Cell-specific expression of the promoters of two nonlegume hemoglobin genes in a transgenic legume, Lotus corniculatus.

The promoters of the hemoglobin genes from the nitrogen-fixing tree Parasponia andersonii and the related nonnitrogen-fixing Trema tomentosa both confer beta-glucuronidase reporter gene expression to the central zone of the nodules of a transgenic legume, Lotus corniculatus. beta-Glucuronidase expression was high in the uninfected interstitial cells and parenchyma of the surrounding boundary layer and was low in the Rhizobium-infected cells. This contrasts with the expression of both the P. andersonii hemoglobin protein in P. andersonii nodules and the endogenous Lotus leghemoglobins that are expressed in the infected cells at very high levels. The expression pattern of the P. andersonii and T. tomentosa hemoglobin promoters in L. corniculatus resembles that of a nonsymbiotic hemoglobin gene from Casuarina glauca, which was introduced into this legume, and suggests that only the nonsymbiotic functions of the P. andersonii promoter are being recognized. Deletion of the distal segments of both the P. andersonii and T. tomentosa promoters identified regions important for the control of their tissue-specific and temporal activity in Lotus. Potential regulatory elements, which enhance nodule expression and suppress nonnodule expression, were also identified and localized to a distal promoter segment. A proximal AAGAG motif is present in the P. andersonii, T. tomentosa, and nonsymbiotic Casuarina hemoglobin genes. Mutation of this motif in the P. andersonii promoter resulted in a significant reduction in both the nodule and root expression levels in L. corniculatus. Some of the regulatory motifs characterized are similar to, but different from, the nodulin motifs of the leghemoglobins.     

植物的血红蛋白

近几年来,植物血红蛋白的研究进展十分迅速,豆科植物中与共生固氮无关的血红蛋白基因和包括禾本科植物在内的许多非豆科植物血红蛋白基因的发现使人们对植物血红蛋白有了新的认识,进而把植物血红蛋白分为共生血红蛋白和非共生血红蛋白两种类型。对这两种血红蛋白的性质、功能、基因结构及表达等方面的研究不仅对共生固氮中植物与微生物的相互关系和固氮工程研究;而且对植物细胞的呼吸代谢和耐涝机理等研究有重要价值。     

Expression of symbiotic and nonsymbiotic globin genes responding to microsymbionts on Lotus japonicus

Leguminous plants have both symbiotic and nonsymbiotic hemoglobin (sym- and nonsym-Hb) genes. Three symbiotic (LjLb1, 2, 3) and one nonsymbiotic (LjNSG1) Hb genes were isolated from a genomic library of Lotus japonicus MG20 Miyakojima. Two motif sequences (AAAGAT and CTCTT) critical for nodule specific expression were conserved on the 5'-upstream sequence of LjLb1, 2 and 3. The 5'-upstream region of LjNSG1 contained the sequence consensus for nonsym-Hb. RT-PCR with specific primer sets for each LjLb gene showed that all the sym-Hb genes (LjLb1, 2, 3) were expressed specifically and strongly in root nodules. The expression of LjLb1, 2 and 3 could not be detected in root, leaf or stem of a mature plant, whereas low level expression was detected in seedlings by RT-PCR. This suggests that sym-Hbs may have another unknown function besides being oxygen transporter for the microsymbiont in symbiotic nitrogen fixation. The expression of LjNSG1, examined with RT-PCR, was detected at low level in root, leaf and stem. The expression of LjNSG1 was enhanced in root nodules, whereas it was repressed in roots colonized by mycorrhizal fungi Glomus sp. R10. The repression of the nonsym-Hb gene was also observed in the roots of Medicago sativa colonized by Glomus sp. R10.     

Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus.

Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.     

Comparison of nodule induction in legume and actinorhizal symbioses: the induction of actinorhizal nodules does not involve ENOD40

Two types of root nodule symbioses are known for higher plants, legume and actinorhizal symbioses. In legume symbioses, bacterial signal factors induce the expression of ENOD40 genes. We isolated an ENOD40 promoter from an actinorhizal plant, Casuarina glauca, and compared its expression pattern in a legume (Lotus japonicus) and an actinorhizal plant (Allocasuarina verticillata) with that of an ENOD40 promoter from the legume soybean (GmENOD40-2). In the actinorhizal Allocasuarina sp., CgENOD40-GUS and GmENOD40-2-GUS showed similar expression patterns in both vegetative and symbiotic development, and neither promoter was active during nodule induction. The nonsymbiotic expression pattern of CgENOD40-GUS in the legume genus Lotus resembled the nonsymbiotic expression patterns of legume ENOD40 genes; however, in contrast to GmENOD40-2-GUS, CgENOD40-GUS was not active during nodule induction. The fact that only legume, not actinorhizal, ENOD40 genes are induced during legume nodule induction can be linked to the phloem unloading mechanisms established in the zones of nodule induction in the roots of both types of host plants.     

Legumes select symbiosis island sequence variants in Bradyrhizobium

Bradyrhizobium strains sampled from 14 legume genera native to eastern North America showed substantial host‐related phylogenetic clustering at three loci in the symbiotic island (SI) region (nodC, nifD, nifH), indicating selection of distinct suites of SI lineages by different legumes. Bacteria assorted consistently with particular legumes across two regions separated by 800 km, implying recurrent assembly of the same symbiotic combinations. High genetic polymorphism of all three SI loci relative to four nonsymbiotic loci supported the inference that a form of multiple‐niche balancing selection has acted on the SI region, arising from differential symbiont utilization by different legume taxa. Extensive discordance between the tree for SI variants and a phylogenetic tree inferred for four housekeeping loci implied that lateral transfer of the symbiosis island region has been common (at least 26 transfer events among 85 Bradyrhizobium strains analysed). Patterns of linkage disequilibrium also supported the conclusion that recombination has impacted symbiotic and nonsymbiotic regions unequally. The high prevalence of lateral transfer suggests that acquisition of a novel SI variant may often confer a strong selective advantage for recipient cells.     

Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China

A total of 115 endophytic bacteria were isolated from root nodules of the wild legume Sphaerophysa salsula grown in two ecological regions of Loess Plateau in China. The genetic diversity and phylogeny of the strains were revealed by restriction fragment length polymorphism and sequencing of 16S rRNA gene and enterobacterial repetitive intergenic consensus-PCR. Their symbiotic capacity was checked by nodulation tests and analysis of nifH gene sequence. This is the first systematic study on endophytic bacteria associated with S. salsula root nodules. Fifty of the strains found were symbiotic bacteria belonging to eight putative species in the genera Mesorhizobium, Rhizobium and Sinorhizobium, harboring similar nifH genes; Mesorhizobium gobiense was the main group and 65 strains were nonsymbiotic bacteria related to 17 species in the genera Paracoccus, Sphingomonas, Inquilinus, Pseudomonas, Serratia, Mycobacterium, Nocardia, Streptomyces, Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus, which were universally coexistent with symbiotic bacteria in the nodules. Differing from other similar studies, the present study is the first time that symbiotic and nonsymbiotic bacteria have been simultaneously isolated from the same root nodules, offering the possibility to accurately reveal the correlation between these two kinds of bacteria. These results provide valuable information about the interactions among the symbiotic bacteria, nonsymbiotic bacteria and their habitats.     

Structural and expression analysis of uricase mRNA from Lotus japonicus

Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.     

Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants.

Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated [Landsmann et al. (1986). Nature 324, 166-168; Bogusz et al. (1988). Nature 331, 178-180]. The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus. Both promoters directed root-specific expression in transgenic tobacco. When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene. The T. tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots. The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes. We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes [Stougaard et al. (1987). EMBO J. 6, 3565-3569].     

Regulation of nonsymbiotic and truncated hemoglobin genes of Lotus japonicus in plant organs and in response to nitric oxide and hormones

? In legumes, symbiotic leghemoglobins facilitate oxygen diffusion to the bacteroids, but the roles of nonsymbiotic and truncated hemoglobins are largely unknown. Here the five hemoglobin genes of Lotus japonicus have been functionally characterized to gain insight into their regulatory mechanisms. ? Plants were exposed to nitric oxide donors, stressful conditions, and hormones. Gene expression profiling was determined by quantitative PCR, and gene activities were localized using in situ hybridization and promoter-reporter gene fusions. ? The LjGLB1-1, LjGLB2, and LjGLB3-1 mRNA expression levels were very high in nodules relative to other plant organs. The expression of these genes was localized in the vascular bundles, cortex, and infected tissue. LjGLB1-1 was the only gene induced by nitric oxide. Cytokinins caused nearly complete inactivation of LjGLB2 and LjGLB3-1 in nodules and induction of LjGLB1-1 in roots. Abscisic acid induced LjGLB1-1 in nodules and LjGLB1-2 and LjGLB2 in roots, whereas polyamines and jasmonic acid induced LjGLB1-1 only in roots. ? The enhanced expression of the three types of hemoglobins in nodules, the colocalization of gene activities in nodule and root tissues with high metabolic rates, and their distinct regulatory mechanisms point out complementary roles of hemoglobins and strongly support the hypothesis that LjGLB1-1, LjGLB2, and LjGLB3-1 are required for symbiosis.     

Cloning of a full-length symbiotic hemoglobin cDNA and in situ localization of the corresponding mRNA in Casuarina glauca root nodule

We have characterized a full-length cDNA ( hb -Cg1F) that represents symbiotic mRNA hemoglobin ( hb ) from Casuarina glauca root nodules. In situ hybridization was used to examine the correlation between hb -Cg1F mRNA and the state of the Frankia infection process. The efficiency of in situ hybridization using DIG-labeled vs [³⁵S]-labeled probes was compared. The expression of hb -Cg1F gene is induced in young infected host cells prior to the detection of Frankia nif H mRNA. Since Frankia does not form vesicles in C. glauca nodules, it is proposed that Hb is necessary to reduce the O₂ concentration in the cytoplasm of the host cells before the nif genes are expressed.     

Lifestyle alternatives for rhizobia: mutualism, parasitism, and forgoing symbiosis

Strains of rhizobia within a single species can have three different genetically determined strategies. Mutualistic rhizobia provide their legume hosts with nitrogen. Parasitic rhizobia infect legumes, but fix little or no nitrogen. Nonsymbiotic strains are unable to infect legumes at all. Why have rhizobium strains with one of these three strategies not displaced the others? A symbiotic (mutualistic or parasitic) rhizobium that succeeds in founding a nodule may produce many millions of descendants. The chances of success can be so low, however, that nonsymbiotic rhizobia can have greater reproductive success. Legume sanctions against nodules that fix little or no nitrogen favor more mutualistic strains, but parasitic strains that use plant resources only for their own reproduction may do well when they share nodules with mutualistic strains.     

The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.