Ice Encasement Injury to Microsomal Membranes from Winter Wheat Crowns : I. Comparison of Membrane Properties after Lethal Ice Encasement and during a Post-Thaw Period

The functional and physical properties of cellular membranes isolated from Triticum aestivum, cvs Norstar and Fredrick, were altered coincident with changes in composition after a lethal ice-encasement stress and further during a 6 hour post-thaw period. Crowns encased in ice for a duration which inhibited regrowth, exhibited enhanced rates of electrolyte leakage. Furthermore, the recovery of total microsomal protein and phospholipid declined, suggesting that some membrane degradation had been induced during the anoxic stress. The microviscosity of microsomes and liposomes prepared from such membranes increased during stress, and this was correlated with a 2- to 4-fold increase in the free fatty acid levels in the microsomal fraction. There was, however, only a relatively minor change in fatty acid unsaturation during the ice-encasement stress. The process continued during a 6 hour aerobic post-thaw treatment, but the pattern was somewhat different. During this phase, the leakage of electrolytes was further increased and the recovery of microsomal protein and phospholipid continued to decline, indicating general degradation; but, in contrast to the anoxic phase, the degree of fatty acid unsaturation declined markedly, indicating lipid peroxidation.     

Histochemical studies on the amphibian opercularis muscle (Amphibia)

Summary Histochemical studies of the opercularis muscle of the bullfrog (Rana catesbeiana) and the tiger salamander (Ambystoma tigrinum) provide evidence that the opercularis muscle of anurans is a specialized, tonic portion of the levator scapulae superior muscle. Staining results for myosin adenosine triphosphatase (ATPase) and succinate dehydrogenase (SDH), combined with measurements of muscle fiber diameters, demonstrate that the opercularis/levator scapulae superior muscle mass of both the tiger salamander and bullfrog consists of an anterior tonic portion, a middle fast oxidative-glycolytic (FOG) twitch portion, and a posterior fast-glycolytic (FG) twitch portion. In R. catesbeiana the tonic fibers represent 57.3% of the fiber total and (because they have relatively narrow diameters) about 29% of the cross-sectional area of the muscle mass, and form that part of the muscle (=opercularis muscle) that inserts on the operculum. In Ambystoma the tonic fibers represent only 8.8% of the fiber total and represent about 4% of the cross-sectional area. In the tiger salamander, the entire levator scapulae superior muscle inserts on the operculum and therefore represents the opercularis muscle. The bullfrog differs from the tiger salamander, therefore, in that the anterior tonic part of the opercularis/levator scapulae superior complex is greatly enlarged and the insertion on the operculum is limited to these tonic fibers. No evidence of a columellar muscle was found in R. catesbeiana. Previous reports of one in this species and in other anurans may be based on the tripartite nature of the opercularis/levator scapulae superior muscle mass. The middle FOG portion of the muscle may have been considered a muscle distinct from the anterior tonic portion (=opercularis muscle) and the posterior FG portion.     

Stress tolerance and stress-induced injury in crop plants measured by chlorophyll fluorescence in vivo: chilling, freezing, ice cover, heat, and high light

The proposition is examined that measurements of chlorophyll fluorescence in vivo can be used to monitor cellular injury caused by environmental stresses rapidly and nondestructively and to determine the relative stress tolerances of different species. Stress responses of leaf tissue were measured by F_R, the maximal rate of the induced rise in chlorophyll fluorescence. The time taken for F_R to decrease by 50% in leaves at 0°C was used as a measure of chilling tolerance. This value was 4.3 hours for chilling-sensitive cucumber. In contrast, F_R decreased very slowly in cucumber leaves at 10°C or in chilling-tolerant cabbage leaves at 0°C. Long-term changes in F_R of barley, wheat, and rye leaves kept at 0°C were different in frost-hardened and unhardened material and in the latter appeared to be correlated to plant frost tolerance. To simulate damage caused by a thick ice cover, wheat leaves were placed at 0°C under N₂. Kharkov wheat, a variety tolerant of ice encapsulation, showed a slower decrease in F_R than Gatcher, a spring wheat. Relative heat tolerance was also indicated by the decrease in F_R in heated leaves while changes in vivo resulting from photoinhibition, ultraviolet radiation, and photobleaching can also be measured.     

Short-term changes in carbon-isotope discrimination identify transitions between C3 and C4 carboxylation during Crassulacean acid metabolism

Short-term measurements of instantaneous carbon-isotope discrimination have been determined from mass-spectrometric analyses of CO₂ collected online during gas exchange for the epiphytic bromeliad Tillandsia utriculata L. Using this technique, the isotopic signature of CO₂ exchange for each phase of Crassulacean acid metabolism (CAM) has been characterised. During night-time fixation of CO₂ (Phase I), discrimination () ranged from 4.4 to 6.6, equivalent to an effective carbon-isotope ratio (¹³C) of –12.3 to –14.5 versus Pee Dee Belemnite (PDB). These values reflected the gross photosynthetic balance between net CO₂ uptake and refixation of respiratory CO₂, characteristic of CAM in the Bromeliaceae. When for the relative proportion of external (p _a ) and internal (p _i) CO₂ is taken into account, calculated p _i/p _a decreased during the later part of the dark period from 0.68 to 0.48. Measurements of during Phase II, early in the light period, showed the transition between C₄ and C₃ pathways, with carboxylation being increasingly dominated by ribulose bisphosphate carboxylase (Rubisco) as increased from 10.5 to 21.2 During decarboxylation in the light period (Phase III), CO₂ leaked out of the leaf and the inherent discrimination of Rubisco was expressed. The value of calculated from on-line measurements (64.4) showed that the CO₂ lost was considerably enriched in ¹³C, and this was confirmed by direct analysis of the CO₂ diffusing out into a CO₂-free atmosphere ( ¹³C = + 51.6 versus PDB). Instantaneous discrimination was characteristic of the C₃ pathway during Phase IV (late in the light period), but a reduction in showed an increasing contribution from phosphoenolpyruvate carboxylase. The results from this non-invasive technique confirm the observations that double carboxylation involving both phosphoenolpyruvate carboxylase and Rubisco occurs during the transient phases of CAM (II and IV) in the light period.Abbreviations and Symbols CAM Crassulacean acid metabolism - H⁺ (dawn-dusk) variation in titratable acidity - ¹³C carbonisotope ratio of plant organic material, relative to Pee Dee Belemnite (vs. PDB) - discrimination against ¹³CO₂, - p _i, p _a internal, external partial pressures of CO₂ - Rubisco ribulose1,5-bisphosphate carboxylase - PAR photosynthetically active radiation - PEPCase phosphoenolpyruvate carboxylase We are grateful for financial support in respect of research grants (GR3/5360, GR3/6419) and a studentship awarded by the Natural Environment Research Council, UK.     

Microbes make the meal: oligolectic bees require microbes within their host pollen to thrive

1. For solitary bees that specialise on select pollen types (oligoleges), larval development depends on the availability of forage pollen from appropriate host plants and the naturally occurring microbiota present therein. While access to host pollen may be critical for the development of oligolectic bees, the extent to which pollen microbiota contribute to their brood success is unknown. 2. To investigate, we used a diet manipulation experiment to rear larvae of the oligolege, Osmia ribifloris, under in-vitro conditions. Larvae were reared either on host pollen provisioned by their mother or on non-host pollen collected by honey bees, in the presence or absence of the respective pollen-associated microbiota. We assessed impacts on components of larval fitness: developmental time, biomass, and survivorship. 3. Our results revealed a significant interaction between pollen type and pollen-associated microbes. The relative effect of microbes on larval performance was substantially greater than that of pollen type. Host pollen substrate produced the fittest larvae but only when combined with its full complement of naturally occurring microbiota. In contrast, host pollen without microbes resulted in a marked decline in fitness components. Larvae consuming non-host pollen showed intermediate fitness, regardless of whether microbes were present or not. 4. These findings imply that the microbiota associated with maternally provisioned host pollen perform critical functions in larval nutrition and survival. For oligoleges in particular, the ability to develop on poorer quality host pollen likely derives from this sustained symbiosis with their microbial exosymbionts, rather than the biochemical characteristics of pollen type alone.     

Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage ϕ29 Capsids

The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

Development of the signalling pathways associated with sperm capacitation during epididymal maturation

As spermatozoa mature within the epididymis they acquire the potential for capacitation and ultimately fertilization. In biochemical terms, the former is reflected in the progressive activation of a signal transduction pathway characterized by cAMP-mediated induction of phosphotyrosine expression on the sperm tail. In this study, we have examined the cellular mechanisms controlling this maturational event. Caput epididymal spermatozoa exhibited tyrosine phosphorylation on the sperm head that was largely unresponsive to cAMP and not significantly impaired by removal of extracellular HCO(3) (-). In contrast, caudal epididymal spermatozoa exhibited low levels of phosphorylation on the sperm head, yet responded dramatically to cAMP by phosphorylating a new set of proteins on the sperm tail via mechanisms that were highly dependent on extracellular HCO(3) (-). The impact of extracellular HCO(3) (-) depletion on caudal cells was not associated with a significant change in the redox regulation of cAMP but could be fully reversed by buffering the intracellular pH with N-Tris[Hydroxymethyl]methyl-3-amino-propanesulfonic acid (TAPS). The pattern of tyrosine phosphorylation was also profoundly influenced by the presence or absence of added extracellular calcium. In the presence of this cation, only caudal spermatozoa could respond to increased extracellular cAMP with tyrosine phosphorylation of the sperm tail. However, in calcium-depleted medium, this difference completely disappeared. Under these conditions, caput and caudal spermatozoa were equally competent to exhibit phosphotyrosine expression on the sperm tail in response to cAMP. These results emphasize the pivotal role played by calcium and HCO(3) (-) in modulating the changes in tyrosine phosphorylation observed during epididymal maturation.