The effect of humic substances on germination and early growth of Lamb’s Quarters (<Emphasis Type="Italic">Chenopodium album</Emphasis> agg.)

In order to understand the biological activity of humic substances (HS), the effect of four humic acids (HA) and one fulvic acid (FA) on seed germination and early growth of cosmopolitan weed Chenopodium album agg. were tested. Humic substances of diverse origin were used, namely purified commercial HA, HA isolated from lignite, cambisol and podzol, and FA from mountain spruce forest soil. Data processing by two-way ANOVA has shown that type of the tested substances was a more important factor on seed development than used concentration. The major differences in germination and length of shoots were found in the first days of the experiment. Commercial and lignite HA stimulated the seed germination and growth, while podzol HA inhibited them. After this initial phase, all humic substances stimulated the seed development, but these stimulations were not significant (P < 0.05). At the end of germination test, the greatest stimulation effect (up to 20%) was achieved with the lignite HA.     

Defining Structural Domains of an Intrinsically Disordered Protein: Sic1, the Cyclin-Dependent Kinase Inhibitor of <Emphasis Type="Italic">Saccharomyces</Emphasis><Emphasis Type="Italic">cerevisiae</Emphasis>

The cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein (IDP) involved in cell–cycle regulation in the yeast Saccharomyces cerevisiae. Notwithstanding many studies on its biological function, structural characterization has been attempted only recently, fostering the development of production and purification protocols suitable to yield large amounts of this weakly expressed protein. In this study, we describe the identification of protein domains by the heterologous expression, purification, and characterization of Sic1-derived fragment. Four C-terminal fragments (Sic1^C-ter) were produced based on functional studies and limited-proteolysis results. The N-terminal fragment (Sic1^1–186) was complementary to the most stable C-terminal fragments (Sic1^Δ186). Both Sic1^1–186 and Sic1^C-ter fragments were, in general, less susceptible to spontaneous proteolysis than the full-length protein. The boundaries of the C-terminal fragments turned out to be crucial for integrity of the recombinant proteins and required two rounds of design and production. Sic1 fragments were purified by a simple procedure, based on their resistance to heat treatment, at the amount and purity required for structural characterization. Circular dichroism (CD) measurements and nuclear magnetic resonance (NMR) spectra of N- and C-terminal fragments confirm their disordered nature but reveal minor structural differences that may reflect their distinct functional roles.     

Effect of transgenic alfalfa plants with introduced gene for Alfalfa Mosaic Virus coat protein on rhizosphere microbial community composition and physiological profile

The aim of this study was to evaluate the effect of transgenic alfalfa (Medicago sativa L.) plants, in comparison to their non-transgenic counterpart, on the density and physiological profiles of aerobic bacteria in the rhizosphere. Plants of transgenic alfalfa expressing the AMVcp-s gene coding for Alfalfa Mosaic Virus coat protein were cultivated in a climatic chamber. Two methods were used to determine the microbial diversity in rhizospheres of transgenic plants. First, the cultivation-dependent plating method, based on the determination of the density of colony-forming bacteria, and second, a biochemical method using the Biolog™ system, based on the utilization of different carbon sources by soil microorganisms. Statistically significant differences in densities of rhizospheric bacteria between transgenic and non-transgenic alfalfa clones were observed in ammonifying bacteria (GTL4/404-1), cellulolytic bacteria (GTL4/404-1, GTL4/402-2, A5-3-3), rhizobial bacteria (GTL4/402-2), denitrifying bacteria (A5-3-3) and Azotobacter spp. (GTL4/402-2). The highest values of substrate utilization by microbial communities and average respiration of C-sources were determined in non-transgenic alfalfa plants of the isogenic line SE/22-GT2. Carbohydrates, carboxylic acids and amino-acids were the most utilized carbon substrates by both Gram-negative and Gram-positive bacteria. Both, the community metabolic diversity and the utilization of C-sources increased in all alfalfa lines with culture time and regardless of transgenic or non-transgenic nature of lines.     

Rotifer digestive enzymes: direct detection using the ELF technique

Hydrolytic enzymes involved in rotifer digestive processes were investigated directly at the sites of enzyme action using the ELF (Enzyme Labelled Fluorescence) technique. After enzymatic hydrolysis of an artificial ELF substrate, the fluorescent product ELF alcohol (ELFA) marked the sites of enzyme action. The time development of ELFA labelling was studied at different incubation times. Phosphatases, β-N-acetylhexosaminidases and lipases were examined in Brachionus angularis, B. calyciflorus, Keratella cochlearis and Lecane closterocerca from fed-batch cultures. We detected activities of all studied enzymes mostly in the stomach and intestine of rotifers. L. closterocerca was the only species showing enzyme activity at the mastax. Lipase activity was observed in the stomach and intestine of all species and in the mastax of L. closterocerca. Phosphatases were frequently located at the corona of B. calyciflorus. In other cases, both phosphatases and β-N-acetylhexosaminidases were rarely detected at the corona, and on the lorica and epidermis of some species. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research