**5. Enzymatic antioxidants**

Plants have developed ROS scavenging mechanisms, which include a variety of nonenzymatic and enzymatic defense systems to protect cellular membranes and organelles from the damaging effects of ROS [49, 50]. Types of antioxidants produced in the plants are represented in **Table 1**. The degree of damage by ROS depends on the balance between the accumulation of ROS products and their detoxification by the antioxidant scavenging system [49].

The efficiency of the antioxidant defense system to scavenge ROS largely decides the plant's sensitivity to chilling [27, 28, 54]. A higher amount of H2O2 produced during stress is detoxified by APX, POD, and CAT in different organelles [48]. Catalase converts H2O2 into O2 and water. Zhao *et al.* [54] reported in tomato cultivars that higher activities of CAT, APX, POX, and SOD could be positively correlated with chilling tolerance. The CAT activity increased in plants under prolonged LT stress [55]. Fahimirad *et al.* [56] recorded an increased CAT activity in canola cultivars in response to LT stress. The increase in activity was higher in winter canola than LT-sensitive spring canola. The LT stress resulted in enhanced peroxidase activity in naked oats (*Avena nuda* L.) [42]. Dai *et al.* [57] observed that after 72 hours of the recovery period, in barley seedlings, the peroxidase activity was significantly higher in the cold-tolerant cultivar (M0103) in comparison to the cold-sensitive cultivar (Chumai). Aydin *et al.* [58] reported that in tomato plants (*Lycopersicum esculentum* L.) highest MDA production occurred after 10 days of stress and SOD enzyme activity gradually increased with increasing exposure to cold stress. Expression of the *SOD* gene and enzyme plays a key role to provide resistance in tomato plants against cold stress. Zhang *et al.* [59] observed that in *C. Sativus,* activities of antioxidant enzymes *viz.* SOD, POD, CAT, and APX were reduced after chilling exposure. Fahimirad *et al.* [56] reported that cold stress exposure enhanced SOD activity by 2.5-fold in winter canola (tolerant) leaves when compared to controls, whereas spring canola (LT sensitive) cultivar showed a 1.7-fold increase. Sun *et al.* [60] reported that in sugar cane seedling roots at 4°C, SOD activity was higher in cold tolerant (GT28) variety than cold-sensitive (ROC22) variety. Various studies showed a similar response to cold stress in wheat [61], strawberries [62], and barley [63]. Hajiboland and Habibi [64] reported that in cold-treated seedlings, the activity of SOD increased significantly, while in the acclimated seedlings, SOD activity did not differ from the control.

CAT and POD are important enzymes that scavenge H2O2 [65]. Generally, there is a positive correlation between stress tolerance and the activity of POD, CAT, and SOD enzymes in plants [65]. Javadian *et al.* [61] reported that cold-tolerant wheat cultivars had higher CAT activity. Fahimirad *et al.* [56] reported that winter canola had a greater increase in CAT activity than LT-sensitive spring canola under LT stress.


**Table 1.**

*List of the different types of enzymatic and nonenzymatic antioxidants of plant.*

Morsy *et al.* [66] reported that under cold stress no change was recorded in peroxidase activity in cold-tolerant as well as cold-sensitive rice seedlings. Liu *et al.* [42] reported that POD activities in naked oats (*Avena nuda* L.) were higher under LT than normal temperature. But with time POD activities decreased greatly, indicating that LT had affected POD enzyme synthesis. Dai *et al.* [57] reported that in two contrasting coldtolerant cultivars of barley, the tolerant cultivar (M0103) had significantly higher peroxidase activity than the sensitive cultivar (Chumai) after 72 h recovery in coldtreated plants. POD activity increased in *Cucumis sativus*, tomato, and canola under LT stress [61, 67, 68]. Sun *et al.* [60] reported that under LT stress at 4°C POD activity was increased in the roots of sugarcane seedlings than in control. The increase in POD activity was higher in the cold-tolerant genotype (GT28) than cold-sensitive genotype (ROC22). Higher POD and SOD activity probably suggest their possible role in mitigating adverse environmental damage. Hajiboland and Habibi [64] reported a slight increase in CAT activity in both wheat cultivars under chilling temperatures with and without acclimation. In contrast, POD activity increased in spring wheat cultivar but not in winter wheat by both temperature treatments. Gong *et al.* [69] reported that in maize seedlings, cold acclimation enhanced the activity of five antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and glutathione reductase (GR). In conclusion, enzymatic antioxidants accumulate under LT stress and are actively involved in the detoxification of ROS thus enhancing the resistance of the plants.

### **6. Nonenzymatic antioxidants**

In plants ascorbic acid (AsA) and glutathione (GSH) are low molecular weights, nonenzymatic antioxidants, abundantly present, and participate in ROS scavenging [28, 70]. The tripeptide glutathione (γ-glutamyl-cysteinyl-glycine) is widely distributed in plant cells and is implicated in the adaptation of plants to environmental stresses, such as extreme temperatures [48]. It is an important antioxidant associated with the regeneration of AsA in the ascorbate-glutathione cycle and participates in the removal of H2O2 [67]. Its antioxidant activity is mainly due to its redox buffer property. It functions to remove toxic peroxides formed in the cell during normal and stressed conditions [70, 71].

$$\text{2GSH} + \text{ROOH} \rightarrow \text{GSSH} + \text{H}\_2\text{O} + \text{R} - \text{OH} \tag{1}$$

Glutathione detoxifies ROS in concert with NADPH. At low nonfreezing temperatures, several plants accumulate GSH and show an increase in GR activity, indicating a possible role in enhancing chilling tolerance and cold acclimation. A differential elevation in GSH has been reported in a number of LT-exposed plants, including cucumber genotypes [72, 73].

Ascorbic acid (AsA) is one of the universal nonenzymatic water-soluble antioxidants having a substantial potential of scavenging ROS in plants both under stressed and non-stressed conditions [74]. Cell cytoplasm constitutes the most abundant pool of ascorbate, while to some extent it is also transported across the plasma membrane (usually 5%) to the apoplast [75, 76]. Ascorbate is a component of the NADPH/glutathione/ascorbate cycle that removes photosynthetically generated O2− and H2O2. It may also directly reduce O2 **−**, quench 'O2 and regenerate reduced tocopherol. Lukatkin

#### *Perspective Chapter: Effect of Low-Temperature Stress on Plant Performance and Adaptation… DOI: http://dx.doi.org/10.5772/intechopen.110168*

and Anjum [77] reported that AsA and GSH have a high potential for sustainably increasing chilling resistance in plants. A significant increase in the levels of these antioxidants as well as the activity of NADPH-generating dehydrogenases have been caused by LT stress [78]. The AsA content was more in tolerant chickpea (*Cicer arietinum*) genotypes after chilling at the reproductive phase [79]. Kim *et al.* [80] reported that changes in GSH content in two rice cultivars were not evident until 10 days of cold stress. Ascorbic acid content increased significantly in stressed IR50 seedlings in comparison to the control while M-202 stressed seedlings showed little or no change. Overexpression of SIGMEs (*Solnaum lycopersicon,* GDP-Mannose 3', 5′-epimerase) was reported to cause AsA accumulation with enhanced cold tolerance in tomatoes [81]. Airaki *et al.* [78] reported that in pepper plants LT stress caused a significant increase in the level of soluble nonenzymatic antioxidants; ascorbate and glutathione. Kader *et al.* [39] reported an increase in GSH and free ascorbate content in 15 days old seedlings of two wheat varieties after cold treatment. Esra *et al.* [82] reported proline accumulation in two pepper variety seedling leaves under cold stress as compared to control counterparts. Yadegari *et al.* [83] reported that proline content increased more under acclimation than non-acclimated seedlings of soybean and hence provide more tolerance. Zuther *et al.* [84] reported that proline content was higher in acclimated leaves of *Arabdopsis thaliana* than in non-acclimated leaves and recovered back to normal levels after de-acclimation. Airaki *et al.* [78] reported that in pepper plants LT stress significantly increased the levels of soluble nonenzymatic antioxidants; ascorbate and glutathione. Kim *et al.* [80] reported that LT stress at 9°C resulted in increased proline and glutathione content in IR50 rice seedlings, compared to controls, and change in glutathione content was evident on the 10th day of LT stress. Kim *et al.* [23] also reported similar changes, under LT stress for proline, glutathione, and ascorbic acid in rice seedlings. Zhang *et al.* [85] reported that to resist the effect of cold stress, resistant sugarcane varieties showed a higher accumulation of proline content in leaves than sensitive varieties. Sun *et al.* [60] reported an accumulation of proline content in sugarcane seedlings under cold stress. Krol *et al.* [86] reported that cold stress caused a decrease in the radical scavenging activity in the leaves of both varieties of grapes and the more-tolerant variety was characterized by better scavenging activity. In conclusion, nonenzymatic antioxidant accumulates in plants under LT stress and are involved in the detoxification of ROS, thus enhancing the resistance of plant against stress.

Phenylalanine ammonia-lyase (PAL) is the key enzyme of the phenylpropanoid pathway, converting L-phenylalanine (substrate) into *trans*-cinnamic acid, a precursor of phenolics. The activity of the PAL enzyme increases in response to LT stress [87] and is considered to be one of the main lines of cell acclimation in plants against stress [88]. Phenolics protect plants against ROS by acting as antioxidants [89, 90]. Christopoulos and Tsantili [91] used a PAL inhibitor to prove the role of PAL in the accumulation of phenolics under cold stress. Chilling stimulates the expression of genes for phenylalanine ammonia-lyase (PAL) in cucumber seedlings [92]. Olenichenko *et al.* [93] studied the effect of cold stress on phenolic compounds in winter wheat (*Triticum aestivum* L.) leaves, which resulted in hardening and detected an increased level of phenolic compounds. In chilling stressed petunia leaves, it was observed that stress led to elevated antioxidant capacity and total phenolic content [94]. Hajiboland and Habibi [64] reported that PAL activity was increased in winter wheat cultivars under acclimation and more phenolic content accumulated in seedling leaves. The transcription level of PAL and phenolic content was higher in acclimated chickpea seedlings than in non-acclimated ones [37]. Chilling stimulates

the enzymatic activities and the expression of genes for phenylalanine ammonia-lyase (PAL) in cucumber seedlings [92]. Pennycooke *et al.* [94] reported that chilling stress leads to elevated total phenolic content and antioxidant capacity in petunia. Krol *et al.* [86] reported that cold stress caused a decrease in the phenolic content in the leaves of two varieties of grapes, though the more-tolerant variety was characterized by higher phenolic contents. Cold acclimation resulted in a higher accumulation of phenolics, which were positively correlated with the antioxidant capacity of plants. Flavonoids are a type of phenolics, that accumulated at higher rates in leaves and stems of LT-stressed plants, which are responsible for enhanced cold tolerance [95]. Ahmed *et al.* [96] reported that anthocyanin (a flavonoid) content increased in *Brassica rapa* under cold stress. Total phenols are also the components of the nonenzymatic antioxidant system and their content has been correlated with the stress tolerance capacity of plants [97]. Esra *et al.* [82] reported that in pepper (*Capsicum annum* L.) phenolics accumulated in seedling leaves under LT stress. In acclimated plants, enhanced PAL activity and accumulation of different phenolics are thought to play an important role in creating cold tolerance [37]. Schulz *et al.* [98] showed that flavonoid accumulation increased in *Arabidopsis thaliana* after cold acclimation and all acclimated plants performed better under cold stress.

Plants accumulate a variety of compatible solutes, including sugars, polyamines, glycine betaine, and proline, in response to cold and other osmotic stresses [13]. In LT-tolerant plants, such as barley, rye, winter wheat, grapevine, potato, chickpea, and *A. Thaliana*, a positive correlation between improved cold tolerance and accumulation of endogenous proline content was observed [99–101]. In order to enhance the stress tolerance level of plants, proline act as a mediator of osmotic adjustment, proteins, and membrane stabilizer, an osmotic stress-related genes inducer, and a ROS scavenger, so that plants can perform better under stress [99, 100, 102]. The most feasible roles of proline are to (a) maintain the acidity of cytosol, (b) maintain the NAD+ /NADH ratio, (c) enhance photosynthetic efficiency of the photosystem II, and (d) inhibit peroxidation of membrane lipids [103, 104]. Proline accumulated in chilling stressed soybean seedlings [83]. Kim *et al.* [80] reported that in two rice cultivars (IR50 and M-202) proline content increased significantly in stressed IR50 seedlings in comparison to control seedlings, whereas in M-202, stressed seedlings showed little or no change. Cold-acclimated plants recovered faster than non-acclimated plants because of the higher accumulation of proline in acclimated plants.

Fernandez *et al.* [105] reported that carbohydrate metabolism has greater LT sensitivity than other photosynthetic components. Although the precise function of soluble sugars remains unclear, their accumulation in plants under a cold acclimation process suggests that sugars probably play an important role as signaling molecules, cryoprotectants, or osmoregulator [106]. Ruelland *et al.* [107] reported that sugars possess a positive correlation with cold stress tolerance. Sugars under LT stress contribute to preventing the water within the plant cells to freeze because of its typical compatible osmolyte property, hence reducing the availability of water for the ice nucleation process in the apoplast. Sugars replaced water molecules in establishing hydrogen bonds with lipid molecules and hence protecting plant cell membranes during cold-induced dehydration [107]. In addition to these, sugars may also play a role in scavenging reactive oxygen species and contribute to enhanced stabilization of membranes [108, 109]. Hormone signaling and sugar signaling are closely associated processes, which contribute to managing plant growth, development, and defensive responses against stress [110]. Seedling resistance against cold was enhanced when rice seedlings were pretreated with fructose or glucose [66]. Trehalose possesses a

#### *Perspective Chapter: Effect of Low-Temperature Stress on Plant Performance and Adaptation… DOI: http://dx.doi.org/10.5772/intechopen.110168*

unique capacity for reversible water absorption and appears to be superior to other sugars in protecting biological molecules from desiccation-induced damage [111]. Transgenic *A. thaliana* plants showed enhanced freezing tolerance due to the accumulation of trehalose during cold treatment [112].

Sucrose accumulated in higher amounts in LT-stressed plants [113]. Sucrose acts as an osmoprotectant, as it maintains the turgor pressure of cells and stabilizes cell membranes by interacting with phosphate in their lipid headgroups, decreasing membrane permeability [106]. In some plants, the increase in sucrose content can be as high as 10-fold. Lower amounts of other free sugars like glucose and fructose also get accumulated under stress. The LT exposure also leads to fructan synthesis in temperate grasses, which were reported to depend upon sucrose accumulation. The effects were studied on a less cold-hardy spring cultivar (pishtaz) and a cold-hardy winter cultivar (CDC-ospray) of wheat under cold acclimation (20 days at 4°C), interrupted by de-acclimation (10 days at 25°C) and then followed by re-acclimation conditions (10 days at 4°C). Hardening conditions induced the accumulation of carbohydrates in both cultivars and the de-acclimated plants exhibited a significant reduction [114]. Total soluble sugars, reducing sugars, and sucrose contents were higher in coldacclimated than those in non-acclimated plants of sweet cherry [115].

Accumulation of carbohydrates under LT may be due to enhanced expression and post-translation activation of enzymes of the sucrose synthesis pathway [116] and fructose-1,6-bisphosphatase [115]. Sun *et al.* [60] also reported that in sugarcane seedling roots, soluble sugar content increased after LT stress and increased to a higher value in a cold-tolerant variety of sugarcane. Hajiboland and Habibi [64] reported that under cold stress and acclimation total soluble sugar content increased equally in winter wheat while sugar content was higher in acclimated spring wheat seedlings than in non-acclimated seedlings. Parteli *et al.* [117] reported in 1-year-old plants of coffee under a cold acclimation period, the soluble sugars accumulated and enhanced cold tolerance. Burchett *et al.* [118] reported that in cold-acclimated (at 5°C) winter barley plants, the sugar concentration was slightly lower than in non-acclimated plants. Sugars had a positive correlation with cold stress tolerance because they act as osmolytes and protect the water within the plant cells and reduce water accessibility for ice formation. Sugars also establish hydrogen bonds with lipids by replacing water molecules and hence protect the membranes during coldinduced dehydration. Sugars also act as ROS scavengers and play role in membrane stabilization [107]. Sucrose synthase (Sus) is one of the key enzymes involved in sucrose synthesis metabolism, especially in non-photosynthetic tissues. The reversible transformation of sucrose and UDP into UDP-glucose and fructose is catalyzed by the sucrose synthase enzyme. Under normal growth conditions, Sus activity has been linked to phloem loading-unloading and nodule function [115]. The differential regulation of stress-responsive *Sus* genes in leaves might represent part of a general cellular response to the allocation of carbohydrates during acclimation processes, such as the synthesis of cell walls and starch. Under normal physiological conditions, sucrose synthase has a very low level of expression and serves no apparent metabolic function. In leaves and various organs of plants, stress resulted in the stimulation of the expression of *Sus* gene(s) and enhanced stress tolerance. Turhan and Ergin [115] studied the effect of cold acclimation in sweet cherry. The activity of sucrose synthase was higher in the non-acclimated stage than those in the cold-acclimated stage. Klotz and Haagenson [119] studied the effect of cold stress on sugar beet roots and reported that sucrose synthase enzyme activity showed several-fold changes. Abdel-Latif [120] reported that cold shock in wheat seedlings caused an increase in sucrose synthase

enzyme activity. The accumulation of sucrose in cane sugar exposed to LT stress supports the role of this sugar as an osmoprotectant that stabilizes cellular membranes and maintains turgor pressure [121]. Yue *et al.* [122] reported that after cold acclimation total soluble sugars and specific sugars, including glucose, sucrose, and fructose, were constantly elevated during cold acclimation and decreased after de-acclimation in tea plants. Cowie *et al.* [123] reported that in *Arabdopsis thaliana* sucrose had a regulatory role in the acclimation of whole plants to cold and this may be important during diurnal dark periods. Zuther *et al.* [84] reported that sucrose content was higher in acclimated leaves of *Arabdopsis thaliana* than in non-acclimated leaves and recovered back to non-acclimated levels after de-acclimation. Burchett *et al.* [118] also observed that in winter barley plants acclimated at 5/−1°C; day/night, and there was a significant increase in the glucose, sucrose, and fructose content. The sucrose content increased by 4-fold in comparison to non-acclimated plants.

Low-temperature stress resulted in the synthesis of different types of proteins [13]. Proteins are the major players in most cellular events and are directly involved in plant LT responses [124]. Cold stress increased soluble protein content in pepper (*Capsicum annum* L.) varieties [82]. Different plant species have shown that cryoprotective proteins are encoded by a range of cold-induced genes. Specific proteins with antifreeze activity (antifreeze proteins, AFPs), accumulated during cold acclimation in the apoplast, thus enhancing plant resistance against freezing stress [125–127]. These AFPs were identified as chitinase-like proteins, β-1,3-glucanase-like proteins, thaumatin-like proteins, and polygalacturonase inhibitor proteins [127, 128]. They were also present in non-acclimated plants, but at different locations and did not exhibit antifreeze activity, which suggested that different isoforms of PR proteins are produced under LT. Until now, no plant has been reported to have constitutive antifreeze activity. However, different studies reported the accumulation of transcripts and translation products of AFP genes during cold acclimation [128]. A number of studies have shown that after exposure to LT, many PR genes get induced and enhanced disease resistance was observed in plants [129].

Xu *et al.* [130] found that frost-sensitive winter wheat cultivars exhibited high levels of ROS and leaf cell death in response to abrupt freezing stress, whereas significant increases in the relative abundance of antioxidant-related proteins were found in frost-tolerant cultivar leaves. Under LT stress in sugarcane seedling roots, the total soluble protein was higher in the cold-tolerant variety than cold-sensitive variety [60] and helped to tolerate LT stress. Moieni-Korbekandi [52] reported in canola (*Brassica napus* L.) seedling leaves that soluble protein content increased under cold stress. Esra *et al.* [82] reported that in two pepper (*Capsicum annum* L*.*) varieties total soluble protein content was higher under cold stress conditions. These proteomic results emphasize the assumption that freezing-tolerant plants are capable of managing ROS-mediated damage more efficiently than sensitive ones. Sarhadi *et al.* [131] investigated the interrelationship between vernalization fulfillment and expression of LT-induced proteins in wheat genotypes differing in freeze tolerance. Their results showed a clear induction of cold-regulated (Cor)/Lea and antifreeze proteins (AFPs) during cold acclimation in the freeze-tolerant genotype, whereas less induction was observed in the semi-hardy genotype. In winter rye seedlings one of the cold-induced thermal hysteresis proteins was β-1,3-glucanase [132]. Consequently, these proteins must possess extensive structural similarities with the pathogen-induced basic β-1,3-glucanase in tobacco. Cryoprotection increased linearly with an increase in β-1,3-glucanase concentration. Chang *et al.* [133] reported that the protein in the cell sap of cold-acclimated mungbean seedlings was 60% higher than control seedlings.

*Perspective Chapter: Effect of Low-Temperature Stress on Plant Performance and Adaptation… DOI: http://dx.doi.org/10.5772/intechopen.110168*

Yadegari *et al.* [83] reported that total protein content increased in both acclimated and non-acclimated seedlings of soybean.

Proteins with antifreeze activity were reported to be accumulated in the apoplast during cold acclimation, thereby offering plant resistance against freezing [127]. Winter rye antifreeze proteins (AFPs) enhance freezing tolerance by preventing physical damage caused by ice crystals and may also function as a barrier to inhibit ice formation [126]. These proteins were identified as β-1,3-glucanase-like proteins, and chitinase-like proteins [127, 128]. Their results interestingly revealed that during the cold acclimation process, the production of ice nucleation substances in both the leaf and the crown was suppressed, correlating with the rapid up-regulation of genes encoding the major antifreeze (chitinases, glucanases, and thaumatin-like proteins) and ice recrystallization inhibition proteins. Antifreeze proteins (AFPs) or ice recrystallization inhibition (IRI) proteins ascribe to a category of proteins in plants that allow their survival in sub-zero situations. Sarhadi *et al.* [131] showed the expression of LT-induced proteins in wheat genotypes differing in freeze tolerance. Their results clearly showed the induction of cold-regulated (Cor)/Lea and antifreeze proteins (AFPs) during cold acclimation in the freezing-tolerant genotype. Comparable results were also observed in diploid wild wheat (*Triticum urartu* L.), where cold acclimation increased the abundance of ROSscavenging proteins, LEA/RAB proteins, and dehydrins [134].

Species adapted by natural selection to LT environments have evolved a number of morphological, physiological, and biochemical means to improve survival under prolonged LT stress periods [135]. Cold-adapted species generally have short stature, small leaf surface area, and a high root /shoot ratio. Seedlings subjected to prolonged LT exposure showed chlorosis, wilting, reduced leaf expansion, necrosis, tissue

**Figure 2.** *Factors affected under low-temperature stress.*

damage, and stunting [23]. Numerous studies indicated that an increase in antioxidants positively correlated with tolerance to LT stress in plants [23, 28, 136]. Fahimirad *et al.* [56] reported that winter canola had higher activity of antioxidant enzymes (SOD, CAT, and APX) and lower levels of MDA as compared to spring canola. The study showed a positive correlation between the activities of antioxidant enzymes and cold tolerance in the canola winter cultivar as compared to the spring cultivar. Sato *et al.* [137] reported that under cold stress, rice plants protected themselves from oxidative damage by increased production of various antioxidant species. Dai *et al.* [57] reported that LT-treated barley cultivars showed an increase in peroxidase activity after 72 hours of the recovery period. The activity of peroxidase in the tolerant cultivar (M0103) was significantly higher than in the sensitive cultivar (Chumai). Liu *et al.* [42] reported that LT tolerance in *Avena nuda* L. was probably due to the higher content of proline, SOD, CAT, and POD activities. Cold stress conditions, caused a slight decrease in Fv/Fm ratio in plants that showed tolerance to cold, but a significant decrease was observed in plants that are sensitive to LT [138, 139]. Zhang *et al.* [140] reported that under 0°C treatment, plantlets of the tolerant genotype of strawberry showed a significant increase in peroxidase activity (**Figure 2**).
