# The glutathione peroxidase family of Theobroma cacao: Involvement in the oxidative stress during witches' broom disease

Akyla Maria Martins Alves, Sara Pereira Menezes Reis, Karina Peres Gramacho, Fabienne Micheli

<figure><img src="image_1.png" /></figure>

## To cite this version:

Akyla Maria Martins Alves, Sara Pereira Menezes Reis, Karina Peres Gramacho, Fabienne Micheli. The glutathione peroxidase family of Theobroma cacao: Involvement in the oxidative stress during witches' broom disease. International Journal of Biological Macromolecules, 2020, 164, pp.3698 - 3708. 10.1016/j.ijbiomac.2020.08.222. hal-03491961

HAL Id: hal-03491961

https://hal.science/hal-03491961

Submitted on 14 Sep 2022

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1

The glutathione peroxidase family of *Theobroma cacao*: involvement in the oxidative

2

stress during witches' broom disease

3

4

Akyla Maria Martins Alves¹, Sara Pereira Menezes Reis¹, Karina Peres Gramacho², Fabienne

5

Micheli¹,³,*

6

7

¹Universidade Estadual de Santa Cruz (UESC), Departamento de Ciências Biológicas (DCB),

8

Centro de Biotecnologia e Genética (CBG), Rodovia Ilhéus-Itabuna, km 16, 45662-900

9

Ilhéus-BA, Brazil. E-mail: akylamma@gmail.com; menezes_sp@yahoo.com.br

10

²Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brasil. Email:

11

karina@cepec.gov.br

12

³CIRAD, UMR AGAP, F-34398 Montpellier, France

13

14

*Corresponding author: Dr Fabienne Micheli, UESC, DCB, Rodovia Ilhéus-Itabuna km16,

15

45662-900, Ilhéus-BA, Brazil. Phone: +55 73 3680 5196. Fax: +55 73 3680 5226. E-mail:

16

fabienne.micheli@cirad.fr

17

18

**Abstract**

19

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system

20

inhibiting the ROS-induced damages of membranes and proteins. In cacao (*Theobroma cacao*

21

L.) genome, five *GPX* genes were identified. Cysteine insertion codons (UGU) were found in

22

*TcPHGPX*, *TcGPX2*, *TcGPX4*, *TcGPX6* and tryptophan insertion codon (UGG) in *TcGPX8*.

23

Multiple alignments revealed conserved domains between *TcGPXs* and other plants and

24

human GPXs. Homology modeling was performed using the *Populus trichocarpa* GPX5

25

structure as template, and the molecular modeling showed that *TcGPXs* have affinity with
selenometionine in their active site. In silico analysis of the *TcGPXs* promoter region revealed the presence of conserved *cis*-elements related to biotic stresses and hormone responsiveness.

The expression analysis of *TcGPXs* in cacao plantlet meristems infected by *M. perniciosa* showed that *TcGPXs* are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that *TcGPXs* may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from *T. cacao*.

34

Keywords: *Moniliophthora perniciosa*; programmed cell death; gene expression

36
## 1. Introduction

Theobroma cacao L. (cacao) is a plant endemic to the rainforests of South America [1] and is grown mainly for the production of cocoa liquor, cocoa butter, and cocoa powder for the chocolate industry [2]. However, cocoa production has been severely damaged by diseases caused by fungi and oomycete [1, 3]. In Brazil, the witches' broom disease caused drastic economic and social changes in the affected areas [4, 5]. This disease is caused by the *Moniliophthora perniciosa* basidiomycete that presents two distinct development phases: a biotrophic phase characterized by a monokaryotic and intercellular mycelium, and a necrotrophic phase characterized by a dikaryotic and intracellular mycelium [6, 7]. In the transition from biotrophic to necrotrophic fungal phase, the plant showed significant accumulation of calcium oxalate crystals and H₂O₂, as well as events of programmed cell death (PCD) [6, 8]. The accumulation of calcium oxalate causes the production of reactive oxygen species (ROS) and therefore the PCD. In cacao plants susceptible to *M. perniciosa*, the PCD occurs initially as a defense mechanism and then is deflected by the fungus for its own benefit, allowing sporulation and further propagation [6]. The transition from the biotrophic to the necrotrophic phase is facilitated by the presence of several compounds, genes, and proteins that induce PCD [8, 9]. In cacao-*M. perniciosa* interaction cDNA library and/or in the cacao genome, several genes potentially related to PCD (possibly involved in its enhancement or inhibition), such as the glutathione peroxidase (GPX; EC 1.11.1.9) were found [10, 11].

GPXs were first discovered in mammals and are enzymes that are part of the cell antioxidant system, inhibiting the ROS-induced damage of cell membrane and proteins [12]. GPXs reduce H₂O₂ to water and lipid hydroperoxides to their corresponding alcohols using reduced glutathione (GSH) as substrate [13]. All mammalian GPXs contain a selenocysteine (SeCys also referred to as the 21st protein amino acid) residue instead of a Cys residue [13-]
15]. The SeCys is encoded by an opal (TGA) stop codon, which is recognized by a special transfer RNA (tRNA); the incorporation of SeCys occurs when the mRNA contains a distinct hairpin mRNA sequence downstream of the UGA codon called SeCys insertion sequence (SECIS) [15, 16]. It is admitted that yeast and plants do not incorporate Sec and, therefore, do not have any selenoproteins [15]. However, some few studies revealed that plant kingdom may also contain selenoproteins: is has been shown that in *Chlamydomonas reinhardtii*, the GPX cDNA contained an internal TGA codon in frame to the ATG [17-19], in *Aloe vera*, native protein purification associated to atomic absorption revealed a molecular complex of four GPX subunits, each one containing one atom of selenium in its structure [20], and that in cranberry, the mitochondrial genome contained two copies of selenocysteine insertion sequence element (SECIS) and tRNA-Sec [21]. It has been shown that the incorporation of SeCys rather than Cys in the active site of the GPX confers better redox activity to these enzymes [16]. In animals, and even more in plants, only little information about the GPX activity regulation is available. In animals, it has been suggested a GPX regulation at mRNA, protein and/or activity level by selenium supply, SECIS-associating factors, but also by some selenium-independent regulator; this regulation may also vary according to the GPX sequence [22]. Some studies also pointed out the role of selenium as cofactor of GPX activity [23, 24]. Finally, it has also been shown the physical interaction between the human GPX1 and selenium-binding protein 1 (SBP1) – which is not a selenoprotein but is able to bind selenium covalently – associated to an inverse regulation of the two corresponding genes [25-27]. The mechanism by which SBP1 and GPX1 regulate each other remains unclear, but a competition for selenium available in the cell has been suggested as a possible mechanism of inverse regulation [28]. Altogether, these data suggested a possible physical interaction between GPX and selenium as possible element of activity regulation. In plants, selenium is absorbed from the soil in the form of selenate (SeO4²⁻) or selenite (SeO3²⁻) that are both toxic, and are used
87 to synthesize Secys and selenomethionine (SeMet) which are further converted to volatile
88 dimethyl(di)selenide (DM(D)Se) [16]. Therefore, SeMet is one of the most abundant and
89 metabolically important forms of selenium in plant, that could be also associated to plant
90 selenium detoxification mechanism [16].

91 *GPX* gene expression has been related with cell protection in various stress conditions
92 such as pathogenic attack, salt treatment, and mechanical stimulation [29]. Interestingly, in
93 humans, the *GPx1* gene was associated to several diseases, including cancer due to disease
94 risk-associated alleles [25, 28, 30]. Several studies have also reported that HsGPx1 proteins
95 were involved in cell cycle regulation, inhibiting apoptosis that allows tumor progression [25,
96 31, 32]. For these reasons, the putative inhibitory potential of PCD associated with the ability
97 to reduce H2O2 and lipid hydroperoxides makes GPXs important for understanding the cacao-
98 *M. perniciosa* pathosystem. Here, we identified and analyzed *in silico* and *in vitro* five GPX
99 sequences from *T. cacao*. TcGPX proteins did not contain SeCys in their primary sequence
100 but interact with SeMet. Expression analysis of *TcGPX* genes during the cacao-*M. perniciosa*
101 interaction showed a higher expression in susceptible cacao plants than in the resistant ones,
102 mainly in disease stages in which oxidative stress and PCD occurred. This data, associated
103 with phylogenetic and location analysis suggested that *TcGPXs* may play a role in protecting
104 cells from oxidative stress, and in inhibiting PCD as a try of disease progression reduction.
105 Moreover, the *TcGPX* expression may be regulated by several hormone pathways known to
106 be involved in the witches' broom disease development, as indicated by the *TcGPX* promoter
107 analysis. To our knowledge, this is the first study of the overall GPX family from *T. cacao*.
108

109 **2. Methods**

110 **2.1 In silico analysis of TcGPXs**
A GPX cDNA was identified in a library of *T. cacao* meristem (genotype TSH1188) infected by *M. perniciosa* [10]. A search in the CocoaGenDB database [11] through local alignment with BLASTn and BLASTp [33] using the GPX cDNA [10] allowed the identification of five homologous sequences named TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and TcGPX8 according to the nomenclature and numeration of the cocoa genome (Supplementary material 1). The complete sequences of the genes and corresponding proteins were obtained from the CocoaGenDB database. The proteins were analyzed using InterProScan [34] and their classification validated by the presence of the GPX domain codes. The MCScanX tool [35] was used to identify possible duplications of the TcGPX genes in the cacao genome. The prediction of theoretical isoelectric point (pI) and molecular weight (MW) was obtained using the Expasy Molecular Biology Server (www.expasy.org). The conserved domain and family protein were analyzed using the Pfam program [36]. Post-translational events were predicted using the NetPhos 2.0 Server to identify putative sites of phosphorylation (Ser/Thr/Tyr) [37] and NetNGlyc 1.0 Server [38] to identify putative sites of N-glycosylation. Predictions of subcellular localization were conducted with the programs TargetP [39] and PSORT [40]. Signal peptide presence was analyzed using the SignalP 4.0 Server [41]. Homologous sequences to TcGPXs were searched using BLAST [33] on the National Center for Biotechnology Information (NCBI) and plants and mammals sequences showing the highest homology levels (> 79%) were selected. Multiple sequence alignment of these sequences was performed with the ClustalW2 software [42], and an unrooted phylogenetic tree was built using the neighbour joining method with the ClustalW2, with 1000 bootstraps [42] and MEGA 6 [43] programs. The search for regulatory sequences of the *TcGPXs* gene was performed in the CocoaGenDB database [11]. A region of 1500 bp upstream the UTR was identified and selected for cis-element analysis. The presence of the
135

cis-regulatory elements in the promoter regions of the *TcGPX* genes was analyzed using the plantCARE (sphinx.rug.ac.be:8080/PlantCARE/cgi/index.html) software [44].

136

137

138

## 2.2 Molecular modeling and docking analysis

139

The prediction of the three-dimensional (3-D) models of the TcGPXs proteins was obtained using the Swiss-Model server and the Automated Protein Homology-modeling, which relies on the high similarity of target-template [45]. The crystal structure of poplar (*Populus trichocarpa*) glutathione peroxidase (PtGPX5; Protein Data Bank code: 2p5q) was used as the template to build the 3-D models of TcGPXs, because of its high identity with TcGPXs (>79%), high coverage of protein sequences and high resolution (2.0 Å). The stereochemical quality of the models was evaluated using the Procheck [46] and Anolea programs [47]. The 3-D model visualization was obtained using the PyMol (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.) and Discovery Studio 4.0. The docking analysis between TcGPXs and SeMet was performed using the AutoDockTools 1.5.6 [48]. SeMet 2D structure was found in PubChem databases (http://pubchem.ncbi.nlm.nih.gov). The 2D structure of this compound was copied into Similies and saved in PDB format using the Marvin program. The ligand and receiver structures were saved in pqbqt format to be used in docking calculations. AutoDock Vina was used to perform Docking Scoring for the complex formed by the receptor - ligand [49]. The reports of each calculation were analyzed for the energy (Kcal / Mol) values of affinity for each binder conformation in its respective complex. The Discovery Studio program was used to verify the ideal complex.

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

## 2.3 Plant material
Plant material was obtained as previously described [50]. Seeds of *T. cacao* genotypes
Catongo (susceptible to *M. perniciosa*) and TSH1188 (resistant to *M. perniciosa*) were
germinated and grown at CEPLAC/CEPEC (Ilhéus, Bahia, Brazil) greenhouses. Twenty to
thirty days after germination, the apical meristems of the plantlets were inoculated by the
droplet method [51] with a basidiospore suspension of *M. perniciosa* (inoculum from isolate
4145 maintained in the CEPLAC/CEPEC phytopathological *M. perniciosa* collection under
number 921 of the WFCC; http://www.wfcc.info/index.php/collections/display). After
inoculation, the plantlets were kept for 24 h at 25 ± 2°C and 100% humidity. Rate of disease
fixation based on presence/absence of symptoms [52] in each genotype, was evaluated 60
days after inoculation (dai); disease rate was 45% and 80% for TSH1188 and Catongo,
respectively. Moreover, the presence of *M. perniciosa* in the plant material was checked by
semi-quantitative RT-PCR using specific *M. perniciosa* actin primers [53]; both genotypes
presented fungus incidence (data not shown) coherent with previous data obtained in the same
conditions of plant culture and inoculation [7]. During the overall experiment, plant
development and presence/absence of symptoms were carefully checked and compared to
previous disease pattern established by our research group [7]. Apical meristems were
harvested at 24, 48 and 72 hours after inoculation (hai), and 8, 15, 30, 45, 60 and 90 dai. Non-
inoculated plants (controls) were kept and harvested under the same conditions at 24 and 72
hai, and **30 and 90** dai. Samples were collected at the same time of day (i.e. time of the
inoculation) to avoid possible fluctuation due to circadian rhythm. For each genotype and at
each harvesting time (for inoculated and non-inoculated plants), 20 samples were collected (1
sample = 1 apical meristem of 1 cacao plantlet). The 20 samples collected from one genotype
at one harvesting time were pooled; thus 9 inoculated and 5 non inoculated (control) samples
were immediately frozen in liquid nitrogen and stored at -80°C until use. Pooling samples
183

before RNA extraction has the advantage of reducing the variation caused by biological
replication and sample handling [54].

184

185

186

**2.4 Reverse transcription quantitative PCR analysis**

187

Samples were ground on liquid nitrogen until obtaining a fine powder. Total RNA was extracted from powdered samples using the RNAqueous Kit® (Ambion) according to the manufacturer's instructions, with modifications as previously described [50]. The RNA quality and quantity were checked using the Nanodrop 2000 spectrophotometer (Thermo Scientific). The synthesis of the first cDNA strand was carried out using Revertaid Fisrt Strand cDNA Synthesis Kit according to the manufacturer's instructions (Thermo Scientific). For the qPCR analysis, two cacao endogenous reference genes were used: the malate dehydrogenase (MDH) and β-actin (ACT), previously identified as *T. cacao* housekeeping genes [55] and tested in our experimental conditions (same plant material, same equipment) [50]. Specific primers and amplified regions containing different size, melting temperature, GC content and GC/AT ratio were defined to avoid cross-reaction between genes from cacao *GPX* family (Supplementary material 2). The expression analysis of *TcPHGPx* and *TcGPx2* was performed using standard settings of the ABI PRISM 7500 and Sequence Detection System (SDS) software, version 1.6.3 (Applied Biosystems).

188

189

190

191

192

193

194

195

196

197

198

199

200

201

The qPCR reaction consisted of 10 ng/µl of cDNA, 1 µM of each primer from reference or targets genes (Supplementary material 2) and 11 µl of Power SYBR Green Master Mix (Applied Biosystems) in a total volume of 22 µl. Cycling conditions were: 50°C for 2 min then 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min and 72°C for 30 s. To verify that each primer pair produced only a single PCR product, a dissociation analysis was carried out from 60°C to 95°C and analyzed with the Dissociation Curve 1.0 program (Applied Biosystems). The expression analysis of *TcGPXs* in meristems of

202

203

204

205

206

207
208 cacao plantlets inoculated or not with *M. perniciosa*, was obtained by RT-qPCR using 3
209 experimental replicates. The relative expression was obtained using the comparative Ct
210 method ($2^{-\Delta\Delta Ct}$) that considered: i) MDH and ACT as endogenous reference genes (average of
211 expression values from both genes); and ii) non-inoculated (control) plants (average of
212 expression values of each *TcGPX* gene in 5 control samples harvested as described above) as
213 calibrator - for this reason the relative expression value of the control (= non-inoculated
214 plants) is always 1.0. Statistical analysis was made using the SASM-Agri software [56] which
215 tested the experiments as a completely randomized design. *t*-test and *F*-test (ANOVA) were
216 applied with a critical value of 0.01. The Scott-Knott ($P\le0.01$) test was employed for mean
217 separation when *F*-values were significant.

218

219 **3. Results**

220 **3.1 In silico analysis of GPX family of T. cacao**

221 In silico analysis on CocoaGenDB revealed the presence of five sequences encoding
222 one *TcPHGPx* and four *TcGPXs* (*TcGPX2*, *TcGPX4*, *TcGPX6* and *TcGPX8*) (Fig. 1). *TcGPX*
223 genes were located on four different chromosomes (chromosome 1/*TcGPX4*, chromosome
224 3/*TcGPX6* and *TcGPX8*, chromosome 9/*TcPHGPX* and chromosome 5/*TcGPX2*; Fig. 1A). No
225 duplication events were found for any of the *TcGPXs* (data not shown). *TcGPX* genes were
226 2,222 to 4,805 bp in length (*TcGPX6* and *TcGPX4*, respectively; Fig. 1A), all contained 6
227 exons and 5 introns (Fig. 1A), and had ORF from 516 to 789 bp (*TcGPX4* and *TcGPX2*,
228 respectively; Table 1). *TcGPXs* encoded proteins from 171 to 262 amino acids (*TcGPX4* and
229 *TcGPX2*, respectively; Table 1), with molecular weight comprised between 19175.9 and
230 29708.3 Da (*TcGPX4* and *TcGPX2*, respectively; Table 1), and putative pI from 5.27 to 9.14
231 (*TcGPX8* and *TcGPX4*, respectively; Table 1). All *TcGPXs* showed putative phosphorylation
232 (from 7 for *TcGPX4* to 22 for *TcGPX6*) and glycosylation (from 1 for *TcPHGPX* and
TcGPX8 to 3 for TcGPX4 and TcGPX6) sites (Table 1). The TcPGXs were predicted to be located in chloroplast (TcPHGPX and TcGPX6), to be secreted (TcGPX2), or did not present any defined subcellular localization (unknown for TcGPX4 and TcGPX8; Table 1). The TcGPXs mature RNA showed the initiation codes (AUG) and cysteine insertion codons (UGU) in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and the tryptophan insertion codon (UGG) in TcGPX8 (Fig. 1B). All TcGPXs showed **at least one** conserved GSH domain (Fig. 1C). TcGPX family showed a cysteine instead of selenocysteine on the active site of TcPHGX, TcGPX2, TcGPX4 and TcGPX6 and a tryptophan on the active site of TcGPH8 instead of a selenocysteine (Fig. 1C). The alignment of TcGPXs sequence using the BLASTP tool revealed high identity with GPXs from other organisms (plant and mammals) and allowed the identification of the conserved region containing a conserved cysteine residue in plants and **SeCys** is mammals (Supplementary material 3). The full-length amino acid sequences of plants and mammalian of GPXs available in the databases were used to build an unrooted phylogenetic tree (Supplementary material 4). Plant and mammalian GPXs were separated and formed two distinct branches in the tree (Fig. 2). The five TcGPXs have **close** phylogenetic relationship with members of the same classification in other plant species (Fig. 2). **In silico** analysis of the TcGPXs promoter region revealed the presence of several conserved **cis**-elements characteristic of different plant species (Table. 2). Elements recognized by the family of WRKY transcription factors (W box) were identified in all TcGPX promoters, and defense and stress responsiveness elements (TC-rich repeats) were identified in TcPHGPX, TcGPX2, TcGPX6 and TcGPX8 (Table 2). Elements related to response to abiotic stress or external stimuli anaerobic induction (ARE), light responsiveness (BOX4), drought-inducibility (MBS), Low-temperature responsiveness (LTR) were also found (Table 2). Finally, several **cis**-elements related to hormone signaling were found such as abscisic acid responsiveness (ABRE), gibberellin-responsive element (GARE-motif),
258 MeJA-responsiveness (TGACG-motif), salicylic acid responsiveness, (TCA-element) and
259 auxin-responsive element (TGA-element) (Table 2).

260

261 **3.2 Homology modeling of GPXs and docking analysis**

262 The alignment of the PtGPX5 (3-D template) amino acid sequence showed 63.92%,
263 69.03%, 59.17%, 75.15% and 84.02% with TcPHGPXs, TcGPX2, TcGPX4, TcGPX6,
264 TcGPX8, respectively (Fig. 3A). The structural modeling of TcPHGPX, **TcGPX4** and
265 **TcGPX6** showed 8 **α**-helices and 6 **β**-strands (Fig.3B, 3D and 3E) while TcGPX2 showed 6 **α**-
266 helices and, 6 **β**-strands (Fig.3C) **and** TcGPX8 7 **α**-helices and 6 **β**-strands (Fig.3F). The
267 Ramachandran plots showed that 100% of the amino acids of the TcGPXs models were
268 located in the most favored regions (Supplementary material 5). The catalytic site of
269 TcPHGPX, TcGPX2, TcGPX4, and TcGPx6 contained the conserved cysteine (Cys114, Cys75,
270 Cys44, and Cys111, respectively) **residue** (Fig. 3A, B, C, D and E). TcGPX8 contained two
271 conserved tryptophan (W44, W151) **residue** (Fig. 3A and F). The docking between TcPHGPXs
272 and SeMet showed conventional hydrogen bond interactions between Lys113, Cys114, Thr163
273 and, carbon hydrogen bond between Pro151 (Fig.4A). The **interaction** between TcGPX4 and
274 SeMet showed conventional hydrogen bond between Gln75, Ser129, Asn108, Arg136 and,
275 unfavorable donor-donor between Arg130 (Fig.4B). The interaction between TcGPX6 and
276 SeMet showed conventional hydrogen bond between Asp196, Arg216, Thr221 and, unfavorable
277 donor-donor between Ala218 (Fig.4C). The interaction between TcGPx8 and SeMet showed
278 Pi-sulfur interaction between Phe58, conventional hydrogen bond between Tyr45, Trp44 and
279 Glu97 and carbon hydrogen between Trp151 (Fig. 4D). No interaction was observed between
280 TcGPX2 and SeMet (data not shown).

281
282

## 3.3 Differential expression of *TcGPXs* in resistant and susceptible cacao genotypes infected by *M. perniciosa*

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

The expression of the *TcPHGPX*, *TcGPX2*, *TcGPX4*, *TcGPX6*, and *TcGPX8* genes was analyzed together for each cacao genotype, i.e. TSH1188 (resistant to witches' broom disease) and Catongo (susceptible), infected or not (control) with *M. perniciosa* (Fig.5A). For both genotypes and for all the harvesting points, the PCR amplification occurred at the same and unique melting temperature for each gene showing that only the corresponding gene was amplified (Supplementary material 6). When we analyzed the gene expression the according to the disease stage (time after inoculation), we verified that, in the resistant variety, the *TcPHGPX* gene did not differ significantly at 24 and 48 hai, with a small repression compared to the non-inoculated control (Fig.5B). At 72 hai, the *TcPHGPX* gene becomes significantly more expressed than the control (Fig.5B). At 8 and 15 dai, the expression of the *TcPHGPX* gene did not differ significantly, however, they were significantly more expressed than the control (Fig.5B). At 30 dai, *TcPHGPX* was significantly less expressed than the control, and at 45 and 90 dai did not differ from the control (Fig.5B). At 60 dai, the *TcPHGPX* gene was overexpressed in the resistant variety (Fig.5B). In the resistant variety, the *TcGPX2* gene did not differ significantly from the control after 24, 48 and 72 hai, as well as after 15, 45, and 60 dai after inoculation (Fig.5B). At 8 dai, the *TcGPX2* gene was repressed compared to the control (Fig.5B). At 30 dai, the *TcGPX2* gene was also less expressed than the control (Fig.5B). At 90 dai, the *TcGPX2* gene was significantly more expressed than in the non-inoculated control (Fig.5B). The *TcGPX4* gene was only significantly more expressed than the control at 60 and 90 dai (Fig.5B). The *TcGPX6* gene was significantly more expressed at 30, 45, and 60 dai, with a peak expression of 60 dai, followed by a 90 dai repression (Fig.5B). The *TcGPX8* gene was significantly more expressed than the control at 45, 60, and 90 dai. In the previous periods they were less expressed than the non-inoculated control
307 (Fig.5B). In the susceptible variety the *TcPHGPX* gene was overexpressed at 24 and 48 hai
308 (Fig.5C). At 72 hai and at 8 and 15 dai, the PHGPX gene was differentially less expressed
309 than the non-inoculated control (Fig.5C). At 30 and 45 dai, the *TcPHGPX* gene did not differ
310 significantly from the non-inoculated control (Fig.5C). The *TcGPX2* gene was repressed
311 within 24 hai after being overexpressed at 48 and 72 hai (Fig.5C). At 30 dai the *TcGPX2* gene
312 was less expressed than the control (Fig.5C). At 24 hai, 8, 15, and 60 dai the *TcGPX4* gene
313 did not differ from the control (Fig.5C). At 48 and 72 hai and 90 dai *TcGPx4* was less
314 expressed than the control (Fig.5C). At 30 and 45 dai the *TcGPX4* gene was overexpressed
315 (Fig.5C). The *TcGPx6* gene was overexpressed at 24 hai and 30, 45 and 90 dai. At 48 and 72
316 hai and 8 and 15 dai the expression of the *TcGPX6* gene did not differ significantly from the
317 non-inoculated control. At 60 dai the *TcGPX6* gene was significantly less expressed than the
318 control (Fig.5C). The *TcGPX8* gene was overexpressed at 24 hai and 30 and 45 dai (Fig.5C).
319 It was significantly less expressed than the control at 48 and 72 hai (Fig.5C). At 60 and 90 dai
320 the *TcGPX8* gene was significantly more expressed than the control (Fig.5C).

321 When comparing the expression of the *TcGPX* family genes, we observed that in the
322 resistant variety, 24 hai there was significant difference between the *TcPHGPX* and *TcGPX8*
323 genes (Fig.5B). At 48 hai there was no significant difference between the genes of the *TcGPX*
324 family (Fig.5B). At 72 hai *TcPHGPX* and *TcGPX6* were significantly more expressed
325 (Fig.5B). *TcGPx4* and *TcGPx8* did not differ significantly after 72 hai (Fig.5B). At 8 dai
326 *TcPHGPX* was significantly more expressed and *TcGPX2* was less expressed (Fig.5B). At 15
327 dai there was no difference between the genes of the *TcGPX* family (Fig.5B). At 30 dai
328 *TcGPX6* was significantly more expressed, whereas *TcPHGPX* and *TcGPx2* were less
329 expressed (Fig.5B). At 43 dai *TcGPX6* and *TcGPX8* were significantly more expressed,
330 respectively (Fig.5B). After 60 dai *TcGPx6* was significantly more expressed, whereas
331 *TcGPX2* was less expressed (Fig.5B). At 90 dai the *TcGPX2*, *TcGPX4* and *TcGPX8* genes
332 were significantly more expressed than the *TcPHGPX* and *TcGPX6* genes (Fig.5B).

333 In the susceptible variety, after 24 hai the *TcPHGPX* gene was overexpressed and
334 *TcGPX2* was less expressed than the control (Fig.5C). At 48 hai *TcPHGPX* and *TcGPX2* were
335 significantly more expressed, whereas *TcGPX4*, *TcGPX6* and *TcGPX8* were less expressed
336 than the control (Fig.5C). At 8 dai *TcGPX2* and *TcGPX4* did not differ from the control,
337 whereas *TcPHGPX*, *TcGPX6* and *TcGPX8* were less expressed than the control
338 (Fig.5C). After 30 dai the *TcGPX4* and *TcGPX8* genes were overexpressed and *TcGPX2* was
339 less expressed than the control (Fig.5C). At 45 dai *TcGPX4*, *TcGPX6* and *TcGPX8* were
340 significantly more expressed than *TcPHGPX* and *TcGPX2* (Fig.5C). After 60 dai the
341 *TcPHGPX*, *TcGPX2*, and *TcGPX6* genes were less expressed than the control. The *TcGPX4*
342 and *TcGPX8* genes did not differ from the control (Fig.5C). At 90 dai the *TcGPX6* gene was
343 significantly more expressed and the *TcGPX4* gene was less expressed than the non-
344 inoculated control (Fig.5C).

345

346 **4. Discussion**

347 **4.1 TcGPX protein family did not contain SeCys but interacted *in silico* with SeMet**

348 Here, we characterized five GPXs (*TcPHGPX*, *TcGPX2*, *TcGPX4*, *TcGPX6* and
349 *TcGPX8*) sequences from *T. cacao* (Fig. 1). **All the TcGPX protein, except TcGPX8,**
350 **contained a conserved GSHPX domain with conserved Cys residues (Fig. 1C), which are**
351 **important for the protein function [13]. Even if it has been related in few studies the possible**
352 **existence of selenoproteins in plants [17, 20, 21], our results corroborate the difference**
353 already observed **in most of the cases** between GPXs from plants and animals: the active site
354 of plant GPXs contains a Cys while the one of animal GPXs contains SeCys [57]. **In the case**
355 **of TcGPX8, the Cys residue was substituted by a tryptophan residue within the GSHPX**
domain. Although tryptophan is an amino acid with antioxidant activity [58] and could act as a hydrogen donor [59], it differs greatly from the chemical structure of cysteine. It could be suggested that TcGPX8 may be a non-functional GPX due to some sequence truncation or mutation. On the other hand, here, we showed that TcGPXs (except TcGPX2) are able to bind in silico the SeMet chemical element (Fig. 4). Some plant GPXs may have increased activity or be selenodependent even without the presence of the UGA codon for incorporation of SeCys [22, 28]. The possibility of GPXs having increased activity when interacting with selenium compounds is due to the fact that selenium serves as a GPX cofactor [23, 24]. Thus, as suggested here by molecular docking assays, the TcGPXs may present PCD inhibition and antioxidant activities if they were associated with selenium compounds (Fig. 4).

366

## 4.2 TcGPXs are ubiquitous protein potentially involved in cell death inhibition

All the TcGPX proteins contained multiple predicted phosphorylation and glycosylation sites (Table 1), which are ubiquitous mechanism for the temporal and spatial regulation of proteins involved in almost every cellular process [60]. The TcGPXs family showed an ubiquitous subcellular localization (Table 1) including chloroplast location (TcHGPX and TcGPX6) where these proteins may use GSH or thioredoxin as a reducing agent to reduce H₂O₂, organic hydroperoxide, and lipid hydroperoxides [61]. Furthermore, in Arabidopsis it has been shown that chloroplastic GPXs play a role in cross talk between photooxidative stress and immune responses [61, 62]. The phylogenetic analysis of TcGPXs with different GPX from plants and animals showed that they were very conserved inclusive for each sub-group of GPX, i.e. PHGPXs vs GPXs (Fig. 2). The sequence and structure conservation of TcGPXs also suggests a conservation of function as indicated by other authors [13]. In mammal signal transduction pathways, it has been suggested that PHGPx functions as an anti-apoptotic agent in mitochondrial death signaling [63]. The antioxidant

368

369

370

371

372

373

374

375

376

377

378

379

380
381

GPX enzymes eliminate ROS and reduce H₂O₂ [64, 65] and in human, are known to be
inhibitors of apoptosis [66]. Functional studies of *Lycopersicon esculentum* PHGPX
(LePHGPx) showed that this gene acted as a cytoprotector in yeast, preventing Bax, hydrogen
peroxide, and heat stress induced cell death, but also protected tobacco leaves from salt and
heat stress and suppressed the apoptotic like features when expressed transiently in this plant
[67]. In addition, stable expression of LePHGPx in tobacco afforded protection against the
necrotrophic fungus *Botrytis cinereal* [67].

382

383

384

385

386

387

388

389

## 4.3 *TcGPXs* may be involved in PCD reduction during *T. cacao* – *M. perniciosa*
interaction

390

391

The expression analysis of *TcGPX* genes by RT-qPCR in cacao plant meristems
infected by *M. perniciosa* showed that these genes are significantly more expressed in the
susceptible variety than in the resistant one (Fig. 5B and C). Moreover, considering the
susceptible variety, *TcGPX* are most expressed during asymptomatic and green broom stages
(Fg.5). During dry broom phase *TcGPx6* is significantly more expressed than the other genes
in the susceptible variety (Fig.5C). *TcGPx2* is significantly more expressed in swelling phase
in susceptible variety (Fig. 5A e C). In previous works, it has been showed that calcium
oxalate crystal amount and H₂O₂ levels in the two cacao varieties present distinct temporal
and genotype dependent patterns [8]. Susceptible variety accumulated more CaOx crystals
than the resistant one [8, 9], the dissolution of these crystals occurred in the asymptomatic and
in the final stages of the disease in the resistant and the susceptible variety, respectively [8, 9].
Considering the role of the GPXs in the antioxidant system inhibiting the ROS-induced
damages, it could be suggested that in the susceptible cacao genotype infected with *M.*
*perniciosa*, *TcGPXs* may play a role in protecting cells from oxidative stress and trying
reducing the progression of the disease by inhibiting PCD. Moreover, the increase of

392

393

394

395

396

397

398

399

400

401

402

403

404

405
406

*TcPHGPX* expression in susceptible variety in the initial stage of witches' broom disease may
be related to protection against pathogens observed in other pathosystems [67].

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

The *TcGPX* expression regulation was led by promoter regions, which contained
several *cis*-elements related to biotic (WRKY transcription factors) and abiotic stress (low-
temperature responsiveness) besides to hormone signaling such abscisic acid, gibberellin,
methyljasmonate-responsiveness, salicylic acid and, auxin-responsive element (Table 2).
Most of these hormones are involved in witches’ broom disease development [10, 68-70].
Several genes related to plant hormone signaling were altered in cacao plants in response to
*M. perniciosa* infection, such ethylene-responsive element, ABA-responsive protein, auxin-
responsive protein, jasmonic acid 2, and brassinosteroid signalling positive regulator [10].
Gibberellin-responsive, ethylene biosynthesis as well as auxin-responsive genes were also
previously identified as up-regulated in green broom [70]. Is has been suggested that the up-
regulation of auxin-responsive genes in infected cacao plants is a consequence of the
biosynthesis of this hormone by *M. perniciosa* [68-70]. In various plant pathogens, the
production of auxin has been correlated to the reduction of the plant defenses mediated by
salicylic acid, and consequently to the pathogenicity and to the biotrophic stage development
[68, 70].

423

424

**5. Conclusion**

425

426

427

428

429

430

Here we showed the first *in silico* and *in vitro* analysis of the GPX family from cacao.
Five *TcGPXs* sequences were analyzed at nucleotide and protein levels, and showed that the
*TcGPX* proteins did not contain *SeCys* in their primary sequence but interacts *in silico* with
*SeMet* as element participating of the *TcGPX* activity. This characteristic, associated to
phylogenetic and location analyses, suggested the participation of *TcGPX* in cell death
inhibition. Finally, the *TcGPX* genes were more expressed in susceptible cacao plants infected
by *M. perniciosa* than in the resistant ones, mainly in the early disease and in the green broom stages, suggesting that *TcGPXs* may play a role in protecting cells from oxidative stress, in inhibiting PCD as a try of disease progression reduction. This expression may be regulated by several hormone pathways known to be involved in the witches' broom disease development.

435

### Availability of supporting data

The data sets supporting the results of this article are included within the article and its additional files.

439

### Abbreviations

ACT: actin; dai: days after inoculation; GPX: glutathione peroxidase; GSH: reduced glutathione; hai: hour after inoculation; pI: isoeletric point; MDH: malate dehydrogenase; MW: molecular weight; ORF: open reading frame; PCD: programmed cell death; PHGPX: phospholipid hydroperoxide glutathione peroxidase; UTR: untranslated regions.

445

### Competing interest

No conflicts of interest to declare.

448

### Authors' contribution

AMMA was responsible for the execution of the all the experimental steps; AMMA, SPMR, and FM analyzed and discussed the data; AMMA and FM wrote the manuscript; SPMR gave support in qPCR experiment; KPG was responsible for plant material production and inoculation with *M. perniciosa*; FM and AMMA were responsible for the conception and design of the experiments; FM was responsible for the financial support of the research and for the advising of AMMA and SPMR.

450

451

452

453

454

455
456

457 Acknowledgements

458 The work of AMMA was supported by the Fundação de Amparo à Pesquisa do Estado da
459 Bahia (FAPESB). The work of SPMR was supported by the Coordenação de
460 Aperfeiçoamento de Pessoal de Nível Superior (CAPES). KPG and FM received o
461 Productivity grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico
462 (CNPq). This research was supported by the FAPESB project (DTE0038/2013) coordinated
463 by FM. The authors thank Francisca Feitosa Jucá (UESC/Ceplac) and Louise Araújo Sousa
464 (Ceplac) for technical help in plant inoculation experiments. The authors thank Raner José
465 Santana Silva (UESC) for advising in modeling, and Dr. Janisete Gomes da Silva Miller
466 (UESC) for correcting English language. This work was developed in the frame of the
467 International Consortium in Advanced Biology (CIBA; https://www.ciba-network.org/).

468

469 References

470 [1] G.A.R. Wood, R.A. Lass, Cocoa, Fourth Edition ed., Blackwell Science Ltd.1985.

471 [2] E.O. Afoakwa, A. Paterson, M. Fowler, A. Ryan, Flavor formation and character in cocoa and
chocolate: a critical review, Crit Rev Food Sci Nutr 48(9) (2008) 840-57.

473 [3] F. Micheli, M. Guiltinan, K.P. Gramacho, M.J. Wilkinson, A.V.d.O. Figueira, J.C.d.M. Cascardo, S.

474 Maximova, C. Lanaud, Chapter 3 - Functional genomics of cacao, in: K. Jean-Claude, D. Michel (Eds.),
475 Advances in Botanical Research, Academic Press2010, pp. 119-177.

476 [4] H.M. Rocha, R.A.C. Miranda, R.B. Sgrillo, R.A. Setubal, Witches' broom in Bahia, Brazil., in: S.A.

477 Rudgard, M. A.C., A. T. (Eds.), Disease and management in Cocoa : Comparative epidemiology of
478 witches' broom, Chapman and Hall, London, 1993, pp. 189-200.

479 [5] L.W. Meinhardt, J. Rincones, B.A. Bailey, M.C. Aime, G.W. Griffith, D. Zhang, G.A. Pereira,

480 *Moniliophthora perniciosa*, the causal agent of witches' broom disease of cacao: what's new from
481 this old foe?, Molecular Plant Pathology 9(5) (2008) 577-88.

482 [6] O. Garcia, J.A. Macedo, R. Tiburcio, G. Zaparoli, J. Rincones, L.M. Bittencourt, G.O. Ceita, F.

483 Micheli, A. Gesteira, A.C. Mariano, M.A. Schiavinato, F.J. Medrano, L.W. Meinhardt, G.A. Pereira, J.C.

484 Cascardo, Characterization of necrosis and ethylene-inducing proteins (NEP) in the basidiomycete

485 *Moniliophthora perniciosa*, the causal agent of witches' broom in *Theobroma cacao*, Mycol Res
486 111(Pt 4) (2007) 443-55.

487 [7] K. Sena, L. Alemanno, K.P. Gramacho, The infection process of *Moniliophthora perniciosa* in cacao,
488 Plant Pathology (2014) 1272-1281.

489 [8] C.V. Dias, J.S. Mendes, A.C. dos Santos, C.P. Pirovani, A. da Silva Gesteira, F. Micheli, K.P.

490 Gramacho, J. Hammerstone, P. Mazzafera, J.C. de Mattos Cascardo, Hydrogen peroxide formation in
cacao tissues infected by the hemibiotrophic fungus *Moniliophthora perniciosa*, Plant Physiol

492 Biochem 49(8) (2011) 917-922.
[9] G. de Oliveira Ceita, J.N.A. Macêdo, T.B. Santos, L. Alemanno, A. da Silva Gesteira, F. Micheli, A.C. Mariano, K.P. Gramacho, D. da Costa Silva, L. Meinhardt, P. Mazzafera, G.A.G. Pereira, J.C. de Mattos Cascardo, Involvement of calcium oxalate degradation during programmed cell death in *Theobroma cacao* tissues triggered by the hemibiotrophic fungus *Moniliophthora perniciosa*, Plant Sci 173(2) (2007) 106-117.
[10] A.S. Gesteira, F. Micheli, N. Carels, A. Da Silva, K. Gramacho, I. Schuster, J. Macedo, G. Pereira, J. Cascardo, Comparative analysis of expressed genes from cacao meristems infected by *Moniliophthora perniciosa*, Ann Bot 100(1) (2007) 129-140.
[11] X. Argout, J. Salse, J.-M. Aury, M.J. Guiltinan, G. Droc, J. Gouzy, M. Allegre, C. Chaparro, T. Legavre, S.N. Maximova, M. Abrouk, F. Murat, O. Fouet, J. Poulain, M. Ruiz, Y. Roguet, M. Rodier-Goud, J.F. Barbosa-Neto, F. Sabot, D. Kudrna, J.S.S. Ammiraju, S.C. Schuster, J.E. Carlson, E. Sallet, T. Schiex, A. Dievart, M. Kramer, L. Gelley, Z. Shi, A. Berard, C. Viot, M. Boccara, A.M. Risterucci, V. Guignon, X. Sabau, M.J. Axtell, Z. Ma, Y. Zhang, S. Brown, M. Bourge, W. Golser, X. Song, D. Clement, R. Rivallan, M. Tahi, J.M. Akaza, B. Pitollat, K. Gramacho, A. D'Hont, D. Brunel, D. Infante, I. Kebe, P. Costet, R. Wing, W.R. McCombie, E. Guiderdoni, F. Quetier, O. Panaud, P. Wincker, S. Bocs, C. Lanaud, The genome of *Theobroma cacao*, Nat Genet 43 (2011) 101-108.
[12] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?, Nature Reviews Drug Discovery 8 (2009) 579.
[13] R. Margis, C. Dunand, F.K. Teixeira, M. Margis-Pinheiro, Glutathione peroxidase family – an evolutionary overview, FEBS Journal 275(15) (2008) 3959-3970.
[14] S. Herbette, P. Roeckel-Drevet, J.R. Drevet, Seleno-independent glutathione peroxidases. More than simple antioxidant scavengers, FEBS J 274(9) (2007) 2163-80.
[15] E. Zoidis, I. Seremelis, N. Kontopoulos, G.P. Danezis, Selenium-Dependent Antioxidant Enzymes: Actions and Properties of Selenoproteins, Antioxidants (Basel) 7(5) (2018) 66.
[16] M. Schiavon, E.A. Pilon-Smits, The fascinating facets of plant selenium accumulation - biochemistry, physiology, evolution and ecology, New Phytol 213(4) (2017) 1582-1596.
[17] L.-H. Fu, X.-F. Wang, Y. Eyal, Y.-M. She, L.J. Donald, K.G. Standing, G. Ben-Hayyim, A Selenoprotein in the Plant Kingdom: MASS SPECTROMETRY CONFIRMS THAT AN OPAL CODON (UGA) ENCODES SELENOCYSTEINE IN CHLAMYDOMONAS REINHARDTII GLUTATHIONE PEROXIDASE, Journal of Biological Chemistry 277(29) (2002) 25983-25991.
[18] S.V. Novoselov, M. Rao, N.V. Onoshko, H. Zhi, G.V. Kryukov, Y. Xiang, D.P. Weeks, D.L. Hatfield, V.N. Gladyshev, Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii, The EMBO Journal 21(14) (2002) 3681-3693.
[19] D.Y. Lee, O. Fiehn, High quality metabolomic data for Chlamydomonas reinhardtii, Plant Methods 4 (2008) 7-7.
[20] F. Sabeh, T. Wright, S.J. Norton, Purification and Characterization of a Glutathione Peroxidase from the Aloe vera Plant, Enzyme and Protein 47 (1993) 92-98.
[21] D. Fajardo, B. Schlautman, S. Steffan, J. Polashock, N. Vorsa, J. Zalapa, The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants, Gene 536(2) (2014) 336-43.
[22] X.G. Lei, W.H. Cheng, J.P. McClung, Metabolic regulation and function of glutathione peroxidase-1, Annu Rev Nutr 27 (2007) 41-61.
[23] S. Chan, B. Gerson, S. Subramaniam, The role of copper, molybdenum, selenium, and zinc in nutrition and health., Clin Lab Med. 18(4) (1998) 673-85.
[24] M. Puccinelli, F. Malorgio, B. Pezzarossa, Selenium Enrichment of Horticultural Crops, Molecules 22(6) (2017).
[25] W. Fang, M.L. Goldberg, N.M. Pohl, X. Bi, C. Tong, B. Xiong, T.J. Koh, A.M. Diamond, W. Yang, Functional and physical interaction between the selenium-binding protein 1 (SBP1) and the glutathione peroxidase 1 selenoprotein, Carcinogenesis 31(8) (2010) 1360-1366.
[26] M. Schott, M.M. de Jel, J.C. Engelmann, P. Renner, E.K. Geissler, A.K. Bosserhoff, S. Kuphal, Selenium-binding protein 1 is down-regulated in malignant melanoma, Oncotarget 9(12) (2018) 10445-10456.
[27] F. Schild, S. Kieffer-Jaquinod, A. Palencia, D. Cobessi, G. Sarret, C. Zubieta, A. Jourdain, R. Dumas, V. Forge, D. Testemale, J. Bourguignon, V. Hugouvieux, Biochemical and Biophysical Characterization of the Selenium-binding and Reducing Site in Arabidopsis thaliana Homologue to Mammals Selenium-binding Protein 1, Journal of Biological Chemistry 289(46) (2014) 31765-31776.

[28] E. Ansong, W. Yang, A.M. Diamond, Molecular cross-talk between members of distinct families of selenium containing proteins, Molecular nutrition & food research 58(1) (2014) 117-123.

[29] W.-J. Li, H. Feng, J.-H. Fan, R.-Q. Zhang, N.-M. Zhao, J.-Y. Liu, Molecular cloning and expression of a phospholipid hydroperoxide glutathione peroxidase homolog in Oryza sativa1, Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1493(1-2) (2000) 225-230.

[30] A. Jerome-Morais, M.E. Wright, R. Liu, W. Yang, M.I. Jackson, G.F. Combs, A.M. Diamond, Inverse Association Between Glutathione Peroxidase Activity and Both Selenium Binding Protein 1 Levels and Gleason Score in Human Prostate Tissue, The Prostate 72(9) (2012) 1006-1012.

[31] C. Huang, G. Ding, C. Gu, J. Zhou, M. Kuang, Y. Ji, Y. He, T. Kondo, J. Fan, Decreased Selenium-Binding Protein 1 Enhances Glutathione Peroxidase 1 Activity and Downregulates HIF-1α to Promote Hepatocellular Carcinoma Invasiveness, Clinical Cancer Research 18(11) (2012) 3042-3053.

[32] J.-Y. Jeong, J.-R. Zhou, C. Gao, L. Feldman, A.J. Sytkowski, Human selenium binding protein-1 (hSP56) is a negative regulator of HIF-1α and suppresses the malignant characteristics of prostate cancer cells, BMB Reports 47(7) (2014) 411-416.

[33] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res 25(17) (1997) 3389-402.

[34] E. Quevillon, V. Silventoinen, S. Pillai, N. Harte, N. Mulder, R. Apweiler, R. Lopez, InterProScan: protein domains identifier, Nucleic Acids Research 33(suppl 2) (2005) W116-W120.

[35] Y. Wang, H. Tang, J.D. DeBarry, X. Tan, J. Li, X. Wang, T.-h. Lee, H. Jin, B. Marler, H. Guo, J.C. Kissinger, A.H. Paterson, MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity, Nucleic Acids Res 40(7) (2012) e49-e49.

[36] S. El-Gebali, J. Mistry, A. Bateman, S.R. Eddy, A. Luciani, S.C. Potter, M. Qureshi, L.J. Richardson, G.A. Salazar, A. Smart, E.L.L. Sonnhammer, L. Hirsh, L. Paladin, D. Piovesan, S.C.E. Tosatto, R.D. Finn, The Pfam protein families database in 2019, Nucleic Acids Research 47(D1) (2019) D427-D432.

[37] N. Blom, S. Gammeltoft, S. Brunak, Sequence and structure-based prediction of eukaryotic protein phosphorylation sites, J Mol Biol 294(5) (1999) 1351-62.

[38] R. Gupta, S. Brunak, Prediction of glycosylation across the human proteome and the correlation to protein function, Pac Symp Biocomput (2002) 310-22.

[39] O. Emanuelsson, S. Brunak, G. von Heijne, H. Nielsen, Locating proteins in the cell using TargetP, SignalP and related tools, Nat. Protocols 2(4) (2007) 953-971.

[40] K. Nakai, P. Horton, PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization, Trends in Biochemical Sciences 24(1) (1999) 34-35.

[41] T.N. Petersen, S. Brunak, G. von Heijne, H. Nielsen, SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat Meth 8(10) (2011) 785-786.

[42] M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H. McWilliam, F. Valentin, I.M. Wallace, A. Wilm, R. Lopez, J.D. Thompson, T.J. Gibson, D.G. Higgins, Clustal W and Clustal X version 2.0, Bioinformatics 23(21) (2007) 2947-2948.

[43] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0, Molecular Biology and Evolution 30(12) (2013) 2725-2729.

[44] M. Lescot, P. Déhais, G. Thijs, K. Marchal, Y. Moreau, Y. Van de Peer, P. Rouzé, S. Rombauts, PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences, Nucleic Acids Research 30(1) (2002) 325-327.

[45] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling, Bioinformatics 22(2) (2006) 195-201.

[46] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK: a program to check the stereochemical quality of protein structures, Journal of Applied Crystallography 26(2) (1993) 283-291.
597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

[47] F. Melo, E. Feytmans, Assessing protein structures with a non-local atomic interaction energy, Journal of Molecular Biology 277(5) (1998) 1141-1152.

[48] M. Sanner, A.J. Olson, J.C. Spehner, Reduced Surface: an Efficient Way to Compute Molecular Surfaces, Biopolymers 38(3) (1996) 305-320.

[49] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry 31(2) (2010) 455-461.

[50] S. Pereira Menezes, E. de Andrade Silva, E. Matos Lima, A. Oliveira de Sousa, B. Silva Andrade, L. Santos Lima Lemos, K. Peres Gramacho, A. da Silva Gesteira, C. Pirovani, F. Micheli, The pathogenesis-related protein PR-4b from *Theobroma cacao* presents RNase activity, Ca²⁺ and Mg²⁺ dependent-DNase activity and antifungal action on *Moniliophthora perniciosa*, BMC Plant Biol 14(1) (2014) 1-17.

[51] G. Frias, L. Purdy, R. Schmidt, An inoculation method for evaluating resistance of cacao to *Crinipellis perniciosa*, Plant Disease 79 (1995) 787-791.

[52] S. Silva, K. Matsuoka, Histologia da interação *Crinipellis perniciosa* em cacaueiros suscetível e resistente à vassoura de bruxa, Fitopatol. Bras. 24 (1999) 54-59.

[53] R.X. Santos, S.C.O. Melo, J.C.M. Cascardo, M. Brendel, C. Pungartnik, Carbon source-dependent variation of acquired mutagen resistance of *Moniliophthora perniciosa*: Similarities in natural and artificial systems, Fungal Genetics and Biology 45(6) (2008) 851-860.

[54] S. Duplessis, P.-E. Courty, D. Tagu, F. Martin, Transcript patterns associated with ectomycorrhiza development in *Eucalyptus globulus* and *Pisolithus microcarpus*, New Phytologist 165(2) (2004) 599-611.

[55] T.T. Pinheiro, C.G. Litholdo Jr., M.L. Sereno, G.A. Leal Jr., P.S.B. Albuquerque, A. Figueira, Establishing references for gene expression analyses by RT-qPCR in *Theobroma cacao* tissues, Genet Mol Res 10(4) (2012) 3291 - 3305.

[56] M.G. Canteri, R.A. Althaus, J.S. das Virgens Filho, E.A. Giglioti, C.V. Godoy, SASM-AGRI - Sistema para analise e separação de médias em experimentos agricolas pelos métodos Scott-Knott, Tukey a Duncan, Revista Brasileira de Agrocomputação 1(2) (2001) 18-24.

[57] T.C. Stadtman, SELENOCYSTEINE, Annual Review of Biochemistry 65(1) (1996) 83-100.

[58] S. Katayama, Y. Mine, Antioxidative activity of amino acids on tissue oxidative stress in human intestinal epithelial cell model, J Agric Food Chem 55(21) (2007) 8458-64.

[59] R.J. Elias, D.J. McClements, E.A. Decker, Antioxidant activity of cysteine, tryptophan, and methionine residues in continuous phase beta-lactoglobulin in oil-in-water emulsions, J Agric Food Chem 53(26) (2005) 10248-53.

[60] Samuel M. Pearlman, Z. Serber, James E. Ferrell, Jr., A Mechanism for the Evolution of Phosphorylation Sites, Cell 147(4) (2011) 934-946.

[61] C.-Z. Zhai, L. Zhao, L.-J. Yin, M. Chen, Q.-Y. Wang, L.-C. Li, Z.-S. Xu, Y.-Z. Ma, Two Wheat Glutathione Peroxidase Genes Whose Products Are Located in Chloroplasts Improve Salt and H(2)O(2) Tolerances in Arabidopsis, PLoS ONE 8(10) (2013) e73989.

[62] C.C.C. Chang, I. Slesak, L. Jordá, A. Sotnikov, M. Melzer, Z. Miszalski, P.M. Mullineaux, J.E. Parker, B. Karpinska, S. Karpinski, Arabidopsis chloroplastic glutathione peroxidases play a role in cross talk between photooxidative stress and immune responses, Plant Physiology 150(2) (2009) 670-683.

[63] K. Nomura, H. Imai, T. Koumura, T. Kobayashi, Y. Nakagawa, Mitochondrial phospholipid hydroperoxide glutathione peroxidase inhibits the release of cytochrome c from mitochondria by suppressing the peroxidation of cardiolipin in hypoglycaemia-induced apoptosis, The Biochemical journal 351(Pt 1) (2000) 183-193.

[64] M.A.R. Milla, A. Maurer, A.R. Huete, J.P. Gustafson, Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways, The Plant Journal 36(5) (2003) 602-615.

[65] Z. Faltin, D. Holland, M. Velcheva, M. Tsapovetsky, P. Roeckel-Drevet, A.K. Handa, M. Abu-Abied, M. Friedman-Einat, Y. Eshdat, A. Perl, Glutathione Peroxidase Regulation of Reactive Oxygen Species Level is Crucial for In Vitro Plant Differentiation, Plant and Cell Physiology 51(7) (2010) 1151-1162.
649

650

[66] R. Franco, J.A. Cidlowski, Apoptosis and glutathione: beyond an antioxidant, Cell Death Differ 16(10) (2009) 1303-1314.

651

652

653

[67] S. Chen, Z. Vaghchhipawala, W. Li, H. Asard, M.B. Dickman, Tomato phospholipid hydroperoxide glutathione peroxidase inhibits cell death induced by Bax and oxidative stresses in yeast and plants, Plant Physiology 135(3) (2004) 1630-1641.

654

655

656

[68] A. Kilaru, B.A. Bailey, K.H. Hasenstein, *Moniliophthora perniciosa* produces hormones and alters endogenous auxin and salicylic acid in infected cocoa leaves, FEMS Microbiology Letters 274(2) (2007) 238-244.

657

658

659

[69] F.C. Chaves, T.J. Gianfagna, Necrotrophic phase of Moniliophthora perniciosa causes salicylic acid accumulation in infected stems of cacao, Physiological and Molecular Plant Pathology 69(1-3) (2006) 104-108.

660

661

662

663

[70] P.J.P.L. Teixeira, D.P.d.T. Thomazella, O. Reis, P.F.V. do Prado, M.C.S. do Rio, G.L. Fiorin, J. José, G.G.L. Costa, V.A. Negri, J.M.C. Mondego, P. Mieczkowski, G.A.G. Pereira, High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa, The Plant Cell 26(11) (2014) 4245-4269.

664
<table><caption>**Table 1.** Gene and protein characteristics of GPX family from *T. cacao*. nd: non determined.</caption><thead><tr><th rowspan="2">Name</th><th colspan="4">Predict gene data</th><th colspan="7">Predict protein data</th></tr><tr><th>Identification*</th><th>Sequence start*</th><th>Sequence end*</th><th>ORF size (bp)</th><th>Size (aa)</th><th>Molecular weight (Da)</th><th>pI</th><th>Subcellular localization</th><th>Phosphorylation sites</th><th>Glycosylation sites</th></tr></thead><tbody><tr><td>TcPHGPX</td><td>Tc09_p001340</td><td>728482</td><td>730367</td><td>720</td><td>239</td><td>26477.2</td><td>9.08</td><td>Chloroplast</td><td>S33 S36 S40 S43 S44 S58 S70 S96 S98 S122 S153 T20 T78 T81</td><td>N120</td></tr><tr><td>TcGPX2</td><td>Tc05_p000210</td><td>101857</td><td>104202</td><td>789</td><td>262</td><td>29708.3</td><td>9.11</td><td>Secretory</td><td>S31 S42 S57 S59 S62 S152 T47 T185 T246 Y44 Y92 Y147</td><td>N165 N227</td></tr><tr><td>TcGPX4</td><td>Tc01_p028750</td><td>24070592</td><td>24074869</td><td>516</td><td>171</td><td>19175.9</td><td>9.14</td><td>nd</td><td>S11 S28 S121 T16 T83 Y52 Y116</td><td>N50 N122 N134</td></tr><tr><td>TcGPX6</td><td>Tc03_p027320</td><td>23147873</td><td>23149733</td><td>780</td><td>235</td><td>25973.7</td><td>8.93</td><td>Chloroplast</td><td>S25 S29 S31 S32 S43 S52 S57 S63 S74 S75 S78 S95 S188 S189 S197 S225 T83 T221 Y118 Y128 Y166 Y183</td><td>N12 N117 N201</td></tr><tr><td>TcGPX8</td><td>Tc03_p027330</td><td>23150262</td><td>23153108</td><td>573</td><td>190</td><td>21600.4</td><td>5.27</td><td>nd</td><td>S5 S11 S28 S50 S159 S173 S176 T16 T54 Y13 Y69 Y79 Y134</td><td>N68</td></tr></tbody></table>

<footnote>* Gene identification and position as indicated in CocoaGenDB database</footnote>
<table><caption>**Table 2.** *Cis*-elements present in the promoter region of *T. cacao* GPXs genes.</caption><thead><tr><th>Gene</th><th>Name of cis-elements</th><th>Organism</th><th>Function</th></tr></thead><tbody><tr><td rowspan="6">TcPHGPX</td><td>ABRE</td><td>Arabidopsis thaliana</td><td>Abscisic acid responsiveness</td></tr><tr><td>ARE</td><td>Zea mays</td><td>Anaerobic induction</td></tr><tr><td>AT-rich</td><td>Pisum sativum</td><td>Maximal elicitor-mediated activation</td></tr><tr><td>Box 4</td><td>Petroselinum crispum</td><td>Light responsiveness</td></tr><tr><td>MBS</td><td>Arabidopsis thaliana</td><td>Drought-inducibility</td></tr><tr><td>TC-rich repeats</td><td>Nicotiana tabacum</td><td>Defense and stress responsiveness</td></tr><tr><td rowspan="6">TcGPX2</td><td>W box</td><td>Arabidopsis thaliana</td><td>WRKY transcription factors</td></tr><tr><td>ABRE</td><td>Arabidopsis thaliana</td><td>Abscisic acid responsiveness</td></tr><tr><td>GARE-motif</td><td>Brassica oleracea</td><td>Gibberellin-responsive element</td></tr><tr><td>MBS</td><td>Arabidopsis thaliana</td><td>Drought-inducibility</td></tr><tr><td>TC-rich repeats</td><td>Nicotiana tabacum</td><td>Defense and stress responsiveness</td></tr><tr><td>W box</td><td>Arabidopsis thaliana</td><td>WRKY transcription factors</td></tr><tr><td rowspan="6">TcGPX4</td><td>Box 4</td><td>Petroselinum crispum</td><td>Light responsiveness</td></tr><tr><td>TGACG-motif</td><td>Hordeum vulgare</td><td>MeJA-responsiveness</td></tr><tr><td>ABRE</td><td>Arabidopsis thaliana</td><td>Abscisic acid responsiveness</td></tr><tr><td>ARE</td><td>Zea mays</td><td>Anaerobic induction</td></tr><tr><td>Box 4</td><td>Petroselinum crispum</td><td>Light responsiveness</td></tr><tr><td>GARE-motif</td><td>Brassica oleracea</td><td>Gibberellin-responsive element</td></tr><tr><td rowspan="6">TcGPX6</td><td>MBS</td><td>Arabidopsis thaliana</td><td>Drought-inducibility</td></tr><tr><td>P-box</td><td>Oryza sativa</td><td>Gibberellin-responsive element</td></tr><tr><td>TCA-element</td><td>Nicotiana tabacum</td><td>Salicylic acid responsiveness</td></tr><tr><td>TGA-element</td><td>Brassica oleracea</td><td>Auxin-responsive element</td></tr><tr><td>W box</td><td>Arabidopsis thaliana</td><td>WRKY transcription factors</td></tr><tr><td>ABRE</td><td>Arabidopsis thaliana</td><td>Abscisic acid responsiveness</td></tr><tr><td rowspan="6">TcGPX8</td><td>ARE</td><td>Zea mays</td><td>Anaerobic induction</td></tr><tr><td>Box 4</td><td>Petroselinum crispum</td><td>Light responsiveness</td></tr><tr><td>AT-rich</td><td>Pisum sativum</td><td>Maximal elicitor-mediated activation</td></tr><tr><td>MBS</td><td>Arabidopsis thaliana</td><td>Drought-inducibility</td></tr><tr><td>O2-site</td><td>Zea mays</td><td>Zein metabolism regulation</td></tr><tr><td>TATC-box</td><td>Oryza sativa</td><td>Gibberellin-responsiveness</td></tr><tr><td>TC-rich repeats</td><td>Nicotiana tabacum</td><td>Defense and stress responsiveness</td></tr><tr><td>TCA-element</td><td>Brassica oleracea</td><td>Salicylic acid responsiveness</td></tr><tr><td>TGA-element</td><td>Brassica oleracea</td><td>Auxin-responsive element</td></tr><tr><td>W box</td><td>Arabidopsis thaliana</td><td>WRKY transcription factors</td></tr></tbody></table>
671 **Figure legends**

672 **Figure 1.** Schematic illustration of the genes, mature RNA and proteins of the GPX
673 family from *Theobroma cacao*. **A.** DNA sequences of GPX family. Untranslated
674 regions, exons and introns are indicated by turquoise, green and salmon squares,
675 respectively. **B.** Mature RNAs from the *TcGPX* genes. Untranslated regions and exons
676 are indicated by turquoise and green squares, respectively. Purple lines represent the
677 initiation codons (AUG), red lines indicate the cysteine insertion codons (UGU) in
678 *PHGPX*, *GPX2*, *GPX4*, *GPX6* and the tryptophan insertion codon (UGG) in GPX8, and
679 black lines indicate the stop codons (UGA). **C.** *TcGPX* protein structure. Black squares
680 represent the conserved GSH domain and yellow lines indicate the presence of cysteine
681 on the active site of *TcPHGX*, *TcGPX2*, *TcGPX4* and *TcGPX6*, and of tryptophan on
682 the active site of *TcGPX8*, instead of selenocysteine.

683 **Figure 2.** Phylogenetic relationships among the nucleotide sequences of TcGPXs and
684 other plants and mammalian GPXs. Only complete sequences were considered. The tree
685 was constructed using the neighbour-joining method of clustalW with 1,000 bootstraps.
686 The amino acid sequences were obtained from GenBank.

687 **Figure 3.** Tridimensional structure of TcPHGPx, TcGPx2, TcGPx4, TcGPx6, and
688 TcGPx8 obtained by homology modeling with PtGPx5 from *Populus trichocarpa*
689 (2P5R.pdb). **A.** Alignment of the TcGPX with PtGPx5. Conserved substitutions,
690 identical amino acids, semi-conserved substitutions and catalytic triad are indicated in
691 grey, dark blue, light blue and purple, respectively. **B.** Secondary structure of
692 TcPHGPX. **C.** Secondary structure of TcGPX2. **D.** Secondary structure of TcGPx4. **E.**
693 Secondary structure of TcGPx6. **F.** Secondary structure of TcGPx8. **B-F.** The catalytic
694 triad of each TcGPx is represented by a solvent surface.
695 **Figure 4.** Docking between TcGPXs and selenomethionine (SeMet). **A.** Interaction between TcPHGPX (blue) and SeMet. **B.** Interaction between TcGPX4 (orange) and SeMet. **C.** Interaction between TcGPX6 (pink) and SeMet. **D.** Interaction between TcGPX8 (salmon) and SeMet.

699 **Figure 5.** Relative expression of *TcPHGPX*, *TcGPX2*, *TcGPX4*, *TcGPX6*, and *TcGPX8* in cacao meristems inoculated or not (control) with *M. perniciosa*. **A.** Representation of plant symptoms and fungus phase during the infection time course in Catongo genotypes. The harvesting times of inoculated plants are indicated on the top of the figure. **B.** Relative expression observed in TSH1188. **C.** Relative expression observed in Catongo. (*) indicates harvesting times of non-inoculated (control) plants. The control, used as calibrator (for this reason is always 1), corresponds to the average of the expression values of each *TcGPX* in 5 non-inoculated samples in each genotype (see also Material and methods section). Different letters indicate significant statistical difference between samples by the Scott-Knott test p(p≤0.01). *t*-test was applied with a critical value of 0.01: upper case letters correspond to statistics between each of the genes on different harvesting times for each genotype while lower case letters correspond to statistics among the genes for each harvesting time. hai: hours after inoculation; dai: days after inoculation.
A DNA

<figure><img src="image_3.png" /></figure>

B Mature RNA

<figure><img src="image_4.png" /></figure>

C Protein

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /></figure>

Theobroma cacao GPXs

<figure><img src="image_7.png" /></figure>

Mammalian GPXs

<figure><img src="image_8.png" /></figure>

Seaweed GPX

<figure><img src="image_9.png" /></figure>

Other plant GPX

<figure><img src="image_10.png" /></figure>

GPX2 Vitis vinifera

GPX2 Populus euphratica

GPX2 Eucalyptus grandis

GPX2 Theobroma cacao

PHGPx Arabidopsis lyrata

PHGPx Jatropha curcas

PHGPx Gossypium arboreum

PHGPx Theobroma cacao

GPX1 Mus musculus

GPX1 Homo sapiens

GPX1 Equus caballus

GPX1 Felis catus

GPX6 Theobroma cacao

GPX6 Malus domestica

GPX8 Solanum lycopersicum

GPX8 Citrus sinensis

GPX8 Theobroma cacao

GPX Glycine max

GPX4 Populus trichocarpa

GPX4 Theobroma cacao

GPX Chlamydomonas reinhardtii
<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>Interactions</figcaption></figure>

<figure><img src="image_17.png" /></figure>
A

<figure><img src="image_18.png" /></figure>

B

<figure><img src="image_19.png" /></figure>

C

<figure><img src="image_20.png" /></figure>