Somatic embryogenesis in leaves and leaf-derived protoplasts of Actinidia deliciosa var. deliciosa cv. Hayward (kiwifruit)

Summary The obtention of embryogenic competence in Actinidia deliciosa var. deliciosa cv. Hayward is reported. Axillary buds from shoots submitted to cold (4°C) and starvation for 1.5 months, developed leaves with embryogenic competence. These leaves, cultured in darkness for 1.5 months on a medium containing zeatin as a sole growth regulator, originated compact structures from which embryos developed. The plating orientation and sectioning of leaves strongly affected the expression of the embryogenic potential. A selected fraction of the protoplasts isolated from these leaves was able to develop in an embryogenic way. The germination of the embryos is still only occasional.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 6-dimethylallyl aminopurine - IAA indole-3-acetic acid - NOA naphthoxyacetic acid - SEM Scanning Electron Microscopy     

Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Effect of electrical fields and external ionic currents on pollen-tube orientation

Summary Agapanthus umbelatus pollen tubes (PTs) display a number of different growth patterns when germinated in an electric field of 750 mV· mm^–1. When pollen is germinated near the cathode (82.44% of orientation to the cathode side) or near the anode (55.35% of orientation to the anode), growth is oriented parallel to the applied field but when germinated at an intermediate position, there is random growth. An increase and decrease in the orientation rates as well as reversion of the polarized growth were observed when the growth conditions were systematically altered. These findings reflect the influence of different ionic currents present in the germination medium. These ionic currents induce the formation of ionic gradients, which were monitored by ion-HPLC. The individual omission of Ca²⁺, K⁺, Mg²⁺ and Cl^– suppresses or alters the oriented growth pattern. The presence of ionic gradients is not by itself suficient to trigger the polarization of tube growth as the presence of an electric field which drives the ionic currents is essential for this to occur.Abbreviations PT Pollen tube - DNS 3,5-dinitro salycilic acid; - TP transient polarization - HPLC high precision liquid chroma tography - DC direct current     

Cytochemical localization of 5′-nucleotidase in the enteric ganglia and in smooth muscle cells of the guinea-pig

Brain Cell Biology - Cytochemical techniques were used to study the localization of 5′-nucleotidase in the enteric ganglia and in smooth muscle cells of the guinea-pig ileum, iris and vas...     

Effect of copper deficiency on photosynthetic electron transport in wheat plants

Copper deficiency in wheat ( Triticum aestivum L. cv. Nazareno Stramppeli) markedly affects photosynthetic activity. Flag leaves of copper-deficient plants showed a 50% reduction of the photosynthetic rate expressed as mg CO₂ dm⁻²h⁻¹. The activities of PSI and PSII, determined for isolated chloroplasts, as well as fluorescence measurements on intact leaves of copper-deficient plants, indicated a low activity of photosynthetic electron transport. Ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity was not affected by copper deficiency but copper deficiency affected the chloroplast ultrastructure, especially at the level of grana, where a disorganization of thylakoids is evident.     

Characterization of the yeast population from traditional corn and rye bread doughs

M.J. ALMEIDA AND C.S. PAIS. 1996. Yeasts were isolated from a variety of home-made bread doughs and identified. A pure culture of Saccharomyces cerevisiae was found in 18% of the doughs. The same species predominated in 80% of the doughs examined whereas Issatchenkia orientalis, Pichia membranaefaciens and Torulaspora delbrueckii were present in about 40% of the samples. About one quarter of the isolates displayed killer activity, strains of P. anomala showing the broadest spectra. Two isolates of S. cerevisiae and three of T. delbrueckii gave biomass values in sucrose medium similar to or higher than those obtained with commercial compressed baker's yeast strains.     

Mutations inside but not outside the peptide binding cleft of the H-2 Ld molecule affects CTL recognition and binding of the nucleoprotein peptide from the lymphocytic chroriomeningitis

In order to investigate the role of residues inside and outside the peptide binding cleft of the L² molecule in peptide presentation to cytotoxic T lymphocytes (CTL), we constructed a series of point mutations in the L ^d gene. We determined the effects of the mutations in the L^d molecule on the binding and recognition of an L^d-restricted CTL epitope derived from the nucleoprotein (NP) of the lymphocytic phoriomeningitis virus (LCMV). Each of the mutations within the L^d peptide binding cleft resulted in a complete loss of CTL recognition. Addition of the LCMV NP peptide to cells expressing these mutants did not increase surface L^d expression, suggesting that the mutations altered peptide binding. Mutations involving pockets D and E within the cleft affected LCMV peptide binding and recognition as drastically as those in pocket B, which was predicted to interact with a main anchor residue of the peptide. In striking contrast, the mutations located outside the cleft did not change either recognition or binding. These results demonstrate that the L^d residues in the peptide binding cleft are the main determinants dictating LCMV NP peptide binding, and that the residues in each of the pockets within the cleft play a role in this interaction. Surprisingly, one mutation outside the peptide binding cleft, T92S, abrogated CTL lysis of target cells treated with the LCMV NP peptide, but not virus-infected cells. These data show that this mutation selectively altered the presentation of the LCMV NP peptide introduced to the cell exogenously, but not endogenously. This implies that the pathway by which peptides associate with class I molecules within the cell differs from that of exogenous peptide binding.     

Litter and soil biodiversity jointly drive ecosystem functions

The decomposition of litter and the supply of nutrients into and from the soil are two fundamental processes through which the above- and belowground world interact. Microbial biodiversity, and especially that of decomposers, plays a key role in these processes by helping litter decomposition. Yet the relative contribution of litter diversity and soil biodiversity in supporting multiple ecosystem services remains virtually unknown. Here we conducted a mesocosm experiment where leaf litter and soil biodiversity were manipulated to investigate their influence on plant productivity, litter decomposition, soil respiration, and enzymatic activity in the littersphere. We showed that both leaf litter diversity and soil microbial diversity (richness and community composition) independently contributed to explain multiple ecosystem functions. Fungal saprobes community composition was especially important for supporting ecosystem multifunctionality (EMF), plant production, litter decomposition, and activity of soil phosphatase when compared with bacteria or other fungal functional groups and litter species richness. Moreover, leaf litter diversity and soil microbial diversity exerted previously undescribed and significantly interactive effects on EMF and multiple individual ecosystem functions, such as litter decomposition and plant production. Together, our work provides experimental evidence supporting the independent and interactive roles of litter and belowground soil biodiversity to maintain ecosystem functions and multiple services.     

Sex determination and optimal development in the Moorish gecko,Tarentola mauritanica

Under temperature sex determination (TSD), sex is determined by temperature during embryonic development. Depending on ecological and physiological traits and plasticity, TSD species may face demographic collapse due to climate change. In this context, asymmetry in bilateral organisms can be used as a proxy for developmental instability and, therefore, deviations from optimal incubation conditions. Using Tarentola mauritanica gecko as a model, this study aimed first to confirm TSD, its pattern and pivotal temperature, and second to assess the local adaptation of TSD and variation of asymmetry patterns across four populations under different thermal regimes. Eggs were incubated at different temperatures, and hatchlings were sexed and measured. The number of lamellae was counted in adults and hatchlings. Results were compatible with a TSD pattern with males generated at low and females at high incubation temperatures. Estimated pivotal temperature coincided with the temperature producing lower embryonic mortality, evidencing selection towards balanced sex ratios. The temperature of oviposition was conservatively selected by gravid females. Asymmetry patterns found were likely related to nest temperature fluctuations. Overall, the rigidity of TSD may compromise reproductive success, and demographic stability in this species in case thermal nest choice becomes constrained by climate change.