Enzymes contributing to the hydrogen peroxide signal dynamics that regulate wall labyrinth formation in transfer cells.

Transfer cells are characterized by an amplified plasma membrane area supported on a wall labyrinth composed of a uniform wall layer (UWL) from which wall ingrowth (WI) papillae arise. Adaxial epidermal cells of developing Vicia faba cotyledons, when placed in culture, undergo a rapid (hours) trans-differentiation to a functional epidermal transfer cell (ETC) phenotype. The trans-differentiation event is controlled by a signalling cascade comprising auxin, ethylene, apoplasmic reactive oxygen species (apoROS), and cytosolic Ca2+. Apoplasmic hydrogen peroxide (apoH2O2) was confirmed as the apoROS regulating UWL and WI papillae formation. Informed by an ETC-specific transcriptome, a pharmacological approach identified a temporally changing cohort of H2O2 biosynthetic enzymes. The cohort contained a respiratory burst oxidase homologue, polyamine oxidase, copper amine oxidase, and a suite of class III peroxidases. Collectively these generated two consecutive bursts in apoH2O2 production. Spatial organization of biosynthetic/catabolic enzymes was deduced from responses to pharmacologically blocking their activities on the cellular and subcellular distribution of apoH2O2. The findings were consistent with catalase activity constraining the apoH2O2 signal to the outer periclinal wall of the ETCs. Strategic positioning of class III peroxidases in this outer domain shaped subcellular apoH2O2 signatures that differed during assembly of the UWL and WI papillae.

Potential of faba bean lipase and lipoxygenase to promote formation of volatile lipid oxidation products in food models.

Faba bean can respond to the need for plant-based proteins for human consumption. The aim of this work was to study the role of lipid-modifying enzymes in faba bean in causing off-flavour compounds during processing. The faba bean exhibited high lipase and lipoxygenase (LOX) activities, with pH optima being 8.0 and 6.0, respectively. Faba bean LOX preferred free fatty acids (FFAs) over triacylglycerols as substrates, and together with other LOX pathway enzymes, it formed specific volatile products, as measured using headspace solid-phase microextraction-gas chromatography. During the preparation of the food models (i.e. the extracts and emulsions), enzymatic lipid oxidation occurred. The inclusion in the emulsions of rapeseed oil, especially of rapeseed oil FFAs, remarkably increased the amounts of volatile products. The largest quantities of products were formed in food models at pH 6.4, which is close to the pH optimum of LOX. Further studies on lipase in food models are needed.

Ecotoxicity and genotoxicity of polystyrene microplastics on higher plant Vicia faba.

Nano- and microplastics have been widely spread in environmental matrices, especially in marine and terrestrial systems. In this study, higher plant Vicia faba root tips were exposed to 5 µm and 100 nm with 10, 50 and 100 mg/L polystyrene fluorescent microplastics (PS-MPs) for 48 h. Root length, weight, oxidative stress and genotoxicity of V. faba were assessed to investigate toxic effects of PS-MPs. The results showed that the biomass and catalase (CAT) enzymes activity of V. faba roots decreased under 5 µm PS-MPs whereas superoxide dismutase (SOD) and peroxidase (POD) enzymes activity significantly increased. Under the 100 nm PS-MPs exposure a significant decrease of growth was observed only at the highest concentration (100 mg/L). However, micronucleus (MN) test and antioxidative enzymes activities showed that 100 nm PS-MPs induce higher genotoxic and oxidative damage to V. faba than 5 µm PS-MPs. Furthermore, the laser confocal scanning microscopy (LCSM) demonstrated that 100 nm PS-MPs can accumulate in V. faba root and most probably block cell connections or cell wall pores for transport of nutrients. These findings provide a new insight into the toxic effects of microplastics on V. faba, and further apply to the ecological risk assessment of microplastics on higher plants.

Phenotypic and molecular characterisation of novel Vicia faba germplasm with tolerance to acetohydroxyacid synthase-inhibiting herbicides (AHAS) developed through mutagenesis techniques.

BACKGROUND: Faba bean (Vicia faba L.) is an important crop in Australian farming systems, however, weed control is a major constraint due to a lack of in-crop broadleaf herbicide options. To address this, we developed acetohydroxyacid synthase (AHAS) inhibitor herbicide tolerance in faba bean using mutagenesis techniques. Dose-response experiments, agronomic field evaluation and DNA sequencing of the AHAS gene were used to quantify and validate tolerance traits. RESULTS: Four M2 faba bean single-plant biotypes (IMI-1, IMI-2, IMI-3 and IMI-4) at a frequency of 3.63 × 10-6 were successfully recovered. Molecular characterisation of the AHAS gene identified two known target site mutations (resulting in protein substitutions Ala205Val and Ser653Asn) conferring tolerance. Phenotypic characterisation found that both mutations conferred high levels of tolerance to the imidazolinone herbicide imazapyr. However, although the Ala205Val substitution showed improved levels of cross-tolerance to a range of sulfonylurea chemistries, the Ser653Asn substitution did not. In the field, IMI-3 showed the highest level of agronomic tolerance across a range of imidazolinone herbicides. CONCLUSIONS: Mutagenesis techniques were successful in the development of tolerance to AHAS inhibitor herbicides in faba bean, and could facilitate the first safe in-crop broadleaf herbicide control option in Australian faba bean production. © 2019 Society of Chemical Industry.

Enzymatic analysis of levan produced by lactic acid bacteria in fermented doughs.

Levans and inulins are fructans with mainly ß-(2→6) and ß-(2→1) linkages, respectively. Levans are produced by many lactic acid bacteria, e.g. during sourdough fermentation. Levans have shown prebiotic properties and may also function as in situ-produced hydrocolloids. So far, levan contents have been measured by acid hydrolysis, which cannot distinguish levans from e.g. inulins. In order to develop a specific analysis for levan in food matrices, a Paenibacillus amylolyticus endolevanase was combined with exoinulinase for levan hydrolysis. A separate endoinulinase treatment was used to detect the possible presence of inulin. Interfering sugars were removed by a pre-wash with aqueous ethanol. Levan content was estimated from fructose and glucose released in the hydrolysis, with a correction made for the residual fructose and glucose-containing sugars. The method was validated using wheat model doughs spiked with commercial Erwinia levan, and tested by analyzing levan content in Leuconostoc mesenteroides DSM 20343-fermented fava bean doughs.

Orobanche foetida resistance in two new faba bean genotypes produced by radiation mutagenesis.

PURPOSE: Broomrape produces serious damage to many legume crops and, particularly, becomes a limiting factor for faba bean (Vicia faba L.) production in the Mediterranean basin. Currently, several traditional methods of control have been developed, but none has proved to be effective for this parasite. However, breeding for resistance to this pest remains as one of the most feasible and environment-friendly methods for managing broomrape, but the mechanisms governing the interaction between the parasite and the host are not yet well understood. Therefore, we studied the behavior and molecular and enzymatic changes associated with the resistance to Orobanche foetida in faba bean mutants, which were obtained through radiation mutagenesis. MATERIALS AND METHODS: Three faba bean genotypes were used in this study, the variety 'Badï', characterized by high productivity in Orobanche-free soils and susceptibility to O. foetida, and two mutant lines P2M3 and P7M3 (derived from radio mutagenesis program), selected for their higher resistance to O. foetida in a field evaluation. The infection progress and the relative changes in the co-culture response, the enzymatic activities changes and the efficiency of the root extract stimulants from the host plant were followed and evaluated in all the genotypes. RESULTS: Experiments showed that low induction of seed germination is a major component of resistance in these lines against O. foetida. This is confirmed by the in vitro experiments with root exudates. The parallel reduction in infection was accompanied by the continuous enhancement of the peroxidase activity, the polyphenol oxidase activity and the phenylalanine ammonia lyase activity in faba bean roots. CONCLUSION: These data suggest the contribution of these enzymes in faba bean resistance to O. foetida broomrape induced by the use of gamma rays. Management of Orobanche by way of crop selection, based on these enzyme systems is a possible option.

Carboxylated multi-walled carbon nanotubes exacerbated oxidative damage in roots of Vicia faba L. seedlings under combined stress of lead and cadmium.

Multi-walled carbon nanotubes (MWCNTs) and heavy metals could be absorbed and bioaccumulated by agricultural crops, implicating ecological risks. Herein, the present study investigated the ecotoxicological effects and mechanisms of individual carboxylated MWCNTs (MWCNTs-COOH) (2.5, 5.0 and 10 mg/L) and their combination with 20 µM Pb and 5 µM Cd (shortened as Pb + Cd) on roots of Vicia faba L. seedlings after 20 days of exposure. The results showed that the tested MWCNTs-COOH induced imbalance of nutrient elements, enhanced isozymes and activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), resulting in accumulation of carbonylated proteins, elevation of endoproteases (EPs) isozymes, and reduction of HSP70 synthesis in the roots. However, the tested MWCNTs-COOH facilitated the enrichment of Cd, Pb and Na elements, contributing to the decrease of SOD, CAT and APX activities, and the reduction of HSP70 synthesis, whereas the elevation of carbonylated proteins, EP activities and cell necrosis in the roots when Pb + Cd was combined in comparison to the treatments of MWCNTs-COOH, or Pb + Cd alone. Thus, the tested MWCNTs-COOH not only caused oxidative stress, but also aggravated the oxidative damage in the roots exposed to Pb + Cd in the culture solution.

Mitogen-activated protein kinases concentrate in the vicinity of chromosomes and may regulate directly cellular patterning in Vicia faba embryos.

MAIN CONCLUSION: Mitogen-activated protein kinases seem to mark genes which are set up to be activated in daughter cells and thus they may play a direct role in cellular patterning during embryogenesis. Embryonic patterning starts very early and after the first division of zygote different genes are expressed in apical and basal cells. However, there is an ongoing debate about the way these different transcription patterns are established during embryogenesis. The presented data indicate that mitogen-activated protein kinases (MAPKs) concentrate in the vicinity of chromosomes and form visible foci there. Cells in the apical and basal regions differ in number of foci observed during the metaphase which suggests that cellular patterning may be determined by activation of diverse MAPK-dependent genes. Different number of foci in each group of separating chromatids and the specified direction of these mitoses in apical-basal axis indicate that the unilateral auxin accumulation in a single cell may regulate the number of foci in each group of chromatids. Thus, we put forward a hypothesis that MAPKs localized in the vicinity of chromosomes during mitosis mark those genes which are set up to be activated in daughter cells after division. It implies that the chromosomal localization of MAPKs may be one of the mechanisms involved in establishment of cellular patterns in some plant species.

Assessing the influence of 1-dodecyl-3-methylimidazolium chloride on soil characteristics and Vicia faba seedlings.

Imidazolium-based ionic liquids (ILs) have attracted increasing attention in recent years. The IL 1-dodecyl-3-methylimidazolium chloride ([C12mim]Cl) has been widely used in the chemical industry. In this study, the influence of [C12mim]Cl on Vicia faba seedlings, soil physicochemical properties and soil enzyme activities was investigated for the first time. Meanwhile, the variation of [C12mim]Cl concentrations in soil was monitored during the exposure period. The present results showed that the concentration of [C12mim]Cl remained stable in the tested soil with a change rate of no more than 10% during the exposure period. The 50% effective concentration (EC50) values for shoot length, root length and dry weight were 188, 69 and 132 mg kg-1, respectively. At 200 mg kg-1 and 400 mg kg-1, [C12mim]Cl had significant influence on soil organic matter content, pH value and conductivity value. At 40 mg kg-1, the reactive oxygen species (ROS) levels were obviously enhanced, resulting in oxidative stress effects in Vicia faba seedling leaves. Additionally, the soil enzyme activities changed significantly at 40 mg kg-1.

Lipid-modifying enzymes in oat and faba bean.

The aim was to study lipase, lipoxygenase (LOX) and peroxygenase (POX) activities in oat and faba bean samples to be able to evaluate their potential in formation of lipid-derived off-flavours. Lipase and LOX activities were measured by spectroscopy, and POX activities via the formation of epoxides. An ultra-high performance liquid chromatography method was developed to study the formation of fatty acid epoxides. The epoxides of esters were measured by gas chromatography. Mass spectroscopy was used to verify the identity of the epoxides. Both oat and faba bean possessed high lipase activities. In faba bean, LOX catalysed the formation of hydroperoxides, whose break-down products are the likely cause of off-flavours. Since oat had low LOX activity, autoxidation is needed to initiate lipid oxidation. Oat had high POX activity, which is able to convert hydroperoxides to epoxy and hydroxy fatty acids that could contribute significantly to off-flavours. POX activity in the faba bean was low. Thus, in faba bean volatile lipid oxidation products could rapidly be formed by LOX, whereas in oat reactions are slower due to the need of autoxidation prior to further reactions.

Proteolytic activities in cortex of apical parts of Vicia faba ssp. minor seedling roots during kinetin-induced programmed cell death.

Programmed cell death (PCD) is a crucial process in plant development. In this paper, proteolytically related aspects of kinetin-induced PCD in cortex cells of Vicia faba ssp. minor seedlings were examined using morphological, fluorometric, spectrophotometric, and fluorescence microscopic analyses. Cell viability estimation after 46 µM kinetin treatment of seedling roots showed that the number of dying cortex cells increased with treatment duration, reaching maximum after 72 h. Weight of the apical root segments increased with time and was about 2.5-fold greater after 96 h, while the protein content remained unchanged, compared to the control. The total and cysteine-dependent proteolytic activities fluctuated during 1-96-h treatment, which was not accompanied by the changes in the protein amount, indicating that the absolute protein amounts decreased during kinetin-induced PCD. N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), and Z-Leu-Leu-Nva-H (MG115), the respective cysteine, serine, and proteasome inhibitors, suppressed kinetin-induced PCD. PMSF significantly decreased serine-dependent proteolytic activities without changing the amount of proteins, unlike NEM and MG115. More pronounced effect of PMSF over NEM indicated that in the root apical segments, the most important proteolytic activity during kinetin-induced PCD was that of serine proteases, while that of cysteine proteases may be important for protein degradation in the last phase of the process. Both NEM and PMSF inhibited apoptotic-like structure formation during kinetin-induced PCD. The level of caspase-3-like activity of ß1 proteasome subunit increased after kinetin treatment. Addition of proteasome inhibitor MG-115 reduced the number of dying cells, suggesting that proteasomes might play an important role during kinetin-induced PCD.

Feeding value of field beans (Vicia faba L. var. minor) with and without enzyme containing tannase, pectinase and xylanase activities for broilers.

Effects of field beans with various tannin content and exogenous enzyme mixture containing tannase, pectinase and xylanase activities on N-corrected dietary apparent metabolisable energy (AMEn), coefficients of dry matter (DMR) and nitrogen retention (NR), fat digestibility, gastrointestinal tract (GIT) development, jejunal villus morphometry, ileal digesta viscosity and sialic acid were examined. Birds' growth performance and energy conversion ratio (ECR) were also measured. Birds were fed one of eight mash diets. The Control diet contained as major ingredients wheat (400 g/kg) and soybean meal (SBM) (127 g/kg and 221 g crude protein/kg and 12.83 MJ AMEn/kg. To reduce nutrient density, the Control diet also contained washed sand at 119 g/kg. Another three diets containing 300 g/kg of each of three experimental field bean cultivar samples in replacement for SBM and sand were also mixed. Each diet was fed to nine pens with two male Ross 308 broilers. Diets high in tannin had low AMEn, ECR, DMR and NR (p < 0.001). Feeding field beans increased (p < 0.001) the weights of the pancreas and the proventriculus and gizzard (PG) of the birds. Supplementing diets with the enzyme mixture improved (p < 0.001) feed conversion efficiency, AMEn and all nutrient utilisation coefficients despite the tannins in diets. The enzyme mixture reduced ileal digesta viscosity (p < 0.001) and the weight of pancreas, total GIT and PG (p < 0.05) of the birds. It can be concluded that the feeding value of field beans with different tannin contents may vary when fed to broilers. The supplementation of the enzyme mixture improved the feeding value of diets for broilers. The beneficial effect of the addition of the enzyme mixture seems to be mediated through reduced ileal digesta viscosity and improved nutrient availability.

Microcystin-LR induces mitotic spindle assembly disorders in Vicia faba by protein phosphatase inhibition and not reactive oxygen species induction.

We aimed to reveal the mechanisms of mitotic spindle anomalies induced by microcystin-LR (MCY-LR), a cyanobacterial toxin in Vicia faba, a well-known model in plant cell and molecular biology. MCY-LR inhibits type 1 and 2A phosphoserine/threonine specific protein phosphatases (PP1 and PP2A) and induces reactive oxygen species (ROS) formation. The cytoskeleton is one of the main targets of the cyanotoxin during cytopathogenesis. Histochemical-immunohistochemical and biochemical methods were used. A significant number of MCY-LR induced spindle alterations are described for the first time. Disrupted, multipolar spindles and missing kinetochore fibers were detected both in metaphase and anaphase cells. Additional polar microtubule (MT) bundles, hyperbundling of spindle MTs, monopolar spindles, C-S- shaped, additional and asymmetric spindles were detected in metaphase, while midplane kinetochore fibers were detected in anaphase cells only. Several spindle anomalies induced mitotic disorders, i.e. they occurred concomitantly with altered sister chromatid separation. Alterations were dependent on the MCY-LR dose and exposure time. Under long-term (2 and mainly 6 days') exposure they were detected in the concentration range of 0.1-20µgmL(-1) MCY-LR that inhibited PP1 and PP2A significantly without significant ROS induction. Elevated peroxidase/catalase activities indicated that MCY-LR treated V. faba plants showed efficient defense against oxidative stress. Thus, although the elevation of ROS is known to induce cytoskeletal aberrations in general, this study shows that long-term protein phosphatase inhibition is the primary cause of MCY-LR induced spindle disorders.

Hydroxyurea-induced replication stress causes poly(ADP-ribose) polymerase-2 accumulation and changes its intranuclear location in root meristems of Vicia faba.

Replication stress induced by 24 and 48h exposure to 2.5mM hydroxyurea (HU) increased the activity of poly(ADP-ribose) polymerase-2 (PARP-2; EC 2.4.2.30) in root meristem cells of Vicia faba. An increase in the number of PARP-2 foci was accompanied by their delocalization from peripheral areas to the interior of the nucleus. Our results indicate that the increase in PARP-2 was connected with an increase in S139-phosphorylated H2AX histones. The findings suggest the possible role of PARP-2 in replication stress. We also confirm that the intranuclear location of PARP-2 depends on the duration of HU-induced replication stress, confirming the role of PARP-2 as an indicator of stress intensity. Finally, we conclude that the more intense the HU-mediated replication stress, the greater the probability of PARP-2 activation or H2AXS139 phosphorylation, but also the greater the chance of increasing the efficiency of repair processes and a return to normal cell cycle progression.

Toxicological effects, mechanisms, and implied toxicity thresholds in the roots of Vicia faba L. seedlings grown in copper-contaminated soil.

Copper (Cu) contamination has become a global concern because of industrial, agricultural, and other anthropogenic activities. In the present experiments, the toxicological effects, mechanisms, and potential toxicity thresholds were investigated in the roots of Vicia faba L. seedlings that were cultivated in Cu-amended soils (0, 6.25, 12.5, 25, 50, 100, 200, 400, and 600 mg kg(-1)) for 20 days, based on an analysis of the soil physicochemical properties, native Cu, available Cu, and root-enriched Cu contents. The superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) isozymes and activities, as well as glutathione (GSH) and heat shock protein 70 (HSP70), changed like biphasic dose-response curves, cooperating to control the redox homeostasis. The APX and POD enzymes exhibited enhanced activities and became H2O2 scavengers primarily when the catalase (CAT) activities tended to decrease. Endoprotease (EP) isozymes and activities might be enhanced to degrade carbonylated proteins and alleviate metabolic disturbance in the roots. Additionally, HSP70 may not be suitable as a biomarker for relatively higher soil Cu concentrations and relatively longer exposure times for the roots. As a result, the isozymes and activities of SOD, CAT, and EP, as well as GSH, can be adopted as the most sensitive biomarkers. The toxicity threshold is estimated as 0.76-1.21 mg kg(-1) of available Cu in the soils or 25.04-36.65 µg Cu g(-1) dry weights (DW) in the roots.

Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L).

Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein.

Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum.

KEY MESSAGE: In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Co-localization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50% of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species.

Peroxidase and polyphenol oxidase activity in moderate resistant and susceptible Vicia faba induced by Aphis craccivora (Hemiptera: Aphididae) infestation.

Two faba bean, Vicia faba L., cultivars, Gazira2 and Misr1, representing cultivars moderately resistant and susceptible to aphids, were analyzed for peroxidase (POD) and polyphenol oxidase (PPO) activities induced by cowpea aphid, Aphis craccivora Koch. infestation. Two tissue types (whole plant [WP] and detached leaf [DL]), two infestation status (infested and uninfested), and three aphid infestation durations (1, 3, and 5 d) were considered in POD and PPO data analysis. Factorial analysis showed that only cultivar factor has a significant effect on both POD and PPO activity, especially on the first day after aphid infestation (P: 0.0003 and 0.001, respectively). Tissue type has no significant effect, indicating either DL or WP can be used for measuring POD and PPO activities. While the unsignificant different of infestation status reflecting a constitutive resistant character in Gazira2. Mann-Whitney U-test showed that POD and PPO activities in Gazira2 were higher significantly when compared with Misr1 with P value 0.0006 and 0.0015 for POD and PPO, respectively. Repeated measures analysis indicates that the POD and PPO activities on Gazira2 were significantly higher when compared with Misr1. Additionally, POD activity changed significantly over the time in 1, 3, and 5 d after aphid infestation. We concluded that higher activity of POD and PPO in cultivar Gazira2 is strongly associated with their resistant characters.

The crucial elements of the 'last step' of programmed cell death induced by kinetin in root cortex of V. faba ssp. minor seedlings.

KEY MESSAGE: Kinetin-induced programmed cell death, manifested by condensation, degradation and methylation of DNA and fluctuation of kinase activities and ATP levels, is an autolytic and root cortex cell-specific process. The last step of programmed cell death (PCD) induced by kinetin in the root cortex of V. faba ssp. minor seedlings was explained using morphologic (nuclear chromatin/aggregation) and metabolic (DNA degradation, DNA methylation and kinases activity) analyses. This step involves: (1) decrease in nuclear DNA content, (2) increase in the number of 4',6-diamidino-2-phenylindole (DAPI)-stained chromocenters, and decrease in chromomycin A3 (CMA3)-stained chromocenters, (3) increase in fluorescence intensity of CMA3-stained chromocenters, (4) condensation of DAPI-stained and loosening of CMA3-stained chromatin, (5) fluctuation of the level of DNA methylation, (6) fluctuation of activities of exo-/endonucleolytic Zn(2+) and Ca(2+)/Mg(2+)-dependent nucleases, (7) changes in H1 and core histone kinase activities and (8) decrease in cellular ATP amount. These results confirmed that kinetin-induced PCD was a specific process. Additionally, based on data presented in this paper (DNA condensation and ATP depletion) and previous studies [increase in vacuole, increase in amount of cytosolic calcium ions, ROS production and cytosol acidification "in Byczkowska et al. (Protoplasma 250:121-128, 2013)"], we propose that the process resembles autolytic type of cell death, the most common type of death during development of plants. Lastly, the observations also suggested that regulation of these processes might be under control of epigenetic (methylation/phosphorylation) mechanisms.

Leaf ion homeostasis and plasma membrane H(+)-ATPase activity in Vicia faba change after extra calcium and potassium supply under salinity.

Salt stress in plants impacts apoplastic ion activities and cytosolic ionic homeostasis. The ameliorating effects exerted by calcium or potassium on compartmentation of ions in leaves under salinity are not fully understood. To clarify how calcium or potassium supply could ameliorate ion homeostasis and ATPase activities under salinity, 5 mM CaSO4 or 10 mM K2SO4 were added with, or without, 100 mM NaCl for 7 d and 21 d to Vicia faba grown in hydroponics. The apoplastic pH was detected with Oregon Green dextran dye in intact second-uppermost leaves by microscopy-based ratio imaging. The cytosolic Ca(2+), Na(+), K(+) activities and pH were detected in protoplasts loaded with the acetoxy methyl-esters of Fura-2, SBFI, PBFI and BCECF, respectively, using epi-fluorescence microscopy. Furthermore, total Ca(2+), Na(+), K(+) concentrations and growth parameters were investigated. The ATPase hydrolyzing activity increased with time, but decreased after long salinity treatment. The activity largely increased in calcium-treated plants, but was depressed in potassium-treated plants after 7 d. The calcium supply increased Vmax, and the ATPase activity increased with salinity in a non-competitive way for 7 d and 21 d. The potassium supply instead decreased activity competitively with Na(+), after 21 d of salinity, with different effects on Km and Vmax. The confirmed higher ATPase activity was related with apoplast acidification, cytosol alkalinization and low cytosolic [Na(+)], and thus, might be an explanation why extra calcium improved shoot and leaf growth.