RESEARCH PROGRAMME
| Carbohydrate-mediated Growth Regulation Work in my group aims to understand how plant growth and development is integrated with the supply of carbohydrate from photosynthesis. All organisms have intricate mechanisms that regulate growth in response to available nutrients that are relatively well characterised. However in plants only a few examples are known of regulatory and signalling systems that regulate cellular responses to carbohydrates. We aim to define new regulatory pathways linking the perception and transduction of metabolic signals to growth and developmental responses using genetic screens in the model plant Arabidopsis. |
Background All organisms need a supply of nutrients to support growth and development. Sugars such as glucose are universal nutrients as they provide carbon skeletons for energy supply, storage and the synthesis of most metabolites. The central importance of sugar supply to cells is reflected in the complex sensing and signalling systems that have evolved to optimise the appropriate supply of sugars for growth and development. In humans and other animals the insulin signalling system integrates sugar supply with use for energy production or storage. In plants sugars are produced in mature photosynthetic tissues and transported to other regions of the plant for use in long-term storage or for growth. Physiological and biochemical evidence suggests the distribution and allocation of resources is carefully controlled, but so far the cellular mechanisms are relatively poorly understood. A thorough understanding of these mechanisms may create new opportunities to design breeding and engineering strategies to enhance the growth and yield potential of crops. |
Strategy We have conducted genetic screens in Arabidopsis to identify mutants affected in glucose- mediated gene expression (Rook et al 2001, Baier et al 2004). These screens were based on fusions of the sugar- responsive ApL3 promoter to reporter genes. Transgenic reporter lines were mutagenised with EMS and screened for reduced expression of the ApL3 transgene suign survival on R7402 proherbicide, and for enhanced expression of a LUC reporter. These screens yielded several impaired sugar induction (isi) mutants and high sugar response (hsr) mutants. In the past 3 years we have used map- based cloning methods to identify mutant genes and characterise their functions. Associated with this work we have performed a detailed analysis of Affymetrix array data to define sugar responsive regulatory elements and some of the transcriptional mechanisms governing glucose- and ABA-mediated gene expression. We developed a promoter classification method, with Gavin Cawley at the Dept of Computing Science at the University of East Anglia, using a Relevance Vector Machine (RVM). |
Results ISI genes. So far we have cloned and characterised 4 ISI genes, and are completing the mapping of isi6 which has a strong phenotype similar to isi1. ISI3 and ISI4 are allelic to the ABA signalling gene ABI4 and ABA biosynthetic genes respectively, and their isolation in our screen provides further evidence that ABA signalling is involved in sugar- responsive gene expression. Mutations in the ISI1 and ISI2 genes lead to reduced ApL3 expression and a variety of whole plant phenotypes that define novel sugar specific responses that are independent of ABA regulation. ISI1 encodes a novel protein with no known homology to other proteins. It is expressed in the vascular system during the developmental transition from sink to source leaf. It functions in the phloem as shown by complementation of phenotypes by an AtSUC2:ISI1 transgene. Phenotypes associated with isi1 alleles include reduced starch and anthocyanin levels, increased chlorophyll, increased sucrose levels and reduced growth and seed set. Our current hypothesis proposes that ISI1 functions as a regulator of carbohydrate perception. In this model loss of ISI1 function leads to the perception that sugar levels are low, leading to reduced growth and carbohydrate storage, and elevated chlorophyll levels. Currently we are testing this model and establishing the cellular processes in which ISI1 functions. Phenotypes associated with mutations in the ISI2 gene include reduced starch and anthocyanin levels. ISI2:GUS fusions are expressed primarily in the vasculature and developing seed during endosperm deposition. Starch levels are reduced in seeds, and seeds also exhibit reduced dormancy. Reduced dormancy may be due to seed coat defects caused by limited transient endosperm starch that is used for seed coat development. ISI2 encodes a protein with a predicted extracellular targeting signal domain and a series of repeated domains that have been implicated in protein-protein interactions. Currently we are determining misexpression phenotypes of ISI2, defining its subcellular location using GFP fusions, and conducting yeast-2-hybrid screens to see what types of proteins bind to the proteins repeats. HSR genes. We have identified 4 HSR genes that all exhibit enhanced expression of an ApL3:LUC transgene in response to low levels of sugars. These mutants also exhibit, to varying extents, enhanced growth in response to low levels of sugars and increased expression of a variety of sugar regulated genes. HSR2 encodes a cytochrome P450 involved in suberin synthesis. hsr2 mutants exhibits a variety of ABA hypersensitive phenotypes such as enhanced water loss (due to a defective cuticle) and altered stomatal closure that may contribute to elevated ApL3 expression. Currently a possible role for P450 function in sugar-specific responses is being pursued. HSR3 and HSR4 encode subunits of the ARP2/3 complex. This functionally conserved complex mediates the formation of characteristic branched actin filaments. Mutations in subunits of the complex cause characteristic trichome and cell expansion defects that are thought to be due to altered cell polarity. The actin cytoskeleton supports and regulates many cellular processes by directing the cellular locations and interactions of proteins through their interaction with actin filaments. Currently we are exploring various scenarios where altered actin polymerisation may contribute to hypersensitive sugar responses. We have recently shown that mutations in the AtNAP and AtPIR genes, which form part of a putative complex that regulates ARP2/3 activity, also exhibit sugar hypersensitive growth. Once the cellular mechanisms regulated by ARP2/3 that contribute to sugar responses have been identified a signal transduction pathway leading from external cues to ARP2/3 can then be constructed. HSR5 encodes an F box protein. We have shown that HSR5 interacts with ASK proteins to form an SCF complex that may target proteins for ubiquitination and proteolysis. The single amino acid change in HSR5 causes a relatively dramatic sugar hypersensitive response, larger seeds, reduced growth and increased expression of sugar regulated genes. Currently we are characterising different alleles of HSR5 and a closely related gene called HSR5-like obtained from TILLING lines, determining phenotypes and genetic interactions with other genes implicated in ubiquitination, and conducting screens for potential target proteins. Regulatory Networks The RVM identified discriminatory features in the promoter sequences of glucose- and ABA-regulated genes (defined by Affymetrix data analysis) that correctly classified the expression patterns of over 70% of the regulated genes according to known and novel promoter sequences. Many of these sequences had known regulatory functions in sugar- and ABA- mediated gene expression and a model of glucose and ABA transcriptional regulatory networks was established. One new promoter motif identified as the strongest classifier of glucose- upregulated gene expression was shown to confer glucose- responsive gene expression. The combination of functional analysis of microarray data and promoter classification establishes a framework for defining models of transcriptional regulatory networks and for understanding the mechanisms governing glucose- and ABA- mediated gene expression. |
References Baier M., Hemmann G., Holman R., Corke F., Card R., Smith C., Rook F., Bevan M. W. (2004) Characterization of mutants in Arabidopsis showing increased sugar-specific gene expression, growth, and developmental responses. Plant Physiology 134, 81-91. Li Y., Sorefan K., Hemmann G., Bevan M. W. (2004) Arabidopsis NAP and PIR regulate Actin-based cell morphogenesis and multiple developmental processes. Plant Physiology 136, 3616-3627. Rook F., Bevan M. W. (2003) Genetic approaches to understanding sugar-response pathways. Journal of Experimental Botany 54, 495-501. Rook F., Corke F., Card R., Munz G., Smith C., Bevan M. W. (2001) Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signalling. Plant Journal 26, 421-433. Li, Y., et al Genome Research (submitted) Saadi,K., Lee, K.K., Cawley, G., and Bevan M. (2005) Predicting sugar regulation in Arabidopsis thaliana using kernel learning methods. International Joint Conference on Neural Networks (IJCNN). |
| Related Grants Related Grants BBSRC Grant 208/P19441 until 01.05.06 CHARACTERISATION OF THE ISI1 GENE, A PHLOEM-LOCALISED REGULATOR OF SUGAR RESPONSES IN ARABIDOPSIS. The ISI1 gene was identified in a screen for reduced gene expression in response to sucrose. The expression pattern of ISI1, the phenotypes caused by over-expression of ISI1 in phloem companion cells of source leaves, and biochemical analysis of isi1 lines, all suggest ISI1 is a phloem- localised regulator of sugar responses. The objective of this work is to define the function of the ISI1 gene using physiological, biochemical and genetic strategies. We will also define other components of the isi1signalling pathway in a genetic screen. This work will lead to better understanding of sugar-mediated signalling, phloem function and resource allocation in plants. BBSRC Grant 208/EGM16126 until 01.03.06 COMPUTATIONAL APPROACHES TO IDENTIFYING GENE REGULATROY NETWORKS IN ARABIDOPSIS Genes encode regulatory information that specifies the time and place of gene expression, and coordinated gene expression resulting from this information is a critical mechanism regulating all aspects of development and environmental responses. This proposal aims to establish and validate a bioinformatics toolkit to interpret the information content of Arabidopsis promoters and initiate the systematic description of gene regulatory networks in Arabidopsis. BBSRC Grant BB/C515620/1 starts 01/07/05 Defining the function and regulation of the SCFHSR5 complex involved in sugar mediated growth control in Arabidopsis We propose to define the function of a novel F box protein in sugar- mediated growth responses in Arabidopsis. This protein, encoded by the High Sugar Response 5 (HSR5) gene, was identified in a screen for Arabidopsis mutants exhibiting elevated expression of sugar- regulated genes and hypersensitive growth responses. We have established that HSR5 interacts with ASK proteins, which are subunits of the SCF complex that targets proteins for ubiquitination and proteasome- mediated degradation. We will establish if the activity of this SCFHSR5 complex is regulated by glucose and two other proteins previously implicated in sugar responses. We will also identify and test potential target proteins in order to establish the cellular processes that may be affected by the SCFHSR5 complex. These analyses will define an important sugar-controlled regulatory network and may also reveal, through a wider definition of the specificity and regulation of proteolysis, a regulatory network involving light, hormones and sugars that integrates growth and development. |