**5. Nutrient deficiency and toxicity of heavy metal**

The micronutrients essential for the normal growth and development of plants, as it is known to be required in several metabolic processes [87]. Deficiency of nutrients such as Zn, Mn, Cu, Fe, Mg, B and K can modify the activities of several antioxidative enzymes [88]. Kosesakal and Unal [89] indicated that Zinc (Zn) is one of the essential micronutrients playing a significant role in many vital metabolic processes. Zinc deficiency is a major global problem hindering plant cultivation, and this problem is especially exacerbated in acidic calcareous soils, which is the most common soil type in arid and semi-arid regions of the world. It is known that magnesium deficiency results in the decrease of chlorophyll amounts in beans. It was observed by Welkie et al. [90] that in peppers, the amounts of chlorophyll and iron in leaves were directly proportional, while zinc was also shown to be beneficial in the synthesis of carotenoids and chlorophyll, thus being proved to be essential in the photosynthetic process in plants. Iron (Fe) is a cofactor of many antioxidant enzymes and could act as a pro-oxidant factor because free or loosely bound it catalyses free radical generation in the presence of reductants and peroxides through the Fenton reaction. The growth of sunflower plants under iron deficiency conditions affects POD isoforms differently, inducing a preferential reduction in activity of those isoforms involved in the detoxification processes [91].

Anderson et al. [83] indicated that the response of ascorbate and glutathione in mesocotyls to acclimation and chilling was generally the same as that in the coleoptile + leaf, although the increase in the total glutathione pools in response to acclimation was not as extensive. Induction of other antioxidants in the mesocotyl may reduce the need for GSH synthesis. In the roots, there was no effect of acclimation or chilling on any of the antioxidants tested. However, the fact that H2O2 levels in the roots were not greatly increased by chilling suggests that a modification of antioxidants was unnecessary to prevent oxidative stress [83]. Likewise, Lee and Lee [84] established that APX is also an important antioxidant enzyme in scavenging or utilising H2O2. Total APX activity increased when chilling stress occurred in the leaves of the cucumbers and seemed to be because of favoured induction of the isozymes APX-5 and APX-4. However, 24 h after the stress, the increase seen in APX activity was because of the favoured expression of the isoform APX-3 [84]. Chilling stress causes many physiological and biochemical changes. Kang and Saltveit [85] investigated that chilling tolerance in cucumber seedling radicals. Chilling seedlings with radicles 20-mm long for 48 h at 2.5°C inhibited subsequent growth by 36%, while it reduced the growth of 70-mm-long radicles by 63%. APX activity was higher in 20-mm-long radicals before chilling than in 70-mm-long radicles. It appears that higher APX, CAT and DPPH (the stable free radical 1,1-diphenyl-2-picrylhydraz)-radical scavenging activities, and sustained APX activity during chilled and during subsequent growth at 25°C following chilling in 20-mm-long radicals corresponds with higher chilling tolerance. The activities of APX, CAT and DPPH appear to be positively correlated

492 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

A study on the effects of chilling stress on two salt- and drought-tolerant and two sensitive pumpkin genotypes in callus culture [18] found that the tolerant genotypes showed lower increase in lipid peroxidation and a greater increase in APX, CAT and GR than the tolerant genotypes under stress conditions. It was observed that even though the increase of glucose, proline and fructose concentration went up with prolonging of the chilling effect in all the pumpkin genotypes, a much more significant increase was observed in the tolerant genotype than that of the sensitive genotype. Thus it can be concluded from these results that in pumpkin genotypes, chilling stress results in an increase in the peroxidation of lipids and in oxidative stress, due to reactive oxygen radical production. Song et al. [86] in their study observed that chilling stress, SOD and CAT activities decreased in some extent in both cultivars, in compar‐ ison to control in tomato cultivars. Compared to control, chilling stress resulted in significantly higher POD activity in cv. Mawa on day 6, whereas no significant changes of POD activity caused by chilling stress were observed in cv. Moneymaker at all time points tested. On the other hands, APX activities were increased in the two cultivars under chilling stress. GR activities increased in cv. Mawa after chilling stress, but almost no change was observed in cv.

The micronutrients essential for the normal growth and development of plants, as it is known to be required in several metabolic processes [87]. Deficiency of nutrients such as Zn, Mn, Cu,

with chilling tolerance [85].

Moneymaker [86].

**5. Nutrient deficiency and toxicity of heavy metal**

Metal toxicities have received widespread attention as large amounts are released into the environment and affect living organisms. Heavy metal intoxication, especially by lead, cadmium, arsenic and mercury, constitutes serious threat to human health [92, 93]. Although information focussed on the relationship between heavy metals and oxidative stress in plants has been available in recent years, it is still difficult to draw a general conclusion about critical toxic metal concentrations in soils [94]. Heavy metals cause oxidative damage to plants, either directly or indirectly through AOS formations which are extremely toxic to living cells. Redox metals such as Cu or Fe appear to act directly on the production of AOS. Copper is among the major heavy metal contaminants in the environment with various anthropogenic and natural sources. Human health risk from heavy metal bioaccumulation in vegetables has been a subject of growing concern in recent years. Excess Cu inhibits plant growth and seed germination, induces chlorophyll degradation and interferes with photosystem activity. At the molecular level, Cu ions generate ROS. These reactive radicals cause oxidative damage of lipids, proteins and nucleic acids. Cu ions also are responsible for alterations of membrane integrity in plant cells. Cu-mediated membrane lipid peroxidation causes membrane damage, thus changing membrane permeability and leading to electrolyte leakage. Plants have evolved several antioxidant defence mechanisms to protect themselves from oxidative damage [95].

Zn is the second most abundant transition metal after iron (Fe) and is involved in various biological processes in organisms. Due to this, the results of the presence of Zinc were investigated, including zinc deficiency, hyperaccumulation and its protective role in plants. However, it is not clear what the implications of zinc stress are on antioxidant responses and the uptake of nutrition, though it is known that excess of zinc is not beneficial and can result in negative symptoms in plants. The symptoms that can be observed at the organism level include prevention of seed germination, of root development and of the growth of the plant, and chlorosis can be seen in the leaves. At the cellular level, excess Zn can significantly alter mitotic activity, affect membrane integrity and permeability and even kill cells. Investigates showed that Zn stress on the activity of many antioxidative enzymes (APX, SOD, POD and CAT) and antioxidant contents (ascorbate and GSH) in plants [96–105].

The availability of manganese (Mn) to plants is governed by redox processes, which depend on soil's Mn reserve, pH and the availability of electrons. However, excess Mn disturbs the metabolism of plants and inhibits the plant growth. Mn causes deficiency of Fe, Mg and Ca and induces inhibition of chlorophyll biosynthesis and a decline in the photosynthetic rate. The toxic effects of heavy metals, both essential and nonessential elements, have been linked to the production of ROS. To quickly get rid of ROS, which result in the disruption of cellular metabolism due to damage by oxidative stress to important molecules, numerous enzymatic and non-enzymatic ways have evolved in living organisms. As Mn2+ plays a role in numerous processes, it is thought that an excess of it results in oxidative stress [106]. Cadmium (Cd) is a non-redox metal unable to participate in Fenton-type reactions. Naturally occurring amounts of Cd are normally low, however, the concentration can be significantly increased by anthro‐ pogenic activities. The impact of the uptake of Cd by living cells has been shown to be drastic, inducing oxidative stress and normally leading to cell death depending on the metal dose and time length of exposure [107]. In general, Cd in plants reduces growth, both in roots and stems, due to suppression of the elongation growth rate of the cells [94]. According to Dinakar et al. [93], cadmium is easily translocated from plant roots to above-ground tissues and potentially threatens human health. Cadmium in plants interferes with physiological processes, resulting in declined productivity. Cadmium can harness photosynthetic activity, chlorophyll content, plant growth and induce oxidative stress. ROS are efficiently eliminated by non-enzymatic (glutathione, ascorbate, a-tocopherol and carotenoids) and enzymatic defence systems such as SOD, APX, POD and GR, which protect plants against oxidative damage. The detoxification of O2 occurs due to the SOD enzyme, while H2O2 is detoxified by the enzymes CAT and PODs and thus OH radicals are not formed. In the detoxification of hydrogen peroxide from different compartments in the cell, glutathione reductase (GR) and APX are key players in the ascorbate– glutathione cycle. Glutathione is also the substrate for the biosynthesis. A constitutively high antioxidant capacity or increase in antioxidant level could prevent oxidative damage and improve tolerance to the oxidative stress established [108, 109]. Sandalio et al. [108] investi‐ gated effects of cadmium on antioxidative enzyme activity in pea. They said that the level of oxygen radicals in cells could be enhanced by a decrease of the enzymatic antioxidants involved in their detoxification, such as SOD.

It was recorded by Schützendübel and Polle [110] that antioxidative enzymes were prevented from functioning and that GSH was depleted for a short period by cadmium and other metals. It was also put forth that hydrogen peroxide accumulation resulted from the depletion in these antioxidants. These results were obtained by accessing models of antioxidative capacity. As more Cd tolerance was observed in plants when more GSH was synthesised, it can be deduced that the decrease in GSH levels is an important step for cadmium sensitivity [110]. Dong et al. [111] investigated that effect of Cd concentration in tomato seedling for antioxidative enzymes. From the results, it can be observed that POD and SOD activities significantly increased in plants that were given Cd with a concentration of 1–10 μM and that MDA levels also showed a significant increase, indicating that oxidative stress response was the result of Cd stress in tomato plants. Tanyolac et al. [112] reported that tolerance and protective mechanisms have evolved to scavenge free radicals such as superoxide, hydroxyl radicals and peroxides generated during various metabolic reactions. Antioxidative enzymes such as APX play a key role in controlling the cellular level of these radicals and peroxides. They found that APX activity was increased with Cu treatment [112]. Zhao et al. [113] investigated the different tolerance mechanisms to Cd stress between YSL189 and HZ903 at the seedling stage. When Cd concentration was >20 μM in the growing medium, the uptake rate of Cd was significantly higher in roots of YSL189 than in the roots of HZ903. When plants were supplied with 50- and 100-μM Cd in the growing medium, there were higher Cd concentration, higher biomass and plant height, shorter roots and higher expression levels of transporter genes natural resistance associated macrophage proteins (Nramp)2, Nramp3 and zinc and iron regulated transporter (ZIP) in roots of YSL 189 compared to HZ903. The high Cd accumulation in YSL189 was partly due to the higher Cd uptake rate and higher expression levels of Nramp2, Nramp3 and ZIP in its roots. At the same time, the degree of cell injury indicated by thiobarbituric acid reactive substance showed no significant differences in roots and stems between the two genotypes. The higher activities of SOD, POD and CAT in roots and stems of YSL189 were compared to HZ903 [113].

The availability of manganese (Mn) to plants is governed by redox processes, which depend on soil's Mn reserve, pH and the availability of electrons. However, excess Mn disturbs the metabolism of plants and inhibits the plant growth. Mn causes deficiency of Fe, Mg and Ca and induces inhibition of chlorophyll biosynthesis and a decline in the photosynthetic rate. The toxic effects of heavy metals, both essential and nonessential elements, have been linked to the production of ROS. To quickly get rid of ROS, which result in the disruption of cellular metabolism due to damage by oxidative stress to important molecules, numerous enzymatic and non-enzymatic ways have evolved in living organisms. As Mn2+ plays a role in numerous processes, it is thought that an excess of it results in oxidative stress [106]. Cadmium (Cd) is a non-redox metal unable to participate in Fenton-type reactions. Naturally occurring amounts of Cd are normally low, however, the concentration can be significantly increased by anthro‐ pogenic activities. The impact of the uptake of Cd by living cells has been shown to be drastic, inducing oxidative stress and normally leading to cell death depending on the metal dose and time length of exposure [107]. In general, Cd in plants reduces growth, both in roots and stems, due to suppression of the elongation growth rate of the cells [94]. According to Dinakar et al. [93], cadmium is easily translocated from plant roots to above-ground tissues and potentially threatens human health. Cadmium in plants interferes with physiological processes, resulting in declined productivity. Cadmium can harness photosynthetic activity, chlorophyll content, plant growth and induce oxidative stress. ROS are efficiently eliminated by non-enzymatic (glutathione, ascorbate, a-tocopherol and carotenoids) and enzymatic defence systems such as SOD, APX, POD and GR, which protect plants against oxidative damage. The detoxification of O2 occurs due to the SOD enzyme, while H2O2 is detoxified by the enzymes CAT and PODs and thus OH radicals are not formed. In the detoxification of hydrogen peroxide from different compartments in the cell, glutathione reductase (GR) and APX are key players in the ascorbate– glutathione cycle. Glutathione is also the substrate for the biosynthesis. A constitutively high antioxidant capacity or increase in antioxidant level could prevent oxidative damage and improve tolerance to the oxidative stress established [108, 109]. Sandalio et al. [108] investi‐ gated effects of cadmium on antioxidative enzyme activity in pea. They said that the level of oxygen radicals in cells could be enhanced by a decrease of the enzymatic antioxidants

494 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

It was recorded by Schützendübel and Polle [110] that antioxidative enzymes were prevented from functioning and that GSH was depleted for a short period by cadmium and other metals. It was also put forth that hydrogen peroxide accumulation resulted from the depletion in these antioxidants. These results were obtained by accessing models of antioxidative capacity. As more Cd tolerance was observed in plants when more GSH was synthesised, it can be deduced that the decrease in GSH levels is an important step for cadmium sensitivity [110]. Dong et al. [111] investigated that effect of Cd concentration in tomato seedling for antioxidative enzymes. From the results, it can be observed that POD and SOD activities significantly increased in plants that were given Cd with a concentration of 1–10 μM and that MDA levels also showed a significant increase, indicating that oxidative stress response was the result of Cd stress in tomato plants. Tanyolac et al. [112] reported that tolerance and protective mechanisms have evolved to scavenge free radicals such as superoxide, hydroxyl radicals and peroxides generated during various metabolic reactions. Antioxidative enzymes such as APX play a key

involved in their detoxification, such as SOD.

Xiong and Wang [95] indicated that Cu phytotoxicity in Brassica pekinensis. Cu treatments increased electrolyte leakage and POD activity, showing a significant correlation between Cu concentration in shoots with electrolyte leakage and POD activity. Oriental melon IVF09 was used as a scion, while the pumpkin Jinxinzhen No. 3 was used as stock to research the physiological characteristics of grafted melon (*Cucumis melo*) seedlings when copper stress was induced. It was observed from the results that copper stress resulted in the inhibition of the physical characteristics of the melon seeds. In the grafted seeds, as opposed to selfrooted seedlings, an increase was seen in the levels of glucose, photosynthetic pigments, fructose, the photosynthetic parameters, biomass, the phosphate and sucrose synthase activities, acid invertase and neutral invertase. When levels of Cu decreased and the levels of P, NA and K increased nutrients were taken up more easily. The concentration of CU in the leaves decreased by 31.3%, while a 15.2% decrease was seen in roots of the grafted seedlings when the levels of copper ion (Cu2+) stress became 800 μM and it was shown that grafting resulted in better endogenous hormone balance in the seedlings. When compared to the control, it was observed that grafted seedlings had a higher concentration of IAA and that POD activity was increased, while concentrations of ABA and maleic dialdehyde and the CAT and SOD activities became less. Thus, it could be deduced that grafting of melon seedlings was beneficial to them when under copper stress and relieved the resulting physiological characteristics from the stress, showing that the resistance of the grafted seedlings to copper stress increased due to grafting [114].

Shi and Zhu [106] indicated that the accumulation of ROS significantly increased in cucumber leaves exposed to excess Mn. It was observed that cucumber leaves in the presence of excess Mn resulted in higher activity of SOD, DHAR, POD and GR while adding SA (salicylic acid) resulted in the inhibition of the activities of APX and CAT, thus showing that different antioxidant enzymes had different changes. When the cucumber leaves were treated with SA, in the presence of excess Mn, the concentrations of the essential glutathione and ascorbate antioxidants increased [106]. Human health risk from heavy metal bioaccumulation in vegetables has been a subject of growing concern in recent years. It was observed by Kiran et al. that when under abiotic stress, mainly heavy metal applications, the Burdur Merkez and Burdur Bucak genotypes that were salt-tolerant had a higher resistance as opposed to the sensitive genotypes. The results also showed that drought, heavy metal and salinity stress resistance was observed to have evolved in similar ways in plants [115]. Another heavy metal lead (Pb) exerts adverse effects on morphology, growth and photosynthetic processes of plants; causes inhibition of enzyme activities, water imbalance and alterations in membrane perme‐ ability; and disturbs mineral nutrition [116]. Wastewater, which is used in agriculture in order to provide growing water demand, might be included heavy metal and trace elements. Lead is one of the most hazardous heavy metals, and it causes an extensive pollution in the envi‐ ronment, and also it has adverse effect on the growing of plants. In the other study was conducted to evaluate the effects of Pb stress in on lettuce (*Lactuca sativa*). It was found that SOD and GR were increased with oxidative stress [115].

#### **6. Conclusions**

In conclusion, both the callus tissue and whole plant studies show a positive correlation between increased antioxidant activity and different abiotic tolerance. Antioxidative enzyme activities play an important role against stress. The tolerance level against salt, drought and chilling stress in callus culture can be utilised as an effective criterion in the plants with other physiological criteria. Therefore, it can be said that antioxidative defence mechanisms and effective working systems in the aspect of tolerance against stress conditions in the plants. The literature suggests that tolerant and sensitive genotypes show different responses under abiotic stress conditions, that antioxidative enzyme activities play a protective role against abiotic stress and that antioxidative defence mechanisms are effective in providing resistance to stress in plants. The results of the studies showed that the young plants of the tolerant genotypes may have better protection against stress by increasing the activity of antioxidant enzymes under different abiotic stresses.

### **Author details**

Sebnem Kusvuran1\*, Sevinç Kiran2 and S. Sebnem Ellialtioglu3

