CONSTRUCTION AND APPLICATIONS OF A MYCORRHIZAL ARBUSCULAR SPECIFIC cDNA LIBRARY.

To exploit the potential benefits of mycorrhizas, we need to investigate the processes that occur in these symbiotic interactions, particularly in the arbuscular compartment where nutrients are exchanged between the plant and the fungus. Progress in this area is restricted due to the intricacy and complexity of this plant-fungus interface and many techniques that have been employed successfully in other plants and animal systems cannot be used. An effective approach to study processes in arbuscules is to examine transcript composition and dynamics. We applied laser capture microdissection (LCM) to isolate approximately 3000 arbuscules from Glomus intraradices colonised Me- dicago truncatula roots. Total RNA was extracted from microdissected arbuscules and subjected to T7 RNA polymerase-based linear amplification. Amplified RNA was then usedfor construction of a cDNA library. The presence and level of enrichment of mycorrhiza-specific transcripts was determined by quantitative Real-time and conventional PCR. To improve enrichment a cDNA library subtraction was performed. Complementation of yeast mutants deficient in the uptake of.potassium, phosphate, sulphate, amino acids, ammonium and of a Mn²âºsensitive strain, demonstrates the functionality of our cDNA library.

Growth and biochemical parameters of Cicer arietinum L. grown on amended fly ash.

Growth and metal accumulation were investigated in two Cicer arietinum L. varieties (var. CSG-8962 and var. C-235) when grown in various combinations of fly ash (FA) amended with garden soil (GS), press mud (PM) or saw dust (SD). In addition, the levels of photosynthetic pigments, nitrate reductase (NR) activity, cysteine, non-protein thiols (NP-SH), and ascorbic acid were studied. FA amended with GS or PM led to a 5-10 times increase in biomass compared to FA control and was most pronounced in the less metal tolerant variety CSG-8962. Amendment of FA with either GS or PM only moderately increased the contents of some essential metals whereas the non-essential Cd and Cr remained similar or decreased slightly compared to FA control. FA combined with either GS or PM increased the amount of photosynthetic pigments and was largely absent when SD was added to FA. Improved nitrogen availability led to increased nitrate reductase (NR) activity with all amendments but less so with SD. Metal stress indicating parameters were generally reduced (cysteine and non-protein thiols) or unchanged (ascorbic acid). In conclusion, of the tested ameliorants both GS and PM greatly improved growth of C. arietinum making FA a suitable component of plant growth substrates.

Sodium uptake in Arabidopsis roots is regulated by cyclic nucleotides.

Sodium uptake from the soil is a major cause of salinity toxicity in plants, yet little is known about the mechanisms that underlie Na(+) influx. We have characterized voltage independent channels (VICs) in Arabidopsis roots that are thought to contribute to Na(+) entry. VICs showed no selectivity among monovalent cations, and their gating was found to be voltage independent. However, VIC open probability showed sensitivity to cyclic nucleotides. The presence of micromolar concentrations of cAMP or cGMP at the cytoplasmic side of the plasma membrane evoked a rapid decrease in channel open probability. In accord with predictions from electrophysiological data, our results show that short-term unidirectional Na(+) influx is also reduced in the presence of cyclic nucleotides. Moreover, addition of membrane permeable cyclic nucleotides during growth assays improved plant salinity tolerance, which corresponded with lower levels of Na(+) accumulation in plants. In summary, these data imply that Arabidopsis plants may contain a cyclic nucleotide-based signaling pathway that directly affects Na(+) transport via VICs.

Phylogenetic relationships within cation transporter families of Arabidopsis.

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.

Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance.

Recently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4DeltaC) from which its C-terminal, with the calmodulin-binding domain and part of the putative cyclic nucleotide-binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full-length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T-DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.

Functional characterization of N-methyl-D-aspartic acid-gated channels in bone cells.

Our recent identification of glutamate receptors in bone cells suggested a novel means of paracrine communication in the skeleton. To determine whether these receptors are functional, we investigated the effects of the excitatory amino acid, glutamate, and the pharmacological ligand, N-methyl-D-aspartic acid (NMDA), on glutamate-like receptors in the human osteoblastic cell lines MG63 and SaOS-2. Glutamate binds to osteoblasts, with a Kd of approximately 10(-4) mol/L and the NMDA receptor antagonist, D(L)-2-amino-5-phosphonovaleric acid (D-APV), inhibits binding. Using the patch-clamp technique, we measured whole-cell currents before and after addition of L-glutamate or NMDA and investigated the effects of the NMDA channel blockers, dizolcipine maleate (MK801), and Mg2+, and the competitive NMDA receptor antagonist, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphoric acid (R-CPP), on agonist-induced currents. Both glutamate and NMDA induced significant increases in membrane currents. Application of Mg2+ (200 micromol/L) and MK801 (100 micromol/L) caused a significant decrease in inward currents elicited in response to agonist stimulation. The competitive NMDA receptor antagonist, R-CPP (100 micromol/L), also partially blocked the NMDA-induced currents in MG63 cells. This effect was reversed by addition of further NMDA (100 micromol/L). In Fura-2-loaded osteoblasts, glutamate induced elevation of intracellular free calcium, which was blocked by MK801. These results support the hypothesis that glutamate plays a role in bone cell signaling and suggest a possible role for glutamate agonists/antagonists in the treatment of bone diseases.

Calcium-induced calcium release mediated by a voltage-activated cation channel in vacuolar vesicles from red beet.

Little is known about the mechanisms underlying calcium-induced Ca2+ release (CICR) in plants. The slow-activating vacuolar (SV) channel is both permeable to, and activated by Ca2+, and is therefore a prime candidate for a role in CICR. Cytosol-side-out vacuolar membrane vesicles loaded with 45Ca2+ showed voltage- and Ca(2+)-dependent Ca2+ release, which was sensitive to the SV channel modulators DIDS, protein phosphatase 2B and calmodulin. Significantly, voltage-dependent Ca2+ release strongly depended on cytoplasmic Ca2+ concentrations. The results support the notion that CICR occurs in plant cells and that the process can be catalysed by the SV channel on the vacuolar membrane.

Plasma membrane transport in context - making sense out of complexity.

Major advances in our understanding of the transport of inorganic nutrient ions across plant plasma membranes have emerged from recent studies on the control of the dominant H+-pumping ATPase and from identification of a range of new transporters for divalent cations, potassium, phosphate and nitrate. In many cases, multiple transporter isoforms have been described. An appreciation of the physiological roles of these transporters demands combined genetic and physiological approaches, which, in the case of an outward rectifying K+ channel, have already been used to yield an intriguing insight into root-mediated K+ release into the xylem. In this review we attempt to place some of those developments in a physiological context.

Cell marking in Arabidopsis thaliana and its application to patch-clamp studies.

Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane-patch voltage-clamp (patch-clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch-clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch-clamp studies and presents a specific example: a comparison of the K+ currents in protoplasts from cortical and stelar cells of Arabidopsis roots. Transgenic Arabidopsis were generated in which the expression of green fluorescent protein (GFP) from Aequoria victoria was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K+ currents in non-fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch-clamp techniques. It was found that both the frequency of observing inward rectifying K+ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K+ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K+ channels. It is concluded that the generation of transgenic Arabidopsis expressing GFP in a cell-specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch-clamp studies.

Kinetics of high-affinity K+ uptake in plants, derived from K(+)-induced changes in current-voltage relationships. A modelling approach to the analysis of carrier-mediated transport.

To investigate coupled, charge-translocating transport, it is imperative that the specific transporter current-voltage (IV) relationship of the transporter is separated from the overall membrane IV relationship. We report here a case study in which the currents mediated by the K(+)-H+ symporter, responsible for high-affinity K+ uptake in Arabidopsis thaliana (L.) Heynh. cv. Columbia roots, are analyzed with an enzyme kinetic reaction scheme. The model explicitly incorporates changes in membrane voltage and external substrate, and enables the derivation of the underlying symport IV relationships from the experimentally obtained difference IV data. Data obtained for high-affinity K+ transport in A. thaliana root protoplasts were best described by a 1:1 coupled K(+)-H+ symport-mediated current with a parallel, outward non-linear K+ pathway. Furthermore, the large predictive value of the model was used to describe symport behaviour as a function of the external K+ concentration and the cytoplasmic K+ concentration. Symport activity is a complex function of the external K+ concentration, with first-order saturating kinetics in the micromolar range and a strong activity reduction when external K+ is in the millimolar range and the membrane depolarises. High cytoplasmic K+ levels inhibit symport activity. These responses are suggested to be part of the feedback mechanisms to maintain cellular K+ homeostasis. The general suitability of the model for analysis of carrier-mediated transport is discussed.

Regulation of K+ absorption in plant root cells by external K+: interplay of different plasma membrane K+ transporters.

Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Details on the structure and function of these K(+) transporters are accumulating, but many prominent questions remain regarding regulation of these uptake pathways in varying physiological conditions. Efficient use of the K(+) absorption capacity requires that the activity of all membrane K(+) conductances interact. In this paper, it is shown how intrinsic properties of the major K(+) transporters in the root plasma membrane generate sufficient inward K(+) flux at varying levels of external [K(+)]. In the high affinity range, uptake proceeds via K(+):H(+) symport and kinetic control prevents outward K(+) leakage through inward rectifying channels. Leakage through outward rectifying channels is minimized due to a combination of kinetic control and intrinsic open channel rectification as predicted by the constant field theory. At millimolar external K(+), symport activity is down regulated by the K(+) induced membrane depolarization. In these conditions, channel-mediated K(+) uptake can only explain the observed unidirectional fluxes in intact tissue if the cell switches from a state where the K(+) conductance dominates (K(+)-state) to one where the primary pumps dominate the membrane conductance (pump-state).

Characterization of csi52, a Cs+ resistant mutant of Arabidopsis thaliana altered in K+ transport.

Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Although details on the structure and function of these transporters are beginning to emerge many prominent questions remain concerning how these proteins function in plants. Such questions can be addressed through the use of well-defined transport mutants. Csi52, a caesium-insensitive mutant of Arabidopsis thaliana which is defective in potassium transport, is further characterized here using conventional electrophysiology, patch-clamp and radiometric approaches to identify the nature of the potassium transport lesion. Rb+ uptake experiments reveal a reduced uptake in csi52 in both the high-and low-affinity uptake range. Patch-clamp analysis indicates that the activity of the predominant inward rectifying channel observed in wild-type cells is extremely low in root protoplasts isolated from csi52, whereas outward rectifying channel activity is comparable between wildtype and mutant. Rb+ uptake studies show that in both wild-type and csi52 the high-affinity uptake pathway is considerably less sensitive to Cs+ than the low-affinity pathway with K1/2 values for Cs+ of around 1.3 and 0.2 mM, respectively. Furthermore, K+ starvation leads to a larger relative increase in high-affinity K+ uptake in the mutant than the wild-type. The results demonstrate the Cs+ sensitivity of each individual uptake pathway is comparable in wild-type and csi52 but the high-affinity pathway is less Cs+ sensitive (in both wild-type and csi52). Therefore, the larger shift toward high-affinity uptake in the mutant compared with the wild-type under K(+)-starvation conditions will endow the mutant with a higher degree of overall Cs+ resistance. The data supply evidence for the hypothesis that the csi52 mutation lies within a gene that regulates the activity of several potassium transport systems and coordinates their relative contribution to overall root K+ uptake.

Contrasting roles in ion transport of two K(+)-channel types in root cells of Arabidopsis thaliana.

Plant roots accumulate K+ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K+ transporters which operate at millimolar and micromolar external [K+]: high-affinity K+ uptake is energised by symport with H+, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K+ uptake, a characterisation of the principal K(+)-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K(+)-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (PCl:PK < 0.025) but less selective amongst monovalent cations (PNa:PK approximately 0.17-0.3). Both the IRC and the ORC were blocked by Ba2+, Cs+ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (EK+), enabling ORCs to conduct mainly outward, but occasionally inward, K+ current. By contrast, gating of the 5-pS IRC was weakly voltage-ependent and IRC gating was invariably restricted to membrane potentials more negative than EK+, ensuring K+ transport was always inwardly directed.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana.

Potassium is a major nutrient in higher plants, where it plays a role in turgor regulation, charge balance, leaf movement, and protein synthesis. Terrestrial plants are able to sustain growth at micromolar external K+ concentrations, at which K+ uptake across the plasma membrane of root cells must be energized despite the presence of a highly negative membrane potential. However, the mechanism of energization has long remained obscure. Therefore, whole-cell mode patch clamping has been applied to root protoplasts from Arabidopsis thaliana to characterize membrane currents resulting from the application of micromolar K+. Analysis of whole cell current/voltage relationships in the presence and absence of micromolar K+ enabled direct testing of K+ transport for possible energization by cytoplasmic ATP and the respective trans-membrane gradients of Na+, Ca2+, and H+. Subtracted current/voltage relations for K(+)-dependent membrane currents are independent of ATP and reverse at potentials that imply H(+)-coupled K+ transport with a ratio of 1 H+:K+. Furthermore, the reversal potential of the K+ current shifts negative as external H+ activity is decreased. K(+)-dependent currents saturate in the micromolar concentration range with an apparent Km of 30 microM, a value in close agreement with previously reported Km values for high-affinity K+ uptake. We conclude that our results are consistent with the view that high-affinity K+ uptake in higher plants is mediated by a H+:K+ symport mechanism, competent in driving K+ accumulation to equilibrium ratios in excess of 10(6)-fold.

Plant membrane transport.

Recent developments in plant membrane transport, particularly concerning the vacuolar and plasma membranes, have increased our understanding of molecular aspects of primary pumps, carrier systems and ion channels.

Inhibition of inward rectifying tonoplast channels by a vacuolar factor: physiological and kinetic implications.

Regulation of ion-channel activity must take place in order to regulate ion transport. In case of tonoplast ion channels, this is possible on both the cytoplasmic and the vacuolar side. Isolated vacuoles of young Vigna unguiculata seedlings show no or hardly any channel activity at tonoplast potentials greater than 80 mV, in the vacuole-attached configuration. When the configuration is changed to an excised patch or whole vacuole, a fast (excised patch) or slow (whole vacuole) increase of inward rectifying channel activity is seen. This increase is accompanied by a shift in the voltage-dependent gating to less hyperpolarized potentials. In the whole vacuole configuration the level of inward current increases and also the activation kinetics changes. Induction of channel activity takes up to 20 min depending on the age of the plants used and the diameter of the vacuole. On the basis of the estimated diffusion velocities, it is hypothesized that a compound with a mol wt of 20,000 to 200,000 is present in vacuoles of young seedlings, which shifts the population of channels to a less voltage-sensitive state.

Patch clamp studies on root cell vacuoles of a salt-tolerant and a salt-sensitive plantago species : regulation of channel activity by salt stress.

Plantago media L. and Plantago maritima L. differ in their strategy toward salt stress, a major difference being the uptake and distribution of ions. Patch clamp techniques were applied to root cell vacuoles to study the tonoplast channel characteristics. In both species the major channel found was a 60 to 70 picosiemens channel with a low ion selectivity. The conductance of this channel for Na(+) was the same as for K(+), P(K) (+)/P(Na) (+) = 1, whereas the cation/anion selectivity (P(K) (+)/P(c1) (-)) was about 5. Gating characteristics were voltage and calcium dependent. An additional smaller channel of 25 picosiemens was present in P. maritima. In the whole vacuole configuration, the summation of the single channel currents resulted in slowly activated inward currents (t((1/2)) = 1.2 second). Inwardly directed, ATP-dependent currents could be measured against a DeltapH gradient of 1.5 units over the tonoplast. This observation strongly indicated the physiological intactness of the used vacuoles. The open probability of the tonoplast channels dramatically decreased when plants were grown on NaCl, although single channel conductance and selectivity were not altered.