Pierre Fourcroy

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.

Differential Regulation of the Expression of Two High-Affinity Sulfate Transporters, SULTR1.1 and SULTR1.2, in Arabidopsis

The molecular mechanisms regulating the initial uptake of inorganic sulfate in plants are still largely unknown. The current model for the regulation of sulfate uptake and assimilation attributes positive and negative regulatory roles to O-acetyl-serine (O-acetyl-Ser) and glutathione, respectively. This model seems to suffer from exceptions and it has not yet been clearly validated whether intracellular O-acetyl-Ser and glutathione levels have impacts on regulation. The transcript level of the two high-affinity sulfate transporters SULTR1.1 and SULTR1.2 responsible for sulfate uptake from the soil solution was compared to the intracellular contents of O-acetyl-Ser, glutathione, and sulfate in roots of plants submitted to a wide diversity of experimental conditions. SULTR1.1 and SULTR1.2 were differentially expressed and neither of the genes was regulated in accordance with the current model. The SULTR1.1 transcript level was mainly altered in response to the sulfur-related treatments. Split-root experiments show that the expression of SULTR1.1 is locally regulated in response to sulfate starvation. In contrast, accumulation of SULTR1.2 transcripts appeared to be mainly related to metabolic demand and is controlled by photoperiod. On the basis of the new molecular insights provided in this study, we suggest that the expression of the two transporters depends on different regulatory networks. We hypothesize that interplay between SULTR1.1 and SULTR1.2 transporters could be an important mechanism to regulate sulfate content in the roots.

Characterization of a Selenate-Resistant Arabidopsis Mutant. Root Growth as a Potential Target for Selenate Toxicity

Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced 35S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.

Iron triggers a rapid induction of ascorbate peroxidase gene expression in Brassica napus

In plants, only ferritin gene expression has been reported to be iron‐dependent. Here it is demonstrated that an iron overload of Brassica napus seedlings causes a large and rapid accumulation of ascorbate peroxidase transcripts, a plant‐specific hydrogen peroxide‐scavenging enzyme. This result documents a novel link between iron metabolism and oxidative stress. The ascorbate peroxidase mRNA abundance was not modified by reducing agents like N‐acetyl cysteine, glutathione and ascorbate or by pro‐oxidants such as hydrogen peroxide or diamide. Furthermore, the iron‐induced ascorbate peroxidase mRNA accumulation was not antagonized by N‐acetyl cysteine. Abscisic acid had no effect on the ascorbate peroxidase gene expression. Taken together these results suggest that iron‐mediated expression of ascorbate peroxidase gene occurs through a signal transduction pathway apparently different from those already described for plant genes responsive to oxidative stress.

Iron-Regulated Expression of a Cytosolic Ascorbate Peroxidase Encoded by the APX1 Gene in Arabidopsis Seedlings

Iron (Fe) is an essential element for living organisms. However, under aerobic conditions, its use is complicated because of its high insolubility and its potential toxicity through reactivity with reduced forms of oxygen. In plants, Fe overload can lead to intracellular concentrations beyond the storage and detoxification capacities of cells. Such a displacement toward a pro-oxidant state can activate antioxidant defenses, including Fe-mediated expression of ascorbate peroxidase genes. In this work, we demonstrate that Fe overload specifically induces the AtAPX1 gene encoding a cytosolic ascorbate peroxidase in Arabidopsis leaves. The strong constitutive expression of the AtAPX1 gene in roots is unaffected by Fe and depends on the first 5′-untranslated region intron. Presence of an AtAPX1 expressed sequence tag in the Arabidopsis database, longer in its 5′ region than what could be predicted from the published AtAPX1transcription initiation site, leads to define a new transcription initiation region for this gene. A minimal promoter sequence enabling Fe-induced expression of the AtAPX1 gene is defined by following expression of various AtAPX1::β-glucuronidase constructs in transformed Arabidopsis plantlets. This 118-bp minimal promoter sequence contains an Fe-dependent regulatory sequence-like cis-element known to be necessary for maize (Zea mays) and Arabidopsis ferritin gene derepression in response to Fe overload. Site-directed mutagenesis of this element within the AtAPX1 promoter sequence does not abolish the Fe-dependent activation of a reporter gene, indicating that it is likely not involved in the Fe-regulated expression of the AtAPX1 gene.

Phytochrome control of gene expression in radish seedlings II. Far-red light mediated appearance of the ribulose 1,5-bisphosphate carboxylase and the mRNA for its small subunit

Abstract The effect of far-red light on the appearance of the radish ribulose 1,5-bisphosphate carboxylase, its subunits and the mRNA for the small subunit has been studied. The immunological analysis of the accumulation of holoenzyme and small subunit showed that there was no pool of free small subunit either in the etiolated seedlings or in the far-red light illuminated seedlings. The precursor to the small subunit has been identified by immunoprecipitation of the in vitro translation products directed by poly(A)-containing RNA of radish cotyledons. The irradiation of radish cotyledons with far-red light led to the apparent increase of the level of the translatable mRNA for the small subunit.

Phytochrome control of gene expression in radish seedlings I. Far-red light mediated stimulation of polyribosome formation and appearance of translatable mRNAs

Illumination of radish seedlings with far-red light brings about an increase of the polyribosome content of cotyledons and hypocotyls. Poly(A)-containing RNA from cotyledons of both etiolated and far-red light-treated radish seedlings were isolated and translated in a rabbit reticulocyte lysate. The comparison of translation products showed that the relative levels of translatable mRNA coding for six abundant polypeptides were higher in the extracts of irradiated seedlings. The comparison of mRNA populations was also investigated with respect to organ specificity, polyribosomal localization and inhibition with the cap analoge, 7-methylguanosine 5′-phosphate (pm7G).

Calmodulin antagonists inhibit the phytochrome-induced appearance of two nuclear encoded transcripts in radish cotyledons.

The effects of two calmodulin antagonists on the phytochrome‐mediated appearance of two nuclear encoded transcripts in radish cotyledons have been investigated. The extent of inhibition of transcript accumulation was dependent of the time ellapsed between the administration of trifluoperazine and the light stimulus. When 1 mM trifluoperazine was administered to the seedlings 8 h before red light irradiation, the inhibition of transcript accumulation was up to 62% for the chlorophyll binding protein mRNA and 56% for the ribulose 1,5‐bisphosphate carboxylase small subunit mRNA. Similar results were obtained with W‐7 (0.1 mM).