**4.5 Antioxidant defense mechanism**

Drought stress triggers cellular dehydration, accumulation of low molecular compounds (osmolytes) like glycine betaine, proline, sugar, alcohols, increased abscisic acid levels, increased expression of genes, excessive generation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide (H2O2) and hydroxyl radical affecting cellular structures and metabolism. Generation of ROS [superoxide radical (O2 •−), the hydroxyl radical (OH− ), hydrogen peroxide (H2O2) and singlet oxygen (1 O2)] is observed as an outcome of the metabolic perturbations caused by osmotic effects of salt or dehydration stress as well as ionic toxicity of salt stress, particularly in chloroplast and mitochondria which ultimately leads to membrane leakage, lipid peroxidation, protein degradation and reduced enzyme activities.

*Physiological and Molecular Adaptation of Sugarcane under Drought vis-a-vis Root System Traits DOI: http://dx.doi.org/10.5772/intechopen.103795*

Elevated production of ROS can seriously disrupt cellular homeostasis and normal metabolisms through oxidative damage to lipids, protein, and nucleic acid. Hydrogen peroxide is considered as one of the potential ROS which inhibits the functioning of the Calvin cycle. To mitigate the ROS-induced oxidative effects, plants have an antioxidant defense system that involves the generation of non-enzymatic and enzymatic antioxidants. Non-enzymatic antioxidants include phenolics, flavonoids, tocopherols, ASC, and GSH. Enzymatic antioxidants include superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), as well as the enzymes of the ascorbate (ASC)–glutathione (GSH) cycle [GSH reductase (GR), ASC peroxidase (APX), monodehydroascorbate dehydrogenase (MDHAR), and dehydroascorbate reductase (DHAR)] that detoxify ROS [66–70].

Ascorbate peroxidase detoxifies hydrogen peroxide using ascorbate for reduction is present in chloroplasts, cytosol, mitochondria, apoplast and peroxisomes. By contrast, CAT is only present in peroxisomes, but it is indispensable for ROS detoxification during stress, when high levels of ROS are produced [71]. In addition, oxidative stress causes the proliferation of peroxisomes [72]. Catalase can be used to reduce hydrogen peroxide levels in the peroxisomes but it is absent in chloroplasts. The role of catalase is filled by specific ascorbate peroxidase. This peroxidase uses ascorbic acid as a hydrogen donor to break down hydrogen peroxide [73]. Water stress (PEG treatment) led to a significant increase in the activity of the antioxidant enzyme like CAT, POX, APX and SOD. Statistically, significant higher SOD activity was observed in salt (by 32%) or PEG (by 27%) stressed plants over the control in sugarcane callus of variety Co 86,032. CAT activity did not differ significantly in stressed and control plants [74]. Cia et al. [75] studied the antioxidant stress response of drought-tolerant (SP 832847 and SP 835073) and drought-sensitive (SP 903414 and SP 901638) sugarcane varieties to water deficit stress, which was imposed by withholding irrigation for 3, 10 and 20 days. SP 832847 exhibited higher CAT and APX activities than the other varieties in the early stage of drought, while the activities of GPOX and GR were the highest in the other varieties at the end of the drought stress period. Boaretto et al. [57] observed that the basal activity of CAT at 70% SAWC was greater in IACSP 95–5000 than in IACSP 94–2094. However, substantial increases in the total CAT activity were observed for both cultivars only at 30% SAWC. Ngamhui et al. [76] reported that under drought stress, tolerant sugarcane variety KK3 accounted 15% and 30% higher activity APX and POX, respectively as compared to variety SP72. Among three antioxidative enzymes, the highest activity of APX and POX was observed as compared to CAT in both the varieties. The activity of ROS content such as the superoxide radical, hydrogen peroxide and hydroxyl radical can cause oxidative stress and consequently membrane injury which leads to leakage of cellular content, peroxidation of membrane lipids, protein degrading, enzyme inactivation, pigment bleaching and disruption of DNA strands and thus cell death [76, 77]. Accumulation of hydrogen peroxide has not only negative consequences on living cells, but it is also involved in stress signaling and mediating the cellular redox status [78, 79]. Arora et al. [80] reported that as plants close the stomata under water deficit and reduce the internal CO2 concentration, the generation of reactive oxygen species seems to stimulate mechanisms that reduce oxidative stress and so it may play an important role in drought tolerance.

Non-enzymatic antioxidant molecules can work synergistically with enzymatic ROS scavenging mechanisms to protect plant cells against oxidative damage. The nonenzymatic system is composed by ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol, carotenoids, phenolics, flavonoids and proline [81, 82]. Proline (Pro)

is an efficient scavenger of OH. and 1 O2. Furthermore, Pro can function as a compatible osmolyte, molecular chaperone and carbon and nitrogen reserve and balances cytosolic pH [82, 83]. During water stress, Pro is accumulated in plants mainly due to increased synthesis and reduced degradation. Pro biosynthesis from glutamate is catalyzed by the enzymes Δ [1]-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR). Alternatively, Pro can be formed from ornithine that is converted into P5C/GSA *via* ornithine-δ-aminotransferase (OAT) [84, 85]. The observations recorded on antioxidative defense system have suggested possible key characteristics of drought tolerance and noted that low ASM levels induced the antioxidative defense system by increasing ROS and the specific activities of antioxidative enzymes, viz. peroxidase, catalase and ascorbate peroxidase [7]. The specific activity of these enzymes increased in varieties Co 0238 and CoS 767 at 60 and 90 DAP. Severe stress of 30% ASM levels also resulted in a sharp rise in total ascorbic acid content (9.36 to 13.14 mg/g), total soluble proteins (from 9.6 to 13.77 mg/g), and the increase was more in varieties Co 0238 and CoS 767.
