Mechanisms of heterosynaptic facilitation in molluscan neurons

This review is focused on the analysis of research data obtained in one of the models of conditioned reflex, heterosynaptic facilitation (HSF), in the molluscan nervous system. Our experiments were performed on identified giant command neurons LS1 and PS1 of the freshwater snail Planorbarius corneus. HSF was elicited during the electrical stimulation of two nerves: pallial (the analog of unconditioned stimulation — US) and one of the cerebral nerves (the analog of the conditioned stimulation — CS). The degree of HSF manifestation depended not on the intensity of the synaptic response of the giant neuron to US, but the efficacy of the connection between the pallial nerve and neurosecretory neurons surrounding the command neuron of the mesocerebrum. It is demonstrated that HSF develops due to the diffuse neurohumoral action of serotonin (5-hydroxytryptamine — 5-HT) on the postsynaptic structures, but not as a result of local synaptic action on the presynaptic mechanism. Approximately 70% of US cases of 5-HT application induced a four- to six-fold increase in amplitude of the excitatory postsynaptic potential (EPSP) and acetylcholine (ACh) response. Both responses are N-cholinergic and depend on the membrane permeability to Na⁺ and K⁺. In 30% of the cases, ACh response diminished simultaneously with EPSP increase. The 5-HT effect on EPSP and ACh responses were mimicked by the action of phosphodiersterase blockers and adenylate cyclase activators. Thus, the activation of the adenylate cyclase system following 5-HT action facilitates the postsynaptic mechanism underlying HSF formation in command neurons of Planorbarius corneus. Dopamine (DA) and noradrenaline (NA) blocked EPSP and simultaneously increased the amplitude of ACh response. These monoamines were also blocked HSF. The wash-out of catecholamines following HSF blockade enhanced the restoration and subsequent prolongation of synaptic facilitation. It is thus concluded that DA or NA may control the HSF intensity and duration under natural conditions of the nervous system in the molluscs.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 224–232, May–June, 1993.     

Conformational behavior of [Trp4, Met5]-enkephalin: comparison of fluorescence parameters at different Ph.

The conformational behavior of the biologically active [Trp⁴,Met⁵]-enkephalin was elucidated by evaluation of intramolecular energy transfer between Tyr¹ and Trp⁴. Identical transfer efficiencies and tyrosine fluorescence quantum yields were observed in aqueous solution at pH 1.5 and 5.5 and the use of these parameters in Förster's equation resulted in the same average Tyr-Trp separation (9.3 Å) under these two conditions. The invariability of these sensitive parameters indicates the existence of very similar types of a folded conformation in the cationic and zwitterionic form of the analog at low concentrations.     

Effects of Lead Ions on the Synaptic Responses of Neurons of the Rat Sympathetic Ganglion

Using intracellular recording, we studied the effects of lead ions on the synaptic responses of neurons of the superior cervical ganglion (SCG) of the rat. Three groups of SCG neurons were found; they differed from each other in directions of the changes evoked by Pb²⁺ applications and in their sensitivity to this agent. In two groups of the phasic SCG neurons, Pb²⁺ decreased the amplitude of EPSP evoked by stimulation of the cervical sympathetic nerve with EC₅₀ of (2.2 ± 0.2) · 10^-6 M and (3.55 ± 0.29) · 10^-5 M, respectively. In the tonic neurons, 2 · 10^-6 M Pb²⁺ increased the EPSP amplitude by 27 ± 2%, on average, as compared with the control values. We postulate that the specificity of responses of these three groups of SCG neurons to the action of Pb²⁺ is determined by the different subunit composition of the nicotinic cholinoreceptors in the membrane of these cells.     

A small post-translocation energy bias aids nucleotide selection in T7 RNA polymerase transcription

The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA to RNA in the absence of additional protein factors. In this work, we present a model of T7 RNAP translocation during elongation. Based on structural information and experimental data from single-molecule force measurements, we show that a small component of facilitated translocation or power stroke coexists with the Brownian-ratchet-driven motions, and plays a crucial role in nucleotide selection at pre-insertion. The facilitated translocation is carried out by the conserved Tyr⁶³⁹ that moves its side chain into the active site, pushing aside the 3′-end of the RNA, and forming a locally stabilized post-translocation intermediate. Pre-insertion of an incoming nucleotide into this stabilized intermediate state ensures that Tyr⁶³⁹ closely participates in selecting correct nucleotides. A similar translocation mechanism has been suggested for multi-subunit RNAPs involving the bridge-helix bending. Nevertheless, the bent bridge-helix sterically prohibits nucleotide binding in the post-transolocation intermediate analog; moreover, the analog is not stabilized unless an inhibitory protein factor binds to the enzyme. Using our scheme, we also compared the efficiencies of different strategies for nucleotide selection, and examined effects of facilitated translocation on forward tracking.     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors

The role of muscarinic receptors in the down‐regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10⁻⁶ M) and atropine (10⁻⁸ to 10⁻⁶ M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10⁻⁶ M) caused a slight depression in the amplitude of responses to ACh pressure‐applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA‐immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10⁻⁶ M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10⁻⁶ M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10⁻⁶ M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 420–431, 1999     

M-channels modulate the intrinsic excitability and synaptic responses of layer 2/3 pyramidal neurons in auditory cortex

Neurons in the auditory cortex are believed to utilize temporal patterns of neural activity to accurately process auditory information but the intrinsic neuronal mechanism underlying the control of auditory neural activity is not known. The slowly activating, persistent K⁺ channel, also called M-channel that belongs to the Kv7 family, is already known to be important in regulating subthreshold neural excitability and synaptic summation in neocortical and hippocampal pyramidal neurons. However, its functional role in the primary auditory cortex (A1) has never been characterized. In this study, we investigated the roles of M-channels on neuronal excitability, short-term plasticity, and synaptic summation of A1 layer 2/3 regular spiking pyramidal neurons with whole-cell current-clamp recordings in vitro. We found that blocking M-channels with a selective M-channel blocker, XE991, significantly increased neural excitability of A1 layer 2/3 pyramidal neurons. Furthermore, M-channels controled synaptic responses of intralaminar-evoked excitatory postsynaptic potentials (EPSPs); XE991 significantly increased EPSP amplitude, decreased the rate of short-term depression, and increased the synaptic summation. These results suggest that M-channels are involved in controlling spike output patterns and synaptic responses of A1 layer 2/3 pyramidal neurons, which would have important implications in auditory information processing.     

Physiological properties of Cantor coding-like iterated function system in the hippocampal CA1 network

Cantor coding provides an information coding scheme for temporal sequences of events. In the hippocampal CA3–CA1 network, Cantor coding-like mechanism was observed in pyramidal neurons and the relationship between input pattern and recorded responses could be described as an iterated function system. However, detailed physiological properties of the system in CA1 remain unclear. Here, we performed a detailed analysis of the properties of the system related to the physiological basis of learning and memory. First, we investigated whether the system could be simply based on a series of on–off responses of excitatory postsynaptic potential (EPSP) amplitudes. We applied a series of three spatially distinct input patterns with similar EPSP peak amplitudes. The membrane responses showed significant differences in spatial clustering properties related to the iterated function system. These results suggest that existence of some factors, which do not simply depend on a series of on–off responses but on spatial patterns in the system. Second, to confirm whether the system is dependent on the interval of sequential input, we applied spatiotemporal sequential inputs at several intervals. The optimal interval was 30 ms, similar to the physiological input from CA3 to CA1. Third, we analyzed the inhibitory network dependency of the system. After GABA_A receptor blocker (gabazine) application, quality of code discrimination in the system was lower under subthreshold conditions and higher under suprathreshold conditions. These results suggest that the inhibitory network increase the difference between the responses under sub- and suprathreshold conditions. In summary, Cantor coding-like iterated function system appears to be suitable for information expression in relation to learning and memory in CA1 network.     

Postsynaptic activity of spinal motoneurons of early postnatal ratsin vitro: Effects of calcium channel blockers

Postsynaptic activity was intracellularly recorded from motoneurons in slices of the spinal cord of early postnatal rats. The amplitude of background EPSP varied within 0.4 to 4.0 mV (a mean of 2.05 ± 0.18 mV); their mean frequency was 11.5 ± 4.9 sec⁻¹. Distributions of the EPSP amplitudes In different cells were unimodal and showed a clear right-side asymmetry. Application of calcium channel blockers (1–2 μM nifedipine, 50–100 μM Cd²⁺, or 500 μM amyloride) considerably modified the EPSP parameters. Nifedipine (3 cells) somewhat enhanced the amplitude of background EPSP (to 108.3 ± 6.6%, on the average) and significantly Increased their frequency (145.6 ± 14.4%). Cadmium (3 cells) and amyloride (7 cells) decreased both the EPSP amplitude (means of 88.9 ±2.8% and 73.1 ± 3.8%, respectively) and their frequency (69.0 ± 5.6% and 61.4 ± 7.8%). All tested agents evoked no dramatic shifts of the membrane potential of motoneurons. It is concluded that the observed modifications of the EPSP amplitudes and frequencies result from modulation of the activity of both pre- and post-synaptically localized high- and low-threshold Ca²⁺ channels. The reason for the opposite direction of nifedipine effects, as compared with those of Cd²⁺ and amyloride, is discussed.     

The mode of action of phenylhydrazine hydrochloride on the locust neuromuscular system

The action of the carbonyl reagent phenylhydrazine hydrochloride (Phen. HCl) on locust excitatory neuromuscular systems was studied by examining the effects of this compound on the mechanical and electrical properties of the retractor unguis and extensor tibiae muscles of the locust Schistocerca gregaria.Low concentrations of Phen. HCl (10^?9 w/v to 2·5 × 10^?5 w/v) potentiated the muscle contractions and the excitatory post-synaptic potential (EPSP), the optimum concentration being about 10^?5 w/v. 10^?8 w/v Phen. HCl increased miniature EPSP frequency, but this increase became less pronounced as the concentration was raised, and no increase at all was observed at 10^?5 w/v. There was no change in miniature EPSP amplitude at any concentration. Higher concentrations of Phen. HCl (> 2·5 × 10^?6 w/v) depressed the neurally evoked contraction, the EPSP, and the response of the muscle to iontophoretically applied l-glutamate. A gradual increase in muscle input conductance was observed on perfusion with these high concentrations of Phen. HCl. The presence of magnesium in the bathing fluid (15 m-moles/l.) reduced the effectiveness of Phen. HCl in potentiating the EPSP and delayed or reduced the increase in input conductance observed on perfusion with high concentrations of Phen. HCl.The results indicate that low concentrations of Phen. HCl act presynaptically, possibly by depolarizing the excitatory nerve terminals. Higher concentrations may act directly on the post-synaptic glutamate receptors.     

Impact of nozzle operation on mass transfer in jet aerated loop reactors. Characterization and comparison to an aerated stirred tank reactor

The impact of mass transfer on productivity can become a crucial aspect in the fermentative production of bulk chemicals. For highly aerobic bioprocesses the oxygen transfer rate (OTR) and productivity are coupled. The achievable space time yields can often be correlated to the mass transfer performance of the respective bioreactor. The oxygen mass transfer capability of a jet aerated loop reactor is discussed in terms of the volumetric oxygen mass transfer coefficient k_La [h^?1] and the energetic oxygen transfer efficiency E [kgO₂ kW^?1 h^?1]. The jet aerated loop reactor (JLR) is compared to the frequently deployed aerated stirred tank reactor. In jet aerated reactors high local power densities in the mixing zone allow higher mass transfer rates, compared to aerated stirred tank reactors. When both reactors are operated at identical volumetric power input and aeration rates, local k_La values up to 1.5 times higher are possible with the JLR. High dispersion efficiencies in the JLR can be maintained even if the nozzle is supplied with pressurized gas. For increased oxygen demands (above 120 mmol L^?1 h^?1) improved energetic oxygen transfer efficiencies of up to 100 % were found for a JLR compared to an aerated stirred tank reactor operating with Rushton turbines.     

Effect of Genomic Location on Horizontal Transfer of a Recombinant Gene Cassette Between Pseudomonas Strains in the Rhizosphere and Spermosphere of Barley Seedlings

The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria. However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids. To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 × 10⁻³ transconjugants/(donors × recipients)^1/2 in the rhizosphere and 4.57 × 10⁻² transconjugants/(donors × recipients)^1/2 in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 × 10⁻³ transconjugants/(donors × recipients)^1/2. Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation. However, transformation did occur by using the same bacterial strains under laboratory conditions. The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level. Received: 21 July 2000 / Accepted: 21 August 2000     

Identification and Application of Plasmids Suitable for Transfer of Foreign DNA to Members of the Genus Gordonia

Gene transfer systems for Gordonia polyisoprenivorans strains VH2 and Y2K based on electroporation and conjugation, respectively, were established. Several parameters were optimized, resulting in transformation efficiencies of >4 × 10⁵ CFU/μg of plasmid DNA. In contrast to most previously described electroporation protocols, the highest efficiencies were obtained by applying a heat shock after the intrinsic electroporation. Under these conditions, transfer and autonomous replication of plasmid pNC9503 was also demonstrated to proceed in G. alkanivorans DSM44187, G. nitida DSM44499^T, G. rubropertincta DSM43197^T, G. rubropertincta DSM46038, and G. terrae DSM43249^T. Conjugational plasmid DNA transfer to G. polyisoprenivorans resulted in transfer frequencies of up to 5 × 10⁻⁶ of the recipient cells. Recombinant strains capable of polyhydroxyalkanoate synthesis from alkanes were constructed.     

The Kinetic Mechanism of 5-Enolpyruvylshikimate-3-Phosphate Synthase from a Gram-Positive Pathogen Streptococcus Pneumoniae

Abstract

The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpqruvyl group from phospho(enol)pqruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may hate potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction. GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P. suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction. GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might he formed between the enzyme. EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.     

Efficient resonance assignment of proteins in MAS NMR by simultaneous intra- and inter-residue 3D correlation spectroscopy

Resonance assignment is the first step in NMR structure determination. For magic angle spinning NMR, this is typically achieved with a set of heteronuclear correlation experiments (NCaCX, NCOCX, CONCa) that utilize SPECIFIC-CP ¹⁵N–¹³C transfers. However, the SPECIFIC-CP transfer efficiency is often compromised by molecular dynamics and probe performance. Here we show that one-bond ZF-TEDOR ¹⁵N–¹³C transfers provide simultaneous NCO and NCa correlations with at least as much sensitivity as SPECIFIC-CP for some non-crystalline samples. Furthermore, a 3D ZF-TEDOR-CC experiment provides heteronuclear sidechain correlations and robustness with respect to proton decoupling and radiofrequency power instabilities. We demonstrate transfer efficiencies and connectivities by application of 3D ZF-TEDOR-DARR to a model microcrystalline protein, GB1, and a less ideal system, GvpA in intact gas vesicles.     

Yield gaps, nutrient use efficiencies and response to fertilisers by maize across heterogeneous smallholder farms of western Kenya

The need to promote fertiliser use by African smallholder farmers to counteract the current decline in per capita food production is widely recognised. But soil heterogeneity results in variable responses of crops to fertilisers within single farms. We used existing databases on maize production under farmer (F-M) and researcher management (R-M) to analyse the effect of soil heterogeneity on the different components of nutrient use efficiency by maize growing on smallholder farms in western Kenya: nutrient availability, capture and conversion efficiencies and crop biomass partitioning. Subsequently, we used the simple model QUEFTS to calculate nutrient recovery efficiencies from the R-M plots and to calculate attainable yields with and without fertilisers based on measured soil properties across heterogeneous farms. The yield gap of maize between F-M and R-M varied from 0.5 to 3 t grain ha^?1 season^?1 across field types and localities. Poor fields under R-M yielded better than F-M, even without fertilisers. Such differences, of up to 1.1 t ha^?1 greater yields under R-M conditions are attributable to improved agronomic management and germplasm. The relative response of maize to N–P–K fertilisers tended to decrease with increasing soil quality (soil C and extractable P), from a maximum of 4.4-fold to ?0.5-fold relative to the control. Soil heterogeneity affected resource use efficiencies mainly through effects on the efficiency of resource capture. Apparent recovery efficiencies varied between 0 and 70% for N, 0 and 15% for P, and 0 to 52% for K. Resource conversion efficiencies were less variable across fields and localities, with average values of 97 kg DM kg^?1 N, 558 kg DM kg^?1 P and 111 kg DM kg^?1 K taken up. Using measured soil chemical properties QUEFTS over-estimated observed yields under F-M, indicating that variable crop performance within and across farms cannot be ascribed solely to soil nutrient availability. For the R-M plots QUEFTS predicted positive crop responses to application of 30 kg P ha^?1 and 30 kg P ha^?1 + 90 kg N ha^?1 for a wide range of soil qualities, indicating that there is room to improve current crop productivity through fertiliser use. To ensure their efficient use in sub-Saharan Africa mineral fertilisers should be: (1) targeted to specific niches of soil fertility within heterogeneous farms; and (2) go hand-in-hand with the implementation of agronomic measures to improve their capture and utilisation.     

Input integration around the dendritic branches in hippocampal dentate granule cells

Recent studies have shown that the dendrites of several neurons are not simple translators but are crucial facilitators of excitatory postsynaptic potential (EPSP) propagation and summation of synaptic inputs to compensate for inherent voltage attenuation. Granule cells (GCs)are located at the gateway for valuable information arriving at the hippocampus from the entorhinal cortex. However, the underlying mechanisms of information integration along the dendrites of GCs in the hippocampus are still unclear. In this study, we investigated the input integration around dendritic branches of GCs in the rat hippocampus. We applied differential spatiotemporal stimulations to the dendrites using a high-speed glutamate-uncaging laser. Our results showed that when two sites close to and equidistant from a branching point were simultaneously stimulated, a nonlinear summation of EPSPs was observed at the soma. In addition, nonlinear summation (facilitation) depended on the stimulus location and was significantly blocked by the application of a voltage-dependent Ca²⁺ channel antagonist. These findings suggest that the nonlinear summation of EPSPs around the dendritic branches of hippocampal GCs is a result of voltage-dependent Ca²⁺ channel activation and may play a crucial role in the integration of input information.     

EPSP synthase inhibitor deisgn III. Synthesis & evaluation of a new 5-oxamic acid analog of EPSP which incorporates a malonate ether as a 3-phosphate mimic

The unusual shikimate oxamic acid analog 3, containing a 3-malonate ether, was synthesized and found to be a potent product-analog inhibitor of EPSP synthase (K_i = 5.2 − 0.2 μM). The potency of 3 confirms that these 3-malonate ethers function as effective 3-phosphate replacements in this system and that the oxamic acid moiety represents an unusual mimetic of the EPSP carboxyvinyl ether group.     

The Mechanism of Inactivation of T4 Bacteriophage by Tritium Decay

Coliphage T4 was used as a model system to study the mechanism of biological inactivation produced by tritium decay. Experimentally, tritiated precursors were incorporated into phage DNA (thymidine-³H) or into phage protein (³H-amino acids). The ratio of killing efficiencies for decays originating in phage DNA to those originating in phage protein was 2.6. Inactivation by decays from labeled amino acids was assumed to occur exclusively from β-particle irradiation of phage DNA. If decays originating in DNA are due solely to irradiation of DNA, then the killing efficiencies reflect the energy transfer paths in phage DNA for decays originating in phage DNA and in the protein coat. The energy transfer paths were determined for the two cases with the help of a computer and found to be very nearly equal to the experimentally determined ratio (2.6). The killing efficiencies for decays originating in phage DNA were 0.12 and for decays originating in protein 0.046.     

Proton pumping by tomato roots. Effect of Fe deficiency and hormones on the activity and distribution of plasma membrane H+-ATPase in rhizodermal cells

The immunocytochemical localization of the plasma membrane H⁺‐ATPase in epidermal cells of tomato roots was studied using a monoclonal antibody raised against purified maize P‐type H⁺‐ATPase. Plants subjected to iron starvation exhibited increased proton extrusion that was confined to the root elongation zones. Immunogold labelling of the H⁺‐ATPase on the plasma membrane was considerably higher in rhizodermal cells within zones with intense proton extrusion than in non‐acidifying areas of the roots. Transfer cells were formed in rhizodermal cells of Fe‐deficient plants. Quantitative determination of immunolabelling revealed that the density of PM H⁺‐ATPase in transfer cells was about twice that of ordinary epidermal cells. In transfer cells, H⁺‐ATPase was most abundant on the plasma membrane lining the labyrinthine invaginations of the peripheral cell wall. While the number of immunologically detectable ATPase molecules in transfer cells was not spatially correlated with proton extrusion activity, the frequency of transfer cells was considerably higher in acidifying root areas relative to non‐active segments. Split‐root experiments indicated that both the steady‐state level of plasma membrane H⁺‐ATPase and proton extrusion activity are systemically regulated, indicating inter‐organ regulation of rhizosphere acidification. Exogenous application of the auxin analog 2,4‐dichlorophenoxyacetic acid and the ethylene precursor 1‐aminocyclopropane‐1‐carboxlic acid caused the formation of transfer cells at a frequency similar to that observed in Fe‐deficient roots. However, the number of proton pumps was not affected by the hormone treatment, suggesting that both responses are regulated independently. It is concluded that transfer cells in the rhizodermis may be important but not crucial for rhizosphere acidification.