Foliar characteristics, cambial activity and wood formation in Azadirachta indica A. Juss. as affected by coal–smoke pollution

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Flora 205 (2010) 61–71

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0367-25

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Foliar characteristics, cambial activity and wood formation in Azadirachtaindica A. Juss. as affected by coal–smoke pollution

Muhammad Iqbal a,b,�, Joanna Jura-Morawiec c, Wies"aw W"och c,d, Mahmooduzzafar a

a Department of Botany, Jamia Hamdard (Hamdard University), New Delhi 110062, Indiab Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, Post Box 2460, Riyadh 11451, Saudi Arabiac Botanical Garden – Centre for Biological Diversity Conservation of the Polish Academy of Sciences, Prawdziwka 2, 02–973, Warsaw 76, Polandd Department of Biosystematics, University of Opole, Oleska 22, 45-052 Opole, Poland

a r t i c l e i n f o

Article history:

Received 24 October 2008

Accepted 14 December 2008

Keywords:

Azadirachta indica

Pollution stress

Stomatal biology

Vascular cambium

Wood characters

30/$ - see front matter & 2009 Elsevier Gmb

016/j.flora.2008.12.003

esponding author at: Department of Botany

ity), New Delhi, 110062, India.

ail address: [email protected] (M. Iqbal).

a b s t r a c t

This study, aimed at elucidating changes in the foliar and cambial behavior in Azadirachta indica (Neem

tree) due to coal-smoke pollution, has revealed inhibitory effects of pollution stress on leaf pigments

concentrations, nitrate reductase activity and the contents of reducing sugars and total N content,

whereas stimulatory effects were given on stomatal index and nitrate and sulphur contents. Under

smoke effects, stomatal conductance was low, leading to a drop in the net photosynthetic rate and a rise

in the internal CO2 concentration of leaf. Cambial reactivation in the stem was delayed at the polluted

site. Although the total span of the cambial activity was reduced, greater amount of wood was observed

to accumulate in the stem axis under heavy pollution stress. Vessel proportion in the wood increased,

whereas size of vessel elements and xylem fibers decreased. ‘‘Vulnerability factor’’ (ratio between mean

vessel diameter and mean vessel abundance) and ‘‘mesomorphic ratio’’ (multiplication product of

vulnerability factor and mean length of vessel element) of the stem–wood, both declined with increase

in the pollution stress, thus indicating a tendency of the species for shifting towards xeromorphy when

grown under stress. Given the opposite trends of photosynthetic rate and wood increment, the carbon-

partitioning pattern rather than the photosynthetic rate seems to have influenced the accumulation of

new wood. The Neem tree proves to be suitable for growing in the polluted areas.

& 2009 Elsevier GmbH. All rights reserved.

Introduction

In major metropolitan cities of India, vehicle exhaust accountsfor over 70% of the total pollutants; most of the remainder iscoming from coal-consuming furnaces, especially from thethermal power plants (Iqbal et al., 2005). On complete fuelburning, the combustion products comprise mainly SO2, NO2, CO2,SO3 and ash, while incomplete combustion produces mainlycarbon monoxide, hydrocarbons, CH4 and C2H4. The pollutantsproduced by coal combustion, including SO2 and NO2, may harmforests as well as crop fields causing tree decline and/or reducedyields. Suspended particulate matters (SPM) may affect pH,electrical conductivity (EC) and the nutrition status of the soil(Iqbal et al., 2000a). Air pollutants may also predispose plants toother biotic as well as abiotic stresses. Exposure to SO2, forinstance, makes plants more susceptible to pathogens such asbacteria, viruses, fungi and nematodes (Khan and Khan, 2000).

H. All rights reserved.

, Jamia Hamdard (Hamdard

Delhi has three thermal power plants (the Badarpur, theInderprastha and the Rajghat), which emit estimated 302 metrictons of pollutants every day (inclusive of particulate matter, SO2,NOx and hydrocarbons). Of the total emissions, about 63% comesfrom the Badarpur thermal power plant (BTPP) alone, which emitssmoke through its 150 m high stacks with a flue gas density of 0 to8 kg/m3 (CPCB, 1995). It runs on a bituminous coal, rich in carbon,sulphur, hydrogen, nitrogen and oxygen, and releases an averageof 12.181 kg of SO2, 277.589 kg of NO2 and 1.574.166 kg of CO2 perhour.

Radial growth of woody plants (formation of secondaryvascular tissues) owes to the activity of vascular cambium, alateral meristem, which produces the wood (secondary xylem) onits inner side and the bark (secondary phloem) on the outer side.In the majority of woody plants, meristematic activity of thecambium is periodic rather than continuous. In the dicotyledo-nous trees of north India, cambial activity generally starts inspring or summer when the atmospheric temperature is moder-ately high, attains its zenith during the humid and warm monsoonseason, and later slows down/stops in cold winters (Iqbal, 1994,1995). The rate of cambial cell division as well as the duration ofthe active phase of the cambium determines the extent of annual

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increment of secondary vascular tissues. All these parameters areaffected by environmental conditions of the habitat (Iqbal, 1995;Pozgaj et al., 1996). Increase in temperature, photoperiod and/orhumidity enhance the cambial growth, provided the availability ofwater is not limiting (Iqbal, 1995; Iqbal et al., 2005). Atmosphericpollutants also affect cambial development (Wali et al., 2007),cambial activity and tissue differentiation (Gupta and Iqbal, 2005;Rajput and Rao, 2005; Tulik, 2001).

Despite extensive studies carried out to understand the impactof environmental pollution on different aspects of plant perfor-mance, plant-pollutant interaction with reference to radial growthin woody species is little documented. This is more so for tropicaland sub-tropical species. The present study was undertaken toinvestigate the impact of emissions of a thermal power plant onfoliar characteristics and the stem cambial activity in Azadirachta

indica A. Juss. (Neem tree, family Meliaceae), a tree known for itsantiseptic and antidiabetic medicinal value: It was the basicassumption that pollution stress may hamper the rate ofphotosynthesis, which in turn would affect the extent of radialgrowth determined largely by wood formation. Such studies ontrees may be helpful in identifying some arboreal species that arecapable to withstand the environmental stress and may thereforebe introduced in the polluted and/or the marginal areas.

Materials and methods

Topography and climate

The state of Delhi, stretching along the western bank ofthe Yamuna River between 281120–281530 north and 761500–771230 east, experiences a dry and tropical monsoon type ofclimate having four different seasons, namely, winter (December–February), pre-monsoon (March–May), monsoon (June–August)and post-monsoon (September–November). In winter, the averageminimum temperature lies around 5 1C, whereas in summer (thepre-monsoon and monsoon periods), the mean maximumtemperature reaches 40 1C. The average rainfall is low during thewinter and pre-monsoon times but often very high during themonsoon, resulting in about 69% relative humidity on average.

Selection of experimental sites

Five sites (I, II, III, IV and V) located about 1/2, 2, 6, 9 and 15 km,respectively, from the BTPP along the canal road in the down-stream (south–east) direction, were selected for this study. Thewind blows frequently in this direction, particularly during themonsoon season. This area was almost free from any local sourceof pollution like vehicle traffic or industrial plants, and possessedcomparable grassland communities. All the study sites had asandy loam type of soil (pH value 7.62–8.23), with a good drainagesystem. The soil was coarse in texture and low in the organicmatter and nitrogen status.

Methods applied

The normal environmental variables and the concentrations ofmajor gaseous pollutants were monitored at each sampling sitewith the help of a portable Weather Station equipped withadditional gas sensors (ELE International Ltd., UK). Concentrationof particles in the atmosphere was determined using a HighVolume Air Sampler. The soil of each site was analyzed for total Nand S and for the available K (ammonium acetate–K) contents,using standard lab. techniques (Kalra and Maynard, 1994).

Growth performance of A. indica was examined on the basis ofleaf characterstics (leaf area, stomatal features, chlorophyllcontents, net photosynthetic rate, nitrate reductase activity andaccumulation of various metabolic products) and radial growthpatterns (cambial activity and wood production), as observed inmonthly collections of leaves and wood blocks obtained from five40-year-old (about 20 m high) trees growing singly (not in stands)at each of the selected sites. Samples to be used for structuralstudies were fixed in formalin–acetic acid–alcohol (FAA), afixative containing 70% alcohol, 40% formaldehyde and glacialacetic acid in a 85:10:5 ratio. Fresh material was used forbiochemical analyses.

Epidermis studies

Epidermal peels of five leaves per plant, collected from fivetrees at each site, were obtained by heating the cut pieces of leafwith 60% nitric acid, as described by Ghouse and Yunus (1972).The peels, dehydrated in the customary ethanol series and stainedwith safranin/Bismarck brown, were mounted in Canada balsamon glass slides for microscopic study. One hundred stomata fromeach slide were measured with an ocular scale under microscope.The stomatal index (S.I.) was calculated following Salisbury(1927). One square centimeter pieces from the central part oflamina of the fully expanded hypostomatic leaves were cleanedwith 0.1 M phosphate buffer (pH 7.4), fixed in a modifiedKarnovsky (1965)’s fluid, post-fixed in 10% OsO4, dehydrated ina graded acetone solution, dried with liquid CO2 and gold-coatedin an inert argon gas atmosphere (Agar Sputter Coater P 7430).The preparations thus obtained were examined under a Leo 435VP (Cambridge, UK) Scanning Electron Microscope operated at15–25 KV.

Photosynthesis and pigment concentration

Leaf area was measured using a Leaf Area Meter with ImageAnalysis System (Sky Instruments Ltd., UK). Stomatal conductance(gs), intercellular carbon dioxide concentration [CO2]i and netphotosynthetic rate (PN) were measured each month by clampingindividual leaves from exposed twigs of the upper crown in theleaf chamber of a portable LI–6200 Photosynthesis MeasuringSystem (LI-COR, Inc. Lincoln, USA) which measured transientexchange rates of water vapor and CO2 in a closed system.Measurements were taken from ten leaves of each of the fiveplants at a given site on clear sunny days around noon to ensurefull sunlight. The chlorophyll and carotenoid contents of freshleaves were estimated by the method of Hiscox and Israelstam(1979), using dimethyl sulphoxide (DMSO). Chlorophyll concen-tration, measured on a DU 640B Spectrophotometer, and calcu-lated by the formulae of MacLachlan and Zalik (1963) andDuxbury and Yentsch (1956), was expressed in mg/g of fresh mass.

N-assimilation-related parameters

Nitrate reductase (NR) activity in leaves, collected in theforenoon (10–11a.m.), was determined by the method of Klepperet al. (1971), treating fresh leaves with phosphate buffer (pH 7.2),KNO3 (0.4 M), sulphanilamide (1% in 1N–HCl) and NEDD [N(1-napthyl)ethylene diamine dihydrochloride] (0.02%), and read-ing the absorbance at 540 nm on a DU 640 B Spectrophotometer.The corresponding concentration of nitrite was determinedagainst a standard curve of sodium nitrite (NaNO2) solution.

Nitrate extraction was done by the method of Grover et al.(1978), which involves treatment of fresh leaves with charcoal,CuSO4–ZnSO4 solution, hydrazine sulphate, 0.1N NaOH anddistilled water. Chilled acetone was used to stop the reactionand remove extra hydrazine sulphate from the aliquot. Sulphani-lamide and NEDD were then added for color development.

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Absorbance was read at 540 nm (Evans and Nason, 1953). Thecorresponding nitrate concentration was measured against astandard curve of sodium nitrate (NaNO3) solution.

The reduced nitrogen content was determined by the methodof Linder (1944), using leaf powder digested in a mixture ofconcentrated sulphuric acid and perchloric acid (2:1) and treatedwith H2O2, NaOH (2.5N), sodium silicate (10%) and Nessler’sreagent. Absorbance was read at 440 nm. The correspondingN concentration was measured against a standard curve ofammonium carbonate solution.

Protein, sugar and sulphur contents

Total soluble protein content of leaves was determined by themethod of Bradford (1976), using 0.2 M phosphate buffer (pH 7.0),0.1 N NaOH, TCA and Bradford reagent. Absorbance was read at595 nm, and protein concentrations were measured by using thebovine serum albumin as standard.

The soluble sugar in leaf was determined by the method of Dey(1990), using alcohol, 5% phenol and analytical grade sulphuricacid. Absorbance was recorded at 485 nm, and sugar concentra-tion was measured against a standard curve of glucose solution.

Estimation of sulphur content in leaf tissues was done usingoven-dried leaf powder digested in HNO3 and HCl, and treating itwith 3% glycerol and 2% BaCl2, as proposed by Chesnin and Yien(1950). Absorbance was recorded at 420 nm, and actual sulphurconcentration was measured with the help of a standard curve ofpotassium sulphate solution.

Table 1Mean values of the seasonal and annual concentrations of SO2, NO2 and suspended

particulate matters (SPM) as obtained from the different study sites (I–V).

Seasons I II III IV V

Concentration of SO2

Winter 46.34 32.55 59.72 31.44 13.11

Pre-monsoon 34.47 23.79 41.22 22.86 10.49

Monsoon 36.86 27.92 44.07 29.06 13.64

Post-monsoon 39.95 27.03 55.31 23.16 14.88

Annual average 36.40 27.82 50.10 26.62 13.03

Concentration of NO2

Winter 31.48 27.85 85.00 45.04 22.34

Pre-monsoon 33.70 28.76 67.05 45.75 21.13

Secondary vascular growth

Blocks of 2 cm3 (containing sapwood, cambium and bark) werechiseled out from tree trunks at breast height. One-year-old twigswere also collected for measuring the annual increment of wood.The collected samples were fixed in FAA for 14 days and thentransferred to a mixture of glycerol and 70% ethanol (V:V) forsoftening and preservation. Twelve micrometer thick transversesections, obtained on a Reichert Sliding Microtome and stainedwith Heidenhains haematoxylin and Bismarck brown (Johansen,1940), were mounted in DPX mountant (a synthetic resinmounting media comprising of distyrene, a plasticizer and xylene)for microscopic study.

To study the variation in size of fibres and vessel elements, thinslices of preserved wood collected from the trunk region weremacerated in hot 90% HNO3, washed with water, stained withsafranin, mounted on glass slides after separating the componentcells with the help of a needle and studied under microscope.Vessel elements and fibres, 100 each per sample, were measuredon a random basis. The area occupied by vessels in transverseplane was calculated using camera lucida drawings of vessellumens on tracing paper of uniform thickness, as described byGhouse and Iqbal (1975). A ‘‘Vulnerability factor of wood’’ wasworked out by dividing the mean vessel diameter by the meanvessel number per square millimeter of transverse area of wood,whereas the ‘‘mesomorphic ratio’’ was determined by multiplyingthe vulnerability value with mean length of vessel elements, asdescribed by Carlquist (1977a).

Monsoon 30.28 27.99 70.27 40.15 21.00

Post-monsoon 29.30 26.26 68.73 40.42 19.73

Annual average 31.19 27.71 72.76 42.84 21.05

Concentration of SPMWinter 179.0 180.2 253.5 158.1 102.0

Pre-monsoon 231.3 223.2 312.2 188.3 138.6

Monsoon 272.8 233.6 295.1 213.1 158.4

Post-monsoon 198.6 194.2 258.6 209.3 148.1

Annual average 212.1 207.8 279.9 192.2 137.6

The values (all in mg m�3) are based on weekly observations.

Data analysis

The data obtained from the monthly collections of materialswere pooled so as to find out the mean values for the winter(December–February), pre-monsoon/summer (March–May), mon-soon (June–August) and post-monsoon (September–November)seasons of a growth year. ANOVA test was applied to the data todetermine the significance of differences between sample means;

values obtained from differently polluted study sites are to becompared with the respective value from the reference site.

Results

Properties of the soil were similar at all the study sites; the N, Sand K contents ranging from 15.2 to 26.2, 7.4 to 10.7 and 40.7 to53.0 ppm, respectively. Monthly monitoring of SO2, NO2 andsuspended particulate matter (SPM) showed that site III was mostpolluted, whereas site V was non-polluted (Table 1). Values ofsome important parameters such as PN, pigment contents andcambial periodicity obtained from this site were similar to thosecollected from a site VI with a clean environment (data not given),thus confirming that the effects of BTPP emissions were nil ornegligible at site V, which could therefore be regarded as thereference site.

Foliar features

At the polluted sites the leaves of A. indica showed a varyingdegree of chlorosis and necrosis. Leaflet size increased withincrease in the degree of the pollution stress. The mean leafletarea was at maximum (about 16 cm2) at site III and minimum(about 12 cm2) at site V.

Pigment concentration

Concentration of photosynthetic pigments (chlorophylls andcarotenoids) was maximum in the monsoon and minimum in thepost-monsoon or winter seasons. In a given season, it declinedwith increase in pollution load, being the lowest at site III. On thewhole, chlorophyll b was more sensitive than chlorophyll a to thepollution stress. Carotenoids occupied an intermediate position(Table 2).

Stomatal and photosynthetic traits

The stomatal size of hypostomatic leaves varied mostly non-significantly and inconsistently in relation to season as well aspollution. However, guard cells were fully turgid at the referencesite, giving way to broader openings (Fig. 1A) in comparison withthose facing the pollution stress (Fig. 1B). The epidermis at the

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Table 2Concentration of chlorophyll a, chlorophyll b and carotenoids (all in mg g�1fr. wt) in the leaves of Azadirachta indica growing under various levels of air pollution (at sites

I–IV) caused by emissions from BTPS, and in a non-polluted environment (at site V), as observed during different seasons of a growth year (see text).

Seasons I II III IV V

Chlorophyll aWinter 0.50f70.05 0.35k70.02 0.26i70.04 0.42ij70.03 0.66c70.12

Pre-monsoon 0.41j70.08 0.42i70.13 0.33k70.09 0.45gh70.14 0.52f 70.13

Monsoon 0.63d70.15 0.73b70.25 0.44hi70.07 0.66c70.14 0.94a70.23

Post-monsoon 0.55e70.11 0.46g70.11 0.34k70.09 0.57e70.14 0.67c70.18

Annual average 0.52f70.05 0.49f70.06 0.34k70.04 0.53f70.05 0.70b70.08

Chlorophyll bWinter 0.38f70.04 0.24j70.02 0.17i70.04 0.33g70.04 0.46c70.07

Pre-monsoon 0.39f70.10 0.32g70.09 0.27i70.08 0.39f70.10 0.46c70.10

Monsoon 0.40e70.10 0.55b70.14 0.29h70.06 0.46c70.08 0.66a70.14

Post-monsoon 0.28hi70.06 0.24j70.002 0.22k70.06 0.29h70.05 0.44c70.10

Annual average 0.36f70.04 0.34g70.03 0.24j70.03 0.37f70.03 0.50c70.05

CarotenoidsWinter 0.45e70.04 0.33i70.02 0.20n70.02 0.39g70.02 0.63b70.06

Pre-monsoon 0.38g70.09 0.30j70.08 0.23m70.53 0.34i70.10 0.43f70.12

Monsoon 0.55d70.009 0.60c70.09 0.36h70.26 0.46e70.01 0.82a70.08

Post-monsoon 0.26lc70.04 0.24m70.01 0.20n70.02 0.27k70.03 0.31j70.05

Annual average 0.41f70.02 0.37g70.02 0.25m70.10 0.39f70.02 0.55d70.04

The values (Mean7SE) are based on five individual readings. Data followed by the same letter within a column are not significantly different at Pr0.05 level as determined

by the Duncan’s multiple range test (DMRT).

F-Value

Chl a Chl b Carotenoids

Site 212.82 219.30 286.57

Season 54.69 5.64 47.21

Interaction 124.19 152.99 223.12

50 µm 50 µm 5 µm

Fig. 1. Morphology of leaf stomata of Azadirachta indica, as seen under SEM: (A) collection from the reference site showing healthy and turgid stomata and (B and C)

collection from the polluted site showing relatively narrow stoamatal apertures and the particulate depositions (A and B at �350; C at �3300).

M. Iqbal et al. / Flora 205 (2010) 61–7164

polluted sites was studded with particulate materials (Fig. 1B–C).The stomata and their apertures were slightly larger during winterand monsoon than in the rest of the year. Stomatal index wascorrelated to pollution level but not to season, and generallyrendered higher values at the polluted sites (Table 3). The PN inleaves varied with season, being the minimum in winters andmaximum in monsoon. It declined consistently with increase indistance from the pollution source, attained its minimum at siteIII (most polluted one), and showed a gradual increase beyond thissite (Fig. 2). The gs varied with season and the pollution load.Maximum in the monsoon as it was, the gs remained extremelylow at sites III and IV. By contrast, [CO2]i of leaves was high at thepolluted sites, showing a maximum at sites III and IV. Changeswith season were irregular (Table 3).

NR activity and N-compounds

The NR activity, minimum at site III, showed an inversecorrelation with pollution load. It was relatively low during winterand pre-monsoon seasons compared to others. Nitrate content ofleaves, showing a positive relationship with pollution, was highduring winter and pre-monsoon seasons (Table 4). The total Ncontent varied with season, having its maxima in monsoons andminima in winters. Within a season, it exhibited inversecorrelation with the pollution load (Table 4).

Protein, sugar and S accumulations

The soluble protein content of the leaf was relatively higherduring the monsoon and post-monsoon periods than in the rest of

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Table 3Functional characterstics of stomata in the leaves of Azadirachta indica experiencing varied levels of air pollution (at sites I–IV) and a non-polluted environment (at site V),

as observed during different seasons of the growth year.

Seasons I II III IV V

Stomata indexWinter 12.61f70.98 12.13e70.51 13.28i70.36 12.86a70.33 12.21b70.32

Pre-monsoon 12.32b70.23 12.85a70.92 14.81j70.89 13.06d70.88 11.23c70.40

Monsoon 11.95g70.39 11.75h70.08 13.16d70.40 11.72h70.31 11.75h70.20

Post-monsoon 11.93g70.29 11.74h70.23 13.29i70.24 12.08e70.55 11.95g70.25

Annual average 11.95g70.24 12.12e70.22 13.63j70.24 12.03e70.26 11.78h70.15

Stomatal Conductance (m mol m�1 s�1)Winter 0.29h70.15 0.70f70.44 0.16j70.06 0.19ij70.07 0.51g70.16

Pre-monsoon 0.19i70.06 0.24hi70.09 0.16j70.06 0.14j70.05 0.21i70.06

Monsoon 1.55c70.48 2.29a70.94 0.76e70.39 0.82d70.44 1.69b70.42

Post-monsoon 0.26h70.03 0.15j70.04 0.14j70.04 0.16j70.05 0.47g70.19

Annual average 0.57g70.09 0.85d70.15 0.30h70.07 0.33h70.004 0.72e70.10

Intercellular CO2 (ppm)Winter 320.16h716.80 368.96e720.47 378.50g75.99 372.50g77.05 310.60d735.11

Pre-monsoon 276.00g718.89 292.27j746.83 326.23d727.06 468.50j743.58 236.50g726.47

Monsoon 349.50f736.05 340.13f75.36 371.00l713.60 284.20i735.09 311.80h725.87

Post-monsoon 298.13j723.68 314.00h724.91 319.27h724.17 296.50i718.88 308.00h729.99

Annual average 310.95d711.90 328.84h712.20 348.75f78.35 355.42f713.07 291.72j714.68

The values (Mean7SE) are based on five individual readings. Data followed by the same letter within a column are not significantly different at Pr0.05, as determined by

the Duncan’s multiple range test (DMRT).

F-Value

Stomatal conductance Intercellular CO2 Stomatal index

Site 1042.62 33.94 998.36

Season 330.85 38.44 85.90

Interaction 621.72 28.10 79.75

0

2

4

6

8

10

12

14

Collection sites

Phot

osyn

thet

ic ra

te (µ

mol

CO

2 m

-2 s

-1) Winter Pre-Monsoon Monsoon

Post-Monsoon Annual average

I II III IV V

Fig. 2. Photosynthetic rate in the leaves of A. indica growing at sites I–V and

experiencing different levels of air pollution caused by emissions from BTPS, as

observed during the pre-monsoon, monsoon, post-monsoon and winter seasons.

Black dots indicate the annual average.

M. Iqbal et al. / Flora 205 (2010) 61–71 65

the year. It varied only marginally up to site III but was markedlylow at site IV (Fig. 3). The quantity of reducing sugars fluctuatedwith season as well as pollution. It was considerably low duringthe post-monsoon and winter, and showed a positive correlationwith pollution stress (Fig. 3). The amount of sulphur was lowestduring the pre-monsoon and highest during the post-monsoonseasons, with the maximum accumulation at site III (Fig. 3). Itincreased gradually up to site III, and declined with furtherincrease in distance from the point source of pollution.

Cambial periodicity

The cambium in A. indica trunk was dormant during winters(Fig. 4A) and consisted of 5–10 cell layers during winter and earlyspring (November–March). The dormant cambium reactivated inApril, beginning with secondary phloem differentiation at all thestudy sites except site III where cambial activity began in Mayonly. Thus, the April and May collections showed phloemformation (Fig. 4B). Accumulation of secondary xylem started inJune at all the collection sites, though a bit late at site III.It continued till September at sites I–III, and till October at sitesIV and V. The cambial zone was relatively broad (8–18 cell layers)during the active phase (Fig. 4C). Dormancy of the cambiumstarted in October (Fig. 4D) and lasted for 5–6 months until themeristematic activity reappeared in the next April (Table 5). Thus,phloem differentiation preceded xylem differentiation by a periodof two months and xylem continued to accumulate for nearly fivemonths. Cambial reactivation was a bit late at site III where thepollution stress was the maximum (Table 5). As compared to thereference site, duration of both phloem and xylem formations wasreduced under heavy pollution stress by one month each, thusshortening the total active phase of the cambium by two months.

Wood characters

The annual wood increment was larger at the polluted sites (upto 1522mm thick at site III) than at the non-polluted one (557mmthick). A similar variation trend was exhibited by vessel density(number per unit transverse area of wood) and vessel proportionin the wood. The width and length of vessel elements exhibited anegative relationship with the degree of pollution. Wood fibreswere also small at the stressed sites (Table 6). The vesselvulnerability factor as well as the mesomorphic ratio was lowunder polluted conditions, showing an inverse variation trend

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Table 4Nitrate reductase activity, nitrate content and nitrogen content in the leaves of Azadirachta indica experiencing varied levels of air pollution (at sites I–IV) and a non-polluted

environment (at site V), as observed during different seasons of a growth year.

Seasons I II III IV V

Nitrate reductase activity (l mol g�1 dry wt. hr�1)Winter 5.31j71.28 4.67k71.37 3.43l71.02 4.80k71.27 6.78i71.73

Pre-monsoon 6.86i72.19 3.60l70.84 3.19i70.72 5.58j71.72 7.79h72.14

Monsoon 17.00b73.65 12.64f73.71 11.88g73.40 15.91cd73.41 18.15a74.01

Post-monsoon 15.52d75.68 12.59f75.50 11.75g75.80 14.85e75.84 16.14c75.63

Annual average 11.17g71.60 8.37h71.43 7.56h71.37 10.28g71.53 12.21f71.69

Nitrate (m mol g�1 dry wt.)Winter 3.99ei70.03 3.28g70.05 3.20g70.05 3.46j70.01 4.65b70.53

Pre-monsoon 4.48a70.55 3.76i70.58 3.50j70.54 4.09h70.84 4.72b70.55

Monsoon 5.40f71.49 3.79i71.68 3.81i71.64 4.76b71.51 5.87d71.37

Post-monsoon 5.36f71.21 4.46a70.61 4.15e70.88 4.22e70.75 5.11c71.39

Annual average 4.81b70.45 3.82i70.36 3.66i70.40 4.13e70.39 5.09c70.48

Nitrogen (m mol g�1 dry wt.)Winter 5.45i70.23 6.95a70.29 8.15d70.30 6.84a70.17 5.27b70.22

Pre-monsoon 6.78a70.27 8.57g70.27 9.04k70.28 7.50l70.26 5.72f70.22

Monsoon 5.93fe70.18 6.36j70.23 7.77h70.23 6.05e70.20 5.98e70.18

Post-monsoon 5.67i70.20 5.82f70.30 6.52j70.13 5.91ce70.27 5.73f70.22

Annual average 5.95fe70.22 6.90a70.27 7.86h70.24 6.57j70.23 5.67f70.21

The values (Mean7SE) are based on five individual readings. Data followed by the same letter within a column are not significantly different at Pr0.05 as determined by

the Duncan’s multiple range test (DMRT).

F-Value

NRA Nitrate Nitrogen

Site 1098.16 1104.67 1090.06

Season 93.70 120.49 111.61

Interaction 93.15 109.17 114.73

M. Iqbal et al. / Flora 205 (2010) 61–7166

with the degree of pollution. The ratio of the lengths of fibres andvessel elements (F/V) was high at the polluted sites (Table 7).

Discussion

Leaf growth and stomatal behavior

The foregoing description of results shows that the pollution aswell as its impact was most severe at the site of intermediatedistance from the pollution source, and not at sites located moreclose to the power plant, seemingly because of the height of thepower plant’s stacks; emissions of stacks came down to settle onvegetation cover after covering a distance of about 6 km in the air.

The phenomena of leaf formation and growth are generallyaffected by the pollution stress. Sometimes, sulphur requirementsof plants are better met in a SO2-rich environment, especiallywhen the soil is sulphur-deficient (Wali et al., 2004, 2007), as isthe case with Delhi, although excessive accumulations are alwaystoxic. The mean leaflet area in A. indica was larger at the pollutedsites, presumably due to a greater allocation of photosynthatestowards the apical meristematic regions. By increasing the leafarea, plants tend to overcome the reduced rate of photosynthesisunder stressful conditions. The present study possibly indicates acase of synergism between low concentration of soil S and highconcentrations of atmospheric N and CO2.

Any changes in the leaf stomatal behavior normally influence(a) the uptake of the pollutants and the photosynthetic CO2,(b) the water loss by transpiration, and (c) the metabolism, thusdisturbing the overall plant growth and yield. Low SO2 doses mayinduce stomatal opening, while higher ones cause a closure; theformer condition could be related to the low turgor of subsidiarycells, whereas the latter to the CO2 accumulation in sub-stomatal

cavities due to reduced photosynthesis (Darrall, 1989; Nighatet al., 2000; Wali et al., 2004). Stomatal density, and hence the SI,may either decrease (an avoidance strategy) or increase (anadaptive trait) in response to environmental stress. A uniquephenomenon of new epidermis formation over the damagedstomata of growing leaves, in defense against acute SO2 stress, hasbeen reported recently for Psoralea corylifolia (Ali et al., 2008). Inthe polluted samples of A. indica, variation in stomatal size,normally a reduction, was inconspicuous and inconsistent withrespect to season or the degree of pollution. However, theirappearance was clearly affected by the harsh environment at thepolluted sites, as evident from SEM images of epidermal surface.Variations in SI, which might emanate from changes in leafgrowth patterns, showed a positive relationship with the pollu-tion load.

Green pigments and photosynthesis

In several plant species, including A. indica, chlorophyll b ismore sensitive to pollution effect than chlorophyll a, possibly dueto induced chlorophyllase activity or inhibited chlorophyll b

synthesis. The sensitivity of carotenoids to coal-smoke pollutionlay between those of chlorophylls a and b. Reduction inchlorophyll concentration without any foliar injury could be dueto a retarded chlorophyll synthesis or oxidation of chlorophyll byfree radicals (Shimazaki et al., 1980). SO2 reportedly causes a localacidity that splits Mg2+ from chlorophyll a molecule, converting itto pheophytin and removes the phytol group of chlorophyll b

molecule, leading to the formation of chlorophyllide b (Iqbal et al.,2005). In A. indica, the maximum concentration of green pigmentsin leaves was recorded during the monsoon, a warm and wetseason that offers the best environment for growth processes.

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Solu

ble

prot

ein

(mg

g-1 d

ry w

t)

Winter Pre-MonsoonMonsoon Post-MonsoonAnnual average

Red

ucin

g su

gar (

µg g

-1 d

ry w

t)

0

1

2

3

4

5

6

7

8

0

1000

2000

3000

4000

5000

6000

0

50

100

150

200

250

300

350

Collection sites

Sulp

hur c

onte

nt (µ

mol

g-1

dry

wt)

I II III IV V

I II III IV V

I II III IV V

Fig. 3. The soluble protein, reducing sugar and sulphur contents in the leaves of A. indica growing at sites I–V and experiencing different levels of air pollution caused by

emissions from BTPS, as observed during the pre-monsoon, monsoon, post-monsoon and winter seasons. Black dots indicate the annual average.

M. Iqbal et al. / Flora 205 (2010) 61–71 67

Within a given season, pigment concentration declined incorrespondence with the degree of the pollution stress.

In A. indica, stomatal conductance at the polluted site wasalways low but [CO2]i was quite high. The pollutant gases mayhave suppressive effects on stomatal conductance even whencombined with CO2 (Kellomaki and Wang, 1997; Kull et al., 1996).The solution of NO2 in the apoplasmic water reduces pH, whichfacilitates the diffusion of SO2 into the cell; the SO2, in turn,

impairs nitrite reductase, thereby hampering the reduction oftoxic nitrite. Therefore, the combination of NO2 and SO2 pollutionoften results in more than just additive effects. An increased[CO2]i, possibly due to a decreased PN, causes stomatal closure,thus lowering the gs, which may then lead to a further decline in[CO2]i and also in photosynthesis.

This study has shown an inverse relationship betweenphotosynthesis and pollution stress. The PN was most inhibited

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Fig. 4. Periodicity of cambial growth in A. indica trunk: The April collection shows that cambial reactivation has taken place at the non-polluted site, as evident from young

and developing derivative cells on the phloem side (A), but the cambium at the polluted site is still dormant and flanked on both sides by mature cells of the derivative

tissues (B). In the October collection, the cambium is still active at the non-polluted site and young derivatives continue to differentiate on the xylem side (C), whereas the

cambium at the polluted site has attained dormancy and is flanked on both sides by mature cells of the derivative tissues (D). SP ¼ Secondary phloem and SX ¼ Secondary

xylem (all at �66).

M. Iqbal et al. / Flora 205 (2010) 61–7168

in winter, and least affected in the monsoon or post-monsoonperiod when growth phenomena were on full swing and theleaves were rich in chlorophylls. Gases and dust may have littleinfluence on plants during the rains, while NOx may act as afertilizer. The gaseous pollutants inhibit photosynthesis possibly(i) by damaging electron transport between photosystems, (ii) bya decrease in phosphoenol pyruvate (PEP) activity and concentra-tion, (iii) through inhibition of RUBP by SO2-derived sulphite ionsor (iv) due to competition of NO2 with NADPH for nitrite reductionand carbon assimilation in the chloroplasts (Ishibashi et al., 1997;Rennenberg and Herschbach, 1996).

NR activity, N compounds and other accumulations

Coal–smoke pollution inhibited NR activity and thus favorednitrate accumulation in A. indica. Mostly, NR activity was at itslowest in winter when nitrate content was normally high. Similarchanges with NR activity and nitrate content have been observedin Peristrophe bicalyculata (Nighat et al., 2008). Inhibition of NRactivity can stem from a reduced supply of NADH or NO3

� to thesite of enzyme synthesis and/or from the failure of detoxifyingmechanisms to operate (Klump et al., 1989). A decline inphotosynthesis may cause inadequate supply of triose phosphates

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M. Iqbal et al. / Flora 205 (2010) 61–71 69

as a source of reductant and NO2 competes for NADPH duringnitrate reduction and carbon assimilation in chloroplasts (Srivas-tava et al., 1975). However, leaves of certain plants accumulatelarge amounts of NO2-derived nitrate in storage pool, which maylater be released to the metabolic pool.

Effect of pollution stress on leaf nitrate content may be eithernegative or positive. Nitrate assimilation is high in fast-growingpioneer species such as Populus deltoides, which have an elevatedNR activity to accommodate an increased available soil nitrate(Soares et al., 1995). Nitrogen content in A. indica leaves graduallydeclined with increase in pollution load, thus showing a negativemutual relationship. It was lowest in the winter and highest in themonsoon.

The amount of soluble proteins in A. indica leaves variedinconsistently with respect to pollution/season. Low SO2 levels mayfavor protein accumulation, though the higher ones fetch inhibitoryeffects (Farooq and Beg, 1988; Sardi, 1981). Reduction in proteincontents could be due to a breakdown of existing proteins, areduction in protein synthesis (Williams and Banerjee, 1995) and/ora decreased photosynthesis (Constantinidou and Kozlowski, 1979).

Table 5Width of cambial zone (in number of cells) and the periodicity of cambial activity

and tissue differentiation during a growth season in trunks of Azadirachta indica

trees growing at sites I–V.

Seasons I II III IV V

January 5d 8c 8c 8c 6e

February 7a 7a 8c 8c 10f

March 9b 10b 8c 9e 9e

April 11(P)d 10(P)d 11d 9(P)e 18(P)h

May 12(P)d 10(P)b 12(P)d 10(P)f 12(P)g

June 10(X)f 8(X)c 10(X)e 10(X)f 13(X)d

July 12(X)e 10(X)d 12(X)c 10(X)f 15(X)h

August 12(X)e 12(X)e 9(X)g 11(X)f 14(X)e

September 10(X)c 8(X)f 8(X)f 10(X)f 15(X)c

October 10c 8f 6d 7(X)d 11(X)b

November 8f 8f 6d 6d 8i

December 8f 8f 7d 7d 7d

Values indicate an average number of cell layers in the cambium as seen in

transections. P and X indicate accumulation of secondary phloem and secondary

xylem, respectively. Values are a mean of 500 individual observations.

Data followed by the same letter within a column are not significantly different at

Pr0.05 as determined by the Duncan’s multiple range test (DMRT).

F-Value

Width of cambial zone

Site 400.34

Season 5.30

Interaction 100.68

Table 6Anatomical features of the wood of Azadirachta indica trees growing at sites I–V.

Parameters I II

Vessel proportion (%) 7.81m70.34 12.50m70.24

Vessel width (mm) 183.33l74.84 170.89l744.20

Vessel element length (mm) 1009.6cd715.22 777.9hi713.38

Fibre length (mm) 1045.5c739.24 999.5de710.93

The values (Mean7SE) on cell size are based on 500 individual readings, while those o

Readings. Data followed by the same letter within a column are not significantly differ

F-Value

Wood width Vessel proportion Vessel wid

224.61 148.49 1.79

Variations in protein contents normally correspond to those ofphotosynthesis, as observed in the present study too.

Higher concentration of reducing sugars in A. indica leavesduring the pre-monsoon or monsoon periods could be due to abreakdown of starch into simple sugars. Decrease in the sugarcontent due to pollution stress was in line with the lowphotosynthetic activity, as reported recently in P. bicalyculata

(Nighat et al., 2008). Bucker and Ballach (1992) have reporteda decrease in the total main non-structural carbohydrates(starch+sucrose+fructose+glucose) in Populus leaves due to ambi-ent air pollution.

The uptake of atmospheric as well as soil sulphur determinesthe leaf sulphur concentration at various stages of plant develop-ment. In SO2-exposed plants, sulphur accumulation occurs mainlyin the aerial plant parts through open stomata on leaves (Iqbalet al. 2005; Mandal, 2006). A consistent increase in S content ofA. indica leaves was indicative of the extent of pollution load. Thismay also be an indicator of failure of detoxifying mechanisms toremove the excess S-derived sulphite and bisulphite ions. Elevatedconcentrations of sulphur can cause negative effects on trees,especially under synergistic influences of other pollutants.However, SO2 should enhance plant growth when sulphur islimiting (Wali et al., 2004, 2007).

Cambial activity and wood development

Impeded cambial activity under pollution stress, resulting in aretarded production of secondary vascular tissues, has beenreported for several softwood (Dmuchowski et al., 1998; Wimmerand Grabner, 1997) as well as hardwood species (Gupta and Iqbal,2005; Iqbal et al., 2000b; Rajput et al., 2008). Inhibitory action ofpollutants on photosynthesis and hormone biosynthesis in leavesis believed to reduce availability of carbohydrates and growthhormones to lower parts of a tree, thus inhibiting woodproduction in trunks (Iqbal et al., 2000b; Kozlowski andConstantinidou, 1986; Rajput et al., 2008). In the present study,the commencement of cambial activity in the trunk of A. indica

trees was relatively late and/or the overall period of the radialgrowth was reduced due to pollution effect, as also reportedrecently for Prosopis spicigera (Rajput et al., 2008).

Wood increments in trees growing in the polluted areas maybe reduced even when there is no visible leaf injury. Woodcomposition and wood cell size may also be affected (Pozgaj et al.,1996). In the present study, annual wood increment was greater ina polluted than in a clean atmosphere, showing that woodformation was not dependent on the photosynthetic rate. Vesselwidth was smaller but vessel density (number per unit area) was

III IV V

13.11m70.30 5.63m70.27 5.41m70.23

167.91l75.94 169.77l75.36 180.95l74.68

754.7i712.48 965.01fg712.48 180.95l74.68

930.62g79.91 970.09ef710.52 1106.08b713.16

n vessel proportion on 250.

ent at Pr0.05 as determined by the Duncan’s multiple range test (DMRT).

th Vessel element length Fibre length

30.90 4.99

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Table 7Variation of some wood characters in trees grown at different sites of study, thus

showing the structural adaptations that have a functional and ecological relevance.

Parameters Site I Site II Site III Site IV Site V

Vulnerability factor 22.90 13.15 12.92 28.33 36.00

Mesomorphic ratio 23119.84 10190.50 9735.63 19668.78 39744.00

F/V ratio 1.03 1.28 1.23 1.00 1.00

M. Iqbal et al. / Flora 205 (2010) 61–7170

greater at the polluted sites, as observed also in the stem of P.

spicigera (Rajput et al., 2008). The shortening of vessel elementsand xylem fibres under pollution stress could be due to a reducedoccurrence of anticlinal divisions in the fusiform cambial initials,as observed by Tulik (2001), or to a reduced capacity ofdifferentiating wood elements to elongate. Studies on mangotrees growing under coal–smoke pollution have suggested thatthe length of vessel elements was more sensitive to pollutantsthan their width, or that the morphogenetic factors controllingthe vessel-element length were the first to be impaired by thepollutants (Gupta and Iqbal, 2005). Trees under stress develop atendency of producing short and narrow vessel elements inabundance. The short elements are more resistant to collapse anddeformation (Carlquist, 1977 b; Zimmermann, 1983), whereasincreased vessel density may help in maintaining the flow of sap,which would otherwise have declined due to decreased vesseldiameter.

Changes in vessel density and vessel size must influencevulnerability factor and mesomorphic ratio of stem wood, whosesignificance in determining the intensity of water stress washighlighted by Carlquist (1977a). The relevance of these ratios inrelation to environmental pollution, however, needs to beassessed. Widening of stomatal aperture under environmentalstress may cause excessive foliar transpiration, creating a water-stressed condition for plants under prolonged exposure. Carlquist(1977a) asserted that the values of the above ratios go counter tothe level of water stress and that the low values of vulnerabilityratio indicate an increased capability of the species concerned towithstand water stress, whereas low values for the mesomorphicratio indicate a tendency towards xeromorphy. Given theseinterpretations, a decrease in vulnerability and mesomorphicratios of wood with increase in pollution stress, as observed in thisstudy, suggests that the species studied is faced with increasingwater stress, which results in a greater degree of vesselsredundancy (narrow vessels in abundance) and a shiftto xeromorphy. Thus, the variations recorded for vulnerabilityand mesomorphic ratios in response to coal–smoke pollutants(Table 7) seem to be a part of histological adaptations of the treein order to minimize its vulnerability to water stress. A gain in F/Vlength ratio is indicative of improvement in the mechanicalstrength of a tree.

Conclusions

Coal–smoke pollution hampers photosynthetic rate of theNeem tree leaves, which in turn affects nitrogen metabolism andthe overall growth processes. The characteristic annual rhythm ofsecondary vascular growth in the stem axis also changes so as toresult in a delayed and shortened span of the seasonal cambialactivity, interestingly, coupled with a higher wood increment. Thegreater accumulation of wood at the polluted site suggests thatwood production may not necessarily be linked to the rate ofphotosynthesis. In fact, whereas the primary growth looks to bedirectly dependent on photosynthetic activity, the secondary

growth seems to be determined by the carbon-partitioningpattern in the species studied. The dimensional changes of woodcells, which are an outcome of changes in growth and structure ofthe fusiform cambial initials, may be supportive in the defensestrategies adopted by trees under stressful conditions. The Neemtree thus comes out to be capable of doing well underenvironmental stress and may, therefore, be preferred forplantation in the polluted areas.

Acknowledgements

This report is based on findings of a research project awardedto the first author by the Ministry of Environment and Forests(Govt. of India). Dr Anjum Arshi and Mr. Zakir A. Siddiqui ofBotany Department, Jamia Hamdard, helped us generously withthe statistics and graphics included in this manuscript.

References

Ali, S.T., Mahmooduzzafar, Abdin, M.Z., Iqbal, M., 2008. Ontogenetic changes infoliar features and psoralen content of Psoralea corylifolia Linn. exposed to SO2

stress. J. Environ. Biol. 29, 661–668.Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein dye-binding.Anal. Biochem. 72, 248–259.

Bucker, J., Ballach, J.H., 1992. Alterations in carbohydrate levels in leaves of Populusdue to ambient air pollution. Physiol. Plant. 86, 512–517.

Carlquist, S., 1977a. Wood anatomy of Onagraceae: additional species andconcepts. Ann. Miss. Bot. Gard. 64, 627–637.

Carlquist, S., 1977b. Ecological factors in wood evolution: a floristic approach.Amer. J. Bot. 64, 887–896.

Chesnin, L., Yien, C.H., 1950. Turbidimetric determination of available sulphates.Proc. Soil Sci. Soc. Amer. 15, 149–151.

Constantinidou, H.A., Kozlowski, T.T., 1979. Effects of SO2 and O3 on Ulmusamericana seedlings. II. Carbohydrates, proteins and lipids. Can. J. Bot. 57,176–184.

C.P.C.B, 1995. Delhi Pollution Statistics. Central Pollution Control Board, New Delhi.Darrall, N.M., 1989. The effect of air pollutants on physiological processes in plants.

Plant Cell Environ. 12, 1–30.Dey, P.M., 1990. Methods in Plant Biochemistry, vol. 2. Academic Press, London

Carbohydrates.Dmuchowski, W., Kurczy �nska, E., Wloch, W., 1998. Chemical composition of

needles and cambial activity of stems of Scots pine trees by air pollutants inPolish forests. USDA Forest Service GTR 166, 197–204.

Duxbury, A.C., Yentsch, C.S., 1956. Plankton pigment nomography. J. Air Pollut.Cont.rol Assoc. 16, 145–150.

Evans, H.J., Nason, A., 1953. Pyridine nucleotide nitrate reductase from extracts ofhigher plants. Plant Physiol. 28, 233–254.

Farooq, M., Beg, M.U., 1988. SO2 resistance of Indian trees. Water Air Soil Pollut. 40,317–326.

Ghouse, A.K.M., Iqbal, M., 1975. A comparative study on the cambial structure ofsome arid zone species of Acacia and Prosopis. Bot. Not. 128, 327–331.

Ghouse, A.K.M., Yunus, M., 1972. Preparation of epidermal peels from leaves ofgymnosperms by treatment with hot 60% HNO2. Stain Technol. 47, 322–324.

Grover, H.L., Nair, T.V.R., Abrol, Y.P., 1978. Nitrogen metabolism of upper three leafblades of wheat at different soil nutrition levels. NR activity and content ofvarious nitrogenous constituents. Plant Physiol. 42, 287–292.

Gupta, M.C., Iqbal, M., 2005. Ontogenetic histological changes in the wood ofmango (Mangifera indica L. cv Deshi) exposed to coal-smoke pollution. Environ.Exp. Bot. 54, 248–255.

Hiscox, J.D., Israelstam, G.F., 1979. A method for the extraction of chlorophyll fromleaf tissue without maceration. Can. J. Bot. 57, 1332–1334.

Iqbal, M., 1994. Structural and operational specializations of the vascular cambiumof seed plants. In: Iqbal, M. (Ed.), Growth Patterns in Vascular Plants.Dioscorides Press, Portland, OR, USA, pp. 211–217.

Iqbal, M., 1995. Structure and behaviour of vascular cambium and the mechanismand control of cambial growth. In: Iqbal, M. (Ed.), The Cambial Derivatives.Borntraeger, Stuttgart, Germany, pp. 1–67.

Iqbal, M., Bano, R., Wali, B., 2005. Plant growth responses to air pollution. In:Chaturvedi, S.N., Singh, K.P. (Eds.), Plant Biodiversity, Microbial Interaction andEnvironmental Biology. Avishkar Publ., Jaipur, India, pp. 166–188.

Iqbal, M., Jacob, M., Mehta, S., Mahmooduzzafar, 2000a. Plant responses toparticulate matters suspended in the air. In: Iqbal, M., Srivastava, P.S., Siddiqi,T.O. (Eds.), Environmental Hazards: Plants and People. CBS Publishers, NewDelhi, India, pp. 80–112.

Iqbal, M., Mahmooduzzafar, Abdin, M.Z., 2000b. Studies on anatomical, physiolo-gical and biochemical responses of trees to coal–smoke pollution around athermal power plant,. Project Report, Department of Environment, Ministry ofEnvironment & Forests (Govt. of India), New Delhi, 322 pp.

ARTICLE IN PRESS

M. Iqbal et al. / Flora 205 (2010) 61–71 71

Ishibashi, M., Sonoike, K., Watanabe, A., 1997. Photo-inhibition of photosynthesisduring the rain treatment: Identification of the intersystem electron-transferchain as the site of inhibition. Plant Cell Physiol. 38, 168–172.

Johansen, D.A., 1940. Plant Microtechnique.. McGraw Hill, New York & London.Kalra, Y.P., Maynard, D.G., 1994. Methods Manual for Forest Soil and Plant Analysis.

Canadian Forest Service, Alberta, Canada.Karnovsky, H.J., 1965. A formaldehyde-glutraldehyde fixative of high osmolarity for

use in electron microscopy. J. Cell Biol. 27, 137A–138A.Kellomaki, S., Wang, K.Y., 1997. Effects of elevated O3 and CO2 on chlorophyll

fluorescence and gas exchange in Scots pine during the third growing season.Environ. Pollut. 97, 17–27.

Khan, M.R., Khan, M.W., 2000. Sulphur dioxide effects on plants and pathogens. In:Iqbal, M., Srivastava, P.S., Siddiqi, T.O. (Eds.), Environmental Hazards: Plantsand People. CBS Publishers, New Delhi, India, pp. 118–136.

Klepper, L.A., Flesher, D., Hageman, R.H., 1971. Generation of reduced nicotinamideadenine nucleotide for nitrate reduction in green leaves. Plant Physiol. 48, 580–590.

Klump, A., Kuppers, K., Guderian, R., 1989. Nitrate reductase activity of needles ofNorway spruce fumigation with different mixtures of ozone, SO2 and NO2.Environ. Pollut. 4, 70–75.

Kozlowski, T.T., Constantinidou, H.A., 1986. Responses of woody plants toenvironmental pollution. For. Abstr. 47, 5–132.

Kull, O., Sober, A., Coleman, M.D., Dickson, R.E., Isebrands, J.G., Gagnon, Z.,Karnosky, D.F., 1996. Photosynthetic responses of aspen clones to simultaneousexposures of ozone and CO2. Can. J. For. Res. 26, 639–648.

Linder, R.C., 1944. Rapid analytical methods for some of the more commoninorganic constituents of plant tissue. Plant Physiol. 19, 70–89.

MacLachlan, S., Zalik, S., 1963. Plastid structure, chlorophyll concentration and freeamino acid composition of a chlorophyll mutant of barley. Can. J. Bot. 41, 1053–1062.

Mandal, M., 2006. Physiological changes in certain test plants under automobileexhaust pollution. J. Environ. Biol. 27, 43–47.

Nighat, F., Mahmooduzzafar, Iqbal, M., 2000. Stomatal conductance, photosyn-thetic rate, and pigment content in Ruellia tuberosa leaves as affected by coal-smoke pollution. Biol. Plant. 43, 263–267.

Nighat, F., Mahmooduzzafar, Iqbal, M., 2008. Coal-smoke pollution modifiesphysio- chemical characteristics of tissues during the ontogeny of Peristrophebicalyculata. Biologia 63, 1124–1130.

Pozgaj, A., Iqbal, M., Kucera, L.J., 1996. Development, structure and properties ofwood from trees affected by air pollution. In: Yunus, M., Iqbal, M. (Eds.), PlantResponse to Air Pollution. John Wiley & Sons, Chichester, UK, pp. 395–423.

Rajput, K.S., Rao, K.S., 2005. Cambial periodicity, structure and formation of woodin Ailanthus excelsa Roxb. growing under the influence of combined airpollutants. Phyton. 45, 51–64.

Rajput, K.S., Rao, K.S., Kim, Y.S., 2008. Cambial activity and wood anatomy inProsopis spicigera (Mimosaceae) affected by combined air pollutants. IAWAJournal. 29, 209–219.

Rennenberg, H., Herschbach, C., 1996. Responses of plants to atmospheric sulphur.In: Yunus, M., Iqbal, M. (Eds.), Plant Response to Air Pollution. John Wiley &Sons, Chichester, UK, pp. 285–293.

Salisbury, E.J., 1927. On the causes and ecological significance of stomatalfrequency with special reference to the woodland flora. Philos. Trans. Roy.Soc. London (B) 216, 1–65.

Sardi, K., 1981. Changes in the soluble protein content of soybean (Glycine max L.)and pea (Pisum sativum L.) under continuous SO2 and soot pollution. Environ.Pollut. 25, 181–186.

Shimazaki, K.I., Sakaki, T., Kondo, N., Sugahara, K., 1980. Active oxygen participationin chlorophyll destruction and liquid peroxidation in SO2-fumigated leaves ofspinach. Plant Cell Physiol. 21, 1193–1204.

Soares, A., Ming, J.Y., Pearson, J., 1995. Physiological indicators and susceptibility ofplants to acidifying atmospheric pollution: a multivariate approach. Environ.Pollut. 87, 159–166.

Srivastava, H.S., Jolliffe, P.A., Runeckles, V.C., 1975. Inhibition of gas exchange inbean leaves by NO2. Can. J. Bot. 53, 466–474.

Tulik, M., 2001. Cambial history of Scots pine (Pinus sylvestris) prior andafter the Chernobyl accident as encoded in the xylem. Environ. Exp. Bot. 46,1–10.

Wali, B., Mahmooduzzafar, Iqbal, M., 2004. Plant growth, stomatal response,pigments and photosynthesis of Althea officinalis as affected by SO2 stress.Indian J. Plant Physiol. 9, 224–233.

Wali, B., Iqbal, M., Mahmooduzzafar, 2007. Anatomical and functional responses ofCalendula officinalis L. to SO2 stress as observed at different stages of plantdevelopment. Flora 202, 268–280.

Williams, A.J., Banerjee, S.K., 1995. Effect of thermal power plant emissions on themetabolic activities of Mangifera indica and Shorea robusta. Environ. Ecol. 13,914–919.

Wimmer, R., Grabner, M., 1997. Effect of climate on vertical resin duct density andradial growth of Norway spruce [Picea abies (L.) Karst.]. Trees: Struct. Funct. 11,271–276.

Zimmermann, M.H., 1983. Xylem Structure and the Ascent of Sap. Springer, Berlin-,Heidelberg-, New York-, Tokyo.