Keywords

12.1 Introduction

Among different abiotic stresses, the effect of heavy metal accumulation is known to be most hazardous factor in environment. Different heavy metals like Zn, Cd, Hg, As, Cu, Pb etc. are the interminable and bio accumulating elements which are highly toxic to living organisms and micro-organisms (Azevedo et al. 2012). These can enter the environment through natural or anthropogenic sources (Zawoznik et al. 2007). Metals can enter the plant roots via appoplastic or symplastic mechanisms which can further be translocated towards the shoot (Lux et al. 2011). They can lead to structural as well as biochemical changes like changing the redox state of the plant thereby inducing the oxidative stress and damaging membrane integrity (Gratao et al. 2009). Moreover, they also undergo physiological changes within the plants such as photosynthesis, mineral uptake and water relations (Gill et al. 2012).

In addition to this, they stimulate ROS production such as hydroxyl radical (OH·), superoxide radical (O2 •) and hydrogen peroxide (H2O2) (Benavides et al. 2005). ROS can stimulate lipid peroxidation by altering membrane lipids and affecting membrane permeability and fluidity (Tian et al. 2012). Accordingly, this causes ROS scavenging mechanisms to get accelerated. Plants possess different mechanisms to protect themselves against metal attack such as through the production of metal chelators and activation of different antioxidative defense mechanisms (Kopittke et al. 2010). Alternatively, some key enzymes involved in ROS detoxification such as superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) get activated (Gratao et al. 2008; Roychoudhury et al. 2012). Other non-enzymatic antioxidants like ascorbic acid, glutathione and secondary metabolites (flavonoids, organic acids, phenols and carotenoids) are also involved in ROS detoxification (Foyer and Noctor 2009).

Plant possess specific mechanism for detoxification and partitioning of metals between roots and shoots which could be directly or indirectly related to sequestration of metals between them (Mendoza-Cozatl et al. 2011). In this condition, they may establish symbiotic relationship with microbes from which they could be benefited by alleviation of the toxic metals along with the elevation of antioxidative responses of the plants (Garg and Chandel 2010; Sousa et al. 2012). These interactions could further be involved in enhancing plant productivities and their adaptive functions (Andrews et al. 2012). Microbial inoculations in metal stressed plants are proved to be involved in improving their growth and antioxidant defense system. According to Islam et al. (2014a, b), Pseudomonas aeruginosa stimulated antioxidant enzymatic activities of SOD, POD and CAT which further decreased MDA and H2O2 levels through ROS scavenging under Zn stressed wheat plants. Similar studies were found in Solanum nigrum under stress where P. aeruginosa (ZGKD5, ZGKD2) upregulated CAT, SOD, POD and APX levels and alleviated oxidative stress generated (Shi et al. 2016). Moreover, improved antioxidative defense system in wheat under Cu stress was reported by Bacillus inoculation through increased activities of SOD, POD, CAT, DHAR, APX by eliminating H2O2 and superoxide radicals in plants (Wang et al. 2013). Further, Madhaiyan et al. (2007) reported that plant growth promoting bacterial strains; Methylobacterium oryzae and Burkholderia sp. isolated from rice enhanced the growth of tomato plant by alleviating Cd stress. For instance, many microbial strains are also involved in modulating the activities of phenolic compounds and PAL (phenylalanine ammonia lyase) under metal stressed conditions (Mollavali et al. 2016). The addition of plant growth promoting bacteria to Cr stressed Zea mays L. induced phenolic contents within the plants. This combinational study showed that phenolic compounds act as metal chelators and elevated upon addition of microbial consortium (Islam et al. 2016a, b). Along with these important aspects, the indirect role of many other metabolites such as low molecular weight organic acids exuded by microbes have been elucidated in nutrient mobilization and metal solubilisation in rhizosphere (Rajkumar et al. 2012). The production of organic acids like oxalic acid, maleic acid, tartaric acid, succinic acid and formic acid by plant associated micro-organisms have gained much more attention in increasing metal solubilisation. Further, studies have been found in which they also increased the levels of enzymatic and non-enzymatic antioxidants under Cr treated B. juncea plants (Mahmud et al. 2017).

Recent studies have demonstrated the importance of arbuscular mycorrhizal fungi (AM fungi) in alleviating metal toxicity effects in plants (Andrade et al. 2010). The presence of AM fungi Glomus vesiforme in Lonicera japonica revealed the correlation between the intensity of mycozzhizal infection and increased stimulation of enzymatic activities of CAT, APX, GR and elimination of oxidative stress generated by Cd (Jiang et al. 2016). Furthermore, AM fungi also enhanced defensive pathways in plants by stimulation of antioxidants. Such type of studies were found by Bhaduri and Fulekar (2012) in which AM fungi elevated enzymatic activities in Cd treated Ipomoea aquatica plants. Moreover, reduced oxidative damage was observed by inoculation of Funneliformis mosseae in Solanum nigrum under Cd stress (Jiang et al. 2016a, b). In addition, Glomusmossae increased the stimulation of glutathione in response to Pb and Cd stress in Cajanus cajan L. The present chapter intends to increase our knowledge on the role of micro-organisms in modulating the antioxidative defense system in plants as well as metabolites leading to plant defence and heavy metal detoxification.

12.2 Heavy Metal Pollution

Rapid rise in world population rate lead to continued industrialization worldwide resulting in enormous environmental problems. Natural resources such as soil, water and air shows contamination of heavy metals, organic solvents, pesticides etc. (Prasad et al. 2010). In nature low concentration of heavy metals are present (Rodriguez Martin et al. 2013) via weathering of parent material of soil, volcanic eruptions, atmospheric deposition, mineralization, erosion etc. (Zhang et al. 2011) but this heavy metals proves fatal when its concentration rises from required amount by anthropogenic activities which include industrialization, mishandling of waste, agricultural chemicals (Zhao et al. 2008; Xu et al. 2014). There are many ways by which heavy metal enters the environment and once they are introduced, they travel great distances from their original source thus contaminating ecosystem far and wide (Oves et al. 2016) resulting in heavy metals deposition in soil which leads to nutrient deficiency and are likely to make land barren. Few reports of different sources of heavy metals are mentioned in Table 12.1. Urbanization and industrialization near coastal areas results in heavy metal pollution (Xu et al. 2016) affecting the flora and fauna of the habitat. Anthropogenic activities increase the frequency of deposition of heavy metals which in turn directly or indirectly affect flora and fauna of the habitat (Wang et al. 2015) and our environment (Sayadi 2014). Enhanced level of heavy metals like chromium, nickel, cadmium, arsenic, mercury, lead, copper, silver, zinc affect human health also by acting as neurotoxic, cytotoxic, mutagenic and carcinogenic agents (Ahmad et al. 2016a, b).

Table 12.1 Different sources of heavy metals
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12.3 Metal Toxicity Effects on Plants

In response to metal toxicity, plethora of physiological and biochemical phenomena are altered (Villiers et al. 2011; Gill 2014). Reduction in growth is the primary visible symptom of metal stress (Sharma and Dubey 2007). Other common symptoms include chlorosis of leaves, change in water balance, decline in rate of seed germination and disruption of photosynthesis as shown in Fig. 12.1 (DalCorso et al. 2010; Kohli et al. 2013, 2017).

Fig. 12.1
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Metal toxicity symptoms in plants

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The severity of metal toxicity is attributed to the chemical composition and behaviour of metal in plants system. The toxicity of metal is dependent upon several factors including: (i) functional sites on protein moieties, (ii) disrupted enzyme functioning, (iii) elevation in synthesis of ROS and (iv) activation of fenton reaction (Halliwell 2006; Keunen et al. 2011). Plants have to maintain homeostasis between essential and hazardous concentration of metal ions in order to attain an ionic balance at cellular, tissue and organ level (Ovecka and Takac 2014). The early symptoms of metal toxicity in plants also include generation of Reactive Oxygen Species (ROS) resulting in oxidative stress (Maksymiec et al. 2007; Sharma and Dietz 2009). Oxidative stress is generally described as imbalance between generation of ROS and their scavenging by antioxidative defense components (Keunen et al. 2011). ROS are synthesized in all aerobic organisms and are one of the imperative signaling molecules in plant system (Foyer and Noctor 2005). The enhancement in ROS levels results in DNA damage, conformational changes in metabolites including protein, carbohydrates and lipids (Borsetti et al. 2005).

O2 (molecular oxygen) in the atmosphere acts the final electron acceptor resulting in formation of ROS such as hydrogen peroxide (H2O2), hydroxyl radicle (OH.) and superoxide anion (O2 ·−) in plants (Temple et al. 2005; Scandalios 2005). Most of the reactive O2, intermediates the oxidation of O2 to singlet oxygen (Triantaphylides et al. 2008). The OH radicle have a non-selective reactivity whereas, O2 − and H2O2 have specific reactivity towards biological macromolecules (D’Autreaux and Toledano 2007). Hydroxyl ions are the most reactive and harmful form of ROS. Another important ROS is nitric oxide (NO), which leads to formation of peroxynitrile radicals (OONO−) by reacting with molecular O2 (Gill and Tuteja 2010). Few other ROS includes peroxy, alkoxy and perhydroxyl radical etc. (Bhattacharjee 2005). The metal ions which are non-redox active in nature don’t increase the production of ROS, although they inhibit activity of certain essential enzymes of the antioxidative defense system (Schutzendubel and Polle 2002). The highly oxidizing nature of ROS and its ubiquitous presence in peroxisomes, mitochondria and chloroplast indicates the predominant effect on photosynthesis and activation of antioxidative defense system (Seth et al. 2012). Figure 12.2 represents diagrammatic representation of heavy metal induced oxidative damage and repair.

Fig. 12.2
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Schematic representation of heavy metal induced oxidative damage and repair

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12.4 Mechanism of Enhanced Antioxidative Defense by Microbes

Plant- microbe interactions have been widely employed in enhancing antioxidant defense systems in plants. They enhance resistance in plants towards abiotic stresses also known as IST (Induced Systemic Tolerance). They have been reported to induce genetic as well as metabolic potential in alleviating abiotic stress in plants. The modulating actions of different microorganisms in plant defense system by alteration of different antioxidative enzymes under abiotic stress have been studied in recent past. However, the inoculation of different microorganisms in the rhizosphere effects the production of different metabolites in the root region as well as inside the plants. The effect of on some of the important metabolites during plant-microbe interaction has been discussed below:

12.4.1 Antioxidant Enzymes

Plant possesses a comprehensive and a complex system of antioxidative enzymes. This system of enzymes ameliorates toxic effect of wide array of stresses (Bano and Ashfaq 2013). The antioxidative enzymes include SOD, POD, APOX, GR, GPOX, GST, CAT, PPO, DHAR and MDHAR. The activities of several antioxidative enzymes are lowered in response to elevation in content of ROS by metal stress (Gill and Tuteja 2010). The enhancement in the metal toxicity results in elevation of levels of ROS consequently leading to disruption of metabolic pathways; cellular structures and functions are irreparably altered (Gratao et al. 2005). One of the most efficient methods for removal of toxic metals from soil includes use of microbes. Microbes play a critical role in remediation of heavy metals pollution from the soil and plant system. There exists a wide range of reports of microbes assisted modulation of antioxidative defense activities in pants under the effect of severe metal toxicity (Pandey et al. 2013; Ahmad et al. 2016a, b; Islam et al. 2016a, b). Few reports have been tabulated in Table 12.2.

Table 12.2 Effect of microbes upon antioxidative enzyme system in metal stressed plants
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Recent developments in the field of microbiology and environment management indicate use of microbes present in the rhizosphere or inoculation in the soil and further elucidate their crucial role in enhancing the antioxidative defense system of plants under heavy metal stress (Ma et al. 2010; Wang et al. 2011; Rajkumar et al. 2012). Bacterial inoculation of Ricinus communis in Helianthus annus plants exposed to Ni stress were reported to have enhanced activity of CAT and POD enzymes (Ma et al. 2010). Certain microbes have an ability to activate the antioxidative defense response endogenously in response to heavy metal stress. One such example is number of Lactococcus spp. and Streptococcus spp. which have been reported to have enhanced activities of Mn SOD (Poyart et al. 2002). Mycorrhizal fungi are an imperative class of heavy metal phytostabilizers. Plants which have an association with mycorrhizal fungi in their roots sequester huge range of metal pollutants in fungal hyphae and vesicles of the root system resulting in their immobilization as well as lowering their inhibitory actions. It was further suggested that, P and other essentials micronutrients uptake was also enhanced resulting in further enhancement in activities of antioxidative defense system including CAT, SOD and POD (Garg and Aggarwal 2011; Bano and Ashfaq 2013). A specific category of rhizobacteria includes plant growth promoting rhizobacteria (PGPR) have been reported to accelerate the phenomena of phytoremediation by modulating bioavailability of metal ions, oxidative damage, synthesis of phytohormones, siderophores interactions and binding of metal ions to the chelators (Ma et al. 2011a, b). A large number of studies suggest synthesis of several antioxidant compounds in response to endophytic fungi inoculation in plants resulting in enhancing the tolerance of plants (Malinowski and Belesky 2006; Yuan et al. 2010). Another report by Huang et al. (2007) suggested elevated antioxidative capacity and phenolic compounds of the host plants by endophytic fungi. This indicated that these endophytic fungi might possibly produce phenolic antioxidants.

12.4.2 Antioxidants

Plants possess different strategies in order to cope with different kinds of metals. The primary response of plants to metal stress is the generation of ROS such as O2 −, H2O2 and HO (Gill et al. 2010; Yadav 2010). As a result, the plant cells respond defensively to oxidative damage by removing ROS and maintaining antioxidant defence compounds at levels that reflect ambient environmental conditions. The antioxidative system consists of both enzymatic and non-enzymatic antioxidants. Among non-enzymatic glutathione (GSH), ascorbic acid (ASA), tocopherol play major role in modulating defensive mechansims within plants (Mittler et al. 2004; Halliwell 2006; Scandalios 2005).

12.4.2.1 Glutathione

Glutathione (GSH; γ-glu-cys-gly) is the main source of non-protein thiols and are biochemically active in plants against different types of stresses. It plays a central role as chelating agent, antioxidant and signalling component and exists in both reduced (GSH) and oxidised (GSSG) forms (Jozefczak et al. 2012). Non enzymatic GSH is the first defence pathway in plants under stressed conditions. GSH after donating electron to ROS gets converted to GSSG and can be regenerated by action of GR at the expense of NADPH. In healthy cells more than 90% of the total GSH pool is in its reduced form but after metal treatment reduced GSH concentration is decreased (Mittler et al. 2004). Oxidation of several antioxidant pathways are interconnected with GSH. ASA-GSH cycle is second defence pathway in which AsA and GSH are oxidized and reduced to permit AsA peroxidise to neutralize H2O2 to H2O as shown in Fig. 12.3 (Jozefczak et al. 2012).

Fig. 12.3
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Oxidised and reduced ASA-GSG forms resulting in conversion of H2O2 to H2O

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Moreover, GSH quenches ROS generated in plants under heavy metal exposure. Along with this, it also enables phytochelatin synthesis in plants (Yadav 2010). It has been reported that GSH synthesis was enhanced in Arabidopsis plants under Cd and Ni stress (Freeman et al. 2004). Studies have been reported in which GSH forms complexes with metals and enables their transport across membranes. In this mechanism, AtATM3 plays important role, that mediates GSH-Cd transport along with mitochondrial membranes in Arabidopsis thaliana (Kim et al. 2006). The interaction between metal hyperaccumulator plants and plant growth promoting bacteria is an emerging technology in heavy metal accumulation and increasing the antioxidative defence system within them (Ma et al. 2016; Xun et al. 2015). Micro-organisms modulate the levels of antioxidants within plants under heavy metal stress conditions (Ahmad et al. 2016). Mycorrhizal fungi in association with the plants undergo sequestration of wide range of heavy metals in fungal hyphae and roots of the plants. This limits their inhibitory effects on growth as well as undergoes immobilization of metal ions (Garg and Kaur 2013). It was further suggested that GSH levels have been increased in Cjanus cajan upon mycorrhizal inoculation of Glomus mosseae under Cd stress which reduced the oxidative stress generated in the plant (Garg and Kaur 2013). Similar reports have been found in which this AM fungi stimulated GSH levels in the Cjanus cajan plant under Zn stress thereby upregulating the defensive mechanism of the plant (Garg and Kaur 2013).

Various reports showing the role of microbes in modulating glutathione activity in plants are summarized in Table 12.3:

Table 12.3 Effect of microbes on glutathione levels in metal stressed plants
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12.4.2.2 Ascorbic Acid

AsA is one of the universal non enzymatic antioxidant for detoxification of ROS (Akram et al. 2017). Ascorbate is physiological active form of AsA that is a resonance stabilized anionic form formed by deprotonation of OH group at C3 carbon and it is water soluble antioxidant within living system (Smirnoff 2000a, b). ASA is involved in activation of antioxidant enzymes and also act as a source of electron for activation of APXs. For eg: APXs are heme containing enzymes that dismutase H2O2 to H2O and molecular oxygen (Mittler et al. 2004; Van Doorn and Ketsa 2014). Ascorbate is significantly involved in the scavenging of O2, OH radicals and reduces H2O2 to H2O respectively in plants exposed to metal toxicities. They are localised in cellular compartments such as chloroplasts and at optimal concentrations enables plant’s defence against metal toxicities (Ray et al. 2016; Fuentes et al. 2016). They are not only restricted to chloroplasts but also plays a crucial role in ROS scavenging in cytosol, mitochondria and peroxisomes (Mittler et al. 2004). Furthermore, soil microorganisms enhance plant growth and quality in mutualistic association by alteration in ascorbic acid levels. They also increase biotic and abiotic stress tolerance of host plant by increasing or decreasing the activity of these antioxidants (Mollavali et al. 2016). Studies were reported in which endophytic bacteria stimulated ascorbic acid content along with glutathione by acting as redox buffers (Ray et al. 2016). It was further observed that ASA levels were increased in the maize plants under Cr stress, which upon PGPB incoulation was further elevated upto certain levels. It signifies that AsA promoted plant growth and minimised H2O2 and MDA content, showing the protective role against oxidative stress tolerance upon bacterial inoculations (Islam et al. 2016). Moreover, bacterial inoculation of Bacillus licheniformis and Pseudomonas flurescens in grape vine exposed to As stress stimulated AsA levels (Pinter et al. 2017). Various reports of microbes modulating ascorbic acid in plants under heavy metal stress are given below in Table 12.4.

Table 12.4 Effect of microbes on AsA content under metal stressed plants
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12.4.3 Phenolic Compounds

Metal toxicity results in oxidative stress which interferes with physiological activities of plants including photosynthesis, respiration and inhibition of imperative enzymes (Shanker et al. 2005; Singh et al. 2013). Various secondary metabolites of plants like flavonoids, proline, total phenols aid in detoxification of heavy metals (Gill and Tuteja 2010). Phenolic compounds (i.e. phenols, phenylopropanoids, phenolic acids and flavonoids) categorized as low molecular weight antioxidants can directly sacavange free radicals. These compounds are an electro-donating agent which act as antioxidants, provides colour and contribute to overall health of plants.

12.4.3.1 Phenols

Phenols are one of the major secondary metabolites found in plants. Phenols also act as metal chelators under heavy metal stress and their metabolism is induced during metal stress depending upon their composition. They are mostly oxidized through H2O2 scavenging or Asc-POX system (Michalak 2006). The content of plant phenols are mostly enhanced in response to heavy metal stress which is also confirmed with the studies of Marquez-Garcia et al. (2012) for Erica andevalensis and Ahmad et al. (2015a, b) in Cannabis sativa. The interaction of plants with microbes is considered as important component of soil ecosystem and this interaction is beneficial for plants in coping up various forms of stresses. The applications of these microorganisms have major impact in tolerating and alleviating heavy metal stress. Increase in the content of total phenolics and activity of phenylalanine ammonia-lyase (PAL) was observed under mycorrhizal symbiosis with plants (Nell et al. 2009; Mollavali et al. 2016). The effect of application of microorganisms on plant phenolics has been listed in the Table 12.5.

Table 12.5 Effect of microbes on phenols in metal stressed plants
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12.4.3.2 Flavonoids

Flavonoids are organic compounds widely distributed in plants as secondary metabolites. More than 9000 naturally occurring flavonoids are known and plays wide range of role, few of which are shown in Fig. 12.4 (Buer et al. 2010). One of the major role of flavonoids are its antioxidative properties which is due to its structure i.e. conjugated double bonds and functional group in the rings (Seyoum et al. 2006).

Fig. 12.4
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Role of flavonoids in plants

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It provides defensive role in plants by protecting them from biotic and abiotic stresses. It reduces the production of reactive Oxygen Species (ROS) while quenching its action by inhibiting the ROS generating enzymes, free radical quenching in lipid peroxidation and regeneration of other antioxidants (Arora et al. 2000; Higdon and Frei 2003). Flavonoids are mostly secreted from root tips and root hair zone as they are the target sites for symbiotic relationship between plant and bacteria Fig. 12.5 (Abdel-Latief et al. 2012). It protects the plant’s photosynthetic machinery from harmful UV-radiations as well as aids plant- bacterial symbiotic relationship. It also plays critical role as specific transmitters legumes secrete luteolin and chrysin as flavonoid which acts as signal for bacteria population to initiate symbiosis (Mierziak et al. 2014). It was further reported that alfalfa roots release large amount of flavonoids due to reduction in nitrogen content. Moreover, in response to alfalfa derived flavonoids, Rhizobium meliloti shows chemotaxis towards luteolin, 4′,7-dihydroxyflavone and 4′,7-dihydroxyflavanone and the chalcone 4,4′-dihydrochalcone (Cohen et al. 2001).

Fig. 12.5
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Role of flavonoids in nodulation (Denarie et al. 1996; Cooper 2004; Reddy et al. 2007)

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Although there are many studies in which micro-organisms enhance plants defensive potential by modulating flavonoid levels in metal stressed plants (Li et al. 2015a, b). It was suggested that independent treatment of PGPB and SA on Cr stressed Zea mays L. plant led to up regulation of phenolic compounds which act as antioxidant and chelator by enhancing metabolic activity of plants (Islam et al. 2016). Furthermore, PGPB and SA combination enhances plant flavonoid content under metal stressed conditions which might be due to the exudation of siderophores or root-microbe interactions (Islam et al. 2009). Similar results have been found by various researchers on phenolic compounds with PGPB inoculation (Saravanakumar et al. 2007; Li et al. 2015). Various reports showing the effect of micro-organisms on phenolic compounds (flavonoids) in metal stressed plants are given below in Table 12.6.

Table 12.6 Effect of microbes on flavoinoid content in metal stressed plants
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12.5 Indirect Role of Siderophores in Enhancing Antioxidative Defence and Heavy Metal Detoxification in Plants

Siderophores are compounds having low molecular mass (400–1000 Da) but high chelation capacity for iron as well as with other metals like Al, Cd Cu, Pb and Zn etc. (Schalk et al. 2011). They are produced by rhizospheric microorganisms in response to iron deficiency. The structural group of siderophores are classified on the basis of their chemical composition depending upon the moieties which donate oxygen ligand for complexation with Fe(III) i.e. α-hydroxy-) carboxylates, hydroxamates and catecholates. Siderophores act as solublizing agents and convert the unavailable heavy metal into soluble forms by forming chelation complexes which are sought to have important role in heavy metal phytoextraction (Braud et al. 2009b; Dimkpa et al. 2009a, b; Rajkumar et al. 2010). Enhanced uptake of Cr, Pb and Cd due to production of siderophores by Pseudomonas aeruginosa and Streptomyces tendae has been reported by Braud et al. (2009a, b) and Dimkpa et al. (2009a, b, c) respectively. Improved phytoextraction in plants by siderophores secreting microorganisms suggests their role in improved uptake by inoculating plants with siderophores. However, antagonistic reports of decreased metal uptake in presence of microbial siderophores have also been reported by Sinha and Mukherjee (2008) in Cucurbita pepo and Brassica juncea and Tank and Saraf (2009) in chickpea. However, the uptake of metal depends upon metal availability, type of plant and translocation of metal to shoots. Increased uptake of heavy metal with siderophore complexes induces oxidative stress in plants due to ROS production (Rajkumar et al. 2010). The siderophore producing microorganisms modulate the heavy metal induced oxidative stress by enhancing the antioxidative defense system of plants. These microorganisms also inhibit the IAA (indole-3-acetic acid) caused oxidative degradation in plants and increase the activity of enzymes like POD (peroxidase), SOD (superoxide dismutase) and carotenoids which have protective role against stress (Dimkpa et al. 2009a, b, c; Sharma and Johri 2003). The enhanced activity of enzymes by siderophores helps to overcome the detrimental effect on plant biomolecules as shown in the Fig. 12.6. The effect of siderophores producing bacteria on growth of mycelium and Pb and Cd uptake as well as antioxidant system was studied by Cao et al. (2012) in Oudemansiella radicata. Results demonstrated that siderophore exposure enhances the growth and Cd (by 26.5% in Bacillus sp.) and Pb accumulation (by 158.9%) in comparison to controls. Decrease in oxidative damage and reduced activity of enzymes like SOD and POD was observed which shows the role of siderophores in declining the toxicity of heavy metals.

Fig. 12.6
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Schemetic representation of siderophore effect on plant antioxidative defense system

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12.6 Indirect Role of Organic Acids in Enhancing Antioxidative Defence and Heavy Metal Detoxification in Plants

Organic acids are CHO comprising compounds distinguished by the occurrence of one or more carboxyl groups with a maximum molecular weight (300 Da) (Jones 1998). In soil, they can bind to metal ions through complexation reaction. The stability of the ligand: metal complexes is reliant on various factors: (a) the nature of organic acids which includes number of carboxylic groups and their position, (b) the binding form of the heavy metals, (c) pH of soil (Jones 1998; Ryan et al. 2001). They might cause the acidification of the rhizosphere by decline pH and then mobilization of insoluble heavy metal chelates in soil and led to enhance their bioavailability (Seshadri et al. 2015). Organic acids are considered as agents of phytoremediation and also act as protectants to enhance tolerance against stress. Maleic acid (MA) increased the content of non-enzymatic antioxidants (AsA, GSH) and the activities of enzymatic antioxidants such as SOD, CAT, APOX, MDHAR, DHAR, GR, GPOX under Cr stress in Brassica juncea plants which further enhanced Cr uptake in the roots, but it slightly decreased the translocation of Cr from roots to shoots at lower concentration of Cr and considerably at higher concentration. MA decreased oxidative damage caused by Cr and improved the chlorophyll content, water status, growth and biomass of the plants (Mahmud et al. 2017).

Over last decades, the low molecular weight organic acids (LMWOAs) exuded by plant-associated microorganisms have gained much attention due to their suggested role in mobilization of mineral nutrients and solubility of heavy metal in the rhizosphere (Rajkumar et al. 2012).

Organic acids produced by plant-associated microorganisms play a significant role in the complexation of toxic and important ions and enhance their mobilization for plant uptake. Moreover, the role of organic acids in antioxidative defense system has been elucidated. It was investigated that, antioxidative defense system of Solanum nigrum L. was improved by supplementation of Paecilomyces lilacinus NH1 and citric acid. It promoted the plant growth under Cd stress and significantly alleviated the oxidative stress experienced by the plant (Gao et al. 2010). It was also reported that citric acid has the ability to enhance heavy metal accumulation in Solanum nigrum L. by enhancing antioxidative activities. It also enhanced chelation of heavy metals and stimulated antioxidant defense system in plants under Cd and Pb treatments (Gao et al. 2012). It was observed that enhanced antioxidative defense in plants was due to the expression of defense related enzymes or specific proteins (Gao et al. 2012). The further study conducted by Saravanan et al. (2007) reported that production of a gluconic acid derivative and 5-ketogluconic acid by Gluconacetobacter diazotrophicus strains further assisted in the solubilization of Zn compounds (ZnO, ZnCO3 or Zn3(PO4)2). It has been demonstrated that P. aeruginosa (CMG 823) had the potential to solubilize large quantity of ZnO and Zn3(PO4)2) that was dependent on production of 2-gluconic acid (Fasim et al. 2002). Soil inoculated with Cd/Zn resistant bacteria showed enhanced water soluble Cd and Zn levels in Sedum alfredii in comparison to uninoculated soil. Increased mobility of heavy metals might be due to enhanced production of organic acids such as acetic acid, formic acid, tartaric acid, oxalic acid and succinic acid (Li et al. 2010). Burkholderia caribensis considerably mobilized P and Fe from phosphorous iron ore because of production of gluconic acid along with exopolysaccharides production which lead to the formation of biofilms that help in the mobilization of P and Fe from the ore (Delvasto et al. 2009). A study conducted by Wani et al. (2007) demonstrated that metal resistant Bacillus strains (PSB 1, PSB 7, and PSB 10) were taken for the mobilization of Pb and Zn and it was found that PSB1 strain had potential to mobilize large quantity of Pb and Zn. Mycorrhizal fungi can also release organic acids into the rihzosphere for the mobilization of heavy metals by forming complexes which further acidify the rhizosphere. It has been reported that ericoid mycorrhizal fungi Oidiodendron maius increased mobility of Zn from insoluble Zno and Zn3(PO4)2 through secretion of Zn chelating malic and citric acid (Martino et al. 2003). Organic acids produced by fungi Beauveria caledonica aided in the solubilization of Zn3(PO4)2 and pyromorphite via acidolysis (protonation) reaction (Fomina et al. 2005). Studies revealed that organic acids secreted by plant associated microorganisms assisted in plant root absorption of metal ions such as Cu (Chen et al. 2005), Pb (Sheng et al. 2008) and Cd and Zn (Li et al. 2010). Pseudomonas fluorescens G10 and Microbacterium sp. G16 (metal resistant endophytic bacteria) were reported to increase the accumulation of Pb in rape through secretion of organic acids (Sheng et al. 2008). Aspergillus niger was capable of mobilize large quantity of Pb and P from pyromorphite with the help of organic acid produced by this fungi. In addition, A. niger significantly increased uptake of Pb and P in Lolium perenne (Sayer et al. 1999). It was revealed that organic acids producing microorganisms have the capacity to improve the phytoextraction technique in metal polluted soils.

Organic acids have been suggested to play an important role in the mechanisms associated with uptake of heavy metals by roots (Han et al. 2006; Panfili et al. 2009). In maize, organic acids such as acetic and malic acids promoted Cd uptake by roots and demonstrated that the organic acid with low stability constant was capable to increase large quantity of Cd accumulation (Han et al. 2006). Maize roots were proficient to detach Cd from Cd-organic acid complex via root surface mediated process and thus led to enhanced Cd uptake. It has been showed that free Cd ions were more easily accessible to maize roots as compared to intact Cd-organic ligand complexes. Similarly, citric acid improved Cd uptake and its distribution among roots of durum wheat (Panfili et al. 2009). Complex formation between metal-organic acid indirectly participate in metal uptake by dissociation of metal from metal-organic acid complexes within diffusion layer or at surface of roots which led to enhance the level of free metal ions (Han et al. 2006; Panfili et al. 2009).

Various plant-associated microbes have the capability to produce organic acids and lead to mobilization of toxic and essential ions (Fomina et al. 2005; Martino et al. 2003; Uroz et al. 2009), a significant question that has yet to be satisfactorily resolved is either they act as sources or sinks of organic acids in the soil. In-vitro studies with soil microbes have elucidated to some level, in which the concentration of organic acid influx is directly normalized by the external concentration (Jones et al. 1996). Soil properties of rhizosphere (sorption, biodegradation, buffering capacity and metal complexation) may change the organic acids profiling, making complex to predict the behaviour of organic acids (Jones et al. 2003; Rajkumar et al. 2012; Mahmud et al. 2017). A comprehensive description of the factors those manage the fate and organic acids behavior in soil such as concentrations requisite for mobilization of metals, efficiency of heavy metal/nutrient-mobilization, biodegradation and sorption to the soil’s solid phase are keys to recognize the exact mechanisms of organic acids produced by microbes in the metal polluted rhizosphere soils. The exact quantification of organic acids in soils and the full sequencing of organic acid producing microorganisms together with biomarker tools like green fluorescent protein-based biosensors, will help to understand the dynamics of transport of organic acid between rhizosphere soils, plants and microbes.

12.7 Conclusion

Micro-organisms associated with plants have the ability to aid the plant growth and metal immobilization or mobilization. Studies have indicated that they exhibit resistance against different heavy metals and protect the plants against adverse effects. Using microbial consortia, different activities of the plants like nodulation and antioxidative capacities are enhanced by reducing oxidative stress. In addition they also promoted heavy metal detoxification within plants by enhancing metal accumulation, translocation and phytoextraction. Further, most of the studies of microbes modulated the secondary metabolites produced within the plants and enhanced their antioxidative defense system. They have the potential to activate the plant enzymatic and non-enzymatic antioxidant system. Moreover, among secondary metabolites, phenolic compounds which aid metal detoxification and directly scavenge free radicals have also been modulated via microbial colonization. Apart from this, micro-organisms producing siderophores and organic acids under metal stressed conditions promoted plant growth by decreasing oxidative stress.

Such studies may enable us to manipulate microbes in improving plant’s performance under metal stressed conditions and utilizing their potential in heavy metal detoxification. We anticipate that exploration of microbial consortium (both bacteria and fungi) possessing plant growth promoting features and their inoculation with hyperaccumulator plants would yield better results for enhancing their anti-oxidative potential as well as metal detoxification.