In this study, we tried to elucidate the biochemical response of T.gallica as a response to various stressors such as arsenic, aluminium, or combined stress (Al / As + 200 mM NaCl).
The accumulation of metal As or Al in T.gallica was described previously in our study (Sghaier et al., 2015; 2019) but here we discussed the accumulation of metal concerning fresh weight. As or Al accumulation in plant increased with metal concentration rising in the medium. This result suggested that T. gallica could absorb and accumulate high As or Al concentrations. 100% of plants exposed to 800 µM As(V) remained alive and As or Al concentrations in roots and shoots still increased till the end of treatment (90 days), demonstrating that the plant was not saturated with toxic ions and was able to accumulate more. However, the salt added in the medium decreased As uptake by the plant at 800 µM. Contrarily, Al showed an enhanced uptake by salt addition in the medium. the rising of Al in the salty medium might be attributed to the increased Al3+ uptake and its translocation in complexed form. Meanwhile, T. gallica is salt cedars and it could exclude toxic ions through special cells presented in the leaves by the salt bladders and those explained the diminution of As uptake. Salinity could modify the bioavailability of heavy metals in soil owing to the reduction of metal sorption from the soil and also the displacement of metals from the below-ground part to the above-ground parts of the plants (Wahla & Kirkham 2008). Most plants considered to be tolerant had mechanisms to keep much of their As load in the root (Leonardi et al., 2021). Salinity reduced Cd2+ absorption and translocation in Kosteletzkya virginica (Han et al., 2012), whereas Cd2+ translocation factor rose with the addition of NaCl in Sesuvium portulacastrum (Ghnaya et al., 2007).
Further, Shoots and roots growth were dramatically affected by arsenic stress alone or in combined stress. Reduction in growth could be provoked by water deficiency and nutrient disproportion. Indeed, the depression in root development under As treatments could be caused by water deficiency and nutrient disproportion. The latter led to an inhibited division of endodermal cells provoked by the rise in doses of apoplastic As and which resulted in a decline in root length (Sarath et al., 2022). However, in Al stress alone, roots were unaffected by high Al concentrations while shoots seemed to be altered by the high metal extent which might lead to a variety of cellular toxic alterations regarding cell division, localization and expression of the nucleolar proteins (Zhang et al., 2014). Aluminium was linked to phosphorus (P) in a reduced utilizable and insoluble form giving rise to P deficiency for plant development. The combined stress enhanced the growth of the shoots and had no effect on root growth. Some salt cedars are known to colonize polluted soils (Conesa et al., 2006) and grow on contaminated soils without showing symptoms of intoxication (Manousaki et al., 2008), this was in line with our experiment which demonstrated that any sign of chlorosis or necrosis was detected after 3 months exposed to high concentrations of metals.
Moreover, it was reported that a least decreased shoot and root development of Suaeda maritima was observed under As exposure alone or combined treatments which hypothesized that As stress did not set serious impacts on nutrient metabolism (Panda et al., 2017). Nevertheless, exist genotypes where root development was unaffected even at very high Al doses, demonstrating that species varied in their Al stress reaction mechanisms at the cellular and tissue level (Bojórquez-Quintal et al., 2017). Further, one of the possible reasons for defunding the enhancement of plant growth provoked by Al was the promotion of nutrient absorption. In hyperaccumulator plants, Al could stimulate or had no impact on nutrient metabolism (Bojórquez-Quintal et al., 2017). Indeed, it had been reported that Al could stimulate channels and Mg transporters in Al-resistant plants (Bojórquez-Quintal et al., 2017).
Our funding suggested that severe metals stress alone engendered oxidative stress determined by the increment MDA content. The results obtained clearly pointed out the installation of membrane lipoperoxidation in T. gallica after exposition to different doses of As or Al which reflected the installation of a state of oxidative stress in the studied species. In addition, it also appeared that salt stress, in turn, stimulated membrane lipoperoxidation (Reginato et al., 2014). The MDA content varied depending on the metals type and doses added to the soil. The MDA content was higher in As stress than Al.
In the case of arsenic stress, the overproduction of membrane lipids might be due to arsenic-induced ROS. These could instantly attack the hydrogen atom of a methylene group next to an unsaturated carbon atom. As could also facilitate lipid peroxidation by disrupting the structure of the membrane (Akter et al., 2005). Parallel to our results, arsenic treatment induced the lipid peroxidation of the membrane in rice seedlings (Gaikwad et al., 2020). It was clear that both treatments damaged the membrane by increasing the MDA content. Plants subjected to salt stress were altered via osmotic stress, nutritional deficiency, ion toxicity, etc., inducing overproduction of ROS (Rahman et al., 2021). Raised ROS production was harmful but played a key role in the expression of stress response genes and also in the activation of the metal resistance approach (Sarath et al., 2022).
Al bound to membranes could instantly cause membrane stiffening and therefore might facilitate the catalysis of membrane peroxidation (Wang et al., 2015). Likewise, Shamsi et al. (2008) had shown that Al treatment indirectly produced ROS which caused a severe imbalance including ROS production and antioxidant defence, resulting in increased lipid peroxidation via stimulation of MDA production (Shahnez et al., 2011). Recent studies demonstrated that some modifications in gene expression might participate in the alleviation of Al toxic effects. For instance, Wu et al. (2015) revealed that overexpression of OsPIN2 would impair the Al-activated formation of ROS and reduced lipid peroxidation (LPO) in rice roots.
In contrast, the constant or attenuated level of the malondialdehydes appeared to be a characteristic of salinity tolerant plants (Reginato et al., 2014). In agreement with our findings, several studies reported that both treatments As and salt had no impact on H2O2 levels. For instance, Atriplex atacamensis (Vromman et al., 2016), Suaeda maritima (Panda et al., 2017) and Kosteletzkya pentacarpos (Zhou et al., 2019).) This supports the hypothesis that salinity succoured plants to overcome HMs stress when salt had no impact like (Zn and Pb) or increased HMs accumulation in the aerial part (Zhou et al., 2019).
It’s noteworthy that As led to inhibit SOD activity in T. gallica notably upon As exposure, the level of decline was variable for As stress alone or in combined stress. Similar results were obtained in an arsenate-tolerant Holcus lanatus, where declined activities of SOD were noticed at high levels of As subjection (Hartley-Whitaker et al., 2001). The reduction in growth was the result of an inadequate enzymatic activity to capture the ROS following high exposure to As.
Sharma et al. (2010) demonstrated that Salicornia brachiata could up-synthesis the activity of SOD towards heavy metals like Cd, Ni and As, whereas, at higher concentrations, SOD activity dropped which marked that the oxygen scavenging function of SOD was altered (Zhang et al., 2007). Vitoria et al. (2001) suggested that the activity of SOD decreased due to the metal ions linked to the active centre of the enzyme at elevated doses of heavy metals. In fact, the inactivation of enzymes by interfering with sulfhydryl groups, interaction with the enzyme-substrate complex, or protein active groups, and denaturation of the enzyme protein were the consequence of As exposure (Reboredo et al., 2021).
The supplemented NaCl diminished SOD activity under moderate and severe Cd stress suggesting that NaCl limited the conversion of O2− radicals to H2O2. Nevertheless, with rising doses of NaCl concentration, the POD activities increased and reduced under moderate and severe Cd stresses indicating the enhanced and inhibited scavenging processes of H2O2 (Wali et al., 2017). In Suaeda maritima subjected to individual effects of salinity, arsenic and mixed impact of As and salinity induced a drop of about 30% SOD activity as compared to untreated plants (Panda et al., 2017). Therefore, a decline in SOD activity under stress conditions designated its application in the sequestration of stress-induced O2∙− radicals (Rangani et al., 2016). The activity of SOD in the leaves was unchanged at a lower Al concentration. Meanwhile, an augmentation was noted at 500 µM and a decline in SOD activity was marked at a high level of Al despite that the same activity as control was observed. The SOD activity was significantly hampered after exposure to the salt, however, an increase in SOD activity was recorded hereafter subjection to the combined stress Al and salt.
The augmentation in SOD activity was interrelated to the overproduction of ROS or the overexpression of genes encoding SOD (Israr et al., 2006). Otherwise, Martınez-Domınguez et al. (2010) reported that Spartina densiflora faced oxidative stress in its habitat and harmonized its antioxidative system depending on the level of metal pollution.
GPX activity was not altered by As treatment while the activity of this enzyme was hampered dropping to near to half under 800 µM As. The addition of the salt in the medium altered dramatically the enzyme’s activity. However, a stimulation in GPX activity at low As concentrations combined with the salt was noticed followed by a decrease at a high level of metal combined with salt. Unchanged GPX activity was noted as a response to Al stress demonstrating that the GPX enzyme reached a high level for the trapping of H2O2 generated by Al stress (Panda et al., 2017). Meanwhile, a sharp reduction was observed in combined stress (Al + salt).
Other studies showed different behaviour in GPX activity as a response to Al stress. Kouki et al. (2021) reported that Al-induced a decrease in GPX activity. However, augmentation of GPX around 31% was observed in Vigna trilobata (L.) Verde after exposure to 6 mM Al (Arundhathi et al., 2016). This was similar to the results observed in Helianthus annus L. (Jouili et al., 2011) subjected to Al stress where there was stimulation in GPX activity. In the cell, the GPX enzyme s known as very sensitive toward metal and it had the ability to change its activity (Kouki et al., 2021).
According to our data, the APX activity was stimulated by 200 µM Al. APX was inhibited after subjection to the salt or 200 µM Al + 200Mm NaCl followed by stimulation at 500, 800 µM combined with salt. Also, there was a significant rise in the transcript extent of all APX encoding genes in rice after 8 hr subjection to 20 ppm Al (Rosa et al., 2010). it was noteworthy that APX and GPX showed simultaneous enhancement and inhibition, which had been shown to work together for As tolerance alone or combined with salt.
APX played a key role in the ROS direction in higher plants during stress and because of a higher APX affinity for H2O2 than GPX (Sofo et al., 2015; Anjum et al., 2016).
Additionally, Ben Amor et al. (2006) noticed an enhancement in APX activity under salt treatment by 20 days in Cakile maritima. In a previous study, APX activity was decreased under low concentrations of arsenic and decreased under high arsenic doses in the wheat seedlings (Li et al., 2007). Also, Shri et al. (2009) mentioned that arsenic treatment increased APX activity in rice plants.
More than that, the application of 200 µM As + 200 mM NaCl induced a 54% rise in POX activity compared to untreated plants highlighting POX’s role in Suaeda maritima, antioxidative defence strategies (Demir et al., 2013). This result demonstrated that both treatments (As + salt) caused a generation of H2O2 which was trapped either by APX or POX, in order to preserve relevant H2O2 quantities for stress persuading (Panda et al., 2017). Indeed, APX was included in the ASH-GSH cycle through H2O2 reduction. Hsu and Kao (2007) reported that the pre-treatment of Oryza sativa with H2O2 induced an enhancement in APX activity and protected rice plants following subjection to Cd.
APX could be recognized from guaiacol peroxidase (GPX) in terms of physiological function. GPX had a crucial role in the biosynthesis of lignin and antioxidant defence by consuming H2O2. The activity of GPX varied considerably according to the plant species and the conditions (Foyer & Noctor, 2003).
Hampered peroxidase activity might be due to blockade of essential functional groups, the substitution of essential metals with trace elements, modification in protein structure or integrity, and disruption of signal transduction of antioxidant enzyme. Enhanced peroxidase activity was interconnected of the phytotoxic metal not linked to cell walls or stored in vacuoles (Hsu & Kao, 2007). Anjum et al. (2016) reported that plants were more tolerant to heavy metal stress through enhancement of APX activity.
The obtained results highlighted the complexity of the synergy between the different antioxidant enzymatic activities in order to regulate the antioxidant metabolism of plants.
Taken all together, the stimulation or deactivation of an enzyme in the defence system was based on metal/metalloid types, doses and extent of subjection and plant species. Under harsh stress conditions, a plant could be too fragile to activate adequate antioxidant enzymes to preserve itself. However, the low activity of the antioxidant enzyme might designate the weak stress which was demonstrated by unchanged MDA values among treatments in combined stress (As/ Al + salt) or under Al stress. Antioxidant enzymes were not the only way to take off the most reactive ROS, (HO·). Hence, assaying antioxidant molecule levels, e.g., proline and glycine betaine were required to have a complete scenario of the scavenging capacity (Kofronová et al., 2020).
In fact, to sustain the ionic equilibrium into vacuoles, the cytoplasm cumulates compatible solutes (Parida & Das, 2005). Among these compatible solutes, proline (Pro) and glycine betaine (GB) were found (Moghaieb et al., 2004).
The higher level of stress applied, the greater proline levels registered (Szabados & Savouré, 2010). Besides its function as osmoprotective, proline was responsible for strengthening the antioxidant system and fighting stress damage as a singlet oxygen deactivator (Lehmann et al., 2010) and hydroxyl radical scavenger (Lehmann et al., 2010). This suggested that this molecule could be accumulated in cells towards Cd, Cu, and other heavy metals (Lefévre et al., 2009).
Recently, a proline cycle was involved in the OH− scan was proposed. In this reaction, the proline first caught OH− by H-abstraction, then, a second H-abstraction, which also caught another OH−, giving the P5C. through P5CR / NADPH enzyme activity, the P5C was recycled to proline (Signorelli et al., 2014). Atriplex halimus under Cd stress showed a twofold enhancement in proline content (Lefevre et al., 2009). Many metals tolerant species such as Armeria maritima, Deschampsia cespitosa, and Silene vulgaris, demonstrated high proline content (Sharma & Dietz, 2006).
Our findings boosted the study of Backor et al. (2004) who reported that proline accumulation was one strategy of heavy metal resistance in plants. Also, Shackira and Puthur (2017) signalled the antioxidant and osmoregulatory roles of proline in A. ilicifolius towards metal stress. Similarly, Pavlik et al. (2010) revealed that the rising in the proline extent of Spinacia oleracea was linked to arsenic stress.
Additionally, heavy metals might stimulate the formation of quaternary ammonium compounds like glycine betaine. GB accumulation protected thylakoids and conserved membrane integrity and preserved other cellular structures. It regulated the quaternary structures of complex proteins and saves chloroplasts and photosystem II (Moghaieb et al., 2004). This was due to its role in osmotic adjustment and limiting the hampered effect of stress resulting in accelerated restoration of harmed PSIIs (Siddiqui et al., 2010).
In Atriplex halimus Cd exposure induced an increase in osmotic adjustment and as consequence the formation of compatible sugars as glycine betaine (Levèvre et al., 2009). T. gallica showed an augmentation in the proline content after treatment with Al and As, whereas, no variation in GB content after exposure to different As and Al concentrations. The unchanged GB content could be related to not enhancing the expression of BADH, a key gene for glycine betaine biosynthesis (Wali et al., 2016). Indeed, a decline in free amino acid like glycine, cysteine and proline could be caused by the generation of stress-sensitive proteins as metallothioneins, phytochelatins (Sarath et al., 2022). Whereas, the exposure to 200 mM NaCl increased the synthesis of GB in Al stress and at high As concentrations. In line with our results, Wali et al. (2016) revealed an increase of about 20% of glycine betaine leaf accumulation after exposure to 200 mM NaCl. Osmotic adjustment in Salicornia europaea and Suaeda maritima was obtained with a significant rise in glycine betaine (Moghaieb et al., 2004).
Otherwise, low doses of salinity did not improve proline and GB levels in plants that were not imposed to heavy metals. The low concentration did not affect the water uptake of the plant confirming that altered water content proceed as one of the signals for osmolyte synthesis (Sarath et al., 2022). Indeed, the combined use of NaCl with As in Chenopodium quinoa improved the metal resistance of this plant. Similarly, the salt associated with CdCl2 raised the Cd resistance ability of the halophyte Bruguiera cylindrica (Sruthi & Puthur, 2021). Obviously, both these examples boosted that the combined application of NaCl and metal stress in halophytes enhanced the metal resistance of the halophytes more than when it was administered individually (Sarath et al., 2022).