As depicted in Fig. 1, different concentrations of S. spectabilis affected antioxidant enzyme activities (superoxide dismutase (SOD), Ascorbate oxidase (APX), Polyphenol oxidase (PPO), catalase (CAT)) of native seedlings; moreover, the effects varied in different native species. ROS are important signalling molecules that stimulate developmental responses in plants. Additionally, they can act as indicators of stress severity in plants under stress conditions, however, their overproduction may interact with normal cellular processes and can destroy cellular compartments or denature DNA (Rahal et al., 2014). The antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbic peroxidase (APX) are known natural control strategies of plants to limit the damage caused by ROS (Huseynova et al, 2014 & Sekmen et al 2012). To reduce the impact of induced oxidative stress, plants activate a complex system of enzymatic antioxidants (SOD, CAT, POD, and APX), which are highly accumulated under stress conditions (Farhoudi and Lee, 2013). Our study showed that SOD level was elevated with the treatment concentration, while simultaneously dropping PPO levels.
In Table 1, Pearson's Correlation Coefficient analysis reveals that CAT demonstrates a positive correlation with SOD, and this correlation is statistically significant (p < 0.01). Conversely, APX exhibits a positive correlation with Formazan, and this correlation is also statistically significant (p < 0.01). It's worth noting that while these correlations are statistically significant, they are not characterized by a strong degree of association. In fact, antioxidants enzymes have different roles in protecting cells by maintaining membrane structures and are responsible for neutralizing the deleterious effects of ROS (Willekens et al., 1995). The recorded increase in antioxidant enzyme activities under allelochemical stress has been previously reported in other plant species (Oracz et al., 2007). In maize, the H2O2 content and the activity of CAT, SOD, and APX enzymes were increased under allelochemical stress of walnut husk (Ciniglia et al., 2015).
Djanaguiraman et al. (2005) showed that allelochemicals present in the leachate of eucalyptus leaves have increased the proline content in sorghum and mung bean. Similarly, Thapar and Singh (2006) reported a stimulation of proline production in the leaves of Parthenium hysterophorus treated by leachate leaves of Cassia tora. Proline accumulation is a general phenomenon in all stressed plants (Parida and Jha, 2010; Shahbaz et al., 2013). Determining the accumulation of proline in plants is a useful assay to monitor and evaluate the physiological tolerance of plants to stress (Abrahám et al., 2010). Proline acts as an electron acceptor and prevents membrane damage (Ain-Lhout et al., 2001). It also provides protection against photosynthetic perturbations induced by ROS (Hare et al., 1998); this could be due to the induction of specific proteins in response to oxidative damage caused by allelochemicals stress (Mishra et al., 2006).
Cellular respiration is a vital phenomenon during germination, providing a supply of ATP to the embryo allowing it to resume its metabolic activities. Reducing respiration in seeds is subjected to the action of plant extracts during germination. The effect of plant extracts or allelochemicals on respiration is reported in the literature (Sampietro et al., 2006; Rashid et al., 2010). Malondialdehyde (MDA) is considered a sensitive marker commonly used to assess the lipid peroxidation of the membrane (Goel and Sheoran, 2003). Polyunsaturated fatty acids constitute the primary lipid constituents of membranes that are prone to peroxidation and degradation. Indeed, the increase in membrane permeability under allelochemicals stress corresponds to an increase in lipid peroxidation estimated by malondialdehyde (MDA) accumulation (Omezzine et al., (2014)). Allelochemicals could eventually damage cell membranes through direct interaction with a constituent of the membrane or as a result of an impairment of some metabolic function necessary to the maintenance of membrane function (Rice, 1984). The increase in lipid peroxidation is also a marker for oxidative stress (Schopfer et al., 2001) and is used as a possible explanation of lipid peroxidation during germination (Schopfer et al., 2001). It was reported previously that the cell membranes of cucumber and sorghum roots were influenced by allelopathic compounds, which was lipid peroxidation determined as MDA content (Zeng et al., 2008). The lipid peroxidation and enzymatic activity in soybeans were markedly correlated under phenolic extract of Brassica napus (Haddadchi and Gerivani, 2009).
Among the allelochemicals identified by GC-MS, (+)-Nerolidol is a well-studied sesquiterpene with documented phytotoxic properties. It has been identified as a potential allelochemical in several studies. For example, Landi et al. (2020) and Kamalrul et al. (2022) reported its presence as a phytotoxic compound. Singh et al. (2023) and Abd et al. (2018) also support these findings, highlighting its role in allelopathy. Neophytadiene, isolated from Nepeta species, has been shown to inhibit the shoot growth of ragweed (Ambrosia artemisiifolia) shoots (Dmitrovi´c et al., 2015). This suggests its role as a potential allelochemical affecting the growth of neighbouring plants. Another allelochemical identified, Lupeol is a triterpene compound naturally occurring in various plant species, and it has garnered significant attention due to its allelopathic properties. The compound has reported ability to exert phytotoxic effects on both weeds and crops, disrupting processes such as seed germination and plant growth. The allelopathic potential of extracts from the leaves and branches of M. eriocarpum and M. hirtum, as well as the lupeol compound itself, was observed in relation to sorghum (S. bicolor L.) seed germination and seedling growth. Experimental studies have demonstrated the phytotoxicity of lupeol and lupenone when applied to weeds like Mimosa pudica and Senna obtusifolia (Luz et al., 2010). Furthermore, lupeol was found to completely inhibit the elongation of etiolated wheat coleoptiles at a concentration of 10 − 3 M, and this bioactivity remained effective even upon dilution (Nebo et al., 2015). Squalene, a triterpene compound, identified has been the subject of research regarding its allelopathic effects, particularly its impact on seed germination and plant growth. Notably, it has been observed that pearl millet, a specific plant species, contains elevated levels of squalene, inhibited the germination of seeds belonging to the epiphytic plant Tillandsia recurvata (Flores-Palacios et al. in 2015). γ-Sitosterol, a type of phytosterol identified in GC-MS, has potential allelopathic properties, especially in its ability to suppress the growth of weeds. It has demonstrated the capacity to hinder the germination of weed seeds and the early growth of weed seedlings. Additionally, within the group of triterpenoids, certain allelochemicals like stigmasterol, γ-sitosterol, and lupeol have been identified, which may also contribute to the phytotoxic response observed in A. pintoi (Thang et al., 2023). Another noteworthy allelopathic compound is phytol, a diterpene alcohol known for its ability to inhibit the germination and growth of various plant species. In the context of the current study, the inhibitory effects of the essential oil from Egyptian C. procera can be attributed to the presence of oxygenated terpenoid compounds, with a particular emphasis on major components such as phytol (Al-Rowaily et al., 2020). Moreover, the essential oil derived from Euphorbia heterophylla has been found to be rich in phytol, and this oil has exhibited allelopathic activity on Cenchrus echinatus (Elshamy et al., 2019)