Complementation Of Yeast Mutants Research Articles

Functional characterization of Fagopyrum tataricum ZIP gene family as a metal ion transporter.

The zinc/iron-regulated transporter-like proteins (ZIP) family acts as an important transporter for divalent metal cations such as Zn, Fe, Mn, Cu, and even Cd. However, their condition is unclear in Tartary buckwheat (Fagopyrum tataricum). Here, 13 ZIP proteins were identified and were predicted to be mostly plasma membrane-localized. The transient expressions of FtZIP2 and FtZIP6 in tobacco confirmed the prediction. Multiple sequence alignment analysis of FtZIP proteins revealed that most of them had 8 putative transmembrane (TM) domains and a variable region rich in histidine residues between TM3 and TM4, indicating the reliable affinity to metal ions. Gene expression analysis by qRT-PCR showed that FtZIP genes were markedly different in different organs, such as roots, stems, leaves, flowers, fruits and seeds. However, in seedlings, the relative expression of FtZIP10 was notably induced under the CdCl2 treatment, while excessive Zn2+, Fe2+, Mn2+ and Cd2+ increased the transcript of FtZIP5 or FtZIP13, in comparison to normal conditions. Complementation of yeast mutants with the FtZIP family genes demonstrate that FtZIP7/10/12 transport Zn, FtZIP5/6/7/9/10/11 transport Fe, FtZIP12 transports Mn and FtZIP2/3/4/7 transport Cd. Our data suggest that FtZIP proteins have conserved functions of transportation of metal ions but with distinct spatial expression levels.

Molecular Cloning, Expression Analysis, and Functional Analysis of Nine IbSWEETs in Ipomoea batatas (L.) Lam

Sugar Will Eventually be Exported Transporter (SWEET) genes play an important regulatory role in plants’ growth and development, stress response, and sugar metabolism, but there are few reports on the role of SWEET proteins in sweet potato. In this study, nine IbSWEET genes were obtained via PCR amplification from the cDNA of sweet potato. Phylogenetic analysis showed that nine IbSWEETs separately belong to four clades (Clade I~IV) and contain two MtN3/saliva domains or PQ-loop superfamily and six~seven transmembrane domains. Protein interaction prediction showed that seven SWEETs interact with other proteins, and SWEETs interact with each other (SWEET1 and SWEET12; SWEET2 and SWEET17) to form heterodimers. qRT-PCR analysis showed that IbSWEETs were tissue-specific, and IbSWEET1b was highly expressed during root growth and development. In addition to high expression in leaves, IbSWEET15 was also highly expressed during root expansion, and IbSWEET7, 10a, 10b, and 12 showed higher expression in the leaves. The expression of SWEETs showed a significant positive/negative correlation with the content of soluble sugar and starch in storage roots. Under abiotic stress treatment, IbSWEET7 showed a strong response to PEG treatment, while IbSWEET10a, 10b, and 12 responded significantly to 4 °C treatment and, also, at 1 h after ABA, to NaCl treatment. A yeast mutant complementation assay showed that IbSWEET7 had fructose, mannose, and glucose transport activity; IbSWEET15 had glucose transport activity and weaker sucrose transport activity; and all nine IbSWEETs could transport 2-deoxyglucose. These results provide a basis for further elucidating the functions of SWEET genes and promoting molecular breeding in sweet potato.

Systematic Identification and Expression Analysis of the Sorghum Pht1 Gene Family Reveals Several New Members Encoding High-Affinity Phosphate Transporters.

Sorghum (Sorghum bicolor) is known to have a more robust capability of phosphorus uptake than many other cereal plants, which could be attributed to its phosphate transporter 1 (Pht1) that has a high phosphorus affinity. There are eleven SbPht1 genes in the sorghum genome, nine of which are expressed in sorghum roots or shoots in response to phosphorus deficiency (low-P). The molecular features of these nine genes were investigated by gene expression analysis, subcellular localization, and a yeast mutant complementation growth assay. They were found to be induced in response to low-P stress in root or shoot. All these SbPht1 proteins were found to be localized on the cell membrane, and SbPht1;8 was also detected in the endoplasmic reticulum. These SbPht1s were able to complement the yeast mutant EY917 that lacks all the functional phosphate transporters, and, among them, SbPht1;5, SbPht1;6 and SbPht1;8 could partially complement the yeast mutant strain EY917 in low-P conditions. Overall, these findings demonstrate that SbPht1;5, SbPht1;6, and SbPht1;8 are high-affinity phosphate transporters. SbPht1;5, in particular, is specifically involved in phosphorus uptake in the roots, whilst SbPht1;6 and SbPht1;8 are key players in both P uptake and P transport in response to low-P stress in sorghum.

Cloning of Nitrate Reductase and Nitrite Reductase Genes and Their Functional Analysis in Regulating Cr(VI) Reduction in Ectomycorrhizal Fungus Pisolithus sp.1.

Assimilatory-type nitrate reductase (NR) and nitrite reductase (NiR) are the key enzymes that involve in nitrate assimilation and nitrogen cycling in microorganisms. NR and NiR with NADH or NADPH and FMN or FAD domains could be coupled to the reduction process of hexavalent chromium [Cr(VI)] in microorganisms. A new assimilatory-type NR gene (named niaD) and a new assimilatory-type NiR gene (named niiA) are cloned, identified, and functionally characterized by 5′ and 3′ RACE, alignment, annotation, phylogenetic tree, and yeast mutant complementation analyses from Pisolithus sp.1, a dominant symbiotic ectomycorrhizal fungi (EMF) that can assist in phytoremediation. Assimilatory-type niaD and niiA were 2,754 bp and 3,468 bp and encode a polypeptide with 917 and 1,155 amino acid residues, respectively. The isoelectric points of NR (Pisolithus sp.1 NR) and NiR (Pisolithus sp.1 NiR) of Pisolithus sp.1 are 6.07 and 6.38, respectively. The calculated molecular mass of Pisolithus sp.1 NR and Pisolithus sp.1 NiR is 102.065 and 126.914 kDa, respectively. Yeast mutant complementation analysis, protein purification, and activities of NR and NiR under Cr treatment suggest that Pisolithus sp.1 NR is a functional NR that mediates Cr(VI) tolerance and reduction. The multiple alignment demonstrates that Pisolithus sp.1 NR is potentially a nicotinamide adenine dinucleotide phosphate-dependent flavin mononucleotide reductase and also Class II chromate reductase. Our results suggest that Pisolithus sp.1 NR plays a key role in Cr(VI) reduction in the EMF Pisolithus sp.1.

Metal tolerance protein MTP6 is involved in Mn and Co distribution in poplar

With the booming demand of the electric vehicle industry, the concentration of manganese (Mn) and cobalt (Co) flowing into land ecosystems has also increased significantly. While these transition metals can promote the growth and development of plants, they may become toxic under high concentrations. It is thus important to understand how Mn and Co are distributed in plants to develop novel germplasms for the remediation of these heavy metals in contaminated soils. Here, an MTP gene that encodes the CDF (cation diffusion facilitator) protein in Populus trichocarpa, PtrMTP6, was screened as the key gene involved in the distribution of both Mn and Co in poplar. The PtrMTP6-GFP fusion protein was co-localized with the mRFP-VSR2, showing that PtrMTP6 proteins are present at the pre-vacuolar compartment (PVC). Yeast mutant complementation assays further identified that PtrMTP6 serves as a Mn and Co transporter, reducing yeast cell toxicity after exposure to excessive Mn or Co. Histochemical analyses showed that PtrMTP6 was mainly expressed in phloem, suggesting that PtrMTP6 probably involved in the Mn and Co transport via phloem in plants. Under excess Co, PtrMTP6 overexpressing poplar lines were more severely damaged than the control due to higher Co accumulations in young tissue. PtrMTP6 overexpressing lines showed little change in their tolerance to excess Mn, although young tissues also accumulated more Mn. PtrMTP6 play important roles in Mn and Co distribution in poplar and further research on its regulation will be important to increase bioremediation in Mn and Co polluted ecosystems.

GhLIP1, a lipoic acid synthase gene, negatively regulates leaf senescence in cotton

Leaves are the key organs of plants that produce photosynthates. Leaf senescence aids nutrient remobilization from source to sink tissues, which dramatically affects crop quality and yield. Although several senescence-associated genes (SAGs) have been recognized in cotton, the function of lipoic acid synthase in leaf senescence remains unclear. Therefore, we isolated a lipoic acid synthase gene named GhLIP1 from cotton (Gossypium hirsutum L.). Protein domain analysis revealed that GhLIP1 had a LIAS-N domain and an Elp3 domain, and yeast mutant complementation experiments demonstrated that GhLIP1 was a lipoic acid synthase. GhLIP1 expression pattern analysis showed that GhLIP1 was vastly expressed in fibres, and the phytohormone IAA (indole-3-acetic acid) induced its transcription. When GhLIP1 was transformed into Arabidopsis, the leaves of overexpression Arabidopsis exhibited a late leaf senescence phenotype compared to wild-type Arabidopsis, Col-0. Moreover, downregulation of GhLIP1 expression using virus-induced gene silencing (VIGS) technology resulted in early leaf senescence in cotton. qRT-PCR analyses revealed that two SAGs, GhWRKY53 and GhNAP, were upregulated in cotton plants with GhLIP1 knock-down. Cell ultrastructure observations found that chloroplasts were degraded in an orderly manner in cotton leaf cells with GhLIP1 knock-down. These results suggest that GhLIP1 plays a significant role in leaf senescence in cotton.

Expression analysis and functional characterization of two PHT1 family phosphate transporters in ryegrass.

The phosphate transporters LpPHT1;1 and LpPHT1;4 have different roles in phosphate uptake and translocation in ryegrass under P stress condition. The phosphate transporter 1 (PHT1) family are integral membrane proteins that operate in phosphate uptake, distribution and remobilization within plants. In this study, we report on the functional characterization and expression of two PHT1 family members from ryegrass plants (Lolium perenne L.) and determine their roles in the specificity of Pi transport. The expression level of LpPHT1;4 was strongly influenced by phosphorus (P) status, being higher under P-starvation condition. In contrast, the expression level of LpPHT1;1 was not correlated with P supply. Yeast mutant complementation assays showed that LpPHT1;4 can complement the growth defect of the yeast mutant Δpho84 under Pi-deficient conditions, whereas the yeast mutant expressing LpPHT1;1 was not able to restore growth. Phylogenetic and molecular analyses indicated high sequence similarity to previously identified PHT1s from other species in the Poaceae. These results suggest that LpPHT1;1 may function as a low-affinity Pi transporter, whereas LpPHT1;4 could acts as a high-affinity Pi transporter to maintain Pi homeostasis under stress conditions in ryegrass plants. This study will form the basis for the long-term goal of improving the phosphate use efficiency of ryegrass plants.

Recretohalophyte Tamarix TrSOS1 confers higher salt tolerance to transgenic plants and yeast than glycophyte soybean GmSOS1

The SOS1 (Salt Overly Sensitive 1) gene has been widely investigated in plants including many glycophytes and a few euhalophytes. The encoded plasma membrane Na+/H+ antiporter, SOS1, is mainly responsible for transporting Na+ outside the cell and reducing the Na+ content in plants under salt stress, and has been considered a key determinant in conferring plant salt tolerance. However, studies on the function of the SOS1 gene from recretohalophyte Tamarix have not yet been reported. In this work, the TrSOS1 gene was first transformed into Arabidopsis wild type and the sos1 mutant mediated by Agrobacterium tumefaciens and the soybean hairy root-composite plant by A. rhizogenes. Then, alongside the glycophyte soybean GmSOS1 gene as a reference, the TrSOS1 gene, including genes derived by site-directed mutagenesis, were transformed into Saccharomyces cerevisiae wild type and mutants. The differences in growth phenotype and physiological responses under salt stress were compared. The results showed that, overexpression of the TrSOS1 gene in Arabidopsis wild type and sos1 mutant could significantly improve transgenic plant growth and reduce Na+ content and Na+/K+ ratio in the shoots. As a result, the secondary oxidative stress was also alleviated by reducing the O2−· production rate, H2O2 and malondialdehyde (MDA) contents in the shoots, thereby displaying enhanced salt tolerance and the complementary effects on the sos1 mutant were more prominent. The overexpression of TrSOS1 in yeast mutants (ena1nha1 or ena1nhx1) also suppressed salt sensitivity by reducing the cellular Na+ content and Na+/K+ ratio, especially for the plasma membrane antiporter NHA1 mutant, ena1nha1. In terms of the yeast mutant complementation, the effects of overexpressing the intact TrSOS1 or mutated TrSOS1A238V gene were superior to those of the GmSOS1 or GmSOS1A241V gene, regardless of the yeast growth phenotype or the regulation of cellular Na+ and K+ levels. This reveals that the recretohalophyte Tamarix TrSOS1 gene may have higher efficiency than the glycophyte soybean GmSOS1 gene in regulating Na+ efflux, maintaining Na+ and K+ homeostasis, and therefore contributing to stronger salt tolerance.

The arbuscular mycorrhizal fungus Rhizophagus irregularis uses a reductive iron assimilation pathway for high-affinity iron uptake.

Arbuscular mycorrhizal (AM) fungi can improve iron (Fe) acquisition of their host plants. Here, we report a characterization of two components of the high-affinity reductive Fe uptake system of Rhizophagus irregularis, the ferric reductase (RiFRE1) and the high affinity Fe permeases (RiFTR1-2). In the extraradical mycelia (ERM), Fe deficiency induced activation of a plasma membrane-localized ferric reductase, an enzyme that reduces Fe(III) sources to the more soluble Fe(II). Yeast mutant complementation assays showed that RiFRE1 encodes a functional ferric reductase and RiFTR1 an iron permease. In the heterologous system, RiFTR1 was expressed in the plasma membrane while RiFTR2 was expressed in the endomembranes. In the ERM, the highest expression levels of RiFTR1 were found in mycelia grown in media with 0.045mM Fe, while RiFTR2 was upregulated under Fe-deficient conditions. RiFTR2 expression also increased in the intraradical mycelia (IRM) of maize plants grown without Fe. These data indicate that the Fe permease RiFTR1 plays a key role in Fe acquisition and that RiFTR2 is involved in Fe homeostasis under Fe-limiting conditions. RiFTR1 was highly expressed in the (IRM), which suggests that the maintenance of Fe homeostasis in the IRM might be essential for a successful symbiosis.

The inositol pyrophosphate synthesis pathway in Trypanosoma brucei is linked to polyphosphate synthesis in acidocalcisomes.

Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP2 ), inositol 1,4,5-trisphosphate (IP3 ), and inositol hexakisphosphate (IP6 ) in T. brucei different stages. Bloodstream forms unable to produce inositol pyrophosphates, due to downregulation of TbIPMK expression by conditional knockout, have reduced levels of polyphosphate and altered acidocalcisomes. Our study links the inositol pyrophosphate pathway to the synthesis of polyphosphate in acidocalcisomes, and may lead to better understanding of these organisms and provide new targets for drug discovery.

Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism.

The amount of cholesterol made by many plants is not negligible. Whereas cholesterogenesis in animals was elucidated decades ago, the plant pathway has remained enigmatic. Among other roles, cholesterol is a key precursor for thousands of bioactive plant metabolites, including the well-known Solanum steroidal glycoalkaloids. Integrating tomato transcript and protein co-expression data revealed candidate genes putatively associated with cholesterol biosynthesis. A combination of functional assays including gene silencing, examination of recombinant enzyme activity and yeast mutant complementation suggests the cholesterol pathway comprises 12 enzymes acting in 10 steps. It appears that half of the cholesterogenesis-specific enzymes evolved through gene duplication and divergence from phytosterol biosynthetic enzymes, whereas others act reciprocally in both cholesterol and phytosterol metabolism. Our findings provide a unique example of nature's capacity to exploit existing protein folds and catalytic machineries from primary metabolism to assemble a new, multi-step metabolic pathway. Finally, the engineering of a 'high-cholesterol' model plant underscores the future value of our gene toolbox to produce high-value steroidal compounds via synthetic biology.

Molecular cloning and functional analysis of a H+-dependent phosphate transporter gene from the ectomycorrhizal fungus Boletus edulis in southwest China

Phosphate transporters (PTs), as entry points for phosphorus (P) in organisms, are involved in a number of P nutrition processes such as phosphate uptake, transport, and transfer. In the study, a PT gene 1632 bp long (named BePT) was cloned, identified, and functionally characterized from Boletus edulis. BePT was expected to encode a polypeptide with 543 amino acid residues. The BePT polypeptide belonged to the major facilitator superfamily and showed a high degree of sequence identity to the Pht1 family. A topology model revealed that BePT exhibited 12 transmembrane helices, divided into two halves, and connected by a large hydrophilic loop in the middle. A yeast mutant complementation analysis suggested that BePT was a functional PT which mediated orthophosphate uptake of yeast at micromolar concentrations. Green fluorescent protein–BePT fusion proteins expressed were extensively restricted to the plasma membrane in BePT transformed yeast, and its activity was dependent on electrochemical membrane potential. In vitro, quantitative PCR confirmed that the expression of BePT was significantly upregulated at lower phosphorus availability, which may enhance phosphate uptake and transport under phosphate starvation. Our results suggest that BePT plays a key role in phosphate acquisition in the ectomycorrhizal fungus B. edulis.

The hybrid four-CBS-domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1.

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy-sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ-like proteins, plants also encode a hybrid βγ protein that combines the Four-Cystathionine β-synthase (CBS)-domain (FCD) structure in γ subunits with a glycogen-binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in-depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast-containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre-CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.

GintAMT2, a new member of the ammonium transporter family in the arbuscular mycorrhizal fungus Glomus intraradices

In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungus delivers mineral nutrients, such as phosphate and nitrogen, to the plant while receiving carbon. Previously, we identified an NH4+ transporter in the AM fungus Glomus intraradices (GintAMT1) involved in NH4+ uptake from the soil when preset at low concentrations. Here, we report the isolation and characterization of a new G. intraradicesNH4+ transporter gene (GintAMT2). Yeast mutant complementation assays showed that GintAMT2 encodes a functional NH4+ transporter. The use of an anti-GintAMT2 polyclonal antibody revealed a plasma membrane location of GintAMT2. GintAMT1 and GintAMT2 were differentially expressed during the fungal life cycle and in response to N. In contrast to GintAMT1, GintAMT2 transcript levels were higher in the intraradical than in the extraradical fungal structures. However, transcripts of both genes were detected in arbuscule-colonized cortical cells. GintAMT1 expression was induced under low N conditions. Constitutive expression of GintAMT2 in N-limiting conditions and transitory induction after N re-supply suggests a role for GintAMT2 to retrieve NH4+ leaked out during fungal metabolism.

Identification of a calmodulin-regulated autoinhibited Ca2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis

In plant cells, the vacuole functions as a major calcium store. Although a calmodulin-regulated Ca2+-ATPase (ACA4) is known to be present in prevacuolar compartments, the presence of an ACA-type Ca2+-ATPase in the mature vacuole of a plant cell has not been verified. Here we provide evidence that ACA11 localizes to the vacuole membrane. ACA11 tagged with GFP was expressed in stable transgenic plants, and visualized in root cells and protoplasts by confocal microscopy. A Ca2+-ATPase function for ACA11 was confirmed by complementation of yeast mutants. A calmodulin binding domain was identified within the first 37 residues of the N-terminal autoinhibitory region.

Development of a set of plasmid vectors for genetic manipulations of the pathogenic yeast Candida parapsilosis

A system for genetic transformation of the yeast Candida parapsilosis, recently developed in our laboratory, opened a venue for investigation of this pathogenic species at the molecular level. In this study we extend the range of available experimental tools by construction of a genomic DNA library suitable for screening and isolation of genes by functional complementation of yeast mutants and a set of replicative plasmid vectors for genetic manipulation of C. parapsilosis cells. The plasmids are based on auxotrophic ( CpGAL1, CpURA3, CpMET2, CpLYS4) and dominant ( CaIMH3) selection markers. In addition, we constructed plasmid derivatives containing reporter genes yEGFP3 and KlLAC4 coding for enhanced version of the green fluorescent protein and Kluyveromyces lactis β-galactosidase, respectively. The vectors facilitate propagation and expression of cloned genes in C. parapsilosis cells and allow intracellular localization of gene products and/or monitoring the activity of promoter sequences.

Molecular Mechanisms of Urea Transport in Plants

Urea is a soil nitrogen form available to plant roots and a secondary nitrogen metabolite liberated in plant cells. Based on growth complementation of yeast mutants and "in-silico analysis", two plant families have been identified and partially characterized that mediate membrane transport of urea in heterologous expression systems. AtDUR3 is a single Arabidopsis gene belonging to the sodium solute symporter family that cotransports urea with protons at high affinity, while members of the tonoplast intrinsic protein (TIP) subfamily of aquaporins transport urea in a channel-like manner. The following review summarizes current knowledge on the membrane localization, energetization and regulation of these two types of urea transporters and discusses their possible physiological roles in planta.

AtSGP1, AtSGP2 and MAP4K alpha are nucleolar plant proteins that can complement fission yeast mutants lacking a functional SIN pathway.

In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is signalled via the septation initiation network (SIN) involving several protein kinases and a GTPase. Arabidopsis thaliana and Brassica napus proteins homologous to fission yeast spg1p (AtSGP1, AtSGP2), cdc7p (AtMAP3K epsilon 1, AtMAP3K epsilon 2, BnMAP3K epsilon 1) and sid1p (AtMAP4K alpha 1, AtMAP4K alpha 2, BnMAP4K alpha 2) exhibit a significant similarity. The plant proteins AtSGP1/2 and BnMAP4K alpha 2 are able to complement the S. pombe mutant proteins spg1-B8 and sid1-239, respectively and to induce mutisepta when overexpressed in wild-type yeast. Yeast two-hybrid assays demonstrated interactions both between plant proteins and between plant and yeast proteins of the SIN pathway. However, the primary structure of the proteins as well as the partial complementation of yeast mutants indicates that plant homologous proteins and their yeast counterparts have diverged during evolution. Real-time RT-PCR studies demonstrated plant SIN-related gene expression in all organs tested and a co-expression pattern during the cell cycle, with a higher accumulation at G(2)-M. During interphase, the plant SIN-related proteins were found to co-localise predominantly in the nucleolus of the plant cells, as shown by fusions to green fluorescent protein. These data suggest the existence of a plant SIN-related pathway.

Physiological and biochemical characterization of metal-phytosiderophore transport in graminaceous species

Physiological studies on the uptake of metal-phytosiderophores in strategy II plants indicated that besides iron-phytosiderophores also other phytosiderophore-chelated metals can be taken up. To verify whether this reflects a property of the recently isolated Fe(Ill)-phytosiderophore transporter ZmYS1, a biochemical study was undertaken using growth complementation of yeast mutants and two-electrode voltage clamp in Xenopus oocytes. These approaches allowed to show that ZmYS1 indeed transports other phytosiderophore-chelated metals, such as Cu, Zn, Co, or Ni, as well as nicotianamine-complexes with Fe(II), Fe(III), and Ni(II). Moreover, it was shown that Fe(III)-DMA is cotransported with protons by ZmYS1, thus allowing substrate transport to be driven by the negative membrane potential. Despite the transport of a broad range of phytosiderophore-chelated heavy metals, neither Zn nor Mn or Cu deficiency led to an upregulation of ZmYS1 transcript levels in leaves or roots of maize. It is therefore concluded that the transporter ZmYS1 itself does not represent a component of the stress response to Zn-, Mn-, or Cu-limiting growth conditions, but that transport of these metal-phytosiderophores represents rather a side activity of ZmYS1.

Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis

Plants directly assimilate minerals from the environment and thus are key for acquisition of metals by all subsequent consumers. Limited bio-availability of copper, zinc and iron in soil decreases both the agronomic productivity and the nutrient quality of crops. Understanding the molecular mechanisms underlying metal homeostasis in plants is a prerequisite to optimizing plant yield and metal nutrient content. To absorb and maintain a balance of potentially toxic metal ions, plants utilize poorly understood mechanisms involving a large number of membrane transporters and metal binding proteins with overlapping substrate specificities and complex regulation. To better understand the function and the integrated regulation, we analyzed in Arabidopsis the expression patterns in roots and in leaves of 53 genes coding for known or potential metal transporters, in response to copper, zinc, and iron deficiencies in Arabidopsis. Comparative analysis of gene expression profiles revealed specific transcriptional regulation by metals of the genes contrasting with the known wide substrate specificities of the encoded transporters. Our analysis suggested novel transport roles for several gene products and we used functional complementation of yeast mutants to correlate specific regulation by metals with transport activity. We demonstrate that two ZIP genes, ZIP2 and ZIP4, are involved in copper transport. We also present evidence that AtOPT3, a member of the oligopeptide transporter gene family with significant similarities to the maize iron-phytosiderophore transporter YS1, is regulated by metals and heterologous expression AtOPT3 can rescue yeast mutants deficient in metal transport.