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.

Glutathione peroxidase expression and activity in barley root tip after short-term treatment with cadmium, hydrogen peroxide and t-butyl hydroperoxide.

The purpose of this study was to analyse the alterations of glutathione peroxidase (GPX) expression and activity during the recovery period after a short-term treatment of barley root tip with cadmium (Cd) and hydrogen peroxide (H(2)O(2)). The transcript level of GPX increased as early as 1 h and GPX activity 3 h after short-term treatment independently of Cd concentration. In 15 µM Cd-treated roots, its expression reached a peak within 2 h and sustained until 3 h, after which it gradually declined. After 6 h of short-term Cd treatment, the activity of GPX was the highest in the 15-µM Cd-treated roots. At higher Cd concentrations, the activity of GPX was lower than in 15 µM Cd-treated roots, but still higher than in control roots. A considerable increase in H(2)O(2) production was observed even after only 1 h of short-term exposure of roots to 30 and 60 µM Cd, while after 15 µM Cd exposure, its production increased 3 h after the treatment. Lipid peroxidation increased even 1 h after short-term treatment in a Cd concentration-dependent manner. A considerable decrease of GPX activity was observed after the exposure of roots to H(2)O(2) or t-butyl hydroperoxide in a concentration-dependent manner despite that its expression increased even 1 h after short-term treatment. Presumable, under high acute Cd stress, rapid accumulation of H(2)O(2) leads to the disturbance of basal metabolic processes affecting also GPX activity. In contrast, high GPX activity under moderate Cd stress maintains cell function despite the high rate of H(2)O(2) metabolism in root tip.

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.

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.

Aluminium toxicity in plants: internalization of aluminium into cells of the transition zone in Arabidopsis root apices related to changes in plasma membrane potential, endosomal behaviour, and nitric oxide production.

The extent of aluminium internalization during the recovery from aluminium stress in living roots of Arabidopsis thaliana was studied by non-invasive in vivo microscopy in real time. Aluminium exposure caused rapid depolarization of the plasma membrane. The extent of depolarization depends on the developmental state of the root cells; it was much more extensive in cells of the distal than in the proximal portion of the transition zone. Also full recovery of the membrane potential after removal of external aluminium was slower in cells of the distal transition zone than of its proximal part. Using morin, a vital marker dye for aluminium, and FM4-64, a marker for endosomal/vacuolar membranes, an extensive aluminium internalization was recorded during the recovery phase into endosomal/vacuolar compartments in the most aluminium-sensitive cells. Interestingly, aluminium interfered with FM4-64 internalization and inhibited the formation of brefeldin A-induced compartments in these cells. By contrast, there was no detectable uptake of aluminium into cells of the proximal part of the transition zone and the whole elongation region. Moreover, cells of the distal portion of the transition zone emitted large amounts of nitric oxide (NO) and this was blocked by aluminium treatment. These data suggest that aluminium internalization is related to the most sensitive status of the distal portion of the transition zone towards aluminium. Aluminium in these root cells has impact on endosomes and NO production.

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.

Evidence for high activity of xylem parenchyma and ray cells in the interface of host stem and Agrobacterium tumefaciens-induced tumours of Ricinus communis.

Rapidly developing tumours at hypocotyls of Ricinus communis, induced by Agrobacterium tumefaciens strain C58, were characterized by strong differentiation of vascular bundles and their functional connection to the host bundles. The stem/tumour interface showed increased xylem, with numerous vessels accompanied by multiseriate unlignified rays. To know how nutrients efficiently accumulate in the tumour sink tissue, cell electropotentials (E(m)) in cross-sections were mapped. The measured cells were identified by injected Lucifer Yellow. Xylem and phloem parenchyma cells and stem/tumour-located rays hyperpolarized to E(m) values of about -170 mV, which suggest high plasma membrane proton pump activities. Rapidly dividing cells of cambia or small tumour parenchyma cells had low E(m). The tumour aerenchyma and the stem cortex cells displayed values close to the energy-independent diffusion potential. The lowest values were recorded in stem pith cells. Cell K(+) concentrations largely matched the respective E(m). The pattern of individual cell electropotentials was supplemented by whole organ voltage measurements. The voltage differences between the tumour surface and the xylem perfusion solution in stems attached to the tumours, the trans-tumour electropotentials (TTP), confirm the findings of respiration-dependent and phytohormone-stimulated high plasma membrane proton pump activity in intact tumours, mainly in the xylem and phloem parenchyma and ray cells. TTPs were inhibited by addition of NaN(3), CN(-) plus SHAM or N(2) gas in the xylem perfusion solution and by external N(2) flushing. The data provide functional evidence for the structural basis of priority over the host shoot in nutrient flow from the stem to the tumour.