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Dissection and manipulation of signal transduction pathways mediating metabolic regulation of gene expression in plant storage organs

Reference	BBS/E/C/00823297
Principal Investigator / Supervisor	Professor Nigel Halford
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	133,493
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

It is clear that complex signal transduction pathways regulate the pathways of plant metabolism, as they do in yeast and animal systems. The elucidation of these pathways is fundamental to gaining an understanding of how nutrient availaility & metabolic status affects plant growth & development and, in particular, storage compound accumulation in seeds, tubers and other storage organs. Clearly, the regulation of carbon and nitrogen metabolism is likely to play a major role in controlling the rates of synthesis of starch and storage proteins and we are investigating signalling pathways which are likely to be involved, focusing on a pathway which is analogous to the SNF1 (sucrose non- fermenting 1) system in yeast, which has been described as a global regulator of carbon metabolism. SNF1 is a protein kinase required for the depression of a number of glucose- repressible genes in response to glucose deprivation. It also regulates glycogen synthesis and plays a part in the regulation of fatty acid metabolism, protein synthesis and the cell cycle. We are identifying and cloning components of this pathway from plants using hybridisation and interaction cloning, and using molecular, biochemical and transgenic techniques to determine their function in cereal seeds, potato tubers and sugar beet storage roots. Objectives 1996 Complete molecular analyses of the potato SNF1-related gene family. Continue biochemical analyses of transgenic potato and sugar beet expressing antisense SNF1-related sequences. Confirm role of SNF1-related protein kinase in regulation of sucrose synthase activity in potato tubers; determine point of action (trascription, translation, post-translation). Perform field trials of transgenic potatoes to confirm and measure effects of antisense SNF1-related gene expression on sprouting time (in collaboration with Advanced Technologies, Cambridge). Continue investigation into the role of SNF1-related protein kinase in the regulation of HMG-CoA reductase (HMGR) activity & the potential of manipulating HMGR activity to increase flux into the isoprenoid biosynthesis pathway. 2 Purify SNF1- related PK from spinach; clone spinach SNF1-related cDNA; measure activity in response to various stresses; purify PK which phosphorylates the SNF1-related PK. Analyse cDNAs isolated by interaction screening of barley endosperm cDNAs with barley SNF1-related protein (BKIN12); determine exact nature of their interaction with BKIN12; explore link with environmental stress (one of the proteins is a small heat- shock protein, another shows some similarity to a cadmium- inducible protein). Use interaction and hybridisation cloning to isolate components of the GCN-related signal transduction pathway; specifically the GCN2-related protein kinase and the GCN4- related transcription factor. 1997 Wheat SnRK1 sequences will be isolated and transformation of wheat (in collaboration with Nickersons Biocem) and barley (in collaboration with Dr. Paul lazzeri, RES) with antisense SnRK1 sequences will begin. Analysis of transgenic potato plants expressing an antisense SnRK1 sequence will continue, focusing on effects on sucrose synthase expression and activity, sink strength, starch accumulation, and expression of other enzymes of carbohydrate metabolism. Attempts will be undertaken to show that SnRK1s regulate HMG-CoA reductase, sucrose phosphate synthase and nitrate reductase (all identified as downstream targets in vitro) in vivo. Field trials of the transgenic potato plants will be repeated (in collaboration with Advanced Technologies, Cambridge). Potato & wheat cDNAs encoding homologues of barley proteins which have been cloned using the two-hybrid system with a SnRK1 as bait will be cloned. The proteins will be tested as substrates for SnRK1s and the transcription factors will be tested for binding to SuSy gene promoters. Specific peptide antibodies will be raised to distinguish between different SnRK1 isoforms in spinach leaves.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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