Regarding the germination criteria, the obtained results show that there was no significant effect of the metal salts (CdCl2 and CuCl2) on the germination rate of zucchini seeds, within the limits of the applied concentrations (100 and 200 µM). While the seed vigor index (SVI) showed a significant decrease with increasing metal stress in the medium. Similar results were obtained by Di Salvatore et al. (2008), who did not record significant differences in the germination rates of different plant species (radish, broccoli, lettuce, and tomato) exposed to concentrations of 0 to 1024 µM of Cd, Cu, Pb, and Ni. In addition, the concentration of 2 mM of AlCl3 has no effect on the seeds germination of barley (Hordeum vulgare L.) (Tamas et al. 2003). On the other hand, the growth of the radicle decreased significantly in the third day of germination, and its growth was completely inhibited on the fourth day.
The phytotoxic effect of cadmium (Cd) and copper (Cu) on seed germination and seedling development has been reported by several authors in different plant species, including zucchini C pepo L. (Labidi et al. 2021), Atriplex A halimus (Bankaji et al. 2017), mung bean P aureus Roxb (Patel and Ramana Rao 2009) and rice O sativa (Ahsan et al. 2007).
Recently, Asadi Aghbolaghi et al. (2022) concluded that C pepo seeds have relative tolerance to the toxic effects of cadmium (200 mg/l) at the early stage of germination, where depletion in protein and lipid of endosperm reserves increased, while seed vigor and the activity of antioxidant enzymes such as CAT, SOD, and POX were significantly reduced in germinated zucchini seeds.
The decrease in the zucchini seed vigor index (SVI) under the influence of environmental metal stress is mainly due to the phytotoxic effect of metal ions (Cd2+, Cu2+) on the enzymatic activity of amylases, proteases, and ribonucleases (Sethy and Ghosh 2013), which results in the inhibition of the seed reserves degradation, alters the various biological processes of germination and thus inhibits the growth of the seedling (Seneviratne et al. 2019).
Moreover, the phytotoxic effect of heavy metals (Cd, Cu) on the growth of zucchini seedlings was also reported in the work of Munzuroglu and Geckil (2002) and Labidi et al. (2021), who showed that the dry weights of embryonic axes (hypocotyl + radicle) were more affected by copper salt (CuCl2) whereas the elongation of young zucchini seedlings was more affected by cadmium salt (CdCl2). The accumulation of Cd2+ ions in the developing embryonic tissues exerts a phytotoxic effect, particularly at the radicle cells level, and leads to the inhibition of its growth (Kalai et al. 2014; Rucińska-Sobkowiak 2016).
The toxic effects of heavy metals on the seed’s metabolic efficiency and consequently on the kinetics of reserves degradation cause a remarkable inhibition of the depletion of endospermic nutrients such as soluble sugars and amino acids, which are essential for the development of young seedlings (Mihoub et al. 2005; Solanki et al. 2011). On the other hand, the high toxicity of Cd+ 2 and Cu+ 2 metal ions inside plant tissues can be attributed to the production of oxidizing radicals, which inhibits cell division and/or elongation, thereby reducing young seedling growth (Datta et al. 2011; Seregin and Kozhevnikova 2006).
Analyzes of oxidative stress parameters revealed an increase in the lipid peroxidation activity (H2O2 and MDA content), and the generation of oxidative processes in the embryonic tissues of zucchini seedlings under the effect of metal treatments (Cd, Cu), which is in agreement with the results of (Labidi et al. 2021) on the same species (C. pepo L.), and with those of Jaouani et al. (2016) on pea and other reports on many botanical species under metal stress (Schützendübel and Polle 2002; Lavid et al. 2001; Hu et al. 2007).
Therefore, the formed and accumulated ROS during heavy metal-induced stress react with the polyunsaturated fatty acid to form the lipid radicals and reactive aldehydes, which cause a decrease in membrane fluidity and the alteration of membrane proteins (Gill and Tuteja 2010). It has been proven that metal-stress leads to the production of malondialdehyde (MDA), following an increase in the polyunsaturated fatty acids content, resulting from the degradation of cell membranes, which is the first damaged site by heavy metals (Chaoui et al. 1997; Aravind and Prasad 2003; Dazy et al. 2009).
On the other hand, increase in MDA content may be due to the inefficiency of the antioxidant system and a disorder of the oxidant/antioxidant balance in favor of ROS (Atamer et al. 2008); this is based on the concept of oxidative stress.
However, analysis of the results relating to variation of enzymatic and non-enzymatic antioxidants showed a decrease in the catalase activity (CAT) of the zucchini seedling tissues under the influence of heavy metals (Cd, Cu) in the medium, and the most important catalase activity of the embryonic axes is associated with higher concentrations of H2O2 (Moussa 2004). Similar results were reported by Labidi et al. (2021) on zucchini and Sandalio et al. (2001) on pea.
According to Del Río et al. (2006), the catalase activity recorded in the embryonic axes of unstressed zucchini seedlings (control) can be explained by the concentration of this enzyme in the peroxisome, and its role in the elimination of the formed hydrogen peroxide, as a result of glycolate oxidase. On the other hand, the decrease in CAT activity in embryonic axes tissues under the effect of metallic treatments of Cd and Cu can be interpreted either by the inhibition of the enzyme by the metallic element or by elimination of ROS in the tissues of zucchini seedlings (Moussa 2004).
Our results show that the variations in the SOD activity are similar to those of CAT. The decrease in SOD activity in zucchini seedlings in response to metal-stress (Cd, Cu) was also reported by Labidi et al. (2021) on zucchini (C. pepo L.), Sandalio et al. (2001) on pea (Pisum sativum L.) and Mobin and Khan (2007) on mustard (Brassica juncea L.).
Glutathione S-transferase (GST) plays a key role in the detoxification mechanism of heavy metals and reactive oxygen species (ROS), as well as the regulation of redox balance (Konings and Penninga 1985; Siritantikorn et al. 2007). This enzyme is defined as a biomarker of heavy metal contamination (Cantú-Medellín et al. 2009). GST isoenzymes of the Tau class (GSTU) protect cells against metal toxicity by catalyzing the conjugation of glutathione (GSH) with metal ions for subsequent vacuolar sequestration (Dixon et al. 2002; Moons 2003).
Kilili et al. (2004) showed that GSTs (LeGSTUs) play a primary role in the defense against oxidative stress, where they transcriptionally control the enhancement of the antioxidant response of organisms under stress conditions, they are able to 'inhibit induced cell death and protect against oxidation of membrane lipids under the influence of H2O2 radicals.
It should also be noted that, according to Edwards et al. (2000), in breeding programs for Triticum species, intense GST expression was linked to resistance to environmental stressors and increased yield in wheat. Additionally, Roxas et al. (1997) confirmed that high expression of the BI-GST enzyme (Tau class homolog) improves resistance to salt stress and cold stress in tobacco seedlings.
The obtained results also show that the variations in the GST activity are similar to those of MDA. The increase in GST activity, in response to metallic stresses, corroborates with the results obtained by Labidi et al. (2021) on zucchini, Jaouani et al. (2016) on pea and Hu et al. (2007) on rice in response to Cadmium. Similarly, Halušková et al. (2009) showed that barley subjected to heavy metals (Cd, Pb, Cu, Hg, Co and Zn) exhibits an increase in GST activity. Indeed, Zhang and Ge (2008) also reported the inhibition of some GST isoenzymes in cadmium-exposed rice seedlings, while some GST isoenzymes were present in control (untreated) seedlings.
Based on these data and on the results of the work carried out by Benhamdi et al. (2014), it is assumed that some GST isoenzymes become inactive and it is only those of the tau class that will be expressed and their activity increases with increasing metallic stress in the environment. This explains the ascending GST activity under the effect of the Cd and Cu salts concentrations, used in this study.
Regarding Glutathione, our results are in agreement with those reported by Ducruix et al. (2006), Nagalakshmi and Prasad (2001) and Gallego et al. (1996), who showed that the glutathione (GSH) content decreases in response to stress-induced by different metal concentrations (Cd, Cu, Fe, Zn, etc.). This decrease in GSH can be explained by the involvement of this compound in detoxification reactions of ROS and metal ions, or by the inhibition of the glutathione synthetase (GS) activity under the effect of the metal (Hossain et al. 2012), and the rapid reduction of oxidized glutathione (GSSG) by the glutathione reductase enzyme (GR) under the effect of metal stress (Sytar et al. 2013). This directly affects the GSH/GSSG redox potential, generating the redox signal in cells exposed to stress (Nocito et al. 2006).
The results relating to the phytochemical parameters, namely the total polyphenols (TPC) and flavonoids (TFC) content and the antioxidant activity (I(%)) of the embryonic axes tissues of zucchini seedlings, reveal that these parameters are induced proportionally and significantly, under the effect of high concentration of copper (200 µM) in the medium, contrary to the case of cadmium (Cd). This confirms the results obtained by Kısa et al. (2016) in maize (Zea mays L) and by Badiaa et al. (2020) in root tissues of tomato plants (Lycopersicum lycopersicum L.) under copper metal stress. The authors report that the increased cellular content of phenolic metabolites sustains the plant under these stressful conditions. Kısa et al. (2016) reveal that phenolic compounds act as metal chelators and potent scavengers of ROS and are able to inhibit the enzymes that produce them. Flavonoids can accumulate both outside and inside cell membranes, and act to maintain membrane integrity by blocking the entry of toxic elements into cells (Agati et al. 2012).
On the other hand, the decrease in total phenols and the regression of the antioxidant activity of the embryonic axes tissues under the influence of 200 µM of cadmium (CdCl2) can be attributed, according to Kısa et al. (2016), to the decrease in the activity of key enzymes linked to the biosynthesis of phenolic compounds. This is in agreement with the results of Okem et al. (2015) on Drimia elata which showed that cadmium (CdNO3) at high concentrations (10 mg/l) significantly reduces the biosynthesis of secondary metabolites (phenols and flavonoids). Cadmium also causes oxidative stress and induces the production of ROS, by modifying the cellular content of enzymatic and non-enzymatic antioxidants (Jawad Hassan et al. 2020). Indeed, the toxicity of Cd2+ on the cellular metabolism of plants causes inhibitory effects on the neoformation of cellular ultrastructures (El Rasafi et al. 2022).