Proteome Analysis of the Inner Integument from Developing

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Proteome analysis of the inner integument from developing seeds of Jatropha curcas L. Emanoella L. Soares, Mohibullah Shah, Arlete A. Soares, José Hélio Costa, Paulo C. Carvalho, Gilberto B. Domont, Fábio C.S. Nogueira, and Francisco A.P. Campos J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr5004505 • Publication Date (Web): 10 Jul 2014 Downloaded from http://pubs.acs.org on July 14, 2014

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Journal of Proteome Research

Proteome analysis of the inner integument from developing Jatropha curcas L. seeds

Emanoella L. Soares1#, Mohibullah Shah1#, Arlete A. Soares2, José H. Costa1, Paulo Carvalho4, Gilberto B. Domont3, Fábio C.S. Nogueira3* and Francisco A.P. Campos3* 1 Department of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Brazil 2 Department of Biology, Federal University of Ceara, Fortaleza, Brazil 3 Proteomic Unit, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 4 Laboratory for Proteomics and Protein Engineering, Carlos Chagas Institute, Fiocruz – Parana, Brazil #Contributed equally *Correspondence: Prof. Francisco A.P. Campos, Department of Biochemistry and Molecular Biology, Federal University of Ceara, 60455-900 Fortaleza, Brazil Email: [email protected] Fax: +55-85-33669829 Prof. Fábio C. S. Nogueira, Proteomic Unit, Institute of Chemistry, Federal University of Rio de Janeiro, 21941-909 Rio de Janeiro, Brazil 1

ACS Paragon Plus Environment


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Email: [email protected] Phone: +55-21-39388862 Fax: +55-21-39387266

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Keywords: label-free quantitative proteomics, programmed cell death, Jatropha curcas, oilseed, seed coat Abstract In this study, we performed a systematic proteomic analysis of the inner integument from developing seeds of Jatropha curcas and further explored the protein machinery responsible for generating the carbon and nitrogen sources to feed the growing embryo and endosperm. The inner integument of developing seeds was dissected into two sections called distal and proximal and proteins were extracted from these sections and from the whole integument and analyzed using an EASY-nanoLC system coupled to an ESI-LTQ-Orbitrap Velos mass spectrometer. We identified 1526, 1192, and 1062 proteins from the proximal, distal and whole inner integuments, respectively. The identifications include those of peptidases and other hydrolytic enzymes that play a key role in developmental programmed cell death (PCD), and proteins associated with the cell wall architecture and modification. As many of these proteins are differentially expressed within the integument cell layers, these findings suggest that the cells mobilize an array of hydrolases to produce carbon and nitrogen sources from proteins, carbohydrates, and lipids available within the cells. Not least, the identification of several classes of seed storage proteins in the inner integument provides additional evidence of the role of the seed coat as a transient source of reserves for the growing embryo and endosperm.

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1. Introduction The seeds of J. curcas are considered to be a potential source of raw material for the production of biodiesel1 and two by-products of the oil extraction viz. the protein-rich press-cake and the seed coat, also offer opportunities for adding value to the crop. The former can potentially be used as animal feed and the latter, representing almost 40% of the seed weight, constitutes a potential source of biomass for the production of second-generation ethanol.2 The full exploitation of such potentials is hindered by a lack of knowledge of the chemical identity and biosynthesis of cell wall components of the seed coat and by the presence, in the seeds, of high concentrations of phorbol esters, tetracyclic diterpenoids known for their tumor-promoting activity, which represent serious risks for humans, animals and for the environment.3 In mature seeds, the highest concentrations of phorbol esters are found in the inner layer of the seed coat, called the tegmen, from where they diffuse to the endosperm.4,5 The seeds of Angiosperms are derived from the ovule, an organ that harbors the female gametophyte which in turn is surrounded by the nucellus and one or two integuments.6 Following fertilization of the egg cell and the central cell, the female gametophyte will differentiate into the diploid embryo and triploid endosperm respectively and the maternally derived diploid seed coat develops from the integuments of the ovule.7 The carbon and nitrogen sources necessary for the developing embryo and endosperm are provided by the long distance transport of metabolites, such as sucrose, amino acids and amides through the maternal vascular tissue. 8 While knowledge of the developmental biology of embryo and endosperm is expanding rapidly9,10 the cellular events underlying seed coat differentiation have received comparatively little attention.11,12 However, it is well established that during early to mid stages of development, the seed coat acts as a maternal conduit to convey nutrients for the developing embryo and endosperm13-15 and at maturity it provides protection, regulates germination and additionally promotes seed dispersal.16 4


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Details of the protein machinery responsible for producing the nitrogen and carbon sources for feeding the embryo and endosperm during seed development are elusive. Although differentiation of the integuments to seed coat is known to involve developmentally regulated programmed cell death (PCD) triggered by vacuole collapse,17,18 very little is known about the synthesis, subcellular location, and site of action of the peptidases, lipases, carbohydrases, and nucleases. These enzymes are needed to digest cell components, including the cell wall, so that the products may be used as energy sources and/or buildings blocks for biosynthetic reactions within the embryo and endosperm. Most of the studies related to the seed coat have been restricted to individual enzymes19-21 or to high throughput transcriptomic analysis.10 Recently Miernyk and Johnston11 published the very first in-depth proteomic analysis of the seed coat, providing a glimpse of the dynamic changes in the proteome of the testa of developing soybean seeds, but similar studies for the seeds of Euphorbiaceae are lacking. In this present work, we capitalized on a detailed description of the histological development of the seed coat of Jatropha curcas22 to guide our proteomic study within the layers of the inner integument during their midstage development phase. This allowed us to report with unprecedented details of the proteome of the enzymatic machinery responsible for executing the developmentally controlled PCD within this tissue.

2. Material and Methods 2.1. Plant materials and histological analysis Seeds (25 days after pollination) were collected from hand-pollinated three year-old plants that were field-grown and irrigated three days per week until the field capacity, according to the measurement of evapotranspiration by a Class A land tank. Histological analyses were performed as described by Rocha et al. 23 Briefly, the inner integument was isolated with a sharp 5



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spatula, dissected into two regions having the vascular bundle as reference – one of these internal to the vascular bundle faced the central cavity (proximal region) and the other external to the vascular bundle, facing the exotegmen (distal region) (Figure 1). The separation of these regions was facilitated by the large size of the inner integument and the easy visualization of the vascular bundle with the aid of a binocular microscope. The middle section between the proximal and distal regions, comprising the vascular bundle, was discarded in order to avoid crosscontamination. In addition to the proximal and distal regions we also collected the whole intact inner integument (here called total integument). For histological analysis tissues were fixed in Karnovsky solution,

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dehydrated in an ethanol series of 10, 20, 30, 40, 50, 60, 70, 80, 90 and

100% (one hour per step) and slowly embedded in historesin LEICA for 20 days. Sections of 5 µm were prepared in a rotatory microtome LEICA 2065, stained with toluidine blue 0.5% in borax 0.12% followed by basic fuchsin 25 (0.5%) and mounted with Tissue Mount.

2.2. Sample preparation and protein determination After separation, the three tissues (whole integument, proximal and distal sections) were immediately frozen in liquid nitrogen and freeze-dried. Subsequently, samples were ground down to powder and 100 mg of each fraction were separately subjected to protein extraction according to Vasconcelos, et al.26 Briefly, powdered tissues were homogenized in pyridine buffer (50 mM pyridine, 10 mM thiourea and 1% SDS, pH 5.0) with polyvinyl−polypyrrolidone (PVPP) in a proportion of 1:40:2 (w/v/w), respectively. The homogenate was stirred for 4 hours at 4º C and centrifuged for 30 minutes at 10000 g. Proteins in the supernatant were precipitated overnight with cold 10% trichloroacetic acid (TCA) in acetone and centrifuged for 15 minutes at 10,000 g. Pellet was washed three times with cold acetone, dried under vacuum and dissolved in 7 M urea/2 M thiourea. Protein concentrations were determined by the Bradford assay.27 Six

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biological replicates were performed for each sample (Figure 2). Each biological replicate was constituted from tissues of approximately 60 seeds collected from approximately 20 fruits.

2.3. LC-MS/MS and data analysis Trypsin digestions were performed using 100 µg of the proteins from each of the three samples as described by Pinheiro et al.

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Peptides were dissolved in 80 µL of 0.5% formic acid

and diluted five times with the same solution. 4 µL of the diluted samples were applied to an EASY II nano LC system (Proxeon Biosystem) coupled online to an ESI-LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Peptides were loaded in a trap column (150 µm × 2 cm) packed in-house with C-18 ReproSil 3 µm resin (Dr. Maisch) and eluted in an analytical column (100 µm x 15 cm) packed with the same material. Peptide separations were performed using a gradient from 100% of A (0.1% formic acid) to 35% of B (0.1% formic acid, 95% acetonitrile) for 150 min, followed by 35% to 90% of B for 15 min, and 90% for 5 min. MS1 spectra were acquired in a positive mode using the data-dependent automatic (DDA) survey MS scan. Each DDA consisted of a survey scan in the m/z range 300−2000 and resolution 60,000 with a target value of 1 × 10-6 ions. The ten most intense ions were subjected to MS2 acquisition in the LTQ using a normalized collision-induced dissociation (CID) of previously fragmented ions were dynamically excluded for 60 s. Raw data were viewed in Xcalibur v.2.1 (Thermo Scientific). Three raw files were generated for each biological replicate of the distal and proximal regions while two raw files were generated for each biological replicate of the total integument, representing three and two technical replicates, respectively (Figure 2). Raw files were converted into MS2 files using PatternLab’s RawReader. ProLuCID v1.329 was used to perform peptide spectrum matching against the J. curcas proteins database30 downloaded from http://www.kazusa.or.jp/jatropha/ 7



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September 2012, combined with J. curcas chloroplast genome encoded proteins, downloaded from NCBI September 2012. Search parameters were: semi tryptic hydrolysis, two missed cleavages, oxidation of methionine as variable and carbamidomethylation as fixed modifications, and a peptide tolerance of 50 ppm. Search results were subsequently filtered/processed through Search Engine Processor31 to achieve a list of identified proteins with 1% false discovery rate (FDR) at protein level. The validity of the Peptide Sequence Matches (PSM) was assessed using the Search Engine Processor v. 2.2.0.2 (SEPro). Identifications were grouped by the charge state (+2 and > +3) and then by tryptic status (tryptic and semi-tryptic), resulting in four distinct subgroups. For each result, the ProLuCID XCorr, DeltaCN and ZScore values were used to generate a Bayesian discriminating function. A cutoff score was established to accept a false-discovery rate (FDR) of 1% based on the number of decoys. This procedure was independently performed on each data subset, resulting in a false-positive rate that was independent of tryptic status or charge state. A minimum sequence length of 6 amino acids was required. Results were post-processed to accept PSM with less than 8 ppm. Proteins were grouped according to the maximum parsimony by the bipartite graph approach.32 Proteins appearing in at least two biological replicates, considered as representatives of that biological sample, were blasted against NCBI non redundant database using Blast2GO annotation tool33 and used for downstream analysis. Spectral counts of the identified proteins were obtained using the PatternLab computational environment.34 Gene ontology (GO) annotation was done using AgBase tools and database (http://agbase.msstate.edu/index.html). Goanna35 was used to retrieve the GO annotations assigned on the basis of sequence similarities and Plant GOSlim was introduced to summarize the sub-categories of the identified proteins.

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Differentially expressed proteins between the distal and proximal regions were pinpointed by using PatternLab’s revised TFold module 36, that uses the spectral count method. Spectral counting is a label-free semi-quantitative measure of the protein abundance and defined as, the total number of spectra identified for a protein.

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Briefly for a given FDR bound, the

revised TFold uses a theoretical FDR estimator (in this case, the Benjamini-Hochberg) to maximize the number of identifications that satisfy both a fold-change cutoff that varies with the t-test P-value as a power law and a stringency criterion that aims to detect lowly abundant proteins. Fold changes were calculated by dividing the average spectral counts obtained in the distal region by those obtained in the proximal region.

2.4 RNA isolation, cDNA synthesis and reverse transcription quantitative real-time PCR (RT-qPCR) Total RNA from proximal and distal regions was isolated from 300mg of tissue, using the Plant RNA Purification Reagent (Invitrogen®) according to the manufacturer’s instructions. RNA was digested with DNase I (Invitrogen) for removal of genomic DNA. The first-strand cDNA synthesis was performed with 0.5 µg of DNase I treated-RNA using the ImProm-II™ Reverse Transcription System (Promega) according to the manufacturer’s instructions and was stored at -20 °C until use. RT-qPCR was carried out using the Fast SYBR Master Mix (Applied Biosystems) with a Mastercycler® ep realplex (Eppendorf AG, Hamburg) under the following conditions: 10 min at 95◦C for activation of the enzyme, followed by 40 cycles of denaturation at 95◦C for 15 s, annealing at 60◦C for 20 s, and extension at 60◦C for 20 s. A melting curve additional step was carried out to check the specificity of the primers. Each reaction was performed with 20 ng of cDNA and 400 nM of each primer in a final volume of 20 µL. The sequences coding for the target genes (arabinofuranosidase - Jcr4S00428.20; aspartic peptidase - Jcr4S06063.10; cysteine 9



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peptidase - Jcr4S01104.40; cysteine peptidase inhibitor - Jcr4S00024.130; serine peptidase Jcr4S01605.60; serpin - Jcr4S00079.140 and subtilisin - Jcr4S01651.50, Jcr4S01323.10) were retrieved from the genome of J. curcas (www.kazusa.or.jp/jatropha/) to design specific primer sets (Supplementary Table I) using the Perl Primer v1.1.19 software.38 Three reference genes (glyceraldehyde-3-phosphatedehydrogenase, polyubiquitin-3 and ubiquitin conjugating enzyme) were used for data normalization.23 Three biological replicates were performed for each sample, each one with two technical replicates in quadruplicate (n=8). The analysis of relative expression was carried out by the 2–∆∆CT method.39

3. Results and Discussion 3.1 Histology of seed development The mature ovule of J. curcas is anatropous, crassinucellar, bitegmic, and the vascular bundles present in the outer integument extend by postchalazal branching from the chalaza through the inner integument.22,40 Both integuments are composed of three layers: endotegmen, mesotegmen and exotegmen in the inner integument and endotesta, mesotesta and exotesta in the outer integument. During development the integuments will differentiate to form the seed coat. At the mid stage of seed development, 25 days after pollination (DAP), different parts of the inner integument are experiencing different changes in its cell features indicative that PCD is occurring in a gradual way (Figure 1). These observations led us to select this stage for proteomic analysis to see the differences in the protein profiles in different regions of the inner integument. At 25 DAP the embryo is at the heart stage, the cellularization of the endosperm is well underway (Figure 1A) and the cell layers closest to the exotegmen are small and become progressively larger and vacuolated as they are positioned closer to the endosperm and those in the immediate vicinity of the endosperm collapse, forming a layer of cell debris (Figure 1B). In 10


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later stages, when cellularization of the nuclear endosperm is complete and the embryo is fully developed, the mesotegmen cells, which are internal to the vascular bundle, are completely consumed, the cells external to the vascular bundle are crushed and those from the exotegmen become sclerified forming the main mechanical barrier of the mature seed coat. At this stage the three layers of the outer integument are differentiated. Figure 1B indicates that cells closer to the exotegmen and those closer to the endosperm are at different metabolic states and are experiencing PCD at different degrees, suggesting the possible existence within the inner integument of a concentration gradient of the proteins involved with the execution of PCD (e.g. peptidases, lipases and carbohydrases). To further investigate this, we took advantage of the large size of the developing seeds to isolate the whole inner integument and to obtain from it proximal and distal sections to the developing endosperm (Figure 1A) and to perform an indepth proteomic analysis of each of the three parts.

3.2 Protein identification and functional classification Our results permitted the identification of 1752, 1405, and 1251 proteins from distal, proximal and total integument, representing 1169, 912, and 823 protein groups, respectively, at a 1% FDR. Supplementary Table II summarizes details about the number of MS, MS2, PSMs, peptides and unique peptides for all tissues and biological/technical replicates. The proteins identified in at least two biological replicates were considered as highly confident; these identifications comprise 1526 (distal), 1192 (proximal), and 1062 (total integument) proteins summing up a total of 1770 (Supplementary Table III). The identification of sucrose synthases and cell wall and apoplastic invertases, which are key players in the control of nutrient supply to the embryo and endosperm,41 indicated that the inner integument of developing J. curcas seeds is metabolically very active. This is further supported by the identification of proteins belonging to major biochemical pathways, such as those related to amino acid and protein biosynthesis, and a 11



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number of transporters (Supplementary Table III). Additionally, the role of the inner integument as a transient storage source of nutrients to the developing embryo and endosperm is supported by the identification of several classes of seed storage proteins such as nutrient reservoir, globulins 11S, legumins, and glutelins (Table 1). Although a number of proteins involved in secondary metabolism were identified, we were unable to identify any casbene synthase or other protein that may be involved in the biosynthesis of phorbol esters, the major toxic components of J. curcas seeds. This result is in agreement with recent findings by Nakano et al.42 which showed that in J. curcas, casbene synthase is expressed in seedlings, mature leaves and in the flesh of developing fruits, but not in developing seeds. Another recent proteome analysis of plastids from developing J. curcas seeds28 reported no evidence for the presence of casbene synthase among more than 1100 proteins. These results indicate that the phorbol esters that are found in the seeds are probably translocated either from the leaves or from the roots, but experimental support for this hypothesis is lacking. Despite the heterogeneity in the total number of the proteins identified in each of the three samples analyzed (Supplementary Table III), we did not observe significant differences in the distribution of these proteins into different Gene Ontology categories (Supplementary Table IV). However, Gene Ontology annotation of the proteins unique to distal and proximal region of the integument revealed a clear difference in their distribution to some of the subcategories of GO Molecular Function and Cellular Component (Supplementary Table V and Figure 3). Proteins related to hydrolase activity of the GO Molecular Function (Figure 3A) were found to comprise the largest functional class in the proximal region as compared to the distal region where proteins related to protein binding (Figure 3A) make up the largest functional class. This indicates that most of the metabolic activity located in the proximal region is geared towards the reallocation to the embryo and endosperm of the catabolic products produced by the digestion of proteins, carbohydrates, lipids, and nucleic acids within the cells undergoing PCD. Moreover, 12


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the percentage of the cell wall sub-category from the GO Cellular Component (Figure 3B) was greater in the proximal region than in the distal region. The proteins mapping within this category are mostly related to the processes of cell wall degradation which further supports a metabolism geared towards the digestion of carbohydrate polymers and proteins that comprise the cell wall.

3.3 Differentially expressed proteins A PCA plot was performed using PatternLab´s Buzios module43; the plot confirms the proximal and distal proteomic profiles to be very distinct (Supplementary Figure I). We used PatternLab’s Approximately Area Proportional Venn Diagram module to pinpoint proteins uniquely identified in distal and proximal regions of the inner integument. The analysis only considered proteins found in two or more biological replicates from a particular sample. Differentially expressed proteins found in two or more biological replicates identified in the distal and proximal regions of the inner integument were discriminated by PatternLab’s TFold module using a q-value of 0.05 (Figure 4). Only proteins that satisfy all statistical tests (blue dots in Figure 4) were considered, and those dots found in the upper section of the plot are upregulated in the distal sample; likewise, those found in the lower section are up-regulated in the proximal sample. We recall that the TFold module uses a theoretical FDR estimator to maximize identifications satisfying both a fold-change cutoff that varies with the t-test p-value as a power law and a stringency criterion that aims to fish out proteins of low abundance that are likely to have had their quantitation compromised. Differentially expressed proteins between distal and proximal regions are present in Supplementary Table VI.

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3.4 Functional classes related to programmed cell death 3.4.1 Peptidases and peptidase inhibitors Of all of the identified proteins, almost 10% belong to one of the four major mechanistic classes of peptidases and their inhibitors or are catalytic subunits of the proteasome or ubiquitination proteins (Supplementary Table VII). The identification of proteasome components and ubiquitination proteins indicates a proper functioning of the proteasome, a conditio sine qua non for the occurrence of PCD in plant cells.44 Many of the identified peptidases are known to have a role in PCD. For example, ϒ-vacuolar processing enzyme (ϒ-VPE), only identified in the distal region, is a homologue to a VPE also identified in castor seeds during the proteomic analysis of nucellus tissue undergoing PCD.45 The VPEs are a sub-class of cysteine peptidases (CP) which possess caspase-1-like activity and are used by the vacuolar-collapse system to promote developmentally controlled PCD in plant tissues.17 The KDEL-tailed CP is another type of CP that has been found to be a hallmark of PCD in plant tissues undergoing PCD.23,45-47 Here we identified a KDEL-tailed CP (Jcr4S01104.40) having a statistically higher expression in the proximal region (Supplementary Table VI); this evidence supports the hypothesis that these proteinases are released into the cytoplasm following the vacuole collapse triggered by the action of the VPEs.21 The spatial and temporal differences in expression of the VPEs and KDEL-tailed CP in the inner integument of J. curcas seeds underline the different roles of these CP in PCD and are in line with recent findings by Rocha et al.23 which indicated that during seed development, transcripts for ϒ-VPE are detected earlier than those for the KDEL-tailed CP. Our results disclose identifications of several serine peptidases (SP) and serine peptidase inhibitors (SPI) (Supplementary Table VII) of which 10 are differentially expressed (Supplementary Table VI). Notably, three subtilisin-like serine peptidases presented spectralcounting fold changes greater than 10 fold higher in the proximal region. These subtilisin-like 14


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serine peptidases are characterized by an aspartate, histidine and serine catalytic triad, have been shown to display caspase like activity, and were seen to be associated with cell death in several plant species.48 In order to further confirm these results, we used qPCR to measure the relative expression of transcripts coding for selected peptidase and peptidase inhibitors in distal and proximal sections of the inner integument. As shown in Table 2, the relative expression of the transcripts analyzed, matches closely the relative abundance of the proteins coding for these transcripts as determined in our proteome analysis. Identification of this remarkable number and diversity of peptidases belonging to the four mechanistic classes of peptidases in tissues undergoing PCD underlines the importance of proteolysis for providing amino acids for protein synthesis in the filial tissues as suggested by Gallardo et al.49 Moreover, such diversity highlights the role of seed integuments in the developmental biology of J. curcas seeds. Miernyk and Johnston11 and Gallardo et al.49 have identified an abundance of peptidase in several stages of the development of the seed coat of soybean and Medicago truncatula, but the possibility that the abundance and variety of these peptidases in the developing seed coat may be related to PCD was not discussed.

3.4.2 Proteins related to cell wall metabolism Besides the peptidases referred to above, we identified proteins involved in the architecture of the cell wall such as several expansins and extensins and various other proteins associated with cell wall modification and degradation processes, such as arabinofuranosidases,

lysosomal

alpha-mannosidases,

pectinesterase,

alpha-

alpha-Land

beta-

glucosidases, beta-xylosidases, alpha-galactosidases, glucanases, polygalacturonases, among others (Supplementary Table III). Both beta-glucosidase and xylosidase are known to be involved in the breakdown or modification of cell wall hemicelluloses and pectins.50,51 Alpha-dgalactosidase is associated with the mobilization of galactomannan, while beta-galactosidase is 15



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involved in the degradation of the galactan side chain.51 Pectinesterases also called pectin methylesterases (PMEs) catalyse the demethyl-esterification of the pectin domains and make it feasible to be degraded by the action of polygalactorunases.52 Additionally, we identified pectinesterase inhibitors (Supplementary Table III) which modulate pectinesterase activity.50 Similarly the enzyme alpha-arabinofuranosidase identified here, in combined action with betagalactosidase and beta-glucuronidase (not identified here) is known to degrade the carbohydrate moieties of arabinogalactan proteins (AGPs).51 Our results show that the expression of many of these proteins is differentially regulated, as is the case of alpha-L-arabinofuranosidase, pectinesterase inhibitor and polygalacturonase that are more expressed in the proximal section than in the distal section of the inner integument (Supplementary Table VI). As measured by qPCR, the relative levels of transcripts coding for the alpha-L-arabinofuranosidase in proximal and distal sections of the inner integument (Table 2), matches closely the relative abundance of the protein coded by this transcript as determined in our proteome analysis (Supplementary Table VI). Additionally, several other proteins such as glucosidases, xylanases, mannosidases and pectinesterase, among others, were identified only in the proximal region of the inner integument (Supplementary Table III). These results indicate that along other possible roles, these polysaccharide acting enzymes together with different kind of peptidases, are acting to liberate all possible carbon and nitrogen sources available in the cell, including those that belong to the cell wall.

3.5.3. Proteins related to lipid catabolism It is noteworthy to point out that our results disclose several proteins involved in the degradation of lipids, viz.: phospholipase D, lipoxygenases, acyl-CoA oxidases, 3-ketoacyl thiolase, among others (Supplementary Table III). Phospholipase is an enzyme that catalyses the 16


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hydrolysis of structural phospholipids into hydroperoxides leading to the loss of membrane integrity, an event associated with PCD.53 This enzyme is known to be involved in the largescale degradation of lipids associated with cell death54 and sometimes contributes to caspasedependent cell death signaling.55 Fatty acids resulting from the degradation of the cell membrane are oxidized by the action of lipoxygenases56 and acyl hydrolases. The appearance of these enzymes along with other hydrolases, reinforce the occurrence of PCD in this maternal tissue.

3.5.3 Comparison with other proteomic analyses of J. curcas So far only a few proteomic studies have been published for J. curcas, concentrating mainly on endosperm, embryo, oilbodies and seed kernel, that collectively produced limited information57-63. We have recently published a first in depth proteomic analysis of J. curcas by analyzing the proteome of plastids isolated from the endosperm of developing J. curcas seeds, which resulted into the identification of 923 proteins28. Comparing the integument and plastids proteomes, we have identified 2153 proteins among which 1230 are unique to integument while 383 are unique to plastids. Proteins unique to the integument proteome consist mainly of different kinds of hydrolases like peptidases and glycosidases, which are involved in the degradation of complex molecules to liberate nutrients to the developing endosperm and embryo. On the other hand, proteins unique to the plastids proteome consist of proteins involved in the lipids metabolism. The other proteomic analyses of J. curcas present in literature57-63 collectively identified 209 non redundant proteins. Comparison of our plastids and integument proteomes to these studies showed, that out 209, only 42 proteins were not identified in our results, which is probably due to the use of different tissues and experimental conditions in each of these different studies.

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4. Conclusion The data presented here demonstrate the role of the inner integument of developing seeds of J. curcas in providing carbon and nitrogen sources for the growing embryo and endosperm. The identification of several classes of peptidases, particularly of ϒ-VPE and KDEL-tailed CP highlights the role of developmental PCD in the developmental biology of seeds of J. curcas. Additionally, the demonstration of the differential expression of these peptidases and other hydrolases within cell layers of the inner integument after the triggering of PCD, suggests that the cells mobilize a whole array of hydrolases to produce carbon and nitrogen sources from proteins, carbohydrates and lipids available within the cells. Not least, the identification of several classes of seed storage proteins in the inner integument is an additional evidence of the role of the seed coat as a transient source of reserves for the growing embryo and endosperm.

Acknowledgements

This work was supported by grants from PETROBRAS, Banco do Nordeste, FUNCAP, CNPq, CAPES and TWAS. Data availability Mass spectrometry raw data and Search Engine Processor (SEPro) file are available at: Chorus repository website (chorusproject.org; Project Name: Proteome analysis of the inner integument from developing Jatropha curcas L seeds) and http://yoda.iq.ufrj.br/mohib2014/.

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Tables Table 1: Classes of seed storage proteins identified in the inner integument of Jatropha curcas seeds. Table 2: Relative expression of selected genes, identified in our proteomic analysis of the inner integument, quantified through qPCR. Positive values represent higher expression in the distal region and negative values represent higher expression in the proximal region. Figures Figure 1: Anatomical structure of J. curcas seed. A. Seed structure 25 DAP. B. Magnified representation of the square area in A showing cell features of the distal and proximal region. Legends: D, distal region; Em, embryo; En, endosperm; II, inner integument; OI, outer integument; P, proximal region; arrowheads indicate cell remnants; arrows indicate vascular bundles. Figure 2: Work flow of the experimental design of the proteomic analysis of the inner integument from J. curcas seeds. Red bar, Proximal region; Yellow bar, Distal region; Green bar, Total inner integument. Figure 3: Gene ontology (GO) categories of the proteins unique to distal and proximal regions of the integument. Plant GO Slim was used to summarize the sub-categories of the identified proteins. Y axes represent percent of proteins. A: GO-Molecular function. B: GO-Cellular component. C: GO-Biological process. Figure 4: TFold analysis pinpointing differentially expressed proteins when comparing the proteomics profiles of the distal and proximal integuments. Each protein is mapped as a dot on the plot according to its –Log2 (p-value) (x-axis) and Log2 (Fold change) (y-axis). Red dots are proteins that satisfy neither the variable fold-change cutoff nor the FDR cutoff α= 0.05. Green dots are those that satisfy the fold-change cutoff but not α. Orange dots are those that satisfy both 24


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the fold-change cutoff and α, but are lowly abundant proteins and therefore most likely have their quantitation compromised. Finally, blue dots are those that satisfy all statistical filters. Dots found in the upper section of the plot are up-regulated in the distal sample; likewise, those found in the lower section are up-regulated in the proximal sample.

Supporting Information - this material is available free of charge via http://pubs.acs.org. Supplementary Material Supplementary Figure I: Principal component analysis generated using PatternLab´s Buzios Module. Each blue dot is representative of one analysis from the distal sample; likewise, each orange dot originates from an analysis obtained from a proximal sample. Supplementary Table I: Primer sequences of the target genes used for gene expression analysis. Supplementary Table II: Details about the number of MS, MS2, PSM, peptides and unique peptides. Supplementary Table III: List of the proteins appearing in at least two biological replicates identified in the distal and proximal regions and intact inner integument of developing J. curcas seeds. Supplementary Table VI: Gene ontology (GO) annotation of the proteins identified in the distal and proximal regions and intact inner integument of developing J. curcas seeds. Plant GO Slim was used to summarize sub-categories of the identified proteins.

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Supplementary Table V: Gene ontology (GO) annotation of the proteins unique to the distal and proximal regions of the inner integument of developing J. curcas seeds. Plant GO Slim was used to summarize the sub-categories of the proteins. Supplementary Table VI: Differentially expressed proteins identified from distal and proximal regions of the inner integument of developing J. curcas seeds. In the column T, positive values show higher expression in distal region while negative values show higher expression in the proximal region of the inner integument. Supplementary Table VII: Peptidases and peptidase inhibitors identified in distal and proximal regions and intact inner integument of developing J. curcas seeds.

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Table 1: Classes of seed storage proteins identified in the inner integument of Jatropha curcas seeds.

Protein ID

Description

R. communis (UNIPROT)

Jcr4S01636.40 Jcr4S00279.60 Jcr4S01636.60 Jcr4S01617.40 Jcr4S08024.20 Jcr4S00423.10 Jcr4S03933.20 Jcr4S01793.30

11S globulin seed storage protein 2-like glutelin type-A precursor legumin B precursor nutrient reservoir nutrient reservoir nutrient reservoir nutrient reservoir late embryogenesis abundant protein

B9SW16 B9T5E7 Q9M4Q8 B9SYN7 B9SYN7 B9SYN7 B9SYN7 B9T526

A. thaliana (TAIR) AT1G03890.1 AT5G44120.3 AT5G44120.3 AT2G28680.1 AT1G07750.1 AT2G28680.1 AT1G07750.1 AT2G44060.1

Spectrum count Distal Proximal Total region region Integument 3 13 5 18 28 46 13 25 6 246 45 66 165 33 43 187 42 42 193 52 45 60 41 25

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Table 2: Relative expression of selected genes, identified in our proteomic analysis of the inner integument, quantified through qPCR. Positive values represent higher expression in the distal region and negative values represent higher expression in the proximal region. Protein ID Jcr4S00428.20 Jcr4S01104.40 Jcr4S00024.130 Jcr4S01605.60 Jcr4S01651.50 Jcr4S01323.10 Jcr4S00079.140

Description Arabinofuranosidase Cysteine peptidase (KDEL) Cysteine peptidase inhibitor Serine peptidase Subtilisin Subtilisin Serpin

Transcript relative expression Mean Fold Change SD CV (%) - 4.14 1.67 40.46 - 16.64 4.61 27.69 - 3.51 1.21 34.36 - 6,55 3.86 58.96 - 48.56 15.78 32.49 - 20.05 4.44 22.16 1.23 0.18 14.91

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Anatomical structure of J. curcas seed. A. Seed structure 25 DAP. B. Magnified representation of the square area in A showing cell features of the distal and proximal region. Legends: D, distal region; Em, embryo; En, endosperm; II, inner integument; OI, outer integument; P, proximal region; arrowheads indicate cell remnants; arrows indicate vascular bundles. 187x71mm (150 x 150 DPI)



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Work flow of the experimental design of the proteomic analysis of the inner integument from J. curcas seeds. Red bar, Proximal region; Yellow bar, Distal region; Green bar, Total inner integument. 151x190mm (150 x 150 DPI)


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Gene ontology (GO) categories of the proteins unique to distal and proximal regions of the integument. Plant GO Slim was used to summarize the sub-categories of the identified proteins. Y axes represent percent of proteins. A: GO-Molecular function. B: GO-Cellular component. C: GO-Biological process. 255x162mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TFold analysis pinpointing differentially expressed proteins when comparing the proteomics profiles of the distal and proximal integuments. Each protein is mapped as a dot on the plot according to its –Log2 (pvalue) (x-axis) and Log2 (Fold change) (y-axis). Red dots are proteins that satisfy neither the variable foldchange cutoff nor the FDR cutoff α= 0.05. Green dots are those that satisfy the fold-change cutoff but not α. Orange dots are those that satisfy both the fold-change cutoff and α, but are lowly abundant proteins and therefore most likely have their quantitation compromised. Finally, blue dots are those that satisfy all statistical filters. Dots found in the upper section of the plot are up-regulated in the distal sample; likewise, those found in the lower section are up-regulated in the proximal sample. 254x151mm (101 x 101 DPI)


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162x86mm (150 x 150 DPI)


Proteome Analysis of the Inner Integument from Developing

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