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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 ¬ion 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.