REVIEWS
pubs.acs.org/jpr
Plant Cell Organelle Proteomics in Response to Abiotic Stress
Zahed Hossain,†,‡ Mohammad-Zaman Nouri,†,§ and Setsuko Komatsu*,†
†
National Institute of Crop Science, Tsukuba 305-8518, Japan
Department of Botany, West Bengal State University, Kolkata-700126, West Bengal, India
§
Rice Research Institute of Iran, Deputy of Mazandaran, Amol 46191-91951, Iran
‡
bS Supporting Information
ABSTRACT:
Proteomics is one of the finest molecular techniques extensively being used for the study of protein profiling of a given plant species
experiencing stressed conditions. Plants respond to a stress by alteration in the pattern of protein expression, either by up-regulating
of the existing protein pool or by the synthesizing novel proteins primarily associated with plants antioxidative defense mechanism.
Improved protein extraction protocols and advance techniques for identification of novel proteins have been standardized in
different plant species at both cellular and whole plant level for better understanding of abiotic stress sensing and intracellular stress
signal transduction mechanisms. In contrast, an in-depth proteome study of subcellular organelles could generate much detail
information about the intrinsic mechanism of stress response as it correlates the possible relationship between the protein
abundance and plant stress tolerance. Although a wealth of reviews devoted to plant proteomics are available, review articles
dedicated to plant cell organelle proteins response under abiotic stress are very scanty. In the present review, an attempt has been
made to summarize all significant contributions related to abiotic stresses and their impacts on organelle proteomes for better
understanding of plants abiotic stress tolerance mechanism at protein level. This review will not only provide new insights into the
plants stress response mechanisms, which are necessary for future development of genetically engineered stress tolerant crop plants
for the benefit of humankind, but will also highlight the importance of studying changes in protein abundance within the cell
organelles in response to abiotic stress.
KEYWORDS: cell organelle, plant proteomics, abiotic stress
’ INTRODUCTION
Abiotic stress is the major constraint that global crop production faces at present. New techniques have been adopted to
dissect the underlying molecular mechanisms of plants’ stress
sensing and tolerance. When a plant encounters abiotic stress, for
example, salinity, drought, flooding, metal toxicity, UV exposure,
high and low temperature, the fine cellular adjustment between
the formation of reactive oxygen species (ROS) and the quenching capacity of plants’ antioxidant molecules get distorted. The
excess ROS produced under abiotic stress leads to oxidative
damages to lipids, proteins and nucleic acids (Figure 1).1,2 To
cope with the stress, plants have evolved complex antioxidant
defense mechanism comprised of both enzymatic and nonenzymatic networks.
r 2011 American Chemical Society
The cellular mechanism of sensing stress and transduction of
stress signals into the cell organelle is of well-known and
represents the initial reaction of plant cells toward stress.3 Stress
signals are first encountered by the outer part of the cell and the
process for sensing environmental changes and activating the
responsive mechanism is highly organized.4 The ROS which are
chiefly formed by the over reduced electron transport chain
during abiotic stress, are recognized as a signal to activate the
plant defense response.5 Transduction of the signal into the cell
Special Issue: Microbial and Plant Proteomics
Received: August 31, 2011
Published: October 26, 2011
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Journal of Proteome Research
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during the stressed condition represent the primary defense
response. In fact, communication between organelles and cytosolic, luminal proteins renders the protein composition of
organelles dynamic.6 Most of the receptor proteins are located
in the plasma membrane, and thus the cell membrane is directly
involved in sensing stress.7 To obtain a comprehensive understanding about the cellular reactions involved in cell defense
mechanisms, the role of cellular organelles should be considered
in stress-related studies.
Proteomics, the cutting edge molecular technique of the
present time, offers several advantages over the genome-based
technologies as it directly deals with the functional molecules
rather than genetic code or mRNA abundance. Identification of
proteins using mass spectrometry has opened a new avenue for
organ and subcellular proteome research. Organelle proteome
analysis provides fundamental information of plant response to a
given stress at the functional level and thus refines our knowledge
about plant stress related signaling pathways. A plethora of
reviews on proteome analysis in plant stress research are available
(Supplementary Table 1, Supporting Information), but review
articles specifically devoted to changes in the organelle proteome
under abiotic stresses are really scant. The present review
provides an overview of the current major findings related to
changes in organelle proteomes in response to abiotic stresses for
better understanding of the abiotic stress tolerance mechanism of
plants at the protein level.
’ ORGANELLE PROTEOME
Analyses of an organelle’s proteins are useful approach to
understand the cell behavior under abiotic stress conditions.
Organelle proteins are primarily nuclear-encoded, but some
organelles, such as mitochondria and chloroplasts, carry their
own genetic material that enables them to synthesize proteins
themselves.6 However, because of the dynamic state of organelles
and their proteins, elucidating the subcellular distribution and
expression of organelle proteins has always been a challenging
job for proteomic researcher.8 Abiotic stress alters interactions
between organelles in plant cells, and this subsequently changes
the regulation and secretion of proteins in cellular organelles and
compartments. Several secretory pathways have been reported to
be involved in targeting proteins in plant cells.9,10 Signal peptides
play a central role in protein targeting, and several amino acid
residues and protein domains involved in specific targeting have
been successfully determined.11 Recent studies reveal that golgito-plastid trafficking of certain chloroplast resident proteins
involve glycosylation before entering the chloroplast. Posttranslational modifications like N-glycosylation and intramolecular
disulfide bridge formation are important determining factors for
correct protein folding and trafficking of target proteins from
secretory pathway to chloroplast.12 15 Thus, identifying organelle proteins involved in the stress response, especially those
with regulatory or protein targeting functions would definitely
enhance the knowledge of the cellular stress response.
A number of proteomics studies have been successfully carried out
on specific cellular organelles and compartments in plants subjected
to abiotic stresses (Table 1), including the mitochondria,16 20
nucleus,21 23 chloroplasts,24 26 cell wall,27 30 and the plasma membrane.31 33 However, the literatures lack critical discussion of the
role played by cooperation between organelles responding to
abiotic stress. This review highlights the current state of plant cell
organelle proteome in response to abiotic stress.
Figure 1. Abiotic stress-induced ROS/antioxidant imbalance and its
cellular impact. Abbreviations: ROS, reactive oxygen species; O2•‑,
superoxide radical; H2O2, Hydrogen peroxide; 1O2, singlet oxygen;
OH•, Hydroxyl radical; SOD, superoxide dismutase; APX, ascorbate
peroxidase; MDAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced
glutathione; AsA, reduced ascorbate.
through cascades alters gene and protein expression levels,
leading to physiological responses. Hence, communication through
intracellular compartments plays a crucial role in stress signal
transduction process. To understand the underlying molecular
mechanism of how a plant cell modulates its protein expression
network to cope with the stress, an in-depth study of the organelle
proteome is of great contribution toward development of stresstolerant crop varieties to meet the increasing demand of food
supply worldwide.
The major subcellular organelles, whose functions get affected
under abiotic stress, are the nucleus, mitochondria, chloroplasts,
peroxisomes, plasma membrane and cell wall. Most of these
organelles have the potential to become a source of ROS. The
intracellular organelles and compartments and their interactions
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39
a
Nucleus
Nucleus
Nucleus
Chloroplast
Chloroplast
Rice
Chickpea
Arabidopsis
Soybean
Arabidopsis
Plasma
Soybean
membrane
Plasma
membrane
Plasma
Cold
Osmotic stress
Flooding
Dehydration
Water deficit
Flooding
Dehydration
Ozone
Low temperature
Ozone
Cold
Dehydration
Dehydration
Salinity
and herbicide
Drought, cold
Oxidative stress
Salinity
Flooding
abiotic stress
IP, Number of identified proteins.
Arabidopsis
Soybean
ECM/Cell
wall
Chickpea
membrane
Cell wall
Maize
Cell wall
Mitochondria
Rice
Soybean
Mitochondria
Pea
Cell wall
Mitochondria
Arabidopsis
Rice
Mitochondria
Wheat
Chloroplast
Mitochondria
Soybean
Poplar
organelle
plant
2-DE/MALDI TOF MS
2-DE/nanoLC MS/MS
nanoLC MS/MS
2-DE/MALDI TOF MS
2-DE/LC TOF MS
2-DE/Q-TOF MS
nanoLC MS/MS
2-DE/MALDI TOF MS
2-DE/LC TOF MS
2-DE/MALDI TOF MS
MALDI TOF MS
2-DE/
2-DE/MALDI TOF MS
2-DE/MALDI TOF MS
2-DE/LC TOF MS
2-DE/LC ESI MS/MS
2-DE/MALDI TOF MS
38
86
14 + 8 = 22
134
152
16
94
27
43
32
184
147
109
8
29
Q-TOF MS
164
LC ESI MS/MS
2DE, BN-PAGE,
68
36 + 16 = 52
IPa
SDS-PAGE/
2-DE/LC MS/MS
MALDI TOF MS
2-DE/BN-PAGE/
methods
major findings
26
25
24
23
22
21
19
20
18
17
16
ref.
osmotic stress, enhancement of CO2 fixation and proteolysis.
Cold responsive proteins are mainly associated with membrane repair, protection of the membrane against
because of up-regulation of plasma membrane H1-ATPase protein.
Under hyperosmotic conditions, calnexin accumulates in the plasma membrane and high ion efflux takes place
and degradation.
up-regulated in plasma membrane; heat shock cognate proteins protect stress induced protein denaturation
Flooding stress induced proteins mostly involved in antioxidative defense system. Cell wall proteins found to be
More than hundred ECM proteins with a variety of cellular functions e.g. cell wall modification, signal transduction,
metabolism, and cell defense and rescue, play crucial roles in dehydration stress sensing and tolerance mechanism.
about the complex mechanisms regulating root growth under water stress.
the apical region of the elongation zone of water stressed maize roots and hence provides novel information
Water stress-responsive proteins were identified and categorized, into 5 groups; apoplastic ROS level increases in
lignifications suppressed under flooding stress.
Two lipoxygenases, germin-like protein precursors, glycoprotein precursors, SOD down-regulated and
cell defense and rescue, cell wall modification, cell signaling and molecular chaperones.
33
32
31
30
29
28
stress, leading to a decrease in abundance of photosystem subunits and other proteins of the chloroplast membranes.
Dehydration-responsive proteins mainly involved in a variety of functions, including carbohydrate metabolism,
27
Under a long-term ozone exposure, the cellular protective measures get exhausted by the oxidative nature of the
stress sensing and signal transduction, presumably helping the plant in cold sensing and acclimatization.
biosynthesis and
Identified proteins chiefly participate in photosynthesis, other plastid metabolic functions, phytohormone
Proteins involved in antioxidant defense and carbon metabolism increased under O3 stress.
pathways and cross-talk.
Proteins with cold stress response-specific motifs/functions detected, mainly involved in RNA-associated functions,
elongation step of protein synthesis, prevents protein misfolding, suggesting involvement of multiple signaling
molecular chaperones, cell signaling, and chromatin remodeling.
Differentially expressed proteins apparently involved in functions related to gene transcription and replication,
remodeling, signaling, gene regulation, cell defense and rescue, and protein degradation.
Dehydration responsive proteins involved in variety of functions e.g. transcriptional regulation and chromatin
ATP synthase may not be the major producer of ATP in mitochondria during the early stage of PCD in rice.
proteins indicates the diversity of response of mitochondria to stresses at the protein level.
metal affinity and varying susceptibility of inactivation by metal ions.
Differential degradation of key matrix enzymes, induction of heat shock proteins and specific losses of other
Metal content of mitochondria is dynamic and changes during oxidative stress. Different proteins have varying
varieties at whole plant level.
Differences in mitochondrial ROS scavenging pathways determine the salinity tolerance of the contrasting wheat
cycle and γ-amino butyrate shunt were up-regulated.
Flooding stress directly impairs electron transport chains, significantly decreased ATP; proteins related to TCA
Table 1. Summary of Published Organelle Proteome Analyses in Response to Abiotic Stress
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metal ions. Mitochondrial respiratory chain pathways and the
matrix enzymes varied widely in terms of their susceptibility
toward metal-induced loss of function and thus exhibiting the
selective oxidation events in the mitochondrial proteome.
In-depth study of mitochondrial proteome during salt stressinduced programmed cell death (PCD) in rice was performed by
Chen et al.19 A total of eight PCD-related proteins were identified
after 2-DE analysis. Out of them, four proteins were up-regulated
after PCD induction, which are glycoside hydrolase, mitochondrial
heat shock protein 70, 20S proteasome subunit, and Cu/Zn-SOD,
and rest four were down-regulated, namely ATP synthase beta
subunit, cytochrome-c oxidase subunit 6b, S-adenosylmethionine
synthetase 2, and transcription initiation factor eIF-3 epsilon.
Proteome study reveals that ATP synthase may not be the major
producer of ATP in mitochondria during the early stage of PCD in
rice. Glycoside hydrolase could be involved in ETC impairment
and ROS burst during salt stress-induced PCD.
Taylor et al.20 analyzed the response of pea mitochondria
under drought, cold and herbicide stresses. Mitochondria isolated from the stressed pea plants maintained their electron
transport chain activity, but changes were apparent in the
abundance of uncoupling proteins, nonphosphorylating respiratory pathways, and oxidative modification of lipoic acid moieties
on mitochondrial proteins. Meticulous analysis of the soluble
proteins of mitochondria by 2D-PAGE and MS reveals differential degradation of key matrix enzymes under chilling and
drought stresses. In addition, differential induction of heat shock
proteins and specific losses of other proteins indicates the
diversity of response of mitochondria to these abiotic stresses
at the protein level.
Mitochondrial Proteome
Study of mitochondrial proteome has revealed new insights of
plants response toward abiotic stress factors. In addition of acting
as a cellular power house, mitochondria also performs numerous
other activities like nucleotide and vitamins synthesis, lipids and
amino acids metabolisms, involvement in the photorespiratory
pathway.34 Under stress, the mitochondrial electron transport
chain become over reduced, favoring the generation of O2•‑ thus
affecting plant growth and development.35
Kruft et al.36 first used the two-dimensional polyacrylamide gel
electrophoresis (2-DE) technique to study the mitochondrial proteins. Thereafter, much attention has been paid to separate proteins
for analyzing plant mitochondrial proteome under stressful condition. Gel-free method of mitochondrial proteome study using
nanoscale 1D and 2D liquid chromatography (LC) offers advantages37 39 over the gel-based techniques, as it allows separation of highly acidic or highly basic proteins, very high and very
low molecular weight proteins as well as low-abundance proteins.
Mitochondria have been a target for subcellular proteomic
study, as most of the abiotic stresses primarily impair mitochondrial electron transport chain resulting excess ROS generation.
A comprehensive analysis of mitochondrial proteins of roots and
hypocotyls of soybean under flooding stress has recently been
performed.16 Mitochondrial matrix and membrane proteins were
separated by 2-DE and blue native-polyacrylamide gel electrophoresis (BN-PAGE), respectively. Differentially expressed proteins and metabolites were identified using MS. Proteins and
metabolites related to the tricarboxylic acid cycle (TCA) and
γ-amino butyrate shunt were increased by flooding stress, while
inner membrane carrier proteins and proteins related to complexes III, IV, and V of the electron transport chains were
decreased. The authors found that the amounts of NADH and
NAD were increased; however, ATP was significantly decreased
under flooding stress. The results led them conclude that flooding
stress directly impairs electron transport chains, although NADH
production increases in the mitochondria through the TCA cycle.
The shoot mitochondrial proteome and differences associated
with salinity tolerance have been extensively investigated in two
contrasting salinity tolerant and sensitive wheat cultivars.17 By
employing reference map and 2D-fluorescence difference gel
electrophoresis (DIGE) technique, they identified five mitochondrial proteins, whose abundance changed under salinity
treatment. These proteins include Mn- SOD, nucleoside diphosphate kinase, cysteine synthase, voltage dependent anion carrier
and alternative oxidase (AOX). Both Mn-SOD and AOX play
pivotal role in scavenging ROS that are produced in excess under
salinity stress.40 The authors suggested that the differences in the
mitochondrial ROS defense pathways observed in the mitochondrial proteomes of the two contrasting cultivars play key role in
salinity tolerance at whole plant level.
Heavy metals have known functions of depleting cellular
glutathione pools, thus causing an impairment of ascorbate
glutathione cycle resulting higher accumulation of ROS in the
cell compartments.41 Inhibition of plant mitochondrial function
under Cd-stress has been reported earlier.42,43 Tan et al.18
investigated the metal homeostasis in Arabidopsis mitochondria
during oxidative stress for better understanding of protein
interactions with metal ions and the associated modulation of
protein functions in plant mitochondria. Their findings demonstrated that the metal content of mitochondria is dynamic and
changes during oxidative stress and the different proteins have
varying metal affinity and varying susceptibility of inactivation by
Nuclear Proteome
Nucleus carries the information necessary for controlled expression of proteins and thus plays essential role in determining plant
response toward abiotic stress. Like the other organelle proteomic
study, nuclear proteome has recently gained importance, as
identification of novel nuclear proteins help us better understand
protein function in conferring cellular stress tolerance. Limited
information on the proteomic study of stress responsive nuclear
protein expression profile in plants is currently available.
A comprehensive nuclear proteome analysis was carried out in
rice for better understanding of molecular mechanisms governing dehydration-responsive adaptation.21 Organellar enrichment
followed by 2-DE based protein identification by LC electrospray
ionization tandem mass spectrometry (ESI MS/MS) techniques
were exploited for proteome determination. The differential display
of nuclear proteome revealed 150 protein spots whose intensities
changed significantly during dehydration period. A total of 109
differentially regulated proteins were identified and expected to be
involved in a variety of functions including transcriptional regulation
and chromatin remodeling, signaling and gene regulation, cell
defense and rescue, and protein degradation. A similar kind of
dehydration responsive nuclear proteome study was performed in
chickpea, providing information about the complex metabolic network operating in the nucleus during dehydration.22 Under stress,
205 protein spots were found to be differentially regulated. Following MS analysis, 147 differentially expressed proteins were identified,
apparently involved in functions related to gene transcription and
replication, molecular chaperones, cell signaling, and chromatin
remodeling. The dehydration responsive nuclear proteome of
chickpea revealed a coordinated response, which involves both
the regulatory as well as the functional proteins. Comparison
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between the dehydration responsive nuclear proteome of rice and
chickpea revealed a crop-specific adaptation mechanism with some
common resident proteins.
Response to cold stress and changes in nuclear protein expressions were thoroughly investigated in model plant Arabidopsis.23
Nuclear proteins were isolated and peptide masses were measured
using 2-DE and matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) MS respectively. Out of the total 184
identified proteins, 54 were up- or down-regulated by more than a
factor of 2 in response to cold stress. In addition, proteins with
defined stress response-specific motifs or functions were also
detected. The function of protein motifs in conferring abiotic
stress tolerance has been well studied in Arabidopsis.44,45 The EARmotif of the Cys2/His2-type zinc finger proteins was found to be
responsible in enhancing tolerance to cold, drought and salinity
stressed conditions. The ZPT2-related proteins primarily function
as transcriptional repressors that down-regulate the trans-activation
activity of other transcription factors.
directly or indirectly by ozone exposure. Findings indicate that
under a long-term ozone exposure, the cellular protective
measures get exhausted by the oxidative nature of the stress,
leading to a decrease in abundance of photosystem subunits and
other proteins of the chloroplast membranes.
Taking all into considerations, more initiatives must be taken
to characterize chloroplast proteome for more comprehensive
understanding of chloroplast’s functional biology in response to
abiotic stress.
Cell Wall Proteome
Plant cell wall plays essential role in stress sensing and signal
transduction between the apoplast and symplast. Hence, in
recent years cell wall proteome science has been the subject of
intense research. For better understanding of the underlying
molecular mechanism of dehydration stress response in rice
seedlings, Pandey et al.27 studied the stress-induced changes in
the extracellular matrix proteins proteome by using two-dimensional
gel electrophoresis. The proteomic analysis led to identification of
about 100 differentially expresses proteins, which might play key
roles in plants’ dehydration tolerance cascade.
Investigation on the function of the soybean cell wall revealed
that 16 out of 204 cell wall proteins responded to flooding
stress.28 Of these, two lipoxygenases, four germin-like protein
precursors, three stem glycoprotein precursors, and one Cu Zn
SOD were reported to be down-regulated. Proteome analysis
suggested that the roots and hypocotyls of soybean caused the
suppression of lignification through decrease of the proteins by
down-regulation of reactive oxygen species and jasmonate biosynthesis under flooding stress.
To achieve a comprehensive understanding of alterations in
the cell wall protein composition of different regions of the maize
root elongation zone to water deficit, a proteomics approach was
initiated to examine water-soluble and loosely ionically bound
cell wall proteins.29 The results revealed region-specific changes
in protein profiles among control and water-stressed roots. All
together, 152 water stress-responsive proteins were identified
and categorized into five groups based on their functions in the
cell wall ROS metabolism, defense and detoxification, hydrolases,
carbohydrate metabolism, and other/unknown. Protein identification reveals that the apoplastic ROS level increases in the apical
region of the elongation zone of water stressed maize roots and
hence provides novel information about the complex mechanisms regulating root growth under water stress.
Comprehensive extracellular matrix proteome analysis of
chickpea under dehydration stress was performed by Bhushan
et al.30 The comparative proteomics study led to the identification of 134 differentially expressed proteins, which include
predicted and some novel dehydration-responsive proteins. This
comparative proteomics study demonstrates that more than 100
extracellular matrix proteins with a variety of cellular functions
like cell wall modification, signal transduction, metabolism, and
cell defense and rescue play crucial roles in dehydration stress
sensing and tolerance mechanism.
Compared to mitochondrial proteomics, only few researches
so far have been carried out on cell wall proteomics. Still, quite a
large number of cell wall proteins remain unidentified. Hence,
there is an urgent need to investigate the functional proteomics
of cell wall proteins under abiotic stress.
Chloroplast Proteome
Environmental stresses like salinity, drought, ozone and high or
low temperatures cause reduction in CO2 fixation; thus, NADP+
regeneration by the Calvin cycle get decreased. As a consequence,
the photosynthetic electron transport chain becomes over reduced, forming superoxide radicals and singlet oxygen in the
chloroplasts.46 Excess ROS impairs chloroplast protein functions
involved in photosynthesis. Only few works so far been carried out
on the chloroplast proteome response to abiotic stress.
Proteome analysis of soybean chloroplasts responding to
ozone stress by Ahsan et al.24 revealed 32 differentially expressed
chloroplast proteins. Proteins involved in photosystem I/II and
carbon assimilation decreased under stress, and this might be one
of the reasons of reduced photosynthetic activity in response to
ozone. In contrast, proteins involved in antioxidant defense and
carbon metabolism increased under stress. The authors came to
the conclusion that not only do the degradation of starch and
higher amounts of sucrose in response to short-term acute ozone
exposure feed the TCA cycle but the availability of sucrose may
also play a pivotal role in oxidative stress signaling and regulation
pathways of antioxidative processes.47,48
Subcellular fractionation and relative protein quantification by
2D-DIGE technique were used to get the insights of the
Arabidopsis chloroplast proteome response to short-term cold
shock and long-term cold acclimation.25 Cold shock resulted in
minimal change in the plastid proteomes, while short-term
acclimation caused major changes in the stromal but few changes
in the lumen proteome. In contrast, long-term acclimation
resulted in modulation of the proteomes of both compartments,
with appearance of new proteins in the lumen and further
changes in protein abundance in the stroma. In total, 43 differentially displayed proteins were identified that participate in
photosynthesis, other plastid metabolic functions, phytohormone biosynthesis and stress sensing and signal transduction,
presumably helping the plant in cold sensing and acclimatization.
Ozone exposure is known to cause chloroplasts to become
smaller and distortion of thylakoids shape and structure.49,50
Effects of ozone stress on the chloroplast membrane proteins was
analyzed using 2D-DIGE.26 Extrinsic photosystem proteins and
ATPase subunits were found to vary in abundance. A decrease
trend in protein abundance was observed under stress, except for
ferredoxin-NADP+ oxidoreductase. This led to the formation of
higher NADPH, which helps in detoxification of ROS generated
Plasma Membrane Proteome
At the cellular level, the plasma membrane is probably the most
diverse form of membrane with a complex protein composition
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Table 2. Classification of Major Defense-related Abiotic Stress Responsive Proteins and Their Subcellular Localization
protein group
Osmoprotectant regulators
proteins
Sucrose synthase, sugar transporter
abiotic stress
subcellular localization
Drought, Osmotic stress
Nucleus, mitochondria, chloroplast,
endoplasmic reticulum, Golgi
apparatus, plasma membrane, cell wall
ROS scavengers
Superoxide dismutase, ascorbate
peroxidase, monodehydroascorbate
Ion transporters
Salinity, Flooding, Drought,
Nucleus, mitochondria, chloroplast,
Chilling, Heat, Radiation stress
peroxisomes, vacuole, endoplasmic
reductase, dehydroascorbate reductase,
reticulum, Golgi apparatus, plasma
glutathione reductase, glutathione
membrane, cell wall
peroxidase, catalase
Na+/H+ antiporters, plasma membrane
Salinity, Osmotic stress
Plasma membrane, Tonoplast
H+-ATPase
Water channels
Aquaporins
Drought, Flooding,
Plasma membrane, Tonoplast
Molecular chaperones
HSPs, calnexin, calreticulin, binding
Drought, Heat, Ozone,
Nucleus, mitochondria, chloroplast,
immunoglobulin protein
Osmotic stress, Heavy metal stress
vacuole, endoplasmic reticulum
Golgi apparatus, plasma membrane,
cell wall
Proteolysis-related
proteins
Ubiquitins
Drought, Salinity
that varies with cell types, developmental stages and environments.51 It acts as a primary interface between the cellular
cytoplasm and the extracellular environment, thus playing a vital
role in cell communication. Change in gene expression at the
protein level is one of the main cellular responses required for
perception of a stress signal and its transduction into the cell,
which mostly happens in the plasma membrane.
To explore the alterations in the plasma membrane proteins of
soybean exposed to flooding stress, small purified plasma membrane proteins were analyzed using gel-based and gel-free
proteomics techniques.31 It is presumed to be the first report
of identifying flood induced plasma membrane proteins in
soybean exploring 2-DE MS/sequencer-based proteomics and
nanoLC MS/MS-based proteomics techniques. A total of 35
stress induced novel proteins were identified, mostly involved in
plants’ antioxidative defense system.
Nouri and Komatsu32 investigated the polyethylene glycol
induced osmotic stress impact on plasma membrane proteome of
soybean. Plasma membranes were purified using a two-phase
partitioning method and their purity was verified by measuring
ATPase activity. Using the gel-based proteomics, four and eight
protein spots were identified as up- and down regulated respectively,
whereas in the nanoLC MS/MS approach, 11 and 75 proteins
were identified as up- and down-regulated respectively under
polyethylene glycol treatment. Osmotic stress responsive proteins,
for example, transporter proteins and proteins with high number of
transmembrane helices as well as low-abundance proteins were
identified by the gel free proteomics. Three homologues of plasma
membrane H+-ATPase, the transporter proteins involved in ion
efflux, were up-regulated under osmotic stress. Among the identified
proteins, seven proteins were mutual in two proteomics techniques,
in which calnexin was the highly up-regulated protein. Findings
suggested that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and high ion efflux takes place because
of up-regulation of plasma membrane H+-ATPase protein.
Mass spectrometric approach was widely used for identification
of putative plasma membrane proteins of Arabidopsis leaves
associated with cold acclimation.33 A significant change in protein
profile was observed after cold acclimation. A total of 38 proteins
Cytoplasm, endoplasmic reticulum, nucleus
were identified using MALDI-TOF MS. The proteins that changed in quantity during the first day of cold acclimation include
those, which are mainly associated with membrane repair, protection of the membrane against osmotic stress, enhancement of CO2
fixation and proteolysis. Plasma membrane proteomics study
describes the functional machinery of membrane proteins and
thus considered to be a core area of investigation in the field of
developmental plant proteomics. All the above-mentioned researches would not only provide the functional information of
individual protein, but also throw light on the protective role of
plasma membrane proteins in response to stress.
Organelle proteome study provides valuable information about
protein functions, interactions and sorting mechanisms; however,
limitations in individual organelle isolation, purification and analysis of
low abundance proteins make the task extremely difficult for studying
abiotic stress tolerance mechanism in endomembrane organelles.52
’ STRESS-RESPONSIVE PROTEINS IN PLANT CELL
ORGANELLES
Exposure of plant cells to abiotic stress leads to a wide range of
changes in protein expression level. Any change in the accumulation of a particular protein in the cell does not necessarily mean
that gene expression has also increased or suppressed, as there is
no certain correlation between gene expression and the final
product of protein synthesis and regulation. Protein expression
can be affected by a number of factors, such as protein targeting or
translocation and post-translational modifications, all of which are
influenced by stress conditions. Secretory pathways and intracellular interactions affect the distribution of proteins in the cell.
Groups of functionally related proteins are often regulated as a
result of exposure to abiotic stress. Some of these proteins, such as
those related to metabolism, storage, and protein synthesis, are not
directly involved in the mechanism of defense. Defense-related
abiotic stress-responsive proteins can be classified into six major
groups according to their functions (Table 2).
Osmoprotectant Regulators
Osmoprotectant regulators are proteins that regulate the
distribution of osmolyte molecules such as sugar, mannitol, or
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amino acids (proline) and their N-methyl derivatives (betaines).
The solutes increase the osmotic pressure in the cell, thereby
preventing further water loss and maintaining turgor.53 Studies
on plants’ response against abiotic stress have revealed that
expression of genes for sugar synthases and sugar transporters
are usually up-regulated under abiotic stress54 and that accumulation of proline55 and glycinebetaine56 helps the plants to
overcome the stressed condition, thus enhancing the tolerance
capacity. Osmoprotectants are found in the cytoplasm and all
organelles except for the vacuoles.53 The osmoprotectant biosynthetic protein Δ1-pyrroline-5-carboxylate synthase as well as
other enzymes such as sucrose synthase and sugar transporters
have been identified in soybean undergoing osmotic stress and
stress affecting the endoplasmic reticulum.57 Adjustment of
osmotic potential is one of the initial reactions of plant cells to
dehydration-related stresses. Osmoprotectants and water channels are usually responsible for preserving the water content of
cells under stress conditions.
enzymes; therefore, proper regulation of ion efflux is vital during
stress conditions.
Water Channels
Adjustment of water content is necessary for plant cells, and it
is critical when plants are undergoing abiotic stress. Since water is
involved in many cellular processes, any change in the water
content of the cell can negatively affect the proper functioning of
the cell. Aquaporins are water channel proteins that reside in the
plasma membrane and vacuoles and facilitate the diffusion of
water and small neutral solutes across the cell membrane.70,71
Aquaporins are divided into two main classes according to their
localization in the cell and sequence homology: plasma membrane-intrinsic aquaporins, and tonoplast-intrinsic aquaporins.
The tonoplast has high water permeability; therefore, the vacuolar space can play a buffering role under osmotic fluctuations.72
The activity of aquaporins is regulated by phosphorylation under
drought stress. In the case of stresses associated with an excess of
water, such as flooding or submergence, the cytoplasmic pH falls,
leading to protonation of the conserved residue of aquaporin.
This process subsequently leads to closure of water channels in
the plasma membrane.73 Water-related stress constitutes the
most frequent and significant environmental factor affecting
plants, and water channel proteins in the tonoplast and plasma
membrane are the primary regulators of water balance in plant
cells. Adjustment of the water content in the cell upon exposure
to water-related stress enables the cell to maintain proper
function of proteins and enzymes in the organelles and other
cellular compartments.
ROS Scavengers
Cells undergoing abiotic stress transiently produce various
ROS, such as hydrogen peroxide, hydroxyl radicals, and superoxide anions, all of which in turn cause oxidative stress. The cell’s
stress response depends on the severity of the oxidative stress,
and may range from activation of antioxidant defense mechanisms to programmed cell death. The production of ROS
scavenger proteins is the primary defense against the oxidative
stress. The major scavenger proteins found in plant cells are
superoxide dismutase, ascorbate peroxidase, glutathione peroxidase, and catalase.58 60 The production of ROS in organelles
such as the mitochondria and chloroplasts is a normal cellular
process. However, stress conditions accelerate the production of
ROS and cause oxidative damage.61 The presence of scavenger
proteins during abiotic stress has been reported in various plant
organelles and subcellular compartments, including the nucleus,23
mitochondria,62 chloroplasts,63 plasma membrane,31 and cell wall.64
The distribution of scavenger proteins throughout the cell suggests
that ROS may serve as signaling molecules in the organelles and
compartments.
Molecular Chaperones
The primary function of molecular chaperones is facilitating
the folding of proteins through binding to newly synthesized
glycoproteins. The chaperone proteins assist in the proper
folding and assembly of secretory proteins74 and they are
normally present in almost all parts of a cell. However, the
majority of chaperone proteins are found in the endoplasmic
reticulum, where newly synthesized proteins are folded.
Calnexin, calreticulin, binding immunoglobulin protein, and
most of the heat shock proteins belong to this group.75 Expression of molecular chaperone proteins prevents protein aggregation and helps the cell to adapt the unfavorable environmental
conditions.76 Several investigations have confirmed changes in
the regulation of chaperone proteins in response to abiotic
stress.31,76,77 The expression of molecular chaperones does not
follow a set of pattern in response to stress. The age of the plant
and the severity and duration of the stress could influence
whether a given protein would be up-regulated or downregulated. Furthermore, the expression of a molecular chaperone
can vary across organelles. For these reasons, studies on the
molecular chaperone subproteome must be carefully planned
with respect to the stress conditions and type of cells and
organelles involved.
Ion Transporters
Changes in the flux of H+, K+, Cl , and Ca2+ ions across the
plasma membrane alters the cytosolic pH and transmembrane
electrical potential. Sun et al.65 reported that accumulation of
these ions in plant tissues could induce stress. The concentrations of Na+ and Cl ions are normally kept low in plant cells,
while nutritionally important elements such as K+ are maintained
at high concentrations. Salinity is a typical abiotic stress that may
upset the homeostasis of Na+, H+, K+, and Cl ions. The plasma
membrane and vacuoles are the two major subcellular components of cells that are involved in maintaining ion balance.65,66
Regulator proteins, such as tonoplast Na+/H+ antiporters and
plasma membrane Na+/H+ antiporters, play important roles
in sequestering Na+ in vacuoles or extruding it to the external
environment, respectively.67,68 Proton pumps associated with
the plasma membrane facilitate stomatal closure under drought
stress by mediating the efflux of K+ and various anions from
guard cells. In this process, Ca2+ regulates the activity of plasma
membrane H+-ATPase.69 Plasma membrane proton pumps are
the integral membrane proteins that are involved in maintaining
the ion balance of the cell under osmotic stresses. Homeostasis
of intracellular ions is important for the activity of many cellular
Proteolysis-Related Proteins
Misfolded, unassembled, or mutated proteins are degraded in
cells through a proteolytic process. The endoplasmic reticulum,
which is the major site of protein folding and quality control, also
has a specific proteolytic system. The protein breakdown mechanism is usually mediated by ubiquitin, although proteolysis
without ubiquitin is possible.78 It was reported that the ubiquitinproteasome system of the endoplasmic reticulum degradation
machinery also extends into the cytoplasm.79 The protein
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Figure 2. Classification of abiotic stress-related proteins and their subcellular distributions. Negative effects of abiotic stress regulate 6 major groups
of defense-related proteins. Examples of proteins are represented in the parentheses and the main locations of the proteins are represented in brackets.
All, all cellular organelles, compartments and cytoplasm; PM, plasma membrane; Ton., tonoplast; ER, endoplasmic reticulum; Cyto., cytoplasm.
consequence of water deficit is enhanced H2O2 production.81
Interestingly, ABA itself stimulates H2O2 production by the
membrane bound enzyme NADPH oxidase,82,83 which basically
catalyzes the production of another free radical superoxide from
oxygen and NADPH.84 However, recent findings suggest that
water stress-induced ABA actually prevents the excessive accumulation of H2O2, by inducing CATB gene expression.85 In the
plant cell, H2O2 acts as a signaling molecule and activates
mitogen-activated protein kinase (MAPK) cascade for stress
responsive genes expression.84,86 Nuclear proteome analyses
lead to the identification of stress signal transduction pathway
components like serine/threonine protein kinase, histidine kinase, tyrosine phosphatase.21,22 In addition, presence of FCA
protein (a known nuclear ABA receptor87) suggests its involvement in the ABA signaling pathway.
Transcription factors (TFs) are the key proteins that facilitate
the transcription process. Several TFs like RF2b, bZIP, HB3,
homeobox-leucine zipper protein were found to be up-regulated
under dehydration stress.21,22 Basically, early stress response
genes are activated first, producing TFs for induction of delayed
stress response genes.88
The superoxide radicals formed as a result of NADPH oxidase
activity need to be scavenged quickly to protect the nucleus from
oxidative damage to DNA. SOD acts as the first level of defense
against ROS as it directly controls the concentrations of two
important ROS namely superoxide radicals and hydrogen peroxide. The excess H2O2 in turn gets nutralized by the activity of
APX or GPX. Drought-induced up-regulation of SOD, APX,
GPX, GST proteins21,22 suggests the presence of well-equipped
antioxidant system in plant nucleus to cope with the abiotic
stresses. Apart from antioxidants, accumulation of molecular
chaperons (HSP20, HSP70, Chap60, dnaK) helps in refolding
of misfolded proteins. In addition, drought induced synthesis of
DHNs proteins further give protection to the membrane from
stress induced damages.89,90 Mitochondria and chloroplasts are
the two potential subcellular sites for ROS generation. Under
water deficit condition, electron transport chains get overreduced resulting formation of superoxide radicals and singlet
oxygen. Mitochondrial proteome study reveals the abundance of
degradation pathways of the ubiquitin-proteasome have also
been found in nucleus.21 Since abiotic stress leads to the
accumulation of misfolded and unfolded proteins, protein breakdown and recycling is an essential feature of the plant response to
environmental stress.80 Proteolytic proteins and molecular chaperones represent two groups of stress-responsive proteins that
directly interact with target proteins. The activity of proteolysisrelated proteins can be considered as the last step in the cell’s
effort to survive under stress conditions.
Organelles are specified to operate cellular functions in
cooperation with other compartments. Role of each organelle
individually or in combination with cytosol or other compartments is described. Abiotic stress has wide range of deleterious
effects on the cell and the survival of plant cell under stress condition highly depends on the interaction among organelles and
compartments. The major negative effects of abiotic stress can be
classified into 4 categories including changes in osmotic potential
or water content, ROS production, ion imbalance and protein
misfolding or aggregation (Figure 2).
’ MODULATION OF ORGANELLE PROTEOME UNDER
DROUGHT STRESS
Plant experiences drought stress when there is a shortage of
water around the root zone. Among the different abiotic stress
factors, drought is the most adverse environmental condition
that negatively affects plant growth, development and yield
throughout the world. Different aspects of plant response toward
dehydration stress are well documented. However, sufficient
information on drought sensing and tolerance mechanism at
the organelle proteome level is not available. In this section,
published proteomic works on dehydration stress mediated
changes in organelle proteomes are summerized for better
understanding of the stress responsive signal pathway and
functional role of defense related proteins in conferring stress
tolerance at organelle level (Figure 3).
Transient increase in endogenous ABA level in response to
drought stress triggers the downstream response that eventually
leads to expression of stress responsive genes. Another inevitable
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Figure 3. Drought stress induced activation of defense related organelle proteins. The scheme is based on the published proteomic works on
dehydration stress mediated changes in organelle proteomes.20 22,26,29 Abbreviations: ABA, abscisic acid; H2O2, hydrogen peroxide; NADP,
Nicotinamide adenine dinucleotide phosphate; SOD, superoxide dismutase; APX, ascorbate peroxidase; GPX, glutathione peroxidase; HSPs, heat
shock proteins; Ser/Thr, serine-threonine; His, histidine; DHNs, dehydrins; ROS, reactive oxygen species; O2.‑, superoxide radical; TF, transcription
factor.
Cu Zn isoform of SOD in drought stressed pea mitochondria.20
Up-regulation of antioxidant enzymes (APX, SOD, MDAR) in
cell wall27,30 further confirms the presence of a robust antioxidative defense system at the organelle level. In addition, abundance of chaperonin in mitochondrial as well as in cell wall
proteomes20,27,30 ensures proper protein folding under dehydration stress.
Survey of articles on organelle proteomics under abiotic stress
has revealed that not much information is available on droughtmediated changes in the chloroplast proteome. In addition, a
large number of stress-induced organelle proteins remain unidentified. So, there is an urgent need of initiating organelle
proteomic research particularly in chloroplast to dissect the stress
responsive proteins in conferring plant stress tolerance.
mechanisms. In-depth analysis of the research works done in
recent times revealed that proteome researcher mainly used
2-DE technique to separate the protein pool while studying the
plant abiotic stress response at organelle level. Interestingly, both
2-DE/MALDI-TOF MS and gel free-LC MS/MS systems were
extensively used for identification of differentially expressed
organelle proteins under stress. To understand the mechanism
of stress tolerance, a detailed study of membrane proteins is
important as they play key role in stress signal perception and
transduction to turn-on the stress responsive genes. As most of
the organelle membrane proteins are hydrophobic in nature,
nanoLC MS/MS gel free system would be the most promising
technique for identification of such proteins. By summerizing
significant contributions related to abiotic stresses and organelle
proteomes, efforts have been made in the review to delineate the
molecular basis of acquisition of stress tolerance mechanism at
the organelle level. This would further enable us to find proteinbiomarkers linked to plants’ abiotic stress tolerance. Instead of
applying the finest proteomics techniques, many organelle
proteins either stress-induced or house-keeping still remain
unclassified. Future initiatives should be taken to identify and
characterize those organelle proteins, which might open a new
avenue for the proteome-based abiotic stress research. Finally,
’ CONCLUDING REMARKS
The present review outlines the impact of various abiotic
stressors on the plant cell organelle proteome. Improved protein
extraction protocols and advance techniques for identification of
novel proteins have been standardized in different plant species
at both cellular and whole plant levels for better understanding of
abiotic stress sensing and intracellular stress signal transduction
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REVIEWS
we do hope that this review would not only provide new insights
into the plants stress response mechanisms, which are necessary
for future development of genetically engineered stress tolerant
crop plants for the benefit of humankind, but would also highlight the importance of studying changes in protein abundance
within the cell organelles in response to abiotic stress.
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Mitochondrial proteome during salt stress-induced programmed cell
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Differential impact of environmental stresses on the pea mitochondrial
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(21) Choudhary, M. K.; Basu, D.; Datta, A.; Chakraborty, N.;
Chakraborty, S. Dehydration-responsive nuclear proteome of rice
(Oryza sativa L.) illustrates protein network, novel regulators of cellular
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8, 1579–1598.
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’ ASSOCIATED CONTENT
bS
Supporting Information
Supplementary Table 1. A summary of published review
articles on plant proteomics (2007 2011). This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Setsuko Komatsu, National Institute of Crop Science, Kannondai
2-1-18, Tsukuba 305-8518, Japan. Tel: +81-298-38-8693. Fax:
+81-298-38-8694. E-mail: skomatsu@affrc.go.jp.
’ ACKNOWLEDGMENT
Z.H. thankfully acknowledges the financial support provided
through the DST-BOYSCAST fellowship programme, government of India for conducting advance research at National
Institute of Crop Science, Japan. M.-Z.N. was supported by
Monbukagakusho (Japanese government) scholarship program.
This work was supported by the grants from National Agriculture
and Food Research Organization, Japan.
’ ABBREVIATIONS:
2-DE, two-dimensional polyacrylamide gel electrophoresis; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;
BN-PAGE, blue native-polyacrylamide gel electrophoresis; MS, mass
spectrometry; MALDI TOF, matrix-assisted laser desorption ionization time-of-flight; Q-Tof, quadrupole time of fligh; LC ESI MS/
MS, liquid chromatography electrospray ionization tandem mass
spectrometry; ROS, reactive oxygen species; TCA, tricarboxylic
acid cycle; SOD, superoxide dismutases; AOX, alternative oxidase;
PCD, programmed cell death; 2D-DIGE, two-dimensional fluorescence difference gel electrophoresis; MudPIT, multidimensional
protein identification technology
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