agronomy
Article
Putrescine Mitigates High Temperature Effects by Modulating
Morpho-Physiological and Biochemical Attributes in
Brassica juncea Seedlings
Parul Sharma 1 , Nita Lakra 2, * , Yogesh Ahlawat 3 , Abbu Zaid 4,5, * , Ahmed M. Abd-ElGawad 6 ,
Hosam O. Elansary 6 and Anita Gupta 5
1
2
3
4
5
6
*
Citation: Sharma, P.; Lakra, N.;
Ahlawat, Y.; Zaid, A.; Abd-ElGawad,
A.M.; Elansary, H.O.; Gupta, A.
Putrescine Mitigates High
Temperature Effects by Modulating
Morpho-Physiological and
Biochemical Attributes in Brassica
juncea Seedlings. Agronomy 2023, 13,
1879. https://doi.org/10.3390/
agronomy13071879
Academic Editor: Cinzia Margherita
Bertea
Received: 17 June 2023
Revised: 11 July 2023
Accepted: 13 July 2023
Published: 16 July 2023
Department of Botany and Plant Physiology, College of Basic Science and Humanities,
CCS Haryana Agricultural University, Hisar 125004, India; parulvats501@gmail.com
Department of Molecular Biology & Biotechnology, College of Biotechnology,
CCS Haryana Agricultural University, Hisar 125004, India
Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA;
ykahlawa@mtu.edu
Plant Physiology and Biochemistry Section, Department of Botany, Aligarh Muslim University,
Aligarh 202002, India
Department of Botany, GGM Science College, Cluster University of Jammu, Jammu 180001, India;
anitagupta94191@gmail.com
Department of Plant Production, College of Food & Agriculture Sciences, King Saud University,
P.O. Box 2460, Riyadh 11451, Saudi Arabia; aibrahim2@ksu.edu.sa (A.M.A.-E.);
helansary@ksu.edu.sa (H.O.E.)
Correspondence: nitahaubotany2019@gmail.com (N.L.); zaidabbu19@gmail.com (A.Z.)
Abstract: A variety of environmental issues are affecting crops all across the world, but rising
temperatures are posing the greatest threat. High temperature has been found to drastically inhibit
seedling emergence and cause leaf necrosis at the seedling stage, which results in poor plant stand
and significantly decreased yields. Polyamines (PAs) are positively charged, low-molecular-weight
aliphatic nitrogenous bases present in all living organisms and are involved in various biological
processes in plant growth and development, including senescence and response to different abiotic
stresses. Putrescine (Put) functions as a master growth regulator that promotes optimal plant
development and greater stress tolerance. Here, the current study aimed to elucidate how Put (1 mM)
functions in reducing the negative impacts of high temperature on four varieties of Brassica juncea (RH1707, RH-1708, RH-1566 and RH-1999-42). Exposure of plants to high temperature resulted in decrease
in growth parameters, chlorophyll content and relative water content. Simultaneously, increases were
found in antioxidant enzymes, electrolyte leakage, lipid peroxidation, hydrogen peroxide content and
stomatal density. High temperature more significantly affected varieties RH-1707 and RH-1708, while
RH-1566 and RH-1999-42 showed lesser effects. Exogenous application of Put mitigated the negative
impacts of high temperature by enhancing growth, chlorophyll content, relative water content and
antioxidant enzyme activities and, simultaneously, it reduces oxidative damage and stomatal density.
This study specifies that varieties RH-1707 and RH-1708 are sensitive whereas RH-1566 and RH-199942 are tolerant of high temperature and provides an insight into the effectiveness of Put in mitigating
the effects of high temperature to a significant extent in B. juncea seedlings.
Keywords: high temperature; putrescine; Brassica juncea; chlorophyll; stomatal density;
antioxidant enzymes
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Indian mustard (Brassica juncea) is an important oilseed crop being produced in tropical
and subtropical areas as a cold season crop (6 ◦ C to 27 ◦ C). It is primarily cultivated in a
variety of conditions, including rainfed and irrigated, early, timely, and late sowing, as well
Agronomy 2023, 13, 1879. https://doi.org/10.3390/agronomy13071879
https://www.mdpi.com/journal/agronomy
Agronomy 2023, 13, 1879
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as solitary or mixed crop [1]. In India, the biggest rapeseed-mustard growing states are
Rajasthan, Uttar Pradesh, Haryana and Madhya Pradesh, contributing 45.5%, 13.1%, 11.8%
and 11.1%, respectively, to the national acreage during the past five years. With an average
yield of 2058 kg ha−1 , Haryana produced 1.25 million tonnes (13.42 percent of all India’s
production) from 0.61 million hectares (9.78 percent of all India’s land) [2].
As a result of global climate change, high-temperature stress is becoming increasingly
important for plant growth and development as it severely affects agricultural productivity [3]. With each degree centigrade rise in average temperature, agricultural productivity
reduces by 17% [4]. Exposure to high temperatures can induce a wide range of morphological, anatomical, physiological and biochemical alterations in plants. These changes
have significant impacts on plant growth and development and can result in a substantial
decline in economic yield [5,6]. Among the essential phases of plant development, the
early stages of seedling growth are one of the most important in stand establishment in
many crops [7]. The exposure of plants to high temperatures during the early stages of
growth retards the overall growth including yield. Complex effects of heat stress on crop
plants include increased seedling mortality, reduced photosynthesis, senescence of the
leaves, decreased pollen production and viability, seed abortion and ultimately lower grain
quantity and weight [8]. High temperature has been found to drastically inhibit seedling
emergence and cause leaf necrosis at the seedling stage, which results in poor plant stand
and significantly decreased yields [9].
Due to their immobility, plants cannot evade stress; however, they employ various
strategies such as early maturation, modifying the composition of membrane lipids, producing stress proteins, osmotic adjustment and the restoration of the redox balance of cells and
homeostasis by modifying the antioxidant system [10]. The application of phytohormones,
osmoprotectants and polyamines (PA) is found to effectively mitigate the effects of heat
stress in plants. Polyamines are a class of small, polycationic and aliphatic metabolites
that are ubiquitously present in all organisms. They participate in a range of physiological
reactions within organisms [11]. In plant cells, the main polyamines are putrescine (Put),
spermidine (Spd) and spermine (Spm) [12]. These polyamines can exist either in their free
form or as conjugates bound to other molecules such as phenolic acids, proteins and nucleic
acids [13]. The cationic nature of polyamines contributes to their biological activity [14,15].
They play a role in various cellular processes, including chromatin condensation, maintaining the structure of DNA, RNA processing, translation and activating proteins [16–18].
PAs work by regulating osmosis and detoxifying the cell by removing ROS by boosting
antioxidant defense capacity or preventing ROS synthesis [19]. The involvement of PAs in
plant growth and development, as well as enhancing their ability to withstand stress, is
widely recognized [20–22].
Putrescine is the central product of the polyamine biosynthesis pathway [23]. It is
produced through the decarboxylation of ornithine and arginine, catalyzed by the enzymes
ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), respectively [24]. It
plays a significant role in providing tolerance to different abiotic stresses. Scavenging
free radicals, controlling ABA levels, avoiding lipid peroxidation, maintaining cellular
pH and ionic equilibrium and regulating cationic channels are among the key reported
processes related to ability of Put to produce abiotic stress tolerance responses [25]. Several studies have shown the significant role of Put in enhancing abiotic stress tolerance
such as drought [26–28], salinity [29,30], cold [31,32] and heat [33–35]. The effectiveness
of polyamines (1 mM) in mitigating the harmful effects of high temperature has been
extensively studied [36–38]. The role of polyamines in mitigating high-temperature stress
in B. juncea is largely unexplored. As a result, this study was undertaken to investigate
the acclimation response of B. juncea to high-temperature stress through the exogenous
application of Put (1 mM). The aim was to understand how the exogenous application of
Put may contribute to enhancing the plant’s tolerance to high temperatures.
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2. Materials and Methods
2.1. Growth Conditions, Plant Materials, and Treatments
Four Indian mustard (Brassica juncea L. Czern. & Coss.) varieties RH-1707, RH-1708,
RH-1566 and RH-1999-42 were utilized for the study. Seeds were obtained from Oilseeds
Section, Department of Genetics and Plant Breeding, CCS Haryana Agricultural University,
Hisar. Uniformly selected seeds were surface sterilized with 0.1% HgCl2 for one minute and
then repeatedly washed with distilled water. Sowing was carried out in pots containing
normal homogenized field soil in a greenhouse. Each variety was sown in triplicates.
Seedlings were allowed to grow under controlled conditions (light- 75 W/m2 , 65% RH,
temp 25 ± 2 ◦ C) and watered regularly for 21 days.
For heat treatment, seedlings were transferred to a growth chamber and exposed
to a gradually elevating temperature of 38–40 ◦ C (RH ~ 45–50%) for 3 h in light. Then
the temperature was decreased to 25 ◦ C (RH ~ 70%) and the entire cycle was repeated
for 7 days. Water was not given during the period of heat stress. A control experiment
was carried out in a greenhouse where optimum temperature (25 ◦ C, RH ~ 70%) was
maintained throughout the experiment. Put (1 mM) was sprayed on both sides of the leaves
of seedlings using a manual sprayer two hours prior to exposing them to heat treatment.
Similarly, control plants were sprayed with an equal volume of double-distilled water
and Put (1 mM). There were four distinct treatments used in the experiment—(1) Control,
(2) Put (1 mM), (3) High temperature (HT) (38–40 ◦ C) and (4) HT + Put (1 mM). Following
seven days of treatment, the treated plants were acclimatized for two days under a normal
environment and leaves from each treatment were collected for subsequent analysis.
2.2. Determination of Growth Attributes
Growth parameters such as seedling length, fresh weight, dry weight and leaf area per
plant were measured. Plants were gently removed from their pots and cleaned to remove
the dirt and dust from the roots. The fresh weight of plants was recorded, and subsequently
they were kept for drying in an oven at 80 ◦ C to record dry weight. Seedling length refers
to combined shoot length and root length which was determined by carefully harvesting
seedlings from pots and measuring length using a standard scale. Leaf area was measured
using a leaf area meter (LI 3000 Area meter, LICOR Ltd., Lincoln, NE, USA).
2.3. Chlorophyll Content
Chlorophyll and carotenoids were extracted by according to the method of [39,40]
using 5 mL of methanol solution for each 50 mg of fresh leaf tissue, and absorbance was read
at 480 nm, 645 nm and 663 nm. Chlorophyll content was calculated using the equations:
Chlorophyll a = (12.21OD663 − 2.81OD645 ) × V/1000
Chlorophyll b = (12.21OD663 − 2.81OD645 ) × V/1000
Total Chlorophyll = Chlorophyll a + Chlorophyll b
Carotenoid = A480 − 2.27 ( A645 ) − 81.4 ( A663 )/227
2.4. Plant Water Status
2.4.1. Relative Water Content
Leaf relative water content was calculated by using the method as described [41]. Leaf
samples were collected and weighed immediately to measure weight. Leaves were placed
separately in petri dishes filled with distilled water. Afterwards, the same leaves (fully
turgid) were weighed again and placed in an oven at 85 ◦ C for 72 h for drying and weighing
and used to calculate relative water content percentage (RWC %) by the formula:
RWC (%) = (Fresh weight − Dry weight/Turgid weight − Dry weight) × 100
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2.4.2. Electrolyte Leakage
Electrolyte leakage was analyzed using a conductivity meter according to the method
described by [42]. Fresh leaves were cut into smaller pieces and then incubated in 20 mL
of deionized water and kept overnight. Next day, the initial electrical conductivity (EC1)
of the medium was measured. The samples were autoclaved for 30 min to release all
the electrolytes, cooled, and then final electrical conductivity (EC2) was measured. The
electrolyte leakage percentage was calculated as follows:
Electrolyte Leakage(%) = (EC1/EC2) × 100
2.5. Antioxidant Enzyme Estimation
2.5.1. Preparation of Enzyme Extract
200 mg of leaf samples were homogenized in a mortar and pestle using a 0.1 M
potassium phosphate buffer with a pH of (7.0). In a chilled centrifuge set at 4 ◦ C, the
homogenate was centrifuged for 30 min at 12,000 rpm. The supernatant was used to
measure the activity of antioxidant enzymes, including superoxide dismutase (SOD) and
catalase (CAT).
2.5.2. Superoxide Dismutase
The potential of SOD to prevent the photochemical reduction of nitro blue tetrazolium
(NBT) was used to measure its activity [43]. Three mL of reaction mixture contained
50 mM of potassium phosphate buffer (pH 7.8), 13 mM of methionine, 25 mM of nitro blue
tetrazolium (NBT), 2 µM of riboflavin, 0.1 mM of EDTA, 50 mM of sodium carbonate and
0.1 mL of enzyme extract. The test tubes were shaken properly to homogenize the reaction
and the mixture was illuminated for 30 min. Absorbance was recorded at 560 nm.
2.5.3. Catalase
Catalase activity was estimated using the protocol given by [44]. Three mL of reaction
mixture contained 0.1 mL of enzyme extract, 1.5 mL of 100 mM phosphate buffer (pH 7.0),
0.5 mL of 75 mM H2 O2 and 950 µL of distilled water. The reduction in H2 O2 absorbance
(=39.4 mM1 cm−1 ) at 240 nm was used to calculate catalase activity. The enzyme activity
was expressed as mM of H2 O2 reduced/min/mg FW.
2.6. MDA Content
Lipid peroxidation was determined by using method [45]. Leaf tissue (500 mg) was
homogenized in 10 mL of 0.1% (w/v) Trichloroacetic acid (TCA) and centrifuged for 20 min
at 10,000 rpm. One ml of the supernatant was mixed with 4 mL of 0.5% Thiobarbituric acid
(TBA) diluted in 20% TCA, incubated in a water bath at 95 ◦ C for 30 min and then cooled
in an ice bath. The absorbance was measured at 532 and 600 nm. Lipid peroxidation was
expressed as µ mols malondialdehyde (MDA)/g FW.
2.7. H2 O2 Content
Hydrogen peroxide levels were determined according to [46]. An amount of 200 mg
of leaf tissues was homogenized in an ice bath with 1 cc of TCA, 0.1%. After centrifuging
the homogenate at 12,000× g for 15 min, 0.5 mL of the supernatant was added to 1 mL of
1 M potassium iodide (KI) and 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0). At
390 nm, the supernatant’s absorbance was measured using the standard curve.
2.8. Histochemical Analysis
The method of Evans blue staining was employed to observe the death of cells in leaf
tissue. This was performed by immersing fresh leaves in a solution consisting of 0.25%
(w/v) aqueous solution of Evans blue [47]. Leaves were kept in the solution overnight
and then bleached with 95% v/v ethanol. The occurrence of cell death could be observed
through the presence of blue patches on the surface of the leaf.
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To analyze the accumulation of superoxide ion (O2 ·− ) through the histochemical
staining method, leaves were stained using nitro blue tetrazolium (NBT) [48]. The samples
from each treatment were immersed into 1 mg/mL of NBT solution prepared in 10 mM of
phosphate buffer (pH 7.8). Leaves were illuminated until dark blue spots became visible.
Subsequently, the leaves were treated with ethanol to remove the staining.
2.9. Stomatal Density
The stomatal density was evaluated by the method described by [49]. A fresh leaf
sample was taken and gently washed with running tap water to remove any kind of dust
particles. A coat of clear nail polish was applied on the abaxial (lower) surface of the
leaf to obtain a suitable replica, then allowed to dry completely for 8–10 min. The replica
was gently peeled off using a forceps and placed on the slide in such a manner that the
imprinted surface was on the upper side. One or two drops of water were put on the slide
and then covered with a coverslip. Similarly, replica preparations were performed for all
the samples and observed under the light microscope. The total number of stomata visible
in the circular view field of the microscope at a given magnification were counted. The
total number of stomata from the lower surface of the leaf were recorded and the area of
the circle under the microscopic view field was calculated by the formula: πr 2, where r is
the radius of the circle (view field), i.e., 12 of the diameter of the circle.
2.10. Statistical Analysis
The data were analysed using a completely randomized block design (CRD). Two factorial analyses of variance were performed on the data using OPSTAT software (CCSHAU,
Hisar, India; http://14.139.232.166/opstat/, accessed on 7 May 2023) to determine the significance of differences among the treatments at p < 0.05. Least significant difference (LSD)
was calculated for the significant data to identify difference in the mean of the varieties
(p < 0.05).
3. Results
3.1. Growth Parameters
3.1.1. Seedling Length
The growth of seedlings in all the studied varieties was reduced by high temperatures.
Exogenous Put application resulted in significant enhancement, counteracting the negative
impact (Figure 1). A significant decrease in seedling length was noticed in RH-1708 (36.01%)
and RH-1707 (33.35%); however, a lesser decrease was shown by RH-1566 (22.94%) and
RH-1999-42 (15.66%). The application of Put under normal temperature showed a slight
increment in the length of seedlings in RH-1708 (10.75%), RH1707 (7.56%), RH-1566 (5.35%)
and RH-1999-42 (4.12%). The application of Put mitigated the effects of high temperature,
leading to improvements in RH-1708 (28.46%), Rh-1707 (26.78%), RH-1999-42 (17.39%) and
RH-1566 (11.32%) (Figure 2).
3.1.2. Fresh Weight
Seedlings subjected to high-temperature stress experienced a decrease in their fresh
weight. The reduction was more pronounced in RH-1708 (65.56%) and RH-1707 (60.39%).
On the other hand, RH-1999-42 (34.1%) and RH-1566 (28.05%) exhibited a lesser decline
in fresh weight. Put application led to a small improvement in fresh weight of seedlings
under normal temperature, i.e., RH-1707 (17.4%), RH-1708 (16.7%), RH-1566 (14.88%) and
RH-1999-42 (13.46%). Under high-temperature conditions, the exogenous application of Put
led to a significant increase in the fresh weight of seedlings in RH-1708 (30.73%), RH-1707
(26.78%), RH-1999-42 (24.05%) and RH-1566 (18.15%) (Figure 3).
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Figure 1. Morphology of Brassica juncea seedlings under the effect of high temperature and Putrescine.
Figure 2. Effect of high temperature and Putrescine on seedling length in Brassica juncea seedlings.
Each bar represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk (*)
indicates a significant difference between the treatments. Different alphabets for each mean show
statistically significant differences between the varieties according to the least significant difference
(LSD) test (p < 0.05).
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Figure 3. Effect of high temperature and Putrescine on fresh weight in Brassica juncea seedlings.
Each bar represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk
(*) indicates significant difference between the treatments. Different alphabets for each mean show
statistically significant differences between the varieties according to the least significant difference
(LSD) test (p < 0.05).
3.1.3. Dry Weight
The effect of high temperature on dry weight of seedlings was comparable to that
on fresh weight. Under high temperature, the most significant reduction was shown by
varieties RH-1708 (59.46%) and RH-1707 (56.87%), whereas varieties RH-1999-42 (39.12%)
and RH-1566 (33.23%) were less affected. Under normal temperature conditions, a minimal
increase in dry weight occurred after the application of Put in all the treated varieties,
as seen in RH-1708 (8.3%), RH-1566 (8.01%), RH-1707 (6.62%) and RH-1999-42 (3.55%).
However, under high temperature, exogenous application of Put led to an increment in
the dry weight of varietiesRH-1708 (25.26%), RH-1707 (23.61%), RH-156 (20.16%) and
RH-1999-42 (15.17%) (Figure 4).
Figure 4. Effect of high temperature and Putrescine on dry weight in Brassica juncea seedlings. Each
bar represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk (*)
indicates significant difference between the treatments. Different alphabets for each mean show
statistically significant differences between the varieties according to the least significant difference
(LSD) test (p < 0.05).
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3.1.4. Leaf Area per Plant
After being exposed to high temperatures, the plants exhibited a visible reduction in
leaf area and displayed symptoms resembling chlorosis. However, the application of Put
helped alleviate these symptoms (Figure 5).
Figure 5. Morphology of Brassica juncea leaves under the effect of high temperature and Putrescine.
High-temperature stress reduces the leaf area per plant substantially. The reduction
was more prominent in RH-1708 (68.96%) and RH-1707 (54.29%) than in RH-1999-42
(27.62%) and RH-1566 (17.73%). Under normal temperature, application of Put led to a
slight increase in leaf area in RH-1708 (13.06%), RH-1707 (12.41%), RH-1566 (9.8%) and
RH-1999-42 (8.76%). The combined treatment of plants with both high temperature and
Put resulted in a reduction in the adverse effects caused by high-temperature stress. This
combined treatment helped to maintain leaf area more efficiently, particularly in variety
RH-1708 (33.07%) followed by RH-1707 (27.92%), RH-1999-42 (19.31%) and RH-1566 (9.98%)
(Figure 6).
−
−
Figure 6. Effect of high temperature and Putrescine on leaf area plant−1 in Brassica juncea seedlings.
Each bar represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk
(*) indicates significant difference between the treatments. Different alphabets for each mean show
statistically significant differences between the varieties according to the least significant difference
(LSD) test (p < 0.05).
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3.2. Chlorophyll Content
High-temperature stress leads to a sharp decline in chlorophyll content in varietiesRH1708 (64.86%) and RH-1707 (55.28%) whereas RH-1566 (24.5%) and RH-1999-42 (21.25%)
were less affected. Under controlled conditions, exogenous application of Put led to a slight
increase in chlorophyll content in RH-1999-42 (12.61%), RH-1566 (9.64%), RH-1707 (6.23%)
and RH-1708 (5.32%). However, combined treatment of both Put and high temperature led
to a considerable improvement in chlorophyll content as was seen in RH-1707 (27.88%),
RH-1708 (20.73%), RH-1999-42 (27.57%) and RH-1566 (18.29%) (Figure 7).
Figure 7. Effect of high temperature and Putrescine on chlorophyll content in Brassica juncea seedlings.
Each bar represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk
(*) indicates significant difference between the treatments. Different alphabets for each mean show
statistically significant differences between the varieties according to the least significant difference
(LSD) test (p < 0.05).
3.3. Plant Water Status
3.3.1. Relative Water Content (RWC)
Under high temperature, the RWC content was significantly reduced, more substantially in varieties RH-1708 (64.86%) and RH-1707 (55.28%) whereas varieties RH-1566
(24.58%) and RH-1999-42 (20.25%) showed a lesser decline. The exogenous application of
Put at normal temperature led to a slight increase in RWC in varieties RH-1999-42 (12.61%),
RH-1566 (9.64%), RH-1707 (6.23%) and RH-1708 (5.32%). However, under high temperature,
Put application shows a more efficient increase in the water status of plants, as was seen in
varieties RH-1707 (27.88%), RH-1708 (20.73%), RH-1566 (18.29%) and RH-1999-42 (17.57%)
(Figure 8).
3.3.2. Electrolyte Leakage
All the studied varieties experienced an elevation in electrolyte leakage, but a more
significant increase was noticed in varieties RH-1708 (61.45%) and RH-1707 (55.87%) than
in RH-1999-42 (32.38%) and RH-1566 (24.17%). The exogenous application of Put at normal
temperature led to a slight increase in electrolyte leakage in varieties RH-1999-42 (13.76%),
RH-1707 (10.21%), RH-1566 (8.93%) and RH-1708 (8.66%). The damage caused to membranes due to high temperature was considerably reduced by exogenous application of
Put, as was seen in varieties RH-1707 (23.29%), RH-1999-42 (21.92%), RH-1566 (8.93%) and
RH-1708 (8.66%) (Figure 9).
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Figure 8. Effect of high temperature and Putrescine on RWC in Brassica juncea seedlings. Each bar
represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk (*) indicates
significant difference between the treatments. Different alphabets for each mean show statistically
significant differences between the varieties according to the least significant difference (LSD) test
(p < 0.05).
Figure 9. Effect of high temperature and Putrescine on electrolyte leakage in Brassica juncea seedlings.
Each bar represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk
(*) indicates significant difference between the treatments. Different alphabets for each mean show
statistically significant differences between the varieties according to the least significant difference
(LSD) test (p < 0.05).
3.4. Antioxidant Enzyme Activity
3.4.1. Superoxide Dismutase
Under high-temperature conditions, the activity of superoxide dismutase (SOD) was
observed to increase in all the stuided varieties. A more significant increase was seen in
varieties RH-1999-42 (44.63%) and RH-1566 (39.88%) than in varieties RH-1707 (28.92%) and
RH-1708 (26.12%). The activity of SOD was higher among plants sprayed with Put in the
absence of heat stress than in control plants, as seen in varieties RH-1708 (19.08%), RH-1707
(19.93%), RH-1999-42 (15.82%) and RH-1566 (11.42%). The activity increased greatly in
seedlings subjected to combined heat stress and Put treatment. RH-1999-42 (50.06%) and
RH-1566 (46.98%) show a more significant increase than RH-1707 (30.92%) and RH-1708
(28.16%) (Figure 10).
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Figure 10. Effect of high temperature and Putrescine on SOD in Brassica juncea seedlings. Each bar
represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk (*) indicates
significant difference between the treatments. Different alphabets for each mean show statistically
significant differences between the varieties according to the least significant difference (LSD) test
(p < 0.05).
3.4.2. Catalase
Under high temperature, the activity of catalase was observed to be enhanced. RH1999-42 showed a more pronounced increase in catalase activity (60.84%) under hightemperature conditions compared to RH-1566 (53.18%), RH-1708 (44.43%) and RH-1707
(40.19%). Under controlled conditions, exogenous application of Put led to a slight increment in catalase content in varieties RH-1999-42 (16.27%), RH-1707 (14.61%), RH-1566
(10.48%) and RH-1708 (8.67%). When Put was applied in combination with high temperature, the maximum increase in catalase activity was observed in RH-1999-42 (70.76%),
surpassing the increase in RH-1566 (59.64%), RH-1708 (56.29%) and RH-1707 (50.24%)
(Figure 11).
Figure 11. Effect of high temperature and Putrescine on catalase in Brassica juncea seedlings. Each bar
represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk (*) indicates
significant difference between the treatments. Different alphabets for each mean show statistically
significant differences between the varieties according to the least significant difference (LSD) test
(p < 0.05).
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3.5. Lipid Peroxidation
Under high-temperature conditions, all the studied varieties exhibited an increase in
lipid peroxidation, with the maximum increase in RH-1708 (71.98%) and RH-1707 (67.14%).
RH-1566 (48.03%) and RH-1999-42 (47.07%) showed an increase but to a lesser extent.
Under normal temperature, the application of Put resulted in a more efficient reduction of
MDA levels in variety RH-1999-42 (30.59%) in comparison to RH-1707 (24.01%), RH-1566
(15.37%), and RH-1708 (7.3%). The exogenous application of Put in combination with
high temperature resulted in a reduction of MDA content in RH-1707 (27.02%), RH-1708
(26.73%), RH-1566 (13.54%) and RH-1999-42 (11.74%) (Figure 12).
Figure 12. Effect of high temperature and Putrescine on MDA in Brassica juncea seedlings. Each bar
represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk (*) indicates
significant difference between the treatments. Different alphabets for each mean show statistically
significant differences between the varieties according to the least significant difference (LSD) test
(p < 0.05).
3.6. H2 O2 Content
Under high temperature, H2 O2 content increased in all four varieties. RH-1708
(51.34%) and RH-1707 (45.81%) showed a more noteworthy increase than RH-1999-42
(30.54%) and RH-1566 (26.21%). Under normal temperature, a decrease in H2 O2 content
was observed in seedlings treated with exogenous Put. Specifically, in RH-1999-42, there
was the maximum reduction (27.25%), followed by RH-1708 (14.85%), RH-1707 (10.03%),
and RH-1566 (9.29%). The application of Put along with high temperature was functional
in reducing the H2 O2 content as was seen in all the varieties, RH-1708 (21.95%), RH-1707
(20.32%), RH-1566 (18.68%) and RH-1999-42 (15.26%) (Figure 13).
3.7. Histochemical Analysis
The staining process using Evans blue dye revealed the rapid initiation of cell death,
which was indicated by the appearance of blue-colored spots on the leaf surfaces. These
spots were more noticeable in leaves exposed to high temperatures compared to the control
group. The presence of blue spots indicated the irreversible uptake of Evans blue stain
by the dying cells. However, when high-temperature conditions were combined with
the application of Put, there were significantly fewer blue spots observed on the leaves
(Figure 14A).
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−
Figure 13. Effect of high temperature and Putrescine on H2 O2 in Brassica juncea seedlings. Each bar
represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk (*) indicates
significant difference between the treatments. Different alphabets for each mean show statistically
significant differences between the varieties according to the least significant difference (LSD) test
−
(p < 0.05).
A
B
Figure 14. Histochemical analysis in leaves of Brassica juncea exposed to high temperature and
Putrescine (1 mM). (A). Cell death visualization using Evans blue staining method (blue color
indicates death of cells), (B). Localization of superoxide radical by NBT staining (blue colored spots
).
reflect reduction of NBT to formazan).
Formation of dark blue colored spots of formazan due to reduction of NBT by O2 ·−
was seen distributed all over the surface of leaves under high temperature, which clearly
marked the accumulation of superoxide ions. When leaves were treated with Put under
normal temperature conditions, there were very few spots observed, suggesting a reduction
Agronomy 2023, 13, 1879
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in the accumulation of superoxide ions. Seedlings that were subjected to the combined
treatment of Put and high temperature exhibited a diminished accumulation of O2 ·− . This
was evidenced by a reduction in the number of dark blue spots (Figure 14B).
3.8. Stomatal Density
Under high-temperature stress, an increase in stomatal density was observed (Figure 15).
RH-1708 exhibited the most substantial increase (37.84%), followed by RH-1707 (29.41%),
while varieties RH-1999-42 (19.63%) and RH-1566 (10.58%) showed a lesser increase. No
significant change in stomatal density was observed in seedlings treated with Put under
normal temperature conditions. However, application of Put under high temperature reduced the density of stomata in leaves to some extent. The maximum percentage reduction
was seen in RH-1708 (18.7%) followed by RH-1707 (9.19%) and RH-1999-42 (6.17%). No
significant change was observed in RH-1566 (0.2%) (Figure 16).
Figure 15. Stomatal density in leaves of Brassica juncea under high temperature and Putrescine.
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Figure 16. Effect of high temperature and Putrescine on stomatal density in Brassica juncea seedlings.
Each bar represents the mean (n = 3) and the error bar indicates the standard deviation. Asterisk
(*) indicates significant difference between the treatments. Different alphabets for each mean show
statistically significant differences between the varieties according to the least significant difference
(LSD) test (p < 0.05).
4. Discussion
Indian mustard (Brassica juncea L.) is one of the most important oilseed crops in
the country and it occupies a considerable acreage among the Brassica group of oilseed
crops. One of the most harmful stresses for plant growth and development is the steadily
rising ambient temperature. Heat stress has a number of effects on biological functions,
either directly or indirectly through the alteration of the environment. In Brassica species,
high temperatures during growth and developmental phases can cause substantial yield
losses [50,51]. There are several pieces of evidence that exogenous application of polyamines
such as Put, spermidine and spermine protects plants against the damage caused by various
types of abiotic stresses [52,53]. Polyamines are the endogenous plant growth regulators,
or they may act as intracellular messengers that encourage a number of physiological and
biochemical processes in response to high temperature. As a result, they increase tolerance
of plants to stresses by modifying growth and development. [54,55].
In the present study, the effects of high temperature on four varieties of B. juncea,
namely RH-1707, RH-1708, RH-1566 and RH-1999-42, were analyzed, and various morphophysiological and biochemical parameters were studied. This study revealed that hightemperature stress significantly reduced various growth-related parameters such as seedling
length, fresh weight, dry weight and leaf area. The most prominent decrease was shown by
variety RH-1708, followed by RH-1707. The impact of exogenous application of Put shows
an improvement in these growth-related parameters which suggests its effectiveness in
ameliorating heat-induced morphological damage. Our results were in agreement with the
findings of El Bassiouny et al. [56] and Hassanein et al. [34] in wheat and Amooaghaie et al.
in soybean [36].
One of the crucial characteristics of plants with improved tolerance and better performance under high temperatures is reportedly their RWC [57]. Elevated temperatures
led to a notable decrease in the RWC across all the examined varieties. However, RH-1566
and RH-1999-42 exhibited a smaller decline. Our results are in agreement with work on
rice [58] and wheat [59]. The decrease in leaf water content might affect plant metabolism
and decrease plant growth and biomass. The decrease in RWC of the leaves may be related
to the reduction in the quantity, mass and development of the roots during heat stress,
which eventually restricts the provision of water and nutrients to the plant’s above-ground
portions [60]. In this study, the exogenous application of Put substantially improved the
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water status of leaves, which is in accordance with the findings of Gupta et al. in wheat
under heat stress [61].
High-temperature stress is known to cause a loss of leaf pigment in plants and seriously
impairs photosynthetic processes. In the present study, it was observed that all four varieties experienced a reduction in chlorophyll content when exposed to high temperatures.
The impact was particularly noticeable in RH-1708 and RH-1707, demonstrating a more
pronounced effect. Chlorophyll breakdown or the suppression of chlorophyll production
may be responsible for the reduction in chlorophyll content [62]. The exogenous application
of Put reduced heat-induced chlorophyll degradation in our study. Put is known to increase
leaf chlorophyll levels and protect thylakoid membranes via a chlorophyll-protein complex
site [63]. Similarly to our findings, Put reduced the heat-induced damage to chlorophyll pigments in wheat [57]. Additionally, the generation of harmful oxygen species and a decrease
in antioxidant defense are the causes of the impact of high temperatures on pigments and
other photosynthetic machinery [64]. Thus, there might be a correlation between decrease
in chlorophyll content and increase EL, MDA and H2 O2 content. Our result revealed
that varieties with higher EL, MDA and H2 O2 content had lower chlorophyll content also.
RH-1707 and RH-1708 showed much higher oxidative damage, which may reveal their
heat sensitivity. However, the other two varieties, RH-1566 and RH-1999-42, had lowered
EL, MDA and H2 O2 content, indicating that these varieties could maintain their membrane
integrity under heat stress. Maintaining membrane stability and integrity under stress
conditions is a key element of tolerance and is necessary for continued photosynthetic and
respiratory function [65]. RH-1566 and RH-1999-42 may be heat-tolerant as a result of lower
oxidative damage. Polyamines play a crucial role in preserving the integrity of the membrane by lowering electrolyte leakage and MDA levels [66]. In the present study, a decline
in the content of EL, MDA and H2 O2 was seen after the application of Put. Our results
correlated with the findings of Islam et al. [67] in Beta vulgaris, Bhattacharjee and Mukherjee
in Amaranthus lividus seedlings [68] and Nagesh and Devaraj in Phaseolus vulgaris [69],
wherein Put-treated plants experienced less oxidative stress than untreated plants, as seen
by decreased electrolyte leakage and lesserH2 O2 and MDA buildup. This is mostly because
of the increased antioxidant enzyme activities that regulate ROS homeostasis.
A significant rise in antioxidant enzyme levels was observed when plants were subjected to high-temperature stress or treated with Put. This can be considered as a mechanism
employed by the plants for the detoxification of ROS. RH-1566 and RH-1999-42 exhibited a
greater increase in antioxidant enzyme concentration under heat stress compared to the
other two varieties. This observation suggests that RH-1566 and RH-1999-42 possess a
greater ability to detoxify oxidative damage. Interestingly, a boost was seen in SOD and
catalase enzyme activity after the application of Put in all the studied varieties. Polyamines
are known to have the ability to increase the activities of ROS-scavenging antioxidants
which results in reducing heat-induced oxidative damage and creates more balanced conditions [70]. Put is known to reduce the oxidative damage by direct scavenging of free
radicals and by indirectly elevating the contents of antioxidants [71]. Similar increases in
antioxidants under high temperature and Put application were reported by Jing et al. [72]
in Triticum aestivum and Zhao et al. [73] in Cabernet Sauvignon seedlings. This indicates that
exogenous application of Put substantially improved high-temperature-induced damage
(Figure 17).
Histochemical analysis was conducted to assess the levels of superoxide ions using
NBT staining, and localized cell death was monitored using Evans blue dye. The leaves
were more extensively stained with blue spots under high-temperature treatment in both the
staining procedures. The staining was more prominent in RH-1707 and RH-1708 showing
more oxidative damage. However, much less accumulation was seen in leaves exposed
to combined treatment of exogenous Put and high temperature. The overproduction of
ROS at high temperatures might be attributed to imbalanced antioxidant enzyme activity
and the expression of associated genes, which has the principal consequence of causing
heat-induced stress [74]. Damage to cellular components such proteins, nucleic acids, lipids
Agronomy 2023, 13, 1879
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and metabolites as a consequence of an imbalance between ROS and antioxidants (both
enzymatic and nonenzymatic) eventually affects cell viability and leads to cell death [75,76].
Figure 17. Schematic diagram displaying the role of Putrescine in mitigating the adverse effects of
high-temperature stress in plants. Application of Putrescine improved plant growth by enhancing
antioxidants, water relation, membrane stabilization and photosynthetic pigments, while reducing
oxidative stress markers.
In response to high temperature, a rapid closure of stomata, an increase in stomatal
density and reduction in cell size have been reported [77]. In the present study, hightemperature stress increased stomatal density in the leaves of B. juncea; consequently
Put application reduced it to some extent. However, no significant difference was seen
in Put-treated and control plants at normal temperature. A more efficient increase in
stomatal density was seen in RH-1707 and RH-1708. Stomatal density increases as external
temperature increases, because to reduce cell and tissue damage caused by heat stress
stomata are newly formed [78]. In maize, an increase in stomatal density was reported,
which is associated with the need of cooling through transpiration, but an excessive increase
can also lead to an increase in leaf temperature [79]. Similar results were obtained from the
study of lettuce seedlings under high-temperature stress. The stomatal density increased
under high temperature treatment and was significantly inhibited after spraying with
exogenous spermidine. This revealed that under high-temperature stress, lettuce seedling
stomatal density rose, and water transpiration loss increased, which is likely to generate
more drought stress. Exogenous spermidine might effectively restrict stomatal density
growth while preventing fast water loss via transpiration. It prevents dehydration in
lettuce seedlings under high-temperature stress, hence preventing drought stress [80]. The
exogenous application of Put exhibited several beneficial effects in mitigating the adverse
Agronomy 2023, 13, 1879
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impacts of high-temperature stress on the growth, physiological, and biochemical processes
of B. juncea. As a result, the plants treated with Put demonstrated improved performance
compared to the untreated plants when exposed to high-temperature stress.
5. Conclusions
High temperature affects growth, morpho-physio and biochemical parameters in
B. juncea. The more pronounced effect was seen in varieties RH-1708 and RH-1707 as
evidenced by reduction in growth rate, water status, membrane stability, more ROS accumulation and increased stomatal density under high temperature. However, varieties
RH-1566 and RH-1999-42 performed better under high temperature by maintaining sufficient membrane integrity and accumulation of antioxidants to reduce oxidative damage.
Thus, RH-1708 and RH-1707 were most sensitive and RH-1566 and RH-1999-42 were
tolerant of high-temperature stress. These varieties can be further used to examine the
molecular mechanisms of heat tolerance in B. juncea. Exogenous application of Put restored
tissue integrity, maximized levels of antioxidant enzymes which have been associated in
reducing oxidants (H2 O2 and O2 ·− ) and their harm in terms of ionic leakage (EL), lipid
peroxidation (MDA) and other oxidative damage. Therefore, exogenously applied Put
could be regarded as an effective bioactive stimulant that mitigates high-temperature stress
damage by minimizing membrane damage, enhancing antioxidative enzyme activities,
resolving ionic imbalance, promoting plant growth and production and, consequently,
promoting production of B. juncea under extreme temperatures.
Author Contributions: Conceptualization, N.L. and P.S.; methodology, P.S.; software, Y.A. and P.S.;
validation, N.L., Y.A. and A.Z.; formal analysis, P.S.; investigation, P.S. and. N.L. resources, A.M.A.E.; and H.O.E.; data curation, P.S.; writing—original draft preparation, P.S., N.L., Y.A. and A.Z.;
writing—review and editing, A.Z. and N.L. and A.G.; visualization, P.S.; supervision, N.L.; project
administration, N.L.; funding acquisition, A.M.A.-E. and H.O.E. All authors have read and agreed to
the published version of the manuscript.
Funding: CSIR-UGC Junior Research Fellowship, and Researchers Supporting Project number
(RSPD2023R676), King Saud University.
Data Availability Statement: All data are present with the article.
Acknowledgments: The author (P.S.) would like to thank CSIR-UGC for Junior Research Fellowship and oilseeds section Department of Genetics and Plant Breeding, CCS Haryana Agricultural
University, Hisar for providing seeds. The authors extend their sincere appreciation to Researchers
Supporting Project number (RSPD2023R676), King Saud University, Riyadh, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Singh, D.; Balota, M.; Collakova, E.; Isleib, T.G.; Welbaum, G.E.; Tallury, S.P. Heat stress related physiological and metabolic traits
in peanut seedlings. Peanut Sci. 2016, 43, 24–35. [CrossRef]
DAC. Agricultural Statistics at a Glance; Directorate of Economics and Statistics, Department of Agriculture, Cooperation and
Farmers Welfare, Ministry of Agriculture and Farmers Welfare, Government of India: New Delhi, India, 2019; pp. 72–73.
Asthir, B. Protective mechanisms of heat tolerance in crop plants. J. Plant Interact. 2015, 10, 202–210. [CrossRef]
Lobel, G.H.; Asner, P.K. Effect of high temperature on crop growth and yield. Prog. Agric. Eng. Sci. 2003, 49, 23–99.
Wahid, A.; Gelani, S.; Ashraf, M.; Foolad, M.R. Heat tolerance in plants: An overview. Environ. Exp. Bot. 2007, 61, 199–223.
[CrossRef]
Porter, J.R. Rising temperatures are likely to reduce crop yields. Nature 2005, 43, 166–174. [CrossRef]
Finch-Savage, W.E.; Bassel, G.W. Seed vigor and crop establishment: Extending performance beyond adaptation. J. Exp. Bot. 2016,
67, 567–591. [CrossRef]
Nadeem, M.; Li, J.; Wang, M.; Shah, L.; Lu, S.; Wang, X.; Ma, C. Unraveling field crops sensitivity to heat stress: Mechanisms,
approaches, and future prospects. Agronomy 2018, 8, 128. [CrossRef]
Azharudheen, T.M.; Yadava, D.K.; Singh, N.; Vasudev, S.; Singh, R.; Prabhu, K.V. A study on the thermo-tolerance at germination
and seedling stage in Indian Mustard [Brassica Juncea (L.) Czern&Coss]. Int. J. Agric. Food Sci. 2013, 4, 589–594.
Valliyodan, B.; Nguyen, H.T. Understanding regulatory networks and engineering for enhanced drought tolerance in plants.
Curr. Opin. Plant Biol. 2006, 9, 189–195. [CrossRef]
Agronomy 2023, 13, 1879
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
19 of 21
Bohra, A.; Sanadhya, D.; Bhatia, D.S. Polyamines: Metabolism and Role in Abiotic Stress Amelioration. J. Plant Sci. Res. 2015, 31,
183–195.
Mustafavi, S.H.; Badi, H.N.; Sekara, A.; Mehrafarin, A.; Janda, T.; Ghorbanpour, M.; Rafiee, H. Polyamines and their possible
mechanisms involved in plant physiological processes and elicitation of secondary metabolites. Acta Physiol. Plant. 2018, 40, 102.
[CrossRef]
Falasca, G.; Franceschetti, M.; Bagni, N.; Altamura, M.M.; Biasi, R. Polyamine biosynthesis and control of the development of
functional pollen in kiwifruit. Plant Physiol. Biochem. 2010, 48, 565–573. [CrossRef] [PubMed]
Galston, A.W.; Kaur Sawhney, R.; Altabella, T.; Tiburcio, A.F. Plant polyamines in reproductive activity and response to abiotic
stress. Acta Bot. 1997, 110, 197–207. [CrossRef]
Bais, H.P.; Ravishankar, G.A. Role of polyamine in the ontogeny of plants and their biotechnological applications. Plant Cell Tissue
Organ Cult. 2002, 69, 1–34. [CrossRef]
Galston, A.W.; Sawhney, R.K. Polyamines in plant physiology. Plant Physiol. 1990, 94, 406–410. [CrossRef] [PubMed]
Alcázar, R.; Altabella, T.; Marco, F.; Bortolotti, C.; Reymond, M.; Koncz, C.; Carrasco, P.; Tiburcio, A.F. Polyamines: Molecules
with regulatory functions in plant abiotic stress tolerance. Planta 2010, 231, 1237–1249. [CrossRef]
Childs, A.C.; Mehta, D.J.; Gerner, E.W. Polyamine-dependent gene expression. Cell. Mol. Life Sci. 2003, 60, 1394–1406. [CrossRef]
Ouyang, J.; Song, C.; Chen, D. Research progress on heat-tolerance mechanism and transports of polyamines in plant. Mol. Plant
Breed. 2017, 15, 3286–3294.
Spormann, S.; Soares, C.; Teixeira, J.; Fidalgo, F. Polyamines as key regulatory players in plants under metal stress—A way for an
enhanced tolerance. Ann. Appl. Biol. 2021, 178, 209–226. [CrossRef]
Antoniou, C.; Zarza, X.; Gohari, G.; Panahirad, S.; Filippou, P.; Tiburcio, A.F.; Fotopoulos, V. Involvement of polyamine
metabolism in the response of Medicago truncatula genotypes to salt stress. Plants 2021, 10, 269. [CrossRef]
Sundararajan, S.; Sivakumar, H.P.; Nayeem, S.; Rajendran, V.; Subiramani, S.; Ramalingam, S. Influence of exogenous polyamines
on somatic embryogenesis and regeneration of fresh and long-term cultures of three elite indica rice cultivars. Cereal Res. Commun.
2021, 49, 245–253. [CrossRef]
Walters, D.R. Polyamines and plant disease. Phytochemistry 2003, 64, 97–107. [CrossRef] [PubMed]
Tiburcio, A.F.; Altabella, T.; Bitrián, M.; Alcázar, R. The roles of polyamines during the lifespan of plants: From development to
stress. Planta 2014, 240, 1–18. [CrossRef] [PubMed]
Gill, S.S.; Tuteja, N. Polyamines and abiotic stress tolerance in plants. Plant Signal. Behav. 2010, 5, 26–33. [CrossRef]
Gupta, S.; Agarwal, V.P.; Gupta, N.K. Efficacy of putrescine and benzyladenine on photosynthesis and productivity in relation to
drought tolerance in wheat (Triticum aestivum L.). Physiol. Mol. Biol. Plants 2012, 18, 331–336. [CrossRef] [PubMed]
Zhu, X.; Wang, L.; Yang, R.; Han, Y.; Hao, J.; Liu, C.; Fan, S. Effects of exogenous putrescine on the ultrastructure of and calcium
ion flow rate in lettuce leaf epidermal cells under drought stress. Hortic. Environ. Biotechnol. 2019, 60, 479–490. [CrossRef]
Hassan, N.; Ebeed, H.; Aljaarany, A. Exogenous application of spermine and putrescine mitigate adversities of drought stress in
wheat by protecting membranes and chloroplast ultra-structure. Physiol. Mol. Biol. Plants 2020, 26, 233–245. [CrossRef]
Quinet, M.; Ndayiragije, A.; Lefèvre, I.; Lambillotte, B.; Dupont-Gillain, C.C.; Lutts, S. Putrescine differently influences the effect
of salt stress on polyamine metabolism and ethylene synthesis in rice cultivars differing in salt resistance. J. Exp. Bot. 2010, 61,
2719–2733. [CrossRef]
Xiong, F.; Liao, J.; Ma, Y.; Wang, Y.; Fang, W.; Zhu, X. The protective effect of exogenous putrescine in the response of tea plants
(Camellia sinensis) to salt stress. HortScience 2018, 53, 1640–1646. [CrossRef]
Mustafavi, S.H.; Shekari, F.; Abbasi, A. Putrescine improve low temperature tolerance of fennel (Foeniculum vulgare Mill.) seeds.
Cercet. Agron. Mold. 2015, 48, 69–76. [CrossRef]
Jankovska-Bortkevic, E.; Gavelienè, V.; Šveikauskas, V.; Mockevičiūtė, R.; Jankauskienè, J.; Todorova, D.; Sergiev, I.; Jurkonienè, S.
Foliar application of polyamines modulates winter oilseed rape responses to increasing cold. Plants 2020, 9, 179. [CrossRef]
Asthir, B.; Deep, A. Thermotolerance and antioxidant response induced by putrescine and heat acclimation in wheat seedlings.
Seed Sci. Biotechnol. 2011, 5, 42–46.
Hassanein, R.A.; El-Khawas, S.A.; Ibrahim, S.K.; El-Bassiouny, H.M.; Mostafa, H.A.; Abd El-Monem, A.A. Improving the
thermotolerance of wheat plant by foliar application of arginine or putrescine. Pak. J. Bot. 2013, 45, 111–118.
Das, A.; Karwa, S.; Taunk, J.; Bahuguna, R.N.; Chaturvedi, A.K.; Kumar, P.; Chinnusamy, V.; Pal, M. Putrescine exogenous
application alleviates oxidative stress in reproductive tissue under high temperature in rice. Plant Physiol. Rep. 2021, 26, 381–391.
[CrossRef]
Amooaghaie, R.; Moghym, S. Effect of polyamines on thermotolerance and membrane stability of soybean seedling. Afr. J.
Biotechnol. 2011, 10, 9673–9679.
Fu, X.Z.; Xing, F.; Wang, N.Q.; Peng, L.Z.; Chun, C.P.; Cao, L.; Ling, L.L.; Jiang, C.L. Exogenous spermine pretreatment confers
tolerance to combined high-temperature and drought stress in vitro in trifoliate orange seedlings via modulation of antioxidative
capacity and expression of stress-related genes. Biotechnol. Biotechnol. Equip. 2014, 28, 192–198. [CrossRef]
Jahan, M.S.; Hasan, M.M.; Alotaibi, F.S.; Alabdallah, N.M.; Alharbi, B.M.; Ramadan, K.M.; Bendary, E.S.; Alshehri, D.; Jabborova,
D.; Al-Balawi, D.A.; et al. Exogenous putrescine increases heat tolerance in tomato seedlings by regulating chlorophyll metabolism
and enhancing antioxidant defense efficiency. Plants 2022, 11, 1038. [CrossRef]
Agronomy 2023, 13, 1879
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
20 of 21
Nobosse, P.; Fombang, E.N.; Mbofung, C.M. Effects of age and extraction solvent on phytochemical content and antioxidant
activity of fresh Moringa oleifera L. leaves. Food Sci. Nutr. 2018, 6, 2188–2198. [CrossRef]
Yang, A.; Larsen, T.W.; Tume, R.K. Carotenoid and retinol concentrations in serum, adipose tissue and liver and carotenoid
transport in sheep, goats and cattle. Aust. J. Agric. Res. 1992, 4, 1809–1817. [CrossRef]
Smart, R.E.; Bingham, G.E. Rapid estimates of relative water content. Plant Physiol. 1974, 53, 258–260. [CrossRef]
Dionisio-Sese, M.L.; Tobita, S. Antioxidant responses of rice seedlings to salinity stress. Plant Sci. 1998, 135, 1–9. [CrossRef]
Dhindsa, R.S.; Plumb-Dhindsa, P.; Thorpe, T.A. Leaf senescence: Correlated with increased levels of membrane permeability and
lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 1981, 32, 93–101. [CrossRef]
Aebi, H. Catalase in vitro. Methods Enzymol. 1984, 105, 121–126. [PubMed]
Heath, R.L.; Packer, L. Photo peroxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch.
Biochem. Biophys. 1968, 125, 189–198. [CrossRef]
Velikova, V.; Yordanov, I.; Kurteva, M.; Tsonev, T. Effects of simulated acid rain on the photosynthetic characteristics of Phaseolus
vulgaris L. Photosynthetica 1998, 34, 523–535. [CrossRef]
Romero-Puertas, M.C.; Rodríguez-Serrano, M.; Corpas, F.J.; Gomez, M.D.; Del Rio, L.A.; Sandalio, L.M. Cadmium-induced
subcellular accumulation of O2 ·− and H2 O2 in pea leaves. Plant Cell Environ. 2004, 27, 1122–1134. [CrossRef]
Wang, J.; Sun, P.P.; Chen, C.L.; Wang, Y.; Fu, X.Z.; Liu, J.H. An arginine decarboxylase gene PtADC from Poncirus trifoliata confers
abiotic stress tolerance and promotes primary root growth in Arabidopsis. J. Exp. Bot. 2011, 62, 2899–2914. [CrossRef]
Paul, V.; Sharma, L.; Pandey, R.; Meena, R.C. Measurements of stomatal density and stomatal index on leaf/plant surfaces. In
Proceedings of the ICAR Sponsored Training Programme for Technical Staff of ICAR Institutes on—Physiological Techniques to
Analyze the Impact of Climate Change on Crop Plants, New Delhi, India, 16–25 January 2017; p. 27.
Angadi, S.V.; Cutforth, H.W.; Miller, P.R.; McConkey, B.G.; Entz, M.H.; Brandt, S.A.; Volkmar, K.M. Response of three Brassica
species to high temperature stress during reproductive growth. Can. J. Plant Sci. 2000, 80, 693–701. [CrossRef]
Morrison, M.J.; Stewart, D.W. Heat stress during flowering in summer Brassica. Crop Sci. 2002, 42, 797–803. [CrossRef]
Todorova, D.; Katerova, Z.; Alexieva, V.; Sergiev, I. Polyamines–possibilities for application to increase plant tolerance and
adaptation capacity to stress. Genet. Plant Physiol. 2015, 5, 123–144.
Chen, D.; Shao, Q.; Yin, L.; Younis, A.; Zheng, B. Polyamine function in plants: Metabolism, regulation on development, and roles
in abiotic stress responses. Front. Plant Sci. 2019, 9, 1945. [CrossRef] [PubMed]
Kusano, T.; Berberich, T.; Tateda, C.; Takahashi, Y. Polyamines: Essential factors for growth and survival. Planta 2008, 228, 367–381.
[CrossRef] [PubMed]
ElSayed, A.I.; Mohamed, A.H.; Rafudeen, M.S.; Omar, A.A.; Awad, M.F.; Mansour, E. Polyamines mitigate the destructive impacts
of salinity stress by enhancing photosynthetic capacity, antioxidant defense system and upregulation of calvin cycle-related genes
in rapeseed (Brassica napus L.). Saudi J. Biol. Sci. 2022, 29, 3675–3686. [CrossRef]
El Bassiouny, H.M.S.; Mostafa, H.A. Physiological responses of wheat plant to foliar treatments with arginine or putrescine. Aust.
J. Basic Appl. Sci. 2008, 2, 1390–1403.
Dhanda, S.S.; Sethi, G.S.; Behl, R.K. Indices of drought tolerance in wheat genotypes at early stages of plant growth. J. Agron. Crop
Sci. 2004, 190, 6–12. [CrossRef]
Fahad, S.; Hussain, S.; Saud, S.; Hassan, S.; Tanveer, M. A combined application of biochar and phosphorus alleviates heat-induced
adversities on physiological, agronomical and quality attributes of rice. Plant Physiol. Biochem. 2016, 103, 191–198. [CrossRef]
Hameed, A.; Goher, M.; Iqbal, N. Heat Stress-Induced Cell Death, Changes in Antioxidants, Lipid Peroxidation, and Protease
Activity in Wheat Leaves. J. Plant Growth Regul. 2012, 31, 283–291. [CrossRef]
Bañon, S.; Fernandez, J.A.; Franco, J.A.; Torrecillas, A.; Alarcón, J.J.; Sánchez-Blanco, M.J. Effects of water stress and night
temperature preconditioning on water relations and morphological and anatomical changes of Lotus creticus plants. Sci. Hortic.
2004, 101, 333–342. [CrossRef]
Gupta, S.; Sharma, M.L.; Gupta, N.K.; Kumar, A. Productivity enhancement by putrescine in wheat (Triticum aestivum L.). Physiol.
Mol. Biol. Plants 2003, 9, 279–282.
Dutta, S.; Mohanty, S.; Tripathy, B.C. Role of Temperature Stress on Chloroplast Biogenesis and Protein Import in Pea. Plant
Physiol. 2009, 150, 1050–1061. [CrossRef]
Besford, R.; Richardson, C.; Campos, J.; Tiburcio, A. Effect of polyamines on stabilization of molecular complexes in thylakoid
membranes of osmotically stressed oat leaves. Planta 1993, 189, 201–206. [CrossRef]
Kumar, S.; Thakur, P.; Kaushal, N.; Malik, J.A.; Gaur, P.; Nayyar, H. Effect of varying high temperatures during reproductive
growth on reproductive function, oxidative stress and seed yield in chickpea genotypes differing in heat sensitivity. Arch. Agron.
Soil Sci. 2013, 59, 823–843. [CrossRef]
Bajji, M.; Kinet, J.M.; Lutts, S. The use of the electrolyte leakage method for assessing cell membrane stability as a water stress
tolerance test in durum wheat. Plant Growth Regul. 2002, 36, 61–70. [CrossRef]
Khalil, S.I.; El-Monem, H.M.S.; Hassanein, R.A.; Mostafa, H.A.; El-Khawas, S.A.; Abd El-Monem, A.A. Antioxidant Defense
System in Heat Shocked Wheat Plants Previously Treated with Arginine or Putrescine. Aust. J. Basic Appl. Sci. 2009, 3, 1517–1526.
Islam, M.J.; Uddin, M.J.; Hossain, M.A.; Henry, R.; Begum, M.K.; Sohel, M.A.T.; Mou, M.A.; Ahn, J.; Cheong, E.J.; Lim, Y.-S.
Exogenous Putrescine Attenuates the Negative Impact of Drought Stress by Modulating Physio-Biochemical Traits and Gene
Expression in Sugar Beet (Beta vulgaris L.). PLoS ONE 2022, 17, e0262099. [CrossRef] [PubMed]
Agronomy 2023, 13, 1879
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
21 of 21
Bhattacharjee, S.; Mukherjee, A.K. Implications of reactive oxygen species in heat shock induced germination and early growth
impairment in Amaranthus lividus L. Biol. Plant. 2003, 47, 517–522. [CrossRef]
Nagesh Babu, R.; Devaraj, V.R. High temperature and salt stress response in French bean (Phaseolus vulgaris). Aust. J. Crop Sci.
2008, 2, 40–48.
Nahar, K.; Hasanuzzaman, M.; Alam, M.M.; Rahman, A.; Mahmud, J.A.; Suzuki, T.; Fujita, M. Insights into spermine-induced
combined high temperature and drought tolerance in mung bean: Osmoregulation and roles of antioxidant and glyoxalase
system. Protoplasma 2017, 254, 445–460. [CrossRef]
Mafakheri, A.; Siosemardeh, A.; Bahramnejad, B.; Struik, P.C.; Sohrabi, Y. Effect of drought stress and subsequent recovery on
protein, carbohydrate contents, catalase and peroxidase activities in three chickpea (‘Cicer arietinum’) cultivars. Aust. J. Crop Sci.
2011, 5, 1255–1260.
Jing, J.G.; Guo, S.Y.; Li, Y.F.; Li, W.H. Effects of polyamines on agronomic traits and photosynthetic physiology of wheat under
higher temperature stress. Photosynthetica 2019, 57, 912–920. [CrossRef]
Zhao, J.; Wang, X.; Pan, X.; Jiang, Q.; Xi, Z. Exogenous putrescine alleviates drought stress by altering reactive oxygen species
scavenging and biosynthesis of polyamines in the seedlings of Cabernet Sauvignon. Front. Plant Sci. 2021, 12, 767992. [CrossRef]
[PubMed]
Rout, J.R.; Sahoo, S.L. Antioxidant enzyme gene expression in response to copper stress in Withania somnifera L. Plant Growth
Regul. 2013, 71, 95–99. [CrossRef]
Xie, X.; He, Z.; Chen, N.; Tang, Z.; Wang, Q.; Cai, Y. The roles of environmental factors in regulation of oxidative stress in plant.
BioMed Res. Int. 2019, 2019, 9732325. [CrossRef] [PubMed]
Davies, K.M.; Jibran, R.; Zhou, Y.; Albert, N.W.; Brummell, D.A.; Jordan, B.R.; Bowman, J.L.; Schwinn, K.E. The evolution of
flavonoid biosynthesis: A bryophyte perspective. Front. Plant Sci. 2020, 11, 7. [CrossRef] [PubMed]
Lin, H.; Chen, Y.J.; Zhang, H.L.; Fu, P.L.; Fan, Z.X. Stronger cooling effects of transpiration and leaf physical traits of plants from a
hot dry habitat than from a hot wet habitat. Funct. Ecol. 2017, 31, 2202–2211. [CrossRef]
Shen, H.F.; Zhao, B.; Xu, J.J.; Liang, W.; Huang, W.M.; Li, H.H. Effects of heat stress on changes in physiology and anatomy in two
cultivars of Rhododendron. S. Afr. J. Bot. 2017, 112, 338–345. [CrossRef]
Zhao, W.; Sun, Y.; Kjelgren, R.; Liu, X. Response of stomatal density and bound gas exchange in leaves of maize to soil water
deficit. Acta Physiol. Plant. 2015, 37, 1704. [CrossRef]
Yang, X.; Han, Y.; Hao, J.; Qin, X.; Liu, C.; Fan, S. Exogenous spermidine enhances the photosynthesis and ultrastructure of lettuce
seedlings under high-temperature stress. Sci. Hortic. 2022, 291, 110570. [CrossRef]
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