In this work, we have analyzed the impact of long-term soil salinity stress on preconditioned and non-preconditioned C. glauca plants, through physiological and biochemical performance and the related expression of key target genes.
Gas exchange, intrinsic water use efficiency, chlorophyll fluorescence, and electrolyte leakage
Gas exchange and chlorophyll fluorescence are the major methods for plant photosynthetic study, particularly under environmental stress. Salinity has a significant impact on photosynthetic carbon metabolism via stomatal closure, which limits CO2 diffusion to carboxylation sites, a decrease in mesophyll conductance to CO2, and metabolic limitations to leaf photochemistry (Loreto et al., 2003). According to the results presented in this study, salt stress significantly reduced stomatal conductance (gs), net photosynthesis (A), transpiration (E), and intercellular CO2 concentration (Ci) in NPrS plants. Batista-Santos et al. (2015) found that increasing NaCl levels in C. glauca resulted in a significant decrease in photosynthesis, stomatal conductance, and internal CO2 concentration, associated with the concomitante reduction of the levels of key photosynthetic and respiratory enzymes (without inactivation). Preconditioning to salinity alleviated the stomatal restriction, enhanced transpiration and intercellular CO2 concentration to a level similar to the plants grown under controlled conditions, and made the decrease in net photosynthesis less pronounced. Therefore, high intrinsic water use efficiency (iWUE) under salt stress indicates that the leaves (specifically, the chloroplasts) struggle to maintain a high photosynthetic performance despite significant stomatal closure. A similar result was observed Batista-Santos et al. (2015) which reported WUE levels > 80 % in plants exposed to 200, 400, and 600 mM NaCl. According to Chaves et al. (2009), a decrease in stomatal conductance (gs) may have stress-protective effects by allowing plant water conservation and improving plant iWUE. The decrease in gas exchange as a whole could be attributed to toxic Na+ and Cl– ions, which reduce photosynthetic electron transport (Ashraf et al., 2012). Photosynthesis inhibition by salinity seems to be partially associated with the PSII complex. Some studies found that salt stress inhibited PSII activity (Netondo et al., 2004; Benzarti et al., 2012), while others found that PSII was highly resistant to salt stress (Lu et al., 2002, Chen et al., 2004; Tarchoune et al., 2012). Our results revealed no effect on the quantum yield of PSII and a slight decrease in the initial fluorescence (Fo) in either treatment which was consistent with the small net photosynthesis (A) decrease, particularly in PrS plants. Similar results were obtained by Batista-Santos et al. (2015) in C. glauca exposed to different NaCl levels. Also, Balti et al. (2021) showed that Fv/Fm was almost not affected by salt stress in eucalyptus. Overall, these findings point to a significant salt tolerance capacity of the C. glauca photosynthetic apparatus. Our results also showed that membrane integrity is maintained in PrS plants, further confirming high salinity tolerance (Mansour, 2013). While we noticed a significant increase in electrolyte leakage (EL) in NPrS plants, Bistgani et al. (2019) showed that EL and Na+ levels in plants have a significant relationship. In fact, higher levels of Na+ in plants result in increased lipid peroxidation and membrane damage (Banu et al., 2009). However, Scotti-Campos et al. (2016), found that C. glauca maintains membrane integrity at salt concentrations up to 400 mM NaCl. The preservation of membrane integrity could be linked to the accumulation of ions in the vacuole and/or the synthesis of compatible solutes in the cytosol, which prevented the loss of cellular turgor (Chaves et al., 2009; Ramalho et al., 2014).
Salt-stressed plants exhibited significantly lower osmotic potential at full (Ψπ100) and zero (Ψπ0) turgor than the control plants. The values of Ψπ100 and Ψπ0 of PrS plants were significantly more negative than those of NPrS and control plants. The current findings are consistent with those of Batista-Santos et al. (2015), who showed that increasing NaCl levels decreased the osmotic potential corrected to full turgor in C. glauca. Navarro et al. (2007) suggested that the decrease in osmotic potential at full turgor may be due to the accumulation of Na+ and Cl- ions or can result from an accumulation of organic and/or inorganic solutes. According to Flowers et al. (2015), osmotic adjustment is possibly the main adaptive mechanism used by plants to limit the osmotic effects of salt stress. Also, Munns (2002) suggested that salt stress frequently causes osmotic adjustment, which is thought to be an important mechanism for maintaining water uptake and cell turgor under stress conditions. This is consistent with our findings showing a substantial increase in OA for PrS plants that was 1.6-fold higher than NPrS ones. A greater decrease in Ψπ100 and Ψπ0 for PrS plants, combined with greater osmotic adjustment, may grant them the ability to take up water, despite the increase in osmotic potential of the soil solution due to salinity (Patakas et al., 2002; Flowers et al., 2015). As a result, PrS can sustain their turgor for a long time before reaching the loss of turgor point (Abidine et al., 1993), which is compatible with reduced RWC0 values and enhanced cell membrane flexibility. This allows gas exchange to be maintained for longer periods (Abidine et al., 1995), as seen in this study. Plants that live in environments with dynamic salinity changes may benefit from flexible cell walls (low ɛ) because cell walls stretch and contract to maintain osmotic equilibrium with the environment (Kirst 1990; Touchette 2014). The fact that PrS has a lower modulus of elasticity (ɛ) than NPrS further supports this concept. This flexibility allows the plant to experience significant variations in the water content of the apoplast without affecting the dynamic structure of the cell walls (Clifford et al., 1998). This is confirmed by the significant increase in the apoplastic water content (AWC) in preconditioned (PrS) plants, compared to the control and the non-preconditioned ones (NPrS).
Chlorophyll, proline, soluble sugars and malondialdehyde (MDA) content
The chlorophyll content was significantly reduced in non-preconditioned and preconditioned plants at least extent in these latter. This result was consistent with the findings of Claver et al. (2020) who found a considerable decrease in chlorophyll concentration in C. equisetifolia at various NaCl levels. The decrease in chlorophyll could be related to the accumulation of Na+ ions, which inhibits particular enzymes involved in the synthesis of photosynthetic pigments (Sayyad et al., 2016), and loss of chloroplast membranes (Ceccarelli et al., 2010), and damage of chloroplasts caused by oxidative stress. (Gill and Tuteja, 2010).
Plants synthesize a range of organic solutes, including proline, soluble sugars, and others, which are referred to as osmolytes. The accumulation of osmolytes in plants exposed to salt stress has been linked to the plants' ability to survive and adapt to salinity conditions (Slama et al., 2008). In fact, osmolytes are osmoprotectant solutes that improve the cell's ability to retain water without interfering with normal metabolism (Singh et al., 2015). Besides, they protect plants from oxidative damage by inhibiting ROS production (Saradhi and Mohanty, 1993). This is consistent with our findings showing a considerable accumulation of soluble sugar and proline under salt stress, with a higher accumulation in PrS plants. The results corroborate the findings of Jorge et al. (2019) showing the importance of the secondary metabolome, namely of the flavonoid-based antioxidant system, in complementing the scavenging of reactive oxygen species (ROS) associated with the ascorbate-glutathione cycle (Scotti-Campos et al., 2016; Jorge et al., 2017a,b, 2021 ). This highlights the importance of both enzymatic and non-enzymatic scavenging components in the control of oxidative stress and, thereby, their key role in stress tolerance enhancement. The high accumulation of proline confirms C.glauca salt tolerance and performance under saline conditions (Jorge et al., 2017a, Claver et al., 2020). It is worth noting that proline preferentially accumulates in the cytoplasm and is involved in the regulation of salt tolerance mechanisms such as cellular enzymes and structural protection and can contribute to osmotic adjustment (Sanchez et al., 2004). Under salt stress, MDA levels increased, although this effect was less noticeable in PrS plants. This may be due to its higher salt tolerance. These results agree with the findings of Scotti-Campos et al. (2016) which showed non-significant variations along the stress imposition in C.glauca plants. With different plant species, Shalata and Tal (1998), and Juan et al. (2005) have concluded that MDA increased considerably in salt-sensitive lines than in salt-tolerant lines. This increase in MDA incidence could be related to photosynthetic impairment, insufficient enzymatic antioxidant activity, and ascorbate declination (non-enzymatic antioxidant) (You et al., 2015; Nxele et al., 2017).
Under salt stress, the number of phenolic compounds in the shoots and roots of both treatments increased. In NPrS plants, the increase was more noticeable. These findings suggest that salinity has a significant impact on the secondary metabolism of C. glauca, possibly as a defense mechanism and biochemical adaptation to environmental stress (Dixon and Paiva, 1995). Increasing phenolic compound content has been observed also in buckwheat sprouts under treatment with various concentrations of NaCl (Lim et al., 2012). Hajlaoui et al. (2009) suggested that NaCl stimulates the synthesis of new polyphenolic derivatives that are potent antioxidants. Phenolic contents are important protective components of plant cells (Ashraf et al., 2010). It has also been reported by Parida et al. (2004) that high levels of phenolic compounds attenuate the ionic effects of NaCl. In previous investigations into numerous plants (e.g. Wahid and Ghazanfar, 2006; Ksouri et al., 2007), salt-resistant cultivars were shown to accumulate more polyphenols than salt-sensitive cultivars.
Effects of salinity and preconditioning on the levels of salt-tolerance gene transcripts
We analyzed the effect of both salt treatments on the expression of a set of genes coding for proteins that have been previously associated with response to salt stress (Duro et al., 2016; Fan et al., 2017; Graça et al., 2020). The expression of Glyceraldehyde 3-phosphate dehydrogenase (CgGAPHD), Ascorbate peroxidase (CgApx) and glutathione peroxidase (CgGPX1) increased significantly under salt stress, particularly in PrS. Plants produce significantly more ROS, including H2O2, in their chloroplasts and peroxisomes when exposed to salinity (del Rio et al., 2006). Ascorbate peroxidase catalyzes the conversion of H2O2 to H2O and O2 and scavenges ROS to protect plants from the toxic effects of ROS accumulation (Chen et al., 2015). The genes that code for APXs are therefore crucial for maintaining ascorbate (AsA) and glutathione (GSH) levels, which are directly or indirectly involved in maintaining high photosynthetic rates in plants under adverse environmental conditions (Foyer et al., 2011). According to Teixeira et al. (2006), the increase in APX mRNA levels in response to salinity stress may maintain the high activity of APX in the cytosol to protect cellular components from ROS-induced oxidative damage. Furthermore, Yin et al. (2019) showed that PtomtAPX is dual-targeted to both the chloroplast and mitochondria of Populus tomentosa and that it exhibits the same expression pattern under salt stress. In French bean seedlings, Nageshbabu et al. (2013) found that the expression of APX-coding genes was up-regulated by salinity and drought stresses, indicating their function in molecular regulation mechanisms. For example, anaerobic stress induces the expression of GAPC3 and GAPC4 in maize (Manjunathet al., 1997), while overexpression of the rice cytosolic gene OsGAPC3 improves salt tolerance (Zhang et al., 2011). In response to salinity stress, numerous plant species have shown an increased accumulation of GPx transcripts probably to detoxify stress-induced ROS (Sreenivasulu et al., 2004; Islam et al., 2015). The accumulation of excess ROS within the cell is a common result of all the stresses that plants are subjected to (Ghosh et al., 2014). Our data showed that succinate dehydrogenase (CgSHD) and copper-zinc superoxide dismutase (CgSOD1) genes were overexpressed in PrS plants. According to Ruth et al. (2002), SOD genes are the first line of defense against oxidative stress. The protective effect of SOD genes against salt stress has been described by Tanaka et al. (1999) and Badawi et al. According to Tanaka et al. (1999), rice transformed with yeast MnSOD was tolerant to salt stress to around 100 mM NaCl. Badawi et al. (2004) transferred rice cytosolic Cu/ZnSOD to tobacco chloroplasts and the transformed tobacco plants could then withstand NaCl stress up to 300 mM. Shafi et al. (2015) demonstrated that SOD genes cause substantial lignin deposition in the vascular system and interfascicular cambium. The enhanced lignification, along with the accumulation of osmoprotectants (proline and soluble sugars), are also crucial mechanisms that boost salt stress tolerance. According to the genetic evidence provided by Gleason et al. (2011), SDH participates in the localization of mitochondrial ROS that regulates plant stress and defense responses. Also, Acevedo et al. (2013) demonstrated that drought up-regulates IpSDH1 expression in a drought-tolerant Ilex paraguariensis genotype and that this up-regulation was associated with a significant increase in succinate dehydrogenase activity in the absence of mitochondrial damage. Altogether the over-expression of these antioxidant genes would explain the maintenance of a high photosynthetic rate and the chlorophyll content in C. glauca plants under saline stress, in particular in preconditioned plants. The results are supported by the proteomic analysis in C. glauca showing a remarkable pattern of accumulation of proteins involved in photosynthetic metabolism, and oxidative stress response, associated to two interacting networks: metabolic pathways and biosynthesis of secondary metabolites; and protein processing and export, carbon metabolism, and peroxisomal metabolism (Graça et al., 2020; Ribeiro-Barros et al., 2022). The overall set of results suggest that under salinity stress conditions, the triggered protective mechanisms might prevent a significant impact at the photochemical efficiency and the biochemical performance levels in C. glauca, explaining its high performance in saline soils.