L. Simova-stoilova - Academia.edu (original) (raw)

Papers by L. Simova-stoilova

In order to evaluate the role of aminopeptidases (APs) in drought response and their potential as... more 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 increas...

One of the most important issues facing health care is the prevention and treatment of different ... more One of the most important issues facing health care is the prevention and treatment of different types of cancer. Among the most frequently encountered types of cancer leading to lethality are lung cancer, breast cancer and colon cancer. The search for new drugs for cancer cells treatment and clarifying the mechanisms of their anti-tumor effect is a scientific challenge and a necessary basis for new more effective methods of cancer treatment. A major role in tumor growth, invasion, angiogenesis and metastasis have different types of proteases whose activities are inhibited by some synthetic drugs. Protease inhibitors of plant origin have the potential to be an alternative or supplement to the treatment with synthetic drugs, but the mechanisms of their anti-tumor effects are poorly understood. In this review the current knowledge of plant protease inhibitors as tumor preventive and suppresive agents, and the perspectives of their use as antitumour drugs are discussed. Abbreviations: ...

Waterlogging has a range of negative effects on plant growth and productivity. The development of... more Waterlogging has a range of negative effects on plant growth and productivity. The development of hypoxia in flooded soils disturbes plant metabolism since roots normally require oxygen for optimal production of adenosine triphosphate. Within the plant, anaerobic respiration leads to the production of ethanol or lactate, causing cellular and membrane damage, impairing nutrient uptake and facilitating pathogen entry. Long-term waterlogging worsens soil mechanical and chemical properties decreasing its agricultural quality. This review focuses on waterlogging stress in Trifolium spp – important fodder legumes with high nutritional value for grazing animals, and its impact on plant proteolysis. The biochemical and functional characteristics of plant proteases are discussed, focusing on the specific proteolytic events observed under excessive water stress. The current knowledge about plant protease inhibitors and their role in control over endogenous proteolysis is briefly reviewed.

Summary. A review is made of the published investigations concerning the structure and conformati... more Summary. A review is made of the published investigations concerning the structure and conformational dynamic changes in the key photosynthetic and photorespiratory enzyme – Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of higher plants. Recent data on the basis of X-ray crystalography, NMR-spectroscopy, site-directed mutagenesis and other techniques are analysed. Despite some differences in the aminoacid sequence of Rubisco from various species, significant conformational changes are not established. The tertiary and quaternary structure of Rubisco is described in detail emphasizing the active and regulatory sites of the enzyme. Possibilities and limitations of the different methods of studying protein structure and conformation are discussed. Some advantages of the immunochemical methods especially the use of monoclonal antibodies for investigations of dynamic changes of protein structures in solution are pointed out.

Four monoclonal antibodies (Mabs) against Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenas... more Four monoclonal antibodies (Mabs) against Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) from Hordeum vulgare L., previously described as inhibiting the enzyme activity, were characterized in respect to their effect on the active site of the enzyme. Two of the Mabs partially prevent Rubisco carbamylation in trapping experiments measuring the amount of 14 CO 2 bound to Rubisco which was rendered non-exchangeable by 2-carboxyarabinitol-1,5-bisphosphate (CABP). Changes in the binding of the same Mabs to Rubisco complexed with some effectors: ribulose-1,5-bisphosphate (RuBP), phosphoenol pyruvate (PEP), fructose-1,6-bisphosphate (FBP), 6-phosphogluconate (6PG), were observed in inhibition ELISA reflecting affinity alteration. A reduced interaction with the complex of activated Rubisco with CABP was revealed for all the Mabs. Although none of the Mabs affect directly the activation or the substrate binding site of the enzyme, Mab affinity for Rubisco is dependent ...

Acta Physiologiae Plantarum, 2021

Radiation mutagenesis could provide new drought-tolerant lines for selection purposes in sustaina... more Radiation mutagenesis could provide new drought-tolerant lines for selection purposes in sustainable agriculture. Drought tolerance and yield stability are closely related to coping with oxidative stress, which occurs at severe/prolonged water deprivation. In this study, the response of a newly generated winter wheat mutant line M181/1338K to severe drought stress at seedling stage (3–4th leaf) was compared to that of two established varieties—Guinness (drought tolerant) and Farmer (drought sensitive). Oxidative damage and antioxidant status analyses were performed on second leaves of control, stressed (45–46% leaf water deficit) and recovered plants. Genotypes exhibited similar pattern of stress response, comprising proline accumulation, rise in hydrogen peroxide content and oxidative damage to membrane lipids, increase in total antioxidant and antiradical activities, in phenolic and flavonoid content, in ascorbate and glutathione pools, mobilization of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) enzyme isoforms. Farmer responded to severe water stress with the highest levels of oxidative damage to membranes, proline accumulation, and glutathione content, and slower normalization of the studied parameters upon recovery. Guinness presented a better control of oxidative membrane damage and it the highest accumulation of flavonoids under drought. The new mutant line M181/1338K had similarities with Guinness in its response to severe water stress, such as the same proline and glutathione levels. Unlike Guinness, the mutant genotype had more pronounced oxidative damage to membranes, along with higher POX activities, and tended to accumulate less flavonoids under drought, which could be regarded as secondary effects of the induced mutagenesis.

Cereal Research Communications, 2016

A high number of protease inhibitors (PI) have been identified in diverse plant species but infor... more A high number of protease inhibitors (PI) have been identified in diverse plant species but information about their role in plant stress responses is still fragmentary. Transcript profiling of six published serine and cysteine protease inhibitor sequences in water-deprived plants from four winter wheat (Triticum aestivum) varieties with varying tolerance was performed in order to outline PIs predominantly accumulating under drought. Expression was analyzed by real time RT-qPCR. Considerable transcript accumulation of Bowman-Birk type PI WALI3 (BBPI) was detected in drought stressed leaves suggesting an important regulatory role of BBPI in adjustment of protein metabolism in leaves under dehydration. Serpin transcripts were less represented in water-deprived plants. Transient accumulation of cystatin transcripts revealed organ-specificity. Under drought cystatin and serpin expression in the leaves of the most drought tolerant variety "Katya" tended to preserve relatively stable levels close to the controls. This preliminary data will serve for future detailed study of regulation of proteolysis in winter wheat subjected to unfavorable environmental factors for development of molecular-based strategies for selection of tolerant varieties.

Progress in Botanical Research, 1998

Little is known about the fragmentation of Rubisco by endogenous and exogenous proteases. More se... more Little is known about the fragmentation of Rubisco by endogenous and exogenous proteases. More sensitive to proteolysis is Rubisco large subunit (LS) and the characteristic pattern of in vivo degrading fragments from Rubisco is different depending on experimental conditions (5, 7, 9, 10). Under limited proteolysis of native spinach Rubisco with trypsin two short peptides were released from the N-terminus and one from the C-terminus of the LS (13). Denatured Rubisco from spinach is digested by different proteases (chymotrypsin, papain and St. aureus V8 protease) and the LS of the enzyme generates about 15–25 bands (1). Barley Rubisco is less sensitive to trypsinolysis compared to wheat and spinach Rubisco (6). The characterization of Mabs against Rubisco applying limited proteolysis of the enzyme has not been made so far. Monoclonal antibodies (Mabs) against Rubisco from barley leaves have been produced and partially characterized (11). All the Mabs recognise only LS in Western blotting. In the present paper an attempt was made to more thoroughly characterize the epitopes on Rubisco molecule recognized by the Mabs, using limited proteolysis.

Summary. The existence of protein/protein interactions between Rubisco (R), Rubisco binding prote... more Summary. The existence of protein/protein interactions between Rubisco (R), Rubisco binding protein (RBP) and Rubisco activase (RA) was proven independently in the presence or in the absence of the ATP-regenerating sys- tem. Sandwich and inhibitory ELISA, immunoprecipitation and immunoblotting methods were used with monoclonal antibodies (Mabs) against barley Rubisco or polyclonal antibodies against barley RBP or RA. The results show

Plant Growth Regulation, 2008

Four wheat varieties differing in their drought tolerance were subjected to severe but recoverabl... more 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

Plant Biology, 2010

Three varieties (cv. Pobeda, Katya and Sadovo) of winter wheat (Triticum aestivum), differing in ... more 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.

Journal of Plant Nutrition, 2006

Barley seedlings (Hordeum vulgare L., cv. ‘Obzor’) were exposed for 5 d to 0, 5, 50, and 500 μM C... more 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.

In order to evaluate the role of aminopeptidases (APs) in drought response and their potential as... more 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 increas...

One of the most important issues facing health care is the prevention and treatment of different ... more One of the most important issues facing health care is the prevention and treatment of different types of cancer. Among the most frequently encountered types of cancer leading to lethality are lung cancer, breast cancer and colon cancer. The search for new drugs for cancer cells treatment and clarifying the mechanisms of their anti-tumor effect is a scientific challenge and a necessary basis for new more effective methods of cancer treatment. A major role in tumor growth, invasion, angiogenesis and metastasis have different types of proteases whose activities are inhibited by some synthetic drugs. Protease inhibitors of plant origin have the potential to be an alternative or supplement to the treatment with synthetic drugs, but the mechanisms of their anti-tumor effects are poorly understood. In this review the current knowledge of plant protease inhibitors as tumor preventive and suppresive agents, and the perspectives of their use as antitumour drugs are discussed. Abbreviations: ...

Waterlogging has a range of negative effects on plant growth and productivity. The development of... more Waterlogging has a range of negative effects on plant growth and productivity. The development of hypoxia in flooded soils disturbes plant metabolism since roots normally require oxygen for optimal production of adenosine triphosphate. Within the plant, anaerobic respiration leads to the production of ethanol or lactate, causing cellular and membrane damage, impairing nutrient uptake and facilitating pathogen entry. Long-term waterlogging worsens soil mechanical and chemical properties decreasing its agricultural quality. This review focuses on waterlogging stress in Trifolium spp – important fodder legumes with high nutritional value for grazing animals, and its impact on plant proteolysis. The biochemical and functional characteristics of plant proteases are discussed, focusing on the specific proteolytic events observed under excessive water stress. The current knowledge about plant protease inhibitors and their role in control over endogenous proteolysis is briefly reviewed.

Summary. A review is made of the published investigations concerning the structure and conformati... more Summary. A review is made of the published investigations concerning the structure and conformational dynamic changes in the key photosynthetic and photorespiratory enzyme – Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of higher plants. Recent data on the basis of X-ray crystalography, NMR-spectroscopy, site-directed mutagenesis and other techniques are analysed. Despite some differences in the aminoacid sequence of Rubisco from various species, significant conformational changes are not established. The tertiary and quaternary structure of Rubisco is described in detail emphasizing the active and regulatory sites of the enzyme. Possibilities and limitations of the different methods of studying protein structure and conformation are discussed. Some advantages of the immunochemical methods especially the use of monoclonal antibodies for investigations of dynamic changes of protein structures in solution are pointed out.

Four monoclonal antibodies (Mabs) against Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenas... more Four monoclonal antibodies (Mabs) against Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) from Hordeum vulgare L., previously described as inhibiting the enzyme activity, were characterized in respect to their effect on the active site of the enzyme. Two of the Mabs partially prevent Rubisco carbamylation in trapping experiments measuring the amount of 14 CO 2 bound to Rubisco which was rendered non-exchangeable by 2-carboxyarabinitol-1,5-bisphosphate (CABP). Changes in the binding of the same Mabs to Rubisco complexed with some effectors: ribulose-1,5-bisphosphate (RuBP), phosphoenol pyruvate (PEP), fructose-1,6-bisphosphate (FBP), 6-phosphogluconate (6PG), were observed in inhibition ELISA reflecting affinity alteration. A reduced interaction with the complex of activated Rubisco with CABP was revealed for all the Mabs. Although none of the Mabs affect directly the activation or the substrate binding site of the enzyme, Mab affinity for Rubisco is dependent ...

Acta Physiologiae Plantarum, 2021

Radiation mutagenesis could provide new drought-tolerant lines for selection purposes in sustaina... more Radiation mutagenesis could provide new drought-tolerant lines for selection purposes in sustainable agriculture. Drought tolerance and yield stability are closely related to coping with oxidative stress, which occurs at severe/prolonged water deprivation. In this study, the response of a newly generated winter wheat mutant line M181/1338K to severe drought stress at seedling stage (3–4th leaf) was compared to that of two established varieties—Guinness (drought tolerant) and Farmer (drought sensitive). Oxidative damage and antioxidant status analyses were performed on second leaves of control, stressed (45–46% leaf water deficit) and recovered plants. Genotypes exhibited similar pattern of stress response, comprising proline accumulation, rise in hydrogen peroxide content and oxidative damage to membrane lipids, increase in total antioxidant and antiradical activities, in phenolic and flavonoid content, in ascorbate and glutathione pools, mobilization of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) enzyme isoforms. Farmer responded to severe water stress with the highest levels of oxidative damage to membranes, proline accumulation, and glutathione content, and slower normalization of the studied parameters upon recovery. Guinness presented a better control of oxidative membrane damage and it the highest accumulation of flavonoids under drought. The new mutant line M181/1338K had similarities with Guinness in its response to severe water stress, such as the same proline and glutathione levels. Unlike Guinness, the mutant genotype had more pronounced oxidative damage to membranes, along with higher POX activities, and tended to accumulate less flavonoids under drought, which could be regarded as secondary effects of the induced mutagenesis.

Cereal Research Communications, 2016

A high number of protease inhibitors (PI) have been identified in diverse plant species but infor... more A high number of protease inhibitors (PI) have been identified in diverse plant species but information about their role in plant stress responses is still fragmentary. Transcript profiling of six published serine and cysteine protease inhibitor sequences in water-deprived plants from four winter wheat (Triticum aestivum) varieties with varying tolerance was performed in order to outline PIs predominantly accumulating under drought. Expression was analyzed by real time RT-qPCR. Considerable transcript accumulation of Bowman-Birk type PI WALI3 (BBPI) was detected in drought stressed leaves suggesting an important regulatory role of BBPI in adjustment of protein metabolism in leaves under dehydration. Serpin transcripts were less represented in water-deprived plants. Transient accumulation of cystatin transcripts revealed organ-specificity. Under drought cystatin and serpin expression in the leaves of the most drought tolerant variety "Katya" tended to preserve relatively stable levels close to the controls. This preliminary data will serve for future detailed study of regulation of proteolysis in winter wheat subjected to unfavorable environmental factors for development of molecular-based strategies for selection of tolerant varieties.

Progress in Botanical Research, 1998

Little is known about the fragmentation of Rubisco by endogenous and exogenous proteases. More se... more Little is known about the fragmentation of Rubisco by endogenous and exogenous proteases. More sensitive to proteolysis is Rubisco large subunit (LS) and the characteristic pattern of in vivo degrading fragments from Rubisco is different depending on experimental conditions (5, 7, 9, 10). Under limited proteolysis of native spinach Rubisco with trypsin two short peptides were released from the N-terminus and one from the C-terminus of the LS (13). Denatured Rubisco from spinach is digested by different proteases (chymotrypsin, papain and St. aureus V8 protease) and the LS of the enzyme generates about 15–25 bands (1). Barley Rubisco is less sensitive to trypsinolysis compared to wheat and spinach Rubisco (6). The characterization of Mabs against Rubisco applying limited proteolysis of the enzyme has not been made so far. Monoclonal antibodies (Mabs) against Rubisco from barley leaves have been produced and partially characterized (11). All the Mabs recognise only LS in Western blotting. In the present paper an attempt was made to more thoroughly characterize the epitopes on Rubisco molecule recognized by the Mabs, using limited proteolysis.

Summary. The existence of protein/protein interactions between Rubisco (R), Rubisco binding prote... more Summary. The existence of protein/protein interactions between Rubisco (R), Rubisco binding protein (RBP) and Rubisco activase (RA) was proven independently in the presence or in the absence of the ATP-regenerating sys- tem. Sandwich and inhibitory ELISA, immunoprecipitation and immunoblotting methods were used with monoclonal antibodies (Mabs) against barley Rubisco or polyclonal antibodies against barley RBP or RA. The results show

Plant Growth Regulation, 2008

Four wheat varieties differing in their drought tolerance were subjected to severe but recoverabl... more 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

Plant Biology, 2010

Three varieties (cv. Pobeda, Katya and Sadovo) of winter wheat (Triticum aestivum), differing in ... more 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.

Journal of Plant Nutrition, 2006

Barley seedlings (Hordeum vulgare L., cv. ‘Obzor’) were exposed for 5 d to 0, 5, 50, and 500 μM C... more 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.