Genetic analysis of cadmium accumulation in lettuce (Lactuca sativa).

This work characterized mechanisms controlling cadmium (Cd) tolerance and accumulation in lettuce at both the physiological and genetic levels. These traits were evaluated in 18 Lactuca accessions representing a large genetic diversity. Cd tolerance and accumulation in roots and shoots as well as Cd translocation from roots to the shoot varied independently, and with a significant range of variation. Analyses of F1 progenies of crosses between cultivars with contrasted phenotypes showed that high tolerance to Cd, low Cd accumulation and low Cd root-shoot translocation were recessive traits. Results of analyses of F2 progenies of different crosses suggest that root Cd concentration and root-shoot Cd translocation were under a complex genetic determinism involving at least two loci. This work thus revealed that limiting both Cd accumulation and Cd root-shoot translocation in lettuce is possible and depends on recessive loci. Differences in the ability to accumulate Cd in roots in the long term could not be linked to differences in short-term 109Cd uptake into, or efflux from, roots. In contrast, the cultivar with the highest root-shoot Cd translocation was the same in the long term and in the short term, which suggests that this trait relies on processes that are implemented quickly (i.e. in less than three days) after the start of Cd exposure.

Over-expression of the bacterial phytase US417 in Arabidopsis reduces the concentration of phytic acid and reveals its involvement in the regulation of sulfate and phosphate homeostasis and signaling.

Phytic acid (PA) is the main phosphorus storage form in plant seeds. It is recognized as an anti-nutrient for humans and non-ruminant animals, as well as one of the major sources of phosphorus that contributes to eutrophication. Therefore, engineering plants with low PA content without affecting plant growth capacity has become a major focus in plant breeding. Nevertheless, lack of knowledge on the role of PA seed reserves in regulating plant growth and in maintaining ion homeostasis hinders such an agronomical application. In this context, we report here that the over-expression of the bacterial phytase PHY-US417 in Arabidopsis leads to a significant decrease in seed PA, without any effect on the seed germination potential. Interestingly, this over-expression also induced a higher remobilization of free iron during germination. Moreover, the PHY-over-expressor lines show an increase in inorganic phosphate and sulfate contents, and a higher biomass production after phosphate starvation. Finally, phosphate sensing was altered because of the changes in the expression of genes induced by phosphate starvation or involved in phosphate or sulfate transport. Together, these results show that the over-expression of PHY-US417 reduces PA concentration, and provide the first evidence for the involvement of PA in the regulation of sulfate and phosphate homeostasis and signaling.

Phosphate/zinc interaction analysis in two lettuce varieties reveals contrasting effects on biomass, photosynthesis, and dynamics of Pi transport.

Inorganic phosphate (Pi) and Zinc (Zn) are essential nutrients for normal plant growth. Interaction between these elements has been observed in many crop plants. Despite its agronomic importance, the biological significance and genetic basis of this interaction remain largely unknown. Here we examined the Pi/Zn interaction in two lettuce (Lactuca sativa) varieties, namely, "Paris Island Cos" and "Kordaat." The effects of variation in Pi and Zn supply were assessed on biomass and photosynthesis for each variety. Paris Island Cos displayed better growth and photosynthesis compared to Kordaat under all the conditions tested. Correlation analysis was performed to determine the interconnectivity between Pi and Zn intracellular contents in both varieties. Paris Island Cos showed a strong negative correlation between the accumulation levels of Pi and Zn in shoots and roots. However, no relation was observed for Kordaat. The increase of Zn concentration in the medium causes a decrease in dynamics of Pi transport in Paris Island Cos, but not in Kordaat plants. Taken together, results revealed a contrasting behavior between the two lettuce varieties in terms of the coregulation of Pi and Zn homeostasis and provided evidence in favor of a genetic basis for the interconnection of these two elements.

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

BACKGROUND: Cadmium (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. RESULTS: The 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. CONCLUSIONS: A 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.

The seed composition of Arabidopsis mutants for the group 3 sulfate transporters indicates a role in sulfate translocation within developing seeds.

Sulfate is required for the synthesis of sulfur-containing amino acids and numerous other compounds essential for the plant life cycle. The delivery of sulfate to seeds and its translocation between seed tissues is likely to require specific transporters. In Arabidopsis (Arabidopsis thaliana), the group 3 plasmalemma-predicted sulfate transporters (SULTR3) comprise five genes, all expressed in developing seeds, especially in the tissues surrounding the embryo. Here, we show that sulfur supply to seeds is unaffected by T-DNA insertions in the SULTR3 genes. However, remarkably, an increased accumulation of sulfate was found in mature seeds of four mutants out of five. In these mutant seeds, the ratio of sulfur in sulfate form versus total sulfur was significantly increased, accompanied by a reduction in free cysteine content, which varied depending on the gene inactivated. These results demonstrate a reduced capacity of the mutant seeds to metabolize sulfate and suggest that these transporters may be involved in sulfate translocation between seed compartments. This was further supported by sulfate measurements of the envelopes separated from the embryo of the sultr3;2 mutant seeds, which showed differences in sulfate partitioning compared with the wild type. A dissection of the seed proteome of the sultr3 mutants revealed protein changes characteristic of a sulfur-stress response, supporting a role for these transporters in providing sulfate to the embryo. The mutants were affected in 12S globulin accumulation, demonstrating the importance of intraseed sulfate transport for the synthesis and maturation of embryo proteins. Metabolic adjustments were also revealed, some of which could release sulfur from glucosinolates.

Identification of three relationships linking cadmium accumulation to cadmium tolerance and zinc and citrate accumulation in lettuce.

Lettuce (Lactuca sativa) is a plant species that shows high accumulation of cadmium, a toxic heavy metal. Lettuce is therefore a good model both for identifying determinants controlling cadmium accumulation in plant tissues and for developing breeding strategies aimed at limiting cadmium accumulation in edible tissues. In this work, 14-day-old plants from three lettuce varieties were grown for 8 days on media supplemented with cadmium concentrations ranging from 0 to 50 microM. Growth, as well as Cd(2+), Zn(2+), K(+), Ca(2+), NO(3)(-), SO(4)(2-), Cl(-), phosphate, malate and citrate root an shoot contents were analyzed. The three lettuce varieties Paris Island Cos, Red Salad Bowl and Kordaat displayed differential abilities to accumulate cadmium in roots and shoots, Paris Island Cos displaying the lowest cadmium content and Kordaat the highest. From the global analysis of the three varieties, three main trends were identified. First, a common negative correlation linked cadmium tissue content and relative dry weight reduction in response to cadmium treatments in the three varieties. Second, increasing cadmium concentration in the culture medium resulted in a parallel increase in zinc tissue content in all lettuce varieties. A common strong positive correlation between cadmium and zinc contents was observed for all varieties. This suggested that systems enabling zinc and cadmium transport were induced by cadmium. Finally, the cadmium treatments had a contrasting effect on anion contents in tissues. Interestingly, citrate content in shoots was correlated with cadmium translocation from roots to shoots, suggesting that citrate might play a role in cadmium transport in the xylem vessels. Altogether, these results shed light on three main strategies developed by lettuce to cope with cadmium, which could help to develop breeding strategies aimed at limiting cadmium accumulation in lettuce.

Sultr4;1 mutant seeds of Arabidopsis have an enhanced sulphate content and modified proteome suggesting metabolic adaptations to altered sulphate compartmentalization.

BACKGROUND: Sulphur is an essential macronutrient needed for the synthesis of many cellular components. Sulphur containing amino acids and stress response-related compounds, such as glutathione, are derived from reduction of root-absorbed sulphate. Sulphate distribution in cell compartments necessitates specific transport systems. The low-affinity sulphate transporters SULTR4;1 and SULTR4;2 have been localized to the vacuolar membrane, where they may facilitate sulphate efflux from the vacuole. RESULTS: In the present study, we demonstrated that the Sultr4;1 gene is expressed in developing Arabidopsis seeds to a level over 10-fold higher than the Sultr4;2 gene. A characterization of dry mature seeds from a Sultr4;1 T-DNA mutant revealed a higher sulphate content, implying a function for this transporter in developing seeds. A fine dissection of the Sultr4;1 seed proteome identified 29 spots whose abundance varied compared to wild-type. Specific metabolic features characteristic of an adaptive response were revealed, such as an up-accumulation of various proteins involved in sugar metabolism and in detoxification processes. CONCLUSIONS: This study revealed a role for SULTR4;1 in determining sulphate content of mature Arabidopsis seeds. Moreover, the adaptive response of sultr4;1 mutant seeds as revealed by proteomics suggests a function of SULTR4;1 in redox homeostasis, a mechanism that has to be tightly controlled during development of orthodox seeds.

Regulation of sulfate uptake and assimilation--the same or not the same?

Plant take up the essential nutrient sulfur as sulfate from the soil, reduce it, and assimilate into bioorganic compounds, with cysteine being the first product. Both sulfate uptake and assimilation are highly regulated by the demand for the reduced sulfur, by availability of nutrients, and by environmental conditions. In the last decade, great progress has been achieved in dissecting the regulation of sulfur metabolism. Sulfate uptake and reduction of activated sulfate, adenosine 5'-phosphosulfate (APS), to sulfite by APS reductase appear to be the key regulatory steps. Here, we review the current knowledge on regulation of these processes, with special attention given to similarities and differences.

Root uptake regulation: a central process for NPS homeostasis in plants.

Homeostasis of nitrogen, phosphorus and sulfur in growing plants requires a sustained intake of these elements into root cells. Under most situations, the adjustment of root N, P or S acquisition to the nutrient demand of the plant is hampered by the limiting and fluctuating availability of these elements in the soil. To cope with this constraint, higher plants modulate their root uptake capacity to compensate for the changes in external concentrations of the N, P or S sources. This adaptive response relies on both physiological and morphological changes in the root system, triggered by nutrient-specific sensing and signalling pathways. The underlying molecular mechanisms now begin to be elucidated. Key root membrane transport proteins have been identified, as well as molecular regulators that control root uptake systems or root system architecture in response to N, P or S availability. Significant but yet poorly understood interactions with carbon or hormone signalling have been unravelled, opening new routes for integrating the mechanisms of nutrient homeostasis into the whole plant.

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.

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 (35)S-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.

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 microm). 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.

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.

Cloning of two contrasting high-affinity sulfate transporters from tomato induced by low sulfate and infection by the vascular pathogen Verticillium dahliae.

Two cDNAs, LeST1-1 (AF347613) and LeST1-2 (AF347614), encoding sulfate transporters have been cloned from tomato (Lycopersicon esculentum Mill.) by reverse transcription-polymerase chain reaction and their expression characterised. Sharing 76% identity at the amino acid level, the transporters are phylogenetically associated with the Group-1, high-affinity plant sulfate transporters. Both were shown to have high affinity for sulfate by uptake kinetic analysis using a yeast (Saccharomyces cerevisiae) sulfate-transporter mutant. Km values of 11.5 microM and 9.8 microM were calculated for LeST1-1 and LeST1-2, respectively, the same order of magnitude as those previously reported for several other Group-1 high-affinity sulfate transporters. In situ hybridisation to S-deficient tomato roots showed LeST1-1 to be expressed in the epidermis and pericycle, whereas LeST1-2 expression was located to the epidermis only. Northern analysis shows that the mRNA abundances of both LeST1-1 and LeST1-2 are upregulated in the root in response to sulfate deprivation. LeST1-1 is specifically expressed in root tissue, a characteristic of Group-1 sulfate transporters. LeST1-2, however, was also detected in tomato leaves and stems and is upregulated and expressed to a similar extent in these tissues under conditions of sulfate deprivation. Induction of LeST1-2 expression was also observed in the vascular tissues of a resistant line of tomato infected with the vascular wilt pathogen Verticillium dahliae.