Review

Proteomics as an approach to the understanding of the

molecular physiology of fruit development and ripening
Jos M. Palma

, Francisco J. Corpas, Lus A. del Ro

Departmento de Bioqumica, Biologa Celular y Molecular de Plantas, Estacin Experimental del Zaidn, CSIC, Apartado 419,
18080 Granada, Spain
A R T I C L E I N F O A B S T R A C T
Article history:
Received 17 December 2010
Accepted 11 April 2011
Available online 16 April 2011
Fruit ripening is a developmental complex process which occurs in higher plants and
involves a number of stages displayed from immature to mature fruits that depend on the
plant species and the environmental conditions. Nowadays, the importance of fruit ripening
comes mainly from the link between this physiological process in plants and the economic
repercussions as a result of one of the human activities, the agricultural industry. In most
cases, fruit ripening is accompanied by colour changes due to different pigment content and
increases in sugar levels, among others. Major physiological modifications that affect
colour, texture, flavour, and aroma are under the control of both external (light and
temperature) and internal (developmental gene regulation and hormonal control) factors.
Due to the huge amount of metabolic changes that take place during ripening in fruits from
higher plants, the accomplishment of new throughput methods which can provide a global
evaluation of this process would be desirable. Differential proteomics of immature and
mature fruits would be a useful tool to gain information on the molecular changes which
occur during ripening, but also the investigation of fruits at different ripening stages will
provide a dynamic picture of the whole transformation of fruits. This subject is furthermore
of great interest as many fruits are essential for human nutrition. Thus far different
maturation profiles have been reported specific for each crop species. In this work, a
thorough review of the proteomic database from fruit development and maturation of
important crop species will be updated to understand the molecular physiology of fruits at
ripening stages.
2011 Elsevier B.V. All rights reserved.
Keywords:
Citrus
Fruit ripening
Grape
Proteomics and crop
Prunus
Tomato
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1231
2. Tomato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233
3. Grape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1234
4. Citrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1236
5. Prunus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1237
6. Apple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1238
J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
Corresponding author. Tel.: +34 958 181600; fax.: +34 958 129600.
E-mail address: jmpalma@eez.csic.es (J.M. Palma).
1874-3919/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2011.04.010
avai l abl e at www. sci encedi r ect . com
www. el sevi er . com/ l ocat e/ j pr ot
7. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1238
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1240
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1240
1. Introduction
The botanical definition of a fruit is a mature ovary and is,
therefore, confined to the Angiosperms. Different parts of the
flower can contribute to the final structure of dry and fleshy
fruits; thus, the final form of the fruit is dependent upon the
number and type of the floral organ components, the position
of the contributing organs, and how the different tissues
within them grow and differentiate [1]. Ripening is the final
phase of fruit development, and involves deep metabolic
changes in the biochemistry, physiology and gene expression
of the fruit such as chlorophyll degradation and pigment
(carotenoids and anthocyanins) biosynthesis, conversion of
starch to simple sugars, accumulation of flavours and cell wall
softening [24], ethylene receptor degradation [5], simple
sugar and organic acid accumulation, volatile production
and flesh softening [6,7]. The pathways involved in the
processes of fruit development and ripening are exclusive
for plants and vary between species. Thus, as an example, in
the maturation of pepper fruits a series of important events
takes place, as indicated in Fig. 1. During development and
ripening of pepper fruits clear visible changes are manifested.
Thus, mature green fruits shift to the final colour either red,
yellow, orange or purple in a process that is accompanied by
intense metabolism, emission of volatile organic compounds,
destruction of chlorophyll and synthesis of new pigments,
formation of pectins, synthesis of proteins, taste alteration
and changes in total soluble reducing equivalents. Overall,
developmental, physiological, anatomical, biochemical and
structural differences contribute to the operation of unique
pathways, genes and proteins [7].
This developmental process seems to be also influenced by
the type of fruit, either climateric or non-climateric, although
no consistent data are available thus far to conclude this
assert. In climateric fruits, which are characterised by a
peculiar burst in the ethylene evolution and the respiration
rate at the onset of ripening, these events are mainly regulated
by the gaseous phytohormone ethylene, which is also
involved in the decrease in flesh firmness typical of many
economically relevant crops like tomato and peach [8]. On the
other hand, ripening of non-climateric fruits such as pepper,
citrus and strawberry is ethylene-independent, although
similar major visual, texture, flavour and metabolic changes
occur as in climacteric fruits. Many of the changes have been
mainly characterised in climacteric-ripening fruits, whereas
non-climacteric fruit ripening is still poorly understood. In
Fig. 2, some climateric and non-climateric fruits are shown.
Interestingly, this physiological behaviour is not linked to
taxonomic groups. Species belonging to the same family, such
as tomato and pepper (Solanaceae) display distinct response to
ethylene. Thus, tomato is a climacteric fruit while pepper is
not.
Taking into account the demand of consumers and agro-
biotechnological companies, more attention was initially paid
to the fruit set and development and post-harvest strategies
than to the fruit maturation itself. In fact, a better compre-
hension of the genetic and molecular mechanisms responsi-
ble for fruit set and development has been gained due to the
major impact of strategies for breeding and crop improvement
in fruit bearing species. The impact of the model plant
Arabidopsis in that field has been relevant, so genetic studies
on this plant species have been proved to be very successful in
the search for key regulatory genes acting in carpel and fruit
development [913]. The study at molecular level of fruit
ripening has been mainly accomplished from a genetic point
of view [13]. The gene expression profiling of fruit develop-
ment and maturation was recently examined [1416]. Thus, an
increasing number of data are now available from large-scale
analysis of the gene expression during the climacteric or non-
climacteric fruit development [1722]. Also the evolution of a
series of metabolites and other molecules during fruit ripening
has been reported. Up until some years ago, only few data on
fruit development proteomics were available [23,24]. However,
the potentialities and the development of proteomics in the
recent past years have triggered a scientific burst in all
biological sciences and, consequently, the fruit proteomics is
now a body of interest not only for biologists but also for
agricultural companies.
Improved understanding of fruit maturation may yield
benefits both for public health and agricultural economy. An
important part of the human nutrition field to assess the
safety of new crop plant varieties is the extensive composi-
tional analysis, including the measurement of all key nutri-
ents and antinutrients in a specific crop. The applicability of
- taste alteration (acidity, pH and astringency)
- intense metabolism
- emitting volatile organic compounds (respiration)
- destruction of chlorophyll
- synthesis of new pigments (carotenoids plus related
xanthophylls, anthocians)
- synthesis of pectins
- protein synthesis
- changes in total soluble reducing equivalents ROS
Fig. 1 Events which take place during ripening of pepper
fruits.
1231 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
omics technologies, such as transcriptomics, metabolomics
and proteomics, as additional tools in this safety assessment
will be crucial to obtain a global picture of the fruit nutrients
and pro-active compounds and their profile throughout
ripening, as it was reported in tomato fruits [25]. Transcrip-
tomics, proteomics and metabolomics provide a wide over-
view of the metabolism at, respectively, the mRNA, protein
and metabolite levels. In theory, the information they supply
in selected samples is largely complementary to other
analytical traits used in human nutrition.
Microarray-based gene expression analysis (transcrip-
tomics) and two-dimensional electrophoresis (2-DE)-based
proteome analysis (proteomics) have the potential to screen
many metabolic pathways simultaneously for alterations in
gene expression and protein levels [25]. Nowadays, the study
of complex biological processes, such as fruit development
and ripening, through comparative proteomics is becoming
increasingly attractive to plant biologists as the rapidly
expanding plant genomic and the availability of EST sequence
databases have provided clear opportunities for protein
identification and functionality [26]. Actually, the proteome
is the full complement of proteins expressed by a genome [27]
at a specific point of time [28]. The proteome of each living cell
is dynamic, being altered in response to the individual cell's
metabolic state and the reception of intracellular and extra-
cellular signal molecules and stimuli [28]. While the genome
enables a prediction of the potential proteome simply as the
sum of the gene products, this cannot be described, in fact, as
the real proteome, since we do not knowwhichgenes and how
they are expressed at any specific moment, and, besides,
many of the proteins which are expressed as gene products
are perhaps post-translationally altered by one or more of the
approximately 200 possible modifications [2932]. Thus, if the
purpose of the proteome analysis is to aid the understanding
of the protein function and interaction, then it is the
identification of the proteins in their final state that is
required [28]. Giving an example, the research carried out on
several antioxidative enzymes from pepper fruits showed
that, whereas important changes took place in the activity
patterns of many enzymes as a consequence of fruit ripening,
the expression levels of transcripts of such antioxidants did
not vary appreciably. Those activity modifications were
usually accompanied by changes in the specific protein
content of the antioxidative enzymes, as revealed by the
western blot analyses [33,34].
Inthis review, the latest data onproteomics of fruit develop-
ment and ripening in a series of important crop species will be
scrolled down. Discussion on the main functional categories of
Pear
(Pyrus)
Banana
(Musa)
Plum
(Prunus)
Peach
(Prunus persica)
Kiwi
(Actinidia)
Tomato
(Solanum lycopersicum)
Orange
(Citrus)
Cherry
(Prunus)
Grape
(Vitis)
Olive
(Olea)
Apple
(Malus)
Melon
(Cucumis)
Cucumber
(Cucumis)
Lemon
(Citrus limon)
Pepper
(Capsicum)
Strawberry
(Fragaria)
Fig. 2 Climateric and non-climateric fruits of nutritional interest.
1232 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
differentially expressed proteins during ripening and the
approaches mostly followed to accomplish the investigation
of fruit proteomes will be addressed.
2. Tomato
Tomato (Solanum lycopersicum) is one of the most worldwide
consumed vegetables playing an important role in the human
diet. Tomato has long served as a model system for plant
genetics, development, physiology, pathology, and fleshy fruit
ripening, resulting in the accumulation of substantial infor-
mation regarding the biology of this economically important
crop [35]. The combination of all those features has led to
consider tomato as one of the main targets to accomplish
proteomic studies. Thus, transcriptomic and proteomic tech-
nologies were used as a guide to scrutinise tomato fruits with
regard to environmental conditions during growth and
harvest, including the ripening stage, as it is stipulated in
international guidance documents for the nutritional and
toxicological assessment of genetically modified plants [25].
Many genomic tools are now available on this Solanaceous
species and have rapidly generated a great amount of genomic
resources, including mapping populations, mapped DNA
markers, bacterial artificial chromosomes, and expressed
sequence tag (EST) collections [36]. There are currently more
than 184,000 tomato available ESTs (above 37,000 fruit ESTs)
that have allowed the identification of approximately 30,000
unigenes across a range of tissues and developmental stages
(http://documents.plant.wur.nl/cgn/pgr/tomato/). Numerous
mutants concerning fruit development and ripening and
genome sequencing are available [37]. Several genetic and
molecular approaches have been developed to increase our
knowledge about the physiological basis of fruit growth. On
the other hand, the increasing interest in plant proteomics
[38], has allowed the number of reports on proteomics of
tomato fruits under different conditions, including chilling
injury and others to grow gradually [39,40].
Recently, changes in gene and protein expression during
tomato ripening have been evaluated. In transcriptomic
analysis, an RDA (representational difference analysis)-based
[41] tomato array was used containing over 2000 EST
sequences that were specific for the red and the green stage
of ripening, respectively [25]. The red-specific EST-library was
assumed to contain ESTs that were related to the nutritional
and perhaps even health protecting properties of the tomato,
while the green-specific EST library was assumed to consist in
part of sequences that were related to antinutritional meta-
bolic routes [25]. In all cases the stage of ripening was the
largest source of variation between samples. The same
tomatoes in the subsequent ripening stages were analysed
for changes in proteome composition by 2-DE. Out of the 655
protein spots that were further analysed, 53 spots were found
to be differentially expressed during ripening. An overall
intensity increase during ripening was detected in 26 spots,
whereas a decrease was seen in 27 spots, and two spots
reached their maximum at the breaker or light red stage [25].
When comparing the proteomics results with the transcrip-
tomics data there was only one identified agreement: acid
beta-fructofuranosidase was analysed in both omics ap-
proaches and found to be upregulated in gene expression in
the breaker stage, downregulated in the subsequent turning
and light red stages and then once again upregulated in the
red stage of ripening [25].
It seems that there is a delayed-phase effect between the
transcriptome and proteome in developmental stages, as the
gene expression profile changes before a changed protein
profile in the same cell system is detected [25,42,43]. Thus, for
this type of analysis, it can be concluded that, for the time
being, the data fromtranscriptomics and proteomics are likely
to be complementary rather than overlapping [25]. Other
studies have confirmed these observations [44].
In parallel studies carried out in three different ripening
stages of tomato (unripe, medium ripened and fully ripened)
and using MALDI-TOF-MS analysis, it was observed that 34-
kDa and 44-kDa proteins were upregulated during fruit
ripening. Peptide mass fingerprinting analysis of those poly-
peptides resulted in the identification of pectinesterase and
heterotrimeric GTP-binding protein fragment homologous to
tobacco [6], which might be implicated in cell wall softening
and changes in firmness. Pectinesterase and the heterotri-
meric GTP-binding protein fragment were proposed as the
ripening specific markers in tomato, since their levels were
upregulated during tomato ripening [6].
The proteome variations associated with cherry tomato
pericarp development and ripening have been also investi-
gated [35]. It was found that protein patterns were markedly
different between stages. Actually, among the 1791 spots of
the master gel, 148 spots (about 8%) were found variable in
intensity throughout the process of fruit development. This
represents a slightly lower percentage than that detected by
transcriptomic analysis [15], where 10% of genes were
differentially expressed in developing tomato pericarp. How-
ever, the majority of proteins that were characterised corre-
sponded to genes known to be regulated during tomato fruit
development. Most of them displayed temporal expression
consistent with the succession of different phases of fruit
development. Based on these stages of development, func-
tional categories of spots that were up- or down-regulated
during fruit development could be identified, and clustered
correlation analysis results pointed out to groups of proteins
with similar expression profiles during fruit development. In
young fruit, spots linked to amino acid metabolism or protein
synthesis were mainly expressed during the cell division stage
and down-regulated later. Some spots linked to cell division
processes could be identified. During the cell expansionphase,
spots associated to photosynthesis and proteins linked to cell
wall formation transiently increased. In contrast, the major
part of the spots related to carbon compounds and carbohy-
drate metabolism or oxidative processes were up-regulated
during fruit development, showing an increase in spot
intensity during development and maximal abundance in
mature fruits. This was also the case for spots related to stress
responses and fruit senescence [35]. In some cases, the
obtained results either boosted or confirmed previous data
on genes/proteins whose expression changed during the
development and maturation of fruits [14,16,4550]. However,
when comparing the reported results with those previously
published on transcriptomic studies, some discrepancies are
still noted, confirming the necessity to carry on proteomic
1233 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
analysis as indicated above, and to go deeper in the analysis of
functional meaning of protein posttranscriptional and trans-
lational modifications [35].
A proteomic analysis has been also carried of tomato fruits
from two ecotypes (regional and commercial) during ripening
[24]. This study was conducted not only to understand the
underlying proteomics of fruit maturation for various eco-
types, in addition to originally clarify proteins/molecular
mechanisms involved in this physiological phenomenon, but
also to contribute to define specific molecular markers for the
selection of breed varieties maintaining original desirable
taste characteristics, while better suited to intensive cultiva-
tion [24]. Almost 57% polypeptides presented overlapping gel
coordinates between the two compared varieties, although
specific proteins were recognised in each ecotype as differen-
tially expressed during ripening. Common variably expressed
proteins in both ecotypes during maturation were associated
to important physiological processes such as redox status
control, defence, stress, carbon metabolism, energy produc-
tion and cellular signalling.
Enzymes associated with the antioxidative ascorbate
glutathione cycle were detected as abundant proteins in the
pericarp of fruits of both tomato ecotypes, namely, ascorbate
peroxidase and dehydroascorbate reductase. Similarly, CuZn-
containing superoxide dismutase was also identified as an
induced protein in both ecotypes, whereas the peptide corre-
sponding to methionine sulphoxide reductase, also known
as fruit-ripening protein E4, was found to be induced in the
regional ecotype during maturation [24]. Other enzymes related
to the reactive oxygen species (ROS) metabolism, including
thioredoxin peroxidase 1 and glutathione S-transferase were
also involved in the ripening of tomato fruits. Several sHSPs,
already described in the cytosol, mitochondria and chloroplasts
[51] were also identified which, in addition to a protective effect
against stresses, may play a pivotal role in plant development
under physiological conditions [24].
Some other proteins, generally related to environmental
stresses (the fruit-ripening protein and the embryo-abundant
EMB protein), to pathogen response (the tobacco stress-induced
gene 1, TSI-1, and the small, pathogen-activated gene STH-2),
organoleptic features (malate dehydrogenase involved in
malic and citric acid accumulation during fruit development
and ripening [52]), glycolysis/gluconeogenesis pathways (UTP-
glucose-1-phosphate uridyltransferase, triosephosphate
isomerase, glyceraldehyde 3-phosphate dehydrogenase, al-
dolase, phosphoglycerate kinase and enolase), and involved in
electron transport/energy production and photosynthetic
apparatus were also identified in both tomato ecotypes [24].
Proteomics of two near isogenic lines differing in their
texture phenotype under storage chilling conditions was also
investigated, rendering 85 differentially expressed proteins
[53]. In that study, it was shown that cold storing decreased
the expression of proteins involved in maturation process,
such as acidic invertase, flavour-related metabolism (terpene
biosynthesis and alcohol dehydrogenase, ADH), and structural
functions (cell wall related proteins). Acidic invertase is a
ripening-related protein, especially expressed from breaker to
red ripe stages of tomato fruits [35]. Likewise, ADHs are
considered as signal proteins for fruit ripening [54]. Several
other proteins were up-regulated that indicated their rela-
tionship to plant freezing tolerance. The proteins included in
this group mainly corresponded to sugar metabolism (enolase
and 6-phosphogluconate dehydrogenase), and oxidative
stress protection (vitamin B6 biosynthesis and small heat
shock proteins) [53].
Proteomic analysis by two-dimensional gels was recently
accomplished in tomato fruits at several ripening stages
subjected to chilling injury, a stress agent to which tomato is
susceptible, displaying physiological disorders characterised
by uneven fruit ripening and colour development, pitting and
decay [40]. In this study, 6% of the detected protein spots
(about 300) changed their expression in response to cold. The
identified proteins were involved in carbon metabolism,
oxidative stress, photosynthesis, and protein processing and
degradation; two proteins were related to cold stress, showing
higher accumulation in non-damaged tissue of chilled fruit.
These proteins corresponded to thioredoxin peroxidase (TPxI)
and glycine-rich RNA-binding protein (GR-RBP). An important
role for these proteins in cold response during tomato fruit
ripening was postulated. TPxI and GR-RBP may be acting
through redox sensing and regulation of gene expression at
low temperature and might be working together to maintain
the cellular homeostasis under cold stress conditions [40].
Lately, due to the important changes occurring in the
plastid population of tomato fruits during ripening, the
analysis of the proteome of these organelles was investigated
by LCMS/MS [4]. In red fruit chromoplasts the presence of 988
proteins corresponding to 802 Arabidopsis unigenes was
revealed, although 209 of them have not been reported as
plastidial protein earlier. Proteins of lipid metabolism, includ-
ing those required for the synthesis of the lipid-derived aroma
volatiles, and trafficking were found. Also, proteins involved
in starch synthesis and degradation co-existed in these
organelles, and they also contained proteins involved in
chlorophyll degradation. On the contrary, chromoplasts lack
proteins related to chlorophyll biosynthesis and those impli-
cated in the thylakoid transport machinery. Interestingly,
chromoplasts contained the entire set of Calvin cycle proteins
including Rubisco, as well as the oxidative pentose phosphate
pathway [4]. However, this study was not accomplished at
different ripening stages, so the dynamics of the protein
turnover in the conversion of chloroplasts into chromoplasts
is a promising subject to be discovered for cell biologists and
plant physiologists.
The research developed in tomato not only provides
continuous data of interest for biologists and tomato seed
companies, but also contributes to fit the platformto carry out
proteomic research in other related Solanaceae fruits such as
pepper, characterised by strong colour changes at maturation.
In Table 1, a summary of the techniques used to investigate
the proteome of tomato fruits is given. A combination of
approaches including 1-DE/LC, 2-DE and high performance
liquid chromatography (HPLC) linked to diverse mass spec-
trometry techniques has been commonly used.
3. Grape
Grape (Vitis vinifera) is also a plant species with high interest
not only from a nutritional point of view, but also from the
1234 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
winery industrial economy. Currently, the basic research
oriented to grapevine genomics has compiled more than
300,000 EST sequences stored in databases from different
grape varieties (http://cropdisease.ars.usda.gov/vitis_at/main-
page.htm), and the investigation of the processes occurring
during ripening has been also the focus of current research.
Grapes are classified as a non-climacteric fruit on the basis of
respiration rates. It has been suggested that abscisic acid may
play a role in the ripening process in grapes, as its concentra-
tion increases as berries ripen [5557]. The onset of maturation
begins at vraison, i.e. the onset of skin colour-change in black
cultivars. Anthocyanin pigment accumulation starts in skin
cells at vraison and continues through the ripening phase.
Ripening is also characterised by an increase in grape size,
softening, and cell expansion resulting in water and sugar
accumulation in the mesocarp cell vacuoles [58,59]. In recent
years, proteomics-based technology has been successfully
applied to grapevine in different cell processes and pathways,
such as herbicide reaction [60], water deficit and salt stress
responses [61,62], single gene transformation-induced protein
changes [63], and somatic embryogenesis-induced protein
changes [64]. The first two-dimensional electrophoresis anal-
ysis of the total polypeptides in ripe red grapevine berries [65]
has launched proteomic studies on grape berries as an
approach of consensus interests among biologists and the
winery industry. The method of protein extraction of grape
berry for proteomic analysis has been the subject of interest,
and this is of great relevance since grape berries are
considered recalcitrant materials in proteomic analysis, due
to the large amounts of secondary metabolites, especially
phenolic compounds, which severely interfere with protein
extraction and electrophoresis separation [66]. Actually,
extraction methods for the proteome study of grape berries
are continuously published [61,6668]. In one of the first
reports on the proteome data of grape berries, Sarry and
colleagues identified 67 mesocarp proteins of ripening berry of
different genotypes [69], and more recently, Giribaldi et al. [70]
investigated the protein expression during different stages of
grape berry development.
The application of iTRAQ (isobaric tag for relative and
absolute quantisation of tryptic peptides) analysis has been
postulated as a very useful tool in the investigation of fruit
biology [71]. Thus, a bioinformatics pipeline for processing EST
data in order to produce a predicted tryptic peptide database
specifically targeted to the wine grape cultivar, and lacking
truncated N- and C-terminal fragments was developed. With
this strategy, the predicted peptide database from MS/MS
applications can be derived fromEST data using advanced and
trimming approaches and successfully implemented for
quantitative proteome profiling in processes such as fruit
development and ripening [71].
The characterisation of skin tissue is apparently an
essential parameter for understanding grape ripening, due to
its key role in developing the main compounds responsible for
wine quality. The skin also constitutes a physical barrier
between the external environment and the inner tissues, and
its integrity is a key factor in preventing pathogen infections
[72].
Proteome maps obtained at three stages of ripening were
compared to assess the extent to which protein distribution
differs in grape skin during ripening. The comparative
analysis showed that numerous soluble skin proteins evolved
during ripening and revealed specific distributions at different
stages. Proteins involved in photosynthesis (Rubisco), carbo-
hydrate metabolism (aconitate hydratase, transketolase,
phosphoenolpyruvate carboxylase, oxalyl-CoA decarboxylase
and aldehyde dehydrogenase), and stress response (HSP17.7)
were identified as being over-expressed at the beginning of
colour-change [72]. At harvest, the dominant proteins were
involved in defence mechanisms. In particular, increases in
the abundance of different chitinase and -1,3-glucanase
isoforms were found as the berry ripened. This observation
could be correlated with the increase of the activities of both of
these enzymes during skin ripening. Thus, the differences
observed in proteome maps clearly showed that significant
metabolic changes occur in grape skin during this crucial
phase of ripening [72]. More recently, by Western blotting
analysis of grape berries separated by 2-D electrophoresis and
using a synthetic antibody raised against 15 amino acid
sequence residing on the surface of the -1,3-glucanase
molecule two major spots were identified by MALDI-TOF
that validates that this enzyme system is present in higher
abundance in berry skins than in pulps, and in red berries than
in white berries [66]. According to these data, it can be
assumed that the use of specific antibodies potentiates the
applicability of proteomics in fruit physiology.
The proteome of grape skin and its evolution throughout
different stages of ripening have also been more lately
accomplished [73]. Eighty spots were differentially expressed
with ripening and applying a two-way hierarchical clustering
analysis, it was found that the most relevant changes occurred
in the first two weeks of ripening. Most variable proteins
were related to response to pathogenesis (chitinase and -1,3-
Table 1 Some of the approaches used in the study of
proteomics of fruit ripening.
Fruits Proteomic approach References
Tomato 1-D/LCMS/MS [4]
2-DE/MALDI-TOF-MS [24,40]
2-DE/electrospray tandem mass
spectrometry
[25]
Multidimensional HPLC/MS/MS [39]
2-DE/HPLMS/MS [52]
Grape
a
2-DE/MALDI-TOF-MS/western blotting [66]
2-DE/LCESI-MS/MS [67,73]
2-DE/MALDI-TOF-MS [68]
iTRAQ/LC-MS/MS [71]
2-DE/LCMS/MS [72,76]
Citrus 2-DE/LCMS/MS [87,89,91]
LC-MS/MS (dMS-SC) [88]
2-DE/MALDI-TOFTOF-MS [92]
Peach 2-DE/LCESI-MS/MS [8]
2-DE DIGE/LCMS/S [94]
2-DE/ESI-Q-TOF-MS/MS [95]
2-DE/ESI-MS/MS/western blotting [20]
Apple 2-DE/MALDI-TOF-MS and
LC-ESI-IT-MS/MS
[98]
2-DE/ESI-MS/MS [99]
a
A full summary of approaches applied to grape proteomics is
provided in [85].
1235 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
glucanase as in [72], and thaumatin-like protein), oxidative
stress (glutathione peroxidase, catalase, and polyphenol oxi-
dase), and carbon and nitrogen metabolisms (transketolase,
NADP-dependent malic enzyme, pyruvate dehydrogenase,
glycine cleavage system P-protein, serine hydroxymethyltrans-
ferase, among others) [73].
Over-expressionof theHSPproteinsmaybearesponsetothe
onset of abscisic acid accumulation in the skin at this stage in
development [74]. The end of colour-change was characterised
by the over-expression of proteins involved in anthocyanin
synthesis (flavanoid 3-o-glucosyltransferase, leucoanthocyani-
din dioxygenase, flavonone 3-hydroxylase, and chalcone
synthase). In Shiraz grape skins, the genes involved in
anthocyanin synthesis are expressed both in early berry
development and during ripening [75]. Therefore, the accumu-
lation of proteins involved in the anthocyanin pathway is
probably correlated with the accumulation of this hormone at
vraison. Actually, although, the control of ripening ingrapevine
fruits is still a matter of debate, several lines of evidence point to
an important role of ABA [76]. Consequently, the effects of ABA
treatments on grape berries before and at vraison were studied
by2-dimensional electrophoresis. Results obtainedshowedthat
a total of 60 proteins displayed significant variations between
control and treated berries. The treatment affected stress-
related proteins (chitinase, lipooxygenase, spermine synthase,
cysteine synthase, among others) and ripening-related proteins
like NADP-dependent malic enzyme (ME), alcohol dehydroge-
nase, xyloglucan endotransglycosylase and glutathione-S-
transferase [76]. The involvement of ME in the ripening of
pepper fruits was also reported recently [34].
As in tomato fruits, the analysis in grape berries has been
also performed at subcellular level, especially in the cell
compartments more affected during ripening. Thus, proteo-
mics of the cell wall fromgrape berries has been accomplished
[67]. Studies dealing with the biochemical and physiological
processes that drive grape berry development have recently
been implemented by characterisation of the transcriptomic
changes that occur. These analyses have revealed alterations
in the expression of hundreds of genes, some of which encode
cell wall proteins and allowed discriminating between apo-
plastic and cell wall proteins [19,22,77]. Although the patterns
were quite similar, some significant differences were observed
[67]. To survey this aspect some spots detected in either both
fractions or present in only one fraction were analysed by
liquid chromatography electrospray ionisation tandem mass
spectrometry (LCESI-MS/MS). Of the 47 spots identified, some
were found to be cell wall proteins, while others were proteins
not traditionally considered as localised in the apoplastic
space [64]. A few of the spots identified contained an N-
terminal signal peptide, which suggested that they enter the
secretory pathway. These were a GRIP 22 precursor protein, a
putative stress-induced protein expressed during ripening of
grape berry [78], a -1,3-glucanase, two proteins belonging to
class IV(endo)chitinase and a protein with unknown function.
A protein disulfide-isomerase precursor containing the signal
peptide was also detected in the cell wall of grapes [67]. Again,
the role of the chitinase and the -1,3-glucanase in the
ripening of grapes was confirmed. On the other hand, most
of the identified proteins retrievable in the cell wall fraction
lack the signal peptide, such as a glyceraldehyde-3-phosphate
dehydrogenase, an enolase, an elongation factor 1a, a copper
zinc superoxide dismutase and a Xaa-Pro aminopeptidase 1
[67]. These results fit with others found previously in cell wall
fractions from other plant species [7982]. In spite of these
interesting results, a deeper approach to this subject with the
development of techniques which allow better yields of the
cell wall protein content is necessary.
Dramatic metabolic changes take place in the cells during
the different berry development stages, especially before and
after vraison. As a boundary of cells, the plasma membrane
(PM) is thought to play a critical role in terms of its barriers,
channels, exchanges, and communication in the cell process-
es. Many essential functions of the PMs are carried out by their
proteinaceous complexes, including molecular transport, cell
cell interactions, ligand binding, signal transduction, and
environmental sensing [83,84]. Then, the investigation of the
proteomics of grape plasma membrane was one of the targets
in grape biology [68].
High purity berry plasma membranes (PMs) of V. vinifera L.
cv. Cabernet Sauvignon were isolated by two-phase partition-
ing of microsome fractions at different stages of berry
ripening. PM proteins resolvable by the detergent cocktail of
CHAPS and ASB-14 were separated by two-dimensional
electrophoresis. A total of 119 protein spots from pre-vraison
berry PMs on 2-D gels detected with silver staining were
subjected to MALDI-TOF mass spectrometry analysis. Sixty-
two spots were identified as putative PM proteins, with 16
predicted transmembrane helices, including true PM proteins
such as ATP synthase, ABC transporters, and GTP-binding
proteins reported in plants. They were then grouped into eight
functional categories, mainly involved in transport, metabo-
lism, signal transduction, proteins associated with cellular
biogenesis and protein synthesis or fate and proteins associat-
ed with metabolism, transcription, and energy protein synthe-
sis. The vraison and post-vraison samples displayed 98 and 86
spots on the gels, respectively. During the berry ripening
process, total PM protein content gradually decreased. Among
all identified proteins, 12 showed differences in terms of their
relative abundance. Increasing ubiquitin proteolysis and
cytoskeleton proteins were observed from pre-vraison to
post-vraison stage. Zeatin o-glucosyltransferase peaked at
vraison, while ubiquitin-conjugating enzyme E221 was
down-regulated at this stage. This proteome research
provided the first information on PMprotein characterisation
during the grape berry ripening process [68]. The main
approaches to study the proteome of grapes are given in
Table 1. The most used approach consists of the combination
of 2-DE and mass spectrometry, although quantitative
shotgun proteome profiling (iTRAQ) has been considered to
be promising to study biological processes such as fruit
development and ripening [71]. A thorough review of the
proteomic perspective on grape and wine reports the main
data and perspective on the ripening of these fruits and the
future aim of this relevant field [85].
4. Citrus
Citrus is another non-climateric important crop species for
human nutrition and agricultural economy with a non-
1236 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
climacteric ripening behaviour and a unique anatomical fruit
structure. In the development of orange (Citrus sinensis) fruits,
three stages have been defined. Stage I (the cell division stage)
starts immediately after fruit setting and lasts for about
90 days after full bloom. Stage II (the cell expansion stage) in
which fruit growth continues, mostly by cell expansion, and
extends until 150180 days after full bloom. And, finally, stage
III (the ripening or maturation stage), which extends until
harvest [86].
In early studies carried in fruits from stage III, the LCMS/
MS analysis of the citrus juice sac proteins resulted in the
detection of 1394 unique proteins by searching in NCBI non-
redundant (green plants) and citrus ESTs databases [http://cgf.
ucdavis.edu; 87]. From the proteins identified, 433 were
associated to ER/Golgi, 502 to plasma membrane, 329 were
related to the tonoplast, 657 were mitochondria-associated,
and 479 were soluble proteins. The proteins were classified
into 12 major functional groups, the most abundant class of
citrus juice sac proteins being those involved in metabolic
processes followed by translation and transport. Thus, several
proteins related to sugar metabolism were found in stage III,
including diverse sugar (glucose, glucose-6-phosphate, su-
crose, and hexose) transporters, sucrose synthase, phospho-
glucomutase, sucrose phosphatase, hexokinase, fructokinase,
the enzymes of the citric acid cycle, among others. A
considerable high number of proteins were classified as
chaperones/heat shock, and others were linked to processing,
trafficking, and signalling [small GTPases, SNARE (soluble N-
ethylmaleimide-sensitive factor attachment protein recep-
tors) proteins and dynamin]. Proteins involved in the reactive
oxygen species (ROS) metabolism were also increased signif-
icantly inthis later developmental stage, including glutathione
peroxidase, glutathione S-transferase, catalase, thioredoxins,
superoxide dismutase, ascorbate peroxidase and others. This
last enzyme takes part in the ascorbate (vitamin C) metabo-
lism, a non-enzymatic antioxidant whichcharacterises orange
fruits. Furthermore, proteins involved in other main cellular
activities, suchas energy and structure were also detected [87].
Very recently, a broader investigation of stages II and III
and an additional early stage II was achieved. This research
involved a label-free LCMS/MS based shot-gun proteomics of
those three stages, and differential mass spectrometry (dMS)
and spectral counting (SC) were used to analyse protein
changes occurring during early and late stages of orange
fruit development [88]. To resolve the bioinformatics limita-
tions, due to the lack of orange databases, the iCitrus database
and interface were created to collect sequences from three
different sources: HarvEST:citrus, NCBI/citrus/unigenes, and
NCBI/citrus/proteins. iCitrus has provided a useful bioinfor-
matics tool for the high-throughput identification of Citrus
proteins and, in fact, Katz and colleagues [88] identified 1500
proteins expressed in orange fruit juice sac cells and
confirmed that most of the up-regulated proteins belonged
to metabolism, oxidative processes, trafficking, transcription
and transport. But, most important, a new and more reliable
approach to quantify changes of the protein expression during
fruit development was issued in this report.
Interestingly, a spontaneous sweet orange (C. sinensis [L.]
Osbeck) mutant Hong Anliu of high value due to the
carotenoid lycopene accumulation in the pulp, has been also
used for proteomic purposes in the investigation of fruit
ripening [89]. The proteomic alterations in the pulp of the
mutant Hong Anliu versus the wild type (WT) at four
maturing stages by using 2-DE combined with MALDI-TOF
TOF MS were analysed. Among the 74 differentially expressed
proteins identified, the majority were predicted to be involved
in stress response, carbohydrate/energy metabolism and
regulation, or protein fate, modification and degradation.
Particularly, expression levels of six antioxidative enzymes
(catalase, peroxidase, ascorbate peroxidase, glutathione re-
ductase and superoxide dismutase) were altered by the
mutation, and assays of their respective enzymatic activities
indicated an enhanced level of oxidative stress in Hong
Anliu, implying a regulatory role of oxidative stress on
carotenogenesis [89]. In fact, the potent antioxidant ascorbate
participates in the synthesis of carotenoids and in the
xanthophyll cycle, a process which usually takes place at the
thylakoidal lumen and implies the regeneration of violaxan-
tine at acidic pH through the violaxantine de-epoxigenase
activity [90].
The proteomic analysis of two orange cultivars with dif-
ferent pigmentation at ripening time has also been performed
by a combination of 2-DE and LCMS/MS [91]. Almost two
thirds of the differentially expressed proteins, which involved
sugar and secondary metabolisms, oxidative processes and
defence, were characteristic of the most pigmented fruits
(blood orange), and this indicated that some of themcould be a
consequence of the high level of anthocyanin accumulation
[91].
Proteomic studies have beendone inlemon(Citrus limonum)
fruit flavedo but no data on how the proteome profile
undergoes with ripening are available. A multistep procedure
has been developed and applied to extracts and purify
proteins. Two-DE, LCESI-MS/MS, and bioinformatics were
used to detect the high abundance of the germin-like
glycoprotein Cit s1, a powerful allergen in humans. Peptides
of Cit s1 were detected in 17 spots ranging from 120 to 20 kDa,
pointing out that in the flavedo of lemon the Cit s1 may be
expressed as several isoforms [92].
The main proteome approaches performed in Citrus are
summarised in Table 1. Most studies combine 2-DE and mass
spectrometry. However, as indicated elsewhere, the use of two
alternative complementary methods, dMS and SC, has pro-
vided to broaden the identification spectrum and strengthen
the identification of trends in protein expression changes
during citrus fruit development [87]. This approach will be a
promising alternative to increase our knowledge in fruit
biology and to develop appropriate proteome fruit databases
since new windows to quantitative proteomics will be opened
(see below in Concluding remarks).
5. Prunus
According to their flesh phenotypes at ripening, peach (Prunus
persica [L.] Batsch) fruits are grouped into melting flesh (MF)
and nonmelting flesh (NMF): they both soften but this event is
more evident in mature MF than in NMF fruits. This behaviour
makes MF fruits soft and juicy, so they are attractive to the
consumers but also extremely susceptible to handling and
1237 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
physical injuries; NMF peaches, on the contrary, are less
appreciated by the consumers but have good keeping quali-
ties. Proteomic approaches were applied to study some
biochemical and physiological features of peach fruit ripening
at the transition from the pre-climacteric to the climacteric
phase [8]. By means of LCESI-MS/MS (Table 1) it was found
that about 53 proteins were involved in different physiological
processes (i.e. sugar metabolism, ethylene evolution, amino
acids metabolism and stress response) typical of fruit devel-
opment and ripening [8].
Little work or peach fruit ripening is available thus far.
Lately, most of reports on proteomics of peach are focussed on
the senescence and post-harvest effects on the proteome
profiles of fruits. Thus, 2-D DIGE analysis was used to
elucidate the possible pathways involved in softening and
chilling injury of fruit at post-harvest conditions. Proteins
such as endopolygalacturonase, catalase, NADP-isocitrate
dehydrogenase, pectin methylesterase and dehydrins were
found to be very important for distinguishing between healthy
and chill injured fruits [93]. The proteins related to response to
stress, cellular homeostasis, metabolismof carbohydrates and
amino acid metabolism were the most affected during the
post-harvest. Besides, some of the proteins that changed
during postharvest were related to peach fruit ripening and
cold stress [93]. This indicates that the proteome analysis may
provide quality markers in fruits and their evolution might be
followed by studying the ripening of fruits at different stages.
The effect of storing peach fruits at different low temper-
atures was investigated by ESI-Q-TOF-MS/MS and proteins
expressed differentially were identified. Four membrane
stability related proteins (enolase, temperature-induced lipo-
calin, major allergen Pru p 1, and type II SK2 dehydrin) were
enhanced, but the proteins related to phenolic compounds
metabolization cinnamyl-alcohol dehydrogenase 5, cinnamyl-
alcohol dehydrogenase 1, and chorismate mutase were
repressed in peach fruits stored at 0 C with regard to storing
at 5 C [94]. The NADPH-generating enzymes glucose-6-
phosphate dehydrogenase, isocitrate dehydrogenase, and
malic enzyme similarly decreased in fruits stored at 0 C.
The overall results indicated that the incubation at 0 C might
regulate the endogenous hydrogen peroxide levels by activat-
ing the transcriptional level of genes which code the proteins
related to membrane stability [94]. Once again, the involve-
ment of ROS in fruit physiology both in ripening and post-
harvest/senescence seems to be a common event. This idea
was confirmed by studies on 2-DE and immunoblotting
performed in peach mitochondria, where levels of the anti-
oxidative enzyme manganese-containing superoxide dismu-
tase (Mn-SOD) were modified in senescent fruits [95].
Additionally in the same experiment, together with modifica-
tions in the expression of the voltage-dependent anion-
selective channel (VDAC) and the tricarboxylic acid cycle
enzymes malate dehydrogenase and aconitase which were
enhanced, an increase in the protein carbonylation level was
detected in senescent fruits [95], a widely used marker of
protein oxidation [96,97].
In apricot (Prunus armenica), another species closely related
to peach, a combination of transcriptomic and proteomics was
developed not only to know the events occurring at ripening,
but also to provide genomic tools for molecular breeding [20].
It was found that during ripening a strong activation of stress-
related proteins and cell wall modifying enzymes was
displayed. In this case, proteomic data confirmed at post-
translational level data obtained from systematic sequencing
strategy from3 end of cDNA, in which about 15,000 ESTs were
generated from cDNA libraries of apricot at three develop-
mental stages (immature, half-ripe and ripe stages) [20].
6. Apple
Apple (Malus domestica) is also one of the most worldwide-
consumed fruits and a number of cultivars, differing in
organoleptic and nutritional characteristics, are available in
the market. Annurca apple is a regional variety from Southern
Italy, which is known for crispness, excellent taste and long
shelf life of fruits. These features have renewed the interest in
the investigation of their genetic potential and different
studies have lead to their partial genetic and metabolic
characterisation. The analysis of the protein repertoire of the
pseudocarp tissues of three accessions of M. domestica Borkh.
cv. Annurca, as the first example of the systematic annotation
of the apple proteome, was reported [98]. Peptide MS and MS/
MS data (Table 1) were searched against publicly available
protein and EST databases, and 44 spots were identified and
associated to 28 different species. They were related to
important physiological processes such as energy production,
ripening and stress response. The occurrence of allergens
causative of widespread food allergy syndromes was also
detected [98]. As in peach, the proteomic analysis of changes
in mitochondrial protein expression during fruit ripening in
apple was performed and compared to that of fruits treated
with high and oxygen concentration. The expression of
enzymes from the tricarboxylic acid cycle, the electron
transport chain, carbon metabolism and membrane carriers
was modified by both senescence and oxygen treatment. The
differential expression of the mitochondrial Mn-SOD and the
increase of oxidised proteins (carbonylated) suggested that, as
in peach, the ROS metabolismis involved in fruit ripening [99].
7. Concluding remarks
In this work, a state of the art of the latest reports on the
proteomic of the most studied fruits during ripening is given,
although not all published data are compiled due to the
increasing number of reports in this field. Proteomics has
contributed to decipher how the metabolism undergoes from
immature to mature fruits, but due to the exclusivity of each
fruit, more research should be devoted to this subject. Thus,
most species share common ripening profiles involving
oxidative metabolism, sugar metabolism, stress-related pro-
teins and others with modified expression during ripening.
Table 2 shows the main functional categories where most
differentially expressed proteins are included for the fruit
crops reported in this work. The analysis of these groups
allows establishing correspondence with the main physiolog-
ical processes already reported to occur in fruit development
and ripening (Fig. 1). Thus, for example, the intense metabo-
lism which takes place at ripening is somehow linked to the
1238 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
changes observed in the carbon, carbohydrates, amino acids,
and lipid metabolism detected by proteomic analyses. Like-
wise, the destruction of chlorophyll and synthesis of new
pigments are associated to photosynthesis, and changes in
total soluble reducing equivalents is clearly linked to proteins
related to response to stress, oxidative stress and redox status
which are differentially expressed during ripening. This leads
to consider proteomics as a very useful tool to the under-
standing of this complex physiological process. On the other
hand, each crop species also display differential events which
give them some peculiarities; i.e.: synthesis of specific caroten-
oid, anthocyanins, flavonoids, terpernes, etc. responsiblefor the
different colours, flavours, and aromas, among other features,
which provide specificity to their respective species. The study
by proteomic approaches of the pathways involved in those
metabolic processes would provide data on the modulation of
such routes during ripening, a subject of great interest to
increase our knowledge but also for breeding and agricultural
and economic strategies. Other important biological functions
where proteins are also differentially expressed during ripening
arerelatedtocross-talkandinterplayamongcells/tissues. Thus,
efforts to investigate protein trafficking as well as signal
transduction and molecular signalling may contribute to more
comprehend the full metabolic picture displayed during fruit
development and ripening.
As the number of vegetables and fruits destined to human
consume is so high, the investigation of fruit development and
ripening by proteomics will undergo a great burst of knowl-
edge. Furthermore, the variety of kinds of fruit and their
changes in the morphology and molecular properties will
make the research in this field doubly enthusiastic. Actually,
the future seems promising and different approaches, includ-
ing the analysis of subproteomes (organelles' proteomes) as
those indicated here [5,19,22,67,68,78,96,99] will be part of the
engine of the field. Within this framework, our group has
started the investigation of the peroxisomal and the mito-
chondrial proteomes from pepper fruits during maturation
[100]. In Fig. 3, a summary of the different approaches that can
be followed to board the proteome of the fruit ripening events
is depicted. The experimental design starts with the selection
of the plant material to be studied. Thus, besides focusing on
the different fruit stages throughout the ripening process, the
specific target has to be set. It implies that the investigation
can be done at the level of the whole fruit, but also to flesh,
seeds and skin. More deeply, the research can be oriented
towards the organelles' proteomes, so chloroplasts, chromo-
plasts, mitochondria, peroxisomes, vacuoles, nuclei and other
cell compartments can be separated by several methods, and
then analysed by the most commonly used proteomic
approaches: combination of protein separation methods (2-
DE, 1-DE, LC, DIGE, and 2-DE plus western blotting) combined
to the different MS tools. The contribution of EST databases
and new revised approaches (iTRAQ, DIGE, immunoblotting,
dMS plus SC, etc.) to future research will shorten the trajectory
to the final objective. The improvement of the techniques
already used in this field combined with more reliable
extraction methods, and the investigation of processes like
fruit development and ripening through the use of comple-
mentary techniques will be also strategy of future work.
Once the main functional categories of proteins which are
Table 2 Functional categories of proteins differentially
expressed in fruits under different developmental and
ripening conditions.
Functional categories Process Species Reference
Carbon/carbohydrate/
amino acid/lipid
metabolisms
Fruit ripening Tomato [24,35]
Grape berry [72,73]
Orange [87,89,91]
Fruit senescence Apple [95]
Fruit storage Ponkan [103]
Chilling Tomato [40]
Response to stress/
oxidative stress
Fruit ripening Tomato [24,25,35]
Grape berry [72,73,76]
Orange [87,89,91]
Apricot [20]
Fruit senescence Apple [95,99]
Fruit storage Ponkan [103]
Chilling Tomato [40,52]
Peach [93]
Transport/
membrane carriers
Fruit ripening Tomato [35]
Grape berry [68]
Fruit senescence Apple [95]
Chilling Peach [93]
Energy Fruit ripening Tomato [24]
Grape berry [68]
Orange [8789]
Chilling Peach [93,94]
Metabolism Fruit ripening Grape berry
Orange
[68] [87]
Fruit senescence Apple [95]
Fruit storage Pear [101,102]
Chilling Peach [93]
Protein fate/synthesis Fruit ripening Tomato [25,35]
Grape berry [68]
Fruit storage Pear [101,102]
Chilling Peach [93,94]
Photosynthesis Fruit ripening Tomato [4,25,35]
Grape berry [72]
Chilling Tomato [40]
Secondary metabolism Fruit ripening Tomato [35]
Grape berry [76]
Orange [91]
Chilling Peach [93,94]
Cell structure/growth Chilling Peach [93,94]
Transcription Fruit ripening Grape berry [68]
Chilling Peach [94]
Defence Fruit ripening Tomato [24,35]
Grape berry [72]
Fruit storage Pear [101,102]
Chilling Tomato [52]
Peach [94]
Signal transduction/
cell signalling
Fruit ripening Tomato [24]
Grape berry [76]
Chilling Peach [93]
Lipid metabolism Fruit ripening Tomato [4,35]
Trafficking/
organelle biogenesis
Fruit ripening Tomato
Orange
[4,35] [87]
Redox status Fruit ripening Tomato [24]
Detoxification Fruit ripening Tomato [35]
Vitamin biosynthesis Fruit ripening Tomato [35]
DNA processing Fruit ripening Tomato [35]
Fruit storage and chilling have been included in the list, although
they could be considered as post-harvest conditions. Nevertheless,
under these situations, most fruits still follow the internal ripening
programme.
1239 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
differentially expressed have been widely documented, the
future interest will be possibly focussed in specific proteins
and their coding genes and their precise role in the physiology
of fruits.
The integration of new and reliable quantitative proteomic
approaches in the current and coming research will be also
focus for studies on fruit biology. The application of 2-DE-
based methods has revealed their limitation due to the
sensitivity and the reproducibility in the analysis of insoluble
and high/low molecular mass proteins. Thus, quantitative
proteomics has been applied in the past few years in order to
compute how complex biological processes undergo [101,102].
Alternative techniques are non-gel LCMS/MS-based shotgun
proteomics, which combined with dMS and SC is, at present,
providing a very promising field in the investigation of fruit
development [88].
Another aspect which will gain interest in the proteomics
of fruits is related to quality and life span of products.
Proteomics could be used as a reference to determine whether
fruits are appropriate and have good features for human
consumption. Works on this direction are appearing more and
more and they are focussed on how proteomes change after
harvest and under several storing conditions [95,103105].
Finally, the complementation of proteomics with other
omics approaches, such as transcriptomics and metabolo-
mics, is now being the best culture media for the scientific
knowledge of ripening to grow.
Acknowledgements
This work was supported by ERDF-cofinanced grant AGL2008-
00834 from the Ministry of Science and Innovation, Spain. The
authors apologise for the many colleagues' reports which have
not been included in this work.
R E F E R E N C E S
[1] Mauseth JD. Botany: an introduction to plant biology.
Third edition. Sudbury, Massachusetts: Jones and Bartlett
Publishers; 2003.
Fig. 3 Strategy to investigate proteomics of fruit ripening. Fruits at different ripening stages can be studied as whole fruits, but
also as separate components (flesh, seeds, and skin), or at subcellular level. Once proteins have been extracted, partially
purified, and characterised, they are subjected to proteomics tools. The confrontation of obtained results with databases
through powerful search engines will lead to the final identification of proteins, and the use of reliable approaches will provide
protein quantification which will be very useful for comparative proteomic analyses.
1240 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
[2] Fray RG, Grierson D. Molecular genetics of tomato fruit
ripening. Trends Genet 1993;9:43843.
[3] Schuch W, Bird CR, Ray J, Smith CJ, Watson CF, Morris PC,
et al. Control and manipulation of gene expression during
tomato fruit ripening. Plant Mol Biol 1989;13:30311.
[4] Barsan C, Sanchez-Bel P, Rombaldi C, Egea I, Rossignol M,
Kuntz M, et al. Characteristics of the tomato chromoplast
revealed by proteomic analysis. J Exp Bot 2010;61:241331.
[5] Kevany BM, Tieman DM, Taylor MG, Dal Cin V, Klee HK.
Ethylene receptor degradation controls the timing of
ripening in tomato fruit. Plant J 2007;51:45867.
[6] Schuch W, Bird CR, Ray J, Smith CJS. Control and
manipulation of gene expression during tomato fruit
ripening. Plant Mol Biol 1989;13:30311.
[7] Fray RG, Grierson D. Molecular genetics of tomato fruit
ripening. Trends Genet 1993;9:43843.
[8] Fedeli C, Negri AS, Prinsi B, Morgutti S, Negrini N, Cocucci M,
et al. Peach fruit ripening: a proteomic comparative analysis
of two cultivars with different flesh firmness characteristics
at the transition from the pre-climacteric to the climacteric
stage. Crop, Animal, Food and Environmental
Biotechnologies. Abstract Book; 2008.
http://www.cnbx.unipg.it/viewabstract.php?id=19.
[9] Fos M, Nuez F. Molecular expression of genes involved in
parthenocarpic fruit set in tomato. Physiol Plant 1996;98:
16571.
[10] Ferrndiz C, Pelaz S, Yanofsky MF. Control of carpel and fruit
development in Arabidopsis. Annu Rev Biochem 1999;68:
32154.
[11] Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL,
Yanofsky MF. SHATTERPROOF MADS-box genes control seed
dispersal in Arabidopsis. Nature 2000;404:76670.
[12] Liljegren SJ, Roeder AHK, Kempin SA, Gremski K, Ostergaard
L, Guimil S, et al. Control of fruit patterning in Arabidopsis by
indehiscent. Cell 2004;116:84353.
[13] Seymour G, Poole M, Manning K, King GJ. Genetics and
epigenetics of fruit development and ripening. Curr Opin
Plant Biol 2008;11:5863.
[14] Fei ZJ, Tang X, Alba RM, White JA, Ronning CM, Martin GB,
et al. Comprehensive EST analysis of tomato and
comparative genomics of fruit ripening. Plant J 2004;40:
4759.
[15] Alba R, Payton P, Fei ZJ, McQuinn R, Debbie P, Martin GB, et al.
Transcriptome and selected metabolite analyses reveal
multiple points of ethylene control during tomato fruit
development. Plant Cell 2005;17:295465.
[16] Lemaire-Chamley M, Petit J, Garca V, Just D, Baldet P,
Germain V, et al. Changes in transcriptional profiles are
associated with early fruit tissue specialization in tomato.
Plant Physiol 2005;139:75069.
[17] Aharoni A, O'Connell AP. Gene expression analysis of
strawberry achene and receptacle maturation using DNA
microarrays. J Exp Bot 2002;53:207387.
[18] Hennig L, Gruissem W, Grossniklaus U, Kohler C.
Transcriptional programs of early reproductive stages in
Arabidopsis. Plant Physiol 2004;135:176575.
[19] da Silva FG, Iandolino A, Al-Kayal F, Bohlmann MC,
Cushman MA, Lim H, et al. Characterizing the grape
transcriptome: analysis of expressed sequence tags from
multiple Vitis species and development of a compendium of
gene expression during berry
development. Plant Physiol 2005;139:57497.
[20] Grimplet J, Romieu C, Audergon JM, Marty I, Albagnac G,
Lambert P, et al. Transcriptomic study of apricot fruit
(Prunus armenica) ripening among 13,006 expressed sequence
tags. Physiol Plant 2005;125:28192.
[21] Moyle R, Fairbairn DJ, Ripi J, Crowe M, Botella JR. Developing
pineapple fruit has a small transcriptome dominated by
metallothionein. J Exp Bot 2005;56:10112.
[22] Terrier N, Glissant D, Grimplet J, Barrieu F, Abbal P, Couture
C, et al. Isogene specific oligo arrays reveal multifaceted
changes in gene expression during grape berry (Vitis vinifera
L.) development. Planta 2005;222:83247.
[23] Sarry JE, Sommerer N, Sauvage FX, Bergoin A, Rossignol M,
Albagnac G, et al. Grape berry biochemistry revisited upon
proteomic analysis of the mesocarp. Proteomics 2004;4:
20115.
[24] Rocco M, D'Ambrosio C, Arena S, Faurobert M, Scaloni A,
Marra M. Proteomic analysis of tomato fruits from two
ecotypes during ripening. Proteomics 2006;6:378191.
[25] Kok EJ, Lehesranta SJ, van Dijk JP, Helsdingen JR, Dijksma
WTP, Van Hoef AMA, et al. Changes in gene and protein
expression during tomato ripening. Consequences for the
safety. Food Sci Technol Int 2008;14:50318.
[26] Rose JKC, Saladi M. Proteomic analysis and fruit
ripeningISHS Acta Horticulturae, 682. Abstract Book; 2008.
[27] Wasinger VC, Cordwell SJ, Cerpapoljak A, Yan JX, Gooley AA,
Wilkins MR, et al. Progress with gene-product mapping of
the Mollicutes: Mycoplasma genitalium. Electrophoresis
1995;16:10904.
[28] Newton RP, Brenton AG, Smith CJ, Dudley D. Plant proteome
analysis by mass spectrometry: principles, problems, pitfalls
and recent developments. Phytochemistry 2004;65:144985.
[29] Jansen RC, Nap JP, Mylnarova L. Errors in genomics and
proteomics. Nat Biotechnol 2002;20:114655.
[30] Liebler DC. Introduction to proteomics: tools for the new
biology. Totowa, NJ: Humana Press; 2002.
[31] Black DL. Mechanisms of alternative pre-messenger RNA
splicing. Annu Rev Biochem 2003;72:291336.
[32] Mann M, Jensen ON. Proteomic analysis of post-translational
modifications. Nat Biotechnol 2003;21:25561.
[33] Mateos, M., Jimnez, A., Romn, P., Romojaro, F., Bacarizo, S.,
van Doorn et al., Antioxidant systems as markers of the
sweet pepper cultivar, fruit ripening stage, and
environmental conditions. Unpublished results.
[34] Mateos RM, Bonilla-Valverde D, del Ro LA, Palma JM, Corpas
FJ. NADP-dehydrogenases from pepper fruits: effect of
maturation. Physiol Plant 2009;135:1309.
[35] Faurobert M, Mihr C, Bertin N, Pawlowski T. Major proteome
variations associated with cherry tomato pericarp
development and ripening. Plant Physiol 2007;143:132746.
[36] Giovannoni J. Genetic regulation of fruit development and
ripening. Plant Cell 2004;16:17080.
[37] Mueller LA, SolowTH, Taylor N, Skwarecki B, Buels R, Binns J,
et al. The SOL genomics network:a comparative resource for
Solanaceae biology and beyond. Plant Physiol 2005;138:
13107.
[38] Cnovas FM, Dumas-Gaudot E, Recorbet G, Jorrn J, MOck HP,
Rossignol M. Plant proteome analysis. Proteomics 2004;4:
28598.
[39] America AHP, Cordewener JHG, van Geffen MHA, Lommen A,
Vissers JPC, Bino RJ, et al. Alignment and statistical
difference analysis of complex peptide data sets generated
by multidimensional LCMS. Proteomics 2006;6:64153.
[40] Vega-Garca MO, Lpez-Espinoza G, Ontiveros JC,
Caro-Corrales JJ, Vargas FD, Lpez-Valenzuela JA. Changes in
protein expresin associated with chilling injury in tomato
fruits. J Am Soc Hortic Sci 2010;135:839.
[41] Lisitsyn N, Lisitsyn N, Wigler M. Cloning the differences
between two complex genomes. Science 1993;259:94651.
[42] Gallie DR. Proteinprotein interactions required during
translation. Plant Mol Biol 2002;50:94970.
[43] Kaufman RJ. Regulation of mRNA translation by protein
folding in the endoplasmic reticulum. Trends Biochem Sci
2004;29:1528.
[44] Metzdorff SB, Kok EJ, Knuthsen P, Pedersen J. Evaluation of a
non-targeted omic approach in the safety assessment of
genetically modified plants. Plant Biol 2006;8:66272.
1241 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
[45] Lawrence SD, Cline K, Moore GA. Chromoplast development
in ripening tomato fruit: identification of cDNAs for
chromoplast targeted proteins and characterization of a
cDNA encoding a plastid localized low-molecular-weight
heat shock protein. Plant Mol Biol 1997;33:48392.
[46] Lw D, Brndle K, Nover L, Forreiter C. Cytosolic
heat-stress proteins Hsp17.7 class I and Hsp17.3 class II of
tomato act as molecular chaperones in vivo. Planta 2000;211:
57582.
[47] Neta-Sharir I, Isaacson T, Lurie S, Weiss D. Dual role for
tomato heat shock protein 21: protecting photosystem II
from oxidative stress and promoting color changes during
fruit maturation. Plant Cell 2005;17:182938.
[48] Jimnez A, Gmez JM, Navarro E, Sevilla F. Changes in the
antioxidative systems in mitochondria during ripening of
pepper fruits. Plant Physiol Biochem 2002;40:51520.
[49] Andrews PK, Fahy DA, Foyer CH. Relationships between fruit
exocarp antioxidants in the tomato (Lycopersicon esculentum)
high pigment-1 mutant during development. Physiol Plant
2004;120:51928.
[50] Carrari F, Baxter C, Usadel B, Urbanczyk-Wochniak E, Zanor
MI, Nunes-Nesi A, et al. Integrated analysis of metabolite
and transcript levels reveals the metabolic shifts that
underlie tomato fruit development and highlight regulatory
aspects of metabolic network behaviour. Plant Physiol
2006;142:138096.
[51] Sun W, van Montagu M, Verbruggen N. Small heat shock
proteins and stress tolerance in plants. Biochim Biophys
Acta 2002;157:19.
[52] Stevens MA. Citrate and malate concentrations in tomato
fruits: genetic control and maturational effects. J Am Soc
Hortic Sci 1972;97:6558.
[53] Page D, Gouble B, Valot B, Bouchet JP, Callot C, Kretzschmar
A, et al. Protective proteins are differentially expressed in
tomato genotypes differing for their tolerance to
low-temperature storage. Planta 2010;232:483500.
[54] Speirs J, Lee E, Holt K, KimY-D, Steel Scott NS, Loveys B, et al.
Genetic manipulation of alcohol dehydrogenase levels in
ripening tomato fruit affects the balance of some flavour
aldehydes and alcohols. Plant Physiol 1998;117:104758.
[55] Antoln MC, Baigorri H, De Luis I, Aguirrezbal F, Geny L,
Broquedis M, et al. ABA during reproductive development in
non-irrigated grapevines (Vitis vinifera L. cv. Tempranillo).
Aust J Grape Wine Res 2003;9:16976.
[56] Geny L, Deytieux C, Donche B. Importance of hormonal
profile on the onset of ripening in grape berries of Vitis
vinifera L. Acta Hort 2004;682:99105.
[57] Okamoto G, Kuwamura T, Hirano K. Effects of water deficit
stress on leaf and berry ABA and berry ripening in
Chardonnay grapevines (Vitis vinifera). Vitis 2004;43:157.
[58] Kanellis AK, Roubelakis-Angelakis KA. In: Seymour G, Taylor
J, Tucker G, editors. Biochemistry of fruit ripening. London:
Chapman and Hall; 1993. p. 189234.
[59] Coombe BG, McCarthy MG. Dynamics of grape berry growth
and physiology of ripening. Aust J Grape Wine Res 2000;6:
1315.
[60] Castro AJ, Carapito C, Zorn N, Magn C, Leize E, Van
Dorsselaer A, et al. Proteomic analysis of grapevine
(Vitis vinifera L.) tissues subjected to herbicide stress. J Exp
Bot 2005;56:278395.
[61] Vincent D, Wheatley MD, Cramer GR. Optimization of
protein extraction and solubilization for mature grape berry
clusters. Electrophoresis 2006;27:185365.
[62] Jellouli N, Ben Jouira H, Skouri H, Ghorbel A, Gourgouri A,
Mliki A. Proteomic analysis of Tunisian grapevine
cultivar Razegui under salt stress. J Plant Physiol 2008;165:
47181.
[63] Sauvage FX, Pradal M, Chatelet P, Tesniere C. Proteome
changes in leaves from grapevine (Vitis vinifera L.)
transformed for alcohol dehydrogenase activity. J Agric Food
Chem 2007;55:2597-03.
[64] Marsoni M, Bracale M, Espen L, Prinsi B, Negri AS, Vannini C.
Proteomic analysis of somatic embryogenesis in Vitis vinifera.
Plant Cell Rep 2008;27:34756.
[65] Tesnires C, Robin JP. Two-dimensional electrophoresis of
the total polypeptides in ripe red grape berries.
Electrophoresis 1992;13:936.
[66] Wang W, Bianchi L, Scali M, Liu L, Bini L, Cresti M. Proteomic
analysis of -1,3-glucanase in grape berry tissues. Acta
Physiol Plant 2009;31:597604.
[67] Negri AS, Prinsi B, Scienza A, Morgutti S, Cocucci M, Espen L.
Analysis of grape berry cell wall proteome: a comparative
evaluation of extraction methods. J Plant Physiol 2008;165:
137989.
[68] Zhang JW, Ma HQ, Feng JD, Zheng L, Wang Z, Chen SW.
Grape berry plasma membrane proteome analysis and its
differential expression during ripening. J Exp Bot 2008;59:
297990.
[69] Sarry JE, Sommerer N, Sauvage FX, Bergoin A, Rossignol M,
Albagnac G, et al. Grape berry biochemistry revisited upon
proteomic analysis of the mesocarp. Proteomics 2004;4:20115.
[70] Giribaldi M, Perugini I, Sauvage FX, Schubert A. Optimization
of protein extraction and solubilization for mature grape
berry clusters. Proteomics 2007;7:315470.
[71] Lcker J, Laszczak M, Smith D, Lund ST. Generation of a
predicted protein database from EST data and application to
iTRAQ analyses in grape (Vitis vinifera cv. Cabernet
Sauvignon) berries at ripening initiation. BMC Genomics
2009;10:50.
[72] Deytieux C, Geny L, Lapaillerie D, Claverol S, Bonneu M,
Doneche B. Proteome analysis of grape skins during
ripening. J Exp Bot 2007;58:185162.
[73] Negri AS, Prinsi B, Rossoni M, Failla O, Scienza A, Corucci M,
et al. Proteome changes in the skin of the grape cultivar
Barbera among different stages of ripening. BMC Genomics
2008;9:378.
[74] Deytieux C, Geny L, Donche B. Relation between hormonal
balance and polygalacturonase activity in grape berry. Acta
Hort 2004;682:16370.
[75] Boss PK, Davies C, Robinson SP. Analysis of the expression of
anthocyanin pathway genes in developing Vitis vinifera L. cv.
Shiraz grape berries and the implication for pathway
regulation. Plant Physiol 1996;111:105966.
[76] Giribaldi M, Gny L, Delrot S, Schubert A. Proteomic analysis
of the effects of ABA treatments on ripening Vitis vinifera
berries. J Exp Bot 2010;61:244758.
[77] Waters DLE, Holton TA, Ablett EM, Lee LS, Henry RJ.
cDNA microarray analysis of the developing grape
(Vitis vinifera cv. Shiraz) berry skin. Funct Integr Genomics
2005;5:4058.
[78] Davies C, Robinson SP. Differential screening indicates a
dramatic change in mRNA profiles during grape berry
ripening. Cloning and characterization of cDNAs encoding
putative cell wall and stress response proteins. Plant Physiol
2000;122:80312.
[79] Chivasa S, Ndimba BK, Simon WJ, Robertson D, Xu XL, Knox
JP, et al. Proteomic analysis of the Arabidopsis thaliana cell
wall. Electrophoresis 2002;13:175465.
[80] Bayer EM, Bottrill AR, Walshaw J, Vigouroux M, Naldrett MJ,
Thomas CL, et al. Arabidopsis cell wall proteome defined
using multidimensional protein identification technology.
Proteomics 2006;6:30111.
[81] Watson BS, Lei Z, Dixon RA, Summer LW. Proteomics of
Medicago sativa cell walls. Phytochemistry 2004;65:170920.
[82] Zhu J, Chen S, Alvarez S, Asirvatham VS. Cell wall proteome
in the maize primary root elongation zone. I. Extraction and
identification of water-soluble and lightly ionically bound
proteins. Plant Physiol 2006;140:31125.
1242 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3
[83] Ephritikhine G, Ferro M, Rolland N. Plant membrane
proteomics. Plant Physiol Biochem 2004;42:94362.
[84] Komatsu S, Konishi H, Hashimoto M. The proteomics of
plant cell membranes. J Exp Bot 2007;58:10312.
[85] Giribaldi M, Giuffrida MG. Heard it through the grapevine:
proteomic perspective on grape and wine. J Proteomics
2010;73:164755.
[86] Katz E, Martinez Lagunes P, Riov J. Molecular and
physiological evidence suggests the existence of a system
II-like pathway of ethylene production in non-climacteric
Citrus fruit. Planta 2004;219:24352.
[87] Kazt E, Fon M, Lee YJ, Phinney BS, Sadka A, Blumwald E.
The citrus fruit proteome: insights into the citrus fruit
metabolism. Planta 2007;226:9891005.
[88] Katz E, Fon M, Eigenheer RA, Phinney BS, Fass JN, Lin DW,
et al. A label-free differential quantitative mass
spectrometry method for the characterization and
identification of protein changes during citrus fruit
development. Proteome Sci 2010;8:68.
[89] Pan ZY, Liu Q, Yun Z, Guan R, Zheng WF, Xu Q, et al.
Comparative proteomics of a lycopene-accumulating
mutant reveals the important role of oxidative stress on
carotenogenesis in sweet orange (Citrus sinensis [L.] osbeck).
Proteomics 2009;9:545570.
[90] Smirnoff N. Ascorbate biosynthesis and function in
photoprotection. Philos Trans R Soc Lond B Biol Sci 2000;355:
145564.
[91] Muccilli V, Licciardello C, Fontaninic D, Russo MP, Cunsola V,
Saletti R, et al. Proteome analysis of Citrus sinensis L. (Osbeck)
flesh at ripening time. J Proteomics 2009;73:13452.
[92] Pignataro V, Canton C, Spadafora A, Mazzuca S. Proteome
from lemon fruit favedo reveals that this tissue produces
high amounts of the Cit s1 germin-like isoforms. J Agric Food
Chem 2010;58:723944.
[93] Nilo R, Saffie C, Lilley K, Baeza-Yates R, Cambiazo V,
Campos-Vargas R, et al. Proteomic analysis of peach fruit
mesocarp softening and chilling injury using difference gel
electrophoresis (DIGE). BMC Genomics 2010;11:43.
[94] Zhang CF, Ding ZS, Xu XB, Wang Q, Qin GZ, Tian SP. Crucial
roles of membrane stability and its related proteins in the
tolerance of peach fruit to chilling injury. Amino Acids
2010;39:18194.
[95] Qin G, Meng X, Wang Q, Tian S. Oxidative damage of
mitochondrial proteins contributes to fruit senescence: a
redox proteomics analysis. J Prot Res 2009;8:244962.
[96] Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG,
et al. Determination of carbonyl content in oxidatively
modified proteins. Methods Enzymol 1990;186:46478.
[97] Romero-Puertas MC, Mccarthy I, Gmez M, Sandalio LM,
Corpas FJ, del Ro LA, et al. Reactive oxygen species-mediated
enzymatic systems involved in the oxidative action of
2,4-dichlorophenoxyacetic acid. Plant Cell Environ 2004;27:
113548.
[98] Guarino C, Arena S, De Simona L, D'Ambrosio C, Santoro S,
Rocco M, et al. Proteomic analysis of the major soluble
components in Annurca apple flesh. Mol Nutr Food Res
2007;51:25562.
[99] Qin G, Wang Q, Liu J, Li B, Tian S. Proteomic analysis of
changes in mitochondrial protein expression during fruit
senescence. Proteomics 2009;9:424153.
[100] Palma JM, Barroso JB, Campos MJ, Ruz C, Bonilla-Valverde D,
del Ro LA, et al. Anlisis funcional del proteoma de
peroxisomas vegetales. IX Reunin de Biologa Molecular de
Plantas; 2008. Abstract # P82.
[101] Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B.
Quantitative mass spectrometry in proteomics: a critical
review. Anal Biochem 2007;389:101731.
[102] Schulze WX, Usadel B. Quantitation in
mass-spectrometry-based proteomics. Annu Rev Plant Biol
2010;61:491516.
[103] Pedreschi R, Hertog M, Robben J, Lilley KS, Karp NA,
Baggerman G, et al. Gel-based proteomics approach to the
study of metabolic changes in pear tissue during storage.
J Agric Food Chem 2009;57:69977004.
[104] Pedreschi R, Hertog M, Robben J, Nobenb JP, Nicola B.
Physiological implications of controlled atmosphere storage
of Conference pears (Pyrus communis L.): a proteomic
approach. Postharv Biol Technol 2008;50:1106.
[105] Yun Z, Li W, Pan Z, Xu J, Cheng Y, Deng X. Comparative
proteomics analysis of differentially accumulated proteins
in juice sacs of ponkan (Citrus reticulata) fruit during
postharvest cold storage. Postharvest Biol Technol 2010;56:
189201.
1243 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 3 0 1 2 4 3