The phloem network is as essential for plants as the vascular system is for humans. This network, assembled by nucleus- and vacuole-free interconnected living cells, represents a long distance transport pathway for nutrients and information. According to the Münch hypothesis, osmolytes such as sucrose generate the hydrostatic pressure that drives nutrient and water flow between the source and the sink phloem (Münch, E. (1930) Die Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, Germany). Although proton-coupled sucrose carriers have been localized to the sieve tube and the companion cell plasma membrane of both source and sink tissues, knowledge of the molecular representatives and the mechanism of the sucrose phloem efflux is still scant. We expressed ZmSUT1, a maize sucrose/proton symporter, in Xenopus oocytes and studied the transport characteristics of the carrier by electrophysiological methods. Using the patch clamp techniques in the giant inside-out patch mode, we altered the chemical and electrochemical gradient across the sucrose carrier and analyzed the currents generated by the proton flux. Thereby we could show that ZmSUT1 is capable of mediating both the sucrose uptake into the phloem in mature leaves (source) as well as the desorption of sugar from the phloem vessels into heterotrophic tissues (sink). As predicted from a perfect molecular machine, the ZmSUT1-mediated sucrose-coupled proton current was reversible and depended on the direction of the sucrose and pH gradient as well as the membrane potential across the transporter.

The Arabidopsis tandem-pore K ⁺ (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker -like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K ⁺ channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K ⁺ currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K ⁺ transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K ⁺ channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.

Summary: Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non‐essential metals such as cadmium (Cd²⁺), which is highly toxic. However, transporters of the natural resistance‐associated macrophage protein (NRAMP) family, which are involved in metal uptake and homeostasis, generally display poor selectivity towards divalent metal cations. In the present study we used a unique combination of yeast‐based selection, electrophysiology on Xenopus oocytes and plant phenotyping to identify and characterize mutations that allow plant and mammalian NRAMP transporters to discriminate between their metal substrates. We took advantage of the increased Cd²⁺ sensitivity of yeast expressing AtNRAMP4 to select mutations that decrease Cd²⁺ sensitivity while maintaining the ability of AtNRAMP4 to transport Fe²⁺ in a population of randomly mutagenized AtNRAMP4 cDNAs. The selection identified mutations in three residues. Among the selected mutations, several affect Zn²⁺ transport, whereas only one, E401K, impairs Mn²⁺ transport by AtNRAMP4. Introduction of the mutation F413I, located in a highly conserved domain, into the mammalian DMT1 transporter indicated that the importance of this residue in metal selectivity is conserved among NRAMP transporters from plant and animal kingdoms. Analyses of overexpressing plants showed that AtNRAMP4 affects the accumulation of metals in roots. Interestingly, the mutations selectively modify Cd²⁺ and Zn²⁺ accumulation without affecting Fe transport mediated by NRAMP4 in planta. This knowledge may be applicable for limiting Cd²⁺ transport by other NRAMP transporters from animals or plants.

Sponges (phylum Porifera) are the phylogenetically oldest metazoan animals, their evolution dating back to 600 million years ago. Here we demonstrate that sponges express ADP-ribosyl cyclase activity, which converts NAD ⁺ into cyclic ADP-ribose, a potent and universal intracellular Ca ²⁺ mobilizer. In Axinella polypoides (Demospongiae, Axinellidae), ADP-ribosyl cyclase was activated by temperature increases by means of an abscisic acid-induced, protein kinase A-dependent mechanism. The thermosensor triggering this signaling cascade was a heat-activated cation channel. Elucidation of the complete thermosensing pathway in sponges highlights a number of features conserved in higher organisms: ( i ) the cation channel thermoreceptor, sensitive to heat, mechanical stress, phosphorylation, and anesthetics, shares all of the functional characteristics of the mammalian heat-activated background K ⁺ channel responsible for central and peripheral thermosensing; ( ii ) involvement of the phytohormone abscisic acid and cyclic ADP-ribose as its second messenger is reminiscent of the drought stress signaling pathway in plants. These results suggest an ancient evolutionary origin of this stress-signaling cascade in a common precursor of modern Metazoa and Metaphyta.

Our research introduces the natural flavonoid naringenin as a novel inhibitor of an emerging class of intracellular channels, Two-Pore Channel 2 (TPC2), as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling, we demonstrated that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1 (negative controls). The ability of naringenin to impair TPC2-dependent biological activities was further explored in an established in vivo model, in which VEGF-containing matrigel plugs implanted in mice failed to be vascularized in the presence of naringenin. Overall, the present data suggest that naringenin inhibition of TPC2 activity and the observed inhibition of angiogenic response to VEGF are linked by impaired intracellular calcium signaling. TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of melanoma, Parkinson’s disease, and Ebola virus infection. The identification of naringenin as an inhibitor of TPC2-mediated signaling provides a novel and potentially relevant tool for the advancement of this field of research.