Localization of p210-related proteins in green flagellates and analysis of flagellar assembly in the green alga Dunaliella bioculata with monoclonal anti-p210

Summary.  Recently, p210 was identified as a component of the flagellar basal apparatus in the green flagellate Spermatozopsis similis. In a search for potential homologues to p210, isolated cytoskeletons of several green flagellates were probed with a monoclonal antibody, BAS4.13, against p210. In Western blots, cross-reacting bands in the molecular-mass range of 210 kDa were detected only in the quadriflagellate Spermatozopsis exsultans. As described earlier for S. similis, the flagellar transition region was decorated in Chlamydomonas reinhardtii and several other green flagellates, whereas in the marine alga Dunaliella bioculata the antigen was present in the proximal part of the axoneme. Double immunofluorescence of D. bioculata with an antitubulin antibody further revealed dotlike signals at sites where the probasal bodies are located. Since most of the antigen in D. bioculata was located in the axoneme, deflagellation offered a possibility to study the kinetics of its incorporation during flagellar regeneration. The antigen was only detected after a flagellum reached a length of 3–4 μm and its integration into the growing flagellar proceeded from proximal to distal. A similar delay in the incorporation of the antigen was also observed during flagellar assembly on new basal bodies during cell division. Thus, the antigen of BAS4.13 was incorporated late and from proximal to distal into the growing flagellum. We conclude that the pace and site by which individual proteins are integrated into the flagellum differ greatly. Received February 18, 2002; accepted May 17, 2002; published on line October 31, 2002 RID="*" ID="*" Correspondence and reprints: Botanisches Institut, Universit?t zu K?ln, Gyrhofstrasse 15, 50931 K?ln, Federal Republic of Germany     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

Identification of possible signal transduction components mediating light induction of the Gsa gene for an early chlorophyll biosynthetic step in Chlamydomonas reinhardtii

 Light-induced expression of the Gsa gene encoding the heme and chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase in Chlamydomonas reinhardtii was previously shown to involve Ca²⁺ and calmodulin (CaM) (C. lm et al. 1996, Plant Cell 8: 2245–2253). To further analyze the signal transduction pathway for light-induced Gsa expression, the effects of several pharmacological agents were examined. Treatment of light-dark synchronized cells with the heterotrimeric G-protein agonist Mas-7 caused partial induction of Gsa in the dark. The phospholipase C inhibitor U73122 inhibited light induction of Gsa. Exposure of cells to light caused a sustained 3-fold increase in cellular d-inositol 1,4,5-trisphosphate (InsP₃) concentration. KN-93, a specific inhibitor of Ca²⁺/CaM-dependent protein kinase II, inhibited light induction of Gsa. In contrast, cyclosporin A, a specific inhibitor of the Ca²⁺/CaM-dependent phosphoprotein phosphatase calcineurin, did not affect light induction of Gsa. These results, together with the earlier results, suggest the involvement of a canonical signal transduction pathway for light-regulated Gsa expression that involves a heterotrimeric G-protein activation, phospholipase C-catalyzed InsP₃ formation, InsP₃-dependent Ca²⁺ release, and activation of a downstream signaling pathway through a Ca²⁺/CaM-dependent protein kinase. Received: 21 October 1999 / Accepted: 3 December 1999     

Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study

In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalancho? species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.     

Phenylcoumaran benzylic ether reductase, a prominent poplar xylem protein, is strongly associated with phenylpropanoid biosynthesis in lignifying cells

 It has previously been shown (D.R. Gang et al., 1999, J Biol Chem 274: 7516–7527) that the most abundant protein in the secondary xylem of poplar (Populus trichocarpa cv. `Trichobel') is a phenylcoumaran benzylic ether reductase (PCBER), an enzyme involved in lignan synthesis. Here, the distribution and abundance of PCBER in poplar was studied at both the RNA and protein level. The cellular expression pattern was determined by immunolocalization of greenhouse-grown plants as well as of a field-grown poplar. Compared to other poplar tissues, PCBER is preferentially produced in the secondary xylem of stems and roots and is associated with the active growth period. The protein is present in all cells of the young differentiating xylem, corresponding to the zone of active phenylpropanoid metabolism and lignification. In addition, PCBER is located in young differentiating phloem fibers, in xylem ray parenchyma, and in xylem parenchyma cells at the growth-ring border. Essentially the same expression pattern was observed in poplars grown in greenhouses and in the field. The synthesis of PCBER in phenylpropanoid-synthesizing tissues was confirmed in a bending experiment. Induction of PCBER was observed in the pith of mechanically bent poplar stems, where phenylpropanoid metabolism is induced. These results indicate that the products of PCBER activity are synthesized mainly in lignifying tissues, suggesting a role in wood development. Received: 28 September 1999 / Accepted: 15 March 2000     

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

 During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents. Received: 14 February 2000 / Accepted: 15 March 2000     

Photosynthetic apparatus organization and function in the wild type and a chlorophyll b-less mutant of Chlamydomonas reinhardtii. Dependence on carbon source

 The assembly, organization and function of the photosynthetic apparatus was investigated in the wild type and a chlorophyll (Chl) b-less mutant of the unicellular green alga Chlamydomonas reinhardtii, generated via DNA insertional mutagenesis. Comparative analyses were undertaken with cells grown photoheterotrophically (acetate), photomixotrophically (acetate and HCO⁻ ₃) or photoautotrophically (HCO⁻ ₃). It is shown that lack of Chl b diminished the photosystem-II (PSII) functional Chl antenna size from 320 Chl (a and b) to about 95 Chl a molecules. However, the functional Chl antenna size of PSI remained fairly constant at about 290 Chl molecules, independent of the presence of Chl b. Western blot and kinetic analyses suggested the presence of inner subunits of the Chl a-b light-harvesting complex of PSII (LHCII) and the entire complement of the Chl a-b light-harvesting complex of PSI (LHCI) in the mutant. It is concluded that Chl a can replace Chl b in the inner subunits of the LHCII and in the entire complement of the LHCI. Growth of cells on acetate as the sole carbon source imposes limitations in the photon-use efficiency and capacity of photosynthesis. These are manifested as a lower quantum yield and lower light-saturated rate of photosynthesis, and as lower variable to maximal (F_v/F_max) chlorophyll fluorescence yield ratios. This adverse effect probably originates because acetate shifts the oxidation-reduction state of the plastoquinone pool, and also because it causes a decrease in the amount and/or activity of Rubisco in the chloroplast. Such limitations are fully alleviated upon inclusion of an inorganic carbon source (e.g. bicarbonate) in the cell growth medium. Further, the work provides evidence to show that transformation of green algae can be used as a tool by which to generate mutants exhibiting a permanently truncated Chl antenna size and a higher (per Chl) photosynthetic productivity of the cells. Received: 10 November 1999 / Accepted: 22 December 1999     

Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: definition of the light environment under which phosphoribulokinase is not in large excess

 To test the hypothesis that the contribution of phosphoribulokinase (PRK) to the control of photosynthesis changes depending on the light environment of the plant, the response of transgenic tobacco (Nicotiana tabacum L.) transformed with antisense PRK constructs to irradiance was determined. In plants grown under low irradiance (330 μmol m⁻² s⁻¹) steady-state photosynthesis was limited in plants with decreased PRK activity upon exposure to higher irradiance, with a control coefficient of PRK for CO₂ assimilation of 0.25 at and above 800 μmol m⁻² s⁻¹. The flux control coefficient of PRK for steady-state CO₂ assimilation was zero, however, at all irradiances in plant material grown at 800 μmol m⁻² s⁻¹ and in plants grown in a glasshouse during mid-summer (alternating shade and sun 300–1600 μmol m⁻² s⁻¹). To explain these differences between plants grown under low and high irradiances, Calvin cycle enzyme activities and metabolite content were determined. Activities of PRK and other non-equilibrium Calvin cycle enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase-oxygenase were twofold higher in plants grown at 800 μmol m⁻² s⁻¹ or in the glasshouse than in plants grown at 330 μmol m⁻² s⁻¹. Activities of equilibrium enzymes transketolase, aldolase, ribulose-5-phosphate epimerase and isomerase were very similar under all growth irradiances. The flux control coefficient of 0.25 in plants grown at 330 μmol m⁻² s⁻¹ can be explained because low ribulose-5-phosphate content in combination with low PRK activity limits the synthesis of ribulose-1,5-bisphosphate. This limitation is overcome in high-light-grown plants because of the large relative increase in activities of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase under these conditions, which facilitates the synthesis of larger amounts of ribulose-5-phosphate. This potential limitation will have maintained evolutionary selection pressure for high concentrations of PRK within the chloroplast. Received: 15 November 1999 / Accepted: 27 January 2000     

Analytical electron microscopical investigations on the apoplastic pathways of lanthanum transport in barley roots

 In transmission electron microscopy studies, lanthanum ions have been used as electron-opaque tracers to delineate the apoplastic pathways for ion transport in barley (Hordeum vulgare L.) roots. To localize La³⁺ on the subcellular level, e.g. in cell walls and on the surface of membranes, electron-energy-loss spectroscopy and electron-spectroscopic imaging were used. Seminal and nodal roots were exposed for 30 min to 1 mM LaCl₃ and 10 mM LaCl₃, respectively. In seminal roots, possessing no exodermis, La³⁺ diffusion through the apoplast was stopped by the Casparian bands of the endodermis. In nodal roots with an exodermis, however, La³⁺ diffusion through the cortical apoplast had already stopped at the tight junctions of the exodermal cell walls resembling the Casparian bands of the endodermis. Therefore, we conclude that in some specialized roots such as the nodal roots of barley, the physiological role of the endodermis is largely performed by the exodermis. Received: 28 July 1999 / Accepted: 24 February 2000     

Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus officinalis plants

Two-year-old rosemary (Rosmarinus officinalis L.) plants were subjected to severe stress by exposure to prolonged drought during a Mediterranean summer. Severely stressed plants recovered completely after the autumn rainfalls although the relative water content remained below 35% for 3 months and the chlorophyll content of leaves was reduced by up to 85% during the drought. In severe stress: (i) α-tocopherol increased 9-fold per g dry weight and 20-fold per unit of chlorophyll; (ii) lutein and β-carotene contents decreased on a dry-weight basis, but an 80% increase in lutein and constant levels of β-carotene were observed on a chlorophyll basis; (iii) there were transient and sustained increases in the de-epoxidation state of the xanthophyll cycle; and (iv) the highly oxidised abietane diterpene isorosmanol increased 8-fold as a result of the oxidation of carnosic acid. With the autumn rainfalls, water status, α-tocopherol and violaxanthin recovered first and the levels of photosynthetic pigments and abietane diterpenes increased later. The photoprotection conferred by the xanthophyll cycle and the antioxidant function of tocopherols, lutein and diterpenes may help to avoid irreversible damage in severe drought, making possible the recovery of functional membranes after the autumn rainfalls. Besides, chlorophyll loss reduces the amount of photons absorbed by leaves, which enhances the photoprotective and antioxidant capacity of leaves per amount of photons absorbed, since the ratios of xanthophylls, α-tocopherol and abietane diterpenes to chlorophyll increase. Received: 12 July 1999 / Accepted: 25 November 1999     

Simulating ozone detoxification in the leaf apoplast through the direct reaction with ascorbate

 This paper presents a mathematical model which enables the semi-quantification of ozone (O₃) detoxification, based upon the direct reaction of the pollutant with ascorbate (ASC) located in the aqueous matrix associated with the cell wall (i.e. the apoplast). The model describes the uptake of ozone into the leaf and its direct reaction with ASC, taking into consideration the regeneration of dehydroascorbic acid in the cytosol, the rate of replenishment of cell wall ASC and the distribution of ASC between sub-cellular compartments – based upon the permeability of biomembranes to the neutral species, ascorbic acid and the pH of various sub-cellular compartments. The importance of various physico-chemical characteristics (e.g. stomatal conductance, mesophyll cell wall thickness and tortuosity, chloroplast volume, apoplast pH, ASC:O₃ reaction stoichiometry) in mediating the flux of ozone to the plasmalemma is analysed. Model simulations, supported by experimental observations, suggest that the ASC concentration in the leaf apoplast is high enough to scavenge a significant proportion of the O₃ taken up into the leaf interior, under environmentally relevant conditions. However, there is considerable variation between taxa in the potential degree of protection afforded by apoplastic ASC, emphasizing the need for an improved understanding of the reaction chemistry of O₃ in the cell wall. Received: 13 May 1999 / Accepted: 5 August 1999     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999     

Immunolocalization of LeAGP-1, a modular arabinogalactan-protein, reveals its developmentally regulated expression in tomato

 Arabinogalactan-proteins (AGPs) are highly glycosylated cell surface proteins that are thought to function in plant growth and development. The developmentally regulated expression of LeAGP-1, a novel and major AGP in tomato, was examined in different organs and tissues of tomato (Lycopersicon esculentum Mill. cv. UC82B) plants with an anti-peptide antibody (i.e. the PAP antibody) directed specifically against the lysine-rich subdomain of the LeAGP-1 core protein. During cell differentiation in tomato plants, LeAGP-1 was associated with cell wall thickening and lignification of particular cell types. Specifically, LeAGP-1 was detected in secondary wall thickenings of maturing metaxylem and secondary xylem tracheary elements in roots and stems, and in thickened cell walls of phloem sieve elements. However, LeAGP-1 was also present in thin-walled, cortical parenchyma cells of seedling roots as well as thick-walled collenchyma cells in young stems, both of which are not lignified. Based on these observed patterns, possible roles for LeAGP-1 in plant growth and development are discussed. Received: 17 August 1999 / Accepted: 7 October 1999     

Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.)

 Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants. Received: 12 July 1999 / Accepted: 16 October 1999     

Light-stress-related changes in the properties of photosystem I

Light-stress-related changes in photosystem I (PS I) were analyzed in photoautotrophic cultured cells of Marchantia polymorpha L. High light treatment (30␣h; 1300 μmol photons · m⁻² · s⁻¹) reduced the PS I-mediated electron-transport rate by more than 50% and the photochemical efficiency of photosystem II (PS II) by about 35%. In photoinhibited cells, 76% of the PS II centers remained closed in low light, which is in agreement with a preferential impairment of PS I. Our data indicate that excessive linear electron transport is a cause of the loss in PS I activity. Two PS I forms could be isolated by sucrose-gradient ultracentrifugation of mildly solubilized thylakoid membranes. After high-light treatment one of these forms, which showed a larger light-harvesting complex (LHC) I antenna and a specific association of LHC IIb, was enriched. The effect could be suppressed by blockage of linear electron transport. It is suggested that PS I inactivation and state transitions caused the change in PS I organisation. Received: 19 August 1996 / Accepted: 24 October 1996     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 2000     

Mutations causing defects in the biosynthesis and response to gibberellins, abscisic acid and phytochrome B do not inhibit vernalization in Arabidopsis fca-1

 The roles of gibberellins, abscisic acid and phytochrome B in the vernalization response were investigated by combining mutations causing defects in their biosynthesis and response with the Arabidopsis thaliana (L.) Heynh. fca-1 mutation. The fca-1 mutation confers a very late-flowering phenotype which can be reversed to wild-type flowering if the seedlings are vernalized. Vernalization was unaffected in ga1-3, gai, abi1-1, abi2-1, abi3-1 and phyB-1 backgrounds, suggesting that gibberellin action mediated via GA1 and GAI, abscisic acid action mediated through ABI1 and ABI2, and phytochrome B, function independently of vernalization. However, the mutations did interact with fca-1 to change flowering time in the absence of vernalization. The abi1 fca-1 and abi2 fca-1 double mutants flowered earlier than fca-1 implying a role for abscisic acid in floral repression. Combination of ga1-3 or gai with fca-1 unexpectedly resulted in opposite interactions, with gai partially suppressing the late flowering of fca-1. Received: 17 July 1999 / Accepted: 11 October 1999     

Inoculation and nitrate alter phytohormone levels in soybean roots: differences between a supernodulating mutant and the wild type

 The levels of different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) in roots of Glycine max [L.] Merr. cv. Bragg and its supernodulating mutant nts382 were compared for the first time. Forty-eight hours after inoculation with Bradyrhizobium, quantitative and qualitative differences were found in the root's endogenous hormone status between cultivar Bragg and the mutant nts382. The six quantified cytokinins, ranking similarly in each genotype, were present at higher concentrations (30–196% on average for isopentenyl adenosine and dihydrozeatin riboside, respectively) in mutant roots. By contrast, the ABA content was 2-fold higher in Bragg, while the basal levels of IAA [0.53 μmol (g DW)⁻¹, on average] were similar in both genotypes. In 1 mM NO₃ ⁻-fed Bragg roots 48 h post-inoculation, IAA, ABA and the cytokinins isopentenyl adenine, and isopentenyl adenosine quantitatively increased with respect to uninoculated controls. However, only the two cytokinins increased in the mutant. High NO₃ ⁻ (8 mM) markedly reduced root auxin concentration, and neither genotypic differences nor the inoculation-induced increase in auxin concentration in Bragg was observed under these conditions. Cytokinins and ABA, on the other hand, were little affected by 8 mM NO₃ ⁻. Root IAA/cytokinin and ABA/cytokinin ratios were always higher in Bragg relative to the mutant, and responded to inoculation (mainly in Bragg) and nitrate (both genotypes). The overall results are consistent with the auxin-burst-control hypothesis for the explanation of autoregulation and supernodulation in soybean. However, they are still inconclusive with respect to the inhibitory effect of NO₃ ⁻. Received: 16 April 1999 / Accepted: 13 December 1999