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
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'VgC liesearcfi Jiwardee
(Department of (Botany JiCigarH 9/LusCim University JiCigarH - 202002, India Off: (0571)2702016 Ta^. +91-0571-2702016 e-maiC: [email protected] %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.
47
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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.
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A P P E IM D I
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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