Polyamine Sparing May Be Involved in the Prolongation of Cell Division Due to Inhibition of Phenylpropanoid Synthesis in Cytokinin-Starved Soybean Cells

Effects on growth, mostly of an inhibitory nature, have been attributed to phenolic compounds in vivo and in vitro. This suggests that l-α-aminooxy-β-phenylpropionic acid (l-AOPP), a competitive inhibitor of phenylalanine ammonia-lyase (PAL), the enzyme controlling the first step in phenylpropanoid synthesis, might stimulate growth in soybean suspension cultures (Glycine max, cv. Acme). The promotive effect of l-AOPP, measured as an increase in cell number, was more clearly detected in the growth-limiting condition of cytokinin starvation. At least one more cell division cycle was completed in the presence of l-AOPP before growth by division ceased and growth continued by expansion only. Phenolic acids are known to conjugate with polyamines, modulating the free levels of these plant growth substances. Thus, the effect of l-AOPP on the titers of free and conjugated polyamines (putrescine, spermidine, and spermine) was investigated by high performance liquid chromatography in the course of cytokinin starvation. An increased level of free putrescine was detected in the presence of l-AOPP relative to controls, especially in the initial period before growth became restricted to cell expansion. The decrease in free putrescine associated with the cessation of cell division was temporarily delayed, suggesting that an interaction between phenolic acids and polyamines is involved in the mechanism of growth promotion by l-AOPP. Received July 30, 1996; accepted January 28, 1997     

Hormonal Effects on Growth and Morphology of Normal and Hairy Roots of Hyoscyamus muticus

Treatment of normal and Agrobacterium rhizogenes-transformed root cultures of Hyoscyamus muticus with three different auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and naphthaleneacetic acid (NAA), revealed that the response varied considerably among auxins, between transformed and normal roots, and depending on the parameter. In normal roots all three auxins provoked abundant branching, with IBA and NAA being the most effective at 2.5 and 0.5 μm, respectively, whereas IAA was most effective at low concentrations (0.05 and 0.1 μm). In transformed roots exogenously supplied auxins were generally inhibitory or, at best, without effect on growth and branching. Only 0.01 μm IAA significantly enhanced lateral root number, whereas at the higher concentrations IBA, although inhibitory, was the least effective auxin. In both root types IBA had little effect on primary root growth, but normal roots were more sensitive to IAA and NAA. These results suggest a different sensitivity to auxins of normal and transformed roots since there was no significant difference in endogenous free and conjugated IAA content nor in IAA uptake capacity. Ethylene production and biosynthesis were approximately threefold higher in hairy roots, but production could be stimulated up to tenfold that of control levels in normal roots by supplying NAA or 1-aminocyclopropane-1-carboxylic acid (ACC). Treatment with 2.5 μm NAA, but not IAA or IBA, also enhanced ethylene biosynthesis in normal roots but not in transformed ones. ACC and malonyl-1-aminocyclopropane-1-carboxylic acid accumulated to detectable levels only after treatment with an auxin (NAA). Received March 3, 1997; accepted May 28, 1997     

Uptake, Distribution, and Metabolism of 1-Naphthaleneacetic Acid and Indole-3-Acetic Acid During Callus Initiation From Actinidia deliciosa Tissues

1-Naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) were required for in vitro callus formation at the basal edge of kiwifruit (Actinidia deliciosa [A. Chev] Liang and Ferguson, cv. Hayward) petioles. The uptake, metabolism, and concentration of NAA and indole-3-acetic acid (IAA) content were examined in the explants during the callus initiation period. After 1, 6, 12, 24, 48, and 96 h of culture in the presence of [H³]NAA, petioles were divided into apical, middle, and basal portions and analyzed. Except for a high IAA level measured at 12 h, IAA content decreased in tissues during a culture period of 96 h. NAA uptake was higher in petiolar edges than in the middle portion, and NAA was rapidly conjugated with sugars and aspartic acid inside the tissues. The amide conjugation was triggered in apical and basal portions from 12 h and in the middle part from 48 h, with α-naphthylacetylaspartic acid being the major metabolite. Free-NAA concentration in cultured petioles achieved an equilibrium with the exogenously applied NAA (0.27 μm) from 12 h, and it remained constant thereafter. The relationships between the role attributed to NAA and BA in the initiation and the maintenance of disorganized growth of callus in kiwifruit cultures are discussed. Received December 21, 1998; accepted July 20, 1999     

Morphogenetic Effect of the Herbicide Cinch on Arabidopsis thaliana Root Development

Cinch is a morphogenetically active herbicide that inhibits primary root growth and induces abnormal ``nodule-like' lateral roots on Arabidopsis thaliana seedlings. Using 200 nm Cinch, the early stages of lateral root formation occurred along the apical half of the root axis; but once emerged, they were inhibited from further growth. Second-order lateral roots formed at the base of stunted first-order lateral roots after 5 days of Cinch treatment. Results from Cinch experiments suggested that pericycle cells are determined in the meristem to be potential sites of lateral root formation, and the developmental transition point between emerged lateral roots and subsequent growth is inhibited. Results using 2,4-dichlorophenoxyacetic acid and 2,3,5-triiodobenzoic acid suggest that Cinch is not a chemical analog of auxin. Received August 8, 1997; accepted February 23, 1998     

Effect of Chlorsulfuron on Morphogenetic and Disordered Cell Division in Cultures of Passiflora edulis

We examined the effects of a sulfonylurea herbicide, chlorsulfuron, which is known as a potent inhibitor of plant cell division, on morphogenetic cell division and disorganized cell division using the culture system of multiple shoot primordia and callus of Passiflora edulis. The multiple shoot primordia tissue treated with chlorsulfuron failed to achieve shoot morphogenesis, and a large part of the tissue was necrotized during the posttreatment culture, even when it was washed and transferred to chlorsulfuron-free medium. The inhibition of Passiflora shoot morphogenesis by chlorsulfuron was not reversed by the simultaneous addition of branched amino acids, which are known to reverse the inhibitory effect of chlorsulfuron. In contrast, the same treatment of chlorsulfuron on the callus did not kill the cells, although the growth resumption was retarded by a prolonged lag period. The addition of branched amino acids enhanced the recovery growth of the chlorsulfuron-treated callus. These results suggest that the inhibition of disorganized cell division (callus growth) by chlorsulfuron is reversible, whereas morphogenetic cell division (shoot morphogenesis), which is under complex regulation, is inhibited irreversibly by chlorsulfuron. Qualitative differences between morphogenetic cell division and disordered simple proliferative cell division are discussed. Received November 17, 1997; accepted June 4, 1998     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

The Binding Specificity of Amino Acid Transport System y+L in Human Erythrocytes is Altered by Monovalent Cations

System y⁺L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na⁺ replacement with K⁺ does not affect lysine transport, but markedly decreases the affinity of the transporter for l-leucine and l-glutamine. This observation suggests that the specificity of system y⁺L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na⁺, K⁺, Li⁺ and guanidinium ion. In agreement with the prediction, the specificity of system y⁺L was altered by the monovalent cations. In the presence of Na⁺, l-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - l-norleucine) strengthened binding. In contrast, bulkiness at the level of the β carbon was detrimental. In K⁺, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li⁺ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na⁺; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards β carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., l-leucine, l-glutamine, l-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations. Received: 22 January 1996/Revised: 26 April 1996     

Volume-regulatory Amino Acid Release from the Protozoan Parasite Crithidia luciliae

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue α-aminoisobutyrate. α-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of α-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated α-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for α-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated α-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mm. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells. Received: 13 May 1996/Revised: 9 July 1996     

Role of the Ring Methyl Groups in Abscisic Acid Activity in Erucic Acid Accumulation in Oilseed Rape (Brassica napus L.)

Modification of the structure of abscisic acid (ABA) has been reported to result in modification of its physiologic activity. In this study we tested the effect of removing methyl groups from the ring and of chirality of ABA on activity in microspore-derived embryos of oilseed rape (Brassica napus L.). The natural (+)-ABA molecule induced growth inhibition and an increase in the amount of erucic acid accumulated in the oil at medium concentrations less than 1 μm. (−)-ABA showed similar effects. Removing the 7′-methyl group resulted in a dramatic decrease in activity: (+)-7′-demethyl-ABA retained some activity as a growth inhibitor; a 10–100 μm concentration of this compound was needed for a response, and (−)-7′-demethyl-ABA was almost completely inactive. Similar effects were observed with regard to elongase activity, which catalyzes erucic acid biosynthesis from oleic acid. Removal of the 8′- and 9′-methyl groups resulted in a more complex response. These compounds all showed intermediate activity; for growth inhibition, the presence of the 9′-methyl was the more important determinant, whereas chirality dominated the response on erucic acid accumulation, with the (+)-enantiomers being more active. Received July 25, 1997; accepted October 31, 1997     

Phenylalanine-Binding RNAs and Genetic Code Evolution

We isolated RNAs by selection–amplification, selecting for affinity to Phe–Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets. Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70% of the 105 sequenced RNAs. The K _D for the strongest sites is ≈50 μM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites. However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 × 10⁻¹¹. Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg, Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during an era of direct RNA–amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during later code evolution.     

Sequential Amino Acid Exchange across b0,+-like System in Chicken Brush Border Jejunum

In the small intestine, cationic amino acids are transported by y⁺-like and b^0,+-like systems present in the luminal side of the epithelium. Here, we report the characterization of a b^0,+-like system in the apical membrane of the chicken jejunum, and its properties as an amino acid exchanger. Analysis of the brush border membrane by Western blot points out the presence of rBAT (protein related to b^0,+ amino acid transport system) in these membranes. A functional mechanism for amino acid exchange across this system was established by kinetic analysis measuring fluxes at varying substrate concentrations both in internal (in) and external (out) vesicle compartments. This intestinal b^0,+-like system functions for l-arginine as an obligatory exchanger since its transport capacity increases 100–200 fold in exchange conditions, thus suggesting an important role in the intestinal absorption of cationic amino acids. The kinetic analysis of Arg_in efflux velocities is compatible with the formation of a ternary complex and excludes a model involving a ping-pong mechanism. The binding affinity of Arg_out is higher than that of Arg_in, suggesting a possible order of binding (Arg_out first) for the formation of the ternary complex during the exchange cycle. A model of double translocation pathways with alternating access is discussed. Received: 23 March 2000/Revised: 29 December 2000     

A Possible Role for Jasmonic Acid in Adaptation of Barley Seedlings to Salinity Stress

The changes caused by NaCl salinity and jasmonic acid (JA) treatment (8 days) on growth and photosynthesis of barley plants (Hordeum vulgare L., var. Alfa) have been studied. Gas exchange measurements and analysis of enzyme activities were used to study the reactions of photosynthesis to salinity and JA. Both 100 mm NaCl and 25 μm JA treatment led to a noticeable decrease in both the initial slope of the curves representing net photosynthetic rate vs intercellular CO₂ concentration and the maximal rate of photosynthesis. The calculated values of the intercellular CO₂ concentration, CO₂ compensation point, and maximal carboxylating efficiency of ribulose-1,5-bisphosphate carboxylase support the suggestion that biochemical factors are involved in the response of photosynthesis to JA and salinity stress. The activities of phosphoenolpyruvate carboxylase and carbonic anhydrase increased more than twofold. Pretreatment with JA for 4 days before salinization diminished the inhibitory effect of high salt concentration on the growth and photosynthesis. The results are discussed in terms of a possible role of JA in increasing salinity tolerance of the barley plants. Received September 8, 1997; accepted May 19, 1998     

Biochemical studies of embryogenic and non-embryogenic callus of Cardiospermum halicacabum L

Some physiological and biochemical changes were measured between embryogenic and non-embryogenic callus obtained from Cardiospermum halicacabum. Combination of auxin with cytokinin was more favourable for high amount of callus formation. 2,4-D played a key role in triggering somatic embryo formation. Embryogenic callus had more total carbohydrate and starch contents, total free amino acids, nucleic acids, phenols and ascorbic acid. Non-embryogenic callus exhibited high chlorophyll content, total soluble sugar, protein, ammonia and enzymes like peroxidase and polyphenol oxidase. Thus, the present study indicated that the process of somatic embryogenesis was characterized by some biochemical and physiological changes induced by plant growth regulators.     

Accumulation of Glutamine by Suspension Cultures of Symphytum officinale

A callus was induced from the veins of a leaf of Symphytum officinale, comfrey, on a medium containing the inorganic elements reported by Murashige and Skoog with addition of 3% sucrose, 0.5 mg/liter 2,4-D and 0.3~3.0 mg/liter kinetin.

Suspension cultures of this cell line obtained from the callus were shown to accumulate a large amount of L-glutamine intracellularly, The effect of growth hormones and nutrients on accumulation of the amino acid has been examined in suspension cultures. The most suitable concentrations of 2,4-D and kinetin for glutamine accumulation were 0.3 mg/liter each. The presence of potassium nitrate as a nitrogen source was beneficial for growth and ammonium nitrate stimulated the accumulation of glutamine. High levels of these nitrogen sources in the medium were required for obtaining a high level of glutamine. The concentration of glutamine accumulated reached to approximately 20% of dry cell weight when S. officinale was incubated in the medium containing 0.495 % of ammonium nitrate and 0.570% of potassium nitrate which corresponded to three times higher levels than those in a Murashige and Skoog’s medium.

Most of the amino acid was found intracellularly but a small amount was excreted into the medium in the later stages of the incubation. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the cultures caused to increase the amount of the amino acid in the culture filtrate.

The contents of free amino acids in leaves of S. officinale were compared with those in the callus. The level of glutamine in the callus was 260 times higher than that in the intact plant.     

Effects of Chemical Growth Retardants on Growth and Development of Sweetpotato (Ipomoea batatas (L.) Lam.) in Vitro

Plant growth retardants were evaluated for their ability to reduce the growth rate of sweetpotato (Ipomoea batatas (L.) Lam.) in vitro. Nodal sections of cv. Jewel were cultured for 30 days on medium containing NDA, ancymidol, phosfon, TIBA, difenzoquat, chlormequat, ACC, mepiquat chloride, or daminozide at 0, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, or 10⁻⁸ m. Difenzoquat, NDA, phosfon, and TIBA, at 10⁻⁴ m, were lethal to axillary bud explants. A low concentration (10⁻⁸ m) of chlorflurenol or NDA stimulated shoot elongation. The effective concentration range for most growth retardants was 10⁻⁵ to 10⁻⁶ m. Small (2- to 4-mm diameter) storage root-like swellings were observed on roots in cultures containing TIBA or ancymidol. The growth-inhibiting effects of ancymidol and NDA were transitory and did not persist through a 180-day culture period. Shoots cultured on medium containing 10⁻⁵ m phosfon, TIBA, or difenzoquat were significantly shorter than control plants after a 180-day culture period. Culture on medium containing TIBA, NDA, ancymidol, or ACC resulted in abnormal leaf and stem development. Plants derived from nodal explants cultured on medium containing either phosfon or chlormequat were near normal in appearance but with some plants exhibiting interveinal chlorosis and reduced root system development. Received May 9, 1997; accepted August 14, 1997     

Exogenous Jasmonic and Abscisic Acids Act Differentially in Elongating Tissues from Oat Stem Segments

Segments can be cut from the peducular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive, auxin-sensitive leaf sheath pulvinus, and the gibberellin-sensitive internodal tissue. These two growth-capable tissues were used to study the effects and interactions of jasmonic acid (JA) and abscisic acid (ABA) in regulating cell elongation. When supplied alone at physiologic concentrations (10⁻⁵, 10⁻⁴ m), JA promoted growth and cell wall synthesis in the internodal tissue, whereas by itself, ABA inhibited internodal elongation and even inhibited JA-promoted growth. When gibberellic acid (GA₃) was used to stimulate internodal elongation, JA and ABA caused similar levels of inhibition and, at certain concentrations, were synergistic. Inhibition by ABA was initiated several hours earlier than inhibition by JA, and only the ABA effect could be partially overcome by 10⁻³ m aminoethoxyvinylglycine. Both JA and ABA inhibited elongation of pulvinar tissue that was induced to grow by gravistimulus or auxin, although here JA was more potent than ABA at equimolar concentrations. When 10⁻⁵ m fusicoccin was used as a general nonphysiologic growth stimulus, JA had no effect on the internode but inhibited the pulvinus, whereas ABA had no effect on the pulvinus but inhibited the internode. These results provide strong physiologic evidence that JA and ABA act by different mechanisms in the regulation of elongation, at least in this representative grass. Received May 28, 1996; accepted November 7, 1996     

Effects of Exogenously Applied Jasmonates on Growth and Intracellular pH in Maize Coleoptile Segments

The effects of jasmonic acid (JA) on elongation growth of coleoptile segments from etiolated maize (Zea mays L.) were investigated in the presence and absence of auxin. When supplied alone, at physiological concentrations (10⁻⁹, 10⁻⁸, and 10⁻⁵ m), JA (or methyl-JA) inhibited growth. JA at a similar range of concentrations also inhibited auxin-induced elongation growth. To determine whether this effect on growth depended on endogenous abscisic acid (ABA), we grew maize coleoptiles in the presence of norflurazon (an inhibitor of carotenoid biosynthesis) that results in reduced endogenous ABA levels. Growth of etiolated coleoptile segments from these plants was inhibited by JA (or methyl-JA) in both the absence and presence of auxin. Previously, we have observed a correlation between elongation growth and cytosolic pH (pH_i), in which auxin lowers pH_i, and growth inhibitors such as ABA raise pH_i. We examined the effect of low concentrations of methyl-JA on pH_i with dual emission dye, carboxy seminaphthorhodafluor-1, and confocal microscopy. To confirm these studies, we also used in vivo ³¹P NMR spectrometry to ascertain the changes in pH_i after addition of jasmonate to maize coleoptiles. Coleoptiles grown in either the absence or presence of norflurazon responded to methyl-JA or JA by increases in pH_i of approximately 0.2 pH unit. This response occurs over a period of 15–20 min and appears to be independent of endogenous ABA. This alkalization induced by JA is likely to form a permissive environment for JA signal transduction pathway(s). Received February 5, 1999; accepted August 25, 1999     

Influence of Polyamines on Growth of Hairy Root Cultures of Witloof Chicory (Cichorium intybus L. cv. Lucknow Local) and Formation of Coumarins

The effect of polyamines (putrescine, spermidine, and spermine) was examined for growth and production of two coumarins, esculetin and esculin, in the hairy roots of chicory (Cichorium intybus L. cv. Lucknow local). Of the polyamines administered, 1.5 mm putrescine alone resulted in a 2.3-fold higher increase in the growth of hairy roots as well as in the production of esculetin and esculin, which was 3.37 times more than that of the control on day 21. The endogenous level of conjugated putrescine was more than fivefold that of free putrescine levels in untreated samples. The production of esculetin and esculin in hairy root cultures strictly correlated with growth in all of the treatments. Putrescine at 1.5 mm resulted in a greater length of primary root (18.29 ± 1.37 cm) compared with the control (10.96 ± 0.82 cm) and more secondary and tertiary roots. This study also provides insight into the morphogenetic changes that occur in roots in response to the external supply of polyamines. Received July 20, 1998; accepted January 19, 1999     

rBAT is an Amino Acid Exchanger with Variable Stoichiometry

The rBAT protein, when expressed in Xenopus oocytes, was previously shown to reproduce the selectivity of the Na⁺-independent neutral and basic amino acid transport system called b^o,+. More recently, the capacity of rBAT to generate a transmembrane current was demonstrated when addition of neutral amino acids stimulated the efflux of cations (presumably basic amino acids) in rBAT-injected oocytes. In the present paper, aminoisobutyric acid (AIB), a neutral amino acid analogue, was shown to induce outward currents (efflux of basic amino acids) through rBAT similar to those caused by alanine in terms of affinity, maximal currents and I-V curves. Despite generating similar currents, the AIB transport rate was more than 30 times lower than that of alanine, thus challenging the assumption that rBAT functions as a classical exchanger. Experiments using a cut-open oocyte voltage clamp demonstrated that AIB was capable of stimulating rBAT-mediated currents from either side of the membrane. AIB, like alanine, was able to stimulate the efflux of radiolabeled alanine and arginine while no rBAT-mediated efflux was measurable in the absence of external rBAT substrates. These results demonstrate that (i) the presence of amino acids is required on both sides of the membrane for rBAT to mediate amino acid flux and thus rBAT must be some type of exchanger but (ii) rBAT-mediated amino acid influx is not stoichiometrically related to the efflux. A model of a ``double gated pore' is proposed to account for these properties of rBAT, which contravene standard models of exchangers and other transporters. Received: 15 June 1995/Revised: 21 September 1995     

Selection and characterization of ethionine-resistant alfalfa (Medicago sativa L.) cell lines

Summary Diploid alfalfa (HG2), capable of plant regeneration from tissue culture, was used to select variant cell lines resistant to growth inhibition due to ethionine (an analog of methionine). Approximately 10⁷ suspension-cultured cells were mutagenized with methane sulfonic acid ethylester and then plated in solid media containing ethionine. Callus colonies formed on media with 0.02 mM ethionine. Of the 124 cell lines recovered, 91 regenerated plants. After six months growth on media without ethionine, 15 of 110 cell lines of callus grew significantly better than HG2 on 1 mM ethionine. Several ethionine-resistant callus cultures were also resistant to growth inhibition due to the addition of lysine + threonine to the media. High concentrations, relative to unselected HG2 callus, of methionine, cysteine, cystathionine, and glutathione were found in some, but not all, ethionine-resistant callus cultures. Cell line R32, which had a ca. tenfold increase in soluble methionine, had a 43% increase in total free amino acids and a 40% increase in amino acids in protein as compared to unselected HG2 callus. Relative amounts of each amino acid in protein were the same in both.Abbreviation LT lysine + threonine in equimolar concentration