Opportunistic Bacteria of Grapevine Crown Galls Are Equipped with the Genomic Repertoire for Opine Utilization.

Young grapevines (Vitis vinifera) suffer and eventually can die from the crown gall disease caused by the plant pathogen Allorhizobium vitis (Rhizobiaceae). Virulent members of A. vitis harbor a tumor-inducing plasmid and induce formation of crown galls due to the oncogenes encoded on the transfer DNA. The expression of oncogenes in transformed host cells induces unregulated cell proliferation and metabolic and physiological changes. The crown gall produces opines uncommon to plants, which provide an important nutrient source for A. vitis harboring opine catabolism enzymes. Crown galls host a distinct bacterial community, and the mechanisms establishing a crown gall-specific bacterial community are currently unknown. Thus, we were interested in whether genes homologous to those of the tumor-inducing plasmid coexist in the genomes of the microbial species coexisting in crown galls. We isolated 8 bacterial strains from grapevine crown galls, sequenced their genomes, and tested their virulence and opine utilization ability in bioassays. In addition, the 8 genome sequences were compared with 34 published bacterial genomes, including closely related plant-associated bacteria not from crown galls. Homologous genes for virulence and opine anabolism were only present in the virulent Rhizobiaceae. In contrast, homologs of the opine catabolism genes were present in all strains including the nonvirulent members of the Rhizobiaceae and non-Rhizobiaceae. Gene neighborhood and sequence identity of the opine degradation cluster of virulent and nonvirulent strains together with the results of the opine utilization assay support the important role of opine utilization for cocolonization in crown galls, thereby shaping the crown gall community.

ALMT-independent guard cell R-type anion currents.

Plant transpiration is controlled by stomata, with S- and R-type anion channels playing key roles in guard cell action. Arabidopsis mutants lacking the ALMT12/QUAC1 R-type anion channel function in guard cells show only a partial reduction in R-type channel currents. The molecular nature of these remaining R-type anion currents is still unclear. To further elucidate this, patch clamp, transcript and gas-exchange measurements were performed with wild-type (WT) and different almt mutant plants. The R-type current fraction in the almt12 mutant exhibited the same voltage dependence, susceptibility to ATP block and lacked a chloride permeability as the WT. Therefore, we asked whether the R-type anion currents in the ALMT12/QUAC1-free mutant are caused by additional ALMT isoforms. In WT guard cells, ALMT12, ALMT13 and ALMT14 transcripts were detected, whereas only ALMT13 was found expressed in the almt12 mutant. Substantial R-type anion currents still remained active in the almt12/13 and almt12/14 double mutants as well as the almt12/13/14 triple mutant. In good agreement, CO2 -triggered stomatal closure required the activity of ALMT12 but not ALMT13 or ALMT14. The results suggest that, with the exception of ALMT12, channel species other than ALMTs carry the guard cell R-type anion currents.

Under salt stress guard cells rewire ion transport and abscisic acid signaling.

Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations. Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.

Differential roles of glucosinolates and camalexin at different stages of Agrobacterium-mediated transformation.

Agrobacterium tumefaciens is the causal agent of crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. Agrobacterium tumefaciens is capable of transferring its T-DNA into different plant parts at different developmental stages for transient and stable transformation. However, the plant genes and mechanisms involved in these transformation processes are not well understood. We used Arabidopsis thaliana Col-0 seedlings to reveal the gene expression profiles at early time points during Agrobacterium infection. Common and differentially expressed genes were found in shoots and roots. A gene ontology analysis showed that the glucosinolate (GS) biosynthesis pathway was an enriched common response. Strikingly, several genes involved in indole glucosinolate (iGS) modification and the camalexin biosynthesis pathway were up-regulated, whereas genes in aliphatic glucosinolate (aGS) biosynthesis were generally down-regulated, on Agrobacterium infection. Thus, we evaluated the impacts of GSs and camalexin during different stages of Agrobacterium-mediated transformation combining Arabidopsis mutant studies, metabolite profiling and exogenous applications of various GS hydrolysis products or camalexin. The results suggest that the iGS hydrolysis pathway plays an inhibitory role on transformation efficiency in Arabidopsis seedlings at the early infection stage. Later in the Agrobacterium infection process, the accumulation of camalexin is a key factor inhibiting tumour development on Arabidopsis inflorescence stalks. In conclusion, this study reveals the differential roles of GSs and camalexin at different stages of Agrobacterium-mediated transformation and provides new insights into crown gall disease control and improvement of plant transformation.

The Nonspecific Lipid Transfer Protein AtLtpI-4 Is Involved in Suberin Formation of Arabidopsis thaliana Crown Galls.

Nonspecific lipid transfer proteins reversibly bind different types of lipid molecules in a hydrophobic cavity. They facilitate phospholipid transfer between membranes in vitro, play a role in cuticle and possibly in suberin formation, and might be involved in plant pathogen defense signaling. This study focuses on the role of the lipid transfer protein AtLTPI-4 in crown gall development. Arabidopsis (Arabidopsis thaliana) crown gall tumors, which develop upon infection with the virulent Agrobacterium tumefaciens strain C58, highly expressed AtLTPI-4 Crown galls of the atltpI-4 loss-of-function mutant were much smaller compared with those of wild-type plants. The gene expression pattern and localization of the protein to the plasma membrane pointed to a function of AtLTPI-4 in cell wall suberization. Since Arabidopsis crown galls are covered by a suberin-containing periderm instead of a cuticle, we analyzed the suberin composition of crown galls and found a reduction in the amounts of long-chain fatty acids (C18:0) in the atltpI-4 mutant. To demonstrate the impact of AtLtpI-4 on extracellular lipid composition, we expressed the protein in Arabidopsis epidermis cells. This led to a significant increase in the very-long-chain fatty acids C24 and C26 in the cuticular wax fraction. Homology modeling and lipid-protein-overlay assays showed that AtLtpI-4 protein can bind these very-long-chain fatty acids. Thus, AtLtpI-4 protein may facilitate the transfer of long-chain as well as very-long-chain fatty acids into the apoplast, depending on the cell type in which it is expressed. In crown galls, which endogenously express AtLtpI-4, it is involved in suberin formation.

Grapevine (Vitis vinifera) Crown Galls Host Distinct Microbiota.

UNLABELLED: Crown gall disease of grapevine is caused by virulent Agrobacterium strains and establishes a suitable habitat for agrobacteria and, potentially, other bacteria. The microbial community associated with grapevine plants has not been investigated with respect to this disease, which frequently results in monetary losses. This study compares the endophytic microbiota of organs from grapevine plants with or without crown gall disease and the surrounding vineyard soil over the growing seasons of 1 year. Amplicon-based community profiling revealed that the dominating factor causing differences between the grapevine microbiota is the sample site, not the crown gall disease. The soil showed the highest microbial diversity, which decreased with the distance from the soil over the root and the graft union of the trunk to the cane. Only the graft union microbiota was significantly affected by crown gall disease. The bacterial community of graft unions without a crown gall hosted transient microbiota, with the three most abundant bacterial species changing from season to season. In contrast, graft unions with a crown gall had a higher species richness, which in every season was dominated by the same three bacteria (Pseudomonas sp., Enterobacteriaceae sp., and Agrobacterium vitis). For in vitro-cultivated grapevine plantlets, A. vitis infection alone was sufficient to cause crown gall disease. Our data show that microbiota in crown galls is more stable over time than microbiota in healthy graft unions and that the microbial community is not essential for crown gall disease outbreak. IMPORTANCE: The characterization of bacterial populations in animal and human diseases using high-throughput deep-sequencing technologies, such as 16S amplicon sequencing, will ideally result in the identification of disease-specific microbiota. We analyzed the microbiota of the crown gall disease of grapevine, which is caused by infection with the bacterial pathogen Agrobacterium vitis. All other Agrobacterium species were found to be avirulent, even though they lived together with A. vitis in the same crown gall tumor. As has been reported for human cancer, the crown gall tumor also hosted opportunistic bacteria that are adapted to the tumor microenvironment. Characterization of the microbiota in various diseases using amplicon sequencing may help in early diagnosis, to serve as a preventative measure of disease in the future.

Regulation of oncogene expression in T-DNA-transformed host plant cells.

Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance cytokinin and auxin levels for further cell proliferation.

Plant responses to Agrobacterium tumefaciens and crown gall development.

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.

Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls.

Agrobacterium tumefaciens-derived crown galls of Arabidopsis (Arabidopsis thaliana) contain elevated levels of unsaturated fatty acids and strongly express two fatty acid desaturase genes, ω3 FATTY ACID DESATURASE3 (FAD3) and STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 (SAD6). The fad3-2 mutant with impaired α-linolenic acid synthesis developed significantly smaller crown galls under normal, but not under high, relative humidity. This strongly suggests that FAD3 plays a role in increasing drought stress tolerance of crown galls. SAD6 is a member of the SAD family of as yet unknown function. Expression of the SAD6 gene is limited to hypoxia, a physiological condition found in crown galls. As no sad6 mutant exists and to link the function of SAD6 with fatty acid desaturation in crown galls, the lipid pattern was analyzed of plants with constitutive SAD6 overexpression (SAD6-OE). SAD6-OE plants contained lower stearic acid and higher oleic acid levels, which upon reduction of SAD6 overexpression by RNA interference (SAD6-OE-RNAi) regained wild-type-like levels. The development of crown galls was not affected either in SAD6-OE or SAD6-OE-RNAi or by RNA interference in crown galls. Since biochemical analysis of SAD6 in yeast (Saccharomyces cerevisiae) and Escherichia coli failed, SAD6 was ectopically expressed in the background of the well-known suppressor of salicylic acid-insensitive2 (ssi2-2) mutant to confirm the desaturase function of SAD6. All known ssi2-2 phenotypes were rescued, including the high stearic acid level. Thus, our findings suggest that SAD6 functions as a Δ9-desaturase, and together with FAD3 it increases the levels of unsaturated fatty acids in crown galls under hypoxia and drought stress conditions.

DNA methylation mediated control of gene expression is critical for development of crown gall tumors.

Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA-encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors.

A role for PSK signaling in wounding and microbial interactions in Arabidopsis.

PSK-alpha is a disulfated peptide that acts as a growth factor in plants. PSK-alpha is derived from preproproteins which are encoded by five PSK precursor genes in Arabidopsis thaliana (L.) Heynh and is perceived by leucine-rich repeat receptor kinases. Arabidopsis has two PSK receptor genes, PSKR1 and PSKR2. Although ligand and receptor are well characterized, the biological functions of PSK signaling are not well understood. Using reporter lines and receptor knockout mutants of Arabidopsis, a role for PSK signaling in biotic interactions and in wounding was analyzed. Treatment of Arabidopsis leaves with the fungal elicitor E-Fol, or the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum resulted in induction of PSK2 and PSKR1 as shown by promoter:GUS analysis. Wounding of hypocotyls or leaves induced PSK3:GUS, PSK5:GUS and PSKR1:GUS expression indicating that PSK precursor genes are differentially regulated in response to specific stresses. The receptor knockout lines pskr1-3 and pskr2-1 showed significantly reduced photosynthesis in response to the fungal elicitor E-Fol which indicates that fungal defence is impaired. pskr1-3 plants further showed reduced growth of crown galls after infection with Agrobacterium tumefaciens. A role for PSK signaling in Agrobacterium tumefaciens tumor growth was supported by the finding that PSK precursor genes and PSKR1 are expressed in crown galls. Overall, the results indicate that PSK signaling may play a previously undescribed role in pathogen or herbivore interactions and is crucial for Agrobacterium-induced cell proliferation in crown gall formation.

Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsis thaliana.

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 Arabidopsis-Agrobacterium 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.

Identification of Arabidopsis thaliana phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments.

SUMMARY: Phloem-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 long-distance RNA signalling mutants.

Is gene activity in plant cells affected by UMTS-irradiation? A whole genome approach.

Mobile phone technology makes use of radio frequency (RF) electromagnetic fields transmitted through a dense network of base stations in Europe. Possible harmful effects of RF fields on humans and animals are discussed, but their effect on plants has received little attention. In search for physiological processes of plant cells sensitive to RF fields, cell suspension cultures of Arabidopsis thaliana were exposed for 24 h to a RF field protocol representing typical microwave exposition in an urban environment. mRNA of exposed cultures and controls was used to hybridize Affymetrix-ATH1 whole genome microarrays. Differential expression analysis revealed significant changes in transcription of 10 genes, but they did not exceed a fold change of 2.5. Besides that 3 of them are dark-inducible, their functions do not point to any known responses of plants to environmental stimuli. The changes in transcription of these genes were compared with published microarray datasets and revealed a weak similarity of the microwave to light treatment experiments. Considering the large changes described in published experiments, it is questionable if the small alterations caused by a 24 h continuous microwave exposure would have any impact on the growth and reproduction of whole plants.

A central role of abscisic acid in drought stress protection of Agrobacterium-induced tumors on Arabidopsis.

Crown gall tumors induced by Agrobacterium tumefaciens represent a sink that has to be provided with nutrients and water by the host plant. The lack of an intact epidermis or cuticle results in uncontrolled loss of water. However, neither the tumor nor the host plant displays wilting. This phenomenon points to drought adaptation in both tumors and the crown gall host plant. To understand the underlying molecular mechanisms of protection against desiccation the gene expression pattern of Arabidopsis (Arabidopsis thaliana) tumors was integrated with the profile of stress metabolites: Arabidopsis tumors accumulated high amounts of abscisic acid (ABA), the ethylene precursor aminocyclopropyl carboxylic acid, osmoprotectants, and form a suberized periderm-like protective layer. Suberization of the outer tumor cell layers most likely is mediated by ABA since external application of ABA induced suberization of Arabidopsis roots. However, the expression level of the classical marker genes, known to respond to drought stress and/or ABA, was lower in tumors. Instead another set of drought and/or ABA-inducible genes was more highly transcribed. Elevated transcription of several ABA-dependent aquaporin genes might indicate that ABA controls the water balance of the tumor. The retarded tumor growth on abi and aba mutant plants underlined the importance of a tumor-specific ABA signaling pathway. Taken together, we propose that ABA is an important signal for protection of tumors against desiccation and thus supports tumor development.

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.

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.

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.

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.

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.