Ján Pavlovkin

Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens.

Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens. Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host. Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment. CO2 release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks. However, tumor water loss contributed little to water flow to host shoots. Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance. Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors. Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization. Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase. Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport. Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment.

Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress.

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E(M)) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%. The E(M) values of cortical cells of Lotus roots varied between -115 and -144mV. Treatment with 250microM of AlCl(3) (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51mV (cv. UFGRS) and 16mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K(+) from Al-treated roots (2mM Al) and resulted in a decrease of the diffusion potential (E(D)). The total amount of K(+) in Al-treated roots dropped from 31.4 to 16.8micromolg(-1) FW in sensitive cv. INIA Draco, or from 26.1 to 22.7micromolg(-1) FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21-34% of the control.

Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species

BACKGROUND AND AIMS Uptake of heavy metals by plant root cells depends on electro-physiological parameters of the plasma membrane. In this study, responses of the plasma membrane in root cells were analysed where early reactions to the metal ion-induced stress are localized. Three different Arabidopsis species with diverse strategies of their adaptation to heavy metals were compared: sensitive Arabidopsis thaliana and tolerant A. halleri and A. arenosa. METHODS Plants of A. thaliana Col-0 ecotype and plants of A. arenosa and A. halleri originating from natural metallicolous populations were exposed to high concentrations of Zn(2+). Plants were tested for root growth rate, cellular tolerance, plant morphology and cell death in the root apex. In addition, the membrane potential (E(M)) of mature cortical root cells and changes in the pH of the liquid culture media were measured. KEY RESULTS Primary roots of A. halleri and A. arenosa plants grew significantly better at increased Zn(2+) concentrations than A. thaliana plants. Elevated Zn(2+) concentrations in the culture medium induced rapid changes in E(M). The reaction was species-specific and concentration-dependent. Arabidopsis halleri revealed the highest insensitivity of the plasma membrane and the highest survival rate under prolonged treatment with extra-high concentrations. Plants were able to effectively adjust the pH in the control, but much less at Zn(2+)-induced lower pH. CONCLUSIONS The results indicate a similar mode of early reaction to Zn(2+), but with different extent in tolerant and sensitive species of Arabidopsis. The sensitivity of A. thaliana and a high tolerance of A. halleri and A. arenosa were demonstrated. Plasma membrane depolarization was lowest in the hyperaccumulator A. halleri and highest in A. thaliana. This indicates that rapid membrane voltage changes are an excellent tool to monitor the effects of heavy metals.

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (Em) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 µM to 1 mM) for several hours to one week were studied. The Em values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (ED) when the Cd concentration exceeded 100 µM. The values of ED ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of Em was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 µM) or complete repolarization (Cd < 100 µM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of Em a continuous concentration-dependent decrease of Em followed at all Cd concentrations. Depolarization of Em was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 µM to 1 mM Cd, with the exception of root cells treated with 1 and 10 µM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 µM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h.All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 µM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H+ ATPase because fusicoccin, an H+ ATPase activator diminished the depolarizing effect of Cd on the Em. The depolarization of Em in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H+ ATPase.

Impact of Agrobacterium tumefaciens -induced stem tumors on NO 3 − uptake in Ricinus communis

Developing tumors induced by Agrobacterium tumefaciens, strain C58, on stems of Ricinus communis L. var. gibsonii cv. Carmencita were shown to be strong metabolic sinks for sucrose and amino acids, thus causing higher nutrient demand in the host plant. However, NO3− uptake and, to a lesser extent, also H2PO4− uptake were strongly inhibited. Correspondingly, NO3− concentration was lower in tumorised than in the control plants. NO3−reductase activity was the same in both plant types, but it was completely suppressed in the tumors. The electrical membrane potential difference of root cells was unaffected in tumorised plants when soil-grown, but significantly lowered when grown hydroponically. Consistent with the low NO3− uptake rate, NO3−-dependent membrane depolarisation at the onset of NO3−/2H+-cotransport was nearly zero. In the phloem sap, sucrose and amino acid concentrations were considerably lower in tumorised than in control plants, and lower below than above the tumor. The qualitative pattern of amino acids of the phloem sap of stems was almost the same in tumorised and control plants. It is concluded that neither the overall amino acid concentration nor special amino acids nor ammonium in the transport phloem suppress NO3− uptake in the roots. Aminocyclopropane-carboxylate, the precursor of ethylene, which is produced in the tumors in high amounts, was low in the stems and the same in both plant types. Thus, ACC and ethylene were ruled out as directly interfering with nutrient uptake in the roots. Root morphology was strongly affected during tumor development. Root fresh weight decreased to 50% of the controls and lateral root development was almost completely prevented. This suggests that the high tumor ethylene production, together with an increasing concentration of phenolic compounds, severely inhibits the basipetal auxin flow to the roots. Auxin accumulation and retention was confirmed by specifically enhanced expression of the auxin-responsive promoter of the soybean gene GH3:GUS in tumors induced in transgenic Trifolium repens L. Hence, root development is poorer and anion uptake inhibited in tumorised plants. This may be aggravated by abscisic acid accumulation in the tumor and its basipetal export into the roots. Moreover, sucrose depletion of the sieve tubes leads to energy shortage at the root level for maintaining energy-dependent anion uptake.

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.

Cellular responses of two Latin-American cultivars of Lotus corniculatus to low pH and Al stress

Toxic effects of acidic root medium and aluminium were evaluated in two forage cultivars of Lotus corniculatus differing in their tolerance to Al stress. The structural response of most of the root cells exposed to low pH without Al3+ differed markedly from that induced by the combined stress. Conspicuous alteration of the nucleus was present only at low pH 4.0 and disintegration of the cytoplasmic components was more drastic than in the roots exposed to acidic solution containing Al3+. Cells exposed to low pH without Al, did not produce wall thickenings. Severely damaged cytoplasm and localized death in some cortical cells or groups of cells contrasting with almost intact cells exposed to Al3+ stress were found. In this respect, a strong correlation between the occurrence of cell wall thickenings and a better preserved structure of the cytoplasm was observed. The frequency of cell damage in the more tolerant cultivar UFRGS was generally lower, significantly more cortical cells capable of maintaining their resting membrane potential were present than in the sensitive INIA Draco. The difference in their tolerance is related rather to the exudation of citrate and oxalate that was higher in UFRGS than to the accumulation of tannins, which increased after Al treatment in both cultivars.

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.

Impact of fusaproliferin on primary roots of maize cultivars differing in their susceptibility to Fusarium

Effects of fusaproliferin (FUS) on membrane potential (EM), electrolyte leakage, enzymes activity and respiration of roots, were studied in two maize cultivars (Zea mays L.), differing in their susceptibility to this toxin. In short-term experiments (≤ 6 h), EM has been rapidly and significantly depolarized by FUS. The rapidity of EM depolarization in tolerant cv. Lucia was more expressive in comparison with susceptible cv. Pavla, but the extent of EM depolarization was higher in cv. Pavla. In both maize cultivars, higher depolarization of EM was registered in cells of root zone I. In long-term experiments after the first EM depolarization, which occurred during the first 6 h of FUS treatment, gradual depolarization continued up to 24 h and was represented not only by the active component (EP) but also by the passive component (ED) of EM. The decrease in EM and ED was followed by a loss of K+ ions from FUS treated roots of both cultivars. The leak of K+ ions from the root cells of both root zones as well as both maize cultivars increased with the time of FUS treatment and was significantly higher in susceptible cv. Pavla than in tolerant cv. Lucia. FUS treatment of maize roots resulted in a significant decrease of root respiration which was higher in susceptible cv. Pavla than in tolerant cv. Lucia.The analysis of enzyme activities revealed that FUS significantly stimulated POD activity in both maize cultivars. SOD activity was significantly increased only in susceptible cv. Pavla, while APX activity was not affected by the presence of FUS. GST activity was specifically induced by FUS only in tolerant cv. Lucia.Due to the observed correlation between the extent of depolarization and the sensitivity of the studied maize cultivars to fusaproliferin, the EM parameters should be used for rapid screening of FUS-resistant cultivars for agricultural practice.

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.