Water Relation Alterations Observed during Hypersensitive Reaction Induced by Bacteria

Upon exposure to pathogenic bacteria, resistant and nonhost plants undergo a hypersensitive reaction (HR) that is expressed as rapid plant cell death. If sufficient concentrations of these bacteria are inoculated to such plant tissue, then that portion of the tissue rapidly collapses and becomes necrotic. As the tissue collapses the water relations of inoculated tissues become markedly disturbed. We measured a decline in the relative water content (RWC) in the leaf-like cotyledons of cotton (Gossypium hirsutum cv Immune 216) within the first 4 h (cut at 1 h) after inoculation with Pseudomonas syringae pv tabaci. However, the decrease in RWC was not caused by a decrease in initial fresh weight but by increased water uptake during incubation in water. By 8 h after inoculation, cotyledons still on the plant had lost turgidity, and their area decreased. K+ efflux was also observed concurrently with the decrease in RWC, providing a reason for the loss of turgidity in the tissue. These observations suggest that cells lose turgor and change shape from cylinders with large intercellular spaces to those of a more tabular shape. During this change cell walls come closer together, providing an avenue for increased water uptake through capillary action. The stomatal diffusive resistance of intact cotyledons increased; hence, water loss through stomata is not the cause of the observed wilting and RWC decline. An increase in K+ per dry weight suggests that phloem loading or movement may also be impaired during bacterially induced HR.     

Billingen (Lower Arenig/Lower Ordovician) acritarchs from the East European Platform and their palaeobiogeographic significance

Billingen (Lower Arenig/Lower Ordovician) sediments of the St. Petersburg region, northwest Russia and the Leba area, northern Poland of the East European Craton yield acritarch assemblages, which are largely homogenous though displaying minor compositional differences that probably reflect a gradient from inner to outer shelf environments. Comparison with coeval acritarch microflora from the Yangtze Platform, South China, shows an overall similarity between Baltoscandian and South Chinese phytoplankton. The widespread uniformity in the fossil microphytoplankton may be related to the extensive global 'evae' sea-level transgression, which characterized the Billingen time. This suggests that during the Tremadoc through early Arenig times, acritarch assemblages displayed essentially an undifferentiated cold-water and oceanic character along the whole margin of Perigondwana in the South, as well as on the South Chinese and Baltic platforms, at middle latitudes (Mediterranean oceanic Realm). Despite this overall similarity, however, some typical taxa of the high-latitude Mediterranean Province (Arbusculidium, Coryphidium and Striatotheca) occur in South China, but are absent in Baltica. This discrepancy is explained as caused by differences in climatic and physiographic conditions that prevailed at the two palaeocontinents at this time. The inferred pattern of oceanic circulation during the Lower Ordovician is consistent with the palynological evidence of a prevailing warmer climate in Baltica than in South China, although the two palaeocontinents occupied the same palaeolatitudinal position.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Transmethylation reactions regulate affinity and functional activity of chemotactic factor receptors on macrophages

Methylation mediated by S-adenosyl-l-methionine is required for the chemotaxis of mononuclear leukocytes. We investigated whether transmethylation reactions are required for normal functioning of chemotactic factor receptors. Three chemoattracrant-mediated functions in macrophages, chemotaxis, the stimulated release of arachidonic acid from membrane phospholipids and superoxide production, are markedly depressed by agents that inhibit cellular methylation reactions. Treatment of macrophages with methylation inhibitors decreased the affinity of the N-formylated chemoattractant receptor present on these cells by a factor of 4.5, but did not significantly alter the total receptor number. These results suggest that the N-formylated chemoattractant receptor on macrophages can exist in more than one affinity state and that an ongoing methylation reaction is required for the maintenance of the receptor in its higher affinity form. Inhibition of methylation lowers the affinity of the receptor and renders it no nfunctional or “uncoupled” in its ability to produce chemotaxis, superoxide and the release of arachidonic acid from leukocyte membranes.     

Red light and auxin effects on rubidium uptake by oat coleoptile and pea epicotyl segments

Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [⁸⁶Rb⁺] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10⁻⁷ molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light-enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [⁸⁶Rb⁺] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [⁸⁶Rb⁺] flux is not a general feature of phytochrome action.     

Phytochrome-controlled Hydrogen Ion Excretion by Avena Coleoptiles

A red light-induced, far red reversible stimulation of proton efflux from apical segments of etiolated Avena sativa L. cv. Victory coleoptiles was observed. The acidification responses to red light and also to auxin were not the consequence of respired CO(2). The response to red light was strongly inhibited by cycloheximide and carbonyl cyanide, m-chlorophenyl hydrazone, but mannitol had a stimulatory effect. Red light and auxin applied together yielded a greater than additive response, in comparison to the effects of the two stimuli applied separately.     

Vitamin D receptor interaction with specific DNA. Association as a 1,25-dihydroxyvitamin D3-modulated heterodimer.

The vitamin D receptor (VDR) is a member of the steroid receptor gene family. In this report, we examine the nature of specific VDR DNA binding utilizing the vitamin D-responsive element derived from the human osteocalcin promoter. Association of the VDR with the human osteocalcin 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) responsive element (VDRE) in vitro was characterized on VDRE affinity columns by both weak and strong interactions. Weak interaction was a property of the VDR itself, monomeric in nature, and determined exclusively by the VDR's DNA-binding domain. Strong interaction, in contrast, was dependent upon an intact receptor molecule as well as a heterologous mammalian cell nuclear accessory factor (NAF). Heteromeric interaction between VDR and NAF was independent of the VDR DNA-binding domain, suggesting the presence of a functional dimerization domain separate from that for DNA binding. Direct association of NAF with immobilized VDR revealed that the interaction does not require the presence of DNA. Most importantly, while occupancy of the VDR by 1,25(OH)2D3 was not required for VDR interactions with either DNA or NAF, the presence of hormone increased the apparent relative affinity of the VDR for NAF approximately 10-fold. These studies suggest that high affinity association of the VDR with DNA requires both the DNA-binding domain as well as an additional independent structure located within the steroid-binding region. This protein subdomain interacts with NAF and is regulated by 1,25(OH)2D3.     

Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7°C and −3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20°C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7°C or −3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.

A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2°C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29°C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7°C (or −3°C) its completion. Because of the present results, however, this interpretation needs to be modified.

Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.

     

Membrane phospholipid phase separations in plants adapted to or acclimated to different thermal regimes

The phase separation temperatures of total leaf phospholipids from warm and cool climate plants were determined in order to explore the relationship of lipid physical properties to a species' thermal habitat. The separation temperatures were determined by measuring the fluorescence intensity and fluorescence polarization of liposomes labeled with the polyene fatty acid probe trans-parinaric acid. To focus on a single climatic region, Mojave Desert dicots (chiefly ephemeral annuals) were examined, with plants grown under identical conditions whenever possible. Winter active species showed lower phase separation temperatures than the summer active species. A group of warm climate annual grasses showed separation temperatures distinctly higher than those of a group of cool climate grasses, all grown from seed under the same conditions. Growth at low temperature seems correlated with (and may require) a low phase separation temperature. Winter active ephemerals appear genetically programmed to synthesize a mixture of phospholipids which will not phase separate in the usual growth conditions. When the lipids of desert perennials were examined in cool and warm seasons, there was a pronounced seasonal shift in the phase separation temperature, implying environmental influences on lipid physical properties. The relationship of these results to high and low temperature tolerance is discussed.