Zinc transport mediated by barley ZIP proteins are induced by low pH

It is estimated that nearly 50% of the world''s population is at risk of zinc (Zn) deficiency. The challenge is therefore to increase the Zn content in edible plant parts in order to improve the nutritional value of staple foods. We recently reported the identification and characterization of three barley genes encoding zinc transport proteins belonging to the ZIP protein family. These proteins are believed to be involved in cellular uptake of Zn²⁺. In this addendum, the Zn²⁺ transport capacity of ZIP proteins isolated from barley roots was investigated in response to various pH levels. We show that a lowering of pH induces a better growth at low Zn²⁺ concentrations of yeast cells expressing ZIP proteins. However, no significant change in transport capacity (V_max) could be observed for HvIRT1, whereas lowering of pH from 5.5 to 4.2 increased the V_max value with 64% for HvZIP5. These results indicate that proton activity has an important role in regulating the Zn²⁺ transport capacity of Zn²⁺ specific ZIP transport proteins. This information will increase the understanding of ZIP proteins and facilitate engineering of genotypes able to grow efficiently on marginal soils.Key words: ZIP proteins, barley, zinc transport, pH     

Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice

The plant vacuole is an important organelle for storing excess iron (Fe), though its contribution to increasing the Fe content in staple foods remains largely unexplored. In this study we report the isolation and functional characterization of two rice genes OsVIT1 and OsVIT2, orthologs of the Arabidopsis VIT1. Transient expression of OsVIT1:EGFP and OsVIT2:EGFP protein fusions revealed that OsVIT1 and OsVIT2 are localized to the vacuolar membrane. Ectopic expression of OsVIT1 and OsVIT2 partially rescued the Fe²⁺‐ and Zn²⁺‐sensitive phenotypes in yeast mutant Δccc1 and Δzrc1, and further increased vacuolar Fe²⁺, Zn²⁺ and Mn²⁺ accumulation. These data together suggest that OsVIT1 and OsVIT2 function to transport Fe²⁺, Zn²⁺ and Mn²⁺ across the tonoplast into vacuoles in yeast. In rice, OsVIT1 and OsVIT2 are highly expressed in flag leaf blade and sheath, respectively, and in contrast to OsVIT1, OsVIT2 is highly responsive to Fe treatments. Interestingly, functional disruption of OsVIT1 and OsVIT2 leads to increased Fe/Zn accumulation in rice seeds and a corresponding decrease in the source organ flag leaves, indicating an enhanced Fe/Zn translocation between source and sink organs, which might represent a novel strategy to biofortify Fe/Zn in staple foods.     

Characterization of a vacuolar zinc transporter OZT1 in rice (Oryza sativa L.)

The CDF family is a ubiquitous family that has been identified in prokaryotes, eukaryotes, and archaea. Members of this family are important heavy metal transporters that transport metal ions out of the cytoplasm. In this research, a full length cDNA named Oryza sativa Zn Transporter 1 (OZT1) that closely related to rat ZnT-2 (Zn Transporter 2) gene was isolated from rice. The OZT1 encoding a CDF family protein shares 28.2 % ~ 84.3 % of identities and 49.3 % ~ 90.9 % of similarities with other zinc transporters such as RnZnT-2, HsZnT-8, RnZnT-8 and AtMTP1. OZT1 was constitutively expressed in various rice tissues. The OZT1 expression was significantly induced both in the seedlings of japonica rice Nipponbare and indica rice IR26 in response to Zn²⁺ and Cd²⁺ treatments. Besides, OZT1 expression was also increased when exposed to other excess metals, such as Cu²⁺, Fe²⁺ and Mg²⁺. Subcellular localization analysis indicated that OZT1 localized to vacuole. Heterologous expression of OZT1 in yeast increased tolerance to Zn²⁺ and Cd²⁺ stress but not the Mg²⁺ stress. Together, OZT1 is a CDF family vacuolar zinc transporter conferring tolerance to Zn²⁺ and Cd²⁺ stress, which is important to transporting and homeostasis of Zn, Cd or other heavy metals in plants.     

Cadmium uptake kinetics in intact soybean plants

The absorption characteristics of Cd²⁺ by 10- to 12-day-old soybean plants (Glycine max cv Williams) were investigated with respect to influence of Cd concentration on adsorption to root surfaces, root absorption, transport kinetics and interaction with the nutrient cations Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺. The fraction of nonexchangeable Cd bound to roots remained relatively constant at 20 to 25% of the absorbed fraction at solution concentration of 0.0025 to 0.5 micromolar, and increased to 45% at solution concentration in excess of 0.5 micromolar. The exchangeable fraction represented 1.4 to 32% of the absorbed fraction, and was concentration dependent. Using dinitrophenol as a metabolic inhibitor, the `metabolically absorbed' fraction was shown to represent 75 to 80% of the absorbed fraction at concentration less than 0.5 micromolar, and decreased to 55% at 5 micromolar. At comparatively low Cd concentrations, 0.0025 to micromolar 0.3, root absorption exhibited two isotherms with K₂ values of 0.08 and 1.2 micromolar. Root absorption and transfer from root to shoot of Cd²⁺ was inhibited by Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺. Analyses of kinetic interaction of these nutrient cations with Cd²⁺ indicated that Cu²⁺, Fe²⁺, Zn²⁺, and possibly Mn²⁺ inhibited Cd absorption competitively suggesting an involvement of a common transport site or process.     

N-terminus of PutCAX2 from <Emphasis Type="Italic">Puccinellia tenuiflora</Emphasis> affects Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript> tolerance in yeast

Cation/H⁺ exchangers (CAXs) are membrane proteins that transport Ca²⁺ and other cations using the H⁺ gradient generated by H⁺-ATPase or H⁺-pyrophosphatase. This study reports the characterization of CAX2 from Puccinellia tenuiflora with respect to molecular and functional properties. PutCAX2 was cloned from a cDNA library of P. tenuiflora seedlings. The expression of PutCAX2 in shoots and roots was induced by Ca²⁺ and Ba²⁺ treatments. A green fluorescent protein (GFP) marker revealed that PutCAX2 was located on the endoplasmic reticulum (ER) membrane. Four yeast transformants were created using GFP fusion PutCAX2 and truncated PutCAX2s, and their growth in the presence of various cations (Fe³⁺, Al³⁺, Mn²⁺, Cu²⁺, Co²⁺, Ni²⁺, Mg²⁺, Zn²⁺, Na⁺, Li⁺, Ca²⁺, and Ba²⁺) was analyzed. The N-terminally truncated PutCAX2 (GFP-ΔNPutCAX2) and the N and C-terminally truncated PutCAX2 (GFP-ΔNCPutCAX2) transformants grew well in the presence of 100 and 150 mM Ca²⁺ or 8 and 20 mM Ba²⁺, whereas the GFP-PutCAX2 and C-terminally truncated PutCAX2 (GFP-ΔCPutCAX2) transformants did not show any tolerance to Ca²⁺ or Ba²⁺. The Ba²⁺ content in whole yeast cells expressing GFP-ΔNPutCAX2 or GFP-ΔNCPutCAX2 was lower than that in other yeast transformants. Moreover, the efflux experiment showed that the Ba²⁺ efflux rate of yeast cells expressing GFP-ΔNPutCAX2 and GFP-ΔNCPutCAX2 was higher than that of other yeast cells. To our knowledge, this is the first report on the molecular and functional characterization of a novel ER-localized CAX protein from a wild halophyte plant; the results suggest that the N-terminus of PutCAX2 acts as an auto-inhibitory domain, which affects the Ca²⁺ and Ba²⁺ tolerance of yeast.     

Functional characterization of the CDF transporter SMc02724 (SmYiiP) in Sinorhizobium meliloti: Roles in manganese homeostasis and nodulation

In bacteria, membrane transporters of the cation diffusion facilitator (CDF) family participate in Zn^2 +, Fe^2 +, Mn^2 +, Co^2 + and Ni^2 + homeostasis. The functional role during infection processes for several members has been shown to be linked to the specificity of transport. Sinorhizobium meliloti has two homologous CDF genes with unknown transport specificity. Here we evaluate the role played by the CDF SMc02724 (SmYiiP). The deletion mutant strain of SmYiiP (ΔsmyiiP) showed reduced in vitro growth fitness only in the presence of Mn^2 +. Incubation of ΔsmyiiP and WT cells with sub-lethal Mn^2 + concentrations resulted in a 2-fold increase of the metal only in the mutant strain. Normal levels of resistance to Mn^2 + were attained by complementation with the gene SMc02724 under regulation of its endogenous promoter. In vitro, liposomes with incorporated heterologously expressed pure protein accumulated several transition metals. However, only the transport rate of Mn^2 + was increased by imposing a transmembrane H⁺ gradient. Nodulation assays in alfalfa plants showed that the strain ΔsmyiiP induced a lower number of nodules than in plants infected with the WT strain. Our results indicate that Mn^2 + homeostasis in S. meliloti is required for full infection capacity, or nodule function, and that the specificity of transport in vivo of SmYiiP is narrowed down to Mn^2 + by a mechanism involving the proton motive force.     

Effect of Fe2+, Mn2+, Zn2+ and Pb2+ on H+/K+ fluxes in excisedPistia stratiotes roots

Pistia stratiotes is used for the epuration of domestic sewage in the Biyem Assi phytopurification station. During the process, Fe²⁺, Mn²⁺, Zn²⁺ and Pb²⁺ are absorbed in substantial amounts by the plant. These metals modify the H⁺/K⁺ exchange system at the root level. H⁺ efflux is inhibited by Fe²⁺ and by Zn²⁺ and enhanced by Mn²⁺ and Pb²⁺. K⁺ influx is inhibited by Fe²⁺, by Zn²⁺ and by Pb²⁺ and enhanced by Mn²⁺. It is shown that the purification capacity ofPistia stratiotes can vary with the composition of the heavy metals in the surrounding medium.     

Free metal activity and total metal concentrations as indices of micronutrient availability to barley [Hordeum vulgare (L.) ‘Klages’]

The form in which a micronutrient is found in the rhizosphere affects its availability to plants. We compared the availability to barley of the free hydrated cation form of Fe³⁺, Cu²⁺, Zn²⁺, and Mn²⁺ versus their total metal concentrations (free ion plus complexes) in chelator-buffered solutions. Free metal ion activities were estimated using the chemical equilibrium program GEOCHEM-PC with the corrected database. In experiment 1, barley was grown in nutrient solutions with different Fe³⁺ activities using chelators to control Fe levels. Chlorosis occurred at Fe³⁺ activities of 10^–18 and 10^–19 M for barley grown in HEDTA and EDTA solutions, respectively. In experiment 2, barley was grown in nutrient solutions with the same calculated Fe³⁺ activity and the same chelator, but different total Fe concentrations. Leaf, root and shoot Fe concentrations were higher from CDTA buffered solutions which had the higher total Fe concentration indicating the importance of the total Fe concentration on Fe uptake. Results from treatments using EDTA or HEDTA, with one exception, were similar to the results from the CDTA treatment. This suggests differences in critical Fe³⁺ activities found in experiment 1 were due to differences in the total Fe concentration and not errors in chelate formation constants used to estimate the critical activities. Results for Cu, Zn, and Mn were similar to Fe; despite solutions with equal free Cu²⁺, Zn²⁺ and Mn²⁺ activities, plant concentrations of these metals were generally greater when grown in the solutions with the greater total amount of Cu, Zn, or Mn. When the free Zn²⁺ activity was kept constant while the total amount of Zn was increased from 4.4 to 49 M, leaf Zn concentration increased from 77 to 146 g g^-1. In order to predict metal availability to barley and other species in chelator-buffered nutrient solutions, both free and total metal concentrations in solution must be considered. The critical Fe³⁺ activities required by barley in this study are much higher than those from tomato and soybean, 10^-28 M, which strongly supports the Strategy 2 model of Fe uptake for Poaceae. This is related to the importance of the Fe³⁺ (barley) and the Fe²⁺ (tomato and soybean) ions in Fe uptake. Fe-stressed barley is known to release phytosiderophores which compete for Fe³⁺ in the nutrient solution, while tomato and soybean reduce Fe³⁺ to Fe²⁺ at the epidermal cell membranes to allow uptake of Fe²⁺ from Fe³⁺ chelates in solution.Abbreviations CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetracetic acid - DTPA diethylenetriaminepentacetic acid - EDTA ethylenediaminetetracetic acid - EDDHA ethylenediamine-di(o-hydroxyphenylacetic acid) - HBED-N,N di(2-hydroxybenzoyl)-ethylenediamine-N,N-diacetic acid - HEDTA-N hydroxyethylenediaminetriacetic acid - MES-2 (N-morpholino)ethanesulfonic acid - NTA nitrilotriacetic acid     

Substrate Profile and Metal-ion Selectivity of Human Divalent Metal-ion Transporter-1

Divalent metal-ion transporter-1 (DMT1) is a H⁺-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluorescence with the metal-sensitive PhenGreen SK fluorophore. We provide validation for the use of PhenGreen SK fluorescence quenching as a reporter of cellular metal-ion uptake. We determined metal-ion selectivity under fixed conditions using the voltage clamp. Radiotracer and continuous measurement of transport by fluorescence assays revealed that DMT1 mediates the transport of several metal ions that were ranked in selectivity by using the ratio I_max/K_0.5 (determined from evoked currents at −70 mV): Cd²⁺ > Fe²⁺ > Co²⁺, Mn²⁺ ≫ Zn²⁺, Ni²⁺, VO²⁺. DMT1 expression did not stimulate the transport of Cr²⁺, Cr³⁺, Cu⁺, Cu²⁺, Fe³⁺, Ga³⁺, Hg²⁺, or VO⁺. ⁵⁵Fe²⁺ transport was competitively inhibited by Co²⁺ and Mn²⁺. Zn²⁺ only weakly inhibited ⁵⁵Fe²⁺ transport. Our data reveal that DMT1 selects Fe²⁺ over its other physiological substrates and provides a basis for predicting the contribution of DMT1 to intestinal, nasal, and pulmonary absorption of metal ions and their cellular uptake in other tissues. Whereas DMT1 is a likely route of entry for the toxic heavy metal cadmium, and may serve the metabolism of cobalt, manganese, and vanadium, we predict that DMT1 should contribute little if at all to the absorption or uptake of zinc. The conclusion in previous reports that copper is a substrate of DMT1 is not supported.     

Transcriptional Regulation,Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn²⁺ and Mn²⁺ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn²⁺ or Mn²⁺ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn²⁺ over Mn²⁺ specificity, the Zn²⁺ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn²⁺-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn²⁺-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn²⁺-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn²⁺ specificity.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K⁺, Na⁺, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Ni²⁺, Zn²⁺, and Li⁺) were analyzed. AtCCX5 expression was found to affect the response to K⁺ and Na⁺ in yeast. The AtCCX5 transformant also showed a little better growth to Zn²⁺. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K⁺ (0.5 mM), and also suppressed its Na⁺ sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K⁺ uptake and was also involved in Na⁺ transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K⁺ uptake and Na⁺ transport in yeast.     

Zinc induces DNA damage in tobacco roots

We applied the alkaline version of the single-cell gel electrophoresis (comet assay) to seedlings of heterozygous tobacco (Nicotiana tabacum L. var. xanthi) treated with zinc acetate dihydrate (20 to 80 mM Zn²⁺ for 2 h or 2 to 12 mM Zn²⁺ for 24 h). A dose dependent increase in DNA damage expressed by the tail moment values were observed in nuclei isolated from the roots after 2 and 24 h Zn²⁺ treatments. In contrast, Zn²⁺ did not induce significant DNA damage to leaf nuclei, with the exception of 10 or 12 mM Zn²⁺ for 24 h. Somatic mutations, identified as dark green, yellow, and dark green/yellow double sectors on the pale green tobacco leaves were not detected after any Zn²⁺ treatments. The accumulation of Zn in roots and shoots was determined by inductively coupled plasma optical emission spectrometry and the Zn content in roots was about three times higher than in shoots.     

Identification and Characterization of Maize and Barley Lsi2-Like Silicon Efflux Transporters Reveals a Distinct Silicon Uptake System from That in Rice

Silicon (Si) uptake has been extensively examined in rice (Oryza sativa), but it is poorly understood in other gramineous crops. We identified Low Silicon Rice 2 (Lsi2)-like Si efflux transporters from two important gramineous crops: maize (Zea mays) and barley (Hordeum vulgare). Both maize and barley Lsi2 expressed in Xenopus laevis oocytes showed Si efflux transport activity. Furthermore, barley Lsi2 was able to recover Si uptake in a rice mutant defective in Si efflux. Maize and barley Lsi2 were only expressed in the roots. Expression of maize and barley Lsi2 was downregulated in response to exogenously applied Si. Moreover, there was a significant positive correlation between the ability of roots to absorb Si and the expression levels of Lsi2 in eight barley cultivars, suggesting that Lsi2 is a key Si transporter in barley. Immunostaining showed that maize and barley Lsi2 localized only at the endodermis, with no polarity. Protein gel blot analysis indicated that maize and barley Lsi2 localized on the plasma membrane. The unique features of maize and barley Si influx and efflux transporters, including their cell-type specificity and the lack of polarity of their localization in Lsi2, indicate that these crops have a different Si uptake system from that in rice.     

The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe²⁺ homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe²⁺ utilization or transport under iron-limiting conditions. To have a more direct test for Mrs3/4p mediated iron uptake into mitochondria we studied iron (II) transport across yeast inner mitochondrial membrane vesicles (SMPs) using the iron-sensitive fluorophore PhenGreen SK (PGSK). Wild-type SMPs showed rapid uptake of Fe²⁺ which was driven by the external Fe²⁺ concentration and stimulated by acidic pH. SMPs from the double deletion strain mrs3/4Δ failed to show this rapid Fe²⁺ uptake, while SMPs from cells overproducing Mrs3/4p exhibited increased Fe²⁺ uptake rates. Cu²⁺ was transported at similar rates as Fe²⁺, while other divalent cations, such as Zn²⁺ and Cd²⁺ apparently did not serve as substrates for the Mrs3/4p transporters. We conclude that the carrier proteins Mrs3p and Mrs4p transport Fe²⁺ across the inner mitochondrial membrane. Their activity is dependent on the pH gradient and it is stimulated by iron shortage.     

Derivatives of the purple phosphatase from red kidney bean: Replacement of zinc with other divalent metal ions

Apoenzyme, containing ⩽0.1 zinc atoms and ⩽0.2 Fe atoms per subunit and with ⩽3% of the phosphatase activity, has been prepared from native red kidney bean purple phosphatase. Treatment of this apoenzyme with Fe³⁺ or Zn²⁺ separately gave very little recovery of activity, whereas treatment with both Fe³⁺ and Zn²⁺ resulted in complete restoration of activity, indicating that both metal ions are essential. ZnFe enzyme with close to one iron and one zinc atom per subunit has been reconstituted by this procedure. Essentially full reactivation was also achieved by addition of Fe³⁺ together with Fe²⁺ or Co²⁺ to the apoenzyme; Fe³⁺ and Cd²⁺ gave 27% restoration of activity, whereas Fe³⁺ with Mn²⁺, Cu²⁺, Ni²⁺ or Hg²⁺ gave little or no increase in activity. Kinetic parameters for the hydrolysis of p-nitrophenyl phosphate and ATP by the FeFe derivative are reported.     

不同浓度Fe2+与Zn2+对羊草幼苗生长和生理特性的影响

以羊草幼苗为研究对象,通过调整全营养培养基(CK,0.05 mmol/L Fe²⁺、0.015 mmol/L Zn²⁺)中铁或者锌含量设置0、10倍、20倍Fe²⁺(Zn²⁺)浓度处理Fe₀(Zn₀)、Fe₁₀(Zn₁₀)、Fe₂₀(Zn₂₀),以及在高铁培养基中单独添加0.15 mmol/L Zn²⁺或同时添加10 mmol/L Ca²⁺、5 mmol/L Mg²⁺、20 mmol/L K⁺处理,测定培养6 d后幼苗生长指标和矿质元素含量、以及高铁(Fe₂₀)处理下幼苗根中抗氧化指标和相关基因表达量,探究不同浓度Fe²⁺、Zn²⁺对羊草幼苗生长、矿质元素吸收积累及抗氧化指标、基因表达的影响。结果表明：(1)缺锌(Zn₀)显著抑制羊草幼苗鲜重的增加和Zn元素的积累,但促进Fe、Mg元素的积累;高浓度锌(Zn₁₀、Zn₂₀)显著促进幼苗叶片生长和Zn元素的积累;缺铁(Fe₀)显著抑制幼苗的根长、鲜重和Fe元素的积累,促进Mg、Zn元素的积累;高浓度铁(Fe₁₀、Fe₂₀)显著抑制羊草幼苗根叶生长、根毛发育和Ca、Zn、Mg、K元素的积累。(2)增加Zn²⁺和Ca²⁺、Mg²⁺、K⁺浓度无法恢复高铁胁迫对幼苗生长的抑制作用。(3)高浓度铁(Fe₂₀)处理羊草幼苗48 h后,根部过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、谷胱甘肽还原酶活性和丙二醛、抗坏血酸、还原型谷胱甘肽含量显著升高;烟酰胺合成酶基因、过氧化物酶基因表达量显著下调,植物类萌发素蛋白基因表达量显著上调。研究发现,羊草幼苗生长发育和矿质元素积累对环境中Zn²⁺浓度变化不敏感,却受到环境中高浓度Fe²⁺的显著抑制,并造成严重的氧化胁迫伤害,这种伤害无法在添加Zn²⁺或同时添加Ca²⁺、Mg²⁺、K⁺的条件下恢复。     

Effects of Metals on 3-Acetyldeoxynivalenol Production by Fusarium graminearum R2118 in Submerged Cultures

The effects of selected metals (Mg²⁺, Mn²⁺, Zn²⁺, and Fe²⁺) on 3-acetyldeoxynivalenol (3-ADN) production by Fusarium graminearum R2118 and on its mycelial growth were investigated by using a two-stage submerged-culture technique. In certain concentrations ranges, Mg²⁺ and Fe²⁺ stimulated growth but suppressed 3-ADN production; at other concentrations, Mg²⁺, Fe²⁺, and Zn²⁺ suppressed growth but stimulated 3-ADN production. In contrast, Mn²⁺ stimulated growth but totally inhibited 3-ADN production at all concentrations tested. In general, the production of 3-ADN was inversely related to the growth rate of the fungus with these metals. Mn²⁺ appears to be a crucial factor regulating the onset of 3-ADN biosynthesis.     

Characterization of a PutCAX1 gene from Puccinellia tenuiflora that confers Ca and Ba tolerance in yeast

The gene for a novel cation/H⁺ antiporter from Puccinellia tenuiflora, PutCAX1, was cloned from a cDNA library. The PutCAX protein was localized in the vacuolar membrane using a GFP marker. Several yeast transformants were created using full-length and truncated form of PutCAX1 and their growths in the presence of various cations (Mg²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Se²⁺, and Ba²⁺) were analyzed. PutCAX1 expression was found to affect the response to Ca²⁺ and Ba²⁺ in yeast. The PutCAX1 and C-terminally truncated PutCAX1 (ΔCPutCAX1) transformants grew in the presence of 70 mM Ca²⁺ as well as in the presence of 8 mM Ba²⁺. However, the ΔCPutCAX1 transformant was able to grow in the presence of 20 mM Ba²⁺ while the PutCAX1 transformant could not. On the other hand, expression of the N-terminally truncated form and the N- and C-terminally truncated form failed to suppress the Ca²⁺ or Ba²⁺ sensitivity of yeast. These results suggest that PutCAX1 can complement the active Ca²⁺ transporters at some level and confer yeast Ba²⁺ tolerance, and that the N- and C-terminal regions of PutCAX1 play important roles in increasing the Ca²⁺ or Ba²⁺ tolerance of yeast.