Vladimír Repka

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.

Auxins in defense strategies

Plant hormones operate in a very complex network where they regulate and control different vital mechanisms. They coordinate growth, development and defense via signaling involving different interactions of molecules. Activation of molecules responsible for regulation of plant immunity is mainly provided by salicylic and jasmonic acid signaling pathways. Similar to the signaling of these defense-associated plant hormones, auxin can also affect resistance to different pathogen groups and disease is manifested indirectly through the effects on growth. The various ways in which auxin regulate growth and plant development and might be closely connected to plant defense, are discussed in this review.

Methyl jasmonate-induced cell death in grapevine requires both lipoxygenase activity and functional octadecanoid biosynthetic pathway

Grapevine (Vitis vinifera L., cv. Limberger) leaf tissues and suspension-cultured cells were induced to undergo programmed cell death (PCD) by exogenously added methyl jasmonate (MeJA). The elicitor signaling pathway involved in MeJA-induced PCD was further investigated using pharmacological, biochemical and histological approaches. Pharmacological dissection of the early events preceding the execution of MeJA-triggered PCD indicated that this process strongly depends on both, de novo protein and mRNA synthesis. Treatment of leaf discs and cell suspensions with lipase inhibitor Ebelactone B and specific lipoxygenase inhibitor Phenidone blocked MeJA-induced PCD. These results suggest that some chloroplast membrane-derived compound(s) is required for MeJA-induced PCD in grapevine. The progression of MeJAtriggered PCD may be further inhibited by the use of metabolic inhibitors of key enzymes of octadecanoid biosynthesis including AOS, AOC, and OPR indicating that the functional jasmonate biosynthetic pathway is an integral part of the MeJA-induced signal transduction cascade that results in the coordinate expression of events leading to PCD. Finally, the activation of the octadecanoid pathway, as a critical point in MeJA-induced PCD, was independently demonstrated with cellulysin, a macromolecular elicitor acting via octadecanoid signaling. The cellulysin was shown to be a very potent enhancer of MeJA-triggered PCD in grapevine cells.

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.

Proteomics and interactomics in grapevine: the next level in the study

Abstract To understand the cellular biology and biochemistry of plant cell behavior, there is not only one approach for studying proteins which are directly responsible for cellular activity. However, despite the enormous quantity of information generated by transcriptome analysis, the picture is still incomplete. The proteomic and interactomic approaches present a new point of view that so far has been missing. Comparative proteomics provide a powerful means to study products of genes and their regulation. On the other side, interactomes of different species can provide information about the evolutionary mechanisms leading to organism diversity. Then, this analysis allows scientists to better understand how complex biological processes are regulated and evolved.

PROTEOMIC INSIGHT INTO THE MOLECULAR PRINCIPLES OF GRAPEVINE HABITUATION

Proteomic Insight Into the Molecular Principles of Grapevine Habituation Two-dimensional gel electrophoresis coupled to protein microarray analysis was used to examine, for the first time, the molecular mechanisms of grapevine (Vitis vinifera L., cv. Limberger) habituation. The examination of 2-D maps derived from control and habituated cell culture revealed the presence of 55 protein spots displaying a differential expression pattern. These facts have provide a molecular evidence suggesting that the habituated cells can be used as a model for study of cell differentiation and plant defense mechanisms. Cell death, extra-cellular alkalinization and expression of genes responsible for the formation of the defense-related proteins were analyzed in suspension cultures with hormonal autonomy (habituation). Results obtained using habituated grapevine cells compared with non-habituated cells were different and strongly depended on the concentration of elicitor applied.

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.