IMPACT OP SULPHUR DIOXIDE ON PHYSIOLOGICAL RESPONSES

OF SOME CROP PLANTS

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OP

iHas ter of $f)tlDS(opi)p in

JBotanp

Sarvajcct Shtgh

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA) 2 0 0 3

PS3614

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J 7 JUL 7D09

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TO MM

a(ZAK)'b uor^G(z

'VgC liesearcfi Jiwardee

(Department of (Botany JiCigarH 9/LusCim University JiCigarH - 202002, India Off: (0571)2702016 Ta^. +91-0571-2702016 e-maiC: naf9@Cycos.com %Jian_na@rediffmaiC. com

(Dated: <^^-7^ 2-^03

Certificate

This is to certify that Mr. Sarvajeet Singh has worked under my supervision

for the M.Phil, degree in Botany. He has fulfilled all conditions required to

supplicate the M.Phil, degree. I, therefore, approve that he may submit his

dissertation entitled Impact of sulphur dioxide on physiological responses of

some crop plants. This is an original work and carried out at the Department

of Botany, Aligarh Muslim University, Aligarh It has not been submitted for

any other degree.

ACKNOWLEDGEMENTS

I bow in r e v e r e n c e t o GOD t h e A l m i g h t y , who b l e s s e d me wi th

an innumerab le favour of academic work .

Like all c r e a t i v e works complet in^g a t h e s i s in any d i s c i p l i n e IS rea l ly a hard work t o a c c o m p l i s h . I have no w o r d s t o e x p r e s s my h e a r t f e l t ^grat i tude t o my e s t e e m e d s u p e r v i s o r Dr. Nafees A. Khan, Depar tment o f Botany, Al i i^arh Musl im University, Alifgarh fo r his e x c e l l e n t g u i d a n c e , va luable and t ime ly s u g g e s t i o n s , s t imu la t i ng d i s c u s s i o n s , c o n s t r u c t i v e c r i t i c i s m and academ\c a l e r t n e s s , sense o f p e r f e c t i o n and p r e c i s i o n which enab led me t o c o m p l e t e t h i s wo rk . His d e v o t i o n t o work was i t se l f enough fo r anyone t o g e t invo lved in t h e work , (nsp i t e o f his t i g h t d e p a r t m e n t a l c o m m i t m e n t s he k e p t his door open w ide f o r me. His a f f e c t i o n a t e i n s t i n c t and constant encouragement were a lways boon t o me. Any error if s t i l l remains is e n t i r e l y my o w n .

1 am e x t r e m e l y o b l i g e d t o Pro f . Samiu l lah , Cha i rman, Department o f B o t a n y , A l igarh Musl im U n i v e r s i t y , A l iga rh fo r p r o v i d i n g n e c e s s a r y f ac i l i t i es fo r t h e work .

I have always been i n s p i r e d by my r e s p e c t e d t e a c h e r s . I du ly a c k n o w l e d g e t he i r moral s u p p o r t . 1 am a lso thankfu l t o my f r i e n d s and c o l l e a g u e s fo r t h e help t h e y e x t e n d e d whenever r e q u i r e d .

I a lso p lace on record t h e a s s i s t a n c e rendered by Dr. M. Khan, Dr. 5 . Javid, Mr . Irfan, Miss Shaila and Mr . Rais A. Khan du r i ng th i s g rue l l i ng t ask .

Last but n o t t h e l e a s t , I wou ld like t o e x p r e s s by p r o f o u n d sense of o b l i g a t i o n t o my p a r e n t s , b r o t h e r s , Dev, Ranjee, inder, Happy and s i s t e r s , Jessie , Sonu and Heena fo r the i r pe renn ia l e n c o u r a g e m e n t , u n d e r s t a n d i n g and p a t i e n c e du r i ng the p r e p a r a t i o n o f d i s s e r t a t i o n .

(SARVAJEET SINGH)

C O N T E N T S

Chapter Page No.

1. INTRODUCTION 1-8

2. SURVEY OF LITERATURE 9-19

Growth characteristics 9 Photosynthetic and biochemical characteristics 11 Antioxidant enzymes and compounds 17 Yield characteristics 18

3. MATERIALS AND METHODS 20-38 Experimentation 20 Plant exposure to sulphur dioxide 20

Exposure chamber 20 Sulphur dioxide generation 21 Sulphur dioxide monitoring 21 Exposure schedule 22

Growth characteristics 22 Carbon assimilation 22 Nitrogen and sulphur assimilation 23 Antioxidative enzymes 23 Yield characteristics 23 Photosynthetic rate, stomatal conductance and intercellular CO2 24 Physiological water use efficiency 24 Carboxylation efficiency 24 Assay of carbonic anhydrase activity 24 Chlorophyll and carotenoid estimation 25 Structural and non-structural carbohydrate 26

Extraction 26 Estimation of soluble carbohydrate 2 7 Estimation of insoluble carbohydrate 2 7 Standard for carbohydrate 27

Starch 28 Procedure 28 Standard for starch 28

Assay of nitrate reductase activity 29 Assay of nitrite reductase activity 30 Nitrogen, phosphorus and sulphur content 30

Digestion 20 Estimation of nitrogen si

Standard for nitrogen Estimation of phosphorus Standard for phosphorus

Estimation of sulphur Digestion Determination of sulphur

Protein estimation Standard for protein

Proline estimation Antioxidants analysis Estimation of ascorbic acid Preparation of extract Estimation of superoxide dismutase, peroxidase and catalase

Estimation of superoxide dismutase Estimation of peroxidase Estimation of catalase

Statistical analysis

RESULTS Growth characteristics Carbon assimilation Nitrogen and sulphur assimilation Antioxidative enzymes Yield characteristics

DISCUSSION Growth characteristics Carbon assimilation Nitrogen and sulphur assimilation Antioxidative enzymes Yield characteristics Conclusive remarks

REFERENCES 50-65

APPENDIX i . jv

C O N T E N T S

31 32 32 33 33 33 34 35 35 36 36 36

37 37 37 37 38

39-41 39 39 40 40 41

42-49 42 43 44 46 47 48

FIGURES AND TABLES

Figure-l. The average trophospheric concentration of S compounds. Figure-2. A Schematic of an exposure chamber. Figure-3. Proposed model for coordination among carbon, nitrogen

and sulphur assimilation and their regulation. Figure-4. Proposed model for SO2 interaction in cell and its

detoxification. Figure-5. Diagrammatic representation of involvement of

detoxificating enzymes. Figure-6. Relationship of SOD, APX and CAT with leaf area of black

gram (Vigna mungo L. Hepper) cv. T-9. Figure-7. Relationship of SOD, APX and CAT with shoot dry mass of

black gram (Vigna mungo L. Hepper) cv. T-9. Figure-8. Relationship of SOD, APX and CAT with photosynthetic

rate (Pn) of black gram (Vigna mungo L. Hepper) cv. T-9. Figure-9. Relationship of SOD, APX and CAT with structural

carbohydrate of black gram {Vigna mungo L. Hepper) cv. T-9.

Figure-l0. Relationship of SOD, APX and CAT with non-structural carbohydrate of black gram (Vigna mungo L. Hepper) cv. T-9.

Figure-l 1. Relationship of SOD, APX and CAT with total protein of black gram (Vigna mungo L. Hepper) cv. T-9.

Figure-12. Relationship of SOD, APX and CAT with biomass of black gram (Vigna mungo L. Hepper) cv. T-9.

Figure-13. Relationship of SOD, APX and CAT with seed yield of black gram (Vigna mungo L. Hepper) cv. T-9.

Table-1. Effect of different doses of sulphur dioxide in the growth characteristics of black gram (Vigna mungo L. Hepper) cv. T-9.

Tabie-2. Effect of different doses of sulphur dioxide in the photosynthetic and carbon assimilation of black gram (Vigna mungo L. Hepper) cv. T-9.

Table-3. Effect of different doses of sulphur dioxide on nitrogen assimilation, protein and proline content of black gram (Vigna mungo L. Hepper) cv. T-9.

Table-4. Effect of different doses of sulphur dioxide on antioxidants of black gram (Vigna mungo L. Hepper) cv. T-9.

TabIe-5. Effect of different doses of sulphur dioxide on yield of black gram (Vigna mungo L. Hepper) cv. T-9.

ABBREVIATIONS

AA App APS APX CA CAT cc Chi Ci cv d Gin Glu gm gs h HSO3-Kg/ha LSD M

ml N NO2" NO3-NiR NR NS NT? O2-P Pn ppm R S SO2 SO3'-S04^-SOD PWUE

Amino acid Appendix Adenosin-5 phosphosulphate Peroxidase Carbonic anhydrase Catalase Cubic centimetre Chlorophyll Intercellular carbon dioxide Coefficient of variation Day Glutamine Glutamate Gram Stomatal conductance Hour Hydrogen sulphite Kilogram per hectare Least significant difference Molar Meter cube per second Microgram per meter cube Millilitre Nitrogen Nitrite Nitrate Nitrite reductase Nitrate reductase Non significant Normal temperature and pressure Superoxide Probability Photosynthetic rate Parts per million Correlation coefficient Sulphur Sulphur dioxide Sulphite Sulphate Superoxide dismutase Physiological water use efficiency

r . •" %0i' -•*' soj -:ir- s o j ' HzO"—--«^ HiSOj

H2S04

I M T R D D U c T I D N

CHAPTER-i INTRDDUCTIDN

T h e life supporting system on this planet consists of air,

water, land, flora and fauna. These are interconnected and also

interdependent. The quest of man and his related activities often

disrupts the intricate balance among the constituents of the life

supporting system. Since the commencement of civilization and

eagerness for urbanization and industrialization, the human

activities have destroyed plant cover (build up meticulously by

nature over millions of years). In the process of industrialization,

air, water and land have become polluted so much that the

development has become synonymous with deforestation and

desertification, and the progress with pollution. Although the

pollution of air, water and land can not be delinked from one

another, but the present account deals only with air pollution with

emphasis on SO2 pollution.

Air pollution is basically the presence of foreign substances

in air and mainly caused by rapid industrialization. Some critics

comment on air pollution as the 'price of industrialization'. Air

pollution caused by automobiles has been described as the 'disease

of wealth' (Rao and Rao, 1995). The combustion of fossil fuel like

coal, oil and gas etc., for utilizing their stored energy, is the

foremost challenging air pollution problem. Historically, air

pollutants have been divided into primary and secondary forms.

A primary pollutant (eg. SO2, HF, NOx or heavy metals) is one

that is injurious in the same chemical form as when it was emitted

into the atmosphere. A secondary pollutant is formed in the

atmosphere through reactions among precursors emitted into the

atmosphere (eg. O3, PAN etc.).

CHAPTER-1 I N T R O D U C T I O N

Sulphur dioxide (SO2) produced primarily during burning of

fossil fuels (Carlson, 1983) is an important primary gaseous air

pollutant, frequently referred to as the major constituent of

"London Smog" which is chemically known as "Reducing Smog".

The developing countries, where coal and wood serve as a major

requirement of energy experience higher levels of ambient SO2

(Khan and Khan, 1993, 1994b). Production, refining and

utilization of petroleum and natural gas produce emissions of

about 2 1 % of the total anthropogenic SO2 (Wood, 1968). There are

other sources of SO2 emission, like coal combustion (more than

6% of total emission), smelting and refining of ores, especially of

Cu, Pb, Zn, Ni and Fe (responsible for about 7%), and

manufacturing and industrial utilization of sulphuric acid and

sulphur. The coal burning power plants represent the most

important single source of SO2 and the amount of SO2 emitted

depends upon the sulphur content of the coal which varies from 1-

6% of the total weight.

The ambient concentration of SO2 at ground levels depends

upon the amount and concentration of emission, distance from the

source and meteorological and topographical conditions. Sulphur

dioxide concentration near point sources such as coal fired power

plants, with no O2 or little pollution control equipments, may be as

high as 1-3 ppm and in large urban areas the SO2 concentration

may vary from 0.05-0.1 ppm. Khan and Khan (1994a, b) recorded

daily mean concentration of SO2 as 0.05 and 0.08 ppm 1 and 2 km

away in windward direction of a coal fire thermal power plant,

Aligarh (Kasimpur) in U.P., India. SO2 problem is a severe

problem in some areas of China, Iran, Pakistan, India, Mexico,

Brazil and some other developing countries with low rainfall and

C H A P T E R - ! I N T R O D U C T I O N

irrigated agriculture (UNEP, 1991). In India, a significant

contribution arises from the use of biomass fuels (Arndt et al.,

1997).

Amount of S in the atmosphere is mostly of anthropogenic

origin. The natural emission mostly as H2S via biological process

is only about 128 tonnes/year whereas anthropogenic emission

through thermal power plants is about 63 million tonnes/year. The

small amount of H2S is also oxidised to SO2 in the troposphere

within a day or two. Even though the residing time of SO2 is only

a few days in the atmosphere, because of lateral movements in

response to meteorological circulation factors, they can move

easily from source area to totally pristine "uncontaminated" areas.

0.7

0.6

0.5

E so

c o I 0.4 • * - > c

g 0.3 o

U '^ 0 7 w. U

^ 0.1-

0 ^ H2S

Compounds

Figure-1. The average trophospheric concentration of S compounds.

The entry of SO2 in plant leaves is through stomata when

they are open during a normal process of gas exchange between

CHAPTER-1 I N T R g P U C T I O N

stomata and atmosphere. Biochemical changes occur in leaves

during long term pollution by SO2 at non-necrotic concentrations

in the air. These metabolic effects can be observed from

concentrations of pollutants lower than those required to produce

ultra-structural changes (Perez-Rodgiguez, 1976). The primary site

for SO2 effects is at the metabolic level. Pollution can be expected

to modify the metabolism in two main ways:

1. by diverting the metabolic operation towards a kind of

pathological pattern through accumulation or consumption of

a specific metabolite or group of substances.

2. by a quantitative modification of the general metabolic

levels in order to achieve a faster metabolization of the

pollutant. Less dramatic, but more insidious, this later

possibility requires the action of mechanisms capable of

coordinating the operation of different levels of metabolic

organization.

Sulphur dioxide once enters in tiie plant system produces

sulphite (SOs^") and bisulphite (HSO3") ions which in turn are

slowly oxidised to sulphate (Pell, 1979; Saxena and Saxena,

1999). Of these sulphite is about thirty time more toxic than

sulphate (Thomas and Handricks, 1956). Conversion of sulphite to

sulphate chemically and biochemically plays a major role in

making the plant tolerant/susceptible. Sulphur dioxide may be a

reducing or oxidising agent depending upon the pH of the medium

in which it exists. In water at a pH of 7.2 (normal for the

cytoplasm of most plants), it exists approximately 50% in the form

of HSO3" and 50% is the form of SOa^" (Cotton and Wilkinson,

1966). At pH 1.8, H2SO3 and HSOa" exist in the ratio of 1:1. In

general acidic solution favours the formation of H2SO3 and HCOs",

CHAPTER-1 I N T R g P U C T I D N

while an alkaline solution favours the formation of SO3 . It may

also be oxidised to SO4 .

SO2 is extremely soluble in water, 22% by weight at 0°C and

9.4% at 24°C. The concentration normally found in polluted areas

dissolve completely upon contact with surface or tissue moisture

of plants. In solution, SO2 establishes the following equilibria,

which has an important bearing on its effects.

SO2 + H2O ^ H2SO3

H2SO3 ^ H" + HSO3-, pK = 1.76

HSO3 V. H^ + SOs^", pK = 7.20

The S03^~ and HSOs" ions in the surface water droplets

probably enter the stomatal openings from where they move into

the guard cells, then across a chain of epidermal cells until they

reach a point where passage into the new cells is possible. SO3

and HSO3", both having a lone pair of electrons (Malhotra and

Hocking, 1976; Thomas and Runge, 1992; Hippeli and Elstner,

1996). This enables them to be readily oxidised by a series of

overlapping reactions which involve formation of free radicals in

the presence of Mn "" and indole acetic acid (lAA) (Yang and

Saleh, 1973). HSO3" is a reductant which accelerates oxidative

processes by reducing peroxide bonds thus producing anions and

radicals. These free radicals of sulphur in the action of light and

reducing agents are capable of forming sulphoxy radicals (S02^")

which persists more longer than most free radicals and may oxides

SO3 " to HS04~ via a radical chain mechanism as indicated below

(Radmer and Kok, 1976; Peiser and Yang, 1978; Miller and

Xerikos, 1979; Hippeli and Elstner, 1996).

H S 0 3 " + 0 2 ^ HSOs* + 02""

H S O 3 - + 2 H " + O2" > H S 0 3 - + H2O2

CHAPTER- 1 I N T R D D U C T I D N

SO3 + O2 > S 0 3 " " + 0 2 '

S O s " + O2 > SOs'"

S O s " + SOa^" > SO4" + S04^"

SO4" + SO3 ' " > SO4 ' " + SO3"

S O 4 - + OH" > S04^" + O H '

OH- + SOs*^- > O H - + S O 3 -

F ina l ly the r eac t ion t e r m i n a t e s as :

OH- + S03 '~ ^ O H " + SO3

S O 3 - + O j - + 2H^ > S03~ + H2O2

O2- + 02"" + 2H" > H2O2 + O2

Plants contain several mechanisms to compensate negative

effects of SO2 exposure. These mechanisms include oxidation of

sulphite to sulphate in the apoplastic space, as well as reductive

conversion of sulphite to sulphide in the chloroplasts combined with

the emission of H2S and the synthesis of organic sulphur compounds.

These compounds may not only be used for growth, but may also be

stored in the shoot and the root because of the sulphur requirement of

plants may be met by direct uptake of SO2 from the atmosphere if it is

present in very low concentration. If the plants initially fail to

detoxify the pollutants, these free radicals then disturb the metabolic

pathways (like photosynthesis, transpiratory, respiratory, growth and

reproductive processes).

Free radicals formation (i.e. reactive oxygen species) is one

major event with high potential of toxicity and injury especially under

SO2 pollution, which are generally more reactive than the pollutant

gases themselves. The reactive oxygen species such as superoxide

anions (02"), hydroxy free radicals (OH") and H2SO3 which are

highly toxic and cause changes in protein and lipids in the cell walls

and membrane organizations. Therefore, many group of compounds

CHAPTER-1 I N T R O D U C T i a N

needs scavanging the free radicals. The enzyme present in the plant

system involved in the scavanging of reactive oxygen species are

superoxide dismutase (SOD), catalase, peroxidase and ascorbic acid

oxidase, these enzymes are called antioxidant enzymes. Also

antioxidants like glutathione, ascorbic acid (Vitamin C), P-carotene

(provitamin A) and phenolics compounds acts as free radical

scavengers. Thus, the plants with robust biochemical defence

mechanisms against reactive oxygen species by the activation or

induction of antioxidant enzymes and by the increase in level of

antioxidant metabolites are necessary.

Sulphur is an indispensable constituent of cysteine, cystine and

methionine that form the protein. The supply of S affects the

availability of N. The supply of S limits the efficiency of soil added

N. To achieve maximum efficiency a proper N : S ratio is to be

maintained. To identify the impact of atmospheric sulphur on the

utilization of nitrogen, a leguminous plant was selected for the study

which may provide in-depth insight into the assimilation of sulphur

and nitrogen. The aspects of soil-derived N and S are although

available in the literature, but interaction of atmospheric S and soil-

derived N has not been reported. Instead of nitrogen input to soil and

its interactive effect with sulphur, the naturally nitrogen fixing plant

was used as an experimental material.

Black gram (Vigna mungo L. Hepper) is a highly prized pulse,

grown in an area of about three million hectares in India. The main

areas of production being M.P., U.P., Punjab, Maharashtra, W.

Bengal, A.P. and Mysore, which contributes 1.3 million tonnes of

grains. The crop prefers water-retentive stiff loaming heavy soil, and

does well on both black cotton soils and brown alluviums. It is grown

as a rainfed crop in the warm plains as well as cool hills, upto an

CHAPTER-1 I N T R D D U C T I O N

altitude of 6,000 feet. The cooking quality of black gram produced

in the hills or in most climates is better. It is very rich source of

vegetable protein containing about 25% protein in its seeds and

some essential minerals and vitamins, important for human body.

It is consumed in the form of dal (whole or split, husked or

unhusked). It is a chief constituent of 'Papad' (poppadam) and

also 'Bari ' (spiced balls) w^hich make delicious curry (singh,

1983). It is known by different names in India (Urd in Hindi, Mash

Kalai in Bengali, Ututtam parappu in Tamil and Udnitele in

Kannada). Black gram is completely self fertile. This would also

enrich the soil fertility by fixing the atmospheric nitrogen. Black

gram var T-9 was developed after the selection from Barielly local

in 1948, is early maturing. It is suitable for entire country, average

yield is 10-12 q/ha.

There has been controversy among taxonomists regarding the

correct botanical name to be assigned to black gram. Until the

middle of the 21^' century, the name Phaseolus mungo (L.)

remained acceptable for black gram. The situation changed with

the transfer of Asiatic Phaseolus species to the genus Vigna on the

basis of certain characters. Finally, Klilczek (1954) named green

gram as Vigna radiata (L.) and black gram as Vigna mungo (L.)

Hepper.

The dissertation reports a research conducted to study the

exposure of SO2 on carbon, nitrogen and sulphur assimilation of

black gram. Growth, physiological, biochemical and yield

characteristics were noted. An attempt was also made to correlate

the events of growth and physiological changes with the activities

of antioxidant enzymes in the plants.

5 U R V E Y

H1SO3

^ m

pH H2SO3 HSO3

D F

L I T E R A T U R E

CHAPTER-z SURVEY OF LITERATURE

Sulphur dioxide! This classic, pre-eminent air pollutant is still

with us. It is produced from burning coal and persists not so much

form home heating as in the furnaces of coal-fired electric-power

generating plants. Sulphur dioxide may be 70-90% controlled in

some countries, but older plants or newer facilities in developing

countries often lack such technology. Even where control exists,

the tremendous amount of coal burned still result in the emission

of significant quantities of sulphur dioxide. The problem is

especially critical in developing countries where control

technology is lacking, or in developed countries where control has

low priority. SO2 pollution is a severe problem in some areas of

China, Iran, Pakistan, India, Mexico, Brazil and some other

developing countries with low rainfall and irrigated agriculture

(UNEP, 1991). The global annual anthropogenic SO2 emission, as

estimated decade back, was around 146 to 187 million tonnes

while the natural SO2 emission was about 5 million tonnes/year

(Freedman, 1989). In India, a significant contribution arises from

the use of biomass fuels (Arndt et al., 1997).

Plants suffer more since they neither can move nor bear

nervous system and physical reorientation, are thus different. The

plants being the producers depending much on gaseous exchange

are remains exposed to the pollutants (i.e. SO2 etc.), attracted

much attention for the impact and effect on their systems.

GROWTH CHARACTERISTICS

Effect of SO2 on the growth of various plants has been

studied extensively. Lalman and Singh (1989) observed that the

lateral spread, plant height, number of leaves and total leaf area,

RGR, LAR, root/shoot ratio and nodulation were reduced in Vigna

CHAPTER-2 S U R V E Y O F L I T E R A T U R E

mungo, when plants were fumigated 2h with SO2 at 0.25 and 1

ppm. Saxe (1983) reported 10-15 [ig m" SO2 reduced leaf area by

promoting senescence, rather than by interfering with leaf

emergence and development. SO2 at a concentration greater than

267 |ig m'"' in open top chambers altered the growth pattern of

Eucalyptus terelicoinis by reducing leaf surface area and weight of

mature and immature leaves (Murray and Wilson, 1988). In open

top chambers, SO2 at 0.8 ppm 2h did not induce foliar injury but

suppressed the root and shoot growth, number and area of leaves

(Pandey and Rao, 1978). SO2 at 0.1, 0.2 and 0.5 ppm 8h for 4

weeks reduced leaf area by 13.5, 16.7 and 45 .1% and plant dry

weight by 15.5, 55.4 and 58.6% respectively in cucumber plants.

Deepak and Agrawal (1999) observed reduction in leaf area and

plant height in wheat plants exposed to 0.6 ppm of SO2. Growth

reduction in Oryza saliva was also noted by Raza and Gouri

(2000). Legumes and solanaceous vegetables have been

extensively tested against SO2. Miller (1979), Ma and Murray

(1991) and Deepak and Agrawal (2001) found that cultivars of

soybean were sensitive to SO2 but the amount of growth reduction

and injury varied with the cultivars. SO2 at 650 and 1300 |ig m"''

induced reduction in fresh weight, dry weight and root/shoot ratio

in Cicer arietinum (Ampily, 1999). SO2 usually at 0.1-0.3 ppm

might cause significant suppressions in growth and yield of

soybean (Carlson, 1983; Klarer et al., 1984; Verma and Agrawal,

1995; Lee ei al., 1997).

Deleterious effect of SO2 have also been observed on other

legumes such as alfalfa (Majstrik, 1980), snapbeans (Rist and

Davis, 1979), pintobean (Davids et al., 1981), navybean, peanut

(Murray and Wilson, 1990), cowpea (Khan and Khan, 1996),

10

CHAPTER-2 S U R V E Y O F L I T E R A T U R E

fenugreek (Krishnayya and Date, 1996), pea (Kumar et al., 1991).

Kasana and Mansfield (1986) reported that more photosynthates

are retained in shoots and less transferred to the roots due to SO2

pollution, which reduced root growth greatly and there are

implications for symbiotic association and water relations of

plants. Contrary to these reports, Kumar and Singh (1986) reported

a slight stimulation in growth at 0.12 ppm SO2 exposure at early

stages of plant growth of Vigna sinensis, however, prolonged

exposure caused significant reduction in growth parameters and

dry weight fractions. As a result of exposure to 0.25 and 0.5 ppm

SO2, shoot and root length in Cajanus cajan and Pisum sativum

was slightly increased. However, it was not accompanied by

increased phytomass of seedlings (Kumar and Prakash, 1990).

Prasad and Rao (1981a) reported reduction in root and shoot

length, number of leaves, nodules and flowers in Phaseolus aureus

exposed to petrocoke pollution. At 320, 667 and 1334 |Lig m'" SO2,

number of leaves, leaf area, fresh and dry weight decreased in

Lycopersicon esculantum (Sharma and Prakash, 1991). Mishra

(1980) exposed plants of Arachis hypogea to 0.06 - 1.00 ppm SO2

for 4h for 6 weeks and observed reduced net primary productivity

in 0.25 ppm and above but slight beneficial effect on plant

productivity was observed in concentrations lower than 0.25 ppm.

PHQTDSYNTHETIC AND BIOCHEMICAL CHARACTERISTICS

Stomatal pores are the main entry ports to internal air spaces

for SO2 under conditions of adequate moisture, when the

surrounding guard cells are turgid and the stomata are open.

Sulphur dioxide affects stomatal behaviour, photosynthesis and

chlorophyll content of green plants. Investigation on plant growth

and crop productivity have shown that quantitative reductions

11

CHAPTER-2 S U R V E Y O F L I T E R A T U R E

occur as a result of various physiological and biochemical

imbalances induced by SO2. The reduction in chlorophyll a,

chlorophyll b, total chlorophyll and carotenoid contents due to

SO2 pollution has been reported by several workers SO2 absorbed

in the plant tissue combines with H2O to form H2SO3 and

dissociates into H" and HSOs" ions which cause degradation of

chlorophyll by displacing Mg^^ ions by free H^ ions and thus

converting them into phaeophytin (Rao and Lee Blanc, 1966).

Verma and Agrawal (1995) and Verma et al. (2002) observed

reduction in chlorophyll content and photosynthetic rate when

wheat and soybean cultivars exposed to SO2. In exposure to 0.2-

0.5 ppm SO2 h"' caused significant reduction in chlorophyll

content in rice (Raza and Gouri, 2000).

Chlorophyll content was found to be sensitive and reduced

significantly even at low concentrations of SO2 as in tomato (Khan

and Khan, 1996; Sharma and Prakash, 1991), some leguminous

crops (Prasad and Rao, 1982; Ma and Murray, 1991; Gammell and

Colls, 1992; Joshi et al., 1993 and Lee et al., 1997). Zeamays

(Jeyakumar et al., 2003), barley (Hagazy and Abd El Hamid, 1998)

and in some trees (Agrawal and Agrawal, 2000). It was reported

that chlorophyll a was more severely affected than chlorophyll b

(Kondo et al., 1980; Prasad and Rao, 1982; Khan and Usmani,

1988; Joshi et al., 1993). In Fagus sylvatica reduction in

chlorophyll a, b and total chlorophyll was observed by Ditmarova

and Kinet (2002). Ampily (1999) exposed Cicer arietinum to 650

and 1300 ng m'^ SO2 and observed reduction in total chlorophyll,

chlorophyll a/b ratio and carotenoid content. Net photosynthesis

being the basic unit of crop productivity is reduced significantly

even at low concentrations of SO2 (Varshney and Garg, 1979;

12

CHAPTER-Z S U R V E Y O F L I T E R A T U R E

Cowling and Koziol, 1978; Sheu, 1994; Miranu el al., 1995,

Nagaya et al., 1998; Sandhu et al., 1992; Junkwon et al., 2000;

Verma et al., 2000). Carlson (1983) and Heck et al. (1986)

reported that SO2 readily penetrates the more delicate leaf

interiors and affects photosynthesis. Pandey and Mansfield (1985)

reported the effect of SO2 on photosynthesis on mungbean. It was

reported that photosynthetic performance in liver wirt Frullania

dilalaia was affected by SO2 (Gimeno and Deltoro, 2000). Ranieri

et al. (2000) also observed decreased photosynthesis in Hordeum

vulgare. Nagaya et al. (1998) tested 67 varieties of winter cereals

against 1 ppni of SO2 for 30 minutes and reported, in all plants

photosynthetic rate was depressed.

Rengua and Paliwal (2002) reported that SO2 concentration

had a significant influence on the internal CO2 which was found to

be increased in plants depend on the interaction on both SO2

concentration and plant age. SO2 might directly induce stomatal

closure by reducing photosynthesis, thereby internal CO2

concentration increased (Winner and Mooney, 1980). Koziol and

Jordan (1978) exposed red kidney bean to various concentrations

of SO2 for 24h and reported decreasing rates of photosynthesis

which were correlated with increasing rates of respiration and the

depletion of sugar and starch levels. Joshi et al. (1993) reported

reduction in carboxylation efficiency and CO2 fixation in Sorghum

bicoUor. Pfanz and Beyschlag (1993) exposed spruce (Picea abies)

in highly SO2 polluted locations and noticed dramatic decrease in

carboxylation efficiency and light-use efficiency from current year

needles to one year old needles.

Sulphur dioxide affects stomatal behaviours and studies have

been made on conductance or resistance of stomata. Biscoe et al.

13

CHAPTER-2 S U R V E Y O F L I T E R A T U R E

(1973) observed decreased stomatal resistance and noticed

suppression in normal diurnal variations in stomatal behaviours m

Viccia faba when exposed to air containing SO2. Khan and Khan

(1993, 1994) and Mc Aunsh et al. (2002) reported that SO2 enters

leaf through stomata and causes the stomata to close only partially

even if guard cells are damaged. Olszyk and Tigney (1985)

observed SO2 at 100 ^g m'' produced 20-25% reduction in

stomatal conductance in Pisum sativum. Black and Unsworth

(1979) studied the stomatal conductance of Viccia faba, Raphanus

sativus, Phaseolus vulgaris, Helianthus annus and Nico liana

tabacum at water vapour deficits (Vpd) ranged from I-O to 1-8

kPG when Vpd was low stomata were open, exposure to SO2

induced rapid and irreversible increase in stomatal conductance.

Studies on the effect of SO2 on guard cells and adjacent epidermal

cells revealed that proportion of surviving adjacent epidermal cells

decreased as SO2 concentration was raised from 50-500 ^g m".

Although the guard cells appeared to be undamaged at

concentrations below 200 ^g m"^, structural disorganization or

death of one or both guard cells was observed frequently at or

above 500 ^g m'^ (Black and Black, 1979). Junkwon et al. (2000)

reported reduction in stomatal responses in various woody plants

at 4-7 ppm SO2 for 7h.

Sulphur dioxide also affects the amino acid contents of

plants. Saxe (1983) reported increase in amino acid in bean plants

exposed to SO2 concentrations above 250 ^g m'^ while Koziol and

Cowling (1978) observed a significant reduction in glycine and

serine contents. Godzik and Linkens (1974) reported that changes

in amino acid concentration in SO2 treated plants leads to protein

reduction. Such reductions might be due to inactivation of

14

CHAPTER-2 S U R V E Y D F L I T E R A T U R E

enzymes responsible for protein synthesis (Cecil and Wake, 1962).

Saxena et al. (2001) reported reduction in protein contents upon

exposure to high doses of SO2. Prasad and Rao (1982), Verma and

Agrawal (1995) and Agrawal and Verma (1997) reported reduction

in protein content and suggested leguminous crops are more

sensitive than cereal crops. Studies on changes in protein contents

as a result of SO2 pollution revealed that soluble protein contents

increased at low levels but decreased at high concentration (Sardi,

1981; Prasad and Rao, 1981b; Saxe, 1983). Lee (2002) also

observed reduction in Rubisco protein in geranium. Bernardi et al.

(2001) studied the effect of SO2 on protein synthesis in Phaseolus

vulgaris. Perez-Soba et al. (1999) recorded lower concentration of

soluble protein, when scot pines exposed to 75 |ig m'' SO2 for 12

weeks. Saraswathi and Rao (1995) also reported reduction in

protein content in Cajanus cajan and Amaranthus paniculatus.

Sulphur dioxide also affects the carbohydrate content. Prasad

and Rao (1982), Koziol and Jordan (1978) reported initial increase

and later decrease in the carbohydrate contents of wheat and red

kidney bean plants exposed to SO2. Koziol and Cowling (1980)

also observed an increase in free and total carbohydrates in

ryegrass at lower concentrations of SO2. Due to SO2 pollution,

maximum decrease in leaf carbohydrate in Vigna sinesis was

reported by Kumar and Singh (1986). Malhotra and Sarkar (1979)

reported an increase in reducing sugars and increase in non-

reducing sugars in pine seedlings as a result of SO2 pollution.

Accumulation of reducing sugars and depletion of starch was

reported in Mangifera indica and Psidium guajava (Kumar and

Singh, 1988). Decrease in carbohydrate content as well as lipid

contents was recorded in Ulmus americana under SO2 pollution

15

C H A P T E R - 2 S U R V E Y O F L I T E R A T U R E

(Constantinidou and Kozlowski, 1979). Borka and Andras (1981)

reported reduction in starch contents of potato tubers exposed to

SO2 and soot emissions. Reduction in leaf starch content of bean

were reported above 250 ig m"- SO2 (Saxe, 1983). Bucker and

Ballach (1992) also reported reduction in the concentrations of

non-structural carbohydrates under pollution. Wheat plants

exposed to 15 ppm of SO2 for 4h shows reduction in starch content

whereas increase in total soluble sugar and reducing sugar levels

was noticed by Agrawal and Verma (1997). Total sugar and starch

content in leaves were increased by exposure to lower

concentrations of SO2 but decreased by the higher concentrations

in red kidney bean (Kaziol and Jordan, 1978).

Mineral contents of plants have been found altered as a

result of SO2 pollution (Mishra, 1980; Sprugel et al., 1980).

Prasad and Rao (1981a) reported decrease in accumulation of N, P,

K and Ca in greengram under SO2 exposure. Perez-Soba et al.

(1994) reported exposure to SO2 resulted in a decreasing activity

of GS and lower contents of P and K in scot pine needles. Prosser

et al. (2001) reported decreased NR activity in spinach plants

exposed to SO2. Gupta et al. (1991) and Sandhu et al. (1992)

reported decrease in specific root nodule nitrogenase activity

(SNA) and foliar nitrogen in soybean. Exposure to 250 ppb SO2

suppressed nitrogenase activity and reduced shoot and root

nitrogen concentrations. Verma et al. (2000) exposed four

cultivars of wheat to SO2 and observed reduction in N content. An

increase in sulphur contents was observed by Case and Krouse

(1980), Cowling and Koziol (1978), Cowling and Andrew (1979)

and Ma and Murray (1991). Liang et al. (1994) exposed Masson

pine seedlings (Pinus massoniana) to 100 ppb SO2 and reported

16

CHAPTER-Z S U R V E Y O F L I T E R A T U R E

that S content of foliage increased by SO2 exposure. Hrdlicka and

Kula (2001) studied the effect of SO2 pollution and observed

increase in S content and decrease in N and P contents. Simoncic

(2001) also reported increased contents of S and nitrogen in the

current year and one year old spruce needles.

ANTIOXIDANT ENZYMES AND coMPauNDs

A reduction in ascorbic acid contents was reported in green

gram exposed to petro coke pollution (Prasad and Rao, 1981a) and

in needles of Picea under the influence of SO2 (Grill el al., 1979).

While increase in Ascorbic acid content in wheat was observed by

Ranieri et al. (1997) to SO2 pollution. Raza and Gouri (2000)

exposed Oryza saliva cv. Tulasi to 5 ppm SO2 for Ih and observed

decrease in ascorbic acid and catalase activity. Many other

workers also reported decrease in ascorbic acid contents (Verma

and Agrawal, 1995; Hagazy et al., 1998 and Agrawal and Agrawal,

2000).

Nandi et al. (1990) exposed Viccia faba to 270+3 2 and

670+45 mg m" SO2 for 1.5h day' ' between 40 and 85d of growth

and observed increased peroxidase activity. Rao (1992) exposed 6,

12, 24 and 48 months old seedlings of potted Cassia siamea and

Delbergia sisso to 96 \ig m'^ SO2 for 6h day' ' for 15 days and

observed enhanced peroxidase and SOD activities. Increase in

SOD activity was observed when soybean seedlings exposed to

0.1-0.5 ppm of SO2 (Qian and Yu, 1991). Karpinski et al. (1992)

exposed mature scot pine to low levels of SO2 and observed the

level of chloroplastic and cytosolic Cu/Zn SOD mRNA which were

significantly greater than control. Ranieri et al. (1997) studied the

isoenzymes pattern of peroxidase and SOD in wheat leaves

exposed to low concentration of SO2. Increased SOD and

17

CHAPTER-Z S U R V E Y O F L I T E R A T U R E

glutathione reductase activities were reported in two cultivars of

soybean, exposed to SO2. Navari-Izzo and Izzo (1994) exposed

two cultivars of barley namely Arda and Plaisant to low

concentration of SO2 (115, 220 and 350 g m'^) for 34d in growth

chambers and observed increase in the activities of catalase, SOD,

glutathione synthase, glutathione reductase, glutathione peroxidase

and glutathione transferase. After fumigation to 350 ^g m' SO2

5% increase in free organic radicals was observed in plaisant.

Suchara (1993) exposed 1 and 2 year old needles of Pinus siorbus

and Toxus baccata and observed that total leaf peroxidase and

catalase activities were significantly more active in 2 years old

than I year old. Ranieri et al. (1997) exposed wheat cv. Mec and

Chiarano to 2, 23, 64 or 96 nl 1"' SO2 for 4 months and observed

that SOD activity was found decreased in Chiarano but remained

unchanged in Mec. Reduction in activities of Lipase, peroxidase,

Catalase enzymes of barley leaves was observed when exposed to

1.00 ppm of SO2 (Hagazy et al, 1998). Bernardi et al. (2001) also

studied the effect of SO2 on SOD activity in bean plants. Lee

(2002) exposed Palargonium graveolens to NaaSOs and observed

total peroxidase activity was increased proportionally with the

amount of Na2S03 while the activities of SOD, glutathione

reductase were enhanced with Na2S03 treatments without

significant alteration of catalase activity.

YIELD CHARACTERISTICS

Sulphur dioxide also affects the yield of certain crop plants.

Deepak and Agrawal (1999, 2001) reported that SO2 doses of 0.06

ppm caused reduction in yield of wheat and soybean. Verma et al.

(2000) also reported reduction in grain yield when wheat plants

exposed to 0.15 ppm SO2. Maly (1974) conducted experiments on

18

CHAPTEW-2 S U R V E Y Q F L I T E R A T U R E

pots where SO2 concentration varied from 3350-3640 |ig m" and

reported decrease in yield of flax, seeds (28.3%) and fibre

(23.8%). Decrease in yield has been reported in several other

crops such as potato (16.2%), maize forage (16.7%), oats grain

(12.2%), oats straw (8.1%) and clover (16.5%). Wartevesiewicz

(1979) showed a good correlation between increase in SO2

pollution and yield decrease. He also reported the reduction in

grain yield of barley. Number of seeds per unit ground area was

more affected than the weight. When field plots of soybean were

exposed to elevated levels of SO2 (0.09-0.79 ppm) periodically,

there was a mean decrease in weight of seeds and number of seeds

per plant. The total yield was reduced by 5-48% and there was also

reduction in harvest ratio (Sprugel et a!., 1980). Kumar and Singh

(1985, 1986, 1987, 1988) reported significant reduction in yield

and yield attributes of different cultivars of Pisum sativum, Vigna

sinensis, Cicer arietinum and Vigna mungo exposed to 0.25 and

0.5 ppm SO2. The reductions in seed yield per plant were upto

50% in V. sinensis, 38% in Cicer arietinum and 42.8% in Vigna

mungo cultivars.

Keeping in view the importance of SO2 as an important

gaseous pollutant and its reducing effects on crop plants, an

attempt in the present study was made to find the level of

reduction and its associated changes in some antioxidant enzymes.

19

' / '

S 0 : ' - 2 1 ^ sqT-Tir- SOi ' H lO ~ — ^

soj- h7/d»v» _5r x , ^ '" :rr..-iiTr^ -ox SOj HJO

H2S03

HiS04

so<

I

S03 HSOj

H ,

2 S04

1 p H r H j S O a HSO3

M A T E R I

A L 5

A N D

M E T H a D 5

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

Black gram (Vigna mungo L. Hepper) cv. T-9 was selected for

the experiment to study the effect of sulphur dioxide on growth

and yield characteristics and physiological changes associated

with it. Experimental details are given below.

EXPERIMENTATION

The seeds obtained from Indian Agricultural Research

Institute, New Delhi, were surface sterilized with 0 .01% HgCh

and washed several times with demineralised water. Healthy seeds

were sown in 23 cm diameter earthen pots filled with sterilized

mixture of compost and soil (1:3). The available S and N of the

soil was 100 mg kg"' soil. After germination and seedlings

establishment, two healthy seedlings were maintained in each pot.

The pots were kept in a green house of the Department of Botany,

Aligarh Muslim University, Aligarh under natural day length

conditions. Plants were exposed to a determined doses of SO2 (0.0,

O.I and 0.2 ppm) for 3h. Each treatment was replicated ten times

( 3 x 1 0 = 30 pots in total).

P L A N T E X P O S U R E T D S U L P H U R D I O X I D E

EXPOSURE CHAMBER

The plants grown in pots were exposed to SO2 in an open top

chamber (Standard Appliance, Varanasi) (Figure-2). The exposure

chamber of 90 x 90 x 12O cm dimension was equipped with a full

size of moveable door, and an exhaust duct (20 x 20 cm) at its top

to carry out the air and gaseous mixture if any. The bottom of the

chamber was double walled. A blower assembly was attached at

the bottom of the chamber. Voltage supply to blower assembly was

regulated by a voltage stabilizer to maintain air flow at a rate of

2.0 ± 0.013 m^'s"', which was fast enough to overcome

Aluminium frame

Transparent fibre glass .

Movable door

Meshed partition

ti'ay

Double layered bottom

Blower assembly

Exhaust duct

Perforation in the upper layer for gas and air entry in the

chamber SO2 Generator

\U

Conti ol panel of the blower assembly to control air flow

rate

Figure-2. A schematic of an exposure chamber.

C H A P T E R - 3 M A T E R I A L S A N D M E T H O D S

aerodynamic resistance. The air (with SO2) inside the chamber was

being replaced approximately seven times in one minute. During

the exposure, the air flow rate was measured at different places in

the exhaust duct (20 x 20 cm) by electronic anemometer.

SULPHUR DIOXIDE OENERATION

SO2 was produced in a generator by reacting sodium sulphite

(NaSOj) with diluted sulphuric acid (10% H2SO4) under controlled

conditions. The amounts of Na2S03 and H2SO4 loaded in the

reagent bottles and mounted over the SO2 generator was

predetermined by collecting the solution through capillary tubes in

a graduated cylinder for a known period of time. The rate in

ml min"' was thus regulated for desired concentration of SO2. It is

known that on complete reaction of IM Na2S03 produces I M SO2

or 126g of NajSOj with 10% H2SO4 produces 64g SO2.

Na jSOs + H2SO4 > Na2S04 + H2O + SO2 (126g) (98g) (142g) (18g) (64g)

(64g SO2 occupy 2 2 , 4 0 0 c c at N . T . P . ) On the basis of this equation, a desired concentration of

Na2S03 solution was prepared to obtain 286 ng m""* (0.1 ppm) and

571 ng m"" (0.2 ppm) SO2 inside the exposure chamber, keeping in

m view the air flow rate (2.0 ± 0.013 m^s"'). The outlet (0.4c

diameter) of the SO2 generator was connected to the blower

assembly of the exposure chamber.

SULPHUR DIOXIDE MONITORING

To assay the actual concentration of SO2 inside the chamber,

a handy air sampler (Kimoto Electricals, Japan) was placed on the

meshed iron tray of the chamber. A 20ml of sodium

tetrachloromercurate solution (absorbing medium for SO2) was

filled into impingers of the sampler. The air was pumped in

through absorbent at a rate of 1.5 litre m"'. After 3h of sampling

21

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

exposure, the liquid absorbent was replaced and analysed spectro-

photonietrically to determine the actual concentration of SO2

(Anonymous, 1986).

EXPOSURE: SCHEDULE

Five pots of ten days old plants were exposed to 0.1 and 0.2

ppm of SO2 for 3h. The plants were exposed on alternate days to

each concentration of SO2 (0.1 and 0.2 ppm) for 23 events i.e.

upto 46 days. The pots of the control set (0.0) were also placed in

an exposure chamber for 3h with an air exchange rate of 2.0 ±

0.013 m•^s"^ This chamber was not connected to an SO2 generator

but received ambient air. Next day of final exposure, three pots

from treatment and control were taken for determining the growth

and yield characteristics. Two pots were selected from each

treatment for photosynthetic characteristics and enzyme analyses.

GROWTH CHARACTERISTICS

1. Shoot length

2. Root length

3. Leaf number plant"'

4. Leaf area plant"'

5. Shoot fresh mass

6. Root fresh mass

7. Root/Shoot length ratio

8. Shoot dry mass

9. Root dry mass

10. Nodule number plant"'

1 1. Nodule fresh mass

12. Nodule dry mass

CARBOfsl ASSIMILATION

1. Net photosynthetic rate

22

C H A P T E R - 3 M A T E R I A L S A N D M E T H O D S

2. Stomatal conductance

3. Physiological water use efficiency

4. Intercellular CO2

5. Carboxylation efficiency

6. Chlorophyll a, b and total chlorophyll

7. Carotenoids

8. Chlorophyll a : Chlorophyll b

9. Total chlorophyll : Carotenoids

10. Carbonic anhydrase activity

11. Soluble and Insoluble carbohydrate

12. Structural and non-structural carbohydrate

13. Starch

NiTRaOEN AND SULPHUR ASSIMILATION

1. Nitrate reductase activity

2. Nitrite reductase activity

3. N, P and S content

4. N/S ratio

5. Soluble, insoluble and total protein

6. Proline content

ANTIOXIDATIVE ENZYMES

1. Superoxide dismutase

2. Catalase

3. Peroxidase

4. Ascorbic acid

YIELD CHARACTERISTICS

1. Bioniass

2. Number of pods plant"'

3. Pod length

4. Number of seeds pod"'

23

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

5. Seed yield

6. Harvest index

7. 100 seed weight

P H D T D S Y N T H E T I C R A T E , S T O M A T A L C O N D U C T A N C E

AND I N T E R C E L L U L A R COg

Photosynthetic rate, stomatal conductance and intercellular

CO2 on fully expanded leaves of the plant was measured using Li-

COR 6200 portable photosynthesis system (Nebraska, Lincoln).

The leaves at fixed nodes on four plants were selected for

photosynthesis measurement. It was done at about 1100

limol m''^s"' saturating photosynthetic active radiation at

1100-1200h.

PHYSIDLDBICAL WATER USE EFFICIENCY

The physiological water use efficiency was calculated by

using the data of photosynthetic rate and stomatal conductance

(Das et al., 1999).

CARBDXYLATIDN EFFICIENCY

Carboxylation efficiency was calculated by using the data of

photosynthetic rate and intercellular CO2.

ASSAY OF CARBONIC ANHYDRASE ACTIVITY

The leaves used for photosynthesis measurement were

divided into two halves. Half of the portion was used for carbonic

anhydrase assay and the other half for chlorophyll estimation.

Carbonic anhydrase (CA) was assayed by adopting the method of

Dwivedi and Randhava (1974). The fresh leaf samples were cut

into small pieces at a temperature below 25°C. 200mg of the leaf

pieces were further cut into smaller pieces in 10ml of 0.2M

cysteine (App., i) and left for 20 minutes at 4°C. The leaf pieces

were taken out from the petriplates and adhering solution was

24

CHAPTER-3 MATERIALS AND METHODS

soaked with the help of blotting paper. These dried samples were

then transferred to a test tube containing 4ml phosphate buffer of

pH 6.8 (App., iii). To this tube 4ml 0.2M sodium bicarbonate

(NaHCO.O in 0.2M sodium hydroxide solution (App., iv) and 0,2ml

of bromothymol blue (0.002%) indicator (App., i) were added. The

tube was shaken gently and left for 20 minutes at 4°C. CO2 .

liberated by catalitic action of CA on NaHCO^ was estimated by

titrating the reaction mixture against 0.0IN hydrochloric acid

(App., ii) using methyl red as an indicator. The control reaction

mixture was also titrated against 0.0 IN HCl. In each case the

quantity of HCl used to neutralize the acid was noted and the

difference was calculated. The activity of the enzyme was

calculated by putting the values in the formula.

V X 22 X N

W Where, V = difference in volume of HCl in control and the

sample

22 = equivalent weight of CO2

N = normality of HCl

W = weight of tissue used

C H L O R D P H Y L L A N D C A R O T E N Q I D E S T I M A T I O N

The chlorophyll (chlorophyll a, chlorophyll b and total

chlorophyll) and carotenoid content of leaves on which

photosynthetic measurements were made, estimated by the method

of Mac Kinney (1941) and Ma Clachlan and Zaiik (1963)

respectively. 100 mg fresh tissue from interveinal areas of leaves

was grind in mortar and pestle in 5ml 80% acetone. The

suspension was filtered with whatman filter paper no . l , collected

in a volumetric flask and final volume was made 10ml with 80%

25

CHAPTER-a M A T E R I A L S A N D M E T H O D S

acetone. The optical density (O.D.) of the solution was read at 645

and 663 for chlorophyll and at 480 and 510nm for carotenoid

estimation on a spectrophotometer (SL-171, Elico, Ahmedabad,

India). The chlorophyll and carotenoid content (mg g ' fresh

tissue) were calculated according to the formula given below:

Chlorophyll a = [0.0127 x 663 (OD) - 0.00269 x 645 (OD)] x Df

Chlorophyll b = [0.0229 x 645 (OD) - 0.00468 x 6 6 3 ( 0 0 ) ] x Df

V X W

Total chlorophyll = 20.2(645) + 8.02(663) x 1000

7.6(OD480) - 1.49(OD510) Carotenoid = xV

d X 1000 X W

OD = optical density at the given wavelengths viz. 645, 663, 480

and 510nm

V = final volume of chlorophyll extract in 80% acetone

W = fresh mass of leaf tissue (g)

d = length of light path (d = 1.4cm)

S T R U C T U R A L AND N D N - S T R U C T U R A L C A R B O H Y D R A T E

For estimation of structural and non-structural carbohydrate,

estimation of soluble, insoluble, total sugar and starch were done.

Soluble and insoluble carbohydrates were extracted according to

the method of Yih and Clark (1965) and estimated by the method

of Dubois et al. (1956).

EXTRACTION

50mg of dried leaf sample powder was transferred to a

centrifuge tube. 5ml of 80% alcohol was added and then heated for

10 minutes on a water bath at 60°C. After cooling, the samples

were centrifuged at 4,000 rpm for 10 minutes. Supernatant was

taken in a 25ml volumetric flask and volume was maintained with

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

80% ethyl alcohol (App., i). This extract was used for soluble

carbohydrate. The residue was used for the estimation of insoluble

carbohydrate. In a centrifuge tube containing the residue, 5ml of

I.5N H2SO4 (App., iv) was added and heated on a water bath for

2h at 90-95°C. After cooling, it was centrifuged at 4,000 rpm for

10 minutes. Supernatant was collected in 25ml volumetric flask

and the volume was maintained with distilled water.

ESTIMATION OF SOLUBLE CARBOHYDRATE

1ml of ethyl alcohol extract was taken in a test tube was

dried on a water bath. The test tube was taken out from the water

bath when alcohol was completely evaporated and cooled by

keeping in chilled water. After cooling, 2ml of distilled water, 1ml

of 5% distilled phenol (App., ii) and 5ml of concentrated H2SO4

were added. The colour of the extract turned yellow orange. It was

cooled for half an hour and read at 490nm on a spectrophotometer

(SL-171, Elico, Ahmedabad, India).

ESTIMATION or INSOLUBLE CARBOHYDRATE

1ml of H2SO4" extract was taken in test tube to which 1ml of

5% phenol and 5ml of concentrated H2SO4 were added and a

yellow orange colour was developed which was read at 490nm on a

spectrophotometer (SL-171, Elico, Ahmedabad, India).

STANDARD FOR CARBOHYDRATE

40mg of glucose was dissolved in 100ml of distilled water.

From this stock solution 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9

and 1.0ml were taken and volume was maintained upto 2ml with

the DDW. This was followed by the addition of 1ml of 5% phenol

and 5ml of concentrated H2SO4. It was kept in chilled water to

cool. After half an hour colour was read at 490nm on a

spectrophotometer (SL-171, Elico, Ahmedabad, India).

27

C H A P T E R - 3 M A T E R I A L S A N D M E T H O D S

S T A R C H

Starch is the storage form of carbohydrate in plants. It was

determined on dry residue left after sugar extraction (McCready el

al., 1950).

PROCEDURE

200mg leaf sample was homogenised in hot 80% ethanol

(App., i) to remove soluble sugar. It was centrifuged and

supernatant was retained. The residue was washed repeatedly with

hot 80% ethanol till the washing did not give colour with anthrone

reagent. The residue was dried well on a water bath. To the

residue 5ml of water and 6.5ml of 52% perchloric acid (App., ii)

were added and extracted at 0°C for 20 minutes. It was centrifuged

and supernatant was saved. The extraction was repeated using

fresh perchloric acid. The supernatant was collected after every

centrifugation and the volume was made to 100ml. 0.1ml of the

supernatant was pipetted and diluted to 1ml with DDW. Following

this 4ml of anthrone reagent (App., i) was added to each tube. It

was heated for 8-10 minutes on a boiling water bath, which was

cooled rapidly. The green colour intensity was read at 630nm on a

spectrophotometer (SL-171, Elico, Ahmedabad, India).

STANDARD FOR STARCH

Stock solution was prepared from glucose. lOOmg of glucose

was dissolved in lOOml of DDW. lOml of stock solution was

diluted to 100ml with double distilled water. From this solution

0.2, 0.4, 0.6, 0.8 and l.Oml of solutions were taken and final

volume made upto 1ml in each test tube with distilled water. To

this 4ml of anthrone reagent was added to each test tube and

heated for 8-10 minutes on boiling water bath and then it was

cooled rapidly. The intensity of green to dark green colour was

28

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

read on a spectrophotometer (SL-171, Elico, Ahmedabad, India) at

630nm.

A S S A Y O F N I T R A T E R E D U C T A S E A C T I V I T Y

NR activity was estimated in fresh leaf. Leaves were cut into

small pieces. The enzyme activity was determined according to the

method by Jaworski (1971). 500mg fresh leaf pieces were placed

in plastic vials. To each, 2.5ml phosphate buffer pH 7.5 (App.,

iii), and potassium nitrate (0.02M) solution (App., iii) were added,

followed by the addition of 2.5ml 5% isopropanoi. Later, two

drops of chloramphenicol solution were added to avoid bacterial

growth in the medium. The vials were incubated for 2h in dark at

28°C. 0.4ml incubated mixture was taken in a test tube to which

0.3ml each of 1% sulphanilamide (App., iv) and 0.02% N-1

nepthylethylenediaminehydrochloride NED-HCl (App., ii) were

added. The solution was left for 20 minutes for maximum colour

development. It was diluted to 5ml with sufficient amount of

double distilled water and optical density was read at 540nm using

a spectrophotometer (SL-171, Elico, Ahmedabad, India). A blank

consisting of 4.4ml double distilled water and 0.3ml each of

sulphanilamide and NED-HCl were used simultaneously for

comparison. A standard curve was plotted by taking known graded

dilution of potassium nitrate from a standard aqueous solution of

this salt. The optical density of the sample was compared with this

calibrated curve and NR activity was expressed as r|mol N02"g''h"'

fresh leaf tissue and 0.02% N-1-naphthylethylenediamine

dihydrochloride. It was centrifuged at 2,000 rpm for 10 minutes.

Supernatant was collected and absorbance was read at 540nm on

spectrophotometer (SL-171, Elico, Ahmedabad, India)

29

CHAPTER-S M A T E R I A L S A N D M E T H O D S

A S S A Y D F N I T R I T E R E D U C T A S E A C T I V I T Y

NiR activity was estimated in fresh leaves according to the

method of Vega et al. (1980). 500 mg leaf tissue was homogenised

with 10ml of Tris-HCl buffer (pH 7.5) in plastic vials kept in ice

and homogenate was filtered through cheese cloth. Filterate was

used as a enzyme source. A reaction mixture was prepared by

mixing 6.25ml of Tris-HCl buffer, 2ml of NaNOs, 2ml methyl

violgen solution (App,, ii) and 14.75ml double distilled water,

1.5ml reaction mixture and 0.3ml of enzyme preparation was taken

into a test tube. Blank was run along with. 0.2ml of freshly

prepared sodium dithionite bicarbonate solution (App., iv) was

added and incubated for 15 minutes at 30°C. Reaction was stopped

by vigorous shaking, blue colour disappears. 20ml aliquot was

taken out for nitrate determination. Reaction was terminated by

the addition of 1ml sulphanilamide (App., iv) followed by 1ml

naphthylethylenediamine reagent (App., ii). Left for 30 minutes

and absorbance was measured at 540nm. A standard curve was

prepared by taking different known aliquots of potassium nitrite

standard solution into a series of test tubes and volume was made

2ml with distilled water. Reaction was terminated by adding 1ml

of sulphanilamide and 1ml naphthylethylenediamine. After 30

minutes, absorbance was read at 540nm. The optical density of the

sample was compared with the calibrated curve.

NITROGEN, PHOSPHDRUS AND SULPHUR CONTENT

DioESTiasi

lOOmg of the oven dried powder from each replicate was

transferred to a 50ml Kjeldahl flask to which 2ml of sulphuric acid

was added. The contents of the flask was heated on temperature

controlled assembly for about 2h to allow complete reduction of

30

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

nitrates present in the plant material by the organic matter itself.

As a result, the content of the flask was turned black After

cooling the flask for about 15 minutes, 0.5ml of 30% H2O2 was

added drop by drop and the solution was heated again till the

colour turns to light yellow. Again after cooling for 30 minutes an

additional 3-4 drops of 30% H2O2 was added, followed by heating

for another 15 minutes. The process was repeated till the contents

of the flask turned colourless. The peroxide digested material was

transferred from Kjeldahl flask to 100ml volumetric flask with

three washings of double distilled water (DDW). The volume of

the flask was made up to the mark with DDW. This peroxide

digested material was used for the estimation of N and P content.

ESTIMATION ar NITROGEN

Nitrogen was estimated according to Lindner (1944). A 10

ml aliquot of the digested material was taken in a 50ml volumetric

flask. To this, 2ml of 2.5N NaOH (App., iv) and 1ml of 10%

NaSi02 solution was added which neutralizes excess of acid and

prevent turbidity. The volume of the solution was made upto the

mark with the distilled water. In a 10ml graduated test tube, 5ml

of this solution was taken and 0.5ml of Nessler 's reagent (App., li)

was added. The final volume was made up with double distilled

water. The content of the tube was allowed to stand for 5 minutes

for maximum colour development. Then the solution was

transferred to a colorimetric tube and optical density (OD) was

read at 525nm with the help of spectrophotometer (SL-171, Elico,

Ahmedabad, India).

STANDARD FOR NITROOEN

50mg ammonium sulphate was dissolved in 1 litre DDW.

From this solution 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and

31

CHAPTER-3 MATERIALS AND METHODS

1.0ml was pipetted to ten different test tubes. The solution in each

test tube was diluted to 5ml with DDW. In each test tube 0.5ml

Nessler 's reagent was added. After 5 minutes, the optical density

was read at 525nm on Spectrophotometer (SL-171, Elico,

Ahmedabad, India). A blank was run with each set of

determination. Standard curve was plotted using different

concentrations of ammonium sulphate solution versus optical

density (OD) and with the help of this standard curve the amount

of nitrogen present in the sample was determined.

ESTIMATION OF PHasPHORUs

The method of Fiske and Subba Row (1925) was used to

estimate the total phosphorus in the digested material. 5ml aliquot

was taken in a 10ml graduated test tube and 1ml of molybdic acid

reagent was carefully added, followed by the addition of 0.4ml of

l-amino-2-naphthol-4-sulphonic acid (App., i). Addition of this

turned the colour of the content blue. Volume was made upto 10ml

with DDW. The solution was shaken for five minutes and

subsequently transferred to a colorimetirc tube. The optical

density was read at 620nm on spectrophotometer (SL-171, Elico,

Ahmedabad, India). A blank was also used simultaneously.

STANDARD FOR PHasPHORUs

351mg of potassium dihydrogen orthophosphate was

dissolved in sufficient DDW to which 10ml of ION H2SO4 was

added and the final volume was made to lOOOml with DDW. From

this solution 0.1, 0.2, 0.3 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and l.Oml was

taken in ten different test tubes. The solution in each test tube was

diluted to 5ml with DDW. In each tube, 1ml of molybdic acid

reagent (App., ii) and 0.4ml l-amino-2-naphthol-4-sulphonic acid

(App., i) was added. After 5 minutes optical density was read at

32

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

620nm on spectrophotometer. A blank was run with each set of

determination.

Standard curve was plotted using different dilutions of

potassium dihydrogen orthophosphate solution versus optical

density. With the help of the standard curve, the amount of

phosphorus present in the sample was determined.

ESTIMATION ar SULPHUR

Total sulphur in plant samples was estimated according to

turbidometric method of Chesnin and Yien (1950).

DIBESTION

lOOmg finely ground samples dried at 80°C was taken in

digestion tubes of 75ml capacity. 4ml of acid mixture and 0.0075g

of selenium dioxide as catalyst was added in each digestion tube.

The acid mixture consists concentrated nitric acid and perchloric

acid in the ratio of 1:1. The digestion was carried out till the

digested solution became colourless. Subsequent to digestion, the

volume was made upto 75ml with DDW. The interference of silica

was checked by filtering the contents of the tubes.

DETERMINATION OF SULPHUR

5ml aliquot was pipetted out from the digested solutions for

turbidity development in 25ml volumetric flasks. Turbidity was

developed by adding 2.5ml gum acacia (0.25%) solution, Ig

barium chloride (BaCb, Sieved through 40-60 mesh) and the

volume was made up to the mark, with double distilled water.

Contents of 25ml volumetric flasks were thoroughly shaken till

BaCh was completely dissolved. Turbidity was allowed to develop

for two minutes. The values were recorded at 415nm with

spectrophotometer within ten minutes, after the turbidity

33

CHAPTE:R-3 M A T E R I A L S A N D M E T H O D S

development. A blank was also run simultaneously after each set

of determination.

PROTEIN ESTIMATION

Protein was estimated following the method of Lowry et al.

(1951). 50mg of dried leaf powder was transferred in a centrifuge

tube to which 5ml of double distilled water was added. The sample

was centrifuged at 4,000 rpm for 10 minutes. Supernatant was

collected in 25ml of volumetric flask. Together with two washings

the residue was preserved for the estimation of insoluble protein.

To the residue 5ml of 5% trichloroacetic acid was added. The

solution was then allowed to stand for 30 minutes at room

temperature with thorough shaking. It was then centrifuged at

4,000 rpm for 10 minutes and supernatant was discarded. A 5ml of

IN NaOH (App., iv) was mixed well with the residue and allowed

to stand on water bath at 80°C for 30 minutes. Solution was

allowed to cool and was centrifuged at 4,000 rpm again.

Supernatant together with three washings with IN NaOH was

collected in 25ml of volumetric flask. Volume was made upto the

mark with IN NaOH and used for estimation of insoluble protein.

1ml of NaOH extract was transferred to 10ml test tube and 5ml of

reagent D (App., iv) was added to it. Solution was mixed well and

allowed to stand for 10 minutes at room temperature and 0.5ml of

reagent E (App., iv) was added rapidly with immediate mixing.

After 30 minutes the intensity of blue colour was measured at

660nm.

For soluble protein 1ml of water extract was transferred to

10ml test tube and 5ml of reagent C (App., iii) was added to it.

The solution was mixed well and allowed to stand for 10 minutes

at room temperature and then 0.5ml of reagent E (App., iv) was

34

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

added rapidly with immediate mixing. After 30 minutes the blue

coloured solution was transferred in a colorimeter tube and

absorbance was read at 660nm on a spectrophotometer (SL-171,

Elico, Ahmedabad, India).

STANDARD FOR PROTEIN

A 50mg of BSA (Bovine Serum Albumin) was dissolved in

double distilled water and volume was made upto 50ml. 10ml of

the stock solution was diluted to 50ml with DDW. Iml of this

solution contains 200^g protein. Of this standard solution 0.2, 0.4,

0.6, 0.8 and 1.0ml were taken out into a series of test tubes. The

volumes were made to 1ml in all test tubes. 5ml of reagent C

(App., iii) for soluble protein and 5ml of reagent D (App., iv) for

insoluble protein was added to each tube. It was mixed well and

allowed to stand for 10 minutes. A 0.5ml of reagent E (App., iv)

was added and mixed well and incubated at room temperature in

the dark for 30 minutes. The intensity of blue colour was measured

at 660nm on a spectrophotometer (SL-171, Elico, Ahmedabad,

India).

PROLINE ESTIMATION

Proline content was determined by following the method of

Bates et al. (1973). 500mg of plant material was homogenized in

10ml of 3% aqueous sulfosalicylic acid (App., i) and the

homogenate filtered through Whatman filter paper no.2. 2ml of

filterate was taken in a test tube and reacted with 2ml of glacial

acetic acid and 2ml acid ninhydrin (App., i) on boiling water bath

for Ih at 100°C. The reaction was terminated by keeping it in an

ice bath. The reaction mixture was extracted with 4ml toluene,

mixed vigorously with a test tube stirrer for 20-40 seconds. The

chromophore containing toluene was taken out from the aqueous

35

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

phase with the help of pipette and warmed to room temperature.

Red colour appears and the absorbance was read at 520nm using

toluene as a blank. Purified proline was used to standardize the

procedure for quantifying sample values. A standard curve was

plotted by taking known graded dilution of pure proline from a

standard aqueous solution of this amino acid. The proline

concentration was determined from a standard curve and

calculated by the formula given below:

[ig proline/ml x ml toluene 5 ^ moles proline g"' tissue = ^

115.5|ig/|i mole g sample

A N T I O X I D A N T S A N A L Y S I S

ESTIMATION OF ASCORBIC ACID

Ascorbic acid was estimated by the visual titration method

based on the reduction of 2, 6 dichlorophenol dye (Mahadevan and

Sridhar, 1996).

PREPARATION or EXTRACT

500mg of leaves were crushed in 2ml of oxalic acid in

mortar and pestle for 5 minutes. The extract was filtered through

two layered cheese cloth and centrifuged at 4,000 rpm for 20

minutes. 5ml of 0.4% oxalic acid was taken in a white porcelein

dish and was titrated against the indophenol reagent (App., ii)

until the solution became pink. Standard was prepared for ascorbic

acid. The indophenol reagent was standardised before use. A 5ml

of standard ascorbic acid solution (App., i) in a white porcelein

dish was titrated against indophenol dye until the solution became

pink and the colour persists for at least 15 seconds.

36

CHAPTER-3 M A T E R I A L S A N D M E T H O D S

E S T I M A T I O N O F S U P E R O X I D E D I S M U T A S E ,

P E R O X I D A S E A N D C A T A L A S E

500mg of leaf tissue was homogenised in 5ml of 50mM

phosphate buffer (pH 7.0) containing 1% polyvinyl pyrrilidone.

The homogenate was centrifuged at 15,000 rpm for 10 minutes at

5°C and the supernatant obtained was used as extract for SOD,

APX and CAT.

ESTIMATION OF SUPEROXIDE: DISMUTASE

The activity of SOD was measured by the method of

Beauchamp and Fridovich (1971). A 3ml of reaction mixture

containing 1ml of 50mM phosphate buffer (pH 7.8), 0.5ml of

13mM methionine (App., ii), 0.5ml of 75mM NBT, 0.5ml of lyM

riboflavin (App., iv), 0.5ml of O.lmM EDTA (App., i) and 0.1ml

of enzyme extract was made. Riboflavin was added in the last. The

absorbance of the reaction mixture was read at 560nm on a

spectrophotometer (SL-171, Elico, Ahmedabad, India).

ESTIMATION OF PEROXIDASE

A 3ml of pyrogallol phosphate buffer (App., iii) and 0.1ml

of enzyme extract, 0.5ml of 1% H2O2 were mixed in a cuvette and

change in absorbance at 20 seconds interval for a period of 3

minutes was read at 420nm on a spectrophotometer (SL-171, Elico,

Ahmedabad, India). The control set was prepared by boiling the

enzyme extract (Chance and Maehly, 1956).

ESTIMATION or CATALASE

The estimation of CAT was done by permanganate titration

method (Chance and Maehly, 1956). For estimation of catalase,

3ml of phosphate buffer (pH 6.8) (App., iii), 1ml of H2O2 and 1ml

of enzyme extract were mixed and this mixture was incubated at

25°C for 1 minute. Then 10ml of H2SO4 was added. The mixture

37

CHAPTER-S M A T E R I A L S A N D M E T H a P S

was titrated against O.IN potassium permanganate (App., ii) to

find the residual H2O2 until a joint purple colour persists for at

least 15 seconds. Similarly, a control set was maintained in which

the enzyme activity was stopped by the addition of H2SO4 prior to

the addition of enzyme extract.

STATISTICAL ANALYSIS

The data were statistically analysed using analysis of

variance (ANOVA). F-value was determined and the data were

declared significant, if the calculated F-value was higher than

tabulated F-value. For significant data least significant difference

(LSD) was calculated to separate the means. The statistical

procedure was adopted as described by Gomez and Gomez (1984).

38

P ^ < L ^ ^ - / '

> ^ ' 807- hr/d»y» 0= n ^ " HjS04

R E 5 U L T 5

C H A P T E R - 4 . ^ R E S U L T 5

Results are presented herein mainly as changes in growth,

yield, physiological and biochemical parameters, as influenced by

different doses of sulphur dioxide.

GROWTH CHARACTERISTICS

The data on the effect of different doses of SO2 on

vegetative growth and nodulation of black gram is presented in

Table-1. Both of the concentrations (i.e. 0.1 and 0.2 ppm) of SO2

reduced the growth characteristics significantly, however, the

reduction being maximum at 0.2 ppm. Significant reductions in

shoot length, root length, leaf number plant"', leaf area plant' ,

shoot and root fresh mass and dry mass and nodule number plant' ,

nodule fresh and dry mass were found to be higher at 0.2 ppm than

0.1 ppm. A high decrease in root length, root fresh and dry mass

was noted in 0.2 ppm SO2 than shoot characteristics. Root length,

root fresh and dry masses were decreased by 34.8, 50.0 and 38.5%

in 0.2 ppm compared to control. The nodule number plant ' ' was

significantly decreased at both the concentrations of SO2. The

nodule fresh and dry mass in treated plants were reduced by 48.28

and 51.52% at 0.2 ppm of SO2 over control.

CARBON ASSIMILATION

Table-2 shows the data recorded on the contents of

chlorophyll and carotenoid, rate of photosynthesis, carbonic

anhydrase activity, carboxylation efficiency, structural and

non-structural carbohydrate and starch content to the application

of SO2 concentrations. At higher SO2 dose, reduction in total

chlorophyll and carotenoids were recorded, which were to the

extent of 29.39 and 18.87% respectively at 0.2 ppm. The

chlorophyll a and chlorophyll b were reduced by 36.75 and 22.44%

Table-1. Effect of different doses of sulphur dioxide in the growth characteristics of black gram {Vigna mungo L. Hepper) cv. T-9. Data followed by the same letter in a row are statistically not different at p<0.05.

Parameters

Shoot length (cm) Root length (cm)

Leaf number plant''

Leaf area plant"' (cm^)

Shoot fresh mass (g)

Root fresh mass (g)

Shoot dry mass (g)

Root dry mass (g)

Shoot/Root dry mass ratio (g)

Nodule number plant''

Nodule fresh mass (g plant:') Nodule dry mass (g plant"')

SO2 concentration (ppm)

0.0

28.1a

18.8a

34.7a

421.0a

16.3a

3.8a

4.7a

0.78a

6.0

11.8a

0.15a

0.07a

0.1

22.5b

14.9b

24.7b

317.9b

13.5b

2.3b

4.1b

0.61b

6.7

9.4b

0.1 lab

0.05b

0.2

21.6b

12.3c

22.3b

296.0c

11.7c

1.9b

3.5c

0.48c

7.1

5.5c

0.08b

0.32c

LSD p < 0.05

4.99

0.72

5.91

14.44

0.48

1.48

0.30

0.02

NS

2.10

0.04

0.003

cv

9.15

1.85

9.58

1.85

1.54

24.54

3.21

1.59

11.92

10.41

17.79

2.95

TabIe-2. Effect of different doses of sulphur dioxide in the photosynthetic and carbon assimilation of black gram (Vigna mungo L. Hepper) cv. T-9. Data followed by the same letter in a row are statistically not different at /?<0.05.

Parameters

Photosynthetic rate (H mol CO2 m" s' ) Stomatal conductance (mol m' s'') Physiological water use efficiency (nmol mol"')

Intercellular CO2 (ppm)

Carboxylation efficiency (%) CA activity (m mol CO2 g'' leaf fresh mass s"') Chlorophyll a (mg g"' fresh tissue) Chlorophyll b (mg g'' fresh tissue) Total chlorophyll (mg g'' fresh tissue) Carotenoid (mg g'' fresh tissue) Chlorophyll a/b ratio (mg g"' fresh tissue) Total Chl/Carotenoid ratio (mg g"' fresh tissue) Soluble sugars (mg g'' dry mass) Insoluble sugars (mg g' dry mass) Structural carbohydrate (mg g'' dry mass) Non-structural carbohydrate (mg g'* dry mass) Starch (mg g"' dry mass)

SO2 concentration (ppm)

0.0

12.5a

0.26a

49.0

286.0

4.4a

1.29a

0.99a

0.35a

1.8a

0.48a

2.8

3.8

23.3a

55.0

78.3a

207.1a

128.8a

0.1

10.0b

0.21b

47.7

309.0

3.2b

1.03b

0.77b

0.29b

1.64b

0.40b

2.7

3.7

18.3b

42.8

61.lb

162.4b

101.3b

0.2

8.6b

0.18c

46.7

336.0

2.5c

0.99c

0.62c

0.27c

1.28c

0.39c

2.3

3.3

12,0c

27.6

36,9c

117.8c

73.4c

LSD p < 0.05

2.14

0.004

NS

NS

0.22

0.03

0.02

0,02

0.04

0.01

NS

NS

1.13

NS

6.25

2.61

1.59

cv

9.10

0.84

3,89

14.25

2.84

1,34

1,25

2,32

0,96

0.61

31,96

18,76

2,8

30,85

4,69

0,71

0,69

CHAPTER-4 R E S U L T S

at 0.2 ppm of SO2. Chlorophyll a/b ratio and total

chlorophyll/carotenoid ratio were found to be non-significant. SO2

treatments reduced net photosynthetic rate, stomatal conductance

at both the concentrations. Increase in the intercellular CO2 at

both the concentrations of SO2 was found to be non-significant.

Soluble, structural and non-structural carbohydrates were reduced

significantly by SO2. Reduction was great at 0.2 ppm than 0.1

ppm. The starch content in exposed plants was reduced by 21.35

and 43 .01% in 0.1 and 0.2 ppm respectively over control.

NITROGEN AND SULPHUR ASSIMILATION

Table-3 shows the data on nitrogen assimilation. Inhibition

of parameters such as contents of protein and total nitrogen were

greater at 0.2 ppm SO2. NR and NiR activities were reduced to the

extent of 37.09, 59.65 and 39.22 and 63.77% in exposure to 0.1

and 0.2 ppm of SO2 respectively. Leaf N and P contents were also

significantly reduced by SO2 at both the concentrations whereas S

content was increased with increasing concentration of SO2.

Reduction in soluble protein was statistically significant while

insoluble and total protein was found to be non-significant.

Proline content in the leaves was significantly increased to the

extent of 16.3 and 29.0% respectively at 0.1 and 0.2 ppm over

control.

ANTIDXIDATIVE ENZYMES

Table-4 comprises the data on antioxidants in response to

exposure to selected doses of SO2. Ascorbic acid was significantly

decreased in treated plants by 19.14 and 31.91% at 0.1 and 0.2

ppm of SO2 over control. Activities of superoxide dismutase,

peroxidase and catalase were increased at 0.1 and 0.2 ppm of SO2.

The extent of increase of SOD, APX and CAT was found to be

40

Table-3. Effect of different doses of sulphur dioxide on nitrogen assimilation, protein and proline content of black gram (Vigna mungo L. Hepper) cv. T-9. Data followed by the same letter in a row are statistically not different at;7< 0.05.

Parameters

NR activity (H mol g'' fresh mass h'') NiR activity (|imol NO2 g'' fresh mass)

LeafN (mgg'')

LeafP(mgg-')

Leafs (mgg"')

N/S ratio (mg g'')

Soluble Protein (mg g'' dry mass) Insoluble Protein (mg g'' dry mass) Total Protein (mg g"' dry mass)

Proline (mg g'')

SO2 concentration (ppm)

0.0

0.46a

3.3a

19.5a

2.7a

1.8a

10.7a

6.5a

14.2

20.7

10.0a

0.1

0.29b

2.0ab

12.8b

1.3b

2.4b

5.2b

4.5b

11.8

16.3

11.6ab

0.2

0.19c

l,2bc

8.9c

l.Obc

2,7c

3.3c

3.2c

9.2

12.6

12.9bc

LSD p<0.05

0.04

1.53

0.004

0.13

0.01

1.19

0.73

NS

NS

2.09

cv

5.06

30,85

0,14

35,73

2.13

8.18

6.77

15,16

16,81

801

Table-4. Effect of different doses of sulphur dioxide on antioxidants of black gram {Vigna mimgo L. Hepper) cv. T-9. Data followed by the same letter in a row are statistically not different atp < 0.05.

Parameters

Ascorbic acid (mg g'' fresh tissue)

Superoxide dismutase (units g'* protein s"')

Peroxidase (units g'' protein s'')

Catalase (m moles O2 released g'' protein s"')

SO2 concentration (ppni)

0.0

0.05a

7.9a

22.3a

58.0a

0.1

0.04b

6.2b

31.5b

40.2b

0.2

0,03c

4.8c

36.5c

21.7c

LSD p < 0.05

0.002

0.42

1.07

1.17

cv

2.34

2.97

1.57

1.29

C H A P T E R - 4 R E S U L T S

29.17, 64.58% and 41.26, 63.68% and 85.25, 167.28% at 0.1 and

0.2 ppm respectively. It was observed that among SOD, peroxidase

and catalase, maximum increase was noticed in the case of

catalase at 0.2 ppm which was 167.28% over control.

YIELD CHARACTERISTICS

Table-5 contains data on the effect of varying doses of SO2

on yield traits. Plants exposed to 0.1 and 0.2 ppm of SO2 showed

significant reduction in biomass, number of pods plant ' ' , number

of seeds pod' ' , seed yield and harvest index. These traits were

decreased by 31.18, 28.13, 27.27, 50.72 and 28.47% at 0.1 ppm

and 52.66, 59.38, 45.45, 82.61 and 63.29% at 0.2 ppm

respectively. Significant effect on 100 seed mass could not be

observed. However, reduction in the number of pods plant"' was

recorded greater than the number of seeds pod"', which was to the

extent of 28.13, 59.38% and 27.27, 45.45% at 0.1 and 0.2 ppm of

SO2 respectively over control.

41

Table-5. Effect of different doses of sulphui" dioxide on yield of black gram {Vigna mungo L. Hepper) cv, T-9. Data followed by the same letter in a row are statistically not different atp < 0.05.

Parameters

Biomass (g plant")

No of pods plant'

Pod length (cm)

No of seeds pod'

Seed yield (g planf')

Harvest index (%)

100 seed weight (g)

SO2 concentration (ppm)

0.0

8.7a

16.0a

4.3

5.5a

2.8a

31.9a

3.1

0.1

6.0ab

11.5ab

3.5

4.0ab

1.4ab

22.8b

3.0

0.2

4.1b

6.5b

2.5

3.0b

0.5b

11.7c

2.5

LSD p < 0.05

3.36

5.02

NS

1.65

1.49

4.94

NS

cv

23.73

19.55

46.32

17.44

42.73

9.85

35.70

' / ' - ^ - ^

SO2 *- S0<

H1SO3

ox

1

1 H

1 SO* 1

H

I i

, „ j 1 HSOa 1 SO3

" 1

1 SO4 1

my«rjr£i c

0

S3 ^-^ Ot '^ 0 i 2 § » § g 7

pHpHzSQj HSO3 SO3 i

D I E C U E E I D IM

CHAPTER-5 D I S C U S S I D N

The experimental findings presented in the preceding chapter

provide a detailed account of the physiological and biochemical

basis of performance of black gram {Vigna mungo L. Hepper)

exposed to SO2. An attempt has been made in this chapter to

examine, evaluate and discuss the cellular mechanism(s) affected

in response to SO2 exposure. Given the importance of nitrogen and

sulphur in seed protein content of leguminous crops, the emphasis

on evaluation of SO2 is given on N and S assimilation together

with carbon assimilation as backbone in providing structural

compounds to N and S assimilatory pathways. An attempt has also

been made on the possible involvement of antioxidative enzymes

in detoxification of toxic compounds of SO2.

GROWTH CHARACTERISTICS

Air pollution is one of the serious threats to the present

environment. SO2 is one of the gaseous pollutants of our

atmosphere and is given more importance because of its peculiar

properties that can not be attributed to any other gaseous

pollutants. Duration of exposure of plants to the pollutant plays a

major role rather than the concentration (Arockia Premalatha et

al., 1997). In the present study, the selected doses of SO2 brought

about an inhibitory effect in all growth parameters studied

(Table-J). The decrease in growth corresponds with the increasing

concentration of SO2. The observed higher reduction in root traits

in comparison to shoot was due to low availability and

translocation of photosynthates to the underground parts. The

available less photosynthates were utilised in the growth of above

ground parts. This resulted in greater reduction in root

CHAPTER-5 D I S C U S S I O N

characteristics. The inhibitory effect of SO2 exposure on the

growth traits of various plants have also been observed by other

workers (Lee et al., 1997; Deepak and Agrawal, 1999; Ampily,

1999; Raza and Gouri, 2000; Singh and Singh, 2003). Reduction in

nodulation, nodule fresh and dry mass was due to the reasons

explained above. It may be emphasised that for good root growth

characteristics, shoot photosynthates should be ample to support

root growth. Besides this, another reason for decrease in root

nodulation is decreased bacterial population due to SO2

precipitation on soil.

CARBON ASSIMILATION

Photosynthetic parameters (chlorophyll a, chlorophyll b,

total chlorophyll and carotenoid contents) were reduced in SO2

exposure to the maximum being 0.2 ppm (Table-2). It has been

suggested that chlorophyll destruction by SO2 is caused either by

conversion to phaeophytin or by the production of superoxide

radicals by the reaction of sulphite with chlorophyll under

illumination (Shimazaki et al., 1980). Further, the loss of chl a

was relatively greater than that of chl b, which affected light

harvesting complex and reduced further process of

photosynthesis. Photosynthesis is the first process to be affected

by SO2 pollution. The argument to this reason is that a competition

between SOs^" and CO2 comes into force for binding to the RuBP

carboxylase. The level of inhibition depends upon the rate of

oxidation of SO2 to sulphate and other sulphuryl metabolites. The

present study showed that SO2 concentration significantly

increased the internal CO2 (Ci) concentration due to stomatal

closure and reduced photosynthesis. Increase in Ci has been

reported by Rengua and Paliwal (2002) in Vigna unguiculata

43

CHAPTER-5 D I S C U S S I O N

exposed to SO2. The reduced carbonic anhydrase activity lowered

the supply of dehydration of HCO3", and therefore, limited the

supply of CO2 to RuBP carboxylase resulting in reduced

photosynthetic rate. The relationship between carbonic anhydrase

activity and photosynthesis has been established (Khan, 1994;

Khan et al., 2003). The carboxylation efficiency decreased with

the decrease in photosynthetic rate even with an increase in

internal CO2 concentration. Low photosynthetic activity of SO2

stressed plant led to reduce carbohydrate content. The possible

reason for decreased carbohydrate content in plants treated with

SO2 might be related to the increased respiration to meet the

energy requirement for repair of SO2 induced damage. This caused

reduction in most of food reserves. A significant decrease in

starch content was observed in SO2 exposed plants, suggesting

decrease starch content was due to reduced photosynthesis which

might have resulted due to reduction in green pigments.

N I T R D G E N A N D S U L P H U R A S S I M I L A T I O N

Reduction in nitrate reductase and nitrite reductase activities

was observed {Table-3). The nitrate reductase enzyme catalyzes

the first step of nitrate assimilation pathway and hence, its product

ultimately finds its destiny in protein. The nitrate reductase

mediates conversion of nitrates to nitrites, which requires

electrons from photosynthetic electrons flow chain reactions.

Similarly, first step of sulphur assimilation is mediated by ATP-

suifurylase, which also requires energy and electrons from

photochemical reaction of photosynthesis. It may be said electrons

used in the assimilation of nitrogen affect generation of reducing

power and ATP. The reducing power used in the reduction of CO2

in the chloroplast stroma may not be generated in sufficient

44

CHAPTER-S D I S C U S S I O N

amount, if electrons and ATP are diverted in nitrogen and sulphur

assimilation. This shows that the three processes of carbon,

nitrogen and sulphur assimilation are interlinked and a proper

integration among the three are required for maintaining high

growth, productivity and protein content. A shift in any one

process may lead to imbalance. The integration of working of

three processes is shown in Figure 2. Reduced nitrate reductase

activity decreased the process of protein synthesis.

In the context the significant increase in S content in leaves

is noteworthy, as SO2 damage is caused by air accumulation of S

compounds within the plants. Excess of S may completely inhibit

the RuBP carboxylase and blocks the carboxylation and disrupted

the balance between incompletely oxidised sulphur compounds and

the sulphydryl groups present in glutathione and cysteine that are

essential for the structural integrity of proteins. The available

literature also shows that SO2 exposure increased the accumulation

of S (Pandey and Rao, 1978; Ma and Murray, 1991; Hidlicka and

Kula, 2001; Simoncic, 2001). SO2 treatment lowered the leaf

protein content at both the concentrations. Reduction in protein

content due to SO2 has been ascribed either to the decrease in

protein synthesis or due to hydrolysis of proteins. It may be

suggested that in the presence of atmospheric sulphur, less energy

is required for the reduction of S04^~ to S but less energy input

could not support the efficient reduction of soil derived nitrogen

to proteins. Although there was increase in S metabolism and S

accumulation in plants, but due to reduced supply of nitrate

reduction protein synthesis was less. It appears that there is

regulation of incorporation of S into cysteine and other S containg

amino acids. Moreover, reduced amino acid formation through N

45

Reductant

> Cysteine/ Methionine

ForN 1- Nitrate reductase 2- Nitrite reductase 3- Glutainini synthetase 4- Glutamate synthetase

ForS 1- ATP-sulphurylase 2- APS-sulphotransferase 3- Sulphite reductase 4-Cysteine synthase

Figure-3. Proposed model for coordination among carbon, nitrogen and sulphur assimilation and their regulation.

CHAPTER-5 D I S C U S S I O N

assimilation pathway could not successfully converted into

cysteine and protein. It is thought that the enzyme responsible for

the reduction of NO3" functioned as regulatory key enzyme for the

reduction of atmospheric SO2 into protein. This argument is shown

in the proposed model for coregulation of N and S assimilation

{Figure-3). Proline is an amino acid and normally it is present in

small amounts in healthy plants. A pronounced effect of SO2 is the

accumulation of the proline in plant tissue. In the present study

proline content was increased by SO2 exposure {Table-3). It is a

known fact that increase in proline is a definite consequence of a

stress. This high accumulation of proline definitely points out that

the plants undergo a stress in the presence of SO2. The findings of

Jeyakumar et al. (2003) also support this view.

ANTIDXIDATIVE ENZYMES

In the present study, significant reduction in ascorbic acid

was noticed {Table-4). The reduction was possibly the result of the

oxidizing property of SO2. SO2 readily diffuses into mesophyll

cells of leaves and produces oxy-radicals. These oxy-radicals

reduce ascorbic acid to hydroxyl ascorbic acid. The decrease in

amount of ascorbic acid makes the plant more sensitive to SO2

(Singh et al, 2003). Plants have inherent defence mechanism for

their survival under any kind of stress and may receive protection

against SO2 stress induced toxicity with the help of certain

specific scavenging molecules like superoxide dismutase,

peroxidase and catalase. A considerable increase in SOD, APX and

CAT were observed. SOD catalyses dismutation of superoxides

and active oxygen species. Similarly, APX and CAT provide

measure to reduce levels of peroxides. Additionally increased

level of these enzymes may induce oxidation of sulphite to

46

CO

o

V

SO4

Atmospheric SO2

Initiation

Cell wall V

HSO/ H2SO3 <-

\/

CHLOROPLAST

>

V

Formation of Superoxide radicals (02 -e -—>02- - )

Scavenging ^ Chain reaction

y -Reactive oxygen species

H2O2, 0H-, O2-

\/ Oxidation of biomolecules

V Increased SOD, APX, CAT synthesis \^

Protein

Enhanced oxidation

V

Chlorophyll

SO3

Oxidation

SO. 2-

H,0

Figure-4. Proposed model for SO2 interaction in cell and its detoxification.

so.

V

Production o f Active Oxygen Species'

\ /

Detoxification

Antioxidative system

(SOD, APX, CAT)

Release of H2S

1. Superoxidase dismutase (SOD; EC 1.15.1.1)

It dismutates superoxide (O2* ~) OH " to H2O2

02"" + 02*" + 2H' SOD

•>H202 + 02

2. Peroxidase (APX; EC 1.11.1.7) and Catalase (CAT; ECl. 11.1.7)

It converts H2O2 to H2O

APX CAT H2O2 + H2O2 ^ > 2H2O + O2

Figure-5. Diagrammatic representation of involvement of detoxificating en2ymes.

CHAPTER-5 D I S C U S S I O N

sulphate. It has also been suggested that an increase in APX

activity after SO2 exposure, promotes oxidative processes and thus

indicates plant 's potential for response to a stressful situation

(Horsman and Wellburn, 1975). The activity of antioxidant

enzymes in plant protection is shown in Figures 4-5. Further the

relationship derived as quadratic regression analysis between

SOD, APX and CAT with leaf area (R^ = 0.979**, R^ = 0.985**,

R2 = 0.990**), Shoot dry mass (R^ = 0.976**, R^ = 0.957**, R^ =

0.967**), photosynthetic rate (R^ = 0.915**, R^ = 0.851**, R^ =

0.851**), structural carbohydrate (R^ = 0.955**, R^ 0.972**, R '

0.981**), non-structural carbohydrate (R^ = 0.991**, R^ =

0.992**, R^ = 0.998**), protein (R^ = 0.822**, R^ = 0.756*, R^ =

0.753*), biomass (R^ = 0.802**, R^ = 0.744*, R^ = 0.743*) and

seed yield (R^ = 0.805**, R^ = 0.828**, R^ = 0.825**) shows that

under exposure of SO2 the antioxidant enzyme activities have

effect on growth, physiological, biochemical and yield

characteristics {Figures-6 to 13).

YIELD CHARACTERISTICS

As for the effect of SO2 for other characteristics, the yield

attributes total plant biomass, number of pods, number of seeds

pod" , seed yield and harvest index reduced on exposure to SO2

{Table-5). For other crops, SO2 also inhibited the yield (Deepak

and Agrawal, 1999, 2001; Verma el al., 2000; Singh and Singh,

2003). The reduction in yield may be attributed to the reduction of

growth and photosynthesis which ultimately reduces the supply of

assimilates for normal growth, thereby diverted carbohydrates

from the site of growth to the damaged part, where repair is

needed. Such diversion of material would adversely affect the

productivity.

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CHAPTER-5 D I S C U S S I O N

As discussed in the preceding pages SO2 competes for the

binding with RuBP carboxylase making the enzyme less efficient

in production of photosynthates. The observed decrease in

carboxylation efficiency is in agreement with this statement.

Moreover, reduced N assimilation also resulted in decrease in

growth and physiological process. Nitrogen role in growth and

development and in maintaining enzyme integrity need not be

overemphasised. These processes resulted in reduced yield in

plants exposed to SO2.

CONCLUSIVE REMARKS

The plants exposed to SO2 concentrations showed decreased

growth and yield characteristics. The reason for decrease in

growth was found to be less availability of photosynthates because

of low carboxylating enzyme activity and carboxylation rate,

reducing photosynthesis and dry mass accumulation.

Exposure of plants also led to decrease in the activities of

nitrate reductase and nitrite reductase, the key enzymes

responsible for nitrogen assimilation. Although there was increase

in sulphur assimilation (observed as increased leaf S content), but

protein content was reduced. It may be said that proper rate of

nitrogen and sulphur assimilation could not be maintained due to

drift of more energy and reductant in sulphur assimilation,

resulting in reduced rate of nitrogen assimilation. Moreover,

failure to incorporation of S into S-containing protein (cysteine)

may also be plausible reason for decrease in protein content.

It may be proposed that under conditions of high SO2

concentration, the plants may be managed to enhance nitrogen

assimilation and incorporation of S into S-containing amino acids.

48

CHAPTER-5 D I S C U S S I O N

This may be achieved by focussing the attention on the following

points.

1. The enzymes nitrate reductase/nitrite reductase may be used

as target enzyme for enhancing their activities and nitrogen

assimilation.

2. The plants may be managed to have high nitrogen-use

efficiency through increase in top growth to provide signals

to roots for greater N absorption.

3. The study on metabolite/enzymes responsible for

incorporation of S into S-containing amino acids may be

looked into.

4. The possibility of release of S as H2S-may also be taken into

consideration.

49

.y^ ' ---

H2SO4

0 1 2 3 4 I g pHrn^SOa HSO3 SO3

R E F E R E N C E 5

REFERENCES

Agrawal, M. and Agrawal, S.B. 2000. Research on air pollution:

Impacts on Indian forests. In: Air Polution and the Forests

of Developing and Rapidly Industrializing Regions. Report

No. 4 of lUFRO Task Force on Environmental Change.

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Wood, F.A. 1968. Sources of plant pathogenic air pollutants.

Phytopathol. 58: 1075-1084.

Yang, S.F. and Saleh, M.A. 1973. Destruction of indole-3-acetic

acid during the aerobic oxidation of sulphite. Phytochem. 12:

1463-1466.

Yih, R.Y. and Clark, H.E. 1965. Carbohydrate and protein

content of boron-deficient tomato root tips in relation to

anatomy and growth. Plant Physiol. 40: 312-315.

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A P P E IM D I

X

APPENDIX

> Acid ninhydrin Warm 1.25g ninhydrin in 30ml glacial acetic acid and 20ml 6M phosphoric acid, with agitation until dissolved. Stored at 4°C and used within 24h.

> Amino napthol sulphonic acid 0.5g l-amino-2-napthol-4-sulphonic acid dissolved in 195ml 15% sodium bisulphite solution which 5ml 20% sodium solution was added.

> Ammonium molybdate solution (a) 25.Og ammonium molybdate dissolved in 175ml double distilled water (b) Add 280ml concentrated H2SO4 to 400ml DDW and cool. Mix the two solutions (a) and (b) and final volume made upto 1 litre with DDW.

> Anthrone reagent 200mg of anthrone was dissolved in 100ml of ice cold 95% sulphuric acid.

> Aqueous sulphosalicylic acid (3%) 3ml of Aqueous sulphosalicylic acid and 97ml DDW.

> Ascorbic acid standard solution To 50ml of 0.4% oxalic acid 50mg of ascorbic acid was added and volume was made to 250ml with oxalic acid.

> Bromothymol blue indicator in ethanol (0.02%) 0.002g bromothymol blue dissolved in 100ml DDW.

> Cysteine hydrochloride solution (0.2M) 48g cysteine hydrochloride dissolved in sufficient DDW and final volume made upto 1000ml with DDW.

> EDTA (O.IM) 2.92g EDTA was dissolved in 100ml of DDW.

> Ethyl alcohol (80%) 80ml ethyl alcohol was mixed with 20ml of DDW.

> Folin phenol reagent lOOg tungstate solution and 25g sodium molybdate dissolved in 700ml DDW to which 50ml 85% phosphoric acid and lOOml concentrated hydrochloric acid were added. The solution was reflexed on a heating mantle for 10 hrs. At the end, 150g lithium sulphate, 50ml DDW and 3-4 drops liquid bromine were added. The reflex condenser was removed and the solution was boiled for 15 minutes to remove excess bromine, cooled and diluted upto 1000ml. The strength of this acidic solution was adjusted to IN by titrating it with IN sodium hydroxide solution.

A P P E N D I X

> H2O2 (O.IM) 0.34ml of H2O2 was added to 100ml of distilled water.

> Hydrochloric acid (O.OIN) 0.86ml pure hydrochloric acid mixed with DDW and final volume made upto 1000ml.

> Indophenol reagent 50mg of sodium 2, 6 dichlorophenol was added to 150ml of distilled water and this was warmed gently on hot water to dissolve the dye. To this 42mg of NaHCOs was added, cooled and the final volume was made to 200ml with distilled water.

> KMn04 (O.OIN) This was made by dissolving 0.162g of KMn04 in 500ml of distilled water.

> Methionine (13mM) It was prepared by dissolving 0.193g of methionine in 100ml of DDW.

> Methyl viologen solution Dissolved 60.1mg methyl viologen in 20ml DDW.

> Molybdic acid reagent (2.5%) 1.25g ammonium molybdate dissolved in 175ml DDW to which 75ml ION sulphuric acid was added.

> Naphthylethylenediamine dihydrochloride (NED-HCI) solution (0.02%) 20mg naphthylethylenediamine dihydrochloride dissolved in sufficient DDW and final volume maintained upto 100ml with DDW.

^ Nessler's reagent 3.5g potassium iodide dissolved in 100ml DDW to which 4% mercuric chloride solution was added with stirring until a slight red precipitate remained. Thereafter, 120g sodium hydroxide with 250ml DDW was added. The volume was made upto 1 litre with DDW. The mixture was decanted and kept in an amber coloured bottle.

> Nitrobluetetrazelium (NBT) (75^M) 6.13mg of NBT was dissolved in 100ml of DDW.

> Perchloric acid (52%) 52ml perchloric acid added with 48ml of DDW

> Phenol (5%) 5ml phenol mixed with 95ml of DDW.

> Phosphate buffer (O.IM) for pH 7.5 (a) 13.6g potassium dihydrogen orthophosphate dissolved in sufficient DDW and final volume made upto 1000ml with

A P P E N D I X

DDW. (b) 17.42g dipotassium hydrogen orthophosphate dissolved in sufficient DDW and final volume maintained upto 1000ml with DDW. 160ml of solution (a) and 840ml of solution (b) were mixed for getting phosphate buffer.

> Phosphate buffer (0.2M) for pH 6.8 This was prepared by dissolving 27.80g sodium dihydrogen ortho-phosphate and 53.65g di-sodium hydrogen ortho-phosphate in sufficient DDW separately and final volume of each was maintained upto 1000ml with DDW. To get pH 6.8, 5ml of monobasic sodium phosphate solution was mixed with 49ml of dibasic sodium phosphate solution and diluted to 200ml with DDW.

> Phosphate buffer (50mM) for pH 7.8 It was prepared by mixing 1.78g Na2HP04 and 1.56g of NaH2P04 in 100ml of DDW separately and mixing 91.5ml of Na2HP04 with 8.5ml of NaH2P04.

> Phosphate buffer (pH 6) 3.54g of Na2HP04 was dissolved in 100ml of DDW and 3.72g of NaH2P04 was added to 100ml of DDW. To this 12.3ml of Na2HP04 was added to 87.7ml of NaH2P04.

> Phosphate buffer (O.IM) for pH 6.8 3.54g of Na2HP04 was dissolved in 100ml of DDW and 3.12g of NaH2P04 was added to 100ml of DDW. 49ml of NaH2P04 was mixed with 51ml of NaH2P04.

> Potassium nitrate solution (0.02M) 2.02g potassium nitrate dissolved in sufficient DDW and final volume maintained upto 1000ml with DDW.

> Pyragallol phosphate buffer It was prepared by mixing 25ml of pyrogallol in 75ml phosphate buffer (pH 6).

> Pyrogallol (0.05M) It was prepared by dissolving 0.63g of pyrogallol in 100ml of DDW.

> Reagent A 2% of sodium carbonate + O.IN sodium hydroxide.

> Reagent B 0.5% copper sulphate + 1% sodium tartarate.

> Reagent C Alkaline copper sulphate solution was obtained by mixing 50ml of reagent A with 1ml of reagent B.

> Reagent D Carbonate CUSO4 solution - same as reagent C except for the omission of NaOH.

Ill

A P P E N D I X

> Reagent E Folin's reagent, diluted with double amount of DDW.

> Riboflavin (2M) 0.732mg of riboflavin was dissolved in 100ml of DDW.

> Sodium bicarbonate solution (0.2M) in 0.02M sodium hydroxide solution 16.8g sodium bicarbonate dissolved in sodium hydroxide solution (0.8g NaOH 1'') and final volume maintained upto 1000ml with the sodium hydroxide solution.

> Sodium dithionite-Bicarbonate solution 250mg each of Na2S204 and NaHCOa dissolved in 10ml DDW.

> Sodium hydroxide solution (O.IN) 40g NaOH dissolved in DDW to make 1000ml solution.

> Sodium hydroxide solution (IN) 4g NaOH dissolved in DDW and final volume made upto 100ml.

> Sodium hydroxide solution (2.5N) lOOg sodium hydroxide dissolved in 1000ml DDW.

> Sodium nitrite solution 43.2mg NaN02 dissolved in 20ml distilled DDW.

> Sulphanilamide solution (1%) Ig sulphanilamide dissolved in 3N hydrochloric acid and final volume made upto 100ml with 3N hydrochloric acid.

> Sulphuric acid (7N) 19.6ml concentrated sulphuric acid added to DDW and the final volume made upto 100ml

> Sulphuric acid H2SO4 (1.5N) 10.4ml H2SO4 was added with 239.5ml of DDW.

> Sulphuric acid H2SO4 (2%) 2ml of H2SO4 was added to 98ml of DDW,

IV