Alessandro Abruzzese

Bacterial communities associated with the rhizosphere of transgenic Bt 176 maize (Zea mays) and its non transgenic counterpart

The effect of transgenic Bt 176 maize on the rhizosphere bacterial community has been studied with a polyphasic approach by comparing the rhizosphere of Bt maize cultivated in greenhouse with that of its non transgenic counterpart grown in the same conditions. In the two plants the bacterial counts of the copiotrophic, oligotrophic and sporeforming bacteria, and the community level catabolic profiling, showed no significant differences; differences between the rhizosphere and bulk soil bacterial communities were evidenced. Automated ribosomal intergenic spacer analysis (ARISA) showed differences also in the rhizosphere communities at different plant ages, as well as between the two plant types. ARISA fingerprinting patterns of soil bacterial communities exposed to root growth solutions, collected from transgenic and non transgenic plants grown in hydroponic conditions, were grouped separately by principal component analysis suggesting that root exudates could determine the selection of different bacterial communities.

Strategy for In Situ Detection of Natural Transformation-Based Horizontal Gene Transfer Events

ABSTRACT A strategy is described that enables the in situ detection of natural transformation in Acinetobacter baylyi BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.

Efflux and active re-absorption of glucose in roots of cotton plants grown under saline conditions.

The effect of growth under saline condition (100 mol m-3 NaCl in the nutrient solution) on the influx and the efflux of glucose from roots of cotton plants was analysed utilising the non metabolisable glucose analogue [14C]-3-O-methylglucose ([14C]3-OMG). Apical segments (1 cm long) excised from cotton roots took up [14C]3-OMG. At each tested concentration (5–500 mmol m-3), the influx was completely inhibited by the presence of the protonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) indicating that it is mediated by a H+-coupled co-transport mechanism. The CCCP-sensitive [14C]3-OMG influx was lower in the root segments excised from plants grown on saline solution than in the controls, and this was particularly evident at lower external concentrations. This difference was not due to a lower H+ apoplastic availability. In fact, the saline condition did not affect the pH of the rhizosphere and indeed the H+–ATPase activity, evaluated in plasma membrane vesicles purified from saline-treated plants, was higher (+23%) than in the controls. The lower uptake of [14C]3-OMG into saline treated root segments was related to an enhanced value of the apparent Km of the carrier for the glucose analogue. This effect is discussed in relation to either the more positive value of the transmembrane electric potential difference (ΔΨ) measured in these root segments, or a competitive inhibition of Na+ on the H+ binding site of the carrier. Growth in saline solution slightly affected the efflux of the [14C]3-OMG preloaded in root segments, changing the membrane permeability to the molecule. The results strongly suggest that the higher (2.5 fold) net exudation of glucose, observed in short-term (4 h) collection experiments, from roots of cotton plants grown in saline condition, is mainly due to an effect of the saline growth condition on the system involved in the reabsorption of the hexose rather than on its efflux.

Changes in the potato (Solanum tuberosum L.) tuber at the onset of dormancy and during storage at 23 °C and 3 °C. I. Biochemical and physiological parameters

SummaryIn the last 30 d of potato (Solanum tuberosum L.) tuber growth metabolic activity decreased. Levels of glucose-6-P and sucrose in whole tuber tissues declined and in tuber slices there was a decrease in the uptake from the medium and in the incorporation into macromolecules of [U-14C]sucrose. During storage at 23°C only the uptake of [U-14C]sucrose increased concomitant with tuber sprouting, indicating a possible involvement of the transport mechanisms in dormancy breaking. At 3°C, levels of reducing sugars and sucrose increased in response to the low temperature and increased release of K+ and malondialdehyde levels indicated cell membrane damage. The cell membrane functionality was restored at sprouting. The sprouting potential of the tubers was evaluated using the sprouting ability of single-bud explants (“seedcores”) in response to water, GA3 or ABA dips. This sprouting potential of tubers changed with stage of tuber growth and storage duration and temperature, indicating that the tissue hormonal state changed strongly throughout tuber life, probably in relation with the “sink” to “source” transition.

Analysis of cadmium translocation, partitioning and tolerance in six barley (Hordeum vulgare L.) cultivars as a function of thiol metabolism

Six barley cultivars widely differing for cadmium (Cd) tolerance, partitioning, and translocation were analyzed in relation to their thiol metabolism. Results indicated that Cd tolerance was not clearly related to the total amount of Cd absorbed by plants, resulting instead closely dependent on the capacity of the cultivars to trap the metal into the roots. Such behaviors suggested the existence of root mechanisms preserving shoots from Cd-induced oxidative damages, as indicated by the analysis of thiobarbituric acid-reactive substances—diagnostic indicators of oxidative stress—whose increases in the shoots were negatively related to Cd root retention and tolerance. Cd exposure differentially affected glutathione (GSH) and phytochelatin (PC) levels in the tissues of each barley cultivar. The capacity to produce PCs appeared as a specific characteristic of each barley cultivar, since it did not depend on Cd concentration in the roots and resulted negatively related to the concentration of the metal in the shoots, indicating the existence of a cultivar-specific interference of Cd on GSH biosynthesis, as confirmed by the existence of close positive linear relationships between the effect of Cd on GSH levels and PC accumulation in both roots and shoots. The six barley cultivars also differed for their capacity to load Cd ions into the xylem, which was negatively related to PC content in the roots. Taken as a whole, these data indicated that the different capacity of each cultivar to maintain GSH homeostasis under Cd stress may strongly affect PC accumulation and, thus, Cd tolerance and translocation.

Changes in some physiological and biochemical parameters during two subcultures in kiwi (Actinidia deliciosa) callus

Abstract Kiwi ( Actinidia deliciosa ) calli were grown for two successive 45-day subcultures on agarised medium in order to evaluate the metabolic changes occurring during the growth of the callus. At different times (10, 35 and 45 days of each subculture), increase in fresh weight, oxygen uptake rate, levels of inorganic cations (K + , Ca 2+ , Mg 2+ and Na + ) and of some metabolites (sucrose, glucose-6-phosphate, malic acid, amino acids and adenosine phosphates) were measured. After an adaptive growth phase (0–6 days) and a phase of high growth (6–12 days), the callus growth rate decreased sharply and then remained essentially constant up to the 45th day. The parameter listed above changed with time following a similar pattern in the two analysed subcultures. In the first 10 days, most of them increased (in particular, oxygen uptake rate and the energetic status of the cells); the levels of Na + decreased. After the first 10 days, the levels of glucose-6-phosphate (glu-6-P) and of the adenylate pool and the availability of the high-energy phosphate bonds of ATP and ADP decreased and the Na + levels began to increase; in this period, the rate of oxygen uptake increased, but this corresponded to neither increased availability of the high-energy phosphate bonds of ATP and ADP nor increased growth rate. In the last period (35–45 days) the main metabolic parameters dropped, with a large increase in Na + levels. Transfer onto new medium restored, after the adaptive period, the maximum growth and the levels of the parameters listed above. The data show that during the subculture periods of Actinidia deliciosa calli important metabolic changes occur. They are probably linked to a different status of suffering (for reduction of nutrient availability, accumulation of catabolic products, and/or variations in oxygen and CO 2 diffusion) with the activation of metabolic mechanisms of adaptation and repair. These metabolic changes appear not severe enough to inhibit the growth of the calli. The results obtained with Actinidia deliciosa calli are discussed in relation to protocols of selection and micropropagation.

Effects of rhizodeposition of non-transgenic and transplastomic tobaccos on the soil bacterial community

The effect of root-released compounds of transplastomic tobacco (Nicotiana tabacum) on the soil bacterial community structure, and their potential to support horizontal gene transfer (HGT) to bacteria have been studied. Soil microcosms were exposed to root-released compounds collected from transplastomic and non-transgenic tobacco cultivars. Cluster analysis of automated ribosomal intergenic spacer analysis (ARISA) profiles of the soil bacterial community after 48 h incubation grouped the transgenic cultivar apart from the non-transgenic, indicating that it had a rhizodeposition pattern different from the parental plants. However, these differences were less than between the two non-transgenic tobacco cultivars studied. NMR characterization of the root-released compounds showed some differences in chemical fingerprinting pattern between the transplastomic and the parental cultivar. However, the effect on bacterial community structure was transient, and tended to disappear after 96 h of incubation. The potential of root-released compounds as a source of transforming DNA for bacteria was investigated by using four potential recipient species. No transformants were obtained following exposure of all the recipients to the root-released compounds. Root-released compounds amended to transgene donor DNA decreased the transformation frequency of Acinetobacter baylyi strain ADP1200, while Azospirillum, Agrobacterium, and Sinorhizobium strains failed to develop competence also in the presence of an external added transgene source. Detection of plastid sequences by PCR suggested that a very low amount of fragmented plastid donor DNA was present in the root-released compounds.

Effect of Hyperosmotic 3-O-methyl-D-Glucose in the Medium on Metabolic Parameters in Actinidia deliciosa Callus

Calli of Actinidia deliciosa were cultured on medium with 30 mM sucrose and 0.2 M 3-O-methyl-D-glucose (3-OMG) and their metabolic adaptation to the hyperosmotic condition was studied. 3-OMG concentration in the tissue reached that of the medium. Experiments with labelled 3-O-methyl-D-U-14C]glucose (3-OM[U-14C]G) showed that it was partially metabolised but not oxidised to CO2. In vivo 31P-NMR showed that in 3-OMG-treated calli a phosphorylated compound, with a chemical shift similar to that of monophosphoesters, was accumulated. The 3-OMG-treated calli showed more negative water (Ψw) and osmotic (Ψπ) potentials than the controls. In the following of subcultures an increase in organic and inorganic osmolytes, together with increase in O2 uptake and decrease in ATP/ADP ratio occurred. The data show that, even if 3-OMG can not be considered a classic osmotic agent due to its permeability, it induced metabolic changes similar to those described for osmotic adaptation, suggesting that the adaptations might be due to a physical or metabolic effect of 3-OMG not linked to a difference between Ψπ and Ψw agar