[Show abstract][Hide abstract] ABSTRACT: Plant defensins (PDF) are cysteine-rich peptides that are major actors in the innate immunity in plants. Besides their antifungal activity, some PDF such as Arabidopsis halleri PDF1.1b confer zinc tolerance in plants. Here we present i) an efficient protocol for the production of AhPDF1.1b by solid-phase peptide synthesis followed by controlled oxidative folding to obtain the highly pure native form of the defensin, and ii) the 3D NMR structure of AhPDF1.1b, the first 3D structure of plant defensin obtained with a synthetic peptide. Its fold is organized around the typical cysteine-stabilized α-helix β-sheet motif, and contains the γ-core motif involved in the antifungal activity of all plant defensins. Based on our structural analysis of AhPDF1 defensins combined with previous biological data for antifungal and zinc tolerance activities, we established the essential role of cis-Pro41 within the γ-core. In fact, the four consecutive residues [Val39-Phe40-Pro41-Ala42] are strictly conserved for plant defensins able to tolerate zinc. We hypothesized that structural and/or dynamic features of this sequence are related to the ability of the defensin to chelate zinc.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
BioMed Research International 06/2014; 2014:548254. DOI:10.1155/2014/548254 · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cadmium (Cd) is a non-essential heavy metal, which is classified as a "known human carcinogen" by the International Agency for Research on Cancer (IARC). Understanding the mechanisms controlling Cd distribution in planta is essential to develop phytoremediation approaches as well as for food safety. Unlike most other plants, tobacco (Nicotiana tabacum) plants translocate most of the Cd taken up from the soil, out of the roots and into the shoots, leading to high Cd accumulation in tobacco shoots. Two orthologs of the Arabidopsis thaliana HMA2 and HMA4 Zn and Cd ATPases that are responsible for zinc (Zn) and Cd translocation from roots to shoots were identified in tobacco and sequenced. These genes, named NtHMAα and NtHMAβ, were more highly expressed in roots than in shoots. NtHMAα was expressed in the vascular tissues of both roots and leaves as well as in anthers. No visual difference was observed between wild-type plants and plants in which the NtHMAα and NtHMAβ genes were either mutated or silenced. These mutants showed reduced Zn and Cd accumulation in shoots as well as increased Cd tolerance. When both NtHMA genes were silenced, plant development was altered and pollen germination was severely impaired due to Zn deficiency. Interestingly, seeds from these lines also showed decreased Zn concentration but increased iron (Fe) concentration.
[Show abstract][Hide abstract] ABSTRACT: Interactions between zinc (Zn) and phosphate (Pi) nutrition in plants have long been recognized, but little information is available on their molecular bases and biological significance. This work aimed at examining the effects of Zn deficiency on Pi accumulation in Arabidopsis thaliana and uncovering genes involved in the Zn-Pi synergy. Wild-type plants as well as mutants affected in Pi signalling and transport genes, namely the transcription factor PHR1, the E2-conjugase PHO2, and the Pi exporter PHO1, were examined. Zn deficiency caused an increase in shoot Pi content in the wild type as well as in the pho2 mutant, but not in the phr1 or pho1 mutants. This indicated that PHR1 and PHO1 participate in the coregulation of Zn and Pi homeostasis. Zn deprivation had a very limited effect on transcript levels of Pi-starvation-responsive genes such as AT4, IPS1, and microRNA399, or on of members of the high-affinity Pi transporter family PHT1. Interestingly, one of the PHO1 homologues, PHO1;H3, was upregulated in response to Zn deficiency. The expression pattern of PHO1 and PHO1;H3 were similar, both being expressed in cells of the root vascular cylinder and both localized to the Golgi when expressed transiently in tobacco cells. When grown in Zn-free medium, pho1;h3 mutant plants displayed higher Pi contents in the shoots than wild-type plants. This was, however, not observed in a pho1 pho1;h3 double mutant, suggesting that PHO1;H3 restricts root-to-shoot Pi transfer requiring PHO1 function for Pi homeostasis in response to Zn deficiency.
[Show abstract][Hide abstract] ABSTRACT: Cadmium (Cd) is a metal pollutant that accumulates in cultivated soils and has detrimental consequences in terms of food safety. Lettuce (Lactuca sativa) can characterised by having a high capacity to accumulate Cd in its tissues. An analysis of Cd tolerance and Cd accumulation was carried out using two varieties of lettuce (‘Divina’ and ‘Melina’). A wide range of CdCl2 concentrations was used (0.0, 0.1, 0.6, 3.0, and 15.0 µM CdCl2). The lowest concentration (0.1 µM CdCl2) stimula’ted growth, while the two highest concentrations resulted in a reduction of the biomass production. Cadmium concentrations were found to be twice as high in roots as in shoots. ‘Divina’ displayed lower concentrations of Cd than ‘Melina’ in nearly all treatments. A strong negative correlation was observed between Cd concentrations and Cd tolerance in the roots and shoots (R² > 0.87) of both ‘Melina’ and ‘Divina’. Lettuce grown in the presence of 15.0 µM CdCl2 had leaf Cd concentrations that were 100 fold higher than the legal maximum level for vegetable products marketed for human consumption, but showed no signs of dehydration, chlorosis, or necrosis. This result is an important alert for lettuce consumers and growers.
Journal of Horticultural Science and Biotechnology 11/2013; 88(6):783–789. · 0.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant defensins are recognized for their antifungal properties. However, a few type 1 defensins (PDF1s) were identified for their cellular zinc (Zn) tolerance properties after a study of the metal extremophile Arabidopsis halleri. In order to investigate whether different paralogues would display specialized functions, the A. halleri PDF1 family was characterized at the functional and genomic levels. Eleven PDF1s were isolated from A. halleri. Their ability to provide Zn tolerance in yeast cells, their activity against Fusarium oxysporum f. sp. melonii, and their level of expression in planta were compared with those of the seven A. thaliana PDF1s. The genomic organization of the PDF1 family was comparatively analysed within the Arabidopsis genus. AhPDF1s and AtPDF1s were able to confer Zn tolerance and AhPDF1s also displayed antifungal activity. PDF1 transcripts were constitutively more abundant in A. halleri than in A. thaliana. Within the Arabidopsis genus, the PDF1 family is evolutionarily dynamic, in terms of gain and loss of gene copy. Arabidopsis halleri PDF1s display no superior abilities to provide Zn tolerance. A constitutive increase in AhPDF1 transcript accumulation is proposed to be an evolutionary innovation co-opting the promiscuous PDF1 protein for its contribution to Zn tolerance in A. halleri.
New Phytologist 07/2013; 200(3). DOI:10.1111/nph.12396 · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Plants are adapted to soils in which the amounts of different nutrients vary widely, like Zn-deficient or Zn-contaminated soils. Exploring the molecular bases of plant adaptation to Zn-contaminated soils is important in determining strategies for phytoremediation. Here, we describe the mapping and characterization of a QTL for Zn tolerance in A. thaliana that underlies the natural variation of the root response to excess Zn. This physiological variation is controlled by different alleles of the AtFRD3 gene, which codes for a citrate transporter that uploads citrate into the xylem sap, hence playing a role in Fe homeostasis. In the Zn-sensitive accession Shahdara, the expression of AtFRD3 is drastically reduced and the protein encoded is unable to efflux citrate in vitro. Less Fe and Zn are found in Shahdara root exudates, and less Fe and Zn are translocated from root to shoot when Zn is in excess. We deduce that a fine-tuned Fe and Zn homeostasis is crucial for Zn tolerance in A. thaliana. Finally, as a range of alleles were identified, some rare, it was possible to define a sequence motif that is a putative metal-responsive cis-element and demonstrate that two amino acids are essential for the function of the FRD3 transporter.
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis thaliana NOK2 accession displays salt tolerance compared to more commonly known A. thaliana accessions, such as Col-0, but the basis of this phenotypic feature is unknown. This work was focused on determining whether salt tolerance in NOK2 plants is affected by calcium supplementation to the growth medium. A. thaliana seedlings were grown in pots containing a mixture of sand and peat under controlled conditions in a low-level Ca(NO3)2 medium supplemented with 0 or 50 mM NaCl with and without amendment with two higher levels of Ca(NO3)2. Calcium amendment was beneficial for salt-treated NOK2 plants, as shown by the increase in dry weight of NOK2 plants with and without NaCl, but had no impact on Col-0 biomass. Sodium accumulation decreased as a function of calcium amendment in NOK2, while Col-0 maintained its high Na levels under these conditions. Leaf K+ content, K+ uptake, and Ca content decreased in NOK2 and Col-0 plants growing in the low-level Ca medium when NaCl was added, but rose in leaves of both accessions with calcium amendment, although K remained low in both accessions in the absence of NaCl. K+/Na+ selectivity increased preferentially in NOK2 with increasing calcium in the presence of NaCl, but when Na was restricted and not under any conditions in Col-0. Preferential effects of calcium were not observed on the transcript accumulation of seven Na+, K+ or Ca2+ transport genes for either of the accessions, except for increased transcription of the CAX4 gene in NOK2 leaves at the highest calcium concentration used (5 mM). Leaf membrane leakage, which increased two-fold higher in Col-0 under salt application compared with the increase in NOK2, declined for both accessions in response to calcium supplementation, and in NOK2 this decline reached no salt levels when Ca2+ amendments were highest. Chlorophyll and carotenoid content dropped two-fold in Col-0 in response to salt, but were unchanged in NOK2 under these conditions. In contrast, leaf anthocyanins, which were normally tenfold higher in Col-0 than in NOK2 in the low-level Ca2+ medium, declined in Col-0 plants as a function of Ca2+ supplementation, but were maintained at low levels in NOK2 leaves regardless of salinity and calcium. In conclusion, NOK2 plants responded positively to calcium supplementation by improving biomass yield during salinity treatment, whereas this amendment only affected Col-0 by reducing its permeability and anthocyanin titre. K+/Na+ selectivity appeared to be an important characteristic of NOK2 response to calcium. The regulation of this response may involve the CAX4 Ca2+/H+ vacuolar transport gene, but does not appear to involve six other common ion transporters.
Acta Physiologiae Plantarum 03/2012; 34(2). DOI:10.1007/s11738-011-0840-7 · 1.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Zinc (Zn) biofortification is considered to be a valuable economical solution to ameliorate Zn malnutrition. Metal Tolerance Protein 1 (MTP1) based on its role in Zn accumulation is considered to be a potential candidate to be expressed in cereals for their Zn biofortification. However, oligomer formation has been shown to be possible for MTP1s which could be a limiting step for successfully manipulating their expression in cereals. In the present study, interaction between different AhMTP1s when present in the same cell was explored. AhMTP1-A2 that displays the highest ability to complement Zn hypersensitivity of zrc1 cot1 yeast mutant was co-expressed with other lesser competent AhMTP1s. Zn assays in yeast indicated the occurrence of an interaction between different AhMTP1s and AhMTP1-A2 could have a positive effect on the functioning of other co-expressed AhMTP1. Our results provide additional argument to use AhMTP1 for Zn biofortification of cereals.
International Journal of Agriculture and Biology 01/2012; 14(4). · 0.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant Zn/Cd/Pb/Co P1B-ATPases (HMAs) play different roles, among which are the control of metal transport from the roots to the shoot and/or from the cytoplasm into the cell vacuole. Transferring the knowledge acquired on HMAs from model species to HMAs from other species requires one to identify orthologues in these other species. Through an extensive screening of the public sequence databases, 96 plant P1B-ATPases showing orthology to any of the AtHMA1, AtHMA2, AtHMA3 or AtHMA4 isoforms were identified from 32 plant species belonging to 15 botanical families. The number of paralogues within a species varied greatly from species to species, even within a specific botanical family, suggesting that gene duplication events occurred after speciation. The phylogenetic tree gathering the Zn/Cd/Pb/Co P1B-ATPases was strongly structured according to the botanical family to which the sequences could be related to. In particular, no strict orthology relationship links the Brassicaceae HMAs to the non-Brassicaceae or the Poaceae ones. Recent data showed that the sole rice HMA characterised to date displays different functional properties from the Arabidopsis HMAs. Altogether, data suggest that it might be risky to directly transfer the knowledge acquired through the study of HMAs in model plant species to HMAs from other species.
[Show abstract][Hide abstract] ABSTRACT: • Apart from their antifungal role, plant defensins have recently been shown to be involved in abiotic stress tolerance or in inhibition of root growth when added in plant culture medium. We studied the subcellular localization of these proteins, which may account for these different roles. • Stable and transient expression of AhPDF1.1::GFP (green fluorescent protein) fusion proteins were analysed in yeast and plants. Functional tests established that the GFP tag did not alter the action of the defensin. Subcellular localization of AhPDF1.1 was characterized: by imaging AhPDF1.1::GFP together with organelle markers; and by immunolabelling AhPDF1.1 in Arabidopsis halleri and Arabidopsis thaliana leaves using a polyclonal serum. • All our independent approaches demonstrated that AhPDF1.1 is retained in intracellular compartments on the way to the lytic vacuole, instead of being addressed to the apoplasm. • These findings challenge the commonly accepted idea of secretion of defensins. The subcellular localization highlighted in this study could partly explain the dual role of plant defensins on plant cells and is of major importance to unravel the mechanisms of action of these proteins at the cellular level.
New Phytologist 06/2011; 192(1):140-50. DOI:10.1111/j.1469-8137.2011.03792.x · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This work reports the first characterization of the natural variation of Zn tolerance and accumulation in Arabidopsis thaliana. Root and shoot growth as well as Zn content were determined for 27 A. thaliana accessions grown in vitro in presence of Zn concentrations ranging from 1 to 250 µm. All traits varied by at least twofold and their broad sense heritability varied from 0.36 to 0.91. Primary and lateral root developments were differently affected by Zn in the different accessions. Remarkably, Zn was for the first time shown to be essential for the development of lateral roots. As a general rule, the different traits showed uncorrelated variations. In particular, variation in Zn tolerance was not linked to either root or shoot Zn contents. The only detectable relationship between different traits linked Zn sensitivity of roots to root-to-shoot Zn translocation but the correlation between variation of these traits was pretty low. This suggests that Zn translocation from root to shoots explains only a part of Zn tolerance. Our analysis opens the way to the characterization of genetic determinants controlling different Zn-related traits through the identification of particular accessions displaying contrasted phenotypes and representing excellent starting material to develop quantitative trait locus (QTL) studies.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Gene duplication is a major mechanism facilitating adaptation to changing environments. From recent genomic analyses, the acquisition of zinc hypertolerance and hyperaccumulation characters discriminating Arabidopsis halleri from its zinc sensitive/non-accumulator closest relatives Arabidopsis lyrata and Arabidopsis thaliana was proposed to rely on duplication of genes controlling zinc transport or zinc tolerance. Metal Tolerance Protein 1 (MTP1) is one of these genes. It encodes a Zn(2+)/H(+) antiporter involved in cytoplasmic zinc detoxification and thus in zinc tolerance. MTP1 was proposed to be triplicated in A. halleri, while it is present in single copy in A. thaliana and A. lyrata. Two of the three AhMTP1 paralogues were shown to co-segregate with zinc tolerance in a BC1 progeny from a cross between A. halleri and A. lyrata. In this work, the MTP1 family was characterized at both the genomic and functional levels in A. halleri. Five MTP1 paralogues were found to be present in A. halleri, AhMTP1-A1, -A2, -B, -C, and -D. Interestingly, one of the two newly identified AhMTP1 paralogues was not fixed at least in one A. halleri population. All MTP1s were expressed, but transcript accumulation of the paralogues co-segregating with zinc tolerance in the A. halleri X A. lyrata BC1 progeny was markedly higher than that of the other paralogues. All MTP1s displayed the ability to functionally complement a Saccharomyces cerevisiae zinc hypersensitive mutant. However, the paralogue showing the least complementation of the yeast mutant phenotype was one of the paralogues co-segregating with zinc tolerance. From our results, the hypothesis that pentaplication of MTP1 could be a major basis of the zinc tolerance character in A. halleri is strongly counter-balanced by the fact that members of the MTP1 family are likely to experience different evolutionary fates, some of which not concurring to increase zinc tolerance.