Guillaume Pilot

Identification and Disruption of a Plant Shaker-like Outward Channel Involved in K+ Release into the Xylem Sap

SKOR, a K+ channel identified in Arabidopsis, displays the typical hydrophobic core of the Shaker channel superfamily, a cyclic nucleotide-binding domain, and an ankyrin domain. Expression in Xenopus oocytes identified SKOR as the first member of the Shaker family in plants to be endowed with outwardly rectifying properties. SKOR expression is localized in root stelar tissues. A knockout mutant shows both lower shoot K+ content and lower xylem sap K+ concentration, indicating that SKOR is involved in K+ release into the xylem sap toward the shoots. SKOR expression is strongly inhibited by the stress phytohormone abscisic acid, supporting the hypothesis that control of K+ translocation toward the shoots is part of the plant response to water stress.

A Shaker-like K + Channel with Weak Rectification Is Expressed in Both Source and Sink Phloem Tissues of Arabidopsis

RNA gel blot and reverse transcription–polymerase chain reaction experiments were used to identify a single K+ channel gene in Arabidopsis as expressed throughout the plant. Use of the β-glucuronidase reporter gene revealed expression of this gene, AKT2/AKT3, in both source and sink phloem tissues. The AKT2/AKT3 gene corresponds to two previously identified cDNAs, AKT2 (reconstructed at its 5′ end) and AKT3, the open reading frame of the latter being shorter at its 5′ end than that of the former. Rapid amplification of cDNA ends with polymerase chain reaction and site-directed mutagenesis was performed to identify the initiation codon for AKT2 translation. All of the data are consistent with the hypothesis that the encoded polypeptide corresponds to the longest open reading frame previously identified (AKT2). Electrophysiological characterization (macroscopic and single-channel currents) of AKT2 in both Xenopus oocytes and COS cells revealed a unique gating mode and sensitivity to pH (weak inward rectification, inhibition, and increased rectification upon internal or external acidification), suggesting that AKT2 has enough functional plasticity to perform different functions in phloem tissue of source and sink organs. The plant stress hormone abscisic acid was shown to increase the amount of AKT2 transcript, suggesting a role for the AKT2 in the plant response to drought.

Amino Acid Homeostasis Modulates Salicylic Acid–Associated Redox Status and Defense Responses in Arabidopsis

This study investigates the relationship between nitrogen metabolism and disease responses in Arabidopsis and shows that knockout of Arabidopsis LHT1, a single amino acid transporter, imparts broad-spectrum resistance to pathogens. The tight association between nitrogen status and pathogenesis has been broadly documented in plant–pathogen interactions. However, the interface between primary metabolism and disease responses remains largely unclear. Here, we show that knockout of a single amino acid transporter, LYSINE HISTIDINE TRANSPORTER1 (LHT1), is sufficient for Arabidopsis thaliana plants to confer a broad spectrum of disease resistance in a salicylic acid–dependent manner. We found that redox fine-tuning in photosynthetic cells was causally linked to the lht1 mutant-associated phenotypes. Furthermore, the enhanced resistance in lht1 could be attributed to a specific deficiency of its main physiological substrate, Gln, and not to a general nitrogen deficiency. Thus, by enabling nitrogen metabolism to moderate the cellular redox status, a plant primary metabolite, Gln, plays a crucial role in plant disease resistance.

Overexpression of GLUTAMINE DUMPER1 Leads to Hypersecretion of Glutamine from Hydathodes of Arabidopsis Leaves

Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants.

A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein–protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r2 = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Amino Acid Export in Plants: A Missing Link in Nitrogen Cycling

The export of nutrients from source organs to parts of the body where they are required (e.g. sink organs) is a fundamental biological process. Export of amino acids, one of the most abundant nitrogen species in plant long-distance transport tissues (i.e. xylem and phloem), is an essential process for the proper distribution of nitrogen in the plant. Physiological studies have detected the presence of multiple amino acid export systems in plant cell membranes. Yet, surprisingly little is known about the molecular identity of amino acid exporters, partially due to the technical difficulties hampering the identification of exporter proteins. In this short review, we will summarize our current knowledge about amino acid export systems in plants. Several studies have described plant amino acid transporters capable of bi-directional, facilitative transport, reminiscent of activities identified by earlier physiological studies. Moreover, recent expansion in the number of available amino acid transporter sequences have revealed evolutionary relationships between amino acid exporters from other organisms with a number of uncharacterized plant proteins, some of which might also function as amino acid exporters. In addition, genes that may regulate export of amino acids have been discovered. Studies of these putative transporter and regulator proteins may help in understanding the elusive molecular mechanisms of amino acid export in plants.

Border control--a membrane-linked interactome of Arabidopsis.

Degrees of Separation Proteins embedded in membranes represent an interesting point of communication between the cell and its environment, but their localization to membranes can make them difficult to study. Jones et al. (p. 711) found an approach to catalog thousands of interactions involving membrane proteins and membrane-associated signaling machinery—including many previously uncharacterized proteins. With a focus on the model plant Arabidopsis, several of the identified interactions fill gaps in important signal transduction chains, while others point to functions for enigmatic unknown proteins. Amembrane and signaling protein interaction network for gene discovery and hypothesis generation is identified in Arabidopsis. Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 106 pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split–green fluorescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

Five-Group Distribution of the Shaker-like K+ Channel Family in Higher Plants

In higher plants, potassium channels of the Shaker family have been shown to play crucial roles in the uptake of K+ from the soil solution and subsequent transport of this ion at the cell, tissue, and organ levels. In the model plant Arabidopsis thaliana, this family is composed of nine members, which are the best characterized among plant channels at the protein, gene, and functional property levels. Plant Shaker channels share a common structure: a hydrophobic core composed of six transmembrane segments, a long cytoplasmic C-terminal region harboring a putative cyclic nucleotide binding domain, and a KHA domain. Many channels also contain an ankyrin domain between the putative cyclic nucleotide binding domain and the KHA domain. The analysis of 44 Shaker channels from plants revealed a five-group classification. The members of each group share high sequence and structure similarities. This grouping also correlates with the diversification of the functional properties of the proteins, as members of an individual group have roughly the same electrophysiological characteristics. Analysis of the intron positions showed that the gene structures are also quite well conserved within the five groups. A correlation linking the evolution of the sequences and the positioning of the introns was established. Finally, a moss sequence provided additional clues about the hypothetical structure of an ancestor of the present channels and suggested that the diversification of plant Shaker channels happened before the separation of monocots and dicots and after the separation of bryophytes and tracheophytes.

pH control of the plant outwardly-rectifying potassium channel SKOR

SKOR, an Arabidopsis depolarisation‐activated K+‐selective channel, was expressed in Xenopus oocytes, and external and internal pH effects were analysed. Internal pH was manipulated by injections of alkaline or acidic solutions or by acid load from acetate‐containing medium. An internal pH decrease from 7.4 to 7.2 induced a strong (ca. 80%) voltage‐independent decrease of the macroscopic SKOR current, the macroscopic gating parameters and the single channel conductance remained unchanged. An external acidification from 7.4 to 6.4 had similar effects. It is proposed that pH changes regulate the number of channels available for activation. Sensitivity of SKOR activity to pH in the physiological range suggests that internal and external pH play a role in the regulation of K+ secretion into the xylem sap.

Regulation of amino acid metabolic enzymes and transporters in plants

Amino acids play several critical roles in plants, from providing the building blocks of proteins to being essential metabolites interacting with many branches of metabolism. They are also important molecules that shuttle organic nitrogen through the plant. Because of this central role in nitrogen metabolism, amino acid biosynthesis, degradation, and transport are tightly regulated to meet demand in response to nitrogen and carbon availability. While much is known about the feedback regulation of the branched biosynthesis pathways by the amino acids themselves, the regulation mechanisms at the transcriptional, post-transcriptional, and protein levels remain to be identified. This review focuses mainly on the current state of our understanding of the regulation of the enzymes and transporters at the transcript level. Current results describing the effect of transcription factors and protein modifications lead to a fragmental picture that hints at multiple, complex levels of regulation that control and coordinate transport and enzyme activities. It also appears that amino acid metabolism, amino acid transport, and stress signal integration can influence each other in a so-far unpredictable fashion.