1. Introduction
Under the current circumstances, where the growing population is exceeding the global food supply, arable land is becoming sparse [
1]. Soil pollution is putting even more restrictions on the availability of qualitative agricultural land. Trace metallic elements, like cadmium (Cd), significantly contribute to this pollution problem, as they are phytotoxic and pose risks to human health via the bioaccumulation in our food chain [
2]. The study of short-term plant responses allows for the identification of the pressure points of a certain stressor and the early challenges that plants face prior to acclimation. Understanding the early stress-induced responses will help improve plant acclimation itself, allowing plants, and in particular crops, to reach their full potential even in suboptimal environments. The latter can be achieved by means of biotechnological and agro-ecological approaches which encompass a.o. genetic modifications and application of soil amendments, respectively.
Cadmium phytotoxicity mainly arises from its bioavailability and chemical similarity to essential elements like zinc, calcium and iron, enabling Cd to hitchhike along transporters for essential elements [
3,
4]. This results in disturbance of the homeostasis of these elements and in their displacement by Cd in biomolecules, rendering them inactive and, at the same time, freeing up redox-active metals like iron [
3,
5,
6,
7]. These redox-active metals directly stimulate reactive oxygen species (ROS) production, while Cd increases ROS production indirectly, via the stimulation of pro-oxidants like NADPH oxidases and the deprivation of the anti-oxidative system [
8]. The anti-oxidative metabolite glutathione (GSH) is one of the prominent defence molecules in the responses to Cd stress and is synthesised in two ATP-dependent steps [
9,
10,
11]. Firstly, glutamate is combined with cysteine, which is catalysed by glutamate cysteine ligase (GSH1) to produce γ-glutamylcysteine (γ-EC) [
12]. Next, the addition of glycine is catalysed by GSH synthetase (GSH2) to form GSH [
13,
14]. The nucleophilic nature of the central thiol group enables GSH and its oligomers, termed phytochelatins (PCs), to chelate Cd and sequester it into the vacuole [
15]. Concurrently, GSH serves to neutralise ROS, like hydrogen peroxide (H
2O
2), directly but mainly through the ascorbate (AsA)-GSH cycle [
16,
17]. In this cycle, the NADPH-dependent enzyme glutathione reductase (GR) serves to maintain the reduced GSH pool [
18]. Moreover, Mhamdi et al. (2010) showed that GR encoded by the
GLUTATHIONE REDUCTASE 1 (
GR1) isoform is crucial in the metabolism of H
2O
2 [
19]. In the apoplastic space, however, it is rather unlikely that GSH functions as major anti-oxidant due to its relatively low abundance [
20]. Nevertheless, it is clear from the literature that apoplastic GSH and its recycling fulfil other important roles that need further consideration [
20,
21,
22,
23,
24]. The recycling of extracellular GSH (eGSH) is accomplished by the activity of γ-glutamyl transpeptidase (GGT) encoded by
γ-GLUTAMYL TRANSPEPTIDASE 1 (
GGT1), that catabolises eGSH into its constituent amino acids [
24]. The hydrolysis of eGSH and glutathione S-conjugates enables the recovery of GSH intracellularly [
24,
25]. Furthermore, it is proposed that GGT plays a role in the redox control of the apoplastic space and serves to mitigate oxidative stress as a result of an unbalanced ROS production [
20,
24,
26]. It is a well-known fact that ROS cannot simply be considered as detrimental compounds, as they often fulfil a signalling role [
27]. Hydrogen peroxide, especially, is considered to be a central component of signal transduction due to its stability and ability to cross membranes [
28]. Because of this double-edged sword, GSH does not serve merely to detoxify H
2O
2, but it is also key in the fine-tuning of H
2O
2-dependent signalling responses [
29,
30].
As demonstrated by Jozefczak et al. (2014), root GSH levels became strongly depleted upon 2 h of Cd (5 µM) exposure in hydroponics, which is attributable to the fact that GSH is allocated to PC synthesis [
10]. This impacts the anti-oxidative capacity of GSH in the early responses to Cd stress. Moreover, the depletion of such a ubiquitous and considerable anti-oxidant will most likely trigger specific signalling events that define the acute responses and acclimation to Cd stress. Besides alterations in the GSH pool and H
2O
2 signalling, other components like phytohormones are key in the responses to environmental stresses. The important stress hormone ethylene was already demonstrated by Schellingen et al. (2015) to serve as key regulator in the responses to Cd stress [
31]. More specifically, ethylene production and signalling are required for the stimulation of leaf GSH metabolism under Cd stress and stimulation of ROS-generating NADPH oxidases in general [
31,
32,
33,
34]. Ethylene production is known to increase under Cd stress and mainly relies on the transcriptional upregulation and post-transcriptional stabilisation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) isoforms ACS2 and ACS6 [
35]. These isozymes catalyse the conversion of S-adenosylmethionine (SAM), derived from methionine, to ACC, the direct precursor of ethylene [
36,
37]. It is known that ACS2 and ACS6 are targeted via the transcription factor WRKY33, by mitogen-activated protein kinase 3 (MPK3) and by MPK6, which, in turn, are phosphorylated by the oxidative-signal inducible 1 (OXI1) kinase, that becomes induced by H
2O
2 [
31,
38,
39]. These findings demonstrate that different signalling pathways are strongly intertwined in a complex network that defines the outcome of stress responses and acclimation thereafter.
In this study, a time-course analysis of different key regulators was conducted in order to unravel the sequence of events in terms of acute Cd-induced responses. The use of a short-term exposure set-up, ranging from 0 h to 24 h of exposure, allowed for the identification of pressure points prior to acclimation. The identification of these pressure points is highly required in order to understand the hurdles a plant needs to overcome before reaching acclimation and contributes to the bigger picture in order to understand the process of acclimation to Cd stress.
3. Discussion
Plants possess a great plasticity and adaptive potential, enabling them to cope with a broad range of environmental stresses and acclimate to changing environments. The process of acclimation encompasses homeostatic adjustments and results in newly established equilibria [
48,
49]. However, acclimation is typically preceded by a stress response, which generally occurs within a time frame of seconds to days, and most often leads to a temporary suboptimal performance [
48]. The study of acute responses to Cd exposure, within the period of 0 h to 24 h, allows us to identify the pressure points of Cd stress before new equilibria are reached. Knowledge concerning such pressure points and acute responses is required to improve our understanding of the triggers and sequence of events that precede acclimation. The obtained knowledge can contribute to the improvement of plant acclimation, enabling plants to reach their full capacity even under stressful conditions. This trait is highly required in the current conditions, since non-polluted arable land is becoming sparse.
While the dual role of GSH under Cd stress, as a chelator and anti-oxidant, was already touched upon by Jozefczak et al. (2014), our data further underline the dilemmas plants encounter in their acute responses to environmentally realistic, sublethal (i.e., 5 µM) Cd stress [
10,
41]. More specifically, the present study further uncovers the Cd-induced trade-offs between (1) GSH as a chelator and anti-oxidative metabolite and (2) ROS signalling and oxidative stress, i.e., an oxidative challenge. From our data, it became clear that these trade-offs were mainly manifested at the root level, especially with regard to GSH, that became strongly depleted within 2 h of Cd exposure (
Figure 2). Jozefczak et al. (2014) indicated that this depletion occurred due to the allocation of GSH to its Cd-chelating oligomers, namely phytochelatins [
10]. The fact that, in the present study, GSH levels were even more strongly depleted after 4 h adds to our knowledge that the effect on free GSH levels becomes more pronounced and persists at least until 4 h after exposure (
Figure 2). Overall, the Cd-induced changes in root GSH concentrations fit the typical stress response curve described by Lambers et al. (1998) [
48]. First of all, the initial alarming phase becomes visible by the rapid depletion of GSH followed by the restitution phase, which is established between 6 h and 24 h, as a full recovery to control levels occurred within this time frame (
Figure 2). As shown previously, overcompensation by increased GSH levels does not occur at the root level under 5 µM of Cd, at least not after 24 h, 48 h and 72 h [
10]. At the leaf level, even though Cd was translocated early on and the leaf Cd concentrations were significantly elevated from 2 h onwards (
Table 1), no changes in GSH concentrations were observed within the considered 24 h time frame (
Figure 2). However, it is known that leaf GSH levels significantly increase after 48 h and 72 h of exposure to 5 µM Cd [
31]. As reviewed by Tausz et al. (2004) and Zagorchev et al. (2013), an increase in GSH concentrations is often observed as an acclimatory response to a range of stresses and hints towards a better stress resistance and a new steady-state [
49,
50]. Note, however, that increased GSH levels, exogenously applied or transgenically enhanced, do not necessarily imply an improved tolerance, especially in the case of Cd stress [
51,
52]. The fact that the manipulation of GSH levels can lead to increased Cd sensitivity underlines the fine-tuning that is required for proper acclimation and emphasizes the importance of the alarming phase, provoked by the rapid and strong GSH depletion at the root level (
Figure 2). Moreover, our data indicate that the depletion and recovery of GSH levels have a relatively fast nature, which further confirms the fact that the pressure points of stress factors are often overlooked when considering longer exposure time frames and underlines the importance of monitoring stresses at different time points. In addition, the fact that responses strongly differ between roots and leaves points out that at least both organs need to be considered when studying plant stress responses and acclimation, especially when the stress is (partly) propagated via the root system.
Even though several studies have indicated that a rapid and transient depletion of GSH occurs after exposure to excess metal concentrations, little is known about the impact of this event on the plant’s responses [
10,
53,
54]. Indeed, depletion of this prominent anti-oxidant could lead to an oxidative challenge at the root level as its anti-oxidative capacity is largely impaired. However, the extent to which this event is detrimental or, on the contrary, contributes to stress signalling—and, ultimately, plant acclimation—remains unclear. Alterations of the GSH pool and generation of the prominent ROS signalling molecule H
2O
2 are both central components of stress-induced signal transduction and often act in concert [
29,
45]. The ratio between oxidising H
2O
2 and the important anti-oxidant GSH allows us to obtain an integrative view of the Cd-induced redox changes and shows that H
2O
2 levels in the roots are most strongly elevated in relation to GSH early on (
Figure 3b). Previous studies have indicated that the changes in GSH status are rather a modulator of the stress-induced increases in H
2O
2 than merely a passive result [
29,
30]. In our study, the Cd-induced H
2O
2 increases are modulated by the depletion of the GSH pool and are not influenced by changes in its redox state, because the redox state of the GSH pool was not affected by Cd exposure and the percentage of reduced GSH remained tightly controlled above 90% (
Table S3). Consistent with these findings, it was shown by Schnaubelt et al. (2015) that buthionine sulfoximine (BSO)-induced depletion of the root GSH level did not necessarily impact its redox state [
30]. Moreover, lowered GSH levels counteract its oxidation [
29]. As GR is key in the recycling of the oxidised GSSG back to its reduced form, an increased GR activity may explain this tight control. However, in our study, GR activity in the roots was only significantly increased after 24 h of exposure (
Table 3). Therefore, it can be concluded that its activity is not contributing to maintain a reduced GSH pool at the early time points when GSH becomes depleted (
Figure 2). As shown by several studies,
GSH1 and
GSH2 are typically induced upon Cd exposure [
9,
10,
55,
56,
57]. In our study, their transcriptional induction coincided with the restoration of GSH levels observed after 6 h and 24 h at the root level (
Figure 2). The catalyser of the first and rate-limiting step of GSH biosynthesis,
GSH1, is regulated at several levels. For example, GSH itself is known to have a negative impact on its own production by inhibition of GSH1 activity [
57]. Hence, the GSH-depleted conditions (
Figure 2) in our study favour an increased activity of GSH1 in the roots. Moreover, at the transcript level, the transcription factor ZAT6 is known to stimulate
GSH1 transcription and its own expression is enhanced upon Cd exposure, which was also observed in our study [
56]. In general, this study is in agreement with the conclusion drawn by Han et al. (2013) that the plant cell redox status is configured in such a way that depletion inhibits GSH oxidation and strong changes in GSH concentration are sufficient to alter the cell’s redox potential and drive GSH accumulation [
29].
It is clear that intracellular GSH is a major modulator of stress responses and the acclimation process thereafter. Noteworthy, however, is the eGSH residing in the apoplastic space, which is largely regulated by the γ-glutamyl cycle [
24,
25]. The fine-tuning of the apoplastic GSH content by this cycle serves in redox, balancing the apoplastic space and recovery of GSH—or, more precisely, its constituent amino acids—into the cell. The driving force of eGSH degradation is the apoplastic GGT enzyme encoded by
GGT1, which catalyses the transfer of the γ-glutamyl group of GSH to a range of acceptors like water or another amino acid [
24]. Considering there is no mechanism to reduce extracellular GSSG, this enzyme prevents the accumulation of GSSG in the apoplastic space, mitigating oxidative stress [
24]. Even though
GGT1 is most strongly expressed in the leaves of
A. thaliana, our data show that the Cd-induced
GGT1 upregulation is more pronounced and occurs faster in the root system (
Table 2) [
25]. Accordingly, enzyme-histochemical analyses showed that GGT activity was very intense in root tips of
Hordeum vulgare and
Zea mays [
26,
58]. Furthermore, Uzilday et al. (2018) observed a strong induction of
GGT1 under endoplasmic reticulum stress, a stress that is also known to be evoked by short-term Cd exposure [
59,
60]. It has been suggested that the γ-glutamyl cycle serves to link the environment to the plant cell and may provide a way to transfer redox information between the apoplast and the symplast [
20]. Other key components known to be involved in the apoplastic redox regulation that bridge the extracellular and intracellular space are NADPH oxidases. In our study, a similar transcriptional profile was observed for
RBOHC, D and
F, which coincided with
GGT1 expression (
Table 2 and
Table 3). The Cd-induced transcription of these prominent NADPH oxidase isoforms (
Table 3) hints at an increased production of superoxide and subsequently H
2O
2 in the roots. An augmented H
2O
2 production, as observed in our study (
Figure 3), could lead to oxidation of the apoplastic GSH pool and activation of the γ-glutamyl cycle. In summary, these data point towards a redox-related signalling event that is in full practice upon 2 h of Cd exposure and persists at least up to 24 h of exposure. Furthermore, as shown by Tolin et al. (2013), apoplastic GGT is an important modulator of the redox response, since the knockout of
GGT1 leads to a constitutive “alert response” even in absence of environmental stimuli [
20,
21]. Therefore, it can be suggested that GGT encoded by
GGT1 also functions as an important modulator in the redox sensing and signalling under Cd stress.
In our study, only a small subset of oxidative stress markers was transcriptionally induced in the root, and only after 24 h of exposure (
Table 4) [
43]. This is in line with our suggestion that the early alterations observed in the H
2O
2/GSH at the root level are required for a proper signalling response under Cd stress, rather than having merely a detrimental oxidative stress effect. In addition, the fact that the transcriptional profile indicating oxidative stress markers in the roots was only induced to a limited extent and delayed, is in line with the findings of Schnaubelt et al. (2015). They suggested that GSH depletion evokes a very specific response as the transcriptome of the
root meristemless 1-1 (
rml1-1) mutant, harbouring only 2.7% of WT GSH levels. This response is different from that of the
catalase 2 (
cat2-1) mutant and lacks an induction of the oxidative stress markers, which might be explained by the absence of a change in the GSH/GSSG ratio [
30]. Another important modulator of ROS signalling is the transcription factor
RRTF1 that was transcriptionally induced in both roots and leaves but only upon 24 h (
Table 4). Matsuo et al. (2015) demonstrated that the expression of
RRTF1 is stimulated by ROS and that RRTF1 itself is responsible for the amplification of ROS generated by a stressor that perturbs basal ROS levels [
61]. Additionally, one of its target genes,
ZAT12, also became significantly upregulated upon 24 h of Cd exposure (
Table 4), which in its turn stimulates the transcription of, for example,
RBOHD, that was increased after 24 h as well (
Table 3) [
61]. Both are implemented in the regulation of ROS signalling under unfavourable conditions, and in this case their upregulation indicates ROS amplification to possibly intensify responses after 24 h when root GSH levels are stabilised. Indeed, at this time point H
2O
2 levels were significantly increased in both root and leaves independently of GSH (
Figure 3a). In this way, the Cd-induced signalling responses might be redirected away from GSH-dependent redox sensing, as root GSH levels are restored to control levels at this later time point (
Figure 2) and can no longer serve as a redox signal.
Our data indicate that the GSH-related leaf responses are delayed and less pronounced in comparison to the roots (
Figure 2 and
Table 2). This is plausible, since the roots are in direct contact with the Cd-containing nutrient solution. However, our data show that Cd is translocated early on to the aerial parts, leading to significantly higher Cd concentrations in the leaves compared to the control already after 2 h of exposure (
Table 1). Nevertheless, no GSH depletion was observed in the leaves (
Figure 2). As mentioned before, Cd-induced GSH depletion is caused by the allocation of GSH to PC synthesis, a process that is also known to occur in leaves after 24 h of exposure to 5 µM Cd [
10]. Hence, since leaf GSH levels are not negatively affected (
Figure 2), leaf signalling responses possibly directly shift to the GSH-independent signalling response, as described above. Our data indicate that Cd-induced leaf GSH stimulation serves to buffer the impact of stresses at the leaf level and that GSH fulfils a protective role rather than a signalling role. This implies, however, that other components are responsible for stress signalling in the leaves in order to reach acclimation.
It is clear from our study that the early depletion in root GSH levels does not stand alone. More specifically, the production of the ethylene precursor and important signalling molecule ACC was already significantly higher from 2 h of exposure onwards at the root level (
Figure 4). As demonstrated by Schellingen et al. (2015), ROS signalling is integrated into the signalling cascade that precedes Cd-induced ethylene biosynthesis by increasing
OXI1 expression, which, in its turn, activates MPK3 and MPK6 [
31,
38]. These kinases target ACS2 and ACS6, leading to an increase in their half-life and a stimulation of their gene expression [
62,
63,
64]. In our study, both
MPK3 and
MPK6 seem to function early in the root responses to Cd stress, and their transcriptional induction (
Table 5) collides with the higher root ACC concentrations (
Figure 4). Moreover, it is known that, in
A. thaliana,
OXI1 gene expression and kinase activity are induced upon exposure to a broad range of H
2O
2-generating stimuli [
38]. One such stimulus could, for example, originate from RBOHC, and indeed the knockout of
RBOHC leads to a decreased induction of
OXI1 in the roots of
A. thaliana [
44]. Correspondingly, in our study, the induction of
OXI1 (
Table 5) peaks in concert with the highest H
2O
2/GSH ratio observed in the roots (
Figure 3b) and possibly leads to the activation of the aforementioned signalling cascade, with the transcriptional induction of
ACS2 and
ACS6 as an end result (
Table 4). The fact that the ACC concentration (
Figure 4) in the roots was already significantly higher after 2 h of exposure, and therefore preceded the transcriptional induction of
ACS2 and
ACS6 (
Table 4), suggests a rapid activation at the protein level, which later on is extended to the transcript level.
Additionally, ethylene and GSH are strongly intertwined in the responses to Cd stress. It was shown by Schellingen et al. (2015) that leaf GSH stimulation under Cd stress depends on ethylene signalling [
31]. More precisely, the considered ethylene insensitive
ein2-1 mutants proved unable to increase their leaf GSH levels upon Cd exposure. This also became apparent at the transcript level, as the induction of the GSH metabolism genes was abolished in these mutants [
31]. It is clear from our point of view that, at least at the transcript level, ethylene signalling (
Table 4) precedes the induction of
GSH1, GSH2 and
GR1 in the leaves (
Table 2). It should be noted, however, that leaf ethylene signalling, assessed by the induction of ethylene-responsive genes such as
ERF1 (
Table 5), precedes the higher leaf ACC concentrations observed after 24 h of Cd exposure (
Figure 4). Therefore, we suggest that the transcriptional induction of
ERF1 is not a result of a de novo ethylene synthesis originating from the leaf, but of ethylene produced at the root level that evokes responses in the leaves. However, cross-talk with other phytohormones, like jasmonate, should not be neglected. Nevertheless, studies found that stress-induced
ERF1 expression was strongly diminished in the leaves of the ethylene biosynthesis double mutant
acs2-6, which further corroborates that ethylene is largely responsible for the induction of
ERF1 in the leaves [
35,
63]. Indeed, at the level of the root, ethylene synthesis, or at least ACC concentration (
Figure 4), was already significantly increased upon 2 h of exposure. Therefore, the root system might serve as a command centre and delivers stress-related signals, like ethylene, to the leaves. The latter will engage in an optimal response that becomes apparent by the stimulation in leaf GSH biosynthesis rather than GSH depletion, which occurs in the roots after similar Cd concentrations are encountered (
Figure 2).