Salinity stress is an extremely complicated problem that interrupts plant growth, development, and thereby productivity (Alaei et al., 2020; Hanafy and Sadak, 2023). In the present investigation, the exposure of flax plants to salt stress significantly impaired the morphological parameters including shoot and root lengths, shoot and root fresh weights, and shoot dry weight (Table 3). These results were in accordance with those obtained by Abdullah et al. (2022). Moreover, salinity reduced stomatal conductance and disrupts photosystems thus promoted the accumulation of ROS such as superoxide, hydrogen peroxide, which stimulated the oxidative degradation of chlorophyll a and b (Table 4) and enhanced membrane peroxidation as evidenced by the significant accumulation of malonaldehyde and causes a greater increase in electrolyte leakage (Fig. 3b & 3c). Meanwhile, the membrane stability index significantly decreased in stressed flax shoots (Fig. 3a). Similar results have been obtained by El-Bassiouny and Sadak, 2016, Alaei et al. (2020), and Abdullah et al. (2022) on flax plants. Salt stress-induced ROS overproduction is one of the major reasons for devastating the morphological, physiological, and biochemical activities of plants. It was reported that salinity reduces stomatal conductance and disrupts photosystems thereby leading to ROS overproduction in plants (Hasanuzzaman et al., 2018). In addition, the decline in chlorophyll contents in salt-stressed flax leaves could be attributed to the destruction of chlorophylls via chlorophyllase enzyme, this destruction or/and the pigment-protein complexes instability (Sachdev et al. 2021 and Hussein and Alshammari, 2022). It was reported that the overproduction of reactive oxygen species disturbs the homeostasis of cells, causing lipid peroxidation and destruction of the membranes thereby increasing membrane leakage and affecting cell viability (Prabha and Negi 2014) and, consequently, reducing crop productivity (Sadak 2022). Meanwhile, the foliar application of either esculin (antioxidant coumarin glycosides) or digitoxin (Na/K inhibitor) significantly nullified the salt stress hazards and restored the growth of flax plants via buffering ROS and thereby maintaining membrane integrity as evidence by reductions of both MDA and EL. The reduction in H2O2 and O•2− levels in flax plants were concomitants with increments in pigment levels and thereby all investigated morphological parameters, particularly in high-dose digitoxin-treated flax plants (Fig. 3d & 3e).
Notably, plants have developed various defense mechanisms to cope with the adverse salt stress effects. The antioxidant defense system is one of the most important mechanisms involved in the mitigation of salinity-induced injuries displayed by ROS (Sadak et al., 2012). The present results showed that salt-stressed flax shoots exhibited high levels of carotenoids and phenols (Table 4 and Fig. 2a) compared with that the unstressed shoots. In addition, salinity stimulated the activities of SOD, CAT, and peroxidase as well as lipoxygenase enzyme in flax shoots (Table 5). Similar results have been reached by Ahmad et al. (2016), and Sadak et al., (2013) on different plant species. Phenylalanine is involved in the defense response of plants, and it is commonly used as stress indicator. PAL is the Key regulatory enzyme in the phenylpropanoids pathway (Abd Elhamid et al., 2016). PAL is involved in “switching” from the primary to the secondary metabolism of the plant and leads to the formation of a wide range of secondary metabolites (Rohde et al., 2004). Imposition of NaCl salt stress results in the stimulation of PAL and TAL activities in flax plants (Table 5). Foliar application of either esculin or digitoxin significantly stimulated the antioxidant enzyme activities including SOD, POX, and catalase as well as PAL and TAL (Table 5) enzymes under salt stress conditions. Recently, it was reported that PAL and TAL contribute to the biosynthesis and accumulation of secondary metabolites as phenols and lignin (Barros et al., 2016). The effect of salinity on PAL and TAL on salt-stress tobacco was also investigated by Mohagheghian and Ehsan Pour, (2021). On contrary, the lipoxygenase activity was markedly decreased in the glycoside-treated flax plants exposed to salt stress as compared with their reference controls (Table 5). The reduction in lipoxygenase activity was positively related to MDA level as well as EL.
In addition, the accumulation of compatible solutes in treated stressed flax plants, such as total soluble augars, free amino acids, and proline (Fig. 1) sustain the ionic balance of vacuole, neutralize ROS and thereby protect macromolecules and plant organelles from the severe effects of the oxidative stress (Rehman et al., 2021; Bellache et al., 2022, Hanafy and Sadak 2023). It was reported that proline has accumulated in many stressed plant species (Khattab, 2007; Bellache et al., 2022). Moreover, esculin or digitoxin-treated flax plants exhibited greater levels of compatible solutes, including carbohydrates, free amino acids, and proline, particularly in stressed plants exposed to the high dose of digitoxin (Fig. 1). Notably, proline can serve as a compatible solute, osmo-protectant, source of nitrogen and carbon, membrane stabilizer, and ROS buffering scavenger (Rehman et al., 2021; Hussein et al., 2022). It was stated also that the accumulated soluble sugars utilized as osmolytes as well as antioxidant compounds (Colak et al., 2020) thereby contribute to the enhancement of cell membrane stability (Colak et al., 2020).
Furthermore, salinity stress stimulated the accumulation of phenols in flax shoots (Fig. 2a). Such increments in phenols were more pronounced in both esculin and digitoxin-treated flax plants exposed to salt stress. The increment in the accumulation of free phenols in salt-stressed plants might be due to the greater stimulation of phenols biosynthesis (Zhou et al., 2018). It was reported that phenols play a crucial role in plant–protection against environmental stress due to their antioxidant activity (Šamec et al., 2021). Furthermore, ion homeostasis particularly K+/ Na+ is necessary for improving salt-stressed tolerance. It was reported that the uptake, efflux, translocation, and compartmentation of toxic ions particularly, Na+ offer the most important issue for salinity tolerance in plants, and consequently crop productivity (Gupta and Huang, 2014). Salinity-induced ionic imbalance in flax shoots is evidenced by the accumulation of greater levels of Na which impaired the accumulation of K, Ca, Mg, and P ions as compared with those of control unstressed flax plants (Table 6). Salinity significantly altered the cation mineral profiles of flax plants (Table 6). The imbalance of Na+ and other cations resulted in undesired ratios of Na+:/ K+ and Na+/Ca2+. Notably, the ionomic profile of flax is affected by element availability, uptake, transport, and environmental stress (Yadav et al., 2022). The accumulation of excess Na+ competitively inhibits the uptake of some other cations, including K+, Ca2+, and Mg+, P thus leading to an imbalance in cellular homeostasis, oxidative stress, and interference with Ca2+ and K+ functions (Kim et al., 2021). Data of the present investigation showed that foliar application of digitoxin increased nutrient levels in salt-stressed flax shoots, particularly K, Ca, and P, however, decreased Na levels thereby inducing ion and nutrient balance and consequently mitigating salt stress hazards in flax plants. The protective roles of esculin were due to its good antioxidant properties which involved in protecting triglycerides against auto-oxidation (Wang et al., 2016), however, digitoxin might be due to its ability to maintain ion homeostasis via inhibition of Na uptake and increases K and thereby improve the ability to maintain stable plasma membrane (PM) potentials (Patel, 2016).
Notably, Na+ interferes with K+ homeostasis, so maintaining a balanced cytosolic Na+/K+ ratio has been used as a crucial salinity tolerance strategy. In particular, the imbalance in Na+/K+ and Na/Ca2+ ratios altered the plant’s physiological traits, including plant growth and photosynthesis (Evelin et al., 2019). It has been indicated that plant tolerance under salt stress requires a high cytosolic K+ /Na + ratio in the cytoplasm. Similar results have been reached by (Kim et al., 2021).A higher Na+/ K + ratio is an indicator of salt sensitivity, and this suggests that Na+-mediated damage to plants (Kim et al., 2021).
In addition, salt reduced endogenous IAA levels in flax shoots (Fig. 2b). This reduction might be due to salt-induced IAA degradation and /or reduction in its biosynthesis (Bano Samina, 2010). Such results are in accordance with those of Sadak et al. (2019). In the contrast, foliar application of either esculin or digitoxin markedly increased the endogenous levels of IAA in stressed and unstressed flax shoots. The increments in IAA levels in unstressed and stressed flax plants treated with esculin and digitoxin might be attributed to glycoside moiety which might be involved in the improvement of biosynthesis of IAA and/or their antioxidant activity which protected IAA from oxidation.
Yield and yield components of Flax plants treated with foliar application of cardiac glycosides digitoxin and antioxidant glycoside esculin are shown in Tables 7 & 8. Digitoxin and esculin treatments markedly stimulated different yield parameters under stress and normal conditions. Such enhanced effects might be due to presence of sugar moiety in these compounds. It was reported that the activity of cardiac glycosides is enhanced several-fold due to the presence of the sugars in these compounds as well as the structure of the sugar (Cornelius, et al., 2013).
Furthermore, esculin and digitoxin significantly improved the quality of flax seeds as estimated by oil%, carbohydrates%, and proteins% (Table 8). The stimulatory effect of esculin and digitoxin might be attributed to the increase in endogenous promoters’ content (particularly IAA) which induces linear growth and development of plants (Sadak et al (2019). Moreover, esculin and digitoxin exhibited antioxidant activity which stimulate the biosynthesis of photosynthetic pigments and thereby metabolites synthesis content which could lead to an increase in seeds weight. In addition, the increases in oil% could be attributed to the increase in the growth in vegetative parameters and nutrient uptake (Khodary, 2004).
Gas-liquid chromatography analysis revealed that salinity stress caused increases in saturated fatty acids including palmitic, Stearic, Behenic, and Lignoceric. Meanwhile, reduced unsaturated fatty acids compared with control plants. These obtained results have been reached also by many investigators who stated that the unsaturated fatty acids content decreases with stress in some oil crops (Ramadan et al., 2019: Bakyani et al, 2022: Hanafy and Sadak 2023). It was reported that salinity stress at the seed development stage was found to decrease photosynthetic assimilation and carbon partitioning to seeds and increase the activities of enzymes involved in fatty acid oxidation (Mohamed et al., 2020). In addition, the exposure of flax plants to either esculin or digitoxin markedly increases the levels of unsaturated fatty acids particularly Oleic and Linoleic acids (Table 10). On the other hand, saturated fatty acids markedly decreased. Ramadan et al (2019) reported that the percentage of unsaturated fatty acids proved the efficiency of desaturation in oil. Indeed, the increments in the total unsaturated/saturated fatty acids ratio (TUS/TS) by esculin and digitoxin in the yielded oil become more favorable for human consumption. Polyunsaturated fatty acid (PUFA) lowers the risk of diseases related to cholesterol oxidation.
It was also noticed that the straw yield significantly increased in response to esculin and digitoxin treatments (Table 9). Notably, the present results also showed that fiber length, width, and fiber percentage were markedly increased in the straw of esculin, and digitoxin-treated plants exposed to normal and salt condition (Table 9). Indeed, the changes in fiber constituents in terms of cellulose and lignin were illustrated in figures (4a & 4b). On contrary, the Application of either esculin or digitoxin significantly reduced the percentage of lignin in flax straw grown in normal and stressed environments (Fig. 4b). The reductions in lignin content of treated straw were positively related to the glycoside concentrations. Meanwhile, cellulose percentage was markedly increased in esculin and digitoxin-treated straw at the two investigated concentrations (Fig. 4a). The reduction in growth and yielded seed percentage as well straw yield in salt-stressed treated plants was concomitant with a decline in different physiological and metabolic processes of stress including reductions in water uptake, chlorophyll content, photosynthesis, transpiration rate, nutrient availability, stomata conductance, and root hydraulic conductance ( Dubey et al., 2020: Sadak et al., 2022).
Recently it has been reported that cardiac glycosides as esculin which belong to coumarin glycoside compounds. These compounds comprise a carbohydrate moiety glycosidically bound to a coumarin moiety. Consequently, esculin showed good antioxidant properties, protecting triglycerides against auto-oxidation (Wang et al., 2016) thus protecting and maintaining cell membrane integrity. Furthermore, cardiac glycosides were utilized as specific inhibitors of Na+ /K+ -ATPase (EC 3.6.3.9), so digitoxin inhibited Na uptake and increased K and Ca, P as well as Mg in flax plants. It was postulated that these compounds contribute to establishing and maintaining the electrochemical gradient across the plasma membrane, which is critical for physiological processes such as osmotic regulation, and ion homeostasis as well as secondary transport of many organic and inorganic substrates (Kaplan, 2002) and thereby improve growth and productivity of flax plants. It was reported that the activity of cardiac glycosides is enhanced several-fold due to the presence of the sugars in these compounds and the structure of the sugar(s) has a dramatic influence on the activity of the cardiac glycoside (Cornelius, et al., 2013).