Germination and Early Seedling Development in Quercus ilex Recalcitrant and Non-dormant Seeds: Targeted Transcriptional, Hormonal, and Sugar Analysis.

Seed germination and early seedling development have been studied in the recalcitrant species Quercus ilex using targeted transcriptional, hormonal, and sugar analysis. Embryos and seedlings were collected at eight morphologically defined developmental stages, S0-S7. A typical triphasic water uptake curve was observed throughout development, accompanied by a decrease in sucrose and an increase in glucose and fructose. Low levels of abscisic acid (ABA) and high levels of gibberellins (GAs) were observed in mature seeds. Post-germination, indole-3-acetic acid (IAA), increased, whereas GA remained high, a pattern commonly observed during growth and development. The abundance of transcripts from ABA-related genes was positively correlated with the changes in the content of the phytohormone. Transcripts of the drought-related genes Dhn3 and GolS were more abundant at S0, then decreased in parallel with increasing water content. Transcripts for Gapdh, and Nadh6 were abundant at S0, supporting the occurrence of an active metabolism in recalcitrant seeds at the time of shedding. The importance of ROS during germination is manifest in the high transcript levels for Sod and Gst, found in mature seeds. The results presented herein help distinguish recalcitrant (e.g., Q. ilex) seeds from their orthodox counterparts. Our results indicate that recalcitrance is established during seed development but not manifest until germination (S1-S3). Post-germination the patterns are quite similar for both orthodox and recalcitrant seeds.

Structure of surface polysaccharides from Aeromonas sp. AMG272, a plant-growth promoting rhizobacterium isolated from rice rhizosphere.

Aeromonas sp. AMG272 is a Gram-negative bacterium that has been isolated from agricultural soil and studied for its plant growth-promoting activities. Structures of the O-specific polysaccharide chain of the AMG272 lipopolysaccharide and its capsular polysaccharide were elucidated using GLC-MS and NMR spectroscopy. The structure of the O-specific polysaccharide, →4)-α-l-Rhap-(1 → 3)-ß-d-GlcpNAc-(1→, has been found in other Aeromonas strains and related bacteria, whereas the structure of the capsular polysaccharide has not been reported before: →6)[ß-d-Fucp3NAc4Ac-(1 → 3)]-α-d-GlcpNAc-(1 → 4)-α-d-Galp-(1 → 3)-α-d-GalpNAc-(1 → 4)-α-d-Galp-(1 → .

Structure of the O-antigen of the lipopolysaccharide isolated from Pantoea ananatis AEP17, a rhizobacterium associated with rice.

The lipopolysaccharide of a Gram-negative bacterium having a putative plant-growth promoting activity (Pantoea ananatis AEP17) has been isolated and subjected to partial hydrolysis. The O-antigen has been studied by mass spectrometry and NMR experiments. On the basis of these experiments it is concluded that the following repeating unit is present in the polysaccharide: →3)-ß-d-GlcpNAc-(1→3)[α-d-GalpAN-(1→2)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→ The occurrence of d-galacturonamide (GalAN) is unusual in bacterial O-polysaccharides. It has only been reported in Escherichia coli O65 [Perry, M. B.; MacLean, L. L. Carbohydr. Res.1999, 322, 57-66].

Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia.

Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant-microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis.

The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea.

In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysaccharides (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ- and rkpU-encoded products are orthologous to Escherichia coli proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium-legume interaction.

Structure of the high-molecular weight exopolysaccharide isolated from Lactobacillus pentosus LPS26.

The strain Lactobacillus pentosus LPS26 produces a capsular polymer composed of a high- (2.0x10(6)Da) (EPS A) and a low-molecular mass (2.4x10(4)Da) (EPS B) polysaccharide when grown on semi-defined medium containing glucose as the carbon source. The structure of EPS A and its deacetylated form has been determined by monosaccharide and methylation analysis as well as by 1D/2D NMR studies ((1)H and (13)C). We conclude that EPS A is a charged heteropolymer, with a composition of D-glucose, D-glucuronic acid and L-rhamnose in a molar ratio 1:2:2. The repeating unit is a pentasaccharide with two O-acetyl groups at O-4 of the 3-substituted alpha-D-glucuronic acid and at O-2 of the 3-substituted beta-L-rhamnose, respectively. -->4)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA4Ac-(1-->3)-alpha-L-Rhap-(1-->4)-alpha-D-GlcpA-(1-->3)-beta-L-Rhap2Ac-(1--> This unbranched structure is not common in EPSs produced by Lactobacilli. Moreover, the presence of acetyl groups in the structure is an unusual feature which has only been reported in L. sake 0-1 [Robijn et al. Carbohydr. Res., 1995, 276, 117-136].

Structure of the O-antigen of the main lipopolysaccharide isolated from Sinorhizobium fredii SMH12.

The lipopolysaccharide of Sinorhizobium fredii SMH12, a wide-range host bacterium isolated from nodulated soybean plants growing in Vietnam, has been studied. Isolation of lipopolysaccharide by the phenol-water method leads to a mixture of two polysaccharides; polyacrylamide gel electrophoresis indicates that both are possibly lipopolysaccharides. The structures of the O-antigen of the main lipopolysaccharide and its deacetylated form are determined by sugar and methylation analysis, partial hydrolysis, lithium degradation, ESI-MS/MS, and NMR studies. Here we show that the fast-growing S. fredii SMH12 produces a lipopolysaccharide whose O-antigen has a repeating unit consisting of the trisaccharide -->4)-alpha-D-Gal pA-(1-->3)-2-O-Ac-alpha-L-Rha p-(1-->3)-2-O-Ac-alpha-D-Man p-(1-->. The position O-6 of the mannose residue in the repeating unit is unsubstituted, acetylated, or methylated in an approximate ratio 1:1:2. The tandem mass spectrometry studies rule out both an alternating and a random distribution of methyl groups and suggest the existence of zones in the polysaccharide rich in methyl groups interspersed with zones without methyl groups.

Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.

Structural determination of the Nod factors produced by Rhizobium gallicum bv. gallicum R602.

Rhizobium gallicum is a fast-growing bacterium found in European, Australian and African soils; it was first isolated in France. It is a microsymbiont which is able to nodulate plants of the genus Phaseolus. Rhizobium gallicum bv. gallicum R602 produces four extracellular signal molecules consisting of a linear backbone of N-acetyl glucosamine, bearing on the nonreducing terminal residue an N-methyl group and different N-acyl substituents. The four acyloligosaccharides terminate with a sulfated N-acetylglucosaminitol. This unit may be also acetylated. These structures were determined using carbohydrate and methylation analysis, mass spectrometric analysis and one-dimensional- and two-dimensional-nuclear magnetic resonance experiments. This work establishes the common structure that a lipochito-oligosaccharide must have so that the Rhizobium that produces and excretes it is able to nodulate plants of Phaseolus vulgaris. The substituents common to all the molecules are an N-methyl group and a C(18:1) fatty acid on the nonreducing terminal residue.

Structural analysis of the capsular polysaccharide from Sinorhizobium fredii HWG35.

We have determined the structure of a capsular polysaccharide from Sinorhizobium fredii HWG35. This polysaccharide was isolated following the standard protocols applied for lipopolysaccharide isolation. On the basis of monosaccharide analysis, methylation analysis, mass spectrometric analysis, one-dimensional (1)H and (13)C NMR, and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the following disaccharide repeating unit: -->6)-2,4-di-O-methyl-alpha-d-Galp-(1-->4)-beta-d-GlcpA-(1-->. Strain HWG35 produces a capsular polysaccharide that does not show the structural motif (sugar-Kdx) observed in those S. fredii strains that, while effective with Asiatic soybean cultivars, are unable to form nitrogen-fixing nodules with American soybean cultivars. Instead, the structure of the capsular polysaccharide of S. fredii HWG35 is in line with those produced by strains HH303 (rhamnose and galacturonic acid) and B33 (4-O-methylglucose-3-O-methylglucuronic acid), two S. fredii strains that form nitrogen-fixing nodules with both groups of soybean cultivars. Hence, in these three strains that effectively nodulate American soybean cultivars, the repeating unit of the capsular polysaccharide is composed of two hexoses, one neutral (methylgalactose, rhamnose, or methylglucose) and the other acidic (glucuronic, galacturonic, or methylglucuronic acid).

NolR regulates diverse symbiotic signals of Sinorhizobium fredii HH103.

We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Str(r)) the influence of the nolR gene on the production of three different bacterial symbiotic signals: Nod factors, signal responsive (SR) proteins, and exopolysaccharide (EPS). The presence of multiple copies of nolR (in plasmid pMUS675) repressed the transcription of all the flavonoid-inducible genes analyzed: nodA, nodD1, nolO, nolX, noeL, rhcJ, hesB, and y4pF. Inactivation of nolR (mutant SVQ517) or its overexpression (presence of pMUS675) altered the amount of Nod factors detected. Mutant SVQ517 produced Nod factors carrying N-methyl residues at the nonreducing N-acetyl-glucosamine, which never have been detected in S. fredii HH103. Plasmid pMUS675 increased the amounts of EPS produced by HH103-1 and SVQ517. The flavonoid genistein repressed EPS production of HH103-1 and SVQ517 but the presence of pMUS675 reduced this repression. The presence of plasmid pMUS675 clearly decreased the secretion of SR proteins. Inactivation, or overexpression, of nolR decreased the capacity of HH103 to nodulate Glycine max. However, HH103-1 and SVQ517 carrying plasmid pMUS675 showed enhanced nodulation capacity with Vigna unguiculata. The nolR gene was positively identified in all S. fredii strains investigated, S. xinjiangense CCBAU110, and S. saheli USDA4102. Apparently, S. teranga USDA4101 does not contain this gene.

A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide.

The pleiotropic phenotype of an auxotrophic purL mutant (SVQ295) of Sinorhizobium fredii HH103 has been investigated. SVQ295 forms colonies that are translucent, produce more slime and absorb less Congo red than those of wild-type strain HH103. SVQ295 did not grow in minimal medium unless the culture was supplemented with thiamin and adenine or with thiamin and AICA-riboside (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), an intermediate of purine biosynthesis. Bacterial cultures supplemented with AICA-riboside or adenine reached the same culture density, although the doubling time of SVQ295 cultures containing AICA-riboside was clearly longer. S. fredii SVQ295 induced pseudonodules on Glycine max and failed to nodulate six different legumes. On Glycyrrhiza uralensis, however, nodules showing nitrogenase activity and containing infected plant cells were formed. SVQ295 showed auto-agglutination when grown in liquid TY medium and its lipopolysaccharide (LPS) electrophoretic profile differed from that of its parental strain HH103-1. In addition, four monoclonal antibodies that recognize the LPS of S. fredii HH103 failed to recognize the LPS produced by SVQ295. In contrast, (1)H-NMR spectra of K-antigen capsular polysaccharides (KPS) produced by SVQ295 and the wild-type strain HH103 were similar. Co-inoculation of soybean plants with SVQ295 and SVQ116 (a nodA mutant derivative of HH103) produced nitrogen-fixing nodules that were only occupied by SVQ116.

Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium giardinii bv. giardinii H152.

Rhizobium giardinii bv. giardinii is a microsymbiont of plants of the genus Phaseolus and produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis. We report here the structures of seven lipochitooligosaccharide (LCO) signal molecules secreted by R. giardinii bv. giardinii H152. Six of them are pentamers of GlcNAc carrying C 16:0, C 18:0, C 20:0 and C 18:1 fatty acyl chains on the non-reducing terminal residue. Four are sulfated at C-6 of the reducing terminal residue and one is acetylated in the same position. Six of them are N-methylated on the non-reducing GlcN residue and all the nodulation factors are carbamoylated on C-6 of the non-reducing terminal residue. The structures were determined using monosaccharide composition and methylation analyses, 1D- and 2D-NMR experiments and a range of mass spectrometric techniques. The position of the carbamoyl substituent on the non-reducing glucosamine residue was determined using a CID-MSMS experiment and an HMBC experiment.

Sinorhizobium fredii HH103 has a truncated nolO gene due to a -1 frameshift mutation that is conserved among other geographically distant S. fredii strains.

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103-suggesting a polar effect of the transposon insertion into nolO over the noel gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.