Call for Papers - Abstract Submission
Please note that abstract submission for this meeting has now closed.
The full programme is now available here.
The abstracts for the meeting are shown below. If you are a speaker and have any corrections to be made to your abstract please email Kate Steel (k.steel@sebiology.org) or Ruth Bastow (ruth@arabidopsis.info) before Wednesday 20th August.
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Talks
L01
The responses of stomata to environmental signals
A Hetherington (University of Bristol), M Panagopulos (University of Bristol), D Worrall (University of Bristol), S Casson (University of Bristol), Y Liang (University of Bristol)
Dr Alistair Hetherington (Alistair.Hetherington@bristol.ac.uk)
Stomata are pores found on the surfaces of plant leaves. They control the uptake of carbon dioxide for photosynthesis and the loss of water vapour during the process of transpiration. The aperture of the stomatal pore is governed by the state of turgor of the two guard cells that surround the stomatal pore. When the guard cells are fully turgid the pore gapes open allowing gas exchange and conversely stomatal closure is associated with a loss of turgor. A wide range of environmental signals and plant hormones contribute to the control of stomatal development and aperture. Underlying changes in guard cell turgor and hence stomatal movements is a complex intracellular signalling network. An increase in the concentration of guard cell cytosolic free calcium ions has been shown to be involved in the response to many different signals. This lecture will discuss evidence that guard cell signalling is organised on a network basis and consider recent data related to how guard cells couple environmental signals to changes in stomatal aperture and development. Reference Hetherington AM & Woodward FI (2003) The role of stomata in sensing and driving environmental change. Nature, 424, 901-908
L02
A high light responsive chloroplast-to-nucleus retrograde signalling pathway in Arabidopsis involves discrete H2O2 sources linked by ABA and is regulated by leaf water status
M J Fryer (University of Essex), G Galvez-Valdivieso (University of Essex), N Smirnoff (University of Exeter), W J Davies (University of Lancaster), J I L Morison (University of Essex), Neil R Baker (University of Essex) and P Mullineaux (University of Essex)
Dr Philip M Mullineaux (mullin@essex.ac.uk)
Keywords: abscisic acid, retrograde signalling, hydrogen peroxide, high light, humidity
In Arabidopsis leaves exposed to a moderate increase in light intensity, bundle sheath cell (BSC) chloroplasts are one of the most prominent sites of hydrogen peroxide (H2O2) accumulation. The H2O2 is formed by the reduction of oxygen at photosystem I in a process driven by changes in linear photosynthetic electron flux (LEF). BSC-produced H2O2 does not promote oxidative stress, but has been shown to participate in signalling that leads to both local and systemic induction of the expression of ASCORBATE PEROXIDASE2 (APX2) and altered responses to further changes in the environment. In both plant and animal cells, a key factor in H2O2-mediated signalling may be its containment at discrete subcellular sites by surrounding antioxidant systems. Therefore, a distinction can be made between H2O2 implicated in eliciting oxidative stress where containment may fail, and spatially discrete H2O2-mediated signalling that leads to recovery from and acclimation to adverse conditions. In a model of BSC chloroplast-to-nucleus retrograde signalling in the high light exposed leaf, containment of H2O2 predicts that a non-ROS molecule must convey a signal out of the chloroplast. In this communication, evidence will be presented that a H2O2-stimulated synthesis of abscisic acid (ABA) in BSC is an essential part this signalling pathway. In all ABA biosynthesising tissues, the pathway is split between the chloroplast and the cytosol. Thus stimulation of ABA biosynthesis by H2O2 would deliver a chloroplast-originated signal to the cytosol. We propose that xanthoxin, the precursor of ABA, synthesised in and exported from the chloroplast, provides a conduit for this retrograde signal. Increased levels of ABA may stimulate a BSC-specific ABA signalling network, which is directed to produce H2O2 at the plasma-membrane. This leads to an observed accumulation of H2O2 in the apoplast between BSC and the vascular strand and provides a possible explanation for the known requirement for an extracellular source of H2O2 for the expression of APX2. The ABA signalling to the plasma-membrane may involve the coordinated action of the ABI1 protein phosphatase 2C (PP2C), the OST1 SNF-related protein kinase, the heterotrimeric G protein complex and AtrbohD/F-encoded NADPH oxidases. Once a plasma-membrane source of H2O2 is established, then the remaining steps to the activation of APX2 expression may involve an ABI2-PP2C regulated protein phosphorylation cascade and transcriptions factors that bind to consensus ABRE cis motifs present in the APX2 promoter. Importantly, the operation of this retrograde signalling pathway is contingent on leaf water status as determined by the prevailing humidity around the leaf. High humidity completely blocks all aspects of this pathway. Thus this paper will discuss how a water status signal that must be transmitted to the chloroplast may control a retrograde signal that exits the chloroplast.
L03
Light and Temperature Crosstalk in Plant Environmental Adaptation
K Franklin (University of Leicester)
Dr Kerry Franklin (kaf5@le.ac.uk)
Light and temperature are amongst the most important environmental signals regulating plant development. One of the greatest threats to plant survival in natural communities is light limitation through vegetational shading. Light reflected from and transmitted through living vegetation is depleted in photosynthetically active red (R) and blue wavelengths and enriched in green and far-red (FR) wavelengths. Plants detect the presence of neighbouring vegetation through monitoring the ratio of R to FR wavelengths (R:FR) in ambient light. Reductions in R:FR are perceived by the photoreversible phytochrome family of plant photoreceptors and initiate a suite of developmental responses termed the shade avoidance syndrome. These include increased elongation growth of stems and petioles, phenotypes which often correlate with reductions in leaf area, leaf thickness and plant biomass. Such responses serve to elevate leaves within a canopy and enable plants to over-top competing vegetation. We have observed that plant adaptation to the threat of vegetational shade is highly plastic and modulated by the integration of multiple signalling pathways. At lower temperatures and permissive photon fluences, plants forage for light by dramatically increasing leaf area, leaf thickness and dry biomass, a strategy which correlates with expression of the ‘CBF regulon’, a suite of genes involved in cold acclimation and enhancement of plant freezing tolerance.
L04
Signalling and gene expression in response to low temperature
H Knight., L Wathagula, D Gao, H Rushton, B Ülker, H Wilkinson and M R Knight (University of Durham)
Prof Marc R Knight (m.r.knight@durham.ac.uk)
Keywords: Low temperature, cold acclimation, calcium, SFR6, CBF/DREB1.
Our work on the molecular basis of plant responses to low temperature has focussed upon the second messenger calcium and the protein SFR6, both components in the cold acclimation response leading to freezing tolerance. We have shown that a cold-induced calcium signal is necessary for the induction of many genes associated with cold acclimation. Most notably, calcium regulates the CBF/DREB1-controlled regulon, which is arguably the most important gene regulon involved in cold acclimation. We have demonstrated that the level of regulation is post-translational and are currently identifying the specific mechanism by which this occurs. We have shown that the SFR6 gene product is necessary for the correct functioning of the CBF/DREB1 regulon: sfr6 mutants show greatly reduced expression of CBF/DREB1 target genes, and consequently reduced ability to cold acclimate. SFR6 regulates CBF transcription factors at the post-translational level. We have recently cloned SFR6 and are using proteomic approaches to understand its function at the molecular level.
L05
Biochemical Scanning of Combinatorial Peptides to Deduce Optimal Phosphorylation Sites of Protein Kinases
J Leung (Centre National de la Recherche Scientifique), F Vlad (Centre National de la Recherche Scientifique), B Turk (Centre National de la Recherche Scientifique), P Peynot (Centre National de la Recherche Scientifique), S Merlot (Centre National de la Recherche Scientifique)
Dr Jeffrey Leung (Leung@isv.cnrs-gif.fr)
Reversible protein phosphorylation is one of the most versatile mechanisms in integrating molecular messages in the cell. In Arabidopsis, there are over 1000 predicted or confirmed kinase genes. Learning about their functions in a physiological context requires, at least in part, identification of their physiological substrates. Several experimental techniques already exist to aid identifying potential targets for protein kinases, including two-hybrid or the more recently developed in vitro phosphorylation of proteins fixed onto a solid phase. Powerful as they may be, verification of hundreds of putative targets by other corroborating evidence can be time-consuming. With this in mind, we had implemented an innovative technology that can accurately predict kinase targets providing that the genome is adequately annotated. This method combines informatics and phosphorylation of semi-degenerate peptides to mathematically deduce a hierarchy of optimal phosphorylation motifs. These motifs are then used to search for their corresponding proteins. In collaboration with Ben Turk [Hutti et al., (2004) Nat. Meth.1, 27-29], we have used this technology to predict targets for the Arabidopsis OPEN STOMATA1 (OST1) kinase, which is involved in drought and ABA signalling. We have predicted that the "preferred" motifs of OST1 exist in the b-ZIP class of transcription factors, for which some ABA-insensitive mutants are already known. This innovative approach, in principle, should expediate the accurate prediction of targets for virtually any kinase at the genome scale.
L06
Rapid hormonal responses in the Arabidopsis shoot apex during light-triggered leaf initiation.
E Lopez-Juez (RHUL), E Dillon (RHUL), Z Magyar (RHUL), S Khan (RHUL), S Hazeldine (RHUL), G Beemster (VIB, Gent), L Bogre (RHUL), H Shanahan (RHUL)
Dr Enrique Lopez-Juez ( e.lopez@rhul.ac.uk)
Dark-grown seedlings exhibit repressed shoot apical meristem activity, and arrested, incipient leaf primordia. Following the first exposure to light, leaves rapidly expand and differentiate. An in-depth examination of this transition should allow the uncovering of phenomena at very early stages in leaf development. We dissected shoot apices of Arabidopsis seedlings grown in the dark, and over a time course following the transfer to light, and carried out microarray analysis using Affymetrix ATH1 arrays. We compared the shoot apical gene expression programme with that of cotyledons. Among early transcriptional responses observed, those associated with plant hormones and with ubiquitination processes were, unexpectedly, most noticeable. Extraction of gene expression signatures associated with the response to the main plant hormones revealed an early shoot apex-specific down-regulation of the response to auxins and ethylene, and an elevation of gibberellin and cytokinin action. The cytokinin action signature followed the drop in auxin response and coincided in time with a highly synchronous initiation of cellular growth and cell cycle activity. These early hormonal responses were transient, with auxin responses becoming again elevated at the time of leaf primordia expansion, and the expression of Auxin Response Factors in successive waves being apparent. We are currently trying to understand the exact location, within the meristem or the primordia, of these changes, and their functional significance in the control of early leaf differentiation and growth.
L07
A key role for Arabidopsis circadian clock genes in temperature sensing and the control of seed germination
S Penfield (University of York), A Hall (University of Liverpool)
Dr Steve Penfield (sdp5@york.ac.uk)
Plant can sense the temperature of their environment and use the information to regulate diverse developmental responses. Yet genetic evidence for the early steps in plant temperature signalling remains elusive. Circadian clocks can be entrained to temperature signals and therefore temperature-responsive, a key feature of a temperature sensor. Here we show that normal circadian clock function is essential for temperature sensing in plant seeds. Seeds are an excellent model system for plant temperature signal transduction because they show quantitative germination responses across the entire biological temperature range, and also respond specifically to alternating temperatures. We show that different clock gene mutants show germination defects in response to low temperature, ambient temperature and alternating temperatures, and that the clock also controls the germination response to dry after-ripening. We show that the transcriptional clock is arrested in an ‘evening’-like state in dry seeds, but in contrast to recent reports, rapidly entrains to light/dark cycles in ambient temperatures upon imbibition. Consistent with a role for the clock in dormancy control, the amplitude of clock-gene expression is strongly affected by dormancy-breaking temperatures and after-ripening, and the control of seed hormone metabolism is perturbed in clock gene mutants. Interestingly, we show that germination is timed to occur at dawn in 12 hour light/dark cycles and that this timing requires clock gene function. We conclude that the circadian clock has a key role in the integration of environmental signalling controlling germination and growth.
L08
Regulatory Network of MicroRNA399 and PHO2 by Systemic Signaling
T Chiou (Academia Snica, Taiwan), S Lin (Academia Snica, Taiwan), S Chiang (Academia Snica, Taiwan), W Lin (Academia Snica, Taiwan), J Chen (Academia Snica, Taiwan), C Tseng (Academia Snica, Taiwan), P Wu (Academia Snica, Taiwan)
Dr Tzyy-Jen Chiou (tjchiou@gate.sinica.edu.tw)
Recently, we showed that microRNA399s (miR399s) control inorganic phosphate (Pi) homeostasis by regulating the expression of PHO2 encoding a ubiquitin-conjugating E2 enzyme (UBC24). Arabidopsis plants overexpressing miR399 or the pho2 mutant overaccumulate Pi in shoots and displayed Pi toxicity symptoms. Pi toxicity was caused by increased Pi uptake and translocation of Pi from roots to shoots, and retention of Pi in the shoots. The association of Pi translocation and co-expression of miR399s and PHO2 in vascular tissues suggests their involvement in long-distance signaling. Reciprocal grafting between wild-type and miR399-overexpressing transgenic plants suggests the movement of miR399 from transgenic scions to wild-type rootstocks where PHO2 expression is suppressed. Suppression of PHO2 with miR399b or c was less efficient than that with miR399f. Of note, findings in grafted Arabidopsis were also found in grafted tobacco plants. The analysis of the pho1 mutant provides additional support for systemic suppression of PHO2 by the movement of miR399 from Pi-depleted shoots to Pi-sufficient roots. We propose that the long-distance movement of miR399s from shoots to roots is crucial to enhance Pi uptake and translocation during the onset of Pi deficiency. Moreover, PHO2 siRNAs mediated by the cleavage of miR399s may function to refine the suppression of PHO2. The regulation of miR399 and PHO2 via long-distance communication in response to Pi deficiency will be discussed
L09
The only constant is change – the yin and yang of nutrient availability and root growth behavior
P Doerner (University of Edinburgh), Y Li (University of Edinburgh), F Lai (University of Edinburgh), H Zhang (University of Edinburgh), X Fu (Chinese Academy Sciences)
Dr Peter Doerner (peter.doerner@ed.ac.uk)
Nutrient distribution in the soil is very heterogeneous, and root growth behavior, which involves changes to apical growth rates, rates of branching, cell expansion and growth and patterning of root hairs, changes specifically in response to differences in their environment. Mineral nutrients cannot be considered just as signals, as a material requirement for them in metabolism also provides direct mechanistic feedback restraints that prevent extreme imbalances between resource availability and growth activities. We are interested in the molecular mechanisms that couple the perception of mineral nutrients to growth behavior: In phosphate (Pi) -limited plants, continued root growth is required for Pi acquisition from new sources, yet meristem activity consumes Pi translocated from the shoot. Thus, the timing, pattern and magnitude of root growth responses have evolved under constraints of ‘cost’ and ‘benefit’. This implies the evolution of a sophisticated growth regulatory network controlling adaptive behavior, which we aim to dissect. We are also dissecting growth responses at the cellular level: When plants are Pi-limited, enhanced root or shoot growth exacerbates, whereas growth inhibition suppresses Pi starvation responses. Our results show that inhibition of cell-cycle activity specifically reduces Pi starvation-responsive gene expression. We propose that cell-cycle activity is the ultimate arbiter for Pi demand in growing organs, and that other factors that influence levels of PSR gene expression do so by affecting growth through modulation of meristem activity. We will present results of our recent spatial and temporal analysis of phosphate-responsive growth control networks.
L10
Post-embryonic cell fate changes of Arabidopsis rhizodermic cells in response to phosphate deficiency
W Schmidt (Academia Sinica), T Yang (Academia Sinica), P Perry (Academia Sinica), W Li (Academia Sinica), N Savage (University of Sheffield)
Prof Wolfgang Schmidt (wosh@gate.sinica.edu.tw)
Acclimation of plants to the prevailing environmental conditions is achieved by reprogramming metabolism and gene expression, gaining new functions of particular cell type(s). The decision of a root epidermal cell to form a hair is dependent on bidirectional signaling circuits among neighboring cells and on positional information from the underlying tissue. In addition, environmental signals are perceived and integrated into the cell specification process and may interact with, or overrule intrinsic programs. In particular, suboptimal availability of phosphate, iron, and manganese causes re-differentiation of rhizodermic cells. The spatial expression of genes determining the cell fate during embryogenesis was found to be unaffected by nutrient signals. Mutants harboring defects in cell specification genes were still responsive to phosphate deficiency, but the mutations affected the number and position of the hairs. We put forward the hypothesis that during post-embryonic development a novel second mechanism becomes dominant over the basic patterning mechanism, conferring cellular plasticity. This supposition is supported by mathematical modeling assuming an inhibitor-activator mechanism downstream of the WER patterning cascade that is sensitive to environmental signals. Candidate genes and processes involved in the change of root hair patterning were investigated by forward genetic mutant screening. The perfect mutant is defective in phosphate sensing and shows a phenotype typical of P-deficient plants in the presence of phosphate. P-deficient perfect plants formed very short root hairs and were impaired in their response to phosphate starvation. PERFECT encodes a ubiquitin-specific protease, underlining the importance of protein turnover in the adaptation to available phosphate.
L11
Temporal regulation of root hair development by RHD6 family genes.
K Yi (John Innes Centre), B Menand (John Innes Centre), L Dolan (John Innes Centre)
Mr Keke Yi (keke.yi@bbsrc.ac.uk)
While many genes that participate in root hair development have been identified, the transcriptional regulation of hair initiation and tip growth is still poorly understood.Previously, we showed that two bHLH transcription factors, RHD6 and RSL1, are required for root hair initiation. Here, we describe four paralogues of RHD6 that form a sister clade to the RHD6 clade. The genetic analysis shows that the sister clade genes function downstream of RHD6 and RSL1 in root hair development. One gene controls both hair initiation and tip growth while the other three members control tip growth in the hair. The expression of RHD6 family genes follows a temporal pattern that reflects the timing of their roles during development. We also showed that the internal (auxin and ethylene) and external (phosphate availability) factors regulate root hair development through these genes that lie downstream of RHD6 and RSL1.In summary, we found a subgroup of key regulator genes in the same bHLH subfamily form a regulatory network to integrate the internal and external signals for root hair initiation and tip growth.
L12
Analysis of natural variation for mineral concentration in Arabidopsis thaliana
A Ghandilyan (Wageningen University), L.O. Barboza (Wageningen University), J. Du (Chinese Academy of Sciences), U.B. Kutman (Sabanci University), S. Tisné (UMR INRA-SUPAGRO), H.-Q. Ling (Chinese Academy of Sciences), I. Cakmak (Sabanci University), C. Granier (UMR INRA-SUPAGRO), M. Koornneef (Wageningen University), H. Schat (Vrije Universiteit) and M.G.M. Aarts (Wageningen University)
Prof Mark Aarts (mark.aarts@wur.nl)
Tight regulation of mineral homeostasis is crucial to growth and development of plants. Plants require more than fifteen mineral elements. These include macronutrient elements like P, K, Ca and micronutrient elements, such as Zn, Fe, Mn and Mg. All of these nutrients are taken up by roots, translocated through the plant and used or stored for later use. Plants or plant derived products can be important sources of essential elements as dietary nutrients for humans and animals. Therefore, knowledge of the genes controlling mineral uptake and distribution in plants will increase our understanding of the mineral homeostasis process and may facilitate the improvement of plant nutrient content with potentially beneficial effects on human and/or animal health as well as on crop yield and quality.We examined the natural variation for seed, leaf and root mineral content in several recombinant inbred line (RIL) populations of Arabidopsis thaliana such as Landsberg erecta (Ler) x Kondara (Kond), Ler x Cape Verde Islands (Cvi), Ler x Antwerp (An) and Ler x Erinsboda (Eri). Plants were grown in several replicates on hydroponic solution or in soil. A quantitative trait loci (QTL) approach was used to identify and unravel the genetic loci controlling seed, leaf or root concentration of P, K, Ca, Mg, Mn, Fe and Zn. For each mineral several QTL were identified in each population. Co-localization of some QTL suggested single loci to be involved in the accumulation of multiple minerals. In many cases, QTL for seed mineral concentration identified in plants grown on soil did not co-localize with QTL for seed mineral concentration identified in plants grown hydroponically, suggesting a strong effect of environment on these traits. Such effect was further genetically examined in plants exposed to abiotic stress conditions, such as induced drought and Zn deficiency. Analysis of the CRY2 and ERECTA loci showed that in addition to environment also plant development has a significant effect on seed mineral status.
L13
Characterisation of the APS kinase gene family in Arabidopsis thaliana
S Mugford (John Innes Centre), N Yoshimoto (Chiba University), M Reichelt (Max Plank Institute for Chemical Ecology), L Hill (John Innes Centre), Y Nakazato (Chiba University), M Noji (Chiba University), R Kramell (Leibniz Institute of Plant Biochemistry), H Takahashi (RIKEN Plant Science Centre), C Wasternack (Leibniz Institute of Plant Biochemistry), J Gershenzon (Max Plank Institute for Chemical Ecology), K Saito (Chiba University), S Kopriva (John Innes Centre)
Dr Sarah Mugford (sarah.mugford@bbsrc.ac.uk)
Upon assimilation in higher plants, sulphur is partitioned into components of primary and secondary metabolism. The branching point is the metabolism of adenosine 5'phosphosulphate (APS). APS can be reduced to sulphite by APS reductase, and after further reduction incorporated into cysteine and other components of primary metabolism, or phosphorylated by APS kinase (APK) to form PAPS. PAPS is the sulphate donor for sulphotransferase enzymes which catalyse the transfer of the sulphate group onto free hydroxyl groups of acceptor molecules. Such sulphated secondary metabolites include the glucosinolates and sulphated hormones, which play important roles in defence against biotic and abiotic stress. How the partitioning of sulphur between primary and secondary metabolism is controlled in plants is poorly understood. By investigating the APK gene family in Arabidopsis we hope to gain insight into the mechanism and control of sulphate partitioning. There are four isoforms of APK in Arabidopsis, which we are characterising using a combination of tools including reverse genetics, web-based and SQRT-PCR expression analysis, promoter::GUS / GFP reporter lines, biochemical analysis and metabolic profiling. Different expression patterns and subcellular localisation suggests distinct roles for individual isoforms within the family. Characterisation of single and multiple knockout mutants of APK is confirming this, with particular attention being paid to the metabolite profiles of the double mutants.
L14
EZ-Rhizo: Integrated Software for Fast and Accurate Measurement of Arabidopsis Root System Architecture
P Armengaud (University of Glasgow), K Zambaux (University of Glasgow), A Hills (University of Glasgow), R Sulpice (Max Planck Institute, Potsdam, Germany), M Blatt (University of Glasgow), A Amtmann (University of Glasgow)
Dr Patrick Armengaud (p.armengaud@bio.gla.ac.uk)
The root system is essential for the growth and development of plants. In addition to anchoring the plant in the ground it is the site of uptake of water and minerals from the soil. Plant root systems show an astonishing plasticity in their architecture, which allows for optimal exploitation of diverse soil structures and conditions. The signalling pathways that enable plants to sense and respond to changes in soil conditions, in particular nutrient supply, are a topic of intensive research, and root system architecture (RSA) is an important and obvious phenotypic output. At present, quantitative description of RSA is labour-intensive and time-consuming, even using currently available software, and the lack of a fast RSA measuring tool hampers forward and quantitative genetics studies. We have developed EZ-Rhizo, a Windows-integrated and semi-automated computer program designed to detect and quantify multiple RSA parameters from plants growing on a solid support medium. The method is non-invasive, enabling the user to follow RSA development over time. Faster and more accurate measurement of RSA will permit a fine dissection of environmental and genetic control of root traits. EZ-Rhizo was applied to investigate Arabidopsis natural variation using principal component analysis of non redundant RSA parameters. This study underscored the importance of measuring different RSA parameters to fully describe RSA variation and allowed us to identify novel RSA determinants.
L15
The molecular basis of cell-cell communication during double fertilization in Arabidopsis
J Escobar-Restrepo (University of Zürich), S Kessler (University of Zürich), N Huck (University of Zürich), H Asano (University of Zürich)
Prof Ueli Grossniklaus (grossnik@botinst.uzh.ch)
Research in our laboratory focuses on the developmental genetics of plant reproduction. Our studies have shown that both genetic and epigenetic mechanisms play a key role in plant reproduction. In this seminar I will focus on cell-cell interactions during double fertilization. We have isolated a female gametophytic mutant, feronia, which disrupts double fertilization: in feronia mutant embryo sacs the pollen tubes, even if wild-type, are unable to release the sperm cells to effect fertilization. This phenotype suggests that the female gametophyte plays a crucial role in pollen tube reception and, thus, controls the behavior of the male gametophyte. The feronia mutant defines novel signaling processes between the male and female gametophytes in the process of double fertilizationFERONIA was shown to encode a receptor-like kinase of a plant-specific subfamily. I will report on the molecular and biochemical characterization of FERONIA and on our search for additional components of this signal transduction process using genetic and biochemical approaches. Interestingly, some interspecific crosses result in phenotypes that are very similar to those observed in theferonia mutant. The evolutionary implications of these findings will be discussed.
L16
Genetic control of male germ line development in flowering plants
D Twell (University of Leicester), L Brownfield (University of Leicester), S Hafidh (University of Leicester), M Borg (University of Leicester), A Sidorova (University of Leicester)
Prof David Twell (twe@le.ac.uk)
Pollen grains represent the highly reduced haploid male gametophyte generation in spermatophyte plants. In flowering plants their role is to nurture and deliver a pair of sperm cells to the embryo sac for double fertilisation. Recent advances in our understanding of landmark events in pollen development are largely based on progress achieved using genetic and transcriptomic approaches. Genome-wide analysis has revealed complex patterns of gene expression during male gametophyte development in Arabidopsis1. There is also evidence for extensive germ cell gene expression in maize and lily2,3 and several male germ cell-specific promoters have been characterized in Arabidopsis4-7. These datasets and molecular tools complement progress in genetic analyses that is beginning to uncover the molecular mechanisms that pattern pollen developmentA central outstanding question in pollen development is how the vegetative cell exits the cell cycle and differentiates to form the pollen tube, while the germ cell divides to form functional twin sperm cells? The identification of a non-germ cell repressor protein, Germline Restrictive Silencing Factor, in lily suggests the involvement of transcriptional derepression in male germ cell specification8. The analysis of a suite of Arabidopsis mutants that block male germ cell division provides further insight. Cyclin Dependent Kinase (CDK) and Chromatin Assembly Factor (CAF1) pathway mutants show that germ cell division can be uncoupled from gamete specification9,10. Recent progress involves the identification of an F-box protein that forms an SCFFBL17 complex in germ cells that targets the CDK inhibitor protein KRP6 for proteasome-dependent degradation. SCFFBL17 acts as a male germ cell proliferation licensing factor, but is also not involved in cell differentiation. However the R2R3 Myb transcriptional regulatory protein DUO15, coordinates these processes by activating germ line-specific gene expression and Cyclin B1:1 to commit differentiating germ cells to mitosis. DUO1 is therefore a key male germ cell fate determinant that regulates twin-sperm cell production and germ cell-specific genes including GCS16, which is essential for gamete fertility and double fertilization.Further progress in understanding the essential switch between germ and non germ cell lineages will continue to benefit from the integration of genetic and genomic technologies, offering new opportunities to build complex functional models of male gametophyte development. 1. Honys D. and Twell D. 2004. Genome Biology 5/11/R85.2. Engel ML, Chaboud A, Dumas C, McCormick S. 2003. Plant J 34:697-707.3. Okada T, Bhalla PL, Singh MB. 2006. Plant Cell Physiol 47:698-705.4. Engel ML, Holmes-Davis R, McCormick S. 2005. Plant Physiol 138:2124-21335. Rotman N, Durbarry A, Wardle A, Yang WC, Chaboud A, Faure JE, Berger F, Twell D. 2005. Curr Biol 15:244-248.6. Mori T, Kuroiwa H, Higashiyama T, Kuroiwa T. 2006. Nat Cell Biol 8:64-71.7. Okada T, Endo M, Singh MB, Bhalla PL. 2005. Plant J 44:557-568.8. Haerizadeh F, Singh MB, Bhalla PL. 2006 Science 313:496-499.9. Nowack MK, Grini PE, Jakoby MJ, Lafos M, Koncz C, Schnittger A. 2006. Nat Genet 38:63-67.10. Chen Z, Tan JL, Ingouff M, Sundaresan V, Berger F. 2008. Development 135:65-73.
L17
Post-genomics approaches to understand mechanisms regulating the completion of germination
M Holdsworth (Agricultural and Environmental Sciences Division, Nottingham University), P Jones (Agricultural and Environmental Sciences Division, Nottingham University), T Holman (CPIB, Nottingham University), A Medhurst (Agricultural and Environmental Sciences Division, Nottingham University), T Gerjets (Agricultural and Environmental Sciences Division, Nottingham University), F Theodoulou (Rothamsted Research)
Dr Michael Holdsworth (michael.holdsworth@nottingham.ac.uk)
Post-genomics approaches, in combination with classical genetics and physiology have been used to analyse how genotype and environment interact to regulate genome expression and phenotype during germination. Novel genetic screens (1) have been used to identify components required for the completion of germination in Arabidopsis thaliana, and this information has been used to study the genetics of ‘candidate genes’ in barley. Physiological analyses have revealed transcriptome components responsive to after-ripening (AR) in Arabidopsis (2,3), and demonstrate that AR capacity is a developmental process that does not require ABA (2). Key regulators underlying the molecular network linking after-ripening status with Abscisic Acid (ABA) function in the imbibed mature seed have been uncovered. Analysis of genome expression responsive to these regulators demonstrates how AR and ABA influence endosperm rupture prior to the completion of germination. These and other approaches are being used to inform research projects addressing the physiological disorder Pre-Harvest Sprouting in wheat (4).References: (1) Russell et al. 2000. Development 127: 3759-3767 (2) Carrera et al. 2008. The Plant Journal. 53 (2), 214–224. (3) Carrera et al.. 2007. Plant Physiol. 143(3): 1669-1679. (4). McKibbin et al. 2002. PNAS. 99(15): 10203-10208.
L18
From Arabidopsis to rice: pathways in pollen development
Dr Zoe Wilson (University of Nottingham)
Abstract not available
L19
Cell fate determination by genetic and hormonal patterning in fruits
L Østergaard (John Innes Centre)
Dr Lars Østergaard (lars.ostergaard@bbsrc.ac.uk)
The fruit of flowering plants provides an excellent system to study cell differentiation and tissue specification, since it is divided into discrete sections with juxtaposed cell types that are dramatically different from each other. The Arabidopsis thaliana fruit has been used extensively as a model to investigate how fruits are formed and several of the key regulators of the process have been identified.Patterning the Arabidopsis fruit involves formation of polarity along the proximo-distal and medio-lateral axes. Getting it right is crucial for proper development and much of the information has been laid down well before fertilisation.The plant hormone auxin plays important roles in organogenesis and tissue patterning. Polar auxin transport ensures regulation of local auxin concentrations, which mediate specification of organ outgrowths, polarity within organs or formation of specific cell types. Although significant leaps have been made in the understanding of auxin distribution and responses, not much is known about the upstream events that are likely to be specific to individual tissues. Our results show that auxin distribution is tightly regulated throughout Arabidopsis fruit development and that the valve margin identity factor INDEHISCENT (IND) negatively regulates polar auxin transport leading to the formation of a local auxin-response minimum and anticlinal cell division. Consistent with these data, ectopic production of auxin specifically in valve margin cells results in inhibition of the specification programme and lack of valve margin formation. Our results firmly embed auxin dynamics within the established regulatory pathway of Arabidopsis fruit patterning.
L20
The Solanum lycopersicum Auxin Response Factor 7 (SlARF7) Regulates Auxin Signaling during Tomato Fruit Set and Development
M de Jong (Radboud University), M Wolters-Arts (Radboud University), R Feron (Radboud University), C Mariani (Radboud University), W Vriezen (Radboud University)
Miss Maaike De Jong (maaike.dejong@science.ru.nl)
Auxin Response Factors (ARFs) are encoded by a gene family of transcription factors that specifically control auxin-dependent developmental processes. A tomato ARF gene, homologous to Arabidopsis NPH4/ARF7 and therefore designated as Solanum lycopersicum ARF7 (SlARF7), was found to be expressed at high level in the unpollinated mature ovaries. More detailed analysis of tomato ovaries showed that the level of SlARF7 transcript increases during flower development, remains at a constant high level in mature flowers, and is down-regulated within 48 hours after pollination, suggesting that this ARF has a regulatory role in fruit set. Transgenic plants with decreased SlARF7 mRNA levels formed seedless (parthenocarpic) fruits. These fruits were heart-like shaped and had a rather thick pericarp due to increased cell expansion, as compared to the pericarp of wild type fruits. The expression analysis, together with the parthenocarpic fruit phenotype of the transgenic lines, suggest that in tomato SlARF7 acts as a negative regulator of fruit set until pollination and fertilization have taken place, and moderates the auxin response during fruit growth.
L21
Senescence in Arabidopsis siliques: a comparison to senescence in other tissues and its role in seed development and nutrition.
C Wagstaff (University of Reading), T Yang (University of Nottingham), T Stead (Royal Holloway, University of London), V Buchanan-Wollaston (University of Warwick), J Roberts (University of Nottingham)
Dr Carol Wagstaff (c.wagstaff@reading.ac.uk)
Senescence of plant organs is a genetically controlled process that regulates cell death to facilitate nutrient recovery and recycling and frequently precedes, or is concomitant with, ripening of reproductive structures. In Arabidopsis the seeds are contained within a silique which undergoes a programme of senescence prior to dehiscence. A transcriptional analysis of the silique wall was undertaken to identify changes in gene expression during senescence and to correlate these events with ultrastructural changes. The study revealed that the most highly up-regulated genes in senescing silique wall tissues encoded seed storage proteins and the significance of this finding is discussed. Global transcription profiles of senescing siliques were compared with those from senescing Arabidopsis leaf or petal tissues using microarray datasets and metabolic pathway analyses. In all three tissues, members of NAC and WRKY transcription factor families were up-regulated whilst components of the shikimate and cell wall biosynthetic pathways were down-regulated. The expression of genes encoding ethylene biosynthesis and action had more similarity between senescing siliques and petals than with leaves. Genes involved in autophagy were highly expressed in the late stages of death of all plant tissues studied, but not always during the preceding remobilisation phase of senescence. Analyses showed that, during senescence, silique wall tissues exhibited more transcriptional features in common with petals than with leaves. The shared and distinct regulatory events associated with senescence in the three organs are evaluated and we present ongoing work exploring the potential for manipulating storage protein accumulation in seeds.
L22
Seasonal control of flowering in annual and perennial plants
G Coupland (Max Planck Institute for Plant Breeding Research), F Turck (Max Planck Institute for Plant Breeding Research), F Fornara (Max Planck Institute for Plant Breeding Research), S Jang (Max Planck Institute for Plant Breeding Research), L Corbesier (Max Planck Institute for Plant Breeding Research), C Vincent (Max Planck Institute for Plant Breeding Research), A Giakountis (Max Planck Institute for Plant Breeding Research), B Agashe (Max Planck Institute for Plant Breeding Research), R.Wang (Max Planck Institute for Plant Breeding Research)
Dr George Coupland (coupland@mpiz-koeln.mpg.de)
Arabis alpina, a member of the Brassicaceae that diverged from Arabidopsis around 30 million years ago. We have studied the effect of vernalization (exposure to low winter temperatures) on the life cycle of Arabis alpina and compare the mechanisms underlying vernalization response in this species with those of Arabidopsis. We propose that there is correlated selection of the vernalization response as species evolve from perennial to annual. The talk will present this comparative approach to study flowering-time control.
L23
The need for winter in the switch to flowering.
C Dean (John Innes Centre, Norwich)
Prof Caroline Dean (caroline.dean@bbsrc.ac.uk)
The Dean laboratory is studying the importance of prolonged cold (winter) for flowering, a process known as vernalization. We have used a molecular genetic analysis in the model plant Arabidopsis thaliana to identify genes involved in determining both the ability to vernalize and the need for vernalization. The pathways we study share a common downstream target, a gene encoding the repressor of flowering, FLC. Mutants attenuating the vernalization response revealed FLC expression is silenced during the cold and this repression remains epigenetically stable through the rest of the plant life-cycle. A Polycomb-based chromatin regulation involving conserved regulators and PHD finger proteins mediates this epigenetic silencing. The autonomous floral promotion pathway affects the need for vernalization. Recent work suggests this pathway links RNA processing, RNAi machinery and chromatin regulation to cause the down-regulation of FLC. It has also been shown to play widespread roles in the epigenetic silencing of the Arabidopsis genome. Functioning antagonistically to both the autonomous pathway and vernalization, FRIGIDA (FRI) causes plants to overwinter in the vegetative state by up-regulating FLC expression. FRI is the major determinant of flowering time variation in Arabidopsis and FRI loss-of-function alleles have been found to be the basis for the evolution of many rapid-cycling Arabidopsis accessions. The talk will describe our current understanding of these pathways and how they have changed in Arabidopsis variants adapted to different climates.
L24
Optimising Flowering Time in Miscanthus for Improved Biomass Yield and Quality
E Jensen (IBERS), S Thomas-Jones (IBERS), K Farrar (IBERS), P Robson (IBERS), J Clifton-Brown (IBERS), I Donnison (IBERS)
Dr Elaine Jensen (fft@aber.ac.uk)
The perennial C4 grass Miscanthus is a leading candidate for sustainable bioenergy production in Europe and N. America. Miscanthus has a broad natural geographical range that provides extensive and currently unexploited genetic diversity. Miscanthus is currently co-fired with coal to produce heat and electricity but may also be chemically converted to liquid fuel. Optimisation of flowering time has been identified as key to improving both the yield and quality of biomass. Early flowering associates with reduced yield and is therefore undesirable. However, as flowering is also a potential trigger for senescence and the subsequent remobilisation of nutrients to the underground rhizome it is important for crop sustainability. Senescence may also improve yield quality as downstream processing is detrimentally affected by residual nutrients in harvested biomass. Genotypes exhibiting late flowering coupled tightly with senescence could therefore optimise the yield, quality and sustainability of the crop. An F1 mapping population, based on a cross between early and late flowering genotypes of M. sinensis, is being used to identify quantitative trait loci associated with flowering time. In addition, homologues of genes controlling flowering time in other species are being identified in Miscanthus. Sequence information from these homologues will be used together with phenotypic data from a genetic collection with wide-ranging flowering times. This data will be used to perform association studies, where single nucleotide polymorphisms will be correlated with flowering phenotype, thereby facilitating marker assisted selection.
L25
Genetic basis of the link between senescence regulation and flowering time control
A Wingler (University College London), S Purdy (University College London), F Chardon (INRA Versailles), C Masclaux-Daubresse (INRA Versailles)
Ms Astrid Wingler ( a.wingler@ucl.ac.uk)
In annual plants, leaf senescence is often linked to reproduction. However, no effect of sterility on senescence was found using mutants of Arabidopsis, suggesting that senescence in Arabidopsis is not regulated by the successful formation of fruits and seeds. Nevertheless, flowering time and senescence are correlated among different accessions of Arabidopsis, indicating that floral initiation and senescence are linked, although the causal relationship remained unresolved. Analysing the effect of sugar supply on senescence in recombinant-inbred lines (RILs) we found that while sugar treatment induced senescence in early-flowering lines, the effect was less pronounced in late-flowering lines. Microarray analysis revealed differences in the expression of flowering genes, such as FLC and SOC1, in the RILs. Quantitative trait locus (QTL) experiments were conducted to determine the genetic basis of this regulation. A major QTL mapped to the top of chromosome IV where FRI, an activator of the floral repressor FLC is localised. Since interaction of functional FRI and FLC alleles results in vernalisation dependent flowering, the effect of vernalisation on senescence was determined in early and late flowering lines. Whereas a clear correlation between flowering and senescence was found without vernalisation, vernalisation abolished differences between the lines, thus supporting the genetic link between the vernalisation-dependent pathway of flowering and senescence regulation revealed by the QTL analysis.
L26
Evolution of regulatory networks controlling floral organ identity: a MIKC blessing
G Theissen, R Melzer, Y Wang, M Mondragón-Palomino, H Simon (Friedrich Schiller University, Jena)
Dr Guenter Theissen (guenter.theissen@uni-jena.de)
Key words: ABC model, floral quartet model, MADS-box gene, macroevolution, homeosis Flower development is controlled by gene regulatory networks (GRNs) that are dominated by MIKC-type MADS-box genes encoding transcription factors (1). Changes in these regulatory networks are underlying the morphological evolution of flowers and hence the generation of floral biodiversity. To better understand the link between the evolution of floral GRNs and the morphology of the flower we study MIKC-type genes and proteins in evolutionary informative spermatophytes. A special focus of our work is on the floral homeotic genes which encode proteins that form multimeric complexes (‘floral quartets’) specifying floral organ identity by activating and repressing the appropriate target genes during the development of floral organs (2). Studies in the gymnosperm Gnetum and in orchids indicate in more and more detail how the origin of new classes of MIKC-type genes by gene or genome duplications contributed to the establishment of morphological novelties, such as the floral perianth, or the orchid’s lip (3, 4). Investigations on grasses such as maize (Zea) and orchids reveal that at least some classes of floral homeotic genes have been highly conserved throughout more than hundred million years of evolution, even though the organs they specify sometimes have been modified dramatically (3, 4). Studies in tulips (Tulipa) demonstrate that class B floral homeotic gene function is also conserved in petaloid monocots. Our findings demonstrate the importance of sub- and neo-functionalization of developmental control genes for the evolutionary origin of morphological novelties. The evolutionary relevance of heterotopic expression of developmental control genes resulting in homeosis, and of the modularity of genes and organisms, are recurrent themes in our investigations (3-5). References 1. Kaufmann, K., et al. (2005) MIKC-type MADS-domain proteins: structural modularity, protein interactions and network evolution in land plants. Gene 347, 183-198. 2. Theissen G. (2001) Development of floral organ identity: stories from the MADS house. Curr. Opin. Plant Biol. 4, 75-85.3. Theissen, G., Melzer, R. (2007) Molecular mechanisms underlying origin and diversification of the angiosperm flower. Ann. Bot. 100, 603-619.4. Mondragón-Palomino M., Theissen G. (2008) MADS about the evolution of orchid flowers. Trends Plant Sci. 13, 51-59. 5. Hintz, M., et al. (2006) Catching a ‘hopeful monster’: shepherd’s purse (Capsella bursa-pastoris) as a model system to study the evolution of flower development. J. Exp. Bot. 57, 3531-3542
L27
Control of floral transition in maize
F Van Kerkhoven (University of Liège), M D’Aloia (University of Liège), C Périlleux (University of Liège)
Mr Fabrizio Van Kerkhoven (f.vankerkhoven@ulg.ac.be)
Flowering time in plants is controlled by a number of environmental factors, among which photoperiod plays a key role. Maize ancestors are short-day (SD) plants, but breeding programs have selected genotypes whose flowering is largely autonomous and occurs after production of a constant number of leaves regardless of photoperiod. Only few flowering time genes have been identified in maize; one of them is INDETERMINATE1 (ID1), cloned from a late-flowering mutant and encoding a zinc finger transcription factor. By contrast, the genetical control of flowering by photoperiod is best understood in the long-day (LD) dicot Arabidopsis and the SD monocot rice. A key regulator is the CONSTANS gene that mediates between the circadian clock – the time-keeper of the plant – and the synthesis of flowering signals. Here we report the analysis of a CONSTANS homolog in maize, ZmCO, in SD and in LD, and in different parts of the plant. Expression of ZmCO was found to be rhythmic and to be higher in young leaf primordia than in mature leaf blades. Striking coincidence was observed with expression of ID1.
L28
Hormonal control of shoot branching
O Leyser (University of York)
Prof Ottoline Leyser (hmol1@york.ac.uk)
Keywords: Auxin, Cytokinin, MAX, bud activation
A unique feature of plant development is the ability to alter body plan in response to environmental conditions. The primary body axis of plants is laid down during embryogenesis with the establishment of the shoot apical meristem at one end and a root apical meristem at the other. Post-embryonically, the meristems elaborate this basic axis, but in addition secondary meristems arise in both the root and shoot, which give rise to new axes of growth- lateral branches. It is this ability to produce lateral branches that gives plants their spectacular plasticity of form. As a model for understanding the role of plant hormones in plant developmental plasticity, we are investigating the hormonal control of shoot branching. We are focusing on two hormones that inhibit branching-auxin, which has been known to regulate branching for 80 years; and a novel hormone, which has not yet been chemically defined. In Arabidopsis, the novel hormone came to light through the analysis of mutants at 4 loci, called MAX1-MAX4, with increased shoot branching. These genes define an additional pathway that interacts with auxin to mediate branch inhibition. Grafting studies have demonstrated that three of these loci are involved in the production of a long-range graft transmissible signal that inhibits bud growth, while the third acts locally in the transduction of this signal, and this is consistent with the molecular identities of these genes. The pathway appears to act by modulating auxin transport capacity in the main stem, suggesting an interesting indirect mechanism for apical dominance and its modulation. A third hormone, cytokinin, that promotes bud activation fits into this network and also interacts with auxin and its transport. Our progress in understanding the operation of this hormonal network in modulating shoot branching in response to the environment will be presented.
L29
To grow or not to grow
N Harberd (University of Oxford), E Belfield (University of Oxford), Y Yasumura (University of Oxford)
Prof Nicholas Harberd (nicholas.harberd@plants.ox.ac.uk)
The DELLA proteins (DELLAs) are a subfamily of the plant-specific GRAS family of putative transcriptional regulators that regulate plant growth in response to the phytohormone gibberellin (GA). The DELLAs restrain growth, and GA promotes growth by opposing DELLA function. Essentially, GA binds to a specific GA-receptor protein (GID1), thus stimulating a GID1-DELLA protein-protein interaction. This interaction itself promotes specific targetting of DELLAs for destruction in the proteasome via the SCFSLY1 E3 ubiquitin ligase. Additional signalling pathways, such as those associated with phytohormones other than GA, and environmental variables such as light, temperature and nutrient status, also influence plant growth via effects on the GA-DELLA growth-regulatory mechanism. The concept that the DELLAs are integrators of multiple plant growth regulatory signalling inputs will be explored, and the broader biological significance of DELLA function will be illustrated, with particular emphasis on the question of how the GA-DELLA growth-regulatory mechanism arose during land-plant evolution. Recent publications: Achard et al. (2006). Science 311: 91-94. Achard et al. (2007). Plant Physiology 143: 1163-1172. Achard et al. (2007). Proceedings of the National Academy of Sciences (USA) 104: 6484-6489. Yasumura et al. (2007). Current Biology 17: 1225-1230.
L30
To divide or not to divide – How ABA affects root growth
D Dietrich (CPIB, University of Nottingham), S Ubeda-Tomas (CPIB, University of Nottingham), R Bhalerao (Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre), M Bennett (CPIB, University of Nottingham)
Dr Daniela Dietrich (daniela.dietrich@cpib.ac.uk)
ABA is primarily known as a stress hormone that relays the plants responses to environmental signals such as drought, salt stress or cold. Increases in ABA concentration lead to inhibition of root growth but it is unclear what the underlying cellular mechanisms are. Detailed growth analyses and cell division markers such as CycB1;1 revealed that ABA regulates root growth by influencing cell division rates.We are also interested where in the root the ABA signal is perceived and used a transactivation approach to investigate whether any root tissue in particular is important for ABA signal perception. Results will be presented that show that there are indeed differences in ABA sensitivity between the different root tissues. In addition, ABA appears to influence root architecture by antagonising auxin in the initiation of lateral roots. Using the same transactivation approach we could show that ABA inhibits the initiation of lateral root primordia in addition to its previously reported inhibitory effect on growth after lateral root emergence (De Smet et al., 2003).In summary we show some of the mechanisms by which ABA integrates environmental responses into the root developmental growth program and thus shapes root growth in response to environmental cues. De Smet et al. 2003, Plant Journal 33, 543-555
L31
Systemic signaling of environmental cues in leaves
P Quick (University of Sheffield), J Gray (University of Sheffield)
Dr Paul Quick (p.quick@sheffield.ac.uk)
Light and carbon dioxide and temperature all affect leaf development. The mechanism by which leaves sense these signals is unclear. We will present evidence that suggests that these signals are systemically transported from mature leaves to developing leaves and that these alter subsequent leaf development. We utilise stomatal guard cells as a model cell type to monitor changes in leaf development as their frequency and density is very sensitive to environmental conditions. We will present evidence to suggest that trehalose metabolism is involved in this process and that the wiring diagram for the vasculature is key in determining which leaves perceive and receive these signals.
L32
PXY signalling induces vascular development
P Etchells (University of Manchester), S Turner (University of Manchester)
Dr Peter Etchells (peter.etchells@manchester.ac.uk)
In plants, stem cells are maintained and regulated in meristems. The cambium and procambium are meristematic tissues that generate the xylem and phloem which are specialised organs for water and nutrient transport. Furthermore, cambial meristems are the main source of plant biomass and as such their regulation has a come under increasing scrutiny as biomass is likely to play an increasing role in generation of renewable energy. One regulator of vascular meristems is PXY, a receptor-like kinase that is required for polar cell divisions within the procambium which are essential for organised vascular tissue development. We have recently identified additional components of the PXY signalling pathway. Over expression of a single PXY dependant component is sufficient to generate dramatic increases in vascular cell number and increase the proportion of undifferentiated cells. Furthermore, over-expression of multiple pathway components results in ectopic vascular tissue development and early onset of secondary growth. The data has identified multiple roles for PXY-dependent signalling. In addition to its role in setting the division plane, PXY forms part of a signalling network that regulates the rate of cambial cell divisions and as a result, the balance between dividing (undifferentiated) and differentiated cells.
L33
Auxin influx carriers are involved in apical hook development
F Vandenbussche (Ghent University), J Petrasek (Charles University, Prague), E Zazimalova (Charles University, Prague), P Zadnikova (Ghent University, VIB), E Benkova (Ghent University, VIB), R Swarup (University of Nottingham), M Bennett (University of Nottingham), D Van Der Straeten (Ghent University)
Dr Filip Vandenbussche (Filip.Vandenbussche@ugent.be)
During skotomorphogenic growth, dicotyledonous plants form an apical hook at the top of the hypocotyl to protect the apical meristem and the cotyledons during growth through soil or mulching.Hook formation, maintenance and exaggeration are supposedly caused by an auxin gradient that induces differential growth. In Arabidopsis, the accumulation of DR5 auxin reporter signal on the concave side of the hook suggests an auxin maximum in this region. The gradient is established by active auxin transport, that is NPA sensitive and auxin efflux regulator dependent. We have found a significant role for a subset of auxin influx carriers in the development of the apical hook. While LAX3 is important for hook development in general, AUX1 is a prerequisite for ethylene induced exaggeration of the hook. Mutant analysis revealed that among the auxin influx regulators these two are playing an important role in steering hook development.
L34
Plant endomembrane system and chemical genomics
N Raikhel (University of California Riverside)
Dr Natasha Raikhel (natasha.raikhel@ucr.edu)
Chemical genomics is an exciting new technology for studying gene functions in the context of living organisms or cell systems. The approach complements existing molecular and genetics tools (e.g. mutagenesis, RNAi) by allowing fine-tunable in vivo modulations of protein functions and cellular processes. For example, lethality and redundancy are common and challenging descriptors in genetic studies of the endomembrane system. Chemical genomics can be used to overcome these challenges and study protein trafficking mechanisms. We have performed a few chemical genomics screens and identified several useful compounds. Effect of these compounds on various markers of the endomembrane system was assessed. Analogs of these chemicals were tested to identify the chemical structures that are responsible for bioactivity of these molecules. Screens for resistant and hypersensitive mutants were carried out with the goal of identifying putative targets or to identify components of the pathway. Information about these targets and pathways will be discussed.
L35
The Cytoskeleton in Plant Cell Morphogenesis and Development
P Hussey (University of Durham)
Prof Patrick Hussey (p.j.hussey@durham.ac.uk)
The plant cytoskeleton governs plant cell morphogenesis and it is composed of microtubules and actin filaments, and a plethora of associated proteins that serve to anchor, cross-bridge or otherwise regulate this fibrous network. These associated proteins are involved in competitive and/or cooperative interactions within cells to adjust the organisation of the cytoskeleton to the cells needs. These associated proteins are often stimulus responsive and are the effectors in signal transduction cascades. This system has evolved so that normally sedentary plant cells can respond to developmental and environmental cues in order to proliferate and grow, to maximise energy production, to take up nutrients from the soil, to reproduce and to protect from pathogen invasion. In all these cases the cytoskeleton has to respond to signals and reorganise so that cells can divide and expand, generate organelle movement, polarise cell growth and thicken the cell wall. Here, some of the main players in the control of cytoskeletal organisation in plant cells will be discussed.
L36
Novel compounds that disrupt actin filaments identified by chemical genetic screen for impairment of the Arabidopsis circadian clock
R Toth (Max Plank Institute for Plant Breeding Research, Cologne, Germany), S Menninger (Max-Planck-Institute of Molecular Physiology, Dortmund, Germany), M Deeks (The Integrative Cell Biology Laboratory, Durham University, UK), I Nougalli-Tonaco (Max Plank Institute for Plant Breeding Research, Cologne, Germany), P Hussey (The Integrative Cell Biology Laboratory, Durham University, UK), H Waldmann (Max-Planck-Institute of Molecular Physiology, Dortmund, Germany), G Coupland Max-Planck-Institute of Molecular Physiology, Dortmund, Germany)
Dr Reka Toth (toth@mpiz-koeln.mpg.de)
To study the Arabidopsis circadian clock a forward chemical genetic screen was performed to identify small molecules that impair circadian rhythms. Here we describe the identification of two small molecules causing shortening of circadian period length. Additionally, the plants treated with the compounds exhibit growth phenotypes associated with microfilament defects. Short treatment with the compounds results in altered actin organization, but does not affect the tubulin filaments. A similar result was obtained in animal cells, indicating that the target of the compounds is highly conserved between the animal and plant Kingdoms. Well-characterized actin inhibitors triggered similar changes in the circadian clock showing that the altered circadian rhythmicity is the result of the impaired actin network. Actin inhibitors have specific, light-dependent effects on circadian oscillations suggesting that the role of actin filaments in the clock is predominantly linked to input pathways.
L37
Intramembrane protein dynamics in the ER and plasma membrane is affected by the actin cytoskeleton.
J Runions (Oxford Brookes University), M Shvedunova (Oxford Brookes University), I Sparkes (Oxford Brookes University)
Dr John Runions (jrunions@brookes.ac.uk)
Proteins within plant cell endomembranes are highly mobile. Their mobility can be quantified using the confocal microscopy technique of photoactivation. What does a description of the speed of membrane protein dispersion tell us? Our initial observations lead us to believe that the structure of ER and plasma membranes is quite different. To mark the membranes, we use known protein transmembrane domains that target respective organelles fused to photoactivatable GFP (PAGFP). The ER is marked by calnexin-PAGFP and the plasma membrane is marked by Low Temperature Inducible protein 6B (LTI6B)-PAGFP. Neither of these proteins is predicted to interact with others and so are useful as a control for non-interacting protein mobility. The ER is a highly dynamic interconnected network of tubules and cisternal sheets. Its motion is actin-cytoskeleton dependant. When the actin is depolymerised by application of latrunculin B, bulk flow and remodelling of the ER ceases but calnexin continues to diffuse. Overexpression of the tail region of a myosin XI also results in cessation of ER movement but has a limiting effect on calnexin diffusion. LTI6B-PAGFP in the plasma membrane moves very slowly when photoactivated. The plasma membrane appears significantly different from the ER in this respect. Interestingly, there is evidence that when the actin cytoskeleton is depolymerised LTI6B becomes unconstrained and can diffuse more quickly within the membrane. This might point to the existence of actin corrals that contribute to protein-domain formation and these domains might be important regulatory structures for plant cells.
L38
The Gene Regulatory Network for Root Epidermal Cell Differentiation
J Schiefelbein (University of Michigan), S Kwak (University of Michigan), Y Panciera (University of Michigan), C Barron (University of Michigan), A Bruex (University of Michigan)
Prof John Schiefelbien (schiefel@umich.edu)
A fundamental aspect of plant development is the specification and differentiation of distinct cell types. The position-dependent formation of the root-hair and non-hair cell types in the Arabidopsis root epidermis is useful as a simple model for studying plant cell differentiation. Cellular, molecular, genetic, and genomic approaches have defined genes and their corresponding proteins involved in this process. By studying these, we have found that transcriptional feedback loops acting within and between adjacent cells are important in establishing the cell type pattern. Specifically, the WER MYB-type protein, the GL3/EGL3 bHLH-type proteins, and the TTG WD-protein appear to interact in a transcriptional complex to positively regulate the GL2, CPC, TRY, and ETC1 genes. The GL2 homeodomain transcription factor regulates genes that generate the non-hair cell type. The CPC, TRY, and ETC1 proteins are structurally-related small MYB transcription factors that appear to move to neighboring cells and inhibit the WER-GL3/EGL3-TTG complex; representing a type of lateral inhibition. In another regulatory loop, the GL3/EGL3 proteins negatively affect their own genes’ expression and likely move to cells in the opposite direction. The position-dependent pattern relies on a LRR-RLK (SCRAMBLED (SCM)), that appears to cause an unequal distribution of the transcriptional regulators in the N and H cell positions. Currently, systems-based approaches and mathematical modelling are being used to construct and analyze the gene regulatory network that controls root epidermal cell patterning and differentiation. These studies are likely to provide insights into the logic of gene regulatory networks and mechanisms of cell specification during development.
L39
Arranging Biological Resource Information on the Semantic Web, An Information Platform Towards Genome Design in Arabidopsis
T Toyoda (RIKEN, Japan)
Dr Tetsuro Toyoda (toyop@gsc.riken.jp)
The Semantic Web is a standard framework for knowledge description and discovery by computer-aided inferences, based on the relationships represented as semantic links among resources including the complete genome sequence along with gene structure, gene products, metabolites, gene expression, resource lines, phenotypes, publications, and any information useful to the research community. As a tool to realize the semantic web, we have developed an internet-based system termed SWF (Semantic Web Folders) which is an information platform that allows people to develop collaboration communities on the semantic web, jointly build various databases, and make the databases available to the public through both the semantic web and the world-wide web. A person acquiring a power user account can set up a new community in SWF, and invite participants to the community to jointly produce a database on the framework of the semantic web, facilitating international collaboration and exchange of comments about individual data elements including annotations and ontology terms jointly defined in SWF. An SWF user can define ontology terms or classes, each of which plays a role as a database or a container of data records, which are connected to each other via semantic links to form an integrated database based on the semantic web. By using SWF we are developing a system allowing a wide range of users to download RIKEN’s omics experimental data as huge as hundreds of terabytes to petabytes and to allow integrative browsing of omics data [Toyoda,T et al., Bioinformatics 23,524-6(2007)] along with predictions such as tiling-array-based predicted genes [Toyoda,T and Shinozaki,K, Plant J. 43,611-21(2005)]. We have established semantic links among Arabidopsis omics resources including the genome, genes and literature and have developed a system to generate inferences based on the data. The system is termed PosMed (Positional Medline), which instantaneously ranks instances of significantly related genes with functions mentioned in literature. This accomplishes not only a direct search but also an indirect search via SWF’s semantic links (pathway relationship, orthologous relationship) for genes not directly related in literature [Kobayashi,N and Toyoda,T. Bioinformatics, in press, 2008]. PosMed is used worldwide to select candidate genes for positional cloning, as it is possible to add a chromosomal interval to the search limiting the ranking to only those genes within the interval. Our future goal is to evolve the entire system into an expert system for genome design or synthetic genomics in Arabidopsis and other plants. Please visit our web site at http://omicspace.riken.jp.
L40
A systems biology approach to identify transcription networks in Arabidopsis leaf senescence
V Buchanan-Wollaston (University of Warwick), E Breeze (University of Warwick), S McHattie (University of Warwick), L Hughes (University of Warwick), A Mead (University of Warwick), D Wild (University of Warwick)
Dr Vicky Buchanan-Wollaston (vicky.b-wollaston@warwick.ac.uk)
Leaf senescence is a programmed event responding to a wide range of external and internal signals including those caused by development, age and environment. Senescence requires de novo gene expression and protein synthesis and is controlled in a tightly regulated manner. Identification of the genes that control senescence has been complicated by the complex combination of signalling pathways that appear to be involved in senescence. Cross talk exists between senescence and stress or pathogen responses and also the hormonal and nutrient signals that are implicated in the control of senescence. We are using Arabidopsis as a model, taking a systems biology approach, to study the genes involved with the control of leaf senescence. Extensive microarray analysis over a detailed time course of development is being used to identify transcriptional networks that operate to control gene expression during developmental leaf senescence. We have two developmental time series, in one the leaf enters senescence primarily due to developmental signals, in the other the leaf is induced to senesce by a combination of age and environment. Using these data sets we are characterising key genes and pathways involved in just one or in both processes. In addition, cross talk between stress related pathways and senescence is being elucidated by the use of mutants, treatments and comparative gene expression analysis. Functional analysis of selected senescence enhanced regulatory genes is underway to pinpoint the key regulatory points within the signalling network.
L.41
Modelling Dynamic Biological Systems at CSBE
A Millar (University of Edinburgh)
Prof Andrew Millar ( andrew.millar@ed.ac.uk)
The Centre for Systems Biology at Edinburgh, CSBE, is a Centre for Integrative Systems Biology funded by the BBSRC and EPSRC and led by co-Directors, Professors Andrew Millar and Igor Goryanin. Modelling is central to systems biology, taking existing knowledge and through static and kinetic models in order to generate new knowledge. The key feature of CSBE is that we place this process at the centre of our endeavour. Through the work of the Centre we aim to make theoretical and practical developments to support all stages of the modelling process. This work is informed by three biological exemplar projects, which have been chosen to exercise all aspects of the modelling process: the circadian clock in Arabidopsis, RNA metabolism in yeast and Interferon signalling in macrophages. The projects represent a range of timescales, dynamics and numbers of biological components. I will use the circadian clock project to illustrate how CSBE's work is advancingthe systems approach.
Firstly, we are establishing an informatics infrastructure that links diverse data types seamlessly to the tools required for modelling, named the Systems Biology Software Infrastructure, SBSI. The modular structure of SBSI will support several means to build models (including biologist-friendly network diagrams) and link the models to data, together with a suite of tools for model optimisation and model analysis. Such large-scale modelling frameworks have not previously been available in the academic setting.
Secondly, we are measuring the biochemical constants that are required to parameterise kinetic models, focussing on the assembly of protein-protein and protein-nucleic acid complexes, protein and RNA degradation and the acquisition of high-quality RNA timeseries. Information about the people and the projects can be found on our regularly updated website http://csbe.ed.ac.uk .
CSBE has members spanning multiple research institutions and representing diverse areas of research expertise. We also have strong links to industry via direct research partnerships, consultancy and our International Science Advisory Board. We welcome further opportunities for collaboration from both the public and private sectors. Our philosophy is strongly collaborative: open source and open access.
L42 Adopting an integrative biology approach to study root growth and development
M Bennett (Centre for Plant Integrative Biology, University of Nottingham)
Prof Malcolm Bennett (malcolm.bennett@nottingham.ac.uk)
The Centre for Plant Integrative Biology (CPIB) at the University of Nottingham aims to create a virtual root which will serve as an exemplar for using Integrative Systems Biology (ISB) to model multi-cellular systems. CPIB brings together biologists, engineers, mathematicians and computer scientists to generate new data, biological resources and virtual models of plant roots that will aid understanding of how they grow and develop. The research programme involves multidisciplinary teams working simultaneously in sub-programmes at the molecular, cellular and organ levels. Our research activities are structured as three overlapping 3-year strands of increasing sophistication. Strand 1 focuses on modelling cell elongation during radicle emergence and primary root growth; Strand 2 focuses on the root apical meristem, the principal site for cell division during primary root growth; whilst Strand3 will examine the initiation, patterning and emergence of lateral roots. Strand4 will integrate these models at different physical scale across the first three strands. The output of the programme will be quantitative observational data, validated models constituting the prototype "virtual root" and proofs of concept which will form the basis for further research programmes. Strand 1 started in March 2007. I will review progress made to date, highlighting the approaches, tools and results obtained so far.
Posters
P1
EFFECT OF INTEGRATED WHITE LIGHT ON THE GERMINATION PERCENT OF FOUR WHEAT
A Abu-Elsaoud (Suez Canal University, Egypt)
Mr Abdelghafar Abu-Elsaoud (Abdoacacia@yahoo.com)
Light quality plays important role in regulating the germination of some seeds. Light requirement is often observed in the small seeds of herbaceous and grassland species, many of which remain dormant, even while hydrated, if they are buried below the depth to which light penetrates. Experiments were carried out to evaluate the integrated white light quantity requirement of four wheat cultivars; 3 Kazakhstanian cultivars and one Egyptian cultivar. Germination energy was recorded at 3, 5, and 7 days after integrated white light illumination and cultivation. Generally, integrated white light illumination had positive effect on the four studied cultivar, dormancy were broken early that early germination of examined seeds were noticed, in addition to enhanced levels of germination energy after illumination were also noticed.
P2
Impact of He-Ne laser seed irradiations on wheat (Triticum aestivum L.) germination and growth
A Abu-Elsaoud (Suez Canal University, Egypt)
Mr Abdelghafar Abu-Elsaoud (Abdoacacia@yahoo.com)
In this research work the influence of He-Ne laser on four wheat cultivars from Kazakhstan and Egypt were carried out in order to enhance their germination percentage and various growth parameters. Seeds were in vitro irradiated to He-Ne laser for 0, 1, 3, 10, 30, 60, 180, 600, 1200 and 1800 seconds. Seeds pre-sowing irradiation with monochromatic He-Ne laser of 632.8 nm and a power intensity of 3 mW.cm-2 have increased the germination percentage of the four wheat cultivars. Maximum germination percentages noticed were mainly after He-Ne-seed pre-sowing irradiation with 1, 60 and 600 seconds. Generally, He-Ne laser enhanced the germination percentage of wheat cultivars after 3 days to reach a maximum of 70.0%3 in cultivar Eretrospermum-350 after irradiation for 60 seconds. He-Ne irradiation of 180 seconds caused a general inhibition in germination and growth in the four studied cultivars with minimum germination percentage of 6.7%3 recorded in sakha-168. Germination percentage at 5 and 7 days showed a similar behavior with maximum germination percentage recorded mainly at 30 seconds of He-Ne seed irradiation. So, seed pre-sowing irradiation with He-Ne laser one-shot for 1second is recommended to be enough for the enhancement of the germination percentage of experimented wheat studied. Various growth parameters were also included concerning shoot and root length, shoot/root ratio,...etc. These changes suggested that the seed pretreatment with He–Ne laser had not only a short-term biological effect, which enhanced inner energy of seeds, but also a long-term effect, which contributed to the acceleration of the growth and development of seedlings. The present study has benefits in enhancing wheat germination, growth and development, as well as in the application of laser in agriculture.
P3
The Molecular Control of in vitro Plant Organogenesis
Y AL-DLAIGAN (ANIMAL & PLANT SCIENCES UNIVERSITY OF SHEFFIELD), A FLEMING (ANIMAL & PLANT SCIENCES UNIVERSITU OF SHEFFIELD Sheffield S10 2TN)
Mr YOUSEF AL-DLAIGAN (y.dlaigan@sheffield.ac.uk)
In vitro organogenesis in plants is the phenomenon of forming new organs (e.g., shoots and roots) from explanted somatic material. It has long been known that de novo organogenesis is mainly controlled by the plant hormones auxin and cytokinin. However, how these hormones trigger stem cell initiation oractivation, leading to adventitious organ formation, is unknown. The aim of this work is to use an inducible gene expression system to investigate the molecular mechanism of in vitro organogenesis by auxin and cytokinin. We have used tetracycline induction of an array of target gene in transgenic tissue to investigate the outcome of altered target gene expression on in vitro regeneration. In particular, we have focused on whether a plant retinoblastoma-related protein (RBR) (an essential G1-S transition cell cycle protein related to a human tumour suppressor protein) is involved in the initiation of new organs via plant hormones. Thus far, we have established a reproducible qualitative and quantitative technique for analyzing in vitro organogenesis and associated gene expression. Furthermore, our preliminary data indicate that overexpressing RBR represses the cell cycle, leading to repression of in vitro root formation. These data are consistent with the theory that RBR is involved in stem cellinitiation/activation.
P4
GIBBERELLIN BIOSYNTHESIS AND ACTION IN ARABIDOPSIS ROOTS
R Barker (Rothamsted ), M Bennett (The University of Nottingham), P Hedden (Rothamsted), S Thomas (Rothamsted)
Mr Richard Barker (richard.barker@bbsrc.ac.uk)
Gibberellins (GAs) are growth promoting hormones that have an essential role in regulating plant growth; mutations in genes that encode certain GA-biosynthetic enzymes are dwarfed with reduced root elongation. GAs act by degrading the growth-repressing DELLA proteins, and it has recently been demonstrated that in the Arabidopsis root DELLA degradation is required only within the endodermis in order to promote root growth. We aim to identify the cellular sites of GA-biosynthesis within the root. We will target expression of genes encoding GA-inactivating enzymes to specific cell types within the root using tissue specific promoters. Inhibition of growth through cell-type specific inactivation of precursor C20-GA or active C19-GAs will indicate the sites of biosynthesis or perception/biosynthesis, respectively. Biosynthesis will be further investigated by attempting to rescue the root elongation phenotype of mutants lacking the enzymes GA 20-oxidase or GA 3-oxidase by cell-type-specific rescue with functional genes. A similar approach will be used to confirm the sites of GA perception, by targeting expression of the GA receptor, AtGID1a in the gid1 triple mutant. The gene expression profiles will be subsequently confirmed using a combination of laser capture microdissection and quantitative real-time PCR. Grafting experiments using GA-deficient mutants will be employed to determine whether root growth is influenced by shoot-derived GAs. In all experiments, root growth will be analysed using semi-automated kinematic computer programs and time lapse photography.
P5
To be confirmed
U Bechtold (University of Essex, UK), P Mullineaux (University of Essex), S Shigeoka (Kinki University, Japan), F Schoffl (Universtiy of Tuebingen, Germany)
Dr Ulrike Bechtold (ubech@essex.ac.uk)
Heat shock transcription factor A1b (HSFA1b or HSF3) and HSFA2 are two of 21 HSF genes in Arabidopsis thaliana and are a key regulators of early responses to heat stress. Compared with yeast and mammals, the plant HSF gene family is highly expanded and suggests that this class of transcription factors may have been recruited to other functions during evolution. HSFA1b and HSFA2 in particular are involved in the excess light and heat dependent expression of ASCORBATE PEROXIDASE 2 (APX2).Independent over expression of HSFA1b (35S:HSF3) and HSFA2 (35S:HSFA2) leads to a constitutive expression of APX2 in Arabidopsis under non stress conditions. Analysis of knockout mutations has shown that HSFA1b and HSFA2 may act independently to promote APX2 expression during heat stress, which may also be modulated by other factors such as light, ABA and H2O2. Interestingly, 35S:HSF3 plants also have a 1.5 to 2 fold increase in foliar H2O2 levels along with enhanced expression of a number of other abiotic and biotic stress associated genes. This suggests that HSFA1b may play a central role in biotic and abiotic stress responses. Testing a variety of virulent pathogens revealed enhanced resistance of the 35S:HSF3 plants to Pseudomonas syringae pv tomato (Pst DC3000), Hyaloperonospora parasitica and turnip crinkle virus. This coincides with enhanced thermotolerance, and improved water use efficiency in 35S:HSF3 plants. A model that explains the mechanism of HSFA1B and HSFA2 during abiotic and biotic stress signaling is currently being developed.
P6
The novel Myb-like protein DUO3 is required for male germline division and sperm cell specification in Arabidopsis
L Brownfield (University of Leicester), S Hafidh (University of Leicester), A Durbarry (University of Leicester), A Sidorova (University of Leicester), D Twell (University of Leicester)
Dr Lynette Brownfield (lrb8@le.ac.uk)
Following meiosis to produce haploid spores, pollen development involves an asymmetric division, giving rise to a large vegetative cell enclosing a smaller germ cell. The germ cell undergoes a second division to produce the two sperm cells required for double fertilization. In Arabidopsis duo pollen mutants germ cell division does not occur giving rise to bicellular pollen containing a single germ cell. In heterozygous duo3 mutants approximately 50% of pollen is bicellular and there is no male transmission of the duo3 mutation. We have identified the DUO3 gene by map-based cloning. The DUO3 protein is unique in Arabidopsis and contains two MYB-like regions and two acidic rich regions. DUO3 is expressed in a number of locations throughout the plant including root tips, apical meristems and vascular tissue as well as both the germ and vegetative cells in pollen. The R2R3 MYB protein DUO1 is also required for germ cell mitosis and we have shown that duo1 pollen does not express a number of germline specific markers. We characterised these markers in duo3 pollen and found expression of some markers reduced while others were unchanged. Thus, DUO1 and DUO3 have overlapping, but distinct roles in male germline specification and our results indicate that DUO1 and DUO3 act on the cell cycle through different mechanisms.
P7
The critical role of the Arabidopsis Circadian Clock at high temperature
N Costa (University of Liverpool), J Hartwell (University of Liverpool), A Hall (University of Liverpool)
Mr Nicola Costa (Nicola.Costa@liverpool.ac.uk)
The circadian clock is an internal mechanism found in most eukaryotes generating a 24h rhythm. It has evolved to anticipate predictable environmental changes and therefore make the best use of resources. In Arabidopsis thaliana circadian clock controls a large number of physiological traits and the expression of about 16% of the plant genes. A clock synchronized with the external environment was found to be important for the growth, performance and fitness of the plant. There is also evidence that plants with a clock period matched to the environment have a substantial advantage over plants with circadian periods differing from their environment (Dodd et al. 2005).In the present study we investigate the importance for Arabidopsis plants of having a functional clock at high temperatures. To address this question we used CCAIox transgenic plants over-expressing the clock gene CCA1 (with an arrhythmic clock) as well as mutant lines whose period is different from the standard 24h of the wild type Col-0. The performance of the plants at different temperatures and photoperiods was measured through the visible leaf area, wet/dry weight and endpoint levels of various metabolites. The results of these experiments will be presented and discussed.
P8
The Histidine Kinase AHK5 Integrates Endogenous and Environmental Signals in Arabidopsis Guard Cells
R Desikan (Imperial College London)
Dr Radhika Desikan (r.desikan@imperial.ac.uk)
Stomatal guard cells monitor and respond to environmental and endogenous signals such that the stomatal aperture is continually optimised for water use efficiency. A key signalling molecule produced in guard cells in response to plant hormones, light, carbon dioxide and pathogen-derived signals is hydrogen peroxide (H2O2). The mechanisms by which H2O2 integrates multiple signals via specific signalling pathways leading to stomatal closure is not known. Here, we identify a pathway by which H2O2, derived from endogenous and environmental stimuli, is sensed and transduced to effect stomatal closure. Histidine kinases (HK) are part of two-component signal transduction systems that act to integrate environmental stimuli into a cellular response via a phosphotransfer relay mechanism. There is little known about the function of the HK AHK5 in Arabidopsis thaliana. Here we report that in addition to the predicted cytoplasmic localisation of this protein, AHK5 also appears to co-localise to the plasma membrane. Although AHK5 is expressed at low levels in guard cells, we identify a unique role for AHK5 in stomatal signalling. Arabidopsis mutants lacking AHK5 show reduced stomatal closure in response to H2O2, which is reversed by complementation with the wild type gene. Over-expression of AHK5 results in constitutively less stomatal closure. Abiotic stimuli that generate endogenous H2O2, such as darkness, nitric oxide and the phytohormone ethylene, also show reduced stomatal closure in the ahk5 mutants. However, ABA caused closure, dark adaptation induced H2O2 production and H2O2 induced NO synthesis in mutants. Treatment with the bacterial pathogen associated molecular pattern (PAMP) flagellin, but not elf peptide, also exhibited reduced stomatal closure and H2O2 generation in ahk5 mutants. Our findings identify an integral signalling function for AHK5 that acts to integrate multiple signals via H2O2 homeostasis and is independent of ABA signalling in guard cells.
P9
An investigation into the role of adenosine methylation in Arabidopsis mRNAs
R Fray (University of Nottingham), H Li (University of Nottingham), S Zhong (University of Nottingham), Z Bodi (University of Nottingham), J Button (University of Nottingham), L Vespa (Université Joseph Fourier ), M Herzog (Université Joseph Fourier )
Dr Rupert Fray (Rupert.fray@nottingham.ac.uk)
N6-methyladenosine is a ubiquitous modification identified in the mRNA of several eukaryotes where it is present within both coding and non-coding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We have shown that Arabidopsis thaliana mRNA contains N6-methyladenosine at levels similar to those previously reported for animal cells. We have also shown that inactivation of the Arabidopsis orthologue of the yeast and human mRNA adenosine methylase (MTA), results in failure of the developing embryo to progress past the globular stage the arrested seeds are deficient in mRNAs containing N6-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems and emerging lateral roots. MTA interacts in vitro and in vivo with AtFIP37, a homologue of the Drosophila protein female lethal 2 D and of human WILMS’ TUMOUR 1-ASSOCIATING PROTEIN - possibly indicating functions in which RNA methylation may be involved.
P10
Effect of Apple Extracts on NF-B Activation in Human Umbilical Vein Endothelial Cells
A Gohil (SSPC)
Mr Ankit Gohil (gohilankit_pharma@yahoo.co.in)
The mechanisms by which foods, such as fruit, are able to reduce the risk of chronic disease are still unclear. Several fruit products, including apples and apple juice, that are flavonoid-rich are reported to increase antioxidant levels in human subjects. This is supported by the finding from our previous studies that the chronic consumption of apple juice by human subjects reduced ex vivo low-density lipoprotein (LDL) oxidation; we hypothesized that this was due to the flavonoid in the apple juice, which, as we reported earlier, reduced in vitro LDL oxidation. To further explore whether the mixture of flavonoids and other phytochemicals in apples are biologically relevant antioxidants, we tested the effects of this flavonoid-rich apple extract (AE) on oxidant-related pathways in a model of the endothelium: human umbilical vascular endothelial cells (HU-VECs). The effects of AE on oxidant-responsive (i.e., tumor necrosis factor [TNF]-–induced) nuclear factor (NF)- B signaling in cell culture were assessed in transfected HUVECs by using a construct that expressed luciferase under the control of NF-B. Incubation of HUVEC for 24 hrs with up to 10 mM (as gallic acid equivalents) of AE demonstrated no cytotoxicity, as determined by lactate dehydrogenase release, caspase 3 activation, and apoptosis marker–based FACS analysis. AE after a 24-hr incubation period at either 200 or 2000 nM showed a complex pattern of decreased basal and TNF-–stimulated NF-B signaling (63% maximal decrease) as assessed by luciferase activity in the transfected HUVECs, as well as by reduced levels of IB protein phosphorylation detected by Western blot analysis. We suggest that AE downregulates NF-B signaling and that this is indicative of an antioxidant effect of the flavonoids present in AE. Key Words: human umbilical vascular endothelial cells • NF-B • antioxidants • apple extract
P11
DELLA-mediated transcriptional control of GA-responsive growth and development in Arabidopsis
A Grønlund (Rothamsted Research), J Griffiths (Rothamsted Research), A Wanchoo (Rothamsted Research), M Wilson (CPIB, University of Nottingham), T Holman (CPIB, University of Nottingham), S Ubeda-Thomas (CPIB, University of Nottingham), M Bennett (CPIB, University of Nottingham), A Phillips (Rothamsted Research), P Hedden (Rothamsted Research), S Thomas (Rothamsted Research)
Dr Anne Grønlund (anne.gronlund@bbsrc.ac.uk)
In recent years there have been major advances in our understanding of how the plant hormone gibberellin (GA) promotes essential processes of plant development. DELLA proteins (DELLAs) are central to the GA signalling cascade where they act as repressors of GA-responsive growth. GA signalling relieves the repression exerted through DELLAs by targeting their degradation. DELLA proteins are nuclear localised and due to their ability to regulate the expression of GA-responsive genes it is suggested they act as transcriptional regulators. However, a canonical DNA-binding domain is absent in DELLAs and it is therefore likely that they regulate the expression of GA-responsive genes through interactions with other transcription factors. This is supported by the recent findings that DELLAs interact with PIF transcription factors to control light-mediated hypocotyls elongation. This work is aimed at improving our understanding of the role of DELLAs and their downstream target genes in regulating GA-responsive growth in Arabidopsis. Microarray experiments have been performed on GA-deficient Arabidopsis mutants, resulting in the identification of many novel GA-responsive genes that are potential primary targets of DELLAs. A reverse genetics-based strategy is subsequently being employed to uncover the role of these potential DELLA target genes in regulating GA-responsive growth. A preliminary study of one of these genes, which encodes a zinc finger transcription factor, suggests that it is a novel regulator of GA metabolism. As an initial approach to understanding how DELLAs regulate expression of these early GA-response genes, yeast two-hybrid assays are being employed to identify DELLA protein interactors.
P12
SPECIFIC LABELLING OF CELL TYPES, STRUCTURES AND DEVELOPMENTAL STAGES IN ARABIDOPSIS
J Grønlund (WHRI, University of Warwick), S Kumar (WHRI, University of Warwick), S Dhondt (VIB, Gent University), G Beemster (VIB, Gent University), P Hilson (VIB, Gent University), J Beynon (WHRI, University of Warwick), V Buchanan-Wollaston (WHRI, University of Warwick)
Dr Jesper Grønlund (j.t.gronlund@warwick.ac.uk)
Although our lives are dependent on plants, our understanding of how they grow and how different levels of organisation (i.e. whole plant, organ, cell, molecular module and molecule) are linked is still not understood. Therefore, the AGRON-OMICS consortium is using existing and novel tools to collect data that enable us to model the growth of the Arabidopsis leaf under non-limiting and limiting environmental conditions (e.g. drought).After initiation of the leaf primordial, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves. However, the Arabidopsis leaf is a complex organ made up of at least 18 individual cell types (10 epidermal, 3 mesophyll and 5 vascular) and 11 structures. At the same time, the growing leaf contains cells at different stages of development with the cells furthest from the petiole being the first to stop expanding and subsequently undergo senescence. Sampling entire leaves can therefore give a distorted view of what is going on in only a subset of the cells.Recently, sectioning and GFP lines, expressing GFP in a cell type specific manner, was used to demonstrate this effect in root tips. It was shown that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes (Birnbaum et al., 2003; Brady et al., 2007). Also, lines containing a cell-type specific GAL4 trans-activation system were used to show that xylem-pole pericycle cells are necessary for lateral root development (Laplaze et al., 2005).The two examples mentioned above show the power of such tools. We are therefore using the LhG4 trans-activation system to develop lines that will allow us to label all the specific cell types, structures and developmental stages in the Arabidopsis leaf. This will allow us to create a high-resolution expression map of the leaf and to specifically over-express or repress transcription of genes in a spatial and temporal manner.
P13
Genomics based analysis of cell wall signalling processes in Arabidopsis thaliana
T Hamann (Imperial College London), M Bennett (Imperial College London), J Mansfield (Imperial College London), C Somerville (Berkeley)
Dr Thorsten Hamann (thamann@imperial.ac.uk)
Development, abiotic and biotic stress impact on the physical architecture and chemical composition of the plant cell wall, making “cell wall stress” (CWS) an integral element of many plant processes. The ability of the plant to respond to CWS is an essential requirement for the maintenance of cell integrity. Over the last years evidence has been mounting that plants have developed a specialised mechanism to perceive CWS and adjust composition and structure of the cell wall in order to ensure cell survival and meet the changing functional requirements during cell differentiation. We have used Arabidopsis seedlings and the cellulose biosynthesis inhibitor isoxaben to characterise the plant´s response to CWS. This work implicates JAR1 and ATRBOH D in regulation of lignin biosynthesis induced by cellulose biosynthesis inhibition (CBI). Both lignin deposition and lesion formation caused by CBI are dependent on the presence of hexoses in the growth media. We will present results characterising the function / relationship of hexoses and JA / ROS based signalling processes during the plant´s response to cell wall stress.
P14
Facilitation of viral RNA trafficking by a plant cellular self-mobile Flowering Locus T RNA
Y Hong (University of Warwick), K Zhang (University of Warwick), X Liu (University of Warwick), C Li (University of Warwick), X Zeng (University of Warwick), S Jackson (University of Warwick), Y Zhou (University of Warwick)
Dr Yiguo Hong (Yiguo.hong@warwick.ac.uk)
RNA trafficking plays an important role in virus-plant interplay and systemic signalling in plant development. Utilizing RNA mobile vectors based on two distinct movement-defective viruses and an agroinfiltration assay, we show that a non-translational Arabidopsis Flowering Locus T (FT) RNA is able to move throughout tobacco and ft-1 Arabidopsis mutant plants, and promote systemic trafficking of GFP, potexvirus and carmovirus RNAs. The results show that a plant host RNA molecule can mediate long-distance trafficking of heterologous viral RNA, indicating movement of cellular RNA and viral RNA may involve a similar mechanism.
P15
New Factors Controlling Stomatal Development
L Hunt (University of Sheffield), J Gray (University of Sheffield)
Dr Lee Hunt (l.hunt@sheffield.ac.uk)
Stomata are pores on the aerial surfaces of plants that facilitate the exchange of CO2 and water vapour with the environment. Each pore is formed by a pair of guard cells which expand and contract in response to environmental signals, to control pore aperture and water loss. Studies of Arabidopsis thaliana mutants have identified a number of genes controlling the pattern of cell divisions leading to stomatal formation and patterning. From analysis of plants with altered expression of these genes it appears that an extracellular signaling pathway involving peptides and processing proteases in combination with LRR receptor complexes activate an intracellular MAPK cascade that inhibits entry to the stomatal lineage by restricting the formation and division of meristemoids. Our recent results suggest several secretory peptides, expressed early in leaf development, limit the capacity of cells to enter the stomatal lineage and alter the patterns of epidermal cells.
P16
Downstream genes of CONSTANS: their roles and interacting partners in Arabidopsis flowering.
S Jang, S Torti and G Coupland (Max-Planck-Institute for Plant breeding research),
Dr Seonghoe Jang (florigen@mpiz-koeln.mpg.de)
CONSTANS(CO) has an important position in the photoperiodic flowering promotion pathway in Arabidopsis thaliana. So far, two different reports have been published on downstream targets of CO: The first one indicating that FT is a major target of CO based on flowering time analyses of p35S:CO ft-10 plants compared with ft-10 alone, whereas the other showed TWINSISTER OF FT (TSF) is also a target of CO independent of FLOWERING LOCUS T (FT). The latter is based on the transcriptional induction of TSF using p35S:COGR co-2 plants and the comparison of expression patterns of FT and TSF. Here, we demonstrate FT and TSF are two independent but genetically redundant downstream targets of CO by transgenic approaches using phloem specific expression of CO in ft and ft tsf double mutant backgrounds. We show that TSF is also a direct target of CO using an inducible system with CO’s own promoter. Additionally, we show mis-expression of FT specifically in minor veins is enough to cause early flowering of Arabidopsis without endogenous FT and TSF. Lastly, proteins interacting with FT and TSF will also be shown and their roles in flowering process in Arabidopsis will be discussed.
P17
Meristem functioning in oil palm and date palm
S Jouannic (IRD Montpellier), H Adam (IRD Montpellier), M Colin (IRD Montpellier), M Marguerettaz (IRD Montpellier), F Aberlenc-Bertossi (IRD Montpellier)
Mr Stefan Jouannic (stefan.jouannic@ird.fr)
Our group studies various aspects of the development of palms, with particular reference to the shoot apex functioning, flowering and sex determination in oil palm and date palm. Elaeis guineensis (African oil palm) and Phoenix dactylifera (date palm) are unbranched plants with a single shoot apical meristem (SAM), which gives rise to pinnate leaves organised in rims. These palms develop unisexual inflorescences from the axillary meristems located at the base of each leaf axis. In oil palm, male and female inflorescences are produced continuously in alternation on the same palm. In contrast, date palm is a dioecious species with seasonal production of inflorescences. A study of the structural organisation of the different meristem types was performed and the characterisation of key regulatory genes was undertaken. KNOX and CUC transcription factors are important regulators of SAM function and leaf morphology by their contribution to dissected leaf development. Palms are of particular interest as they produce dissected leaves by a mechanism different to eudicots. We investigated whether KNOX, CUC-related genes and their microRNA regulator miR164 might be involved in meristem functioning and leaf dissection in palms. Our results so far will be presented. In parallel, a study of flowering meristem activity and its molecular determination was undertaken in date palm through the characterisation of marker genes expression patterns
P18
Sensitivity to freezing 6 (sfr6) has a conditional circadian phenotype that is dependent upon sucrose.
H Knight (Durham University ), A Thompson (University of Edinburgh), H McWatters (University of Oxford)
Dr Heather Knight (p.h.knight@durham.ac.uk)
The sensitive to freezing 6 (sfr6) mutant of Arabidopsis thaliana is late flowering in long days. This can be attributed to reduced expression of components in the photoperiodic flowering pathways, including FKF1, GIGANTEA, CONSTANS and FLOWERING LOCUS T, in long day photoperiods. Microarray analysis of gene expression showed that a circadian clock-associated motif, the evening element, was overrepresented among genes down-regulated in sfr6 plants. We investigated the effect of sfr6 upon clock gene expression using QRT-PCR on timecourses collected from plants grown in free-running and entrained conditions. Expression of the morning clock component CCA1 was reduced in sfr6 seedlings; rhythmic expression of the evening clock genes GIGANTEA and TOC1 showed damped amplitude and delayed phase. We therefore investigated the effects of the sfr6 mutation upon behaviour of the free-running circadian clock. We found sfr6 plants showed a significant long period phenotype that was dependent upon the presence of sucrose in the growth medium. The circadian period of wild type plants shortened when they were grown with sucrose; this was not so for sfr6 seedlings, suggesting that this mutation alters the clock’s ability to respond to sucrose. Analysis of sfr6 circadian behaviour and gene expression imply that, contrary to the predictions of the current model, large changes level and timing of clock gene expression may have little effect upon clock outputs. Moreover, our data suggest that sucrose, despite causing relatively minor changes in clock gene expression, acts upon the Arabidopsis clock. The implications for clock regulation will be discussed.
P19
Investigating effects of temperature on the circadian clock in plants.
J Kusakina (University of Liverpool), A Hall (University of Liverpool), J Hartwell (University of Liverpool)
Miss Jelena Kusakina (jvk@liv.ac.uk)
A vast majority of eukaryotic organisms require a properly functioning circadian clock that allows them to anticipate daily environmental changes and subsequently prepare and adjust their physiological processes. The core oscillator of any clock is made up of interlocking loops of a series of genes and proteins. In Arabidopsis thaliana, a model plant, CCA1 (Circadian Clock Associated 1) and LHY (Late Elongated Hypocotyl) form a part of the main transcriptional negative feedback loop. The two genes encode MYB transcription factors with a high molecular similarity and high functional redundancy. Using LUCIFERASE reporter gene and quantitative RT-PCR techniques, the redundant roles of CCA1 and LHY were separated in plants grown at high (27ºC) and low temperatures (12ºC), with LHY buffering the clock against high temperatures and CCA1 against low. The exact mechanism of the temperature compensation of the clock, however, is still not known. We are currently working on understanding how temperature differentially regulates the expression, transcript and protein stability of CCA1 and LHY. This work should give us a key insight in how temperature is sensed by the plant and how it feeds into the clock.
P20
A tomato HD-Zip homeobox protein, LeHB-1, is involved in control of ethylene synthesis, floral organogenesis and ripening
Z Lin (University of Nottingham), Y Hong (University of Warwick), M Yin (Northwest A & F University), C Li (University of Warwick), K Zhang (University of Warwick), D Grierson (University of Nottingham)
Dr Zhefeng Lin (zhefeng.lin@nottingham.ac.uk)
Ethylene is required for climacteric fruit ripening. Inhibition of ethylene biosynthesis genes, ACC synthase and ACC oxidase, prevents or delays ripening, but it is not known how these genes are modulated during normal development. LeHB-1, a previously uncharacterised tomato homeobox protein, was shown by gel retardation assay to interact with the promoter of LeACO1, an ACC oxidase gene expressed during ripening. Inhibition of LeHB-1 accumulation in tomato fruit using virus-induced gene silencing greatly reduced LeACO1 mRNA levels and inhibited ripening. Conversely, ectopic overexpression of LeHB-1 by viral delivery to developing flowers elsewhere on injected plants triggered altered floral organ morphology, including production of multiple flowers within one sepal whorl, fusion of sepals and petals, conversion of sepals into carpel-like structures that grew into fruits and ripened. Our findings suggest that LeHB-1 is not only involved in control of ripening but also plays a critical role in floral organogenesis. Putative LeHB1 –binding sites are also present in the promoters of the ripening regulatory genes RIN and NOR, which have been suggested to operate upstream of ethylene. We propose that LeHB-1 is part of a regulatory network controlling ethylene synthesis, fruit development and ripening.
P21
LeTPR1 interacts with the ethylene receptors NR and LeETR1, and modulates ethylene and auxin responses
Z Lin (University of Nottingham), L Arciga-Reyes (Bayer de Mexico), S Zhong (University of Nottingham), L Alexander (Waltham Centre for Pet Nutrition), R Hackett (IGD), I Wilson (University of the West of England), D Grierson (University of Nottingham)
Dr Zhefeng Lin (zhefeng.lin@nottingham.ac.uk)
The gaseous hormone ethylene is perceived by a family of ethylene receptors which interact with the Raf-like kinase CTR1. LeTPR1 encodes a novel TPR (tetratricopeptide repeat) protein from tomato that interacts with the ethylene receptors NR and LeETR1 in yeast two-hybrid and in vitro protein interaction assays. Overexpression of LeTPR1 in tomato resulted in ethylene-related pleiotropic effects including reduced stature, delayed and reduced production of inflorescences, abnormal and infertile flowers, epinasty, reduced apical dominance, inhibition of abscission, altered leaf morphology, parthenocarpic fruit and altered fruit morphology. Similar phenotypes were also seen in Arabidopsis plants overexpressing LeTPR1. LeTPR1 overexpression did not increase ethylene production but caused enhanced accumulation of mRNA from the ethylene responsive gene ChitB and the auxin-responsive gene LeSAUR1-like, and reduced expression of the auxin early responsive gene LeIAA9, which is known to be inhibited by ethylene and to be associated with parthenocarpy. It is suggested that LeTPR1 overexpression enhances a subset of ethylene and auxin responses by interacting with specific ethylene receptors. LeTPR1 shares features with human TTC1, which interacts with heterotrimeric G-proteins and Ras, and competes with Raf-1 for Ras binding. Models for LeTPR1 action are proposed involving modulation of ethylene signalling or receptor levels.
P22
Statistical analysis of next generation sequencing data with application to a ChIP-SEQ experiment in Arabidopsis
J M (IGR-PAN), K Kaufmann (Wageningen UR), P Krajewski (IGR-PAN)), G Angenent (Wageningen UR)
Mr Jose M Tbc (jmui@igr.poznan.pl)
Next generation sequencing technologies offer unique opportunities and challenges for a broad range of applications. In this poster, we focus on the genome-wide analysis of binding sites for a particular transcription factor involved in flower development in Arabidopsis thaliana using Chromatin Immunoprecipitation in combination with the Solexa sequencing-by-synthesis technology (ChIP-SEQ). A new single-nucleotide resolution statistical approach is presented for the analysis of this type of data sets and the methodology is compared with the alternative ChIP-CHIP method.
P23
Acropetal auxin transport mediates root patterning in plants
V Mironova (Institute of Cytology and Genetics, Russia), V Likhoshvai (Institute of Cytology and Genetics, Russia), N Omelyanchuk (Institute of Cytology and Genetics, Russia), E Mjolsness (Institute of Genomics and Bioinformatics, University of California, Irvine, USA), N Kolchanov (Institute of Cytology and Genetics, Russia)
Miss Victoria Mironova (kviki@bionet.nsc.ru)
Recent studies indicated that the plant hormone auxin may regulate the rates of its own transport by influencing gene expression and stability of PIN family efflux transporters [1-2]. Based on these data we created a mathematical model for mechanism of root patterning along its longitudinal axis. We demonstrated in silico that known genetic regulation of PIN1 expression is necessary and sufficient condition for formation and maintaining of the pattern of auxin distribution in root. Furthermore, our simulation demonstrate that formation of root structure in development, its maintaining and restoration after damage might be the different outcomes of the same mechanism orchestrated by auxin. The following three types of auxin concentration maxima were experimentally detected in plants. The terminal auxin maximum is always located in the root tip [3]. The internal auxin maxima are temporally observed in the provascular tissue preceding the initiation of lateral root meristems [4]. As well as a hypocotyl auxin maxima precede to adventitious root meristems. In our model increasing auxin flow from the shoot resulted in reiterative appearance of proximal auxin maxima. The model potential for additional maxima formation varied depending on efficiency of PIN1 expression inhibition. These facts indicated that the difference in the auxin acropetal transport regulation may be the main factor responsible for plant diversity in root architecture. 1. Vieten et al. (2005) Development. 132(20): 4521-4531.2. Sauer et al. (2006) Genes&Dev. 20: 2902-2911. 3. Sabatini et al. (1999) Cell. 99(5): 463–472. 4. de Smet et al. (2007) Development, 134, 681-690.
P24
From images and shapes to modeling root hair initiation
R Morris (John Innes Centre), S Grandison (John Innes Centre), R Leggett (John Innes Centre), S Takeda (John Innes Centre), L Dolan (John Innes Centre)
Dr Richard Morris (richard.morris@bbsrc.ac.uk)
We present a novel methodology based on image processing techniques, minimal energy surfaces and the calculus of variations, which allows us to infer the mechanical properties of growing Arabidopsis thaliana root hairs from raw time-lapse image data. Computational simulation of root hair emergence, modeled as a large set of interconnecting springs, allows for different growth models to be validated against experimental data. With this approach we demonstrate that root hair initiation is consistent with elastic growth. Independent data from growing cells show that simulation and experiment are in excellent agreement. Furthermore, we are able to provide supporting evidence to discriminate one growth model over the other.
P25
Cytokinins and abscisic acid may be involved in the regulation of age-related changes in the reproductive strategy of the Mediterranean shrub, Cistus albidus L.
S Munné-Bosch (Universitat de Barcelona), M Oñate (Universitat de Barcelona)
Prof Sergi Munné-Bosch (smunne@ub.edu)
To better understand aging in perennials, the role of phytohormones in vegetative and reproductive growth in the Mediterranean shrub, Cistus albidus L. was evaluated. Two groups of different ages (5- and 10-y-old), both at advanced developmental stages but with similar size, were compared. Total plant biomass, biomass produced per apical meristem and levels of cytokinins, zeatin and zeatin riboside, abscisic acid and jasmonic acid in leaves and flower buds, as well as flower production, were measured. No differences in vegetative growth (photosynthetic and non-photosynthetic biomass) and levels of phytohormones in leaves were observed between 5- and 10-y-old plants. However, they showed significant differences in flower bud development. At stage I (starting flower organ formation), contents of zeatin and abscisic acid were 82% and 41%, respectively, smaller in 10- than 5-y-old plants. At stage II (with all flower organs formed) differences were smaller (61% and 29% for zeatin and abscisic acid, respectively). Age-related changes in phytohormones may be associated with loss of flower bud vigour, the oldest plants showing 58% smaller number of flowers reaching anthesis. Nevertheless, the total number of flowers produced per individual was similar in both plant groups. The results suggest that, although 5- and 10-y-old plants do not show differences in vegetative growth, plants change their reproductive strategy at advanced developmental stages in a process that appears to be regulated by phytohormones. However, an age-related loss in reproductive vigour at the organ level does not lead to a decrease in flower production at the whole plant level.
P26
Salicylic acid deficiency in NahG transgenic lines and sid2 mutants delays senescence and increases seed yield in the annual plant Arabidopsis thaliana
S Munné-Bosch (Universitat de Barcelona), M Abreu (Universitat de Barcelona)
Prof Sergi Munné-Bosch (smunne@ub.edu)
Salicylic acid-deficient NahG transgenic lines and sid2 mutants were used to evaluate the role of this compound in the development of the short-lived, annual plant Arabidopsis thaliana, with a particular focus on the interplay between salicylic acid and other phytohormones. Low salicylic acid levels during the transition to flowering led to smaller abscisic acid levels and reduced damage to PSII (as indicated by Fv/Fm ratios) in leaves of NahG transgenic lines and sid2 mutants, thus resulting in a delay of senescence, compared to wild type plants. Furthermore, salicylic acid deficiency caused increases in seed yield by 4.4 and 3.5-fold in NahG transgenic lines and sid2 mutants, respectively, compared to wild type. Salicylic acid deficiency also improved seed composition in terms of antoxidant vitamin contents, seeds of salicylic acid-deficient plants showing higher levels of a- and g-tocopherol (vitamin E) and b-carotene (pro-vitamin A) than seeds of wild type plants. It is concluded that (i) the sid2 gene, which encodes for isochorismate synthase, plays a central role in salicylic acid biosynthesis during plant development in A. thaliana, (ii) salicylic acid plays a role in the regulation of leaf senescence and seed production, (iii) there is a cross-talk between salicylic acid and other phytohormones during plant development, and (iv) the contents of antioxidant vitamins in seeds may be influenced by the endogenous levels of salicylic acid in plants.
P27
Regulation of heterotrimeric G-protein complexes in Arabidopsis
H Okamoto (University of Oxford), R Reid (University of Oxford), S Harris (University of Oxford)
Dr Haruko Okamoto (haruko.okamoto@plants.ox.ac.uk)
Heterotrimeric G-proteins, comprising α, β, and γ subunits, are common signalling molecules in multicellular organisms across the phyla. The Arabidopsis genome encodes a single copy each of Gα and Gβ and two Gγ subunits. The heterotrimeric complex dissociates upon activation and the Gα subunit binds to its effectors that in turn initiate downstream signalling that enable plants to adapt to their environment. In order to understand G-protein signalling, it is important to know what proteins the G-protein subunits are associated with in different tissue types and in response to different signals. We have taken two approaches to addressing this. Firstly, we have analysed protein complexes containing the Gα subunit. We have found that membrane-associated Gα subunit is primarily in a 150 kDa complex when WT seedlings are grown in the absence of light. In contrast, in seedlings grown in the light, the Gα subunit is associated with protein complexes of approximately 600, 400, 250, and 100 kDa. The 600, 250, and 100 kDa Gα protein complexes were all detectable in a Gβ loss of function mutant indicating that Gα association with these complexes does not require the Gβ subunit. The occurrence of Gα complexes is also tissue-dependent with only the 600, 400 and 100 kDa complexes present in meristematic tissue. To identify potential components of these complexes we have also investigated Gα interacting proteins by yeast 2-hybrid screening. The role of these Gα-interacting proteins in Gα complexes and the possible function of these complexes will be discussed.
P28
The evolution of nuclear auxin signaling
I Paponov (University of Freiburg), W Teale (University of Freiburg), D Lang (University of Freiburg), M Paponov (University of Freiburg), R Reski (University of Freiburg), S Rensing (University of Freiburg), K Palme (University of Freiburg)
Dr Ivan Paponov (ivan.paponov@biologie.uni-freiburg.de)
Genomic sequencing has enabled thorough and far reaching comparative analyses of many important cellular processes. Recently, genomes of two non-seed plants, Physcomitrella patens and Selaginella moellendorffii, have been completely sequenced, enabling a detailed inference of key processes over 450 million years of divergent evolution. One such process is auxin signaling, which has a pervasive effect on flowering plant growth and development. Genes encoding auxin signaling proteins are ubiquitous in land plants. However, there are significant differences among them; most notably in the AUXIN RESPONSE FACTOR (ARF) and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) gene families, encoding transcription factors and their repressors. Comparative analysis of the P. patens, S. moellendorffii and Arabidopsis thaliana genomes suggests that the rapid transcriptional response to auxin, observed in flowering plants, evolved in vascular plants after their divergence from the last common ancestor shared with mosses. An N-terminally truncated ARF transcriptional activator is encoded by the genomes of P. patens and S. moellendorffii, revealing a supplementary mechanism of nuclear auxin signaling absent in flowering plants. Site-specific analyses of positive Darwinian selection (PDS) gave insights into the evolution of ARFs and Aux/IAAs. Relatively high rates of synonymous substitution in the A. thaliana ARFs of classes IIa (and their closest orthologous genes in poplar) and Ib, suggest that neofunctionalization in important functional regions has driven the evolution of auxin signaling in flowering plants. Primary auxin responsive gene families (GH3, SAUR, LBD) show different phylogenetic profiles in P. patens, S. moellendorffii and flowering plants, highlighting genes for further study.
P29
An FLC-like gene is controlled by vernalization in root chicory
C Périlleux (University of Liège), A Pieltain (University of Liège), M DAloia (University of Liège)), O Maudoux (Cosucra Groupe Warcoing), S Lutts (Université catholique de Louvain), J Kinet (Université catholique de Louvain)
Prof Claire Périlleux (cperilleux@ulg.ac.be)
Vernalization is known to promote flowering in Arabidopsis via the repression by cold of the floral inhibitor gene FLOWERING LOCUS C (FLC). For long, FLC homologs have been found in Brassicaceae only but it was recently reported that in sugar beet, the FLC-like gene BvFL1 functions as a repressor of flowering and is downregulated in response to cold. We describe here the cloning of CiFL1 from root chicory (Cichorium intybus var. sativum). Expression patterns were studied in two cultivars, differing in their sensitivity to vernalization. Transcript level analyzes were performed during the vernalization treatment of the seedlings and in different post-vernalization conditions. Our results give further support to conservation of the biological function of FLC-like genes in eudicot species.
P30
Arabidopsisvacuolar Ca2+/H+ exchangers CAX1 and CAX3 play a role in abiotic stress tolerance
J Pittman (University of Manchester), C Edmond (University of Manchester), S Acton (Lancaster University), K North (Lancaster University), J Zhao (Baylor College of Medicine), K Hirschi (Baylor College of Medicine), M McAinsh (Lancaster University)
Dr Jon Pittman (jon.pittman@manchester.ac.uk)
Abiotic stress can significantly perturb plant growth, thus plants have specific adaptive responses which are activated following stress signal perception and signal transduction. A common response to abiotic stress is the generation of cytosolic Ca2+ elevations with specific dynamics which are thought to elicit a specific response. It is unclear what components are involved in generating these Ca2+ signatures. Arabidopsis vacuolar Ca2+/H+ exchangers encoded by CAX genes mediate high capacity Ca2+ transport into the vacuole. Analysis of cax1 and cax3 knockout mutants suggest that these transporters are involved in specific abiotic stress responses including low temperature, salinity and oxidative stress. Furthermore, CAX1 and CAX3 are both regulated in response to abiotic stress. CAX1 and CAX3 are transcriptionally up-regulated following stress treatments. In addition, stress-dependent phosphorylation was observed for both proteins; however, the stress-dependent regulation profile differs between CAX1 and CAX3 with respect to specific stresses. Inhibition of vacuolar Ca2+/H+ exchange activity affects the generation of stress-induced Ca2+ signatures. Alterations in the dynamics of cold- and H2O2-induced cytosolic Ca2+ signatures were observed in the cax1 knockout. These results suggest that CAX1 and CAX3 may be central components in controlling cytosolic Ca2+ dynamics under specific stress conditions.
P31
The Role of the GA20ox Gene Family in Arabidopsis Reproductive Development
A Plackett (Rothamsted Research), P Hedden (Rothamsted Research), S Thomas (Rothamsted Research), A Phillips (Rothamsted Research), Z Wilson (University of Nottingham)
Mr Andrew Plackett (andrew.plackett@bbsrc.ac.uk)
Reproductive development represents a complex and interesting facet of plant biology due to its determinate nature and the integration of many different environmental and physiological signals. The phytohormone gibberellin (GA) has an important role in plant male fertility: mutants in the GA biosynthesis pathway display defects in stamen development that encompass pre-anthesis filament elongation, anther dehiscence and pollen development. Recent advances in our understanding of the underlying mechanism of GA signalling, as well as genetic pathways involved in floral developmental processes, have provided the tools to address the question of how GA regulates stamen development. The GA 20-oxidases (GA20ox) are functionally similar enzymes that catalyse multiple steps late in the GA biosynthetic pathway. In Arabidopsis they are encoded by a family of five genes that differ in their expression patterns. This project aims to explore the role of GA in stamen development through studying the GA20ox gene family. Past work with GA20ox1 and GA20ox2 suggests redundancy of function during stamen development that includes at least one more of the remaining GA20ox genes. Mutant lines carrying knockouts of multiple GA20ox genes will identify which paralogues are important in this process, whilst GA20ox spatial and temporal expression patterns in individual tissues will be refined through transgenic GUS reporter lines. GA biosynthesis and signalling will be manipulated in target tissues and at critical time points via transgenic elements to investigate directly the roles GA plays within the stamen developmental programme.
P32
Post-translational regulation enters the three-loop mathematical model of circadian clock in Arabidopsis
A Pokhilko (Edinburgh University, School of Biological Sciences), A J. Millar (Edinburgh University, School of Biological Sciences)
Dr Alexandra Pokhilko (apokhilk@staffmail.ed.ac.uk)
Daily plant rhythms are driven by cyclic expression of the circadian clock genes. The core structure of circadian gene network consists of three feedback loops, which represent connected morning and evening oscillators. Here we extend our previous model of circadian gene expression (Locke at. al. Mol. Syst. Biol. 2: 59, 2006), firstly based on recently-published data. This introduces post-translational regulation of the evening protein TOC1 by the F-box protein ZEITLUPE (ZTL), ZTL’s regulation by light, and stabilization of ZTL by its interaction with GIGANTEA (GI). GI’s role in the clock model has thus been revised according to the data: GI promotes inhibition of TOC1 protein through positive regulation of ZTL. Our simulations show that this results in the observed long period of the ztl mutant and fast dampening of rhythms in the lhy/cca1/gi triple mutant. Secondly, we are using models to propose specific hypotheses in advance of experimental studies. The connection between morning and evening oscillators has been significantly modified, inspired by existing data. We propose a new circuit structure for the circadian clock network, including a novel, mutual inhibition of morning and evening oscillators. We add more mechanistic detail to account for the specific waveforms of clock gene expression, particularly of the morning elements LHY and CCA1. The new structure retains the good match of our previous 3-loop model to the large volume of kinetic data on rhythms in mutant plants, extends this realism to recapitulate additional mutations, and makes predictions that are currently being tested experimentally
P33
ArabidopsisSPA proteins in regulation of plant growth and development
A Ranjan (Botanical Institute, University of Cologne, Germany), U Hoecker (Botanical Institute, University of Cologne, Germany)
Mr Aashish Ranjan (aranjan@uni-koeln.de)
Light is one of the most important environmental factors affecting various stages of plant growth and development. Arabidopsis SPA- and COP1 proteins act as repressors of seedling photomorphogenesis in darkness. Members of the SPA protein family (SPA1-SPA4) likely act as cofactors for the E3 ubiquitin ligase COP1 to target activators of light signal transduction for degradation in darkness. SPA proteins have partly redundant but also distinct functions throughout development of Arabidopsis thaliana. SPA1 and SPA2 predominate in seedling development in darkness, whereas SPA1 is the key player in seedling development in the light. SPA1 and SPA2 are further important for the arrested shoot differentiation and root development in darkness, indicating possible communication between shoot differentiation and root development. SPA3 and SPA4 are the predominant regulators in adult plant growth and leaf development. SPA1 is necessary and sufficient for normal photoperiodic flowering by controlling the stability of the floral inducer CONSTANS. To investigate the involvement of SPA proteins in non-cell autonomous regulation of plant development, we expressed SPA1 under the control of various tissue-specific promoters in a spa mutant background. The early-flowering phenotype of short day-grown spa1 mutant plants was fully complemented by phloem-specific expression of SPA1, but not by mesophyll- or meristem-specific expression of SPA1. This indicates that SPA1 acts in the phloem to regulate photoperiodic flowering. Expression of SPA genes under various other tissue-specific promoters during different developmental stages is currently being analysed to have a broader overview of cell-cell communication in SPA-regulated plant development.
P34
Jasmonate signalling in far-red light responses in Arabidopsis
F Robson (University of East Anglia), H Okamoto (University of Oxford), E Patrick (University of East Anglia), S Harris (University of Oxford), J Turner (University of East Anglia)
Dr Fran Robson (f.robson@uea.ac.uk)
The plant hormone jasmonate (JA) plays important roles in plant defence, growth and fertility. coi1 mutants flower earlier than wild-type under both long- and short-days and have elongated petioles and hyponastic leaves; phenotypes reminiscent of plants undergoing shade avoidance responses. We found that the JA signalling mutant, coi1, is partially insensitive to far-red light with respect to hypocotyl growth inhibition, has altered expression of the far-red light-regulated transcription factor genes HAT4, HFR1 and PIL1 and, similar to the photoreceptor mutant, phyA, is partially resistant to the far-red-mediated block of greening due to elevated NADPH-protochlorophyll oxidoreductase A (PORA) gene expression in cFR light. coi1 mutants also display exaggerated shade avoidance responses in low, but not high, red:far-red light regimes. COI1 is a component of an SCF E3 ubiquitin ligase and is a receptor for JA and its bioactive derivatives. The JA biosynthesis mutant, aos, also shows far-red insensitivity with respect to hypocotyl growth inhibition at a level similar to that of coi1 mutants, as do the JA signalling mutants jin1/myc2 and jai3/jaz3 indicating that, in addition to COI1, JA biosynthesis and signalling modulate plant responses to far-red light. The JAZ1 protein, a target of the COI1 E3 ligase, fused to GUS, is degraded by far-red light in a COI1-dependent manner. We also found that in far-red light, the phyA mutant has altered expression of a number of genes involved in JA biosynthesis and signalling. These data indicate that JA biosynthesis and signalling play a role in far-red light responses.
P35
The molecular defect of the epibrassinolide-responsive dwarf bashful mutant of Arabidopsis
P Rocha (I.S.A.-C.A.S), X Xia (I.S.A.-C.A.S), R Zhang (I.S.A.-C.A.S.), K Lindsey (U. of Durham)
Dr P. Rocha (procha_ac@yahoo.co.uk)
Brassinosteroids are phytohormones involved in a wide range of plant biological processes including the regulation of physiological responses and of developmental programs. Here we report progress in the identification and characterization of a candidate locus and molecular defect associated with bashful, a previously isolated epibrassinolide-responsive dwarf mutant of A. thaliana. In the bashful mutant genome this chromosome 3 locus is tagged by two T-DNA insertions separated by 1.4 kb in a region containing two annotated transcription units encoding putative proteins of yet unknown function.
P36
The NtMPK4 MAP kinase is essential for CO2 responses in guard cells of Nicotiana tabacum
R Roelfsema (Julius Maximilians University Wuerzburg)
Dr Rob Roelfsema (ROELFSEMA@BOTANIK.UNI-WUERZBURG.DE )
Light-induced stomatal opening in C3- and C4 plants is mediated by two signaling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on Photosynthetic Active Radiation (PAR). Here the role of NtMPK4 on light-induced stomatal opening was studied, since silencing of this MAP kinase stimulates stomatal opening. Stomata of NtMPK4-silenced plants do not close in elevated CO2 and show a reduced response to PAR. However, stomatal closure can still be induced by ABA. Measurements with multi-barreled intracellular micro electrodes showed that CO2 activates plasma membrane anion channels in wild type Nicotiana tabacum guard cells, but not in NtMPK4-silenced cells. Anion channels were also activated after switching off PAR in wild type guard cells. In approximately half of these cells, this response was accompanied by a rise in the cytoplasmic free calcium concentration. The activity of anion channels was higher in cells displaying a parallel rise in cytosolic calcium, than in those with steady calcium levels. Both the darkness-induced anion channels activation and calcium signals were repressed in NtMPK4 silenced guard cells. These data show that NtMPK4 plays an essential role in CO2-and darkness-induced activation anion channels, through calcium-dependent and -independent signaling pathways.
P37
A MUTANT AFFECTED IN THE CLP R4 SUBUNIT OF CLP PROTEASE REVEALS A ROLE IN CHLOROPLAST DEVELOPMENT
G Saini (Institute of Plant Science,. ETH ), K Apel (Boyce Thompsone Institute of Plant Research)
Dr Geetanjali Saini (saing@ethz.ch)
Norflurazon treatment in plants leads to photobleached chloroplasts, in which the light induced transcription of nuclear genes encoding plastid proteins (such as Lhcb) is suppressed. This suggests the role of a plastid-derived signal for regulating the expression of nuclear genes encoding plastid proteins. In Arabidopsis thaliana, a terapyrrole dependent signaling (gun mutants) has already been proposed, using Norflurazon treated seedlings grown at 5uM NF and exposed to moderate light intensities. Under these conditions the seedlings are under stress as indicated by plastid damage and anthocyanin accumulation. Under these conditions bleaching of NF-treated seedlings may primarily result from photodamage. In order to minimize this possible cytotoxic impact and to optimize conditions for a new suppressor screen of NF treated seedlings, NF concentrations and light intensities were reduced. Under these conditions seedlings were still bleached and retained nonfunctional chloroplasts, but were devoid of anthocyanins. Under these mild NF growth conditions a novel class of mutants could be identified that showed chlorophyll accumulation in the presence of NF. These have been dubbed as happy on norflurazon (hon). In these mutants Lhcb expression was de-repressed with much higher levels of Lhcb transcript as compared to the wildtype seedlings (white and bleached). Map-based cloning, sequencing and complementation of one of these mutants, hon5 has been completed. The hon5 mutation has been located on chromosome 4 within the ClpR4 gene. ClpR4 forms part of an ATP-dependent multi-subunit proteolytic complex in chloroplasts consisting of five ClpP proteases, four non-catalytic ClpP-related ClpR homologs, three ClpAAA chaperones and three members with unknown function (Peltier et al., 2004).The hon5 mutation leads to a splicing defect and as a consequence the concentration of the ClpR4 protein is drastically reduced relative to the wild-type control. This in turn leads to a reduction in the accumulation of the ClpPRS protease complex. Based on the functions of this protease and its substrates in the chloroplast, we are trying to dissect why the mutation in this gene restores chloroplast development. References1.Peltier J-B, Ripoll DR, Friso G, Rudella A, Cai Y, Ytterberg J, Giacomelli L, Pillardy J, and van Wijk KJ (2004). Clp protease complexes from photosynthetic and non-photosynthetic plastids and mitochondria of plants, their predicted 3-D structures and functional implications. J. Biol. Chem 279: 4768-4781.
P38
Arabidopsis MYB26/MALE STERILE 35gene regulates secondary cell wall thickening in the anther endothecium and controls anther dehiscence.
J Song (University of Nottingham), C Yang (University of Nottingham), Z A Wilson (University of Nottingham)
Miss Jie Song (sbxjs1@nottingham.ac.uk)
Anther dehiscence is a two-phase process involving lytic opening of the stomium and then retraction of the anther wall. During microspore maturation, cellulose and lignified thickenings are deposited in the anther endothecium, which is important in generating the forces required for dehiscence. MYB26/MALE STERILE 35 (MS35) acts as a key regulator in the process of lignified secondary thickening in the endothecium and subsequent dehiscence. The ms35 mutant fails to produce endothecial secondary thickening. MYB26 expression occurs early during endothecial development and is maximal during pollen mitosis I and the bicellular stage. Over-expression of MYB26 results in ectopic secondary thickening and changes in expression in several genes linked to secondary thickening, including IRREGULAR XYLEM 1 (IRX1), IRX3, IRX8, IRX12, NAC SECONDARY WALL PROMOTING FACTOR1 (NST1) and NST2. This indicates that MYB26 functions in a regulatory role in determining endothecial cell development and acts upstream of the lignin biosynthesis pathway. Immunolocalisation of pectin and xylan has provided further evidence for the role of MYB26 in the regulation of secondary wall development in the anther. AffymetrixTM microarray analysis was performed using isolated, precisely staged anthers from the ms35 mutant compared with the wild type, and, revealed a coordinated down-regulation of genes previously linked to secondary cell wall biosynthesis. It also suggested that a number of additional genes are involved, including several transcription factors and genes in ubiquitin-dependent protein degradation and phytohormone related pathways. These data and the potential role for MYB26 will be discussed.
P39
Ca2+ - dependent activation of S-type anion channels in guard cells of N. tabacum
A Stange (University of Würzburg), R Roelfsema (University of Würzburg), R Hedrich (University of Würzburg)
Ms Annette Stange (A.Stange@botanik.uni-wuerzburg.de)
Stomata are responsive to various stimuli such as ABA and CO2. Several of these stimuli have been suggested to alter the cytoplasmic free Ca2+ concentration. However, ABA can also activate anion channels in the plasma membrane of guard cells without a rise in the cytoplasmic free Ca2+ concentration. In this studies, we impaled guard cells of intact N.tabacum plants with triple barrelled electrodes, to iontophoretically load the Ca2+ sensitive dye FURA-2 and to stimulate the cells by different voltages. Hyperpolarizing the membrane to -180mV lead to a rise in the cytoplasmic free Ca2+ concentration. After returning to the holding potential of -100mV transient inward currents are activated. In 8 out of 13 cells a peak cytoplasmic free Ca2+ concentration of more than 400nM correlates with these inward currents. Voltage pulses to -60mV revealed, that these inward currents are slowly deactivating, a feature that is typical for S-type anion channels. We conclude that cytosolic concentrations of Ca2+ above 400nM are capable of activating S-type anion channels. Future studies will be aimed to elucidate the mechanism by which Ca2+ activates S-type anion channels in the plasma membrane of guard cells.
P40
Receptor Like Kinases Involved In Secondary Cell Wall Synthesis.
N Taylor (University of York)
Dr Neil Taylor (ngt2@york.ac.uk)
Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, UK. Cellulose is central to plant development and is synthesized at the plasma membrane by an organized protein complex that contains three different cellulose synthase proteins (Taylor et al 2003). The ordered assembly of these three catalytic subunits is essential for normal cellulose synthesis. The way in which the cell regulates cellulose synthesis is currently unknown though it is clear that there must be some form of communication between the extracellular cell wall and the intracellular site of polymerization of glucan chains. Trans-membrane signaling by receptor like kinases (RLKs) is one mechanism by which this could occur. RLKs contain an extracellular ‘sensing’ domain linked by a trans-membrane domain to an intracellular kinase domain which is capable of phosphorylating proteins to regulate their activity, stability or to activate a signal transduction pathway. Recent work has shown that at least two of the cellulose synthases responsible for secondary cell wall cellulose synthesis in Arabidopsis thaliana, AtCesA4 and AtCesA7, are phosphorylated in vivo. Analysis of phosphorylation sites by mass spectrometry has identified some of these in vivo phosphorylation sites which occur in a region of hyper-variability between the CesA proteins (Taylor 2007). Current work is focused on the identification of the kinases that phosphorylate the cellulose synthase proteins, and the latest results from this work will be presented. The identification of the receptor like kinases involved in regulating cellulose synthesis would clearly represent a major step forward in our understanding of cellulose synthesis. The identification of regulatory mechanisms controlling cellulose synthesis will allow a unique opportunity to manipulate the content and composition of cell walls, a developmentally and economically important target for biotechnology. Taylor et al (2003). PNAS 100:1450-1455.Taylor (2007). Plant Molecular Biology, 64:161-171.
P41
Genetic control of water use efficiency in Arabidopsis
A Thompson (University of Warwick), I Taylor (University of Nottingham), J Lynn (University of Warwick), P White (Scottish Crops Research Institute), G Farquhar (Australian National University), J Deswarte (University of Warwick)
Dr Andrew Thompson (a.j.thompson@warwick.ac.uk)
Soil degradation, climate change and depletion of aquifers will reduce water availability for agriculture at a time when food production must increase to meet demand. Breeding for crop varieties with improved water-use efficiency (WUE) is one solution to this dilemma. The genetic control of WUE, the ratio between biomass accumulated and water transpired, is a complex and intractable trait with a large interaction with environment; the extent to which WUE can be improved without reducing yield potential (or biomass) is controversial. Here we have studied the genetic control of WUE in Arabidopsis. Investigation of natural variation in 96 accessions indicated that WUE and biomass varied independently and that variation in stomatal conductance, rather than biomass accumulation, was the main source of variation in WUE. We used two recombinant inbred line (RIL) populations to identify five QTL for WUE, including three novel QTL; some co-localised with QTL for flowering time or biomass. We performed association mapping and identified markers strongly associated within two of the RIL QTL. Such markers provide leads for the identification of causative genes; we are currently constructing near-isogenic lines in these regions. We compared the magnitude of improvements in WUE due to transgenic enhancement of abscisic acid biosynthesis to the degree of natural variation in WUE in Arabidopsis. Transgenic lines in a Col-0 background displayed 30-50% higher WUE than WT, and had a greater WUE than any natural accession tested. The high ABA plants showed improved biomass under dry conditions, and only a mild penalty when well-watered.
P42
A one-loop model of the Ostreocuccus tauri circadian clock
C Troein (University of Edinburgh), A Millar (University of Edinburgh), F Bouget (Université Paris VI)
Dr Carl Troein (carl.troein@ed.ac.uk)
The circadian clock of plants in general - and Arabidopsis in particular -is a system of great importance to the fitness of the organism. Thisoscillator is known to regulate the expression of a large number of genes,and modelling and experiments have revealed much about how it works. Themajor components of the clock are genes with many homologues, and togetherthey form a highly non-trivial system of interconnected feedback loops,making accurate modelling a great challenge. In contrast, in Ostreococcustauri, a tiny alga with an exceptionally small genome, the only homologuesto known Arabidopsis clock genes are a single homologue of each of the genesTOC1 and CCA1. The organism is nonetheless capable oftemperature-compensated entrainment to light/dark cycles in both long andshort days, as would be expected from a proper circadian clock. We havemodelled the Ostreococcus clock as a single negative feedback loop betweenTOC1 and CCA1, and shown that this model fits with data from luciferasepromoter and protein fusion experiments, both under light/dark cycles andin constant light.
P43
Transcriptomics and metabolomics implicate aromatic secondary metabolites in the basal and systemic immunity of Arabidopsis to bacterial infection.
W Truman (University of Exeter), S Forcat (Imperial College London), M Bennett (Imperial College London), V Perera (University of Exeter), M Grant (University of Exeter)
Dr William Truman (W.M.Truman@exeter.ac.uk)
In nature successful pathogen infections of plants are relatively rare. Although plants lack an adaptive immune system they deploy a series of preformed and induced defence strategies for protection. Recognition of microbe associated molecular patterns (MAMPs) by the plant elicits basal resistance responses, which successful invading bacteria overcome by the secretion of Type III effector proteins (TTE) and phytotoxins. Recognition of avirulence factors amongst the TTEs can result in the plant mounting a hypersensitive response (HR) and systemic acquired resistance (SAR). SAR offers broad spectrum and long lasting protection against typically virulent pathogens. We have conducted transcriptional profiling, using full genome microarrays, of key time-points during the development of both the basal resistance and SAR responses of Arabidopsis to Pseudomonas syringae. Significant overlaps were identified in these two responses that implicate the co-ordinated regulation of the biosynthetic pathways of aromatic secondary metabolites, including lignins and flavonoids, in defence against infection. Both the basal resistance response and SAR have now been subject to global metabolite profiling using liquid chromatography mass spectroscopy (LC/MS); significant changes in abundance were observed for several metabolites. Commonalities have also been identified amongst the metabolites accumulating during basal defence and SAR. The polar transport of auxin, mediated by AUX1, has been shown to be essential for the establishment of SAR. Metabolite profiling of the systemic response to avirulent infection in the aux1 mutant has revealed its impact on at least two classes of indolic compounds that may be involved in defence.
P44
Root growth in Arabidopsis requires gibberellin/DELLA signalling in the endodermis
S Ubeda-Tomas (University of Nottingham)
Dr Susana Ubeda-Tomas (SUSANA.UBEDA-TOMAS@NOTTINGHAM.AC.UK)
Gibberellins (GAs) are key regulators of plant growth and development.They promote growth by targeting the degradation of DELLA repressorproteins; however, their site of action at the cellular, tissue or organlevels remains unknown. To map the site of GA action in regulating rootgrowth, we expressed gai, a non-degradable, mutant DELLA protein, inselected root tissues. Root growth was retarded specifically when gaiwas expressed in endodermal cells. Our results demonstrate that theendodermis represents the primary GA responsive tissue regulating organgrowth and that endodermal cell expansion is rate-limiting forelongation of other tissues and therefore of the root as a whole.
P45
Nucleosome Positioning In Arabidopsis
S Usher (Rothamsted Research), S Kurup (Rothamsted Research), G Barker (Warwick HRI), G King (Rothamsted Research)
Miss Sarah Usher (sarah.usher@bbsrc.ac.uk)
Epigenetic variation is defined as mitotically and meiotically heritable changes in gene function not associated with changes in DNA sequence. Mechanisms regulating epigenetic variation include changes to chromatin structure. Nucleosomes consist of approximately 147 bp DNA wrapped around a histone octomer, separated by linker DNA and a linker-associated histone. While no consensus DNA sequence exists for nucleosome positioning, sequence preferences influence nucleosome position. Repeating dinucleotide sequences confer curvature and flexibility to a DNA molecule and are often found in nucleosomal DNA sequences. Since genome architecture and sequence composition differs between species, it is likely that nucleosome positioning sequence preferences do also. The aims of this study are to obtain a set of nucleosome positions in Arabidopsis and test the data to determine sequence biases, and to use genome tiling microarrays to investigate global patterns of nucleosome occupancy. The hypotheses being tested are: 1. Arabidopsis has specific biases for nucleosome positioning. 2. Nucleosome positioning in plants differs from other taxa due to differences in genome architecture. Dinucleosome DNA libraries have been constructed and approximately 500 clones sequenced by conventional Sanger methods. Fourier analysis of linker-length distribution indicates that there may be bias of periodicity in Arabidopsis (7-8 bp) which differs from previously established linker lengths in yeast, chicken and human chromatin. Cross-linked mononucleosome DNA fragments have successfully been hybridised to the Affymetrix Arabidopsis GeneChip® 1.0R tiling array, and analyses are currently underway. Ongoing work involves high-throughput sequencing in order to identify translational variation in nucleosome positions within Arabidopsis dinucleosomes.
P46
UVR8 in Arabidopsis regulates UV-B specific leaf photomorphogenesis and other aspects of whole-plant development
J Wargent (Lancaster University), V Gegas (John Innes Centre), G Jenkins (University of Glasgow), J Doonan (John Innes Centre), N Paul (Lancaster University)
Dr Jason Wargent (j.wargent@lancaster.ac.uk)
UV-B radiation is widely held to be a globally significant environmental phenomenon, although responses specific to UV-B wavelengths are still poorly understood, both in terms of initial signalling and effects on morphogenesis. Arabidopsis (Arabidopsis thaliana) UV RESISTANCE LOCUS8 (UVR8) is the only known UV-B specific signalling component and regulates a range of UV-protective gene expression responses. However, there is little knowledge regarding the role of UVR8 in regulating whole-plant responses to UV-B. We have examined the regulatory effects of UVR8 on leaf morphogenesis and other aspects of plant development at a range of UV-B doses expected to occur in natural conditions. We have observed that UVR8 is required for a UV-B stimulated compensatory increase in epidermal cell size, whilst reductions in epidermal cell number in response to UV-B are substantially independent of UVR8, thus demonstrating that UVR8 regulates leaf growth through the control of epidermal cell development. We also report that UVR8 is required for normal progression of endocycle in response to UV-B and has a regulatory role in stomatal differentiation. Our findings show that UVR8 has a more extensive role in UV-B responses than previously recognised and that in addition to regulating UV-protective gene expression responses UVR8 is involved in controlling aspects of leaf morphogenesis. This work extends our understanding of how UV-B adaptation is orchestrated at the whole-plant level and provides an opportunity to develop our understanding of how UV-B may interact with other abiotic and biotic factors at an ecosystem level.
P47
Manganese deficiency alters the patterning and development of roothairs in Arabidopsis
T Yang (academia sinica), P Perry (Academia Sinica), S Pandian (Academia Sinica), W Schmidt (Academia Sinica)
Dr Thomas Yang (thomasjwyang@gmail.com)
Manganese (Mn) is an essential micronutrient for metabolism. We report here the acclimation to low Mn availability in Arabidopsis seedlings subjected to Mn deficiency. This deficiency alters the arrangement and characteristics of the root epidermal cells. Extra hairs were formed in atrichoblasts, indicating postembryonic cell fate re-programming. When plants were grown under high light intensity in the presence of Mn, root hair elongation was inhibited, whereas Mn-deficient seedlings displayed stimulated root hair development. GeneChip analysis revealed several genes with potential roles in reprogramming of rhizodermic cells. None of the genes function in epidermal cell fate specification was affected, indicating that the normal patterning mechanisms have been bypassed. This assumption is supported by the partial rescue of the hairless cpc mutant by Mn deficiency. Inductively coupled plasma optical emission spectroscopy analysis revealed that Mn deficiency also caused an increase in iron concentration, which was mirrored by a decreased transcript level of the iron transporter IRT1. A number of T-DNA mutants was identified from a forward screening and named manic. They either show an exaggerated, or a lack of response under Mn deficiency, some also showed a differential response to light. Cloning of the flanking sequence of one candidate showed the insert resides upstream of an RNA polymerase transcriptional regulation mediator subunit, resulting in reduced number of hairs under Mn deficiency. In the second mutant, who produced more hairs, the disrupted sequence encodes a putative acyltransferase. A more detailed study of these mutants is underway to elucidate their role.
P48
KISS OF DEATH (KOD) is a novel peptide-encoding-gene in Arabidopsis thaliana which regulates Programmed Cell Death (PCD) of the suspensor during embryogenesis
B Young (Manchester University), R Blanvillain (University of California), P Gallois (University of Manchester)
Mr Bennett Young (b.young@postgrad.manchester.ac.uk)
We have identified a transcript encoding a peptide of 25 amino acids which has the capability of inducing PCD when constitutively over expressed in plants. PCD was also induced in protoplasts treated with the synthetic peptide; a C23S substitution within a putative killer domain abolished this lethality. Homozygous knockout plants for the KOD gene showed a delay in the induction of PCD in suspensor cells when compared to wildtype plants. PCD inhibitors are being used in cell death assays to shed light on the mode-of-action of KOD. This approach has suggested that KOD is upstream of caspase-like activity. KOD appears to be a plant specific regulator of PCD.
P49
PDK1 localisation and partners in Arabidopsis thaliana
C Zalejski (School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, Surrey TW20 0EX, United Kingdom), P BINAROVA (Institut of Microbiology AVCR, Videnska 1083, 14200 Prague 4, Czech Republic), A JONES (The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom), L BOGRE (School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, Surrey TW20 0EX, United Kingdom)
Dr Christine Zalejski (christine.zalejski@rhul.ac.uk)
Plants are constantly being attacked by pathogens which can cause significant crop losses. Thus understanding signalling mechanisms used to elicit counteracting responses to biotic stresses is a major goal for plant breeding and fundamental plant science. Plants basal defence response intiated by pathogens using elicitors pathways in Arabidopsis is one aspect of our research. Recently, the phospholipid second messenger: phosphatidic acid (PA) has been implicated in elicitor signalling. PA, specifically produced by phospholipases D (PLD), activates phosphoinositide-dependent protein kinase-1 (PDK1). PDK1 physically interacts with the downstream protein kinase target: OXI1 which is activated by PA via PDK1 and by elicitors. OXI1 is required for activation of MAP-kinases, such MPK6. MPK6, localised to cortical microtubules, is activated by PA and xylanase only in presence of both PDK1 and OXI1. To elaborate further this signalling, we focus our research on the localisation of PDK1 and its regulation by PA. We fused, via Gateway® technology, RFP marker in N-terminal of PDK1 and also PH domain of PDK1 under control of their own promoter. Some preliminary data on Arabidopsis transformed cells showed that PDK1 and MAPK6 are both localised on cortical microtubules. Constructs will be used to transform Arabidopsis suspension cells and plants which possess GFP-tubulin marker and the regulation of PDK1 localisation will be analysed by treating with elicitors and PA. In parallel, we started to extract microtubules of Arabidopsis suspension cells with or no elicitors treatment in order to identify by mass spectrometry new phosphorylated partners of PDK1 signalling pathway.