Molecular basis of salicylic acid-phytohormone crosstalk in regulating stress tolerance in plants

Abstract

Due to their sessile nature, plants are often subjected to a plethora of abiotic (drought, heat, cold, salt and metal toxicity) and biotic (bacteria, virus, fungi and nematode) stress conditions, which constitute an immense danger to plant life and significantly impact crop growth, development, metabolism and production. It is well known that phytohormones are effective metabolites for reducing the negative impacts of abiotic and biotic stresses on agricultural plants over time. Salicylic acid is a powerful phenolic signalling biomolecule and a versatile plant growth regulator that has a key role in growth, metabolic and defence system of plants, thereby coordinating responses to pathogen attack and abiotic stress. This metabolite is crucial for the development of systemic acquired resistance (SAR) in plants because it causes the expression of genes associated with defence. Exogenous administration of SA promotes seed germination, development and blooming, up-regulates photosyn-thesis and boosts the activity of antioxidants that are enzymatic and non-enzymatic. Salicylic acid is an efficient signalling molecule that can modify physiological and metabolic processes in plants and can thus help in reducing environmental stress in plants over a long period of time. The less addressed issue, however, is the detailed investigation about the interaction of SA with major phytohormones as well as combined action of SA and other phytohormones that can support and influence the fundamental biochemical/physiological and molecular processes of plants against different forms of stress conditions (biotic and abiotic). The current review aims to document the detailed crosstalk of SA with other phytohormones (auxin, gibberellin, cytokinin, ethylene, abscisic acid, jasmonic acid, polyamines, melatonin, brassinosteroids and strigolactones) that confers protection and helps in the recovery of plants from different stress conditions.

Full text

Vol.:(0123456789)
Brazilian Journal of Botany
https://doi.org/10.1007/s40415-024-00983-3
BIOCHEMISTRY & PHYSIOLOGY - REVIEW ARTICLE
Molecular basis ofsalicylic acid–phytohormone crosstalk inregulating
stress tolerance inplants
PujaGhosh1· AryadeepRoychoudhury2
Received: 7 August 2023 / Revised: 30 September 2023 / Accepted: 17 January 2024
© The Author(s), under exclusive licence to Botanical Society of Sao Paulo 2024
Abstract
Due to their sessile nature, plants are often subjected to a plethora of abiotic (drought, heat, cold, salt and metal toxicity)
and biotic (bacteria, virus, fungi and nematode) stress conditions, which constitute an immense danger to plant life and sig-
nificantly impact crop growth, development, metabolism and production. It is well known that phytohormones are effective
metabolites for reducing the negative impacts of abiotic and biotic stresses on agricultural plants over time. Salicylic acid is a
powerful phenolic signalling biomolecule and a versatile plant growth regulator that has a key role in growth, metabolic and
defence system of plants, thereby coordinating responses to pathogen attack and abiotic stress. This metabolite is crucial for
the development of systemic acquired resistance (SAR) in plants because it causes the expression of genes associated with
defence. Exogenous administration of SA promotes seed germination, development and blooming, up-regulates photosyn-
thesis and boosts the activity of antioxidants that are enzymatic and non-enzymatic. Salicylic acid is an efficient signalling
molecule that can modify physiological and metabolic processes in plants and can thus help in reducing environmental stress
in plants over a long period of time. The less addressed issue, however, is the detailed investigation about the interaction of
SA with major phytohormones as well as combined action of SA and other phytohormones that can support and influence
the fundamental biochemical/physiological and molecular processes of plants against different forms of stress conditions
(biotic and abiotic). The current review aims to document the detailed crosstalk of SA with other phytohormones (auxin,
gibberellin, cytokinin, ethylene, abscisic acid, jasmonic acid, polyamines, melatonin, brassinosteroids and strigolactones)
that confers protection and helps in the recovery of plants from different stress conditions.
Keywords Biotic and abiotic stress· Crosstalk· Jasmonic acid· Metabolism· Phytohormones
Abbreviations
ABA Abscisic acid
BR Brassinosteroids
CK Cytokinin
GA Gibberellic acid
ICS Isochorismate synthase
JA Jasmonic acid
PA Polyamine
PAL Phenylalanine ammonia lyase
SA Salicylic acid
SL Strigolactone
TF Transcription factor
1 Introduction
Plants are regularly exposed to a wide range of different
stress conditions due to their sessile nature which results in
reduction of their productivity. Abiotic stress that includes
salinity, drought, extreme temperature, heavy metal toxicity,
radiation, flooding, etc., can be either physical or chemi-
cal, whereas biotic stress like infection caused by bacteria,
fungi, oomycetes, virus, nematodes and herbivores is mainly
manifested in the form of diseased symptoms (Gull etal.
2019). Abiotic stress conditions like water logging, drought,
heat, cold, salt and metal toxicity have negative impacts
on crop in terms of plant growth, development, yield and
seed quality. Future experts believe that when freshwater
would become more scarce, abiotic stress will become more
* Aryadeep Roychoudhury
aryadeep.rc@gmail.com
1 Department ofBiotechnology, St. Xavier’s College
(Autonomous), 30, Mother Teresa Sarani, Kolkata,
WestBengal700016, India
2 Discipline ofLife Sciences, School ofSciences, Indira
Gandhi National Open University, Maidan Garhi,
NewDelhi110068, India

P.Ghosh, A.Roychoudhury
intensely prominent. To maintain food security and safety
in the upcoming years, it is urgent to create crop types that
are resistant to abiotic stress. The roots of a plant act as its
first line of defence against abiotic stress. If the soil in which
the plant is growing is healthy and biologically varied, the
likelihood of the plant surviving adverse conditions will be
high. Biotic stress is mainly caused by microbes and other
living organisms that generate pre- and post-harvest losses.
Plants can withstand biotic stress even if they lack an adap-
tive immune system by adapting via certain sophisticated
techniques. The genetic code housed in plants regulates the
defence systems that respond to various challenges. The
plant genome has hundreds of genes that are resistant to
various biotic stressors (Gull etal. 2019).
Salicylic acid (SA) is a phytohormone that mediates plant
growth, development and defence against environmental
challenges in a variety of ways. Salicylic acid is named from
the Latin word "salix" and refers to a simple beta-hydroxy
phenolic acid that was initially isolated from willow. SA is
present at an amount between 0.1 and 10g per g of fresh
weight in diverse plant species. The majority of SA is main-
tained in glucosylated and/or methylated forms (Liu etal.
2022). Studies over the past 20years have revealed that SA
plays significant roles in the regulation of plant growth and
development, including plasmodesmatal closure, seed ger-
mination, flowering and thermogenesis, interactions with
other organisms, responses to environmental stress, as well
as induction of plant disease resistance (Zhao etal. 2017).
There are 36 plants from a variety of groups that can be clas-
sified as the natural sources of SA (Raskin etal. 1990). More
studies have indicated that SA has a significant signalling
role in plants. There are two potential routes for SA bio-
synthesis. Phenylalanine, t-cinnamic acid and benzoic acid
(BA), which are generated by the action of phenylalanine
ammonia lyase (PAL), are used by the plants to synthesise
SA from chorismate (Rohde etal. 2004). Both molecular
suppression of PAL activity in Arabidopsis and silencing of
PAL genes in tobacco lessen the pathogen-induced accumu-
lation of SA. The activity of isochorismate synthase (ICS)
in Arabidopsis catalysed the other routes used by plants to
synthesise SA from chorismate via isochorismate. By glu-
cosylating, methylating or hydroxylating the aromatic ring,
free SA in plants is converted to a variety of conjugate forms
(Zhao etal. 2017).
In the last decades, there has been a lot of interest in
the use of phytohormones to reduce the effects of major
forms of stress conditions. A complex system regulated by
the phytohormones exists for avoiding stress at multiple
levels, including physiological, molecular and biochemical
ones. Thus, phytohormones play a vital role in plant growth
and development. Numerous phytohormones, including
melatonin, jasmonic acid (JA), brassinosteroids (BRs) and
strigolactones (SLs), have a substantial role in enhancing
plant ability to withstand stress conditions (Zheng etal.
2023). The present review provides detailed analysis of
reported case studies and aims to portray one of the novel
crosstalks between SA and phytohormones, highlighting the
diverse mechanisms by which SA regulates different forms
of biotic and abiotic stress mitigation by influencing differ-
ent other phytohormones and growth regulators.
2 Biosynthesis ofsalicylic acid
It is well known that plants may produce SA from choris-
mate through the isochorismate synthase (ICS) and PAL
pathways (Lefevere etal. 2020). There are several enzymes
that catalyse these processes, although not all of them have
been found in plants. In various plant species, the signifi-
cance of these routes for the production of SA differs. The
ICS system is crucial in Arabidopsis, but PAL pathway in
rice appears to be more crucial for SA accumulation. As is
the case with soybeans, it is also possible for both the routes
to contribute equally. Additionally, the control of SA biosyn-
thesis within the plant may differ. For instance, in rice, basal
SA levels in shoots are much greater than those in roots (Sil-
verman etal. 1995; Duan etal. 2014). The principal source
for the SA biosynthesis route is chorismate, the by-product
of the shikimate pathway (Mishra and Baek 2021; El-Sherif
etal. 2022). The first reaction of the shikimate pathway
involves two metabolites, phosphoenolpyruvate (PEP) and
erythrose 4-phosphate (erythrose-4-P) for the initiation of
the reaction, which results in the first intermediate product
3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), the
starting point of the shikimate pathway. This route com-
prises of seven reactions that are catalysed by six different
enzymes to produce the metabolite chorismate, which is then
used by chorismate mutase to produce prephenate (Ghosh
etal. 2023). Through the PAL pathway, phenylalanine (Phe)
is transformed into trans-cinnamic acid (t-CA). Trans-cin-
namic acid is converted to SA by either the intermediary
ortho-coumaric acid (OA) or benzoic acid (BA), depend-
ing on the type of plant species. It has been proposed that
the step involving the conversion of BA to SA is catalysed
by the enzyme BA 2-hydroxylase. The isochorismate (IC)
route presumably evolved so that the plants may make SA
via a process similar to that of some bacteria. Isochorismate
synthase (ICS), the enzyme that converts chorismate into
IC, may be encoded by a gene. However, no gene from a
plant was discovered that functions similarly to the bacterial
enzyme isochorismate pyruvate lyase (IPL), which converts
IC to SA and pyruvate. Following its production, Arabidop-
sis ICS1 is transported into the chloroplast stroma, where SA

Molecular basis ofsalicylic acid–phytohormone crosstalk inregulating stress tolerance in…
synthesis takes place (Dempsey and Klessig 2017; El-Sherif
etal. 2022).
The two primary forms of SA found in plants are the
active free form (SA glucoside or SAG) and the inactive
vacuolar storage form (SA glucose ester or SGE). SAG and
SGE build-up significantly in the cell vacuoles and can be
hydrolysed to create active, useful forms. When a pathogen
attacks, the total SA (SA + SAG/SGE) level dramatically
rises, activating the defence mechanism that is dependent on
systemic acquired resistance (SAR). Additionally, methyla-
tion of SA produces methyl salicylate, a volatile form of SA
that contributes to enhanced membrane permeability. Methyl
salicylate can play a significant role in both SAR signalling
and plant–insect interactions. Moreover, 2,3-dihydroxyben-
zoic acid (2,3-DHBA) is produced by the hydroxylation of
SA (Mishra and Baek 2021) (Fig.1).
3 Salicylic acid mediated regulation
towardsbiotic andabiotic stress
conditions
Numerous diseases such as bacteria, viruses, fungus, oomy-
cetes and nematodes have the capacity to live on plants.
However, they are also capable of recognising an invasive
pathogen and mounting a successful defence response
through effector-triggered immunity (ETI), which is a
method of direct or indirect interaction between the products
of host resistance (R) genes and their corresponding patho-
gen-encoded effector protein (Jones and Dangl 2006). If the
plant lacks these related genes, it will be more vulnerable to
pathogen invasion since it would not be able to immediately
activate its defence mechanisms. Moreover, plants contain
a surface receptor-based pathogen-recognition mechanism
known as pathogen-associated molecular pattern (PAMP)-
triggered immunity (PTI) (Zhao etal. 2017). It is necessary
to recognise pathogen-derived components and subsequently
establish local resistance in the infected region as well as
systemic resistance in the entire plant for the small phenolic
compound SA to play an important signalling role in the
activation of both ETI and PTI in plant defence signalling
(Loake and Grant 2007).
Endogenous SA may accumulate as a result of patho-
gen infections, treatment with pathogen elicitors or other
abiotic stress situations. As a result, plants with defects
in endogenous SA accumulation or synthesis would have
weaker pathogen defences and be more vulnerable to them
(Martinez-Medina etal. 2011). Furthermore, exogenous
administration of SA and its functional counterparts can
control the response of plant immune system to a variety of
pathogen-mediated illnesses (Vallad and Goodman 2004).
Researchers discovered long back that administering SA
or its derivative aspirin might improve tolerance against
tobacco mosaic virus (TMV) (Antoniw and White 1980).
Since then, the use of SA and its analogues, including
2,6-dichloroisonicotinic acid (INA) and benzothiadiazole
Fig. 1 Biosynthesis of salicylic
acid in plants; AAO aldehyde-
oxidase, BA2H benzoic acid
2-hydroxylase, CM chorismate
mutase, ICS isochorismate
synthase, IPL isochorismate
pyruvate lyase, PAL phenylala-
nine ammonia lyase, SAG SA
glucoside, SGE SA glucose
ester, MeSA methyl salicylate
and 2, 3-DHBA: 2, 3-dihy-
droxybenzoic acid

P.Ghosh, A.Roychoudhury
S-methyl ester (BTH), has also induced the expression of
pathogenesis related (PR) genes and enhanced resistance
to bacterial, oomycete and fungal pathogens in a variety of
plants (Zhao etal. 2017). Later research found that over-
expressing salicylate hydroxylase (NahG), a SA-degrading
enzyme produced by the Pseudomonas putida NahG gene,
in tobacco or Arabidopsis reduced SA build-up following
pathogen infection by impairing both ETI and SAR (Delaney
etal. 1994).
Plants are frequently subjected to a variety of environ-
mental stressors, particularly as a result of the consequences
of global climate change. Hence, it is crucial to find chemi-
cals that might lessen vulnerability of plants to stress. SA
was one of the first chemicals shown to be involved in the
plant reaction to abiotic stressors (Fragniere etal. 2011).
When plants are subjected to abiotic challenges, their levels
of endogenous SA rise, and treating plants with SA at the
proper concentration makes different plant species more
tolerant to stress. Clarifying the role of SA in the induction
of plant abiotic stress tolerance has been possible with the
use of mutants and transgenic plants that are involved in its
production, accumulation or translocation.
In the absence of sufficient water, plants are subjected
to drought stress, which is the most frequent environmen-
tal stress having a significant impact on agricultural output.
Effective strategies for drought resistance include develop-
ing long roots to access deep soil groundwater or reducing
transpirational water loss to preserve water. Several plant
species have been shown to accumulate endogenous SA dur-
ing drought stress. During drought, the SA level in Phillyrea
angustifolia L. leaves was gradually elevated up to five times
(Munne-Bosch and Penuelas 2003). Under drought stress,
the SA concentration in barley roots increased by around a
factor of two (Bandurska and Stroinski 2005). The soil of
arid and semiarid regions frequently suffers from salinity,
and at least 20% of irrigated lands also have salt stress. In
response to salt stress, rice seedlings accumulated endog-
enous SA along with induced SA biosynthesis enzyme,
viz., benzoic acid 2-hydroxylase, indicating that SA may
be involved in salt stress responses (Sawada etal. 2006).
The study showed that in an SA-deficient transgenic line
expressing NahG gene, the extensive necrotic lesions were
induced following NaCl or mannitol application (Borsani
etal. 2001). Cold stress reduces the amount and rate of
nutrient and water absorption, which causes cell desiccation
and starvation. Extreme cold stress, also known as freez-
ing stress, results in the production of ice crystals in cell
fluids, which causes dehydration and plant death. Salicylic
acid is implicated in the control of cold responses since low-
temperature encourages the formation of endogenous free
SA and glucosyl SA in wheat, grape berries and Arabidop-
sis shoots (Miura and Tada 2014). Pre-treatment with SA
defends the plants against harmful metals as lead, mercury
and cadmium. Application of SA improved plant growth and
photosynthesis, while mitigating Cd stress in Brassica jun-
cea. Additionally, SA supplementation stabilises the plant
membrane and strengthens the antioxidant defence mecha-
nism in plants, reducing the amounts of reactive oxygen spe-
cies (Faraz etal. 2020). Thus, the primary purpose of SA
in plants is to promote protection against different forms
of stressors, be it environmental hazards or attack by phy-
topathogens or animals.
4 Crosstalk ofSA withdierent
phytohormones
Crosstalk with auxin In a plant cell, hormonal crosstalk is a
natural occurrence that controls the action of the transcrip-
tion factor under both stress-free and stressful situations.
Salicylic acid is frequently used to control plant ability to
withstand biotic and abiotic stress. In order to provide plant
defence against a variety of environmental challenges, SA
interacts either positively or antagonistically with a num-
ber of other plant hormones (Tiwari etal. 2020). An ear-
lier study reported that the SA-mediated disease-resistance
mechanism included an inhibitory effect on auxin signalling
due to the antagonistic behaviour of auxin and SA against
stress responses. Jasmonic acid and SA play a crucial role
in poplar through an integrated transcriptome machinery,
which integrates numerous hormone signalling pathways
during fungal infection to control the balance between
growth and defence responses (Luo etal. 2019). Further-
more, Qiao etal. (2020) showed that auxin pre-treatment
affects the endogenous auxin homeostasis in rice, which is
favourably connected to improving sheath blight resistance.
The MYB TF family is a large and functionally signifi-
cant class of proteins that are involved in the modulation
of most of the biological processes in plants (Tiwari etal.
2020). The R1-type MYB is a functionally essential class
of MYB transcription factor, since it plays a vital role in
surviving abiotic and biotic stress by regulating a variety
of defence mechanisms in many plant species (Erpen etal.
2018). The variable region controls the regulation of protein
activity, whereas the conserved MYB repeats/SANT domain
is involved in DNA binding and protein interactions. The
three-dimensional structure of SANT domain consists of
three alpha-helix structures that resemble the DNA-binding
MYB type helix-turn-helix (HTH) domain. Helix-turn-helix
(HTH) structure made up of the second and third helices
attaches to the main groove of DNA (Du etal. 2010). Previ-
ous reports of in silico analysis state that the OsMYB-R1
gene exhibited up-regulation upon being exposed to hexa-
valent chromium [Cr(VI)] (Dubey etal. 2010). In addition,

Molecular basis ofsalicylic acid–phytohormone crosstalk inregulating stress tolerance in…
examination of the expression of OsMYB-R1 under various
abiotic stresses revealed that the gene is similarly suscepti-
ble to drought. Because it reduces crop yield, the effect of
limited or non-existent water supply is considerably more
harmful in case of rice (Lanceras etal. 2004). An intrigu-
ing finding from the OsMYB-R1 promoter study was the
presence of cis-acting elements that are responsive to SA,
defence, stress and fungal elicitor in the promoter region.
This further suggested that the OsMYB-R1 gene may play a
role in abiotic as well as biotic stress tolerance in addition
to Cr (VI) responsiveness (Singla etal. 2016). In a study
conducted by Tiwari etal. (2020), the hormonal crosstalk
of wound-triggering and stress-responsive OsMYB-R1 tran-
scription factor was investigated based on its potential to
fight abiotic stress conditions like heavy metal (hexavalent
chromium) and drought along with biotic stress (represented
by the phytopathogen Rhizoctonia solani). Upon being
exposed to the fungal biotic stressor R. solani, the wound-
inducible promoter of the gene OsMyb-R1 displayed signifi-
cant GUS activity in the roots. Microscopic analysis showed
that the fungal load was much larger in knockdown lines in
comparison to wild type and over-expressing lines. Hence,
the results showed that lines over-expressing OsMYB-R1 can
effectively handle fungal stress. The plant samples that were
over-expressing OsMYB-R1 gene exhibited significant lateral
root formation, which can be considered as a result of the
increased tolerance upon being exposed to chromium and
drought stress. The elevation in the levels of auxin was noted
in the OsMYB-R1 up-regulated lines that further highlighted
the protective function of lateral roots under stress. Salicylic
acid signalling molecules are over-represented in RNA-Seq
data, which likely activates downstream stress-responsive
genes encoding peroxidases, glutathione S-transferases,
osmotins, PR proteins and heat shock proteins. Catalase,
guaiacol peroxidase and superoxide dismutase activities
were found to be enhanced in the over-expressed line, con-
firming the OsMYB-R1-driven strong antioxidant system.
The experimental data along with the RNA-Seq data analy-
sis clearly indicated towards a prominent crosstalk between
SA and auxin. Salicylic acid and auxin treatment dramati-
cally elevated the OsMYB-R1 transcript. OsMYB-R1 is a SA-
responsive TF that controls SA biosynthesis, SA degradation
and SA-responsive genes in order to counteract the stressful
state. When OsMYB-R1 was constitutively over-expressed,
it activated SA signalling and prevented SA production
since excess of SA might cause an oxidative burst through
the accumulation of H2O2, which could be fatal to plants.
OsMYB-R1 over-expressing lines maintain optimal SA lev-
els and may enhance its signalling via activating calcium
dependent protein kinase (CDPK) to activate downstream
genes that respond to stress (Tiwari etal. 2020).
Crosstalk with gibberellin As a crucial phytohormone, gib-
berellic acids (GAs) are members of the tetracyclic diter-
penoid carboxylic acid group. Gibberellins can stimulate
growth, promotion of seed germination, blooming, fruit rip-
ening, leaf expansion and inhibition of trichome formation
(Yamaguchi etal. 2008). As tiny plant growth molecules,
they have an impact on growth and development throughout
the whole life cycle. Gibberellins are crucial for promot-
ing cell elongation and cell division. They also aid in the
formation of transitional phases. They contribute to the ger-
mination and release of dormancy in seeds, as well as the
adult and juvenile growth phases, where seedlings that are
unable to recognise or synthesise GAs grow into plants with
stunted growth. Gibberellins have a role in plant defence
mechanisms that increase tolerance to abiotic stress, which is
characterised by encouraging plant growth and development
under adverse conditions (Emamverdian etal. 2020). Earlier
studies demonstrated that the most active form of gibberel-
lin, GA3, improved plant tolerance to the trace amounts of
the heavy metals, viz., lead (Pb) and cadmium (Cd) in the
green alga Chlorella vulgaris (Chlorophyceae) (Falkowska
etal. 2011). Additionally, in maize, GA3 enhanced mem-
brane permeability effectiveness and nutrient levels to
increase seed germination and seedling establishment under
metal stress (Tuna etal. 2008). Furthermore, GAs play a
significant role in controlling the rate of sink-source transfer
under various growth conditions, allowing them to modify
assimilate translocation through a variety of mechanisms,
including altered sink and source formation, controlling
resource mobilisation and increasing photosynthetic rates
(Emamverdian etal. 2020).
Recent research has revealed that Arabidopsis DELLA
proteins, which serve as negative regulators of GA signal-
ling, modulate SA- and JA-dependent defensive responses
to regulate plant immunological responses (Navarro etal.
2008). The fungal necrotrophic pathogens Alternaria bras-
sicicola and Botrytis cinerea are particularly sensitive to the
Arabidopsis quadruple-della mutant, which lacks the four
DELLA genes (gai-t6, rga-t2, rgl1-1, and rgl2-1). However,
the mutant is more resistant to the biotrophic infections
caused by Pst DC3000 and Hyaloperonospora arabidopsidis
(Navarro etal. 2008). Moreover, compared to Pst DC3000-
challenged wild-type plants, the expression of the JA/ET
marker gene, PDF1.2 (Plant defensin 1.2) was significantly
delayed in the quadruple-della mutants. In contrast, DELLA
over accumulating mutants like ga1-3, gai and sly1-10 were
less resistant to Pst DC3000 and more vulnerable to A. bras-
sicicola. These findings imply that DELLA proteins increase
resistance to necrotrophs by activating JA/ET-dependent
defence responses, but decrease vulnerability to biotrophs
by suppressing SA-dependent defence responses in Arabi-
dopsis. Thus, SA and JA/ET-based plant defence response

P.Ghosh, A.Roychoudhury
pathways seem to be integrated by DELLA proteins (Bari
and Jones 2009).
Salicylic acid and gibberellin are involved in the con-
trol of several plant responses. The interaction between GA
and SA clearly indicates that under stressful circumstances,
plant resilience to abiotic stress is increased. It has been
demonstrated that the FaGASA4 transgenic line of Arabi-
dopsis is significantly more resistant to abiotic stress, such
as oxidative and salt stress. Incubating Arabidopsis seeds
in 50 µM GA3 resulted in a 2.0-fold increase in SA lev-
els than in seeds that were incubated in water for 24 hours
(Alonso-Ramirez etal. 2009). Using knockout mutants, it
was previously established that there is a functional relation-
ship between lower GA signalling and decreased disease
severity. sRNA network was diminished in the transgenic
counterpart NahG-Désirée that manifested severe illness
signs and lacked SA, validating again that this regulation
is SA-dependent. The increase of transcripts like miR164,
miR167, miR169, miR171, miR319, miR390 and miR393 in
tolerant Désirée as well as the down-regulation of GA sig-
nalling revealed significant parallels to responses seen in
mutualistic symbiotic associations. The interplay of several
regulatory networks demonstrated how disease symptom
development, stress signalling and developmental signalling
are tightly regulated (Kriznik etal. 2017).
Crosstalk with cytokinin Cytokinins (CKs) are significant
growth hormones that encourage vital functions like cell
proliferation, nutrient mobilisation and leaf life span in
plants. Previous reports have stated that CKs have an impact
on plant disease resistance as well (Pertry etal. 2009).
Numerous plant pathogens release CKs or cause host plants
to synthesise CKs. The hemibiotrophic actinomycete Rho-
dococcus fascians possesses an isopentenyl transferase (ipt)
gene in the fas operon and generates CKs. The CKs derived
from R. faciens are identified by two component histidine
kinases 3 and 4 (AHK3 and AHK4) that are needed for the
development of symptoms (Pertry etal. 2009). Cytokinins,
which may be connected to green islands on infection sites,
are present in the spores of biotrophic rust and powdery mil-
dew (Kiraly etal. 1966, 1967). Necrotrophic fungi such as
Penicillium expansum and B. cinerea, do not, however, syn-
thesise CKs (Walters and McRoberts 2006). It is therefore
conceivable that CK-releasing biotrophs or hemibiotrophs
modify CK signalling to control the host cell cycle and nutri-
tion distribution, and processes required for pathogenesis
(Choi etal. 2010). Earlier reports revealed that increased
levels of endogenous CK in tobacco result in resistance to
TMV, the induction of SA in the wounding response, or
increased activities of the PR proteins, viz., chitinase in
extracellular spaces (Sano etal. 1996; Synkova etal. 2004).
According to a study by Choi etal. (2010), plant derived-
CKs confer resistance to Arabidopsis against the pathogenic-
ity of Pseudomonas syringae pv. tomato DC3000 (Pst). In
CKX- or IPT-over-expressing plants, as well as in ahk2 and
ahk3 mutants, modified resistance was linked with changed
CK levels or signalling activity. In actuality, Pst resistance
is especially facilitated by the CK-activated transcription
factor, ARR2. When TGA-binding cis-elements in the
Pr1 promoter were altered, CK- and ARR2-dependent Pr1
activation was eliminated. TGA3 is a SA response factor
that binds ARR2. Due to the absence of the CK-dependent
activation of Pr1, CK administration did not result in an
increase in pathogen resistance in tga3 plants. Furthermore,
SA signalling improved the ability of ARR2/TGA3 to bind
to the Pr1 promoter. All of these findings demonstrate how
CKs alter SA signalling to increase resistance to Pst, which
is accomplished through the interaction of TGA3 and ARR2.
Jiang etal. (2013) also noted the interaction between SA
and CK in rice plants infected with Magnaporthe oryzae
(blast fungus). They observed that rice and blast fungus
evolved in tandem, and infection with the blast fungus ele-
vated the CK levels in rice for its own benefits in the form
of nutrient efflux. This is a response against infection and
defence mechanism is elicited as a result of its synergis-
tic interaction with SA. CK and SA were found to function
co-operatively to stimulate the expression of defence genes.
This is accompanied with the dramatically elevated levels
of OsPR1b and PBZ1 in leaf blades after treatment with
100mM SA and 100mM CK (Kaya etal. 2023).
Cytokinins control a number of physiological processes
that are involved in plant growth and development. Cyto-
kinins are often responsible for increasing tolerance towards
abiotic stress stimuli such as high salinity and high tempera-
ture (Székács etal. 2000; Javid etal. 2011). For instance,
it was found that raising CK levels enhanced the resistance
of cereals to salinity stress (Kuiper etal. 1990). In a study
conducted by Torun etal. (2022), the concentrations of 16
distinct CK metabolites in the leaves and roots of barley
seedlings were raised under diverse experimental settings.
Under salt stress, roots had lower total CK concentrations
than leaves. Under non-saline circumstances, SA pre-treat-
ment did not result in any appreciable alterations in the CK
levels of barley roots and leaves. SA pre-treatment raised CK
levels at a moderate NaCl concentration (150mM), particu-
larly in leaves. The levels of all CK groups were reduced by
SA pre-treatment at 300mM NaCl. The build-up of IAA and
CKs is thus affected by salt in opposite ways. These findings
imply that SA functions as a CK antagonist in extremely
saline environment (Torun etal. 2022).
Crosstalk with ethylene Several different abiotic stressors
have been shown to cause ethylene synthesis, where SA has

Molecular basis ofsalicylic acid–phytohormone crosstalk inregulating stress tolerance in…
been shown to suppress the same (Khan etal. 2015; Peng
etal. 2021). Exogenous SA, for instance, reduces heat stress
by boosting proline metabolism and limiting the production
of Eth in heat-stressed plants by decreasing the 1-aminocy-
clopropane 1 carboxylate synthase (ACS) activity in wheat
(Khan etal. 2013). Similar to mustard, mung bean and sweet
pepper, the SA-mediated ACS activity suppression increases
plant tolerance to salt and drought stress (Khan etal. 2014;
Nazar etal. 2015; Ahmed etal. 2020). Furthermore, it has
been shown that in pear suspension cells, exogenous SA
may limit the 1-aminocyclopropane 1 carboxylate oxidase
(ACO) activity, preventing the conversion of 1-aminocy-
clopropane 1 carboxylate to Eth (Leslie etal. 1986). When
exposed to ozone (O3) stress, ethylene and SA work together
to control cell death in Arabidopsis and tobacco leaves (Rao
etal. 2002; Ogawa etal. 2005). By increasing the expres-
sion of the SA biosynthetic genes encoding CHORISMATE
MUTASE and PAL, O3-induced Eth encourages SA produc-
tion (Ogawa etal. 2005). While Eth overproduction aggra-
vates cell death, inhibition of either SA or Eth biosynthesis
in Arabidopsis recovers the O3-induced hypersensitive cell
death phenotype (Rao etal. 2002). A chemical or genetic
alteration of SA signalling in Arabidopsis, on the other hand,
prevents the formation of Eth in response to O3, indicating
that SA is necessary for O3-induced Eth biosynthesis (Rao
etal. 2002). Future research should explore the fascinating
hypothesis that SA and Eth may use some shared machin-
ery to control downstream genes because of the synergistic
effects of Eth and SA in abiotic stress tolerance (Chen etal.
2021).
Ethylene is a low molecular weight signalling molecule
that participates in defence during environmental stress, just
as SA and JA. Salinity raised the Eth levels in barley seed-
lings in one investigation (Torun etal. 2022). Under non-
salty and moderately saline circumstances, SA pre-treatment
likewise boosted Eth levels; however, it was decreased under
extremely saline conditions. This is in line with the find-
ings of Tirani etal. (2013), who discovered that SA and
Eth exhibited an antagonistic interaction in canola plants.
Numerous investigations have demonstrated that SA and
abscisic acid (ABA) are inhibited by the synergistic action of
jasmonate and Eth (Anderson etal. 2004; He etal. 2017). SA
elevated ABA and Eth levels at moderate salt concentrations
(150mM NaCl), but decreased the two phytohormones at
high salt concentration (300mM NaCl) (Torun etal. 2022).
Salicylic acid crosstalk with ABA Abscisic acid (ABA) is a
key plant hormone that performs important signalling func-
tions in a variety of processes related to plant growth and
development as well as in the way plants react to various
abiotic challenges. ABA has also become a vital signal-
ling molecule in interactions between plants and pathogens
(Jiang etal. 2010). A number of plant species have been
shown to be more vulnerable to bacterial and fungal patho-
gens when exogenous ABA is applied or accumulated due
to genetic defects (Fan etal. 2009; Jiang etal. 2010). On
the other hand, ABA-deficient mutations in tomato led to
increased resistance to several diseases (Achuo etal. 2006).
The result of the mutation causing ABA-deficiency led to
an improved resistance to several diseases. These mutant
plants exhibited SA-dependant defence reactions that are
more potent than those of wild-type plants, indicating that
the antagonistic interaction of ABA with SA signalling
pathway causes ABA to have a detrimental impact on plant
defence mechanisms. This idea was further confirmed by a
recent report that revealed antagonistic interaction between
SA-mediated induction of SAR and ABA-mediated abiotic
stress responses (Yasuda etal. 2008). A previous report
demonstrated the mechanism by which P. syringae type III
effectors (T3Es) hijack the Arabidopsis ABA biosynthesis
pathway to inhibit plant basal defence and antagonise SA
levels, indicating a critical role of ABA in pathogenicity
(de Torres etal. 2009). However, another research indicated
that ABA benefits the plant defence mechanisms by per-
forming functions such as the regulation of stomatal closure
(Melotto etal. 2006) and priming callose deposition (Flors
etal. 2008). As a result, ABA seems to have varied defensive
functions depending on the type of phases of the infections
and pathosystems (Jiang etal. 2010).
The detrimental effects of ABA on rice defence system
against Magnaporthe grisea have also been documented
(Koga etal. 2004). Infection by a suitable race of M. grisea
was shown to increase the sensitivity of the rice genes to
ABA and dehydration stressors (Ribot etal. 2008). Accord-
ing to Bailey etal. (2009), the antagonistic interaction of
ABA with the Eth signalling pathway raises the blast sus-
ceptibility in a favourable rice-M. grisea interaction. These
studies confirm that ABA participates in the interaction
between rice and M. grisea. In a study conducted by Jiang
etal. (2010), exogenous administration of ABA affected the
rice plants severely and reduced resistance in both com-
patible and incompatible M. grisea strains, demonstrating
that ABA has a deleterious impact on both basal and blast
resistance mediated by resistance genes. The SA, benzo-
thiadiazole or blast infection-induced transcriptional up-
regulation of WRKY45 and OsNPR1, the two essential SA
signalling pathway components in rice, were significantly
reduced by ABA. The increased blast susceptibility caused
by ABA was significantly reversed by over expression of
WRKY45 or OsNPR1, indicating that ABA operates in the
rice SA pathway, upstream of WRKY45 and OsNPR1. In

P.Ghosh, A.Roychoudhury
the compatible rice-blast interaction, ABA-responsive genes
were elevated during blast infection in a manner opposite to
that of WRKY45 and OsPR1b. These findings imply that
the interaction between rice and M. grisea was significantly
influenced by the balance of SA and ABA signalling (Jiang
etal. 2010).
The transcription factor MYB96 is thought to enhance SA
biosynthesis and induce drought tolerance and has been dis-
covered to mediate the interaction between SA and ABA in
response to both biotic and abiotic circumstances (Ku etal.
2018). The superactive mutant myb96-1d, which is more
responsive to ABA-mediated signalling than wild type, has
been demonstrated to have elevated levels of SID2, the gene
responsible for SA production via the ICS enzyme. These
mutant plants have the same dwarf phenotype as plants with
enhanced ABA production. This dwarfism is inhibited by
crossing with NahG plants, which have a SA hydroxylase
gene that converts SA to catechol (Friedrich etal. 1995),
suggesting that SA regulates the level of ABA that results
from dwarfism (El-Sheriff 2022). In many instances, ABA
and SA regulate mutual biosynthesis. In a variety of envi-
ronmentally stressful situations, SA behaves in a synergistic
way with ABA (Singh and Roychoudhury 2023). Accord-
ing to Prodhan etal. (2020), npr1 mutants or mutants with
a deficiency in SA production effectively displayed ABA-
mediated stomatal closure. Prodhan etal. (2018) previously
reported that the mutant for ABA production demonstrated
efficient SA-triggered stomatal closure. Salicylic acid and
ABA signalling pathways operate independently of one
another, but several molecular studies have demonstrated
that following calcium accumulation and the generation of
reactive oxygen species (ROS) during stressful conditions,
SA and ABA signalling are integrated by the MPK12 and
MPK9-mediated mitogen-activated protein kinase (MAPK)
pathway and the calcium dependent protein kinase 9 (CPK9)
and CPK3-mediated pathway (Jammes etal. 2009; Prodhan
etal. 2018).
Exogenous application of SA in drought-stressed and
cadmium-stressed tomato and wheat seedlings, respec-
tively, resulted in the elevation of the synthesis of ABA,
which imparted stress tolerance (Munoz-Espinoza etal.
2015; Shakirova etal. 2017). Freezing tolerance in wheat
was triggered by exogenous application of SA that led to
successful interaction of ABA and H2O2 (Wang etal. 2018).
Another example of the interaction between ABA and SA
during heavy metal stress was found when priming with
SA reduced lead toxicity by promoting the accumulation
of ABA and JA precursors in Zygophyllum fabago (L'opez-
Orenes etal. 2020).
Crosstalk with polyamines Until now, it was thought that
polyamines (PAs) are only direct protective substances. They
are able to interact with negatively charged macromolecules
in a reversible manner because of their cationic nature at
physiological pH, stabilising their structure, particularly
under stressful circumstances. They can attach to the phos-
pholipid head groups of membranes, changing the properties
that determine how permeable they are. They can attach to
chromatin, altering the availability of genomic sites to DNA
or RNA polymerases, changing DNA and RNA synthesis,
and bind to a variety of proteins non-specifically, stabilising
their structure and changing their activity and/or function
(Pal etal. 2015).
Polyamines are crucial for plant defence against patho-
gens, but little is known about how hormone-mediated
defence signalling pathways regulate PA metabolism. Exog-
enous administration of SA was combined with the use of
mutants for PA biosynthesis and SA synthesis/signalling to
examine the control of PA metabolism in Arabidopsis (Rossi
etal. 2021). Salicylic acid significantly altered the metabo-
lism of PAs, principally by causing putrescine to accumulate
in both whole-plant extracts and apoplastic fluids. Putrescine
was accumulated due to increased arginine decarboxylase 2
activity and reduced copper amine oxidase oxidation. The
signalling kinases MKK4 and MPK3 as well as the regu-
latory protein Non-Expressor of Pathogenesis Related 1
(NPR1) were not necessary for increasing the putrescine lev-
els induced by SA, although MPK6 was mandatory for the
same. Plant infection with Pseudomonas syringae pv. tomato
DC3000, on the other hand, caused putrescine accumulation
in a manner that was SA-dependent. Hence, the work exhib-
ited a distinct relationship between SA signalling and plant
PA metabolism in Arabidopsis and helped to understand how
SA modifies PA levels during plant–pathogen interactions
(Rossi etal. 2021).
In a study conducted by Nemeth etal. (2002), it was
depicted that addition of 0.5mM SA to the hydroponic
solution of maize improved tolerance to low-temperature
stress, as demonstrated in a prior study (Janda etal. 1999).
In this study, the impact of SA and cold treatments on the PA
content of the leaves was examined using high performance
liquid chromatography (HPLC) method. Cold treatment and
0.5mM SA both significantly raised the putrescine levels.
Spermidine only increased in response to low-temperature
stress after SA addition. However, SA and low-temperature
treatments resulted in a reduction in the spermine concen-
tration. In maize and wheat, the concurrent use of 0.5mM
SA and 15% polyethylene glycol (PEG) resulted in a sig-
nificant increase in electrolyte leakage and a drop in several
photosynthetic parameters. Thus, it was concluded that pre-
treatment with 0.5mM SA enhanced freezing tolerance, but

Molecular basis ofsalicylic acid–phytohormone crosstalk inregulating stress tolerance in…
increased the susceptibility to drought (Nemeth etal. 2002).
In a study conducted by Hao etal. (2018), the exogenous
application of SA in tomato plants triggered the expression
of SlPAO2-4 genes, while a decrease in the expression of
SlPAO6-7 genes was noted. Expression of CsSAMS1 and
CsSAMS2 in leaves of cucumber was induced when SA was
applied exogenously (He etal. 2019).
Salicylic acid crosstalk with jasmonic acid The interac-
tion between SA and jasmonic acid (JA) has been most
elaborately explained among all the phytohormones stud-
ied. The reaction of the plant to stress conditions has been
fine-tuned by a network of hormonal signalling cascades
that are activated under stress situations. For instance, the
defensive signalling pathways controlled by the SA and
JA might act either synergistically or antagonistically (El-
Sherif 2022). SA-targeted gene PR1 and the JA marker gene
PDF1.2 were both synergistically expressed after low-dose
SA and JA therapy, whereas the same genes were antago-
nistically expressed after high-dose SA and JA treatment
(Mur etal. 2006). SA and JA are two significant hormones
in plant defence response against pathogenic attacks (Tang
etal. 2022; Li and Ahammed 2023). Plant immunity against
biotrophic diseases is favourably triggered by SA signal-
ling, but resistance against necrotrophic pathogens is mostly
provided by the JA pathway (Yang etal. 2014). The SAR
against pathogens in plants is mediated by SA (Peng etal.
2021), whereas JA signalling is involved in induced sys-
temic resistance against microorganisms like Rhizobacte-
ria (Yu etal. 2022). Global warming is mostly caused by
rising atmospheric CO2. Due to extensive human activities
including industrialization and deforestation, atmospheric
CO2 levels have risen from 280 parts per million (ppm) to
400ppm during the past century. By the end of this cen-
tury, it is predicted that the atmospheric CO2 concentration
would range between 730 and 1000ppm (IPCC 2022; Li
and Ahammed 2023). It is well known that elevated CO2
(eCO2, commonly defined as twice that of ambient levels, for
example, 800ppm) has positive effects on agricultural yield
in C4 plants (Foyer and Noctor 2020). The production of SA
and JA and their signalling are impacted by eCO2 (Kazan
2018). For instance, eCO2 often favours the SA biosynthe-
sis and signalling pathways, while suppressing the JA path-
way (Foyer and Noctor 2020). Furthermore, the presence of
eCO2 also affects SA/JA crosstalk. Plant defence responses
vary significantly at eCO2 depending on the plant–patho-
gen system because variations in SA and JA signalling are
typically connected to differences in how pathogens interact
with host plants (Li and Ahammed 2023). There are strong
antagonistic interactions between SA and JA pathways at
elevated CO2, which suggests that resistance against certain
pathogens, mediated by SA pathways, can decrease the
impact of JA signalling pathway and make the host plants
more vulnerable to various other diseases, including attacks
by fungal necrotrophs that mostly depend on JA-regulated
defence mechanism. For instance, eCO2 elevates the endog-
enous SA contents, while decreasing the JA contents in
tomato plants, resulting in increased resistance to P. syrin-
gae and TMV but decreased resistance to B. cinerea in an
NPR1-dependent manner. NPR1 is a key node mediating the
crosstalk between SA and JA signalling (Zhang etal. 2015).
In Arabidopsis, exposure to eCO2 increases the expression
of the SA-responsive defence genes, viz., PR1, PR2 and
PR5, as well as the ISOCHORISMATE SYNTHASE1 (ICS1)
gene, which encodes an enzyme implicated in the produc-
tion of SA. Additionally, in Arabidopsis, the expression of
JA-responsive defence genes, such as PAD3, LOX3, OPR3,
JAZ10 and PDF1.2, increases with eCO2 (Mhamdi and Noc-
tor 2016). As a result, simultaneous activation of the SA and
JA pathways confers increased resistance to the necrotrophic
fungus B. cinerea and the hemibiotrophic bacteria P. syrin-
gae in Arabidopsis (Kazan 2018; Bazinet etal. 2022).
In defective tomato plants where NPR1 expression was
suppressed due to virus-induced gene silencing, eCO2
increased the expression levels of JA-dependent defence
genes, viz., PI 1 and PI II, thereby increasing resistance
to B. cinerea. This further suggested that eCO2 increases
SA-JA antagonism in tomato plants (Li and Ahammed
2023). Elevated CO2 simultaneously stimulated both the
SA and JA signalling pathways in Arabidopsis, and the
interaction between P. syringae and Arabidopsis at eCO2
resulted in increased resistance to both P. syringae and B.
cinerea (Mhamdi and Noctor 2016). In contrast, the inter-
action between P. syringae and tomato at eCO2 led to the
promotion of SA, but suppression of JA pathway. Addition-
ally, when exposed to eCO2, mustard (Brassica juncea L.)
plants produced greater amounts of SA and JA and exhibited
improved resistance to the necrotrophic fungus Alternaria
brassicae (Mathur etal. 2018). In light of these latter find-
ings, the theory that the SA and JA pathways are mutually
hostile cannot be supported (Gupta etal. 2020). Hence, plant
reaction to intracellular oxidative stress brought upon by
culture at eCO2 is likely to be the cause of activation of these
pathways at eCO2 (Mhamdi and Noctor 2016).
JA controls a variety of physiological processes, includ-
ing root inhibition, anthocyanin accumulation, trichome
initiation, male fertility, leaf senescence and responses to
biotic and abiotic stress (Hu etal. 2017). The antagonis-
tic regulation of WRKY53, a TF that responds to senes-
cence, by JA and SA in Arabidopsis suggested that these
two phytohormone pathways interact with one another dur-
ing senescence (Miao and Zentgraf 2007). SA substantially

P.Ghosh, A.Roychoudhury
induced the expression of WRKY53 that altered SA-medi-
ated responses (Miao etal. 2004; Hu etal. 2012). In the
nucleus, WRKY53 interacts with the JA-inducible protein
ESR (EPITHIOSPECIFYING SENESCENCE REGULA-
TOR), and this interaction has a detrimental effect on the
ability of WRKY53 to bind DNA. These findings imply that
the interaction between ESR and WRKY53 proteins dur-
ing leaf senescence, which is most likely controlled by the
JA and SA balance, mediates the negative JA/SA crosstalk
(Miao and Zentgraf 2007; Hu etal. 2017).
In a study conducted by Torun etal. (2022), salt stress
was found to enhance endogenous JA levels in experiments
on tomato (Pedranzani etal. 2003) and rice (Kurotani etal.
2015). Additionally, according to Pedranzani etal. (2003),
the elevated JA levels exhibited in salt-tolerant cultivars in
saline conditions could be a factor for this trait. These find-
ings imply that JA accumulation could be a part of defence
mechanism against salt stress. In the mentioned work, pre-
treatment with SA decreased JA levels in leaves but dramati-
cally elevated them in roots under non-saline circumstances.
In contrast, under salinity, SA pre-treatment significantly
decreased the levels of JA in both the vegetative organs. This
suggested that under salt stress, SA and JA have a hostile
interaction.
Crosstalk with melatonin Plant researchers are concentrating
on melatonin (N-acetyl-5-methoxytryptamine), a relatively
"new" chemical found in plants, because of its enormous
potential as a regulator of numerous physiological processes.
Application of melatonin encourages plant development
and growth while also making plant cells more resilient to
stress (Rafique etal. 2023). Melatonin controls the genes
connected to plant hormones by acting as a receptor with
intricate multifunctional behaviours. It is currently regarded
as a contemporary plant hormone (Arnao and Hernández-
Ruiz 2018, 2019). In a previous report, the synthesis of
melatonin and its impact on plant defence systems using
Arabidopsis knockout mutants, deficient in functional sero-
tonin N-acetyltransferase (SNAT) gene, the penultimate gene
in melatonin biosynthesis, was examined. The phenotype of
the Arabidopsis snat knockout mutants exhibited reduction
in melatonin synthesis during pathogen attack, leading to
increase in the vulnerability to the avirulent pathogen Pseu-
domonas syringae pv. tomato DC3000 expressing the effec-
tor avrRpt2 (Pst-avrRpt2). The observation was correlated
with reduced levels of SA and reduction in the activation
of defence gene, associated with the pathogen-susceptible
phenotype of the snat mutant lines (Lee etal. 2015). In a
recent noteworthy work, Zhang etal. (2017) showed that
melatonin reduced the severity of Phytophthora infestans
that induces late blight of potato. Melatonin reduced the
mycelial development and altered the expression of several
genes linked to stress and virulence and thus, increased plant
innate immunity against fungal infection. Hence, melatonin
up-regulates genes involved in pathogenesis that are SA-
and Eth- dependent; this impact was inhibited in mutants
deficient in SA and Eth signalling. Nitric oxide (NO) and
SA-related genes were also up-regulated by melatonin, and
this was followed by a decrease in the sensitivity of the cells
to the pathogen. In addition to having decreased levels of SA
and melatonin, snat knockout mutants also showed increased
susceptibility to the infection (Hernández-Ruiz and Arnao
2018).
Melatonin is a pleiotropic chemical that can affect a vari-
ety of functions in plants. The present study investigated
how melatonin regulated the antioxidant system in Linum
album Kotschy ex Boiss. cell culture (Esmaeili etal. 2023).
The findings demonstrated that whereas H2O2 content was
enhanced at high quantities of melatonin, it did not change
at lower values. It can be explained by the fact that modest
melatonin doses have the ability to eliminate large amounts
of H2O2, but high melatonin dosages have hazardous con-
sequences. Contrarily, the NO augmentation occurred at 50
µM melatonin, suggesting that it plays a part in inducing
defensive reactions in response to melatonin. NO caused
oxidative stress in cells treated with 50 µM melatonin. Mela-
tonin therapy stimulated the antioxidant enzyme peroxidase,
while SOD responded in the opposite manner, which could
explain variations in the H2O2 content. Additionally, the phe-
nolics profile demonstrated that an increase in PAL enzyme
activity led to an increase in the concentrations of phenolic
acids, flavonoids and lignans. SA-dependent mechanism may
be involved in the action of melatonin in defensive responses
in L. album cells, as shown by the elevated levels of the
phenolic hormone SA. In summary, it appears that melatonin
can stimulate the activity of antioxidant enzymes and the
formation of phenolics, notably lignans, in L. album cells
through altering NO and SA levels (Esmaeili etal. 2023).
Crosstalk with brassinosteroids The steroid hormone named
brassinosteroid (BR) is necessary for the growth and devel-
opment of plants. The essential functions like development
of stomatal cell, cell growth, xylem differentiation, photo-
morphogenesis and development of reproductive organs are
regulated by BRs (Kim etal. 2022). The transcription fac-
tors BRASSINAZOLE-RESISTANT 1 (BZR1) and BRI1-
EMS-SUPPRESSOR 1 (BES1) are activated by BR through
sequential signal relay. The glycogen synthase kinase 3
(GSK3)-like kinase, BRASSINOSTEROID-INSENSITIVE
2 (BIN2) constitutively inhibits BZR1 and BES1 by phos-
phorylation when BR is not present (Kim and Wang 2010;
Clouse 2011; Nolan etal. 2020). When BR binds to the BR

Molecular basis ofsalicylic acid–phytohormone crosstalk inregulating stress tolerance in…
receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1),
BRI1 interacts with BRI1-ASSOCIATED KINASE 1
(BAK1) to form a receptor complex that transmits the signal
to receptor-like cytoplasmic kinases (RLCKs) like BRASSI-
NOSTEROID SIGNALLING KINASE 1 (BSK1) and CON-
STITUTIVE DIFFERENTIAL GROWTH1 (CDG1) (Kim
etal. 2022).
In an investigation carried out by Kim etal. (2022), it
was revealed that BR suppresses the redox-sensitive clade
I TGAs in Arabidopsis by activating BR-INSENSITIVE
2 (BIN2) to elevate SA responses. It was discovered that
BR together with BIN2 inhibitor bikinin synergistically
enhanced the physiological responses mediated by SA, such
as conferring resistance against Pst DC3000. The biochemi-
cal and genetic analyses of the investigation revealed that,
in contrast to other TGAs, TGA1 and TGA4 functionally
interacted with BIN2. Additionally, the study showed that
Ser-202 of TGA4 is phosphorylated by BIN2, which desta-
bilises TGA4 and suppresses its redox-dependent interaction
with NPR1. Overall, the findings indicate towards a unique
crosstalk mechanism through which BR signalling orches-
trates the SA responses carried out by redox-sensitive clade
I TGAs (Kim etal. 2022).
Crosstalk with strigolactones Recent studies have demon-
strated that strigolactones (SLs) have a role in plant reac-
tions to biotic and abiotic stressors. In a study conducted by
Kusajima etal. (2022), a thorough investigation was done
on the ways SLs affected SAR triggered by SA-mediated
signalling. It was revealed that in mutants with defects in
SL signalling and synthesis, SAR was induced. However,
compared to the wild-type plants, SL signalling-deficient
max2 mutants had lower levels of endogenous SA and lower
levels of SA-responsive gene expression. The SL analogue
GR24 improved disease resistance in both the wild-type and
SL biosynthesis-deficient mutants, but treatment with a SL
biosynthesis inhibitor decreased disease resistance in the
wild type. Treatment with GR24 did not activate defence-
related genes in wild-type plants, prior to the introduction
of pathogenic bacteria; nevertheless, SA-responsive defence
genes were quickly activated following pathogenic infection.
These results imply that SLs prime Arabidopsis thaliana
for disease resistance through SA induction (Kusajima etal.
2022).
One of the most important abiotic stress factors that
affects plant growth and development and limits the output
of cherries (Cerasus spp.) is drought. It has been shown
that SA and SLs participate in a variety of biotic and abi-
otic stressors (Xu etal. 2023). The effects of SLs and SA
on cherry rootstocks under drought stress were examined in
the current study. Under drought stress, the relative water
content, net photosynthetic rate and maximum quantum
yield of photosystem II were all higher in the groups that
were pre-treated with rac-GR24 and SA than in the well-
watered group, but the activity of the antioxidant enzymes
were found to be reduced, indicating that they significantly
increased drought tolerance. There were 837, 755, 586 and
489 differentially expressed genes (DEGs), respectively,
identified by RNA-Seq analysis of leaf samples treated with
drought stress or drought stress combined with 1mM SA
(T2), 1µM rac-GR24 (T3), and 5µM rac-GR24 (T4) pre-
treatments. Many of the DEGs, according to transcriptome
analysis, were associated with ABA-responsive proteins
(PavNCED and PavPP2C51), stress responsive transcrip-
tion factors (PavWRKY and PavMYB), antioxidant enzymes
(PavGST and PavPOD) and components associated with
osmotic adjustment (PavUGTs, PavSUSs and PavSPS1).
Drought treatment substantially induced the proteins
PavDBP2D, PavABCG15-like, PavPUP9, PavLOG1-like
and PavENT8, while rac-GR24 or SA pre-treatment reduced
the expression level of these genes under drought stress con-
dition. Thus, the current research offers fresh insights into
the regulatory mechanisms that control drought tolerance in
woody plants as well as provides a fundamental knowledge
of drought tolerance in cherry rootstocks controlled by SA
and SLs (Xu etal. 2023). In a similar study performed on
tomato under drought conditions, the combined impact of
GR24 and SA triggered the recovery in plants by decreas-
ing lipid peroxidation with the help of increased antioxidant
potential and increased tolerance to drought conditions (Bal-
tacier etal. 2023).
Table1 provides an overall summary of the interaction of
SA with different phytohormones during different stress epi-
sodes in plants.
5 Future perspectives
The ability to manipulate plants to increase stress tolerance
and productivity depends on our ability to understand the
molecular mechanisms behind their reactions to abiotic and
biotic stress conditions. Plant hormones regulate intricate sig-
nalling networks that control growth and plant responses to
biotic and abiotic environmental stressors. Finding the essen-
tial elements and comprehending plant hormone signalling,
particularly SA, JA and Eth, as well as plant defence reac-
tions, have advanced significantly. There is evidence from a
number of recent researches that additional hormones, includ-
ing ABA, auxin, GA, CK and BR, are involved in the signal-
ling pathways for plant defence. When some hormones are
administered to plants, the host metabolism, gene expression
and plant defence mechanisms against microbial attack are

P.Ghosh, A.Roychoudhury
Table 1 Crosstalk between SA and other phytohormones during different stress conditions
Plant Type of stress agents Category of stress Effect on phytohormone Mechanism/Pathway Reference
Arabidopsis P. syringae pv. maculicola Biotic Salicylic acid induced repression of auxin-related
genes, thus inducing SAR in infected tissues
SA analogue, viz., benzothiadiazole S-methyl ester,
resulted in the repression of a number of auxin-
responsive genes, viz., auxin receptors TIR1
and AFB1, auxin exporter PIN7, auxin importer
AUX1, auxin inducible SAUR and the Aux/IAA
family of genes
Wang etal. (2007)
Potato Potato virus Y Biotic Salicylic acid regulated the sRNA-gibberellin net-
work and impacted the disease symptoms
The sRNA-gibberellin pathway participated in
reducing the disease symptoms that were noted
in SA-depleted transgenic NahG Desiree variety,
thus increasing the symptoms of the disease
Kriznik etal. (2017)
Arabidopsis Botrytis cinerea Biotic CAT2 was found to be engaged in SA-mediated
antagonistic control of JA biosynthesis via
interacting with ACX2 and ACX3, the two JA-
biosynthetic enzymes
SA reduced JA production by inhibiting the activity
of CAT2, whereas CAT2 promoted the activ-
ity of acyl-CoA oxidase (ACX) involved in JA
biosynthesis to boost plant immunity against
necrotrophic fungus B. cinerea
Zhang etal. (2021)
Brassica napus Drought Abiotic SA and melatonin acted synergistically to induce
resistance towards drought stress
Simultaneous priming of B. napa seeds with mela-
tonin and foliar application of SA could overcome
stress by regulating growth criteria, yield compo-
nents and osmotic potential
Rafique etal. (2023)
Cucumber Freezing Abiotic Exogenous application of SA helped in improve-
ment of grafting-induced chilling tolerance in
cucumber by triggering the biosynthesis of ABA
ABA induced the production of hydrogen peroxide
which further enhanced resistance to chilling
stress by decreasing the electrolyte leakage and
chilling injury index
Zhang etal. (2022)
Potato Phytophthora infestans Biotic Activation of both ethylene and SA pathways is
necessary for the positive modulation of a potato
StMKK5-SIPK for resistance against immunity to
P. infestans
The kinase activity of both StMKK5 and SIPK
appeared necessary for introducing point muta-
tions that further triggered cell death
Yang etal. (2023)

Molecular basis ofsalicylic acid–phytohormone crosstalk inregulating stress tolerance in…
reprogrammed. Different hormones have opposing effects on
distinct biotrophic and necrotrophic pathogens, depending on
the nature of plant–pathogen interactions. While auxin mostly
interacts antagonistically with SA, BRs exhibit a synergis-
tic relationship with SA. Such crosstalks open new avenues
for enhanced resistance in plants towards any form of stress
conditions by applying a combination of phytohormones as
required. Additionally, the complicated network caused by
phytohormone signalling will open up new opportunities for
genetic improvement ofcrops which, in the face of climate
change, can meet and fulfil the food demands of an expanding
population in future, thereby ensuring food security.
Acknowledgements Financial assistance from Science and Engi-
neering Research Board, Government of India through the grant
[EMR/2016/004799] and Department of Higher Education, Science
and Technology and Biotechnology, Government of West Bengal,
through the grant [264(Sanc.)/ST/P/S&T/1G-80/2017] to Prof. Ary-
adeep Roychoudhury is gratefully acknowledged.
Author contributions PG drafted the manuscript. AR critically
reviewed the manuscript, incorporated necessary modifications, pro-
vided valuable suggestions and supervised the overall work.
Declarations
Conflict of interest The authors declare that there is no conflict of in-
terest in publishing this manuscript.
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