Eva Johannes

Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum.

A genetic approach was used to increase phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKIα in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P2 100-fold. In vivo studies revealed that the rate of 32Pi incorporation into whole-cell PtdIns(4,5)P2 increased >12-fold, and the ratio of [3H]PtdInsP2 to [3H]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [3H]inositol trisphosphate and [3H]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] binding assay showed that total cellular Ins(1,4,5)P3/g fresh weight was >40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKIα increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.

The N-terminal Membrane Occupation and Recognition Nexus Domain of Arabidopsis Phosphatidylinositol Phosphate Kinase 1 Regulates Enzyme Activity

The type I B family of phosphatidylinositol phosphate kinases (PIPKs) contain a characteristic region of Membrane Occupation and Recognition Nexus (MORN) motifs at the N terminus. These MORN motifs are not found in PIPKs from other eukaryotes. To understand the impact of the additional N-terminal domain on protein function and subcellular distribution, we expressed truncated and full-length versions of AtPIPK1, one member of this family of PIPKs, in Escherichia coli and in tobacco cells grown in suspension culture. Deletion of the N-terminal MORN domain (amino acids 1–251) of AtPIPK1 increased the specific activity of the remaining C-terminal peptide (ΔMORN) >4-fold and eliminated activation by phosphatidic acid (PtdOH). PtdOH activation could also be eliminated by mutating Pro396 to Ala (P396A) in the predicted linker region between the MORN and the kinase homology domains. AtPIPK1 is product-activated and the MORN domain binds PtdIns(4,5)P2. Adding back the MORN peptide to ΔMORN or to the PtdOH-activated full-length protein increased activity ∼2-fold. Furthermore, expressing the MORN domain in vivo increased the plasma membrane PtdInsP kinase activity. When cells were exposed to hyperosmotic stress, the MORN peptide redistributed from the plasma membrane to a lower phase or endomembrane fraction. In addition, endogenous PtdInsP kinase activity increased in the endomembrane fraction of hyperosmotically stressed cells. We conclude that the MORN peptide can regulate both the function and distribution of the enzyme in a manner that is sensitive to the lipid environment.

An auxin-inducible gene from loblolly pine (Pinus taeda L.) is differentially expressed in mature and juvenile-phase shoots and encodes a putative transmembrane protein.

We have isolated a gene from loblolly pine, 5NG4, that is highly and specifically induced by auxin in juvenile loblolly pine shoots prior to adventitious root formation, but substantially down-regulated in physiologically mature shoots that are adventitious rooting incompetent. 5NG4 was highly auxin-induced in roots, stems and hypocotyls, organs that can form either lateral or adventitious roots following an auxin treatment, but was not induced to the same level in needles and cotyledons, organs that do not form roots. The deduced amino acid sequence shows homology to the MtN21 nodulin gene from Medicago truncatula. The expression pattern of 5NG4 and its homology to a protein from Medicago involved in a root-related process suggest a possible role for this gene in adventitious root formation. Homology searches also identified similar proteins in Arabidopsis thaliana and Oryza sativa. High conservation across these evolutionarily distant species suggests essential functions in plant growth and development. A 38-member family of genes homologous to 5NG4 was identified in the A. thaliana genome. The physiological significance of this redundancy is most likely associated with functional divergence and/or expression specificity of the different family members. The exact biochemical function of the gene is still unknown, but sequence and structure predictions and 5NG4::GFP fusion protein localizations indicate it is a transmembrane protein with a possible transport function.

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K+ and Ca2+ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca2+ channels. Respective K+ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl− efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 μM), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 μM), which inhibits K+ fluxes and part of the initial Ca2+ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.

Calcium-dependent membrane depolarisation activated by phytochrome in the moss Physcomitrella patens

In caulonemal filaments of Physcomitrella patens which had been preincubated in the dark for 24 h, irradiation with red light (640 nm, fluence rate 85 μmol · m−2 · s−1) evoked (i) the development of side branch initials and (ii) a rapid, but transient, depolarisation of the plasma membrane by 90 ± 13 mV from a resting potential of -178 ± 13 mV. This was followed by a transient hyperpolarisation to a value 21± 8 mV more negative than the original membrane potential. The refractory period for the transient depolarisation was between 12 and 15 min. The fluence rate of red light required to evoke maximal depolarisation was about 80 μmol · m−2 · s−1 for a 1-min pulse. At this fluence rate, a depolarising response could be recorded for pulse lengths as small as 7 s. The transient depolarisation was insensitive to 3-(3′,4′dichlorophenyl)-1,1-dimethyl urea (DCMU) and was unchanged in plants bleached by growth on norflurazon (SAN 9789). Furthermore, the electrical response could be blocked by simultaneous application of far-red light. These results suggest the involvement of the photoreceptor phytochrome in the response. Removing Ca2+ from the external medium or replacing Ca2+ with Mg2+ blocked the depolarisation. The depolarisation could also be blocked by the K+ channel-blocker tetraethylammonium (10 mM) and the Cl− channel-blocker niflumic acid (1 μM). Conversely, although calcium channel-antagonists such as nifedipine and lanthanides, applied at a concentration of 100 μM, also altered the response, they did not block it. A possible ionic mechanism for the membrane potential transient is advanced, and the physiological significance discussed in the context of early events in the phytochrome signalling pathway.

The use of weak acids as physiological tools: A study of the effects of fatty acids on intracellular pH and electrical plasmalemma properties of Riccia fluitans rhizoid cells

Abstract The mode of action of fatty acids on electrical membrane properties and on cytoplasmic pH has been investigated on rhizoid cells of the aquatic liverwort, Ricchia fluitans , by means of pH-sensitive microelectrodes and standard electrophysiology. (1) Riccia rhizoids have a cytoplasmic pH of 7.3 ± 0.13 and a vacuolar pH of 4.8 ± 0.23 . In the undisturbed cell, the cytoplasmic buffer capacity was determined to be 60–80 mM H + /pH unit, which is reduced by roughly 50% when the cytoplasm is acidified to 6.3 and is exhausted below pH 6. (2) Depending on concentration and on the carbon chain length of the tested acids. the cell first hyperpolarizes (short chains), but then depolarizes (longer chains). No hyperpolarizations are observed when the pump is deactivated with (CN − ). (3) All acids tested acidify the cytoplasm according to the concentrations of their protonated form, HA. In the presence of acetic, propionic and butyric acids, the cytoplasmic pH partly recovers, while hexanoic and heptanoic acids, rapidly deplete the electromechanical pH gradient across the plasmalemma. (4) All fatty acids show a concentration-dependent and reversible decrease of the membrane resistance, heptanoic acid having an approx. 10-times stronger effect that acetic acid. It is suggested that weak acids induce a net current across the plasmalemma of Riccia rhizoids , one portion of which is active (pump current) while the other, of opposite sign, is passive and unspecific. Since the protonmotive force remains essentially constant in the presence of lower concentrations of the shorter-chained acids, or even increases with acetic or propionic acids, we assume the lipid solubilities of the unprotonated acid (A − ) to be negligible. For hexanoic and heptanoic acids, the lipid solubilities of A − seem high enough for them to be effective as uncouplers. It is further concluded that the hyperpolarizations are the result of a stimulated H + -extrusion pump, whereas the depolarizations derive from (a) decreased membrane resistance, (b) strongly shifted internal pH, and (c) uncoupled mitochondria.

Implications for cytoplasmic pH, protonmotice force, and amino-acid transport across the plasmalemma of Riccia fluitans

By means of pH-sensitive microelectrodes, cytoplasmic pH has been monitored continuously during amino-acid transport across the plasmalemma of Riccia fluitans rhizoid cells under various experimental conditions. (i) Contrary to the general assumption that import of amino acids (or hexoses) together with protons should lead to cytoplasmic acidification, an alkalinization of 0.1–0.3 pHc units was found for all amino acids tested. Similar alkalinizations were recorded in the presence of hexoses and methylamine. No alkalinization occurred when the substrates were added in the depolarized state or in the presence of cyanide, where the electrogenic H+-pump is inhibited. (ii) After acidification of the cytoplasm by means of various concentrations of acetic acid, amino-acid transport is massively altered, although the protonmotive force remained essentially constant. It is suggested that H+-cotransport is energetically interconnected with the proton-export pump which is stimulated by the amino-acid-induced depolarization, thus causing proton depletion of the cytoplasm. It is concluded that, in order to investigate H+-dependent cotransport processes, the cytoplasmic pH must be measured and be under continuous experimental control; secondly, neither ΔpH nor the protonmotive force across a membrane are reliable quantities for analysing a proton-dependent process.

Red light-induced membrane potential transients in the moss Physcomitrella patens: ion channel interaction in phytochrome signalling

In caulonemal filaments of the moss Physcomitrella patens red light (fluence rate 85 µmol m(-2) s(-1)) triggers within 2-15 s a transient membrane depolarization, and 3 d later the development of side branch initial cells. Both the rapid electrical events at the plasma membrane and the morphological response are Ca(2+)-dependent, phytochrome-mediated and effectively inhibited by the cation channel blocker TEA (10 mM) and the anion channel blocker niflumic acid (1 µM). This suggests that both responses are connected. Current voltage analyses of the red light-induced current combined with ion flux measurements revealed that Ca(2+), K(+) and anion-permeable channels are open at the peak of the depolarization. While Ca(2+) influx and anion efflux coincide with the depolarizing phase, K(+) entry occurs during the first 30 s only and is followed by a dramatic transient K(+) efflux leading to repolarization. Patch clamp studies have revealed the presence of a class of depolarization-activated outward rectifying cation channel with a likely role in the rapid repolarization. Furthermore, the channel has a small permeability for Ca(2+) and could provide an additional mode of Ca(2+) entry. The sequence of events which underlie the red light-induced membrane potential transient is discussed with respect to ion channel interaction during phytochrome signalling.

Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel

The patch clamp technique has been used to investigate ion permeation and Ca2+-dependent gating of a voltage-sensitive Ca2+ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca2+ ≈ Sr2+ ≈ Ba2+ > Mg2+ ≫ K+ which is inversely related to the size of the unitary conductances K+ ≫ Mg2+ ≈ Ba2+ > Sr2+ ≈ Ca2+, suggesting that ion movement is not independent. In the presence of Ca2+, the unitary K+ current is reduced in a concentration- and voltage-dependent manner by Ca2+ binding at a high affinity site (K0.5 = 0.29 mm at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca2+ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca2+ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca2+ at −41 mV predicts that two Ca2+ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.

Transport of basic amino acids in Riccia fluitans: evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess α-aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [14C]-basic amino acids. iv) Uptake of [14C]-glutamic acid took place only at acidic pHs. v) [14C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.