Aluminium Fluoride induced changes in chlorophyll a fluorescence, antioxidants and psb A gene expression of Brassica juncea cultivars

ABSTRACT In present study, the effects of combined Aluminium and Fluoride (AlF) stress on chlorophyll a fluorescence, photosynthetic pigments, antioxidant system and psb A gene expression are first time reported in four Brassica juncea cultivars (CS-14, Pusa-Tarak, Bio-902 and Laxmi). Each cultivar was exposed to soil supplemented with AlF (0, 50 + 25, 100 + 50 and 150 + 75 mgkg−1). Lowest decline in the chlorophyll content, saturating photosynthetically active photon flux density, maximum apparent electron transport rate and effective quantum yield (PSII) under AlF was observed in Pusa-Tarak followed by CS-14, Bio-902 and Laxmi. The improved performance of the cultivar Pusa-Tarak under AlF stress was accompanied by an increase in proline level and enzymes activity of catalase and ascorbate peroxidase. However, significant increase in superoxide dismutase activity was observed in cultivar Laxmi. We also observed that AlF inhibits psb A gene expression to a lesser extent in tolerant cultivar Pusa-Tarak in comparison to susceptible cultivar Laxmi.


Introduction
About half of the world's agricultural soil is acidic (Divya Sri et al. 2016;Gupta et al. 2013). Soil acidification mainly occurs naturally when basic cations leach from soil and also increase by acidic rain (Ryan and Delhaize 2012). Aluminium (Al) toxicity is a key limiting factor for agriculture in acidic soil (Gupta et al. 2013). In soil, Al is one of the plentiful metals and contributes 7% (approx) of the earth's crust. Excessive Al content in the soil induces higher ROS (reactive oxygen species) formation, which leads to oxidative damage in both crop vegetation and plants (He et al. 2014;Yamamoto et al. 2001;Zheng and Yang 2005).
Fluoride (F) is a highly reactive halogen which forms complexes with many cations and is readily adsorbed by the soil. Atmospheric F particles and excessive usage of phosphate fertilizers in farming enhance F contamination in soil (Stevens et al. 1997). F toxicity causes foliar damage in plants and also influences enzyme activities and physiological processes such as photosynthesis and respiration (Baunthiyal and Ranghar 2014). Generally, it has been noticed that Al 3+ has the highest binding affinity for F − in comparison to other 60 metal ions (Foster 2004). Owing to this affinity, Al and F form AlFx complex in acidic conditions. The accumulation of AlFx complex in plant affects many physiological, biochemical processes and molecular changes (H + -ATPase of root) (Rai et al. 1996;Rocha-Fachna and Okorokova-Facanha 2002).The formation of AlF − 4 , being a PO 3− 4 analogue, might compete with PO 3− 4 for binding sites of H + -ATPase and create starvation condition in plants (Alia 2004).Therefore, Al and F together are one of the major problems in acidic soil globally.
Despite huge crop losses due to AlF toxicity, currently no effective strategies are available for AlF removal and improved crop production in AlF contaminated soil. In this context, identification and cultivation of AlF tolerant cultivars may be an effective solution to improve agricultural production in acidic soil. Therefore, the study of growth parameters, antioxidant enzyme activity, gene expression and tolerance ability of the plants are necessary before claiming the cultivar as 'tolerant'. During stressful environment, ROS accumulate and cause oxidative damage in plants, which may induce cell death (Dietz 2005). Electron transport chain occurs in mitochondria and chloroplast, thus high oxidative damage takes place in these organelles (Sairam and Tyagi 2004). The scavenging potential of ROS is associated with antioxidant enzymes's activity viz. superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.1.1.1.11), catalase (CAT, EC 1.1.1.1.6) and other enzymes in plants (Passardi et al. 2007). Accumulation of osmolytes occurs in stressful conditions to provide defense and enhance tolerance ability in cultivars (Caverzan et al. 2016).
Chlorophyll a fluorescence is also an appropriate parameter for measuring growth and tolerance potential of crop cultivars as it is more precise and sensitive in comparison to other ordinary methods. The physiological impact of abiotic stress such as salt (Mittal et al. 2012), drought (Batra et al. 2014) and fluoride (Baunthiyal and Sharma 2014) has been studied using chlorophyll a fluorescence technique in plants.
Several studies also demonstrated that genes associated with photosystem I and II are sensitive under adverse environment viz. drought (Degenkolbe et al. 2009;Hayano-Kanashiro et al. 2009;Zhang et al. 2015), salt (Chaves et al. 2009) and light (Murchie et al. 2005). Proteins associated to photosynthesis were also repressed in plants under heat stress (Ahsan et al. 2010). Han et al. (2009) andHasanuzzaman et al. (2013) reported that heat stress and high temperature decrease the activity & quantum yield of PS II. Stressful environment slows down the photosynthetic phenomenon in plants via changing the chloroplast structure and reducing the level of photosynthetic pigments. Light energy absorbed by chlorophyll is not properly used by photosystems (PS I & PS II) in adverse conditions resulting in Chl a fluorescence phenomenon (Maxwell and Johnson 2000). Thus, growth parameters such as antioxidant enzyme activity, gene expression and chlorophyll a fluorescence can be used to evaluate the AlF tolerance in cultivars.
The most significant factor to enhance agricultural yield in contaminated soil is the identification and cultivation of AlF tolerant cultivars. India is the 2 nd largest producer of Brassica juncea in the world (Shah 2007). Family Brassicaceae also represents a potential and promising group of plants to be used for phytoremediation (Goswami and Das 2015).
The study was carried out to assess the chlorophyll a fluorescence, biochemistry and psb A gene expression in four cultivars of Brassica juncea cultivars viz.  in the presence of AlF stress. We also investigated the effect of AlF on anti-oxidative systems to identify the correlation among AlF stress and cultivar tolerance.

Plant material and their growth
The seeds of four Brassica juncea (L.) Czern cultivars (CS-14, Laxmi, Pusa-Tarak and Bio-902) were obtained from Krishi Vigyan Kendra (Banasthali Vidyapith, India). The seeds of the selected cultivars were sterilized by using 0.01% HgCl 2 . The seeds were sown in petri dishes and 4 days old seedlings were transferred into pots. Three seedlings were transferred in each pot. The original pH of pots soil at room temperature was 7.2 and was maintained 5.2 by the addition of ammonia solution 25% by volume. The pH was determined by using water and soil suspension (2.5:1) by glass electrode (Jackson 1973). pH maintained pots were kept in sunlight for approx 20 days to allow evaporation of excessive water. The type and texture of soil was sandy and general light brown in color. The organic of the soil was determined by Walkley and Black's (1934) wet digestion method and was found 0.12 g kg −1 . The pots were kept in a culture room at 27 ± 2°C and photoperiod of 16 h. These cultivars of Brassica juncea were treated with different AlF concentration i.e. 0, 50 + 25, 100 + 50 and 150 + 75 mg kg −1 where 0 mg kg −1 served as control. All the experiments were done at vegetative (60 days) and reproductive (120 days) growth stages of Brassica juncea cultivars. The sources used for Al and F were AlCl 3 and NaF respectively.

Screening of tolerance of Brassica juncea cultivars for Al + F
Various morphological parameters, viz. the germination %, shoot length (cm), number of flowers on main raceme, number of siliquae on main raceme, number of seeds per siliquae and test weight (wt. of 1000 seeds) were studied (Rath et al. 2010).

Measurement of photosynthetic pigments
The level of total Chl, Chl a, Chl b and Car was calculated using Arnon's method (1949). The absorbance of sample was taken at wavelengths 480, 645 and 663 nm. The content of the photosynthetic pigments was calculated in mg g −1 fresh weight of the sample.

Chlorophyll a fluorescence measurements
Chlorophyll a fluorescence was measured using DUAL PAM 100 of H. Walz (Effeltrich, Germany) using the method given by Klughammer and Schreiber (2008). The fluorescence measurements were performed on vegetative and reproductive stages of plants. The quantum efficiency of photosystem II (PS II) and the rate of electron transport (ETR) through PS II were measured simultaneously. The effective quantum yield of PS II (ΔF/Fm ' ) was calculated using the formula (ΔF/Fm ′ ) = (Fm ′ -F)/ Fm ′ , where F is minimal fluorescence and Fm ′ is maximal fluorescence under a saturating light pulse i.e. 800 ms (Genty et al. 1989). Apparent ETR was then obtained as ETR = ɸPSII × PPFD × 0.5, where 0.5 accounts for equal excitation in photosystems I and II, photosynthetic photon flux density (PPFD) (µ mol photon m −2 s −1 ) is absorbed light; ɸPSII is the quantum yield of PSII (Genty et al. 1989

Measurement of proline
The proline content was determined in leaves of Brassica juncea cultivars by following Bates et al. (1973) method.

Catalase activity (CAT)
The enzyme activity was carried out in each cultivar (Pusa-Tarak, CS-14, Bio-902 and Laxmi) by following the method of Aebi (1984).

Ascorbate peroxidase activity (APX)
The analysis of APX activity was carried out by using the method of Asada (1984).

Superoxide dismutase activity (SOD)
The method of Beauchamp and Fridovich (1971) was used to analyze the activity of SOD in terms of ability to inhibit the nitroblue tetrazolium (NBT) reduction to formazon.

Gene expression level
Expression of psbA gene was detected in two selected cultivars (Pusa-Tarak and Laxmi) under combined stress of Aluminium and Fluoride using qRT-PCR. The level of gene expression was determined by using the method of Sharma et al. (2014). Relative gene expression was calculated in terms of fold change using the following formula: Primer pairs specific to Brassica juncea psb A gene (NCBI accession numbers AOAOPOKT86) cDNA (sense primer, 5 ′ -TAC GTT CRT GCA TAA CTT CC-3 ′ and antisense primer 5 ′ -CGA AGC TCC ATC TAC AAA TGG-3 ′ ) were used. Primer pairs of Brassica juncea ubiquitin 9 gene (UBQ9) (sense primer, 5 ′ -GAA GAC ATG TTC CAT TGG CA-3 ′ , antisense primer 5 ′ -ACA CCT TAG TCC TAA AAG CCA CCT-3 ′ ) were used as a reference gene for internal control (Chandna et al. 2012). The expression level of psb A and ubiquitin 9 genes were determined at vegetative and reproductive stages.

Statistical analysis
The data has been presented as Mean ± SD (n = 3). The statistical analysis was performed by using SPSS (version 17.0). The analysis was conducted by compare means test, using One Way Analysis of Variance (ANOVA) and then by applying post-hoc-Tukey's test. One-way ANOVA analysis was used to compare four Brassica juncea cultivars (Bio-902, CS-14, Pusa-Tarak and Laxmi). Sigma Plot 12.0 software was used to plot the graphs. Chlorophyll a fluorescence and pigments results are represented as percentage (%) change after statistical analysis.

Results
The experiments were performed to study the effect of combined Al and F (AlF) stress at both vegetative and reproductive growth stages of Brassica juncea cultivars (Bio-902, CS-14, Pusa-Tarak and Laxmi). Each cultivar of Brassica juncea was exposed to AlF stress and revealed significant variation in sensitivity towards toxicity of AlF. The AlF sensitive cultivar Laxmi did not grow at a concentration of Al + F stress i.e. 150 + 75 mg kg −1 respectively.

Effect of Al + F on various growth parameters of Brassica juncea
Eight cultivars of mustard (NRCDR-2, CS-14, Laxmi, Vasundhra, Swarn Jyoti, Pusa-Tarak, Bio-902,  were treated with (Al + F) and four months old plants were screened on the basis of growth and reproductive parameters such as germination %, dry biomass (%), shoot length, number of flowers on main raceme, number of siliquae on main raceme, number of seeds per siliquae and test weight (wt. of 1000 seeds). Based on initial screening, four cultivars (CS-14, Pusa-Tarak, Bio-902 and Laxmi) were selected to analyse their phytoremediation andphotosynthetic efficiency,antioxidant activities and psb A gene expression under treatment of Al + F during vegetative (60 days) as well as reproductive (120 days) growth stages. The results showed that as the concentration of (Al + F) increased, all the morphological parameters were affected. The parameters such as germination percentage, dry biomass %, shoot length, number of siliquae on main raceme (Yadav et al. 2018), number of flowers on main raceme ( Figure S1), number of seeds per siliquae ( Figure S1) and test weight (wt. of 1000 seeds) ( Figure S1) were found maximum in Pusa-Tarak followed by CS-14, Bio-902 and Laxmi under AlF.
The comparative analysis of total Chl content between all the cultivars at different AlF concentration was performed using SPSS at p < 0.05. The chlorophyll content of Bio-902, CS-14 is not significantly different at different concentrations with respect to Pusa-Tarak indicating that these three cultivars have same potential for growth and chlorophyll formation under control conditions. However, chlorophyll content of Laxmi cultivar is significantly different under AlF stress with respect to Pusa-Tarak. When Chl content of Pusa-Tarak and Laxmi at AlF concentration 50 + 25 and 100 + 50 mg kg −1 was compared, significant difference was observed between both the cultivars indicating that Laxmi has less potential for Chl formation under AlF stress. As the cultivar Laxmi did not grow at higher AlF concentration of 150 + 75 mg kg −1 , a comparison could not be made between the cultivar Pusa-Tarak and Laxmi.
3.2. Effect of AlF on chlorophyll a fluorescence 3.2.1. Vegetative stage When four cultivars of Brassica juncea were grown in absence of AlF stress, the ɸ PSII values observed at zero PPFD was 0.8 or approx 0.8 in all cultivars. At zero PPFD, the ɸ PSII values decreased with respect to increase in AlF stress in each cultivar however, the drastic inhibitory effect of AlF on ɸ PSII values was observed in cultivar Laxmi followed by Bio-902, CS-14 and Pusa-Tarak. At 100 + 50 mg kg −1 concentration of AlF, the ɸ PSII values reduced to 52%, 33%, 23% and 17% in cultivars viz. Laxmi, Bio-902, CS-14 and Pusa-Tarak respectively (Figure 1).

Reproductive stage
At the reproductive stage, it was found that ɸ PSII values and cardinal points (ETRmax, PPFDsat, ½PPFDsat, ΔF/Fm ′ sat and ½ΔF/Fm ′ sat) declined under AlF stress in each cultivar (Pusa-Tarak, CS-14, Bio-902 and Laxmi) (Figure 2 and Table 4). However, the reduction was again in the order Pusa-Tarak <CS-14 < Bio-902 < Laxmi. The inhibitory effect of AlF on photosynthetic performance increased gradually with rise in AlF stress.

Effect of AlF on antioxidant enzymes
The effect of AlF on the activity of CAT, APX and SOD in four cultivars viz. Bio-902, CS-14, Pusa-Tarak and Laxmi are shown in Figures 3 and 4. At 100 + 50 mg kg −1 AlF, the activities of CAT and APX enzymes increased by 3.1 folds and 2.2 folds respectively at vegetative stage of AlF-tolerant cultivar i.e Pusa-Tarak. However, in other cultivars (CS-14, Bio-902 and Laxmi), the activity of CAT and APX is also enhanced by the AlF stress but to a lower level with respect to Pusa-Tarak (Figure 3). The highest increase in SOD activity was observed in Laxmi (2.6 folds) followed by Bio-902 (2.1 folds), CS-14 (1.6 folds) and Pusa-Tarak (1.4 folds) at AlF (100 + 50 mg kg −1 ) (Figure 3).
At reproductive stage, the CAT activity significantly induced up to 100 + 50 mg kg −1 AlF concentration in each cultivar with highest increase by 2.2 folds in Pusa-Tarak and thereafter, the activity of enzyme reduced gradually but was still highest in the tolerant cultivar Pusa-Tarak followed by CS-14, Bio-902 and Laxmi (Figure 4). Similar to vegetative stage, the activity of APX was again higher in the cultivar Pusa-Tarak as compared to other cultivars viz. CS-14, Bio-902 and Laxmi. Highest increase in SOD activity was again observed in AlF sensitive cultivar Laxmi and lowest in AlF tolerant cultivar Pusa-Tarak (Figure 4). Table 3. Cardinal points of the regression lines of PPFD dependencies of apparent electron transport rate (ETR) and effective quantum yield(ΔF/Fm ′ ) in 60 days old cultivars (Bio-902, CS-14, Pusa-Tarak and Laxmi) of Brassica juncea treated with different concentrations of AlF (0 + 0, 50 + 25, 100 + 50 and 150 + 75 mg kg −1 ). Indicates critical difference at 0.01%. Table 4. Cardinal points of the regression lines of PPFD dependencies of apparent electron transport rate (ETR) and effective quantum yield(ΔF/Fm ′ ) in 120 days old cultivars (Bio-902, CS-14, Pusa-Tarak and Laxmi) of Brassica juncea treated with different concentrations of AlF (0 + 0, 50 + 25, 100 + 50 and 150 + 75 mg kg −1 ). The values are expressed as mean ± S.D for triplicates (n = 3). The values in parentheses indicate percentage change.

Cultivars of Brassica juncea
a, b and c shows the presence of homozygocity in results (p < 0.001). *Shows statistical significance (p < 0.001).

Effect of AlF on psb A gene expression level
The most tolerant (Pusa-Tarak) and sensitive (Laxmi) cultivars of Brassica juncea showed variable response in psb A gene expression to AlF stress at both the growth stages (vegetative and reproductive) ( Figure 5). The expression of psb A gene was down regulated in both the cultivars i.e Pusa-Tarak and Laxmi during combined stress of AlF, however, down regulation was higher in the cultivar Laxmi at both the growth stages. During vegetative stage the exposure of AlF stress (100 + 50 mg kg −1 ) resulted in down regulation of the psb A gene expression by 2.38 and 5.24 folds in AlF tolerant cultivar Pusa-Tarak and sensitive cultivar Laxmi respectively. Similarly during reproductive stage, the expression of psb A gene was down regulated lesser in the cultivar Pusa-Tarak (1.78 folds) in comparison to the cultivar Laxmi (4.0 folds) at 100 + 50 mg kg −1 concentration of AlF ( Figure 5).

Discussion
Eight cultivars of Brassica juncea (NRCDR-2, CS-14, Laxmi, Vasundhra, Swarn Jyoti, Pusa-Tarak, Bio-902 and RGN-46) were screened for their response to various growth parameters under AlF stress. The cultivar Pusa-Tarak showed lowest reduction in all the growth parameters such as germination percentage, biomass, shoot length, no. of siliquae on main raceme (Yadav et al. 2018), no. of flowers, no. of seeds per siliquae and wt. of 1000 seeds ( Figure S1). Further cultivar Pusa-Tarak showed highest accumulation of Al and F, when subjected to AlF stress. Even the accumulation of Al and F is highest, the reduction in the growth parameters was lowest in Pusa-Tarak compared to other cultivars. Therefore the cultivar Pusa-Tarak is considered as tolerant cultivar. As the cultivar Laxmi showed lowest accumulation of Al and F and largest reduction in growth parameters under AlF stress, it can be considered as the most sensitive cultivar. The accumulation of Al and F as well as some growth parameters viz. germination percentage, shoot length, no. of siliquae and biomass has been investigated earlier by us (Yadav et al. 2018). When comparison was made between the cultivars, we observed that the accumulation of pollutants positively correlated with the biomass of the cultivars (Szczyglowskaet al. 2011, Yadav et al. 2018. In this study, we have demonstrated the adverse effects of AlF stress on antioxidant enzyme activity, photosynthetic pigments, chlorophyll a fluorescence and psb A gene expression in four Brassica juncea cultivars (Bio-902, CS-14,     Rao 2008;Pukachi 2000;Reddy and Kaur 2008;Sabal et al. 2006) cause significant reduction in photosynthetic performance and photosynthetic pigments of plants. Jain et al. (2012) reported that chlorophyll content was reduced in flora under combined stress of AlF. It was observed that photosystem I and II were highly sensitive to AlF − 4 followed by AlF 3 and AlCl 3 in algae (Nostoclinckia) (Rai et al. 1996). However, in plants no such study which shows the effect of AlF on photosynthetic performance, gene expression and antioxidant enzyme activity has been conducted. From this study we have concluded that AlF causes decline in Chl and Car content, ΔF/Fm ′ sat, ETRmax and PPFDsat values of PSII in all the cultivars. The lowest decline for combined AlF stress was observed in Pusa-Tarak followed by CS-14 and Bio-902 whereas maximum decline was observed in the cultivar Laxmi (Tables 1-4). Variation in the chlorophyll a fluorescence parameters and photosynthetic pigment levels of selected cultivars (Bio-902, CS-14, Pusa-Tarak and Laxmi) may be due to the intrinsic differences between the cultivars. It can be assumed from our results that the reduction in photosynthetic performance of all the cultivars is due to the damage of photosynthetic machinery (PSI and PSII) (Mittal et al. 2012) or due to deviation of biochemical processes involving in PSII reaction centers or due to decline in the catalytic activity of Rubisco or due to inhibition of chlorophyll synthesis as it is a key component of photosynthesis in plants (Batra et al. 2014;Haider et al. 2018). The elevation in temperature also results in reduced maximal quantum yield (PSII) of plants (Hasanuzzaman et al. 2013) such as tomato (Morales et al. 2003), Populus (Ferreira et al. 2006) and rice (Han et al. 2009;Vani et al. 2001).
Along with this, the decrease in Chl content can either be considered as a result of its degradation and inhibition of its synthesis (Baunthiyal and Sharma 2014;Mittal et al. 2012) or increase in activity of chlorophyll degrading enzymes and reduction in Fe 2+ ions which are essential for chlorophyll synthesis (Elloumi et al. 2005;Ram et al. 2014). This study concludes that as the concentration and duration of the stress increases, the chl a and b ratio also increases (Tables 1 and  2). Mittal et al. (2012) also reported that the chl a and b ratio increases with an increase in stress concentrations. The ratio of chl a and b indicates the change in PSI and PSII ratio in plants under stressful conditions (Anderson 1986).
The scavenging potential of ROS is associated with antioxidant enzymes activity and the level of mannitol and proline (Xiong et al. 2002). Proline accumulates in response to abiotic stress and it is a source of carbon and nitrogen (Babaei et al. 2017). It provides energy for growth and survival to recover cells (Mittal et al. 2012; and improves stress response of plants (Vardharajulaet al. 2011); thus it is considered as an important non-enzymatic antioxidant. Similar to our results it is also observed that proline accumulation was more in tolerant cultivar in comparison to sensitive cultivar under stress conditions (Rahneshan et al. 2018). Antioxidant enzymes viz. CAT, APX and SOD are efficient scavengers of ROS (Vasconcelos et al. 2009). H 2 O 2 is considered as a stable signal ROS molecule in plants. CAT and APX enzymes can efficiently scavenge H 2 O 2 induced in plants due to stress. However, APX has higher scavenging affinity for H 2 O 2 in comparison to CAT (Chalanika de silva and Asaeda 2017). A significant increase is observed in the activities of ROS enzymes (SOD, POD, CAT, GPX and APX) with respect to abiotic stress (Haider et al. 2018;Vardharajulaet al. 2011). In this study, each cultivar of Brassica juncea also showed a continuous rise in antioxidant activity. The highest proline content, CAT and APX activity was found in AlF-tolerant cultivar i.e. Pusa-Tarak followed by Bio-902, CS-14 and Laxmi (Figures 3 and 4). A significant increase in the activity of SOD was reported in the sensitive cultivar Laxmi followed by Bio-902, CS-14 and Pusa-Tarak (Figures 3 and 4). It may be due to higher production of O 2−· in the cultivar Laxmi in comparision to other cultivars under stress condition, and the conversion of O 2−· into H 2 O 2 by SOD. The scavenging of this H 2 O 2 is not fully possible due to lesser proline content, and the APX and CAT activities in the cultivar Laxmi as the latter are responsible for scavenging of H 2 O 2 . Thus, the accumulated H 2 O 2 causes strong oxidative damage in the cultivar Laxmi. Thus it also seems to be one of the contributing factors towards sensitive behavior of cultivar Laxmi. Our results regarding the activity of SOD are similar with the results of Mittal et al. (2012). During reproductive stage, decrease in CAT activity at highest concentration of AlF i.e. 150 + 75 mg kg −1 was due to the fact that this enzyme is photosensitive (Panda et al. 2003). An increase in activity of CAT, SOD and APX in response to abiotic stress was studied in Cajanus cajan (Divya Sri et al. 2016), tea plants (Ghanati et al. 2005) and maize seedlings (Hamada AbdElgawad et al. 2016). However, highest antioxidant activity such as CAT and APX in response to adverse environment was also shown by tolerant variety (Liu et al. 2008;Mittal et al. 2012).
We also observed the down regulation of psbA gene in Brassica juncea cultivars under AlF stress. The synthesis of D 1 protein of PS II in plants is regulated by psbA gene (Mulo et al. 2012). The down regulation of psbA gene in Pusa-Tarak and Laxmi cultivars might be due to the aggregation of ROS species under adverse conditions, which led to the reduction of net photosynthetic rate and decreased PSII activity ( Figure 5). Earlier studies have also reported that the genes related to photosystems I and II, RuBisCO subunits and Calvin cycle were repressed under adverse environment such as salt (Chaves et al. 2009), drought (Chaves et al. 2009;Degenkolbe et al. 2009;Moumeni et al. 2011) and light (Kimura et al. 2003;Murchie et al. 2005).

Conclusion
AlF toxicity is of major concern in acidic soils worldwide as it decreases crop yield. However, prior to our study, no research related to AlF stress was carried out in plants. On the basis of our results, chlorophyll a fluorescence, enzymatic and non-enzymatic antioxidant activity and gene expression can be used as sensors to monitor and validate tolerant cultivars and can also be applied in ecophysiological studies to estimate crop damage. As evaluated from several parameters such as chlorophyll a fluorescence, proline accumulation, activity of antioxidants and psb A gene expression, the cultivar Pusa-Tarak has been assessed as the most tolerant among the selected four cultivars whereas the cultivar Laxmi is most susceptible to AlF stress. Thus, it can be suggested that the cultivar Pusa-Tarak may possibly be helpful in remediation of Al without reduction in agricultural productivity. Also, the cultivar Pusa-Tarak can be recommended in breeding programs as a germplasm to develop new tolerant cultivars against AlF.