A protocol for direct somatic embryogenesis of Protea cynaroides L. using zygotic embryos and cotyledon tissues

We describe a protocol for somatic embryogenesis of Protea cynaroides, with potential for high frequency production of this important horticultural species. Somatic embryos formed directly on both P. cynaroides mature zygotic embryos and excised cotyledons cultured on MS medium without growth regulators. The addition of growth regulators such as naphthalene acetic acid (NAA) (5; 13 and 27 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (5; 11 and 23 μM), in combination with thidiazuron (TDZ) (1 μM), benzylaminopurine (BAP) (1 μM) or kinetin (1 μM) suppressed the formation of somatic embryos. After eight weeks in culture, formation of somatic embryos was observed. Zygotic explants formed the most embryos when cultured in a 12-h photoperiod in comparison to explants cultured in the dark. Up to 83% of these embryos germinated after transferal to the germination medium containing 0.3 μM GA₃. Significantly fewer embryos germinated in MS medium with no growth regulators, or supplemented with higher concentrations of GA₃, while low germination percentages were also observed in MS media containing casein hydrolysate and coconut water. The germination of normal somatic embryos (two separate cotyledons and a single radicle) was observed only in media containing either no growth regulators, 0.3 μM GA₃ or 1 μM GA₃. All embryos that germinated in high concentrations of GA₃ were malformed.     

Winning by a neck: tall giraffes avoid competing with shorter browsers

With their vertically elongated body form, giraffes generally feed above the level of other browsers within the savanna browsing guild, despite having access to foliage at lower levels. They ingest more leaf mass per bite when foraging high in the tree, perhaps because smaller, more selective browsers deplete shoots at lower levels or because trees differentially allocate resources to promote shoot growth in the upper canopy. We erected exclosures around individual Acacia nigrescens trees in the greater Kruger ecosystem, South Africa. After a complete growing season, we found no differences in leaf biomass per shoot across height zones in excluded trees but significant differences in control trees. We conclude that giraffes preferentially browse at high levels in the canopy to avoid competition with smaller browsers. Our findings are analogous with those from studies of grazing guilds and demonstrate that resource partitioning can be driven by competition when smaller foragers displace larger foragers from shared resources. This provides the first experimental support for the classic evolutionary hypothesis that vertical elongation of the giraffe body is an outcome of competition within the browsing ungulate guild.     

Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture

AIMS: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. METHODS AND RESULTS: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. CONCLUSIONS: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. SIGNIFICANCE AND IMPACT OF THE STUDY: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.     

A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgipl gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.     

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc⁻) DT40 cells arrest in G₂ phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc⁻ cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G₂ checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G₂ arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G₂ phase.     

The effects of gender and obesity on myocardial tolerance to ischemia

Obesity is increasing at an alarming rate globally. Several studies have shown that premenopausal women have a reduced risk of CV disease and a reduced myocardial susceptibility to ischemia/reperfusion injury. The effect of obesity on myocardial tolerance to ischemia in women has not been established. To determine how obesity affects myocardial susceptibility to ischemia/reperfusion injury in both males and females, we fed male and female Wistar rats a high caloric diet (HCD) or a control rat chow diet (CD) for 18 weeks. Rats were subsequently fasted overnight, anesthetized and blood was collected. In separate experiments, 18-week-fed (HCD and CD) rats underwent 45 min in vivo coronary artery ligation (CAL) followed by 2 hours reperfusion. Hearts were stained with TTC and infarct size determined. Both male and female HCD fed rats had increased body and visceral fat weights. Homeostasis model assessment (HOMA) index values were 13.95+/-3.04 for CD and 33.58+/-9.39 for HCD male rats (p<0.01) and 2.98+/-0.64 for CD and 2.99+/-0.72 for HCD fed female rats. Male HCD fed rats had larger infarct sizes than CD fed littermates (43.2+/-9.3 % vs. 24.4+/-7.6 %, p<0.05). Female HCD and CD diet fed rats had comparable infarct sizes (31.8+/-4.3 % vs. 23.9+/-3.3 %). We conclude that male rats on the HCD became viscerally obese, dyslipidemic and insulin-resistant, while female HCD fed rats became viscerally obese without developing dyslipidemia or insulin resistance. Obesity increased myocardial infarct size in males but not the females.     

Proteomic analysis of grape berry cell cultures reveals that developmentally regulated ripening related processes can be studied using cultured cells

Background

This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first.

Methodology/Principal Findings

In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions.

Conclusions

The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening.