Genomics

Review
Proteomics applied on plant abiotic stresses: Role of heat shock

proteins (HSP)
Anna Maria Timperio, Maria Giulia Egidi, Lello Zolla
Department of Environmental Sciences University of Tuscia, Largo dell'Universit snc, 01100 Viterbo, Italy
A R T I C L E D A T A A B S T R A C T
Article history:
Received 13 May 2008
Accepted 15 July 2008
The most crucial function of plant cell is to respond against stress induced for self-defence.
This defence is brought about by alteration in the pattern of gene expression: qualitative and
quantitative changes in proteins are the result, leading to modulation of certain metabolic
and defensive pathways. Abiotic stresses usually cause protein dysfunction. They have an
ability to alter the levels of a number of proteins which may be soluble or structural in
nature. Nowadays, in higher plants high-throughput protein identification has been made
possible along with improved protein extraction, purification protocols and the
development of genomic sequence databases for peptide mass matches. Thus, recent
proteome analysis performed in the vegetal Kingdom has provided new dimensions to
assess the changes in protein types and their expression levels under abiotic stress. As
reported in this review, specific and novel proteins, proteinprotein interactions and post-
translational modifications have been identified, which play a role in signal transduction,
anti-oxidative defence, anti-freezing, heat shock, metal binding etc. However, beside
specific proteins production, plants respond to various stresses in a similar manner by
producing heat shock proteins (HSPs), indicating a similarity in the plant's adaptive
mechanisms; in plants, more than in animals, HSPs protect cells against many stresses. A
relationship between ROS and HSP also seems to exist, corroborating the hypothesis that
during the course of evolution, plants were able to achieve a high degree of control over ROS
toxicity and are now using ROS as signalling molecules to induce HSPs.
2008 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
2. Proteomic tools for investigating abiotic stresses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
3. Proteomic application on abiotic stresses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
3.1. High temperature stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
3.2. Low temperature stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
3.3. Drought stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
3.4. Light stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
J O U R N A L O F P R O T E O M I C S 7 1 ( 2 0 0 8 ) 3 9 1 4 1 1
This work was supported by the Italian Ministry for University and research (MIUR-Pron 2006) and by MIPAF GENZOOT 2006 projects.
Corresponding author. Tel.: +39 0761 357100; fax: +39 0761 357179.
E-mail address: zolla@unitus.it (L. Zolla).
1874-3919/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2008.07.005
avai l abl e at www. sci encedi r ect . com
www. el sevi er. com/ l ocat e/ j pr ot
3.5. Heavy metals stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
3.6. Salt stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
3.7. Ozone stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
4. Abiotic stresses and heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
5. Concluding remarks and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
1. Introduction
Maintaining proteins in their functional conformations and
preventing the aggregation of non native proteins are
particularly important for cell survival under stress. There is
widespread interest in the cellular mechanisms utilized by an
organism to cope with a disruption in homeostasis. Much
evidence has demonstrated that mammalian species have
developed different ways to deal with stress if compared to
plants. Examples at the cellular level include temporary
modifications in gene expression to survive changing envir-
onments, as well as altering cellular structure and function to
deal with more permanent adverse conditions. Plants, as
sessile organisms, rely on proteomic plasticity to remodel
themselves during periods of developmental change, to
endure varying environmental conditions, and to respond to
biotic and abiotic stresses. The latter are the primary cause of
crop loss worldwide, reducing average yields for most major
crop plants by more than 50% [1]. Some abiotic stresses, such
as drought, salinity, extreme temperatures, chemical toxicity
and oxidative stress are serious threats to agriculture and
result in the deterioration of the environment. Abiotic stresses
usually cause protein dysfunction. Different families of
proteins are known to be associated with a plant's response
to stresses by being newly synthesized, accumulating or
decreasing. Among other things, these proteins are involved
in signalling, translation, host-defence mechanisms, carbohy-
drate metabolismand amino acid metabolism. Nowit is a well
known fact that proteins mediate these features by playing a
role in directing the genome and ultimately physical features
(such as in xerophytes) or encountering stressors directly
(such as antioxidant enzymes and chaperonins) or indirectly
(such as a key enzyme in osmolyte synthesis). Thus, elucidat-
ing the various mechanisms of plant response to stress and
their roles in acquired stress tolerance is of great practical and
basic importance [2].
Unfortunately, genome sequence information alone is
insufficient to reveal the facts concerning gene function,
developmental/regulatory biology, andthe biochemical kinetics
of plants toadapt under stresses andconsequentlytodetermine
the exact responsive mechanism. To investigate these facts,
more comprehensive approaches that include quantitative and
qualitative analyses of gene expression products are necessary
at the transcriptome, proteome, and metabolome levels.
For example, environmental stresses that result in cellular
dehydration, such as freezing, salt and water stress, often lead
to similar changes in plant gene expression and metabolism
[37], and there exists cross-talk in their signalling pathways
[8,9], but it is not clear if similar over- or under expressed
proteins are also detectable. On the other hand, through
transcriptome analysis, mRNA and protein levels cannot be
correlated due to inability of total mRNA to translate into
protein [10,11]; protein levels are not directly correlated with
the number of transcripts in the cell, and post translational
modification is not visible by examination of transcriptome,
making proteomics the study of the real players in organisms.
Moreover, the proteome is not a static entity, being it affected
by multiple modifications such as cell cycle, changes of
external conditions, kind of tissue examined, and particular
physiological states. For these reasons, proteomics is becom-
ing a powerful tool to analyse biochemical pathways and the
complex response of plants to environmental stimuli. In
particular, comparative proteomic investigations of plants
before and after specific or interactive stresses allow us to
obtain information on how defensive mechanisms are
adopted from plants. Proteomics also provides an essential
link between the transcriptome and metabolome [3,12],
complementing genomics research. Only by grouping all this
information together is it possible to achieve a comprehensive
and exhaustive analysis of the strategies induced by the cells
as response to stress.
2. Proteomic tools for investigating abiotic
stresses
Although more advanced proteomic technologies are being
developed on model systems including human, yeast and
bacterial proteomes, they may not be directly applied to plant
tissues. Sample preparation is the most critical step for
proteomic analysis specially for plant cells due to the rigidity
of plant cell walls, and to the presence of large amounts of
secondary compounds such as polyphenols, polysaccharides,
accumulated in the central vacuole, which can lead to protein
precipitationwhentissues are disrupted[13]. For these reasons
vegetal cells are considered recalcitrant tissues for proteomic
analysis and it is often difficult to obtain high quality protein
suitable for 2-DE analysis from them. Fruit analysis requires
the most challenging proteomic approach, because of the low
protein content [14] and the presence of a great amount of
interfering substances such as pigments, carbohydrates, salts,
polyphenols, polysaccharides and starch [15]. Finally, some
tissues contain highly abundant proteins, e.g. RuBisCO in
leaves and storage proteins in seeds, which can dominate in
proteomic analysis. Because of heterogeneity of protein
abundance, molecular weight, charge, hydrophobicity, PTM,
interaction with other molecules, an ideal protocol, for total
protein extraction is difficult to obtain. Many protocols for the
preparation of protein extracts from plant tissue [1618] and
some from fruit tissues [19,20] have been reported, but,
392 J O U R N A L O F P R O T E O M I C S 7 1 ( 2 0 0 8 ) 3 9 1 4 1 1
because plants can be very different from one another, it is
not possible to use the same extraction protocol, and it is
necessary to modify extracting conditions for different
species. The ability to extract large numbers of proteins in a
single sample necessitates new technologies for the rapid
separation and identification of proteins of interest.
However difficult the task is, two main protein extraction
procedures are useful for vegetable samples: TCA/acetone and
a phenol based procedure. In the case of TCA/acetone,
typically, plant tissues are ground to a powder in liquid N
2
followed by acetone or acetone plus 510% trichloroacetic acid
(TCA) precipitation of proteins [21]. The proteins are then
solubilised in a buffer. To this regard, while the precipitation
step is critical for removing interfering compounds, the
composition the extraction buffer is also important to
solubilize the maximum number of proteins without interfer-
ing with the subsequent IEF procedure. It has been reported
that the high SDS binding capacity of proteins, particularly at
an elevated temperature of 95 C, improves the solubilization
of membrane proteins and the recovery of soluble proteins
[22]. Hot SDS can also inhibit protease activity during cell
disruption and protein extraction, thus avoiding potential
artefacts [23]. However, SDS has an anionic charge that may
cause proteins to precipitate in IEF gels [24]. Heated SDS was
used to increase the solubility of proteins in the initial
extraction procedure and then the negative effects of SDS on
IEF were reduced by precipitating the proteins with a TCA/
acetone [20]. According to D'Amici et al. [25], instead of hot
SDS, thylakoid membranes were solubilized and washed with
washing buffer. The combination of SDS extraction with TCA/
acetone precipitation enhanced the overall quantity and size
range of proteins present in the final sample and removed
other interfering compounds such as polyphenolics and
polysaccharides. For protocols using phenol, it has been
reported that it is suitable for the extraction of low concentra-
tions of protein in vegetative plant tissues rich in contaminat-
ing components that interfere with electrophoresis [26].
Phenol extraction of protein from tomato and banana fruit
was found to be comparable to protein precipitation with
acetone and it demonstrated that more protein spots and
glycoproteins may be present in phenol extracts [27].
A study comparing proteins precipitate found that both
procedures were effective in extracting and purifying a large
number of proteins, but the phenol extraction was slightly
better in removing interfering compounds fromdifficult tissues
suchas ripe fruit [27]. For recalcitrant plant tissues, a systematic
comparison [28] showed that a phenol-based protein extraction
protocol should be used as a standard procedure, while a SDS
andacetone/TCAprotocol withor without a clean-upprocedure
may be used as an effective alternative protocol [29].
Once proteins are extracted and purified, they need to be
re-hydrated in a buffer that keeps all the proteins in solution
thorough the first and second dimension electrophoresis
procedures. A buffer published by Khoudoli and colleagues
[30] improved protein quality and reduced streaking in 2-DE
gels. Additional modification of re-hydration buffers may
further improve the solubility of membrane and total cell
content proteins [31,32].
Fig. 1 summarizes up the main conditions generally used
for the preparation of protein extracts from plant tissues that
are suitable for the successive analysis. However, overall, this
study demonstrated the complexity of protein extraction
from vegetable tissues and how the protein solubilization is
critical to the production of gels with discrete protein spots
that are suitable for MS analysis. In our experience, although
phenol extractionis considered time-consuming, it generates
high-quality protein extract from a large variety of plant
species.
Regarding the separation and identification of proteins, it is
now possible through techniques such as bidimensional
electrophoresis (2-DE) or liquid chromatography coupled with
tandem mass spectrometry (LC-MS/MS) [33,34] (see Fig. 2). 2-DE
is used for separating and displaying the components of large
protein complexes. It shows as main advantages the simplicity,
reproducibility, awidesizerange (10 kDato500 kDa) andthe fact
that both moderately hydrophobic and very acidic-basic pro-
teins can be isolated and visualized. It is important to point out
that the limitations of gel based techniques such as low
abundance proteins, limitedpI range andabsence of membrane
proteins may limit the broad mapping of proteins in samples.
Moreover, pertaining to limitations of 2DE-gel, enormous
manual editing is required to achieve precision specially for
comparativeproteomics. The HPLCtechniques, giventheir wide
versatility, relative ease of use, reproducibility, fast analysis
time and high resolution, may be considered the most valuable
tools for characterization of virtually any hydrophobic protein
and in the case of isoform proteins, where pI is very close.
Furthermore, LC-MS/MS allows the protein identification by
comparisonof the intact mass measurement of the proteinwith
that expected from the DNA sequence. Intact mass measure-
ment performed by RP-HPLC-ESI-MS is less expensive and less
technically demanding than SDS-PAGE electrophoresis, where
it can be done by the time consuming antibody titration. RP-
HPLC-ESI-MS resulted successful for thylakoid component
separation and mitochondrial membrane [35]. In addition to 2-
DE, another technique, referred to as blue-native PAGE (BN-
PAGE) [36], has been developed to study hydrophobic and/or
membrane proteins. BN/CN-PAGE allows a simple global over-
view of stable complexes and is somewhat equivalent to a 2-DE
but does not introduce all the biases linked to 2-DE (e.g., bias
against high MW proteins).The BN/CN-PAGE method has been
used to investigate complexes in the tomato respiratory chain
[37], and was recently extended to both mixtures of membrane
and membrane-associated proteins [38] and to soluble com-
plexes [39] inplastids. Recently, a new3Dnative electrophoretic
protocol has been proposed for an exhaustive separation and
identificationof thylakoidmembraneproteins [25]. It isbasedon
a native liquid phase isolectrofucusing (N-LP-IEF) of protein
complexes in the first dimension, followed by a blue native
electrophoresis (BN-PAGE) inthe seconddimension, whereboth
the pI and the molecular masses of protein complexes (2-DE N-
LP-IEF-BN) were used for their separation in native form. This
procedure has improved resolution by reducing the limitation
and difficulty in resolving membrane proteins.
Regarding protein identification it can be done only after
tryptic digestion of spots cut out from 2DE maps, as well as
after tryptic digestion of the protein(s) contained into a HPLC
peak, and subsequent mass spectrometry analysis. This can
be performed either by PMF (peptide mass finger printing) or
by MS/MS analysis, although nowadays, to enhance proteome
resolution it is often necessary to use multiple techniques that
provide complementary results (see Fig. 2).
A PMF database search is usually employed following
MALDI TOF mass analysis. The premise of peptide mass finger
printing is that every unique protein will have a unique set of
peptides and hence unique peptide masses. This technique
does well with 2DE gel spots where the protein purity is high.
PMF protein identification can run into difficulties with
complex mixtures of proteins. Low level identification also
becomes difficult due to common place contamination by
keratin, and is not suitable for those organisms with little
genomic information [40,41]. Moreover, since Peptide Mass
Fingerprinting is performed by matching their constituent
fragment masses (peptide masses) to the theoretical peptide
masses generated from a protein or DNA database, it can only
be used for species where the genome is known. Unfortu-
nately, genetic and genomic information for crops and
vegetables is still limited despite the complement of genome
of Arabidopsis, rice and Poplar is rapidly advancing.
Regarding the MALDI/TOF analysis, a limitation is that the
measured signal intensity is not linear with the quantity of
introduced sample, and therefore the method is not applicable
for determining concentrations of peptides/proteins in a
mixture. Strengths of MALDI-TOF, instead, are its high sensi-
tivity, broad mass range, relative tolerance to salts/buffers and
suitability for the analysis of relatively complex mixtures.
HPLC and LC coupled to ESI-MS/MS presently dominate
the field of protein identification by tandem mass spectro-
metry and database searching. ESI-MS/MS offers definitive
information about protein structure; in some cases amino acid
sequence and specific chemical modifications can be unequi-
vocally deduced from the pattern of product ions generated. It
is indicated for screening and structural characterization of
complex mixtures of peptides, giving more reliable results for
protein identification. MS/MS methods can be employed to
identify unknown proteins and posttranslational modifica-
tions. Additionally, since the technique uses soft ionization, it
is possible to observe labile species, e.g., nitrosothiols, protein
multimers and even biologically native non-covalent interac-
tions which would be destroyed and therefore undetected in a
MALDI-TOF MS experiment, such as sites of non-covalent
association with small molecules or other proteins. Limitations
of ESI-MS/MS are the need for relatively high purity protein
samples and a poor tolerance for electrolytes and detergents.
However, in plant proteomics a big challenge is errors in
protein identification that are associated with i) the low
percentage of annotated genes (20% in Arabidopsis) whose
functions have been determined experimentally [42]; ii) the
quality of registered genomic sequences, which significantly
impacts protein identifications that rely on database searching
[43]. In addition, current plant genome annotation has intro-
duced a large number of errors in assignments of intron/exon
boundaries and prediction of N- and C-termini, which may be
partially solved by future improvements in bioinformatics tools
for gene discovery and annotation. For species without regis-
tered database, homology searching may yield putative or
promiscuous identification which makes it very difficult to
infer functions. There is no doubt that the development of
vegetable genomics will lead to a greater impact on proteomics
and its application to vegetable research.
3. Proteomic application on abiotic stresses
As assessedabove, there is widespreadinterest inknowing the
cellular mechanisms utilized by an organism to cope with a
Fig. 1 Conditions for the preparation of protein extracts from plant tissues that are suitable for 2-DE.
Fig. 2 Different strategies of separation and identification of proteins and peptides. 3
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disruption in homeostasis and to check if different stresses
may induce similar defence mechanisms. One reason for this
interest, and the complexity associated with the topic, is
evidence demonstrating that mammalian species have devel-
oped many different ways to deal with stress. In plants a direct
result of stress-induced cellular changes is the enhanced
accumulationof toxic compounds incells that include reactive
oxygen species (ROS); the latter mediated by NADPH oxidases
[44]. Upon several stress novel protein, protein-protein inter-
action and post-traslation modification have been also identi-
fied. In the last decade, technical improvements have allowed
comparative proteomic investigations of plants under stress
which in turn have allowed us to analyse biochemical path-
ways and the complex response of plants to environmental
stimuli, revealing interesting modulation of protein candidate
to further investigations [45]. Recent update on plant proteo-
mics has been reported in other reviews [4648], therefore, in
this review, besides reporting specific proteins inducedby each
stress, particular emphasis will be placed on the HSP family,
yielded under different stresses by using genomic and
proteomic approaches. It will be also stressed how plant
adaptation consists of a high presence of heat shock proteins
inside the cell.
3.1. High temperature stress
Plants must face temperature stress, at present increasing year
by year, because of greenhouse gas emission. Worldwide,
extensive agricultural losses are attributed to heat, often in
combination with drought or other stresses. Rice grain yields
decline by 10%for each 1 C increase in minimumtemperature,
during the growing-season, so the gravity of this problem is
easily understood[49]. Heat stress disturbs cellular homeostasis
and can lead to severe retardation in growth and development,
andevendeath. Temperature stress canhave also a devastating
effect on plant metabolism, acting first on protein complexes,
being the quaternary structure the first folding lost during
heating; one of the side effects is the uncoupling of pathways,
most of which involve electron transfer, resulting in transition
of electron in high-energy state, with concomitant formation of
ROS [50]. Heat stress causes oxidative damage that manifests in
lipid peroxidation [5153]. The detoxification enzymes such as
glutathione S-transferase, catalases, superoxide dismutase and
ascorbate peroxidases (APX) are involved in protection from
reactive singlet oxygen species and finally proteins involved in
regulatory functions and in signal transduction, including
various protein kinases and transcriptional factors have a
broader role in governing stress responses. Moreover, several
studies have indicated that HSFs are involved in the sensing of
ROS. The works of Mittler and Zilinskas [54] and Storozhenko et
al. [55] have revealed the presence of a HSF-binding sequence at
the promoter region of the gene encoding the H
2
O
2
-scavenging
enzyme APX1.
3.1.1. Genomic approach
In terms of gene expression, the response of plant to heat
stress was also studied by analysis of Arabidopsis transcrip-
tome [5659]. Some genes appeared up-regulated. The pro-
ducts of these genes included Hsp100/ClpB (eukaryotic/
Escherichia coli nomenclature), Hsp90/HtpG, Hsp70/DnaK,
Hsp60/GroEL and small heat shock proteins (smHSPs), con-
firming proteomic studies. These HSPs are proposed to act as
molecular chaperones in protein quality control. Transgenic
plants expressing a dominant negative variant of (heat shock
factor) HSF21 showed suppressed expression of Zat12, a H
2
O
2
-
responsive zinc finger protein required for expression of APX1,
and APX1 [60]. Heat shock provokes a rapid reprogramming of
gene expression to favour translation of HSPs in which
translation factors could play a role. Gallie and coworkers [61]
analysed translation initiation factors in wheat heat-shocked
seedlings and during seed development.
3.1.2. Proteomic approach
When plants are exposed to high temperatures they synthe-
size both high molecular mass HSPs (from 60 to 110 kDa) and
small HSPs (from 15 to 45 kDa) [62,63]. Lee and coworkers [64]
with the same unbiased to investigate the responses of
abundant and low-abundant proteins of rice leaves upon
heat stress, performed 2-DE on PEG-fractionated supernatant
and pellet samples of rice leaf proteome. A total of 48
differentially expressed proteins in samples taken after 12 or
24 h of heat exposure compared to controls were revealed, 18
of which were HSP (HSP70, dnak-type molecular chaperone
BiP, HSP100, Cpn60, smHSPs).
Studies carried out by Polenta and colleagues [65] regarding
isolation and characterization of relevant HSPs from plant
tissues induced in tomato pericarp by thermal treatment,
highlighted the presence of two major (HSPC1, HSPC2) and two
minor (HSPC3 and HSPC4) class I smHSPs, identified and
characterized by using monospecific polyclonal antiserum
and MS/MS analysis of tryptic peptides. In this case the
authors claimed that smHSPs accumulation is an appropriate
parameter for monitoring treatment intensities and conse-
quently for preventing chilling injury.
3.1.3. Plant adaptation
Heat shock proteins are known to be induced not only in
response to short-term stress, but their production is a
necessary step in plant heat acclimation. It is important to
note, in fact, the early production of Hsp101 chaperone by
plant exposed to heat stress, but there are also many other
pathways involved in adaptation to higher temperatures. The
studies of expression made by Larkindale and Vierling [66]
demonstrated that a better acclimation occurs when plants
are gradually exposed to increasing temperatures, without
changing parameters suddenly. They also examined clusters
of genes whose expression increased during heat stress; in
particular, cluster 45 contains 18 HSPs (Hsp101, 14 small HSPs,
3 Hsp70s).
3.2. Low temperature stress
Low temperature has a great impact on plant productivity,
mostly because it alters significantly plant metabolism and
physiology [6769]. Cold acclimation is the term used to define
the increasing of freezing tolerance by plants exposed to low
non freezing temperatures [69,70], whereas the term freezing
stress is used to define a plant exposition to temperatures near
to 0 C which cause formation of both inter and intracellular ice
crystals.
3.2.1. Cold stress
3.2.1.1. Genomic approach. Different plant species have built
diverse molecular machineries to face cold stress [71]. It
involves distinct changes in gene expression, protein expres-
sion and metabolites [4,7276]. However, the mechanisms of
coping with low-temperature stress are complex and multi-
genic [70,77].
About 20 years ago, Guy [69] clarified the biochemical and
physiological bases of cold acclimation, demonstrating that
exposure to low temperatures in plants causes modification of
membrane lipid content, increases the level of soluble proteins
and of cryoprotectants molecules such as sugars and proline
[69]. Later on, the first evidence that changes ingene expression
occur during cold acclimation was provided [7882]. In fact,
within a few hours after exposure to low temperatures, several
genes are turned on, most of which have similar structure: COR
(cold-regulated), KIN (cold-induced), LTI (low temperature
induced), and also RD (responsive to dehydration). The last
one suggests a link between genes expressed during cold stress
and those during drought stress, clarifying the similar response
of plants facing different stresses.
3.2.1.2. Proteomic approach. Investigations on rice leaves
made by Yan and coworkers [83] led to the identification of
eighty-five differentially expressed proteins. Most of them
could not be associated to a particular functional class, but
had a significant homology with proteins from other species
involved in cellular signalling, RNA processing, translation,
protein processing, redox cascades, photosynthesis and
carbon nitrogen, sulphur cycles. In a proteomic study of pea
(P. sativum L.) mitochondria, 33 proteins showed either up- or
down regulation under different stress conditions, 20 of which
appeared to respond to low temperature (4 C for 36 h) [84]. In
leaves of poplar seedlings exposed to 4 C during 2 weeks, 26
proteins were identified using a 46 pH gradient that were
COR, of which 21 were over-expressed and five repressed [74].
In a study conducted by Hashimoto and Komatsu [85], a
proteomic approach was utilized to evaluate changes in rice
protein expression. After depletion of RuBisCO LSU (large
subunit) from leaf blades sample, it was possible to detect low
abundant cold stress-responsive proteins; one of them
(referred as LB-a) was identified by ESI-MS/MS analysis as
Hsp70 and appeared to decrease in cold stressed samples; this
was probably due to degradation of chloroplasts under these
conditions.
Under cold stress many authors [8689] conclude that an
accumulation of HSPs and chaperonins especially for the
HSP90, HSP70 and small HSPs have been found. Similar results
have also been obtained from cold stress studies using 2DE
where both HSPs (HSP90 and HSP70; 22 and 20 kDa), as well as
chaperonins (chaperonin 60 and 20), have been found to be
induced by low temperature [74,84,90,91].
3.2.2. Freezing stress
The most evident plant symptoms upon freezing stress are
growth impairment, lesions, chlorosis, wet leaves. The
mechanisms that plants use to survive sub-zero temperatures
have been subject to intense research for decades and recently
modern technologies have elucidated some aspects of the
molecular basis of plant frost tolerance. More extreme low
temperature conditions resulting in freezing injury also target
membranes. Freezing-induced dehydration destabilizes
plasma membranes resulting in the formation of inverted
hexagonal phase membrane structure [92]. To face freezing
temperatures, plants must modify enzymatic and membrane
composition, altering metabolism to synthesize cryoprotec-
tants such as polyols and sugars to lower freezing point in
tissues, fight desiccation and suppress metabolic rate.
3.2.2.1. Genomic approach. Most of the studies regarding
freezing stress are carried out on genetic investigations, being
microarrays used by numerous groups to identify cold-
inducible genes in Arabidopsis. Xin and coworkers [93]
demonstrated that an Arabidopsis gene, called ESK1 (Eskimo1),
has a great influence on freezing tolerance in Arabidopsis,
being a negative regulator of tolerance to low temperatures. In
fact, esk1-1 plants with loss-of-function mutations in ESK1
survive at temperatures lower than their corresponding wild
types. The present mutation turns on specific genes, among
which At5g43480 and At3g24520 were interestingly found,
both belonging to heat shock transcription factor family.
Induction of many, but not all, cold responsive genes is
mediated by CBF/DREB [80]. CBF/DREB transcription factors
belong to a small gene family in Arabidopsis consisting of three
sub-groups. Of the three, the group of CBF/DREB1 members was
specifically induced by cold. Microarray technology has enabled
the analysis of genome-wide gene expression [94]. By using a
larger microarray containing approximately 1300 full-length
cDNAs, Seki and colleagues [7] identified 19 cold inducible
cDNAs and Fowler and Thomashow [95] concluded that 4% of
the genome may be altered during low temperature exposure.
Manyof thegenes involvedincoldacclimationbelong toseveral
families; among them, there are some which encode AOE
(antioxidant enzymes) and shock proteins, that make the so
called UPR (unfolded protein response). GRP78 (glucose-related
protein) is a chaperone of the subfamily of Hsp70, involved in
protein stabilization, that shows an increase in expression
during freezing stress.
3.2.2.2. Proteomic approach. Using temperatures around
0 C, proteomic analyses are not allowed because of drastic
modification in the hydration of proteins [96]. In fact, when
temperature decreases, water molecules move from cell to
intercellular space by osmosis, causing water stress within the
cell. For the above reasons, studies have been carried out
between 3 or 5 C [97].
Several mini-reviews have been written using proteomics
analysis to study response to low temperature stress. The
mini-review by de la Fuente van Bentem and coworkers [98]
outlines an exciting new proteomics approach to study
phosphoprotein dynamics in response to stress situations
that is certain to reveal important new insights regarding
post-translational regulatory mechanisms of plants. The
mini-review by Kaplan and colleagues [99] focused on the
function of a specific gene, b-amylase, which has been linked
with cold stress responses in a variety of transcriptome
studies dealing with cold and temperature stress. Uemura
and colleagues [100] describe studies that employ lipid
profiling and proteomic approaches to understand the
dynamics of changes that occur during acclimation and
freezing. Reviews by Murata and Los [101], Ensminger and
coworkers [102] and Suzuki and Mittler [50] examine different
biochemical and physiological possibilities that could be
important in sensing and signalling cold stress or the
consequences of low temperatures on cellular processes.
The most important proteins involved in plant defence
from freezing damage are anti-freezing proteins (AFPs), which
have multiple hydrophilic ice-binding domains that absorb on
the surface of ice crystals and modify their growth; some of
them are homologous to the pathogenesis-related proteins,
avoiding both freezing and disease. These proteins have the
ability to decrease the temperature at which ice is formed by
inhibiting ice crystal growth. AFPs accumulate in the apoplast
of many winter cereals, and accumulation of AFPs has been
correlated with freezing tolerance in winter rye and wheat
[103]. Moreover, studies conducted by Lopez-Matas and
colleagues [88] analyzing the occurrence of smHSPs in
vegetative organs of the freezing tolerant Castanea sativa
demonstrated the constitutive expression of a smHSPs with
seasonal expression modulations, identified by MALDI spec-
trometry and internal peptide sequencing as CsHSP17.5, a
cytosolic class I smHSPs previously described in cotyledons.
These results were confirmed by cDNA cloning and reverse
transcription-PCR.
3.2.2.3. Plant adaptation. One strategy for freezing tolerance
is super-cooling or decreasing the temperature of ice forma-
tion. However, prevention of ice formation is usually the most
successful strategy at relatively high freezing temperatures. A
contrasting strategy is to allow extra-cellular ice formation
and tolerate the resulting cellular dehydration and ice masses.
In higher plants, low-temperature resistance is age-depen-
dent and tissue-specific, being roots most sensitive to low
temperatures. Moreover, plants differ significantly in their
ability to withstand low temperature [104]. The consequences
of exposure, in fact, vary depending on species, threshold of
temperature reached and duration of exposure. Some tender
species are damaged by exposure to chilling temperatures.
These plants, known as chilling-sensitive, are damaged when
temperatures decrease below 12 C. They include economic-
ally important species such as maize, rice and tomato.
Tropical and subtropical plants are always sensitive to chilling
and lack capacity for cold acclimation [105]. Chilling-tolerant
plants can endure chilling temperatures but are susceptible to
freezing. Lastly, freeze tolerant plants can withstand tem-
peratures below 0 C [106]. Temperate plants are capable of
developing freezing tolerance when they are exposed to low
non-freezing temperature. This adaptation involves repro-
gramming of gene expression and metabolism.
3.3. Drought stress
Water deficiency leads invariably to a decrease in photosyn-
thetic rate, although levels of tolerance may vary in different
plant species [107110]. Among the factors that contribute to
this photosynthesis reduction, stomatal closure can be
considered as a direct response to leaf water potential
reduction induced by drought [111,112]. Stomatal conductance
reduction limits CO
2
supplying, lowering intercellular CO
2
concentration and consequently constraining net CO
2
assim-
ilation [113,114,110], which decreases plant growth and
productivity. During exposure to drought stress carbon
metabolism and relations between sink and source organs
are perturbed, as well as the metabolism of elements that are
normally absorbed with water. Cellular responses include
osmotic adjustment, regulation of water circulation (aqua-
porins), protection or degradation of proteins, and protection
against oxidative stress.
In terms of genomic investigations, under droughty condi-
tions, the expression of HSP70 among molecular chaperones
has been examined by detecting HSP70 RNA and protein levels
[115119]. Cho and Hong [120] investigated the effect of HSP70
in tobacco transgenic lines expressing a tobacco HSP70,
NtHSP70-1 (AY372069), in either the sense or anti-sense
orientation, to gain information about the molecular mechan-
ismfor response and adaptation to stress by this protein. Their
results agreed with the hypothesis of the important role of
Hsp70 in maintaining stress-tolerant phenotype under condi-
tions of water deficit. In an analogous study made by Alvim
and coworkers [121] it has been shown that transgenic tobacco
(Nicotiana tabacum) plants constitutively expressing elevated
levels of BiP (an endoplasmic reticulum (ER)-localized HSC70
homologue) were resistant to drought stress, supplying
evidence of direct correlation between HSP70 and water stress
tolerance. However, except for BiP, function of HSP70 is not
well understood for in vivo plant systems [122].
3.3.1.1. Proteomic approach. At the protein level, a signature
of desiccation tolerance is the accumulation of heat-shock
proteins (HSP) and late embryogenesis abundant (LEA) proteins;
they mostly accumulate upon water, salinity, and high
temperature stress, playing the function to protect cellular
components during seed desiccation. Gazanchian and collea-
gues [123] studied defence mechanisms of tall wheatgrass
undergoing vegetative stage drought stress and then recovery
after re-watering. Comparative two-dimensional electrophor-
esis revealed 58 protein spots that were reproducibly and
significantly changed during drought stress and recovery,
among about 600 protein spots detected in each gel. A large
number of proteins were up-regulated only under severe
drought stress; seven proteins were identified as chaperone
proteins and oxidative defence enzymes which allow plants to
survive water deficit. A new protein was identified as a heat
shock protein. Proteomic approach was used also by Hajheidari
and coworkers [124] to reveal modifications of proteome in
sugar beets exposed to drought stress compared to controls,
analyzing modulation of protein expression in three wheat
genotypes in response to drought. During exposure to drought
stress, plants respond activating the expression of proteins
involved in stress signalling pathways, protection against
oxidative stress, protein folding and degradation [124]. They
identified an up-regulation of HSP17 in drought resistant
members, while it was down-regulated in the susceptible
genotypes, proving evidence of the crucial role of this protein
in defence mechanisms. Moreover, three HSP70 were down-
regulated in the susceptible genotype, called Afghani. Their
down regulation would probably cause impairments in protein
structure and function. Larrainzar and colleagues [125] applied
a non-gel proteomic approach based on liquid chromatography
coupled to tandem mass spectrometry on Medicago truncatula
nodule tissue under water deficit. The analysis allowed the
identification of protein belonging to several functional classes,
12% of which were redox- and stress-related.
3.3.1.2. Plant adaptation. The so called desiccation toler-
ance is a complex trait, therefore it is not surprising that a
large spectrum of pathways are involved in the protection and
repair of cellular structures against dehydration, survival in
the dry state, and imbibitions. Mostly plants acclimated to
drought by osmotic adjustment. The loss of metabolic activity
occurred only at severe stress conditions. The increased
stomatal resistance under stress levels indicates the efficiency
of the species to conserve water [126,127]. Panaia Kron and
coworkers [128], tested the hypothesis that previous non-
lethal water deficit applied in different developmental stages
in soybean plants could enables them to improve the
tolerance to environmental perturbations. They concluded
on soybean plants submitted to water deficit in different
stages of plant development, that water deficit induces more
suitable response, enabling plants to develop a process of
tolerance improvement to a further water shortage period,
probably through a reduction of growth, which maintains a
conservative strategy of energy use.
3.4. Light stress
Light is a very important environmental factor for plants,
enabling them to make photosynthesis and determining
growth and productivity. However, it is well known that
excess light could damage plant tissue structures, from
impairing main physiological pathways to causing plant
death. Light stress interferes with oxygenic photosynthesis,
a phenomenon known as photoinhibition [129], which, con-
sists in the inhibition of the repair of PSII by the oxidative
stress resulting from absorption of the excess energy [130].
When light exceeds photosynthetic capacity, ROS are gener-
ated in the chloroplasts and cause oxidative damage. Plant
responses to high light include synthesis of carotenoids to
dissipate light energy in the form of heat, and the synthesis of
proteins involved in ROS scavenging. Chloroplasts are the
primary targets of damage caused by high light.
Hewezi andcolleagues [131] analysedtranscriptome profiles of
leaves and immature seeds in sunflower plants subjected to
HT (high temperature) and HL (high light), as well as to a
combination of HL and HT stress using cDNA microarrays to
identify genes whose expression is regulated by these stress
factors. Data analysis revealed that a major response of
sunflower to HL involves down-regulation gene expression,
as previously reportedalso inArabidopsis subjectedto HL stress
[132]. Down-regulation in response to HL is a general phenom-
enon. Most of the genes, in fact, with potential roles in primary
metabolism, energy, DNA processing and translation have
been shown to be repressed, such as fructose-bisphosphate
aldolase, S-adenosyl-L-methionine synthetase and GTP cyclo-
hydrolase II [131]. Interestingly, two subunits of PSI were found
withopposite expressionpatterns: PSI-Vwas induced whereas
PSI-D was repressed. The authors [131] have been also found
two cDNA clones encoding putative 11S seed storage globulin
among the highly down-regulated genes in immature seeds.
Frster and colleagues [133] performed comparative 2DE of
wild-type and two very high light (VHL)-resistant mutants,
called VHLR-S4 and VHLR-S9, of the green alga Chlamydomonas
reinhardtii, after high light or very high light treatment to
assess proteome-wide adjustments of protein abundance and
to identify proteins with a potential role in photo-protection
and survival in excess light. This study revealed deep
modifications in proteomes; in particular, chaperonines and
HSP were differentially expressed among mutants and wild-
type exposed to different kinds of light for different periods.
Among chaperonins, CPN60, CPN23 and CPN20 showed the
most relevant changes in expression, whereas HSP70, out of
all heat shock proteins, appeared to be up-regulated in very
high light-resistant mutants S4 exposed to excess light,
compared to wild type. It is interesting to note that HSP70
changes in expression are consistent with transcriptional
profiles of the same protein in other studies on high-light (HL)-
induced gene expression in Arabidopsis [132,134].
De-etiolation could also be regarded as a stress for etiolated
young plants, causing global changes in protein expression.
Yang and coworkers [135] analysed proteomic profiles of two-
week-old dark-grown rice-seedlings exposed to white light.
Again, the presence of differentiallyexpressedHspwere shown.
Giacomelli and coworkers [136] analyzed the quantitative
response of the thylakoid-associated proteome of Arabidopsis
thaliana wild type and the ascorbate-deficient mutant vtc2-2
after transition to high light (1.000 mmol Photons m
2
s
1
). At
different times of high light exposure qualitative and quantita-
tive changes inproteinexpressionwereobserved, eveninHSP70
members, which appear up-regulated or down-regulated. Nam
and coworkers [137] performed proteomic analysis of the
responses of Panax ginseng leaves to high light. Two types of
smHSPs appeared to be up-regulated, rapidly increasing in
concentration. It was hypothesized that H
2
O
2
generated by HL
acts as a signal for the inductionof sHSP. Thus, again, inorder to
prevent damaging consequences of light stress, plants evolved
mechanisms to face photo-inhibition and photo-damage set-
ting new parameters of homeostasis such as HSPs.
However, ROS production is the first and the main damage
commonly reported upon light stress. Absorption of high light,
in fact, leads to the formation of unwanted and potentially
dangerous chlorophyll triplet excited states (
3
Chl) which can
interact with oxygen (a triplet in its ground state) to formsingle
oxygen [138]. This could result in the formation of extremely
reactive single oxygen that can damage pigments and proteins
of any chlorophyll protein [139] especially with regards to
proteins of the photosynthetic apparatus, producing truncated
proteins deriving from native ones, fragments or aggregates
[140]. Reduced cellular ascorbate content within the cell could
alter chloroplasts response and improve light tolerance [136].
Acclimation of photosynthetic light reactions to high light
requires bothstructural and functional dynamics, whichoccur
in the timescale from seconds to several days. In high light
(HL), feedback de-excitation (qE) is a well known photopro-
tective mechanism that dissipates excess excitation energy in
the light-harvesting antenna of photosystemII (PSII) [141]. The
xanthophylls zeaxanthin (Z) and lutein (L) function in qE, but
also have roles as antioxidants. elisko and coworkers [142],
by using a reversed genetic approach, identified a chloroplast-
targeted protease FtsH6 as responsible for the degradation of
the PSII light-harvesting antenna (LHC II), that is a regulator
under various environmental conditions, e.g., light stress, to
prevent photochemical damage to the reaction centre.
3.5. Heavy metals stress
At physiological concentrations, ion metals are an important
factor for plant growth and seed maturation, but above this
level, toxic effects occur, while belowphysiological concentra-
tions, plants suffer deficiency of enzymes involved in impor-
tant cellular pathways [143]. The two effects are worth
discussing separately.
3.5.1. Metals accumulation
The most evident effect of ion metal excess in plants is
impairments in growth and development; at molecular level,
damage is made by ROS (reactive oxygen species) generated by
radical chain reactions characterized by an excess of free
metals. There is considerable evidence that ROS production in
turn induces expression of HSP and chaperones, providing a
protective function against oxidative stress.
Iron: Fe is an essential nutrient for plants; it functions
accepting and donating electrons in redox reactions, and also
plays important roles in the electron-transport chains of
photosynthesis and respiration. The exposure of plants to iron
excess results in oxidative damage, leading to high production
of O
2
and OH free radicals, via Fenton reaction, which can

damage lipids, proteins and DNA [143].
Cadmium: Cd has been classified as a carcinogen for human
species, and causes severe impairments in plants too. In
higher plants, cadmiumis readily taken up by roots and trans-
located into aerial organs where it can accumulate to high
levels [144]. The most visible effects in plants are chlorosis and
impairments in growth. Cd
2+
might inactivate different metal-
containing enzymes by replacing it randomly to other ions
into prosthetic group of some proteins [145,146]. Cadmium
may also affect cellular processes by formation of active
oxygen redox status [147149]. Many investigations have
reported that in higher plants, upon cadmium pollution, an
increased accumulation of phytochelatins occurs inside
leaves. They are a family of GSH-derived peptides which are
involved in heavy metal detoxification by chelating positive
metal ions [146].
Copper: is an essential micronutrient at low concentration,
but becomes toxic and damaging if its level increases. The
main effect is, like cadmium, inhibition of plant growth and
development causing impairment of many physiological and
biochemical pathways.
3.5.1.3. Proteomic approach. Sarry and coworkers [147] per-
formed 2DE PAGE on proteome extracted from A. thaliana
suspension cultures treated with different amounts of CdCl
2
for 24 h. Interestingly, among other proteins, HSP were up-
regulated in stressed plants; in particular, MS analysis
identified a putative chaperonin protein, similar to T-complex
protein 1, zeta subunit HSP70, mitochondrial chaperonin
hsp60 and protein disulfide-isomerase, also involved in
protein folding. Their role during cadmium exposure is
preventing oxidative damage and irreversible unfolding of
proteins made by ROS. Similarly Ireland et al., [148] found that
Hsp70 was produced by exposure to cadmium in marine
macroalgae and fresh water plant species. Similarly Ahsan
and coworkers [150], utilizing proteomic approach to investi-
gate changes of germinating rice seeds under copper stress,
found fifty proteins differentially expressed in response to
copper treatment; among them, MALDI-TOF analysis identi-
fied stress related proteins such as DnaK-type molecular
chaperon BiP, member of a subfamily of HSP 70 preventing
protein unfolding mainly in endoplasmic reticulum. Hence,
one spot showed high homology with chaperone protein
HchA, another member of plant defence mechanism against
stress. Copper effects in plants was also investigated by Bona
and colleagues [151] on root proteome of C. sativa, where its
excess induced the suppression of two proteins, the down-
regulation of seven proteins (among which 60S rpL12,
thioredoxin peroxidase, putative peroxidase, enolase, elicitor
inducible protein, cyclophilin, GRPs, ABC transporter substrate
binding protein), while five proteins were up-regulated (aldo/
keto reductases, formate dehydrogenase, 40S ribosomal
protein S20).
3.5.2. Metal deficiency
Metal deficiency causes a hypo-function of most enzymes and
consequently an over expression of some helping proteins.
Deficiency symptoms of low mobile nutrients (Fe, Zn, and Mo)
appear initially and primarily on upper leaves or leaf tips,
while deficiency symptoms of mobile nutrients (B and Mn)
appear primarily on lower leaves [152].
Molybdenum: if not present or not uptaken at proper
concentration, it causes impairment in plant growth and
development, and even death. Mo-deficiency in plants causes
lesions and altered morphology of leaves [153].
Molybdenum is utilized by specific plant enzymes to
participate in reduction and oxidative reactions; this metal
itself is not biologically active but has to be complexed by a
pterin compound, forming the molybdenum cofactor (Moco),
in order to gain biological activity [154]. Enzymes involved in
primary nitrogen assimilation such as nitrate reductase (NR),
and the nitrogen-fixing enzyme nitrogenase found in bacter-
oids of legume nodules are examples of molybdoenzymes.
Other plant enzymes needing molybdenum in their structure
are xanthine dehydrogenase/oxidase, aldehyde oxidase and
sulphite oxidase [154,155]; although molybdenum participate
in many reactions as cofactor, up to now no proteomic studies
have been performed.
Iron: higher plants affected by this kind of deficiency exhibit
intercostal chlorosis, principally on young leaves, and mor-
phological changes in roots [156]. In cyanobacteria the
adaptation is characterized by the enhanced expression of
two major iron-regulated proteins, IdiA (iron deficiency
induced protein A) and IsiA (iron stress induced protein A)
[157160]. In higher plants, an elevated glycolysis and a
decreased expression of the small and large subunits of
RuBisCO is the main response to Fe deficiency [161]. In a
recent paper [162], iron deficiency induced significant reduc-
tion in the relative amounts of electron transfer protein
complexes, whereas those of proteins participating in leaf
carbon fixation-linked reactions were increased. Timperio and
colleagues [163] reported that no new protein were expressed
and no proteins were missing suggesting that, in higher
plants, only changes in the monomer-trimer equilibrium of
major PSII antenna was observed, which resulted in a first
adaptive adjustment to iron deficiency, and may eventually
play a role in light dissipation mechanisms.
Experiments carried out by O'Rourke and coworkers [164], have
identified genes involved in the soybean iron deficiency
chlorosis response under irondeficient conditions. Root mem-
brane bound reductase capacity is often correlated with iron
efficiency. Genes were up-regulated that point toward an
increased transport of molecules through membranes. Genes
associatedwithreactiveoxidative species andanROS-defensive
enzyme were also induced. The up-regulationof genes involved
in DNArepair and RNAstability reflect the inhospitable cellular
environment resulting from iron deficiency stress. Other genes
were induced that are involved in protein and lipid catabolism,
while the under-expressionof a keyglycolitic gene mayresult in
the iron-inefficient genotype being energetically challenged to
maintain a stable cellular environment.
3.6. Salt stress
Some aspects of salt stress responses are intimately related to
drought and cold stress responses [165]. This is possibly due to
the fact that sub-lethal salt-stress condition is ultimately an
osmotic effect, which is apparently similar to that brought in
by water deficit and to some extent by cold as well as heat
stresses. Salinity acts like drought on plants, preventing roots
from performing their osmotic activity where water and
nutrients move from an area of low concentration into an
area of high concentration.
Salinity causes impairments in plant growth, and its effect
can be devastating both in short and long term. The short term
effects are osmotic stress, lowering of external water potential
and reduction of water uptake by plants, similar to the situation
under drought [166]; the long term effect is ion toxicity which
occurs if ions cannot be compartmentalized properly. Cells, in
fact, remove sodium from the cytoplasm, where it is toxic, and
sequester it in the vacuole. If salt concentration in the root
medium increases, Na
+
and Cl
are taken up; the main effect is

displacement of mineral nutrients like K
+
, Ca
++
andnitrate, with
serious consequences to plant survival. Ion excess introduced
into cells causes formation of ROS (reactive oxygen species),
which disrupt cellular homeostasis and turn on expression of
genes involved in defence mechanisms. In some cases, salinity
also has a toxic effect on plants because of the high concentra-
tion of certain salts in the soil. Salinity prevents the plants from
taking up the proper balance of nutrients they require for
healthy growth [167]. A high salt level interferes with the
germination of new seeds. As soil salinity levels increase, the
stress on germinating seedlings also increases.
It is difficult to evaluate the importance of particular genes or
groups of genes in plant salt-tolerance with genomic or
trascriptomic techniques, because in response to high con-
centrations of NaCl, up to a few hundred of genes are turned
on [6,168170].
Many research groups had used proteomic approach for the
identification of salt-responsive proteins in several plants.
Salekdeh and coworkers [171] identified several salt-respon-
sive proteins in root proteome of salt-tolerant and salt-
sensitive rice varieties, including ABA- and stress-responsive
proteins, ascorbate peroxidase, and many others. Several
proteins were found to be modulated in expression by salt
concentration in a coordinated manner [172]. These proteins
were involved in photosynthesis, photorespiration, signal
trasduction, metabolism regulation, oxidative stress defence,
control of ion channels, and protein folding. Two-dimensional
electrophoresis was used also by Dooki and colleagues [173] to
reveal changes in protein expression of rice in response to salt
stress at seedling stage; they identified several salt responsive
proteinsincludinganABA- andstress-responsiveprotein(ASR1),
ascorbate peroxidase, and caffeoyl CoA O-methyltransferase.
Regarding HSPs many authors [174178] confirmed that
protein sti1 appeared to be up-regulated in response to salt
stress; this protein contains two heat shock chaperonin-
binding motif (STI1), three tetratricopeptide repeat (TPR) and
two Sti1 domains. The up-regulation of this regulatory protein
may decrease the sterility of pollen during another develop-
ment. It is believed that Hsp90 interacts with TPR-containing
proteins to modulate diverse cellular processes through
proteinprotein interaction. Studies were carried out on Zea
mays, here, mitochondrial smHSPs improved mitochondrial
electron transport during salt stress, mainly by protection of
the NADH: ubiquinone oxidoreductase activity (Complex I),
but it failed to protect enzymes associated with Complex II.
As demonstratedfor most stresses takeninto account inthis
review, Hsp 70 is confirmed as biomarker of stress produced by
NaCl in marine macroalgae and fresh water plant species [148],
emphasized its role in protecting plants against stress.
Dual channel imaging and warping of 2-DE protein gels were
also used to visualize global changes in the protein synthesis
pattern of cells in response to osmotic stress (6% NaCl). Many
vegetative enzymes were repressed in response to salt stress
and derepressed after resumption of growth. The enzymes
catalyzing the metabolic steps from glucose to 2-oxoglutarate,
however, were almost constantly synthesized during salt stress
despite the growth arrest. This indicates an enhanced need for
the proline precursor glutamate. The synthesis of enzymes
involved in sulfate assimilation and in the formation of FeS
clusters was also induced, suggesting an enhanced need for the
formation or repair of FeS clusters in response to salt stress
[179].
Plant species vary in how well they tolerate salt-affected
soils: some plants will tolerate high levels of salinity while
others can tolerate little or no salinity. Perennial plants seem
to handle salinity better than annual plants. In general,
molecules not highly charged, but polar and highly soluble,
having a larger hydration shell, such as glycerol and sucrose
were discovered to protect biological macromolecules against
the damaging effects of salinity. [180].
Plants acclimatedtosalinity stress showedaccumulationof
polyamines, and particularly putrescine. Recent studies with
transgenic carrot cells over-expressing ornithine decarboxy-
lase (ODC) cDNA showed that these cells were significantly
more tolerant to both salt stress as well as water stress [181].
During salt-stress in it was noted an overexpression of a
protein involved in K+ uptake in callus of M. crystallinum [182].
Moons and colleagues [183] demonstrated that both abscis-
sic acid (ABA) and ABA-responsive proteins are present in rice
roots at higher levels in salt tolerant varieties with respect to
controls.
3.7. Ozone stress
Ozone is a destructive gaseous pollutant withserious impact on
humanandanimal respiration[184] as well as causingextensive
damage to both natural and cultivated plant populations. O
3
enters intoplant throughleaf stomata, subsequentlyreacts with
cell wall and membrane components [185,186], leading to the
production of reactive oxygen species (ROS) either by contact
with water, plasmalemma or other cellular components, with
damaging consequences for cells. The precise mechanisms of
the injury process and the plant defence systems against ozone
attacks remain poorly understood.
Miles and coworkers found that [187] in Arabidopsis thaliana,
ROS-induced signalling had been shown to flow through a
protein phosphorylation cascade involving the mitogen-acti-
vated protein kinases (MAPKs) AtMPK3 (MPK3) and AtMPK6
(MPK6). They found moreover, that RNAi-mediated silencing of
MPK6 rendered the plant more sensitive to ozone, as deter-
mined by visible leaf damage. The MPK6-RNAi genotype also
displayed a more intense and prolonged activation of MPK3
compared to that of WT plants. To understand better the plant
response to ozone, Saji and coworkers [188] isolated and
characterized an ozone-sensitive (ozs1) mutant strain from a
set of T-DNA-tagged Arabidopsis thaliana ecotype Columbia.
The mutant plants show enhanced sensitivity to ozone, desic-
cation and sulfur dioxide, but have normal sensitivity to
hydrogen peroxide, low temperature and high light levels.
The T-DNA was inserted at a single locus which is linked to
ozone sensitivity. The authors conclude that OZS1 helps to
close stomata, being not involved in the responses to these
signals.
Agrawal [189] performed two-dimensional electrophoresis
(2-DE) together with amino acid sequencing and immunoblot
analysis, examining, for the first time, the effect of ozone on rice
seedling proteins. 52 protein spots were visually identified as
differentially expressed over controls. Ozone caused drastic
reductions in the major leaf photosynthetic proteins, including
the abundantly present ribulose-1,5-bisphosphate carboxylase/
oxygenase (RuBisCO), and induction of various defence- and
stress-related proteins. The latter were identified as pathogen-
esis related (PR) class 5 protein, PR 10 class protein, ascorbate
peroxidase(s), superoxide dismutase, calcium-binding protein,
calreticulin, and a protease with 90% of homology to an ATP-
dependent CLPproteaseproteolytic subunit, aheat shockprotein
from E. coli. It is interesting to note that CIpA, the proteolytic
subunit of CLP, is an Hsp100-class protein of E.coli, involved in
plant defence fromstress, having a prominent knownrole indis-
aggregation of protein upon increasing temperatures. Control-
ling intracellular proteolysis is, in fact, essential for cellular
function, providing removal of damaged and misfolded poly-
peptides and of short-lived regulatory proteins. This is a
fundamental mechanism in normal cells and it is of particular
relevance in many disease states. Interestingly, E. coli ClpP is a
serineproteasewithringstructuresimilar totheproteasomeand
the GroESL chaperonincomplex [190,191]. The active formof this
protease are chaperones with ATPase activity, constituting part
of a proteinquality control systeminwhichchaperones promote
folding of folding competent chains and proteases eliminate
aberrant polypeptide chains [192].
Torres and colleagues [193] investigated bean and maize
response to ozone stress by using proteomic approach.
Superoxide dismutase (SOD) decreased, while ascorbate
peroxidase (APX, 25 kDa), small heat shock protein HSP,
33 kDa increased. In maize, expression levels of HSPs (24 and
30 kDa) were strongly increased in O
3
-stressed younger leaves.
Their functions were correlated with important cellular path-
ways such as glycolysis, photosynthesis, antioxidant- and
pathogen-related defence. Recently Bohler and coworkers
[194] investigated the effects of ozone in young poplar trees
with 2-D DIGE technology. Several enzymes from the carbon
metabolism resulted down-expressed while two isoforms of
Hsp70 increased, confirming their main role in protein folding
during ozone stress.
Because ozone toxicity is an oxidative stress, one would
expect that plants having higher concentrations and proper
kinds of antioxidants would be more tolerant. In agreement,
Burkey and Eason [195] found that ozone tolerance in snap
bean is associated with elevated ascorbic acid in the leaf
apoplast. It has the potential to limit ozone injury by
participating in reactions that detoxify ozone and reactive
oxygen intermediates and thus prevent plasma membrane
damage. Moreover, the large changes in carbon metabolism
could play a central role in the strategy of the foliar cells in
response to chronic ozone exposure, participating in the
supply of reducing power and carbon skeletons for repair
and detoxification, and modifying the stomatal mode of
functioning. To reinforce the accuracy of the definition of
the threshold for ozone risk assessment, it was proposed by
Dizengremel and coworkers [196], to also consider the redox
pool (NAD(P)H), the ratio between carboxylases and the water
use efficiency as indicators of the differential ozone tolerance
of plants.
4. Abiotic stresses and heat shock proteins
Most abiotic stresses taken into account in this review have
shown that plants, besides over-expressing specific proteins as
consequence of each stress, respond in similar manner to
various stresses and there is a similarity in the plant's adaptive
mechanisms (see Table 1). They are expressed in different part
of cell (see Fig. 3). A direct result of stress-induced cellular
changes is the enhanced accumulation of toxic compounds in
cells that include reactive oxygen species (ROS); the latter
mediated by NADPHoxidases [197]. The intensity, duration and
localization of the different ROS signals are determined by
interplay between the ROS-producing and ROS-scavenging
pathways of the cell (superoxide dismutase SOD, ascorbate
peroxidase APX, catalase CAT, glutathione peroxidase
GPXandperoxiredoxinPrxR). ROS provide a crucial linkinthe
cross-talktodifferent responses [198]. It has beensuggestedthat
plant cells sense ROS via redox-sensitive transcription factors,
such as nitrogen permease reactivator (NPR1) or heat shock
transcription factors (HSFs) [199], which in turn activate HSPs
expression. Thereby, most HSP are thought to be intimately
associated with ROS [50]. ROS levels, as well as ROS signals, are
thought to be controlled by the ROS gene network of plants,
playing a key role in mediating important signal transduction
events. This network is highly dynamic and redundant, and
encodes ROS-scavenging and ROS-producing proteins. In
Arabidopsis, a network of at least 152 genes is involved in
managing the level of ROS. It is likely that inplants this network
is interlinked with the different networks that control stress
acclimationandtolerance [198,199]. The use of ROSas signalling
molecules by plant cells suggests that, during the course of
evolution, plants were able to achieve a high degree of control
over ROS toxicity andare nowusing ROS as signalling molecules
to control more specialized processes such as plant growth and
defence, hormonal signalling, and development.
Along this review, the role of HSPs in directing defence
mechanisms within cell fighting has been found in several
stresses (see Table 1). They are, in fact, induced by a broad spec-
trum of stresses such as low and high temperatures, drought,
salinity or flooding [1,8789,200], as well as osmotic, coldandsalt
stresses were demonstrated to be the strongest inducers of heat
shock gene expression. However, whereas upon heat stress HSP
role as molecular chaperones is without doubt, their function in
non-thermal stress could be different: unfolding of proteins is
not the main effect and protection from damage could occur in
an alternative way apart from ensuring the maintenance of
correct protein structure. Most HSPs have strong cytoprotective
effects, maintaining proteins in their functional conformations,
preventing aggregation of non-native proteins, refolding of
denatured proteins to regain their functional conformation and
removal of non-functional but potentially harmful polypeptides
(arising from misfolding, denaturation or aggregation). Thereby,
Table 1 List of the major specific and aspecific proteins expressed upon several stresses in plant
The first column refers to specific protein typical of each stress, the second and the third refer to the common response pathways, involving both
antioxidant enzymes and HSPs.
HSPs ensure maintenance of homeostasis [201], protect cells and
help to return to equilibrium during recovery.
Members of Hsp70 chaperones are expressed in response to
environmental stress conditions such as heat, cold and
drought, as well as to chemical and other stresses [202204].
The tobacco plants with higher Hsp70 BiP protein levels were
more resistant to drought stress and to tunicamycin than the
antisense and control plants [120]. The overexpression of
Hsp70 genes correlates positively with the acquisition of
thermotolerance [205] and results in enhanced tolerance to
salt, water and high-temperature stress in plants [120,206
208]. For this, Hsp70 has been proposed as potential biomarker
[148]. Hsp100 is also developmentally regulated and is induced
by different environmental stresses, such as heat, cold,
dehydration, high salt or dark-induced etiolation [209212].
Similarly, expression of Hsp90 is developmentally regulated
and responds to heat, cold, salt stress, heavy metals,
phytohormones and light and dark transitions [213,214].
Small heat-shock proteins (smHSPs) which are usually unde-
tectable in plant cells under physiological conditions, are
induced upon stress and plant tolerance to stress, including
drought, salinity, oxidized species and low temperatures
[200,215221]. The high diversification of plant smHSPs, 19
open reading frames in Arabidopsis, probably reflects a
molecular adaptation to stress conditions that are unique to
plants. The smHSPs are not themselves able to refold non-
native proteins, but they bind to partially folded or denatured
substrates proteins, preventing irreversible unfolding or
wrong protein aggregation [222]. Recent findings showed
that the smHSP 18.1 isolated from Pisum sativum, as well as
Hsp 16.6 from Synechocystis sp. PCC6803 under in vitro
conditions, binds to unfolded proteins and allows further
refolding by Hsp70/Hsp100 complexes [223].
In conclusion, there is much evidence that in response to
several stresses, plants synthesize different classes of Hsps/
chaperones which act in concert in cellular protection and
play complementary and sometimes overlapping roles in the
protection of proteins from stress. This supports the hypoth-
esis that the plant multiple stress tolerance mechanism
consists in a complex network by which several pathways
overlap and interact with each other [74,84,90,91].
5. Concluding remarks and perspectives
Plants have particular advantages for biological study, as they
are easily handled and enjoy an enormous breadth of genetic
diversity, both within and between species. Comprehensive
quantitative comparative studies of dynamic protein profiling
during developmental or stress responses, and functional
analysis and characterization of regulatory processes are now
needed to understand plant physiology and how plants
interact with and adapt to the environment.
At present, the progress inplant proteomics has beenlargely
made by 2DE-based proteomic approaches, although it is of
limited use for the identification of low-abundance proteins.
Thus we do not know if many other specific proteins or many
other HSPs are expressed upon abiotic stresses. This is a big
probleminthe vegetable Kingdomwhere most samples suchas
seeds, fruits and leaves contain abundant similar proteins:
reserve protein, photosynthetic apparatus, etc. RuBisCO (ribu-
lose bisphosphatedecarboxylase/oxygenase) is oneof the major
factors hindering the high resolution of 2-DE due to limitations
of the loading capacity of IPG gels: the low-abundant proteins
present (or induced) in leaf samples remain difficult to analyse.
Further, more specific classes of proteins are excluded or
underrepresented, including very acidic or basic proteins,
exclusively large or small proteins and membrane proteins. It
is without doubt that in vegetal sample, removal of the most
abundant proteins for gaining access tothedeepproteome is a
necessity. To detect low abundant proteins, time-consuming
methodological tricks, such as subcellular fractionation,
Fig. 3 Localization of the major stress-related proteins in the plant cell. Protein belonging to the specific class and those
belonging to the common response pathway upon stresses are listed together.
affinity-purification of samples or the use of zoom gels (which
are used in 2DE-PAGE to cover narrow pH ranges and to give
better resolution, as well as higher sensitivity), are necessary.
Moreover one of the major drawbacks is the restricted loading
capacity of the first dimension facing the enormous dynamic
range of protein abundance. Shotgun proteomics has recently
beenintroducedas a complementaryapproachtoaddress these
technical limitations [224,225]. The use of DIGE technology has
also allowed for rigorous statistical analyses of quantitative
changes in protein abundance [226]. Recently, the usage of
combinatorial ligand libraries for capturing and amplifying
the low-abundance proteome [227], has represented a promis-
ing challenge.
Another interesting aspect emerging from the proteomic
investigation on vegetal cells, is that most new specific
proteins induced from abiotic stress are peptides or, however,
short proteins which can escape from the common 2DE
electrophoresis separation. In this context, the liquid chro-
matography coupled with mass spectrometer should be taken
into consideration for investigating peptides and peptide
pools, the so-called peptidomics.
However, PTMs, and interactomics, the real thermometer of
the proteomics status in a field, still remains a major challenge.
Inthe case of heat shock proteins, since they interact withother
cell proteins, to maintain proteins intheir functional conforma-
tions, or prevent aggregation of non-native proteins and refold
denatured proteins to regain their functional conformation, it is
expected that proteinsprotein interaction, the so called inter-
actome, plays a main role in abiotic-stress proteomics research.
HSPs should be investigated not as singular entities but as
multicomponent complexes. Very little is known about what
interactions exist for any HPS in cell upon stress and whether
they hold biologic relevance. A more detailed understanding of
the proteinprotein interactions constitutes a challenging task.
Inthis regardBlue-Native (BN) andCNgels is promising or better
coupling of native liquid phase isoelectrofocusing and Blue
Native polyacrylamide gel electrophoresis [25]. Regarding the
PTM research, which is indicative of the final players in most
abioticresponses, it is limitedtophosphorylationinArabidopsis
[33,47]. The need for a fully sequenced genome for the positive
identificationof PTMs is themajor obstacle preventingextension
to other species. It is clear that plant proteomics will progress in
harness with genomic progress, and the expectation is that the
coming years will see an increasing number of fully sequenced
and annotated plant genomes. Integration of proteomic and
genomicdatawill deliver muchof therawinformationnecessary
topredict whichproteinforms, PTMs, andproteincomplexes are
present at a specific moment in a given tissue upon stress.
Quantitative comparisons during development or in
response to stress, robust predictions of the role(s) of
particular proteins in metabolic and signal pathways, and
large-scale functional testing of candidate proteins, will serve
to fill the gaps in genetically defined pathways. Significant
newdiscoveries have already been made in the vegetable field
by using metabolomics alone or in combination with other
omics disciplines [47]. In the future, we will think about more
studies that include metabolomics as an integral part of the
systems biology approach to study plant response to a variety
of stress conditions. Progress in these directions will lead to
the modelling of entire metabolic pathways in the coming
years, and thus usher in an era of predictive biology. This will
represent a giant step for biotechnology, allowing it to
contribute significantly to the design of genetic solutions to
the ever-present threats of biotic and abiotic stress. Surely,
integrating data from transcriptomics, proteomics, and meta-
bolomics will allow for a more precise knowledge of how
changes in gene expression lead to changes in metabolism.
Unfortunately, few studies have combined proteomics with
the quantitative analysis of transcripts. This is related to the
fact that, in general, analysis of transcripts only partially
confirms the results observed with proteins. In addition to
problems relating to the existence of multigene families,
discordance can be related to differences in turnover between
RNAs and proteins. This can be especially important when
dynamic processes such as the study of response to stress are
analysed [228,229]. However, despite being a relatively new
approach in plant biology, application of integrated Omic
technologies is becoming one of the major tools in studying
plant stress responses. It is worthwhile that more novel
proteomic techniques make possible improvements in this
context and in the near future, a large-scale transcriptome
analysis coupled with metabolomic analysis of plants and
integration of results obtained from these studies with the
development of computer models [197,230] and bioinfor-
matics tools [231233] should enable us to gain a system-
level understanding of ROS metabolism in plants, their
interrelationship with HSP and how both offer defence to
abiotic stresses. This will have a substantial impact on
agriculture and through this, on human health.
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Genomics

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Review

Proteomics applied on plant abiotic stresses: Role of heat shock

Anna Maria Timperio, Maria Giulia Egidi, Lello Zolla

and OH free radicals, via Fenton reaction, which can

are taken up; the main effect is

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