An update on nutrient transport processes in ectomycorrhizas

Nutrient transport, namely absorption from the soil solution as well as nutrient transfer from fungus to plant and carbon movement from plant to fungus are key features of mycorrhizal symbiosis. This review summarizes our current understanding of nutrient transport processes in ectomycorrhizal fungi and ectomycorrhizas. The identification of nutrient uptake mechanisms is a key issue in understanding nutrition of ectomycorrhizal plants. With the ongoing functional analysis of nutrient transporters, identified during sequencing of fungal and tree genomes, a picture of individual transport systems should be soon available, with their molecular functions assessed by functional characterization in, e.g., yeast mutant strains or Xenopus oocytes. Beyond the molecular function, systematic searches for knockout mutants will allow us to obtain a full understanding of the role of the individual transporter genes in the physiology of the symbionts. The mechanisms by which fungal and plant cells obtain, process and integrate information regarding nutrient levels in the external environment and the plant demand will be analyzed.     

Molecular insights into the structure and function of plant K(+) transport mechanisms

Our understanding of plant potassium transport has increased in the past decade through the application of molecular biological techniques. In this review, recent work on inward and outward rectifying K(+) channels as well as high affinity K(+) transporters is described. Through the work on inward rectifying K(+) channels, we now have precise details on how the structure of these proteins determines functional characteristics such as ion conduction, pH sensitivity, selectivity and voltage sensing. The physiological function of inward rectifying K(+) channels in plants has been clarified through the analysis of expression patterns and mutational analysis. Two classes of outward rectifying K(+) channels have now been cloned from plants and their initial characterisation is reviewed. The physiological role of one class of outward rectifying K(+) channel has been demonstrated to be involved in long distance transport of K(+) from roots to shoots. The molecular structure and function of two classes of energised K(+) transporters are also reviewed. The first class is energised by Na(+) and shares structural similarities with K(+) transport mechanisms in bacteria and fungi. Structure-function studies suggest that it should be possible to increase the K(+) and Na(+) selectivity of these transporters, which will enhance the salt tolerance of higher plants. The second class of K(+) transporter is comprised of a large gene family and appears to have a dual affinity for K(+). A suite of molecular techniques, including gene cloning, oocyte expression, RNA localisation and gene inactivation, is now being used to fully characterise the biophysical and physiological function of plants K(+) transport mechanisms.     

The Arabidopsis HKT1 gene homolog mediates inward Na(+) currents in xenopus laevis oocytes and Na(+) uptake in Saccharomyces cerevisiae

The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters. The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.     

Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters

Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K(+) uniporter, Arabidopsis AtHKT1-mediated Na(+) uniporter and wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.     

Comparative functional features of plant potassium HvHAK1 and HvHAK2 transporters

Plant K(+) transporters of the HAK family belong to four rather divergent phylogenetic clusters, although most of the transporters belong to clusters I or II. A simple phylogenetic analysis of fungal and plant HAK transporters suggests that an original HAK gene duplicated even before fungi and plants diverged, generating transporters that at present fulfill different functions in the plant. The HvHAK1 transporter belongs to cluster I and mediates high-affinity K(+) uptake in barley roots, but no function is known for the cluster II transporter, HvHAK2, which is not functional in yeast. The function of HvHAK2 was investigated by constructing HvHAK1-HAK2 chimeric transporters, which were not functional even when they included only short fragments of HvHAK2. Then, amino acids characteristic of cluster II in the N terminus and in the first transmembrane domain were introduced into HvHAK1. All of these changes increased the Rb(+) K(m), introducing minimal changes in the Na(+) K(m), which suggested that HvHAK2 is a low-affinity, Na(+)-sensitive K(+) transporter. Using a K(+)-defective Escherichia coli mutant, we functionally expressed HvHAK2 and found that the predicted characteristics were correct, as well as discovering that the bacterial expression of HvHAK2 is functional at pH 5.5 but not at 7.5. We discuss whether HvHAK2 may be a tonoplast transporter effective for vacuolar K(+) depletion in K(+) starved plants.     

Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K⁺) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K⁺ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K⁺ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem‐pore outward‐rectifying K⁺ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two‐electrode voltage‐clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K⁺ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K⁺ from the fungus towards the plant .     

Potassium transporters in plants--involvement in K+ acquisition, redistribution and homeostasis

Potassium is a major plant nutrient which has to be accumulated in great quantity by roots and distributed throughout the plant and within plant cells. Membrane transport of potassium can be mediated by potassium channels and secondary potassium transporters. Plant potassium transporters are present in three families of membrane proteins: the K(+) uptake permeases (KT/HAK/KUP), the K(+) transporter (Trk/HKT) family and the cation proton antiporters (CPA). This review will discuss the contribution of members of each family to potassium acquisition, redistribution and homeostasis.     

Yeast SMF1 mediates H(+)-coupled iron uptake with concomitant uncoupled cation currents

Yeast membrane proteins SMF1, SMF2, and SMF3 are homologues of the DCT1 metal ion transporter family. Their functional characteristics and the implications of these characteristics in vivo have not yet been reported. Here we show that SMF1 expressed in Xenopus oocytes mediates H(+)-dependent Fe(2+) transport and uncoupled Na(+) flux. SMF1-mediated Fe(2+) transport exhibited saturation kinetics (K(m) = 2.2 microM), whereas the Na(+) flux did not, although both processes were electrogenic. SMF1 is also permeable to Li(+), Rb(+), K(+), and Ca(2+), which likely share the same uncoupled pathway. SMF2 (but not SMF3) mediated significant increases in both Fe(2+) and Na(+) transport compared with control oocytes. These data are consistent with the concept that uptake of divalent metal ions by SMF1 and SMF2 is essential to yeast cell growth. Na(+) inhibited metal ion uptake mediated by SMF1 and SMF2 expressed in oocytes. Consistent with this, we found that increased sensitivity of yeast to EGTA in the high Na(+) medium is due to inhibition of SMF1- and SMF2-mediated metal ion transport by uncoupled Na(+) pathway. Interestingly, DCT1 also mediates Fe(2+)-activated uncoupled currents. We propose that uncoupled ion permeabilities in metal ion transporters protect cells from metal ion overload.     

植物菌根共生磷酸盐转运蛋白

大多数植物能和丛枝菌根（arbuscular mycorrhiza, AM）真菌形成菌根共生体。AM能够促进植物对土壤中矿质营养的吸收,尤其是磷的吸收。磷的吸收和转运由磷酸盐转运蛋白介导。总结了植物AM磷酸盐转运蛋白及其结构特征,分析其分类及系统进化,并综述了AM磷酸盐转运蛋白介导的磷的吸收和转运过程及其基因的表达调控。植物AM磷酸盐转运蛋白属于Pht1家族成员,它不仅对磷的吸收和转运是必需的,而且对AM共生也至关重要,为进一步了解菌根形成的分子机理及信号转导途径提供了理论基础。     

Functional Characterization of the Amanita muscaria Monosaccharide Transporter, AmMst1

Abstract: Fungal carbohydrate nutrition is an important aspect of ectomycorrhizal symbiosis. At the plant/fungus interface, fungal and root cortical cells compete for monosaccharides, generated from plant-derived sucrose. Therefore, the kinetic properties of the monosaccharide uptake systems are decisive for the monosaccharide yield of each partner. For the functional characterization of a hexose transporter (AmMst1) of the ectomycorrhizal fungus Amanita muscaria, the entire cDNA was expressed in a Saccharomyces cerevisiae strain unable to take up hexoses. Uptake experiments with ¹⁴C-labelled monosaccharides resulted in K_M values of 0.46 mM for glucose and 4.20 mM for fructose, revealing a strong preference of AmMst1 for glucose as substrate. Glucose uptake by AmMst1 was strongly favoured even in the presence of a large excess of fructose. Comparable affinities of AmMst1 for glucose, 3-O-methyl glucose and mannose were obtained. In contrast, AmMst1 imported galactose with a much lower efficiency, revealing that this transporter distinguishes pyranoses by steric hindrance at the C-4 position. While yeast contains numerous hexose transporter genes, the AmMst1 gene seems to be the main, if not the only, hexose transporter that is expressed in A. muscaria, as concluded from the comparison of hexose import properties of A. muscaria protoplasts and AmMst1 expressed in yeast.     

Functional characterization of a Na(+)-coupled dicarboxylate carrier protein from Staphylococcus aureus

We have cloned and functionally characterized a Na(+)-coupled dicarboxylate transporter, SdcS, from Staphylococcus aureus. This carrier protein is a member of the divalent anion/Na(+) symporter (DASS) family and shares significant sequence homology with the mammalian Na(+)/dicarboxylate cotransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na(+)-dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na(+) (K(0.5) of 2.7 mM) binding before dicarboxylate (K(m) of 4.5 microM). Because this transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.     

Trk1 and Trk2 define the major K(+) transport system in fission yeast

The trk1(+) gene has been proposed as a component of the K(+) influx system in the fission yeast Schizosaccharomyces pombe. Previous work from our laboratories revealed that trk1 mutants do not show significantly altered content or influx of K(+), although they are more sensitive to Na(+). Genome database searches revealed that S. pombe encodes a putative gene (designated here trk2(+)) that shows significant identity to trk1(+). We have analyzed the characteristics of potassium influx in S. pombe by using trk1 trk2 mutants. Unlike budding yeast, fission yeast displays a biphasic transport kinetics. trk2 mutants do not show altered K(+) transport and exhibit only a slightly reduced Na(+) tolerance. However, trk1 trk2 double mutants fail to grow at low K(+) concentrations and show a dramatic decrease in Rb(+) influx, as a result of loss of the high-affinity transport component. Furthermore, trk1 trk2 cells are very sensitive to Na(+), as would be expected for a strain showing defective potassium transport. When trk1 trk2 cells are maintained in K(+)-free medium, the potassium content remains higher than that of the wild type or trk single mutants. In addition, the trk1 trk2 strain displays increased sensitivity to hygromycin B. These results are consistent with a hyperpolarized state of the plasma membrane. An additional phenotype of cells lacking both Trk components is a failure to grow at acidic pH. In conclusion, the Trk1 and Trk2 proteins define the major K(+) transport system in fission yeast, and in contrast to what is known for budding yeast, the presence of any of these two proteins is sufficient to allow growth at normal potassium levels.     

RGD-CAP ((beta)ig-h3) is expressed in precartilage condensation and in prehypertrophic chondrocytes during cartilage development

The TRK-HKT family of K(+) transporters mediates K(+) and Na(+) uptake in fungi and plants. In this study, we have investigated the molecular mechanism involved in the movement of alkali cations through the TRK1 transporter of Saccharomyces cerevisiae. The model that best explains the activity of ScTRK1 is a cotransport of two K(+) or Rb(+), both of which bind the two binding sites of ScTRK1 with very high affinities in K(+)-starved cells. Na(+) can be transported in the same way but it exhibits a much lower affinity for the second binding site. Therefore, only at critical concentration ratios between K(+) and Na(+), or Rb(+) and Na(+), the transporter takes up Na(+) together with K(+) or Rb(+). Mutation analyses suggest that the two binding sites are located in the P fragment of the first MPM motif of the transporter, and that Gln(90) is involved in these binding sites. ScTRK1 can be in two states, medium or high affinity, and we have found that Leu(949) is involved in the oscillation of the transporter between these two states. ScTRK1 mediates active K(+) uptake. This is not Na(+)-coupled and direct coupling of ScTRK1 to a source of chemical energy seems more probable than K(+)-H(+) cotransport.     

Partial deletion of a loop region in the high affinity K+ transporter HKT1 changes ionic permeability leading to increased salt tolerance

HKT1 is a high affinity K(+) transporter protein that is a member of a large superfamily of transporters found in plants, bacteria, and fungi. These transporters are primarily involved in K(+) uptake and are energized by Na(+) or H(+). HKT1 is energized by Na(+) but also mediates low affinity Na(+) uptake and may therefore be a pathway for Na(+) uptake, which is toxic to plants. The aim of this study was to identify regions of HKT1 that are involved in K(+)/Na(+) selectivity and alter the amino acid composition in those regions to increase the ionic selectivity of the transporter. A highly charged loop was identified, and two deletions were created that resulted in the removal of charged and uncharged amino acids. The functional changes caused by the deletions were studied in yeast and Xenopus oocytes. The deletions improved the K(+)/Na(+) selectivity of the transporter and increased the salt tolerance of the yeast cells in which they were expressed. In light of recent structural models of members of this symporter superfamily, it was necessary to determine the orientation of this highly charged loop. Introduction of an epitope tag allowed us to demonstrate that this loop faces the outside of the membrane where it is likely to facilitate the interaction with cations such as K(+) and Na(+). This study has identified an important structural feature in HKT1 that in part determines its K(+)/Na(+) selectivity. Understanding the structural basis of the functional characteristics in transporters such as HKT1 may have important implications for increasing the salt tolerance of higher plants.     

Characterization and regulation of PiDur3, a permease involved in the acquisition of urea by the ectomycorrhizal fungus Paxillus involutus

Urea, which is known to be a source of nitrogen for the growth of many organisms, represents an important fertilizer in forest soils. Since most trees form symbiotic associations with ectomycorrhizal fungi, the capacities of these symbionts to take up and assimilate urea would determine the efficiency of urea nitrogen salvaging by plants. We showed that Paxillusinvolutus, an ectomycorrhizal basidiomycete, is capable of using urea as sole nitrogen source. We report the molecular characterization of an active urea transporter (PiDur3) isolated from this fungus. We demonstrated that the import of urea is a minor event on ammonium condition, since the expression of PiDUR3 is repressed by the high intracellular glutamine pool. Interestingly, on urea nutritive condition, the uptake of urea is rather mediated by the intracellular urea pool and particularly by urease efficiency.     

Characterization and regulation of PiDur3, a permease involved in the acquisition of urea by the ectomycorrhizal fungus Paxillus involutus.

Urea, which is known to be a source of nitrogen for the growth of many organisms, represents an important fertilizer in forest soils. Since most trees form symbiotic associations with ectomycorrhizal fungi, the capacities of these symbionts to take up and assimilate urea would determine the efficiency of urea nitrogen salvaging by plants. We showed that Paxillusinvolutus, an ectomycorrhizal basidiomycete, is capable of using urea as sole nitrogen source. We report the molecular characterization of an active urea transporter (PiDur3) isolated from this fungus. We demonstrated that the import of urea is a minor event on ammonium condition, since the expression of PiDUR3 is repressed by the high intracellular glutamine pool. Interestingly, on urea nutritive condition, the uptake of urea is rather mediated by the intracellular urea pool and particularly by urease efficiency.