Mechanisms of solute efflux from seed coats: whole-cell K+ currents in transfer cell protoplasts derived from coats of developing seeds of Vicia faba L.

In developing seed ofVicia faba L., solutes imported throughthe phloem of the coats move symplastically from the sieve elementsto a specialized set of cells (the thin-walled parenchyma transfercells) for release to the seed apoplast. Potassium (K⁺) is thepredominant cation released from the seed coats. To elucidatethe mechanisms of K⁺ efflux from seed coat to seed apoplast,whole-cell currents across the plasma membranes of protoplastsof thin-walled parenchyma transfer cells were measured usingthe whole-cell patch-clamp technique. Membrane depolarizationelicited a time-dependent and an instantaneous outward current.The reversal potential (E_R of the time-dependent outward currentwas close to the potassium equilibrium potential (E_K and itshifted in the same direction as E_K upon changing the externalK⁺ concentration, indicating that this current was largely carriedby an efflux of K⁺. The activation of the time-dependent outwardK⁺ current could be well fitted by two exponential componentsplus a constant. The instantaneous outward current could alsobe carried by K⁺ efflux as suggested by ion substitution experiments.These K⁺ outward rectifier currents elicited by membrane depolarizationare probably too small to represent the mechanism for the normalK⁺ efflux from seed coat cells. Membrane hyperpolarization morenegative than –80 mV activated a time-dependent inwardcurrent. K⁺ influx was responsible for the inward current asthe current reversed at membrane voltage close to E_K and shiftedin the same direction as E_K when external [K⁺] was varied. Activationof this K⁺inward rectifier current was well fitted with twoexponential components plus a constant. A regulating functionfor this current is suggested. Key words: Potassium outward rectifier, potassium inward rectifier, transfer cell protoplast, seed coat, Vicia faba L     

Fast activation of a time-dependent outward current in protoplasts derived from coats of developing Phaseolus vulgaris seeds

An outward current that appeared to activate instantaneously in response to depolarising voltage pulses at low sampling frequencies predominated in the plasma membrane of ground-parenchyma protoplasts derived from coats of developing Phaseolus vulgaris L. (cv. Redland Pioneer) seeds. However, the outward current showed time-dependent activation when higher sampling frequencies were used to measure the current. Activation of the current was best described as a double-exponential time course with the fast and slow time constants being 1 and 20 ms, respectively. The current also exhibited a rapid deactivation that followed a double-exponential time course with time constants of approximately 2 and 30 ms, respectively. “Tail-current” analysis allowed us to show that this current exhibited a low selectivity between K⁺ and Cl⁻ (P _K:Cl=1.8). Such a fast-activating current may account for some of the reports of time-independent, instantaneous currents that have been observed in plasma membranes of plant cells digitised at low sampling frequencies. Therefore, when “instantaneous” currents appear it is advisable to characterise these currents using higher sampling frequencies with correspondingly higher filtering frequency cut-offs. Received: 12 May 2000 / Accepted: 26 June 2000     

Pulsing Cl- channels in coat cells of developing bean seeds linked to hypo-osmotic turgor regulation

Seed coat cells in the developing seeds of grain legumes release nutrients to the developing embryo. This occurs into an apoplastic space that separates the maternal (seed coat) and filial (embryo) generations. Protoplasts of seed coat cells from coats of Phaseolus vulgaris L. seeds were isolated and whole-cell current across their plasma membranes was characterized using the patch-clamp technique. A pulsing inward current that displayed a spontaneous activation and voltage-dependent inactivation was observed. The frequency and magnitude of the current pulses were positively dependent on cytoplasmic Cl(-) concentrations and independent of external cations. The pulse current was inhibited by DIDS and La(3+), but not by Gd(3+). Single channel events (conductance=18 pS) could be identified with the inactivating phase of the pulses. Together, these findings are consistent with the current being carried by a burst of Cl(-) efflux through Cl(-)-permeable channels that activate almost simultaneously. Neomycin caused a reversible inhibition of the pulsed current, suggesting that its activation is likely to be modulated by an IP(3)-dependent intracellular Ca(2+) release. The pharmacological profiles of Cl(-) efflux from excised seed coats were comparable with those of the Cl(-) channels in the whole cell configuration, suggesting that the Cl(-) channels may underpin Cl(-) efflux from the seed coats. Efflux of Cl(-) from the seed coats was also stimulated by hypo-osmotic treatment as was the frequency and magnitude of Cl(-) channel in whole-cell patch clamp experiments. This implies that the Cl(-) channels responsible for the pulsed Cl(-) currents are likely to be a component of the turgor-regulatory mechanism in developing bean seeds.     

Labeling and isolation of plasma membranes from corn leaf protoplasts

A plasma membrane-enriched fraction has been isolated from corn leaf mesophyll protoplasts and its identity confirmed with the aid of an external label, diazotized [¹²⁵I]iodosulfanilic acid. Gentle cell disruption enabled internal organelles to be maintained intact and thus facilitated separation from the plasma membrane. The plasma membrane-enriched fraction was devoid of chloroplast or mitochondrial markers, whereas markers for the endoplasmic reticulum and golgi indicated minimal contamination. The highly enriched plasma membrane fraction contained a Mg²⁺-dependent, K⁺-stimulated ATPase with a pH optimum near neutrality. The position of the membranes on sucrose density gradients indicates that the plasma membranes have characteristics similar to other plasma membrane fractions.     

A plasma membrane-enriched fraction isolated from the coats of developing pea seeds contains H(+)-symporters for amino acids and sucrose

Aqueous polymer two-phase partitioning was used to obtain a plasma membrane-enriched fraction from coats of developing pea (Pisum sativum L.) seeds in the filling stage. Uptake of amino acids and sucrose by vesicles from this fraction was determined after imposition of gradients of proton concentration (DeltapH, inside alkaline) and electrical potential (Deltapsi, inside negative) across the vesicle membrane. The uptake of sucrose and the amino acids L-valine, L-lysine, and L-glutamic acid was stimulated by the imposition of DeltapH. The imposition of Deltapsi, either in the presence or in the absence of DeltapH, stimulated the uptake of L-valine and L-lysine, but had no detectable effect on the uptake of sucrose and L-glutamic acid. The proton-motive-force-driven uptake of all four substrates was abolished by the protonophore carbonylcyanide m-chlorophenyl-hydrazone (CCCP). The results demonstrate the presence of H(+)-symporters for sucrose and amino acids in pea seed coats. This is running counter to the previously reported finding that their uptake by isolated pea seed coats was insensitive to CCCP, and that the uptake of sucrose, L-valine, and L-glutamic acid displayed linear kinetics. Possible causes of this discrepancy will be discussed.     

Lipids and fatty acid composition of developing winged bean seeds

The moisture, lipids and fatty acid composition of developing winged bean (Psophocarpus tetragonolobus) seeds were studied. The moisture content decreased steadily as the seeds matured. The lipid content increased gradually and reached a maximum ca 6 weeks after flowering (WAF). In the early stage (2 WAF) of the developing seeds there were more polar lipids (glycolipids and phospholipids) than neutral lipids but, as the seeds developed, neutral lipids gradually accumulated while the polar lipids decreased until 6 WAF. Thereafter, both the neutral lipid and polar lipid levels remained little changed. The amounts of palmitic and stearic acids decreased, but the level of behenic acid increased as the seeds matured. On the other hand, the oleic acid content increased while that of linolenic acid decreased rapidly as the seeds matured. The concentration of linoleic acid, however, fluctuated during the development of the seeds.     

Differences in release of endogenous sugars and amino acids from attached and detached seed coats of developing pea seeds

The effect of p -chloromercuribenzenesulfonic acid (PCMBS), carbonylcyanide- m -chlorophenylhydrazone (CCCP) and a high apoplastic pH (pH 7.5 compared with pH 5.5) on the release of sugars (sucrose and glucose) and amino acids from attached and detached seed coats of Pisum sativum L. cv. Marzia into a bathing solution was measured by means of the 'empty seed coat technique'. PCMBS reduced the release of sugars and amino acids from attached as well as from detached seed coats, suggesting that carrier-mediated transport might be involved. CCCP reduced sugar release from attached seed coats while amino acid release was hardly affected. In experiments with detached seed coats CCCP had no effect on release of either sugar or amino acids, suggesting that it is not energy-dependent. Raising the pH of the bathing solution from pH 5.5 to pH 7.5 slightly increased sugar release from both attached and detached seed coats while amino acid release was not affected. This might indicate a role of the apoplastic pH in regulating sugar release from the seed coat via a retrieval mechanism. The presented data indicate that there are important differences between sugars and amino acids with respect to transport processes in the seed coat. This is supported by the observation that the rate of amino acid release from the seed coat was higher than the rate of sugar release. The release data of detached seed coats were subjected to compartmental analysis in order to calculate rate constants for release from cell compartments. In the case of sugars, the half-times for emptying the cytoplasmic and vacuolar compartment were 0.8 h and 12.5 h. respectively. For amino acids the half-times were 0.5 h for emptying the cytoplasmic and 3.8 h for emptying the vacuolar compartment.     

Free and conjugated indole-3-acetic Acid in developing bean seeds

The changes in conjugated indole-3-acetic acid (IAA) levels compared to the levels of free IAA have been analyzed during the development of bean (Phaseolus vulgaris L.) seed using quantitative mass spectrometry. Free and ester-linked IAA levels are both relatively high in the early stages of seed development but drop during seed maturation. Concomitantly, the amide-linked IAA becomes the major form of IAA present as the seed matures. In fully mature seed, amide IAA accounts for 80% of the total IAA. The total IAA pool in the seed is maintained at approximately the same level (150-170 nanograms/seed) once the level of free IAA has attained its maximum. Thus, the amount of amide IAA conjugates that accumulate in mature seed is closely related to the amounts of free and ester-linked IAA that disappeared from the rapidly growing seed. Analysis of developing bean pods, from which the seeds were taken for analysis, showed very low levels of both ester and amide-linked IAA conjugates. The pattern of changes seen in the levels of free and conjugated IAA in developing bean seed supports our prior hypothesis suggesting a role of IAA conjugates in the storage of the phytohormone in the seed.     

Differential chain-length specificities of two isoamylase-type starch-debranching enzymes from developing seeds of kidney bean

Plant isoamylase-type starch-debranching enzymes (ISAs) hydrolyze alpha-1,6-linkages in alpha-1,4/alpha-1,6-linked polyglucans. Two ISAs, designated PvISA1/2 and PvISA3, were purified from developing seeds of kidney bean by ammonium sulfate fractionation and several column chromatographic procedures. The enzymes displayed different substrate specificities for polyglucans: PvISA1/2 showed broad chain-length specificities, whereas PvISA3 liberated specific chains with a DP of 2 to 4.     

A comparative spin-label study of isolated plasma membranes and plasma membranes of whole cells and protoplasts from cold-hardened and nonhardened winter rye

Lipid-lipid and lipid-protein interactions in the plasma membranes of whole cells and protoplasts and an isolated plasma membrane fraction from winter rye (Secale cereale L. cv Puma) have been studied by spin labeling. Spectra were recorded between −40°C and 40°C using the freely diffusing spin-label, 16-doxyl stearic acid, as a midbilayer membrane probe. The probe was reduced by the whole cells and protoplasts and reoxidized by external potassium ferricyanide. The reoxidized probe was assumed to be localized in the plasma membrane. The spectra consisted of the superposition of a narrow and a broad component indicating that both fluid and immobilized lipids were present in the plasma membrane. The two components were separated by digital subtraction of the immobilized component. Temperature profiles of the membranes were developed using the percentage of immobilized lipid present at each temperature and the separation between the outermost hyperfine lines for the fluid lipid component. Lipid immobilization was attributed to lipid-protein interactions, lipid-cell wall interactions, and temperature-induced lipid phase transitions to the gel-state. Temperature profiles were compared for both cold-hardened and nonhardened protoplasts, plasma membranes, and plasma membrane lipids, respectively. Although cold-hardening extended the range of lipid fluidity by 5°C, it had no effect on lipid-protein interactions or activation energies of lipid mobility. Differences were found, however, between the temperature profiles for the different samples, suggesting that alterations in the plasma membrane occurred as a consequence of the isolation methods used.     

Ion channels in the plasma membrane of protoplasts from the halophytic angiosperm Zostera muelleri

Patch clamp studies show that there may be as many as seven different channel types in the plasma membrane of protoplasts derived from young leaves of the halophytic angiosperm Zostera muelleri. In whole-cell preparations, both outward and inward rectifying currents that activate in a timeand voltage-dependent manner are observed as the membrane is either depolarized or hyperpolarized. Current voltage plots of the tail currents indicate that both currents are carried by K⁺. The channels responsible for the outward currents have a unit conductance of approximately 70 pS and are five times more permeable to K⁺ than to Na⁺. In outside-out patches we have identified a stretch-activated channel with a conductance of 100 pS and a channel that inwardly rectifies with a conductance of 6 pS. The reversal potentials of these channels indicate a significant permeability to K⁺. In addition, the plasma membrane contains a much larger K⁺ channel with a conductance of 300 pS. Single channel recordings also indicate the existence of two Cl^– channels, with conductances of 20 and 80 pS with distinct substates. The membrane potential difference of perfused protoplasts showed rapid action potentials of up to 50 mV from the resting level. The frequency of these action potentials increased as the external osmolarity was decreased. The action potentials disappeared with the addition of Gd³⁺, an effect that is reversible upon washout.We would like to thank K. Morris and D. McKenzie for technical assistance and the Australian Research Council for financial support.     

Isolation of intact plastids from protoplasts from castor bean endosperm

Protoplasts were prepared from castor bean (Ricinus communis) endosperm by treatment with a mixture of the commercial enzymes Macerozyme R-10 and Cellulose “Onozuka” R-10. The protoplasts were gently ruptured by forcing the suspension through a hypodermic needle and the homogenate centrifuged on a linear sucrose gradient. From such a homogenate the mitochondria are recovered at their typical isopycnic density of 1.18 g/ml, but the glyoxysomes are retained, with other membranes, at a density of 1.13. The plastids reach their typical density of 1.22 on the gradient and are thus clearly separated from other organelles. Moreover, since essentially all of the ribulose bisphosphate carboxylase activity on the gradient is present in this fraction it can be concluded that the plastids are intact and have been recovered in high yield.     

Sugar retrieval by coats of developing seeds of Phaseolus vulgaris L. and Vicia faba L

Influxes of glucose, fructose and sucrose were characterised for coat cells of developing seeds of Phaseolus vulgaris L. and Vicia faba L. by monitoring uptake of [(14)C]sugars into excised seed-coat halves and two different protoplast populations derived from seed coats. Sugar influxes by the two populations of protoplasts were similar for each sugar species [sucrose > (fructose approximately glucose)] and hexoses competed with sucrose. Concentration-dependent influxes of all three sugars by excised seed coats could be described by a simple directly proportional relationship between concentration ([S]) and uptake rate (v) in the physiological range of sugar concentrations (v approximately A.[S]). Alternatively, with the exception of fructose influx by Vicia, all could be fitted to a Michaelis-Menten relationship, as could sucrose uptake by Vicia protoplasts. Apparent K(m) values were high ( approximately 100-500 mM) compared with those reported for other systems. Sucrose transport was distinct from glucose and fructose transport in both species. Sugar influx was decreased by p-chloromercuribenzenesulfonic acid, carbonylcyanide m-chlorophenylhydrazone and erythrosin B. These responses are consistent with sugar/H(+) symport acting to retrieve photoassimilates leaked to the apoplasm during post-sieve element transport within seed coats.     

Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots

The aim of the present work was to characterize Na(+) currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca(2+) by digesting tissue in elevated Ca(2+). Experiments in whole-cell and outside-out modes were carried out. We found that Na(+) currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium(+) and verapamil, had no time-dependent activation (permanently opened or completely activated within 1-2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K(+) (1.49) > NH(4)(+) (1.24) > Rb(+) (1.15) approximately equal to Cs(+) (1.10) approximately equal to Na(+) (1.00) > Li(+) (0.73) > tetraethylammonium(+) (0.47). Arabidopsis root NSCCs were blocked by H(+) (pK approximately equal to 6.0), Ca(2+) (K(1/2) approximately equal to 0.1 mM), Ba(2+), Zn(2+), La(3+), Gd(3+), quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca(2+)-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na(+) influx to the cell and probably having other important roles in physiological processes of plants.     

An in vivo technique for the study of Phloem unloading in seed coats of developing soybean seeds

A technique has been developed which permits mechanistic studies of phloem unloading in developing seeds of soybean (Glycine max cv Clark) and other legumes. An opening is cut in the pod wall and the embryo surgically removed from the seedcoat without diminishing the capacity of that tissue for assimilate import, phloem unloading, or efflux. The sites of phloem unloading were accessible via the seedcoat apoplast and were challenged with inhibitors, solutes, buffers, etc., to characterize the unloading process.

Unloading is stimulated by divalent metal chelators and diethylstilbestrol, and inhibited by metabolic uncouplers and sulfhydryl group modifiers. Solutes released from the seed coat had a carbon/nitrogen ratio of 31 milligrams carbon per milligram nitrogen; sucrose represented 90% of the carbon present and various nitrogenous solutes contributed the remaining 10%. Unloading could be maintained for up 8 hours at rates of 0.5 to 1.0 micromoles per hour, providing a valid, convenient in vivo technique for studies of phloem unloading and seed growth mechanisms.

     

Potassium currents across the plasma membrane of protoplasts derived from rye roots: a patch-clamp study

Epidermal-cell protoplasts from rye (Secale cereale L.) rootswere voltage-clamped in both the whole-cell and outside-outmembrane-patch modes. Time-dependent inwardly-rectified (IR)and outwardly-rectified (OR) K⁺-currents were recorded, as wellas a ubiquitous, timeindependent (instantaneous) K⁺-current. The IR current activated at voltages more negative than —100mVwith two exponentially rising components. The time-constantof the shorter component was voltage-independent, whereas thetime-constant of the longer component was voltage-dependent,increasing as the activating voltage became more negative. TheIR current showed no inactivation. The IR current deactivatedwith a single exponential timecourse. The steady-state IR currentcould be fitted to a Boltzmann function with —135 mV asthe voltage at which the current was half-maximal and a minimalgating charge of 1.93. These parameters were insensitive tochanges in E_K. One component of the IR current was K ⁺ , butother ions were also permeable. The IR current was inhibitedby extracellular Ca²⁺ , Ba²⁺ , Cs⁺, and TEA⁺, but was insensitiveto quinine. Single channels with unitary conductances of 56pS and 110 pS (in c.100 mM K⁺) were recorded at negative voltages. Two OR currents were observed. One had sigmoidal activationkinetics and activated at low positive voltages. The other activatedmore rapidly, with apparently exponential kinetics, at voltages50–100 mV more positive than the first. Neither currentshowed inactivation and deactivation of OR currents followeda double exponential time-course. Unitary-conductances of thechannels mediating these OR currents were 24 pS and 57 pS (inc.100 mM K⁺), respectively. Only the first type of OR currentwas studied in detail. This current activated with a sigmoidaltime-course, which could be described using a Hodgkin-Huxleyfunction with the activation variable raised to the second power.Its voltage-dependence was modulated in response to changesin E^K and analysis of single-channel recordings indicated thatthe channel was K⁺-selective. The current was inhibited by Ba²⁺and TEA+, but not Ca²⁺, Cs⁺ or quinine. The instantaneous current was selective for monovalent cationsand K⁺ , Na⁺ and Cs⁺ were all permeant. It was inhibited byextracellular quinine and the instantaneous inward K⁺-currentwas reduced by extracellular Ca²⁺, Ba²⁺ and TEA⁺, as well asby competing permeant monovalent cations. The kinetics and pharmacology of these currents are comparedwith K⁺-currents across the plasma membrane of protoplasts fromother root-derived cells and with K⁺ channels in the plasmamembrane of rye roots studied following incorporation into artificial,planar lipid bilayers. Key words: Ionic currents, patch-clamp, pharmacology, potassium, K⁺, rye, Secale cereale L     

Comparison of the redox activities in plasma membranes from roots and shoots of etiolated bean seedlings

Summary Plasma membrane (PM) vesicles were purified in parallel from the roots and shoots of 6-day-old etiolated bean (Phaseolus vulgaris L.) seedlings, grown in water culture at 25 °C, by aqueous polymer two-phase partitioning. The purity of PM fractions was determined by measuring the activity of known marker enzymes (vanadate-sensitive Mg-ATPase, 1,3--glycan synthase, latent ID-Pase, cytochromec oxidase, and antimycin-A-insensitive cytochromec reductase). NADH-(acceptor) oxidoreductase activities were determined with the following electron acceptors: ferricyanide, cytochromec, duroquinone, monodehydroascorbate, Fe³⁺-EDTA, and oxygen. Cytochromeb content was also determined. In general, results show that redox activities are higher in the root PM than in the shoot PM which follows the glycan synthase II (PM marker) pattern. The relative activities of the distinct redox enzymes measured (activity profile) are remarkably similar in the root and shoot PM preparations. The cytochromeb content and level of ascorbate reduction were also similar in both plant organs. However, the ratio of NADH-(acceptor) oxidoreductase activity to cytochrome content was signifcantly higher in roots when compared to the shoots.Abbreviations CCO cytochromec oxidase - CCR cytochromec reductase - GSII 1,3--glycan synthase - MF microsomal fraction - N-CC-OR NADH-cytochromec oxidoreductase - N-DQ-OR NADH-duroquinone oxidoreductase - N-FC-OR NADH-ferricyanide oxidoreductase - N-FE-OR NADH-Fe³⁺-EDTA oxidoreductase - N-MDA-OR NADH-monodehydroascorbate oxidoreductase - PM plasma membrane     

Calcium-regulated anion channels in the plasma membrane of Lilium longiflorum pollen protoplasts

? Currents through anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts were studied under conditions of symmetrical anionic concentrations by means of patch-clamp whole-cell configuration. ? With Cl(-) -based intra- and extracellular solutions, three outward-rectifying anion conductances, I(Cl1) , I(Cl2) and I(Cl3) , were identified. These three activities were discriminated by differential rundown behaviour and sensitivity to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), which could not be attributed to one or more channel types. All shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for positive potentials. All showed modulation by intracellular calcium ([Ca(2+) ](in) ), increasing intensity from 6.04 nM up to 0.5 mM (I(Cl1) ), or reaching a maximum value with 8.50 μM (I(Cl2) and I(Cl3) ). ? After rundown, the anionic currents measured using NO(3) (-) -based solutions were indistinguishable, indicating that the permeabilities of the channels for Cl(-) and NO(3) (-) are similar. Additionally, unitary anionic currents were measured from outside-out excised patches, confirming the presence of individual anionic channels. ? This study shows for the first time the presence of a large anionic conductance across the membrane of pollen protoplasts, resulting from the presence of Ca(2+) -regulated channels. A similar conductance was also found in germinated pollen. We hypothesize that these putative channels may be responsible for the large anionic fluxes previously detected by means of self-referencing vibrating probes.