Iron deficiency induces sulfate uptake and ... - CSIRO Publishing

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Functional Plant Biology, 2006, 33, 1055–1061

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Iron deficiency induces sulfate uptake and modulates redistribution of reduced sulfur pool in barley plants Stefania AstolfiA,E , Sabrina ZuchiA , Stefano CescoB , Luigi Sanità di ToppiC , Daniela PirazziA , Maurizio BadianiD , Zeno VaraniniB and Roberto PintonB A Dipartimento

di Agrobiologia e Agrochimica, Università della Tuscia, via S. C. de Lellis, 01100 Viterbo, Italy. di Scienze Agrarie e Ambientali, Università di Udine, Viale delle Scienze 208, 33100 Udine, Italy. C Dipartimento di Biologia Evolutiva e Funzionale, Universit` a di Parma, Viale delle Scienze 11 / A, 43100 Parma, Italy. D Dipartimento di Biotecnologie per il Monitoraggio Agro-alimentare ed Ambientale, Università Mediterranea di Reggio Calabria, Loc. Feo di Vito, 89129 Reggio Calabria, Italy. E Corresponding author. Email: sastolfi@unitus.it B Dipartimento

Abstract. We studied the possibility that the sulfur (S) assimilatory pathway might be modulated by iron (Fe) starvation in barley, as a consequence of plant requirement for an adequate amount of reduced S to maintain methionine and, in turn, phytosiderophore biosynthesis. Barley seedlings were grown with or without 100 µM FeIII – EDTA, at three S levels in the nutrient solution (S2 = 1200, S1 = 60, and S0 = 0 µM sulfate) in order to reproduce conditions of optimal supply, latent and severe deficiency, respectively. Fe deprivation increased root cysteine content irrespective of the S supply. However, this increase was not associated with either higher rates of 35 SO4 2− uptake or increased expression of the gene for the high-affinity sulfate transporter, HvST1, and these roots failed to increase their activities of ATP sulfurylase (ATPS) and O-acetylserine(thiol) lyase (OASTL). We observed a significant increase in 35 SO4 2− uptake rate (+76%) only in Fe-deficient S1 plants and we found an increase in root ATPS activity only in S0 plants. We observed an increase of ATPS enzyme activity in leaves of S1 and S2 plants, most likely suggesting increased S assimilation followed by translocation of thiols (Cys) to the root. Taken together, our results suggest that Fe deficiency affects the partitioning from the shoot to the root of the reduced S pool within the plant and can affect SO4 2− uptake under limited S supply. Keywords: iron deficiency, iron uptake, phytosiderophores, Strategy II, sulfur deficiency, thiols.

Introduction Plants subjected to Fe deficiency respond in two different ways (Römheld 1987; Marschner and Römheld 1994). Strategy I plants (all families except Gramineae) favour Fe mobilisation either by converting Fe3+ to Fe2+ in the soil solution via rhizosphere acidification or by releasing reducing compounds, such as phenolics, or by enhancing the reducing capacity of the roots. Moreover Romera and Alcantara (2004) proposed that Fe-deficiency in Strategy I plants causes an increase in ethylene production by roots and that this hormone might trigger some Fe-deficiency stress responses. Strategy II plants (Gramineae) release chelating compounds from roots, the phytosiderophores (PS), which are able to form stable complexes with cationic micronutrients. The most

common PS are mugineic acid (MA), deoxymugineic acid (DMA) and epi-hydroxymugineic acid (epi-HMA). Mori and Nishizawa (1987) identified methionine (Met) as their sole precursor. In higher plants, Met biosynthesis involves two pathways: S assimilation and aspartate metabolism (Ravanel et al. 1998; Hesse and Hoefgen 2003). Plants use inorganic sulfate taken up from the soil as a source of S. To enter the assimilatory pathway, sulfate must be activated by the enzyme ATP sulfurylase (ATPS) before being reduced. The synthesis of Cys by condensation of O-acetylserine and sulfide, catalysed by O-acetylserine(thiol) lyase (OASTL), represents the final step in the reductive sulfate assimilation pathway. Apart from being incorporated into proteins, Cys acts as a donor of

Abbreviations used: ATPS, ATP sulfurylase; Cys, cysteine; DMA, deoxymugineic acid; DTNB, 5,5 -dithio-bis(2-nitrobenzoic acid); epi-HMA, epi-hydroxymugineic acid; GSH, glutathione; MA, mugineic acid; Met, methionine; NS, nutrient solution; OASTL, O-acetylserine(thiol) lyase; PS, phytosiderophores. © CSIRO 2006

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reduced S in the synthesis of both Met, which leads to Sadenosyl methionine and S-methyl methionine derivatives, and glutathione (γ -Glu-Cys-Gly; GSH), which is a major reservoir of non-protein reduced S and a multifunctional metabolite in plants. Therefore Met, being the precursor of Sadenosyl methionine, plays also a central role in polyamines, ethylene and PS biosynthesis. Over recent years, S deficiency in soils has become widespread because of both a strong decrease in atmospheric SO2 emissions and the use of high-analysis, low-S fertilisers (McGrath and Zhao 1995). Consequently, there has been renewed research interest in S biology in plants aimed at a better understanding of yield and quality responses to S application. However, under field conditions availability of a nutrient for plants is often the result of a network of interactions with many environmental factors, including excess or deficiency of other nutrients, both beneficial or potentially toxic (e.g. heavy metals), or soil alterations such as salinity (Neves-Piestun and Bernstein 2005). As a consequence, plant responses are complex and this complexity often hinders the identification of the deficiency symptoms. In particular, interactions between S and Fe are assumed to occur, as recently reported in literature (Kuwajima and Kawai 1997; Astolfi et al. 2003, 2004; Bouranis et al. 2003), but little research has been focused so far on this aspect and the interaction mechanisms involved await clarification. Our previous study (Astolfi et al. 2003) showed that leaf Fe content was lower in maize (Zea mays L.) plants grown under S deficiency than in corresponding plants grown in the presence of S. Furthermore, Fe deprivation resulted in an increase in the level of non-protein thiol compounds in both S conditions (+S and −S), which is realistically explained by assuming an increased demand of reduced S for methionine and, consequently, PS synthesis. The rationale for the present study was the possibility that the S assimilatory pathway is modulated by Fe starvation in Strategy II species, because of the plant requirement for an adequate amount of reduced S to maintain PS biosynthesis (Astolfi et al. 2006). To this purpose, levels of S metabolites (i.e. GSH and Cys) as well as extractable activities of ATPS and OASTL, the first and the last enzyme of the S assimilation pathway, respectively, were evaluated in leaves and roots of barley plants subjected to different Fe and S supply. Furthermore, we report changes in SO4 2− uptake and expression of a high-affinity sulfate transporter (HvST1) in roots (Smith et al. 1997). Materials and methods Growing conditions Barley (Hordeum vulgare L. cv. Europe) seeds were germinated on moistened paper in the dark at 26◦ C for 3 d. Seedlings were then transferred to plastic pots (18 seedlings per pot) and were cultured hydroponically for 14 d, being exposed to three S application levels

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(S0 = 0, S1 = 60 and S2 = 1200 µM sulfate). Pots contained 2.5 L of a nutrient solution (NS; pH 6.2), having the following composition for the S2 condition (mM): K2 SO4 , 0.7; MgSO4 , 0.5; Ca(NO3 )2 , 2.0; KCl, 0.1; KH2 PO4 , 0.1; H3 BO3 , 10−3 ; MnSO4 , 10−3 ; CuSO4 , 2.5 × 10−4 ; (NH4 )6 Mo7 O24 , 10−5 ; ZnSO4 , 10−3 (Zhang et al. 1991) with (+Fe) or without (−Fe) 100 µM FeIII –EDTA. In S1 and S0 NSs sulfate salts (K+ , Mn2+ , Zn2+ , Cu2+ ) were replaced by appropriate amounts of the corresponding chloride salts (K+ , Mn2+ , Zn2+ , Cu2+ ). NS was continuously aerated and changed every 2 d. Plants were grown in a climate chamber under 200 µmol m−2 s−1 PPF and 14 / 10-h day / night regime (27 / 20◦ C day / night temperature cycling; 80% relative humidity). Both leaves and roots were harvested 14 d after sowing, frozen in N2 and stored at −80◦ C until used. Cysteine and GSH quantification Root and leaf samples (300 mg each) were homogenised in a mortar in the presence of ice-cold 5% (w / v) 5-sulfosalicylic acid containing 6.3 mM diethylenetriaminepentaacetic acid, following the method of De Knecht et al. (1994). After centrifugation at 10 000 g for 10 min at 4◦ C, the supernatants were filtered through Minisart RC4 0.45-µm filters (Sartorius, Goettingen, Germany) and immediately assayed by HPLC (model 200, Perkin-Elmer, Norwalk, CT). Cysteine (Cys) and GSH were separated through a reverse-phase Purosphere C18 column (Merck GmbH, Darmstadt, Germany), by injecting 200 µL of each extract. Cys and GSH separation was achieved isocratically by using 2% (v / v) aqueous acetonitrile containing 0.05% (v / v) trifluoroacetic acid at a flow rate of 0.7 mL min−1 . Cys and GSH were determined by post-column derivatisation with 300 µM Ellman’s reagent (DTNB) and detected at 412 nm (Model 430 UV-VIS detector, Kontron Instruments S.p.A, Milano, Italy). For Cys and GSH quantitation, a calibration curve for standard SH groups was set up by means of pure Cys and GSH standards (Merck GmbH, Darmstadt, Germany). Measurements of 35SO4 2– uptake Roots of 14-d-old intact barley plants were washed with micronutrientand sulfate-free NS for 30 min and then transferred to beakers containing 200 mL of a freshly prepared micronutrient- and sulfate-free NS; 35 SO4 2− (specific activity 2.1 KBq µmol−1 35 SO4 2− ) was added in order to give a final SO4 2− concentration of 600 µM. The uptake solution was buffered at pH 6.0 with 10 mM Mes–KOH and the uptake period was 1 h. After the uptake period, the plants were transferred to an ice-cold desorption solution containing 1 mM CaSO4 and 10 mM Mes– KOH (pH 6.0) for 5 min. Roots and shoots were oven-dried at 60◦ C, weighed, ashed at 550◦ C, and suspended in 1% (w / v) HCl for 35 SO4 2− determination by liquid scintillation counting. The 35 SO4 2− uptake rate, measured as µmol 35 SO4 2− , refers to the whole plant (root + shoot) and is presented per g DW of root per h. Enzyme extraction and assays Frozen tissue (approximately 1 g FW) was ground to a fine powder in a pre-chilled mortar under liquid nitrogen. Cold extraction buffer containing 50 mM HEPES–KOH (pH 7.4), 5 mM MgCl2 , 1 mM EDTA, 10% (v / v) glycerol, 0.1% (v / v) Triton X-100, 5 mM DTT, 1 mM PMSF and 1% (w / v) PVP was added in a ratio of 1 : 7 (w / v). The brei was filtered through four layers of cheesecloth and the homogenate was centrifuged at 1000 g for 5 min at 4◦ C. The resulting supernatant was desalted at 4◦ C on a Sephadex G-25 column (PD-10, Pharmacia, Uppsala, Sweden) pre-equilibrated with extraction buffer minus Triton X-100. The desalted extract was then centrifuged at 30 000 g for 5 min at 4◦ C. The supernatant was divided into 300-µL aliquots, which were then frozen in liquid nitrogen and stored at −80◦ C until analysis. Adenosine triphosphate sulfurylase (ATPS; EC 2.7.7.4) activity was assayed by the bioluminescence technique (Schmutz and Brunold 1982). The ATP production during the enzyme reaction is coupled to the

Relationship between sulfur supply and iron deficiency responses


light-producing reaction catalysed by firefly luciferase (E.C. 1.13.12.7). The reaction mixture contained in a total volume of 0.25 mL was: 16 mM Tris-acetate buffer pH 7.75, 8 µM APS, 68 µM Na4 P2 O7 , 40 µL of firefly luciferase (ATP Monitoring Reagent, ThermoLabSystems) and 5 µL of sample. Light emission was measured with LKB 1250 luminometer. O-acetylserine(thiol)lyase (OASTL; EC 4.2.99.8) was determined following the procedures described by Ferretti et al. (1993).

Isolation of total RNA from the roots of barley plants was performed with the Trizol reagent system according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). DNase-treated RNA (1 µg) was reverse-transcribed by M-MLV (H-) Reverse Transcriptase (Invitrogen) to synthesise the first strand of cDNA. Oligo(dT) 12-primed first-strand cDNA samples (1 µL) were used for PCR, with reverse and forward primers designed on the basis of the published HvST1 gene sequence (accession number Q43482), which amplify a fragment of 699 bp. Primers were as follows: HvST1-1 primer, 5 -CGGATTCTTCAGGCTAGGGT-3 and HvST1-2 primer, 5 -GCCACCATTTCTTTGTTCCC-3 . The level of the 18S gene expression was used as control for quantification. PCR conditions were: 93 and 55◦ C for 1 min each and 72◦ C for 2 min for each cycle, plus a final extension time of 72◦ C for 7 min. The number of PCR cycles was adjusted to obtain detectable amounts of amplicons without reaching signal saturation, which was accomplished with 40 and 30 cycles for HvST1 and 18S (control), respectively. All semiquantitative RT–PCRs were performed in duplicate. RT–PCR amplification products were separated electrophoretically on 1% (w / v) agarose gels and stained with ethidium bromide.

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Fig. 1. Shoot (a) and root (b) dry weight of barley plants grown for 14 d at three S concentrations in the nutrient solution (S0 = 0, S1 = 60 and S2 = 1200 µM sulfate) with (+Fe) or without (−Fe) 100 µM FeIII – EDTA. Inset: shoot : root ratio. Data are means from four independent replications. Error bars indicate s.e.

Other measurements and statistics To determine total S concentration, 1 g of each leaf or root sample was dried at 105◦ C and then ashed in a muffle furnace at 600◦ C. The ashes were dissolved in 10 mL of 3 N HCl and filtered through Whatman No. 42 paper. In contact with BaCl2 , a BaSO4 precipitate is formed which is determined turbidimetrically (Bardsley and Lancaster 1960). Protein content was determined according to Bradford (1976) using BSA as standard. Data reported represent the mean of measurements carried out at least in triplicate and obtained from at least three independent experiments ± s.e.

As previously reported (Astolfi et al. 2006), after 14 d of culture under S0 conditions barley plants showed typical S starvation symptoms, such as reduced plant dry weight (Fig. 1) and decreased photosynthetic rate and chlorophyll content (data not shown). No significant difference in shoot dry weight between the other two treatments (S1 and S2 , 60 and 1200 µM sulfate, respectively) was observed. Root dry weight increased with reduced S supply and consequently shoot : root ratio was lower in S0 and in S1 plants, compared with S2 plants. Fe deprivation caused a significant decrease in shoot dry weight irrespective of the S supply; root dry weight was negatively affected by Fe deprivation only in S1 and in S0 treatments. Analysis of the leaf and root mineral composition showed that the S concentration in S0 and S1 plants was very low compared with S2 plants (−96 and −48% respectively, in leaves; −75 and −70%, respectively, in roots) (Fig. 2).

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Fig. 2. Total S concentration in leaves (a) and roots (b) of 14-d-old barley plants. Plants were grown at three S concentrations in the nutrient solutions (S0 = 0, S1 = 60 and S2 = 1200 µM sulfate) with (+Fe) or without (−Fe) 100 µM FeIII –EDTA. Statistics as for Fig. 1.


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Imposition of Fe deficiency resulted in an increase in the leaf S concentration which was significantly higher than that of the Fe-sufficient plants, showing ∼45 and 60% increase with respect to the control in S0 and S1 plants, respectively. Also in S2 plants Fe deficiency resulted in an increase in leaf S concentration, although to a lesser extent than in plants grown at 60 and 0 µM sulfate. Total S concentration of roots was enhanced by Fe deprivation only in the S1 treatment. Decreased S supply favoured Cys accumulation in roots, and decreased leaf Cys content only in S1 plants (Fig. 3). Furthermore, we observed that Cys content increased at root level when plants were under Fe deficiency, regardless of the S supply level. In particular, under the S1 treatment, root Cys content reached its highest value and it was 5-fold higher than the Fe-sufficient control, whereas under S0 and S2 conditions the measured Cys levels were approximately 2-fold higher than those of the Fe-sufficient controls. At the leaf level, in contrast, Cys content was depressed by Fe starvation, except in S1 plants. Although GSH content was clearly related to S supply in both roots and leaves, it was unaffected by Fe deprivation at any S treatment (Fig. 3). In order to evaluate whether or not changes in the level of Cys were related to changes in sulfate uptake capacity, 35 SO4 2− uptake was measured in plants treated with different levels of S and Fe. Table 1 shows that when sulfate was completely omitted from the NS (S0 ), plants showed increased rates of 35 SO4 2− uptake, similar to previous reports

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(Hawkesford and Smith 1997; Buchner et al. 2004; Kataoka et al. 2004). Fe deprivation further increased 35 SO4 2− uptake rates by 76% in barley plants treated with 60 µM sulfate (S1 ), but did not modify the capability for uptake of 35 SO4 2− in plants subjected to S0 and S2 treatments. In accordance with other reports (Smith et al. 1997; Hawkesford and Wray 2000), greater HvST1 transcript abundance was observed in roots of S-starved (S0 ) plants than in those of S1 and S2 plants (Fig. 4). HvST1 transcript abundance was increased by Fe deficiency in S1 roots, while being unaffected or even decreasing in S0 and S2 roots, respectively. We further investigated the influence of Fe availability on S metabolism through evaluation of changes in activities of ATPS and OASTL, the first and the last enzymes of the S assimilation pathway, respectively (Fig. 5). ATPS and OASTL activities increased in response to S starvation (S0 ) in both roots and leaves but no significant difference between S1 and S2 treatments was found in root and foliar activities of either enzyme. The increase in enzyme activity induced by S starvation was abolished in plants exposed to Fe starvation. Furthermore, Fe deficiency affected ATPS and OASTL activities differently. In particular, Fe deficiency under the lowest S dosage significantly increased the root ATPS activity (+77% of the control; Fig. 5b). We observed an increase in activity of this enzyme in leaves only in S1 and S2 plants (+35 and +60%, respectively) (Fig. 5a). In contrast, OASTL activity in both roots and leaves decreased

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Fig. 3. Cysteine content in leaves (a) and roots (b) and GSH content in leaves (c) and roots (d) of barley plants grown for 14 d at three S concentrations in the NS (S0 = 0, S1 = 60 and S2 = 1200 µM sulfate) with (+Fe) or without (−Fe) 100 µM FeIII –EDTA. Statistics as for Fig. 1.



Table 1. Uptake rate of 35 SO4 2− by barley plants grown hydroponically for 14 d at three S concentrations in the NS (S0 = 0, S1 = 60 and S2 = 1200 ␮M sulfate) with (+Fe) or without (−Fe) 100 ␮M FeIII –EDTA Data are means of four measurements ± s.e.

S0

28.20 ± 0.25 4.11 ± 0.63 3.22 ± 0.49

S0 S1 S2

29.56 ± 0.63 7.24 ± 1.05 2.62 ± 0.49

when plants were subjected to Fe deficiency, the percentage of inhibition varying from 10 to 30% of the Fe-sufficient controls, depending on S treatment (Fig. 5c, d ). Discussion The efficacy of the S deprivation protocol used in the present work is reflected in the production of dry biomass, with plants grown under the S0 treatment showing the lowest shoot biomass accumulation. No significant difference was found in shoot dry mass between S1 and S2 treatments suggesting that the S1 dosage might provide a sufficient S supply. However, it must be considered that both S0 and S1 treatments resulted in higher root dry mass, producing a lower shoot : root ratio than that observed for S2 plants (Fig. 1). The decrease in the shoot : root ratio is considered to be a classical response

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Fig. 4. RT–PCR expression analysis of sulfate transporter gene HvST1 in roots of barley plants. Plants were grown for 14 d at three S concentrations in the nutrient solution (S0 = 0, S1 = 60 and S2 = 1200 µM sulfate) with (+Fe) or without (−Fe) 100 µM FeIII –EDTA. An 18S PCR product was used as a cDNA calibration control.

to S supply, as extensively reported (Robinson 1994). Thus, we regard the S2 level as optimal supply and the S1 and S0 treatments as suboptimal and deficient, respectively. Although total leaf S concentration was decreased with increasing severity of S deficiency, it was increased in leaves of plants grown in the absence of Fe. This result suggests that Fe deficiency might induce an adjustment of S concentration at the leaf level, where S is effectively assimilated (Herschbach et al. 2002). Barley plants possess the Strategy II Fe uptake system and thus release chelating compounds, PS, in response to Fe deficiency. Since PS biosynthesis requires Met (Mori and Nishizawa 1987; Ma et al. 1995), Fe starvation could

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Fig. 5. Changes in (a, b) ATPS (nmol ATP min−1 mg−1 protein) and (c, d ) OASTL (µmol min−1 mg−1 protein) activities in leaves (a, c) and roots (b, d ) of barley plants. Shoots and roots were sampled from plants grown for 14 d at three S concentrations in the NS (S0 = 0, S1 = 60 and S2 = 1200 µM sulfate) with (+Fe) or without (−Fe) 100 µM FeIII –EDTA. Statistics as for Fig. 1.

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conceivably play an important role in regulating the size of the reduced S pool. This is indeed suggested by the observed higher levels of ATPS activity in leaves of S1 and S2 plants and of Cys content in roots of Fe-deficient barley plants. This hypothesis is also consistent with our previous report showing that lowering S supply in NS results in a sharp decrease in the release of PS by Fe-deficient barley plants (Astolfi et al. 2006). While GSH content remained at constitutive levels in plants under Fe stress conditions, a preferential allocation of Cys to the roots was observed, regardless of the level of S supply, suggesting a role for the thiol-containing amino acid in Fe deficiency response. Increased amounts of Cys in roots of Fe-deficient plants grown in S1 and S2 conditions may be supplied either by a greater capacity to take up sulfate from the external medium and / or by translocation of thiol compounds from the shoot. It seems, however, that the adaptation of barley plants to Fe deficiency does not involve enhanced assimilation of sulfate at root level because the activity of two enzymes of the assimilatory pathway (ATPS and OASTL) did not increase. Increase in leaf ATPS activity rather indicates an increase of S assimilation followed by thiols (Cys) translocation to the root. As Met coming from the shoot is not used to synthesise mugineic acid in roots (Nakanishi et al. 1999), our results indicate that thiol compounds produced in the upper part of the plant might be transferred from the shoot to the root and used as the source of reduced sulfide for the synthesis of Met and its derivatives (PS) in the root. In S0 plants the increase in root Cys content could well be sustained by enhanced assimilation (ATPS activity) in the root and translocation from the shoot. Increase in root Cys content of Fe-deficient plants is parallelled by higher rates of 35 SO4 2− uptake only in S1 plants. In fact, data from sulfate uptake experiments show that Fe deficiency clearly enhances uptake rate of 35 SO4 2− only in S1 plants. These results indicate that at suboptimal S supply (S1 ) Fe deprivation enhances sulfate uptake, leaf assimilation and thiols translocation from the leaves to the roots. These changes would poise the root tissue for adequate synthesis of PS. Plants contain several sulfate transporter genes, which may be subdivided into four different groups depending on their specific function. Group 1 sulfate transporters are considered to be responsible for the primary uptake of sulfate by the roots and have a high affinity for sulfate (Hawkesford 2003). Nutrient uptake and transcription of transporter genes generally respond to the nutritional status of plants. In fact the HvST1 gene was clearly up-regulated in S-starved plants (S0 treatment) compared with S1 and S2 plants. However imposition of Fe deficiency caused contrasting effects, with HvST1 gene expression being down-regulated in S0 and S2 plants and up-regulated in S1 plants.

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While the behaviour of plants grown under S0 and S2 treatments might reflect a general means of maintaining a balanced uptake of nutrients (Smith et al. 1999) in conditions of non-limiting (S2 ) or severely limited (S0 ) S supply, it appears that in S1 Fe-deficient plants, which also showed an increased SO4 2− uptake rate, a greater amount of S is needed, possibly in order to sustain adequate PS biosynthesis (Astolfi et al. 2006). These results support the view that the overall nutritional status of plant may influence transcription of nutrient transporters (Smith et al. 2003). In conclusion, this study provides strong evidence that S assimilatory pathway and SO4 2− uptake can be modulated by Fe availability. Our data support the idea that Fe deficiency affects the partitioning of reduced S pool, inducing the translocation of thiols from the shoots to the roots, in order to sustain PS synthesis. Furthermore, it appears that the development of the biochemical responses to severe S deficiency could be prevented by a limited Fe availability. Such mutually influencing relationships between S and Fe should be taken into consideration under several different circumstances encountered in the field, including the occurrence of Fe-retentive soils, cultivated species having a particularly high S requirement, or crops for which S is an important determinant of marketable quality. Acknowledgments Research was supported by grants from Italian M.I.U.R.COFIN 2004. References Astolfi S, Zuchi S, Passera C, Cesco S (2003) Does the sulfur assimilation pathway play a role in the response to Fe deficiency in maize (Zea mays L.) plants? Journal of Plant Nutrition 26, 2111–2121. doi: 10.1081/PLN-120024268 Astolfi S, Zuchi S, Cesco S, Varanini Z, Pinton R (2004) Influence of iron nutrition on sulfur uptake and metabolism in maize (Zea mays L.) roots. Soil Science and Plant Nutrition 50, 1079–1083. Astolfi S, Cesco S, Zuchi S, Neumann G, Roemheld V (2006) Sulfur starvation reduces phytosiderophores release by Fe-deficient barley plants. Soil Science and Plant Nutrition 52, 80–85. Bardsley CE, Lancaster JD (1960) Determination of reserve sulfur and soluble sulfate in soils. Soil Science Society American Proceedings 24, 265–268. Bouranis DL, Chorianopoulou SN, Protonotarios VE, Siyannis VF, Hopkins L, Hawkesford MJ (2003) Leaf response of young ironinefficient maize plants to sulfur deprivation. Journal of Plant Nutrition 26, 1189–1202. doi: 10.1081/PLN-120020364 Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. doi: 10.1016/00032697(76)90527-3 Buchner P, Stuiver EE, Westerman S, Wirtz M, Hell R, Hawkesford MJ, De Kok LJ (2004) Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H2 S and pedospheric sulfate nutrition. Plant Physiology 136, 3396–3408. doi: 10.1104/pp.104.046441


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Manuscript received 19 July 2006, accepted 4 September 2006

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