Auxin and physical constraint exerted by the perianth promote androgynophore bending in Passiflora mucronata L. (Passifloraceae).

The androgynophore column, a distinctive floral feature in passion flowers, is strongly crooked or bent in many Passiflora species pollinated by bats. This is a floral feature that facilitates the adaptation to bat pollination. Crooking or bending of plant organs are generally caused by environmental stimulus (e.g. mechanical barriers) and might involve the differential distribution of auxin. Our aim was to study the role of the perianth organs and the effect of auxin in bending of the androgynophore of the bat-pollinated species Passiflora mucronata. Morpho-anatomical characterisation of the androgynophore, including measurements of curvature angles and cell sizes both at the dorsal (convex) and ventral (concave) sides of the androgynophore, was performed on control flowers, flowers from which perianth organs were partially removed and flowers treated either with auxin (2,4-dichlorophenoxyacetic acid; 2,4-D) or with an inhibitor of auxin polar transport (naphthylphthalamic acid; NPA). Asymmetric growth of the androgynophore column, leading to bending, occurs at a late stage of flower development. Removing the physical constraint exerted by perianth organs or treatment with NPA significantly reduced androgynophore bending. Additionally, the androgynophores of plants treated with 2,4-D were more curved when compared to controls. There was a larger cellular expansion at the dorsal side of the androgynophores of plants treated with 2,4-D and in both sides of the androgynophores of plants treated with NPA. This study suggests that the physical constraint exerted by perianth and auxin redistribution promotes androgynophore bending in P. mucronata and might be related to the evolution of chiropterophily in the genus Passiflora.

Overexpression of the CBF2 transcriptional activator in Arabidopsis suppresses the responsiveness of leaf tissue to the stress hormone ethylene.

The plant hormone ethylene affects myriad developmental processes ranging from seed germination to organ senescence, and plays a crucial role in plant resistance to environmental stresses. The C-repeat/dehydration-responsive element binding factor genes (CBF1-3) are transcriptional activators involved in plant low-temperatures responses; their overexpression enhances frost tolerance, but also has various pleiotropic effects on growth and development, mainly growth retardation and delay of flowering and senescence. We found that overexpression of CBF2 in Arabidopsis suppressed leaf tissue responsiveness to ethylene as compared with wild-type plants, as manifested in significantly delayed senescence and chlorophyll degradation. In wild-type plants, exposure to ethylene at 0.1 microl.l(-1) for 48 h caused 50% reduction in chlorophyll levels as compared to leaves held in air alone, whereas CBF2-overexpressing plants required an ethylene concentration of 10.0 microl.l(-1) to cause the same effect. Furthermore, continuous exposure to ethylene at 1.0 microl.l(-1) reduced chlorophyll content in wild-type leaves by 50% after 42 h but took 72 h in CBF2-overexpressing plants. Transcript profiling of ethylene receptors and signal transduction genes in leaves of wild-type and CBF2-overexpressing plants, by means of the Affymetrix ATH1 genome array, revealed only minor differences in gene expression patterns - insufficient to explain the observed responsiveness differences. Nevertheless, we found that overexpression of CBF2 significantly increased transcript levels of 17 ABA biosynthetic and responsive genes and, thus, may have affected leaf responsiveness to ethylene via contrasting interactions with other hormones, mainly ABA. Overall, the current findings suggest that overexpression of the CBF2 transcriptional activator in Arabidopsis may, at least in part, contribute to the observed delay of leaf senescence and enhanced plant fitness by suppressing leaf responsiveness to stress-regulated ethylene.

The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals.

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

Photoperiodism: the consistent use of CONSTANS.

Photoperiodic induction of flowering in the long-day plant Arabidopsis is mediated by the circadian regulated CONSTANS gene. New evidence suggests that CONSTANS-like genes have a similar role in short-day induction of flowering of rice and Pharbitis.

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.

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.

The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem.

Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ-identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F-box protein that interacts with Arabidopsis SKP1-like proteins, both in the yeast two-hybrid system and in vitro. In yeast and other organisms both F-box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F-box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains.

Flowering of Arabidopsis is promoted by long days and delayed by short days. Mutations in the GIGANTEA (GI) gene delay flowering under long days but have little or no effect under short days. We have now isolated the GI gene and show that it encodes a novel, putative membrane protein. By comparing the sequence of the Arabidopsis gene with that of a likely rice orthologue and by sequencing mutant alleles, we identify regions of the GI protein that are likely to be important for its function. We show that GI expression is regulated by the circadian clock with a peak in transcript levels 8-10 h after dawn. The timing, height and duration of this peak are influenced by daylength. We analysed the interactions between GI and the LHY, CCA1 and ELF3 genes, previously shown to affect daylength responses; we show that the rhythmic pattern of GI expression is altered in the elf3, CCA1-OX and lhy genotypes, and that CCA1 and LHY expression are reduced by gi mutations. Our results are consistent with the idea that GI plays an important role in regulating the expression of flowering time genes during the promotion of flowering by photoperiod.

The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering.

The dominant late elongated hypocotyl (lhy) mutation of Arabidopsis disrupted circadian clock regulation of gene expression and leaf movements and caused flowering to occur independently of photoperiod. LHY was shown to encode a MYB DNA-binding protein. In wild-type plants, the LHY mRNA showed a circadian pattern of expression with a peak around dawn but in the mutant was expressed constantly at high levels. Increased LHY expression from a transgene caused the endogenous gene to be expressed at a constant level, suggesting that LHY was part of a feedback circuit that regulated its own expression. Thus, constant expression of LHY disrupts several distinct circadian rhythms in Arabidopsis, and LHY may be closely associated with the central oscillator of the circadian clock.

Divergence of function and regulation of class B floral organ identity genes.

Regulatory mechanisms controlling basic aspects of floral morphogenesis seem to be highly conserved among plant species. The class B organ identity genes, which are required to establish the identity of organs in the second (petals) and third (stamens) floral whorls, are a good example of such conservation. This work compares the function of two similar class B genes in the same genetic background. The DEFICIENS (DEF) gene from Antirrhinum, including its promoter, was transformed into Arabidopsis and compared in function and expression with the Arabidopsis class B genes APETALA3 (AP3) and PISTILLATA (PI). The DEF gene was expressed in the second, third, and fourth whorls, as was PI. Functionally, DEF could replace AP3 in making petals and stamens. The DEF gene's AP3-like function and PI-like expression caused transformation of fourth-whorl carpels to stamens. Like AP3, all aspects of DEF function in Arabidopsis required a functional PI protein. Surprisingly, DEF could not replace the AP3 protein in properly maintaining AP3 transcripts (autoregulation). Our data allow us to revise the current model for class B autoregulation and propose a hypothesis for the evolution of class B gene expression in dicotyledonous plants.

The BELL1 gene encodes a homeodomain protein involved in pattern formation in the Arabidopsis ovule primordium.

Ovule development in Arabidopsis involves the formation of three morphologically defined proximal-distal pattern elements. Integuments arise from the central pattern element. Analysis of Bell 1 (Bel 1) mutant ovules indicated that BEL1 was required for integument development. Cloning of the BEL1 locus reveals that it encodes a homeodomain transcription factor. Prior to integument initiation, BEL1 RNA localizes to the central domain, providing molecular evidence for a central pattern element. Therefore, proximal-distal patterning of the ovule involves the regulated expression of the BEL1 gene that controls integument morphogenesis. A model for BEL1 function is evaluated with regard to new data showing the expression pattern of the floral homeotic gene AGAMOUS (AG) early in wild-type and BEL1 ovule development.

Expression of an amino acid biosynthesis gene in tomato flowers: developmental upregulation and MeJa response are parenchyma-specific and mutually compatible.

The gene coding for threonine deaminase (TD), the enzyme which catalyzes the first committed step in the biosynthesis of isoleucine, was isolated from tomato as a consequence of its unusual 500-fold upregulation in floral organs. It was subsequently shown that TD is induced in potato leaves in response to wounding, abscisic acid and methyl jasmonate (MeJa). Detailed analysis presented here, reveals an intricate developmental regulation pattern of gene expression in flowers that is operating solely in parenchyma territories. Yet, despite its high pre-existing expression level, TD in flowers can be further induced by MeJa. Induction of TD in flowers as well as in leaves is effective only in the parenchyma domains, irrespective of the prior expression levels. TD is neither expressed nor induced in epidermal, vascular or sporogenous tissues. Promoter analysis in transgenic tomato plants indicates that induction of TD follows identical kinetics in flowers and leaves. Furthermore, the 'conditioning' of developmental upregulation in flowers, the response to MeJa in flowers and leaves, and the parenchyma-specific expression are all mediated by the cis-elements within the proximal 192 bp of the promoter. Promoter elements regulating the correct organ-specific expression are located, however, further upstream. The promoter constructs used in this study can serve as useful tools for expressing extremely high levels of transgenes in specific cells. A scheme explaining tissue-specific response to MeJa, in conjunction with developmental control, is discussed.

Biosynthetic threonine deaminase gene of tomato: isolation, structure, and upregulation in floral organs.

The gene encoding the plant biosynthetic threonine deaminase (Td; EC 4.2.1.16) has been cloned as a result of its unusual upregulation in tomato flowers. The Td gene of tomato encodes a polypeptide of 595 residues, the first 80 of which comprise a putative two-domain transit peptide cleaved at position 51. Comparison of the amino acid sequence with the corresponding enzymes from yeast and bacteria reveals a near identity of the important catalytic regions and greater than 40% overall similarity. The Td gene is unique in the tomato genome and its coding region is interrupted by eight introns. Its expression is greater than 50-fold higher in sepals and greater than 500-fold higher in the rest of the flower than in leaves or roots. Its overexpression, however, is strictly confined to the parenchymal cells of the floral organs. In young tomato leaves, the chloroplast-bound enzyme is found almost exclusively in the subepidermal spongy mesophyll cells.

Fate of peritoneal progesterone in the rabbit.

The efficiency of peritoneal absorption of progesterone was investigated by following the appearance of 3H-progesterone and its metabolites in the circulation after intravenous or intraperitoneal administration. Tritium rapidly entered the peripheral circulation when given by either route. The percentage of tritium appearing as modified progesterone was substantially lower following intraperitoneal administration. This difference can be largely attributed to splanchnic absorption and hepatic metabolism of intraperitoneal steroids. The potentially large amount of progesterone secreted into the peritoneal compartment may not contribute significantly to the peripheral circulating pool of progesterone.