Plant gene expression during effective and ineffective nodule development of the tropical stem-nodulated legume Sesbania rostrata

The expression of plant genes during symbiosis of Sesbania rostrata with Rhizobium sp. and Azorhizobium caulinodans was studied by comparing two-dimensional PAGE patterns of in vitro translation products of poly(A)⁺ RNA from uninfected roots and stems with that of root and stem nodules. Both types of nodules are essentially similar, particularly when stem nodules are formed in the dark. We detected the specific expression of at least 16 genes in stem and root nodules and observed the stimulated expression of about 10 other genes in both nodules. Six of the nodule-specific translation products (apparent molecular masses around 16 kDa) cross-react with an antiserum raised against leghemoglobin purified from Sesbania rostrata stem nodules. During stem nodule development, most of the nodule-stimulated genes are expressed concomitantly with leghemoglobin at day 12 after inoculation. However, some genes are already stimulated at days 6–7, some others later in development (day 18), and some are transiently activated. Patterns of root nodules induced by either Azorhizobium caulinodans strain ORS571, capable of effective root and stem nodulation, or Rhizobium sp. strain ORS51, capable of effective root nodulation only, are very similar except for a specific 37.5 kDa polypeptide. Several types of ineffective stem and root nodules were studied; in every case the amount of leghemoglobin components appeared reduced together with most of the nodule-stimulated polypeptides.     

Nodule-enhanced protease inhibitor gene: emerging patterns of gene expression in nodule development on Sesbania rostrata

A novel marker for the early stages of nodulation of Sesbania rostrata was found to encode a putative member of the Kunitz family of protease inhibitors (SrPI1). Its expression was enhanced during nodulation, and was not up-regulated by wounding or upon infection with wide host-range pathogens. In situ expression patterns resembled those previously described for functions that may be implicated in delimiting infected nodule tissues from the rest of the plant. Thus, SrPI1 may be a component of a multi-layered barrier that restrains the invading rhizobia.     

Growth of Sesbania rostrata (Brem) with stem nodules under controlled conditions

A series of experiments was carried out in an attempt to produce nodulated plants of Sesbania rostrata with qualities more closely resembling those in the wild than has been achieved to date. When groups of five plants were grown in a controlled climate chamber in pipes containing ～12dm³ modified Jensen's medium with 6mol m^?3 nitrate, the daily growth in height reached 5 cm and at 30 d the plants were ～40cm high. At this time, the stems were inoculated with Azorhizobium caulinodans ORS 571 and the medium replaced with Jensen's medium without nitrate. In the subsequent 19-d period ～300 nodules (representing >50% of the potential infection sites) developed on each stem. The nodules increased linearly in size over this time to ～15mg. Specific acetylene reduction activity, ARA ((μmol C₂H₄ mg^?1 h^?1) rose to 45 between days 5 and 10 after inoculation and plateaued; total ARA rose to ～200 μmol C₂H₄ plant^?1 h^?1. Under the conditions described the plants grew vigorously, and reproducibly uniform yields of nodules with high ARA activities were obtained. As outlined, the procedure offers a standard system in which, within a 2-week period after inoculation, individual strains of bacteria can be quantitatively compared in their ability to induce nodulation and N₂-fixation. Physiological and biochemical aspects of the nodulated system can be much more readily approached than with plants producing only root nodules. The inhibitory effects of stem nodules induced by wild type and two mutant strains of Azorhizobium on the development and activity of root nodules are described.     

Sesbania rostrata root and stem nodule leghemoglobins: purification, and relationships among the seven major components

By anion-exchange chromatography, the nitrogen fixing photosynthetic stem nodules and nonphotosynthetic root nodules of Sesbania rostrata are shown to contain the same seven major components of leghemoglobin (Lb), numbered LbI-LbVII in order of elution, although in different proportions. No novel component was found in photosynthetic nodules. All components of Sesbania Lb are monomeric, with molecular weights varying between 15,000 and 17,000, and at least six of them are separate gene products. It is suspected that variable conjugation with nonprotein moieties might be partially responsible for the molecular weight differences and anomalous behavior observed between isoelectric focusing and anion-exchange chromatography.     

Translocation of iron to the N2-fixing stem nodules of Sesbania rostrata (Brem)

Labelled iron (applied as ⁵⁵FeCl₃) moves rapidly from the soilto the shoots and stem nodules of S. rostrata. Time-course experimentsare described that investigate the movement of iron throughdifferent plant parts, detailing the route of iron translocation.The results show that iron moves first to the mature leavesand is subsequently translocated to the vegetative buds andstem nodules. Phloem girdling the stem nodules almost completelystops the movement of labelled iron into the nodules. We concludethat iron is supplied to stem nodules in the phloem; these resultssupport other work suggesting there is no inward xylem flowinto legume nodules, which probably receive almost all nutrientsand most of their water via phloemmediated transport. Key words: Translocation, nutrient, Fe, iron, stem nodules, legume, nitrogen fixation     

Initial stages in the morphogenesis of nitrogen-fixing stem nodules of Sesbania rostrata.

Morphogenesis of stem nodules in Sesbania rostrata was studied over a period of 6 days after inoculation with an appropriate species of Rhizobium. Nodulation sites were initially slightly raised, circular areas 0.3 to 0.6 mm in diameter and 4 to 5 mm apart in vertical rows along the length of the stem. Each site was underlaid by an adventitious root primordium. A site became susceptible to infection by a specific Rhizobium sp. when the root primordium broke through the epidermis, leaving a fissure. Rhizobia multiplied within this fissure and colonized the exposed intercellular spaces. The infection extended inward as narrow, branched intercellular threads moved into a cortical meristematic zone, where cell division was initiated, and invagination of infection thread branches into adjacent plant cells followed. Rhizobia were released into the plant cells and surrounded immediately by plant membrane. Intracellular rhizobia divided actively, leading to bacteroid-filled cells. Infected areas enlarged and coalesced as the nodule matured.     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.     

Detection of lectin-related sequences in the genome of Sesbania rostrata

The extensive homologies between the amino-acid sequences of lectins extracted from the seeds of Leguminous plants have been confirmed by using cDNAs corresponding to the lectin genes from Pea, French bean, and Soybean. These cDNAs, used as probes, were hybridized with restriction fragments of genomic DNA from Pea. Good cross-hybridizations were observed. This strategy was extended to another plant: Sesbania rostrata, a tropical legume for which the presence of lectins has not been reported yet. Several genomic fragments hybridize with the lectin cDNA probes, indicating the existence of lectin-related sequences.     

Occurrence of Two Pathways for Malate Oxidation in Bacteroids Isolated from Sesbania rostrata Stem Nodules during C(2)H(2) Reduction

Malate oxidation supported C₂H₂ reduction by bacteroids isolated from Sesbania rostrata stem nodules. Optimal activity reached 7.5 nanomoles per minute per milligram of dry weight and was in the same order of magnitude as that observed with succinate but always required a lower O₂ tension. Malate dehydrogenase (EC 1.1.1.37), purified 66-fold from bacteroids, actively oxidized malate (K_m = 0.19 millimolar). Malic enzyme (EC 1.1.1.39) from Sesbania bacteroids had a lower affinity for malate (K_m = 2.32 millimolar). Both enzymes exclusively required NAD⁺ as cofactor and required an alkaline pH for optimal activity. 2-Oxoglutarate and oxalate, inhibiting malate dehydrogenase and malic enzyme, respectively, were used to specifically block each malate oxidation pathway in bacteroids. The predominance of malate dehydrogenase activity to support bacteroid N₂ fixation was demonstrated. The inhibition of O₂ consumption by 2-oxoglutarate confirmed the importance of the malate dehydrogenase pathway in malate oxidation. It is proposed that the utilization of malate, with regard to O₂, is important in a general strategy of this legume to maintain N₂ fixation under O₂ limited conditions.     

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5-Srglb3-uidA-3nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.