Nitrogen Biofertilizer Alleviates the Inhibitory Effect of Cadmium on Physiology
and Nitrogen Assimilation in Maize Plants
Hanan Mahmoud Abou-Zeid1, Nabil
El-Sayed Saber1, Basma Ibrahim Abdelrahim2
and Ghada Saber Mohamed Ismail1*
1Botany and Microbiology
Department, Faculty of Science, Alexandria University, Egypt
2Botany Department, Faculty of
Science, Benghazi University, Libia
*For correspondence: ghada5f@yahoo.com
Received 11 April 2020; Accepted 05 September 2020; Published 10
December 2020
Abstract
The present study investigated the role of inoculation
with Nitrobien biofertilizer (N-Bio, Azospirillum and azotobacter spp.)
on the response of maize plants to cadmium-toxicity (applied as 2 and 10 mM
CdSO4). Cd-stress caused a significant reduction in the fresh and
dry biomass of leaves and roots as well as a marked disturbance in the
anatomical features of roots and stomatal structure and behavior. Cd-stress
significantly depressed the total photosynthetic pigments, photochemical
efficiency of PS II, total carbohydrates, and proteins content. Furthermore, increasing Cd level prompted oxidative stress measured in terms of malondialdehyde and H2O2
contents in maize plants. Application of N-Bio improved these attributes in
Cd-stressed maize plants. Moreover, NO3- uptake and its assimilating
enzymes (nitrate reductase, NR; glutamine synthase, GS; and, glutamate
dehydrogenase GDH) were significantly increased in N-Bio-pretreated Cd-stressed
plants than Cd- stressed ones and that was associated with a decrease of NH4+
content. N-Bio pretreatment also stimulated the accumulation of amino acids and
markedly increased endogenous phytohormone content (IAA, GA3) of
Cd-stressed maize plants. These results revealed the potentiating effect of
N-Bio pretreatment in regulating Cd-induced damages in maize plants. © 2021
Friends Science Publishers
Keywords: Biofertilizer; Cadmium stress; Root anatomy; Phytohormone; Nitrate reductase
Introduction
Plants are exposed to several abiotic and biotic stresses. In the present era, heavy metals have been widely distributed; being
one of the major outstanding apprehensions for sustainable agriculture and
human welfare (Edelstein and Ben-Hur 2018). Cadmium (Cd) is a non-essential
heavy metal that occurs naturally in the environment
in traces; however, its concentration is continuously increased due to
extensive industrial processes, dispersal of sewage sludge and usage of
phosphate fertilizers in agriculture (Liu et al. 2007). Furthermore, Cd
uptake alters plant growth and development, and affects human health by its
accumulation in the consumable parts of crop plants.
The
processes related to Cd toxicity in plants are very complicated as it can
affect several morphological and physiological processes even at low
concentrations. Excessive amounts of Cd frequently elicits many stress symptoms
in plants, such as decrease of carbon assimilation, generation of oxidative
stress, inhibition of chlorophyll synthesis, reduction in nutrient uptake,
impairment of photosynthesis and at last bringing about stunted growth,
chlorosis, leaf epinasty, alterations in chloroplast ultrastructure, induction
of lipid peroxidation, alterations in nitrogen (N) metabolism and interruption
of antioxidant machinery (Shah et al. 2017; Farooq et al. 2020).
It is well known that Cd is more easily absorbed by plants than any other heavy
metals and more than 90% of the Cd is accumulated in roots (Hussain et al.
2021). Although most studies had focused on the impact of heavy metals on
visible symptoms on aerial parts and root morphological characters, few studies
had recorded toxic symptoms on the anatomical parameters of several plants
grown under cadmium stress including maize (Gowayed and Almaghrabi 2013) and
rice (Li et al. 2014). It is well known that stomata play an important
role in adjustment of plant water balance and gas exchange. Alterations in
stomatal structure and behavior have been observed due to different heavy
metals toxicity as Cd (Mondal et al. 2013) and Pb (Divyajyothi and
Sujatha 2019).
It has
been shown that nitrate uptake and its assimilation by plants are differently
affected under Cd treatment (Chaffei et al. 2004). Nikolić et al.
(2017) reported that increasing Cd levels markedly decline NO3-
uptake and its assimilation as indicated by suppressing nitrate reductase (NR)
and glutamine synthetase (GS) activities. Singh and Prasad (2017) found a
marked decline in glutamine synthetase–glutamate synthase (GS/GOGAT) activities
in tomato plants imposed to Cd treatment. They suggested that the decline of
both GS and GOGAT activities could result in the accumulation of NH4+
and a decrease of growth. On the other hand, other researchers (Chaffei et
al. 2004; Skopelitis et al. 2006) have recorded an increase of
glutamate dehydrogenase (NADH-GDH) activity under Cd stress.
The
plants exposed to toxic elements tend to accumulate specific amino acid (AAs),
which may have valuable functions and play various roles in plants (Xu et al.
2012; Zemanová et al. 2017). It is well recognized that long-term application of chemical
fertilizers contributes to the pollution of soil and ground water with various
heavy metals, soil degradation and destruction of soil microflora (Rashid et
al. 2016). On the other hand, biofertilizers
are different types of living population microorganisms, such as bacteria,
fungi and cyanobacteria, live in plants root vicinity or rhizosphere and have
the ability to stimulate plant growth through different modes of actions such
as nitrogen fixation, degradation of organic materials and secretion of
phytohormones (Sinha et al. 2014).
Recent studies have also
demonstrated that application of various strains of plant growth promoting
rhizobacteria (PGPR) can promote and improve several plants subjected to
various environmental stresses (Naveed et al. 2014; Gouda
et al. 2018). Bacteria of the genus Azospirillum and Azotobacter are among the best researched PGPR detected in the
rhizosphere of many crop plants like wheat and tomato (Agami et al.
2017; Reddy et al. 2018). They exert their roles through N2
fixation, secretion of several components as vitamins,
plant growth regulators and several natural products as secondary metabolites
in the rhizosphere (Vejan et al. 2016). Such capabilities accordingly
result in enhanced growth of plants under various stresses like drought and
heavy metal stress (Agami et al. 2017; Rezvi and Khan 2018). Previously, Pacwa-Płociniczak et al. (2011)
stated that bacterial biosurfactants can bind toxic heavy metals, hence remove
them from soil and increase plant tolerance.
Maize (Zea mays L.) is one of the most economically important cereal
crops utilized for grain, silage, and biofuel goals (Tejada et
al. 2016). An exponential increment in the
world populace would request a higher crop production and hence more
utilizations of chemical fertilizers. Therefore, the aim of the
current investigation was to study the impact of Cd
stress on Zea mays and evaluate the role of Nitrobien biofertilizer
applied by seed inoculation in the response of maize plants to Cd stress. The
modifications in some growth parameters, photosynthetic efficiency, stomatal
behavior, and root anatomical structures were followed. In
addition, nitrate, ammonia, free amino acids and hormonal content as well as
nitrogen assimilating enzymes activity were evaluated.
Materials and Methods
Plant material, growth conditions and treatments
Maize
(Zea mays L. cv. Nevertity) seeds were obtained from the Agricultural
Research Center, Giza, Egypt. Nitrobien biofertilizer (N-Bio) containing a
combination of nitrogen-fixing bacteria; Azotobacter spp. and Azospirillum
spp. were kindly supplied by
biofertilizers Unit, General Organization of Agriculture Equalization Fund,
Agriculture Research Centre, Ministry of Agriculture, Giza, Egypt. After
surface-sterilization with 4% sodium hypochlorite for 10 min, the seeds were
washed with distilled water several times, soaked for 24 h at 25oC
in aerated water and then transferred to weighed plastic pots filled with
acid-washed quartz sand and clay (3:1). The pots were divided into four groups
and each group consists of 3 replicates. The first group was left as a control
without any treatment and irrigated with one tenth strength modified Hoagland
solution (Epstein 1972). The second group was irrigated with one tenth strength
modified Hoagland solution supplemented with 2 and 10 mM CdSO4.
In the third group the soaked seeds were inoculated with N-Bio; seed
inoculation was performed by mixing maize seeds with the nitrobien using Arabic
gum as adhesive material. The coated seeds were then air dried in shade for 30
min and the seeds were sown immediately in pots and irrigated with one tenth
strength modified Hoagland solution. In the fourth group the seeds were
inoculated with N-Bio and irrigated with one tenth strength modified Hoagland
solution supplemented with 2 and 10 mM CdSO4. The pots were
placed in an environmentally controlled growth chamber under a 16-h photoperiod at an irradiance of about 23 µmol m-2 s-1 (cool
white fluorescent tubes) and 31/28 ± 2°C light/dark temperature and irrigated
with the treatment solutions every two-day interval throughout the whole
experimental period. After 21 days, homologous plants were harvested, washed
thoroughly from adhering soil particles, gently plotted, dissected to shoots
and roots and quickly saved for estimation of the various growth parameters and
chemical analyses. All chemical analyses were performed on roots and leaves.
Growth parameters
The roots and leaves were
separated and taken for determination of fresh (FM) and dry biomass (DM). Shoot
height was measured.
Light and scanning electron microscopy
The
fragments of Zea mays L. (cv.
Nevertity) roots from control and treated samples were fixed in a mixture of 2%
formaldehyde and 2.5% glutaraldehyde in cacodylate buffer at pH 7.4 for 2 h,
thoroughly washed in the same buffer and then post fixed with 1.0% (w/v) osmium
tetraoxide in the same buffer for 2 h at room temperature. Subsequently, the
samples were transferred to re-distilled water and stained with a 0.5% aqueous
solution of uranyl acetate. After passing through increasing concentrations of
ethanol and embedded in Spurr's resin at 70°C (Spurr 1969). Semi-thin sections
(1 µm) were observed with light
microscope (Olympus, Japan) after staining with 2% uranyl acetate and lead
acetate solutions (Venable and Coggeshall 1965). Samples preparation,
visualization and photographing were carried out at the Electron Microscopic
Unit, Faculty of Science, Alexandria University.
Scanning electron microscopy
Small
pieces of fresh specimens of maize leaves from both control and treated samples
were removed and fixed by immersing immediately in 4F1G in phosphate buffer
solution (pH 7.2) at 4°C for 3 h. Specimens were then post fixed in 2% OSO4
in the same buffer at 4°C for 2 h. Samples were washed in the buffer and
dehydrated at 4°C through a graded series of ethanol. The samples were then
dried by means of the critical point method, mounted using carbon paste on an
Al-stub and coated with gold up to a thickness of 400A in a sputter-coating
unit (JFC-1100 E). Observations of leaf morphology in the coded specimens were
performed in a Jeol JSM-5300 scanning electron microscope operated between 15
and 20 KeV.
Estimation of photosynthetic pigments and quantum yield
of PSII (Fv/Fm)
The
photosynthetic pigments were determined according to methods described by Moran
(1982) using N, N-dimethyl formamide (DMF). Absorbance was measured at two
wavelengths of 646.8 and 663.8 nm using spectrophotometer (JENWAY, 6305,
UV/Vis). Measurement of chlorophyll fluorescence was performed with OS-30P
pulse modulated chlorophyll fluorimeter (Opti-sciences, Hudson, and USA)
following the procedure described by Kooten and Snel (1990).
Estimation of lipid peroxidation and H2O2
content
Hydrogen peroxide content was determined according to
the method of Velikova and Loreto (2005). The tissue was homogenized in 0.1% (w/v) TCA, 0.5 mL of
the supernatant was mixed with 0.5 mL of 10 mM potassium phosphate
buffer (pH 7.0) and 1 mL of 1 M KI,
and the absorbance was read at 390 nm. Lipid peroxidation was
monitored by spectrophotometric determination of malondialdehyde (MDA) using
thiobarbituric acid (TBA) as described in Wang et al. (2009). The
content of MDA was calculated on a fresh weight basis using the following
formula:
MDA (μmol g-1 FM) = [6.45(OD532-OD600)
-0.56(OD450)×1000]/wt.
Estimation of total carbohydrate, total protein, nitrate
and ammonia contents
The quantification of total available carbohydrate (TAC)
was done following Murata et al. (1968). About 100 mg of finely powdered
oven-dry plant material was hydrolyzed using 0.7 N HCl then assayed as glucose
by phenol sulphoric acid method (Dubois et al. 1956). Absorbance was
read at 490 nm using a UV-Vis Spectrophotometer (JENWAY, 6305, UV/Vis) with
reference to known concentration of glucose.
Total protein (TP) content was determined according to the method of Hatree (1972)
using Folin-phenol reagent. Absorbance was recorded
spectrophotometrically at 650 nm using bovine serum albumin as a standard.
Nitrate contents were measured from an aqueous extraction of
0.2 g dried leaves or roots in 10 mL Millipore-filtered water. A 5-mL aliquot
was dried in an air-drying oven at 60°C to complete dryness, after which 2 mL
of the phenoldisulphonic acid reagent were added, the absorbance of the
solution was measured with a spectrophotometer at 420 nm according to Johnson
and Ulrich (1950)
Ammonium was extracted by homogenizing leaf and root segments in
borate buffer (pH 8) containing 1.0% acetic acid. The homogenate was
centrifuged for 10 min at 16 000 × g and the supernatant was used for determination of ammonium using phenol-hypochlorite method as
described by Solorzano (1969) Absorbance was measured at 630 nm with reference
to known concentrations of ammonium sulphate.
Nitrogen assimilating enzymes Assay
Nitrate reductase (NR, E.C. 1.6.6.1) was
extracted and assayed following the method adopted
by Saber et al. (1989). Fresh plant material
(leaves or roots) was homogenized in 0.05 M Tris – HCl pH 7.5 followed by the addition of potassium nitrate and incubation at 30°C for 2 min. The reaction was
started by addition of 0.6 µmol
NADH.H and allowed to proceed for 15 min at 30şC, then stopped by adding 0.1 M zinc sulphate solution and 95% ethanol. The
NR activity was assayed according to the method adopted by Saber et al. (1989). The produced nitrite was estimated by measuring the absorbance at 290 nm
and the NR specific activity was expressed as µmol NO2-
produced mg-1 protein min-1.
Glutamine synthetase (GS, EC.
6.3.1.2): was
extracted as described by O’Neal and Joy (1973). Plant
material was homogenized in 25 mM
tris-HCl buffer (pH 7.8), 1 mM MgCl2, 14 mM
β-mercaptoethanol and 1% (w/v) polyvinyl pyrrolidone (PVP). GS specific
activity was determined using hydroxylamine as substrate and the formation of
γ-glutamylhydroxamate (γ-GHM) was determined colorimetrically at 540
nm after complexion with acidified ferric chloride (Canovas et al. (1991). The GS specific activity was expressed in µmol glutamyl hydroxamate mg-1 protein min-1.
The NADH-dependent glutamate dehydrogenase
(NADH-GDH, EC.1.4.1.2) was extracted according to Turano et al. (1996). Frozen samples were homogenized using a cold mortar and pestle with
grinding medium consisting of 100 mM
Tris–HCl (pH 7.5), 14 mM β-mercaptoethanol and 1% (w/v) PVP.
NADH-GDH specific activity assays were carried out according to Groat and Vance
(1981). The oxidation of NADH was measured by a UV-vis Spectrophotometer
(TU-1901, Purkinje General, Beijing, China) at 340 nm for 7–10 min and the
specific activity of GDH in units of μmol of NADH oxidized mg-1 protein min-1 was calculated using an extinction coefficient
for NADH at 340 nm.
High-performance liquid chromatography (HPLC) analysis
Plant phytohormons: Endogenous phythormones, namely
auxins (as Indole-3-butyric acid), abscisic acid (ABA) and gibberellic acid (as GA3) were
estimated by HPLC. The plant roots and leaves (1 g) were thoroughly extracted
in 80% methanol containing 0.1% butylhydroxytoluene (Kettner and Dorffling
1995). The extract was centrifuged at 5000 g for 5 min at 4°C and the
supernatant was reduced to aqueous phase using rotary evaporator. The pH of aqueous
phase was adjusted to 2.5–3.0 and extracted four times with half volume of
ethyl acetate. The ethyl acetate was dried completely using rotary evaporator
and the dried sample was re-dissolved in 1 mL of methanol (100%). 50 μL
of methanol extract was analyzed using HPLC system (Agilent technologies 1200
series and UV/VIS detector 200 LC, U.S.A.) equipped with a 5-μm column (Exclipse XDB-18; 4.6 X
150 mm; Brownlee). The solvent used was methanol- 2% acetic acid and H2O
(40:20:20) as the mobile phase, run isocratically at flow rate of 1 mL min-1.
The detector was set at 254 nm for the integration of peak areas after
calibration with the external standard.
Free amino acids: For estimation of free amino
acids, samples were homogenized in 1:10 (w/v) glass distilled water and the homogenate was centrifuged at 5000 rpm for 15 min at 4°C. The supernatant was treated with methanol 1:1
(v/v), centrifuged at 10,000 rpm for 5 min and
collected for analysis using the previously mentioned HPLC system. Free amino
acids were determined as their stable OPA derivative (Williams 1986). The
mobile phase consisted of Solvent A (sodium
acetate buffer pH 6.8) and solvent B
(glacial acetic acid and methanol), run isocratically at flow rate
of 1 mL min-1. The detector was set at excitation
230 and emission 450 nm for the integration of peak
areas after calibration with the external standards.
Statistical analysis
Statistical analysis of the
results was carried out according to Duncan’s multiple range tests using
SPSS-20. Data were subjected to one-way ANOVA following the method of Sokal and
Rohlf (1995). Differences between treatment-means were considered statistically
significant at P ≤ 0.05
Results
Growth parameters
The N-Bio pretreatment enhanced
the biomass accumulation of maize seedlings as well as shoot height. Increasing Cd concentration in the nutrient solution resulted in a
significant decline of FM, DM and shoots height of N-Bio pretreated and
untreated maize plants compared to the control; but the attained values of the
former were greater than the latter (Table 1). The same
trend was observed for water status of leaves and roots. At the end of the
experimental period, the reduction in FM of leaves and roots of 10 mM
Cd-stressed plants was 88 and 92% respectively, compared to control. The
corresponding values for N-Bio pretreatment were 68 and 78%, respectively. In
10 mM Cd-treated roots, the water content raised from 39 to 82% upon
application of N-Bio. The shoot height in 10 mM
Cd-stressed maize plants in presence of N-Bio was 3.4-fold the value of 10 mM
Cd-stressed ones.
Light microscope of root cross sections
Cd stress had a strong negative impact on the anatomical structure
of roots in comparison to control plants (Fig. 1). The epidermal and cortical cells were severely ruptured
with shrinkage and disturbance of pith parenchyma cells as well as a decrease in the diameter and number of metaxylem elements. In addition, there was a marked disorganization and
crimple structures of xylem and phloem elements. Obviously, N-Bio pretreatment of
maize seeds mostly improved the adverse effect of Cd on the root anatomical
structure.
Scanning electron microscope
A clear stomatal structure in control and
N-Bio-pretreated plants were observed, whereas marked variations in stomatal
opening and guard cell shape were noticed in response to Cd stress and N-Bio
application (Fig. 2). The stomata of the 10 mM Cd treated maize
seedlings were highly defective with their completely collapsed, irregularly
thickened guard cells and their stomatal opening almost remained closed. On the
other hand, N-Bio- pretreatment of Cd-stressed leaves increased the stomatal opening compared to Cd- stressed ones which might reveal the
role of N-Bio in enhancing the CO2 diffusion.
Photosynthetic pigments and photosynthetic efficiency
Contamination of nutrient solution with various Cd
levels significantly decreased Chl a, b and total photosynthetic pigments content as well as Fv/Fm values. The carotenoid
Table 1: Effect of biofertilizer application on
fresh and dry biomasses, shoot height and water content in the leaves of maize seedlings grown at 2-
and 10-m Cd for 21 d
Treatments |
Cd conc. (mM) |
FM (mg plant-1) |
DM (mg plant-1) |
Shoot
Height (cm) |
Water contents (%) |
|||
Roots |
Leaves |
Roots |
Leaves |
Roots |
Leaves |
|||
-Bio |
0 |
5.64 ± 0.63c |
19.83 ± 1.65c |
0.71 ± 0.06bc |
1.75 ± 0.13cd |
8.8 ± 0.73 b |
87
± 6.69a |
91 ± 7.00a |
2 |
3.95 ± 0.40d |
14.01 ± 1.56d |
0.64 ± 0.05cd |
1.68 ± 0.15de |
6.5 ± 0.54c |
84 ± 9.33a |
88 ± 8.00a |
|
10 |
0.44 ± 0.03e |
2.35 ± 0.24c |
0.27 ± 0.02e |
0.71 ± 0.06f |
1.7 ± 0.14d |
39 ± 3.90c |
70 ± 7.00b |
|
+ Bio |
0 |
12.94 ± 1.18a |
36.57 ± 3.32a |
1.12 ± 0.11a |
3.71 ± 0.37a |
15.4 ± 1.71a |
91 ± 8.27a |
93 ± 8.45 a |
2 |
9.66 ± 0.97b |
33.10 ± 3.01a |
0.91 ± 0.08ab |
2.48 ± 0.21b |
14.2 ± 1.29b |
91 ± 8.27a |
93 ± 10.33a |
|
10 |
2.75 ± 0.23d |
11.52 ± 0.96b |
0.49 ± 0.03d |
1.53 ± 0.14e |
6.8 ± 0.62c |
82 ± 9.11b |
87 ± 9.67b |
|
P = 0.05 |
0.003* |
0.0087* |
0.005* |
0.003* |
0.017* |
0.039* |
0.04 |
Values are means of 3 independent replicates ±SE. means
followed by different letters are significantly different at P ≤ 0.05 according to the least
significant difference (LSD)
Fig. 1: Scanning electron
microscopy (SEM) of abaxial leaf surface from 21-day-old maize plants in
response to Cd-stress and biofertilizer application. (A): untreated control, (B):
10 mM Cd, (C): biofertilizer and (D):
biofertilizer +10 mM Cd
contents were insignificantly
changed compared to control (Table 2). Inoculation of maize seeds with N-Bio significantly
increased total pigments content and Fv/Fm values in
Cd-stressed leaves versus those of non-inoculated ones. It is interesting to
demonstrate that in absence of Cd, inoculation of N-Bio significantly increased
the photosynthetic pigments content compared to non-inoculated control.
H2O2 content and Lipid peroxidation
After prolonged exposure of
maize plants to Cd stress, there was a significant increment in H2O2
content in a concentration dependent aspect; it was about 5.4- and 4.6-folds
the value of control in response to 10 mM Cd in the leaves and roots,
respectively (Fig. 3). Inoculation with N-Bio resulted in a significant
depression in H2O2 accumulation compared to
non-inoculated treatment. At N-Bio-10 mM Cd-
treatment, the decrease in H2O2
content was
Fig. 2: Light
microscope photography showing transverse sections of 21-day-old maize roots in
response to Cd-stress and biofertilizer application (A): untreated control, (B):
10 mM Cd, (C): biofertilizer and (D):
biofertilizer +10 mM Cd. (Ep) Epidermis,
(End) Endodermis, (Cor) Cortex, (Ph) Phloem, (Mxy) Metaxylem, (Pxy) Protoxylem
and (P) Pith
41 and 55% in the leaves and
roots, respectively compared to the value at 10 mM Cd alone.
In
parallel with changes in H2O2, Cd treatment significantly increased MDA content, indicting lipid peroxidation in the
leaves and roots of maize plants compared to control. However, inoculation of
maize seeds with N-Bio reduced the Cd-induced MDA accumulation (Fig. 3).
Carbohydrate, protein, nitrate
and ammonia contents
Increasing Cd- levels significantly reduced TAC and TP
in the leaves and roots of maize seedlings (Table 3). At
10 mM Cd treatment, the TAC content of leaves and
roots were 49 and 38% of control, respectively. The corresponding values for TP
were 36 and 30%, respectively. The interactive effect of N-BiO and Cd displayed
a significant
Table 2: Effect of biofertilizer application on
photosynthetic pigments (mg g-1 FM) and quantum yield of PSII
(Fv/Fm) in the leaves of maize seedlings
grown at 2- and 10-m Cd for 21 d.
Treatments |
Cd conc. (m) |
Pigment content (mg g−1
FM) |
Quantum yield of PSII (FV/FM) |
|||
Chl. a |
Chl. b |
Carot. |
Total |
|||
-Bio |
0 |
22.86 ±
2.54b |
12.08 ± 1.10c a |
5.75 ±
0.52d |
40.68 ±
3.39a |
0.807 ± 0.09a |
2 |
14.94 ±
1.15c |
7.54 ±
0.66d |
6.40 ±
0.71d |
28.88 ±
2.72c |
0.730 ± 0.08bc |
|
10 |
5.12 ±
0.47d |
2.38 ±
0.20d |
7.46 ±
0.42cd |
14.96 ±
1.08d |
0.562 ± 0.04d |
|
+ Bio |
0 |
34.92 ±
2.49a |
10.03 ±
0.63b |
9.56 ±
0.87bc |
54.51 ±
6.06a |
0.815 ± 0.07a |
2 |
28.78 ±
3.20ab |
14.22 ± 1.19ac |
10.79 ±
0.98bc |
53.79 ±
4.48a |
0.780 ± 0.07b |
|
10 |
12.63 ±
1.64c |
6.60 ±
0.96d |
17.54 ±
1.75a |
36.77 ±
4.25b |
0.721 ± 0.07c |
|
P = 0.05 |
0.019* |
0.009* |
0.035* |
0.028* |
0.007* |
Values are means of 3 independent replicates ±SE. means
followed by different letters are significantly different at P ≤ 0.05 according to the least
significant difference (LSD)
Table 3: Effect
of biofertilizer application on nitrate (mg g-1 DM), ammonium (µg g-1
DM), total available carbohydrates (mg g-1 DM) and total protein
contents (mg g-1 DM) in the leaves and roots of maize seedlings grown at 2-
and 10-mM Cd for 21 d.
Treatments |
Cd conc. (mM) |
NO3-
content (mg- g-1 DM) |
NH4+
content (µg g-1 DM) |
TAC (mg g-1 DM) |
TP (mg g-1 DM) |
||||
Roots |
Leaves |
Roots |
Leaves |
Roots |
Leaves |
Roots |
Leaves |
||
-Bio |
0 |
1.50 ± 0.045a |
2.53 ± 0.281c |
3.38 ± 0.307 d |
10.74 ± 1.193dc |
66.79 ± 6.07b |
91.32 ± 10.15b |
19.43 ± 1.49b |
42.51 ± 4.72bc |
2 |
0.82 ± 0.047c |
1.74 ± 0.158d |
12.64 ± 1.149c |
21.16 ± 1.924c |
62.69 ± 5.70b |
81.08 ± 9.01c |
16.91 ± 1.30c |
37.88 ± 3.16cd |
|
10 |
0.38 ± 0.072e |
0.48 ± 0.051f |
54.06
± 5.691a |
77.22 ± 8.128a |
25.47 ± 1.96d |
45.04 ± 4.50d |
5.76 ± 0.58d |
15.16 ± 1.26e |
|
+ Bio |
0 |
1.48 ± 0.053a |
4.59 ± 0.399a |
4.21 ± 0.468d |
6.08 ± 0.676f |
74.65 ± 6.22a |
101.65 ± 8.47a |
24.64 ± 2.46a |
51.11 ± 4.26a |
2 |
1.09 ± 0.041b |
3.06 ± 0.269b |
6.99 ± 0.583cd |
8.83 ± 0.803ef |
73.52 ± 7.35a |
94.57 ± 7.88b |
23.26 ± 2.11a |
48.38 ± 4.03ab |
|
10 |
0.55 ± 0.058d |
1.06 ± 0.112c |
25.36 ± 2.669b |
35.56 ± 3.743b |
42.52 ± 3.54c |
78.32 ± 6.02c |
12.53 ± 1.14c |
32.27 ± 3.23d |
|
P = 0.05 |
0.001* |
0.003* |
0.010* |
0.009* |
0.017* |
0.011* |
0.025* |
0.009* |
Values are means of 3 independent replicates ±SE. means
followed by different letters are significantly different at P ≤ 0.05 according to the least
significant difference (LSD)
Fig. 3: Effect of N-biofertilizer application on hydrogen
peroxide (H2O2) and malondialdehyde (MDA) in the roots and leaves of maize seedlings grown at 2-
and 10-mM Cd for 21 d
Values are means of 3 independent replicates ±SE. means
followed by different letters are significantly different at P ≤ 0.05 according to the least
significant difference (LSD)
increment in TAC and TP contents compared to those of
Cd treated-plants. The TAC and TP contents in N-Bio inoculated leaves in
presence of 10 mM Cd were 1.7- and 2.1-folds, respectively compared to
those of non-inoculated ones. The corresponding values in roots were 1.9- and 2.5-folds,
respectively.
Supplementing the nutrient
solution with various Cd levels significantly suppressed NO3- content
in leaves and roots of maize plants either pretreated or untreated with N-Bio,
compared to their controls; but attained values of the latter were markedly
higher than the former (Table 3). The increase in the NO3- content
in the leaves and roots of N-Bio inoculated plants grown at 10 mM Cd was
121 and 45% compared to those of non inoculated ones.
Conversely to the NO3- trend, there
was a marked accumulation of NH4+ content in leaves and
roots of maize plants with increasing Cd concentrations in growth media in
absence or presence of the N-Bio, but the attained values in the latter were
lower than those in the former. On the other hand, N-Bio-
pretreatment of Cd-stressed plants decreased the NH4+
accumulation under Cd stress. The NH4+ content in leaves and
roots of N-Bio-10 mM Cd-treated plants decreased
by 54 and 53%, respectively compared to N-Bio-pretreated plants alone.
Changes in phytohormones and
free individual amino acids
The
10 mM Cd stress brought about significant decline of IAA and GA3
contents in leaves and roots of N-Bio inoculated and non-inoculated maize
plants, whereas ABA significantly increased in comparison to controls (Table 4). The decrease of IAA and GA3 contents in leaves of
Cd-stressed plants were 49 and 66% respectively compared to the control. The
corresponding values for roots were 66 and 44% respectively.
Contrarily, the increase of ABA in leaves
Table 4: Effect of
biofertilizer application on plant phytohormons (ng g-1 FM) in the
leaves and roots of maize seedlings grown at 2- and 10-mM Cd for 21 d. (ABA):
Abscisic acid; (IAA): indole acetic acid and (GA3): gibberellic acid.)
Treatment |
Cd conc. (mM) |
Hormone
Content (ng g-1 FM) |
|||||
IAA |
GA3 |
ABA |
|||||
Roots |
Leaves |
Roots |
Leaves |
Roots |
Leaves |
||
-Bio |
0 |
18.14 ± 1.64b |
22.05 ± 2.20c |
36.15 ± 3.27c |
48.11 ± 4.80c |
9.09 ± 0.75c |
10.08 ± 0.83c |
10 |
6.22 ± 0.46c |
11.30 ± 0.92d |
20.10 ± 2.00d |
16.21 ± 1.33d |
23.06 ± 1.92a |
35.10 ± 3.89a |
|
+ Bio |
0 |
31.11 ± 2.38a |
46.09 ± 4.18a |
51.14 ± 5.67a |
106.11 ± 10.60a |
9.09 ± 0.75c |
11.11 ± 1.00b |
10 |
17.09 ± 1.31b |
32.12 ± 2.91b |
44.12 ± 4.00b |
70.10 ± 7.78b |
16.08 ± 1.60b |
16.10 ± 1.78b |
|
P |
0.021* |
0.029* |
0.012* |
0.009* |
0.015* |
0.011* |
Values are means of 3 independent replicates ±SE. means
followed by different letters are significantly different at P ≤ 0.05 according to the least
significant difference (LSD)
Table 5: Effect of biofertilizer application on free individual
amino acid content (mg 100 g-1 DM) in the roots and
leaves of maize seedlings grown at 2- and 10-mM Cd for
21 d.
Amino acids (mg 100 g-1 DM) |
Treatment |
|||
Control |
Bio |
10 mM Cd |
10 mM + Bio |
|
Aspartic acid |
1.49 |
2.07 |
2.46 |
3.00 |
Glutamic acid |
1.98 |
5.40 |
0.61 |
3.43 |
Glutamine |
1.16 |
0.88 |
0.07 |
0.19 |
Arginine |
1.35 |
1.17 |
2.31 |
1.02 |
Lysine |
1.19 |
0.98 |
0.38 |
0.33 |
Alanine |
0.34 |
0.95 |
0.16 |
1.29 |
Glycine |
0.34 |
0.72 |
0.04 |
0.34 |
Isoleucine |
0.23 |
0.47 |
0.12 |
0.17 |
Leucine |
1.45 |
1.06 |
0.57 |
0.87 |
Serine |
0.19 |
0.92 |
0.10 |
0.13 |
Theronine |
1.39 |
4.51 |
0.23 |
2.63 |
Tyrosine |
0.58 |
0.18 |
0.34 |
0.46 |
Phenylalanine |
0.51 |
1.20 |
1.40 |
0.33 |
Methionine |
0.41 |
0.37 |
0.15 |
1.20 |
Cysteine |
0.34 |
0.44 |
0.09 |
0.32 |
Tryptophan |
1.44 |
3.46 |
0.17 |
0.98 |
Total |
14.39 |
24.78 |
9.02 |
16.69 |
and roots of 10 mM Cd-stressed maize plants was
248 and 154% compared to control, respectively. N-Bio pretreatment alone or in
combination with Cd stress had a stimulatory effect on the endogenous IAA and GA3 while it decreased the ABA content, compared to their respective
controls.
Among 16 detected free amino acids, 10 mM Cd resulted in a marked increase in arginine (Arg, 71%),
aspartic acid (Asp, 65%) and phenylalanine (Phe, 176%) compared to control
(Table 5). Other amino acids were markedly decreased such as glutamic acid
(Glut, 69%), glycine (Gly, 88%) and cysteine (Cys, 74%). Application
of N-Bio in presence of 10 mM-Cd mainly led to suppression in the level
of 3 amino acids namely Arg, Lyc and Phe while the other detected amino acids
were markedly increased compared to non-inoculated Cd-stressed leaves.
Enzyme activities
Generally, prolonged exposure to Cd-stress in presence
or absence of N-Bio significantly decreased both NR and GS specific activity
in leaves and roots of maize plants in comparison to their controls (Fig. 4). The decrease of NR and GS specific activity in severely Cd-stressed leaves was 79 and 75%,
respectively compared
Fig. 4: Effect of N-biofertilizer application on NR, GS and GDH specific activities in the roots and leaves of maize seedlings grown at 2- and 10-mM Cd for 21 d
Values are means of 3 independent replicates ±SE. means
followed by different letters are significantly different at P ≤ 0.05 according to the least
significant difference (LSD)
to control. The corresponding values for roots were 81
and 88%, respectively. The same trend was observed upon
N-Bio pretreatment but the attained values for both NR and GS specific
activities were much less than in Cd-treated plants.
In contrast to NR and GS trends,
contaminating the nutrient media with various Cd levels significantly increased
GDH specific activity in leaves and roots of N-Bio-untreated or treated maize
plants (Fig. 4). At 10 mM Cd stress, the GDH specific activity in leaves
and roots was 2.3-and 6-folds respectively, compared to untreated control. The
corresponding values in N-Bio-pretreated leaves and roots were 1.5- and
1.8-folds, respectively.
Discussion
Cd-stress negatively influenced
several growth parameters of maize plants. Several studies have shown that
growth biomarkers have been depressed due to Cd-stress in a number of plants
including Solanum melongena L. (Singh and Prasad 2014) and Zea
mays L. (Liu et al. 2007). It has been reported that increasing Cd
accumulation in plant organs could enhance the generation of ROS which caused
the damage of plasma membranes, photosynthetic pigments, and various cellular
components, leading to reduction of growth (Liu et al. 2007). In
accordance with these views, the current study demonstrated that Cd stress
induced a significant accumulation of H2O2 and MDA in
leaves and roots of maize plants and disturbed the root anatomical structure causing
immature xylogenesis and dysfunctions of phloem which reflect the decrease of
water and photosynthates allocation. Moreover, the disturbance of the
anatomical feature of maize roots was accompanied with a marked accumulation of
Cd in roots (Data not shown) and a significant decline of IAA and GA3
contents indicating the inhibitory effect of Cd on auxins biosynthesis and
disordering the mitotic divisions leading to suppression of cell divisions and
elongation and hence the growth. Soudeh and Zarinkamar (2012) reported that
inhibition of root growth may be the result of decreased cell division and or
disorderliness in the activity and contents of phytohormones like auxins in
response to heavy metals stress. Cd-stress seems to provoke a series of
structural alterations with possible functional implications in the maize plant
such as shrinkage of root diameter (Li et al.
2014) and reduction in the metaxylem vessels diameter (Gowayed
and Almaghrabi (2013) which is considered as important factor affecting root
capacity as translocation conduits.
Comstock
(2002) reported that the regulation of guard cell has become a crucial model
system for explaining the regulatory signals that control stomatal behavior. Cd
exposure profoundly alters the behavior of stomata in maize leaves (Fig. 1).
Similar observations were reported for many plant species grown under different
heavy metals stress (Mondal et al. 2013; Divyajyothi and Sujatha 2019). The
defective stomata probably might have lost a functional closing mechanism, and
therefore were unable to regulate the exchange of water vapor and CO2,
which decreases both transpiration and photosynthesis (Fatemy et al. 1985).
Furthermore, this adverse stomatal closure may be due to the loss of turgor of
the guard cells and the damage to the guard subsidiary cells (Priyadarshini and
Sujatha 2011).
In the present study, Cd stress
triggered a significant reduction in the photosynthetic pigments content and the quantum yield of PSII (Fv/Fm).
These observations are in accordance with those reported for coriander (Haneef
et al. 2013) and tomato (Singh and prasad 2017). The suppression in photosynthetic pigments content has been reported to
be related to the inhibitory effect of Cd on specific enzymes responsible for their
synthesis and induction of some degradative enzymes such as chlorophyllase as
well as destruction of the photosynthetic machinery and reduction in the
chlorophyll proteins content (Singh and Prasad 2017). In addition, Singh and Prasad (2014) suggested that the decrease of
Fv/Fm values in plants under Cd stress could be related to the decline in the active reaction centers and inability of PSII to reduce the primary acceptor
(QA) resulting in a decrease of electron transport and photosynthetic activity.
Therefore, the decline of chlorophyll contents in Cd-stressed maize plants
could be related to the enhancement of lipid peroxidation of thylakoids and
chloroplast membranes as indicated by increasing MDA content in the leaves.
Moreover, the decrease of TP content, due to increase of oxidative stress by
generated ROS (H2O2) might inhibit the biosynthesis and
content of pigment-protein complexes of photosystems, hence reduce the
photosynthetic activity and growth.
The results of this study
apparently demonstrated that N-Bio pretreatment significantly increased the
growth of Cd-stressed maize plants. Fukami et al. (2017) reported that
spraying cells or metabolites of Azosperillum brasilense Ab-V5 and Ab-V6
enhanced the growth of maize plants. Gothandapani et al. (2017) have
reported that application of various plant growth promoting microorganisms
(PGPMs) induces the growth biomarkers in several plants. Earlier, Sokhangoy
et al. (2012) concluded that biofertilizers might enhance the nutrient
availability to plants causing an increase of growth. Whereas, Etesami (2018)
suggested that secretion of plant hormones (e.g., IAA) by biofertilizers
might induce the plant growth. Therefore, the increase of growth
biomarkers in N-Bio-pretreated maize plants under Cd stress could
be attributed to increasing the absorption of plant hormones (IAA) and
nutrients including NO3- from the rhizophere. Data
recoded in this study (Table 3 and 4) pointed out that, there was a marked
increase of NO3- content and plant growth regulators (IAA
and GA3) in leaves and roots of N-Bio-pretreated maize plants under Cd stress
in
comparison to untreated ones. These observations were accompanied with a marked
increase of TAC and TP contents, revealing the stimulation of growth. Several
studies in Eruca sativa (Kamran et al. 2015) and Zea mays
(Roychowdhury et al. 2017) grown under Cd stress showed a marked
reduction in growth in response to Cd stress, while PGPR application could
contribute to improved growth indices.
Furthermore, N-Bio treatment
markedly increased the photosynthetic pigment contents and Fv/Fm ratio of
Cd-treated maize leaves compared to non-treated plants. Similarly, Khanna et
al. (2019) found that plant growth promoting microorganism’s inoculation
resulted in an increase of photosynthesis and growth of Cd-stressed tomato
plants. The findings in this study might be explained by decreasing the
generation of ROS and oxidative damage of chloroplasts and thylakoids membranes
and enzymatic proteins as well as increasing the availability of essential
elements introducing in chlorophyll biosynthesis (as indicated by the increase
of NO3 content). Moreover, inoculation of maize seeds with N-Bio
significantly declined ABA content that was associated with an increase of
stomatal opening. These observations might reflect the increase of CO2
diffusion and hence increase photosynthesis. Haneef et al. (2013)
reported that PGPMs are capable of shifting off the toxic effect of heavy
metals through enhancing the mobilization of elements such as N, K, P, Mg and
Fe.
Nitrogen (N) is one of the
essential nutrients involved in biosynthesis of various cell components such as
amino acids, protein and chlorophylls that reflect its essential role in
sustaining the growth of plants. Decreased TP content in the maize plants grown
on excess Cd was in accordance with earlier observations in several plants such
as chamomile plants (Kovacik and Backor 2017). It has been reported that the
decrease of protein content under Cd stress may be related to enhancement of
protein hydrolysis and/or decrease of protein synthesis in addition to the
suppression in amino acids biosynthesis (Xu et al. 2012). It can be
suggested that the decrease of TP content in Cd-stressed maize plants, in this
study, might be attributed to increasing ROS generation, as indicated by
increase of H2O2 accumulation, which cause oxidative
damage for protein. Moreover, the decrees of C-skeleton, due to decline of TAC
and inhibition of NO3- uptake and its assimilation, via
NR and GS (Fig. 4), could result in a marked decrease of amino acids
biosynthesis and hence protein synthesis.
It is well documented that
plants exposed to different abiotic stresses modulate total and amino
composition (Zemanová et al. 2017). The findings in this study indicated
an increase of some amino acids while others were decreased. These variations
might reveal the participation of various amino acids in the biosynthesis of
secondary metabolites involved in strategy mechanism (Chaffei et al.
2004; Xu et al. 2012). The decrease of arginine and phenylalanine could
be involved in the biosynthesis of polyamine and phenolic compounds, while the
decrease of methionine, cysteine, glutamic and glycine content might be
introduced in the synthesis of phytochelatine and thiol compounds such as GSH
and thiol-non proteins. On the other hand, the decrease of tryptophan content
might reflect the suppression of IAA concentration in Cd-stressed maize plants.
Costa and Spitz (1997) reported that increasing of asparagine accumulation in
Cd-treated in vitro lupin tissues culture could participate in the
synthesis of chelate peptide for detoxification of the Cd toxicity. It is
interesting to demonstrate that methionine content decreased markedly in 10 mM
Cd-stressed plants while it increased upon application of N-Bio compared to
stressed non-inoculated plants. These findings might reflect the role of N-Bio
in the protection of maize growth from the inhibitory effect of ethylene via
secreting of 1-amino cyclopropane 1- carboxylate (ACC). Ahmad et al.
(2013) reported that N-Bio can produce several protective enzymes such as ACC
deaminase. Kang et al. (2010) postulated that ACC, the precursor of
ethylene is synthesized from methionine via ACC synthase activity, and
then produced ethylene could inhibit the growth. Thus, the decreased methionine
content in Cd-treated leaves, in this study, was accompanied with a marked
reduction of growth, and that might be explained by enhancing ethylene
production under Cd stress causing a marked inhibitory effect on maize growth.
It has been firmly established
that NO3- is taken up via specific channel systems
and ATP-dependent plasma membranes-associated carriers (Forde 2000). The
suppression of NO3- content and NR activity in leaves and
roots of maize plants imposed to Cd stress might be correlated with Cd-induced
disturbance of plasma membranes that might disordered the specific NO3-
channels and plasma membrane-associated NO3- carriers.
Moreover, the disturbance of cortical cells and vascular system of Cd-stressed
roots could lead to suppression of NO3- transportation
through xylem elements from roots to leaves. Furthermore, the decline of TAC,
the requisite C-skeleton and source of H-donors for nitrogen assimilation might
indirectly inhibit NR activity. Besides, the reduction of NR activity in
Cd-treated maize plants could be related to the oxidative damage of NR enzyme
and NO3- carrier proteins, by generated ROS and
interaction of functional SH groups of the enzyme with Cd as well as
suppression of NR gene expression (Erdal and Turk 2016).
It is noteworthy that parallel
to the suppression of NO3- content and NR activity in
leaves and roots of maize plants imposed to Cd stress, there was a significant
increase of NH4+content and induction of NADH-GDH. Similarly,
several studies have demonstrated a notable increment of NADH-GDH activity under
Cd stress (Chaffei et al. 2004; Skopelitis et al. 2006).
Singh and Prasad (2017) reported that the suppressed GS and GOGAT activities in
Cd-treated tomato seedlings could resulted in a marked disturbance in NH4+
assimilation process, and that leads to an increase of NH4+
accumulation and a decrease of protein content. Britto and Kronzucker (2002)
suggested that NAD(P)H-GDH activity (an alternative enzyme for NH4+
assimilation) might be enhanced when GS/GOGAT cycle is inhibited to protect the
cell damage caused by accumulated NH4+. Thus, it is
possible that the increase of NADH-GDH activity was to compensate the decrease
of GS activity to sustain NH4+ assimilation resulting in
a marked accumulation of NH4+. Application of N-Bio
partially shifted off the inhibitory consequences of Cd on both processes as
indicated by the significant increase of NR, GS and GDH activities as well as
NO3- content in N-Bio-pretreated Cd-stressed plants
compared to those of Cd-stressed ones. The increase of NO3-
uptake and NO3- transportation and its assimilating
enzymes in N-Bio-pretreated maize plants might be attributed to decreased
Cd-bioavailability to root system via increase of Cd adsorption on
organic matter (Pacwa-Płociniczak et al.
2011) and reduction of oxidative damage, hence controlling plasma membrane
integrity.
Conclusion
This
study pointed out that N-Bio treatment (Azotobacter spp. and Azospirillum
spp.) was able to mitigate Cd toxicity in maize plants causing an
increase in growth biomarkers. However, N-Bio pretreatment
may have restored the growth of maize plants via increasing of NO3-
uptake (content) and modulation of its assimilating enzyme activities (NR, GS,
and GDH) as well as suppression of oxidative damage of plasma membranes and
improvement of Cd-induced alteration in root anatomical structure. Moreover,
inoculation with N-Bio may have enhanced the uptake of N-Bio secreted
phytohormones (IAA, GA3) which was accompanied with a decrease of ABA content
leading to an increase of stomatal opening, CO2 diffusion and
finally increasing the
photosynthetic efficiency.
Author Contributions
The authors confirm contribution to the paper as follows:
study conception and design: Saber N.E., Abou-Zeid H.M., Ismail G.S.M.,
following lab experiments: Abdelrahim B.I., Saber N.E., Abou-Zeid H.M., Ismail
G.S.M. Data analysis and interpretation: Saber N.E., Abou-Zeid H.M., Ismail
G.S.M., Abdelrahim, B.I., draft manuscript preparation: Ismail G.S.M.,
Abou-Zeid H.M., critical revision of the article and final approval of the
version to be published: Saber N.E., Abou-Zeid H.M., Ismail G.S.M.
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