On the capacity of putative plant odorant-binding proteins to bind volatile plant isoprenoids

Plants use odors not only to recruit other organisms for symbioses, but to ‘talk’ to each other. Volatile organic compounds (VOCs) from “emitting” plants inform the “receiving” (listening) plants of impending stresses or simply of their presence. However, the receptors that allow receivers to perceive the volatile cue are elusive. Most likely, plants (as animals) have odorant bind proteins (OBPs), and in fact few OBPs are known to bind “stress-induced” plant VOCs. We investigated whether OBPs may bind volatile constitutive and stress-induced isoprenoids, the most emitted plant VOCs, with well-established roles in plant communication. First, we performed a data base search that generated a list of candidate plant OBPs. Second, we investigated in silico the ability of the identified candidate plant OBPs to bind VOCs by molecular simulation experiments. Our results show that monoterpenes can bind the same OBPs that were described to bind other stress-induced VOCs. Whereas, the constitutive hemiterpene isoprene does not bind any investigated OBP and may not have an info-chemical role. We conclude that, as for animal, plant OBPs may bind different VOCs. Despite being generalist and not specialized, plant OBPs may play an important role in allowing plants to eavesdrop messages sent by neighboring plants.


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Plants synthesize a variety of volatile organic compounds (VOCs) that are important for reproduction 48 and defense, and in general to communicate with other organisms (Ninkovic et al., 2020). Insects and 49 generalist herbivores, or carnivore insects that are also attracted by the volatile "cry for help" released 50 by plants upon herbivore attacks, are all able to sense plant volatiles (Dicke and Loreto, 2010). 51 Whether volatiles are also important in plant-plant communication is a more fascinating, yet 52 controversial, issue (Vickers et al., 2009). Growing reports show that volatiles are able to influence 53 plant-plant relationships (Baldwin et al., 2002;Erb, 2019;Ninkovic et al., 2019), and that volatiles 54 elicited in "emitting" plants by abiotic or biotic stresses prime defensive responses in non-elicited 55 "receiving" plants (Zuo et al. 2019;Frank et al. 2021). However, no study has so far looked for the 56 primary events in such elusive plant-plant interaction, i.e. the receptors by which plants may perceive 57 the volatiles emitted from neighboring plants are largely unknown. 58 Recently, it has been proposed that the passage of VOCs across the plasma membrane relies on their 59 active transport. In particular, the presence of an ABC carrier protein involved in active transport into interactions. TPL and TPL-related (TPR) proteins are transcriptional co-repressors (also toward JA-74 mediated signaling). Interestingly, only the capacity to bind -caryophyllene was tested with emitting 75 and receiving (eavesdropping) plants (Nagashima et al., 2019). 76 These three cases need confirmation, and all other plant volatiles (at least 1700 known so far, Dicke 77 and Loreto (2010)) wait for receptor recognition (if any). We report here an in-silico study based on 78 current knowledge of plant protein structure, especially aiming at selecting best candidates as plant 79 OBPs for plant volatiles whose receptors are still unknown/unavailable. We particularly focused on

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We looked at plant OBPs following a two-level structural approach (identification of candidate plant 86 OBPs, and validation by in silico molecular simulations of OBP capacity), as detailed in the Method 87 section below (see also Figure 1 It is interesting to note that all plant protein sequences reported in Table 1

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The results suggest that the three monoterpenes tested (α-pinene, β-myrcene and limonene) may bind  Table S3). In our docking simulations, the best results for -pinene were obtained 149 with SABP2 and the complete JA receptor, with binding energy value of -6.03 Kcal/mol and -5.92 150 Kcal/mol, and predicted Ki of 37 M and 38,55 μM, respectively. In both cases, this is a better 151 interaction than with the reference complex. In the case of GA receptor and heading date 3A, -152 pinene has binding energy values very similar to the value in the reference complex.

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Similarly to -pinene, -myrcene binds SABP2, GA receptor and the JA receptor better than the 154 reference complex. Among the other candidate proteins, protein heading date 3A, FT, and tfl1 showed 155 binding energy values similar to the reference complex for -myrcene.

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In the case of the other reference complex, the ligand reported in the literature (limonene 1,2 epoxide) 157 is a modified form of the plant volatile used in our simulation experiments (the monoterpene 158 limonene). Therefore, we used this reference complex with less confidence. In any case, the energy 159 values were similar to the reference complex only for SAPB2.

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Results obtained for -caryophyllene, isoprene, and linalool could not be compared to a reference

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Interestingly, monoterpenes seem to bind more efficiently with OBPs that are also reported to bind 177 other plant volatiles. In particular, SABP2, the SA-binding protein that strongly binds the stress-178 induced volatile MeSA, also seems to be a candidate for the three monoterpenes tested. Protein 179 heading date 3A and tfl1, GA receptor and FT may also bind, perhaps more specifically, the three 180 monoterpenes. However, our results indicate that, as reported for the OBPs from animals and insects,     Overall, our study confirms that plant OBPs may exist, and that they may be structurally and 214 functionally similar to OBPs described in animals. As in the case of animal OBPs, also plant OBPs 215 seem to be able to bind different VOCs in the same binding site, using the same amino acid sequences.

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The search for potential OBP proteins in plants was performed following the procedure schematized 222 in Figure 1, and consisted of two levels of investigation.

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The first level was about amino acid sequences to search for plant proteins with potential OBP 224 function. The second level was about experimental 3D structures of the candidate plant OBPs, to 225 validate by molecular simulations their potential ability to bind volatile molecules.

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In detail for the first level, three steps (named a, b, and c, see Figure 1) were followed.
Step "a" was 227 a screening for proteins of interest performed on the UniProt (http://www.uniprot.org) and NCBI 228 (http://www.ncbi.nih.nlm.gov) protein databases. Initial screening was performed by using the 229 protein name and entry annotations, with the query "odorant binding protein". Five plant proteins 230 were found, annotated as "predicted proteins", which means that they were obtained by nucleotide 231 sequence translation, without evidence at protein or transcript levels, and the name was assigned to 232 the proteins by similarity to other proteins. In the case of the Uniprot Entry A0A1D1ZDX5, named 233 general odorant-binding protein 56d (OBP56d), from Anthurium amnicola, the annotations revealed 234 that the protein is included into the "Pheromone/general odorant-binding protein superfamily" of the 235 InterPro database (http://www.ebi.ac.uk/interpro/). In the case of UniProt entry A0A1D1Z329, 236 named putative odorant-binding protein A10_1 (OBPA10_1), from Anthurium amnicola, the 237 annotations revealed that the protein is included into the "Insect odorant-binding protein A10" protein 238 family of the InterPro database. These observations may explain the OBP annotation for these 239 proteins.

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The protein sequence selected were further investigated by BLAST searches for similar sequences, 241 by using the BLAST interfaces at the database web sites. Standard BLAST search parameters were 242 used, which means that results with E-value <10 are reported in the output. This setting leaves a wide 243 possibility of including results not significant, being the E-value < 0.001 considered the reference 244 threshold for similarity.

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Step "b" was of a search for plant proteins similar to the 432 OBPs from animal sources available in 246 the protein databases. BLAST searches performed for the 432 OBPs, identified plant proteins and 247 protein families with similarity to known OBPs.
Step "c" was about collecting information for plant 248 proteins whose OBP function has been experimentally tested (see Introduction).

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The second level of investigation concerned the study by molecular simulations of the interaction of

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To validate the docking simulation experimental protocol, we applied a re-docking procedure to the 277 reference complexes, following the procedure in use in our laboratory (Scafuri et al., 2016(Scafuri et al., , 2020. 278 We depleted the ligand from the complex ligand-protein and then the ligand-depleted complex (the 279 protein alone) was used to simulate the ligand docking. The re-docking experiments were carried out 280 for the protein-ligand reference structures selected above. This approach allowed us to check that the 281 simulation procedure located correctly the ligand in the expected binding site, and gave us the 282 reference value of the binding energy expected in the true protein-ligand complex.