Modeling the Blood-Brain Barrier Permeability of Potential Heterocyclic Drugs via Biomimetic IAM Chromatography Technique Combined with QSAR Methodology.

Penetration through the blood-brain barrier (BBB) is desirable in the case of potential pharmaceuticals acting on the central nervous system (CNS), but is undesirable in the case of drug candidates acting on the peripheral nervous system because it may cause CNS side effects. Therefore, modeling of the permeability across the blood-brain barrier (i.e., the logarithm of the brain to blood concentration ratio, log BB) of potential pharmaceuticals should be performed as early as possible in the preclinical phase of drug development. Biomimetic chromatography with immobilized artificial membrane (IAM) and the quantitative structure-activity relationship (QSAR) methodology were successful in modeling the blood-brain barrier permeability of 126 drug candidates, whose experimentally-derived lipophilicity indices and computationally-derived molecular descriptors (such as molecular weight (MW), number of rotatable bonds (NRB), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), topological polar surface area (TPSA), and polarizability (α)) varied by class. The QSARs model established by multiple linear regression showed a positive effect of the lipophilicity (log kw, IAM) and molecular weight of the compound, and a negative effect of the number of hydrogen bond donors and acceptors, on the log BB values. The model has been cross-validated, and all statistics indicate that it is very good and has high predictive ability. The simplicity of the developed model, and its usefulness in screening studies of novel drug candidates that are able to cross the BBB by passive diffusion, are emphasized.

Jasmonic Acid Effect on Cucumis sativus L. Growth Is Related to Inhibition of Plasma Membrane Proton Pump and the Uptake and Assimilation of Nitrates.

When plants are exposed to environmental stress, their growth is inhibited. Under such conditions, controlled inhibition of growth is beneficial for plant survival. Jasmonic acid (JA) is a well-known phytohormone that limits plant growth, which has been confirmed in several species. However, its role in cucumber seedlings has not yet been comprehensively investigated. For this reason, we aimed to determine the involvement of JA in the regulation of proteins crucial for growth including plasma membrane proton pump (PM H+-ATPase), PM nitrate transporters, and nitrate reductase (NR). Treatment of cucumber seedlings with JA not only limited their growth but also increased the H2O2 content in their roots. The main sources of ROS generated for signalling purposes are PM NADPH oxidase (RBOH) and superoxide dismutase (SOD). Exposure of seedlings to JA induced the expression of some CsRBOH and SOD encoding genes, suggesting that ROS signalling can be activated by JA. As a consequence of JA exposure, the activity of all analysed proteins was inhibited and the expression of their genes was modified. The results indicate that reduction of PM H+-ATPase activity and the related decrease in nitrate uptake and assimilation are responsible for the root growth retardation of JA-treated plants.

Exogenous Putrescine Modulates Nitrate Reductase-Dependent NO Production in Cucumber Seedlings Subjected to Salt Stress.

Polyamines (PAs) are small aliphatic compounds that participate in the plant response to abiotic stresses. They also participate in nitric oxide (NO) production in plants; however, their role in this process remains unknown. Therefore, the study aimed to investigate the role of putrescine (Put) in NO production in the roots of cucumber seedlings subjected to salt stress (120 mM NaCl) for 1 and 24 h. In salinity, exogenous Put can regulate NO levels by managing NO biosynthesis pathways in a time-dependent manner. In cucumber roots exposed to 1 h of salinity, exogenous Put reduced NO level by decreasing nitrate reductase (NR)-dependent NO production and reduced nitric oxide synthase-like (NOS-like) activity. In contrast, during a 24 h salinity exposure, Put treatment boosted NO levels, counteracting the inhibitory effect of salinity on the NR and plasma membrane nitrate reductase (PM-NR) activity in cucumber roots. The role of endogenous Put in salt-induced NO generation was confirmed using Put biosynthesis inhibitors. Furthermore, the application of Put can modulate the NR activity at the genetic and post-translational levels. After 1 h of salt stress, exogenous Put upregulated CsNR1 and CsNR2 expression and downregulated CsNR3 expression. Put also decreased the NR activation state, indicating a reduction in the level of active dephosphorylated NR (dpNR) in the total enzyme pool. Conversely, in the roots of plants subjected to 24 h of salinity, exogenous Put enhanced the NR activation state, indicating an enhancement of the dpNR form in the total NR pool. These changes were accompanied by a modification of endogenous PA content. Application of exogenous Put led to an increase in the amount of Put in the roots and reduced endogenous spermine (Spm) content in cucumber roots under 24 h salinity. The regulatory role of exogenous Put on NO biosynthesis pathways may link with plant mechanisms of response to salt stress.

Structural and Functional Diversity of Two ATP-Driven Plant Proton Pumps.

Two ATP-dependent proton pumps function in plant cells. Plasma membrane H+-ATPase (PM H+-ATPase) transfers protons from the cytoplasm to the apoplast, while vacuolar H+-ATPase (V-ATPase), located in tonoplasts and other endomembranes, is responsible for proton pumping into the organelle lumen. Both enzymes belong to two different families of proteins and, therefore, differ significantly in their structure and mechanism of action. The plasma membrane H+-ATPase is a member of the P-ATPases that undergo conformational changes, associated with two distinct E1 and E2 states, and autophosphorylation during the catalytic cycle. The vacuolar H+-ATPase represents rotary enzymes functioning as a molecular motor. The plant V-ATPase consists of thirteen different subunits organized into two subcomplexes, the peripheral V1 and the membrane-embedded V0, in which the stator and rotor parts have been distinguished. In contrast, the plant plasma membrane proton pump is a functional single polypeptide chain. However, when the enzyme is active, it transforms into a large twelve-protein complex of six H+-ATPase molecules and six 14-3-3 proteins. Despite these differences, both proton pumps can be regulated by the same mechanisms (such as reversible phosphorylation) and, in some processes, such as cytosolic pH regulation, may act in a coordinated way.

Interactions of Polyamines and Phytohormones in Plant Response to Abiotic Stress.

Numerous environmental conditions negatively affect plant production. Abiotic stresses, such as salinity, drought, temperature, and heavy metals, cause damage at the physiological, biochemical, and molecular level, and limit plant growth, development, and survival. Studies have indicated that small amine compounds, polyamines (PAs), play a key role in plant tolerance to various abiotic stresses. Pharmacological and molecular studies, as well as research using genetic and transgenic approaches, have revealed the favorable effects of PAs on growth, ion homeostasis, water maintenance, photosynthesis, reactive oxygen species (ROS) accumulation, and antioxidant systems in many plant species under abiotic stress. PAs display a multitrack action: regulating the expression of stress response genes and the activity of ion channels; improving the stability of membranes, DNA, and other biomolecules; and interacting with signaling molecules and plant hormones. In recent years the number of reports indicating crosstalk between PAs and phytohormones in plant response to abiotic stresses has increased. Interestingly, some plant hormones, previously known as plant growth regulators, can also participate in plant response to abiotic stresses. Therefore, the main goal of this review is to summarize the most significant results that represent the interactions between PAs and plant hormones, such as abscisic acid, brassinosteroids, ethylene, jasmonates, and gibberellins, in plants under abiotic stress. The future perspectives for research focusing on the crosstalk between PAs and plant hormones were also discussed.

Water and Nutrient Recovery for Cucumber Hydroponic Cultivation in Simultaneous Biological Treatment of Urine and Grey Water.

Water and nutrient deficiencies in soil are becoming a serious threat to crop production. Therefore, usable water and nutrient recovery from wastewater, such as urine and grey water, should be considered. In this work, we showed the possibility of using grey water and urine after processing in an aerobic reactor with activated sludge in which the nitrification process takes place. The resulting liquid (nitrified urine and grey water, NUG) contains three potential factors that can adversely affect plant growth in a hydroponic system: anionic surfactants, nutrient deficits, and salinity. After dilution and supplementation with small amounts of macro- and micro-elements, NUG was suitable for cucumber cultivation. Plant growth on this modified medium (enriched nitrified urine and grey water, NUGE) was similar to that of plants cultivated on Hoagland solution (HS) and reference commercial fertilizer (RCF). The modified medium (NUGE) contained a significant amount of sodium (Na) ions. Therefore, typical effects of salt stress were observed in cucumber plants, including reduced chlorophyll levels, slightly weaker photosynthesis parameters, increased H2O2 levels, lipid peroxidation, ascorbate peroxidase (APX) activity, and proline content in the leaves. In addition, reduced protein levels were observed in plants treated with recycled medium. At the same time, lower nitrate content in tissues was found, which may have resulted from their intensive use by nitrate reductase (NR), the activity of which significantly increased. Although cucumber is a glycophyte, it grew very well in this recycled medium. Interestingly, salt stress and possibly anionic surfactants promoted flower formation, which in turn could positively affect plant yield.

Urine and grey water based liquid fertilizer - Production and the response of plants.

Plant cultivation is a key aspect of future long-distance space missions, and the creation of an efficient food system will not be possible without it. The production of fertilizer in space is based on the recovery of water and nutrients from wastewater, such as urine and grey water. In this study, the fertilizer production process was conducted in an aerobic, activated sludge reactor, where nitrification and the process of carbon removal take place. Treated streams have three potential factors that could affect the plants growth in a hydroponic system (anionic surfactants, nutrients deficiencies, high salinity). The effect of these factors was examined for two hydroponic configurations. Their influence on lettuce yield, quality parameters and stress response were investigated and compared to the control cultivation. The results showed that the main cause of a decrease (up to 24%) in the yield productivity of plants grown on nitrified urine and grey water is oxidative stress originated from a deficiency of elements, not from used anionic surfactant. Enrichment with nutrients resulted in the restoration of proper protein synthesis and an increase in the activity of antioxidant enzymes, which was positively reflected in the qualitative and quantitative parameters of the enriched cultivation (fresh leaves mass equal to 103% of the control). Results also show that Sodium Methyl Cocoyl Taurate (SMCT) surfactant itself after biological treatment used in plant cultivation has no negative effects reflected in lettuce yield or quality.

Plant Plasma Membrane Proton Pump: One Protein with Multiple Functions.

In plants, the plasma membrane proton pump (PM H+-ATPase) regulates numerous transport-dependent processes such as growth, development, basic physiology, and adaptation to environmental conditions. This review explores the multifunctionality of this enzyme in plant cells. The abundance of several PM H+-ATPase isogenes and their pivotal role in energizing transport in plants have been connected to the phenomena of pleiotropy. The multifunctionality of PM H+-ATPase is a focal point of numerous studies unraveling the molecular mechanisms of plant adaptation to adverse environmental conditions. Furthermore, PM H+-ATPase is a key element in plant defense mechanisms against pathogen attack; however, it also functions as a target for pathogens that enable plant tissue invasion. Here, we provide an extensive review of the PM H+-ATPase as a multitasking protein in plants. We focus on the results of recent studies concerning PM H+-ATPase and its role in plant growth, physiology, and pathogenesis.

Combined Micellar Liquid Chromatography Technique and QSARs Modeling in Predicting the Blood-Brain Barrier Permeation of Heterocyclic Drug-like Compounds.

The quantitative structure-activity relationship (QSAR) methodology was used to predict the blood-brain permeability (log BB) for 65 synthetic heterocyclic compounds tested as promising drug candidates. The compounds were characterized by different descriptors: lipophilicity, parachor, polarizability, molecular weight, number of hydrogen bond acceptors, number of rotatable bonds, and polar surface area. Lipophilic properties of the compounds were evaluated experimentally by micellar liquid chromatography (MLC). In the experiments, sodium dodecyl sulfate (SDS) as the effluent component and the ODS-2 column were used. Using multiple linear regression and leave-one-out cross-validation, we derived the statistically significant and highly predictive quantitative structure-activity relationship models. Thus, this study provides valuable information on the expected properties of the substances that can be used as a support tool in the design of new therapeutic agents.

Role of Plasma Membrane NADPH Oxidase in Response to Salt Stress in Cucumber Seedlings.

Plasma membrane NADPH oxidases (RBOHs, EC 1.6.3.1) are known as the main ROS generators involved in plant adaptation to stress conditions. In the present work, regulation of NADPH oxidase was analyzed in cucumber (Cucumis sativus L. var. Krak) seedlings exposed to salinity. RBOH activity and gene expression, as well as H2O2 content, were determined in the roots of plants treated with 50 or 100 mM NaCl for 1 h, and 50 mM NaCl for 1 or 6 days. It was found that enzyme activity increased in parallel with an enhancement in the H2O2 level in roots exposed to 100 mM NaCl for 1 h, and to 50 mM NaCl for 1 day. The expression of some CsRboh genes was induced by salt. Moreover, an increase in the activity of G6PDH, providing the substrate for the NADPH oxidase, was observed. In seedlings subjected to salinity for a longer time, antioxidant enzymes-including superoxide dismutase, catalase, and ascorbate peroxidase-were activated, participating in maintaining a steady-state H2O2 content in the root cells. In conclusion, NADPH oxidase and endogenous H2O2 up-regulation seem to be early events in cucumber response to salinity.

Quantitative Retention (Structure)-Activity Relationships in Predicting the Pharmaceutical and Toxic Properties of Potential Pesticides.

The micellar liquid chromatography technique and quantitative retention (structure)-activity relationships method were used to predict properties of carbamic and phenoxyacetic acids derivatives, newly synthesized in our laboratory and considered as potential pesticides. Important properties of the test substances characterizing their potential significance as pesticides as well as threats to humans were considered: the volume of distribution, the unbonded fractions, the blood-brain distribution, the rate of skin and cell permeation, the dermal absorption, the binding to human serum albumin, partitioning between water and plants' cuticles, and the lethal dose. Pharmacokinetic and toxicity parameters were predicted as functions of the solutes' lipophilicities and the number of hydrogen bond donors, the number of hydrogen bond acceptors, and the number of rotatable bonds. The equations that were derived were evaluated statistically and cross-validated. Important features of the molecular structure influencing the properties of the tested substances were indicated. The QSAR models that were developed had high predictive ability and high reliability in modeling the properties of the molecules that were tested. The investigations highlighted the applicability of combined chromatographic technique and QS(R)ARs in modeling the important properties of potential pesticides and reducing unethical animal testing.

Involvement of Diamine Oxidase in Modification of Plasma Membrane Proton Pump Activity in Cucumis sativus L. Seedlings under Cadmium Stress.

Cucumber (Cucumis sativus L.) is a crop plant being the third most-produced vegetable developed as a new model plant. Heavy metal pollution is a serious global problem that affects crop production. An industrial activity has led to high emissions of Cd into the environment. Plants realize adaptive strategies to diminish the toxic effects of Cd. They can remove excess toxic ions of heavy metals from the cytoplasm to the outside of cells using the metal/proton antiport. The proton gradient needed for the action of the antiporter is generated by the plasma membrane (PM) H+-ATPase (EC 3.6.3.14). We have shown that treatment of cucumber plants with Cd stimulated the diamine oxidase (DAO, EC 1.4.3.6) activity in roots. Under cadmium stress, the PM H+-ATPase activity also increased in cucumber seedlings. The stimulating effect of Cd on the PM H+-ATPase activity and expression of three genes encoding this enzyme (CsHA2, CsHA4, CsHA8) was reduced by aminoguanidine (AG, a DAO inhibitor). Moreover, we have observed that H2O2 produced by DAO promotes the formation of NO in the roots of seedlings. The results presented in this work showed that DAO may be an element of the signal transduction pathway, leading to enhanced PM H+-ATPase activity under cadmium stress.

Antisense oligonucleotide technology as a research tool in plant biology.

An antisense oligonucleotide (ASO) is a short single-stranded deoxyribonucleotide complementary to the sense strand of a selected nucleic acid. As a result, an ASO can modulate gene expression through several mechanisms. The technology based on ASO has already been applied in studies on gene function in mammalian cells and selective therapeutic strategies for many diseases. The conceptual simplicity and low cost of this method, and the developments in the field of plant genome sequencing observed in the last decades, have paved the way for the ASO method also in plant biology. It is applied in gene function analysis as well as the development of non-invasive plant production technology involving gene modifications without transgenesis. Therefore, the first part of this review provides a comprehensive overview of the structure, mechanism of action and delivery methods of ASOs in plants and shows the most important features essential for the proper design of individual experiments. We also discuss potential issues and difficulties that may arise during practical ASO implementation. The second part of this article contains an analysis of ASO applications in various studies in the field of plant biology. We presented for the first time that ASOs were also successfully applied in cucumber.

Predicting Pharmacokinetic Properties of Potential Anticancer Agents via Their Chromatographic Behavior on Different Reversed Phase Materials.

The Quantitative Structure-Activity Relationship (QSAR) methodology was used to predict biological properties, i.e., the blood-brain distribution (log BB), fraction unbounded in the brain (fu,brain), water-skin permeation (log Kp), binding to human plasma proteins (log Ka,HSA), and intestinal permeability (Caco-2), for three classes of fused azaisocytosine-containing congeners that were considered and tested as promising drug candidates. The compounds were characterized by lipophilic, structural, and electronic descriptors, i.e., chromatographic retention, topological polar surface area, polarizability, and molecular weight. Different reversed-phase liquid chromatography techniques were used to determine the chromatographic lipophilicity of the compounds that were tested, i.e., micellar liquid chromatography (MLC) with the ODS-2 column and polyoxyethylene lauryl ether (Brij 35) as the effluent component, an immobilized artificial membrane (IAM) chromatography with phosphatidylcholine column (IAM.PC.DD2) and chromatography with end-capped octadecylsilyl (ODS) column using aqueous solutions of acetonitrile as the mobile phases. Using multiple linear regression, we derived the statistically significant quantitative structure-activity relationships. All these QSAR equations were validated and were found to be very good. The investigations highlight the significance and possibilities of liquid chromatographic techniques with three different reversed-phase materials and QSARs methods in predicting the pharmacokinetic properties of our important organic compounds and reducing unethical animal testing.

Predicting the Blood-Brain Barrier Permeability of New Drug-Like Compounds via HPLC with Various Stationary Phases.

The permeation of the blood-brain barrier is a very important consideration for new drug candidate molecules. In this research, the reversed-phase liquid chromatography with different columns (Purosphere RP-18e, IAM.PC.DD2 and Cosmosil Cholester) was used to predict the penetration of the blood-brain barrier by 65 newly-synthesized drug-like compounds. The linear free energy relationships (LFERs) model (log BB = c + eE + sS + aA + bB + vV) was established for a training set of 23 congeneric biologically active azole compounds with known experimental log BB (BB = Cblood/Cbrain) values (R2 = 0.9039). The reliability and predictive potency of the model were confirmed by leave-one-out cross validation as well as leave-50%-out cross validation. Multiple linear regression (MLR) was used to develop the quantitative structure-activity relationships (QSARs) to predict the log BB values of compounds that were tested, taking into account the chromatographic lipophilicity (log kw), polarizability and topological polar surface area. The excellent statistics of the developed MLR equations (R2 > 0.8 for all columns) showed that it is possible to use the HPLC technique and retention data to produce reliable blood-brain barrier permeability models and to predict the log BB values of our pharmaceutically important molecules.

Two novel classes of fused azaisocytosine-containing congeners as promising drug candidates: Design, synthesis as well as in vitro, ex vivo and in silico studies.

Searching for new less toxic anticancer drug candidates is a big challenge from a medical point of view. The present investigation was aimed at describing two independent synthetic approaches based on isosteric replacements, spectroscopic characteristics, in vitro anticancer and ex vivo antihaemolytic activities of novel molecules (9-22) and correlations between their standardised lipophilicity indices, computed log Paverage values and pharmacokinetic descriptors. Two novel protocols for annelation of the triazinone template on hydrazinylideneimidazolidines (1-8) (showing a high reactivity towards electrophilic reagents, such as ethyl trifluoropyruvate and ethyl 3-methyl-2-oxobutyrate) were developed for the first time, giving rise to two original classes of highly conjugated azaisocytosine-containing molecules (9-16 and 17-22). Both syntheses proceeded under basic conditions to yield the most probable intermediates (e.g. hemiaminals and imines), which in refluxing two-component solvent mixtures or a suitable solvent cyclised through closing the triazinone ring on functionalised imidazolidines in both cases. All fused azaisocytosine-containing congeners were investigated with the purpose of preselecting possible drug candidates with a better selectivity that could be suitable for further more detailed drug development studies. The majority of test molecules revealed strong antiproliferative effects in most tumour cell cultures and they were more cytotoxic against tumour cells than anticancer drug - pemetrexed. These cytotoxicities may be associated with the activation of initiator and executioner caspases (confirmed for compound 12) which are inducers of apoptosis. Simultaneously, three bioisosteres bearing the trifluoromethyl moiety at the C-3 and the ortho substitution at the phenyl ring (10, 12 and 13) proved to be the most promising in terms of selectivity as they were less or equally toxic to normal cells as pemetrexed. It was shown that isosteric replacement of the ethyl group in antitumour active congeners by the trifluoromethyl or isopropyl group was favourable for the selectivity of the designed drug-like molecules. Almost all new compounds revealed the protective effects in an ex vivo model of oxidatively stressed rat erythrocytes (better or comparable than that of ascorbic acid/Trolox), proving that they are safe to red blood cells. The statistically significant and predictive QSAR equations were derived that describe relationships between some pharmacokinetic descriptors (such as log Ka, HSA, fu, brain, Caco-2, log Kp) and lipophilicity parameters of test molecules. Among all molecules with anticancer profile, the possible drug candidates seem to be 10, 12, 13, 19 and 21 which are the least toxic for normal cells, deprived of haemolytic effects on oxidatively-stressed red blood cells and have the optimum pharmacokinetic descriptors in terms of their lipophilicity parameters. Because of a high development potential they should be utilised in further more extended in vivo investigations aimed at developing novel less toxic anticancer agents.

Interaction between the signaling molecules hydrogen sulfide and hydrogen peroxide and their role in vacuolar H+ -ATPase regulation in cadmium-stressed cucumber roots.

Vacuolar H+ -ATPase (V-ATPase; EC 3.6.3.14) is the main enzyme responsible for generating a proton gradient across the tonoplast. Under cadmium (Cd) stress conditions, V-ATPase activity is inhibited. In the present work, hydrogen sulfide (H2 S) and hydrogen peroxide (H2 O2 ) cross-talk was analyzed in cucumber (Cucumis sativus L.) seedlings exposed to Cd to explain the role of both signaling molecules in the control of V-ATPase. V-ATPase activity and gene expression as well as H2 S and H2 O2 content and endogenous production were determined in roots of plants treated with 100 µM CdCl2 and different inhibitors or scavengers. It was found that H2 S donor improved photosynthetic parameters in Cd-stressed cucumber seedlings. Cd-induced stimulation of H2 S level was correlated with the increased activities of the H2 S-generating desulfhydrases. Increased H2 O2 and lowered H2 S contents in roots were able to reduce V-ATPase activities similar to Cd. H2 O2 and H2 S-induced modulations in V-ATPase activities were not closely related to the transcript level of encoding genes, suggesting posttranslational modifications of enzyme protein. On the other hand, exogenous H2 O2 raised H2 S content in root tissues independently from the desulfhydrase activity. Although treatment of control plants with H2 S significantly stimulated NADPH oxidase activity and gene expression, H2 S did not affect H2 O2 accumulation in roots exposed to Cd. The results suggest the existence of two pathways of H2 S generation in Cd-stressed cucumber roots. One involves desulfhydrase activity, as was previously demonstrated in different plant species. The other, the desulfhydrase-independent pathway induced by H2 O2 /NADPH oxidase, may protect V-ATPase from inhibition by Cd.

Involvement of NR and PM-NR in NO biosynthesis in cucumber plants subjected to salt stress.

Nitrate reductase (NR) mainly reduces nitrate to nitrite. However, in certain conditions it can reduce nitrite to NO. In plants, a plasma membrane-associated form of NR (PM-NR) is present. It produces NO2- for nitrite NO/reductase (Ni-NOR), which can release NO into the apoplastic space. The effect of 50 mM NaCl on NO formation and the involvement of NR in NO biosynthesis were studied in cucumber seedling roots under salt stress. In salt-stressed roots, the amount of NO was higher than in control. The application of tungstate abolished the increase of NO level in stressed roots, indicating that NR was responsible for NO biosynthesis under the test conditions. The involvement of other molybdoenzymes was excluded using specific inhibitors. Furthermore, higher cNR and PM-NR activities were observed in NaCl-treated roots. The increase in NR activity was due to the stimulation of CsNR genes expression and posttranslational modifications, such as enzyme dephosphorylation. This was confirmed by Western blot analysis. Moreover, the increase of nitrite tissue level in short-term stressed roots and the nitrite/nitrate ratio, with a simultaneous decrease of nitrite reductase (NiR) activity, in both short- and long-term stressed roots, could promote the production of NO by NR in roots under salt stress.

Assay of Plasma Membrane H+-ATPase in Plant Tissues under Abiotic Stresses.

Plasma membrane (PM) H+-ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H+-ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.

The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress.

The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H+-ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H+-ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H+-ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H+-ATPase and NADPH oxidase are key factors.