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Modulation of plant proteome composition is an inevitable process to cope with the environmental challenges including heavy metal (HM) stress. Soil and water contaminated with hazardous metals not only cause permanent and irreversible health problems, but also result substantial reduction in crop yields. In course of time, plants have evolved complex mechanisms to regulate the uptake, mobilization, and intracellular concentration of metal ions to alleviate the stress damages. Since, the functional translated portion of the genome plays an essential role in plant stress response, proteomic studies provide us a finer picture of protein networks and metabolic pathways primarily involved in cellular detoxification and tolerance mechanism. In the present review, an attempt is made to present the state of the art of recent development in proteomic techniques and significant contributions made so far for better understanding the complex mechanism of plant metal stress acclimation. Role of metal stress-related proteins involved in antioxidant defense system and primary metabolism is critically reviewed to get a bird’s-eye view on the different strategies of plants to detoxify HMs. In addition to the advantages and disadvantages of different proteomic methodologies, future applications of proteome study of subcellular organelles are also discussed to get the new insights into the plant cell response to HMs.
High-throughput OMICS techniques are extensively being exploited in recent times to dissect plants molecular strategies of heavy metals (HMs) stress tolerance. Plants growing in HMs contaminated environment have developed coordinated homeostatic mechanisms to regulate the uptake, mobilization, and intracellular concentration of toxic metal ions to alleviate stress damages. As the functional translated portion of the genome play a key role in plant stress response, proteomic studies provide us a finer picture of protein networks and metabolic pathways primarily involved in cellular detoxification and tolerance mechanism against HM toxicity.
By definition, elements having specific gravity above five are considered as HMs. Nevertheless, the term HM commonly refers to toxic metals, e.g., cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), zinc (Zn) as well as hazardous metalloids viz., arsenic (As), boron (B), which exert negative effects on plant growth and development (
Most of the HMs get their entry into plant root system via specific/generic ion carriers or channels (
Over the last decade, extensive research on plants response to HM stress has been conducted to unravel the tolerance mechanism. Genomics technologies have been useful in addressing plant abiotic stress responses including HM toxicity (
Plant response to HM stress has been reviewed extensively over the past decade (
Current review represents the state of art of recent developments in proteomic techniques and significant contributions made so far to strengthen our knowledge about plants HM-stress response cascade at protein level. Special emphasis is given to highlight the role of metal stress-related proteins engage in HM ions sequestration, antioxidant defense system, and primary metabolism for deeper understanding of coordinated pathways involve in detoxification of HM ions within plant cells. Furthermore, future applications of proteome study of subcellular organelles are discussed to get the new insights into the plant cell response to HMs.
Conventional two-dimensional gel electrophoresis (2-DE) approach coupled with protein identification by mass spectrometry (MS) has been the most widely used proteomic technique for investigation of HM-induced alteration of plant proteome composition (
Summary of functional proteomic analyses in response to heavy metal stress (2007–2012).
Metal | Plant (tissue) | Protein extraction buffer + precipitation | Protein solubilization/lysis buffer | Proteomic methodologies | IP | Major findings | Reference |
---|---|---|---|---|---|---|---|
Cd | 10% TCA, 0.07% 2-ME in acetone | 8 M urea, 2 M thiourea, 5% CHAPS, 2 mM TBP, ampholytes (pH 3–10) | IPG, 2-DE, nanoLC-MS/MS, MALDI-TOF MS | 32 (HL), 26 (FL), 44 (CL), 16 (R) | Activation of SOD, APX, and CAT ensures cellular protection from ROS mediated damages under cadmium stress; enhanced expression of molecular chaperones help in stabilizing protein structure and function, thus maintain cellular homeostasis. | ||
10% TCA, 0.07% 2-ME in acetone | 8 M urea, 2 M thiourea, 5% CHAPS, 2 mM TBP, ampholytes (pH 3–10) | IPG, 2-DE, nanoLC-MS/MS, MALDI-TOF MS | 78 | High abundance of Hsp70 helps BABA-primed plants to maintain normal protein functions; higher abundance of Prx indicates BABA potentiated antioxidant defense system to combat Cd stress. | |||
0.5 M Tris–HCl (pH 8.0), 2 mM EDTA, 2 mM DTT, 0.25 M sucrose, 1 mM PMSF + Tris–HCl saturated phenol | 8.5 M urea, 2.5 M thiourea, 5% CHAPS, 1% DTT, 1% Triton X-100, 0.5% Biolyte (pH 5–8) | IPG, 2-DE, nanoLC-MS/MS | 22 | Up-regulation of proteins associated with Cd-chelating pathways and increased lignification of xylem vessels lead to low root-shoot translocation of Cd in cv. Enrei. | |||
phenol-saturated Tris–HCl 0.1 M (pH 8.0), 5 mM ME | 8 M urea, 2% (w/v) CHAPS, 50 mM DTT, 2 mM PMSF, 0.2% (v/v) 3–10 ampholytes | IPG, 2-DE, MALDI-TOF-MS, LIFT TOF–TOF | 27 (low Cd), 33 (high Cd) | Low Cd treatment (10 µM) activates glycolysis, TCA cycle and respiration; at high Cd (100 µM) major decreases in growth, a shutdown of the carbohydrate metabolism and decreases in respiration takes place. | |||
0.5 M Tris–HCl (pH 8.0), 50 mM EDTA, 900 mM sucrose, 100 mM KCl, 2% ME, 1 mM PMSF + Tris-buffered phenol (pH 8.0) | 7 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF, 50 mM DTT, 0.5% IPG buffer | IPG, 2-DE, MALDI-TOF MS | 18 (R) |
ROS scavengers (GST, APX, NADH-ubiquinone oxidoreductase) primarily up-regulated in roots under Cd treatment, indicates prompt antioxidative response against oxidative stress damages. | |||
Tonoplast proteins dissolved in iTRAQ dissolution buffer | – | iTRAQ labeling, MALDI-TOF/TOF MS | 56 | Candidate proteins like CAX1a and MRP-like ABC transporter play significant role in vacular Cd2+ transport, hence Cd2+ detoxification. | |||
Tris-buffered phenol (pH 8.8) and 600 mL of 0.1 M Tris–HCl with 10 mM EDTA, 0.4% v/v 2-ME, 0.9 M sucrose | DIGE solubilization buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 0.2% w/v SDS, 10 mM Tris, pH 8.5), and 0.5 M bicine pH 8.4 with 0.09% w/v SDS (for iTRAQ Label) | IPG, 2-D DIGE, iTRAQ, nanoLC-MS/MS | 102 (DIGE), 585 (iTRAQ) | ||||
20% TCA and 0.1% (w/v) DTT in ice-cold acetone | Labeling buffer | IPG, 2-D DIGE, MALDI-TOF-TOF MS | 125 | Up-regulation of mitochondrial respiration provides energy and reducing power to cope with met al stress, photosynthesis comparatively less affected. | |||
Cu | 1.5% w/v PVP, 0.7 M sucrose, 0.1 M KCl, 0.5 M Tris–HCl (pH 7.5), 250 mM EDTA, protease inhibitor, 2% v/v ME, 0.5% w/v CHAPS + phenol saturated Tris–HCl (pH 7.5) | 7 M Urea, 2 M Thiourea, 4% w/v CHAPS, 60 mM DTT, 20 mM Tris–HCl (pH 8.8), Biolytes (pH 3–10) | IPG, 2-DE, MALDI-TOF MS | 10 (Es32) |
Copper stress leads to up-regulation of photosynthesis (PSII Mn-stabilizing protein of OEC33), glycolysis, and pentose phosphate metabolism; higher accumulation of HSP70 and vBPO for proper protein folding and ROS detoxification respectively. | ||
50 mM Tris–HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol (DTT), and 1 mM PMSF + ice-cold |
8 M urea, 4% CHAPS, 65 mM DTT, 0.2% (w/v) Biolytes (pH 3–10) | IPG, 2-DE, MALDI-TOF MS | 16 | First proteomic evidence that met allothionein and CYP90D2 (a putative small cytochrome P450) are Cu-responsive proteins in plants. | |||
0.5 M Tris–HCl (pH 7.5), 0.7 M sucrose, 50 mM EDTA, 0.1 M KCl, 10 mM thiourea, 2 mM PMSF/DMSO, 2% v/v ME + phenol saturated Tris–HCl (pH 8.8) | 9 M urea, 4% w/v CHAPS, 0.5% Triton X-100, 20 mM DTT, 2% v/v IPG Buffer | IPG, 2-DE, LC-MS/MS | 20 | Copper induced aldo/keto reductase acts as copper chaperone reduce copper ions to Cu (I), promote PCs-mediated vacuolar transport; Suppression/no change in ROS scavenging enzymes. | |||
0.5 M Tris–HCl (pH 8.3), 2% v/v NP-40, 20 mM MgCl2, 2% v/v ME, 1 mM PMSF, 1% w/v PVP + acetone | 9.5 M urea, 2% v/v NP-40, and 2.5% v/v pharmalytes (pH 3–10: pH 5–8: pH 4–6.5 = 1:3.5:2.5) | IEF gel (tube gel), 2-DE, MALDI-TOF MS | 25 | Excess Cu induces oxidative stress thus hampering metabolic processes; up-regulation of antioxidant and stress-related regulatory proteins (glyoxalase I, peroxiredoxin) help to maintain cellular homeostasis. | |||
B | 0.06 M DTT, 10% (w/v) TCA in cold acetone with 0.06 M DTT | 2 M thiourea, 7 M urea, 4% (w/v) CHAPS, 0.4% (v/v) TritonX-100, 0.06 MDTT, and 1%(v/v) IPG buffer 3–10 NL | IPG, 2-DE, LC-MS/MS | 128 | Proteins associated with energy (glycolysis, TCA cycle, oxidation–reduction), cell division, protein metabolic processes suppressed under B deficiency. | ||
50 mM phosphate buffer (pH 7.5), 20 mM KCl, 0.5 M Suc, 10 mM DTT, 0.2 mM PMSF, 10 mM EDTA, 10 mM EGTA + 10% (w/v) TCA in acetone | 0.5 M TEAB (pH 8.5) containing 0.1% SDS | iTRAQ peptide tagging, MS/MS | 139 | Higher abundance of Iron deficiency sensitive2 [IDS2], IDS3, and methylthio-ribose kinase observed in B-tolerant barley is linked to siderophore production | |||
As | 10 mM Tris–HCl (pH 8.0), 1.5 mM MgCl2, 10 mM KCl + 10% (w/v) TCA in acetone | 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, and 1.0% IPG buffer (4–7) | IPG, 2-DE, MALDI-TOF, and LC-MS | 45 | Up-regulations of PGK, FBA II, FBPase, TK, ATP synthase, Prx, Trx, oxidoreductase help to maintain normal glycolysis, PPP, and turnover rate of Calvin cycle, protect cells from oxidative stress, thereby helping As-stress acclimation. | ||
0.5 M Tris–HCl (pH 8.3), 2% (v/v) NP-40, 20 mM MgCl2, 2% (v/v) ME, 1 mM PMSF, 0.7 M sucrose + acetone precipitation | 8 M urea, 1% CHAPS, 0.5% (v/v) IPG buffer pH 4–7, 20 mM DTT | IPG, 2-DE, MALDI-TOF MS, ESI-MS/MS | 12 | Energy and metabolism related proteins over expressed indicating higher energy demand under As stress; down-regulation of RuBisCO and chloroplast 29 kDa ribonucleoproteins lead to decreased photosynthesis. | |||
As (V and III) | Glacial acetone containing 0.07% (v/v) 2-ME, 0.34% (w/v) plant protease inhibitor, and 4% (w/v) PVP | 4% (w/v) CHAPS, 7 M urea, 2 M thiourea, 2% (w/v) DTT, 1% (w/v) pharmalytes pH 3–10, 1% (w/v) resolytes pH 6–9.5 | IPG, 2-DE, MALDI-TOF MS | 31 | As treatment resulted in partial disruption of the photosynthetic processes with prominent fragmentation of the RubisCO. | ||
0.5 M of Tris–HCl (pH 8.3), 2% v/v NP-40, 20 mM MgCl2, 2% v/v ME, 1 mM PMSF, 0.7 M sucrose + acetone precipitation | 8 M urea, 1% CHAPS, 0.5% v/v IPG buffer pH 4–7, 20 mM DTT | IPG, 2-DE, MALDI-TOF MS | 23 | Energy, primary metabolic pathways suppressed under stress; higher GSH content coupled with enhanced expressions of GR, SAMS, GSTs, CS, GR mitigate As-induced oxidative stress. | |||
Mn | 700 mM sucrose, 500 mM Tris, 50 mM EDTA, 100 mM KCl, and 2% v/v ME + water saturated phenol | 8 M urea, 2% w/v CHAPS, 0.5% v/v IPG buffer pH 3–11, 50 mM DTT | IPG, 2-DE, Nano-LC-MS/MS, ESI MS/MS | 8 | Lower abundance of chloroplastic proteins involved in CO2 fixation and photosynthesis indicate channelizing metabolic energy to combat the Mn-stress; coordinated interplay of apoplastic and symplastic reactions essential for stress response. | ||
Cr | 0.5 M Tris–HCl, pH 8.3, 2% (v/v) NP-40, 20 mM MgCl2, 1 mM PMSF, 2% (v/v) ME, and 1% (w/v) PVP | 8 M urea, 1% CHAPS, 0.5% (v/v) IPG buffer pH 4–7, 20 mM DTT | IPG, 2-DE, MALDI-TOF MS, MALDI-TOF/TOF MS | 36 | Novel accumulation of chromium-responsive proteins (e.g. IMPase, nitrate reductase, adenine phosphoribosyl transferase, formate dehydrogenase, putative dihydrolipoamide dehydrogenase) observed; Cr toxicity is linked to heavy met al tolerance and senescence pathways. | ||
500 mM Tris–HCl (pH 8), 700 mM sucrose, 10 mM EDTA, 4 mM ascorbate, 0.4% ME, 0.2% Triton X-100 10%, 1 mM PMSF, 1 µM Leupeptin, 0.1 mg/mL Pefabloc + water saturated phenol | 7 M urea, 2 M thiourea, 4% CHAPS, 50 mg/mL DTT | IPG, 2-DE, LC-ESI-MS/MS | 16 | Cr-stress target photosynthetic proteins (RuBisCO, RuBisCO activase, Light Harvesting Chla/b protein complex, stress related Chl a/b binding protein) identified; Cr also induces modulation of proteins involved in amino acids metabolism. | |||
Al | 10% (w/v) TCA in acetone containing 0.07% (w/v) DTT, 1% PVP | 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 1% (w/v) DTT, and 2% Pharmalyte pH 3–10 | IPG, 2-DE, MALDI-TOF MS | 30 | Chaperones, PR 10, phytochrome B, GTP-binding protein, ABC transporter ATP-binding protein either newly induced or up-regulated, facilitate stress/defense, signal transduction, transport, protein folding, gene regulation, primary metabolisms. | ||
40 mM Tris-base, 5 M urea, 2 M thiourea, 2% w/v CHAPS, 5% w/v PVP, and 50 mM DTT + ice-cold acetone with 0.07% (w/v) DTT | 5 M urea, 2 M thiourea, 4% w/v CHAPS, 2% v/v IPG buffer, 40 mM DTT | IPG, 2-DE, MALDI-TOF/TOF MS, MALDI-TOF-MS | 17 | Antioxidation and detoxification lead by up regulation of Al-responsive proteins (Cu–Zn SOD, GST, SAMS 2), ultimately related to sulfur metabolism. CS, a novel Al-induced protein, play key role in Al resistance. |
As compared to classical staining procedure of 2-DE gel using CBB or silver staining, advanced fluorescence two-dimensional difference gel electrophoresis (2-D DIGE) proteomic approach is now being used which allows comparison of the differentially expressed proteins of control and HM-stressed tissue on one single gel (
In course of time, higher plants have evolved sophisticated mechanisms to regulate the uptake, mobilization, and intracellular concentration of HM ions (
One of the important plant strategies of detoxifying HMs within cell is to synthesize low molecular weight chelators to minimize the binding of metal ions to functionally important proteins (
Phytochelatins synthesized from glutathione (GSH) by the enzyme PC synthase readily form complexes with HM in the cytosol and to facilitate their transport into vacuoles (
The PC biosynthetic pathway has been finely dissected in Cd-exposed
Unlike PCs, proteomics-based report on HM-induced alterations of MTs is very limited.
The final step of HM detoxification involves sequestering of either free HMs or PCs-HMs complexes into cell vacuoles (
Cellular ROS generation gets accelerated upon exposure to HM stress. HMs (Cu, Fe, Cr) that are directly involved in cellular redox reaction lead to ROS generation known as redox active, while redox inactive HMs (Cd, Al, As, Ni) trigger oxidative stress by depleting cells major thiol-containing antioxidants and enzymes, disrupting electron transport chain or by inducing lipid peroxidation (
Most of the proteomic research done so far on HM-related toxicity revealed positive correlation between tolerance and increased abundance of scavenger proteins. Within plant cells, SOD constitutes the first line of defense against ROS. It plays pivotal role in cellular defense against oxidative stress, as its activity directly modulates the amount of
The abundance of another antioxidant enzyme of ascorbate–GSH cycle, monodehydroascorbate reductase (MDAR) was found to be increased in response to Cd (
Plants are also equipped with some additional defense proteins, shown to be up-regulated by HM stress. This group includes thioredoxin (Trx), Trx-dependent peroxidase, NADP(H)-oxido-reductase and glyoxylase I (Gly I). Trx is known to suppress apoptosis as well as supplies reducing equivalents to antioxidants (
Methylglyoxal (MG), a cytotoxic by-product of glycolysis generally accumulated in cell in response to environmental stresses including HM (
Plants tolerance against HMs is often attributed to steady state of GSH pool for its multifunctional activities in PC synthesis, MG detoxification, ROS scavenging through ascorbate–GSH cycle, GSTs mediated decomposition of toxic compounds as well as stress signaling (
Proteomic analyses strongly indicate that accumulation of defense proteins chiefly enzymatic components of ascorbate–GSH cycle, POD, CAT, GSH, GSTs, Gly I, Prx, Trx help cells to mitigate HM-induced oxidative stress by scavenging ROS.
Protein dysfunction is an inevitable consequence of a wide range of adverse environmental conditions including HM toxicity. Molecular chaperones/heat-shock proteins (HSPs) are responsible for protein stabilization, proper folding, assembly, and translocation under both optimum and adverse growth conditions (
Down-regulation of photosynthetic machinery is a known phenomenon of Cd stress. Low abundance of proteins involved in photosynthetic electron transport chain and Calvin cycle has been reported in Cd-exposed
To maintain the normal growth and development under stressed environment, plants need to up regulate metabolic pathways such as glycolysis and tricarboxylic acid (TCA) cycle. Detailed analysis of HM toxicity-related proteomic works has shown higher abundance of glycolytic enzymes phosphoglycerate mutase (PGM), glucose-6-phosphate isomerase (G6PI), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), enolase (ENO), and pyruvate kinase (PK) in response to Cd (
Like glycolysis, enzymes of TCA cycle citrate synthase (CS), succinate dehydrogenase (SD), malate dehydrogenase (MDH), aconitase (ACO), aconitate hydratase (AH) were found to be up-regulated under Cd stress (
Plant cells trigger some common defense machineries whenever they encounter a biotic or abiotic stress. Accumulation of PR proteins is one of such plant defense strategies and often associated with systemic acquired resistance (SAR) against a wide range of pathogens (
The present review outlines the impact of HMs stresses on plant proteome constituents. Most of the investigations done so far primarily highlighted the differential expression of proteins involved in plant defense and detoxification pathways, namely ROS scavenging, chelation, compartmentalization. In addition, accumulation of PR proteins and modulation of plants vital metabolic pathways CO2 assimilation, mitochondrial respiration in maintaining steady state of reducing power and energy required for combating HM-induced stress has been discussed in detail. Careful analysis of published proteomic works on HM toxicity has revealed that classical 2-DE coupled with MS-based protein identification has been the most widely used proteomic technique in investigating plant HM tolerance at organ/whole plant level. These proteomic findings have enriched us for deeper understanding plants HM tolerance mechanism.
The cellular mechanism of sensing stress and transduction of stress signals into the cell organelle represent the initial reaction of plant cells toward any kind of stress including HM. Communication through intracellular compartments plays a significant role in stress signal transduction process that finally activates defense gene cascade (
As the PCs mediated HM-ion detoxification pathway ends in sequestration of PC-HM complexes into vacuole through various transporter proteins present in tonoplast membrane, more research on vacuole proteome needs to be undertaken for identification and characterization of novel metal transporter proteins responsible for cytoplasmic efflux of transition metal cations into vacuole. Legendary work by
Plants response to multiple HMs would be another interesting area of future proteomic research (
Heavy metal-induced protein oxidation study through redox proteomic approach has been the focus of much interest. More initiatives in this topic need to be taken as PTM/redox modification of proteins provides fundamental information about HM toxicity mechanism and biomarker discovery (
In summary, we believe that more research on sub-proteome-based HM approach would provide new insights into plants HM-stress response mechanism. HM-induced novel marker proteins would further enable us to design HM-tolerant transgenic crops.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thankfully acknowledge support from the Department of Science and Technology, Government of India, through DST-BOYSCAST Fellowship Programme and National Agriculture and Food Research Organization, Japan.
coomassie brilliant blue
two-dimensional polyacrylamide gel electrophoresis
glutamine synthetase
glutathione
glutathione
isoelectric focusing
liquid chromatography
mass spectrometry
metallothioneins
phytochelatins
isoelectric point
pathogenesis related
reactive oxygen species
superoxide dismutase.