Effect of hypoxic acclimation on anoxia tolerance in Vitis roots: response of metabolic activity and K+ fluxes.

The effect of a hypoxic pre-treatment (HPT) on improving tolerance to prolonged anoxia conditions in two contrasting Vitis species (V. riparia, anoxia tolerant; V. rupestris, anoxia sensitive) was evaluated. The energy economy of root cells was studied by measuring heat production, the activity of pyruvate decarboxylase (PDC) and alcohol dehdrogenase (ADH), ethanol and ATP production, and K(+) fluxes. The results showed that HPT is an effective tool in order to maintain a sustainable metabolic performance in both the species under anoxia conditions, especially in sensitive species such as V. rupestris. Our results showed that the improved tolerance was mainly driven by: (i) an enhanced activity of key enzymes in alcohol fermentation (ADC and PDC); (ii) the capability to maintain a higher level of respiration, evidenced by a lesser decrease in heat development and ATP production; and (iii) the maintenance of a better ion homeostasis (highlighted by measurement of K(+) fluxes) and K(+) channel functionality.

Phospholipase dzeta2 drives vesicular secretion of auxin for its polar cell-cell transport in the transition zone of the root apex.

Auxin (IAA) is versatile signalling molecule of plants, currently classified as plant hormone. But there are data suggesting that auxin is acting also as plant-specific morphogen, electric-responses inducing transmitter, and as general signalling molecule used for plant-bacteria communication. Our previous data revealed that auxin is associated with secretory endosomes and also highly enriched within cell walls of cells active in transcellular auxin transport. Our present data, based on in vivo non-invasive auxin flux recordings, reveal that auxin is secreted out of synaptic-like domains specialized for efflux of auxin in root apex cells highly active in polar cell-cell transport of auxin. We obtained both genetic and pharmacological evidence that phospholipase Dzeta2 drives vesicular secretion of auxin for its polar transcellular transport in the transition zone of the root apex. Secretion of auxin via secretory vesicles has far-reaching consequences not only for our understanding of cell-cell auxin transport but also for plant sciences as a whole.

Mixture design in the optimization of a microemulsion system for the electrokinetic chromatographic determination of ketorolac and its impurities: method development and validation.

Microemulsion EKC (MEEKC) was used for the determination of ketorolac and its three impurities. The microemulsion system was optimized, for the first time in the literature, using a multivariate strategy involving a mixture design. A 13-run experimental plan covering an experimental domain defined by the components aqueous phase (10 mM borate buffer pH 9.2), oil phase (n-heptane) and surfactant/cosurfactant (SDS/n-butanol) was carried out. Good results were obtained with all microemulsions tested considering as responses analysis time and resolution, and according to the desirability function the best microemulsion system was constituted by 90.0% 10 mM borate buffer, 2.0% n-heptane, 8.0% of SDS/n-butanol in 1:2 ratio. Finally, with the aim of reducing analysis time, a response surface study was carried out in the experimental domain defined by the process variables temperature and voltage and the best values were 17 degrees C and -17 kV, respectively. Applying the optimised conditions, a complete resolution among the analytes was obtained in about 3 min using the short-end injection method. The method was validated for both drug substances and drug product and was applied to the quality control of ketorolac in coated tablets. A comparison of MEEKC, MEKC and CEC for assaying ketorolac and its related substances has been made.

Adaptative response of Vitis root to anoxia.

The effect of anoxia on the energy economy of root cells was studied by measuring heat production, ethanol and ATP production, K(+) fluxes and electrical activity in two Vitis species, V. riparia and V. rupestris, that differ in their tolerance to anoxia. Anoxia triggered a marked decrease of metabolic activity (measured by microcalorimetry) and of ATP levels in both species. In V. riparia after the first 2 h of anoxia, the decrease in the rate of heat production was not associated with a further significant decrease in ATP content, whereas in V. rupestris the ATP level continued to decrease until very low values were reached. The concomitant increase in the rate of ethanol production did not compensate for the decreased aerobic ATP supply. In V. rupestris, anoxia typically led to energy deficit and ATP imbalance, together with the subsequent disruption of ion homeostasis and cell death. In V. riparia, the strong decrease in K(+) membrane permeability together with the fast down-regulation of the electrical signals allowed the cells to avoid severe ion imbalances during prolonged anoxic episodes.

Noninvasive and continuous recordings of auxin fluxes in intact root apex with a carbon nanotube-modified and self-referencing microelectrode.

Auxin (also known as indole-3-acetic acid, IAA) represents an ancient signaling molecule of plants that also exerts bioactive actions on yeast and animal cells. Importantly, IAA emerges as a new anticancer agent due to the ability of oxidatively activated IAA to selectively kill tumor cells. IAA acts as a pheromone-like molecule in brown algae, whereas the hormone concept of IAA dominates current plant biology. However, recent advances also favor the morphogen- and transmitter-like nature of IAA in plants, making this small molecule one of the most unique molecules in the eukaryotic superkingdom. Here, we introduce new technology for the continuous measuring of IAA fluxes in living cells, tissues, and whole organs that is based on a carbon nanotube-modified and self-referencing microelectrode specific for IAA. This technique not only will advance our knowledge of how IAA regulates plant development but will also be applicable in medicine for its potential use in cancer therapy.

Different pathways of the oxygen supply in the sapwood of young Olea europaea trees.

Gaseous transport through lenticels is widely accepted to be the main pathway for oxygen supply to the parenchymatous tissues of the wood. Circumstantial evidence exists that the oxygen required for respiration by these living cells can be obtained from the transpiration stream. However, there has been no functional confirmation of this role. To address this problem and to quantify the contribution of the different pathways to the oxygen supply of the sapwood, we have developed a three-electrode miniaturized oxygen-selective sensor to be implanted into the sapwood for long-term determination of the oxygen concentration. In spring, during the active growing season, the oxygen concentration of the sapwood of young olive (Olea europaea L.) trees changed from 80-90 micromol O(2) l(-1) around midday to 20-30 micromol O(2) l(-1) in the night. These concentrations correspond to a deficit of oxygen for the sapwood between 65-70% and 88-90% of an aqueous solution saturated with air. In the daylight hours, almost all the oxygen present in the sapwood was delivered by the transpiration stream, driven by the soil-plant-atmosphere water-potential gradient. During the night the diffusion of oxygen via the sap-filled lumina of the tracheids and vessels (xylary diffusion in the aqueous phase) accounted for about 87% of all the oxygen present, whereas only the remaining 13% was assessed as supplied by radial diffusion in the aqueous or gaseous phase.