ollege of Horticulture, Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, PR China
r t i c l e i n f o
rticle history:eceived 29 January 2012eceived in revised form 28 April 2012ccepted 8 May 2012
eywords:-DEapanese apricot
ALDI-TOF/TOFistil abortion
a b s t r a c t
The phenomenon of pistil abortion widely occurs in Japanese apricot and has seriously affected theyield in production. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) approaches to identifythe differentially expressed proteome between perfect and imperfect flower buds in Japanese apricot.More than 400 highly reproducible protein spots (P < 0.05) were detected and 27 protein spots showeda greater than two-fold difference in their expression values. The proteins identified were classifiedinto eight functional classifications and ten process categories, according to the Gene Ontology (GO).Acetyl-CoA produced by ATP citrate lyase (ACL) as a structural substance during formation of the cellwall could regulate pistil abortion in Japanese apricot. S-adenosylmethionine (SAM), xyloglucan endo-
roteome transglucosylase/hydrolases (XTHs) and caffeoyl-CoA-O-methyl transferase (CCoAOMT) could promotecell wall formation in perfect flower buds of Japanese apricot, greatly contributing to pistil development.Spermidine hydroxycinnamoyl transferase (SHT) may be involved in the O-methylation of spermidineconjugates and could contribute to abnormal floral development. The identification of such differentiallyexpressed proteins provides new targets for future studies that will assess their physiological roles andsignificance in pistil abortion.
Floral organs play an essential role in plant sexual reproduc-ion. However, in most floral plants, only a few of the flowers andvules that are initiated actually give rise to mature seeds and fruitsArathi et al., 1999). Several different mechanisms have been pro-osed to explain the phenomenon of female sterility, including
istil abortion. The most often discussed causal factors of femaleterility are thought to be triggered by environmental and nutri-ional conditions (Zinn et al., 2010; Beppu and Kataoka, 2011), low
Abbreviations: 2-DE, two-dimensional gel electrophoresis; 2D-PAGE, two-imensional polyacrylamide gel electrophoresis; ACL, acetyl-CoA produced byTP citrate lyase; CCoAOMT, caffeoyl-CoA O-methyl transferase; CHAPS, 3-[(3-holamidopropyl)dimethylamonio]-1-propanesulphonate; DTT, dithiothreitol; GA,ibberellin; GPX, glutathione peroxidase; HCCA, �-cyano-4-hydroxycinnamc acid;EF, isoelectric focusing; kDa, kilodalton; MALDI-TOF/TOF, matrix-assisted laseresorption/ionization time of flight/time of flight; MS, mass spectrometry (ic);/z, mass-to-charge ratio; OD, optical density; PPA database, a local entire peachroteome library (http://www.rosaceae.org/node/355); pI, isoelectric point; PVP,olyvinylpyrrolidone; SAM, S-adenosylmethionine; SDS, sodium dodecyl sulphate;HT, spermidine hydroxycinnamoyl transferase; TCA, trichloroacetic acid; TFA, tri-uoroacetic acid; XTHs, xyloglucan endotransglucosylase/hydrolases.∗ Corresponding author. Tel.: +86 02584395724; fax: +86 02584395724.
sink strength (Morio et al., 2004; Reale et al., 2009), influence ofpathogens (Kocsis and Jakab, 2008), occurrence of sporophytic orgametophytic mutations (Wang et al., 2008), ABCDE model andother related genes (Jofuku et al., 1994; Causier et al., 2003; Penget al., 2008), and phytohermone (Olkamoto and Omori, 1991; Elliset al., 2005; Lim et al., 2010; Kumar et al., 2011). Morphologicalstudies have shown that pistil development of staminate flowersin the olive is interrupted after differentiation of the megasporemother cell. At that stage, no starch was observed in the pistils ofthe staminate flowers; the plastids had few thylakoid membranesand grana and the staminate flowers appeared very similar to pro-plastids (Reale et al., 2009). In Arabidopsis, heat stress reduced thetotal number of ovules and increased ovule abortion (Whittle et al.,2009). Early ovule degeneration was also caused by high temper-atures in sweet cherry, and ovule development was regulated bygibberellin (GA) in sweet cherry flowers (Beppu and Kataoka, 2011).GA suppressed the development of the embryo sac and shortenedits longevity in grapes (Olkamoto and Omori, 1991).
Japanese apricot (Prunus mume Sieb. et Zucc) originated in Chinaand is an important economical fruit crops in China and Japan
(Chu, 1999). Owing to its charming flowers and flavorsome fruit,it has been widely planted as an ornamental plant and an eco-nomic fruit tree (Shi et al., 2009). The fruit of Japanese apricothas consistently been a valuable processing material used in the
ood and vintage industry and is believed to contain many phys-ochemical substances which are beneficial to human health (Xiat al., 2010, 2011). However, the phenomenon of imperfect flowersidely occurs and has seriously affected the production yield (Gao
t al., 2006). The imperfect flowers are characterized by either pis-ils below the stamens, withered pistils or an absence of pistils, andence fail to bear fruits (Hou et al., 2010). Comparative proteomicnalysis has been performed for perfect and imperfect flowersnd the different proteins have been analyzed in both perfect andmperfect flowers for the different stages of young bud, matureud and blossom flower; moreover, glucose metabolism, starchetabolism and photosynthesis related to pistil abortion were
ound (Wang, 2008). More recently, real-time quantitative reverseranscription polymerase chain reaction and in situ hybridizationave shown that PmAG mRNA was highly expressed in the sepals,arpel and stamens, and a weak signal was detected in the seed andutlet. No expression was detected in the leaves or petals, but noignificant differential was expressed in perfect and imperfect flow-rs (Hou et al., 2010). However, the molecular mechanism involvedn pistil abortion remains unknown for Japanese apricot.
Recently, global expression profiling approaches have been uti-ized to investigate the mechanisms of plant development (Hajducht al., 2005; Xu et al., 2010; Soitamo et al., 2011). Similar to genexpression profiling, proteomics based on two-dimensional poly-crylamide gel electrophoresis followed by matrix-assisted laseresorption/ionization time of flight/time of flight (MALDI-TOF/TOF)
s able to simultaneously analyze changes and classify temporalatterns of protein accumulation that occur in vivo (Katz et al.,007). Proteomics has established itself as an increasingly usedxperimental tool for the investigation of complex cellular pro-esses, including seed, ovule, embryo, and endosperm developmentGallardo et al., 2003; Chen et al., 2006; Bai et al., 2010; Kwon et al.,010; Liu et al., 2010; Martínez-García et al., 2012; Prassinos et al.,011).
In the present study we investigated the perfect and imper-ect flower buds at the proteomic level using two-dimensionalel electrophoresis (2-DE) techniques. The differentially expressedroteins identified were analyzed using MALDI-TOF/TOF combinedith a database search. The characterization of these proteins
learly reflected differences between perfect and imperfect floweruds.
aterials and methods
lant materials
The ratio of imperfect flowers of Japanese apricot cultivarsDaqiandi’ is about 76%. While the pistil of a perfect flower contin-es to differentiate and develop perfectly, the pistil of an imperfectower stops differentiation in early December and finally disin-egrates. We chose two types of flower buds of this period fromDaqiandi’ trees grown in the ‘National Field Genbank for Japanesepricot’, Nanjing, Jiangsu Province, China (Fig. 1). All the sam-les were collected and immediately frozen in liquid nitrogen andtored at −80 ◦C.
rotein extraction and quantification
Protein extraction was performed according to therichloroacetic acid/acetone precipitation method describedy with some modifications. The flower buds were transferred into
mortar and ground into a fine powder with 10% polyvinylpyrroli-one in liquid nitrogen. The powder was homogenized in coldcetone (containing 10% trichloroacetic acid and 0.07% dithio-hreitol (DTT)) and then precipitated at −20 ◦C overnight. The
Fig. 1. Photos of perfect and imperfect flowers. Left is perfect flower and right isimperfect flower, bar scale represents 1 cm.
homogenate was centrifuged at 15 000 × g for 0.5 h at 4 ◦C. Thepellet was washed with 5 mL cold acetone (containing 0.07%DTT) and centrifuged again at 15 000 × g for 0.5 h at 4 ◦C. Theabove-mentioned steps were repeated until the supernatant wascolorless, and then the pellet was air dried at 4 ◦C and stored at−70 ◦C until further use. The protein powder was weighed and thenresuspended in a sample rehydration buffer (7 M urea, 4% (w/v)3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulphonate,65 mM DTT, 0.2% (v/v) 3–10 and 4–7 ampholytes (Amersham,Uppsala, Sweden), 2 M thiourea and 0.001% bromophenol blue).The supernatant was collected after centrifugation at 15 000 × g for0.5 h at 4 ◦C. The protein concentration was determined accordingto the method described by Bradford (1976).
Two-dimensional gel electrophoresis (2-DE) and staining
The first dimensional gel electrophoresis was performed on17 cm ReadyStrip IPG Strips (Bio-Rad) with a linear pH gradient ofpH 4–7 in a PROTEANIEF system (Bio-Rad). Strips were rehydratedin 350 �L of rehydration buffer containing 1.3 mg protein at 50 V,19 ◦C for 12 h, before being transferred to a strip tray. The voltagewas set at 200 V for 0.5 h, 500 V for 0.5 h, 1000 V for 1 h, 2000 V for1 h, 8000 V for 5 h and then was run at 8000 V until the final volt-hours (60 kVh) were reached. All separations were performed at19 ◦C.
Before SDS–PAGE, the IPG strips were equilibrated for 15 min inequilibration buffer (6 M urea, 0.375 M Tris–HCl, pH 8.8, 2% (w/v)sodium dodecyl sulphate (SDS), 20% (v/v) glycerol, 2% (w/v) DTT),followed by a further 15 min in equilibration buffer containing 2.5%(w/v) iodoacetamide instead of DTT. After transferring the stripsonto vertical 10% SDS–PAGE gels, electrophoresis was undertakenat 16 ◦C in running buffer (20 mM Tris–HCl, 192 mM glycine, 0.1%SDS) using an Ettan DALT-six System (GE Healthcare). The gels wererun at 1 W/gel for 1.5 h, and then at 15 W/gel until the bromophe-nol blue reached the bottom of the plate. After electrophoresis, thegels were washed for 15 min with double-distilled water and thenstained in a staining buffer (0.12% Coomassie brilliant blue G-250,10% ammonium sulphate, 10% phosphoric acid, and 20% methanol)for 2 h and subsequently decoloured using double-distilled wateruntil the background was clear. To ensure data reliability, sam-ple preparation (both perfect and imperfect flower buds) and 2-DEwere performed in triplicate.
Image acquisition and data analysis
2-DE gels were scanned using a Versdoc 3000 scanner (Bio-Rad). Spot detection of the stained gels, gel matching and group
T. Shi et al. / Journal of Plant Physiology 169 (2012) 1301– 1310 1303
F se aprs flowe
aewriotcaassnia
Pd(
wi2t5epfvtwAa
MTwaubn8T
buds. Those regions of the 2-DE maps shown in Fig. 2 that indicated
ig. 2. Representative 2-DE patterns of perfect and imperfect flower buds in Japanepot numbers correspond to those listed in Table 1 and Additional file 1. (A) Perfect
nalysis of six gels (three independent analytical replicate gels forach developmental stage) were performed using PDQuest 8.0 soft-are (Bio-Rad) and, when needed, spot detection was manually
efined. Quantitative analyses were carried out after normaliz-ng the spot quantities (as spot optical density) in all gels inrder to compensate for non-expression related variations, andhe individual protein spot quantities were normalized as a per-entage of the total quantity of valid spots present in the gelnd expressed as ‘percent’. Quantitative comparisons of the aver-ged gels in each developmental stage were used to determineignificant differentially expressed spots, and only the spots thathowed at least a two-fold change and which were statistically sig-ificant in a one-way ANOVA (P < 0.05) for reproducible changes
n three analytical replicates were considered for successivenalysis.
rotein in-gel digestion and identification by matrix-assisted laseresorption/ionization time of flight/time of flightMALDI-TOF/TOF)
Protein spots of interest were excised from the gels and cleanedith double-distilled water before being transferred to steril-
zed Eppendorf tubes. The protein spots were then washed with5 mM NH4HCO3 followed by dehydration with 50% acetoni-rile (ACN) in 25 mM NH4HCO3, reduction with 10 mM DTT in0 mM NH4HCO3 for 1 h at 56 ◦C and alkylation in 55 mM iodoac-tamide in 50 mM NH4HCO3 for 1 h at room temperature. Therotein spots were washed several times with 50 mM NH4HCO3ollowed by dehydration with ACN before finally being dried in aacuum centrifuge and digested overnight at 37 ◦C by the addi-ion of 1.5 mL trypsin. The resulting peptides were extracted byashing the protein spots with 0.1% trifluoroacetic acid in 67%CN. The supernatants were gathered and stored at −20 ◦C untilnalysis.
The resulting peptides were air dried and analyzed by a 4800ALDI-TOF/TOF Proteomics Analyzer (Applied Biosystems, USA).
he UV laser was operated at a 200 Hz repetition rate with aavelength of 355 nm, and the accelerated voltage was oper-
ted at 20 kV. The �-cyano-4-hydroxycinnamc acid matrix wassed for the mass spectrometry (MS) analyses. Protein digestedy trypsin was used to calibrate the mass instrument as an inter-
al calibration mode. Parent mass peaks with a mass range of00–4000 Da and minimum S/N 20 were selected for tandemOF/TOF analysis.
icot. The proteins identified are marked with arrows and numbers, and the proteinr buds and (B) imperfect flower buds.
Database search and bioinformatics analysis
An MS/MS Ion search was performed using GPSExplorerTM software v3.5 (Applied Biosystems) in a locallibrary built from the entire peach proteome database(http://www.rosaceae.org/node/355) (PPA database) using theMASCOT search engine v3.5 (Matrix Science Ltd., London). If nocredible candidate was matched, the NCBInr 20110419 database(13767831 sequences, 4728199773 residues) was then searched.Compared with the former database, the NCBInr protein is tax-onomically restricted to Viridiplantae, and the other parameterswere the same with one missed cleavage, 50 ppm mass tolerancein MS and 0.2 Da in MS/MS, cysteine carbamidomethylation as afixed modification, and methionine oxidation as a variable mod-ification. A total ion score in the PPA database of more than 95%or individual ion scores of more than 35 in the MOWSE databaseindicated successful protein identification. If a spot was identifiedin both of the databases, we used the higher MOWSE score toidentify the protein and/or peptide positively. Only significanthits defined by the MASCOT probability analysis (P < 0.05) wereaccepted.
Results
Proteomic maps of perfect and imperfect flower buds of Japaneseapricot
To identify protein involved in the development of the flowersof Japanese apricot, we used a 2-DE technique on the proteome pro-file of the perfect and imperfect flower buds. The results showeda consistent pattern of protein expression levels on the gels, andimage analysis revealed about 400 highly reproducible proteinspots that were consistently observed in all replicates (Fig. 2). Theseproteins cover the isoelectric point (pI), range from 4 to 7, andtheir MW (molecular weight) ranged from 15 to 70 kDa. In gen-eral, the proteome patterns were very similar for all 2-DE images,indicating that most proteins were accumulated at comparable lev-els in perfect and imperfect flower buds. However, quantitativeimage analysis revealed a total of 27 protein spots that signifi-cantly changed in abundance between perfect and imperfect flower
differentially expressed protein spots are enlarged and presentedin Fig. 3. The calculated MW of differentially expressed proteinsranged from 9.90 to 71.10 kDa, and the calculated pI range was from
1304 T. Shi et al. / Journal of Plant Physiology 169 (2012) 1301– 1310
shown
4f
2ra1tsid
P
fie
eotng((ipmttbA
D
fP
regulated in a spatiotemporal manner by ACL activity (Ratledgeet al., 1997). Fatty acids and sterols play important roles in manycellular and developmental processes, such as the generation of
Fig. 3. Magnified views of some of the differentially abundant proteins
.61 to 9.43, which was close to the experimental data as judgedrom the location of the spots on the 2-DE gels (Table 1).
Among 27 protein spots, 16 up-regulated protein spots (spots 1,, 3, 4, 5, 6, 7, 8, 10, 11, 14, 15, 16, 17, 18, and 19) and seven down-egulated protein spots (spots 20, 21, 22, 23, 24, 25, and 27) showed
more than two-fold difference, and four protein spots (spots 9,2, 13, and 26) were specifically expressed. We also found thathree protein spots (spots 9, 12, and 13) showed specific expres-ion in perfect flower buds and one spot (spot 26) was specific tomperfect flower buds, which might be closely related to the pistilevelopment and female sterility.
rotein identification and functional categorization
All the 27 differently expressed spots (P < 0.05) were selectedor excision and analyzed using MALDI-TOF/TOF and identifiedn the peach database. The identified results of the differentiallyxpressed proteins are listed in Table 1.
In order to understand the function of these differentiallyxpressed proteins, we categorized them by GO analysis. Genentology categories were assigned to all 27 proteins according toheir molecular function, biological processes and cellular compo-ent (Fig. 4 and Additional file 1). Based on molecular function, theenes were finally classified into eight categories: enzyme activity14), unclassified (4), binding (3), molecular function (2), signaling1), transcription factor activity (1), cell structure (1) and oxidiz-ng reaction (1), as shown in Fig. 4A. Additionally, ten biologicalrocesses were identified: stress response and defence (8), energyetabolism (4), biosynthetic process (4), unclassified (2), signal
ransduction (2), protein metabolism (2), plant development (2),ranscription (1), oxidation reduction process (1) and microtubule-ased process (1) (Fig. 4B). The component categories were indditional file 1.
iscussion
Comparative analysis of the 2-DE maps of perfect and imper-ect flower bud proteins in Japanese apricot was performed usingDQuest software. More than 400 highly reproducible protein spots
in Fig. 2. Left is perfect flower buds and right is imperfect flower buds.
(P < 0.05) were detected, and 29 protein spots showed a more thantwo-fold difference in expression values, of which 27 were confi-dently identified according to the databases.
Acetyl-CoA produced by ATP citrate lyase (ACL) is used mainly infatty acid and sterol biogenesis (Suh et al., 2001). Several genes ofthe fatty acid biosynthesis pathway of Brassica napus are tightly
Fig. 4. Gene ontology of 27 differentially expressed proteins. Categorizationof proteins was performed according to the molecular function (A) and bio-logical process (B). This categorization was based on electronic annotation(http://www.geneontology.org/) and the literature.
24 Histidine kinase-, DNAgyrase B-, and HSP90-likeATPase family protein[Prunus persica]
ppa016752 2 65.42/9.07 70.35/6.39 49 10.80
00.20.40.60.81
1 2
25 Transketolase familyprotein[Prunus persica]
ppa007259 4 40.11/5.48 49.74/5.47 264 32.53
0
0.2
0.4
0.6
0.8
1 2
26 glutathione peroxidase 6[Prunus persica]
ppa012416 2 19.24/4.98 24.11/4.77 47 3.170
0.4
0.8
1.2
1.6
1 2
27 general regulatory factor 2[Prunus persica]
ppa010141 4 29.46/4.78 40.30/4.75 320 30.35
00.51
1.522.53
1 2
a Numbering corresponds to the 2-DE gel in Fig. 1.b Names and species of the proteins obtained via MASCOT software from the PPA database and the NCBInr 20110419 database.c Accession numbers from the PPA database and the NCBInr 20110419 database.d The total number of peptides identified.e MOWSE score probability (protein score) for the entire protein.f Sequence coverage.g The X-axis denotes the four stages of dormancy, and 1 and 2 represent perfect and imperfect flower buds, respectively, and the Y-axis denotes the relative protein
e mean
bFHs2dbpsmttoa
xpression levels (normalized volume of spots), and the values are expressed as the
iomembranes, hormones, and secondary messengers (Wang andaust, 1988; Brett and Müller-Navarra, 1997; Wang et al., 2004;an et al., 2010). In plants, the formation of pollen grains and
eeds is closely correlated with lipid production (Mandaokar et al.,006). Investigation of the time course of ACL expression in Sor-aria macrospora suggests that ACL is specifically induced at theeginning of the sexual cycle and produces acetyl-CoA, which mostrobably is a prerequisite for fruiting body formation during latertages of sexual development, and ACL is essential for fruiting bodyaturation (Nowrousian et al., 1999). In our study, ACL family pro-
ein (spot 4) expression in perfect flower buds was higher thanhat in imperfect flower buds, which indicated that the reductionf fatty acid and sterol biosynthesis increased the ratio of pistilbortion.
of three replicates ± the standard deviation.
Our research found that the expression of S-adenosylmethionine (SAM) synthetase 1 (spots 1 and 3) andSAM synthetase family protein (spot 2) in perfect flower budswas higher than that in imperfect flower buds. SAM, a directproduct of Met catabolism, is a substrate in numerous trans-methylation reactions, including several reactions that occur inthe biosynthesis of lignin (Campbell and Sederoff, 1996; Shenet al., 2002). Lignin, a complex phenolic polymer, is importantfor mechanical support, water transport, and defence in vascularplants (Campbell and Sederoff, 1996). p-Coumaroyl shikimate
3′-hydroxylase (C3′H) catalyzes the ring meta-hydroxylationreaction leading to the biosynthesis of lignin units, necessary forthe biosynthesis of both G and S lignin units (Vanholme et al.,2010). Various alleles of the C3′H-deficient reduced epidermal
Physio
flAsifDHPc(tspfppsti
nteptatt
wotingsi
ipSsafirlaXsaTr
otdiaoelwgtbb
T. Shi et al. / Journal of Plant
uorescence 8 (ref8) mutants exhibit severe female sterility inrabidopsis (Weng et al., 2010). Highly lignified tissues such astem tissue might be expected to have increased levels of SAMSn the Arabidopsis (Shen et al., 2002). SAM is the key compoundor all transmethylation reactions such as methylation of pectin,NA, RNA, histones and polyamine synthesis (Moffatt et al., 2002).igh level of SAM is also needed for pectin synthesis of cell walls.ectin is transported as a highly methylated molecule into theell wall and must be demethylated by pectin methyl esterasePME) prior to insertion into the cell wall. Due to decreasedransmethylation capacity, the cell wall and especially pectinynthesis may have been affected (Soitamo et al., 2011). Ourrevious research found the pistil of imperfect flower stopped dif-erentiation in early December and finally disintegrated, while theistil of perfect flowers continued to differentiate and developederfectly (Shi et al., 2011). This phenomenon might result from thetrain formation of cell walls by SAM. This could be responsible forhe lower expression of SAM and SAM synthetase family proteinn imperfect than in perfect flower buds.
During flower development, spermidine hydroxycin-amoyl transferase (SHT) has been shown to be involved inhe O-methylation of spermidine conjugates (Grienenbergert al., 2009). Martin-Tanguy (1997) finds that elevated freeolyamine and water-soluble polyamine levels (located inhe ovaries) contribute to abnormal floral development, butmine conjugates (via transferases) have important func-ions in floral induction, floral evocation and reproduction inobacco.
Xyloglucan endotransglucosylase/hydrolases (spot 11) are cellall enzymes that catalyze the cleavage and molecular grafting
f xyloglucan chain functions in loosening and rearrangement ofhe cell wall (Hyodo et al., 2003; Imoto et al., 2005). As they arenvolved in the modification of the load-bearing cell-wall compo-ents, they are believed to be very important in the regulation ofrowth and development (Maris et al., 2011). Hyodo et al. (2003)howed that XTH9 tends to be expressed strongly in rapidly divid-ng and expanding tissues in Arabidopsis.
Caffeoyl-CoA O-methyl transferase (CCoAOMT) (spot 13) is anmportant enzyme and is involved in an alternative methylationathway in lignin biosynthesis (Ye et al., 1994; Campbell andederoff, 1996). Tissue print hybridization showed that the expres-ion of the CCoAOMT gene is temporally and spatially regulatednd that it is associated with lignification in xylem and in phloembers in Zinnia organs (Ye et al., 1994). Lignin analysis showed thateduction in CCoAOMT alone resulted in a dramatic decrease inignin content; the reduction in CCoAOMT also led to a dramaticlteration in lignin composition in tobacco (Nicotiana tabacum cvanthi) (Zhong et al., 1998). The levels of G lignin were mosttrongly reduced in line with the greatest decrease in CCoAOMTctivity in Alfalfa (Medicago sativa cv Regen SY) (Guo et al., 2001).hese differentially expressed proteins might play an importantole in pistil abortion in Japanese apricot.
Peroxidase was one of the main enzymes that eliminated activexygen in the plant cell (Welinder, 1992). It has been reported thathere is a close relationship between peroxidase (spot 7) and flowerevelopment (Sun et al., 2005; Sood et al., 2006). Genes encod-
ng enzymes that detoxify reactive oxygen species (ROS), includingscorbate peroxidase and peroxidase, were down-regulated aftervules committed to abort (Sun et al., 2005). In our study, thexpression of peroxidase (spot 7) in imperfect flower buds wasower than that in perfect flower buds. This result is consistent
ith previous reports. Although glutathione peroxidase (spot 26,
lutathione peroxidase (GPX)) belongs to the peroxidase family,here is no expression of glutathione peroxidase in perfect floweruds; in contrast, its expression is very high in imperfect floweruds. In general, the abundance of GPX increased upon treatment
logy 169 (2012) 1301– 1310 1309
with various stresses (Eshdat et al., 1997; Jiang et al., 2007).Therefore, we hypothesized that the forming of an imperfect floweris an actual adversity process, and the higher expression of GPXmight be prepared to abort the pistil.
Conclusion
Our results showed that the majority of the proteins identi-fied were mainly related to stress response and defense (8), energymetabolism (4) and biosynthetic processes (4). The regulation andexecution of Japanese apricot pistil abortion is a complex networkof biochemical and cellular processes, and the differentially abun-dant proteins are involved in multiple metabolic pathways.
The conclusions of the present study were as follows: ACL,SAM, XTH and CCoAOMT could promote the formation of cellwalls in perfect flower buds in Japanese apricot, which greatlycontributes to pistil development. SHT may be involved in theO-methylation of spermidine conjugates and could contributeto abnormal floral development. The identification of such dif-ferentially expressed proteins provides a new target for futurestudies to assess their physiological roles and significance in pistilabortion.
Acknowledgements
We gratefully acknowledge the Special Fund for Agro-ScientificResearch in the Public Interest of the Ministry of Agricultureof China (201003058), the National Science Foundation of China(31101526) and the Natural Science Foundation of Jiangsu Province(BK2011642) for providing financial support, and the PriorityAcademic Program Development of Jiangsu Higher Education Insti-tutions (PAPD) for funding part of this study.
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.jplph.2012.05.009.
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