Guard Cell Protoplasts Of Vicia Faba Research Articles

Extracellular Ca 2+ alleviates NaCl-induced stomatal opening through a pathway involving H 2O 2-blocked Na + influx in Vicia guard cells

To gain further insights into the function of extracellular Ca²+ in alleviating salt stress, Vicia faba guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that 100 mM NaCl clearly induced Na+ influx across the plasma membrane in GCPs and promoted stomatal opening. Extracellular Ca²+ at 10 mM efficiently blocked Na+ influx and inhibited stomatal opening, which was partially abolished by La³+ (an inhibitor of plasma membrane Ca²+ channel) or catalase (CAT, a H₂O₂ scavenger), respectively. These results suggest that the plasma membrane Ca²+ channels and H₂O₂ possibly mediate extracellular Ca²+-blocked Na+ influx in GCPs. Furthermore, extracellular Ca²+ activated the plasma membrane Ca²+ channels under NaCl stress, which was partially abolished by CAT. These results, taken together, indicate that hydrogen peroxide (H₂O₂) likely regulates Na+ uptake by activating plasma membrane Ca²+ channels in GCPs. In accordance with this hypothesis, H₂O₂ could mimic extracellular Ca²+ to activate Ca²+ channels and block Na+ influx in guard cells. A single-cell analysis of cytosolic free Ca²+ ([Ca²+](cyt)) using Fluo 3-AM revealed that extracellular Ca²+ induced the accumulation of cytosolic Ca²+ under NaCl stress, but had few effects on the accumulation of cytosolic Ca²+ under non-NaCl conditions. All of these results, together with our previous studies showing that extracellular Ca²+ induced the generation of H₂O₂ in GCPs during NaCl stress, indicate that extracellular Ca²+ alleviates salt stress, likely by activating the H₂O₂-dependent plasma membrane Ca²+ channels, and the increase in cytosolic Ca²+ appears to block Na+ influx across the plasma membrane in Vicia guard cells, leading to stomatal closure and reduction of water loss.

Crosstalk of Nitric Oxide with Ca 2+ in Stomatal Movement in Vicia faba Guard Cells

For gaining further insights into Ca 2+ function in nitric oxide (NO)-regulated stomatal movement, Vicia faba guard cell protoplasts were patch-clamped in a whole-cell configuration. In the absence of extracellular Ca 2+, sodium nitroprusside (SNP, the NO donor) inhibited inward rectifying K + current at a concentration of 10 or 100 μmol L −1, but had little effect on outward rectifying K + current. SNP significantly activated outward rectifying K + current when CaCl 2 (0.1 mmol L −1) was added to the bath solution, and LaCl 3 could alleviate this effect of NO. In contrast, 0.1 mmol L −1 CaCl 2 alone had little effect on inward or outward rectifying K + current. Extracellular Ca 2+ significantly inhibited inward rectifying K + current and activated outward rectifying K + current at the concentration of 10 mmol L −1, and this effect was not alleviated by 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline (c-PTIO). When monitoring Ca 2+ and NO levels in single cells by Fluo 3-AM and 4,5-diaminofluorescein diacetate (DAF-2DA), the specific fluorescence probes for Ca 2+ and NO, respectively, SNP at 100 μmol L −1 evidently induced the accumulation of Ca 2+ in guard cells, and the induction could be partially alleviated by LaCl 3. However, CaCl 2 at 0.1 mmol L −1 or 10 mmol L −1 had small effect on the accumulation of NO in the guard cells. These results indicated that NO promoted influx of Ca 2+ into cytoplasm through Ca 2+ channels to activate outward rectifying K + channels and promotes K + efflux. Alternatively, NO inhibited inward rectifying K + channels and blocks K + influx, thus inhibiting stomatal opening and preventing excessive loss of water in plants. In addition, extracellular Ca 2+ at a concentration of 10 mmol L −1 modulated stomatal movement and plasma membrane K + channels of V. faba guard cells in a NO-independent signaling pathway.

Phosphorylation of the inward-rectifying potassium channel KAT1 by ABR kinase in Vicia guard cells.

A 48-kDa protein kinase was detected in Vicia faba guard cell protoplasts by an in-gel protein kinase assay using a recombinant peptide (KAT1C) of the carboxyl-terminus of an inward-rectifying voltage-dependent K+ channel cloned from Arabidopsis thaliana, KAT1. This protein kinase (ABR* kinase) was activated by pretreatment of guard cell protoplasts with ABA, but not by pretreatment with IAA, 2,4-D, kinetin or GA3. The activation of ABR* kinase was dependent on the time and concentration of ABA. The kinase activity was sensitive to staurosporine and K-252a, protein kinase inhibitors, and insensitive to Ca2+. No ABR* kinase activity was detected in mesophyll cell protoplasts. These characteristics of ABR* kinase are consistent with those of an ABA-responsive protein kinase (ABR kinase) reported previously [Mori and Muto (1997), Plant Physiol. 113: 833]. These results indicate that ABR* kinase phosphorylates the inward-rectifying K+ channel in response to treatment of stomatal guard cells with ABA. The data reported here provide evidence that this ABA-responsive protein kinase may promote ABA signaling by directly phosphorylating guard cell ion channels.

Regulating role of acetylcholine and its antagonists in inward rectified K+ channels from guard cell protoplasts ofVicia faba

The inward rectified potassium current of Vicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K(+) current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K(+) current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%. However, if guard cell protoplasts are treated with d-Tub and Atr together, the inward K(+) current is inhibited by 60% -75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K(+) channels has no effect on the inward K(+) current regulated by ACh, suggesting that there are inward K(+) channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.

Tonoplast inorganic pyrophosphatase in Vicia faba guard cells

Lysed guard-cell protoplasts of Vicia faba L. exhibited hydrolytic activity characteristic of tonoplast inorganic pyrophosphatase (V-PPase; EC 3.6.1.1). Activity was inhibited by the specific V-PPase inhibitor aminomethylenediphosphonate, stimulated by K+ (K m = 51 mM) and inhibited by Ca2+ (80 nM free Ca2+ was required for 50% inhibition at 0.27 mM free Mg2+). Patch-clamp measurements of electrogenic activity confirmed enzyme localisation at the tonoplast. This is the first report of V-PPase activity in guard cells; its possible involvement in stomatal opening is discussed.

Abscisic Acid Activates a 48-Kilodalton Protein Kinase in Guard Cell Protoplasts

A 49- and a 46-kD Ca2+-independent protein kinase and a 53-kD Ca2+-dependent protein kinase were detected in Vicia faba guard cell protoplasts (GCPs) by an in-gel protein kinase assay using myelin basic protein as a substrate. A 48-kD protein kinase designated as abscisic acid (ABA)-responsive protein kinase (ABR kinase) appeared when GCPs were treated with ABA. The activation of ABR kinase was suppressed by the protein kinase inhibitor staurosporine, indicating that a putative activator protein kinase phosphorylates and activates ABR kinase. The treatment of GCPs with 1,2-bis(o-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid, a calcium chelator, suppressed the activation of ABR kinase, suggesting that an influx of extracellular Ca2+ is required for the activation. Staurosporine and K-252a inhibited both the activity of ABR kinase and the stomatal closure induced by ABA treatment of V. faba epidermal peels. These results suggest that ABR kinase and its activator kinase may consist of a protein kinase cascade in a signal transduction pathway linking ABA perception to stomatal closure. The mobility of the 53-kD Ca2+-dependent protein kinase in sodium dodecyl sulfate-polyacrylamide gel was shifted upon Ca2+ binding to the enzyme, thus exhibiting the characteristics of a Ca2+-dependent or calmodulin-like domain protein kinase. This kinase may be the activator of ABR kinase.

Interconversion of fast and slow gating modes of GCAC1, a Guard Cell Anion Channel

For guard-cell protoplasts of Vicia faba L. we elucidated whether the slow (S-type) and rapid (R-type) activating anion channels represent different gating modes of GCAC1, the Guard Cell Anion Channel. In the whole-cell configuration of the patch-clamp technique, GCAC1 was activated in a Ca2+- and nucleotide-dependent manner. Cell-free outside-out membrane patches were isolated to determine the relative contribution of different gating modes or channel types to the overall anion current in this cell type. Within 2–15 min after the loss of cytoplasmic control through patch excision, depolarization-activated 38-pS channels characterized by flickering openings in the millisecond range convert into a channel of similar conductance but prolonged open times (hundreds of milliseconds) and lack of pronounced voltage-dependence. The rapid (R-type) and slow (S-type) gating modes exhibited similar ion selectivity but different susceptibility towards block by the stilbene derivative DNDS (4,4′-dinitrostilbene-2,2′-disulfonic acid). On R-type channels DNDS caused a flickering block and a shift in the threshold potential of activation whereas S-type channels remained unaffected. Because of the striking similarities of both channels with respect to single-channel conductance and relative permeability sequence on the one hand, but time-dependent conversion of R- into S-type gating after patch-excision on the other, we conclude that the mode of action of GCAC1 is under the control of cytoplasmic factors.

External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba.

Previous studies reveal that the pH of the apoplastic solution in the guard cell walls may vary between 7.2 and 5.1 in closed and open stomata, respectively. During these aperture and pH changes, massive K+ fluxes cross the cellular plasma membrane driving the osmotic turgor and volume changes of guard cells. Therefore, we examined the effect of extracellular pH on the depolarization-activated K channels (KD channels), which constitute the K+ efflux pathway, in the plasma membrane of Vicia faba guard cell protoplasts. We used patch clamp, both in whole cells as well as in excised outside-out membrane patches. Approximately 500 KD channels, at least, could be activated by depolarization in one protoplast (density: approximately 0.6 micron-2). Acidification from ph 8.1 to 4.4 decreased markedly the whole-cell conductance, GK, of the KD channels, shifted its voltage dependence, GK-EM, to the right on the voltage axis, slowed the rate of activation and increased the rate of deactivation, whereas the single channel conductance was not affected significantly. Based on the GK-EM shifts, the estimated average negative surface charge spacing near the KD channel is 39 A. To quantify the effects of protons on the rates of transitions between the hypothesized conformational states of the channels, we fitted the experimental macroscopic steady state conductance-voltage relationship and the voltage dependence of time constants of activation and deactivation, simultaneously, with a sequential three-state model CCO. In terms of this model, protonation affects the voltage-dependent properties via a decrease in localized, rather than homogeneous, surface charge sensed by the gating moieties. In terms of either the CO or CCO model, the protonation of a site with a pKa of 4.8 decreases the voltage-independent number of channels, N, that are available for activation by depolarization.

Regulation of blue-light-induced proton pumping by Vicia faba L. guard-cell protoplasts: Energetic contributions by chloroplastic and mitochondrial activities

An initial response during signal transduction in guard cells, following absorption of blue light, is the extrusion of protons. Translocation of protons across the guard-cell plasmalemma is an energy-requiring activity. The present study has investigated the energetic contribution from guard-cell chloroplasts and mitochondria to blue-light-induced proton pumping by Vicia faba guard-cell protoplasts. The addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea to the protoplast suspension had a minimal effect on rates of acidification when oxygen concentrations of the medium were maintained close to near-saturating levels. Under the same conditions, oligomycin reduced both the rates of blue-light-induced acidification and total proton efflux. Lowering the oxygen concentration of the suspending medium to approximately 20 μM resulted in complete inhibition of blue-light-induced acidification activity. Swelling of protoplasts induced by blue light was also inhibited by low oxygen levels. Levels of ATP from whole-protoplast extracts were reduced by about 64% when exposed to low levels of oxygen. Increasing oxygen levels to near-saturating levels restored both blue-light-induced acidification rates and swelling of the protoplasts within a 60-min recovery period. Levels of ATP also increased during the recovery period. Addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or oligomycin to the suspending medium prior to increasing the oxygen concentration caused a reduction in acidification rates after the recovery period by 40 and 80%, respectively. Levels of ATP in guard-cell protoplasts were also reduced by both inhibitors after a 60-min recovery period. The results demonstrate that both guard-cell chloroplasts and mitochondria contribute energetically to blue-light-induced proton pumping by guard-cell protoplasts. Furthermore, both energy sources are inhibited by low oxygen concentrations, suggesting coordinated metabolic regulation between photo- and oxidative phosphorylation in guard cells.

Hodgkin-Huxley analysis of whole-cell outward rectifying K+-currents in protoplasts from tobacco cell suspension cultures

The voltage and time dependence of outward-rectifying K+ currents (IK,out) measured in protoplasts from tobacco cell suspension cultures in the whole-cell configuration of the patch-clamp technique are quantitatively analyzed. The voltage and time dependence was described according to the Hodgkin and Huxley model for IK,out currents in the squid giant axon, and to allow comparison, in analogy with the quantitative analysis of IK,out currents in Vicia faba guard cell protoplasts as described by Schroeder (J. Membrane Biol., 107:229-235, 1989). The IK,out from tobacco could be described by a similar model as the IK,out from guard cell protoplasts (i.e., sigmoid activation time course, activation variable raised to second power, single exponential deactivating tail currents, absence of inactivation). However, in contrast to guard cells, both the activation and deactivation time constants were strongly voltage dependent in tobacco protoplasts. The voltage dependence of the transition rates for channel opening and channel closing was slightly asymmetrical and inverse to the asymmetry found in guard cells. The data presented show that the voltage-dependent kinetic properties of the IK,out conductance of tobacco protoplasts are different from these properties in guard cell protoplasts. This analysis provides a basis for the study of IK,out conductance function and modulation.

A slow anion channel in guard cells, activating at large hyperpolarization, may be principal for stomatal closing

Slowly activating anion channel currents were discovered at micromolar ‘cytoplasmic’ Ca 2+ during patch-clamp measurements on guard-cell protoplasts of Vicia faba and Xanthium strumarium. They activated at potentials as low as −200 Mv, with time constants between 5 and 60 s, and no inactivation. The broad voltage dependence exhibited a current maximum near −40 mV. The single-channel open time was in the order of seconds, and the unitary conductance was 33 ps, similar to that of the already described ‘quick’ anion channel of guard cells. Because of its activity at low potentials, the slow anion channel may be essential for the depolarization of the plasmalemma that is required for salt efflux during stomatal closing.

Stretch-activated chloride, potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L.

Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl-, K+ and Ca2+ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.

Diacylglycerols induce both ion pumping in patch-clamped guard-cell protoplasts and opening of intact stomata.

Stomatal guard cells in leaves regulate the apertures of microscopic pores through which photosynthetic gas exchange and water vapor loss occur. Environmental signals, including light, high humidity, and low CO2 concentrations, open stomata by increasing the volume of guard cells. Activation of a plasma membrane H+ pump initiates K+ and Cl- influx, accompanied by malate synthesis, resulting in osmotic water flow into the guard cells, a bowing apart of the guard-cell pair, and consequent stomatal opening. Physiological and electrophysiological techniques were employed to investigate the possibility that a second-messenger lipid, 1,2-diacylglycerol, is involved in the transduction of opening stimuli. The synthetic diacylglycerols 1,2-dihexanoylglycerol and 1,2-dioctanoylglycerol enhanced light-induced stomatal opening in Commelina communis and induced stomatal opening under darkness, whereas an isomer with no known second-messenger role, 1,3-dioctanoylglycerol, did not affect stomatal responses. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, the enzyme typically activated by 1,2-diacylglycerol in animal cells, inhibited light-stimulated stomatal opening and enhanced dark-induced stomatal closure. N-[(2-Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), which inhibits cyclic nucleotide-dependent protein kinases preferentially over lipid-dependent protein kinases such as protein kinase C, had little effect on stomatal apertures. Whole-cell patch clamping of guard-cell protoplasts of Vicia faba revealed that 1,2-dihexanoylglycerol and 1-oleoyl-2-acetylglycerol activated an ATP-dependent, voltage-independent current, suggesting activation of an electrogenic ion pump such as the H+ pump. Diacylglycerol or functionally similar lipids may act through protein phosphorylation to provide the intracellular signals that mediate H+-ATPase activation and stomatal opening in response to light or other opening stimuli.

Modulation of the activity of phosphoenolypyruvate carboxylase during potassium-induced swelling of guard-cell protoplasts of Vicia faba L. after light and dark treatments

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) activity was found to be modulated by light and darkness when measured in the presence of K(+), which had been added to induce swelling of guard-cell protoplasts (GCPs) from Vicia faba L., whereas no modulation was detected in the absence of K(+) (PEPcase activity remained constant at 1.5±0.15 pmol PEP metabolized · GCP(-1) ·h(-1); subsequently, pmol GCP(-1) ·h(-1) will be used). The activity of PEPCase increased by 100% (from 1.5 to 3 pmol·protoplast(-1)·h(-1)) in darkness and by 200% (from 1.7 to 5 pmol·protoplast(-1)· h(-1)) in light and oscillations in activity of these magnitudes were repeated at intervals of 2 min (dark) and 2.5 min (light) for a period of 10 min during K(+)-induced increase in the volume of GCPs. The oscillations were reflected in changes in malate-pool sizes determined in plastids, mitochondria and the supernatant fraction (consisting of the cytosol and the vacuole). Malate probably functioned as a mitochondrial substrate, thus supplying ATP for K(+) uptake and the swelling of the protoplasts. On the basis of the present paper and previous results (H. Schnabl and B. Michalke 1988, Life Sci. Adv. Plant Physiol. 7, 203-207) involving adenine nucleotidepool sizes in fractionated GCPs, a model is proposed to explain the cause-effect relationship between K(+), PEPCase, the cytosolic and mitochondrial malate levels and ATP levels during the K(+)-induced increase of GCP volume.

Voltage-dependent anion channels in the plasma membrane of guard cells

WHEN plants assimilate CO2 from the atmosphere, water vapour escapes. This exchange of gases can be moderated by the plant through volume changes of guard cells, which in pairs surround each stomatal pore in the epidermis and act as turgor-operated valves. When light intensity diminishes or water stress develops, guard cells shrink and stomata close. This process requires the release of ions from the guard cells. Voltage-dependent K+ chan-nels1,2 provide a path for cations. The required corresponding passage for anions, particularly chloride and malate3, is not known at the molecular level. We have applied the patch-clamp technique4 to guard-cell protoplasts of Vicia faba and found strongly voltage-dependent activities of single anion channels in the plasma mem-brane (plasmalemma). These channels have a conductance of 39 pS in 100 mM KC1 and are permeable for chloride and malate. They become active at membrane potentials positive to −80 mV. Their maximum activity occurs at −40 mV. The resulting outward Cl– currents of individual guard cells are at least 25 pA. These voltage–dependent anion channels provide a mechanism for the control of salt efflux from guard cells.

Red light stimulates an electrogenic proton pump in Vicia guard cell protoplasts.

Stomatal opening in response to light has a component that matches the absorption spectrum of chlorophyll; however, the intervening sensory transduction steps are not well understood. To study this process, we illuminated Vicia faba guard cell protoplasts with red light and simultaneously recorded current flow across the plasma membrane, utilizing the patch clamp technique in the whole cell configuration. We report evidence that under voltage clamp conditions, red light (1 mmol of photons.m-2.S-1) stimulated an outward current. This response required ATP (2.5 mM) and orthophosphate (1 mM) at the cytoplasmic side of the membrane. Both red-light-stimulated currents and currents activated in the dark by the proton pump agonist fusicoccin (10 microM) were abolished by the protonophore carbonylcyanide m-chlorophenylhydrazone at 10 microM, indicating that these responses were carried by protons. Pump currents were inhibited by orthovanadate applied to the cytoplasmic side of the membrane (50% inhibition at 3.5 microM), implicating a H+ -ATPase. Elimination of the current by the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, in the presence of saturating concentrations of ATP, pointed to a requirement for photosynthetically active chloroplasts. We conclude that red light stimulates an electrogenic proton pump at the plasmalemma of Vicia guard cells and that chloroplasts modulate this response.

Effects of abscisic acid on K + channels in [formula omitted] guard cell protoplasts

Potassium channels were resolved in Vicia faba guard cell protoplasts by patch voltage-clamp. Whole-cell currents and single K + channels had linear instantaneous current-voltage relations, reversing at the calculated Nernst potential for K +. Whole cell K + currents activated exponentially during step depolarizations, with half-activation times of 400–450 msec at +80 mV and 90–110 msec at +150 mV. Single K + channel conductance was 65 ± 5 pS with a mean open time of 1.25 ± 0.30 msec at 150 mV. Potassium channels were blocked by internal Cs + and by external TEA +, but they were insensitive to external 4-aminopyridine. Application of 10 uM abscisic acid increased mean open time and caused long-lasting bursts of channel openings. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology.

Blue light-dependent proton extrusion by guard-cell protoplasts of Vicia faba

In plant leaves, light regulation of stomatal movement, and hence of gas exchange1, involves a non-photosynthetic photosensory system sensitive to blue light2–6, which transduces many other plant movement responses. The exact nature of the primary photo-reactions of this system is still not known7. Stomatal opening is, in general, based on swelling of the guard cells driven by ion uptake1, and it has been suggested that energy for such uptake may be provided by a chemi-osmotic extrusion of protons across the plasmalemma1,8,9. We report here that guard-cell protoplasts from Vicia faba, irradiated continuously with photosynthetically saturating, high-fluence-rate red light, extrude H+ ions in response to a short pulse (30s) of blue light. The response is relatively long-lived, such that a high rate of H+ extrusion continues for several minutes after the pulse stimulation, indicating that continuous input of blue light energy is not required for a sustained response. The kinetic relation between pulse input and response output matched closely that found for the stomatal response in intact leaves6. In agreement with the chemi-osmotic hypothesis, activation of an electrogenic plasmalemma H+-ATPase is postulated as the cause of blue light-induced H+ extrusion.

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K(+) concentration within 2-3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K(+) ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the Km(PEP) and Vmax values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.

Effect of Fusicoccin on Dark 14CO2 Fixation by Vicia faba Guard Cell Protoplasts

When Vicia faba guard cell protoplasts were treated with fusicoccin, dark (14)CO(2) fixation rates increased by as much as 8-fold. Rate increase was saturated with less than 1 micromolar fusicoccin. Even after 6 minutes of dark (14)CO(2) fixation, more than 95% of the incorporated radioactivity was in stable products derived from carboxylation of phosphoenolpyruvate (about 50% and 30% in malate and aspartate, respectively). The relative distribution of (14)C among products and in the C-4 position of malate (initially more than 90% of [(14)C]malate) was independent of fusicoccin concentration. After incubation in the dark, malate content was higher in protoplasts treated with fusicoccin. A positive correlation was observed between the amounts of (14)CO(2) fixed and malate content.It was concluded that (a) fusicoccin causes an increase in the rate of dark (14)CO(2) fixation without alteration of the relative fluxes through pathways by which it is metabolized, (b) fusicoccin causes an increase in malate synthesis, and (c) dark (14)CO(2) fixation and malate synthesis are mediated by phosphoenolpyruvate carboxylase.