3.1. Cadmium accumulation reduced maize root growth and modified root nutrient composition
The presence of Cd in the hydroponic solution reduced maize root growth by about 70% in length and 45% in biomass (Table 1), in line with previous reports (Xu et al. 2014; Anjum et al. 2016b; Li et al. 2020a), and Cd accumulation in maize root was clearly dose-dependent (Fig. 1). However, a similar degree of growth impairment was observed under both Cd concentrations tested. Laboratory soil-less systems abolish the complex physicochemical interactions that take place under natural field conditions and may alter nutrients’ and pollutants’ bioavailability. Among the soil properties that govern Cd diffusion flux towards the root surface, soil pH, clay content, metal oxides, cation exchange capacity, organic matter content, and Ca2+ concentration have been reported, and also total Cd content impacts on Cd uptake (Liu et al. 2015a; Lin et al. 2016; Yi et al. 2020).
Table 1
Effect of Cd on root length and biomass. Maize seedlings were grown in a hydroponic system containing diluted (1/10) Hoagland’s nutrient solution without (control, C) or with 50 and 100 µM of CdCl2, and root length, fresh weight (FW), and dry weight (DW) were determined 72 h later.
|
Control
|
µM CdCl2
|
|
|
50
|
100
|
Length (cm)
|
8.0 ± 1.2a
|
2.7 ± 0.3b
|
2.4 ± 0.7b
|
FW
|
558 ± 79a
|
269 ± 65b
|
337 ± 52b
|
DW
|
38 ± 12a
|
17 ± 6b
|
17 ± 6b
|
Data are expressed in mg/10 seedlings; means ± SEM of five independent experiments, with three biological replicates per treatment, are shown. Different letters within rows indicate significant differences (P < 0.05), according to Tukey’s multiple range test. |
Plants have not developed any specialized uptake system for cadmium because this element has no biological function. Nevertheless, this metal can be easily taken up by plant roots through membrane transporters of essential nutrients (Sterckeman y Thomine 2020). Current evidence indicates that Cd root symplastic influx in maize is controlled by high- and low-affinity transport systems (Redjala et al. 2009, 2010). Furthermore, cadmium can be strongly adsorbed on the maize cell wall, resulting in a large amount of Cd2+ being retained in the root apoplast (Redjala et al. 2009).
As Table 2 shows, Cd accumulation in emerging maize roots was accompanied by significant decreases in Ca, Fe, Mg, and Mn contents. A reduction of 48% and 68% in Ca level was determined for 50 and 100 µM Cd, respectively. For both Cd concentrations assayed, the reduction in Mg level was close to 60%, and for Fe and Mn, similar decreases of about 38% were detected. On the other hand, Zn was incremented by 16% over the control only under 50 µM Cd, and Cu content doubled that of the control in the roots of seedlings subjected to 100 µM Cd.
Table 2
Effect of Cd on root chemical composition. Maize seedlings were grown in a hydroponic system containing diluted (1/10) Hoagland’s nutrient solution without (control, C) or with 50 and 100 µM of CdCl2. After 72 h of treatment, roots were harvested and used for analytical determinations. Element concentrations are expressed in mg kg-1 of dry weight.
|
Cu
|
Ca
|
Fe
|
K
|
Mg
|
Mn
|
P
|
S
|
Zn
|
C
|
30 ± 10b
|
2135 ± 326a
|
79 ± 09a
|
15367 ± 1436a
|
1372 ± 460a
|
8 ± 1a
|
9871 ± 420a
|
1688 ± 398a
|
59 ± 1b
|
50 µM
|
35 ± 4b
|
1103 ± 94b
|
49 ± 3b
|
19474 ± 1611a
|
525 ± 9b
|
5 ± 1b
|
10868 ± 165a
|
1723 ± 124ª
|
69 ± 4a
|
100 µM
|
73 ± 9a
|
700 ± 49b
|
50 ± 2b
|
17927 ± 178a
|
654 ± 12b
|
5 ± 1b
|
9299 ± 268a
|
1644 ± 196ª
|
55 ± 2b
|
Data are mean ± SEM of three independent experiments, with three biological replicates per treatment. Different letters within columns indicate significant differences (P < 0.05), according to Tukey’s multiple range test. |
Change in nutrient absorption/distribution patterns is one of the most recognized cadmium harmful effects and has been mainly attributed to competition with divalent cation transporters (Huang et al. 2020). Ca and Mg (typically the most abundant divalent cations in plants) reductions could have affected normal growth and development. In this sense, it has been pointed out that growth restriction under Cd stress would be a nutrient deficiency symptom and the result of homeostatic balance loss between these cations (Tang y Luan 2017; Thor 2019; Kleczkowski y Igamberdiev 2021). Similarly, Cd reduced Fe and Mn contents in maize root. According to several reports, Cd shares similar plant entry routes with these relevant nutrients, so that the decreases found can be the outcome of Cd competition with Fe and Mn transporters (Thomine et al. 2000; Wu et al. 2016; Chen et al. 2017b; Chang et al. 2020). On the other hand, it has been demonstrated that the external addition of Ca, Mg, Fe, or Mn to the nutrient solution restricted Cd uptake and translocation, resulting in alleviation of Cd stress (Paľove-Balang et al. 2006; Sterckeman et al. 2011; Liu et al. 2013; Kudo et al. 2015; Rahman et al. 2016; Huang et al. 2017; Chen et al. 2017a; Hussain et al. 2020).
A complex interaction between Cd and Zn has been documented before, and it was proposed that uptake/translocation of Zn would increase in the presence of Cd (Nan et al. 2002). Moreover, it was demonstrated that induction of several genes belonging to the ZIP family–a group of proteins that mediate Zn and Cd transport–depends on the Zn:Cd ratio in the growing medium (Barabasz et al. 2016; Palusińska et al. 2020).
Cu increase and Mn decrease could account for cell redox homeostasis disruption under Cd stress. Cu is a redox-active metal and Mn, apart from having free radical scavenging capacity (Coassin et al. 1992), acts as a cofactor of an important enzymatic antioxidant, superoxide dismutase (Mn-SOD); Ca is also a signaling messenger intimately interconnected with ROS (Mazars et al. 2010; Steinhorst y Kudla 2013). Thus, the nutrient imbalance could be part of the indirect mechanisms by which Cd induces oxidative stress in maize roots.
3.2. Cadmium differentially affected peroxidase activities along the root and disrupted ascorbate homeostasis
In maize seminal root, CAT and APX activities were mostly localized in the root tip (Ap), while GPX activity was predominantly in the remaining tissue (Rt) (Fig. 2). Among peroxidases, CAT catalyzes the dismutation of H2O2 in the absence of electron donors. Its activity is largely found in subcellular compartments with H2O2 generation, such as peroxisomes, and also in mitochondria, chloroplasts, and the cytosol (Sharma y Ahmad 2014). CAT activity increased in the Ap under 100 µM Cd2+ (130% over the control), but in the Rt, CAT activity increased by 67% under 50 µM Cd2+ and decreased by 42% under 100 µM Cd2+ compared to the control. An increase in CAT activity may be interpreted as a cell-protective strategy against the detrimental effect of H2O2. On the contrary, a decrease in CAT activity deprives cells of their normal antioxidant capacity and results in oxidative stress. Catalase inactivation by metals has been associated with the oxidation of the protein structure (Pena et al. 2011) and the suppression of CAT gene expression (Ye et al. 2014).
To counteract an excessive H2O2 formation in plant tissues, non-specific peroxidases acting on one- or two-electron donors (including phenolic compounds such as guaiacol) are usually induced. In plants, GPX activity is mainly located in vacuoles and cell walls but not in organelles (Asada 1992). Under both concentrations, Cd increased GPX activity by about 70% in the Ap, while in the Rt, increases over the control of 47% and 72% for 50 and 100 µM Cd2+, respectively, were recorded (Fig. 2). GPX activity rise during Cd stress would be involved not only in the control of H2O2 levels but also in the modulation of plant growth and development through the control of hormonal and cell wall metabolism (Jouili et al. 2011).
Ascorbate peroxidase reduces H2O2 to H2O using ascorbate as the specific electron donor. Different APX isoforms are located in chloroplasts, cytosol, mitochondria, and peroxisomes, as well as in the apoplastic space (Gill y Tuteja 2010; Hasanuzzaman et al. 2019). In maize root apex, APX activity was not affected by Cd treatment, in line with previous observations in barley root tips (Bocova et al. 2012), but the activity of this enzyme was particularly impaired in the Rt, dropping to near to half under both Cd concentrations (Fig. 2). Because of a higher APX affinity for H2O2 than CAT and GPX, it has been suggested that this enzyme has a more crucial role in the scavenging of ROS during abiotic stress (Sofo et al. 2015; Anjum et al. 2016a).
In both root portions, total ASC (ASC plus DHAs) levels augmented under Cd treatment due to a pronounced rise in DHAs content, resulting, at the same time, in the reduction of ASC/DHAs ratio (Table 3). This finding suggests that Cd altered the adequate functioning of the ASC-GSH cycle.
Table 3
Effect of Cd on ascorbate (ASC) and dehydroascorbate (DHAs) content. Maize seedlings were grown in a hydroponic system containing diluted (1/10) Hoagland’s nutrient solution without (control, C) or with 50 and 100 µM of CdCl2 for 72 h. Concentrations are expressed in nmol g-1 of fresh weight.
|
Ap
|
Rt
|
|
ASC
|
DHAs
|
ASC / DHAs
|
ASC
|
DHAs
|
ASC / DHAs
|
C
|
218 ± 10a
|
475 ± 31c
|
0.5
|
915 ± 50A
|
310 ± 60B
|
2.9
|
50 µM
|
248 ± 5a
|
2265 ± 81a
|
0.1
|
1090 ± 20A
|
590 ± 40B
|
1.8
|
100 µM
|
151 ± 5b
|
1600 ± 69b
|
0.1
|
1010 ± 60A
|
1030 ± 120A
|
1.0
|
Data are means ± SEM of five independent experiments, with three biological replicates per treatment. Different letters within columns indicate significant differences (P < 0.05), according to Tukey’s multiple range test. |
3.3 Cadmium-induced accumulation of oxidatively damaged proteins was prevented by 20S proteasome increased activity
3.4. Cadmium altered hormonal root homeostasis
Cadmium enhanced IAA and ABA levels in the entire root tissue, whereas SA content increased only in the Rt portion (Table 4). IAA increments by Cd in rice roots were related to the overexpression of the biosynthetic genes OsASA2 and OsYUCCA1 (Ronzan et al. 2019). Also, it has been described that Cd affects not only IAA content but also its distribution, metabolism, and transport (Chmielowska-Bak et al. 2014), suggesting an eventual switch to an alternative morphogenic root program to counteract metal stress (Hu et al. 2013; Fattorini et al. 2017; Piacentini et al. 2020). Also, numerous reports indicate that exogenous application of IAA, as well as the IAA precursor indole-3-butyric acid (IBA), reduced Cd toxicity in plants (Agami y Mohamed 2013; Li et al. 2020b; Zhang et al. 2020; Zhou et al. 2020; Piacentini et al. 2020; Demecsová et al. 2020). However, more information is needed to know if endogenous IAA levels reached in maize root during Cd stress can induce a similar effect compared to that observed when IAA is exogenously added.
Table 4
Effect of Cd on hormone content. Extracts were obtained from root apex (Ap) and the remaining root tissue (Rt) of maize seedlings subjected to hydroponic culture without (control, C) or with 50 and 100 µM of CdCl2 for 72 h.
Hormone
|
Ap
|
Rt
|
content
|
C
|
CdCl2 (µM)
|
C
|
CdCl2 (µM)
|
(ng g− 1 FW)
|
|
50
|
100
|
|
50
|
100
|
IAA
|
10.8 ± 1.7a
|
13.9 ± 0.6b
|
20.5 ± 0.7c
|
8.1 ± 2.3A
|
23.6 ± 2.6B
|
36.3 ± 5.7C
|
ABA
|
1.94 ± 0.15a
|
5.46 ± 0.61b
|
3.49 ± 0.12b
|
1.67 ± 0.49A
|
8.65 ± 0.34B
|
12.51 ± 0.63B
|
SA
|
9.3 ± 0.2a
|
17.3 ± 1.6b
|
5.6 ± 0.5c
|
16.6 ± 1.7A
|
25.6 ± 1.6B
|
25.0 ± 1.0B
|
GA20
|
3.1 ± 0.3a
|
3.0 ± 0.3a
|
4.9 ± 0.1b
|
2.7 ± 0.2A
|
3.7 ± 0.6A
|
6.4 ± 0.2B
|
GA7
|
146 ± 9a
|
123 ± 4b
|
125 ± 6b
|
146 ± 4A
|
148 ± 8A
|
156 ± 3A
|
GA3
|
5.1 ± 0.8a
|
3.7 ± 0.3b
|
5.3 ± 0.3a
|
3.3 ± 0.6B
|
5.8 ± 0.7A
|
3.9 ± 0.7B
|
GA4
|
58.6 ± 14.3a
|
25 ± 2 b
|
18.2 ± 2c
|
65.7 ± 10.0A
|
38 ± 10B
|
35 ± 1.8B
|
JA
|
61.2 ± 10.7a
|
64.2 ± 5.8a
|
38.4 ± 2.5b
|
82.8 ± 12.1A
|
71.8 ± 1.9A
|
89.3 ± 8.5A
|
JA-Ile
|
47.6 ± 5.7a
|
9.5 ± 2.8b
|
3.7 ± 0.2b
|
57.9 ± 9.6A
|
14.8 ± 0.3B
|
20.9 ± 3.1B
|
Data are means ± SEM of three independent experiments, with three biological replicates per treatment. Values represent means ± SEM. Different letters within rows indicate significant differences (P < 0.05), according to Dunnett’s multiple comparisons test. |
In plants, ABA is recognized as a modulator of the adaptive abiotic stress responses (Cutler et al. 2010) and a key player in alleviating heavy metal stress (Hu et al. 2020). Hsu and Kao (2003) reported a close relationship between endogenous ABA content and Cd tolerance in rice seedlings. Also, it was described that exogenous ABA application would partially relieve Cd toxic effects by increasing GSH and phytochelatins biosynthesis (Chen et al. 2016; Song et al. 2016), as well as restrict Cd uptake and distribution (Han et al. 2016; Shen et al. 2017; Tang et al. 2020).
SA increase in the Rt may be involved as a mechanism to counteract oxidative stress induced by Cd. It has been well established that SA application improves plant acclimation to Cd excess by reducing the metal uptake and/or promoting plant antioxidant capacity (Popova et al. 2009; Hayat et al. 2010; Agami y Mohamed 2013; Shakirova et al. 2016; Guo 2019). In accordance, an Arabidopsis SA-deficient mutant resulted in negative effects on Cd tolerance, mainly due to the lowered GSH status (Guo et al. 2016).
Cadmium increased the root concentration of GA20, the precursor of the active form 13-hydroxylated GA3. Interestingly, the total root content of GA3 remained similar to the control at 100 µM Cd but decreased in the Ap and increased in the Rt at 50 µM Cd. On the other hand, the contents of non-13-hydroxylated GA7 and GA4 were reduced under both Cd treatments. A similar drastic decrease in GA4 content was reported during copper stress (Matayoshi et al. 2020).
The mechanism by which metals affect GA4 homeostasis could involve interference with hormone biosynthesis but also with subsequent gibberellin transformations. Liu et al. (2015b) reported up-regulation of two genes encoding GA2-oxidase, a major enzyme for deactivating bioactive gibberellins, in response to Cd stress.
The presence of Cd had a dramatic consequence on the active form JA-Ile, whose concentration was strongly diminished under the metal treatment. JA-Ile is considered the most metabolically active jasmonate (Fonseca et al. 2009), and, although the exogenous application of JA or methyl jasmonate (MJ) has been shown to alleviate Cd-toxic effects in plants (Singh y Shah 2014; Siddiqi y Husen 2019; Lei et al. 2020), little attention has been paid to Ile-JA regarding cadmium stress. Kurotani et al. (2015) suggested that deactivation of JA-Ile results in enhanced salt tolerance in rice. It would be of special interest to evaluate the turnover of JA-Ile in the context of Cd stress in future studies.