Lyudmila Simova-Stoilova

Proteolytic activity and cysteine protease expression in wheat leaves under severe soil drought and recovery

The involvement of acidic proteases in soil drought response of winter wheat (Triticum aestivum L.) at seedling stage in three cultivars differing in water stress tolerance was studied. Withholding irrigation for seven days resulted in severe drought stress corresponding to 60% leaf water deficit. Stressed plants were recovered by providing optimal water supply for 3 days. Reversible changes in leaf pigment and protein content were registered, being least expressed in the drought-resistant cultivar Katya. Protein loss was inversely related to the increase in total proteolytic activity at pH 5 and in aminopeptidase activity at pH 7. Quantitative differences among the cultivars were established only for azocaseinolytic activity (pH 5). The drought-resistant cultivar (Katya) showed relatively little increase in acid protease activity whereas the highest values of this activity were detected in cultivar Pobeda. In-gel staining for cysteine-activated proteases revealed four to five separate activity bands. The upper band, specifically inhibited by E-64, was raised at severe drought. Transcript abundance of two wheat cysteine proteases -Ta.61026 putative thiol protease, and WCP2 peptidase of papain type was analyzed by RT-PCR. Gene expression of the cysteine proteases under study was suppressed in the drought-tolerant cultivar, while in the less resistant ones it remained unchanged or augmented. The results suggest that lower proteolytic activity and decreased expression of certain cysteine protease genes under water deficit during early developmental stage could be regarded as an indicator for drought resistance of winter wheat cultivars.

Heat stress effects on ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco binding protein and Rubisco activase in wheat leaves

Changes in chlorophyll content, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) binding protein (RBP), Rubisco activase (RA), Rubisco large (LS) and small (SS) subunits, and electrolyte leakage were investigated in wheat leaf segments during heat stress (HS) for 1 h and for 24 h at 40 °C in darkness or in light, as well as after recovery from heat stress (HSR) for 24 h at 25 °C in light. The 24-h HS treatment in darkness decreased irreversibly photosynthetic pigments, soluble proteins, RBP, RA, Rubisco LS and SS. An increase in RA and RBP protein contents was observed under 24-h HS and HSR in light. This increase was in accordance with their role as chaperones and the function of RBP as a heat shock protein.

Antioxidative protection and proteolytic activity in tolerant and sensitive wheat ( Triticum aestivum L.) varieties subjected to long-term field drought

Field drought studies were performed in order to assess oxidative stress, proteolytic activity and yield loss under natural stress conditions. Flag leaves of two drought-tolerant (Yantar and Zlatitsa) and two drought-sensitive (Miziya and Dobrudjanka) winter wheat varieties were analyzed. Stress intensity was assessed by relative electrolyte leakage and proline accumulation. Senescence progression was followed by loss of chlorophyll and protein. Lipid peroxidation, H2O2 content, activities of superoxide dismutase (SOD), catalase (CAT), and non-specific peroxidase (GPX) isoforms, as well as proteolytic activities were analyzed from heading throughout grain filling. Weakening of membrane integrity and oxidative damage to lipids were more pronounced in the sensitive varieties under field drought. The activities of Fe- and Cu/Zn SOD isoforms decreased in the controls, but remained high in drought-treated plants. The activities of MnSOD isoforms and CAT were enhanced towards grain filling, especially in the sensitive varieties under drought. GPX activities were rised under drought but progressively diminished. Accelerated senescence under field drought was linked to higher proteolytic activity with variety specific differences in the protease response, but without a clear correlation to drought resistance or sensitivity. Field drought led to higher oxidative stress more pronounced for drought sensitive varieties, especially during the grain filling period.

Rubisco and some chaperone protein responses to water stress and rewatering at early seedling growth of drought sensitive and tolerant wheat varieties

Four wheat varieties differing in their drought tolerance were subjected to severe but recoverable water stress at seedling stage. Growth parameters, leaf water deficit (WD) and electrolyte leakage (EL) were used to evaluate the stress intensity and the extent of recovery. The physiological response of the varieties was quite similar under severe drought. Leaf protein patterns and levels of some individual proteins relevant to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) maintenance were studied in control, stressed and recovering plants by electrophoresis and immunoblotting. The bands representing Rubisco large subunit (RLS), N- and C-terminus of RLS, Rubisco activase (RA) and Rubisco binding protein (RBP, cpn 60), as well as the chaperone and proteolytic subunits of the Clp protease complex were identified using polyclonal antibodies. Under drought conditions RLS, Clp proteases and especially RBP were enhanced, whereas the RA band was only slightly affected. The drought tolerant varieties had higher RBP content in the controls and drought treated plants. Its concentration could be a potential marker for drought tolerance.

Variety-specific response of wheat (Triticum aestivum L.) leaf mitochondria to drought stress.

The main objective of the present work was to examine leaf respiratory responses to dehydration and subsequent recovery in three varieties of winter wheat (Triticum aestivum L.) known to differ in their level of drought tolerance. Under dehydration, both total respiration and salicylhydroxamic acid (SHAM)-resistant cytochrome (Cyt) pathway respiration by leaf segments decreased significantly compared with well-watered plants. This decrease was more pronounced in the drought-sensitive Sadovo and Prelom genotypes. In contrast, the KCN-resistant SHAM-sensitive alternative (Alt) pathway became increasingly engaged, and accounted for about 80% of the total respiration. In the drought-tolerant Katya variety, increased contribution of the Alt pathway was accompanied by a slight decrease in Cyt pathway activity. Respiration of isolated leaf mitochondria also showed a variety-specific drought response. Mitochondria from drought-sensitive genotypes had low oxidative phosphorylation efficiency after dehydration and rewatering, whereas the drought-tolerant Katya mitochondria showed higher phosphorylation rates. Morphometric analysis of leaf ultrastructure revealed that mitochondria occupied approximately 7% of the cell area in control plants. Under dehydration, in the drought-sensitive varieties this area was reduced to about 2.0%, whereas in Katya it was around 6.0%. The results are discussed in terms of possible mechanisms underlying variety-specific mitochondrial responses to dehydration.

Cadmium Stress in Barley: Growth, Leaf Pigment, and Protein Composition and Detoxification of Reactive Oxygen Species

ABSTRACT 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.

Abscisic acid and late embryogenesis abundant protein profile changes in winter wheat under progressive drought stress

Three varieties (cv. Pobeda, Katya and Sadovo) of winter wheat (Triticum aestivum), differing in their agronomic characteristics, were analysed during progressive soil water stress and recovery at early vegetation stages. Changes in abscisic acid content, SDS-PAGE and immunoblot profiles of proteins that remained soluble upon heating were monitored. Initially higher ABA content in control Pobeda and Katya corresponded to earlier expression of the studied late embryogenesis abundant (LEA) proteins. A combination of higher ABA content, early immunodetection of dehydrins, and a significant increase of WZY2 transcript levels were observed in drought-stressed leaves of the tolerant variety Katya. One-step RT-PCR analyses of some acidic dehydrin genes (WCOR410b, TADHN) documented their relatively constant high expression levels in leaves under drought stress during early vegetative development. Neutral WZY2 dehydrin, TaLEA2 and TaLEA3 transcripts accumulated gradually with increasing water deficit. Delayed expression of TaLEA2 and TaLEA3 genes was found in the least drought-tolerant wheat, Sadovo. The expression profile of WZY2 revealed two distinct and separate bands, suggesting alternative splicing, which altered as water stress increased.

Involvement of the leaf antioxidant system in the response to soil flooding in two Trifolium genotypes differing in their tolerance to waterlogging

A comparative study of the response to waterlogging in a tolerant (Trifolium repens L., white clover cultivar Rivendel) and susceptible (Trifolium pratense L., red clover cultivar Raya) plants was undertaken to reveal the possible link between plant performance and oxidative stress protection mechanisms in leaves. Two weeks of soil waterlogging induced visible leaf damage in the susceptible genotype. In the tolerant one, signs of stress suffering appeared a week later. Waterlogging induced hydrogen peroxide accumulation in both clover species. The content of lipid hydroperoxides markedly increased in the sensitive plants along with stress prolongation, while in the tolerant ones their initial rise was followed by return to control levels. In the leaves of both genotypes ascorbic acid content increased following treatment, accompanied by transient increase in oxidized ascorbate. Superoxide dismutase (SOD) isoforms responded differently to the treatment, CuZn SOD isoforms being inhibited; catalase activity diminished while peroxidase activity increased and a new peroxidase isoform was detected after prolonged waterlogging in both clover species. Results support more pronounced oxidative secondary stress in red clover leaves as a result of waterlogging with progressively increased oxidative membrane injury, protein loss, and peroxidase activity enhancement. White clover presented relative protein stability and earlier and more active ascorbate involvement in the antioxidative protection.

Antioxidant response to drought in red and white clover

Antioxidant response to drought in red (Trifolium pratense L., cv. “Start”) and white clover (Trifolium repens L, cv. “Haifa” and cv. “Debut”) grown as soil cultures was evaluated in water-deprived and recovered plants. Drought provoked oxidative stress in leaves confirmed by the considerable changes in electrolyte leakage, malondialdehyde, hydrogen peroxides and proline contents. Immunoblot of Δ-1-pyrroline-5-carboxylate synthetase (P5CS), which catalyzes the first two steps in proline biosynthesis, revealed strong induction of the enzyme in red clover plants submitted to drought. Water-deprived white clover plants exhibited distinct P5CS profiles. This was related to different drought tolerance of the studied T. repens cultivars. Isoenzyme analyses of superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) demonstrated certain differences in antioxidant defence among the tested varieties. It was confirmed that MnSOD (in both T. repens and T pratense) and FeSOD (in T. repens) isoforms were the most affected by drought. The red clover cultivar “Start” exhibited the lowest FeSOD and POX activities which could contribute to its poor performance under water deprivation.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Specific Proteolysis in Barley Chloroplasts During Dark Induced Senescence

Intact chloroplasts were isolated from dark-senescing primary barley (Hordeum vulgare L.) leaves in order to study selective ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) degradation by the stromal and membrane fractions. RuBPCO specific degradation was estimated and characterised applying sensitive avidin-biotin ELISA method with non-modified or oxidatively modified biotinylated RuBPCO (BR) as substrates. Distinct proteolytic activities were detected. They differed in ATP and divalent metal ion dependence, protease inhibitory profile, and dynamics in the time-course of dark-induced senescence. The results supported involvement of ATP- and metal ion-dependent serine type proteolytic activity against non-modified BR early in induced senescence and appearance of ATP-independent activity at later stage. Active oxygen-modified BR was degraded by ATP-independent serine-type protease probably containing essential SH-groups and requiring divalent metal ions.