Influence of iron status on cadmium and zinc uptake by different ecotypes of the hyperaccumulator Thlaspi caerulescens

We have previously identified an ecotype of the hyperaccumulator Thlaspi caerulescens (Ganges), which is far superior to other ecotypes (including Prayon) in Cd uptake. In this study, we investigated the effect of Fe status on the uptake of Cd and Zn in the Ganges and Prayon ecotypes, and the kinetics of Cd and Zn influx using radioisotopes. Furthermore, the T. caerulescens ZIP (Zn-regulated transporter/Fe-regulated transporter-like protein) genes TcZNT1-G and TcIRT1-G were cloned from the Ganges ecotype and their expression under Fe-sufficient and -deficient conditions was analyzed. Both short- and long-term studies revealed that Cd uptake was significantly enhanced by Fe deficiency only in the Ganges ecotype. The concentration-dependent kinetics of Cd influx showed that the V(max) of Cd was 3 times greater in Fe-deficient Ganges plants compared with Fe-sufficient plants. In Prayon, Fe deficiency did not induce a significant increase in V(max) for Cd. Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes. These results are in agreement with the gene expression study. The abundance of ZNT1-G mRNA was similar between the Fe treatments and between the two ecotypes. In contrast, abundance of the TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions. The present results indicate that the stimulatory effect of Fe deficiency on Cd uptake in Ganges may be related to an up-regulation in the expression of genes encoding for Fe(2+) uptake, possibly TcIRT1-G.     

Soil strength influences wheat root interactions with soil macropores

Deep rooting is critical for access to water and nutrients found in subsoil. However, damage to soil structure and the natural increase in soil strength with depth, often impedes root penetration. Evidence suggests that roots use macropores (soil cavities greater than 75 μm) to bypass strong soil layers. If roots have to exploit structures, a key trait conferring deep rooting will be the ability to locate existing pore networks; a trait called trematotropism. In this study, artificial macropores were created in repacked soil columns at bulk densities of 1.6 g cm⁻³ and 1.2 g cm⁻³, representing compact and loose soil. Near isogenic lines of wheat, Rht-B1a and Rht-B1c, were planted and root–macropore interactions were visualized and quantified using X-ray computed tomography. In compact soil, 68.8% of root–macropore interactions resulted in pore colonization, compared with 12.5% in loose soil. Changes in root growth trajectory following pore interaction were also quantified, with 21.0% of roots changing direction (±3°) in loose soil compared with 76.0% in compact soil. These results indicate that colonization of macropores is an important strategy of wheat roots in compacted subsoil. Management practices to reduce subsoil compaction and encourage macropore formation could offer significant advantage in helping wheat roots penetrate deeper into subsoil.     

Mechanical Impedance and Nutrient Acquisition in Rice

The aim of this work was to examine the effect of mechanical impedance on nutrient acquisition and gene expression in rice (Oryza sativa L.). Roots were mechanically impeded in a sand-core apparatus to vary impedance independently of aeration and water status. The effect of impedance on plant growth, anion concentration and expression of genes for anion transporters was compared for six varieties with differences in root penetration ability. Impedance decreased shoot growth more than root growth in all varieties, resulting in increased root/shoot ratios. Impedance substantially increased shoot tissue nitrate concentration in all varieties but only caused a small increase in shoot sulphate and phosphate concentrations. High impedance increased expression of the sulphate transporter OsST1 in five varieties, which was associated with decreased sulphate concentration in root tissues. In contrast, impedance decreased expression of the phosphate transporter OsPT2 expression in all varieties, which was associated with decreased phosphate concentration in root tissues. Localisation of expression of the sulphate transporter by in situ hybridisation indicated high levels of expression in lateral bud primordia. It was suggested that the decreased root phosphate concentrations of impeded roots were caused by low phosphate transporter gene expression, while the increase in sulphate transporter gene expression was due to a derepression mechanism of control.     

Elemental Sulfur and Thiol Accumulation in Tomato and Defense against a Fungal Vascular Pathogen

The occurrence of fungicidal, elemental S is well documented in certain specialized prokaryotes, but has rarely been detected in eukaryotes. Elemental S was first identified in this laboratory as a novel phytoalexin in the xylem of resistant genotypes of Theobroma cacao, after infection by the vascular, fungal pathogen Verticillium dahliae. In the current work, this phenomenon is demonstrated in a resistant line of tomato, Lycopersicon esculentum, in response to V. dahliae. A novel gas chromatography-mass spectroscopy method using isotope dilution analysis with 34S internal standard was developed to identify unambiguously and quantify 32S in samples of excised xylem. Accumulation of S in vascular tissue was more rapid and much greater in the disease-resistant than in the disease-susceptible line. Levels of S detected in the resistant variety (approximately 10 microg g-1 fresh weight excised xylem) were fungitoxic to V. dahliae (spore germination was inhibited >90% at approximately 3 microg mL-1). Scanning electron microscopy-energy dispersive x-ray microanalysis confirmed accumulation of S in vascular but not in pith cells and in greater amounts and frequency in the Verticillium spp.-resistant genotype. More intensive localizations of S were occasionally detected in xylem parenchyma cells, vessel walls, vascular gels, and tyloses, structures in potential contact with and linked with defense to V. dahliae. Transient increases in concentrations of sulfate, glutathione, and Cys of vascular tissues from resistant but not susceptible lines after infection may indicate a perturbation of S metabolism induced by elemental S formation; this is discussed in terms of possible S biogenesis.     

Markedly different gene expression in wheat grown with organic or inorganic fertilizer

Nitrogen is the major determinant of crop yield and quality and the precise management of nitrogen fertilizer is an important issue for farmers and environmentalists. Despite this, little is known at the level of gene expression about the response of field crops to different amounts and forms of nitrogen fertilizer. Here we use expressed sequence tag (EST)-based wheat microarrays in combination with the oldest continuously running agricultural experiment in the world to show that gene expression is significantly influenced by the amount and form of nitrogenous fertilizer. In the Broadbalk winter wheat experiment at Rothamsted in the United Kingdom and at three other diverse test sites, we show that specific genes have surprisingly different expression levels in the grain endosperm when nitrogen is supplied either in an organic or an inorganic form. Many of the genes showing differential expression are known to participate in nitrogen metabolism and storage protein synthesis. However, others are of unknown function and therefore represent new leads for future investigation. Our observations show that specific gene expression is diagnostic for use of organic sources of nitrogen fertilizer and may therefore have useful applications in defining the differences between organically and conventionally grown wheat. [The sequences reported in this paper have been deposited in the GenBank database (accession nos. AL 208216-AL 831324).]     

Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana

The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.     

Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants

Sulfur is an essential macro-element in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.     

Separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach

Cysteine synthase (O-acetylserine (thiol) lyase, EC 4.2.99.8) and -cyanoalanine synthase (EC 4.4.1.9) have been isolated from leaves of Spinacea oleracea L. and separated by anion-exchange chromatography. Further separation of one minor and two major isoforms of cysteine synthase was achieved by hydrophobic interaction chromatography and high resolution native electrophoresis (PAGE). Analysis of root material indicated that amongst the multiple isoforms present, a single isoform predominated. Subcellular fractionation studies indicated that one of the major leaf forms, cysteine synthase B, was located in the chloroplast and the other, cysteine synthase B, occurred in the cytoplasm. No specific isoform of cysteine synthase was resolved in the mitochondria, while cyanoalanine synthase was predominantly located in the mitochondrial fraction. Sulphur deprivation decreased cysteine synthase activity, but not cyanalanine synthase activity in both young and mature leaves, although cysteine synthase activity in the roots increased slightly. A selective decrease in cystein synthase B (chloroplastic abundance was observed in mature leaves. Patterns of expression of cysteine synthase in response to S-availability are discussed in relation to possible roles for this enzyme in controlling S-flux through the S-assimilatory pathway.Key words: Cysteine synthase isoforms (expression of), Spinacea oleracea L., sulphur deficiency.      

Approaches to cloning genes encoding for nutrient transporters in plants

Pairs of fungi were incubated on wheat straw in microcosms for 10 weeks. Release of Na⁺, K⁺ and NH₄ ⁺-N was similar from all combinations, but Ca²⁺, Mg²⁺ and PO₄ ^3--P release depended on the species. In Agrocybe gibberosa/Chaetomium globosum and Sphaerobolus stellatus/Chaetomium globosum combinations, there was evidence of interactions which suppressed the predicted rate of phosphate release, and in all the mixed species combinations there were interactions which increased the rate of fungal respiration above that of the more combative fungus in pure culture. ei]{gnR}{fnMerckx}