Pierre Berthomieu

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.

Structural and Functional Analysis of the C-terminal STAS (Sulfate Transporter and Anti-sigma Antagonist) Domain of the Arabidopsis thaliana Sulfate Transporter SULTR1.2

The C-terminal region of sulfate transporters from plants and animals belonging to the SLC26 family members shares a weak but significant similarity with the Bacillus sp. anti-anti-sigma protein SpoIIAA, thus defining the STAS domain (sulfate transporter and anti-sigma antagonist). The present study is a structure/function analysis of the STAS domain of SULTR1.2, an Arabidopsis thaliana sulfate transporter. A three-dimensional model of the SULTR1.2 STAS domain was built which indicated that it shares the SpoIIAA folds. Moreover, the phosphorylation site, which is necessary for SpoIIAA activity, is conserved in the SULTR1.2 STAS domain. The model was used to direct mutagenesis studies using a yeast mutant defective for sulfate transport. Truncation of the whole SULTR1.2 STAS domain resulted in the loss of sulfate transport function. Analyses of small deletions and mutations showed that the C-terminal tail of the SULTR1.2 STAS domain and particularly two cysteine residues plays an important role in sulfate transport by SULTR1.2. All the substitutions made at the putative phosphorylation site Thr-587 led to a complete loss of the sulfate transport function of SULTR1.2. The reduction or suppression of sulfate transport of the SULTR1.2 mutants in yeast was not due to an incorrect targeting to the plasma membrane. Both our three-dimensional modeling and mutational analyses strengthen the hypothesis that the SULTR1.2 STAS domain is involved in protein-protein interactions that could control sulfate transport.

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.

Unequal functional redundancy between the two Arabidopsis thaliana high‐affinity sulphate transporters SULTR1;1 and SULTR1;2

* In Arabidopsis, SULTR1;1 and SULTR1;2 are two genes proposed to be involved in high-affinity sulphate uptake from the soil solution. We address here the specific issue of their functional redundancy for the uptake of sulphate and for the accumulation of its toxic analogue selenate with regard to plant growth and selenate tolerance. * Using the complete set of genotypes, including the wild-type, each one of the single sultr1;1 and sultr1;2 mutants and the resulting double sultr1;1-sultr1;2 mutant, we performed a detailed phenotypic analysis of root length, shoot biomass, sulphate uptake, sulphate and selenate accumulation and selenate tolerance. * The results all ordered the four different genotypes according to the same functional hierarchy. Wild-type and sultr1;1 mutant plants displayed similar phenotypes. By contrast, sultr1;1-sultr1;2 double-mutant plants showed the most extreme phenotype and the sultr1;2 mutant displayed intermediate performances. Additionally, the degree of selenate tolerance was directly related to the seedling selenate content according to a single sigmoid regression curve common to all the genotypes. * The SULTR1;1 and SULTR1;2 genes display unequal functional redundancy, which leaves open for SULTR1;1 the possibility of displaying an additional function besides its role in sulphate membrane transport.

Direct amplification of plant genomic DNA from leaf and root pieces using PCR

The screening of transformed plants for integration of foreign DNA is a relatively timeconsuming procedure which involves genomic DNA preparation [ 1 ] and Southern blot analysis of these preparations. In order to speed up this process, we have developed a one-step procedure based on PCR (polymerase chain reaction) technology. This allowed us to directly amplify a genomic target (a resident gene or an inserted foreign DNA) using small leaf or root pieces placed in the PCR reaction mixture. Blue or yellow tips were used to punch plant leaves and roots and to deposit the resulting plant piece in a 100/A PCR reaction mixture kept at 4 °C and made up with 200/~M of each dNTP, 30 pmol of each primer, 2.5 units of Taq polymerase (Promega) in PCR buffer (50 mM KC1, 10mM Tris-HC1 pH 9.0 at 25 °C, 1.5mM MgC12, 0.01~o (w/v) gelatin, 0.1~o (v/v) Triton X100). The mixture was then overlaid with paraffin oil and submitted to denaturation for 5 min at 94 °C in a Perkin-Elmer Cetus thermal cycler and subsequently to 30 cycles of amplification (1 min at 94 °C, 2 min at 60 °C, 2 min at 72 °C). After that, the PCR reaction was directly loaded onto a 1.5 ~o agarose gel for electrophoretic analysis. To check the efficiency and the reliability of this method we first amplified, using leaf pieces, a resident genomic target, namely the first intron of the two homeologous nitrate reductase genes in tobacco [2]. Using a pair of primers flanking this intron we were able to obtain two bands of the expected size corresponding to the two different genes (Fig. 1). As a positive control for PCR am-

sas1, an Arabidopsis Mutant Overaccumulating Sodium in the Shoot, Shows Deficiency in the Control of the Root Radial Transport of Sodium

A recessive mutation of Arabidopsis designated sas1 (for sodium overaccumulation in shoot) that was mapped to the bottom of chromosome III resulted in a two- to sevenfold overaccumulation of Na+ in shoots compared with wild-type plants. sas1 is a pleiotropic mutation that also caused severe growth reduction. The impact of NaCl stress on growth was similar for sas1 and wild-type plants; however, with regard to survival, sas1 plants displayed increased sensitivity to NaCl and LiCl treatments compared with wild-type plants. sas1 mutants overaccumulated Na+ and its toxic structural analog Li+, but not K+, Mg2+, or Ca2+. Sodium accumulated preferentially over K+ in a similar manner for sas1 and wild-type plants. Sodium overaccumulation occurred in all of the aerial organs of intact sas1 plants but not in roots. Sodium-treated leaf fragments or calli displayed similar Na+ accumulation levels for sas1 and wild-type tissues. This suggested that the sas1 mutation impaired Na+ long-distance transport from roots to shoots. The transpiration stream was similar in sas1 and wild-type plants, whereas the Na+ concentration in the xylem sap of sas1 plants was 5.5-fold higher than that of wild-type plants. These results suggest that the sas1 mutation disrupts control of the radial transport of Na+ from the soil solution to the xylem vessels.

Coordination between zinc and phosphate homeostasis involves the transcription factor PHR1, the phosphate exporter PHO1, and its homologue PHO1;H3 in Arabidopsis

Summary Phosphate overaccumulates in shoots in response to Zn deprivation. Results shown in this article suggest key roles of PHR1 and PHO1 and a counteractive function of PHO1;H3 in controlling root-to-shoot phosphate translocation in Arabidopsis.

Routine transformation of rapid cycling cabbage (Brassica oleracea) : molecular evidence for regeneration of chimeras

Abstract We have developed a transformation procedure using both disarmed and wild-type Agrobacterium tumefaciens strains for a rapid-cycling cabbage genotype ( Brassica oleracea var. capitata ). This method is based on the fact that the wild-type A. tumefaciens strain (82.139) can induce shooty tumors in rapid-cycling cabbage. No special regeneration medium was required and no selection pressure was exerted at any stage of the transformation procedure; the transformants were identified by screening for β-glucuronidase (GUS) activity with a histological assay. Southern analyses ascertained that the GUS-expressing plants contained the T-DNA carried by the disarmed strain but not the T-DNA of the wild-type A. tumefaciens strain. One transgenic plant was obtained for an average of 25 plants inoculated. Southern analysis showed that most of the transgenic plants, under these non-selective conditions, proved to be chimeric. Regeneration was established to be of pluricellular origin. The transgenic plants transmitted their T-DNA inserts to the progeny.

Phosphate and zinc transport and signalling in plants: toward a better understanding of their homeostasis interaction

Inorganic phosphate (Pi) and zinc (Zn) are two essential nutrients for plant growth. In soils, these two minerals are either present in low amounts or are poorly available to plants. Consequently, worldwide agriculture has become dependent on external sources of Pi and Zn fertilizers to increase crop yields. However, this strategy is neither economically nor ecologically sustainable in the long term, particularly for Pi, which is a non-renewable resource. To date, research has emphasized the analysis of mineral nutrition considering each nutrient individually, and showed that Pi and Zn homeostasis is highly regulated in a complex process. Interestingly, numerous observations point to an unexpected interconnection between the homeostasis of the two nutrients. Nevertheless, despite their fundamental importance, the molecular bases and biological significance of these interactions remain largely unknown. Such interconnections can account for shortcomings of current agronomic models that typically focus on improving the assimilation of individual elements. Here, current knowledge on the regulation of the transport and signalling of Pi and Zn individually is reviewed, and then insights are provided on the recent progress made towards a better understanding of the Zn-Pi homeostasis interaction in plants.

Comparative effect of potassium on K and Na uptake and transport in two accessions of Arabidopsis thaliana during salinity stress

Potassium-sodium interaction was compared in two natural accessions of Arabidopsis thaliana, Columbia-0 and NOK2. Seedlings were grown in the presence of 0 or 50 mM NaCl and 0.1; 0.625 or 2.5 mM K(+). At the lowest K(+) concentration, salt treatment inhibited both K(+) uptake and growth. Increasing the K(+) availability did not modified salt response in Columbia-0, but restored nearly normal net K(+) uptake in NaCl condition and alleviated NaCl growth reduction in NOK2. The effect of K(+) and NaCl on transcript level of several K(+) and Na(+) transporters in both shoots and roots was assessed using semi-quantitative RT-PCR. The mRNA abundance of the NHX1 and SOS1 Na(+)/H(+) antiporters was significantly increased by 50 mM NaCl in the two accessions. NHX1, which is responsible for Na(+) sequestration into vacuoles, was more up-regulated in NOK2 leaves than in Columbia-0s in NaCl stress condition. AKT1, which is the major channel involved in K(+) absorption, was down-regulated in salt stress condition, but was not responding to K(+) treatments. Only in NOK2, SKOR and AKT2, which respectively control xylem and phloem K(+) transport, were markedly up-regulated by 2.5 mM K(+) in both roots and shoots, independently of NaCl. Phenotypic and gene expression analyses suggest that the relative salt tolerance of NOK2 is mainly due to a high ability to sequester Na(+) in the vacuole and to take up and transport K(+). Up-regulation of SKOR and AKT2 by K(+), and of NHX1 by NaCl could participate in determining this phenotype.