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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 micromol (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-.

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Experimental conditions were optimized for hemp, a difficult to transform plant, to be effectively infected with either Ri or Ti plasmid-bearing agrobacteria and to establish stably transformed tissues. Hypocotyl of intact seedlings was the most responsive material and the response depended on both bacterial strain and plant variety. Transformed tissues, hairy roots and tumors, were cultured and stabilized in vitro and showed the characteristic traits of fast and phytohormone-independent growth as well as high incidence of lateral branching and abundance of root hairs in the case of roots. They all contained T-DNA of the corresponding Ri or Ti plasmid as revealed by PCR analysis with specific primers and further hairy roots induced by AR10GUS strain showed normal pattern of b-glucuronidase positive staining. To our knowledge, this represents the first reported protocol for the establishment of Cannabis sativa hairy root cultures.

Previously, we reported (a) a positive correlation between the nitrate concentrations in growth medium and ethylene evolved from uninoculated and inoculated alfalfa (Medicago sativa) roots and (b) a negative correlation between ethylene evolution and nodulation. Here, we report that the inhibitory effect of N03-on nodulation of alfalfa can be eliminated by the ethylene inhibitor aminoethoxyvinylglycine (AVG). This effect was probably related to the strong inhibition (90%) of ethylene biosynthesis caused by AVG in these inoculated and N03-treated roots. These results support our hypothesis that the inhibitory effect of N03-is mediated through the phytohormone ethylene. A possible role of endogenous ethylene in the autoregulation of nodulation also is discussed. AVG at 10 micromolar significantly (P < 0.05) increased total nitrogenase activity (acetylene reduction) in 2.5 and 5 millimolar N03-fed plants probably as a result of the very high stimulation of nodulation.The formation of a nitrogen-fixing root nodule is the consequence of a series of interactions between host plant and microsymbiont. The process is regulated by internal plant factors, which are termed autoregulation factors (5, 24) and environmental factors, of which NO3-is a major component in the inhibition of both nodulation and N2 fixation (8,28). The effect of nitrate is exerted soon after inoculation, inhibiting both initial cortical cells divisions and infection thread formation (21). Moreover, an interaction between NO3-and the autoregulation signal during

We previously reported that inhibition of ethylene biosynthesis with aminoethoxyvinylglycine (AVG) eliminated the inhibitory effect of NO 3 -on nodulation of alfalfa (Medicago sativa L. cv. Aragon) plants grown aeroponically. In this work, the effect of Ag + , as an inhibitor of ethylene action, has been studied in plants growing aeroponically or in darkened tubes with vermiculite, and low-nitrate or high-nitrate solution. Vermiculite-grown plants developed up to 3 times as many nodules as did those growing aeroponically. Nodule formation was mirrored by dry-matter accumulation. High (10 mol m -3 ) NO 3 -applied from planting inhibited nodulation to an equal extent (c. 50%) in the two growth conditions. In contrast, Ag + treatment increased nodule formation at all NO 3 -concentrations assayed under the two growth conditions, with the stimulation being higher in plants grown aeroponically. Finally, no effect of Ag + (10 mmol m -3 ) on plant growth was observed in either of the growth conditions. The effectiveness of NO 3 -as a nodulation inhibitor and enhancer of ethylene biosynthesis in roots of alfalfa was also studied. Within 24 h after inoculation, 10 mol m -3 NO 3 -exerted most of its inhibitory effect on nodulation. At the same time, both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and ethylene evolution rates markedly increased in inoculated and uninoculated alfalfa roots treated with NO 3 -. Support for a role of endogenous ethylene in the control of nodule formation in legumes is discussed.Key-words: Medicago sativa; endogenous ethylene; growth system; nitrate; nodulation control. INTRODUCTIONNodule formation in legumes is tightly controlled by an internal mechanism called autoregulation, but the establishment and function of the symbiosis is also regulated by fixed nitrogen (mainly nitrate) in the soil (Streeter 1988;Carroll & Mathews 1990;Gresshoff 1993). Basically, autoregulation involves early infection events that systemically suppress the development into nodules of subsequent infected cell-division foci (Caetano-Anollés & Gresshoff 1990); the result is restriction of nodulation to the root crown. The shoot is believed to be the source of the autoregulation inhibitor (Delves et al. 1986). Supernodulating mutants, which lack this autoregulatory response and show prolific nodulation, have been generated for several legume species. It is feasible that such a regulatory mechanism operates in other nodulated legumes, although variation in the mode of action may also exist (see Postma, Jacobsen & Fenstra 1988;Gremaud & Harper 1989;Akao & Kouchi 1992;Sagan & Gresshoff 1996). For example, nodule detachment experiments in alfalfa and soybean revealed both additional regulatory mechanisms controlling nodule number and different stages at which autoregulation could be exerted (CaetanoAnollés & Gresshoff 1991).Nitrate inhibition of nodulation is a common phenomenon among legume species; however, considerable variation in the degree of regulation exists even between cultivars or accessions with...

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Acetylene reduction activity (ARA) and leghemoglobin (Lb) content in nodules were sigificantly reduced when pea (Pisum sativum L. cv. Lincoln) plants were subjected to 50 mM sodium chloride stress for 3 weeks. C2H2 reduction activity by bacteriods isolated from pea nodules was drastically inhibited by saline stress, and malate appeared to be a more appropriate substrate than glucose or succinate in maintaining this activity. Salt added directly to the incubation mixture of bacteriods or to the culture medium of plants inhibited O2 uptake by bacteroids. Nodule cytosolic phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and bacteriod malate dehydrogenase (MDH; EC 1.1.1.37) activities were strongly enhanced by salt stress. Under these conditions, malate concentration was depressed in bacteroids and cytosol, whereas total soluble sugar (TSS)content slightly increased in both fractions. The effect of salt stress on TSS and malate content suggests that the utilization of carbohydrate within nodules could be inhibited during salt stress. The inhibitory effect of NaCl on N2 fixation activity of bacteroids and to the decrease in bacteroid respiration. The stimulation of fermentative metabolism induced by salinity suggests some reduction in O2 availability within the nodule. Salt stress was also responsible for a decrease of the cytosolic protein content, specifically of leghemoglobin, in the nodules.