Instrumental Approach to the Determination of Ozone Phytotoxicity

Ozone (O 3 ) phytotoxicity is a serious concern all over the globe. O 3 can cause both biochemical and physiological changes to plants that could lead to reduction in growth and yield. Its effect on agricultural crops is usually detected as foliar injury. Although foliar injury has frequently served as physiological basis for monitoring performance of agricultural crops and assessing air quality, its visibility depends on period of exposure, concentration of ozone, and environmental conditions. The present study focused on the determination of biochemical indicators of ozone phytotoxicity including Rubisco (biochemical indicator for carbon assimilation), photo-pigments [carotenoids and chlorophyll (a & b)], and chlorophyll photochemical efficiency on a judiciously selected plant-Nicotiana tabacum (Tobacco), variety: Little Havanna. An enclosed computer controlled growth chamber was used for cultivation of the experimental plant groups (ozone exposed and control). Spectrophotometric measurements of the biochemical indicators were carried out to evaluate the exposure-response behaviour of the plant. Our results showed a significant (P< 0.05) decrease of the biochemical indicators that is reproducible even when foliar injury is not detected. Besides, experiments also showed that chlorophyll a is more sensitive to ozone than chlorophyll b. The instrumental readings were supported by a reduction in total organic nitrogen and starch content and ultimate decrease in growth rate and root: shoot ratio. While significant variations in the intensities of foliar injury can be detected among plants within a group, a reproducible exposure-response data were obtained from instrumental measurements. Such measurements can also serve as basis for the development of implantable sensors for in-situ monitoring of ozone pollution in sensitive crops.


INTRODUCTION
Tropospheric ozone, a photo-oxidant produced from nitrogen oxides and volatile organic compounds in the presence of sunlight, is toxic to sensitive plant species at relatively low concentrations. It is one of the most phytotoxic air pollutants that cause considerable damage to vegetation throughout the world. The effects of O 3 on plants are numerous and vary with the intensity and duration of exposure, concentration, and presence of compounding stresses, such as drought, competition, nutrient deficiency, and pathogens. O 3 enters plants through stomatal pores and reacts with plant cells, generates other reactive oxygen species and causes oxidative stress [1]. It causes biochemical and physiological changes which lead to decrease in photosynthetic rate and crop yield [1][2][3][4][5]. There can be visible foliage symptoms associated with ozone injury, including chlorotic mottle, tip or margin burn, stipple, and necrosis [6][7]. Such symptoms have served as a basis for monitoring performance of agricultural crops and assessing air quality. However, foliar ozone injury symptoms are only an indication of previous exposure to elevated ozone concentrations for a certain time period, and when environmental conditions were conducive to ozone uptake and cellular injury. There is, therefore, continuing interest to establish more reliable methods for monitoring performance of agricultural crops and assessing air quality related to O 3 . Studies have shown that exposure to ozone often leads to reduction in growth and yield of a plant even if signs of any visual injury are not observed [8]. Both growth and yield results from optimum photosynthetic rate and chlorophyll photochemical efficiency. Photosynthetic rate is linked to carbon dioxide assimilation. Exposure to O 3 causes reduction in assimilation and increased respiratory loss of carbon dioxide. The diminished assimilation of carbon dioxide is induced by a decline in Ribulose 1,5bisphosphate carboxylase/oxygenase (Rubisco) and causes impairment of the processes of photosynthesis and alter allocation of carbon to different organs and leads to altered growth responses of these organs [9][10][11]. In agricultural crops, this results in reduced grain or seed yield, and restricts the plant's ability to tolerate additional stresses, such as stress due to drought or low temperatures. Measurements of CO 2 gas exchange, carboxylation rate of ribulose bisphosphate by Rubisco and chlorophyll photochemical efficiency could thus serve as a basis to monitor performance of agricultural crops.
The objective of this work was to determine the impact of ozone exposures on selected plant cultivars through measurement of CO 2 gas exchange, Rubisco, chlorophyll photochemical efficiency and related parameters such as biomass accumulation, stomatal conductance, carotenoids, total organic nitrogen and starch contents. Tobacco variety, Little Havanna, SR1 (Nicotiana tabacum) was selected because of its sensitivity to ozone. Experiments were carried out in enclosed computer controlled growth chambers. Such chambers provide climatic conditions that are similar to those outside and provide the best available source of data for plant responses to O 3 exposure owing to rapid air exchange. In addition to a reliable exposureresponse data, instrumental measurements reported herein can also serve as a basis for the development of implantable sensors for in-situ monitoring of ozone pollution in sensitive crops.

Qualitative (Visible) Observations
O 3 exposed tobacco plants exhibited foliar injury during the 8 days exposure. Specific characteristics recorded for leaves included abnormal coloration (especially chlorosis), mottling, necrosis, pigmented stipple, curling, and senescence. The appearance of visible injury was evaluated at the end of ozone exposure from the onset of lesion development and served as basis for subsequent experiments.

Gas exchange measurement
Net photosynthetic rate (P n ) and stomatal conductance (g s ) of ozone exposed and of control tobacco plants were assessed using an open gas exchange system (LICOR -6400). Three fully developed leaves from each tobacco plant were selected and measured under saturating irradiance. Photosynthetic photon flux density (PPFD) was fixed at 1200 µmol photons/m 2 /sec, using a red-blue LED light source built into the leaf cuvette, though other environmental factors, such as air humidity was not controlled, i.e. natural variation was permitted to replicate the conditions that occur in the real environment.

Chlorophyll fluorescence measurement
The chlorophyll fluorometer, a multi-purpose tool which provides dark adaptation for the sample (required for the measurement of maximum photochemical efficiency) was used to measure the maximal apparent efficiency of PSII i.e. F v /F m = (F m -F 0 ) /F m , where F 0 is minimal fluorescence yield and F m is maximal fluorescence yield of dark adapted leaves. Four fully expanded leaves were selected from each plant (four plants, two from each treatment) and a dark leaf clip was placed on the leaf for 20 minutes prior to the measurements. The instrument utilized a time resolved method to determine F 0 with lightemitting diodes that also delivered a saturating pulse of 6000 µmol photons/m 2 /sec to obtain F m . It is vitally important that the illumination should not vary in intensity during measurement otherwise the F 0 and peak fluorescence will have been determined using different light levels and therefore cannot be compared. By measuring the yield of chlorophyll fluorescence, information about changes in the efficiency of photochemistry and heat dissipation can be gained. Dark-adapted values of F 0 , F m and F v /F m were obtained prior to the first light period.

Determination of chlorophyll concentration, total carotenoids and rubisco
Chlorophyll extraction and total carotenoids were determined in 80% acetone from three leaf discs of 2 cm 2 diameter(Ca. 70 mg altogether) according to the method described by [12], with modifications by [13]. Leaf discs were carefully removed from non-necrotic areas of leaves, weighed and immediately frozen in liquid N 2 and then stored at -80ºC. The leaf discs were ground and the carotenoids and chlorophyll pigments were extracted by immersion of the crude extract in 80% acetone (1600 µl of 100% acetone and 400 µl crude extract makes 80% acetone). Absorbance of extracts was read by spectrophotometer (Shimadzu UV-2401PC, UV-VIS recording spectrophotometer) at 646 nm, 663 nm and 470 nm. Chlorophyll a (mg/cm 2 ), chlorophyll b (mg/cm 2 ) and total chlorophyll content (mg/cm 2 ) were calculated according to Lichtenthaler, and Welburn equations [13].
The concentration of Rubisco was determined by native polyacrylamide gel electrophoresis using Thermo EC120 Mini Vertical Gel System [14], using purified spinach Rubisco with a known concentration as a standard. With this procedure, the concentrations of leaf Rubisco can be determined from a fresh leaf sample in a single crude extract. Fresh tobacco leaf samples were used, with three leaf discs of 2 cm 2 (ca. 70 mg altogether) per 2 ml of extraction buffer.

Determination of starch
Determination of starch concentration is based on enzyme reactions in which starch is first broken down to glucose, glucose then converted to gluconate-6-phosphate, and on light absorption of NADPH (at 340 nm) formed in the reactions. Boehringer Mannheim Kit describes how to analyse starch (common polysaccharides in plants) concentration with enzymatic methods from freeze-dried plant material. The leaf discs were ground in liquid nitrogen with a mortar and pestle. Ca. 300 mg powder, 20 ml DMSO and 5 ml 8 M HCl were taken respectively to a to 100 ml Erlenmeyer flask with wide opening. Flasks were capped with a piece of Para film and, placed into a water bath (60ºC) for 30 min. The flasks were allowed to cool down to room temperature and ca. 50ml ultra purified water was added. pH was adjusted to 4-5, and the sample was moved to 100 ml volumetric flask. Water was added to the volume mark and well mixed.
The sample was filtered with suction bottle and Büchner funnel. Sample solution and the solutions from the enzyme kit (bottles 1, 2 and 3) were pipetted to the cuvettes as follows, 0.10 ml of sample solution into sample and sample blank and 0.2 ml of solution 1 into sample. The cuvettes were capped with Para film, well mixed and kept in the water bath at 60°C for 15 min.1.00 ml and 1.20 ml of water was added to sample and sample blank respectively. Then 1.00 ml of solution 2 was added to both sample and sample blank. Absorbance was measured with the Shimadzu UV-2401PC, UV-VIS recording spectrophotometer.

Determination of total organic nitrogen
Total organic nitrogen is often analysed from leaf samples to assess the general nitrogen (and nutrient) status of the plants. Total organic nitrogen was determined by the Kjeldahl method followed by digestion, steam distillation and titration.

Qualitative Results
A preliminary examination of ozone effect on the Nicotiana tabacum (Tobacco), variety Little Havanna, plant was investigated through qualitative experiments involving observation of foliar symptoms of ozone exposure vis-à-vis control.
Our results showed significant physiological changes including necrotic and chlorotic spots, and acceleration of leaf senescencewith 85% of the ozone fumigated plant groups and the intensities of such effects varied among plants within the group. However, the intensity of the observed symptoms depended on the period of exposure. No visible deleterious effects of ozone were observed with the remaining 15% of the plants. The plants remained in similar vigor and growth rate as those in the control group.

Photosynthetic rate, stomatal conductance, and chlorophyll photochemical efficiency
Quantitative determination of the biochemical indicators were carried with both groups (ozone fumigated and control) as well as those with which physiological changes were not observed after ozone exposure. Photosynthetic rate, stomatal conductance, and chlorophyll photochemical efficiency are interdependent and are determined by health state of the plant. In this study, the above parameters were measured to determine the phytotoxic effect of ozone and results are summarized in Table 1. Table 1, reduction in all of the three parameters was recorded with ozone treated plant groups as compared to that of control group. The results can be explained based on interaction of ozone with constitutive plant cells and consequent effect on biochemical processes. Photosynthetic rate is usually measured by CO 2 gas exchange rate [15]. In this study, a decrease in gas exchange was observed (Table 1) and suggested a decrease in photosynthetic rate. The other secondary response to ozone is a reduction in stomatal conductance, as the stomata close in response to increased internal CO 2 that occurs because of the reduced photosynthetic activity caused by the ozone [16][17][18]. It has been suggested that the decrease in stomatal conductance caused by O 3 is similar in magnitude to the 10% decrease caused by CO 2 increases since preindustrial conditions [19]. A reduction of net photosynthesis and a reduction in stomatal conductance observed in this study, agrees with the results of other researchers, who also found reduced photosynthesis and reduced stomatal conductance in O 3 exposed plants [20]. Chlorophyll photochemical efficiency is measured by chlorophyll fluorescence. Chlorophyll fluorescence is a red and far-red light emitted by photosynthetic tissue when it is excited by a light. O 3 -induced reduction in photosynthetic rate can thus indicate reduction in chlorophyll activity and/or quantity. A decrease in chlorophyll fluorescence in turn indicates decreased concentration of chlorophyll (Beer's law). Therefore photosynthetic rate and chlorophyll fluorescence are interrelated [21,22] and their measurement can serve as a basis to monitoring O 3 pollution. Interestingly, all the O 3 treated plants including those which did not show foliar injury responded to the exposure.

Rubisco, Chl a, Chl b, and carotenoid concentrations
Results from instrumental measurements of the carbon assimilation indicator (Rubisco), and photosynthetic pigments (chl a, chl b, and carotenoids) for O 3 pollution are summarized in Table 2. A general decrease in concentration of Rubisco and the pigments can be observed with all of the plants previously exposed to ozone as compared to that of the control. While the decrease in Rubisco concentration in O 3 polluted plants could be due to O 3 -induced oxidation which enhances its susceptibility to proteolytic degradation [23] that of chlorophyll and carotenoid is explained by their redox reaction with ozone. Reductions in stomatal conductance (g s ), net photosynthetic CO 2 assimilation, and carboxylation efficiency have all been associated with O 3 exposure [24][25][26][27]. These reductions reveal a loss of ribulose-1, 5-bisphosphate carboxylase/ oxygenase (Rubisco), and decreased Rubisco activity [28,29]. Conditions where additional synthesis should be minimal showed a substantial decline in Rubisco content suggesting that the primary cause of the decline in Rubisco due to O 3 exposure was enhanced degradation rather than reduced production. Rubisco activity and/or quantity is thus a good bioindicator for oxidative stresses in plants. Accelerated loss of Rubisco in mature leaves reflect O 3 -stimulated proteolytic degradation.
Chlorophyll a (Chl a) is universally found in all photosynthesizing cells and plays an active role in photosynthesis functioning as a photoenzyme that converts and stores incoming excitation energy in the form of an oxido-reduction potential. Chl b is the second common but not universal kind of chlorophyll, in addition to chl a, and represents a locally oxidized derivative of chl a. The observed decrease in chl a concentrations is explained by the oxidative damage to the chlorophyll by reaction with O 3 . Experiments also showed that chlorophyll a is more sensitive to ozone than chlorophyll b (Table 2).
Carotenoids [carotenes (non-oxygenated) and carotenoids referred to as xanthophylls (oxygenated)], on the other hand, are long chain conjugated olefinic molecules synthesized by a wide variety of photosynthetic (plants, algae) and non-photosynthetic (some fungi and bacteria) organisms. Carotenoids are involved in several aspects of photosynthesis, notably light absorption, energy transfer to photosynthesis system, and protection of the photosynthetic apparatus from damage by strong illumination. Previous studies have shown that carotenoids are also susceptible to oxidative damage by O 3 [30]. Thus the observed decrease in the

Total nitrogen and starch concentrations
Generally, reduction in photosynthesis would lead to a reduction in starch and total organic nitrogen production. This is verified in our experiment and result is indicated in Fig. 1.
As can be seen in the Fig. 1, a decrease in total nitrogen and starch content was observed with O 3 fumigated plant groups. The decrease in starch content expected from a decrease in photosynthetic rate due to O 3 . The degree of O 3 induced accelerated senescence could be responsible for decline in N availability. Previous studies [31,32] suggested that when available organic N in the plant is declines, the intensity of O 3 induced accelerated senescence may be increased and vice versa. Data reported herein support these observations.

Biomass accumulation
Allocation of carbon to different organs may be altered under conditions of reduced assimilate supply through photosynthesis leading to altered growth responses of these organs. This results in reduced root:shoot weight ratios or in a reduction of the ratio between seed yield and total biomass production. In order to verify this experimentally, measurements were conducted and results are summarized in Fig. 2.
As can be seen from the Figure a decrease in both total dry mass and root: shoot ratio was recorded. The observed reduction in the dry weight was probably the consequence of reduced P n . The greater reduction of P n in the lower leaves is much more important for plant growth than an equal percentage reduction in the upper leaves, since, due to their greater size, their contribution to plant assimilation is greater.
The reduction of the root to shoot ratio suggests that, although the root was not directly exposed to ozone, it was the most severely affected part of the exposed plant. This observation is in agreement with those of [33] who found that 'the respiratory activity of roots began to decrease well before visible injury appeared on the leaves, and the percent of reduction of respiration was much greater than the percent of leaf injury. The number of healthy looking leaves per plant was In contrast, old, yellow leaves (%), leaves with brown, grey, yellow and white stippling (%) and leaves with necrosis (%) were increased significantly in O 3 exposed plants.

CONCLUSIONS
In conclusion, a reliable exposure-response data were obtained by instrumental measurement of biochemical indicators [Rubisco, carotenoids and chlorophyll (a & b), and chlorophyll photochemical efficiency] of ozone pollution on a judiciously selected plant-Nicotiana tabacum (Tobacco), variety: Little Havanna. While significant variations in the intensities of foliar injury can be observed among plants within a group, a reproducible exposure-response data were obtained from instrumental measurements of the biochemical indicators. Exposure to ozone caused a decrease in photosynthetic rate and concentrations of biochemical indicators (Rubisco, Chl a, Chl b, and carotenoid). The observed decrease in bioaccumulation, root: shoot ratio, total nitrogen, and starch were also consistent with the above findings. Experiments also showed that chlorophyll a is more sensitive to ozone than chlorophyll b. Further research at the biochemical level using sensor technologies is needed to decipher the mechanism and improve sensitivities of the measurement for field applications and in situ monitoring.