Transformation of barley scutellum protoplasts: regeneration of fertile transgenic plants

 A system for barley transformation via polyethyleneglycol-mediated DNA uptake into protoplasts isolated directly from scutella and the regeneration of transgenic plants is reported. Scutellum protoplasts (cv. Clipper, an Australian malting cultivar) were co-transformed with plasmids Act 1-DGUS, containing the marker uidA gene, and pCaIneo, which contains the selectable marker neomycin phosphotransferase gene. Protoplast-derived calluses were selected on medium containing the antibiotic G418 (25 and 15 mg.l^–1) and macroscopic antibiotic resistant colonies were recovered. Fertile plants were regenerated from a callus line and molecular analysis confirmed transgene integration. Received: 11 October 1999 / Revision received: 11 February 2000 / Accepted: 11 February 2000     

Increasing sensitivity of Ca2+ spark detection in noisy images by application of a matched-filter object detection algorithm

Microscopic calcium (Ca²⁺) events (such as Ca²⁺ sparks) are an important area for study, as they help clarify the mechanism(s) underlying intracellular signaling. In the heart, Ca²⁺ sparks occur as a result of Ca²⁺ release from the sarcoendoplasmic reticulum, via ryanodine receptor channels. Measurement of Ca²⁺ spark properties can provide valuable information about the control of ryanodine receptor channel gating in situ, but requires high spatiotemporal resolution imaging, which produces noisy datasets that are problematic for spark detection. Automated detection algorithms may overcome visual detection bias, but missed and false-positive events can distort the distribution of measured Ca²⁺ spark properties. We present a sensitive and reliable method for the automated detection of Ca²⁺ sparks in datasets obtained using confocal line-scanning or total internal reflection fluorescence microscopy. This matched-filter detection algorithm (MFDA) employs a user-defined object, chosen to mimic Ca²⁺ spark properties, and the experimental dataset is searched for instances of the object. Detection certainty is provided by nonparametric statistical testing. The supplied codes can also refine the search object on the basis of those detected to further increase detection sensitivity. In comparison to a commonly used, intensity-thresholding algorithm, the MFDA is more sensitive and reliable, particularly at low signal/noise ratios. The MFDA can also be easily adapted to other signal-detection problems in noisy datasets.     

Optimization studies for the bioconversion of Jerusalem artichoke tubers to ethanol and microbial biomass

Summary A total of 8 yeast and microbial cultures have been grown in the extract derived from the tubers of Jerusalem artichoke (Helianthus tuberosus) and screened according to the following optimization criteria: rates and yields of ethanol production, rates and yields of biomass production, and % of original sugars utilized during fermentation. Batch growth kinetic parameters were also determined for the cultures studied.     

Latitudinal patterns of magnitude and interannual variability in net ecosystem exchange regulated by biological and environmental variables

Over the last two and half decades, strong evidence showed that the terrestrial ecosystems are acting as a net sink for atmospheric carbon. However the spatial and temporal patterns of variation in the sink are not well known. In this study, we examined latitudinal patterns of interannual variability (IAV) in net ecosystem exchange (NEE) of CO₂ based on 163 site-years of eddy covariance data, from 39 northern-hemisphere research sites located at latitudes ranging from ∼29°N to ∼64°N. We computed the standard deviation of annual NEE integrals at individual sites to represent absolute interannual variability (AIAV), and the corresponding coefficient of variation as a measure of relative interannual variability (RIAV). Our results showed decreased trends of annual NEE with increasing latitude for both deciduous broadleaf forests and evergreen needleleaf forests. Gross primary production (GPP) explained a significant proportion of the spatial variation of NEE across evergreen needleleaf forests, whereas, across deciduous broadleaf forests, it is ecosystem respiration (Re). In addition, AIAV in GPP and Re increased significantly with latitude in deciduous broadleaf forests, but AIAV in GPP decreased significantly with latitude in evergreen needleleaf forests. Furthermore, RIAV in NEE, GPP, and Re appeared to increase significantly with latitude in deciduous broadleaf forests, but not in evergreen needleleaf forests. Correlation analyses showed air temperature was the primary environmental factor that determined RIAV of NEE in deciduous broadleaf forest across the North American sites, and none of the chosen climatic factors could explain RIAV of NEE in evergreen needleleaf forests. Mean annual NEE significantly increased with latitude in grasslands. Precipitation was dominant environmental factor for the spatial variation of magnitude and IAV in GPP and Re in grasslands.     

Impact of rainfall manipulations and biotic controls on soil respiration in Mediterranean and desert ecosystems along an aridity gradient

Spatially heterogeneous ecosystems form a majority of land types in the vast drylands of the globe. To evaluate climate‐change effects on CO₂ fluxes in such ecosystems, it is critical to understand the relative responses of each ecosystem component (microsite). We investigated soil respiration (R_s) at four sites along an aridity gradient (90–780 mm mean annual precipitation, MAP) during almost 2 years. In addition, R_s was measured in rainfall manipulations plots at the two central sites where ～30% droughting and ～30% water supplementation treatments were used over 5 years. Annual R_s was higher by 23% under shrub canopies compared with herbaceous gaps between shrubs, but R_s at both microsites responded similarly to rainfall reduction. Decreasing precipitation and soil water content along the aridity gradient and across rainfall manipulations resulted in a progressive decline in R_s at both microsites, i.e. the drier the conditions, the larger was the effect of reduction in water availability on R_s. Annual R_s on the ecosystem scale decreased at a slope of 256/MAP g C m^?2 yr^?1 mm^?1 (r²=0.97). The reduction in R_s amounted to 77% along the aridity gradient and to 16% across rainfall manipulations. Soil organic carbon (SOC) decreased with declining precipitation, and variation in SOC stocks explained 77% of the variation in annual R_s across sites, rainfall manipulations and microsites. This study shows that rainfall manipulations over several years are a useful tool for experimentally predicting climate‐change effects on CO₂ fluxes for time scales (such as approximated by aridity gradients) that are beyond common research periods. Rainfall reduction decreases rates of R_s not only by lowering biological activity, but also by drastically reducing shrub cover. We postulate that future climate change in heterogeneous ecosystems, such as Mediterranean and deserts shrublands will have a major impact on R_s by feedbacks through changes in vegetation structure.     

Cloning of a pea cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I using a monoclonal antibody

A monoclonal antibody (MAb UB42) is described that binds to thylakoids in pea chloroplasts, as shown by EM-immunogold labelling. The antibody recognised proteins of ca. 23–29 kDa in western blots of a pea leaf homogenate. A cDNA library was prepared from pea epidermal cells in the vector ZAP II, and immunoscreening of the library with UB42 led to the isolation of a clone, pUB42. This was sequenced and had an open reading frame of 269 codons encoding a predicted polypeptide of 28.9 kDa. The sequence showed extensive homology with three closely related polypeptides belonging to a family of chlorophyll a/b-binding proteins from the light harvesting complex of photosytem I (LHCI). Collectively, the results suggest that MAb UB42 recognises an epitope on the type II chlorophyll a/b-binding protein from LHCI and that clone pUB42 encodes this protein.     

Forest refugia revisited: nSSRs and cpDNA sequences support historical isolation in a wide‐spread African tree with high colonization capacity,Milicia excelsa (Moraceae)

The impact of the Pleistocene climate oscillations on the structure of biodiversity in tropical regions remains poorly understood. In this study, the forest refuge theory is examined at the molecular level in Milicia excelsa, a dioecious tree with a continuous range throughout tropical Africa. Eight nuclear microsatellites (nSSRs) and two sequences and one microsatellite from chloroplast DNA (cpDNA) showed a deep divide between samples from Benin and those from Lower Guinea. This suggests that these populations were isolated in separate geographical regions, probably for several glacial cycles of the Pleistocene, and that the nuclear gene pools were not homogenized despite M. excelsa’s wind‐pollination syndrome. The divide could also be related to seed dispersal patterns, which should be largely determined by the migration behaviour of M. excelsa’s main seed disperser, the frugivorous bat Eidolon helvum. Within Lower Guinea, a north–south divide, observed with both marker types despite weak genetic structure (nSSRs: F_ST = 0.035, cpDNA: G_ST = 0.506), suggested the existence of separate Pleistocene refugia in Cameroon and the Gabon/Congo region. We inferred a pollen‐to‐seed dispersal distance ratio of c. 1.8, consistent with wide‐ranging gene dispersal by both wind and bats. Simulations in an Approximate Bayesian Computation framework suggested low nSSR and cpDNA mutation rates, but imprecise estimates of other demographic parameters, probably due to a substantial gene flow between the Lower Guinean gene pools. The decline of genetic diversity detected in some Gabonese populations could be a consequence of the relatively recent establishment of a closed canopy forest, which could negatively affect M. excelsa’s reproductive system.     

Hormonal correlates of parental behavior in yellow-eyed penguins (Megadyptes antipodes)

Penguins show varying degrees of brood reduction behavior, from obligate brood reducers to brood maximizers, and we hypothesize that this is associated with differences in prolactin secretion. To address this hypothesis, we determined the breeding season prolactin profile of the yellow-eyed penguin (Megadyptes antipodes) for comparison with those of other penguin species found in the literature. We also measured sex steroid plasma concentrations to better characterize the reproductive cycle of the species. Plasma concentrations of prolactin increased from early in the season, reaching a peak during late incubation, and remained elevated up to the guard period. This general pattern was similar to that of other penguins for which we have corresponding data. However, we found that throughout the laying period, prolactin titers in yellow-eyed penguins remained elevated while they fell to basal levels after the laying of the first egg in macaroni penguins, which corresponds to differences in incubation behavior during this time. We conclude, therefore, that differences in the brood reduction behavior in penguins, may be reflected in the pattern of PRL concentrations around the time of egg laying.