Gian Attilio Sacchi

Heavy Metal Stress and Sulfate Uptake in Maize Roots

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 ± 0.4 μm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.

Cadmium-Induced Sulfate Uptake in Maize Roots

The effect of cadmium (Cd) on high-affinity sulfate transport of maize (Zea mays) roots was studied and related to the changes in the levels of sulfate and nonprotein thiols during Cd-induced phytochelatin (PC) biosynthesis. Ten micromolar CdCl2 in the nutrient solution induced a 100% increase in sulfate uptake by roots. This was not observed either for potassium or phosphate uptake, suggesting a specific effect of Cd2+ on sulfate transport. The higher sulfate uptake was not dependent on a change in the proton motive force that energizes it. In fact, in Cd-treated plants, the transmembrane electric potential difference of root cortical cells was only slightly more negative than in the controls, the external pH did not change, and the activity of the plasma membrane H+-ATPase did not increase. Kinetics analysis showed that in the range of the high-affinity sulfate transport systems, 10 to 250 μm, Cd exposure did not influence the K m value (about 20 μm), whereas it doubled theV max value with respect to the control. Northern-blot analysis showed that Cd-induced sulfate uptake was related to a higher level of mRNA encoding for a putative high-affinity sulfate transporter in roots. Cd-induced sulfate uptake was associated to both a decrease in the contents of sulfate and glutathione and synthesis of a large amount of PCs. These results suggest that Cd-induced sulfate uptake depends on a pretranslational regulation of the high-affinity sulfate transporter gene and that this response is necessary for sustaining the higher sulfur demand during PC biosynthesis.

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.

Root Uptake and Xylem Translocation of Pesticides from Different Chemical Classes

A pressure-chamber technique was used to study the root uptake and xylem translocation of some fungicides, herbicides and an insecticide from different chemical classes in detopped soybean roots. Physiological parameters such as K + leakage from roots, K + concentrations in the xylem sap, and protein and ATP levels in the root cells were measured so as to evaluate any potential damage of this technique to the root system. HPLC was used to quantify the compounds in the xylem sap. The pressure-chamber technique has proved useful to study the root uptake and translocation of pesticides, does not damage the root system, and allows one to obtain appreciable volumes of xylem sap that can be analysed directly by HPLC, thus avoiding dependence on the availability of radio-labelled compounds. The concentration of each pesticide in the xylem sap showed a steady-state kinetic profile. Non-linear regression analysis was used to calculate the steady-state concentration and the time required to achieve 50% of the steady-state concentration (TSSC 50 ). TSSC 50 was well correlated with log K ow ; the more lipophilic the compound the more time was required to reach the steady-state concentration. The efficiency of translocation was assessed by the transpiration stream concentration factor (TSCF) and a non-linear relationship between TSCF and log K ow was observed. The highest TSCF values were measured for those compounds with log K ow values around 3, a lipophilicity value similar to that reported earlier in an analogous experiment with detopped soybean plants but slightly higher than that reported in earlier experiments with intact barley plants. Lower TSCF values were obtained with chemicals with log K ow values below as well as above 3.

Implication of organic acids in the long-distance transport and the accumulation of lead in Sesuvium portulacastrum and Brassica juncea

The implication of organic acids in Pb translocation was studied in two species varying in shoot lead accumulation, Sesuvium portulacastrum and Brassica juncea. Citric, fumaric, malic and α-cetoglutaric acids were separated and determined by HPLC technique in shoots, roots and xylem saps of the both species grown in nutrient solutions added with 200 and 400 μM of Pb(II). The lead content of the xylem saps was determined by ICP-MS. Results showed that S. portulacastrum is more tolerant to Pb than B. juncea. Lead concentration in xylem sap of the S. portulacastrum was significantly greater than in that of B. juncea. For both species, a positive correlation was established between lead and citrate concentrations in xylem sap. However minor relationship was observed for fumaric, malic and α-cetoglutaric acids. In the shoots lead treatment also induced a significant increase in citric acid concentration. Both observations suggest the implication of citric acid in lead translocation and shoot accumulation in S. portulacastrum and B. juncea. The relatively high accumulation of citric acid in xylem sap and shoot of S. portulacastrum could explain its high potential to translocate and accumulate this metal in shoot suggesting their possible use to remediate Pb polluted soils.

Comparative Ni tolerance and accumulation potentials between Mesembryanthemum crystallinum (halophyte) and Brassica juncea: Metal accumulation, nutrient status and photosynthetic activity.

Saline soils often constitute sites of accumulation of industrial and urban wastes contaminated by heavy metals. Halophytes, i.e. native salt-tolerant species, could be more suitable for heavy metal phytoextraction from saline areas than glycophytes, most frequently used so far. In the framework of this approach, we assess here the Ni phytoextraction potential in the halophyte Mesembryanthemum crystallinum compared with the model species Brassica juncea. Plants were hydroponically maintained for 21 days at 0, 25, 50, and 100μM NiCl2. Nickel addition significantly restricted the growth activity of both species, and to a higher extent in M. crystallinum, which did not, however, show Ni-related toxicity symptoms on leaves. Interestingly, photosynthesis activity, chlorophyll content and photosystem II integrity assessed by chlorophyll fluorescence were less impacted in Ni-treated M. crystallinum as compared to B. juncea. The plant mineral nutrition was differently affected by NiCl2 exposure depending on the element, the species investigated and even the organ. In both species, roots were the preferential sites of Ni(2+) accumulation, but the fraction translocated to shoots was higher in B. juncea than in M. crystallinum. The relatively good tolerance of M. crystallinum to Ni suggests that this halophyte species could be used in the phytoextraction of moderately polluted saline soils.

Cadmium induces acidosis in maize root cells.

* Cadmium (Cd) stress increases cell metabolic demand for sulfur, reducing equivalents, and carbon skeletons, to sustain phytochelatin biosynthesis for Cd detoxification. In this condition the induction of potentially acidifying anaplerotic metabolism in root tissues may be expected. For these reasons the effects of Cd accumulation on anaplerotic metabolism, glycolysis, and cell pH control mechanisms were investigated in maize (Zea mays) roots. * The study compared root apical segments, excised from plants grown for 24 h in a nutrient solution supplemented, or not, with 10 microM CdCl(2), using physiological, biochemical and (31)P-nuclear magnetic resonance (NMR) approaches. * Cadmium exposure resulted in a significant decrease in both cytosolic and vacuolar pH of root cells and in a concomitant increase in the carbon fluxes through anaplerotic metabolism leading to malate biosynthesis, as suggested by changes in dark CO2 fixation, metabolite levels and enzyme activities along glycolysis, and mitochondrial alternative respiration capacity. This scenario was accompanied by a decrease in the net H(+) efflux from the roots, probably related to changes in plasma membrane permeability. * It is concluded that anaplerotic metabolism triggered by Cd detoxification processes might lead to an imbalance in H(+) production and consumption, and then to cell acidosis.

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.

Cadmium exposure and sulfate limitation reveal differences in the transcriptional control of three sulfate transporter ( Sultr1;2 ) genes in Brassica juncea

BackgroundCadmium (Cd) exposure and sulfate limitation induce root sulfate uptake to meet the metabolic demand for reduced sulfur. Although these responses are well studied, some aspects are still an object of debate, since little is known about the molecular mechanisms by which changes in sulfate availability and sulfur metabolic demand are perceived and transduced into changes in the expression of the high-affinity sulfate transporters of the roots. The analysis of the natural variation occurring in species with complex and highly redundant genome could provide precious information to better understand the topic, because of the possible retention of mutations in the sulfate transporter genes.ResultsThe analysis of plant sulfur nutritional status and root sulfate uptake performed on plants of Brassica juncea – a naturally occurring allotetraploid species – grown either under Cd exposure or sulfate limitation showed that both these conditions increased root sulfate uptake capacity but they caused quite dissimilar nutritional states, as indicated by changes in the levels of nonprotein thiols, glutathione and sulfate of both roots and shoots. Such behaviors were related to the general accumulation of the transcripts of the transporters involved in root sulfate uptake (BjSultr1;1 and BjSultr1;2). However, a deeper analysis of the expression patterns of three redundant, fully functional, and simultaneously expressed Sultr1;2 forms (BjSultr1;2a, BjSultr1;2b, BjSultr1;2c) revealed that sulfate limitation induced the expression of all the variants, whilst BjSultr1;2b and BjSultr1;2c only seemed to have the capacity to respond to Cd.ConclusionsA novel method to estimate the apparent kM for sulfate, avoiding the use of radiotracers, revealed that BjSultr1;1 and BjSultr1;2a/b/c are fully functional high-affinity sulfate transporters. The different behavior of the three BjSultr1;2 variants following Cd exposure or sulfate limitation suggests the existence of at least two distinct signal transduction pathways controlling root sulfate uptake in dissimilar nutritional and metabolic states.