The Survival of Transplants of Rare Ligularia sibirica is Enhanced by Neighbouring Plants

Reintroduction programs are often initiated to restore the viability of endangered plant populations, whose decline is usually caused by loss of suitable habitats. Ligularia sibirica is a species associated with wetlands. It is endangered in Europe and has declined considerably in Estonia since the cessation of traditional management and, in addition, drainage leading to the overgrowth of habitats. The purpose of this work was to estimate the extent of the impact of competition from neighbouring plants and habitat change caused by overgrowing on the survival of transplants of L. sibirica and thereby assess the effectiveness of reinforcement. Laboratory-grown transplants were planted back into their original populations in plots arranged in a two × two (vegetation intact or removed × open or overgrown habitat) factorial experimental design, and their survival was followed for three years. The survival differed notably among populations, but the percentage of surviving plants per plot was on average higher in plots with intact vegetation and in open habitats. The latter indicates that overgrowing indeed decreases habitat quality for this species, despite the fact that plants of L. sibirica can often be found in forested habitats. The lower survival in plots where vegetation had been removed can largely be explained by increased damage caused by animals. In intact plots, by contrast, neighbouring plants provide shelter and protection. Our results stress the importance of restoration and preservation of habitat quality for the protection of this rare species, for which reinforcement may be effective.     

How to Preserve Plant Samples for Carbohydrate Analysis? Test of Suitable Methods Applicable in Remote Areas

Nonstructural carbohydrates (NSC) are frequently studied both by ecologists and plant physiologists because they serve as fundamental carbon and energy sources in plant metabolism. Because of the rapid enzymatic hydrolysis of NSC after harvest, plants are usually placed into liquid nitrogen until subsequent analyses in the laboratory. Nevertheless, when the research is performed in poorly accessible regions such as high mountains, tropical forests or deserts, the use of heavy containers containing vaporizing liquid nitrogen is problematic for logistical reasons. These places are particularly interesting as they harbor plant species with interesting physiological adaptations. In our study we aimed to develop a suitable storage method for plants intended for NSC analyses, which would require minimal equipment. These demands resulted in the idea of using the first step in NSC analyses – extraction in ethanol. Samples were extracted in 50 % and 96 % boiling or non-boiling ethanol and then stored for one month; they were compared with samples immediately frozen in liquid nitrogen. We discovered that for samples containing starch, fructans, soluble sugars and sugar alcohols, the best pretreatment for subsequent storage is extraction in 50 % or 96 % boiling ethanol. For total nonstructural carbohydrates (TNC) assessment, only extracting with 50 % ethanol without boiling gave very good results. Finally, we developed a method that could be used in any remote place without bulky laboratory equipment.     

Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: DISPARATE SUSCEPTIBILITIES DUE TO THE REPAIR OF TYR35 RADICAL BY CYS83 IN Fe-SODB THROUGH INTRAMOLECULAR ELECTRON TRANSFER*

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10⁴ m⁻¹ s⁻¹ and 4.3 ± 0.4 × 10⁴ m⁻¹ s⁻¹ at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr³⁵. Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys⁸³ mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys⁸³ present in Fe-SODB acts as an electron donor that repairs Tyr³⁵ radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.     

Adaptive transgenerational plasticity in the perennial Plantago lanceolata

Phenotypes of plants, and thus their ecology and evolution, can be affected by the environmental conditions experienced by their parents, a phenomenon called parental effects or transgenerational plasticity. However, whether such effects are just passive responses or represent a special type of adaptive plasticity remains controversial because of a lack of solid tests of their adaptive significance. Here, we investigated transgenerational effects of different nutrient environments on the productivity, carbon storage and flowering phenology of the perennial plant Plantago lanceolata, and whether these effects are influenced by seasonal variation in the maternal environment. We found that maternal environments significantly affected the offspring phenotype, and that plants consistently produced more biomass and had greater root carbohydrate storage if grown under the same environmental conditions as experienced by their mothers. The observed transgenerational effects were independent of the season in which seeds had matured. We therefore conclude that transgenerational effects on biomass and carbon storage in P. lanceolata are adaptive regardless of the season of seed maturation.     

Switching substrate specificity of AMT/MEP/ Rh proteins

In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH₄⁺, NH₃ + H⁺, NH₄⁺ + H⁺ or NH₃, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH₃ transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH₃ across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH₃ transport through the central subunit pore is suggested.     

Influence of Trichobilharzia regenti (Digenea: Schistosomatidae) on the Defence Activity of Radix lagotis (Lymnaeidae) Haemocytes

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2–36 h post exposure (p.e.) to the parasite. At later time points, 44–92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae.     

The distribution of lead in duckweed (Lemna minor L.) root tip

While considerable information on lead distribution in the cells of terrestrial plants has been collected, little is known about lead localization in the cells of the aquatic plant. Lemna minor L. (duckweed) roots were examined using X-ray microanalysis. After 1-h treatment with lead, its concentration was the highest in small vacuoles. After 6 and 12 h, the lead content of cell walls gradually increased. The changes of lead level between vacuoles and cell walls may result from redistribution of this metal from symplast to cell walls or it may reflect increased apoplastic transport. Lead was not found in the ground cytoplasm of any variants of the experiments. This fact and presence of lead in small vesicles suggests that endocytosis may play the role in lead uptake in Lemna.     

Seasonal Changes of Selected Parameters of CO2 Fixation Biochemistry of Norway Spruce Under the Long-Term Impact of Elevated CO2

Twelve-year-old Norway spruce (Picea abies [L.] Karst.) trees were exposed to ambient (AC) or elevated (EC) [ambient + 350 µmol(CO₂) mol^-1] CO₂ concentrations in open-top-chamber (OTC) experiment under the field conditions of a mountain stand. Short-term (4 weeks, beginning of the vegetation season) and long-term (4 growing seasons, end of the vegetation season) effects of this treatment on biochemical parameters of CO₂ assimilation were evaluated. A combination of gas exchange, fluorescence of chlorophyll a, and application of a mathematical model of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activity was used. The analysis showed that the depression of photosynthetic activity by long-term impact of elevated CO₂ was mainly caused by decreased RuBPCO carboxylation rate. The electron transport rate as well as the rate of ribulose-1,5-bisphosphate (RuBP) formation were also modified. These modifications to photosynthetic assimilation depended on time during the growing season. Changes in the spring were caused mainly by local deficiency of nitrogen in the assimilating tissue. However, the strong depression of assimilation observed in the autumn months was the result of insufficient carbon sink capacity.