**1. Introduction**

Salicylic acid (SA, 2-hydroxybenzoic acid) is a phenolic plant hormone. SA has a well- documented role in plant responses to environmental stresses including chilling [1], freezing [2,3], heat [4], heavy metals [5], salt [6], drought [7] and, notably, reactions to biotrophic pathogens [8]. Indeed, SA has a major role in plant innate immunity and systemic acquired resistance (SAR)—a whole-plant resistance triggered by a local infection [9]. SA also plays a role in the regulation of stomatal closure [10] and seed germination [11], among others. Despite these beneficial effects, constitutive over-accumulation of SA stunts plant growth [12,13].

SA is accumulated in plants following stress exposure. In *Arabidopsis thaliana sid2* mutants, SA accumulation induced by *Pseudomonas syringae* pv. *tomato* (Pst) DC3000 expressing *avrRpt2* was abolished [14]. These plants were characterized as isochorismate synthase 1 (ICS1)-deficient [15]. ICS1 is a component of SA biosynthesis pathways (Figure 1). The second ICS-coding gene (*ICS2*) provides a much lower contribution to SA synthesis in Arabidopsis. Both ICS1 and ICS2 proteins are found in chloroplasts [16]. Suppression of the barley *HvICS* gene by RNAi led to an impaired SA accumulation induced by *Fusarium graminearum* infection [17]. Recently, ICS1 was suggested to be post-translationally activated via direct interaction with PHB3—a member of the prohibitin protein family in Arabidopsis [18]. Following the production of isochorismate by ICS, it is believed to be converted to SA by isochorismate pyruvate lyase (IPL). However, the coding gene has not yet been cloned in plants. Recently, an alternative route for the processing of isochorismate was

suggested. It could be converted to isochorismate-9-glutamate by avrPphB Susceptible 3 (PBS3, also known as Gretchen Hagen 3.12, GH3.12); isochorismate-9-glutamate would lead to SA either via a passive decay [19] or by Enhanced Pseudomonas Susceptibility 1 (EPS1)—an enzyme with isochorismoyl-glutamate A pyruvoyl-glutamate lyase activity [20] (Figure 1). Interestingly, the activity of GH3.12/PBS3 is inhibited by SA in vitro (see below).

**Figure 1.** SA biosynthesis pathways in plants. Solid arrows represent single enzymatic steps. Dashed arrows represent multiple consecutive enzymatic steps. AA-SA, amino acid-SA conjugate; BA2H, benzoic acid 2-hydroxylase; DHBA, dihydroxybenzoic acid; EPS1, Enhanced Pseudomonas Susceptibility 1; GH3.5, Gretchen Hagen 3.5; ICS, isochorismate synthase; IC-Glu, isochorismate-9-glutamate; IPL, isochorismate pyruvate lyase; MeSA, methyl-salicylate; MES, methyl-salicylate esterase; MT, metyl transferase; PAL, phenylalanine ammonia-lyase; PBS3, avrPphB susceptible 3; SAG, salicylic acid glucoside; SAH, salicylic acid hydroxylase; SGE, salicylic acid glucose ester; SOT12, sulfotransferase 12; UDP-GST, UDP-glycosyltransferase; UGT76D1, UDP-glycosyltransferase 76D1; \*, enzyme not cloned in plants; \*\*, non-enzymatic decay.

Another pathway for SA synthesis involves the phenylalanine ammonia lyase (PAL) converting phenylalanine (Phe) to *trans*-cinnamic acid. This pathway occurs in the cytosol. Four PAL-encoding genes are found in *A. thaliana* [21]. The PAL pathway was shown to be active in poplar infected with *Botryosphaeria dothidea* fungus [22]. Yet, another route for SA biosynthesis involving mandelonitrile, a Phe derivative, was proposed to be functional in peach plants (Figure 1). Mandelonitrile-treated peach plants accumulated more SA and were more resistant to *Plum pox virus* [23]. The synthesis of SA through this pathway occurs through a benzoate intermediate. The exact role of mandelonitrile in SA accumulation is still a matter of discussion.

SA can be converted to a number of derivatives. These molecules have roles as either transportable forms of SA (such as methyl-salicylate, MeSA) or inactive/storage forms of SA (Figure 1). Among the latter are glucosylated SA derivatives—salicylic acid glucoside (SAG) and salicylic acid glucose ester (SGE) [24]. These molecules are stored in the vacuole and can be reversely converted to SA [25]. Hydroxylated SA derivatives—2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA—are catabolic SA forms in the cytosol [26]. Yet, glycosylation of these molecules by UGT76D1 (an UDP-glycosyltransferase) was shown to be a part of the immune response to Pst DC3000 in *A. thaliana* and regulated ICS1-dependent SA production [27]. In *A. thaliana*, SA can also be sulfonated by cytosolic SOT12 sulphotransferase. SA sulfonation has a similar positive feedback on SA accumulation in stressed plants [28]. Aspartyl amino acid conjugates of SA are thought to attenuate SA signaling, and they are formed by GH3.5 in *A. thaliana* [29].

Biotic stress-induced SA accumulation is controlled by three protein regulators: Enhanced Disease Susceptibility 1 (EDS1), Phytoalexin Deficient 4 (PAD4) and Senescence Associated Gene 101 (SAG101) [30]. It is known that EDS1 binds at least several TIR-NB-LRR receptors (used by the plant to sense the presence of pathogens) [31] and interacts with either PAD4 or SAG101 [32]. EDS1, PAD4, and SAG101 are all lipase/esterase-like proteins. However, the exact mode of their input to SA synthesis is yet to be established and does not necessarily rely on these enzymatic activities. Recently, an EDS1-PAD4 complex was found to inhibit MYC2 [33], which is a positive regulator of jasmonic acid (JA) pathway signaling that itself antagonizes SA (for review see Ref. [34]). This is thought to be an evolution-selected mechanism to block the action of coronatine—a JA-mimicking compound produced by the bacteria to subdue SA-driven defenses. EDS1 and PAD4 could also have a role independent of SA. For instance, the activation of *FLAVIN-DEPENDENT MONOOXYGENASE1* (*FMO1*) expression in Arabidopsis in response to thaxtomin A (a bacterial toxin) was dependent of EDS1 and PAD4 but independent of ICS1-generated SA [35].

Basal SA accumulation is controlled by multiple inputs. In many SA-overaccumulating mutants reviewed in Ref. [13], the connection between the function of the protein encoded by the altered gene and SA pathway signaling is vague. In rice, the knocking-down of *SEC3A*—coding for an exocyst protein complex component that guides exocytic vesicles to the plasma membrane—led to SA over-accumulation [36]. The *pi4k*β*1*β*2* double mutant, altered in two phosphatidylinositol-4-kinases, also accumulates high SA levels, but the reason for this is still being investigated [12].

After SA is produced, it interacts with Nonexpressor of Pathogenesis-related protein 1 (NPR1)—a key transcriptional regulator of SA signaling. SA affects NPR1 in at least two ways: i. NPR1 directly binds SA [37]; and ii. SA induces redox changes in the cell that conditions NPR1 monomerization [38]. Monomeric NPR1 shuttles to nuclei where it interacts with TGA transcription factors leading to the expression of *PATHOGENESIS-RELATED* (*PR*) genes involved in the set-up of plant immune defenses [13]. The regulation of PR gene transcription by NPR1 has been shown to involve histone acetylation via CBP/p300-family histone acetyltransferases [39].

Intriguingly, the mechanisms of SA perception are not fully understood. For instance, some signaling events are triggered upstream of NPR1 monomerization and/or shuttling into the nucleus. This is the case of the cell redox change, but also of phosphatidylinositol 4-kinase (PI4K) and phospholipase D (PLD) activation [40–42]. Moreover, some responses to SA are observed in NPR1-deficient mutants [43–45]. Therefore, NPR1 cannot be the only SA-binding protein (SABP) in plants. NPR1 paralogs, NPR2 [46], NPR3 and NPR4 [47,48], also bind SA. Moreover, high-throughput biochemical screens have provided a list of almost 100 candidate SABPs [49,50]. Many of these proteins are important enzymes of primary carbon metabolism. This challenges the classical paradigm of hormonal signaling where a ligand is recognized by a single/few receptor(s). Are these SABPs real SA receptors or are they false positive of high through-put omics techniques? If they really bind SA, what are the roles of such binding in SA-signaling pathways? And why do plant cells have so many SABPs. The role(s) of such a multitude of SABPs is still unexplained and offers a new paradigm for hormonal regulation in plants.

#### **2. SA Binding Proteins**

The multiplicity of SABPs has already been highlighted as an intriguing phenomenon [51]. For many of the SABPs, the physiological outcome of their interaction with SA is unclear. Moreover, it is not established whether and how the SABPs are integrated into the conventional NPR1 pathway. At least several plant SABPs have their animal orthologs that also bind SA [51]. In animals, SA and its derivatives (e.g., aspirin) act not as hormones but as therapeutic compounds that target proteins and prevent their role as disease components. How animals have developed such sophisticated responses to substances originating from plants is an interesting question by itself. Nevertheless, this suggests that SA can clearly act in a NPR1-free environment [51]. Here, we present an up-to-date vision of these

problems, characterise the canonical and newly identified plant SABPs, and discuss how they can be integrated into a unified SA-signaling network.

#### *2.1. SABP1—Catalase*

SABP1 was purified from tobacco as a soluble cytosolic protein that binds SA with a *K*<sup>d</sup> of 14 μM [52]. *SABP1* was cloned and the resulting 57 kDa protein was characterized as catalase [53]. Its activity was drastically inhibited by SA but not by inactive SA analogues in vitro [54]. This effect corresponds well to the results obtained using tobacco cell suspensions where SA could inhibit the total catalase activity [55].

In *A. thaliana*, catalase 2 (CAT2) shares 78% sequence identity with tobacco SABP1 (UniProt P49319). SA inhibited CAT2 catalase activity in vitro and total catalase activity in leaves of SA-pre-treated plants. However, the direct binding of SA to CAT2 has not been tested [56]. CAT2 was demonstrated to have a role in SA-mediated resistance to a biotrophic pathogen (Pst DC3000) by inhibiting indole-3-actic acid (IAA, an auxin) and JA accumulation [56]. Inhibition of CAT2 by SA leads to a H2O2 increase in plants upon pathogen infection. This promotes sulfenylation (sulfenic acid conjugation) of tryptophan synthetase b subunit1 (TSB1) at Cys308 leading to the inhibition of its activity. This enzyme acts in the IAA biosynthesis pathway and as a result, SA antagonizes IAA accumulation. In parallel, SA diminished the stimulatory effect of CAT2 protein on the in-vitro activity of acyl-CoA oxidases (ACX2/ACX3) implicated in jasmonic acid (JA) biosynthesis. It is not clear if this effect is due to the catalase activity of CAT2. A direct interaction between CAT2 and ACX2/ACX3 has been observed. This interaction was impaired in SA-pre-treated plants [56].

Unlike wild-type (WT) plants, *sid2* mutants accumulated JA in response to Pst DC3000 (reflecting the antagonism of SA against JA accumulation). Such JA accumulation was diminished in *sid2cat2* double mutants, suggesting that CAT2 is indeed a positive regulator of JA production and thus plays a part in the SA-JA antagonism in plants [56]. The *cat1* (78% identity to SABP1) and *cat3* (76% identity to tobacco SABP1) Arabidopsis mutants were not altered in the biotic stress-induction of neither IAA nor JA accumulation [56].

The way SA binds SABP1 or catalase orthologues, from a molecular point of view, has not been deciphered.

#### *2.2. SABP2—MeSA Esterase*

SABP2 was similarly purified from tobacco and had a much higher affinity to SA (*K*<sup>d</sup> = 90 nM) when compared to SABP1 [57]. A corresponding ORF encoding a 260-residue α/β fold hydrolase superfamily protein with a calculated molecular mass of 29 kDa was cloned and characterized. Its lipase activity towards *para*-nitrophenyl palmitate (measured by the release of *p*-nitrophenol) was drastically stimulated by SA in vitro. The silencing of SABP2 using the RNAi technique resulted in the lowering of SA-induced *PR1* expression and resistance level against *Tobacco mosaic virus* (TMV) [58]. Later, it was established that SABP2 activity was that of a methyl salicylate (MeSA) esterase, converting MeSA into SA, an end-product inhibitor of such activity. The co-crystallization of SABP2 with SA revealed that it is positioned carboxylate group-first in the active site [59]. The inhibition of methyl esterase (MES) activity of SABP2 by SA could be a mechanism to fine-tune active SA concentration in the cell.

SABP2 can also convert acibenzolar-S-methyl (a functional analogue of SA) into acibenzolar. Silencing of SABP2 in tobacco results in the loss of an acibenzolar-S-methyl effect on the induction of *PR1* expression and the onset of SAR [60].

MeSA (produced by salicylate carboxyl methyltransferases) is inactive but is more hydrophobic than SA and it easily penetrates cell membranes. Therefore, MeSA is, in line with other molecules such as pipecolic acid [61], considered to be a mobile signal of the SAR. Produced in infected cells, MeSA reaches distant leaves by phloem transport. In these so-called systemic tissues, it is converted into active SA via MES activity. The SA thus produced triggers-preventive defense responses in these distal leaves. SABP2 was established as a key enzyme of SAR in tobacco [62,63]. In potato, an orthologue of SABP2, StMES1, has been cloned. The enzymatic activity of StMES1 was inhibited by SA in vitro and its role was, in a similar way, linked to SAR development [64].

Two methyl esterase-encoding genes were identified in poplar [65]. In *A. thaliana*, 18 SABP2 orthologues were identified and at least five proteins (AtMES1,-2,-4,-7,and -9) were shown to possess an esterase activity acting on MeSA that was inhibited by SA in vitro [66]. In these two species, MES is also an important component of SAR [22,66].

#### *2.3. SABP3—*β *Carbonic Anhydrase*

SABP3 was identified in tobacco chloroplasts as β carbonic anhydrase (βCA). SABP3 binds SA with moderate affinity (*K*<sup>d</sup> = 3.7 μM) [67]. CAs are ubiquitous and evolutionary-conserved enzymes that catalyse the interconversion of CO2 and bicarbonate (HCO3 −). CAs have roles in photosynthesis, respiration, stomata movements, and lipid biosynthesis among others [68]. From an immunity point of view, SABP3 was required for the hypersensitive response (HR) in tobacco leaves infiltrated with *A. tumefaciens* expressing *Pto*:*avrPto* (R-avr gene pair). Two recombinant tobacco proteins—SABP3/βCA1 and βCA2—were shown to bind SA but not 4-hydroxybenzoic acid (4-HBA, an inactive isomer). Based on the fact that SABP3 could complement the phenotype of an oxidative stress-sensitive strain of *Saccharomyces cerevisiae* [67], it was suggested that SABP3 might have antioxidative properties.

In *A. thaliana*, the affinity of AtSABP3/βCA1 (a SABP3 orthologue) to SA and its CA activity is diminished in the presence of S-nitrosoglutathione (GSNO), a NO donor. These effects were not observed in a C280S AtSABP3-mutated protein, suggesting that they are due to the nitrosylation of Cys280 [69]. AtSABP3 knockout mutants have their resistance compromised towards Pst DC3000 (*avrB*). The resistance phenotype was restored when complemented by AtSABP3 but not by C280S AtSABP3 [69]. In this manner, AtSABP3 nitrosylation is clearly a requirement for its role in immunity. In contrast, the role of the binding of SA to AtSABP3 requires further research.

Indeed, there are contrasting data about SA influence on CA activity in plants. SA did not affect the CA activity of purified SABP3 from tobacco chloroplasts at physiological concentrations. The inhibition only occurred at concentrations as high as 3 mM, while binding occurred at lower concentrations [67]. A significant increase in CA activity was reported in leaves of SA-treated peppermint (*Mentha piperita*) [70]. In contrast, in *A. thaliana*, CA activity was reported to decrease following treatment with SA or with benzothiadiazole (a functional SA analogue), or after inoculation with *P. syringae* [71].

SABP3 orthologs from Arabidopsis and *Chenopodium quinoa* were found to physically interact with HCPro—a viral protein of *Turnip mosaic virus* (TuMV). This protein has RNA silencing suppressor (RSS) activity and counteracts host's anti-viral RNA interference. The transient expression of HCPro antagonized AtSABP3 transcripts and protein accumulation in Arabidopsis [72]. AtSABP3 was thus established as a component of anti-viral defense. Whether this role of AtSABP3 is dependent on SA binding is still unknown. Unexpectedly, both AtSABP3-knockout and AtSABP3-overexpressing lines of Arabidopsis were compromised for their resistance to TuMV [72].

AtSABP3 bearing no signal peptide was shown to interact with NPR1 in a yeast two-hybrid assay. Intriguingly, this result was observed only when growth plates were supplemented with SA (but not with inactive isomer 4-HBA), suggesting that this interaction was SA dependent [71]. An AtSABP3-NPR1 interaction was also demonstrated in tobacco leaves in planta. Bimolecular fluorescence complementation revealed that this interaction occurred in the nucleus and the perinuclear region. The transient expression of GFP-NPR1 and MBP-AtSABP3 constructs led to co-purification of GFP-NPR1 together with MBP-AtSABP3 on amylose resin [71]. In the same study, interactions with NPR1 and NRB4 (a protein that is perhaps involved in SA perception), were similarly reported for at least several other cloned fragments/alternative splice variants of proteins representing the βCA family in Arabidopsis. These data suggest that βCA family members could be a part of SA signalling. However, an Arabidopsis β*ca1* mutant deficient in AtSABP3 (or quintuple β*ca1,2,3,4,6* mutant for that matter) was only partially insensitive to exogenous SA as suggested by pathogen resistance and

*PR1* expression responses. The homozygous mutation in β*CA5* could not be tested due to plants sterility [71].

Note that amino acid residues required for SA binding to SABP3 and orthologues have not yet been identified.

#### *2.4. NPR1*/*2*/*3*/*4—Signalling Proteins*

NPR1 binds SA with high affinity in vitro (*K*<sup>d</sup> = 140 nM) [37]. SA binding to NPR1 is Cu2+-dependent, implicates Cys521 and Cys529, and results in conformational changes of NPR1—a mechanism that could stand behind its role as a transcription cofactor with TGA [37].

NPR1 exists in the cytosol as an oligomer due to disulphide bridges between Cys82 and Cys216 of different subunits. In order to shuttle to the nucleus and act on regulating gene expression, NPR1 has to be monomerized [38]. This monomerization requires the reduction of the disulphide bonds. SA binding has been shown to facilitate de-oligomerization of recombinant NPR1. Yet, SA alone is not sufficient to trigger NPR1 monomerization [37]. The upstream SA-driven redox events that allow the reduction of the disulphide bonds in NPR1 are not fully understood. Two cytosolic thioredoxins, TRX-h3 and TRX-h5, have been shown to interact with NPR1 since they were pulled-down by the immobilised His-tagged N-terminal part of NPR1. Furthermore, co-immunoprecipitation of TRX-h5 with a NPR1-TAP fusion protein was stimulated by SA [73]. Thioredoxins are redox regulators and can reduce disulphide bridges [74,75]. Co-incubation of cell lysates with recombinant TRX-h5 led to an increase in NPR1-GFP monomers, thus suggesting that TRX-h5 plays a part in NPR1 monomerization. Both *trx-h3* and *trx-h5* mutants were compromised in *PR1* induction by exogenous SA [73]. In contrast, NPR1 monomerization is negatively regulated by S-nitrosylation at Cys156 [73].

In Arabidopsis, NPR3, NPR4 (both having 39% identity to NPR1) [48] and NPR2 (62% identity to NPR1) [46] are paralogues of NPR1. They have all been shown to bind SA. The affinities of NPR3 (*K*<sup>d</sup> = 176nM) and of NPR4 (*K*<sup>d</sup> = 23nM) to SA are known [76]. NPR3 [77], NPR4 [78] and NPR2 [46], similarly to NPR1, act in the nucleus. Interestingly, NPR3 and NPR4 were demonstrated to bind NPR1 in a SA-dependent manner in vitro. Based on the fact that the ability of cullin 3 (CUL3, a component of protein E3 ligase complex) to pull-down NPR1-GFP was reduced in a *npr3 npr4* genetic background, it was suggested that NPR3 and NPR4 act as adaptors for SA-dependent proteasome-mediated degradation of NPR1 [48]. In a similar manner, NPR3 and NPR4 were suggested to mediate the proteasome-dependent degradation of EDS1 [79].

NPR3 and NPR4 have been shown to have roles as transcriptional co-repressors that function in parallel to NPR1. They act as negative regulators of immunity. *SARD1* and *WRKY70* genes are, for instance, under the negative transcriptional control of NPR3/NPR4 [76]. Co-transformation of Arabidopsis protoplasts expressing a luciferase reporter gene under the control of either SARD1 or WRKY70 promoters with plasmids overexpressing either NPR3 or NPR4 resulted in the inhibition of luciferase expression. This effect was diminished when TGA-binding motifs were mutated in either SARD1 or WRKY70 promoter regions. SARD1 and WRKY70 expression were also partly de-repressed in TGA2/TGA5/TGA6-deficient plants. This suggests that TGA transcription factors are implicated in the negative control of their expression by NPR3 or NPR4. More importantly, SA could antagonise the observed transcriptional repression activity of NPR4 [76].

In potato, the StNPR3L (NPR3-like protein) was shown to interact with the transcription factor StbZIP61 and inhibit its transcriptional activation activity in an SA-dependent manner [80]. This could have a role in the regulation of SA accumulation in infected plants since the expression of *StICS1*, a SA biosynthesis gene, positively correlated to *StbZIP61* expression as suggested by RNAi and mutant studies [80].

NPR2 physically interacts with NPR1 in vitro, and in planta the overexpression of NPR2 could partly complement the *NPR1*-deficient phenotype [46]. The role of SA binding in this interaction is unknown.

#### *2.5. Glutathione S-transferase*

Several *A. thaliana* glutathione *S*-transferase (GST) isoenzymes (GSTF2, GSTF8, GSTF10, GSTF11) have been shown to bind SA [81]. The enzymatic activity of GSTF10, GSTF11 and that of GSTF8 (to a lesser extent) were inhibited by SA in vitro. GSTs comprise a large group of enzymes that catalyse at least several reactions in connection to glutathione conjugation. Promoters of many GST-encoding genes contain disease-related W-boxes and WT-box cis-regulatory elements. GSTs are a part of plant immunity with roles in glucosinolate (antimicrobial compound) metabolism and detoxification of mycotoxins among others [82].

#### *2.6. Thioredoxins*

The chloroplastic thioredoxin-m1 (TRXm1) was shown to bind SA in a high-throughput screen and later confirmed by surface plasmon resonance (SPR) analysis [50]. Reduced thioredoxins can reduce disulphides of target proteins [74,75]. The reduction of TRX arises either from NADPH through NADPH thioredoxin reductases or from ferredoxin via ferredoxin thioredoxin reductases. The effect of SA binding on TRXm1 activity has not been yet established. As mentioned earlier, the activity of other thioredoxins, the cytosol located TRX-h3 and TRX-h5, is required for NPR1 monomerization [73]. It could be interesting to investigate whether these cytosolic TRXs are also SA-binding proteins.

#### *2.7. GH3—Acyl Acid Amido Synthetase*

GH3.12/PBS3 from *A. thaliana* conjugates specific amino acids to acyl substrates (e.g., 4-substituted benzoates) in an Mg2+- and ATP-dependent manner. This enzyme binds SA in a ternary complex with AMP as shown by its crystallographic structure [83]. Due to the observed position in the active pocket, SA was suggested not to be a substrate but an inhibitor of AtGH3.12/PBS3. This is consistent with the fact that SA indeed inhibits AtGH3.12/PBS3 activity in vitro [84]. Conjugation of amino acids to plant hormones, such as jasmonic acid or auxins, is a common strategy aimed at controlling their active level in plant cells [85]. PBS3 was also shown to be an important enzyme of SA biosynthesis [19,20]. The role of AtGH3.12/PBS3 in SA signalling is, however, unclear. The *P. syringae*-induced free SA accumulation in *pbs3* mutants was actually higher than that of WT plants, but it was accompanied by the reduced pathogen resistance and retarded *PR1* expression in such plants. These effects were accompanied by the drastic diminution of SA-*O*-β-glucoside (SAG) accumulation [86]. The connection between SAG production and AtGH3.12/PBS3 has not been established yet.

Recently, PBS3 was shown to directly interact with EDS1—one of the three key protein regulators of SA pathway signalling (see above). In tobacco leaves, the interaction occurred both in the nucleus and cytoplasm as revealed by bimolecular fluorescence complementation assays. No interaction was observed for PBS3 and PAD4 or SAG101. Using an inhibitor approach, authors concluded that PBS3 could control EDS1 abundance in a post-translational manner by preventing its proteasome degradation—most likely by the 26S proteasome [79]. The role of SA binding to PBS3 was not investigated in this context.

It was reported that another GH3 family member, AtGH3.5, could accept SA as a substrate. In AtGH3.5-overexpressing Arabidopsis lines, a significant increase in the content of SA-aspartyl was registered [29]. The very same enzyme also produces inactive aspartyl conjugates of indole-3-acetic acid (IAA)—an auxin hormone. In this manner, AtGH3.5 could play a part in SA-IAA crosstalk.

#### *2.8. GAPDH*

Several isoforms (subunits) of *A. thaliana* GAPDH (glyceraldehyde 3-phosphate dehydrogenase)—GAPA-1, GAPA-2, GAPC-1, and GAPC-2—bind SA as demonstrated using SPR [87]. Among the detected SA-binding GAPDH isoforms, some are cytosolic (GAPC-1, GAPC-2) while others are plastidial (GAPA-1, GAPA-2) enzymes. They play essential roles in glycolysis (cytosolic) and Calvin cycle (plastidial), respectively. However, the effect of SA binding on GAPDH function is unknown.

Some GAPDH isoenzymes have been shown to be multifunctional. AtGAPC-1 is required for *Tomato bushy stunt virus* (TBSV) asymmetric replication via its direct association with a negative RNA strand of the virus. The binding of SA to AtGAPC-1 inhibits the association of the enzyme to the virus RNA. This was suggested to be a part of plant anti-viral defenses [87].

Interestingly, human GAPDH also binds SA and is similarly implicated in the regulation of the replication of some viruses [88]. SA binding suppresses HsGAPDH translocation to the nucleus. Whether a similar mechanism affecting GAPDH localization is employed in plants is yet to be established.

#### *2.9. Alpha-ketoglutarate Dehydrogenase—Krebs Cycle Enzyme*

The E2 subunit of the α-ketoglutarate dehydrogenase (αKGDE2) enzyme complex acts in the tricarboxylic acid (Krebs) cycle in mitochondria. This protein was shown to bind SA in two independent assays (photoaffinity labelling and SPR) both in Arabidopsis [81] and tomato [89]. The αKGDE2 activity was reduced almost by half in isolated mitochondria sampled from tomato leaves pre-treated with SA for 24 h. This could be due to either a transcriptional or translational regulation by SA and not to a direct effect of the binding of SA on the protein. The silencing of αKGDE2 resulted in the increase of tomato resistance to TMV. Interestingly, the treatment with SA could similarly induce resistance to TMV in WT plants but it could not enhance the resistance phenotype of αKGDE2-silenced plants [89]. Based on these facts, a suppression of αKGDE2 by SA was suggested to be a part of plant antivirus defenses.
