Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants

Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2β*A and INH2β*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2β mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability

Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance.

The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (KD) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The KD measured for the binding of wild type and His6-Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased KD by 200-300-fold, whereas the A173T substitution increased KD by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein

Reorganization of terminator DNA upon binding replication terminator protein: implications for the functional replication fork arrest complex.

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers

Effects of environmental factors and management on dynamics of mixed calcareous forests under climate change in Central European lowlands

Mixed lowland forests reserved for natural succession are sparse in the Czech Republic. However, their development provides essential insights into the natural processes of these forests and recommendations for forest management in a changing climate. The research describes the dynamics, productivity, structure, diversity, dead wood, and radial growth of hornbeam-oak groves and calcareous beech-dominated forests in the Karlštejn National Nature Reserve (Czechia) based on inventory in 2002, 2008, 2014 and 2020. The objective was to evaluate changes in differently managed stands (high forest, coppice with standards, and coppice) after leaving the stands to spontaneous development in 2004. The tree density increased by 2–10% from 2002 to 2014 and decreased by 6–18% in 2020. In the high forest, an increase in the stand volume was observed during the whole period, while in the coppice with standards and coppice, only until 2014. The stand volume ranged from 190 (coppice) to 630 (high forest) m³ ha−1 in 2020 and increased by an average of 28% over 18 years. Overall diversity of tree layer showed an uneven structure in the high forest and a substantially diverse structure in the other variants. The deadwood volume has been steadily increasing (18–35 m³ ha−1 in 2020), accumulating an average of 1 m³ ha−1 yr−1. A lack of precipitation and high temperatures from June to August were the main limiting factors of the radial growth of tree species, while the number of negative pointer years has increased in the last decade. European beech (Fagus sylvatica L.) was the most sensitive tree species to climate compared to the resilient European ash (Fraxinus excelsior L.). The lowest fluctuations in the diameter increment were recorded in Norway maple (Acer platanoides L.) and the highest in beech in the temperature cycles of 7–15 years. Over the last 20 years, sessile oak [Quercus petraea (Matt.) Liebl.] showed an increase in radial growth by 7%, while other tree species reported a decrease with a maximum in beech (by −38%). The forest stands managed as high forest, characterized by a higher production potential and lower diversity, had slower dynamics when compared to coppice with standards and coppice

Data on liquid gated CNT network FETs on flexible substrates

This article presents the raw and analyzed data from a set of experiments performed to study the role of junctions on the electrostatic gating of carbon nanotube (CNT) network field effect transistor (FET) aptasensors. It consists of the raw data used for the calculation of junction and bundle densities and describes the calculation of metallic content of the bundles. In addition, the data set consists of the electrical measurement data in a liquid gated environment for 119 different devices with four different CNT densities and summarizes their electrical properties. The data presented in this article are related to research article titled “Metallic-semiconducting junctions create sensing hot-spots in carbon nanotube FET aptasensors near percolation” (doi:10.1016/j.bios.2018.09.021) [1]

Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins

Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions. Immunocytochemistry experiments in Drosophila S2 cells imply that Or22a has seven transmembrane domains but that its membrane topology is opposite to that of GPCRs, with a cytoplasmic N-terminus and extracellular C-terminus. To investigate Or signalling mechanisms, we expressed Or43b in Sf9 and HEK293 cells, and show that inhibitors of heterotrimeric G proteins (GDP-beta-S), adenylate cyclase (SQ22536), guanylyl cyclase (ODQ), cyclic nucleotide phosphodiesterases (IBMX) and phospholipase C (U73122) have negligible impact on Or43b responses. Whole cell patching Or43b/Or83b-transfected HEK293 cells revealed the opening of plasma membrane cation channels on addition of ligand. The response was blocked by lanthanum and by 2-APB, but not by Ruthenium red or SKF96365. Based on these data, we conclude that Drosophila Ors comprise a novel family of seven transmembrane receptors that in HEK293 cells signal by opening cation channels, through a mechanism that is largely independent of G proteins. (c) 2008 Elsevier Ltd. All rights reserved

Dendrochronological data from twelve countries proved definite growth response of black alder (Alnus glutinosa [L.] Gaertn.) to climate courses across its distribution range

Black alder (Alnusglutinosa [L.] Gaertn.) is an important component of riparian and wetland ecosystems in Europe. However, data on the growth of this significant broadleaved tree species is very limited. Presently, black alder currently suffers from the pathogen Phytophthora and is particularly threatened by climate change. The objective of this study was to focus on the impact of climatic variables (precipitation, temperature, extreme climatic events) on the radial growth of alder across its geographic range during the period 1975-2015. The study of alder stands aged 46-108 years was conducted on 24 research plots in a wide altitude range (85-1015 m) in 12 countries of Europe and Asia. The most significant months affecting alder radial growth were February and March, where air temperatures are more significant than precipitation. Heavy frost and extreme weather fluctuations in the first quarter of the year were the main limiting factors for diameter increment. Within the geographical setting, latitude had a higher effect on radial growth compared to longitude. However, the most important variable concerning growth parameters was altitude. The temperature's effect on the increment was negative in the lowlands and yet turned to positive with increasing altitude. Moreover, growth sensitivity to precipitation significantly decreased with the increasing age of alder stands. In conclusion, the growth variability of alder and the number of negative pointer years increased with time, which was caused by the ongoing climate change and also a possible drop in the groundwater level. Riparian alder stands well supplied with water are better adapted to climatic extremes compared to plateau and marshy sites

Symmetry and Secondary Structure of the Replication Terminator Protein of Bacillus-Subtilis - Sedimentation Equilibrium and Circular Dichroic, Infrared, and Nmr Spectroscopic Studies

We have used analytical ultracentrifugation in combination with a number of spectroscopic techniques to analyze the symmetry and secondary structure of the DNA-binding replication terminator protein (RTP) of Bacillus subtilis. Sedimentation equilibrium studies confirm that RTP is a dimer in solution under the conditions used for spectroscopic analysis, whereas the number of cross peaks displayed in H-1-N-15 HSQC NMR spectra of uniformly N-15-labeled RTP are consistent with the primary structure of the monomer. These two results in combination lead to the conclusion that RTP is a symmetric dimer in solution. Circular dichroic and Fourier-transform infrared spectra reveal, in contrast to the results obtained from a number of commonly used secondary structure prediction algorithms, that RTP contains 20-30% alpha-helical and 40-50% beta-sheet/beta-turn secondary structure and that the conformation of the protein remains unchanged over the pH range 5-8. It is proposed on the basis of protein folding-class prediction algorithms, in combination with various physical properties of RTP, that it belongs to the alpha + beta protein-folding class