Project Leader
Lawes Trust Studentship: Gibberellin biosynthesis and action in Arabidopsis roots
The aim of this project is to examine how gibberellin (GA) plant hormones control plant growth and development, including root elongation. Although Gas are essential for root growth there is little information on the mechanisms involved or in which cells Gas are synthesised or act. This project will address these issues by investigating GA-mediated root elongation in the model plant, Arabidopsis. Two complementary strategies will be used to determine the cellular sites of GA biosynthesis and action in the Arabidopsis root:
1. The expression of genes encoding GA-inactivating enzymes to specific cell types within the root will be targetted. Inhibition of growth by expressing enzymes that are specific for active Gas or their biosynthetic precursors in specific cells will indicate sites of action/biosynthesis or biosynthesis, respectively. Sites of GA action will be further investigated by restoring root growth to a mutant lacking GA receptors by cell-specific expression of a GA receptor gene.
2. Sites of expression of endogenous GA-biosynthetic and receptor genes will be determined using reporter lines, a combination of laser capture microdissection with quantitative real-time PCR and protein localisation in root tissues using specific antibodies.
Understanding how DELLA-mediated transcription controls GA-responsive growth and development in Arabidopsis
In recent years there have been major advances in our understanding of how the plant hormone, gibberellin (GA) is perceived and this signal transduced, leading to changes in growth and development. The DELLA proteins (DELLAs) are central to this signalling cascade, and act as repressors of GA-responsive growth. GA signalling relieves the repression exerted through DELLAs by targeting their degradation through an ubiquitin-proteasome mediated process, which is well understood. In contrast, our understanding of the role of DELLAs in repressing GA-responsive growth is very limited. Although it is thought that DELLAs act as transcriptional regulators, their mode of action is not clear and no target genes have been identified. The work outlined in this proposal is aimed at improving our understanding of the role of DELLAs and their downstream target genes in regulating GA-responsive growth in Arabidopsis. Our proposed strategy involves using a combination of microarray and chromatin immunoprecipitation experiments to identify early GA-responsive genes that are the primary targets of the DELLAs. We will focus on specific GA-responsive tissues, rather than the whole plant. To uncover the role of these DELLA target genes in regulating GA-responsive growth, we will use a reverse genetics-based strategy. Based on the probable absence of a canonical DNA-binding domain in the DELLAs, it is likely that they regulate the expression of GA-responsive genes through their interaction with other transcription factors. To identify these components we will perform yeast two-hybrid screens using a DELLA bait construct. Putative DELLA interactors will be confirmed by testing the interactions in vivo. Furthermore, their roles in GA signalling will be assessed by analysing the phenotype of knock-out mutants.
Member
BBSRC Quota Studentship: The role of Gibberellin signalling in reproductive development
Objectives:
1. To conduct a functional analysis of the different GA20ox and GID1 paralogues in reproductive development.
We have preliminary evidence that AtGA20ox4 and AtGA20ox5 are expressed at very low levels in reproductive tissues and we will therefore focus on AtGA20ox1, -2 and -3. We have shown that expression of AtGA20ox3 is strongly up-regulated in the ga20ox1/ga20ox2 double mutant due to feedback regulation and believe this gene enables sufficient GA synthesis for partial floral development in the mutant. This will be tested by producing a triple ga20ox knock-out, as well as the remaining two double knock-outs. Crossing the AtGA20ox3 line containing a point mutation that produces a truncated protein with the ga20ox1/ga20ox2 double mutant will provide the three remaining ga20ox mutants, which will be characterised for defects in reproductive development. If necessary, ga20ox4 and ga20ox5 mutants will also be selected and crossed with the triple mutant. The mutants will be analysed for changes in bioactive GAs, precursors and inactivation products using GC-MS. Their phenotypes, particularly in relation to stamen and pollen development, will be correlated with gene expression profiles, by determining transcript abundance and localisation using a combination of qRT-PCR, and analysis of promoter: GUS reporter lines, which will be prepared.
2. To identify the source of GAs for floral development and the sites of GA action in floral tissues.
We will use tissue-specific promoters to drive expression of GA2ox genes encoding enzymes that inactivate C20-GA precursors (to determine sites of synthesis) or active C19-GAs (to target sites of synthesis and action). In addition, sites of GA action will be specifically targeted by tissue-specific expression of a dominant negative form of Gai that blocks GA signal transduction and by rescue of the gid1 mutants by tissue-specific expression of a functional GID1 gene.
Enhancing wheat field performance and response to abiotic stress with novel growth-regulatory allelles
The "Green Revolution" dwarfing (Rht) alleles that increase wheat yields under high input conditions are orthologues of the Arabidopsis GAI gene and encode mutant DELLA proteins. DELLAs are repressors of plant growth that are degraded in the presence of gibberellin (GA) whereas the gai/Rht mutants are insensitive to GA. Most UK wheat varieties carry the semi-dwarfing Rht2 (Rht-D1b) allele but variation in height between genotypes suggests that other loci play a role in determining stature. We aim to identify these loci through co-localisation of quantitative stature traits identified in UK wheat germplasm with genes in the GA-DELLA pathway. Based on functional analyses in vitro and performance in the field alleles will be identified for use in wheat breeding.
Aims:
1. Identify genetic loci responsible for variation in wheat stem height in elite UK cultivars through co-localisation of genes encoding components of GA biosynthesis and signalling with height QTLs. Identify allelic sequence variation in GA-DELLA genes within this material. Develop intragenic, allele-specific markers for these loci and validate by crossing into elite germplasm.
2. Assess the utility of known alternative dwarfing alleles by introgression into modern, high-yielding backgrounds.
2. Assess the performance and stress tolerance of the introgressed alleles under controlled environment and field conditions for a range of characters including germination rate, seedling vigour, plant stature, straw strength, tillering, grain number and individual grain weights.
Regulation of gibberellin biosynthesis
The gibberellins (GAs) are a class of polycyclic diterpenoids that control a wide range of developmental processes throughout the life cycle of plants. These processes include seed germination, petiole and stem elongation, flower development and seed growth. They also mediate responses to environmental signals such as day-length and light quality. The aim of this project is to understand the control of GA levels in model and crop species by endogenous and environmental effectors. This is being pursued using a variety of analytical techniques including promoter-reporter gene fusions, transcript analysis, immunodetection and GA analysis. In addition, the production of transgenic plants with modified expression of GA-biosynthetic and GA-catabolic genes allows the identification of regulatory steps in GA metabolism and the production of crop plants with modified GA levels for agronomic benefit.
Includes studentships.
The role of gibberellin inactivation in the control of plant development
The aim of the work is to establish the role of GA 2-oxidases, enzymes involved in the inactivation of GAs, in the regulation of plant development. We will focus on Arabidopsis stem elongation and flowering in short days as models of GA action. A key objective is to test the hypothesis that GA inactivation is not only important in GA homeostasis but is also essential in maintaining tissue-specific action of GAs. There will be a high degree of interaction with existing externally-funded projects on GA 20-oxidases and GA 3-oxidases, and with work in other laboratories, that will provide information on the co-ordination of GA biosynthesis and turnover in the control of developmental processes. We will identify of the patterns of expression of the five GA2ox genes using existing promoter: GUS fusions and RT-PCR. We will also study the fine detail of expression of the genes in the leaves, stem and apex during GA-regulated stem elongation and flowering, using in situ hybridization and immunolocalization, and propose roles for GA 2-oxidases in these processes. This will be correlated with analysis of GA levels in these tissues and with expression of key GA-regulated genes such as LEAFY (in collaboration with SLU Umeå, Sweden). We will test hypotheses about the roles of GA2ox genes in the regulation of flowering and stem elongation through the analysis of knockout mutants and the generation of RNAi lines. The GA status of apical and surrounding tissues will also be manipulated by expression of the GA desaturase gene from Gibberella fujikuroi that will produce GAs resistant to inactivation and thereby test the importance of GA turnover in these processes and in GA homeostasis.
Understanding the signalling pathways that impact on plant architecture
Plant architecture, including stem height and girth, numbers and size of branches, leaf size and shape, root length and numbers of secondary roots, has a major influence on crop yields and is an important factor in cultivation practices. Reduced stem growth, allowing, for example, improved stem stability, high planting densities, or a more effective canopy structure for optimal light energy capture, is a major target in breeding programmes in many crop species and is commonly achieved through application of growth retardants. Among the plant hormones that regulate the growth of organs such as stems, leaves and roots the gibberellins (GAs) are of particular relevance as the target for most growth retardants. Furthermore, many important semi-dwarf varieties have lesions in the GA biosynthesis or signal transduction pathway. The project aims to elucidate and manipulate signalling pathways involved in the developmental and environmental control of plant architecture, with particular emphasis on the GA signalling pathway, including biosynthesis, perception and signal transduction. Crop and model species will be utilised to identify components of these pathways, using reverse genetics and ectopic expression to study their physiological function and determine their contribution to relevant physiological processes. Their sites of expression will be determined using reporter genes and laser capture microdissection. Sites of GA biosynthesis and action relevant to the physiological processes underlying architecture will be investigated by tissue-specific manipulation of the signalling pathways. Interactions between GA, environmental and other hormonal signalling pathways will be investigated. Results obtained with model species will inform experiments with crop species, principally, but not exclusively wheat. The project will also examine GA signalling in relation to reproductive development in so far as this impacts on plant architecture, as in, for example, sugar beet.
1. Hedden P and Thomas SG. Gibberellin biosynthesis and its regulation (2012) Biochem. J. 444, 11-25
2. Plackett AR, Powers SJ, Fernandez-Garcia N, Urbanova T, Takebayashi Y, Seo M, Jikumaru Y, Benlloch R, Nilsson O, Ruiz-Rivero O, Phillips AL, Wilson ZA, Thomas SG, Hedden P. Analysis of the Developmental Roles of the Arabidopsis Gibberellin 20-Oxidases Demonstrates That GA20ox1, -2, and -3 Are the Dominant Paralogs. Plant Cell. 2012 Mar 16.
3. Pearce S, Saville R, Vaughan SP, Chandler PM, Wilhelm EP, Sparks CA, Al-Kaff N, Korolev A, Boulton MI, Phillips AL, Hedden P, Nicholson P, Thomas SG. Molecular characterization of Rht-1 dwarfing genes in hexaploid wheat. Plant Physiol. 2011 Dec;157(4): 1820-31.
4. Plackett AR, Thomas SG, Wilson ZA, Hedden P. Gibberellin control of stamen development: a fertile field. Trends Plant Sci. 2011 Oct;16(10): 568-78.
5. Mutasa-Gottgens, E, Qi, AM, Mathews, A, Thomas, S, Phillips, A, Hedden, P (2009) Modification of gibberellin signalling (metabolism & signal transduction) in sugar beet: analysis of potential targets for crop improvement. TRANSGENIC RESEARCH 18, 301-308
6. Rieu I, Eriksson S, Powers SJ, Gong F, Griffiths J, Woolley L, Benlloch R, Nilsson O, Thomas SG, Hedden P, Phillips AL. “Genetic analysis reveals that C19-GA 2-oxidation is a major gibberellin inactivation pathway in Arabidopsis.” Plant Cell. 2008 Sep;20(9): 2420-36.
7. Ivo Rieu, Omar Ruiz-Rivero, Nieves Fernandez-Garcia, Jayne Griffiths, Stephen J. Powers, Fan Gong, Terezie Linhartova, Sven Eriksson, Ove Nilsson, Stephen G. Thomas, Andrew L. Phillips and Peter Hedden.”The gibberellin biosynthetic genes AtGA20ox1 and AtGA20ox2 act, partially redundantly, to promote growth and development throughout the Arabidopsis life cycle.” Plant J. 2008 Feb;53(3): 488-504. Epub 2007 Dec 6.
8. Dijkstra C, Adams E, Bhattacharya A, Page AF, Anthony P, Kourmpetli S, Power JB, Lowe KC, Thomas SG, Hedden P, Phillips AL, Davey MR.”Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species.” Plant Cell Rep. 2008 Mar;27(3): 463-70. Epub 2007 Nov 13.
9. Zentella R, Zhang ZL, Park M, Thomas SG, Endo A, Murase K, Fleet CM, Jikumaru Y, Nambara E, Kamiya Y, Sun TP. “Global analysis of DELLA direct targets in early gibberellin signaling in Arabidopsis.” Plant Cell. 2007 Oct;19(10): 3037-57.
10. Appleford NE, Wilkinson MD, Ma Q, Evans DJ, Stone MC, Pearce SP, Powers SJ, Thomas SG, Jones HD, Phillips AL, Hedden P, Lenton JR. “Decreased shoot stature and grain alpha-amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat.” J Exp Bot. 2007;58(12): 3213-26.
11. Ivo Rieu, Omar Ruiz-Rivero, Nieves Fernandez-Garcia, Jayne Griffiths, Stephen J. Powers, Fan Gong, Terezie Linhartova, Sven Eriksson, Ove Nilsson, Stephen G. Thomas, Andrew L. Phillips and Peter Hedden.”The gibberellin biosynthetic genes AtGA20ox1 and AtGA20ox2 act, partially redundantly, to promote growth and development throughout the Arabidopsis life cycle.” Plant J. 2008 Feb;53(3): 488-504. Epub 2007 Dec 6.
12. Jayne Griffiths, Kohji Murase, Ivo Rieu, Rodolfo Zentella, Zhong-Lin Zhang, Stephen Powers, Fan Gong, Andrew L Phillips, Peter Hedden, Tai-ping Sun and Stephen G Thomas. “Genetic Characterization and Functional Analysis of Gibberellin Receptors in Arabidopsis.” Plant Cell (2006) 18: 3399-3414
13. Thomas, S.G. and Hedden, P. “Gibberellin metabolism and signal transduction.” Annual Plant Reviews-Plant Hormone Signaling. 2006. 24: 147-184.
14.Thomas, S.G., Rieu, I. and Steber, C.M. “Gibberellin metabolism and signalling.” Vitamins and Hormones-Advances in Research and Applications. 2005. 72: 289-338
15.Muangprom, A., Thomas, S.G., Sun, T.-p. and Osborn, T.C. “A novel dwarfing mutation in a ‘Green Revolution’ gene from Brassica rapa.” Plant Physiol. 2005 137(3): 931-938.
16.Thomas, S.G. and Sun, T.-p. “Update on gibberellin signaling. A tale of the tall and the short.” Plant Physiol. (2004) 135: 668-676.
17.Tyler, L., Thomas, S.G., Hu, J., Dill, A., Alonso, J.M., Ecker, J.R. and Sun, T.-p. “DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis.” Plant Physiol. (2004) 135: 1008-1019.
18.Dill, A., Thomas, S.G., Hu, J., Steber, C.M. and Sun, T.-p. “The Arabidopsis F-Box Protein SLEEPY1 Targets Gibberellin Signaling Repressors for Gibberellin-Induced Degradation.” Plant Cell (2004) 16: 1392-1405.
19.McGinnis, K.M., Thomas, S.G., Soule, J.D., Strader, L.C., Zale, J.M., Sun, T.-p. and Steber C.M. "The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase." Plant Cell (2003) 15: 1120-1130
20.Curtis, I.S., Ward, D.A., Thomas, S.G., Phillips, A.L., Davey, M.R., Power, J.B., Lowe, K.C., Croker, S.J., Lewis, M.J., Magness, S.L. and Hedden, P. "Induction of dwarfism in transgenic Solanum dulcamara by over- expression of a gibberellin 20-oxidase cDNA from pumpkin." Plant J. (2000) 23: 329-338
21.Thomas, S.G., Phillips, A.L. and Hedden, P. "Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation." Proc. Natl. Acad. Sci. USA (1999) 96: 4698-4703
22.Hedden, P., Phillips, A.L., Coles, J., Thomas, S., Appleford, N., Ward, D., Beale, M. and Lenton, J. "Gibberellin biosynthesis: Genes, regulation and genetic manipulation." RIKEN Review (1999) 21: 29-30
23.Hedden, P., Coles, J.P., Phillips, A.L., Thomas, S.G., Ward, D.A., Curtis, I.S., Power, J.B., Lowe, K.C., Davey, M.R., Gray, D., Seymour, G.B. and Thomas, B. "Modification of plant morphology by genetic manipulation of gibberellin biosynthesis." In: Genetic and Environmental Manipulation of Horticultural Crops, K. E. Cockshull (Ed.) (1998) 205-217
24.Henderson, L.M., Thomas, S., Banting, G. and Chappell, J.B. (1997). The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox. Biochemical Journal 325, 701-705.
25.Soriano, S., Thomas, S., High, S., Griffiths, G., Dsantos, C., Cullen, P. and Banting, G. "Membrane association, localization and topology of rat inositol 1,4,5-trisphosphate 3-kinase B: Implications for membrane traffic and Ca2+ homoeostasis." Biochemical Journal (1997) 324: 579-589
26.Thomas, S., Soriano, S., dSantos, C. and Banting, G. "Expression of recombinant rat myo-inositol 1,4,5-trisphosphate 3-kinase B suggests a regulatory role for its N-terminus." Biochemical Journal (1996) 319: 713-716
27.Thomas, S., Brake, B., Luzio, J.P., Stanley, K. and Banting, G. (1994). Isolation and sequence of a full-length cDNA encoding a novel rat inositol 1,4,5-trisphosphate 3-kinase. Biochimica et Biophysica Acta 1220, 219-222.
28.Ellis, N.A., Tippett, P., Petty, A., Reid, M., Weller, P.A., Ye, T.Z., German, J., Goodfellow, P.N., Thomas, S. and Banting, G. (1994). PBDX is the XG blood-group gene. Nature Genetics 8, 285-290.
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Rothamsted Insect Survey
