Indian Journal of Biochemist ry & Biophysics Vol. 37. December 2000. pp. 498-505

Oxidati ve stress in cucumber {C cumis sativusT~ seedlings treated with . acitluorfen

Illora Gupta and Baishnab Charan Tripa thy*

School of Li fe Sc iences. Jawaharlal Nehru University. ew Delhi 110067. India

Recei\'ed 31 D ecelllber 1999: accepled 31 May 2000 -Treatment of diphenyl ether herbicide acilluorfen-Na (A F-Na) to intac t cucumber (ell clllllis S(ll i l'IIS L ev Poinsette)

se dl ings induced ove raccumulation of plOtoporphyrin IX in li gh t (75 pmole m'~ S·I). The extra-p!astidic protopo rphyrin IX acc umulated during the li ght exposure disappea red wi thin t\Vo hours of transfer of acifluorolcn-treated seed li ngs to darkness. The dark disappearance was due to re-e ntry of migrated pro toporph yri n IX into the plastid and ItS subsequent conversion to protoch lorophyll ide. In ligh t. protoporphyrin IX acted as a photosensi ti ze r and caused gene ration of active oxygen species. The I:lller caused damage to the ce llul ar membranes by peroxidati on of membrane lipids that resu lted in production of ma londialdehyde. Damage to the plastidic membrancs resulted in damage to photosystem I and photosystem II reaction s. Dark-incubation of hcrbicide-sprayed pl ants before their exposure to li gh t enhanced photody namic damage due to diffusion of the herbicide to the site of ac tion. Comparcd to control. III treated samples the cation-induced increases in variab le flu orescence/maxi mum fluore scence ratio and increase in photosystelll II acti vity was lower due to reduced grana stacking in herbicide- treated and light-exposed plants:') ._) \ ( I

./ .' Oxidati ve stress is widespread in nature. Several biotic and abiot ic st resses are ultimately manifested through ox idati ve ac tion of acti ve oxygen species on a cell. Even programmed cell dea th is ul timately mediated th rough ox idation reac ti ons. One of direct manifestations of oxidati ve stress is herbicida l ac ti on of 5-a minolevuli nic ac id (ALA) and diphenyl ether (DPE) and allied compounds that generate porphyric compounds as photosen i!i zers respons ibl e for production of active oxygen species caus ing cel l dea th. Acc umulat ion of excess porphyrins in case of peop le suffering from porphyria is one of the examples of a human di sease caused by oxidative stress. Porphyrin biosy nthesis from ALA is responsible for the synthesis of heme and chlorophyll (ChI). Enzymes of the porphyrin pathway, from ALA to protoporphyrin IX (Proto IX). probably ex ist in fo rm of multi-enzyme complexcs l.2. Such compiexes are capable of cata lyzing sequentia l enzymatic react ions on a sub -trate. It preven ts the intermediates 0f Proto IX biosynthetic pathway from being III

~'Au thor for correspondence: E- ma il: bCl@jnuniv.ernet.in .\hhrcviatiolls: AF-I a: acilluorlcn-sodium: ALA: 5-all;i noicvulinic acid: Chi: ch lorophyll: DC IP: 2.6-dichlorophenol Inuophenol: HEAR: hexane extracted acetone resiuue solve nt mixture: MDA: malond ialdehyde: MV : methyl vio logen: Pchlide: pmtoL'illoroph yllide : PD: p-phcnylenediami ne: Proto IX: protoporphyrin IX: Protogen IX : protoporph yri nogen IX: Protox: protopmph yrinoge n IX oxidase: TBA : Thioharbituric acid

diffusion equilibrium with identical molec ul es in the same ce ll compartment. Porphyrin intermed iates of the Ch I and heme bi osynthet ic pathway are tox ic to all li ving things because they produce active oxygen spec ies which reac t with cellular components incl uding membrane lipids wh ich are essenti al for life.

Synthesis of tetrapyrrol ic intermed iates of heme and ChI biosynthetic pathway i. c .. Proto IX and protoc hlorophyllide (Pch!ide) is tigh tl y regu lated. Pch lide and Proto IX act as feedbac k inh ibitor of biosy nthesis of ALA , the precursor of porphyri ns".). Foliar app lication of ALA to green plants bypasscs the regulatory feedback inhibition of the Pchl ide pooi and ind uces excess accu mu la tion of Mg­tetrapyrroles5

•7

. ALA-induced accumulation of no~­phototransformable Pchlide in presence of light generates the active oxygen spec ies i.e., singlet oxygen ( 102) through type II photosensiti zation reaction, which damages the plants.9.

In contrast to ALA-induced photodynamic damage where Pchlide ac ts as a photosensiti zer. treatment of DPE and allied herbici des leads to the inhibition or protoporphyri nogen oxidase (Protox) and light­induced excess acc ulllulati on of the photosensiti zer Proto IX7

.tO

•1,. Definite proof for in volvement of

tetrapyrroles in DPE mode of acti on came from the observation that inhibitrOl of biosynthes is of ALA,

GU PTA & TRIPATHY: OXIDATI VE STRESS IN CUCUMB ER SEEDLI GS 499

the precursor of tetrapyrrol es, by gabaculine and 4,6-dioxoheptanoic in OPE-treated plants led to non­accumulation of tetrapyrroles and consequent nullification of the herbicidal activityl 4. Over­accumulated Proto IX absorb sunli ght to produce triplet exci ted states that interact with molecular oxygen via type II photosensiti zation reaction to generate the acti ve oxygen spec ies (1 0 2) that cause severe photodynamic damage to pl antsls.

The present study aims to characteri ze the mechani sm of ac ti on of AF-Na-induced Proto IX­sensiti zed ox idati ve stress on the photosynthetic membrane. The acc umulati on of the photosensitizer Proto IX and its poss ible conversion to Pchlide in the plast id are discussed.

Materials and Methods

Plant lIlaterial Cucumber (ClI cll ll! is sativlI s L. cv Poinsette)

seedlings were grown on moist germination paper in pet ri pl ates (14.5 cm diameter) under continuous cool­whi te-flu orescent li ght. Temperature was maintained at 25°C. Five-day-old seedlings were used in all expcri ments.

Herbicide treatlllent The technica l grade materi als of a OPE herbi cide,

AF-Na was a gift from Rohm and Haas Bay Port Inc. SA. The herbicide was diluted with wa ter + 0.5 %

Tween-20 and each petri plate hav ing a populati on of 25 seed lings was sprayed with 10 ml of 3 )..lM of AF-

a. A glass sprayer (atomi zer) attac hed to a rubber bulb was used fo r sprayi ng AF-Na. Control seedlings were sprayed with disti lled water hav ing 0.5% Tween-20. After sprayi ng the seedlings were covered with alumi niu m fo il and kept for 14 hI' in darkness at 2YC, before they were exposed to light. Seedlings were exposed to cool-white-fluorescent light at 25°C (75 ~t mole m-~ S-I) . Light intensity was measured using LJ-COR Quantum meter Model U -1 85B with a Ll- J90SB qua ntum sensor (Ll-COR Inc., L incoln , USA).

Piglllent estilllmion Ch i content was es timated in 80% acetone l6.

/so lmion of thylakoid il/elllbrones Thylakoid membranes were isolated from

cucumber cotyledons at 4"(', under safe green light. by hand-homogenizing the ti ssue in an isolation

medium consisting of 0.4 M sucrose, 10 mM NaCl and 25 mM Hepes-NaOH (pH 7.6). The homogenate was passed through 2 layers of Mira cloth and was centrifuged at 3,000 g for 5 min to sediment the thylakoid membranes. Thylakoids were suspended in the isolation buffer?

Electron transport Assays of electron transport acti vity of the whole

chain , photosystem II (PSII) and photosystem I (PSI) were carried out using a glass cuvette fitted within a Clark-type oxygen electrode (YSI, Yell ow Springs, USA). The reaction mi xture was maintained at 25°C by using a temperature controlled water bath and was illuminated for 20 s with white light from a tu ngs ten I ight source at a photon flux rate of 1500 )..lmole m -2 s - I .

The whole chain electron transport from H20 to methyl viologen (MY) was monitored as O2 uptake. The reac tion mi xture (3 ml) consisted of 50 mM Hepes/NaOH (pH 7.6), 3 mM MgCl2• 10 mM aCI. 0.5 mM MY, 2 mM NH.jC, and 0.5 mM NaN}. The reaction mi xture (3 ml ) fo r p-phenylenedi amine (PD)­supported and PSlI-medi ated O2 evolut ion consisted of 50 mM Hepes/NaOH (PH 7.6), 3 mM MgClc. lO mM NaCI, 1 mM K3Fe(CN)6 and 0.3 mM fresh ly prepared PD8

. Thylakoids were added at a concentra tion of 10 )..lg Chi ml- I to the assay medium. The parti al elec tron transport chain th rough PSI was measured as oxygen consumption17

. Electron flow from psn was blocked by 3-(3,4-di chl orophenyl ) 1. 1-dimethyl urea (DCMU). Ascorbate/ DCIP couple was used as an elect ron donor to PSI and MY was used as an electron acceptor. Assay medium (3 ml) contained thylakoids (1 0 )..lg Chi ml- I

), 50 mM Hepes (pH 7.6). 3 mM MgCl:!, 10 mM NnCI, 1 mM NH.jCl . I mM NaN3, 0.5 mM MY, 20 )..lM DCMU, 1 mM sodium ascorbate and 100 )..lM DCTP.

CM a f luorescence transient Fluorescence transients of thylakoids isolated from

control and AF-Na-t reated seedl ings were monitored by pul se amplitude mod ul ated (PAM) Chi fluorometer (PAM 101 , Heinz Walz, Germany). For measurement of FQ, mod ul ated light of 1.6 KHz (0.075 )..lmole m-:! S- I) was used . For measurement of Fill> modul ated light of 100 KHz along with actinic light (900 )..lmole m-:! S- I) was used whi le keeping the other settings same as that for Fo measurement. Light was commu nicated from the source to th sample through a Walz liber optic cable (type 1O IF). Thylakoid

500 INDIAN J BIOCH EM BIOPHYS, VOL. 37, DECEMBER 2000

membranes were dark adapted for 15 min before fluorescence transients were recorded. For measuring transients of thylakoid samples , isolated thylakoids at a concentration of 10 Ilg Chi ml- I were suspended in a buffer containing 50 mM sucrose and 10 mM Hepes (PH 7.6).

M easurelllelll of lIlalolldialdehyde (M DA) production

Thiobarbituric acid (TBA) test was used to spectrophotometricall y determine the extent of lipid peroxidation l 8

. Lipid peroxides decompose to MDA during the acid-heating stage of the test. Two molecules of TBA react with one molecule of MDA to form a brownish coloured pigment having maximum absorption at around 532 nm in acid solution. Absorbance measured at 532 nm was corrected for non-specific turbidity at 600 nm. Sucrose was not added to the buffer as it interferes with the assay. Quantity of MDA was calculated using extinction coefficient of 155 mlf2 cm- I.

Estimation of tetrapyrroles Leaf tissue was hand-homogenized in cold

ammoniacal acetone (9: 1 v/v, acetone: O. IN N~OH),

under safe green light. Fully esterified tetrapyrroles were extracted with equal volume of hexane. While the mono- and di-carboxylic tetrapyrroles remained in the hexane extracted acetone residue solvent mixture (HEAR) fraction, the fully esterified tetrapyrroles were transferred to hexane phase. Quantitative estimation of Proto IX and Pchlide from their mixture was carried out spectrofluorometrically' 9.

Fluorescence spectra from HEAR fraction were recorded in the ratio mode using a computer-driven SLM AMINCO 8000 spectrofluorometer and corrected for photomultiplier tube senSItivIty. Rhodamine B was used in the reference channel as a quantum counter. A tetraphenylbutadiene block was used to adjust the voltage in the sample as well as reference channels, to 20,000 counts/sec at excitation and emission wavelengths of 348 and 422 nm respectively. Spectra were recorded at excitation and emission bandwidths of 4 nm. Emission spectra were recorded from 580 to 700 nm.

Results

Visual observations of photodynamic damage Light was mandatory for herbicidal action as there

was no damage in AF-Na-sprayed plants kept in dark

for even up to 72 hI'. AF-Na-treated plants incubated in dark for 14 hr and subsequently transferred to cool white fluorescent light (75 Ilmole m-:! S- I), developed necrotic spots within 12 hr and were severe ly damaged by 48 hr, because of oxidative stress. There was enough water in petri plates when plants were exposed to light. Therefore, the bleaching of plants was not due to water stress rather it was caused by photodynamic damage. When portions of trea ted leaves were shaded from light by loosely covering them with pieces of thin and light aluminium foil , bleaching took place only in the light exposed portions of leaves. The shaded pOltion of the leaf was not bleached (picture not shown).

Tetrapyrrole acculIlulation ill the cotyledons of AF­Na-treated and light-exposed plallts

AF-Na-treated and 14 hr dark incubated plants did not accumulate Proto IX in dark . Only when treated plants were exposed to light (75 Ilmole m-2 S- I), large quantity of Proto IX accumulated compared to none in control (Fig. 1). This showed that Proto IX accumulation in AF-Na-treated plants was induced by light. Maximum Proto IX accumul ated in treated

30~----------------------------------.

.... .J-...

~20 u.. 0> -Q.)

o E c C/)

~10 E .Ql a..

--0-- Proto IX control

Prot,,, IX Treated

.... 0- ..• Pchlide control

- - 6-- Pchlide Treated

O~~A~~~~~====r=dh=-~---O--,---~~ o 10 20 30 40 50

Light exposure (hr)

Fig. I - Accumulation of Proto IX and Pchl ide in control and AF-Na-treated plants upon li ght (75 !-lmole m·2

S· I) exposure. [Error bars represent SO and mi ssing error bars indi cate that they are smaller than the symbol)

GUPTA & TRIPATHY: OXIDATIV E STRESS IN CUCUMBER SEEDLI NGS 501

plants after 6 hr of light treatment. After 24 hI' Proto IX level decreased and remained almost constant up to 48 hr of light exposure. Pchlide content in AF-Na­sprayed plants after 14 hr dark period was approximately twice of that of controls. Exposure of plants to light caused Pchlide to get phototransformed to ChI ide and resulted in a decrease in the amount of Pchlide in both control and treated plants.

Dark disappearance of Proto IX was due to its conversion to PchLide

AF-Na-treated plants exposed to light (75 Ilmole m-2

S- I) for 1.5 hr (Fig. 2) accumulated 4 nmole of

Proto IX (g Fwtr l. This light-induced Proto IX in

treated plants was reduced to nearly control levels (ie., near zero) wi thin 2 hI' of transferring AF-Na­treated plants to dark. Re-exposure of plants to light after 24 hr dark treatment induced re-synthesis of Proto IX although total amounts of Proto IX synthesized in the second illumination was 25% less

5~---------------------------------.

-{}- Proto IX

4 T

...->-..

~ LL3 T Ol --Q)

0 E C2

X 0 -0 "-0..1

O~--~~-----.--~~.-----.-----~ o 1 0 20 30 40 50 ~~I --------------~I ~I --------------~I

light dark light

Treatment duration (hr)

Fig. 2 - Dark di sappearance of Proto IX in AF-Na-treated pl ants. [AF-Na-treated and 14 hI' dark-incubated plants were exposed to light (75 llmole m·2

5.1) for 3 hr. Subsequently, they were returned

to dark for 24 hr and then were re-exposed to light for further 24 hr. Leaves were harvested at the time periods shown in the figure and Proto lX content was estimated as described in ' Materials and Methods'. Error bars represent SO and missing error bars indicate that they are smaller than the symbol]

than that synthesized in the first illumination regime. The dark disappearance of Proto IX may be due to

enzymatic dark destruction of Proto IX in dark or its conversion to Pchlide. To probe the latter possibility, control and AF-Na-sprayed plants were exposed to light (75 Ilmole m-2

S-I) for 4 hI' and then transferred to dark for 6 hr (Fig. 3). Proto IX and Pchlide contents were estimated from leaf samples at different time points. Control and AF-Na-treated plants, incubated in dark for 14 hr, accumulated 12 and 26 nmole of Pchlide (g Fwtr l

, respectively. As shown in Fig. 1, they did not acc umulate Proto IX in dark. After exposure of these plants to light (75 Ilmole m-2

S- I)

for 4 hr, Pchlide content in control and treated samples fell to approximately 2 nmole (g Fwtr l due to its phototransformation to Chlide. During thi s light treatment period Proto IX content in treated plants rose from 0 to 14 nmole (g Fwtr l

. After subsequent transfer of plants to 6 hr dark, there was a sharp fall in Proto IX content from 14 nmole (g Fwtr l to zero. However, Pchlide content in treated plants increased to 22.5 nmole (g Fwtr l

, and that of control increased

30.--------------------------------,

..­-25

~ 20 LL Ol --Q)

015 E C

en ~ 10 E Ol a::

5

-0- Proto IX control

-_.+--- Proto IX treated

--··0·-- Pchlide control

---.!\--- Pchlide treated

T 1

T 1

O~--~.---~----_.----~----~--~ o 2 4 6 8 10 r-- LIGHT --I I DARK I

Treatment period (hr)

Fig. 3 - Over accumul ation of Pchlide in dark in AF-Na-treated plants was due to conversion of Proto IX to Pchlide. [AF- la­treated and 14 hr dark- incubated plants were exposed to light (75 llmole m-2

S-I) for 4 hr after which they were returned to dark for 6 hr. Leaves were harvested at the time periods shown in the figure and Proto IX and Pchlide contents estimated as described in ' Materials and Methods'. Error bars represent SO]

502 INDI A J BIOCH EM BIOPHYS. VOL. 37, DECEMBER 2000

to 8 nmole (g Fwtr'. Thus about 14.5 (22.5-8) nmole (g Fwtr ' ex tra Pchlide accumulated in treated plants. This amount was approximately equal to the amount of Proto IX accumulated i.e. , 14 nmole (g Fwtr ' in AF-Na-sprayed plants exposed to light for 4 hr. Though, unambi guous conclusions about precursor­prod uct relationship cannot be drawn from thi s ex periment, the data suggests that Proto IX synthes ized during light treatment of AF-Na-treated plants was converted to Pchlide in dark. Thi s implies that in additi on to conversion of intra-plastidic Proto IX to Pchlide in dark, ex tra-plastidic Proto IX that mi grated out of the chloroplasts during light ex posure') could re-enter the chl oroplasts and get converted to Pchlide via Mg branc h of tetrapyrrole biosynthetic pathway.

MDA production MDA is a cytotox ic, perox idati on product of lipids.

A time course of MDA producti on revea led that there was a substantial increase in MDA production in AF­. a-treated plants after 48 hI' and 72 hr of li ght (75 ~lmo l e m-2 s- ') ex posure (Fig. 4). This suggests that membrane lipids were peroxidi sed by AF-Na-induced photodynamic reacti ons.

125.----------------------------------,

100

~ 50 o ~

25

-0- conlro'

- _ .• -._. Irealed

/ ; ,

! /

/

o+-------.--------,-------,------~ o 20 40 60 80

Light exposure (hr) Fig. 4 - MDA production in control and AF-Na-trcatcd pl ants upon li ght (75 pmole m-2

S- I) ex posu re. [MDA cOlllelll in treated plants was correctcd for decrease in fresh wcight. Error bars reprcsent SO and missing error bars indieatc thatthcy arc small er th an the symbol. ]

Dark-preincubation prior to light exposure enhances photodynal/lic dal/lage

When AF-Na-sprayed cucumber cotyledons were put overni ght (14 hr) in darkness before exposi ng them to light, the photodynamic damage was greater than when the AF-Na-sprayed plants were exposed to light (75 )..lmole m-2 s- t) immediately afte r spraying.

Fig. 5 shows differences in the activi ties of whole chai n (A), PSI! (B) and PSI (C) reactions of plants after AF-Na spray, without or with 14 hI' dark incubati on, prior to ex posure to light. The ex tent of photodynamic damage to the thylakoid membrane­linked function s i.e., whole chain , PSII and PSI activities in pl ants pre- incubated in dark was higher than those which were expos d to light immedi ately after AF-Na treatment. After 48 hr of li ght exposure, in the dark pre-incubated plants, damage to the whole chain react ion was 64% whereas damage to the same was 33% in plants immediately exposed to light. Similarly, after 48 hI' light ex posure in pre-incubated

80 ,-----------------------------~~

f whole chain T

60

40

-0- control '";"

20 L- - +--- exposed directly J::

preincubated in dark · · · ·0 '";"

J:: 0 U PSII 0) 80 E

T r-~=--~---~T~------i

~ 60 1.

ro ...... §- 40

-.... c:: o 20 ~ :::J

-0- controt

---+-- exposed directly

··· ·0 ·· · · preincubaled in dark

B

o O+-------_.--------._------_.------~

~ PSI C 0 250 ~'=:---_t_---__J,.T ___ --IT

1 1 _<12 200 o [150

100 1

___ +_ exposed directly T .1 T

-0- control ~

50+-__ " ' _' 0_"'_' ~p_re.in_cu_b_aled __ in_d_a_rk._------_.----~J.~~

o 20 40 60 80 Light exposure (hr)

Fig. 5 - Comparison bctwcen changes in whole ehain r A]. PS II LB] and PSI rC] reactions in thylakoid mcmbranes isolated from AF-Na-treated pl :lIl ts wi thout or with 14 hI' dark-pre-treatment prior to li ght (75 ~tmo l c m·2

S· I) exposu re. [Error bars represc nt SO]

GUPTA & TRIPATHY: OXIDATIVE STRESS IN CUCUMBER SEEDLINGS 503

Table 1- Erfeet o f Mg2+ on spill over o f excitation energy measured as increase in FJ FIII due to addition o f 3.6 mM MgCl z to thylakoid membranes isolated rrom 48 hr li ght-exposed control and AF-Na- treated plants.[± represents SD of 3 replicates]

Sample Fluorescence _Mg2+ +M g2+ % Increase of FJFIII transient

Control Fo 2.5±0. 11 2 .5±0.12 Fill 7.5±0.4 12.5±0.65 Fv 5.0±0.3 10.0±0.78 FJFIII 0 .66±0.004 0 .8±0.OO5 21.2

Treated Fo 3 .0±0.14 3.0±0.12 Fill 6.8±0.4 8.0±0.5

F" 3.8±0.3 5.0±0.4 F/FIII 0 .56±0.2 0.62±0.2 10.7

and non-pre-incubated plants, the PSII was inhibited by 55 %, 30% and PSI was impaired by 61 %, 30% respectively. In subsequent experiments AF-Na­sprayed plants were 14 hI' dark incubated before light exposure.

Effect of photodynamic dOll/age on grana stacking Cations are known to induce grana stacking and

regulate the excitation transfer between photosystems"o. Low sa lt medium leads to dissociation of grana stacks to thylakoids and there is a simultaneous decrease in PSII photochemistry and Fv. Addition of sa lt to low­salt-chloroplast-suspension (- Mg2+) reverses the process and induces reconstitution of grana which results in an increase in the exc itation transfer in favour of PSII and consequent increase in PSII activity and Fv. Fo levels were not affected by the addition of Mg2+ (Table I). In presence of Mg2+, the FlFm ratio increased by 2l.2% in control and by 10.7% in treated sa mples (Table 2). Tn the same vein, due to additi on of Mg"+ there was 25 .3% increase in PSII rates of control and 14.6% increase in PSII rates of treated samples (Table 2).

Discussion DPEs are a group of commercial herbicides that

kill plants via tetrapyrrole mediated photodynamic reactions. Unlike in ALA-treated plants where Pchlide

I I . . '; 8-9 ' 1-'1 I d ' was t le p lotosensltlzer" ' .- _. , t le pre omlnant tetrapyrrole that accumulated in response to DPE treatment was Proto IX. Light was essential for DPE ac ti on. Shading part of a leaf maintained the ChI content of that portion , whereas the li ght exposed portion was bleac hed. This demonstrated that AF-Na­induced de-pi gmentation was dependent upon light.

Damage to plant tissues by ox idative stress was due to accumulat ion of the photosensi tizer Proto IX. In treated samp les, Proto IX accumul ated to its

Table 2 - Mg2+-induced enhancement of PSII act ivi ty of thylakoid membranes isolated from control and AF-Na-treated

plants exposed to light (75 ~molc m·2 S·I) for 48 hr. [± represents

SD of 3 replicates]

Sample PSII activity % increase in (~mo l e O2 mg Chr l hr- I

) PSII activity _Mg2+ +M g2+

Control 59.0±3.29 73.9±4.85 25 .3 Treated 33 .2±2.65 38.0±2.86 14.6

maximum level after 6 hr of exposure to light (Fig. 1). Proto IX concentration dec lined upon further illumination (24-48 hr). Decline of Proto IX was probably due to the reduced ability of treated pl ant tissue to synthesize Proto IX and photo destruction of the photosensitizer. AF-Na-treated plants accumulated twice the amount of Pch lide as compared to control in darkness suggesting that treatment of AF-Na stimulated the synthesis of tetrapyrroles. Subsequent ly, after 8 hr of illumination, the Pchlide content in both control and treated leaves declined to the same level, due to the photo transformation of Pchlide to Ch!ide.

The exact mechanism of li ght-induced accumu­lation of Proto IX is not known and could not be ex plained only on the bas is of increased ALA biosynthesi s in l ight2~,25 . Plast idic protox was shown to be sensitive to DPEs whereas the plasma membrane bound Protogen IX ox idi ser is insensitive to DPEs' 3,25.26. Incubation of intact plastids with ALA and AF-Na in light caused extraplastidic migration of Protogen IX 15. It was shown that out of Proto IX that accumulated in the cotyledons of AF-Na-treated and 1 hr-light-expos d plants, 22% was located within the chloroplasts and 78% was outside chloroplasts' S. This demonstrated that most of Protogen IX that was synthes ized in the chloroplast mi grated out of the plastid and thi s confirmed the hypothesis regardi ng mi grati on of Protogen IX to extraplas tidic locations.

504 INDI AN J BIOC HEM BIO PH YS. VOL. 37, D ECEM BER 2000

Proto IX content in treated plants increased in light but almost disappeared within 2 hI' of transferring the plants to darkness (Fig. 2). Thi s dark di sappearance could be either due to e nzymatic degradation of Proto IX in dark o r due to re-entry o f Proto IX into the pl astid and its subsequent conversion by Mg­tetrapyrrole bi osynthetic pathway to Pchlide. The convers ion of light-accumul ated Proto IX to Pchlide in da rk is a di stinct poss ibility (Fig. 3). Migration of Proto IX out of the plastid and its re-entry into the plastid may be constantly occurring both in light and dark. However, the phe nomenon of re-entry o f ex tra­p l::t stidic Proto IX to plastidic Mg-tetrapyrrolic bi osynthetic pathway was not detected in light because the ex tra-pl as tidic Proto IX that re-entered chl oroplasts and was converted to Pchlide was instantly phototransformed to ChI ide.

Perox idation of me mbrane lipids has been found to be one of the phytotox ic consequences of herbic ides27 . L ipid perox idat ion is a complex process where oxygen reacts direc tly with polyunsaturated fatty ac ids of the cellul ar me mbranes to form free radical intermedi ates and produces semi stable peroxides28. MDA production is cons idered as an index of lipid perox idati on. ALA-induced ox idati ve stress caused lipid peroxidation whic h resulted in accumulati on of inc reased MDA in leaf-ti ssues (Fig. 4 ). Membrane lipid peroxidation resulted in damage to pl ant ti ssues.

Dark-incubation of AF-Na sprayed plants be fore their exposure to light, enhanced oxidati ve stress and res ulted in the loss of thylakoid me mbrane linked­photosynthetic functions of PSI, psn and whole chain e lectron transport reactions (Fig. 5) . Dark-incubation mi ght be needed fo r di ffus ion of AF-Na to the s ite of ac tion and for priming the plants for accumulation of excess photosensitizer. Dark pre- incubation also enhanced the ALA-induced photodynamic damage6

.

Addition of Mg2+ to thylakoid membranes suspended in low salt medium caused stacking of the thylako id s. This resulted in exc itation transfer in favo ur of PSII and could be monitored by increase in FJFm rati020

. In treated samples compared to control , the increase in F JFm ratio due to addition of M g2+, dec lined (Table 1). Increase in PSII activity due to cat ion-induced g rana stacking a lso declined in lhylakoid membranes isolated fro m treated pl ants (Table 2). T hi s suggests that in AF-Na-trea ted pl ants thy lakoids had reduced ability to stack probably owi ng to partial damage to the light-harvesting prote in complex II .

Results shown above demonstrate that DPE herbicide acifluorfe n induce overaccumulation of Proto IX in light and not in dark. The herbic ide bl oc ks plastidic protox that results in accumulati on and subsequent transport of its substrate protoporphyrinogen IX (Protogen IX) to ex tra-plastidic location. M igrated Protogen IX is ox idi zed to Proto IX by the extra­plastidic plasma membrane-bound and herbic ide­insensiti ve Protogen IX ox idi ser. T he extra-plastidic Proto IX migrates back to the pl astid and is conven ed to Mg-tetrapyrroles. As Proto IX biosynthesis is up­regulated by light in herbicide-treated pl an ts, the Proto IX that rema in s unutili sed in the plastids as we ll as that which fail s to re-enter the plastid act as photosensitizer and damage the ce ll ular membranes. Da mage to the plas tidic membranes results in damage to PSI, PSII and whole chain reac tions and reduced stacking of thylakoids to form grana.

Acknowledgement Thi s work was supported by grants fro m the

Department of Atomic Energy (98/37/27/BRNS) to BCT.

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