proteomic analysis of “moncada” mandarin leaves with contrasting fruit load

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Plant Physiology and Biochemistry 62 (2013) 95e106

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Plant Physiology and Biochemistry

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Research article

Proteomic analysis of “Moncada” mandarin leaves with contrasting fruit load

Natalia Muñoz-Fambuena a, Carlos Mesejo a, Manuel Agustí a, Susana Tárraga b, Domingo J. Iglesias c,Eduardo Primo-Millo c, M. Carmen González-Mas c,*

a Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, C/Ingeniero Fausto Elio, E-46022 Valencia, Spainb Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Edificio 8E, E-46022 Valencia, SpaincCentro de Citricultura y Producción Vegetal, Instituto Valenciano de Investigaciones Agrarias, E-46113 Moncada, Valencia, Spain

a r t i c l e i n f o

Article history:Received 31 July 2012Accepted 9 October 2012Available online 14 November 2012

Keywords:Alternate bearingCitrusLeavesPrimary metabolismProteomicRedox state

Abbreviations: CHAPS, 3-[(3-cholamidoproppropanesulfonate; 2D DIGE, two-dimensional differedithiothreitol; IPG, immobilized pH gradient; LC-MScoupled with tandem mass spectrometry; MALDdesorption/ionization-mass spectrometry; pI, isoelemethylsulfonyl fluoride; Q-TOF, quadrupole time-of-flPAGE, sodium dodecyl sulphate-polyacrylamide gel e* Corresponding author. Tel.: þ34 96 3424000; fax

E-mail address: [email protected] (M.C. Gonz

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.10.020

a b s t r a c t

A proteomic approach was used to knowmore about the molecular mechanism related to Citrus alternatebearing. To this end, we researched protein expression differences between on-crop and off-crop“Moncada” [Clementine ‘Oroval’ (Citrus clementina Hort ex Tanaka) x ‘Kara’ mandarin (Citrus unshiuMarc. x Citrus nobilis Lou.)] mandarin leaves. This variety usually shows a remarkable behaviour inalternate production. Samples were collected in the period during which the fruit affect floweringinduction. From 2D DIGE gel, 110 spots were isolated: 43 showed increased expression in the off-cropsamples compared to on-crop samples, while 67 showed increased expression in the on-crop samplesagainst off-crop samples. These spots were identified by MALDI-MS or LC-MSeMS. According to the up-expressed proteins in off-crop leaves such as proteins related to nutrient reservoir activity or to thepentose phosphate pathway, the primary metabolism was more active in off-crop trees than in on-croptrees. In contrast, the proteins up-expressed in on-crop samples such as catalase were related to theoxidoreductase activity and, therefore, the redox state seemed different for off-crop and for on-cropleaves. Other proteins with unknown functions were isolated, which could be also related to the alter-nate bearing and to the flowering induction.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Many cultivars of Citrus tend to alternate bearing. It is a majorproblem in Citrus production worldwide, especially for lateripening mandarin cultivars [1]. The alternate bearing in Citrus isknown to be due to a reduced flower production in the springfollowing a heavy on-crop year [2,3] this effect also depending onthe time the fruit remains on the tree [4]. In addition, developingfruits exert a significant inhibitory effect on vegetative growth,reducing the number of summer/fall shoots and thereby decreasingthe number of nodes that can bear flowers during the following

yl) dimethylammonio]-1-nce gel electrophoresis; DTT,-MS, liquid chromatographyI-MS, matrix-assisted laserctric point; PMSF, phenyl-ight mass spectrometer; SDS-lectrophoresis.: þ34 96 3424001.ález-Mas).

son SAS. All rights reserved.

spring [5]. This alternation in crop load makes orchard manage-ment difficult and has a negative economic impact.

The use of genetic andmolecular approaches hasmade it possibleto identify genes in leaves that regulate flower initiation and devel-opment in Citrus [6e8]. The isolation of FLOWERING LOCUS T (FT) andits ectopic expression conferring early flowering in Poncirus trifoliataand its repression by fruit load in ‘Moncada’ hybrid mandarin[Clementine ‘Oroval’ (Citrus clementina Hort ex Tanaka) x ‘Kara’mandarin (Citrus unshiu Marc. x Citrus nobilis Lou.)] suggest a flow-ering-inducing role in Citrus [8,9]. All of these studies measuredlevels of gene expression, allowing a deeper understanding of themolecularbasis offloral inductionprocess. Besides, severalproteomicCitrus studies have been recently donewith different purposes [10e12]. However, there are no studies about which proteins are up-expressed at the floral induction time in Citrus leaves and how thepresence of the fruit may affect the levels of these proteins.

In this work, we researched protein expression differencesbetween on-crop and off-crop Citrus trees during the period inwhich the fruit affects floral induction. Moreover, we conducted anontology analysis to know the molecular functions that thesedifferential proteins can normally carry out and the biologicalprocesses that these proteins are involved in. As far as we know, this

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N. Muñoz-Fambuena et al. / Plant Physiology and Biochemistry 62 (2013) 95e10696

paper is the first published proteome-wide experiment on Citrusleaves with contrasting fruit load and therefore provides informa-tion in an undocumented area that could assist in alternated bearingcontrol. The cultivar selected for this studywas the ‘Moncada’hybridmandarin, a strong alternate bearing variety, whose mature crop isnormally still on the tree during floral induction.

2. Results

2.1. Comparative proteome analysis

The aim of this work was to compare the leaf proteome frommandarin trees with contrasting crop. We identified the proteinspots that were up- and down-regulated in off-crop treescomparing with on-crop trees. Therefore, leaf samples from off andon-crop trees were analyzed by 2DE. Gels were of high quality withreproducible protein patterns among replicates of the samesamples (Fig. 1).

Approximately 1436 spots in gel images from samples wereresolved. To assess the global differences in the expression levelsbetween off and on-crop samples, gels were compared and quan-tified using the DeCyder� Differential Analysis Software. Amongthe total proteins, 176 protein spots showed a significant quanti-tative differential accumulation (t-text < 0.05) between on and off-crop samples. 111 spots were confirmed with a good match anda sufficient volume for subsequent identification by mass spec-trometry. To reliably determine quantitative changes in proteinexpression and therefore overcome error imposed by technical andbiological variations, proteins were identified as up-regulated inoff-crop samples if they were found to have an average expressionlevel at least 1.10 higher than those of on-crop samples and as up-regulated in on-crop samples if they were found to have an averageexpression level at least �1.10 higher (absolute value) than those ofoff-crop samples. Among the 110 proteins, 43 had increasedexpression in the off-crop samples compared to on-crop samples(Av ratio þ), while 67 showed decreased expression in the off-cropsamples (Av ratio �).

2.2. Identification of differentially expressed proteins

We could manually excise the 110 proteins with a good matchfrom a preparative 2DE gel to further identify 90 of them by

Fig. 1. Representative 2D DIGE gel of proteins extracted from “Moncada” mandarin leaves. Egreen) and internal standard (Cy2, blue) were loaded in the same gel. (A) Proteins up-expresin red and proteins unaffected appear in yellow. (B) Proteins selected for the analysis by m

MALDI-MS analysis and the other 20 proteins by LC-MS/MSanalysis. Table 1 provides the spot number, the function of eachprotein together with the putative protein name, the accessioncode, the organism based on the protein has been identified, thehomologue in C. clementina established by the database in www.phytozome.net, the homologue in Arabidopsis thaliana estab-lished by the database in www.arabidopsis.orgs, the values fortheoretical and experimental pI and molecular mass, the expres-sion ratio and p-value, and the MASCOT score together with thesequence coverage and peptides matched.

2.2.1. Classification of identified proteinsThe identified proteins could be classified into seven groups

according to its biological function: (i) primary metabolism (33spots: 26 spots being associated with photosynthesis and carbo-hydrate metabolism, 4 spots related to Krebs cycle, 1 spot related topentose phosphate pathway, and 2 spots related to nutrientreservoir activity); (ii) oxidoreductase activity (8 spots: one ofthem, the spot 79, up-regulated in off-crop samples, presented thehighest ratio among all spots, and 5 spots were catalase, all of themup-regulated in on-crop samples, with spots 381, 384, 386 and 394matching the same EST sequence); (iii) stress responses (3 spots, allof them up-regulated in on-crop samples); (iv) signal transduction(1 spot); (v) protein synthesis and degradation (6 spots); (vi)expansins (1 spot); (vii) other proteins (58 spots: it is the largestgroup, most proteins of the last group with an unknown function).The relative percentages of proteins both in on-crop leaves and inoff-crop leaves appear in Fig. 2.

On the one hand, some of these spots were identified as theidentical protein such as catalase, for spots 381, 384, 386 and 394(oxidoreductase group); NADP-isocitrate dehydrogenase, for spots515 and 516 (Krebs cycle subgroup, up-regulated in on-cropsamples); RuBisCO large subunit-binding protein subunit betachloroplast, for spots 300, 301, and 307; granule-bound starchsynthase Ib precursor, for spots 327 and 328 (Fig. 3); putativecinnamoyl-CoA reductase, for spots 743, 744, and 748. The lastthree groups of proteins are related to primary metabolism and allof them are up-regulated in off-crop samples. On the other hand,some of the spots were identified as the same protein, but displayeddifferent pI and molecular mass values and might account for iso-forms or post-translationally modified forms of these proteins.Examples of the latter spots aremiraculin-like protein 1 (spots 1016

qual amounts (50 mg) of on-crop leaves sample (Cy5, red), off-crop leaves sample (Cy3,sed in off-crop leaves appear in green, those down-expressed in off-crop leaves appearass spectrometry. Spot numbers correspond to the same ones indicated in Table 1.

http://www.phytozome.net

http://www.phytozome.net

http://www.arabidopsis.orgs

Table 1List of identified proteins.

Spot Function/protein Accession no. Species Homologue in Citrusclementina

Homologue inArabidopsis thaliana

Mol. mass(kDa) Theo/Exp

pI Theo/exp Ratio p-value Score/sequencecoverage (%)/peptides matched

(i) Related to primary metabolismPhotosynthesis and carbohydratemetabolism

238 NADP-dependent malic enzyme 12712UC37CL5763Contig3

Vitis vinifera clementine0.9_005680m AT1G79750.1 75.79/81.50 8.58/7.45 �1.27 0.050 110/20/15

267 ATP-binding cassette transportera 98077UC452943 Pteris vittata clementine0.9_009053m AT5G60790.1 88.65/80.00 8.62/7.60 �1.22 0.033 233/6/5288 Putative t-complex protein 1 theta

chain144062UC3813154 Magnoliophyta clementine0.9_006345m AT3G11830.1 79.87/79.50 8.55/8.70 1.23 0.025 82/27/17

300 RuBisCO large subunit-bindingprotein subunit betab

13227UC37CL6180Contig2

Brassicaceae clementine0.9_005361m AT5G56500.2 74.41/79.00 6.73/6.10 1.83 0.034 172/28/17




302 Phosphoglucomutasea,b 46090UC37041305 Brassicaceae(Arabidopsisthaliana)

clementine0.9_004855m AT5G51820.1 30.55/78.00 7.86/7.50 �1.19 0.042 319/31/8

303 3,4-dihydroxy-2-butanone kinasea 15551UC37CL8203Contig1

Solanumlicopersicum

clementine0.9_005619m AT3G17770.1 67.21/79.50 6.46/7.40 �1.23 0.012 607/15/11

306 RuBisCO large subunit-bindingprotein subunit betaa,b

136270UC385359 Brassicaceae clementine0.9_005361m AT1G55490.2 80.16/80.00 8.93/6.20 1.70 0.025 1463/33/26




330 Ribulose-1.5-bisphosphatecarboxylase/oxygenase large subunit

gij21634083 Dichondrabrachypoda

clementine0.9_008884m ATCG00490.1 51.69/70.00 6.09/7.60 1.19 0.025 104/26/14

362 Ribulose-1.5-biphosphate-carboxylase gij6634076 Citrus paradisi clementine0.9_008884m ATCG00490.1 52.03/70.00 6.19/8.00 �1.15 0.046 185/31/18417 Ribulose bisphosphate carboxylase

large chainRBL_CITSI Citrus sinensis clementine0.9_008884m ATCG00490.1 53.00/68.50 6.29/8.80 �1.28 0.025 339/38/24

420 Nadp-dependent glyceraldehyde-3-phosphate dehydrogenase

99276UC454142 Medicagotruncatula

clementine0.9_008326m AT2G24270.4 63.30/68.50 8.73/9.45 1.23 0.029 204/44/14

437 Nadp-dependent glyceraldehyde-3-phosphate dehydrogenase


Medicagotruncatula

clementine0.9_008326m AT2G24270.4 61.03/65.50 8.76/9.00 1.20 0.021 177/21/11

438 AlaT1 14418UC37CL7192Contig2

Vitis vinifera clementine0.9_008926m AT1G70580.4 67.99/62.00 8.56/7.55 1.17 0.029 112/21/14

445 Ribulose-1.5-bisphosphatecarboxylase/oxygenase large subunit

gij296277499 Atalantia spinosa clementine0.9_008884m ATCG00490.1 48.05/61.50 6.30/5.85 �1.16 0.022 235/32/20

462 ADP-glucose pyrophosphorylasesmall subunit

gij111660950 Citrus sinensis clementine0.9_007503m AT5G48300.1 57.33/62.00 6.73/7.05 1.24 0.007 235/33/16

554 Alcohol dehydrogenase 12873UC37CL5895Contig1

Citrus paradisi clementine0.9_036084m AT1G77120.1 57.84/56.50 8.30/8.20 �1.22 0.018 77/18/8

743 Putative cinnamoyl-CoA reductase 2277UC37CL154Contig7

Arabidopsisthaliana

clementine0.9_027444m AT2G02400.1 39.14/48.00 8.38/7.90 1.20 0.033 155/24/7

744 Putative cinnamoyl-CoA reductasea 2277UC37CL154Contig7

Arabidopsisthaliana

clementine0.9_027444m AT2G02400.1 39.14/49.00 8.38/8.00 1.30 0.023 105/8/3

748 Putative cinnamoyl-CoA reductase 2277UC37CL154Contig7

Arabidopsisthaliana

clementine0.9_027444m AT2G02400.1 39.14/49.00 8.38/8.40 1.45 0.019 209/34/11

847 Carbonic anhydrasea 1035UC37CL1Contig1036

Populus tremulax Populustremuloides

clementine0.9_015268m AT3G01500.2 43.81/46.00 7.95/8.00 1.22 0.033 247/19/6

894 Transketolase. chloroplastic TKTC_MAIZE Zea mays clementine0.9_003238m AT3G60750.1 73.35/45.00 5.47/6.25 �1.40 0.030 59/7/6970 Putative ribose 5-phosphate

isomerasea2390UC37CL176Contig5

Arabidopsisthaliana

clementine0.9_018380m AT3G04790.1 40.34/43.00 9.22/5.60 �1.18 0.044 349/21/11

1016 Miraculin-like protein 1a 902UC37CL1Contig903

Citrus jambhiri clementine0.9_020626m AT1G17860.1 35.05/42.50 9.50/9.00 1.74 0.016 422/29/13

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Table 1 (continued )

Spot Function/protein Accession no. Species Homologue in Citrusclementina

Homologue inArabidopsis thaliana

Mol. mass(kDa) Theo/Exp

pI Theo/exp Ratio p-value Score/sequencecoverage (%)/peptides matched

1049 Miraculin-like protein 1a 184UC37CL1Contig184

Citrus jambhiri clementine0.9_020626m AT1G17860.1 35.58/41.50 9.38/8.25 1.75 0.015 454/28/13

Krebs cycle500 Citrate synthase CISY_CITMA Citrus maxima clementine0.9_009277m AT2G44350.1 52.43/59.00 6.90/8.45 �1.27 0.027 212/26/14515 NADP-isocitrate dehydrogenase gij5764653 Citrus limon clementine0.9_006693m AT1G65930.1 46.82/58.00 6.49/8.50 �1.24 0.025 79/23/10516 NADP-isocitrate dehydrogenase gij5764653 Citrus limon clementine0.9_006693m AT1G65930.1 46.82/58.00 6.49/8.70 �1.22 0.011 164/31/15764 Malate dehydrogenase, glyoxysomal

precursor143397UC3812489 Citrullus lanatus clementine0.9_011590m AT2G22780.1 49.76/48.50 9.43/8.80 1.25 0.021 95/17/6

Pentose phosphate pathway454 6-phosphogluconate dehydrogenase 3692UC37CL541

Contig4Arabidopsisthaliana

clementine0.9_008389m AT1G64190.1 67.85/62.00 8.52/8.70 1.19 0.025 99/18/10

Related to nutrient reservoir activity327 Granule-bound starch synthase Ib

precursor12888UC37CL5907Contig1

Phaseolusvulgaris

clementine0.9_005261m AT1G32900.1 82.61/74.00 8.87/7.75 1.40 0.012 221/25/16

328 Granule-bound starch synthase Ibprecursor


Phaseolusvulgaris

clementine0.9_005261m AT1G32900.1 82.61/74.00 8.87/7.80 1.56 0.005 107/21/14

(ii) Oxidoreductase activity79 Putative monocopper

oxidase precursor100961UC455827 Arabidopsis

thalianaclementine0.9_034025m AT4G12420.2 83.18/89.0 9.12/9.30 2.94 0.005 128/23/16

381 Catalase 11460UC37CL4806Contig1

Prunus persica clementine0.9_008455m AT4G35090.1 67.93/69.50 8.86/8.70 �1.26 0.011 362/28/18

382 Catalase 147681UC3816774 Prunus persica clementine0.9_008455m AT4G35090.1 68.54/69.00 8.99/8.75 �1.40 0.015 345/36/27384 Catalase 11460UC37CL4806

Contig1Prunus persica clementine0.9_008455m AT4G35090.1 67.93/69.00 8.86/8.80 �1.40 0.011 385/33/25





474 Monodehydroascorbatereductase


Solanumlycopersicum

clementine0.9_008242m AT1G63940.2 60.85/63.00 8.77/8.50 �1.26 0.025 231/35/20

1135 Fe-superoxide dismutasea 1143UC37CL6Contig5

Lotus japonicus clementine0.9_018576m AT5G51100.1 41.15/37.50 8.86/7.35 �1.43 0.011 352/17/8

(iii) Stress responses717 Clone C31705D02 gij218832267 Citrus clementina clementine0.9_014588m AT3G03080.1 27.31/49.50 6.92/8.40 �1.24 0.010 187/37/101249 Stress-related proteina 96234UC451100 Citrus sinensis clementine0.9_023848m AT5G45860.1 26.39/30.00 8.87/6.95 �1.81 0.012 489/35/121259 Stress-related proteina 12135UC37CL5321

Contig1Citrus sinensis clementine0.9_023848m AT5G45860.1 27.07/30.50 9.34/7.45 �1.39 0.039 305/19/5

(iv) Signal transduction736 Putative plastidic

cysteine synthase 1105791UC4510657 Oryza sativa clementine0.9_013305m AT2G43750.2 49.14/49.00 9.00/6.70 �1.17 0.036 222/44/22

(v) Protein synthesis anddegradation

104 Protein disulphide isomerasea 4082UC37CL670Contig2

Elaeis guineensis clementine0.9_031693m AT5G60640.1 36.28/88.00 5.38/5.40 1.30 0.042 534/31/12

126 Protein disulphide isomerasea 41019UC37AZ Elaeis guineensis clementine0.9_031693m AT5G60640.1 32.97/86.50 5.35/5.30 1.24 0.036 413/28/11358 Chaperonin subunit putative 101818UC456684 Arabidopsis


933 Tudor; Staphylococcusnuclease subtypea


Medicagotruncatula

clementine0.9_001246m AT5G07350.2 39.58/43.50 6.16/9.00 1.38 0.023 196/7/4

1018 Cysteine proteinase-like protein 8069UC37CL2571Contig1

Ipomoea batatas clementine0.9_022372m AT3G49340.1 41.17/42.00 5.67/5.20 �1.57 0.016 78/19/7

1264 Adenylate isopentenyltransferase8, chloroplastic

IPT8_ARATH Arabidopsisthaliana

clementine0.9_014843m AT3G19160.1 37.58/30.00 8.75/5.40 1.26 0.016 61/45/15

(vi) Expansins

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871 Xyloglucan endotransglycosylase 98350UC453216 Gossypiumhirsutum

clementine0.9_017721m AT5G65730.1 47.70/45.00 7.90/8.05 1.63 0.008 132/22/9

(vii) Other proteins263 T13D8.29 proteina 31101UC37 Arabidopsis


428 Tubulin alpha-1 chain 4507UC37CL828Contig2

Arabidopsisthaliana

clementine0.9_030036m AT5G19780.1 64.11/67.00 5.64/5.90 �1.21 0.021 80/19/11

545 Putative pectinesterasea 19469UC37CL12030Contig1

Oryza sativa clementine0.9_008076m AT4G33230.1 34.41/57.00 9.18/10.25 1.65 0.011 214/13/4

561 Clone C05136B10 gij63065066 Citrus sinensis clementine0.9_009673m AT1G30580.1 27.71/56.00 8.96/9.20 �1.14 0.030 234/70/19621 Clone IC0AAA48BC11 gij218838992 Citrus clementina clementine0.9_010996m AT2G20420.1 31.93/54.00 5.91/6.65 �1.24 0.025 126/42/13677 Clone STG2_1309 50 gij209935988 Citrus unshiu clementine0.9_011770m AT5G50850.1 24.53/50.00 5.21/6.70 �1.10 0.042 90/34/8703 Clone UCRPT02-33B05-D10-1-6.b gij57873050 Citrus trifoliata clementine0.9_020100m AT2G38380.1 31.89/50.50 9.32/9.50 1.34 0.008 95/29/10706 Clone IC0AAA46BA07 gij218790269 Citrus clementina clementine0.9_014847m AT1G71695.1 31.79/50.50 8.93/10.80 �2.51 0.005 133/35/8709 Beta-1,3-glucanase class IIIa 13154UC37CL6125

Contig2Citrus clementina clementine0.9_014533m AT3G57260.1 43.33/50.00 5.68/5.80 �1.29 0.048 412/19/9

749 IC0AAA37AD07RM1 CitNFL gij110857582 Citrus clementina clementine0.9_020100m AT2G02400.1 47.64/49.00 9.09/8.65 1.46 0.014 171/20/10770 PT11-C9-005-009-C08-CT.F gij188225533 Poncirus trifoliata clementine0.9_011590m AT2G22780.1 29.28/48.00 9.38/10.00 1.60 0.027 108/43/9792 Clone IC0AAA10DG03 gij218812293 Citrus clementina clementine0.9_016626m AT4G39230.1 34.13/47.50 9.61/9.00 �2.33 0.011 80/34/9801 Clone KN0AAH3CD04 gij218820391 Citrus clementina clementine0.9_016626m AT4G39230.1 29.06/47.00 9.51/9.50 �2.78 0.012 358/50/14812 Clone IC0AAA10DG03 gij218812293 Citrus clementina clementine0.9_016626m AT4G39230.1 34.13/47.00 9.61/9.90 �2.86 0.005 417/56/18825 Clone IC0AAA10DG03 gij218812293 Citrus clementina clementine0.9_016626m AT4G39230.1 34.13/46.50 9.61/10.10 �3.22 0.005 114/33/9909 KN0AAP9YD08FM1 Fruit-TF gij110851549 Citrus clementina clementine0.9_021318m AT1G09560.1 30.66/44.00 8.78/10.40 �1.64 0.025 138/14/5941 Clone UCRCS04_ 030_T3_C11 gij55936798 Citrus sinensis clementine0.9_006827m AT5G66140.1 27.71/43.00 8.69/9.90 1.20 0.042 155/29/9947 DC884625 ANT gij209925742 Citrus unshiu clementine0.9_019708m AT3G48420.1 28.53/43.00 6.73/6.10 �1.28 0.017 96/14/3972 Clone A1650001_IIF_C01 50 gij45451762 Citrus sinensis clementine0.9_019618m AT3G09640.2 32.25/42.50 6.19/6.65 �1.62 0.044 175/43/14978 Clone UCRCS08-3E10-J19-1-5.g gij56588623 Citrus sinensis clementine0.9_012723m AT1G49970.1 27.09/42.00 5.40/5.95 �1.18 0.030 94/65/14979 Clone C05808B11 gij63065913 Citrus sinensis x C.

trifoliataclementine0.9_020467m AT5G02790.1 27.01/42.50 5.58/6.20 �1.27 0.025 159/57/12

1006 IC0AAA29DA06RM1 CitNFL gij110851709 Citrus clementina clementine0.9_003575m AT1G21680.1 37.05/42.00 5.63/10.10 �1.75 0.023 123/28/71009 Clone VPE-39_H09 50 gij71597421 Citrus sinensis clementine0.9_020761m AT1G17100.1 26.39/40.50 5.17/5.35 �1.61 0.016 86/31/51010 Clone C31102C11 gij218825114 Citrus reshni clementine0.9_018172m AT2G32520.1 24.79/42.50 8.59/7.60 �1.24 0.012 107/38/71012 UCRCR01_06K12_f gij38032410 Citrus reticulata clementine0.9_014505m AT4G09010.1 20.38/42.00 6.34/8.75 �1.20 0.026 91/49/81014 KN0AAL1BE04FM2 KCl-Salt1 gij110886813 Citrus reshni clementine0.9_035067m AT3G22110.1 39.67/42.00 6.52/8.30 1.14 0.043 134/19/61015 Hypothetical protein At2g31670 142854UC3811946 Arabidopsis thaliana clementine0.9_019220m AT2G31670.1 30.98/42.00 8.88/6.7 �1.34 0.018 86/42/101045 CR05-C1-100-016-F12-CT.F gij188356206 Citrus reticulata clementine0.9_000163m AT3G01500.2 32.88/41.50 8.86/7.05 �1.40 0.036 74/39/91047 PT11-C1-900-008-E04-CT.F gij188446025 Poncirus trifoliata clementine0.9_016969m AT3G26340.1 32.80/42.00 8.47/9.00 1.24 0.017 119/33/121058 At1g76020 23150UC37CL15808

Contig1Arabidopsis thaliana clementine0.9_034879m AT1G76020.1 27.52/41.50 10.19/8.70 �1.49 0.007 208/26/7

1061 Chitinase CHI1a 14249UC37CL7048Contig1

Citrus sinensis clementine0.9_020680m AT3G54420.1 41.25/41.00 8.42/8.95 �1.48 0.015 269/20/6

1064 Clone UCRCS09-27B08-D16-1-5.g gij56533662 Citrus sinensis clementine0.9_027803m AT3G14290.1 31.80/40.50 4.75/5.30 �1.27 0.003 123/34/121071 CS00-C1-650-010-H07-CT.F gij188298712 Citrus sinensis clementine0.9_015268m AT3G01500.2 31.80/39.00 8.97/6.85 �1.22 0.025 123/53/141076 CS00-C1-650-010-H07-CT.F gij188298712 Citrus sinensis clementine0.9_015268m AT3G01500.2 30.96/39.50 8.97/7.15 �1.30 0.016 150/45/91080 Clone GSA0864 50 gij209929778 Citrus unshiu clementine0.9_020733m AT3G54420.1 28.52/40.00 6.64/5.90 �1.83 0.030 114/28/61081 Clone C04010B03 gij63060920 Citrus clementina clementine0.9_020733m AT3G54420.1 22.68/40.00 9.55/6.10 �1.81 0.027 110/36/61094 PT11-C9-005-004-C03-CT.F gij188333275 Poncirus trifoliata clementine0.9_020475m AT3G12490.2 35.07/39.00 6.69/7.85 1.27 0.048 90/39/91102 Clone F80DAB0001_ IVF_D04 50 gij45449729 Citrus sinensis clementine0.9_021222m AT2G47730.1 24.55/39.00 6.38/8.30 �1.16 0.025 134/42/91104 Clone C34003B06 gij218845933 Citrus clementina clementine0.9_021364m AT1G78380.1 28.36/40.00 7.07/9.15 1.47 0.005 103/28/111108 Clone UCRPT02-45F01-L2-1-5.g gij57874981 Citrus trifoliata clementine0.9_020929m AT2G15220.1 27.65/40.00 6.65/8.75 �1.87 0.025 155/41/131150 Clone CS_REb0004P08 gij46209000 Citrus sinensis clementine0.9_022586m AT1G33140.1 30.01/37.00 9.19/10.30 1.17 0.013 229/46/111166 Clone KN0AAP4YL20 gij218837597 Citrus clementina clementine0.9_022102m AT3G22630.1 25.51/35.50 6.34/7.55 �1.42 0.017 282/55/181167 Clone A1650001_IIF_A05 50 gij45451803 Citrus sinensis clementine0.9_017849m AT3G10920.1 33.39/35.50 8.52/7.80 �1.34 0.023 285/43/141188 Clone 724 50 gij229045058 Citrus sinensis clementine0.9_021335m AT1G17860.1 28.23/34.50 8.75/5.85 �1.47 0.048 171/44/131192 Clone 724 50 gij229045058 Citrus sinensis clementine0.9_021335m AT1G17860.1 28.23/34.00 8.75/6.70 �1.79 0.041 293/54/131201 5 Y 1069 106422UC4511288 clementine0.9_020989m AT2G32645.1 39.45/34.00 9.35/5.40 2.12 0.016 125/26/121266 Clone 724 50 gij229045058 Citrus sinensis clementine0.9_021335m AT1G17860.1 28.23/29.50 8.75/6.70 �1.45 0.026 88/46/101280 Clone VPE-18_C10 50 gij71598314 Citrus sinensis clementine0.9_025138m AT1G65980.1 19.39/28.00 5.61/6.80 1.20 0.011 109/50/81285 Clone CS_REb0002M11 gij46207565 Citrus sinensis clementine0.9_020920m AT5G10160.1 28.92/29.00 8.78/7.75 1.27 0.011 138/23/6

(continued on next page)

N.M

uñoz-Fambuena

etal./

PlantPhysiology

andBiochem

istry62

(2013)95

e106

99

Table

1(con

tinu

ed)

Spot

Function/protein

Accession

no.

Species

Hom

olog

uein

Citrus

clem

entina

Hom

olog

uein

Arabido

psisthaliana

Mol.m

ass

(kDa)

Theo

/Exp

pITh

eo/exp

Ratio

p-va

lue

Score/sequ

ence

cove

rage

(%)/

pep

tides

match

ed

1325

Clone72

450

gij229

0450

58Citrus

sine

nsis

clem

entine0

.9_0

2133

5mAT1

G17

860.1

28.23/27

.00

8.75

/8.75

�1.66

0.01

180

/32/3

1361

CloneKN0A

AI3DE0

8gij218

8253

85Citrus

clem

entina

clem

entine0

.9_0

2149

5mAT2

G25

640.1

26.76/24

.00

9.01

/6.90

�1.66

0.01

120

9/40

/10

1384

PT11

-C1-90

0-02

1-E1

0-CT.F

gij188

3209

71Po

ncirus

trifo

liata

clem

entine0

.9_0

2641

8mAT2

G47

710.1

34.79/24

.00

9.53

/8.90

�1.21

0.02

311

8/22

/614

09CloneUSD

A-FP_

0228

150

gij216

5235

9Citrus

sine

nsis

clem

entine0

.9_0

2069

6mAT5

G63

310.1

19.29/21

.00

6.21

/7.05

�1.35

0.01

112

1/43

/10

1421

IC0A

AA87

DD12

RM1CitNFL

gij110

8769

70Citrus

clem

entina

clem

entine0

.9_0

2057

4mAT4

G05

180.1

29.05/21

.50

9.32

/10.25

�1.39

0.04

933

8/49

/15

1428

CloneKN0A

AP8

YO03

gij218

8426

40Citrus

clem

entina

clem

entine0

.9_0

1996

2mAT4

G11

600.1

27.03/33

.00

8.93

/6.95

�1.43

0.02

112

3/31

/814

29CR05

-C3-70

0-06

2-E0

9-CT.F

gij188

4073

33Citrus

reticu

lata

clem

entine0

.9_0

3422

8mAT2

G45

790.1

31.75/42

.50

9.20

/8.30

1.46

0.01

698

/30/8

1432

CloneKN0A

AM3C

H09

gij218

7992

86Citrus

sine

nsis

clem

entine0

.9_0

1579

4mAT1

G24

020.2

26.67/31

.00

9.34

/7.55

�1.57

0.02

111

0/53

/11

1433

Putative

spindle

disassembly

relatedprotein

CDC48

gij989

6249

7Nicotiana

taba

cum

clem

entine0

.9_0

2978

3mAT5

G03

340.1

90.64/89

.00

5.13

/6.00

1.22

0.01

617

0/22

/20

aProteiniden

tified

byLC

-MS/MS.

bChloroplast

precu

rsor.

A

B

Fig. 2. Functional classification of the proteins identified and found to be upregulatedin on-crop (A) or in off-crop (B) leaves. The relative percentages of proteins in eachcategory are shown.


and 1049) or Nadp-dependent glyceraldehyde-3-phosphate dehy-drogenase (spots 420 and 437), both of them related to carbohy-drate metabolism, or protein disulphide isomerase (spots 104 and126, belonging to the protein synthesis and degradation group), allof them up-regulated in off-crop samples.

2.3. Species from which the identified proteins proceed

Fifty-nine spots were identified by matching against Citrussequences, the number of matched peptides being between 3 and24, with 14e70% of sequence coverage. Results of a similar orderwere found for the other 51 proteins, identified by homology tosequences from other species such as Arabidopsis thaliana (15spots), Prunus persica (5 spots), Medicago truncatula (3 spots), Vitisvinifera (2 spots), Phaseolus vulgaris (2 spots) or Solanum lyco-persicum (2 spots).

2.4. Gene ontology analysis

Total amounts of protein isolatedwere analyzed separately in twogroups established according to ratio expression, by using the Webtool FatiGO (http://babelomics.bioinfo.cipf.es) [13,14]. The databasein www.arabidopsis.orgs was used to search Arabidopsis proteinshomologous to proteins identified in this study (Table 1). The estab-lishment of these homologies allowedus to know themain biologicalprocesses in which the identified proteins are involved. The largest

http://babelomics.bioinfo.cipf.es

http://www.arabidopsis.orgs

Fig. 3. Representative proteins analyzed using DeCyder Software (spots 327, 328, 381, 462, 784 and 1018). Differential expression analysis in on-crop and off-crop leaves ofrepresentative spots from Fig. 1.

N. Muñoz-Fambuena et al. / Plant Physiology and Biochemistry 62 (2013) 95e106 101

groups of the proteins with AV ratio þ were composed of proteinsinvolved in carbohydrate and starch biosynthesis, carbohydratemetabolism, protein folding and response to metal ion (Fig. 4). Thelargest group of the proteins with AV ratio e was composed ofproteins involved in hydrogen peroxide catabolic process and inresponse to stress, but many other biological processes are related tothe up-expressed proteins in the on-crop samples (Fig. 5).

3. Discussion

Wereportchangesintheleafproteomeofthe ‘Moncada’mandarin,differinginitscropload(offandon-crop).Usingaproteomicapproachweaimedatabetterunderstandingof thebiologicalprocesses relatedto the alternated bearing, and how it is related to the flowering.

3.1. Proteins up-expressed in the off-crop samples

3.1.1. Proteins related to primary metabolismAccording to our ontology study, for proteins up-expressed in

the off-crop samples compared to the on-crop sample, the largestgroups of proteins are involved in carbohydrate and starch

biosynthesis, both of them related to primary metabolism. Amongthem, the following proteins may be highlighted: Granule-boundstarch synthase Ib precursor (GBSS, spot 327 and 328), malatedehydrogenase glyoxysomal precursor (MD, spot 764) and ADP-glucose pyrophosphorylase small subunit (AGP, spot 462) (Fig. 3).The activation state of these proteins is correlated with the accu-mulation of starch and soluble sugar in several tissues from rice,wheat or tomato [15e17]. In fact, we found that the starch level wassignificantly higher in leaves of off-crop trees than in leaves of on-crop trees, being these results consistent with the up-expression ofGBSS in off-crop leaves (Fig. 6). The same behaviour has beendescribed for other biennial-bearing species. In pistachio, duringnut development, various organs of “off” trees began to accumulatea greater concentration of soluble sugars and starch and surpassedthe amount measured in those of “on” trees [18]. Also, in biennial-bearing mango trees, the on-crop trees had lower starch content inthe shoots than off-crop trees, during floral-inductive period [19].Since starch accumulation in the Citrus shoots seemed to parallelflower induction [20], the carbohydrate reserves in leaves in‘Moncada’ mandarin off-trees could act like an active sink [3].Moreover, in olive (Olea europaea) another study suggests a strong

OFF Biological process

Response to

stimulus

Cellular process

Response to

metal ion

Response to

cadmium ion

Starch

biosynthetic

process

Protein folding

Cellular

carbohydrate

metabolic process

Cellular glucan

metabolic process

Carbohydrate

biosynthetic

process

Metabolic process

Fig. 4. Biological process related to proteins up-regulated in off-crop leaves accordingto FatiGO study. Terms at higher levels of the hierarchy describe more generalprocesses while terms at lower levels are more specifics. Colour intensity in the graphsquares means the level of contribution in the total biological processes (the greaterthe colour intensity, the higher the level of contribution). (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version ofthis article.)


correlation between flower starch content and functional pistildevelopment [21].

The more important enzymes related to photosynthesis werealso up-expressed in off-crop samples in general. Among them, wenote the RuBisCO large subunit-binding protein subunit beta (spots300, 301, 306, and 307) or NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (spots 420 and 437). Both of them areinvolved in the photosynthetic Calvin cycle [22]. This suggests thatthe light-independent reactions for photosynthesis are moreenhanced in the off-crop samples than the on-crop samples.

ON Biologic

Cellular processMetabolic process

Photosynthesis, dark

reaction

Cellular respiration

Reductive pentose-

phosphate cycle

Oxygen and

reactive oxygen

species metabolic

process

Superoxide

metabolic

process

Response to

cold

Carbon utilization by

fixation of carbon dioxide

Response

per

Response to

saltstress

Response to

temperature stimulus

Response t

str

Fig. 5. Biological process related to proteins up-regulated in on-crop leaves according to Fatiterms at lower levels are more specifics. Colour intensity in the graph squares means the levhigher the level of contribution). (For interpretation of the references to colour in this figu

Another notable up-expressed protein in the off-crop samplesrelated to primary metabolism was 6-phosphogluconate dehy-drogenase (6PGD; spot 454). This protein is a key enzyme of thepentose phosphate pathway, a part of the central metabolism.Mutants of 6PGD genes are more sensitive to hydrogen peroxidecompared with the wild-type, suggesting a role in the oxidativestress tolerance [23]. Likewise 6PGD is able to co-operatebetween the cytosolic and plastidic oxidative pentose phos-phate pathway in the provision of NADPH for biosynthesis [24].Moreover, the up-expression of 6PGD in the off-crop samples isaccording to its behaviour in Dioscorea esculenta tubers andCurcuma longa rhizomes, where its activity increases before thevisible appearance of sprouting and reaches its maximum duringsprouting [25].

3.1.2. Other noteworthy up-expressed proteins in the off-cropsamples

These proteins were chaperonin subunit (spot 358), putativepectinesterase (spot 545) and adenylate isopentenyltransferase 8,chloroplastic (spot 1264). With respect to the first protein, chap-eronins are a type of molecular chaperones that occur inprokaryotes and in the mitochondria and plastids of eukaryotes[26]. Maeda et al. (2006) showed that in plumules of Pharbitis nilthe up-regulation of a 60 kDa chaperonin b-subunit may havea role in modulating other proteins during flower evocation [27];also, Zabaleta et al. (1994) observed that in tobacco plant withanti-sense Chaperonin 60 b the flowering was inhibited [28]. Onthe other hand, the up-expression of pectinesterase for off-cropsamples can be explained by its implication in important physio-logical processes associated with reproductive plant development,including microsporogenesis and pollen tube growth [29]. Lastly,adenylate isopentenyltransferase catalyzes the initial step in thebiosynthesis of cytokinin in higher plants [30]. Several studiessuggested the cytokinin content in primordial organs is importantto control the progression of floral meristem development [31e33].

al process

Response to stimulus Multi-organism process

Response to other

organism

Response to

reactive oxygen

species

Response to

metal ion

Response to

bacterium

Defense response to

bacterium

Response to

cadmium ion

Hydrogen peroxide

catabolic process

Defenseresponse

Response to

oxidative stress

Response to biotic stimulus

to hydrogen

oxide

o osmotic

ess

GO study. Terms at higher levels of the hierarchy describe more general processes whileel of contribution in the total biological processes (the greater the colour intensity, there legend, the reader is referred to the web version of this article.)

Starch concentration

0

50

100

150

200

250

300

ON-crop leaves

OFF-crop leaves

B

A

Catalase activity

0

20

40

60

80

100

120

140

160

ON-crop leaves

OFF-crop leaves

Fig. 6. A. Starch concentration in on-crop and off-crop leaves expressed in mg g�1 dryweight. Data are means of six independent replicates (n ¼ 6). There are significantdifferences between on-crop and off-crop leaves (P � 0.05). B. Catalase activity in on-crop and off-crop leaves expressed in mmol H2O2 consumed g�1 protein min�1. Data aremeans of six independent replicates (n ¼ 6). There are significant differences betweenon-crop and off-crop leaves (P � 0.05).


3.2. Proteins up-expressed in the on-crop samples

3.2.1. Proteins with oxidoreductase activityFor proteins up-expressed in the on-crop samples, the largest

groups are those involving a hydrogen peroxide catabolic process,such as catalase (spots 381, 382, 384, 386, and 394) or mono-dehydroascorbate reductase (spot 474) (Fig. 3). This is consistentwith studies in apple that showed how catalase activity remainedhigh during stages of fruit growth [34]. To validate it in Citrus, thecatalase activity was measured in on- and off-crop leaves; theresults of this measurement confirmed that catalase activity in onsamples was twice as high that in off samples, approximately(Fig. 6).

Contrary to what is observed in the on-crop samples, in theoff-crop samples, generally, proteins with oxidoreductase activityare down-regulated (Table 1). Since in the spring following thecollection of these samples, the flowering for off-crop “Moncada”trees was significantly higher than for on-crop “Moncada” trees[8], it is possible to establish a correlation between flowerinduction and a decline in oxidoreductase activity in leaves. Thislater possible correlation is according to the association observedin Arabidopsis between the developmental transition to floweringwith a decline in the activity of its leaf ascorbate peroxidase [35].Only putative monocopper oxidase precursor was up-expressedin off-crop samples. Copper oxidase genes belong to a largefamily and the main subgroups are ferroxidase, ascorbate oxidaseand laccases. These proteins are involved in plant growthprocesses such as cell wall lignification or cotyledon vascularpatterning [36].

3.2.2. Other noteworthy proteins up-expressed in the on-cropsamples

Between them, citrate synthase (spot 500) and NADP-isocitratedehydrogenase (spots 515 and 516) are remarkable. Both areresponsible for fruit acidity during its growth, increasing and thenreaching a peak prior to ripening [37,38]. Another importantprotein up-expressed in the on-crop sample is the cysteineproteinase-like protein (spot 1018), which has an important role inplant senescence and programmed cell death (Fig. 3); its inhibitionis related to an increased number of flowers and seeds [39,40]. Thedown-expression of cysteine proteinase in off-crop samples couldpromote the flower development the following year.

In conclusion, more different proteins seem to be involved in thealternated bearing. As far as this study shows, in the period offlowering induction the primary metabolism is more active in off-crop trees than in on-crop trees, according to the proteins up-expressed in off-crop leaves. In contrast, in this same period theproteins up-expressed in on-crop samples compared to off-cropsamples are more related to the oxidoreductase activity; there-fore, the redox state is different for off-crop and for on-crop leavesand it seems to be crucial to the subsequent floral induction.However, we isolated other proteins with unknown functionswhich could also be related to the alternate bearing and the flowerinduction. To clearly establish the proteins related to theseprocesses, it would be necessary to compare this differential pro-teome of leaves with that of other tissues linked also to alternatebearing such as buds.

4. Material and methods

4.1. Plant material

Twelve-year-old ‘Moncada’Mandarin trees [Clementine ‘Oroval’(C. clementina Hort ex Tanaka) x ‘Kara’ mandarin (C. unshiu Marc. xC. nobilis Lou.)] on ‘Carrizo’ citrange (Citrus sinensis x Poncirustrifoliate) were used in this experiment. The experimental orchardis located at the Experimental Station of the Instituto Valenciano deInvestigaciones Agrarias, Moncada, Valencia, Spain, under a Medi-terranean climate. It involved 28 healthy trees of uniform canopysize, half of which were on-crop and the other half were off-crop.Twelve trees were randomly selected for the experiment, 6 ofeach type. To get 6 completely off-crop trees, the fruit was removedin July 2009 for 6 trees.

4.2. Biological material

Spring flush leaves of 8 month old were used as biologicalmaterial and were randomly collected from 12 trees (6 on-crop and6 off-crop) in autumn, which is when the fruit affects floralinduction (November 2010) [8]. In spring 2011 the flowering for off-crop trees was significantly higher than for on-crop trees [8]. Allleaves were harvested the same day at 11:00 a.m. and wereimmediately stored frozen at �80 �C.

4.3. Measurements

4.3.1. Protein extractionFrozen Moncada leaves of each tree were separately pulverized

in liquid nitrogen with 0.05% PVP (twelve samples). Then, sampleswere homogenized in extraction buffer (50 mM TriseHCl, pH 7.5,1 mM PMSF, 0.2% b-mercaptoethanol) using a pestle and mortar.Homogenates were centrifuged for 20 min at 20,000 g. Thesupernatants were mixed with an equal amount of cold 20% TCA.The mixtures were incubated for 1 h at 4 �C and centrifuged at20,000 g for 15 min at 4 �C. The protein pellets were washed three


times with acetone. After the last centrifugation at 20,000 g for15 min at 4 �C, pellets were re-suspended in lysis buffer (7 M urea,2 M thiourea, 4% CHAPS). Protein aliquots analyzed by 2D electro-phoresis were stripped of non-protein contaminants using a 2DClean-Up Kit following the manufacturer’s instructions (GEHealthcare). The cleaned protein was re-solubilised in a lysis bufferfor the conventional 2D analysis or in a Tris-buffered solution (7 Murea, 2 M thiourea, 4% CHAPS, 20 mM TriseHCl pH 8.5) for 2D DIGEanalysis. Protein concentration was determined with the Bio-Radprotein assay using bovine serum albumin (BSA) as standard.

4.3.2. Fluorescent labellingProtein samples were labelled using the CyDyes DIGE fluors

(Cy2, Cy3 and Cy5) according to the manufacturer’s instructions(GE Healthcare). Three different protein samples (internal standard,on and off samples) were labelled individually with the three dyes.The internal standard was created by pooling an aliquot of allbiological samples analyzed in the experiment and it was alwayslabelled with Cy2. Six biological replicates were analyzed in thisexperiment then twelve biological samples were used to make theinternal standard. The sample of each on-crop tree never wasmixed with samples of other on-crop trees (the same for off-cropsamples). The on sample and the off sample are labelled with Cy3or Cy5, alternatively, depending on the biological replicate, thusavoiding the label effect. Equal amounts (50 mg) of on (Cy3, forexample), off (Cy5) and internal standard (Cy2) samples of thesame biological replicate were pooled. Lysis buffer was added tomake up the volume to 40 mL. Then, the sample was mixed with40 mL of isoelectrofocusing (IEF) rehydration buffer (8 M urea, 4%CHAPS, 0.005% bromophenol blue) containing 65 mM DTT and 1%IPG buffer pH 3e11 and loaded in the gel (one gel for each biologicalreplicate).

4.3.3. 2D electrophoresisFor 2D analysis, strips of 24 cm in length with immobilized pH

gradient of 3e11 were hydrated overnight at room temperaturewith 450 mL of IEF rehydration buffer, containing the reagentsDestreak and Pharmalyte pH 3e10, according to the manufacturer’sinstructions (GE Healthcare). CyDyes labelled samples (150 mg ofprotein) were loaded in hydrated strips. IEF was performed on anIPGphor unit (GE Healthcare) at 20 �C and a maximum currentsetting of 50 mA per strip, using the following settings: 300 V for 1 h,an increasing voltage gradient to 1000 V over 6 h, an increasingvoltage gradient to 8000 V over 3 h, before finally holding at 8000 Vfor a total of 32,000 V h. After IEF, each strip was equilibratedseparately for 15min in 10mL equilibration solution I (0.05M TriseHCl buffer, pH 8.8 containing 6 M urea, 30% glycerol, 2% SDS,200 mg DTT per 10 mL buffer) followed by equilibration solution II(substituting DTT for 250 mg iodoacetamide per 10 mL buffer andadding 0.01% bromophenol blue) before being applied directly tothe second dimension 12.5% SDS-PAGE gels. Six gels were runsimultaneously at 20 �C, applying 2 W/gel for 30 min and 20 W/gelfor the remaining 5e6 h, using an Ettan DALTsix unit (GE Health-care). A running buffer of 25 mM Tris pH 8.3, 192 mM glycine and0.2% SDS was used. Each gel showed the differential proteinexpression between an on sample (from a single on-crop tree) andan off sample (from a single off-crop tree).

4.3.4. Gel imaging and data analysisAfter SDS-PAGE, CyDye-labelled proteins were visualized by

scanning using a Typhoon Trio scanner (GE Healthcare) with therelevant wavelengths for each CyDye. Cy2 images were scannedusing a blue laser (488 nm) and an emission filter of 520 nm bandpass (BP) 40. Cy3 images were scanned using a green laser (532 nm)and a 580 nm BP 30 emission filter. Cy5 images were scanned using

a red laser (633 nm) and a 670 nm BP 30 emission filter. All gelswere scanned at 200 mm (pixel size) resolution. The photo-multiplier tube (PMT) was set between 500 and 600 V by usingnormal sensitivity. The scanned gels were than directly transferredto the ImageQuant V5.2 software package (GE Healthcare). Imagegel analysis was carried out using the DeCyder 2D Software V6.5following the manufacturer’s instructions (GE Healthcare). Theimages were exported to the DeCyder Batch Processor module andDIA (Differential in-gel analysis) and BVA (Biological Variationanalysis) modules were made automatically. DIA module was usedfor spot detection, spot volume quantification, and volume rationormalization of different samples in the same gel. BVA modulewas used to match protein spots among different gels and toidentify protein spots that exhibit significant difference. Manualediting was performed in the biological variation analysis moduleto ensure that spots were correctly matched between different gelsand were not contaminated with artifacts, such as streaks or dust.The paired t-test was used for statistical analysis of the data. A falsediscovery rate (FDR) correction was applied to eliminate falsepositives. Protein spots that showed a statistically significantchange in abundance between on and off samples using a Student’st-test (p < 0.05) were considered as being differentially expressed.

4.3.5. Protein identification by mass spectrometry (MALDI, MS/MS)analysis

For picking spots of interest, gels were first stained with SilverStaining Kit, Protein (GE Healthcare). Proteins of interest weremanually excised from analytical gels and were distained with two5-min washes with acetonitrile (ACN)/water (1:1, v/v), followed byrehydration with 50 mM ammonium bicarbonate for 5 min and25 mM ammonium bicarbonate in 50% (v/v) ACN for 15 min. Gelpieces were then digested with sequencing grade trypsin (Prom-ega) as described elsewhere [41], and subject to PMF and/or LC/MS/MS analyses.

The digestion mixture was dried in a vacuum centrifuge,resuspended in 7 mL of 0.1% TFA (trifluoroacetic acid, Sigma), and1 mL was spotted onto the MALDI target plate. After air-drying thedroplets at room temperature, 0.5 mL of matrix (5 mg/mL CHCA) (a-cyano-4-hydroxycinnamic acid, Sigma) in 0.1% TFA-ACN/H2O (1:1,v/v) was added and allowed to air-dry at room temperature. Theresulting mixtures were analyzed in a 4700 Proteomics Analyzer(Applied Biosystems, Foster City, USA) in positive reflectron mode(2000 shots each position). Five of the most intense precursors(according to the threshold criteria: minimum signal-to-noise: 10,minimum cluster area: 500, maximum precursor gap: 200 ppm,maximum fraction gap: 4) were selected for every position for theMS/MS analysis. And, MS/MS data were acquired using the default1 kV MS/MS method.

The MS and MS/MS information was sent to MASCOT via theProtein Pilot software (Applied Biosystems). Database searches onNCBI, Swiss-Prot, and HarvESTs: Citrus databases were performedusing MASCOT search engine (Matrix-Science). HarvEST: Citruscontains best BLASTX hits from UniProt, the Arabidopsis genomeand Phytozome version Poptr1.1 (http://harvest.ucr.edu). Searcheswere performed with tryptic specificity allowing one missedcleavage and a tolerance on the mass measurement of 100 ppm inMS mode and 0.6 Da for MS/MS ions. Carbamidomethylation of Cyswas used as a fixed modification factor, while oxidation of Met anddeamidation of Asn and Gln as variable modifications.

The samples without a positive identification were analyzed byLC/MS/MS. Peptide separation by LC-MS/MS was performed usingan Ultimate nano-LC system (LC Packings) and a QSTAR XL Q-TOFhybrid mass spectrometer (AB Sciex). Samples (5 mL) were deliv-ered to the system using a FAMOS autosampler (LC Packings) at30 mL/min, and the peptides were trapped onto a PepMap C18

http://harvest.ucr.edu


pre-column (5 mm � 300 mm i.d.; LC Packings). Peptides were theneluted onto the PepMap C18 analytical column (15 cm� 75 mm i.d.;LC Packings) at 300 nL/min and separated using a 30 min gradientof 5e45% ACN. The QSTAR XL was operated using an information-dependent acquisition mode, in which a 1-s TOF MS scan from400 to 2000 m/z was performed, followed by 3-s product ion scansfrom 65 to 2000 m/z on the three most intense doubly or triplycharged ions.

The MS/MS information was sent to MASCOT via the MASCOTDAEMON software (MATRIX SCIENCE). The search parameters weredefined as for MSeMS/MS analysis.

4.3.6. Starch analysisLeaves (2 g) were dried in the oven (60 �C) and then were

treated with 80% ethanol. The remaining pellets were gelatinizedby autoclaving and then sodium-acetate buffer and amyloglucosi-dase were added to the gelatinized extracts, according to Schafferet al. [42]. Enzymatic digestions were performed for 2 h at 55 �C.After filtration, released glucose was quantified with aWaters HPLCsystem equippedwith a carbohydrate column (4.6� 250mm, 5 mm,Tracer Carbohydrat Tecknokroma, Barcelona, Spain) and a 2410differential refractometer. A binary isocratic phase consisting inACN:water 75:25 (v/v) was used and the retention time for glucosewas 11.5 min. Quantification was performed by external standardcalibration. Starch content was expressed in mg g�1 dry weight.

4.3.7. Catalase activityLeaves (2 g) were homogenized in a Polytron 3100 (Kinematica,

Lucerne, Switzerland) using 10 mL of 50 mM phosphate buffer, ph7.0, containing 2 mM EDTA and 2% polyvinylpolypyrrolidone (PVPP,Sigma, Barcelona, Spain). The crude extract was centrifuged at12,000 rpm at 4 �C for 30 min, and the supernatant was filtered(0.45 mm; Nylon) and used for the catalase assay within 1 h. Theprotein concentration in the supernatant was determined in theTCA precipitate using bovine serum albumin as standard [43].

The reaction medium (2 mL) contained 100 mM phosphatebuffer pH 7.0, and 100 mL of the supernatant. The reaction wasstarted by adding 100 mL of 10 mM H2O2. Catalase activity wasspectrophotometrically determined by the decrease in hydrogenperoxide [44]. The reaction was monitored at 240 nm in a spectro-photometer UV-1610 (Shimadzu Corp., Kyoto Japan), at roomtemperature. The molar extinction coefficient used was43.6 M�1 cm�1. Catalase activity was expressed as mmol H2O2consumed g�1 protein min�1 after 3 min reaction.

Statistical analyses for catalase activity and starch concentrationdata. These data correspond to the mean of six independentreplicates (from six on-crop trees and from six off-crop trees).ANOVA and regression analysis were performed with StatgraphicsPlus for Windows, version 5.1 (Statistical Graphics, EnglewoodCliffs, NJ, USA).

Acknowledgements

M.C. González was recipient of a contract by the FundaciónAgroAlimed (Conselleria d’Agricultura, Pesca i Alimentació, Gen-eralitat Valenciana). This work was supported by the InstitutoNacional Investigaciones Agrarias, Spain (RTA2009-00147,RTA2011-00114-00-00). The DIGEwere performed in the ProteomicService of the Instituto de Biologia Molecular y Celular de Plantas(UPV-Valencia). Mass analyses were performed in the ProteomicLaboratory of CIPF (member of ProteoRed_ISCIII; the Spanish Pro-teomics Network); special thanks are due to Dr. L. Valero and E.Dionís, for assistance and advice on Mass analyses. The authorshave declared no conflict of interest.

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proteomic analysis of “moncada” mandarin leaves with contrasting fruit load

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