MDA con-

7 fold in treated incubation.

How-

same

cnange in tne MDA content was seen during dark incuperiod. The light induced production of MDA in chloro-

plast preparations nation chloroplasts

has also been reported earlier (14, 18). Upon illumihave been reported to generate superoxide, hydrogen

bation for the

peroxide

and hydroxyl

radicals

(17)

which

in turn

are known to bring

iipid peroxidation (14). The rise in the MDA level in both the types of thylakoids wnen exposed to light was reduced when proline was added to the incubation mixture: The reduction in the level of MDA with proline reflects tne reduction in lipid peroxidation (18). This seems to about

be related

to its capacity

to reduce the production

1242

of free radicals

and/or

Vol.

BIOCHEMICAL

181, No. 3, 1991

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1.0 -

,

Proline

I

6

z 22

+ Prollnr

‘0 0.6-

I

,

” it 5 -D ”

O-G-

40.2s

0 0

Pig.

its

4.

ability

show for

to

the

of thylakoids lipid

0 min

50 Time,

0

50

50 Time,

0 min

50

Changes in malondialdehyde content in thylakoid membranes isolated from cotyledonary leaves of Rta66ica juncea seedlings raisad in (A) tne absence and (B) the presence of N&l, and after incubating them in strong wnite light of 650b;fzr~ Intensity ( I ) or in dark ( 0 ) for 50 min witnout and witn ld proiine. Thylakoids equivalent to 10 in medium containing 175 mM NaCl in Pfl were incubated 50 mM Tris - iiC1 buffer (pH d-0) at 2521°C. Vertical lines represent standard deviation of the mean value of three independent experiments.

XC

as a free

first

time

possibly

peroxidation

by

radical

that

free

arresting

proline

induced

(6).

Our

results,

enhances photochemical

proline

reducing

and thus

II. The mechanism of elucidated (Fig. 4).

scavenger

radicals

the

which

activities

induce thylakoid

photoinhibitory

protection

therefore,

damage to

PS

of PS 11 remains to be

Acknowledgments We thank Dr.S.C.Gulati for providing seeds of &abbica juncea One' of us (Alia) is also grateful to University Grants' Commission for providing Senior Research Fellowship. PM thanks the Dean, School of Life Sciences, JNU, for support from UGC Departmental Special Assistance Scheme. REFERENCES 1. 2. 3. 4. 3. 6.

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181, No. 3, 1991

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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