Plant Biosystems - An International Journal Dealing with

all Aspects of Plant Biology

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Morphophysiological responses of Aechmea

blanchetiana (Bromeliaceae) to excess copper
during in vitro culture

João Paulo Rodrigues Martins, Luiz Carlos de Almeida Rodrigues, Thayna dos
Santos Silva, Franciele Pereira Rossini, Andreia Barcelos Passos Lima Gontijo
& Antelmo Ralph Falqueto

To cite this article: João Paulo Rodrigues Martins, Luiz Carlos de Almeida Rodrigues, Thayna
dos Santos Silva, Franciele Pereira Rossini, Andreia Barcelos Passos Lima Gontijo & Antelmo
Ralph Falqueto (2020): Morphophysiological responses of Aechmea�blanchetiana (Bromeliaceae)
to excess copper during in vitro culture, Plant Biosystems - An International Journal Dealing with all
Aspects of Plant Biology, DOI: 10.1080/11263504.2020.1756976

To link to this article: https://doi.org/10.1080/11263504.2020.1756976

Accepted author version posted online: 17

Apr 2020.
Published online: 13 May 2020.

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PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY
https://doi.org/10.1080/11263504.2020.1756976

Morphophysiological responses of Aechmea blanchetiana (Bromeliaceae)

to excess copper during in vitro culture
Jo~ao Paulo Rodrigues Martinsa,b , Luiz Carlos de Almeida Rodriguesc , Thayna dos Santos Silvab,
Franciele Pereira Rossinib , Andreia Barcelos Passos Lima Gontijob and Antelmo Ralph Falquetoa
a
Plant Ecophysiology Laboratory, Federal University of Espırito Santo, S~ao Mateus, ES, Brazil; bPlant Tissue Culture Laboratory, Federal
University of Espırito Santo, S~ao Mateus, ES, Brazil; cFaculty of Dentistry, Federal University of Alfenas, Alfenas, MG, Brazil

ABSTRACT ARTICLE HISTORY

The use of plants as bioindicators can be effective for environmental monitoring, mainly related to Received 18 July 2019
heavy metals such as copper (Cu). In vitro conditions are more easily controlled, to enable isolating Accepted 27 March 2020
the stress factors that can interfere in plants’ responses. The aim was to investigate the morphophysio-
KEYWORDS
logical responses of Aechmea blanchetiana submitted to excess Cu, and to verify the degree of toler-
Bromeliad; chlorophyll a
ance and potential as a bioindicator of this species. Previously cultured shoots of A. blanchetiana were fluorescence; environmental
transferred to agar-solidified MS medium. After 60 days, the tank system was recreated, with the add- bioindicator plant anatomy;
ition of stationary liquid MS medium with different concentrations of Cu (0, 25, 50 and 100 lM). After plant physiology
90 days with excess Cu, anatomical analysis along with quantification of the contents of nutrients,
photosynthetic pigments and chlorophyll a fluorescence were carried out. Alterations in the stomatal
density and chlorenchyma thickness were observed in the plants grown with excess Cu. There was an
increase in the Cu content in the plants in function of the Cu levels. However, the adjustments in the
content of the other nutrients were essential for tolerance to excess Cu. Aechmea blanchetiana toler-
ates high Cu concentrations and has potential for use as a bioindicator.

Introduction are related to reduction of the content of chlorophyll and

Environmental contamination has been increasing steadily
due to anthropic actions. In the agricultural area, this is due
mainly to the indiscriminate use of agrochemicals containing
heavy metals, such as zinc (Zn), iron (Fe), copper (Cu), man-
ganese (Mn), cobalt (Co) and magnesium (Mg) (Amari et al.
2017). However, at low concentrations, these metals are also
considered to be essential nutrients due to their participation
in metabolic pathways that are fundamental to plants’ devel-
opment (Emamverdian et al. 2015). Therefore, the reaction of
a plant to the accumulation of heavy metals in its tissues
depends on the bioavailability of these elements and the
plant’s efficiency to absorb, translocate and store them in its
structure (Qian et al. 2012).
Copper acts as a structural component in regulatory pro-
teins, participating in the transport of electrons during
photosynthesis, mitochondrial respiration, responses to oxi-
dative stress, cell wall metabolism and hormone signaling
(Mackie et al. 2012; Lange et al. 2017). However, both the
deficiency and excess of Cu inhibit the growth of plants and
alter important biochemical processes (Martins et al. 2016).
The limit for Cu deficiency of various plant species is nor-
mally lower than 5 mg kg
1 of dry weight of the aerial part.
Below this level, plants’ growth is reduced and other symp-
toms of deficiency can appear (Marschner 1995). On the
other hand, excess Cu can cause chlorosis and necrosis and
carotenoids (Costa et al. 2018). These conditions can indicate
damages to the chloroplasts and impairment of photochem-
ical reactions (Martins et al. 2018b).
Knowledge of the response mechanisms of plants to
excess metals allows determining their potential use as bioin-
dicators. Besides directly providing information about the
bioavailable fraction of an element in the environment, bio-
indication also indirectly gives an alert of the degradation
potential of trophic interactions of humans and ecosystems
(Bonanno et al. 2017; Farias et al. 2018).
The literature contains various reports of plant species,
including bromeliads, that can be used for environmental
biomonitoring (Giampaoli et al. 2016; Sanchez-Chadi 2016;
Piazzetta et al. 2019) due to alterations in their morphophysi-
ology in reaction to pollutants. The bromeliads can be par-
ticularly useful in this respect due to the formation of a tank
system at the base of the leaf rosette (Schreck et al. 2016),
which can accumulate a large quantity of detritus from the
surrounding environment, resulting in prolonged exposure
to metals. The tank has the basic role of storing and supply-
ing water along with organic and inorganic compounds from
the environment. In bromeliads, the uptake of the nutrients
stored in the tank occurs via trichome scales. The root sys-
tem also has the function of absorbing and translocating
nutrients to the aerial part, but on a lesser scale (Leroy et al.

CONTACT Jo~ao Paulo Rodrigues Martins jprmartinss@yahoo.com.br Plant Ecophysiology Laboratory, Federal University of Espırito Santo, S~ao Mateus,
ES, Brazil
ß 2020 Societa Botanica Italiana

Published online 13 May 2020

2 J. P. R. MARTINS ET AL.
2019). For the majority of bromeliad species growing in the
wild, the roots have the main function of attachment to the
substrate (Pedroso et al. 2010).
Some studies have demonstrated that the responses to
exposure to heavy metals involve modifications of the leaf
and/or root anatomy (Martins et al. 2016; Pereira et al. 2016;
Ribeiro et al. 2019), as well as changes in the levels of
chlorophyll (Bazihizina et al. 2015; Rodrigues et al. 2017;
Wiszniewska et al. 2017) and in the physiology due to
oxidative stress, caused mainly by the production of reactive
oxygen species (ROS), which in excess act to inhibit photo-
system II (PSII) and other metabolic pathways (Fryzova
et al. 2017).
The use of chlorophyll a fluorescence techniques can
make a significant contribution to studies of the reaction of
plants to stress. These techniques reveal quickly and pre-
cisely the level of stress of plants by determining the per-
formance of the photosynthetic apparatus, mainly of PSII
(Kalaji et al. 2016). The measurement of chlorophyll a fluores-
cence to quantify the performance of the photosynthetic
apparatus has been used in plants grown in vitro (Martins
et al. 2018a, 2018b, 2019), including under stress conditions
caused by heavy metals, to verify the potential of plant spe-
cies to act as bioindicators (Kalaji et al. 2018).
Some studies have been published involving analysis of
controlled microenvironmental conditions (in vitro) to dem-
onstrate the anatomical and physiological alterations induced
by Cu in bromeliads, such as Billbergia zebrina (Helbert)
Lindley (Martins et al. 2016) and Aechmea blanchetiana
(Baker) LB SM (Giampaoli et al. 2012). Those studies were
based on observing the effects of excess Cu added to a
semi-solid culture medium on the roots’ uptake and trans-
location of mineral elements to the aerial part. Studies have
also been conducted with a more physiological approach
under controlled microenvironmental conditions (Giampaoli
et al. 2012; Martins et al. 2016; Rodrigues et al. 2017; Pinto
et al. 2019; Silvestri et al. 2020). The manipulation of those
conditions is advantageous by allowing efficient isolation of
the real effects caused by metals in the plant.
The aim was to analyze the anatomical and physiological
responses of A. blanchetiana submitted to excess Cu in
controlled microenvironmental conditions and to verify the
degree of tolerance and bioindication potential of this species.
Material and methods
Plant material and in vitro culture conditions
Side shoots of A. blanchetiana previously established in vitro
were used as initial explants. These were transferred to
268 mL flasks containing 50 mL MS culture medium
(Murashige and Skoog 1962) with 10 lM 6-benzylaminopurine
(BAP) and 30 g L
1 sucrose (Rosa et al. 2018). After culture for
60 days, the explants that had formed multiple shoots were
subcultured in 268 mL flasks containing 50 mL MS medium
supplemented with 30 g L
1 sucrose, without the addition of
plant growth regulators. These steps were performed with five
explants per flask. The pH of all culture media was adjusted
to 5.8, followed by autoclaving for 20 minutes at a
temperature of 120  C. The inoculation procedures were car-
ried out under aseptic conditions in a laminar flow cabinet.
After inoculation, the plant material was kept in a growth
room with temperature of 26 ± 2  C and 16:8 hour active
photoperiod, provided by fluorescent lamps (EmpaluxV FT8
R
HO, 36 W/6400K), with 90 lmol m
2 s
1 of photosynthetically
active radiation (PAR).
In vitro cultivation and exposure to Cu
After subculture for 60 days, the lateral shoots with approxi-
mate length of 4 ± 1 cm obtained in the previous step were
individualized with a scalpel and transferred to 268 mL flasks
containing 25 mL MS medium supplemented with 30 g L
1
sucrose, 2 lM naphthalene acetic acid and solidified with
6 g L
1 agar (Martins et al. 2018a). Each flask received five
explants in this step. After 60 days for establishment of these
explants, the tank system was recreated with the addition of
25 mL stationary liquid MS medium modified with different
concentrations of Cu (CuSO4.5H2O) (0, 25, 50 and 100 lM
Cu), without addition of sucrose. The established explants
were cultured in biphasic medium (semi-solid þ stationary
liquid) for 90 more days. The pH of all culture media was
adjusted to 5.8, followed by autoclaving for 20 minutes
at 120  C. The inoculation procedures and environmental
conditions in the growth room were the same as previ-
ously described.
Anatomical analysis
For anatomical characterization, five plants per treatment
were randomly collected after cultivation for 90 days with dif-
ferent concentrations of Cu. The plant material was fixed in
FAA (formaldehyde, acetic acid and ethanol at 70%; 0.5/0.5/
9, v/v) for 72 h, followed by storage in 70% ethanol
(Johansen 1940). Transversal and paradermal sections were
prepared from the first and second completely expanded
leaf of the rosette central region, respectively. The sections
were clarified in 10% (v/v) sodium hypochlorite (2.5% active
chlorine) and stained with solutions of safranin and astra-
blue. The stained sections were fixed on slides with liquid
glycerin (50% v/v) and the images were observed by light
microscopy (Bioval, L-2000A-Flur) and captured with an
attached digital camera (Leica ICC50 HD, Wetzlar, Germany).
The photomicrographs were analyzed using the UTHSCSA-
ImagetoolV software calibrated with a microscope ruler. The
R
following anatomical traits were examined: stomatal density
(0.01 mm2) and stomatal index (%), density of epidermal cells
(0.01 mm2), thickness of hydrenchyma (lm) and chloren-
chyma (lm), number of xylem vessels, and diameter of xylem
vessels (lm).
Quantification of the bioaccumulated mineral nutrients
The bioaccumulated mineral nutrients were quantified in the
aerial part of A. blanchetiana plants that were previously
washed in distilled water, followed by forced-circulation dry-
ing (70  C). Then the material was ground, weighed (1 g per
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY
Table 1. Abbreviations of the parameters, formulas and description of the data derived from the transient fluorescence of chlorophyll a.
Fluorescence parameters
Extracted fluorescence parameters
F0
FM
Ft
F20 ms
F300 ls
FJ ¼ F2ms
FI ¼ F30ms
FP ¼ F 300ms  FM
Calculated parameters
Fv ¼ FM – F0
FV/F0
M0 ¼ dV/dt0 ¼ 4  (F300ls –F0)/(FM –F0)]
VJ ¼ (F2ms
F0)/(Fm
F0)
Vt ¼ (Ft
F0)/(FM
F0)
Specific energy fluxes
ABS/RC ¼ M0  (1/VJ) . (1/uP0)
TR0/RC ¼ M0  (1/VJ)
ET0/RC ¼ M0  (1/VJ)  w0
DI0/RC ¼ (ABS/RC) – (TR0/RC)
Quantum yields and probabilities
uP0 ¼ TR0/ABS ¼ [1 – (F0/FM] ¼ FV/FM
uE0 ¼ ET0/ABS ¼ [1 – (F0/FM)]  w0 ¼ uP0  w1
uD0 ¼ 1 – uP0 ¼ (F0/FM)
WE0 ¼ ET0/TR0 ¼ (1 – VJ)
RC/ABS ¼ 1 – (F0/FM) / (VJ /M0)
Performance indice
PIABS ¼ RC/ABS  uP0/(1
uP0)  wE0/(1
wE0)
For review see Strasser et al. (2004) and Goltsev et al. (2016).
sample) and subdivided into three independent samples per
treatment. The concentrations of N, P, K, Mg, S, Cu, Mn and
Fe were determined according to the method described by
Malavolta et al. (1997).
Analysis of the photosynthetic pigments
The photosynthetic pigments were quantified in seven plants
for each treatment, collected at random. The pigments were
extracted from 0.04 g of fresh matter obtained from the third
completely expanded leaf from the central rosette. The
material was placed in test tubes containing 5 mL 80% (v/v)
acetone and maintained for 72 hours in the dark at a con-
stant temperature of 4  C. Then the absorbances were read
with a spectrophotometer (GenesysTM 10S UV-Vis/Thermo
Fisher Scientific) at k ¼ 470, 645 and 665 nm for carotenoids
(Car), chlorophyll b (Chl b) and chlorophyll a (Chl a) respect-
ively. The readings were used to calculate the contents as
proposed by Arnon (1949) and Wellburn (1994). The contents
were expressed as micrograms of pigment per gram of fresh
weight (lg g
1 FW).
Analysis of chlorophyll a fluorescence
The readings of chlorophyll a fluorescence of each treatment
were performed with a portable fluorimeter (Handy PEA,
Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK). The
measurements were performed on 14 plants per treatment,
using the third fully expanded leaf from the central rosette.
The leaves were adapted to the dark for 30 minutes with the
help of leaf clips (Hansatech), after which a light ray with
intensity of 3000 lmol (photons) m
2 s
1 was emitted. The
3
Description
Initial fluorescence.
Maximum fluorescence.
Fluorescence at time t after start of actinic illumination.
Minimum fluorescence signal measured at 20 ms (corresponds to F0).
Fluorescence intensity at 300 ms.
Fluorescence intensity at 2 ms.
Fluorescence intensity at 30 ms.
Fluorescence intensity at 300 ms.
Variable fluorescence
Ratio of photochemical to nonphotochemical quantum efficiencies (PSII potential activity)
Net rate of photosystem II closure.
Relative variable fluorescence at 2 ms (point J).
Relative variable fluorescence at time t.
Absorption flux per reaction center (RC) at t ¼ 0.
Trapping flux (leading to QA reduction) per RC at t ¼ 0.
Electron transport flux (further than QA-) per RC at t ¼ 0.
Dissipated energy flux per RC at t ¼ 0.
Maximum quantum yield of primary photochemistry at (t ¼ 0).
Quantum yield of electron transport (at t ¼ 0)
Quantum yield of energy dissipation (at t ¼ 0)
Probability (at t ¼ 0) that a trapped exciton moves an electron into
the electron transport chain beyond QA-
Total number of active reaction center per absorption.
Performance index based on absorption.
return emission of transient fluorescence was recorded and
the parameters of the JIP test (Table 1) were analyzed
(Strasser et al. 2004; Stirbet and Govindjee 2011).
Growth characteristics
To evaluate the growth of the plants cultivated in vitro, 30
plants from each treatment were collected at random, div-
ided into six portions and weighed on a semi-analytic preci-
sion balance. The fresh weight (FW) was determined in
grams (g) of the roots and aerial part separately.
Statistical analysis
The experimental design was completely randomized and
the data were submitted to analysis of variance (ANOVA) and
the means were compared by the Tukey test at 5% probabil-
ity. Besides, to verify significant adjustments to trend models,
the data were subjected to regression analysis.
Results
Anatomical analysis
The culture conditions with excess Cu influenced the forma-
tion of stomata. The A. blanchetiana plants had greater sto-
matal density and stomatal index in function of rising
concentrations of Cu until concentrations greater than
50 lM, after which there was a reduction of the values of
stomatal density (R2 ¼ 0.96) and index (R2 ¼ 0.97), in detri-
ment of a decrease of the number of stomata per area. The
4 J. P. R. MARTINS ET AL.

Figure 1. Paradermal sections (a–d) and cross-sections (e–l) of leaves of Aechmea blanchetiana plants as a function of concentration (0, 25, 50 and 100 lM) of Cu
during in vitro culture. Ad: adaxial epidermis; ab: abaxial epidermis; chl: chlorenchyma; fb: fibers; hy: hydrenchyma; ph: phloem; xl: xylem; vb: vascular bundles.
Bars ¼ 100 lm.

density of epidermal cells was similar in all the treatments
(Figures 1 and 2).
The hydrenchyma thickness also was similar among the
treatments. However, the thickness of the chlorenchyma was
greater in the plants cultured with 50 and 100 lM Cu. With
respect to the characteristics of the xylem vessels, there was
an increase in diameter in the plants grown with 50 and
100 lM Cu, but the number was not affected by the treat-
ments (Figures 1 and 3).
Quantification of bioaccumulated mineral nutrients
The content of bioaccumulated nutrients in the aerial part of
the plants was influenced by the treatments. In general, the
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 5

plants grown with 100 lM Cu presented the highest values
for the contents of macro and micronutrients. The concentra-
tion of Cu increased linearly (R2 ¼ 0.97) in function of the
concentrations of Cu supplied in the culture medium
(Table 2).
Analysis of photosynthetic pigments
The levels of total chlorophyll (Chl total) and chlorophyll a
(Chl a), as well as carotenoids (Car) were highest in the
plants cultured with 100 lM Cu. On the other hand, when
the plants did not receive Cu, the contents of these pig-
ments were 30% lower on average than in the plants grown
with 100 lM. The contents of Chl b were statistically similar,
irrespective of the concentration of Cu. Likewise, the Chl a/b
ratio did no change in function of the treatments (Figure 4).

Analysis of chlorophyll a fluorescence

Figure 2. Density of stomata and epidermal cells (0.01 mm2) and stomatal
index (%) of Aechmea blanchetiana grown in vitro in function of concentrations
of Cu (lM). For each anatomical characteristic, means (±SE) followed by the
same letter do not differ significantly by the Tukey test at 5% probability.
Among the technical parameters obtained from the JIP test,
the initial (F0), maximum (FM) and variable (FV) fluorescence
did not differ among the treatments, with average values of
462.94, 1769.10 and 1306.41, respectively. Likewise, the FV/F0
ratio was similar among the treatments. However, the net
closing rate of the PSII (M0) and variable fluorescence related
to point J (VJ) were significantly lower in the plants grown
with 50 and 100 lM Cu (Figure 5).
The parameters related to the specific energy fluxes per
reaction center (RC) were similar regarding absorption (ABS/
RC), trapping (TR0/RC) and dissipation (DI0/RC). Only the spe-
cific flux of electron transport per RC (ET0/RC) showed an
increase, in the plants cultured with 50 and 100 lM Cu
(Figure 5). Similarly, the other parameters that describe the
transport quantum efficiency (uE0) and probability of a
trapped exciton moving an electron to the electron transport
chain (WE0) were also higher in the plants supplied with 50
and 100 lM Cu. The other quantum efficiency parameters
(uP0 and uD0) and total number of active reaction centers
per absorption center (RC/ABS) were similar to each other
(Figure 5). The performance index (PI(ABS)) was statistically
similar in all the treatments, with average value of 1.06.

Figure 3. Anatomical structures of Aechmea blanchetiana plants grown in vitro
in function of concentrations of Cu (lM). For each anatomical characteristic,
means (±SE) followed by the same letter do not differ significantly by the
Tukey test at 5% probability.
Growth characteristics
After exposure to excess Cu for 90 days the plants did not
present visual symptoms that could indicate toxicity, such as
leaf chlorosis or necrosis. Besides this, no mortality of the
plants occurred. In general, the plants cultured with high Cu
concentrations were more compact, but the fresh weights of
the aerial part and roots were similar in all the treatments
(Figure 6).

Table 2. Contents of macro and micronutrients bioaccumulated in the aerial part of Aechmea blanchetiana plants in function of concentrations of Cu (0, 25, 50
and 100 lM) during in vitro culture.
Contents of macro and micronutrients
Cu (lM) N (%) P (%) K (%) Mg (%) S (%) Cu (ppm) Mn (ppm) Fe (ppm)
0 3.11 ± 0.08ab 0.24 ± 0.00b 4.57 ± 0.22a 0.27 ± 0.00bc 0.18 ± 0.06b 6.37 ± 2.22c 283.6 ± 3.28a 277.4 ± 11.32b
25 2.67 ± 0.21b 0.20 ± 0.01b 3.10 ± 0.21b 0.23 ± 0.01c 0.30 ± 0.03ab 17.44 ± 2.39c 210.9 ± 14.73b 235,7 ± 14.10c
50 3.45 ± 0.03a 0.29 ± 0.00a 3,33 ± 0.50ab 0.31 ± 0.01ab 0.39 ± 0.01a 44.87 ± 1.75b 298.6 ± 1.32a 332.6 ± 5.84a
100 3.37 ± 0.05a 0.30 ± 0.00a 4.54 ± 0.97a 0.33 ± 0.01a 0.36 ± 0.02a 70.70 ± 4.29a 300.6 ± 3.36a 316.9 ± 10.59ab
Means (±SE) followed by the same letter in the column do not differ significantly by the Tukey test at 5% probability. N ¼ nitrogen; P ¼ phosphorus,
K ¼ potassium; Mg ¼ magnesium; S ¼ sulfur; Cu ¼ copper; Mn ¼ manganese; Fe ¼ iron.
6 J. P. R. MARTINS ET AL.
Figure 4. Contents of photosynthetic pigments of Aechmea blanchetiana plants
grown in vitro in function of concentrations of Cu (lM). For each photosyn-
thetic pigment, means (±SE) followed by the same letter do not differ signifi-
cantly by the Tukey test at 5% probability.
Figure 5. Parameters of the JIP test of Aechmea blanchetiana plants grown
in vitro in function of concentrations of Cu (lM). For each parameter, means fol-
lowed by an asterisk () differ significantly by the Tukey test at 5% probability.
The means of each parameter were normalized in relation to the control (0 lM
of Cu ¼ 1). ns ¼ no significant.
Discussion
This study involved examination of the physiological and
anatomical mechanisms of A. blanchetiana grown under con-
trolled microenvironmental conditions with excess Cu. The
plants presented morphophysiological alterations that
allowed their growth even under conditions that could
induce symptoms of toxicity, since the exposure to excess
Cu was directly on the aerial part, i.e., on the leaves. This
organ is very important for absorption of water and nutrients
in the majority of bromeliad species, besides photosynthesis.
The absorption by leaves of bromeliads occurs mainly by
means of trichome scales located on the leaf surface. But it
should be borne in mind that the root system of bromeliads
also plays a role in the uptake of nutrients (Vanhoutte et al.
Figure 6. Fresh weight of the aerial parts and roots of Aechmea blanchetiana
plants grown in vitro in function of concentrations of Cu (lM). Means (±SE) fol-
lowed by the same letter do not differ significantly by the Tukey test at 5%
probability.
2017; Leroy et al. 2019). Nevertheless, unlike the leaves, the
roots have mechanisms that can diminish the uptake of
excessive heavy metals through modifications of the tissues,
such as formation of thicker cell walls of the exodermis, act-
ing as an apoplastic barrier against the free translocation of
mineral elements (Martins et al. 2016).
The leaf anatomy of A. blanchetiana is similar to that of
other bromeliads (Hermes et al. 2018; Silva et al. 2018) and
has already been described for this species under in vitro
conditions (Martins et al. 2018a, 2018b, 2019). Alteration of
the number of stomata per area is considered to be a com-
mon response of plants under stressful conditions, such as
excess salt, lack of water or exposure to heavy metals (Vile
et al. 2012; Ali et al. 2017; Andrade Ju nior et al. 2019).
However, the tendency for increase or decrease of the sto-
matal density can vary among species and this response
appears to be dose-dependent (Martins et al. 2016; Andrade
nior et al. 2019). The large decrease in the number of sto-
Ju
mata due to exposure to high concentrations of heavy met-
als can be related to a mechanism that regulates
transpiration, which characterizes a tolerance strategy, by
reducing the uptake of these metals and maintaining water
in the tissues (Andrade Ju nior et al. 2019). This affects the
stomatal conductance, since the stomatal density is also
related to adjustment of the influx of carbon (Tanaka et al.
2013). According to Boer et al. (2016), the investment of leaf
epidermal space to stomata is closely related to the balance
between the plant’s needs for carbon and the efficient use
of water. Under the culture conditions in our study, the A.
blanchetiana plants presented increased stomatal density
due to greater investment in these structures in relation to
the total number of epidermal cells, which was also
expressed as a higher stomatal index. This greater stomatal
density can act positively to increase the stomatal conduct-
ance, because a greater investment in stomata per area can
positively influence the uptake and bioaccumulation of
nutrients by means of mass flow (Hepworth et al. 2015).
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY
However, it was also possible to note anatomical mecha-
nisms that can regulate the mass flow and uptake of exces-
sive metals, mainly when the plants were grown with
100 lM Cu, due to the pronounced decrease in the stomatal
density and index values.
The increase in the number and/or diameter of the xylem
vessels in the leaves can also have a significant effect on the
uptake, translocation and accumulation of nutrients in the
plant tissues and organs (Martins et al. 2019). These charac-
teristics of the xylem vessels are key factors that influence
the water conductance. That statement is based on the
Hagen-Poiseuille law, according to which even small changes
in the diameter of the xylem vessels have exponential effects
on the specific water conductance (Scholz et al. 2013).
Therefore, greater water conductance can increase the
absorption of nutrients, mainly through the roots, and thus
distribute the minerals from the culture medium to the
plants’ aerial parts. This finding is in line with our results,
because the A. blanchetiana plants with larger xylem vessel
diameter also presented greater accumulation of nutrients in
the aerial part.
Alterations in the chlorenchyma thickness in reaction to
exposure to excess metals, mainly an increase, have been
reported in other plant species (Pereira et al. 2016; Ribeiro
et al. 2019). This appears to be related to a trade-off mech-
anism, in which the loss of leaf area (more compact leaves)
is offset by increased thickness of the chlorenchyma (Martins
et al. 2016). Therefore, the photosynthetic efficiency can be
maintained or even increased in detriment to larger thick-
ness of this tissue, as verified by Pereira et al. (2016).
According to Terashima et al. (2006), the quantity of chloren-
chyma thickness in the leaves can be related to the photo-
synthetic capacity of plants. These adjustments of the leaf
anatomy appear to create favorable conditions for mainten-
ance of the photosynthetic tissues (Ribeiro et al. 2019).
The adjustments of the contents of nutrients in the plants
were essential to allow tolerance to the excess Cu. The
higher concentration of Cu in the tissues of A. blanchetiana
might have induced an imbalance in the content of
nutrients, among other physiological disturbances, mainly
regarding the formation of reactive oxygen species (ROS)
(Adrees et al. 2015). The interaction of the mineral elements
absorbed and translocated is important for the adjustment
to the deficiency and toxicity of these elements in plants
(Hermans et al. 2011; Rahman et al. 2016a, 2016b). The con-
tents of N, Mg and Mn in plants have a direct impact on
their growth, since these are structural components of
chlorophyll molecules, among others. However, these ele-
ments also act in other plant metabolism processes, such as
in the antioxidant system.
The increase in the concentration of N can be related to
the formation of amino acids that are necessary in the
enzymatic antioxidant processes [e.g., superoxide-dismutase
(SOD) and catalase (CAT)], or the non-enzymatic processes
for protection against excess Cu. Responses of the antioxi-
dant system of A. blanchetiana plants grown in vitro with
excess Cu were previously described by Giampaoli et al.
(2012). An increase of amino acids, such as free proline, can
7
contribute to these responses to exposure to heavy metals
(Annunziata et al. 2017), among them Cu (Vijayarengan and
Maheswari 2018). Proline plays an important role in the
adjustment to osmotic stresses and is related to changes in
the water status of plants under stress from heavy metals.
This alleviation of water stress induced by metals, by raising
the concentration of proline, is fundamental for tolerance to
heavy metals (Annunziata et al. 2017). Moreover, proline also
has been reported to be involved in the elimination of free
radicals (Signorelli et al. 2015).
The content of K in the leaves is also significantly corre-
lated with higher content of proline (Upadhyaya and Panda
2013). It has been suggested that an improved K status of
plants can greatly reduce the production of ROS, conferring
benefits when plants are subjected to abiotic stress (Cakmak
2005; Ashley et al. 2006). This osmotic adjustment promoted
by K is an essential characteristic in the acclimatization of
plants in function of mineral elements (Martins et al. 2019).
Mn plays a vital role as a cofactor and contributes to the
antioxidant defense (Mn-SOD and Mn-CAT), besides contribu-
ting to the homeostasis of ions (Rahman et al. 2016a). In
turn, Mg can help relieve the intoxication caused by excess
heavy metals, such as Cu, by diminishing the oxidative stress,
inducing a substantial decline in the quantity of hydrogen
peroxide H2O2) and malondialdehyde (MDA) in plant tissues
(Singh et al. 2016). Besides this, the endogenous contents of
Mn and Mg are mutually regulated, and an imbalance
between these two minerals can induce oxidative damages
(Farzadfar et al. 2016). That response was observed in A.
blanchetiana, because the increase of these elements
occurred concomitantly in function of the treatments.
Cu can also influence the homeostasis of Fe (Bernal et al.
2012). According to Kochoni and Fortin (2019), Fe acts to
protect the plant against excess uptake and toxicity of Cu.
The interaction between Fe and Cu in plants can be directly
associated with the chloroplasts. In the chloroplasts, Fe and
Cu are part of the structural conformation of the SODs that
capture ROS. Both the SODs containing Fe (Fe-SODs) and Cu
(Cu-SODs) function similarly (Pilon et al. 2011). This physio-
logical role of alternation between Fe-SODs and Cu-SODs is
important regarding the capacity to neutralize oxidative
stress (Waters and Armbrust 2013). Besides this, Fe also is a
cofactor of peroxidase (POX) and CAT (Feng et al. 2013).
The availability of P in plant tissues can be useful as a
strategy to deal with exposure to heavy metals (Lo pez-Bucio
et al. 2014). A higher P content can alleviate the stress
induced by heavy metals and at the same time permit their
accumulation in the tissues (Bargaz et al. 2013). Low avail-
ability of P can result in imbalance of the antioxidant
defense system with alterations of the components of the
mitochondrial membrane and restriction to the transport of
electrons through the cytochrome respiratory chain (Juszczuk
et al. 2001). On the other hand, S can control the accumula-
tion of Cu in the tissues. The addition of S favors the content
of MDA in the leaves, indicating alleviation of oxidative
stress. This can lead to reduction of the activities of SOD and
CAT, as well as the content of glutathione (GSH), alleviating
oxidative stress (Ren et al. 2017).
8 J. P. R. MARTINS ET AL.
The nutritional balance contributed positively to the bio-
synthesis of the photosynthetic pigments, favoring an
increase of Chl a and Car in the A. blanchetiana plants due
to the treatments. The greater availability of Mg, Mn and N
in the leaf tissues can promote an increase in the photosyn-
thetic pigments of bromeliads, even under in vitro conditions
(Martins et al. 2019). Plants under stress caused by Cu, as
well as other heavy metals, can have low pigment content
due to the action of the oxidative stress induced by ROS
(Bazihizina et al. 2015; Rodrigues et al. 2017). This reduction
of the content of pigments indicates disturbances in the
photosynthetic apparatus of plants. The carotenoids are part
of the essential structures of the photosynthetic apparatus
(Ustin et al. 2009; Yaroshevich et al. 2015). Besides their dir-
ect role in photosynthesis, Car are involved in mechanisms
for defense against oxidative stress (Gill and Tuteja 2010).
They are able to directly capture ROS, protecting cells
against membrane degradation and destruction of Chl medi-
ated by ROS (Mostofa et al. 2017). In the A. blanchetiana
plants we did not observe a decrease in the Car content in
function of the Cu treatments, indicating a very low level of
stress or even its absence. The morphophysiological adjust-
ments were effective against the deleterious effects of excess
Cu, as confirmed by the Chl a/b ratio, since alterations of this
ratio can reflect stress (Martins et al. 2018b). An increase in
the Chl a/b ratio indicates physiological disturbances,
because this can be a response to conversion of Chl b into
Chl a (Zrig et al. 2015).
The clearest signs that the morphophysiological altera-
tions mentioned were effective in adjustment to the excess
Cu were confirmed by the parameters of the JIP test. Among
the parameters that normally decline in response to stress
caused by heavy metals is FV/FM (uP0) (Dai et al. 2016;
Huang et al. 2019). Normally, this response induces increases
in the energy dissipation (DI0/RC and uD0). In our study, due
to the stability and functionality of the photosynthetic appar-
atus, the plants presented values of uP0 greater than 0.74
and of uD0 lower than 0.27. Plants considered free of stress
typically have values of uP0 between 0.75 and 0.85 (Bolhar-
Nordenkampf et al. 1989).
In A. blanchetiana plants grown under in vitro with excess
Cu, the transfer of electrons in PSII as well as quinone A (QA)
was improved, and this physiological response resulted in a
small decrease of VJ According to Martins et al. (2019), this is
interpreted as slightly higher electron transport rates
between QA and quinone B (QB). Therefore, point J indicated
that the excess of Cu positively affected the flow of electrons
from QA to QB or QB-, without accumulation of QA-. Further
regarding the flow of electrons between QA and QB, the
decrease of M0 indicated that the excess of Cu did not
induce any inhibition on the receptor side. A significant
increase of M0 could suggest a reduction of the number of
active RCs, and hence an increase in the number of closed
reaction centers (Einali and Shariati 2015). In our study, alter-
ations of the number of RCs for absorption (RC/ABS) were
not observed, indicating the absence of stress in the range
of concentrations tested.
We observed small increments on the average values of
ET0/RC, uE0 and WE0. This response might be involved in the
role of Cu, because it is an essential element in the transport
of electrons due to its participation in the electron transport
chain (Droppa et al. 1984). However, these increments did
not lead to an increase of the performance index (PI(ABS)) of
PSII, since other parameters influence the values of PI(ABS),
such as uP0 and RC/ABS. This is in line with the growth of
the plants, because the accumulation of fresh weight in the
aerial part and roots was similar. The results clearly showed
that the A. blanchetiana plants were able to adjust to the
excess Cu conditions without suffering deleterious physio-
logical effects, proving their tolerance to the metal.
Conclusion
The microenvironmental conditions with excess copper
induced alterations in the anatomy and physiology of the A.
blanchetiana plants. The anatomical responses permitted the
plants to tolerate the conditions imposed. However, the
adjustments and interactions among the mineral elements
absorbed and translocated were important to the adjust-
ments to excess Cu. Those adjustments were efficient to
enable tolerance of excess Cu and to maintain the stability
and functioning of the photosynthetic apparatus. The species
A. blanchetiana tolerates high concentrations of Cu and has
potential for use as a bioindicator.
Acknowledgments
The authors would like to acknowledge the scholarship awarded by the
CNPq (Brazilian National Council for Scientific and Technological
Development), the CAPES (Coordination for the Improvement of Higher
Education Personnel), and the FAPES (Espırito Santo State Research
Foundation). The authors are also grateful to Elizangela Rodrigues
Santos for her technical assistance.
Disclosure statement
The authors declare no conflict of interest.
ORCID
Jo~ao Paulo Rodrigues Martins http://orcid.org/0000-0003-0554-6793
Luiz Carlos de Almeida Rodrigues http://orcid.org/0000-0001-
6336-0247
Franciele Pereira Rossini http://orcid.org/0000-0003-1235-831X
Andreia Barcelos Passos Lima Gontijo http://orcid.org/0000-0003-
3422-4398
Antelmo Ralph Falqueto http://orcid.org/0000-0002-0488-7441
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