The role of calcium in ABA-induced gene expression and stomatal movements

There is much interest in the transduction pathways by which abscisic acid (ABA) regulates stomatal movements (ABA-turgor signalling) and by which this phytohormone regulates the pattern of gene expression in plant cells (ABA-nuclear signalling). A number of second messengers have been identified in both the ABA-turgor and ABA-nuclear signalling pathways. A major challenge is to understand the architecture of ABA-signalling pathways and to determine how the ABA signal is coupled to the appropriate response. We have investigated whether separate Ca2+-dependent and -independent ABA-signalling pathways are present in guard cells. Our data suggest that increases in [Ca2+]i are a common component of the guard cell ABA-turgor and ABA-nuclear signalling pathways. The effects of Ca2+ antagonists on ABA-induced stomatal closure and the ABA-responsive CDeT6-19 gene promoter suggest that Ca2+ is involved in both ABA-turgor signalling and ABA-nuclear signalling in guard cells. However, the sensitivity of these pathways to alterations in the external calcium concentration differ, suggesting that the ABA-nuclear and ABA-turgor signalling pathways are not completely convergent. Our data suggest that whilst Ca2+-independent signalling elements are present in the guard cell, they do not form a completely separate Ca2+-independent ABA-signalling pathway.     

Photoreceptors in species of the Macrostomida (Plathelminthes): ultrastructural findings and phylogenetic implications

The submicroscopic anatomy of intracerebral and pericerebral photoreceptors in six species of the Macrostomida is described. Cylindromacrostomum notan-dum, Paramyozonaria simplex and Macrostomum hystricinum marinum possess two rhabdomeric intracerebral photoreceptors each consisting of two pigmented cup cells and three (C. notandum and P. simplex) or two sensory cells (M. hystricinum marinum). In C. notandum and P. simplex two of the sensory cells are equal in size, while the third one is much smaller. This organisation is hypothesised as an autapomorphy of the Dolichomacrostomidae. Photoreceptors with two mantle cells are also known for Microstomum spiculifer. Since only one cup cell exists in representatives of nearly all other high-ranked taxa of the Rhabditophora, it is concluded that the characteristic ”two cup cells in rhabdomeric photoreceptors” has evolved in the stem lineage of the taxon Macrostomida or Macrostomorpha, respectively. In Myozona purpurea and Psammomacrostomum turbanelloides rhabdomeric intracerebral photoreceptors of a special type were encountered. These light-sensing organs consist of numerous cells forming an ellipsoid. The surface membranes of these cells are elongated to form filiform extensions which are tightly intertwined with each other. Pericerebral ciliary aggregations consisting of cells with an internal cavity into which axonemata of modified cilia project were observed in all species mentioned above and in Bradynectes sterreri as well. Such putative light-perceiving organs are widespread within taxa of the Plathelminthes Rhabditophora and have been hypothesised either as homologous characteristics or as analogous ones. With increasing examples being described it becomes likely that pericerebral ciliary aggregations are an apomorphic ground pattern characteristic of the Rhabditophora. Accepted: 22 January 2001     

CAC3(MSI1) suppression of RAS2(G19V) is independent of chromatin assembly factor I and mediated by NPR1

Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA. CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism. We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function. CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function. Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm. In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway. Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway. Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p. Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p. Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway. Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.     

An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression

The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12–14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.     

Increased contractility and altered Ca(2+) transients of mouse heart myocytes conditionally expressing PKCbeta

Activation of protein kinase C (PKC) in heart muscle signals hypertrophy and may also directly affect contractile function. We tested this idea using a transgenic (TG) mouse model in which conditionally expressed PKCbeta was turned on at 10 wk of age and remained on for either 6 or 10 mo. Compared with controls, TG cardiac myocytes demonstrated an increase in the peak amplitude of the Ca(2+) transient, an increase in the extent and rate of shortening, and an increase in the rate of relengthening at both 6 and 10 mo of age. Phospholamban phosphorylation and Ca(2+)-uptake rates of sarcoplasmic reticulum vesicles were the same in TG and control heart preparations. At 10 mo, TG skinned fiber bundles demonstrated the same sensitivity to Ca(2+) as controls, but maximum tension was depressed and there was increased myofilament protein phosphorylation. Our results differ from studies in which PKCbeta was constitutively overexpressed in the heart and in studies that reported a depression of myocyte contraction with no change in the Ca(2+) transient.     

Sensory deficits induced by cadmium in banded kokopu, Galaxias fasciatus, juveniles

The potential for cadmium to produce sensory deficits in the mechanosensory lateral line and olfactory systems was examined in migratory Galaxias fasciatus juveniles or whitebait. Using a two-choice chamber apparatus, three groups of whitebait were tested for a known attraction to adult pheromones and then exposed to either 0.1, 0.5 or 1g Cd⁺² l^-1 for 48h and retested. The attraction to adult pheromones had been eliminated after exposure to concentrations of 0.5 and 1g Cd⁺² l^-1, indicating these levels of cadmium exposure had impaired olfactory function. Rheotaxis trials were conducted to determine the level of cadmium exposure which would inhibit lateral line function. The lateral line system was not blocked until a concentration of 2g Cd⁺² l^-1. The blocking of the lateral line and olfactory sensory systems was reversible. After 14 days recovery in clean freshwater both rheotaxis and the attraction to adult pheromones had returned. Whitebait were also tested for a preference/avoidance response at 2g Cd⁺² l^-1. Fish showed neither a preference for, or an avoidance of, a concentration which would disable both the lateral line and olfactory sensory systems. The disabling of these sensory systems may render migratory cues undetectable, affecting habitat selection by whitebait, which may ultimately affect the distribution of banded kokopu populations.     

Fate map of early avian cardiac progenitor cells

Cardiogenic fate maps are used to address questions on commitment, differentiation, morphogenesis and organogenesis of the heart. Recently, the accuracy of classical cardiogenic fate maps has been questioned, raising concerns about the conclusions drawn in studies based on these maps. We present accurate fate maps of the heart-forming region (HFR) in avian embryos and show that the putative cardiogenic molecular markers Bmp2 and Nkx2.5 do not govern the boundaries of the HFR as suggested in the literature. Moreover, this paper presents the first fate map of the HFR at stage 4 and addresses a void in the literature concerning rostrocaudal patterning of heart cells between stages 4 and 8.     

Biliverdin reductase-induced phytochrome chromophore deficiency in transgenic tobacco

Targeted expression of mammalian biliverdin IXalpha reductase (BVR), an enzyme that metabolically inactivates linear tetrapyrrole precursors of the phytochrome chromophore, was used to examine the physiological functions of phytochromes in the qualitative short-day tobacco (Nicotiana tabacum cv Maryland Mammoth) plant. Comparative phenotypic and photobiological analyses of plastid- and cytosol-targeted BVR lines showed that multiple phytochrome-regulated processes, such as hypocotyl and internode elongation, anthocyanin synthesis, and photoperiodic regulation of flowering, were altered in all lines examined. The phytochrome-mediated processes of carotenoid and chlorophyll accumulation were strongly impaired in plastid-targeted lines, but were relatively unaffected in cytosol-targeted lines. Under certain growth conditions, plastid-targeted BVR expression was found to nearly abolish the qualitative inhibition of flowering by long-day photoperiods. The distinct phenotypes of the plastid-targeted BVR lines implicate a regulatory role for bilins in plastid development or, alternatively, reflect the consequence of altered tetrapyrrole metabolism in plastids due to bilin depletion.     

Local striatal infusion of MPP+ does not result in increased hydroxylation after systemic administration of 4-hydroxybenzoate

In vivo bilateral microdialysis in the rat striatum was used to investigate hydroxyl radical formation under basal conditions and after intrastriatal administration of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). After a short equilibration period, 4-hydroxybenzoate (4HBZ), which scavenges hydroxyl radicals to produce 3,4-dihydroxybenzoate (34DHB), was injected intraperitoneally 15 min before infusion of MPP+. To evaluate the enzymatic contribution to hydroxyl radical formation, two other series of microdialyses were performed following administration of monoamine oxidase B inhibitors, either 1-deprenyl (selegiline) or MDL 72,974A [(E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride]. Microdialysate samples were analyzed by high-performance liquid chromatography for catecholamines, 3,4-dihydroxyphenylacetate (DOPAC), homovanillate (HVA), along with the hydroxyl radical adduct, 34DHB and its precursor, 4HBZ. MPP+ administration resulted in a massive release of dopamine along with a decrease in DOPAC and HVA in all three groups. A striking effect in all three groups was noted in which MPP+ resulted in a decrease in interstitial 4HBZ to < 50% of the non-MPP+ -treated side. In absolute terms, the amount of 34DHB produced was low but similar in all three groups, even after unilateral MPP+ infusion. When 34DHB was normalized to 4HBZ release to account for differences in precursor availability, there were no significant differences in the 34DHB/4HBZ ratios either with or without MAO inhibitor treatment or after local MPP+ infusion. Systemic 4HBZ administration appears to result predominantly in intra-cellular sampling of hydroxyl radicals which produces different results from local infusion of trapping agents such as salicylate.