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Plant Physiology and Biochemistry 98 (2016) 162e170

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Research article

Characterization of the legumains encoded by the genome of Theobroma cacao L Juliano Oliveira Santana a, Laís Freire a, Aurizangela Oliveira de Sousa a, Virgínia Lúcia Fontes Soares a, Karina Peres Gramacho b, Carlos Priminho Pirovani a, * a b

Biotechnology and Genetics Center, State University of Santa Cruz, 45662-900 Ilh eus, BA, Brazil Cocoa Research Center, CEPLAC/CEPEC, P.O. Box 7, 45600-970 Itabuna, BA, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 July 2015 Received in revised form 29 October 2015 Accepted 16 November 2015 Available online 2 December 2015

Legumains are cysteine proteases related to plant development, protein degradation, programmed cell death, and defense against pathogens. In this study, we have identified and characterized three legumains encoded by Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression pattern in different tissues and as a response to the inoculation of Moniliophthora perniciosa fungus. The three proteins were named TcLEG3, TcLEG6, and TcLEG9. Histidine and cysteine residue which are part of the catalytic site were conserved among the proteins, and they remained parallel in the loop region in the 3D modeling. Three-dimensional modeling showed that the propeptide, which is located in the terminal C region of legumains blocks the catalytic cleft. Comparing dendrogram data with the relative expression analysis, indicated that TcLEG3 is related to the seed legumain group, TcLEG6 is related with the group of embryogenesis activities, and protein TcLEG9, with processes regarding the vegetative group. Furthermore, the expression analyses proposes a significant role for the three legumains during the development of Theobroma cacao and in its interaction with M. perniciosa. © 2015 Universidade Estadual de Santa Cruz, CNPJ: 40738999/0001-95. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Keywords: Cysteine-proteinase Cocoa Modeling qPCR Gene expression Witches' broom

1. Introduction Legumains are cysteine proteases in the C13 family (EC 3.4.22.34) and are found in plant tissues (Santos-Silva et al., 2012), parasites (Klinkert et al., 1989), and mammals (Chan et al., 2009). These proteins are also called Vacuolar Processing Enzymes (VPEs), due to their ability to recognize and cleave asparagine (Asn) or aspartic acid (Asp) residue in polypeptides (Hara-Nishimura et al., 1995; Müntz and Shutov, 2002). VPEs are synthesized as inactive prolegumains, and they are transferred to the vacuoles or to the cell wall. Once they reach those compartments under acid pH conditions, prolegumains undergo activation through self-cleavage in the propeptides that are located in the amino and carboxy-terminal. Then they become active legumains in order to play their biological role in plant tissues (Dall and Brandstetter, 2012; Li et al., 2003). In plants, those proteins were initially isolated from mature castor beans (Ricinus communis) and from soybean cotyledons (Glycine max) (Hara-Nishimura et al.,

* Corresponding author. E-mail address: [email protected] (C.P. Pirovani).

1991; Scott et al., 1992), and they have conserved the Cys and His catalytic residues which are preceded by a block of four hydrophobic amino acids (Chen et al., 1998). In plants, VPEs are involved in several physiological processes, and they are classified in three groups, according to the tissues where they are expressed the most. VPEs in from the vegetative group are involved in programmed cell death (PCD) regulation (Hatsugai et al., 2004), senescence (Donnison et al., 2007), and pathogen response (Rojo et al., 2004). VPEs from seeds are related to the processing and mobilizing of reserve proteins during seed germination (Nakaune et al., 2005; Radchuk et al., 2011). VPEs in the embryogenesis group seem to play a role in the formation process of the external tegument of seeds (Nakaune et al., 2005). In Arabidopsis thaliana, three legumain genes were found, and they are referred to as aVPE, bVPE, and gVPE (Kinoshita et al., 1995a, 1995b). Later, on the same plant, a fourth protein was reported through the observation of its expression in young developing seeds, called dVPE (Gruis et al., 2002). Studies regarding the expression of VPE genes showed that aVPE and gVPE are inserted in the vegetative group, showing higher expression in roots, senescent leaves, and in PCD tissues (Kinoshita et al., 1999). bVPE and dVPE were cataloged in seed VPEs; the first of which was mainly

http://dx.doi.org/10.1016/j.plaphy.2015.11.010 0981-9428/© 2015 Universidade Estadual de Santa Cruz, CNPJ: 40738999/0001-95. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

J.O. Santana et al. / Plant Physiology and Biochemistry 98 (2016) 162e170

expressed in dry seeds, embryonic axis, and cotyledon, and dVPE was expressed in developing seeds (Hara-Nishimura et al., 2005; Nakaune et al., 2005). Cocoa tree (Theobroma cacao L.) is highly important economically as source of almonds, raw material for chocolate and sweets, motivating several biotechnological studies which led to the complete sequencing of its genome (Argout et al., 2011). Fungal diseases are responsible for significant losses in the production of almonds, especially witches' broom, caused by the basidiomycete fungus Moniliophthora perniciosa (Aime and Phillips-Mora, 2005). This fungus is hemibiotrophic and its life cycle is divided into two phases, a biotrophic and a necrotrophic phase, causing histological, physiological, morphological alterations, and death of infected tissues in plants (Ceita et al., 2007; Orchard et al., 1994; Scarpari et al., 2005). In this study, we have identified and characterized the three VPEs (TcLEG3, TcLEG6 e TcLEG9) that are coded by the Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression patterns in different tissues, and propose a significant role of VPEs during plant development and in response against the M. perniciosa pathogen, which causes witch's broom in the cocoa tree. 2. Materials and methods 2.1. Primary in silico analysis The primary amino acid sequences that were predicted for the VPEs which are coded by the Theobroma cacao genome were obtained from the CocoaGenDB database (http://cocoagendb.cirad.fr/ ). The three identified VPEs were submitted to different bioinformatics analyses: sequence alignment e ClustalW (http://www.ebi. ac.uk/Tools/msa/clustalw2/); signal peptide prediction e SignalP 4.1 Sever (http://www.cbs.dtu.dk/services/SignalP/) (Bendtsen et al., 2004); tracking of functional domains e Pfam (http://pfam. xfam.org/search/sequence), and analysis on MEROPS (http:// merops.sanger.ac.uk/). The phylogenetic dendrogram was built with the help of MEGA 6 software (Kumar et al., 2004), using Neighbor-Joining method and the confidence of the branching order was verified by making 1000 bootstrap replicates. The dendrogram was built with 31 sequences of homologous proteins that were distributed in eleven plant species, as follows: Nicotiana tabacum (tobacco), Solanum lycopersicum (tomato), Oryza sativa (rice), Vitis vinifera (grape), R. communis (castor oil plant), Triticum aestivum (wheat), Hordeum vulgare (barley), Cucumis sativus (cucumber), Sesamum indicum (sesame), A. thaliana, and Theobroma cacao (cocoa). The searches for homologous sequences were conducted in NCBI database (National Center for Biotechnology Information - http://www.ncbi.nlm.nih.gov/ BLAST), and the sequences identified with their access numbers. One of the cystatins (TcCys1) of cocoa, which is a competitive inhibitor of cysteine protease, described by Pirovani et al. (2010), was used as an outlier. 2.2. Three-dimensional modeling Homology modeling was used to obtain the active and inactive three-dimensional structures from the three T. cacao VPEs. The searches for template structures as resolved through x-ray crystallography or nuclear magnetic resonance (NMR) were carried out by the PDB database (http://www.rcsb.org/pdb/home/home.do). Modeller 9.12 software (Eswar et al., 2007) was used in the three-dimensional modeling of the structures. The scripts for processing were generated by the Notepadþþ software (Version 6.5.1), with one “.ali” extension file (Dynamics AX Label Index File) and

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five “.py” extension files (Python). The “.ali” contains information regarding the target protein sequence, whereas the scripts in the “.py” format have modeling commands and three-dimensional structures of template proteins selected in a “.pdb” format. The generated protein structures were refined and validated by softwares (Charmm, Anolea, and Procheck) which analyze possible structural errors in specific regions and stereochemical parameters (Johnson et al., 1994) (Figs. S1eS3). 2.3. Tissues and fruits analyzed Plants of the CCN51 variety were used to analyze the genetic expression in different tissues (root, tegument, seed, leaf and stalk). They were grown under natural lighting, drip irrigation, and room temperature. The seed cotyledons of the variety “Parazinho” were germinated in a growth chamber at a 27 ± 2  C temperature, with photoperiod of 16 h of light and 8 h of darkness, with 36 mmol m2 s1 irradiance. Samples were collected at ten different germination stages, as follows: zero hour (quiescent seed), 24 h, 48 h, 72 h, day 4, day 6, day 8, day 12, day 20, and day 30, after the germination. Fruits of the Parazinho variety were collected in four different development stages in order to analyze the expression of VPEs in the seed development phase (Fig. S4). Fruits were measured with the aid of a caliper ruler, and weighed through precision scales. After collection tissues were immediately frozen in liquid nitrogen, lyophilized, and stored at 80  C, until DNA extraction. 2.4. Germination conditions of infected tissues Two hundred seeds of the CCN51 variety were planted and kept in the greenhouse under natural light, spray irrigation, and room temperature conditions. After 20 growth days, seedlings were taken to a moist chamber, making up for a total 100 plants infected by M. perniciosa and 100 control plants inoculated with 0.3% agarwater. Inoculation was conducted through the spraying of 5.0  105 basidiospores/mL of M. perniciosa in a 0.3% agar-water suspension (Surujdeo-Maharaj et al., 2003). The seedlings, after the inoculation, were stored at 23  C for 48 h, under relative humidity >97%, and controlled by an automated nebulization system. Afterwards, seedlings returned to the greenhouse. Biological sample collections (meristems) were conducted on different days, as follows: 0 h (right after the inoculation), 24 h, 48 h, 72 h, 15 days and 45 days after the inoculation. The collected samples were immersed in liquid nitrogen and lyophilized prior to extraction. 2.5. Criteria for primer design The oligonucleotides that were used for the qPCR (Table S1) were designed with the v.3.0 Primer Express® software (Applied Biosystems®). In order to generate the design of primers for the VPEs (TcLEG3, TcLEG6, and TcLEG9), the following criteria were considered: CG percentage, size between 18 and 25 bp, melting temperature Tm (58e60  C), and amplicon size (50e150 pb). Actin (ACT), malate dehydrogenase (MDH), and glyceraldehyde 3phosphate dehydrogenase (GAPDH) genes were selected as endogenous controls, since they presented homogeneous expression in the analyzed tissues (Pinheiro et al., 2011). The relative expression levels of genes TcLeg3, TcLeg6, and TcLeg9 were calculated according to the delta Ct (DCt) method, and analyzed in two biological replicates (different tissues), five biological replicates (developing seed), ten biological replicates (infected tissues). All tissues were analyzed in experimental quintuplicates. After Ct values were obtained, the average value of the Ct of each target gene was normalized with the Ct value from the endogenous control that was established by the NormFinder

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software (Claus et al., 2004), which calculated the best stability value among the genes that were used as endogenous controls. The relative expression values of the target genes in plant tissues were expressed as powers of two (2DCt). In order to analyze the genetic expression in T. cacao plant tissues, the 2DDCt (Livak and Schmittgen, 2001) method was used (Table S2). The statistical analyses of data were conducted by the BioEstat 5.0 software, applying Dunnett's statistical test with a 95% confidence interval.

amino acid residues. In the dendrogram, the VPEs of Theobroma cacao have been separated into three groups: a vegetative type (a/gVPE), a seed type (bVPE) and an embryogenesis type (dVPE). The TcLEG3 in group bVPE, TcLEG6 in group dVPE and TcLEG9 in group a/gVPE (Fig. 2). 3.2. Three-dimensional modeling

The tissues of T. cacao were sprayed, and 100 mg used to extract RNA with the help of the Zymo Research kit, according to the manufacturer's recommendations. The RNA of infected tissues (meristem) and developing seeds were purified with the RNAqueous reagent (Ambion), and then submitted to ultrasonication (3 pulses of 2 s each, 70% range, with a 10 s interval) due to the excess mucilage. Afterwards, the RNA was treated with DNAse I (Thermo Scientific). RNA integrity was analyzed in 1% agarose gel stained with GelRed™, and using the NanoDrop 2000 spectrophotometer (Thermo Scientific). cDNA was synthesized with RvertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer's recommendations and quantified and diluted to 10 pmol/mL. For qPCR, the “Maxima® Sybr Green/Rox” kit (Thermo Scientific), was used. Each reaction was performed in a 22 mL volume, containing 10 mL cDNA, 11 mL Sybr Green, and 0.5 mL of each oligonucleotide, which were diluted at 10 pmol/mL. The amplification was conducted in an Mx 3005P device (Agilent Technologies), according to the following conditions: UDG incubation for 2 min at 50  C, polymerase activation for 10 min at 95  C, followed by 40 15 s cycles at 95  C and 1 min at 60  C. The dissociation curve that was obtained in the reaction was used to evaluate the confidence of the amplified region, and to observe the presence of unspecific amplification (Figs. S5eS7).

Three-D models of the three legumains, TcLeg3, TcLeg6, and TcLeg9 in their active and inactive forms, were obtained with the three dimensional modeling. The template structure of Homo sapiens (4n6o, 4awb, and 4fgu) (Dall and Brandstetter, 2013, Dall et al., 2015) and Mus musculus (4noj) (Zhao et al., 2014) were the best models with a degree of identity greater than 46%. Models 4n6o, 4fgu, and 4awb were selected for the construction of threedimensional model of TcLEG3 and structures 4n6o, 4fgu, and 4noj, were selected as models for TcLEG6 and TcLEG9. Small differences were found in the superficial molecular areas and in the number of atoms in the structures of the three active VPEs (without the propeptides). In TcLEG3, the superficial area was 27.157.854 Å, with 2.620 atoms; in TcLEG6, it was 26.952.984 Å with 2.625 atoms; and in TcLEG9, an area of 26.825.139 Å with 2.572 atoms. The number of segments with conforming b-leaves, a-helices, and loops were equally distributed among the structures, and they were 8, 9, and 17, respectively (Fig. 3). The distances between the catalytic residues of histidine (His) and cysteine (Cys) in the loop regions of the three VPEs were similar - 5.5 Å for TcLEG3, 6.3 Å for TcLEG6 and 5.1 Å for TcLEG9. The structure validation process was shown to have a Ramachandran plot with 100% of residues in energetically favorable regions for all three generated VPEs. The three-dimensional structures of the three inactive VPEs (Fig. 3) showed that the C-terminal pro-peptide is positioned in the upper portion of the catalytic domain (His and Cys), blocking the catalytic cleft and preventing the coupling of the active site substrates or inhibitors.

3. Results

3.3. Genetic expression

3.1. In silico analysis of VPE sequences

3.3.1. Different tissues Gene TcLeg3 was found to have an accumulation of transcripts for all analyzed tissues (Fig. 4a); it was least expressed in the root, which was used as calibrator (1.00X), and most expressed in the seed (21.7X) and tegument (21.5X). Still on the same gene, statistically significant differences are observed in the expression of the tegument (21.5X), seed (21.7X), leaf (9.9X), and stalk (17.8X), and those values were higher than the expression in the root (1.0X) (calibrator). The TcLeg9 gene showed expression in all organs and tissues analyzed (Fig. 4b). The leaf was the organ with the highest expression (25.5X) as compared to the calibrator (root). Statistically significant differences were observed in the tegument (4.7X), leaf (25.6X), and stalk (19.0X) as compared to the expression in the root (1.00X). Unlike for the TcLeg3 gene, the TcLeg9 gene in the seed presented less expression (0.7X), with no significant differences as compared to the calibrator. Gene TcLeg6 expression was not identified in any of organs or tissues (seed, tegument, root, stalk, and leaf) analyzed.

2.6. RNA extraction, cDNA synthesis, and RT-qPCR

In CocoaGenDB, the T. cacao genome database, three VPE cysteine proteases were found in loci Tc03_g025550, Tc06_g006030, and Tc09_g004580 (Fig. S8). Those three proteins were named TcLEG3, TcLEG6, and TcLEG9. Pfam software revealed that the three identified sequences in the blast correspond to peptidases from the C13 class; that is, from the VPE family. The alignment conducted among cocoa tree VPEs and a legumain (gVPE) from A. thaliana, as described by Kinoshita et al. (1995b), found several blocks of conserved amino acids and revealed a high degree of identity (61.4%, 57.8%, and 77.1% in TcLEG3, TcLEG6, and TcLEG9, respectively). The signal peptide was observed in the amino-terminal regions of the three analyzed legumains (Fig. 1). In TcLEG3, the signal peptide cleavage sites were between A27 and A28. In TcLEG6, between the S21 and the E22 residue. In contrast, for TcLEG9, the cleavage site was between the A21 and the G22 residue. TcLEG9 presented the motif LPS (L32, P33, and S34) in the N-terminal pro-peptide. Two conserved Asp residues indicate the N-terminal and C-terminal propeptide cleavage sites in two active proteins - D51 and D422 for TcLEG3, and D46 and D420 for TcLEG9. However, in TcLEG6, the cleavage residues were located in N49 and D426 cleavage sites. The probable His catalytic sites (H175, H173, and H170 in TcLEG3, TcLEG6, and TcLEG9, respectively) Cys catalytic sites (C217, C215, and C212 in TcLEG3, TcLEG6, and TcLEG9, respectively), remained conserved among the analyzed proteins, and both residues are preceded by a block of four hydrophobic

3.3.2. Cotyledon expression during germination In the cotyledons of germinating seeds, the accumulation of transcripts of gene TcLeg3 decreased from the initial time (quiescent seed) to the thirtieth day (1.00e0.018X) (Fig. 5a), a reduction of approximately 90-fold. The results point towards statistically significant differences from the first to the thirtieth germination day. Still on the same tissue, TcLeg9 expression rose from time zero to

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Fig. 1. a: Alignment among T. cacao VPEs and a VPE from A. thaliana (NP_195020). Conserved amino acids among VPEs are indicated by an asterisk. Signal peptides are shaded. Box I indicates the vacuolar motif that was described by Jackson et al. (2007). The arrows indicate the cleavage site of propeptides in N and C-terminal regions. Boxes II and IV indicate the four hydrophobic residues which precede the catalytic residues that are indicated in box III and V. b: Global identity among amino acid sequences, the three cocoa tree VPEs, and the gVPE from A. thaliana.

the twentieth day, with an approximate 64-fold increase as compared to the control (0 time - quiescent seed) (Fig. 5b), undergoing a high reduction of its relative expression, from 64.10X to 20.34X in the thirtieth day. Results also pointed toward statistically significant differences from the second to the thirtieth germination day for this gene, in comparison to the control. Gene TcLeg6 was not expressed in any of the stages in the germinating cotyledon tissues. 3.3.3. Developing seeds The TcLeg3 genes TcLeg6 and TcLeg9 showed transcripts in all

stages of seed development (Fig. 6). Gene TcLeg3 did not present significant expression variation among the analyzed stages, from 1.0X in the first stage (calibrator) to 0.8X in second, 0.8X third, and 1.4X in last stage. In contrast, gene TcLeg6 expression increased from the first stage of seed development to the third stage (1.00e8.9X), remaining practically unaltered in the third and fourth stages (8.9e8.7X). In same tissue, the transcripts of gene TcLeg9 did not show significant variation for the first three stages, from 1.0X in first stage to 1.2X in second, 0.7X in third, and finally a significant increase of 2.5X in the fourth stage.

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Fig. 2. Dendrogram of VPEs of ten plant species. Black triangle: cocoa legumains. VPEs were separated into three groups: bVPE (brown), a/gVPE (green), and dVPE (yellow). The analysis was conducted through MEGA 6 software. Signal peptides were excluded from the sequences. Access numbers on NCBI are within parentheses. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3.4. Infected tissues The analysis of the relative expression in infected tissues by M. perniciosa showed low accumulation of gene TcLeg3 transcripts (Fig. 7a) for all days after inoculation. Significant variation was only obtained in the plant initial necrosis period (45 days) as compared to its calibrator (non-inoculated plant e 45 days) shifting from 0.76X to 0.23X. Gene TcLeg9 (Fig. 7b) in the same tissue presented significant expression variation on first day (1.00X to 2.93X) and on the forty-fifth day after inoculation (1.00X to 1.82X). Gene TcLeg6 was not expressed in the analyzed days in plants that were infected by M. perniciosa fungus basidiospores, and neither in control plants. 4. Discussion 4.1. TcLEG3, TcLEG6, and TcLEG9 are bVPE, dVPE, and a/gVPE, respectively Cocoa VPEs are synthesized as prolegumains, with the presence of signal peptides and amino and carboxy-terminal which depend on cleavage processing (Fig. 1). They are probably transported to their destination sites in inactive forms, which can be self-activated under acidic conditions, as so happens with the castor oil plant VPEs (R. communis) (Hiraiwa et al., 1997), A. thaliana (Kuroyanagi et al., 2002), and humans (Chen et al., 2000). The amino acid sequence of TcLEG9 has the LPS motif (Fig. 1) located on the N-terminal propeptide, which according to Jackson et al. (2007) is a probable signal for the transportation of that enzyme to the lytic vacuole. His and Cys residues remained

conserved among the VPEs. Those residues, according to Hiraiwa et al. (1997), are part of the catalytic site. The three VPEs also present conserved residues of Asp, which indicate the cleavage site of N and C-terminal propeptides during the activation of the protein. However, TcLEG6 in the N-terminal regions has the Asn residue replacing the Asp. TcLEG3, TcLEG6, and TcLEG9 VPEs were separated in the dendrogram (Fig. 2) into three distinct groups: bVPE, dVPE, and a/ gVPE, respectively. According to Christoff et al. (2014) the groups are related to the specificity of biological roles of VPEs during a plant life cycle. It is important to point out that TcLEG6 is inserted in the dVPE group, and was the last group to be specified (Nakaune et al., 2005). Those proteins are of the embryogenesis type and their expression takes place before the synthesis of storage proteins (bVPE). That protein is involved in the process in removing the internal layers that are required for the formation of external tegument of developing seeds (Yamada et al., 2005). Homology modeling is a reliable in silico technique for the prediction of protein structures (Chothia and Lesk, 1986; MartiRenom et al., 2000). The three active VPEs modeled revealed that the catalytic residues are arranged on the surface of the loop region along folds, which facilitates the interaction between two molecules (Bode and Huber, 1992). The modeling of inactive structures showed how the propeptide which is located in the C-terminal region of the VPEs blocks the catalytic cleft (Fig. 3), being positioned in the upper part of the catalytic domain (His e Cys), preventing its interaction with the substrate or inhibitor, at the same time, giving enzyme latency and conformational stability to the protein (Dall and Brandstetter, 2013; Zhao et al., 2014).

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Fig. 3. Three-dimensional structure of T. cacao VPEs. Active VPEs e a: TcLEG3, c: TcLEG6, e: TcLEG9. Inactive VPEs e b: TcLEG3, d: TcLEG6, f: TcLEG9. Blue (a-helices), orange (bleaves), green (Loops). Highlighted loops (red) are the (His and Cys) catalytic residues. The propeptide which is located in the terminal C region, in yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Expression pattern of TcLeg3 (a) and TcLEG9 (b) genes in different tissues of T. cacao. Values were normalized through GAPDH endogenous enzyme. Error bars represent the average of two replicates (n ¼ 2). The asterisks indicate the significant values (p < 0.05) as compared to the calibrator (root) according to Dunnett's method.

4.2. Genetic expression The expression of VPE genes was detected in different levels in T. cacao tissues. Genes TcLeg3 and TcLeg9 were found to have detectable expression levels in all analyzed tissues, as well as significant alterations in cotyledons during germination. In contrast, bgene TcLeg6 was only expressed in the stages of seed development. 4.2.1. TcLeg3 is related to specific roles in seeds The relative expression of TcLeg3 in different plant tissues (Fig. 4a) was shown to have great accumulation of transcripts, especially in the tegument and in the seed. In germinating cotyledons (Fig. 5a), the expression pattern decreased from the initial

time to the thirtieth germination day, a reduction of approximately 90 times, which corroborates with the data from Kinoshita et al. (1999), in which the expression of gene bVPE in Arabidopsis decreased after the seed germinated, and kept gradually reducing with seedling growth. In the tissue that was infected by the M. perniciosa fungus, gene TcLeg3 presented accumulation of transcripts reduced on the fortyfifth day after the inoculation, when the plant necrosis process begins (Fig. 7a) and the disease changes to the saprophytic phase (Ceita et al., 2007; Frias et al., 1991), with a 53X reduction in the transcript, which suggests that this gene is not related to the defense against the M. perniciosa pathogen, senescence and tissue degradation. The obtained expression profile suggests that VPE TcLeg3 is

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Fig. 5. Expression pattern of TcLeg3 (a) and TcLEG9 (b) genes in germinating cotyledons of T. cacao. Values were normalized through MDH endogenous enzyme. Error bars represent the average of two replicates (n ¼ 2). The asterisks indicate the significant values (p < 0.05) as compared to the calibrator (quiescent seed), according to Dunnett's method.

gradual, since no transcripts of TcLeg6 gene were detected in the analysis of the mature seed (quiescent) and germinating cotyledon. According to the dendrogram (Fig. 2) TcLEG6 is inserted in the dVPE group. That type of VPE is expressed in developing seeds before bVPE (TcLeg3), and it is inserted in the embryogenesis-type group, being involved in the process in of removing the internal layers that are required for the formation of the external seed tegument (Christoff et al., 2014; Hara-Nishimura and Hatsugai, 2011; Nakaune et al., 2005).

Fig. 6. Expression pattern of TcLeg3, TcLeg6, and TcLeg9 genes in developing seeds of T. cacao. Values were normalized through ACT endogenous protein. Error bars represent the average of five replicates (n ¼ 5). The asterisks indicate the significant values (p < 0.05) as compared to the calibrator (first development stage), according to Dunnett's method.

related to specific roles in seeds. That is corroborated through the dendrogram analysis (Fig. 2) which includes the TcLEG3 in the bVPE group and in the developing seeds during the expression (Fig. 6), in which no significant differences occurred, and reinforces the idea that such VPE must play an important role in the mobilization of proteins during the initial germination step (Müntz and Shutov, 2002; Nakaune et al., 2005; Radchuk et al., 2011). 4.2.2. TcLeg6 is related to embryogenesis TcLeg6 gene was only expressed in the developing seeds, when the transcript was observed to continuously increase from the first to the third seed development stage. Afterwards, a slight reduction of the expression took place. That transcript reduction must be

4.2.3. TcLeg9 is a VPE related to development and defense against pathogens The TcLeg9 gene was the most expressed in leaves and stalk, as compared to the calibrator (root) (Fig. 4b). In the germinating cotyledons (Fig. 5b), the expression pattern increased from the initial time to its overexpression in the twentieth day, decreasing right after the thirtieth day. The expression reduction on the thirtieth day may be related to the period marked by the senescence of cotyledons. In the tissue that was infected by M. perniciosa, the expression of gene TcLeg9 was shown to be high on the first day after the inoculation, and on the forty-fifth day, when the plant necrosis process started (Fig. 7b), which suggests that this gene may be related to the defense against pathogens or PCD. The significant expression increase on the first day may be related to the increased hydrogen peroxide production in that period, which was not enough to characterize a hypersensitivity response, but instead a biochemical mechanism against M. perniciosa infection (Dias et al., 2011). In response to the increased peroxide production, in fungus-resistant cocoa genotypes, increased ascorbate peroxidase (APx) activity was observed in infected tissues (Camillo et al., 2013). In the first signs of the fungus attack, the calcium oxalate druses are converted into carbon dioxide, oxygen, and hydrogen peroxide, but they do not

Fig. 7. Expression pattern of TcLeg3 (a) and TcLEG9 (b) genes in tissue (meristem) after inoculation of M. pernicious. Values were normalized through MDH endogenous enzyme. Error bars represent the average of ten replicates (n ¼ 10). The asterisks indicate the significant values (p < 0.05) as compared to the calibrator (control), according to Dunnett's method.

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cause cell death (Dias et al., 2011). In contrast, the significant expression increase on the forty-fifth day may have been induced by the tissue senescence and degradation, because the initial symptoms of witches' witch are observed in this period after inoculation, and the lengthening of the axillary buds and swelling of the stalk (Ceita et al., 2007). The expression profile of TcLeg9 the VPE suggests that is related to the type vegetative functions. This is also corroborated through its location in the dendrogram (Fig. 2), where it is included in the a and gVPE group, which suggests it is involved in the plant development (Kinoshita et al., 1995a e 1995b). According to Müntz and Shutov (2002), during the germination of seeds, reserve proteins that are deposited in the storage vacuoles are mobilized and used for plant growth. Even though genes TcLeg3 and TcLeg9 are respectively inserted in the seed and vegetative groups and have been expressed in several tissues, the classification of VPEs continues to be performed based on the functions that these enzymes play during the plant life cycle (Gruis et al., 2002; Kinoshita et al., 1999; Yamada et al., 2005). 5. Conclusion We have presented the identification and characterization of the three VPEs that are coded by the T. cacao genome, which are named TcLEG3, TcLEG6, and TcLEG9. Three-dimensional modeling revealed that the propeptide that is located in the C-terminal regions of VPEs blocks the enzyme catalytic cleft, which prevents interactions and causes enzyme latency. VPEs are separated in three groups: seed (TcLeg3), embryogenesis (TcLeg6), and vegetative (TcLeg9) according to where they are located in the dendrogram and how genes are expressed in the various tissues of plants. TcLeg3 may be related to the mobilization of reserve proteins, whereas TcLeg9 may be involved in the vegetative phase during the life cycle of plants and processes regarding senescence and response against the M. perniciosa pathogen. In turn, TcLeg6 only accumulates transcripts in the developing seeds, and it may be related to the formation of the seed external tegument. Contributions from the authors Juliano Santana performed the experiments and wrote the manuscript. Laís Freire and Aurizangela Oliveira contributed with the data analysis and with the execution of some experiments. Virgínia Lúcia, advice and guidance. Karina Gramacho contributed with the experiment design and plant inoculations. Carlos Priminho, since the first idea, has supervised the investigation efforts and contributed with reagents and materials. Conflict of interest The authors state that no conflict of interest exists. Authors contributions Experimental design: Juliano Oliveira Santana, Virgínia Lúcia Fontes Soares and Carlos Priminho Pirovani. Data collect: Juliano Oliveira Santana, Laís Freire, Aurizangela Oliveira de Sousa, and Karina Peres Gramacho. Data analysis: Juliano Oliveira Santana, Laís Freire, Aurizangela Oliveira de Virgínia Lúcia Fontes Soares, Carlos Priminho Pirovani. laboratory infrastructure: Carlos Priminho Pirovani and Karina Peres Gramacho writing of the manuscript: Juliano Oliveira Santana and Carlos Priminho Pirovani.

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