Cells expressing ENOD2 show differential spatial organization during the development of alfalfa root nodules

We have used in situ hybridization to examine the spatial organization of cells expressing the early nodulin gene (ENOD2) during the development of alfalfa root nodules. ENOD2 gene expression was found in the nodule parenchyma, uninfected cells surrounding the symbiotic region of both effective and ineffective nodules. However, in empty nodules, ENOD2 gene expression was found in a mass of parenchyma cells at the base of the nodule. Similar results were also observed in 11-day-old nodules that contained infected cells but that had not yet begun to express leghemoglobin. Although early events of nodulation result in the induction of ENOD2 expression in cells at the nodule base, the pattern of cells expressing ENOD2 during nodule growth appears to be correlated with the development of other peripheral tissues.     

Root, root hair, and symbiotic mutants of the model legume Lotus japonicus

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod- (Ljsym70 to Ljsym73), Hist- (alb1-1, alb1-2, and Ljsym79), and Fix- (fen1, Ljsym75, and Ljsym81).     

Expression of LjENOD40 genes in response to symbiotic and non-symbiotic signals: LjENOD40-1 and LjENOD40-2 are differentially regulated in Lotus japonicus

Nitrogen fixation in nodules provides leguminous plants with an ability to grow in nitrogen-starved soil. Infection of the host plants by microsymbionts triggers various physiological and morphological changes during nodule formation. In Lotus japonicus, expression of early nodulin (ENOD) genes is triggered by perception of bacterial signal molecules, nodulation factors (Nod factors). We examined the expression patterns of ENOD40 genes during the nodule formation process. Two ENOD40 genes of L. japonicus were specifically expressed in the nodule formation process, but they showed different expression patterns upon infection. Each ENOD40 gene demonstrates an individual specificity and regulation with regard to rhizobial infection.     

Expression of the early nodulin, ENOD40, in soybean roots in response to various lipo-chitin signal molecules

The lipo-chitin (LCO) nodulation signal (nod signal) purified from Bradyrhizobium japonicum induced nodule primordia on soybean (i.e. Glycine soja) roots. These primordia were characterized by a bifurcated vascular connection, cortical cell division, and the accumulation of mRNA of the early nodulin gene, ENOD40. A chemically synthesized LCO identical in structure to the Nod signal purified from B. japonicum cultures showed the same activity when inoculated on to soybean roots. Surprisingly, synthetic LCO or chitin pentamer, inactive in inducing root hair curling (HAD) or cortical cell division (NOI) in G. soja, induced the transient accumulation of ENOD40 mRNA. In roots inoculated with such LCO, ENOD40 mRNA was abundant at 40 h after inoculation but decreased to the background levels 6 days after inoculation. In contrast, nod signals active in inducing HAD and NOI induced high levels of ENOD40 accumulation at 40 h and 6 days after inoculation. In situ hybridization analysis showed that ENOD40 mRNA accumulated in the pericycle of the vascular bundle at 24 h after root inoculation with nod signal. At 6 days post-inoculation with nod signal, ENOD40 expression was seen in dividing subepidermal cortical cells. These results provide morphological and molecular evidence that nodule induction in soybean in response to purified or synthetic nod signal is similar, if not identical, to nodule formation induced by bacterial inoculation. Surprisingly, ENOD40 mRNA accumulation occurs in response to non-specific chitin signals. This suggests that, in the case of ENOD40, nodulation specificity is not determined at the level of initial gene expression.     

ENOD12, an early nodulin gene, is not required for nodule formation and efficient nitrogen fixation in alfalfa.

To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medicago sativa subspecies were tested for the presence or absence of ENOD12 alleles. In M. s. ssp coerulea w2 (Mcw2), two ENOD12 genes were detected, whereas in M. s. ssp quasifalcata k93 (Mqk93) only one gene was present. In both plants, the ENOD12 genes were expressed in nodules induced by Rhizobium meliloti. The nucleotide sequence of the ENOD12 genes showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M. s. ssp sativa. ENOD12 from Mqk93 was similar to the corresponding gene found in M. truncatula. From the aligned ENOD12 sequences, an evolutionary tree was constructed. Genetic analysis of the progenies of a cross between Mqk93 and Mcw2 showed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only lacking the ENOD12 gene appeared. Surprisingly, the ENOD12-deficient plants were similar to their wild-type parents in viability, nodule development, nodule structure, and nitrogen fixation efficiency. Therefore, we concluded that in Medicago the ENOD12 gene is not required for symbiotic nitrogen fixation. Furthermore, we proposed that the heterozygous nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the construction of plant mutants deficient in these genes.     

The Lotus japonicus Sen1 gene controls rhizobial differentiation into nitrogen-fixing bacteroids in nodules

A Lotus japonicus mutant, Ljsym75, which forms ineffective symbiotic nodules and defines a new locus involved in the process of nitrogen fixation, was characterized in detail in order to identify the stage of developmental arrest of the nodules. No nitrogen-fixing activity was detectable in Ljsym75 nodules at any stage during plant development, and plant growth was markedly retarded. Ljsym75 plants formed twice as many nodules as the wild-type Gifu, and this phenotype was not influenced by the application of low concentrations of nitrate. Although the ineffective nodules formed on Ljsym75 were anatomically similar to effective Gifu nodules, Ljsym75 nodules senesced prematurely. Microscopic examination revealed that bacteria endocytosed into Ljsym75 nodules failed to differentiate into bacteroids. Moreover, the bacteria contained no nitrogenase proteins, whereas leghemoglobin was detected in the cytosol of the nodules. These results indicate that Ljsym75 is required for bacterial differentiation into nitrogen-fixing bacteroids in nodules, and thus the Ljsym75 gene was renamed sen1 (for stationary endosymbiont nodule). Linkage analysis using DNA markers showed that Sen1 is located on chromosome 4.     

Nodulin gene expression in effective alfalfa nodules and in nodules arrested at three different stages of development

Nodulin gene expression was analyzed in effective and ineffective root nodules of alfalfa (Medicago sativa L. cv Iroquois) elicited by three different Rhizobium meliloti mutants: an exoB mutant having defective acidic exopolysaccharide that does not fluoresce on plates containing the fluorescent brightener Calcofluor; fix21, a spontaneous mutant that has defective lipopolysaccharide and is Calcofluor bright; and a Rhizobium mutant resulting from a Tn5 insertion in the nifH gene of the nif operon. The ineffective nodules elicited by these various mutant rhizobia are blocked at different stages of nodule development and have unique phenotypes. A distinctive pattern of nodulin gene expression as determined by in vitro translations of total nodule RNA characterizes each nodule phenotype. Seventeen nodulins are found in effective nodules including five leghemoglobins. Only one nodulin gene is expressed in the bacteria-free nodules elicited by the exoB mutant. Other nodulin genes (leghemoglobin and nine others) are expressed in fix21-induced nodules. The genes for nodule-enhanced glutamine synthetase as well as for all the other nodulins are expressed in nodules induced by the nifH mutant. The expression of genes for the nodulins, including leghemoglobin, is independent of the nitrogen-fixing ability of the nodule and appears to correlate with the differentiation of densely cytoplasmic host cells in the nodule and, to some extent, with bacterial release from infection threads.     

The aberrant cell walls of boron-deficient bean root nodules have no covalently bound hydroxyproline-/proline-rich proteins.

B-deficient bean (Phaseolus vulgaris L.) nodules examined by light microscopy showed dramatic anatomical changes, mainly in the parenchyma region. Western analysis of total nodule extracts examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one 116-kD polypeptide was recognized by antibodies raised against hydroxyproline-rich glycoproteins (HRGPs) from the soybean (Glycine max) seed coat. A protein with a comparable molecular mass of 116 kD was purified from the cell walls of soybean root nodules. The amino acid composition of this protein is similar to the early nodulin (ENOD2) gene. Immunoprecipitation of the soybean ENOD2 in vitro translation product showed that the soybean seed coat anti-HRGP antibodies recognized this early nodulin. Furthermore, we used these antibodies to localize the ENOD2 homolog in bean nodules. Immunocytochemistry revealed that in B-deficient nodules ENOD2 was absent in the walls of the nodule parenchyma. The absence of ENOD2 in B-deficient nodules was corroborated by performing hydroxyproline assays. Northern analysis showed that ENOD2 mRNA is present in B-deficient nodules; therefore, the accumulation of ENOD2 is not affected by B deficiency, but its assembly into the cell wall is. B-deficient nodules fix much less N2 than control nodules, probably because the nodule parenchyma is no longer an effective O2 barrier.