Nucleotides provide a voltage-sensitive gate for the rapid anion channel of arabidopsis hypocotyl cells.

The rapid anion channel of Arabidopsis hypocotyl cells is highly voltage-dependent. At hyperpolarized potentials, the channel is closed, and membrane depolarization is required for channel activation. We have previously shown that channel gating is regulated by intracellular nucleotides. In the present study, we further analyze the channel gating, and we propose a mechanism to explain its regulation by voltage. In the absence of intracellular nucleotides, closure at hyperpolarized voltages is abolished. Structure-function studies of adenyl nucleotides show that the apparent gating charge of the current increases with the negative charge carried by nucleotides. We propose that the fast anion channel is gated by the voltage-dependent entry of free nucleotides into the pore, leading to a voltage-dependent block at hyperpolarized potentials. In agreement with this mechanism in which intracellular nucleotides need to be recruited to the channel pore, kinetic analyses of whole-cell and single-channel currents show that the rate of closure is faster when intracellular nucleotide concentration is increased, whereas the rate of channel activation is unchanged. Furthermore, decreasing the concentration of extracellular chloride enhances the intracellular nucleotide block. This result supports the hypothesis of a mechanism in which blocking nucleotides and permeant anions interact within the channel pore.

Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata.

Oligomeric degradation products of alginate elicited a respiratory and oxidative burst in the sporophytes of the kelp Laminaria digitata. The generation of activated oxygen species (AOS), O(2)(-), and H(2)O(2) was detected at the single cell level, using nitroblue tetrazolium precipitation and a redox-sensitive fluorescent probe, respectively. The oxidative burst involved diphenyleneiodonium-sensitive AOS-generating machinery and its amplitude depended on the type of tissue. After a first elicitation plants were desensitized for about 3 h. The activity of alginate oligosaccharides was dose dependent, saturating around 40 microM. It was also structure-dependent, with homopolymeric blocks of alpha-1,4-L-guluronic acid, i.e. the functional analogs of oligogalacturonic blocks in pectins, being the most active signals. The perception of oligoguluronate signals resulted in a strong efflux of potassium. Pharmacological dissection of the early events preceding the emission of AOS indicated that the transduction chain of oligoguluronate signals in L. digitata is likely to feature protein kinases, phospholipase A(2), as well as K(+), Ca(2+), and anion channels.

Characterization of a nitrate-permeable channel able to mediate sustained anion efflux in hypocotyl cells from Arabidopsis thaliana.

We have characterized a new anionic current in Arabidopsis hypocotyl cells. This current, activated by membrane depolarization, has slow activation and deactivation kinetics in the 10 sec range. It presents many distinct properties from the rapid-type anion current already described on the same membrane. The slow-type channel is highly permeable to nitrate with a PNO3-/PCl- close to 20, but totally impermeable to sulphate. Activation of the channel requires cytosolic ATP and the slow current is partially inhibited by staurosporin, suggesting that channel regulation involves protein phosphorylation. The slow anion channel displays a unique pharmacological profile different from that of the rapid channel: the slow channel is inhibited by DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid) with an IC50 of 26 microM. The slow and rapid anion channels are probably dedicated to specific functions: the first is able to mediate sustained anion efflux, while the second is a good candidate to be involved in fast electrical signalling.

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells.

Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [14C]2,4-D at concentrations of less than 10 microM. Most failed to interfere with [3H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed.

Sulfate is both a substrate and an activator of the voltage-dependent anion channel of Arabidopsis hypocotyl cells.

On the basis of the anion content of in vitro-cultured Arabidopsis plantlets, we explored the selectivity of the voltage-dependent anion channel of the plasma membrane of hypocotyl cells. In the whole-cell configuration, substitution of cytosolic Cl(-) by different anions led to the following sequence of relative permeabilities: NO(3)(-) (2.6) >/= SO(4)(2-) (2.0) > Cl(-) (1.0) > HCO(3)(-) (0.8) >> malate(2-) (0.03). Large whole-cell currents were measured for NO(3)(-) and SO(4)(2-), about five to six times higher than the equivalent Cl(-) currents. Since SO(4)(2-) is usually considered to be a weakly permeant or non-permeant ion, the components of the large whole-cell current were explored in more detail. Aside from its permeation through the channel with a unitary conductance, about two-thirds that of Cl(-), SO(4)(2-) had a regulatory effect on channel activity by preventing the run-down of the anion current both in the whole-cell and the outside-out configuration, increasing markedly the whole-cell current. The fact that the voltage-dependent plasma membrane anion channel of hypocotyl cells can mediate large NO(3)(-) and SO(4)(2-) currents and is regulated by nucleotides favors the idea that this anion channel can contribute to the cellular homeostasis of important metabolized anions.

Biological activity of reducing-end-derivatized oligogalacturonides in tobacco tissue cultures.

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspension-cultured N. tabacum cv Xanthi cells, and (d) elicit H2O2 accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [14C]2,4-dichlorophenoxyacetic acid and [3H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [3H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [14C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.

Anion-channel blockers interfere with auxin responses in dark-grown Arabidopsis hypocotyls.

Anion channels are thought to participate in signal transduction and turgor regulation in higher plant cells. The regulation of hypocotyl cell elongation is a situation in which these channels could play important roles because it involves ionic fluxes that are implicated in turgor control and orchestrated by various signals. We have used a pharmacological approach to reveal the contribution of anion channels in the regulation of the development of hypocotyls by auxins. Auxins induce an inhibition of elongation, a disintegration of the cortical cell layers, and the formation of adventitious roots on Arabidopsis thaliana hypocotyls grown in the dark. Anion-channel blockers such as anthracene-9-carboxylic acid, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid, 4-acetamido-4'-isothiocyanato-stilbene-2-2'-disulfonic acid, and R(+)-methylindazone; indanyloxyacteic acid-94, which produce little or no stimulation of hypocotyl elongation by themselves, are able to counteract the inhibition and the disintegration induced by auxins with various efficiencies. This interference appears to be specific for auxins and does not occur when hypocotyl elongation is inhibited by other growth regulators such as ethylene or cytokinins. The putative involvement of anion channels in auxin signal transduction is discussed.

Voltage-dependent anion channel of Arabidopsis hypocotyls: nucleotide regulation and pharmacological properties.

Plasma membrane anion channels are thought to play important roles in osmoregulation and signal transduction in higher plant cells. Knowledge of their pharmacology and regulation is of importance to unravel their physiological functions. In this study, we explore the pharmacological properties and the nucleotide regulation of the voltage-dependent anion channel of Arabidopsis hypocotyls. The pharmacological profile of this channel is characterized by a low sensitivity to most anion channel blockers. It is inhibited by niflumic acid with an IC50 of 80 microM, but poorly sensitive to IAA-94 and NPPB and insensitive to 9-AC and DIDS. Nucleotides alter the amplitude, the kinetics and the voltage-dependence of the channel. The main effect of nucleotides is a shift of the voltage-dependent gate of the channel toward depolarized potentials leading to a strong reduction of the current amplitude. This regulation does not require ATP hydrolysis as nonhydrolyzable ATP analogues-AMPPNP and ATP gamma S-also regulate the anion current. This suggests that a nucleotide binding site is involved in the regulation. The study of the properties of this putative nucleotide binding site reveals that (i) ATP regulates the channel with an EC50 of 0.7 mM, (ii) adenyl nucleotides modulate the channel with the following order of effectiveness: ATP > ADP > > AMP, and (iii) thiophosphate nucleotide analogues are the most potent agonists with EC50 in the range of 80 microM. The hypothesis that this regulation may couple the electrical properties of the membrane with the metabolic status of the cell is discussed.

Purified vesicles of tobacco cell vacuolar and plasma membranes exhibit dramatically different water permeability and water channel activity.

The vacuolar membrane or tonoplast (TP) and the plasma membrane (PM) of tobacco suspension cells were purified by free-flow electrophoresis (FFE) and aqueous two-phase partitioning, with enrichment factors from a crude microsomal fraction of >/=4- to 5-fold and reduced contamination by other cellular membranes. For each purified fraction, the mean apparent diameter of membrane vesicles was determined by freeze-fracture electron microscopy, and the osmotic shrinking kinetics of the vesicles were characterized by stopped-flow light scattering. Osmotic water permeability coefficients (Pf) of 6.1 +/- 0.2 and 7.6 +/- 0.9 microm . s(-1) were deduced for PM-enriched vesicles purified by FFE and phase partitioning, respectively. The associated activation energies (Ea; 13.7 +/- 1.0 and 13.4 +/- 1.4 kcal . mol(-1), respectively) suggest that water transport in the purified PM occurs mostly by diffusion across the lipid matrix. In contrast, water transport in TP vesicles purified by FFE was characterized by (i) a 100-fold higher Pf of 690 +/- 35 microm . s(-1), (ii) a reduced Ea of 2.5 +/- 1.3 kcal . mol(-1), and (iii) a reversible inhibition by mercuric chloride, up to 83% at 1 mM. These results provide functional evidence for channel-mediated water transport in the TP, and more generally in a higher plant membrane. A high TP Pf suggests a role for the vacuole in buffering osmotic fluctuations occurring in the cytoplasm. Thus, the differential water permeabilities and water channel activities observed in the tobacco TP and PM point to an original osmoregulatory function for water channels in relation to the typical compartmentation of plant cells.

Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense.

Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley.

Identification of calreticulin-like protein as one of the phosphoproteins modulated in response to oligogalacturonides in tobacco cells.

Oligogalacturonide-induced modifications of protein phosphorylation in cells of Nicotiana tabacum L. were investigated by in-vitro phosphorylation of plasma-membrane-enriched fractions and electrophoretic analysis on two-dimensional gels. About 100 polypeptides were resolved; among these 40 phosphoproteins were detected and their 33P-labelling quantified. Most of the phosphorylations were inhibited by staurosporine and several proteins were hyperphosphorylated in the presence of okadaic acid, indicating the presence of protein phosphatase(s) in addition to staurosporine-susceptible protein kinase(s) in the plasma-membrane-enriched fraction. In the presence of oligogalacturonides, phosphorylation of seven acidic polypeptides ranging from 15 to 65 kDa was strongly enhanced. A twofold enhancement of the phosphorylation of 24-kDa protein and a two- to threefold decrease in the phosphorylation of acidic proteins of MrS 62, 65, 80 and 84 was also observed in response to oligogalacturonides. One of the oligogalacturonide-modulated phosphoproteins was identified as calreticulin by direct nucleotide sequencing after preparative two-dimensional electrophoresis and comparison with protein database sequences. Decreased phosphorylation of calreticulin was also observed in vivo, shortly after addition of oligogalacturonides to tobacco cells, confirming the biological relevance of the modification. Although the presence of calreticulin, an abundant reticuloplasmin with high calcium-binding capacity, has been reported in both mammalian and plant cells, its function is as yet largely unknown. Modulation of the phosphorylation of a plant calreticulin-like protein by oligogalacturonides is shown here, suggesting a role in the early transduction steps of this signal.

Function and regulation of seed aquaporins.

The discovery of water channel proteins named aquaporins has shed new light on the molecular mechanisms of transmembrane water transport in higher plants. As with their animal counterparts, plant aquaporins belong to the large MIP family of transmembrane channels. An increasing number of aquaporins is now being identified on both the vacuolar and plasma membranes of plant cells, but their integrated function remains unclear. Aquaporin α-TIP is specifically expressed in the membrane of protein storage vacuoles in seeds of many plant species. α-TIP was previously shown to undergo phosphorylation in bean seeds. The functional significance of this process was further investigated after heterologous expression of the protein in Xenopus oocytes. Using site-directed mutagenesis of α-TIP and in vitro and in vivo phosphorylation by animal cAMP-dependent protein kinase, it is shown that, in oocytes, direct phosphorylation of α-TIP occurs at three distinct sites and stimulates its water channel activity. In addition to aquaporin phosphorylation, other mechanisms that target aquaporin function are used by living cells to regulate their membrane water permeability. These are the fine control of aquaporin gene expression and, in animal cells only, the regulated trafficking of water channel-containing vesicles. The present work and studies by others on the phosphorylation of nodulin-26, an ion channel protein homologous to α-TIP, provide novel insights into the mechanisms of plant membrane protein regulation. These studies might help identifying and characterizing novel membrane-bound protein kinases and phosphatases. Finally, an integrated function for seed vacuolar aquaporins is discussed. During germination, the rehydration of seed cells, the drastic changes in vacuole morphology, the breakdown and the mobilization of storage products from the vacuole may create osmotic perturbations in the cytoplasm. The fine tuning of TIP aquaporin activity may help control the kinetics and amplitude of osmotic water flows across the tonoplast to achieve proper cytoplasm osmoregulation and control of vacuolar volume.

Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway.

Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date.

Cloning and functional expression of a plant voltage-dependent chloride channel.

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants.

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls.

Although Arabidopsis is the object of many genetic and molecular biology investigations, relatively few studies deal with regulation of its transmembrane ion exchanges. To clarify the role of ion transport in plant development, organ-and tissue-specific ion channels must be studied. We identified a voltage-dependent anion channel in epidermal cells of Arabidopsis hypocotyls, thus providing a new example of the occurrence of voltage-dependent anion channels in a specific plant cell type distinct from the stomatal guard cell. The Arabidopsis hypocotyl anion channel is able to function under two modes characterized by different voltage dependences and different kinetic behaviors. This switch between a fast and a slow mode is controlled by ATP. In the presence of intracellular ATP (fast mode), the channels are closed at resting potentials, and whole-cell currents activate upon depolarization. After activation, the anion current deactivates rapidly and more and more completely at potentials negative to the peak. In the absence of ATP, the current switches from this fast mode to a mode characterized by a slow and incomplete deactivation at resting potentials. In addition, the whole-cell currents can be correlated with the activity of single channels. In the outside-out configuration, the presence of ATP modulates the mean lifetimes of the open and closed states of the channel at hyperpolarized potentials, thus controlling its open probability. The fact that ATP-dependent voltage regulation was observed in both whole-cell and outside-out configurations suggests that a single type of anion channel can switch between two modes with distinct functional properties.

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular determinants of the species selectivity of neurokinin type 1 receptor antagonists.

Most nonpeptide neurokinin (NK)1 antagonists display a marked difference in affinity for rat versus human NK1 receptors. The molecular basis for the species selectivity of RP67580 and CP96,345 has been previously addressed [J. Biol. Chem. 267:25668-25671 (1992); J. Biol. Chem. 268:2319-2323 (1993)]. We are extending these previous results to additional NK1 antagonists, which are members of different chemical families. Included is a new perhydroisoindolol, RPR100893, which unlike its parent compound (RP67580) is human receptor selective. Chimeric rat/human NK1 receptors, as well as rat and human mutant NK1 receptors, were constructed and expressed in COS-1 cells, and affinities for substance P and the various antagonists were determined in binding studies. With human receptor-selective antagonists, the rat R290(S-->I) mutation was the most effective in increasing antagonist affinity (from 7- to 23-fold). Combination with the R116(L-->V) mutation led to an additional increase in affinity for trans-4-hydroxy-1-(1H-indol-3-ylcarbonyl)-L-prolyl-N- methyl-N-(phenylmethyl)-L-tyrosineamide (a derivative of FK888) and to nearly full human receptor affinity for RPR100893 and (+/-)-CP99,994. Based on the gains in affinities, these results confirm and extend the role of residues 116 and 290 of the NK1 receptor in the species selectivity of these three new human receptor-selective NK1 antagonists. In comparison, the affinity of RP67580, the least selective molecule, was most affected by changes at position 116, and combination with mutations at either position 97 (V-->E) or position 290 led to the human receptor phenotype. For the heterosteroid KAN610857, modifications of the rat receptor at positions 97 and 290, and to a lesser degree position 116, were the most effective in reducing affinity. Two double-mutants [R(97,290) and R(116,290)], although different from those identified for RP67580, also displayed human receptor-like affinity. Therefore, the molecular determinants of the species selectivity appear to be different, in part, between rat and human receptor-selective compounds, even between closely related chemical families.

Alterations of auxin perception in rolB-transformed tobacco protoplasts. Time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin.

Expression and physiological effects of the root-inducing rolB gene of Agrobacterium rhizogenes T-DNA were studied simultaneously in tobacco (Nicotiana tabacum) mesophyll protoplasts. The kinetic study of the expression of rolB mRNA following exogenous auxin application showed that auxin transiently stimulated rolB expression, with mRNA levels starting to accumulate 6 to 9 h after auxin was supplied and increasing 300-fold after 12 to 18 h. The parallel study of the auxin sensitivity of rolB-transformed protoplasts, as assayed by their electrical response to the hormone, showed that the auxin treatment generated an increase in sensitivity by a factor of up to 100,000, whereas in untransformed protoplasts the same auxin treatment induced an increase in auxin sensitivity that never exceeded 30- to 50-fold. This reflects a strong cooperative effect of auxin and rolB in transformed protoplasts. Surprisingly, the maximal increase in sensitivity was observed several hours before the maximal accumulation of rolB mRNA, suggesting that the dramatic control of auxin sensitivity by auxin in rolB-transformed protoplasts requires only low levels of rolB expression. Antibodies directed against ZmER-abp1, the major auxin-binding protein from maize, differentially altered the auxin sensitivity of the electrical response of rolB-transformed and normal protoplasts. This suggests that alterations of the auxin reception-transduction pathway at the plasma membrane of rolB-transformed protoplasts may account for their increased auxin sensitivity.

The rolB Gene of Agrobacterium rhizogenes Does Not Increase the Auxin Sensitivity of Tobacco Protoplasts by Modifying the Intracellular Auxin Concentration.

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-[beta]-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.