Réjane Pratelli

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 Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition in Arabidopsis

A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K+ channel subunit that assembles with different inward-rectifying K+ channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K+ channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K+ channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inward-rectifying K+ current in Arabidopsis root epidermal protoplasts as well as K+ acquisition and growth in seedlings when channel-mediated K+ uptake was limiting. That SYP121 should be important for gating of a K+ channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE–ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K+ uptake coordinately with membrane expansion for cell growth.

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.

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.

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.

A Grapevine Gene Encoding a Guard Cell K Channel Displays Developmental Regulation in the Grapevine Berry

SIRK is a K+ channel identified in grapevine (Vitis vinifera), belonging to the so-called Shaker family. The highest sequence similarities it shares with the members of this family are found with channels of the KAT type, although SIRK displays a small ankyrin domain. This atypical feature provides a key to understand the evolution of the plant Shaker family. Expression inXenopus laevis oocytes indicated that SIRK is an inwardly rectifying channel displaying functional properties very similar to those of KAT2. The activity of SIRK promoter region fused to the GUS reporter gene was analyzed in both grapevine and Arabidopsis. Like other KAT-like channels,SIRK is expressed in guard cells. In Arabidopsis, the construct is also expressed in xylem parenchyma. Semiquantitative reverse transcriptase-polymerase chain reaction experiments indicated that SIRK transcript was present at low levels in the berry, during the first stages of berry growth. After veraison, the period of berry development that corresponds to the inception of ripening and that is associated with large biochemical and structural modifications, such as evolution of stomata in nonfunctional lenticels and degeneration of xylem vasculature, the transcript was no longer detected. The whole set of data suggests that in the berriesSIRK is expressed in guard cells and, possibly, in xylem tissues. The encoded channel polypeptide could therefore play a role in the regulation of transpiration and water fluxes in grapevine fruits.

Stimulation of Nonselective Amino Acid Export by Glutamine Dumper Proteins

Phloem and xylem transport of amino acids involves two steps: export from one cell type to the apoplasm, and subsequent import into adjacent cells. High-affinity import is mediated by proton/amino acid cotransporters, while the mechanism of export remains unclear. Enhanced expression of the plant-specific type I membrane protein Glutamine Dumper1 (GDU1) has previously been shown to induce the secretion of glutamine from hydathodes and increased amino acid content in leaf apoplasm and xylem sap. In this work, tolerance to low concentrations of amino acids and transport analyses using radiolabeled amino acids demonstrate that net amino acid uptake is reduced in the glutamine-secreting GDU1 overexpressor gdu1-1D. The net uptake rate of phenylalanine decreased over time, and amino acid net efflux was increased in gdu1-1D compared with the wild type, indicating increased amino acid export from cells. Independence of the export from proton gradients and ATP suggests that overexpression of GDU1 affects a passive export system. Each of the seven Arabidopsis (Arabidopsis thaliana) GDU genes led to similar phenotypes, including increased efflux of a wide spectrum of amino acids. Differences in expression profiles and functional properties suggested that the GDU genes fulfill different roles in roots, vasculature, and reproductive organs. Taken together, the GDUs appear to stimulate amino acid export by activating nonselective amino acid facilitators.

The Ubiquitin E3 Ligase LOSS OF GDU2 Is Required for Glutamine Dumper1-Induced Amino Acid Secretion in Arabidopsis

Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1’s role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

Altered amino Acid metabolism in glutamine dumper1 plants.

Amino acid metabolism lies at the crossroad between nitrogen assimilation, carbon fixation and secondary metabolism. Because of this central position in plant metabolism, amino acid metabolism is tightly regulated by numerous factors to match both demand from the organs and availability of reduced carbon and inorganic nitrogen. While the amino acid biosynthesis enzymes have been shown to be regulated at the transcriptional and protein levels, the genes involved in amino acid sensing, signal transduction and regulation have not yet been identified. The overexpression of Glutamine Dumper1 leads to a large increase in the amino acid content of the plant and, as we show here, to insensitivity to externally applied amino acids. This phenotype is reminiscent of that of the pig1-1 mutant proposed to display a deregulated metabolism. These data suggest that GDU1 is involved in the regulation of amino acid metabolism and transport. As published previously, the analysis of deletion mutants proves that GDU1s VIMAG domain is important for the function of the protein. The present data show furthermore that other regions participate to this function.