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1.
Plant J ; 50(3): 401-13, 2007 May.
Article in English | MEDLINE | ID: mdl-17425714

ABSTRACT

Potassium is an inevitable component of plant life, and potassium channels play a pivotal role in plant growth and development. The role of potassium and of K(+) channels in plant cell division and cell-cycle progression, however, has not been determined so far. K(+) channel blocker studies with synchronized tobacco BY-2 cells revealed that K(+) uptake is required for proper cell-cycle progression during the transition from G(1) to S phase. Electrophysiological studies (patch-clamp and voltage-clamp techniques) showed a cell-cycle dependency of K(+) channel activities and reduced driving force for K(+) uptake in dividing cells. Among the four Shaker-like K(+) channel genes expressed in BY-2 cells, NKT1 represents an inwardly rectifying K(+) channel that mediates K(+) uptake. NKT1 is transcriptionally induced during G(1) phase, while transcripts of the outward-rectifier NTORK1 dominate S phase. Elongating BY-2 cells appeared hyperpolarized (-101 +/- 11 mV), and had elevated osmotic pressure and approximately twice the turgor pressure when compared with depolarized (-64 +/- 8 mV) dividing cells. This indicates that cells have to gain a threshold K(+) level to re-enter the cell cycle. Based on these findings, turgor regulation through modulation of K(+) channel density in plant cell division and cell-cycle progression is discussed.


Subject(s)
Cell Cycle/physiology , Nicotiana/cytology , Nicotiana/metabolism , Potassium/metabolism , Cells, Cultured , Gene Expression Regulation, Plant , Molecular Sequence Data , Plant Proteins/genetics , Plant Proteins/metabolism , Potassium Channel Blockers/pharmacology , Potassium Channels/genetics , Potassium Channels/metabolism , Nicotiana/drug effects , Nicotiana/genetics
2.
Planta ; 225(5): 1179-91, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17146665

ABSTRACT

Heterologous expression of plant genes in yeast and animal cells represents a common approach to study plant ion channels. When expressed in Xenopus oocytes and COS cells the Arabidopsis Shaker-like K+ channel, AKT2 forms a weakly voltage-dependent channel, blocked by Ca2+ and protons. Channels with these characteristics, however, were not found in AKT2-expressing Arabidopsis cell types. To understand this phenomenon, we employed Agrobacterium-mediated transient transformation to functionally characterise Arabidopsis thaliana channels in Nicotiana benthamiana mesophyll cells. In this expression system we used AtTPK4 as a control for voltage-independent A. thaliana channels. Agrobacteria harbouring GFP-tagged constructs with the coding sequences of AKT2 and AtTPK4 were infiltrated into intact tobacco leaves. With quantitative RT-PCR analyses channel transcripts of AKT2 and AtTPK4 were determined in transformed leaves. These results were confirmed by Western blots with V5 epitope-tagged AKT2 and AtTPK4 proteins, showing that the channel protein was indeed synthesised. For functional analysis of these channels, mesophyll protoplasts were isolated from infiltrated leaf sections. Patch-clamp studies revealed that AKT2 channels in mesophyll protoplasts retained Ca2+ and pH sensitivity, characteristics of the heterologously expressed protein, but displayed pronounced differences in voltage-dependence and kinetics. AKT2-transformed mesophyll cells, displayed inward-rectifying, rather than voltage-independent K+ channels, initially characterised in AKT2-expressing animal cells. In contrast, AtTPK4 showed the same electrophysiological characteristics both, in oocytes and plant cells. Our data suggest that heterologous systems do not always possess all regulatory components for functional expression of plant channels. Therefore, transient expression of plant proteins in planta provides an additional research tool for rapid biophysical analysis of plant ion channels.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/physiology , Nicotiana/physiology , Potassium Channels/physiology , Arabidopsis Proteins/genetics , DNA, Plant/genetics , Patch-Clamp Techniques , Plant Leaves/cytology , Plant Leaves/physiology , Plants, Genetically Modified , Potassium Channels/genetics , Protoplasts/physiology , Reverse Transcriptase Polymerase Chain Reaction , Rhizobium/genetics , Nicotiana/cytology , Nicotiana/genetics , Transcription, Genetic
3.
Plant Physiol ; 143(1): 487-94, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17114272

ABSTRACT

Living organisms are capable of discriminating thermal stimuli from noxious cold to noxious heat. For more than 30 years, it has been known that plant cells respond to cold with a large and transient depolarization. Recently, using transgenic Arabidopsis (Arabidopsis thaliana) expressing the calcium-sensitive protein aequorin, an increase in cytosolic calcium following cold treatment was observed. Applying the patch-clamp technique to Arabidopsis mesophyll protoplasts, we could identify a transient plasma membrane conductance induced by rapid cooling. This cold-induced transient conductance was characterized as an outward rectifying 33 pS nonselective cation channel. The permeability ratio between calcium and cesium was 0.7, pointing to a permeation pore >3.34 A (ø of cesium). Our experiments thus provide direct evidence for the predicted but not yet measured cold-activated calcium-permeable channel in plants.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Calcium Channels/metabolism , Calcium/metabolism , Cold Temperature , Arabidopsis/cytology , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Calcium Channels/genetics , Cell Membrane/metabolism , Mutation , Patch-Clamp Techniques , Permeability , Plant Leaves/cytology , Plant Leaves/metabolism , Plant Leaves/physiology , Protoplasts/metabolism
4.
J Gen Physiol ; 125(5): 483-92, 2005 May.
Article in English | MEDLINE | ID: mdl-15824192

ABSTRACT

Inward-rectifying K+ channels serve as a major pathway for Ca2+-sensitive K+ influx into guard cells. Arabidopsis thaliana guard cell inward-rectifying K+ channels are assembled from multiple K+ channel subunits. Following the recent isolation and characterization of an akt2/3-1 knockout mutant, we examined whether the AKT2/3 subunit carries the Ca2+ sensitivity of the guard cell inward rectifier. Quantification of RT-PCR products showed that despite the absence of AKT2 transcripts in guard cells of the knockout plant, expression levels of the other K+ channel subunits (KAT1, KAT2, AKT1, and AtKC1) remained largely unaffected. Patch-clamp experiments with guard cell protoplasts from wild type and akt2/3-1 mutant, however, revealed pronounced differences in Ca2+ sensitivity of the K+ inward rectifier. Wild-type channels were blocked by extracellular Ca2+ in a concentration- and voltage-dependent manner. Akt2/3-1 mutants lacked the voltage-dependent Ca2+ block, characteristic for the K+ inward rectifier. To confirm the akt2/3-1 phenotype, two independent knockout mutants, akt2-1 and akt2::En-1 were tested, demonstrating that the loss of AKT2/3 indeed affects the Ca2+ dependence of guard cell inward rectifier. In contrast to AKT2 knockout plants, AKT1, AtKC1, and KAT1 loss-of-function mutants retained Ca2+ block of the guard cell inward rectifier. When expressed in HEK293 cells, AKT2 channel displayed a pronounced susceptibility toward extracellular Ca2+, while the dominant guard cell K+ channel KAT2 was Ca2+ insensitive. Thus, we conclude that the AKT2/3 subunit constitutes the Ca2+ sensitivity of the guard cell K+ uptake channel.


Subject(s)
Arabidopsis Proteins/physiology , Calcium/metabolism , Plant Leaves/physiology , Potassium Channels/physiology , Potassium/metabolism , Arabidopsis , Arabidopsis Proteins/genetics , Cell Line , Gene Expression , Humans , Ion Channel Gating/physiology , Kidney/cytology , Membrane Potentials/physiology , Mutagenesis , Potassium Channels/genetics
5.
Plant J ; 41(4): 606-14, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15686523

ABSTRACT

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.


Subject(s)
Arabidopsis/physiology , Ion Channel Gating/physiology , Potassium Channels/physiology , Sodium/physiology , Vacuoles/physiology , Calcium/physiology , Cell Culture Techniques , Membrane Potentials/physiology
6.
Planta ; 221(2): 212-21, 2005 May.
Article in English | MEDLINE | ID: mdl-15599592

ABSTRACT

Potassium ions constitute the most important macronutrients taken up by plants. To unravel the mechanisms of K+ uptake and its sensitivity to salt stress in the model plant rice, we isolated and functionally characterized OsAKT1, a potassium channel homologous to the Arabidopsis root inward rectifier AKT1. OsAKT1 transcripts were predominantly found in the coleoptile and in the roots of young rice seedlings. K+ channel mRNA decreases in response to salt stress, both in the shoot and in the root of 4-day-old rice seedlings. Following expression in HEK293 cells, we were able to characterize OsAKT1 as a voltage-dependent, inward-rectifying K+ channel regulated by extracellular Ca2+ and protons. Patch-clamp studies on rice root protoplasts identified a K+ inward rectifier with similar channel properties as heterologously expressed OsAKT1. In line with the transcriptional downregulation of OsAKT1 in response to salt stress, inward K+ currents were significantly reduced in root protoplasts. Thus, OsAKT1 seems to represent the dominant salt-sensitive K+ uptake channel in rice roots.


Subject(s)
Oryza/metabolism , Plant Proteins/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Sodium Chloride/metabolism , Animals , Cell Line , Gene Expression Regulation, Plant , Oryza/genetics , Plant Epidermis/metabolism , Plant Proteins/genetics , Plant Roots/metabolism , Potassium Channels, Inwardly Rectifying/genetics , Seedlings/metabolism
7.
Plant J ; 37(6): 815-27, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14996216

ABSTRACT

The transcript abundance of the K+-channel gene ZMK1 (Zea mays K+ channel 1) in maize coleoptiles is controlled by the phytohormone auxin. Thus, ZMK1 is thought to function in auxin-regulated coleoptile elongation, as well as during gravitropism and phototropism. To investigate related growth phenomena in the dicotyledonous plant Arabidopsis thaliana, we screened etiolated seedlings for auxin-induced K+-channel genes. Among the members of the Shaker-like K+ channels, we thereby identified transcripts of the inward rectifiers, KAT1 (K+ transporter of Arabidopsis thaliana) and KAT2, to be upregulated by auxin. The phloem-associated KAT2 was localised in cotyledons and the apical part of etiolated seedlings. In contrast, the K+-channel gene KAT1 was expressed in the cortex and epidermis of etiolated hypocotyls, as well as in flower stalks. Furthermore, KAT1 was induced by active auxins in auxin-sensitive tissues characterised by rapid cell elongation. Applying the patch-clamp technique to protoplasts of etiolated hypocotyls, we correlated the electrical properties of K+ currents with the expression profile of K+-channel genes. In KAT1-knockout mutants, K+ currents after auxin stimulation were characterised by reduced amplitudes. Thus, this change in the electrical properties of the K+-uptake channel in hypocotyl protoplasts resulted from an auxin-induced increase of active KAT1 proteins. The loss of KAT1-channel subunits, however, did not affect the auxin-induced growth rate of hypocotyls, pointing to compensation by residual, constitutive K+ transporters. From gene expression and electrophysiological data, we suggest that auxin regulation of KAT1 is involved in elongation growth of Arabidopsis. Furthermore, a role for KAT2 in the auxin-controlled vascular patterning of leaves is discussed.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/drug effects , Arabidopsis/genetics , Genes, Plant/drug effects , Indoleacetic Acids/pharmacology , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Electrophysiology , Gene Expression Regulation, Plant/drug effects , Gene Targeting , Hypocotyl/drug effects , Hypocotyl/metabolism , Mutation , Plant Proteins , Potassium Channels/chemistry , Potassium Channels, Voltage-Gated , Protein Subunits , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , Seedlings/drug effects , Seedlings/metabolism
8.
Plant J ; 36(6): 931-45, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14675456

ABSTRACT

K+ channels control K+ homeostasis and the membrane potential in the sieve element/companion cell complexes. K+ channels from Arabidopsis phloem cells expressing green fluorescent protein (GFP) under the control of the AtSUC2 promoter were analysed using the patch-clamp technique and quantitative RT-PCR. Single green fluorescent protoplasts were selected after being isolated enzymatically from vascular strands of rosette leaves. Companion cell protoplasts, which could be recognized by their nucleus, vacuole and chloroplasts, and by their expression of the phloem-specific marker genes SUC2 and AHA3, formed the basis for a cell-specific cDNA library and expressed sequence tag (EST) collection. Although we used primers for all members of the Shaker K+ channel family, we identified only AKT2, KAT1 and KCO6 transcripts. In addition, we also detected transcripts for AtPP2CA, a protein phosphatase, that interacts with AKT2/3. In line with the presence of the K+ channel transcripts, patch-clamp experiments identified distinct K+ channel types. Time-dependent inward rectifying K+ currents were activated upon hyperpolarization and were characterized by a pronounced Ca2+-sensitivity and inhibition by protons. Whole-cell inward currents were carried by single K+-selective channels with a unitary conductance of approximately 4 pS. Outward rectifying K+ channels (approximately 19 pS), with sigmoidal activation kinetics, were elicited upon depolarization. These two dominant phloem K+ channel types provide a versatile mechanism to mediate K+ fluxes required for phloem action and potassium cycling.


Subject(s)
Arabidopsis/genetics , Membrane Transport Proteins/genetics , Plant Proteins/genetics , Potassium Channels/genetics , Potassium/metabolism , Promoter Regions, Genetic/genetics , Arabidopsis/physiology , Base Sequence , DNA Primers , Genetic Markers , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Potentials/physiology , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Patch-Clamp Techniques , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/physiology , Potassium Channels/physiology , Protoplasts/physiology , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction
9.
Plant Physiol ; 133(2): 528-37, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12972665

ABSTRACT

Guard cell chloroplasts are unable to perform significant photosynthetic CO2 fixation via Rubisco. Therefore, guard cells depend on carbon supply from adjacent cells even during the light period. Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata. Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture. This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells. Expression of the AtSTP1 H+-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum. Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday. This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose. Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/physiology , Gene Expression Regulation, Plant/physiology , Nuclear Proteins/genetics , RNA-Binding Proteins , Saccharomyces cerevisiae Proteins , Transcription Factors , Arabidopsis/genetics , Arabidopsis/radiation effects , Chloroplasts/physiology , Chloroplasts/radiation effects , Circadian Rhythm , Gene Expression Regulation, Plant/radiation effects , In Situ Hybridization , Light , Nuclear Proteins/deficiency , Photosynthesis/radiation effects , Ribulose-Bisphosphate Carboxylase/metabolism
10.
Plant J ; 34(6): 778-87, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12795698

ABSTRACT

After completion of the Arabidopsis genome-sequencing programme, crown galls induced by Agrobacterium tumefaciens may become a model system to study plant tumour development. The molecular mechanisms of nutrient supply to support tumour growth and development are still unknown. In this study, we have identified a unique profile of Shaker-like potassium channels in agrobacteria-induced Arabidopsis tumours. Comparing the gene expression pattern of rapidly growing tumours with that of non-infected tissues, we found the suppression of shoot in favour of root-specific K+ channels. Among these, the upregulation of AKT1 and AtKC1 and the suppression of AKT2/3 and GORK were most pronounced. As a consequence, K+ uptake and accumulation were elevated in the tumour (163 mm) compared to control tissues (92 mm). Patch clamp studies on tumour protoplasts identified a population expressing the electrical properties of the AKT1 K+ channel. Furthermore, plants lacking a functional AKT1 or the AKT2/3 phloem K+ channel gene did not support tumour growth. This indicates that the delivery of potassium by AKT1 and the direction of assimilates, triggered by AKT2/3, are essential for tumour growth.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Plant Tumors , Potassium Channels/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cations , Gene Expression Regulation, Neoplastic , Gene Expression Regulation, Plant , Membrane Potentials , Mutation , Patch-Clamp Techniques , Plant Stems/genetics , Plant Stems/metabolism , Plant Tumors/microbiology , Potassium/metabolism , Potassium Channels/genetics
11.
Proc Natl Acad Sci U S A ; 99(6): 4079-84, 2002 Mar 19.
Article in English | MEDLINE | ID: mdl-11904452

ABSTRACT

Ion channels in roots allow the plant to gain access to nutrients. The composition of the individual ion channels and the functional contribution of different alpha-subunits is largely unknown. Focusing on K(+)-selective ion channels, we have characterized AtKC1, a new alpha-subunit from the Arabidopsis shaker-like ion channel family. Promoter-beta-glucuronidase (GUS) studies identified AtKC1 expression predominantly in root hairs and root endodermis. Specific antibodies recognized AtKC1 at the plasma membrane. To analyze further the abundance and the functional contribution of the different K(+) channels alpha-subunits in root cells, we performed real-time reverse transcription-PCR and patch-clamp experiments on isolated root hair protoplasts. Studying all shaker-like ion channel alpha-subunits, we only found the K(+) inward rectifier AtKC1 and AKT1 and the K(+) outward rectifier GORK to be expressed in this cell type. Akt1 knockout plants essentially lacked inward rectifying K(+) currents. In contrast, inward rectifying K(+) currents were present in AtKC1 knockout plants, but fundamentally altered with respect to gating and cation sensitivity. This indicates that the AtKC1 alpha-subunit represents an integral component of functional root hair K(+) uptake channels.


Subject(s)
Arabidopsis/metabolism , Plant Roots/metabolism , Potassium Channels/chemistry , Potassium Channels/metabolism , Potassium/metabolism , Amino Acid Sequence , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/isolation & purification , Arabidopsis Proteins/metabolism , Base Sequence , Blotting, Western , Cloning, Molecular , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Molecular Sequence Data , Organ Specificity , Patch-Clamp Techniques , Plant Roots/cytology , Plant Roots/genetics , Potassium Channels/genetics , Potassium Channels/isolation & purification , Protein Subunits , Protoplasts/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism
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