Negative impacts of elevated ozone on dominant species of semi-natural grassland vegetation in Indo-Gangetic plain

Ecotoxicology and Environmental Safety 182 (2019) 109404

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Negative impacts of elevated ozone on dominant species of semi-natural grassland vegetation in Indo-Gangetic plain

T

Tsetan Dolker, Madhoolika Agrawal* Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, 221005, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Ozone Ischaemum rugosum Malvastrum coromandelianum Stomatal conductance Photosynthesis Biomass

Increasing tropospheric ozone (O3) concentrations in most regions of the world have led to significant phytotoxicity to all types of vegetation. Indo-Gangetic Plains of India is one of the hot spot areas with high O3 concentrations throughout the year although O3 phytotoxicity on grassland species in this region is not explored. Therefore the present study was conducted to assess the responses of a dominant species, Ischaemum rugosum Salisb, a C4 grass and a co-dominant species Malvastrum coromandelianum (L.) Garcke, a C3 forb under future elevated O3 (non filtered ambient + 20 nl l−1; NFA+) concentration compared to non filtered ambient (NFA; 48.7 nl l−1, 8 h mean) for 9 weeks from 15th May to 15th July 2016 in mix-culture using open-top chambers (OTCs). Plants were assessed for physiological, biochemical and growth parameters including biomass accumulation during vegetative and reproductive stages to assess the O3 induced responses. Under NFA+, higher reductions were observed in physiological parameters, growth and total biomass accumulation in M. coromandelianum compared to I. rugosum while both the species suffered membrane damage. Enhancement in contents of ascorbic acid and tannin in I. rugosum while proline and total phenolics in M. coromandelianum led to more protection of former species compared to later from oxidative damage. No significant change in stomatal conductance in I. rugosum while significant increase in M. coromandelianum might have led to more accumulation of O3 inside the plant, thus more negatively affecting the performance of later species. The present study concludes that M. coromandelianum (C3 photosynthetic pathway) will be relatively more negatively affected compared to I. rugosum (C4 photosynthetic pathway) under future O3 concentrations.

1. Introduction Tropospheric ozone (O3) is a most potent phytotoxic secondary air pollutant, formed by photochemical reactions between the precursors like nitrogen oxides (NOx), non-methane volatile organic carbons (NMVOCs), methane (CH4) and carbon monoxide (CO) in the presence of sunlight (Sicard et al., 2017). O3 concentration has been increasing due to rapid urbanization and industrialization and is predicted to increase in the future under all scenarios of development (Ainsworth et al., 2019). By 2030 or 2050 worldwide and particularly in India, Southeast Asia and some regions of Africa, AOT40 (accumulation of O3 over the threshold of 40 nl l−1) value for O3 may increase by 80–100% under ‘business-as-usual’ scenario (RCP 8.5) (Dentener et al., 2010). The present-day scenario provides more favorable conditions for O3 formation and average O3 concentrations in Asia are predicted to be high enough to cause injury to vegetation. For the most part of Asia, both NOx and VOCs are limiting factors for O3 formation (Tie et al., 2007). However, in India and China, NOx acts as a main limiting factor

*

for O3 formation (Sinha et al., 2014). India is the second largest populated country, highly industrialized, urbanized and has witnessed a tremendous increase in vehicles, which introduced more O3 precursors in the urban areas causing high ground level O3 formation downwind. Using the regional chemical transport model (REMOCTM), Roy et al. (2008), recognized the Indo-Gangetic Plain (IGP) to experience higher O3 compared to other parts of India (Pandey et al., 2014). Tropospheric O3 has been considered as one of the important air pollutants affecting forests, natural and semi-natural grassland communities due to higher O3 influx potential of vegetation (Guerreiro et al., 2014), 2011; Hardacre et al. (2015). Many studies were performed to understand the effects of ground-level O3 on grassland species based on an evaluation of different parameters. To understand the effects of elevated O3 at the community level, the study of dominant or co-dominant species might be valuable initial steps to understand the consequences at a complex community level. O3, a strong oxidant generates reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals and superoxide after entering

Corresponding author. E-mail addresses: [email protected] (T. Dolker), [email protected], [email protected] (M. Agrawal).

https://doi.org/10.1016/j.ecoenv.2019.109404 Received 7 March 2019; Received in revised form 14 June 2019; Accepted 29 June 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 182 (2019) 109404

T. Dolker and M. Agrawal

2. Materials and methods

into plant leaves through the stomata (Ainsworth, 2017). The strong oxidizing environment due to O3 causes damage to photosynthetic apparatus, leading to decline in mesophyll conductance (gm), rubisco carboxylation rate (Vcmax), quantum yield and CO2 assimilation rate (Mills et al., 2018; Xu et al., 2019). O3 concentration in the apoplastic region depends upon stomatal conductance (gs) which in-turn affects photosynthesis (Masutomi et al., 2019). Previous studies have found stomatal closure at elevated O3 concentrations (Li et al., 2017) or even incomplete closure of stomata (Hoshika et al., 2013). As a result O3 uptake and carbon assimilation are reduced that ultimately lead to reduction in total plant biomass (Yendrek et al., 2017). Most of the studies on grassland species are performed based on leaf injury symptoms and growth parameters (Feng et al., 2015), while few studies are based on effects on physiological and biochemical parameters (Su et al., 2017; Dai et al., 2019). The leaf injury symptoms often do not give a proper interpretation of O3 effect, as leaf injury has not always been correlated with plant growth reduction (Davison and Barnes, 1998). Leaf symptoms and growth reductions occur after chronic exposure to O3 concentrations. Therefore, physiological and biochemical assessments could depict early responses of plants and species sensitivity towards elevated O3 concentrations. Most of the studies of O3 effects on grassland species have been conducted in European countries with single species or multiple species in mix-culture (Hayes et al., 2011) showing a wide range of O3 effects. The studies with two species mixtures were conducted with Trifolium repens and Lolium perenne in mesocorm (Hayes et al., 2009) and in natural field conditions (Mills et al., 2011). The critical levels of O3 concentrations were developed in Europe for all types of vegetation including natural and semi-natural grasslands. These studies led the policy makers to develop strategies to check the O3 precursors leading to a decrease in peak O3 concentration in European countries (Mills et al., 2009). The most fertile IGP in India, once flourished with natural vegetation, is now facing a high burden of ground-level O3 which has the potential to affect the natural vegetation (Oksanen et al., 2013). In India, most of the researches related to O3 have been focused on the cultivable crops and no studies have been done on wild or grassland species of the tropical region. Studies conducted to assess the responses of C4 (Maize) and C3 (wheat) crop plants to elevated O3 (ambient + 30 nl l−1 O3) in Indo-Gangetic region showed induction of nonenzymatic and enzymatic antioxidants and reductions in yield (Singh et al., 2014; Fatima et al., 2018). More reductions in yield under elevated O3 were recorded in wheat compared to maize plants. As grasslands are already under threat due to habitat change and variable climates and no reports are available in relation to the response of grassland species under elevated O3 in tropical areas, the present study was conducted to assess the responses of Ischeamum rugosum salisb and Malvestrum coromandelianum (L.) Garcke to future O3 concentration. The selected plant species represents C4 and C3 photosynthetic pathways, respectively, in I. rugosum and M. coromandelianum. The hypothesis proposed for the present study is that future O3 concentration will differentially alter the physiological, biochemical and growth parameters of representative grass (I. rogusum) and forb (M. coromandeliamum) species. To test the hypothesis, following objectives were proposed: I. To assess the effects of elevated O3 concentration on photosynthetic pigments, membrane damage, antioxidants and metabolites of the test species.

2.1. Experimental site The study was carried out from May to July 2016 in the Botanical Garden of Department of Botany, Banaras Hindu University (BHU), Varanasi (25.28° N, 82.95° E, and 76 m above mean sea level), situated in the eastern Gangetic plains of India. Soil at the experimental site was sandy loam in texture, pale yellow with 27, 28 and 45% of clay, silt and sand. pH of soil ranged between 7.2 and 7.4. Total soil nitrogen and organic carbon contents were 0.2% and 1.4%, respectively. 2.2. Experimental design The experiment was performed in open-top chambers (OTCs) having three replicates for non filtered ambient (NFA; control; n = 3) and three for non filtered ambient with supplemented ozone (Ambient + 20 nl l−1; NFA+; treatment; n = 3). O3 dose was decided based on the report of Yadav et al. (2019) predicting a 20–40% increase in O3 concentration in the northern hemisphere by 2050 if current rising trends continue. OTCs having 1.5 m diameter and 1.8 m height were connected with a high-speed blower (168 ls−1) which circulated air three times per minute inside the chamber, maintaining the near natural conditions (Mishra and Agrawal, 2015). The schematic presentation showing the distribution of OTCs and arrangement of plants inside OTCs (Fig. S1). Seeds were collected from patches of semi-natural grassland community. Six OTCs were installed in the well-prepared field. Seeds were sown on 1st May 2016 in a mix-culture of both the plants in OTCs. Homogeneity of space in the OTCs was maintained by thinning the plants to keep a distance of 15 cm between the plants. OTCs having three each of NFA and NFA+ were distributed following a randomized block design (Fig. S1). Elevated O3 was given for 5 h daily between 10.00 and 3.00 h for two months from 15th May to 15th July with the help of ozone generator (A1G, Faraday, India). The timing of exposure of elevated O3 was based on higher O3 concentrations during these hours. 2.3. Meteorological parameters The meteorological parameters at the study site were collected from the Indian Meteorological Division, B.H.U. station, Varanasi (Table S1). The maximum mean temperature ranged from 33.0 to 39.8 °C, while minimum mean temperature ranged from 26.0 to 29.1 °C during the experimental period. The maximum relative humidity ranged from 61.8 to 89.7%, while minimum values ranged between 38.6 and 81.2%. The monthly total rainfall also varied with a minimum of 18.5 mm in May and a maximum of 423 mm in July. Mean sunshine hour was 3.5 in July and 8.6 in May. 2.4. Ozone monitoring Ambient O3 was monitored in NFA and NFA+ chambers by using non-dispersive UV absorption photometric ozone analyzer (Model APOA-370, HORIBA Ltd., Kyoto, Japan) for 8 h from 9.00 a.m. to 5.00 p.m. daily during the study period. The air sample was collected at the canopy level from the chambers with the help of an inert Teflon tube (0.35 cm diameter). Exceedance hours over 40, 50, 60, 70, 80, 90, 100, 110 and 120 nl l−1 were calculated from the hourly O3 values.

i. To evaluate the various physiological parameters (photosynthetic rate, stomatal conductance and AQ and ACi derived parameters) of selected species under elevated O3 concentration compared to ambient. ii. To evaluate the effects of elevated level of O3 concentration on different growth parameters and biomass accumulation of test species.

2.5. Plant material For the present study, two plants i.e. I. rugosum (grass) and M. coromandelianum (forb) growing extensively in IGP region and indigenous to the study area, were selected. I. rugosum grow robustly in many natural habitats and also in crop fields posing a serious weed problem (Bakar and Nabi, 2003). I. rugosum belonging to family 2

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Continuous monitoring of different physiological parameters was conducted during June end. Photosynthesis measurements were performed by using a portable gas exchange system (Model 6400, LICOR Lincoln, NE, USA). The measurement was done on a clear sunny day from 7.00 a.m. to 11.00 a.m. IST (Indian Standard Time). At the time of measurement, leaf temperature and flow rate were fixed to 25.0 °C and 300 μmol s−1, respectively. Photosynthesis (A) and gs was measured at ambient CO2 and photosynthetically active radiation (PAR). Light response curve (AQ curve) was measured at ambient CO2 and PAR values were controlled by decreasing in descending order from 2000 to 0 μmol m−2 s−1 in 13 steps (2000, 1800, 1600, 1400, 1200, 1000, 800, 600, 400, 200, 100, 50, 0). Some important parameters such as dark respiration (Rdark), apparent quantum yield (Φ) and light compensation point (LCP) were measured from the data of AQ curves after fitting in Light Response Curve Fitting 1.0. ACi curve: Net CO2 assimilation rate (A) versus calculated substomatal CO2 concentration (Ci) was measured at fixed PAR of 1400 μmol m−2 s−1 for grass and 1200 μmol m−2 s−1 for forb. The CO2 supplied was 400 ppm which was reduced to 50 ppm and then stepwise increased to 1400 ppm in 14 steps (400, 300, 200, 100, 50, 400, 400, 500, 700, 800, 900, 1000, 1200 and 1400 ppm). ACi curve data for C3 plants were used for M. coromandelianum to fit in A/Ci curve fitting utility version 2007.1 (Sharkey et al., 2007) model, which provides many important parameters like rubisco carboxylation rate (Vcmax), RuBP regeneration limited photosynthesis (Jmax) and triose phosphate use (TPU). For I. rugosum (C4 grass), the Von Caemmerer (2000) C4 photosynthesis model with small modifications was used to determine the enzyme limited and electron transport-limited photosynthesis (Massad et al., 2007; Yin et al., 2011; Ge et al., 2014). Enzymes limited photosynthesis gives the parameters of Vcmax and maximum PEP carboxylation (Vpmax) as functions of the rate of PEP (Vp) and Rubisco (Vc) carboxylation. To determine these parameters, one needs to calculate other parameters like Cm, Cs and Os and the various formulae used for the calculation of parameters are given below and the constant values and definition for gi, Rm, Rd, gbs, α, θ, x, y*Os, Ko, Kc, and Kp are described in Table 1. Mesophyll CO2 partial pressure (Cm) was calculated from every Ci and A measured in ACi curve, assuming a value for the mesophyll cell conductance (gm) (Yin et al., 2011) as given below.

Poaceae, is an annual (130–140 days), 2–3 m tall tufted grass with silt roots, rooting at the lower nodes and is commonly known as Murano grass. The leaf blade is hairy, linear-lanceolate and flowering starts in the last week of June. It is an opportunistic and strong competitor (Santos and Marenco, 1999). Being an opportunistic and effective colonizer, it readily grows in disturbed, open and clear areas and protects soil from erosion. M. coromandelianum, a medicinal plant having antiinflammatory and analgesic effect is used by tribal people to cure many diseases (Mathur et al., 2016). M. coromandelianum is a co-dominant species with high importance value in many sub-tropical communities in Asia (Shabbir and Bajwa, 2006). M. coromandelianum belonging to family Malvaceae is an annual, upright woody, attaining a height up to 1 m, commonly known as yellow meadow. Leaves are ovate with a serrated margin and taproot system. M. coromandelianum flowers singly in leaf axial with light yellow colour during June end. 2.6. Assessment of plant parameters 2.6.1. Growth parameters Plants were randomly selected in triplicate at both vegetative (50 DAS [days after sowing]) and reproductive (90 DAS) stages. Plants were dug carefully with intact roots by digging monolith of 10 × 10 × 25 cm3. Growth parameters such as root and shoot length, numbers of leaves, senescent leaves and tillers (only for grass species) were measured. Leaf area was measured by using a portable leaf area meter (Model Li- 3100, Li-COR, Inc., USA). Plants were separated into root, shoot and leave and then kept in the oven (80 °C) until constant weights were achieved. Dry weight was taken and cumulative addition of all plant parts gave the total biomass as g plants−1. Leaf area ratio (LAR) was calculated as a ratio of total leaf area vs total biomass and the shoot-root ratio (SR) as a ratio of shoot vs root biomass. 2.6.2. Photosynthetic pigments, membrane damage, antioxidants and metabolites A random sampling of healthy leaves devoided of any injury symptoms was done from the OTCs for biochemical analysis after 50 DAS. Photosynthetic pigments (total chlorophyll and carotenoids) were measured by slight modification in methods of Maclachlan and Zalik, 1963and Duxbury and Yentsch, 1956 formulae as given in Takshak and Agrawal (2018). The biochemical parameters like AsA, total phenolics, proline, anthocyanin and tannin contents were estimated following methods given by Keller and Schwager (1977), Bray and Thorpe (1954), Bates et al. (1973), Gitelson et al. (2001) and Khomdram and Singh (2011), respectively. Membrane damage was quantified in form of malondialdehyde (MDA) content as described by Heath and Packer (1968).

A= gm (Ci − Cm)

(1)

The bundle-sheath CO2 partial pressure (Cs) was calculated from the formula (Von Caemmerer, 2000) as:

Cs = Cm + 2.6.3. Physiological measurements and gas exchange Three plants were tagged in each OTC and the third leaf from the top in case of I. rugosum and fourth leaf from the top in case of M. coromandelianum were assessed for physiological measurements.

Vp −A−Rm gbs

(2)

The bundle-sheath O2 partial pressure (Os) was measured from the formula (Von Caemmerer, 2000; Yin et al., 2011) as:

Table 1 Values for constants used in the calculations of photosynthetic parameters in the model of C4 grasses along with brief definition and source of constant values. Symbol

Values/Units

Definition

Resource

Vcmax Kc Ko Kp gbs Rm Rd gi x

60 μmol m−2 s−1 650 μbar 450 m bar 80 μbar 3 mmol m−2 s−1 0.5Rd 0.01V cmax 2 mol m−2 s1 0.4 mol mol−1

θ

0.7

Maximum rubisco activity Michaelis constant of rubisco for CO2 Michaelis constant of rubisco for O2 Michaelis constant of PEP carboxylase for CO2 Bundle-sheath conductance to CO2 Mesophyll mitochondrial respiration Leaf mitochondrial respiration Mesophyll conductance to CO2 Partitioning factor of JATP between C4 activity Vp (PEP regeneration) and C3 activity Vc + Vo(reductive pentose phosphate pathway and photorespiratory cycle) Empirical curvature factor

Von Caemmerer (2000) Von Caemmerer (2000) ” Bauwe (1986) Von Caemmerer (2000) ” ” Massad et al., 2007 Von Caemmerer (2000) Ge et al., 2014 Massad et al., 2007

3

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T. Dolker and M. Agrawal

Table 2 Numbers of hours O3 concentrations exceeded 40, 50, 60, 70, 80, 90,100,110 and 120 nl l−1 and cumulative exceedance hours in OTCs under non filtered ambient (NFA) and non filtered elevated (NFA+) O3 chambers during the experimental period.

A=

Exceedance hours Ozone (ppb)

NFA

xJ tm − gbs(Cs − Cm) − Rm 2

A=

(1 − Y* ) (1−x)Jts − Rd 3 (1 + 7Y* ) Os Cs

Os Cs

Os =

May

June

July

Total

Mav

June

July

Total

214 200 172 117 87 44 27 10 4 875

201 170 112 50 32 8 2 0 0 575

14 2 0 0 0 0 0 0 0 16

429 372 284 167 119 52 29 10 4 1466

214 202 187 151 124 68 39 12 4 1001

220 214 200 162 109 49 33 7 3 997

56 39 13 2 0 0 0 0 0 110

490 455 400 315 233 117 72 19 7 2108

αA + Om 0.047gbs

CmVpmax − gbs(Cs − Cm) − Rm Cm + Kp

2.7. Statistical analysis The significant differences in different parameters between NFA and NFA+ were calculated through one way ANOVA. Turkey test was performed as a post hoc to assess the significant difference between NFA and NFA+ for both species. For identifying specific association among the various biochemical, physiological and growth parameters, principal component analysis (PCA) was performed for both the test plants at 50 DAS. PCA enables in reductions of nth numbers to some important variables which were largely affected by elevated O3 treatment during the study. All the statistical analysis was done using the statistical package SPSS software (SPSS Inc, Version 16.0).

(3)

3. Results 3.1. Ozone concentrations The monthly 8 hourly mean O3 concentrations in May, June and July were 72.3 ± 16.5, 62.5 ± 15.7 and 32.1 ± 6.3 nl l−1 in NFA and 93.5 ± 16.7, 82.5 ± 15.9 and 51.5 ± 6.7 nl l−1 in NFA+, respectively (Table S1). Ozone concentrations reached the highest value of 120 nl l−1 in the month of May and June in NFA+ chambers and only in May in NFA chambers. The cumulative exceedance hours above 40 nl l−1 were 1466 h in NFA and 2108 h in NFA+ chambers, respectively during the study period (Table 2). 3.2. Biochemical parameters Lipid peroxidation measured as MDA content was significantly increased in both I. rugosum (p < 0.01) and M. coromandelianum (p < 0.05) under NFA+ compared to NFA (Fig. 1). Significant variations were also observed between both the species under NFA+ compared to NFA (Fig. 1). Anthocyanin significantly varied between the species with higher content in I. rugosum under both NFA and NFA+ (Fig. 1). Significant (p < 0.001) reductions in total chlorophyll and carotenoids were recorded in I. rugosum under NFA+ compared to NFA (Fig. 1). No significant change in total chlorophyll content between the species under NFA+ was recorded, while significant variations between species for carotenoids was observed (Fig. 1). Under NFA, AsA content was lower in I. rugosum compared to M. coromandelianum while contrasting results were obtained under NFA+. Under elevated O3 exposure (NFA+), AsA significantly increased in I. rugosum (p < 0.001) but decreased in M. coromandelianum (p < 0.001) (Fig. 2). Post hoc analysis revealed significant variations in AsA content between the species under both NFA and NFA+. No significant changes in total phenolics content was recorded in case of I. rugosum, while significant (p < 0.001) increase in M. coromandelianum was found in NFA+ compared to NFA. Proline content decreased significantly in I. rugosum (p < 0.001) while increased in M. coromandelianum (p < 0.001) under NFA+. Tannin content significantly increased in I. rogusum (p < 0.001) and declined in M. coromandelianum (p < 0.01) under NFA+ compared to NFA (Fig. 2). The results of the post hoc test showed significant variations in total phenolics, proline and tannin contents between the species under NFA+ (Fig. 2). Higher contents of total phenolics and proline were observed in

(4)

Equation (5) given below was used to derive Cs:

Cs =

Y*Os+ Kc(1 + Os/Ko)(( A + Rd)/ Vcmax ) 1 − (Ac + Rd)/Vcmax

(5)

The maximum Rubisco rate of CO2 assimilation (Vcmax) was calculated as:

A=

CsVcmax

(

Cs+Kc 1 +

Os Ko

)

⎛1 − Y*Os ⎞ − Rd Cs ⎠ ⎝

(9)

where x is a partitioning factor of electron transport rate.

2.6.3.1. Calculation of Vcmax and Vpmax. ACi curve was used for the measurement of Vcmax and Vpmax. The experimental ACi curve we obtained by splitting into two parts because of the fact that the initial linear slope with low Ci values is valid for the estimation of PEP carboxylase activity, while rest parabolic curve with high Ci values is valid for calculation of Rubisco activity. Former part was converted into a linear regression line while later was converted to three-degree polynomial, was best fit for different point’s curve. The equations obtained from these best-fit regression lines help in checking the various values of A with respect to different values of Ci. The Ci values lower and above 70 ppm were used for the calculations of Vpmax and Vcmax, respectively. The grand mean Ci values from the linear and polynomial lines were used for the calculations of important parameters from given equations. For the measurements of Vpmax and Vcmax, we first estimated the apparent Vpmax and Vcmax (Yin et al., 2011). The initial linear part of low and later parabolic part of high Ci values with high assimilation rate (A) of ACi curve was used for the calculation of apparent Vpmax and Vcmax, respectively (Yin et al., 2011). The Vpmax was measured (Massad et al., 2007) as:

A=

(8)

The total electron transport as a whole was calculated as:

NFA+

Jt = Jm + Js 40 50 60 70 80 90 100 110 120 Total

(7)

(6)

2.6.3.2. Calculations of Jt. ACi curve was used for the calculation of electron transport rate of mesophyll compartments (Jm), electron transport of bundle-sheet (Js) and total electron transport rate (Jtotal). The total electron transport rate of mesophyll compartments (Jtm) (Eqn (7)) and the total electron transport expressions for bundlesheath (Jts) (Eqn (8)) (Massad et al., 2007; Von Caemmerer, 2000) and the respective values were put in another formulae Jm = xJtm and Js = (1-x) Jts, where x = 0.4 (Von Caemmerer, 2000) to calculate the values of Jm and Js. The summation of these two values gives Jtotal (Eqn (9)). 4

Ecotoxicology and Environmental Safety 182 (2019) 109404

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Fig. 1. Total chlorophyll, carotenoids, MDA equivalent and anthocyanin contents in I. rugosum and M. coromandelianum under non filtered ambient (NFA) and non filtered elevated (NFA+) O3 levels. Values are mean ± SE. Different letters on the bars showing significant difference between NFA and NFA+ (Capital letters) in same species based on one way ANOVA and among each group (NFA and NFA+) in both species (small letters) based on post hoc test at *p ≤ 0.05 significant level.

lengths at 90 DAS in M. coromandelianum were recorded under NFA+ compared to NFA (Table 3). As compared to NFA, number of leaves in I. rugosum decreased significantly (p < 0.001) at 90 DAS and at vegetative (p < 0.05) and reproductive (p < 0.01) stages in M. coromandelianum under NFA+ (Table 3). Leaf area decreased significantly at vegetative (p < 0.05) and reproductive (p < 0.001) stages in M. coromandelianum in NFA+ (Table 3). LAR increased significantly (p < 0.001) under elevated O3 at reproductive stage in I. rugosum, but increased at vegetative (p < 0.01) and decreased at reproductive (p < 0.001) stage in M. coromandelianum (Table 3). Total biomass reduced significantly (p < 0.001) in I. rugosum and M. coromandelianum (p < 0.01) at both stages under NFA+ compared to NFA (Table 3). Based on post hoc analysis, significant changes were observed between species in all above parameters at vegetative stage under NFA+. Likewise, variations between species were also recorded in all the parameters at the reproductive stage but significant changes were only observed in leaf area under NFA+.

M. coromandelianum compared to I. rogusum. 3.3. Physiological parameters 3.3.1. Photosynthetic rate (A) and stomatal conductance (gs) Photosynthetic rate (A) decreased significantly in both the species, while gs increased significantly (p < 0.01) only in M. coromandelianum in NFA+ compared to NFA (Fig. 2). The post hoc analysis showed significant variation in gs between the species under NFA+. 3.3.2. AQ and ACi derived parameters The Rdark increased significantly (p < 0.05) in M. coromandelianum while decreased significantly (p < 0.01) in I. rugosum under NFA+ compared to NFA. Apparent quantum yield (Φ) showed significant (p < 0.01) decrease in I. rugosum under NFA+ compared to NFA. LCP increased significantly (p < 0.001) in both the species under NFA+ (Table S2). Significant variations in Rdark, Φ and LCP were also observed between NFA+ in both the species (Table S2). The Vcmax, Jmax and TPU decreased significantly (p < 0.001; 0.01) in M. coromandelianum under NFA+ compared to NFA (Table S2; Fig. S2). The Vcmax and Jtotal reduced significantly (p < 0.05) at 1400 PAR in I. rugosum in NFA+ compared to NFA (Table S2, Fig. 3).

3.5. Principal component analysis (PCA) Two principal components were extracted with 53.7 and 22.5% variance in I. rugosum and 74.0 and 13.0% variance in M. coromandelianum (Table S3). In I. rugusum, PC1 showed higher loadings of total chlorophyll, carotenoids, proline, A, total biomass, MDA content, AsA, tannin, LCP, no. of tillers, LAR, no. of leaves, anthocyanin, Φ, Rdark, root length and leaf area, which explained 53.7% variance. PC2 explained 22.5% variance, loaded with parameters like Vpmax, Vcmax and Jtotal, SR ratio, Φ and anthocyanin in I. rugosum (Fig. 4A). In M. coromandelianum, high loadings of parameters in PC1 were AsA, tannin, A, Vcmax, Jmax, no of leaves, total biomass, MDA content, total phenolics, proline, gs, LCP and LAR, shoot length, leaf area, SR ratio, Rdark, anthocyanin, total chlorophyll, carotenoids, TPU and root length which explained 74.0% variance (Fig. 4B). PC2 explained 13.0% variance with parameters like anthocyanin, Φ, Jmax, total chlorophyll and carotenoids in M. coromandelianum. PCA biplot showed a strong association of total chlorophyll, carotenoids, A, total biomass and proline on one axis and AsA, tannin, MDA content and LCP on opposite axis in I

3.4. Growth parameters During the vegetative stage, early senescence (yellowing of leaves followed by abscission) in case of M. coromandelianum and leaf injury (stripling), coloration, necrosis of a particular area of leaf followed by whole leaf senescence in I. rugosum were observed under NFA+. Plant leaves were counted as senescent when more than 25% senescence was observed. In I. rugosum, numbers of senesced leaves were 23.1% more in NFA+ compared to NFA at 50 DAS. In I. rugosum, the shoot and root lengths showed non-significant change at vegetative stage (50 DAS) under NFA+ compared to NFA change, while significant (p < 0.05) decreases were observed at reproductive stage (90 DAS) (Table 3). Significant (p < 0.05) decrease in shoot length at 50 DAS and in shoot (p < 0.05) and root (p < 0.01) 5

Ecotoxicology and Environmental Safety 182 (2019) 109404

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Fig. 2. Ascorbic acid, tannin, total phenolics and proline contents, A and g in I. rugosum and M. coromandelianum under non filtered ambient (NFA) and non filtered elevated (NFA+) O3 levels. Values are mean ± SE. Different letters on the bars showing significant difference between NFA and NFA+ (Capital letters) in same species based on one way ANOVA and among each group (NFA and NFA+) in both species (small letters) based on post hoc test at *p ≤ 0.05 significant level.

concentrations declined during July and August due to scavenging of O3 precursor gases by wet scavenging in monsoon season (Tiwari et al., 2008; Oksanen et al., 2013). The trend of increasing O3 concentration since the last decade (2004–2015) along IGP in Indian sub-continent remained highest concentration in the pre-monsoon season (May–June) are attributed to continuous rise in anthropogenic activities and biomass burning (Rupakheti et al., 2018). During the present study, O3 concentrations exceeding 90 nl l−1 were recorded for 68 and 49 h, respectively during May and June when mean concentrations were 93.5 and 82.5 nl l−1 (Table 2). Chaudhary and Agrawal (2014b; 2015) also recorded high O3 concentrations during May (90.8 nl l−1 in 2012; 85.4 nl l−1 in 2011 and 71.0 nl l−1 in 2010) at the same study site. The trend of increasing O3 concentration clearly suggests a serious risk to vegetation damage in the region. Both the plants showed significant increments in MDA content

rugosum (Fig. 4A). Higher association among Vcmax, AsA, Jmax, tannin, A, total biomass, no. of leaves, leaf area and shoot length on one axis and total phenolics, proline, LCP, MDA content, LAR ratio and gs on opposite axis in M. coromandelianum on biplot (Fig. 4B). 4. Discussion The trend of O3, in the present study showed the highest concentrations during the month of May in both NFA and NFA+ followed by June and July. O3 concentrations were correlated with favorable meteorological conditions such as high temperature, low humidity and longer sunshine hours, which enhanced O3 formation during summer months. Earlier studies have also reported high monthly mean O3 concentrations in May and June due to favorable meteorological conditions at the same study site (Tiwari et al., 2008), thereafter 6

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Fig. 3. Photosynthetic rate (A) as a function of intercellular CO2 concentration (Ci) (ACi) under non filtered ambient (NFA) (3a) and non filtered elevated (NFA+) (3b) for I. rugosum with fitted regression line to calculate Vpmax (3a(i) and 3b(i)); three-degree polynomial to calculate Vcmax (3a(ii) and 3b(ii)) and Jtotal at 1400 PAR. Levels of significant differences: *p ≤ 0.05; **p ≤ 0.01; ns: non significant.

7

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Table 3 Growth parameters in I. rogusum and M. coromandelianum under non filtered ambient (NFA) and non filtered elevated (NFA+) O3 levels at different developmental stages. Values are mean ± SE. Different letters showing significant difference between NFA and NFA+ (Capital letters) in same species based on one way ANOVA and among each group (NFA and NFA+) in both species (small letters) based on post hoc test at *p ≤ 0.05 significant level. Parameters

Shoot length (cm) Root length (cm) No. of tiller (plant−1) No. of leaves (plant−1) Leaf area (cm2) Total biomass (g plant−1) LAR (cm−2 g−1) SR ratio (g g−1)

Species

I. rogusum M. coromendelianum I. rogusum M. coromendelianum I. rogusum I. rogusum M. coromendelianum I. rogusum M. coromendelianum I. rogusum M. coromendelianum I. rogusum M. coromendelianum I. rogusum M. coromendelianum

Vegetative

Reproductive +

NFA

NFA

NFA

NFA+

93.3 ± 3.5A,a 45.3 ± 3.3A,b 16.7 ± 2.2A,a 12.3 ± 0.7A,ab 6.7 ± 0.3A 62.3 ± 6.2A,a 16.0 ± 1.2A,b 836.0 ± 56.9A,a 102.0 ± 7.7A,b 26.8 ± 0.5A,ab 3.1 ± 0.3A,bc 32.0 ± 2.0A,a 31.9 ± 7.2A,b 4.7 ± 0.6A,ab 4.2 ± 0.6A,ab

96.7 ± 6.0 A,a 29.3 ± 3.2B,b 14.0 ± 1.0 A,ab 9.7 ± 1.7A,b 4.3 ± 0.9A 49.3 ± 7.8A,a 10.3 ± 0.3B,b 684.0 ± 65.2A,a 66.6 ± 9.8B,b 11.3 ± 1.1B,ac 1.1 ± 0.15B,bc 62.5 ± 7.2B,b 57.0 ± 5.5B,a 5.1 ± 0.6A,a 2.1 ± 0.7A,b

203 ± 5.3A,abc 95.67 ± 2.3A,acd 34.7 ± 3.0A,ab 17.50 ± 0.8A,c 10.7 ± 0.3A 92.3 ± 1.5A,abc 27.0 ± 0.6A,acd 677.0 ± 28.7A,a 69.8 ± 1.4A,b 34.7 ± 0.3A,abc 7.7 ± 0.3A,acd 20.3 ± 0.5A,abd 9.5 ± 0.4A,acd 5.0 ± 0.1A,c 3.0 ± 0.4A,a

178.3 ± 3.2B,abd 53.47 ± 1.2B,bcd 23.33 ± 0.9B,ac 13.1 ± 0.6B,bc 8.7 ± 0.3A 66.3 ± 1.2B,abd 17.7 ± 0.9B,bcd 628 ± 26.6A,a 27.4 ± 1.7B,b 22.0 ± 0.6B,abd 4.33 ± 0.33B,bcd 28.5 ± 0.9B,abc 6.1 ± 0.3B,acd 5.6 ± 0.6B,ab 2.5 ± 0.4A,ab

under NFA+ compared to NFA, suggesting induction of membrane damage due to oxidative stress caused by elevated O3. Species wise comparison showed higher increment in MDA content in I. rugosum, which also resulted in significant reductions in carotenoids contents. Carotenoids showed significant variations between species under elevated O3, while response of chlorophyll did not vary significantly. A significantly higher reduction in carotenoids in I. rugosum may have led to more loss of chlorophyll in this species as carotenoids protect chlorophyll from oxidative stress. Scebba et al. (2006) reported reductions in total chlorophyll and carotenoids in several grassland species under acute exposure of 150 nl l−1 for 3 h. Significant increments in MDA content of grassland species were also recorded in Plantago major and Sonchus oleraceus after exposure to 85 ± 5 nl l−1 O3 for 9 h day−1 for 30 days in OTCs (Su et al., 2017). Dai et al. (2019) also reported increase in MDA content in tobacco, soybean and poplar plants after O3 exposure. O3, being an oxidizing agent directly or indirectly (through ROS) interacts with unsaturated membrane’s lipids leading to lipid peroxidation which results in formation of lipid hydroperoxides and various lipid based signaling molecules (Marchica et al., 2019). The reduction in total chlorophyll under NFA+ led to more leaf senescence in I. rugosum. M. coromandelianum also showed leaf senescence despite non significant reduction in total chlorophyll. Enhancement of senescence in mix-culture of Festuca ovina, Agrostis capillaries and Galium saxatile species of upland grassland was also reported under 35 nl l−1 background O3 concentration with occasional peak of 85 nl l−1 O3 (Hayes et al., 2010). Premature senescence leads to decrease in assimilation during growing season which also affects its ability to withstand other natural extremes or stresses (Hayes et al., 2010). Microbial community and C - N cycling in ecosystem could also be disturbed indirectly due to early senescence (Manninen et al., 2009). In the present study, non enzymatic antioxidants such as AsA, total phenolics and proline showed significant variations between the species as well as under elevated O3 exposure. AsA is the first line defense against O3 and scavenge ROS in the apoplastic region of the cell which helps in overcoming the initial O3 effects after its entry. The induction in AsA under EO in I rugosum showed that species could overcome the O3 effects, however, no such response was recorded for M. coromandelianum. The remaining ROS or O3 could either interact or traverse through plasma membrane resulting in cascades of signaling molecules (Wang et al., 2015). Increase in cellular ROS will lead to biosynthesis of various other metabolites like proline, phenolics and anthocyanin in response to O3 (Calzone et al., 2019). Apart from AsA, stress response is further regulated by other non-enzymatic antioxidants such as proline, phenolics and anthocyanin. A strong induction in

proline content in M. coromandelianum showed that species was under stress. Induction in proline content was correlated with the synthesis of several others metabolites like phenolics and anthocyanin in plants under O3 stress (Calzone et al., 2019). Similarly for M. coromandelianum, significant inductions in proline and phenolics indicated the responses to elevated O3 through induction of non-enzymatic antioxidants. However, AsA played major role in detoxification of O3 in I rugosum in the present study. Differential induction of antioxidants under the same stress is a unique mechanism in many plant species which depends upon the balance between carbon and nitrogen allocation to defense and vital growth processes (Shang et al., 2019). Under elevated O3 exposure, significant increments in AsA were reported in S. oleraceus and P. major (Su et al., 2017), in six varieties of Vigna radiata and T. alexandrinum (Chaudhary and Agrawal, 2014a and b) and of proline content in Medicago sativa, S. oleraceus and Senecio vulgaris (ElKhatib, 2003). Previous studies have showed that O3 induces the phenylpropanoid biosynthesis resulting into secondary metabolite formation such as phenol (Andersen, 2003), anthocyanin and tannin (Bellincontro et al., 2017), which also functions as antioxidant molecules. In the present study, tannin content also increased in I. rugosum under elevated O3 exposure, however, a contradictory trend was observed for M. coromandelianum which showed higher induction of total phenolics. No significant change in anthocyanin content in both species under NFA+ indicates that anthocyanin did not respond to O3 during the present study. The trend suggested that AsA and tannins provided tolerance against O3 induced oxidative stress in I. rugosum while proline and phenolics played a major role as antioxidant in M. coromandelianum. The current study showed that photosynthetic rate (A) under NFA+ was reduced in both the species, but stomatal conductance (gs) remained unchanged in I. rugosum and increased in M. coromandelianum. Increase in gs was reported in C3 plants such as Dactylis glomerata and Ranunculus acris at daily mean O3 levels ranging from 16.2 to 89.5 nl l−1 after 63 days of treatment under solardomes (Wagg et al., 2013). An increase in gs under elevated O3 was related to the species sensitivity towards O3 (Temple et al., 1992). The magnitude of damage to plant is often related to stomatal flux of O3 accumulation inside the plants (Mills et al., 2011). The reduction in photosynthesis due to elevated O3 may also result due to non-stomatal factors like reduced CO2 enrichment at carboxylation site or decrease in Rubisco activity, less energy availability for RuBP regeneration or photo-system oxidation (Sun et al., 2014). The initial enzymes which fixed the CO2 during photosynthesis in C4 plant are PEPc (activity measured as Vpmax in I. rugosum) and Rubisco in C3 plants (activity measured as Vcmax in M. 8

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Fig. 4. A, B: Principal component analysis extract two principal components (PC1 and PC2) respectively with 53.7 and 22.5% of the variance in I. rugosum and 74.0 and 13.0% of total variance in M. coromandelianum loadings on the biplots under elevated O3 compared to ambient at vegetative stage (50 DAS). SL shoot length, RL root length; LN no. of leaves; LA leaf area; TB total biomass; SR SR ratio; LAR leaf area ratio; TPU triose phosphate use; LCP light compensation point; AQY (Φ) apparent quantum yield; Rdark dark respiration; A photosynthetic rate; gs stomatal conductance; AsA ascorbic acid; MDA malondialdehyde content; antho anthocyanin; caro carotenoids.

of the most important factors causing a reduction in photosynthesis in soybean where a decline of 6% was recorded at 10 nl l−1 increase of O3 concentration (Sun et al., 2014). Vcmax (Rubisco activity) and Jtotal (the sum total of electron transport in mesophyll and bundle sheath in the C4 pathway) in I. rugosum were negatively affected in NFA+ compared to NFA. Ozone negatively affected the small and large subunits of

coromandelianum). The current study showed a significant decrease in Vcmax in M. coromandelianum but no significant change in Vpmax was observed in I. rugosum. This result might be due to more induction of anaplerutic PEPc, which obscure the negative effect on photosynthetic PEPc. The anaplerutic PEPc in C3 plant was reported to be induced in response to O3 stress (Dizengremel, 2001). The Vcmax was found as one 9

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increment while an opposite trend was observed at reproductive stage which may be due to increase in the photosynthetic surface area during early growth stage to support high demand for carbon in defense which was later compensated by higher biomass allocation under lower O3 stress during the later growth stage. In general reductions in photosynthetic rate, leaf area and no. of leaves and/or diversion of assimilate towards defense against ROS productions are attributed for reductions in total biomass of herbaceous plants at elevated O3 (Wilkinson and Davies, 2010). Hayes et al. (2012) found a significant decrease in total biomass of a grass species, D. glomerata at O3 concentrations varying from 16.2 to 89.5 nl l−1 for 20 weeks. In the present study, no significant pattern was recorded for SR ratio as both shoot and roots were similarly affected by O3 in both the species. Reductions in growth parameters were more at the reproductive compared to the vegetative stage in I. rugosum while were more or less similar at both stages in M. coromandelianum except root length. Differences in growth responses between the two species are mostly due to its antioxidative and resource allocation strategy. In I. rugosum, lower input in metabolites might have led to higher allocation towards growth in early stage while in M. coromandelianum more induction of antioxidative metabolites under O3 stress may have lowered the biomass accumulation. The increases in total phenolics and proline contents in M. coromandelianum might use more ATP for their synthesis, consequently more reductions in growth parameters and total biomass were recorded. The decrease in growth during later stage may be ascribed to carry over of the effect of early stress response and more allocation towards reproduction. The carry-over effect was noticed for many grassland species under O3 stress (Hayes et al., 2011). The current study showed higher negative effects of O3 on M. coromandelianum (a C3 forb) compared to I. rugosum (a C4 grass) with respect to growth parameters. Similar results were also reported in two mix-culture (T. repens – L. perenne) study, where an increase in the grass: forb ratio in response to O3 was found (Wilbourn et al., 1995; Hayes et al., 2009). Studies with 10 species mix-culture in calcareous grassland (Ashmore et al., 1995) and species from lowland hay meadow (Ramo et al., 2006) also showed reduction in forb fraction. The differential sensitivity of the test plants towards O3 might resulted in a decrease in the proportion of forb (C3 plant) with an increase in grass (C4 plant) species, demonstrating that increasing O3 concentration may cause change in species composition in the community and species which are tolerant to O3 may dominate leading to community simplification. Negative effects of ozone were more in growth parameters of M. coromandelianum, a medicinal plant with higher socioeconomic implications for tribal community suggesting that future increase in O3 will substantially influence its exploitation. Principle component analysis (PCA) showed that more variance was explained by PC2 in I. rugosum compared to M. coromandelianum with high loadings of physiological parameters like Vpmax, Vcmax and Jtotal which might be due to differential response of photosynthetic pathway to O3 stress. While in M. coromandelianum, all the physiological parameters were loaded in PC1 along with other biochemical and growth parameters. Most of the biochemical parameters behaved oppositely to growth parameters and vice versa with more distinct separation in M. coromandelianum compared to I. rugosum (Fig. 4A and B). The strong association of parameters like total chlorophyll, carotenoids, A, total biomass and proline suggested that decrease in A was due to reductions in total chlorophyll and carotenoids consequently leading to lower biomass in I. rugosum (Fig. 4A). High association among AsA, tannin, MDA content and LCP might be due to less ROS scavenging activity resulting in membrane damage in spite of high AsA and tannin contents in I. rugosum. On the other hand in M. coromandelianum, Vcmax, Jmax, AsA, tannin, A, total biomass, no. of leaves, leaf area and shoot length were grouped in PC1 axis suggesting a decrease in A leading to lowering of most of the growth parameters (no. of leaves, leaf area and shoot length) and total biomass. More dark respiration took place to compensate ATP while A was already reduced, leading to more

Rubisco by decreasing mRNA levels as well as biosynthesis by increasing degradation of enzymes (Zheng et al., 2002). On the other hand in M. coromandelianum, RuBP regeneration is directly related to TPU via Pi cycle and synthesis of sucrose and starch. Therefore, in the present study Vcmax, Jmax and TPU decreased in M. coromandelianum while Vcmax and Jtotal in I. rugosum suggesting more negative effects in M. coromandelianum compared to I. rugosum, leading to reduced photosynthates formation in the former species. In M. coromandelianum, more percent reduction in Vcmax compared to Jmax was recorded. The reduction in Jmax might be due to a decrease in activity of regenerative enzymes in the C3 cycle (Zheng et al., 2002). Both Vcmax and Jmax decreased in L. perenne and T. repens after 10 weeks of O3 exposure (Hayes et al., 2009). Reductions in Vcmax, Jmax and TPU in soybean were reported in response to elevated O3 concentration (Sun et al., 2014) as found in M. coromandelianum. Zheng et al. (2002) also found decreases in Vcmax, Jmax, and TPU in P. major under 15–75 nl l−1 O3 treatment for 33 days. Like I. rugosum, reduction in Vcmax in Zea mays, a C4 plant has also been reported at ambient +80 nl l−1 O3 treatment in OTCs (Leitao et al., 2007). Spartina alternifolia (C4 plant) showed more tolerance compared to Phragmites australis (C3 plant) with less effect on Vpmax, Vcmax and Jmax under salinity stress (Ge et al., 2014). Under future climate scenario, C4 plants are predicted to flourish more compared to C3 plants (Bernacki, 2012). Under NFA+ treatment, O3 induced LCP and reduced Φ in both the plants which are familiar responses to O3 (Neufeld et al., 2018). The decrease in Φ in both species may be due to less photon absorption because of damage to chlorophyll pigments. A similar result was found in Nicotiana tabacum varieties under 135 nl l−1 O3 exposure for 8 h daily for 20 days (Saitanis et al., 2001). M. coromandelianum showed an increase in Rdark under NFA+ treatment which is a common response to O3 stress (Runeckles et al., 1992). The respiration and assimilation are adjusted to help plants cope-up from the stress condition. The increase in LCP in M. coromandelianum may be due to an increase in dark respiration because A is decreased which could lead to less availability of ATP. Therefore more respiration is needed for plants to cope-up from stress. The Rdark decreased in I. rugosum which may be because of comparatively lesser damage or lower ATP requirement. This suggests that the C4 photosynthetic pathway in I.rugosum is less affected compared to the C3 photosynthetic pathway in M. coromandelianum under elevated O3 stress. At vegetative stage, all the growth parameters except root length significantly declined due to elevated O3 exposure in M. coromandelianum, which also showed inductions of proline and total phenol contents. As more photosynthates were allocated towards defense, growth was more reduced in M. coromandelianum. In I rugosum, all the growth parameters except total biomass showed non-significant variations under O3 stress suggesting tolerance of this plant during the early growth stage. Higher induction of AsA synthesis in I rugosum at early growth stage might have reduced the overall O3 impact and hence other antioxidant metabolites were not induced leading to higher allocation of carbon and nitrogen to growth instead of defense. At later growth stage, both the test plants showed significant reductions in most of the growth parameters indicating age-dependent response of plants to prolonged O3 exposure independent of early antioxidative response. The responses of photosynthetic parameters are substantiated by the pattern of growth and total biomass accumulation in both plants under elevated O3. Higher negative effects on photosynthetic parameters in M. coromandelianum were reflected in terms of greater reduction in growth and total biomass accumulation under NFA+. The LAR narrates the allocation and leaf morphology (Poorter and Remkes, 1990). LAR showed a contrasting response in the test plants indicating differential strategy under similar stress condition. I. rugosum showed a significant increase in LAR at later growth stage suggesting a decrease in biomass accumulation as a result of prolonged O3 stress. In M. coromandelianum, at an early growth stage, LAR showed significant 10

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References

LCP in M. coromandelianum (Fig. 4B). Reductions in AsA and tannin were also associated with Vcmax and Jmax which may lead to more ROS production in M. coromendelianum. Higher associations of parameters such as total phenolics, proline, LCP, MDA, LAR ratio and gs suggested that high gs allowed more O3 entry into sub-stomatal space which may have caused a high production of ROS and more biosynthesis of total phenolics and proline to overcome the negative effects of O3 in M. coromandelianum (Fig. 4B). Based on the observations, the hypothesis of the present study is reasonably fulfilled as both the representative grass (I. rogusum) and forb (M. coromandeliamum) species showed remarkable variations in physiological, biochemical and growth parameters under future O3 concentration and the variations were species specific. More studies are required with different O3 concentrations to assess the range of responses under natural conditions to better understand the responses of grassland vegetation to future increase in O3 concentrations.

Ainsworth, E.A., 2017. Understanding and improving global crop response to ozone pollution. Plant J. 90 (5), 886–897. Ainsworth, E.A., Lemonnier, P., Wedow, J.M., 2019. The influence of rising tropospheric carbon dioxide and ozone on plant productivity. Plant Biol. https://doi.org/10. 1111/plb.12973. Andersen, C.P., 2003. Source-sink balance and carbon allocation below ground in plants exposed to ozone. New Phytol. 157 (2), 213–228. Ashmore, M.R., Thwaites, R.H., Ainsworth, N., Cousins, D.A., Power, S.A., Morton, A.J., 1995. Effects of ozone on calcareous grassland communities. Water Air Soil Pollut. 85 (3), 1527–1532. Bakar, B.H., Nabi, L.N.A., 2003. Seed germination, seedling establishment and growth patterns of wrinklegrass (Ischaemum rugosum Salisb). Weed Biol. Manag. 3 (1), 8–14. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39 (1), 205–207. Bauwe, H., 1986. An efficient method for the determination of Km values for HCO3- of phosphoenolpyruvate carboxylase. Planta 169 (3), 356–360. Bellincontro, A., Catelli, C., Cotarella, R., Mencarelli, F., 2017. Postharvest ozone fumigation of Petit Verdot grapes to prevent the use of sulfites and to increase anthocyanin in wine. Aust. J. Grape Wine Res. 23 (2), 200–206. Bernacki, Z., 2012. Changes in the balance between C3 and C4 plants expected in Poland with the global change. Ecol. Quest. 16, 59–68. Bray, H.G., Thorpe, W.V., 1954. Estimation of phenols. Methods Biochem. Anal. 1, 27–52. Calzone, A., Podda, A., Lorenzini, G., Maserti, B.E., Carrari, E., Deleanu, E., Hoshika, Y., Haworth, M., Nali, C., Badea, O., Pellegrini, E., 2019. Cross-talk between physiological and biochemical adjustments by Punica granatum cv. Dente di cavallo mitigates the effects of salinity and ozone stress. Sci. Total Environ. 656, 589–597. Chaudhary, N., Agrawal, S.B., 2014a. Cultivar specific variations in morphological and biochemical characteristics of mung bean due to foliar spray of ascorbic acid under elevated ozone. Acta Physiol. Plant. 36 (7), 1793–1803. Chaudhary, N., Agrawal, S.B., 2014b. Role of gamma radiation in changing phytotoxic effect of elevated level of ozone in Trifolium alexandrinum L. (Clover). Atmospheric Pollution Research 5 (1), 104–112. Chaudhary, N., Agrawal, S.B., 2015. The role of elevated ozone on growth, yield and seed quality amongst six cultivars of mung bean. Ecotoxicol. Environ. Safe. 111, 286–294. Dai, L., Feng, Z., Pan, X., Xu, Y., Li, P., Lefohn, A.S., Harmens, H., Kobayashi, K., 2019. Increase of apoplastic ascorbate induced by ozone is insufficient to remove the negative effects in tobacco, soybean and poplar. Environ. Pollut. 245, 380–388. Davison, A.W., Barnes, J.D., 1998. Effects of ozone on wild plants. New Phytol. 139 (1), 135–151. Dentener, F., Keating, T., Akimoto, H. (Eds.), 2010. Hemispheric Transport of Air Pollution 2010: Part A: Ozone and Particulate Matter. UN, New York, pp. 304. Dizengremel, P., 2001. Effects of ozone on the carbon metabolism of forest trees. Plant Physiol. Biochem. 39 (9), 729–742. Duxbury, A.C., Yentsch, C.S., 1956. Plankton pigment nomographs. Journal of Marine Research 15, 93–101. El-Khatib, A.A., 2003. The response of some common Egyptian plants to ozone and their use as biomonitors. Environ. Pollut. 124 (3), 419–428. Fatima, A., Singh, A.A., Mukherjee, A., Agrawal, M., Agrawal, S.B., 2018. Variability in defence mechanism operating in three wheat cultivars having different levels of sensitivity against elevated ozone. Environ. Exp. Bot. 155, 66–78. Feng, Z., Paoletti, E., Bytnerowicz, A., Harmens, H., 2015. Ozone and Plants. Environmental Pollution, vol. 202. pp. 215–216. Ge, Z.M., Zhang, L.Q., Yuan, L., Zhang, C., 2014. Effects of salinity on temperature-dependent photosynthetic parameters of a native C3 and a non-native C4 marsh grass in the Yangtze Estuary, China. Photosynthetica 52 (4), 484–492. Gitelson, A.A., Merzlyak, M.N., Chivkunova, O.B., 2001. Optical properties and nondestructive estimation of anthocyanin content in plant leaves¶. Photochem. Photobiol. 74 (1), 38–45. Guerreiro, C.B., Foltescu, V., De Leeuw, F., 2014. Air quality status and trends in Europe. Atmos. Environ. 98, 376–384. Hardacre, C., Wild, O., Emberson, L., 2015. An evaluation of ozone dry deposition in global scale chemistry climate models. Atmos. Chem. Phys. 15 (11), 6419–6436. Hayes, F., Mills, G., Ashmore, M., 2009. Effects of ozone on inter-and intra-species competition and photosynthesis in mesocosms of Lolium perenne and Trifolium repens. Environ. Pollut. 157 (1), 208–214. Hayes, F., Mills, G., Harmens, H., Wyness, K., 2011. Within season and carry-over effects following exposure of grassland species mixtures to increasing background ozone. Environ. Pollut. 159 (10), 2420–2426. Hayes, F., Mills, G., Jones, L., Ashmore, M., 2010. Does a simulated upland grassland community respond to increasing background, peak or accumulated exposure of ozone? Atmos. Environ. 44 (34), 4155–4164. Hayes, F., Wagg, S., Mills, G., Wilkinson, S., Davies, W., 2012. Ozone effects in a drier climate: Implications for stomatal fluxes of reduced stomatal sensitivity to soil drying in a typical grassland species. Glob. Chang. Biol. 18 (3), 948–959. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125 (1), 189–198. Hoshika, Y., Omasa, K., Paoletti, E., 2013. Both ozone exposure and soil water stress are able to induce stomatal sluggishness. Environ. Exp. Bot. 88, 19–23. Keller, T., Schwager, H., 1977. Air pollution and ascorbic acid. For. Pathol. 7 (6), 338–350. Khomdram, S.D., Singh, P.K., 2011. Polyphenolic compounds and free radical scavenging activity in eight Lamiaceae herbs of Manipur. Not. Sci. Biol. 3 (2), 108. Leitao, L., Bethenod, O., Biolley, J.P., 2007. The impact of ozone on juvenile maize (Zea

5. Conclusions The present study suggests that both the grassland species of IGP are negatively affected at a future concentration of O3. The negative effects of O3 on photosynthetic pigments, antioxidants and metabolites were variable between the species. AsA acted as a strong antioxidant in I. rugosum while proline and total phenolics in M. coromandelianum. The present study found more percent reductions in Vcmax, Jmax and TPU in M. coromandelianum (C3 plant) and comparatively less in Vcmax, Jtotal with no significant change in Vpmax in I. rugosum (C4 plant), suggesting more negative effects on former compared to later under future O3 concentrations. More alteration in growth parameters and total biomass accumulation at both stages were found in M. coromandelianum compared to I. rugosum under O3 stress. The more negative effects on M. coromandelianum than I. rugosum under future O3 concentrations suggest their differential response strategy under elevated O3 exposure. I. rugosum partitioned the biomass judiciously between growth and defense and hence maintained growth and development under elevated O3 concentration. Thus, a differential response of pattern was observed in both the grassland species at future O3 concentration, suggesting a shift in community structure and function in natural conditions, leading to a risk of disappearance of sensitive species. There is a need of long term studies on grassland species of tropical areas under elevated O3 levels to assess their vulnerability under natural conditions to understand the changes in community structure, which have long lasting impact on the sustainability of tropical grassland ecosystems. Conflicts of interest Authors declare that they have no conflict of interest. Acknowledgements Authors are thankful to the Head, Department of Botany and Coordinators DST-FIST, CAS in Botany, DST PURSE and ISLS, B.H.U for the field and instrumental facilities. University Grants Commission, Rajiv Gandhi National Fellowship, New Delhi, is greatly acknowledged for providing fellowship to Tsetan Dolker. The authors were supported by Research Council of Norway through the CiXPAG project (grant no. 244551). Authors are thankful to the learned reviewers for their valuable suggestions which helped in better presentation of our manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109404. 11

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belonging to natural ecosystems. Environ. Exp. Bot. 57 (1), 89–97. Shabbir, A., Bajwa, R., 2006. Distribution of parthenium weed (Parthenium hysterophorus L.), an alien invasive weed species threatening the biodiversity of Islamabad. Weed Biol. Manag. 6 (2), 89–95. Shang, B., Xu, Y., Dai, L., Yuan, X., Feng, Z., 2019. Elevated ozone reduced leaf nitrogen allocation to photosynthesis in poplar. Sci. Total Environ. 657, 169–178. Sharkey, T.D., Bernacchi, C.J., Farquhar, G.D., Singsaas, E.L., 2007. Fitting photosynthetic carbon dioxide response curves for C3 leaves. Plant Cell Environ. 30 (9), 1035–1040. Sicard, P., Anav, A., Marco, A.D., Paoletti, E., 2017. Projected global ground-level ozone impacts on vegetation under different emission and climate scenarios. Atmos. Chem. Phys. 17 (19), 12177–12196. Singh, A.A., Agrawal, S.B., Shahi, J.P., Agrawal, M., 2014. Investigating the response of tropical maize (Zea mays L.) cultivars against elevated levels of O3 at two developmental stages. Ecotoxicology 23 (8), 1447–1463. Sinha, V., Kumar, V., Sarkar, C., 2014. Chemical composition of pre-monsoon air in the Indo-Gangetic Plain measured using a new air quality facility and PTR-MS: High surface ozone and strong influence of biomass burning. Atmos. Chem. Phys. 14 (12), 5921–5941. Su, B., Zhou, M., Xu, H., Zhang, X., Li, Y., Su, H., Xiang, B., 2017. Photosynthesis and biochemical responses to elevated O3 in Plantago major and Sonchus oleraceus growing in a lowland habitat of northern China. J. Environ. Sci. 53, 113–121. Sun, J., Feng, Z., Ort, D.R., 2014. Impacts of rising tropospheric ozone on photosynthesis and metabolite levels on field grown soybean. Plant Sci. 226, 147–161. Takshak, S., Agrawal, S.B., 2018. Interactive effects of supplemental ultraviolet-B radiation and indole-3-acetic acid on Coleus forskohlii Briq.: Alterations in morphological-, physiological-, and biochemical characteristics and essential oil content. Ecotoxicol. Environ. Safe. 147, 313–326. Temple, P.J., Riechers, G.H., Miller, P.R., 1992. Foliar injury responses of ponderosa pine seedlings to ozone, wet and dry acidic deposition, and drought. Environ. Exp. Bot. 32 (2), 101–113. Tie, X., Madronich, S., Li, G., Ying, Z., Zhang, R., Garcia, A.R., Lee-Taylor, J., Liu, Y., 2007. Characterizations of chemical oxidants in Mexico City: A regional chemical dynamical model (WRF-Chem) study. Atmos. Environ. 41 (9), 1989–2008. Tiwari, S., Rai, R., Agrawal, M., 2008. Annual and seasonal variations in tropospheric ozone concentrations around Varanasi. Int. J. Remote Sens. 29 (15), 4499–4514. Von Caemmerer, S., 2000. Biochemical Models of Leaf Photosynthesis. Csiro publishing, Collingwood. Wagg, S., Mills, G., Hayes, F., Wilkinson, S., Davies, W.J., 2013. Stomata are less responsive to environmental stimuli in high background ozone in Dactylis glomerata and Ranunculus acris. Environ. Pollut. 175, 82–91. Wang, L., Pang, J., Feng, Z., Zhu, J., Kobayashi, K., 2015. Diurnal variation of apoplastic ascorbate in winter wheat leaves in relation to ozone detoxification. Environ. Pollut. 207, 413–419. Wilbourn, S., Davison, A.W., Ollerenshaw, J.H., 1995. The use of an unenclosed field fumigation system to determine the effects of elevated ozone on a grass–clover mixture. New Phytologist 129 (1), 23–32. https://doi.org/10.1111/j.1469-8137. 1995.tb03006.x. Wilkinson, S., Davies, W.J., 2010. Drought, ozone, ABA and ethylene: New insights from cell to plant to community. Plant Cell Environ. 33 (4), 510–525. Xu, Y., Feng, Z., Shang, B., Dai, L., Uddling, J., Tarvainen, L., 2019. Mesophyll conductance limitation of photosynthesis in poplar under elevated ozone. Sci. Total Environ. 657, 136–145. Yadav, D.S., Rai, R., Mishra, A.K., Chaudhary, N., Mukherjee, A., Agrawal, S.B., Agrawal, M., 2019. ROS production and its detoxification in early and late sown cultivars of wheat under future O3 concentration. Sci. Total Environ. 659, 200–210. Yendrek, C.R., Erice, G., Montes, C.M., Tomaz, T., Sorgini, C.A., Brown, P.J., McIntyre, L.M., Leakey, A.D., Ainsworth, E.A., 2017. Elevated ozone reduces photosynthetic carbon gain by accelerating leaf senescence of inbred and hybrid maize in a genotype‐specific manner. Plant Cell Environ. 40 (12), 3088–3100. Yin, X., Sun, Z., Struik, P.C., Van der Putten, P.E., Van Ieperen, W.I.M., Harbinson, J., 2011. Using a biochemical C4 photosynthesis model and combined gas exchange and chlorophyll fluorescence measurements to estimate bundle‐sheath conductance of maize leaves differing in age and nitrogen content. Plant Cell Environ. 34 (12), 2183–2199. Zheng, Y., Shimizu, H., Barnes, J.D., 2002. Limitations to CO2 assimilation in ozone‐exposed leaves of Plantago major. New Phytol. 155 (1), 67–78.

mays L.) plant photosynthesis: Effects on vegetative biomass, pigmentation, and carboxylases (PEPc and Rubisco). Plant Biol. 9 (04), 478–488. Li, S., Harley, P.C., Niinemets, U., 2017. Ozone‐induced foliar damage and release of stress volatiles is highly dependent on stomatal openness and priming by low‐level ozone exposure in Phaseolus vulgaris. Plant Cell Environ. 40 (9), 1984–2003. Maclachlan, S., Zalik, S., 1963. Plastid structure, chlorophyll concentration, and free amino acid composition of a chlorophyll mutant of barley. Canadian Journal of Botany 41 (7), 1053–1062. Manninen, O., Stark, S., Kytoviita, M.M., Lampinen, L., Tolvanen, A., 2009. Understorey plant and soil responses to disturbance and increased nitrogen in boreal forests. J. Veg. Sci. 20 (2), 311–322. Marchica, A., Lorenzini, G., Papini, R., Bernardi, R., Nali, C., Pellegrini, E., 2019. Signalling molecules responsive to ozone-induced oxidative stress in Salvia officinalis. Sci. Total Environ. 657, 568–576. Massad, R.S., Tuzet, A., Bethenod, O., 2007. The effect of temperature on C4‐type leaf photosynthesis parameters. Plant Cell Environ. 30 (9), 1191–1204. Masutomi, Y., Kinose, Y., Takimoto, T., Yonekura, T., Oue, H., Kobayashi, K., 2019. Ozone changes the linear relationship between photosynthesis and stomatal conductance and decreases water use efficiency in rice. Sci. Total Environ. 655, 1009–1016. Mathur, M., Nama, K.S., Choudhary, K., 2016. Economic and ethnomedicinal importance of the floral diversity on ancient walls of kota district, Rajasthan. International Journal Pure and Applied Bioscience 4 (4), 167–173. Mills, G., Hayes, F., Simpson, D., Emberson, L., Norris, D., Harmens, H., Büker, P., 2011. Evidence of widespread effects of ozone on crops and semi‐natural vegetation in Europe (1990–2006) in relation to AOT40‐and flux‐based risk maps. Glob. Chang. Biol. 17 (1), 592–613. Mills, G., Hayes, F., Wilkinson, S., Davies, W.J., 2009. Chronic exposure to increasing background ozone impairs stomatal functioning in grassland species. Glob. Chang. Biol. 15 (6), 1522–1533. Mills, G., Sharps, K., Simpson, D., Pleijel, H., Broberg, M., Uddling, J., Jaramillo, F., Davies, W.J., Dentener, F., Van den Berg, M., Agrawal, M., 2018. Ozone pollution will compromise efforts to increase global wheat production. Glob. Chang. Biol. 24 (8), 3560–3574. Mishra, A.K., Agrawal, S.B., 2015. Biochemical and physiological characteristics of tropical mung bean (Vigna radiata L.) cultivars against chronic ozone stress: An insight to cultivar-specific response. Protoplasma 252 (3), 797–811. Neufeld, H.S., Johnson, J., Kohut, R., 2018. Comparative ozone responses of cutleaf coneflowers (Rudbeckia laciniata var. digitata, var. ampla) from Rocky Mountain and Great Smoky Mountains National Parks, USA. Sci. Total Environ. 610, 591–601. Oksanen, E., Pandey, V., Pandey, A.K., Keski-Saari, S., Kontunen-Soppela, S., Sharma, C., 2013. Impacts of increasing ozone on Indian plants. Environ. Pollut. 177, 189–200. Pandey, A.K., Majumder, B., Keski-Saari, S., Kontunen-Soppela, S., Pandey, V., Oksanen, E., 2014. Differences in responses of two mustard cultivars to ethylenediurea (EDU) at high ambient ozone concentrations in India. Agric. Ecosyst. Environ. 196, 158–166. Poorter, H., Remkes, C., 1990. Leaf area ratio and net assimilation rate of 24 wild species differing in relative growth rate. Oecologia 83 (4), 553–559. Ramo, K., Kanerva, T., Nikula, S., Ojanperä, K., Manninen, S., 2006. Influences of elevated ozone and carbon dioxide in growth responses of lowland hay meadow mesocosms. Environ. Pollut. 144 (1), 101–111. Roy, S., Beig, G., Jacob, D., 2008. Seasonal distribution of ozone and its precursors over the tropical Indian region using regional chemistry‐transport model. J. Geophys. Res.: Atmosphere 113 (D21). Runeckles, V.C., Chevone, B.I., Lefohn, A., 1992. Surface level ozone exposures and their effects on vegetation. In: Lefohn, A.S. (Ed.), Crop Responses to Ozone. Lewis Publishers, Inc, Chelsea, Michigan, pp. 189–270. Rupakheti, D., Kang, S., Rupakheti, M., Tripathee, L., Zhang, Q., Chen, P., Yin, X., 2018. Long-term trends in the total columns of ozone and its precursor gases derived from satellite measurements during 2004–2015 over three different regions in South Asia: Indo-Gangetic Plain, Himalayas and Tibetan Plateau. Int. J. Remote Sens. 39 (21), 7384–7404. Saitanis, C.J., Riga-Karandinos, A.N., Karandinos, M.G., 2001. Effects of ozone on chlorophyll and quantum yield of tobacco (Nicotiana tabacum L.) varieties. Chemosphere 42 (8), 945–953. Santos, R.V.C.D., Marenco, R.A., 1999. Wrinkled grass and rice intra and interspecific competition. Rev. Bras. Fisiol. Vegetal 11 (2), 107–111. Scebba, F., Giuntini, D., Castagna, A., Soldatini, G., Ranieri, A., 2006. Analysing the impact of ozone on biochemical and physiological variables in plant species

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