Plant Proteome in Response to Abiotic Stresses

Abstract

Abiotic stresses, including drought, heat, salinity, waterlogging, and toxic metal stress, can negatively impact plant growth, leading to reduced crop yield and quality. Plants employ two key strategies, avoidance and tolerance, to adapt to such stress, making cellular and metabolic adjustments to withstand adverse conditions. Acclimatization results in significant changes in a plant’s proteome, crucial for its stress response. Proteins encoded by a plant’s genome play a vital role in environmental adaptation, supporting biotechnological advancements in plant breeding, utilizing data from proteomic investigations. Proteomics provides unique insights into post-translational modifications and their impact on crop yield. Subcellular proteomics aids in understanding cellular responses and interactions during growth and responses to biotic and abiotic stresses. Proteomic tools, like mass spectrometry, liquid chromatography, protein microarrays, and antibody-based assays, are integral to proteomic studies, contributing to our understanding of protein functions and interactions. Developing stress-tolerant crops is crucial for enhancing crop productivity and growth.

1. Introduction

Abiotic stress encompasses various environmental factors such as drought, salt, temperature fluctuations, waterlogging, and nutritional imbalances, all of which significantly impede plant growth and yield. These stresses create unfavourable conditions for plants. Predictions indicate that approximately half of all crop losses are attributed to abiotic stresses. Profound changes in the physiological, molecular, and biochemical processes within plants limits their distribution, alter growth patterns, and diminishes yield. Drought, a major abiotic stress, triggers oxidative stress through the overproduction of reactive oxygen species (ROS), leading to cell membrane damage and activating various stress-signalling pathways. Key responses to drought stress include root development, stomatal closure, photosynthesis, hormone production, and ROS scavenging. Salt stress negatively impacts plant growth and reproduction through nutritional and hormonal imbalances, ion toxicity, oxidative and osmotic stress, and increased susceptibility to diseases. The three major ways plants are damaged by salt stress involve altered soil properties causing water stress, destabilization of cell membranes, and protein degradation due to toxic ion effects. Physiological and metabolic changes occur, affecting seed germination, photosynthesis, biosynthetic processes, and overall growth reduction. Different crops respond differently to salinity, with glycophytes experiencing growth and yield reduction, while halophytes thrive. Plants cope by accumulating compatible solutes, redistributing ions, and increasing endogenous abscisic acid content, resulting in changes in genetic expression. Elevated levels of ions, such as Na⁺ and Cl⁻, induce ionic toxicity, disrupting ion homeostasis and limiting essential nutrient availability for plant growth and metabolism [1].

High temperatures exert a detrimental impact on plant yield, particularly during the vulnerable reproductive stage, causing a substantial reduction in seed set and overall crop yield. This vulnerability is especially pronounced in food crops like rice, wheat, soybean, maize, cotton, sorghum, and tomato. Intense high temperatures contribute to pollen abortion, leading to incomplete pollination and hindering successful reproduction. The delicate balance between pollen and pistil interactions is disrupted under these conditions, further compromising the reproductive process [2]. During waterlogging reduced light intensity, restricted gas diffusion, nutrient leaching, and hypoxia are pronounced effects. Plants exhibit active responses to stress, with the nature of their reaction contingent upon the intensity and duration of the stress.

Kosova et al. (2018) delineated five phases characterizing plant responses to stress. The initial alarm phase occurs when plants first encounter stress, followed by the acclimation phase during which biological processes adapt to stress, leading to increased tolerance. Subsequently, the maintenance phase ensues, where plants sustain their tolerance levels. The exhaustion phase follows, marked by a decline in tolerance due to prolonged stress and the failure to maintain homeostasis achieved during acclimation. The final stage is the recovery phase, wherein plants, upon stress withdrawal, regain normal homeostasis [3]. Each phase may be distinguished by a unique protein composition. Acclimation is an energy-intensive, dynamic process, with metabolic adjustments reflected at both the metabolic and proteomic levels in response to stress [4, 5].

In recent decades, research employing diverse Omics approaches has put forth numerous abiotic stress-related mechanisms as potential contributors to the creation of resilient plant varieties. The comprehensive utilization of functional Omics plays a crucial role in elucidating the intricate connection between an organism’s genome and its observable characteristics in varying environmental conditions. Among all the Omics approaches, proteomics offers a comprehensive exploration of the structural, functional, abundance and interactions of proteins at a specific point of time. This technique holds an advantage over other “omics” tools, given that proteins play a pivotal role in the majority of cellular processes. Beyond complementing changes observed at the genomic and transcriptome level, proteomics has the capacity to identify translational and post-translational regulations, providing additional insights into intricate biological phenomena like abiotic stress responses in plants. This chapter delves in to the proteomics studies focused on plant responses to drought, salinity, flooding, and waterlogging.

1.1 Proteome

Marc Wilkins coined the term “proteome” in 1994 to refer to the complete set of proteins in a given organism during a specific time period, essentially representing the protein complement of the genome. In contrast to the genome, a static structure inherited from parents that defines the plant genotype, alterations in the plant epigenome, transcriptome, proteome, and metabolome collectively influence the plant phenotype.

Proteins play a direct role in the plant stress response, serving as both structural components and regulators of the plant epigenome, transcriptome, and metabolome. Additionally, the functionality of a protein is not solely determined by its molecular structure; it is also influenced by factors such as cellular localization, post-translational modifications, and interactions with other proteins. These aspects collectively contribute to the nuanced and multifaceted role that proteins play in the intricate mechanisms of plant stress adaptation and response. Proteomics, a state-of-the-art molecular technique, provides distinct advantages over genome-based technologies by directly focusing on functional molecules rather than the genetic code or mRNA abundance [6].

1.2 Importance of proteomics for abiotic stress

The field of plant stress proteomics is a dynamic discipline focused on investigating the plant proteome and the biological functions of proteins in plants that are subjected to various stress conditions. This area of study aims to unravel the complex and dynamic changes occurring at the protein level in response to stress, providing insights into the molecular mechanisms underlying a plant’s adaptive responses to environmental challenges [7].

In plant abiotic stress studies, it is common to analyse proteomes by contrasting stressed plants with controls to correlate changes in protein accumulation with phenotypic responses. Additionally, comparing genotypes with varying stress sensitivity (sensitive vs tolerant) is crucial for understanding the influence of differentially abundant proteins in tolerant genotypes.

2. Proteomic tools to address abiotic stresses in plants

In response to abiotic stress, plants activate specific cellular processes leading to the accumulation of protective proteins and adjustments in metabolism and development. This involves substantial alterations in their gene expression patterns, which are regulated by various transcription factor (TF) families and signal-sensing networks, including phosphorylation/dephosphorylation events and other post-translational modifications (PTMs). These PTMs can either stabilize TFs or spot them for degradation. However, detecting and quantifying these elements through proteomics is tricky due to the typically low abundance of TFs and the rapid, transient nature of protein PTMs like phosphorylation and cysteine oxidation/reduction [8].

Quantitative proteomic methods can be categorized into gel-based or gel-free approaches, as well as “label-free” or “label-based” methods. The latter can be further divided into various types of labelling approaches, including chemical and metabolic labelling.

2.1 Low throughput-based approaches

2.1.1 Antibody-based methods

Techniques like ELISA (enzyme-linked immunosorbent assay) and western blotting rely on the presence of antibodies specifically directed at particular proteins or epitopes. These methods are utilized to identify proteins and quantify their expression levels.

2.1.2 Gel based approaches

2.1.2.1 Two-dimensional gel electrophoresis (2D-DIGE)

Since 1975, 2-DE has undergone various advancements, emerging as a primary method for protein separation and the preferred choice for analysing differential protein expression. In this technique, proteins undergo isoelectric focusing (IEF) based on their net charge at different pH values, followed by a second-dimensional separation based on molecular weight (MW) in an orthogonal direction.

Two types of reagents are integral to 2-DE buffers for protein solubility and denaturation. Chaotropes like urea and thiourea, employed at high concentrations, unfold proteins by weakening noncovalent bonds. The second type involves ionic detergents, exemplified by SDS (sodium dodecyl sulfate), which, with its long hydrocarbon chain, binds to hydrophobic amino acids, promoting denaturation.

2D-DIGE has proven effective in examining symbiosis and pathogenesis-related proteins in Medicago truncatula. Additionally, it has been employed to investigate the effects of abiotic stresses, including drought in oak, frost in Arabidopsis, as well as ozone and heavy metals in poplar. In context with the field crops, Natarajan et al., 2013 [9] analysed proteome of common bean and depicted that, 2D-PAGEfacilitated the identification of a significant number of low abundant proteins, demonstrating its efficacy in dissecting the common bean proteome with high resolution and sensitivity.

Limitations: less reproducibility, poor representation of less abundant proteins, hydrophobicity and problems in automation of the gel-based techniques.

2.1.2.2 Electrophoretic separations of innate proteins

This method facilitates the separation of protein complexes under native conditions, followed by the separation of individual proteins under denaturing conditions. This dual approach provides valuable insights into the stoichiometry of the complexes.

Recently, this approach was effectively employed to analyse the proteome of wheat chloroplast protein complexes.

Limitations: less resolution and difficult to optimize

2.1.2.3 One dimensional gel electrophoresis (1-DE)

One-dimensional gel electrophoresis (1-DE) has become the most widely adopted method, primarily serving two purposes: rapid determination of protein molecular weight (MW) and assessment of protein purity. This widely used technique finds applications in various areas, including comparing protein compositions of different samples, analysing the number and size of polypeptide subunits, conducting western blotting coupled with immunodetection, and, of course, serving as a second dimension in 2-DE maps.

The 1-DE approach has found extensive application in plant studies, as exemplified by research on M. truncatula plasma membrane alterations in response to arbuscular mycorrhizal symbiosis and investigations into the Arabidopsis thaliana chloroplast envelope. More recently, this method has been employed to construct a protein expression map of Arabidopsis roots, offering insight into the identity and cell type-specific localization of nearly 2,000 proteins.

2.1.2.4 Chromatography-based methods

Chromatography-based approaches are effective for the separation and purification of proteins from intricate biological mixtures like cell lysates. Ion-exchange chromatography, for instance, segregates proteins according to their charge, while size exclusion chromatography distinguishes proteins based on their molecular size. Affinity chromatography utilizes reversible interactions between specific affinity ligands and target proteins, such as the use of lectins to purify IgM and IgA molecules. These methods serve the dual purpose of purifying and identifying proteins of interest and preparing them for subsequent analysis, such as downstream mass spectrometry (MS).

2.2 High-throughput methods:

2.2.1 Mass spectrometry (MS)

This is a technique used to determine the molecular weight of proteins by measuring their mass-to-charge ratio (m/z). The process involves three steps. First, molecules need to be transformed into the gas phase. In the second step, ions are separated based on their m/z values in the presence of a mass analyzer. Finally, the separated ions and the quantity of each species with a specific m/z value are measured. Common ionization methods include matrix-assisted laser desorption ionization (MALDI), surface-enhanced laser desorption/ionization (SELDI), and electrospray ionization (ESI).

2.2.1.1 Mass spectrometry-based proteomics

Various “gel-free” methods exist for protein separation, such as isotope-coded affinity tag (ICAT), stable isotope labeling with amino acids in cell culture (SILAC), and isobaric tags for relative and absolute quantitation (iTRAQ). These approaches facilitate both quantitation and comparative/differential proteomics. Less quantitative techniques like multidimensional protein identification technology (MudPIT) are faster and simpler. Additionally, gel-free chromatographic methods for protein separation encompass gas chromatography (GC) and liquid chromatography (LC).

Tandem Mass Tag (TMT) is a sensitive and quantitative proteomic technique that enables the simultaneous identification and determination of the relative abundances of peptides from multiple samples. This method utilizes isobaric labeling in conjunction with tandem mass spectrometry (MS/MS) for analysis [10].

2.2.1.2 Isobaric tags for relative and absolute quantification (iTRAQ)

It is a multiplex protein labeling technique used for protein quantification, relying on tandem mass spectrometry. This method involves labeling proteins with isobaric tags, available in 8-plex and 4-plex configurations, for quantification purposes. The N-terminus and side chain amine groups in proteins are labeled and subsequently fractionated using liquid chromatography. The final analysis is conducted through Mass Spectrometry. iTRAQ is a suitable method that allows for the simultaneous identification and quantification of proteins [11]. Liu et al. (2020) assessed maize seed proteome, they explored the differential protein expression profiles between the two groups comparing transgenic and natural variation shedding light on the impact of genetic modification on the maize seed proteome. Their findings provide valuable insights into the molecular mechanisms underlying transgenic traits in maize seeds, offering potential implications for agricultural biotechnology and crop improvement strategies [12].

2.2.1.3 Reversed-phase chromatography (RP)

This is the most commonly used liquid chromatography method in proteomics, enabling the separation of neutral peptides based on their hydrophobicity. The process relies on the analyte’s partition coefficient between the polar mobile phase and the hydrophobic (non-polar) stationary phase. Yu et al. (2013) effectively utilized RP-UPLC to swiftly separate and characterize water-soluble proteins in wheat grains through optimized conditions. This method ensures fast, high-resolution, and reproducible separations with minimal resource consumption, showcasing its potential for rapid analysis in wheat breeding and fundamental genetic studies. RP-UPLC emerges as a robust tool for exploring wheat grain proteins, promising valuable insights for agricultural research and germplasm screening [13].

2.2.1.4 Two-dimensional liquid chromatography (2D-LC)

To mitigate sample complexity and enhance proteome coverage, multidimensional analytical methods with orthogonal separation capabilities are essential. The separation of peptide mixtures in 2D-LC has been achieved through various orthogonal combinations, including anion exchange coupled to reversed-phase (AX/RP), size exclusion chromatography coupled to reversed-phase (SEC/RP), and affinity chromatography coupled to reversed-phase (AFC/RP).

2.2.1.5 OFFGEL electrophoresis (OGE)

Orthogonal Gel Electrophoresis (OGE) boasts high loading capacity and resolution power. In contrast to LC fractionation, OGE offers additional physicochemical information, including peptide isoelectric point (pI). This information proves highly valuable for corroborating MS results, sorting false positive rates, and enhancing the reliability of the identification procedure.

2.2.1.6 Stable isotope labeling by amino acids in cell culture (SILAC)

This is a quantitative proteomic method that employs labeled isotopically heavy amino acids, such as 13C6,15N2-lysine and 13C6,15N4-arginine. These labeled amino acids are metabolically incorporated into the entire proteome, enabling multiplexing for quantitative analysis.

2.2.1.7 Nuclear magnetic resonance (NMR) spectroscopy

NMR spectroscopy is a discipline within structural biology that utilizes NMR spectroscopy to gather information about the structure and dynamics of proteins, nucleic acids, and their complexes. The phases encompass sample preparation, measurements, the application of interpretive approaches, and the calculation and validation of the protein structure. Studies by Mazzei and Piccolo (2017) NMR technique offers high-resolution spectroscopy for semi-solid samples, bridging solid- and liquid-state NMR advantages [14]. Its rapid spinning and sample orientation minimize anisotropic processes, ensuring enhanced spectral quality. The method employs edited pulse sequences, providing simultaneous information on polar and non-polar components without sample extraction. With applications expanding into agricultural chemistry, HRMAS NMR proves valuable in characterizing soil components, plant tissues, and agrofood products.

2.2.2 Protein microarrays

Microarray technology involves the miniaturization of numerous assays on a single small plate. This concept originated from the earlier ambient analyte immunoassay introduced by Roger Ekins in 1989. Over the subsequent decade, this concept evolved into the DNA microarray, a technology capable of simultaneously determining the mRNA expression levels of thousands of genes.

2.2.2.1 Analytical protein microarrays

These are also known as antibody arrays, come in different formats. The “analyte-labelled” format detects proteins after antibody capture through direct labelling, revealing alterations in protein expression in various cell types. However, it has limitations in specificity and sensitivity. Another model, the “sandwich” assay, uses two antibodies for higher sensitivity, successfully distinguishing between samples like blood plasma and serum with minimal volume. While resembling a multiplexed ELISA, these arrays can detect dozens of analytes simultaneously due to potential cross-reactivity between antibodies.

2.2.2.2 Functional protein microarrays

These play a crucial role in understanding protein functionality at the proteome level. These microarrays, made with individually purified proteins, allow the exploration of various biochemical properties, including interactions like protein-protein, protein-DNA, and enzyme-substrate relationships. Zhu et al. (2001) pioneered their use in determining the substrate specificity of protein kinases, and since then, these microarrays have found applications in basic and clinical research [15]. They have been instrumental in providing the entire proteome of organisms on arrays, leading to significant biological discoveries. Additionally, protein microarrays facilitate the study of post-translational modifications, contributing vital insights into cellular protein synthesis and function.

2.2.2.3 Reverse-Phase Protein Microarrays

This technique employs an opposite format to traditional protein microarrays, offer expanded applications in analyzing samples obtained at different states by directly spotting tissue, cell lysates, or fractionated cell lysates on a glass slide. Poetz et al., 2005 successfully detected microscopic transition stages of pro-survival checkpoint proteins in different prostate cancer stages, correlating phosphorylation status with cancer progression [16].

2.2.3 X-ray protein crystallography

It is a technique that enables the determination of the three-dimensional positions of each atom within a protein. Protein crystallography involves multiple steps, starting with the production of protein using Escherichia coli and cloning the gene of interest into an expression plasmid. After inducing protein expression, cells are lysed, and the protein is purified. Crystallization is then attempted using various conditions, and a drop diffusion technique is commonly used. Once suitable crystals are obtained, X-ray diffraction data is collected by bombarding the crystals with X-rays. This data is analyzed computationally to infer the three-dimensional positions of atoms in the protein, producing an electron density map through a process called phasing. Brief workflow for crop improvement using proteomic tools (Figure 1).

Figure 1.

A brief and schematic depiction of proteomic tools for different abiotic stresses.

3. Studies exploring changes in the plant proteome in response to various abiotic stress factors.

In this chapter, we offer an update on the contribution of proteomic studies in elucidating the mechanisms that underlie stress responses in plants. The detailed view of plant proteome responses to various stresses are briefly discussed below:

3.1 Drought or water deficit stress

Water is essential for plant life, serving as the primary compound in cytosol for biochemical reactions and influencing cell expansion. However, water stress, caused by factors like drought or waterlogging, poses a significant challenge to global crop production. Plants vary in their optimal environments and responses to water availability extremes.

Proteins play a crucial role in conferring resistance to drought stress by participating in stress signalling, transcription regulation, cellular detoxification, and protection of macromolecules. Proteomic methods, studying the dynamically translated part of the genome, offer insights into molecular events not revealed by DNA or mRNA analysis (Table 1). The application of proteomic technologies has been instrumental in identifying mechanisms associated with drought stress and tolerance in various plant species. Some of the illustrations are provided below (Table 2):

Stress	Specific response	Aspecific response
		Antioxidant enzymes	HSPs
Drought	Osmotin, Dehydrin, Aquaporin, LEA proteins	Aldose/Aldehyde reductase, Met synthase	smHSPs, HSP70, HSC70
Heat	Galactinol synthase, Choline kinase, Peptidyl prolyl isomerase, Glutaredoxin, Thaumatin	APX2, GST, Catalase, SOD	HSP110, HSP100, HSP90, HSP70, HSP60, smHSPs, CPN60, BiP
Salt	Osmotin, Dehydrin, Remorin1, HIR protein, GF14a, GF14b, ABP	COX6b-1, Triosephosphate isomerase, Enolase, UGPase, GST, GPX, Methyltransferase	HSP70, sti1, HSP90, smHSPs
Cold	Osmotin, Dehydrin, Glycine-rich protein, RNA binding protein CP29	APX, CA, Met synthase, GST, Thx	smHSPs, HSP70, HSP90, CPN20, CPN60, GRP78
Heavy metals	MT and PC proteins, Phytochelatins	GSH-derived peptides	HSP70

Table 1.

List of the major specific and aspecific proteins expressed upon several abiotic stresses in plant [17].

Crop	Stress treatment	Proteomics approach	Differentially expressed protein spots (identified proteins)	References
Drought
Soybean cv Taegwang	Root – 4 d and recovery	2-DE MALDI-TOF	45 (28)	[18]
Arachis hypogea	Leaf proteins	1 and 2-DE/MALDI-TOF-MS	49	[19]
Pea cv Green Feast	7 d of water withdrawal	2-DE LC–MS/MS	33 (32)	[20]
Maize (line FR697	primary root elongation zone – cell wall proteome ψw −1.6 MPa (48 h)	2-DE LC–ESI-MS TOF	152	[21]
Chickpea gen JG-62	nuclear fraction 0, 24, 48, 72, 96, 120, 144 h of water withdrawal	2-DE LC–MS/MS	205 (147)	[22]
Populus euphratica	35, 24, 8% rel. soil water and recovery (10 d after reirrigation)	2D-DIGE MALDI-TOF/TOF	375 (39)	[23]
Oryza sativa L.	cv.CT9993, cv. IR2266 leaves	2DE/MALDI-TOF-MS	42	[24]
Triticum aestivum L.	Ningchun 4, Chinese spring, grain	2DE/MALDI-TOF-MS	28	[25]
Hordeum vulgare Golden promise	Basrah roots and leaves	DIGE/MALDI-TOF-MS	24 (L)	[26]
Arabidopsis thaliana	Plastid	2DE-DIGE/MALDI-TOF-MS	18	[27]
Sugarcane	Stop watering	1-DE, 2-DE	30	[28]
Grapevine (Vitis sylvestris)	water deficit stress for 16 days	2D-PAGE	18	[29]
Maize (tolerant ND476 and sensitive ZX978)	water deficit (drought) treatment conditions for 12 days at the grain filling stage	iTRAQ analysis	165	[30]
Pandanus amaryllifolius	withholding water for 4, 7, 10, and 14 days	TMT-labelled LCMS/MS	74	[31]
Tomato (Solanum lycopersicum; cultivar M82)	Five-leaf stage withhold water	HPLC fractionation and LC-MS/MS analysis	294	[32]
Heat
Common wheat (T. aestivum) cvs Fang	grain 40/25°C (72 h)	2-DE MALDI-TOF	48 (7)	[33]
Populus euphratica	leaf 42/37°C (3 d)	2-DE MALDI-TOF/TOF	62 (51)	[34]
Rice cv Dongjin	seedling leaf 42°C (12, 24 h)	2-DE MALDI-TOF	73 (48)	[34]
Carissa spinarum	leaf 42/35°C (5 d)	2-DE MALDI-TOF/TOF	49 (30)	[35]
Spinach (Spinacia oleracea L.)	temperature regime of 22/18°C, 10/14 h day/night cycle	Trypsin Digestion and iTRAQ Labeling	45	[36]
Chick pea	27/18°C Day/night temperatures till flowering	LCMS analysis, fractionation, MS parameters	82	[37]
Sorghum (ICSB338)	40°C for 72 h	iTRAQ Labelling	31	[38]
Clematis foridaThun (CfT)	38°C for 3 h; air humidity 65%	LC-MS/MS	17	[39]
Pepper		iTRAQ	91	[40]
Salinity
Rice	150 mM NaCl	2-DE	63	[41]
Wheat	Mitochondria	2-DE/LC MS/MS	68	[42]
Tobacco (Nicotiana tabacum) cv Petit Havana	SR1 – leaf apoplast 100 mM NaCl (20 d)	2-DE LC–MS/MS	30 (20)	[43]
Durum wheat (T. durum)	leaf 100 mM NaCl (2 d)	2-DE MALDI-TOF	(38)	[44]
Soybean cv Enrei	root, hypocotyl, leaf 40 mM NaCl (7 d)	2-DE Edman sequencing MALDI-TOF	14 – root; 22 – hypocotyl; 19 – leaf	[45]
Rice cv Nipponbare	seedling 3 rd leaf 130 mM NaCl (4 d)	2-DE nanoESI-LC–MS/MS	55 (33)	[46]
Rapeseed (Brassica napus L.)	0, 150, and 300 mM NaCl	2-DE	44	[47]
Waterlogging
Grapevine rootstock (V. berlandieri × V. riparia) plants	waterlogging at the sixth-leaf stage for 0 days (T0d), 10 days (T10d), and 20 days (T20d)	LC–MS/MS	1057	[48]
Peanut (Arachis hypogaea L.)	flooding depth of 1 cm for different duration time (3, 6, 9, 12, and 15 days)	HPLC Fractionation and LC MS/MS	6285	[49]
Wheat (Triticum aestivum L.)	Flowering stage for 15 days	iTRAQ and HPLC-MS techniques	710	[50]
Cold temperature
Rapeseed (Brassica napus L.)	four-leaf stage were subjected to cold treatments (8°C, 16 h of light/4°C, 8 h of dark) for 7 days	iTRAQ and LC-MS techniques	241	[51]
Arabidopsis	4–5 expanded leaf stage, plants were transferred to − 4°C for 24 h)	SDS-PAGE	(21)	[52]
Citrus junos fruits	−7°C as a cold stress condition	TMT labelling and LC MS/MS	413	[53]
Coconut (Cocos nucifera L.)	8°C for 2 and 5 days	iTRAQ and LC-ESI-MS/MS	104	[54]
Saffron crocus (Crocus sativus)	16°C, non-flowering phenotype) for 30 days	iTRAQ	201	[55]
Toxic metals
Arsenic; B. napus	200 μmol L^−1	MS/MS and iTRAQ labeling	117	[56]
Cadmium; Wheat	50 μM for 24 h	TMT and LC-MS/MS	532	[57]

Table 2.

Summary of proteomic analysis of plant responses to abiotic stress.

In rice, upregulation of three proteins namely S-like RNase homologue, actin depolymerizing factor, and Rubisco activase and downregulation of isoflavone reductase-like protein was observed under drought stress. In barley, greater regulation of ROS-homeostasis has been observed. In soyabean, the enzyme caffeoyl-CoA O-methyltransferase, crucial in lignin synthesis, exhibited significant upregulation. This upregulation is likely linked to the increased accumulation of lignin, thereby associated with growth inhibition. In tomato plants subjected to osmotic stresses, there was an increase in levels of ABA (abscisic acid), phospholipid signalling, and mitogen-activated protein kinases (MAPKs) [58].

Drought stress triggers the activation of Ca²⁺-binding proteins, such as calmodulin, in plants, serving as crucial transducers for Ca²⁺ signals. This involvement of Ca²⁺⁻binding proteins, alongside Ca²⁺⁻dependent protein kinases, calcineurin B–like proteins, and SOS₃, highlights their significant role in mediating ABA-dependent stress responses in plants. The functions of numerous proteins induced in higher plants during stress remain unknown, requiring inference based on available information from model plants [59].

3.2 Flooding or waterlogging stress

Waterlogging, caused by excessive soil water, is a major abiotic stress affecting about half of global crops annually, leading to a 25% yield reduction. This stress restricts oxygen availability to plant roots, triggering anaerobic metabolism and processes like glycolysis and pyruvate fermentation. In higher plants, the cell wall is the initial organelle to respond to flood stress signals, transmitting them to the cell’s interior and influencing the cell signalling cascade, stress tolerance, or intolerance [60].

Proteins within the cell walls play essential roles in signal transduction, maintaining structural integrity, managing metabolism, and facilitating cell enlargement in response to waterlogging stress. Upregulation of several proteins viz., fructose-bisphosphate aldolase, alcohol dehydrogenase, UDP-glucose pyro phosphorylase and GAPDH involved in the fermentation and glycolysis pathways. Under waterlogging stress in tomato leaves, a comparative proteomic analysis showed increased ion leakage, lipid peroxidation, and H₂O₂ content, coupled with a decrease in chlorophyll levels. Proteome profiling in different crops has demonstrated that prolonged waterlogging stress led to an elevation in glycolytic flux, meeting increased ATP demands [59].

3.3 Imbalances in mineral nutrition

Plants necessitate a minimum of 17 essential nutrients to successfully complete their life cycle. The absorption of mineral elements from the soil solution by plant roots and their subsequent distribution within the plant has been extensively studied for decades. Deficiencies in any of these mineral elements significantly limit crop growth and yield, while higher soil concentrations can adversely affect plant growth and development.

Plants employ various adaptations, including proton exudation, enzyme release, and structural changes in roots, to enhance phosphorus (P) uptake efficiency from the soil. Under P deficiency, specific proteins associated with root cell cycle and division, such as GTP-binding nuclear protein, mini-chromosome maintenance protein, and glycogen synthase kinase-3 homolog MsK-3, are up-regulated. Phosphorus (P) deficiency induces oxidative stress in plants, leading to an increase in reactive oxygen species (ROS) [59].

Proteomic analysis revealed the up-regulation of P deficiency-responsive proteins involved in oxidative stress defense, including superoxide dismutase (SOD), 2-Cys peroxiredoxin, ascorbate peroxidase (APX), and 1,4-benzoquinone reductase. Nitrogen (N) deficiency in plants leads to down-regulation of proteins involved in photosynthesis, such as Rubisco activase, Rubisco LS, and SS, particularly in leaves. N deficiency triggers complex responses in carbon (C) metabolism, affecting various C metabolism-related proteins. Additionally, it accelerates leaf senescence, leading to the breakdown of proteins like RuBisCO, enabling the remobilization of N to active tissues. This condition also induces an increase in reactive oxygen species (ROS), with up-regulation of antioxidant proteins like 2-Cys peroxiredoxin, superoxide dismutase (SOD), and ascorbate peroxidase (APX) to mitigate oxidative stress [61].

3.4 Toxic metal stress

Metals play vital roles in plant metabolism, serving as essential micronutrients for various protein functions. However, an excess of these essential metals can lead to detrimental effects on plant health. Additionally, nonessential metals such as cadmium, chromium, lead, mercury, and arsenic, even in trace amounts, can negatively impact plants. The influence of these nonessential metals on plants is complex, and their adverse effects are determined by biological availability rather than the absolute concentration in the soil. This highlights the delicate balance required for optimal metal levels to ensure proper plant growth and development.

Metal toxicity occurs when ionic forms of metals form complexes or ligands with biomolecules. The presence of redox-active metals can lead to the production of reactive oxygen species (ROS) through Haber-Weiss and Fenton reactions, contributing to metal toxicity [59]. NADP(H)-oxido-reductase plays a crucial role in protecting cells from the harmful effects of toxic metals.

3.5 Salinity stress

Salinity adversely affects plant growth through osmotic water-deficit and ionic stress/toxicity. The osmotic effect results from a decrease in soil water osmotic potential due to dissolved salt ions, limiting water uptake and causing physiological drought or cellular dehydration. Ionic stress involves the entry of salts, particularly Na and Cl₂, into plant cells, leading to cell damage, growth impairment, and secondary stresses like oxidative damage [62]. Salinity-induced consequences include slow growth, developmental delays, reduced yields, economic losses, soil erosion, ecological imbalances, and potential harm to human health through the introduction of toxic elements into the food chain.

Proteins responsive to salt stress are involved in key processes such as stress signal transduction, transcription, protein metabolism, osmotic homeostasis, ion homeostasis, ROS homeostasis, photosynthesis, carbohydrate and energy metabolism, and cytoskeleton and cell wall dynamics. Salt-responsive proteins are identified in various plant organs, including vegetative organs (seeds, seedlings, radicles, hypocotyls, roots, shoots, leaves), reproductive organs (panicles, anthers, grains), and other tissues (callus and cell suspension cultures). Proteome studies also target subcellular organelles and membrane systems, including the plasma membrane, microsome, apoplast, mitochondria, and chloroplast [59].

3.6 Heat stress

High temperature stress is a prominent abiotic stress affecting plant growth, leading to enzyme denaturation and membrane fluidity issues. Protein synthesis is generally suppressed, while the induction of Heat Shock Proteins (HSPs) occurs. Proteomic studies on heat stress, including details on tissues, varieties, temperature duration, and analysis methods, have been extensively studied.

Plants have an optimal temperature range for optimal growth and development. Temperature extremes, both high and low, can impact plant sensitivity and growth. High-temperature treatment increases the abundance of low-molecular-weight sHSPs (small heat shock proteins). The expression of high-molecular-weight HSPs is minimally affected by high-temperature treatment [8]. The expression level of glyceraldehyde-3-phosphate dehydrogenase, an enzyme in the glycolytic pathway, is influenced by high temperatures.

3.7 Cold stress

Chilling or cold stress is a significant environmental factor affecting crop growth and productivity. Cold stress poses a serious limitation to crop development, particularly in various regions around the world.

Chilling temperatures directly impact plants by slowing metabolic processes and causing the loss of membrane functions, leading to cold stress. Plants are categorized into chilling-sensitive, chilling-tolerant/resistant, and freezing-tolerant types based on their ability to survive different temperature conditions. Chilling-sensitive plants experience metabolic dysfunction at temperatures slightly below the optimum, chilling-tolerant plants endure lower non-freezing temperatures, and freezing-tolerant plants can survive below freezing conditions [59].

UDP-glucose pyrophosphorylase and sucrose synthase are proteins linked to UDP-glucose formation. These proteins exhibit up-regulation in the leaf blade of Japonica rice seedlings in response to short-term cold treatment. Four antioxidant enzymes – APX, glutathionine S-transferase, thioredoxin h, and thioredoxin peroxidase – are induced by oxidative stress caused by ROS. A RING zinc finger protein-like, identified as a new cold-induced protein, exhibits marked responsiveness only to extreme cold stress.

Plant proteome analysis requires various bioinformatics tools as discussed in Table 3.

Crop	Proteome database	References
Rice	http://oryzapg.iab.keio.ac.jp/	[63]
	http://gene64.dna.affrc.go.jp/RPD/	[64]
Soybean	http://proteome.dc.affrc.go.jp/Soybean	[65]
	http://www.oilseedproteomics.missouri.edu/soybean.php	[66]
Maize	http://ppdb.tc.cornell.edu	[67]
Wheat	http://www.proteome.ir	[68]
	http://www.gramene.org/protein	[69]
Chickpea	http://bioinfo.noble.org/manuscript-support/legumedb	[70]
Pea	http://bioinfo.noble.org/manuscript-support/legumedb	[70]
Alfalfa	http://bioinfo.noble.org/manuscript-support/legumedb	[70]
Barley	http://www.gramene.org/protein	[69]
Sorghum	http://www.gramene.org/protein	[69]

Table 3.

Proteome databases helpful in the protein identification of different crops.

4. Conclusion

Proteomics serves as a fundamental frontier in the realm of biological research, offering a holistic perspective on the proteins within living systems. By systematically analysing the entire proteome of a cell, tissue, or organism, proteomics enables a comprehensive exploration of protein structure, function, and regulation. One of its key contributions lies in unravelling the functional roles of proteins, shedding light on their involvement in diverse biological processes, signalling pathways, and cellular functions. Proteomics is particularly adept at uncovering post-translational modifications (PTMs), providing crucial insights into the dynamic regulatory mechanisms that influence protein activity and stability. Furthermore, it plays a crucial role in translational research and clinical applications. In the broader context of environmental responses, such as the plant’s reaction to abiotic stress, proteomics becomes indispensable in delineating the specific proteins involved in stress adaptation and understanding the molecular intricacies underlying these adaptive processes. As an integral component of systems biology, proteomics collaborates with genomics, transcriptomics, and metabolomics to provide a holistic understanding of biological systems, paving the way for advanced breeding of crop plants for abiotic stress and biotic stress at the molecular level.