Fabio F. Nocito

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.

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.

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.

Oxidative stress and senescence-like status of pear calli co-cultured on suspensions of incompatible quince microcalli.

This work presents a simple in vitro system to study physiological, biochemical and molecular changes occurring in a pear callus (Pyrus communis L., cv. Beurré Bosc) grown in close proximity to spatially separated undifferentiated homologous (pear) or heterologous (quince; Cydonia oblonga Mill., East Malling clone C) cells in its neighboring environment. After a 7-day co-culture period, the presence of heterologous cells produced negative effects on the pear callus, whose relative weight increase and adenylate energy charge decreased by 30 and 24%, respectively. Such behavior was associated with a higher O(2) consumption rate (+125%) which did not seem to be coupled to adenosine triphosphate synthesis. Analyses of alternative oxidase and enzymatic activities involved in reactive oxygen species (ROS) detoxification strongly suggested that the higher O(2) consumption rate, measured in the pear callus grown in the heterologous combination, may probably be ascribed to extra-respiratory activities. These, in turn, might contribute to generate metabolic scenarios where ROS-induced oxidative stresses may have the upper hand. The increase in the levels of 2-thiobarbituric acid reactive metabolites, considered as diagnostic indicators of ROS-induced lipid peroxidation, seemed to confirm this hypothesis. Moreover, reverse transcription polymerase chain reaction analysis revealed that the expression levels of a few senescence-associated genes were higher in the pear callus grown in the heterologous combination than in the homologous one. Taken as a whole, physiological and molecular data strongly suggest that undifferentiated cells belonging to a pear graft-incompatible quince clone may induce an early senescence-like status in a closely co-cultured pear callus.

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.

Kinetic Analysis of Zinc/Cadmium Reciprocal Competitions Suggests a Possible Zn-Insensitive Pathway for Root-to-Shoot Cadmium Translocation in Rice.

BackgroundAmong cereals, rice has a genetic propensity to accumulate high levels of cadmium (Cd) in grains. Xylem-mediated root-to-shoot translocation rather than root uptake has been suggested as the main physiological factor accounting for the genotypic variation observed in Cd accumulation in shoots and grains. Several evidence indicate OsHMA2 – a putative zinc (Zn) transporter – as the main candidate protein that could be involved in mediating Cd- and Zn-xylem loading in rice. However, the specific interactions between Zn and Cd in rice often appear anomalous if compared to those observed in other staple crops, suggesting that root-to-shoot Cd translocation process could be more complex than previously thought. In this study we performed a complete set of competition experiments with Zn and Cd in order to analyze their possible interactions and reciprocal effects at the root-to-shoot translocation level.ResultsThe competition analysis revealed the lack of a full reciprocity when considering the effect of Cd on Zn accumulation, and vice versa, since the accumulation of Zn in the shoots was progressively inhibited by Cd increases, whereas that of Cd was only partially impaired by Zn. Such behaviors were probably dependent on Cd-xylem loading mechanisms, as suggested by: i) the analysis of Zn and Cd content in the xylem sap performed in relation to the concentration of the two metals in the mobile fractions of the roots; ii) the analysis of the systemic movement of 107Cd in short term experiments performed using a positron-emitting tracer imaging system (PETIS).ConclusionsOur results suggest that at least two pathways may mediate root-to-shoot Cd translocation in rice. The former could involve OsHMA2 as Zn2+/Cd2+ xylem loader, whereas the latter appears to involve a Zn-insensitive system that still needs to be identified.

The Sulfate Supply Maximizing Arabidopsis Shoot Growth Is Higher under Long- than Short-Term Exposure to Cadmium

The processes involved in cadmium detoxification in plants deeply affect sulfate uptake and thiol homeostasis and generate increases in the plant nutritional request for sulfur. Here, we present an analysis of the dependence of Arabidopsis growth on the concentration of sulfate in the growing medium with the aim of providing evidence on how plants optimize growth at a given sulfate availability. Results revealed that short-term (72 h) exposure to a broad range of Cd concentrations (0.1, 1, and 10 μM) inhibited plant growth but did not produce any significant effects on the growth pattern of both shoots and roots in relation to the external sulfate. Conversely, long-term (22 days) exposure to 0.1 μM Cd significantly changed the pattern of fresh weight accumulation of the shoots in relation to the external sulfate, without affecting that of the roots, although their growth was severely inhibited by Cd. Moreover, under long-term exposure to Cd, increasing the sulfate external concentration up to the critical value progressively reduced the inhibitory effects exerted by Cd on shoot growth, indicating the existence of sulfate-dependent adaptive responses protecting the shoot tissues against Cd injury. Transcriptional induction of the high-affinity sulfate transporter genes (SULTR1; 1 and SULTR1; 2) involved in sulfate uptake by roots was a common adaptive response to both short- and long-term exposure to Cd. Such a response was closely related to the total amount of non-protein thiols accumulated by a single plant under short-term exposure to Cd, but did not showed any clear relation with thiols under long-term exposure to Cd. In this last condition, Cd exposure did not change the level of non-protein thiols per plant and thus did not alter the nutritional need for sulfur. In conclusion, our results indicate that long term-exposure to Cd, although it induces sulfate uptake, decreases the capacity of the Arabidopsis roots to efficiently absorb the sulfate ions available in the growing medium making the adaptive response of SULTR1; 1 and SULTR1; 2 “per se” not enough to optimize the growth at sulfate external concentrations lower than the critical value.

Protein profiling and tps23 induction in different maize lines in response to methyl jasmonate treatment and Diabrotica virgifera infestation

Plant responses to herbivore insects involve direct and indirect defense with the production of signal molecules including jasmonic acid (JA) and its derivatives (e.g. methyl jasmonate, MeJA). In maize (Zea mays), root feeding by Diabrotica virgifera larvae activates an indirect defense mechanism, through enthomopathogenic nematodes that are recruited after Terpene Synthase 23 (tps23) upregulation and (E)-β-caryophyllene root emission. In order to gain insight into the correlation between JA signaling and response to Diabrotica attack, we analyzed tps23 expression and protein profiles in maize roots in response to MeJA treatment and insect infestation. Similar to herbivore feeding, MeJA treatment was found to increase tps23 transcript accumulation, with consistent variations for both treatments in maize lines differing in (E)-β-caryophyllene production. Analysis of root protein profiles showed specific alterations leading to the identification of three proteins that were induced by MeJA treatment. We focused on a peroxidase-like protein (Px-like) showing that the corresponding transcripts accumulated in all tested lines. Results show that exogenous application of MeJA upregulates tps23 expression and specifically alters protein patterns in maize roots. Parallel effects on tps23 transcript accumulation were observed upon hormone exposure and insect infestation in different maize lines. In contrast, Px-like transcript profiling showed differences between treatments. These results support the possible involvement of MeJA in mediating the upregulation of tps23 in response to Diabrotica attack.

Monitoring Plant Nutritional Status

Methods and techniques effective in achieving yield objectives, optimizing the use of resources and preventing environmental contamination are defined as agronomic Best Management Practices (BMPs). Considering fertilisations BMPs pursue the aims to match mineral nutrient supply with crop requirements, minimizing their losses from the field. If spatial and temporal information about crop needs were available, precision fertilisation approaches could be planned in order to increase fertiliser use efficiency and to improve some economic and environmental aspects related to the crop systems. The state of the art and the research perspectives on the fine tuning of optical devices allowing proximal or remote sensing at sub-field scale of crops traits related to plant nutritional status, as well as on the exploitation of the gene fusion concept in developing transgenic bioindicators monitoring the nutritional status of the plants are here reviewed and discussed. Concerning the latter aspect particular attention is paid to the development of synthetic promoters conferring to the biondicator nutrient specificity and also able to target the expression of the associated reporter genes in organs in which their signals should be early and easily detectable.