Physiological and Molecular Adaptation of Sugarcane under Drought vis-a-vis Root System Traits

Abstract

Among various abiotic stresses, water is reported as a rare entity in many parts of the world. Decreased frequency of precipitation and global temperature rise will further aggravate the situation in future. Being C4 plant, sugarcane requires generous water for the proper growth. Plant root system primarily supports above-ground growth by anchoring in the soil and facilitates water and nutrients uptake from the soil. The plasticity and dynamic nature of roots endow plants for the uptake of vital nutrients from the soil even under soil moisture conditions. In sugarcane, the major part of root system are generally observed in the upper soil layers, while limited water availability shifts the root growth towards the lower soil layer to sustained water uptake. In addition, root traits are directly related to physiological traits of the shoot to cope up with water limited situations via reduction in stomatal conductance and an upsurge in density and deep root traits, adaptations at biochemical and molecular level which includes osmotic adjustment and ROS detoxification. Under stressed conditions, these complex interactive systems adjust homeo-statically to minimize the adverse impacts of stress and sustain balanced metabolism. Therefore, the present chapter deals with physiological and biochemical traits along with root traits that helps for better productivity of sugarcane under water-limited conditions.

1. Introduction

Global climate change along with abiotic stresses are one of the major constraints limiting factors to crop productivity that influences various agronomic characteristics, such as biomass and other growth traits, phenology, and yield-contributing traits, of various crops. Depletion of water resources, irregular rainfall pattern, impermanent periods of low water availability, moisture-holding capacity of the soil, water losses through evapotranspiration and poor groundwater quality pushing agriculture closer to the water scarcity situations. Generally, the damage to crop plants due drought is unpredictable, but plants experience drought when either the water supply to the roots is limited or the loss of water through transpiration is very high. Severe droughts cause a substantial decline in crop yields through negative impacts on plant growth, physiology, and reproduction. The plant response to drought varies from species to species and cultivars, phenological stages of the plant, and the duration of plant exposure to the stress. Under drought, along with nutrient and water relations, vital physiological traits viz. chlorophyll content, photosynthesis, stomatal conductance, chlorophyll fluorescence, assimilate partitioning, canopy temperature depression, membrane stability, impaired radiation use efficiency and reduced absorption of photosynthetically active radiations were seriously disrupted. At the same time, several biochemical and metabolic processes contributing to general growth and development were constrained along with the production of reactive oxygen species (ROS) that negatively affect cellular homeostasis, expression of genes, synthesis of hormones. Sugarcane, a C₄ plant, with a long life cycle is highly sensitive to water deficit and divided into four major phenophases, i.e., germination, formative/tillering, grand growth and maturity. Among these, the formative stage is considered as highly sensitive to drought stress [1, 2] as it required 550 mm of rainfall [3], causing a significant reduction in cane yield up to 50% [4]. Water is the major constituent of cane and approximately 2.97 lakh ha of cane area is prone to the drought that distributed cellular osmotic balance, decreased turgor, inhibited photosynthesis, inhibition of enzyme activities and cellular processes, root architecture and morphology and leading to a reduction in yield [5, 6, 7]. In point of these, this book chapter mainly focuses on the importance of physiological, biochemical, molecular traits along with root traits interventions related to drought for better management.

2. Effect of drought stress on sugarcane production

Sugarcane, the world’s major industrial C₄ crop mainly grows in the tropic/subtropic regions and used for sugar and bioenergy. In India, the tropical regions (Maharashtra, Andhra Pradesh, Tamil Nadu, Karnataka, Gujarat, Madhya Pradesh, Goa, Pondicherry and Kerala) shared about 45% and 55% and the Sub-tropical region (Uttar Pradesh, Bihar, Haryana and Punjab) accounted for about 55% and 45% of the total sugarcane area and production in the country, respectively. Prevailing climatic factors particularly atmospheric CO₂, temperature, precipitation are the key factors for sugarcane production worldwide. Among these, the availability of water in addition to atmospheric CO₂ is considered to have been a major driving force for the evolution and the ecological success of C₄ plants [8, 9]. Drought or prolonged dry periods are limiting crop yields in tropical worldwide areas, including sugarcane production by 40–60% [10, 11]. Sugarcane is a globally important crop for food and industrial input production, accounts for 60% of world sugar output as well as bio-ethanol and energy generation [12]. Sugarcane is also known as water guzzler consumes about 22.5 million liters of water per hectare during its long growing cycle. In tropical India, the total water requirement of the crop for optimum growth varies from 2000 to 3000 mm inclusive of rainfall, while in Sub-tropical India, the water requirement is 1400–1800 mm. Drought severely depresses cane yield to the tune of 40–60%, whereas, the sucrose formation and sucrose recovery are reduced up to 5%. The severe drought causes the complete failure of crop and sucrose recovery. India is the largest consumer and the second-largest producer of sugar in the world. The average annual production of sugarcane is around 35.5 crore tonnes which is used to produce 3 crore tonnes of sugar. In India, most of the country’s irrigation facilities are utilized by paddy and sugarcane, depleting water availability for other crops. Pressure on water due to sugarcane cultivation in States like Maharashtra has become a serious concern, calling for more efficient and sustainable water use through alternative cropping pattern. This is especially important in regions where groundwater use has reached a critical and overexploited stage or where more than 50% surface water is used for irrigating sugarcane alone. Drought is a serious problem, but under production processing. There are various strategies to solve this obstacle, C₄ plants are often considered to have mastered the art of drought control particularly as they are able to maintain leaf photosynthesis with closed stomata. Generally, C₄ plants have high water use efficiencies (WUEs), and the presence of the CO₂-concentrating mechanisms makes C₄ photosynthesis more competitive in conditions that promote carbon loss through photorespiration, such as high temperatures, high light intensities, and decreased water availability [13]. C₄ photosynthesis is characterized by the presence of a metabolic CO₂ pump that concentrates CO₂ in the vicinity of the main enzyme of carbon dioxide fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase [13, 14]. This confers several important advantages in terms of WUE because it allows high rates of photosynthesis to occur even when stomata are closed while limiting flux through the photo-respiratory pathway [14]. Therefore, an emergent choice for sustainable sugarcane production is the identification of water-efficient cultivars or providing water for irrigation.

3. Root Biology

Roots provide anchorage and facilitate the acquisition of water and nutrients from the soil, hence understanding the multi-faceted aspects of root biology are key to plant water management [15]. Plants exhibit a high degree of root plasticity and can transiently adapt to various environmental stresses. Sugarcane is a deep-rooted crop, owing to its long growth cycle. Sugarcane roots may be highly branched superficial roots, downward oriented buttress roots or deeply penetrating agglomerations of vertical roots known as rope roots. Sugarcane root systems may reach depths of about 1.5 to 6.0 m [16]. Nevertheless, drought stress usually leads to the formation of deeper root systems, which aid in extracting water from Sub-surface soil [17]. Sugarcane genotypes with drought-tolerant mechanisms cope better under water-limited environments by maintaining high water status and investing a higher proportion of assimilates for root growth under stress. Maintenance of high-water uptake under drought stress is also facilitated by improved root/shoot ratio in drought-tolerant cultivars [18]. Namwongsa et al. [19] observed that roots of sugarcane genotypes showed reduced growth in the upper soil layers (0 to 30 cm) in response to drought stress, whereas growth in the lower soil layers (below 30 cm) increased substantially. Under water-limited conditions, assimilated partitioning towards the roots area relatively higher than to shoots [20]. With the decrease in the soil moisture content, the roots alter their distribution patterns, proliferating more into deeper soil layers for extracting and engaging a larger soil volume for water uptake. As moisture at the soil surface and top soil profile is reduced under drought, deep roots take up water from the deeper profile [21]. Such deep root schemes are characteristic features under drought and is an important consequence to soil drying, allowing some roots to continue their lifecycle under stress. Hence, root architecture and distribution strongly depend upon the moisture content of various soil layers.

Jangpromma et al. [18] noticed that drought stress significantly reduced root length, root surface area, root volume and root dry weight, with negligible effect on the root/shoot ratio. Most of the root morphological traits could fully recover when plants were re-watered following drought stress during the formative phase. The unchanged root/shoot ratio might explain that sugarcane invested in roots under drought conditions to mine more water from deeper soil layers. Drought stress reduced root growth of sugarcane by 50 to 80% when the soil water status reduced to a water potential of −0.07 MPa [22]. Endres et al. [23] reported a drought-tolerant sugarcane genotype with higher root length density and better field performance under stress. Vantini et al. [24] observed differentially expressed genes between tolerant and sensitive sugarcane varieties across different time intervals (1, 3, 5, and 10 days after withholding water) in root tissues. At the beginning of the stress (1 and 3 days), genes encoding proteins with protection function (chaperones, heat shock proteins, antioxidant enzymes and protease inhibitor proteins) were induced in the tolerant variety. Gene encoding a protein involved in ABA-response, a trehalose-phosphatase synthase (enzyme involved in the synthesis of trehalose) and serine/threonine kinase receptors also showed higher expression in the tolerant variety, revealing differences between sugarcane genotypes for water stress protection and adaptive mechanisms. Hence, root systems are more important sink as compared to above ground organs under drought stress, especially during the active growth vegetative stages. Root growth might be indicative of a drought resistance mechanism under water-limited conditions. The positive relationship between root length and soil water content at the end of the drought period in 40 cm soil layers re-emphasizes the advantage of deeper roots for extracting water over extended drought periods. Nonetheless, the association between root length and physiological responses to plant water status are very complicated. Several root systems are considered to be essential in sustaining plant productivity under drought. Overall root growth, branching and distribution pattern is crucial to improve the acquisition of water and nutrients from the soil, and are positively associated with drought resistance and yield performance under stress.

4. Physiological responses of sugarcane under drought

4.1 Plant water relations

Plant–water relations under stress conditions explain how plants control or maintained the hydration of their cells up to an optimal level because it has important implications in the physiological and metabolic processes. It controls almost all metabolic activities within the cell which are dependent on the availability of sufficient amount of water present. Relative water content signifies as an indicator of plant water balance [25] and it indicates the level of cellular and tissues hydration which is imperative for the physio biochemical processes [26]. Plants under water deficit condition tends to have lesser RWC which triggers stomatal closure resulting in decreased CO₂ uptake [27]. Generally, the tolerant sugarcane genotype displays a higher RWC than the susceptible clones and Silva et al. [26] have described that the tolerant clones maintain better RWC (~87%) than the susceptible genotypes (~80%). Almeida et al. [28] also reported a 50% decline in RWC particularly in RB 943365 (drought-sensitive sugarcane genotype) while the tolerant clone RB 72910 (drought-tolerant sugarcane genotype) remained constant at 86% RWC upon exposure to the water stress for 30 days. Medeiros et al. [29] witnessed significant decline in RWC with average values of 88.7 and 90.7% in the varieties RB 867515 and RB 962962 varieties owing to decrease of water in the soil, correspondingly.

Water potential (ψ_w) and Osmotic potential (ψ_s) are the physiological parameter used for recording the extent of stress level in plant. Water deficit induced decrease in leaf water potential led to interfere with plants’ ability to extract water from the soil and maintain turgor [30]. Water potential controls stomatal conductance, which affects transpiration and photosynthesis, and affects root water uptake driven by the potential difference between leaf and soil water. Leaf water potential (ψ_w) was significantly reduced by 34.50% due to drought stress. However, the reduction in water potential was comparatively less in resistant genotypes viz., Co 99,004 and Co 99,012 (26.96 and 30.15%) while the reduction was more than 50% in sensitive genotypes viz., CoVc 93,136 and Co 99,014 [31]. The RB 72910 variety was able to maintain higher values Ψw when compared to RB 943365 variety under water deficit [28]. The water potential (ψ_w) was significantly reduced during stress treatment and values around 11-fold times smaller than those found in the control treatment, on average − 1.19 and − 0.78 MPa in the RB 867515 and the RB 962962 varieties, respectively. On rewatering treatment, a recovery of the plant water status was observed, as expected, with no difference among treatments, the average values of that treatment were − 0.16 MPa in the RB 867515 variety and −0.14 MPa in the RB 962962 variety [29]. Osmotic potential (ψ_s) is another physiological parameter used for recording the extent of stress level in plants. Reduction in osmotic potential of leaves has been observed under drought stress in sugarcane cultivar [32]. Basra et al. [33] studied the water relations in drought sensitive (BL-4) and drought tolerant (CP 43/33) sugarcane varieties by exposing to 200 mol m⁻³ mannitol solution and found that decline in turgor was faster in BL-4 than in CP 43/33 with time. Bulk leaf osmotic pressures and cell wall solutes were higher in BL-4 than in CP 43/33. Biancos et al. [34] observed the maximum difference in osmotic potential, between well-watered and stressed plants (i.e., the maximum osmotic adjustment) of about −0.5 and − 0.6 MPa for mature leaves and shoot tips in sugarcane, respectively. Pooja et al. [35] noted a significant decline in leaf RWC, leaf osmotic potential and leaf water potential in four sugarcane varieties exposed to drought stress. In another study, Pooja et al. [2] found that sugarcane clones Co 05011 (78.77%) and Co 0238 (76.88%) showed/maintained better RWC under stress conditions with a mean reduction of 16.70% over Co-canes RWC.

4.2 Growth analysis

The leaf area fairly gives a good idea of the photosynthetic capacity of the plant. The reduction in leaf area index (LAI) under water stress was due to reduced leaf area and a number of green leaves per stalk. Water stress had a greater effect on the expansive growth of lamina than its dry mass. Among different sugarcane varieties, water deficits reduced LAI and the biomass of sugarcane throughout the growing period. Inman-Bamber [36] observed that the specific leaf area (SLA) was lower in drought-treated plants as compared to control plants. da Silva & da Costa [37] has observed a significantly higher reduction in LAI under rainfed conditions than in irrigated conditions. Gomathi et al. [31] reported that the drought treatment caused an average reduction of 27.4 and 17.4% in leaf area and LAI, respectively in sugarcane. Varieties Co 99,004 and Co 99,008 transpired less water and showed a relatively higher photosynthetic rate with significant improvement in growth attributes, viz., shoot leaf production and LAI. LAI showed significant variation among the genotypes and treatments indicating the sensitivity of these parameters to drought treatment. The genotypes Co 99,004, Co 99,012 and Co 99,006 recorded higher LAI of 3.53, 3.40 and 3.06, respectively even under drought conditions indicating their adaptability for leaf area production. Farooq et al. [38] reported a substantial reduction of LAI with increasing drought levels in a sugarcane cultivar.

It is well-established fact that the plant infrastructure is decided by the growth parameters such as, CGR, RGR and NAR. This concept not only involves the final crop yield and its components, but also probes into the physiological events that have occurred early in the growth stages causing variation in yield potential [39]. Singh & Singh [40] reported that 20 days and 40 days irrigation intervals in different sugarcane varieties caused significant loss in dry matter production viz., RGR, NAR, and CGR during stress period when all other conditions were favorable for the remaining period of growth. Ramesh [41] evaluated four commercial sugarcane varieties (Co 8021, Co 419, Co 8208 and Co 6304) under three levels of drought stress (severe, moderate and control) during the formative phase (60–150 days after planting) and found a higher reduction in NAR, LAI, CGR especially under water-limited drought conditions. Farooq et al. [38] reported that NAR was lower at 80 and 60% irrigation coefficient as compared to 100% irrigation coefficient and variety NSG-59 showed higher NAR. Leaf area and cane length exhibited 37.3% and 26.53% reduction under drought conditions in comparison to the control [2].

4.3 Gas exchange traits

One of the more immediate responses of the drought stress is a reduction in the water potential of plant tissues leading to diminished stomatal aperture [42, 43] and consequent reduction in transpiration rate and photosynthesis, as well as longer-term responses such as growth inhibition, and accumulation of osmolytes [44]. In C₄ plants some evidences demonstrate that photosynthesis is highly sensitive to water deficit [45]. Moreover, these plants present low recovery capacity mainly when water deficit exceeds the plant recovery capacity limiting the photosynthesis metabolic pathways [46]. Sugarcane plants when subjected to decreased soil water content under moderate (42%) and severe stress (22%) caused changes in all photosynthetic apparatus, such as stomatal closure, reduction of transpiration and photosynthetic rate, as well as in RWC, photochemical efficiency of photosystem II (PS II), and increase in leaf temperature [47, 48]. The photosynthetic rate and stomatal conductance decreased significantly in drought-tolerant (SP 83–2847 and CTC 15) and sensitive sugarcane cultivars (SP 86–155), when submitted to water deficit however higher reduction percent, was recorded in sensitive cultivar [49]. Medeiros et al. [29] also reported that when young sugarcane plants of two varieties RB 867515 and RB 962962 were subjected to irrigation suspension until total stomata closure, and then rewatered, a significant reduction on stomatal conductance, transpiration rate, and net photosynthesis were observed. RB 867515 showed a faster stomatal closure while RB 962962 slowed the effects of drought on the gas exchanges parameters with a faster recovery after rewatering. Farooq et al. [38] that maximum water use efficiency was observed under 60% irrigation coefficient as compared to 80% and 100% irrigation coefficient and under 60% irrigation coefficient maximum water use efficiency was recorded in variety NSG followed by HSF-240. Water stress also caused a reduction in gas exchange traits and the associated pigments by 56.57% in stomatal conductance (gS), 56.55% in photosynthetic rate (pN), 38.21% in transpiration rate (E), 28.01% in internal CO2 (Ci) and 16.86% in the chlorophyll content [2]. Maximum water use efficiency (pN/E) under drought stress was recorded in Co 0238 (4.12) and Co 98,014 (3.93).

4.4 Osmotic adjustments

The cellular response to turgor reduction is an osmotic adjustment. The osmotic adjustment is achieved in these compartments by the accumulation of compatible osmolytes and osmoprotectants. Through the process of osmotic adjustment, higher plants can survive in dry and saline conditions. In this process, an accumulation of organic and inorganic solutes that reduce cellular osmotic potential and a reduction in the hydraulic conductivity of the membranes occurs, possibly by decreasing the number of water channels (aquaporins). Once the turgor is recovered, growth can be restored. The accumulation of compatible solutes is often regarded as a basic strategy for the protection and survival of plants under salt stress. Osmolytes are the organic compounds that play role in maintaining fluid balance as well as cell volume. In situations where increased external osmotic pressure tends to rupture the plant cells, certain osmotic channels are switched on to allow the efflux of certain osmolytes. As these osmolytes move outside, they carry water with themselves preventing the cell from bursting out. Sugars, alcohols, amino acids, polyols, tertiary and quaternary ammonium and sulphonium compounds are some examples of such osmolytes. A variety of compounds such as amino acids and amides (e.g., proline), ammonium compounds (e.g., betaine) and soluble carbohydrates act as compatible solutes. Proline, which is widely found in higher plants, accumulates in stressed plants in larger amounts than other amino acids [50]. Proline is a strong source to store carbon, nitrogen and a purifier of free radicals. Proline also maintains the structure of cell membrane and proteins [51, 52] and contributes to membrane stability [53, 54, 55]. It may also act as a signaling regulatory molecule able to activate multiple responses that are components of the adaptation process [56]. Boaretto et al. [57] reported that leaf proline content in IACSP 96–2042 sugarcane genotype was significantly increased about 2.3 to 2.7 times as compared to SP 87–365 under severe water-deficient conditions. Among the varieties, the differential accumulation of proline may be due to the response of a variety towards the environment [58]. The overproduction of proline may also mean a greater stress impact in Co 86,032 as compared to CoC 671, thus, rendering higher salt tolerance in CoC 671 than Co 86,032. Proline has also been reported to accumulate to maintain the osmotic potential of the plant cell under stress. Medeiros et al. [29] reported that free proline content was significantly increased in drought-affected sugarcane plants, i.e., 81.2% in RB 867515 variety and 72% in RB 962962 variety as compared to control plants. After rewatering, these values returned to normal levels.

Sugars were the main solutes that contributed to osmotic adjustment (OA) particularly in growing leaves. According to Zhou & Yu [59], these changes are related to the activation of responses to cope with this adverse environmental condition, to assist in the maintenance of cell water relations. The accumulation of soluble carbohydrates during water-deficient is considered a plant response to maintain hydration of the shoot and also protect enzyme and membrane system through the stabilization of proteins and lipids [60, 61]. Drought caused increases of soluble sugars content (SS) in sugarcane variety IACSP 96–2042 and IACSP 94–2094 sugarcane cultivar. On the other hand, water withholding increased non-structural carbohydrate content (NSCC) in IACSP 94–2094 and IACSP 96–2042. Under well-hydrated conditions, SP 87–365 had the highest NSCC when compared to the others, which did not vary due to water deficit [57]. Medeiros et al. [29] reported that soluble carbohydrate increased under water suppression, drought treatment increase was 51.2% in RB 867515 variety (sugarcane) and 28% in RB 962962. After rewatering, these values returned to normal levels, in the carbohydrates content of the RB 962962 variety, which did not differ from the water suppression and control treatment, such as in the RB 867515 variety that did not differ from the water suppression treatment. The alteration of protein synthesis or degradation is one of the fundamental metabolic processes that affect drought tolerance. Medeiros et al. [29] reported that amino acids and protein were significantly increased in drought-affected plants by 23.5 and 27% in sugarcane variety RB 867515 variety and 51.1 and 31.82% in variety RB 962962, respectively as compared to control plants. After rewatering, these values returned to normal levels. With other osmoregulatory, proteins were the major contributors to the osmoregulation of both varieties. Jangpromma et al. [62] reported accumulation of an 18 kDa protein was K86–161 sugarcane line which was subjected to progressive water stress for 20 days. Ngamhui et al. [63] reported a 16.9 kDa class 1 heat shock protein and two isoform elongation (EF-Tu) proteins, which are associated with heat tolerance under moisture stress in sugarcane variety Khon Kaen 3. Pooja et al. [35] observed approx. Two-fold increase in total soluble carbohydrates, four folds in proline and two-fold increase in lipid peroxidation under severe water stress conditions of 30% available soil moisture (ASM).

Potassium is a major ion which helps in the regulation of osmotic pressure, providing water maintenance at cells and plants textures, activation of various enzymes and coordination of opening or closing of stomata which may cause more air and plant evaporation. Ge et al. [64] observed that drought stress-induced sharp decreases in total K content and its uptake in maize organs at different developmental stages and, in particular, detrimentally affected the nutrient uptake capability of roots. Severe drought stress caused more deleterious effects than moderate drought stress on total K uptake by plant organs. Errabii et al. [65] reported that K content decreased in mannitol-induced stressed in sugarcane calli and it shows that inorganic solutes seemed to have no contribution in the osmotic adjustment in mannitol-induced stressed sugarcane calli. Such disruptions were due to the water outflow and the leakage of essential ions such as potassium and calcium content in sugarcane calli. Pooja et al. [7] observed significantly reduced K⁺ content in leaves (2.93 to 1.83%) at ASM levels 30% and 40% as compared to 50% ASM level.

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 (¹O₂)] 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. 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, 67, 68, 69, 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 non-enzymatic 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 ¹O₂. 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.

5. Molecular responses of sugarcane under drought

During drought stress, the molecular responses include regulation of various signaling molecules, transcription factors (TFs) and drought induced genes (DIGs), which interact with each other and confer the drought tolerance potential to individual genotypes. Some of the major genes involved in drought tolerance are presented in Table 1. The genes are classified into two major categories: genes encoding functional proteins, and genes encoding regulatory proteins. Functional proteins play an important role in the protection of cells against dehydration, including late embryogenesis abundant (LEA) proteins, aquaporins (AQP), heat shock proteins (HSPs), ion transporters and metabolic enzymes. Regulatory proteins comprise calcium-binding proteins, protein kinases, transcription factors and signaling factors which can cause changes in plant physiology through signal transduction pathways, and regulate the expression of downstream genes [96]. LEA proteins exhibit important dehydrating protective functions during the late stage of embryo development in seeds. They play a key role in dehydration tolerance by capturing enough water into the cell. LEA proteins are composed of a high proportion of polar amino acids which makes them hydrophilic in nature, and can scavenge reactive oxygen species [96]. Transcription factors are one of the master regulators under stress, causing a significant change in gene expression. Manipulation of these regulatory elements may be beneficial for the enhancement of drought tolerance in sugarcane. It is now well established that both the transcription activators and repressors are involved in the drought stress tolerance [97, 98]. The major TF families involved in drought stress responses are WRKY, MYB, bZIP, NAC and DREB, which are perfect choices for genetic engineering to enhance stress tolerance. The most extensively studied TF family is WRKY which helps in regulating different physiological and metabolic processes [99]. The WRKY TF binds to the conserved DNA cis-element W-box to regulate further downstream processing of plant defense. Liu et al. [90] isolated and characterized the expression of WRKY TF of sugarcane using E. coli vector. It was observed that with the increase in the duration of stress, the relative expression level of WRKY increased, hence conferring drought tolerance in sugarcane.

S.No.	Genes	Predicted function	References
1.	LEA protein	Protection of macromolecules such as membranes	Liu et al. [86]
2.	Chaperones		Cagliari et al. [87]
3.	Dehydrin	Protection of cell against dehydration	Hayati et al. [88]
4.	Early responsive to dehydration protein (ERD)	Drought responsive genes	Devi et al. [89]
5.	WRKY	Drought induced Transcription factors	Liu et al. [90]
6.	ABRE-binding factor	Drought induced Transcription factors	Devi et al. [89]
7.	DRE-binding protein 2 (DREB2)	Drought induced Transcription factors	Reis et al. [91]
8.	NAC1 transcription factor	Drought induced Transcription factors	Devi et al. [89]
9.	Ethylene-responsive transcription factor (ERF3 gene)	Drought induced Transcription factors	Devi et al. [89]
10.	Trehalose 6-phosphate synthase	Signaling and trehalose metabolism	Hu et al. [92]
11.	Invertase	Plant development and gene expression regulation	Devi et al. [89]
12.	Sucrose phosphate synthase	ABA signaling and sucrose metabolism	Devi et al. [89]
13.	Calmodulin	Calcium binding protein and signaling molecule	Liu et al. [93]
14.	ABAR	ABA receptor	Devi et al. [89]
15.	P5CS	Proline metabolism and osmotic adjustment	Li et al. [94]
16.	BADH	Glycine betaine metabolism and osmotic adjustment	Chengmu et al. [95]

Table 1.

Important genes involved in the drought stress tolerance.

To better understand the molecular basis of the physiological responses of sugarcane under stress conditions, high throughput gene expression studies have been conducted [48, 100, 101, 102]. Drought stress induces extensive signal transduction networks, comprising TFs, protein kinases and phosphatases [103, 104, 105, 106, 107]. As some of the plant responses under drought are ABA-dependent, both water stress and exogenous ABA treatment lead to several differentially expressed genes [108, 109, 110]. Free proline accumulation was correlated to drought stress tolerance in different sugarcane cultivars [111]. Transgenic sugarcane plants expressing a heterologous P5CS (encoding a proline biosynthetic enzyme), showed a positive correlation between enhanced proline content, increased biomass yield and photochemical efficiency of photosystem II under drought stress [112]. Iskandar et al. [113] indicated that a strong correlation between the expression of DIGs with increasing drought stress. Under severe drought stress, the expression of dehydrin proteins was induced [102], although there is no significant relation to sucrose accumulation [101, 113].