Characterization of the mitochondrial genome of the MAX1 type of cytoplasmic male-sterile sunflower

Background

More than 70 cytoplasmic male sterility (CMS) types have been identified in Helianthus, but only for less than half of them, research of mitochondrial organization has been conducted. Moreover, complete mitochondrion sequences have only been published for two CMS sources – PET1 and PET2. It has been demonstrated that other sunflower CMS sources like MAX1, significantly differ from the PET1 and PET2 types. However, possible molecular causes for the CMS induction by MAX1 have not yet been proposed. In the present study, we have investigated structural changes in the mitochondrial genome of HA89 (MAX1) CMS sunflower line in comparison to the fertile mitochondrial genome.

Results

Eight significant major reorganization events have been determined in HA89 (MAX1) mtDNA: one 110 kb inverted region, four deletions of 439 bp, 978 bp, 3183 bp and 14,296 bp, respectively, and three insertions of 1999 bp, 5272 bp and 6583 bp. The rearrangements have led to functional changes in the mitochondrial genome of HA89 (MAX1) resulting in the complete elimination of orf777 and the appearance of new ORFs - orf306, orf480, orf645 and orf1287. Aligning the mtDNA of the CMS sources PET1 and PET2 with MAX1 we found some common reorganization features in their mitochondrial genome sequences.

Conclusion

The new open reading frame orf1287, representing a chimeric atp6 gene, may play a key role in MAX1 CMS phenotype formation in sunflower, while the contribution of other mitochondrial reorganizations seems to appear negligible for the CMS development.

     

Structural basis for assembly of the CBF3 kinetochore complex

Eukaryotic chromosomes contain a specialised region known as the centromere, which forms the platform for kinetochore assembly and microtubule attachment. The centromere is distinguished by the presence of nucleosomes containing the histone H3 variant, CENP‐A. In budding yeast, centromere establishment begins with the recognition of a specific DNA sequence by the CBF3 complex. This in turn facilitates CENP‐A^Cse4 nucleosome deposition and kinetochore assembly. Here, we describe a 3.6 Å single‐particle cryo‐EM reconstruction of the core CBF3 complex, incorporating the sequence‐specific DNA‐binding protein Cep3 together with regulatory subunits Ctf13 and Skp1. This provides the first structural data on Ctf13, defining it as an F‐box protein of the leucine‐rich‐repeat family, and demonstrates how a novel F‐box‐mediated interaction between Ctf13 and Skp1 is responsible for initial assembly of the CBF3 complex.     

E-NTPDase and E-ADA activities are altered in lymphocytes of patients with indeterminate form of Chagas' disease

Trypanosoma cruzi infection triggers a chronic inflammatory process in human host and purinergic system ecto-enzymes play an important role in modulating the inflammatory and immune responses. In this study, it was investigated ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and ecto-adenosine deaminase (E-ADA; EC 3.5.4.4) activities in lymphocytes from patients with indeterminate form of Chagas' disease (IFCD). Twenty-five IFCD patients and 25 healthy subjects (control group) were selected. The peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Adenine nucleotides and adenosine levels were determined in serum by HPLC and the E-NTPDase1 expression in lymphocytes by Western blot analysis. E-NTPDase (ATP and ADP as substrates) and E-ADA (adenosine as substrate) activities were decreased in lymphocytes from IFCD patients (P<0.05 and P<0.01, respectively), while the E-NTPDase1 expression presented no changes in these patients. Serum ATP levels showed to be decreased (P<0.05) and both AMP (P<0.01) and adenosine (P<0.001) levels were increased in the IFCD group. The enzymatic alterations observed are in agreement with the immune response against T. cruzi infection in IFCD patients, since the decreased extracellular ATP and the increased adenosine levels trigger a Th2 anti-inflammatory response, which it is associated to adaptation of host to parasite, preventing clinical progress of disease.     

Mapping of DNA Sex-Specific Markers and Genes Related to Sex Differentiation in Turbot (Scophthalmus maximus)

Production of all-female populations in turbot can increase farmer's benefits since sexual dimorphism in growth in this species is among the highest within marine fish, turbot females reaching commercial size 3-6?months earlier than males. Puberty in males occurs earlier than in females, which additionally slows their growth. Thus, elucidating the mechanisms of sex determination and gonad differentiation is a relevant goal for turbot production. A ZZ/ZW sex determination mechanism has been suggested for this species, and four sex-related quantitative trait loci (QTL) were detected, the major one located in linkage group (LG) 5 and the three minor ones in LG6, LG8, and LG21. In the present work, we carried out a linkage analysis for several sex-related markers: (1) three anonymous sex-associated RAPD and (2) several candidate genes related to sex determination and gonad differentiation in other species (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2, Cyp19a, Cyp19b). We focused our attention on their co-localization with the major and minor sex-related QTL trying to approach to the master sex-determining gene of this species. Previously described growth-related QTL were also considered since the association observed between growth and sex determination in fish. Amh, Dmrta2, and one RAPD were located in LG5, while Sox9 and Sox17 (LG21), Cyp19b (LG6), and a second RAPD (LG8) co-mapped with suggestive sex-related QTL, thus supporting further analyses on these genes to elucidate the genetic basis of this relevant trait for turbot farming.     

Classical anticytokinins do not interact with cytokinin receptors but inhibit cyclin-dependent kinases

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.     

A chemically induced new pea (Pisum sativum) mutant SGECdt with increased tolerance to, and accumulation of, cadmium

BACKGROUND AND AIMS: To date, there are no crop mutants described in the literature that display both Cd accumulation and tolerance. In the present study a unique pea (Pisum sativum) mutant SGECd(t) with increased Cd tolerance and accumulation was isolated and characterized. METHODS: Ethylmethane sulfonate mutagenesis of the pea line SGE was used to obtain the mutant. Screening for Cd-tolerant seedlings in the M2 generation was performed using hydroponics in the presence of 6 microm CdCl2. Hybridological analysis was used to identify the inheritance of the mutant phenotype. Several physiological and biochemical characteristics of SGECd(t) were studied in hydroponic experiments in the presence of 3 microm CdCl2, and elemental analysis was conducted. KEY RESULTS: The mutant SGECd(t) was characterized as having a monogenic inheritance and a recessive phenotype. It showed increased Cd concentrations in roots and shoots but no obvious morphological defects, demonstrating its capability to cope well with increased Cd levels in its tissues. The enhanced Cd accumulation in the mutant was accompanied by maintenance of homeostasis of shoot Ca, Mg, Zn and Mn contents, and root Ca and Mg contents. Through the application of La(+3) and the exclusion of Ca from the nutrient solution, maintenance of nutrient homeostasis in Cd-stressed SGECd(t) was shown to contribute to the increased Cd tolerance. Control plants of the mutant (i.e. no Cd treatment) had elevated concentrations of glutathione (GSH) in the roots. Through measurements of chitinase and guaiacol-dependent peroxidase activities, as well as proline and non-protein thiol (NPT) levels, it was shown that there were lower levels of Cd stress both in roots and shoots of SGECd(t). Accumulation of phytochelatins [(PCcalculated) = (NPT)-(GSH)] could be excluded as a cause of the increased Cd tolerance in the mutant. CONCLUSIONS: The SGECd(t) mutant represents a novel and unique model to study adaptation of plants to toxic heavy metal concentrations.     

Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis

Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 ( http://www.brachypodium.org ) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium -mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium ; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO₄ prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T₀) and their progeny (T₁). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.     

Enantioselective determination of metoprolol acidic metabolite in plasma and urine using liquid chromatography chiral columns: applications to pharmacokinetics

Enantioselective separations on chiral stationary phases with or without derivatization were developed and compared for the HPLC analysis of (+)-(R)- and (-)-(S)-metoprolol acidic metabolite in human plasma and urine. The enantiomers were analysed in plasma and urine without derivatization on a Chiralcel OD-R column, and in urine after derivatization using methanol in acidic medium on a Chiralcel OD-H column. The quantitation limits were 17 ng of each enantiomer/ml plasma and 0.5 microgram of each enantiomer/ml urine using both methods. The confident limits show that the methods are compatible with pharmacokinetic investigations of the enantioselective metabolism of metoprolol. The methods were employed in a metabolism study of racemic metoprolol administered to a patient phenotyped as an extensive metabolizer of debrisoquine. The enantiomeric ratio (+)-(R)/(-)-(S)-acid metabolite was 1.1 for plasma and 1.2 for urine. Clearances were 0.41 and 0.25 l/h/kg, respectively, for the (+)-(R)- and (-)-(S)-enantiomers. The correlation coefficients between the urine concentrations of the acid metabolite enantiomers obtained by the two methods were >0.99. The two methods demonstrated interchangeable application to pharmacokinetics.