Salicylic acid accumulation: emerging molecular players and novel perspectives on plant development and nutrition

Abstract

Salicylic acid (SA) is a central phytohormone that orchestrates genetic and physiological responses involving defense mechanisms against pathogens. This review presents cutting-edge research on emerging molecular players identified within the past 5 years contributing to SA accumulation. Furthermore, we delve into two relatively underexplored domains: the dynamic production of SA throughout the plant life cycle, with a specific focus on senescence, and the intricate interplay between SA, nutrition, and its multifaceted implications on plant development and defense response. This synthesis aims to provide a contemporary and comprehensive understanding of the diverse roles of SA in plant biology.

Recent research on endogenous production of SA in plants is reviewed, emphasizing newly described molecular players. We delve into how developmental and nutritional cues play pivotal roles in SA biosynthesis regulation and accumulation.

Introduction

Salicylic acid (SA) stands as a pivotal phytohormone in plant pathology, primarily recognized for its crucial role in defense against pathogens. When a plant detects a potential threat, it triggers an increase in SA accumulation, activating genetic programs and physiological responses in order to display a defense response to control proliferation of the pathogen, restricting disease progression (Peng et al., 2021). Pharmacological or genetic disruption of SA accumulation results in a deficient defense response against a wide variety of pathogens and other stressful conditions. On the other hand, plants that hyperaccumulate SA display a resistance phenotype but this is often accompanied by a reduction in plant fitness, affecting developmental programs (van Butselaar and Van den Ackerveken, 2020) and generating detrimental phenotypes such as dwarfism or premature senescence (Chan, 2022).

The hormone has been studied from different points of view, and interesting recent reviews have been released exploring its biosynthesis mechanisms (Peng et al., 2021; Lim, 2023; Ullah et al., 2023), its involvement in the response against pathogens (Zhang and Li, 2019; Ding and Ding, 2020; Bauters et al., 2021), its participation in abiotic stress (Castroverde and Dina, 2021; Sangwan et al., 2022; Rossi et al., 2023; Yang et al., 2023), its involvement in plant interaction with non-pathogenic organisms (Benjamin et al., 2022), its intricate interplay with other phytohormones (Hou and Tsuda, 2022; Kaya et al., 2023), and oxidative stress (Herrera-Vásquez et al., 2015; Saleem et al., 2021; Kohli et al., 2022).

In this review, we embark on an in-depth exploration of facets that have yet to receive extensive scrutiny: the production of SA throughout the plant life cycle in non-stressed conditions with a special emphasis on senescence. Furthermore, we aim to unravel the complex interplay between two main nutritional elements nitrogen and sulfur, SA production, and the multifaceted actions of this hormone. Additionally, we turn our attention to the latest advancements in understanding the molecular determinants driving SA accumulation, incorporating it into the classically described pathways, and the breakthroughs achieved within the past 5 years. By delving into these uncharted territories, we aspire to provide a comprehensive and contemporary perspective on the roles and regulation of SA in plant development and physiology.

Emerging regulators: unveiling new molecular contributors to salicylic acid accumulation

Under normal conditions, SA concentrations remain low but detectable. However, these levels increase when plants detect some environmental conditions, such as microorganisms acting as pathogens (Peng et al., 2021), abiotic stress such as UV light (Yalpani et al., 1994), elevated CO₂ (Mhamdi and Noctor, 2016), nutritional deficit (Criollo-Arteaga et al., 2021; Xing et al., 2023), and even clues that trigger developmental programs such as senescence (Morris et al., 2000; Zhang et al., 2017). SA accumulation under biotic stress is one of the main fields of study, though details of this complex regulation network could cast light on the mechanisms involved in other cellular processes. The primary source of SA in response to pathogens is the iso-chorismate (IC) pathway (Wildermuth et al., 2001), although, depending on the plant species, the phenylalanine ammonia-lyase (PAL) pathway may also contribute significantly (Ullah et al., 2023).

Several key proteins have been identified as central players in the IC pathway (Fig. 1). CAM-BINDING PROTEIN 60-LIKE G (CBP60g) and SAR DEFICIENT 1 (SARD1) (Zhang et al., 2010a) serve as master transcriptional regulators controlling the expression of ISOCHORISMATE SYNTHASE 1 (ICS1) (Zhang et al., 2010a), which encodes the enzyme responsible for catalyzing the conversion of chorismate into IC (Garcion et al., 2008). Additionally, CBP60g and SARD1 positively regulate the expression of ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5) (Sun et al., 2015), a gene coding for a transporter that facilitates the movement of IC from the chloroplast to the cytoplasm (Peng et al., 2021). Once in the cytoplasm, IC undergoes a two-step conversion to SA. First, avrPphB SUSCEPTIBLE 3 (PBS3), which is also positively regulated by SARD1 and CBP60g (Sun et al., 2015), catalyzes the conversion of IC to IC-9-glutamate (IC-9G), which can then spontaneously convert into SA or be further transformed into SA by action of the ENHANCED PSEUDOMONAS SUSCEPTIBILTY 1 (EPS1) enzyme (Rekhter et al., 2019; Torrens-Spence et al., 2019).

Fig. 1.

Regulatory network of salicylic acid (SA) biosynthesis. The figure depicts the well-known iso-chorismate (IC) pathway of SA synthesis, alongside the less extensively studied beta-oxidation and phenylalanine lyase (PAL) pathway. It illustrates the recently described genetic regulation controlling these pathways. The evidence supporting these interactions ranges from direct evidence including genetic studies, biochemical assays, and transcriptomic analyses. It should be noted that while the figure incorporates evidence from various species, specific regulatory interactions may vary among different plant species. The most highly connected nodes, the ICS1, EDS5, and SARD1 genes, underscore their crucial roles in SA synthesis and regulation. ICS1 and EDS5 are the rate-limiting enzymes of SA biosynthesis in the IC pathway, which is the main pathway in Arabidopsis thaliana, while SARD1 is a key regulator of the network. The abbreviations of each molecular factor are described in the main text. Continuous lines indicate a regulation tested by direct evidence, while segmented lines indicate inferred information. Blank lines indicate the biochemical flux for SA production, the green lines indicate a regulatory relationship, and gray lines connect the genes and their products.

In recent years, additional participants have been identified as regulators within the SA biosynthesis pathway. These regulators exert their influence by either directly impacting the master regulators or by affecting other different components of the pathway. Consequently, they play a crucial role in finely tuning the accumulation of SA, adding a layer of complexity to the regulatory network.

In this section, we will review recent literature that describes these components, with a primary focus on the regulation of the key players involved in SA accumulation (Fig. 1).

Controlling the controllers: CBP60g and SARD1 regulation

In Arabidopsis, the CBP60 protein family is composed of eight genes coding for transcription factors that act as positive and negative regulators of plant immunity (Truman et al., 2013). CBP60g and SARD1 are two closely related members of this family (Truman et al., 2013). They are induced by pathogens and pathogen-associated molecular patterns (PAMPs), and are known as positive regulators of SA biosynthesis (Zhang et al., 2010a) by regulating the expression of several genes that participate in SA production (Sun et al., 2015). The CBP60g gene has been identified as a direct target of the transcription factor Trihelix GT2-like 1 (GTL1), which was initially described as a regulator of the cell cycle in trichomes (Breuer et al., 2009). Additionally, GTL1 binds to the EDS5 promoter, exerting control over their expression under basal conditions (Fig. 1; Völz et al., 2018). Furthermore, GTL1 contributes to up-regulating ICS1 expression in response to the elicitor Flagelin-22 (flg22). As a result, gtl1 mutant plants exhibit increased susceptibility compared with wild-type plants when challenged with the pathogen Pseudomonas syringae pv. maculicola (Psm) due to impaired SA accumulation (Völz et al., 2018). In contrast, CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) exerts direct transcriptional repression on CBP60g (Fig. 1; Sun et al., 2020). Along with its family members, CAMTA1 and CAMTA2, CAMTA3 was identified years ago as a mediator between Ca²⁺ signaling and the negative regulation of SA accumulation (Du et al., 2009; Kim et al., 2013). CAMTA3 also regulates SARD1 expression. Nevertheless, ChIP assays suggest that, unlike the direct control on CBP60g, CAMTA3 indirectly suppresses SARD1 expression (Sun et al., 2020). Moreover, CAMTA3 is implicated in the modulation of pipecolic acid (Kim et al., 2020), a crucial molecule for basal immunity and systemic acquired resistance that acts in concert with SA (Hartmann and Zeier, 2019).

The SARD1 gene is a key player in the finely tuned regulation of SA accumulation, with several proteins contributing to its control. Among these, WRKY DNA-BINDING PROTEIN 70 (WRKY70) binds to the promoter of SARD1 and basally represses its expression (Fig. 1; M. Zhou et al., 2018). This suppression is not observed in plants challenged with pathogens such as Psm (M. Zhou et al., 2018); nevertheless, WRKY70 together with WRKY54 act as positive regulators of SARD1 and also CBP60g in an ICS1-dependent manner when the defense response is activated (S. Chen et al., 2021).

The basic leucine zipper (bZIP) transcription factor TGACG SEQUENCE-SPECIFIC BINDING PROTEIN 1 (TGA1) also plays a pivotal role by directly binding to the SARD1 promoter, thereby positively regulating its expression (Fig. 1; Sun et al., 2018). In Arabidopsis thaliana, TGA1, along with TGA4, belongs to clade I of the TGA protein family (Gatz, 2012), known for its involvement in the defense response, as it can interact with the SA receptor and transcriptional co-activator NONEXPRESSER OF PR GENES 1 (NPR1), an essential mediator of the downstream genetic response to SA (Després et al., 2003; Lindermayr et al., 2010). Notably, the tga1/tga4 double mutant plants accumulate significantly less SA than single tga1 or tga4 mutants and wild-type plants in response to Psm, indicating that these proteins are positive regulators of SA accumulation (Sun et al., 2018). Additionally, TGA1 and TGA4 are required for the PAMP-triggered immunity-mediated induction of CBP60g, SARD1, and their target ICS1 (Sun et al., 2018). In Solanum tuberosum (potato) plants, the transcription factor StbZIP61 contributes to the defense response against Phytophthora infestans (Zhou et al., 2018). StbZIP61 binds to the StICS1 promoter, enhancing its expression. This positive regulation is inhibited by the NPR1-LIKE PROTEIN 3 (StNPR3L) in an SA-dependent manner, which physically interacts with StbZIP61 (Fig. 1). The authors suggest that StbZIP61 might regulate SA synthesis in potato plants similarly to how SARD1 and CBP60g do in Arabidopsis (Zhou et al., 2018).

AtTGA2 is another bZIP transcription factor that binds to the SARD1 promoter under basal conditions (Fig. 1; Ding et al., 2018). TGA2 belongs to the clade II TGA protein family in Arabidopsis, which also includes TGA5 and TGA6 (Gatz, 2012). These proteins act as repressors of SA accumulation in the defense response triggered by the avirulent Pseudomonas syringae pv. tomato (Pst) AvrRPM1 (Fonseca et al., 2022). Triple tga256 mutant plants exhibit increased expression of SA biosynthetic genes, including SARD1, ICS1, EDS5, and PBS3, and consequently an elevated SA accumulation in response to UV-C radiation and Pst AvrRPM1 infection (Fonseca et al., 2022). These phenotypes in tga256 plants are reversed by the expression of TGA2 (Fonseca et al., 2022). In rice, OsTGA5 is the closest homolog of AtTGA2. Loss-of-function mutations in OsTGA5 lead to increased SA accumulation and up-regulation of defense-related genes compared with wild-type plants in response to the pathogen Magnaporthe oryzae (Niu et al., 2022). These findings suggest that the repressive effect of TGA2 on SA accumulation is a conserved function. It has been proposed that TGA2-mediated negative regulation of SA accumulation occurs through the transcriptional repression of SARD1 by TGA2 in conjunction with the transcriptional co-regulator NPR1-LIKE PROTEIN 4 (NPR4) in Arabidopsis (Ding et al., 2018). In rice, the SA accumulation in leaves mainly depends on the PAL pathway (Xu et al., 2023), so the specific molecular mechanism of the negative regulation of AtTGA2/OsTGA5 on SA accumulation remains unclear.

CBP60b, a member of the same protein family as CBP60g and SARD1, also plays a regulatory role in SARD1 expression (W. Huang et al., 2021; Li et al., 2021). This regulation was described by using mutant plants lacking the CBP60g gene and the autoimmune mutant suppressor of npr1 constitutive-2 (snc2-1), which possesses a gain-of-function mutation in the SNC2 gene coding for a pattern recognition receptor belonging to the receptor-like protein (RLP) family (Zhang et al., 2010b). The double mutant snc2-1/cbp60g displays elevated SA concentrations (W. Huang et al., 2021). Notably, the cbp60b mutation in the snc2-1/cbp60g genetic background suppresses this characteristic increase of the hormone (W. Huang et al., 2021). Additionally, ChIP and transcriptional reporter assays show the SARD1 promoter as a target of CBP60b (Fig. 1; W. Huang et al., 2021). Likewise, NAC-LIKE ACTIVATED BY AP3/PI (AtNAP) positively regulates SARD1 expression and binds to its promoter (Fig. 1; Wang et al., 2022). Its binding site is essential for SARD1 induction. Consequently, atnap mutant plants accumulate less SA compared with wild-type plants, while gain-of-function mutations lead to an increase in the hormone concentration (Guo and Gan, 2006; Wang et al., 2022). These SA differences observed in knockout and overexpressor plants are displayed in senescent but not in young leaves. Notably, atnap mutant plants exhibit bacterial growth comparable with wild-type plants when challenged with Pst, indicating that the AtNAP gene does not significantly contribute to the defense response against pathogens (Wang et al., 2022). Remarkably, AtNAP not only positively regulates SA accumulation but is also induced in response to the hormone, participating in a positive feedback regulatory loop of SA accumulation, particularly in late developmental stages (Guo and Gan, 2006; Wang et al., 2022).

The regulation of SARD1 is not limited to transcriptional control. SMALL AND GLOSSY LEAVES1 (SAGL1) is a Kelch repeats-containing F-box (KFB) protein that mediates the proteolytic turnover of enzymes through the ubiquitin–26S proteasome system (K. Yu et al., 2022). SAGL1 physically interacts with SARD1, inducing its proteasome-mediated degradation (Fig. 1; K. Yu et al., 2022). Consequently, sagl1 mutant plants accumulate more SA under basal and defense-induced conditions (K. Yu et al., 2022). In rice, the CASEIN KINASE 2 (CK2) protein phosphorylates OsTGA5, mitigating its inhibitory effect on defense-responsive genes (Niu et al., 2022). Likewise, in Arabidopsis, TGA2 is phosphorylated by CK2 (Fig. 1), affecting its DNA binding capacity (Kang and Klessig, 2005). Furthermore, the BRASSINOSTEROID-INSENSITIVE 2 (BIN2) protein, known for its role as a suppressor of the brassinosteroid response (He et al., 2002), interacts with and phosphorylates TGA4 (Fig. 1), modulating the SA downstream response (Y.-W. Kim et al., 2022). Given the positive contribution of TGA4, along with TGA1, in SA accumulation through the regulation of SARD1 (Sun et al., 2018), it is plausible that BIN2 indirectly influences SA accumulation through post-translational modification of TGA4 participating in the growth–defense trade-off (Y.-W. Kim et al., 2022). Similarly, the phosphorylation of OsTGA5/AtTGA2 by OsCK2/AtCK2 could disrupt the repressive function of these transcription factors on SA accumulation, although further research is needed to validate this hypothesis.

Controlling iso-chorismate biosynthesis: ICS1 regulation

The ICS1 gene represents another crucial checkpoint in SA accumulation. In Arabidopsis, the absence of ICS function significantly suppresses SA accumulation by ~90% in response to pathogens (Wildermuth et al., 2001). Alongside the previously mentioned control mechanisms over the ICS1 gene involving SARD1 and CBP60g, NAC DOMAIN CONTAINING PROTEIN 3 (NAC3) transcription factor also contributes to SA accumulation by directly and positively affecting ICS1 regulation (Fig. 1; Liu et al., 2022). NAC3 is an H₂O₂-responsive gene known to participate in Arabidopsis senescence processes (Balazadeh et al., 2011). Plants lacking this protein exhibit reduced SA accumulation and consequently display increased susceptibility to the avirulent Pst AvrRPT2 (Liu et al., 2022). Furthermore, SALICYLIC ACID BIOGENESIS CONTROLLER 1 (SABC1), a long non-coding RNA (lncRNA), represses the basal expression of the NAC3 transcription factor by modulating the H3K27 methylation status on the NAC3 promoter (Fig. 1; Liu et al., 2022). When plants activate the defense against pathogens, SABC1 is down-regulated, increasing NAC3 expression (Liu et al., 2022). Consequently, the indirect regulation of ICS1 by SABC1, mediated through epigenetic repression of NAC3, plays a significant role in controlling SA accumulation and its downstream defense responses (Liu et al., 2022).

AtWRKY42 is another recently described transcription factor that positively controls the expression of ICS1 (Fig. 1; Niu et al., 2020). ChIP assays showed that it binds to the ICS1 and the PBS3 promoter (Niu et al., 2020). wrky42 mutant plants accumulate significantly less SA compared with wild-type plants (Niu et al., 2020). Conversely, the transgenic up-regulation of WRKY42 elevates the transcript abundance of SARD1, CBP60g, ICS1, PBS3, and EDS5 genes, resulting in increased SA accumulation (Niu et al., 2020). WRKY75 is a different WRKY protein that also positively regulates ICS1 expression (Fig. 1; Guo et al., 2017). Its binding to the ICS1 promoter was demonstrated using EMSAs, reporter transactivation assays, and also by ChIP-qPCR (Guo et al., 2017). In contrast to the positive regulation of these WRKY proteins, the transcription factor MYB DOMAIN PROTEIN 59 (MYB59) plays a suppressive role in SA accumulation (He et al., 2023). MYB59 also binds to the ICS1 and PAL2 promoters (Fig. 1), but reporter assays suggest a repressive transcriptional activity on its target genes (He et al., 2023). A similar effect was described for the transcription factor NAC DOMAIN CONTAINING PROTEIN 90 (ANAC090), which also binds to the ICS1 promoter (Fig. 1; Kim et al., 2018). Both myb59 and anac090 mutant plants exhibit higher SA levels under basal conditions compared with wild-type plants (Kim et al., 2018). Moreover, MYB59 transcript is up-regulated by SA in leaves, which suggests a feedback control mechanism orchestrating the hormone accumulation (He et al., 2023). Notably, the MYB59 SA-dependent induction is not observed in other tissues, such as roots (Wiśniewska et al., 2021), suggesting differential tissue-specific regulatory mechanisms, as has been described for development-associated hormones such as auxins and cytokinins (Wong et al., 2023). These findings complement our understanding of SA regulation by adding to the previously described controllers such as NTM1-LIKE 9 (NLT9), CCA1 HIKING EXPEDITION (CHE) (Zheng et al., 2015), and NPR3/4 (Ding et al., 2018), whose functions in modulating SA accumulation have been reviewed in earlier studies.

ICS1 is also post-translationally regulated. PROHIBITIN 3 (PHB3) controls SA accumulation by regulating the stability of the ICS1 protein (Fig. 1; Seguel et al., 2018). In addition to its presence in mitochondria (Van Aken et al., 2007), PHB3 localizes in the chloroplast and interacts with ICS1. phb3 mutant plants display reduced SA accumulation in response to UV-C radiation and infection with an avirulent strain of Pst (Seguel et al., 2018). These differences were not accompanied by changes in the ICS1 transcript, suggesting a mechanism of SA accumulation regulation mediated by ICS1 protein stabilization (Seguel et al., 2018).

Regulation of the PAL pathway

In plant species such as rice and tobacco, the PAL pathway plays a significant role in basal and pathogen-induced SA accumulation (Pallas et al., 1996; Tonnessen et al., 2015), where several proteins have been identified as regulators of this specific SA biosynthetic pathway.

In rice, knockout mutant plants for the OsMYB30 transcription factor show a decrease in SA accumulation, ultimately enhancing disease symptoms in response to the insect brown planthopper (Nilaparvata lugens). OsMYB30 exerts positive control over the expression of OsPAL6 and OsPAL8 through direct binding to their promoters (Fig. 1). This positive regulation supports SA accumulation mediated by the PAL pathway in a MYB30-dependent manner (He et al., 2020).

In tobacco, the hypersensitivity-related (HSR) genes HSR201 and HSR203J encode a benzoyl-CoA:benzyl alcohol benzoyl transferase (BEBT) and an enzyme with esterase activity, respectively (Baudouin et al., 1997; D’Auria et al., 2002). NbSARD1 and NbCBP60g transcriptionally control these genes, and their silencing compromises SA accumulation in response to the fungal elicitor INF1 (Baudouin et al., 1997; D’Auria et al., 2002). Although the exact biochemical reactions by which these proteins contribute to SA accumulation remain unknown, comparative analysis with other species suggests that HSR201 may act downstream of peroxisomal β-oxidation, converting benzoyl-CoA into an intermediate of SA biosynthesis that enters into the PAL biosynthetic pathway (Takagi et al., 2022). Accordingly, in rice, ABNORMAL INFLORESCENCE MERISTEM (AIM1)-dependent β-oxidation is critical for the basal accumulation of SA (Xu et al., 2023), since aim1 mutant plants accumulate only 10% of SA in basal conditions compared with wild-type plants (Xu et al., 2017).

The E2F transcriptional repressor DP-E2F-LIKE 1 (DEL1) also negatively regulates SA accumulation. DEL1 binds to the EDS5 promoter (Fig. 1), repressing its expression under basal conditions, thereby restricting SA accumulation in leaves (Chandran et al., 2014). Additionally, SA accumulation in roots increases in del1 mutant plants in response to the pathogen Meloidogyne incognita (Nakagami et al., 2020). Accordingly, lignin levels and transcripts associated with lignin biosynthesis are elevated (Nakagami et al., 2020). Among the up-regulated genes, PAL1 and PAL2 have been identified as players in PAL-mediated SA biosynthesis (Huang et al., 2010; Nakagami et al., 2020). Thus, DEL1 modulates basal SA accumulation through EDS5 repression in leaves and may also contribute to modulating pathogen-triggered SA accumulation by affecting the PAL pathway in roots (Huang et al., 2010). The transcription factor VqWRKY31 from Vitis quinquangularis also positively regulates SA accumulation (Yin et al., 2022). Its overexpression up-regulates the expression of SA-responsive genes and promotes the accumulation of resistance-related secondary metabolites such as stilbene and flavonoid derivatives from the PAL pathway, contributing to the resistance to the biotrophic fungus Erysiphe necator (Yin et al., 2022).

Other regulators of SA biosynthesis

The transcriptional Mediator complex also contributes significantly to SA accumulation. This multiprotein complex bridges transcription factors and RNA polymerase II (Mathur et al., 2011). Among its components, cyclin-dependent kinase 8 (CDK8) from the kinase module of the complex has garnered attention (Huang et al., 2019). Mutations in the CDK8 gene lead to compromised basal expression of ICS1 and EDS5, resulting in reduced basal SA levels compared with wild-type plants (Fig. 1; Huang et al., 2019). Interestingly, defense responses against Hyaloperonospora arabidopsidis and Psm are also compromised in these mutants (Huang et al., 2019). Surprisingly, the SA accumulation in cdk8 mutants remains unaltered when challenged with Psm compared with wild-type plants, suggesting that CDK8 positively regulates SA accumulation only under uninfected conditions (Huang et al., 2019). Additionally, mutations in the MEDIATOR 12 (MED12) gene, encoding another subunit of the Mediator complex, yield phenotypes akin to cdk8 mutants, upholding the complex’s positive role in regulating SA accumulation and basal defense (Huang et al., 2019). This is not the only MED gene implicated in SA-mediated defense. MED14, MED15, and MED16 were previously described as having a role as positive regulators of SA accumulation and downstream signaling (Canet et al., 2012; Wathugala et al., 2012; Zhang et al., 2012, 2013b). Further expanding our understanding of the Mediator complex in SA regulation, recent studies have shown that GUANYLATE BINDING PROTEIN-LIKE 3 (GBPL3), a key component involved in Arabidopsis immunity, interacts with MED15, MED16, CDK8, and other MED subunits (S. Huang et al., 2021). The recruitment and binding of GBPL3 to these subunits and the CBP60g promoter highlight its role in modulating SA accumulation (Fig. 1; J.H. Kim et al., 2022).

Cysteine-rich receptor-like kinases (CRKs) are integral components of the plant signaling network, facilitating communication between cells and their external environment. Typically located in the plasma membrane, CRKs possess extracellular domains capable of recognizing PAMPs and other stimuli, initiating signaling cascades that govern the cell’s response (Zeiner et al., 2023). Several CRK proteins have been described as defense regulators (Zeiner et al., 2023), and some of them in particular as players in SA accumulation (Acharya et al., 2007; Zhang et al., 2013a). Recently, CRK5 and CRK22 were identified as new regulators of SA accumulation and signaling in response to Verticillium dahliae toxins in Arabidopsis (Zhao et al., 2022). CRK5- and CRK22-deficient plants exhibit diminished SA levels and fail to induce SA-responsive genes (Zhao et al., 2022). This effect can be attributed to their potential involvement upstream of the MITOGEN-ACTIVATED PROTEIN KINASE 3 and 6 (MPK3/6) proteins, which are positively regulated by the CRKs (Zhao et al., 2022). Furthermore, MPK3/6 interact with the transcription factor WRKY70, resulting in the positive regulation of the expression of TGA2 and TGA6 (Fig. 1; Zhao et al., 2022) and possibly SARD1 and CBP60g (S. Chen et al., 2021).

The involvement of certain genes in SA accumulation has yielded conflicting results. A notable example is the ZINC TRANSPORTER OF ARABIDOPSIS THALIANA 1 (ZAT18) gene (Gao et al., 2022; W. Li et al., 2022). This C2H2 transcription factor harbors a functional EAR motif known as a transcriptional repressor module (Kagale and Rozwadowski, 2011). According to Gao et al. (2022), ZAT18 is induced by Pst to suppress the SA-mediated defense response in a MYC2-dependent manner. ZAT18 induction directly represses EDS1 expression, subsequently reducing SA accumulation. Conversely, W. Li et al. (2022) demonstrate that the zat18 mutation leads to the down-regulation of SA signaling transcripts, increasing susceptibility in response to Psm ES4326 (W. Li et al., 2022). The differing conclusions between these research groups may stem from variations in the Pseudomonas strains used in the respective studies and differences in infection methods; for instance, W. Li et al. (2022) conducted infections by spraying the Psm ES4326 strain, whereas Gao et al. (2022) performed direct leaf infiltration with Pst. The apparently contradictory result suggests a differential mechanism contingent on the attacking pathogen.

While several genes play significant roles in SA accumulation, the intricate molecular mechanisms underlying some of their functions justify further investigation. For instance, APETALA2/ETHYLENE RESPONSIVE FACTOR in cotton (Gossypium hirsutum), GhTINY2, has emerged as a positive regulator of SA accumulation, whose up-regulation bolsters resistance against the pathogenic fungus V. dahliae (Xiao et al., 2021). To dissect the molecular basis of this phenotype, researchers demonstrate that in a heterologous system, GhTINY2 exerts positive control over the expression of the Arabidopsis gene WRKY51, a positive transcriptional regulator of SA accumulation (Gao et al., 2011). This suggests a potential role for GhTINY2 in regulating the expression of the Arabidopsis homolog GhWRKY51 in cotton, thus influencing SA accumulation (Fig. 1; Xiao et al., 2021).

ACT DOMAIN REPEATS 11 (ACR11) is a chloroplast protein that lacks any known catalytic domain (Singh et al., 2018). Interestingly, mutations in this gene lead to a mimic-lesion phenotype characterized by an increase in reactive oxygen species (ROS) levels, elevated SA concentrations, and the activation of SA downstream genes. Consequently, acr11 mutant plants exhibit enhanced resistance to Pst (Singh et al., 2018). These results support the link between the imbalance of ROS in chloroplast signaling and SA accumulation (Bali et al., 2023).

Another intriguing example is the PLANT NATRIURETIC PEPTIDE A (PNP A), which was identified as a negative regulator of the SA response (Lee et al., 2020). PNP A is secreted into the apoplastic space, where it interacts with its putative receptor PNP-R2 (Fig. 1), thereby suppressing SA signaling in the lesion simulating disease 1 (lsd1) mutant background, known for increased SA accumulation in response to stress (Lv et al., 2019). Notably, the pnp-a mutation in the lsd1 mutant background amplifies SA accumulation under light stress compared with the single lsd1 mutant. Conversely, overexpression of PNP-A suppresses SA accumulation (Lee et al., 2020). Moreover, PNP-A is transcriptionally up-regulated in response to various stress conditions, encompassing both biotic and abiotic stress sources (Lee et al., 2020). This intriguing finding suggests that this gene might exert regulatory control over plant physiological responses that involve SA accumulation facing a spectrum of environmental factors (Lee et al., 2020). Nevertheless, the downstream mechanisms following PNP-A perception remain a subject of ongoing investigation.

In summary, elucidating the intricate network of molecular participants in SA biosynthesis provides a foundational understanding of how plants modulate accumulation of this phytohormone to orchestrate diverse aspects of plant physiology, particularly in stress response. Beyond its established role in defense against pathogens, SA has emerged as a multifaceted regulator with far-reaching implications in plant biology. Subsequent sections of this review will explore intersections between SA accumulation and plant development, exploring their effects on processes such as floral transition and senescence (Fig. 2). Additionally, we will describe the dynamic interplay between plant nutrition and SA accumulation, shedding light on how nutrient availability influences SA biosynthesis and, reciprocally, how SA modulates nutrient utilization. Through these discussions, we aim to provide a comprehensive perspective on the pivotal role of SA in shaping the intricate tapestry of plant growth, development, and adaptation.

Fig. 2.

Regulatory network of salicylic acid (SA) production in senescence. This scheme illustrates the relationship between SA, oxidative status, growth, and senescence, along with key molecular players. The main points depicted include the gradual increase of SA from flowering time onwards, regulated by the feedback loop of WRKY75 and the IC pathway. Additionally, it highlights changes in oxidative status indicated by reactive oxygen species (ROS) accumulation and the GSH/GSSG index. Moreover, it illustrates the suppression of gibberellin signaling leading to growth inhibition and the irreversible progression towards senescence mediated by the feedback loop of WRKY46 and associated genetic reprogramming. These effects collectively represent the outcomes of SA signaling. Lines connecting nodes indicate regulation, while black arrows indicate activation and red lines indicate repression.

A life without stress: SA production in plant development

Several studies have explored the effects of SA on plant development in different species. Typically, these studies employ models featuring stress-induced SA accumulation, mutant plants with altered SA production, or exogenous SA treatments (Xie et al., 2007; Alonso-Ramírez et al., 2009; Rivas-San Vicente and Plasencia, 2011; Xu et al., 2017; Pasternak et al., 2019). Under these experimental conditions, SA has been positively and negatively related to cell division and expansion processes, also acting in a cross-regulation with other hormones canonically associated with development (Rivas-San Vicente and Plasencia, 2011; Bagautdinova et al., 2022; A. Li et al., 2022). This is the case of the recently described SA inhibition of gibberellic acid (GA) signaling through the NPR1-mediated GA INSENSITIVE DWARF 1 (GID1) degradation, which modulates plant growth (Yu et al., 2022).

SA concentration in non-stimulated plants varies according to the plant species, even though external stimuli can induce changes in the SA accumulation in all of them, suggesting different functions and SA sensitivity in plant development. For example, compared with Arabidopsis which possesses 1 µg SA g^–1 fresh tissue in basal conditions (Wildermuth et al., 2001; Mateo et al., 2006), plants such as rice and potato possess up to one order of magnitude more, reaching 15 µg SA g^–1 fresh tissue (Coquoz et al., 1998; Pál et al., 2020). These differences make establishing a general rule of SA effects on non-stressed plants difficult, considering contrasting developmental effects depending on SA concentration (Pasternak et al., 2019).

Throughout Arabidopsis development, there is an increase in SA accumulation in leaves, particularly during the floral transition, reaching approximately twice the amount compared with the early developmental stages (2 µg SA g^–1 fresh weight) (Abreu and Munné-Bosch, 2009). This increase depends on the IC pathway, which is also responsible for the SA accumulation in seeds where wild-type plants reach 0.3 µg g^–1 seed (Abreu and Munné-Bosch, 2009). The deficit in SA accumulation positively influences the plant biomass and the number of siliques, seeds, and shoots (Abreu and Munné-Bosch, 2009), and delays flowering induction in Arabidopsis (Martínez et al., 2004). Accordingly, applying the pharmacological inhibitor of the PAL pathway, aminooxyacetic acid (AOA), reduces floral induction by low nutrient availability in Pharbitis nil (Wada et al., 2010). At the same time, the use of AOA in combination with SA restores the normal phenotype (Wada et al., 2010). These results suggest SA participation in floral induction. This hypothesis is also supported by evidence showing that exogenous application of SA induces flowering in Arabidopsis (Martínez et al., 2004) and other plant species such as duckweed (Fu et al., 2020), apple (Shah et al., 2022), and saffron (Rastegari et al., 2022). On the other hand, SA accumulation, which is induced in long-day conditions in Chenopodium ficifolium plants, does not correlate with flowering time, that is induced under short-day conditions where SA is not detected in the same species (Gutierrez-Larruscain et al., 2022). This effect could be explained by the interaction of SA with other phytohormones, such as abscisic acid (ABA), which is also induced in long-day conditions (Gutierrez-Larruscain et al., 2022) and blocks SA-triggered flowering (Fu et al., 2020).

Interestingly, in wild-type Arabidopsis plants, flowering time coincides with a phenomenon known as age-related resistance (ARR) (Rusterucci et al., 2005). ARR refers to increased plant resistance against pathogens at an advanced ontological age (reviewed in Hu and Yang, 2019). Even though the increase in SA accumulation and the onset of ARR correlates with the flowering transition, mutation on genes that influence flowering time, such as CONSTANCE 9 (CO9) or FLOWEING LOCUS T (FT), does not affect ARR (Lyons et al., 2015; Gutierrez-Larruscain et al., 2022), indicating that floral transition is not directly linked to the onset of ARR. However, the SHORT VEGETATIVE PHASE (SVP) gene controls ARR in leaves by repressing the expression of SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1; Wilson et al., 2017) whose particular expression in the shoot apical meristem initiates flowering by activating LEAFY (LFY), a master regulator of the process (Lee et al., 2008; Liu et al., 2008; Siriwardana and Lamb, 2012). Thus, while the SVP gene molecularly links the phenomena of flowering and ARR, its tissue-specific regulation functionally separates them. ARR requires SA accumulation (Kus et al., 2002), but it acts in an NPR1-independent fashion since npr1-1 mutant plants display age-dependent resistance similar to wild-type plants (Kus et al., 2002; Cameron and Zaton, 2004). The evidence indicates that the apoplastic SA acts as a molecule with antibiotic activity itself, affecting the proliferation of microorganisms (Cameron and Zaton, 2004).

SA has also been implicated in pre- and post-harvest traits in fruit (Chen et al., 2023). These relationships have been primarily explored through exogenous applications of SA (Chen et al., 2023). A recent study, however, revealed a correlation between endogenous SA accumulation in fruit and post-harvest maturation traits, including ethylene evolution and extended shelf life (Changwal et al., 2021). These data suggest the hormone’s involvement in this process. Nevertheless, further research is required to definitively attribute these post-harvest traits to SA.

A natural developmental stage in which SA accumulation has been extensively studied and represents a pivotal event is senescence (Morris et al., 2000; Zhang et al., 2017). Senescence in plants involves the execution of a genetically programmed process that possesses essential functions such as nutrient recycling and energy conservation (Woo et al., 2019). Various internal and external factors can influence this process, including hormonal regulation and environmental conditions (Woo et al., 2019). SA accumulation contributes to the expression of senescence-associated genes (SAGs) and phenotypes such as chlorophyll catabolism (Morris et al., 2000). Current knowledge indicates that the SA accumulated by the IC pathway plays a substantial role in the senescence phenotype, as evidenced by delayed chlorotic symptoms in the ics1 mutant compared with Arabidopsis wild-type plants (Morris et al., 2000; Guo et al., 2017). Accordingly, transcriptional regulators such as MYB59, NAP, NAC3, and WRKY42 are positively regulated in senescent tissue, and they modulate the IC pathway as described above (Figs 1, 2) (Niu et al., 2020; Liu et al., 2022; Wang et al., 2022). Among them, the NAP transcription factor possesses the particular characteristic that seems to participate in SA accumulation in senescence but not in the defense response against pathogens (Wang et al., 2022).

The inhibition of SA accumulation, achieved through using ics1 mutant plants or applying aminobenzotriazole to block SA biosynthesis, leads to a delayed senescence phenotype (Zhang et al., 2023). A similar phenotype is observed in plants lacking the SA receptor and transcriptional co-activator NPR1, highlighting the significance of SA downstream signaling (Zhang et al., 2023). Furthermore, the SA-dependent senescence process relies on a signaling cascade mediated by the MKK4/5 and MPK1/2 proteins (J. Zhang et al., 2020), of which MPK1 phosphorylates NPR1, mediating its monomerization (J. Zhang et al., 2020) which is required for the activation of the NPR1 downstream effects (Mhamdi, 2019). Additionally, NPR1 interacts with the transcription factor WRKY46, positively regulating the expression of the WRKY46 and WRKY6 genes, thereby positively influencing leaf senescence (Fig. 2; Zhang et al., 2021). Surprisingly, unlike Arabidopsis, where the increase in SA accumulation is required for senescence, aspen trees initiate the metabolic process of senescence through enhanced SA catabolism, which implies a reduction in SA levels (Lihavainen et al., 2023). Considering these findings and others, the authors have concluded that induced and sustained SA levels can delay the onset of senescence in aspen trees under field conditions (Lihavainen et al., 2023). This contrasts with Arabidopsis and other annual species studied under controlled conditions (Lihavainen et al., 2023).

SA acts in a coordinated and synergistic manner with ethylene during the senescence process (C. Wang et al., 2021; Yu et al., 2021). This orchestrated interplay involves the interaction between NPR1 and the key transcriptional regulator of the ethylene response, ETHYLENE-INSENSITIVE3-LIKE 1 (EIL1). In the presence of SA, this interaction enhances the expression of the genes NON-YELLOWING 1 (NYE1) and ORESARA 1 (ORE1), which positively regulates developmental senescence (Fig. 2; Qiu et al., 2015; C. Wang et al., 2021; Yu et al., 2021).

The process of autophagy is notably intertwined with senescence. Plants with genetically compromised autophagy, such as autophagy 2 (atg2) and atg5 mutants, display an early senescent phenotype dependent on SA (Yoshimoto et al., 2009). Interestingly, under senescence induced by carbon starvation conditions, SA inhibits autophagy through NPR1 (Fig. 2; Zhang et al., 2023). Surprisingly, SA also promotes autophagy via NPR3/NPR4 (Wang et al., 2016). Thus, the equilibrium between these co-regulators is fundamental for the coordination between autophagy and senescence, as well as the immune response (Ding et al., 2018). It is important to consider that chlorotic phenotypes associated with senescence could be related to cell death propagation, which is well described in the defense response as a consequence of the increase in SA (Yoshimoto et al., 2009).

The signals that trigger SA accumulation in developmental- and/or carbon deprivation-induced senescence remain elusive. However, ROS emerge as an important candidate due to their close association with SA production and the cellular redox balance (Herrera-Vásquez et al., 2015; Saleem et al., 2021). In senescent tissue, a significant ROS increase is supported by a reduction in ROS-detoxifying enzyme activities, such as catalase (CAT), that correlates with a decrease in the expression of CAT-coding genes, such as CATALASE 2 (CAT2) (Zimmermann et al., 2006), that contribute to 90% of CAT activity in Arabidopsis leaves (Queval et al., 2007; Mhamdi et al., 2010). Furthermore, SA inhibits CAT2 and the total CAT activity in plant tissues (Yuan et al., 2017), providing additional evidence of its role in modulating ROS levels during senescence.

Mutation of CAT2 induces ROS accumulation and cell death, resulting in a senescent-like chlorosis phenotype dependent on the photoperiod (Queval et al., 2007; Mhamdi et al., 2010). Accordingly, cat2 mutant plants accumulate more SA compared with wild-type plants (Yuan et al., 2017) and display a light regime-dependent chlorosis that is genetically reversed by the ics1 mutation (Chaouch et al., 2010), emphasizing the pivotal role of SA in this tissue degeneration and death. Moreover, the early senescent phenotype seen in nac domain containing protein 75 (nac075) mutant plants can be rescued by CAT2 overexpression (Kan et al., 2021). Similarly, the premature senescence observed in Arabidopsis plants overexpressing the transcription factor WRKY75 is suppressed by CAT2 overexpression, which mitigates the heightened ROS accumulation resulting from WRKY75 overexpression (Guo et al., 2017). Notably, the ics1 mutation also suppresses the early senescent phenotype of WRKY75 overexpressor plants (Guo et al., 2017).

Glutathione (GSH), one of the primary redox buffers that control the ROS increase in stressed and non-stressed conditions (Dorion et al., 2021), undergoes modifications in senescent tissue (Ding et al., 2016). In standard growth conditions, older leaves exhibit a reduction in the total GSH content as well as a decreased ratio of the reduced to oxidized GSH form (GSH/GSSG ratio), indicating a shift towards a more oxidative cellular status (Fig. 2; Ding et al., 2016). Additionally, mutation in the GLUTATHIONE REDUCTASE-2 (GR2) gene, involved in converting oxidized glutathione (GSSG) to GSH, leads to premature leaf senescence (Ding et al., 2016). Despite an increased total GSH content (GSH+GSSG), these plants exhibit an enhanced and early decrease in the GSH/GSSG ratio, supporting the early senescence onset (Ding et al., 2016). These results indicate that the alteration of GSH homeostasis influences the onset of senescence. In line with this, cat2 mutant plants display an increased oxidized form of GSH compared with wild-type plants (Queval et al., 2007; Mhamdi et al., 2010). Notably, the increased GSH/GSSG ratio in the cat2 mutant is similar to the ratio in the cat2/ics1 double mutant (Chaouch et al., 2010), suggesting that the SA increase in senescent tissue is more a consequence than a cause of the redox imbalance.

SA has also been associated with root development (Xu et al., 2017; Pasternak et al., 2019; Wang et al., 2021). The root architecture is modified by SA in a concentration-dependent fashion in Arabidopsis (Pasternak et al., 2019). Additionally, SA modulates the activation of cell division in the root meristem of rice (Xu et al., 2017) and also the quiescent center in Arabidopsis (Wang et al., 2021). All these observations have been made in experiments with exogenous SA treatment. Interestingly, some evidence shows a possible modulation of SA accumulation in response to soil nutritional status. This suggests a potential bidirectional relationship between plant nutrition and SA accumulation, wherein nutritional conditions could modulate SA levels, influencing root architecture.

Additionally, nutrient imbalances profoundly impact the intricate interplay between plant growth and stress defense responses, which has been called the ‘growth–defense trade-off’ (Chan, 2022). The effects depend on each element’s concentration in the plant, which is directly linked to their availability in the soil. The plant’s nutritional status significantly influences and sometimes even defines its ability to develop and withstand adverse environmental conditions. In the following section, our search focused on uncovering updated evidence directly linking nutrient availability to the accumulation of SA. Our findings predominantly show that the synthesis and accumulation of SA are directly impacted by the nutritional status of sulfur (S) and nitrogen (N).

Have sulfur and nitrogen something to say in the SA pathway?

Sulfur stands as a crucial element that plants must assimilate. The uptake form of this element is sulfate (SO₄^2–), which is incorporated into and mobilized through the plant by specialized transporters (Takahashi, 2019). Once sulfate enters the plant cell, it undergoes a series of reduction steps, leading to the production of sulfite (SO₃^2–) and, finally, sulfide (S^2–). The reaction between S^2– and O-acetyl-serine (OAS) results in the formation of Cys, which is the final S assimilation product (Watanabe et al., 2010; Künstler et al., 2020). The reduction of SO₄^2– to S^2– occurs in plastids (mainly in the chloroplast), but the synthesis of Cys takes place in chloroplasts, mitochondria, and the cytosol (Abadie and Tcherkez, 2019; Li et al., 2020). The production of Cys plays a central and pivotal role in orchestrating the production of S-containing biomolecules. Thus, S insufficiency negatively impacts the synthesis of Cys and, as a consequence, the synthesis of GSH, methionine antimicrobial agents, and signaling peptides fulfilling essential functions in plant development, growth, and defense responses against pathogens (Krueger et al., 2010; Hu et al., 2018). The direct influence of soil sulfur on SA synthesis has been relatively underexplored. However, evidence suggests that the availability of this nutrient impacts the accumulation of SA and subsequently influences the plant’s defense response (Criollo-Arteaga et al., 2021). Supporting this idea, Arabidopsis wild-type plants grown in S deprivation showed constitutive expression of the PATHOGENESYS RELATED 1 (PR1) gene in an SA-dependent manner (Criollo-Arteaga et al., 2021). Accordingly, these plants display a resistance phenotype after the challenge with Pst DC3000, compared with plants in standard S concentration (Criollo-Arteaga et al., 2021). The opposite effect is observed in plants supplemented with S in excess (Criollo-Arteaga et al., 2021). These results show that the lack of S activates the defense response in an SA- and NPR1-dependent manner. In this study, Cys and GSH/GSSG levels were not addressed (Criollo-Arteaga et al., 2021); however, S deprivation on Brassica napus roots showed a down-regulation of genes involved in GSH metabolism, which was reflected in a decrease in the general GSH content, suggesting an uncontained ROS accumulation under S deprivation (Courbet et al., 2021). These results are consistent with the findings presented by Henriquez-Valencia et al. (2018), who analyzed transcriptomic data from several published experiments involving S treatments. They found that the most consistent biological functions differentially regulated by S treatment include the Gene Ontology classification of ‘response to biotic stimulus’ and also ‘glutathione metabolism’ along with ‘sulfate assimilation and metabolism’ as expected (Henríquez-Valencia et al., 2018). Thus, these results suggest that SA accumulation by deprivation of S might result from the imbalance of Cys and GSH, resulting in an impairment of ROS containment due to the lack of one of the main antioxidant cellular buffers (Zechmann, 2020).

An optimal balance of Cys is paramount for plant immunity, as illustrated by Álvarez et al. (2012a). Mutant plants lacking functional cytosolic l-Cys DESULFHYDRASE (DES1), an enzyme involved in the catabolism of cytosolic Cys into sulfide, ammonia, pyruvate, and H₂S, show hyperaccumulation of cytosolic Cys (Álvarez et al., 2012a, b). This accumulation is accompanied by elevated total GSH content and SA accumulation, as well as the up-regulation of PR1, WRKY54, and RPM1 genes. Consequently, this mutant displays an increased resistance to pathogen challenges (Álvarez et al., 2012a). The des1 mutant also displays an early senescent phenotype accompanied by the up-regulation of the senescence-associated transcription factor gene SENESCENCE-ASSOCIATED GENE 21 (SAG21) and the NAC transcription factor gene NAP (Fig. 1), responses which support the increase of SA concentration (Álvarez et al., 2012a). In contrast, the phytoalexin deficient 2 (pad2-1) Arabidopsis mutant accumulates five times more Cys than the wild-type plants and displays an enhanced susceptibility to Psm E4326 (Parisy et al., 2007). Although des1 and pad2-2 mutant backgrounds have higher Cys levels than wild-type plants, only des1 exhibits higher total GST (Parisy et al., 2007). PAD2 encodes a γ-GLUTAMYL-CYSTEINE SYNTHASE 1 (GSH1) enzyme, part of the GSH synthesis pathway (Parisy et al., 2007). Indeed, GSH levels correlate with plant resistance to several pathogens. For instance, the artificial increase of GSH content by overexpressing GSH1 (Ghanta et al., 2011) or by directly infiltrating GSH in tobacco plants (Künstler et al., 2019) increases the SA accumulation and shows a resistance phenotype to P. syringae pv. tabaci and tobacco mosaic virus (TMV), respectively.

GSH is also able to react with nitric oxide (NO) to produce S-nitrosogluthathione (GSNO), a crucial molecule that facilitates S-nitrosylation (SNO) on reactive Cys residues on target proteins (Lindermayr, 2018; T. Zhang et al., 2020). Regarding the SA signaling, SNO regulates NPR1, promoting its oligomeric state and retaining it in the cytosol, as well as TGA1, enhancing its DNA binding capacity (Lindermayr et al., 2010). Recently, T. Zhang et al., (2020) demonstrated that the activation of the SA pathway dependent on intracellular H₂O₂ relies on GSH to maintain the S-nitrosoglutathione reductase 1 (GSNOR1) gene up-regulated, thereby ensuring an optimal level of SNO-modified proteins.

Hydrogen sulfide (H₂S) is a gas produced by the plant when S is assimilated and, as a consequence of the excess of S fertilization, it has become a contaminant gas present in the environment (Álvarez et al., 2012a; Ausma and De Kok, 2019). Initially, it was thought that its production was the plant’s way to eliminate the excess S in their tissue. Yet, many reports in the literature support this molecule as a player in the stress defense response. This fact was primarily proposed when environmental regulations reduced H₂S emissions in Europe, and the crops started to suffer diseases caused by fungi (Sehar et al., 2021; Khan et al., 2022). There is evidence connecting H₂S and SA production; Shi et al. (2015) modified the endogenous production of H₂S in Arabidopsis by modulating the expression of l-CYSTEINE DESULFHYDRASE (LCD) and d-CYSTEINE DESULFHYDRASE 1 (DCD), encoding enzymes which participate in the H₂S synthesis pathway in Arabidopsis (Khan et al., 2022). Plants overexpressing the LCD or the DCD gene showed an increase in the endogenous concentration of H₂S and an increased resistance to Pst (Shi et al., 2015). Expression analysis showed an up-regulation in the SA-responsive genes PR1, PR3, PR2, and also EDS1 and PHYTOALEXIN DEFICIENT 4 (PAD4) genes, which participate in upstream SA signaling, suggesting an increase in SA accumulation (Shi et al., 2015). Consistently, treatment with exogenous H₂S showed improved resistance to Pst DC3000, and the lcd and dcd knockout lines showed the opposite phenotype (Shi et al., 2015).

Similarly, plants also respond to other oxidized forms of S, such as sulfur dioxide (SO₂), which is also an air pollutant derived mainly from volcanoes and the combustion of S-containing fossil fuels (Fioletov et al., 2016). SO₂-pre-treated Arabidopsis plants are more resistant to Botrytis cinerea, and PAL, PROTOPORPHYRINOGEN OXIDASE (PPO), PR2, and PR3 genes are induced, as well as their enzymatic activity, compared with unprimed plants (Fioletov et al., 2016). Although the up-regulation of these genes suggests a potential involvement of SA, the authors propose that the augmented defense response is also attributed to the repression of the auxin signaling pathway induced by SO₂ exposure (Xue and Yi, 2018). Conversely, it has been shown that Zea mays (maize) plants pre-treated with SO₂ improve the heat shock response in an SA-dependent manner (Xue and Yi, 2018). When SO₂-pre-treated plants were exposed to heat stress, the amount of ROS [H₂O₂ and malondialdehyde (MDA)] was reduced, consistent with the increased activity of antioxidant enzymes such as SUPEROXIDE DISMUTASE (SOD) and CAT, and higher levels of SA, compared with plants without SO₂ pre-treatment (Li et al., 2023). The SA accumulation correlates with the up-regulation of ZmICS1 and ZmPAL genes. Plants treated with paclobutrazol (PAC), an SA synthesis inhibitor, fail to show SO₂-induced heat tolerance (Li et al., 2023). These results agree with the well-established role for SA in improving the thermotolerance response and the role of SA in controlling the ROS burst (Rai et al., 2020; Sangwan et al., 2022). Thus, the described results suggest that Cys, H₂S, and SO₂ could prime the plant stress response through the SA signaling pathway.

The influence of nitrogen availability in the defense response to pathogens and its connection with SA accumulation is not straightforward. Whether the plant defense response improves with high or low N depends on the host, the pathogen, and even the form of N used to fertilize the plant (Ballini et al., 2013; Vega et al., 2015; Mur et al., 2017). The primary source of nitrogen in dry land is nitrate (NO₃^–), while in flooded or acidic soils, it is ammonium (NH₄⁺) (Xuan et al., 2017). The assimilation forms of N influence the defense response (Ding et al., 2021). This issue has been explored recently in tomato plants where their defense response to Pst DC3000 and Ralstonia solanacearum was analyzed using both main forms of soil N, NH₄⁺ and NO₃^–, in high and low concentrations. Tomato plants were shown to be more resistant to Pst DC3000 when nitrogen was limited, regardless of the form of N supplied (NO₃^– or NO₃^–/NH₄⁺), although plants supplied with NO₃^– alone showed more resistance than plants fed with the mix NO₃^–/NH₄⁺, when keeping the same low concentration (Ding et al., 2021). Moreover, defense in low N concentrations correlates with increased SA accumulation and up-regulation of the PAL1, PR2, and PR4 genes (Ding et al., 2021).

Nitrogen uptake from soil and its incorporation into the plant cell is mediated by transporters regulated at both transcriptional and post-translational levels (Vidal et al., 2020). NO₃^– and NH₄⁺ uptake is mediated by the nitrate peptide family/transporters (NPF/NRTs) and ammonium transporters, respectively, located in the roots (O’Brien et al., 2016). Notably, NO₃^– treatment induces NRT2.2, a high-affinity N transporter, as well as NTR1.1, a dual-affinity nitrate transporter and putative NO₃^– sensor (Vidal et al., 2020). Furthermore, NO₃^– also triggers the expression of TGA1/4 genes, which are partially responsible for the positive regulation of the transcription of both transporters. The tga1/4 double mutant plants show a differential nitrate-dependent lateral root phenotype compared with wild-type plants (Alvarez et al., 2014). Interestingly, TGA1/4 transcription factors positively regulate the expression of SARD1 and, consequently, SA accumulation (Fig. 1) (Sun et al., 2018).

Once NO₃^– is transported into the root cells, it is mobilized to the shoot and converted to NH₄⁺ by the enzymes NITRATE REDUCTASE (NR) and NITRITE REDUCTASE (NiR) (Chamizo-Ampudia et al., 2017). Both sources of NH₄⁺ (the naturally absorbed and the reduced form NO₃^–) are further assimilated into the organic repertoire by the glutamine synthase/glutamate synthase cycle (Xing et al., 2023). This cycle provides glutamine and glutamate amino acids and the initial substrates synthesizing all the N-containing biomolecules in the plant (Lam et al., 1996). Glu, a pivotal molecule primarily associated with proteins and a source of N, is directly linked to SA synthesis and response pathways (Toyota et al., 2018; Tsuruda and Yoshida, 2023). Glu and IC are substrates of PBS3 in the SA synthesis pathway to produce the intermediary IC-9G (Fig. 1). Glu is also one of the three amino acids making up GSH. Tsuruda and Yoshida (2023) demonstrated that the exogenous application of l-Glu induces the accumulation of SA and up-regulates the expression of ICS1 and PR1 genes in wild-type Arabidopsis plants. This effect is not observed in glutamate receptor-like 3 (glr3.1), npr1.1, and ics1 mutant plants (Tsuruda and Yoshida, 2023). Previous studies have shown that the ion channel glutamate receptor-like family protein is involved in Ca²⁺ signaling, suggesting that an increase in cytosolic Ca²⁺ could be involved in the Glu-induced SA accumulation (Toyota et al., 2018).

While transcriptional regulation of genes is the main mechanism for modulating transcript abundance, the alternative splicing and polyadenylation of mRNAs could be additional levels of expression modulation (Wu et al., 2023). For instance, the classical 3'-untranslated region (UTR) polyadenylation could also occur within introns, coding regions, in more than one place within the 3'-UTR and even in 5'-UTRs, a phenomenon known as non-canonical polyadenylation or alternative polyadenylation (APA) (Wu et al., 2023). APA has been reported as an important feature of plant stress response, especially associated with abiotic stress, producing truncated proteins and changes in mRNA stability (Wu et al., 2023). In their 2020 study, Conesa et al. assessed the influence of N availability on APA by using a mutant allele of the FACTOR INTERACTING WITH POLY(A) POLYMERASE 1 (FIP1) protein involved in APA (Téllez-Robledo et al., 2019; Conesa et al., 2020). Their findings in Arabidopsis plants subjected to nitrogen deficiency stress revealed significant alterations in the APA patterns of genes primarily associated with nitrogen assimilation and stress response pathways, especially in the SA signaling pathway (Conesa et al., 2020). The authors report that in plants grown under low nitrogen conditions (50 µM), SA accumulates considerably in shoots and roots, compared with plants grown under control conditions (2.5 mM nitrogen) (Conesa et al., 2020).

Further analysis of APA patterns indicated shifts in polyadenylation site choice for genes such as that encoding the SA receptor and transcriptional co-regulator NPR3 and the transcription factors that regulate the SA biosynthesis TGA5, CAMTA1, and CAMTA3 (Fig. 1; Conesa et al., 2020). The authors hypothesized that these APA changes potentially modulate the activity of these proteins, thereby influencing the accumulation of SA (Conesa et al., 2020). These findings add another regulation level in SA production, connecting SA production, N availability, development, and defense response.

The effects of N on SA accumulation have also been observed in cotton plants. In this experimental system, plants grown under N deprivation demonstrated a notable increase in SA accumulation, observed in both the primary root and lateral roots, in contrast to plants cultivated under N-sufficient conditions. Notably, an excess of N also led to SA accumulation, although this effect was less pronounced than in N-deprived plants and confined to the main root (Chen et al., 2021).

In this context, it is intriguing to highlight the involvement of SA in various developmental processes. Notably, in roots, SA plays a pivotal role in mediating the interaction between plants and soil microorganisms (Lebeis et al., 2015; Lemarié et al., 2015; Benjamin et al., 2022), but it also contributes to determining root architecture. While extensive research has investigated the effects of SA on this process, establishing a universal rule is challenging because the morphological effects depend on treatment intensity and plant species. This topic has been extensively reviewed by Bagautdinova et al. (2022). Given that plant nutrition primarily relies on acquiring molecules from the soil, modification of root architecture becomes a critical factor in optimizing nutrient uptake. Consequently, SA could play a role in shaping the plant’s nutritional status by influencing this process. This field remains relatively unexplored, presenting an intriguing and promising study area with substantial potential for future research.

Discussion

This comprehensive review on SA biosynthesis and its regulatory mechanisms in plants elucidates several important aspects of SA accumulation, revealing the intricate interplay between developmental processes, nutrient signaling, and stress responses.

One of the key themes that emerged is the accumulation of SA fulfilling different roles depending on the specific biological outcome, which can be observed particularly in defense and development. The dynamic nature of SA biosynthesis across various stages of plant development, especially during flowering (Martínez et al., 2004; Fu et al., 2020; Shah et al., 2022), senescence (Morris et al., 2000; Guo et al., 2017; Lihavainen et al., 2023), and in response to nutrient status (Conesa et al., 2020; Criollo-Arteaga et al., 2021), underscores the hormone’s critical role in modulating plant physiology. The discovery of new molecular regulators at different levels, such as transcription factors and post-translational regulators detailed in our review (Fig. 1), enhances our understanding of SA regulation, particularly how these factors contribute to its synthesis and accumulation under different physiological conditions. Interestingly, so far, core components seem to participate in all processes involving SA accumulation, such as ICS1 (Wildermuth et al., 2001) or its transcriptional regulator SARD1 (Zhang et al., 2010a). Although traditionally recognized for their roles in defense, these proteins also trigger senescence and respond to nutritional status (Morris et al., 2000; Zhang et al., 2017; Criollo-Arteaga et al., 2021). This involvement across different physiological processes suggests the existence of a shared group of proteins governing SA dynamics. Intriguingly, the regulators upstream of these components may be process specific, suggesting that while a core part of the pathway is shared, other aspects are uniquely tailored to specific physiological events, thus conferring specificity to SA production.

An interesting example of this is the NAP transcription factor which modulates SARD1 expression (Fig. 1; Wang et al., 2022). NAP participates in SA production during senescence, but it does not participate in the defense response against pathogens (Wang et al., 2022).

Additionally, tissue-specific regulation can influence SA function. Specific stimuli sensed by different organs may lead to distinct regulatory processes, as illustrated by the gene MYB59, which suppresses SA production (Fig. 1) and exhibits differential regulation in roots or leaves in response to SA (Wiśniewska et al., 2021; He et al., 2023). Similarly, the gene SVP regulates flowering when expressed in the meristem and controls ARR when expressed in leaves (Wilson et al., 2017), presenting a compelling candidate for linking flowering and ARR through SA modulation. These ideas collectively suggest a sophisticated regulatory architecture where shared and unique components interplay, fine-tuning SA roles across various physiological landscapes, thus reinforcing its essential function in plant adaptability and survival. Most of the upstream regulators of these core components are described predominantly within single processes, and their roles in other processes that involve SA accumulation remain largely unexplored. Additionally, while the IC pathway and its regulation are the most thoroughly described (because of its relevance in Arabidopsis), the PAL pathway crucial for plants such as rice and tobacco (Pallas et al., 1996; Huang et al., 2010; Tonnessen et al., 2015) is almost underexplored in terms of its regulators. This gap in our understanding of regulation of the PAL pathway, combined with the limited exploration of the broader roles of upstream regulators, represent significant areas where further research could be immensely beneficial in understanding the regulatory landscape of SA accumulation.

The diverse outcomes that arise from the accumulation of SA could be explained by the interaction with other phytohormones. For example, SA has a combined effect with auxin on the modification of the root meristem (Pasternak et al., 2019; Bagautdinova et al., 2022), or it can act in conjunction with ethylene to influence the senescence process (Yu et al., 2021). NAP and SAG21, marker genes of senescence, are up-regulated in des1 plants, which have a high level of Cys, early senescent phenotype, accumulation of SA, and a greater resistance to pathogens (Álvarez et al., 2012a, b). This suggests that although there are common players involved in both responses, the final outcome has to be influenced by others factors such as the activation of pathways of other hormones. In this particular case, senescence involves the activation of the ethylene pathway, while Cys abundance is related to the availability of S. Cys availability also indirectly influences the immunity response since it is abundant in the Cys-rich receptor-like kinases and GSH.

An essential element involved in controlling SA accumulation is the interaction with ROS and oxidative balance (Herrera-Vásquez et al., 2015). This dimension crosses all the physiological processes where SA is accumulated, including defense response, developmental processes, and nutritional imbalance. In senescence, the signal that induces SA accumulation remains elusive; however, ROS is a strong candidate due to its increase as a product of the diminished ROS-scavenging enzymatic activities, and SA would be a signal to the positive feedback loop enhancing its production (Zimmermann et al., 2006; Herrera-Vásquez et al., 2015; Yuan et al., 2017). Additionally, the role of redox balance in floral development has been recognized, linking oxidative cues with the regulation of flowering time (Schippers et al., 2016). Given the modulation of SA levels during these developmental stages, it is plausible that SA interacts with redox signaling pathways to influence these processes. On another note, changes in GSH levels triggered by sulfur starvation (Krueger et al., 2010; Kopriva et al., 2019; Li et al., 2020) could lead to oxidative imbalances, subsequently triggering SA accumulation driven by nutritional deficits. Despite ROS being almost universally produced under various stress conditions, not all of these conditions result in the accumulation of SA, indicating that other signals different from ROS must be integrated to induce SA increase.

Nutritional status involving nitrogen or sulfur has a less explored role in modulating SA accumulation. We summarize some evidence supporting this link, for instance, the straightforward modulation by nitrogen of APA of core enzymes for SA accumulation (Conesa et al., 2020). Furthermore, key regulators of nitrogen metabolism, such as the transcription factors TGA1 and TGA4 (Alvarez et al., 2014), are identified as positive regulators of SA accumulation (Fig. 1; Sun et al., 2018). Despite these findings, detailed molecular mechanisms connecting N nutrition and SA accumulation remain largely unexplored. Similarly, in conditions of sulfur deficiency, although solid evidence supports an increase in SA (Criollo-Arteaga et al., 2021), the underlying molecular mechanisms are still elusive. These observations underscore significant gaps in our understanding of how nutrient availability influences SA accumulation and, importantly, the physiological functions associated with these hormonal changes.

Conclusion

In conclusion, SA emerges as a multifaceted player in plant physiology, influencing diverse aspects of plant development, senescence, and stress responses. From its concentration-dependent effects on root architecture to its intricate involvement in floral transitions, SA exhibits a nuanced relationship with developmental processes. Moreover, SA’s pivotal role in senescence, where it orchestrates a cascade of genetic and biochemical events, highlights its significance in nutrient recycling and energy conservation. The interplay of SA with other phytohormones, such as ethylene, auxin, and abscisic acid, further underscores its complex regulatory network. Intriguingly, the regulators upstream of these components may be process specific, suggesting that while a core part of the pathway is shared, other aspects are uniquely tailored to specific physiological events, thus conferring specificity to SA production depending on the physiological process that triggers the hormone increase. This specificity underscores the need to investigate the molecular components involved in controlling the nutritional response, particularly how it might relate to the defense response and the maintenance of ROS balance as a result of an increase in SA.

Nevertheless, despite significant strides in understanding SA’s contributions, intriguing questions remain surrounding its precise signaling mechanisms and interactions with other regulatory pathways. Continued research in this area promises to unveil even deeper insights into the intricate role of SA in plant biology. More knowledge comes with more possibilities for plant biotechnology, opening up new avenues for enhancing crop resilience and productivity in an ever-changing environment.

Author contributions

PS and AH-V: conceptualization, investigation, writing—original draft, review and editing; SV: visualization, review and editing; AH-V: funding acquisition, supervision.

Conflict of interest

The authors declare no conflict of interest.

Funding

This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico [ANID-FONDECYT Iniciación 11200944 and FONDECYT regular 1141029 and 1240785] and Programa Iniciativa Científica Milenio [Instituto Milenio de Biología Integrativa (iBio) ICN17_022].