2 1. Ion channels are ubiquitous in plant membranes An action potential as seen in the large internode cells of some algae
3 Minosa pudica leaf evokes an action potential that cause the pulvinus to lose turgor. Some insectivorous plants also use action potentials to couple the sensing of prey.
4 Channels are now known to be present in all plant cell types, : at the PM and vacuoles and all other membranes as well.
5 2. Ion channels are studied with electrophysiological techniques Path Clamp: detecting tiny electrical currents, resolving activity of single protein molecules.Planar Lipid Bilayers: recording activity of channels at intracellular membranes.Voltage Clamp: classical approach, keeping cell wall and regulatory machinery within cell intact.
6 Path Clamp: detecting tiny electrical currents, resolving activity of single protein molecules.
8 Planar Lipid Bilayers: recording activity of channels at intracellular membranes. Voltage Clamp: classical approach, keeping cell wall and regulatory machinery within cell intact.
9 Ion flow through channels is passive. 3.5.3 Ionic fluxes through channels are driven solely by electrochemical potential differencesIon flow through channels is passive.
10 Current-voltage (I-V) curve for a single channel.
11 4. Ion channels exhibit ionic selectivity Cation channelsmonovalent: K+, H+divalent: Ca2+,Anion channelsCl-, No3-, organic acidsThe fact that channels are selective implies that binding sites capableof distinguishing specific ions must be located within the channel pores.
12 5. Ion channels are gated, often by voltages or ligands, through changes in open state probability.
15 Voltage dependent K+ channels at the PM stabilize Vm and allow controlled K+ uptake and lossOutward rectifying K+ channels: decrease K+ in cytosolInward rectifying K+ channels: increase K+ in cytosol
16 Whole cell currents in the plant cell Current-voltage relationshipWhole cell currents in the plant cellcurrents flow across the PM in both directions, into and out of the cytosol
17 K+ outward rectifier stabilize Vm cytosol100 mM K+1 mM K+EK=-120mVIf hyper-depolarized, outward rectifying channels opencytosolEK >-120mVK+
18 K+ inward rectifiers take up K+ from the soil and the apoplast cytosolK+ < 1mMaccumulatedby ion coupled carrierK+K+In guard cell,Outward rectifiers are activated by the small increase in cytosolic pH (elicited by ABA)Inward rectifiers are subject to modulation by G-protein and are inhibited by increased free cytosolic Ca+ and by PP2B
20 2. Plant cell inward rectifiers are members of the Shaker family of votage-gated channelsShaker familySuperfamily of voltage-gated K+, Ca2+, Na+Plant inward rectifiersS4 domain : positively charged residues every third residuevoltage sensor that open the channel in response to a permissive voltage
21 K+-selective inward rectifiers Products of multigene familyTissue-specific expression of plant inward rectifiersKAT1 expressed in guard cellAKT1 found in roots and hydathodesmature root lateral root hydathodes
22 Radioactive tracer flux analysis 86Rb+ uptake in WT and akt1-1 roots akt1-1 shows the reduced K+ uptake and less growth in media with low K+ concentrations
23 The outward rectifier Outward rectifying K+ channel, KCO1 Member of the two-pore K+ channel familyFour transmembrane spans in each subunitTwo-EF hands reside toward the C-terminus: sensitive to the concentrations of cytosolic Ca2+highly conserved P-domain motif TxGYGD
24 Hyperpolarizing voltage conformational change : opening the gate screw out of the membraneconformational change: opening the gate
25 Ca2+ dependence of the KCO1 : KCO1 mediated currents are potentiated by cytosolic free Ca2+
26 4. Voltage – insensitive cation channels may be a major pathway for Na+ uptake across the plasma membrane and for salt release to the xylemChannels :- constitute a major pathway for Na+ uptake into plant cells- important implications for salinity tolerance.- partially blocked by external Ca2+2) Less selective channels :- observed in the plasma membrane of xylem- almost as permeable to anions as to cations.- perhaps this channel could provide a pathway for release of salts from the symplasm into the xylem.
27 Na+-dependent currents across the plasma membrane of protoplasts.
28 (B) I-V relationship for the currents in A showing substantial influx of Na+
29 Monovalent cation channels at the vacuolar membrane are Ca2+-sensitive and mediate vacuolar K+ mobilization.Vacuoles : - organelles for accumulation and storage of solutes- require massive solute loss in some circumstancesHypo-osmotic stress, during stomatal closure,: mobilization of ions from vacuoles to restore normal cell turgor3) Guard cell has two different classes of K+-permeable channels in releasing K+ from the vacuole.- the fast vacuolar (FV) channel- the vacuolar K+ (VK) channel
30 The fast vacuolar (FV) channel Little selectivity among monovalent cationsInhibited when cytosolic Ca2+ concentrations exceed 1μMActivated when cytosolic pH increasesThe vacuolar K+ (VK) channelHighly selective for K+ over other monocovalent cationsActivated by cytosolic Ca2+ in the nanomolar to low micromolar concentration rangeInhibited by increasing cytosolic pH
31 Calcium-permeable channels in the plasma membrane provide potential routes for entry of Ca2+ to the cytosol during signal transduction.Ca2+ : Ca2+ increase is central to signal transduction.activation of down stream targets, initial signal into the end response.2) Ca2+ permeable channels : activation of increase in cytosolic Ca2+,upstream elements in Ca2+ - based signal transduction pathway,3) Voltage-gated Ca2+_ permeable channels are activated by membrane depolarization.
32 Calcium-based signal transduction in a typical plant cell. Receptor is perceived by signal.2) Changed the activity of Ca2+ -ATPases or plasma membrane Ca2+ channel3) Change on cytosolic free calcium concentration.4) release of Ca2+ from internal stores.5) 2nd messenger is activation6) increased free Ca2+ changes the activity of Ca2+ - binding proteins7) final cellular response.
33 External Signals Diverse Cellular Responses [Ca2+]cyt Amplitude (AM) Light (red, blue)Cold stressHeat shockMechanical stresses(touch, wind &wounding)PathogensPhytohormones(Auxin, ABA, GA)GravityCa2+-SignalDecoderDiverseCellularResponses[Ca2+]cytAM, DU, FMCalmodulinsCDPKs, CBLsOther CBPsAmplitude (AM)Let me remind you about calcium signaling in plant. Many kinds of external signals such as light, cold, heat, mechanical stresses, pathogens, phytohormones, and gravity can increase many different forms of cytosolic Ca2+ concentration. Different Ca2+ spike with different amplitude, duration and frequency can be formed dependent on the external signals. Different Ca2+ spike are recognized by Ca2+ decoders such as CaMs, CDPKs, CBLs and other CBPs. Ca2+ decoders modulate downstream enzyme and gene expression and results in appropriate cellular responses.Duration (DU)Frequency (FM)33
34 Encoding and Decoding Calcium Signaling InfluxEncodingCa2+ spikesStimulusEffluxDecodingDecodersRegulates enzymesChanges gene expressionResponsesCa2+ signals are generated by the influx of Ca2+ through Ca2+ channels.Maintenance of low Ca2+ is archived by Ca2+ efflux though Ca2+ active transporters34
35 Activation of a wheat root plasma membrane Ca2+ channel by voltage. The channel activates strongly as Vm and is fully activated at -100mV.
36 7. Calcium-permeable channels in endomembranes are activated by both voltage and ligands
37 Diagram illustrating channel activities at the guard cell vacuolar membrane during stomatal closure During plasma membrane-based signal transduction
38 Activity of the SV channel increases with increasing cytosolic concentration of Ca2+ (A) Slow activation of the SV channel in barley aleurone vacuoles in response to positive voltages(B) Ca2+-dependence of whole-vacuole channel activity. Increasing free calcium above approximately 1μM increasing the activity of the channel. Ca2+ is thought to interact with calmodulin associated with the channel or a channel regulatory protein
39 8. Plasma membrane anion channels facilitate salt release during turgor adjustment and elicit membrane depolarization after stimulus perception
40 Anion channels in guard cell Current-voltage relationship for rapidly activating(R-type)anion channels.Current-voltage relationship for slowly activating(S-type)channels
41 Anion channels function 팽압(turgor)을 조절 기능특별한 자극에 의해 Depolarization되어 Ca2+ channel activation되게 함Membrane hyperpolarization으로 activation되어 Vm의 조절에 관여Guard cell을 포함한 cell type에서 ATP, protein Kinase로 부터 activatione됨
42 3.6.9 Vacuolar malate channels participate in malate sequestration
43 Current-voltage relationship for vacuolar uptake of malate through time-dependent anion channel in the tonoplast.Malate uptake by anion channel is strongly promoted by negative membrane otentials and increases with cytosolic malate concentration. In this figure, cytosolic malate concentration were 10mM(filled squares), 20mM(open squares), 50mM(open circles), and 100mM(filled triangles)- all with a vacuolar malate concentration of 10mM. Malate uptake with equal concentration of malate(50mM) presente on both side of the membrane is indicated by stars.
44 Accumulation of malate in the root of CAM plants Accumulation of malate in the root of CAM plants. Malate2- is thought to enter the vacuole though malate-selective channels.These channel are strongly inward rectifying and do not allow substantial malate2- efflux. Once inside the vacuole,malate2- is protonated to H.malate and H2.malate0. This maintains the effective concentration difference for malate2- across the membrane.
45 Integrated channel activity at the vacuolar and plasma membranes yields sophisticated signaling systems
46 Ca2+ signaling coordinates the activities of multiple ion channels and H+-pumps during stomatal closure. In this model,perception of ABA by a receptor(R) results in an increase in cytosolic free Ca2+ through Ca2+influx or Ca2+ release from internal stores. Increased cytosolic Ca2+ promotes opening of plasma membrane anion and K+out channels and inhibits opening of K+in channels. As more ions leave the cell than enter it, water follows, turgor is lost, and the stomatal pore is closed
48 Jv = Lp(▲p- σ▲п) ▲p: hydrostatic pressure ▲п : osmotic pressure 3.7.1 Directionality of water flow is determined by osmotic and hydraulic forcesJv = Lp(▲p- σ▲п)▲p: hydrostatic pressure▲п : osmotic pressureJv: the flux of water across the membraneσ: Expresses the ability of the osmotically relevant solutes to permeate the membrane relative to water
49 Membrane permeability to water can be measure in two ways: 3.7.2 Membrane permeability to water can be defined with either an osmotic coefficient (Pf) or a diffusional coefficient (Pd)Membrane permeability to water can be measure in two ways:+ The imposition of an osmotic or a hydrostatic pressure (Pf) difference across the membrane can be used to generate a water flow.Pf = Lp RT/Vw+ The assessing water permeability relies on measuring the diffusional permeability (Pd) with isotopic water.
51 3.7.3. The nonequivalence of Pf and Pd provides evidence for water channels Pf involves net flow of water. Each water molecule entering the channel form the left will knock out one molecule on the right.In the diffusion flow case, a molecule of labeled water entering the channel from the left can diffuse back into the solution on the left.
52 Water movement across biological membranes occurs through both the lipid bilayer and the pores formed by water channels.Model for water flow through a single-life, multiple occupancy aquaporin
53 Aquaporins are members of the major intrinsic protein family, which can form water channels when expressed in heterologous systemsWater channels or aquaporins are highly expressed in plant membrane. Member of a family of transmembrane channels known as the major intrinsic protein MIP family and these proteins are about 25 to 30 kDa, probably span the bilayer six times and contain an internal repeat sequence indicative of origins from a gene duplication and fusion.Aquaporins are also characterized by the highly conserved NPA (Asn-Pro-Ala) residue in the N and C terminal.In plant the aquaporins have been identified in both vacuolar (known as tonoplant intrinsic protein TIP) and plasma membrane (plasma membrane intrinsic protein PIP).
54 Structure of an aquaporin showing the six transmembrane helices and two conserved NPA (Asn-Pro-Ala) residue
55 Three-dementional structure of aquaporin-1 from human erythrocytes Three-dementional structure of aquaporin-1 from human erythrocytes. Extracellular view of eight asymmetrical subunits that form two tetramers. One of the monomers of the central tetramer is colored gold.
56 Environmental stimuli (blue light, ABA, GA, cold & drought) 3.7.5 Aquaporin activity is regulated transcriptionally and posttranslationallyH2OAquaporinTranscriptionPosttranslationEnvironmental stimuli (blue light, ABA, GA, cold & drought)Phosphorylation by Ca2+ dependent protein kinase
57 Figure . Schematic representation of putative mechanisms involved in plant aquaporin egulation. (a) Control of transcription and protein abundance. Drought and salinity, as other environmental stimuli, are known to act on aquaporin gene transcription and possibly interfere with aquaporin translation and degradation, thereby determining protein abundance. Theformation of PIP hetero-tetramers was demonstrated in Xenopus oocytes (Fetter et al. 2004) and is still hypothetical in plant cells. This mechanism might favour transfer of PIP1 homologues to the plasma membrane (PM). (b) Sub-cellular relocalization. The redistribution of a TIP aquaporin, from the tonoplast (TP) to small intracellular vesicles, was demonstrated in Mesembryanthemumcrystallinum suspension cells exposed to a hyperosmotic treatment (Vera-Estrella et al. 2004). The occurrence of a similar relocalization mechanism for PIP aquaporins is shown but remains hypothetical.
58 3.7.6 Plasma membrane aquaporins may play a role in facilitating transcellular water flowvacuoleTonoplant intrinsic protein(TIP)Plant waterchannelPlasma membranePlasma membrane intrinsic protein(PIP)vacuolePlasma membrane
59 Transpiration apoplast symplast Requirement for a low-resistance pathway
60 3.7.7Differential water permeabilities of the vacuolar and plasma membranes can prevent large changes in cytoplasmic volume during water stressThe water permeability of the vacuolar membraneThe water permeability of the plasma membrane
61 Requirment for maintenance of cytosolic volume during osmoticstress