MATERIALS AND METHODS

PLANTS
G r e e n gram ("@"a radiata L. Wilczek cv. KM-21,a nitrogen fixing grain legume was procured from Pondicherry Agro Service Industries Corporation (PASIC), Pondicherry. Viable seeds were selected for uniform colour, slze and weight and used In the experiments. Plants were grown In earthenware pots 125 x 25 cm) or polybags filled with a mlxture of sand, red soil and farmyard manure ( 2 : l : l vlv). Ten seeds were sown at equal dtstances at a depth of 2 cm in each pot. The posttion of each pot was randomised at four days intervals to minimize spatial effects and were maintained under natural photoperiod~c conditions (day temperature: maximum 38 relative humidity 6 0

* 2'C;

night temperature: minimum 18 2'C;

* 5%;

maximum irradiance (PAR) 1,400 pmol m 2 s'; photoperiod

12 to 1 4 h.

UV-B RADIATION SET-UP

Supplementary UV-B radiation was provtded by two UV-B lamps (Philips TL 20W/12 Sunlamps, The Netherlands) which were suspended horizontally over the plant rows. UV-B dose was maintained by adjusting the dtstance (1ml between plant canopy and the lamps (Plate 1). The lamps were wrapped with cellulose d~acetate filters 10.076 mm) to filter UV-C radiation 1 < 290 nm). The filters were changed periodically to maintain uniform

1.Experimental set-up for growlng plants under normal ambience and under su~plernentalUV-B rad~ation.(A] Full vtew of the w~re-meshcage designed to exclude rodents and Insects (B) Intertor vlew showing he~ght An adlustable lamps hanging over the Plants. (C) Plants under randomised arrangement (Control).

PLATE

optical properties. UV-B exposure was glven for t w o hours dally from 10 to 11 and 1 5 to

16 h starting from
UV-B dose IUV-B,,)

sthday

after sowing (5 DAS). Plants received a biologically effective

of 12.2 kJ m 2d ' equivalent to an anticipated 20% ozone depletion at

pondicherry (12.Z0NIndia). The control plants, grown under natural solar radiation, received UV-B,,

10 kJ m ' d

'

(Caldwell 1971 ) .

TRIAD TREATMENT
Triadimefon, the trlazole fungicide under the brand name Bayleton was procured from the market. Preliminary tr~alsconfirmed that 2 0 mg L ' of TRIAD suppl~ed through seed-soaking w a s Ideal ~n amellorating UV-B stress. A booster dose of TRIAD as soil-drench on 15130 DAS, w h ~ c h was found to enhance the effects was also incorporated (Rajendiran and Ramanujam 2000a).

EXPERIMENTAL DESIGN (Table 4)

Plants were divlded lnto t w o lots

one for growing under normal ambience

(control) and another for receiving supplementary UV-B ( + U V - 6 ) . Each lot was agaln subd~v~ded t w o groups, where, one was treated w ~ t h lnto tr~adlmefon(+TRIAD) while the other was treated w ~ t h water to serve as reference. Table 4 Exper~mental des~gn Treatments 1 : Control (normal ambience) Tr~ad~rnefon appl~catlon(20 mg L ' ) 1 : Seed soak~ng(overn~ght) 2
: Booster : lrrigat~on wlth 200 ml pot ' 115 I 3 0 DASi

2
3
4

: UV-B (supplementary1 : UV-B

Trladlmefon 120 mg L ' )

: Triadimefon (20mg L ' i only

MORPHOMETRlC MEASUREMENTS
Ten plants from each treatment were carefully uprooted on 15, 30, 4 5 and 6 0 DAS and thelr axial growth (root and shoot length and plant height) and fresh biomass were measured. They were then d r ~ e d an oven at 8 0 " for 48 h and weighed agaln for in ~ mass measurements. Alongs~de, morpholog~caland developmental abnormalltles,

if any, caused by UV-B radiation were also recorded.

Assessment of growth
Growth and yield attributes of green gram at appropriate stages were calculated following standard methods as follows:

Total leaf area

Ten plants were selected at random from each of the treatments. The leaf area (the leaflets from all the nodes) was determined at various stages using Area meter (Analytical Development Corporation, UK, model AM100). The total leaf area per plant was obtained
by summing up the area of the leaves from all the nodes of the plant.

Leaf area index (LAI) (Williams 1946)

LA1 = Leaf area of the plant Icrn'l Ground area occup~ad (crn'l

Specific leaf weight (SLW) (Pearce et at. 1968)

SLW =
Leaf dry weight 1gl Leaf area lrn'l

Relative growth rate (RGR) (Williams 1946)

RGR =
h, i

w ;
; t

Log. t,

w,

where, W, and W, are dry mass of whole plants at t, and t, (time in days) respectively.

ShootlRoot ratio (Racey et at. 1983)

SIR ratlo =
Shoot welght lgl Root weight Ig)

Net productivity (Jain et at. 1999)

The net productivity was calculated by subtracting the values of biomass of the Specific stage from that of subsequent stage. Total sum of positive values of each Plant-part gives the total net productivity of the plant (TNPI during the growth season.

ASSESSMENT OF FLOWERING AND YIELD

Onset of flowering began'from the day on which the first plant formed floral buds. The number of floral budslflowers/fruits in each plant and also the number of plants per treatment bearing flowers was recorded on every alternate day. The day on which 50% of the plants flowered IF,) was calculated from the pooled data.

Fruit harvest
Mature fruits were harvested periodically from each plant and the length and weight of the pod, number of seeds per pod and weight of 100 seed lot were recorded.

Harvest index (Mohan et a/. 1992)

Harvest index was calculated by the following formula:
Harvest ~ndex= Yield of the plant lgl Biomass of the plant Ig)

loo

Potential yield (Mark and Tevini 1997)

Potential yield = No. of buds

+ No. of flowers + No. of fruits plant-'

Shelling percentage (Francis et a/. 1978)

Shelling percentage = Seed wt. plant' Fruit w t . plant'

EPIDERMAL STUDIES Stornatal characteristics

The size and number of epidermal cells, stomata and trichomes were recorded using a calibrated light microscope. Stomatal frequency was determined by examining the leaf impressions on polystyrene plastic film. The plastic medium ( 1 g of polystyrene in 100 ml of xylol) was applied on the leaf uniformly as a thin layer. After drying, the material was carefully removed and observed under magnification. Stomatal counts were made randomly from ten regions on the adaxial/abaxial surfaces. Since the stomatal frequencies vary according to cell size, Salisbury (1928) recommended the 'stomatal index' (SII which relates the number of stomata per unit leaf area to the number of epidermal cells in the same area.

Stornatal index (SI)

-S S+E

100

where, S = number of stomata per unit leaf area, E = number of epidermal cells per unit leaf area.

Leaf thickness
Cuticle, mesophyll and leaf thickness were measured using stage and ocular micrometers end the values were expressed in pm.

Mesophyll volume
Mesophyll thickness (mm) was multiplied by 100 to calculate the mesophyll volume in cm3 dm-' of leaf area as recommended by Patterson e t a / . (1978).

SCANNING ELECTRON MICROSCOPY (SEM)

Leaf bits were first dehydrated in graded series of ethanol (70% to 100%) and 3 mmz pieces were cut from the middle of lamina adjacent to the midvein. Each bit was mounted on an aluminium stub and then coated with gold (20A) in a Hitachi HUS-5GB vacuum coater and viewed and photographed with Hitachi 5-450 Scanning Electron Microscope operated at 10 kV.

ANALYSIS OF CUTICULAR WAX (Kohlen and Gulz 1976)

Fresh leaf discs 10.1 g) were punched out with a cork-borer (1 cm dia.) and immediately dipped in 10 ml of redistilled chloroform for 10 seconds. The wax extract was evaporated t o dryness and was redissolved in 2 ml of redistilled chloroform and read at 275 nm in a dual wavelength spectrophotometer (Shimadzu, Japan, Model 160). The amount of total cuticular wax was determined w i t h reference to a pure paraffin wax standard graph (A 0.1 OD at 275 nm is equivalent to 6.0 pg of wax) and expressed in Pg g" fw.

Thin Layer Chromatography (TLC)

The wax extract was further separated into single wax classes by TLC. Aliquots 1100 PI) of wax extract was spotted on activated silica gel (Merck Kieselgel-G) plates and

developed in benzene as the solvent. The lipids were stained with iodine vapour and the ~f values were calculated and compared with standard Rf values reported by Steinmuller and Tevini ( 1 9821.

Infrared (IR) spectrometry

Epicuticular waxes were analysed in lnfrared (IR) spectrophotometer (FT-IR Vector 22, Bruker, Japan) using KBr pellets. One mg of wax sample and 50 mg of KBr were ground together, dried to remove moisture and pressed at room temperature under high pressure into a small disc. The functional groups were identified from a standard chart.

Gas chromatography

- Mass spectrometry

(GC-MS)

The wax classes were further separated in a gas chromatograph GC-MS QP5000 Shimadzu (Japan) instrument operated with the following specifications: column length x inner diameter - 30m x 0.25mm; film thickness - 0.25 pm; binding material - bonded, poly (100%) dimethyl silioxane; temperatures of column injector

260C (from 70C @ 10'C min')

- 250C and interface - 300".

The sample in chloroform 0.2 p1 was injected

w~th Helium as carrier gas at a flow rate of 1.4 ml min'. The peaks were identified with reference to the retention times of the peaks of known wax analytical data IWiley 139 Lib).

BIOCHEMICAL ANALYSIS
Biochemical estimations were made from the fully expanded trifoliate leaves of the second node from the top of the shoot on the 15, 3 0 and 45 DAS.

Estimation of photosynthetic pigments Chlorophylls (Moran and Porath 1980)

Fresh leaf discs ( 5 0 mg) were cut and placed in a test tube containing 10 ml of

N, N-dimethyl formamide (DMF) and stored for 24 h at 4OC. The absorbance of the coloured
supernatant was read at 647nm and 666nm in a spectrophotometer (Shimadzu, Japan, Model 160) with DMF as blank. The contents of Chl a, b and total Chl were calculated Using the following formulae (Inskeep and Bloom 1985) :

where, A =absorbance, a = length of light path in the cell (1 cm), V=volume of the extract in rnl and W = fresh weight of the sample in g.

Carotenoids (Ikan 1969)

Absorbance values of the DMF pigment extract at 480, 647 and 666 nm were used to find the corrected optical density (OD) for carotenoids which was used for further calculations.
Corrected OD = A,+I(0.114 x A,,)

- 10.638 x A,,,)]
1

Carotenoid (rng g -' = Corrected OD x s fw)

where, A = absorbance, V = volume of the extract In ml and W = fresh weight of the sample in g.

Estimation of anthocyanins (Mancinelli et a/. 1975)

The leaves ( 1 0 0 m g ) were placed in a vial c o n t a i n i n g 10 m l of methanol:water:conc. HCI (80:20:1 vlv) and placed on a shaker in dark at 4C. After 48 h. the extract was filtered through Whatman No. 1 filter paper, made up to 12 rnl with the same solution and the absorbance was read at 530 and 657 nm. Anthocyanin concentration (A) was determined by the following formula:
A = A,,-

0.3A,,,

and expressed as absorbance (A) units g-' fw.

Preparation of alcohol extract for biochemical estimations

Fresh leaves ware oven dried (80C, 48 h) and powdered using a mortar and Pestle. Dried leaf powder ( 5 0 mg) was boiled in 10 ml of 80% ethyl alcohol on a Water-

bath. The homogenate was first cooled and then centrifuged at 600 rpm for 15 min. The supernatant was saved and made up to 20 ml with 80% ethyl alcohol. This extract was used for quantitative estimations of amino acids, flavonoids, sugars and total phenols. The residue was saved for starch estimation.

Estimation of flavonoids (Shinoda 1928)

To 1 m l of alcohol extract, 100 mg of magnesium powder and 1 ml of distilled water were added. The mixture was heated in a boiling water-bath for 10 min and cooled. Conc. HCI (2 ml) was added drop by drop with care and finally the volume was made up to

5 ml with distilled water. The colour intensity was read at 370 nm in a spectrophotometer.
Flavonoid content was calculated from the catechin hydrate standard.

Estimation of reducing sugars (Nelson 1944)

To 1 ml of ethanolic extract, 1 ml of fresh copper reagent prepared by mixing copper tartarate and copper sulphate solution ( 2 5 : l v/v) was added. The mixture was heated for 20 min in a boiling water-bath and cooled. One ml of Arsenomolybdate reagent was then added and the contents incubated for 15 min. The solution was then diluted to

25 m l w i t h distilled water and the colour intensity was read at 500 nrn in a
spectrophotometer. The reducing sugar content was calculated using the standard graph for glucose and expressed in mg g ' dw.

Coppertafiaratesdution(A): 25 g of anhydrous sodium carbonate, 25 g of sodium potassium

tartarate, 20 g of sodium bicarbonate, 200 g of anhydrous sodium sulphate were dissolved in 800 ml of distilled water, diluted to 1 litre, then filtered and stored in a brown bottle.

Copper sulphete solution (B): 15 g of copper sulphate was added to 100 ml of distilled
water. One or t w o drops of conc. H, SO, was added to this reagent

B.

Copper reegent: 25 ml of reagent A and 1 ml of reagent B were mixed. Arsenomolybdate reagent: To 450 ml of distilled water, 25 g of ammonium molybdate was
dissolved. To this, 21 ml of cone. H,SO, was added. 3 g of sodium arsenate d~ssolvedIn

25 ml of distilled water was also added to the above mixture. This was incubated at 37'C

or 48 h. The reagent was stored in a glass stoppered brown bottle.

istimation of non-reducing sugars

The amount of non-reducing sugars was determined by following the formula suggested by Loomis and Shull I1 937)and expressed in mg g ' dw. Non-reducing sugars = (total sugars - reducing sugars) x 0.95

Estimation of total sugars (Dubois et at. 1956)

Cold anthrone reagent 1 ml) was added to 1 ml of ethanolic extract. This 4 mixture was shaken vigorously and boiled for 1 min in a boiling water-bath. After cooling 0 In running tap water, the absorbance was read at 620 nm in a spectrophotometer. The reference to a glucose standard. amount of total sugar was estimated w ~ t h
Anthrone reagent: To 40 ml of distilled water, 100 mi of conc. H,SO,

was added. To 100

ml of the above mixture, 200 mg of enthrone was added and thoroughly mixed until a golden yellow colour appeared.

Estimation of OD phenols (Johnson and Shoal 1952)

To 1 ml of alcoholic extract, 1 ml of 0.5 N HCI and 1 ml of Arnow's reagent

0 were added. To this, 2 rnl of 1 N NaOH and 1 ml of distilled water were added. A pink
colour appeared immediately on adding NaOH. The colour intensity was reduced by diluting ~tto 25 m l with distilled water and the absorbance read at 5 5 nm. The OD phenol content 1 was calculated using a standard curve with catechol and expressed in m g g ' dw.
Amow's reagent: 10 g of sodium nitrite and 1 g of sodium molybdate were mixed in 100 0

ml of distilled water and stored in a brown bottle.

Estimation of total phenols (Bray and Thorpe 1954)

002697

To 1 m l of alcoholic extract, 1 ml of folin-ciocalteau reagent and 2 ml of 20% sodium carbonate were added and shaken well. The mixture was heated in a boiling water-bath for 1 min and cooled under running tap water. The blue solution was diluted to

25 ml w i t h distilled water and read at 650 nm in a spectrophotometer. Phenols were

quantified using cetechol as standard and the results were expressed in mg g ' dw.

~olin-ciocaIte0u reagent: Commercial folin-ciocalteau was diluted with distilled water in

1 :2 ratio.

Estimation of total free amino acid (Moore and Stein 1948)

Ethanol extract (1 ml) was taken in a 25 ml test tube and neutralized with 0.1 N NaOH using methyl red indicator to which 1 ml ninhydrin reagent was added. The contents were boiled in a boiling water-bath for 20 min after which 5 ml of diluting solution was added, cooled and made up to 25 ml with distilled water. The absorbance was read at 570 nm in a spectrophotometer against an appropriate blank. The amino acid content was calculated from the standard graph for leucine and expressed as mg g ' dw.
Ninhydrin reagent: 8 0 m g of stannous chloride in 5 0 rnl citrate buffer at pH 5.0 and 2 g of

ninhydrin in 5 0 ml methyl cellosolve, were mixed freshly.

Diluting reagent: Mixture of distilled water and n-propanol (1:1 vlv).

Estimation of starch (McCready et a/. 1950)

The residue left behind after alcoholic extraction of the leaf materials was (PCA) for 1 h. The mixture was filtered through dissolved in 5 ml of 5 2 % perchloric a c ~ d Whatman No. 42 filter paper and the filtrate was made up to 100 ml with distilled water. To 1 ml of the PCA extract, 4 ml of distilled water and 10 ml of freshly prepared cold anthrone reagent were added carefully along the side of the tube. The contents of the tubes were shaken vigorously and heated in a boiling water bath for 7.5 min. The tubes were then cooled immediately in running tap water and shaken well before reading the colour intensity at 630 nm. The starch content was calculated with reference to glucose standard and multiplied by 0 . 9 and expressed in mg g ' dw.
Anthrone reagent: Anthrone ( 2 0 0 mg) was dissolved in 100 ml of cold 95% H,SO,. Perchloricecid: To 18 ml of distilled water, 52 rnl of commercial perchloric acld (70%) was

added to get 52% perchloric acid.

Estimation of soluble proteins (Lowry et el. 1951)

Extraction Fresh leaf material (300 mg) was macerated in a pre-chilled mortar and pestle with 10 m l of 20% TCA. The homogenate was centrifuged for 15 min at 600 rpm. The supernatant was discarded. To the pellet was added 5 ml of 0.1 N NaOH, stirred well and centrifuged again for 15 min. The supernatant was saved as protein fraction. Estimation To 0.5 ml of protein extract 5 ml of reagent C was added and allowed to stand for 10 min in dark. Then. 0.5 ml of folin-ciocalteau reagent was added to this solution and again kept in dark for 3 0 min. The absorbance was read at 660 nm in a spectrophotometer. The protein content was calculated with reference to the standard curve for bovine serum albumin and expressed in mg g-' fw. Alkaline sodium carbonate (A): Sodium carbonate (2 gl was dissolved in 0.1 N NaOH and made up to 100 ml. Copper sulphate end sodium potassium tartarate (8):Copper sulphate (1%) was mixed with equal volume of 2% sodium potassium tartarate solution prepared freshly. Reagent C: 5 0 ml of reagent A was freshly mixed with 1 ml of reagent B. Fdinciocehursagent: Commercial folin-ciocalteau was diluted with glass distilled water
(1 :2 vlv).

Estimation of proline (Bates et a/. 1973)

Extraction 5 0 0 rng of fresh plant material was homogenized in a mortar and pestle with 10 rnl of 3% aqueous sulfoselicylic acid. The homogenate was filtered through Whatman No. 1 filter paper and the residue was re-extracted. The extracts were pooled and made up to 20 ml with aqueous sulfosalicylic acid and used for the estimation. Estimation To 2 ml of the extract, 2 ml of acid ninhydrin and 2 ml of glacial acetic acid were

added. The mixture was incubated for 1 h at 100C in boiling water-bath. Then, the test tubes were transferred to an ice-bath'to terminate the reaction. Then, 4 rnl of toluene was added end mixed vigorously using a test tube stirrer for 15 to 2 0 seconds and the toluene containing the chrornophore was separated with the help of separating funnel and the absorbance was measured at 520 nm in a spectrophotometer against an appropriate blank. The proline content was determined from a standard curve prepared with proline and expressed in rng g ' dw. Acid ninhydrh reagent : 1.25 g of ninhydrin was dissolved in 3 0 rnl warm glacial acetic acid and t o it 2 0 ml of 6 M phosphoric acid was added with agitation.

Estimation of glycine betaine (Grieve and Grattan 1983)

Extraction 5 0 0 m g of finely ground dry leaf material was mechanically shaken with 2 0 ml of deionized water for 24 h at 25'C. Time required for this step was determined by extracting the plant samples for 4, 8, 16, 24 and 48 h. The samples were then filtered through Whatmen No. 1 filter paper and the filtrates were used for analysis. Estimation The extract was diluted with 2 N H, SO, (1:l vlv) and 0.5 rnl of the acidified extract was cooled in ice water for 1 h. Later, 0.2 ml of cold potassium tri-iodide solution was added and mixed in a vortex mixture and the tubes were stored at 4OC for 15 min and a centrifuged at 10,000 rpm for 15 min. The supernatant was aspirated w ~ t h f~ne-tipped glass t u b a The per iodide crystals were dissolved in 9 ml of 1,2-dichloroethane with vigorous shaking. After 2.5 h, the absorbance was measured at 3 6 5 n m in a Spectrophotometer. The glycine betaine content was determined from a standard curve Prepared w i t h glycine betaine in 1 N H, SO, and expressed in pg g dw. btassium tri-iodide reagent: 15.7 g of iodine and 20 g of potassium iodide were d~ssolved In 100 rnl of distilled water and stirred in a vortex mixture.

'

Extraction of nucleic acids (Schneider 1945)

After removing midribs, 2 g of the lamina was homogenised with 10 ml of ice cold 10% trichloro acetic acid (TCA) and centrifuged at 2500 rpm for 10 min. The supernatant was discarded. The above step was repeated twice with 5 ml of 10% TCA followed by centrifugation. The residue was washed twice with 5 ml of absolute ethanol and centrifuged again. 2.5 ml of 10% TCA was then added t o the precipitate, mixed and again centrifuged at 2500 rpm for 10 min. The supernatant was stored. To the precipitate, 5 ml of 5 % TCA was added, mixed and kept in a boiling water-bath for 15 min. The tubes were cooled and centrifuged at 3000 rpm for 15 min. This step was repeated and supernatant obtained each time was pooled with the first one. The supernatant containing nucleic acids was used for the estimation of DNA and RNA.

Estimation of DNA (Burton 1956)

1.5 ml of the nucleic acid extract was transferred to a clean test tube. 3 ml of
diphenylamine reagent was added to the tube which was later corked and placed on the boiling water-bath for 15 min. After cooling, the absorbance of the solution was read at 610 nm. The concentration of the DNA sample was determined by comparison with a standard curve prepared with standard DNA in the range of 2 0 to 100 pg ml-'. The DNA content was expressed as pg g ' fw.
Diphanylsmine nwgent: 1.5 g of diphenylamine was dissolved in 100 ml of glacial acetic

acid and 1.5 m l of conc. H,SO,.

Estimation of RNA (Rawal 1977)

To 0.5 ml of the nucleic acid extract, 3 ml of orcinol reagent was added and the tube was corked and put on the boiling water-bath for 20 rnin. After cooling, absorbance of the solution was measured at 660 nm. The concentration of RNA content was determined with reference to a standard curve prepared with a set of standards of RNA in the range Of 20 to 100 p g m f ' and expressed as pg g" fw.

6% Orcinol (A/: 6 g of orcinol was dissolved in 100 ml of methanol.

Femic Chloride IB): 0.25 g of ferric chloide was dissolved in 100 ml of conc. HCI.
Orcinolreagent: 3.5 ml of solution A was added to solution B.

Estimation of carbon and nitrogen

The carbon and nitrogen content of the plant parts (leafheed) were estimated through Automatic Elemental Analyser (Carlo Erba 1108, 1991 Model, Japan) operating on 'dynamic flush combustion1method which converts all organic and inorganic substances into combustion products. The resulting combustion gases pass through a reduction furnace and are swept into the chromatographic column by the carrier gas (Helium) where they are separated and detected by a thermal conductivity detector. The signals are proportional to the concentration of the individual components of the mixture and are expressed as percent composition.

PHOTOSYNTHETIC MEASUREMENTS Functional attributes

Net photosynthetic rate (P,), transpiration rate IT,), intercellular CO,

concentration (Ci), stomata1 conductance (gs) and leaf chamber temperature

(L,) were

measured on the first fully expanded leaves from the top of plants at 3 0 DAS. Gas exchange measurements were determined by using lnfra Red Gas Analyser (IRGA) connected to Perkinson 1 litre leaf chamber analyser (Analytical Development Corporation ADC, UK. Model LCA 2). The chamber position after inserting the leaf was identical to the natural position of the leaf. At the time of measurements, the relative humidity (RH) and temperature were 6 0 % and 28.3OC respectively. The external CO, concentration was about 4 7 0 y mol

m-2 s-', the leaf to air vapour pressure difference of 2.5 to 3.5 KPa, leaf temperature
28-30C and photosynthetically active irradiance was 1400 p mol m-' s-'. Ten readings were taken for each sample at 5s intervals.

Measurement of chlorophyll fluorescence

In vivo Chla fluorescenoe induction was followed in intact leaves after excitation

with the broad-band blue radiation 1100 W m", 400-460 nm; Corning 511 3) as described by Kulandaivelu and Daniell 11980).

he photon flux density was 700 p~m-2s-'.Leaves

were dark incubated a t 28OC for 10 min before fluorescence measurements. The photomultiplier IHamamatzu R 375) placed at 90 to the excitation beam was protected by an interference filter I5 max 690 nm, half band width 12 nm, Schott, Germany). A leaf bit was inserted in the bleck plexiglass frame and placed diagonally in a standard 4 ml glass cuvette. Photophone Slide Projector was used to produce the excitation light. An electromagnetic shutter with a short opening time (ms) was used to control the excitation beam. The signal from the photomultiplier was directly displayed on a Hitachi recorder (Model 0 5 6 ) or stored in a digital oscilloscope Ilwatzu, Model SS-58021. The variable (F,) to maximal (, chlorophyll fluorescence ratios IF, / F were determined from the transients. F) ),

Oxygen evolution
PS II mediated 0,evolution was continuously monitored at 25

* 1C from leaf

discs using a leaf disc 0,electrode [Hansatech, UKI. The assay procedure was the same as that of Delieu and Walker (1981 ). The capillary matting used to floor the leaf discs was wetted in 1p1 HCO, buffer. The leaf discs were exposed to a saturating light intensity of 600 p mol m" s-'

Isolation of chloroplasts
Fresh leaf tissue was homogenised in semifrozen isolation medium, containing 50mM KH, PO,, pH 7.8,5mM pg CI, lOmM NaCl and 400 m M sucrose The homogenate was filtered through four layers of cheese cloth and was centrifuged at 500 g for 1 min. The supernatant was carefully collected and centrifuged at 2,000 g for 3 min. The pellet was washed once with the isolation medium and resuspended in the same. All the above operations were carried out at 4C.

Measurement of electron transport activities Whole chain electron transport (H,O

-+

MVJ (Armond et el. 19781

The rate of whole chain electron tranSpOR (H,O

+ MV) in the isolated chloroplasts

(mesophyll cells) was measured as 0, uptake at 25OC using a Hansatach oxygen electrode (Hansatech, U.K).White actlnlc light (900pE, rn-' s-') from a slide projector was passed through a 10 cm water-bath before illumlnatlng the sample. The reaction mixture in a total volume of 1 m l contained 2 0 m M phosphate (pH 7.5), 5 m M MgCI,, 10 m M NaCI, lOOmM sucrose, 0.1 m M NaN,. 5mM NH,CI and mesophyll cells equivalent to 2 0 pg chlorophyll.

PS I1 electron transport (H,O

+ BQ)

PS II mediated 0, evolution was continuously monitored at 25OC in the presence

of BQ using the same electrode set-up described above. The reaction mixture in e total volume of 1 m l contained 2 0 m M phosphate (pH 7.51, 5 m M MgCI,, 0.5 m M 00,100 m M sucrose and chloroplasts (mesophyll cells) equivalent to 20 pg chlorophyll.

Room temperature absorption spectra

Room temperature absorption spectra were recorded directly using a double beam spectrophotometer (Hitachi, Model 557). Chloroplasts were suspended in a medium containing 2 0 m M Tr~s-HCI(pH 7.51, 100 m M sucrose, 10 m M NaCI, 5 m M MgCI, and glycerol t o a final concentration of 60% (vlv). Both the reference and sample cuvettes were placed in the cuvette-holder with their ground surface facing the light beams, so as to scatter beams equally. The spectra were normalised at 720 nm.

SDS-PAGE analysis of chloroplast polypeptides (Laernrnli 1970)

SDS-PAGE was performed using a polyacrylamide gradient of 7.5-15% gel. Chloroplast proteins, both stromal and thylakoid, were isolated after lysing the chloroplast using 10 m M Tris-HCI buffer (pH 7.8) containing 4 m M MgCI,. The lysate was spun at 10,000 g for 10 min and thylakoids in this pellet were resuspended in the lysis buffer and again pelleted by centrifugation. The supernatants representing soluble and membrane fractions were mixed with 8 0 % acetone end left at 20C for 3 0 min. The proteins were separated by centrifugation and the pigments were washed off repeatedly in acetone. The pellet was treated with 10% TCA and the precipitated protein was separated by Centrifugation. It was again washed with 80% acetone and diethyl ether, respectively by

centrifugation and the dry powder was finally suspended in a small volume of 5% SDS. After determining the protein content by the method of Lowry et el. (1951), aliquots of each sample were mixed with an equal volume of sample buffer and heated in boiling water-bath for 2 min. It was then plunged into ice water for cooling immediately and spun in a microfuge for 5 min at 15,000 g before loading on to the gel.

Preparation of SDS-PAGE
A 7.5-1 5 % acrylamide linear gradient gel was prepared from solutions A and B
using a gradient marker. Before casting, the glass plates with spacers (1 mm) were clipped and the sides and the bottom were sealed with a 3 % agar solution. The bottom of the gel was plugged with a solution containing 2 ml of solution B, 20 pl of APS and 3 p1 of TEMED. The mixture was slowly let into the chamber between the glass plates. A layer of isopropanol was laid over the gel solution slowly and the gels were allowed to polymerize for 3 0 min. The isopropanol was washed off with water. The gel comb was then inserted leaving a gap of 1 cm above the gel and then the stacking gel solution was poured in slowly. After complete polymerisation in about 2 to 3 h, the bonom spacers and the comb were removed gently. The wells were washed with water and the gels placed in a glass tank containing running buffer. Care was taken to avoid air bubbles in the system. The protein concentration of all samples were equalised and 300 kg of each sample was loaded in the respective slot. The run was started at 6 0 mV end increased to 125 mV subsequently and was continued until the marker dye reached the bottom. The glass plates along with the gel were removed, the gels were slowly slid into a glass trey, washed once with water and then placed in the Coomassie brilliant blue staining solution for 3-4 h. The gels were then destained. Protein standards were coeiectrophoressed with green gram samples to determine the relative molecular weights of the polypeptide bands.

Reagents: Solution A Solution 6

17.5% acrylarnide) 40% acrylarnide solutlon 1 5 M Tr16-HCIpH 8.8 . 10% SDS

H2

(1 5% acrylarnide)

5.7 rnl 7.5 rnl 0.6 rnl 14.0ml 0.2rnl 10.0rnl 38.0rnl

11.4ml 7 5 rnl . 0.6 ml 6.3rnl 0.2 ml 10.0rnl 36.0rnl

10% APS
TEMED

ASP was prepared freshly. The last two reagents were added just before casting the gel. The sample buffer conta~ned 125 rnM Tris-HCI, pH 6.8;25% glycerol Ivlv); P-mercaptoethanol and 0.1% brornophenol blue. Stacking gel solution

25%

0.5 M Tr~s-HCI,pH 6.8 40% acrylam~de 10% SDS

3.8rnl 1.8rnl 0.3 ml

10% APS
TEMED Water

0.25 rnl 15 PI 8.85rnl

Running lwffer conta~ned 6 g of Tris, 28.8 g glycine and 1 g SDS in 1 litre of water. Staining s d u t h contained 200 rng Coornassie brilliant blue, 50 rnl ethanol, 7 rnl acetic acid and 43 ml water. Destalner contained 20 rnl ethanol, 7 rnl acetic acid and 73 rnl water.

ENZYMES ASSAYS Adenosine triphosphatase (ATPase, EC 3.6.1.3) (Evans 1969)

Extraction of Fresh plant rnater~al 1 g was placed in a precooled mortar and pestle and ground w i t h 5 rnl of 0.1 M PIS-HCI buffer at pH 7.5 at 4OC. The extract was passed g 0 through cheese cloth and centrifuged at 17,000 for 1 min In a refrigerated centrifuge at

4oC. The final volume of the supernatant was made up to 10 ml with 0.1 M Tris-HCI buffer (pH 7.5) and this extract was used for assaying ATPase activity. Assay The incubation mixture in a total volume of 2 ml contained 1 ml of 0.1 M Tris-HCI buffer (pH 7.51, 0.1 ml of 0.0075 M ATP solution, 0.1 ml of each 0.1 M MgCI,, 0.1 M NaCI, 0.1 M KCI, 0.1 M CaCI, and 0.1 ml of enzyme extract. The mixture was incubated at 37OC for 20 min and the reaction was terminated by the addition of 1 ml of 10% TCA. The control tubes received 1 ml of 10% TCA solution before the addition of the enzyme extract. The suspension was centrifuged and the inorganic phosphorus content of the supernatant was assayed as described by Jaffe and Galston (1966). The enzyme activity (one unit) was defined as the amount of enzyme liberating one p mole Pi min' mg-' protein.

a-amylase (Danielson 1947) and P-amylase (Englard and Singer 1950)

Extraction Fresh leaf tissue (1g) was homogenized in a prechilled mortar and pestle with 20 ml of distilled water. The homogenate was centrifuged at 24,000 rpm for 3 0 rnin at 4OC in a refrigerated centrifuge. The supernatant was saved and used as the enzyme source.

Assay for a-amylase (EC 3.2.1.I 1

To 0.5 rnl of the enzyme extract, 1 ml of 0.1 M citrate buffer (pH 5.0) and 0.5 ml of 2% soluble starch were added. The reaction was allowed for 5 min after the addition of starch at 30C. After 5 min the reaction was stopped by adding 2 ml of the colour reagent. The mixture was boiled for 5 min in a water-bath at 50C. After cooling, the final volume of the solution was made up to 10 ml with distilled water. The absorbance was read at 540 nm in a spectrophotometer (Shimadzu, Model 160). The activity was expressed in units min-' mg" protein.

Assay for P-amylase (EC 3.2.1.2)

pamylase activity was assayed by adding 1 ml of 0.1 M citrate buffer (pH 3.4) and 0.5 m l of 2 % starch to the 0.5 ml of the enzyme extract. The reaction was allowed for

5 min and then stopped by adding 2 ml of the colour reagent. The final volume was
made up to 10 ml with distilled water and the colour intensity was read at 6 4 0 nm in a spectrophotometer (Shimadzu, Model 160). The activity was expressed in units min1mg' protein.
Reagents: 1. Citrate buffer pH 5.0 and pH 3.4.

2. Colourreegent: 1 g of 3, 5-dinitrosalicylic acid was dissolved in 2 0 m l of 2 N NaOH

and 30 g of Rochelle's salt. The final volume was made up to 100 rnl with distilled water. 3. 2 N NaOH.

ANTIOXIDANT ENZYMES
Extraction (Joshi et a/. 1984) Leaf tissue (1 gl was homogenized with 10 rnl of ice-cold extraction medium containing 2 m M MgCI,, 1 rnM EDTA, 10 mM P-mercaptoethanol, 7% PVP and 10 rnM sodium metabisulphite. The hornogenate was strained through t w o layers of cheese cloth and centrifuged at 10,000 g for 15 min. The supernatant was made up to 10 ml with the same buffer and used as the source of enzymes. The extraction was carried out at 4OC.

Peroxidase (Kurnar and Khan 1982)

(Donor: Hydrogen-peroxide oxidoreductase, EC 1 .11.1.7) Assay Assay mixture of peroxidase contained 2 ml of 0.1 M phosphate buffer (pH 6.81, 1 ml of 0.01 M pyrogallol, 1 ml of 0.005 M hydrogen peroxide and 0.5 ml of enzyme extract. The solution was incubated for 5 min at 25OC after which the reaction was terminated by adding 1 ml of 2.5 N H,SO,. The amount of purpurogallin formed was

determined by measuring the absorbance at 420 nm against a blank prepared by adding

the extract after the addition of 2.5 N H,SO, at zero time. The activity was expressed in units

I = 0.1

A min m g ' protein).

'

Polyphenoloxidase (Kumar and Khan 1982)

(0-diphenol: 0, oxidoreductase, EC 1.10.3.1) Assay Assay mixture for polyphenoloxidase contained 2 ml of 0.1 M phosphate buffer (pH 6.01, 1 ml of 0.1 M cetechol and 0.5 ml of enzyme extract. This was incubated for

5 min at 25OC, after which the reaction was stopped by adding 1 ml of 2.5 N H,SO,.
The absorbancy of the purpurogallin formed was read at 495 nm. To the blank 2.5 N H,SO, was added at the zero time in the same assay mixture. The enzyme activity was expressed In units (1 unit =0.1 A min ' rng ' protein).

Superoxide dismutase (SOD) (EC 1.15.1.1 ) (Beauchampand Fridovich 1971)

The reaction mixture contained 1.1 7 X 10" M riboflavin, 0.1 M methionine, 2 X l o - ' M potassium cyanide and 5.6 X 10.' M nitroblue tetrazolium salt (NET) dissolved In 3 ml of 0.05 M sodium phosphate buffer (pH 7.8). Three rnl of the reaction medium was added to 1 rnl of enzyme extract. The mixture was placed under light provided by t w o sets of Philips 4 0 W fluorescent tubes with a set of tube lights on either side of the row of test sample tubes. Illumination was started to initiate the reaction at 30C and continued for 1 h. Identical solutions kept under dark served as blanks. The absorbence was read at 560 nm in the spectrophotometer against the blank. SOD activity was expressed in units. One unit is defined as the amount of change in the absorbance by 0.1 h-' mg protein (Cherry, 19631. Giannopolitis and Ries 11977) found no detectable amount of the reaction occurring under room light during preparation of the solution and spectrophotometric measurements.

'

NODULATION AND NITROGEN METABOLISM

Ten plants from each treatment and control were carefully uprooted from the

soil at 30,45 and 6 0 DAS and the number and fresh weight of nodules were recorded after removing the soil particles by washing them repeatedly and blotting to dryness. The nitrate and nitrite levels, nitrogenase and nitrate reductase activity (NRA) of the nodules were recorded at 3 0 DAS since nodulation was at its peak level during this period. The nitrate and nitrite levels and NRA of the third trifoliate leaves were also recorded at 3 0 DAS.

Estimation of nitrate and nitrite (Wooley et a/. 1960)

5 0 mg of shade-dried and powdered material (leaf I nodule) was boiled for 10 rnin in 5 rnl of distilled water. one ml of this aqueous extract was added to 9 ml of 20% (vlv) acetic acid solution containing 0.2 ppm of CuSO,. Salt mixture (1 g) as described by Nelson et a/. (19541 was added to each sample. A blank containing all but the extract was also run simultaneously. Tubes containing the assay mixture were shaken at least thrice at 3 min intervals and finally centrifuged at 3000 rpm for 10 min. Absorbance of the clear supernatant was read at 520 nm against the reagent blank. The procedure was repeated for another batch of samples omitting Zn, MnSO, and CuSO,. The second run gave the quantity of nitrite alone present in the sample.

The first value minus the second gave the quantity of nitrate present in the samples. The amounts of nitrate and nitrite were calculated from standard graphs for potassium nitrate and sodium nitrite respectively and expressed in mg g" dw.

Sah mlxlure: It was made by mixing thoroughly the finely ground chemicals viz. barium
sulphate 100 g, citric acid 7 5 g, manganous sulphate 10 g, sulphanilic acid 4 g, zinc powder 2 g, and 1-napthyl amlne 2 g.

Nitrate reductase activity (NRA) - in vivo

NRA activity was assayed by the method of Jaworski (1971) with modiflcattons Suggested by Muthuchelian et el. (1993). Fresh leaf bits Inodules (100 mg) were Incubated In vials containing 5 ml of incubation medlum prepared by mixlng 0.1 N KNO, (1 mll, 0.1 M

phosphate buffer of pH 7.5 (3.75 ml), 0.1 % of Triton X-100 (0.01 ml) and 1% propanol 10.25 ml). Incubation was carried out in dark for 1 h a t room temperature (28

* 2OC) with

occasional shakings. Aliquots of 0 . 2 m l from the incubation mixture were analysed for nitrite after 6 0 min. To 0.2 m l of incubation medium, 1.8 ml of distilled water, 1 ml of 3% sulphanilamidein 3N HCI and 1 ml of 0.02% N-(1-naphthyl) ethylene-diamine dihydrochloride were added in quick succession. This was incubated for 15 min in darkness for colour development and absorbance was read a t 5 4 0 n m w i t h a suitable blank i n a spectrophotometer .The amount of nitrite formed was expressed as 'n' moles of nitrite produced rnin' rng" f w using a sodium nitrite standard curve.

Nitrogenase activity
Nitrogenase activity of roots and nodules were determined by the acetylene reduction technique as described by Stewart et al.(1967). Enzyme assays were carried out in 7 ml capacity bottles fitted with rubber serum stoppers. 500 mg sample was placed in each bottle and stoppered tightly and the assay was initiated by withdrawing 10% of gas phase and injecting 0.6 m l acetylene gas using a disposable hypodermic syringe. The bottles ware incubated in light for 30 min and the reaction was then terminated by injecting 0.2 ml of 10% TCA. One ml of the gas phase was withdrawn and the ethylene formed was measured in a gas chromatograph (Aimil Nucon 5700 with

FID using poropak T column

model). Nitrogen was used as the carrier gas and the flow rate was adjusted to 4 0 m l min'. Ethylene standard was used as reference. Nitrogenase activity was calculated using the following formula:
Nkmpanara activity = n moles of ethylene x 60 x volume of gas phaae fresh weight x 30 x injectionvolume

where, 3 0 = incubation time in rnin, 6 0 = unit time (1h) for enzyme activity.
'n'moles of ethylrna = ethylene peak height Irnrn) x attenuation sensitiv~ty

,4

where, attenuation = 32, sensitivity = 1000. Nitroganasa activity was expressed as

P moles of acetylene reduced h-'g ' fw.

PHOTOGRAPHY
Leaf surfaces were v~ewed through Nlkon Labophot stereo
microscope

under

lnc~dentand translucent l ~ g h tand photographed Whole plants and plant parts were uslng a Pentax (K-1000, Japan) camera fltted w ~ t h appropriate photographed In dayl~ght close-up accessories

STATISTICAL ANALYSIS
At least flve replicates were maintamed for all treatments and control The experiments were repeated to conflrm the trends As the depth of s~gnlflcance between the treatments could be brought out clearly by a rnultlple range testlng programme only,

difference the means were compared by least s~gn~flcance

test (TMRT) (P=O 0 5 ) Ear, 1984)

(LSD) Tukey's rnult~ple range