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Keywords:

  • YSL;
  • iron;
  • nicotianamine;
  • transport;
  • seed;
  • circulation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The Arabidopsis Yellow Stripe 1-Like (YSL) proteins have been identified by homology with the maize (Zea mays) Yellow Stripe 1 (YS1) transporter which is responsible for iron–phytosiderophore (PS) uptake by roots in response to iron shortage. Although dicotyledonous plants do not synthesize PS, they do synthesize the PS precursor nicotianamine, a strong metal chelator essential for maintenance of iron homeostasis and copper translocation. Furthermore, ZmYS1 and the rice (Oryza sativa) protein OsYSL2 have metal-nicotianamine transport activities in heterologous expression systems. In this work, we have characterized the function of AtYSL1 in planta. Two insertional loss-of-function ysl1 mutants of Arabidopsis were found to exhibit increased nicotianamine accumulation in shoots. More importantly, seeds of both ysl1 knockouts contained less iron and nicotianamine than wild-type seeds, even when produced by plants grown in the presence of an excess of iron. This phenotype could be reverted by expressing the wild-type AtYSL1 gene in ysl1 plants. ysl1 seeds germinated slowly, but this defect was rescued by an iron supply. AtYSL1 was expressed in the xylem parenchyma of leaves, where it was upregulated in response to iron excess, as well as in pollen and in young silique parts. This pattern is consistent with long-distance circulation of iron and nicotianamine and their delivery to the seed. Taken together, our work provides strong physiological evidence that iron and nicotianamine levels in seeds rely in part on AtYSL1 function.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Iron (Fe) is an essential element for living organisms, and plants play a major role in its entry into the food chain. Because Fe bioavailability is often low in soils, plants have evolved efficient uptake strategies that can be classified into two types (Curie and Briat, 2003; Römheld and Marschner, 1986). Non-grass plants have adopted the so-called strategy I, in which Fe(II) transport is coupled to a Fe(III)-chelate reduction step. This strategy is sensitive to alkaline pH and is therefore less efficient in calcareous soils, which represent up to 30% of arable land world-wide. Molecular components of this uptake strategy have been characterized in Arabidopsis. The plasmalemma root ferric-chelate reductase FRO2 reduces soil Fe(III) (Robinson et al., 1999) and provides Fe(II) for IRT1, a major metal transporter that takes up Fe(II) into the root epidermis (Eide et al., 1996; Henriques et al., 2002; Varotto et al., 2002; Vert et al., 2002). The FRO2 and IRT1 genes are co-regulated in response to plant Fe status at both the transcriptional and the post-transcriptional levels, through complex pathways integrating signals such as local Fe concentration and uncharacterized long-distance signaling molecules (Connolly et al., 2002, 2003; Eide et al., 1996; Robinson et al., 1999; Vert et al., 2003). Grasses, or strategy II plants, such as maize (Zea mays), are less susceptible to chlorosis on calcareous soils because their uptake mechanism relies on the efficient chelation/solubilization of Fe(III). In response to Fe deficiency, grasses synthesize non-proteinogenic amino acids of the mugineic acid family, derived from S-adenosyl methionine and commonly called phytosiderophores (PS; Takagi et al., 1984). PS are secreted in the rhizosphere where they chelate various metals, including Fe(III), with a very strong affinity. Fe(III)–PS complexes are then taken up into the root by a specific transporter encoded by the Yellow Stripe 1 (YS1) gene in maize (Curie et al., 2001; von Wiren et al., 1994). Expression of ZmYS1 in heterologous systems has shown that YS1 functions as a proton-coupled symporter for phytosiderophore-chelated metals (Curie et al., 2001; Roberts et al., 2004; Schaaf et al., 2004).

Distribution of Fe to the various plant organs involves its long-distance transport through the sap. In the xylem, Fe is thought to be mainly chelated to organic acids such as citric or malic acid. In the phloem, which supplies micronutrients to roots and developing organs, the transport of Fe causes problems because of the high pH (between seven and eight) of this compartment and the propensity of Fe ions to precipitate in alkaline solution. In Ricinus communisseedlings, a binding partner of Fe(III) has been identified as a small protein of the late embryogenesis abundant (LEA) family, called the iron transport protein (ITP; Krüger et al., 2002). Nicotianamine (NA), a strong chelator of transition metals (von Wiren et al., 1999), is likely to chelate Fe(II) in the phloem as Fe(II)–NA is very stable at pH 7 or above and as NA is abundant in phloem sap. NA is a precursor and a structural analog of PS, which is present in both strategy I and strategy II plants. The role of NA in Fe homeostasis is illustrated by the numerous Fe metabolism defects presented by the tomato (Lycopersicon esculentum) NA-less mutant chloronerva, which is chlorotic despite the presence of a high concentration of Fe in its leaves and constitutive activity of its root Fe uptake system, and which fails to flower if not supplied with exogenous NA (Higuchi et al., 1996; Ling et al., 1999; Stephan et al., 1996). In addition, a tobacco plant (Nicotiana tabacum) deficient in NA was engineered via overproduction of the barley (Hordeum vulgare) nicotianamine amino transferase (NAAT) enzyme which consumes NA (Takahashi et al., 2003). This naat plant presents pleiotropic phenotypes, including interveinal chlorosis of young leaves, abnormally shaped and mostly sterile flowers, and rare and incompletely developed seeds, all of these defects being related to a shortage in transition metal and more specifically Fe delivery to plant organs.

Schaaf et al. (2004) have shown that, in addition to metal–PS chelates, maize YS1 can transport metals chelated to NA in heterologous expression systems. As eight YS1-like (YSL) gene sequences sharing a high level of homology with ZmYS1 were found in Arabidopsis (Curie et al., 2001), we postulated that YSL proteins may transport metal–NA chelates in strategy I plants.

Several laboratories have recently reported work on Arabidopsis and rice YSL2 (DiDonato et al., 2004; Koike et al., 2004; Schaaf et al., 2005). In Arabidopsis, DiDonato et al. (2004) showed that expression of AtYSL2 is able to restore growth of the fet3fet4 Fe uptake-defective yeast mutant, specifically when Fe is provided as Fe(II)–NA but not as Fe(III)–NA. These authors therefore concluded that AtYSL2 is a Fe(II)–NA transporter in planta. We have obtained contradictory results, as we did not observe any Fe–NA-dependent complementation of the fet3fet4 mutant by AtYSL2, and furthermore established that DiDonato et al.'s growth restoration was independent of Fe or NA supply (Schaaf et al., 2005). AtYSL2 also failed to mediate Fe(II)–NA-inducible currents in Xenopus laevisoocytes (Schaaf et al., 2005). These two reports reached the same conclusions regarding the expression of AtYSL2 in the vasculature, at the level of xylem-associated cells in roots, and also regarding AtYSL2 downregulation in response to Fe deficiency. We have additionally observed that AtYSL2 expression strongly decreases upon zinc starvation (Schaaf et al., 2005). In transgenic plants expressing the AtYSL2::GFP fusion protein, fluorescence was observed on the lateral sides of the xylem parenchyma plasma membrane (DiDonato et al., 2004). This prompted the authors to propose a role for AtYSL2 in the lateral movement of metals from the vasculature. In rice, expression of OsYSL2, one of 18 putative OsYSL genes in this species, is upregulated by Fe deficiency in the leaf, where it is particularly strong in the phloem, and is also observed in developing seeds (Koike et al., 2004). Based on OsYSL2 localization in the plasma membrane and on electrophysiological experiments in X. laevis oocytes, the authors have proposed that OsYSL2 may be required for long-distance transport of Fe(II)–NA and Mn(II)–NA through the phloem and for their loading into the grain.

In this study, we show that a functional AtYSL1 gene is required to produce seeds containing wild-type levels of Fe and NA, and suggest that AtYSL1 could contribute to the long-distance transport of these compounds via the xylem. AtYSL1 was expressed in the xylem parenchyma of the leaf, as well as in various parts of the developing flower and silique. AtYSL1 expression increased in response to an Fe excess. Knocking out AtYSL1 resulted in disturbances in the levels of NA in leaves and seeds, a decrease in the amount of Fe in seeds, and a slow germination rate. An increased Fe supply rescued germination but did not restore wild-type levels of Fe or NA in mutant seeds, which strongly suggests that at least part of Fe feeding into seeds requires the simultaneous mobilization of NA.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Phylogeny and structural organization of AtYSL1

The Arabidopsis genome contains eight YSL gene sequences encoding putative transmembrane proteins with high homology to ZmYS1, the maize founding member of the family (Curie et al., 2001). Eighteen YSL genes have also been reported in rice, and we found a nineteenth member of the family (Os1g61390) in the databases (Figure 1). AtYSL1, AtYSL2 and AtYSL3 are most similar to ZmYS1 (around 60% similarity) and together form a subfamily among the YSL proteins that includes OsYSL2 as well as three other rice homologs (DiDonato et al., 2004; Koike et al., 2004; Schaaf et al., 2005) (Figure 1). The YSL proteins are related to the oligopeptide transporter (OPT) family (Yen et al., 2001), but quite distantly as they only share an average of 10% similarity and do not contain the OPT consensus domain (Koh et al., 2002). The open reading frame of AtYSL1 was amplified from reverse-transcribed Arabidopsis total RNA and cloned into a pBluescript vector. It encodes a 673 amino acid-long protein containing 12–14 predicted transmembrane domains and presenting an N-terminal domain predicted to be cytoplasmic (http://psort.nibb.ac.jp).

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Figure 1. Phylogenetic tree of proteins of the YSL family from Arabidopsis thaliana (AtYSL1-8), Zea mays (ZmYS1) and Oryza sativa (OsYSL2 and Os accession numbers) generated using the phylogenetic tree printer Phylodendron (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html).

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AtYSL1 expression depends on Fe status

The hypothesis that the YSL proteins are involved in metal mobilization prompted us to analyze the pattern of expression of the AtYSL1 gene in response to metal content variations, with the hope of obtaining an indication on the physiological role of AtYSL1. Five-week-old hydroponically grown Arabidopsis plants were transferred to medium lacking or not lacking one of the metal ions Fe, manganese (Mn), zinc (Zn) or copper (Cu) and grown for three additional days. Total RNA extracted from these plants was blotted and hybridized with an AtYSL1-specific probe (see Experimental procedures). Compared with control conditions, we observed no change in the amount of AtYSL1 mRNA in Mn, Zn or Cu deficiency (data not shown). Upon Fe-deficiency treatment, however, accumulation of AtYSL1 transcripts was slightly but significantly reduced (data not shown). This prompted us to test whether AtYSL1 expression is inducible by an Fe overload. Six-week-old plants were therefore treated with 500 μm Fe following a 5-day period of Fe starvation to promote the Fe overload response (Lobreaux et al., 1992). The kinetic of accumulation of AtYSL1 mRNA was assayed by Northern blot in roots and shoots separately [Figure 2(a,b)]. Expression of the ferritin-encoding gene AtFER1, which is specifically induced by an Fe excess, was used as a control for the Fe treatment. AtFER1 mRNA accumulation was detected 6 h after transfer to Fe excess conditions in both roots and shoots and decreased within 12–24 h of treatment. AtYSL1 mRNA was only detected in shoots where, although detectable in control growth conditions (0 h), its amount increased from 6 h to reach a maximum at 12 h post-treatment, like AtFER1. However, unlike AtFER1, expression of which decreased after 12 h in shoots, AtYSL1 gene expression stayed on over the next 3 days of treatment [Figure 2(b)]. Moreover, the induction factor calculated for AtYSL1 was 10 times smaller (2.5-fold) than that for AtFER1 (25-fold). We concluded that the AtYSL1 gene is specifically expressed in shoots and rapidly although moderately induced by Fe excess. Although Cu is a likely ligand for NA, and accumulates in roots of chloronerva, we observed no change in AtYSL1 gene expression in response to Cu deficiency or excess (data not shown).

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Figure 2. AtYSL1 gene expression in response to iron nutrition. (a) RNA gel blot showing the kinetics of accumulation of AtYSL1 mRNA in plant shoots in response to excess iron. Fifteen micrograms of total RNA extracted from 6-week-old plants treated with an excess of 500 μm iron-ethylenediaminetetraacetic acid (Fe-EDTA) for the indicated length of time was blotted and hybridized successively to AtYSL1, AtFER1 and AtFE-1α specific probes. Ethidium bromide (EtBr) staining is shown. (b) Quantification of the RNA gel blot shown in (a) using Image J (http://rsb.info.nih.gov/ij/). AtYSL1 and AtFER1 transcripts were normalized by the AtEF-1α signal. (c) Enzymatic GUS assay for 12 independent transgenic lines expressing an AtYSL1 promoter–GUS fusion and grown in the absence of added iron (−Fe) or in the presence of 300 μm of added Fe-EDTA. No significant activity was detected in wild-type plant extracts (data not shown). MU, methylumbelliferone.

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In order to quantify the AtYSL1 promoter response to Fe excess, we generated transgenic Arabidopsis plants expressing the uidA gene, encoding β-Glucuronidase (GUS) gene under the control of 1.5 kb of AtYSL1 sequences located upstream of the ATG. GUS activity was monitored in shoots of 12 independent transgenic lines selected on kanamycin in vitro for 10 days, and then transferred for 5 days onto media either lacking Fe (−) or supplemented with 300 μm Fe (++). The mean of the enzymatic GUS activities, assayed by fluorometry, was around 0.04 nmol MU−1 min mg−1 protein in control conditions, while it reached 0.16 nmol MU−1min mg−1 protein in high Fe conditions, indicating a 4-fold induction of AtYSL1 promoter activity in response to Fe excess [Figure 2(c)]. This result is consistent with the 2.5-fold induction of AtYSL1 mRNA accumulation measured by Northern blotting.

Tissue-specific expression of AtYSL1

To gain insight into the biological function of AtYSL1, we looked at the tissues and cell types expressing AtYSL1. Histochemical analyses were performed using the above-mentioned Arabidopsis transgenic lines expressing an AtYSL1 promoter–GUS fusion. Plants were grown in vitro for 2 weeks in the presence of various amounts of Fe as indicated in the legend to Figure 3. Staining of entire plantlets confirmed that the AtYSL1 promoter was active in shoots but not in roots (data not shown). In leaves, GUS activity was restricted to the veins and, although visible in the absence of Fe in the medium [Figure 3(a)], it increased with Fe concentration, spreading to neighboring mesophyll cells at 300 μm Fe [Figure 3(b,c)]. A cross-section of the basal part of a leaf treated as in Figure 3(b) showed that the staining was restricted to the xylem pole of primary and secondary veins and more specifically to the xylem parenchyma surrounding xylem tubes [Figure 3(d,e)]. In flowers, we observed GUS activity in the vasculature of sepals and petals as well as in pollen grains [Figure 3(f–h)]. A faint staining was generally visible on the style, underneath the stigmatic papillae [Figure 3(g)]. Staining of pollen grains was observed in young flowers, when anthers are still positioned well below the stigma, and disappeared at stages with fully elongated anthers [Figure 3(f)]. Counter-staining of blue pollen grains with 4, 6-diamidino-2-phenylindole (DAPI) showed the presence of three nuclei, one vegetative and two generative, indicating that pollen grains expressing GUS have already reached the mature stage [inset of Figure 3(h)]. In senescing stamens, coloration of the anthers disappeared whereas strong GUS staining was seen in the vascular tissue of the filament [Figure 3(i)].

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Figure 3. Cell type-specific localization of AtYSL1 gene expression. (a–e) GUS histochemical staining of leaves of 15-day-old transgenic plantlets grown on various iron-ethylenediaminetetraacetic acid (Fe-EDTA) concentrations: (a) no added Fe; (b, d, e) 50 μm Fe; (c) 300 μm Fe. (d, e) Cross-section through the base of a leaf; (e) higher magnification of the midvein; (f) the inflorescence; (g) the immature flower; (h) pollen grains, with inset of 4, 6-diamidino-2-phenylindole (DAPI) staining showing one large vegetative nucleus and two small dense generative nuclei; (i) the senescing stamen. ep, epidermis; mv, midvein; p, parenchyma; st, stomata; sv, secondary vein. Bar, 10 μm in (h) and 200 μm in (d) and (e).

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The AtYSL1 promoter is also active in siliques (Figure 4). We observed strong staining in the petiole next to the abscission zones of the sepals, petals and stamens where it increased upon maturation of the silique [Figure 4(a)]. A close-up view of the abscission region revealed coloration of the vessels connected to each flower whorl [Figure 4(c)]. A cross-section of the petiole indicated that the GUS activity was restricted to the xylem parenchyma [Figure 4(f)]. In addition to the petiole, staining was observed in very young siliques at the level of the carpel veins, in the style underneath the stigmatic papillae [Figure 4(e)] and in a small region of the developing seed [Figure 4(d)]. Only the staining in the style persisted in mature siliques [Figure 4(a)]. A cross-section of a very young silique containing seeds at a very early stage of development showed GUS activity in cells surrounding xylem tubes in the silique envelope [Figure 4(g)]. In addition, inside the locule, GUS activity was detected in the vascular tissue of the funiculus and at the basis of the funiculus in the posterior pole of the ovule, a region corresponding to the chalazal endosperm [Figure 4(g)].

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Figure 4. AtYSL1 gene expression in siliques. GUS histochemical staining of siliques removed from soil-grown plants is shown. (a) Binocular observation of siliques at different developmental stages. (b–e) Early-stage siliques observed using an optical microscope. (b) The entire silique reconstituted from several images obtained with an optical microscope. (c, d, e) Boxed regions are shown at higher magnification (c, petiole; d, central part; e, style and stigmate). (f) A 5-μm cross-section of a silique petiole near the abscission zone. (g) A 5-μm cross-section of the young silique shown in (b). f, funiculus; ds, developing seed; c, chalaza; v, vein; x, xylem. Bar, 500 μm (a), 100 μm (c, d, e) or 50 μm (f, g).

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Characterization of an Arabidopsis ysl1-1 knockout plant

We and others have previously shown that expression of the first member of the YS gene family, the maize YS1 gene, can functionally complement the yeast Fe uptake defect when Fe is provided as an Fe–PS chelate (Curie et al., 2001; Schaaf et al., 2004). However, we did not succeed in showing such heterologous complementation with Arabidopsis YSL2 (Schaaf et al., 2005). We also failed to show growth restoration mediated by AtYSL1 expression on media containing Fe(II)–NA, Fe(III)–NA or Fe(III)–PS (data not shown). We therefore addressed the biological function of AtYSL1 in planta by scrutinizing the physiological alterations resulting from knocking out the AtYSL1 gene.

The release of flanking sequence tags (FST) for Arabidopsis T-DNA insertion lines from the Versailles-Institut National de la Recherche Agronomique (INRA) laboratory (Versailles, France) and from the SIGnAL program (Salk Institute, La Jolla, CA, USA) identified two insertion lines in the AtYSL1 gene. Line DRM20 in the ecotype Wassilewskija (Ws), named ysl1-1, and line SALK_034534 in the ecotype Columbia (Col-0), named ysl1-2, contain a single T-DNA inserted respectively in the first intron and in the fourth exon of the gene in the orientation indicated in Figure 5(a). Using PCR on reverse-transcribed mRNA prepared from the ysl1-1 and ysl1-2 lines, we amplified an AtYSL1-specific fragment with wild-type Ws or Col-O samples but none with ysl1-1 or ysl1-2 samples, whereas the control aquaporin AtPIP2;1 gene-specific band was obtained with both wild-type and mutant plants [Figure 5(b)]. The absence of AtYSL1 transcripts thus confirmed that ysl1-1 and ysl1-2 are null alleles of AtYSL1.

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Figure 5. Characterization of two AtYSL1 gene knockout lines. (a) Schematic representation of the T-DNA integration site in the AtYSL1 genomic sequence for ysl1-1 and ysl1-2 mutant lines. Exons are represented by boxes and introns by lines. Orientation of the T-DNA copies is shown. (b) Absence of AtYSL1 transcript in ysl1-1 and ysl1-2 plants. An ethidium bromide-stained DNA gel is presented showing reverse transcriptase–polymerase chain reaction (RT-PCR) amplification fragments corresponding to AtYSL1 or Arabidopsis Plasma membrane Aquaporin 2;1 (AtPIP2;1) (control) transcripts in wild-type and mutant alleles. Arrowheads indicate the position of AtYSL1 primers. (c) Iron (Fe), zinc (Zn) and manganese (Mn) content determined by atomic absorption spectrometer (AAS) in shoots or roots of wild-type and mutant alleles of AtYSL1 grown as above. (d) Nicotianamine content measured by high-performance liquid chromatography (HPLC) in shoots of wild-type and mutant plants. Plants were grown for 5 weeks in hydropony, washed, transferred for 5 days to Fe-less medium and then transferred again for 3 days to either low iron-containing medium (20 μm, +) or high iron-containing medium (500 μm, ++). Values are the mean of three samples (c, d).

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ysl1 homozygous mutants did not show any change in macroscopic phenotype, either on soil or in vitro. Moreover, variation of the metal content, i.e. an excess or deficiency of Fe, Cu, Mn or Zn, did not reveal any growth defect (data not shown). We then measured the metal content in the two mutant lines and in their parental wild-type background. Five-week-old hydroponically grown plants were transferred, after a 5-day period of Fe starvation, to either low Fe-containing medium (20 μm) or excess Fe (500 μm) for 3 days. Measurement of metal ions was performed using an atomic absorption spectrometer (AAS) on shoots and roots harvested separately. We found no significant variation in Fe, Zn or Mn content between wild-type and mutant plants, either in shoots or in roots [Figure 5(c)].

If the physiological role of AtYSL1 is to transport NA–metal complexes into or out of the xylem, one might expect to see a modification of NA distribution between shoots and roots. NA was detected by high-performance liquid chromatography (HPLC) analysis on shoots of the tissue samples previously subjected to AAS analysis. Upon treatment with 20 μm Fe, shoots of both ysl1 mutants were found to contain around 40% more NA than their respective wild type [Figure 5(d)]. At high Fe concentration, we observed an increase of NA content in the mutant, the extent of which was difficult to evaluate because of elevated standard errors, although this increase was smaller than that in low Fe [Figure 5(d)]. We concluded that AtYSL1 moderately but significantly contributes, directly or indirectly, to NA content in leaves.

Knocking out the AtYSL1 gene resulted in decreased Fe and NA content in seeds

The fact that GUS expression is in part localized in the young silique, both in the vascular tissue and at the level of the chalazal end of the seed, suggests that AtYSL1 could be involved in Fe loading via Fe–NA transport into the seed. We therefore asked whether NA content is modified in the seeds of the ysl1 mutants. Using HPLC analysis, we found an almost 2-fold decrease in the amount of NA in ysl1-1 and a 4-fold decrease in ysl1-2 [Figure 6(a)]. We previously showed that addition of FeEDDHA to the growth solution can fully rescue the severe Fe deficiency of an IRT1-less mutant in Arabidopsis (Vert et al., 2002). However, this defect in NA accumulation was not rescued when seeds were harvested from ysl1-1 plants watered with 0.6 mm FeEDDHA [Figure 6(b)]. Furthermore, compared with the wild type, the Fe content of ysl1 seeds was altered for both mutant alleles, although to a different extent: ysl1-1 seeds contain 65% less iron than Ws seeds whereas ysl1-2 seeds contain 30% less iron than Col-0 seeds [Figure 6(c)]. FeEDDHA watering of plants barely increased the Fe content in wild-type seeds (by <20%), and did not rescue Fe deficit in ysl1-1 seeds [Figure 6(d)]. Therefore, exogenous addition of Fe during plant growth did not correct deficiencies in the NA or Fe content of ysl1 seeds. Finally, the metal concentration in seeds of both ysl1 alleles, determined by inductively coupled plasma mass spectrometry (ICP-MS), revealed no variations in Cu and Zn content, but a significant accumulation of Mn [Figure 6(e)], which could result from the deregulation of another metal transporter in response to the change of seed Fe status.

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Figure 6. Disorders of seed physiology in ysl1 mutants. (a, b) Nicotianamine content in ysl1 and wild-type seeds set by plants irrigated with water (a) or with 600 μm FeEDDHA (b). (c, d) Iron content in seeds obtained as in (a) and (b), respectively. (e) Metal content in seeds of wild-type and mutant plants measured by inductively coupled plasma mass spectrometry (ICP-MS). Values are the mean of three samples (c–e).

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To further confirm that a defective AtYSL1 gene was responsible for the physiological disorders observed in ysl1-1 and ysl1-2 seeds, we investigated whether expressing the wild-type AtYSL1 gene in ysl11-1 could rescue its phenotype. Eight such transgenic ysl1-1:AtYSL1 plants were generated and their Fe and NA contents were determined in seeds (Figure 7). We found the levels of Fe and NA in the different complemented lines to spread roughly between the values measured in knockout and wild-type seeds. This heterogeneity is consistent with the position effect observed in independent transgenic lines and probably explains why one complemented mutant had a NA concentration in seeds that was up to 30% higher than that of the wild type [Figure 7(b)]. Thus, expression of a wild-type AtYSL1 gene in a ysl1 knockout can efficiently rescue disorders in its seed physiology.

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Figure 7. Expression of AtYSL1 in ysl1-1 mutant restores its seed physiological disorders. (a) Seed nicotianamine content. (b) Seed iron content. Values are the mean of three measurements.

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ysl1.1 germination is slower on Fe-deficient medium

Finally, given that seeds of ysl1-1 knockout plants are Fe-deficient, we looked for a defect in seed germination on media containing a range of Fe concentrations. Seeds of Ws and ysl1-1, produced by water- or FeEDDHA-irrigated plants, were sown on agar plates supplemented with 0, 50 or 350 μm Fe. The number of germinated seeds, i.e. seeds with an emerging radicle, counted once a day from 48 to 120 h post-sowing is represented as a percentage of the total number of seeds [Figure 8(a,b)]. On Fe-deficient plates, <40% of the ysl1-1 seeds had germinated at 48 h compared with >65% of the Ws seeds [Figure 8(a)]. This difference decreased over time and had almost vanished at 96 h after sowing. Seeds from FeEDDHA-watered plants showed the same curve of germination [Figure 8(a)]. In the presence of a high concentration of Fe in the plate, however, mutant and wild-type seeds did not show a significant difference [Figure 8(b)]. An intermediate response of the mutant was obtained at 50 μm Fe, as 45% of the mutant seeds had germinated at 48 h and the percentage germination had already caught up with that of the wild type at 72 h (data not shown).

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Figure 8. Germination defect of ysl1-1 mutant seeds. (a, b) Germination rate between 48 and 120 h after sowing on iron-deprived medium (a) and 350 μm iron-containing medium (b). Results are presented as a percentage of germinated seeds. Filled symbols, Wassilewskija (Ws); open symbols, ysl1-1; squares, seeds obtained from water-irrigated plants; triangles, seeds obtained from FeEDDHA-irrigated plants. (c) Seedlings with the Ws (a, c) or ysl1-1 allele (b, d) germinating for 72 h on medium either lacking iron (a, b) or containing 350 μm iron (c, d).

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On Fe-starved plates, compared with Ws, ysl1-1 plants exhibited a shorter radicle, bleached cotyledons and a lack of anthocyanin-characteristic pink stain [Figure 8(c), panels (a) and (b)]. On plates containing high Fe, however, the wild type and mutant were undistinguishable [Figure 8(c), panels (c) and (d)]. We concluded that mutation in AtYSL1 results in a transient defect of germination, which can be rescued by Fe supply during germination, but not by the accumulation of Fe during the vegetative life of the plant.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this work, we have established that the AtYSL1 transporter is necessary for correct loading of Fe and NA into Arabidopsis seeds, as seeds of two independent knockout plants lacking a functional AtYSL1 gene were found to have reduced levels of NA and Fe, and as normal levels could be restored by expression of a wild-type AtYSL1 gene. This strongly suggests that AtYSL1 participates in Fe loading of the seed via its transport as an Fe–NA chelate. Such a function is supported by the strong expression of AtYSL1 in tissues surrounding xylem vessels in the silique as well as in the chalazal endosperm of the seed, a region that adjoins the maternal vascular tissue to the seed integuments and which could be involved in the transfer of nutrients to the embryo (Nguyen et al., 2000) and in mineral storage (Otegui et al., 2002). Direct evidence of the transport activity and substrate of AtYSL1 is still lacking, in contrast to the situation for two other YS family members, ZmYS1 and OsYSL2, which have been shown to transport Fe–NA chelates in heterologous expression systems (Koike et al., 2004; Schaaf et al., 2004). Nevertheless, based on reverse genetics, our study provides in planta evidence of the involvement of YSL transporters in NA allocation in a specific plant organ. We found the ratio between Fe decrease and NA decrease to vary between the two mutant alleles and between two experiments with one allele (Figures 6 and 7). Such fluctuations may be explained by the fact that the concentrations of metabolites and nutrients are modulated by environmental cues and according to the physiological state of the plant. However, in all our experiments, NA and Fe concentrations in seeds showed the same tendency to decrease in both ysl1 mutants, while this tendency was either reduced or inverted in the complemented mutant lines.

The fact that growth of plants in the presence of 600 μm FeEDDHA did not allow recovery of normal Fe levels in the ysl1-1 mutant seeds supports the hypothesis that Fe loading in the seed requires that Fe is provided as a specific chelate. This is consistent with earlier data showing that the pea (Peasum sativum) E107 mutant, which contains 36 times more Fe in its leaves than wild-type Sparkle pea, does not translocate more Fe in seeds than the wild type, suggesting that a specific chelate must be loaded in the phloem to feed the seed (Grusak, 1994). The possibility exists that AtYSL1 could transport a chelate other than Fe–NA. As the Arabidopsis NA synthase-encoding genes are differentially regulated by Fe nutrition (N. K. Nishizawa, Laboratory of Plant Biology, University of Tokyo, Tokyo, Japan, personal communication), the decrease in seed NA content could in fact result from the deregulation of its synthesis by the nicotianamine synthases (NAS). However, except for the AtNAS2 gene, whose transcript was not detectable in shoots, we found no change in AtNAS1, AtNAS3 or AtNAS4 gene expression in the ysl1 mutants (data not shown). Alternatively, AtYSL1 could transport NA alone. One consequence of its absence would thus be a reduction of NA content in ysl1-1 seeds, which could in turn modulate the Fe content, as one of the proposed functions for NA is to scavenge free cellular Fe. However, this is very unlikely because YS transporters have been shown to discriminate between various metal chelates (DiDonato et al., 2004; Koike et al., 2004; Schaaf et al., 2004), implying that their substrate is a metal–NA chelate rather than NA alone. Physicochemical characterization of the Fe chelates contained in mutant and wild-type seeds should give a clear-cut answer to the question of whether the decrease in Fe measured in the mutant corresponds to a decrease in Fe–NA content.

ysl1-1 seeds showed a slower rate of germination. In contrast to the Fe and NA content of the seed, this phenotype was efficiently rescued by an exogenous supply of Fe. Therefore, the germination defect is likely to result from the Fe-deficient state of the seeds rather than from a defect in Fe–NA transport during germination, which implies that seed germination does not rely on AtYSL1 activity. Two additional symptoms of Fe deficiency in ysl1 germinating seedlings were (i) the yellow color of the cotyledons, which is consistent with the paleness of ysl1-1 seeds (data not shown), and (ii) the lack of pink stain corresponding to anthocyanins, which may result from a loss of activity of the iron-requiring anthocyanin synthase, which requires Fe (De Carolis and De Luca, 1994). Both defects were restored when seedlings were germinated in the presence of a high amount of Fe.

AtYSL1 may also play a role in leaves, as we observed a moderate increase in the quantity of NA in leaves of ysl1-1 mutants and as the AtYSL1 gene is strongly expressed in the vasculature of leaves. Physiological data obtained on tomato chloronerva and tobacco naat plants demonstrate the essential role of NA in the unloading of Fe from vessels into leaves in order to provide Fe to intercostal regions (Scholz, 1989; Takahashi et al., 2003). Our data on the localization of AtYSL1 expression in the xylem parenchyma, its modulation by Fe nutrition and the accumulation of NA in ysl1-1 mutant leaves suggest that AtYSL1 could participate in the delivery of Fe–NA from the xylem to the intercostal regions of the leaves.

The induction of AtYSL1 expression in shoots in response to increased Fe supply suggests that AtYSL1 participates in the plant detoxification strategy. Upon treatment with an excess of Fe, AtYSL1 expression spreads to mesophyll cells surrounding the leaf vein. As Fe(II)–NA is a rather Fenton-inactive form of Fe (von Wiren et al., 1999), its transport by AtYSL1 into tissues with detoxification capacity or into specific cellular compartments may protect the cell from oxidative damage. A role of AtYSL1 in the mechanism of response to Fe excess is further supported by the fact that AtYSL1 and AtFER1, which encodes the storage protein ferritin, are quickly and coordinately induced by excess Fe treatment. Furthermore, publicly available databases indicate that AtYSL1 expression is stronger in senescent leaves. During senescence, dismantling of chloroplasts and degradation of macromolecules lead to the release of Fe, which catalyzes the production of reactive oxygen species and produces oxidative stress. Interestingly, a ferritin gene has been shown to be upregulated in senescent leaves of Brassica napus (Buchanan-Wollaston and Ainsworth, 1997), where it could participate in the detoxification of cellular Fe. Similarly, AtYSL1 may protect the cell from cellular damage by Fe–NA compartmentalization or by transporting Fe–NA to specialized tissues. In pea Fe-overaccumulating mutants, the NA level was shown to parallel the Fe level and NA to accumulate in the vacuole in response to Fe overload, suggesting that NA may detoxify excess Fe through vacuolar sequestration (Pich et al., 2001). It would also be interesting to determine in future experiments whether AtYSL1 in senescent leaves is expressed in intercostal regions in addition to the xylem parenchyma.

The presence of AtYSL1 in pollen and more importantly in the vasculature of the anther filament suggests that it also plays a role in the delivery of Fe to pollen grains. The role of NA in reproductive organs and more specifically in flowers has been nicely illustrated by the physiological characterization of NA-deficient tobacco naat plants with very abnormal and essentially sterile flowers (Takahashi et al., 2003). We found that the capacity of ysl1-1 pollen to germinate was not impaired in in vitro germination assays (data not shown). We also assessed the fertility of ysl1-1 flowers by counting the number of seeds in siliques but observed no significant difference between mutant and wild type (data not shown). Further work will be needed to elucidate the physiological role of AtYSL1 in pollen.

The main finding of this work is the fact that knocking out the AtYSL1 gene seriously affected the Fe and NA content of the seed. In contrast to tomato chloronerva or transgenic tobacco naat plants, which both show pleiotropic phenotypes and profound developmental alterations, including fertility defects, the targeted mutation of AtYSL1 did not affect the metal content or development of the rest of the plant, allowing us to pinpoint the significant role of a YSL transporter in the loading of Fe into seeds. As Fe entry into the seeds seems to be a rate-limiting step, strategies aimed at increasing seed Fe content via overexpression of a ferritin gene are likely to be insufficient. Indeed, transgenic rice seeds accumulate relatively little Fe despite a very high production of ferritin (Qu et al., 2005). In the future, one might envisage combining these approaches with the overproduction of Fe loading systems to improve seed quality.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant growth

For hydroponic cultures, plants were grown under short-day conditions (8 h at 300 μE m−2 sec−1, 20°C) in a medium containing (in mm) 1.25 KNO3, 1.5 Ca(NO3)2, 0.75 MgSO4, 0.5 KH2PO4 and 0.1 Na2O3Si, and (in μm) 50 H3Bo3, 12 MnCl2, 0.7 CuSO4, 1 ZnCl2, 0.2 MoO4Na2 and 100 Fe-ethylenediaminetetraacetic acid (Fe-EDTA). The medium was changed weekly. After 5 weeks of culture, roots were washed for 3 min in a solution containing 0.3 mm bathophenantroline-disulfonic acid (B-1375; Sigma, St. Louis, MO, USA) and 5.7 mm Na2S2O4 (Art 6507; Merck, Darmstadt, Germany), then rinsed three times with deionized water. Plants were then grown on the medium described above without Fe-EDTA. After 5 days of Fe deprivation, 300–500 μm of Fe-EDTA was added to the medium and the plants further grown for the length of time indicated in Figures 2 and 4.

For the GUS assay on young plantlets, transgenic seeds were surface-sterilized and grown on Petri dishes in half-strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) with 30 mm sucrose [pH adjusted to 5.2 with 2-Morpholino-ethane-sulfonic acid (MES)-KOH] containing 50 mg ml−1 kanamycin. Plants were incubated in long-day conditions (16 h at 150 μmol sec−1 m−2, 21°C). After 10 days, resistant plantlets were transferred onto Fe-deficient (no Fe added to the media) or Fe-excess (300 μm NaFe-EDTA) plates for 5 days. Production of seeds by water- and Fe-irrigated plants: plants were cultivated on soil (Humin Substrate N2 Neuhaus; Klasmann-Deilmann, Geeste, Germany) in a greenhouse at 23°C and irrigated with water or with a solution of 600 μm FeEDDHA (0.5 g l−1 Sequestrene, Fertiligene, Ecully, France) for 6 weeks.

For Arabidopsis transformation, constructs were introduced into the MP90 strain of Agrobacterium tumefaciens, which was then used to transform Arabidopsis (ecotype Columbia) following the ‘floral dip’ method (Clough and Bent, 1998). Transformed lines expressing GUS fusions were selected on kanamycin, whereas complemented ysl1-1 mutant lines were selected on hygromycin.

Identification of two AtYSL1 knockout alleles

The mutant lines DMR20 and SALK_034534 from the Versailles–INRA and SIGnal (Salk Institute, La Jolla, CA, USA) collections of T-DNA insertion mutants, respectively, were identified through the T-DNA insertions sequence databases. Homozygous mutated plants were isolated by PCR screening using a T-DNA left border-specific primer and an AtYSL1 gene-specific primer (for the ysl1-1 mutant: forward: 5′-CAGTCTCCATGGAAATAGAGC-3′; reverse: 5′-CGGGTACCAGACAACACATATGTCTTATGG-3′: for the ysl1-2 mutant: 5′-GGCTTAATGTGGCCTCTTC-3′ and LB-b1: 5′-GCGTGGACCGCTTGCTGCAACT-3′). Reverse transcriptase–polymerase chain reactions (RT-PCRs) were performed on RNA prepared from leaves of wild-type and ysl1-1 and ysl1-2 mutants using AtYSL1 gene-specific primers on each side of the T-DNA insertions (ysl1-1: forward: 5′-CCGCTCGAGTTACGGCATCGCTGTCGG-3′; reverse: 5′-CGGGTACCAGACAACACATATGTCTTATGG-3′; ysl1-2: forward: 5′-GGCTTAATGTGGCCTCTTC-3′; reverse: 5′-GGACTAGTCCTATGAAGCTAAGAACTTC-3′) which amplify a fragment in the wild-type allele, but not in the mutant [Figure 5(b)]. PIP2;1 primers (forward: 5′-TGCGAAAGGATGTGGCAGCCGTTCCCGGA-3′; reverse: 5′-CAACGCATAAGAACCTCTTTGA-3′) amplifying the aquaporin PIP2;1 were used as a control. Segregation on kanamycin revealed a 3:1 ratio characteristic of a single T-DNA insertion in both alleles.

Plasmid constructions

AtYSL1 cDNA cloning.  The AtYSL1 cDNA was amplified by PCR from reverse-transcribed RNA prepared from shoots of plants grown in hydropony, as described above, and treated with 500 μm NaFe-EDTA for 12 h. The PCR reaction was performed using thermostable DNA polymerase (pfu) polymerase (Promega, Madison, WI, USA) and the following primers: ysL1-ATG (5′-CAGTCTCCATGGAAATAGAGC-3′), containing a NcoI restriction site, and ysL1-STOP (5′-GGACTAGTCCTATGAAGCTAAGAACTTC-3′), containing a SpeI restriction site. The NcoI-SpeI-digested AtYSL1 cDNA was cloned into a modified pBlueskript KS+ (Eyal et al., 1995) previously digested with the same enzymes and completely sequenced. This vector was named pBK-YSL1.

Plasmid for GUS activity analyses.  A 1.5-kb fragment of the AtYSL1 promoter was amplified from the BAC clone T19F6 containing the AtYSL1 gene using pfu DNA polymerase and the oligonucleotides YSL1-1 (5′-TAGGAAGCTTATATCAAAAATAAGGTGAGAC-3′), containing a HindIII restriction site, and YSL1-2 (5′-CTCTATTTCCATGGAGACTG-3′), containing a NcoI restriction site. This HindIII-NcoI fragment was cloned in frame with the uidA gene previously inserted in pBKS+ modified as described in Eyal et al. (1995). The HindIII-XbaI fragment containing the AtYSL1-GUS fusion was excised from the resulting plasmid and ligated into the pBIN19 vector (Bevan, 1984) previously digested by the same enzymes. The resulting plasmid was named pBIN19-YSL1-GUS.

Plasmid for ysl1-1 complemented lines.  An AtYSL1 gene fragment containing 1.5 kb of promoter sequence upstream of the ATG and 0.37 kb of the 3′ untranslated region was amplified from the BAC clone T19F6 using the pfu polymerase and the primers YSL1-1 (see above) and 5′-CCCGAGCTCTAAAGGCAAGGATTTCTTCC-3′, containing a HindIII site and a SacI site, respectively. The HindIII-SacI restriction fragment was subcloned into the pGreen179 binary vector digested by the same enzymes and the resulting construct was sequenced.

Metal content determination

The metal (Fe, Zn, Mn and Cu) concentrations of samples of mineralized roots and leaves were determined by atomic absorbance spectrometry. Prior to mineralization, root samples were washed as indicated in the section ‘Plant growth’. All samples were ground with a mortar and pestle in liquid nitrogen. About 100 mg of dry matter was completely digested in 70% NO3 at 120°C and solubilized in 2 ml of 70% HNO3 and 18 ml of H2O. The metal content was determined using a Varian SpectrAA220-FS (Varian; Palo Alto, CA, USA). In seeds, Fe content was measured as described in Lobreaux and Briat (1991), while Zn, Cu and Mn contents were measured by ICP-MS.

Nicotianamine measurement

Nicotianamine was extracted and measured as described in Neumann et al. (1999) with some modifications. Plant tissues were ground in liquid nitrogen and approximately 100 mg was extracted with 300 μl of H2O at 80°C for 20 min and then centrifuged (18 000 g) for 10 min. Fifty microliters of supernatant was derivatised with an equal volume of a o-phthlaldialdehyde (OPA) solution (12.8 mg of OPA, 2.5 ml of methanol, 10.5 ml of 0.2 m borate buffer, pH 9.9, containing 0.1 m KCl, and 25 μl of mercaptopropionic acid), incubated in the dark for 1 min. One microliter of a sulfosalicylic acid solution [50%, weight/volume (w/v)] was then added, just before injection. HPLC separation was carried out on a PrepStar Solvant Delivery Module (Varian) using a binary gradient: solvent A, phosphate-citrate buffer pH 3; solvent B, methanol/tetrahydrofuran 80%/20% (v/v) gradient at a flow rate of 0.7 ml min−1 on a C18 Nucleodur column (250 mm × 4.6 mm; Macherey-Nagel, Düren, Germany). Gradient parameters were 0–20 min, 0–100% B, 20–22 min, 100% B, 22–25 min, 100–0% B. Fluorescence of OPA derivatives was measured with a ProStar Fluorescence detector (Varian; excitation 350 nm; emission 455 nm), and quantification was carried out using pure chemically synthesized NA (T-Hasegawa Co., Tokyo, Japan) as external standard.

Gene expression analysis

Tri-Reagent (T9424; Sigma) was used to extract total RNA from roots and shoots of plants cultivated hydroponically as described above.

For Northern blot experiments, 15 μg of total RNA was denatured and electrophoresed on a 1.2% 3-(N-morpholino)-propane-sulfonic acid/formaldehyde/agarose gel before transfer to a nylon Hybond-N+ membrane (Amersham, Buckinghamshire, UK). The hybridizations were performed in Church buffer (Church and Gilbert, 1984). The AtYSL1-specific probe was generated by amplification of a 1.5-kb fragment of the cDNA including 378 bp of 3′-untranslated region (3′-UTR) sequences using the following primers : forward: 5′-ggCTTAATgTggCCTCTTC-3′; reverse: 5′-CCCgAgCTCTAAAggCAAggATTTCTTCC-3′. The AtFER1-specific probe was obtained by fusing 281 bp of 5′-UTR located immediately upstream of the ATG with 188 bp of 3′-UTR located immediately downstream of the stop codon and was a gift from F. Gaymard (Biochime et Physiologie, moléculaire des plantes, CNRS, Montpellier, France). The AtEF-1α probe was amplified by RT-PCR on total RNA prepared from Arabidopsis suspension cells using the following gene-specific primers : forward: 5′-CCACCACTGGTGGTTTTGAGGCTGGTATC-3′; reverse: 5′-CATTGAACCCAACGTTGTCACCTGGAAG-3′. Membranes were exposed to a BAS imaging plate (BAS-SR2025; Fujifilm, Tokyo, Japan) for 3, 1 and 1 days for AtYSL1, AtFER1 and AtEF-1α, respectively. The signal was revealed using BAS 5000 (Fujifilm).

GUS expression analysis

For enzymatic GUS assay, roots and shoots of 12 kanamycin-resistant T1 independent lines were harvested separately and ground in Eppendorf tubes in GUS extraction buffer (Jefferson et al., 1987). GUS activity was measured fluorometrically using 1 mm 4-methylumbelliferyl-β-d-glucuronide as substrate (Euromedex, Mundolsheim, France). The total protein content of the samples was determined according to Bradford (1976) and used to correct for GUS activity. GUS histochemical staining was performed either on 15-day-old plantlets grown on plates or on flowers and siliques of hydroponically grown plants. 5-bromo-4-chloro-3-indolyl β-d-glucuronide was used as substrate (Euromedex). Stained leaves of plantlets grown in plates containing 50 μm NaFe-EDTA and stained siliques harvested from soil-grown plants were embedded in hydroxyethylmethacrylate (Technovit 7100; Heraus-Kulzer, Werheim, Germany) and cut into thin cross-sections (4 μm for leaves and 5 μm for siliques) using a Leica RM 2165 microtome (Leica, Nussloch, Germany). Cross-sections were counter-stained with Schiff dye and observed with an Olympus BH2 microscope (Olympus, Tokyo, Japan).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Geneviève Conejero for assistance with histology, Laurent Ouerdane for performing ICP-MS analysis, and Frederic Gaymard (CNRS) for sharing AtFER1 and EF-1α probes. The work of MLJ was supported by a thesis fellowship from the Ministère de I'Éducation Nationale, de la Recherche et de la Technologie, and the work of AS was supported by an INRA postdoctoral fellowship. This work was funded by Centre National de la Recherche Scientifique and INRA and by the Toxicologie Nucléaire program of the Réseau Inter-Organismes (RIO).

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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