It has previously been shown (Demecsová et al., 2020) that in the barley root tips, mild Cd stress (10 µM Cd for 30 min) evokes increased IAA synthesis and accumulation, and this change in IAA level is partially responsible for both morphogenic and defense responses. Meanwhile, severe (60 µM Cd for 30 min) Cd stress induces a rapid decrease of IAA level. During moderate Cd -stress (30 µM Cd for 30 min), there is a transient depletion of IAA in the root tips, and then its level increases compared to the untreated seedlings, in a similar fashion as observed during mild Cd stress. The involvement of IAA in Cd-induced response was supported also by the observation that Cd up-regulated the tryptophan synthase gene transcription, resulting in an elevated tryptophan content, the main precursor of IAA, in Arabidopsis seedlings (Sanjaya et al., 2008). Moreover, the authors also observed that exogenously applied tryptophan or the overexpression of the tryptophan synthase gene provide enhanced Cd tolerance, which suggests the possible involvement of tryptophan in the plant defense response to Cd. Additionally, direct IAA level modulation in plant tissue has a considerable effect on drought stress (Shi et al., 2014). While mutants in genes involved in IAA synthesis (with lower IAA level) showed increased sensitivity, transgenic lines with higher endogenous IAA content exhibited enhanced tolerance to water deficit. Besides the depletion of IAA level observed in moderately and severely stressed seedlings, Cd also induced superoxide generation as a very early symptom of stress in the root tips (Tamás et al., 2016). It has recently been suggested that this Cd-mediated superoxide generation, similarly to AA-mediated superoxide accumulation, is originated from the mitochondrial complex III (Zelinová et al., 2019). Based on the results in these experiments, we compared the Cd-, IAA- and AA-induced alteration in gene expression in barley root tips. The concentrations of IAA and AA were chosen so that all short-term treatments, already at the lowest used concentration, evoked a considerable but similar inhibition of primary root growth, which further increased with increasing concentrations of Cd, IAA or AA (Fig. 1).
It is a widely accepted fact that AOX has a key role in reducing the superoxide generation in plant mitochondria. Antisense suppression of AOX caused a buildup of ROS in cells, whereas the cells where AOX was overexpressed had lower ROS formation (Maxwell et al., 1999). In our experiments, western blot analysis revealed that the AOX protein levels increase in a Cd concentration-dependent manner 3 h after the short-term Cd treatment (Fig. 2). This early increase of AOX levels in root tip cells indicates that the reduction of ROS generation by inhibition of mitochondrial complex III and IV activity is an important component of barley root response to Cd stress. In a highly Cd-tolerant Euglena, enhanced alternative respiration accounted for 69 % of total respiration in the presence of Cd (Castro-Guerrero et al., 2008). On the contrary to Cd treatment, a 2.5 µM concentration of IAA had no effect on the AOX protein levels, although this concentration evoked a marked inhibition of root growth. However, in roots that were exposed to higher IAA concentrations considerable higher AOX levels were observed than in control root tips (Fig. 2). In turn, a marked increase in AOX protein abundance was observed already at 5 µM AA treatment, and only a slight additional increase was detected at higher AA concentrations. An antagonistic relationship between IAA and mitochondrial ROS generation has been described in Arabidopsis, where AA treatment caused an inhibition of auxin signaling, and vice versa, auxin diminished the AA-induced response, including AOX expression (Ivanova et al., 2014; Kerchev et al., 2014). Therefore, the detected increase in AOX protein levels after the transient treatment of roots with high IAA concentrations is probably a consequence of an IAA-induced response, such as ROS generation or ABA synthesis, but not a direct activation of AOX by IAA. Increased IAA level in the roots of Cd-exposed tall fescue seedlings was accompanied by elevated H2O2 content and increased antioxidant enzyme activities (Han et al., 2020). It is supported also by the observation that the reduction of IAA synthesis by PPBo did not affect the AOX protein levels either in control or in Cd-treated roots (Fig. 3). Previous research carried out with Arabidopsis implied that the inhibition of complex I in mitochondria by rotenone did not induce an expected oxidative stress or cell death, but rather numerous metabolic pathways were redirected, including activation of AOX (Garmier et al., 2008). Likewise, we also detected a substantial increase in the AOX protein levels after the inhibition of mitochondrial complex I by rotenone in both control roots and roots treated with Cd (Fig. 3). This robust activation of AOX may contribute to the alleviating effect of rotenone on the extensive cell death in barley root tips observed during severe Cd stress due to the considerable reduction of Cd-induced generation of superoxide at complex III (Tamás et al., 2016).
Involvement of MYB TF in the early response of roots to Cd has been previously described in soybean seedlings, where the level of MYBZ2 expression was increased within 3 h after Cd treatment (Chmielowska-Bak et al., 2013). In barley root tips, the MYB1 mRNA transcripts were increased in Cd-treated roots already within 1 h after the transient 10 µM Cd treatment, and further increased slightly with time and with rising Cd concentrations (Fig. 4). On the one hand, these TFs are part of the mechanism that regulates Cd uptake and transport, leading to increased Cd accumulation (Zhang et al., 2019; Zhu et al., 2020). But on the other hand, overexpression of the Cd-induced MYB genes resulted in a markedly increased Cd tolerance, while loss of function mutant lines showed enhanced sensitivity to Cd (Agarwal et al., 2020; Zhu et al., 2020). This very early activation of MYB1 expression indicates the key role of MYB1 during Cd stress response in barley roots as a regulator of early gene expression. Furthermore, transgenic barley lines that overexpress the HvMYB1 gene were more tolerant to osmotic and drought stress, compared to the wild type, probably due to the constitutively high level of DHN, GPX and APX gene expression (Alexander et al., 2019). Similarly to barley transgenic lines, in Arabidopsis MYB overexpression lines, the enhanced Cd tolerance is associated both with enhanced protection against oxidative stress and with increased expression of genes encoding phytochelatin synthase and metallothioneins involved in Cd detoxification of plants (Agarwal et al., 2020). While the expression of MYB1 was markedly increased by IAA treatment of roots, AA application did not influence its expression (Fig. 5). In turn, both PPBo and rotenone slightly attenuated its elevated expression in the Cd-treated roots (Fig. 6). The expression of MdSIMYB1 was upregulated by IAA in apple, while in transgenic tobacco, the overexpression of this gene increased the tolerance of seedlings to salt, drought and cold stresses owing to the induction of stress-responsive genes expression (Wang et al., 2014). In addition, MYB genes have been characterized as an important component of auxin signaling in the regulation of lateral root induction in Arabidopsis seedlings (Shin et al., 2007). These and our results indicate that MYB TFs have a crucial role in the activation of root morphogenic and defense responses to stresses mediated by IAA signaling.
The level of AOC mRNA was increased in roots treated with 10 µM Cd, causing mild stress, already 1 h after the transient Cd treatment, and a slight increase was present 2 h after moderate, 30 µM Cd-induced stress (Fig. 4). In both 10 and 30 µM Cd-treated roots, a considerable increase of AOC transcript abundance was detected 3 h after transient treatment. In contrast, severe stress, induced by 60 µM Cd treatment, did not activate AOC expression. This pattern of changes in AOC expression is very similar to the changes detected in IAA level in barley root tip treated with the same concentrations of Cd (Demecsová et al., 2020). Moreover, exogenously applied IAA activated the expression of the AOC gene, whereas AA did not affect its expression (Fig. 5). While PPBo markedly inhibited the expression of AOC in control as well as in Cd-treated roots, rotenone had only a minor inhibitory effect on Cd-induced AOC expression (Fig. 6). These results imply that IAA signaling is involved, through the induction of AOC expression, in the activation of JA synthesis and accumulation during mild and moderate Cd stresses in barley root tips. Similarly to our result, JA synthesis genes expression was considerably increased within 1 h of Cd treatment, eventuating in an elevated JA level in the Arabidopsis root (Lei et al., 2020). In Arabidopsis, JA biosynthesis genes were upregulated by IAA, and this induction was strongly impaired in the auxin-signaling mutant (Tiryaki and Staswick, 2002). In addition, JA interacts with auxin, affecting its homeostasis and root system remodeling during the response of seedlings to metal excess (Ronzan et al., 2019). In accordance with these results, tomato JA-deficient mutant showed enhanced sensitivity to Cd due to the decreased antioxidant enzymes activity resulting in increased ROS formation (Zhao et al., 2016). By contrast, AOC overexpressing Arabidopsis seedlings exhibited increased copper tolerance (Wang et al., 2015), supporting the function of JA in defense mechanisms of plants against excess metal-mediated toxicity.
Similarly to Cd-induced changes in AOC expression, increased NOXB1 expression was a characteristic feature of root tips during mild and with lower intensity during moderated Cd stresses (Fig. 4). The expression of NOXB1 was strongly activated within 1 h and further increased 2 and 3 h after the transient treatment with 10 µM Cd. The induction of NOXB1 expression in barley root tip mainly under mild Cd stress further support the idea that NOX is involved in different defense responses but not in an uncontrolled generation of toxic superoxide (Jakubowska et al., 2015; Tamás et al., 2016). The role of NOX in auxin signaling pathway has previously been suggested in root development (Müller et al., 2012). Authors observed that knock-down NOX expression lines exhibited a strong seedling root phenotype resembling phenotypes of mutant lines defective in auxin-regulated processes. In agreement with these results, in our experiments exogenous application of IAA evoked an increase of NOXB1 mRNA levels, whereas AA inhibited its expression in a dose-dependent manner (Fig. 5). Rotenone did not influence the elevated NOXB1 expression in roots exposed to 10 or 30 µM Cd (Fig. 6). In turn, PPBo reduced the level of NOXB1 transcripts in both control and Cd-treated roots, suggesting that NOXB1 expression is affected by the depletion of IAA, and an elevated IAA level is required for the activation of NOXB1 in Cd stressed roots to develop appropriate defense responses. In turn, high Cd or AA concentrations- generated ROS probably take part in the attenuation of auxin signaling through the oxidation of IAA, leading to an inactive oxiIAA molecule (Peer et al., 2013).
On the contrary, early changes in the DHN6 transcripts level, within an hour following the transient Cd treatment, were found in the moderately and severely stressed tips of barley roots (Fig. 4). This strong increase of DHN6 transcripts 1 h after 60 µM Cd treatment remained elevated even up to 3 h after the severe stress- inducing treatment. The changes induced by 30 µM Cd were similar to those described for 60 µM Cd treatment but with a lower intensity 1 h after the treatment. When a lower Cd (10 µM) concentration was used, the DHN6 expression activation was detected only 2 and 3 h after the treatments. These results indicate that the endogenous IAA is not a major signal for the induction of DHN6 expression throughout the Cd stress, because as a previous study has shown 1 h after the exposure of roots to Cd, the IAA accumulated only in root tips exposed to mild Cd stress, whereas severely stressed roots had a depleted level of IAA (Demecsová et al., 2020). In spite of this fact, exogenous IAA induced DHN6 expression; however, this effect decreased with increasing IAA concentrations (Fig. 5). AA at 10 and 20 µM concentrations strongly downregulated its expression 3 h after the transient treatment (Fig. 5). While PPBo reduced the Cd-induced activation of DHN6 expression only at the lower 10 µM Cd concentration, evoking mild Cd stress, rotenone inhibited the activation of its expression during all analyzed Cd concentrations (Fig. 6). In barley, several abiotic stress-responsive elements were observed in the promoter region of DHNs, including MYC and MYB TFs binding sites, dehydration-responsive element and abscisic acid-responsive elements (Abedini et al., 2017). Therefore, the expression of several DHNs is activated under various stresses; however, heavy metals specifically activate some of them and probably have a key role during metal detoxification as well as during the reduction of metal-induced damages in cells (Zhang et al., 2006). It has been observed in wheat seedlings that DHN accumulation constitutes a key component in the protection against Cd toxicity induced by salicylic acid (Shakirova et al., 2016). A crucial function of DHNs was observed in metal hyperaccumulator species as well (Xu et al., 2008). Inhibition of expression of the gene encoding DHN in antisense Brassica juncea seedlings led to the increased sensitivity to heavy metal stress besides the reduced accumulation of Cd in comparison with wild type seedlings. In turn, transgenic tobacco seedlings overexpressing this gene were more tolerant to Cd or Zn than control seedlings.
Expression of both APX1 and GPX1 was upregulated in the root tips of Cd-treated roots in a Cd dose-dependent manner, and it increased with incubation time after the transient treatment (Fig. 4). While GPX was activated as soon as 1 h after the transient, mild stress-evoking, 10 µM Cd treatment, APX expression was induced only with severe stress-causing 60 µM Cd treatment. This early, mild Cd stress-activated GPX expression accompanied by IAA accumulation indicates the IAA involvement in the signaling process leading to this increased GPX expression in the barley roots. Indeed, while PPBo did not influence the increased APX1 expression either under mild or severe Cd stress, its application reduced the elevated expression of GPX1 under mild Cd stress. In turn, rotenone had only a slight effect on the Cd-induced GPX1 expression, whereas markedly attenuated Cd-induced APX1 expression (Fig. 6), suggesting that in the induction of APX expression, a crucial role is played by ROS. In agreement with these results, it has previously been reported that H2O2 level within cells has a key role in the activation of APX expression (Morita et al., 1999). Additionally, both IAA and AA caused an increase in APX1 and GPX1 mRNA levels in the root apices (Fig. 5). However, it is a well-known fact that exogenously applied IAA, at concentrations evoking RGI, induces a considerable ROS formation in the tips of plant roots (Ivanchenko et al. 2013), which leads to an increased expression of several antioxidant enzymes, including APX and GPX. Both the transgenic Arabidopsis line with increased endogenous IAA level and wild type plants had increased activity of several antioxidant enzymes during drought stress after IAA application; due to the positive IAA effect on ROS homeostasis (Shi et al., 2014).
The expression of C-Prot was activated to a similar extent in both mildly and severely Cd-stressed roots within 1 h after the transient treatment and increased further with incubation time (Fig. 4). Both IAA and AA activated the expression of C-Prot in a dose dependent manner; however, AA activated its expression more intensively in comparison with IAA (Fig. 5). In mildly and moderately stressed roots, both PPBo and rotenone reduced the Cd-induced increased expression of C-Prot (Fig. 6), suggesting that both increased IAA and ROS levels upregulated the expression of C-Prot in barley root tips. In salt and Cd tolerant strain (W80) of Chlamydomonas, isolated from seawater, C-Prot activity was markedly increased both under oxidative and Cd stress (Usui et al., 2007). The significance of the rapid recycling of unamendable proteins is further supported by the findings that the C-Prot gene was upregulated during the early stage of stress responses to low- or high- temperature conditions and drought (Stroeher et al., 1997).
The Cd-evoked accumulation of BAXI-1 mRNA was characteristic for moderately and severely stressed roots (Fig. 4). While mild Cd stress caused only a slight activation of BAXI-1, moderate and severe Cd stress strongly activated its expression within 1 h after the transient treatment of roots with Cd. The abundance of BAXI-1 mRNA was considerably increased by both IAA and AA treatment in the root tips (Fig. 5). Whereas PPBo did not affect its expression, rotenone markedly reduced the Cd-induced expression of BAXI-1 (Fig. 6). Using yeast-based cDNA survival screening technique, numerous Cd-tolerant genes have been identified, including BAXI-1 (Wang et al., 2020b). It has been reported that BAXI-1 plays a crucial role also in the tolerance mechanisms to lead (Kobylińska and Posmyk, 2016). Authors in this study observed that melatonin had a protective effect against lead toxicity in tobacco suspension cells which was attributed not only to its own antioxidative properties but also to the marked induction of BaxI-1 gene leading to the considerable restriction of cell death. In addition to increased expression of BaxI-1 during several stress conditions, BaxI-1-overexpressing transgenic tobacco seedlings showed markedly increased tolerance to high temperature, salt and water stresses (Isbat et al., 2009). In rice suspension cells, BAXI-1 overexpression did not influence the menadione-induced oxidative stress but markedly altered the metabolic components of several defense pathways against oxidative stress (Ishikawa et al., 2010). Possibly, BaxI-1 suppresses the spreading of cell death in the early stage of stresses to enable the activation of defense and repair mechanisms inevitable for the survival of cells during unfavorable conditions.