Mariela Odjakova

A Central Role for Thiols in Plant Tolerance to Abiotic Stress

Abiotic stress poses major problems to agriculture and increasing efforts are being made to understand plant stress response and tolerance mechanisms and to develop new tools that underpin successful agriculture. However, the molecular mechanisms of plant stress tolerance are not fully understood, and the data available is incomplete and sometimes contradictory. Here, we review the significance of protein and non-protein thiol compounds in relation to plant tolerance of abiotic stress. First, the roles of the amino acids cysteine and methionine, are discussed, followed by an extensive discussion of the low-molecular-weight tripeptide, thiol glutathione, which plays a central part in plant stress response and oxidative signalling and of glutathione-related enzymes, including those involved in the biosynthesis of non-protein thiol compounds. Special attention is given to the glutathione redox state, to phytochelatins and to the role of glutathione in the regulation of the cell cycle. The protein thiol section focuses on glutaredoxins and thioredoxins, proteins with oxidoreductase activity, which are involved in protein glutathionylation. The review concludes with a brief overview of and future perspectives for the involvement of plant thiols in abiotic stress tolerance.

The Role of Plant Cell Wall Proteins in Response to Salt Stress

Contemporary agriculture is facing new challenges with the increasing population and demand for food on Earth and the decrease in crop productivity due to abiotic stresses such as water deficit, high salinity, and extreme fluctuations of temperatures. The knowledge of plant stress responses, though widely extended in recent years, is still unable to provide efficient strategies for improvement of agriculture. The focus of study has been shifted to the plant cell wall as a dynamic and crucial component of the plant cell that could immediately respond to changes in the environment. The investigation of plant cell wall proteins, especially in commercially important monocot crops revealed the high involvement of this compartment in plants stress responses, but there is still much more to be comprehended. The aim of this review is to summarize the available data on this issue and to point out the future areas of interest that should be studied in detail.

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L.

Abstract The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (EGSSG/2 GSH) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (ECySS/2 Cys). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a ‘thiol–disulphide redox environment’ (Ethiol–disulphide), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of ECySS/2 Cys to Ethiol–disulphide in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.

Non-Enzymatic Glycosylation and Deglycating Enzymes

ABSTRACT Biological amines react with reducing sugars to form a heterogeneous group of compounds, called advanced glycation end products, a process known as the Maillard reaction. Glycation of proteins starts with formation of Shiffs base, which rearrange into Amadori product. The Amadori products then undergo a series of chemical modifications to form advanced glycation end products (AGEs). Many advanced glycation end products are capable of forming cross-links between proteins and many of them are fluorophores. The formation of AGEs is an irreversible process and glycation is a major cause of spontaneous damage to proteins in physiological systems. AGEs accumulate in tissues with age and their rate of accumulation is accelerated in diabetes. Hyperglycemia in diabetes causes accelerated AGEs formation and has been linked to various diabetic complications like nephropathy, retinopathy, angiopathy, and neuropathy. Nature has several defense mechanisms to protect tissues from AGEs accumulation. Among them are amadoriases, which can remove the Amadori product and interrupt the glycation cascade in the early steps of the Maillard reaction. To date three classes of deglycating enzymes were found-fructosamine oxidases, fructosamine-3-kinases, and fructoselysine 6-phosphate deglycase. These enzymes are known to occur in mammalian, fungal and other eukaryotic and prokaryotic cells.

Hydroxyproline Rich Proteins in Salt Adapted Embryogenic Suspension Cultures of Dactylis Glomerata L.

ABSTRACT Plant cell wall proteoglycans are involved in almost every substantial process in plant growth and development and are considered as possible biochemical markers for embryogenic potential, abiotic stress resistance, etc. Hydroxyproline-rich glycoproteins (HRGPs) are of particular interest due to their heterogeneity and abundance in all plant species. In the present study we characterized the distribution of HRGPs recognized by AGPs directed JIM8 and JIM13 and extensins directed JIM12 in salt adapted embryogenic suspension cultures of Dactylis glomerata L. The results obtained from Western blot analysis revealed that HRGPs are predominantly localized in the growth medium in salt treated cultures and are observed at the top of the separating gel with MW above 200 kDa. The presence of 43 kDa fraction, specific for JIM12 only in growth medium from salt-treated cultures is considered as a molecular marker for stress adaptation. The protein fractions detected by all antibodies in growth medium of salt treated cultures after SDS-PAGE in reducing conditions suggest the availability of non classical AGPs. Immunolocalization showed cell type specific and salt affected distribution of the JIM8 and JIM13 epitopes on the cell surface and implied that AGPs might have a specific role in the stress response and determination of the cell fate during somatic embryogenesis.

Мicroscopic investigations (LM, TEM and SEM) and identification of Chlorella isolate R-06/2 from extreme habitat in Bulgaria with a strong biological activity and resistance to environmental stress factors

An extremophilic Chlorella strain R-06/2, isolated from a geothermal spring (+42 °C) in the region of Rupite village (SW Bulgaria), was investigated for species identification. This was done by observation of the cell morphology, reproduction and ultrastructure by light microscopy, scanning electron microscopy and transmission electron microscopy, and by investigation of the cell-wall chemistry. The pyrenoid ultrastructure with a double-layered thylakoid traversing the matrix, the shape of the starch envelope, as well as the cell wall, composed of glucosamine and developed around young autospores, were the features that allowed us to classify the thermophilic strain Chlorella R-06/2 as Chlorella vulgaris Beijerinck 1890.

Ascorbate–Glutathione Cycle: Controlling the Redox Environment for Drought Tolerance

Drought impact on plants, similar to other abiotic stresses, is accompanied by increased levels of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (.OH) and superoxide anion (O2−). Survival strategies of plants under stress conditions often involve antioxidant defense activation. The nonenzymatic antioxidants ascorbate (AsA) and glutathione (GSH), their respective oxidized forms—dehydroascorbate (DHA) and glutathione disulphide (GSSG) and the enzymes that continuously recycle them constitute the major ROS scavenging system in plants—the ascorbate–glutathione cycle. This system operates in different cell compartments and genes, encoding its components are differentially regulated according to the current needs. The ability to effectively regulate the ascorbate–glutathione cycle discriminates between drought-sensitive and drought-tolerant cultivars. Enzymes from the cycle are also central for the mechanisms of drought adaptation and tolerance in xerophyte plants. Overexpression or transgenesis of genes encoding the ascorbate–glutathione cycle enzymes could significantly improve the drought tolerance of economically important crop plants. Recent experiments also revealed the importance of ascorbate–glutathione cycle in resurrection plant metabolism, in successful rehydration after desiccation. The current chapter aims to summarize the recent data on the role of the ascorbate–glutathione cycle for the drought stress response and tolerance of plants. Special attention is paid to the compartmentalization and gene expression control of the enzymatic machinery of the cycle.

Changes in protein thiols in response to salt stress in embryogenic suspension cultures of Dactylis glomerata L.

The aim of the present study is to assess the rate of protein disulphide formation and the activity of NADPH-dependent thioredoxin and glutaredoxin systems, responsible for the reverse reduction of protein and mixed protein-glutathione disulphides, in embryogenic suspension cultures of Dactylis glomerata, subjected to salt stress. Two concentrations of NaCl previously established as enhancing (0.085 mol/L) and inhibiting (0.17 mol/L) somatic embryogenesis were used. The quantitative (by colour reaction with Ellmans reagent) and qualitative (by diagonal gel electrophoresis) analyses showed a significant increase in protein disulphide formation in salt-treated cultures compared to controls. The ratio of disulphides to free thiols is higher in 0.17 mol/L NaCl-treated cultures. The activity of the thioredoxin–thioredoxin reductase system has been increased accordingly in 0.085 mol/L NaCl-treated cultures but decreased at the higher salt concentration. The activity of glutaredoxins was also estimated, by using glutathionylated bovine serum albumin as substrate and following the decrease of NADPH absorbance at 340 nm in the presence of glutathione and glutathione reductase. Mild salt (0.085 mol/L NaCl) treated cultures again showed the highest activity compared to controls and 0.17 mol/L NaCl-treated cultures. Based on these observations it was suggested that salt treatment resulted in increased protein disulphide formation and thioredoxin and glutaredoxin systems are important regulators of this process, strongly involved in salt stress response. The highest activity at 0.085 mol/L NaCl may be also related to the regulatory mechanisms, involved in the potentiating of somatic embryogenesis at this salt concentration.

Substrate Specificity of the Escherichia Coli FRLB Amadoriase

ABSTRACT Amadoriases are discovered in distant groups of organisms ranging from bacteria to humans. These enzymes catalyze the degradation of Amadori products which are formed in the early stage a non-enzymatic reaction between reducing sugars and primary amines called the Maillard reaction or glycation. The physiological role of amadoriases is debatable and perhaps not constrained to one cellular process only. Among other functions amadoriases are proposed to ensure enzymatic defense against the deleterious consequences of protein glycation. A decade ago an amadoriase enzyme (the FrlB amadoriase) has been discovered in Escherichia coli catalyzing the removal of Amadori products form the ϵ-amino group of free lysine. The E. coli FrlB enzyme has been suggested to catabolize glycated lysine released in the human intestine upon digestion of food proteins. In the present study we demonstrate that the E. coli FrlB amadoriase is capable of catalyzing in the reverse reaction the formation of Amadori products on a number of free amino acids and on polypeptides as well. Based on these results we suggest that in the forward reaction the FrlB enzyme might serve to deglycate E. coli proteins thus helping bacterial cells to withstand the protein glycation burden.

In Vitro Effect of some Pyrimidine Analogues on Embryogenic Response of Dactylis-glomerata Leaf Explants

Summary Five pyrimidine analogues (4,6-dihydroxy-5-nitropyrimidine, picolinic acid hydrazide, 2-hydrazinouracile, 2-thio-4-hydrazinouracile and D, L-dihydroorotic acid hydrazide) were tested for their ability to influence in vitro response of leaf explants from a highly embryogenic genotype of Dactylis glomerata L. Three of them induced a stimulatory effect on indirect somatic embryo formation after explant pretreatment for 6 h. 2-thio-4-hydrazinouracile at 50 mg·L -1 showed an apparently selective action on direct and indirect somatic embryogenesis.