R.C. van den Bos

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH4NO3 on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteroids in the nodule tissue were studied. The addition of NH4NO3 decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [35S]sulfate into the components I and II of nitrogenase was similarly not affected. The addition of NH4NO3 decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.

Modelling the effect of water-table management on CO2 and CH4 fluxes from peat soils

Drainage of peatlands for agriculture causes an increase of CO 2 flux from peat decomposition, contributing to national CO 2 emission. The reverse process, i.e. for re-creation of wetlands, reduces the CO 2 flux, but increases the CH 4 flux. We developed a process model (PEATLAND) to simulate these fluxes from peat soils subject to different water-table management scenarios. The model combines primary production, aerobic decomposition of soil organic matter (including the soil-parent material, peat), CH 4 formation, oxidation, and transport. Model input requires specification of water table and air temperature data sets, vegetation parameters such as primary production and parameters related to gas transport, and basic soil physical data. Validation using closed flux-chamber measurements of CO 2 and CH 4 from five different sites in the western Netherlands shows that seasonal changes in fluxes of CO 2 and CH 4 are correctly modelled. However, the CO 2 submodel underestimates peat decomposition when peat decomposition rates obtained from laboratory incubation experiments are used as input. Field decomposition rates are considerably higher. This is attributed to enhancement of decomposition by the addition of easily decomposable material from root exudation (’priming effect’). Model experiments indicate that 1) drainage increases the CO 2 production from peat decomposition strongly; 2) restoring a high water table may decrease the total greenhouse gas flux by a small amount although the CH 4 flux increases strongly; 3) a warmer climate may cause higher greenhouse gas fluxes from peat soils resulting in a positive feedback to climate warming, and 4) high vegetation productivity in fen meadows may stimulate peat decomposition by the priming effect.

The size and the location of the ribosomal RNA segments in ribosomal precursor RNA of yeast.

Abstract The order of the different segments of the 42-S ribosomal precursor RNA of yeast was investigated. For that purpose first the lengths of the rRNA stretches were measured. These were inferred from the relative amounts of oligonucleotides present in complete pancreatic ribonuclease digests of the two large rRNA components, and in this way were found to be 1600 and 3200 nucleotides, respectively. Next the influence of ultraviolet irradiation on the synthesis of both rRNA components in yeast cells was determined. It was found that the synthesis of 26-S rRNA is about 1.65 times more sensitive to ultraviolet damage of the DNA than the synthesis of 17-S rRNA. On the basis of these data the construction of the 42-S ribosomal precursor RNA was derived.

Characteristics of the methylation in vivo of ribosomal RNA in yeast

Abstract The characteristics of the methylation in vivo of ribosomal RNA (rRNA) in yeast has been studied by examining the pattern of incorporation of labeled methyl groups into rRNA and into ribosomal precursor RNA. 1. 1. Steady-state labeling with [ Me - 14 C]methionine showed that the degree of methylation for 17-S and 26-S rRNA is very similar, being about 1 methyl group per 70 nucleotides for both rRNAs. The methylation of yeast rRNA was found to be mainly ribose methylation, amounting to 74 and 83% of the total methylation for 17-S and 26-S rRNA, respectively. Chromatographic analysis of the alkaline hydrolysates of the two separate rRNAs revealed that the distribution of the methyl groups along the polynucleotide chains is distinctly different for the two rRNA components. 2. 2. The kinetics of methylation was studied by simultaneous pulse-labeling with [ Me - 3 H]methionine and [ 14 C]uracil. The results showed that methylation starts already at the level of the first ribosomal RNA precursor, immediately after or at the time of its transcription. However, from the ratio of the incorporation of [ 3 H]methyl and [ 14 C]uracil, it could be inferred that additional methylation takes place at later stages of the maturation process of rRNA, presumably after the conversion of ribosomal precursor RNA to rRNA. 3. 3. By analysis of the distribution of the methyl label in rRNA after increasing periods of labeling with [ Me - 3 H]methionine, it could be demonstrated that the additional methylation is base methylation, occurring in both 26-S and 17-S rRNA.

Secondary methylation of yeast ribosomal RNA

The biosynthesis of ribosomal RNA (rRNA) in yeast starts with the transcription of a 42 S common precursor molecule which is subsequently converted in a number of steps into 26 S and 17 S rRNA [ 1,2]. The processing includes the stepwise removal of nonconserved RNA to produce an intermediate precursor RNA which is cleaved into two separate precursors, namely 29 S and 18 S RNA, that eventually are converted into 26 S and 17 S rRNA, respectively [2-51. In this maturation process methylation of the rRNA precursors seems to play some, hitherto unknown, role. In a previous paper [6] it was shown that the attachment of methyl groups to predominantly ribose moieties occurs at the level of 42 S precursor RNA, immediately after or at the time of its transcription. However, we also obtained indirect evidence for an additional methylation of the heterocyclic bases at a later stage of rRNA formation. In this communication we present a direct proof for the occurrence of a secondary base methylation of yeast rRNA in a final step of the maturation process. In an enzymatic digest of 17 S rRNA a methylated oligonucleotide, containing the sequence rn! Apm;Ap, is found, which is lacking in the corresponding digest of 18 S RNA, the immediate precursor of 17 S rRNA. Thus, the final step in the formation of the small ribosomal subunit in yeast is accompanied by the introduction of at least four methyl groups into its RNA constituent.

The Sequence of Appearance of Leghaemoglobin and Nitrogenase Components I and II in Root Nodules of Pisum sativum

The sequence of appearance of nitrogenase components I and II and leghaemoglobin in root nodules of pea plants inoculated with Rhizobium leguminosarum (strain PRE) was studied using radioimmunoassays. Leghaemoglobin was detected before nitrogenase activity. The MoFe component (I) of nitrogenase was detected at the same time as leghaemoglobin, while the Fe component (II) of nitrogenase and nitrogenase activity appeared 1 and 2d later, respectively. The same order of appearance of nitrogenase activity and leghaemoglobin was found in root nodules of cowpea plants inoculated with Rhizobium sp. 32H1.

Development of the nitrogen-fixing and protein-synthesizing apparatus of bacteroids in pea root nodules

Some aspects of root nodule development of Pisum sativum inoculated with Rhizobium leguminosarum were examined. 1. Nitrogenase activity (measured as acetylene reduction) appears to be preceded by leghemoglobin synthesis (measured immunologically). 2. Syntheses of component I and component II of nitrogenase are not strictly coordinated. Synthesis of component I starts before component II. 3. Plant and bacteroid protein synthesis (measured by [35S]sulfate labeling) in root nodules declines rapidly during nodule development. Corresponding with this decline is a decrease in quantity and quality of rRNA.

Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum

Bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum are enclosed by a plant-derived peribacteriod membrane (PBM). The contents of the interstitial peribacteroid space (PBS) between bacteroid membrane and PBM were isolated by a controlled osmotic shock of PBM-enclosed bacteroids and analysed by two-dimensional gel electrophoresis. Silver staining revealed approximately 40 PBS polypeptides. Ex planta35S-methionine labeling of PBM-enclosed bacteroids revealed that about 90% of the PBS proteins are synthesized by the bacteroid. Approximately 30% of the PBS polypeptides are common between the PBS and the periplasmic space of free-living bacteria; one (38kDa) PBS protein is also excreted by free-living bacteria in the bacterial culture medium. At least four bacteroid-encoded PBS polypeptides were clearly identified as symbiosis-specific.

Analysis of DNA content, nitrogenase activity and in vivo protein synthesis of Rhizobium leguminosarum bacteroids on sucrose gradients

SUMMARY: Bacteroids of Rhizobium leguminosarum (strain PRE) isolated from pea root nodules (Pisum sativum) were separated by sucrose density gradient centrifugation, according to their stage of development from bacteria to mature bacteroids. The DNA content per cell, as measured by cytofluorometry, increased with development. Nitrogenase components in soluble bacteroid proteins were present in highest concentrations in mature bacteroids. The ratio of the amounts of Mo-Fe protein to Fe protein was not constant in different stages of development. Incorporation of 35SO2- 4 into soluble bacteroid proteins in the nodule was maximal in the youngest stages. In mature bacteroids nitrogenase was synthesized preferentially although less 35SO4 2- was incorporated into total soluble protein. Nitrogenase activity, measured as ATP- and S2O4 2--dependent acetylene reduction by EDTA/toluene treated bacteroids, was high in mature bacteroids and low in the youngest stages.

A rapid and simple method for separation and quantitation of ribonucleosides

Abstract A simple and rapid method for the quantitative determination of ribonucleosides is described. It is based on gel filtration on polyacrylamide gels at high pH (10.6). The method is particularly useful in the quantitative analysis of ribonucleic acid end groups.