Geochemical fingerprints and controls in the sediments of an urban river: River Manzanares, Madrid (Spain)

The geochemical fingerprint of sediment retrieved from the banks of the River Manzanares as it passes through the City of Madrid is presented here. The river collects the effluent water from several Waste Water Treatment (WWT) plants in and around the city, such that, at low flows, up to 60% of the flow has been treated. A total of 18 bank-sediment cores were collected along the course of the river, down to its confluence with the Jarama river, to the south–east of Madrid. Trace and major elements in each sample were extracted following a double protocol: (a) “Total” digestion with HNO3, HClO4 and HF; (b) “Weak” digestion with sodium acetate buffered to pH=5 with acetic acid, under constant stirring. The digests thus obtained were subsequently analysed by ICP-AES, except for Hg which was extracted with aqua regia and sodium chloride-hydroxylamine sulfate, and analysed by Cold Vapour-AAS. X-ray diffraction was additionally employed to determine the mineralogical composition of the samples. Uni- and multivariate analyses of the chemical data reveal the influence of Madrid on the geochemistry of Manzanares' sediments, clearly manifested by a marked increase in the concentration of typically “urban” elements Ag, Cr, Cu, Pb and Zn, downstream of the intersection of the river with the city's perimeter. The highest concentrations of these elements appear to be associated with illegal or accidental dumping of waste materials, and with the uncontrolled incorporation of untreated urban runoff to the river. The natural matrix of the sediment is characterised by fairly constant concentrations of Ce, La and Y, whereas changes in the lithology intersected by the river cause corresponding variations in Ca–Mg and Al–Na contents. In the final stretch of the river, the presence of carbonate materials seems to exert a strong geochemical control on the amount of Zn and, to a lesser extent, Cu immobilised in the sediments. This fact suggests that a variable but significant proportion of both elements may be susceptible to reincorporation in the aqueous phase under realistic environmental conditions.

Schizosaccharomyces selective differential media

This study discusses the optimisation of a selectiv e and differential medium which would facilitate the isolation of Schizosaccharomyces (a genus with a low incidence compared to other microorganisms) to select individuals from this genus for industrial purposes, especially in light of the recent approval of the use of yeasts from this genus in the wine industry by the International Organisation of Vine and Wine, or to detect the presence of such yeasts, for those many authors who consider them food spoilers. To this end, we studied various selective differential agents based on the main thephysiological characteristics of this species, such as its high resistance to high concentrations of sugar, sulfur dioxide, sorbic acid, benzoic acid, acetic acid or malo ethanolic fermentation. This selective medium is based on the resistance of the genus to the antibiotic actidione and its high resistance to inhibitory agents such as benzoic acid compared to possible microorganisms which can give rise to false positive results. Malic acid was used as a differential fact or due to the ability of this genus to metabolise it to ethanol, which allows detecting of the degradation of this compound. Lastly, the medium was successfully used to isolate strains of Schizosaccharomyces pombe from honey.

New Schizosaccharomyces applications in red wines alcoholic fermentation

The Schizosaccharomyces strains consumed less primary amino nitrogen and produced less urea and more pyruvic acid than other Saccharomyces species. Further, three of the four Schizosaccharomyces strains completed the breakdown of malic acid by day 4 of fermentation. The main negative effect of the use of Schizosaccharomyces was strong acetic acid production. The Schizosaccharomyces strains that produced most pyruvic acid (938 and 936) were associated with better ?wine? colour than the remaining yeasts. The studied Schizosaccharomyces could therefore be of oenological interest.

Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.

YUCCA8 and YUCCA9 overexpression reveals a link between auxin signaling and lignification through the induction of ethylene biosynthesis.

Auxin is associated with the regulation of virtually every aspect of plant growth and development. Many previous genetic and biochemical studies revealed that, among the proposed routes for the production of auxin, the so-called indole-3-pyruvic acid (IPA) pathway is the main source for indole-3-acetic acid (IAA) in plants. The IPA pathway involves the action of 2 classes of enzymes, tryptophan-pyruvate aminotransferases (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1(TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR)) and flavin monooxygenases (YUCCA). Both enzyme classes appear to be encoded by small gene families in Arabidopsis consisting of 5 and 11 members, respectively. We recently showed that it is possible to induce transcript accumulation of 2 YUCCA genes, YUC8 and YUC9, by methyl jasmonate treatment. Both gene products were demonstrated to contribute to auxin biosynthesis in planta.1 Here we report that the overexpression of YUC8 as well as YUC9 led to strong lignification of plant aerial tissues. Furthermore, new evidence indicates that this abnormally strong secondary growth is linked to increased levels of ethylene production.

Combine use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production

Most red wines commercialized in the market use the malolactic fermentationprocess in order to ensure stability from a microbiological point of view. In this secondfermentation, malic acid is converted into L-lactic acid under controlled setups. Howeverthis process is not free from possible collateral effects that on some occasions produceoff-flavors, wine quality loss and human health problems. In warm viticulture regions suchas the south of Spain, the risk of suffering a deviation during the malolactic fermentationprocess increases due to the high must pH. This contributes to produce wines with highvolatile acidity and biogenic amine values. This manuscript develops a new red winemakingmethodology that consists of combining the use of two non-Saccharomyces yeast strains asan alternative to the traditional malolactic fermentation. In this method, malic acid is totallyconsumed by Schizosaccharomyces pombe, thus achieving the microbiological stabilizationobjective, while Lachancea thermotolerans produces lactic acid in order not to reduce andeven increase the acidity of wines produced from low acidity musts. This technique reducesthe risks inherent to the malolactic fermentation process when performed in warm regions.The result is more fruity wines that contain less acetic acid and biogenic amines than thetraditional controls that have undergone the classical malolactic fermentation.

Estudio de la estabilidad genética asociada a los procesos de crioconservación de ápices de menta

La crioconservación se ha descrito como una técnica de conservación ex situ a largo plazo que ha sido aplicada con éxito a numerosas especies, y resulta especialmente importante en aquellas con propagación vegetativa, infértiles o amenazadas, en las que sistemas de conservación ex situ más sencillos, como los bancos de semillas, no son posibles. También presenta ventajas frente a la conservación in vitro, ya que logra disminuir o eliminar problemas como la excesiva manipulación del material, evitando los subcultivos periódicos y disminuyendo así el riesgo de contaminaciones y de aparición de variación somaclonal. Sin embargo, someter al material vegetal a los procedimientos que implica la crioconservación provoca distintos estreses. Entre ellos, el estrés oxidativo puede potencialmente producir daños en membranas, proteínas, carbohidratos y en el ADN. En este trabajo se han evaluado diversos sistemas de crioconservación en ápices de Mentha × piperita L., híbrido estéril entre Mentha aquatica L. y Mentha spicata L. Se han utilizado ápices de dos genotipos (‘MEN 186’y ‘MEN 198’) en los cuales se compararon dos técnicas de crioconservación, encapsulación-deshidratación y vitrificación-droplet. El análisis de la supervivencia y capacidad de regeneración del material sometido a los tratamientos de crioconservación, junto con el análisis de la estabilidad genética de dicho material mediante marcadores moleculares (RAPD y AFLP) han permitido comparar los distintos protocolos y tratamientos establecidos. El estudio sobre el tipo de protocolo empleado reveló una mayor variabilidad genética en la técnica de encapsulación-deshidratación, especialmente en el genotipo ‘MEN 186’, ya que ‘MEN 198’ resultó ser más estable en todos los análisis. La inestabilidad encontrada en esta técnica no fue exclusiva de aquellos explantos crioconservados, sino que los pasos previos a la inmersión en nitrógeno líquido (NL) también provocaron variaciones en el ADN. Según el tipo de muestra analizada se encontraron diferencias en la estabilidad: muestras provenientes de callos presentaron una mayor inestabilidad que aquellas de hojas (brotes). Se utilizaron tres medios para la recuperación de los ápices tras la crioconservación con el uso de diferentes combinaciones de reguladores de crecimiento: “Reed” (0,5 mgL-1 6-bencilaminopurina, BAP), “Senula” (0,5 mgL-1 6-dimetilalilamino-purina, 2-iP + 0,1 mgL-1 ácido α-naftalen-acético, ANA) y “Nudos” (0,5 mgL-1 BAP + 0,1 mgL-1ANA). El medio “Reed” produjo un aumento en la supervivencia y recuperación de los ápices en ambos genotipos y técnicas, y disminuyó la formación de callo. Sin embargo, no tuvo un efecto significativo en la estabilidad genética. El medio “Senula” provocó una mayor estabilidad genética en el genotipo más inestable, ‘MEN 186’. Para reducir el daño oxidativo producido durante la encapsulación-deshidratación, e incrementar la recuperación de los ápices manteniendo su estabilidad genética, se comparó el efecto de añadir sustancias antioxidantes en el precultivo de los ápices (ácido ascórbico, vitamina E y glutatión). No se obtuvo la respuesta esperada y estos tratamientos no presentaron efectos significativos tanto en la estabilidad como en la recuperación. Para entender mejor qué sucede durante todo el proceso de encapsulación-deshidratación, se evaluó cada paso del protocolo por separado y su efecto en la estabilidad y la recuperación. Además, se determinó el estado de oxidación en cada etapa mediante la cuantificación de malondialdehído y la detección de la formación de radicales libres (mediante el ensayo del ácido tiobarbitúrico, y sondas fluorescentes específicas, respectivamente). Se determinó que a partir de los primeros pasos se genera estrés oxidativo, el cual aumenta a medida que se avanza por el protocolo hasta la inmersión en nitrógeno líquido. Esto se ve reflejado en la disminución progresiva tanto de la recuperación como de la estabilidad genética. Con el uso de antioxidantes en el precultivo (ácido ascórbico y vitamina E) no se obtuvo un efecto positivo en el mantenimiento de la estabilidad genética, y tan sólo con el uso de vitamina E se observó una recuperación mayor en uno de los pasos estudiados (después de la desecación). Sin embargo, cuando se utilizó ácido ascórbico durante el precultivo o la deshidratación osmótica se consiguió disminuir de forma significativa la formación de MDA y la acumulación del radical superóxido (O2•-) en la mayoría los pasos analizados, aunque esta reducción no parece tener un efecto directo en la estabilidad genética del material recuperado. ABSTRACT Cryopreservation has been described as an effective technique for the long term of ex situ conservation that has been successfully applied to numerous species, and is of especial relevance for those with vegetative propagation, infertile or endangered, in which simpler systems of ex situ conservation, such as seed banking, are not feasible. It also has advantages over in vitro conservation, as it reduces or eliminates excessive material handling, avoids periodic subcultures and thus limits the risk of contamination and the appearance of somaclonal variation. However, plant material is subjected to different treatments involved in the cryopreservation procedures, which impose several stresses. Among them, oxidative stress can potentially cause damage to membranes, proteins, carbohydrates and DNA. In this work, two cryopreservation techniques have been evaluated in Mentha × piperita L. shoot tips, sterile hybrid between Mentha aquatica L. and Mentha spicata L. Two genotypes ('MEN 186' and 'MEN 198') were used to compare two techniques: encapsulation-dehydration and droplet-vitrification. The analysis of survival and recovery capacity of the material after the cryopreservation treatments, and the analysis of the genetic stability by molecular markers (RAPD and AFLP) have enabled the comparison between protocols and treatments. The study of the two cryopreservation procedures revealed a higher genetic variability in the encapsulation-dehydration technique, especially in genotype 'MEN 186', as 'MEN 198' was more stable in all analyses. The instability generated in this technique was not exclusive of cryopreserved explants, pretreatments prior to immersion in NL also caused DNA variations. The type of sampled plant material revealed also differences in the stability: callus samples showed greater instability than shoots. Three different culture media were used for the recovery of shoot tips after cryopreservation, using different combinations of growth regulators: "Reed" (0.5 mgL-1 6-benzylaminopurine, BAP), "Senula" (0.5 mgL-1 6-dimetilalilamino-purine, 2-iP + 0.1 mgL-1 α-naphthalene acetic acid, ANA) and "Nodes" (0.5 mgL-1 BAP + 0.1 mgL-1 ANA). "Reed" medium increased survival and recovery of shoot tips in both genotypes and techniques and decreased callus formation. However, it didn`t have a significant effect on genetic stability. "Senula" medium caused a higher genetic stability in the most unstable genotype, 'MEN 186'. To reduce oxidative damage during encapsulation-dehydration, and increase shoot tip recovery and maintain genetic stability, the effect of added antioxidants (ascorbic acid, vitamin E and glutathione) in the shoot tip preculture medium was studied. These treatments had no significant effect on both stability and recovery. To better understand the events during the encapsulation-dehydration process, the effect of each step of the protocol on stability and recovery was evaluated separately. Moreover, the oxidation level was determined by quantifying malondialdehyde (MDA) formation and detecting free radical accumulation (using the thiobarbituric acid assay, and specific fluorescent probes, respectively). The oxidative stress was detected from the first steps and increased throughout the protocol until the immersion in liquid nitrogen. This was also reflected in the gradual decline of recovery and genetic stability. The use of antioxidants (ascorbic acid and vitamin E) in the shoot tip preculture medium had no effect in maintaining genetic stability; only vitamin E increased recovery in one of the steps studied (after desiccation). However, when ascorbic acid was used during the preculture or during the osmotic dehydration, a significantly decrease was observed in MDA formation and superoxide radical accumulation in most of the steps analyzed, although this reduction did not seem to have a direct effect on the genetic stability of recovered material.

Influence of sequential fermentations with Torulaspora delbrueckii and Saccharomyces cerevisiae on wine quality

Torulaspora delbrueckii is a non-Saccharomyces yeast with interesting metabolic and physiological properties of potential use in oenology. This work examines the fermentative behaviour of five strains of T. delbrueckii in sequential fermentations with Saccharomyces cerevisiae, analysing the formation of aromatic compounds, polyalcohols and pigments. The fermentative power of these five strains ranged between 7.6 and 9.0% v/v ethanol; the associated volatile acidity was 0.2e0.7 g/l acetic acid. The production of glycerol was inferior to that of S. cerevisiae alone. The mean 2,3-butanediol concentration reached in single-culture S. cerevisiae fermentations was 73% higher than in the five sequential T. delbrueckii/S. cerevisiae fermentations. However, these fermentations produced larger quantities of diacetyl, ethyl lactate and 2-phenylethyl acetate than single-culture S. cerevisiae fermentation. 3-ethoxy propanol was produced only in the sequential fermentations. The five sequential fermentations produced smaller quantities of vitisin A and B than single-culture S. cerevisiae fermentation. In tests performed prior to the addition of the S. cerevisiae in the sequential fermentations, none of the T. delbrueckii strains showed any extracellular hydroxycinnamate decarboxylase activity. They therefore produced no vinyl phenolic pyranoanthocyanins.

High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis

Physiological studies with excised stem segments have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of cell elongation. Supporting evidence from intact plants has been somewhat more difficult to obtain, however. Here, we report the identification and characterization of an auxin-mediated cell elongation growth response in Arabidopsis thaliana. When grown in the light at high temperature (29°C), Arabidopsis seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20°C. This temperature-dependent growth response is sharply reduced by mutations in the auxin response or transport pathways and in seedlings containing reduced levels of free IAA. In contrast, mutants deficient in gibberellin and abscisic acid biosynthesis or in ethylene response are unaffected. Furthermore, we detect a corresponding increase in the level of free IAA in seedlings grown at high temperature, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response. Consistent with this possibility, high temperature also stimulates other auxin-mediated processes including auxin-inducible gene expression. Based on these results, we propose that growth at high temperature promotes an increase in auxin levels resulting in increased hypocotyl elongation. These results strongly support the contention that endogenous auxin promotes cell elongation in intact plants.

Thioredoxin-linked mitigation of allergic responses to wheat

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S—S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the α and β fractions were most potent, followed by the γ and ω types. Thioredoxin mitigated the allergenicity associated with the major protein fractions—i.e, the gliadins (including the α, β, and γ types) and the glutenins—but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP–thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously—i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

Fusion of Endosomes Involved in Synaptic Vesicle Recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca2+ chelator EGTA. Endosome fusion was restored by the addition of Ca2+ with an optimum at a free Ca2+ concentration of 0.3 × 10−6 M. Other divalent cations did not substitute for Ca2+. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca2+. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca2+ from the endosomal interior.

Nociception in cyclooxygenase isozyme-deficient mice

Prostaglandins formed by cyclooxygenase-1 (COX-1) or COX-2 produce hyperalgesia in sensory nerve endings. To assess the relative roles of the two enzymes in pain processing, we compared responses of COX-1- or COX-2-deficient homozygous and heterozygous mice with wild-type controls in the hot plate and stretching tests for analgesia. Preliminary observational studies determined that there were no differences in gross parameters of behavior between the different groups. Surprisingly, on the hot plate (55°C), the COX-1-deficient heterozygous groups showed less nociception, because mean reaction time was longer than that for controls. All other groups showed similar reaction times. In the stretching test, there was less nociception in COX-1-null and COX-1-deficient heterozygotes and also, unexpectedly, in female COX-2-deficient heterozygotes, as shown by a decreased number of writhes. Measurements of mRNA levels by reverse transcription–PCR demonstrated a compensatory increase of COX-1 mRNA in spinal cords of COX-2-null mice but no increase in COX-2 mRNA in spinal cords of COX-1-null animals. Thus, compensation for the absence of COX-1 may not involve increased expression of COX-2, whereas up-regulation of COX-1 in the spinal cord may compensate for the absence of COX-2. The longer reaction times on the hot plate of COX-1-deficient heterozygotes are difficult to explain, because nonsteroid anti-inflammatory drugs have no analgesic action in this test. Reduction in the number of writhes of the COX-1-null and COX-1-deficient heterozygotes may be due to low levels of COX-1 at the site of stimulation with acetic acid. Thus, prostaglandins made by COX-1 mainly are involved in pain transmission in the stretching test in both male and female mice, whereas those made by COX-2 also may play a role in the stretching response in female mice.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Platelets modulate gastric ulcer healing: Role of endostatin and vascular endothelial growth factor release

Bleeding and delayed healing of ulcers are well recognized clinical problems associated with the use of aspirin and other nonsteroidal antiinflammatory drugs, which have been attributed to their antiaggregatory effects on platelets. We hypothesized that antiplatelet drugs might interfere with gastric ulcer healing by suppressing the release of growth factors, such as vascular endothelial growth factor (VEGF), from platelets. Gastric ulcers were induced in rats by serosal application of acetic acid. Daily oral treatment with vehicle, aspirin, or ticlopidine (an ADP receptor antagonist) was started 3 days later and continued for 1 week. Ulcer induction resulted in a significant increase in serum levels of VEGF and a significant decrease in serum levels of endostatin (an antiangiogenic factor). Although both aspirin and ticlopidine markedly suppressed platelet aggregation, only ticlopidine impaired gastric ulcer healing and angiogenesis as well as reversing the ulcer-associated changes in serum levels of VEGF and endostatin. The effects of ticlopidine on ulcer healing and angiogenesis were mimicked by immunodepletion of circulating platelets, and ticlopidine did not influence ulcer healing when given to thrombocytopenic rats. Incubation of human umbilical vein endothelial cells with serum from ticlopidine-treated rats significantly reduced proliferation and increased apoptosis, effects reversed by an antibody directed against endostatin. Ticlopidine treatment resulted in increased platelet endostatin content and release. These results demonstrate a previously unrecognized contribution of platelets to the regulation of gastric ulcer healing. Such effects likely are mediated through the release from platelets of endostatin and possibly VEGF. As shown with ticlopidine, drugs that influence gastric ulcer healing may do so in part through altering the ability of platelets to release growth factors.

Evidence That Auxin-Induced Growth of Tobacco Leaf Tissues Does Not Involve Cell Wall Acidification1

Interveinal strips (10 × 1.5 mm) excised from growing tobacco (Nicotiana tabacum L. cv Xanthi) leaves have an auxin-specific, epinastic growth response that is developmentally regulated and is not the result of ethylene induction (C.P. Keller, E. Van Volkenburgh [1997] Plant Physiol 113: 603–610). We report here that auxin (10 μm naphthalene acetic acid) treatment of strips does not result in plasma membrane hyperpolarization or detectable proton efflux. This result is in contrast to the expected responses elicited by 1 μm fusicoccin (FC) treatment, which in other systems mimics auxin growth promotion through stimulation of the plasma membrane H+-ATPase and resultant acid wall loosening; FC produced both hyperpolarization and proton efflux in leaf strips. FC-induced growth was much more inhibited by a strong neutral buffer than was auxin-induced growth. Measurements of the osmotic concentration of strips suggested that osmotic adjustment plays no role in the auxin-induced growth response. Although cell wall loosening of some form appears to be involved, taken together, our results suggest that auxin-induced growth stimulation of tobacco leaf strips results primarily from a mechanism not involving acid growth.