**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 [53–55]. 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<sup>+</sup> content in leaves (2.93 to 1.83%) at ASM levels 30% and 40% as compared to 50% ASM level.
