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Chlorophyll, nitrogen and antioxidant activities in Cumaru (Dipteryx odorata (Aubl.) Willd) (Fabaceae) in two water regimes

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Vol. 15(44), pp. 2480-2489, 2 November, 2016
DOI: 10.5897/AJB2016.15528
Article Number: 84A594461394
ISSN 1684-5315
Copyright © 2016
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJB
African Journal of Biotechnology
Full Length Research Paper
Chlorophyll, nitrogen and antioxidant activities in
Cumaru (Dipteryx odorata (Aubl.) Willd) (Fabaceae) in
two water regimes
Bruno Moitinho Maltarolo1,2*, Ellen Gleyce da Silva Lima1,2, Vitor Resende do Nascimento2,
Kerolém Prícila Sousa Cardoso1,2, Ana Ecídia de Araújo Brito1,2, Tamires Borges de Oliveira2,
Glauco André dos Santos Nogueira1,2, Karollyne Renata Souza Silva2,3, Wander Luiz da Silva
Ataíde1,2, Cândido Ferreira de Oliveira Neto1,2, Waldemar Viana De Andrade Junior4 and
Benedito Gomes dos Santos Filho2
1Institute of Agricultural Sciences Program in Forest Sciences, UFRA - Federal Rural University of the Amazon, BEL
66.077-830, Brazil.
2Federal Rural University of the Amazon, BEL 66.077-830, Brazil.
3Agronomy student at the Federal Rural University of Amazon, Brazil.
4Department of Vegetal physiology, UFRA- Federal Rural University of Amazonia, Brazil.
Received 14 June, 2016; Accepted 21 September, 2016
The Cumaru (Dipteryx odorata (Aubl.) Willd.) is a species used by traditional populations and industries
using timber and non-timber forest products. This study aimed to analyze the levels of chlorophyll A, B,
total ammonia levels, nitrate, proline, electrolyte leakage and activity of oxidative enzymes in evaluation
to tolerance of cumaru plants subjected to drought for 21 days of stress. The experiment was
conducted in a greenhouse at the Federal Rural University of Amazonia (UFRA), Belém, Pará, in the
period from March to July 2015. The results showed a significant decrease in the relative water content
of 50.8 and 55% for chlorophyll b, 45% to total chlorophyll and an increase in proline to the plants under
drought. There was no significant difference to chlorophyll a, ammonium and nitrate. Increases in
electrolyte leak with 22.74% for roots and 39.55% for leaves were observed. The enzyme catalase (CAT)
showed a significant increase from the 14th day of the experiment, while changes in superoxide
dismutase (SOD) and ascorbate peroxidase (APX) activities were observed from the 7th day of the
experiment. Cumaru plants are not drought tolerant over 21 days; also, young plants of cumaru
respond negatively to conditions of low water availability in the soil.
Key words: Drought, oxidative stress, chlorophyll, tolerance, Dipteryx odorata.
INTRODUCTION
Cumaru (Dipteryx odorata (Aubl.) Willd.) is a species
used by traditional populations and industries using timber and non-timber forest products such as oils for
medicinal and cosmetic properties, as well as with the
*Corresponding author. E-mail: bruno.maltarolo@hotmail.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
reforestation of degraded areas, which in addition to the
ecological benefits, increase the supply of wood from
reforestation in the region, increasing the income on the
farm and reducing the pressure on the remaining natural
forest dependent of water resources (Shimizu, 1998).
As water resources become scarce, the commercial
exploration of plants tolerant to drought becomes a
priority for obtaining high yields (Matos et al., 2012). The
impact of drought in the forestry and agricultural activities
is an important socioeconomic consequence that affects
millions of people around the world (Elliott et al., 2013).
Among the various factors affecting the production plant,
the water deficit occupies a prominent position, as well as
affect the water balance in plants by altering their
metabolism, is a phenomenon that occurs in large
extensions of arable areas (Nogueira et al., 2001).
Among the many implications of drought on plant
development, the restriction on the acquisition of nutrients
and water is commonly recognized (Manivannan et al.,
2008). Evidence suggests that drought causes oxidative
stress in various plants, in which reactive oxygen species
(ROS) such as superoxide radical (O2-), hydroxyl radical
(OH-), hydrogen peroxide (H2O2) and singlet oxigen (1O2),
are produced (Jaleel et al., 2007).
To minimize the cytotoxic effects of ROS, the plants
causes a complex antioxidant system where specific
enzymes act by neutralizing the action of these radicals,
starting with the superoxide dismutase (SOD), which
inmute radical O2 to H2O2. This, in turn, undergoes action
of various enzymes such as catalase (CAT), responsible
for the conversion of H2O2 to H2O and O2, and
peroxidase, ascorbate peroxidase (APX) reducing the
H2O2 to H2O (Apel and Hirt, 2004). Besides, in water
restriction, the plants should be able to handle ROSs
particularly to prevent oxidative damage to lipids, proteins
and nucleic acids; if there is an inability to adequately
handle ROSs, oxidative damage may result in cell death
(Demidchik, 2015).
Several methods are adopted by researchers to identify
species tolerant to water stress, being more common
selection through ecological descriptors associated with
physiological and biochemical descriptors. According
Pincelli (2010) water deficiency is one of the
environmental stresses responsible for the reduction of
pigments in the leaves, making the plant life cycle
changes. Among these, related to the antioxidant system
and osmotic adjustment are supported substantially in
identifying promising species, and consequently, the
progress of culture of works for improving drought
resistance (Azevedo Neto et al., 2009).
The antioxidant enzyme activity is usually enhanced to
promote better elimination of ROSs and promote
increased cellular protection against oxidative damage
(Jaleel et al., 2009). Considering then that collaboration
between antioxidant enzymes should provide better
protection against the deleterious effects of ROS,
minimizing oxidative damage (Blokhina et al., 2003).
Maltarolo et al. 2481
Given the above, the study aimed to analyze the content
of chlorophyll A and B, ammonium, nitrate and proline as
well as the activity of oxidative enzymes in evaluation
tolerance of cumaru plants subjected to drought.
MATERIALS AND METHODS
Location and experimental conduction
The experiment was conducted in a greenhouse at the Federal
Rural University of Amazonia (UFRA) belonging to the Institute of
Agricultural Sciences (IAS), located in Belém, Pará, in the period
from March to July 2015. The seedlings of cumaru (Dipteryx
odorata (Aubl.) Willd.), from seeds were provided by AIMEX
(Association of Industries Exporters of Wood in Pará) with four
months old, they were placed in plastic pots with a capacity of 3.6
L. The substrate consisted of yellow dystrophic Latosol (EMBRAPA,
2013). Before the start of treatment, all plants were irrigated daily
for three months, corresponding to the acclimation time. 5 ml of
solution cocktail containing macro and micronutrients (Table 1) was
added to all the samples at the start of acclimation, in the form of
nutrient solution (Hoagland and Arnon, 1950), modified in
Biodiversity Studies Laboratory in Higher Plants (EBPS), UFRA.
The plants were subjected to two water regimes: Irrigated
(control) and water deficit, in which the imposition of water deficit
was obtained by suspension of irrigation in 21 days, and the time 0
(zero days of drought), time 1 (7 days of drought), time 2 (14 days
of drought) and 3 time (21 days of drought). During the period of
analysis, control plants were irrigated daily to replace the water lost
by evapotranspiration. There was also the weed control manually. It
was not detected occurring nutritional deficiency symptoms, as well
as the attack of pests and pathogens.
Experimental design and statistical analysis
The experimental design was completely randomized in split plot in
time (four times evaluation and two water conditions: Control and
drought), with 5 repetitions, totaling 40 experimental units, each
experimental unit was composed of a plant/pot. Analysis of
variance of the results was applied and when there was a
significant difference, the means were compared by Tukey test at
5% significance level. Moreover, the standard deviations were
calculated for each treatment, and statistical analyzes performed by
Assistant Version 7.7 Beta program.
Relative water content (RWC)
The RWC was determined at 06:00h a.m in each collect. The
method used was that described by Slavick (1979). Results were
expressed as a percentage, according to the formula:
RWC = (FM1 - DM)/(FM2 - DM) × 100 (%)
Where, FM1 = Fresh mass 1; FM2 = Fresh mass 2 with saturation;
DM = Dry mass.
Determining the ammonium content
50 mg of previously lyophilised leaves and roots were weighed and
put in a test tube containing 400 ml of total extract + 2.5 ml of
solution A (5 g phenol + 0.025 g of sodium nitroprusside / 500 ml
2482 Afr. J. Biotechnol.
Table 1. Solution cocktail containing macro and micronutrients.
Composition
Concentration (M)
KNO3
1
Ca (NO3)2
1
NH4NO3
1
KH2PO4
1
MgSO4
1
Fe-EDTA
1. FeSO4. 7H2O
0.1
2. Na2 (EDTA)
0.08
Micronutrientes
1. H3BO3
0.04
2. MnCl2. 4 H2O
0.009
3. CuSO4. 5H2O
0.0003
4. ZnSO4. 7 H2O
0.0007
5. Na2MoO4. 2 H2O
0.0001
CoCl2. 6 H2O
0.004
Al2 (SO4)3 . 18 H2O [(50 mM) pH= 4.0]
0.3
Al2 (SO4)3 . 18 H2O [(100 mM) pH= 4.0]
0.3
Al2 (SO4)3 . 18 H2O [(150 Mm) pH= 4.0]
0.3
distilled water) and homogenized by vortexing, adding 2.5 ml of
Solution B (2.5 g NaOH + 12.6 ml of sodium hypochlorite / 500 ml
distilled water), respectively. The free ammonium concentrations of
the total extract were estimated from the standard curve constructed
with (NH4)2SO4 p.a. (Sigma) according to the method described by
Weatherburn (1967).
Determination of nitrate
50 mg each of previously lyophilized leaves and roots was weighed
and mixed with extract containing 100 mL + 200 of salicylic acid 5%
solution (w / v) in concentrated sulfuric acid. After stirring vigorously
in a vortex stirrer was slowly added 4700 uL of 2N NaOH. The
concentration of nitrate was obtained from a standard curve with
increasing concentrations of NO3 (0, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0
μmol ml-1) according the method described by Cataldo et al. (1975).
Determining the proline content
50 mg were weighed of previously lyophilised leaves and roots by
adding in the test tubes the total extract, 1 ml of ninhydrin acid and
1 ml of glacial acetic acid 99.5%. It was determined through a
standard calibration curve using proline and proline contents in
samples were extrapolated from the curve and expressed in mmol
g-1. The dry matter (DM) was determined according to Bates et al.
(1973).
Determination of photosynthetic pigments
The determination of photosynthetic pigments was realized accor-
ding to Lichtenthaler (1987). The concentrations of chlorophyll A, B
and total (mg. L-1) were calculated using the formulas:
Chlorophyll A = 12.25 × L(662) 2.79 × L(644) Chlorophyll B = 21.5 ×
L(644) - 5.1 × L(662)
Total chlorophyll = 7.15 × L(662) + 18.71 × L(644)
Membrane integrity (leak electrolytes)
The degree of membrane integrity was estimated by electrolyte leak
according Blum and Ebercon (1981). The electrolyte leak was
estimated by the following equation:
EL (%) = (C1/C2) × 100
Enzymatic activity
Superoxide dismutase (SOD)
The SOD activity was determined by inhibition of photoreduction of
nitroblue tetrazolium chloride (NTC) according to Giannopolitis and
Ries (1977).
Catalase (CAT)
CAT activity was determined by the method of Beers Jr. and Sizer
(1952) with modifications.
Ascorbate peroxidase (APX)
The APX activity was determined by the method of Nakano and
Asada (1981).
Figure 1. Relative water content in young plants Cumaru
subjected to water deficit. Capital letters show statistical
differences between water conditions and lower statistical
differences between the collections time. Tukey test p < 0.05
probability was used for comparison.
RESULTS AND DISCUSION
Relative water content (RWC)
The relative water contents present in the leaves of
cumaru under water stress decreased as the weeks went
by, on average control plants showed water percentage
between 87.7 and 85.5%, and plants under drought
between 88.3 and 37.5%, representing a decrease of
50.8%.
The relative water content present in the leaves
represent the water availability in the soil as well as the
efficiency of the plant in pick up water in adverse
conditions and maintaining water in the system reducing
losses. Cumaru seedlings have the high water content in
the leaf under normal conditions but had a sharp
decrease due to lack of water. The decrease was
significant from the 7th day of water suspension and
decreasing over the 21 days of stress, as shown in Figure
1.
Photosynthetic pigments
The chlorophyll A contents do not vary significantly
throughout the experiment (Tukey test at 5% significance
level), while the chlorophyll B and total had a significant
reduction in plants under drought compared to control
Maltarolo et al. 2483
plants. Mean values for chlorophyll A were 3.31 mmol.m-
2.s-1 for the control plants and 2.8 mmol.m-2.s-1 for plants
under drought. Chlorophyll B shows the mean values of
3.05 and 2.27 mmol.m-2.s-1, while total chlorophyll was
6.37and 5.08 mmol.m-2.s-1 in control plants and plants
under drought, respectively (Figure 2). Representing a
decrease of 34% to chlorophyll A, 55% for chlorophyll B
and 45% for total chlorophyll compared the two water
conditions on the 21st day of the experiment. According
to Morais et al. (2007), chlorophylls A and B are
interconverted in the chlorophyll cycle and form
complexes of chlorophyll-protein, that are important in the
regulation and organization of the photosystem.
Chlorophylls play an important role in photosynthesis, are
responsible for capturing light energy, especially
chlorophyll A as the main pigment of complex light
collectors (LHC) for the photochemical reactions (Taiz
and Zeiger, 2013).
Under reduced stomata conductance and consequently
lower influx of CO2 proceeds in reduction of net
assimilation rate, which directly affects the biochemistry
of photosynthesis and reduces the photochemical energy
consumption (Carmo et al., 2014). In these situations
there is constant production of reactive oxygen species
and other chlorophyll degradation agents (Matos et al.,
2012). Chlorophyll degradation occurs according to the
level of stress in the plants are submitted and the
implication is leaf senescence, occurrence found in this
study (Carmo et al., 2014).
In this work the chlorophyll A showed no significant
difference, a fact that may be in accordance with the
statement of Dinakar et al. (2012), in which the
chloroplasts are particularly susceptible to oxidative
damage and when it comes to tolerance to drought
periods as well as the production of antioxidants,
chlorophyll content is maintained after the drying, to
prevent the formation of reactive oxygen species (ROS’s).
Ammonium, nitrate and proline content
The ammonium and nitrate concentrations had no
significant change throughout the experiment in plants
under drought and the control plants. Proline already had
a significant increase from the 14th day in the leaves and
21th day in roots. The values for ammonium in the last
day of collection were 11.2 and 11.5 mmol of NH4+.Kg-1
DM in roots and 7.2 and 6.4 mmol of NH4+.Kg-1 DM in
leaves, control and drought, respectively (Figure 3A).
Nitrate was of 0.07 and 0.08 mmol from NO3-.Kg-1 DM in
roots and 0.06 and 0.06 mmol of NO3-.Kg-1 DM in leaves,
control and drought, respectively (Figure 3B). Proline was
of 3.8 and 20.8 mmol of Pro. g-1 DM in roots and 2.3 and
29.8 mmol of Pro.g-1 DM in leaves, control and drought,
respectively (Figure 3C).
Most plants have a preference for nitrate ion as a
nitrogen source, so it is common their levels were lower
2484 Afr. J. Biotechnol.
Figure 2. Chlorophyll content A (A), chlorophyll B (B) and chlorophyll total (C) in young plants Cumaru subjected to water
deficit. Capital letters show statistical differences between water conditions and lower statistical differences between the
collections time. Tukey test p < 0.05 probability was used for comparison.
than those found ammonium levels (Martinelli, 2003;
Araújo et al., 2004), corroborating with these results.
The ammonium and the nitrate are the main forms of
nitrogen available to plants, reduction processes and
nitrogen assimilation can be absorbed both in the leaves
and in the roots simultaneously or between these bodies
becoming an essential process for the plant, since it is
through it that It is controlled growth and development of
the plant (Shan et al., 2012).
As a result, various forms of N available in the
substrate can affect the morphological, physiological and
biochemical plant, possibly in root growth, photosynthetic
rates and catalytic activity of several enzymes (Li et al.,
2013). In studies comparing the nutrition with nitrate
(NO3-) or ammonium (NH4+) show that these nitrogen
sources can induce different metabolic responses
(Patterson et al., 2010).
The accumulation of soluble solutes in plant cells
provides a type of response to water deficit, called
osmotic adjustment, which allows more negative water
Maltarolo et al. 2485
Figure 3. Ammonium levels (A), nitrate (B) and proline (C) in young plants Cumaru subjected to water deficit.
Capital letters show statistical differences between water conditions and lower statistical differences between
the collections time. Tukey test p < 0.05 probability was used for comparison.
potential in leaves, thus helping to keep the movement of
water to the leaves (Silva et al., 2014). Proline has been
highlighted as a compatible solute occurs in plants in
response to environmental stresses that solute
accumulates variety of plant species in response to
stresses such as drought, heavy metals, extreme
temperatures, salinity and ultraviolet radiation
(Siripornadulsil et al., 2002).
2486 Afr. J. Biotechnol.
Figure 4. Electrolyte leak in young plants Cumaru subjected to water deficit. Capital letters show statistical differences
between water conditions and lower statistical differences between the collections time. Tukey test p < 0.05 probability was
used for comparison.
It is possible to note a proline increased much more
significant in the leaves than the roots that this fact can
be given by the need of the plant to have a more negative
potential in the leaves so that the water can reach the
highest parts of the plant. Proline contents only had
increased from 14 days even if there is already a
significant reduction in the RWC on the 7th day, this may
be because the proline can be a compatible solute
(osmoprotectors organic compound and osmoregulator)
more secondary role in Cumaru species as it was the
case in the study of Pinhão-manso (Sousa et al., 2012),
which highlighted the glycine main osmoregulator and
osmoprotectors.
Electrolyte Leak
Results show that there was a significant increase in both
the leaves and the roots that were under water deficit
with values of 12.2 to 29.58% and 12.2 to 60.73% for the
leaves (plant control and drought, respectively). As well
as 24.56 to 28.55% and 24.6 to 51.29% for the roots
(plants control and drought), respectively (Figure 4) with
a 39.55% increase in percentage for leaves and roots
22.74% to the 21 day of experiment. Lack of water
causes a decrease in liquid photosynthesis and in this
case the sharp reduction of water in the cumaru plants
probably caused this decrease in liquid photosynthetic
rate and to produce more O2- and H2O2 in chloroplasts
(Blokhina et al., 2003; Reddy et al., 2004). The increased
cellular leak in plants under drought is strongly related to
the damage caused by free radicals O2- that attacks
different parts of the plant as lipids and membrane
proteins, nucleic acids and others causing cell death.
Enzymatic activity
Superoxide dismutase (SOD)
Plants subjected to drought showed a significant increase
when compared to the control plants over the 21 days of
experiment (Figure 5). The values for the roots were
49.86 to 50.85 mg-1.protein, and of 49.79 to 58.59 mg-
1.protein (control plants and under drought), respectively.
For the leaves the results were similar with values of
41.06 to 40.35 mg-1.protein and of 40.91 to 49,2 mg-
1.protein (control plants and under drought), respectively.
Plants have enzymatic systems of defense against
reactive oxygen species, including SOD, CAT, APX.
Activation of genes encoding these enzymes in response
to oxidative stress was observed, for examples, tobacco
(Bowler et al., 1991), soybean (Lee et al., 1999), and
peanut (Sankar et al., 2007). Thus, increased activity of
these enzymes is directly related to differential
expression of the genes belonging to the antioxidant
system, having as one of its functions to prevent H2O2
accumulation in cells (Eyidogan and Oz, 2007;
Vaidyanathan et al., 2003).
Catalase (CAT)
The enzyme catalase showed significant difference from
the 14th day of the experiment (Figure 6), with values for
the roots of 0.042 mg-1.protein (control plants) and of
0.042 to 0.054 mg-1.protein (under drought), respectively.
The leaves presents values of 0.043 to 0.042 mg-1.protein
and of 0.043 to 0.073 mg-1.protein (control plants and
under drought), respectively.
Maltarolo et al. 2487
Figure 5. Superoxide dismutase enzyme activity in young plants Cumaru subjected to water deficit. Capital letters
show statistical differences between water conditions and lower statistical differences between the collections time.
Tukey test p < 0.05 probability was used for comparison.
Figure 6. Enzyme catalase activity in young plants Cumaru subjected to water deficit. Capital letters show statistical
differences between water conditions and lower statistical differences between the collections time. Tukey test p <
0.05 probability was used for comparison.
Akcay et al. (2010), who studied the CAT activity in
creeping and erect peanut, found that the enzyme activity
increased significantly when subjected to higher stress
levels, confirming the results of this work. According to
these authors, the CAT is one of the most effective
defense enzymes in oxidative processes, since, in the
resistant plants enables the integrity of the cell even
when the stress is in a more rigorous stage. These
results are reported in previous studies of water stress,
salinity and other stresses, which reported that there is a
reduced production of ROS in tolerant genotypes than in
susceptible genotypes (Karabal et al., 2003; Chaitanya et
al., 2002; Bhoomika et al., 2013). According to Sankar et
al. (2007), as can be seen in his work, where an average
increase of up to 230% of activity was obtained at the
earliest material, when subjected to 10 days of water
suppression.
Ascorbate peroxidase (APX)
The values of APX enzyme showed significant difference
after 7 days of the experiment in plants were subjected to
drought, when compared with control plants. The
increase for the roots was from 0.0298 to 0.032
mmol.min-1 and of 0.0293 to 0.0376 mmol.min-1 in control
plants and under drought, respectively. The leaves show
values of 0.0315 to 0.0322 mmol.min-1 and of 0.0309 to
0.0405 mmol.min-1 in control plants and under drought,
respectively (Figure 7). These results highlight that
2488 Afr. J. Biotechnol.
Figure 7. Enzyme peroxidase activity in young plants Cumaru subjected to water deficit. Capital letters show
statistical differences between water conditions and lower statistical differences between the collections time.
Tukey test p < 0.05 probability was used for comparison.
2013).
Thus, the potential oxidative damage due to drought
was adequately mitigated by the constitutive activity APX
(Cruz et al., 2013). Possibly, this gives rise to the
triggering of multiple strategies antioxidants (Silva et al.,
2015).
The species studied presents different mechanisms to
overcome the drought periods, either by maintaining low
RWC values and photosynthetic pigments, or by the
increased activity of oxidative enzymes which are
variables that can be used as water stress sensitivity
indicator.
Young plants of cumaru are not tolerant to more than
21 days of water stress, and respond very negatively to
the conditions of low water availability in the soil.
Conflict of Interests
The authors have not declared any conflict of interests.
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