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P5—Flowering
Comparative Biochemistry and Physiology, Part A 143 (2006) S163 – S171 www.elsevier.com/locate/cbpa
Society for Experimental Biology Annual Main Meeting 2nd–7th April 2006, University of Kent at Canterbury, UK
P5–FLOWERING Organised by N. Battey (University of Reading) Sponsored by: Journal of Experimental Botany
P5.2 Keynote Paper: The flowering process G. Bernier, (University of Liege) During several decades following World War II a very large knowledge base concerning the physiology of flowering was produced in the world. My main contribution comes from work with Sinapis alba where ‘‘florigen’’ was found to be multifactorial and to induce precisely timed events at the meristem. I have edited the Flowering Newsletter from 1989 to 2005, a period that will probably be remembered as the ‘‘golden’’ age of the genetical/ molecular study of the flowering process. This period started in 1990 with the cloning of the first floral homeotic genes DEFICIENS and FLORICAULA from snapdragon and AGAMOUS from Arabidopsis, and with the proposal in 1991 of the ABC model of flower organogenesis. Culmination of this period might have been reached in 2004 – 2005 with the claim that a specific component of the long-tracked ‘‘mythical’’ florigen in Arabidopsis is the transcript or the protein of the FLOWERING LOCUS T gene. Work during this last period was performed mainly with Arabidopsis and secondarily with few other species. At this stage an important challenge is to achieve integration between these two very large and potentially complementary bodies of information. Although some progress has been recorded recently in this area, a serious problem is that the plants best suited for the genetical/ molecular work are usually different from the best model plants for the physiological work. Further integration will require imaginative efforts from researchers of both sides. Suggestions for progress towards this goal will be discussed.
P5.3 Vernalization and the epigenetic memory of winter R. Amasino, (Wisconsin) Certain plants, such as biennials or winter annuals, require relatively long periods of cold exposure during winter to initiate flowering the doi:10.1016/j.cbpa.2006.01.054
following spring. Cold exposure renders the meristem of such coldrequiring species competent to flower, and this acquisition of competence is known as vernalization. A vernalization requirement ensures that flowering does not occur prematurely before the onset of winter. A similar cold response is bud dormancy; in many species that grow in temperate climates, bud dormancy is not broken until a the plant has ‘‘counted’’ a sufficient number of days of cold to ensure that any subsequent warn weather actually indicates that spring has arrived. Our studies of vernalization in Arabidopsis have revealed that meristem competence is a function of the expression level of certain MADS-box genes such as FLOWERING LOCUS C (FLC) that act as repressors of flowering. Exposure to prolonged cold causes an epigenetic switch of these MADS box genes to an unexpressed state, thus rendering the shoot apical meristem competent to flower. This epigenetic switch is caused by covalent modifications to histones of the chromatin of the flowering repressors.
P5.4 The autonomous pathway: Linking RNA processing, RNAi and chromatin regulation C. Dean, F. Liu, P. Crevillen, I. Baurle, (John Innes Centre) The timing of the floral transition has significant consequences for the reproductive success of plants. Plants need to gauge when both environmental and endogenous cues are optimal before undergoing the switch to reproductive development. To achieve this, a complex regulatory network has evolved consisting of multiple pathways that quantitatively and antagonistically regulate the genes whose activity causes the transition of the meristem to reproductive development. The Dean group has focused on a set of pathways that regulate the strong floral repressor, FLC. FLC expression needs to be effectively shut-down both for the timely transition to flowering and for normal flower development. One pathway that represses FLC expression involves FCA, an RNAbinding protein which interacts with SWI3b and FY, a highly
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conserved protein known as Pfs2p in S. cerevisiae. Pfs2p is an essential protein which functions to regulate polyadenylation and 3V processing of mRNAs. In Arabidopsis, FY and FCA have also been demonstrated to regulate mRNA 3Vend processing in that they regulate poly A site choice in the FCA transcript. FY is required for the negative feedback regulation of FCA and null mutations are embryo lethal. In order to determine how FCA and FY regulate FLC, we have undertaken a genetic approach and have identified sof1 as a suppressor of FCA overexpression. sof1 carries a mutation in FLD, a regulator of FLC chromatin structure. FLD was reported as mediating histone deacetylation at FLC but a human homologue LSD1 has recently been described as the first histone lysine 4 demethylase. We have also shown that DCL3, previously shown to be required for RNAdependent DNA methylation and heterochromatin formation, is required for FCA repression of FLC. These data suggest a model whereby FCA and FY cause changes in FLC RNA processing that trigger an RNAi-mediated chromatin regulation involving histone demethylation.
P5.5 Light signals in flowering B. Thomas, A. Massiah, S. Jackson, K. Morris, S. Adams, (U. Warwick) Plants modify their flowering behaviour in response to a range of changes in the light environment. The best known of these is the effect of timing of light signals in photoperiodic responses. Here, light acts to entrain the circadian rhythm underlying the daylength response and also interacts with the output of the clock to generate flowering signals. In addition, light can modify flowering times separately from its action on the photoperiodic pathway through quality and irradiance-dependent responses and can also have a major influence on the onset flowering through effects on the duration of the juvenile phase of development. In all cases, responses to light are mediated through specific light receptors. These could include known photoreceptors such as phytochromes and cryptochromes, photosynthetic pigments or other light-sensing molecules with specific functions in flowering. In this talk, evidence for several light-dependent pathways that lead to altered flowering time will be presented and the roles of different light sensors discussed.
P5.6 Mechanistic modelling of flowering regulators based on molecular data A. Millar, P. Brown, T. Saithong, (University of Edinburgh); D. Salazar, J. Locke, I. Carre, D. Rand, (University of Warwick) Day-length (photoperiod) measurement depends on the circadian clock, which thereby affects seasonal rhythms including flowering. Flowering is regulated by numerous other environmental and developmental factors. With collaborators in the IPCR at Warwick, we developed the first mathematical models of the Arabidopsis clock gene network, incorporating 6 genes in two, interlocking negative feedback loops. Experiments based upon predictions of the model identified a novel and central function for GIGANTEA (GI)
in the clock network (Locke et al., Molecular Systems Biology 2005). Such successful prediction of a genetic network structure is a goal of many systems biology groups, and is clearly possible for these plant gene networks. We have recently extended the clock model to include the photoperiod-responsive switch formed by CO and FT. These show that the simple external coincidence model has been considerably elaborated in Arabidopsis. Results from alternative model structures will be discussed, showing, amongst other points, that the photoperiod-dependent entrainment of the circadian clock is important in explaining the molecular rhythms. The models allow us to quantify phase-specific modifiers of the external coincidence mechanism, using timeseries data only from wild-type plants: modelling adds value to timeseries data even without experimental manipulations. We have also extended our experimentalist-friendly simulation software, Circadian Modelling, to simulate flowering time experiments; the software is available at www.amillar.org.
P5.7 Control of GIGANTEA gene expression by light and the clock F. Cremer, R. To´th, A. Giakountis, I. Bu¨rstel, G. Coupland, (Max Planck Institute for Plant Breeding Research) The photoperiodic pathway of floral induction is controlled by the interaction of light signals with the circadian clock. GIGANTEA (GI), CONSTANS (CO)and FLOWERING LOCUS T (FT)are part of a gene cascade controlled by the clock and inducing the floral transition of Arabidopsis in response to long days. GI is expressed with an evening phase and has a positive effect on the amplitude of the rhythmic pattern of CO transcription. Independently of its effect on floral induction, GI is also involved in other light signaling pathways and affects the circadian clock. We have used promoter fusions to luciferase to show that GI transcription is induced by blue, red and far-red light and that this response is gated by the circadian clock, with a higher induction at peak times of circadian GI expression than at trough times. The diurnal peak of GI expression also occurs earlier in short than in long days and the acute light response follows the same pattern. In order to understand the control of the timing of GI expression, we are analyzing its promoter using deletions and phylogenetic shadowing. In another approach, we are studying the pattern of GI expression under different photoperiods in relation to flowering time. This last approach has been extended to the study of natural variation in patterns of GI expression in a group of a hundred accessions. These and further results will be presented at the meeting.
P5.8 Crucial role of outgroups in inferring the likely reproductive characteristics of the first angiosperm R. Bateman, (Natural History Museum); J. Hilton, (University of Birmingham); P. Rudall, (Royal Botanic Gardens Kew) Recent attempts to address the long-debated ‘‘origin’’ of the angiosperms depend on a phylogenetic framework derived from a matrix of taxa versus characters, and assume that empirical rigour
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is proportional to the size of the matrix. Sequence-based approaches increase the number of characters (nucleotides) in the matrix but are confined to the highly restricted spectrum of extant species, whereas morphology-based approaches increase the number of phylogenetically informative taxa (including fossils) at the expense of accessing only a restricted spectrum of phenotypic characters. The two approaches are currently delivering strongly contrasting hypotheses of relationship. Most molecular studies indicate that all extant gymnosperms form a natural group, suggesting surprisingly early divergence of (pre-)angiosperms, whereas morphology-only phylogenies indicate that a succession of (mostly extinct) gymnosperms preceded a later angiosperm origin. Causes of this conflict include: (1) the vast phenotypic and genotypic lacuna, largely reflecting preTertiary extinctions, that separates early-divergent living angiosperms from their closest relatives among the living gymnosperms; (2) profound uncertainty regarding which (a) extant and (b) extinct angiosperms are most closely related to gymnosperms; and (3) profound uncertainty regarding which (a) extant and (b) extinct gymnosperms are most closely related to angiosperms, and thus best serve as ‘‘outgroups’’ dictating the perceived evolutionary polarity of character transitions among the early-divergent angiosperms. These factors still permit a remarkable range of hypotheses regarding the order in which the many phenotypic characters that distinguish ‘‘classic’’ angiospermy from ‘‘classic’’ gymnospermy were acquired. Focusing on reproductive characters, we will explore some of the more strongly contrasting hypotheses.
P5.9 Novel genes and their putative functions in grasses E. Kellogg, (University of Missouri-St. Louis) A central problem in evolutionary biology is the diversification of form. This question is the basis of the field of evolutionary developmental biology, which links phylogenetic studies with developmental genetics, often in species that are not model organisms. Recent work in this field can generally be placed in one of two categories. First is the direct, genetic approach, which uses experimental mapping populations to identify quantitative trait loci (QTL) controlling the difference between crossable species, and may in some cases lead to identification of genes. Second is a candidate gene approach, which chooses genes whose mutant phenotype in a model organism is similar to the natural phenotype of a wild organism, and investigates the naturally occurring variation in that gene. Combining the two approaches has proven particularly fruitful in studies of grasses (Poaceae), in which numerous genes have been cloned that control aspects of inflorescence architecture. Because of a long and complex history of gene duplication, many inflorescence genes are unique to the grasses; the presence of multiple novel genes correlates with the presence of multiple morphological novelties in the family. QTL studies in many grass species, particularly the cereal crops, have identified loci that control particular aspects of inflorescence form. For some species, candidate genes have been identified that fall in the QTL. Association analysis shows that some of these genes could be important for inflorescence evolution among all grasses.
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P5.10 Flower-like terminal structures in racemose inflorescences: a tool in morphogenetic and evolutionary research D. Sokoloff, M. Remizowa, (Moscow State University); P. Rudall, (Royal Botanic Gardens, Kew) Terminal flower-like structures (TFLS) occur in many angiosperms that possess (partial) inflorescences such as spikes, racemes, or spadices. Using both original and review data, we consider the occurrence, morphology, and some developmental aspects of TFLS in early-divergent angiosperms and monocots. We explore the hypothesis that structures with long-debated pseudanthial or euanthial interpretation in various angiosperm groups could have evolved as a result of overlap in developmental programs of a typical flower and inflorescence. Spikes and spadices are typical for both Chloranthaceae and perianthless Piperales among earlydivergent angiosperms, but whereas TFLS occur in some Piperales such as Saururaceae and a few Piperaceae, they are absent from Chloranthaceae. Within the monocot clade, TFLS are here described in some early-divergent families such as Acoraceae, Aponogetonaceae, Potamogetonaceae and Ruppiaceae. Thus, essentially similar TFLS occur in wide range of taxa. Similar TFLS with obscure organ identity have been found in mutants of model organisms such as Arabidopsis. TFLS can often be interpreted as pseudanthia (close aggregations of reduced flowers), but in some cases the entire terminal pseudanthium is very similar to a true flower. Elaborated TFLS could therefore give rise to what we normally term true flowers. Furthermore, unusual structures frequently occur at or near the inflorescence tip; for example, the entire inflorescence apex or an individual flower primordium can be transformed into either a filamentous or tubular structure, or an intermediate form. This indicates that pseudanthium formation can provoke morphological novelties, perhaps due to new patterns of overlap between expression zones of regulatory genes.
P5.11 The evolution and development of petaloidy in Aizoaceae (Caryophyllales) S. Brockington, (Florida Museum of Natural History); P. Rudall, (Royal Botanic Gardens Kew); M. Frohlich, (Natural History Museum); D. Soltis, (University of Florida); P. Soltis, (Florida Museum of Natural History) An understanding of the genetic program controlling floral organ identity in eudicot species such as Arabidopsis thaliana and Antirrhinum majus has led to new approaches in the study of floral diversity and petal evolution. More specifically, it has been proposed that MADS-box genes may play an important role in generating floral diversity through changes in gene expression pattern. The family Aizoaceae provides a good opportunity to test this hypothesis because within this clade significant differentiation has occurred in the development of different floral organs as attractants for pollinators. In the early divergent subfamilies Sesuvioideae and Aizooideae the tepals of the uniseriate perianth are petaloid on the adaxial side,
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whereas in subfamilies Mesembryanthoideae and Ruschoideae the tepals are not petaloid and instead outer whorls of stamens are sterile and showy. In advance of gene expression analysis we have characterised the floral development of several species of Aizoaceae including some abnormal floral variants. Examination of the epidermal and sub-epidermal features of the petaloid tepals and petaloid staminodes reveals few morphological similarities between the different kinds of petaloid organ. The development of the petaloid tepals is similar to that of a monocot foliage leaf: the lamina is formed from the leaf base region. Abnormal floral variants suggest that specification of petaloid tissue is dissociable from tepal morphogenesis in development. We speculate on the significance of this unusual mode of tepal development for the evolution of petaloidy and make specific hypotheses regarding expected patterns of MADS-box gene expression.
P5.12 Capsella as a model system to study the evolution of flower development G. Theissen, M. Hintz, P. Nutt, J. Ziermann, C. Bartholmes, (University of Jena); B. Neuffer, (University of Osnabrueck) Capsella is a small genus within the mustard family (Brassicaceae). It has only three species which nevertheless show many evolutionary trends also found in other Brassicaceae (including Arabidopsis) and far beyond, including transitions from a diploid, self-incompatible, obligatory outcrossing species with comparatively large and attractive flowers but a quite restricted area of distribution, to a polyploid, self-compatible, predominantly selfing, invasive species with floral reductions. All these transitions may have contributed to the fact that Capsella bursa-pastoris (Shepherd’s purse) became one of the most widely distributed flowering plants on our planet. In addition, Capsella bursa-pastoris shows a very rare phenomenon which, nevertheless, could be of great evolutionary importance, i.e. the occurrence of a homeotic variety in quite stable populations in the wild. Several lines of evidence suggest that homeotic changes played a considerable role during the evolution of flowers, but how floral homeotic varieties are established in natural populations has remained a highly controversial topic among evolutionary biologists. Due to its close relationship to the model plant Arabidopsis thaliana numerous experimental tools are available for studying the genus Capsella, and more tools are currently being developed. Capsella thus provides great opportunities to investigate the evolution of flower development from molecular developmental genetics to field ecology and biogeography, and from morphological refinements to major structural transitions.
P5.13 The role of the FT mRNA in photoperiodic regulation of plant growth and development
gene CONSTANS (CO). We have shown that local induction of FT in a single Arabidopsis leaf is sufficient to trigger flowering and that the FT mRNA is transported to the shoot apex, where downstream genes are activated. These data suggest that the FT mRNA is an important component of the elusive ‘‘florigen’’ signal that moves from leaf to shoot apex. In contrast to annual plants like Arabidopsis, forest trees display a perennial growth behaviour characterized by a very extended juvenile phase before flowering, and, in temperate regions of the world, an annual cycling between growth and dormancy. We show here that the CO/FT regulatory module is functionally conserved in the aspen tree where it controls the timing of flowering. However, unexpectedly, it also controls the short-day induced growth cessation and bud set normally occuring in the fall. This suggests that FT is not a specific regulator of flowering, but might have a more general role in regulating biological processes controlled by variations in day length.
P5.14 Analysis of the expression pattern of FT protein during flowering L. Corbesier, C. Vincent, I. Searle, F. Fornara, G. Coupland, (Max Planck Institute for Plant Breeding Research) Flower development at the shoot apex is initiated in response to environmental cues. Among these, daylength is one of the most important and is perceived by the mature leaves. When exposed to inductive photoperiodic conditions, these leaves produce endogenous signals that are transported to the shoot apical meristem where they cause the transition from leaf to flower morphogenesis. In Arabidopsis, genetic analysis identified a pathway of genes required for the initiation of flowering in response to daylength. The nuclear zincfinger protein CONSTANS (CO) plays a central role in this pathway and, in response to long days, activates the transcription of FLOWERING LOCUS T (FT), which encodes a RAF-kinase inhibitor-like protein. Since CO has been shown to trigger flowering when expressed in the phloem but not when expressed in the meristem itself, it was proposed that CO regulates the synthesis or the transport of a floral signal in the phloem and this involves partly the cell autonomous activation of FT. A product of FT might comprise this signal and recently FT mRNA was proposed to move from the leaves to the shoot apex. The FT protein is only 23 kilodaltons, and we have tested whether this can move in the phloem to the meristem. We will present our work aiming to test FT protein translocation with the help of tissue-specific promoters driving FT:GFP constructs, confocal microscopy, in situ immunolocalisation and in situ hybridization.
P5.15 The tomato FT gene triggers conserved systemic flowering signals that regulate termination and substitute for light and photoperiodic stimuli
O. Nilsson, (Umea˚ Plant Science Centre) Day length controls flowering time in many plants. The daylength signal is perceived in the leaf, but how this signal is transduced to the shoot apex where floral initiation occurs has not been known. In Arabidopsis, the day-length response depends on the induction of the FLOWERING LOCUS T (FT) gene by the
E. Lifschitz, T. Eviatar, A. Rozman, A. Shalit, (Technion); A. Goldshmidt, Z. Amsellem, J. Alvarez, Y. Eshed, (Weizmann Institute of Science) The FLORIGEN model implies a mechanism that is common to all plants but is activated by different flowering stimuli in different
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species. During transition to flowering in annuals with a monopodial habit like Arabidopsis, the replacement of the vegetative phase by a single reproductive phase signals the completion of the life cycle. Growth habits of other, particularly perennial plants require the cohabitation of vegetative and floral buds along their shoots and thus reiterated vegetative-reproductive transitions. Tomato plants are perennial in their native habitat, flowering is independent of photoperiodism, and the sympodial shoots, inflorescence shoots and leaves are compound, consisting of determinate morphogenetic duplications. This modular organization permits the analysis of multiple flowering events in a single plant and affords an architectural flexibility which is reflected in a unique range of developmental variants. SINGLE FLOWER TRUSS, a regulator of flowering time and morphogenesis, encodes the tomato ortholog of FT (TFT). We show that SFT-stimulated signals abide by all tenets of the florigen hypothesis. Grafting and leaf-specific expression show that SFT generates systemic signals which substitute for light and photoperiodic stimuli in Arabidopsis and tobacco and for innate morphogenetic signals in tomato. SFT and its interacting proteins are co-expressed in leaves and the SFT protein is localized to leaf nuclei. In tomato grafts, the donor SFT-RNA could not be detected in flowering receptor shoots. The role of SFT in growth and termination and its contribution to the vegetative/reproductive balance and plant architecture in tomato and Arabidopsis will be discussed.
P5.16 FT signalling in the floral transition P. Wigge, (John Innes Centre) The floral transition is a critical decision in plant development. Reflecting this, there are numerous pathways that sense both environmental and endogenous signals in order to control flowering time. A critical integrator of signalling information in the floral decision is FT. FT is a potent inducer of flowering when overexpressed. Early genetic studies have shown that an FT acts in parallel with the transcription factor LEAFY to promote flowering. FT has homology at the amino acid sequence level to small soluble proteins found in animals and fungi called PEBPs or RKIPs. We and others have been able to show that FT signalling is dependent on the bZIP transcription factor FD. An important target of FT signalling appears to be the transcription factor AP1, as this is precociously upregulated in 35S FT plants. We have shown that this activation of AP1 is FD dependent. Furthermore, knock-out lines for this transcription factor are late flowering and these are able to partially suppress the overexpression of 35S FT. We show that FD activation of the AP1 promoter is strongly dependent on the presence of FT, and moreover FD is expressed in the young flowers, suggesting that it provides the spatial information for FT signalling. We are investigating the role of relatives of FT and FD in control of the vegetative to floral transition.
P5.17 Protein trafficking of FT/TFL1 and flowering signal transmission K. Goto, (Res. Inst. Biological Sciences) FT and TFL1 of Arabidopsis are highly homologous proteins but their functions in flowering pathway are opposite; while FT promotes, TFL1
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retards flowering. In addition, TFL1 functions to maintain inflorescence meristem (IM) and repress floral meristem (FM) development. For this function, TFL1 is required in the whole region of IM, but its transcript was localized inner region of L3 layer of IM. We have revealed this noncell-autonomous function of TFL1 is conferred by protein trafficking. We also found that FT moves among cell layers of IM. The endogenous FT is expressed in the vascular bundle of leaf apical region, and recent study revealed that FT functions with FD in the shoot apical meristem (SAM). Taken together, the following scenario can be deduced; after long distance travel from leaves via vascular bundle, FT is unloaded near the SAM (vascular bundle is undifferenciated in the SAM) and then FT protein moves to spread into the SAM to promote phase transition. Another recent study suggests that FT RNA moves from the leaf to the SAM. To confirm this, we developed grafting system using tobacco. Since transgenic tobacco carrying Arabidopsis FT showed early flowering, we used it as a rootstock and wild type scion was grafted. We found the scions acquired early flowering phenotype. Now we are investigating FT RNA and/or protein are transmitted from stock to scion.
P5.19 Protein complexes make the flower G. Angenent, S. de Folter, I. Nougalli, R. Immink, (Plant Research International) The MADS box transcription factor family is one of the most important families of regulatory proteins in plants. The genes are involved in many developmental processes, such as root development, flowering, organ identity specification, and fruit and seed formation. We studied members of this family in Petunia and Arabidopsis. The MADS box transcription factors form dimers and it has become apparent recently that higher order complexes between several MADS box proteins form the basis for floral organ specification. Most likely all MADS box proteins form these multiprotein complexes. To unravel a part of the network of protein complexes in which MADS proteins play a role, we performed a comprehensive yeast two- and three-hybrid screen between all Arabidopsis MADS box proteins using a matrix-based approach. The obtained interactions confirmed known interactions and revealed many novel interactions. These interactions and data about target genes of these MADS box factors suggest that an extensive network exists in which MADS box complexes regulate their own expression by positive and negative autoregulatory loops. Preliminary work indicates that flowering genes, e.g. SOC1, AGL24, SVP, are negatively regulated by floral organ identity proteins. Currently, Chromatin Immuno Precipitation (CHIP) and yeast one-hybrid experiments are being performed to confirm these models.
P5.20 Flower development in rice M. Kater, L. Dreni, N. Pelucchi, L. Colombo, (University of Milan) Flower development has been intensively studied in the dicot plant species Antirrhinum majus and Arabidopsis thaliana. Analysis of homeotic mutants resulted in the beginning of the nineties in the formulation of the ABC model which postulates that three classes of genes control floral organ identity in a combinatorial way. Isolation and characterisation of these ABC genes showed that most of them belong to the MADS-box family of transcription factors. Although the floral
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structure of the rice flower is different, especially in respect to the perianth organs, genes homologous to the ABC genes of dicots have also been identified in rice. Functional analysis of several of them showed that to a certain extent their function seems to be conserved in rice. In subsequent years the ABC model has been extended with class D and E genes. D genes have shown to control ovule identity and class E genes (or SEPALLATA genes) are necessary for the function of class A, B, C and D genes. Recently we have started with the characterisation of class D genes of rice. An overview of flower development in rice will be presented and the latest results concerning rice class D genes will be discussed.
P5.21 Characterisation of the HAWAIIAN SKIRT gene, which encodes a novel F-box protein invloved in sepal boundary development Z. Gonzalez-Carranza, J. Roberts, (The University of Nottingham) Boundary regulation in plants plays an important role in the formation of floral organs. Recently we have mapped the location of Hawaiian skirt, a novel mutant from Arabidopsis that exhibits aberrant sepal separation, and characterised the gene that it encodes. The HAWAIIAN SKIRT gene encodes a novel F-box protein that exhibits a low level of amino acid homology to the mutant UFO. Fbox proteins have been shown to form a key component of the SCF complex and have been proposed to target the degradation of specific substrates by the 26S proteasome (Ni et al., 2004). Characterisation of the temporal and spatial expression of the HS gene by RT-PCR, and fusion of its promoter to the reporters GUS or GFP, has revealed that transcript accumulation takes place in both root and shoot tissues and as a consequence of wounding. The possible role of this gene during Arabidopsis development is discussed. 1. Ni, W., Xie, D., Hobbie, L., Feng, B., Zhao, D., Akkara, J., Ma, H., 2004. Regulation of flower development in Arabidopsis by SCF complexes. Plant Physiology 134, 1574 – 1585.
P5.22 Reproductive meristem fates in Gerbera T. Teeri, M. Kotilainen, R. Laitinen, H. Help, P. Elomaa, (University of Helsinki); V. Albert, (University of Oslo); A. Uimari, (University of Kuopio) Flowering plants go through several phases between regular stem growth and the actual production of flower parts. The stepwise conversion of vegetative into inflorescence and floral meristems is usually unidirectional, but under certain environmental or genetic conditions, meristems can revert to an earlier developmental identity. Vegetative meristems are typically indeterminate, producing organs continuously, whereas flower meristems are determinate, shutting down their growth after reproductive organs are initiated. Inflorescence meristems can show either pattern. We have investigated flower and inflorescence development in Gerbera hybrida, an ornamental plant in the sunflower family, Asteraceae. Unlike the common model species used to study flower development (Arabidopsis, Antirrhinum and Petunia), Gerbera bears determinate inflorescences, and the architecture of the flower
differs in that Gerbera ovaries are inferior. We have shown that floral meristem determinacy and identity are spatially and genetically distinct in Gerbera, and that a single SEPALLATAlike MADS domain factor controls both flower and inflorescence meristem fate in the plant. Although these phenomena have not been directly observed in Arabidopsis, the integrative role of the SEPALLATA function in reproductive meristem development may be generalizable to all flowering plants.
P5.23 Carpel and fruit patterning in Arabidopsis C. Ferrandiz, (Instituto De Biologia Molecular Y Celular De Plantas. CSIC-UPV) The fruit is a major evolutionary acquisition of the angiosperms, basically derived from the flower gynoecium, which elongates and differentiates after fertilization of the ovules to protect and disperse seed. Fruits display a great variety of morphological structures among the angiosperms and we are beginning to get some insight into the genetic basis of this diversity. In the model plant Arabidopsis thaliana, genetic analyses have identified a small set of regulatory genes that establish the initial patterning of gynoecium development along the longitudinal and transverse axes. In addition, auxin gradients appear to play a key role in establishing apical-basal patterning in the developing Arabidopsis gynoecium, but the genetic pathways involved remain unclear. In this talk we will discuss the current models on fruit patterning and the contribution of our lab to the characterization of a small subfamily of transcription factors, the TOWER-OF-PISA genes, that could work to coordinate development along these two main axes. We will also discuss some of our recent work on comparative fruit development. We are trying to address how general are the proposed models and, for that purpose, we are beginning to explore these questions in legumes with different fruit morphologies (such as pea or Medicago) by cloning and functionally characterizing orthologous genes to those with a defined role in Arabidopsis gynoecium development.
P5.25 Effects of light level on the juvenile phase in Antirrhinum B. Thomas, S. Adams, V. Valdez, A. Jackson, A. Massiah, (U. Warwick) Plants raised from seed usually pass through a stage of development where they are incapable of initiating flowers, irrespective of environmental treatments. This is referred to as the juvenile phase and may last for weeks or, in some cases, years. After passing through this stage with progression to the adult phase, many plants become competent to flower but will not do so unless they receive an additional environmental stimulus such as a permissive photoperiod and/or a period at a particular temperature, to initiate flowers. The juvenile to adult phase change within the vegetative phase and vegetative – reproductive phase change are distinct processes that influence each other. In Antirrhinum the length of the juvenile phase can be modified by the light levels at which plants are grown. This is not simply because of slower development as plants at lower light levels do not become competent to respond to inductive conditions
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until they are bigger, with more leaves than plants grown at higher light levels. In order to understand further this response, we will describe the effect of light level on assimilates, apical development, growth and gene expression in Antirrhinum during the juvenile phase.
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mutation. Mass spectrometry methods are being used to elucidate the protein identity and database searching utilised to determine potential protein function.
P5.29 P5.26 Molecular characterisation of the day neutral flowering (dnf) mutant of Arabidopsis S. Thornber, S. Jackson, K. Morris, L. Codrai, (Warwick HRI) Arabidopsis is a facultative long day plant and as such flowers under both long and short days but sooner under long day conditions. To look for novel flowering mutants of the photoperiodic pathway a TDNA tagged population of Arabidopsis was screened for altered flowering times in long and short days. A novel Arabidopsis mutant, termed day neutral flowering (dnf) was isolated as a result of this screen and displays no short-day inhibition of flowering. The dnf mutant, therefore, flowers at the same time under both long and short day conditions as WT does under LD conditions. Successful complementation using the wild-type DNF gene under the control of its endogenous promoter confirmed that the dnf mutation is responsible for the observed phenotype. The DNF gene is 141 amino acids in length and contains a predicted membrane spanning domain between residues 13 and 33 and a putative RING finger domain between residue 79 and 121. Many RING finger proteins are E3 ubiquitin ligases that target specific proteins for degradation by the 26S proteasome. In an attempt to identify the target(s) of the DNF RING finger protein we have adopted an in vivo pull-down approach and have produced plants expressing a TAP tagged DNF protein. Recent progress and the hypothesised role of DNF in the control of flowering will be discussed.
P5.27 Development of protein analysis methods for MALE STERILITY 1 (MS1) protein in Arabidopsis thaliana E. Hewitt, (University of Nottingham) The MALE STERILITY1 gene in Arabidopsis encodes a nuclear localised protein expressed in the tapetal cells of the anther. It is proposed to be a transcriptional regulator of male gametogenesis, which is required for pollen maturation and contains a PHD finger motif thought to participate in protein-protein interactions. It has therefore been suggested that the MS1 protein acts as part of a protein complex that is vital in the process of male gametophyte development. We are currently investigating the role of the MS1 protein and the effects of its mutation on the male gametophyte developmental pathway. To this end a reproducible 2-dimensional (2-D) protein gel electrophoresis method has been developed which can be used to study the expression of anther proteins. Total protein has been isolated from buds and anthers of wild type plants for 2D gel analysis. Relatively high yields of good quality total protein could be isolated from anther tissue and this was therefore selected as the source tissue for the comparative analysis. Total protein isolated from anthers of wild type plants has been compared to that from ms1 mutant anthers to identify alterations in protein expression associated with the ms1
Micropropagation of Chrysanthemum: securing novel mutations M. Davey, P. Anthony, L. Cheetham, B. Power, K. Lowe, (University of Nottingham) Floriculture is globally a major industry driven by consumer demands for novel flower architecture and colour. Genetic diversity within some ornamental plants has produced commercially-important variants (Fsports_), particularly in Chrysanthemum (Dendranthemum). However, the ease with which such sports can be secured by cuttings depends upon the extent of mutation. Micropropagation can be exploited to secure mutations limited to petals and/or sectors of petals. Petals of the spray Chrysanthemum cv. White Enbee Wedding [flower colour White Group 155A; RHS Colour Chart and Flower Council of Holland] that had sported to Yellow Group 13B were removed from influorescences, surface sterilized and transferred to 50 ml of MSbased medium supplemented with 2.0 mg l 1 a-naphthaleneacetic acid and 0.5 mg l 1 benzylaminopurine contained in 175 ml glass jars. Cultures were maintained at 25 T 2 -C in the light (50 mmol m 2 sec-1; 16 h photoperiod) for 100 days. Regenerated shoots were transferred to MS-based medium, lacking growth regulators for rooting and, subsequently, to compost. Plants were grown to flowering and assessed over 2 years for flower colour and morphology. Flowers from regenerants exhibited a colour index of Yellow Group 12A, 12A/ B or 12B. In 15% of regenerants, the mean influorescence diameter was significantly (P < 0.05) greater than control. In contrast, the mean number of petals per flower was significantly (P < 0.05) reduced, but the mean number of flowers per stem was unaltered. Interestingly, several plants developed flowers with altered floral architecture owing to superimposed somaclonal variation.
P5.30 Genetical approaches towards understanding flowering time regulation in tomato M. Quinet, H. Batoko, J. Kinet, (Universite´ catholique de Louvain, Belgium) Our aim through the study of flowering in tomato, a sympodial plant, is to contribute to enlarge the knowledge from Arabidopsis to other species. We investigated different late flowering mutants and double mutants, generated from these single mutants, all having as a common parent the uniflora mutant which has the most delayed flowering. All double mutants were late flowering. uniflora: blind and uniflora :self pruning had a flowering time intermediate between their two parents. jointless: uniflora and compound inflorescence: uniflora flowered later than uniflora. They also developed strong lateral shoots at node levels grossly corresponding with the level where their parent cultivars initiated their first reproductive structure, which is a typical trait of uniflora. This suggests that these plants underwent partial evocation but that they were unable to complete floral transition. This phenotype could also be indicative of the occurrence of processes upstream of UF that direct the primary shoot SAM into reproductive growth and of the
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absence of influence of the genes we investigated upon the timing of events that initiate floral transition of this SAM. Our results also suggest that, from that time point, the different genes were interacting to regulate flowering time. Unravelling the regulation of floral transition in tomato is thus complicated by the fact that tomato has a sympodial growth habit and that the process is apparently not regulated in the same way in primary shoot and sympodial segments.
P5.31 Control of floral transition in maize F. Van Kerkhoven, N. Jenne`s, C. Pe´rilleux, (University of Lie`ge) Maize (Zea mays L.) is a quantitative short-day plant, although floral induction is largely autonomous in modern genotypes. Maize is a major crop but, despite being largely studied, little is known about the physiological and molecular mechanisms involved in the regulation of its floral transition. One outstanding fact is that - in reproductible growing conditions - the apical meristem produces a predictable total leaf number (TLN) before floral transition occurs. This led to the hypothesis that leaf counting is the basis of flowering time measurement. In this rationale, a change in leaf initiation rate should directly delay or accelerate flowering. To test this, we used hormonal treatments to modify the leaf initiation rate and regularly dissected the plants to determine the time of floral transition. Plants were grown in phytotronic chambers at 400 AE m 2 s 1, 16 h photoperiod and 24/18 -C day/night temperature. Benzyladenine 10 3 M (BA), gibberellic acid 10 3 M (GA3) or 2,4-dichlorophenoxyacetic acid 2.10 5 M (2,4-D) were applied in the centre of the leaf roll when plants were 15-day old. BA increased the leaf initiation rate and the TLN but delayed flowering. GA3 increased the leaf initiation rate but TLN was lower and floral transition was advanced. 2,4-D decreased the leaf initiation rate and the TLN but floral transition was unchanged. So, it appears that floral transition in maize is not simply determined by leaf counting. This work is supported by the FFe´de´ration Nationale de la Production des Semences de Maı¨s et de Sorgho’ (F.N.P.S.M.S., France); F.VK is grateful to the FFonds pour la formation a` la Recherche dans l’Industrie et l’Agriculture_ (F.R.I.A.) for the award of a PhD fellowship.
P5.32 Cloning of CONSTANS and FLOWERING LOCUS T in Sinapis alba’ K. Tamseddak, M. D’Aloia, C. Pe´rilleux, (University of Lie`ge) Flowering in photoperiodic species is known to require a Ffloral stimulus_ moving from the leaves- where photoperiod is perceived- to the shoot apical meristem. Although the nature of the floral stimulus has long remained elusive, molecular and genetic studies in Arabidopsis thaliana, a qualitative long day (LD) plant, showed that the product of the FLOWERING LOCUS T (FT) gene is a crucial signal that follows the floral stimulus route after FT has been activated in LD by the transcription factor CONSTANS (CO). On the other hand, changes in the translocation of nutrients and hormones have also been described during the transition to flowering. In Sinapis alba, flowering can be obtained by a single LD and a Fshoot-to-root-to-shoot_ physiological loop has been described which involves sucrose and cytokinin as components of the floral stimulus. In order to integrate the genetic data into this physiological network, we cloned CO and FT homologues in Sinapis and analysed their expression patterns. Cloning of SaCO was
performed by screening a cDNA library with an AtCO probe, and cloning of SaFT was done by RT-PCR with degenerated primers. The sequences obtained showed very high identity with Arabidopsis cDNAs and the expression patterns observed by semi-quantitative RT-PCR analyses also followed the kinetics reported in Arabidopsis. This work is supported by the FInteruniversity Attraction Poles Programme – Belgian State – Federal Office for Scientific, Technical and Cultural Affairs_ P5/13; M.D. is grateful to the FFonds pour la formation a` la Recherche dans l’Industrie et l’Agriculture_ (F.R.I.A.) for the award of a PhD fellowship.
P5.33 The FLC-dependent vernalisation pathway in Sinapis alba M. D’Aloia, P. Tocquin, C. Pe´rilleux, (University of Lie`ge) Arabidopsis, the vernalisation pathway was shown to promote flowering via the repression of the FLOWERING LOCUS C (FLC) gene, which encodes a repressor of flowering. As far as we know, the genetic control of flowering is conserved among Brassicaceae, and we have reported elsewhere cloning of flowering time genes of the photoperiodic pathway in Sinapis alba, based on sequence similarity with Arabidopsis. However, little is known about vernalisation in Sinapis. We therefore undertook a physiological and molecular study of this process. Plants of Sinapis were grown in non-inductive short days and vernalised at 7 -C, at the seedling stage. Vernalisation was found to accelerate flowering and an increasing effect was observed for vernalisation treatments longer than 2 weeks. We cloned an FLClike sequence (SaFLC) by screening a cDNA library, and used it as a probe to perform expression analyses. We observed that SaFLC was almost completely repressed after 1 week of vernalisation, but repression was stable only after 2 weeks, which is consistent with the fact that 2-week is the minimal duration of vernalisation that promotes flowering. Hence the molecular mechanisms of vernalisation seem to be conserved in Sinapis and Arabidopsis. M.D. is grateful to the FFonds pour la formation a` la Recherche dans l’Industrie et l’Agriculture_ (F.R.I.A.) for the award of a PhD fellowship.
P5.34 Genetical control of sympodial growth and flowering in tomato J. Thouet, S. Ormenese, C. Pe´rilleux, (University of Lie`ge) Tomato is a day neutral plant with an indeterminate growth habit: after production of the first inflorescence, growth is taken over by the sympodial meristem, that is the last axillary meristem formed before floral transition. Several genes, like SELF-PRUNING (SP), JOINTLESS (J), SINGLE FLOWER TRUSS (SFT) and FALSIFLORA (FA), have been identified whose mutation modifies flowering time and morphogenesis. Our aim is to contribute to a better understanding of the interactions between these genes, and to compare this network with models elaborated for monopodial species, such as Arabidopsis thaliana and Antirrhinum majus. The SP gene is thought to control sympodial growth, since sp mutants show a determinate growth habit (Pnueli et al., 1998). We performed in situ hybridizations to study SP expression in vegetative
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and floral shoots. In a wild type background (Ailsa Craig), SP transcripts were detected in sympodial and other axillary meristems, but not in the apical meristem. Hence SP does not control the destiny of lateral meristems on its own. The expression pattern was unchanged in a j:sp mutant, suggesting that expression of SP does not require J function.
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This work is supported by the FFonds de la Recherche Fondamentale Collective_ (F.R.F.C. no. 2.4534.05); J.T. is grateful to the FFonds pour la formation a` la Recherche dans l’Industrie et l’Agriculture_ (F.R.I.A.) for the award of a PhD fellowship. References: Pnueli et al., 1998. Development 125, 1979 – 1989.
Society for Experimental Biology Annual Main Meeting 2nd–7th April 2006, University of Kent at Canterbury, UK
P5–FLOWERING Organised by N. Battey (University of Reading) Sponsored by: Journal of Experimental Botany
P5.2 Keynote Paper: The flowering process G. Bernier, (University of Liege) During several decades following World War II a very large knowledge base concerning the physiology of flowering was produced in the world. My main contribution comes from work with Sinapis alba where ‘‘florigen’’ was found to be multifactorial and to induce precisely timed events at the meristem. I have edited the Flowering Newsletter from 1989 to 2005, a period that will probably be remembered as the ‘‘golden’’ age of the genetical/ molecular study of the flowering process. This period started in 1990 with the cloning of the first floral homeotic genes DEFICIENS and FLORICAULA from snapdragon and AGAMOUS from Arabidopsis, and with the proposal in 1991 of the ABC model of flower organogenesis. Culmination of this period might have been reached in 2004 – 2005 with the claim that a specific component of the long-tracked ‘‘mythical’’ florigen in Arabidopsis is the transcript or the protein of the FLOWERING LOCUS T gene. Work during this last period was performed mainly with Arabidopsis and secondarily with few other species. At this stage an important challenge is to achieve integration between these two very large and potentially complementary bodies of information. Although some progress has been recorded recently in this area, a serious problem is that the plants best suited for the genetical/ molecular work are usually different from the best model plants for the physiological work. Further integration will require imaginative efforts from researchers of both sides. Suggestions for progress towards this goal will be discussed.
P5.3 Vernalization and the epigenetic memory of winter R. Amasino, (Wisconsin) Certain plants, such as biennials or winter annuals, require relatively long periods of cold exposure during winter to initiate flowering the doi:10.1016/j.cbpa.2006.01.054
following spring. Cold exposure renders the meristem of such coldrequiring species competent to flower, and this acquisition of competence is known as vernalization. A vernalization requirement ensures that flowering does not occur prematurely before the onset of winter. A similar cold response is bud dormancy; in many species that grow in temperate climates, bud dormancy is not broken until a the plant has ‘‘counted’’ a sufficient number of days of cold to ensure that any subsequent warn weather actually indicates that spring has arrived. Our studies of vernalization in Arabidopsis have revealed that meristem competence is a function of the expression level of certain MADS-box genes such as FLOWERING LOCUS C (FLC) that act as repressors of flowering. Exposure to prolonged cold causes an epigenetic switch of these MADS box genes to an unexpressed state, thus rendering the shoot apical meristem competent to flower. This epigenetic switch is caused by covalent modifications to histones of the chromatin of the flowering repressors.
P5.4 The autonomous pathway: Linking RNA processing, RNAi and chromatin regulation C. Dean, F. Liu, P. Crevillen, I. Baurle, (John Innes Centre) The timing of the floral transition has significant consequences for the reproductive success of plants. Plants need to gauge when both environmental and endogenous cues are optimal before undergoing the switch to reproductive development. To achieve this, a complex regulatory network has evolved consisting of multiple pathways that quantitatively and antagonistically regulate the genes whose activity causes the transition of the meristem to reproductive development. The Dean group has focused on a set of pathways that regulate the strong floral repressor, FLC. FLC expression needs to be effectively shut-down both for the timely transition to flowering and for normal flower development. One pathway that represses FLC expression involves FCA, an RNAbinding protein which interacts with SWI3b and FY, a highly
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conserved protein known as Pfs2p in S. cerevisiae. Pfs2p is an essential protein which functions to regulate polyadenylation and 3V processing of mRNAs. In Arabidopsis, FY and FCA have also been demonstrated to regulate mRNA 3Vend processing in that they regulate poly A site choice in the FCA transcript. FY is required for the negative feedback regulation of FCA and null mutations are embryo lethal. In order to determine how FCA and FY regulate FLC, we have undertaken a genetic approach and have identified sof1 as a suppressor of FCA overexpression. sof1 carries a mutation in FLD, a regulator of FLC chromatin structure. FLD was reported as mediating histone deacetylation at FLC but a human homologue LSD1 has recently been described as the first histone lysine 4 demethylase. We have also shown that DCL3, previously shown to be required for RNAdependent DNA methylation and heterochromatin formation, is required for FCA repression of FLC. These data suggest a model whereby FCA and FY cause changes in FLC RNA processing that trigger an RNAi-mediated chromatin regulation involving histone demethylation.
P5.5 Light signals in flowering B. Thomas, A. Massiah, S. Jackson, K. Morris, S. Adams, (U. Warwick) Plants modify their flowering behaviour in response to a range of changes in the light environment. The best known of these is the effect of timing of light signals in photoperiodic responses. Here, light acts to entrain the circadian rhythm underlying the daylength response and also interacts with the output of the clock to generate flowering signals. In addition, light can modify flowering times separately from its action on the photoperiodic pathway through quality and irradiance-dependent responses and can also have a major influence on the onset flowering through effects on the duration of the juvenile phase of development. In all cases, responses to light are mediated through specific light receptors. These could include known photoreceptors such as phytochromes and cryptochromes, photosynthetic pigments or other light-sensing molecules with specific functions in flowering. In this talk, evidence for several light-dependent pathways that lead to altered flowering time will be presented and the roles of different light sensors discussed.
P5.6 Mechanistic modelling of flowering regulators based on molecular data A. Millar, P. Brown, T. Saithong, (University of Edinburgh); D. Salazar, J. Locke, I. Carre, D. Rand, (University of Warwick) Day-length (photoperiod) measurement depends on the circadian clock, which thereby affects seasonal rhythms including flowering. Flowering is regulated by numerous other environmental and developmental factors. With collaborators in the IPCR at Warwick, we developed the first mathematical models of the Arabidopsis clock gene network, incorporating 6 genes in two, interlocking negative feedback loops. Experiments based upon predictions of the model identified a novel and central function for GIGANTEA (GI)
in the clock network (Locke et al., Molecular Systems Biology 2005). Such successful prediction of a genetic network structure is a goal of many systems biology groups, and is clearly possible for these plant gene networks. We have recently extended the clock model to include the photoperiod-responsive switch formed by CO and FT. These show that the simple external coincidence model has been considerably elaborated in Arabidopsis. Results from alternative model structures will be discussed, showing, amongst other points, that the photoperiod-dependent entrainment of the circadian clock is important in explaining the molecular rhythms. The models allow us to quantify phase-specific modifiers of the external coincidence mechanism, using timeseries data only from wild-type plants: modelling adds value to timeseries data even without experimental manipulations. We have also extended our experimentalist-friendly simulation software, Circadian Modelling, to simulate flowering time experiments; the software is available at www.amillar.org.
P5.7 Control of GIGANTEA gene expression by light and the clock F. Cremer, R. To´th, A. Giakountis, I. Bu¨rstel, G. Coupland, (Max Planck Institute for Plant Breeding Research) The photoperiodic pathway of floral induction is controlled by the interaction of light signals with the circadian clock. GIGANTEA (GI), CONSTANS (CO)and FLOWERING LOCUS T (FT)are part of a gene cascade controlled by the clock and inducing the floral transition of Arabidopsis in response to long days. GI is expressed with an evening phase and has a positive effect on the amplitude of the rhythmic pattern of CO transcription. Independently of its effect on floral induction, GI is also involved in other light signaling pathways and affects the circadian clock. We have used promoter fusions to luciferase to show that GI transcription is induced by blue, red and far-red light and that this response is gated by the circadian clock, with a higher induction at peak times of circadian GI expression than at trough times. The diurnal peak of GI expression also occurs earlier in short than in long days and the acute light response follows the same pattern. In order to understand the control of the timing of GI expression, we are analyzing its promoter using deletions and phylogenetic shadowing. In another approach, we are studying the pattern of GI expression under different photoperiods in relation to flowering time. This last approach has been extended to the study of natural variation in patterns of GI expression in a group of a hundred accessions. These and further results will be presented at the meeting.
P5.8 Crucial role of outgroups in inferring the likely reproductive characteristics of the first angiosperm R. Bateman, (Natural History Museum); J. Hilton, (University of Birmingham); P. Rudall, (Royal Botanic Gardens Kew) Recent attempts to address the long-debated ‘‘origin’’ of the angiosperms depend on a phylogenetic framework derived from a matrix of taxa versus characters, and assume that empirical rigour
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is proportional to the size of the matrix. Sequence-based approaches increase the number of characters (nucleotides) in the matrix but are confined to the highly restricted spectrum of extant species, whereas morphology-based approaches increase the number of phylogenetically informative taxa (including fossils) at the expense of accessing only a restricted spectrum of phenotypic characters. The two approaches are currently delivering strongly contrasting hypotheses of relationship. Most molecular studies indicate that all extant gymnosperms form a natural group, suggesting surprisingly early divergence of (pre-)angiosperms, whereas morphology-only phylogenies indicate that a succession of (mostly extinct) gymnosperms preceded a later angiosperm origin. Causes of this conflict include: (1) the vast phenotypic and genotypic lacuna, largely reflecting preTertiary extinctions, that separates early-divergent living angiosperms from their closest relatives among the living gymnosperms; (2) profound uncertainty regarding which (a) extant and (b) extinct angiosperms are most closely related to gymnosperms; and (3) profound uncertainty regarding which (a) extant and (b) extinct gymnosperms are most closely related to angiosperms, and thus best serve as ‘‘outgroups’’ dictating the perceived evolutionary polarity of character transitions among the early-divergent angiosperms. These factors still permit a remarkable range of hypotheses regarding the order in which the many phenotypic characters that distinguish ‘‘classic’’ angiospermy from ‘‘classic’’ gymnospermy were acquired. Focusing on reproductive characters, we will explore some of the more strongly contrasting hypotheses.
P5.9 Novel genes and their putative functions in grasses E. Kellogg, (University of Missouri-St. Louis) A central problem in evolutionary biology is the diversification of form. This question is the basis of the field of evolutionary developmental biology, which links phylogenetic studies with developmental genetics, often in species that are not model organisms. Recent work in this field can generally be placed in one of two categories. First is the direct, genetic approach, which uses experimental mapping populations to identify quantitative trait loci (QTL) controlling the difference between crossable species, and may in some cases lead to identification of genes. Second is a candidate gene approach, which chooses genes whose mutant phenotype in a model organism is similar to the natural phenotype of a wild organism, and investigates the naturally occurring variation in that gene. Combining the two approaches has proven particularly fruitful in studies of grasses (Poaceae), in which numerous genes have been cloned that control aspects of inflorescence architecture. Because of a long and complex history of gene duplication, many inflorescence genes are unique to the grasses; the presence of multiple novel genes correlates with the presence of multiple morphological novelties in the family. QTL studies in many grass species, particularly the cereal crops, have identified loci that control particular aspects of inflorescence form. For some species, candidate genes have been identified that fall in the QTL. Association analysis shows that some of these genes could be important for inflorescence evolution among all grasses.
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P5.10 Flower-like terminal structures in racemose inflorescences: a tool in morphogenetic and evolutionary research D. Sokoloff, M. Remizowa, (Moscow State University); P. Rudall, (Royal Botanic Gardens, Kew) Terminal flower-like structures (TFLS) occur in many angiosperms that possess (partial) inflorescences such as spikes, racemes, or spadices. Using both original and review data, we consider the occurrence, morphology, and some developmental aspects of TFLS in early-divergent angiosperms and monocots. We explore the hypothesis that structures with long-debated pseudanthial or euanthial interpretation in various angiosperm groups could have evolved as a result of overlap in developmental programs of a typical flower and inflorescence. Spikes and spadices are typical for both Chloranthaceae and perianthless Piperales among earlydivergent angiosperms, but whereas TFLS occur in some Piperales such as Saururaceae and a few Piperaceae, they are absent from Chloranthaceae. Within the monocot clade, TFLS are here described in some early-divergent families such as Acoraceae, Aponogetonaceae, Potamogetonaceae and Ruppiaceae. Thus, essentially similar TFLS occur in wide range of taxa. Similar TFLS with obscure organ identity have been found in mutants of model organisms such as Arabidopsis. TFLS can often be interpreted as pseudanthia (close aggregations of reduced flowers), but in some cases the entire terminal pseudanthium is very similar to a true flower. Elaborated TFLS could therefore give rise to what we normally term true flowers. Furthermore, unusual structures frequently occur at or near the inflorescence tip; for example, the entire inflorescence apex or an individual flower primordium can be transformed into either a filamentous or tubular structure, or an intermediate form. This indicates that pseudanthium formation can provoke morphological novelties, perhaps due to new patterns of overlap between expression zones of regulatory genes.
P5.11 The evolution and development of petaloidy in Aizoaceae (Caryophyllales) S. Brockington, (Florida Museum of Natural History); P. Rudall, (Royal Botanic Gardens Kew); M. Frohlich, (Natural History Museum); D. Soltis, (University of Florida); P. Soltis, (Florida Museum of Natural History) An understanding of the genetic program controlling floral organ identity in eudicot species such as Arabidopsis thaliana and Antirrhinum majus has led to new approaches in the study of floral diversity and petal evolution. More specifically, it has been proposed that MADS-box genes may play an important role in generating floral diversity through changes in gene expression pattern. The family Aizoaceae provides a good opportunity to test this hypothesis because within this clade significant differentiation has occurred in the development of different floral organs as attractants for pollinators. In the early divergent subfamilies Sesuvioideae and Aizooideae the tepals of the uniseriate perianth are petaloid on the adaxial side,
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whereas in subfamilies Mesembryanthoideae and Ruschoideae the tepals are not petaloid and instead outer whorls of stamens are sterile and showy. In advance of gene expression analysis we have characterised the floral development of several species of Aizoaceae including some abnormal floral variants. Examination of the epidermal and sub-epidermal features of the petaloid tepals and petaloid staminodes reveals few morphological similarities between the different kinds of petaloid organ. The development of the petaloid tepals is similar to that of a monocot foliage leaf: the lamina is formed from the leaf base region. Abnormal floral variants suggest that specification of petaloid tissue is dissociable from tepal morphogenesis in development. We speculate on the significance of this unusual mode of tepal development for the evolution of petaloidy and make specific hypotheses regarding expected patterns of MADS-box gene expression.
P5.12 Capsella as a model system to study the evolution of flower development G. Theissen, M. Hintz, P. Nutt, J. Ziermann, C. Bartholmes, (University of Jena); B. Neuffer, (University of Osnabrueck) Capsella is a small genus within the mustard family (Brassicaceae). It has only three species which nevertheless show many evolutionary trends also found in other Brassicaceae (including Arabidopsis) and far beyond, including transitions from a diploid, self-incompatible, obligatory outcrossing species with comparatively large and attractive flowers but a quite restricted area of distribution, to a polyploid, self-compatible, predominantly selfing, invasive species with floral reductions. All these transitions may have contributed to the fact that Capsella bursa-pastoris (Shepherd’s purse) became one of the most widely distributed flowering plants on our planet. In addition, Capsella bursa-pastoris shows a very rare phenomenon which, nevertheless, could be of great evolutionary importance, i.e. the occurrence of a homeotic variety in quite stable populations in the wild. Several lines of evidence suggest that homeotic changes played a considerable role during the evolution of flowers, but how floral homeotic varieties are established in natural populations has remained a highly controversial topic among evolutionary biologists. Due to its close relationship to the model plant Arabidopsis thaliana numerous experimental tools are available for studying the genus Capsella, and more tools are currently being developed. Capsella thus provides great opportunities to investigate the evolution of flower development from molecular developmental genetics to field ecology and biogeography, and from morphological refinements to major structural transitions.
P5.13 The role of the FT mRNA in photoperiodic regulation of plant growth and development
gene CONSTANS (CO). We have shown that local induction of FT in a single Arabidopsis leaf is sufficient to trigger flowering and that the FT mRNA is transported to the shoot apex, where downstream genes are activated. These data suggest that the FT mRNA is an important component of the elusive ‘‘florigen’’ signal that moves from leaf to shoot apex. In contrast to annual plants like Arabidopsis, forest trees display a perennial growth behaviour characterized by a very extended juvenile phase before flowering, and, in temperate regions of the world, an annual cycling between growth and dormancy. We show here that the CO/FT regulatory module is functionally conserved in the aspen tree where it controls the timing of flowering. However, unexpectedly, it also controls the short-day induced growth cessation and bud set normally occuring in the fall. This suggests that FT is not a specific regulator of flowering, but might have a more general role in regulating biological processes controlled by variations in day length.
P5.14 Analysis of the expression pattern of FT protein during flowering L. Corbesier, C. Vincent, I. Searle, F. Fornara, G. Coupland, (Max Planck Institute for Plant Breeding Research) Flower development at the shoot apex is initiated in response to environmental cues. Among these, daylength is one of the most important and is perceived by the mature leaves. When exposed to inductive photoperiodic conditions, these leaves produce endogenous signals that are transported to the shoot apical meristem where they cause the transition from leaf to flower morphogenesis. In Arabidopsis, genetic analysis identified a pathway of genes required for the initiation of flowering in response to daylength. The nuclear zincfinger protein CONSTANS (CO) plays a central role in this pathway and, in response to long days, activates the transcription of FLOWERING LOCUS T (FT), which encodes a RAF-kinase inhibitor-like protein. Since CO has been shown to trigger flowering when expressed in the phloem but not when expressed in the meristem itself, it was proposed that CO regulates the synthesis or the transport of a floral signal in the phloem and this involves partly the cell autonomous activation of FT. A product of FT might comprise this signal and recently FT mRNA was proposed to move from the leaves to the shoot apex. The FT protein is only 23 kilodaltons, and we have tested whether this can move in the phloem to the meristem. We will present our work aiming to test FT protein translocation with the help of tissue-specific promoters driving FT:GFP constructs, confocal microscopy, in situ immunolocalisation and in situ hybridization.
P5.15 The tomato FT gene triggers conserved systemic flowering signals that regulate termination and substitute for light and photoperiodic stimuli
O. Nilsson, (Umea˚ Plant Science Centre) Day length controls flowering time in many plants. The daylength signal is perceived in the leaf, but how this signal is transduced to the shoot apex where floral initiation occurs has not been known. In Arabidopsis, the day-length response depends on the induction of the FLOWERING LOCUS T (FT) gene by the
E. Lifschitz, T. Eviatar, A. Rozman, A. Shalit, (Technion); A. Goldshmidt, Z. Amsellem, J. Alvarez, Y. Eshed, (Weizmann Institute of Science) The FLORIGEN model implies a mechanism that is common to all plants but is activated by different flowering stimuli in different
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species. During transition to flowering in annuals with a monopodial habit like Arabidopsis, the replacement of the vegetative phase by a single reproductive phase signals the completion of the life cycle. Growth habits of other, particularly perennial plants require the cohabitation of vegetative and floral buds along their shoots and thus reiterated vegetative-reproductive transitions. Tomato plants are perennial in their native habitat, flowering is independent of photoperiodism, and the sympodial shoots, inflorescence shoots and leaves are compound, consisting of determinate morphogenetic duplications. This modular organization permits the analysis of multiple flowering events in a single plant and affords an architectural flexibility which is reflected in a unique range of developmental variants. SINGLE FLOWER TRUSS, a regulator of flowering time and morphogenesis, encodes the tomato ortholog of FT (TFT). We show that SFT-stimulated signals abide by all tenets of the florigen hypothesis. Grafting and leaf-specific expression show that SFT generates systemic signals which substitute for light and photoperiodic stimuli in Arabidopsis and tobacco and for innate morphogenetic signals in tomato. SFT and its interacting proteins are co-expressed in leaves and the SFT protein is localized to leaf nuclei. In tomato grafts, the donor SFT-RNA could not be detected in flowering receptor shoots. The role of SFT in growth and termination and its contribution to the vegetative/reproductive balance and plant architecture in tomato and Arabidopsis will be discussed.
P5.16 FT signalling in the floral transition P. Wigge, (John Innes Centre) The floral transition is a critical decision in plant development. Reflecting this, there are numerous pathways that sense both environmental and endogenous signals in order to control flowering time. A critical integrator of signalling information in the floral decision is FT. FT is a potent inducer of flowering when overexpressed. Early genetic studies have shown that an FT acts in parallel with the transcription factor LEAFY to promote flowering. FT has homology at the amino acid sequence level to small soluble proteins found in animals and fungi called PEBPs or RKIPs. We and others have been able to show that FT signalling is dependent on the bZIP transcription factor FD. An important target of FT signalling appears to be the transcription factor AP1, as this is precociously upregulated in 35S FT plants. We have shown that this activation of AP1 is FD dependent. Furthermore, knock-out lines for this transcription factor are late flowering and these are able to partially suppress the overexpression of 35S FT. We show that FD activation of the AP1 promoter is strongly dependent on the presence of FT, and moreover FD is expressed in the young flowers, suggesting that it provides the spatial information for FT signalling. We are investigating the role of relatives of FT and FD in control of the vegetative to floral transition.
P5.17 Protein trafficking of FT/TFL1 and flowering signal transmission K. Goto, (Res. Inst. Biological Sciences) FT and TFL1 of Arabidopsis are highly homologous proteins but their functions in flowering pathway are opposite; while FT promotes, TFL1
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retards flowering. In addition, TFL1 functions to maintain inflorescence meristem (IM) and repress floral meristem (FM) development. For this function, TFL1 is required in the whole region of IM, but its transcript was localized inner region of L3 layer of IM. We have revealed this noncell-autonomous function of TFL1 is conferred by protein trafficking. We also found that FT moves among cell layers of IM. The endogenous FT is expressed in the vascular bundle of leaf apical region, and recent study revealed that FT functions with FD in the shoot apical meristem (SAM). Taken together, the following scenario can be deduced; after long distance travel from leaves via vascular bundle, FT is unloaded near the SAM (vascular bundle is undifferenciated in the SAM) and then FT protein moves to spread into the SAM to promote phase transition. Another recent study suggests that FT RNA moves from the leaf to the SAM. To confirm this, we developed grafting system using tobacco. Since transgenic tobacco carrying Arabidopsis FT showed early flowering, we used it as a rootstock and wild type scion was grafted. We found the scions acquired early flowering phenotype. Now we are investigating FT RNA and/or protein are transmitted from stock to scion.
P5.19 Protein complexes make the flower G. Angenent, S. de Folter, I. Nougalli, R. Immink, (Plant Research International) The MADS box transcription factor family is one of the most important families of regulatory proteins in plants. The genes are involved in many developmental processes, such as root development, flowering, organ identity specification, and fruit and seed formation. We studied members of this family in Petunia and Arabidopsis. The MADS box transcription factors form dimers and it has become apparent recently that higher order complexes between several MADS box proteins form the basis for floral organ specification. Most likely all MADS box proteins form these multiprotein complexes. To unravel a part of the network of protein complexes in which MADS proteins play a role, we performed a comprehensive yeast two- and three-hybrid screen between all Arabidopsis MADS box proteins using a matrix-based approach. The obtained interactions confirmed known interactions and revealed many novel interactions. These interactions and data about target genes of these MADS box factors suggest that an extensive network exists in which MADS box complexes regulate their own expression by positive and negative autoregulatory loops. Preliminary work indicates that flowering genes, e.g. SOC1, AGL24, SVP, are negatively regulated by floral organ identity proteins. Currently, Chromatin Immuno Precipitation (CHIP) and yeast one-hybrid experiments are being performed to confirm these models.
P5.20 Flower development in rice M. Kater, L. Dreni, N. Pelucchi, L. Colombo, (University of Milan) Flower development has been intensively studied in the dicot plant species Antirrhinum majus and Arabidopsis thaliana. Analysis of homeotic mutants resulted in the beginning of the nineties in the formulation of the ABC model which postulates that three classes of genes control floral organ identity in a combinatorial way. Isolation and characterisation of these ABC genes showed that most of them belong to the MADS-box family of transcription factors. Although the floral
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structure of the rice flower is different, especially in respect to the perianth organs, genes homologous to the ABC genes of dicots have also been identified in rice. Functional analysis of several of them showed that to a certain extent their function seems to be conserved in rice. In subsequent years the ABC model has been extended with class D and E genes. D genes have shown to control ovule identity and class E genes (or SEPALLATA genes) are necessary for the function of class A, B, C and D genes. Recently we have started with the characterisation of class D genes of rice. An overview of flower development in rice will be presented and the latest results concerning rice class D genes will be discussed.
P5.21 Characterisation of the HAWAIIAN SKIRT gene, which encodes a novel F-box protein invloved in sepal boundary development Z. Gonzalez-Carranza, J. Roberts, (The University of Nottingham) Boundary regulation in plants plays an important role in the formation of floral organs. Recently we have mapped the location of Hawaiian skirt, a novel mutant from Arabidopsis that exhibits aberrant sepal separation, and characterised the gene that it encodes. The HAWAIIAN SKIRT gene encodes a novel F-box protein that exhibits a low level of amino acid homology to the mutant UFO. Fbox proteins have been shown to form a key component of the SCF complex and have been proposed to target the degradation of specific substrates by the 26S proteasome (Ni et al., 2004). Characterisation of the temporal and spatial expression of the HS gene by RT-PCR, and fusion of its promoter to the reporters GUS or GFP, has revealed that transcript accumulation takes place in both root and shoot tissues and as a consequence of wounding. The possible role of this gene during Arabidopsis development is discussed. 1. Ni, W., Xie, D., Hobbie, L., Feng, B., Zhao, D., Akkara, J., Ma, H., 2004. Regulation of flower development in Arabidopsis by SCF complexes. Plant Physiology 134, 1574 – 1585.
P5.22 Reproductive meristem fates in Gerbera T. Teeri, M. Kotilainen, R. Laitinen, H. Help, P. Elomaa, (University of Helsinki); V. Albert, (University of Oslo); A. Uimari, (University of Kuopio) Flowering plants go through several phases between regular stem growth and the actual production of flower parts. The stepwise conversion of vegetative into inflorescence and floral meristems is usually unidirectional, but under certain environmental or genetic conditions, meristems can revert to an earlier developmental identity. Vegetative meristems are typically indeterminate, producing organs continuously, whereas flower meristems are determinate, shutting down their growth after reproductive organs are initiated. Inflorescence meristems can show either pattern. We have investigated flower and inflorescence development in Gerbera hybrida, an ornamental plant in the sunflower family, Asteraceae. Unlike the common model species used to study flower development (Arabidopsis, Antirrhinum and Petunia), Gerbera bears determinate inflorescences, and the architecture of the flower
differs in that Gerbera ovaries are inferior. We have shown that floral meristem determinacy and identity are spatially and genetically distinct in Gerbera, and that a single SEPALLATAlike MADS domain factor controls both flower and inflorescence meristem fate in the plant. Although these phenomena have not been directly observed in Arabidopsis, the integrative role of the SEPALLATA function in reproductive meristem development may be generalizable to all flowering plants.
P5.23 Carpel and fruit patterning in Arabidopsis C. Ferrandiz, (Instituto De Biologia Molecular Y Celular De Plantas. CSIC-UPV) The fruit is a major evolutionary acquisition of the angiosperms, basically derived from the flower gynoecium, which elongates and differentiates after fertilization of the ovules to protect and disperse seed. Fruits display a great variety of morphological structures among the angiosperms and we are beginning to get some insight into the genetic basis of this diversity. In the model plant Arabidopsis thaliana, genetic analyses have identified a small set of regulatory genes that establish the initial patterning of gynoecium development along the longitudinal and transverse axes. In addition, auxin gradients appear to play a key role in establishing apical-basal patterning in the developing Arabidopsis gynoecium, but the genetic pathways involved remain unclear. In this talk we will discuss the current models on fruit patterning and the contribution of our lab to the characterization of a small subfamily of transcription factors, the TOWER-OF-PISA genes, that could work to coordinate development along these two main axes. We will also discuss some of our recent work on comparative fruit development. We are trying to address how general are the proposed models and, for that purpose, we are beginning to explore these questions in legumes with different fruit morphologies (such as pea or Medicago) by cloning and functionally characterizing orthologous genes to those with a defined role in Arabidopsis gynoecium development.
P5.25 Effects of light level on the juvenile phase in Antirrhinum B. Thomas, S. Adams, V. Valdez, A. Jackson, A. Massiah, (U. Warwick) Plants raised from seed usually pass through a stage of development where they are incapable of initiating flowers, irrespective of environmental treatments. This is referred to as the juvenile phase and may last for weeks or, in some cases, years. After passing through this stage with progression to the adult phase, many plants become competent to flower but will not do so unless they receive an additional environmental stimulus such as a permissive photoperiod and/or a period at a particular temperature, to initiate flowers. The juvenile to adult phase change within the vegetative phase and vegetative – reproductive phase change are distinct processes that influence each other. In Antirrhinum the length of the juvenile phase can be modified by the light levels at which plants are grown. This is not simply because of slower development as plants at lower light levels do not become competent to respond to inductive conditions
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until they are bigger, with more leaves than plants grown at higher light levels. In order to understand further this response, we will describe the effect of light level on assimilates, apical development, growth and gene expression in Antirrhinum during the juvenile phase.
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mutation. Mass spectrometry methods are being used to elucidate the protein identity and database searching utilised to determine potential protein function.
P5.29 P5.26 Molecular characterisation of the day neutral flowering (dnf) mutant of Arabidopsis S. Thornber, S. Jackson, K. Morris, L. Codrai, (Warwick HRI) Arabidopsis is a facultative long day plant and as such flowers under both long and short days but sooner under long day conditions. To look for novel flowering mutants of the photoperiodic pathway a TDNA tagged population of Arabidopsis was screened for altered flowering times in long and short days. A novel Arabidopsis mutant, termed day neutral flowering (dnf) was isolated as a result of this screen and displays no short-day inhibition of flowering. The dnf mutant, therefore, flowers at the same time under both long and short day conditions as WT does under LD conditions. Successful complementation using the wild-type DNF gene under the control of its endogenous promoter confirmed that the dnf mutation is responsible for the observed phenotype. The DNF gene is 141 amino acids in length and contains a predicted membrane spanning domain between residues 13 and 33 and a putative RING finger domain between residue 79 and 121. Many RING finger proteins are E3 ubiquitin ligases that target specific proteins for degradation by the 26S proteasome. In an attempt to identify the target(s) of the DNF RING finger protein we have adopted an in vivo pull-down approach and have produced plants expressing a TAP tagged DNF protein. Recent progress and the hypothesised role of DNF in the control of flowering will be discussed.
P5.27 Development of protein analysis methods for MALE STERILITY 1 (MS1) protein in Arabidopsis thaliana E. Hewitt, (University of Nottingham) The MALE STERILITY1 gene in Arabidopsis encodes a nuclear localised protein expressed in the tapetal cells of the anther. It is proposed to be a transcriptional regulator of male gametogenesis, which is required for pollen maturation and contains a PHD finger motif thought to participate in protein-protein interactions. It has therefore been suggested that the MS1 protein acts as part of a protein complex that is vital in the process of male gametophyte development. We are currently investigating the role of the MS1 protein and the effects of its mutation on the male gametophyte developmental pathway. To this end a reproducible 2-dimensional (2-D) protein gel electrophoresis method has been developed which can be used to study the expression of anther proteins. Total protein has been isolated from buds and anthers of wild type plants for 2D gel analysis. Relatively high yields of good quality total protein could be isolated from anther tissue and this was therefore selected as the source tissue for the comparative analysis. Total protein isolated from anthers of wild type plants has been compared to that from ms1 mutant anthers to identify alterations in protein expression associated with the ms1
Micropropagation of Chrysanthemum: securing novel mutations M. Davey, P. Anthony, L. Cheetham, B. Power, K. Lowe, (University of Nottingham) Floriculture is globally a major industry driven by consumer demands for novel flower architecture and colour. Genetic diversity within some ornamental plants has produced commercially-important variants (Fsports_), particularly in Chrysanthemum (Dendranthemum). However, the ease with which such sports can be secured by cuttings depends upon the extent of mutation. Micropropagation can be exploited to secure mutations limited to petals and/or sectors of petals. Petals of the spray Chrysanthemum cv. White Enbee Wedding [flower colour White Group 155A; RHS Colour Chart and Flower Council of Holland] that had sported to Yellow Group 13B were removed from influorescences, surface sterilized and transferred to 50 ml of MSbased medium supplemented with 2.0 mg l 1 a-naphthaleneacetic acid and 0.5 mg l 1 benzylaminopurine contained in 175 ml glass jars. Cultures were maintained at 25 T 2 -C in the light (50 mmol m 2 sec-1; 16 h photoperiod) for 100 days. Regenerated shoots were transferred to MS-based medium, lacking growth regulators for rooting and, subsequently, to compost. Plants were grown to flowering and assessed over 2 years for flower colour and morphology. Flowers from regenerants exhibited a colour index of Yellow Group 12A, 12A/ B or 12B. In 15% of regenerants, the mean influorescence diameter was significantly (P < 0.05) greater than control. In contrast, the mean number of petals per flower was significantly (P < 0.05) reduced, but the mean number of flowers per stem was unaltered. Interestingly, several plants developed flowers with altered floral architecture owing to superimposed somaclonal variation.
P5.30 Genetical approaches towards understanding flowering time regulation in tomato M. Quinet, H. Batoko, J. Kinet, (Universite´ catholique de Louvain, Belgium) Our aim through the study of flowering in tomato, a sympodial plant, is to contribute to enlarge the knowledge from Arabidopsis to other species. We investigated different late flowering mutants and double mutants, generated from these single mutants, all having as a common parent the uniflora mutant which has the most delayed flowering. All double mutants were late flowering. uniflora: blind and uniflora :self pruning had a flowering time intermediate between their two parents. jointless: uniflora and compound inflorescence: uniflora flowered later than uniflora. They also developed strong lateral shoots at node levels grossly corresponding with the level where their parent cultivars initiated their first reproductive structure, which is a typical trait of uniflora. This suggests that these plants underwent partial evocation but that they were unable to complete floral transition. This phenotype could also be indicative of the occurrence of processes upstream of UF that direct the primary shoot SAM into reproductive growth and of the
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absence of influence of the genes we investigated upon the timing of events that initiate floral transition of this SAM. Our results also suggest that, from that time point, the different genes were interacting to regulate flowering time. Unravelling the regulation of floral transition in tomato is thus complicated by the fact that tomato has a sympodial growth habit and that the process is apparently not regulated in the same way in primary shoot and sympodial segments.
P5.31 Control of floral transition in maize F. Van Kerkhoven, N. Jenne`s, C. Pe´rilleux, (University of Lie`ge) Maize (Zea mays L.) is a quantitative short-day plant, although floral induction is largely autonomous in modern genotypes. Maize is a major crop but, despite being largely studied, little is known about the physiological and molecular mechanisms involved in the regulation of its floral transition. One outstanding fact is that - in reproductible growing conditions - the apical meristem produces a predictable total leaf number (TLN) before floral transition occurs. This led to the hypothesis that leaf counting is the basis of flowering time measurement. In this rationale, a change in leaf initiation rate should directly delay or accelerate flowering. To test this, we used hormonal treatments to modify the leaf initiation rate and regularly dissected the plants to determine the time of floral transition. Plants were grown in phytotronic chambers at 400 AE m 2 s 1, 16 h photoperiod and 24/18 -C day/night temperature. Benzyladenine 10 3 M (BA), gibberellic acid 10 3 M (GA3) or 2,4-dichlorophenoxyacetic acid 2.10 5 M (2,4-D) were applied in the centre of the leaf roll when plants were 15-day old. BA increased the leaf initiation rate and the TLN but delayed flowering. GA3 increased the leaf initiation rate but TLN was lower and floral transition was advanced. 2,4-D decreased the leaf initiation rate and the TLN but floral transition was unchanged. So, it appears that floral transition in maize is not simply determined by leaf counting. This work is supported by the FFe´de´ration Nationale de la Production des Semences de Maı¨s et de Sorgho’ (F.N.P.S.M.S., France); F.VK is grateful to the FFonds pour la formation a` la Recherche dans l’Industrie et l’Agriculture_ (F.R.I.A.) for the award of a PhD fellowship.
P5.32 Cloning of CONSTANS and FLOWERING LOCUS T in Sinapis alba’ K. Tamseddak, M. D’Aloia, C. Pe´rilleux, (University of Lie`ge) Flowering in photoperiodic species is known to require a Ffloral stimulus_ moving from the leaves- where photoperiod is perceived- to the shoot apical meristem. Although the nature of the floral stimulus has long remained elusive, molecular and genetic studies in Arabidopsis thaliana, a qualitative long day (LD) plant, showed that the product of the FLOWERING LOCUS T (FT) gene is a crucial signal that follows the floral stimulus route after FT has been activated in LD by the transcription factor CONSTANS (CO). On the other hand, changes in the translocation of nutrients and hormones have also been described during the transition to flowering. In Sinapis alba, flowering can be obtained by a single LD and a Fshoot-to-root-to-shoot_ physiological loop has been described which involves sucrose and cytokinin as components of the floral stimulus. In order to integrate the genetic data into this physiological network, we cloned CO and FT homologues in Sinapis and analysed their expression patterns. Cloning of SaCO was
performed by screening a cDNA library with an AtCO probe, and cloning of SaFT was done by RT-PCR with degenerated primers. The sequences obtained showed very high identity with Arabidopsis cDNAs and the expression patterns observed by semi-quantitative RT-PCR analyses also followed the kinetics reported in Arabidopsis. This work is supported by the FInteruniversity Attraction Poles Programme – Belgian State – Federal Office for Scientific, Technical and Cultural Affairs_ P5/13; M.D. is grateful to the FFonds pour la formation a` la Recherche dans l’Industrie et l’Agriculture_ (F.R.I.A.) for the award of a PhD fellowship.
P5.33 The FLC-dependent vernalisation pathway in Sinapis alba M. D’Aloia, P. Tocquin, C. Pe´rilleux, (University of Lie`ge) Arabidopsis, the vernalisation pathway was shown to promote flowering via the repression of the FLOWERING LOCUS C (FLC) gene, which encodes a repressor of flowering. As far as we know, the genetic control of flowering is conserved among Brassicaceae, and we have reported elsewhere cloning of flowering time genes of the photoperiodic pathway in Sinapis alba, based on sequence similarity with Arabidopsis. However, little is known about vernalisation in Sinapis. We therefore undertook a physiological and molecular study of this process. Plants of Sinapis were grown in non-inductive short days and vernalised at 7 -C, at the seedling stage. Vernalisation was found to accelerate flowering and an increasing effect was observed for vernalisation treatments longer than 2 weeks. We cloned an FLClike sequence (SaFLC) by screening a cDNA library, and used it as a probe to perform expression analyses. We observed that SaFLC was almost completely repressed after 1 week of vernalisation, but repression was stable only after 2 weeks, which is consistent with the fact that 2-week is the minimal duration of vernalisation that promotes flowering. Hence the molecular mechanisms of vernalisation seem to be conserved in Sinapis and Arabidopsis. M.D. is grateful to the FFonds pour la formation a` la Recherche dans l’Industrie et l’Agriculture_ (F.R.I.A.) for the award of a PhD fellowship.
P5.34 Genetical control of sympodial growth and flowering in tomato J. Thouet, S. Ormenese, C. Pe´rilleux, (University of Lie`ge) Tomato is a day neutral plant with an indeterminate growth habit: after production of the first inflorescence, growth is taken over by the sympodial meristem, that is the last axillary meristem formed before floral transition. Several genes, like SELF-PRUNING (SP), JOINTLESS (J), SINGLE FLOWER TRUSS (SFT) and FALSIFLORA (FA), have been identified whose mutation modifies flowering time and morphogenesis. Our aim is to contribute to a better understanding of the interactions between these genes, and to compare this network with models elaborated for monopodial species, such as Arabidopsis thaliana and Antirrhinum majus. The SP gene is thought to control sympodial growth, since sp mutants show a determinate growth habit (Pnueli et al., 1998). We performed in situ hybridizations to study SP expression in vegetative
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and floral shoots. In a wild type background (Ailsa Craig), SP transcripts were detected in sympodial and other axillary meristems, but not in the apical meristem. Hence SP does not control the destiny of lateral meristems on its own. The expression pattern was unchanged in a j:sp mutant, suggesting that expression of SP does not require J function.
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This work is supported by the FFonds de la Recherche Fondamentale Collective_ (F.R.F.C. no. 2.4534.05); J.T. is grateful to the FFonds pour la formation a` la Recherche dans l’Industrie et l’Agriculture_ (F.R.I.A.) for the award of a PhD fellowship. References: Pnueli et al., 1998. Development 125, 1979 – 1989.