Biomedical subjects

Rosalia Deeken

Publications and source records attributed to Rosalia Deeken.

6 recordsLinked to original sources

PubMed2006

An integrated view of gene expression and solute profiles of Arabidopsis tumors: a genome-wide approach.

Transformation of plant cells with T-DNA of virulent agrobacteria is one of the most extreme triggers of developmental changes in higher plants. For rapid growth and development of resulting tumors, specific changes in the gene expression profile and metabolic adaptations are required. Increased transport and metabolic fluxes are critical preconditions for growth and tumor development. A functional genomics approach, using the Affymetrix whole genome microarray (approximately 22,800 genes), was applied to measure changes in gene expression. The solute pattern of Arabidopsis thaliana tumors and uninfected plant tissues was compared with the respective gene expression profile. Increased levels of anions, sugars, and amino acids were correlated with changes in the gene expression of specific enzymes and solute transporters. The expression profile of genes pivotal for energy metabolism, such as those involved in photosynthesis, mitochondrial electron transport, and fermentation, suggested that tumors produce C and N compounds heterotrophically and gain energy mainly anaerobically. Thus, understanding of gene-to-metabolite networks in plant tumors promotes the identification of mechanisms that control tumor development.

Arabidopsis↗

PubMed2006

In planta AKT2 subunits constitute a pH- and Ca2+-sensitive inward rectifying K+ channel.

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.

Arabidopsis↗

PubMed2005

AKT2/3 subunits render guard cell K+ channels Ca2+ sensitive.

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.

Arabidopsis↗

PubMed2003

Tumour development in Arabidopsis thaliana involves the Shaker-like K+ channels AKT1 and AKT2/3.

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.

Arabidopsis↗

PubMed2003

Isolation of AtSUC2 promoter-GFP-marked companion cells for patch-clamp studies and expression profiling.

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.

Arabidopsis↗

PubMed2002

Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis.

Members of the AKT2/3 family have been identified as photosynthate-induced phloem K(+) channels. Here we describe the isolation and characterisation of an AKT2/3 loss-of-function mutant (akt2/3-1) from Arabidopsis thaliana (L.) Heynh. Microautoradiography following (14)CO(2) incubation in the light revealed that a major fraction of (14)CO(2)-derived photosynthates leaking out of sieve tubes appears not to be effectively reloaded (retrieval) into the phloem of the mutant. Using the aphid stylectomy technique we showed that the phloem sap of the mutant, lacking the phloem channels of the AKT2/3 type, contained only half the sucrose content of the wild type. Furthermore, the akt2/3-1 mutant exhibited a reduced K(+) dependence of the phloem potential. Xenopus oocytes expressing the phloem sucrose/proton symporter depolarise upon sucrose application. When, however, the phloem channel was co-expressed - mimicking the situation in the sieve tube/companion cell complex - depolarisation was prevented. From our studies we thus conclude that AKT2/3 regulates the sucrose/H(+) symporters via the phloem potential.

Animals↗