Brazilian Journal of Biological Sciences, 2015, v. 2, n. 4, p. 287-294. ISSN 2358-2731
Air pollution impact on micromorphological and biochemical response of Tabernaemontana divaricata L. (Gentianales: Apocynaceae) and Hamelia patens Jacq. (Gentianales: Rubiaceae)
L. Amulya, N. K. Hemanth Kumar* and Shobha Jagannath
Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore, India. *Email: [email protected].
Abstract. In the present investigation an attempt was been made to assess the air pollution effects on micromorphological and Received biochemical parameters of Tabernaemontana divaricata June 27, 2015 (Gentianales: Apocynaceae) and Hamelia patensi (Gentianales: Rubiaceae). In polluted area the number of stomata and clogged Accepted stomata were found to be higher then control, where as the number of August 12, 2015 unclogged stomata are found to be very less in the control site. The stomatal breadth and pore length were found to be decreased in Released polluted area in both the plants when compared to control. The December 31, 2015 number of subsidiary cells, trichome length values was found to be Open Acess less then control plants. However trichomes are absent in Full Text Article T. divaricata. The stomatal index was found to be higher in both the plants is when compared to control. The chlorophyll a, chlorophyll b, and total chlorophyll content were found to be maximum in control samples when compare to polluted samples. But ascorbic acid, relative water content, pH and Air Pollution Tolerance Index (APTI) were found to be maximum in polluted samples when compare to control. Based on the present study both the plant species were categorized as tolerant to air pollution.
Keywords: Pollution, Air Pollution Tolerance Index, APTI, Micromorphology, Stomata.
Introduction monoxide, hydrocarbons (benzene, methane and ethylene), oxides of nitrogen (NOx), Urban air pollution is a serious sulphur dioxide (SO2), lead (Pb) and other problem in both developing and developed suspended particulate matter (SPM) like countries. Vehicles exhaust emissions are a smoke, metal and inert dust are the major dominant feature of urban environments pollutants emitted by the automobile and are widely believed to have detrimental vehicles (Ho and Tai, 1988). All these effects on plants (Honour et al., 2009). The pollutants are released at the ground level booming vehicular pollution is a global and their upward movement is restricted phenomenon which has completely due to tall buildings and congested transformed the socio-economic scenario in thorough fares. Therefore, high build up of urban areas all over the world. Though the pollutants frequently occur causing adverse number of vehicles in India may be less in effects on the environmental quality of comparison to those in more advanced urban areas. The gasoline passenger cars nations, the environmental pollution is quite and two and three wheelers are the principal formidable due to predominance of old and source of carbon monoxide (CO), the two - poorly maintained vehicles, and narrow, and three - wheelers also contribute about uneven and bumpy roads. Carbon 70% of the total hydrocarbon emissions.
ISSN 2358-2731/BJBS-2015-0127/2/4/11/287 Braz. J. Biol. Sci. http://revista.rebibio.net 288 Amulya et al.
The diesel vehicles are principle Materials and methods contributors of soot particles (Pundir, 1994). Leaves of T. divaricata and Air pollution directly affects plants H. patens were collected from Chikkahalli via leaves or indirectly via soil and K. R. Circle which served as control acidification. When exposed to air borne and polluted area respectively. K. R. Circle pollutants, most plants experienced which is located in the central part of physiological changes before exhibiting Mysore city is one of the busiest areas of visible damage to leaves (Liu and Ding, the city with a very high traffic density. 2008). The atmospheric SO2 adversely Chikkahalli which is 10 km away from the affects various morphological and city served as control area as it had physiological characteristics of plants. High negligible traffic. soil moisture and high relative humidity aggravated SO2 injury in plants (Tankha Micromorphological studies and Gupta, 1992). Pollutants can cause leaf Leaf samples collected from both injury, stomatal damage, premature polluted and control areas were processed senescence, decreased photosynthetic following the method of Ahmed and Yunus activity, disturb membrane permeability (1974). The matured leaf samples were cut and reduce growth and yield in sensitive into small bits and taken in separate test plant species (Tiwari et al., 2006). Air tubes. 30% nitric acid solution was added to pollution stress leads to stomatal closure, each test tube. The mixture was boiled in a which reduces CO2 availability in leaves water bath for 3 h till the leaf bits became and inhibits carbon fixation. Net transparent. The leaf bits were washed in photosynthetic rate is a commonly used distilled water and stained with 2% indicator of impact of increased air safranin. Excess stain was removed by pollutants on tree growth (Woo et al., washing with distilled water and mounted 2007). The interaction between plants and using gelatin jelly. A small amount of the different types of pollutants were influence jelly was taken on a slide and gently of environmental pollution focus on warmed using Bunsen burner. The leaf bits physiological and ultrastructural aspects were placed to the jelly and a coverslip was (Psaras and Christodoulakis, 1987; mounted over it and observed under light Velikova et al., 2000). microscope. Micromorphological charac- The use of plants as monitors of Air teristics such as frequency of stomata, pollution has long been established as trichomes, size of the stomata, stomatal plants are the initial acceptors of air index and trichome length were studied in pollutants. They act as the scavengers for 10 microscopic fields selected at random many air borne particulates in the from each side and measured using ocular atmosphere (Joshi and Swami, 2007). Air micrometer. Photomicrographs were taken pollution has the potential to reduce yield with a digital camera at different and the nutritional quality of crop plants magnifications. (Jager et al., 1993). Thus plants play a vital role as indicators of pollution. The emission Biochemical studies of air pollutants from the various industrial Leaf samples were washed and and social activities has a large impact upon collected in polythene bags until further the vegetation growing in these areas. The process. Fresh leaves were used for the main sectors emitting air pollutants are road estimation of chlorophyll a, chlorophyll b, transport, power and heat production total chlorophyll, and total ascorbic content. sectors and industry. Therefore, the present work is Chlorophyll estimation: The designed for the study of air pollution effect chlorophyll content of leaves was estimated on leaf characteristics of two plant species following the procedure described by (Tabernaemontana divaricata L. and Arnon (1949). The volume of solvent Hamelia patens Jacq.) in Mysore, extraction was made up to 100 mL by Karnataka State, India. adding 80% acetone. The absorbance was
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Air pollution impact on Tabernaemontana divaricata L. and Hamelia patens Jacq. 289
measured by using spectrophotometer at and deposition of dust on the leaves, when 645 nm, and 663 nm. compared to control. The leaves of T. divaricata appeared pale green in colour. Ascorbic acid content: The total It was more pronounced in T. divaricatum ascorbic content was estimated using the compared to H. patens. Changes in the method of Behrens and Madere (1994). micromorphological characteristics such as 0.2 mL of supernatant was taken and 1 mL stomatal number, subsidiary cell number, of 5% TCA was added and mixed well. stomatal pore length and breadth, stomatal 1 mL of 2% DNPH reagent was added to index, trichome number, length and breadth this mixture and incubated on a water bath in leaf samples from control and polluted for 15 min to get the precipitate. 7 mL of areas is presented in Table 1. 80% H2SO4 was added and the absorbance The stomatal number increased in was measured at 540 nm spectrophotome- the leaf samples of both the species trically. The total ascorbic acid content was growing at polluted areas. The stomatal determined with standard curve by using pore size was found to be reduced in ascorbic acid. polluted sample (Figure 1, C and D) when compared to control (Figure 1, A and B). Relative water content The stomatal number increased to a mean Relative water content of leaf value of 50.9 and 61.9 in polluted leaf sample was calculated by the method samples of T. divaricata and H. patens, described by the Singh and Rao (1986). The respectively. The length and breadth of relative water content of leaf sample was stomatal pore however was reduced in the estimated as percentage moisture on over leaf samples of both the species (4.3 and dry wt. basis. 0.13, and 3.4 and 0.3, respectively) growing at polluted area (Figure 1). Most of the stomata in leaf samples of both the species
from polluted area were found to be pH clogged (Figure 2, C and D). The subsidiary pH of aqueous leaf abstract was cells were found to be decreased in number determined using digital pHmeter. in both the species growing at polluted area (7.3 and 6.7) compared to control area (9.2 APTI and 7.3). While an increase in the stomatal Air Pollution Tolerance Index index was observed in both the plants. (APTI) was determined by calculating the Trichomes were observed only in H. patens ascorbic acid content, total chlorophyll of the present investigation. A threefold content, pH of leaf extract and relative decrease in the length of the trichomes and water content of leaf. APTI was calculated was observed in leaves from polluted area by the method described by Singh and Rao (Figure 3 C and D (compared to control (1983): area while the breadth of the trichomes increased in leaves growing at polluted area and compared to leaves from control area (Figure 3 A and B). The number of trichomes in leaves growing at polluted site Where: was found to be less when compared to leaf A = Ascorbic acid (mg g-1 FW); samples from control site Figure 3 A and B. T = Total chlorophyll (mg g-1 FW); Biochemical parameters chloro- P = Leaf extract pH; and phyll a, b, and total chlorophyll, ascorbic R = Relative water content (%) of acid content, relative water content and pH the leaves. were presented in Table 2. There was no significant variation in the chlorophyll a Results content in the leaves of T. divaricata growing in K. R. Circle compared to leaves The two shrub species i.e. of control area; while it showed only a T. divaricata and H. patens growing at slight reduction in the leaves of H. patens K. R. Circle, showed decrease in leaf size from 0.63 to 0.43 mg/g. Chlorophyll b and
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Table 1. Micromorphological characteristics of T. divaricata and H. patens from polluted and control site.
Mean ± SD followed by same superscript are not statistically significant between the concentrations when subjected to SPSS package ver. 14.0 according to Tukey’s mean range test at 5% level. C = Control; P = Polluted.
Figure 1. Stomata in leaf samples T. divaricata and H. patens from control and polluted areas. Note reduction in stomatal pore size in polluted leaf. (A) T. divaricata from control area; (B) H. patens from control area; (C) T. divaricata from polluted area reduced pore length; and (D) H. patens from polluted area reduced pore length.
Figure 2. Stomata in leaf samples T. divaricata and H. patens from control and polluted areas. Note clogged stomata in leaf sample from polluted area. (A) T. divaricata from control area unclogged stomata; (B) H. patens from control area unclogged stomata; (C) T. divaricata from polluted area clogged stomata; and (D) H. patens from polluted area clogged stomata.
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Figure 3. Trichomes of leaf samples T. divaricata and H. patens from control and polluted areas. Note clogged stomata in leaf sample from polluted area. (A) T. divaricata from control area; (B) H. patens from control area; (C) T. divaricata from polluted area reduced length and number; (D) H. patens from polluted area reduced length and number.
Table 2. Air pollution tolerance index of plant species growing in polluted site and control site.
Mean ± SD followed by same superscript are not statistically significant between the concentrations when subjected to SPSS package ver. 14.0 according to Tukey’s mean range test at 5% level. C = Control, P = Polluted, Chl a = Chlorophyll a; Chl b = Chlorophyll b; RWC = relative water content; TCH = Total chlorophyll content; AA = Ascorbic acid content; APTI = Air Pollution Tolerance Index.
Table 3. Ambient air quality monitoring at K. R. Circle.
SL No. Month (2012) SO2 (ug/m³) NO2 (ug/m³) SPM (ug/m3) RSPM (ug/m3) 1. Feb 11.1 21.5 115 58 2. Mar 11.1 22.5 138 67 Average at KSRTC building K. R. Circle, Mysore.
total chlorophyll reduced in the leaves of leaf samples of K. R. Circle showed an both the species growing at K. R. circle increase over control plants from 5.4 to 6 in when compared to control. Chl b was found T. divaricatum and from 4.5 to 5 in case of to be reduced in the leaves of H. patens. A two fold increase in the T. divaricatum from 0.35 to 0.28 mg/g and ascorbic acid content (40.1 and 50, in H. patens it reduced from 0.78 to respectively) was observed in the leaves of 0.43 mg/g. Total chl reduced from 0.91 to T. divaricatum and H. patens growing at 0.81 mg/g in T. divaricatum and from 1.5 to K. R. Circle. The ambient air quality 0.84 mg/g in case of H. patens. pH in the monitoring data at K. R. Circle was
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obtained from KSPCB, Mysore (Table 3). are unable to remove by wind, rain and The concentration of SO2, NOx and SPM physical washing. Kulshreshtha and Rai were within the permissible limits of (2005) also reported stomata clogged in CPCB. Both the plant species growing at Nyctanthes, Quisqualis and Terminalia. K. R. Circle showed an increased APTI Decrease in trichome number and length in value of 35.27 and 36.42 respectively in polluted population has been observed in T. divaricatum and H. patens. Based on the Lantana (Kamala et al., 1994) and APTI values both the plant species were Calatropis (Kulshreshtha et al., 1994b). On categorized as tolerant to air pollution. the contrary increase in trichome number has been observed by Sharma and Tyree Discussion (1973). According to Sharma (1977) trichomes help trap the particulate matter Leaf surface characteristics are falling directly on the leaf surface which sensitive to the air pollution. Response of otherwise block the stomata pores and leaf character to air pollution will indicate adversely affect the process of gaseous the adverse effects of air pollution, which exchange. Thus increased number of thus can be used as bioindicator. trichomes seems to be another adaptation of T. divaricata and H. patens in the present the stress of air pollution providing an investigation showed a reduction in leaf outline defense. size compared to populations growing in Reduction in the concentration of control area. Similar reduction in leaf area chlorophyll content in leaves of polluted was also observed in leaves of Newbouldia area was observed in both the plant species. laevis (Kayode and Otoide, 2007) and Similar changes in concentration of Albizia lebbek under the stress of air pigments were also observed in leaves of pollution (Seyyednejad et al., 2009). six tree species expose to air pollution due Reduction of leaf area and petiole length to vehicle emission (Joshi and Swami, under pollution stress was reported by 2009). Leaves from polluted area had Tiwari et al. (2006). Reduction in leaf size significantly lower chlorophyll content than is an adaptation by the plants exposed to air control (Tripati and Prajapathi, 2008; pollution to minimize the entry of Stevvovic et al., 2010). In present study, poisonous gases into the leaves Ascorbic acid concentration was increased (Zarinkamar et al., 2013). in T. divaricata and H. patens ascorbic acid In the present investigation increased with the increase of dust clogging was observed in most of the deposition (Tripati and Prajapathi, 2008). stomata in the leaf samples of polluted area. Being a very important reducing agent, Kulshreshtha et al. (1994b) had reported ascorbic acid also plays a vital role in cell significant decrease in size of epidermal wall synthesis, defense and cell division cells, stomata and trichome per unit area in (Conklin, 2001). Increase in ascorbic auto exhaust polluted population of concentration with respect to the control Calotropis procera and Nerium indicum leaves also reported by Jyothi and Jaya collected from busiest roadside and also (2010). The leaf pH values increased in the Kulshreshtha and Rai (2005) revealed that, polluted area compared with that of control as compared to the leaves from control, the similarly increased in pH values in polluted frequency of epidermal cells and stomata site observed (Patel and Kousar, 2011). had increased considerably in plants of In present study, increased in polluted area and also Saadabi (2011) also relative water content was observed in reported that, in polluted sites leaves polluted site similarly increased in relative become smaller with reduced length and water content was observed (Patel and width and stomatal index per leaves area. In Kousar, 2011). Among two shrub species this study, stomata were found clogged in investigated for APTI H. patens showed T. divaricata and H. patens. Kulshreshtha et more tolerance to air pollution than al. (1994a) revealed that the diesel exhaust T. divaricata. Jyothi and Jaya (2010) particles are very small and they are readily observed that among the three shrubs deposited on both surfaces of leaves and species studied, C. infortunatum showed remained adhered to it for long time as they highest APTI values and found to be more
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