Cadmium is one of the most widespread contaminants that can be readily absorbed by plants and reduce the yield and safety of plant products. The cadmium content of plant tissues is highly correlated to the Cd concentration in the soil (Luo et al. 2016). In the present study, the Cd content of barley seedlings increased under Cd exposure. The roots of Cd-treated barley plants accumulated a considerably higher content of Cd as compared to the shoots, which suggests a limitation in long-distance translocation of Cd in barley. Consistent with our results, it has been well established that in most plants, the main portion of absorbed Cd (70–85%) remains in the root and is prevented from reaching the aerial parts (Pirseloya et al. 2016). Accumulation of Cd can lead to the impairment of several physiological processes in plants, including mineral nutrition. It has been reported that the absorption and distribution of nutrients, particularly N, Ca, Mg, and K, are adversely affected by Cd toxicity (Ismael et al. 2019; Yang et al. 2021). Our results demonstrated that the Ca and Mg content of barley seedlings decreased under Cd treatment. Cd is taken up via cation transport systems normally involved in the uptake of essential nutrients. Being a divalent cation, Cd competes with Ca and Mg cations for their binding sites in transport systems across the membrane and diminishes their uptake (Nazar et al. 2012). The content of N in the roots of barley seedlings was also significantly reduced as a result of the high Cd concentration. Similar to this result, a significant reduction in N content was reported in tomato under different Cd concentrations (Khan et al. 2016).
Cd toxicity can lead to a higher generation of ROS including superoxide (O2.−) and hydrogen peroxide (H2O2) through indirect activation of membrane NADPH oxidases (Asopa et al. 2017; Nazar et al. 2012). Furthermore, the interconversion of O2.− to H2O2 catalyzed by the superoxide dismutase (SOD) enzyme, to ensure safe ROS metabolism can also contribute to the higher content of H2O2 in plant cells. In the present study, the imposed Cd stress increased the specific activity of the SOD enzyme as well as higher H2O2 content in barley seedlings. In addition to inducing oxidative damage to biological macro-molecules, H2O2 is considered to be a key signaling molecule, activating a variety of defense mechanisms under stressful conditions (Nazir et al. 2020; Ghotbi-Ravandi et al. 2021). Recent studies have demonstrated an intricate interplay between H2O2 and phytohormones under stress conditions. The phytohormone balance is central to the outcome of plant tolerance against environmental stresses.
ABA is an essential phytohormone organizing stress-induced responses in plants. Our findings showed that the ABA content of barley seedlings significantly increased under Cd stress. Consistent with our results, Perez Chaca et al (2014) reported that the ABA content of Glycine max significantly increased at different time points of Cd exposure. Similarly, higher levels of endogenous ABA were found in plants such as chickpea, cucumber, potato, rice, and wheat under heavy metal stress, indicating that ABA plays a crucial role in alleviating the negative impacts of metal toxicity (Atici et al. 2005; Munzuro et al. 2008; Stroinski et al. 2010; Kim et al. 2014; Wang et al. 2014).
SA is linked to mediating numerous responses during exposure to abiotic and biotic stresses. Several studies have shown that resistance to salinity, drought, thermal stresses, nutrient deficiency, and metal toxicity can be regulated by SA (Shimakawa et al. 2012; Okuma et al. 2014; Khanna et al. 2016; Emamverdian et al. 2020; Kaya et al. 2020; Saleem et al. 2021). However, Zhao et al (2021) reported that the nahG (naphthalene hydroxylase G)-transformed Arabidopsis (with a reduced endogenous SA) showed an improved tolerance, while the mutant snc1 (with a high endogenous SA) was more susceptible to Cd stress, as compared with the control group.
Consistently, Cd toxicity led to a decrease in the SA content of barley seedlings in the present study. It has been demonstrated that ABA downregulates the biosynthesis of SA and inhibits SA responses under pathogen attack and drought stress (de Torres Zabala et al. 2009; La et al. 2019). The reduction in SA content observed in the present study was consistent with the prominent accumulation of ABA in response to Cd treatment. Despite the reduction of SA content of barley seedlings in the present study, the transcripts of the SAR pathway genes, PR3 (Chitinase) and PR13 (Thionin), significantly increased in response to Cd stress. It has been reported that in barley, the induction of the SAR pathway against pathogens was not associated with local accumulation of SA, and alternatively, systemic immunity was induced by ABA accumulation (Dey et al. 2014). PR proteins, as part of the SAR pathway, are accumulated in plants in the event of a pathogen attack. However, it has been demonstrated that abiotic stresses can also mediate the expression of PR genes (Ali et al. 2017). Consistent with our results, Pilaisangsuree et al (2020) showed that the genes encoding PR proteins, including PR4, PR5, PR10, and PR3 were upregulated in peanut hairy roots under Cd stress. Similarly, it has been reported that salinity and water deficit stresses significantly increased the expression of PR proteins in Arabidopsis (Seo et al. 2008; Liu et al. 2013). Similarly, Su et al (2016) reported that the PR protein (beta-1,3-glucanase) is induced by ABA, H2O2, and CdCl2 stresses in sugarcane. In addition, PR proteins like PR3 possess antifreeze properties and protect plant cells during cold stress (Janska et al. 2010). Cold stress significantly upregulated the expression of PR12 and PR13 in Oxytropis (Fabaceae) and winter wheat (Gaudet et al. 2003; Archambault and Strömvik 2011).
JA and ET are other signaling molecules that are synthesized in response to biotic and abiotic stress such as pathogen attack, wounding, osmotic and heavy metals, especially cadmium (Schellingen et al. 2014; Farhangi-Abriz and Ghassemi- Golezani 2019). As a signaling molecule, JA regulates the various physiological processes in plants, including tolerance to environmental stresses (Siddiqi and Husen 2019; Yu et al. 2019). JA diminishes the deleterious effects of heavy metals primarily by enhancing the antioxidant machinery capacity, increasing thiol metabolism, and protecting the photosynthetic apparatus (Farooq et al. 2016; Per et al. 2016). In tomato, a JA-deficient mutant was found to be more sensitive to Cd than the control, indicating that JA may play a role in alleviating heavy metal stress (Zhao et al. 2016). Our results demonstrated that the content of JA significantly increased as a result of Cd stress. Our results corroborated the findings of Maksymiec et al (2005), that reported an accumulation of JA in Arabidopsis thaliana and runner bean plants under Cd exposure.
The critical involvement of ET in the adaptation of plants to various environmental stresses such as heavy metals has been reported (Iqbal et al. 2017). Steffens (2014) demonstrated that ET modulates both ROS production and the activity of antioxidant enzymes under metal exposure. In tomato, ET improved the tolerance against Cd-induced oxidative stress (Nawaz et al. 2017). Our results demonstrated that Cd exposure leads to an increase in the ET content of barley seedlings. Similar to our results, Schellingen et al (2014) reported an increase in ET release after Cd exposure in A. thaliana.
The ISR pathway is dependent on the signals of JA acid and ET. In the present study, the expression of genes for phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX) involved in the ISR pathway significantly increased in barley seedlings in response to Cd stress. Consistently, Pawlak-Sprada et al (2011) demonstrated that the expression of PAL significantly increased in soybean seedlings treated with different concentrations of Cd. LOX represents a suitable marker for upregulation of the JA/ET pathway (Seltmann et al. 2010). Consistent with our results, elevated activity of the LOX enzyme has been detected in barley roots, after 3 hours of Cd exposure (Liptáková et al. 2013). Consistent with our results, the up-regulation of LOX activity was observed in finger millet under nickel stress, affecting antioxidant enzymes and alleviating stress effects (Kotapati et al. 2017).
The nonexpressor of pathogenesis-related 1 (NPR1), is an essential element required for the plant immune system and confers the SAR pathway to protect plant cells from a wide spectrum of pathogens. NPR1 is a master key in plant defense signaling networks and acts as an intermediary cross-talk between SA and JA/ET signals (Baker et al. 2019). In addition to its involvement in defense mechanisms against biotic pathogenesis, NPR1 has been reported to be a crucial component of abiotic stress responses. In the present study, Cd exposure led to a significant increase in NPR1 transcripts in barley seedlings. Consistently, it is reported that salt stress causes a rapid accumulation of NPR1, which elicits many adaptive responses in plants (Seo et al. 2020). Sarisoy et al (2018) reported an increase in expression of the NPR1 gene in tolerant and susceptible soybean cultivars under salt stress. Seo et al (2020) stated that overexpression of NPR1 in tobacco under salt stress reduced stress-induced ROS formation and enhanced antioxidant activity. In addition, NPR1 is proven to play an important role in plant thermotolerance mediated by 24-epibrassinolide (Divi et al. 2010).