Freeze-drying vegetative mycelium of Laccaria fraterna and its subsequent regeneration

Laccaria fraterna, a basidiomycetous, filamentous, non-sporulating (in vitro) fungus, was subjected to freeze-drying and tested for viability after rehydration. The optimization of the freeze-drying protocol included selection of the candidate fungus; optimizing physiological growth conditions and age; standardizing the protectant type and concentration; optimizing the pre-freezing method, freeze-drying run and extent of drying; choice of the rehydrant and the extent of rehydration. The culture retained its viability after lyophilization and when subjected to quality assurance tests gave consistent results similar to the non-lyophilized culture, indicating the stability of the product and the applicability of the developed process to freeze-dry vegetative mycelium of filamentous non-sporulating fungi.     

Rapid cooling triggers forisome dispersion just before phloem transport stops

Phloem transport stops transiently within dicot stems that are cooled rapidly, but the cause remains unknown. Now it is known that (1) rapid cooling depolarizes cell membranes giving a transient increase in cytoplasmic Ca²⁺, and (2) a rise of free calcium triggers dispersion of forisomes, which then occlude sieve elements (SEs) of fabacean plants. Therefore, we compared the effects of rapid chilling on SE electrophysiology, phloem transport and forisomes in Vicia faba. Forisomes dispersed after rapid cooling with a delay that was longer for slower cooling rates. Phloem transport stopped about 20 s after forisome dispersion, and then transport resumed and forisomes re‐condensed within similar time frames. Transport interruption and forisome dispersion showed parallel behaviour – a cooling rate‐dependent response, transience and desensitization. Chilling induced both a fast and a slow depolarization of SE membranes, the electrical signature suggesting strongly that the cause of forisome dispersion was the transient promotion of SE free calcium. This apparent block of SEs by dispersed forisomes may be assisted by other Ca²⁺‐dependent sealing proteins that are present in all dicots.     

The influence of cyclic heating and cooling on <Emphasis Type="Italic">Escherichia coli</Emphasis> survival

Repeated heating and cooling in lethal (2–52°C) and nonlethal (2–37°C) temperature ranges resulted in cell death of Escherichia coli B/r and E. coli B_S?1 suspended in 0.01 M phosphate buffer, pH 7.0 at varying osmotic pressure, but not in cow’s milk. The lethal effect increased with the rate of heating and cooling and with increasing suspension media tonicity; it may be caused by the temperature destabilization of cellular osmotic homeostasis.     

Automated robotic harvesting of protein crystals—addressing a critical bottleneck or instrumentation overkill?

One of the critical steps in high throughput crystallography that so far has evaded automation is the actual harvesting of the delicate crystals from the mother liquor in which they are growing. The late-stage operation of harvesting is presently a most risky and loss-intensive procedure, compounded by its tight integration with the critical steps of cryo-protection and cryo-quenching. Recent advances in micromanipulation robotics and micro-fabrication have made it possible to seriously consider automation of protein crystal harvesting. Based on the experience gained during the development of an operator-assisted (and now operator-assisting) universal micromanipulation robot (UMR) prototype, we discuss the challenges ahead for the design of a fully autonomous, integrated system capable of the reliable harvesting of protein microcrystals. Experience from participation in NIH structural genomics projects and feedback from bottleneck workshops indicates that genuine demand exists in the high throughput community as well as in pharmaceutical production pipelines, justifying the effort and resources to develop autonomous harvesting robotics.     

Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than −180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues.Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H₂O g⁻¹ dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s⁻¹ or programmed cooling at 3·3 °C s⁻¹. Samples were thawed rapidly (177 °C s⁻¹) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.Key Results Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth.Conclusions Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2–0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.     

Performance evaluation of native plants suited to extensive green roof conditions in a maritime climate

Assessing plant species performance on extensive green roofs can inform about and improve green roof functioning, aesthetics, longevity and the diversity of plant palettes available for the green roof industry. In this study, we evaluate survival, cover, roof cooling and stormwater retention properties of 15 plant species native to coastal regions of Atlantic Canada in extensive green roof monocultures. After a complete growing season (May-October 2009), all but one species had greater than 80% survival, and 10 species reached greater than 90% groundcover. Over the growing season, the top performing species reduced roof surface temperature by an average of 3.44 °C and increased solar reflectivity by 22.2% over the growing-medium only controls. Moreover, the best species retained 75.3% of experimentally added stormwater. Our results demonstrate that several species (mainly graminoids) performed better than creeping shrubs and forbs for most functions, although significant variation existed within life-form groups.     

Electrophysiological identification of cold receptors on the antennae of the ground beetle Pterostichus aethiops

Abstract.  In single-sensillum extracellular electrophysiological recordings, terminal campaniform sensilla at the tip of antennae of the ground beetle Pterostichus aethiops (Pz., 1797) show action potentials of three sensory cells, A-, B- and C-cells, distinguished by differences in their spike amplitudes. Only the A-cell, with the largest spike amplitude, is highly sensitive to temperature fluctuations, showing remarkable changes in its firing rate induced by changes in temperature of 0.1 °C. The firing rate of A-cells at 23 °C varies from 15–52 Hz among different beetles. Mean impulse frequency of A-cells is found to be a function of steady temperature, the firing rate decreasing with temperature increase. A-cells respond to a rapid temperature drop with a strong phasic-tonic reaction; larger decreases in temperature evoke higher peak frequency values. Maximum peak frequencies, varying from 380–630 Hz in different beetles, are induced by temperature decreases of 3–10 °C, whereas temperature rise strongly inhibits impulse activity of the A-cell. The first manifestation of rapid warming in the nerve impulse sequence is a very long interspike period, followed by diminished activity. Both the length of the long interspike period and the rate of following impulse activity are functions of temperature change; hence, A-cells respond to temperature changes as typical cold receptors, similar to coeloconic and short hair-like sensilla in other insects.     

Fire‐induced tree mortality in a neotropical forest: the roles of bark traits,tree size,wood density and fire behavior

Large‐scale wildfires are expected to accelerate forest dieback in Amazônia, but the fire vulnerability of tree species remains uncertain, in part due to the lack of studies relating fire‐induced mortality to both fire behavior and plant traits. To address this gap, we established two sets of experiments in southern Amazonia. First, we tested which bark traits best predict heat transfer rates (R) through bark during experimental bole heating. Second, using data from a large‐scale fire experiment, we tested the effects of tree wood density (WD), size, and estimated R (inverse of cambium insulation) on tree mortality after one to five fires. In the first experiment, bark thickness explained 82% of the variance in R, while the presence of water in the bark reduced the difference in temperature between the heat source and the vascular cambium, perhaps because of high latent heat of vaporization. This novel finding provides an important insight for improving mechanistic models of fire‐induced cambium damage from tropical to temperate regions. In the second experiment, tree mortality increased with increasing fire intensity (i.e. as indicated by bark char height on tree boles), which was higher along the forest edge, during the 2007 drought, and when the fire return interval was 3 years instead of one. Contrary to other tropical studies, the relationship between mortality and fire intensity was strongest in the year following the fires, but continued for 3 years afterwards. Tree mortality was low (≤20%) for thick‐barked individuals (≥18 mm) subjected to medium‐intensity fires, and significantly decreased as a function of increasing tree diameter, height and wood density. Hence, fire‐induced tree mortality was influenced not only by cambium insulation but also by other traits that reduce the indirect effects of fire. These results can be used to improve assessments of fire vulnerability of tropical forests.