Response of Blackgram Cultivars to Elevated Tropospheric Ozone

As a secondary pollutant, Tropospheric ozone is inadvertently increasing every year, thereby causing severe loss to agricultural crops. The present study aimed at evaluating the response of tropospheric ozone against blackgram varieties. Eight ruling blackgram varieties (V.B.N. 1, V.B.N. 2, V.B.N. 3, V.B.N. 5, V.B.N. 6, V.B.N. 7, V.B.N. 8 and CO 6) were exposed to elevated tropospheric ozone concentration (50 ppb) in an open-top chamber. The exposure was given during 31 days after sowing (D.A.S.) to 40 D.A.S. for seven hours (10.00 h-17.00 h). The changes in physiological, biochemical, growth, and yield traits were observed by comparing them with control (ambient condition). Results indicate that physiological, biochemical, growth, and yield traits significantly differed under ozone stress. Amongst all varieties, the reduction of all observed traits was higher in V.B.N. 3 and least in V.B.N. 8. The reduction of photosynthetic rate, stomatal conductance, and chlorophyll content was higher in V.B.N. 3 (33.57, 29.17 and 35.67 %) and least in V.B.N. 8 (26.23, 22.92 and 31.78 %). In the case of biochemical traits, in V.B.N. 3, the malondialdehyde and proline content increased twice and ascorbic acid declined by 39.85 %. However, in V.B.N. 8, malondialdehyde and proline content increased by 78.26 and 89.01 %; while ascorbic acid decreased by 36.31 % only. Similarly, 100-grain weight reduced in V.B.N. 3 by 8.69 % while it was only 5.37 % in V.B.N. 8. The current investigation revealed that V.B.N. 3 is highly sensitive, while V.B.N. 8 is tolerant to ozone stress.


INTRODUCTION
Tropospheric ozone (O 3 ) has become one of the world's most widely dispersed toxic pollutants in the last several decades (I.P.C. C. 2007;Booker et al., 2009;Brauer et al., 2016), exerting severe impact on humans, plants, and animals (Cho et al., 2011;Ainsworth, 2017;Agathokleous et al., 2018;Osborne et al., 2019). This secondary pollutant, under bright sunshine, is produced by a series of photochemical reactions involving carbon monoxide (CO), methane (CH 4 ), nitrogen oxides (NOx), as well as volatile organic compounds (V.O.C.s) (Collins et al., 1997;Monks et al., 2015). Besides anthropogenic activities and other industrial emissions, and transportation also contributes to ozone production in metropolitan areas. Ground-level ozone is an issue in rural places as well. Although being hundreds or thousand miles away from the original source, higher concentration of tropospheric ozone is experienced in rural areas wherein a majority of land is devoted to agricultural operations (Prather et al., 2003;Agrawal et al., 2006;Williams et al., 2016). In recent decades, the background O 3 concentration has risen by between 0.5 and 2 percent every year (Vingarzan, 2004). The projected trends in ozone precursors emission increases the global average tropospheric ozone concentration by 20-25% between 2015 and 2050, further raising by 40-60% by 2100 (Meehl et al., 2007). Crop yield losses are predicted throughout the world by the year 2030, and India would be suffering the worst situation when it comes to relative yield (Van Dingenen et al., 2009). It has been estimated that current levels of O 3 can result in economic losses of $ 14-26 billion, with China and India alone accounting for 40% (Van Dingenen et al., 2009;Danh et al., 2016;Ashrafuzzaman et al., 2017;Harmens et al., 2018;Mills et al., 2018).
India, being one of the fastest expanding countries in terms of economy in the Asian region, endures a significant increase in tropospheric ozone concentration (Berntsen et al., 1996;Brasseur et al., 1998;Oksanen et al., 2013). Agriculture in India is the country's most populous economic sector, and it ranks second in the world (Ghude et al., 2014;Brauer et al., 2016). Increasing levels of O 3 pollution are a serious concern for India's agriculture, which is home to a fifth of the world's hungry people. As time goes on, reports indicate that the ambient levels of O 3 are growing in the northern region of India (Pandey et al., 1992;Agrawal et al., 2002;Tiwari et al., 2008;Sarkar and Agrawal 2010), which might significantly result in crop yield loss (Agrawal et al., 2005;Singh and Agrawal, 2010;Agathokleous et al., 2015;Sugai et al., 2018). In Tamil Nadu, various regions like Chennai (Pulikesi et al., 2006;Padma et al., 2014;Muthulakshmi et al., 2017;Prabakaran et al., 2017;Mohan and Saranya, 2019), Ooty (Udayasoorian et al., 2013), Suchindrum (Sharma et al., 2012), Kanyakumari (Sharma and Nagaveena, 2016), Tirunelveli (Usha et al., 2018) were reported to experience higher concentration of ground-level ozone.
Pulses, one of the vital agricultural crops, occupies 252.29 lakh hectares of agricultural land with a production of 16.47 million tonnes (D.A.C. & F.W., 2018). They are susceptible to tropospheric ozone, and a critical level of 40 ppb is required for 5% yield reduction . Blackgram is one of the vital pulse crop containing 26% protein and plays a major role in the Indian diet. Studies using Open-top chambers (OTC) have been extensively studied over the last 50 years to determine the yield losses owing to different air pollutants, including O 3 . This study investigates the response of O 3 against eight ruling blackgram varieties since it is one of the vital pulses in India and provides a major protein requirement in the human's diet.

Study Location and plant materials
The study was conducted at wetland (11.00° N, 76.92° E at an elevation of 426.72 m amsl) of Tamil Nadu Agricultural University, During the experimental period, the maximum temperature ranged from 29.9 to 30.8 ºC; while the minimum temperature varied between 17.5 and 18.2 ºC. The maximum relative humidity was from 71 to 85% and the minimum relative humidity varied from 48 to 54%. The study area is located in the under semi -arid tropics. The soil was deep clay loam with taxonomical classification as Typic Haplustalf. The pH was found to be 8.05 with an E.C. of 0.39 dS m -1 . The available nitrogen was found to be low (217 kg ha -1 ) with medium available phosphorus (11.8 kg ha -1 ) and high available potassium (295 kg ha -1 ). The soil had low organic carbon content (0.48%).

Experimental design
A pot experiment was conducted in two Open-Top Chamber (OTC) of standard size (3.5 × 3.5 m), which were installed in the experimental site and consisted of two treatments -Control -nonfiltered chamber (N.F.C.) and N.F.C. + elevated O 3 (50 ppb). The elevated ozone was given using an ozone generator (A4G, Faraday, India). An ozone feed rate of 0.6 mg/min was maintained throughout the experimental period (Van Leeuwen, 2015) and the inlet oxygen flow was maintained at 12 L min -1 to regulate the ozone flow. The plants were exposed to an elevated ozone concentration of 50 ppb in the open-top chamber during the flowering stage from 31 D.A.S. (days after sowing) to 40 D.A.S. for 7 hours (10.00 h -17.00 h) with an AOT40 value of 0.714 ppm. h. The emission was given 30 cm above the canopy of the plant. In the control chamber, the ozone concentration was less than 7 ppb.

Plant sampling
Random plants were sampled in nine replications from each OTC for each blackgram variety (n = 8 variety x 9 replications) and analyzed. Sampling was done after 10 days of ozone exposure and was used to analyze all physiological and biochemical traits. Sampling was done during the harvest or crop maturity stage for growth and yield traits.

Plant analysis
Leaf injury percentage (L.I.P.) ranging from 0 to 100 was given to all the blackgram varieties . Physiological parameters like photosynthetic rate (A), stomatal conductance (gs), and chlorophyll content were measured at different points of the leaf during day light (09.30 AM to 12.00 PM) before and after 10 days of ozone exposure. The youngest leaf was chosen to measure the physiological traits. A portable photosynthetic system (A.D.C. Bio Scientific LCpro-SD System, U.K.) was used to measure A and gs; while chlorophyll content meter (CCM-200+, U.S.A.) was used to measure the chlorophyll content. Biochemical traits like malondialdehyde (MDA), proline, and ascorbic acid (AsA) were also analyzed. The standard protocol given by Heath and Packer (1968) was used to measure; while proline was measured using the procedure given by Bates et al. (1973) and for ascorbic acid (AsA) content the method given by Keller and Schwager (1977) was followed.

Statistical analysis
The significant difference amongst the cultivars and treatment were statistically evaluated using one-way ANOVA test, while their interactions were done by two-way ANOVA test. S.P.S.S. (Ver. 16.0.0), a statistical tool was used to perform the tests. The variation among the treatment means was studied using Tukey method and Pearson's correlation coefficient was used to determine the degree of correlation.

Leaf injury percentage
Exposure to elevated ozone exhibited various leaf injury symptoms, and the leaf injury percentage (L.I.P.) varied among the cultivars. The symptoms were observed to intensify with an increasing exposure period. Younger leaves with early necrotic symptoms were identified in V.B.N. 1, V.B.N. 2, V.B.N. 3, and CO6, indicating its sensitivity towards ozone stress. The L.I.P. for the above-mentioned varieties was found to be 40.00, 40.00, 50. Furthermore, cultivar-specific variation reported in this study demonstrated that our test blackgram cultivars have varying resistance levels to high ozone exposure. This tendency might be explained by a more significant rise in reactive oxygen species (ROS) in comparison to an increase in ozone levels. According to Weadow et al. (2021), superoxide is produced under ozone exposure, causing leaf damage. , revealed that extended ozone exposure intensified the leaf injury in mung bean cultivars and that foliar L.I.P. can demonstrate different levels of ozone sensitivity of test cultivars. Similarly, Mishra and Agrawal (2015) found that ROS buildup inside the plant system caused foliar and cellular damage.

Physiological traits
Elevated ozone exposure significantly reduced the physiological traits like photosynthetic rate, stomatal conductance, and chlorophyll content in all blackgram varieties. The photosynthetic rate varied from 13.04 to 15.02 µmol CO 2 m -2 s -1 . Similarly, stomatal conductance varied between 0.33 to 0.39 mol H 2 O m -2 s -1 and chlorophyll content between 18.34 and 20.67 (Table 1). V.B.N. 3 recorded the highest reduction in all physiological traits among the varieties, while V.B.N. 8 recorded the least reduction. This indicates that V.B.N. 3 is highly sensitive to ozone stress while V.B.N. 8 is tolerant. Like L.I.P., the varietal difference was also observed in physiological traits. Generally, the opening and closing of stomata regulate the entry of tropospheric ozone into the apoplast of the plant system (Tingey and Hogsett, 1985;Daszkowska-Golec and Szarejko, 2013;Rai, 2020). In this study, the reduction in photosynthetic rate and stomatal conductance is associated with the offset mechanism to prevent pollutant entry through stomatal closure (Fiscus et al., 2005;Betzelberger et al., 2010;Ghosh et al., 2020).
Moreover, variation in the partial pressure of the guard cells and loss of osmotic potential might also lead to the closure of stomata under ozone stress. With stomatal closure, the ability to uptake CO 2 declines, thereby decreasing the photosynthetic rate. Similar results were reported in mung bean (Mishra and Agrawal, 2015) and soybean (Sun et al., 2014;Rai et al., 2015;Ramya et al., 2021 a,b). The decline in chlorophyll content due to ozone stress might be attributed to the destruction of the chloroplast structure, thereby suppressing chlorophyll synthesis (Castagna et al., 2001;Biswas and Jiang, 2011;Jing et al., 2016). Moreover, the decline in the carotenoid pigments might also reduce the chlorophyll content (Salvatori et al., 2013). The results corroborate with studies given by Tetteh et al. (2016). The relative physiological characteristics of blackgram varieties varied among each other with a mean value of 70.80, 71.39, and 66.63% for stomatal conductance, photosynthetic rate, and chlorophyll content (Fig.1). The greater deviation was observed in stomatal conductance indicating that stomatal conductance is highly responsive to ozone stress compared to the photosynthetic rate and chlorophyll content.

Biochemical traits
In all blackgram varieties, malondialdehyde content (MDA) significantly increased under ozone stress compared to control and a varietal variation was also observed. The MDA content ranged from 1.07 to 1.49 µmol g -1 F.W. in control and from 2.27 to 2.97 µmol g -1 F.W. under 50 ppb ozone stress. The entry of tropospheric ozone into the plant system induces the generation of reactive oxygen species (ROS), thereby damaging the membrane components like lipids, chloroplast, nucleic acids, and proteins (Blokhina et al., 2003;Hasanuzzaman et al., 2012;Saxena et al., 2019). The induction in MDA content under ozone stress is related to its sensitivity suggesting greater lipid peroxidation of the membrane compared to ambient conditions. Furthermore, the destruction of membrane components due to ROS generation inhibits the scavenging ability of the plant cell (Sanmartin et al., 2003). The results corroborate with the findings of Mishra and Agrawal (2015) observed a 30.8 and 21% increase in MDA content of mung bean cultivars under 68.9 ppb ozone stress. Significant reduction in biochemical traits was also observed in cauliflower (Sethupathi et al., 2018). garlic (Gayathri et al., 2019) and rice (Ramya et al., 2021a).  Figure 3. Relative growth traits of blackgram varieties under 50 ppb ozone stress Unlike MDA content, the ascorbic acid content was found to decline in all blackgram cultivars under ozone stress compared to control. The ascorbic acid varied between 1.33 to 1.72 mg g -1 F.W. in control and between 0.78 and 1.03 mg g -1 F.W. in elevated ozone condition. This decline might be due to the non-enzymatic defense mechanism of blackgram varieties under ozone stress. Antioxidants are produced to nullify the ROS toxicity (Caregnato et al., 2013) and the redox condition of AsA becomes unstable, eventually resulting in insufficient detoxification by AsA (Tetteh et al., 2016). The proline content increased under ozone stress varying from 4.81 to 6.08 µmol g -1 F.W. under ambient conditions and from 10.15 to 13.46 µmol g -1 F.W. under 50 ppb ozone stress. This increased proline content might be attributed due to the scavenging ability of proline under ozone stress (Gill and Tuteja, 2010;Rejeb et al., 2014). The relative biochemical traits showed variation with mean values of 201.01, 60.11 and 202.00% in MDA, AsA and proline content (Fig.2). The number of nodules per plant ranged between 92.50 and 100. Similarly, V.B.N. 8 recorded the highest number of pods per plant (11.00), number of seeds per pod (5.67), pod length (4.83 cm), number of leaves per plant (59.00), 100-grain weight (4.23 g), and plant weight (18.60 g). The reduction was relatively higher in V.B.N. 3 in most growth and yield traits, signifying its sensitivity to ozone stress. Reduced photosynthetic capacity under elevated ozone conditions might, in turn, decrease the plant biomass by altering the allocation of photosynthates to several parts of the plant (Sarkar and Agrawal, 2010;Feng et al., 2011;Ruiz-Vera et al., 2017;Ghosh et al., 2020). Similar results were observed in mung bean where the plant height and number of leaves per plant decreased by 25.7 and 24% under 70.9 ppb ozone stress (Chaudhary and Agarwal, 2015). The reduction in yield traits might be due to extended closure of stomata and decline in carbon fixation thereby reducing the availability of assimilates to its reproductive parts. Alterations in physiological and biochemical characteristics under ozone stress might in turn mighthave altered the growth and yield traits in blackgram varieties. V.B.N. 3 and V.B.N. 1 exhibited senescence due to ozone stress which might be ascribed due to the induction of genes associated with senescence (Miller et al., 1999). Similar results were also observed by Chaudhary and Agarwal (2015) in mung bean and by Ghosh et al. (2020) (Fig.3). The relative number of pods per plant, number of seeds per pod, pod length, and 100-grain weight had a mean value of 69.73, 93.93, 97.93, and 91.85%, respectively (Fig.4). The Pearson's correlation depicted a negative correlation between leaf injury percentage and other characteristics (Table 2). Similarly, a positive correlation was observed between stomatal conductance and photosynthetic rate, between chlorophyll content and photosynthetic rate, and between chlorophyll content and stomatal conductance. Likewise, linear relationship was observed between photosynthetic rate and growth traits (number of seeds per plant and pod length).

CONCLUSION
Understanding the influence of elevated tropospheric ozone on blackgram is highly imperative for global food security. The current study exhibited varietal variation among blackgram cultivars to ozone stress. Results indicate that elevated ozone concentration increased the leaf injury percentage, while all physiological traits like photosynthetic rate, stomatal conductance, and chlorophyll content declined significantly. Similarly, the malondialdehyde and proline content increased, while ascorbic acid content decreased under ozone stress. Significant reduction in the growth and yield traits were also observed in all blackgram varieties under ozone stress. This indicates that, among the blackgram varieties under study, V.B.N. 3 is sensitive and V.B.N. 8 is tolerant to ozone stress. Hence, this finding serves as a preliminary base to evaluate and assess the choice of cultivars to regions experiencing high tropospheric ozone concentration and for future breeding programmes. Varietal-specific alterations in plant attributes under ozone stress might pave the way for developing ozone-tolerant blackgram cultivars using genome mapping and quantitative trait loci (QTL)-based approaches, thereby boosting global production. Significant genetic heterogeneity in ozone sensitivity and tolerance across test cultivars might be helpful in creating models to anticipate ozone-induced yield loss and adaptation techniques for long-term cultivation.

Funding and Acknowledgment
The authors sincerely thank Tamil Nadu Agricultural University, Coimbatore for extending facilities and support in performing the experiments. Sincere thanks to Dr. S. Karthikeyan, Professor (Microbiology), T.N.A.U. for providing all infrastructure facilities to carry out the pot experiments.

Ethics statement
No specific permits were required for the described field studies because no human or animal subjects were involved in this research.

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Authors ensure the originality of the manuscript and the work and/or words of others, has been appropriately cited. We acknowledge that plagiarism in all its forms constitutes unethical publishing behavior and is unacceptable.

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Competing interests
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Data availability
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