Drought stress response in winter wheat varieties – changes in leaf proteins and proteolytic activities

Radiation mutagenesis has been used in sustainable agriculture as a tool for increasing plant variability and providing new lines for selection. This necessitates a comparison, by using suitable stress markers, of the newly created lines with some well-established varieties, which are stress tolerant or susceptible. Drought is one of the most frequently encountered stresses with deleterious effects on plant performance and crop yield. Winter wheat seedlings (soil cultures at 3–4th leaf stage) from one mutant line (M181/1338K), one drought-tolerant (Guinness) and one sensitive variety (Farmer) were subjected to severe drought stress by water withholding, followed by recovery. Changes in leaf protein profiles, the amount of Rubisco large subunit (RLS), some specific chloroplast proteins such as Rubisco binding protein (RPB), Rubisco activase (RA), the chaperone subunit clpA/C of clp protease, as well as the activities of exo- and endo-proteases were analyzed. At the protein level, some differences were found in the drought response of genotypes – stability of RLS and RBP in M181/1338K and Guinness, diminution of RLS and increase in RBP in Farmer. RA presented strong up-regulation at recovery in Guinness but decreased in content under drought in M181/1338K and Farmer. Increase in ClpA/C level was found in all compared varieties under stress. Strong increase in total proteolytic activity was detected under drought only in Farmer. Inhibitory analysis revealed a predominance of cysteine and serine protease types. Aminopeptidase activities remained higher at recovery in M181/1338K and Farmer. Results are discussed in terms of genotype-linked different stress coping strategies

2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage

Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress

Holm oak proteomic response to water limitation at seedling establishment stage reveals specific changes in different plant parts as well as interaction between roots and cotyledons

Quercus ilex is a dominant tree species in the Mediterranean region with double economic and ecological importance and increasing use in reforestation. Seedling establishment is extremely vulnerable to environmental stresses, particularly drought. A time course study on physiological and proteomic response of holm oak to water limitation stress and recovery during early heterotrophic growth is reported. Applied stress led to diminution in plant water content and root growth, oxidative stress in roots and some alterations in the anti-oxidative protection. Plant parts differed substantially in soluble sugar and free phenolic content, and in their changes during stress and recovery. Proteomic response in holm oak roots and cotyledons was estimated using combined 1-DE/2-DE approach and protein identification by MALDI TOF-TOF PMF and MS/MS. A total of 127 differentially abundant protein species (DAPs) were identified. DAPs related to starch metabolism, lipid to sugar conversion, reserve proteins and their mobilization were typical for cotyledons. DAPs in roots were involved in sugar utilization, secondary metabolism and defense, including pathogenesis related proteins from PR-5 and PR-10 families. Results emphasize specific proteome signatures of separate plant parts as well as importance of sink-source interaction between root and cotyledon in the time course of stress and in recovery

Selection and Breeding of Suitable Crop Genotypes for Drought and Heat Periods in a Changing Climate: Which Morphological and Physiological Properties Should Be Considered?

Selection and breeding of genotypes with improved drought/heat tolerance become key issues in the course of global change with predicted increased frequency of droughts or heat waves. Several morphological and physiological plant traits must be considered. Rooting depth, root branching, nutrient acquisition, mycorrhization, nodulation in legumes and the release of nutrients, assimilates or phytohormones to the shoot are relevant in root systems. Xylem embolism and its repair after a drought, development of axillary buds and solute channeling via xylem (acropetal) and phloem (basipetal and acropetal) are key processes in the stem. The photosynthetically active biomass depends on leaf expansion and senescence. Cuticle thickness and properties, epicuticular waxes, stomatal regulation including responses to phytohormones, stomatal plugs and mesophyll resistance are involved in optimizing leaf water relations. Aquaporins, dehydrins, enzymes involved in the metabolism of compatible solutes (e.g., proline) and Rubisco activase are examples for proteins involved in heat or drought susceptibility. Assimilate redistribution from leaves to maturing fruits via the phloem influences yield quantity and quality. Proteomic analyses allow a deeper insight into the network of stress responses and may serve as a basis to identify suitable genotypes, although improved stress tolerance will have its price (often lowered productivity under optimal conditions)

Cadmium stress in barley: growth, leaf pigment and protein composition and detoxification of reactive oxygen species

Barley seedlings (Hordeum vulgare L., cv. ‘Obzor’) were exposed for 5 d to 0, 5, 50, and 500 μM CdCl2 in nutrient solution. Cadmium (Cd) treatment caused a reduction of plant length, biomass, and leaf pigment content. The level of soluble leaf proteins was not changed significantly. SDS-PAGE revealed a slight diminution of Rubisco subunits and the appearance of a new low molecular mass band after exposure to 50 or 500 μM Cd. The antioxidative protection in leaves under Cd toxicity was studied in its complexity. Slightly diminished superoxide dismutase, enhanced catalase, and drastically increased total peroxidase activities were found at the highest Cd level. Ascorbate peroxidase activity was not changed significantly. The isoenzyme patterns of the antioxidant enzymes under study were only slightly altered without synthesis of new isoforms. The content of oxidized ascorbate increased during exposure to 50 and 500 μM Cd. The level of H2O2 rose only at 500 μM Cd without accumulation of malondialdehyde and oxidized proteins. Non-protein thiol groups increased up to four-fold after exposure to 50 and 500 μM Cd. The results are in accordance with the induction of mechanisms allowing an immobilization and sequestration of Cd in barley leaves, and suggest only minor effects via oxidative damage

The response of four winter wheat (Triticum aestivum L.) varieties to field water deficit: leaf anti-oxidative protection and proteolytic activity

Introduction: Drought is one of the most significant factors that limit plant productivity. Oxidative stress is a secondary event in many unfavorable environmental conditions. Intracellular proteases have a role in the metabolism reorganisation and nutrient remobilization under stress. In order to under stand the relative significance of oxidative stress and proteolysis in the yield reduction under drought, four varieties of Triticum aestivum L. with different field drought resistance were examined. Methods: A two-year field experiment was conducted. Analyses were performed on the upper most leaf of control plants and plants under water deficitat the stages most critical for yield reduction under drought (from jointing till milk ripeness). Leaf water deficit and electrolyte leakage, malondyaldehyde level, activities and isoenzymes of superoxide dismutase, catalase and peroxidase, leaf protein content and proteolytic activity were studied. Yield components were analyzed. Results: A general trend of increasing the membrane in stability and accumulation of lipid hydroperoxides was observed with some differences among varieties, especially under drought. The anti-oxidative enzyme activities were progressively enhanced, as well as the azocaseinolytic activities. The leaf protein content decreased under drought at the last phase. Differences among varieties were observed in the parameters under study. They were compared to yield components` reduction under water deprivation

Aminopeptidase activities in roots and leaves of drought stressed winter wheat seedlings

In order to evaluate the role of aminopeptidases (APs) in drought response and their potential as protein markers to distinguish between stress tolerant and sensitive varieties, various AP activities were studied in roots and leaves of winter wheat seedlings, subjected to severe but recoverable soil drought stress. Two varieties with contrasting drought tolerance – Yantar (drought tolerant) and Miziya (sensitive) were compared. Activity changes under severe water stress and subsequent recovery were related to changes in the pools of the major redox buffers ascorbate and glutathione, changes in protein profiles and total proteolysis in roots and leaves. Glutathione was responsive to drought both in roots and in leaves, with increased total pool and transient rise in the oxidized form; stronger response in the roots of Yantar was observed. The sensitive variety had higher ascorbate content in leaves under stress. Severe drought led to reversible changes in protein profiles and increase in major protease bands in leaves but not in roots. AP activities were partly independent from the predominant endoprotease activities. Highest activities in roots were detected with substrates releasing terminal leucine, lysine and methionine. In stressed leaves AP activities toward most of the substrates increased under drought, without clear differences comparing varieties. Activities tested with Gly-pNA were raised in leaves only in recovery from stress. In roots, the tolerant variety Yantar presented increased AP activities under stress with most of the substrates used except Leu-pNA and Phe-pNA, whereas the sensitive variety Miziya had almost unchanged AP activities. Based on activity profile changes, at least two different AP enzymes should exist in wheat. It remains to be established which activities towards different substrates reflect distinct aminopeptidases