The diversity of stem-associated bacteria of non-nodulated (Nod À ), wild-type nodulated (Nod þ ) and hypernodulated (Nod þ þ ) soybeans were evaluated by clone library analyses of the 16S ribosomal RNA gene. Soybeans were dressed with standard nitrogen (SN) fertilization (15 kg N ha -1 ) and heavy nitrogen (HN) fertilization (615 kg N ha -1 ). The relative abundance of Alphaproteobacteria in Nod þ soybeans (66%) was smaller than that in Nod À and Nod þ þ soybeans (75-76%) under SN fertilization, whereas that of Gammaproteobacteria showed the opposite pattern (23% in Nod þ and 12-16% in Nod À and Nod þ þ soybeans). Principal coordinate analysis showed that the bacterial communities of Nod À and Nod þ þ soybeans were more similar to each other than to that of Nod þ soybeans under SN fertilization. HN fertilization increased the relative abundance of Gammaproteobacteria in all nodulation phenotypes (33-57%) and caused drastic shifts of the bacterial community. The clustering analyses identified a subset of operational taxonomic units (OTUs) at the species level in Alpha-and Gammaproteobacteria responding to both the nodulation phenotypes and nitrogen fertilization levels. Meanwhile, the abundance of Betaproteobacteria was relatively constant in all libraries constructed under these environmental conditions. The relative abundances of two OTUs in Alphaproteobacteria (Aurantimonas sp. and Methylobacterium sp.) were especially sensitive to nodulation phenotype and were drastically decreased under HN fertilization. These results suggested that a subpopulation of proteobacteria in soybeans is controlled in a similar manner through both the regulation systems of plant-rhizobia symbiosis and the nitrogen signaling pathway in plants.

SUMMARYZinc (Zn) depletion adversely affects plant growth. To avoid lethal depletion of cellular Zn, plants have evolved mechanisms to adjust the expression of genes associated with Zn homeostasis, the details of which are poorly understood. In the present study, we isolated an Arabidopsis thaliana T-DNA insertion mutant that exhibited hypersensitivity to Zn depletion. By monitoring root development under Zn-deficient conditions, we isolated a single mutant lacking the basic-region leucine-zipper transcription factor gene bZIP19. To identify proteins whose expression is affected by bZIP19, an iTRAQ-based quantitative proteomics analysis was performed using microsomal proteins from wild-type and the bzip19 mutant A. thaliana roots grown on Basal and Zn-deficient media. Of the 797 proteins identified, expression of two members of the Zrt-and Irt-related protein family, ZIP3 and ZIP9, and three defensin-like family proteins was markedly induced in wild-type but not in the bzip19 mutant under Zn-deficient conditions. Furthermore, selected reaction monitoring and quantitative real-time PCR revealed that ZIP9 expression is mediated by bZIP19 and may be partly supported by bZIP23, a homolog of bZIP19. Mutant analysis revealed that ZIP9 is involved in uptake of Zn by the roots, and the mutant lacking ZIP9 was significantly more sensitive to Zn depletion than the wild-type. These results demonstrate that bZIP19 mainly contributes to expression of genes, such as ZIP9, under Zn-deficient conditions.

A model system developed to produce N2O emissions from degrading soybean nodules in the laboratory was used to clarify the mechanism of N2O emission from soybean fields. Soybean plants inoculated with nosZ-defective strains of Bradyrhizobium japonicum USDA110 (ΔnosZ, lacking N2O reductase) were grown in aseptic jars. After 30 days, shoot decapitation (D, to promote nodule degradation), soil addition (S, to supply soil microbes), or both (DS) were applied. N2O was emitted only with DS treatment. Thus, both soil microbes and nodule degradation are required for the emission of N2O from the soybean rhizosphere. The N2O flux peaked 15 days after DS treatment. Nitrate addition markedly enhanced N2O emission. A 15N tracer experiment indicated that N2O was derived from N fixed in the nodules. To evaluate the contribution of bradyrhizobia, N2O emission was compared between a nirK mutant (ΔnirKΔnosZ, lacking nitrite reductase) and ΔnosZ. The N2O flux from the ΔnirKΔnosZ rhizosphere was significantly lower than that from ΔnosZ, but was still 40% to 60% of that of ΔnosZ, suggesting that N2O emission is due to both B. japonicum and other soil microorganisms. Only nosZ-competent B. japonicum (nosZ+ strain) could take up N2O. Therefore, during nodule degradation, both B. japonicum and other soil microorganisms release N2O from nodule N via their denitrification processes (N2O source), whereas nosZ-competent B. japonicum exclusively takes up N2O (N2O sink). Net N2O flux from soybean rhizosphere is likely determined by the balance of N2O source and sink.

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The diversities leaf-associated bacteria on nonnodulated (Nod ؊ ؉؉ soybeans (46% to 76%) and, conversely, increased those of Gammaproteobacteria and Firmicutes in these mutant soybeans. In the Alphaproteobacteria, cluster analyses identified two operational taxonomic units (OTUs) (Aurantimonas sp. and Methylobacterium sp.) that were especially sensitive to nodulation phenotypes under SN fertilization and to nitrogen fertilization levels. Arbuscular mycorrhizal infection was not observed on the root tissues examined, presumably due to the rotation of paddy and upland fields. These results suggest that a subpopulation of leaf-associated bacteria in wild-type Nod ؉ soybeans is controlled in similar ways through the systemic regulation of autoregulation of nodulation, which interferes with the impacts of N levels on the bacterial community of soybean leaves.)Although diverse microorganisms reside in the phytosphere as endophytes, epiphytes, and rhizosphere bacteria, many questions about the driving forces and ecological rules underlying the relationships between these microbes and plants remain unanswered (18,39). During their evolution, legumes have developed two systems for attaining mutual symbioses with rhizobia and mycorrhizae. One of the systems genetically required for rhizobial and arbuscular mycorrhizal interactions in plants overlaps in a common signaling pathway (CSP) leading to successful symbioses (24). Plants also have a control system for regulating the degree of nodulation and mycorrhization on roots by rhizobia and mycorrhizae, respectively. This autoregulatory system occurs through long-distance signaling between shoots and roots (33). Leguminous plants deficient in the CSP and autoregulation systems develop nonnodulated (Nod Ϫ ) and hypernodulated (Nod ϩϩ ) roots, respectively. However, the degree to which plants use similar or identical systems, such as CSP and autoregulation, for interactions with other microorganisms in the phytosphere remains unclear.Recently, it was shown that the bacterial and fungal community structures in the roots of symbiosis-defective mutants of Medicago truncatula (32) and soybean (22) differ from those in the roots of wild-type host plants; it was also shown that certain microbes preferentially associate with arbuscular mycorrhizal roots (41) and nodulated (Nod ϩ ) roots (22). However, unexpectedly, analyses of the rhizosphere community in soybeans have revealed that the bacterial community in nonnodulated soybeans is more similar to that in hypernodulated soybeans than to that in wild-type soybeans (22).The autoregulation of nodulation occurs through long-distance signaling between shoots and roots (33), and a heavy supply of nitrate to the roots of leguminous plants inhibits nodulation through autoregulation (6, 34). Thus, it is possible that the nodulation phenotype and host nitrogen status affect the structure of the microbial community in aboveground tissues, such as stems and leaves, as has been observed in roots (22). Indeed, the results of our previous study o...