Differential proteomic analysis of developmental stages of Acca sellowiana somatic embryos

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ORIGINAL PAPER

Differential proteomic analysis of developmental stagesof Acca sellowiana somatic embryos

Gabriela Claudia Cangahuala-Inocente Æ Andrea Villarino Æ Daniela Seixas ÆEliane Dumas-Gaudot Æ Hernan Terenzi Æ Miguel Pedro Guerra

Received: 9 May 2008 / Revised: 31 October 2008 / Accepted: 28 November 2008 / Published online: 14 January 2009

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2008

Abstract Feijoa (Acca sellowiana, Myrtaceae), a native

fruit species from southern Brazil and northern Uruguay, is

considered to constitute a reference system for somatic

embryogenesis in woody dicots. This in vitro regenerative

pathway is an efficient micropropagation method, and a

suitable model system for studies in plant developmental

physiology. This study attempts to detect and identify

proteins that are expressed during the different develop-

mental stages of somatic embryos of A. sellowiana. Using

high resolution two-dimensional polyacrylamide gel elec-

trophoresis (2-DE), a high degree of similarity between

protein profiles of the assayed somatic embryos was

observed. Of the 74 different protein spots extracted for

analysis, 60 were identified by means of 2-DE/MALDI-

TOF/MS. Twelve proteins were expressed in all the

assayed stages. Ten proteins were expressed in the initial

stages and 22 proteins were expressed in the mature

developmental stages of somatic embryos. Only one pro-

tein was expressed exclusively in the torpedo stage,

whereas four were expressed in the pre-cotyledonary, and

none in the cotyledonary stage. The proteins identified

were involved in the synthesis of phenylalanine ammonia-

lyase, a conspicuous polyphenol present in the induction of

feijoa embryogenic cultures. The presence of essential

proteins of nitrogen metabolism, such as the cytosolic

glutamine synthetase protein, was also observed. The

physiological implications of these findings are discussed.

Keywords Feijoa sellowiana � Embryogenesis �Protein patterns � Phenol extraction � 2-DE

Introduction

Somatic embryogenesis may be considered as a model

developmental system supporting the basis of cellular

totipotency in higher plants. This in vitro morphogenetic

route allows for the investigation of the whole process of

differentiation from a single cell to a complete plant, with

certain advantages compared with zygotic embryogenesis,

since (1) the process of embryogenesis is easily monitored,

(2) the environmental and developmental phases of somatic

embryos can be controlled, and (3) large numbers of

embryos can be obtained (Komamine et al. 2005).

During the embryogenic induction of cells, there occurs

a clear differential gene expression phenomenon, resulting

Communicated by M. Stobiecki.

G. C. Cangahuala-Inocente � M. P. Guerra

Laboratorio de Fisiologia do Desenvolvimento e Genetica

Vegetal, Centro de Ciencias Agrarias, Universidade Federal de

Santa Catarina, Florianopolis, Brazil

A. Villarino � H. Terenzi

Laboratorio de Expressao Genica, Departamento de Bioquımica,

Centro de Ciencias Biologicas, Universidade Federal de Santa

Catarina, Florianopolis, Brazil

D. Seixas

Departamento de Bioquımica e Biologia Molecular,

Universidade Federal do Parana, Parana, Brazil

E. Dumas-Gaudot

UMR 1088 INRA/CNRS 5184/UB

(Plante–Microbe–Environnement), INRA-CMSE,

BP 86510, Dijon Cedex 21065, France

M. P. Guerra (&)

Departamento de Fitotecnia, Centro de Ciencias Agrarias,

Universidade Federal de Santa Catarina,

Rod Ademar Gonzaga 1345, Itacorubi, Florianopolis,

SC CEP 88440-000, Brazil

e-mail: [email protected]

123

Acta Physiol Plant (2009) 31:501–514

DOI 10.1007/s11738-008-0259-y

Page 2

in the synthesis of novel mRNAs and proteins in each

developmental stage (Chugh and Khurana 2002). For

example, the late embryogenesis abundant (LEA) protein

family has been observed both in zygotic and somatic

embryogenesis (Zimmerman 1993). Heat shock protein

accumulation has also been demonstrated during the

somatic embryogenesis, and has been used to characterize

particular steps of this process (Gyorgyey et al. 1991).

During embryo maturation, subsequent developmental

pathways are established which determine whether these

steps are critical points for evaluating the potential quality

and viability of somatic embryo-derived seedlings (Lippert

et al. 2005). Few studies have dealt with comparisons of

each maturation step of the embryo at the proteome level,

especially in woody plants (Dodeman and Ducreux 1996;

Lippert et al. 2005; Chivasa et al. 2006; Gallardo et al.

2006). However, this data reveals that the induction and

development of somatic embryos follows a complex

pathway and involves a number of unknown proteins, some

of which are associated with the developmental pathways

leading to the formation of complete somatic embryos

(Dodeman et al. 1997).

Feijoa (Acca sellowiana, Myrtaceae) is a native fruit

species of southern Brazil and northern Uruguay. This

species is considered as a reference system for somatic

embryogenesis in woody dicots (Guerra et al. 2001). The

route of somatic embryogenesis in A. sellowiana was first

described by Cruz et al. (1990). Subsequently, several

studies were carried out to improve the protocol of somatic

embryogenesis (Canhoto and Cruz 1996; Guerra et al.

2001; Stefanello et al. 2005), as well as to detect histo-

logical and biochemical alterations occurring in the

development of somatic embryos (Cangahuala-Inocente

et al. 2004, 2007).

2-DE-gel-based proteomic analysis attempts to evaluate

the total protein profiles of a given cell, organelle, or tissue

(Blackstock and Weir 1999), and may be employed in

order to evaluate the biochemical properties of proteins,

such as post-translational modification and interactions

with other biomolecules (Fitzgerald 2001).

Recently, proteomic approaches have been applied in

order to gain insight into the process of somatic embryo-

genesis. Dodeman and Ducreux (1996) identified several

proteins related to the induction of somatic embryogenesis

and expressed in different developmental stages of Daucus

carota somatic embryogenesis, where ten proteins were

specific to the cell suspension stage, and might be assigned

to the induction of either callogenesis or embryogenesis.

Six proteins specific to somatic embryos were shown to

fluctuate at different developmental stages and therefore

represent morphogenesis markers. Dupire et al. (1999)

analyzed global protein expression during the induction of

somatic embryogenesis in wild type and mutant Asparagus

officinalis. The 116 proteins observed were organized into

20 groups potentially related to somatic embryogenesis.

Six polypeptides specific to the mutant phenotype were

related to competence in entering the somatic embryo-

genesis pathway; 11 proteins detected in the wild type

tissues were related to an inhibition of somatic embryo-

genesis ability.

In an effort to better characterize the somatic embryo-

genetic processes and decrypt the effectors that control it,

the main purpose of the present work is to detect and

identify proteins that are expressed during the different

developmental stages of somatic embryos of A. sellowiana.

Material and methods

Plant material

Mature fruits of A. sellowiana plants were maintained in

the germplasm collection of the EPAGRI Experimental

Station of Sao Joaquim, Santa Catarina State, Brazil.

Zygotic embryos were excised and inoculated in a culture

medium according to the protocol described by Cangahuala-

Inocente et al. (2007). The incubation of the embryogenic

cultures was performed in a room chamber at 25�C, in the

dark. After 70 days of culture, torpedo, pre-cotyledonary

and cotyledonary stages somatic embryos (as shown in

Fig. 1) were collected and stored in a freezer at -20�C.

For identification of the somatic embryo developmental

stages, the following criteria were employed: (1) Torpedo:

elongated somatic embryos less than 3 mm, the histologi-

cal analysis revealing well-defined procambium, and

conspicuous apical caulinar meristem and root apical

meristem. (2) Pre-cotiledonary: white-translucent somatic

embryos longer than 3 mm showing elongated cotyledon-

ary leaves; (3) Cotiledonary: white somatic embryos with

expanded cotyledonary leaves.

Protein extraction and quantification

Fresh tissue (300 mg) was ground in a mortar with liquid

nitrogen following the procedure described by Carpentier

et al. (2005). Extracts were re-suspended in 500 lL of an

ice-cold extraction buffer (50 mM Tris–HCl pH 8.5, 5 mM

EDTA, 100 mM KCl, 1% w/v DTT, 30% w/v sucrose;

protease inhibitors as indicated by the manufactorer (Pro-

tease Inhibitor Mix, GE Healthcare) and vortexed for 30 s.

Five hundred microlitres of ice-cold Tris-buffered phenol

(pH 8.0) were added and the sample was vortexed for

15 min at 4�C. After centrifugation (3 min, 6,0009g, 4�C),

the lower phenolic phase was collected, re-extracted with

500 lL of extraction buffer, and vortexed for 30 s. After

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Fig. 1 Somatic embryogenesis

of Acca sellowiana. a Somatic

embryos developed from

cotyledonary stage of the

zygotic embryo.

b, d, f Developmental stages of

somatic embryos.

c, e Histological sections of

different developmental stages

of somatic embryos.

b, c Torpedo stage somatic

embryo; d, e Pre-cotyledonary

stage somatic embryos, and

f Cotyledonary stage somatic

embryo. Bars a, b, d, e, f: 1 cm;

c 500 lm

Acta Physiol Plant (2009) 31:501–514 503

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centrifugation (3 min, 6,0009g, 4�C) the phenolic phase

was collected and precipitated overnight with five volumes

of 100 mM ammonium acetate in methanol at -20�C. The

pellet was air-dried, re-suspended in 100 lL lysis buffer

[7 M urea, 2 M thiourea, 4% CHAPS, 0.8% IPG-buffer

(Amersham Biosciences), 1% DTT], and vortexed for 1 h

at room temperature. The protein concentration was

determined according to the colourimetric method of

Bradford (1976), using bovine serum albumin (BSA) as the

standard.

Two-dimensional electrophoresis (2-DE)

An aliquot of the sample prepared as described, containing

100 lg of total protein, was precipitated with TCA (tri-

chloroacetic acid), and further acetone extracted following

the recommendation of the 2-D Clean Up Kit (Amersham

Biosciences). The protocol to run 2D electrophoresis gels

was as described by Carpentier et al. (2005). Briefly,

samples were diluted with a rehydration buffer (6 M urea,

2 M thiourea, 0.5% CHAPS, 10% glycerol, 0.002% bro-

mophenol blue, 0.5% IPG-buffer, 0.28% DTT) in 125 lL.

IPG Drystrips (7 cm pH 3–10; Amersham Biosciences) for

12 h. Isoelectrofocusing (IEF) was carried out on an IPG-

phor III system (Amersham Biosciences) at 20�C, with the

current limited to 1 mA/strip. Prior to the second dimen-

sion, each strip was equilibrated for 15 min in 5 mL

equilibration solution (6 M urea, 30% glycerol, 2% SDS,

0.002% bromophenol blue, 50 mM Tris pH 8.8) containing

1% w/v DTT, and subsequently for 15 min in a 5 mL

equilibration buffer containing 4.5% w/v iodoacetamide.

The second dimension was performed on a MiniProtean III

system (BioRad) in 1.5 mm SDS polyacrylamide gels

(12% acrylamide).

Protein staining and gel analysis

Proteins were stained with colloidal coomassie blue G250

[20% methanol, 0.1% w/v CBB G-250, 1.6% o-phos-

phoric acid, and 8% w/v ((NH4)2SO4)] for 12 h and

destained with 50% (v/v) ethanol in water with 10%

acetic acid. Gels were kept in 1% acetic acid at 4�C.

Stained gels were scanned and calibrated with Labscan 5

Software (Amersham Biosciences) using a precision

scanner. Image analysis was performed with Image

Master 2-D Platinum 6.0 (Amersham Biosciences). Indi-

vidual protein spots were quantified using the percentage

volume parameter. Only protein spots that were repro-

ducibly found in at least three biological replicates were

included in further examinations. The protein spots found

among classes (i.e. common protein spots for torpedo,

pre-cotyledonary and cotyledonary stages) were analyzed

by means of Statistica v. 7 software. In pairwise

comparisons of the proteomes of different tissues, the

relative abundance of proteins was monitored.

Trypsin digestion fingerprinting

Selected protein spots were manually excised from the

gels, and in-gel digested by trypsin was achieved according

to the protocol of Westermeier and Naven (2002). Briefly,

protein spots were destained once with a solution of 50%

acetonitrile and 25 mM ammonium bicarbonate, pH 8, for

1 h at room temperature. Subsequently, gel plugs were

dehydrated by adding 100% acetonitrile for 5 min and then

drying on a ‘‘Speedvac’’ centrifuge concentrator (Ther-

moSavant, Milford, USA) for 15 min. Gel plugs were re-

hydrated in 10 lL of a solution containing 10 lg mL-1 of

Trypsin (Promega, Madison, USA), prepared in 25 mM

ammonium bicarbonate and digested overnight at 37�C.

Peptides were extracted three times with a solution of 50%

acenotrile and 5% trifluoroacetic acid (TFA), and then

vortexed for 30 min.

Mass spectrometry analysis and data interpretation

Samples were pooled and dried under vacuum apparatus

for 1 h at room temperature, and re-suspended in 2 lL

0.1% TFA v/v. Samples for MALDI-TOF/MS were pre-

pared as follows: 1 lL of peptide solution was mixed with

an equal volume of matrix (a-cyano-4-hydroxycinnamic

acid) solution, deposited on the instrument target plate

(Bruker Daltonics), and air-dried at room temperature.

Mass spectra measurements were obtained with an Auto-

flex/MS matrix-assisted laser desorption ionization time-

of-flight mass spectrometer (MALDI-TOF/MS, Bruker

Daltonics). External calibration was performed using

the standard proteins Angiotensin II [M ? H]? mono

1046.5418, Angiotensin I [M ? H]? mono 1296.6848,

Substance P [M ? H]? mono 1347.7354, Bombesin

[M ? H]? mono 1619.8223 and ACTH clip (18–39)

[M ? H]? mono 2465.1983. The list of peaks obtained

was analyzed by means of FlexAnalysisTM

version 2 soft-

ware (Bruker Daltonics) and the algorithm SNAP. The

peptide mass fingerprint was submitted to identification

using MASCOT on line (Matrix Science, London, UK) and

the MCDB and NCBI databases. Hits were considered

significant according to MASCOT score (P \ 0.05). The

parameters used for the acceptance or rejection of identi-

fication were: taxonomy—viridiplantae, enzyme—trypsin,

missed cleavages—1; modifications—carbamidomethyl,

mass values—MH?, peptide mass tolerance—100 ppm

and monoisotopic mass values. In addition, searches were

performed without constraining protein Mr (relative

molecular weight) and pI.

504 Acta Physiol Plant (2009) 31:501–514

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Results

Developmental stages of somatic embryogenesis

The protocol of Cangahuala-Inocente et al. (2007) was

efficient for the induction of somatic embryos which

develop from the cotyledonary leaves used as explants

(Figure 1a). Starting from 45 days in cultures supple-

mented with 2,4-D, we observed the presence of globular

and heart stages somatic embryos (data not shown). After

70 days in culture, a large number of somatic embryos

were observed in the torpedo (Fig. 1b), pre-cotyledonary

(Fig. 1d) and cotyledonary (Fig. 1f) stages.

Histological sections revealed the main features of each

stage. Thus, torpedo stage somatic embryos showed a

conspicuous protoderm and a well-defined apex, besides

root regions connected by procambial meristematic strands,

already seen at the globular stage (Fig. 1c). Pre-cotyle-

donary stage somatic embryos were elongated with well-

defined cotyledonary leaves (Fig. 1e). Cotyledonary stage

somatic embryos showed expanded cotyledonary leaves

(Fig. 1f), their cells being different from those present in

the embryonal axis. We also observed protein bodies and

starch grains in cotyledonary leaves (data not shown).

Expressed protein dynamics

The increasing cellular complexity of the developing

embryos was evident at the molecular level. Measurement

of protein content in these samples revealed a threefold

increase in total protein content based on fresh weight

between the early and late stages of development (Fig. 2).

Using 2D electrophoresis, 58, 66 and 57 protein spots

were detected in the torpedo, pre-cotyledonary and coty-

ledonary stages somatic embryos respectively, which were

reproducibly found in three independent replicate gels

(Fig. 3). Twelve of these protein spots were expressed in

all assayed developmental stages, from which 11 were

statistically significant (P \ 0.05). Eleven protein spots

were expressed in the heart, torpedo, pre-cotyledonary and

cotyledonary stages somatic embryos, from which nine

were statistically significant. One protein spot was

expressed in the heart, torpedo and pre-cotyledonary stages

somatic embryos. Three protein spots were expressed in the

globular, heart and torpedo stages somatic embryos, from

which two were statistically significant. Ten protein spots

were expressed in the globular and heart stage somatic

embryos, from which six were statistically significant.

Among the heart and torpedo stages somatic embryos, four

protein spots were expressed and all of them were statis-

tically significant (Table 1).

An increase and/or decrease in the protein expression

pattern tentatively assigned to the development and matu-

ration of the somatic embryo was observed (Fig. 3).

Twenty-two protein spots were detected simultaneously in

the three developmental stages assayed (torpedo, pre-

cotyledonary and cotyledonary), from which 14 were

statistically significant. These protein spots were assigned

to four different groups. Group 1 contained protein spots

with a decreased expression along the evolution of somatic

embryos, for example spot 3 (Fig. 3). Group 2 contained

protein spots with increased protein expression along the

development of somatic embryos (spot 5). Group 3 repre-

sented protein spots ubiquitous to the three developmental

stages (spot 19). Finally, group 4 contained protein spots

that were preferentially expressed in the pre-cotyledonary

stage (Table 2, spot 16).

In the torpedo and pre-cotyledonary stages somatic

embryos, four protein spots appeared simultaneously, from

which two were statistically significant (Table 1). The

expression of these protein spots decreased as the embryos

developed, disappearing in the cotyledonary stage. In the

pre-cotyledonary and cotyledonary stages, 12 coincident

protein spots were observed, five of which were statisti-

cally significant (Table 1). Out of the identified protein

spots, four increased their expression as the embryos

developed (e.g. spot 36, Table 2).

Comparing the developmental stages analyzed, protein

spot 23 was expressed only in torpedo-staged somatic

embryos (Table 1). In the pre-cotyledonary stage, the

expression of four protein spots was observed: 28, 29, 30

and 31 (Table 2).

Protein identification

Seventy-four differentially expressed proteins (2-DE spots)

were selected for MALDI-TOF/MS analysis to determine

their identities following fingerprinting, but the analysis

was unable to identify fourteen of them.

0

1

2

G H T PC C

Stages of somatic embryos

pro

tein

co

nce

ntr

atio

n

µg

/mg

FW

Fig. 2 Total protein (lg/mg FW) in different developmental stages

of A. sellowiana somatic embryos (SE). G globular, H heart, Ttorpedo, PC pre-cotyledonary, C cotyledonary. Mean of three

replicates with the standard error

Acta Physiol Plant (2009) 31:501–514 505

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Spectra were manually inspected, and in each case at

least one autolytic fragment of trypsin could be detected,

indicating that the sample had been exposed to protease

and that the mass spectrometer was operating correctly.

The experimental mass of the tryptic polypeptides was

compared with a data bank using Mascot software, and, as

no data from ESTs or genomic sequences existed for A.

sellowiana, we used ‘green plants’ as the source of theo-

retical tryptic peptides (http://www.expasy.ch). The

proteins identified by this method are listed in Table 2,

which also shows the relative expression of proteins eval-

uated by means of the ‘spot volume’ criteria from the

software Image Master Platinum (GE Healthcare) at dif-

ferent developmental stages. One spot, 4CC-1, showed a

significance that was higher than 65%, according to the

parameters utilized from the Mascot analysis, coinciding

with the experimental pI (Fig. 3; Table 2), and 15 proteins

presented a sequence coverage greater than 20% (Table 2).

During the various developmental stages, protein spot

23 was expressed only in torpedo stage somatic embryos

(Table 1). This protein may be similar to the hypothetical

protein At5g50240 of Arabidopsis thaliana. In the pre-

cotyledonary stage, the expression of four protein spots

was observed: 28, 29, 30 and 31, with homology to NmrA-

like proteins of Medicago truncatula, osmotin-like proteins

of Capsicum annuum, auxin-induced proteins of Helian-

thus annuus, and nitrate reductase of M. sativa,

respectively (Table 2).

Theoretical Mr and pI were obtained from Mascot

analysis and compared with the experimental values that

were estimated directly from the gel images, using Im-

ageMaster software (GE Healthcare; Fig. 4). There was a

well-defined correlation between the theoretical and

experimental Mr values, as indicated by the overlap of the

calculated data trendline (dashed line) and the ideal cor-

relation (solid line). On the other hand, the plot of

theoretical and experimental pIs demonstrated that while

there was a positive co-relation between the theorical and

measured values, it was much further from the ideal. The

R2 values for the two plots were calculated (Fig. 4), indi-

cating that there was significantly more scatter in the pI

data as compared with the Mr data. In general,

Fig. 3 2-DE gels from different developmental stages of A. sellowi-ana somatic embryos: a Torpedo stage with 58 spots. b Pre-

cotyledonry stage with 66 spots. c Cotyledonary stage with 57 spots.

Gels stained with coomassie brilliant blue G250, protein concentra-

tion 100 lg. Gels were prepared in three replicates

506 Acta Physiol Plant (2009) 31:501–514

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experimental pI values must be estimated from the shape of

the pH gradient within a batch of IPG strips. As suggested

by Lippert et al. (2005), this gradient is susceptible to

perturbation by contaminants within the sample as well as

by the protein samples by themselves. As such, the deter-

mination of pI is normally found to be less accurate than

Mr. Interestingly, the strong co-relation between theoretical

and experimental Mr sustains the protein identification

within this dataset.

Classification of identified proteins

The first step for interpretation of proteomic expression

data is to cluster proteins on the basis of gene ontology

(GO). The most complete GO classification for plants is

that from A. thaliana (Rhee et al. 2003). Thus, we were

able to classify the identified proteins in a list of associated

ontological terms, displayed graphically in Fig. 5. There

are three basic classes of ontological terms as defined by

the gene ontology consortium (Ashburner et al. 2000).

Molecular function (Fig. 5a) refers to the task(s) performed

by an individual gene product. Biological process (Fig. 5b)

refers to the broad biological role(s) or pathway(s) in which

a protein is involved. Cellular location (Fig. 5c) refers to

the subcellular location or macromolecular complex with

which proteins are associated.

Thus, considering somatic embryogenesis proteins dif-

ferentially expressed, most of them showed catalytic and

binding activities (43 and 25%, respectively). Also, when

considering the biological processes in which they were

involved, we identified proteins related to metabolism

(21%), as well as those related to energy-producing bio-

chemical processes (28%). Finally, proportionally more

proteins located in the cytoplasm (12%) were detected,

followed by those proteins located in various organelles

(8%), for instance, chloroplast, mitochondria, and endo-

plasmic reticulum. This is in complete agreement with the

extraction and 2D separation protocol we used. The num-

ber of proteins without an assigned function or biological

process in which they are involved, and those from which

the organelle where they act is not known, was quite high

(21, 41, and 61% respectively; Fig. 5). In the proteomic

analysis of Picea glauca somatic embryogenesis, Lippert

et al. (2005) reported the presence of membrane and

nuclear proteins, these being involved in metabolism and

energy-producing biochemical processes.

Discussion

Somatic embryogenesis in A. sellowiana

Brazil is considered as a country of megadiversity pre-

senting a variety of native plants with potential use, but as

yet undomesticated. Among these there has been a recent

focus on A. sellowiana (Myrtaceae), commonly known as

Table 1 Proteins detected on 2-DE and identified by MALDI-TOF analysis in different developmental stages of A. sellowiana somatic embryos

Characteristics Differential spots

detected by 2-DE

Differential proteins

identified by MALDI-

TOF/MS

Spot labeled

Present in all assayed stages 12 12 5SE-1, 5SE-2, 5SE-3, 5SE-4, 5SE-5, 5SE-6,

5SE-7, 5SE-8, 5SE-9, 5SE-10, 5SE-11,

5SE-12

Present in the heart, torpedo, pre-cotyledonary

and cotyledonary stages

11 10 4CC-1, 4CC-2, 4CC-3, 4CC-4, 4CC-5, 4CC-6,

4CC-7, 4CC-8, 4CC-9, 4CC-10, 4CC-11

Present in the globular, heart and torpedo stages 3 3 3GT-1, 3GT-2, 3GT-3

Present in heart, torpedo, and pre-cotyledonary

stages

1 1 3CP-1

Present in heart, and torpedo stages 4 0 2CT-1, 2CT-2, 2CT-3, 2CT-4

Present in globular, and heart stages 10

Present in torpedo, pre-cotyledonary,and cotyledonary stages

22 20 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22

Present in torpedo and pre-cotyledonary stages 4 3 24, 25, 26, 27

Present in pre-cotyledonary, and cotyledonarystages

12 6 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44

Exclusive of torpedo stage 1 1 23

Exclusive of pre-cotyledonary stage 4 4 28, 29, 30, 31

Exclusive of cotyledonary stage 0 0

Stages of development analyzed at work correspond are in italics

Acta Physiol Plant (2009) 31:501–514 507

123

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508 Acta Physiol Plant (2009) 31:501–514

123

Page 9

‘‘pineapple guava’’ or ‘‘feijoa’’, a fruit species native to the

southern Brazilian highlands. This species shows high

genetic variability among natural populations (Nodari et al.

1997), and its cultivation occurs in several countries (Thorp

and Bieleski 2002). In southern Brazil a domestication and

breeding program of A. sellowiana is in progress, with

successful results. Biotechnological techniques associated

to somatic embryogenesis are valuable tools in order to

capture the genetic gains and mass propagate plants from

elite germoplasm (Guerra et al. 2001).

In the present study, patterns of protein expression

associated with the development of A. sellowiana somatic

embryos were investigated using 2-DE technology coupled

to MALDI-TOF/MS and protein sequence database

searches. The 2-DE technique coupled to MS analysis is

well adapted for high abundant protein, such as storage

proteins and the sensitivity and scale of MS-based pro-

teomic approaches is expected to lead to the identification

of many proteins directly involved in somatic embryo

developmental programming (Lippert et al. 2005; Gallardo

et al. 2006; Winkelmann et al. 2006). Perturbations in the

expression profiles can therefore be indicative of aberrant

developmental processes, in such a manner that the gen-

erated profiles can be used as biochemical markers of the

quality of somatic embryo developmental processes

(Dodeman et al. 1997; Lippert et al. 2005). Additionally,

this type of analysis allows for the evaluation of the various

stresses intrinsically generated by tissue culture

procedures.

2-DE Protein profiles during somatic embryogenesis

Somatic embryos normally mimic the morphological and

metabolic features of zygotic embryos. In our study, rela-

tively few proteins were expressed in the late

developmental stages of somatic embryos, especially in the

cotyledonar stage. Imin et al. (2004) established a proteo-

mic reference map for globular stage somatic embryos of

M. truncatula, and resolved more than 2,000 proteins. As

stated by Winkelmann et al. (2006), the differences in

resolution would be ascribed to different extraction proto-

cols, 2-DE separations and staining procedures used by

different authors such as gradient of focalisation and gel

size. However, different species control is likely to be the

main reason for discrepancy. In Cyclamen persicum,

Winkelmann et al. (2006) detected 200 protein spots of

zygotic and somatic embryos using the protocol of Gal-

lardo et al. (2002). In the present work, using the protocol

of Carpentier et al. (2005), 181 protein spots were detected

in the diverse developmental stages of A. sellowiana

somatic embryos, which is in good agreement with the

study of Winkelmann et al. (2006).

Protein accumulation during somatic embryo develop-

ment of the carrot was studied by Racusen and Schiavone

(1988). They showed that a number of proteins were stage-

and tissue-specific, a few proteins being detected in

embryogenic calli and at certain embryo stages. Three

proteins found in embryogenic calli disappeared at early

stages, but they appeared again at the torpedo stage. Sim-

ilar results were also observed in D. carota (Dodeman and

Ducreux 1996), A. officinalis (Dupire et al. 1999) and C.

persicum (Winkelmann et al. 2006).

In the present work, one protein was expressed exclu-

sively in the torpedo stage, four were expressed in the pre-

cotyledonary stage, and none in the cotyledonary stage.

This suggests that few genes are involved in the control of

hystodifferentiation and morphogenesis of A. sellowiana

6,2

5,8

5,4

5,1

6,0

6,7

6,4

8,6

6,0

5,55,25,8

8,6

6,8

5,26,8

9,3

8,47,6

6,6

7,6

6,1

6,05,3

9,08,8

5,25,5

5,1

5,2 6,7

7,0

8,5

5,5

6,75,9

6,4

7,7

9,4

6,3

5,3

5,3

8,36,2

8,77,5

6,4

8,6

8,46,7

5,2

7,7

5,0

5,0 6,7

6,1

6,05,1

7,7

5,7

9,2

6,5

y = 0,3611x + 3,9452R

2 = 0,141

3

4

5

6

7

8

9

A

B

10

3 4 5 6 7 8 9 10

Theorical pI

Exp

erim

enta

l pI

*

7062

51

74

5457

514743

4062

3535

38

2623

271820221918

2330

7376

52

55

39

2619 25

18

26

24

34

60

14

61

4439

41

363739

27 37

2618

57

26

14

36

19

14

18

37

17

14

77

25

19

y = 0,9469x + 1,4067R

2 = 0,9425

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90 100

Theoretical Mr

Exp

erim

enta

l Mr

Fig. 4 Theoretical and experimental a pI and b molecular weight

comparison for each protein identified in different developmental

stages of A. sellowiana somatic embryos. The ideal co-relation is

indicated by the solid line, and those calculated from experimental

data by dashed lines. The linear regression analysis equation obtained

is indicated as an insert in each figure

Acta Physiol Plant (2009) 31:501–514 509

123

Page 10

somatic embryos, and that gene expression occurs prior to

morphological modifications.

Torpedo stage somatic embryos

Protein spot 23, only found in the torpedo stage somatic

embryo, matches the hypothetical protein At5g50240, that

presents similar features to L-isoaspartyl-O-methyltrans-

ferase (PIMT), an enzyme able to repair abnormal

isoaspartyl (isoAsp) residues in proteins under normal

physiological conditions (Thapar et al., 2002). In plants,

L-iso-Asp methyltransferase activity has been identified in

monocots, dicots, and green algae (Mudgett and Clarke

1994), although some PIMT enzymatic activity is

detectable in a few non-seed tissues (Thapar et al. 2002).

Interestingly, the greatest PIMT specific activity is found

in seeds where non-enzymatic protein damage occurs

during dehydration and quiescence (Mudgett and Clarke

1994).

Pre-cotyledonar stage somatic embryos

In this developmental stage, protein spot 28 is homologous

with an osmotin-like protein. This protein belongs to the

pathogenesis-related (PR) proteins, a category of proteins

which may be induced by various agents ranging from

ethylene to pathogens. So-called PR proteins have also

been shown to accumulate in healthy plants (Regalado and

Ricardo 1996), in particular development programmes,

such as germination (Casacuberta et al. 1992), senescence

(Hanfrey et al. 1996) and flowering (van Eldik et al. 1996).

Helleboid et al. (2000) identified three classes of PR pro-

tein: b 1,3 glucanases (38 kDa), chitinase (32 kDa) and

osmotin-like (25 kDa), secreted into the culture medium of

embryogenic Chicorium hybrida 474. These embryogenic

cultures accumulate the proteins by eightfold as compared

with the non-embryogenic line, suggesting that PR protein

could be associated with the competence for somatic

embryogenesis. We observed that this protein was not

Molecular functionbinding

25%transport

1%

transcription3%

reserve3%

defense1%

unlocalized3%

unknown21%

catalytic activity43%

Biological Process

biosynthesis11%

catabolism8%

response to stimulus3%

cell organization2%

cell morto2%

cell transport5%

sexual reproduction2%

translation2%

unlocalized3%

unknown41%

metabolism21%

Cellular Componentcytoplasm

12%

membrane

2%

nucleus

6%

vacuole

2%

organela

8%

cell wall

2%

extracellular matrix

2%

unknown

61%

unlocalized

3%

cytoskeleton

2%

C

A B

Fig. 5 Ontology classification of differentially expressed proteins from A. sellowiana somatic embryos. a Biochemical function, b Biological

process, and c cellular component. Searches were performed using GO software (http://ca.expasy.org)

510 Acta Physiol Plant (2009) 31:501–514

123

Page 11

expressed either in the torpedo stage or in the cotyledonary

stage. Previous studies had shown that A. sellowiana cot-

yledonary stage somatic embryos presented low conversion

rates (Cangahuala-Inocente et al. 2007). Thus, we

hypothesized that this protein could be marker of the

conversion ability of somatic embryos.

Protein spot 29 is an auxin-induced protein and it

belongs to a family of aldo-keto reductase (potential

members of the aldo-keto reductase superfamily). The

auxin indole-3-acetic acid (IAA) regulates a range of cel-

lular and developmental processes in plants, including cell

division, expansion, differentiation and patterning of

embryo responses (Kulaeva and Prokoptseva 2004). The

literature on auxin biosynthesis, metabolism, and transport

in embryos has grown out of extensive analysis which

shows that auxin plays important roles both in induction

of embryo formation in culture and in the subsequent

elaboration of proper morphogenesis during embryo

development (Feher et al. 2003; Quint and Gray 2006).

Somatic embryogenesis follows a unique developmental

pathway regulated by temporal and spatial patterns of gene

expression, which is triggered very early during the auxin

induction phase (Singla et al. 2007). In the present work,

we expected this protein to appear in all the assayed

developmental stages, being more greatly expressed in the

early developmental stages as suggested by Prewein et al.

(2006). Thus, the detection of these protein in the pre-

cotyledonary stages, together with its non-expression in the

other developmental stages, could suggest an anomalous

gene expression in mature somatic embryos showing

morphologic abnormalities, as revealed by Pescador et al.

(2008), or a low rate of embryo conversion to plantlets as

shown by Cangahuala-Inocente et al. (2007).

Protein spot 30 is a homologous of nitrate reductase

protein (NR), a key enzyme in nitrate assimilation in higher

plants, green algae and fungi (Pelsy and Caboche 1992).

The activity of NR in roots and cotyledons of Gossypium

hirsutum L. increases rapidly on germination, reaching a

maximum after 1 day of imbibition, and then declining

until emergence and greening of the cotyledons (Radin

1974). Fukuoka et al. (1996) detected NR mRNA 14 days

after culture initiation, corresponding to the heart/torpedo-

shaped stage. These results suggest the unique regulation of

NR in embryogenesis in which NR mRNA transcription is

activated in a developmental stage-specific manner. In the

present study, this protein was also present in the pre-cot-

yledonary somatic embryos, at a stage in which a high

conversion rate had been observed in a previous study

(Cangahuala-Inocente et al. 2007).

We also identified protein spot 31 as a homologous of

NmrA-like protein, a transcription repressor involved in the

regulation of nitrogen metabolism in Aspergillus nidulans

(Lamb et al. 2003). Interestingly, such protein reported to

occur in the extra-radical mycelium of Glomus intrara-

dices, a mycorrhizal fungus associated with the roots of D.

carota, has been proposed to help in nitrogen metabolism

(Dumas-Gaudot et al. 2004).

Functional relationship of differentially expressed

proteins

Protein spots 5SE-1 and 10 were proteins detected with a

statistically significant expression in every assayed devel-

opmental stage as an orthologue of phenylalanine

ammonia-lyase protein (PAL) of M. domestica (Fig. 3;

Table 2). PAL protein is involved in the synthesis of lignin,

suberin, and other wound-induced polyphenolic barriers

(Hahlbrock and Scheel 1989). All phenylpropanoids are

derived from cinnamic acid, which is formed from phen-

ylalanine by the action of PAL. The phenylpropanoid

compounds are induced in plants by various biotic and

abiotic stresses (Dixon and Paiva 1995). During the direct

somatic embryogenesis of Myristica fragrans, the somatic

embryos synthesize chlorophyll and phenolics, and exhibit

phenylalanine ammonia-lyase activity (Iyer et al. 2000). In

the growth of cell suspension culture of Vitis labrusca,

programmed cell death is accompanied by the coordinated

activation of phenylalanine ammonia-lyase, cinnamic acid

4-hydroxylase, and stilbene synthase gene expression, as

well as by the accumulation of stilbenes and other phenolic

compounds (Chen et al. 2006).

The peptidyl prolyl cis/trans isomerases (PPIases) cata-

lyze the cis/trans isomerization of the proline-preceding

peptide bond, which is an intrinsically slow process

(Landrieu et al. 2002). PPIases are highly functionally

conserved in yeast, animal, and plant species (Yao et al.

2001). In the present work, this protein appeared in the

form of three isomers and possibly as a homologous of

protein spots 5SE-2, 5SE-10 and 34, their expression being

statistically significant, except for 5SE-10. The isomer

5SE-10 had an enhanced expression (7%) in torpedo stage

somatic embryos. Protein spot 5SE-2 revealed enhanced

expression (3.9%) in the cotyledonar stage and spot 34 was

detected at the same level of expression in pre-cotiledonary

and cotyledonary stages somatic embryos. Expression of

AtPIN1 (A. thaliana) and MdPIN1 (M. domestica) tran-

scripts associated with cell division suggests that plant

Pin1-type PPIases could be associated with cell division

(Landrieu et al. 2002; Yao et al. 2001).

Another of the most highly expressed proteins in the

somatic embryos was protein spot 4CC-1 (Fig. 3; Table 2),

showing homology with the Dnak-type molecular chaper-

one Nthsp 70. This protein spot was significantly present

starting from the heart stage, and it showed enhanced

expression in the pre-cotyledonary and cotyledonary stages

somatic embryos in which several proteins are required for

Acta Physiol Plant (2009) 31:501–514 511

123

Page 12

the establishment of the embryo longitudinal axis, as well

as for the synthesis of storage proteins. The role of 70-kDa

heat shock proteins (Hsp70 s) in the folding of non-native

proteins can be divided into three related activities: pre-

vention of aggregation, promotion of folding to the native

stage, and solubilization and re-folding of aggregated

proteins (Mayer and Bukau 2005). In the proteomic anal-

ysis of M. truncatula somatic embryogenesis, the

chaperone proteins (DnaK-type hsp70 and luminal binding

proteins) decrease in 8-week-old cultures, showing that a

higher level of expression of the chaperones is required for

the maintenance of cells during early culture (Imin et al.

2005).

Another protein displayed as two isoforms (4CC2 and

35) is possibly homologous with a cytosolic glutamine

synthetase protein (fragment). The first isoform was sig-

nificantly expressed starting from heart stage somatic

embryos, and it was more greatly expressed in the torpedo

stage, while the other isoform was present only in the pre-

cotyledonary and cotyledonary stages. Glutamine synthe-

tase (GS) plays a central role in the nitrogen metabolism of

higher plants, being responsible for the primary assimila-

tion of ammonia (1) in root cells, (2) generated by nitrite

reduction in chloroplasts, and (3) produced by nitrogen

fixation in root nodules (McNally and Hirel 1983). In

higher plants, GS is an octameric enzyme 310–350 kD that

occurs as a number of isoenzymes, located both in the

plastids (GS2) and in the cytosol (GS1) (Li et al. 1993).

Higashi et al. (1998) investigated the expression pattern of

three carrot cDNA clone codings for three isoforms of the

enzyme glutamine synthetase during somatic as well as

zygotic embryogenesis. Transcription levels of CGS102

and CGS201 increased during the early stages of somatic

embryogenesis and developing seeds, whereas CGS103

was expressed only in the later stages of seed development

and in senescent leaves, being absent in somatic embryos

and young leaves. The expression of CGS102 and CGS201

declined in culture medium supplemented with glutamine

as a nitrogen source, indicating transcriptional regulation

of GS activity. Taken together, these results suggest a

common regulatory system in somatic and zygotic

embryogenesis with regard to the nitrogen metabolism.

Protein spot 21 was associated with another protein

expressed in the three assayed developmental stages, and it

was a homologous of the porin-like protein. This protein

showed a significant expression in the pre-cotyledonary

stage in which the embryo underwent an increase in size

and mass. Porin-like proteins belong to the so-called

voltage-dependent anion channels (VDACs), also known as

mitochondrial porins, which are pore-forming proteins

found in the outer mitochondrial membrane of eucaryotes

(Sampson et al. 1997). Wu et al. (2005) found a protein in

peroxisomes of Bromus inermis that bears identity with

various channel-forming proteins or porins of other plant

species. The precise function of the protein was not

established, but its expression was found to vary in

response to cold and drought stress, ABA, and during

embryogenesis.

Protein spots 26 and 36 matches a yippee-like protein, a

protein that contains a putative zinc-finger-like metal

binding domain, and it is the first characterized member of

a conserved gene family of proteins present in diverse

eukaryotic organisms (Roxstrom-Lindquist and Faye

2001). In our work, this protein represented two isoforms

appearing significantly in different developmental stages.

Protein spot 26 was observed in the torpedo and pre-cot-

yledonary stages at quite similar expression levels, while

protein spot 36 was observed in the pre-cotyledonary and

cotyledonary stages with an increased expression as the

somatic embryos evolved (Table 2).

Conclusions

A high resolution 2-DE proteome reference map was

generated for different developmental stages of Acca sel-

lowiana somatic embryos. Of the 74 different protein spots

analyzed, 60 were identified by means of 2-DE/MALDI-

TOF/MS. A high similarity in the profiles of the assayed

somatic embryos was observed, suggesting that only a few

specific genes are involved in the different developmental

stages, and that gene expression occurs prior to mor-

phological changes. The expression of phenylalanine

ammonia-lyase protein in all the assayed developmental

stages confirmed the synthesis and accumulation of several

phenolic compounds observed during the induction of

embryogenic cultures and the development of somatic

embryos. The presence of cytosolic glutamine synthetase

protein and Nmr-like proteins in the somatic embryos

revealed the activation of nitrogen metabolism, observed

particularly in the later developmental stages in which the

accumulation of storage compounds (mostly in the coty-

ledonary leaves) is enhanced. To date, our results represent

an advance towards a better understanding of the events

that precede the formation of the somatic embryo, in a

plant species for which very few genomic resources are yet

available. Although proteome analyses are still signifi-

cantly less representative in the literature than those based

on genomic approaches, the integration of the expressed

protein data, together with transcriptome and even metab-

olome data, will provide the most comprehensive and

informative clues on zygotic and somatic embryogenesis in

plants.

Acknowledgement We thank Drs. Emanuel Maltempi de Souza

and Fabio Pedrosa for their help with tandem mass spectrometry

512 Acta Physiol Plant (2009) 31:501–514

123

Page 13

analysis. This work was financed by CNPq, CAPES, and Rede Pro-

teoma de Santa Catarina-MCT/FINEP/FAPESC.

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