plants
Article
Elevated CO2 Can Improve the Tolerance of Avena sativa to
Cope with Zirconium Pollution by Enhancing
ROS Homeostasis
Mahmoud M. Y. Madany 1, * , Hamada AbdElgawad 2 , Doaa A. Galilah 3 , Ahmed M. A. Khalil 4
and Ahmed M. Saleh 4
Citation: Madany, M.M.Y.;
AbdElgawad, H.; Galilah, D.A.;
Khalil, A.M.A.; Saleh, A.M. Elevated
CO2 Can Improve the Tolerance of
Avena sativa to Cope with Zirconium
Pollution by Enhancing ROS
Homeostasis. Plants 2023, 12, 3792.
https://doi.org/10.3390/
plants12223792
Academic Editor:
Daniel Sánchez-Mata
Received: 29 August 2023
Revised: 23 September 2023
Accepted: 16 October 2023
Published: 7 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Plants 2023, 12, 3792. https://doi.org/10.3390/plants12223792
1 Biology Department, Faculty of Science, Taibah University, Al-Madinah Al-Munawarah 41411, Saudi Arabia
2 Botany and Microbiology Department, Faculty of Science, Beni-Suef University, Beni-Suef 62521, Egypt
3 Botany Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
4 Biology Department, Faculty of Science at Yanbu, Taibah University, King Khalid Rd., Al Amoedi,
Yanbu El-Bahr 46423, Saudi Arabia
* Correspondence: madany@cu.edu.eg
Abstract: Zirconium (Zr) is one of the toxic metals that are heavily incorporated into the ecosystem
due to intensive human activities. Their accumulation in the ecosystem disrupts the food chain,
causing undesired alterations. Despite Zr’s phytotoxicity, its impact on plant growth and redox status
remains unclear, particularly if combined with elevated CO2 (eCO2 ). Therefore, a greenhouse pot
experiment was conducted to test the hypothesis that eCO2 can alleviate the phytotoxic impact of
Zr upon oat (Avena sativa) plants by enhancing their growth and redox homeostasis. A complete
randomized block experimental design (CRBD) was applied to test our hypothesis. Generally,
contamination with Zr strikingly diminished the biomass and photosynthetic efficiency of oat plants.
Accordingly, contamination with Zr triggered remarkable oxidative damage in oat plants, with
concomitant alteration in the antioxidant defense system of oat plants. Contrarily, elevated levels of
CO2 (eCO2 ) significantly mitigated the adverse effect of Zr upon both fresh and dry weights as well as
the photosynthesis of oat plants. The improved photosynthesis consequently quenched the oxidative
damage caused by Zr by reducing the levels of both H2 O2 and MDA. Moreover, eCO2 augmented
the total antioxidant capacity with the concomitant accumulation of molecular antioxidants (e.g.,
polyphenols, flavonoids). In addition, eCO2 not only improved the activities of antioxidant enzymes
such as peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) but also boosted the
ASC/GSH metabolic pool that plays a pivotal role in regulating redox homeostasis in plant cells. In
this regard, our research offers a novel perspective by delving into the previously unexplored realm
of the alleviative effects of eCO2 . It sheds light on how eCO2 distinctively mitigates oxidative stress
induced by Zr, achieving this by orchestrating adjustments to the redox balance within oat plants.
Keywords: antioxidants; ascorbate/glutathione cycle; Avena sativa; oxidative damage; toxic metals
1. Introduction
Growing environmental and health apprehensions have been raised regarding toxic
metals, primarily due to their extensive dissemination throughout the ecosystem. The
accumulation of such toxic metals in the ecosystem imposes rigorous imperilment on both
human health and agricultural productivity. There are several reasons for the introduction
of such toxic metals to the environment. Anthropogenic endeavors such as mining activities,
the application of pesticides and industrial wastes are the common sources of such toxic
metals [1]. Once they have been introduced to the ecosystem, they are incorporated into
the food chain, causing undesired alterations [2]. Although plants need some of these
metals for their growth and development, the existence of these metals in high levels
causes a tremendous disruption in plant physiology and metabolism [3]. Like other toxic
https://www.mdpi.com/journal/plants
Plants 2023, 12, 3792 2 of 21

metals, Zirconium (Zr) is widely distributed in the Earth’s crust [4]. It is considered a
reactive metal but is more resistant to corrosion than many other metals. This is due to
the formation of a thin layer of zirconium dioxide (ZrO2 , also known as zirconia) that
acts as a protective layer for the underlying metal against further oxidation [5]. Zr and its
alloys are widely used in different human activities. It is commonly applied in the nuclear
industry due to its high resistance to corrosion, in addition to being used as cladding for
nuclear fuel rods in reactors [5]. Furthermore, Zr compounds can be utilized in various
sectors of industries such as ceramics, refractories, and electronics. More interestingly,
ZrO2 is a material that can be utilized in various applications, including dental implants,
catalysts, and solid oxide fuel cells. The widespread occurrence of Zr in various activities
has led to its extensive presence in our surroundings. This situation has raised alarm
within the scientific community due to the potential detrimental effects it poses on human
health and agricultural productivity. Zr and its compounds are toxic to human health,
so proper handling and safety precautions should be taken to prevent its dispersion [6].
Concerning plants, although Zr is not an essential element for plant growth, excessive
exposure to Zr and its compounds may slow plant growth and development and change
the soil structure [7]. Nonetheless, when it comes to understanding the effects of Zr on
plants, there has been relatively limited research compared to more extensively studied
elements. The excessive buildup of Zr within plant tissues has the potential to interfere with
nutrient absorption and crucial metabolic processes, ultimately resulting in a significant
decline in plant growth [6]. Shaid et al. [6] also declared that Zr can interfere with the
availability of some essential nutrients, such as Ca, Mg, and K which are vital for most plant
metabolic processes, something that can hinder overall plant growth and development.
Moreover, excessive exposure to Zr has been reported to alter the photosynthetic pigments
of Chlorella pyrenoidosa, leading to a negative impact on culture mass and yield [8]. Both root
function and development can also be affected by higher exposure to Zr. The disturbance
in root function and growth, in turn, can impact the plant’s capacity to absorb nutrients
and water, diminishing its ability to acquire vital resources, leading to stunted growth and
decreased vitality [9]. Furthermore, Fodor et al. [9] declared that plant exposure to Zr may
trigger plant oxidative stress that causes an imbalance between the production of ROS and
the ability to detoxify them. This oxidative damage can disrupt the cellular components
and slow different metabolic pathways in plants [10]. It is crucial to understand that
the detrimental impacts of Zr on plants can be influenced by various factors, including
the type and the concentration of Zr in the soil, soil conditions and the specific plant
species involved. Nonetheless, the precise mechanisms through which Zr influences plant
metabolism remain incompletely elucidated, underscoring the need for further research to
comprehensively assess the impact on plant growth and metabolic processes. To this end,
researchers should explore environmentally friendly and economically viable agricultural
approaches to mitigate the potential harmful effects of excessive Zr accumulation. One of
these tools is by enriching the atmosphere of plants with carbon dioxide.
The intensive increase in atmospheric CO2 levels is a major environmental challenge.
This elevation is expected to further modify soil properties, altering the growth and devel-
opment of economically important crops. Progressive industrial activities have caused a
rapid increase in CO2 concentration from 280 ppm to 400 ppm [11]. Prognostications have
shown that this increment will continue to increase as a consequence of immense human
industrial impact [12]. In point of fact, elevation in CO2 within a physiological range has
been proven to enrich plant growth, enhancing photosynthetic carbon metabolism with
consequent boosting in assimilation [10]. The enhancement in carbon metabolism will
accordingly be reflected in the partitioning of plant carbon and nitrogen [11]. Furthermore,
several studies showed that eCO2 could alleviate the detrimental effects of a variety of
environmental constraints upon plant growth and metabolism [10,13,14]. This potential of
eCO2 is attributed to its effective changes in stomatal conductance, which play a crucial
role in enhancing plant water uptake [15]. More interestingly, eCO2 could mitigate the
imposed stress by modulating the redox homeostasis through regulating ROS production
Plants 2023, 12, 3792 3 of 21

and scavenging [16]. Therefore, the scientific community should pay attention to the impact
of eCO2 in boosting plant tolerance under different environmental challenges, especially for
economically important crops such as oat plants. In this regard, eCO2 raised the tolerance
level of two barley cultivars grown under salinity stress [17]. Furthermore, Mhamdi and
Noctor [18] suggested that Arabidopsis subjected to eCO2 treatment exhibits increased
resistance against bacterial and fungal invasions. Likewise, it was found that treatment
with eCO2 enhanced the defense system of Arabidopsis against different leaf and root
pathogenic fungi [19]. Furthermore, tomato plants treated with eCO2 can cope with heat
stress by enhancing photosynthesis and redox homeostasis [20]. Overall, these investi-
gations and more were directed to study the combined effect of both eCO2 and different
biotic and abiotic stresses. Nevertheless, the effect of raising atmospheric CO2 upon a toxic
metal like Zr has not yet been considered. Therefore, our study was conducted to closely
investigate the impact of two levels of CO2 (ambient, 420 ppm, and elevated, 710 ppm) on
the growth and redox status of oat (Avena sativa) grown under the curb of Zr pollution.
We further aim to verify the hypothesis that eCO2 amplifies carbohydrate portioning by
bolstering photosynthetic efficiency. This, in turn, fortifies the antioxidant defense system,
enabling it to counteract the oxidative damage caused by Zr.

2. Material and Methods

2.1. Plant Materials and Treatment
In order to gain a deeper insight into the impact of Zr on the plants, this study was
performed with conditions that approached environmentally realistic conditions that closely
mimic the environmental scenarios. The seeds of Avena sativa were homogeneously selected
and sterilized with Na-hypochlorite (5% v/v, 20 min) and then washed thoroughly with
distilled water. The sterilized seeds were sown in 20 cm diameter, 30 cm height polyvinyl
chloride (PVC) tubes containing sandy soil with adjusted pH (7.6). The soil was irrigated
daily to maintain soil capacity at 68%. The cultivation of plants took place within chambers
that were illuminated by sunlight and maintained under controlled temperature and CO2
levels. The upper parts of these sunlit chambers were constructed with a transparent
polycarbonate plate to permit light entry. The temperature was set to 26 ◦ C during the day
and 20 ◦ C at night, and Photosynthetic Active Radiation was detected using an SDEC, type
JYP1000 quantum sensor. The pots were divided into four groups, each with 4 replicates:
(1) ambient CO2 (aCO2 , 420 ppm); (2) aCO2 + Zr (400 mg/kg soil); (3) future climate CO2
(eCO2 , 710 ppm); and (4) eCO2 + Zr (400 mg/kg soil). Zr was introduced to the soil as
ZrSO4 .4H2 O (Sigma-Aldrich, Germany). The soil was consistently mixed with the desired
amount of Zr. After 8 weeks, the rhizosphere soil as well as plant tissues were collected
and kept in −80 ◦ C for further biological analyses. The fresh and dry weights of oat plants
were recorded.

2.2. Determination of Zr in Plant Tissues

To extract Zr, the plant samples, which had been dried at 70 ◦ C, were subjected
to a treatment involving 13 M nitric acid at a temperature of 185 ◦ C for a duration of
25 min. Subsequently, the concentration of elements was determined using a quadrupole
inductively coupled plasma mass spectrometry (ICP-MS; model 820-MS) setup, linked
with a glass nebulizer operating at a flow rate of 0.4 mL/min. To generate calibration
curves, external standards were prepared at concentrations ranging from 1 to 600 µg/L.
Additionally, yttrium was included as an internal standard during the extraction process to
calibrate the efficiency of the nebulizer. Standard mineral samples were prepared using
0.23 M nitric acid.

2.3. Photosynthesis and Photosynthetic Related Parameters

Photosynthetic and stomatal conductance measurements were carried out following
the methodologies detailed in Ainsworht and Rogers [15]. To determine photosynthesis
under saturating light conditions (Asat, µmol CO2 m−2 s−1 ), a LI-COR LI-6400 instrument
Plants 2023, 12, 3792 4 of 21

(LI-COR Inc., Lincoln, NE, USA) was used. Each step of the measurement included a leaf
equilibration period of at least 5 min before recording data. The LI-COR leaf chamber was
configured with conditions set at either 400 or 620 ppm CO2 , a block temperature of 22 ◦ C,
and a saturated photon flux density of 1500 µmol m−2 s−1 . Stomatal conductance (gs, mol
CO2 m−2 s−1 ) was assessed on the abaxial surface of fully developed leaves utilizing a leaf
porometer (Model SC-1, Decagon Devices, Inc., Hopkins, Pullman, WA, USA). The average
vapor pressure deficit and leaf temperature were maintained at 0.37 ± 0.02 and 20 ± 2.02,
respectively. Chlorophyll fluorescence was gauged on fully expanded leaves that had been
dark-acclimated, using an FMS-2 pulse-modulated fluorometer (Hansatech Instruments,
Norfolk, UK). For leaves adapted to darkness for 30 min, minimal fluorescence (F0) and
maximal fluorescence (Fm) were recorded. The photochemical efficiency of Photosystem II
(PSII) (Fv/Fm) for these dark-adapted leaves was computed, where Fv (maximal variable
fluorescence) was calculated as Fm−F0. Chlorophyll a, chlorophyll b and carotenoid
concentrations were assessed in oat shoots homogenized using acetone [21]. RuBisCo
activity was quantified using a non-radioactive microplate-based method that involved the
enzymatic cycle between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate
oxidase to measure the product, 3-phosphoglycerate (3-PGA) [22].

2.4. Determination of Oxidative Stress Markers

Lipid peroxidation was assessed using the thiobarbituric acid–malondialdehyde (TBA–
MDA) assay according to the method outlined by Senthilkumar et al. [23]. For this, 0.3 g of
frozen oat tissue was homogenized in 80% ethanol using a mortar and pestle. Centrifuge
the extract (10,000× g for 10 min). Take 0.5 mL of the clear supernatant then add 2.5 mL
of the TBA and 0.5 mL butylated hydroxytoluene (BHT) to prevent further oxidation
during heating. Incubate in a boiling water bath for 30 min, centrifuge and measure the
absorbance at 532 nm. MDA concentration was calculated using the molar extinction
coefficient of MDA (1.53 mM−1 cm−1 ). The quantification of H2 O2 levels was performed
using the xylenol orange-based FOX1 technique [22]. Mix 0.1 mL of plant extract with
0.9 mL FOX1 reagent (4.4 mM xylenol orange, 2.6 mM sorbitol, 25 mM sulfuric acid, and
0.2 mM ferrous ammonium sulfate) then add 0.05 mL of ascorbic acid. Incubate the mixture
at room temperature for 30 min in the dark, then centrifuge (10,000× g for 5 min). Measure
the absorbance of the colored complex at 560 nm. H2 O2 levels were calculated using a
standard curve with known concentrations of H2 O2 .

2.5. Determination of Antioxidants’ Secondary Metabolites

Polyphenols and flavonoids were measured employing the Folin–Ciocalteu and alu-
minum chloride assays, respectively, as outlined in the reference [24]. The separation
and quantification of tocopherols were achieved through High-Performance Liquid Chro-
matography (HPLC) using normal phase conditions with a Shimadzu system based in
Hertogenbosch, Netherlands. The HPLC setup involved a Particil Pac column (5 µm
column material, length 250 mm, i.d. 4.6 mm). Total Antioxidant Capacity (TAC) was
evaluated following the Ferric Reducing Antioxidant Power (FRAP) method described by
Benzie and Strain [25], wherein Trolox (Sigma-Aldrich, St. Louis, MO, USA) was employed
as an internal standard.

2.6. Estimation of Enzymatic Antioxidants

For the determination of antioxidant enzyme activity, proteins were extracted using
K-phosphate extraction buffer (50 mM, pH 7.0) supplemented with 10% PVPP (w/v), 0.25%
Triton X-100 (v/v) and 1 mM PMSF. Peroxidase (POX) activity was assessed through the
oxidation of pyrogallol at 430 nm [26], while superoxide dismutase (SOD) enzyme activities
were determined by monitoring the inhibition of NBT reduction at 560 nm [27]. A known
weight of frozen tissue (0.5 g) was homogenized in 1 ml extraction buffer (0.1 M phosphate
buffer, pH 6.0), then centrifuged (4000× g, for 10 min at 4 ◦ C). Then, 1 ml clear extract
was taken and mixed with 1 ml assay mixture (20 M pyrogallol; 20 mM guaiacol; 30%
Plants 2023, 12, 3792 5 of 21

H2 O2 ). After mixing well, the produced color was measured at 420 nm. Dehydroascorbate
reductase (DHAR), glutathione reductase (GR), ascorbate peroxidase (APX), and monode-
hydroascorbate reductase (MDHAR) were evaluated spectrophotometrically based on the
method described by Murshed et al., [28] by utilizing 0.05 M MES/KOH as the buffer.
Catalase (CAT) activity was quantified by observing the rate of H2 O2 decomposition at
240 nm [29]. A known fresh weight of plant tissue was homogenized in 0.1 M potassium
phosphate buffer (pH 7.0), then centrifuged at low speed (3000 g) for 10 min at 4 ◦ C. Then,
100 µL clear extract was mixed with 2 mL assay mixture (0.1 M KH2 PO4 buffer, pH 7.0; 30%
H2 O2 ). The change in color at 240 nm was measured over time. Glutathione peroxidase
(GPX) activity was measured by monitoring the reduction of NADPH at 340 nm [30]. For
GPX activity, a known weight of powdered fresh tissue was homogenized in ice-cold 0.1 M
KH2 PO4 buffer (pH 7.0). After centrifugation, 50 µL of the clear extract was mixed with
the assay mixture (0.1 M KH2 PO4; 1 M GSH; 1 mM NADPH). The change in color was
monitored at 340 nm, after adding 30% H2 O2 to initiate the reaction. The total soluble
protein concentration was determined using the Lowry technique [31].

2.7. Determination of AsA/GSH Metabolites

Reduced ascorbate (ASC) and reduced glutathione (GSH) levels were measured us-
ing High-Performance Liquid Chromatography (HPLC) with a Shimadzu SIL10-ADvp
system and a C18 column (Spherisorb ODS2, 5 µm particle diameter, 4.6 × 250 mm, Wa-
ters). The concentrations of total ascorbate (ASC + DHA) and glutathione (GSH + GSSG)
were determined after reduction with dithiothreitol (DTT), following the methodology
outlined by Sinha et al. [32]. Enzyme activities including superoxide dismutase (SOD),
glutathione peroxidase (GPX), ascorbate peroxidase (APX), glutathione reductase (GR),
monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and
glutathione-S-transferase (GST) were assessed according to the procedures detailed in our
previous research [33].

2.8. Statistical Analysis

The outcomes of the measured parameters are presented as the average of four biolog-
ical replicates (n = 4). Statistical evaluation was executed utilizing one-way ANOVA and
two-way ANOVA within the SPSS 21 software (Inc., Chicago, IL, USA). Mean distinctions
were ascertained through Fisher’s least significance difference (LSD) test. The normality
of data distribution and the equality of variances were assessed using the Kolmogorov–
Smirnov test and Levene’s test, respectively. Furthermore, a comparative evaluation of
pollution severity between Zr and Zr + eCO2 was conducted through LSD, with a signifi-
cance threshold set at p < 0.05.

3. Results
3.1. Effect of eCO2 on Zr Cumulation and the Growth of Oat Plants
Our results showed that the levels of Zr in contaminated oat plants was very high;
however, the cotreatment of oat plants with both Zr and elevated levels of eCO2 caused a
remarkable reduction in the levels of Zr (~40% reduction) when compared with oat plants
grown in contaminated soils (Figure 1A). This significant reduction in the levels of Zr due
to the application of eCO2 prompted us to measure other growth parameters stand on the
mitigative impact of eCO2 . In this regard, oat plants experienced a noticeable reduction
in the biomass (fresh and dry weights) in response to Zr pollution when compared with
uncontaminated control plants (Figure 1B,C). On the other hand, treatment with eCO2
significantly enriched the biomass of oat plants (p < 0.05) as compared with normal control
plants. Further, the co-application of both Zr and CO2 noticeably enhanced both fresh and
dry weights of oat relative to plants contaminated with Zr. A two-way ANOVA revealed
that both Zr and eCO2 significantly interacted to affect both fresh (p < 0.001) and dry
(p < 0.05) weights of oat plans (Table 1).
Plants 2023, 12, x FOR PEER REVIEW 7 of 23
Plants 2023, 12, 3792 6 of 21

Figure
Figure 1. Thecombined
1. The combinedeffect
effectof of
bothboth
eCOeCO 2 and/or
2 and/or Zr upon
Zr upon (A)
(A) Zr Zr accumulation,
accumulation, (B)
(B) fresh fresh weight
weight
and
and (C) dry weight
(C) dry weightofofoat
oatplants.
plants. Four
Four biological
biological replicates
replicates werewere
used used to investigate
to investigate the response.
the response.
The
The vertical errorbars
vertical error barsare
arethethe standard
standard error
error (SE).(SE). Fisher’s
Fisher’s LSD(ptest
LSD test (p <n0.05,
< 0.05, n = used
= 4) was 4) wasforused for
pairwise comparison between groups. The different le ers indicate significant differences between
pairwise comparison between groups. The different letters indicate significant differences between
the
themeans
means ofof each
eachgroup.
group.

3.2. Photosynthesis of Oats as Affected by Zr and/or eCO2

The enrichment in oat growth due to the application of eCO2 in combination with Zr
encouraged us to investigate its effect upon the photosynthetic efficiency (Figure 2). As
intended, the delay in the biomass of oat in response to Zr pollution was associated with
a concomitant reduction in the photosynthetic machinery. This reduction was more obvi-
 Plants 2023, 12, 3792 7 of 21

Table 1. Two-way ANOVA on the significance of interaction between the two independent variables
(Zr and eCO2 ) upon Zr content and the biomass and photosynthesis in oat plants (ns = non-significant;
* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Dependent Type III Sum of Mean

df F Sig.
Variables Squares Square
Zr content 178.230 1 178.230 51.630 ***
FW 0.152 1 0.152 31.423 ***
DW 0.002 1 0.002 8.790 *
Photosynthesis 0.194 1 0.194 0.231 ns
CHLa 0.000 1 0.000 4.014 ns
CHLb 0.000 1 0.000 6.258 *
CHLab 0.000 1 0.000 5.429 **
Carotenoids 0.000 1 0.000 0.958 ns
gs 0.005 1 0.005 0.001 ns
CHLflourec 0.009 1 0.009 80.172 ***
Rubisco 7.279 1 7.279 0.989 ns

3.2. Photosynthesis of Oats as Affected by Zr and/or eCO2

023, 12, x FOR PEER REVIEW
The enrichment in oat growth due to the application of eCO2 in combination with Zr
encouraged us to investigate its effect upon the photosynthetic efficiency (Figure 2). As
intended, the delay in the biomass of oat in response to Zr pollution was associated with a
concomitant reduction in the photosynthetic machinery. This reduction was more obvious
in the levels of Chl a, Chl b and Chl (a + b), and accordingly the rate of photosynthesis, which
decreased by 61%, 71%, 50%, and 62%, respectively, in contaminated oat as compared with
uncontaminated control plants (Figure 2A,E–G). This could be attributed to the significant
decline in both stomatal and non-stomatal parameters such as stomatal conductance and
RuBisco that exhibited about 50% reduction relative to uncontaminated control plants
(Figure 2C,D). On the other hand, the individual treatment with eCO2 significantly affected
the rate of photosynthesis in oat plants. More interestingly, the application of eCO2 along
with Zr recovered the adverse effect of Zr upon photosynthetic parameters. For instance,
stomatal conductance and RuBisco increased by about 60% and 40%, respectively, when
compared with oat grown under aCO2 and Zr conditions (Figure 2C,D). More obviously,
total chlorophyll content experienced a three-fold increase relative to plants grown in
aCO2 and Zr conditions (Figure 2G). The two-way ANOVA showed different interactive
responses between the two independent variables (eCO2 and Zr) for all photosynthetic
parameters (Table 1). The co-application of eCO2 and Zr (eCO2 + Zr) significantly
9 of 23 enhanced
the chlorophyll (a + b) and carotenoids (p < 0.05) as well as chlorophyll fluorescence
(p < 0.001).

Figure 2. Cont.
Plants 2023, 12, 3792 8 of 21

Figure 2. The combined effect of eCO and/or Zr upon photosynthetic efficiency of oat plants.
Figure 2. The combined effect of eCO2 and/or Zr upon 2photosynthetic efficiency of oat plants. (A)
(A) rate(B)
rate of photosynthesis, ofchlorophyll
photosynthesis, (B) chlorophyll
fluorescence, fluorescence,
(C) stomatal (C) (D)
conductance, stomatal conductance,
RuBisco activity, (D) RuBisco
(E) chlorophyll a, (F) chlorophyll b, (G) total chlorophyll, and (H) carotenoids. Four biological rep-
activity, (E) chlorophyll a, (F) chlorophyll b, (G) total chlorophyll, and (H) carotenoids. Four biological
licates were used to investigate
replicates the response.
were used to investigateThe the
vertical error The
response. barsvertical
are theerror
standard error
bars are the(SE).
standard error (SE).
Fisher’s LSD testFisher’s
(p < 0.05,
LSDn =test
4) was
(p < used
0.05, nfor= pairwise
4) was usedcomparison between
for pairwise groups.between
comparison The different
groups. The different
le ers indicate significant differences
letters indicate between
significant the means
differences of each
between thegroup.
means of each group.

3.3. Influence of3.3.

eCOInfluence
2 on the Zr-Induced
of eCO2 on Oxidative Damage
the Zr-Induced in Oat Plants
Oxidative Damage in Oat Plants
The accumulation of lipid peroxidation
The accumulation (MDA) and (MDA)
of lipid peroxidation H2O2 isandconsidered one of theone of the main
H2 O2 is considered
main markers for oxidative damage in plants [34]. In this context, the individual applica-
markers for oxidative damage in plants [34]. In this context, the individual application of Zr
strikingly increased the levels of H O
tion of Zr strikingly increased the levels of H2O2 (a2 two-fold
2 (a two-fold increase) in oat plants
increase) in oat plants (Figure (Figure 3A). It is
3A). It is likely that the levels of MDA exhibited a noticeable accumulation in oat plants in in response
likely that the levels of MDA exhibited a noticeable accumulation in oat plants
to treatment with Zr (87% increase) relative to plants grown in eCO2 -uncontaminated
conditions. Additionally, individual treatment with eCO2 reduced the levels of both H2 O2
and MDA a little bit. The results showed that eCO2 alone reduced the levels of H2 O2
and MDA in oat by about 15% and 25%, respectively, as compared with the counter
control plants treated with aCO2 (Figure 3). More interestingly, the application of eCO2
in combination with Zr caused a tremendous reduction in the levels of H2 O2 and MDA.
The reduction was more pronounced in the levels of MDA that exhibited a 47% reduction
relative to Zr-treated control plants (Figure 3B). Overall, a significant interaction was found
between eCO2 and Zr for both H2 O2 and MDA (p < 0.01 and p < 0.001, respectively)
(Table 2).
Plants 2023, 12, x FOR PEER REVIEW 11 of 23
Plants 2023, 12, 3792 9 of 21

Figure 3. The combined effect of eCO and/or Zr on the levels of oxidative stress markers of oat
Figure 3. The combined effect of eCO22 and/or Zr on the levels of oxidative stress markers of oat
plants. (A)hydrogen
plants. (A) hydrogenperoxide
peroxide(H2O2)
(H2O2)andand (B)
(B) malondialdehyde
malondialdehyde (MDA).
(MDA).Four
Fourbiological
biologicalreplicates
replicates
are used to investigate the response. The vertical error bars are the standard error (SE). Fisher’s LSD
are used to investigate the response. The vertical error bars are the standard error (SE). Fisher’s LSD
test
test(p
(p<<0.05,
0.05,nn==4)4)was
wasused
usedfor
forpairwise
pairwisecomparison
comparisonbetween
betweengroups.
groups.The
Thedifferent
differentleletters
ers indicate
indicate
significant
significantdifferences
differences between
between thethe means
means of
of each
each group.
group.

3.4.eCO
3.4. eCO22and
andNon-Enzymatic
Non-EnzymaticAntioxidants
AntioxidantsininZr-Contaminated
Zr-ContaminatedOat
OatPlants
Plants
To contend
To contend with
with the
the oxidative
oxidative damage,
damage, plants
plants augmented
augmented their
their antioxidant
antioxidant defense
defense
arsenal to keep cell viability against the phytotoxic impact of Zr. Therefore, we aimed
arsenal to keep cell viability against the phytotoxic impact of Zr. Therefore, we aimed to to
measure the total antioxidant capacity (FRAP) as well as some non-enzymatic
measure the total antioxidant capacity (FRAP) as well as some non-enzymatic antioxi- antioxidants
(polyphenols and flavonoids) in oat plants treated with eCO and/or Zr (Figure 4). Our
dants (polyphenols and flavonoids) in oat plants treated with2eCO2 and/or Zr (Figure 4).
results revealed that Zr significantly increased the levels of FRAP in oat (~36% increase)
Our results revealed that Zr significantly increased the levels of FRAP in oat (~36% in-
compared to aCO2 -treated oat plants (Figure 4A). This increment was associated with
crease) compared to aCO2-treated oat plants (Figure 4A). This increment was associated
a remarkable increase in the levels of both polyphenols and flavonoids. Polyphenols
with a remarkable increase in the levels of both polyphenols and flavonoids. Polyphenols
experienced a two-fold increase in response to individual treatment with Zr relative to
experienced a two-fold increase in response to individual treatment with Zr relative to
aCO -treated control plants (Figure 4B). Similarly, treatment with Zr increased the levels
aCO22-treated control plants (Figure 4B). Similarly, treatment with Zr increased the levels
of flavonoids of oat plants by about 72% as compared with aCO -treated control plants
of flavonoids of oat plants by about 72% as compared with aCO22-treated control plants
(Figure 4C). Except for flavonoids, eCO2 caused a significant elevation in FRAP as well as
(Figure 4C). Except for flavonoids, eCO2 caused a significant elevation in FRAP as well as
polyphenols (Figure 4A,B). More interestingly, the application of both Zr and eCO2 FRAP
polyphenols (Figure 4A,B). More interestingly, the application of both Zr and eCO2 FRAP
caused a two-fold increase in both FRAP and flavonoids of oat plants relative to those
caused a two-fold increase in both FRAP and flavonoids of oat plants relative to those
grown under aCO2 . To a greater extent, polyphenols exhibited about a three-fold increment
grown under grown
in oat plants aCO2. To a greater
under extent,
both Zr and polyphenols
eCO2 if they exhibited about awith
were compared three-fold incre-
those grown
ment in oat plants grown under both Zr and eCO2 if they were compared with those
Plants 2023, 12, 3792 10 of 21

under aCO2 . According to the two-way ANOVA, the interaction between Zr and eCO2
had varying significance on the responses. The significance of interaction between factors
in polyphenols was higher (p < 0.001) than that of both FRAP and flavonoids (p < 0.05)
(Table 2).

Table 2. Two-way ANOVA for the significance of interaction between the two independent variables
(Zr and eCO2 ) upon oxidative damage, as well as the enzymatic and non-enzymatic antioxidants in
oat plants (ns = non-significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Dependent Type III Sum of Mean

df F Sig.
Variables Squares Square
H2 O2 5903.0 1 5903.0 12.62 *
MDA 4.114 1 4.114 48.04 ***
FRAP 18.639 1 18.639 6.267 *
Polyphenol 3.157 1 3.157 2.338 ***
Flavo 0.030 1 0.030 0.002 *
POX 0.226 1 0.226 10.63 *
CAT .001 1 0.001 4.171 ns
SOD 1428.6 1 1428.6 37.0 ***
APX 0.000 1 0.000 .000 ns
DHAR 0.217 1 0.217 14.90 **
MDHAR 0.000 1 0.000 4.004 ns
GR 0.006 1 0.006 1.939 ns
GPX 0.044 1 0.044 5.253 *

3.5. eCO2 Affecting the Enzymatic Antioxidants of Zr-Contaminated Oat Plants

Antioxidant enzymes with those of the ASC/GSH cycle play a pivotal role in ROS
homeostasis. In our study, we were concerned with investigating the activities of peroxidase
(POX), superoxide dismutase (SOD), GSH-peroxidase (GPX) and catalase (CAT), in addition
to the enzymes involved in ASC/GSH metabolism in oat plants treated with Zr and eCO2 ,
either alone or in combination (Figure 5). Although the treatment of oat plants with Zr
alone had no significant effect upon DHAR, and MDHAR, it caused a slight increase
in the activities of some other enzymes. For example, SOD activity increased by 39%
in response to the treatment of oats with Zr (Figure 5B). Moreover, the application of
Zr upon oat plants increased the activity of both CAT and GPX up to 45% and 54%,
respectively, when compared with control (aCO2 ) uncontaminated plants (Figure 5D). It is
likely that the activity of glutathione reductase (GR) in oat plants exhibited a 33% increase
due to the contamination with Zr if compared with plants grown under aCO2 conditions
(Figure 5H). To a greater extent, POX activity showed an about two-fold increment in
response to Zr treatment relative to control plants grown under aCO2 conditions (Figure 5A).
Compared to aCO2 , eCO2 had no significant impact upon the activities of the enzymatic
antioxidants, except for CAT, whose activity increased by 46% relative to plants grown
under aCO2 conditions (Figure 5E). Contrarily, eCO2 highly enhanced the enzymatic
antioxidants in oat plants grown in Zr pollution conditions. In this regard, the activities
of SOD and DHAR were augmented (30% and 50% increase, respectively) in oat plants in
response to the co-application of both eCO2 and Zr if compared with aCO2 -uncontaminated
control plants (Figure 5B,C). A further increase was observed in the activities of both GPX
and CAT (91% increase for each) due to the treatment of oat with both eCO2 and Zr
(Figure 5D,E). Additionally, the treatment of oat plants with both eCO2 and Zr caused
a two-fold enhancement in the activities of both APX and GR in comparison with aCO2
uncontaminated control plants (Figure 5F,H). More interestingly, POX activity was increased
Plants 2023, 12, x FOR PEER REVIEW 12 of 23
Plants 2023, 12, 3792 11 of 21

grown under aCO2. According to the two-way ANOVA, the interaction between Zr and
eCOby about four-fold in oat plants treated with eCO2 and
2 had varying significance on the responses. The
Zr relativeoftointeraction
significance aCO2 -uncontaminated
between
control plants (Figure 5A). A two-way ANOVA declared that a differential
factors in polyphenols was higher (p < 0.001) than that of both FRAP and flavonoids interactive
(p effect
<
existed between
0.05) (Table 2). the two independent variables (Zr and eCO 2 ) upon SOD (p < 0.001), DHAR
(p < 0.01) and GPX (p < 0.05) (Table 2).

Figure
Figure The combined
4. combined
4. The effecteffect
of eCO of2 eCO 2 and/or
and/or Zr upon Zrthe
upon theoflevels
levels of total antioxidant
total antioxidant capacity capacity
and
molecular antioxidants of oat plants. (A) total antioxidant capacity (FRAP), (B) polyphenols,
and molecular antioxidants of oat plants. (A) total antioxidant capacity (FRAP), (C)
(B) polyphenols,
flavonoids. Four biological
(C) flavonoids. replicates
Four biological were used
replicates were to investigate the response.
used to investigate The vertical
the response. Theerror barserror
vertical
are the
barsstandard error (SE).
are the standard Fisher’s
error LSD testLSD
(SE). Fisher’s (p < test
0.05,(pn<=0.05,
4) was
n =used forused
4) was pairwise comparison
for pairwise compar-
between groups. The
ison between different
groups. le ers indicate
The different letters significant differences
indicate significant between the
differences meansthe
between of means
each of
group.
each group.
Plants 2023, 12,2023,
Plants x FOR PEER REVIEW
12, 3792 1421of 23
12 of

Figure 5. The combined effect of eCO2 and/or Zr upon the activities of antioxidant oat plants. (A) per-
Figure 5. The combined effect of eCO2 and/or Zr upon the activities of antioxidant oat plants. (A)
oxidase, (B) superoxide dismutase, (C) dehydroascorbate reductase, (D) glutathione peroxidase,
peroxidase, (B) superoxide dismutase, (C) dehydroascorbate reductase, (D) glutathione peroxidase,
(E) catalase, (F) ascorbate peroxidase, (G) monodehydroascorbate reductase, and (H) glutathione
(E) catalase, (F) ascorbate peroxidase, (G) monodehydroascorbate reductase, and (H) glutathione
reductase.
reductase. Four
Four biologicalreplicates
biological replicates were
wereused
usedtoto
investigate the response.
investigate The vertical
the response. error bars
The vertical are bars
error
the standard error (SE). Fisher’s LSD test (p < 0.05, n = 4) was used for pairwise comparison
are the standard error (SE). Fisher’s LSD test (p < 0.05, n = 4) was used for pairwise comparison between
groups.
between The different
groups. letters indicate
The different le erssignificant differences between
indicate significant the means
differences of each
between thegroup.
means of each
group.

3.6. Effect of Zr and/or eCO2 on the AsA/GSH Metabolic Pool of Oat Plants

The ascorbate/glutathione metabolic pathway is involved in different plant metabolic

Plants 2023, 12, 3792 13 of 21

3.6. Effect of Zr and/or eCO2 on the AsA/GSH Metabolic Pool of Oat Plants
The ascorbate/glutathione metabolic pathway is involved in different plant metabolic
reactions, including those elicited by environmental cues. Therefore, measuring its metabolic
components is of great importance for the effectiveness of eCO2 in mitigating the phytotoxic
effect of Zr. Although Zr had no significant effect on the levels of ascorbate (Figure 6A), it
greatly reduced the glutathione levels in oat plants (Figure 6C). In addition, Zr caused a
three-fold reduction in the GSH/GSSG ratio in polluted oat relative to aCO2 -unpolluted
control plants (Figure 6H). Contrarily, oat plants grown in soils polluted with Zr exhibited
a noticeable increment in the levels of tASC and tGSH (Figure 6B,D). A further elevation
was observed in the levels of DHA and GSSG, which increased by 65% and 59% higher
than aCO2 -uncontaminated control plants (Figure 6E,G). Moreover, GSSG experienced
a more than two-fold increase in oats due to contamination with Zr that could explain
the remarkable reduction in GSH/GSSG ratio (Figure 6G,H). Except for the GSSG and
ASC/DHA ratio, the individual application of eCO2 significantly enhanced the components
of ascorbate/glutathione metabolic pathway in oat plants relative to aCO2 control plants
(Figure 6). For instance, tGSH showed a 25% increase in oats treated with eCO2 alone
when compared with aCO2 control plants (Figure 6C). More interestingly, eCO2 further
enhanced the levels of ascorbate/glutathione metabolites in oats when grown under Zr-
contamination conditions. In this context, the treatment of oat plants with both eCO2 and
Zr enhanced the levels of tGSH by about 28% higher than those treated with Zr alone
(Figure 6D). Moreover, eCO2 improved GSH and DHA by about 66% and 65% in oat plants
grown in Zr compared with those grown in Zr alone (Figure 6C,E). A further increase was
observed in GSSG, as well as in the ratio of both ASC/DHA and GSH/GSSG (two-fold and
three-fold increase, respectively) in response to the treatment of oat plants with both eCO2
and Zr, as compared with those treated with Zr alone (Figure 6F–H). Two-way ANOVA
indicates presence of interactive effect of both Zr contamination and eCO2 treatment that
was apparent on the activities of DHA, GSH, TGSH (p < 0.05, 0.01, 0.001), in addition to the
levels of GSH and GSSG (p < 0.01, 0.001) (Table 3).

Table 3. Two-way ANOVA for the significance of interaction between the two independent vari-
ables (Zr and eCO2 ) upon ascorbate/glutathione metabolic pool in oat plants (ns = non-significant;
* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Dependent Type III Sum of Mean

df F Sig.
Variables Squares Square
ASC 0.000 1 0.000 0.159 ns
TASC 0.000 1 0.000 0.551 ns
DHA 0.000 1 0.000 9.152 *
ASC_DHA 0.163 1 0.163 36.236 ***
GSH 0.000 1 0.000 9.543 **
TGSH 0.027 1 0.027 81.929 ***
GSSG 0.020 1 0.020 131.102 ***
GSH_GSSG 0.038 1 0.038 42.503 ***
Plants 2023, 12, 3792 14 of 21
Plants 2023, 12, x FOR PEER REVIEW 16 of 23

Figure 6. The combined effect of eCO2 and/or Zr upon the ascorbate/glutathione metabolic
Figure
pool 6. The
of oat combined
plants. effect ofacid,
(A) ascorbic eCO(B)
2 and/or
total Zr upon the(C)
ascorbate, ascorbate/glutathione
glutathione, (D) total metabolic pool of
glutathione,
oatdehydroascorbate,
(E) plants. (A) ascorbic acid, (B) total ascorbate, (C)ratio,
(F) ascorbate/dehydroascorbate glutathione, (D) total
(G) glutathione glutathione,
disulfide (E)glu-
and (H) dehy-
droascorbate, (F) ascorbate/dehydroascorbate
tathione/glutathione ratio,
disulfide ratio. Four biological (G) glutathione
replicates were used todisulfide
investigate and
the(H) glutathi-
response.
one/glutathione
The vertical error disulfide ratio.
bars are the Four biological
standard replicates
error (SE). Fisher’swere
LSD used
test (pto<investigate
0.05, n = 4)the
was response.
used forThe
vertical error
pairwise bars arebetween
comparison the standard error
groups. The(SE). Fisher’s
different LSD
letters test (p <significant
indicate 0.05, n = 4)differences
was used for pairwise
between
comparison
the between
means of each groups. The different le ers indicate significant differences between the means
group.
of each group.

4. Discussion
Upon being incorporated into the food chain, Zr becomes a threat to human health
as it can be easily taken in by the human body through food or water. Concerning plants,
Plants 2023, 12, 3792 15 of 21

4. Discussion
Upon being incorporated into the food chain, Zr becomes a threat to human health as
it can be easily taken in by the human body through food or water. Concerning plants, Zr
was found to inhibit the germination of seeds and the growth of plant shoots and roots [35].
Nevertheless, there has been a lack of information about its impact upon plant physiology
and metabolism. Therefore, our study aims to investigate the phytotoxic impact of Zr upon
the growth, photosynthesis and ROS homeostasis of oat plants (Avena sativa) and how
elevated CO2 could ameliorate this phytotoxic effect.

4.1. eCO2 Ameliorated the Growth Reduction and Oxidative Burst in Oat That Was Initiated by Zr
Our results showed a remarkable reduction in the growth as well as photosynthesis of
oat plants in response to contamination with Zr. These findings highlight the phytotoxic
impact of Zr upon plant growth and development. Comparable findings were noted in
the case of algae by Simon et al. [8], who reported an adverse impact of Zr on the growth
and photosynthesis of Chlorella pyrenoidosa. Moreover, the dry matter of maize shoots
and roots were dramatically decreased by increasing the concentration of Zr [35]. It is
likely that treatment with Zr not only inhibits the germination of Triticum aestivum but also
adversely inhibits the growth of their shoots and roots [9]. Generally, soil contamination
with toxic metals adversely delays the growth and development of plants. In this context,
the growth of Oryza sativa was highly delayed when grown in soils contaminated with
Indium [36]. A similar observation was opined by Kopittke et al. [37], who found that
contamination with trace metals such as Ga, In, Hg and Ru significantly reduced the growth
of cowpea roots and caused cell rupture. This phytotoxic effect could be explained by
the high binding tendency of these elements to the cell wall, increasing the cell rigidity,
delaying the cell growth and finally causing the rupturing of cells [33]. It is important to
recognize that the biogeochemical behavior of toxic elements like Zr in the environment and
their phytotoxic impact on plants are primarily contingent on their speciation. Speciation
refers to the existence of metals in diverse chemical forms, which is greatly influenced
by environmental factors such as soil pH and interactions with soil organic matter [6].
Regarding Zr, it exists in the soil in different chemical forms that have a wide variety of
solubility and bioavailability [38]. This speciation could explain the adverse effect of Zr
upon plant growth and biomass.
On the other hand, our study revealed that the phytotoxic effect of Zr was obviously
mitigated by the application of eCO2 . This ameliorative effect was embodied in the recovery
of the plant biomass and photosynthetic efficiency relative to contaminated plants. In line
with our findings, the growth of both maize and barley contaminated with As2 O3 and HgO,
respectively, exhibited a noticeable recovery in both growth and photosynthetic efficiency
in response to treatment with eCO2 [39]. This advantageous effect might be ascribed to the
ability of eCO2 , within the physiological range, to enhance plant biomass by stimulating
its photosynthetic machinery [33]. This, in turn, will modulate the photosynthetic carbon
metabolism and so improve carbohydrate partitioning [10]. Furthermore, the enhancement
in photosynthesis due to eCO2 can also be attributed to the elevated levels of CO2 at the
RuBisco site, which reduces the availability of NADPH and ATP for photorespiration, thus
facilitating CO2 assimilation in plants [40]. Kaiser et al. [41] reported that eCO2 increased
the relative carbon gain with concomitant enhancements in the photosynthesis of tomato
by initiating carboxylation reaction and slowing oxygenation reaction at RuBisco. Likewise,
the photosynthesis in lettuce was improved in response to eCO2 , the thing that reflected
on its growth due to the availability of the carbon skeleton, resulting in higher levels of
carbohydrates [42]. In conclusion, it is hypothesized that eCO2 could provide protection to
significant crops against various environmental threats, including metal pollution like Zr,
by enhancing their growth and photosynthesis.
Plants 2023, 12, 3792 16 of 21

4.2. eCO2 Highly Quenched the Oxidative Damage Caused by Zr in Oat Plants
Improvement in plant growth and photosynthesis is associated with modulating the
plant redox homeostasis, including the regulation of reactive oxygen species under various
environmental cues [33]. Our results declared that Zr triggered oxidative stress in oat plants
by increasing the levels of H2 O2 and MDA. In this regard, the treatment of wheat seedlings
with Zr caused an oxidative damage by altering the activities of the antioxidant enzymes [9].
Similarly, Urtica dioica grown in heavy-metal-contaminated soils experienced remarkable
oxidative stress with concomitant DNA damage [43]. Likewise, soil contamination with Zn
and Pd provoked noticeable oxidative damage in sorghum plants [44]. Oxidative stress was
triggered in both wheat and soybean plants treated with As [45]. Moreover, cereal crops
treated with tungsten nanoparticles exhibited a remarkable accumulation in both H2 O2 and
MDA, the main cause of oxidative damage in plants [46]. Plants respond to metal stress by
triggering diverse signaling pathways, including those related to ROS metabolism, as tools
to cope with the different environmental challenges [47]. The adverse effects of toxic metals,
one of the environmental challenges, on the growth and oxidative status of plants could be
due to direct or indirect reasons. The direct toxic impact lies in the ability of heavy metals
to inhibit the cytoplasmic enzymes and the destruction of cell structures in response to
oxidative damage [48]. Conversely, toxic metals can indirectly influence plant growth and
oxidative balance by inhibiting the functions of soil microorganisms. This, in turn, impacts
the decomposition of organic matter, leading to adverse effects on soil nutrient levels [49].
Furthermore, essential metabolic enzymes may be hampered due to the interference of the
toxic metals with the soil microorganisms [50]. Toxic metals may also accumulate H2 O2 by
impairing photorespiration, one of most important H2 O2 -generating pools in plants [51].
Additionally, toxic metals caused ABA accumulation, leading to the hyperaccumulation
of H2 O2 and the slowing of cell division [52]. These detrimental effects, when combined,
can impede plant growth and initiate plant oxidative damage. On the other hand, the
co-application of eCO2 with Zr obviously reduced the oxidative damage caused by Zr
alone by reducing the levels of H2 O2 and MDA in oat plants. Similar results were obtained
by AbdElgawad et al. [53], who proclaimed that the growth of both wheat and soybean
in an atmosphere enriched with CO2 reduced the oxidative damage caused by As stress.
The authors also reported that eCO2 enhanced the antioxidative defense system in cereals
grown in As-contaminated soils [45]. Moreover, eCO2 obviously quenched the oxidative
damage triggered in both wheat and maize treated with both NiO and HgO by diminishing
the accumulation of H2 O2 and MDA [13,54]. This mitigative impact of eCO2 could be
attributed to its potency to reduce the chance of oxygenation reaction on RuBisco [55].
This will increase the rate of carboxylation reaction that, in turn, will increase the carbon
assimilation [11]. Moreover, high levels of CO2 can reduce the activity of the enzymes of
photorespiration, leading to a reduction in the accumulated H2 O2 , accordingly [56].

4.3. eCO2 Raised Oat Tolerance to Zr Contamination by Regulating the Antioxidant

Defense System
In plants, the antioxidant defense system includes low molecular weight non-enzymatic
antioxidants (ascorbate, glutathione, polyphenols, flavonoids, etc.) and antioxidant en-
zymes that function in a coordinated manner to restrain the over-production of ROS in
plants [57]. Superoxide dismutase (SOD) is the key enzyme in the conversion of superoxide
ions into H2 O2 , which is further turned into H2 O by CAT, GPX, or APX [34]. Therefore,
the elevation in the activities of such enzymes in response to Zr treatments is an attempt
by the plant to rein in the overproduction of H2 O2 , one of the main causes of oxidative
damage. We also opined an elevation in the ascorbate/glutathione pathway that functions
mainly in scavenging H2 O2 in oat plants under contamination conditions [58]. Pollution
with Zr, like other environmental stresses, causes oxidative damage in oat plants. Likewise,
we previously found that tungsten nanoparticles caused an increment in the components
of both enzymatic and non-enzymatic antioxidants [46]. Moreover, the growth of Camel-
lia sinensis in media containing Cd enhanced the accumulation of phenolics including
Plants 2023, 12, 3792 17 of 21

flavans [59]. Similarly, oilseed rape treated with Cd showed a noticeable increase in the
levels of non-enzymatic antioxidants [60]. Also, the application of Zn caused a noticeable
increase in lipid peroxidation in Brassica juncea due to the hyperaccumulation of ROS [61].
Analogous findings were documented in the moss Taxithelium nepalense, wherein exposure
to Cr and Pd led to the generation of reactive oxygen species (ROS), accompanied by a
simultaneous reduction in the antioxidant defense system [62]. In rice, the application
of Cu caused an over-accumulation of ROS and an observable decline in the antioxidant
defense arsenal [63]. In this context, ascorbic acid was induced in wheat shoots subjected
to nickel stress, indicating its pivotal role in ROS manipulation [64]. Moreover, the redox
statuses of ASC (ASA/DHA) and GSH (GSH/GSSG) in oat plants were reduced in response
to Zr contamination, the thing that delayed the antioxidant defense system in oat plants.
Overall, plants activate the production of secondary metabolites such as phenylpropanoids
in response to different environmental cues [65]. This process provides the cell with a
wide array of phenolic metabolites by activating the phenylpropanoid pathway [66]. By
activating the phenylpropanoid pathway, non-enzymatic antioxidants were synthesized to
enhance plant tolerance against environmental stresses such as toxic metals [67].
On the other hand, eCO2 not only augmented the detoxification of ROS, but also kept
the balance of both GSH/GSSG and ASC/DHA in Zr-polluted oat plants. In line with our
findings, AbdElgawad et al. [68] declared that eCO2 caused a remarkable improvement
in the growth of cereals grown in soils contaminated with As via augmenting the ascor-
bate/glutathione metabolism. It is worth noting that the ASC/DHA metabolic pool keeps
the redox homeostasis in the photosystem by maintaining high ratios of GSH/GSSG and
ASC/DHA, which is important for plant tolerance against different environmental chal-
lenges [69]. Therefore, the concurrent suppression of photorespiration and improvement
of photosynthesis highlights the crucial contribution of eCO2 in mitigating the phytotoxic
effect of Zr-contaminated oats. In other words, the mitigative role of eCO2 could be mainly
due to its ability to increase carboxylation at the expense of the oxygenation reaction in
RuBisco [10]. More interestingly, eCO2 can provide the cells with the C skeleton and energy
needed for the growth of stressed plants [70]. This increment in carbon availability will
supply the cells with antioxidant molecules, so raising the plant tolerance against the stress-
induced oxidative damage [40]. In our study, the observed elevation in total antioxidant
capacity (FRAP) due to treatment with eCO2 indicates an awakening within the plant to
tolerate the ROS generated by Zr. In accordance, we opined a noticeable increment in the
levels of polyphenols and flavonoids in response to eCO2 in plants contaminated with
Zr (Figure 4). Indeed, this increment could quench the toxicity induced by Zr pollution
as an adaptive response to environmental stresses [71]. Another perspective is that the
sugars accumulated in response to enhanced photosynthesis can scavenge free radicals
generated by ROS leading to alleviation of the oxidative damage caused by environmental
stresses [72]. To this end, our study, for the first time, shed light on the positive role of
increasing atmospheric CO2 in modulating the ROS homeostasis in oat plants to cope with
pollution with Zr.

5. Conclusions
This investigation improves our knowledge about how eCO2 differentially alleviates
the phytotoxic effect of Zr on the growth, photosynthesis, and redox status of Avena
sativa plants. Contamination with Zr greatly reduced the photosynthesis of oat plants
with concomitant reduction in their biomass. Moreover, contamination with Zr triggered
remarkable oxidative stress in oats. On the other hand, eCO2 significantly recovered
the adverse effect of Zr on the biomass and redox status of oats. Furthermore, eCO2
modulated the redox homeostasis of oat plants by enhancing their photosynthetic efficiency.
Accordingly, enhanced photosynthesis can improve the plant biomass under both clean and
contaminated conditions. This beneficial effect of eCO2 extends beyond photosynthesis and
biomass; it also mitigates the severe oxidative damage in oats by decreasing the levels of
H2 O2 and MDA induced by Zr pollution. Moreover, eCO2 ameliorated the negative impact
Plants 2023, 12, 3792 18 of 21

of Zr upon the non-enzymatic antioxidants by improving the total antioxidant capacity

(FRAP) and then the levels of the non-enzymatic antioxidants (polyphenols and flavonoids).
Moreover, eCO2 caused a noticeable activation in all measured antioxidant enzymes that
were inhibited in oats contaminated with Zr. In conclusion, our study, for the first time,
introduces a new insight on the ability of eCO2 in harnessing the redox homeostasis of oat
plants to cope with the phytotoxic effect imposed by toxic metals, particularly Zr.

Author Contributions: M.M.Y.M. and H.A.: conceptualization, methodology, resources, data cu-
ration, and writing—original draft preparation. M.M.Y.M., H.A. and A.M.S.: software and data
validation. A.M.S. and H.A.: formal analysis. A.M.S., H.A., M.M.Y.M. and A.M.A.K.: investigation.
M.M.Y.M. and A.M.S.: writing—review and editing. A.M.A.K. and D.A.G.: visualization. A.M.S.,
M.M.Y.M. and H.A.: project administration. A.M.S. and M.M.Y.M.: funding acquisition. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Deputyship for Research & Innovation, Ministry of
Education in Saudi Arabia [grant number 445-9-379].
Data Availability Statement: Data presented in this study is included in this article.
Acknowledgments: The authors extend their appreciation to the Deputyship for Research and
Innovation, Ministry of Education, in Saudi Arabia for funding this research work through the project
number 445-9-379.
Conflicts of Interest: The authors declare no conflict of interest.

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