Citric Acid As Soil Amendment in Cadmium Removal by Salix Viminalis L., Alterations On Biometric Attributes and Photosynthesis
Citric Acid As Soil Amendment in Cadmium Removal by Salix Viminalis L., Alterations On Biometric Attributes and Photosynthesis
Citric Acid As Soil Amendment in Cadmium Removal by Salix Viminalis L., Alterations On Biometric Attributes and Photosynthesis
To cite this article: Danijela Arsenov, Milan Župunski, Milan Borišev, Nataša Nikolić, Andrej
Pilipovic, Saša Orlovic, Marko Kebert & Slobodanka Pajevic (2019): Citric acid as soil amendment
in cadmium removal by Salix�viminalis L., alterations on biometric attributes and photosynthesis,
International Journal of Phytoremediation, DOI: 10.1080/15226514.2019.1633999
Article views: 19
a
Faculty of Sciences, Department of Biology and Ecology, University of Novi Sad, Novi Sad, Serbia; bInstitute of Lowland Forestry and
Environment, University of Novi Sad, Novi Sad, Serbia
ABSTRACT KEYWORDS
During the past decade, the target in cleaning polluted sites is an application of chelating agents, Cd accumulation; chelating
such as citric acid (CA), which is proposed as a good candidate in the promotion of phytoremedia- agent; photosynthetic
tion. Among heavy metals, cadmium (Cd) is one of the most common and dangerous elements, performances; phytoreme-
diation; willow
which strongly disturbs morphophysiological properties in plants. A pot experiment was assessed
to evaluate the influence of CA in Cd phytoremediation in alkaline soil by Salix viminalis (clone
SV068). The effects of CA on Cd bioavailability, mobility, and distribution in plants, various mor-
phometric measurements, and physiological performances as photosynthesis, transpiration, water
use efficiency, and pigment content were tested. The highest Cd accumulation was evident after
60 days of growing, in plants subjected to combined treatment of CA with a higher dose of Cd.
Application of CA showed a beneficial effect in maintaining the photosynthetic rate as well as gas
exchange capacity in willows grown in Cd-contaminated soil. Furthermore, CA slightly increased
plant growth and biomass production, depending on applied Cd dose and harvest period. A che-
lating agent like CA showed strong influence in plant response to combat Cd toxicity.
CONTACT Danijela Arsenov danijela.arsenov@dbe.uns.ac.rs Faculty of Sciences, Department of Biology and Ecology, University of Novi Sad, Novi
Sad, Serbia.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bijp
Supplemental data for this article can be accessed here
ß 2019 Taylor & Francis Group, LLC
2 D. ARSENOV ET AL.
approach is strongly criticized, because excessive and uncon- the data analyzing efficiency of CA application in phytore-
trolled use of synthetic chelates may have a toxic effect on mediation trials using woody species, including willows, are
plants and can adversely affect the number and variety of still lacking.
rhizosphere microorganisms, possessing a threat to the over- Taking into account everything aforementioned, the main
all environment (Zhao et al. 2011). With respect to this, low goal of this research was to test CA efficiency as a soil
molecular weight organic acids (LMWOAs), such as citric amendment in Cd removal from contaminated soil by Salix
acid (CA), have been proposed as an alternative to synthetic viminalis. In context to that, CA was added in single, dou-
chelators (Nascimento and Xing 2006; Muhammad et al. ble, and multiple doses and removal of Cd from mild to
2009). Several studies support the fact that organic acids moderate contaminated soil was analyzed depending on
have a role as a chelating agent and can modify heavy metals experimental duration. Photosynthetic activity, pigment con-
mobilization without a negative impact on the environment, tent, and proline (Pro) accumulation were also addressed.
thus LMWOAs have a greater social acceptance (Freitas et al.
2013; Song et al. 2018). Enhanced metal uptake, accumula-
tion, and translocation in various plants due to the applica- Material and methods
tion of CA are well documented (Gao et al. 2010; Ehsan Experimental set-up
et al. 2014; Shakoor et al. 2014). On contrary to above men-
tioned, several authors emphasized the weak effectiveness of The 1-year-old cuttings of S. viminalis L. (clone SV068) were
LMWOAs in the phytoextraction process, which is provoked selected from the Institute of Lowland forestry and
by their rapid biodegradation and mineralization by soil Environment, Serbia. The pot culture experiment was set-up
microorganisms (Lesage et al. 2005; Meers et al. 2008). This in the glasshouse. Willow cuttings and soil were obtained
inconstancy might be due to different influences of CA from a woody plantation near Novi Sad (45.3025 N,
depending on plant species, type, and concentration of heavy 19.9385 E). Plants were grown in natural photoperiod, the
metals, soil properties, and therefore, further studies are temperature was in range 20–30 C night/day, while plants
mandatory aiming to define the efficiency of CA as a soil were watered periodically (drinking water, pH 7.82, CaCO3
amendment in assisted phytoremediation. 75–150 mg L1, EC 425 mS cm1) to maintain optimal soil
Numerous studies highlight that fast-growing trees pos- moisture content of 60% (400 mL of water per pot per week).
sess desirable characteristics for phytoremediation based on Stem cuttings were rooted in Mitscherlich pots
several points, that is, high biomass production, rapid (“Mitscherlich containers” for greenhouse experiments, size
growth, deep root system, energy production, and so on 15 cm diameter by 19 cm tall and having a double bottom,
(Lux et al. 2004; Zacchini et al. 2009; Nikolic et al. 2017). A into which excess fertilizer flow) and soil used for experi-
number of studies confirmed the high efficacy of willows ment was mixed with peat, thus each pot contains 3 kg of
(Salix spp.) in phytoremediation, especially toward Cd-con- total substrate, 2.5 kg of soil, and 0.5 kg of peat, which was
taminated soil and water (Yang et al. 2015; Mleczek et al. homogenized before treatment exposure. The physicochemi-
2018). Furthermore, Cd effect on the growth rate, biomass cal properties of soil are given in Table 1. Cd was applied as
production, and photosynthetic attributes in Willows has Cd(NO3)2 4H2O and sprayed onto the soil, the substrate
been a subject of numerous studies (Pietrini et al. 2009; was mixed thoroughly, and left 10 days for stabilization
Zacchini et al. 2009). Willows are widely distributed, can be before plantation. Cd was applied in two concentrations:
easily propagated and hybridized, which allows clones selec- 3 mg kg1 (Cd3) and 6 mg kg1 dry soil (Cd6). The Cd che-
tion and targeted breeding for various environmental condi- lation was provided by the addition of CA in water solution
tions that attend to enhance phytoextraction capacity and it was applied on soil surface a week before plants har-
(Zalesny and Bauer 2007). Besides previously mentioned, vest. CA was added in concentrations of 20 mmol kg1 of
willows are often used in short rotation coppices, as a good soil and its application was performed in three repetitions
source of renewable bioenergy due to the large biomass yield after 30 days, following the same after 60 and 90 days of Cd
and high planting density, thus it can be established on mar- exposure. In total, six treatments included as follows: control
ginal land (Casta~no-Dıaz et al. 2018). Furthermore, willows (soil without Cd, neither CA) and two doses of Cd (Cd3
are favorable candidates for decontamination of polluted and Cd6), followed by same with CA addition (CA without
sites due to their facilities to grow fast, produce high bio- Cd) and combined treatments with Cd (CA þ Cd3 and
mass yield, and possess the ability to adapt in a new envir- CA þ Cd6). Each treatment was set-up in three replicates,
onment as a pioneer species (Mleczek et al. 2018). However, with six plants per pot. Plants were harvested three times,
1 week after the single, double, and multiple additions of On the basis of Cd content in plant tissue and soil, the
CA; in total, 54 pots were analyzed. bioaccumulation factor (BAF) of the root (BAF root) and
the aerial plant parts (BAF aerial parts) was calculated as
mean of Cd concentration of both YL and OL, and stem
Morphometric measurements and evaluations of
and translocation factor (TF) was calculated according to
biometric parameters
Zacchini et al. (2009).
Before analyses, plant material was washed in deionized
water, dried, and biomass of different organs (root, stem,
Proline content
YL, and old leaves (OL)) was measured to calculate different
parameters. Separations of leaves to young and old were The content of Pro amino acid was measured in YL and OL
obtained according to our previous research Arsenov et al. by the modified method of Bates et al. (1973). Fresh leaves
(2017). Young leaves (YL) contained a group of four fully were homogenized in 3% (w/v) sulfosalicylic acid and centri-
developed youngest leaves and the other leaves are defined fuged at 3500 rpm for 10 min. The supernatant was mixed
as OL. The organ mass ratio (MR) was calculated according with acid ninhydrin reagent and glacial acetic and incubated
to Iori et al. (2017), as a ratio of leaves (LMR), stem (SMR), for 15 min at 100 C. The reaction was stopped by cooling,
and roots (RMR) to total plant biomass, while roots to followed with the addition of toluene, and absorbance was
shoots ratio (RSR) was calculated by dividing roots to measured at k ¼ 520 nm. Pro concentration was expressed in
aboveground biomass (leaves þ stem). milligram per gram of fresh weight.
Leaf area of both YL and OL was measured by leaf area
meter “ADC Bioscientific Ltd. AM350”. The specific leaf
area (SLA) was calculated according to Dijkstra and Statistical analysis
Lambers (1989) as a ratio of leaf area to its biomass. The obtained data were expressed as a mean of three inde-
The tolerance index (TI) was calculated according to pendent measurements (n ¼ 3) ± standard deviation (SD).
Yang et al. (2015) as a ratio of plant biomass of Cd-treated Mean values between treatments and experimental duration
plants to biomass of controlled plants. were analyzed using two-way analysis of variance at a sig-
nificance level of p .05. Multiple comparisons between
Photosynthetic pigment content and gas observed parameters were performed using the Fisher LSD
exchange parameters post hoc test. Different letters indicate significant differences
at p .05.
The chlorophyll and carotenoids concentrations were A comparison between SLA, biomass production of YL,
assayed according to modified method by Von Wettstein OL, total biomass, and photosynthetic (PN), and transpir-
(1957) following extraction in absolute acetone and meas- ation (E) rate was conducted by the Pearson correlation
ured by UV-VIS spectrophotometer (Beckman DU-65, CA). coefficient for p .05. Principal component analysis (PCA)
Total chlorophylls were calculated as a sum of chlorophyll was performed to analyze the influence of Cd concentration
a þ b. Concentrations of photosynthetic pigments are in various plants organs to different characteristics (biomass
expressed in mg g1 fresh weight. production, chlorophylls, carotenoids, photosynthetic rate,
Gas exchange parameters such as net assimilation rate of transpiration rate, WUE, intercellular CO2 concentration,
CO2 (PN), transpiration (E), water use efficiency (WUE), stomatal conductance, and Pro content). Analyses were per-
and stomatal conductance (gs) were carried out on YL, using formed by STATISTICA 13.5 (StatSoft).
infrared gas analyzer (LCProþ, ADC Bioscientific, UK). The
analyses were performed between 10 and 12 a.m., at a tem-
perature of 25 to 30 C. The photosynthetic active radiation Results and discussion
(PAR) was set to 1000 mmol m2 s1, while the flow of ambi-
Soil properties
ent air in the chamber was 100 mmol s1. The temperature,
air humidity, and the concentration of CO2 were depending The soil was mixed with peat, aiming to reduce the pH
on their ambient values. WUE was calculated as the ratio of value of the soil, and we observed a reduction in pH value
net CO2 exchange and transpiration rate (PN/E) from 8.16 to 7.88 (in dH2O) and from 7.62 to 7.18 (in KCl).
Measurements were performed on six plants per treatment. At the same time, peat addition resulted in the reduction of
CaCO3 content and elevation of humus content and total
nitrogen (Table 1). These changes in soil properties could
Cd content and phytoremediation factors
have an impact on the production of biomass without affect-
Separated plant organs (roots, stem, YL, and OL) and soil ing Cd uptake and accumulation. According to Trevisan
samples were dried in an oven at 70 C for 48 h, followed by et al. (2010), the addition of peat which is characterized by
complete mineralization to dry-ash at 450 C with the add- a low pH value (pH value is about 3.5) can affect the bio-
ition of 30% of hydrogen peroxide and extraction with 25% availability of metal cations and contribute to their removal
HCl. Cd content was determined using an atomic absorption from the soil. Furthermore, Stanislawska-Glubiak et al.
spectrophotometer with a flame atomizer (FSAAS240, (2012) recorded increased adsorption of Cd and Pb due to
Varian, Canada). the addition of peat in sandy soils, compared with the same
4 D. ARSENOV ET AL.
treatment without peat. Cd content in the soil at the begin- as a consequence of stress-avoidance strategy. In line with
ning and at the end of experiment (90 days) was presented our findings, Iori et al. (2017) reported a reduction in RMR
in Table 1. The highest reduction in Cd content was by 65% in Eucalyptus plants exposed to 50 mmol CdSO4
observed under CA þ Cd6 treatment and was 10.2% com- grown hydroponically for 3 weeks. Morphological alterations
pared with the initial Cd concentration. induced by CA application under heavy metal stress is well
established in many studies (Li et al. 2014; Shakoor et al.
2014). According to Melo et al. (2008), both single or mul-
CA and Cd influence on morphological traits
tiple application of 5 mmol kg1 soil of CA in contaminated
The biomass production and its partitioning as a plant soil has no negative effect to shoot biomass. A similar obser-
adjustment to heavy metal stress are vital in the selection of vation was reported by Freitas et al. (2013) who noted no
favorable candidates for phytoremediation (Audet and changes in dry biomass production of maize and vetiver
Charest 2008). Biomass production (YL, OL, stem, and plants grown under Pb stress with the addition of
roots) showed variation depending on Cd dosage (3 and 40 mmol kg1 soil CA. On contrary to this, the beneficial
6 mg kg1), and CA applications (CA or CA addition) and role of CA in the alleviation of plant growth inhibition
experimental duration (Table S1). Similarly, the results of induced by heavy metal stress was evident in various plants
this study revealed variation in plant biomass partitioning (Ehsan et al. 2014; Farid et al. 2017; Gao et al. 2010).
depending on experimental duration and applied treatment Within biomass production and growth performances,
(Figure 1). The LMR was the highest after 30 days of Cd SLA is an important factor in defining plant tolerance to
exposure, RMR, and RSR after 60 days, while SMR showed heavy metal stress, since it can be correlated to photosyn-
no significant differences regardless of treatments or time thetic rate, relative growth rate, and nitrogen content in
exposure to Cd (Figure 1). Furthermore, after 30 days, RMR leaves (Sharma et al. 2007; Iori et al. 2017). Based on the
was considerably decreased by Cd3 and Cd6, that is, 43 and Pearson correlation coefficient, a significant positive correl-
32% as compared to control. The adverse effect of Cd was ation was observed between SLA and photosynthetic rate as
alleviated with CA addition, thus combined treatments did well as with biomass production (Table S2). In this study,
not cause significant changes in RMR in comparison with SLA tended to maintain on control level under both Cd
CA treatment. The exception of this trend was recorded doses applied, thus no significant changes were observed
after 60 days when combined treatments caused a significant regardless leaves maturity or experimental duration
decrease in RMR (25% and 20% by CA þ Cd3 and (Figure 2). The exception of this pattern was observed in
CA þ Cd6, respectively) compared with CA treatment. mature leaves after 90 days, when both Cd doses induced a
Nevertheless, CA addition elevated RSR in respect to Cd reduction in SLA by 34 and 36% compared with control
applied alone, with exception after 60 days (Figure 1D). (Figure 2B). Similar findings were observed by Iori et al.
Variations in plant biomass partitioning during time could (2017), who reported no significant changes in SLA between
be the result of intrinsic ontogeny, as well as Cd content in control and Cd-treated plants of Eucalyptus (velino clone)
plant tissue. According to Audet and Charest (2008), fast- grown hydroponically. Meanwhile, the CA application
growing species tend to shift biomass allocation and metal caused changes in SLA depending on Cd doses applied,
distribution depending on heavy metals enrichment in soil, leaves maturity, and experimental duration. After 30 days,
Figure 1. Leaves biomass ratio (A), shoot biomass ratio (B), roots biomass ratio (C), and roots to shoots ratio (D) of S. viminalis depending on Cd dosage, CA applica-
tions, and experimental duration. Different letters indicate significant differences at p .05.
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 5
no significant changes in SLA was found neither to Cd nor under CA þ Cd6 treatment is probably attributed to high Cd
combined treatment applied. Furthermore, after double CA accumulation and reduced net photosynthetic rate.
application (60 days), CA þ Cd6 significantly reduced SLA in
YL by 19% in comparison with Cd6 treatment (Figure 2A).
Cd accumulation and phytoextraction potential
The same trend was observed in mature leaves, and
CA þ Cd6 reduced SLA by 28% as compared to Cd6. The metal allocation among plant organs is a useful tool in
Meanwhile, multiple additions of CA (90 days) increased plant species selection for phytoremediation and can play a
SLA in YL by 19% under CA þ Cd3 and 31% under role as an indicator of detoxification mechanism under
CA þ Cd6 treatment, in comparison with respective treat- heavy metal stress (Shamshad et al. 2018). Significant differ-
ment without CA, while this trend is not observed in mature ences in Cd concentrations were evident depending on Cd
leaves. Correlation coefficient showed a negative correlation dose applied, the experimental duration and the presence of
between SLA and applied treatment (Table S2). Reduction CA (Figure 3). Cd accumulation showed a dose-dependent
in SLA might be the result of a higher accumulation of pattern with decreasing order, respectfully, YL, followed by
carbohydrate compounds (Dijkstra and Lambers 1989). roots, OL, and stem. The highest Cd content (25.23 mg g1)
According to Borowiak et al. (2015), enchanted accumula- was noticed in YL under CA þ Cd6 treatment after 60 days
tion of carbohydrates under heavy metal stress can disturb and was increased by 67% in comparison with Cd6 applied
photosynthesis and lead to changes in water balance within alone. Besides that, after 60 days, the almost linear elevation
a plant, and consequently inhibit normal growth and devel- of Cd amount in all tested plant organs with increasing Cd
opment. Therefore, the lowest SLA after 60 days of treatment in the soil was observed, whereas Cd content tends to
Figure 2. SLA of YL (A) versus OL (B) of S. viminalis depending on Cd dosage, CA applications, and experimental duration. Different letters indicate significant differ-
ences at p .05.
Figure 3. Cd content in YL (A), OL (B), stem (C), and roots (D) of S. viminalis depending on Cd dosage, CA applications, and experimental duration. Different letters
indicate significant differences at p .05.
6 D. ARSENOV ET AL.
Figure 4. BAF of the root (A), aerial plant parts (B), TF (C), and TI (D) in S. viminalis depending on Cd concentration, CA applications, and experimental duration.
Different letters indicate significant differences at p .05.
diminish after 90 days of growing (Figure 3). However, Cd aerial parts) were calculated. In general, lower BAF was
content in roots reached a maximum after 90 days under recorded in aerial plant parts (1.03–2.43) relative to roots
Cd6 and CA þ Cd6 treatments and was almost twofold (1.42–3.79) (Figure 4A and B). Besides that, Cd chelation by
higher with respect to Cd3 and CA þ Cd3. Combined treat- CA showed promoting role to BAF in both roots and aerial
ments elevated Cd concentration in roots, regardless of the parts depending on treatment and experiment exposure
time of exposure, or Cd content in the soil (Figure 3D). In (Figure 4). Chelating effect of CA and its beneficial role
contrast to these results, in our previous research Arsenov on heavy metal uptake and accumulation was reported by
et al. (2017), we observed no significant changes in Cd con- numerous research (Muhammad et al. 2009; Farid et al.
tent in roots of S. viminalis exposed to combined treatment 2017; Song et al. 2018). Melo et al. (2008) recorded a
of 6 mg kg1 Cd with a single dose of 20 mmol kg1 CA lower metal accumulation by a single CA application
compared with the same treatment without CA. In the line compared with its successive application. The authors
with these results, Song et al. (2018) reported that the appli- found that Cd accumulation in velvet bean increased by
cation of organic acids in the soil can promote Pb uptake by 32% in the case of threefold CA addition in relation to
Larix olgensis, but their effectiveness depends on the acid individual treatment.
type, concentration, and duration of metal exposure. Various literature data revealed that Cd distribution
Similarly, Melo et al. (2008) observed that multiple applica- within a plant relies on selected clone properties, thus con-
tion of CA showed more prominent effects in phytoextrac- trasting findings among willow clones ability to accumulate
tion because it can alleviate the negative effect of its and translocate Cd from roots to shoots and leaves occurred
biodegradation. Differences in Cd accumulation depending (Lux et al. 2004; Yang et al. 2015). TF showed value higher
on the time of experimental duration can be explained by than 1, indicating that the selected clone represents a good
changes in Cd availability which occurs over time. Zhang candidate for phytoextraction. Meanwhile, TF in Cd-treated
et al. (2018) noted rapid decreasing in EDTA-extractable Cd plants decreased in the dose-dependent pattern (Figure 4C).
in the soil after 30–60 days, while after 90 days, its content CA addition significantly elevated Tf under CA þ Cd6 after
tends to be stable. This strongly implies the need for taking 30 and 60 days, while under CA þ Cd3 after 90 days, in com-
into consideration the aging time of Cd in soil when apply- parison with respective non-chelated Cd treatment (Figure
ing phytoremediation. Additionally, Wang et al. (2017) 4C). The effectiveness of organic acids in the phytoextrac-
observed that water-soluble Cd forms and metal-organic tion is reflected by their ability to form complexes with
acid complex are easily mobile and have a high adverse heavy metals that are easily transported to the above-ground
impact on plants. plant parts (Song et al. 2018). The beneficial role of CA to
To evaluate the plant ability to accumulate Cd in a par- translocate Cd from roots to shoots was observed in differ-
ticular tissue, the most common factors such as BAF of the ent plants, radish (Chen et al. 2003), Typha angustifolia
root (BAF root) and the above-ground plant parts (BAF (Muhammad et al. 2009), and Brassica juncea (Mahmud
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 7
Figure 5. Total chlorophylls in young (A) versus old (B) leaves and carotenoids in YL (C) versus OL (D) in S. viminalis depending on Cd dosage, CA applications, and
experimental duration. Different letters indicate significant differences at p .05.
et al. 2018). Organic acids can bind heavy metal ions and have an influence on plant tolerance to heavy metal stress
thus promote their mobilization through xylem sap for by chelating metal ions, forming organometallic complexes
long-distance transport (Wang et al. 2017). Mahmud et al. and sequestrated them in the vacuole, thereby reducing the
(2018) observed that metal soluble fractions of Cd and negative impact of these pollutants on fundamental physio-
metal-organic complex migrate rather than protein-inte- logical processes in plants (Shakoor et al. 2014; Guo
grated Cd complex, which mainly remains in cell walls. et al. 2017).
Consequently, it can be concluded that plants differently dis-
tribute Cd, depending on the chemical form of the analyzed
metal, its concentration in the plant tissue, as well as on the CA and Cd influence on pigment content and leaf gas
time of Cd exposure. exchange parameters
Metal tolerance is an essential feature in plant evaluation Cd hampers plant productivity causing a disturbance in fun-
with high phytoremediation capacity, since the accumulation damental plant processes such as photosynthesis causing
and tolerance are independent traits (Yang et al. 2015). reduction in CO2 fixation and gaseous exchange, transpir-
According to Lux et al. (2004), TI > 0.60 is defined as a
ation, as well as inhibition of chlorophyll biosynthesis
threshold value for defining willow species as a highly toler-
(Pietrini et al. 2009; Parmar et al. 2013). Chlorophyll pro-
ant to heavy metal stress. The analyzed S. viminalis clone
duction can indicate the physiological and nutritional status
showed TI > 0.6 under both Cd doses applied (Figure 2D),
of plants as well as the occurrence of heavy metal toxicity,
which strongly support its usage in decontamination of mild
therefore, it is a significant trait in estimations of plant abil-
to moderate Cd-polluted soil. Relatively high TI may be
ity to cope with heavy metal stress (Borowiak et al. 2015).
related to willows ability to adapt to different environmental
conditions such as heavy metal stress as well as changed soil In this research, lower Cd dose applied (Cd3) mainly did
properties due to the addition of chelating agents. However, not cause significant changes in photosynthetic pigments
the lowest TI (0.63) was detected after 60 days under regardless to control, neither in YL nor OL, whereas Cd6
CA þ Cd6 treatment and was 17% reduced in comparison significantly decreased pigments content (Figure 5).
with the same treatment without CA (TI ¼ 0.76). Lowered Furthermore, Cd6 treatment-induced reduction in chloro-
tolerance is directly linked to high Cd concentrations phylls in YL by 17%, 14%, and 48% after 30, 60, and
observed in plants grown 60 days under combined treatment 90 days, while in OL considerable reduction in total chloro-
CA þ Cd6, indicating that high Cd content in plant tissue phyll content was evident only after 90 days, 21% in com-
consequently can reduce plant tolerance to Cd stress. On parison with control (Figure 5A and B). Reduced
the other hand, no visual symptoms, such as chlorosis and chlorophyll production and its enhanced breakdown are one
necrosis, were found due to the application of CA, even if of the prime consequences of Cd stress (Kaur et al. 2017).
the Cd content in plant tissue was higher. These results In line with our results, Borisev et al. (2016) reported a sig-
could be attributed to CA involvement in Cd detoxification nificant reduction in photosynthetic pigments in YL of S.
mechanism and metal compartmentalization in subcellular viminalis exposed to Cd, while their content in mature
structures. Several studies have shown that organic acids leaves tends to maintain on control level. It was previously
8 D. ARSENOV ET AL.
Figure 6. Photosynthetic rate (A), transpiration rate (B), WUE (C), and stomatal conductance (D) in S. viminalis depending on Cd dosage, CA applications, and
experimental duration. Different letters indicate significant differences at p .05.
reported that Cd2þ can substitute Mg2þ ions, disturbing application (90 days), while after 60 days of growing it was
chlorophyll structure and its functionality, resulting in recorded reduction in PN values by 14%, in comparison with
graduate inhibition of photosynthesis (Parmar et al. 2013). Cd6. Similarly, CA increased transpiration rate (E) after 30
The time-responses of Cd and CA influence on pigment and 90 days of growing on both Cd doses applied, while
content showed that total chlorophylls and carotenoids after 60 days, combined treatment provoked decreases in
reached a maximum after 90 days of growing under CA transpiration rate by 27 and 42% under CA þ Cd3 and
treatment (Figure 5). Moreover, after 90 days combined CA þ Cd6 treatment compared with respective treatment
treatments induced significant elevation of total chlorophylls, without CA addition (Figure 6B). Furthermore, WUE
regardless leaves maturity, compared with Cd applied alone. showed variation depending on the time of experiment
Exogenous application of CA under heavy metal stress exposure, Cd doses applied, as well as on the presence of
increased chlorophyll content in diverse plant species (Ehsan CA (Figure 6C). The highest elevation of WUE (80%) was
et al. 2014; Kaur et al. 2017; Song et al. 2018). Possibly, CA recorded after 60 days under CA þ Cd3 compared with Cd3
can preserve the chloroplast ultra-structure and thylakoid treatment. Both Cd doses did not affect stomatal conduct-
membrane integrity in stress conditions by limiting Cd ance (gs) after 30 and 60 days, whereas after 90 days, Cd
transport in the cytosol (Li et al. 2014; Mahmud et al. induced a significant reduction in gs by 32 and 42% under
2018). CA can act as a Cd chelating agent leading to metal Cd3 and C6 treatment relative to control (Figure 6D).
accumulation in the vacuole, thereby preserving primary Stomatal conductance reduction due to Cd exposure is prob-
metabolism and accelerating plant ability to overcome stress. ably induced by stomatal closure or decrease in stomatal
In contrary to our study, Shamshad et al. (2018) reported aperture size which is related to restriction water vapor dif-
lower pigment contents and less toxicity in younger leaves fusion and gases exchange (Nikolic et al. 2017).
in comparison with OL in pea plants subjected to Cd stress Furthermore, Cd chelation with CA stress did not affect the
with CA addition. stomatal conductance after single nor double application in
Excess heavy metals content can affect photosynthesis comparison with the same treatment with no CA addition,
causing damages in photosynthetic apparatus, inhibition of while after multiple application (90 days) gs values were
electron transport, carbon fixation capacity, reduction in sto- reduced by 33 and 47% in comparison with Cd3 and Cd6.
matal conductance, and transpiration rate (Borowiak et al. Gas exchange attributes can be modified by different
2015; Nikolic et al. 2017). Results of this study noted heavy metals and can lead to a reduction in plant growth
decreasing order in gas exchange attributes such as net and biomass production. The inhibition of net photosyn-
assimilation rate, transpiration rate, WUE, as well as stoma- thetic rate induced by excessive heavy metals is provoked by
tal conductance with an elevation of Cd in plant tissue disturbed CO2 assimilation through reduced stomatal con-
(Figure 6). Net assimilation rate of CO2 showed Cd dose- ductance of CO2 (Pietrini et al. 2009). Meanwhile, the appli-
dependent decreasing, whereas CA addition diminishes cation of natural chelating agents in contaminated soils can
adverse effect of Cd, thus PN was higher under combined accelerate the efficiency of water use, plant growth, thus sta-
treatments in comparison with Cd applied alone (Figure bilizing photosynthesis and transpiration processes at a high
6A). Combined treatment CA þ Cd6 elevated PN by 20% level (Ehsan et al. 2014; Li et al. 2014). In context of that,
after single CA application (30 days), 23% after multiple CA changes in photosynthetic performances revealed in this
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 9
Table 2. Pro content in YL versus OL of S. viminalis depending on Cd dosage, CA applications, and experimental duration.
Young leaves Old leaves
30 days 60 days 90 days 30 days 60 days 90 days
Control 6.7 ± 1.6c 9.7 ± 2.9ghi 6.7 ± 1.2c 10.7 ± 4.4fgh 8.8 ± 2.6c 6.3 ± 0.9hi
Cd3 20.6 ± 6.1b 11.9 ± 2.1efg 9.3 ± 1.8c 22.1 ± 1.0b 10.1 ± 0.4c 7.3 ± 0.4ghi
Cd6 38.3 ± 6.0a 16.3 ± 1.9cde 8.4 ± 1.9c 32.2 ± 4.1a 10.2 ± 0.7c 5.9 ± 0.3hi
CA 9.1 ± 3.4c 15.1 ± 3.0def 9.4 ± 0.2c 5.4 ± 2.1i 8.3 ± 0.1c 7.7 ± 1.1ghi
CA þ Cd3 10.4 ± 1.1c 16.5 ± 2.2cd 11.2 ± 3.5c 10.1 ± 0.1ghi 8.9 ± 0.7c 7.1 ± 0.4ghi
CA þ Cd6 18.6 ± 5.9b 21.3 ± 7.3b 9.8 ± 0.7c 17.5 ± 2.5bcd 8.7 ± 0.8c 8.5 ± 1.ghi
Data were expressed as mean ± SD (n ¼ 3). Different letters indicate significant differences at p .05 applying Fisher LSD post hoc test.
Figure 7. PCA of morphophysiological traits in Salix viminalis grown in soil culture under different Cd dosage (3 and 6 mg kg1), CA applications (CA addition or no
CA) and experimental duration. CA, citric acid addition of 20 mmol kg1 of soil; Acc: accumulation of Cd; YL: young leaves; OL: old leaves; Chl: chlorophyll; Car: caro-
tenoids; PN: photosynthetic rate; E: transpiration rate; WUE: water use efficiency; ci: intercellular CO2 concentration; gs: stomatal conductance.
study under combined treatment with CA may be a result role as a compatible osmolyte, ROS scavenger, and in buf-
of plant adjustment to overcome Cd stress. This acclimatiza- fering cytosolic pH (Verbruggen and Hermans 2008).
tion enables the plant to cope with heavy metal stress that Therefore, the accumulation of this amino acid has been
consequently leads to enhanced plant tolerance to these pol- considered a useful marker for assessing plant tolerance to
lutants. In the line to our findings, Farid et al. (2017) heavy metal stress. In this study, the highest Pro accumula-
reported the positive role of CA in mitigation of heavy met- tion was recorded after 30 days, in plants exposed to Cd6, in
both YL and OL, and was almost six- and threefold higher
als to photosynthetic performances. Similarly, Guo et al.
in comparison with control (Table 2). The increased synthe-
(2017) reported that malic acid can mediate photosynthetic
sis of this amino acid under heavy metal stress can be con-
rate and stomatal conductance in Miscanthus sacchariflorus sidered as a non-enzymatic defense strategy due to its role
plants exposed to Cd stress. in scavenging of OH radicals and 1O2, indicating the anti-
oxidant role of this metabolite (Xu et al. 2009). However,
CA and Cd influence on Pro content chelation of Cd by CA-induced lower production of Pro in
plants subjected to the combined treatment with respect to
Pro accumulation has been reported to occur in different Cd applied alone. At the same time, after 60 days, the add-
species mainly as a response to osmotic stress influenced by ition of CA caused elevation of Pro accumulation in YL, by
drought, high temperature, salt, or heavy metals (Xu et al. 38 and 31% under CA þ Cd3 and CA þ Cd6, respectively,
2009; Mahmud et al. 2018). High Pro content is proposed to while in OL combined treatments only slightly reduced Pro
have an important role in plant stress tolerance, due to its accumulation, with no significant differences in relation to
10 D. ARSENOV ET AL.
the same treatments without CA (Table 2). The possible rea- adaptation and reduction” III43007, financed by the Ministry of
son is a significantly higher amount of Cd in YL exposed to Education and Science of the Republic of Serbia.
combined treatment with respect to Cd applied alone.
Contrary to this, after 90 days, no significant differences ORCID
were found irrespective of applied treatment or leaves
maturity (Table 2). Obtained results indicate that Pro accu- Danijela Arsenov http://orcid.org/0000-0002-1200-5023
Milan Zupunski http://orcid.org/0000-0002-8576-1292
mulation strongly depends on Cd accumulation, presence of Milan Borisev http://orcid.org/0000-0002-1833-002X
soil chelating agents, plant organs and maturity, as well as Natasa Nikolic http://orcid.org/0000-0002-7326-589X
on the experimental duration. According to Verbruggen and Andrej Pilipovic http://orcid.org/0000-0002-9458-0581
Hermans (2008), Pro accumulation varied among plant spe- Sasa Orlovic http://orcid.org/0000-0002-2724-1862
cies, maturation stage, and leaf age. Authors declared that
genes involved in Pro biosynthesis are predominantly References
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