Identification and characterization of simple sequence repeat markers from a glandular Origanum vulgare expressed sequence tag

Oregano (Origanum vulgare) and marjoram (Origanum majorana) are two sensorial distinct spices within the genus Origanum (Lamiaceae). Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) of essential oil glands of O. vulgare. Thirteen EST-SSR loci were evaluated using 20 individual plants of O. vulgare and 19 plants of Origanum majorana. The number of alleles per locus ranged from one to four. All loci developed from O. vulgare successfully cross-amplified in O. majorana.     

Ratios of cis- and trans-Sabinene Hydrate in Origanum majorana L. and Origanum microphyllum (Bentham) Vogel

(+)-cis-Sabinene hydrate and (+)-trans-sabinene hydrate are the main monoterpenes found in marjoram (Origanum majorana), but can also be found in other Origanum species as well, as in e.g. Melaleuca alternifolia. The synthesis of sabinene hydrate in marjoram (Origanum majorana) is performed by sabinene hydrate synthase. It is claimed, that both, (+)-cis- and (+)-trans-sabinene hydrate are produced by the same enzyme in an exact ratio of 10:1. To verify this in vitro result in vivo, we analysed single plants of 20 different genotypes of Origanum majorana and of three different populations of Origanum microphyllum and calculated the ratios of (+)-cis- to (+)-trans-sabinene hydrates. In Origanum majorana a constant ratio of 20:1 could be found, whereas in Origanum microphyllum the ratio did not prove to be constant.     

Inhibitory effect of ursolic acid purified from Origanum majorana L on the acetylcholinesterase

We screened 139 herbal spices in search of the acetylcholinesterase (AChE) inhibitor from natural resources. AChE inhibitors, which enhance cholinergic transmission by reducing the enzymatic degradation of acetylcholine, are the only source of compound currently approved for the treatment of Alzheimer's Disease (AD). Among these herbs, edible plants and spices, the ethanol extract from Origanum majorana L. showed the highest inhibitory effect on AChE in vitro. By sequential fractionation of Origanum majorana L. the active component was finally identified as ursolic acid (3 beta-Hydroxyurs-12-en-28-oic acid). The ursolic acid of Origanum majorana L. inhibited AChE activity in a dose-dependent and competitive/non-competitive type. The Ki value (representing the affinity of the enzyme and inhibitor) of Origanum majorana L. ursolic acid was 6 pM, and that of tacrine was 0.4 nM. The concentration required for 50% enzyme inhibition of the active component (IC50 value) was 7.5 nM, and that of tacrine was 1 nM. This study demonstrated that the ursolic acid of Origanum majorana L. appeared to be a potent AChE inhibitor in Alzheimer's Disease.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-pinene and (+)- and (-)-camphene

Cyclase I from Salvia officinalis leaf catalyzes the conversion of geranyl pyrophosphate to the stereo-chemically related bicyclic monoterpenes (+)-alpha-pinene and (+)-camphene and to lesser quantities of monocyclic and acyclic olefins, whereas cyclase II from this plant tissue converts the same acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene and (-)-camphene as well as to lesser amounts of monocyclics and acyclics. These antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H]Linalyl pyrophosphate (3H:14C = 5.14) was tested as a substrate with both cyclases to determine the configuration of the cyclizing intermediate. This substrate with cyclase I yielded alpha-pinene and camphene with 3H:14C ratios of 3.1 and 4.2, respectively, indicating preferential, but not exclusive, utilization of the (3R)-enantiomer. With cyclase II, the doubly labeled substrate gave bicyclic olefins with 3H:14C ratios of from 13 to 20, indicating preferential, but not exclusive, utilization of the (3S)-enantiomer in this case. (3R)- and (3S)-[1Z-3H]linalyl pyrophosphate were separately compared to the achiral precursors [1-3H]geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. With cyclase I, geranyl, neryl, and (3R)-linalyl pyrophosphate gave rise exclusively to (+)-alpha-pinene and (+)-camphene, whereas (3S)-linayl pyrophosphate produced, at relatively low rates, the (-)-isomers. With cyclase II, geranyl, neryl, and (3S)-linalyl pyrophosphate yielded exclusively the (-)-isomer series, whereas (3R)-linalyl pyrophosphate afforded the (+)-isomers at low rates. These results are entirely consistent with the predicted stereochemistries and additionally revealed the unusual ability of these enzymes to catalyze antipodal cyclizations when presented with the unnatural linalyl enantiomer.     

Activity of essential oils and individual components against acetyl- and butyrylcholinesterase

We have tested acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of nineteen essential oils obtained from cultivated plants, namely one from Anethum graveolens L. (organic fertilizer), two from Foeniculum vulgare Mill. collected at fully-mature and flowering stages (organic fertilizer), two from Melissa officinalis L. (cultivated using organic and chemical fertilizers), two from Mentha piperita L. and M. spicata L. (organic fertilizer), two from Lavandula officinalis Chaix ex Villars (cultivated using organic and chemical fertilizers), two from Ocimum basilicum L. (green and purple-leaf varieties cultivated using only organic fertilizer), four from Origanum onites L., O. vulgare L., O. munitiflorum Hausskn., and O. majorana L. (cultivated using organic fertilizer), two from Salvia sclarea L. (organic and chemical fertilizers), one from S. officinalis L. (organic fertilizer), and one from Satureja cuneifolia Ten. (organic fertilizer) by a spectrophotometric method of Ellman using ELISA microplate-reader at 1 mg/ml concentration. In addition, a number of single components widely encountered in most of the essential oils [gamma-terpinene, 4-allyl anisole, (-)-carvone, dihydrocarvone, (-)-phencone, cuminyl alcohol, cumol, 4-isopropyl benzaldehyde, trans-anethole, camphene, iso-borneol, (-)-borneol, L-bornyl acetate, 2-decanol, 2-heptanol, methyl-heptanol, farnesol, nerol, iso-pulegol, 1,8-cineole, citral, citronellal, citronellol, geraniol, linalool, alpha-pinene, beta-pinene, piperitone, iso-menthone, menthofurane, linalyl oxide, linalyl ester, geranyl ester, carvacrol, thymol, menthol, vanilline, and eugenol] was also screened for the same activity in the same manner. Almost all of the essential oils showed a very high inhibitory activity (over 80%) against both enzymes, whereas the single components were not as active as the essential oils.     

Ursolic acid of Origanum majorana L. reduces Abeta-induced oxidative injury

Amyloid beta protein (Abeta) increases free radical production and lipid peroxidation in PC12 nerve cells, leading to apoptosis and cell death. The effect of ursolic acid from Origanum majorana L. on Abeta-induced neurotoxicity was investigated using PC12 cells. Pretreatment with isolated ursolic acid and vitamin E prevented the PC12 cell from reactive oxygen species (ROS) toxicity that is mediated by Abeta. The ursolic acid resulted in decreased Abeta toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and trypan blue assay. Thus, treatment with these antioxidants inhibited the Abeta-induced neurotoxic effect. Therefore, these results indicate that micromolar Abeta-induced oxidative cell death is reduced by ursolic acid from Origanum majorana L.     

Chemical composition and fumigant toxicity of some essential oils against Ephestia kuehniella

The chemical constituents of some essential oils extracted from aromatic plants (savory, Satureja thymbra L.; Turkish oregano, Origanum onites L.; myrtle, Myrtus communis L.; marjoram, Origanum majorana L.; laurel, Laurus nobilis L.; lemon, Citrus limon L.; sticky goosefoot, Chenopodium botrys L.; and tansy, Tanecetum armenum [DC.] Suchultz Bip.) were analyzed by gas chromatography-mass spectrometry. Fumigant toxicity of volatile compounds was tested against Mediterranean flour moth, Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae), eggs and adults. Percentage of mortality and longevity of E. kuehniella adults were estimated after essential oil treatments. LC50 and LC99 values were determined for each exposure time for each essential oil. Marjoram and lemon oils were the most effective of all the essential oils tested. The major components were linalool, 1.8-cineole, citral, 2-(4a.8-dimethyl-1.2.3.4.4a.5.6.7-octahydro-naphthalen-2-yl)-prop-2-en-l-ol, and p-cymene for marjoram, laurel, lemon, goosefoot, and tansy, respectively. The LC50 and LC99 values were estimated as 3.27 and 5.13 microl liter(-1) air for marjoram and 4.05 and 5.57 microl liter(-1) air for lemon essential oils at the longest exposure time. Decreasing longevity effect of marjoram and lemon was more prominent compared with other essential oils. We suggest that essential oils obtained from certain aromatic plants have potential as fumigants for stored product pests.     

A comparative study of cytotoxic, membrane and DNA damaging effects of Origanum majorana’s essential oil and its oxygenated monoterpene component linalool on parental and epirubicin-resistant H1299 cells

In this study, cytotoxic, membrane and DNA damaging effects of the essential oil from Origanum majorana and its oxygenated monoterpene component linalool were tested on parental and epirubicin-resistant (drug-resistant) human lung cancer cell lines (H1299). Essential oil’s and linalool’s cytotoxicities were examined and parental cells were found more sensitive to the essential oil’s and linalool’s cytotoxicities than drug-resistant cells. O. majorana essential oil had more effective membrane damaging effect than linalool on parental cells, while in drug-resistant H1299 cells, linalool had more effective membrane damaging effect than the essential oil. O. majorana essential oil possessed more effective DNA damaging effect than linalool on both parental and drug-resistant cells. The conclusions from this study suggest that O. majorana essential oil and linalool exhibit cytotoxic, membrane and DNA damaging effects. They thus need further investigation as potential therapeutic agents for human lung cancer.     

Phenolic antioxidants from herbs and spices

Spices and herbs are recognized as sources of natural antioxidants and thus play an important role in the chemoprevention of diseases resulting from lipid peroxidation. Our studies on spices and herbs have given us over a hundred compounds, known and new, having high antioxidant activity. From the Labiatae family, Rosmarinus officinalis, Thymus vulgaris, Origanum vulgare and O. majorana gave 26 active comopounds. Over 40 antioxidative compounds from Zingiber officinale, 26 compounds from Curcuma domestica = C. longa, C. xanthorrhiza and Z. cassumunar were determined, these belonging to the family Zingiberaceae. From the family Myrtaceae, 25 compounds from the berries of Pimenta dioica were determined and 3 carbazoles were isolated from Murraya koenigii. Structure-activity relationships of some of the isolated compounds were also discussed.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-bornyl pyrophosphate

Enzymes from Salvia officinalis and Tanacetum vulgare leaf epidermis catalyze the conversion of the acyclic precursor geranyl pyrophosphate to the cyclic monoterpenes (+)- and (-)-bornyl pyrophosphate, respectively. The antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H] Linalyl pyrophosphate (3H:14C = 5.22) was tested as a substrate with the cyclases from both sources to determine the configuration of the cyclizing intermediate. This substrate yielded (-)-bornyl pyrophosphate with 3H:14C ratio greater than 31, indicating specific utilization of (+)-(3S)-linalyl pyrophosphate as predicted. With the (+)-bornyl pyrophosphate cyclase, the 3H:14C ratio of the product was about 4.16, indicating a preference for the (-)-(3R)-enantiomer, but the ability also to utilize (+)-(3S)-linalyl pyrophosphate. (3R)- and (3S)-[1Z-3H]Linalyl pyrophosphate were separately compared to the achiral precursors [1-3H] geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. All functional precursors afforded optically pure (-)-(1S,4S)-bornyl pyrophosphate with the T. vulgare-derived cyclase (as determined by chromatographic separation of diastereomeric ketals of the derived ketone camphor), and (+)-(3S)-linalyl pyrophosphate was the preferred substrate. With the (+)-bornyl pyrophosphate cyclase from S. officinalis, geranyl, neryl, and (-)-(3R)-linalyl pyrophosphates gave the expected (+)-(1R,4R)-stereoisomer as the sole product, and (-)-(3R)-linalyl pyrophosphate was the preferred substrate. However, (3S)-linalyl pyrophosphate yielded (-)-(1S,4S)-bornyl pyrophosphate, albeit at lower rates, indicating the ability of this enzyme to catalyze the anomalous enantiomeric cyclization.     

Ontogenic diet shifts and scale-eating in Roeboides dayi, a Neotropical characid

Scale feeding (lepidophagy) has been documented for a variety of Neotropical fish taxa, including the characid genus Roeboides. Ontogenetic differentiation of jaws and snout teeth allow larger Roeboides to remove scales, however, the less specialized tooth/jaw structure in Roeboides dayi, indicates that it may be a facultative scale feeder. Population dynamics and diets of R. dayi in a Venezuelan lowland swamp/creek and a piedmont stream were compared over an annual cycle. Juvenile R. dayi consumed aquatic insect larvae and microcrustacea, and although spawning was year-round at both sites, most reproduction occurred during the wet season when the availability of these resources was greatest for juveniles. At both sites, larger R. dayi fed on a combination of invertebrate prey and fish scales, the former being more important at the piedmont site, and the latter being especially important during initial low water conditions at both sites. In the lowland stream, the reduction of aquatic habitat during the early dry season created higher fish densities and a more profitable environment for scale-feeders. Insectivory probably was less profitable during this early low water period due to interspecific competition for reduced aquatic insect stocks.     

Tracking structural features leading to resistance of activated protein C to alpha 1-antitrypsin

Activated protein C (APC) is a multi-modular anticoagulant serine protease, which degrades factor V/Va and factor VIIIa. Human APC (hAPC) is inhibited by human alpha 1-antitrypsin (AAT), while the bovine enzyme (bAPC) is fully resistant to this serpin. Structural features in the catalytic domains between the two species cause this difference, but detailed knowledge about the causal molecular difference is missing. To gain insight into the APC-AAT interaction and to create a human protein C resistant to AAT inhibition, we have used molecular modeling and site-directed mutagenesis. First, a structural model for bAPC based on the Gla-domainless X-ray structure of hAPC was built. Screening the molecular surface of the human and bovine APC enzymes suggested that a hAPC molecule resistant to AAT inhibition could be constructed by substituting only a few amino acids. We thus produced recombinant hAPC molecules with a single mutation (S173E, the numbering follows the chymotrypsinogen nomenclature), two mutations (E60aS/S61R) or a combination of all these substitutions (E60aS/S61R/S173E). Amidolytic and anticoagulant activities of the three mutant APC molecules were similar to those of wild-type hAPC. Inhibition of wild-type hAPC by AAT was characterized by a second-order rate constant (k2) of 2.71 M-1 s-1. The amino acid substitution at position 173 (S173E mutant) led to partial resistance to AAT (k2 = 0.84 M-1 s-1). The E60aS/S61R mutant displayed mild resistance to AAT inhibition (k2 = 1.70 M-1 s-1), whereas the E60aS/S61R/S173E mutant was inefficiently inactivated by AAT (k2 = 0.40 M-1 s-1). Inhibition of recombinant APC molecules by the serpin protein C inhibitor (PCI) in the presence and absence of heparin was also investigated.     

A study of the larvicidal activity of Origanum (Labiatae) species from southwest Turkey.

The insecticidal activity of essential oils isolated from two species of Origanum (Origanum onites L. and Origanum minutiflorum (O. Schwarz and P.H. Davis) (Labiate)) was investigated against 3rd and 4th instar larvae of the mosquito, Culex pipiens L. (Diptera: Culicidae). The LC50 and LC90 values were 24.8 and 61.3 ppm, and 73.8 and 118.9 ppm, respectively. This activity was attributed to the carvacrol, the major constituent of these oils.     

Ontogenetic diet shifts in Roeboides affinis with morphological comparisons

Fishes of the characid genus Roeboides are documented as lepidophagous, but the degree of specialization on scales varies between species. This study examines ontogenetic changes in morphology and diet of Roeboides dayi and Roeboides affinis from Venezuela. Juveniles of both species feed on microcrustacea and insects during the wet season, when availability of these resources is greatest. As aquatic habitat decreases during the dry season, insect and microcrustacea availability decreases and fish densities increase. As the fish densities increase, scale consumption becomes more profitable. Interspecific differences in the degree of specialization of the teeth and jaws was associated with the degree of scale feeding. Adult R. dayi consumed scales in approximately equal proportions to insects. Adult R. affinis consumes approximately 100% scales, and has more specialized head morphology than R. dayi.     

Floral Scent Production in Clarkia (Onagraceae) (I. Localization and Developmental Modulation of Monoterpene Emission and Linalool Synthase Activity)

The flowers of many plants emit volatile compounds as a means of attracting pollinators. We have previously shown that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of approximately 8 to 12 volatile compounds[mdash]three monoterpenes and nine benzoate derivatives (R.A. Raguso and E. Pichersky [1994] Plant Syst Evol [in press]). Here we report that the monoterpene alcohol linalool is synthesized and emitted mostly by petals but to a lesser extent also by the pistil and stamens. Two linalool oxides are produced and emitted almost exclusively by the pistil. These three monoterpenes are first discernible in mature unopened buds, and their tissue levels are highest during the first 2 to 3 d after anthesis. Levels of emission by the different floral parts throughout the life span of the flower were correlated with levels of these monoterpenes in the respective tissues, suggesting that these monoterpenes are emitted soon after their synthesis. Activity of linalool synthase, an enzyme that converts the ubiquitous C10 isoprenoid intermediate geranyl pyrophosphate to linalool, was highest in petals, the organ that emits most of the linalool. However, linalool synthase activity on a fresh weight basis was highest in stigma and style (i.e. the pistil). Most of the linalool produced in the pistil is apparently converted into linalool oxides. Lower levels (0.1%) of monoterpene emission and linalool synthase activity are found in the stigma of Clarkia concinna, a nonscented relative of C. breweri, suggesting that monoterpenes may have other functions in the flower in addition to attracting pollinators.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of linalyl pyrophosphate to (-)-endo-fenchol

The conversion of geranyl pyrophosphate to (-)-endo-fenchol is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. To test this stereochemical scheme, phosphatase-free preparations of (-)-endo-fenchol cyclase from fennel (Foeniculum vulgare M.) fruit were repeatedly incubated with a sample of (3RS)-[1-3H2]linalyl pyrophosphate until approximately 50% of this precursor was converted to the bicyclic monoterpenol end product. The residual linalyl pyrophosphate was isolated and enzymatically hydrolyzed to the free alcohol, linalool, which was resolved by chiral phase capillary gas-liquid chromatography of the derived threo and erythro mixture of 1,2-epoxides. The predominance of the (3S)-enantiomer in the residual substrate indicated that the (3R)-enantiomer was preferred for the cyclization to (-)-(1S)-endo-fenchol. This conclusion was subsequently confirmed by the preparation and direct testing of (3R)-1Z-[1-3H] linalyl pyrophosphate, which afforded a Km value lower than that observed for geranyl pyrophosphate and a relative velocity nearly three times higher. (3S)-1Z-[1-3H]Linalyl pyrophosphate was not an effective substrate for (-)-endo-fenchol biosynthesis but did, by an anomalous cyclization, give rise to low levels of the enantiomeric (+)-(1R)-endo-fenchol as well as to other products. These results support the proposed stereochemical model and also suggest that the isomerization step is rate limiting in the coupled isomerization-cyclization of geranyl pyrophosphate to (-)-endo-fenchol.     

Aromatic amino acid aminotransferase activity and indole-3-acetic acid production by associative nitrogen-fixing bacteria

In this work, we report the detection of aromatic amino acid aminotransferase (AAT) activity from cell-free crude extracts of nine strains of N(2)-fixing bacteria from three genera. Using tyrosine as substrate, AAT activity ranged in specific activity from 0.084 to 0.404 micromol min(-1)mg(-1). When analyzed under non-denaturating PAGE conditions; and using tryptophan, phenylalanine, tyrosine, and histidine as substrates Pseudomonas stutzeri A15 showed three isoforms with molecular mass of 46, 68 and 86 kDa, respectively; Azospirillum strains displayed two isoforms which molecular mass ranged from 44 to 66 kDa and Gluconacetobacter strains revealed one enzyme, which molecular mass was estimated to be much more higher than those of Azospirillum and P. stutzeri strains. After SDS-PAGE, some AAT activity was lost, indicating a differential stability of proteins. All the strains tested produced IAA, especially with tryptophan as precursor. Azospirillum strains produced the highest concentrations of IAA (16.5-38 microg IAA/mg protein), whereas Gluconacetobacter and P. stutzeri strains produced lower concentrations of IAA ranging from 1 to 2.9 microg/mg protein in culture medium supplemented with tryptophan. The IAA production may enable bacteria promote a growth-promoting effect in plants, in addition to their nitrogen fixing ability.     

Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation.

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.     

The involvement of aspartate aminotransferases in ammonium assimilation in lupin nodules

Aspartate aminotransferase (AAT) activity has been detected in the plant and bacteroid fractions of lupin nodules, and in free-living Rhizobium lupini. Two electrophoretically distinct forms of AAT were detected in the plant fraction of the nodule and a third form in the bacteroid fraction. AAT activity increased in the plant fraction during nodule development and this increase may be due to an increase in the activity of one of the AAT forms in this fraction. The single form of AAT detected in the bacteroid fraction had the same electrophoretic mobility as that detected in free-living R. lupini. The nodulated roots of lupins, grown in a media supplemented with nitrate and ammonium, had a 3- and 4-fold lower activity of AAT and nitrogenase activity respectively, compared to the nodulated roots of plants grown in the absence of added nitrogen. A role for the plant AAT in ammonium assimilation in lupin nodules is proposed.