Effect of root interaction on nodulation characteristics and isoflavonoid mechanism of alfalfa in the simulated alfalfa/oat in pots

Rational intercropping is capable of promoting nodulation and facilitating the fixation and utilization of nitrogen (N) of legumes. Isoflavonoid is critical to form root nodules of legumes, whereas the regulation of isoflavonoid over nodulation and N fixation in legume/cereal intercropping remains unclear. In the present study, nutrient solution of different root partitions (e.g., no barrier (A-O), nylon mesh barrier (NA-O), plastic barrier (PA-O) and sole alfalfa (SA)) and nitrogen levels (e.g., 21 and 210 mg L-1 N) were used to delve into the variations of nodulation, N fixation, isoflavonoid content, isoflavonoid synthase (IFS) and nodulation-signaling pathway (NOD) genes in alfalfa. As suggested from the results, the parameters of nodulation and N fixation ability with A-O were all evidently higher than those with PA-O and SA. Formononetin and genistein with A-O had noticeably higher contents than those with PAT and SA. Daidzein and luteolin with A-O had remarkably higher contents than those with NA-O in the root. IFR 1, IFR 4, NOD 1 and NOD 2 with A-O and NA-O had noticeably higher relative expressions than those with SA and PA-O. IFR 2 and IFR 3 with AT and NA-T had significantly lower relative expressions than those with PA-O and SA. The nodulation and isoflavonoids met significant positive associations. IFR and NOD genes and TNN and ENN followed significant positive associations. NOD genes and formononetin exhibited extremely significant positive associations. The conclusion was drawn that the closer root interaction between alfalfa and oat, the more effective the nodulation, N fixation ability and isoflavoids content of alfalfa will be. Isoflavonoids primarily affected nodulation and N fixation. The mechanism of isoflavonoids on nodulation and N fixation in alfalfa/oat intercropping was explained that root interaction results in the reduction of environmental N, thereby regulating the relative expressions of IFR genes, elevating the isoflavonoids contents, and up-regulating the relative expressions of NOD genes; as a result, the nodulation and N fixation ability in alfalfa are facilitated.

Using nod genes control system to create rhizospheric microorganisms with regulated gene expression

Inducible vector containing the full-sized nodD gene and the promoter region of the nod-box under the control of which was cloned the gfp gene was constructed. Modified bacteria R. galegae in which the synthesis of GFP protein was activated by plant flavonoids were obtained.

The Modification of the Flavonoid Naringenin by Bradyrhizobium sp. Strain ORS285 Changes the nod Genes Inducer Function to a Growth Stimulator

As inducers of nodulation (nod) genes, flavonoids play an important role in the symbiotic interaction between rhizobia and legumes. However, in addition to the control of expression of nod genes, many other effects of flavonoids on rhizobial cells have been described. Here, we show that the flavonoid naringenin stimulates the growth of the photosynthetic Bradyrhizobium sp. strain ORS285. This growth-stimulating effect was still observed for strain ORS285 with nodD1, nodD2, or the naringenin-degrading fde operon deleted. Phenotypic microarray analysis indicates that in cells grown in the presence of naringenin, the glycerol and fatty acid metabolism is activated. Moreover, electron microscopic and enzymatic analyses show that polyhydroxy alkanoate metabolism is altered in cells grown in the presence of naringenin. Although strain ORS285 was able to degrade naringenin, a fraction was converted into an intensely yellow-colored molecule with an m/z (+) of 363.0716. Further analysis indicates that this molecule is a hydroxylated and O-methylated form of naringenin. In contrast to naringenin, this derivative did not induce nod gene expression, but it did stimulate the growth of strain ORS285. We hypothesize that the growth stimulation and metabolic changes induced by naringenin are part of a mechanism to facilitate the colonization and infection of naringenin-exuding host plants.

Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes

Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.