Phytotoxic effects of tropospheric ozone altered by elevated levels of CO2 in Indian mustard for Sustaining production

doi:10.21203/rs.3.rs-4880728/v1

Tropospheric ozone is the most important air pollutant of global concern since it affects crops negatively by causing oxidative damage. The increased levels of carbon dioxide positively impacted the C₃ plants. The impacts of elevated O₃ and CO₂ on most crops have been studied, but the information in relation to the interactive effects of O₃ and CO₂ is still limited and elusive in Indian mustard. Thus, the study was aimed at quantifying the impacts of elevated O₃(Ambient + 25±5 ppb) and CO₂ (550±10 ppm) along with their interaction on different winter mustard varieties, to elucidate their response towards growth and yield parameters as well as photosynthetic activity and stomatal behaviour for two seasons (2020-21 & 2021-22). The photosynthetic activity in mustard declined by about 16%, crop index by 15% and seed yield by 24% under elevated O₃treatments. Contrarily, the CO₂ enrichment treatments nullified the O₃ effects on yield by a maximum of 17% in PDZM 31 followed by 14% in Pusa Bold and 13% in PM 30. The elevated levels of ozone reduced stomatal conductance, and the photosynthetic activity offsetted by elevated CO₂ acts as a defensive mechanism to avoid the entry of O₃ into leaf tissues and restrict the production of reactive oxygen species. Thus, the current interaction studies revealed that the strong oxidative damage caused by elevated O₃ was reduced by elevated CO₂ in mustard varieties and discussed in detail.

Biological sciences/Plant sciences

Earth and environmental sciences/Climate sciences

Earth and environmental sciences/Environmental sciences

Tropospheric Ozone

Elevated carbon dioxide

Crop growth

Seed yield

Photosynthetic rate

The phenomenon of increasing tropospheric ozone (O₃) and carbon dioxide (CO₂) concentrations presents a critical challenge for global agriculture, particularly under the evolving climatic conditions of diverse agroecosystems. The rise in tropospheric O3, a detrimental secondary air pollutant and a key greenhouse gas, is primarily attributed to human-induced factors such as accelerated industrialization, expanding urbanization, and increasing vehicle emissions. This upsurge in O₃ levels, estimated to escalate by approximately 20–25% by the year 2050, poses a significant threat to plant health and agricultural productivity worldwide (Chan et al., 2008; Jaggard et al., 2010). Ozone's phytotoxic nature manifests through the induction of oxidative stress in plants, caused by the generation of reactive oxygen species (ROS), leading to visible leaf injury, impaired photosynthetic processes, and ultimately, a reduction in crop yields (Yadav et al., 2019; Zhang et al., 2019).

In parallel, the issue of rising atmospheric CO₂ concentrations, a primary contributor to global climate change, has garnered considerable attention. Since the dawn of the industrial era, atmospheric CO₂ levels have experienced a substantial increase of at least 35%, with projections indicating a potential rise to between 540–970 ppm by the end of the 21st century (Pachauri and Reisinger, 2007). Elevated CO₂ concentrations have been observed to enhance photosynthetic activity in C₃ plants, leading to increased carbon assimilation and potential improvements in crop productivity. However, the implications of this elevation in CO₂ levels extend beyond photosynthesis, influencing various aspects of plant growth, development, and nutritional quality. The interaction of CO₂ with other environmental factors, such as O₃, further complicates its impact on plant physiology and crop yields, particularly in C₃ crops like wheat, rice, and soybeans (Toreti et al., 2020; Kimball, 2016).

In the Indian subcontinent, mustard holds a position of significant agricultural importance, especially in the northern regions. As a major oilseed crop, mustard contributes substantially to the national vegetable oil industry, accounting for about 27% of total vegetable oil production annually. The crop covers an extensive area, encompassing approximately 6.8 million hectares, which represents around 25.1% of the total oilseed cultivation area in India (MOAFW, 2022). Recent studies, such as that by Mukherjee et al. (2020), highlight mustard's heightened sensitivity to O₃ stress, surpassing even that of wheat, a major staple crop. This sensitivity underscores the urgent need for research focused on developing mustard varieties that are not only high-yielding and of superior quality but also resilient to the dual challenges posed by elevated O₃ and CO₂ levels.

The current research endeavor aims to fill a critical void in our understanding of how increased levels of tropospheric O₃ and CO₂ interact and impact the growth and productivity of mustard crops. Specifically, the study focuses on the double zero mustard varieties, which are of particular interest due to their reduced concentrations of glucosinolates and erucic acid, making them more beneficial for human health and livestock. This research comprehensively investigates the growth rate, seed yield, physiological, and biochemical responses of mustard to elevated O₃ and CO₂ levels. By delving into these aspects, the study offers invaluable insights into the adaptive mechanisms of mustard under altered environmental conditions and contributes to the broader discourse on sustainable agriculture in the face of climate change. The findings from this research are expected not only to advance our scientific understanding of plant-environment interactions but also to inform the development of resilient crop varieties, thus ensuring food security and agricultural sustainability in a changing world.

2.1 Study area

This study was conducted at the Division of Environment Sciences, Indian Agricultural Research Institute, New Delhi, India, in the Genetic-H field (28.64˚N, 77.16˚E). Employing Free Air Carbon Dioxide Enrichment (FACE), Free Air Ozone Enrichment (FAOE), and combined FACE and FAOE (FACOE), the research aimed to replicate elevated atmospheric CO₂ and O₃ conditions. Data collection occurred over two cropping seasons, October 2020 to March 2021 and October 2021 to March 2022, tracking daily minimum and maximum temperatures, relative humidity, sunshine hours, and evapotranspiration.

Ozone levels were monitored using a TONGDY Gas Monitor/Transmitter/Controller, providing daily readings of both ambient and elevated ozone. Carbon dioxide concentrations were similarly recorded using a SenseAir sensor for both ambient and elevated levels. This comprehensive environmental monitoring was critical for assessing the impact of increased CO₂ and O₃ on mustard crop growth and development.

2.2 Plant material

For the experimental study, we utilized breeder seed materials of Indian mustard, Brassica juncea (L.) Czern. & Cross. The specific varieties selected were Pusa Double Zero Mustard 31 (PDZM 31), Pusa Bold, and Pusa 30 (LES 43). These seeds were obtained from the Genetics Division and the Division of Seed Science and Technology at the ICAR-Indian Agricultural Research Institute (IARI), New Delhi. A detailed description of these mustard varieties, including their specific characteristics and relevance to the study, is provided in the Supplementary file (Table SI 1). This selection of varieties was intended to encompass a range of responses to the environmental conditions simulated in the study.

2.3 Treatment details

This study spanned two rabi seasons from mid-October to mid-March in 2020–2021 and 2021–2022, using three Indian mustard varieties. Four treatments were applied: T₁- FAOE (Free Air Ozone Enrichment) with elevated ozone at 65 ± 5 ppb, T₂- FACE (Free Air Carbon Dioxide Enrichment) with elevated CO₂ at 550 ± 10 ppm, T₃- FAOCE (combined elevated ozone and CO₂), and T₄- Ambient (control), each replicated four times.

Ambient ozone and CO₂ levels were recorded at 25–30 ppb and 403–412 ppm, respectively, for the 2020–2021 season, and 23.8–30 ppb and 396–410 ppm for 2021–2022, as shown in Figs. 1(a) and 1(b). The crops were exposed to these conditions daily for 7 hours (9am to 4 pm) after emergence until harvest, focusing on the flowering to seed-filling stage, roughly 75 days post-sowing. This timeframe was critical for evaluating the impact of the treatments on the physiological development and eventual yield of the mustard crops.

2.4 Canopy temperature

Canopy temperature (Tc) was recorded for both seasons using a FLUKE 62 MAX infrared thermometer. Measurements were taken between 12 PM and 2 PM under clear skies, from four directions (north, south, west, and east) to mitigate radiation effects and ensure comprehensive data. The average of these readings represented the Tc for each treatment. This method provided a balanced assessment of the plants' thermal response under different environmental conditions, crucial for understanding their reaction to elevated CO₂ and ozone levels.

2.5 Leaf gas exchange parameters

Leaf gas exchange parameters—photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E), and water use efficiency (WUE = Pn/E)—were accurately measured using the LI-6400XT IRGA portable photosynthesis system (LICOR, USA). These parameters were recorded every five seconds at mustard's critical stages: from 50% flowering to the seed-filling stage (75 DAS). Data collection was conducted during the morning hours, between 9.00 AM and 11.00 AM, across two growth seasons (2020–2021 and 2021–2022). This approach provided valuable insights into the plants' physiological adaptations to the experimental treatments under study.

2.6 Free proline, antioxidant enzymes and lipid peroxidation (MDA)

In this study, leaf samples from mustard plants (specifically, the fully expanded uppermost second leaf) were collected at the critical flowering to seed-filling stage (75 DAS). These samples were immediately flash-frozen in liquid nitrogen and stored at -80°C to preserve their enzymatic integrity for subsequent antioxidant enzyme analysis.

For proline estimation, 0.5 g of the mustard leaf samples, particularly those exposed to elevated levels of O₃ and CO₂, were homogenized in 5 ml of 3% sulfosalicylic acid. The concentration of proline in these samples was then determined by comparison with a standard curve of known proline concentrations (Bates et al., 1973).

The preparation of enzyme extracts for superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POX), and catalase (CAT) involved initially freezing 1 g of leaf samples in liquid nitrogen. This step was crucial to inhibit proteolytic activity. The frozen samples were then ground with 10 ml of an extraction buffer. This buffer comprised 0.1 M phosphate buffer (pH 7.5) with 0.5 mM EDTA for SOD, CAT, glutathione reductase (GR), and POX. For APX, the buffer additionally contained 1 mM ascorbic acid.

CAT enzyme activity was quantified based on the concentration of hydrogen peroxide reduced per minute per mg of protein (Aebi, 1984). The activity of APX was determined by measuring the concentration of ascorbic acid oxidized per minute per mg protein (Nakano and Asada, 1981). SOD activity was estimated by its absorbance at 560 nm, with a unit of enzyme activity defined as the amount of enzyme that inhibits the rate of nitroblue tetrazolium reduction by 50%.

The peroxidase (POX) activity in the mustard leaf samples was determined by measuring the absorbance due to tetra-guaiacol formation at 470 nm. The enzyme activity was calculated based on the extinction coefficient of its oxidation product, tetra-guaiacol (26.6 mM^− 1 cm^− 1). This activity is expressed in terms of µmol tetra-guaiacol formed per minute per gram of fresh weight or per mg of protein (Catillo et al., 1984). Additionally, the level of lipid peroxidation in the treated mustard leaves was assessed by measuring the content of thiobarbituric acid reactive substances (TBARS), as described by Heath and Packer (1968). This involved recording the absorbance of the supernatant at 532 nm. The TBARS content was then calculated using its specific extinction coefficient (155 mM^− 1 cm^− 1), with adjustments made for non-specific absorbance at 600 nm. This method provides a quantitative measure of lipid peroxidation, indicative of oxidative stress in the plant tissues under the different treatment conditions. Such measurements are vital for understanding the impact of elevated O₃ and CO₂ levels on the oxidative stability and overall health of mustard plants.

2.8 Growth observations

2.8.1 Leaf area index (LAI): The leaf area was measured by using a leaf area meter during 45, 60, 75 and 90 DAS of the cropping season (2020–2021 and 2021–2022). Thus, the leaf area index was calculated by using the formula Eq. (1)

$$\:LAI=\frac{Leafarea\left({m}^{2}\right)}{Ground\:area\left({m}^{2}\right)}\dots\:\dots\:\left(1\right)$$

2.8.2 Relative growth rate (RGR): The relative growth rate was obtained by measuring the plant dry matter for 15 days intervals from 45 DAS to 90 DAS for both cropping year and incorporated in the formula Eq. (2)

$\:RGR=\frac{lnW2-lnW1}{t2-t1}\dots\:\dots\:\left(2\right)$ g/m²/day

Where W₁ & W₂ are dry weight at times t1& t2.

2.8.3 Crop index (CI): The crop index is estimated after the harvest of the crop from economic yield (gm^− 2) to the shoot yield (gm^− 2) for both seasons. The formula (Eq. (3)) is

$$\:Crop\:Index\left(CI\right)=\frac{Economic\:yield\left(g{m}^{-2}\right)}{Shoot\:yield\left(g{m}^{-2}\right)}х100\dots\:\dots\:\left(3\right)$$

2.9 Seed yield: The three different varieties of mustard (2020–2021 & 2021–2022) are harvested on per meter square basis and seed yield was quantified.

2.10 Statistical analysis

The obtained results were statistically analyzed by three-way ANOVA using SPSS version 16.0 statistical software to determine the individual treatment and between treatment, year, and varietal effects at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001. The box - whisker graphs were obtained by GraphPad Prism 9.3.1 version statistical tool. To statistically analyze the significant performance among the treatments, years, and varieties, multivariate correlation analysis, as well as the linear discriminant analysis between treatments and between varieties, were performed using JMP Pro 16 version statistical software from the discovery of SAS.

3.1. Ambient and elevated CO₂ and O₃

The ambient CO₂ was 399 to 400 ppm during the crop growing period of 2020–2021 and 400–401 ppm during 2021–2022. The elevated CO₂ was maintained at 590 to 595 ppm, and 592 to 596 ppm during the crop-growing period of 2020–2021 and 2021–2022 respectively. Similarly, the recorded ambient ozone level was 21–24 ppb, and 22–25 ppb during the crop growing period of 2020–2021 and 2021–2022 respectively. The maintained elevated O₃ levels were 61–63 ppb and 60–62 ppb during the crop growing period of 2020–2021 and 2021–2022.

Changes in canopy temperature (Tc) and gas exchange parameters

The canopy temperature (Tc) for the three mustard varieties (PDZM 31, PB, and PM 30) remained relatively consistent across the two seasons (2020–2021 and 2021–2022), irrespective of the elevated conditions. This suggests that Tc may not be as sensitive to changes in O₃ and CO₂ levels as other physiological parameters.

However, the photosynthetic rate (Pn) of these varieties exhibited significant variability under different treatments as shown in Fig. 2a. In the first season (2020–2021), ambient conditions yielded the highest Pn, particularly in the PM 30 variety, followed by PDZM 31 and PB. This outcome indicates that ambient conditions might be more conducive to photosynthesis in mustard. In contrast, elevated O₃ (eO₃) conditions led to a substantial reduction in Pn, with the most pronounced effect seen in PDZM 31, demonstrating its sensitivity to ozone stress. Interestingly, elevated CO₂ (eCO₂) conditions enhanced Pn across all varieties, with PB showing the most significant increase. This enhancement under eCO₂ conditions aligns with the expected response of C₃ plants to increased CO₂ levels, suggesting a potential mitigating effect on the detrimental impacts of ozone. The combined treatment effects of elevated CO₂ and O₃ (eCO₂*eO₃) generally resulted in a decreased photosynthetic rate, with the most notable effect observed in PM 30. This indicates that the positive effect of CO₂ enrichment on photosynthesis can be offset by the simultaneous increase in ozone levels. The second season (2021–2022) followed a similar trend, with ambient conditions favoring higher Pn. The negative impact of eO₃ was evident again, particularly in the PB variety, which showed the most significant decline.

Stomatal conductance (gs) was also significantly impacted under elevated O₃ and CO₂ conditions and their interaction, as depicted in Fig. 2b. The highest gs was observed under ambient conditions, particularly in PM 30. Elevated O₃ caused a marked decline in gs across all varieties, with PB showing the greatest decrease. This decline under eO₃ conditions highlights the stress imposed by ozone on the stomatal functioning of mustard plants.

Transpiration rate (E) in the Fig. 2c, mirrored the trends observed in gs, with reductions under ambient conditions and increases under elevated O₃. This relationship between E and gs is expected, as stomatal conductance directly influences transpiration. Elevated CO₂ conditions resulted in the lowest percentage loss of transpiration rate, especially in the PM 30 variety, suggesting that increased CO₂ may lead to more efficient water use in mustard plants.

Water use efficiency (WUE) varied significantly across treatments and seasons, as shown in Fig. 2d. Elevated O₃ conditions generally resulted in a loss of WUE, particularly in PDZM 31 and PB, while eCO₂ conditions improved WUE, notably in PB and PM 30. However, PDZM 31 showed a negative response to eCO₂ in the first season, suggesting variety-specific responses to elevated CO₂. The interaction of eCO₂ and eO₃ often led to a loss in WUE, with the least impact observed in PM 30. This indicates that while CO₂ enrichment can improve WUE, its combination with elevated O₃ levels may negate this benefit.

These results collectively demonstrate that elevated CO₂ and O₃ levels have complex and varied impacts on mustard physiology, influenced by both environmental conditions and plant variety. The differential responses of PDZM 31, PB, and PM 30 to these treatments provide valuable insights into the adaptive mechanisms of mustard plants to changing atmospheric conditions.

3.2 Changes in free proline, antioxidant enzymes and MDA activity

In the first season, the exposure of mustard varieties to elevated conditions notably influenced the concentration of free proline compared to ambient conditions. Free proline, often accumulated in plants under stress as a protective measure, saw the most significant reduction in PDZM 31, followed by PM 30 and PB. This decrease under elevated conditions suggests that these varieties might be employing other stress-response mechanisms over proline accumulation. Intriguingly, under elevated O₃ (eO₃) conditions, all varieties showed an increase in proline concentration, indicating an O₃-induced stress response. PB, in particular, displayed a lesser increase, suggesting a possible inherent resilience or different stress response mechanism in this variety.

Across both seasons, antioxidant enzyme activities demonstrated interesting patterns. Catalase (CAT), a key enzyme in detoxifying reactive oxygen species (ROS), showed negligible effects under elevated conditions in all varieties. However, under eO₃, there was a marked increase in CAT activity, especially in PDZM 31. This could be indicative of enhanced oxidative stress in plants due to ozone exposure, prompting a stronger antioxidant response. Elevated CO₂ (eCO₂) conditions, on the other hand, led to a decreased CAT activity compared to eO₃, suggesting that eCO₂ might be less stressful or that it modulates the oxidative stress differently. The interaction of eCO₂ and eO₃ showed an increase in CAT activity from ambient levels but a decline compared to eO₃ alone, indicating a complex interplay of these gases on oxidative stress responses.

Other antioxidant enzymes like peroxidase (POX), ascorbate peroxidase (APX), and superoxide dismutase (SOD) also exhibited significant increases under elevated conditions, further underscoring the elevated oxidative stress experienced by the plants. PM 30 demonstrated a broader range of antioxidant protection, followed by PDZM 31 and PB. This variation among varieties suggests different capacities or strategies to counteract oxidative stress.

MDA content, an indicator of lipid peroxidation and cellular damage, significantly increased under eO₃ conditions across all varieties, reflecting oxidative damage. The similar trends of MDA content under eCO₂ and eO₃*eCO₂ conditions suggest that these conditions either exacerbate or do not mitigate the oxidative stress induced by eO₃.

The analysis of these biochemical parameters across two seasons and under various elevated conditions (as depicted in Figs. 3 and 4) highlights the differential responses of mustard varieties to environmental stress. It underscores the complex interplay of external conditions on plant physiology and the varying coping mechanisms among different varieties. This understanding is crucial for predicting plant behavior under changing climatic conditions and for breeding more resilient crop varieties.

3.3 Changes in Growth Parameters

The study's exploration of growth parameters, including Leaf Area Index (LAI) and Relative Growth Rate (RGR), alongside yield parameters like crop index and seed yield, revealed significant variances under different treatment conditions (eO₃, eCO₂, eO₃*eCO₂, and control). These findings, detailed in Table 1, provide a nuanced understanding of how elevated environmental conditions impact mustard crop growth and productivity.

Table 1

Growth and seed yield characteristics of mustard varieties grown in elevated ozone, elevated carbon dioxide and their interaction
2020–2021
Treatment	Stages	eO₃			eCO₂			eO₃*eCO₂			Ambient				SEm	CD (0.05)
Parameter	Genotype	PDZM 31	PB	PM 30	PDZM 31	PB	PM 30	PDZM 31	PB	PM 30	PDZM 31		PB	PM 30	SEm	CD (0.05)
LAI	45 DAS	0.58^f	1.26^e	1.33^e	2.20^a	2.00^b	2.10^ab	1.48^d	1.58^d	1.52^d	1.81^c	1.86^c		1.80^c	0.042	0.129
	60 DAS	1.63ⁱ	2.58^fg	2.46^fg	3.04^cd	3.64^ab	3.94^a	1.97^h	2.91^de	2.70^ef	2.35^g	3.35^bc		3.35^bc	0.102	0.328
	75 DAS	1.69^g	2.44^e	2.16^f	2.85^bc	3.05^a	2.96^ab	1.83^g	2.63^d	2.34^e	2.35^e	2.78^cd		2.64^d	0.047	0.154
	90 DAS	0.79^f	0.93^e	0.74^f	1.48^bc	1.59^a	1.52^ab	0.96^e	1.02^e	0.93^e	1.32^d	1.38^cd		1.36^d	0.033	0.107
RGR (g/m²/day)	45–60 DAS	0.05^e	0.05^de	0.05^de	0.05^cd	0.06^ab	0.06^a	0.05^de	0.05^cd	0.06^abc	0.05^de	0.06^bc		0.06^ab	0.001	0.004
	60–75 DAS	0.04^bcd	0.04^d	0.04^cd	0.05^a	0.04^abcd	0.04^ab	0.04^abc	0.04^bcd	0.04^bcd	0.05^ab	0.04^abcd		0.04^bcd	0.002	0.007
	75–90 DAS	0.02^ef	0.02^f	0.03^def	0.03^abcd	0.04^ab	0.04^a	0.02^ef	0.03^def	0.03^bcde	0.03^cde	0.04^abc		0.04^abc	0.003	0.008
Crop index	Harvest	30.56^e	35.89^de	38.87^bcd	38.84^bcd	45.89^ab	46.45^a	34.83^de	37.69^cde	42.17^abcd	36.61^de	41.85^abcd		44.96^abc	2.307	7.524
Seed yield (g/m²)	Harvest	153.00^f	202.50^e	247.50^cd	240.00^d	344.00^a	326.75^b	172.25^f	249.50^cd	262.25^c	203.75^e	294.75^b		295.75^b	6.728	19.38
2021–2022
Treatment	Stages	eO₃			eCO₂			eO₃*eCO₂			Ambient				SEm	CD (0.05)
Parameter	Genotype	PDZM 31	PB	PM 30	PDZM 31	PB	PM 30	PDZM 31	PB	PM 30	PDZM 31		PB	PM 30	SEm	CD (0.05)
LAI	45 DAS	0.41^h	1.20^g	1.23^fg	2.34^a	1.95^bc	2.07^b	1.43^e	1.41^ef	1.48^e	1.48^e		1.71^d	1.82^cd	0.062	0191
	60 DAS	1.78^f	2.48^de	2.38^de	3.01^c	3.60^a	3.60^a	1.93^f	3.19^bc	2.67^d	2.35^e		3.19^bc	3.45^ab	0.088	0.301
	75 DAS	1.61^h	2.17^f	2.17^f	2.76^bc	2.81^b	2.93^a	1.79^g	2.47^d	2.29^e	2.32^e		2.65^c	2.66^c	0.039	0.120
	90 DAS	0.69^f	0.98^de	0.89^e	1.32^c	1.48^ab	1.57^a	0.96^de	1.05^d	1.01^d	1.32^c		1.48^ab	1.41^bc	0.036	0.123
RGR (g/m²/day)	45–60 DAS	0.05^ef	0.05^f	0.05^cd	0.05^cd	0.06^bcd	0.06^a	0.05^ef	0.05^ef	0.06^bc	0.05^de		0.05^cd	0.06^ab	0.001	0.004
	60–75 DAS	0.03^c	0.03^c	0.03^d	0.04^ab	0.04^a	0.04^bc	0.03^c	0.04^bc	0.03^cd	0.04^bc		0.04^ab	0.03^c	0.001	0.004
	75–90 DAS	0.02^bcd	0.02^bcd	0.02^d	0.03^abc	0.03^a	0.03^ab	0.02^bcd	0.03^abc	0.02^cd	0.03^abc		0.03^ab	0.03^bcd	0.002	0.006
Crop index	Harvest	36.82^d	38.46^cd	38.86^bcd	48.51^a	47.76^a	46.53^ab	38.63^cd	38.28^cd	41.78^abcd	44.66^abc		44.24^abcd	45.10^abc	2.238	7.767
Seed yield (g/m²)	Harvest	148.00^h	215.00^f	247.75^de	241.25^e	383.25^a	324.00^b	174.00^g	252.25^de	261.75^d	212.00^f		286.75^c	303.00^c	6.803	19.64
eO₃- elevated Ozone, eCO₂- elevated Carbon dioxide, eO₃eCO₂- elevated Ozone & elevated Carbon dioxide interaction* PDZM 31- Pusa Double zero mustard 31, PB- Pusa Bold, PM 30- Pusa Mustard 30 45 DAS- Vegetative stage, 60 DAS- Flowering stage, 75 DAS- Peak flowering and seed filling stage and 90 DAS- Physiological maturity stage LAI- Leaf area index, RGR- Relative growth rate

Exposure to elevated ozone (eO₃) and its combination with elevated CO₂ (eO₃*eCO₂) notably reduced LAI and RGR compared to plants exposed only to elevated CO₂ (eCO₂) and control conditions. This outcome indicates that ozone stress negatively impacts leaf area development and overall plant growth, a trend commonly observed in plants subjected to oxidative stress caused by elevated O₃ levels. The impact of ozone on LAI and RGR can be attributed to its interference with photosynthesis and stomatal conductance, leading to reduced carbon assimilation and growth.

The fluctuating pattern of LAI across different developmental stages (45, 60, 75, and 90 DAS) under all treatments points to a dynamic response of mustard plants to changing environmental conditions. The initial decrease in LAI at 45 DAS, followed by an increase at 60 DAS and subsequent decrease at 75 and 90 DAS, suggests that the plant's growth responses are stage-specific. PM 30 showed resilience at the initial growth stage (45 DAS), while Pusa Bold fared better at later stages under eO₃ stress.

Interestingly, eCO₂ conditions facilitated an increase in LAI, particularly noticeable in PDZM 31 and PM 30 at the earlier growth stages (45 and 60 DAS) and in PB at later stages (75 and 90 DAS). This increase under eCO₂ conditions aligns with the expected response of C₃ plants, which typically exhibit enhanced growth and leaf expansion due to increased availability of CO₂ for photosynthesis.

The interaction treatment (eO₃*eCO₂) revealed an improvement in LAI, particularly in PB, suggesting a mitigating effect of elevated CO₂ on the detrimental impact of ozone. This finding is significant as it indicates that increased CO₂ levels might help buffer the negative effects of ozone on plant growth.

RGR, a crucial indicator of overall plant growth efficiency, was highest under eCO₂ conditions, especially in PM 30, which was comparable with PB. This suggests that elevated CO₂ positively impacts mustard's growth rate, likely due to enhanced photosynthetic activity and carbon assimilation. Conversely, a significant decline in RGR under eO₃ conditions across both seasons reaffirms the inhibitory impact of ozone on plant growth.

3.4 Yield Parameters

The crop index and seed yield vary significantly between treatments, varieties and two seasons. In both seasons the crop index, the elevated O₃ decreased by 16-17.5%, 13–14% and 13.6% in PDZM 31, PB and PM 30 varieties, respectively, at elevated CO₂, the crops responded to increase (6.1–8.6%, 7.9–9.7% and 3.2–3.3% in PDZM 31, PB and PM 30) as compared to ambient condition. The seed yield decreased by 14.3–22%, 12.5–13% and 16.7% in the varieties respectively, at elevated O₃ levels as compared to ambient, but increased significantly at elevated CO₂ (Table 1). However, the ozone and carbon dioxide interactions in crop index and seed yield showed a reduced percentage decrease in interaction treatments in different varieties of mustard.

Three-way ANOVA (Table SI 2) was carried out to obtain the significant levels of canopy temperature (Tc) and gas exchange parameters (Pn, gs, E and WUE). In terms of the year, a significant difference was observed in photosynthetic rate (Pn) and stomatal conductance (gs) with respect to p ≤ 0.05 significant levels, the treatment effects of Pn, gs, E and WUE were highly significant to the levels p ≤ 0.001 followed by Tc which was slightly significant to the level p ≤ 0.05. The varietal effects concerning photosynthetic rate (Pn) were highly significant to the levels p ≤ 0.001. The treatment*varietal effects under two-way ANOVA were significantly concerning in WUE p ≤ 0.01. With respect to gas exchange parameters and canopy temperature, there was no significant difference observed.

A significant variation was observed from three-way ANOVA for antioxidant enzymes between treatment, years and varieties in Table SI 3. A highly significant difference with respect to year was observed in Catalase and Superoxide dismutase to the levels p ≤ 0.001. In terms of treatment and variety, the antioxidant enzyme activity namely proline, catalase, ascorbate peroxidase, peroxidase, superoxide dismutase and lipid peroxidation were highly significant towards the significant levels p ≤ 0.001. The two-way interaction of Treatment and variety was highly significant in considering APX, POX and SOD to the significant levels of p ≤ 0.001 whereas the effects concerning year*varieties were significant towards SOD to the significant levels p ≤ 0.01 and least significant towards CAT to the levels p ≤ 0.05. The three-way ANOVA was significant to superoxide dismutase (SOD) to P ≤ 0.01 significant levels.

Likewise, under different treatments, varieties of mustard with two seasons (2020–2021 & 2021–2022), (Table SI 4) signified that the LAI at 45 DAS, 75 DAS, RGR at 60–75 and 75–90 DAS showed a very high significance of p ≤ 0.001 and crop index with high significance (p ≤ 0.01) for the two seasons. The treatment effect is very highly significant in LAI, RGR, crop index and seed yield. The varietal effects were highly significant under LAI, RGR at 45–60 DAS and in seed yield (p ≤ 0.001), and high significance was observed in crop index (p ≤ 0.01) and RGR at 60–75 DAS was p ≤ 0.05 significant. A very high performance (p ≤ 0.001) of treatment and varietal interaction was found in LAI at 45, 75 DAS and in seed yield. The significant levels under the variety and year interaction were observed in LAI at 75 DAS (p ≤ 0.001), RGR (p ≤ 0.01) and crop index (p ≤ 0.05)

Differences in canopy temperature, gas exchange parameters, free proline, antioxidant enzyme activity, MDA, growth and seed yield among the different treatments (elevated levels of O₃, CO₂ and O₃*CO₂), among different varieties (PDZM 31, PB and PM 30) were analysed by LDA (Linear Discriminant Analysis). Furthermore, LDA exhibited significant effects on mustard varieties (Fig. 5) and treatments (Fig. 6) with the measured parameters. From the results, it was evident that the elevated O₃*CO₂ interaction altered the effects caused by elevated O₃ with respect to canopy temperature, gas exchange parameters, free proline, antioxidant enzyme activity, MDA, growth and seed yield.

4.1 Change in crop canopy temperature and gas exchange parameters

Our results revealed that the phytotoxic effects of O₃ influenced the different varieties of mustard crop physiological and biochemical parameters (Gupta et al., 2018). The canopy temperature (Tc) was non-significant in both seasons, revealing that Tc in ambient conditions affected PDZM 31 and PB varieties, when compared to other treatments (eO₃, eCO₂ and eO₃*eCO₂). The increased levels of O₃ negatively influenced photosynthesis and affected plant growth by reducing stomatal conductance, which increased Tc (Yue et al., 2017; Singh et al., 2021). The negative effects of eO₃ on gas exchange parameters (Pn, gs, E and WUE) of Indian mustard supported by Diksaityte et al. (2019); Zaltauskaite et al. (2022), and they found that reduced stomatal conductance and transpiration rates (E) in wild mustard. The rise in the uptake of atmospheric CO₂ concentrations of plants would inhibit oxygenation and suppress photorespiration resulting in temperature rise (Diksaityte et al., 2019). The reduction in gs was observed under elevated levels of CO₂ (Saha et al., 2012; Singh et al., 2021) and elevated levels of O₃ (Pandey et al., 2018; Hoshika et al., 2015; Tomer et al., 2015). The reduced transpiration rate of crops due to eO₃, and eCO₂ increased the canopy temperature (reduces the cooling effect) (Bhatia et al., 2013; Singh et al., 2021). The of reduction in transpiration was observed in our study, which was in line with the findings of Booker et al. (2004). Many studies have reported that Pn and WUE are linked straight away to each other, which is more crucial to osmotic imbalance in crop response. The highest stomatal conductance was observed in chickpea crops under elevated ozone and carbon dioxide levels at the same time reduced transpiration rate corroborated (Bhatia et al., 2021) when compared to ambient conditions. When the soybean was exposed to elevated levels of ozone at 30–79 ppb, about 17.5% gs (stomatal conductance) reduction was observed by Morgan et al. (2003). In our experiment under the interaction of elevated O₃ and CO₂, the gas exchange parameters decreased by 7.5–14.6% in Pn, 20–31.2% in gs, 20–35.6% in E and 2–25% in WUE, which showed a decline in loss of gas exchange parameters when compared to elevated O₃. Negative effects of eO₃ were reduced by eCO₂ under interaction treatment of eCO₂^*eCO₂ this could be due to reduction in stomatal opening and minimize the fluctuation of O₃ in the apoplast (Oikawa and Ainsworth, 2016). CO₂ enrichment benefits the C₃ plant rather no significant difference or negligible increase was observed in C₄ plants like maize (Yadav et al., 2021) and under O₃-induced exposure, the C₄ plants were less prone to oxidative damage when compared to C₃ plants like rice, wheat, mustard etc. The changes in the stomatal aperture of crops under eCO₂ were the result of the decline in stomatal conductance, moreover, while the CO₂ level increases, the stomata need not expand as the adequate quantity of CO₂ enters the leaves for CO₂ fertilization (Ainsworth and Long, 2020; Bhatia et al., 2021).

Kobayakawa and Imai, (2011) revealed that the photosynthetic mechanism and its processes were damaged in agricultural crops by photochemical oxidants like ozone, which led to a reduction in the yield. However, the negative effects of ozone are ameliorated by a significant increase of elevated CO_2, which causes a reduction in ozone injury of leaves when there are escalating levels of elevated O₃. The CO₂ enrichment studies protect against the negative effects of O₃ damage to crops and necessitate the photosynthates availability by increasing it by prolonging the plant growth phases that are used for oxidative injury repair and in the process of detoxification (Booker et al., 2007).

4.2. Change in free proline, antioxidant enzymes and lipid peroxidation

The highest proline activity was observed under elevated ozone conditions in our experiments which in line with many previous studies. Under elevated O₃ conditions By storing osmolytes, plants could defend themselves against stress to some extent (Shinde and Thakur, 2015; Bhatia et al., 2021) and thus, a substantial increase in proline levels illustrated that the crops were under stress (Dolker and Agrawal, 2019). Proline accumulation took place in response to any abiotic stress to protect the protein structure of plant cells and also to prevent oxidative damage in plants, which maintained the ROS (Hayat et al., 2012; Daripa et al., 2016). The antioxidant enzyme activity occurs in plants during oxidative stress that causes oxidative damage, which is varied between the varieties and at elevated O₃ and CO₂. The antioxidant activity was increased under elevated O₃, eO₃*eCO₂ in three mustard varieties. Despite the increased activity sequence of enzymes, which varied between the varieties, the interaction of eO₃*eCO₂ results implied that the eCO₂ would suppress the negative effects of eO₃ levels. The mustard varieties for both seasons implied an increase in the activities of CAT, POX, APX, and SOD under eO₃, eCO₂ and their interaction, when compared to ambient conditions. This significant increase of antioxidant enzyme activity in the elevated levels disclosed the increased plant defence mechanism from oxidative damage against ROS and thus similar results were depicted in potatoes (Kumari et al., 2015), wheat (Pandey et al., 2018; Fatima et al., 2019; Maurya et al., 2020), rice (Surabhi et al., 2022), wild mustard (Zaltauskaite et al., 2022), soybean (Li et al., 2020). Gupta et al. (2018) also reported that increased activities of APX enzymes along with higher amounts of SOD and CAT in wheat helped to detoxify reactive oxygen species more efficiently. The SOD is a metalloenzyme, which is produced by plants to protect them from the toxic effects of elevated ROS levels in crops.

The performance of three mustard varieties under eO₃ treatment showed enhanced antioxidant activity with a mild increase in elevated CO₂ conditions when compared to eO₃ (Table SI 5). The mustard variety PDZM 31 showed an increase in antioxidant enzyme activity such as SOD, CAT and APX (the ROS scavengers), which played a vital role in scavenging superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), whereas the least performance of antioxidant activity was observed in PB under eCO₂, eO₃*eCO₂ and higher under eO₃. Under the elevated CO₂ and interaction studies, the variety PDZM 31 exhibited increased levels of antioxidants enzymes followed by Pusa bold (PB) and the performance of PM 30 under these treatments were sensitive to antioxidant capacity (CAT, POX, APX and SOD) and similar results were observed in rape and wild mustard (Kaciene et al., 2019). Higher activities of hydrogen peroxide scavenging enzymes are one of the promising mechanisms in reducing the wild mustard competitiveness under O₃ conditions. The future increase in the modified levels of elevated ozone will inhibit growth and development by altering the physiological responses of crops (Kaciene et al., 2019).

Another indicator for oxidative stress is lipid peroxidation (MDA content) content and also MDA content is a product of lipid peroxidation and an important measure of membrane injury, caused by oxidative damage in plants (Surabhi et al., 2022) which was increased in the PM 30 and indicated that the variety was highly sensitive to elevated levels of O₃, CO₂ and also the interaction (eO₃*eCO₂) mediated damage. Our results revealed that the MDA content at the critical stage of three mustard varieties indicated increased stress levels since the crops are being exposed to elevated levels. The escalating concentration of lipid peroxidation i.e., MDA content is directly correlated with increased ozone levels and similarly, this was observed by Kumari et al. (2015). The mustard variety-wise comparison revealed a higher increment in MDA content under elevated O₃ levels and similar results were obtained in Ischaemum rugosum (C₄ grass) and a co-dominant species Malvastrum coromandelianum (L.) (C₃ forb) in non-filtered O₃ treatments (Dolker and Agrawal, 2019) suggested that elevated O₃ (oxidative stress) induce membrane damage by increasing ROS.

The physiological processes to protect plants from oxidative stress due to elevated CO₂ on ozone damage were poorly understood and require further explanation. The primary causes of this protective mechanism such as the exclusion of ozone improved tolerance against ozone-induced oxidative stress, and reduction of ROS levels by enhanced CO₂ concentrations (AbdElgawad et al., 2016).

4.3 Change in growth and yield Parameters

The LAI of mustard varieties increased from 7.5–29.4% under elevated CO₂ treatment. Liu et al. (2021) observed that LAI was increased by 5–8% in rice due to increased concentration of CO₂. Many studies revealed that due to reduced light compensation point, extended period for leaf growth and increased number of leaves were responsible for increased LAI under eCO₂ (Doughty et al., 2008). The more reduction (26–68% at 45 DAS, 27–31%, at 60 DAS, 18–21% at 75 DAS and 32–45% at 90 DAS) of LAI was observed in the mustard varieties. The reduction of LAI was due to the decline of chlorophyll a and b content, which led to less photosynthesis and reduced CO₂ assimilation under eO₃ (Feng et al., 2019; Liu et al., 2021; Kovilpillai et al., 2023). The eCO₂ is nullifying (11–47%) the negative effect of eO₃ on LAI, during the interaction studies. There was a 3.5% increment in RGR in mustard varieties due to eCO₂ and 9.1% reduction in RGR under eO₃ treatment. Hacour et al., 2002 observed that 12% reduction in RGR under eO₃ treatments. The same trend was also observed in the crop index also. The seed yield of mustard varieties was also greatly negatively affected by eO₃ and positively affected by eCO₂. The increased potato tuber yield (16%) was observed by Craigon et al., 2002 under the eCO₂ level. The same results were observed in rice (26% reduction in yield under eO₃) was reported by Kumar et al. (2021).

The experimental study concluded that the detrimental effects of the air pollutant i.e., tropospheric ozone and its response to mustard crop growth, yield and physiology. The increase in O₃levels of about 24.7 ppmhr and 23.3 ppmhr (AOT 40) highly affected the crop photosynthesis and led to oxidative damage by causing oxidative stress thus ultimately declining the crop growth by 17.6%, 13% and 13.8% and seed yield by 27.5%, 28% and 17.3% in PDZM 31, Pusa Bold and PM 30. In the increased CO₂ levels Moreover, the deleterious effects of elevated ozone on mustard crops were alleviated by the elevated CO₂ under combined interaction (eO₃*eCO₂)treatment. Generally, Indian mustard varieties are high in erucic acid and glucosinolate levels, which are at high risk for consumption. Among the varieties, PDZM 31 and the conventional variety Pusa Bold were highly sensitive to elevated levels of ozone and carbon dioxide effects, when compared to a single zero variety (PM 30). Therefore, our experiment disclosed that crop growth, seed yield, photosynthesis and stomatal behavior of Indian mustard were susceptible to future changing climatic conditions of increased levels of ozone. However, elevated carbon dioxide levels ameliorated the seed yield decline in the eO₃ to the extent of 15%. Though the interaction study implied the amelioration effects of CO₂ on O₃,still more work on molecular and physiological aspects needs to be documented. Furthermore, studies are required to document the biochemical changes of crops under ozone and carbon dioxide interaction effects under changing climate on yield and quality.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The authors are grateful to the Post Graduate School, ICAR-IARI, for offering the IARI-SRF throughout the study period and provided necessary field and laboratory facilities to carry out the entire work. The financial assistance provided by the NICRA project, ICAR, Govt. of India is duly acknowledged.

Author Contribution:

G.J. Investigation, Writing - original draft, Formal analysis, Visualization

D.K.S. and A.B. Conceptualization, Methodology, Supervision

K.B. and D. A. Writing - review & editing, Formal analysis, Funding acquisition

S.K. Data Curation and Formal analysis

M.P., S.S.S. and V.D. Formal Analysis

Data Availability Statement:

All data are provided within the manuscript or supplementary information files.

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No competing interests reported.

Supplementaryfiles.docx

Phytotoxic effects of tropospheric ozone altered by elevated levels of CO2 in Indian mustard for Sustaining production

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Study area

2.2 Plant material

2.3 Treatment details

2.4 Canopy temperature

2.5 Leaf gas exchange parameters

2.6 Free proline, antioxidant enzymes and lipid peroxidation (MDA)

2.8 Growth observations

2.10 Statistical analysis

3. RESULTS

3.1. Ambient and elevated CO₂ and O₃

3.2 Changes in free proline, antioxidant enzymes and MDA activity

3.3 Changes in Growth Parameters

3.4 Yield Parameters

4. DISCUSSION

4.1 Change in crop canopy temperature and gas exchange parameters

4.2. Change in free proline, antioxidant enzymes and lipid peroxidation

4.3 Change in growth and yield Parameters

5. CONCLUSIONS

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Phytotoxic effects of tropospheric ozone altered by elevated levels of CO2 in Indian mustard for Sustaining production

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Study area

2.2 Plant material

2.3 Treatment details

2.4 Canopy temperature

2.5 Leaf gas exchange parameters

2.6 Free proline, antioxidant enzymes and lipid peroxidation (MDA)

2.8 Growth observations

2.10 Statistical analysis

3. RESULTS

3.1. Ambient and elevated CO2 and O3

3.2 Changes in free proline, antioxidant enzymes and MDA activity

3.3 Changes in Growth Parameters

3.4 Yield Parameters

4. DISCUSSION

4.1 Change in crop canopy temperature and gas exchange parameters

4.2. Change in free proline, antioxidant enzymes and lipid peroxidation

4.3 Change in growth and yield Parameters

5. CONCLUSIONS

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

3.1. Ambient and elevated CO₂ and O₃