Effect of Salicylic Acid on the Growth, Photosynthetic Efficiency and Enzyme Activities of Leguminous Plant under Cadmium Stress

The present study was designed with an objective to elucidate the effect of the exogenous application of 10 M of salicylic acid (SA) to the foliage of the chickpea plants exposed to 0, 25, 50 or 100 mg cadmium (Cd) per kg of soil. The foliage of the plants grown in soil amended with varying doses of Cd was sprayed with 10 M of SA at 30 days after sowing (DAS). The plant samples were collected at 60 and 90 DAS to assess various parameters. The damage caused due to the Cd exposure was partially overcome by the exogenous application of SA. The SA treatment resulted in a significant increase of growth, photosynthesis, yield and the activity of antioxidative enzymes viz. catalase, peroxidase and superoxide dismutase in the plants exposed to 0, 25 or 50 mg Cd per kg of soil. However, the treatment did not prove to be fruitful in alleviating completely the stress generated by 100 mg Cd per kg of


Introduction
Metal contamination issues are becoming increasingly throughout the world (Foy et al., 1978), with many documented cases of metal toxicity including in mining and agriculture practices. Metals are a natural part of terrestrial systems occurring in soil, rock, air, water and organisms. A few metals, including Cu, Mn and Zn, are however essential to plant metabolism in trace amounts. It has potential to become toxic to plants when metals are present in bioavailable forms at excessive levels. On the other hand, Cd is a non-essential trace element for plants, and has strong toxicity at low concentrations with the ability to readily absorb by roots (Sanita di Toppi and Gabbrielli, 1999). It can suppress the elongation growth of plant cell, inhibit oxidative mitochondrial phosphorylation, induce oxidative stress, inhibit the activities of several antioxidative enzymes, affect photosynthesis by inhibiting ferrous reductase or damaging photosynthesis apparatus, and cause the alteration of chromatin and the change of plasma membrane ATPase activity (Tran and Popova, 2013). Cadmium also causes various direct and indirect effects on plant growth and metabolism (Hasan et al., 2009) by forming complexes with O, N and S ligands (Van Assche and Clijsters, 1990). It interferes with protein metabolism,membrane functioning and water relations (Irfan et al., 2013). Cadmium inhibited net photosynthesis in Cajanus cajan (Sheoran et al., 1990) and Phaseolus vulgaris (Krupa et al., 1993), O2 evolution in Anacystis nedulens and photosystem II (PS II) in isolated chloroplasts of maize and spinach (Bazzas et al., 1974). Moreover, they cause metabolic disturbance by altering essential biochemical reactions (Hermans et al., 2004). Exposure to Cd stress disrupt cellular homeostasis and enhances the production of several activated oxygen species designated as reactive oxygen species (ROS) such as superoxide (O2 ·− ), hydroxyl radicals (OH · ), singlet oxygen ( 1 O2), and hydrogen peroxide (H2O2) that are produced continuously by plants (Foyer and Noctor, 2003). ROS have been shown to damage cell membranes, proteins, lipids, and DNA (causing inter alia DNA base oxidation, DNA protein cross-links, DNA gaps, and breaks) resulting in lipid peroxidation (Skórzyńska-Polit and Krupa, 2006), developmental defects, and genetic instability in plant species (Bal et al., 2000). However, plants possess very efficient enzymatic and non-enzymatic antioxidant defense systems that allow scavenging of ROS and protection of cell to oxidative damage (Gratão et al., 2005).
Salicylic acid (SA) is a phenolic compound synthesized throughout the plant kingdom via the phenylpropanoid pathway (Métraux, 2002). Over the past decade, various workers documented that this molecule plays vital roles in many physiological processes of plants and also have ability to confer tolerance against various abiotic stresses Hayat et al., 2013). SA application at a concentration of 0.1 or 0.2 mM reduced the inhibitory effect of Pb 2+ and Hg 2+ on the seed germination and seedling growth of two rice (Oryza sativa L.) cultivars (Mishra and Choudhuri, 1997). SA increased the fresh and dry mass of shoots and roots in both cultivars under heavy metal stress conditions. The higher concentration of SA was more effective, as evident from a better recovery from metal-induced growth inhibition. SA moderated the inhibitory effect of lead on the activity of the nitrate reductase enzyme in maize (Zea mays L.) plants (Sinha et al., 1994). This suggests that SA has some role in the tolerance of plants to heavy metal stress, though other authors found no increase in the endogenous SA content as the result of Cd treatment, or any difference in the SA content of sensitive and resistant plants of Salix viminalis in the control (Landberg and Greger, 2002).
On the basis of above reports, the primary objective of this work was to examine whether or not SA could mitigate the Cd-induced oxidative stress in legumes by regulating the antioxidant defense system and also dissect the efficiency of photosynthetic machinery under Cd stress in the presence as well as absence of SA.

Materials and methods
The certified seeds of Cicer arietinum L. cv. 'Avarodhi' were purchased from the Chola Beejh Bhandar, Aligarh, India. The seeds were surface sterilized with 0.01% mercuric chloride solution followed by inoculation with Rhizobium and were sown in five sets of earthen pots (10 inch diameter) filled with sandy loam soil and farmyard manure (6:1) arranged under a simple randomized block design. At the start of the experiment, out of these five sets of prepared pots, four sets were supplemented with different doses (0, 25, 50 or 100 mg per kg of soil) of Cd in the form of CdCl2 respectively and one set of pots was left untreated serving as control. At the stage of 30 DAS, the foliage of the resulting plants was sprayed with 10 -5 M of salicylic acid (SA), except control which received double distilled water (DDW) instead of SA. The concentration of SA was selected on the basis of our earlier experiments (Hayat et al., 2010). The plant samples were collected at 60 and 90 DAS to assess various growth and physiological parameters.

Plant growth analysis
The plants were uprooted and washed under running tap water. The root and shoot length of these plants was measured with the help of a graduated scale. These plants were blotted in blotting sheets to remove the adhering water and weighed on electronic balance to record their fresh mass. These plants were kept in an oven run at 80 °C for 72 hours and then weighed to obtain their dry mass.

Carbonic anhydrase (CA) activity
The CA activity in the leaves was measured by following the method described by Dwivedi and Randhawa (1974).

Assay of antioxidative enzymes
The activities of peroxidase (POX) and catalase (CAT) were analysed as per the method described by Chance and Maehly (1956) whereas the superoxide dismutase (SOD) was measured by the method of Beauchamp and Fridovich (1971).

Proline content in leaves
The proline content in the fresh leaf sample was measured following the method described by Bates et al. (1973).

Yield Characteristics
At harvest (160 DAS), three plants from each treatment were randomly sampled and counted for the number of pods per plant. The pods from five plants, representing each treatment, were crushed, cleaned to assess the seed weight per plant and 100 seed mass.

Seed protein content
The total protein content in the dry seeds, at harvest, was estimated by adopting the method of Lowry et al. (1951).

Statistical analysis
Each observation was replicated three times. The treatment means were compared by analysis of variance using SPSS software version 10 (SPSS, Chicago, IL, USA). Least significant difference (LSD) was calculated at 5% level of probability. Standard error (SE) due to replicates was also calculated.

Length of root and shoot
Foliar application of 10 -5 M of SA significantly increased the length of root and shoot by 33.3%, 40.0% and 29.4%, 30.2% in unstressed plants, 22.5%, 26.1% and 16.7%, 18.0% in the plants exposed to Cd at the rate of 25 mg per kg of soil and 13.0%, 14.5% and 7.5%, 8.2% in the plants exposed to a Cd stress of 50 mg per kg of soil, at 60 and 90 DAS, respectively, over their controls (Tab. 1). However, the spray proved to be inefficient in alleviating the stress generated by Cd, supplemented at the rate of 100 mg per kg of soil.
Fresh and dry mass per plant Exogenous application of 10 -5 M of SA resulted in a significant increase of 44.4%, 46.9% and 64.5%, 64.9% in the fresh and dry mass of unstressed plants and 17.8%, 21.2% and 19.3%, 13.5% in those exposed to Cd supplemented at the rate of 25 mg per kg of soil, at 60 and

Carbonic anhydrase (CA) activity in leaves
The plants received 100 mg Cd and also sprayed with SA exhibited a lower activity of CA compared to the control, at both the sampling stages (Tab. 3). The application of SA to the foliage, however, resulted in a significant increase of 47.3%, 58.0% (unstressed); 28.1%, 33.9% (25 mg Cd) and 12.9%, 13.9% (50 mg Cd) at the two sampling stages, respectively, in the activity of CA compared to the control (Tab. 3).

Photosynthetic parameters
The foliar application of 10 -5 M of SA resulte Fig. d    Antioxidative enzyme activities Application of 10 -5 M of SA to the foliage of unstressed plants resulted in a significant increase of 14.9%, 17.1% (CAT); 30.3%, 34.9% (POX) and 27.7%, 30.0% (SOD) at 60 and 90 DAS, respectively, compared to the control (Tabs. 6-7). Further, when SA was applied exogenously to the Cd stressed plants, a sharp increase in the activity of these antioxidant enzymes was observed that was proportionate with the concentration of the metal. The activity of CAT, POX and SOD was found to be highest under the influence of exogenous SA in the plants fed with Cd at the rate of 100 mg per kg of soil, showing a significant increase of 33.4%, 38.1%; 85.6%, 95.2% and 61.3%, 70.0% at 60 and 90 DAS, respectively, compared to the control. The values revealed for these parameters, in the plants fed with Cd at the rate of 25 mg per kg of soil followed by a foliar spray of SA was found to be significantly higher by 20.5%, 23.8%; 40.9%, 43.8% and 36.5%, 40.0% at 60 and 90 DAS, respectively, over control (Tabs. 6-7).

Proline content in leaves
The exogenous application of SA significantly increased the endogenous level of proline over control, irrespective of the Cd concentration used (Tab. 7). The endogenous proline level in unstressed plants was found to be 50.0% and 48.2% higher at 60 and 90 DAS, respectively, compared to the control, in response to exogenous SA. Further, the proline level in response to the foliar applied SA, increased linearly with increasing concentration of Cd and was found to be 57.0%, 56.8% (25 mg Cd); 68.1%, 62.0% (50 mg Cd) and 92.4%, 90.6% (100 mg Cd) higher, compared to the control at 60 and 90 DAS, respectively (Tab. 7).
Seed yield, 100 seed mass and seed protein content The exogenous foliar application of 10 -5 M of SA significantly increased the seed yield, 100 seed mass and seed protein content in unstressed plants by 108%; 56.8% and 14.0% respectively, over control (Tab. 8). Further, the SA treatment to the plants already fed with Cd (25 or 50 mg per kg soil) significantly increased the values of seed yield, 100 seeds mass and seed protein content over that of control. The percent increase recorded in these parameters was found to be 53.9%, 40.9% and 8.0% (Cd 25 mg) and 15.0%, 20.3% and 5.0% (Cd 50 mg), respectively, over that of the control. However, the foliar spray of SA to the plants already supplemented with 100 mg Cd exhibited 20.0%, 5.0% and 8.8% reduction in seed yield, 100 seeds mass and seed protein content, respectively, compared to the control (Tab. 8).
Beside this promising response of SA on photosynthesis, SA has ability to conferred tolerance to barley seedlings and maize plants against oxidative stress induced by Pq (Paraquat) and Cd respectively (Krantev et al., 2008). Accumulation of reactive oxygen species i.e. oxidative stress that cause extensive damage including lipid peroxidation, chlorophyll breakdown, loss of photosynthetic activity and membrane integrity, as well as electrolyte leakage. However, Krantev et al. (2008) observed in maize plants that pretreated with 0.5 mM SA before exposure to 10-25 mM cadmium, protection of photosynthesis conferred by SA could be the result of a very rapid detoxification of ROS. It has been demonstrated in different plants species that pretreatment with low concentrations of SA enhances tolerance toward most kinds of abiotic stresses due to an enhanced antioxidant capacity . Similar response was observed in the present study where SA (10 -5 M) significantly increased the antioxidant enzymes (CAT, POX, and SOD) compared to control (Tabs. 6-7) in the presence and absence of Cd along with increased accumulation of proline in SA treated plants (Tab. 7). Moreover, proline plays vital role in wide range of protective responses including osmotic adjustment, stabilizer for cellular structure and reducing damage to photosynthetic apparatus (Nounjan et al., 2012). Hossain et al. (2011) demonstrated that exogenous proline provided a protective action against abiotic stress-induced oxidative damage by reducing H2O2 and lipid peroxidation level and by enhancing antioxidant defense and methylglyoxal detoxification systems. In addition to this, exogenous proline also prevents protein aggregation and stabilization of M4 lactate dehydrogenase during extreme temperatures, protection of nitrate reductase during heavy metal and osmotic stress (Szabados and Savouré, 2010).
The accelerated photosynthesis and its related attributes, enhanced proline accumulation coupled with the increased activity of antioxidative enzymes in Cd stressed plants in the presence of exogenously applied 10 -5 M of SA is naturally expected to increase the growth of plants which was reflected in the form of increased length, fresh and dry mass of root and shoot per plant (Tabs. 1-2). Further, the enhanced growth characteristics of Cd fed plants sprayed with 10 -5 M of SA might also be due to the fact that SA acts at the level of transcription and/or translation thereby increasing the activity of various other enzymes necessary for growth of plants (Hayat et al., 2010). Moreover, SA also

Number of pods per plant
Application of SA to the foliage of unstressed plants resulted in a significant increase of 57.8% (number of pods) over control. Further the treatment also proved to be fruitful in increasing the number of pods in the plants fed with Cd (25 or 50 mg) and also received SA where a significant increase of 32.4% and 13.0% in the number of pods per plant was observed respectively over control. Further, like other parameters the foliar application of SA proved to be inefficient in alleviating the stress generated by Cd (100 mg), where 11.7% reduction in this parameter was observed compared to the control (Tab. 8).
Tab. 8. Effect of 10 -5 M of salicylic acid on the Cd (0, 25, 50 or 100 mg per of soil) induced changes in yield characteristics in chickpea. Data are the mean of three independent replicates. LSD is the least significant difference promotes cell division and cell enlargement . This increased growth under the influence of exogenous SA is likely to increase the yield characteristics (Tab. 8). The plausible reason for increasing the crop yield might be due to delayed senescence of plant organs (particularly leaves and flowers) in response to exogenous SA (Imran et al., 2007) that will automatically help the plant in extending the duration of photosynthetically active sites and also prevent the premature loss of flowers and fruits. This consequently resulted in the observed increase in the number of pods per plant (Tab. 8). It gets additional support from the observations of Marschner (2012) that phytohormones increase the degree of sink at the level of seeds, directing the flow of metabolites to the developing seeds increasing the seed protein content (Tab. 8) consequent to an improvement in the seed mass (Tab. 8) and seed yield per plant at harvest (Tab. 8).

Conclusions
The present investigation revealed that exogenously applied 10 -5 SA plays significant role to protect the photosynthetic machinery under Cd-stressed plants. In addition to this, SA also enhanced the activities of antioxidant enzymes and proline accumulation that confer tolerance to Cd-stressed plants and enhanced the yield characteristics.