Journal of Crop Science and Biotechnology (2023) 26:63–75

https://doi.org/10.1007/s12892-022-00162-4

ORIGINAL RESEARCH

Exogenously applied plant growth regulator protects rice

from heat‑induced damage by modulating plant defense mechanism
G. Lakshmi2 · R. Beena1 · K. B. Soni2 · M. M. Viji1 · Uday Chand Jha3

Accepted: 23 June 2022 / Published online: 12 July 2022

This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2022

Abstract
This study was undertaken to investigate the effect of biostimulants such as spermidine (1 mM, 2 mM), indole-3-acetic acid
­(10–3 M, ­10–5 M), brassinolide (1 mg/L, 2 mg/L) and boron (50 mg/L, 100 mg/L) on protecting rice plants from heat-induced
damage. Two rice genotypes, Nagina 22 (heat tolerant) and Manu Ratna (heat susceptible), primed with biostimulant solution
were germinated, 14-day-old seedlings were further foliar sprayed with respective treatment solutions prior to the induction of
heat stress (42 °C, 48 h). Heat stress-induced fatal damages to non-biostimulant-treated rice seedlings, whereas biostimulant-
treated ones were protected from heat stress-induced damages, marked by lower levels of malondialdehyde (MDA), H ­ 2O 2
coupled with increased levels of seedling vigour index (SVI), chlorophyll and proline contents. Results showed that 1 mM
spermidine (Spd) and ­10–5 M indole-3-acetic acid (IAA)-treated rice seedlings showed increased heat tolerance and were
evaluated for its effect at reproductive stage stress in a pot culture study. At the panicle initiation stage, plants were pretreated
with 1 mM Spd and 1­ 0–5 M IAA followed by the induction of heat stress, keeping it in a temperature-controlled polyhouse
(42.1 °C, till the time of harvest). Spd- and IAA-pretreated heat-exposed plants exhibited yield improvement over control
by altering physiological, biochemical and molecular mechanisms. Therefore, we conclude from our results that foliar IAA
or Spd applications can be regarded as an agronomic strategy for mitigating the adverse effects of heat stress conditions on
rice plants.

Keywords  Spermidine · Brassinosteroid · Boron · Indole-3-acetic acid · Polyamines · Antioxidant enzymes

Abbreviations Pro Proline

Spd Spermidine Chl Chlorophyll
IAA Indole-3-acetic acid B Boron
ROS Reactive oxygen species
SOD Superoxide dismutase
CAT​ Catalase Introduction
MDA Malondialdehyde
CMSI Cell membrane stability index Heat stress negatively affects the growth of plants. Accord-
SVI Seedling vigor index ing to the IPCC (Intergovernmental Panel on Climate
HT High temperature Change) report 2021, the global average temperature is
expected to rise by 5–7 °C by the end of the century (IPCC
2021). The increase in temperature negatively affects the
* R. Beena growth and development of food crops such as rice (Beena
beena.r@kau.in
2013). Rice being the staple food for more than half of the
1
Department of Plant Physiology, College of Agriculture world’s population, a reduction in its production may lead
Vellayani, Kerala Agricultural University, to severe food scarcity (Janni et al. 2020).
Thiruvananthapuram, Kerala 695522, India Temperature above 32 °C has been shown to have a nega-
2
Department of Agricultural Biotechnology, College tive impact on rice plant growth throughout their lifecycle
of Agriculture Vellayani, Kerala Agricultural University, (Mittler et al. 2012; Jagadish et al. 2012). Severe heat stress
Thiruvananthapuram, Kerala 695522, India
may lead to the accumulation of reactive oxygen species
3
Department of Plant Breeding and Genetics, Indian Institute (such as singlet oxygen, superoxide, hydrogen peroxide
of Pulse Research, Kanpur, India

13
Vol.:(0123456789)

64
­(H2O2) and hydroxyl radicals), which leads to oxidative
damage inside the cell. ROS negatively affects the growth
of plants by reacting with various cellular components (Zhao
et al. 2018). ROS accumulation leads to the breakdown of
membrane lipids by peroxidation. Lipid peroxidation of
the membrane results in the accumulation of MDA, which
serves as an indicator for the extent of membrane damage
and electrolyte leakage (Prasanth et al. 2017). ROS reacts
with proteins, leading to in situ amino acid modification,
fragmentation, or alteration of its electric charge. When ROS
interacts with genetic material, the sugar and base moieties
in DNA are more prone to oxidation, leading to base deg-
radation, single-strand breaks, or mutations. Sailaja et al.
(2015) suggest that ROS may act as a signalling molecule
for the induction of apoptosis, which ultimately leads to the
death of the plant.
Rice plants are more prone to heat stress injury at the
reproductive stage as compared to other growth stages
(Jagadish et al. 2010). High temperatures at the time of
anthesis reduce anther dehiscence, pollen shedding, stigma
receptivity, pollen germination, and pollen tube penetration,
which results in a reduction of pollen viability and spikelet
fertility percentage (Mittler et al. 2012; Beena et al. 2018).
Biostimulant seed priming and foliar application have been
shown to improve heat tolerance in rice plants by increasing
the production of antioxidant enzymes and osmoprotectants
(Raghunath et al. 2021). Auxin, brassinosteroids (Br), sper-
midine (Spd) and boron (B) used in this study are known to
play a significant role in plant growth and development and
are associated with thermo-tolerance in plants and protec-
tion against oxidative damage (Mostofa et al. 2014; Sharma
et al. 2018; Raghunath and Beena 2021). The protective role
of auxin (Zhang et al. 2018; Sharma et al. 2018), brassi-
nosteroids (Holá et al. 2010; Thussagunpanit et al. 2013),
spermidine (Chen et al. 2021; Fu et al. 2019) and boron
(Calderón-Páez et al. 2021) against different abiotic stresses
has been reported.
Auxin, a phytohormone involved in growth and reproduc-
tive development, helps in reducing the heat stress injury
that occurs during the reproductive stages of rice. Indole-
3-acetic acid, a naturally occurring auxin, is formed by the
two-step conversion of tryptophan into indole-3-pyruvate,
which is subsequently converted to IAA catalysed by the
action of the YUCCA encoding flavin monooxygenase
enzyme (Zhao 2011). The upstream regulators of the
YUCCA​gene were downregulated during heat stress, which
impairs reproductive development in plants (Sharma et al.
2018). Thus, the exogenous application of IAA increases its
production endogenously by enhancing the upstream control
elements of YUCCA​genes (Sharma et al. 2018).
13
Journal of Crop Science and Biotechnology (2023) 26:63–75
Brassinosteroids (BRs) are naturally occurring steroid
hormones in plants. It regulates cell growth and develop-
ment by promoting stem elongation, pollen tube growth,
fruit development, and proton pump activity (Castorina
and Consonni 2020). They also confer tolerance to a
wide variety of abiotic stresses such as high temperature,
drought stress, and salinity, by inducing the antioxidant
defensive machinery in stressed plants, resulting in the
reduction of reactive oxygen species (ROS) such as hydro-
gen peroxide, singlet oxygen, the superoxide radical, and
the hydroxyl radical (Ogweno et al. 2008; Nayyar et al.
2014). BRs have been used in agriculture to ameliorate
stress injuries and increase plant yields by enhancing cell
membrane stability index (CMSI), photosynthetic rate
(Pn), stomatal conductance (Gs), Fv/Fm ratio, and chlo-
rophyll stability index (CSI) (Raghunath and Beena 2021).
Polyamines are naturally occurring molecules involved
in a wide variety of physiological and developmental
processes like cell division, cell wall formation, fruit set-
ting, and provide tolerance to biotic and abiotic stresses
in plants (Kusano et al. 2008). Polyamines such as sper-
midine, spermine and putrescine accumulates during abi-
otic stress confers tolerance by increasing the antioxidant
enzyme activities such as superoxide dismutase (SOD),
catalase (CAT) and osmoprotectants such as beta glycine
and proline. Mostofa et al. 2014 reported that the exog-
enous application of polyamines renders heat stress toler-
ance to rice seedlings by modulating the antioxidant and
glyoxalase systems.
Proper plant nutrition alleviates the negative effects
caused by high temperature. The application of boron
(B) significantly reduces the heat stress-induced damage
by involving in various physiological and biochemical
processes in plant growth. B also involved in cell wall
formation, sugar translocation and reproductive organ
development (Guru et al. 2016). The application of boron
to heat-stressed plants increases the production of antioxi-
dant enzymes by scavenging the ROS; thus confers, heat
tolerance. B application also increased pollen germination
and pollen tube formation thus increased spikelet fertility
in rice. Boron deficiency has been identified as one of the
most important factors causing sterility in cereals because
of poor development of anthers and pollen and failure of
pollen germination (García-Hernández and López 2005;
Raghunath and Beena 2021).
Hence, this study was conducted to analyse the impact
of foliar application of various biostimulants on heat toler-
ance based on physiological, biochemical, molecular and
yield traits.
Journal of Crop Science and Biotechnology (2023) 26:63–75 65
Materials and methods
Experiment 1: Preliminary screening of rice
seedlings for heat tolerance using seed priming
and foliar application of biostimulants
A preliminary experiment was conducted to determine
two effective treatments from the following treatments:
spermidine (1  mM and 2  mM), indole-3-acetic acid
­(10–3 M, ­10–5 M), brassinolide (1 mg/L, 2 mg/L) and boron
(50 mg/L, 100 mg/L) based on heat tolerance. The experi-
ment was laid out in a completely randomized design with
five replicates. Each replication consists of three plants.
Supplementary Table 1 lists the characteristics of each
active ingredient of biostimulants sprayed in this study.
Seeds of both varieties, Nagina 22 (heat tolerant) and
Manu Ratna (heat susceptible), were soaked (16 h) with
this biostimulant solution followed by shade drying for
seed priming. Primed seeds were germinated in a paper
towel. After 14 days of germination, the seedlings were
sprayed with the respective biostimulant solution prior to
heat exposure (42 °C) in an incubator. After 48 h of heat
stress, SVI, ­H2O2, MDA, Pro, and Chl content were meas-
ured using the fully expanded leaves of rice seedlings.
Malondialdehyde content (Heath and Packer 1968) was
estimated to measure the extent of lipid peroxidation using
thiobarbituric acid (TBA). 0.5 g leaf samples were homog-
enized in 3 mL of 5.0% (w/v) trichloroacetic acid (TCA).
After centrifugation at 12000 rpm (12 min), 1 ml of super-
natant was collected and mixed with 4 mL of 20% TCA
containing 0.5% TBA, heated at 95 °C for 30 min, and
cooled immediately on ice. This solution was again centri-
fuged at 12000 g for 10 min. The difference in absorbance
at 532 and 600 nm was used to calculate the MDA content
(extinction coefficient-155 ­mM−1 ­cm−1).
Hydrogen peroxide was estimated according to the
method proposed by Yu et al. (2003). 0.5 g of fresh leaves
was homogenized in 3 mL of 50 mM potassium phosphate
buffer (pH 6.5), followed by centrifugation at 12,000 g
for 10 min. 1 ml of supernatant was collected and mixed
with 1 mL 0.1% ­TiCl4 in 20% ­H2SO4. This solution was
again centrifuged at 12000 g for 10 min. ­H2O2 content was
calculated by taking absorbance at 410 nm (extinction co-
efficient: 0.28 µmol−1 ­cm−1).
The seedling vigour index (SVI) was calculated accord-
ing to the formula proposed by Abdul-Baki and Ander-
son (1973). SVI was calculated by multiplying germina-
tion percentage by the seedling length. Seedling vigour
index (SVI) = germination percentage × seedling length
(root + shoot), where germination percentage = (number
of germinated seeds/total number of seeds) × 100.
Proline content was calculated according to the method
proposed by Bates et al. (1973). For 12 mints, 0.5 g of fresh
leaf samples was ground with 5 mL of 3% aqueous sulfos-
alicylic acid and centrifuged at 10,000 g. 2 ml of the super-
natant was collected and mixed with 2 mL of glacial acetic
acid and 2 mL of acid ninhydrin solution. The mixture was
then boiled at 100 °C for 1 h and cooled on ice to stop the
reaction. The resulting red solution was extracted in 2 ml of
toluene and the absorbance at 520 nm was measured. The
proline content in the sample was calculated using a standard
curve of L-Pro.
The chlorophyll content of leaves was measured based
on the procedure proposed by Arnon (1949). 0.5 g of fresh
leaf samples was cut into small bits and put into test tubes
containing 10 ml of DMSO:80% acetone mixture (1:1 v/v)
and were incubated overnight at room temperature. The col-
oured solution obtained was then transferred to a measuring
cylinder and made up to 25 ml with the DMSO–acetone mix.
The absorbance was taken at 645 and 663 nm spectrophoto-
metrically. Total chlorophyll content (mg ­g−1 FW) = (20.2 9
­A645 + 8.02 9 ­A663) × (V/1,000) × sample weight.
Experiment II: Evaluation of contrasting rice
varieties for reproductive stage heat tolerance
using selected two treatments from experiment I
Seeds of Nagina 22 and Manu Ratna varieties were sown in
pot trays (25.5 cm length × 10.2 cm width × 2.7 cm height)
with potting compost (coir pith compost and vermicompost
@ 2:1 ratio). 18 days after sowing the seedlings were trans-
planted to mud pots (45 cm length × 20 cm diameter) with
potting mixture (soil, sand and cow dung). All the plants
were grown under control (ambient) environment until the
panicle initiation stage. The high-temperature stress was
induced from panicle initiation to harvest by keeping the
pots in a temperature-controlled polyhouse at Department
of Plant Physiology, College of Agriculture, Kerala Agri-
cultural University, Kerala, situated at 8°5′ N latitude and
76°9′ E longitude and an altitude of 29 m above mean sea
level from February to May 2021. Environmental conditions
inside polyhouse were 42.1 °C (max) and 26 °C (min) with
relative humidity (RH) 96% (max) and 70.2% (min), whereas
in control condition, it was 32.2 °C (max) and 24.6 °C (min)
with RH 92.6% (max) and 60.2% (min). Package of practices
(Kerala Agricultural University) of rice was followed for
fertilizer application.
In the present study, foliar growth regulators were
applied twice. To prepare plants for environmental stress,
the first set of foliar plant hormone spray was applied 5 days
before heat stress treatment and second set after 5 days
of heat stress induction for inducing tolerance. The following
were the foliar growth regulator concentrations used: 1­ 0–5 M
auxins (IAA) and 1 mM spermidine (Spd). These treatments
13

66
were selected because they showed a positive response and
increased plant tolerance to heat stress in the previous pre-
liminary screening experiment. A hand sprayer was used
for spraying. The application volume per plant was 20 mL,
which was used to wet the upper and lower leaf surfaces.
The following groups of treatments were obtained at
the end of the experiments: normal temperature + distilled
water (NT), heat stress + distilled water (HT), heat stress
condition + 1 mM Spermidine (Spd) and heat stress con-
dition + ­10–5  M Indole-3-acetic acid (IAA). Both varie-
ties (Nagina 22 and Manu Ratna) received these treatment
groups. Each replicate consisted of three plants, and all treat-
ments were set up in a completely randomized design with
five replicates. Readings of the variables determined at the
end of the experiment were taken from each plant.
Biometric observations were taken on plant height, pro-
ductive tiller number, spikelet fertility percentage, pollen
viability percentage, and yield per plant.
The plants' heights were measured using a tape measure
from stem base to shoot tip at the time of harvest of both
control and treatment plants and were expressed in cm. Pro-
ductive tiller was calculated at reproductive stage by manual
counting.
Spikelet fertility was measured at the time of harvest
by manually counting the total number of spikelets, and
number of complete and partially filled spikelets. (Spike-
let fertility percentage = (filled spikelets/total number of
spikelets) × 100.)
The pollen viability test was done by the IKI (iodine
potassium iodide) staining method proposed by Baker and
Baker (1979). IKI (iodine potassium iodide) solution was
prepared by dissolving 1 g of potassium iodide and 0.5 g of
iodine in 100 ml of distilled water. Pollens were stained with
IKI solution and observed under a compound microscope
after 5 min. Live pollen grains absorb the dye and become a
dark red or brown colour, whereas dead pollen grains remain
colourless. (Pollen viability percentage = (number of live
pollens/total pollen count) × 100.)
The weight of all the grains collected per plant was taken
at the time of harvest and expressed in g.
Estimation of malondialdehyde, cell membrane
stability index (CMSI), chlorophyll a/b ratio
and proline content
MDA content was estimated according to Heath and Packer
(1968). Cell membrane stability index (CMSI) was calcu-
lated according to the procedure proposed by Sairam et al.
(1997). After thorough washing, 100 mg of leaf samples
was taken in two sets, each containing 10 ml of double-
distilled water. One set was heated at 40 °C for 30 min,
while the other set was boiled at 100 °C for 10 min. Then
13
Journal of Crop Science and Biotechnology (2023) 26:63–75
the conductivity of both sets was measured as C1 and C2,
respectively. (CMSI% = [1 – (C1/C2)] × 100).
The chlorophyll a/b ratio was calculated using Arnon's
(1949) method, while proline content was calculated using
Bates et al.'s (1973) method.
Enzyme extraction and assay of activities
of antioxidant enzymes
Superoxide dismutase activity was calculated according to
the method proposed by Kono (1978). Enzyme extract solu-
tion was prepared by grinding 0.5 g of fresh leaf samples
in 50 mM potassium phosphate buffer (pH 6.5) and was
centrifuged at 10,000 g for 12 mints. The supernatant thus
collected was used as enzyme extract. In a test tube, 1.3 ml
of 50 mM potassium phosphate buffer, 500 µl nitroblue
tetrazolium (NBT), and 100 µl of Triton-X 100 were taken.
The reaction for the superoxide radical production was ini-
tiated by adding 100 µl of hydroxylamine hydrochloride
to the aforementioned mixture. After 2 min, 70 µl of the
enzyme extract was added and absorbance was calculated
at 540 nm. An increase in absorbance was noted at a time
interval of 1 min. The SOD activity was expressed as units
(amount of enzyme required to inhibit NBT reduction by
50%) ­min−1 ­mg−1 protein, which was then converted to U
­mg−1 FW.
Catalase (CAT) activity was calculated according to the
method proposed by Aebi (1974). Enzyme extract solu-
tion was prepared by grinding 0.5 g of fresh leaf samples
in 50 mM potassium phosphate buffer (pH 6.5) and was
centrifuged at 10,000 g for 12 mints. The supernatant thus
collected was used as an enzyme extract. The enzyme
extract (100 µl) was diluted in 50 mM potassium phosphate
buffer (1 ml); the reaction was initiated by adding 100 µl
of 100 mM ­H2O2. The change in absorbance was calcu-
lated at 240 nm every 15 s for 2 min. The CAT activity was
expressed as ­units−1  ­min−1  ­mg−1 protein, which was then
converted to U ­mg−1 FW.
Gene expression analysis of spermidine and auxin
biosynthetic genes
Gene expression analysis was done in two genotypes, viz.
Nagina22 (N22) and Manu Ratna, subjected to HT stresses
at the panicle initiation stage. The flag leaves (three biologi-
cal replicates) were pooled for RNA isolation. Total RNA
was extracted from fresh leaves using a modified TRIzol
reagent method (Yin et al. 2016). cDNA was synthesized
using the iScript cDNA synthesis Kit (Biorad). The details
of the primers used in this study are given in Supplemen-
tary Table 2. An expression study was done by real-time
quantitative PCR with 2X SYBR Green qPCR Mix (G-Bio-
sciences, USA) in the CFX Mastero™ Real-Time PCR
Journal of Crop Science and Biotechnology (2023) 26:63–75 67
system (Bio-Rad). Following amplification, the relative fold
change in gene expression was determined using the com-
parative Ct method (Livak and Schmittgen 2001), with rice
β-actin serving as an internal control. The results represent
an average of three technical replicates.
Statistical analysis
The data were subjected to one-way analysis of variance
(ANOVA) and the mean differences were compared by least
significant difference test (LSD) using GRAPES. Data are
represented as means ± SD from three independent experi-
ments. Different letters within the same column indicate
significant differences between treatments at p < 0.05.
Results
Preliminary screening for heat tolerance
A preliminary experiment was conducted to evaluate the
effects of different biostimulants on heat tolerance. Upon
the application of various biostimulants, both varieties
exhibited enhanced heat tolerance, while Nagina 22 being
a heat-tolerant variety showed a better tolerance to heat as
compared to the heat-susceptible Manu Ratna. Malondial-
dehyde (MDA), hydrogen peroxide (­ H2O2) and proline (Pro)
content increased significantly, whereas chlorophyll content
and seedling vigour index (SVI) reduced significantly dur-
ing heat stress, which led to fatal damage to rice seedlings.
In both Nagina 22 and Manu Ratna, the percentage
increase in SVI was maximum for plants treated with 1 mM
Spd (59 and 51%) followed by ­10−5 M IAA treatment (45
and 46%) (Fig. 1a).
Percentage reduction of MDA content under stressed
condition was maximum in plants treated with 1 mM sper-
midine (– 34%) of Nagina 22 and 1­ 0–5 M IAA (-56%) of
Manu Ratna varieties. While minimum percentage reduc-
tion was observed during 2 ppm brassinosteroid (+ 17%) and
100 ppm boron (– 7%) treatments in Nagina 22 and Manu
Ratna, respectively (Fig. 1b).
In both Nagina 22 and Manu Ratna, the percentage reduc-
tion in H­ 2O2 content was maximum for plants treated with
­10–5 M auxin (– 67 and – 44%) followed by 1 ppm brassinos-
teroid treatment (– 50 and – 56%), while minimum percent-
age reduction was observed during 2 mM spermidine treat-
ment in both Nagina 22 (+ 17%) and Manu Ratna (+ 11%)
varieties (Fig. 1c).
Percentage increase of proline content under stressed con-
dition was maximum in plants treated with 1 mM spermidine
in both Nagina 22 (100%) and Manu Ratna (96%) varieties
(Fig. 1d).
Percentage increase in chlorophyll content under
stressed condition was maximum in plants treated with
­10–5 M IAA in both Nagina 22 (106%) and Manu Ratna
(126%) varieties, while minimum percentage increase
was observed during ­10–3 M IAA (– 12%) and 100 ppm
boron (– 12%) treatments in Nagina 22 and 100 ppm boron
(– 50%) treatment in Manu Ratna (Fig. 1e).
Evaluation of contrasting rice varieties
for reproductive stage heat tolerance using selected
two treatments from experiment I
MDA content increased significantly during heat stress.
While the application of IAA and Spd prior to the induc-
tion of heat stress significantly decreased MDA content
in both varieties. The application of IAA increased pro-
line concentration by 33% (Nagina 22) and 46% (Manu
Ratna), while Spd application showed an increase of 34%
(Nagina 22) and 51% (Manu Ratna) with respect to its
normal temperature control plants. Minimum percentage
increase was observed in high-temperature control plants
of both Nagina 22 (22%) and Manu Ratna (27%) varieties
(Fig. 2a).
In contrast to this, CMSI decreased significantly during
heat stress in both Nagina 22 and Manu Ratna varieties. Spd-
and IAA-pretreated plants significantly increased CMSI in
both varieties. The application of Spd increased CMSI by
– 10% (Nagina 22) and – 17% (Manu Ratna), while Spd
application showed an increase of – 12% (Nagina 22) and
– 18% (Manu Ratna) with respect to its normal temperature
control plants (Fig. 2b).
Proline content significantly increased during heat-
stressed condition. Proline levels further increased in IAA-
and Spd-treated plants as compared to heat-stressed ones.
The application of IAA decreased MDA concentration by
53% (Nagina 22) and 71% (Manu Ratna), while Spd appli-
cation showed an decrease of 30% (Nagina 22) and -48%
(Manu Ratna) with respect to its normal temperature control
plants (Fig. 2c).
Under heat-stressed condition chlorophyll a/b ratio
decreased in both Nagina 22 and Manu Ratna. Application
of IAA and Spd prior to the induction of heat stress signifi-
cantly increased chlorophyll a/b ratio in both Nagina 22 and
Manu Ratna. The application of IAA increased chlorophyll
a/b ratio by – 17% (Nagina 22) and – 15% (Manu Ratna),
while Spd application showed an increase of – 20% (Nagina
22) and -21% (Manu Ratna) with respect to its normal tem-
perature control plants (Fig. 2d).
Spermidine application was found to enhance CMSI by
lowering MDA accumulation. In contrast, Spd and IAA
application was found to produce significantly similar effect
on Chl a/b ratio and proline content.
13

68 Journal of Crop Science and Biotechnology (2023) 26:63–75

Fig. 1  Graph showing a seedling vigor index, b malondialdehyde (MDA), c hydrogen peroxide (­ H2O2), d proline (Pro) and e chlorophyll content
of rice under different treatment conditions

13
Journal of Crop Science and Biotechnology (2023) 26:63–75 69
Fig. 2  Graph showing a MDA, b CMSI, c Pro and d chlorophyll a/b ratio of rice under different treatment conditions (NT normal temperature,
HT high temperature, IAA indole-3-acetic acid, Spd spermidine)
Effect of high temperature on plant height,
productive tiller number, spikelet fertility
percentage, pollen viability percentage and yield
per plant
Inside polyhouse, the plant height slightly increased in both
varieties, the application of IAA and Spd prior to the induc-
tion of heat stress significantly increased plant height in both
varieties. Percentage increase of plant height under stressed
condition with respect to normal temperature control was
maximum in plants treated with 1 mM spermidine in both
of Nagina 22 (8%) and Manu Ratna (9%) varieties. Mini-
mum percentage increase was observed in high-temperature
control plants of both Nagina 22 (3%) and Manu Ratna (5%)
varieties (Fig. 3a).
Similarly, IAA and Spd pretreatment significantly
increased productive tiller number than non-treated heat-
stressed plants of both varieties. Percentage increase of
number of productive tillers under stressed condition with
respect to normal temperature control was maximum in
plants treated with ­10–5 M IAA in Nagina 22 (3%) and 1 mM
spermidine in Manu Ratna (4%) varieties. Minimum per-
centage increase was observed in high-temperature control
plants of both Nagina 22 (– 6%) and Manu Ratna (– 23%)
varieties (Fig. 3b).
High temperature during flowering and grain filling stage
caused a reduction in pollen viability and spikelet fertility
percentage of both Nagina 22 and Manu Ratna. The IAA and
Spd pretreatment prior to the induction of heat stress signifi-
cantly increased both pollen viability and spikelet fertility per-
centage in both varieties. The application of IAA increased
spikelet fertility percentage by – 11% (Nagina 22) and – 28%
(Manu Ratna) followed by Spd application showed an increase
of – 13% (Nagina 22) and – 31% (Manu Ratna) with respect
to its normal temperature control plants(Fig. 3c). Percentage
increase in pollen viability percentage was maximum during
the application of IAA in both Nagina 22 (– 11%) and Manu
Ratna (– 24%). Minimum percentage increase was observed
in high-temperature control plants of both Nagina 22 (– 21%)
and Manu Ratna (– 37%) varieties (Fig. 3d).
Under high temperature, the reduced pollen fertility %
and spikelet fertility % result in remarkable reduction of
grain yield in both Nagina 22 and Manu Ratna. Application
of IAA and Spd prior to the induction of heat stress signifi-
cantly increased yield in both varieties. During IAA applica-
tion the yield per plant observed was 9.5 gm (Nagina 22) and
7.8 gm (Manu Ratna), while Spd application showed 8.9 gm
(Nagina 22) and 6.1 gm (Manu Ratna). Percentage increase
was recorded maximum during the application of IAA in
both Nagina 22 (– 23%) and Manu Ratna (– 45%), followed
by Spd application showed an increase of – 28% (Nagina 22)
and – 57% (Manu Ratna). Minimum percentage increase was
observed in high-temperature control plants of both Nagina
22 (– 34%) and Manu Ratna (-61%) varieties (Fig. 3e).
13

70 Journal of Crop Science and Biotechnology (2023) 26:63–75

Fig. 3  Graph showing a plant height, b productive tiller number, c spikelet fertility percentage, d pollen viability percentage and e yield per
plant of rice under different treatment conditions (NT normal temperature, HT high temperature, IAA indole-3-acetic acid, Spd spermidine)

Maximum plant height and productive tillers were
observed during Spd application while spikelet fertility per-
centage, pollen viability percentage and yield were improved
during IAA application.
Effect of IAA and Spd application on the activities
of ROS detoxifying enzymes under reproductive
stage heat stress in contrasting rice varieties
Under heat stress conditions, superoxide dismutase (SOD)
activity increased in both varieties. In contrast, SOD activ-
ity in Spd and IAA pretreated was significantly higher

Fig. 4  Graph showing a superoxide dismutase (SOD) and b catalase activity of rice under different treatment conditions (NT normal tempera-
ture, HT high temperature, IAA indole-3-acetic acid, Spd spermidine)

13
Journal of Crop Science and Biotechnology (2023) 26:63–75 71
during heat stress. The application of IAA increased SOD
activity by 51% (Nagina 22) and 67% (Manu Ratna), while
Spd application showed an increase of 72% (Nagina 22)
and 54% (Manu Ratna) with respect to its normal tempera-
ture control plants (Fig. 4a).
Similarly, catalase activity increased significantly during
heat stress in both Nagina 22 and Manu Ratna. Applica-
tion of IAA and Spd prior to the induction of heat stress
significantly further increased catalase activity in both vari-
eties. The application of IAA increased catalase activity
by 95% (Nagina 22) and 113% (Manu Ratna) while, Spd
application showed an increase of 77% (Nagina 22) and 94%
(Manu Ratna) with respect to its normal temperature control
plants. IAA application increased the activity of antioxidant
enzymes such as SOD and CAT as compared to Spd applica-
tion (Fig. 4b).
Expression of biosynthetic genes
OsYUC 1 (flavin-containing monooxygenase like enzyme)
and SPDSYN 1 gene expression were investigated using
OsActin as a control gene. The expression of both OsYUC
1 and SPDSYN 1 genes decreased during heat stress. It was
observed that the exogenous application of IAA and sper-
midine to heat-stressed rice plants increases the expression
of OsYUC 1 and SPDSYN 1 genes, respectively (Fig. 5). The
expression of OsYUC 1 decreased during heat stress by 0.3
(Nagina 22)- and 0.5 (Manu Ratna)-fold, while the exog-
enous IAA application prior to the induction of heat stress
significantly increased the expression of OsYUC 1 by 4.4
(Nagina 22)- and 5.1 (Manu Ratna)-fold as compared to its
normal temperature control. However, spermidine applica-
tion during heat stress does not show any effect on expres-
sion of OsYUC 1 gene (Fig. 5a).
The expression of OsSPDSYN 1 decreased during heat
stress by 0.4 (Nagina 22)- and 0.6 (Manu Ratna)-fold, while
the exogenous spermidine application prior to the induc-
tion of heat stress significantly increased the expression of
Fig. 5  Relative fold change of a OsYUC 1 gene under IAA treatment; b OsSPDSYN 1 gene under spermidine treatment with respect to control
OsSPDSYN 1 by 1.6 (Nagina 22)- and 1.8 (Manu Ratna)-
fold as compared to its normal temperature control. Also,
the application of IAA during heat stress does not showed
any effect on the expression of OsSPDSYN 1 gene (Fig. 5b).
Discussion
In this study, we found that exogenous spermidine (1 mM)
and indole-3-acetic acid (­ 10–5 M) pretreatments improved
seedling vigour index, which may be due to the breakdown
of reserve food material, increased cell division or expansion
of embryonic axis (Basra et al. 2005). This was similar to the
findings made by Muhammad et al. (2008) who found that
Spd priming could significantly improve seed germination
and enhance seed vigor, which was similar to our results.
Furthermore, Mostofa et al. 2014 reported that under high
temperature, the rice seeds primed with spermidine showed
increased seedling vigor index. Guangwu and Xuwen (2014)
reported that exogenous application of 1­ 0–4 M and 1­ 0–5 M
IAA significantly promoted seed germination in Chinese red
pine, Pinus massoniana Lamb.
Heat stress results in the accumulation of reactive oxy-
gen species (ROS) leading to oxidative stress, which results
in the overproduction of ­H2O2 inside cells. ­H2O2 acts as
an inducer of lipid peroxidation of membranes (Yin et al.
2008; Dai et al. 2012) and produce malondialdehyde as a
byproduct whose level inside cell served as an indicator for
the extent of oxidative damage (Quintero-Calderon et al.
2021). Our results shows that at seedling stage 50 mg/l
boron, ­10−5 M IAA and 1 mg/L brassinolide treatment low-
ers the levels of ­H2O2 in stressed conditions of both the rice
varieties. These findings were supported by the findings of
Sharma et al. (2018), Das et al. (2019), and Calderón-Páez
et al. (2021), reported that exogenous application of boron,
IAA and brassinolide ameliorates the negative effect of
heat stress by reducing the H ­ 2O2 production during stressed
conditions. Low levels of MDA contents were observed on
13

72
seedlings treated with 1 mM spermidine, 1 mg/l brassinos-
teroid and ­10−5 M IAA as compared to other treatments in
both varieties. These results were in accordance with early
findings of Mostofa et al. (2014) and Sharma et al. (2018)
who observed that foliar application of spermidine and IAA
lowers lipid peroxidation in heat-stressed plants.
Increase in concentration of osmoprotectants such as gly-
cine betaine, total soluble proteins (TSPs) and proline in
heat-stressed plants alleviates negative effects of heat stress
injury by osmotic adjustments. Lower production of these
compounds triggers ROS accumulation which is detrimental
for the survival of the plant (Gadallah 1999). The proline
concentration of rice seedlings treated with 1 mM spermi-
dine, ­10−5 M IAA, 2 mg/L brassinosteroid and 100 mg/l
boron increased significantly as compared to control plants
at 42 °C. Kumar et al. (2012) reported that prolonged expo-
sure of rice to temperature greater than 40 °C causes reduc-
tion in proline concentration leading to growth inhibition.
Proline accumulation increases the efficiency of K ­ + uptake
resulting in increased chlorophyll content in plants (Gadallah
1999). During heat-stressed condition, the proline concen-
tration declines along with increasing H ­ 2O2 levels causing
degradation of chloroplast and thylakoid membrane by trig-
gering lipid peroxidation, resulting in lowering the chlo-
rophyll levels that results in yellowing of leaves (Camejo
et al. 2006). The concentration of chlorophyll decreases in
stressed plants while increases on 1 mM spermidine, 1­ 0−3 M
IAA, ­10−5 M IAA and 50 mg/L boron treatment. Our find-
ings were similar to that of Mostofa et al. (2014), Calderón-
Páez et al. (2021) and Sharma et al. (2018) who reported that
exogenous application of spermidine, boron, IAA increases
chlorophyll content in stressed leaves of rice.
The cell membrane stability index (CMSI) decreases due
to heat stress; because of lipid peroxidation, the stability
of the cell membrane is lost (Niu and Xiang 2018) and the
production of malondialdehyde is increased (Savchenko
et al. 2002). Spd was known to prevent biofilms and mac-
romolecules and also maintains organelle integrity under
stress (Diao et al. 2015). Under salt stress, spd application
reduced MDA content in rice seedlings (Aryadeep et al.
2011). Transgenic over expression of AtYUC6 improved
transgenic potato's  drought tolerance by lowering ROS
and MDA levels and increasing the expression of genes
encoding ROS detoxification (Kim et al. 2013). Our results
showed that with spermidine or IAA treatment, the CMSI
increased by lowering MDA content in rice. Similar results
were reported by Sharma et al. (2018) and Fu et al. (2019)
who reported that IAA or spermidine reduces membrane
damage due to lipid peroxidation by enhancing different
antioxidant machineries in the cell.
Heat stress causes the accumulation of ROS (reactive oxy-
gen species) and damages various cellular components. Most
common ROS includes superoxide anions, peroxide and
13
Journal of Crop Science and Biotechnology (2023) 26:63–75
hydroxyl ions (Bahuguna et al. 2015). H ­ 2O2 produced during
stressed condition also promotes ROS accumulation. Hence,
plants adopt different adaptive tactics to reduce ROS accu-
mulation (Sailaja et al. 2015). The activity of antioxidant
enzymes such as superoxide dismutase (SOD), catalase, and
peroxidase increased during stressed condition reduced ROS
levels in cells (Zhao et al. 2018). Exogenous Spd has been
shown to lower MDA levels, scavenge ROS in tomato cells,
and participate in the stress response of antioxidant enzyme
systems, primarily, boosting the activity of SOD (superoxide
dismutase) and CAT (catalase) (Diao et al. 2015). Tang et al.
(2018) discovered that after flowering, leaves sprayed with
1 mM Spd increased SOD and POD activity while decreas-
ing MDA content in traditional rice varieties. Abdel Latef
et al. (2021) reported that during salt stress the exogenous
application of IAA significantly increased the activities of
SOD, CAT, POD, and APX in V. faba plants. The results of
the present study revealed that during the exogenous applica-
tion of IAA or spermidine there was a remarkable increase
activity of SOD and catalase enzymes. This was similar to
the finding made by Zhang et al. (2014) and Fu et al. (2019)
who reported that antioxidant machinery in heat-stressed
plant was induced by the application of IAA or spermidine.
At reproductive and grain filling stage, elevated tempera-
ture affects anthesis the most, which leads to lowered pol-
len viability, spikelet fertility and ultimately the yield of the
plant (Matsui and Omasa 2002; Prasad et al. 2017; Beena
et al. 2018). During high temperature, the pollen viability
decreases due to impaired pollen germination and pollen
tube growth (Fu et al. 2016; Xu et al. 2020; Amrutha et al.
2021a). In our study, pollen viability percentage increased
by the application of IAA and spermidine. This finding is
supported by the study of Sharma et al. (2018) reporting
increased pollen viability % by the exogenous application of
IAA. Zhou et al. (2020) reported that the exogenous applica-
tion spermidine and spermine increases pollen viability in
rice during normal and high temperatures. Spikelet sterility
caused by elevated temperature may be due to the impaired
mobilization of carbohydrates form vegetative organs such
as leafs to the grains (Shi et al. 2018; Pravallika et al. 2020;
Amrutha et al. 2021b; Beena et al. 2021). The spikelet fertil-
ity percentage increases by the application of IAA or sper-
midine in the present study. The same results were obtained
by Sharma et al. (2018) in rice. Furthermore, Zhou et al.
(2020) reported that the exogenous application spermidine
and spermine increases spikelet fertility in rice during nor-
mal and high temperatures. Increase in pollen viability and
spikelet fertility percentage due to the Spd and IAA contrib-
utes to enhanced yield under heat-stressed condition.
The exogenous application of IAA or spermidine
enhances the expression of its biosynthetic genes. YUCCA​
genes were involved in the biosynthesis of IAA from
tryptophan. YUCCA​ gene codes for a flavin-containing
Journal of Crop Science and Biotechnology (2023) 26:63–75 73
monoxygenase enzyme which involved in the conversion
of indole pyruvic acid to indole-3-acetic acid (Zhao 2011).
Till now, 11 YUCCA​ family genes were identified in rice
(Matthes et al. 2019) of which YUC 1, YUC 2, YUC 4, and
YUC 6 were expressed in shoots and leaves (Chen et al.
2014). During heat stress, the endogenous levels of IAA
decreases because of the downregulation of upstream control
elements of YUCCA​gene such as NGATHA (NGA), SHORT-
INTERNODES (SHI)/STYLISH1 (STY1), and TERMINAL
FLOWER 2 (TFL2)/HETEROCHROMATIN PROTEIN1
(HP1) (Sohlberg et al. 2006; Trigueros et al. 2009; Eklund
et al. 2010; Rizzardi et al. 2011). The exogenous application
of IAA was found to enhance the YUCCA​ gene expression
by enhancing regulatory elements (Sharma et al. 2018). IAA
application coupled with heat stress triggers the expression
of auxin-responsive genes responsible for enhancing repro-
ductive development and antioxidant machinery such as (1)
HAF (HALF FILLED)/CESTA (CES), regulates reproduc-
tive tract development and fertilization efficiency; (2) SPO-
ROCYTELESS (SPL) for ovule development; (3) OsANN1
regulate the activity of catalase and SOD (Crawford and
Yanofsky 2011). In present study, the exogenous IAA appli-
cation to heat-stressed plants at flowering stage increases the
expression of its biosynthetic gene OsYUC 1 in flag leaf.
Similar findings were reported by Zhang et al. (2018) that
pistils sprayed with 10 µmol/L NAA (naphthalene acetic
acid) were reported to increase the expression of OsYUC 1
as compared to control plants.
During heat stress, the exogenously applied spermidine
enters the cells through PUT (polyamine uptake transport-
ers) present in the plasma membrane (Vaishali et al. 2012).
The exogenously applied Spd enhanced the expression of
spermidine synthase (SPDSYN) gene which was down-
regulated during high temperature. Increased endogenous
levels of Spd induce OsSAP 5 (a stress-associated Zn fin-
ger protein) (Vij and Tyagi 2006) which further regulate
the expression of TFs such as MYC binding protein (MBP)
(Chen et al. 2021) and dehydration responsive element bind-
ing protein (DREB) (Hozain et al. 2012; Kang et al. 2011).
These TFs induce several stress-responsive genes involved
in the production of osmoprotectants, antioxidant enzymes
and heat-shock proteins. Spd application also enhanced the
grain quality by enhancing the expression of soluble starch
synthase II-3 (SS II-3) and granules bound starch synthase
I (GBSSI) (Fu et al. 2019). Duan et al. (2008) reported the
exogenous application of 0.1 mM Spd to salt-stressed plants
increases production of spermidine and spermine. The result
of present study provides evidence that the application of
spermidine exogenously to the heat-stressed rice plants dou-
bles the expression of its biosynthetic gene, OsSPDSYN 1 as
compared to control. Analogous observations were made by
Fu et al. (2019) who reported that the exogenous application
of 1.5 mM spermidine to heat-stressed plants enhances the
expression of its biosynthetic gene OsSPDSYN 1. The induc-
tion of stress-responsive genes responsible for the produc-
tion of antioxidant enzymes and osmoprotectants was used
to improve stress tolerance by exogenous application of IAA
and spermidine. ROS that accumulate during stressful situ-
ations were detoxified by increased activity of antioxidant
enzymes and compatible solutes.
This study showed that heat stress negatively affects the
growth and survival of rice plants by inducing oxidative
and osmotic damage. Exogenous spermidine application
was found to reduce these damages by inducing the pro-
duction of antioxidant enzymes and osmoprotectants along
with enhancement of various yield components under heat-
stressed condition. On assessing these physiological, bio-
chemical, molecular and yield parameters, we can conclude
that exogenous application of ­10–5 M IAA and 1 mM Spd
can be utilized to improve heat tolerance in rice plants. How-
ever, more field experiments would be needed, for the long-
term crop production under changing climatic conditions.
Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 12892-0​ 22-0​ 0162-4.
Acknowledgements  The authors thank Kerala Agricultural University
for providing all the facilities.
Author contributions  All authors contributed to the study conception
and design. Material preparation, data collection and analysis were
performed by (Lakshmi G.), (Dr.Beena R.), (Dr.Soni K.B..), (Dr.Viji,
M.M.) and Dr.Uday Chand Jha). The first draft of the manuscript
was written by (Lakshmi G.) and all authors commented on previous
versions of the manuscript. All authors read and approved the final
manuscript.
Declarations 
Conflict of interest  The authors do not have any conflict of interest in
publishing this article.
References
Abdel Latef AAH, Tahjib-Ul-Arif M, Rhaman MS (2021) Exogenous
auxin-mediated salt stress alleviation in faba bean (Vicia faba L.).
Agron 11(3):547–568
Abdul-Baki AA, Anderson JD (1973) Vigor determination in soybean
seed by multiple criteria 1. Crop Sci 13(6):630–633
Aebi H (1974) Catalase. Methods of enzymatic analysis. Elsevier, NY,
pp 673–684
Amrutha V, Shanija S, Beena R, Sarada S, Sajitha RT, Roy S, Manju
RV, Viji MM (2021a) High temperature induced changes in qual-
ity and yield parameters of tomato (Solanum lycopersicum L) and
similarity coefficients among genotypes using SSR markers. Heli-
yon. https://​doi.​org/​10.​1016/j.​heliy​on.​2021.​e05988
Amrutha V, Shanija S, Beena R, Nithya N, Jaslam MPK, Soni KB, Viji
MM (2021b) Population structure analysis and marker trait asso-
ciation in selected set of Indian tomato (Solanum lycopersicum
13

74
L.) varieties under high temperature condition. Gen Resour Crop
Evol. https://​doi.​org/​10.​1007/​s10722-​021-​01216-2
Arnon DT (1949) Copper enzymes in isolated chloroplast polyphenol
oxidase in Beta vulgaris. Plant Physiol 24:1–15
Aryadeep R, Supratim B, Sengupta DN (2011) Amelioration of salin-
ity stress by exogenously applied spermidine or sermine in three
varieties of indica rice differing in their level of salt tolerance. J
Plant Physiol 168:317–328
Baker HG, Baker I (1979) Starch in angiosperm pollen grains and its
evolutionary significance. Am J of Bot 66(5):591–600
Basra SMA, Farooq M, Tabassum R, Ahmad N (2005) Physiological
and biochemical aspects of seed vigor enhancement treatments
in fine rice (Oryza sativa L.). Seed Sci and Technol 33:623–628
Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free
proline for water-stress studies. Plant Soil 39:205–207
Beena R (2013) Research paradigm and inference of studies on high
temperature stress in rice (Oryza sativa L.). Adv Plant Physiol
14:497–511
Beena R, Vighneswaran V, Sindumole P, Narayankutty MC, Voleti SR
(2018) Impact of high temperature stress during reproductive and
grain filling stage in rice. Oryza, an Int J on Rice 55(1):126–133
Beena R, Veena V, Jaslam MPK, Nithya N, Adarsh VS (2021) Germ-
plasm innovation for high-temperature tolerance from tradi-
tional rice accessions of Kerala using genetic variability, genetic
advance, path coefficient analysis and principal component analy-
sis. J of Crop Sci and Biotechnol 24:555–556
Calderón-Páez SE, Cueto-Niño YA, Sánchez-Reinoso AD, Garces-
Varon G, Chávez-Arias CC, Restrepo-Díaz H (2021) Foliar boron
compounds applications mitigate heat stress caused by high day-
time temperatures in rice (Oryza sativa L.) Boron mitigates heat
stress in rice. J Plant Nutr 44(17):2414–2527
Camejo D, Jiménez A, Alarcón JJ, Torres W, Gómez JM, Sevilla F
(2006) Changes in photosynthetic parameters and antioxidant
activities following heat-shock treatment in tomato plants. Funct
Plant Biol 33(2):177–187
Castorina G, Consonni G (2020) The role of brassinosteroids in con-
trolling plant height in Poaceae: a genet perspective. Int J Mol
Sci 21:1191
Chen Q, Dai X, De-Paoli H, Cheng Y, Takebayashi Y, Kasahara H,
Kamiya Y, Zhao Y (2014) Auxin overproduction in shoots can-
not rescue auxin deficiencies in Arabidopsis roots. Plant and Cell
Physiol 55(6):1072–1079
Chen M, Fu Y, Mou Q, An J, Zhu X, Ahmed T, Zhang S, Basit F, Hu J,
Guan Y (2021) Spermidine induces expression of stress associated
proteins (SAPs) genes and protects rice seed from heat stress-
induced damage during grain-filling. Antioxidants 10(10):1544
Crawford B, Yanofsky MF (2011) Half-filled promotes reproductive
tract development and fertilization efficiency in Arabidopsis thali-
ana. Dev 138:2999–3009
Dai AH, Nie YX, Yu B, Li Q, Lu LY, Bai JG (2012) Cinnamic acid
pretreatment enhances heat tolerance of cucumber leaves through
modulating antioxidant enzyme activity. Environ Exp Bot 79:1–10
Das S, Mohanty S, Dash D, Muduli KC (2019) Enhancement of growth
and seed yield of rice (Oryza sativa L.) through foliar spray of
osmoprotectants under high temperature stress. Indian J of Tradit
Knowl 19(1):92–100
Diao Q, Song Y, Qi H (2015) Exogenous spermidine enhances chill-
ing tolerance of tomato (Solanum lycopersicum L.) seedlings via
involvement in polyamines metabolism and physiological param-
eter levels. Acta Physiol Plant 37:1–15
Duan J, Li J, Guo S, Kang Y (2008) Exogenous spermidine affects
polyamine metabolism in salinity-stressed Cucumis sativus roots
and enhances short-term salinity tolerance. J of Plant Physiol
165(15):1620–1635
13
Journal of Crop Science and Biotechnology (2023) 26:63–75
Eklund DM, Staldal V, Valsecchi I (2010) The Arabidopsis thaliana
STYLISH1 protein acts as a transcriptional activator regulating
auxin biosynthesis. Plant Cell 22:349–363
Fu G, Feng B, Zhang C, Yang Y, Yang X, Chen T, Zhao X, Zhang X,
Jin Q, Tao L (2016) Heat stress is more damaging to superior
spikelets than inferiors of rice (Oryza sativa L.) due to their dif-
ferent organ temperatures. Front Plant Sci 7:1637
Fu Y, Gu Q, Dong Q, Zhang Z, Lin C, Hu W, Pan R, Guan Y, Hu J
(2019) Spermidine enhances heat tolerance of rice seeds by modu-
lating endogenous starch and polyamine metabolism. Molecules
24(7):1395
Gadallah MAA (1999) Effects of proline and glycinebetaine on Vicia
faba responses to salt stress. Biol Plant 42(2):249–257
García-Hernández EDR, López GIC (2005) Structural cell wall pro-
teins from five pollen species and their relationship with boron.
Brazilian J Plant Physiol 17:375–381
Guangwu Z, Xuwen J (2014) Roles of gibberellin and auxin in promot-
ing seed germination and seedling vigor in Pinus massoniana.
Forest Sci 60(2):367–373
Guru T, Kunta RT, Rao P (2016) Improving pollen viability and stigma
receptivity by boron application as a physiological approach for
high yields in rice. Bioscan 11(1):79–83
Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts:
I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Bio-
chem Biophys 125:189–198
Holá D, Benešová M, Honnerová J, Hnilička F, Rothová O, Kočová
M, Hniličková H (2010) The evaluation of photosynthetic param-
eters in maize inbred lines subjected to water deficiency: can these
parameters be used for the prediction of performance of hybrid
progeny? Photosynth 48(4):545–558
Hozain MD, Abdelmageed H, Lee J, Kang M, Fokar M, Allen RD,
Holaday AS (2012) Expression of AtSAP5 in cotton up-regulates
putative stress-responsive genes and improves the tolerance to
rapidly developing water deficit and moderate heat stress. J of
Plant Physiol 169(13):1261–1270
IPCC, Masson-Delmotte V, Zhai P, Pirani A, Connors SL, Péan C,
Berger S, Caud N, Chen Y, Goldfarb L, Gomis MI, Huang M
(2021) Climate change 2021: The Phys sci basis contribution of
working group I to the sixth assess. Rep. of the intergovernmental
panel on clim. change.
Jagadish SVK, Cairns J, Lafitte R, Wheeler TR, PriceAH and Craufurd
PQ, (2010) Genetic analysis of heat tolerance at anthesis in rice.
Crop Sci 50:1633–1641
Jagadish SVK, Septiningsih EM, Kohli A, Thomson MJ, Ye C, Redona
E, Kumar A, Gregorio GB, Wassmann R, Ismail AM, Singh RK
(2012) Genetic advances in adapting rice to a rapidly changing
climate. J Agron Crop Sci 198(5):360–373
Janni M, Gullì M, Maestri E, Marmiroli M, Valliyodan B, Nguyen
HT, Marmiroli N, Foyer C (2020) Molecular and genetic bases
of heat stress responses in crop plants and breeding for increased
resilience and productivity. J Exp Bot 71:3780–3802
Kang M, Fokar M, Abdelmageed H, Allen RD (2011) Arabidop-
sis SAP5 functions as a positive regulator of stress responses
and exhibits E3 ubiquitin ligase activity. Plant Mol Biol
75(4–5):451–466
Kim JI, Baek D, Park HC (2013) Overexpression of Arabidopsis
YUCCA6 in potato results in high-auxin developmental phe-
notypes and enhanced resistance to water deficit. Mol Plant
6:337–349
Kono Y (1978) Generation of superoxide radicals during auto-oxida-
tion of hydroxyl-amine hydrochloride an assay for SOD. Arch
Biochem Biophys 186:189–195
Kumar S, Gupta D, Nayyar H (2012) Comparative response of maize
and rice genotypes to heat stress: status of oxidative stress and
antioxidants. Acta Physiol Plant 34(1):75–86
Journal of Crop Science and Biotechnology (2023) 26:63–75 75
Kusano T, Berberich T, Tateda C, Takahashi Y (2008) Polyamines:
essential factors for growth and survival. Plant 228(3):367–381
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression
data using realtime quantitative PCR and the 2−ΔΔCT method.
Methods 25:402–408
Matsui T, Omasa K (2002) Rice (Oryza sativa L.) cultivars tolerant to
high temperature at flowering: anther characteristics. Ann of Bot
89(6):683–687
Matthes MS, Best NB, Robil JM, Malcomber S, Gallavotti A, McS-
teen P (2019) Auxin EvoDevo: conservation and diversification
of genes regulating auxin biosynthesis, transport, and signaling.
Mol Plant 12(3):298–320
Mittler R, Finka A, Goloubinoff P (2012) How do plants feel the heat?
Trends Biochem Sci 37:118–125
Mostofa MG, Yoshida N, Fujita MJ (2014) Spermidine pretreatment
enhances heat tolerance in rice seedlings through modulating anti-
oxidative and glyoxalase systems. Plant Growth Regul 73:31–44
Muhammad F, Shahzad M, Basra H (2008) Seed priming with polyam-
ines improves the germination and early seedling growth in fine
rice. J New Seeds 9:145–155
Nayyar H, Kaur R, Kaur S, Singh RS (2014) γ-aminobutyric acid
(GABA) imparts partial protection from heat stress injury to rice
seedlings by improving leaf turgor and upregulating osmoprotect-
ants and antioxidants. J Plant Growth Regul 33:408–419
Niu Y, Xiang Y (2018) An overview of biomembrane functions in
plant responses to high-temperature stress. Front Plant Sci 9:915
Ogweno JO, Song XS, Shi K, Hu WH, Mao WH, Zhou YH, Yu JQ,
Nogués S (2008) Brassinosteroids alleviate heat-induced inhibi-
tion of photosynthesis by increasing carboxylation efficiency and
enhancing antioxidant systems in Lycopersicon esculentum. J
Plant Growth Reg 27(1):49–57
Prasad PVV, Bheemanahalli R, Jagadish SVK (2017) Field crops and
the fear of heat stress-opportunities, challenges and future direc-
tions. Field Crop Res 200:114–121
Prasanth VV, Babu MS, Basava RK, Tripura VGN, Venkata SK, Man-
grauthia SR, Voleti S, Neelamraju, (2017) Trait and marker asso-
ciations in Oryza nivara and O. rufipogon derived rice lines under
two different heat stress conditions. Front Plant Sci 8:1819
Pravallika K, Arunkumar C, Vijayakumar A, Beena R, Jayalekshmi VG
(2020) Effect of high temperature stress on seed filling and nutri-
tional quality of rice (Oryza sativa L.). J Crop Weed 16(2):18–23
Quintero-Calderon EH, Sanchez-Reinoso AD, Chavez-Arias CC,
Garces-Varon G, Restrepo-Diaz H (2021) Rice seedlings
showed a higher heat tolerance through the foliar application
of biostimulants. Notulae Bot Hortic Agrobot Cluj-Napoca
49(1):12120–12120
Raghunath MP, Beena R (2021) Manipulation of flowering time to
mitigate high temperature stress in rice (Oryza sativa L.). Indian
J Agric Res 1:4
Raghunath M, Beena R, Mohan V, Viji M, Manju R, Stephen R (2021)
High temperature stress mitigation in rice (Oryza sativa L.): Foliar
application of plant growth regulators and nutrients. J Crop Weed
17(1):34–47
Rizzardi K, Landberg K, Nilsson L, Ljung K, Larsson AS (2011)
TFL2/LHP1 is involved in auxin biosynthesis through positive
regulation of YUCCA genes. Plant J 65:897–906
Sailaja B, Subrahmanyam D, Neelamraju S, Vishnukiran T, Rao YV,
Vijayalakshmi P, Voleti SR, Bhadana VP, Mangrauthia SK (2015)
Integrated physiological, biochemical, and molecular analysis
identifies important traits and mechanisms associated with dif-
ferential response of rice genotypes to elevated temperature. Front
Plant Sci 6:1044
Sairam RK, Deshmukh PS, Shukla DS (1997) Tolerance of drought
and temperature stress in relation to increased antioxidant enzyme
activity in wheat. J Agron Crop Sci 178(3):171–178
Savchenko GE, Klyuchareva EA, Abramchik LM, Serdyuchenko EV
(2002) Effect of periodic heat shock on the inner membrane sys-
tem of etioplasts. Russian J Plant Physiol 49(3):349–359
Sharma L, Dalal M, Verma RK, Kumar SV, Yadav SK, Pushkar S,
Kushwaha SR, Bhowmik A, Chinnusamy V (2018) Auxin protects
spikelet fertility and grain yield under drought and heat stresses in
rice. Environ and Exp Bot 150:9–24
Sohlberg JJ, Myrenas M, Kuusk S, Lagercrantz U, Kowalczyk M, Sand-
berg G, Sundberg E (2006) STY1 regulates auxin homeostasis
cts apical-basal patterning of the Arabidopsis gynoecium. Plant
J 47:112–123
Tang S, Zhang H, Li L, Liu X, Chen L, Chen W, Ding Y (2018) Exog-
enous spermidine enhances the photosynthetic and antioxidant
capacity of rice under heat stress during early grain-filling period.
Funct Plant Biol 45:911–921
Thussagunpanit J, Jutamanee K, Kaveeta L, Chai-arree W, Pankean
P, Suksamrarn A (2013) Effects of brassinosteroid and ecdysone
analogue on pollen germination of rice under heat stress. J Pestic
Sci 38:105–111
Trigueros M, Navarrete-Gomez M, Sato S, Christensen SK, Pelaz S,
Weigel D, Yanofsky MF, Ferrandiz C (2009) The NGATHA genes
direct style development in the Arabidopsis gynoecium. Plant Cell
21:1394–1409
Vaishali M, Marcus C, Chibucos Vipaporn P, Paul F, Morris, (2012)
Kinetic and phylogenetic analysis of plant polyamine uptake trans-
porters. Funct Plant Biol 236(4):1261–1273
Vij S, Tyagi AK (2006) Genome-wide analysis of the stress associated
protein (SAP) gene family containing A20/AN1 zinc-finger (s)
in rice and their phylogenetic relationship with Arabidopsis. Mol
Genet Genomics 276(6):565–575
Xu J, Henry A, Sreenivasulu N (2020) Rice yield formation under high
day and night temperatures—a prerequisite to ensure future food
security. Plant Cell Environ 43:1595–1608
Yin CC, Ma B, Wang W, Xiong Q, Zhao H, Chen SY, Zhang JS (2016)
RNA extraction and preparation in rice (Oryza sativa). Curr Pro-
tocols in Plant Biol 1(2):411–418
Yu CW, Murphy TM, Lin CH (2003) Hydrogen peroxide-induces chill-
ing tolerance in mung beans mediated through ABA independent
glutathione accumulation. Funct Plant Biol 30:955–963
Zhang C, Bai MY, Chong K (2014) Brassinosteroid-mediated regula-
tion of agronomic traits in rice. Plant Cell Rep 33:683–696
Zhang C, Li G, Chen T, Feng B, Fu W, Yan J, Islam MR, Jin Q, Tao L,
Fu G (2018) Heat stress induces spikelet sterility in rice at anthe-
sis through inhibition of pollen tube elongation interfering with
auxin homeostasis in pollinated pistils. Rice 11(1):1–12
Zhao Y (2011) Auxin biosynthesis: a simple two-step pathway converts
tryptophan to indole-3-acetic acid in plants. Mol Plant 5:334–338
Zhao Q, Zhou L, Liu J, Cao Z, Du X, Huang F, Pan G, Cheng F (2018)
Involvement of CAT in the detoxification of HT-induced ROS
burst in rice anther and its relation to pollen fertility. Plant Cell
Rep 37:741–757
Zhou R, Hu Q, Pu Q, Chen M, Zhu X, Gao C, Zhou G, Liu L, Wang Z,
Yang J, Zhang J (2020) Spermidine enhanced free polyamine lev-
els and expression of polyamine biosynthesis enzyme gene in rice
spikelets under heat tolerance before heading. Sci Rep 10(1):1–9
Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
13