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ABSTRACT The application of flumioxazin and imazethapyr can affect the interactions between rhizobia and soybean in the rhizosphere. However, it remains unclear the effect of both herbicides, even when applied in mixture, on early nodulation and biological N fixation (BNF) in soybean, mainly related to some important biochemical traits in nodules. This study assessed the effect of flumioxazin, imazethapyr and their mixture on early nodulation and BNF in rhizosphere of soybean. The plant emergence, early nodulation, growth, and N accumulation were evaluated in soybean grown in sandy and clayey soils. The leghemoglobin content and glutamine synthetase (GS) activity were also assessed in fresh nodules. Individually, flumioxazin and imazethapyr did not affect the plant emergence, nodulation and N accumulation in soybean, while the mixture decreased significantly these parameters. Also, the values of leghemoglobin content and GS activity did not decrease with the application of the herbicides individually. The application of herbicides imazethapyr and flumioxazin separately negatively influenced the growth of soybean, while they do not influence the nodulation and N accumulation. However, the use of these herbicides in mixture can negatively influence the early nodulation and N fixation by native rhizobia.

The application of flumioxazin and imazethapyr can affect the interactions between rhizobia and soybean in the rhizosphere. However, it remains unclear the effect of both herbicides, even when applied in mixture, on early nodulation and biological N fixation (BNF) in soybean, mainly related to some important biochemical traits in nodules. This study assessed the effect of flumioxazin, imazethapyr and their mixture on early nodulation and BNF in rhizosphere of soybean. The plant emergence, early nodulation, growth, and N accumulation were evaluated in soybean grown in sandy and clayey soils. The leghemoglobin content and glutamine synthetase (GS) activity were also assessed in fresh nodules. Individually, flumioxazin and imazethapyr did not affect the plant emergence, nodulation and N accumulation in soybean, while the mixture decreased significantly these parameters. Also, the values of leghemoglobin content and GS activity did not decrease with the application of the herbicides individually. The application of herbicides imazethapyr and flumioxazin separately negatively influenced the growth of soybean, while they do not influence the nodulation and N accumulation. However, the use of these herbicides in mixture can negatively influence the early nodulation and N fixation by native rhizobia. [ABSTRACT FROM AUTHOR]

Previous studies have shown that the Nodulin 26-like intrinsic membrane protein (NIP) Lsi1 (OsNIP2;1) is involved in arsenite [As(III)] uptake in rice (Oryza sativa). However, the role of other rice NIPs in As(III) accumulation in planta remains unknown. In the present study, we investigated the role OsNIP3;2 in As(III) uptake in rice. When expressed in Xenopus laevis oocytes, OsNIP3;2 showed a high transport activity for As(III). Quantitative real-time RT-PCR showed that the expression of OsNIP3;2 was suppressed by 5 pM As(III), but enhanced by 20 and 100 pM As(III). Transgenic rice plants expressing OsNIP3;2pro-GUS showed that the gene was predominantly expressed in the lateral roots and the stele region of the primary roots. Transient expression of OsNIP3;2:GFP fusion protein in rice protoplasts showed that the protein was localized in the plasma membrane. Knockout of OsNIP3;2 significantly decreased As concentration in the roots, but had little effect on shoot As concentration. Synchrotron microfocus X-ray fluorescence showed decreased As accumulation in the stele of the lateral roots in the mutants compared with wild-type. Our results indicate that OsNIP3;2 is involved in As(III) uptake by lateral roots, but its contribution to As accumulation in the shoots is limited. [ABSTRACT FROM AUTHOR]

Members of the aquaporin (AQP) family have been suggested to transport aluminum (Al) in plants; however, the Al form transported by AQPs and the roles of AQPs in Al tolerance remain elusive. Here we report that NIP1;2, a plasma membrane-localized member of the Arabidopsis nodulin 26-like intrinsic protein (NIP) subfamily of the AQP family, facilitates Al-malate transport from the root cell wall into the root symplasm, with subsequent Al xylem loading and root-to-shoot translocation, which are critical steps in an internal Al tolerance mechanism in Arabidopsis. We found that NIP1;2 transcripts are expressed mainly in the root tips, and that this expression is enhanced by Al but not by other metal stresses. Mutations in NIP1;2 lead to hyperaccumulation of toxic Al3+ in the root cell wall, inhibition of root-to-shoot Al translocation, and a significant reduction in Al tolerance. NIP1;2 facilitates the transport of Al-malate, but not Al3+ ions, in both yeast and Arabidopsis. We demonstrate that the formation of the Al-malate complex in the root tip apoplast is a prerequisite for NIP1;2-mediated Al removal from the root cell wall, and that this requires a functional root malate exudation system mediated by the Al-activated malate transporter, ALMT1. Taken together, these findings reveal a critical linkage between the previously identified Al exclusion mechanism based on root malate release and an internal Al tolerance mechanism identified here through the coordinated function of NIP1;2 and ALMT1, which is required for Al removal from the root cell wall, root-to-shoot Al translocation, and overall Al tolerance in Arabidopsis. [ABSTRACT FROM AUTHOR]

Silicon (Si), although mostly ignored by plant nutritionists and ecologists, is now gaining more attention because of its beneficial role in plant fitness under stress environment imposed by a diverse range of biotic and abiotic factors. Si appears to systematically confer benefits to plants as long as a given species can absorb the element. Here, we review recent developments regarding the molecular mechanisms, evolution, regulation and structural specificity of influx transporter proteins involved in Si uptake by plants., Si absorption is facilitated by specific nodulin 26-like intrinsic proteins ( NIPs). The Si transporter NIPs have evolved a unique amino acid selective filter ( SF), which is one of the required features to regulate the influx of Si. While Si accumulation in plants requires the dual action of both an influx transporter and an efflux transporter, it appears that the presence of the former is the indispensable key for a plant to be able to absorb Si., Based on sequence analyses and comparisons, influx transporters appear to have conserved features across all species that allow to discriminate between plants that are Si competent or not. While it is unclear how and why plants have acquired or lost this trait, genomic data now offer a reliable tool to predict with accuracy which plant species are predisposed to benefit from Si. This will undoubtedly result in a better understanding of Si role in many fundamental aspects of ecology regarding plant fitness under stress. [ABSTRACT FROM AUTHOR]

Allotetraploid rapeseed ( Brassica napus L., AnAnCnCn, 2 n = 4x = 38) is extraordinarily susceptible to boron (B) deficiency, a ubiquitous problem causing severe losses in seed yield. The breeding of B-efficient rapeseed germ plasm is a cost-effective and environmentally friendly strategy for the agricultural industry; however, genes regulating B efficiency in allotetraploid rapeseed have not yet been isolated. In this research, quantitative trait locus (QTL) fine mapping and digital gene expression (DGE) profiling were combined to identify the candidate genes underlying the major-effect QTL qBEC-A3a, which regulates B efficiency. Comparative phenotype analyses of the near-isogenic lines (NILs) indicated that qBEC-A3a plays a significant role in improving B efficiency under B deficiency. Exploiting QTL fine mapping and DGE analyses revealed a nodulin 26-like intrinsic protein (NIP) gene, which encodes a likely boric acid channel. The gene co-expression network for putative B transporters also highlighted its central role in the efficiency of B uptake. An integration of whole-genome re-sequencing (WGS) with bulked segregant analysis (BSA) authenticated the emerging availability of QTL-seq for the QTL analyses in allotetraploid rapeseed. Transcriptomics-assisted QTL mapping and comparative genomics provided novel insights into the rapid identification of quantitative trait genes (QTGs) in plant species with complex genomes. [ABSTRACT FROM AUTHOR]

Background: Albumin 1b peptides (A1b) are small disulfide-knotted insecticidal peptides produced by Fabaceae (also called Leguminosae). To date, their diversity among this plant family has been essentially investigated through biochemical and PCR-based approaches. The availability of high-quality genomic resources for several fabaceae species, among which the model species Medicago truncatula (Mtr), allowed for a genomic analysis of this protein family aimed at i) deciphering the evolutionary history of A1b proteins and their links with A1b-nodulins that are short non-insecticidal disulfide-bonded peptides involved in root nodule signaling and ii) exploring the functional diversity of A1b for novel bioactive molecules. Results: Investigating the Mtr genome revealed a remarkable expansion, mainly through tandem duplications, of albumin1 (A1) genes, retaining nearly all of the same canonical structure at both gene and protein levels. Phylogenetic analysis revealed that the ancestral molecule was most probably insecticidal giving rise to, among others, A1b-nodulins. Expression meta-analysis revealed that many A1b coding genes are silent and a wide tissue distribution of the A1 transcripts/peptides within plant organs. Evolutionary rate analyses highlighted branches and sites with positive selection signatures, including two sites shown to be critical for insecticidal activity. Seven peptides were chemically synthesized and folded in vitro, then assayed for their biological activity. Among these, AG41 (aka MtrA1013 isoform, encoded by the orphan TA24778 contig.), showed an unexpectedly high insecticidal activity. The study highlights the unique burst of diversity of A1 peptides within the Medicago genus compared to the other taxa for which full-genomes are available: no A1 member in Lotus, or in red clover to date, while only a few are present in chick pea, soybean or pigeon pea genomes. Conclusion: The expansion of the A1 family in the Medicago genus is reminiscent of the situation described for another disulfide-rich peptide family, the "Nodule-specific Cysteine-Rich" (NCR), discovered within the same species. The oldest insecticidal A1b toxin was described from the Sophorae, dating the birth of this seed-defense function to more than 58 million years, and making this model of plant/insect toxin/receptor (A1b/insect v-ATPase) one of the oldest known. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) in plants have been shown to facilitate the transport of water and other small molecules through biological membranes. This activity is essential for plant stress management during drought. Here, we describe and characterize a novel aquaporin gene AcNIP5;1 ( Atriplex canescens nodulin 26-like intrinsic protein 5;1) isolated from an A. canescens cDNA library. qRT-PCR results indicated that the transcript level in leaf tissue of AcNIP5;1 was upregulated by 20 % PEG 6000 and downregulated by 400 mM NaCl. To further understand the function of AcNIP5;1, we generated transgenic Arabidopsis thaliana plants that expressed AcNIP5;1 under control of the CaMV35S promoter. The overexpression of AcNIP5;1 in these transgenic Arabidopsis plants resulted in higher drought tolerance and sensitivity to salinity compared to wild-type plants on both MS plate and pot experiments. AcNIP5;1 protein tagged with GFP was localized to the cell plasma membrane in Nicotiana benthamiana. Our results suggest that AcNIP5;1 might play a role in drought tolerance in A. canescens. [ABSTRACT FROM AUTHOR]

Metalloids are a group of physiologically important elements ranging from the essential to the highly toxic. Arsenic, antimony, germanium, and tellurium are highly toxic to plants themselves and to consumers of metalloid-contaminated plants. Boron, silicon, and selenium fulfill essential or beneficial functions in plants. However, when present at high concentrations, boron and selenium cause toxicity symptoms that are detrimental to plant fitness and yield. Consequently, all plants require efficient membrane transport systems to control the uptake and extrusion of metalloids into or out of the plant and their distribution within the plant body. Several Nodulin 26-like intrinsic proteins (NIPs) that belong to the aquaporin plant water channel protein family facilitate the diffusion of uncharged metalloid species. Genetic, physiological, and molecular evidence is that NIPs from primitive to higher plants not only transport all environmentally important metalloids, but that these proteins have a major role in the uptake, translocation, and extrusion of metalloids in plants. As most of the metalloid-permeable NIP aquaporins are impermeable or are poorly permeable to water, these NIP channel proteins should be considered as physiologically essential metalloido-porins. [ABSTRACT FROM AUTHOR]

Until now very few plant genes with possible regulatory functions during nodule development have been isolated. We have used a modified cold-plaque screening method to identify new transcripts expressed at low levels that are induced during nodulation. Several clones were isolated and characterized by their mRNA expression patterns during nodule development and in spontaneous nodules. Sequence homology with known genes of other organisms indicated that transcripts corresponded to (i) “basic” genes probably required during the growth of the nodule organ (e.g., structural proteins), (ii) genes related to the metabolic adaptations taking place during nodule morphogenesis and function (e.g., carbonic anhydrase), and (iii) genes containing regulatory motifs and/or homologies (three clones out of the 20 identified). The latter genes encode a zinc-finger-containing protein, a putative protein kinase, and a Wilm's tumor (WT) suppressor homologue, respectively. Expression of the kinase and WT suppressor homologues was induced early in nodulation, although the latter was activated transiently. Accumulation of the Zn-finger gene transcripts was detected at a later stage of development and seems to be regulated in a complex manner. Hence, using a cold-plaque screening procedure, we could identify genes that may play regulatory roles in the signal transduction pathways activated during nodule development.

Plant genes whose expression is induced in legumes by Rhizobium bacteria upon nodulation were initially referred to as nodulins. Several of them play a key role in the establishment of symbiosis. Yet, nodulin-like proteins are also found in non-nodulating plant species such as Arabidopsis, rice, maize or poplar. For instance, 132 are predicted in the Arabidopsis thaliana Col-0 genome. Recent studies now highlight the importance of nodulin-like proteins for the transport of nutrients, solutes, amino acids or hormones and for major aspects of plant development. Interestingly, nodulin-like activities at the plant–microbe interface are also important for pathogens to enhance their fitness during host colonization. This work presents a genomic and functional overview of nodulin-like proteins in non-leguminous plant species, with a particular focus on Arabidopsis and rice. [ABSTRACT FROM AUTHOR]

Cathepsin B is an ancient family of eukaryotic cysteine proteases. We describe PsCat1, a plant cathepsin B-like transcript, identified as an expressed sequence in Rhizobium-induced, nitrogen-fixing root nodules of pea. In situ hybridization studies in root nodules showed strong, extremely localized expression of PsCat1 in individual cells associated with the central infected tissue. Restriction fragment polymorphism mapping of the PsCat1 locus in pea shows no correlation with existing mutant lines defective in symbiosis.

Nodulation is an extraordinary symbiotic interaction between leguminous plants and nitrogen-fixing bacteria (rhizobia) that assimilate atmospheric nitrogen (in root nodules) and convert it into compounds suitable for the plant host. A class of plant hormones called cytokinins are involved in the nodulation process. In the model legume Medicago truncatula, nodulin 13 (MtN13), which belongs to the pathogenesis-related proteins of class 10 (PR-10), is expressed in the outer cortex of the nodules. In general, PR-10 proteins are small and monomeric and have a characteristic fold with an internal hydrophobic cavity formed between a seven-stranded antiparallel β-sheet and a C-terminal α-helix. Previously, some PR-10 proteins not related to nodulation were found to bind cytokinins such as trans-zeatin. Here, four crystal structures of the MtN13 protein are reported in complexes with several cytokinins, namely trans-zeatin, N6-isopentenyladenine, kinetin and N6- benzyladenine. All four phytohormones are bound in the hydrophobic cavity in the same manner and have excellent definition in the electron-density maps. The binding of the cytokinins appears to be strong and specific and is reinforced by several hydrogen bonds. Although the binding stoichiometry is 1:1, the complex is actually dimeric, with a cytokinin molecule bound in each subunit. The ligand-binding site in each cavity is formed with the participation of a loop element from the other subunit, which plugs the only entrance to the cavity. Interestingly, a homodimer of MtN13 is also formed in solution, as confirmed by small-angle X-ray scattering (SAXS). [ABSTRACT FROM AUTHOR]

Background: Legume-rhizobium symbiosis is a complex process that is regulated in the host plant cell through gene expression network. Many nodulin genes that are upregulated during different stages of nodulation have been identified in leguminous herbs. However, no nodulin genes in woody legume trees, such as black locust (Robinia pseudoacacia), have yet been reported. Methodology/Principal findings: To identify the nodulin genes involved in R. pseudoacacia-Mesorhizobium amorphae CCNWGS0123 symbiosis, a suppressive subtractive hybridization approach was applied to reveal profiling of differentially expressed genes and two subtracted cDNA libraries each containing 600 clones were constructed. Then, 114 unigenes were identified from forward SSH library by differential screening and the putative functions of these translational products were classified into 13 categories. With a particular interest in regulatory genes, twenty-one upregulated genes encoding potential regulatory proteins were selected based on the result of reverse transcription-polymerase chain reaction (RT-PCR) analysis. They included nine putative transcription genes, eight putative post-translational regulator genes and four membrane protein genes. The expression patterns of these genes were further analyzed by quantitative RT-PCR at different stages of nodule development. Conclusions: The data presented here offer the first insights into the molecular foundation underlying R. pseudoacacia–M. amorphae symbiosis. A number of regulatory genes screened in the present study revealed a high level of regulatory complexity (transcriptional, post-transcriptional, translational and post-translational) that is likely essential to develop symbiosis. In addition, the possible roles of these genes in black locust nodulation are discussed. [ABSTRACT FROM AUTHOR]

To identify the genes involved in nodule formation and to increase usable molecular probes, a cDNA library of Astragalus sjnicus genes specifically expressed in infected roots by Mesorhizobium huakuii 7653R is generated using a PCR-based suppressive subtractive hybridization (SSH) technique with two mRNA populations of infected and uninfected control roots. Two nodule-specific genes, AsllC259 and AsG2511, are identified from infected roots of A. sinicus. The amino acid sequences deduced from the open reading frames (ORFs) reveal that AsllC259 and AsG2511 encodes a polypeptide with 134 and 58 amino acids, respectively. A signal peptide sequence is predicted with high probability at the N-termini of the AsllC259 and AsG2511. The motif searches show that the deduced polypeptide of AsllC259 contains two N-glycosylation sites, a cAMP- and cGMP-dependent protein kinase phosphorylation site and a casein kinase II phosphorylation site. BLASTP searches reveal that AsllC259 putative protein displays a low degree of similarity to a unique nodulin from Lupinus luteus nodules. No significant identity is displayed over the predicted polypeptides of AsG2511 with any published sequences. Virtual Northern blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicate that the two genes are expressed exclusively in inoculated roots and that their expression is 2-4 d later than that of the leghaemoglobin (Lb) gene during nodule development. [ABSTRACT FROM AUTHOR]

Abstract: Numerous nodule-specific genes, which are involved in the root nodule development and function, have been known and are still being discovered. Here, we reported the structure, expression, and genetic map location of two novel nodule-specific genes. First, two EST groups, one obtained from a nodule library and the other from all aboveground tissue libraries, were clustered with regard to in silico expression profiles. We compiled a pool of 103 putative nodule-specific sequence clusters. Then, two representative ESTs were selected for further experimental analyses. According to the full-length cDNA sequences, one was an EST of a novel nodule-specific polygalacturonase gene, GmPGN, and the other was an EST of a new short nodule-specific gene, GmEKN. The results of expression analyses of the GmPGN cDNAs indicated that GmPGN expression was not detectable in any of the soybean tissues except in the nodule tissue and may be regulated via alternative splicing. GmEKN expression was the most strongly detected in the nodule. The predicted GmEKN protein is both glutamic acid- and lysine-rich, and is also highly hydrophilic. Genetic mapping located GmPGN near the known quantitative trait locus conferring resistance to soybean cyst nematode on soybean molecular linkage group (MLG) B1, and GmEKN on MLG A2. These results provide useful information for the use of these genes in research on the orchestration of numerous genes in nodule development and function. [Copyright &y& Elsevier]

cDNA markers differentially expressed in mature and senescent soybean (Glycine max [L.] Merr. cv. Labrador) nodules were isolated by mRNA differential display. Three clones were confirmed to have a differential expression by dot-blot analysis. Two of them were down-regulated during senescence and showed a total identity with the nodulin 22 precursor cDNA sequence. Nodulin 22 has been suggested to be involved in metal exchanges between the symbiotic partners; the down-regulation of this gene suggests that these exchanges are reduced during the senescence process. The third clone (DD15) was up-regulated: no transcripts were detected in mature functioning nodules but senescent ones exhibited a high level of expression. This clone exhibited high homology with several soybean cDNA ESTs from the USDA-ARS soybean genetics and genome database. This gene family appears to encode proteins involved in the remobilisation occurring during natural or stress induced senescence or the utilisation of reserves during the germination process. On the other hand, DD15 also exhibits significant homology with an EST from the model plant Medicago truncatula, which corresponds to a gene expressed in cell cultures upon elicitor induction. Our results suggest that DD15 could constitute a good marker of soybean nodule senescence. [Copyright &y& Elsevier]

A cDNA clone (URcot-35) was isolated from a soybean cotyledonary cDNA library using a cDNA clone (UR-nod-35) for nodule uricase II as a probe. URcot-35 was a 1,170-bp cDNA with an open reading frame that encoded a protein of putative 309 amino acids with a molecular mass of 35,137 Da. The nucleotide sequence of URcot-35 was identical to that of URnod-35.Expression of the URcot-35 gene in cotyledons was investigated by Northern dot-blot hybridization, by the reverse transcription-polymerase chain reaction with subsequent hybridization assays with the cDNA for nodule uricase II as a probe, and by immunoblotting analysis with a monoclonal antibody that was specific to nodule uricase II. The results suggested that the transcript of URcot-35 was present in developing cotyledons and that uricase II accumulated during the pod-filling stage. This is the first report of the isolation of cDNA for uricase II from non-symbiotic tissue and the results demonstrate that uricase II in soybean cotyledons is identical to that in soybean nodules. [ABSTRACT FROM AUTHOR]

Immunoblot analysis showed that uricases in nonureide-transporting determinate nodules (Canavalia gladiata and Lotus japonicus) did not react with a monoclonal antibody against soybean nodule uricase, suggesting different immunological reactivities from those of uricases of ureide-transporting legumes. [ABSTRACT FROM AUTHOR]

The objective of this review is to summarise current knowledge about the structure and function of the peribacteroid membrane from the root nodules of leguminous plants. The information is presented in terms of development of this symbiotic membrane from its origin, through proliferation and in the mature state. There are clear indications that the peribacteroid membrane has a distinct structure and function at each developmental stage. The mature peribacteroid membrane has been the most intensively studied. The lipid and protein content of the mature peribacteroid membrane is discussed with particular emphasis on genetic and functional studies of the proteins. The mechanism and control of peribacteroid membrane biogenesis is also discussed. There is evidence for a specific biogenetic pathway for this membrane which requires both symbiotic partners for its correct functioning. [ABSTRACT FROM AUTHOR]

The symbiotic root nodule, an organ formed on leguminous plants, is a product of successful interactions between the host plant and the soil bacteria, Rhizobium spp. Plant hormones play an important role in the genesis of this organ. The hormonal balance appears to be modulated by the signals produced by bacteria. Many host genes induced during nodule organogenesis and the symbiotic state have been identified and characterized from several legumes. These genes encode nodule-specific proteins (nodulins) which perform diverse functions in root nodule development and metabolism. Formation of a subcellular compartment housing the bacteria is essential to sustain the symbiotic state, and several nodulins are involved in maintaining the integrity and function of this compartment. The bacteroid enclosed in the perbacteroid membrane behaves as an ‘organelle,’ completely dependent on the host for all its requirements for carbon, nitrogen and other essential elements. Thus it seems likely that the nodulins in the peribacteroid membrane perform specific transport functions. While the function of a few other nodulins is known (e.g. nodulin-100, nodulin-35), a group of uncharacterized nodulins exists in soybean root nodules. These nodulins share structural similarities and seem to have been derived from a common ancestor. Induction of nodulin genes occurs prior to and independent of nitrogen fixation, and thus is a prelude to symbiosis. Although some of the early nodulin genes are induced prior to or during infection, induction of late nodulins requires endocytotic release of bacteria. [ABSTRACT FROM AUTHOR]

Soybean [Glycine max (L.) Merr. cv. Amsoy 71] plants were inoculated with either the vesicular‐arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum, with a strain of Bradyrhizobium japonicum. or with both endophytes together. Non‐inoculated plants were fertilized with levels of N and P that resulted in plant growth similar to that following infection by Bradyrhizobium or Glomus, respectively. In general, plants colonized by Glomus contained more pinitol (1D‐3‐O‐methyl‐chiro‐inositol) but less starch than corresponding P‐fertilized soybeans, while nodulated soybeans contained more pinitol and sucrose in the roots than corresponding N‐fertilized plants. Compared to nutrient‐amended soybeans, increases or decreases in free amino acids in symbiotically grown plants tended to follow transient changes in N content and endophyte activity. The exception to this was the elevated levels of methionine in nodulated roots or aspartate and arginine in Glomus‐inoculated roots regardless of N level. There were both quantitative and qualitative changes in the proteins found in inoculated soybeans, especially in infected roots where nodulins (nodule‐specific proteins) and endomycorrhizins (mycorrhiza‐specific proteins) were detected by polyacrylamide gel eletrophoresis. Five nodulins (11.8% of root proteins) were detected with molecular weights of 14, 34, 40, 41 and 100 kDa. Five endomycorrhizins (5.1% of root proteins) were detected with molecular weights of 16, 17, 18, 22 and 30 kDa. Differences in nutrient uptake and allocation in symbiotic plants could not be duplicated by fertilizer input alone, although nutrient addition could lead to similar growth rates and dry weight accumulation. These findings suggest that the quality and quantity of plant carbohydrates, proteins and amino acids shift in response to the physiological changes resulting from infection by N2‐fixing bacteria or endomycorrhizal fungi. [ABSTRACT FROM AUTHOR]

In roots of the lupine (Lupinus luteus L. cv. Ventus) glutamate dehydrogenase (EC L4.1.2-4) was present in a single, electrophoretically homogeneous form (GDHR), while in root nodules eight forms of the enzyme (GDHN) were detected. Highly purified fractions of glutamate dehydrogenase from roots and nodules were used to prepare antisera in rabbits. The two antisera recognized extracts of glutamate dehydrogenase from both roots and nodules. Low concentrations of the antiserum raised against nodule glutamate dehydrogenase precipitated GDH from the nodule extract (the homologous antigen) effectively but decreased the activity of GDH from the root extract only slightly. At high concentrations, however, the antiserum totally precipitated both enzyme extracts. Ouchterlony's double diffusion test showed that seven forms of root nodule glutamate dehydrogenase arose as the result of random association of two subunits in a hexameric complex, while the eighth form probably consisted of totally distinct subunits. The question of whether the new glutamate dehydrogenase forms, present exclusively in root nodules, can be classified as nodulins is discussed. [ABSTRACT FROM AUTHOR]

Root nodule organogenesis is described. Plant regulated aspects of nodulation and N2 fixation are reviewed and discussed. Since the effective N2 fixing symbiosis requires the interaction of the host plant and bacterium in an appropriate environment (the rhizosphere and the root nodule) it is essential that research aimed at improving N2 fixation involve a knowledge and understanding of the plant genes that affect nodule development, growth, and function. Current knowledge of host plant genes involved in N2 fixation is summarized. Various experimental approaches to the study of the host plant's contribution to nodulation are noted. The functions of nodule specific proteins (nodulins) in symbiosis are delineated. Future areas of research are suggested. [ABSTRACT FROM AUTHOR]

Fifteen nodulins and several nodule-stimulated gene products were expressed in effective, nitrogen-fixing root nodules of white sweetclover (Melilotus alba Desr. cv. U389), as determined by two-dimensional gel electrophoresis of in vitro translation products. The number and gel position of eight leghaemoglobin (Lb) products, as well as a product tentatively identified as nodule-stimulated glutamine synthetase (GS), was similar to previous reports of alfalfa (Medicago sativa L. cv. Iroquois) nodulins. Three mutants of Rhizobium meliloti, including an exoH mutant, a lipopolysaccharide mutant, and a nifH mutant, elicited ineffective sweetclover nodules blocked at empty (bacteria-free), partially infected, or fully infected stages of nodule development, respectively. In these ineffective nodules, the nodulin Nma30 and nodule-stimulated NSTma42 were expressed early in development, while a group of four nodulins and two nodule-stimulated products were intermediate in order of expression. Lb, GS and the late nodulin Nmal2a were expressed later, following infection. The exoH mutant, Rm7154, appeared to be a leaky mutant, as a small percentage of the plants developed nitrogen-fixing nodules about 4 weeks after inoculation. The sequential expression of a large number of nodulins and nodule-stimulated products, as well as the availability of sweetclover nodulation mutants indicates that sweetclover is a useful diploid system for analysis of host genes essential to the Rhizobium/legume symbiosis. [ABSTRACT FROM PUBLISHER]

The early nodulin gene, PsENOD7, is expressed in pea root nodules induced by Rhizobium leguminosarum bv. viciae, but not in other plant organs. In situ hybridization showed that this gene is transcribed during nodule maturation in the infected cells of the proximal part of the prefixation zone II. At the transition of zone II into interzone II-III, the level of PsENOD7 mRNA drops markedly. PsENOD7 has no significant homology to other genes. RFLP mapping studies have shown that PsENOD7 is located in linkage group I between the leghaemoglobin genes and sym2. [ABSTRACT FROM AUTHOR]

The pea late nodulin gene PsNOD6 has been cloned and sequenced. PsNOD6 is homologous to the pea early nodulin genes PsNOD3 and PsENOD14. In situ hybridization experiments showed that, like the PsENOD3 and PsENOD14 genes, the PsNOD6 gene is only expressed in the infected cell type. The PsNOD6 gene is first expressed at the transition of the pre-fixation zone II into the interzone II-III (the amyloplast-rich zone preceding the fixation zone III), whereas the early nodulin genes PsENOD3 and PsENOD14 are already induced in the pre-fixation zone II. Thus these nodulin genes encoding homologous proteins are induced at consecutive stages of nodule development. The expression of the late nodulin genes encoding leghaemoglobin precedes the expression of the late nodulin gene PsNOD6. Therefore these late nodulin genes have to be regulated by different mechanisms despite the fact they are expressed in the same cell type. This conclusion is consistent with the fact that PsNOD6 lacks one of the conserved regions occurring in the promoters of all other late nodulin genes studied. [ABSTRACT FROM AUTHOR]

More than 600 potentially nodule-specific clones have been detected by differential hybridization of a broadbean cDNA library constructed from root nodule poly(A) RNA. These isolated cDNAs belong to at least 28 different clone groups containing cross-hybridizing sequences. The number of clones within a clone group varies from about 200 to only one single clone. Northern hybridization experiments revealed nodule-specific transcripts for 14 clone groups and markedly nodule-enhanced transcripts for another 7 clone groups. Sequence homologies indicate that three transcript sequences code for different leghemoglobins. Two other transcripts encode a nodule-specific sucrose synthase and a nodule-enhanced asparagine synthetase, respectively. Four deduced gene products are proline-rich, two of them being the homologues of PsENOD2 and PsENOD12. The third proline-rich protein (PRP) is composed of similar amino acid repeats as the nodule-specific PsENOD12 but is expressed in nodules and roots in comparable amounts. The fourth PRP is a nodule-enhanced extensin-type protein built up by Ser-Pro repeats. Two further nodule-specific transcripts encode gene products showing some similarity to structural glycine-rich proteins. Additionally, transcripts could be identified for broadbean homologues of the nodulins MsNOD25, PsENOD3 and PsENOD5 and transcripts specifying a nodule-enhanced lipoxygenase and a translation elongation factor EF-1 α, which is expressed in all broadbean tissues tested. [ABSTRACT FROM AUTHOR]

To study how iron-rich nodules concentrate and store iron, ferritin (mRNA, protein) was analyzed in developing soybean nodules and compared to nitrogenase (mRNA/activity) and leghemoglobin (mRNA, protein, heme). Both ferritin mRNA and protein concentrations increased early in nodulation. Later in nodulation ferritin protein declined, in contrast to the mRNA, as nitrogenase (mRNA and activity) increased and leghemoglobin (mRNA and protein) accumulated. A precursor/product relationship between iron stored in ferritin and iron in nitrogenase or leghemoglobin is suggested. The uncoordinated changes in ferritin mRNA and protein during nodulation contrast with nitrogenase mRNA and nitrogenase activity suggesting possible translational and posttranscriptional effects on ferritin expression. [ABSTRACT FROM AUTHOR]

Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin ( lbc3) promoter region and the chloramphenicol acetyl transferase ( cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata g lb3 ( Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7: 1265-1271, 1988). Subfragments of the S. rostrata g lb3 ( Srg lb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181-191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100°C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srg lb3 5′ upstream region were fused to the β-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase ( nos) promoters. These constructs were used to generate transgenic Lotus corniculatus plants and their expression was measured in different plant tissues. The Srg lb3 CAAT and TATA box region was found to be required for nodule-specific expression and several upstream enhancer-type regions were identified. [ABSTRACT FROM AUTHOR]

The identification of some nodule-specific host proteins (nodulins) from common bean ( Phaseolus vulgaris L.), a tropical ureide-transporting legume, is described. Particularly, the existence and developmental expression of several abundant nodule-specific transcripts of P. vulgaris are shown, including leghemoglobin, nodulespecific uricase and a group that in vitro translates into a cluster of about 30 kDa products. The expression pattern of nodulins in effective (Fix) nodules compared to ineffective (Fix) ones is also presented. The modified expression of main nodulins observed between these nodules indicates that different levels and/or factors associated with their regulation are involved. The intracellular infection by Rhizobium as a decisive step in the induction of some P. vulgaris nodulins is discussed. [ABSTRACT FROM AUTHOR]

Glutamine synthetase expression was studied in developing root-nodules of common bean with regard to the time-course of specific activity, antigen accumulation, polypeptide composition and in vitro translation products. This analysis shows that the nodule-specific GS polypeptide (GS-gamma) is detected prior to the nitrogenase acetylene-reducing activity, and that its accumulation together with that of the GS-alpha and GS-beta polypeptides vary with nodule age. GS-gamma is present in ineffective nodules, although in a lower ratio to GS-beta than in wild-type nodules. Comparisons of in vitro translated and in vivo synthesized GS polypeptides suggest no post-translational modifications. The possible factors and mechanisms involved in the regulation of expression of GS polypeptides are discussed. [ABSTRACT FROM AUTHOR]

Powdery mildew (PM) is considered a major cause of yield losses and reduced quality in cucumber worldwide, but the molecular basis of PM resistance remains poorly understood. A segment substitution line, namely, SSL508-28, was developed with dominant PM resistance in the genetic background of PM-susceptible cucumber inbred line D8. The substituted segment contains 860 genes. An iTRAQ-based comparative proteomic technology was used to map the proteomes of PM-inoculated and untreated (control) D8 and SSL508-28. The number of differentially regulated proteins (DRPs) in SSL508-28 was almost three times higher than that in D8. Fourteen DRPs were located in the substituted segment interval. Comparative gene expression analysis revealed that nodulin-related protein 1 (NRP1) may be a good candidate for PM resistance. Gene Ontology enrichment analysis showed that DRPs functioning in tetrapyrrole biosynthetic process, sulfur metabolic process and cell redox homeostasis were specifically enriched in the resistant line SSL508-28. DRPs categorized in the KEGG term photosynthesis increased in both lines upon PM infection, suggesting that the strategies used by cucumber may be different from those used by other crops to react to PM attacks at the initial stage. The measurement of hydrogen peroxide and superoxide anion production and net photosynthetic rate were consistent with the changes in protein abundance, suggesting that the proteomic results were reliable. There was a poor correlation between DRPs measured by iTRAQ and the corresponding gene expression changes measured by RNA-seq with the same experimental design. Taken together, these findings improve the understanding of the molecular mechanisms underlying the response of cucumber to PM infection. [ABSTRACT FROM AUTHOR]