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.     

The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: nucleotide sequence,expression in different organs and uptake into isolated chloroplasts

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M _r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M _r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

Complementation of a pleiotropic Nif-Gln regulatory mutant of Rhodospirillum rubrum by a previously unrecognized Azotobacter vinelandii regulatory locus

A spontaneous pleiotropic Nif^- mutation in Rhodospirillum rubrum has been partially characterized biochemically and by complementation analysis with recombinant plasmids carrying Azotobacter vinelandii DNA in the vicinity of ORF12 [Jacobson et al. (1989) J. Bacteriol 171:1017–1027]. In addition to being unable to grow on N₂ as a nitrogen source the phenotypic characterization of this and other metronidazole enriched spontaneous mutants showed (a) no nitrogenase activity, (b) the absence of NifHDK polypeptides, (c) a slower growth rate on NH _inf4 ^sup+ , (d) approximately 50% higher glutamine synthetase (GS) activity than the wild-type, which was repressible, (e) an inability to switch-off GS activity in response to an NH _inf4 ^sup+ up-shift, and (f) an inability to modify (³²P-label) the GS polypeptide. The apparent relationship between the absence of nifHDK expression and the absence of GS adenylylation cannot be explained in terms of the current model for nif gene regulation. However, R. rubrum transconjugants receiving A. vinelandii DNA which originated immediately upstream from nifH, restored all aspects of the wild-type phenotype. These data suggest a here-to-fore unrecognized relationship between nif expression and GS switch-off (adenylylation) activity, and the existence of a previously unidentified regulatory locus in Azotobacter that complements this mutation.     

Glutamine synthetase isoenzymes of Phaseolus vulgaris L.: subunit composition in developing root nodules and plumules

In the legume Phaseolus vulgaris L., glutamine synthetase (GS) (EC.6.3.1.2.) occurs as three cytosolic polypeptides, , and , and a plastidic polypeptide, . This paper describes the subunit composition of active octameric GS isoenzymes from root nodules and plumules using ionexchange high-performance liquid chromatography followed by two-dimensional denaturing gel electrophoresis and Western immunodetection. Root nodules contained four separable GS activities, three of which were composed mainly of cytosolic , / and GS polypeptides, whereas the fourth activity, consisted of plastidic GS polypeptides. The increase in GS activity during nodulation was due largely to the appearance of -containing isoenzymes, and to a lesser extent on the isoenzyme, whereas the -isoenzyme activity remained approximately constant throughout. Plumule GS from imbibed seeds was found to be composed of separate and isoenzymes, but 2 d after germination, plumule GS consisted of a mixture of , / and isoenzymes. The results from both nodules and plumules indicate that different cytosolic GS polypeptides in P. vulgaris are able to assemble into both homo-octameric and heterooctameric isoenzymes. Moreover, the changes in the patterns of isoenzymes observed during nodule development and plumule growth are interpreted to be caused both by temporal changes in the denovo synthesis of the polypeptides and also by their spatial separation in different cell types.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_s GS semibiosynthetic activity - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules.

We have characterized the distinct polypeptides, primary translation products and mRNAs encoding glutamine synthetase (GS) in the various organs of pea. Western blot analysis of soluble protein has identified five distinct GS polypeptides which are expressed at different relative levels in leaves, roots and nodules of pea. Of the two GS polypeptides in leaves (44 and 38 kd), the 44-kd GS polypeptide is predominant and is localized to the chloroplast stroma. In roots, the predominant GS polypeptide is 38 kd. Upon Rhizobium infection of roots, three 37-kd GS polypeptides increase in abundance in the nodules relative to uninfected roots. cDNA clones encoding three different GS mRNAs have been characterized. Hybrid-select translation has identified three different GS primary translation products (49, 38 and 37 kd). Two cDNA clones (pGS134 and pGS341) are homologous to GS mRNAs most abundant in nodules which encode the 38- and 37-kd GS primary translation products. A third cDNA (pGS197) corresponds to a larger GS mRNA species specific to leaf poly(A) RNA, which encodes a 49-kd putative precursor to the mature chloroplast GS polypeptide. cDNA sequence analysis and Southern blot analysis of pea nuclear DNA identifies at least three genes encoding GS in pea which are related but distinct in structure and in vivo pattern of expression.     

Expression of Two Different Glutamine Synthetase Polypeptides during Root Development in Phaseolus vulgaris L

Immunoprecipitation and two-dimensional gel electrophoresis analysis of the glutamine synthetase (GS) polypeptides (α and β) during Phaseolus vulgaris root development shows that the α polypeptide is the main component of the enzyme in the embryo and in up to 5 day old roots. From 5 days on, the β polypeptide becomes the root predominant GS monomer. The α/β ratio of the in vitro translated GS polypeptides from the total polysomal RNA isolated at different root ages correlates with the α/β ratio observed in the root extracts. These results suggest that the two root GS polypeptides are encoded by different mRNA species in Phaseolus vulgaris.     

Glutamine synthetases of green and etiolated leaves ofSinapis alba

Studies on the glutamine synthetases (GS, EC 6.3.1.2) of green (GS₂) and etiolated leaves (GS_et) ofSinapis alba L. (cv. Steinacher) revealed striking similarities between the respective enzyme proteins. The enzymes showed corresponding chromatographic properties, both on dimethylaminoethyl-Sephacel and on hydroxylapatite columns. The purified GS proteins were also identical with regard to the molecular weight of their subunits. Isoelectrofocusing of pure GS_et yielded two distinct polypeptide bands in the pH 5.6 region of the gels. This pattern corresponded to the two strong bands of GS₂. Two charge variants of GS polypeptides could be detected by Western-blot analysis of the soluble protein of green leaves using antibodies against mustard GS₂. In immunoprecipitation experiments, the holoenzymes of GS₂ and GS_et were recognized with identical affinities by this antiserum. We conclude that strong similarities exist between the proteins of the GS enzymes in green and etiolated leaves of mustard. Most probably only one GS form, namely the plastidic enzyme, can be found in the epigeal organs ofSinapis. The polypeptides of the GS₂ subunits showed no differences in the hydrophobicity of the polypeptide chains. Neither glucosyl nor mannosyl residues could be detected. Dedicated to Professor Dr. H. Mohr on the occasion of his 60th birthday     

Changes in polypeptide patterns in tobacco roots colonized by two Glomus species

Changes in gene expression were studied during the establishment of arbuscular mycorrhizal symbiosis in tobacco roots from an amphidiploid hybrid Nicotiana glutinosa x N. debneyi. Polypeptide patterns from control roots and from roots infected by Glomus mosseae or G. intraradices were resolved by two-dimensional polyacrylamide gel electrophoresis and followed in a time-course analysis. Arbuscular mycorrhizal infection led to significant modifications in polypeptide patterns with: (a) decreased amounts of some polypeptides, (b) increased accumulation of others, and (c) appearance of newly-induced polypeptides. Comparisons made during infection development by the two Glomus species demonstrated that protein modifications changed in relation to the mycorrhizal state of the tobacco roots.     

HtpG,the prokaryotic homologue of Hsp90, stabilizes a phycobilisome protein in the cyanobacterium Synechococcus elongatus PCC 7942

HtpG, a homologue of HSP90, is essential for thermotolerance in cyanobacteria. It is not known how it plays this important role. We obtained evidence that HtpG interacts with linker polypeptides of phycobilisome in the cyanobacterium Synechococcus elongatus PCC 7942. In an htpG mutant, the 30 kDa rod linker polypeptide was reduced. In vitro studies with purified HtpG and phycobilisome showed that HtpG interacts with the linker polypeptide as well as other linker polypeptides to suppress their thermal aggregation with a stoichiometry of one linker polypeptide/HtpG dimer. We constructed various domain‐truncated derivatives of HtpG to identify putative chaperone sites at which HtpG binds linker polypeptides. The middle domain and the N‐terminal domain, although less efficiently, prevented the aggregation of denatured polypeptides, while the C‐terminal domain did not. Truncation of the C‐terminal domain that is involved in the dimerization of HtpG led to decrease in the anti‐aggregation activity, while fusion of the N‐terminal domain to the middle domain lowered the activity. In vitro studies with HtpG and the isolated 30 kDa rod linker polypeptide provided basically similar results to those with HtpG and phycobilisome. ADP inhibited the anti‐aggregation activity, indicating that a compact ADP conformational state provides weaker aggregation protection compared with the others.     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans

Summary A cDNA clone (pcPvNGS-01) to glutamine synthetase (GS) mRNA from root nodules of Phaseolus vulgaris showed cross-hybridization to GS and mRNA from soybean root nodules, thus allowing its use as a probe to study the expression of GS genes during root nodule development in soybeans. Hybrid-select translation of root and nodule RNA of soybean with DNA from pcPvNGS-01, followed by 2D gel electrophoresis, showed six peptides in the root and an additional four peptides in the nodule which represent nodule-specific glutamine synthetase (GS_n) gene products. The GS_n gene products appeared for the first time between day 11 and 12 after infection, either concomitant with the onset of nitrogenase activity or immediately following it. The levels of expression of the GS_n and leghemoglobin genes were not affected in young Fix^- nodules formed by Bradyrhizobium japonicum strains that are defective in nitrogenase activity, suggesting that the induction of these two sets of host genes take place independent of nitrogenase activity. However, in Fix^- nodules that are incapable of maintaining the peribacteroid membrane, GS_n gene products were not detected while 1ba, 1bc₂ and 1bc₃ appeared. In both the timing of appearance during root nodule development and the effect of different bacterial mutations on the expression, GS_n genes differ from most other nodulin genes examined (30), suggesting different regulatory mechanisms.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.