Plant growth promoting rhizobacteria induced Cd tolerance in Lycopersicon esculentum through altered antioxidative defense expression
Introduction
Cadmium is a highly toxic heavy metal that exists in the environment due to its bioaccumulation in the food chain (Gall et al., 2015). It enters the environment through man-made sources such as the weathering of rocks, fertilisers, sewage sludge, the burning of fossil fuels, mining and smelting (Zoffoli et al., 2013). Cd triggers physiological changes by causing negative effects on growth, chlorophyll content, mineral uptake, seed germination, photosynthesis, transpiration, oxidative phosphorylation and water transportation (Gallego et al., 2012, Azevedo et al., 2012, Gratao et al., 2009). It also inhibits the electron transport chain and membrane integrity and may lead to the depolarisation of cytoplasmic membranes resulting in disturbance of cellular homeostasis (Fojtova and Kovarik, 2000; Chen et al., 2001a, Chen et al., 2001b). These series of events occur primarily due to the production of ROS species leading to lipid peroxidation, disruption of membrane integrity, inhibition of various enzymatic activities and nuclear damages (Dourado et al., 2015, Gratao et al., 2015). The adverse effects of ROS can be overcome by cellular defence systems within plants that are comprised of antioxidant enzymes related to the Asada Haliwell pathway and non-enzymatic antioxidants (Ahmad et al., 2010, Ahmad et al., 2011, Boaretto et al., 2014). Quantitative and qualitative studies on various plants revealed the active involvement of antioxidants in combating metal stress. Gene expression analysis further supported the participation of antioxidants. Studies on rye grass under Cd-induced toxicity revealed an overexpression of various antioxidative genes (Mn/SOD, FeSOD POD, CAT, APX and GR) (Luo et al., 2011). Moreover, overexpression of FeSOD genes (FSD1, FSD2 and FSD3), CuSOD genes (CSD1, CSD2 and CSD3) and MnSOD gene (MSD1) were also reported during abiotic stress in A. thaliana (Kliebenstein et al., 19990). Furthermore, studies with enhanced expression of OsAPX1, OsAPX2, OsAPX3, OsAPX4, OsAPX5, OsAPX6, OsAPX7 and OsAPX8 genes was reported in O. sativa plants exposed to salt stress (Hong et al., 2007). In addition, non-enzymatic antioxidants such as ascorbic acid (ASA), tocopherol and glutathione (GSH) were found to participate in the internal detoxification of ROS in cells (Noctor and Foyer, 1998, Dai et al., 2012).
The soil consists of a plethora of PGPRs (plant growth-promoting rhizobacteria) that exert favourable effects on plant growth and development (Kloepper and Schroth, 1978). Recent studies in the area of environmental management advocate the use of PGPRs to ameliorate heavy metal stress in plants (Ma et al., 2010, Sousa et al., 2012, Rajkumar et al., 2012). PGPRs develop resistance or tolerance towards these metals through intrinsic or induced mechanisms that enable them to grow under elevated concentrations of metals (Lakzian et al., 2002, Giller et al., 1998). PGPRs have evolved various mechanisms by which they mobilise and solubilise the metals (Madhaiyan et al., 2007). They primarily include exclusion, extrusion, accommodation (complex formation), biotransformation, methylation and demethylation of metals (Kao et al., 2006, Umrania, 2006). These mechanisms allow the microbes to survive and actively function in metal-stressed conditions, rendering them metal resistant. They also enhance the production of different phytohormones, such as auxins, cytokinins and gibberellins (Gupta et al., 2002). In addition, they produce siderophores and enhance phosphate solubilisation within plants (Zaidi and Khan, 2007). The Burkholderia genus is known to symbiotically act in the rhizosphere and promote plant growth by competing against pathogens (Compant et al., 2010), bioremediating xenobiotics and enhancing phosphate solubilisation in plants (Luvizotto et al., 2010, Payne et al., 2006). In addition, the Pseudomonas genus also plays a critical role in the detoxification of different contaminants present in the environment, thereby improving plant growth and metabolism (Wasi et al., 2013).
In addition, the associations of these microbes resulted in enhancing plant productivity, as well as their defence systems, by modulating their antioxidant potential (Garg and Aggarwal, 2012, Sathyapriya et al., 2012). PGPRs can induce detoxification mechanisms by increasing root hair production, metal availability and its solubilisation along with an improvement in the antioxidative defence system of plants under stressful conditions (Sessitsch et al., 2013). Consequently, the abilities of PGPRs to resist metal stress in plants make them most suitable candidates for antioxidative defence studies.
Lycopersicon esculentum (tomato) is an important commercially consumed crop that is the second largest in the world. Different PGPRs reside symbiotically within the rhizosphere of tomato plants and tend to improve the growth characteristics under usual as well as unusual conditions. These microbes also perform the bio-amelioration of stress and alleviate stress-induced changes within the plants by strengthening their antioxidative potential (Hashem et al., 2016).
The objectives of the present study were to examine the stress protective potential of Pseudomonas aeruginosa and Burkholderia gladioli in enhancing the antioxidant potential of L. esculentumseedlings grown under Cd stress. The oxidative stress generated by Cd was assessed through the accumulation of superoxide anions, hydrogen peroxide and malondialdehyde. In addition, different levels of antioxidants (enzymatic as well as non-enzymatic), total antioxidant capacities (water soluble and lipid soluble) were also assessed. Moreover, gene expression profiling of 10-day-old L. esculentum seedlings exposed to Cd metal stress (0.4 mM) was also done using qRT-PCR to characterise the role of PGPRs in antioxidative defense expression.
Section snippets
Microbial inoculations
Two different bacterial strains (Pseudomonas aeruginosa (M1); MTCC7195) and Burkholderia gladioli (M2); MTCC10242) obtained from IMTECH (Mohali, India) were cultured independently in conical flasks containing 50 mL of nutrient broth (13 gL-1) media for 24–48 h at 28 °C in a BOD incubator (Caltan (Deluxe Automatic), New Delhi, India). These isolates were sub-cultured and maintained for additional applications. One millilitre of a pure-culture of bacteria (109 cells mL−1) was cultivated for
Oxidative damage
In this study, oxidative stress damage was estimated using the superoxide anion, H2O2, along with the MDA levels. Cd toxicity resulted in a dramatic increase in the levels of the superoxide anion (136.9%), H2O2 (378.5%) and MDA content (135%) in 0.4 mM Cd-treated plants in contrast to the control plants. Supplementation with P. aeruginosa (M1) reduced the levels of the superoxide anion (17.79%), H2O2 (12.87%) and MDA (16.53%) in Cd-treated tomato seedlings. In addition, augmentation with
Discussion
Plants possess specific characteristics to combat the oxidative stress generated by heavy metals through direct or indirect sequestration and modulating their antioxidative potential (Mendoza-Cozatl et al., 2011, Anjum et al., 2015, Dheeba et al., 2014). Under these conditions, they form mutualistic associations with PGPRs that lead to the mitigation of heavy metal stress along with the modulation of their antioxidative potential (Madhaiyan et al., 2007, Garg and Chandel, 2010, Islam et al.,
Conclusion
Cd toxicity in tomato resulted in oxidative stress and the up/downregulation of antioxidative defence responses, including enzymatic and non-enzymatic antioxidants. However, this oxidative damage caused by Cd can be mitigated by the association of microorganisms that can promote heavy metal detoxification. In addition, these microorganisms amend the activities of ROS-quenching and balance their homeostasis, and therefore, preclude plant dysfunction and increases its survival under stressful
Conflicts of interest
No conflict of interest exists among the authors.
Acknowledgements
The research work was supported by the University Grant Commission, Government of India, GOI (UPE-scheme) and DST-FIST, of GOI. The authors would like to extend their sincere appreciation to the Deanship of Scientific Research, King Saud University for its funding to the Research Group number (RG-1435-014).
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