Plant Proteomics Research Articles

Modulation of Abiotic Stress Responses in Rice by E3-Ubiquitin Ligases: A Promising Way to Develop Stress-Tolerant Crops.

Plants are unable to physically escape environmental constraints and have, therefore, evolved a range of molecular and physiological mechanisms to maximize survival in an ever-changing environment. Among these, the post-translational modification of ubiquitination has emerged as an important mechanism to understand and improve the stress response. The ubiquitination of a given protein can change its abundance (through degradation), alter its localization, or even modulate its activity. Hence, ubiquitination increases the plasticity of the plant proteome in response to different environmental cues and can contribute to improve stress tolerance. Although ubiquitination is mediated by different enzymes, in this review, we focus on the importance of E3-ubiquitin ligases, which interact with the target proteins and are, therefore, highly associated with the mechanism specificity. We discuss their involvement in abiotic stress response and place them as putative candidates for ubiquitination-based development of stress-tolerant crops. This review covers recent developments in this field using rice as a reference for crops, highlighting the questions still unanswered.

A sulfated polysaccharide κ-carrageenan induced antioxidant defense and proteomic changes in chloroplast against leaf spot disease of tomato

Ecofriendly approaches to control plant diseases using biopolymers are gaining momentum due to their non-toxic nature to the environment. The present study elucidates the potency of a sulfated polysaccharide, κ-carrageenan, obtained from the red seaweed Kappaphycus alvarezii, as an inducer of antioxidant defense and also chloroplast proteome against leaf spot disease in tomato. к-carrageenan (0.3%) treatment was very effective in reducing disease scores in tomato plants. Fluorescent microscopy of tomato leaves stained with Uvitex 2B (a chitin specific stain) revealed that foliar application of κ-carrageenan (0.3%) reduced colonization of the pathogen Septoria lycopersici. In addition, κ-carrageenan pretreatment followed by pathogen infection recorded a higher accumulation of calcium oxalate crystals. The treatment of κ-carrageenan resulted in the induction of H2O2 and superoxide anion compared to control or S. lycopersici-infected plants. The tomato leaves treated with κ-carrageenan showed higher activity of peroxidase enzyme. Furthermore, chloroplast proteome analysis by 2D-PAGE of control, S. lycopersici-infected, κ-carrageenan-treated, and к-carrageenan treatment followed by S. lycopersici-infected leaves of tomato showed differential expression of 31 proteins, of which 11 proteins were upregulated including acid phosphatase 1-like, C- terminal processing peptidase, GATA transcription factor 5, Calvin cycle protein CP12-1, Photosystem I reaction center subunit PSAK, and subunit of cytochrome B6-F complex iron-sulfur centers. These results indicated that κ-carrageenan could be a promising inducer of antioxidant defense and modulator of chloroplast proteome in tomato plants.

Mapping the plant proteome: tools for surveying coordinating pathways.

Plants rapidly respond to environmental fluctuations through coordinated, multi-scalar regulation, enabling complex reactions despite their inherently sessile nature. In particular, protein post-translational signaling and protein–protein interactions combine to manipulate cellular responses and regulate plant homeostasis with precise temporal and spatial control. Understanding these proteomic networks are essential to addressing ongoing global crises, including those of food security, rising global temperatures, and the need for renewable materials and fuels. Technological advances in mass spectrometry-based proteomics are enabling investigations of unprecedented depth, and are increasingly being optimized for and applied to plant systems. This review highlights recent advances in plant proteomics, with an emphasis on spatially and temporally resolved analysis of post-translational modifications and protein interactions. It also details the necessity for generation of a comprehensive plant cell atlas while highlighting recent accomplishments within the field.

Network Topological Analysis for the Identification of Novel Hubs in Plant Nutrition.

Network analysis is a systems biology-oriented approach based on graph theory that has been recently adopted in various fields of life sciences. Starting from mitochondrial proteomes purified from roots of Cucumis sativus plants grown under single or combined iron (Fe) and molybdenum (Mo) starvation, we reconstructed and analyzed at the topological level the protein–protein interaction (PPI) and co-expression networks. Besides formate dehydrogenase (FDH), already known to be involved in Fe and Mo nutrition, other potential mitochondrial hubs of Fe and Mo homeostasis could be identified, such as the voltage-dependent anion channel VDAC4, the beta-cyanoalanine synthase/cysteine synthase CYSC1, the aldehyde dehydrogenase ALDH2B7, and the fumaryl acetoacetate hydrolase. Network topological analysis, applied to plant proteomes profiled in different single or combined nutritional conditions, can therefore assist in identifying novel players involved in multiple homeostatic interactions.

Phloem Sap Proteins Are Part of a Core Stress Responsive Proteome Involved in Drought Stress Adjustment.

During moderate drought stress, plants can adjust by changes in the protein profiles of the different organs. Plants transport and modulate extracellular stimuli local and systemically through commonly induced inter- and intracellular reactions. However, most proteins are frequently considered, cell and organelle specific. Hence, while signaling molecules and peptides can travel systemically throughout the whole plant, it is not clear, whether protein isoforms may exist ubiquitously across organs, and what function those may have during drought regulation. By applying shotgun proteomics, we extracted a core proteome of 92 identical protein isoforms, shared ubiquitously amongst several Medicago truncatula tissues, including roots, phloem sap, petioles, and leaves. We investigated their relative distribution across the different tissues and their response to moderate drought stress. In addition, we functionally compared this plant core stress responsive proteome with the organ-specific proteomes. Our study revealed plant ubiquitous protein isoforms, mainly related to redox homeostasis and signaling and involved in protein interaction networks across the whole plant. Furthermore, about 90% of these identified core protein isoforms were significantly involved in drought stress response, indicating a crucial role of the core stress responsive proteome (CSRP) in the plant organ cross-communication, important for a long-distance stress-responsive network. Besides, the data allowed for a comprehensive characterization of the phloem proteome, revealing new insights into its function. For instance, CSRP protein levels involved in stress and redox are relatively more abundant in the phloem compared to the other tissues already under control conditions. This suggests a major role of the phloem in stress protection and antioxidant activity enabling the plants metabolic maintenance and rapid response upon moderate stress. We anticipate our study to be a starting point for future investigations of the role of the core plant proteome. Under an evolutionary perspective, CSRP would enable communication of different cells with each other and the environment being crucial for coordinated stress response of multicellular organisms.

Labeling and label-free quantitative proteomics in plant biology

Proteins not only construct cellular structures but also play regulatory functions in plant development and growth. Accurate quantification of protein dynamics, turnover rates, and post-translational modifications provide crucial insights into the molecular mechanisms of biological systems. While early studies only consisted of listing proteins identified in given organs or under specific conditions. There is growing need to apply quantitative proteomic techniques to be biased or unbiased studies of plant protein dynamic changes. With rapid improvements in mass spectrometry (MS) technologies, various proteomic quantification methods have been developed and widely used in biological research. In the meantime, MS-based quantitative proteomics has emerged as a powerful approach for gaining insights into functional aspects of proteins in biological systems. MS is not inherently quantitative; thus, several strategies have been developed to assist MS for protein quantification. These strategies can be distinguished not only by the method of quantification, such as labeled or label-free method, but also by the quantitative goal, such as relative or absolute quantification. They can be also distinguished by the scopes of the experiments performed, such as unbiased or targeted studies. Even within these different research goals, numerous options exist, since each strategy has advantages and disadvantages. For example, labeling methods can tag proteins/peptides with chemical, metabolic, or enzymatic reagents, whereas label-free quantitation methods use spectra ion counting, or peak intensity-based analysis. In comparison with labeling strategies, label-free methods are widely used because of the dynamic quantitative range, costing less, and without labeling steps. However, label-free methods are limited in the sample multiplex capacity and quantitative accuracy. For labeling-based quantitation, materials or samples are labeled by different isotope reagents, the paired MS spectra ions are extracted for relative or absolute quantification. In combination with high accuracy MS instruments, various labeling strategies can be used in quantitative proteome analysis, these methods can be distinguished by quantification ions, such as precursor ion-based, reporter ion-based, peptide-coupled reporter ion-based, and fragment ion-based quantitation. For stable isotope labeled methods, proteins or peptides labeled with different stable isotopes are combined into one vial for LC-MS analysis. These strategies have gained popularity due to the diversity and flexibility of stable isotope reactions, the power to analyze multiple samples simultaneously, and the high quantification accuracy. In this review, we discuss the most widely used label-free and stable isotope-labeling-based proteomic strategies for relative and absolute quantitative proteomics in plants. We compare their strengths and weaknesses in terms of quantitation accuracy, proteome coverage and robustness. We summarize the most promising recent developments and their applications for solving plant biological complexities.

Plant Proteomics and Systems Biology.

Proteome analysis of model and non-model plants is a genuine scientific field in expansion. Several technological advances have contributed to the implementation of different proteomics approaches for qualitative and quantitative analysis of the dynamics of cellular responses at the protein level. The design of time-resolved experiments and the emergent use of multiplexed proteome analysis using chemical or isotopic and isobaric labeling strategies as well as label-free approaches are generating a vast amount of proteomics data that is going to be essential for analysis of protein posttranslational modifications and implementation of systems biology approaches. Through the target proteomics analysis, especially the ones that combine the untargeted methods, we should expect an improvement in the completeness of the identification of proteome and reveal nuances of regulatory cellular mechanisms related to plant development and responses to environmental stresses. Both genomic sequencing and proteomic advancements in the last decades coupled to integrative data analysis are enriching biological information that was once confined to model plants. Therewith, predictions of a changing environment places proteomics as an especially useful tool for crops performance.

Quantitative Proteomics and Phosphoproteomics Support a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis

Protein phosphorylation is one of the most prevalent posttranslational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to environmental stimuli. The Mut9-like kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs.

Membrane Proteomic Responses in Sugar Beet Roots to Salt Stress

Salt stress as one of the major abiotic stresses limits the world crop production. Sugar beet monosomic addition line M14 is a unique germplasm with salt stress tolerance, and root is directly exposed to salt stress in the soil. Here we report changes in root membrane proteome of the M14 plants in response to 0, 200 mM and 400 mM NaCl treatment using iTRAQ LC-MS/MS quantitative proteomics. A total of 115 differentially expressed membrane proteins (96 increased and 19 decreased) were identified with a significant fold change of >2.0. The proteins were mainly involved in the processes of transport, signaling, stress and defense, energy, protein degradation, and transcription. Transcriptional changes of 10 genes encoding the differential membrane proteins were analyzed using real-time PCR, and seven genes showed a positive correlation between transcriptional and protein levels. These results have revealed interesting mechanisms underlying the M14 root response to the salt stress, which may have potential applications toward improving crop salt tolerance through genetic engineering and molecular breeding.

Advances in the elucidation of nuclear proteins in the model plant Arabidopsis thaliana: based on protein interactions and bioinformatics analysis

ABSTRACT Plant protein–protein interaction (PPI) networks provide valuable information for the understanding of plant biological processes. In this study, we summarized and constructed the nuclear-protein interactome of Arabidopsis based on PPIs, and preliminarily analyzed the relationships among the main pathways involving these protein interactions. These proteins are involved mainly in the plant hormone signal transduction and plant-pathogen interaction, and these pathways are linked closely by co-pathway and nodal proteins. The key nodal proteins play important roles in communication among hormonal pathways. Further analysis revealed that all of these key nodal proteins were localized in multiple organelles. High-frequency protein multi-localization analysis suggested that the multi-location proteins may play key roles in organelle communication and plant growth. Thus, the screening and study of multi-location proteins may be an important research direction. Our findings can inform new research based on protein interactions and provide novel directions for plant proteome research.

Isolation of Apoplastic Fluid from Woody Plant Leaves: Grapevine and Coffee as a Case Study.

Proteomics is one of the key approaches to understand plant cell physiology involving the regulation of expression of many genes and metabolite production. Technical advances allowed a deeper characterization of plant proteomes, highlighting the need to study cellular compartments. The apoplast is the cellular compartment external to the plasma membrane including the cell wall, where a broad range of processes take place including intercellular signaling, metabolite transport, and plant-microbe interactions. Due to the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves while maintaining cell integrity, which is particularly true for woody plants. Here, we describe the vacuum infiltration-centrifugation (VIC) method for the extraction of the apoplastic fluid compatible with high-throughput proteomic approaches and biochemical analysis from different woody plants.

Responses of Phragmites australis to copper stress: A combined analysis of plant morphology, physiology and proteomics.

Few relevant research attempts have been made to determine heavy metal resistance mechanisms of rhizomatous perennial plants. Thus, it is pertinent to investigate the physiological and biochemical changes in Phragmites australis under metal-stressed conditions to facilitate the development of strategies to enhance copper (Cu) tolerance. We measured parameters related to plant growth and development, metal translocation and physiological responses of P. australis subjected to Cu stress. In addition, the differentially expressed proteins (DEP) were evaluated using the isobaric tag for relative and absolute quantification (iTRAQ) system. A large amount of copper accumulates in the roots of P.australis, but the growth parameters were not sensitive to Cu. However, the high concentration of Cu reduced the content of chlorophyll a and chlorophyll b, and the expression of important photosynthesis proteins PsbD, PsbO and PsaA were all down-regulated, so photosynthesis was inhibited. In contrast, the content of ascorbic acid and proline both increased with the increase of copper stress. P.australis fixed a large amount of Cu in its roots, limiting the migration of Cu to other parts of the plant. Moreover, Cu stress can affect photosynthesis by inhibiting the activity of PSI, PSII and LHCII. In addition, P.australis synthesizes ascorbic acid through the D-mannose/L-galactose pathway, and synthesizes proline through the ornithine pathway. Ascorbic acid and proline can increase Cu tolerance and protect photosynthesis. These results provide a theoretical basis for understanding the tolerance and repair mechanisms of plants in response to heavy metal pollution.

Bringing New Methods to the Seed Proteomics Platform: Challenges and Perspectives.

For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins, and carbohydrates in their seeds, distinctly dominate modern agriculture, thus play an essential role in food industry and fuel production. Therefore, seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, seed development and germination as well as age- and stress-related alterations in seed vigor, longevity, nutritional value, and safety can be addressed by a broad panel of analytical, biochemical, and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes accompanying plant development, senescence, and response to biotic or abiotic stress. Among individual post-genomic methodological platforms, proteomics represents one of the most effective ones, giving access to cellular metabolism at the level of proteins. During the recent decades, multiple methodological advances were introduced in different branches of life science, although only some of them were established in seed proteomics so far. Therefore, here we discuss main methodological approaches already employed in seed proteomics, as well as those still waiting for implementation in this field of plant research, with a special emphasis on sample preparation, data acquisition, processing, and post-processing. Thereby, the overall goal of this review is to bring new methodologies emerging in different areas of proteomics research (clinical, food, ecological, microbial, and plant proteomics) to the broad society of seed biologists.

Expression dynamics of glyoxalase genes under high temperature stress in plants

Heat stress, owing to the recent climate change events, has emerged as one of the major environmental factors posing a grave threat to plant survival and crop productivity. Oxidative damage due to increased generation of reactive oxygen and dicarbonyl species in response to heat, is one of the serious consequences of high temperature stress. Methylglyoxal (MG), is one such cytotoxic dicarbonyl metabolite whose levels are known to increase in response to high temperature and various other stress conditions in plants. In fact, it is considered as a general consequence of stress in plants. The synergistic co-activation of reactive oxygen species scavenging and the MG detoxification pathways thus, plays a key role in regulation of stress responses for plant acclimation. Glyoxalase enzymes are the major MG detoxifying proteins in the living organisms. In plants, glyoxalases are encoded as multigenic families, known to be involved in plant growth and development, although, chiefly associated with multiple stress inducible behaviour and tolerance. While their role in salinity, drought and heavy metal stresses has been well-studied, their significance under high temperature stress has received limited coverage. In this review, we emphasize on the dynamic role of glyoxalases under high temperature stress for conferring thermotolerance. Through analysis of heat responsive transcriptomes and proteomes of various plants, we provide evidence for a hitherto, unexplored role of glyoxalases as a mechanism of mounting thermotolerance responses. We also, show that ‘priming’ induced stress/cross stress tolerance in plants involves the activation of glyoxalase pathway.

Arabidopsis Response to Inhibitor of Cytokinin Degradation INCYDE: Modulations of Cytokinin Signaling and Plant Proteome.

Cytokinins are multifaceted plant hormones that play crucial roles in plant interactions with the environment. Modulations in cytokinin metabolism and signaling have been successfully used for elevating plant tolerance to biotic and abiotic stressors. Here, we analyzed Arabidopsis thaliana response to INhibitor of CYtokinin DEgradation (INCYDE), a potent inhibitor of cytokinin dehydrogenase. We found that at low nanomolar concentration, the effect of INCYCE on seedling growth and development was not significantly different from that of trans-Zeatin treatment. However, an alteration in the spatial distribution of cytokinin signaling was found at low micromolar concentrations, and proteomics analysis revealed a significant impact on the molecular level. An in-depth proteome analysis of an early (24 h) response and a dose-dependent response after 168 h highlighted the effects on primary and secondary metabolism, including alterations in ribosomal subunits, RNA metabolism, modulations of proteins associated with chromatin, and the flavonoid and phenylpropanoid biosynthetic pathway. The observed attenuation in stress-response mechanisms, including abscisic acid signaling and the metabolism of jasmonates, could explain previously reported positive effects of INCYDE under mild stress conditions.

A Phylogenetic Rate Parameter Indicates Different Sequence Divergence Patterns in Orthologs and Paralogs.

Heterotachy-the change in sequence evolutionary rate over time-is a common feature of protein molecular evolution. Decades of studies have shed light on the conditions under which heterotachy occurs, and there is evidence that site-specific evolutionary rate shifts are correlated with changes in protein function. Here, we present a large-scale, computational analysis using thousands of protein sequence alignments from animal and plant proteomes, representing genes related either by orthology (speciation events) or paralogy (gene duplication), to compare sequence divergence patterns in orthologous vs. paralogous sequence alignments. We use sequence-based phylogenetic analyses to infer overall sequence divergence (tree length/number of sequences) and to fit site-specific rates to a discrete gamma distribution with a shape parameter α. This inference method is applied to real protein sequence alignments, as well as alignments simulated under various models of protein sequence evolution. Our simulations indicate that sequence divergence and the α parameter are positively correlated when sequences evolve with heterotachy, meaning that inferred site rate distributions appear more uniform as sequences diverge. Divergence and α are also positively correlated in both orthologous and paralogous genes, but the average increase in α (as a function of divergence) is significantly higher in paralogous protein alignments than in orthologous alignments. This result is consistent with the widely held view that recently duplicated proteins initially evolve under relaxed selective pressure, promoting functional divergence by accumulation of amino acid replacements, and hence experience more evolutionary rate fluctuations than orthologous proteins. We discuss these findings in the context of the ortholog conjecture, a long-standing assumption in molecular evolution, which posits that protein sequences related by orthology tend to be more functionally conserved than paralogous proteins.

Parallel global profiling of plant TOR dynamics reveals a conserved role for LARP1 in translation.

Target of rapamycin (TOR) is a protein kinase that coordinates eukaryotic metabolism. In mammals, TOR specifically promotes translation of ribosomal protein (RP) mRNAs when amino acids are available to support protein synthesis. The mechanisms controlling translation downstream from TOR remain contested, however, and are largely unexplored in plants. To define these mechanisms in plants, we globally profiled the plant TOR-regulated transcriptome, translatome, proteome, and phosphoproteome. We found that TOR regulates ribosome biogenesis in plants at multiple levels, but through mechanisms that do not directly depend on 5' oligopyrimidine tract motifs (5'TOPs) found in mammalian RP mRNAs. We then show that the TOR-LARP1-5'TOP signaling axis is conserved in plants and regulates expression of a core set of eukaryotic 5'TOP mRNAs, as well as new, plant-specific 5'TOP mRNAs. Our study illuminates ancestral roles of the TOR-LARP1-5'TOP metabolic regulatory network and provides evolutionary context for ongoing debates about the molecular function of LARP1.

Towards an understanding of the role of intrinsic protein disorder on plant adaptation to environmental challenges.

Intrinsic protein disorder is an interesting structural feature where fully functional proteins lack a three-dimensional structure in solution. In this work, we estimated the relative content of intrinsic protein disorder in 96 plant proteomes including monocots and eudicots. In this analysis, we found variation in the relative abundance of intrinsic protein disorder among these major clades; the relative level of disorder is higher in monocots than eudicots. In turn, there is an inverse relationship between the degree of intrinsic protein disorder and protein length, with smaller proteins being more disordered. The relative abundance of amino acids depends on intrinsic disorder and also varies among clades. Within the nucleus, intrinsically disordered proteins are more abundant than ordered proteins. Intrinsically disordered proteins are specialized in regulatory functions, nucleic acid binding, RNA processing, and in response to environmental stimuli. The implications of this on plants' responses to their environment are discussed.

Effects of Space Flight on Expression of Key Proteins in Rice Leaves

As a unique form of abiotic stress, the environmental conditions of outer space are expected to induce changes in plant genomes, proteomes and metabolic pathways. However, the effect of outer space conditions on the overall physiology of plants at the protein level has yet to be reported. To investigate the effects of outer space conditions on the growth- and development-related physiological processes and metabolic pathways of rice different stages, the seeds of rice variety DN423 were sent into orbit for 12.5 d aboard the SJ-10 Returning Satellite, and then the seedlings of both treated and control rice were compared at the three-leaf stage (TLS) and tillering stage (TS). In addition to comparing plant growth and reactive oxygen species (ROS) levels, seedling proteomes were also compared using isobaric tags for relative and absolute quantitation (iTRAQ). Space flight increased TLS plant height by 20%, reduced and increased ROS levels of the TLS and TS seedlings, respectively, and affected the expression of 36 and 323 proteins in TLS and TS leaves, respectively. Furthermore, the functions of the differentially abundant proteins were mainly associated with metabolism, energy, and protein synthesis and degradation. These results suggested that the exposure of seeds to outer space conditions affects the subsequent abundance of key signaling proteins, gene expression, and the processes of protein synthesis and degradation, thereby affecting metabolic processes and promoting adaptation to the abiotic stress of outer space. As such, the present study sheds light on the effects of space flight on plants and contributes to a more comprehensive understanding of extraterrestrial biology.

Wheat Line "RYNO3936" Is Associated With Delayed Water Stress-Induced Leaf Senescence and Rapid Water-Deficit Stress Recovery.

Random mutagenesis was applied to produce a new wheat mutant (RYNO3926) with superior characteristics regarding tolerance to water deficit stress induced at late booting stage. The mutant also displays rapid recovery from water stress conditions. Under water stress conditions mutant plants reached maturity faster and produced more seeds than its wild type wheat progenitor. Wild-type Tugela DN plants died within 7 days after induction of water stress induced at late booting stage, while mutant plants survived by maintaining a higher relative moisture content (RMC), increased total chlorophyll, and a higher photosynthesis rate and stomatal conductance. Analysis of the proteome of mutant plants revealed that they better regulate post-translational modification (SUMOylation) and have increased expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) proteins. Mutant plants also expressed unique proteins associated with dehydration tolerance including abscisic stress-ripening protein, cold induced protein, cold-responsive protein, dehydrin, Group 3 late embryogenesis, and a lipoprotein (LAlv9) belonging to the family of lipocalins. Overall, our results suggest that our new mutant RYNO3936 has a potential for inclusion in future breeding programs to improve drought tolerance under dryland conditions.