Roderik Fiala

Metal uptake, antioxidant status and membrane potential in maize roots exposed to cadmium and nickel

Root growth of the seedlings of maize cultivars Premia and Blitz exposed to 2 μM cadmium (Cd), nickel (Ni) or both metals acting simultaneously (Cd + Ni) for 72 h was significantly reduced but not ceased. The effect was more pronounced in the seedlings of the cv. Blitz. The heavy metals (HMs) contents increased significantly in the roots. Simultaneous application of metals had an antagonistic effect on either Cd or Ni uptake in Premia but not in Blitz. In control roots the contents of ascorbic acid (AsA) and dehydroascorbic acid (DHA) were lower and gluthatione (GSH) content was higher in Premia than in Blitz. A decrease of AsA content was induced by all metal treatments in Premia but only by Cd + Ni in Blitz while an increase was induced by single metals in this cultivar. All metal treatments increased DHA contents in both cultivars. GSH content decreased significantly in Premia treated with Cd or Cd + Ni, and in Blitz treated with Ni. Unlike the contents of AsA, DHA and GSH, the increased metal concentrations in root cells did not affect the membrane potential (EM). The changes in antioxidant contents depended on both, maize genotypes and HMs treatments. Nevertheless, the results indicated a role of antioxidative system in minimizing the effects of oxidative stress and protecting cell membranes in both maize cultivars.

Partially resistant Cucurbita pepo showed late onset of the Zucchini yellow mosaic virus infection due to rapid activation of defense mechanisms as compared to susceptible cultivar

Zucchini yellow mosaic virus (ZYMV) is an emerging viral pathogen in cucurbit-growing areas wordwide. Infection causes significant yield losses in several species of the family Cucurbitaceae. To identify proteins potentially involved with resistance toward infection by the severe ZYMV-H isolate, two Cucurbita pepo cultivars (Zelena susceptible and Jaguar partially resistant) were analyzed using a two-dimensional gel electrophoresis-based proteomic approach. Initial symptoms on leaves (clearing veins) developed 6–7 days post-inoculation (dpi) in the susceptible C. pepo cv. Zelena. In contrast, similar symptoms appeared on the leaves of partially resistant C. pepo cv. Jaguar only after 15 dpi. This finding was confirmed by immune-blot analysis which showed higher levels of viral proteins at 6 dpi in the susceptible cultivar. Leaf proteome analyses revealed 28 and 31 spots differentially abundant between cultivars at 6 and 15 dpi, respectively. The variance early in infection can be attributed to a rapid activation of proteins involved with redox homeostasis in the partially resistant cultivar. Changes in the proteome of the susceptible cultivar are related to the cytoskeleton and photosynthesis.

Antibody Microarray Expression Profiling Of Maize Roots Treated With Cadmium And Nickel

Abstract Here, we report the effectiveness of antibody microarray expression profiling (AbMEP) procedure to determine similarities and differences between two maize cultivars (Premia and Blitz) exposed to various concentration of cadmium (Cd), nickel (Ni), as well as simultaneous exposure to both metals (Cd + Ni) for 0, 12 and 24 h. After protein extraction from control (untreated) and heavy metals (HM)-treated root pairs and their fluorescent labelling, the protein extracts were used for the AbMEP procedure and western blot analysis. The results from the microarray were analysed using an internally normalised ratio. Using this highly parallel AbMEP-approach, the timing, dynamics and regulation of the expression of 101 specific genes in untreated and HM-treated roots of maize cultivars were determined. The microarray results revealed 23, 15 and 10 up-regulated/down-regulated proteins in Cd, Ni and Cd + Ni treated cv. Premia roots, respectively. In contrast, the microarray results revealed that 15, 11 and 7 proteins were up-regulated/down-regulated in Cd, Ni and Cd + Ni treated cv. Blitz roots, respectively. The data obtained from the AbMEP array experiment were validated by western blot analyses with more than 89% (the Pearson’s correlation coeficient Rr = 0.78) correlation between the two methods.

The effect of cadmium-nickel interactions on superoxide production, cell viability and membrane potential (EM) in roots of two maize cultivars

Effects of CdCl2, NiCl2 or both on superoxide production, viability and membrane potential (EM) of root cells in meristematic (MZ) and differentiation (DZ) zones of two maize cultivars (cv. Premia and cv. Blitz) were studied. Plants were supplied with 10 and 100 μM concentrations of heavy metals (HM). The responses in the studied parameters to HM were concentration- and time-dependent, and were found only in the cells of MZ. The treatment of roots with Cd-stimulated massive superoxide production, although to different extent depending on the cultivar, root zone, and metal concentration. The stimulating effect of Ni on oxidative burst in Cd-treated maize roots was related to an increased Cd-induced superoxide production. The cell death appeared between 24 and 48 h and between 12 and 24 h of the 10 μM and 100 μM metal treatments, respectively. This was in accordance with Cd-induced ROS (superoxide) production and the EM decline in the corresponding time periods. Cell viability, EM changes and partially superoxide production indicate that the impact of the metals on the studied parameters declined in the order Cd+Ni > Cd > Ni and that cv. Blitz tends to respond more sensitively than cv. Premia.

Responses of maize root cells to zearalenone and its derivatives α-zearalenol and β-zearalenol

The extent of mycotoxin-induced responses in living roots of two maize (Zea mays L.) cultivars differing in their susceptibility to Fusarium was studied. Application of mycotoxin zearalenone (ZEN) or its derivates α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) caused a rapid depolarization of plasma membrane potential (EM). The extent of EM depolarization was dependent on the type of mycotoxin that have been used and showed concentration dependence. Interestingly, ZEN, but not its derivatives α-ZEL and β-ZEL, significantly decreased respiration of maize root cells. Electrolyte leakage increased with the duration of toxins treatment and was significantly higher in susceptible cv. Pavla than in resistant cv. Lucia. A strong superoxide dismutase insensitive nitro blue tetrazolium (NBT) reduction activity was identified in the root tips of control plants. This activity was rapidly inhibited by mycotoxin application in the meristematic/distal transition zones of roots in both cultivars examined. The level of recovery was a function of the mycotoxin concentration. Moreover, mycotoxin treatment also resulted in the onset and the progression of root cell death which was dependent on both, the type and concentration of mycotoxin.

Role of ethylene and phospholipid-mediated signalling in mycotoxin-induced programmed cell death in the apical part of maize roots

Abstract Maize (Zea mays L. cv. Thermo) root segments were treated for 24 h with 100 μg mL-1 of zearalenone and its derivatives α- and β-zearalenol. The mycotoxin treatment resulted in cell death which was evident by Evans blue staining and was accompanied by DNA release/fragmentation. Mycotoxin-induced programmed cell death (MPCD) was abolished by sub-micromolar concentrations of caspase-specific peptide inhibitors pointing to a MPCD mechanism similar to animal apoptosis. Here we demostrate that exogenous ethylene and ethylene precursor (aminocyclopropane-1-carboxylic acid; ACC) substantially blocked MPCD while the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) did not markedly reduce cell death rate. In addition, Western blot analyses revealed that MPCD was induced via ethylene-regulated expression of DAD1 protein. Pre-treatment of root segments with inhibitors of phospholipase C and D signalling pathway intermediates significantly reduced the rate of MPCD. Treatment with mastoparan and lyso-phosphatidic acid (L-PA), G protein activator and analogue of the lipid second messenger phosphatidic acid (PA), respectively, stimulated cell death. Furthermore, application of lipid and protein kinase inhibitors (wortmannin, Go 6983, staurosporine) also reduced cell death, indicating that various kinases are a part of signalling cascade involved in MPCD. Taken together, the results presented in this paper provide direct evidence that MPCD exhibits formal apoptotic-like features, involves caspase-mediated pathway and is regulated via ethylene and phospholipid signal transduction pathways.

Effects of ZnCl2 on ROS generation, plasma membrane properties, and changes in protein expression in grapevine root explants

Abstract This study is aimed at the responses of grapevine adventitious root explants to zinc (Zn2+) excess. Within 24 h Zn2+ induced oxidative burst in concentration-dependent manner. The time course analysis revealed biphasic response in superoxide (O2–·) production. Hydrogen peroxide (H2O2) accumulation rose gradually within 24 h. Enhanced Zn2+ concentrations did not induce progression of cell death. Immediately upon Zn2+ addition to the perfusion solution, root epidermal cells exhibited hyperpolarization of their electrical membrane potential (EM), that was transient and independent of Zn2+ concentration. The subsequent, transient depolarization of EM was concentration-dependent and its magnitude increased with increasing Zn2+ concentration. After 24 h the EM in treated roots recovered and its values were identical with those of control roots. Membrane permeability of root cells increased in the roots treated with 5 mM Zn2+ within 24 h while the lower concentrations did not show any impact on membrane permeability. Differences in protein expression pattern identified by proteomic approach involving antibody microarray expression profiling revealed Zn2+-induced upregulation of apoptosis-related protein dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit (DAD1), extracellular signal-regulated kinase 1/2 (ERK1/2), some antioxidant enzymes and structural proteins in the roots. Moreover, the proteins involved in plant defense mechanisms endochitinase I (CHIT 1) and phenylalanine ammonia-lyase (PAL) were down-regulated indicating a cross-talk between defense and heavy metal signaling pathways. Taken together, these results showed that the grape cultivar Limberger is highly Zn2+-responsive and could be used as a model plant for studying physiological and molecular responses to heavy metal excess.

Physiological Responses to Cadmium, Nickel and their Interaction in the Seedlings of Two Maize (Zea mays L.) Cultivars

Abstract In the leaves of maize seedlings, cultivars Premia and Blitz, the relatively low 2 μmol/L concentration of cadmium (Cd), nickel (Ni), or both metals acting simultaneously (Cd +Ni) for 72 h, induced a significant metal accumulation, decrease in total K+ content, reduction of light-induced membrane electrical potential (EM) repolarisation in mesophyll cells and changes of ascorbic acid (AsA), dehydroascorbic acid (DHA) and glutathione (GSH) content. Shoot growth and the values of resting EM did not change significantly. Increased K+ leakage, from the leaves, and lipid peroxidation accompanied by increase of TBA-reactive substances (TBARS) were found only in cv. Blitz exposed to Cd + Ni. This indicates a capability of high leaf-cell anti-oxidant pool to ameliorate the toxic effects on plasma membrane of single ions in both cultivars, and of Cd + Ni only in cv. Premia. The decreased total content of K+ in leaves in all variants indicated repressing the K+ uptake and/or distribution to the shoots. Under anoxia, the magnitude of the repolarisation obtained after switching on the light was smaller in Cd-treated cultivar Premia than in the controls, and this also occurred in Ni- and Cd + Ni-treated cultivar Blitz.

Impact of nickel on grapevine (Vitis vinifera L.) root plasma membrane, ROS generation, and cell viability

Abstract The present study investigated the impact of nickel (Ni2+) on trans-membrane electrical potential (EM) and permeability properties of plasma membrane (PM) in epidermal cells of adventitious grapevine roots. The relationship between disturbances of membrane functionality and the production of superoxide anion, hydrogen peroxide and cell viability after the exposure of roots to Ni2+ was also studied. Treatments with 0.1-5 mmol L-1 NiCl2 induced a concentration-dependent transient PM depolarization, which was recovered to the initial resting potential within 50-70 min in the presence of Ni2+. Longer (up to 24 h) exposure of roots to 1 mmol L-1 of Ni2+ hyperpolarized the EM by approximately 17 mV. Application of the highest 5 mmol L-1 concentration of Ni2+ during longer treatments (up to 48 h) resulted in the increase of membrane permeability; however the EM, cell viability, and superoxide content remained unaffected. The increase in the formation of hydrogen peroxide was time- and concentration- dependent and maximum production was recorded after 180 min of Ni2+ treatment. We can conclude that oxidative stress resulting from an imbalance in the generation and/ or removal of hydrogen peroxide in the root tissues of grapevine was the major cause of Ni2+ toxicity.