For Peer ReviewA first insight into Citrus clementina leaf proteome

Journal: PROTEOMICS

Manuscript ID: proteo-2007-00959

Wiley - Manuscript type: Rapid Communication

Date Submitted by the Author:

15-Oct-2007

Complete List of Authors: Maserti, Biancaelena; CNR-IBF, Istituto di BioFisica, Dipartimento Terra & Ambiente Centeno, Delphine; INRA-UR1199 Laboratoire de Protéomique Fonctionnelle Hirtz, Christophe; INRA-UR1199 Laboratoire de Protéomique Fonctionnelle Sommerer, Nicolas; INRA-UR1199 Laboratoire de Protéomique Fonctionnelle Della Croce, Clara Maria; CNR-IBBA, Istituto di Biologia e Biotecnologia Agraria, Dipartimento Agro-Alimentare Podda, Alessandra; CNR-IBF, Istituto di BioFisica, Dipartimento Terra & Ambiente Migheli, Quirico; University of Sassari, Department of Plant Protection Luro, François; INRA - CIRAD, SRA, San Giuliano, Corse Morillon, Raphael; INRA - CIRAD, SRA, San Giuliano, Corse Froelicher, Yan; INRA - CIRAD, SRA, San Giuliano, Corse Ollitrault, Patrick; INRA - CIRAD- UPR Multiplication Végétative, Talon, Manuel; IVIA - Instituto Valenciano de Investigaciones Agrarias Gambale, Franco; CNR- IBF, Istituto di BioFisica, Dipartimento Materiali e Dispositivi Rossignol, Michel; INRA-UR1199 Laboratoire de Protéomique Fonctionnelle

Key Words: Citrus clementina, Stress, Leaf proteome, LC-MS/MS, 2D-PAGE

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A first insight into Citrus clementina leaf proteome. 1

B.E. Maserti1*, D. Centeno2., C. Hirtz2, N. Sommerer2. , C.M. Della Croce1,8, A. Podda1, Q. 2Migheli3, F. Luro4, R. Morillon4,6, Y. Froelicher4, P. Ollitrault5, P. M. Talon6, F. Gambale7, M. 3Rossignol2.41. CNR - IBF, Istituto di BioFisica Dipartimento Terra & Ambiente, Area della Ricerca, Via 5Moruzzi 1- I- 56124 Pisa. 62. INRA - UR1199 Laboratoire de Protéomique Fonctionnelle, F- 34060 Montpellier 73. Università di Sassari, Dipartimento di Protezione delle Piante , Via Enrico De Nicola 9 , 8I - 07100 Sassari. 94. INRA - CIRAD, SRA, F- San Giuliano, Corse. 10 5. CIRAD - UPR Multiplication Végétative, Avenue Agropolis. F- 34398 Montpellier Cedex 5. 11 6. IVIA - Instituto Valenciano de Investigaciones Agrarias, Moncada E- 46113 Valencia. 12 7. CNR - IBF, Istituto di Biofisica, Dipartimento Materiali e Dispositivi, Area della Ricerca,Via De 13 Marini 6, I- 00100 Genova. 14 8. CNR- IBBA, Istituto di Biologia e Biotecnologie Agrarie, Area della Ricerca , Via Moruzzi 1, I- 15 56124 Pisa. 16 17 18 Corresponding author: bianca.elena.maserti@pi.ibf.cnr.it19 20 21 Keywords: Citrus clementina, stress, leaf proteome, LC-MS/MS, Two-dimensional electrophoresis. 22 23 24

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Abstract 25 The present study reports the first investigation of the leaf proteome of Citrus clementina. On the 26 basis of a previously published extraction protocol, proteins were first separated using 2-DE, and 27 then identified through LC-MS/MS. A total of 92 different protein accessions was identified and 28 functionally categorised. About 50 % of the proteins were involved in energy production and 29 metabolism. A noticeable number of proteins belonging to stress/defence class was also identified, 30 opening interesting perspectives for future comparative proteomic studies of the response of Citrus 31 to abiotic and biotic stress. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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Citrus fruits represent the most widely fruit production, with 109 million tons produced annually in 48 the world, where the Mediterranean basin accounts for 20% of the total by itself. Nevertheless, this 49 crop is facing increasing biotic and abiotic stresses and consequently, the generation of new 50 varieties and rootstocks combining tolerance to major biotic and abiotic stresses and good quality 51 characteristics constitutes a major challenge of the current Citrus Industry. To this end, the 52 molecular characterization of the physiological and genetic determinants of selected characteristics 53 appears to be mandatory, since among other reasons, Citrus exhibit a very long juvenile phase. A 54 proteomic approach providing knowledge about the gene expression at protein level could help to 55 complement the genomic data. Nevertheless, only little effort was devoted to investigate the 56 proteome of Citrus. Thus, a proteomic approach was initially used to evaluate the expression of 57 parental proteome in Citrus somatic allotetraploid hybrid [1] and two protocols for the extraction 58 and 2-DE separation of citrus proteins were optimized [2, 3]. In addition, a deep study into the 59 Citrus fruit proteome [4] and a detailed survey on proteins changes in the albedo citrus fruits have 60 recently been presented [5]. 61

In the framework of a collaboration among international and interdisciplinary groups involved in 62 Citrus research, the characterization of the proteomes and subproteomes of Citrus clementina was 63 started, with the aim to evaluate the potential of proteomics as a tool for Citrus improvement against 64 adverse environmental stimuli. Citrus clementina is the main small Citrus variety and it is 65 particularly important for the Mediterranean Basin Citrus industry. Clementine has recently been 66 chosen by the International Citrus Genome Consortium, as a model system for whole genome 67 sequencing from an haploid line. Furthermore, important genomic resources such as ESTs database 68 [6] and BAC libraries [7] are available and genetic and physical maps are currently implemented 69 [8]. In this work we report the first insight into the leaf proteome of a Citrus species. We show the 70 presence of several proteins potentially involved in response/defence mechanisms to environmental 71

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stimuli that opens the way to dedicated proteomic investigation of Citrus tolerance to biotic and 72 abiotic stress. 73

Two years old Citrus clementina plants ( SRA 535 IP.P) were grown under controlled conditions 74 (T: 28 °C; photoperiod: 16/8 hours; relative humidity 60-80%). Four biological replicates of tissue 75 samples were prepared based on nine pooled leaves collected from a plant each and analyzed twice. 76 Each replicate was processed according to previously published protocol [3] with slight 77 modifications. 600 µg of protein in 350 µL of rehydration buffer (7 M urea, 2 M thiourea, 4% 78 CHAPS, 10% isopropanol, 0.5% IPG Buffer 3-11 NL, and 1.5% DeStreak Reagent (GE-79 Healthcare) were loaded on 18 cm IPG strips (GE-Healthcare). IPGs were focused for 100 kV.h on 80 a Protean IEF system (BioRad). The second dimension was performed by SDS-PAGE in vertical 81 slab of acrylamide (13% T, 2.6% C) using a Protean XL cell (BioRad). Gels were stained in 82 colloidal Coomassie Blue for three days. About 400 proteins spots were detected by the Image 83 Master Platinum 6.0 software (GE-Healthcare). A set of 100 spots (present in at least six of the 84 eight gels), was picked randomly to ensure a good representation in terms of pH, molecular weight 85 and protein abundance (Figure 1). 86

87 FIGURE 1 88 89 Spots were then processed using a MultiProbe II liquid handling automate (PerkinElmer). Briefly, 90 after extensive wash with ammonium bicarbonate and acetonitrile, proteins were digested using 91 trypsin (0.1 µg) and, after peptides extraction with ammonium carbonate and acetonitrile, the 92 volume of digests was reduced to ca 10 µL. LC MS experiments were performed using an Agilent 93 1200 LC chain fitted with the Chip Cube (Agilent) and coupled to an Esquire-HCT ion trap mass 94 spectrometer (Bruker). Tryptic peptides (2 µL) were resolved, on a Chip containing and enrichment 95 precolumn and a 43 mm x 75 µm RP column, using a linear gradient (7 min) from 3% to 45% of a 96

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80% acetonitrile mobile phase in 0.1% formic acid. The overall system performance was assessed 97 by introducing a BSA sample (25 fmol) every 10 samples. Raw data were processed using the 98 DataAnalysis software (Bruker) to generate files for database searching with the Mascot software 99 (Matrix Sciences). The Viridiplantae taxonomy was selected to search the MSDB database 100 (20063108 release) using the following parameters: one missed cleavage allowed, 101 carboxymethylated cystein as fixed modification, mass tolerance of ± 0.6 Da for both precursor and 102 product ions. Ion scores above 38 were significant (p < 0.05). 103 104

The molecular weight of the visualized proteins ranged from 10 to 80 kDa, and pI between 3.8 and 105 10. These ranges are in agreement with data reported for the reference leaf proteomes of Quercus 106 ilex [9], Triticum aestivum [10] and Arabidopsis thaliana [11] and the observation of Candiano et 107 al. [12] who reported that current commercially available 4% total acrylamide IPG gel strips 108 disfavour the recovery of high molecular weight proteins. Overall (Supplementary Table 1, ), ca 109 significant 120 hits were obtained in MSDB and no identification could be obtained for less than 110 10% of spots, a global result indicative of co-migration. These hits allowed the identification of 92 111 different protein accessions, suggesting the occurrence of post-translational modifications or of 112 protein degradation in some cases. Not unexpectedly because of the low amount of Citrus data in 113 databases, only one quarter of accessions was identified in species from the Citrus genus. However, 114 interestingly, a number of accessions came from quite exotic genomes showing a closer taxonomic 115 distance to Citrus than well-known model genomes. Collectively, the identified accessions defined 116 more than 50 protein functions that could be classified according to accepted functional criteria in 117 five major classes (Figure 2) (Supplementary Table 2) . (Supplemental material will be 118 accessible online at http://www.pi.ibf.cnr.it/Citrus_Biotech) .119

FIGURE 2 120

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121

Fragments of Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RubisCO-LSU), 122 were detected in several spots and the location of the RubisCO-LSU fragments on gels was not only 123 close to the theoretical mass and pI (52 kDa and pH 6.5), but was distributed over a large area 124 across the entire gel. Although intriguing, such finding is in agreement with recent observations of 125 several authors. In Arabidopsis proteome leaf [11], RubisCO-LSU fragments represented 13% of all 126 assigned spots, spreading on a large area on the gels. In Medicago truncatula leaf, about 26% of the 127 identified proteins were attributed to RubisCO large subunit [13]. Two possible explanations for 128 this phenomenon could be proposed. As suggested by Zhao et al [14], such behaviour could result 129 from normal turnover rates of RubisCO. Alternatively, this could reveal protein degradation during 130 sample preparation. Overall, this latter hypothesis cannot be ruled and could concern here as well 131 some RubisCO-LSU spots as the 2 lipoxygenases (spot n. 85) for which only 1 peptide was 132 identified. However, for other proteins showing molecular weight lower than expected (like allergen 133 len c, convicilin, legumins, translation elongation factor), the discrepancy in MW cannot be 134 attributed to C-ter or N-ter truncations as the observed peptides covered a main part of the proteins 135 (70%-90%). 136

Most of the Clementine proteins (50%) were involved in energy production and metabolism, as it 137 would be expected in plant leaf tissue [9-11]. Within this class, several proteins have been reported 138 to be implicated in the response to salt/drought stresses in plants. Fructose bisphosphate aldolase 139 (spot n. 43), one of the key regulatory enzyme of glycolysis pathway and photosystem II (PSII) 140 oxygen evolving complex protein (spots n. 49, 50, 53, 91) accumulated in response to salt stress in 141 the leaf of Oryza sativa [15]. Glyceraldehyde-3-phosphate dehydrogenase (spots n. 40, 66, 67), 142 dihydrolipoamide dehydrogenase (spot n. 20) and malate dehydrogenase (spots 39, 40), three 143 enzymes essential for ATP production, were found up-regulated in young rice panicles exposed to 144 salt stress [16], probably due to the need of energy during external stress. Glyoxalase (spot 70) was 145

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observed to play an important role in conferring tolerance to osmotic stress in rice [17]. A 146 contradictory behaviour was reported on some enzymes belonging to RubisCO complex. Down-147 regulation of RubisCO large subunit (spots 18-23) and RubisCO activase (spots n. 25, 26) was 148 observed in the leaf of Quercus ilex L. under drought stress [18], while accumulation of RubisCO 149 large subunit and RubisCO activase was reported in drought-stressed rice plants [19]. Such 150 contradictory results could be due to the methodology used, but also suggest that the genotypic 151 variability and the intensity and duration of the stress will constitute important factors to take into 152 account in future proteomic comparative research. 153

Proteins participating to oxidative-response processes (15%) were also identified. In plants it has 154 been shown that environmental stress can increase the production of ROS resulting from 155 photosynthesis, respiration and NADPH oxidase. Superoxide dismutase (SOD) (spots n. 79, 90) 156 acts as the first line of defence converting superoxides to less toxic hydrogen peroxide molecules. 157 Increase in SOD expression was observed in salt-stressed rice leaf sheath [15], in rice young 158 panicles exposed to salinity [16] and in rice leaf blades exposed to bacterial infection [20]. 159 Detoxification of H2O2 is accomplished by ascorbate peroxidase (spots n.74, 75) and catalase (spots 160 n. 95, 96), whose expression was activated by salt-stress in rice young panicles [16]. Interestingly, 161 superoxide dismutase and ascorbate peroxidase are also two of the most abundant proteins of the 162 albedo of citrus fruits [5]. Thus, collectively, previous data and present results indicate that the 163 approach used here is convenient to address important targets in the response of Citrus to oxidative 164 stress. 165 Noteworthy the Clementine leaf contained a noticeable percentage (24%) of proteins involved in 166 defence against biotic and abiotic stress (Table 1). These proteins protect plants against pathogenic 167 fungi, bacteria and viruses and adverse environmental conditions such as drought, salinity, cold. 168 169 TABLE 1 170

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Among them, the miraculin-like protein 1 and 2 ( spots n. 50, 89, 99) and the acidic class II 171 chitinase (spots n. 47, 56) were of particular relevance, in agreement with previous observation that 172 miraculin and chitinase cDNAs are widespread and very abundant in practically all citrus organs 173 [5]. Miraculin-like protein is a plant protein that can modify sour taste in sweet taste in berries. Two 174 cDNA encoding putative miraculin-like proteins were characterized in Citrus jambhiri, and 175 overtranscription of miraculin-mRNA after wounding or fungal infection was reported [21], 176 suggesting that such proteins might have defensive function against pathogens. Thus an intriguing 177 topic for future research will be the in-depth analysis of the role of miraculin-like protein in 178 defence of Citrus. Plant chitinase is one of the PR proteins and accumulates around fungal cell in 179 planta, where there is no obvious natural substrate other than the cell wall of invading fungal 180 pathogens [22]. Two cDNAs encoding acidic chitinase 1 were identified from Citrus jambhiri, and 181 accumulation of the transcript in leaves within 2 h after wounding or microbe attack was reported 182 [23]. Furthermore, recently, genetic evidence for a possible role of a chitinase-like proteins in 183 Arabidopsis salt tolerance was also found (24). Therefore, a comparative proteomic survey might 184 confirm the role of those proteins in defence mechanisms and lead to a better understanding of the 185 molecular basis of the response of Citrus to environmental stress. 186 187 In conclusion the approach used here appears to be powerful to raise proteomic information in 188 citrus. Although the genome sequence is not yet available, we were able to identify relevant 189 proteins putatively involved in the defence against abiotic and biotic attacks. This set of data, to our 190 knowledge the first one describing the Citrus leaf proteome, represents the starting point to generate 191 a large-scale protein reference maps of Citrus clementina, that might be used later in ongoing 192 integrated functional genomic studies of Citrus response to environmental stimuli. 193 194 195

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Acknowledgements: Work at IBF lab was carried out with financial support from the Provincia di 196 Livorno (Italy) with Mis 3.1 – PIC INTERREG IIIA- Italia – Francia - Isole 2000/2006, research 197 program: CITRUS: Citrus as a model system for the Mediterranean area (study on varieties resistant 198 to biotic and abiotic stresses). Work at MT lab was supported by INIA grants RTA04-013 and 05-199 247. BEM offers special thanks to Mrs C. Petrongolo, G. Tocchini, C. Neri, S. de Robertis for the 200 technical support. 201 202 References:203 [1] Gancel, A.L., Grimplet, J., Sauvage, F.X., Ollitrault, P. et al. Predominant expression of 204 diploid mandarin leaf proteome in two citrus mandarin-derived somatic allotetraploid hybrids. J. 205 Agric. Food Chem. 2006, 54, 6212-6218. 206 [2] Zukas, A.A. and Breska III, A.P., Extraction methods for analysis of citrus leaf proteins by 207 two-dimensional gel electrophoresis. J. Chromatogr. A. 2005, 1078, 201-205. 208 [3] Maserti, B.E., Della Croce, C.M., Luro, F., Morillon, R. et al., A general method for the 209 extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up. J. Chromatogr. 210 B. 2007, 849, 351-356. 211 [4] Katz, E., Fon, M., Lee, Y.J., Phinney, B.S., et al., The citrus fruit proteome: insights into citrus 212 fruit metabolism. Planta 2007, 226, 989-1005. 213 [5] Lliso, I, Tadeo, F.R., Phinney, B.S., Wilkerson, C.G. et al. Protein changes in the albedo of 214 citrus fruits on post-harvesting storage, J. Agric. Food Chem 2007 (in press). 215 [6] Terol, J.F., Conesa, A., Colmenero, J.M., Cercos M. et al. Analysis of 13000 unique Citrus 216 clusters associated with fruit quality, production and salinity tolerance. BMC Genomics. 2007, 8,217 31 (online paper). 218

[7] Terol, J., Naranjo M.A. , Talon, M., Plant & Animal Genomes XV Conference January 13-17, 219 2007, San Diego, CA.W80: Citrus. 220

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[8] Luro, F. , Costantino, G. , Billot, C., Froelicher, Y. et al. Plant & Animal Genomes XV 221 Conference January 13-17, 2007, San Diego, CA. P 487. 222 [9 ] Jorge, I., Navarro, R.M., Lenz, C., Ariza, D. et al., The Holm oak leaf proteome: Analytical and 223 biological variability in the protein expression level assessed by 2-DE and protein identification 224 tandem mass spectrometry de novo sequencing and sequence similarity searching. Proteomics 2005,225 5, 222-234. 226 [10] Donnelly, B.E., Madden, R.D., Ayouby, P., Porter, D.R. et al. The wheat (Triticum aestivum 227 L. ) leaf proteome. Proteomics 2005, 5 , 1624-1633. 228 [11] Giavalisco, P. Nordhoff, E., Kreitler, T, Kloppel, K-D., et al., Proteome analysis of 229 Arabidopsis thaliana by two-dimensional gel electrophoresis and matrix-assisted laser 230 desorption/ionisation-time of flight mass spectrometry. Proteomics 2005, 5, 1902-1913. 231 [12] Candiano, G., Musante, L., Bruschi, M., Ghiggeri, G.M., et al., Two-dimensional maps in soft 232 immobilized pH gradient gels: A new approach to the proteome of the Third Millenium. 233 Electrophoresis 2002, 23, 292-297. 234 [13] Watson, B.S., Asirvatham, V.S., Wang, L. Sumner, L.W., Mapping the proteome of Barrel 235 Medic. Plant Physiol. 2003, 131, 1104-1123. 236 [14] Zhao, C., Wang, J., Cao, M., Zhao, K., et al., Proteomic changes in rice leaves during 237 development of field-grown rice plants. Proteomics 2005, 5, 961-972. 238 [15] Abbasi F.M., Komatsu S., A proteomic approach to analyze salt-responsive proteins in rice 239 leaf sheat. Proteomics, 2004, 4, 2072-2081. 240 [16 Dadashi Dooki, A., Mayer-Posner, F.J., Askari, H., Zaiee, A. et al. Proteomic responses of rice 241 young panicles to salinity. Proteomics 2006, 6, 6498- 6507.242 [17] Zang X. and Komatsu, S., A proteomics approach for identifying osmotic-stress-related 243 proteins in rice. Phytochemistry 2007, 68, 426-437. 244

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[18] Jorge I. , Navarro, R. M., Lenz, C., Ariza, D. et al. Variation in the holm oak leaf proteome at 245 different plant developmental stages, between provenances and in response to drought stress. 246 Proteomics 2006, 6, S207 –S214. 247 [19] Salekdeh, Gh.H., Siopongco, J., Wade, L.J., Ghareyazie, B. et al. Proteomic analysis of rice 248 leaves during drought stress and recovery. Proteomics 2002, 2, 1131- 1145. 249 [20] Mahmood, T., Jan, A., Kakishima, M., Komatsu, S., Proteomic analysis of bacterial-blight 250 defense-responsive proteins in rice leaf blades. Proteomics 2006, 6, 6053-6065. 251 [21] Tsukuda S., Gomi, K.., Yamamoto, H., Akimitsu, K., Characterization of cDNAs encoding 252 two distinct miraculin-like proteins and stress-related modulation of the corresponding mRNAs in 253 Citrus jambhiri. Plant Mol. Biol. 2006, 60, 125-136. 254 [22] Wubben, J.P., Joosten, M.H.A.J., Van Kan, J.A.L., de Wit, P.J.G.M., Subcellular localization 255 of plant chitinase and 1,3-b-glucanases in Cladosporium fulvum (syn. Fulvia fulva)-infected tomato 256 leaves. Physiol. Mol. Plant Pathol. 1992 , 41, 23-32 257 [23] Gomi, K., Itoh, N., Yamamoto, H., Akimitsu, K., Characterization and functional analysis of 258 Class I and II acidic chitinase cDNA from rough lemon. J. Gen. Plant Pathol. 2002, 68, 191-199. 259 [24] Kwon, Y.R, Kim, S.H., Jung M.S., Kim, M.S. et al., Arabidopsis hot2 encodes an 260 endochitinase-like protein that is essential for tolerance to heat, salt and drought stresses. Plant J. 261 2007, 49, 184-193. 262 263 264 265 266 267 268 269 270

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Table 1. Summary of proteins related to stress response in Clementine leaf. 271 Spot number Mascot MW

(kDa) pI

Protein (other spots) Species Accession Score (theor./obs.)

Heat shock protein, 70K 2 Cucumis sativus T10248 460 75 / 75 5.1 / 4.8 Probable chaperonin 60 beta chain 6 Pisum sativum T06412 701 63 / 67 5.9 / 5.2 Putative heat shock 70 KD protein 7 Oryza sativa Q8GTB0 237 71 / 72 5.4 / 5.2 Chaperonin groEL 8 Ricinus communis HHCSBA 481 52 / 64 4.7 / 5.0 Lectin-related protein 48 (1, 3, 69) Citrus paradisi Q9FQ12 177 29 / 33 5.1 / 4.9 Miraculin-like protein 1 75 (50, 89, 99) Citrus jambhiri Q2HXG9 223 26 / 32 8.1 / 5.7 Miraculin-like protein 2 89 Citrus paradisi Q9FQ16 67 26 / 21 6.1 / 5.6 Allergen Len c 85 Lens culinaris Q84UI1 596 48 / 11 5.3 / 5.9 Convicilin 85 Lens culinaris Q9M3X8 504 60 / 11 5.6 / 6.0 Legumin B4 precursor 85 Vicia faba A24942 146 55 / 11 6.0 / 6.0 Legumin A2 precursor 85 Vicia faba S14393 136 57 / 11 6.2 / 6.0 Plant lipoxygenase 85 Medicago trunculata Q1SCS9 85 97 / 11 5.9 / 6.0 Lipoxygenase 85 Pisum sativum S01142 42 98 / 11 6.1 / 6.0 COR15 98 C. clementina x C. reticulata Q6VTH3 42 15 / 22 6.5 / 6.3 Acidic class II chitinase 47 (56) Citrus jambhiri Q8H985 64 32 / 33 5.1 / 4.8

272

273274

275276

277278

279280

281282

283284

285286

287288

289290

291292

293

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Figure legends 294

295

Figure 1. 2-DE protein profile of colloidal Coomassie-stained leaf proteins from Citrus 296 clementina. Proteins (600 µg) were separated on pH 3-11 non linear IPG strips and PAGE-SDS. 297 The relative Mr is given on the left, while the pI is given at the top of the figure. 298 299 300 301 Figure 2. Functional distribution of Citrus clementina leaf proteins. 302 303 304

305 306

307

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Figure 1. 2-D profile of colloidal Coomassie-stained leaf proteins from Citrus clementina.Proteins (600 g were separated on first-dimensional ph 3-11 non linear IPG gels and second-dimensional 13% vertical polyacrylamide gels. The relative Mr| is given on the

left, while the pI is given at the top of the figure. 254x190mm (96 x 96 DPI)

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Figure 2. Functional distribution of Citrus clementina leaf proteins 254x190mm (96 x 96 DPI)

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