Nitrogen-fixing Symbiosis Research Articles

Abscisic acid utilizing rhizobacteria disturb nitrogen-fixing symbiosis of pea <em>Pisum sativum</em> L.

Rhizosphere bacteria are capable of utilizing various phytohormones (particularly auxins) as nutrients and thereby affect plant growth, nutrition and interactions with symbiotic microorganisms. Here, for the first time we evaluated the effects of rhizosphere bacteria Novosphingobium sp. P6W and Rhodococcus sp. P1Y capable of utilizing abscisic acid (ABA) on growth and nitrogen-fixing symbiosis of pea (Pisum sativum L.) line SGE and its Cd-insensitive mutant SGECdt using hydroponic culture. The plants were co-inoculated with the ABA-utilizing bacteria and nodule bacterium Rhizobium leguminosarum bv. viciae RCAM1066. Treatment with cadmium (Cd) was applied as an inducer of ABA biosynthesis in plants. In the presence of only nodule bacteria, Cd significantly inhibited the growth of roots and shoots and also decreased the nodule number and nitrogen-fixing activity in SGE peas, but not in the SGECdt mutant. Inoculation with ABA-utilizing bacteria also inhibited biomass production, nodulation and nitrogen-fixation of Cd-untreated SGE plants. This negative effect of bacteria on the SGECdt mutant was less pronounced. Contrary to this, ABA-utilizing bacteria had no effect on SGE plants treated with Cd, but decreased shoot biomass and nitrogen-fixing activity of the SGECdt mutant. Inoculation with ABA-utilizing bacteria had no effect on shoot Cd and nutrient content of both pea genotypes, suggesting that bacterial effects on plants were not associated with the plant nutrient status. We propose that the bacteria counteracted the increased ABA concentrations in SGE roots caused by Cd due to utilization of this phytohormone. However, opposite processes aimed at inhibiting and stimulating growth and legume–rhizobia symbiosis can be caused by the ABA-utilizing bacteria.

Patterns for Pea-Rhizobium symbiosis efficiency response to pedological and varietal variations in Tunisia

Pedological constrains and varietal recommendations are serious causes of Pea-Rhizobium symbiosis efficiency deviations in Tunisia limiting pea crop adaptation to varying environments. In order to explore variation patterns of this symbiosis in contrasting soils and varieties and appreciate its contribution to plant adaptation, nine locations with potential pea crop vocation are prospected for collection of soils. Two pea varieties with different cycle length are randomly sown on triplicate irrigated containers and placed on natural condition during two seasons. The results have shown that soil fertility is mostly low and highly variable. It is mainly affected by high active limestone content and pH or by salinity. The studied factors (Soil type, Variety) have presented highly significant effect on native rhizobia efficiency, pea root nodulation, shoot and root biomass production, and shoot nitrogen accumulation (p < 0.0001). The long cycle variety gave the highest nodulation and adaptability to pedological variations. The multivariate data analysis indicates that Pea-Rhizobium symbiosis efficiency variability is mainly due to soil microbial biomass, phosphorus, organic matter, and electrical conductivity differences between soils. However, Principal Coordinate Analysis has confirmed the determining effect of these variables on rhizobia effectiveness, pea nodulation, growth and nitrogen accumulation. These results confirm that nitrogen-fixing symbiosis does not contribute in adaptation of pea crop to pedological constrains in Tunisia. Rhizobial inoculation and sepecific fertilization managment have become an urgent need to improve the production of this crop.

Antioxidant ability of glutaredoxins and their role in symbiotic nitrogen fixation in Rhizobium leguminosarum bv. viciae 3841.

Glutaredoxins (Grx) are redoxin family proteins that reduce disulfides and mixed disulfides between glutathione and proteins. Rhizobium leguminosarum bv. Viciae 3841 contains three genes coding for glutaredoxins: RL4289 (grxA) codes for a dithiolic glutaredoxin, RL2615 (grxB) codes for a monothiol glutaredoxin, while RL4261 (grxC) codes for a glutaredoxin-like NrdH protein. We generated mutants interrupted in one, two, or three glutaredoxin genes. These mutants had no obvious differences in growth phenotypes from the wild type RL3841. However, while a mutant of grxC did not affect the antioxidant or symbiotic capacities of R. leguminosarum, grxA-derived or grxB mutants decreased antioxidant and nitrogen fixation capacities. Furthermore, grxA mutants were severely impaired in rhizosphere colonization, and formed smaller nodules with defects of bacteroid differentiation, whereas nodules induced by grxB mutants contained abnormally thick cortices and prematurely senescent bacteroids. The grx triple mutant had the greatest defect in antioxidant and symbiotic capacities of R. leguminosarum and quantitative proteomics revealed it had 56 up-regulated and 81 down-regulated proteins relative to wildtype. Of these proteins, twenty-eight are involved in transporter activity, twenty are related to stress response and virulence, and sixteen are involved in amino acid metabolism. Overall, R. leguminosarum glutaredoxins behave as antioxidant proteins mediating root nodule symbiosis.IMPORTANCE Glutaredoxin catalyzes glutathionylation/deglutathionylation reactions, protects SH-groups from oxidation and restores functionally active thiols. Three glutaredoxins exist in R. leguminosarum and their properties were investigated in free-living bacteria and during nitrogen-fixing symbiosis. All the glutaredoxins were necessary for oxidative stress defense. Dithiol GrxA affects nodulation and nitrogen fixation of bacteroids by altering deglutathionylation reactions, monothiol GrxB is involved in symbiotic nitrogen fixation by regulating Fe-S cluster biogenesis, and GrxC may participate in symbiosis by an unknown mechanism. Proteome analysis provides clues to explain the differences between the grx triple mutant and wild-type nodules.

The role of cytokinins in Legume-Rhizobium symbiosis

Legume plants have a unique ability to form associations with nitrogen-fixing rhizobial species. An exchange of specific signaling molecules between host and microsymbiont constitutes the first step required both for bacterial infection and the formation of new, root-derived organs, called nodules. Since these two events occur in different root tissues, namely in the rhizodermis and in the underlying cortical cells, their strict regulation and coordination have to exist. An essential role of plant hormones, especially cytokinins, in the modulation of nitrogen-fixing symbiosis has been widely postulated. Activation of the cytokinin signaling pathway in the root cortex, by an unknown signal, is thought to be a key event of the infection process. As a consequence bioactive cytokinins are biosynthesized in the root susceptible zone, and they are a part of a positive feedback loop to trigger cortical cell division and sustain nodule organogenesis. Understanding the genetic mechanisms underlying events that occur in the inner layers of the root is one of the key challenges emerging in the study of symbiotic processes.

Plant Nitrate Reductases Regulate Nitric Oxide Production and Nitrogen-Fixing Metabolism During the Medicago truncatula–Sinorhizobium meliloti Symbiosis

Nitrate reductase (NR) is the first enzyme of the nitrogen reduction pathway in plants, leading to the production of ammonia. However, in the nitrogen-fixing symbiosis between legumes and rhizobia, atmospheric nitrogen (N2) is directly reduced to ammonia by the bacterial nitrogenase, which questions the role of NR in symbiosis. Next to that, NR is the best-characterized source of nitric oxide (NO) in plants, and NO is known to be produced during the symbiosis. In the present study, we first surveyed the three NR genes (MtNR1, MtNR2, and MtNR3) present in the Medicago truncatula genome and addressed their expression, activity, and potential involvement in NO production during the symbiosis between M. truncatula and Sinorhizobium meliloti. Our results show that MtNR1 and MtNR2 gene expression and activity are correlated with NO production throughout the symbiotic process and that MtNR1 is particularly involved in NO production in mature nodules. Moreover, NRs are involved together with the mitochondrial electron transfer chain in NO production throughout the symbiotic process and energy regeneration in N2-fixing nodules. Using an in vivo NMR spectrometric approach, we show that, in mature nodules, NRs participate also in the regulation of energy state, cytosolic pH, carbon and nitrogen metabolism under both normoxia and hypoxia. These data point to the importance of NR activity for the N2-fixing symbiosis and provide a first explanation of its role in this process.

Nitrate reductases and hemoglobins control nitrogen-fixing symbiosis by regulating nitric oxide accumulation.

The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship between plant and bacteria. This is characterized by the formation of a new organ, the nodule, which facilitates the fixation of atmospheric nitrogen (N2) by nitrogenase through the creation of a hypoxic environment. Nitric oxide (NO) accumulates at each stage of the symbiotic process. NO is involved in defense responses, nodule organogenesis and development, nitrogen fixation metabolism, and senescence induction. During symbiosis, either successively or simultaneously, NO regulates gene expression, modulates enzyme activities, and acts as a metabolic intermediate in energy regeneration processes via phytoglobin-NO respiration and the bacterial denitrification pathway. Due to the transition from normoxia to hypoxia during nodule formation, and the progressive presence of the bacterial partner in the growing nodules, NO production and degradation pathways change during the symbiotic process. This review analyzes the different source and degradation pathways of NO, and highlights the role of nitrate reductases and hemoproteins of both the plant and bacterial partners in the control of NO accumulation.

The Impacts of Domestication and Breeding on Nitrogen Fixation Symbiosis in Legumes.

Legumes are the second most important family of crop plants. One defining feature of legumes is their unique ability to establish a nitrogen-fixing root nodule symbiosis with soil bacteria known as rhizobia. Since domestication from their wild relatives, crop legumes have been under intensive breeding to improve yield and other agronomic traits but with little attention paid to the belowground symbiosis traits. Theoretical models predict that domestication and breeding processes, coupled with high−input agricultural practices, might have reduced the capacity of crop legumes to achieve their full potential of nitrogen fixation symbiosis. Testing this prediction requires characterizing symbiosis traits in wild and breeding populations under both natural and cultivated environments using genetic, genomic, and ecological approaches. However, very few experimental studies have been dedicated to this area of research. Here, we review how legumes regulate their interactions with soil rhizobia and how domestication, breeding and agricultural practices might have affected nodulation capacity, nitrogen fixation efficiency, and the composition and function of rhizobial community. We also provide a perspective on how to improve legume-rhizobial symbiosis in sustainable agricultural systems.

The Exopolysaccharide Cepacian Plays a Role in the Establishment of the Paraburkholderia phymatum - Phaseolus vulgaris Symbiosis.

Paraburkholderia phymatum is a rhizobial strain that belongs to the beta-proteobacteria, a group known to form efficient nitrogen-fixing symbioses within root nodules of several legumes, including the agriculturally important common bean. The establishment of the symbiosis requires the exchange of rhizobial and plant signals such as lipochitooligosaccharides (Nod factors), polysaccharides, and flavonoids. Inspection of the genome of the competitive rhizobium P. phymatum revealed the presence of several polysaccharide biosynthetic gene clusters. In this study, we demonstrate that bceN, a gene encoding a GDP-D-mannose 4,6-dehydratase, which is involved in the production of the exopolysaccharide cepacian, an important component of biofilms produced by closely related opportunistic pathogens of the Burkholderia cepacia complex (Bcc), is required for efficient plant colonization. Wild-type P. phymatum was shown to produce cepacian while a bceN mutant did not. Additionally, the bceN mutant produced a significantly lower amount of biofilm and formed less root nodules compared to the wild-type strain with Phaseolus vulgaris as host plant. Finally, expression of the operon containing bceN was induced by the presence of germinated P. vulgaris seeds under nitrogen limiting conditions suggesting a role of this polysaccharide in the establishment of this ecologically important symbiosis.

GmVTL1a is an iron transporter on the symbiosome membrane of soybean with an important role in nitrogen fixation.

Legumes establish symbiotic relationships with soil bacteria (rhizobia), housed in nodules on roots. The plant supplies carbon substrates and other nutrients to the bacteria in exchange for fixed nitrogen. The exchange occurs across a plant-derived symbiosome membrane (SM), which encloses rhizobia to form a symbiosome. Iron supplied by the plant is crucial for rhizobial enzyme nitrogenase that catalyses nitrogen fixation, but the SM iron transporter has not been identified. We use yeast complementation, real-time PCR and proteomics to study putative soybean (Glycine max) iron transporters GmVTL1a and GmVTL1b and have characterized the role of GmVTL1a using complementation in plant mutants, hairy root transformation and microscopy. GmVTL1a and GmVTL1b are members of the vacuolar iron transporter family and homologous to Lotus japonicus SEN1 (LjSEN1), which is essential for nitrogen fixation. GmVTL1a expression is enhanced in nodule infected cells and both proteins are localized to the SM. GmVTL1a transports iron in yeast and restores nitrogen fixation when expressed in the Ljsen1 mutant. Three GmVTL1a amino acid substitutions that block nitrogen fixation in Ljsen1 plants reduce iron transport in yeast. We conclude GmVTL1a is responsible for transport of iron across the SM to bacteroids and plays a crucial role in the nitrogen-fixing symbiosis.

Metabolomic profiling of wild-type and mutant soybean root nodules using laser-ablation electrospray ionization mass spectrometry reveals altered metabolism.

The establishment of the nitrogen-fixing symbiosis between soybean and Bradyrhizobium japonicum is a complex process. To document the changes in plant metabolism as a result of symbiosis, we utilized laser ablation electrospray ionization-mass spectrometry (LAESI-MS) for in situ metabolic profiling of wild-type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl-acyl carrier protein desaturase (sacpd-c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix+ ) and ineffective (nifH mutant, fix- ) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd-c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI-MS for high-throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.

The role of microRNAs in the legume-Rhizobium nitrogen-fixing symbiosis.

Under nitrogen starvation, most legume plants form a nitrogen-fixing symbiosis with Rhizobium bacteria. The bacteria induce the formation of a novel organ called the nodule in which rhizobia reside as intracellular symbionts and convert atmospheric nitrogen into ammonia. During this symbiosis, miRNAs are essential for coordinating the various plant processes required for nodule formation and function. miRNAs are non-coding, endogenous RNA molecules, typically 20-24 nucleotides long, that negatively regulate the expression of their target mRNAs. Some miRNAs can move systemically within plant tissues through the vascular system, which mediates, for example, communication between the stem/leaf tissues and the roots. In this review, we summarize the growing number of miRNAs that function during legume nodulation focusing on two model legumes, Lotus japonicus and Medicago truncatula, and two important legume crops, soybean (Glycine max) and common bean (Phaseolus vulgaris). This regulation impacts a variety of physiological processes including hormone signaling and spatial regulation of gene expression. The role of mobile miRNAs in regulating legume nodule number is also highlighted.

The Hybrid Histidine Kinase HrmK Is an Early-Acting Factor in the Hormogonium Gene Regulatory Network.

Filamentous, heterocyst-forming cyanobacteria belonging to taxonomic subsections IV and V are developmentally complex multicellular organisms capable of differentiating an array of cell and filament types, including motile hormogonia. Hormogonia exhibit gliding motility that facilitates dispersal, phototaxis, and the establishment of nitrogen-fixing symbioses. The gene regulatory network (GRN) governing hormogonium development involves a hierarchical sigma factor cascade, but the factors governing the activation of this cascade are currently undefined. Here, using a forward genetic approach, we identified hrmK, a gene encoding a putative hybrid histidine kinase that functions upstream of the sigma factor cascade. The deletion of hrmK produced nonmotile filaments that failed to display hormogonium morphology or accumulate hormogonium-specific proteins or polysaccharide. Targeted transcriptional analyses using reverse transcription-quantitative PCR (RT-qPCR) demonstrated that hormogonium-specific genes both within and outside the sigma factor cascade are drastically downregulated in the absence of hrmK and that hrmK may be subject to indirect, positive autoregulation via sigJ and sigC Orthologs of HrmK are ubiquitous among, and exclusive to, heterocyst-forming cyanobacteria. Collectively, these results indicate that hrmK functions upstream of the sigma factor cascade to initiate hormogonium development, likely by modulating the phosphorylation state of an unknown protein that may serve as the master regulator of hormogonium development in heterocyst-forming cyanobacteria.IMPORTANCE Filamentous cyanobacteria are morphologically complex, with several representative species amenable to routine genetic manipulation, making them excellent model organisms for the study of development. Furthermore, two of the developmental alternatives, nitrogen-fixing heterocysts and motile hormogonia, are essential to establish nitrogen-fixing symbioses with plant partners. These symbioses are integral to global nitrogen cycles and could be artificially recreated with crop plants to serve as biofertilizers, but to achieve this goal, detailed understanding and manipulation of the hormogonium and heterocyst gene regulatory networks may be necessary. Here, using the model organism Nostoc punctiforme, we identify a previously uncharacterized hybrid histidine kinase that is confined to heterocyst-forming cyanobacteria as the earliest known participant in hormogonium development.

Reprogramming of Root Cells during Nitrogen-Fixing Symbiosis Involves Dynamic Polysome Association of Coding and Noncoding RNAs.

Translational control is a widespread mechanism that allows the cell to rapidly modulate gene expression in order to provide flexibility and adaptability to eukaryotic organisms. We applied translating ribosome affinity purification combined with RNA sequencing to characterize translational regulation of mRNAs at early stages of the nitrogen-fixing symbiosis established between Medicago truncatula and Sinorhizobium meliloti Our analysis revealed a poor correlation between transcriptional and translational changes and identified hundreds of regulated protein-coding and long noncoding RNAs (lncRNAs), some of which are regulated in specific cell types. We demonstrated that a short variant of the lncRNA Trans-acting small interference RNA3 (TAS3) increased its association to the translational machinery in response to rhizobia. Functional analysis revealed that this short variant of TAS3 might act as a target mimic that captures microRNA390, contributing to reduce trans acting small interference Auxin Response Factor production and modulating nodule formation and rhizobial infection. The analysis of alternative transcript variants identified a translationally upregulated mRNA encoding subunit 3 of the SUPERKILLER complex (SKI3), which participates in mRNA decay. Knockdown of SKI3 decreased nodule initiation and development, as well as the survival of bacteria within nodules. Our results highlight the importance of translational control and mRNA decay pathways for the successful establishment of the nitrogen-fixing symbiosis.

EFFECT OF GOAT’S-RUE RHIZOBIA ON THE FORMATION AND FUNCTIONING OF THE SOYBEAN – BRADYRHIZOBIUM JAPONICUM 634B SYMBIOSIS

Objective. Study the peculiarities of nodule formation upon the formation of the symbiotic sys-tem soybean-Bradyrhizobium japonicum 634b, as well as the symbiotic nitrogen-fixation ability and plant growth and development under the influence of goat’s-rue rhizobia. Methods. Microbiologi-cal, physiological, statistical, gas chromatography. Results. In green house experiments, using sand as a substrate for growing plants, the mixed microbial cultures combining soybean nodule bacteria B. japonicum 634b and goat’s-rue nodule bacteria R. galegae 0702 or R. galegae 0703 in the ratio of 1 : 1 differed from the monoculture bradyrhizobium by their influence on the nodulation, nitro-gen-fixation ability of soybean-rhizobial symbiosis and development of soybean plants (variety Almaz). Increased nodulation activity in the primordial leaf and budding phases, as well as a signif-icant decrease in the level of symbiosis nitrogen fixation during budding, were observed when used in binary bacterial compositions of strain R. galegae 0703. These rhizobia of goat’s-rue suppressed the development of the root system of soybeans, but had no significant effect on the formation of the aerial part of the plants throughout the observation period. R. galegae 0702 strain slightly slowed the formation of nodules by bacteria in the primordial leaf phase, which caused a decrease in the number of soybean plants that formed symbiosis with B. japonicum 634b. Goat’s-rue nodule bacte-ria R. galegae 0702 improved the formation of the root system, and stimulated the growth and de-velopment of the aerial part of the macro symbiont in the phase of two trigeminal leaves. Conclu-sion. Combined inoculation of the rhizobia of goat’s-rue with nodule bacteria B. japonicum 634b showed a multidirectional effect on the formation of symbiosis by soybean plants of variety Almaz and functioning of soybean rhizobial symbiosis. The nature of the influence of R. galegae depended on their strain affiliation.

Evolving together, evolving apart: measuring the fitness of rhizobial bacteria in and out of symbiosis with leguminous plants.

Most plant-microbe interactions are facultative, with microbes experiencing temporally and spatially variable selection. How this variation affects microbial evolution is poorly understood. Given its tractability and ecological and agricultural importance, the legume-rhizobia nitrogen-fixing symbiosis is a powerful model for identifying traits and genes underlying bacterial fitness. New technologies allow high-throughput measurement of the relative fitness of bacterial mutants, strains and species in mixed inocula in the host, rhizosphere and soil environments. I consider how host genetic variation (G×G), other environmental factors (G×E), and host life-cycle variation may contribute to the maintenance of genetic variation and adaptive trajectories of rhizobia - and, potentially, other facultative symbionts. Lastly, I place these findings in the context of developing beneficial inoculants in a changing climate.

A Toolkit for High Resolution Imaging of Cell Division and Phytohormone Signaling in Legume Roots and Root Nodules.

Legume plants benefit from a nitrogen-fixing symbiosis in association with rhizobia hosted in specialized root nodules. Formation of root nodules is initiated by de novo organogenesis and coordinated infection of these developing lateral root organs by rhizobia. Both bacterial infection and nodule organogenesis involve cell cycle activation and regulation by auxin and cytokinin is tightly integrated in the process. To characterize the hormone dynamics and cell division patterns with cellular resolution during nodulation, sensitive and specific sensors suited for imaging of multicellular tissues are required. Here we report a modular toolkit, optimized in the model legume Lotus japonicus, for use in legume roots and root nodules. This toolkit includes synthetic transcriptional reporters for auxin and cytokinin, auxin accumulation sensors and cell cycle progression markers optimized for fluorescent and bright field microscopy. The developed vectors allow for efficient one-step assembly of multiple units using the GoldenGate cloning system. Applied together with a fluorescence-compatible clearing approach, these reporters improve imaging depth and facilitate fluorescence examination in legume roots. We additionally evaluate the utility of the dynamic gravitropic root response in altering the timing and location of auxin accumulation and nodule emergence. We show that alteration of auxin distribution in roots allows for preferential nodule emergence at the outer side of the bend corresponding to a region of high auxin signaling capacity. The presented tools and procedures open new possibilities for comparative mutant studies and for developing a more comprehensive understanding of legume-rhizobia interactions.

A common mechanism for efficient N2 O reduction in diverse isolates of nodule-forming bradyrhizobia.

Bradyrhizobia are abundant soil bacteria, which can form nitrogen-fixing symbioses with leguminous plants, including important crops such as soybean, cowpea and peanut. Many bradyrhizobia can denitrify, but studies have hitherto focused on a few model organisms. We screened 39 diverse Bradyrhizobium strains, isolated from legume nodules. Half of them were unable to reduce N2 O, making them sources of this greenhouse gas. Most others could denitrify NO3 - to N2 . Time-resolved gas kinetics and transcription analyses during transition to anaerobic respiration revealed a common regulation of nirK, norCB and nosZ (encoding NO2 - , NO and N2 O reductases), and differing regulation of napAB (encoding periplasmic NO3 - reductase). A prominent feature in all N2 -producing strains was a virtually complete hampering of NO3 - reduction in the presence of N2 O. In-depth analyses suggest that this was due to a competition between electron transport pathways, strongly favouring N2 O over NO3 - reduction. In a natural context, bacteria with this feature would preferentially reduce available N2 O, produced by themselves or other soil bacteria, making them powerful sinks for this greenhouse gas. One way to augment such populations in agricultural soils is to develop inoculants for legume crops with dual capabilities of efficient N2 -fixation and efficient N2 O reduction.

Evolution of Angiosperm Pollen. 7. Nitrogen-fixing Clade

Nitrogen-fixing symbiosis in root nodules is known in only 10 families, which are distributed among a clade of four orders and delimited as the nitrogen-fixing clade. As the seventh in a series that examines pollen morphological distribution and evolution in the angiosperms, this paper focuses on pollen morphological character states of the nitrogen-fixing clade. To illustrate the palynological diversity of the clade, we first examined pollen grains from 26 species with light electron, scanning electron, and transmission electron microscopy. Second, we used a reduced data matrix from Li et al. (2015) to reconstruct a maximum likelihood tree and then optimized 18 pollen character states onto the tree using Fitch parsimony, maximum likelihood, and hierarchical Bayesian inference. Finally, 12 plesiomorphic states for the nitrogen-fixing clade were inferred unambiguously under all methods, and more than 40 clades (or lineages) at or above familial level were characterized by unambiguous pollen character state changes in at least one of the optimizations. We found a number of evolutionary trends for changes in pollen character states. These include increasing grain size, increasing aperture number accompanied by concomitant changes in aperture position (from equatorial to global) and aperture shape (from colpate to colporate), and increasing complexity of tectum ornamentation. There was a strong correlation between some pollen characters (prolate shape class, lobe outline in polar view, colpate ectoaperture, lalongate and lolongate endoaperture, absent supratectal element, reticulate tectum) and insect pollination, while other pollen characters—simple aperture structure, porate ectoaperture, circular endoaperture, present and gemmate or echinate supratectal element, and imperforate tectum—were strongly correlated with wind pollination. In addition, rugulate tectum was significantly correlated with shrub habit while larger pollen size was significantly correlated with vine habit; the helophytic habitat was significantly correlated with having two apertures. Our study provides rich evidence for the phylogenetic significance of pollen morphological diversity in the nitrogen-fixing clade.

Stable carbon and nitrogen isotopes in woody plants and herbs near the large copper smelting plant

Variations of stable carbon (13С and 12С) and nitrogen (15N and 14N) isotopic composition are analyzed in forest plants subjected to the emissions of large copper smelting plant. The studies were carried out in pine forests at ten test plots near the Karabash copper smelting plant and in the Ilmen State Reserve at South Urals. The 13С/12С and 15N/14N isotopic ratios were analyzed in leaves of plants of different functional groups (with ecto-, ericoid, or arbuscular mycorrhiza; with nitrogen-fixing symbiosis, and non-mycorrhizal). The 13С/12С ratio did not change under technogenic pollution. The low isotopic 15N/14N ratio was established in ectomycorrhizal trees, while the high ratio was found in herbs with arbuscular mycorrhiza, nitrogenfixing symbiosis, and non-mycorrhizal groups. As compared to nonpolluted habitats, the 15N content in leaves near the copper smelting plant increases by 2.7‰ in the ectomycorrhizal trees and by 3.4‰ in undershrubs with ericoid mycorrhiza, and by 2.2‰ in herbs with arbuscular mycorrhiza. This indicates a significant change in conditions of mineral feeding of plants under heavy metal pollution of natural ecosystems.

Delineation of the integrase-attachment and origin-of-transfer regions of the symbiosis island ICEMlSymR7A

Integrative and conjugative elements (ICEs) are chromosomally-integrated mobile genetic elements that excise from their host chromosome and transfer to other bacteria via conjugation. ICEMlSymR7A is the prototypical member of a large family of “symbiosis ICEs” which confer upon their hosts the ability to form a nitrogen-fixing symbiosis with a variety of legume species. Mesorhizobial symbiosis ICEs carry a common core of mobilisation genes required for integration, excision and conjugative transfer. IntS of ICEMlSymR7A enables recombination between the ICEMlSymR7A attachment site attP and the 3′ end of the phe-tRNA gene. Here we identified putative IntS attP arm (P) sites within the attP region and demonstrated that the outermost P1 and P5 sites demarcated the minimal region for efficient IntS-mediated integration. We also identified the ICEMlSymR7A origin-of-transfer (oriT) site directly upstream of the relaxase-gene rlxS. The ICEMlSymR7A conjugation system mobilised a plasmid carrying the cloned oriT to Escherichia coli in an rlxS-dependent manner. Surprisingly, an in-frame, markerless deletion mutation in the ICEMlSymR7A recombination directionality factor (excisionase) gene rdfS, but not a mutation in intS, abolished mobilisation, suggesting the rdfS deletion tentatively has downstream effects on conjugation or its regulation. In summary, this work defines two critical cis-acting regions required for excision and transfer of ICEMlSymR7A and related ICEs.