Functional characterisation of an intron retaining K(+) transporter of barley reveals intron-mediated alternate splicing.

Intron retention in transcripts and the presence of 5' and 3' splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K(+) transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K(+) uptake restored growth in medium containing hygromycin in the presence of different concentrations of K(+) and mediated hypersensitivity to Na(+) . HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K(+) and Na(+) concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel.

Cyclic nucleotide-gated (cng) non-selective cation channels have been cloned from a number of animal systems. These channels are characterized by direct gating upon cAMP or cGMP binding to the intracellular portion of the channel protein, which leads to an increase in channel conductance. Animal cng channels are involved in signal transduction systems; they translate stimulus-induced changes in cytosolic cyclic nucleotide into altered cell membrane potential and/or cation flux as part of a signal cascade pathway. Putative plant homologs of animal cng channels have been identified. However, functional characterization (i.e. demonstration of cyclic-nucleotide-dependent ion currents) of a plant cng channel has not yet been accomplished. We report the cloning and first functional characterization of a plant member of this family of ion channels. The Arabidopsis cDNA AtCNGC2 encodes a polypeptide with deduced homology to the alpha-subunit of animal channels, and facilitates cyclic nucleotide-dependent cation currents upon expression in a number of heterologous systems. AtCNGC2 expression in a yeast mutant lacking a low-affinity K(+) uptake system complements growth inhibition only when lipophilic cyclic nucleotides are present in the culture medium. Voltage clamp analysis indicates that Xenopus laevis oocytes injected with AtCNGC2 cRNA demonstrate cyclic-nucleotide-dependent, inward-rectifying K(+) currents. Human embryonic kidney cells (HEK293) transfected with AtCNGC2 cDNA demonstrate increased permeability to Ca(2+) only in the presence of lipophilic cyclic nucleotides. The evidence presented here supports the functional classification of AtCNGC2 as a cyclic-nucleotide-gated cation channel, and presents the first direct evidence (to our knowledge) identifying a plant member of this ion channel family.

Evaluation of functional interaction between K(+) channel alpha- and beta-subunits and putative inactivation gating by Co-expression in Xenopus laevis oocytes.

Animal K(+) channel alpha- (pore-forming) subunits form native proteins by association with beta-subunits, which are thought to affect channel function by modifying electrophysiological parameters of currents (often by inducing fast inactivation) or by stabilizing the protein complex. We evaluated the functional association of KAT1, a plant K(+) channel alpha-subunit, and KAB1 (a putative homolog of animal K(+) channel beta-subunits) by co-expression in Xenopus laevis oocytes. Oocytes expressing KAT1 displayed inward-rectifying, non-inactivating K(+) currents that were similar in magnitude to those reported in prior studies. K(+) currents recorded from oocytes expressing both KAT1 and KAB1 had similar gating kinetics. However, co-expression resulted in greater total current, consistent with the possibility that KAB1 is a beta-subunit that stabilizes and therefore enhances surface expression of K(+) channel protein complexes formed by alpha-subunits such as KAT1. K(+) channel protein complexes formed by alpha-subunits such as KAT1 that undergo (voltage-dependent) inactivation do so by means of a "ball and chain" mechanism; the ball portion of the protein complex (which can be formed by the N terminus of either an alpha- or beta-subunit) occludes the channel pore. KAT1 was co-expressed in oocytes with an animal K(+) channel alpha-subunit (hKv1.4) known to contain the N-terminal ball and chain. Inward currents through heteromeric hKv1. 4:KAT1 channels did undergo typical voltage-dependent inactivation. These results suggest that inward currents through K(+) channel proteins formed at least in part by KAT1 polypeptides are capable of inactivation, but the structural component facilitating inactivation is not present when channel complexes are formed by either KAT1 or KAB1 in the absence of additional subunits.

Molecular cloning and expression characterization of a rice K+ channel beta subunit.

K+ channel proteins native to animal membranes have been shown to be composed of two different types of polypeptides: the pore-forming alpha subunit and the beta subunit which may be involved in either modulation of conductance through the channel, or stabilization and surface expression of the channel complex. Several cDNAs encoding animal K+ channel beta subunits have been recently cloned and sequenced. We report the molecular cloning of a rice plant homolog of these animal beta subunits. The rice cDNA (KOB1) described in this report encodes a 36 kDa polypeptide which shares 45% sequence identity with these animal K+ channel beta subunits. and 72% identity with the only other cloned plant (Arabidopsis thaliana) K+ channel beta subunit (KAB1). The KOB1 translation product was demonstrated to form a tight physical association with a plant K+ channel alpha subunit. These results are consistent with the conclusion that the KOB1 cDNA encodes a K+ channel beta subunit. Expression studies indicated that KOB1 protein is more abundant in leaves than in either reproductive structures or roots. Later-developing leaves on a rice plant were found to contain increasing levels of the protein with the flag leaf having the highest titer of KOB1. Leaf sheaths are known to accumulate excess K+ and act as reserve sources of this cation when new growth requires remobilization of K+. Leaf sheaths were found to contain higher levels of KOB1 protein than the blade portions of leaves. It was further determined that when K+ was lost from older leaves of plants grown on K+-deficient fertilizer, the loss of cellular K+ was associated with a decline in both KOB1 mRNA and protein. This finding represents the first demonstration (in either plants or animals) that changes in cellular K+ status may specifically alter expression of a gene encoding a K+ channel subunit.

Physical association of KAB1 with plant K+ channel alpha subunits.

K+ channel proteins contain four alpha subunits that align along a central axis perpendicular to membranes and form an ion-conducting pore. Recent work with K+ channels native to animal membranes has shown that at least some members of this protein family also have four beta subunits. These structural components of the holoenzyme each form tight associations with the cytoplasmic portion of an alpha subunit. We have cloned an Arabidopsis cDNA (KAB1) that encodes a polypeptide sharing 49% amino acid identity with animal K+ channel beta subunits. In this study, we provide experimental evidence that the KAB1 polypeptide forms a tight physical association with the Arabidopsis K+ channel alpha subunit, KAT1. An affinity-purified KAB1 fusion protein was immobilized to a support resin and shown to sequester selectively the KAT1 polypeptide. In addition, polyclonal antibodies raised against KAB1 were shown to immunoprecipitate the KAT1 polypeptide as a KAT1-KAB1 protein complex. Immunoblot analysis demonstrated that KAB1 is expressed in Arabidopsis seedings and is present in both membrane and soluble protein fractions. The presence of KAB1 (a soluble polypeptide) in both soluble and membrane protein fractions suggests that a portion of the total amount of native KAB1 is associated with an integral membrane protein, such as KAT1. The presence of KAB1 in crude protein fractions prepared from different Arabidopsis plant organs was evaluated. High levels of KAB1 protein were present in flowers, roots, and leaves. Immunoblot analysis of protein extracts prepared from broad bean leaves indicated that the KAB1 expression level was 80-fold greater in guard cells than in mesophyll cells. Previous studies of the in situ transcription pattern of KAT1 in Arabidopsis indicated that this alpha subunit is abundantly present in leaves and, within the leaf, exclusively present in guard cells. Thus, KAB1 was determined to be expressed in plant organs (leaves) and cell types (guard cells) that are sites of KAT1 expression in the plant. The in situ expression pattern of KAB1 suggests that it may associate with more than one type of K+ channel alpha subunit. Sequence analysis indicates that KAB1 may function in plant K+ channels as an oxidoreductase. It is postulated that beta subunits native to animal K+ channels act as regulatory subunits through pyridine nucleotide-linked reduction of alpha polypeptides. Although the KAB1 primary structure is substantially different from that of animal beta subunits, amino acid motifs critical for this catalytic activity are retained in the plant beta subunit.

Characterization of a TTX-sensitive Na+ current in pacemaker cells isolated from rabbit sinoatrial node.

A tetrodotoxin (TTX)-sensitive Na+ current (iNa) was investigated in single pacemaker cells after 1-4 days in culture. Ruptured-patch and perforated-patch whole cell recording techniques were used to record iNa and spontaneous electrical activity, respectively. For seven cells exposed to 20 mM Na+ (22-24 degrees C) and held at -98 mV (25% of the channels inactivated), the uncorrected maximum iNa was -39 +/- 10 pA/pF at -29.1 +/- 2.4 (SE) mV, maximum conductance was 0.9 +/- 0.2 nS/pF (1.6 +/- 0.2 mS/cm2). Half-activation and inactivation potentials were -41.4 +/- 2.0 and -90.6 +/- 2.5 mV, and the corresponding slope factors were 6.0 +/- 0.4 and 6.4 +/- 0.6 mV. Inactivation and recovery from inactivation were best fit by sums of two exponentials. During action potential clamp, a TTX-sensitive compensation current accounted for 55% of the upstroke velocity. The results suggest that iNa contributes significantly to the action potential in some nodal pacemaker cells, and the characteristics of iNa are similar to those of atrial and ventricular myocytes.

Evidence that plant K+ channel proteins have two different types of subunits.

Plant K+ channel proteins have been previously characterized as tetramers of membrane-spanning alpha subunit polypeptides. Recent studies have identified a 39-kD, hydrophilic polypeptide that is a structural component of purified animal K+ channel proteins. We have cloned and sequenced an Arabidopsis thaliana cDNA encoding a 38.4-kD polypeptide that has a sequence homologous to the animal K+ channel beta subunit. Southern and northern analyses indicate the presence of a gene encoding this cDNA in the Arabidopsis genome and that its transcription product is present in Arabidopsis cells. To our knowledge, this is the first report to document the presence of K+ channel beta subunits in plants.

Molecular and Physiological Analysis of a Thylakoid K+ Channel Protein.

Transport studies identified a K+ channel protein in preparations of purified spinach (Spinacea oleracea) thylakoid membrane. This protein was solubilized from native membranes and reconstituted into artificial proteoliposomes with maintenance of functional integrity. A 33-kD thylakoid polypeptide was identified as a putative component of this thylakoid protein. This identification was made using an antibody raised against a synthetic peptide representing a highly conserved region of K+ channel proteins. K+ channel activity co-migrated with the immunoreactive 33-kD polypeptide when solubilized thylakoid membrane protein was fractionated on a Suc density gradient. The antibody was used to immunoprecipitate the 33-kD polypeptide. Physiological function of this thylakoid membrane protein was elucidated by measuring photosynthetic electron transport of thylakoid preparations in the presence and absence of a K+ channel blocker. Results indicated that K+ efflux from the thylakoid lumen through this channel protein is required for the optimization of photosynthetic capacity. The effect this protein has on photosynthetic capacity is likely due to the requirement for K+ efflux from the thylakoid lumen to charge-balance light-induced proton pumping across this membrane.

Development of a K(+)-channel probe and its use for identification of an intracellular plant membrane K+ channel.

Polyclonal antibodies were generated against a 9-amino acid, synthetic peptide corresponding to the selectivity filter in the pore region of K(+)-channel proteins. The sequence of amino acids in the ion-conducting pore region of K+ channels is the only highly conserved region of members of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was immunoreactive with known K(+)-channel proteins and (ii) to demonstrate the usefulness of the antibody by employing it to identify a newly discovered K(+)-channel protein. Anti-channel pore peptide was immunoreactive with various K(+)-channel subtypes native to a number of different species. Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K(+)-channel proteins. Studies also suggested that the anti-channel pore peptide antibody did not immunoreact with membrane proteins other than K+ channels. The anti-channel pore peptide antibody was used to establish the identity of a 62-kDa chloroplast inner envelope polypeptide as a putative component of a K(+)-channel protein. It was concluded that an antibody generated against the conserved pore region/selectivity filter of K+ channels has broad but selective affinity for this class of proteins. This K(+)-channel probe may be a useful tool for identification of K(+)-channel proteins in native membranes.

Characterization of a chloroplast inner envelope K+ channel.

A K(+)-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.

Use of Transgenic Plants with Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Antisense DNA to Evaluate the Rate Limitation of Photosynthesis under Water Stress.

The biochemical lesion that causes impaired chloroplast metabolism (and, hence, photosynthetic capacity) in plants exposed to water deficits is still a subject of controversy. In this study we used tobacco (Nicotiana tabacum L.) transformed with "antisense" ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) DNA sequences to evaluate whether Rubisco or some other enzymic step in the photosynthetic carbon reduction cycle pathway rate limits photosynthesis at low leaf water potential ([psi]w). These transformants, along with the wild-type material, provided a novel model system allowing for an evaluation of photosynthetic response to water stress in near-isogenic plants with widely varying levels of functional Rubisco. It was determined that impaired chloroplast metabolism (rather than decreased leaf conductance to CO2) was the major cause of photosynthetic inhibition as leaf [psi]w declined. Significantly, the extent of photosynthetic inhibition at low [psi]w was identical in wild-type and transformed plants. Decreasing Rubisco activity by 68% did not sensitize photosynthetic capacity to water stress. It was hypothesized that, if water stress effects on Rubisco caused photosynthetic inhibition under stress, an increase in the steady-state level of the substrate for this enzyme, ribulose 1,5-bisphosphate (RuBP), would be associated with stress-induced photosynthetic inhibition. Steady-state levels of RuBP were reduced as leaf [psi]w declined, even in transformed plants with low levels of Rubisco. Based on the similarity in photosynthetic response to water stress in wild-type and transformed plants, the reduction in RuBP as stress developed, and studies that demonstrated that ATP supply did not rate limit photosynthesis under stress, we concluded that stress effects on an enzymic step involved in RuBP regeneration caused impaired chloroplast metabolism and photosynthetic inhibition in plants exposed to water deficits.

Magnesium, potassium flux and photosynthesis.

Photosynthetic capacity of chloroplasts is regulated by stromal pH. The pH of the stroma must be maintained higher than external pH during illumination in order to optimize photosynthetic carbon reduction cycle activity. Stromal pH is affected by K+/H+ counterexchange across the chloroplast envelope. Magnesium ion external to the chloroplast affects K+/H+ counterfluxes and hence stromal pH and photosynthesis. K+/H+ counterfluxes across the envelope are facilitated by K+ and H+ conducting ion channels, and an H+ pumping envelope ATPase. External magnesium affects K+/H+ exchange by binding to negative surface charges on the envelope membrane. Magnesium binding restricts currents through K+ channels. Magnesium block of K+ uptake through this channel impairs H+ efflux, inhibiting photosynthesis. Under conditions which lead to net K+ efflux through the channel, envelope-bound magnesium restricts K+ efflux, reducing the driving force for electroneutral H+ uptake. External magnesium has a profound effect on chloroplast photosynthetic capacity by an indirect control of H+ movement across the envelope and hence of stromal pH.

K+-conducting ion channel of the chloroplast inner envelope: functional reconstitution into liposomes.

Potassium flux between the chloroplast stroma and cytoplasm is known to be indirectly linked to H+ countertransport and, hence, stromal pH and photosynthetic capacity. The specific molecular mechanism that facilitates K+ flux across the chloroplast envelope is not known and has been a source of controversy for well over a decade. The objective of this study was to elucidate the nature of this envelope protein. To this end, solubilized protein in detergent extracts of purified chloroplast inner envelope vesicles was reconstituted into artificial liposomes, and cation fluxes into these proteoliposomes were measured. Results of inhibitor studies and counterflux experiments indicated that a K+-conducting ion channel was solubilized and functionally reconstituted into the proteoliposomes. This transport protein may be a nonspecific monovalent cation channel. This report represents a direct demonstration of ion channel activity associated with the limiting (inner) membrane of the chloroplast envelope.

Chloroplast Inner-Envelope ATPase Acts as a Primary H+ Pump.

The stromal pH of the chloroplast must be maintained higher than that of the surrounding cytosol for photosynthetic carbon assimilation to occur. Experimental evidence demonstrating how this is accomplished in the plant cell is lacking. In the experiments reported here, we studied H+ and K+ flux across membranes of purified chloroplast inner-envelope vesicles. We were able to demonstrate ATP-dependent transport of both cations across the membranes of these vesicles. The data presented document the presence of an H+-pump ATPase in the chloroplast envelope. Energy-dependent K+ flux across these membranes occurs as a consequence of primary H+ pumping. The H+-pumping activity demonstrated in this report is consistent with a model involving the activity of this envelope ATPase as a primary mechanism facilitating a stroma:cytosol [delta]pH.

K stimulation of ATPase activity associated with the chloroplast inner envelope.

Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg(2+) and Mg.ATP complex effects on ATPase activity revealed that any Mg(2+) stimulation of activity was likely a function of the use of the Mg.ATP complex as a substrate by the enzyme; free Mg(2+) may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K(+). This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K(+). K(+) stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K(+) effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the K(m) increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K(+)-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K(+) effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H(+)-ATPase). Such ATPases are thought to be indirectly involved in active K(+) uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K(+) effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo.

Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon.

The extent and occurrence of water stress-induced "patchy" CO(2) uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [(14)C]CO(2) before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to -1.1 megapascals during a 4-day period did result in heterogenous CO(2) assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO(2) assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO(2)] in stressed bean leaves. When extent of heterogenous CO(2) uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to -1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO(2) assimilation patterns when stressed to a leaf water potential of -2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits.

Stromal pH and Photosynthesis Are Affected by Electroneutral K and H Exchange through Chloroplast Envelope Ion Channels.

Potassium movement across the limiting membrane of the chloroplast inner envelope is known to be linked to counterex-change of protons. For this reason, K(+) efflux is known to facilitate stromal acidification and the resultant photosynthetic inhibition. However, the specific nature of the chloroplast envelope proteins that facilitate K(+) fluxes, and the biophysical mechanism which links these cation currents to H(+) counterflux, is not characterized. It was the objective of this work to elucidate the nature of the system regulating K(+) flux linked to H(+) counterflux across the chloroplast envelope. In the absence of external K(+), exposure of spinach (Spinacia oleracea) chloroplasts to the K(+) ionophore valinomycin was found to increase the rate of K(+) efflux and H(+) influx. These data were interpreted as suggesting that H(+) counterexchange must be indirectly linked to movement of K(+) across the envelope. Studies using the K(+) channel blocker tetraethylammonium indicated that K(+) likely moves, in a uniport fashion, into or out of the stroma through a monovalent cation channel in the envelope. Blockage of K(+) efflux from the stroma by exposure to tetraethylammonium was found to restrict H(+) influx, further substantiating an indirect linkage of these cation currents. Further studies comparing the effect of exogenous H(+) ionophores and K(+)/H(+) exchangers suggested that K(+) uniport through this ion channel likely is the main endogenous pathway for K(+) currents across the envelope. These experiments were also consistent with the presence of a proton channel in the envelope. Movement of H(+) through this channel was speculated to be regulated and rate limited by an electroneutral requirement for K(+) countercurrents through the separate K(+) uniport pathway. K(+) and H(+) fluxes across the chloroplast envelope were envisioned to be interrelated via this mechanism. The significant effect of cation currents across the envelope, as mediated by these channels, on photosynthetic capacity of the isolated chloroplast was also demonstrated.

Lidocaine and ATPase inhibitor interaction with the chloroplast envelope.

Photosynthetic capacity of isolated intact chloroplasts is known to be sensitive to K(+) fluxes across the chloroplast envelope. However, little is known about the system of chloroplast envelope proteins that regulate this K(+) movement. The research described in this report focused on characterizing some of the components of this transport system by examining inhibitor effects on chloroplast metabolism. Digitoxin, an inhibitor of membrane-bound Na(+)/K(+) ATPases, was found to reduce stromal K(+) at a range of external K(+) and inhibit photosynthesis. Scatchard plot analysis revealed a specific protein receptor site with a K(m) for digitoxin binding of 13 nanomolar. Studies suggested that the receptor site was on the interior of the envelope. The effect of a class of amine anesthetics that are known to be K(+) channel blockers on chloroplast metabolism was also studied. Under conditions that facilitate low stromal pH and concomitant photosynthetic inhibition, the anesthetic, lidocaine, was found to stimulate photosynthesis. This stimulation was associated with the maintenance of higher stromal K(+). Comparison of the effects on photosynthesis of lidocaine analogs which varied in lipophilicity suggested a lipophilic pathway for anesthetic action. The results of experiments with lidocaine and digitoxin were consistent with the hypothesis that a K(+) channel and a K(+)-pumping envelope ATPase contribute to overall K(+) flux across the chloroplast envelope. Under appropriate assay conditions, photosynthetic capacity of isolated chloroplasts was shown to be much affected by the activity of these putative envelope proteins.

Surface Charge-Mediated Effects of Mg on K Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis.

Studies of Spinacia oleracea L. were undertaken to characterize further how Mg(2+) external to the isolated intact chloroplast interacts with stromal K(+), pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed hypothesis that millimolar levels of external, unchelated Mg(2+) result in lower stromal K(+), which somehow is linked to stromal acidification. Stromal acidification directly results in photosynthetic inhibition. These effects were attributed to Mg(2+) interaction (binding) to negative surface charges on the chloroplast envelope. Chloroplast envelope-bound Mg(2+) was found to decrease the envelope membrane potential (inside negative) of the illuminated chloroplast by 10 millivolts. It was concluded that Mg(2+) effects on photosynthesis were likely not mediated by this effect on membrane potential. Further experiments indicated that envelope-bound Mg(2+) caused lower stromal K(+) by restricting the rate of K(+) influx; Mg(2+) did not affect K(+) efflux from the stroma. Mg(2+) restriction of K(+) influx appeared consistent with the typical effects imposed on monovalent cation channels by polyvalent cations that bind to negatively charged sites on a membrane surface near the outer pore of the channel. It was hypothesized that this interaction of Mg(2+) with the chloroplast envelope likely mediated external Mg(2+) effects on chloroplast metabolism.