| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada
Correspondence
Roger R. Lew
planters{at}yorku.ca
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
40 min) and magnitude. Prior to turgor recovery, the hyperosmotic shock caused a rapid transient depolarization of the membrane potential, followed by a sustained hyperpolarization that occurred concomitant with increased H+ efflux, indicating that the plasma membrane H+-ATPase was being activated. These changes also occurred in the wild-type. Net fluxes of Ca2+ and Cl– during turgor recovery were similar to those in the wild-type, but K+ influx was attenuated in the cut mutant. The similar turgor recovery can be explained by the ion uptake, since glycerol did not accumulate in the cut mutant within the time frame of turgor recovery (but did accumulate in the wild-type). The results suggest that turgor regulation involves multi-faceted coordination of both ion flux and glycerol accumulation. Ion uptake is activated by a MAP kinase cascade, while CUT is required for glycerol accumulation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). There are a number of mechanisms that may be used by a fungus to adjust its internal osmolarity, and therefore its turgor. One of these mechanisms is accumulation of osmotically active metabolites. Alcohol sugars (glycerol and erythritol) accumulate to high levels in Aspergillus nidulans grown in high salt (NaCl or KCl) concentrations (Beever & Laracy, 1986); glycerol levels begin to increase within an hour of hyperosmotic treatment. In Neurospora crassa, mannitol and glycerol accumulate within 22 h of treatment with high NaCl concentrations (Ellis et al., 1991). Slightly lower accumulation is observed in the os-1 mutant, which is defective in a histidine kinase that is homologous to the osmosensing SLN-1 histidine kinase upstream of the high osmolarity glycerol (HOG) pathway in yeast (Alex et al., 1996; Schumacher et al., 1997). The yeast HOG pathway is an MAP kinase cascade which is activated by high osmolarity and causes the accumulation of glycerol. It is found in numerous fungal species, on the basis of comparative genomics (Krantz et al., 2006). Kinetic models relate activation of the kinase members of the MAP kinase cascade to gene expression induced by Hog1p [the last of the three kinases in the MAP kinase cascade; the corresponding gene in N. crassa is os-2 (Zhang et al., 2002)]. Gene expression then results in the appearance of enzymes required for the biochemical changes that effect glycerol production (Klipp et al., 2005). Glycerol production is not associated solely with hyperosmotic stress. The HOG pathway is also activated by low temperatures, since glycerol can confer freezing tolerance as a cryoprotectant (Panadero et al., 2006). Glycerol synthesis is also activated at the level of gene expression in the homologous osmotic response MAP kinase cascade in N. crassa (Noguchi et al., 2007).
An alternative to the production and accumulation of osmotically active metabolites is the accumulation of osmotically active ions from the external environment. This can be measured directly with an ion-selective self-referencing probe (Newman, 2001; Smith et al., 1999). Net changes in ion influx that can explain turgor recovery after hyperosmotic treatment have been observed in higher plants (Arabidopsis thaliana) (Shabala & Lew, 2002) and N. crassa (Lew et al., 2006).
In addition to MAP kinase cascade-mediated turgor regulation [the sensor OS-1 (Miller et al., 2002), MAPKKK OS-4 and MAPKK OS-5 (Fujimura et al., 2003), and MAPK OS-2 (Zhang et al., 2002)], there are other proteins that function in response to osmotic stress in N. crassa which are not homologues of MAP kinase cascade members. The osmosensitive mutant cut is one example. The cut mutant was originally isolated as a morphological mutant which exhibits a flat, as if it had been cut, conidiation zone when grown in slants (Kuwana, 1953). Temperature, pH and carbon source do not affect the mutant, but growth is affected by humidity (normal morphology is observed at 100 % relative humidity) and osmotic pressure (it is unable to grow at high osmolarity) (Kuwana, 1953). The gene has been cloned. It is allelic to ovc (overaccumulator of carotenoids) and encodes a putative phosphatase (Youssar et al., 2005). The expression of the cut-1 gene increases after hyperosmotic and heat-shock treatment (Youssar & Avalos, 2006). Other osmosensitive mutants are known [e.g. os-8, os-9, os-10 and os-11 (Perkins et al., 2001)] but the genes have not yet been identified. Scumbo (sc) and the semi-colonial mutants smco-8 and smco-9 are also osmosensitive and resistant to dicarboximide fungicides (Grindle & Temple, 1983). Mutations in the os genes encoding members of the osmoresponsive MAP kinase cascade in N. crassa are insensitive to dicarboximide (Grindle & Temple, 1982) and phenylpyrrole fungicides (Zhang et al., 2002). The OS-1 family of histidine kinases apparently plays a central role in fungicide sensitivity, since its expression in yeast (normally insensitive to the fungicides) confers sensitivity to the dicarboximide, phenylpyrrole and aromatic hydrocarbon fungicides (Motoyama et al., 2005).
In previous work, we have examined the role of ion transport in turgor regulation in wild-type and os mutants of N. crassa, and demonstrated turgor recovery within 60 min directly with a pressure probe, as in Fig. 1 of the current study. Before the onset of turgor recovery, the membrane potential responds by depolarizing transiently, followed by a sustained hyperpolarization which is attributed to direct activation of the plasma membrane H+-ATPase (Lew et al., 2006). Subsequently, there is net uptake of K+ and Cl– sufficient to explain turgor recovery. The electrical changes are not observed in the osmosensitive os-1 and os-2 mutants, leading to the conclusion that the HOG-like MAP kinase cascade functions as more than a gateway to changes in gene expression, and activates ion transport directly during turgor regulation.
|
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Stock cultures were maintained on Vogel's (plus 1.5 %, w/v, sucrose and 2.0 %, w/v, agar) medium (VM) (Vogel, 1956). We confirmed the phenotype of the cut mutant: an inability to grow on VM+4 % (w/v) NaCl (Perkins et al., 2001).
Culture preparation for experiments.
Cultures were grown at 28 °C (or room temperature, 21–24 °C) overnight by transferring conidia onto strips (2.5x6 cm) of dialysis tubing that overlaid the VM in Petri dishes. The dialysis tubing was cut with a razor blade to a size of about 1x3 cm, which included the growing edge of the colony. The strip of dialysis tubing was placed inside the cover of a 30 mm Petri dish, and immobilized on the bottom with masking tape or double-sided sticky tape. The culture was flooded with 3 ml buffer solution (BS) containing KCl (10 mM), CaCl2 (1 mM), MgCl2 (1 mM), sucrose (133 mM) and MES (10 mM), pH adjusted to 5.8 with KOH, or, for some of the ion-flux measurements, a modified BS containing CaCl2 (0.05 mM), MgCl2 (0.05mM) and sucrose (150 mM), which was unbuffered. Growth of hyphae at the colony edge normally resumed within 15 min. In experiments comparing wild-type and mutants, we normally interspersed measurements of each of the strains.
Electrical measurements.
For electrophysiology measurements, large-trunk hyphae (10–20 µm diameter) 0.5 cm behind the colony edge were impaled. After a stable membrane potential was obtained, the hyphae were treated in hyperosmotic conditions by adding 0.5 ml BS+1 M sucrose to 3 ml BS in the dish (a net increase of 155 mosmol kg–1). In other experiments, hyphae were treated with fludioxonil by adding 0.3 ml well-mixed 0.8 mM fludioxonil. The electrophysiological techniques have been described in detail previously (Lew, 1996). Briefly, double-barrel micropipettes (Lew, 2006) were used to voltage-clamp the hyphae, using a bipolar staircase of resting-potential clamps and alternating positive and negative voltages of 50 ms duration. Measurement of current density in the hyphae is complicated by the cable properties of the hyphae. Voltage is attenuated 67 % at a distance of 200 µm from the site of micropipette impalement (R. R. Lew and N. N. Levina, unpublished data). Correcting for current spread along the hyphae requires multiple impalements, which is technically challenging because vibration during treatment may dislodge the micropipettes. Thus, input currents are shown.
Turgor measurements.
Impalements were made on large trunk hyphae (Fig. 1
), similar to those for electrophysiological measurements. The technique has been described in detail previously (Lew et al., 2004, 2006). Large-aperture micropipettes were fabricated using a double-pull technique. The micropipette was filled with low-viscosity silicone oil (polydimethylsiloxane, 1.5x10–6 m2 s–1; Dow Corning), and mounted in a holder attached to a micrometer-driven piston by thick-wall teflon tubing. Pressure was measured with a transducer (XT-190-300G; Kulite Semiconductor Products) mounted on the holder. After impalement, the internal hydrostatic pressure pushed the silicone oil per cell sap meniscus into the micropipette; the pressure required to bring the meniscus back to the tip was a measure of the turgor (Fig. 1). After turgor measurements for 6 min, 0.5 ml BS+1 M sucrose was added to the dish containing 3 ml BS for a hyperosmotic increase of 155 mosmol kg–1. If the micropipette tip became plugged during the turgor measurements, every attempt was made to re-impale the same hypha, or another one nearby, with a fresh micropipette.
Ion-flux measurements.
Large-trunk hyphae were selected for measurements of ion fluxes in situ. Construction of the ion-selective probe has been described in detail previously (Lew, 1999; Lew et al., 2006). The following ion-selective cocktails were used (all from Sigma–Aldrich): H+, Fluka hydrogen ionophore II-cocktail A, catalogue no. 95297; Ca2+, calcium ionophore I-cocktail A, catalogue no. 21048; K+, potassium ionophore I-cocktail B, catalogue no. 60398; and Cl–, chloride ionophore I-cocktail A, catalogue no. 24902. The concentration of the selected ion was measured at two positions: as near to the hyphal wall as possible, and 20 µm away, at a frequency of 0.3 Hz. To confirm the generation of steady-state diffusive gradients, and thus the ability to infer ion fluxes from the diffusive ion-concentration gradients, measurements were performed using K+ and Cl– point sources. The point sources were micropipettes pulled to a fine tip and filled with 3 M KCl, so that K+ and Cl– diffused from the tip into the solution. The gradients and corresponding ion fluxes were measured 10 to 50–100 µm from the point source, and exhibited an exponential shape consistent with the generation of a diffusive gradient. The differences in ion concentrations 0 and 20 µm from the hypha were converted to ion flux with the following equation, which accounts for the cylindrical geometry of the hyphae: J (nmol cm–2 s–1)=(D/r)[(c2–c1)/ln(r2/r1)], where D is the diffusion coefficient (H+, 9.31x10–5 cm2 s–1; Ca2+, 0.4x10–5 cm2 s–1; K+, 1.96x10–5 cm2 s–1; Cl–, 2.03x10–5 cm2 s–1); r is the hyphal radius; c2 and c1 the concentrations at the two excursion points; and r2 and r1 the distances from the hyphal centre to the two excursion points (Henriksen et al., 1992). The ion-selective cocktail for Cl– registered a higher than expected Cl– concentration in the modified BS. However, the addition of Cl– to the solution caused the predicted voltage change, indicating that there was interference, probably caused by an organic substance, which affected the voltage offset, but not the slope of voltage versus Cl– concentration. To correct for this, the concentration was normalized to the expected concentration of 0.2 mM Cl– for flux calculations.
Immediately after addition of 0.5 ml modified BS+1 M sucrose to the dish containing 3 ml modified BS, the hyphae were observed to shrink and occasionally undergo incipient plasmolysis, followed by the disappearance of incipient plasmolysis and reswelling. These observations corroborated the process of turgor recovery during measurements of ion fluxes.
Glycerol measurements.
Colonies were grown overnight on a 7x7 cm strip of dialysis tubing overlaying VM. The dialysis tubing with the mycelial colony was transferred to a 100 mm Petri dish, which was flooded with 15 ml BS. Either 2.5 ml BS+1 M sucrose, or 2.5 ml 1 M NaCl was added to the dish to commence hyperosmotic treatments. The controls involved the addition of 2.5 ml BS. To harvest the mycelium, the strip of dialysis tubing with overlaying colony was transferred to a paper towel; the mycelium was scraped off the tubing, blotted, and placed in liquid N2 in a mortar. After freezing, the mycelium was ground to a powder with a pestle, the powder was transferred to an Eppendorf tube, 0.5 ml distilled water was added to the tube, and the tube was vortex-mixed, heated in a water bath at 80–90 °C for 20 min and stored at –20 °C. Mycelium yield was determined by measuring protein by the Bradford technique (Bradford, 1976). Glycerol was assayed using a commercially available kit (catalogue no. 10 148 270 035, Boehringer Mannheim/R-Biopharm). The assay comprised glycerokinase-mediated phosphorylation of glycerol, using ATP; ATP consumption was quantified by measuring NADH oxidation in the coupled reaction of pyruvate kinase (ADP+PEP
ATP+pyruvate) and lactate dehydrogenase (pyruvate+NADH+H+lactate+NAD+), where PEP stands for phosphoenolpyruvate.
Statistical analysis.
Data are shown as means±SD (sample size), unless stated otherwise. Independent two-tailed t tests were performed in either SYSTAT version 5.03 (Systat) or Excel (Microsoft).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The difference in turgor was not statistically significant (t test, P=0.684). Thus, the turgor poise under non-stressed conditions was similar. Since osmosensitivity may be reflected in the response of the cut mutant to hyperosmotic stress, we examined the presence and time-course of turgor recovery.
|
) similar to that in the wild-type (Lew et al., 2004; Fig. 3). The recovery time was similar in both strains, with turgor recovering to near the initial level within 60 min. One mechanism of turgor recovery involves ion uptake, which may manifest itself in electrical changes.
|
). The sustained hyperpolarization is absent in the os-1 and os-2 mutants of the osmotic response MAP kinase cascade (Lew et al., 2006). Unlike the os-1 and os-2 mutants, the electrical response of the cut mutant was the same as that of the wild-type (Fig. 4A). The initial potentials for wild-type (–174±23 mV, n=11) and cut (–169±17 mV, n=11) depolarized to the same extent within 1–2 min after hyperosmotic treatment (–65±23 and –54±17 mV, respectively), followed by similar sustained hyperpolarization (–189±33 and –182±24 mV, respectively) after 3–4 min. The electrical changes for the two strains were statistically the same, based on t tests (0.367<P<0.926, n=11). Current–voltage relations for the cut mutant revealed a higher conductance compared to that for the wild-type (Fig. 4B), but the responses to hyperosmotic treatment were similar. Thus, the cut mutant maintained the same array of electrical responses to hyperosmotic stress as did the wild-type, unlike the os-1 and os-2 mutants, for which sustained hyperpolarization is absent (Lew et al., 2006).
|
), it appears that cut is not part of the cascade. To corroborate this, we examined the effect of the phenylpyrrole fludioxonil on the electrical properties of the wild-type and the cut and os–2 mutants. Fludioxonil has been reported to activate the MAP kinase cascade, based upon the insensitivity of MAP kinase cascade mutants (including os-2) to fludioxonil, and the induction of glycerol synthesis and hyphal bursting in the wild-type by fludioxonil (Fujimura et al., 2000). Similar to hyperosmotic treatment, fludioxonil caused hyperpolarization of the potential of both the wild-type and the cut mutant, but not the os-2 mutant (consistent with os-2 insensitivity to the fungicide fludioxonil) (Fig. 5A, B). Hyphal tip growth was inhibited within the same time frame as the hyperpolarization in the wild-type and cut, but not the os-2 mutant (Fig. 5C).
|
Net ion-flux responses in the cut mutant
In response to hyperosmotic treatment, there was an initial H+ influx into the hyphae, which corresponded to nearly plasmolytic conditions (incipient plasmolysis was often observed). Within 5 min, both the wild-type and the cut mutant shifted to H+ efflux (corresponding to the disappearance of incipient plasmolysis) (Fig. 6). The time-course of H+ efflux is similar to that of the sustained electrical hyperpolarization, suggesting that H+-ATPase activation is responsible for both (Lew et al., 2006). Transient changes in Ca2+ (influx), K+ (efflux) and Cl– (a net shift to efflux) were also observed during the initial hyperosmotic shock (0–10 min). During the time interval associated with turgor recovery (10–40 min), Ca2+ efflux, and K+ and Cl– influx, occurred. Similar changes were observed in the wild-type and the cut mutant during turgor recovery, but the magnitudes varied. Compared to the wild-type, H+ efflux was similar and Ca2+ efflux was lower in the cut mutant; there was a shift towards net K+ influx 20–40 min after treatment, relative to the initial flux in cut, and Cl– influx was similar to wild-type (Fig. 6). Other factors, such as glycerol production, may contribute to turgor recovery.
|
). Even in the absence of hyperosmotic treatment (BS control), wild-type mycelia contained low levels of glycerol, while the cut mutant did not. Hyperosmotic treatment (either sucrose or NaCl) caused accumulation of glycerol in the wild-type, but not in the cut mutant. Glycerol accumulation by the wild-type varied within the three replicate experiments. After 80 min of hyperosmotic treatment, it ranged from 0.40 to 4.5 g glycerol (g protein)–1, but it was always higher than that in the cut mutant [0.0–0.3 g glycerol (g protein)–1], significantly so, after 80 min of treatment with either sucrose (P=0.013) or NaCl (P=0.004).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Bohnert & Jensen, 1996)]. This mechanism relies on modulation of biosynthetic pathways to divert carbon to osmolyte production. An alternative mechanism, ion uptake, depends upon the availability of ions in the extracellular milieu.
Osmolyte accumulation has garnered much of the attention. In addition to a role in long-term acclimation of the organism to high external osmotic stress, osmolytes also confer osmoprotection to cytoplasmic constituents, and are even accumulated in response to other stresses, such as freezing (Panadero et al., 2006). In contrast, ion uptake and accumulation cannot play a dual role of regulating both turgor and osmoprotection. Nevertheless, ion uptake does play a role in immediate responses to hyperosmotic conditions, and appears to be well-correlated with rapid turgor recovery in higher plants (Shabala & Lew, 2002) and fungi (Lew et al., 2006).
In functional characterization of fast turgor regulation in N. crassa, it has become clear that the osmotic MAP kinase cascade not only regulates glycerol production, but also acts directly on ion transport, activating the H+-ATPase and mediating an increase in net ion influx to cause turgor recovery within 60 min of hyperosmotic stress (Lew et al., 2006). In the present work, we extended our characterization of turgor regulation and ion transport to include the cut mutant, which is osmosensitive, but is defective in a pathway that is separate and distinct from the osmotic MAP kinase cascade.
In the absence of a functional cut gene, the initial turgor in BS was the same as that in the wild-type. After hyperosmotic treatment, both turgor recovery and electrical changes were very similar to those in the wild-type. The sustained hyperpolarization observed after hyperosmotic treatment can be attributed to activation of H+-ATPase, based upon the similar time-courses of hyperpolarization and increased H+ efflux after hyperosmotic treatment. H+-ATPase activation is absent in the os-1 and os-2 mutants of the osmotic MAP kinase pathway, since they do not exhibit sustained hyperpolarization, and the os-1 mutant (os-2 has not been examined) does not exhibit H+ efflux (Lew, 2006). The role of ion uptake in the wild-type and the cut mutant was corroborated by examining the effects of the fungicide fludioxonil.
Fludioxonil, reported to activate the osmotic MAP kinase cascade (Fujimura et al., 2000), also causes sustained hyperpolarization of the potential in the wild-type and the cut mutant, but not in the os-2 mutant, consistent with activation of H+-ATPase by the osmotic MAP kinase cascade. Fludioxonil inhibited hyphal elongation of the wild-type and the cut mutant (but not os-2) within 5 min of treatment, similar to the time-course of the sustained hyperpolarization. This result suggests that there is a regulatory link between the osmotic MAP kinase cascade and tip growth. However, fludioxonil may have multiple targets. In the basidiomycete Cryptococcus neoformans, fludioxonil causes growth cessation before cell swelling, and mutations in the calcineurin pathway confer heightened sensitivity to fludioxonil (Kijima et al., 2006). In addition, the cut mutant exhibits less sensitivity to fludioxonil than the wild-type or the allelic ovc mutant, which overproduces carotenoids in the light (Youssar et al., 2005) (we did not examine the concentration-dependence of the effect of fludioxonil). Thus, fludioxonil may inhibit tip growth by a mechanism distinct from activation of the MAP kinase cascade, although a direct link between the MAP kinase cascade and tip growth seems more likely. If so, the inhibitor may offer insight into the linkages between turgor and tip growth.
After hyperosmotic stress, the cut mutant did not accumulate glycerol. Our results confirm a previous comprehensive characterization of glycerol accumulation in os and cut mutants, in which no glycerol accumulation was observed in the cut mutant (Fujimura et al., 2000) when the osmotic stress was 4 % NaCl, and accumulation was measured after 4 h. The alternative mechanism for turgor regulation that we examined is ion uptake. The cut mutant did take up ions from the extracellular medium, although the magnitude varied with respect to the wild-type. Of the two major osmotically active ions, Cl– influx was similar to that in the wild-type, but only a shift towards net K+ influx was observed in the cut mutant. Thus, we cannot ascribe turgor recovery in the cut mutant solely to ion uptake, although it will certainly contribute significantly.
Our results suggest a cause for the osmosensitivity of the cut mutant. The os-1 and os-2 mutants of the osmotic MAP kinase cascade maintain a lower turgor than the wild-type under non-stressed conditions (Lew et al., 2006). It is easy to attribute their osmosensitivity to an inability to maintain a significantly high turgor poise by uptake of ions and accumulation of glycerol during osmotic stress. In the absence of glycerol accumulation in the cut mutant, steady-state turgor poise is the same as that in the wild-type, as is turgor recovery, presumably due to ion uptake. However, long-term survival may not be possible in the absence of the osmoprotectant glycerol, hence the osmosensitivity of the cut mutant.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: N. L. Glass
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Beever, R. E. & Laracy, E. P. (1986). Osmotic adjustment in the filamentous fungus Aspergillus nidulans. J Bacteriol 168, 1358–1365.
Bohnert, H. J. & Jensen, R. G. (1996). Strategies for engineering water-stress tolerance in plants. Trends Biotechnol 14, 89–97.[CrossRef]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]
Ellis, S. W., Grindle, M. & Lewis, D. H. (1991). Effect of osmotic stress on yield and polyol content of dicarboximide-sensitive and -resistant strains of Neurospora crassa. Mycol Res 95, 457–464.
Fujimura, M., Ochiai, N., Ichiishi, A., Usami, R. & Yamaguchi, I. (2000). Sensitivity to phenylpyrrole fungicides and abnormal glycerol accumulation in os and cut mutant strains of Neurospora crassa. J Pestic Sci (Nihon Noyaku Gakkai shi) 25, 31–36.
Fujimura, M., Ochiai, N., Oshima, M., Motoyama, T., Ichiishi, A., Usami, R., Horikoshi, K. & Yamaguchi, I. (2003). Putative homologs of SSK22 MAPKK kinase and PBS2 MAPK kinase of Saccharomyces cerevisiae encoded by os-4 and os-5 genes for osmotic sensitivity and fungicide resistance in Neurospora crassa. Biosci Biotechnol Biochem 67, 186–191.[CrossRef][Medline]
Grindle, M. & Temple, W. (1982). Fungicide-resistance of os mutants of Neurospora crassa. Neurospora Newsl 29, 16–17.
Grindle, M. & Temple, W. (1983). Fungicide resistance of smco mutants of Neurospora crassa. Neurospora Newsl 30, 7–8.
Henriksen, G. H., Raj Raman, D., Walker, L. P. & Spanswick, R. M. (1992). Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. II. Patterns of uptake along the root axis and evaluation of the microelectrode flux estimation technique. Plant Physiol 99, 734–747.
Jennings, D. H. (1995). Water relations and salinity. In The Physiology of Fungal Nutrition, pp. 398–446. Cambridge: Cambridge University Press.
Kijima, K., Bahn, Y.-S. & Heitman, J. (2006). Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans. Microbiology 152, 591–604.
Klipp, E., Nordlander, B., Kruger, R., Gennemark, P. & Hoffmann, S. (2005). Integrative model of the response of yeast to osmotic shock. Nat Biotechnol 23, 975–982.[CrossRef][Medline]
Krantz, M. E., Becit, E. & Hoffmann, S. (2006). Comparative genomics of the HOG-signaling system in fungi. Curr Genet 49, 137–151.[CrossRef][Medline]
Kuwana, H. (1953). Studies on the morphological mutant "cut" in Neurospora crassa. Cytologia 18, 235–239.
Lew, R. R. (1996). Pressure regulation of the electrical properties of growing Arabidopsis thaliana L. roothairs. Plant Physiol 112, 1089–1100.[Abstract]
Lew, R. R. (1999). Comparative analysis of Ca2+ and H+ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa. Eur J Cell Biol 78, 892–902.[Medline]
Lew, R. R. (2006). Use of double barrel micropipettes to voltage-clamp plant and fungal cells. In Plant Electrophysiology – Theory and Methods, pp. 139–154. Edited by A. G. Volkov. Berlin & New York: Springer.
Lew, R. R., Levina, N. N., Walker, S. K. & Garrill, A. (2004). Turgor regulation of hyphal organisms. Fungal Genet Biol 41, 1007–1015.[CrossRef][Medline]
Lew, R. R., Levina, N. N., Shabala, L., Anderca, M. I. & Shabala, S. N. (2006). Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi. Eukaryot Cell 5, 480–487.
McCluskey, K. (2003). The Fungal Genetics Stock Center: from molds to molecules. Adv Appl Microbiol 52, 245–262.[CrossRef][Medline]
Miller, T. K., Renault, S. & Selitrennikoff, C. P. (2002). Molecular dissection of alleles of the osmotic-1 locus of Neurospora crassa. Fungal Genet Biol 35, 147–155.[CrossRef][Medline]
Motoyama, T., Ohira, T., Kadokura, K., Ichiishi, A., Fujimura, M., Yamaguchi, I. & Kudo, T. (2005). An Os-1 family histidine kinase from a filamentous fungus confers fungicide-sensitivity to yeast. Curr Genet 47, 298–306.[CrossRef][Medline]
Newman, I. A. (2001). Ion transport in roots: measurement of fluxes using ion-selective microelectrodes to characterize transporter function. Plant Cell Environ 24, 1–14.[Medline]
Noguchi, R., Banno, S., Ichikawa, R., Fukumori, F., Ichiishi, A., Kimura, M., Yamaguchi, I. & Fujimura, M. (2007). Identification of OS-2 MAP kinase-dependent genes induced in response to osmotic stress, antifungal agent fludioxinil, and heat shock in Neurospora crassa. Fungal Genet Biol 44, 208–218.[CrossRef][Medline]
Panadero, J., Pallotti, C., Rodríguez-Vargas, S., Randez-Gil, F. & Prieto, J. A. (2006). A downshift in temperature activates the high osmolarity glycerol (HOG) pathway, which determines freeze tolerance in Saccharomyces cerevisiae. J Biol Chem 281, 4638–4645.
Perkins, D. D., Radford, A. & Sachs, M. S. (2001). The Neurospora Compendium: Chromosomal Loci. San Diego: Academic Press.
Schumacher, M. M. C. S., Enderlin, C. S. & Selitrennikoff, C. P. (1997). The osmotic-1 locus of Neurospora crassa encodes a putative histidine kinase similar to osmosensors of bacteria and yeast. Curr Microbiol 34, 340–347.[CrossRef][Medline]
Shabala, S. & Lew, R. R. (2002). Turgor regulation in osmotically stressed Arabidopsis thaliana epidermal root cells: direct support for the role of inorganic ion uptake as revealed by concurrent flux and cell turgor measurements. Plant Physiol 129, 290–299.
Smith, P. J. S., Hammar, K., Porterfield, D. M., Sanger, R. H. & Trimarchi, J. R. (1999). Self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux. Microsc Res Tech 48, 398–417.
Vogel, H. J. (1956). A convenient growth medium for Neurospora. Microb Genet Bull 13, 42–46.
Youssar, L. & Avalos, J. (2006). Light-dependent regulation of the gene cut-1 of Neurospora, involved in the osmotic stress response. Fungal Genet Biol 43, 752–763.[Medline]
Youssar, L., Schmidhauser, T. J. & Avalos, J. (2005). The Neurospora crassa gene responsible for the cut and ovc phenotypes encodes a protein of the haloacid dehalogenase family. Mol Microbiol 55, 828–838.[CrossRef][Medline]
Zhang, Y., Lamm, R., Pillonel, C., Lam, S. & Xu, J.-R. (2002). Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue. Appl Environ Microbiol 68, 532–538.
Received 3 November 2006;
revised 4 January 2007;
accepted 9 January 2007.
This article has been cited by other articles:
|
|
 |
|
  R. R. Lew, Z. Abbas, M. I. Anderca, and S. J. Free Phenotype of a Mechanosensitive Channel Mutant, mid-1, in a Filamentous Fungus, Neurospora crassa Eukaryot. Cell, April 1, 2008; 7(4): 647 - 655. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
