MATERIALS & METHODS

MATERIALS AND METHODS

Phnt material

Seedlings of gunyule (Parthenium argentatum Gray cv. 'Cal-l', 'USS2X' and

'1 1591') were obtained from National Botanical Rescarch Institute (NBRI),

Lucknow. Guayule cultivars were grown in 30 cm pots under natural (12 h)

photopcriod in the University botanical garden. The average maximum incident

photosynthetic photon flux ana density (PPFD, 400-700 nm) available at the top of

the canopy was about 1600 pE m" s-' on a clear day. Daily average maximum and

minimum air tcmpcrahucs during the growth were 33 O C and 24 O C nspectively. The

plants were well watered and periodically fertilized with Hoagland nutrient solution.

Young and fully expanded leaves from three-year-old plants were used for all the

experiments in this investigation.

M a w Ficus elastica Roxb. plants were purchased from the Pondcherry

Agro &nice and Industries Corporation Lld (PASIC), Pondichcny and grown in

the natural climatic conditions in the University botanical garden. Ibt latex was

collected in tubes on ice from the apical portions of the plant by malring a cut using a

razor blade. Latex was later stored on ice until used.

Exporimonhi Dalgn

The axperimcntrl dc&gu of the present study consists of four anas of w o k

1. Rubbw fomulod and ~ b b a tmnahsc wtivity.

30

2. Isolation, purikation and characterization of Rubber Particle Proteins (RPP) from

guayule stem bark tissues.

3. Comparison of guayule rubber particles with those isolated h m Ficus elmica.

4. Photosyn~is , carbohydrate metabolism and nitrogen metabolism, proli i and

antioxidant defensc mechanism.

The influence of different environmental variables like low night ternpermre,

light intensity and low water regimes on the following aspects have been determined:

!i+ Rubber formation and rubber hmsferase activity.

D Isolation, purification and characterization of rubber particle proteins and rubber

transfcrase.

>. lmmunodetection studies using RPP.

> Comparison of guayule and Ficus rubber particles.

Z Photosynthetic C a by guayule leaves, photosystun activities, enzymes relattd to

carbon assimilation and carbohydrates.

D Quantification of biomolecules of adaptive significance in leaves.

k Evaluation of the antioxidative system under different environmental stress

conditions.

Conbdkd-Environment OrovlRh Chrmkr Experiments

l'k inw plants wm subjected to different stresses -y in a

control led^^ growth (Labline, model 7 0 4 A - 2 S D W Illinois.

USA). Ths plants wsrr subjectad to low night tempaatun treatmmt (LNT) st 15 %

for 60 cycles (12 h daily; cach night h 1800 h to 0600 h; day tempuatw was

30 'C). Varying growth light intensity to the plants inside the growth cabinet was

provided by adjusting the required light intensity (from 450 pE m" 6' to 1500

pE m" s") using the artificial light source. Drought stress (DS) was imposed by

supplying d c t c d amount of water. All other growth parameters were as described

above.

Water status measurement

Meamnment of leaf water potentials were made psychrometrically on leaf

discs using a pressure chamber (Skye IRFhumenls, SKPM 1400, Powys, Wales, UK).

STUDIES ON RUBBER BlOSYN7YfESIS

Rubber content

Stem portions of guayule wen cut into pieces and dried in an oven at 70 OC

until constant weights wcn obtained. The dried tissue was ground in a mill through

40-mtsb scncn, the powder was thoroughly mixed, and samples were placed in

cellulose extraction thimbles (whaman) and cxtra&d with acetone for 16 h in a

Soxhlct app~nuus to remove resins. The e m t i o n thimbles were then dried and

further attrreted for 16 h in tbe Soxhlet apparatus. Hexane exhwts con- the

dissolved rubba mn prepad and wen made up to 25 ml in a volumetric flask.

A 2 ml Iliguot w tdlten into 8 cuvcttc rad 6 ml of acidified ethanol was added as a

prsdpi-~gant. ~bc~wcpe@Wanda l lowedmshubdforZOmin .Tbe

percent bansmittam of the samples was measured at 750 nm (Naqvi et ul., 1984).

Transmittance values we& compared with a standard curve pnparad with pure

aatural rubber.

Isolation of washed rubber particles (WRPs)

Rubber particles were isolated from 40 g of guayule stem bark following the

method of Cornish and Backhaus (1990) with slight modifications. Guayule stembmrk

(80 g), peeled from green stems was homogenized in 400 ml i m l d extraction

buffer containing 100 mM Tris-HCI (pH 7.5); 50 mM KF, 1% ascorbic acid; 5 mM

MgSQ; 5 mM 2-mtl~~ptoetbanol, 0.1 mM phenylmethylsulphonyl fluoride, 17.5 p1

Antifom A and 30 g PVP-40. The homogenate was filtered through four layers of

cheese clotb and centrifuged at 5000 g and 4 "C for 8 min. The creamy layer of

unwashed rubber particles were scooped from the tubes and suspended in in 100 mM

Tris-HCI (pH 7.5) wntaining 2 mM MgSO4 and 5 mM DTT. The WRPs were

prepared by resuspending the unwashed particles, isolated as described above, in 170

ml ice-cold buffer and wac centrifuged at 2500 g and 4 O C for 8 min. Thc rubber

particles were scooped h m the tubes and suspended in ice-cold wash buEet. The

WRP suspensions were stored on ice and used on the same day.

Rubber Tmr).fmnse (RUT)

Extnrction

Stem portions mn rinsrd with distilled watu and homogcnised in a p

cooled bleadol with 100 mM Tris-HCI buffer @H 7.5) which contained. 2 mM

33

k i d 0 4 and 0.1 mM OSH. The homogenate was filtered through eight layers of

cheesecloth and centrifuged at 30,000 g for 45 min at 2 OC. The upa an at ant was

fractionated with solid ammonium sulphate and the protein that precipitated between

40 and 60 % saturation was collected by cen&ging at 25,000 g. The protein was

dissolved in a small volume of extraction buffer and desalted by passing through a

column of Scphadex G-25, precquilibrated with the extradon buffer. The

fractions wuc pooled and assayed for rubber transf-.

Enzyme assay

Rubber transferase (RUT) was assayed accordiig to Cornish and Backhaw

(1990) by measuring the incorporation of '"CPP (Ammham Pharmacia Biotech

Intl.. UK) into newly synthesized rubber molecule in prrsmce of an allylic

diphosphate and cofactor. MC. The reaction mixture (1 ml) contain& Tris-HC1

buffer (100 pmoles; pH 8.2), MgSO. (5 pmol), GSH (6 pmol), FPP (0.23 pmol),

0.8 pCi (I-"C)IPP (50 nmol, specific activity 55 mCi/mmol), washed rubber

particles (WRP. 50 mg) and the uuyme protein (100 pg). Different allylic

dipbosphate initiators like dimethyl ally1 diphosphatc (DMAPP), geranyl &phosphate

(GPP), h c s y l diphosphate (FPP) or g&yl gaanyl diphosphate (GGPP) at 20 pM

concentration wae used undcr di&rcnt urperimeneal conditions. Incubations wac

carried out for 1 h ot 30 OC and termiaated by the addition of 0.5 ml of 0.2 M EDTA.

Thccontatsiatberartiontubsswaedriedinastnamofairat7OOC. Thtrubbcr

N m s w m s @ e d & ~ r s ~ b c d b y ~ ~ 1 1 a a d & n a d i c t ( 1 9 8 4 ) .

'~hc colprlrte - 1 d in 2 ml of I % TCA io t01-. ~ c h t i h t rmf

34

addad to give a total volumc of 6 ml and the solutions were counted for radioactivity

in a scintillation counter. Protein concentrations wm dctmnined by the mcthod of

Bradford (1976) using BSA as the standard protein. Thc effect of EDTA and the

recovery of "c-incorporation into the WRPs were also detcnnined.

pH optima

RUT was measured by incubating the puritied sample in different buffers,

having different pH ranges, such as 0.1 M each of Mops @H 6.75 - 7.25), Hcpes (PH

7.3 - 7.75) and tris-HC1 @H 8.0 - 8.5) which wen adjusted to the desired pH to find

out the pH optima The RUT assay was carried out as described above.

SDS-PAGE of rubber particles

Analytical PAGE with SDS was performed using the method of Laemmli

(1970). Rubber particle samples were solubilized in 2X-SDS sample buffer

containing 63 mM Tris-HCl (pH 6.8), 5 % (wlv) SDS, 1 mM PMSF, 2 % (wlv) 2-

m n c a p t o e ~ l , 10 % glycerol and 0.01 % (wlv) bromophenol blue and heated at

70 OC for 3 min Aliquots of the dcnatud WRP samples in SDS sample bu&r

wue then resolved by SDS-PAGE. The resolving gel was made up of 12.5 % T (total

acrylamidc concentration and 2.6% cross l i i c r using methylene-bis(acrylamide; %

CBIS). The apparent molecular mass of proteins wen estimated by c o m e n with

mobility of stdml proteins (Banealo~ Genie Ltd, Bangalon, India). A I k

e l u m m the gels wen stlriwd with Coomassie Brilliant Blue following the

W&rd . protocol (Simbmk et d. 1989).

IMMUNOLOGICAL ANALYSIS

Propantion of guryule rubber particle soluble antigens

Rubber particles were isolated and purified as described earlier. The

supernatant containing soluble proteins wen denoted as "Crude antigen" and used for

further d y s i s .

Antiserum to guayule RPP in nbblt

lmmunizstion : 0.5 mg of guayule rubber particle antigens were mixed with

an quai volume of Freund's complete adjuvant to make a fine water-in-oil emulsion.

The emulsion was then administered subcutantously to albino rabbits (= 1 kg body

weight). The same amount of antigen in Freund's complete adjuvant was

administered as a booster dose after 3 weeks following primary immunization.

Collection and storege of Anti-sere : The blood was collected 10 days

&r the booster dose was given. Bleeding was done from the marginal ear vein of

the rabbit. For control serum, blood was collected from an unimmunizzd rabbit

maintained for the purpose. The blood was allowed to clot at room tunpcmtm for 1

h. The clot was then separated h m the walls of the tube using a sterile glass rod.

The clot was then left for aevaal hours at 4 T to retract to half its original volume.

The antiserum obtained was transferred to a fnsh tube and was mtrihrgad at 8000

p m for 5-10 min at room tanpaaane to remove any debris. The clot was discad&

a n d a l l ~ o f t & r n t i s e n r m w a s s t o d a t - 2 0 ~ i n a o u n d i l 1 1 t a d f o r m f o r ~

use. The antibody titrc in.& antisera to the wmspond'hg antigen was found out by

ELISA.

Detection of rubber particle mtlgen~pecllc antibodies in polyclonal

antism raised against guayule rubber particle antigens by EUSA

Reagents

9 Purified 50 kDa rubber particle protein fktion - I pg 1 100 pl

9 Test suum-polyclonal antiserum raised against rubber particle antigens in

albino rabbit.

9 0.05 M carbonate-bicarbonate buffer (pH 9.6)

9 0.01 M phosphate buffered saline (PBS) @H 7.4)

> 0.05 % PBS Tween

)z 1% BSA in PBS-T-Blocking buffer

9 02% BSA in PBS-t-Antibody diluting buffer

9 Goat anti-rabbit IgG-HMO

b Orchophenylenc diamine (OPD)

9 Cieate phosphate buffer (pH 5.0)

Urea&&

9 3 M %SO4

Bulrorsu08dCOIEUSA

~~ bu' 1M @H 9.6) [Coating b g e r stock sdntioa]

Solution A : 1.0 M sodium bioubonate

8.4 g.of NaHCa was d i i l v e d in 100 ml of distilled water

Solution B : 1.0 M sodium carbonate

10.6 g of Na2CO3 was dissolved in 100 ml of distilled water

40 ml of solution A was mixed with IS ml of solution B to give a stock solution of

1M carbonate-bicarbonate buffer (pH 9.6).

Blocking Buffer : 1 % B!L4 in 0.05% PBS-T

PBS (lM,pH 7.4) - 1 ml

Tween-20 - loop1

BS A - lg(O.l%)

Made up to 100 ml with distilled water

Antibody diluting buffer - 0.2% BSA in 0.05% PBS-T

PBS (IM,pH 7.4) - I ml

Tween 20 - loop1

BSA - 0.2 g

Msde up to 100 ml with distilled wtcr

Wash B e u s

PBS - 0.1 M,pH 7.4

PBS (1 M, pH 7.4) - I0 ml

Mdeuptol lifnwithdistilledwater

PBS-been

PBS (0.01 M, pH 7.4) - 1 L

Method

1. Coating of antigen was effected by incubating 100 p1 of antigen at 1 pg/100 p1

wnctntration in carbonate-bicarbonate buffer (pH 9.6) in the wells of a

microtitre plate (Tarsons) overnight in a humid chamber.

2. The antigen-coated plates were blocked with 1% BSA for 4 h at mom

tempturc.

3. The wells were washed once with PBST and once with PBS.

4. 100 p1 of diluted test sera (diluted in 0.2% BSA in PBS-T) was added to the

wells and incubated for 4 h.

5. The wells wen washed thrice with PBST and thrice with PBS.

6. 100 p1 per well of goat anti-rabbit IgG HRPO at 1:1000 dilution was added to

each well and incubated for 1 h.

7. The platcs were washed thrice with PBS-T and thrice with PBS.

8. 100 p1 of substrate solution (OPD - 0.4 mg dl, citrate phosphate buffer, pH

5.0 and una-Hz& - 0.4 mg dl) was added to each well and incubated in

dark till wlour developed.

9. The reaction was stopped with 3 M HaSO4.

10. The absorbance was mad at 490 nm in the ELlSA Reads (hhdti~cope,

bbpt*m, Fi).

WESTERN BLOT OF RPP

Reegents

P Tnasfa buffer (39 mM glycine, 48 mM tris base, 0.037% SDS md 20%

mathanol)

9 Tween20

9 Nitroblue Tctnrzolium (NBT)

> BClP

9 A W i phosphatase bu& (100 mM NaCI, 5 mM MgC12 and lOOmM

Tris.CI, pH 9.5).

Method

1. The undestained slab gel was washed in distilled water.

2. Tk gel was transferred to the "transfer buffer'' for 15-30 min.

3. Pieces of Whatmann No. 3 filter paper and nitrocellulose transfer membmes

(Schleickr & SchwU, USA), cut to the size of the PAGE gel was immersed

in distilled wata and soaked in the transfer buffer (15-30 min).

4. A sandwich of Paspac pad, Whahnsna No.3 filter papa, auylamide gel and

nitrocallulo~e ahett (ac) was assanbled and the traosfer of peptides was

efktcd.

5. The 'nc' simt was removed aad air dried. Thc 'nc' membrane was then

~2-3 t imsrWithTBS~2Otachfor lOminwi thshs l ing .

6. Ths 'w' man- w mshed 2-3 times with TBS-Tween 20 each for 10

7. Ihe 'nc' was incubed with the primar antibody for 3 h and then washed 2-3

times with TBS d T w m 20 each for 10 min with shaking.

8. The 'nc' was then h h t c d with the 8cc0mhy antibody (Goat anti-rabbit

IgG) with TBS 3% milk powder for 3-4 h under shaking. It was later washed 2

times with TBS d Tween 20.

9. The washed 'ac' membrane was tmnsfcmd to a shallow tray and 0.1 ml of the

chromogenic substrate mixhue (BCIPMBT) and incubated at room

temperatun with gentle agitation.

10. Whm bands of d d i intensity (dense blue colour) were- obtained (-30 min),

the 'nc' was h m f d to a tray containing 200 pl of 0.5 M EDTA (pH 8.0)

and 50 ml PBS.

FICUS RUBBER PAR77CLES

Extraction of Ficus rubber particles

Latex was tapped from stems and petioles of one-yearald Ficus plants by

removing the apicel portion with an angled cut using a m r blade. Latex was

coUsaedintubsrmdrtondonicelmtiluscd.

T& latex wr, cenuihgd at 15,000 g for IS min at 4 'C. Thc pellet was

discarded md the supanrtua was r e s m in 2.5 mM tris-HCI buffex @H 8.0)

containiap 2.5 mM MgSO,; 5 mM KF; 0.1 mM PMSF and 12% glycerol. Th

~ i r P l u u ~ r t 2 5 O O g f w l O m i n u t c s a t 4 ~ . Thiscsatdfh~tioa

41

p m w war &uc twiw. Pftg the d ccntxXugati04 the -on of ru&x

particles that floated to the top (Buopnt pcr*ticIed) were collected and the

supematants was decanted. The sedimmted particles (Heavy particles) were also

collected.

Purification of Buoyant and Heavy Ficus Rubber Particles

The buoyant particles were resuspended in the sample buffer and cent&@.

At 2500 g for 10 min at 4 OC. This washing procedure was repeated twice. The

buoyant particles were collected and nsupaded in a Wush Buffw (100 mM tris-

HCI at pH 8.0 containing 2 mM MgSO' and 5 mM Dm. The exeact was

fractionated with solid ammonium sulphate and the protein that precipitated between

40 and 60%. W o n was collected by centrifuging at 10,000 g. The extract was

desalted on a Scphaduc G-25 column equilibrated with the extraction buffer (2.5 mM

tris-HC1, pH 8.0, containing 2.5 mM MgSOa 5 mM KF and 0.1 mM PMSF) and the

flow through was collcctad in one bulk. The column was then washed with the

equilibrating buffa until the washes mhibitcd less than 0.005 A at 280 nm. The

eluate was dialysed at 4 OC, with three changes of the dialysate. This dialysant was

used to determine the rubber particle p1otsh.s in the buoyant rubber particle fiactioa

Tbe h v y rubber puticle fraction was resuspGndcd in the cxhctiOn bu8Rz

(2.5 mM oLHCI, pH 8.0 containing 2.5 mM MgSOb 5 mM KF and 0.1 mM PMSF)

aad~csnhi f i rOsdrt25Wgfor lOmin~4 .C. lh i spPoctdun~reper ted~

and bsrny rubko @dm wwe fidonatd with solid ammoDium wrl*

42

desalted and dialyd as S s b e d earlier to get the piiied heavy rubber particle

fraction that was suspended in wash buffex ac described above and stored on ice until

uscd.

Rubber transfemse assay

Rubber bansferase was assayed in heavy and buoyant rubber particle M o n s

of Ficus according to the method of Cornish and Backhaus (1990) by following the

incorporation of "c-IPP into the newly synthesized rubber molecule in the presence

of an allylic pyrophosphatc and cofactor, M$ as described earlier for guayule rubba

particles.

SDS-PAGE analysis of Ficus rubber particles

Ficus rubber particles were electrophoresed on 12% discontinuous SDS-

polyacrylamide gel as alnady described for guayule rubber particles.

PurHlcrtion of Phosphoribosylpyrophosphate Synthetase (PRS) from

Ficus latex

Purification of PRS was done following the procedure of Gallois et al. (1997)

with slight modifications. The latex was centrifuged at 20,000 g for 15 min at 4 O C .

The supanatant was rntrifitged to separate the rubber particles from the cytosol. 'Thc

pellet, composed of lutoids and chromoplast-like organelles (Fw-Wysslins

particles), wu mmpadd in r M u containing 23 mM tris-HCl, pH 8.3 toll-

5 mM IvlgCh; 3 mM EDTA urd 0.02% sodium wide (NaN,) [ B e A]. lhis d

as tlla ths onde B q d volumc oftaturated -04 aoluti~a ww added slowly

to Ule crude PRS cxhct witb gemtle atking and then kept for 1h. Tbe precipitate was

collected by centdbptioo at 15,000 g for 10 min at 4 OC. The pellet was

nswpmded in 16 ml of Buffer A and then dc4tcd by passiog through a column of

s e p W G-25. About 5 g of blue Sepharose was praequilibrated by stiniog in

Buffa A for two hours. Eluation was &oe at 70 ml h-' with 25 mM KC1 pnpared in

the same buffer. All these operations were carried out at 4 OC.

Assay of Phosphoribosylpymphosphate Synthetase (PRS)

PRS activity was determined by eazymatic measMment of AMP as M b e d

by Gallois el al. (1996) with catain modifications. Tbe reaction mixture (1 ml)

contakd 25 mM HEPES - Tris (pH 7.5); 50 pl sample; 1 mM mM

P h o s p h o c n o 1 ~ (Sigma); 10 mM MgSO4; 120 mM KCI; 0.32 mM NADH and

0.12 mM ATP (Sigma). Thc AMP f d during incubation was mcesurad by

monitoring NADH doction at 320 urn afta adding 33.4 nkat each of pyruvatc

K i m (PK; ATP: 2 - 0 - p ~ o s p h o t t a n s f ~ EC 2.7.1.40, Sigma) and lactate

dchydrogenase (LDH; Llactatc: NAD oxidorcductse, EC 1.1.1.27, Sigma).

the rpgmhlc ahown in Fig. 1 (Rilrmaohndra Rddyt 1980). The apperatus coasirtad

44

Fig. 1: "C02 feeding appatatua for steady stab incorporation by leaves.

of 500 ml wide mouth (A) that antaid 10 ml of watcr to maintain the

f r c h w for Itaves. It in fitted with four-bolded Nbbg stoppa and insatcd through

the holm were a mcrvoir (B), r tkmomctef (C), thistle fuoncl 0) and a &livery

tube (E) with a stop cock (F2). The other end of the delimy tube was arded in 20%

(wh) potasail8111 hydroxide solution taken in a 250 ml Erlenmeyer flask (0) with a

side arm (H) for sctbg suction. Radiocarbon in the form of N ~ H ' ~ C O ~ (0.1 mCi

specific activity 47.0 mCi mmorl) was taken into a polythene cwette (I) amd lmached

to the end of the thistle funnel stem with an adhesive plestn. The leaves or twigs

from control and treated plants wue cut under watcr and were allowed to equilibrate

in light for 20 minutes. During the period of equilibration, suction was applied

through the side arm (H) of the Erlenmeyer flask and atmospheric air was allowed to

flow (500 d m i n ) through the assimilation chamber by opening the stop cocks FI and

F2. Aftcr the nquircd preequilibration, suction was disconnected and the stop cocks

FI and F2 mn c l o d .

"c@ was generated inside the chamber by allowing 3 ml of 3N HCI h m the

thistle funnel @) into the vial containing N~H'~c@ (T) and immediately the stop

cocL (F) was closed. The final concentnition of carbon dioxide inside the chamber

was 0.04%. 80% ethanol ( m l y b o i i was kept in the m i r (B) just btfore

mpuirsd. AAa aeIW psriodc of "c@ assimWon by the leavts, hot ethanol was

allowed to flow into tbe clumbsr by opming FI until the lea- wen amplately

~ M d a r b a t ~ L ~ h e m i d u d ~ ~ ~ i n t b e ~ o n ~ b e r w a s

mnowd'rad mppsd kto 20% (wk) KOH solution by applying suction throulgh the

side arm. The leaves vme removed from the assimilation chamber along with

ethanol. Tht amount of "c& incorporated by the leaves was dctamincd by

extracting the leaf tissue with 80,M and Wh (vlv) ethanol.

" ~ - i n c ~ ~ r a t i o n in the ethanol soluble organic compounds was determined

by trausfeming the contents separately into a scintillation vial and a dioxinc-based

scintillation cocktail (containing 0.5 % PPO, 0.001% POPOP, 6% naphthalene, 10%

ethanol and 2% ethelcne glycol) was added. The radioactivity meawments were

made using LKB Wallac 1209 RackBeta Liquid Scintillation Counter (LSC) after

malung proper background corrections and quenching corrections.

PHOTOCHEMICAL ACnVlnES

Isolation of chloroplasts

The leaves of both control and treated plants were washed with tap water

followed by a rinsing with deionized water. The material was blotted dry and the

leaves were chilled at 0 'C and held in a cold condition. The leaves were cut into

strips and bomogcnkd in a semi-from grin- medium that contained: 0.33 M

sorbitol, 10 mM N4P207, 5 mM MgCL2, 1% polyvinyl pymlidone. 0.5 mM

dithiothrritol and 2 mM sodium ancaate. The crude extract was quickly squeued

through two layers of cheesccloth and the filtrate was cen-ed at 2500 g for 5 min

to nm0w the dimcat that consistad of whole cells and cell debris. The gnen

supcmaturt was tbm cenhifUOed at 2500 g for 10 min. The pellet containing

chloroplasts war suspended in the cold incubation medium that contained: 0.33 M

sobitol, 2 mM EDTA, 1 mM MgCI2,l mM MnCI2 and 50 mM HEPES (pH 7.6). A

portion of this chloroplast prepamtion was layend on to a sucrose gradient

comprising 1.5, 1.0 and 0.75 M sucrose in 10 mM tricineKOH (pH 7.6) and

centrifuged at 2500 g for 15 min. The chloroplasts at the interface between 1.0 and

1.5 M sucrose were diluted with a suspension medium consisting of 0.33 M sorbitol,

50 mM HEPES (pH 7.6), 2 mM EDTA, 1 mM MgClr and 1 mM MnCb. This

suspension was centrifuged at 5000 g for 5 min to yield a pellet of intact purified

chloroplasts. The intactness of the purified chloroplasts used in the present study was

80 to 85 % accord'i to Lilley el d. (1975). The photochemical activities in isolated

chloroplasts were determined spectrophotometrically as described by Raghavendra

and Das (1 976).

Dlchlorophenol Indophenol reduction

Ihe photoreduction of DCPIP by isolated chloroplasts was m d

following the decrease in absorbance at 620 nm. The reaction mixture (3 ml)

contained: 0.05 M phosphate buffer, pH 7.5; 20 pM 26-dichlorophenol indophmol;

ImM MgCh; 20mM NaCl and chloroplasts (about 15 pg dl). The reaction mixture

was placed in I cm diameter cuvettes and illuminated laterally at 25 T with

incandement light souire (500 fiE ma s", 400-700 nm) for 3 min. The reaction was

terminated by tuning off the light. The rate of dye reduction was calcuiatad with the

help of a g m d d c u m pcpurd for DCPIP under identical conditions.

Ferrlcymidr nductlon

The photoreduction of ferricyanidc by isolated chloroplasts was measured

following the deueasc in absorbance at 420 nm. The d o n mi* (3 ml)

contained: 15 mM tris-HCI buBer, pH 7.8; 0.5 mM ferricyanide; ImM MgCI2; 20mM

NaCl and chloroplasts (about IS pg d'). The reaction was stopped by adding 0.3 ml

of 20°h (wlv) TCA and s'multancously turning off the source of illumination.

Ferricyanide reduction in the samples was calculated by following deueasc in

absorbance at 420 nm after ccntrifupg to remove precipitated proteins. A standard

curve for faricyanide was prrpand for calculations.

NADP reduction

The rates of NADP reduction was followed with the reaction mixture (3 ml)

having: 10 mM phosphate buffer, pH 7.8; 20mM NaCl; ImM MgC12; 0.5 mM NADP;

1.25 pM DCMU; 60 pM DCPIP; 2.5 mM ascorbate, 5 mM spinach ferredoxin and

chloroplasts (10 pg ml"). Soon after switching off the lights, 0.3 ml of 1N NaoH was

added to the d o n vessels to avoid the reoxidation of NADPH formed. NADP

reduction was calculated by the increase in absorbance at 340 nm using the extinction

coefficient of 632 x lo6 an2 MI.

PIGMENT COMPOSmON OF THE LEAVES

Total Chlorophyll

Tbe toorl chlorophyll mtan of tke leaves was estimatad acco* to Amon

(1949). Obc g m ~ of l e i s q d e a wu au into d pi- and rrmxmd with 80%

(v/v) acetone, with little sand ad a p i i h of calcium carbonate. The homogenate was

centrihpi at 3000 g for 10 min and the supanatant was made up to a known volume

with 80% acetone. The optical density of pen supematant was dctennined at 645

and 663 nm in a W-Visible Spectrophotometer 11 1 (@stronics, India) against 80%

acetone blank. All the procedures were carried out in dim light.

The total chlorophyll content was calculated using the following formula:

(20..2 x 4 5 ) + (8.02 x A663)

Total chlorophyll (mg g l fw) = ------- x V 1000 x w x a

Whm

A - Absorbance at specific wavelength (run)

W - Fresh weight of the sample (g)

V - Volume of the sample (ml)

a - Length of the light path in the cell (1 cm)

Total Carobnoids

The carotcnoid content in the leaf extracts was determined using the method

of Ikm (1969) by following the absorbance at 480,645 and 663 MI.

A - Absorbance of specific wavelength (nm)

V - Volumeoftheexhnct(ml)

W - Fresh weight of the sample (g)

a - Length of the light path in the cell (1 cm)

ENZYME COMPLEMENT OF THE LEAVES

Prepatstion of crude etuyme extrocta

Approximately 2 g of leaf tissue was homogenized in 50 mM tris-HC1 buffer,

pH 8.0 that contained. 30 mM Umemptoethanol; 5 mM D m , 5 mM MgCl*; 1 mM

EDTA and 1 % PVP-40 in a waring blendor for 2 min at full speed at 4 OC. The

homogenate was filtered through two layers of cheesecloth and an aliquot was set

aside for chlorophyll estimation (Arnon, 1949). The extracts were spun at 20,000 g

for 20 min in a refrigerated centrifuge. The following enzymes were assayed at

30 T.

RuBP Carboxylase (EC 4.1.1.39)

The enzyme was assayed as the amount of "C incorporated into acid stable

products (Lorima el ul., 1977) in a reaction mixture (3.0 ml) that contained. 50 mM

trie-HCl buffer, pH 8.0; 5 mM DTT, 10 mM MgC12; 10 mM N~H"CO, (0.3 mCi

mmol"); 0.5 mM RuBP and the cmyme extract. After pnincubation for 5 min, the

-on ms stopped with 1 ml of 4N HCI and measured for radioactivity.

Purifcation of the Ce&oxylating System

The crude enzyme was purified for esthting the Linetic chwtc&ies

( lbm&dm Reddy, 1980). All operations wen carried out at 0 T.

Exfmction : The leaf tissue (25 g) was homogenized at I11 speed with 3

volumes of 0.1 M tris-HC1 buffer, pH 7.6 that contained: 2 mM EDTA; 5 mM Dm, 5

mM magaesium acetate; 170 mM B-mercaptocthaml and 1.5 % (wlv) PVP-40. The

homogenate was passed through muslin cloth and the extract was centrifuged at

10,000 g for IS min in a refrigerated centrifuge.

DUE-CeIIuIose Adsorption : 25 g of DEAE-ceUulose was added to it and

the solution was s t i d wcll for 15 min. The solution was filtered off to remove the

cellulose and washed twice with the extraction medium used above.

Ammonium sulphate frectionafion : 40 g of solid (NHk SO4 was added to

the filtmte obtained above and stirred continuously for 10 min. The light precipitate

was rmroved by ccntdughg at 2500 g for 15 min and more solid (NHk SO, was

added to the mpcmtmt. Thc solution was stirred wcU and centrii%ged at 20,000 g

for 10 min. The v ip i t a t e was dissolved in 50 mM tris-HCI buffer, pH 7.8 that

contain& 2 mM EDTA and 1 mM DTT. 002684

Advatbn of RuSP cahmyi8se by Sephadex G-25 Tmatments:

quo t r of th above prepad011 ware prused through a column (10 by 1 cm) of

sq&adcx 0-25, equilibrated with 100 mM tris-HCI buffer, pH 8.0 that contained:

ImM Dm, 10 rnM NaHCO3 and 20 mM MgC12. The eluate was collected and stored

until use.

The purified enzymes were assayed with the reaction mixtures as already

described.

F~ctore-l,6-bi.ph~phatpre (EC 3.1.3.1 1 )

Fmtose-1,6-bisphosphatase was assayed according to Ziennan et al.

(1978).

Extraction of the enzyme

The leaf tissue (10 g) was homogenized with 100 mh4 his-HCI buffer, pH 7.8

that contained: 5 mM DTT; 10 mM MgC12; 1 mM EDTA; 5 mM magnesium acetate

and 1.5 % PVP-40. The homogenate was squeezed through four laym of

cheesecloth and then cenmfuged at 10,000 g for 10 min. 25 g of DEAE-cellulose was

added to it and the solution was stirred well for I5 min. The solution was filtered off

to remove the cellulose and ~ ~ e d twice with the extraction medium used above.

The protein was precipitated with 75% (wlv) solid (N* So4 and spun at 30,000 g

for 30 min. The precipitate was dissolved in 50 mM tris-HCI buffer, pH 7.8 that

containad: 2 mM EDTA and 1 mM Dm. The preparation wae applied to a column

of Sephadex G-25, equilibnted with 100 mM hs-HCI buffer, pH 8.0 that contab&

ImM Dm, 10 mM N a H m and 20 mM MgCh and 0.2 mM NADPH. The eluate

wss colloot#l and stored until use.

Enzyme esJey

The reaction mixtun containod: SO mM tris-HCI, pH 8.0; 10 mM MgC12; S

mM Dm, 1 mM EDTA; 0.5 mM NADP; 1 mM fiwtosel,6-bisphosphate, 10 units

each of glucose phosphate isomgese and glucose-6-phosphate dchydrogenase, and

the enzyme extract. The reaction (total volume 1 ml) was initiated with extract

containing 1-5 pg of chlorophyll.

Sucme Phosphate Synthare (EC 2.4.1.14)

Sucmse phosphate synthase (SPS) was assayed by the method of H u h

(1981).

Reagents

1. Exrmction bufler : The buffer contained: 100 mM HEPES; S mM magnesium

chloride, 1 mM ethylenediaminetetnacetic acid; 25 mM &mmaptoethol; 1

mM Phenyl methyl sulphonyl fluoride and 0.02 % Triton-X 100 at pH 7.4 .

2. Assq b@r for V, ucfivify : Thc buffer contained: SO mM HEPES; 15 mM

magnesium chloride, 4 mM 6uctose-6-phosphate, S mM UDP glucose and 20

mM glucosa-6-phosphate at pH 7.5.

3. Asmy b m r for V k activity : The bu&r contained: SO mM HEPES; IS mM

mgmaium chloride; S mM UDPO, 2 mM f i u c t o ~ ~ ; 10 mM

( I l d p h o s p W rad lOmM potassium dibydro~enphospbate at pH 7.5.

4. Ant- Rrogrnt (0.1 4 96) : 40 40 of conccntratcd sdphuric soid ma ddsd

~lOmlofdictilladwrtaradlrra7Omgofllnthro11~w~sdissolve&

5. Potapsium hydroxide (3099) : 30 gm of potassium hydroxide was dissolved in

l o o m l o f ~ c d w r t a .

Extmclion of the enzyme

The leaf material was hornogmated in 10 volumes of the extraction buffer in a

mortar and pestle. The homogenate was filtered through muslin cloth and the filtrate

was centrihged at 13,000 g for 10 minutes. The supematant was desalted on a

Sephadcx (3-25 column, equilibrated with the extraction b e without Triton-X 100.

The eluate was centifuged. The supernatant was stored for the assay.

Enzyme Assay

50 p1 of the enyme extract was added to 100 pl of the assay buffer and

Incubated at 25 "C for 20 minutes. Then the reaction was terminated by adding 100 fi

of 30 % potassium hydroxide. The tubes were placed on a boiling water bath for 10

minutes to destroy the un-reacted fiuctosed-phosphate. After cooling, 1.0 ml of

anthrone w e n t was added. The tubes me incubated at 40°C for 20 minutw on a

watu bath. The absorbance of the solution was read at 620 nm. For the 'control', the

reaction was terminated at '0' minute with 30 % potassium hydroxide. Thc above

reaction was CMied out with assay buffers for both Vm and Vt, activities. The

activity of sucroae pb- aynthasc was detumincd using the standmd cwe

o b t a i d with known concentration of sucrose ranging between 10 to 50 pg. Thc

activity of SPS vias apnssad as pmol mg chl" h". The protein content in the

Quyme txbrct vias detsrmined by the method of Bradford (1976).

54

Probin b u y

Total leaf protein content was estimated by the method of Bradford (1976 ).

Bractford reagent: 100 mg of Coomassie Brilliant Blue G-250 was dissolved in 50 ml

of ethanol and 100 m! of 85% phosphoric acid was added and the total volume was

made up to 1 L with distilled water.

0.1 ml of the leaf extract was made upto Iml volume with 0.1 M phosphate

buffer (pH 7.5). 5 ml of Bradford reagent to the tubes and mixed thoroughly. The

measurement of absorbance of the sample solution was taken in the

spectrophotometcr at wavelength of 595 nm against the reagent blank. The

concentration of the protein sample was determined by means of the analytical curve.

CARBOHYDRATE METABOLISM

Alcoholic Extraction

Leaves were collected from the control and stressed plants and dried in a Hot

air oven at 60°C and then powdered. 25 mg of the powdered sample was extracted in

10 ml of 80 % ethanol using a waterbath, at 80°C. The homogenate was centifuged at

600 g for 15 min. Thc supernatant was saved and made up to 20 ml with 80 %

ethanol. This alcoholic extract was used for the quantitative estimation of reducing

sugars. ma-reducing sugars and total sugars. The residue was saved for starch

-on.

Reagents

1. Anthrone Reagent : 200 mg of anthrone was dissolved in 100 ml of cold 95 %

concentrated sulphuric acid.

2. Dinitrosalicylatc reagent : 1 gm of 3,5dinitrosalicylate, 30 gm of sodium

potassium tartrate and 1.6 gm of sodium hydroxide were dissolved in water and

made up to 100 ml.

3. Perchloric acid (52 % ) : 52 ml of commercial perchloric acid (70 % ) was added

to 18 ml of distilled water .

4 Resorcinol reagent : 1 g resorcinol and 0.25 g thiourea added to 100 mi glacial

acetic acid.

Starch and Sucrose

Starch and sucrose contents in the leaf tissues were estimated according to

Ramachandra Reddy et 01. (1996). For starch content, eight leaf discs (1 cm diameter)

wen selected at random fmm the leaves and extracted 4 to 5 times with 80 % ethanol

for 15 min at 80 O C . The exhracts were combined and stored for estimating sucrose.

The ethanol-extracted leaf discs were suspended in I ml of 0.2 M KOH and boiled for

0.5 h. Thc tubes were cooled to room temperature, 0.2 ml of 1 M acetic acid was

added to each tube and reacted for 0.5 h at 55 'C to hydrolyze starch, the reaction was

stopped by rnaGng for 60 sec at 100 O C . The contents were cooled and brought to a

known volume (6 ml). Two-tenths ml aliquot of the extract was added to 0.3 ml of

distilled water and 1 ml of glucosbcnyme reagent (Sigma 115). The tuba wen

incubatad at 37 O C far 20 mio nnd the absorbance was rcad at 492 nm. For the

56

estimation of sucrose content, .the ethanol extracts, previously described, were used.

The extract (0.2 ml) was addcd to 0.3 ml of glucose reagent and incubatcd for 0.3 h at

37 O C . Invertwe (75 pl, Sigma 1 4753) was addcd and incubated for 0.5 h at 37 O C .

The absorbancc was read at 492 nm. This reading is proportional to the original plus

glucose liberated via invertase action on sucrose. To determine the sucrose content,

the assays were run simultaneously (lackmg invertase) for glucose content. Sucrose

concentration was determined by comparing the difference in the absorbance of the

two samples with that obtained from the sucrose standards.

NITROGEN METABOUSM

Foliar Nitrogen Content

The total nitrogen content of the leaves was estimated in dry leaf powders of

control and treated plants accordq to Kjeldahl method using the KJEL PLUS System

(Pelican, India).

The method involves thru stages :

1. Digestion

2. Distillation

3. Titration

Reegents

1. Hydrochloric acid (0.1 N) :0.82 ml of concentrated hydrochloric acid was

dded to 99.18 ml of distilled water.

2. Digestion Activator : 25 g of Potrmssium sulphate . 5 g of Copper sulphate .0.5

g of Selenium were mixed.

3. Sodium hydroxide (40 %) : 40 gm of Sodium hydroxide was dissolved in 100

ml of distilled water.

4. Mixed indicator : 30 mg of Bromocresol green and 20 mg of Methyl red were

dissolved in 40 ml of 90 % ethanol.

Digestion

Leaves were dried and powdered after removing the midribs. 500 mg of the

powder and 3 gm of digestion activator were weighed and added to the digestion tube

of the XJEL PLUS Digestion Block System. To this, 10 ml of concentrated sulphuric

acid was added. The tubes were loaded on to the KJEt PLUS Digestion Block

System and the kmperature set at 350 "C. The samples were digested for 1 hour.

Distillation

The digestion tube was placed inside the KlEL PLUS DISTIL-M chamber

through the alkali hose. The alkali hose at the back panel was immersed in to the

bottle containing 40 % NaOH solution and the volume of the alkali was fixed. The

receiver end of the hose was i m m d into a conical flask containing 20 ml of boric

acid and 2 to 3 drop of the mixed indicator. 30 ml of the alkali was added to the

digestion tuk. The distillation time was fixed at 6 min and the distillation process

started.

T!tr8tion

The solution collected in the conical flask was titrated against 0.1N

Hydrochloric acid. The titre value was noted. The percentage of Nitrogen was

calculated using the following formula :

(Titre Value) x (Normality of HCI) x (Nitrogen factor) %of E ------I----.----------- Nz Weight of the sample

whm Nitrogen factor = 1.401.

The Nitrogen content was expressed as pacentage of nitrogen per gram f.w.

Nitrate Roductarr (EC 1.6.6.3)

Sliced leaf material (300 mg) was taken into 50 ml Erlenmayer flask that

contained: 10 ml of infiltration medium with 25 mM potassium phosphate buffer (pH

7 5) and 10 mM K N 4 (Lin and Kao, 1980). The flask was evacuated at 6 mm Hg

for 3 scc. The flask was then w v d with a black cloth as the enzyme is

photosensitive (Klepper, 1976). After incubating at 30 O C for 30 min, an aliquot (1.5

ml) was taken from the flask to which 0.75 mi of 0.2% (wlv) sulphanilamide and 0.75

ml of 0.02% (wlv) N-(1-Naphthyl) ethylene diamine hydrochloride (NEDA) were

added. The mixnvc was kept in dark for 30 min and the absorbance was read at 540

Nn.

PROUNE METABOUSM .

Prolino

The extraction and csthation of proline was done according to Bates el 01.

(1973).

Extraction

The midribs of a leaf were removed and 500 mg of the leaf tissue was

weighed. It was homogcniscd with 10 ml of 3% sulphosalicylic acid in a mortar and

pestle. The homogenate was filtered through a Whatmann No. 2 filter paper. The

procedure was repeated with the residue and the filtrates were p l e d

Reagents

1. Aqueous Sulpho salicylic acid (3%) : 3 g of sulphosalicylic acid was dissolved

in I00 ml of distilled water.

2. Acid Ninhydrin : 1.25 g of Ninhydrin was dissolved in a warm mixture of 30 ml

of glacial acetic acid and 20 ml of 6M Phosphoric acid with agitation. The

reagent was stable for 24 hours when stored at 4 "C.

3. Staadard P r o l i : 5 mg of proline was dissolved in 10 ml of 0.1 N

Hydrochloric acid.

Estimetian

2.0 ml of the Rltmtc wns taken and 2.0 ml of acid ninhydrin and 2.0 ml of

glacial d c r i d mn added. T& tubas wcn incubated for 1 h at 100 O C on a

watduth. The hrbes were transferred to an iccbath to terminate the d o n . 4.0 ml

of toluene was added and mixed vigorously for 15 to 20 sec. The chromophore

containing toluene was aspirated from the aqueous phase. It was allowed to reach

room temperature and the absorbance measured at 575 nm. A reagent blank was

maintained. A standard curve was obtained using a known concentration of authentic

proline. Roline content was expnssed as mg of prolie per gram d.w.

Proline dohydrogenare (PDH; EC 1.5.1.2)

Enzyme prepamtion

200 mg of leaf tissue was homogenized in 10 ml of chilled 0.1 M potassium

phosphate buffer (pH 7.8) with pinch of washed fine sand. Homogenatts were

centrifuged for 20 min at 12,000 g at 4 "C and the supernatants were passed through a

Sephadex G-25 column. The eluants were used for enzyme assays.

PDH activity was assayed by following the reduction of NAD at 340 nm

(Miler and Stewatt, 1976). The reaction mixture (3 ml) contained: 0.15 M NazCa-

HCI b& (pH 10.3); 2.67 mM L-prolie; 0.01 M NAD and the enzyme extract. The

assay was initiated by the addition of NAD at 25 OC and the reaction was followed for

over 5 min at 340 nm. The activity was expressed as Units mg proteio".

ANllOXlDAUT ENIMWES

Su~~roxidr dkmutru (SOD, EC 1.16.1.1)

Th, activity of supaoxide dismutax was dacnnined by the method of

61

Beauchamp and Fridovich (1971) as modified by Dhindsa and Matowe (1981) by

following the photo-reduction of nitroblue tetrszolium.

Extraction of the enzyme

The leaf material was homogenized in a mortar and pestle with 5 volumes of

the exmction buffer that contained: 20 mM tris-HCl, pH 7.5; 5 mM MgCh and 10

mM NaCl. The homogenate was centrifuged at 6000 g for 90 sec. The pellet was

washed with extraction buffer and again centrifuged. The supernatants were pooled

and used for the enzyme assay.

Enzyme assay

The d o n mixture (1 ml) contained: 50 mM phosphate buffer (pH 7.8), 0.1

rnM EDTA, 13 mM methonine, 75 pM nitroblue tetrazolium (NTB), 2 pM

riboflavin and 100 p1 of the supernatant. Riboflavin was added asthe last

component and the reaction was initiated by placing the tubes under two 15-W

fluorescent Lamp. The reaction was terminated after 10 min by removing the

reaction tuba from the light source. Nonilluminted and illuminated reactions without

supernatant Jervad as calibration standards and the absorbance was measured at 560

nm. Ihe volume of th supcrnamnt corresponding to 50% inhibition of the reaction

was asrig& a value of 1 cnyw unit. The activity was expressed as number of units

per mg chlorophyll per minute.

Catalarr (CAT, EC 1.11.1.6)

Extraction of the enzyme

The leaf material was homogenized in a pre-cooled mortar and pestle with 5

volumes of the extraction buffer 50 mM phosphate buffer. The homogenate was

centrifuged at 8000 g for 20 sec at 4 O C . The pellet was stirred with cold phosphate

and allowed to stand with occasional stirring. The combined supmatanta were used

for the assay.

Enzyme assay

A modified method of Luck (1974) was used for assaying Catalasc. 50 pl of

the enzyme extract was added to 3 ml of hydrogen-peroxide phosphate bu8Ecr (pH

7.0). The time requid for the decrease in absorbance at 240 nm h m 0.45 to 0.40

was noted. Enzyme solution containing hydrogen peroxide-free phosphate buffer was

used as control. The activity was expressed as mmoles per mg chlorophyll per

minute.

Ascorbate poroxIdase (APX, EC 1.11.1.11)

Extmction of the enzyme

The leaf tissue was homogenized in a pre-cooled mortar and pestle with the

extraction medium that contained 1 mM phosphate buffer, pH 7.0; 1 mh4 m r b a t e ;

20% sorbitol; 1 mM EDTA and 0.1% PMSF. The homogwzate was s q u d h u g h

four layas of chetsecloth and then centrifuged at 20,000 g for 30 min. The

supematmtts wsre used for the enzyme assay.

Enzyme essay

ASC&WC peroxidasc wu -dly assayed following a

decrease in absohm at 265 nm (Asada, 1994). The assay mixme contain& 0.25

M ~scorbate and 1 mM H l q in 50 mM phosphate buffer (pH 7.0) with 37.5 pl of

enzyme extract. The hydrogen peroxide-dependent oxidation of the ascorbate was

followed by derrease in the absorbance at 290 nm. Comctions were made for low

rates of ascorbate disappeanncc due to nonenzymatic and Hz@-independent

oxidation. Rate of 8 9 ~ 0 - disappearance was determined during hear phase of the

reaction. The activity was expressed as pmoles per mg chlorophyll per minute.

Peroxidare (POD, EC 1 .I1 .I .7)

Extrection of the enzyme

1 g of fresh leaf tissue was homogenized with 3 ml of 0.1 M phosphate buffer,

pH 7.0 by grinding in a precooled mortar and pestle. The homogenate was

centrifuged at 18, 000 g for 15 min and the supanatant was used as the enzyme

source.

Enzyme assay

Paoxidase activity was determined specifically with guaiacol at 436 nm

following the mahod of Puttu (1974). 1 ml of the mymc extract was added to the

reaction mixtun 0.05 mi piawl solution end 0.03 ml hydrogen paoxide

solution in 3 ml phosphate buffer (pH 7.0). The solution was mixed well and waited

until tha ab&antx at 436 nm read 0.05 in the s p c c t r ~ p h o m . T i e was then

notad for the absorbance to incmsc by 0.1. The enzyme activity was calculated using

the &notion coefficient of guaiacol dehydrogenation product under the conditions

specified. The enzyme activity was measured as p o l e s per mg chlorophyll per

minute.

Glutathione reductase (GR, EC 1.6.4.2)

Extraction of the enzyme

The leaf tissue was homogenized in a pre-cooled mortar and pestle with the

extraction mixture that contained: 50 mM phosphate buffer, pH 7.0; 1 mh4 EDTA;

0.05 % Triton X-100; 2% P V P 4 and 1 rnM ascorbic acid. The homogenate was

centrifuged at 17,000 g for 20 min and the supernatant was used as the enzyme assay.

Enzyme essay

Glutathione reductase activity was determined by following the rate of

NADPH oxidation at 340 run (Foyer and Halliwell, 1976). 1 ml of assay mixture

contained: 0.1 M tris b d e r (pH 7.8); 2 mM EDTA; 50 pM NADPH; 0.5 mM GSSG

and 20 p1 of the extract. The reaction was initiated by the addition of NADPH at 25

"C and the naction was followed for over 5 min at 340 nm. Chlorophyll content was

detenninad spectrophotometrically according to the method of Amon (1949). The

enzyme activity was cxpmscd as p o l e s per mg chlorophyll per hour.

Sbtl8tlwl Analysis .

All thc data in thc present study are expressed as Mean 5 SE. The data was

analyzed using Students 't' test to see the differences between means of each group.

The 'p' value of less than 0.05 was considered as significant.