NAD+-malic enzyme affects nitrogenase activity of Mesorhizobium loti bacteroids in Lotus japonicus nodules

Abstract

A gram-negative bacterium, Mesorhizobium loti, contains a NADP ϩ -malic enzyme (mlr5329) and a malate oxidoreductase (mlr0809) in the genome. We have screened transposon-induced mutants from the signature-tagged mutant library to survey their roles in nodule nitrogenase activity. The nodules induced by malate oxidoreductase mutants failed to fix N 2 , although NADP ϩ -malic enzyme (NADP ϩ -ME) mutants induced nodules exhibiting no change in nodule nitrogenase activity. When malate-degrading enzyme activities were compared between malate oxidoreductase mutants and wild-type M. loti, NAD ϩ -malic enzyme (NAD ϩ -ME) activity was decreased significantly in malate oxidoreductase mutants, suggesting it is an NAD ϩ -ME mutant. We found that NADP ϩ -ME was not required for N 2 fixation. The fact that significant accumulations of sucrose, starch granules and malate were observed in the nodules induced by malate oxidoreductase mutant suggests that low nitrogenase activity of the nodules resulted in photosynthate accumulation. These data suggest that this malate oxidoreductase has a similar function to NAD ϩ -ME in both Bradyrhizobium japonicum and Sinorhizobium meliloti. Plant Biotechnology 27, 311-316 (2010) Original Paper Transposon insertion mutants, generated using signature-tagged mutagenesis (STM), are a powerful technique that can be used to identify the genes required for symbiotic nitrogen fixation function in root nodules (Shimoda et al. 2008). We have selected STM mutants that had a transposon inserted in the malic enzyme genes from the collection of transposon mutants and studied the function of these genes. Key words: Lotus japonicus, Mesorhizobium loti, NAD Materials and methods Bacterial strains and mediaTransposon insertion mutants (STM mutant) of M. loti MAFF303099 were obtained from Kazusa DNA Research Institute (Shimoda et al. 2008). M. loti strains were cultured in tryptone-yeast extract (TY) liquid medium (Beringer 1974) at 28ºC. Antibiotics were added to the media in the following concentrations: spectinomycin 100 µg mL Ϫ1, streptomycin 100 µg mL -1 and phosphomycin 100 µg mL Ϫ1. Plant materialsSeeds of L. japonicus B-129 Gifu (Handberg and Stougaard 1992) were sterilized with a sodium hypochlorite solution, rinsed with sterile water, and allowed to germinate in sterile vermiculite with liquid B&D medium (Broughton and Dilwoth 1971) in double Magenda jars. Then 1ϫ10 9 cells mL Ϫ1 was added in the jars. These plants were grown in an artificially growth cabinet controlled at 22ºC (16 hr/8hr, light/dark). Acetylene reduction activityFor acetylene reduction activity, plants at 35 days after infection were used. The nodules were placed in a 20 ml vial and incubated at 25ºC by adding 2.6 ml of acetylene. After 30 min, the amount of ethylene formed was measured by gas chromatography (Shimazu GC-8A) to determine ethylene production as described previously (Kouchi et al. 1989) Light microscopyNodules were fixed in 4% paraformaldehyde and 25% glutaraldehyde in 0.5 M Sodium phosphate buffer (pH 7.2...

Cell autonomous sanctions in legumes target ineffective rhizobia in nodules with mixed infections

“…1–3; Appendix S1), and (3) maintained intact symbiosomes with relatively stable bacteroid populations (Table 3). These latter features are typical of effective nodules in legumes with determinate nodule development (Chua et al, 1985; Banba et al, 2001; Schumpp and Deakin, 2010; Thapanapongworakul et al, 2010; Chungopast et al, 2014). The features associated with the hypothesized induced cellular senescence appeared to initiate slightly earlier in L. japonicus hosts (∼5 wpi) compared to A. strigosus (∼6 wpi) consistent with the early‐flowering phenotype and faster development of the L. japonicus MG‐20 accession (Kawaguchi, 2000).…”

Transport and Metabolism in Legume-Rhizobia Symbioses

“…Sinorhizobium sp. NGR234 also uses both of these pathways on a variety of hosts, and M. loti and B. japonicum USDA110 may also use both pathways, on the basis that dme mutants retain some nitrogen fixation (26,142). Dme is a complex protein with N-terminal, malic enzyme, and C-terminal phosphotransacetylase (Pta) domains.…”

NAD(P) + -Malic Enzyme Mutants of Sinorhizobium sp. Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N 2 Fixation Capabilities

“…The NGR234 dme mutants were found to form Fix ϩ nodules with various N 2 fixation efficiencies on various host plants (see below). Moreover, while this work was under way, dme mutants of B. japonicum, R. leguminosarum, and Mesorhizobium loti were shown to form Fix ϩ nodules on soybean, pea, and Lotus, respectively (8,36,48). Together, these findings demonstrate that the N 2 fixation phenotype of dme mutants is host plant dependent and also suggest that the completely Fix Ϫ phenotype of S. meliloti dme mutants is unusual and perhaps unique to the S. meliloti-alfalfa symbiosis.…”

“…In a symbiotic mutant screen of A. caulinodans ORS571, two insertion mutants with mutations in a malic enzyme-like gene (azc3656) were found to form Fix Ϫ nodules (47). In a subsequent study, transcription of a second malic enzyme-like gene (azc0119) was found to be much higher in A. caulinodans bacteroids than in free-living cells (48). The interpre- tation of these results was hindered by an absence of functional data on either the azc3656 or azc0119 gene product.…”