Cloning of the β2-microglobulin gene in the zebrafish

The ₂-microglobulin (₂m) is a protein found in the serum in a free form and on the cell surface in a form noncovalently associated with the chain of the class I major histocompatibility complex (Mhc) molecules. In mammals, the ₂m-encoding gene (B2m) is found on a chromosome different from the Mhc proper. We have isolated and characterized the B2m gene of the zebrafish, Brachydanio rerio, family Cyprinidae. We obtained both cDNA and genomic clones of the Brre-B2m gene. The cDNA clones contained the entire coding sequence, the entire 3 untranslated (UT) region, and at least part of the 5UT region. The genomic clone contained the entire Brre-B2m gene. The coding sequence specifies 97 amino acid residues of the mature protein so that the zebrafish ₂m is two residues shorter than human and one residue shorter than cattle, fowl, or turkey ₂m (codons at positions 85 and 86 have been deleted in the Brre-B2m. gene). The amino acid and nucleotide sequence similarities between zebrafish and human ₂m (B2m) are 45% and 59%, respectively. Approximately 24% of the positions are invariant and an additional 9% show only conservative substitutions in comparisons which include all known 2m sequences (fish, avian, and mammalian). Most of the conserved positions are in the strands (some 47% of the -strand positions are conserved in the three vertebrate classes). The Brre-B2m gene consists of four exons separated by three introns. All of the introns are considerably shorter than the corresponding introns in the mammalian B2m genes. The coding sequences of the cDNA and the genomic clones are almost identical but the sequences of the 3'UT regions differ at 1.7% of the sites, suggesting that the genes borne by these clones might have diverged at least 0.7 million years (my) ago. In contrast to the human B2m gene, the Brre-B2m gene shows no bias in the distribution of the CpG dinucleotides: the dinucleotides are distributed evenly along the entire available sequence. The haploid genome of the zebrafish contains only one copy of the B2m gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L05383 (B2M) and L05384 (B2RG). Correspondence to: J. Klein.     

Proteasomes in the brain of β2-microglobulin knockout mice

MHC class I molecules play an important role in synaptic plasticity of the mammalian nervous system. Proteolytic complexes (proteasomes) produce oligopeptides that are presented on cell surfaces in complexes with MHC class I molecules and regulate many cellular processes beside this. The goal of the present work was to study peculiarities in functioning of proteasomes and associated signaling pathways along with evaluation of NeuN and gFAP expression in different sections of the brain in mice with knockout of β2-microglobulin, a constituent of MHC class I molecules. It was found that the frontal cortex and the brainstem, structures with different ratio of NeuN and gFAP expression, are characterized by opposite changes in the proteasome pool under constant total proteasome levels in B2m-knockout mice in comparison with those in control animals. ChTL-activity as well as expression of LMP7 immune subunit and PA28 regulator of proteasomes was elevated in the cortex of B2m-knockout mice, while these indicators were decreased in the brainstem. The concentrations of the signaling molecules nNOS and HSP70 in B2m-knockout mice were increased in the cortex, while being decreased in the brainstem, and this indicates the possibility of control of expression of the LMP7 subunit and the regulator PA28 by these molecules. Changes in the proteasome pool observed in striatum of B2m-knockout mice are similar to those observed in the brainstem. At the same time, the cerebellum is characterized by a specific pattern of proteasome functioning in comparison with that in all other brain structures. In cerebellum the expression of immune subunits LMP7 and LMP2 and the regulator PA28 was increased, while expression of regulator PA700 was decreased. Deficiency of NeuN and gFAP was revealed in most brain compartments of B2m-knockout mice. Thus, increased expression of the above-mentioned immune subunits and the proteasome regulator PA28 in the cortex and cerebellum may compensate disturbances revealed in the brain structures and the absence of MHC class I molecules. Apparently, this promotes production of peptides necessary for cell-to-cell interactions and maintains nervous system plasticity in B2m-knockout mice.     

Localization of β2-microglobulin in the term villous syncytiotrophoblast

The non-covalent association of beta 2-microglobulin with MHC class I molecules and MHC class I-type molecules such as FcRn or the hemochromatosis protein (HFE) is of major importance for their function, i.e., antigen presentation, IgG transport, and regulation of iron uptake, respectively. In the human hemochorial placenta, the syncytiotrophoblast forms a continuous epithelial layer covering the villous trees, where it directly contacts maternal blood and, among many other functions, mediates uptake of maternal IgG and iron. The villous syncytiotrophoblast lacks MHC class I molecules but expresses FcRn and HFE. Since data on beta 2-microglobulin synthesis and localization in the term villous syncytiotrophoblast were contradictory, we investigated the subcellular localization of beta 2-microglobulin by immunoelectron microscopy. Synthesis in the trophoblast is demonstrated by colocalization of beta 2-microglobulin with protein disulfide isomerase, a marker protein of the endoplasmic reticulum. The presence of beta 2-microglobulin at the apical plasma membrane corresponds to the recently observed association of beta 2-microglobulin with HFE and FcRn. Localization of beta 2-microglobulin in late endosomes/lysosomes, labeled with antibodies to lysosome membrane antigen LAMP 2, suggests also a degradative route of beta 2-microglobulin internalized by fluid-phase from the maternal blood.     

Primordial linkage of β2-microglobulin to the MHC

β2-Microglobulin (β2M) is believed to have arisen in a basal jawed vertebrate (gnathostome) and is the essential L chain that associates with most MHC class I molecules. It contains a distinctive molecular structure called a constant-1 Ig superfamily domain, which is shared with other adaptive immune molecules including MHC class I and class II. Despite its structural similarity to class I and class II and its conserved function, β2M is encoded outside the MHC in all examined species from bony fish to mammals, but it is assumed to have translocated from its original location within the MHC early in gnathostome evolution. We screened a nurse shark bacterial artificial chromosome library and isolated clones containing β2M genes. A gene present in the MHC of all other vertebrates (ring3) was found in the bacterial artificial chromosome clone, and the close linkage of ring3 and β2M to MHC class I and class II genes was determined by single-strand conformational polymorphism and allele-specific PCR. This study satisfies the long-held conjecture that β2M was linked to the primordial MHC (Ur MHC); furthermore, the apparent stability of the shark genome may yield other genes predicted to have had a primordial association with the MHC specifically and with immunity in general.     

Proteomics of β2-microglobulin amyloid fibrils

Knowledge on the chemical structure of β2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of β2-microglobulin extracted from non-osteotendinous tissues. β2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to β2-microglobulin derived from fibrils but is not observed in the β2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar β2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1–2% of the intact molecule.     

Frequent loss of heterozygosity in the β2-microglobulin region of chromosome 15 in primary human tumors

Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.     

A substitution of cytosine for thymine in codon 110 of the human β-globin gene is a novel cause of β-thalassemia phenotypes

Summary We have described a novel human globin gene mutation that produced in a Japanese family the -thalassemia phenotype through a post-translational mechanism. Substitution of proline for leucine at position 110 in the G-helix of the -globin chain greatly reduced the molecular stability of the -globin subunit, leading to total destruction of the variant globin chains by proteolysis and hence to the -thalassemia phenotype. The mutation could be identified after MspI digestion. This detection of the mutation on the gene level is valuable for diagnostic purposes.     

Structure and dynamics of oligomeric intermediates in β2-microglobulin self-assembly

β₂-Microglobulin is a 99-residue protein with a propensity to form amyloid-like fibrils in vitro which exhibit distinct morphologies dependent on the solution conditions employed. Here we have used ion mobility spectrometry-mass spectrometry to characterize the oligomeric species detected during the formation of worm-like fibrils of β₂-microglobulin at pH 3.6. Immediately upon sample dissolution, β₂-microglobulin monomer and oligomers—the latter ranging in size from dimer to hexamer—are present as a pool of rapidly interconverting species. Increasing the ionic strength of the solution initiates fibril formation without a lag-phase whereupon these oligomers become more stable and higher-order species (7-mer to >14-mer) are observed. The oligomers detected have collision cross-sectional areas consistent with a linearly stacked assembly comprising subunits of native-like volume. The results provide insights into the identity and properties of the transient, oligomeric intermediates formed during assembly of worm-like fibrils and identify species that differ significantly from the oligomers previously characterized during the nucleated assembly of long, straight fibrils. The data presented demonstrate the interrelationship between different fibril-forming pathways and identify their points of divergence.     

Contribution of gene conversion in the evolution of the human β-like globin gene family

Gene conversion is referred to as one of two types of mechanisms known to act on gene families, mainly to maintain their sequence homogeneity or, in certain cases, to produce sequence diversity. The concept of gene conversion was established 20 years ago by researchers working with fungi. A few years later, gene conversion was also observed in the human genome, i.e. the γ-globin locus. The aim of this article is to emphasize the role of genetic recombination, particularly of gene conversion, in the evolution of the human β-like globin genes and further to summarize its contribution to the convergent evolution of the fetal globin genes. Finally, this article attempts to re-examine the origin and spread of specific mutations of the β-globin cluster, such as the sickle cell or β-thalassemia mutations, on the basis of repeated gene conversion events. Received: 13 February 1997 / Accepted: 15 May 1998     

Immunofluorescence and ultrastructural localization of β2-microglobulin in human renal allografts

Summary The localization of 2-microglobulin (2m) was studied in renal biopsies from 18 patients with pathological transplanted kidney using immunofluorescence and electron-immunohistochemical techniques; the renal biopsies of 4 cases with normal kidneys were used as controls. Using immunofluorescence, 2m was not observed in the normal kidneys, 2m was found in the glomeruli (7 cases) and the tubular epithelium (16 cases) of the transplanted kidneys. Using immunoelectron microscopy, some labelling of the normal kidneys was observed mainly along the cell coat of foot processes and in tubular-epithelial lysosomes. In the glomeruli of transplanted kidneys, particularly in cases of acute or chronic rejection, 2m was most frequently localized on the outer layer of the basement membrane and along the cell coat of foot processes. The brush border of the proximal tubules and the lysosomal structures were intensely labelled. Although immunoelectron-microscopy studies are unable to discriminate between the localization of 2m in normal and transplanted kidneys, these findings nevertheless suggest the glomerular filtration of 2m and its metabolism in the tubular epithelium.     

Implication of Porins in β-Lactam Resistance of Providencia stuartii

An integrative approach combining biophysical and microbiological methods was used to characterize the antibiotic translocation through the outer membrane of Providencia stuartii. Two novel members of the General Bacterial Porin family of Enterobacteriaceae, named OmpPst1 and OmpPst2, were identified in P. stuartii. In the presence of ertapenem (ERT), cefepime (FEP), and cefoxitin (FOX) in growth media, several resistant derivatives of P. stuartii ATCC 29914 showed OmpPst1-deficiency. These porin-deficient strains showed significant decrease of susceptibility to β-lactam antibiotics. OmpPst1 and OmpPst2 were purified to homogeneity and reconstituted into planar lipid bilayers to study their biophysical characteristics and their interactions with β-lactam molecules. Determination of β-lactam translocation through OmpPst1 and OmpPst2 indicated that the strength of interaction decreased in the order of ertapenem ≫ cefepime > cefoxitin. Moreover, the translocation of these antibiotics through OmpPst1 was more efficient than through OmpPst2. Heterologous expression of OmpPst1 in the porin-deficient E. coli strain BL21(DE3)omp8 was associated with a higher antibiotic susceptibility of the E. coli cells to β-lactams compared with expression of OmpPst2. All our data enlighten the involvement of porins in the resistance of P. stuartii to β-lactam antibiotics.     

Characterization of the functional domain of β2-microglobulin from the Asian seabass, Lates calcarifer

Background

β2-Microglobulin (β₂M) is the light chain of major histocompatibility class I (MHC I) that binds non-covalently with the α heavy chain. Both proteins attach to the antigen peptide, presenting a complex to the T cell to be destroyed via the immune mechanism.

Methodology/Principal Findings

In this study, a cDNA sequence encoding β₂M in the Asian seabass (Lates calcarifer) was identified and analyzed using in silico approaches to predict and characterize its functional domain. The β₂M cDNA contains an open reading frame (ORF) of 351 bases with a coding capacity of 116 amino acids. A large portion of the protein consists of the IG constant domain (IGc1), similar to β₂M sequences from other species studied thus far. Alignment of the IGc1 domains of β₂M from L. calcarifer and other species shows a high degree of overall conservation. Seven amino acids were found to be conserved across taxa whereas conservation between L. calcarifer and other fish species was restricted to 14 amino acids at identical conserved positions.

Conclusion/Significance

As the L. calcarifer β₂M protein analyzed in this study contains a functional domain similar to that of β₂M proteins in other species, it can be postulated that the β₂M proteins from L. calcarifer and other organisms are derived from a common ancestor and thus have a similar immune function. Interestingly, fish β₂M genes could also be classified according to the ecological habitat of the species, i.e. whether it is from a freshwater, marine or euryhaline environment.     

Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils

Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of β(2)-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized β2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of β2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of β2m fibril solution. 0.5 mM SDS completely prevented β2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes.     

Dynamics of free versus complexed β2-microglobulin and the evolution of interfaces in MHC class I molecules

In major histocompatibility complex (MHC) class I molecules, monomorphic β₂-microglobulin (β₂m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. β₂M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed β₂m and to understand the evolution of the interfaces between β₂m and different HC. Using human β₂m and the HLA–B*27:09 complex as a model system, a comparison of free and HC-bound β₂m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of β₂m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved β₂m–HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that β₂m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.     

Population study of a sequence polymorphism in intron 2 of the human β-globin gene

The distribution of a nucleotide polymorphism in intron 2 of the -globin gene (IVS-2 nt 666 C > T was examined in populations in southern Germany and Cameroon. The allelic frequencies were 0.86 for T and 0.14 for C in southern Germany and 0.87 for T and 0.13 for C in Cameroon, respectively.     

Association of polymorphisms in the β2-adrenoreceptor gene with higher levels of parasitic infection

The diminishing incidence of parasitic infection in westernised societies has been suggested to result in an increased prevalance of asthma. Asthma is a polygenic disease and genome screens have shown that genes on chromosome 5q31–33 are strongly linked to the disease. The gene for the β₂-adrenoreceptor is located in this region and two polymorphisms have been identified that result in amino acid changes at positions 16 (ArgGly) and 27 (GlnGlu). To determine whether these polymorphisms influence asthma and parasitic infection, a genotype/phenotype study has been performed on a cohort of 126 children from Coche Island in Venezuela. There is a high incidence of asthma on the island and intestinal helminthiasis is endemic. Genotyping for both polymorphisms was carried out by using the polymerase chain reaction and allele-specific oligonucleotide hybridisation. Genotype frequencies in this cohort were consistent with other studies and both polymorphisms were in significant linkage disequilibrium. Individuals who were homozygous for Arg16 had significantly higher levels of specific IgE to Ascaris lumbricoides (P=0.002), significantly higher A. lumbricoides egg counts (P=0.001) and significantly larger wheal sizes following skin-prick testing with A. lumbricoides allergen (P=0.008). There was no association between either polymorphism and total serum IgE or asthma in this population. A combination of mast cell degranulation and the lung migratory phase of A. lumbricoides larvae may result in bronchoconstriction in infected individuals. These results suggest that the Gly 16 allele confers resistance to high levels of parasitic infection in this population. An alternative explanation for the association is that it may be the result of linkage disequilibrium with other genes in the chromosome 5q31–33 region. Received: 25 November 1998 / Accepted: 30 January 1999     

Functional Implication of β-Carotene Hydroxylases in Soybean Nodulation

Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative β-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.Legume-Rhizobium spp. symbiosis results in the formation of the root nodule, in which rhizobia fix atmospheric nitrogen. Nodule development requires diverse events, such as Nod factor synthesis in the rhizobia, perception of the Nod factor on plant roots by receptor-like kinases, endocytosis of rhizobia into plant cells, and so on (Stacey et al., 2006; Oldroyd et al., 2011; Singh and Parniske, 2012). Sequential expression of numerous plant genes occurs during nodulation, contributing to different stages including nitrogen fixation. Arbuscular mycorrhizal (AM) symbiosis exhibits many similarities to the nodulation process (Oldroyd et al., 2009). For example, SymRK, the receptor-like kinase gene, is required for both rhizobial and AM symbioses (Stracke et al., 2002). Similarly, the signal transduction pathways following perception are also in part the same, and the genes common to the two pathways have been referred to as the common symbiosis (SYM) genes (Kistner et al., 2005). These similarities may reflect common mechanisms for host plant cells to respond to symbionts, although the commonality is not globally defined yet.Plant carotenoids are mostly C₄₀ tetraterpenoid pigments with a series of double bonds (DellaPenna and Pogson, 2006; Lu and Li, 2008). They play essential roles in photosynthesis. The phytohormone abscisic acid (ABA) is synthesized from xanthophylls, oxygenated derivatives of carotenoids. The beneficial effects of carotenoids for human disease prevention and health promotion are well established and are based on their antioxidant activities (Kopsell and Kopsell, 2006; Rao and Rao, 2007; von Lintig, 2010). Metabolic engineering approaches have produced crop plants with enhanced carotenoid contents and improved nutritional value (Giuliano et al., 2008). For example, enhancement of β-carotene, provitamin A, by engineering the carotenoid biosynthetic pathway resulted in the development of cv Golden rice (Oryza sativa; Ye et al., 2000; Paine et al., 2005; Ha et al., 2010).The initial step of carotenoid biosynthesis is the production of phytoene by the enzyme phytoene synthase (Fig. 1; DellaPenna and Pogson, 2006; Cazzonelli and Pogson, 2010). The subsequent activities of desaturases, isomerase, and cyclase convert phytoene into lycopene and further into β-carotene. Xanthophyll synthesis begins with the action of β-carotene hydroxylase (BCH) on β-carotene, producing initially β-cryptoxanthin and thereafter zeaxanthin (Kim et al., 2009). Overexpression of BCH has been found to confer tolerance to light stress (Davison et al., 2002). The subsequent steps catalyzed by zeaxanthin epoxidase (ZEP) and neoxanthin synthase lead to the synthesis of ABA (Takaichi and Mimuro, 1998).Open in a separate windowFigure 1.The biosynthetic pathway of carotenoids in plants. GGPP, Geranylgeranyl diphosphate; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTISO, carotene isomerase; LCYB, lycopene β-cyclase; CYP97A3 and CYP97C1, cytochrome P450 enzymes; NSY, neoxanthin synthase; LCYE, lycopene ε-cyclase; CRTR-E, ε-carotene hydroxylase. Enzymes in red were examined in this study.Various carotenoid cleavage dioxygenases (CCDs) catalyze the formation of apocarotenoids with functions as hormones, flavors, and pigments (Auldridge et al., 2006b; Strack and Fester, 2006; Tsuchiya and McCourt, 2009; Walter et al., 2010). Recently, CCD7 and CCD8 were shown to control the synthesis of strigolactones, newly discovered hormones that inhibit shoot branching (Gomez-Roldan et al., 2008; Umehara et al., 2008; Vogel et al., 2010; Ruyter-Spira et al., 2013). In addition, carotenoid cleavage products have been discovered in plant roots colonized by AM fungi (Strack and Fester, 2006). During AM symbiosis, roots synthesize apocarotenoids at the same time as activating plant genes for carotenoid metabolism. Although RNA interference (RNAi)-mediated inhibition of apocarotenoid synthesis suggests that apocarotenoids are functionally significant (Snowden et al., 2005; Floss et al., 2008), their role in AM symbiosis is unknown.In a search for genes differentially induced during soybean (Glycine max)-Rhizobium spp. symbiosis, several antioxidant genes, including a gene encoding a putative BCH, were identified. In this report, we describe genes (GmBCHs) encoding a putative BCH whose expression increased in soybean root nodules. Therefore, the biochemical activities of BCHs were investigated. RNAi inhibition of GmBCH expression interfered with nitrogen fixation as well as nodule development. Subsequent analysis of the expression and biochemical activities of GmCCDs in root nodules led us to hypothesize that GmCCD8 could be involved in the synthesis of apocarotenoids from zeaxanthin in these nodules.