Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

Regulation of pollen tube growth by Rac-like GTPases

Plant Rac-like GTPases have been classified phylogenetically into two major groups-class I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen- and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.     

Phlorizin inhibits hexose transport across the plasmalemma of Riccia fluitans

The effect of phlorizin has been tested on hexose transport and hexose-induced changes of electrical potential (_m) and conductance (g _m) across the plasmalemma of rhizoid and thallus cells of the aquatic liverwort Riccia fluitans. The decrease of _m (depolarization) and g _m induced by 1 mM 3-oxymethyl-D-glucose (3-OMG) is substantially inhibited by simultaneous addition of 2 mM phlorizin, whereas no significant response was observed when phlorizin was added alone or several minutes after the sugar. Current-voltage data show that the 3-OMG-generated electrical inward current of 0.016 A m^-2 drops to 0.010 A m^-2 when phlorizin is present. Uptake as well as efflux of [¹⁴C]-3-OMG is strongly and reversibly inhibited by phlorizin between 0.2 and 5 mM. The results are consistent with our hypothesis that the hexose carrier has one binding site with competitive inhibition of glucose uptake by phlorizin (k _i=0.08 mM). The electrical data indicate that phlorizin affects an _m step of the carrier transport cycle.Abbreviation 3-OMG 3-oxymethyl-D-glucose     

Membrane transport of hydrogen peroxide

Hydrogen peroxide (H₂O₂) belongs to the reactive oxygen species (ROS), known as oxidants that can react with various cellular targets thereby causing cell damage or even cell death. On the other hand, recent work has demonstrated that H₂O₂ also functions as a signalling molecule controlling different essential processes in plants and mammals. Because of these opposing functions the cellular level of H₂O₂ is likely to be subjected to tight regulation via processes involved in production, distribution and removal. Substantial progress has been made exploring the formation and scavenging of H₂O₂, whereas little is known about how this signal molecule is transported from its site of origin to the place of action or detoxification. From work in yeast and bacteria it is clear that the diffusion of H₂O₂ across membranes is limited. We have now obtained direct evidence that selected aquaporin homologues from plants and mammals have the capacity to channel H₂O₂ across membranes. The main focus of this review is (i) to summarize the most recent evidence for a signalling role of H₂O₂ in various pathways in plants and mammals and (ii) to discuss the relevance of specific transport of H₂O₂.     

Membrane transport of hydrogen peroxide

Hydrogen peroxide (H2O2) belongs to the reactive oxygen species (ROS), known as oxidants that can react with various cellular targets thereby causing cell damage or even cell death. On the other hand, recent work has demonstrated that H2O2 also functions as a signalling molecule controlling different essential processes in plants and mammals. Because of these opposing functions the cellular level of H2O2 is likely to be subjected to tight regulation via processes involved in production, distribution and removal. Substantial progress has been made exploring the formation and scavenging of H2O2, whereas little is known about how this signal molecule is transported from its site of origin to the place of action or detoxification. From work in yeast and bacteria it is clear that the diffusion of H2O2 across membranes is limited. We have now obtained direct evidence that selected aquaporin homologues from plants and mammals have the capacity to channel H2O2 across membranes. The main focus of this review is (i) to summarize the most recent evidence for a signalling role of H2O2 in various pathways in plants and mammals and (ii) to discuss the relevance of specific transport of H2O2.     

Regulation of cathepsin K activity by hydrogen peroxide

Although cysteine cathepsins, including cathepsin K, are sensitive to oxidation, proteolytically active forms are found at inflammatory sites. Regulation of cathepsin K activity was analyzed in the presence of H2O2 to gain an insight into these puzzling observations. H2O2 impaired processing of procathepsin K and inactivated its mature form in a time- and dose-dependent mode. However, as a result of the formation of a sulfenic acid, as confirmed by trapping in the presence of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol, approximately one-third of its initial activity was restored by dithiothreitol. This incomplete inactivation may partially explain why active cysteine cathepsins are still found during acute lung inflammation.     

蛋白质可逆磷酸化对花粉管生长的调控作用

花粉管极性生长受多种信号与代谢过程的调控,主要包括Rop GTPase信号途径、磷脂酰肌醇信号通路、Ca²⁺信号途径、肌动蛋白动态变化、囊泡运输、细胞壁重塑等,这些过程都受到蛋白质可逆磷酸化作用的调节。如：(1) Rop调节蛋白(GEF、GDI和GAP)的可逆磷酸化可以改变其活性,从而调节Rop GTPase;同时,蛋白激酶还可能作为Rop下游的效应器分子参与Rop下游信号途径的调节;(2) 蛋白质可逆磷酸化作用既能够激活/失活质膜上的Ca²⁺通道或Ca²⁺泵,又参与调节胞内贮存Ca²⁺的释放,从而调控花粉管尖端Ca²⁺梯度的形成;此外,蛋白激酶还作为Ca²⁺信号的感受器,磷酸化相应的靶蛋白,参与Ca²⁺信号下游途径的调节;(3) 肌动蛋白结合蛋白(ADF和Profilin)的活性也受到蛋白质可逆磷酸化的调节,进而调控肌动蛋白聚合与解聚之间的动态平衡;(4) 蛋白质磷酸化作用调节胞吞/胞吐相关蛋白的活性,并调控质膜的磷脂代谢,从而参与调控囊泡运输过程;(5) 胞质丝氨酸/苏氨酸蛋白激酶和蔗糖合酶的可逆磷酸化可以调节其在花粉管中的功能与分布模式,参与花粉管细胞壁重塑;(6) 转录调节蛋白与真核生物翻译起始因子的可逆磷酸化可以改变其活性,从而调控RNA转录与蛋白质合成。文章主要综述了花粉管生长过程中重要蛋白质的可逆磷酸化作用对上述关键事件的调节。     

Regulation of ion transport proteins by membrane phosphoinositides

Over the past decade, there has been an explosion in the number of membrane transport proteins that have been shown to be sensitive to the abundance of phosphoinositides in the plasma membrane. These proteins include voltage-gated potassium and calcium channels, ion channels that mediate sensory and nociceptive responses, epithelial transport proteins and ionic exchangers. Each of the regulatory lipids is also under multifaceted regulatory control. Phosphoinositide modulation of membrane proteins in neurons often has a dramatic effect on neuronal excitability and synaptic transmitter release. The repertoire of lipid signalling mechanisms that regulate membrane proteins is intriguingly complex and provides a rich array of topics for neuroscience research.     

Characteristics of sulfate transport across plasmalemma and tonoplast of carrot root cells

Compartmental analysis of ³⁵SO₄²⁻ exchange kinetics is used to obtain SO₄²⁻ fluxes and compartment contents in carrot (Daucus carota L.) storage root cells, where 2 to 5% of the SO₄²⁻ taken up is reduced to organic form. The necessary curve fitting is verified by (a) consistency between `content versus time' and `rate versus time' plots of washout data; (b) agreement between loading and washout kinetics; and (c) correct identification of the fastest exchange phase as being from extracellular spaces.

Sulfate is actively transported up an electrochemical potential gradient at both plasmalemma and tonoplast. The plasmalemma influx is from 2 to 10 times higher than the tonoplast influx, is much greater than the SO₄²⁻ reduction rate, and would not limit the rate of either. This is consistent with the finding that the plasmalemma influx is not regulated by internal SO₄²⁻ or cysteine (Cram 1982 Plant Sci Lett, in press).

Both SO₄²⁻ influxes rise with only limited saturation as the external SO₄²⁻ concentration increases up to 50 millimolarity. Both effluxes appear to be passive, with extensive recycling in the plasmalemma influx pump. SO₄²⁻ permeability is about 10⁻¹¹ meter per second at both membranes.

The high, nonlimiting fluxes of SO₄²⁻ at the plasmalemma relative to the tonoplast (found also in Lemna; Thoiron, Thoiron, Demarty, Thellier 1981 Biochim Biophys Acta 644: 24-35) contrasts with SO₄²⁻ fluxes in bacteria and with Cl⁻ fluxes in plant cells. Their implications for work on characteristics and regulation of SO₄²⁻ uptake in roots and tissue cultures are discussed.

     

Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Regulation of morphological and functional properties of astrocytes by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, and menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H₂O₂ at 5 × 10^?7?1 × 10^?8 M concentrations acted as a regulator of morphological and functional properties of astrocytes, inducing their reactivation, which is manifested as cell body hypertrophy and an increase of proliferative activity and menadione-induced production of superoxide anion radicals (O ₂ ^?? ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 × 10^?6 M on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O ₂ ^?? under the effect of menadione. Use of lipopolysaccharide B as a functional stimulator has shown that H₂O₂ modifies signaling pathways leading to an increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-dependent chemiluminescence of astrocytes under the action of menadione to the level of control values.     

Regulation of morphologicaland functional properties of astroglial cells by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H2O2 at 1 x 10(-8) - 5 x 10(-7) M concentrations acts as a regulator of morphological and functional properties of astrocytes by inducing their reactivation that is manifested as a cell body hypertrophy and an increase of proliferative activity and of menadione-dependent production of superoxide (O2- ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 microM on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O2- under menadione action. Using lipopolysaccharide B as a functional stimulator it has been shown that H2O2 modifies signaling pathways leading to the increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-enhanced chemiluminescence of astrocytes under menadione action to the level of control values.     

Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation

The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H(2)O(2) was determined in Caco-2 cell monolayer. Incubation with H(2)O(2) (20 microM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170-250 kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H(2)O(2). However, H(2)O(2) reduced 80-85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H(2)O(2) reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H(2)O(2)-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H(2)O(2) inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.     

Regulation of ion and water transport across the eel intestine: effects of acetylcholine and serotonin

Summary Both acetylcholine (ACh) and serotonin (5-HT) lowered the serosa-negative transepithelial potential difference (PD) and the short-circuit current (I_sc), accompanied by a decrease in NaCl and water absorption across the eel intestine. These inhibitory effects of ACh and 5-HT were blocked by atropine, a muscarinic receptor antagonist, and ICS-205930, a 5-HT₃ receptor antagonist, respectively. Even after blocking the ACh receptor with atropine, 5-HT inhibited the PD and I_sc, and ACh lowered them after blocking the 5-HT receptor with ICS-205930, indicating that ACh and 5-HT act independently. Similar inhibition in the PD and the I_sc was observed after electrical field stimulation (EFS) which is expected to release endogenous regulators. These effects of EFS were reduced by 70% after simultaneous addition of atropine and ICS-205930. Since atropine and ICS-205930 block ACh and 5-HT receptors, respectively, these results suggest that endogenous ACh and 5-HT are released by EFS.Abbreviations ACh acetylcholine - EFS electrical field stimulation - 5-HT serotonin - I _sc short-circuit current - PD transepithelial potential difference - R _t tissue resistance - TTX tetrodotoxin     

Potassium ion channels in the plasmalemma

The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K⁺ transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K⁺ equilibrium potential (E_K) – membrane potential (V_m), or secondary active by coupling through a carrier to the inward driving force of H⁺ or Na⁺. Known K⁺ channels are permeable to monovalent cations, a permeability order being K⁺ > Rb⁺ > NH₄⁺ > Na⁺≥ Li⁺ > Cs⁺. The macroscopic K⁺ currents across a cell or protoplast surface commonly show rectification, i.e. a V^m-dependent conductance which in turn, may be controlled by the cytosolic activity of Ca²⁺, of K⁺, of H⁺, or by the K⁺ driving force. Analysis by the patch clamp technique reveals that plant K⁺ channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K⁺ channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.     

Hydrogen peroxide affects ion channels in lily pollen grain protoplasts

Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model – protoplasts obtained from lily pollen grains at the early germination stage – to reveal the effect of H₂O₂ on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS‐sensitive currents on the pollen grain plasma membrane: the hyperpolarisation‐activated calcium current, which is strongly enhanced by H₂O₂, and the outward potassium current, which is modestly enhanced by H₂O₂. We used low concentrations of H₂O₂ that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining.     

Regulation of pollen tube polarity: Feedback loops rule

Targeted delivery of immotile sperm through growing pollen tubes is a crucial step in achieving sexual reproduction in angiosperms. Unlike diffuse-growing cells, the growth of a pollen tube is restricted to the very apical region where targeted exocytosis and regulated endocytosis occur. The plant-specific Rho GTPases, Rops, are central organizers for pollen tube polarity. Through effector binding, Rops regulate the tip-focused Ca²⁺ gradient and the actin cytoskeleton during pollen tube growth. Therefore, understanding the spatiotemporal regulation of Rop activity would reveal how establishment and maintenance of tube polarity as well as re-orientation of the growth axis are accomplished. Recent findings indicated that two feedback loops may be fundamental in maintaining a fine-tuned and dynamic Rop activity. The concerted activities of RopGAP and RopGDI prevent lateral diffusion of activated Rop, restricting Rop activity to the apical plasma membrane. Conversely, pollen receptor kinases (PRKs) and RopGEFs positively feedback regulate Rop activity through protein binding and membrane recruitment. Feedback loops would also be essential for pollen tube re-orientation. Shallow extracellular cues amplified by concerted activities of feedback loops would lead to asymmetric activation of Rop and result in tube re-orientation.Key words: Rho GTPases, Rop, GEF, GAP, GDI, receptor kinase, feedback loop