Root nodulation of Sesbania rostrata.

The tropical legume Sesbania rostrata can be nodulated by Azorhizobium caulinodans on both its stem and its root system. Here we investigate in detail the process of root nodulation and show that nodules develop exclusively at the base of secondary roots. Intercellular infection leads to the formation of infection pockets, which then give rise to infection threads. Concomitantly with infection, cortical cells of the secondary roots dedifferentiate, forming a meristem which has an "open-basket" configuration and which surrounds the initial infection site. Bacteria are released from the tips of infection threads into plant cells via "infection droplets," each containing several bacteria. Initially, nodule differentiation is comparable to that of indeterminate nodules, with the youngest meristematic cells being located at the periphery and the nitrogen-fixing cells being located at the nodule center. Because of the peculiar form of the meristem, Sesbania root nodules develop uniformly around a central axis. Nitrogen fixation is detected as early as 3 days following inoculation, while the nodule meristem is still active. Two weeks after inoculation, meristematic activity ceases, and nodules then show the typical histology of determinate nodules. Thus, root nodule organogenesis in S. rostrata appears to be intermediate between indeterminate and determinate types.     

A gfp reporter plasmid to visualize Azorhizobium caulinodans during nodulation of Sesbania rostrata

Compared with other labeling techniques, the use of the green fluorescent protein (GFP) is advantageous to visualize bacteria because observations can be performed in real time. This feature is particularly interesting to study invasion events of rhizobia during nodule development on their legume host plant. To investigate the symbiotic interaction between Azorhizobium caulinodans ORS571 and Sesbania rostrata, we constructed two plasmids, pMP220-hem-gfp5 and pBBR5-hem-gfp5-S65T, that carry a modified gfp gene, the expression of which is controlled by the constitutive hem promoter. Introduction of either of these plasmids into A. caulinodans allowed the visualization of single bacteria. Determination of the plasmid stability in cultured bacteria and in nodules demonstrated that pBBR5-hem-gfp5-S65T is more stable than pMP220-hem-gfp5. The plasmid pBBR5-hem-gfp5-S65T can be used to study early invasion events during nodule development on hydroponic roots of S. rostrata.     

Nod factor requirements for efficient stem and root nodulation of the tropical legume Sesbania rostrata

Azorhizobium caulinodans ORS571 synthesizes mainly pentameric Nod factors with a household fatty acid, an N-methyl, and a 6-O-carbamoyl group at the nonreducing-terminal residue and with a d-arabinosyl, an l-fucosyl group, or both at the reducing-terminal residue. Nodulation on Sesbania rostrata was carried out with a set of bacterial mutants that produce well characterized Nod factor populations. Purified Nod factors were tested for their capacity to induce root hair formation and for their stability in an in vitro degradation assay with extracts of uninfected adventitious rootlets. The glycosylations increased synergistically the nodulation efficiency and the capacity to induce root hairs, and they protected the Nod factor against degradation. The d-arabinosyl group was more important than the l-fucosyl group for nodulation efficiency. Replacement of the 6-O-l-fucosyl group by a 6-O-sulfate ester did not affect Nod factor stability, but reduced nodulation efficiency, indicating that the l-fucosyl group may play a role in recognition. The 6-O-carbamoyl group contributes to nodulation efficiency, biological activity, and protection, but could be replaced by a 6-O-acetyl group for root nodulation. The results demonstrate that none of the studied substitutions is strictly required for triggering normal nodule formation. However, the nodulation efficiency was greatly determined by the synergistic presence of substitutions. Within the range tested, fluctuations of Nod factor amounts had little impact on the symbiotic phenotype.     

毛萼田菁和其它茎瘤固氮植物(综述)

近来,豆科植物茎瘤固氮研究取得很大进展,这种双重固氮能力在农业上具有很大应用潜力。茎瘤固氮植物主要有三个属,即田菁属（Ｓｅｓｂａｎｉａ）、合萌属（Ａｅｓｃｈｙｎｏｎｍｅｎｅ）和假含羞草属（Ｎｅｐｔｕｎｉａ）。本文论述它们的生长特性、茎瘤的形成、固氮及在农业上的应用。     

长喙田菁-Azorhizobium caulinodans共生固氮体系在华南地区的生长、结瘤、固氮和种子生产

研究了长喙田菁－Ａｚｏｒｈｉｚｏｂｉｕｍｃａｕｌｉｎｏｄａｎｓ共生固氮体系在华南地区的生长、结瘤、固氮和种子生产．结果表明,长喙田菁－Ａ．ｃａｕｌｉｎｏｄａｎｓ共生固氮体系在华南地区生长正常,并具旺盛的茎根瘤结瘤作用．经６５天的生长,其在湛江地区的单位面积生物量（干物质）和单位面积Ｎ产量分别为２８７５２和６８１ｋｇ·ｈｍ－２,远优于普通田菁的１６５２０和３５２ｋｇ·ｈｍ－２;茎瘤菌Ａ．ｃａｕｌｉｎｏｄａｎｓ品系ＡＲ１１１和ＡＲ５６在华南地区混合接种效果良好,植株茎瘤结瘤率达到１００％,平均单株茎瘤个数为１８２个,单株茎瘤鲜重约为１．２ｇ,茎瘤生物量在茎根瘤总生物量中所占比重为７０％,而其根瘤生物量略高于普通田菁;长喙田菁在华南地区能够正常开花结实,在栽植密度为４×１０４株·ｈｍ－２的条件下,其种子产量达３２００ｋｇ·ｈｍ－２．     

Micropropagation of Sesbania rostrata from the Cotyledonary Node

Multiple shoots were induced from the cotyledonary nodes derived from seedling of Sesbania rostrata on Nitsch (1969; N) medium supplemented with various concentrations of benzyladenine (BA). 1 mg dm⁻³ BA proved to be the best, eliciting 5.8 ± 1.0 shoots per explant in 100 % cultures. The elongation of shoots was best at 2.0 mg dm⁻³ BA. The shoot proliferation capacity increased to 7.5 shoots per explant following transfer of explants to the fresh shoot multiplication medium (MS + 1.0 mg dm⁻³ BA), after an initial incubation of 30 d. To further enhance number of shoots per explant an alternative strategy of cultivation of mother explant on fresh shoot multiplication medium after excision of shoots was adopted. Following the repeated harvesting of shoots an average of 33 shoots per explant could be obtained. The in vitro regenerated shoots produced roots when transferred to half-strength MS medium supplemented with 3 % sucrose and 1 mg dm⁻³ IBA. The developed plantlets were planted in the soil and transferred to the field after an acclimatization period of 3 – 4 months. These plants produced flowers and fruits in the field and exhibited the development of prominent and more organized stem nodules as compared to the in vivo raised plants of the same age. This revised version was published online in July 2006 with corrections to the Cover Date.     

Photosynthetic oxygen evolution within Sesbania rostrata stem nodules

The tropical wetland legume, Sesbania rostrata Brem. forms N₂-fixing nodules along its stem and on its roots after infection by Azorhizobium caulinodans . The N₂-fixing tissue is surrounded by a cortex of uninfected cells which, in the stem nodules (but not the root nodules), contain chloroplasts. The photosynthetic competence of these chloroplasts was assessed through a novel technique involving image analysis of chlorophyll a fluorescence. Calculation of the quantum efficiency of photosystem II (PS II) photochemistry from these images indicated that most of the chloroplasts with potential for non-cyclic photosynthetic electron transport were concentrated within the mid- and inner-cortex, close to the edge of the N₂-fixing tissue. PS II activity in the cortical cells was confirmed in vivo using O₂-specific microelectrodes which showed that the concentration of O₂ (pO₂) in the outer cortex could rise from less than 1% up to 23.4% upon increased irradiance of the nodule, but that the pO₂ of the inner cortex and infected tissue remained less than 0.0025%. Nitrogenase activity of stem nodules, as measured using a flow-through acetylene reduction assay (no H₂ evolution was evident), showed a reversible increase of 28% upon exposure of the nodules to supplemental light. This increase resembled that obtained with stem nodules upon their exposure to an external pO₂ of 40%.     

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.     

Photosystem II and oxygen regulation in Sesbania rostrata stem nodules

The tropical wetland legume Sesbania rostrata Brem. produces nitrogen-fixing stem nodules which are green and contain chlorophyll, the chloroplasts being concentrated in a hand in the inner and mid-cortex close to the nitrogen-fixing cells. The photosystem II thylakoid membrane proteins D₁, D₂ and PsbO, which are essential for photo-synthetic O₂ evolution, were shown by immunoblotting to be present in extracts of leaves and stem nodules. Immunogold labelling confirmed their presence on stem nodule thylakoids and showed that labelling was most intense in well-developed chloroplasts in the mid-cortex and least intense in the smaller, less-abundant chloroplasts adjacent to the nitrogen-fixing cells. Concentrations of the oxygen-carrying protein leghaemoglobin (Lb) did not differ between stem and S. rostrata root nodules, and Lb was localized in bacteroid-containing cells, including those immediately adjacent to the cortex, in both nodule types. Moreover, nitrogenase component 2 was localized in bacteroids within the outermost layers of infected cells, suggesting that a low pO₂ was maintained, despite the nearby chloroplasts. Nodule extracts examined by ELISA and immunoblots, using the monoclonal antibody MAC265, showed greatly enhanced expression of a 139 kDa glycoprotein in stem compared to root nodules. Immunogold labelling showed that material containing the MAC265 antigen occluded intercellular spaces, and was present in cell walls, throughout the cortex of stem nodules (particularly in the chloroplasl-rich inner and mid-cortex), but was considerably less evident in root nodules.     

Xylem colonization of the legume Sesbania rostrata by Azorhizobium caulinodans

A novel pathway of invasion of the legume Sesbania rostrata by Azorhizobium caulinodans is described that involves colonization of the root xylem, possibly following entry into the natural fissures created during emergence of lateral roots. Azorhizobia were detected microscopically, and their presence confirmed by the expression of a lacZ reporter gene. We have shown that rhizobial Nod factors are not required for either xylem colonization or for crack-entry of lateral roots. We discuss the extent to which this discovery of xylem colonization by azorhizobia is likely to improve our understanding of both symbiosis and of pathogenicity in plant–bacterial interactions.     

Root-to-shoot primordium conversion on Sesbania rostrata Brem. stem explants

The morphogenetic responses of cultured stem explants of Sesbaniarostrata Brem. from various positions along the stem axis wereanalysed after treatment with four growth regulators (BAP, NAA,kinetin, and GAJ. Internodal explants formed adventitious shootbuds when cultured on a Murashige and Skoog basal medium withoutadded growth regulators. Histological studies of regenerated shoot buds revealed thatapproximately 30% of the buds resulted from the conversion ofa preformed root primordium (characteristic of this species)into a shoot bud without a callogenesis phase. Each bud whichoriginated from a single root primordium grew into a leafy shoot.Preformed root primordia of stem explants of Sesbania rostratamay constitute an excellent model for physiological researchon plant differentiation. Key words: Organogenesis, adventitious bud, preformed root primordium, conversion, Sesbania rostrata     

Effect of Sesbania rostrata on Hirschmanniella oryzae in Flooded Rice

Microplot experiments on flooded soil infested with Hirschmanniella oryzae were conducted to investigate the influence of the legum Sesbania rostrata as a rotation crop with rice, Oryza sativa L. cv. Moroberekan. To avoid a green manure effect from S. rostrata, all aerial parts were removed at harvest. The dry weight of paddy, culms and leaves, and number of culms of rice following Sesbania were 214%, 158%, and 121% greater, respectively, than those following rice. Ripening of the paddy occurred earlier if rice followed Sesbania. The beneficial effect of Sesbania may have been due to the trap-crop action of Sesbania against H. oryzae.     

长喙田菁植物螯合肽合成酶基因在烟草中的表达

以pHANNIBAL及pART27为基础,构建了CaMV35S启动子驱动的长喙田菁(Sesbania rostrata)植物螯合肽合成酶SrPCS1基因植物超量表达载体pAM22,采用电击转化方法将pAM22导入根癌农杆菌EHA105,并用该菌株对烟草进行了转化,通过抗生素筛选、PCR及Northern-blotting检测了目的基因的表达水平,并用ClustalW对SrPCS1进行了进化分析.结果表明:SrPCS1编码233个氨基酸与豆科植物PCSs的同源性较高;得到27株抗性植株,23株PCR阳性植株,Northern-blotting证明得到了超量表达该基因的烟草14株,但基因的表达水平不同.     

An Azorhizobium caulinodans ORS571 locus involved in lipopolysaccharide production and nodule formation on Sesbania rostrata stems and roots.

Azorhizobium caulinodans ORS571 is able to nodulate roots and stems of the tropical legume Sesbania rostrata. An ORS571 Tn5 insertion mutant, strain ORS571-X15, had a rough colony morphology, was nonmotile, and showed clumping behavior on various media. When this pleiotropic mutant was inoculated on roots or stems of the host, no nodules developed (Nod-). Compared with the wild type, strain ORS571-X15 produced lipopolysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a different O-antigen structure with a lower degree of polymerization. A cosmid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was relieved and nodulation was almost completely restored, but the strain was still nonmotile. LPS preparations from these complemented strains resembled the wild-type LPS, although minor quantitative and qualitative differences were evident. The sequence of the locus hit by the Tn5 in ORS571-X15 (the oac locus) revealed a striking homology with the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosynthesis. The Tn5 insertion position was mapped to the oac3 gene, homologous to rfbA, encoding dTDP-D-glucose synthase. Biochemical assaying showed that ORS571-X15 is indeed defective in dTDP-D-glucose synthase activity, essential for the production of particular deoxyhexoses. Therefore, it was proposed that the O antigen of the mutant strain is devoid of such sugars.     

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.     

Functional nodFE genes are present in Sinorhizobium sp. strain MUS10, a symbiont of the tropical legume Sesbania rostrata

We have cloned the nodFE operon from Sinorhizobium sp. strain MUS10. MUS10 NodF shows sequence homology to acyl carrier protein and enables an S. meliloti nodF mutant to effectively nodulate alfalfa. Our results demonstrate the occurrence of nodFE in a symbiont that nodulates a legume host not belonging to the galegoid group.