Biological Networks Underlying Abiotic Stress Tolerance in Temperate Crops--A Proteomic Perspective.

Abiotic stress factors, especially low temperatures, drought, and salinity, represent the major constraints limiting agricultural production in temperate climate. Under the conditions of global climate change, the risk of damaging effects of abiotic stresses on crop production increases. Plant stress response represents an active process aimed at an establishment of novel homeostasis under altered environmental conditions. Proteins play a crucial role in plant stress response since they are directly involved in shaping the final phenotype. In the review, results of proteomic studies focused on stress response of major crops grown in temperate climate including cereals: common wheat (Triticum aestivum), durum wheat (Triticum durum), barley (Hordeum vulgare), maize (Zea mays); leguminous plants: alfalfa (Medicago sativa), soybean (Glycine max), common bean (Phaseolus vulgaris), pea (Pisum sativum); oilseed rape (Brassica napus); potato (Solanum tuberosum); tobacco (Nicotiana tabaccum); tomato (Lycopersicon esculentum); and others, to a wide range of abiotic stresses (cold, drought, salinity, heat, imbalances in mineral nutrition and heavy metals) are summarized. The dynamics of changes in various protein functional groups including signaling and regulatory proteins, transcription factors, proteins involved in protein metabolism, amino acid metabolism, metabolism of several stress-related compounds, proteins with chaperone and protective functions as well as structural proteins (cell wall components, cytoskeleton) are briefly overviewed. Attention is paid to the differences found between differentially tolerant genotypes. In addition, proteomic studies aimed at proteomic investigation of multiple stress factors are discussed. In conclusion, contribution of proteomic studies to understanding the complexity of crop response to abiotic stresses as well as possibilities to identify and utilize protein markers in crop breeding processes are discussed.

Proteome analysis of cold response in spring and winter wheat (Triticum aestivum) crowns reveals similarities in stress adaptation and differences in regulatory processes between the growth habits.

A proteomic response to cold treatment (4 °C) has been studied in crowns of a frost-tolerant winter wheat cultivar Samanta and a frost-sensitive spring wheat cultivar Sandra after short-term (3 days) and long-term (21 days) cold treatments. Densitometric analysis of 2-D differential in gel electrophoresis (2D-DIGE) gels has resulted in the detection of 386 differentially abundant protein spots, which reveal at least a two-fold change between experimental variants. Of these, 58 representative protein spots have been selected for MALDI-TOF/TOF identification, and 36 proteins have been identified. The identified proteins with an increased relative abundance upon cold in both growth habits include proteins involved in carbohydrate catabolism (glycolysis enzymes), redox metabolism (thioredoxin-dependent peroxidase), chaperones, as well as defense-related proteins (protein revealing similarity to thaumatin). Proteins exhibiting a cold-induced increase in the winter cultivar include proteins involved in regulation of stress response and development (germin E, lectin VER2), while proteins showing a cold-induced increase in the spring cultivar include proteins involved in restoration of cell division and plant growth (eIF5A2, glycine-rich RNA-binding protein, adenine phosphoribosyltransferase). These results provide new insights into cold acclimation in spring and winter wheat at the proteome level and enrich our previous work aimed at phytohormone dynamics in the same plant material.

The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress.

Drought and low temperature are the two most significant causes of abiotic stress in agricultural crops and, therefore, they pose considerable challenges in plant science. Hence, it is crucial to study response mechanisms and to select genes for identification signaling pathways that lead from stimulus to response. The assessment of gene expression is often attempted using real-time RT-PCR (qRT-PCR), a technique which requires a careful choice of reference gene(s) for normalization purpose. Here, we report a comparison of 13 potential reference genes for studying gene expression in the leaf and crown of barley seedlings subjected to low temperature or drought stress. All three currently available software packages designed to identify reference genes from qRT-PCR data (GeNorm, NormFinder and BestKeeper) were used to identify informative sets of up to three reference genes. Interestingly, the data obtained from the separate treatment of leaf and crown have led to the recommendations that HSP70 and S-AMD (and possibly HSP90) to be used as the reference genes for low-temperature stressed leaves, HSP90 and EF1α for low-temperature stressed crowns, cyclophilin and ADP-RF (and possibly ACT) for drought-stressed leaves, and EF1α and S-AMD for drought-stressed crowns. Our results have demonstrated that the gene expression can be highly tissue- or organ-specific in barley and have confirmed that reference gene choice is essential in qRT-PCR. The findings can also serve as guidelines for the selection of reference genes under different stress conditions and lay foundation for more accurate and widespread use of qRT-PCR in barley gene analysis.

Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression.

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

Proteins involved in distinct phases of cold hardening process in frost resistant winter barley (Hordeum vulgare L.) cv Luxor.

Winter barley is an economically important cereal crop grown in higher latitudes and altitudes where low temperatures represent an important environmental constraint limiting crop productivity. In this study changes in proteome of leaves and crowns in a frost tolerant winter barley cv. Luxor in relation to short and long term periods of cold followed by a brief frost treatment were studied in order to disclose proteins responsible for the cold hardening process in distinct plant tissues. The mentioned changes have been monitored using two dimensional difference gel electrophoresis (2D-DIGE) with subsequent peptide-mapping protein identification. Regarding approximately 600-700 distinct protein spots detected on 2D gels, there has been found at least a two-fold change after exposure to low temperatures in about 10% of proteins in leaves and 13% of proteins in crowns. Protein and nitrogen metabolic processes have been influenced by low temperature to a similar extent in both tissues while catabolism, carbohydrate metabolism and proteins involved in stress response have been more affected in crowns than in leaves. The range of changes in protein abundance was generally higher in leaves and chloroplast proteins were frequently affected which suggests a priority to protect photosynthetic apparatus. Overall, our data proved existence of slightly different response strategies to low temperature stress in crowns and leaves, i.e., tissues with different biological role. Moreover, there have been found several proteins with large increase in accumulation, e.g., 33 kDa oxygen evolving protein of photosystem II in leaves and "enhanced disease susceptibility 1" in crowns; these proteins might have potential to indicate an enhanced level of frost tolerance in barley.

Proteomics of wheat and barley cereals in response to environmental stresses: Current state and future challenges.

Wheat and barley genera represent a wide range of genotypes from Triticeae group grown around the globe. The broad plasticity of Triticeae phenotypes mirrors the robustness of their genomes revealing a high level of gene homeology. Publication and annotation of the reference genome sequences for spring barley Morex and Chinese Spring wheat represents an important milestone enabling the researchers to precisely identify and annotate nearly all proteins. Due to the broad range of environments used for wheat and barley cultivation and their economical importance, proteomic studies focused on their responses to environmental stresses including combined stress treatments. Most of the Triticeae stress proteomics studies are comparative ones aimed at determination of differentially abundant proteins (DAPs) between two or more genotypes with contrasting stress tolerance. Studies focused on subcellular fractions and protein posttranslational modifications (PTMs) are still relatively rare although PTMs play a crucial role in modulation of protein biological function. Functional and interactomics studies are needed although gene homeology and the resulting protein functional redundancy practically excludes the utilization of knock-out mutants. The alternatives could represent either gene overexpression in a heterologous system such as A. thaliana or transient posttranscriptional gene silencing using RNAi. Publication of complete reference genome sequences together with novel technological approaches such as pQTL mapping boost the Triticeae proteomics studies not only to provide data but also to contribute to designing novel genotypes with improved adaptations to ever changing environments.

Plant Proteoforms Under Environmental Stress: Functional Proteins Arising From a Single Gene.

Proteins are directly involved in plant phenotypic response to ever changing environmental conditions. The ability to produce multiple mature functional proteins, i.e., proteoforms, from a single gene sequence represents an efficient tool ensuring the diversification of protein biological functions underlying the diversity of plant phenotypic responses to environmental stresses. Basically, two major kinds of proteoforms can be distinguished: protein isoforms, i.e., alterations at protein sequence level arising from posttranscriptional modifications of a single pre-mRNA by alternative splicing or editing, and protein posttranslational modifications (PTMs), i.e., enzymatically catalyzed or spontaneous modifications of certain amino acid residues resulting in altered biological functions (or loss of biological functions, such as in non-functional proteins that raised as a product of spontaneous protein modification by reactive molecular species, RMS). Modulation of protein final sequences resulting in different protein isoforms as well as modulation of chemical properties of key amino acid residues by different PTMs (such as phosphorylation, N- and O-glycosylation, methylation, acylation, S-glutathionylation, ubiquitinylation, sumoylation, and modifications by RMS), thus, represents an efficient means to ensure the flexible modulation of protein biological functions in response to ever changing environmental conditions. The aim of this review is to provide a basic overview of the structural and functional diversity of proteoforms derived from a single gene in the context of plant evolutional adaptations underlying plant responses to the variability of environmental stresses, i.e., adverse cues mobilizing plant adaptive mechanisms to diminish their harmful effects.

COR/LEA Proteins as Indicators of Frost Tolerance in Triticeae: A Comparison of Controlled versus Field Conditions.

Low temperatures in the autumn induce enhanced expression/relative accumulation of several cold-inducible transcripts/proteins with protective functions from Late-embryogenesis-abundant (LEA) superfamily including dehydrins. Several studies dealing with plants grown under controlled conditions revealed a correlation (significant quantitative relationship) between dehydrin transcript/protein relative accumulation and plant frost tolerance. However, to apply these results in breeding, field experiments are necessary. The aim of the review is to provide a summary of the studies dealing with the relationships between plant acquired frost tolerance and COR/LEA transcripts/proteins relative accumulation in cereals grown in controlled and field conditions. The impacts of cold acclimation and vernalisation processes on the ability of winter-type Triticeae to accumulate COR/LEA proteins are discussed. The factors determining dehydrin relative accumulation under controlled cold acclimation treatments versus field trials during winter seasons are discussed. In conclusion, it can be stated that dehydrins could be used as suitable indicators of winter survival in field-grown winter cereals but only in plant prior to the fulfilment of vernalisation requirement.

Relationship between WCS120 Protein Family Accumulation and Frost Tolerance in Wheat Cultivars Grown under Different Temperature Treatments.

Frost tolerance (FT) is generally acquired after exposure of plants to low, but non-freezing temperatures, where it is associated with the accumulation of COR proteins. The aim of the study was to reveal the effect of different temperature treatments (25, 17, 9 and 4 °C) on accumulation of cold-regulated dehydrins, dry weight content, and the development of FT in five wheat cultivars of different frost-tolerances in detail. The levels of cold-regulated dehydrins, WCS120 proteins in wheat were determined by immunoblot analysis, probed with an anti-dehydrin antibody. The lower the growth temperature: the higher the level of frost tolerance, dry weight content, and dehydrin accumulation, in all cultivars. There was a significant correlation between the level of induced FT and the accumulation of WCS120 proteins in cultivars grown at lower temperatures (9 and 4 °C). Moreover, the highly frost-tolerant wheat cultivars (as opposed to the lower-tolerant) accumulated higher levels of WCS120 proteins at 17 °C, a temperature at which it was not possible to differentiate between them via a frost test. Here, we demonstrated the possibility to distinguish differently frost-tolerant cultivars grown at different temperatures by the accumulation of different members of WCS120 family.