Specific Nodule Activity Research Articles

Ancient CO2 levels favor nitrogen fixing plants over a broader range of soil N compared to present

Small inreases in CO2 stimulate nitrogen fixation and plant growth. Increasing soil N can inhibit nitrogen fixation. However, no studies to date have tested how nitrogen fixing plants perform under ancient CO2 levels (100 MYA), when nitrogen fixing plants evolved, with different levels of N additions. The aim of this study was to assess if ancient CO2, compared to present, favors nitrogen fixers over a range of soil nitrogen concentrations. Nitrogen fixers (Alnus incana ssp. rugosa, Alnus viridis ssp. crispa, and Alnus rubra) and their close non-nitrogen fixing relatives (Betula pumila, Betula papyrifera, Betula glandulosa) were grown at ancient (1600 ppm) or present (400 ppm) CO2 over a range of soil N levels, equivalent to 0, 10, 50, and 200 kg N ha−1 year−1. The growth of non-N fixing plants increased more than N fixing plants in response to the increasing N levels. When grown at an ancient CO2 level, the N level at which non-nitrogen fixing plant biomass exceeded nitrogen fixing plant biomass was twice as high (61 kg N ha−1 year−1) as the N level when plants were grown at the ambient CO2 level. Specific nodule activity was also reduced with an increasing level of soil N. Our results show there was a greater advantage in being a nitrogen fixer under ancient levels of CO2 compared with the present CO2 level.

The use of Tn5-mutants Bradyrhyzobium japonicum for regulation of prooxidant-antioxidant processes in soybean plants under drought condition

Aim. To investigate the use of soybean seed inoculation by nodule bacteria obtained by transposon mutagenesis to ensure the effective formation and functioning of symbiotic systems by regulating prooxidant - antioxidant processes and reducing the negative effects of drought on crop productivity. Methods. Microbiological, physiological, biochemical methods, gas chromatography and spectrophotometry. Results. It has been proved that due to the activation of protective antioxidant enzymes of catalase, ascorbate and guaiacol peroxidase in soybean roots and root nodules, adaptive rearrangements of plant metabolism occur aimed at stabilizing the content of prooxidants, hydrogen peroxide, in drought conditions. At the same time, the specific nitrogen-fixation activity of soybean root nodules undergoes no significant changes and indicates the preservation of the effective functioning of the symbiotic apparatus, is the result of activation of protective antioxidant processes and adaptation of the soybean symbiotic system with the participation of Tn-5 mutant Bradyrhizobium japonicum B1-20 to dehydration conditions. Conclusions. The use of inoculation of soybean seeds with the Tn-5 mutant Bradyrhizobium japonicum B1-20 leads to regulation of prooxidant - antioxidant protective processes in plants, helps to increase their nitrogen-fixation potential and maintain grain yield under prolonged exposure to drought. Keywords: soybean (Glycine max (L.) Merr.), hydrogen peroxide, catalase, ascorbate peroxidase, guaiacol peroxidase, drought.

Co-existence of Rhizobia and Diverse Non-rhizobial Bacteria in the Rhizosphere and Nodules of Dalbergia odorifera Seedlings Inoculated with Bradyrhizobium elkanii, Rhizobium multihospitium–Like and Burkholderia pyrrocinia–Like Strains

Rhizobia induce root nodules and fix atmospheric N2 for most legume species in exchange for carbon. However, the diverse endophytic non-rhizobial bacteria in legume nodules that co-exist with rhizobia are often ignored because they are difficult to cultivate using routine cultivation approaches. To enhance our understanding of the incidence and diversity of legume–bacteria associations, a high-throughput sequencing analysis of bacterial 16S rRNA genes was used to examine the bacterial community in the rhizospheres and root nodules of Dalbergia odorifera seedlings that were uninoculated or inoculated with Bradyrhizobium elkanii H255, Rhizobium multihospitium–like HT221, or Burkholderia pyrrocinia–like H022238, in two growth media (nitrogen [N]-supplied soil or N-omitted potting mix). Seedlings inoculated with Bradyrhizobium had significantly more nodules than seedlings in the other inoculation conditions, regardless of growth media. Using the 15N natural abundance method, it was shown that the inoculated plants had significantly higher N2 fixation efficiency (48–57%) and specific nodule activity [269–313 μg N mg−1 of dry weight (dwt) nodule] compared to the uninoculated plants (203 μg N mg−1 dwt nodule). The 16S rRNA gene analysis showed that there was generally a higher bacterial diversity in the rhizosphere than in the nodules in the corresponding condition. Both rhizobial inoculation and media status significantly altered the bacterial communities in the rhizospheres and nodules (P < 0.05), with the exception of the inoculated soil rhizospheres. Regarding non-rhizobial bacteria, three genera, i.e., Lactococcus, Bacillus, and Pseudomonas, were consistently enriched in the rhizosphere and Bradyrhizobium, Chloroplast norank (which belongs to Cyanobacteria), and Lactococcus were commonly found in the nodules. In contrast, common rhizobial genera (including Rhizobium, Mesorhizobium, and Burkholderia) were only present in the nodules at low relative abundances (0.01–3.41%). Regarding non-rhizobial bacteria, 32 genera were found in the nodules, with non-rhizobial bacteria being predominant in the N omitted potting mix (with a relative abundance of 56–87%). This study suggests that legume nodules are inhabited by a high diversity of non-rhizobial bacteria, which may play a vital role in nodulation and N2 fixation in the host plants.

Biosolids differently affect seed yield, nodule growth, nodule-specific activity, and symbiotic nitrogen fixation of field bean

The main aim of this research was to verify whether mineral nitrogen (N) continuously released by organic fertilisers during the field bean growth cycle may be sufficiently high to enhance plant growth and seed yield but sufficiently low that it does not negatively affect nodulation and symbiotic N2 fixation. Plants were grown without N fertilisation, and with mineral and organic N (biosolids) fertilisation. All plant parts were collected and dry matter, N content, %Ndfa, and N2 fixed were measured at 8th node, flowering, and maturity stages. Nodule specific activity, N derived from soil, and N remobilisation were estimated. The nitrate concentration of soil was also determined. Biosolids reduced nodule growth, nodule fixation activity, and N2 fixation during the vegetative but not the reproductive phase. During seed filling, nodule fixation activity increased and N2 fixation was roughly twice that of the Control plants. Biosolids increased seed yield by removing the imbalance between N demand and N supply for pod growth. This may be related to an increase in nodule-specific activity due to the reduction in mineral N in the soil.

Enhancing nitrogen fixation in soybean (Glycine max L.) by composting under saline stress condition

Soybean is susceptible to saline stress and reduced yield loss. Organic fertilization has several beneficial effects on agricultural crop fields. To minimize the deleteri­ous effects of the salinity organic fertilization is using which can provide economic yield under saline conditions. Therefore, a greenhouse research was conducted at the Department of Environmental Dynamics and Management, Hiroshima University, Japan to find out the role of compost in alleviating the adverse effects of salinity stress on soybean. The nitrogen-fixing activity was estimated by using gas chromatograph. The results revealed that salinity stress reduced the biological nitrogen fixation and specific nodule activity. Our results show that, there was a corresponding improve in nitrogen fixation with compost application.

Nod factor supply under water stress conditions modulates cytokinin biosynthesis and enhances nodule formation and N nutrition in soybean

ABSTRACTNod factors (NF) are molecules produced by rhizobia which are involved in the N2-fixing symbiosis with legume plants, enabling the formation of specific organs called nodules. Under drought conditions, nitrogen acquisition by N2-fixation is depressed, resulting in low legume productivity. In this study, we evaluated the effects of NF supply on nitrogen acquisition and on cytokinin biosynthesis of soybean plants grown under drought. NF supply to water stressed soybeans increased the CK content of all organs. The profile of CK metabolites also shifted from t-Z to cis-Z and an accumulation of nucleotide and glucoside conjugates. The changes in CK coincided with enhanced nodule formation with sustained nodule specific activity, which ultimately increased the total nitrogen fixed by the plant.

Roles of compost fertilizer on nitrogen fixation in soybean (Glycine max L.) under water deficit conditions

Presently, increasing nitrogen fixation in soybean is critical because of widespread increase in soil degradation in Egypt. Therefore, a greenhouse research was conducted at the laboratory of Plant Nutritional Physiology, Hiroshima University, Japan for assessing the impact of compost in alleviating the adverse effects of water stress on soybean. The nitrogen-fixing activity was estimated by using gas chromatograph. The results showed that water deficit stress reduced biological nitrogen fixation and specific nodule activity than normal irrigation conditions. Application of compost increased the biological nitrogen fixation and specific nodule activity under water stress conditions. As the results indicate that compost application could help in improving nitrogen fixation in soybean.

Pea nodule gradients explain N nutrition and limited symbiotic fixation in hypernodulating mutants

Legumes fix atmospheric nitrogen by symbiosis with soil bacteria in root nodules. Legume yields are limited by the low capacity of N2 fixation. Hypernodulating mutants have been selected decades ago to try to increase nodule number. However, literature data show that N fixation of hypernodulating mutants was not increased compared to parental lines. Here, we study the functional basis of limited N fixation associated to hypernodulation. We grew two wild type genotypes and nine hypernodulating mutants of pea in hydroponics in three greenhouse experiments. We measured the following traits related to N nutrition during the vegetative period: nodule number, plant N uptake, nodule-specific activity, and plant and nodule concentrations. Genetic and environmental variations induced nodule gradients. These gradients were used to set quantitative relationships between N nutrition traits and nodule number. We compared the relationships obtained for hypernodulating and for wild types. N nutrition traits were analysed together with C nutrition traits, through correlation networks. Our results show that higher nodule number of hypernodulating mutants is correlated with lower levels of nodule activity, from −25 to −60 %, by comparison to the wild type. Higher nodule number of hypernodulating mutants is also correlated with lower total N uptake by symbiotic fixation, from −0 to −60 %, by comparison to the wild type. Findings demonstrate that N nutrition is not a factor limiting growth in hypernodulating mutants, as shown by N nutrition index higher than 1, indicating N nutrition in excess. The correlations suggest that limited N2 fixation in hypernodulating mutants arises from restricted shoot growth, which limits the plant capacity to accumulate N. Furthermore, symbiotic efficiency decreased with increasing nodule number, down to a minimal value for hypernodulating mutants. Thus, to overcome the trade-off between N benefits from N2 fixation and carbon nodulation costs, the hypernodulation trait should be associated with high shoot growth capacity in breeding programs.

Soybean is less impacted by water stress using Bradyrhizobium japonicum and thuricin-17 from Bacillus thuringiensis

Climate change is forecasted to induce more drought stress events. Water scarcity is already the most limiting abiotic stress for crop production. With higher food demand, there is a need for sustainable solutions to cope with the loss of productivity due to water stress. It is known that plant growth-promoting rhizobacteria (PGPR) can colonize plant roots and increase plant growth. However, there is actually no sustainable method to decrease the impact of water stress. Therefore, we hypothesized that an application of thuricin-17, a molecule produced by the PGPR Bacillus thuringiensis, could enhance soybean tolerance to water stress. We grew soybean plants for 1 month in growth chambers in order to evaluate their response to thuricin-17 root application under drought, in association with the inoculation of N2-fixing Bradyrhizobium japonicum. We measured traits reflecting root architecture: number of tips, root diameter, root length, number of nodules; water fluxes: water potential, stomatal conductance; carbon nutrition: leaf area, photosynthetic rate, biomass and carbon partitioning; nitrogen nutrition: nitrogen partitioning and hormone signalling: abscisic acid concentration during the vegetative growth period. Our results show that thuricin-17 application under water stress increased plant biomass by 17 %, thus masking drought impact. This effect is due to modifications of below-ground structures, with 37 % increase of root and 55 % increase of nodule biomass, and to slight increases of leaf area and photosynthetic rate. We also observed that application of thuricin-17 induced a 30 % increase of root abscisic acid, an increase of root length and of leaf water potential. Finally, thuricin-17 induced an activation of nodule formation by 40 %, a partial restoration of nodule-specific activity, nodule growth and consequently, an increase by 17 % of the total nitrogen amount in the plant. Overall, our findings reveal a new method to decrease the negative impact of water stress. Results also demonstrate that the plant restored an adequate water and N balance by changing its root structure.

Photosynthetic down-regulation in N2-fixing alfalfa under elevated CO2 alters rubisco content and decreases nodule metabolism via nitrogenase and tricarboxylic acid cycle

Although responsiveness of N2-fixing plants to elevated CO2 conditions have been analyzed in previous studies, important uncertainties remain in relation to the effect enhanced CO2 in nodule proteomic profile and its implication in leaf responsiveness. The aim of our study was to deepen our understanding of the relationship between leaf and nodule metabolism of N2-fixing alfalfa plants after long-term exposure to elevated CO2. After 30-day exposure to elevated CO2, plants showed photosynthetic down-regulation with reductions in the light-saturated rate of CO2 assimilation (A sat) and the maximum rate of rubisco carboxylation (Vcmax). Under elevated CO2 conditions, the rubisco availability limited potential photosynthesis by around 12 %, which represented the majority of the observed fall in Vcmax. Photosynthetic down-regulation has been associated with decreased N availability even if those plants are capable to assimilate N2. Diminishment in shoot N demand (as reflected by the lower rubisco and leaf N content) suggests that the lower aboveground N requirements affected negatively nodule performance. In this condition, specific nodule activity was reduced due to an effect on nodule metabolism that manifested as a lower amount of nitrogenase reductase. Moreover, the nodule proteomic approach also revealed that nodule functioning was altered simultaneously in various enzyme quantity apart from nitrogenase. At elevated CO2, the tricarboxylic acid cycle was also altered with a reduced amount of isocitrate synthase protein. The nodule proteome analysis also revealed the relaxation of the antioxidant system as shown by a decline in the amount of catalase and isoflavone reductase protein.

Evaluación de la tolerancia al estrés de fósforo en caupí (Vigna unguiculata L. Walp) en Cuba. I. cultivo en solución nutritiva.

Phosphorus deficiency is a major limitingfactor for symbiotic nitrogen fixation and grain yield oflegume crops in the tropics. Eight cowpea genotypes of wideuse in Cuba, were grown in hydroponics solutions; supplyingfrom 50 to 500 µM of P/plant/week. Nitrogen nutrition wasdependent upon symbiotic N2-fixation on adding of 4mmoles NO3/plant/week. Shoot growth was severely limitedby P deficiency, while dry weight of roots increased in bothgroups of plants. Nodule formation was severely decreasedby P deficiency, while specific nodule activity was increasedin the atmospheric N2 fixing plants. Growth response of N2plants was higher than that of NO3- fed plants due to P useefficiency, and revealed a the great large N2-fixationpotential of the specie.

Approaches for enhancement of N2 fixation efficiency of chickpea (Cicer arietinum L.) under limiting nitrogen conditions

Chickpea (Cicer arietinum) is an important pulse crop in many countries in the world. The symbioses between chickpea and Mesorhizobia, which fix N₂ inside the root nodules, are of particular importance for chickpea's productivity. With the aim of enhancing symbiotic efficiency in chickpea, we compared the symbiotic efficiency of C-15, Ch-191 and CP-36 strains of Mesorhizobium ciceri in association with the local elite chickpea cultivar 'Bivanij' as well as studied the mechanism underlying the improvement of N₂ fixation efficiency. Our data revealed that C-15 strain manifested the most efficient N₂ fixation in comparison with Ch-191 or CP-36. This finding was supported by higher plant productivity and expression levels of the nifHDK genes in C-15 nodules. Nodule specific activity was significantly higher in C-15 combination, partially as a result of higher electron allocation to N₂ versus H⁺. Interestingly, a striking difference in nodule carbon and nitrogen composition was observed. Sucrose cleavage enzymes displayed comparatively lower activity in nodules established by either Ch-191 or CP-36. Organic acid formation, particularly that of malate, was remarkably higher in nodules induced by C-15 strain. As a result, the best symbiotic efficiency observed with C-15-induced nodules was reflected in a higher concentration of the total and several major amino metabolites, namely asparagine, glutamine, glutamate and aspartate. Collectively, our findings demonstrated that the improved efficiency in chickpea symbiotic system, established with C-15, was associated with the enhanced capacity of organic acid formation and the activities of the key enzymes connected to the nodule carbon and nitrogen metabolism.

Inhibition and recovery of symbiotic N2 fixation by peas (Pisum sativum L.) in response to short-term nitrate exposure

The design of more sustainable cropping systems requires increasing N-input from symbiotic N2 fixation (SNF). However, SNF can be inhibited by nitrate exposure (e.g., soil N-mineralization). Although the effect of nitrate on SNF has been extensively investigated at the cell scale, few studies have highlighted the impact of nitrate exposure on nodule number and biomass, nodule activity of the SNF apparatus or its ability to recover. Pea plants were grown in greenhouse conditions in a N-free nutrient solution and exposed to nitrate (5 mM NO 3 − L−1) for one week during either early vegetative growth, flowering or seed filling. After nitrate removal, the plants were grown either under natural light or shade. Nitrate exposure reduced the rate of nodule establishment during vegetative growth, whereas it caused damage to existing nodules when applied during the reproductive stages. Nitrate decreased the specific activity of nodules regardless of the stage of the exposure. After nitrate removal, an extra wave of nodulation was observed on plants grown under natural light but only when nitrate exposure occurred before the seed filling stage. The recovery of SNF activity after nitrate removal depended on the amount of carbon available to nodules.

Phloem‐derived γ‐aminobutyric acid (GABA) is involved in upregulating nodule N2 fixation efficiency in the model legume Medicago truncatula

Nitrogen fixation in legumes is downregulated through a whole plant N feedback mechanism, for example, when under stress. This mechanism is probably triggered by the impact of shoot-borne, phloem-delivered compounds. However, little is known about any whole-plant mechanism that might upregulate nitrogen fixation, for example, under N deficiency. We induced emerging N-deficiency through partial excision of nodules from Medicago truncatula plants. Subsequently, the activity and composition of the remaining nodules and shifts in concentration of free amides/amino acids in the phloem were monitored. Furthermore, we mimicked these shifts through artificial feeding of γ-aminobutyric acid (GABA) into the phloem of undisturbed plants. As a result of increased specific activity of nodules, N(2) fixation per plant recovered almost completely 4-5 d after excision. The concentration of amino acids, sugars and organic acids increased strongly in the upregulated nodules. A concomitant analysis of the phloem revealed a significant increase in GABA concentration. Comparable with the effect of nodule excision, artificial GABA feeding into the phloem resulted in an increased activity and higher concentration of amino acids and organic acids in nodules. It is concluded that GABA might be involved in upregulating nodule activity, possibly because of its constituting part of a putative amino acid cycle between bacteroids and the cytosol.

The importance of nodule CO2 fixation for the efficiency of symbiotic nitrogen fixation in pea at vegetative growth and during pod formation.

Nodule CO2 fixation is of pivotal importance for N2 fixation. The process provides malate for bacteroids and oxaloacetate for nitrogen assimilation. The hypothesis of the present paper was that grain legume nodules would adapt to higher plant N demand and more restricted carbon availability at pod formation through increased nodule CO2 fixation and a more efficient N2 fixation. Growth, N2 fixation, and nodule composition during vegetative growth and at pod formation were studied in pea plants (Pisum sativum L.). In parallel experiments, 15N2 and 13CO2 uptake, as well as nodule hydrogen and CO2 release, was measured. Plants at pod formation showed higher growth rates and N2 fixation per plant when compared with vegetative growth. The specific activity of active nodules was about 25% higher at pod formation. The higher nodule activity was accompanied by higher amino acid concentration in nodules and xylem sap with a higher share of asparagine. Nodule 13CO2 fixation was increased at pod formation, both per plant and per 15N2 fixed unit. However, malate concentration in nodules was only 40% of that during vegetative growth and succinate was no longer detectable. The data indicate that increased N2 fixation at pod formation is connected with strongly increased nodule CO2 fixation. While the sugar concentration in nodules at pod formation was not altered, the concentration of organic acids, namely malate and succinate, was significantly lower. It is concluded that strategies to improve the capability of nodules to fix CO2 and form organic acids might prolong intensive N2 fixation into the later stages of pod formation and pod filling in grain legumes.

The efficiency of nitrogen fixation of the model legume Medicago truncatula (Jemalong A17) is low compared to Medicago sativa

Medicago truncatula (Gaertn.) (barrel medic) serves as a model legume in plant biology. Numerous studies have addressed molecular aspects of the biology of M. truncatula, while comparatively little is known about the efficiency of N 2 fixation at the whole plant level. The objective of the present study was to compare the efficiency of N 2 fixation of M. truncatula to the genetically closely related Medicago sativa (L.) (alfalfa). The relative growth of both species relying exclusively on N 2 fixation versus nitrate nutrition, H 2 evolution, nitrogen assimilation, the concentration of amino acids and organic acids in nodules, and 15N 2 uptake and distribution were studied. M. truncatula showed much lower efficiency of N 2 fixation. Nodule-specific activity was several-fold lower when compared to M. sativa, partially as a result of a lower electron allocation to N 2 versus H +. M. truncatula or M. sativa plants grown solely on N 2 fixation as a nitrogen source reached about 30% or 80% of growth, respectively, when compared to plants supplied with sufficient nitrate. Moreover, M. truncatula had low %N in shoots and a lower allocation of 15N to shoots during 1 h 15N 2 labeling period. Amino acid concentration was about 20% higher in M. sativa nodules, largely as a result of more asparagine, while the organic acid concentration was about double in M. sativa, coinciding with a six-fold higher concentration of malate. Total soluble protein in nodules was about three times lower in M. truncatula and the pattern of enzyme activity in that fraction was strongly different. Sucrose cleaving enzymes displayed higher activity in M. truncatula nodules, while the activity of phosphoenolpyruvate carboxylase (PEPC) was much lower. It is concluded that the low efficiency of the M. truncatula symbiotic system is related to a low capacity of organic acid formation and limited nitrogen export from nodules.

Variability of nitrogen-fixing Frankia on Alnus species

Plants maintain mutualistic symbioses with multiple symbiont genotypes that differ in the benefits they provide. To investigate differences in the effect of nitrogen-fixing Frankia on Alnus species, spore-producing (sp+) nodules from Alnus rubra Bong. and Alnus incana subsp. rugosa (Du Roi) Clausen and non-spore-producing (sp–) nodules from Alnus viridis subsp. crispa (Ait.) Turrill, A. rubra, and A. incana subsp. rugosa were collected from each of four different populations and used to inoculate all three Alnus species. As expected, sp+ Frankia produced significantly more nodules on all three species. However, A. crispa, which normally does not have sp+ nodules in the field, was more susceptible to a high level of infection by sp+ Frankia in general, and by any source of sp+ Frankia in particular, whereas A. incana subsp. rugosa, which has the highest abundance of sp+ in the field, was less susceptible to high levels of infection. This suggests that A. incana subsp. rugosa develops resistance to high levels of infection. The infectivity of an sp+ Frankia source on A. viridis subsp. crispa and A. rubra was positively correlated with the proportion of sp+ nodules on the site it was collected from, suggesting that the variation in the abundance of sp+ in the field is caused by sp+ Frankia with different levels of infectivity. There was no effect of Frankia sources on nodule allocation. Plant growth was positively correlated with the specific nodule mass and the specific nodule activity, and negatively correlated with the nodule number per plant. Sp+ Frankia resulted in significantly smaller plants in A. rubra. While there was no overall sp+ type effect on the growth of A. viridis subsp. crispa, the largest plants always resulted when they were inoculated with sp–, and the smallest with sp+ Frankia. Neither spore type nor inoculum source had any effect on the performance of A. rugosa. These results suggest that Alnus species remain susceptible to infection by both Frankia spore types, but are able to modulate the effectiveness of these spore types when they are the common symbionts in the field.

Changes in Phosphate Fractions Extracted From Different Organs of Phosphorus Starved Nitrogen Fixing Pea Plants

ABSTRACT The present work investigates the impact of phosphorus (P) starvation on plant growth, symbiotic nitrogen fixation, and internal P status (determined as extracted P fractions) of leaves, roots, and nodules of 27-days–old pea (Pisum sativum L) plants inoculated with Rhizobium leguminosarum bv viciae strain D293. The procedure of separation of organic and inorganic P compounds in 10% perchloric acid (HCLO4) and the absorption of nucleotides in active charcoal gave several fractions, containing different phosphorus compounds, which were extracted and determined as inorganic phosphate after combustion. These are acid soluble and insoluble P, sugar P, nucleotide P, and inorganic P. The P starvation of plants inhibited significantly plant dry mass accumulation, nodulation rate and specific nitrogenase activity of nodules. These results were accompanied with lower quantities of total P per plant, acid soluble and acid non-soluble P fractions in all plant organs. The inhibited accumulation of P in the acid soluble P fraction was associated with decrease of sugar, nucleotide and inorganic P in all plant organs. The most negatively affected were all P fractions extracted from nodules and leaves. The low content of inorganic P in the stressed plant tissues was regarded as primary reason for induced alterations in the content of analyzed P fractions.

The promoter of the leghaemoglobin gene VfLb29: functional analysis and identification of modules necessary for its activation in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots.

In this study the further characterization of the Vicia faba leghaemoglobin promoter pVfLb29 is presented that was previously shown to be specifically active in the infected cells of root nodules and in arbuscule-containing cells of mycorrhizal roots. Using promoter studies in transgenic hairy roots of the Pisum sativum mutant RisNod24, disabled in the formation of functional arbuscules, VfLb29 promoter activity is assigned to later stages of arbuscule development. In order to narrow down the regions containing cis-acting elements of pVfLb29, the activity of five VfLb29 promoter deletions (-797/-31 to -175/-31 in relation to the start codon) fused to the gusAint coding region were tested in transgenic V. hirsuta hairy roots. The results specify a promoter region ranging from position -410 to -326 (85 bp) as necessary for gus expression in arbuscule-containing cells, whereas this segment is not involved in the nodule-specific activity. Sequence analysis of the pVfLb29 fragment -410/-326 (85 bp) revealed sequence motifs previously shown to be cis-acting elements of diverse promoters. To investigate the autonomous function of pVfLb29 regions for activation in arbuscule-containing cells, different regions of pVfLb29 from positions -410 to -198 were used to prepare chimeric promoter constructs for trans-activation studies. These fragments alone did not activate the mycorrhiza inactive promoter of the Vicia faba leghaemoglobin gene VfLb3, showing that the activation of pVfLb29 in arbuscule-containing cells is governed by a complex regulatory system that requires at least two modules located between position -410 and -31 of the VfLb29 gene.

The use of temperature gradient tunnels for studying the combined effect of CO2, temperature and water availability in N2 fixing alfalfa plants

Summary Atmospheric CO 2 concentration and temperature are increasing as a consequence of human activity. Periods of low water availability are expected to increase in Mediterranean ecosystems. Temperature gradient tunnels were used to provide near to ambient conditions and conditions simulating predicted increases in CO 2 and temperature for an alfalfa crop. The performance, construction, and running costs of the tunnels are reported and discussed. Two levels of water supply were included in the treatments. Plants were grown in large, 13 L pots, keeping a fixed soil volumetric water content and with nodule fixation as the only nitrogen source for the plant. Regardless of water regime, the effect of elevated CO 2 on plant growth was temperature dependent. Dry matter was enhanced when elevated CO 2 and temperature were combined. Plant yield improvement was partly a result of increased photosynthetic rates. There were no effects on plant N concentration. Decreased specific nodule activity might suggest that lack of differences in plant N were a consequence of reduced nodule enzyme activity.