Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway. Received: 15 August 1996 / Accepted: 23 October 1996     

Oxygen consumption in the foraging honeybee depends on the reward rate at the food source

Oxygen consumption of the honeybee Apis mellifera ligustica was measured as a function of the flow rate supply of sucrose solution at an automatic feeder located inside a respirometric chamber. Trained bees freely entered the respirometric chamber and collected the sucrose solution supplied. The mean value of the O₂ consumption rate per visit increased with the sucrose flow rate, and for a given flow rate, with increasing locomotor activity. However, when no locomotor activity was displayed, O₂ consumption also increased with increasing nectar flow rate. Crop load attained at the end of the visit showed a positive relationship with the nectar flow rate; however, for a given flow rate, O₂ consumption showed either no correlation or a negative one with the final crop load attained. It is concluded that the energy expenditure of the foraging bee is controlled by a motivational drive whose intensity depends on the reward rate at the food source. Accepted: 30 July 1996     

Neural correlates of habituation of the proleg withdrawal reflex in larvae of the hawk moth, Manduca sexta

The larval proleg withdrawal reflex of the hawk moth, Manduca sexta, exhibits robust habituation. This reflex is evoked by deflecting one or more mechanosensory planta hairs on a proleg tip. We examined neural correlates of habituation in an isolated proleg preparation consisting of one proleg and its segmental ganglion. Repeated deflection of a single planta hair caused a significant decrease in the number of action potentials evoked in the proleg motor nerve (which carries the axons of proleg retractor motor neurons). Significant response decrement was seen for interstimulus intervals of 10 s, 60 s and 5 min. Response decrement failed to occur in the absence of repetitive stimulation, the decremented response recovered spontaneously following a rest, and electrical stimulation of a body wall nerve facilitated the decremented response (a neural correlate of dishabituation). Adaptation of sensory neuron responses occurred during repeated hair deflections. However, when adaptation was eliminated by direct electrical stimulation of sensory neurons, the response in the proleg motor nerve still decreased significantly. Muscle recordings indicated that the response of an identified proleg retractor motor neuron decreased significantly during habituation training. Thus, habituation of the proleg withdrawal reflex includes a central component that is apparent at the level of a single motor neuron. Accepted: 20 December 1996     

A comparison of the response to induced exercise in red- and white-blooded Antarctic fishes

The physiological responses to forced exercise were studied in yellowbelly and marbled rockcod (Notothenia coriiceps and N. rossii), and the haemoglobinless icefish (Chaenocephalus aceratus), from blood samples obtained via indwelling catheters. The maximal exertion tolerable by N. coriiceps was 3–5 min, although N. rossii was not fully exhausted by this effort, and it proved difficult to elicit sustained maximal activity in C. aceratus. Arterial O₂ tension reflected the relative degree of exhaustion, showing a significant fall in the case of N. coriiceps, little change in N. rossii, and even a rise in C. aceratus as a result of hyperventilation. Such changes in the red-blooded species were not caused by altered O₂ carrying capacity, as there was no change in haematocrit. In Notothenia spp. the decrease in arterial pH was better correlated with a rise in arterial CO₂ tension than with blood lactate concentration, which is reflected in a modest net metabolic acid load. In contrast, the icefish showed an attenuated hypercapnia and a more pronounced lactacidosis, but an insignificant net metabolic acid load. Disturbance in ionoregulation following exercise was limited to an elevated [Cl⁻] in Notothenia, while circulating catecholamine levels remained unusually low in all specimens. The response to stress appears to reflect lifestyle and/or endemic speciation, rather than specific adaptations to the stenothermal environment. Accepted: 9 August 1996     

Adaptive reconfiguration of a reflex circuit during different motor programmes in the locust

The cuticle strain which develops in the hindleg tibiae when a locust prepares to kick, or when the tibia thrusts against an obstacle, is detected by two campaniform sensilla, which reflexly excite the fast extensor tibiae motoneuron, some of the flexor tibiae motoneurons and nonspiking interneurons. The reflex excitation is adaptive for the extensor motoneuron during both co-activation and thrusting, but is only adaptive for the flexor motoneurons during co-activation, and is maladaptive during thrusting. We show that the femoral chordotonal organ, which monitors tibial position, controls the efficacy of the strain feedback. The campaniform sensilla-induced depolarization in the extensor motoneuron is about twice as large when the tendon is in mid position (reflecting a tibial-femoral angle of 90°) than when fully stretched (reflecting tibial flexion), while in the flexors the reverse is true. The amplitudes of excitatory postsynaptic potentials evoked by single campaniform sensilla spikes, are, however, not affected. Our data suggests that the chordotonal organ modulates the gain of the strain feedback onto the motoneurons by exciting interneuronal circuits whose output sums with the former. Thrusting typically occurs with the tibia partially extended, therefore the actions of the chordotonal organ support the production of a maximal thrusting force. Accepted: 27 December 1996     

Physiological characterization of antennular flicking reflexes in the crayfish

The cellular substrates of antennular flicking behavior in the crayfish Procambarus clarkii were investigated. Flicking involves fast downward movements of the external filament of each biramous antennule (1st antenna), and is mediated by phasic contractions of a short muscle, the external filament depressor. Phasic contractions of the external filament depressor depend upon stereotyped impulse bursts in a single motorneuron (P1). These bursts have a characteristic impulse frequency profile that is consistent upon successive occurrences. The temporal characteristics of the impulse burst suggest that the central depolarizations generating each burst may be similar to driver potentials described for motor neurons in crustacean cardiac ganglia. Responses of the external filament to odorants have a long latency and are characterized by repetitive bursts and tonic activity in some external filament depressor fibers. Tonic activity in a slowly contracting muscle, the antennular depressor muscle, is also evoked by chemical stimulation. Flicking is consistently evoked only by mechanical or hydrodynamic stimulation of the cephalothorax, antennae and antennules. The sensitivity and short latency of the hydrodynamic antennule-generated flick reflex is consistent with the sensitivity of rapidly conducting, hydrodynamically activated mechanoreceptor neurons in both antennular filaments. I propose that antennular flicking, which has been shown to enhance the dynamic response characteristics of olfactory receptor neurons on the external antennular filament, has evolved as a response to the turbulence associated with fluid movement, within which chaotic odorant concentration fronts may be imbedded. Accepted: 23 October 1996     

Sexual dimorphism and species differences in the neurophysiology and morphology of the acoustic communication system of two neotropical hylids

We examined auditory tuning and the morphology of the anatomical structures underlying acoustic communication in female Hyla microcephala and H. ebraccata and compared our findings to data from a previous study (Wilczynski et al. 1993) in which we showed species differences in the traits that in males relate to differences in the species-typical calls. Female species differences in the best excitatory frequency (BEF) of the basilar papilla (BP) were similar to the differences seen in males, and females had a significantly lower BEF in H. ebraccata, but not H. microcephala. In both species, females had lower BP thresholds. Snout-vent length, head width, and tympanic membrane diameters were sexually dimorphic in both species and larger in females, whereas laryngeal components were sexually dimorphic and larger in males. Middle and inner ear volumes were not sexually dimorphic. Despite the significant species differences in laryngeal morphology seen in males, female larynges are not significantly different. Furthermore, the interaction of species and sex differences resulted in significantly different degrees of sex dimorphism in the species, particularly for the larynx, which is more sexually dimorphic in H. microcephala, and measures of body size, which are more dimorphic in H. ebraccata. Accepted: 6 December 1996     

Multiple excitatory receptor types on individual olfactory neurons: implications for coding of mixtures in the spiny lobster

The aim of our paper was to investigate whether single olfactory receptor neurons (ORNs) of the spiny lobster Panulirus argus functionally express more than one type of receptor, examine the consequences of this on coding of mixtures, and compare principles of odorant mixture coding by spiny lobsters with that by the channel catfish, which has been studied extensively using the same experimental and analytical procedures (Caprio et al. 1989; Kang and Caprio 1991). We examined responses of individual taurine-sensitive ORNs to binary mixtures of excitatory compounds, either competitive agonists (taurine, β-alanine, hypotaurine) or non-competitive agonists (taurine, l-glutamate, ammonium chloride, adenosine-5′-monophosphate). Responses to mixtures were compared to two indices: mixture discrimination index (MDI) and independent component index (ICI). Binary mixtures of competitive agonists had MDI values close to 1.0, as expected for competitors. Mixtures of non-competitive agonists had ICI values averaging 0.83, indicating the effects of the components are not independent. We conclude that individual olfactory cells of spiny lobsters can express more than one type of receptor mediating excitation, one of which typically has a much higher density or affinity, and that spiny lobster and catfish olfactory cells encode mixtures of two excitatory agonists using similar rules. Accepted: 20 December 1996     

Responses to object approach by a wide field visual neurone, the LGMD2 of the locust: Characterization and image cues

The LGMD2 belongs to a group of giant movement-detecting neurones which have fan-shaped arbors in the lobula of the locust optic lobe and respond to movements of objects. One of these neurones, the LGMD1, has been shown to respond directionally to movements of objects in depth, generating vigorous, maintained spike discharges during object approach. Here we compare the responses of the LGMD2 neurone with those of the LGMD1 to simulated movements of objects in depth and examine different image cues which could allow the LGMD2 to distinguish approaching from receding objects. In the absence of stimulation, the LGMD2 has a resting discharge of 10–40 spikes s⁻¹ compared with <1 spike s⁻¹ for the LGMD1. The most powerful excitatory stimulus for the LGMD2 is a dark object approaching the eye. Responses to approaching objects are suppressed by wide field movements of the background. Unlike the LGMD1, the LGMD2 is not excited by the approach of light objects; it specifically responds to movement of edges in the light to dark direction. Both neurones rely on the same monocular image cues to distinguish approaching from receding objects: an increase in the velocity with which edges of images travel over the eye; and an increase in the extent of edges in the image during approach. Accepted: 23 October 1996     

Rhythmic components of photoperiodic time measurement in the pitcher-plant mosquito, Wyeomyia smithii

Photoperiodic time measurement regulating larval diapause in the pitcher-plant mosquito, Wyeomyia smithii, varies in a close relationship with latitude. The critical photoperiod mediating the maintenance and termination of diapause is positively correlated with latitude (r ² = 0.977) among six populations from southern (30–31° N), intermediate (40° N), and northern (46–49° N) latitudes in North America. The developmental response to unnaturally short and to unnaturally long photoperiods declines with increasing latitude, so that longer critical photoperiods are associated with a downward rather than a lateral shift in the photoperiodic response curve. Exotic light and dark cycles of varying period (T) with a short (10 h) photophase and a scotophase ranging from 14 (T = 24) to 62 (T = 72) h, reveal two geographic patterns: a decline in perturbability of the photoperiodic clock with increasing latitude, and no change with latitude in the 21-h period of rising and falling development with increasing T. These results show (1) that there is a rhythmic component to photoperiodic time measurement in W. smithii, (2) that the period of this rhythm is about 21 h in all populations, and (3) that more northern populations show decreasing responsiveness to photoperiod and increasing stability against perturbation by exotic period lengths (T > 24). Previous studies on W.␣smithii indicate that this single temperate species of a tropical and subtropical genus has evolved from south to north. We therefore conclude that the evolution of increasing critical photoperiod in W. smithii during its adaptive radiation into North America has more likely involved the amplitude and not the period of the underlying circadian pacemaker. Received: 22 July 1996 / Accepted: 30 September 1996     

Post-dispersal seed predation and the establishment of vertebrate dispersed plants in Mediterranean scrublands

The post-dispersal fate of seeds and fruit (diaspores) of three vertebrate-dispersed trees, Crataegus monogyna, Prunus mahaleb and Taxus baccata, was studied in the Andalusian highlands, south-eastern Spain. Exclosures were used to quantify separately the impact of vertebrates and invertebrates on seed removal in relation to diaspore density and microhabitat. The three plant species showed marked differences in the percentage of diaspores removed, ranging from only 5% for C. monogyna to 87% for T. baccata. Although chaffinches (Fringilla coelebs) fed on diaspores, rodents (Apodemus sylvaticus) were the main vertebrate removers of seed and fruit. Two species of ant (Cataglyphis velox and Aphaenogaster iberica) were the only invertebrates observed to remove diaspores. However, the impact of ants was strongly seasonal and they only removed P. mahaleb fruit to any significant extent. While removal of seed by rodents was equivalent to predation, ants were responsible for secondary dispersal. However, their role was limited to infrequent, small-scale redistribution of fruit in the vicinity of parent trees. Rodents and ants differed in their use of different microhabitats. Rodents foraged mostly beneath trees and low shrubs and avoided open areas while the reverse was true of ants. Thus, patterns of post-dispersal seed removal will be contigent on the relative abundance and distribution of ants and rodents. Studies which neglect to quantify separately the impacts of these two guilds of seed removers may fail to elucidate the mechanisms underlying patterns of post-dispersal seed removal. The coincidence of both increased seed deposition by the main avian dispersers (Turdus spp.) and increased seed predation with increasing vegetation height suggested that selection pressures other than post-dispersal seed predation shape the spatial pattern of seed dispersal. Rather than providing a means of escaping post-dispersal seed predators, dispersal appears to direct seeds to microhabitats most suitable for seedling survival. Nevertheless, the reliance of most vertebrate-dispersed trees on regeneration by seed and the absence of persistent soil seed banks imply that post-dispersal seed predators may exert a strong influence on the demography of the plants whose seeds they consume. Even where microsites are limited, the coincidence of the most suitable microhabitats for seedling establishment with those where seed predation is highest provide a means by which selective seed predators can influence community composition. Received: 19 August 1996 / Accepted: 25 January 1997     

c-fos expression in the putative avian suprachiasmatic nucleus

c-fos induction was investigated as a potential component in the avian photic entrainment pathway and as a possible means of locating the central pacemaker in birds. In both quail (Coturnix coturnix japonica) and starlings (Sturnus vulgaris) exposure to 1 h of light induced Fos-lir in the visual suprachiasmatic nucleus but not in the medial suprachiasmatic nucleus. However, the degree of c-fos induction in the visual suprachiasmatic nucleus was similar at different circadian times despite the fact that the light pulses caused differential phase shifts in the locomotor rhythm. For golden hamsters the same experiment resulted in significantly different levels of Fos-lir in the suprachiasmatic nucleus, as well as different phase shifts. Starlings and hamsters were also entrained to T-cycles that caused a large daily phase shift (T = 21.5 h in starlings, T = 22.67 hours in hamsters), or no daily phase shift (T = free running period). No difference in the induced levels of Fos-lir in the visual suprachiasmatic nucleus region was observed between the two groups of starlings, but in hamsters there were significantly different levels of Fos-lir in the suprachiasmatic nucleus between the two groups. Accepted: 15 November 1996     

Application of a 15N tracer to simulate and track the fate of atmospherically deposited N in the coastal forests of the Waquoit Bay Watershed, Cape Cod, Massachusetts

The central grassland region of the United States encompasses major gradients in temperature and precipitation that determine the distribution of plant life forms, which in turn may influence key ecosystem processes such as nutrient cycling and soil organic matter dynamics. One such gradient is the threefold increase in precipitation from the eastern Colorado shortgrass-steppe, in the rain shadow of the Rocky Mountains, to the tallgrass prairie in eastern Kansas. We investigated the relative roles of plant species and plant cover in influencing soil C and N cycling in three sites along this gradient. Plant cover (i.e., the presence or absence of an individual plant) was relatively more important than plant species in explaining variability in soil properties at the dry site, the Central Plains Experimental Range in␣northeastern Colorado. However, plant species explained relatively more of the variability in soil properties than did plant cover at the two wetter sites, Hays and Konza, in central and eastern Kansas. The wetter sites had more continuous plant cover, resulting in less plant-cover-induced variation in soil C and N, than did the dry site, which had distinct patches of bare ground. Plant species at the wetter sites had higher and more variable levels of tissue C:N than plant species at the dry site, due to both within species changes and changes in species composition. Aboveground tissue C:N was better correlated with net nitrogen mineralization rates at the wet sites than the dry site. Thus, tissue chemistry appears to exert more control on nitrogen dynamics at the wet than the dry sites. The results suggest that plant species traits that are relevant to nutrient cycling (e.g., tissue C:N ratios, spatial patterns, productivity) reflect environmental limitations as well as species' physiological potentials. Furthermore, a dominant environmental driver such as precipitation may ameliorate or exaggerate the importance of individual species traits for nutrient cycling. Received: 11 July 1996 / Accepted: 5 December 1996     

Invasive capacity of Tamarix ramosissima in a Mojave Desert floodplain: the role of drought

Tamarix ramosissima (Tamaricaceae) is a woody phreatophyte that has invaded thousands of hectares of floodplain habitat in the southwestern U.S. In this study, we examined the response of gas exchange and stem sap flow of Tamarix and three co-occurring native phreatophytes (Pluchea sericea (Asteraceae), Prosopis pubescens (Fabaceae) and Salix exigua (Salicaceae)) to drought conditions in an early successional floodplain community in the Mojave Desert of southern Nevada. In an analysis of a size/age series of each species across the whole floodplain (both mature and successional stands), stem growth rate was lowest for Tamarix. However, along the same successional chronosequence, Tamarix came to dominate the 50+ year old stands with dense thickets of high stem density. Xylem sap flow, when expressed on a sapwood area basis, was highest in Tamarix under early drought conditions, but comparable between the four species toward the end of the summer dry season. Multivariate analysis of the gas exchange data indicated that the four species differentiated based on water use under early drought conditions and separated based on plant water potential and leaf temperature (indices of drought effects) at the end of the summer dry season. This analysis suggests that the invasive Tamarix is the most drought tolerant of the four species, whereas Salix transpires the most water per unit leaf surface area and is the least tolerant of seasonal water stress. Therefore, Salix appears to be well adapted to early successional communities. However, as floodplains in this arid region become more desiccated with age, Tamarix assumes greater dominance due to its superior drought tolerance relative to native phreatophytes and its ability to produce high density stands and high leaf area. Received: 8 August 1996 / Accepted: 29 January 1997     

Multisegmental motor activity in the segmentally restricted gin trap behavior in Manduca sexta pupae

Stimulation of sensory neurons innervating hairs in the gin traps on the abdomen of Manduca sexta pupae evokes a rapid bending of the abdomen that is restricted to one or more of the three articulating posterior segments. However, electrical stimulation of the gin trap sensory nerve in an isolated abdominal nerve cord evokes characteristic motor neuron activity in every abdominal segment. To determine if the segmentally distributed motor activity also occurred in intact animals and how it contributed to the segmentally restricted reflex movement, mechanical stimulation of the sensory hairs in intact animals was used to evoke reflex responses that were recorded as electromyograms synchronized with video recordings of the behavior. Motor activity was monitored during movements to determine if there was activity in many segments when the movement was restricted to one segment. Coordinated muscle activity was evoked throughout the abdomen in response to stimulation of any of the three gin traps, even when movement was restricted to one segment. Differences in the timing of ipsilateral and contralateral motor activity among segments allowed the closing of gin traps to be segmentally restricted. These findings suggest that the neural circuit underlying the gin trap reflex is distributed throughout the abdominal nerve cord. This network generates a complex, yet coordinated, motor pattern with muscular activity in many abdominal segments that produces a localized bending reflex. Accepted: 10 January 1997     

The role of carbohydrates in the induction of flowering in Arabidopsis thaliana: comparison between the wild type and a starchless mutant

In order to test whether an increased export of carbohydrates by leaves and starch mobilization are critical for floral transition in Arabidopsis thaliana, the Columbia ecotype as well as its starchless mutant pgm and starch-in-excess mutant sex1 were investigated. Induction of flowering was achieved by exposure of plants to either one long day (LD) or one displaced short day (DSD). The following conclusions were drawn: (i) Both the pgm and sex1 mutants have a late-flowering phenotype in days shorter than 16 h. (ii) When inductive treatments cause a large percentage of induced plants, there is always a large, early and transient increase in carbohydrate export from leaves. By contrast, when an inductive treatment results in only a low percentage of induced plants (pgm plants exposed to one DSD), the export of carbohydrates from leaves is not increased, supporting the idea that phloem carbohydrates have a critical function in floral transition. (iii) Starch mobilization is not required to obtain an increased carbohydrate export when induction is by one LD (extended period of photosynthesis), but is absolutely essential when induction is by one DSD (period of photosynthesis unaffected). (iv) Floral induction apparently increases the capability of the leaf phloem-loading system. Received: 27 August 1997 / Accepted: 6 March 1998     

Alignment of the genetic and physical maps in the dpy-5 bli-4 (I) region of C. elegans by the serial cosmid rescue of lethal mutations

Lethal mutations in the 0.5 map unit region between dpy-5 and bli-4 on chromosome I in Caenorhabditis elegans were serially rescued using cosmid-containing transgenic strains. All the lethal mutations analyzed came from a set of 495 EMS-induced, sDp2-rescued lethals described previously. Germline transformation with cosmid DNA was used to create 25 transgenic strains bearing heritable extrachromosomal arrays. These arrays were used as small duplications for the fine-scale mapping of essential genes, via the rescue of lethal mutations. Lethal mutations in 13 essential genes have been phenotypically rescued, allowing the alignment of the genetic and physical maps in this region. Extrachromosomal arrays were found to be transmitted 2- to 7-fold less frequently in oocytes than in hermaphrodite sperm for 12 of the 16 arrays that were examined. Three of these strains showed a subsequent 4- to 13-fold increase in array stability in oocytes. This phenomenon may be influenced by cosmid sequences. Early mitotic loss of the arrays was observed in all 17 transgenic strains examined, suggesting that loss of the array can occur at any time during development when cell divisions are occurring. As a result of this work, 13 of the essential loci positioned between dpy-5 and bli-4 are anchored to the physical map, thereby providing links between the physical and genetic maps on average every 85 kb. Received: 8 May 1996 / Accepted: 27 January 1997     

Highly independent olfactory receptor sites for naturally occurring bile acids in the sea lamprey, Petromyzon marinus

(1) Electro-olfactogram recording was used to determine whether the olfactory epithelium of adult sea lamprey is specifically sensitive to bile acids, some of which have been hypothesized to function as pheromones. Ten bile acids were selected from 38 which had already been pre-screened for olfactory activity. These compounds were first tested on their own, then as adapting stimuli, and finally as components of mixtures (2) The lamprey-specific bile acids, petromyzonol sulfate and allocholic acid, were the most potent compounds tested. Five other bile acids were also detectable at picomolar concentrations. Petromyzonol sulfate had a distinctive dose-response curve. (3) Cross-adaptation demonstrated that sensitivity to bile acids is attributable to at least four independent classes of olfactory receptor sites and that both the nature and position of conjugating group(s) are critical to receptor specificity. Notably, petromyzonol sulfate has its own highly specific and independent receptor site. The situation for unconjugated bile acids was more complex and there appeared to be several sub-classes of receptor sites for these compounds. (4) Mixture studies largely confirmed the cross-adaptation results, describing receptor site independence for the same four sets of odorants. Mixture enhancement was also seen when expected and there was no evidence of mixture suppression. (5) Together, these data demonstrate that conspecific bile acids are discriminated by the olfactory epithelium of the sea lamprey, supporting the possibility that these compounds may function as migratory pheromones. Accepted: 23 November 1996     

Rhythmicity of torpor in a marsupial hibernator, the mountain pygmy-possum (Burramysparvus), under natural and laboratory conditions

Circadian rhythms have been observed in most mammals, but their importance and function remain controversial with respect to daily cycles during hibernation. We investigated the timing of arousals from and entries into hibernation for both free-living and captive mountain pygmy-possums (Burramys parvus). Under both natural and laboratory conditions most arousals and entries were entrained with the light-dark cycle. Entries occurred mainly during the night and arousals preferably around dusk, which coincides with the onset of the normal activity phase for the nocturnal pygmy-possums. This entrainment prevailed throughout the hibernation season although only the laboratory animals were constantly subjected to photoperiodic stimuli, whereas under natural conditions hibernacula are shielded from photic cues and diurnal temperature fluctuations. Nevertheless, possums left their hibernacula frequently throughout winter and were occasionally trapped close to the snow surface suggesting that during the periods of post-arousal normothermia they can be exposed to environmental stimuli. It thus appears that the synchronisation with the photocycle was governed by a temperature-compensated circadian clock which was reset periodically during short activity periods. For the mountain pygmy-possum, entrainment with the photocycle probably has two functions: 1. Entrainment ensures that foraging bouts during the hibernation season remain synchronised with the dark phase. 2. Information about the prevailing climatic conditions sampled during short activity periods enables them to time final spring emergence from hibernation when snow melt begins and ensures that the breeding season can commence as early as possible. Accepted: 26 August 1998     

Molecular evolution and functional relevance of the chalcone synthase genes of pea

We have isolated seven genomic chalcone synthase (CHS) genes and six classes of CHS cDNA from elicitor-treated pea tissues. Comparison of the nucleotide sequences of the coding regions revealed the existence of eight members of the CHS gene family in pea. These can essentially be divided into three groups (PSCHS1, 2 and 8; PSCHS3, 4 and 5; and PSCHS6 and 7) on the basis of nucleotide and/or amino acid sequence comparisons of the coding regions, introns and promoter regions. We previously reported that the accumulation of CHS mRNAs is induced by elicitor treatment. Accumulation of CHS mRNA was observed mainly in roots and very little was found in floral organs. To specifically detect expression of each CHS gene in various types of pea cells, S1 nuclease protection assays were performed. Interestingly, the classification of the eight members of the CHS gene family based on the sequence identity was found to reflect their expression patterns as determined by the S1 nuclease protection assay. The first group of CHS genes, PSCHS1, 2 and 8, was strongly induced not only by elicitor treatment and UV irradiation but is also constitutively expressed in root and flower tissues. The second group, PSCHS3, 4 and 5, was also strongly induced by elicitor treatment and UV irradiation but is constitutively expressed only in root. Expression of the third group, PSCHS6 and 7 was barely detectable in any of the organs tested and was not influenced by environmental stimuli such as elicitor or UV. Furthermore, sequence analysis of the promoter region of each member of the CHS gene family revealed that putative cis-regulatory elements, such as Box-I, Box-II and G-Box, were conserved only in PSCHS1, 2, 3, 4 and 5. From these results we propose that an ancestral CHS gene might have given rise to defense response-related (UV irradiation- and elicitor-responsive) and -unrelated (unresponsive) genes at an early stage of evolution, followed by divergence within these subclasses based upon the developmental program in pea. Received: 5 November 1996 / Accepted: 6 February 1997