1. INTRODUCTION

Rapid industrialization, growing cities, and increasing vehicular load have caused serious environmental pollution and have affected plant life [1–3]. Ozone is a secondary air pollutant in the tropospheric atmosphere, formed by the reaction of sunlight and originator gases, including SO_X, NO_X, and volatile organic carbon, generated through anthropogenic activities [4,5]. The rapid increase in ground-level ozone concentration has come to be a global concern due to its direct phytotoxic effect or indirect effect through global warming [6–8]. Researches from the past four to five decades acknowledge the detrimental effects of ozone on plants. A higher ozone concentration could constrain photosynthesis [9], reduce yields and biomass [10], and also change the allocation of photosynthesis in plant organs. Ozone is also reported to cause foliar injury [11,12] and changes to stomatal conductance [13].

Ozone primarily enters the plant through stomatal pores [14] and generates oxidative stress [7,15]. To counteract this stress, the plant develops a series of defense response. However, most of the studies describing plants defense response to ozone are based on antioxidative efficiency and/or stomatal conductance. However, previously it was shown that balancing the density of stomata and cell division in developing leaves is directly connected with the plant’s response to environmental stress [16–18]. The study of Navea et al. [19] reveals a specific defense mechanism in case of drought stress in which plants turn down the number of stomata and refer to it as ‘stomata abortion’. Chaudhary and Rathore [3], Paakkönen et al. [20], and Aasamaa et al. [21] found decreased stomatal pores or aperture size due to the abiotic stress. Studies on plant response to air pollution, including ozone, have observed an increased stomatal density of plants [22,23]. Ferdinand et al. [23] observed an ozone-sensitive clone Prunus serotina, which has larger stomatal density and a lesser ratio of palisade per spongy parenchyma. The authors also asserts that resistance can be described by separate gas permeability, which is based on extending the structure of the leaf, as gas resistance of palisade tissue is larger than that of spongy tissue.

Alteration to stomata and abundance as a response to stress is also expressed as a major pollutant absorption controlling mechanism [24]. Stengleina et al. [25] stated that the density of stomata is basically affected by both the beginning of stomatal development and the build-up of epidermal cells (EC). The build-up of EC turns into a function of various environmental and developmental variables; the altitude of the experimental site can even influence stomatal index (SI) and its density. The redox state of plant cells gets unbalanced due to ozone, which leads to modifications in metabolic processes and gene expression, which affects cell growth and development. Cell division on the whole is sensible to the redox state of plant cells whereby oxidative stress hinders the cell cycle and hence proliferation [26]. Ozone sensitivity was dissimilar among plant species. However, a lot of unanswered questions come forward regarding the possible mainspring of ozone sensitivity or resistance. Furthermore, the effects of ozone on internal tissues of plants are also missing.

Therefore, the present experiment was conducted to assess the effects of ozone on plant development, micromorphology, and anatomy of groundnut (Arachis hypogaea L.) cultivars and their possible role in plant defense. This study hypothesized that the stomatal distribution and movement influence plant sensitivity to ozone.

2. MATERIALS AND METHODS

2.3. Micro-Morphological and Anatomical Characteristics

2.3.1. Microscopic observation

Micro-morphological characteristics of the leaf were measured using a digital microscope (Milton Instruments, Mumbai, Maharashtra) and scanning electron microscopy (SEM) (Model: EVO 18, Make: Carl ZEISS). The sample for micro-morphological studies was collected and shifted to the laboratory in desiccators to avoid the effect of humidity. For stomatal study through a compound microscope, leaf abaxial surfaces were peeled off using dissecting needles and forceps and were finally washed with clean water. After that, each specimen was stained with safranin (1% aqueous) for 3–10 minutes and excess stains were washed using deionized water and then the stained cuticle was mounted on glycerine jelly and detected under a microscope. The number of stomata and EC and its index was calculated as per the equation of Salisbury [27], i.e., (SI = S × 100/S + E) using a compound microscope.

Figure 1: Monthly average ambient ozone (ppb) and temperature (°C) during the study periods of groundnut cultivars. (Mean ± standard deviation of three replicates presented by thin vertical bars.) [Click here to view]

where SI = stomatal index, S = No. of stomata/unit leaf area, and E = number of EC/unit leaf area.

2.3.2. SEM analysis

For SEM [Model: EVO 18, Make: Carl ZEISS] analysis, the specimen of the collected leaf sample was cut in 2–4 mm pieces and fixed (Primary fixation) to 2.5% Glutaraldehyde/Karnovsky’s fixative for 6 hours at 4°C. After that, the samples were washed in 0.1 M phosphate buffer, for three changes each of 15 minutes at 4°C. After primary fixation, the same sample was ready for post-fixation.

For post-fixation, 1% osmium tetroxide was used for 2 hours at 4°C.

With regard to washing and dehydration, the sample was washed in 0.1 M phosphate buffer for three changes each of 15 minutes at 4°C to remove the unreacted fixative. After that, the specimen was dehydrated using increasing concentrations of acetone to remove water by the following procedure:

For dehydration, increasing concentrations of acetone, such as 30%, 50%, 70%, and 90%, were used for 30 minutes of each step, and finally 100% (dry acetone) was used.

After that, the specimen was dried by air and critical point drying (critical point, i.e. 31.5°C at 1,100 p.s.i.) and kept in desiccators.

With regard to specimen mounting and coating, the specimen was mounted on aluminum stubs with a carbon tape. The sample was coated using a sputter coater to make the sample conductive.

3. RESULTS

3.1. Visible Injury

Foliar injuries on the plants are the first visible symptoms of the ozone pollution. Interveinal chlorosis and necrotic stippling were observed on the adaxial surface of the leaves of enhanced ozone-treated groundnut plants (Table 1 and Fig. 2). Chlorotic spots were found to be higher than the necrotic spots in all the selected cultivars. Injury was identified as ozone-like because it was either absent or very less in ambient ozone (data not mentioned). Ozone visible injury was higher in older leaves than in younger leaves. The number of injured leaves was increased with the duration of treatment and was found to be maximum at 60 DAS in all the cultivars tested. The maximum number of injured plants was noted in cultivar Dh-86 (12.66 m⁻²) and the minimum injury was noted in cultivar TAG-24 (6.66 m⁻²). The number of injured leaves was also higher in cultivar Dh-86 (22 plant⁻¹) at 60 DAS and the minimum number of injured leaf was found in cultivar TPG-41 (5.33 plant⁻¹) at 20 DAS. The trends of injury among the selected cultivars were GG-20 > Dh-86 > TG-37A > TPG-41 > TAG-24 under the acute level of ozone (Fig. 2) at all the sampling durations.

Table 1: Ozone-like injury such as total number of plants injured, number of leave injured, chlorosis per plant, and necrosis of groundnut cultivars. [Click here to view]

Figure 2: Ozone-like visible injury on selected groundnut cultivars. (A) ambient ozone, (B) chlorosis and necrotic spots, and (C) visible injury on TG-37A, TPG-41, TAG-24, GG-20, and Dh-86. [Click here to view]

3.2. Plant Morphology

An acute level of ozone changed the morphology of tested groundnut cultivars. At the initial growth stage, the number of leaves of all the cultivars remained similar for enhanced ozone and control plants; however, it was highly affected by an enhanced level of ozone at latter growth stages. Maximum variation in the total number of leaves per plant was noticed in cultivar Dh-86 (−40%) at 40 DAS, while minimum reduction was found in cultivar TPG-41 (−6.52%) at 60 DAS due to the enhanced level of ozone (Table 2).

Similar to the number of leaves, shoots and root branching of selected groundnut cultivars were also affected by enhanced ozone exposure (Table 2). The effect of ozone on branching groundnut cultivars was higher during the early growth stage. Branching of the shoot was highly affected in cultivar TAG-24 (−66.66%) at 40 DAS and cultivar TPG-41 showed minimum reduction in the number of shoot branches (−16.75%) at 60 DAS. A higher reduction in root branching was found in cultivar TG-37A (−36.60%) at 60 DAS, while minimum root branching reduction was found in cultivars GG-20 (−2.30%) at 40 DAS.

Table 2: Number of roots, number of leaves, and number of branches of groundnut cultivars under enhanced ozone and ambient ozone. [Click here to view]

3.5. Regression and Three-Way ANOVA Test

Data were analyzed by regression and ANOVA (three factors) test for significant variation between treatments, age, and cultivars of selected parameters. Pearson’s correlation analysis with R² values nearest to one shows a strong relationship with the number of injured plants per plot (Fig. 7). The number of stomata and EC shows a strong relationship when compared to other parameters, while the number of injured leaves per plant was highly correlated with chlorosis of leaves per plant in cultivar TG-37A. The highest R² (37%) value was found in the number of injured leaves per plant. In cultivar TPG-41, the highest R² (75%) value was noted in necrotic spots per plant. Chlorosis (CHLO) and necrotic showed a strong relationship with each other, while the number of injured leaves slightly correlated with these parameters. EC, chlorosis, and the number of stomata highly correlated in cultivar TAG-24 with R² (95%) values are same in all three parameters, while the number of injured leaves is slightly correlated (Fig. 7). For cultivar GG-20, the plant necrotic spots showed higher R² values (99%) and chlorosis of the plant was slightly correlated with R² value (73%). The higher R² value of cultivar Dh-86 was found in SI (92%) and the lowest R² value was for the number of EC (10%). The number of injured leaves, stomata, and chlorosis was slightly correlated with SI. Overall, sensitive cultivars showed a strong relationship with the number of injured plants per plot. In sensitive cultivars, the number of injured plants per plot was higher; therefore, the number of injured leaves per plant and chlorosis necrosis was also higher (Fig. 7).

Figure 3: Microscopic observation. (A) Number of stomata, (B) number of EC, and (C) SI (%) of groundnut cultivars under enhance and ambient ozone-treated plants. (Mean ± standard deviation of three replicates presented by thin vertical bars.) [Click here to view]

Three-way ANOVA test (three-factor ANOVA) confirmed the significant levels of treatment, DAS, and cultivars (Table 3). Cultivars wise the number of stomata significantly varied at p < 0.01 level and the level of significance of SI was p < 0.05, while the number of EC showed no significant levels. Selected parameters, such as the number of stomata, EC, and SI of groundnut, were highly significant with treatments (p < 0.001). The level of significance of the number of stomata and EC showed the same values (p < 0.01), while the significant level of SI was p < 0.05. Selected parameters’ relationship with Cult.*Treat. Cult.*DAS, Treat.* DAS, and Cult.*Treat.*DAS showed no significant level, except the number of EC; the significant level of the number of EC was p < 0.05.

4. DISCUSSION

The present study shows that the enhanced level of ozone caused a negative effect on groundnut cultivars. The micro-morphological and anatomical modifications observed in selected cultivars show higher variability in the injuries of plants. On the basis of morphological, micro-morphological, and anatomical modification, ozone-sensitive cultivars were highly affected than ozone-tolerant cultivars. Besides having global warming potential, ozone is one of the major gaseous pollutants that directly affected plant growth and productivity [7,9,10,28]. However, its effect depends on the genotype and prevailing environmental condition [29,30].

Foliage is the primary plant organ that is exposed to ozone and shows visible symptoms. Foliar injury is generally the first visible sign of injury to plants from ozone exposure and indicates lessened physiological processes in the leaves [31]. However, these symptoms vary with the genotypes and are restricted to sensitive species and the plant site exposed to the ozone [32]. All the groundnut cultivars tested in the present experiment showed injury under enhanced ozone exposure. However, the total number of plant affected and the number of leave affected in each plant was varied. Leung et al. [28] explained that the sensitive species had a higher injury than resistant species. A higher number of chlorosis and necrotic spots were found in cultivar Dh-86, suggesting its higher sensitivity to ozone, while cultivar TPG-41 showed reduced sensitivity to ozone with the least number of chlorosis and necrotic spots. Similar to this study, Basahi et al. [33] and Islam et al. [34] also reported visible ozone injury under ambient ozone to olive and mung bean plants, respectively. Hayes et al. [35] reported increased ozone-induced leaf injury in Phaseolus vulgaris with increased ozone exposure. In the present experiment, we also found that the ozone-induced injury was higher in mature leaves. This may be due to the longer duration to ozone exposure to older leaves than younger ones.

Figure 4: Microscopic observation of the number of stomata, number of EC, and stomatal opening of groundnut cultivar TPG-41. (A) Enhanced ozone, (B) ambient ozone and cultivar Dh-86, (C) enhanced ozone, and (D) ambient ozone. [Click here to view]

The reduction in the number of leaves per plant is an indicator of an unhealthy and stressed condition. The reduction in leaves per plant reduced total carbon assimilation and NPP of the ecosystem. The reduction in leave was seen in all the groundnut cultivars, despite their sensitivity to ozone. However, a higher sensitive cultivar Dh-86 had a higher reduction and tolerant cultivar TPG-41 had the least reduction in leaves. Consistent production of new leaves can be a reason for lesser leaf reduction in tolerant cultivars. Leung et al. [28] reported the production of new leaves in tolerant cultivar of Phaseolus vulgaris which reduces the percentage of ozone-affected leaves. Furthermore, the least affected branching of tolerant species also maintained the number of leaves. Similar to our study, Cotrozzi et al. [36] also reported 25%–60% phylloptosis in Quercus cultivars under ozone exposure. The minimum reduction in the number of branches per plant in cultivar TPG-41 is also an indication of tolerance of this cultivar than other cultivars. Tsukahara et al. [37] reported ozone-induced reduction in rachis branches in two cultivars of rice and explained this as the effect of ozone on genes near RM3430 markers. However, a surprising reduction in branching of root under enhanced ozone cannot be explained as ozone effect as the effect of ozone on underground plant part is not direct [38].

Stomata development during cell differentiation is confirmed to be regulating by genes that are simultaneously regulating physiological parameters, such as stomatal conductance. The present study found an increase in stomata as well as SI under enhanced ozone exposure in all the groundnut cultivars, suggesting a reduced size of EC under ozone pollution. Islam et al. [34] reported that the ozone pollution interferes with stomatal functioning causing increases in conductance, sluggish stomatal response to environmental factors, or stomatal closure, depending on the species and ozone exposure. Wang et al. [39] and Taiz and Zeiger [26] suggested that between sensitive leaves, young mature ones that developed after the plant had been exposed to significant cumulative doses of ozone stopped cell division of EC earlier than the old ones did. From another aspect, leaves which developed later maintained generous cell growth for a longer duration. This phenomenon may be due to ozone-convinced oxidative stress, which changes the redox state of cells and hence proliferation. The expansive growth of cell decreased the stomata number per mm² of epidermis in sensitive strain and increased EC size. Guard cells development changed similarly after the plants had been exposed to the climate of the summer season and high cumulative ozone doses; the recently developed leaves of sensitive plants had higher-sized guard cells and thus found the larger stomatal apparatus. Cultivar TPG-41 showed maximum increase in EC suggesting its adaptability to enhanced ozone by reducing epidermal cell size and stomatal pores to reduce conductance. The idea of SI normalizes the epidermal cell expansion effect on the density of stomata [25]. SI (Fig. 2) of sensitive strain was mainly lower, but this difference was more significant in resistant genotypes, which developed an increase in stomata numbers, while sensitive plants had a lesser number of stomata either as an attempt to reimburse the adverse effects of ozone or as a consequence of ozone which would have caused the change in cell division and developments. If the purpose of this change had been to lower the accession of the contaminant, it should have also resulted in lower gas condition and/or transpiration rate. The reason of the different stomatal developments could have possibly been ozone-inhibited cell division and/or increased expansive growth of cells, which comparatively decreased the stomata numbers per mm² epidermal area. Chaudhary and Rathore [2] and Qadir et al. [40] reported higher stomata indexes due to oxidative stresses.

Figure 5: Micro-morphological modification identified by SEM of selected groundnut cultivars (A) TG-37A, (B) TPG-41, (C) TAG-24, (D) GG-20, and (E) Dh-86 under enhanced and ambient ozone. [Click here to view]

Figure 6: Cross-section of groundnut ozone-tolerant cultivar TPG-41. (A) Enhanced ozone, (B) ambient ozone and ozone-sensitive cultivar Dh-86, (C) enhanced ozone, and (D) ambient ozone. [Click here to view]

Moreover, the present experiment also found that the enhanced ozone leads to closure of stomata of all the experimental groundnut cultivars. This would reach a reduction in evapotranspiration and water use efficiency, including reduced capability to uptake soil water and an increase in sensible heat flux as seen in soybean by VanLoocke et al. [41] and Bou Jaoudé et al. [42]. Although stomatal closure seems to be the principal response in crops, previous studies have proposed that under chronic ozone exposure, ozone-induced eminent production of stress ethylene can lead to a reducing of the abscisic acid signal [43, 44], which would normally lead to decreases in gas to conserve water in dry soils [45]. This could result in the crop losing control of stomatal closure, impairing water loss, and enhancing ozone uptake that would otherwise be limited by soil water stress, thus generating a feedback loop that enhances ozone damage. The present study also found that the elevated ozone caused stomatal and EC modifications and caused succulents guard cells under exposure of enhanced ozone.

Figure 7: Pearson’s correlation analysis between the total number of injured plants at 60 DAS of plant in response to number of leave injured per plant (IL), CHLO, Necrosis (NECRO), number of stomata (S), number of EC, and SI of groundnuts cultivars (A) TG-37A, (B) TPG-41, (C) TAG-24, (D) GG-20, and (E) Dh-86. Coefficient (R2) value close to 1 expressed the strong positive correlation in between the subsets. [Click here to view]

Table 3: F ratio and level of significance of number of stomata, number of EC, and SI of groundnut cultivars obtained by ANOVA test. [Click here to view]

Ozone-induced anatomical changes in groundnut cultivars were also observed in the present study. Mitu et al. [46] reported changes in spongy parenchyma, epidermis, and vascular bundles of leaves and stems of mango, mahogany, and koroi due to continuous exposure of pollutants. Reduced vessel size in leaves of groundnut suggested reduced water transport and can be correlated with stomatal closure. It would be vital to emphasize that a more detailed anatomical assessment is needed with variable species and cultivars to justify the effect of ozone on internal tissues of plants.

5. CONCLUSION

Ozone is a toxic gaseous pollutant that causes a negative effect on the micromorphology and anatomy of groundnut plants. The study observed ozone-like visible injury symptoms on all the groundnut cultivars using OTC. The results of the study showed that the visible injury was maximum in cultivar Dh-86 and minimum was in cultivar TPG-41. Micro-morphological characteristics, such as the number of stomata, EC, and SI, were increased due to elevated levels of ozone. The higher SI was found in cultivar TPG-41, while cultivars TAG-24 > TG-37A > and Dh-86 showed moderate values and the lowest SI was noted in cultivar GG-20. The enhanced level of ozone injured the leaves, micromorphology, and anatomy of all the groundnut cultivars. The results also confirmed that the stomatal closure and anatomical characteristics such as xylem, phloem, collenchyma, and mesophyll cells disorganization are important characteristics for the identification of ozone-resistant variety. On the basis of stomatal movement and distribution, anatomical changes in cultivar TPG-41 were shown as the most ozone-resistant groundnut variety among the tested cultivars. However, more studies with variable plant species and different cultivars are needed for a substantial conclusion.

Acknowledgments

The authors are grateful to the School of Environment and Sustainable Development, Central University of Gujarat, for providing necessary laboratory and field facilities, and Central Instrumentation Facility, Central University of Gujarat, for instrumentation support. The authors are also thankful to Science and Engineering Research Board (SERB), New Delhi, for financial support in the form of project (File No. YSS/2015/000724).

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SUPPLYMENTRY FIGURES

Figure S1: Modification of micro-morphological characteristics such as number of stomata, number of EC, and stomatal opening of groundnut cultivars (A) TG-37A, (B) TPG-41, (C) TAG-24, (D) GG-20 and (E) Dh-86 under enhanced ozone and ambient ozone. [Click here to view]

Figure S2: Cross-section of anatomical characteristic changes such as epidermis, xylem, phloem, parenchyma and palisade mesophyll cells of groundnut cultivars (A) TG-37A, (B) TPG-41, (C) TAG-24, (D) GG-20 and (E) Dh-86 under enhanced ozone and ambient ozone stress. [Click here to view]

GRAPHICAL ABSTRACT

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