Comparative cytogenetic analysis of the genomes of the model grass Brachypodium distachyon and its close relatives

Background and Aims

Brachypodium is a small genus of temperate grasses that comprises 12–15 species. Brachypodium distachyon is now well established as a model species for temperate cereals and forage grasses. In contrast to B. distachyon, other members of the genus have been poorly investigated at the chromosome level or not at all.

Methods

Twenty accessions comprising six species and two subspecies of Brachypodium were analysed cytogenetically. Measurements of nuclear genome size were made by flow cytometry. Chromosomal localization of 18–5·8–25S rDNA and 5S rDNA loci was performed by dual-colour fluorescence in situ hybridization (FISH) on enzymatically digested root-tip meristematic cells. For comparative phylogenetic analyses genomic in situ hybridization (GISH) applied to somatic chromosome preparations was used.

Key Results

All Brachypodium species examined have rather small genomes and chromosomes. Their chromosome numbers and genome sizes vary from 2n = 10 and 0·631 pg/2C in B. distachyon to 2n = 38 and 2·57 pg/2C in B. retusum, respectively. Genotypes with 18 and 28 chromosomes were found among B. pinnatum accessions. GISH analysis revealed that B. pinnatum with 28 chromosomes is most likely an interspecific hybrid between B. distachyon (2n = 10) and B. pinnatum (2n = 18). Two other species, B. phoenicoides and B. retusum, are also allopolyploids and B. distachyon or a close relative seems to be one of their putative ancestral species. In chromosomes of all species examined the 45S rDNA loci are distally distributed whereas loci for 5S rDNA are pericentromeric.

Conclusions

The increasing significance of B. distachyon as a model grass emphasizes the need to understand the evolutionary relationships in the genus Brachypodium and to ensure consistency in the biological nomenclature of its species. Modern molecular cytogenetic techniques such as FISH and GISH are suitable for comparative phylogenetic analyses and may provide informative chromosome- and/or genome-specific landmarks.     

Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana

A novel peptidasome PreP is responsible for degradation of targeting peptides and other unstructured peptides in mitochondria and chloroplasts. Arabidopsis thaliana contains two PreP isoforms, AtPreP1, and AtPreP2. Here we have characterized single and double prep knockout mutants. Immunoblot analysis of atprep1 and atprep2 mutants showed that both isoforms are expressed in all tissues with the highest expression in flowers and siliques; additionally, AtPreP1 accumulated to a much higher level in comparison to AtPreP2. The atprep2 mutant behaved like wild type, whereas deletion of AtPreP1 resulted in slightly pale-green seedlings. Analysis of the atprep1 atprep2 double mutant revealed a chlorotic phenotype in true leaves with diminished chlorophyll a and b content, but unchanged Chl a/b ratio indicating a proportional decrease of both PSI and PSII complexes. Mitochondrial respiratory rates (state 3) were lower and the mitochondria were partially uncoupled. EM pictures on cross sections of the first true leaves showed aberrant chloroplasts, including less grana stacking and less starch granules. Mitochondria with extremely variable size could also be observed. At later developmental stages the plants appeared almost normal. However, all through the development there was a statistically significant decrease of ~40% in the accumulated biomass in the double mutant plants in comparison to wild type. In mitochondria, deletion of AtPreP was not compensated by activation of any peptidolytic activity, whereas chloroplast membranes contained a minor peptidolytic activity not related to AtPreP. In summary, the AtPreP peptidasome is required for efficient plant growth and organelle function particularly during early development.     

Biophysical properties of Saccharomyces cerevisiae and their relationship with HOG pathway activation

Parameterized models of biophysical and mechanical cell properties are important for predictive mathematical modeling of cellular processes. The concepts of turgor, cell wall elasticity, osmotically active volume, and intracellular osmolarity have been investigated for decades, but a consistent rigorous parameterization of these concepts is lacking. Here, we subjected several data sets of minimum volume measurements in yeast obtained after hyper-osmotic shock to a thermodynamic modeling framework. We estimated parameters for several relevant biophysical cell properties and tested alternative hypotheses about these concepts using a model discrimination approach. In accordance with previous reports, we estimated an average initial turgor of 0.6 ± 0.2 MPa and found that turgor becomes negligible at a relative volume of 93.3 ± 6.3% corresponding to an osmotic shock of 0.4 ± 0.2 Osm/l. At high stress levels (4 Osm/l), plasmolysis may occur. We found that the volumetric elastic modulus, a measure of cell wall elasticity, is 14.3 ± 10.4 MPa. Our model discrimination analysis suggests that other thermodynamic quantities affecting the intracellular water potential, for example the matrix potential, can be neglected under physiological conditions. The parameterized turgor models showed that activation of the osmosensing high osmolarity glycerol (HOG) signaling pathway correlates with turgor loss in a 1:1 relationship. This finding suggests that mechanical properties of the membrane trigger HOG pathway activation, which can be represented and quantitatively modeled by turgor.     

High‐performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the acyl‐CoA‐dependent biosynthesis of triacylglycerol, the predominant component of seed oil. In some oil crops, including Brassica napus, the level of DGAT1 activity can have a substantial effect on triacylglycerol production. Structure–function insights into DGAT1, however, remain limited because of the lack of a three‐dimensional detailed structure for this membrane‐bound enzyme. In this study, the amino acid residues governing B. napus DGAT1 (BnaDGAT1) activity were investigated via directed evolution, targeted mutagenesis, in vitro enzymatic assay, topological analysis, and transient expression of cDNA encoding selected enzyme variants in Nicotiana benthamiana. Directed evolution revealed that numerous amino acid residues were associated with increased BnaDGAT1 activity, and 67% of these residues were conserved among plant DGAT1s. The identified amino acid residue substitution sites occur throughout the BnaDGAT1 polypeptide, with 89% of the substitutions located outside the putative substrate binding or active sites. In addition, cDNAs encoding variants I447F or L441P were transiently overexpressed in N. benthamiana leaves, resulting in 33.2 or 70.5% higher triacylglycerol content, respectively, compared with native BnaDGAT1. Overall, the results provide novel insights into amino acid residues underlying plant DGAT1 function and performance‐enhanced BnaDGAT1 variants for increasing vegetable oil production.     

The frequency of MMS-induced,umuDC-dependent,mutations declines during starvation in Escherichia coli

It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D′)C proteins. The frequency of argE(ochre)→Arg⁺ mutations (which occur predominantly by AT→TA transversions) and Rif^S→Rif^R mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD′₂C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.     

Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases

Background  

SPOUT methyltransferases (MTases) are a large class of S-adenosyl-L-methionine-dependent enzymes that exhibit an unusual alpha/beta fold with a very deep topological knot. In 2001, when no crystal structures were available for any of these proteins, Anantharaman, Koonin, and Aravind identified homology between SpoU and TrmD MTases and defined the SPOUT superfamily. Since then, multiple crystal structures of knotted MTases have been solved and numerous new homologous sequences appeared in the databases. However, no comprehensive comparative analysis of these proteins has been carried out to classify them based on structural and evolutionary criteria and to guide functional predictions.     

Are seeds suitable for flow cytometric estimation of plant genome size?

BACKGROUND: Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G0/G1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. METHODS: The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C = 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants. RESULTS: For Anethum graveolens, Beta vulgaris, and Zea mays, cytometrically estimated genome size was the same in seeds and leaves. For Helianthus annuus, different values for DNA amounts in seeds and in leaves were obtained when using all but one of four nuclei isolation buffers. For Brassica napus var. oleifera, none of the applied nuclei isolation buffers eliminated differences in genome size determined in the seeds and leaves. CONCLUSIONS: The genome size of species that do not contain compounds that influence fluorochrome accessibility appears to be the same when estimated using specific seed tissues and young leaves. Seeds can be more suitable than leaves, especially for species containing staining inhibitors in the leaf cytosol. Thus, use of seeds for FCM nuclear DNA content estimation is recommended, although for some species a specific seed tissue (usually the radicle) should be used. Protocols for preparation of samples from endospermic and endospermless seeds have been developed.     

137Cs and 40K isotopes in forest and wasteland soils in a selected region of eastern Poland 20 years after the Chernobyl accident

The vertical ¹³⁷Cs profile of forest and wasteland soils was analyzed in the south of the Podlasie Lowland area (Eastern Poland) about 20 years after the Chernobyl accident. In addition, the concentration of ⁴⁰K in soils of the investigated area was measured. Below the litter layer (mean thickness 3 cm), the soil samples were collected up to a depth of 12 cm and then divided into three layers: 0–3, 3–7, 7–12 cm. The behavior of ¹³⁷Cs and ⁴⁰K isotopes in soils was analyzed depending on the depth from which the soil samples were collected, as well as on the content of organic carbon, pH of soil and its granulometric composition. It was established that the density of ¹³⁷Cs in the litter layer equals 2.17 kBq m⁻²; it is the highest in layer 0–3 cm where it equals 3.44 kBq m⁻², and it decreases with the depth to the value of 0.76 kBq m⁻² in layer 7–12 cm. No similar pattern was observed in wasteland soils. The concentrations of ⁴⁰K in forest and wasteland soils did not change significantly with depth.