Juan M. Caba

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

Abstract. 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 roots 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−.

Detoxification of semisolid olive-mill wastes and pine-chip mixtures using Phanerochaete flavido-alba

Semisolid olive-mill residues, pine chips, and mixtures of both residues contain phytotoxic components capable of inhibiting germination and vegetative growth in plants. Solid-state cultures of Phanerochate flavido-alba on pine chips or mixtures of both residues reduce these phytotoxic effects in fermented substrates. The phenol and lipid contents in cultures detoxified by this fungus also decreases.

Nitrate- and Inoculation-enhanced Ethylene Biosynthesis in Soybean Roots as a Possible Mediator of Nodulation Control

Summary Root ethylene biosynthesis has been studied in soybean (Glycine max [L.] Merr.) cv. Bragg plants and its supernodulating (nts382 and nts1007) and non-nodulating (nod49 and nod139) mutants. Regardless of NO3 treatment, inoculation with Bradyrhizobium japonicum significantly increased root ethylene evolution rate, reaching a plateau between 24 and 48 h after inoculation, with the rates being significantly higher in 8 mmol L−1 fed roots (high) than in those given 1 mmol L-1 (low) during the time of experiment. This Inoculation Stimulated Ethylene Release (ISER) response appears to be related to the infection process and nodule development, as treatment with Ag+ (an inhibitor of ethylene action) at the moment of inoculation markedly increased nodule number of Bragg plants under both high and low NO3 concentrations. Compared with the parental Bragg, the near-isogenic nodulating mutants used in this study showed normal ethylene biosynthesis ability (ethylene evolution and ACC oxidase activity), although significant quantitative differences were detected among them. Whether these differences are causally related to the nodulation phenotype is not known. Our previous observations on the involvement of endogenous ethylene in the control of nodule number in alfalfa are therefore also applicable to soybean, a determinate nodule type legume. The results further suggest that effects other than an alteration of ethylene biosynthesis might have also been caused by the nts mutations.

Genotypic differences in nitrogen assimilation in Vicia faba: Effect of nitrate

Five inbred lines and a commercial cultivar of field-bean (Vicia faba) were inoculated with a strain of Rhizobium leguminosarum bv. viciae and grown, with or without nitrate, under axenic conditions in a growth chamber. There were significant (p<0.01) differences between lines in N yield, both in N-free solution (N2 fixed, fully symbiotic) and with added NO3−. Increase in the N yield of NO3−-fed plants compared with fully symbiotic plants also varied between genotypes, with line VF109 being the least responsive to NO3−. This may indicate nitrate tolerance in relation to N2 fixation. The indication was supported by the observations that nodule development and nodule activity (acetylene reduction) were less inhibited in VF109 in the presence of 8 mM NO3− than in the other lines of Vicia faba. Glutamine synthetase and glutamate synthase (GOGAT) activities appeared to be related to genotypic differences in symbiotic efficiency. This finding suggests that assays of nodule glutamine synthetase and NADH-glutamate synthase might be used in Vicia faba breeding programs to indicate capacity for symbiotic N2 fixation, particularly in the presence of NO3−. ei]Section editor: B G Rolfe

Agrobacterium infection of hemp (Cannabis sativa L.): establishment of hairy root cultures

Abstract 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 β-glucuronidase positive staining. To our knowledge, this represents the first reported protocol for the establishment of Cannabis sativa hairy root cultures.

Genotypic variability of nitrogen metabolism enzymes in nodulated roots of Vicia faba

Abstract Twenty-nine genotypes of Vicia faba were grown in the presence or absence of NO3− to study variability in root and nodule nitrate reductase (NR, EC 1.6.6.1) and nodule cytosolic glutamine synthetase (GS, EC 6.3.1.2) activities. These V. faba lines apparently lack constitutive root NR activity (NRA), whereas inducible activity was detected in all of them except line VF17. Although a marked genotypic variability was found for this activity (F-value = 400), the major factor affecting its expression was nitrate (95% of the total effect observed). Most of the lines, however, showed appreciable rates of constitutive NRA in their nodules, that further declined with 4 mM NO3−. High genotypic variability was also found for the level of GS activity from the plant fraction of V. faba nodules. This activity, little affected by 2 mM NO3−, was generally declined by 4 m m for a week at the end of the experiment. No correlation was found between the activities measured here and measurements of symbiotic performance. Nodule NR, however, correlated inversely with both GS and nodule protein content.

NITROGEN FIXATION AND CARBON METABOLISM IN VICIA FABA ROOT NODULES OVER ONTOGENY

The evolution of growth, nodulation, nitrogen fixation, and activities of root-nodule enzymes related to sucrose breakdown (sucrose synthase, alkaline invertase), pentose phosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase), malate dehydrogenase, phosphoenolpyruvate carboxylase, and NADP-dependent isocitrate dehydrogenase, were followed over the complete life-cycle of faba bean in a growth chamber. The aim was to study the ontogenic changes of these parameters to obtain information on the carbon metabolism in nodules of Vicia faba , an amide-exporting legume. The maximum values of the relative growth rate and the specific acetylene-reducing activity were registered during the vegetative period. At pod-filling, the specific and total acetylene-reducing activity per plant declined in parallel with the enzyme activities of carbon metabolism. Contrary to reports for other legume species, in V. faba root nodule activity of sucrose synthase exceeded alkaline invertase values by 2-fold or more during the vegetative period. The activity of the two enzymes was similar at flowering and pod formation. The enzymes of carbon metabolism registered two maxima, one before and one after a trough (day 32), which marked the change from the vegetative to the reproductive period.

Hairy Root Culture as a Biotechnological Tool in C. sativa

Hemp (Cannabis sativa L.), a difficult to transform plant, has been effectively infected with either Ri or Ti plasmid-bearing agrobacteria and several transformed tissues (tumors and hairy roots) were established and its transgenic nature confirmed. Hypocotyl of intact seedlings was the most responsive material and the response depended on both bacterial strain and plant variety. Transformed tissues 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. The presence of some nitrogen compounds, metabolites of pharmaceutical implication, has been assayed in these transformed roots. Atropine, choline and muscarine were detected at quantitative levels in transformed roots and untransformed plant material of C. sativa. Further, the three compounds are present in hairy roots at concentrations quantitatively higher than in untransformed control tissues. Finally, hemp transformed roots exhibited a high callusing response, with calli that grew vigorously and developed compact and green nodular areas on the surface, a priori indicative of organogenesis capacity, but that were unable of shoot regeneration.

Nitrate uptake by Vicia faba L. Plants: A physiological approach 1

Abstract This study was conducted to obtain information on the properties of the nitrate (NO3 ‐) uptake system in Vicia faba. The results showed that in plants grown in the absence of NO3 ‐, they cannot absorb it upon initial exposure to this anion. When this plant makes contact with environmental NO3 ‐, NO3 ‐uptake by the roots is progressively induced. However, in NO3 ‐‐grown plants, the uptake system is yet induced so they had the ability to take up NO3 ‐ at high rates from the beginning. The Michaelis’ constant (K,M) and maximum uptake rate (Vmax) were estimated from measurements of NO3 ‐ depletion in the uptake medium. The apparent KM value for net NO3 ‐ uptake in induced V. faba plants was 60 μM and the corresponding Vmax 0.44 umol NO3 ‐ (g FW)‐1 h‐1. This last value was low compared with that of other species, while KM is in the range for other higher plants. From the results of our experiments at low and high NO3 ‐ concentrations, we discuss the possibility of the existence of two NO3 ‐ uptake sys...

Effect of combined nitrogen supply and nodulation on nitrate reductase activity and growth of pea plants

Abstract Nitrate reductase activity (NRA; EC 1.6.6.1) was measured in leaves, stems and roots of Pisum sativum cv. Lincoln supplied with different nitrate concentrations and inoculated with selected Rhizobium leguminosarum strains. As a control, noninoculated plants were grown in the same nutrient medium. NRA was determined by an in vivo‐nitrate assay. Although differences in tissue NRA were mainly related to nitrate concentration in the growing medium, nodulation much affected the NRA in the different plant organs, especially in root. An increased proportion of total plant NRA occurred in the leaves and stems as nitrate concentration was increased. Nitrate accumulation in leaves, stems and roots was correlated with the concentration of this ion in the nutrient solution. Nodulation also affects the nitrate accumulation in the different plant organs.