*2.12. HMGB3—DAMP Protein*

High Mobility Group Box 3 (HMGB3) was shown to bind SA using SPR analysis [94]. The binding affinity of this protein to the immobilized 3-aminoethyl SA was very high (*K*<sup>d</sup> = 1.5 nM). This protein was found to be a DAMP (damage-associated molecular pattern molecules) acting via BAK1 and BKK1 receptor kinases. Exogenous application of purified recombinant HMGB3 induced plant immune responses and was enough to improve Arabidopsis resistance to *B. cinerea*. HMGB3, when applied together with 1 μM SA, lost its effectiveness as a DAMP.

#### **3. Response of SABPs to Treatments Linked to SA**/**Biotic Stress**

To sum up the above sections and to find common regulatory patterns, if any, among SABPs, we mined transcriptomics data for genes encoding the SABPs described above (Figure 2). In panel A, we show the effect of SA on the protein, when it is known. In our list are proteins from *A. thaliana*. However, the effects of SA on CAT2, MES9 and βCA1 were extrapolated from those observed for tobacco orthologs (SABP1, SABP2 and SABP3, respectively). SA treatment led to an inhibition of enzymatic activity of a number of SABPs in vitro. The activity of NtSABP3 was not affected by SA while the effect of SA on transcriptomic activities of NPR proteins was not considered. No example of SA activating a SABP enzymatic activity in vitro is currently available to us.

In panel B (Figure 2), we show two sets of transcriptomics data. A first set was used to draw the dendrogram showing the hierarchical clustering of SABPs based on their expression across 111 conditions. In these experiments, *A. thaliana* plants were challenged with bacterial (*Pseudomonas* spp.), fungal (*Sclerotinia sclerotiorum*, *Golovinomyces orontii*, *Hyaloperonospora arabidopsidis*) and viral (TuMV) pathogens. All of these conditions should directly implicate SA signaling responses. We could separate three clusters. The alpha cluster represents the genes whose expression was mostly inhibited by the treatments; the gamma clusters represent genes whose expression was induced by the treatments while the beta cluster represents genes exhibiting an intermediate situation.

In the second set of transcriptomics data (heat map), we illustrate how the expression of SABPs change when different aspects of SA signalling are stimulated. Here we included the SA treatment per se, but also treatments with methyl jasmonate (MeJa, a SA-antagonist hormone), model pathogens, and a flg22 elicitor—a fragment of flagellin, a protein from bacterial flagellum that triggers immunity responses. *NPR3* and *NPR4* are two genes early stimulated by SA (3 h). After a longer time (24 h), SA could both stimulate (*NRP* genes, *GH3.12*) and inhibit (*GSTF11*, *CA1*, *GAPA-2*) the expression of SABPs. No SABPs were reactive to MeJa. We could not observe significant differences in expression patterns of SABPs following treatments with Pst and Psm bacteria—the latter bearing the *avrRMP1* avirulence gene that activates effector-triggered immunity (ETI). The transcriptional responses to *S. sclerotiorum* fungus and TuMV were a tad divergent. In virus-treated plants, a negative effect on *GAPA-2*, *TRX-m1* and *CA1* expression was relieved. The same can be said for plants treated with flg22. It should be noted that across all modelled infections, the stimulation of *NPR* genes and *GH3.12* was quite consistent.

Therefore, similarly to what was observed in the clustering analysis, binding to SA concerns proteins that are both positively and negatively expressed in response to elicitation related to biotic stresses. All the SABP-coding genes behaved differently, but at least some SABPs are well synchronised at the transcriptomic level. For instance, NPR proteins, especially NPR2 and NPR3, appear to cluster together (gamma2), thus suggesting that they are involved in the same signaling cascade. Indeed, NPR2, NPR3 and NPR4 have been shown to interact physically with NPR1 [46,48]. The same transcriptomic connections are true for βCA1, GAPA-1 and TRXm1 in the alpha cluster, and KGDE2 with GAPC1 in the gamma1 cluster. How these connections at the transcriptomic level translate into interplay between protein functions is yet to be established.

**Figure 2.** Effect of SA on the in-vitro catalytic activity of SABPs from *A. thaliana* (**A**) and transcriptional responses of corresponding coding genes (**B**). \*, effect observed in tobacco orthologs. Transcriptomic data was mined using Genevestigator [95]. Experiment IDs: SA 3 h, AT-00113; SA 24 h, AT-00320; MeJA 3 h, AT-00110; Psm, AT-00406; Pst avrRpm1, AT-00106; *S. sclerotiorum*, AT-00681; TuMV, AT-00324; flg22, AT-00392. Note that the hierarchical clustering was performed on more experiments than the one used for the heat map (see the main text).

It is also interesting to note that for GAPDH family members, some appear to be strongly inhibited in responses to immunity-related stresses (such as the plastidial GAPA2) while others are induced (such as cytosolic GAPC1). These enzymes are likely not to have the same role in SA-signaling pathways. Besides, there is no strong correlation between the effect of SA on the protein (panel A) and the way their genes react to immunity-related inputs (panel B): Proteins that are inhibited by SA are found in alpha and gamma clusters.

Interestingly, a human GAPDH gene is commonly used as a "housekeeping" reference gene in quantitative RT-PCR analyses [96]. The data presented in this paper, however, show that the same cannot be translated to GAPDH isogenes in Arabidopsis since a strong transcriptomic reaction to either viral (GAPC-1) or bacterial/fungal infections (GAPA-2) was observed.

#### **4. Molecular Mechanisms of SA-Protein Interactions**

Some plant SABPs have been crystallized with SA. Since no conserved SA binding motif is known, the analysis of crystallography data could help to better understand how binding occurs. Here we have focused on two plant proteins, AtGH3.12 and NtSABP2 (Figure 3).

**Figure 3.** Molecular interactions between SA and selected SABPs. Black dashed links represent hydrogen bonds. Green dashed lines represent pi-pi stacking. Green solid lines represent hydrophobic interactions. Molecular graphic images were produced using UCSF Chimera [97].

In AtGH3.12, SA binding occurs in the active site. The carboxyl group of SA forms hydrogen bonds with side chains of Arg123 and Tyr120. Using the UCSF Chimera software [97], we could identify that SA also forms contacts (likely nonpolar interactions) with Gly326 and the side chain of Ile217 (Figure S1). While SA was co-crystallized with AMP in the active site of AtGH3.12, there is no direct interaction between SA and this cofactor (Figure S1).

For NtSABP2, two SA-binding sites have been found; an inner pocket, in the active site and a surface pocket that has been suggested to be a crystallographic artefact [59]. In the inner pocket, the carboxyl group of SA forms hydrogen bonds with Ala13, Ser81 and His238 residues (Figure 3). An interaction between the SA carboxyl group carbon chain and the carbon chain of Ser81 was predicted using UCSF Chimera (Figure S1). As for the surface-binding pocket, the carboxyl group of SA forms a hydrogen bond with Lys159, while interactions were predicted with Lys159, Leu132 and His158. As for the latter, the aromatic ring of SA is in a parallel plane to the imidazole ring of His158 (Figure S1).

Interestingly, many animal and human proteins have been co-crystallized with SA (or, alternatively, with acetyl SA—an aspirin). Similar mechanisms are apparently involved in the binding. Bovine milk xanthine dehydrogenase binds SA via hydrogen bonds formed between Thr1010, Arg880 and the carboxyl group of SA. Moreover, clear π–π stacking is formed between the aromatic ring of SA and aromatic rings of Phe914 and Phe1009 (Figure 3). In human ferrochelatase (FECH), however, it is a

side hydroxyl group, and not the carboxylic group, that forms a hydrogen bond to Ser281 (Figure 3). In this protein (a homodimer), SA binds directly at the dimer interface, implicating hydrophobic amino acid residues from both monomers (Figure S1). It is worth mentioning that SA inhibits FECH activity in vitro. This could be due to induced conformational changes since the gel filtration elution profile was altered in the presence of SA [98].

Interestingly, for NPR1, SA binding was suggested to involve Cys521 and Cys529 based on protein mutation studies and requires the presence of Cu2<sup>+</sup> ions. However, NPR1 has not yet been crystallized with SA [37].

In conclusion, SA binds to proteins both in plants and animals using similar mechanisms. As a common rule, binding occurs by hydrogen bond formation between the carboxyl group of SA and side chains of various amino acid residues (Arg, Tyr, Ala, Ser, His). Such interaction could be strengthened by nonpolar/π-π contacts made by the aromatic ring of SA. The side hydroxyl group of SA can also be involved. The change in its relative position is enough to alter the binding affinity of SA/4-HBA to proteins [81,89].

Many of the SABPs were identified by SPR analysis, a method where an immobilized 3-animoethyl SA is used as a ligand. This implies that interactions should occur on the surface of the proteins, at least in the SABPs identified with this technique.

#### **5. Discussion and Conclusions**

SA interacts with multiple plant proteins. These include the canonical SA receptor, NPR1, and its paralogs, but also many other proteins with diverse roles in cell regulation. The role of these interactions is still not known. NPR1 is, without a doubt, a bottleneck of SA signaling, since in NPR1-deficient plants, SA-induced responses, e.g., the expression of PR genes [99], are drastically altered. The goal of this review was to give a physiological reasoning for SA binding to other plant proteins as a mode of multinodal input (Figure 4). In a canonical situation (panel A), a hormone binds a receptor (one protein or proteins of the same family) and activates a signaling cascade, leading to cell responses. This is true for many plant hormones (e.g., auxins and jasmonates). Based on available data, the SA-signaling pathway appears to be different (panel B). A number of structurally unrelated receptors exist in parallel. Each receptor is a node that will lead either to the same cascade, acting synergistically, or activate separate cascades.

**Figure 4.** Model of conventional hormonal input via a single receptor (R) leading to downstream responses (**a**); model of SA multinodal input via multiple SABPs acting as independent receptors (**b**). Some receptors in model B may be in a functional connection (e.g., a putative SA-binding TRX acting on NPR1) and act in a single reception pathway.

This model is based on the fact that many SABPs (acting as potential SA receptors) are important enzymes where, at least for some of them, SA binding results in a modification of their activity in vitro (Figure 2). In most, if not all examples available to us where SA inhibits enzymes, SA is likely to obstruct substrate–enzyme interactions or take the place of a necessary cofactor.

SABPs are often connected to plant immunity. For instance, the silencing of αKGDE2 (a SABP) led to an increase in plant-virus resistance [89]. However, since many SABPs are crucial enzymes of basal metabolism, knocking them out could result in unspecific growth aberrations. Instead, point mutations that preserve enzymatic function but disrupt SA interactions should be introduced in planta to test the role of SA binding. Finding SA interaction sites in proteins with no crystallization data available is an intriguing task. This approach would also help to validate SA binding to SABPs in planta.

SABPs could play one of the two probable roles: 1.) Act in support to the NPR1 pathway or 2.) Act in parallel to NPR1 (Figure 5). Regarding the first option, the mechanisms of NPR1 monomerization are indeed not fully understood, although we know that they require upstream redox changes to occur. The proteins involved in this process are still a matter of further investigation. Thioredoxins are perfect candidates for disulphide bond reduction in NPR1 oligomers. TRXm1 has been found to be an SABP, however, this protein is in chloroplasts, while cytosolic TRXh5, implicated in NPR1 monomerization, is not a SABP. AtCAT2 is a homolog of tobacco SABP1—both enzymes could also be a part of cell redox regulation (Figure 5). At some point, NPR1 has to be degraded to attenuate the response, and this involves NPR3 and NPR4 [48], but, in addition, TOP1, TOP2 and TPPII—all proteolytic SABPs—could arguably play a part. Another question is what receptor allows the SA-driven activation of PI4K and PLD, which are both linked to NPR1 [40–42].

**Figure 5.** Schematic representation of putative functional roles of SABPs in cell metabolism. Phe, phenylalanine; DAMP, damage-associated molecular pattern; ROS, reactive oxygen species. See the main text for protein abbreviations. Solid arrows represent single enzymatic steps. Dashed arrows represent multiple consecutive enzymatic steps. Hollow arrows represent functional connections to cell activities.

Alternatively, SABPs could act independently of NPR1. Indeed, at least some of the plant reactions to SA occur in an *npr1* genetic background [43–45], but our knowledge of the molecules involved in the NPR1-independent pathway is next to none.

At least two enzymes directly involved in energy metabolism have been found to bind SA—GAPDH (glycolysis) and alpha-ketoglutarate dehydrogenase (Krebs cycle). This could be a way for SA to control the stress-to-growth transition of cell metabolism. Alternatively, since cytosolic GAPDH has been shown to be a dual activity enzyme [87], SA could be a trigger for the transition.

SA has an activity in pollen tubes and in isolated organelles. In these systems, the NPR1 pathway is absent. In Arabidopsis, exogenous SA and MeSA had an opposite effect on pollen tube elongation. The inhibitory effect of SA could be due to changes in clathrin-mediated endocytosis. SA inhibited the internalization of FM4-64 dye while this effect was abolished in *chc2-2* (clathrin heavy chain)

mutants [45]. MeSA methylesterase- and SA methyltransferase-GFP constructs were both located in growing pollen tips, suggesting that SA is employed in the control of polarized growth.

SA stimulates the activity of mitochondrial succinate dehydrogenase (SDH, respiratory Complex II) and H2O2 accumulation in isolated mitochondria [100]. The authors concluded that SA could act at the ubiquinone binding site of respiratory Complex II. Since SDH-deficient plants had diminished SA responses and SA-induced ROS production—this can be the bona fide mechanism of SA perception. At the moment, no SABPs have been formally identified to mediate the above-mentioned effects of SA.

Affinity to SA can differ by up to 1000-fold in SABPs (e.g., SABP1 *K*<sup>d</sup> = 14 μM; NPR4 *K*<sup>d</sup> = 23 nM). So, at any given moment, based on current cell SA concentration, SA will interact with a limited set of SABPs. In this manner, a regulatory input of SA will differ depending on SA concentration. In Arabidopsis, the basal SA level is around 1 μM, thus some SABPs will bind SA even at basal concentrations, while others will interact only when SA levels rise following stress exposure. The abundance of SABPs (e.g., by transcriptional regulation) is also subjected to regulation (Figure 2).

The uncertainty of a role for SABPs in SA signaling stipulates the need to study SA–protein interaction in planta. In such experiments, the use of isotope-labeled SA is preferred while alternatively photoaffinity labelling [81] could be adopted for protoplast experiments.

Intriguingly, components of the SA-signaling network are still being revealed as we speak. In recent publications, a role for GH3.12/PBS3 was highlighted. PBS3, whose enzymatic activity is regulated by SA [84], was shown to be both an important enzyme of SA biosynthesis [19,20] and signaling (Figure 5), controlling EDS1 [79].

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/18/ 4377/s1.

**Author Contributions:** I.P. wrote the manuscript and generated the figures. V.K. corrected and added insights to the manuscript. E.R. supervised and reviewed the manuscript.

**Funding:** This research was funded by NAS of Ukraine, grant number 2.1.10.32-15 and PHC DNIPRO program. I.P. benefitted from a Marie Curie/Université Paris-Est Créteil post doc fellowship. We would like to thank Michael Hodges, CNRS UMR9213 Institute of Plant Sciences Paris-Saclay, for proofreading of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

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