Introduction
In recent times, a serious concern has developed among
researchers and environmentalists regarding the metals and their harmful
association with the plants. Although some metals are known to be beneficial
for plants when applied in specific quantities, most of the metals are
detrimental to plant growth overall (Zeng et al. 2008). Usually they cause Hindrance in growth, development and metabolic processes and
accelerate senescence in plants (Sah et al. 2016). Thus, the metal pollution
in soil becomes an increasing risk for human well-being and the environment (Yáñez et al.
2002). In this context, the whole world should worry about the metals
pollution (He et al. 2008). The
commonly contaminating metals are cadmium (Cd), chromium (Cr), copper (Cu),
mercury (Hg), Pb and zinc (Zn), while burning of
fossil fuels, industrial residues, waste water, sewage sludge, and phosphate
fertilizers are their major sources. These sources have considerably enhanced
the level of these metals in the soil (Khan et
al. 2008). The Pb2+
is a hazardous trace element because of its greater solubility and toxicity in
the soil (Wani et
al. 2015). Plant roots simply absorbed Pb2+ and then uptake it to above ground plant parts, entering
the food chain via consumption of
vegetables and causing risks to human fitness (Cao et al. 2010).
The Pb2+
negatively affects various biological processes in plants. Reacting to Pb2+
stress, plants over generate reactive oxygen species (ROS) (Zulfiqar
et al. 2019) that cause oxidative
stress, which leads to increase in nonspecific lipids oxidation, membrane
permeability, Chl. degradation,
damages to proteins and DNA (Hasanuzzaman et al. 2018). Moreover, it activates
downstream signaling for transcriptional regulation (Vinocur
and Altman 2005), possibly through up-regulation of mitogen-activated protein
kinases (Hamel et al. 2006). In
Plants, ROS signaling and calmodulin (CaM) and CaM-like
proteins/signaling pathways seem to be the leading players in transcriptional
regulation under abiotic stresses (Zeng et al. 2015). One of the ways of plant
defense from ROS, the plants possess protective antioxidant enzymes and low
molecular weight antioxidants (Cuypers et al. 2016). Ascorbate-glutathione
(AsA-GSH) cycle and GHS metabolism are involved in
the detoxification of H2O2 in various cell compartments (López-Climent et al.
2014). The degree of oxidative damage is assessed in the form of MDA, which is
the product of lipid peroxidation in plasma membranes (Grotto et al. 2009). Further, activities of
antioxidant enzymes like CAT, SOD, APX and GR rise in response to Pb2+
stress (Caverzan et
al. 2012).
Tomato (Lycopersicon lycopersicum L.) plants serve as a rich source of minerals,
vitamins and lycopene (Wilcox et al.
2003), and can grow under all climatic regions of the globe. Nevertheless,
ecological stress factors limit its production (Gerszberg
and Hnatuszko-Konka 2017). In Pakistan, tomato
is 2nd primary vegetable crop (FAO
2009). Metals, particularly Pb2+ have harshly hindered the
production of tomato (Mengel et al. 2001). Irrespective of better nutritious value, its yield
per hectare is very less. However, breeding or transgenic strategies have
revealed significant results in improving plant stress tolerance (El-Esawi and Alayafi 2019) Further, plants can acquire tolerance
to abiotic stresses through general and specific stress components (Sung et al. 2003). Plants adapt to abiotic
stresses by regulating the expression of several stress-responsive genes (Jiang
et al. 2007). Accumulation of Pb2+
contents in various plant parts and its effect on fruit quality and production
is seldom studied. However, little information is available in the literature
about the impact of Cat2 gene in Pb2+
tolerance in tomato plants and its role in nutritive value of tomato fruit as
well. Therefore, in this study, two contrasting tomato cultivars were grown
under different Pb2+ regimes with the hypothesis that Pb2+
stress might differently modulate key physio-chemical
and molecular processes to affect growth and fruit quality in tomato.
Materials
and Methods
Experiment site, and growth conditions
This study was conducted in experimental research
site of Department of Botany, Government College University Faisalabad,
Pakistan during winter season (November-2018 to March-2019). Seedlings of two tomato cultivars
having differential metal tolerance capacity (Hussain
et al. 2017), namely, Nagina (tolerant) and Roma (sensitive) were collected from vegetable section, Ayub Agriculture Research Institute, Faisalabad, Pakistan.
The plants were grown under 11 h/13 h light/dark period and 24°C/12°C day/night
temperatures, average relative humidity (54%), and photosynthetically
active radiation (PAR 687–948 μmol m−2 s−1) during
the whole study. Selected three seedlings of Nagina
and Roma were maintained in each pot (dimension 30 cm high, circumference 50 cm
at bottom and 60 cm at top). At vegetative stage, plants were subjected to
different Pb2+
regimes (160, 320, 640 and 1280 μM) for three weak. For this purpose, Pb2+
solutions were prepared in Hoagland nutrient solution and applied to each pot
in an increment of 40, 80, 160 and 320 μM each day until desired Pb2+ level were achieved. Lead nitrate was used as Pb2+ source. An equivalent
amount of NO3 (as calcium nitrate) was added to the control plants
to compensate for NO3 content of Pb2+-treated plants in nutrient solution. Experiment
was laid out in completely randomized design (CRD) in triplicates. Fresh leaf
samples were harvested three weeks after the application of Pb2+ treatments and kept in
a freezer at -20oC for determination of different growth and
biochemical attributes. Fruit quality attributes were obtained from mature but
green tomato fruits.
Data collection
Growth attributes: The growth parameters including shoot length, leaf area,
shoot and fruit biomasses of two plants per replicate were studied. Leaf area
was determined by multiplying the maximum length (L) and width (W) of leaf with
the correction factor (6.45) obtained by using graph squares.
Leaf area (cm2) = L × W × 6.45 (Hussain et al.
2017)
Analysis of photosynthetic
pigments
Tomato fresh leaves (0.1 g) were
used for pigments extraction in 80% acetone. The mixture
was centrifuged at 4000 rpm for 10 min. The optical density (OD) was assessed
at 480, 645 and 663nm spectrophotometrically (Lichtenthaler 1987) and Chl.
pigments were calculated by using the following formulas.
Chl. a (mg g-1 FW) = 12.7 (OD663 –2.69 (OD645) × (V/1000 × W)
Chl. b (mg g-1 FW) = 12.9 (OD645–4.68 (OD663) ×
(V/1000 × W)
Total Chl. (mg g-1 FW)
= 20.2 (OD645–8.02 (OD663) × (V/1000 × W)
Car. (mg g-1 FW) = OD480
+ (0.114 × OD663) - (0.638 × OD645)
Estimation of H2O2 contents
Determination of H2O2
content in fresh leaves was done according to the method of Velikova
et al. (2000). For this purpose, 0.2
g fresh leaves were grinded in 0.1% TCA (5 mL) and then centrifuged for 15 min
at 12000 g, the supernatant mixed with potassium phosphate buffer (10 mM; pH 7.0) and
potassium iodide (1 mL; 1 M). Optical
density was determined at 390 nm.
Analysis of malondialdehyde
(MDA) contents
Tomato leaves (0.2
g) were grinded in 5.0 mL of 5% (w/v) TCA, and centrifuged. The MDA was
estimated at 532 and 600 nm spectrophotometrically
using thiobarbituric acid assays (Heath and Packer
1968).
Analysis of ascorbic acid content
Ascorbic acid was
measured by using the method of Mukherjee and Choudhuri
(1983). Tomato fresh sample (0.2 g) was grinded in ice bath with 5.0 mL of TCA
(6%) and centrifuged. Two mL of supernatant was dissolved with 2,4-DNP hydrazine (1 mL). Then one drop of thiourea (10%) was added to the mixture and heated (95°C)
for 20 min, then cool down the mixture at 25°C. Thereafter, cool 80% H2SO4
(2.5 mL) was added to the mixture placed on ice bath and the absorbance was
read at 530 nm.
Protein and antioxidant enzymes assays
The total soluble
proteins in fresh leaves of tomato were estimated by Bradford method using Coomassive blue dye (Bradford 1976). The reaction mixture
contained 100 μL
of protein sample (leaf extract) and 5 mL of Bradford reagent while the blank
had 100 μL
of distilled water. These test tubes were incubated for 20 min in dark and then
absorbance measured at 595 nm. For the antioxidant enzyme, fresh leaf material
homogenized with 10 mL of chilled potassium phosphate buffer (50 mM; pH 7.5) was
poured down. The mixture was centrifuged at 12,000 g at 4°C. The
supernatant was collected and used for the determination of following antioxidant enzyme
activities.
Estimation of POD activity
The
reaction volume (3 mL) consisted of phosphate buffer (50 mM; pH 7.5),
guaiacol (20 mM) and H2O2 (5.9 mM) and enzyme extract (100 μL). The POD contents were assessed at 470 nm after every 20 seconds for 2 min spectrophotometrically (Chance and Maehly 1955)
Estimation of CAT activity
The CAT activity
was analysed with method of Chance and Maehly (1955).
The reaction solution contained 1 mL H2O2 (5.9 mM), phosphate buffer (50
mM; pH 7.5)
and enzyme extract (100 μL).
The change in absorbance of reaction mixture was noted at 240 nm after every 20
seconds for 2 min.
Estimation of APX activity
The reaction
volume for APX (1 mL) contained phosphate buffer
(50 mM;
pH 7.5), AsA (0.5 mM), H2O2
(0.1 mM)
and enzyme extract (200 μL).
The readings of the mixture were taken at 290 nm after every 20 seconds for 2
min (Nakano and Asada 1987).
Cat gene
expression with RTq-PCR analysis
Fresh leaves (0.1
mg) were weighed from each sample, and obtained using the total RNA extraction
kit (Takara Bio Inc. Japan). The RNA
purity and integrity was confirmed by using micro-spectrophotometer (Nano Drop
ND-1000) and agarose gel electrophoresis,
respectively after extraction. The cDNA was
synthesized from total RNA (1 µg) using a Prime Script RT Reagent Kit
(Takara Bio Inc.) and subjected to RT-PCR. The KAPA SYBR FAST qPCR Kit (Kapa Bio, Boston, MA, USA)
was used for amplification to observe the Cat2 gene expression level by using RTq-PCR (Bio-Rad, Hercules, CA, USA). The
amplification conditions were: pre-denaturation at 95°C for 30 sec;
denaturation at 95°C for 5 sec; annealing at 60°C for 20 sec; extension at 72°C
for 10 sec, 40 cycles. After the completion of reaction, verified the
amplification curve and melting curve, and Ct value was calculated from the
amplification curve. Actin (NM_00111149) was used as a control or data
normalization. Transcript level of
the specific gene was calculated
using the 2-△△Ct method (Livak and Schmittgen 2001). Sequences
of Cat 2 and Actin primers are
given in Table 1.
Fruit moisture and ash contents determination
The moisture
contents in fruit were calculated after drying using air forced draft oven and
ash content by the method (44-15A and 8-1) explained in AACC (2000),
respectively.
Fruit total protein and fibre contents determination
The nitrogen
contents in fruit were calculated with Kjeldhal’s
procedure and crude fibre according to method (46-10A and 32-10) described in
AACC (2000), respectively. The protein content in fruits measured by their
total N content and multiplied by factor 5.7 (N×5.7) to obtain protein content
in crude form (Jones 1931)
Estimation of mineral nutrients from tomato fruits
Fruit samples (500
mg) of each tomato variety were digested with HNO3 and HCLO3
(3:1) at hot plate for 2–3 hours. Digested material was used to measure the
nutrients with the procedure of Yoshida et
al. (1976). Calcium (Ca2+), potassium (K+) and sodium
(Na+) contents in the fruit of tomato were measured using atomic
absorption spectrophotometer (Hitachi, Z-2000, Tokyo, Japan). Similarly, Pb2+
concentration was determined in root, shoot, leaves and fruit samples of
tomato.
Table 1: Primer for real-time PCR
Plant |
Accession No. |
Gene |
Amplicon size (bp) |
Sequences (5´→3´) |
Tomato |
NM_001247257.1 |
Cat 2 |
185 |
F 5′- ctt tcc tct tcg
acg ata ttg gta R 5′- gtg att tgc tcc
tcc gac tc |
|
NM_00111149 |
Actin |
160 |
F 5′- tct gtt tcc cgg
ttt tgc tat tat R 5′- tgc atc agg cac
ctc tca ag |
Table 2: Mean square values from ANOVA of data for growth,
chlorophyll pigments, oxidative defense system, catalase gene expression and
fruit quality in tomato subjected to different Pb2+ regimes
SOV |
df |
SDW |
RDW |
SL |
LA |
Chl. a |
Chl. b |
Tot. Chl. |
Car. |
MDA |
H2O2 |
AsA |
Lead regimes (T) |
4 |
9.75 *** |
0.33 *** |
74.64 *** |
6940.48 ***
|
0.57 *** |
0.087 *** |
1.104 *** |
0.015 *** |
279.96 *** |
3.6624
*** |
5.677 *** |
Cultivars (C) |
1 |
2.58 ** |
0.28 *** |
1219.2 *** |
633.88 *** |
0.36 *** |
0.038 *** |
0.637 *** |
0.009 *** |
25.357 ** |
13.668 *** |
5.502 *** |
T×C |
4 |
0.05 ns |
0.00 ns |
5.38 ns |
3.81 ns |
0.007 ns |
0.000 ns |
0.007 ns |
0.000 ns |
6.839 ns |
1.325 *** |
0.396 *** |
Error |
20 |
0.47 |
0.03 |
8.81 |
35.78 |
0.012 |
0.002 |
0.017 |
0.000 |
3.848 |
0.254 |
0.142 |
SOV |
df |
TSP |
CAT |
APX |
POD |
Cat2 |
Moisture
in fruit |
Ash in fruit |
Fiber
in fruit |
Protein
in fruit |
Glucose
in fruit |
Fructose
in fruit |
Lead regimes (T) |
4 |
11.28 *** |
5989.0 *** |
614.809 *** |
33697.3 *** |
7.679 *** |
10.47 *** |
0.047 *** |
0.074 ** |
0.036 *** |
0.027 *** |
0.032 *** |
Cultivars (C) |
1 |
1.981 *** |
11901.7 *** |
686.634 *** |
27686.3 *** |
1.843 *** |
5.70 ** |
0.007 ** |
0.129 ** |
0.001 ** |
0.000 ns |
0.004 *** |
T×C |
4 |
0.153 *** |
44.6 ns |
21.853 *** |
2235.8 *** |
0.187 ns |
0.260 ns |
0.017 *** |
0.026 ns |
0.005 *** |
0.004 ns |
0.001 *** |
Error |
20 |
0.0196 |
129.7 |
5.179 |
144.1 |
0.144 |
1.700 |
0.001 |
0.019 |
0.000 |
0.004 |
0.032 |
SOV |
df |
FFW |
FDW |
Na+
in fruits |
K+
in fruits
|
Ca2+
in fruits |
Pb2+
in roots |
Pb2+
in shoots |
Pb2+
in leaves |
Pb2+
in fruits |
|
|
Lead regimes (T) |
4 |
706.049 *** |
4.269 *** |
1.018 *** |
1342.47 *** |
2.066 *** |
74809.4 *** |
25421.3 *** |
40607.1 *** |
0.230 *** |
|
|
Cultivars (C) |
1 |
192.027 *** |
0.358 *** |
0.100 *** |
11.19 * |
1.756 ** |
1182.1 *** |
218.4 *** |
860.6 *** |
0.343 *** |
|
|
T×C |
4 |
13.724 ns |
0.077 ns |
0.477 *** |
97.65 *** |
0.194 ns |
155.7 *** |
52.9 *** |
153.6 *** |
0.009 ns |
|
|
Error |
20 |
8.330 |
0.108 |
0.001 |
3.05 |
0.302 |
27.1 |
6.3 |
10.7 |
0.007 |
|
|
Data analysis
Experiment was conducted in CRD with triplicate. Obtained data
were subjected to two-way analysis of variance (ANOVA) using COSTAT 6.2,
(Cohort software, 2003, Montery, CA, USA). Difference
among means was ascertained using least significant difference (LSD) at P ≤ 0.05.
Results
Growth attributes
Interaction between tomato cultivars and Pb2+ regimes had
significant effect on morphological attributes including shoot and root
biomasses, shoot length and leaf area of tomato (Table 2). The exposure of
tomato plants to Pb2+
stress (160–1280 μM)
produced a considerable decline in various morphological attributes including biomass of tomato shoot and root, shoot length and leaf area (Fig. 1a–d). The response of tomato
cultivars was diverse regarding to shoot and root dry weights. The shoot dry
weight was reduced in both tomato cultivars, though cv. Roma (10–43%) showed a
more decline than cv. Nagina (15–41%) under different
Pb2+ regimes (160–1280
μM)
over control plants (Fig. 1a). Lead
stress (160–1280 μM)
decreased the root dry weight in both cv. Nagina (20–39%)
and Roma (18–50%) over control plants, respectively (Fig. 1b). Lead inhibited (>8%) elongation of
shoot growth was more evident as compared to controls, though cv. Nagina showed a less shoot length reduction (1–8%) than cv.
Roma (1–10%) at 160–1280 μM
level of Pb2+,
respectively (Fig. 1c). Lead stress
(160–1280 μM)
reduced the leaf area in both cv. Nagina (23–52%) and
Roma (23– 54%) over control plants, respectively (Fig. 1d). Lead stress induced
a visible decline in plant dry weights, shoot length and leaf area in the
present study, and this Pb2+
induced decreased was more in cv. Roma (Fig. 1a–d).
Chlorophyll (Chl.) and carotenoid (Car.) contents
In case of Chl.
pigments and Car. contents,
no significant (P > 0.05)
interaction of Pb2+
regimes and cultivar was noted (Table 2). The Pb2+ regimes differentially modulated the contents of Chl. and total Car. in
both tomato cultivars. Although the two cultivars showed similar trends, the
degree of increase or decrease in photosynthetic pigment content was not the
same, and the change in degree of decrease or increase in Roma
was significantly higher than that in Nagina.
Lead stress significantly
reduced the Chl. a content (11–37% and 5–41%), Chl. b
content (9–37% and 8–42%) and total Chl.
(10–37% and 6–41%) in both cv. Nagina and cv. Roma,
respectively (Fig. 1e–g). Nevertheless, the reduction in Chl. content was higher in cv. Roma than that in cv. Nagina. In contrast, cultivar’s response was similar
regarding Car. contents. Between two
cultivars, lesser contents of total Car. (13–34%) were recorded in cv. Nagina under Pb2+
stress (160–1280 μM)
leaves (Fig. 1h).
Fig. 1: Changes in growth characteristics and photosynthetic
pigments in two tomato cultivars subjected to different Pb2+ regimes
(n=3± SD). Bars expressed with different letters are significantly different
according to using least significant difference (LSD) at P ≤ 0.05
Oxidative stress
and ascorbic acid (AsA) contents
Lead stress led to enhance in membrane disruption
reflected by higher MDA content. Interaction between tomato cultivars and Pb2+ regimes had
non-significant (P > 0.05) effect
in MDA contents (Table 2). When tomato plants were subjected to Pb2+ stress (160–1280 μM), the MDA
content of cv. Roma and Nagina increased by (43–122%)
and (9–86%), respectively as compared to control plants (Fig. 2a). For H2O2,
the interaction of Pb2+
regimes and cultivars were more significant (P < 0.001). The H2O2 content also markedly
increased under Pb2+
stress as well as during plant senescence. The Pb2+ stress (160–1280 μM) in growth medium caused
a significant increase in H2O2 contents in cv. Nagina (9.45–84.13%) and Roma (10.48–96.06%) as shown in Fig.
2b.
Fig. 2: Changes in malondialdehyde
(MDA), hydrogen peroxide (H2O2) and ascorbic acid
contents in two tomato cultivars subjected to different Pb2+ regimes
(n =3± SD). Bars expressed with different letters are significantly different
according to using least significant difference (LSD) at P ≤ 0.05
Interaction between tomato cultivars and Pb2+ regimes had
significant (P < 0.05) effect on
ascorbic acid contents (Table 2). A substantial decrease in ascorbic acid
levels was present in tomato plants subjected to Pb2+ stress in growth medium. Toxic effect of Pb2+ stress (160–1280 μM) also
triggered a significant decline in ascorbic acid contents of cv. Nagina (4–28%) and Roma (14–65%), but this reduction was
more apparent in cv. Nagina (Fig. 2c).
Fig. 3: Changes in antioxidant enzymes activities (CAT, APX and
POD) and transcript level of Cat2
gene in two tomato cultivars subjected to different Pb2+ regimes (n
=3± SD). Bars expressed with different letters are significantly different
according to using least significant difference (LSD) at P ≤ 0.05
Total soluble protein (TSP) contents
Interaction between Pb2+ regimes and tomato
cultivars had significant (P < 0.001)
effect on TSP contents of tomato fruits (Table 2). Total soluble protein
content was significantly affected
due to Pb2+ regimes. Lead
stress markedly increased the TSP in fruit of Nagina
(100–278%) and Roma (52–274%) over control plants, but this enhancement was
more in cv. Nagina (Fig. 2d).
Antioxidant enzyme
activity and Cat gene expression
Interaction
between tomato cultivars and Pb2+
regimes had significant (P < 0.001)
effect APX and POD activities of tomato cultivars except for CAT enzyme and Cat2 gene (Table 2). After Pb2+ treatments on tomato,
the activities of antioxidant enzymes of the both cultivars increased first and
then decreased, and these changes were different between cultivars. Plants of
cv. Nagina showed a rise (7–74%) in CAT activity,
while Roma (13–94%) was superior in this context under various Pb2+ regimes (160–1280 μM) (Fig. 3a).
Lead stress caused a markedly
increase in APX contents of cv. Nagina (60–166%) and
Roma (45–135%), respectively over control plants (Fig. 3b; Table 2). In this
study, we have recorded Pb2+
(160–1280 μM)
caused a considerable decline in POD activity of both cv. Nagina
(2–50%) and cv. Roma (14–120%) can be seen in Fig. 3c. Compared with control,
after Pb2+ treatment
(320 μM),
the relative expression of Cat2 gene
of cv. Nagina and Roma increased by 316 and 241%, respectively,
but the degree of change of cv. Nagina is greater
than Roma (Fig. 3d).
Moisture contents
and Ash contents of tomato fruit
Plants subjected to Pb2+ stress had non-significant (P > 0.05) effect in fruit moisture contents of both tomato
cultivars (Table 2). The Pb2+
stress affected the quality of tomato fruit. In this regard, Pb2+ (160–1280 μM) induced
a steady decrease in moisture contents in cv. Nagina
(2–3%) and Roma (3–4%), while interaction of Pb2+ regimes and cultivars (P < 0.001) was significant in ash contents (Table 2). The Pb2+ stress was increased
the ash contents (13–40%) more significantly in plants of Nagina
when subjected to Pb2+
regimes (Fig. 4a–b). Moisture contents
are important to storage life of the fruits. The results of present study also
exhibited that lower moisture
contents in fruit of tomato were recorded in Nagina
plants (Fig. 4a).
Total fiber and
total protein contents of tomato fruit
Lead
caused non-significant (P > 0.05)
effect on fruit total fiber contents of both tomato cultivars (Table 2). A dose
dependent Pb2+
toxicity decline the total fiber (0.5–5% and 2.87–19%) under different Pb2+ regimes (160–1280 μM) in both
Roma and Nagina fruits, respectively. The interaction
of Pb2+ regimes and
cultivars had significant (P < 0.001)
effect on fruit protein contents in both tomato cultivar
(Table 2). Lead stress markedly
increased the total protein content in fruit of Nagina
(7–35%) and Roma (5–23%).
Glucose contents
and fructose contents of tomato fruit
Interaction
between tomato cultivars and Pb2+
regimes had non-significant (P > 0.05)
effect on glucose contents of both tomato cultivar
(Table 2). Glucose contents were increased in Nagina
(4.65 and 4.65%) and Roma (9.41 and 14.12%) fruits under 320 and 1280 μM Pb2+ levels, respectively (Fig.
4e). The interaction between Pb2+
regimes and cultivars was significant (P <
0.001) for fructose contents. Fructose content was also affected significantly
due to Pb2+ regimes.
High concentration of fructose contents (14%) were found in cv. Roma at 1280 μM (Fig. 4f).
Fresh and dry
biomass of tomato fruit
Interaction between tomato cultivars and Pb2+ regimes had
non-significant (P > 0.05) effect
on fruit biomass (Table 2). Lead
toxicity also resulted in a substantial decrease in biomasses of tomato fruit. Nagina cultivar showed maximum reduction in fruit fresh and
dry weight (43–68% and 38–53%) than Roma (49–66% and 29–65%) under different Pb2+ regimes (160–1280 μM),
respectively (Fig. 4g–h).
Mineral (Na+,
K+ and Ca2+) contents in tomato fruits
Interaction between tomato cultivars and Pb2+ regimes had
significant (P < 0.001) effect on
Na+ and K+, while non-significant (P > 0.05) on Ca2+ contents of both tomato cultivar (Table 2). The
response of both cultivars was inconstant regarding Na+ contents.
Lead stress (160–1280 μM)
substantially increased the Na+ content in the fruit of cv. Nagina (3–7%), respectively and this increase was 6% in
Roma at 320 μM
of Pb2+ (Fig. 5a). In
contrast, Pb2+
toxicity increased the K+ content significantly in cv. Roma (7–20%)
than of Nagina (1–15%) under higher Pb2+ levels (640–1280 μM),
respectively (Fig. 5b). The Ca2+
contents were reduced in response to Pb2+
toxicity and this reduction was being more maximum in Roma (13–26%) at 160–1280
μM of Pb2+ (Fig. 5c).
Fig. 4: Changes in the fruit quality and biomass of two tomato
cultivars subjected to different Pb2+ regimes (n =3± SD). Bars
expressed with different letters are significantly different according to using
least significant difference (LSD) at P ≤
0.05
Lead (Pb2+)
uptake and accumulation in root, shoot, leaf and fruit of tomato
Fig. 5: Changes in mineral ions content (Na+, K+
and Ca2+) in fruit of two tomato cultivars subjected to different Pb2+
regimes (n=3± SD). Bars expressed with different letters are significantly different
according to using least significant difference (LSD) at P ≤ 0.05
Fig. 6: Changes in Pb2+ contents in roots, shoot,
leaves and fruit of two tomato cultivars subjected to different Pb2+
regimes (n=3±SD). Bars expressed with different letters are significantly
different according to using least significant difference (LSD) at P ≤ 0.05
A significant increase in root, shoot, and leaf (P < 0.001) Pb2+ content
was apparent in tomato cultivars under Pb2+ regimes. The differences
in fruit Pb2+ content were not significant (P > 0.05) between cultivars and Pb2+ regimes (Table
2). After Pb2+
treatments (160–1280 μM),
compared with control, the accretion of Pb2+
contents in root, shoot leaf and fruit was more significant in tomato plants
under Pb2+ stress.
Lead contents were increased in root (2750–7114% and 2596–6758%), shoot (569–4265%
and 536–4073%) and leaves tissues (863–2467% and 817–2288%) of both Nagina and Roma cultivars under Pb2+ regimes (160–1280 μM Pb2+), respectively. After Pb2+ treatments, 7–20% Pb2+ contents accumulation was increased in cv. Nagina and 1–15% in cv. Roma fruit (Fig. 6a–d).
Discussion
In the
present study, Pb2+
toxicity reduced shoot fresh and dry masses, and this Pb2+-induced decrease was more prominent in the
sensitive cultivar. A number of reports are available in the literature where Pb2+ stress induced
decrease in plant growth (Zhou et al.
2018). The Pb2+ has
the ability to affect the nutrients uptake, photosynthesis, and disturb the
structure and properties of membrane (Hadi and Aziz
2015). It is well known that Pb2+
stress causes increased generation of ROS that in turn may provoke oxidation
damage (Lopes et al. 2016). In this
study, reduction in dry weights, shoot length and leaf area of plant might also
be the consequence of oxidative stress. Likewise, Pb2+ toxicity inhibits the growth of plant by limiting
the water uptake that altered the extensibility of cell wall, which in turn
decreased the growth in plants (John et
al. 2008).
Lead toxicity considerably declined the Chl. a, b and total Car. contents
being lesser in the tolerant cultivar. This decline in Chl. and total Car. contents might be due to increase in chlorophyllase
activities under metal toxicity and/or photo-oxidation (Malar et al. 2016). There are several reasons
that Pb2+ induced
decline in the Chl. contents. In this
perspective, Pb2+
stress motivates chloroplast disorganization; thus caused decline in Chl. contents. The Pb2+ toxicity also
decreased Chl. contents by reducing
uptake and buildup of certain nutrient contents such as Mg2+, Zn2+,
Fe2+ and Cu2+ that take part in the synthesis of Chl. contents (Malar et al. 2016). Under Pb2+ stress, Mg2+
in Chl. content is substituted by Pb2+
is the main reason for degradation of Chl. contents in plants grown-up in metal polluted area (Iqbal et al.
2017). This replacement of Mg2+ by Pb2+ hinders the capacity of Chl. pigment to catch light that declines the photosynthetic
process. Moreover, excessive production of H2O2 may
interrupt the process of photosynthesis (Gopal and Khurana 2011).
Under diverse environmental constraints, plants produce
reactive oxygen species, which cause oxidative stress that damages proteins,
DNA, membranes, pigments and enhance lipid peroxidation causing cell
dysfunction and death (Xie et al. 2019). Similarly, greater MDA content was ascribed in
chickpea and Macrotyloma uniflorum
subjected to Pb2+
stress (Reddy et al. 2005). This
study also indicated that increasing concentration of Pb2+ mediated more MDA and H2O2
in both tomato cultivars, respectively. Kumar and Prasad (2015) reported that Pb2+ stress interrupts the
photosystem (PSII), which results in the production of more ROS and
peroxidation of lipid. Ascorbic acid is an essential component of non-enzymatic
antioxidant system and carries out detoxification of ROS, especially of H2O2
in plants under Pb2+ stress. Our results also exhibited a remarkable
rise in the endogenous levels of ascorbic acid in plants under abiotic
stresses. Similar results are also described in water deficit wheat plants (Roy
et al. 2017). Ghorbanli
et al. (2013) described the increase
in ascorbic acid in tomato plants under drought. Similarly, Pb2+
stress exhibited a significant increase in ascorbic acid in wheat plants (Alamri et al.
2018). Plants display substantial accumulation of osmolytes
to improve cell turgor and circumvent injuries to cell membranes and proteins
from Pb2+ induced
oxidative stress (Sahoo et al. 2015). This study also indicated TSP in tomato plants
increased under Pb2+ stress regimes. Further, our results manifested
a significant negative correlation between growth attributes and oxidative
stress markers (MDA and H2O2) under drought (Fig. 1a–d
and 2a–b).
In general, to overcome oxidative damage caused by
increased cellular levels of H2O2 and MDA, plants display
variations in antioxidant enzyme activities (Gupta
et al. 2009). In this study,
higher CAT, APX and POD activity was seen in the tolerant cultivar while the
sensitive cultivar displayed lower POD activity due to Pb2+ stress. Our results are consistent with earlier
reports in radish (He et al. 2008), Jatropha seedlings (Shu et al. 2012), cotton (Bharwana et al.
2013) and okra (Tiwari and Lata 2018).
Numerous genes are induced by metal stress. Modifications in the level of
catalase gene have been detected in several plant species when subjected to
metal stress (Aydin et al.
2016). In this situation, Azpilicueta et al. (2008) assessed the gene
expression of catalase (CATA1 to CATA4) in sunflower under metal stress.
These authors also further described that CATA1
and CATA2 accumulated more cadmium in
cotyledon and roots and result preventing plant growth. In our study, we
documented that qRT-PCR analysis of Cat2 gene shows upregulation
of the Cat2 in shoot of cv. Nagina under Pb2+
stress.
In this perspective, fruit
having higher water content may not be saved for longer period being perishable
(Miranda et al. 2019). In this study, higher ash contents was seen in cv. Nagina, while cv. Roma showed lower ash contents due to Pb2+ stress. Our results are mirrored in the findings by
Garuba et al.
(2018), who described that ash
content of tomato fruit gives evidence about good mineral contents. It is well documented that greater fiber
contents, less vitamin and mineral contents in the diet of infants and children
can cause trouble in stomachs (Eromosele and Hamagadu 1993).
Lead toxicity reduced the fiber content in the fruit of tomato and this lessens
the quality of fruit more in cv. Roma. In this case, more fiber contents
were observed in the tolerant cultivar.
Furthermore, higher fiber content in the food of adult are also
considered better for anti-constipation (Bae 2014). Protein contents are important part of
food, which are necessary for humans and animals health. Proteins provides
ample amount of amino acids during metabolism (Zhang et al. 2017). Our results also revealed marked increase
in the accumulation of proteins contents in the tolerant cultivar.
Results of this study
highlighted a significant enhancement in fructose and glucose contents in
fruits of tomato plants, and this increase was more significant in the
sensitive cultivar. Lead stress brought a significant decline in grain yield in
rice (Ashraf et al. 2017). Results of this study are correlated with Hung et al.
(2014), who reported that Pb2+ stress caused substantial decline in
the yield of okra plants. However, in the present study, we found a
considerable decline in fruit biomasses of both cultivars and this decline was
superior in the sensitive than tolerant cultivar.
Lead structurally resembles
with some nutrients, and therefore, it strongly competes with the uptake of P,
K+, Mg2+, and Ca2+ contents (Pourrut et
al. 2011). Decrease in K+
content induced by Na+ is a well-known competitive process exhibited
by plants under stress condition (Ashraf et al. 2018). In this study, a significant decrease in uptake of K+ and Ca2+
contents and increase of Na+ content in cv. Roma fruits exposed to
Pb2+ stress in nutrient solution. These results are correlated with Lamhamdi et al.
(2013), who reported Pb2+-induced reduction in the uptake of
macronutrients (K+, Ca2+, Mg2+ and P) and
micronutrients (Fe, Zn, and Cu) in wheat
plants. Excess Pb2+
activated its accumulation in the tissues of tomato plants. The toxicity of Pb2+
stress depends on Pb2+ accumulated plant species, which influences
the uptake, accumulation, and translocation of Pb2+ contents (Akinci et al.
2010). Although the Pb2+ concentration elevated, the order of Pb2+
distribution within the plant are as followed root>shoot>leaf>fruits.
Akinci et al.
(2010) and Dahmani et al. (2000) found similar results in tomato and sea thrift,
respectively. They stated that differences in Pb2+ accumulation in roots, shoots, leaves and fruits
showed significant restriction of the interior transport of heavy metals from
roots to fruit tissues. Furthermore, Gothberg et al. (2004) indicated that maximum Pb2+ contents accumulation
occurred in the roots, followed by shoots. In agreement with these findings, we
found that Pb2+
contents accumulated more in the roots, shoots, leaves and fruit tissues,
respectively in tomato plants.
Conclusion
Taken together, results
indicated a significant reduction in growth attributes, photosynthetic
pigments, yield attributes, and calcium ions uptake in tomato cultivars under
lead stress, although the damaging effects were more evident in the sensitive
cultivar. Moreover, lead stress enhanced oxidative damage in terms of higher malondialdehyde and hydrogen peroxide contents in tomato
plants. Nonetheless, lead stress decreased
fruit quality, whereas the tolerant tomato cultivar displayed upregulation of Cat2
gene expression, and thus had less malondialdehyde and
hydrogen peroxide contents, and thus showed higher lead stress tolerance
than the sensitive one. Further, the tolerant cultivar showed better
antioxidant system, lesser degradation of chlorophylls and better growth under
lead stress.
Acknowledgements
The data presented in this manuscript is part of Ph.D.
thesis of Ms. Zarbakht Afzaal
and financially supported by GCUF-RSP
(4325-4359) and Project Code:
48-Bot-7.
Author Contributions
ZA, IH and MRA planned the research work and IH
supervised the whole research work. ZA performed the experiment, done sampling
and analyses. MAA, RR, SA, MTJ and MI contributed in data analysis. IH and ZA
write up the paper. All authors read and approved the final paper.
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