J. Crop Sci. Biotech.

2019 (September) 22 (3) : 253 ~ 264 253

DOI No. 10.1007/s12892-019-0097-0

RESEARCH ARTICLE

Impact of Zinc Stress on Biochemical and Biophysical

Parameters in Coffea Arabica Seedlings
Jacqueline Oliveira dos Santos1*, Cinthia Aparecida Andrade1, Kamila Rezende Dázio de Souza1, Meline de Oliveira Santos2,
Isabel Rodrigues Brandão1, Jose Donizeti Alves1, Iasminy Silva Santos1

1
Setor de Fisiologia Vegetal, Departamento de Biologia, Universidade Federal de Lavras, Lavras, Minas Gerais, Brasil
2
Empresa de Pesquisa Agropecuária de Minas Gerais/ EPAMIG SUL, Campus UFLA, Lavras, Minas Gerais, Brasil

Received: March 24, 2019 / Revised: April 30, 2019 / Accepted: May 08, 2019
Ⓒ Korean Society of Crop Science and Springer 2019

Abstract
Zinc is an essential micronutrient for the healthy development of plants, since its insufficient and supraoptimal doses can
disrupt the metabolism and biomass production. We aimed to investigate the physiological responses of coffee seedlings to
Zn deficiency and excess. Six-month-old seedlings were transferred to plastic pots containing a nutrient solution. The
treatments were control (0.03 ppm), zinc deficiency (0.00 ppm), and zinc excess (0.12 ppm). The evaluations were performed
in leaves and roots at the beginning of the treatments and after 30 and 60 d of treatments. Zn deficiency and excess increased
the production of hydrogen peroxide, antioxidant enzymes activity, ascorbate, and lipid peroxidation contents. The imbalance
in zinc nutrition reduced total chlorophyll content and increased carotenoids content throughout the experimental period.
Lower biomass and proline accumulation were observed only for deficient seedlings at the end of the experiment. The
characteristics analyzed showed that zinc deficiency caused greater damage to the Coffea arabica plants of (Catuaí cultivar)
than zinc excess.

Key words : Plant nutrition, coffee, antioxidant metabolism, proline, plant growth

Introduction
In Brazil, the predominant coffee farm species is Coffea
arabica, due to its broad acceptance in the world markets,
and Catuaí is the most widely cropped variety covering 65%
of the cultivated area (Fernandes et al. 2012), with high grain
yield and plant vigor as well as excellent drink quality (Malta
et al. 2008). The importance of studies that assess the
tolerance of coffee cultivars under conditions of low avail-
ability or excess in the soil, addressing the physiological
characteristics is well known (Pedrosa et al. 2014; Zabini et
al. 2007). The elucidation of differences in nutritional require-
ments of cultivars, allows cultivation areas to be increased,
allocating less demanding zinc progenies on soils that are
poorer in Zn or vice versa, maintaining the productive
potential and reducing expenses with agricultural inputs and
fertilizers (Pedrosa et al. 2014).
The need for an adequate zinc supply for growth and grain
Jacqueline Oliveira dos Santos ()
Email: jack_oliver3@hotmail.com
production of coffee is widely known, especially for plants
growing in clayey soils and those poor in zinc which are
common in Brazil (Baker and Pilbeam 2015; Martinez et al.
2013), or in areas where zinc toxicity is related to soil nutrient
uptake (Lopes et al. 2014). As zinc has practically no mobility
into the deeper layers in clayey soils, zinc toxicity is common
in the replacement of old coffee plantations, which have
received zinc applications for many years (Paiva et al. 2012).
In this scenario, zinc accumulation occurs on the soil surface,
but when using plowing techniques, zinc in depth is absorbed
by the roots, in which it accumulates, and can reach toxic levels.
Zinc excess and deficiency are separated by a narrow
concentration range since it is a micronutrient. Furthermore,
mistakes can frequently be made when visually trying to
detect zinc deficiency and toxicity in plants, since the
symptoms are the same (Faquim 2002). Therefore, it is
common to add more Zn to the soil, believing it is deficient,
when actually the soil is already saturated with this element
at toxic levels. Despite being a micronutrient, zinc can affect

The Korean Society of Crop Science

254 Zinc Stress in Coffea Arabica Seedlings
growth and normal metabolism of plant species when found
in toxic levels in the environment (Vangrosveld et al. 2009).
Zn stress can inhibit photosynthetic activity through
different mechanisms. Zn has been shown to have a specific
impact on photosystem activities (Paunov et al. 2018) and
Calvin cycle (Vassilev et al. 2011) and zinc deficiency and
toxicity in plants enhance the production of reactive oxygen
species (ROS) causing oxidative damage to plants. This is
done by generating active biological forms of oxygen such
as superoxide (O2•-) and hydroxyl (•OH) that together with
the singlet oxygen (O21) (Cakmak 2000; Islam et al. 2014)
are extremely reactive and cytotoxic to all organisms. They
inactivate enzymes and cause cell damage (Karuppanapandian
et al. 2011) which directly affect plant growth and pro-
ductivity. The main cause of this damage is lipid peroxidation
of the mitochondrial endomembrane system, endoplasmic
reticulum, lysosomes, and peroxisomes among others (Noctor
et al. 2018).
Regarding the Catuaí cultivar there are no studies which
approach the effect of Zn deficiency and toxicity on the
physiological characteristics in coffee plants, and especially
on the antioxidant metabolism. thus, the present study was
undertaken to investigate the responses of the cultivar Catuaí
144 to the nutritional efficiency of zinc, in the seedling phase
through evaluating the effect of zinc supply on biophysical
and biochemical characteristics under conditions of disability
and excess zinc.
Materials and Methods
Plant culture and Zn treatments
The experiment was conducted in a greenhouse at the
Federal University of Lavras-UFLA, Lavras-MG, Brazil.
Coffea arabica L. seedlings from cultivar Catuaí 144 were
cultivated for six months in of 500 mL polypropylene bags
filled with subsoil and cattle manure in a 2:1 proportion, plus
potassium chloride and superphosphate in the proportion of
1:10 (Guimarães et al. 2002). After selection for uniformity
in size and vigor, seedlings were transferred to 10 L plastic
containers (33 x 31 x 38 cm - WxHxD) containing nutrient
solution (Hoagland and Arnon 1950).
The plants were maintained in a greenhouse with a cooling
wall by water, with automatically controlled temperature
(25°C ± 2). Plants were acclimated for 21 d when solutions
with increasing concentrations were used: ¼ of total con-
centration for 7 d, ½ of total concentration for 7 d, and full
concentration for 7 d.
After acclimation, seedlings were submitted to three
treatments consisting of applying increasing doses of Zn, as
follows: zinc deficiency treatment (0.0 ppm/ 0 µmol L-1),
plants grown without Zn addition; For control treatment
(0.03 ppm) the original concentration of Zn and all nutrients
were used. For Zn excess treatment (0.12 ppm) Zn solutions
for each treatment were prepared using Hoagland’s solution
with zinc sulfate (ZnSO4.7H2O).
Zn concentration in the Zn excess treatment was determined
based on preliminary tests. In those tests, different concen-
trations of zinc were used to determine the concentration of
excess zinc that caused visual symptoms in the seedlings.
Nutrient solutions were exchanged weekly and maintained
under constant aeration throughout the experimental period.
The pH of the solution was adjusted daily to 5.5 ± 0.5 with
NaOH and/or HCl solution (1 M), and the volume of the
solution was filled with distilled water whenever necessary.
Evaluations were performed on leaves and roots at the
beginning of the experiment and after 30 and 60 d of
treatment. The experimental design was completely randomized
using a 3x3 factorial scheme: three treatments (control, Zn
deficiency, and Zn excess) and three evaluation times (0, 30,
and 60 d), totaling nine treatments with five replications.
Each experimental plot consisted of five seedlings. The
harvesting periods were determined according to visual
analysis of symptoms of zinc deficiency and excess. In each
harvesting time, roots and shoots were collected for bio-
chemical analysis and for dry mass quantification.
At 0, 30, and 60 d after the imposition of treatments, fully
expanded leaves of the third and fourth pairs and the root
system of five plants were collected. For biochemical
analyses, plant material was harvested, divided into leaves
and roots, placed in liquid nitrogen and then stored in an
ultra-freezer at -80°C until the subsequent analysis. For
determination of shoot and root dry weight the seedlings
were collected and divided into roots and shoots. The plant
material was then dried at 70°C in a forced-air circulation
oven until constant weight and the dry weight measured.
Statistical analysis
The statistical analyses were performed using the statistical
program SISVAR 4.3 (System Analysis of Variance for
Balanced Data) (Ferreira 2011). The data presented were
means of five replicates. Differences between Zn treatments
were quantified using analysis of variance (ANOVA), and
all the data was presented as mean ± standard error (SE).
Data was first tested for normality with the Shapiro-Wilk test
and for homogeneity of variance with the Welch and Brown-
Forsythe test. Significant test results were followed by
application of the Scott and Knott test (1974) test at P ≤ 0.05.
Graphic work was carried out using Sigma Plot 8.0.
Determination of Zn content
Leaf and root zinc contents were determined according to
Malavolta (1997) 500 mg of leaf dry weight were ground
and placed in digestion tubes, to which were added 6 mL of a
mix of HNO3 and HClO4 2:1 (v/v). The digestion tubes were
then placed in a digestion block and temperature was increased
gradually until 160°C and kept until volume of the solution
was reduced to half. Temperature was then increased to 210°C
and kept until white HClO4 fumes were obtained and the
extract became colorless. After cooling, the final volume
was made up to 50 mL through the addition of deionized
water. The concentrations of the samples were determined

using the concentrations and readings of standard solutions
as reference. The zinc content in the extracts was determined
by standard curves through atomic absorption spectrometry.
Gas-exchange parameters
Gas exchange parameters were measured in the morning
(9:00-11:00 a.m.), using five replicates for each treatment in
completely expanded leaves. Two leaves per plant were
analyzed, with five biological replicates. Net photosynthesis
(Anet), stomatal conductance (gs), Transpiration (E), and
intercellular CO2 concentration were measured using an
open infrared gas analyzer (IRGA; LI-COR 6400 system,
LI-COR Inch, Lincoln, NE, USA). The IRGA chamber was
irradiated with a photosynthetic photon flux density (PPFD)
of 1000 μmol m− 2 s− 1, which gas exchange parameters were
measured under a PPFD of approximately 1000 μmol m− 2 s− 1
(equivalent to natural light).
Photosynthetic pigments content
Extraction and quantification of leaf pigments were carried
out under green safe light according to the methodology
proposed by Lichtenthaler and Buschmann (2001). We used
0.1 g of fresh weight of the first pair leaves from each
treatment, grinding them with 80% (v/v) acetone. The final
volume of the extract was completed to 10 mL using 80%
acetone. The resulting extracts were used to read the absorbance
at wavelengths of 645, 663, and 445 nm corresponding to
concentrations of chlorophylls a and b and total carotenoids,
respectively. The extracts were analyzed using a UV-visible
spectrophotometer (UV-1000).
Proline and ascorbat content determination
Proline analysis was performed according to Torello and
Rice (1986) with some modifications. For this, 0.5 g of leaves
and roots were immediately homogenized in 10 mL of 3%
sulfosalicylic acid solution, and the homogenate filtered.
Afterwards, the homogenate was centrifuged at 6250 g for
20 min. In test tubes containing 2 mL of the supernatant, 2
mL of acidic ninhydrin and 2 mL of glacial acetic acid were
added. The samples were boiled at 95°C for 1 h. After
cooling by immersion in an ice bath, the color intensity was
measured at 520 nm. The absorbances obtained were com-
pared with the standard curve of proline.
Ascorbate concentration was determined as described by
Arakawa et al. (1981). Fifty milligrams of tissue (fresh weight)
was macerated in liquid nitrogen, added to PVPP and
homogenized in 1500 μL of 5% trichloroacetic acid (TCA)
(m/v). The homogenate was then centrifuged at 13000 g for
15 min at 4°C. Aliquots of the supernatant were added to the
reaction media composed of 5% TCA (m/v), 99.8% ethanol
(v/v), 0.4% phosphoric acid (H3PO4) in ethanol (v/v), 0.5%
bathophenanthroline in ethanol (m/v), and 0.03% ferric chloride
(FeCl3) in ethanol (m/v). The mix was homogenized thor-
oughly and incubated in a water bath at 30°C for 90 min.
Readings were performed at 534 nm. Ascorbate content was
determined based on a standard curve of ascorbate.
JCSB 2019 (September) 22 (3) : 253 ~ 264 255
Antioxidant enzymes activity
The extract for the determination of activity of SOD, CAT,
and APX was obtained according to Biemelt et al. (1998).
Tissue amounts of 0.2 g (fresh weight) were macerated in
liquid nitrogen and PVPP and then homogenized in 1.5 mL
of extraction buffer containing 400 mM potassium phosphate
buffer (pH 7.8), 10 mM EDTA, 200 mM ascorbic acid and
water. The extract was centrifuged at 13000 g for 10 min at
4°C, and the supernatant was collected and stored at -20°C
during the analysis period.
SOD activity was measured according to Giannopolitis
and Ries (1977). Aliquots (15 μL) of enzymatic extract were
added to incubation media containing 100 mM potassium
phosphate (pH 7.8), 70 mM methionine, 0.1 µM EDTA, water,
1mM nitroblue tetrazolium (NBT), and 2 mM riboflavin.
Tubes containing the reaction medium and 10 μL of the
sample were illuminated for 7 min with a 20 W fluorescent
lamp. The same reaction medium without a sample was
illuminated as a control. Readings were taken at 560 nm and
calculation of enzyme was done with the following equation:
% inhibition = (A560 sample with enzyme extract – A 560
control without enzyme extract)/(A 560 control without
enzyme). One unit of SOD is able to inhibit 50% of NBT
photoreduction under the assay conditions.
CAT activity was evaluated according Havir and McHale
(1987): aliquots of enzymatic extract were added to the
incubation medium containing 200 mM potassium phosphate
buffer (pH 7.0), 0.1 mM disodium ethylenediamine tetraacetic
acid (Na2EDTA), water, and 12.5 mM hydrogen peroxide,
previously incubated in a water bath at 28°C. Enzyme
activity was determined by the decrease in absorbance at 240
nm every 15 s for 3 min, monitored by the consumption of
hydrogen peroxide. The molar extinction coefficient used
was 36 mM-1cm-1.
APX activity was determined by monitoring of the rate of
oxidation of ascorbate at 290 nm every 15 s for 3 min. For
this, aliquots of enzymatic extract were added to an incubation
buffer consisting of 200 mM potassium phosphate (pH 7.0),
10 mM ascorbic acid, water, and 2 mM hydrogen peroxide,
previously incubated at 28 °C (Nakano and Asada 1981).
The molar extinction coefficient was 2.8 mM-1cm-1.
Hydrogen peroxide and lipid peroxidation
The H2O2 content was determined according to Velikova
et al. (2000). Two hundred milligrams of tissue (fresh weight)
was macerated in liquid nitrogen and added to polyvinyl-
polypyrrolidone (PVPP) and homogenized in 1500 μL of 0.1%
trichloroacetic acid (TCA) (m/v). The homogenate was cen-
trifuged at 12,000 g for 15 min at 4°C. The H2O2 was
determined by measuring the absorbance at 390 nm in a
reaction medium containing 45 μL of extract, 45 μL of a 10
mM potassium phosphate buffer (pH 7.0), and 90 μL of 1 M
potassium iodide.
Measurement of malondialdehyde (MDA) content MDA
content was determined by the thiobarbituric acid reaction
(Peever and Higgins 1989). A 1.0 g sample of fresh leaves
256 Zinc Stress in Coffea Arabica Seedlings

was homogenized in 5 ml 0.1% (w/v) trichloroacetic acid
(TCA). The homogenate was centrifuged at 10 000 g for 5 min
and 4 ml of 20% TCA containing 0.5% (w/v) thiobarbituric
acid (TBA) were added to 1 ml of the supernatant. The mixture
was heated at 95 °C for 30 min and then quickly cooled on ice.
The contents were centrifuged at 10,000 g for 15 min and
absorbance of the supernatant at 532 and 600 nm was read.
After subtracting the non-specific absorbance at 600 nm, the
MDA concentration was calculated by the following equation:
MDA=(A535-A600)÷(ξ. b), where: ξ(extinction coefficient
=1.56 x10−5 cm–1); b (optical length=1).
Results
Shoot and root dry weight
An increase in shoot (Fig. 1A) and root (Fig. 1B) biomass
of control plants and under excess zinc over the days of
treatment was observed. The treatments influenced plant
biomass accumulation differently only at 60 d, when higher
shoot and root biomass were found in control plants and 0.12
ppm of zinc, followed by zinc-deficient seedlings.
Zinc content
Zinc contents in leaves and roots of deficient seedlings
were 12.3 and 21.5 ppm, respectively, at the end of the
experiment. Plants grown under conditions of Zn excess
showed an increase in Zn concentration throughout the
experimental period, reaching final values of 50.3 ppm in the
leaves and 95.6 ppm in the roots. In the control, the values
remained constant in leaves (Fig. 2A) and roots (Fig. 2B)
and within the critical Zn concentration range described by
Malavolta et al. (1997).
Gas Exchange
Zn treatment caused a notable decline in photosynthetic

Fig. 1. Dry weight of shoots (A) and roots (B) of Coffea arabica L. seedlings submitted to different concentrations of zinc, ± S.E (the results are the
average of five replications). Capital letters compare treatments (0.03, 0 and 0.12 ppm of zinc) at each sampling time, and lowercase letters
compare the effect between times (0, 30 and 60 days) within each treatment. Different letters on top of bars indicate significantly different means
at P ＜ 0.05 (Scott and Knott test).

Fig. 2. Zinc content in shoots (A) and roots (B) in Coffea arabica L. seedlings submitted to different concentrations of zinc, ± S.E (the results are
the average of five replications). Capital letters compare treatments (0.03, 0 and 0.12 ppm of zinc) at each sampling time and lowercase effect
between times (0, 30 and 60 days) within each treatment. Different letters on top of bars indicate significantly different means at P ＜ 0.05 (Scott
and Knott test).
 JCSB 2019 (September) 22 (3) : 253 ~ 264 257

Fig. 3. Net photosynthesis rate (Anet) (A),and stomatal conductance (gs) (B) of Catuaí Coffea arabica L. seedlings submitted to different
concentrations of zinc, ± S.E (the results are the average of five replications). Capital letters compare treatments (0.03, 0 and 0.12 ppm of zinc) at
each sampling time, and lowercase letters compare the effect between times (0, 30 and 60 days) within each treatment. Different letters on top of
bars indicate significantly different means at P ＜ 0.05 (Scott and Knott test).

Fig. 4. Transpiration (E) (A) and CO2 internal concentration (Ci) (B) of Catuaí Coffea arabica L. seedlings submitted to different concentrations of
zinc, ± S.E (the results are the average of five replications). Capital letters compare treatments (0.03, 0 and 0.12 ppm of zinc) at each sampling time,
and lowercase letters compare the effect between times (0, 30 and 60 days) within each treatment. Different letters on top of bars indicate
significantly different means at P ＜ 0.05 (Scott and Knott test).

rates (Fig. 3A). Leaves under zinc deficiency displayed higher
functional impairment than those of the control and 0.12
ppm Zn. At the end of the experiment, for the 0 ppm of zinc
treatment significant declines were observed, with photo-
synthetic rates dropping to near zero. Throughout the experiment
there were no significant differences between control and
0.12 ppm Zn. The Zn effect on gs (Fig. 3B), was evident
after 30 d under zinc deficiency treatment, with a significant
reduction of stomatal conductance. After 60 d this reduction
was 89% lower than in the control.
The time and zinc deficiency affected the transpiration in
coffee seedlings (Fig. 4A), At the end of the experiment,
plants with 0 ppm had a significant decrease of 89%. Zn
treatments affected the concentration of substomatal CO2
(Ci) (Fig. 4B), indicating a limitation of photosynthesis in
zinc-deficient seedlings. CO2 substomatal concentration was
lower than 50% when compared to control plants. Plants
with 0.12 ppm zinc had the same standard for gs and for Ci
there were no differences between the control during the
experimental period.
Photosynthetic pigments content
Pigment content was influenced by zinc deficiency (Fig. 5).
On the 60th day, total chlorophyll content (Fig. 5A) was
lower in deficient plants than at 0.12 ppm Zn and control
plants. Plants under 0.12 ppm of zinc maintained their pigment
content in the experimental period and this did not differ for
the control treatment.
As for carotenoids (Fig. 5B) at 30 d, lower levels were
observed in deficient plants and under Zn excess than in the
control plants. At the end of the evaluation period, there was
higher content in deficient plants and those under Zn excess
when compared to the control. The nutritional imbalance of
Zn caused a reduction in total chlorophyll content and
increased carotenoid content throughout the experimental
period.
258 Zinc Stress in Coffea Arabica Seedlings

Fig. 5. Total chlorophyll (A) and carotenoids (B) content in Coffea arabica L. seedlings submitted to different concentrations of zinc, ± S.E (the
results are the average of five replications). Capital letters compare treatments (0.03, 0 and 0.12 ppm of zinc) at each sampling time, and
lowercase letters compare the effect between times (0, 30 and 60 days) within each treatment. Different letters on top of bars indicate
significantly different means at P ＜ 0.05 (Scott and Knott test).

Fig. 6. Proline (A) and ascorbate (B) content in Coffea arabica L. seedlings submitted to different concentrations of zinc, ± S.E (the results are the
average of five replications). Capital letters compare treatments (0.03, 0 and 0.12 ppm of zinc) at each sampling time and lowercase letters
compare the effect between times (0, 30 and 60 days) within each treatment. Different letters on top of bars indicate significantly different means
at P ＜ 0.05 (Scott and Knott test).

Proline and ascorbate content Antioxidant enzymes activity

The proline level was elevated in the 0.12 ppm Zn treat-
ment in leaves (Fig. 6A). In the last evaluation, Zn deficiency
and 0.12 ppm Zn resulted in increases in proline con-
centration of 110 and 125%, respectively. Plants under Zn
excess showed higher proline content than deficient plants
(Fig. 6A).
The ascorbate content in leaves (Fig. 6B) increased in
plants under zinc deficiency and in plants at 0.12 ppm Zn
over the experimental period. In leaves, deficient plants
showed a progressive increase in ascorbate content, being
112% higher than in the control plants at 60 d of treatment.
Plants submitted to 0.12 ppm of Zn also showed an increase
in their ascorbate content but in a lower proportion when
compared to deficient plants, being 69% higher than the
control at the end of the experiment.
The SOD activity in leaves and roots (Table 1) decreased
in plants under zinc deficiency and increased in plants at
0.12 ppm Zn over the experimental period. In leaves,
deficient plants showed a progressive reduction in SOD
activity, being 43% lower than in the control plants at 60 d of
treatment. On the other hand, in plants submitted to 0.12
ppm Zn, there was a progressive increase in the activity of
this enzyme, being 60% higher than in the control at the end
of the experiment. In roots, at the end of the experiment, the
SOD activity decreased by 18% in the treatment with
deficiency and increased by 29.33% in plants under excess
zinc when compared to the control group.
For catalase, there was an increase in the activity in leaves
and roots (Table 1) due to Zn deficiency and 0.12 ppm Zn. In
leaves, at the end of the experiment, CAT activity was 98%
higher in the deficient plants and 50% in those with 0.12
ppm Zn when compared to the control. In roots, deficient
 JCSB 2019 (September) 22 (3) : 253 ~ 264 259

Table 1. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activity and lipid peroxidation in leaf and root of Coffea
arabica L. seedlings submitted to different concentrations of zinc, ± S.E (the results are the average of five replications). Capital letters compare
treatments (0.03, 0 and 0.12 ppm of zinc) at each sampling time, and lowercase letters compare the effect among times (0, 30 and 60 days) within
each treatment. Different letters indicate significantly different means at P ＜ 0.05 (Scott and Knott’s test).
SOD CAT
U g-1 FW µmol H2O2 min-1 g-1 FW
Zn
Days ppm Leaf Root Leaf Root
0.03 865 Aa ± 23.62 491 Aa ± 12.56 4 Aa ± 0.67 3 Aa ± 0.28
0 0.00 865 Aa ± 23.62 491 Aa ± 12.56 4 Aa ± 0.67 3 Aa ± 0.28
0.12 865 Aa ± 23.62 491 Aa ± 12.56 4 Aa ± 0.67 3 Ab ± 0.28

0.03 958 Ba ± 18.34 500 Aa ± 28.52 4 Ca ± 0.19 3 Ba ± 0.16

30 0.00 785 Cb ± 20.20 470 Aa ± 29.62 7 Ab ± 0.28 5 Ab ± 0.27
0.12 1234 Ab ± 25.81 492 Ab ± 27.26 6 Ba ± 0.20 5 Aa ± 0.13

0.03 988 Ba ± 24.81 545 Ba ± 21.78 4 Ca ± 0.18 3 Ca ± 0.21

60 0.00 487 Cc ± 20.74 398 Cb ± 15.36 8 Aa ± 0.03 6 Aa ± 0.47
0.12 1389 Aa ± 25.51 635 Aa ± 10.44 6 Ba ± 0.08 4 Ba ± 0.37
APX Lipid Peroxidation
mmol AsA min-1 g-1 FW nmol g-1 FW
Leaf Root Leaf Root
0.03 21 Aa ± 0.61 17 Aa ± 0.59 430 Aa ± 17.18 81 Aa ± 7.97
0 0.00 21 Ab ± 0.61 17 Ac ± 0.59 430 Ab ± 17.18 81 Ac ± 7.97
0.12 21 Ab ± 0.61 17 Aa ± 0.59 430 Ab ± 17.18 81 Ab ± 7.97

0.03 14 Bb ± 0.93 19 Ba ± 0.75 470 Aa ± 22.55 101 Aa ± 12.06

30 0.00 39 Aa ± 3.18 23 Ab ± 1.37 495 Ab ± 27.62 167 Ab ± 27.52
0.12 36 Aa ± 2.96 19 Ba ± 0.93 475 Ab ± 19.31 125 Ab ± 4.90

0.03 22 Ba ± 0.31 17 Ba ± 0.81 400 Ba ± 23.62 112 Ca ± 6.03

60 0.00 45 Aa ± 2.09 31 Aa ± 1.09 699 Aa ± 18.88 248 Aa ± 21.14
0.12 31 Aa ± 1.30 16 Ba ± 0.37 713 Aa ± 24.10 173 Ba ± 14.70

and excess plants showed an increase of 100 and 33.3%,
respectively, in the activity of this enzyme in relation to the
control.
Ascorbate peroxidase activity increased in leaves of
plants under deficiency or 0.12 ppm of zinc, whereas in roots
the increase occurred only in deficient plants (Table 1), In
leaves, at the end of the experiment, this activity was 138
and 92% higher under the deficiency and 0.12 ppm Zn
conditions, respectively, in relation to the control. In roots,
zinc deficient plants showed a progressive increase over
time, reaching values 72% higher than in the control. Plants
at 0.12 ppm of zinc maintained the activity during the
experimental period and did not differ from the control.
Hydrogen peroxide and Malondialdehyde content
Both Zn deficiency and 0.12 ppm Zn resulted in an
increase in H2O2 production (Fig. 7). This increase started on
the 30th day in leaves and on the 60th day in roots. At the
60th day, the increase in foliar H2O2 production (Fig. 7A)
due to Zn exclusion and 0.12 ppm Zn of zinc were 75 and
38%, respectively. In the root tissue, the increase in hydrogen
peroxide production was 206% in deficient seedlings and
111% in seedlings exposed to 0.12 ppm of Zn (Fig. 7B).
MDA levels (Table 1) were higher in leaves and roots of
plants under 0 and 0.12 ppm Zn since the 30th day. At the
60th day, this increase in leaves was 201% in treatment of 0
ppm Zn and 124% in 0.12 ppm Zn. On the other hand, the
lipid peroxidation level increased by 132% in roots of plants
under 0 ppm Zn and 80% in plants exposed to 0.12 ppm Zn.
Discussion
Due to Zn deficiency and 0.12 ppm Zn, coffee seedlings
(Catuaí cultivar) showed an increase in ROS production
(Fig. 7), which stimulated enzymatic antioxidant systems
activity. Despite the highest production of antioxidants, their
performance was not enough to neutralize ROS at 60 d of
stress, when higher levels of lipid peroxidation were observed.
The oxidative stress resulted in plants with physiological
responses characterized by reduction in chlorophyll content
and alteration in plant growth under Zn deficiency.
Zinc concentrations in plants generally range from 3 to
150 mg kg-1 (Epstein and Bloom 2006). In coffee leaves,
260 Zinc Stress in Coffea Arabica Seedlings
Fig. 7. Hydrogen peroxide content (H2O2) in leaves (A) and roots (B) in Coffea arabica L. seedlings submitted to different concentrations of zinc, ±
S.E (the results are the average of five replications). Capital letters compare treatments (0.03, 0 and 0.12 ppm of zinc) at each sampling time and
lowercase letters compare the effect between times (0, 30 and 60 days) within each treatment. Different letters on top of bars indicate
significantly different means at P ＜ 0.05 (Scott and Knott test).
zinc concentrations vary between 15 and 30 mg kg-1 (Malavolta
et al. 1997). Considering the zinc concentration range in
coffee leaves, the plants in this study were deficient in Zn at
30 d (12.3 mg kg-1) and under Zn excess at 60 d (50.26 mg
kg-1) (Fig. 2A).
The zinc content varied between the plant organs evaluated
according to the applied treatment, with greater variations in
the roots. In the case of deficiency, there was a greater
micronutrient relocation from the storage organs to those
with more active metabolism, which explains the smaller
variations in the foliar zinc content in relation to the control
plants (Zabini et al. 2007). On the other hand, the greater
accumulation of zinc in plant roots under the 0.12 ppm of
this nutrient can be related to the fact that the roots constitute
the zinc storage organ, since it is common that accumulating
plants store this nutrient in the roots (Ozdener and Aydin
2009).
Exposure of coffee plants to different Zn concentrations
altered cellular homeostasis, increasing ROS generation in
plants under Zn deficiency or 0.12 ppm of Zn, but these
effects were more pronounced only in the case of deficiency.
In general, stress caused by nutrient excess or deficiency in
plants is related to the increased activity of the antioxidant
system (Li et al. 2013). The higher activity of antioxidant
enzymes is attributed to the role of these enzymes in the
neutralization of ROS excess produced by the occurrence of
stress, aiming at the maintenance of cellular homeostasis
(Höller et al. 2014; Sharma and Dietz 2006). In plant species
under stresses, the increased performance of the antioxidant
system is related to reduction in ROS levels and to cell
membrane integrity (Chu et al. 2018). In the case of coffee
seedlings, the variation in Zn contents led to the highest
ascorbate content, and altered activity in the SOD, CAT, and
APX enzymes as well as the proline contents in leaves.
Coffee seedlings showed variations in SOD activity in
response to Zn deficiency or excess. SOD activity is increased
by an excess of Zn (Li et al. 2013; Michael and Krishnaswamy
2011) and reduced by its deficiency (Höller et al. 2014).
Clemente et al. (2018) reported similar results concerning
adult Coffea arabica plants, when evaluating the feasibility
of Zn supply in response to productivity and beverage
quality terms. In this work, the SOD activity increased with
Zn rates and decreased with Zn deficiency. The reduction in
SOD activity may have occurred due to the fact that low Zn
concentrations led to a structural imbalance in the isoforms,
reducing their activity (Ozdener and Aydin 2009). Also, the
high H2O2 production may be related to the inhibition of this
enzyme by its product (Ozdener and Aydin 2009).
The increased CAT activity in leaves and roots and APX
in the leaves and roots for deficient plants may be related to
H2O2 neutralization, avoiding cellular damage. Similar results
were found in roots and leaves of Saccharum officinarum
under excess Zn (Jain et al. 2010). However, maintenance of
APX activity observed in coffee roots under excess Zn was
contrary to that observed by Jain et al. (2010). This maintenance
of APX activity may be related to the low levels of ascorbate
detected in this organ, since ascorbate is a cofactor of this
enzyme (Noctor et al. 2014). Regarding Zn deficiency re-
sponses, increased CAT activity and APX were observed in
leaves of Oriza sativa (Frei et al. 2010). With respect to CAT
activity, it reduced in response to excess Zn in leaves and
roots of Eruca sativa (Ozdener and Aydin 2010).
Together with enzymatic antioxidants, the non-enzymatic
antioxidants are low molecular weight compounds that
neutralize ROS. Among these compounds, ascorbate is an
antioxidant or an enzymatic cofactor in the ascorbate-glutathione
cycle (Gill and Tuteja 2010), helping to maintain cellular
homeostasis under different stresses. The increase in AsA
concentration was found in leaves and roots of Oriza sativa
under Zn deficiency (Höller et al. 2014) and leaves of Eruca
sativa under Zn excess (Ozdener and Aydin 2010).
Another mechanism used by plant cells under stress con-

ditions is the synthesis of low molecular weight compounds
that, besides acting on osmoregulation, may present antioxidant
action (Liang et al. 2013). Among these compounds, proline is
produced in cases of Zn stress (Li et al. 2013). In our study,
plants under Zn excess showed higher proline content in
leaves.
Similar results were observed in Triticuma estivum (Li et
al. 2013), Brassica juncea, Cajanus cajan (Prasad and Saradhi
1995), and Phaseolus vulgaris (Andrade et al. 2009). The
increase in proline content was also found in Phaseolus
vulgaris (Michael and Krishnasway 2011) under Zn deficiency.
Higher proline levels are associated with better performance
of plants subjected to stress caused by excess metal (Rizvi
and Khan 2018), salinity (Nazarbeygi et al. 2011), and drought
(Bandurska et al. 2017). However, in our study there was a
significant reduction in proline content in Zn deficient coffee
plants. The reduction of proline levels may be due to deg-
radation of this amino acid, whose reactions are catalyzed by
mitochondrial enzymes such as proline dehydrogenase (Kishor
et al. 1995; Pavlikova et al. 2008) under various stress con-
ditions. In addition to induction or activation of proline enzyme
biosynthesis or decreased proline oxidation to glutamate,
proline accumulation under stress conditions may be caused
by decreased utilization of proline in protein synthesis and
enhanced protein turnover (Hildebrandt et al. 2015).
The morphological and physiological responses of plants
to stress depend on the species, tissue, stage of development,
time, intensity of stress (Jaleel et al. 2008) and they are also
affected by the morphology of the root system and its
significant capacity to uptake water and nutrients (Alvarez-
Flores et al. 2014). In Coffea arabica seedlings submitted to
deficiency and to 0.12 ppm of Zn, the antioxidant system
response contained cellular damage up to 30 d of treatment,
despite the activity of the antioxidant system, it was not
enough to avoid damage to the cellular components, culmi-
nating with high MDA formation (Table 1). This higher MDA
formation, a product of lipid peroxidation, was observed for
different plant species under several concentrations of Zn
(Feigl et al. 2014),
In plants, the photosynthetic apparatus is sensitive to the
Zn stress, which has been reported to inhibit or damage
chlorophyll synthesis in the PSII, the oxygen-evolving
complex, the plastoquinone pool, PSI, and Rubisco (Caldelas
et al. 2010; Prasad 2004). In general, Zn stress affects the
assimilation process directly, by limiting and inhibiting the
Calvin cycle enzyme activities, and indirectly reducing the
carboxylation by stomatal closure (Vaillant et al. 2005).
At 0 ppm Zn, Ci was reduced in coffee seedlings indicating
that CO2 availability was the limiting factor in this treatment
(Zn), suggesting stomatal limitation of photosynthesis for
deficient plants. Zn deficiency might be associated with
limitation of gs and intercellular CO2 concentration as
suggested by Sharma et al. (1994) and Vaillant et al. (2005).
The results of this study showed that Zn deficiency also
resulted in a reduction in leaf transpiration efficiency, Zn
may be involved in the stomatal opening, favoring main-
JCSB 2019 (September) 22 (3) : 253 ~ 264 261
tenance and absorption of K+ and also acting on membrane
integrity in guard cells (Vaillant et al. 2005). Zn deficiency
reduces the carbonic anhydrase activity in some plants, and
this directly affects a variety of physiological processes and
that also contribute to reduce the photosynthetic rate and
CO2 fixation under limited CO2 conditions (Soltanghei et al.
2014).
The induced stress from metals may affect photosynthetic
rate and pigment contents in plants. Metal treatment usually
decreased the total chlorophyll in various plants (Cherif et al.
2010; Hashemi 2018; Jiang et al. 2019; Santos et al. 2017).
This study has shown that at 30 d, plants under Zn deficiency
and 0.12 ppm of Zn did not have their total chlorophyll
content altered. However, at the end of the experiment, the
total chlorophyll was significantly reduced, in plants with Zn
restriction as well as those under 0.12 ppm of Zn. The Zn
stress may lead to the decrease in chlorophyll synthesis and
inhibition of photosynthetic electron transport (Vassilev et
al. 2011). Our results also suggest that the high levels and
deficiency of Zn cause chloroplast disorganization and
photosynthesis alteration. These results are in agreement
with those observed in Malus domestica (Fu et al. 2015),
Coffea Arabica (Clemente 2014), and Oryza sativa (Lee et
al. 2017). The photosynthetic apparatus is one of the targeted
sites of action of Zn in plants, since at toxic concentrations it
disrupts the chloroplast envelope and the integrity of the
thylakoid membranes (Cherif et al. 2010). In addition, either
condition, Zn deficiency or excess, may inhibit the transport
of electrons from photosystem II (PSII) with a concomitant
decrease in chlorophyll biosynthesis (Monnet et al. 2001).
Regarding carotenoids, there was higher production of
these pigments in deficient plants or under excess Zn at the
end of the experiment. Similar results were reported in
progenies of Coffea arabica and in Ceratophyllum demersum
(Aravind and Prasad 2004; Zabini et al. 2007), while in
Lycopersicum esculentum, carotenoid levels remained constant
even under Zn deficiency (Cherif et al. 2010). This increase
of carotenoids may have occurred as a form of defense
against oxidative stress, since they play a key role in the
control of free radicals, directly acting on the protection of
photosynthetic membranes (Havaux 2013).
At the end of the experiment, Zn deficiency resulted in
lower accumulation of biomass. However, excess Zn did not
affect the biomass of the Coffea arabica seedlings. In the
literature, lower growth is reported for different species under
varying concentrations of Zn (Blasco et al. 2015; Mattiello et
al. 2015). Growth reduction is attributed to changes in gas
exchange, chlorophyll fluorescence, and oxidative stress.
Moreover, Zn may affect ionic homeostatic systems, interfering
with the uptake, transport and regulation of ions, leading to a
misrepresentation of plant homeostasis (Cakmak et al. 1998;
Rout and Das 2009).
Coffea arabica seedlings were deficient in Zn at 30 d of
treatment, while the excess of this element in the tissues was
observed only at 60 d. However, growth reduction was
observed only for deficient plants at the end of the experiment.
262 Zinc Stress in Coffea Arabica Seedlings
Although Zn nutritional imbalance caused changes in the
antioxidant metabolism and in the pigment contents of Coffea
arabica seedlings, seedlings under Zn excess showed better
performance, which can be verified by shoot biomass and
roots similar to control seedlings. Therefore, Zn deficiency
causes greater damage to Coffea arabica seedlings (Catuaí
cultivar) than the excess of this element.
Acknowledgements
We especially thank the Fundação de Amparo à Pesquisa
do Estado de Minas Gerais (FAPEMIG), Conselho Nacional
de Desenvolvimento Científico e tecnológico (CNPq), Coor-
denação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) for funding and Dr. João Paulo R. Alves Delfino
Barbosa for assisting us in this project.
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