It is generally accepted that K ؉ uptake into guard cells via inwardrectifying K ؉ channels is required for stomatal opening. To test whether the guard cell K ؉ channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1::En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K ؉ uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription-PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K ؉ channels AKT1, AKT2͞3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inwardrectifying K ؉ currents across the plasma membrane of both wildtype and KAT1::En-1 plants. This study demonstrates that multiple K ؉ channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.

The potassium-channel gene, AKT3, has recently been isolated from an Arabidopsis thaliana cDNA library. By using the whole-mount and in situ hybridization techniques, we found AKT3 predominantly expressed in the phloem. To study the physiological role of this channel type, AKT3 was heterologously expressed in Xenopus oocytes, and the electrical properties were examined with voltage-clamp techniques. Unlike the plant inward-rectifying guard cell K ؉ channels KAT1 and KST1, the AKT3 channels were only weakly regulated by the membrane potential. Furthermore, AKT3 was blocked by physiological concentrations of external Ca 2؉ and showed an inverted pH regulation. Extracellular acidification decreased the macroscopic AKT3 currents by reducing the single-channel conductance. Because assimilate transport in the vascular tissue coincides with both H ؉ and K ؉ f luxes, AKT3 K ؉ channels may be involved in K ؉ transport accompanying phloem loading and unloading processes.The plant vascular system, which consists of xylem and phloem, is specialized for long-distance solute and water transport. In both tissues, potassium represents one of the major mineral nutrients and is likely to assist in osmotic homeostasis. After uptake from the soil, K ϩ ions circulate between roots and shoots through the xylem and phloem to adopt the specific demands for this cation in the various tissues (1, 2). Recently, in vivo and in vitro analysis demonstrated the presence of K ϩ uptake and release channels in xylem parenchyma cells (3-6). In comparison, information about K ϩ transport across the plasma membrane of phloem cells, the underlying mechanisms, and the physiological role in long-distance solute transport is still limited. Phloem loading with assimilates is accompanied by ionic movements (7). Protons are pumped into the apoplast by a H ϩ -ATPase, generating transmembrane gradients in electropotential and pH that in turn enable the uphill transport of assimilates into the phloem through assimilate/ H ϩ -cotransporters (8-10). The phloem loading coincides with an increase in the symplastic K ϩ concentration likely to maintain electrical neutrality that is required for creating the pH gradient (2, 7). In addition, the K ϩ concentration in the sieve tube may affect the volume flow rate in the phloem (11). Furthermore, the membrane potential that transiently changes during phloem-propagating action potentials is possibly reestablished on K ϩ release from the sieve tube (12). Evidence for the expression of K ϩ channels in the phloem was recently provided (P.A., K. Philippar, and R.H., unpublished data) to support the idea that K ϩ channels may also mediate the transmembrane K ϩ fluxes in the phloem.In the present paper, we localized the cloned Arabidopsis K ϩ channel AKT3 to the phloem and revealed its unique dependence on voltage, Ca 2ϩ , and pH properties, which are well suited for meeting its supposed role in processes associated with the phloem. MATERIALS AND METHODSRNA Extraction and Northern Blot Analysis. Total RNA was isolate...

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the ArabidopsisAgrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria.

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.

Distribution of K, Ca, Cl, S, and P in freeze-dried sections of Arabidopsis flower stalk was analyzed by energy dispersive x-ray imaging. Concentrations of these elements in different cell types were quantified by microanalysis of single-cell samples and phloem exudates. Results showed a differential pattern of distribution for all five elements. K concentration was found to be highest in the parenchymatous tissue around vascular bundles. Ca and Cl were present mainly in the central part of the flower stalk. P was largely located in the bundles and in the parenchyma surrounding them. S signal was extraordinary high in groups of cells (S-cells) situated between the phloem of every vascular bundle and the endodermis. Enzymatic hydrolysis by thioglucosidase of cell sap collected from S-cells using a glass microcapillary resulted in the release of glucose, indicating that these cells contain glucosinolates at high (> 100 mm) concentration, which is consistent with the concentration of S (> 200 mm) estimated by x-ray analysis of cell sap samples. Since their position outside of the phloem is ideally suited for protecting the long-distance transport system from feeding insects, the possible roles of these cells as components of a plant defense system are discussed.

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 (∼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.

SummaryPhloem-mobile signals play a major role in plant nutrition, development and communication. In the latter context, phloem-mobile RNAs have been associated with signalling between plant tissues. In this study, we focused on the identification of transcripts in the shoot phloem of the model plant Arabidopsis thaliana. To isolate transcripts expressed in phloem parenchyma cells and in companion cell-sieve element complexes, we used laser microdissection coupled to laser pressure catapulting (LMPC). Mobile transcripts in sieve elements were isolated from leaf phloem exudates. After optimization of sampling and fixation, RNA of high quality was isolated from both sources. The modifications to the RNA amplification procedure described here were well suited to production of RNA of sufficient yield and quality for microarray experiments. Microarrays hybridized with LMPC-derived phloem tissue or phloem sap RNA allowed differentiation between phloem-expressed and mobile transcript species. Using this set of phloem transcripts and comparing them with microarrays derived from databases of light, hormone and nutrient treatment experiments, we identified phloem-derived RNAs as mobile, potential long-distance signals. Our dataset thus provides a search criterion for phloem-based signals hidden in the complex datasets of microarray experiments. The availability of these comprehensive phloem transcript profiles will facilitate reverse-genetic studies and forward-genetic screens for phloem and longdistance RNA signalling mutants.

Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumors. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide (“omic”) approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumor formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic, and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant.