CSIRO PUBLISHING

Functional Plant Biology

http://dx.doi.org/10.1071/FP16153

Early interconnectivity between metabolic and defense

events against oxidative stress induced by cadmium
in roots of four citrus rootstocks

Griselda Podazza A, Marta Arias B and Fernando E. Prado C,D

A
Instituto de Ecología, Fundación Miguel Lillo, Miguel Lillo 251, CP 4000, Tucumán, Argentina.
B
Cátedra de Anatomía Vegetal, Facultad de Ciencias Naturales e IML, Miguel Lillo 205, CP 4000,
Tucumán, Argentina.
C
Cátedra de Fisiología Vegetal, Facultad de Ciencias Naturales e IML, Miguel Lillo 205, CP 4000,
Tucumán, Argentina.
D
Corresponding author. Email: fepra@csnat.unt.edu.ar

Abstract. The effect of cadmium on roots of four citrus rootstocks was studied to assess the relationships between
oxidative stress, carbohydrates, phenolics and antioxidant responses. Swingle citrumelo (SC), Rangpur lime (RL), Troyer
citrange (TC) and Volkamer lemon (VL) genotypes were exposed to 0, 5 and 10 mM Cd over 7 days, after which Cd
accumulation was markedly higher in roots compared with stems and leaves. Malondialdehyde (MDA) and lipoxygenase
(LOX) activity increased in Cd-treated SC and RL roots, suggesting that a lipid peroxidation is the main driver of
plasma membrane damage. In contrast, in TC and VL genotypes, LOX-mediated lipid peroxidation does not appear to play a
key role in Cd-induced lipid peroxidation, but H2O2 accumulation seems to be responsible of less plasma membrane
damage. Catalase (CAT), superoxide dismutase (SOD) and guaiacol and syringaldazine peroxidases (G-POD and S-POD
respectively) were differentially affected by Cd. Lipid profile and ATPase-dependant proton extrusion indicated higher
disfunctionalities of root plasma membrane in SC and RL genotypes than in TC and VL genotypes. Differences in
carbohydrates and phenolic compounds were also observed. Histochemical analysis of G-POD activity and lignin and
suberin deposition revealed differences among genotypes. A model to explain the relationships among carbohydrates,
soluble phenolics, lipid peroxidation and H2O2 accumulation in Cd-exposed roots was proposed.

Additional keywords: cadmium, defence, genotype, metabolism, oxidative stress, root.

Received 10 August 2015, accepted 1 June 2016, published online 30 June 2016

Introduction (Tran and Popova 2013). Cd-induced ROS accumulation is

The increased use of fertile lands for human activities other than driven by both non-enzymatic and enzymatic mechanisms
agricultural practices has pushed crop cultivation to less (Tamás et al. 2010). The main members of the ROS family
include free radicals such as O2– (superoxide) and OH
*

productive lands and even polluted areas. In these conditions *

an increased application of fertilisers to soils is required to (hydroxyl), as well as non-radicals like 1O2 (singlet oxygen)
improve the productivity of plants. Phosphate mineral is the and H2O2 (hydrogen peroxide) (Sharma et al. 2012). Major
most used fertiliser worldwide, but contains cadmium (Cd) as H2O2-generating enzymes induced by Cd include, among
a contaminant at levels varying from trace amounts to as much as others, plasma membrane-bound NADPH oxidase (NOX),
300 mg kg–1 on a total DW basis (Alloway and Steinnes 1999). It xanthine oxidase (XOD), polyamine oxidase (PAO) and
represents the major source of Cd input into agricultural lands apoplastic peroxidase (POD) (Cheeseman 2007). Lipid
(Irfan et al. 2013). Cd is a non-essential metal for plants and peroxidation catalysed by the lipoxygenase enzyme (LOX) is
animals: it is one of the most widespread pollutants and is the of particular concern in plants exposed to Cd ions (Skórzy nska-
most ubiquitous heavy metal in the environment (Kabata- Polit and Krupa 2006). LOX are ubiquitous enzymes in both
Pendias 2011). Phytotoxic effects of Cd are related to animal and plant organisms. In plants, they play important roles in
disturbances of basic physiological processes such as the biotic and abiotic stress defence mechanisms and also in different
inhibition of root growth and decrease in both photosynthesis morphological processes and the jasmonate signalling pathway
and biomass accumulation (Benavides et al. 2005). It also (Liavonchanka and Feussner 2006). Many studies have reported
affects the normal balance between generation and scavenging a close connection between increased LOX activity and lipid
of reactive oxygen species (ROS), and causes oxidative stress peroxidation induced by different heavy metals such as Cd, Hg,
Journal compilation  CSIRO 2016 www.publish.csiro.au/journals/fpb
B Functional Plant Biology G. Podazza et al.

Zn, Cu, Pb and Cr (Skórzy nska-Polit and Krupa 2006; Zhou that variability in responses of citrus rootstocks to Cd stress is
et al. 2008; Prado et al. 2010). In plants exposed to transitions a key factor for better understanding the performance of
metals, both non-enzymatic and LOX-dependant lipid scion–rootstock cultivars growing in soils treated with mineral
peroxidation may be initiated in parallel, which results in a phosphate fertilisers. The aim of this study was to evaluate inter-
severe lipid peroxidation and increased level of lipid peroxides. genotype relationships among H2O2 accumulation, lipid
Although LOX activity is increased first in plants exposed to Cd peroxidation, antioxidant responses, soluble carbohydrates and
(non-transition metal), non-enzymatic lipid peroxidation begins soluble phenolics occurring in four citrus rootstocks exposed to
as a result. However, transition metals produce a stronger different Cd concentrations. For this, internal accumulation of
stimulation of non-enzymatic lipid peroxidation than that H2O2, MDA, soluble phenolics (SP), soluble sugars (sucrose,
induced by Cd (Gallego et al. 1996). Furthermore, lipid glucose and fructose), and lipids (phospholipids, sterols and
peroxidation also depends on the amount and kind of metal, glycolipids), as well as LOX, CAT, SOD, G-POD, and S-POD
individual part of plant, plant age and plant species or genotype activities and plasma membrane proton extrusion were
(Skórzynska-Polit 2007). measured in roots of Swingle citrumelo (SC), Rangpur lime
To alleviate Cd-induced oxidative stress, plants have a set (RL), Troyer citrange (TC) and Volkamer lemon (VL)
of enzymatic and non-enzymatic antioxidant molecules seedlings. Cd accumulation in roots, stems and leaves was
(Benavides et al. 2005). Antioxidant enzymes such as superoxide determined. Histochemical analyses were performed to detect
dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate localised G-POD activity, lignin and suberin deposition in roots
peroxidase (AsP), glutathione peroxidase (GP), glutathione of both Cd-untreated (control) and Cd-treated seedlings.
reductase (GR), monodehydroascorbate reductase (MDHAR)
and dehydroascorbate (DHAR) reductase, as well as reduced
Materials and methods
glutathione (GSH), ascorbic acid (AsA), a-tocopherol,
metallothioneins (MTs) and phytochelatins (PCs), participate Plant material
actively in the scavenging of ROS of plant cells (Mohamed et al. Seedlings of Swingle citrumelo (Citrus paradise
2012). Since Cd is absorbed easily by roots, plants have evolved Macf.
Poncirus trifoliata (L.) Raf.), Rangpur lime
different strategies to avoid its toxicity. Some species use an (Citrus
limonia Osbeck), Troyer citrange (Citrus sinensis
avoidance strategy to retain most Cd on roots (Rascio et al. (L.) Osb.
Poncirus trifoliata (L.) Raf.), and Volkamer lemon
2008), whereas others use internal mechanisms to cope Cd (Citrus volkameriana V. Ten. & Pasq.) genotypes were used as
toxicity (Zhang et al. 2010). The cell wall is the major plant material. Seeds were provided by CITRUSVIL SA
environment–plant exchange interface in roots and constitutes a (Tucumán, Argentina). Before sowing, seeds were soaked
barrier to Cd entry. Binding Cd to functional groups of the cell overnight in distilled water at 45C, and then surface-sterilised
wall such as carboxyl, phenolyl, hydroxyl and amino acyl leads with 10% sodium hypochlorite for 15 min. Sterilised seeds
to the formation of stable complexes, which play a pivotal role were thoroughly washed with distilled water, sown on wet
to block the entry of Cd in root cells (Parrotta et al. 2015). In this vermiculite in plastic boxes (15
20
5 cm) and transferred
regard, increases in both POD activity and lignin synthesis to a germination chamber at 30C under darkness. To avoid
have been reported as common responses of Cd-exposed roots water loss by evapotranspiration, plastic boxes were sealed with
(Elobeid et al. 2012; Podazza et al. 2012; Rui et al. 2016). Inter- plastic film. After 7 days, uniform seedlings were carefully
and intra-genotype variability in anatomical and physiological selected, planted in 250 mL plastic pots filled with wet
responses against Cd toxicity has been frequently found (Gaudet vermiculite (three seedlings per pot), and transferred to growth
et al. 2011; Gill et al. 2011); however, little is known about the chamber under 180 mmol m–2 s–1 photon flux density, 30/
biochemical mechanisms underlying such variability in woody 25C day/night temperature, 12 h photoperiod and 70%
plants. RH. Pots were supplied with 1/4 strength Hoagland’s nutrient
Since commercial citrus plants are scion–rootstock solution (25 mL per pot) each week. After 4 weeks, seedlings
combinations, selection of suitable rootstocks becomes a key with two leaves and similar root length were washed thoroughly
strategy to improve the performance of scion cultivars under with distilled water to eliminate residual vermiculite and
unfavourable conditions (Ribeiro et al. 2014). Responses of transferred to 600 mL plastic pots (six seedlings per pot, 15 pots
citrus rootstocks to some environmental stresses such as salinity, per treatment) containing 500 mL of different Cd solutions
drought, flooding, low temperature and boron, were studied prepared in distilled water. Next, pots were transferred to a
extensively; however, heavy metal effects have not been growth chamber for 7 days under similar conditions. We choose
similarly analysed (Arbona et al. 2008; Balal et al. 2012; Chen a 7 day treatment period based on a previous study conducted in
et al. 2012; Merlin et al. 2012; Liao et al. 2015). Studies on the our laboratory, which showed that citrus seedlings are able to
effects of Cd have been focussed on Cd-induced changes of grow and stay healthy, without visible chlorosis symptoms, in
non-enzymatic and enzymatic antioxidants, but the relationships distilled water without nutrient supply for at least 9 days (G
between Cd-induced oxidative stress and primary (carbohydrates) Podazza, unpubl. data). Cd solutions were prepared from a
and secondary (phenolic compounds) metabolisms has not been 500 mM stock solution. Hoagland’s nutrient solution was not
analysed. As far as we know, studies related to this subject on used to prepare treatment solutions to avoid competition
Cd-exposed citrus rootstocks have been made only in our between Cd and cations of the Hoagland’s solution either by cell
laboratory (Podazza et al. 2012), thus, specifics relating to inter- wall binding sites and/or plasma membrane ion transporters
genotype cross-talk between oxidative stress and primary and (Sterckeman et al. 2011). The use of nutrient solution could
secondary metabolisms remains unreported. We hypothesised also result in algal growth that can interfere with root Cd uptake.
Cd stress responses in citrus rootstocks Functional Plant Biology C

Selected Cd concentrations were 5 and 10 mM, which are Proton extrusion

equivalent to 0.62 and 1.24 mg Cd kg–1 soil DW (Kabata- Proton extrusion from Cd-untreated and Cd-treated roots was
Pendias 2011). These Cd concentrations are found frequently in measured as described by Zenoff et al. (1994) and expressed as
soils added with phosphate mineral fertiliser or sewage sludge DpH% by assuming that maximum DpH (100%) correspond
(Alloway and Steinnes 1999). Treatment solutions were aerated to Cd-untreated (control) roots. To assess the involvement of
every day by bubbling filtered air for 6 h and were changed every plasma membrane H+ ATPase activity in proton extrusion, DpH
2 days to avoid Cd depletion during the experiment. Distilled of control roots was also measured in presence of Na3VO4, a
water was used as a control solution (0 mM Cd). At the end of specific inhibitor of the plasma membrane H+ ATPase activity.
Cd treatment, seedlings were harvested and used for both
biochemical and histochemical studies. For enzymatic analyses, Quantification of soluble sugars
plant material was stored at –20C.
Sucrose, glucose and fructose were extracted and determined
as described by Prado et al. (2000). Sugar concentrations were
Biomass accumulation and Cd determination expressed as mmol g–1 FW.
After harvest, seedlings with similar height (30 per treatment)
were washed with deionised water, blotted dry onto absorbent Histochemical localisation of G-POD, lignin and suberin
paper, and sectioned into roots, stems and leaves. Sectioned For histochemical analyses, roots (10 per treatment) were
organs were weighed to obtain the FW and dried in a hot air washed thoroughly with distilled water and blotted dry onto
oven at 60C for 48 h to obtain the DW. Biomass accumulation absorbent paper. Next, transversal freehand sections were cut
was expressed as g DW. After that, dry samples were ground to ~2-cm from root apex from both control and Cd-treated seedlings.
fine powder in an electrical mill. Powdered samples (roots, Visualisation of G-POD activity, lignin and suberin was
stems and leaves) were digested in concentrated HNO3 : HClO4 performed according to Podazza et al. (2012). Microscopic
(4 : 1, v/v) mixture, following a modified USEPA 3051 protocol observations were performed using a binocular light
(USEPA 1994). Cd content was determined by atomic absorption microscope and photomicrographs were taken with a digital
spectrophotometry (Perkin-Elmer 373 Spectrophotometer) and camera (Olympus BX51 Microscope and DP70 Digital
expressed as mg g–1 DW. Data presented are the mean values of Camera Systems).
triplicates. The ratio between Cd accumulated in root and Cd
accumulated in shoot, (Cdroot : Cdshoot), was calculated as Statistics
follows:
For chemical and enzymatic determinations, at least three
Cdroot =Cdshoot ¼ R=ðS þ LÞ; ð1Þ replicates were analysed and two independent experiments
were performed. Results were analysed by one-way ANOVA
where R is root Cd content
root DW, S is stem Cd using the Sigma Stat program for Windows, ver. 3.5 (Systat
content
stem DW and L is leaf Cd content
leaf DW. Software). Significant differences in numerical results between
treatments were tested using the Tukey’s multiple comparison
Enzyme activities tests at P < 0.05. Data in tables and figures are presented as mean
SOD, CAT and LOX activities were extracted and assayed as values  s.e.
described by Prado et al. (2010). SOD and CAT activities were
expressed as UE min–1 g–1 FW whereas LOX activity was Results
expressed as DA234 min–1 g–1 FW. G-POD and S-POD Biomass and Cd accumulation in seedling organs
activities were extracted and assayed according to work by Table 1 shows both biomass accumulation and Cd content in
Peyrano et al. (1997). Enzyme activities were expressed as roots, stems and leaves of SC, RL, TC and VL seedlings exposed
DA470 min–1 g–1 FW (G-POD) and DA530 min–1 g–1 FW to different Cd concentrations. Root, stem and leaf DW were
(S-POD) respectively. quickly affected by increasing Cd concentrations in almost all
genotypes, but whereas root DW decreased significantly in RL
Quantification of H2O2, MDA and SP genotype (21.1%), in VL genotype a significant increase (30.8%)
H2O2, MDA and SP were extracted and determined according to occurred. Cd accumulation increased under increasing Cd
work by Prado et al. (2010). Concentrations were expressed as concentrations in all studied genotypes. Accumulation of Cd
mmol g–1 FW (H2O2), nmol g–1 FW (MDA), and mmol phenol was markedly higher in roots compared with stems and leaves.
equivalents g–1 FW (SP) respectively. There were no significant inter-genotype differences in root Cd
content, but in stem and leaf contents significant differences were
observed. Under low Cd exposure, metal content in roots was
Determination of phospholipids (PL), sterols (ST) and between 40- (TC) and 124-fold (SC) higher than in stems,
glycolipids (GL) whereas under high Cd concentration was between 45- (TC)
Root lipids were extracted with methanol : chloroform : H2O, and 122-fold (SC) higher. For all assayed genotypes the pattern of
(50 : 50 : 10), mixture and determined as described by Zenoff Cd accumulation was: roots > stems > leaves. At the whole-plant
et al. (1994). Lipid concentrations were expressed as mmol Pi g–1 level, metal accumulation was higher in VL and TC genotypes
FW (PL), mmol stigmasterol g–1 FW (ST) and mmol glucose g–1 under 5 mM Cd, but there were no significant difference at 10 mM
FW (GL). Cd. Higher and lower values of the Cdroot : Cdshoot ratio occurred
 D
Functional Plant Biology

Table 1. Biomass accumulation, Cd content and tolerance index (Ti) in roots, stems and leaves of SC, RL, TC, and VL seedlings exposed to 0, 5 and 10 mM Cd during 7 days
Root Cd accumulation to shoot (stem + leaves) Cd accumulation ratio (Cdroot : Cdshoot) is also showed. Different lowercase letters indicate significant differences inside each genotype for each evaluated
parameter. Different uppercase letters indicate significant differences among genotypes for each Cd concentration and each evaluated parameter. Values are means  s.e. (n = 6)

Concentration SC RL TC VL
BiomassA Cd Ti BiomassA Cd Ti BiomassA Cd Ti BiomassA Cd Ti
(g DW) (mg g–1 DW) (g DW) (mg g–1 DW) (g DW) (mg g–1 DW) (g DW) (mg g–1 DW)

Root
0 mM 0.16 ± 0.02aB – – 0.19 ± 0.02aA – – 0.19 ± 0.03aA – – 0.16 ± 0.01bC – –
5 mM 0.13 ± 0.02bB 632.2 ± 57bB 0.81 ± 0.04bB 0.15 ± 0.01bB 582.1 ± 65bB 0.79 ± 0.06bB 0.22 ± 0.03aA 730.1 ± 75bA 1.16 ± 0.12aA 0.18 ± 0.01bA 766.8 ± 100bA 1.13 ± 0.12aA
10 mM 0.15 ± 0.02aB 1332.3 ± 118aA 0.93 ± 0.07bB 0.16 ± 0.01bB 1320.3 ± 165aA 0.84 ± 0.06bB 0.22 ± 0.04aA 1213.8 ± 146aA 1.16 ± 0.14aA 0.20 ± 0.02aA 1316.7 ± 187aA 1.23 ± 0.12aA
Stem
0 mM 0.20 ± 0.03aA – – 0.15 ± 0.02bB – – 0.19 ± 0.03aA – – 0.19 ± 0.02aA – –
5 mM 0.23 ± 0.04aA 5.1 ± 0.8dD 1.15 ± 0.10aA 0.20 ± 0.02aA 7.3 ± 1.1dC 1.25 ± 0.11aA 0.22 ± 0.05aA 18.3 ± 2.1dA 1.16 ± 0.10aA 0.20 ± 0.02aA 12.7 ± 0.9dB 1.05 ± 0.10aA
10 mM 0.23 ± 0.04aA 10.9 ± 0.6cC 1.15 ± 0.09aA 0.19 ± 0.01aA 18.8 ± 1.4cB 1.19 ± 0.10aA 0.22 ± 0.04aA 26.7 ± 1.9cA 1.11 ± 0.09aA 0.18 ± 0.02aA 26.4 ± 2.1cA 1.00 ± 0.12aA
Leaf
0 mM 0.22 ± 0.04aB – – 0.20 ± 0.03aB – – 0.28 ± 0.06aA – – 0.18 ± 0.01aC – –
5 mM 0.24 ± 0.06aB 1.1 ± 0.1fB 1.09 ± 0.08aA 0.23 ± 0.02aB 1.2 ± 0.1fB 1.15 ± 0.13aA 0.27 ± 0.06aA 1.5 ± 0.2fA 0.96 ± 0.10aA 0.19 ± 0.02aC 1.8 ± 0.2fA 1.05 ± 0.10aA
10 mM 0.23 ± 0.05aB 3.0 ± 0.1eB 1.04 ± 0.10aA 0.20 ± 0.02aB 3.6 ± 0.3eB 1.00 ± 0.09aA 0.27 ± 0.04aA 3.7 ± 0.4eA 0.96 ± 0.09aA 0.18 ± 0.02aC 4.4 ± 0.3eA 1.00 ± 0.11aA
Cdroot : Cdshoot
5 mM 57.2 ± 4.9aA 47.5 ± 3.4aB 36.2 ± 3.7aC 45.2 ± 3.1aB
10 mM 62.5 ± 6.1aA 49.3 ± 5.1aB 38.8 ± 3.9aC 47.5 ± 4.4aB
A
Corresponds to a single plant.
G. Podazza et al.
Cd stress responses in citrus rootstocks Functional Plant Biology E

in SC and TC genotypes compared with VL and RL genotypes SC RL TC VL

respectively. There were no significant differences between VL
and RL genotypes. Attempts to detect Cd in control seedlings 200 (a)
failed in all genotypes. The tolerance index (Ti), which was

LOX (A234 min–1 g–1 FW)

calculated as the ratio between DW of plant growth in Cd 160 aA
aA

solution and DW of plant grown in control solution, ranged aB

between 0.79 and 1.25 in roots, stems and leaves. Ti was aB
120 bC
significantly higher (P < 0.05) in roots of TC and VL genotypes bA bA
bA

compared with roots of SC and RL genotypes, whereas in stems

80 cB
and leaves there were no significant differences among
cC
genotypes. For each genotype there was no significant cC
cD
difference in Ti values between the two Cd concentrations 40

(Table 1).
0
(b)
Effect of Cd on LOX activity, and MDA and H2O2
20
accumulation aA

MDA (nmol g–1 FW)

bA
LOX activity was differentially affected by Cd treatment: in SC aB
15
and RL roots LOX activity increased markedly under increasing
Cd concentrations. Maximum increases of 3.5-fold (SC) and bB
78.8% (RL) occurred at 10 mM Cd. In contrast, in TC and VL 10 cA
roots it decreased by 61.4% in the former (5 mM Cd) and 68.7% cB aC aC
aC
in the latter (10 mM Cd) (Fig. 1a). Cd treatment also induced aC aD

changes in the concentration of MDA. At 10 mM Cd, MDA 5

bD
increased significantly in SC (2.4-fold) and RL (58.9%) roots.
In contrast, in both TC and VL roots MDA content was not 0
affected by Cd treatment, but decreased significantly (73.3%) in (c)
6.0 aA
TC roots exposed to 10 mM Cd (Fig. 1b). H2O2 concentration aA
bA
increased in Cd-treated genotypes, being significantly higher in
3.0
VL and TC roots (Fig. 1c). Maximum increases were observed in
H2O2 (µmol g–1 FW)

VL (10.5-fold) and TC (2.6-fold) genotypes under 10 mM Cd. In 2.0

SC and RL genotypes, highest increases (71.2% and 28.7%) cA aB
aB
occurred at 5 mM Cd. 1.5 aC
bB
bD bC
Effect of Cd on antioxidant enzymes of seedling roots 1.0
cC
cD
Except G-POD activity, SOD, CAT and S-POD activities were 0.5
differently affected by Cd in all studied genotypes (Fig. 2).
Enzyme activities were either increased or decreased under 0.0
0 5 10
increasing Cd concentrations when compared with Cd-
Cd (µm)
untreated controls. SOD activity decreased by 40.6% in TC
roots exposed to 5 mM Cd, but displayed a non-significant Fig. 1. Effect of Cd on lipoxygenase (LOX) activity, and H2O2 and
increase at 10 mM Cd. By contrast in SC, RL and VL roots, malondialdehyde (MDA) concentrations of Swingle citrumelo (SC),
SOD activity increased markedly at 5 and 10 mM Cd respectively. Rangpur lime (RL), Troyer citrange (TC) and Volkamer lemon (VL) roots.
Maximum increases 5.1-fold (VL) and 2.9-fold (RL) were Bars indicate means  s.e. (n = 6). Different lowercase letters on bars indicate
observed at 10 mM Cd (Fig. 2a). CAT activity increased significant differences for each measured parameter and for each genotype.
Different uppercase letters on bars indicate significant differences among
significantly in both SC (12.5-fold) and VL (9.7-fold) roots
genotypes, for each Cd concentration and each measured parameter.
exposed to 5 mM Cd but sharply decreased at 10 mM Cd,
reaching even much lower values than that control values. In
an opposite trend, in TC and RL roots CAT activity showed a Cd- increase of S-POD activity (23.4%) occurred in TC roots under
dependant increment, reaching maximum values of 83.3% (TC) 10 mM Cd (Fig. 2d).
and 165.9% (RL) under 10 mM Cd (Fig. 2b). G-POD showed a
similar activity pattern in all rootstocks with significant increases Effect of Cd on root lipids
occurring at 5 mM Cd. Maximum increases were 11.6-fold (VL) Phospholipids (PL), sterols (ST) and glycolipids (GL) were
and 8.7-fold (RL and TC) respectively. At 10 mM Cd, G-POD differentially affected by Cd in all genotypes (Table 2). PL
activity decreased in all genotypes (Fig. 2c). Excepting in TC showed a Cd-dependant reduction in both SC and RL roots.
roots, S-POD activity decreased in Cd-treated SC, RL and VL Maximum reductions (31.5% and 27.6%) were observed at
roots. Maximum reductions were 58.3% (VL) and 22.9% (RL), 10 mM Cd. In contrast, there was no significant change in PL
respectively. Lowest values of S-POD activity were observed at concentrations in TC and VL roots. ST decreased in SC and RL
both 5 mM (SC, VL) and 10 mM (RL) Cd respectively. Maximum roots, but was more pronounced in the first one. Maximum
F Functional Plant Biology G. Podazza et al.

SC RL TC VL

600.0 (a) (b)

30.0

500.0 aA
25.0
aA
SOD (UE min–1 g–1 FW)

20.0

CAT (UE min–1 g–1 FW)

400.0
aB 15.0
bA bA
bA
300.0 bA
10.0
bB
aB
aC
bB aC cB aC
5.0
200.0 aD
aC

bD 1.0
100.0 bB bC
cC bC 0.5
cC
cC cC
0.0 0.0
(c) (d )
aA
aA
G-POD (A470 min–1 g–1 FW)

S-POD (A530 min–1 g–1 FW)

0.09 aB 0.09
bA
aA

bB
cB
cC bC
aA
aA
aA
0.06 0.06
cC bC

cD

aB

0.03 0.03

bA bA
cA bA bA
cB bB
cC

0.00 0.00
0 5 10 0 5 10
Cd (µm)

Fig. 2. Effect of Cd on superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (G-POD) and
syringaldazine peroxidase (S-POD) activities in Swingle citrumelo (SC), Rangpur lime (RL), Troyer citrange
(TC) and Volkamer lemon (VL) roots. Bars indicate means  s.e. (n = 6). Different lowercase letters on bars
indicate significant differences for each enzyme activity and for each genotype. Different uppercase letters on
bars indicate significant differences among genotypes, for each Cd concentration and each enzyme activity.

reductions (45.1 and 24.6%) occurred at highest Cd Effect of Cd on root proton extrusion
concentration. Further, in TC and VL genotypes, ST content Root proton extrusion decreased at 10 mM Cd in all genotypes,
was increased by 16.5 and 61.0% at 5 and 10 mM Cd respectively. but was higher in SC and RL roots. Maximum reductions were
GL content decreased in Cd-exposed roots of SC (14.4%) and 52.4% (RL), 50.3% (SC), 37.9% (VL) and 37.1% (TC). Proton
RL (25.0%) genotypes under 10 mM Cd, whereas no significant extrusions of control roots were strongly decreased by Na3VO4,
changes occurred in TC roots. In VL roots GL was increased by but there were no significant differences with Cd-induced
22.8% at 10 mM Cd. Total lipids (PL + ST + GL) were decreased decreases (Fig. 3a–d). At 5 mM Cd less decreases of proton
by Cd treatment in SC and RL genotypes whereas in VL one was extrusion occurred (data not shown).
increased by 25.0%. There was no significant change of total
lipids in TC roots. In Cd-exposed roots the ST/PL ratio decreased
by 21.1% in SC genotype, but in TC and VL genotypes increased
by 20.9 and 28.6% respectively. No significant changes were Effect of Cd on root soluble phenolics (SP)
observed in RL roots. The GL : PL ratio increased by 15.3% in the SP content was increased in Cd-exposed roots of all genotypes,
SC genotype, but in the rest of genotypes there were no significant being higher at 5 mM Cd concentration. Maximum increases
changes. were 155.2% (VL), 90.6% (TC), 42.9% (SC) and 23.3% (RL).
Cd stress responses in citrus rootstocks Functional Plant Biology G

Different lowercase letters indicate significant differences for each genotype and for each lipid type. Different uppercase letters indicate significant differences among genotypes, for each Cd concentration
Table 2. Phospholipids (PL), sterols (ST), glycolipids (GL) and total lipids (PL+ST+GL), and ST : PL and GL : PL ratios in roots of SC, RL, TC and VL rootstocks exposed to 0, 5 and 10 mM

15.22 ± 1.43aA 13.43 ± 1.23aA 13.02 ± 1.43aA 14.12 ± 1.40aA 11.76 ± 1.20bB 10.59 ± 1.05bB 10.96 ± 1.21aB 10.29 ± 1.09aB 10.82 ± 1.00aB 10.54 ± 1.10aB 11.80 ± 1.00bB 12.94 ± 1.30aA

18.65 ± 1.72aA 15.82 ± 1.43bA 15.05 ± 1.50bA 16.92 ± 1.65aA 14.01 ± 1.22bB 12.67 ± 1.22bB 12.87 ± 1.16aB 12.40 ± 1.15aC 12.60 ± 1.12aB 12.15 ± 1.23cB 13.84 ± 1.43bB 15.19 ± 1.33aA

14.09 ± 1.32bB 15.26 ± 1.53bB 17.59 ± 1.78aA 13.44 ± 1.45aB 14.34 ± 1.42aB 13.93 ± 1.32aB 18.89 ± 2.11aA 18.37 ± 1.76aA 21.64 ± 2.10aA 17.29 ± 1.80aA 17.10 ± 1.71aA 20.22 ± 2.00aA
0.64 ± 0.06aB

1.61 ± 0.12aB

2.52 ± 0.20aB
At 10 mM Cd concentration, increases were 73.9, 57.4, 30.3

Cd (10 mM)
and 15.7% respectively (Fig. 4).

Effect of Cd on soluble sugar accumulation

0.69 ± 0.05aB

1.43 ± 0.11bA 1.32 ± 0.17bA 1.33 ± 0.12bC 1.55 ± 0.20aA 1.28 ± 0.10bC 1.00 ± 0.10bD 1.35 ± 0.13aB

1.96 ± 0.19aB
Exogenous Cd caused changes in sucrose, glucose and fructose

Cd (5 mM)
concentrations in SC, TC, RL and VL roots. Sugars were
VL differentially affected by Cd treatment, and showed different
distribution patterns among genotypes (Fig. 5). The sucrose
0.61 ± 0.06aB pattern revealed that in SC, TC, RL and VL roots a Cd-

2.77 ± 0.25aA 2.56 ± 0.21aA 1.96 ± 0.20aB

Cd (0 mM)

dependant sucrose reduction occurred, being more pronounced

in TC and VL genotypes. Maximum reductions occurring at
10 mM Cd were 61% (TC), 52.4% (VL), 45.2% (RL) and 27.5%
(SC) (Fig. 5a). The fructose pattern was similar in all genotypes
with higher reductions occurring under 5 mM Cd. Maximum
0.50 ± 0.04aB
Cd (10 mM)

decreases were 46.5% (VL), 44.1% (TC), 20.8% (RL) and

12.9% (SC) respectively (Fig. 5b). Glucose accumulation did
not show a similar pattern in all genotypes. In SC and RL roots
glucose displayed Cd-dependant increases with maximum
0.56 ± 0.07aB

values of 69.3% (RL) and 57.4% (SC) occurring at 10 mM Cd.

Cd (5 mM)
TC

In contrast, in TC and VL genotypes glucose contents decreased

under low Cd concentration, but was greater than control values
at 10 mM Cd. Maximum decreases occurring at 5 mM Cd were
and each lipid type. Values are means  s.e. (n = 6)

1.05 ± 0.11aA 0.82 ± 0.08bA 0.76 ± 0.06bA 0.58 ± 0.04aB

2.29 ± 0.23aB

39.1% (TC) and 37.7% (VL) respectively (Fig. 5c).

Cd (0 mM)

Histochemical localisation of lignin, suberin and G-POD

activity
Cd during 7 days

G-POD activity and lignin and suberin depositions were

1.74 ± 0.17aC
Cd (10 mM)

observed in both control and Cd-treated roots, but staining, in

general, was more intense in the latter (Fig. 6a–x). At 5 mM Cd
colour intensity was higher in TC and VL roots compared with
SC and RL ones. Under 10 mM Cd fewer differences among
1.67 ± 0.14aD 1.74 ± 0.18aC

genotypes were observed. All attempts to detect S-POD activity

Cd (5 mM)

failed.
RL

Discussion
1.75 ± 0.12aB

Biomass and Cd accumulation

Cd (0 mM)

Many woody plant species retain large amounts of Cd in roots

without transporting it to aerial parts (Gogorcena et al. 2011).
A high accumulation of Cd has been observed in roots of Cd-
2.35 ± 0.20aA 1.51 ± 0.15bA 1.29 ± 0.17bB

1.74 ± 0.14bC

exposed Cleopatra mandarin and Carrizo citrange genotypes

1.08 ± 0.09aA 0.88 ± 0.07bA 0.74 ± 0.04cA
Cd (10 mM)

(López-Climent et al. 2014). Data of the present work show a

markedly higher content of Cd in roots of all studied citrus
rootstocks than in stem and leaf contents (Table 1). At 5 mM
Cd, higher Cd accumulation occurs in roots, stems and leaves
1.72 ± 0.16bC

of TC and VL genotypes than in SC and RL ones. Since the

Cd (5 mM)

Cdroot : Cdshoot ratio did not show a similar trend among

SC

genotypes, it may be assumed that there exists a complex

heterogeneity in the mechanism that regulates the transport of
Cd towards aerial part in Cd-treated roots. Further, the difference
2.18 ± 0.18aB
Cd (0 mM)

in the ratio may also indicate a metal-induced disruption of

the root-to-shoot transport mechanism, as well as reflect a
genotypic difference in metal translocation strength. Indeed,
since at a 5 mM Cd concentration both DW and Ti changes
(mmol Pi g–1 FW)

were similar to Cd accumulation, it can be assumed either that

(mmol glucose
stigmasterol

a higher threshold of Cd toxicity or a more efficient detoxification

Phospholipids

Sterols (mmol

Glycolipids

Total lipids
g–1 FW)

g–1 FW)

mechanism, or even both processes, could be operating in TC

GL : PL
ST : PL

and VL genotypes regarding to SC and RL ones. However,

there were no significant genotypic differences in root Cd
H Functional Plant Biology G. Podazza et al.

Cd (0 µM) Cd (10 µM) Na3 VO4 (1.5 mM)

(a) SC (b) RL
aA aA aA aA
100 100

bA
bA
80 80

cA dA
60 60
aB aB
aB aB aB
aB
aB
aB
40 bB bB 40
dA dA
bB cA bB
cB
cC cC
Proton extrusion (%)

Proton extrusion (%)

20 dA dA dA dA 20

0 0
(c) TC (d) VL
aA aA
aA aA
100 100
bA bA

80 80
cA cA
aA aA aA aA
aA aA
aA
aA
60 60
bA
bA

bA bA
40 40
dA dA cB cA
cB
cB

dA dA
20 dA dA 20

0 0
5 10 20 30 40 5 10 20 30 40

Time (min)

Fig. 3. Effect of Cd and Na3VO4 on the temporal evolution of proton extrusion from roots of Swingle citrumelo (SC), Rangpur lime
(RL), Troyer citrange (TC) and Volkamer lemon (VL) seedlings. Bars indicate means  s.e. (n = 6). Different lowercase letters on bars
indicate significant differences for each measurement condition and for each genotype. Different uppercase letters on bars indicate
significant differences among genotypes, for each Cd concentration and each measurement condition.

accumulation under the highest Cd concentration. Although this Krupa 2006). Heavy metal-induced lipid peroxidation in plants
fact may be related to saturation of Cd-binding sites onto cell wall mainly occurs by enzymatic (LOX-dependant) or non-enzymatic
(Nocito et al. 2011), other unknown processes could also be (H2O2-dependant) mechanisms, or by both (Sharma et al. 2012).
occurring. In this regard, both accumulation and translocation of LOX activity and H2O2 content in roots of citrus rootstocks were
metals in plants are often not clearly identifiable entities because affected differently by Cd treatment. In SC and RL genotypes
they depend upon species/genotype, metal concentration and LOX activity increased strongly whereas H2O2 increased less
metal speciation, and also may be the results of complex markedly (Fig. 1a, c). These results could indicate that both
interactions of metal ion with other essential or non-essential enzymatic and non-enzymatic lipid peroxidation took place in SC
ions and with several metabolic events (internal factors), and even and RL genotypes, but a LOX-mediated oxidative event would be
with the environment (external factors) (Ernst 2006). It has also primarily responsible of plasma membrane damage. In agreement
been suggested that root-to-shoot Cd transport is a genetically with this assumption is that the accumulation of MDA, an
regulated process, rather than one dependent on external factors indicator of lipid peroxidation, strongly increased in these
(Obata and Omebayashi 1997). Additional experiments are being genotypes. In contrast, in TC and VL Cd-treated roots, H2O2
planned to clarify this subject and will be reported elsewhere. increased markedly whereas both MDA and LOX activity
decreased significantly, indicating that in these genotypes
Lipid peroxidation and LOX activity LOX-mediated lipid peroxidation is not a key event, being
Cd, like the transition metals, is able to induce lipid peroxidation accumulated H2O2 responsible of Cd-induced oxidative stress.
of polyunsaturated fatty acids (PUFAs) (Skórzy nska-Polit and Also supporting this assumption, it has been demonstrated that
Cd stress responses in citrus rootstocks Functional Plant Biology I

SC RL TC VL SC RL TC VL

4.0 14 (a) aA
aB
aB
12
Soluble phenolics (µmol phenol equ. g–1 FW)

bA

Sucrose (µmol g–1 FW)

aA 10 bB bB
aC
3.0 aC cB cB cB
aB 8
bA bC

6
bB
2.0 aC 4
bC
cA
aD
cB 2
bD
cC cC
0
1.0 20 (b)
aA
aA

Fructose (µmol g–1 FW)

aB
15 aA
aC aA
bA
0.0 bA bB
bB bB
0 5 10
10 cB
Cd (µm)

Fig. 4. Effect of Cd on soluble phenolic contents of Swingle citrumelo

(SC), Rangpur lime (RL), Troyer citrange (TC) and Volkamer lemon (VL) 5
roots. Bars indicate means  s.e. (n = 6). Different lowercase letters on bars
indicate significant differences for each genotype. Different uppercase
0
letters on bars indicate significant differences among genotypes for each aB
2.00 (c) aA
Cd concentration. bA aB aB
cA
1.50 bB
Glucose (µmol g–1 FW)

bB bB
the increase of LOX activity is involved in Cd-induced 1.25 bC
cD
oxidative stress responses but not in harmful lipid peroxidation
1.00 cC
in barley roots (Liptáková et al. 2013). Furthermore, in Cd-treated
leaves of Phaseolus coccineus, LOX activity was similar to Cd- 0.75
untreated plants, whereas non-enzymatic lipid peroxidation was
significantly increased (Skórzy nska-Polit and Krupa 2006). 0.50
Moreover, lipid peroxidation also depends on plant species or 0.25
genotype. Because we did not measure the time progress of
LOX activity and both H2O2 and MDA accumulation, we 0.00
0 5 10
cannot fully confirm our hypothesis.
Cd (µm)

H2O2 and SP accumulation Fig. 5. Effect of Cd on sucrose, fructose and glucose concentrations of
1 Swingle citrumelo (SC), Rangpur lime (RL), Troyer citrange (TC) and
High levels of both non-radical (e.g. H2O2, O2) and radical Volkamer lemon (VL) roots. Bars indicate means  s.e. (n = 6). Different
(e.g. O2 –, OH) oxygen species are commonly associated to
*
*
lowercase letters on bars indicate significant differences for each genotype and
cellular oxidative damage (Sharma et al. 2012). According with for each sugar. Different uppercase letters on bars indicate significant
biomass yield and H2O2 accumulation found in TC and VL roots, differences between genotypes and for each sugar.
this trait seems not to be occurring in these genotypes. This could
indicate that in TC and VL roots efficient mechanisms exist to
remove the excess of H2O2. In this way, excess of H2O2 could be higher in TC and VL roots. Additionally, histochemical
accumulated and detoxified inside a safe cell compartment such observations of Cd-treated roots also showed higher G-POD
as the vacuole (Yamasaki et al. 1997), or channelled to H2O2- activity and lignin and suberin deposition in TC and VL
dependant synthesis of phenolic compounds (Kovácik and genotypes than SC and RL genotypes (Fig. 6). In agreement
Klejdus 2008). Agreeing with this last suggestion is the result with our results, in a recent study conducted with two varieties of
that the highest levels of SP were observed in TC and VL roots Vicia sativa, a differential increase of both G-POD activity and
(Fig. 4). In metal stressed plants, SP consume H2O2 in both POD- lignin deposition in roots of Cd-exposed plants has been
dependant vacuolar ROS scavenging and POD-dependant demonstrated (Rui et al. 2016). Although both G-POD and S-
synthesis of cell wall protective compounds, i.e. lignin and POD activities have been related to protective compounds
suberin (Michalak 2006). Increased POD activity and SP synthesis (Prado et al. 2011), our results indicate that G-POD
accumulation were found in all studied genotypes, but were is the main activity involved in lignin and suberin accumulation
J Functional Plant Biology G. Podazza et al.

SC SC RL RL TC TC VL VL
(0 µM Cd) (5 µM Cd) (0 µM Cd) (5 µM Cd) (0 µM Cd) (5 µM Cd) (0 µM Cd) (5 µM Cd)
(a) (b) (c) (d) (e) (f ) (g) (h)

(i) (j) (k) (l) (m) (n) (o) (p)

(q) (r) (s) (t) (u) (u) (v) (w) (x)

Fig. 6. Histochemical visualisation of guaiacol peroxidase (G-POD) activity (a–h), lignin (i–p), and suberin (q–x) in Swingle citrumelo (SC), Rangpur
lime (RL), Troyer citrange (TC) and Volkamer lemon (VL) roots exposed to 0 (control) and 5 mM Cd during 7 days. Arrowheads indicate enzyme activity,
lignin and suberin deposition in both exodermis and vascular cylinder. Scale bar = 50 mm.

in citrus rootstock roots. However, higher levels of H2O2 in TC (2014), decreases of both total lipids and PL are related to less
and VL roots than in SC and RL roots can be faster channelled plasma membrane stability against Cd toxicity. Thus, root plasma
through another H2O2-dependant signal pathway to orchestrate membranes of TC and VL genotypes would be less affected by
an early Cd-stress response. This assumption could be supported Cd toxicity. In contrast though, ST modulates the physical state
considering the hypothesis proposed by Petrov and Van of lipid bilayer by restricting the motion of fatty acid chains,
Breusegem (2012). According to these authors a low level of and also regulating the fluidity of cell membranes (Hartmann
H2O2 creates a signal for mild stress, whereas a higher amount of 1998). Moreover, ST becomes crucial for structural and
H2O2 produces a message for more severe stress. Based on this functional properties of plant membranes (Roche et al. 2008).
assumption, roots of TC and VL could respond faster to Cd- Further increasing values of ST : PL ratio have been related to
induced oxidative stress and then will be able to cope with stress tolerance (Wu et al. 1998). Data of this study show that ST
oxidative stress better than SC and RL roots. and ST : PL ratio increased in Cd-treated roots of TC and VL
Regarding detoxification of Cd-induced radical oxygen genotypes. Together, data related to lipid content seem indicate
species, both SOD and CAT activities responded differently a higher tolerance of plasma membrane against cadmium
to increasing Cd concentrations among studied genotypes, toxicity in TC and VL roots compared with SC and RL roots.
indicating that in Cd-exposed roots of citrus rootstocks can be Plasma membrane fluidity is determined by both unsaturated and
acting through different antioxidant mechanisms. Further polyunsaturated fatty acids, which, in turn, are highly dependent
research is needed to gain new insights in this topic. of LOX activity. Since LOX activity decreased in Cd-treated TC
and VL roots, higher plasma membrane fluidity is expected to
occur in these genotypes. Supporting this assumption, proton
Lipids and plasma membrane proton extrusion extrusion, which is frequently used as an indicator of membrane
Decreases of PL in root cell membranes dramatically affect both fluidity, showed less decreases in TC and VL roots in absence and
fluidity and activity of intrinsic membrane enzymes such as H+ presence of Na3VO4 (specific inhibitor of the plasma membrane
ATPase and H+ PPase (Fodor et al. 1995). Total lipids and PL in H+ ATPase activity). Additionally, proton extrusion of Cd-
SC and RL roots were significantly reduced by Cd treatment, untreated roots did not show significant inter-genotypical
wheras in TC and VL roots total lipids were increased, but in PL differences. Hence, plasma membrane functionality seems to
there were no significant changes. According to Elloumi et al. be less affected by Cd treatment in TC and VL roots.
Cd stress responses in citrus rootstocks Functional Plant Biology K

However, both H+ ATPase and H+ PPase activities involved free fructose and strong increase of SP were observed. By
in plasma membrane proton extrusion were not measured in contrast, in SC and RL genotypes, a smaller decrease of
this work, so caution should be taken before accepting this fructose and less synthesis of SP were observed (Figs 4, 5b).
assumption. The decreases observed in glucose content at the highest Cd
concentration in all genotypes probably reflect a generalised
metabolic reduction induced by metal toxicity. Beyond their
Soluble sugars metabolic functions, soluble sugars (mainly sucrose and
Under heavy metal stress a high demand for sucrose occurs to glucose) also act as signal molecules, playing a central role in
support the respiratory metabolism and synthesis of stress-related both regulation and fine tuning of source–sink relationships and
metabolites (e.g. phenolic compounds, amino acids, organic heavy metal stress responses (Bolouri-Moghaddam et al. 2010).
acids, glutathione, phytochelatins, metallothioneins and stress- Figure 7 summarises a possible model of interactions among
related proteins) (Prado et al. 2011). In this regard, all genotypes oxidative stress, H2O2 accumulation, lipid peroxidation, soluble
showed significant reductions in sucrose under increasing Cd carbohydrates and soluble phenolic compounds occurring in
concentrations, indicating a high metabolic demand to cope Cd root cells of citrus rootstocks exposed to Cd: in both SC and
toxicity. However, decreased levels of sucrose can also reflect a RL roots, Cd enters cells and triggers oxidative stress with
Cd-induced photosynthate flux reduction from leaves. Although increased accumulation of ionic ROS through increased LOX
in this study did not include measurement of gas-exchange activity (high lipid peroxidation) and also, but to a lesser extent,
parameters, in a previous study performed on Carrizo citrange intrinsic H2O2 accumulation produced by Cd-induced both
and Cleopatra mandarin rootstocks exposed to 30 and 150 mM apoplastic and plasma membrane-bound H2O2-generating
Cd during 85 days, López-Climent et al. (2014) demonstrated enzymes (POD, NOX, XOD, PAO) (Cheeseman 2007). High
only a slight decrease in CO2 assimilation in Cd-treated plants. lipid peroxidation concomitantly drives a plasma membrane
Hence, higher decreases occurring in sucrose contents of TC and damage, which, in turn, is accompanied by a possible
VL roots (Fig. 5a) could be related to higher release of fructose to dysfunction of the plasma membrane H+ ATPase activity
increase the synthesis of phenolic compounds. Fructose, through (lower proton extrusion). Furthermore, the lower consumption
its phosphorylated derivative fructose-6-phosphate (F-6-P) of fructose occurring in Cd-exposed SC and RL roots compared
produces erythrose-4-phosphate (E-4-P), enters into shikimate with TC and VL ones can also drive a decrease in synthesis of
and phenylpropanoid pathways to produce phenolic compounds both soluble and polymerized-derived phenolic compounds
(Mustafa and Verpoorte 2007). Supporting this assumption in (lower lignin and suberin deposition on cell walls). In contrast,
Cd-treated roots of TC and VL genotypes, a strong decrease of in cells of TC and VL roots, accumulated Cd does not trigger

SC, RL TC, VL
(high) (low) (low) (high)
PM damage H+ extrusion NOX, POD, PAO, XOD PM damage H+ extrusion NOX, POD, PAO, XOD

LOX LOX
Signaling Signaling
H2O2
Lignin, Suberin

H2O2

Lignin, Suberin
ROS (high)
(low) ROS
(low)

G-POD

(high)
G-POD
Cd Cd
OXIDATIVE Phenolics OXIDATIVE
STRESS (low) STRESS Phenolics
(high)
Cd
Photosynthate

Cd
Photosynthate

Antiox. enzymes Fructose

Antiox. enzymes Fructose
Phenolics
Vacuole Sucrose Sucrose
Non-enzym. antiox.
Glucose Non-enzym. antiox. H2O H2O2 Glucose
POD
Respiration Respiration
Signaling ?
Early stress responses Signaling

Fig. 7. Hypothetical scheme of major metabolic and defence events involving soluble carbohydrates, soluble phenolics, H2O2 and antioxidant enzymes
that occurs in Cd-exposed roots of Swingle citrumelo (SC), Rangpur lime (RL), Troyer citrange (TC) and Volkamer lemon (VL) seedlings. Abbreviations:
PM, plasma membrane; LOX, lipoxygenase; NOX, plasma membrane NADPH oxidase; POD, apoplastic and vacuolar peroxidase; PAO, polyamine oxidase;
XOD, xanthine oxidase; G-POD, guaiacol peroxidase. In this figure denotes high contribution; denotes low contribution; = = = = denotes
probable effect.
L Functional Plant Biology G. Podazza et al.

LOX-induced lipid peroxidation, and then less severe plasma Elloumi N, Zouari M, Chaari L, Jomni C, Marzouk B, Ben Abdallah F (2014)
membrane damage and less H+ ATPase dysfunction occurs Effects of cadmium on lipids of almond seedlings (Prunus dulcis).
(higher proton extrusion). However, Cd-toxicity triggers Botanical Studies 55, 61. doi:10.1186/s40529-014-0061-7
Elobeid M, Göbel C, Feussner I, Polle A (2012) Cadmium interferes with
higher intrinsic H2O2 accumulation (non-ionic ROS), possibly,
auxin physiology and lignification in poplar. Journal of Experimental
via enhanced activities of H2O2-generating enzymes.
Botany 63, 1413–1421. doi:10.1093/jxb/err384
Furthermore, increased H2O2 would trigger a higher synthesis Ernst WHO (2006) Evolution of metal tolerance in higher plants. Forest
of secondary defence compounds through G-POD-catalysed Snow and Landscape Research 80, 251–274.
reactions that use fructose-derived phenolics as substrates Fodor E, Szabó-Nagy A, Erdei L (1995) The effects of cadmium on the
(higher SP accumulation and increased lignin and suberin fluidity and H+-ATPase activity of plasma membrane from sunflower
deposition on cell walls). Additionally, increased synthesis of and wheat roots. Journal of Plant Physiology 147, 87–92. doi:10.1016/
SP in TC and VL roots can also contribute to eliminate the excess S0176-1617(11)81418-5
of H2O2 inside the vacuole through a vacuolar POD-catalysed Gallego SM, Benavides MP, Tomaro ML (1996) Effect of heavy metal ion
reaction (Yamasaki et al. 1997). Beyond these H2O2-dependant excess on sunflower leaves: evidence for involvement of oxidative stress.
Plant Science 121, 151–159. doi:10.1016/S0168-9452(96)04528-1
reactions, H2O2 in TC and VL root cells could also act as
Gaudet M, Pietrini F, Beritognolo I, Iori V, Zacchini M, Massacci A,
secondary signal molecule to trigger other early stress
Mugnozza GS, Sabatti M (2011) Intraspecific variation of
responses to counteract metal toxicity. Together, these traits physiological and molecular response to cadmium stress in Populus
would allow to TC and VL genotypes get greater fitness to nigra L. Tree Physiology 31, 1309–1318. doi:10.1093/treephys/tpr088
tolerate Cd-induced stress. Gill SS, Khan NA, Tuteja N (2011) Differential cadmium stress tolerance in
five Indian mustard (Brassica juncea L.) cultivars. An evaluation of the
Conclusions role of antioxidant machinery. Plant Signaling & Behavior 6, 293–300.
doi:10.4161/psb.6.2.15049
Overall results of this study indicate that different metabolic
Gogorcena Y, Larbi A, Andaluz S, Carpena RO, Abadía A, Abadía J (2011)
events linked to Cd tolerance occur in SC, RL, TC and VL
Effects of cadmium on cork oak (Quercus suber L.) plants grown in
roots. Moreover, this study also represents a good approach to hydroponics. Tree Physiology 31, 1401–1412. doi:10.1093/treephys/
understand how low Cd concentrations in agricultural soils could tpr114
affect the performance of citrus rootstocks. Hartmann MA (1998) Plant sterols and the membrane environment.
Trends in Plant Science 3, 170–175. doi:10.1016/S1360-1385(98)
Acknowledgements 01233-3
Irfan M, Hayat S, Ahmad A, Alyemeni MN (2013) Soil cadmium enrichment:
We thank to Dr Eduardo Pagano (Facultad de Agronomía, Universidad de
allocation and plant physiological manifestations. Saudi Journal of
Buenos Aires) for Cd determination. This work was supported by grants from
Biological Sciences 20, 1–10. doi:10.1016/j.sjbs.2012.11.004
Consejo de Investigaciones de la Universidad Nacional de Tucumán (26/
Kabata-Pendias A (2011) ‘Trace elements in soils and plants.’ (4th edn)
G437) and Consejo Nacional de Investigaciones Científicas y Técnicas (PIP
(CRC Press: Bocca Raton, FL, USA).
11/265). FEP is a researcher of the Consejo Nacional de Investigaciones
Kovácik J, Klejdus B (2008) Dynamics of phenolic acids and lignin
Científicas y Técnicas.
accumulation in metal-treated Matricaria chamomilla roots. Plant Cell
Reports 27, 605–615. doi:10.1007/s00299-007-0490-9
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