Divergence of annual and perennial species in the Brassicaceae and the contribution of cis-acting variation at FLC orthologues.

Variation in life history contributes to reproductive success in different environments. Divergence of annual and perennial angiosperm species is an extreme example that has occurred frequently. Perennials survive for several years and restrict the duration of reproduction by cycling between vegetative growth and flowering, whereas annuals live for 1 year and flower once. We used the tribe Arabideae (Brassicaceae) to study the divergence of seasonal flowering behaviour among annual and perennial species. In perennial Brassicaceae, orthologues of FLOWERING LOCUS C (FLC), a floral inhibitor in Arabidopsis thaliana, are repressed by winter cold and reactivated in spring conferring seasonal flowering patterns, whereas in annuals, they are stably repressed by cold. We isolated FLC orthologues from three annual and two perennial Arabis species and found that the duplicated structure of the A. alpina locus is not required for perenniality. The expression patterns of the genes differed between annuals and perennials, as observed among Arabidopsis species, suggesting a broad relevance of these patterns within the Brassicaceae. Also analysis of plants derived from an interspecies cross of A. alpina and annual A. montbretiana demonstrated that cis-regulatory changes in FLC orthologues contribute to their different transcriptional patterns. Sequence comparisons of FLC orthologues from annuals and perennials in the tribes Arabideae and Camelineae identified two regulatory regions in the first intron whose sequence variation correlates with divergence of the annual and perennial expression patterns. Thus, we propose that related cis-acting changes in FLC orthologues occur independently in different tribes of the Brassicaceae during life history evolution.

Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis.

In plants, flowering is triggered by endogenous and environmental signals. CONSTANS (CO) promotes flowering of Arabidopsis in response to day length. Four early target genes of CO were identified using a steroid-inducible version of the protein. Two of these genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and FLOWERING LOCUS T (FT), are required for CO to promote flowering; the others are involved in proline or ethylene biosynthesis. The SOC1 and FT genes are also regulated by a second flowering-time pathway that acts independently of CO. Thus, early target genes of CO define common components of distinct flowering-time pathways.

Differential effects of light-to-dark transitions on phase setting in circadian expression among clock-controlled genes in Pharbitis nil.

The circadian clock is synchronized by the day-night cycle to allow plants to anticipate daily environmental changes and to recognize annual changes in day length enabling seasonal flowering. This clock system has been extensively studied in Arabidopsis thaliana and was found to be reset by the dark to light transition at dawn. By contrast, studies on photoperiodic flowering of Pharbitis nil revealed the presence of a clock system reset by the transition from light to dark at dusk to measure the duration of the night. However, a Pharbitis photosynthetic gene was also shown to be insensitive to this dusk transition and to be set by dawn. Thus Pharbitis appeared to have two clock systems, one set by dusk that controls photoperiodic flowering and a second controlling photosynthetic gene expression similar to that of Arabidopsis. Here, we show that circadian mRNA expression of Pharbitis homologs of a series of Arabidopsis clock or clock-controlled genes are insensitive to the dusk transition. These data further define the presence in Pharbitis of a clock system that is analogous to the Arabidopsis system, which co-exists and functions with the dusk-set system dedicated to the control of photoperiodic flowering.

Genetic and environmental control of flowering time in Arabidopsis.

Plants detect fluctuations in environmental conditions and in response to these changes they promote or delay flowering. Some of the genes that are required to regulate flowering time in response to environmental changes were identified recently and cloned from Arabidopsis. The study of these genes is establishing connections between plant physiology and the expression of the homeotic genes that are required for floral development.

Response of plant development to environment: control of flowering by daylength and temperature.

The transition from vegetative growth to flowering is often controlled by environmental conditions and influenced by the age of the plant. Intensive genetic analysis has identified pathways that regulate flowering time of Arabidopsis in response to daylength or low temperature (vernalization). These pathways are proposed to converge to regulate the expression of genes that act within the floral primordium and promote floral development. In the past year, genes that confer the responses to daylength or vernalization have been cloned and have enabled aspects of the genetic models to be tested at the molecular level.

Analysis of natural allelic variation at flowering time loci in the Landsberg erecta and Cape Verde Islands ecotypes of Arabidopsis thaliana.

We have analyzed the flowering behavior of two Arabidopsis ecotypes: the laboratory strain Landsberg erecta (Ler) and an ecotype from the tropical Cape Verde Islands (Cvi). They differ little in their flowering phenotypes and in their responses to photoperiod length changes and to vernalization treatment. However, segregating populations derived from crosses between them showed a much larger variation. An approach of quantitative trait locus (QTL) mapping in recombinant inbred lines (RILs) grown under three environments differing in day-length and/or vernalization treatment has been used to detect and locate flowering loci. Four main QTLs were identified, designated early day-length insensitive (EDI), flowering F, G, and H (FLF, FLG, and FLH, respectively), to which most of the flowering behavior differences could be attributed. To further characterize the individual loci, near isogenic lines were constructed by introgressing Cvi early alleles of EDI and FLH into the Ler genetic background. EDI-Cvi alleles produce earliness under both long- and short-day photoperiods, rendering Ler plants almost day-length neutral. In addition, RILs were selected to analyze FLF and FLG. These loci interact epistatically and RILs carrying late alleles at FLF and FLG were very responsive to vernalization and showed an increased response to photoperiod length changes. The possible role of these loci for the control of flowering is discussed in the context of the current Arabidopsis model.

The family of CONSTANS-like genes in Physcomitrella patens.

The CONSTANS (CO) gene plays a central role in the regulation of flowering time in Arabidopsis, and is a member of a family of 17 CO-like genes. CO and CO-like genes have been found in all flowering plants, but not in yeast and animals. To address the question of the origin of CO, we analysed this gene family in the moss Physcomitrella patens, a phylogenetically distant organism. Database searches in EST libraries that almost completely covered the Physcomitrella transcriptome, and Southern blotting, identified only three genes that had all of the hallmarks of CO. Further analysis demonstrated that these are most similar to CO-like genes AtCOL3/AtCOL4/AtCOL5, a group of Arabidopsis genes closely related to, but distinct from CO, suggesting that the CO branch of the AtCOL phylogeny does not exist in the Physcomitrella genome. Since 17 COL genes occur in Arabidopsis and only three closely related and two distantly related genes were found in Physcomitrella, the family of CO-like proteins appears to be smaller in Physcomitrella than in Arabidopsis, in agreement with observations made with other gene families. The data also indicate that CO-like genes must have existed in the common ancestor of bryophytes and flowering plants, and that CO originated in the group of CO-like genes represented by AtCOL3/AtCOL4/AtCOL5. Furthermore, expression of the three closely related Physcomitrella homologues is regulated by light, suggesting that the role of CO in flowering time control was probably derived from an ancestral function in light signal transduction.

Ultrasonic indications to explore the common bile duct.

A prospective comparison between operative cholangiography and operative choledochosonography has been made in 100 patients with cholelithiasis and potential choledocholithiasis. Thirty-one common bile ducts were explored, and 25 of them contained stones. Operative cholangiography was attempted in every case, but technical difficulties prevented examination of 12 common bile ducts, two of which contained stones. Both of these stones were detected by operative ultrasonography. Of the remaining 88 ducts, the sensitivity of the operative cholangiogram was 96% and the specificity 96%. Operative ultrasonography was performed for all 100 patients, with a sensitivity of 96% and a specificity of 93%. The ultrasonic findings of a sonically dense particle, shadowing, dilatation of the duct, and the persistence and meniscus signs are described with their relative incidence in the 100 patients. The time required for choledochosonography (3 to 5 minutes) compared favorably with the time needed for operative cholangiography (10 to 15 minutes). Operative bile duct scanning is a rapid, reliable technique for detecting the presence of choledocholithiasis at the time of routine cholecystectomy.

Peritoneoscopy--use in assessment of intra-abdominal malignancy.

Peritoneoscopy was performed on 236 patients between 1971 and 1980 to assess intraabdominal malignancy or to determine its presence when doubt existed. The diagnostic rate was 86%. The main causes of failure to reach a diagnosis were adhesions arising from previous surgery and tumor adhesions. It was a useful means of obtaining a tissue diagnosis, and the complication rate was low--4.5%. Peritoneoscopy demonstrated the cause of hepatomegaly in 90.9% of patients, the pathology of abdominal masses in 82.5%, and of ascites in 80.5%. It was also useful in assessing abdominal ultrasound and isotope liver scan abnormalities. The procedure is safe, cheap, and allows adequate assessment of intra-abdominal malignancy in a high proportion of patients.

Operative ultrasonic features of insulinomas.

The technique of operative pancreaticosonography is described as a method of localizing occult insulinomas. The specific ultrasonic features of two small but palpable insulinomas are compared with those of other small lesions in the pancreas. Insulinomas are ultrasonically hypoechoeic and well-defined, with smooth borders, they deform but do not infiltrate surrounding structures, and they cause some dilatation of the secondary pancreatic ducts. Real-time, high resolution, intraoperative ultrasonic pancreatography is suggested as an adjunct to assist in the localization of insulinomas at surgery.

Genetic structure at range edge: low diversity and high inbreeding in Southeast Asian mangrove (Avicennia marina) populations.

Understanding the genetic composition and mating systems of edge populations provides important insights into the environmental and demographic factors shaping species' distribution ranges. We analysed samples of the mangrove Avicennia marina from Vietnam, northern Philippines and Australia, with microsatellite markers. We compared genetic diversity and structure in edge (Southeast Asia, and Southern Australia) and core (North and Eastern Australia) populations, and also compared our results with previously published data from core and southern edge populations. Comparisons highlighted significantly reduced gene diversity and higher genetic structure in both margins compared to core populations, which can be attributed to very low effective population size, pollinator scarcity and high environmental pressure at distribution margins. The estimated level of inbreeding was significantly higher in northeastern populations compared to core and southern populations. This suggests that despite the high genetic load usually associated with inbreeding, inbreeding or even selfing may be advantageous in margin habitats due to the possible advantages of reproductive assurance, or local adaptation. The very high level of genetic structure and inbreeding show that populations of A. marina are functioning as independent evolutionary units more than as components of a metapopulation system connected by gene flow. The combinations of those characteristics make these peripheral populations likely to develop local adaptations and therefore to be of particular interest for conservation strategies as well as for adaptation to possible future environmental changes.

Regulation of flowering time: Arabidopsis as a model system to study genes that promote or delay flowering.

The time that plants flower is often tightly regulated and adapted to the locations in which they grow. The basis of this regulation has been analysed using genetic and physiological approaches since the early decades of this century. The study of flowering time in the model plant species Arabidopsis thaliana has allowed many genes involved in regulating flowering time to be identified as mutations, and for the genetic interactions between these mutations to have been studied. Furthermore, two genes required to promote flowering of Arabidopsis have recently been isolated, and their sequences have provided some insight into the identity of proteins involved in regulating flowering time.

The presence of enhancers adjacent to the Ac promoter increases the abundance of transposase mRNA and alters the timing of Ds excision in Arabidopsis.

Two copies of domain B of the CaMV 35S promoter were inserted ca. 300 bp upstream of the transcriptional start site of the Ac transposase gene. Four independent Arabidopsis transformants containing this fusion (35SenhAc::TPase) were made and the abundance of transposase mRNA in each of them was determined. The presence of the enhancers increased the abundance of the transposase mRNA by about 12-fold compared to that found in plants containing an Ac promoter fusion to the transposase gene (Ac::TPase). Hybrid plants carrying 35SenhAc::TPase and a Ds element inserted in a streptomycin phosphotransferase (SPT) gene were constructed and the frequency with which Ds excision occurred in the developing cotyledons was measured. Moreover, the number of progeny of these hybrid plants which inherited an SPT gene activated by Ds excision was studied in individual F2 families. Those derived from 35SenhAc::TPase often contained higher proportions of streptomycin-resistant (strepR) F2 progeny than those derived from Ac::TPase. These high frequencies of strepR seedlings were comparable to those previously detected after activation of Ds by a CaMV 35S promoter fusion to transposase (35S::TPase), but occurred in fewer families. The higher frequency with which this occurred in families derived from 35SenhAc::TPase compared to Ac::TPase suggests that the presence of enhancers adjacent to the native Ac promoter can influence transposase gene expression, and in this case often results in earlier excision of Ds during plant development.

Time measurement and the control of flowering in plants.

Many plants are adapted to flower at particular times of year, to ensure optimal pollination and seed maturation. In these plants flowering is controlled by environmental signals that reflect the changing seasons, particularly daylength and temperature. The response to daylength varies, so that plants isolated at higher latitudes tend to flower in response to long daylengths of spring and summer, while plants from lower latitudes avoid the extreme heat of summer by responding to short days. Such responses require a mechanism for measuring time, and the circadian clock that regulates daily rhythms in behaviour also acts as the timer in the measurement of daylength. Plants from high latitudes often also show an extreme response to temperature called vernalisation in which flowering is repressed until the plant is exposed to winter temperatures for an extended time. Genetic approaches in Arabidopsis have identified a number of genes that control vernalisation and daylength responses. These genes are described and models presented for how daylength might regulate flowering by controlling their expression by the circadian clock. BioEssays 22:38-47, 2000.