Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca 2+ binding sites have been characterized in cytosolic proteins. However, little is known about Ca 2+ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca 2+ . However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca 2+ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca 2+ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.

Although the concept of botanical carnivory has been known since Darwin's time, the molecular mechanisms that allow animal feeding remain unknown, primarily due to a complete lack of genomic information. Here, we show that the transcriptomic landscape of the Dionaea trap is dramatically shifted toward signal transduction and nutrient transport upon insect feeding, with touch hormone signaling and protein secretion prevailing. At the same time, a massive induction of general defense responses is accompanied by the repression of cell death–related genes/processes. We hypothesize that the carnivory syndrome of Dionaea evolved by exaptation of ancient defense pathways, replacing cell death with nutrient acquisition.

SignificanceThe Venus flytrap Dionaea muscipula has been in the focus of scientists since Darwin’s time. Carnivorous plants, with their specialized lifestyle, including insect capture, as well as digestion and absorption of prey, developed unique tools to gain scarce nutrients. In this study, we identified the molecular nature of the uptake machinery for prey-derived potassium and the posttranslational regulation. For the first time, to our knowledge, we functionally characterize DmHAK5 here—a KUP/HAK/KT family member as activated by a CBL-CIPK kinase complex. Detailed electrophysiological characterization identified DmHAK5 as a proton-driven, high-affinity potassium transporter with a weak selectivity. Working hand-in-hand with the low-affinity, high-capacity K+-channel DmKT1 activated by the same kinase, the transporters allow the Venus flytrap to take up prey-derived potassium.

In Escherichia coli K-12, the major glucose transporter with a central role in carbon catabolite repression and in inducer exclusion is the phosphoenolpyruvate-dependent glucose:phosphotransferase system (PTS). Its membrane-bound subunit, IICB Glc , is encoded by the gene ptsG; its soluble domain, IIA Glc, is encoded by crr, which is a member of the pts operon. The system is inducible by D-glucose and, to a lesser degree, by L-sorbose. The regulation of ptsG transcription was analyzed by testing the induction of IICB Glc transporter activity and of a single-copy ⌽(ptsGop-lacZ) fusion. Among mutations found to affect directly ptsG expression were those altering the activity of adenylate cyclase (cyaA), the repressor DgsA (dgsA; also called Mlc), the general PTS proteins enzyme I (ptsI) and histidine carrier protein HPr (ptsH), and the IIA Glc and IIB Glc domains, as well as several authentic and newly isolated UmgC mutations. The latter, originally thought to map in the repressor gene umgC outside the ptsG locus, were found to represent ptsG alleles. These affected invariably the substrate specificity of the IICB Glc domain, thus allowing efficient transport and phosphorylation of substrates normally transported very poorly or not at all by this PTS. Simultaneously, all of these substrates became inducers for ptsG. From the analysis of the mutants, from cis-trans dominance tests, and from the identification of the amino acid residues mutated in the UmgC mutants, a new regulatory mechanism involved in ptsG induction is postulated. According to this model, the phosphorylation state of IIB Glc modulates IIC Glc which, directly or indirectly, controls the repressor DgsA and hence ptsG expression. By the same mechanism, glucose uptake and phosphorylation also control the expression of the pts operon and probably of all operons controlled by the repressor DgsA.In Escherichia coli K-12, D-glucose (Glc) is taken up and concomitantly phosphorylated either by the glucose-specific enzyme II (EII) transporter (II Glc ) or the mannose-specific EII transporter (II Man ) (genes manXYZ) of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) (for reviews, see references 10 and 21). As for most other PTS carbohydrates, the phosphoryl groups are sequentially transferred from PEP through two common intermediates, enzyme I (EI; gene: ptsI) and the phosphohistidine carrier protein (HPr; gene: ptsH), to sugar-specific EII (IICB Glc ; see below) and to glucose (for a review see reference 41). II Glc consists of two subunits, IIA Glc (crr [catabolite repression resistance]) and membrane-bound IICB Glc (ptsG) (8). The crr gene is part of the ptsHI crr operon (46) separated from the ptsG gene, which maps at 25.0 min (4). IIA Glc is a small hydrophilic protein which has, in addition to its transport function, a central regulatory role in carbon catabolite repression and inducer exclusion (for a review, see reference 22). The IICB Glc subunit is composed of an amino-terminal, hydrophobic IIC Glc domain, which large...

Understanding seasonality and longevity is a major challenge in tree biology. In woody species, growth phases and dormancy follow one another consecutively. In the oldest living individuals, the annual cycle may run for more than 1,000 years. So far, however, not much is known about the processes triggering reactivation from dormancy. In this study, we focused on wood rays, which are known to play an important role in tree development. The transition phase from dormancy to flowering in early spring was compared with the phase of active growth in summer. Rays from wood samples of poplar (Populus 3 canescens) were enriched by laser microdissection, and transcripts were monitored by poplar whole-genome microarrays. The resulting seasonally varying complex expression and metabolite patterns were subjected to pathway analyses. In February, the metabolic pathways related to flower induction were high, indicating that reactivation from dormancy was already taking place at this time of the year. In July, the pathways related to active growth, like lignin biosynthesis, nitrogen assimilation, and defense, were enriched. Based on "marker" genes identified in our pathway analyses, we were able to validate periodical changes in wood samples by quantitative polymerase chain reaction. These studies, and the resulting ray database, provide new insights into the steps underlying the seasonality of poplar trees.

Sulphate uptake and its distribution within plants depend on the activity of different sulphate transporters (SULTR). In long-living deciduous plants such as trees, seasonal changes of spatial patterns add another layer of complexity to the question of how the interplay of different transporters adjusts S distribution within the plant to environmental changes. Poplar is an excellent model to address this question because its S metabolism is already well characterized. In the present study, the importance of SULTRs for seasonal sulphate storage and mobilization was examined in the wood of poplar (Populus tremula ¥ P. alba) by analysing their gene expression in relation to sulphate contents in wood and xylem sap. According to these results, possible functions of the respective SULTRs for seasonal sulphate storage and mobilization in the wood are suggested. Together, the present results complement the previously published model for seasonal sulphate circulation between leaves and bark and provide information for future mechanistic modelling of whole tree sulphate fluxes.