**4. Chilling stress**

Low-temperature, or chilling, stress (damage caused by low, but above-freezing temperatures) has been recognised as a unique environmental impact on crop plant physiology [66]. The damage resulting from the symptoms of the chilling stress includes a decrease in growth and yield of the plant. These symptoms consist of the prevention of metabolic processes, rise in the permeability and seepage through the cell membrane due to alterations in the order of the molecules or in the physical form, wilting and chlorosis [67]. Prasad et al. [68] has suggested that mitochondria are critica1 organelles in the metabolic production of energy in the cell. The competence and the stability of mitochondria are very important for the seedlings to survive low-temperature stress, especially during early seedling growth. Low temperature induces oxidative stress in the cell [69]. Under aerobic conditions, superoxide radicals and H2O2 are found to be normal metabolites of plant cells [70, 71] as well as animal cells [72, 73] and are kept at low, steady-state levels by the action of antioxidant enzymes such as SOD, CAT, GSH POD and APX located in the organelles and cytosol [74–76].

Active oxygen species (AOS) has been proposed to be responsible for cold-induced injury because they are produced at higher concentration during cold stress and may initiate degradative reactions, causing lipid peroxidation, membrane deterioration, protein degrada‐ tion and chlorophyll quenching. An efficient antioxidant activity is essential in order to maintain the concentration of AOS at relatively low levels [67]. On the other hand, the damage that occurs during chilling stress accompanying illumination was thought to be mediated by an oxygen radical. The defence mechanisms of the cell against oxidative stress involve antioxidants that can be found in many plant organs in large amounts to perform vital biological functions. These include the enzyme systems CAT, SOD and numerous PODs, e.g. APX and guaiacol peroxidases (POX) [69]. During photosynthesis, superoxide and hydrogen peroxide are produced as side products and need to be removed. This is achieved by the SOD enzyme and the enzymes GSH and ascorbate from the ascorbate–glutathione cycle. It was shown by Aroca et al. [77] that due to ROS being produced under chilled environments during light-induced photo-oxidation, the major damage from chilling stress occurs during this time. The reason for ROS production under these circumstances is because of the slowing down of the enzymes involved in the Calvin–Benson cycle, thus resulting in the limitation of the NADP + supplements receiving the electrons from the electron transport chain and inducing oxygen to absorb more energy than needed. To decrease photo-oxidation under chilling, there are three important mechanisms. The first one involves avoiding production of ROS by diminishing electron transport chain; the second one involves scattering surplus energy in the form of heat via violaxanthin de-epoxidation, and the third one involves scavenging ROS produced by antioxidant compounds and enzymes. Additionally, the water-water cycle in the chloroplast, where electrons flow in photosystem II from water to photosystem I to reduce oxygen without O2 levels having a net change, is said to be an active mechanism that can disperse energy from over excitation when there is an environmental stress. SOD, GR and APX are also some of the antioxidant enzymes involved in the function of this cycle [77]. The enzyme SOD is located in the cytoplasm, chloroplast, mitochondrion and peroxisome and acts as the first line of defence mechanism against ROS by dismutating O2 into H2O2 [78]. Furthermore, the dismutation of superoxide radicals into H2O2 and oxygen is an important step in protecting the cell, and in that conversion, SOD is considered a key enzyme [69]. CAT also played a significant role in chilling tolerance and is especially important for removal of H2O2 in C3 plants. Exposure to low temperature may increase the amount of AOS not only in cold-sensitive but also in coldtolerant plants. There was a correlation between the reduction in CAT activity and H2O2 accumulation [79].

Drought tolerance in black pepper is attained through osmotic adjustment and better ROS scavenging machinery, functioning through different antioxidant enzymes. The activities of antioxidant enzymes suchas SODandPODbecomehigherduringstress intolerantvariety[65].

Low-temperature, or chilling, stress (damage caused by low, but above-freezing temperatures) has been recognised as a unique environmental impact on crop plant physiology [66]. The damage resulting from the symptoms of the chilling stress includes a decrease in growth and yield of the plant. These symptoms consist of the prevention of metabolic processes, rise in the permeability and seepage through the cell membrane due to alterations in the order of the molecules or in the physical form, wilting and chlorosis [67]. Prasad et al. [68] has suggested that mitochondria are critica1 organelles in the metabolic production of energy in the cell. The competence and the stability of mitochondria are very important for the seedlings to survive low-temperature stress, especially during early seedling growth. Low temperature induces oxidative stress in the cell [69]. Under aerobic conditions, superoxide radicals and H2O2 are found to be normal metabolites of plant cells [70, 71] as well as animal cells [72, 73] and are kept at low, steady-state levels by the action of antioxidant enzymes such as SOD, CAT, GSH

Active oxygen species (AOS) has been proposed to be responsible for cold-induced injury because they are produced at higher concentration during cold stress and may initiate degradative reactions, causing lipid peroxidation, membrane deterioration, protein degrada‐ tion and chlorophyll quenching. An efficient antioxidant activity is essential in order to maintain the concentration of AOS at relatively low levels [67]. On the other hand, the damage that occurs during chilling stress accompanying illumination was thought to be mediated by an oxygen radical. The defence mechanisms of the cell against oxidative stress involve antioxidants that can be found in many plant organs in large amounts to perform vital biological functions. These include the enzyme systems CAT, SOD and numerous PODs, e.g. APX and guaiacol peroxidases (POX) [69]. During photosynthesis, superoxide and hydrogen peroxide are produced as side products and need to be removed. This is achieved by the SOD enzyme and the enzymes GSH and ascorbate from the ascorbate–glutathione cycle. It was shown by Aroca et al. [77] that due to ROS being produced under chilled environments during light-induced photo-oxidation, the major damage from chilling stress occurs during this time. The reason for ROS production under these circumstances is because of the slowing down of the enzymes involved in the Calvin–Benson cycle, thus resulting in the limitation of the NADP + supplements receiving the electrons from the electron transport chain and inducing oxygen to absorb more energy than needed. To decrease photo-oxidation under chilling, there are three important mechanisms. The first one involves avoiding production of ROS by diminishing electron transport chain; the second one involves scattering surplus energy in the form of heat via violaxanthin de-epoxidation, and the third one involves scavenging ROS produced by antioxidant compounds and enzymes. Additionally, the water-water cycle in the chloroplast, where electrons flow in photosystem II from water to photosystem I to reduce oxygen without

POD and APX located in the organelles and cytosol [74–76].

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

**4. Chilling stress**

Oxidative free radicals canbehighlyreactive towards cell components,andtherefore,theability of the cell to remove these undesirable species might be viewed as an important feature in improved resistance to chilling stress. The increases in the activities of CAT3 provide evi‐ dence for the increased production of superoxide and H2O2 in mitochondria of maize seed‐ lings. Increases in superoxide and H2O2 can be expected in cases in which there is either high O2 uptake or decreased ability of the electron transport pathway, which increases potential for higher electron leakage to O2 for subsequent production of superoxide and H2O2 [68].

One major antioxidant that plays a role in the detoxification of ROS and plant protection against oxidative damage is glutathione. There are two versions in which glutathione can exist in which are the oxidised disulphide version (GSSG) and the reduced version (GSH). The function of glutathione as an antioxidant is mainly assigned to its reduced (GSH) version as this form is oxidised to form the oxidised (GSSG) version during its function as an antioxidant. Therefore, keeping the concentration of reduced glutathione, from the ratio GSH/GSSG, high is important for plants. The production of GSH can occur both in cytosol and the chloroplast in the leaves of the plant. Furthermore, in the ascorbate–glutathione cycle, GR catalyses GSSG reduction into GSH via donation of electrons from NADPH molecules. ROS detoxification in the chloroplast is known to be mostly carried out by the ascorbate–glutathione cycle, which is thus accepted as the main pathway in this process. In this cycle, ascorbate is also considered to be a major antioxidant in addition to GSH [80]. According to Prasad [81], rapidity with which GR enzyme was induced during the early stages of acclimation and remained induced during chilling and recovery clearly suggests that acclimation uniquely induces the antioxidant defence mechanism that is necessary for protecting the seedling from oxidative stress injury.

Chilling-sensitive pepper cultivars were investigated for SOD, CAT and POD enzyme activity under chilling stress condition. The results showed that the activity of CAT decreased, and both SOD and POD activities raised in two cultivars, However, permeability of plasma membrane was positively related to MDA content, SOD and POD activity and also negatively related to CAT activity variation. As low temperature treating was extended, permeability of plasma membrane, MDA content and POD activity increased and SOD and CAT activity decreased in two cultivars: Xiza No. 7 (less chilling-sensitive cv.) could maintain a higher protective enzyme activity, and permeability of plasma membrane and MDA content were low; Hajiao No. 1 (chilling-sensitive cv.) was quite the contrary [82].

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 with chilling tolerance [85].

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. Moneymaker [86].
