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The role of branched chain sugars in the regulation of photosynthesis

Reference	BBS/E/C/00011492
Principal Investigator / Supervisor	Professor Martin Parry
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	238,713
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

Much of the control of photosynthetic rate, under most conditions, lies in Rubisco. Control of Rubisco activity is complex. Rubisco activity is regulated by reversible carbamylation which is determined by the concentrations of carbon dioxide and magnesium, the pH, (dependent on energization of the thylakoids) and Rubisco activase which facilitates carbamylation by a mechanism involving hydrolysis of ATP. Catalytic activity of Rubisco is also regulated by the tight binding of naturally occurring inhibitors. 2 Total activity of Rubisco, isolated from leaves, can be enhanced, by up to 80%, if measures are taken to remove inhibitors from the catalytic site (maximal activity). At night Rubisco activity in many species is reduced by the transition state analogue, 2- carboxyarabinitol-1-phosphate (CA1P) which binds to carbamylated enzyme. CA1P released from Rubisco in the light is rendered non-inhibitory by CA1P- phosphatase which removes its phosphate group to produce 2-carboxyarabinitol (CA). Curiously, large quantities of CA are found in some species which contain little CA1P. Pure CA1P-phosphatase has been isolated. The biosynthetic pathway for CA1P is unclear but both radiolabelled CA and the branched chain sugar, 2- hydroxymethyl ribose (hamamelose) fed to Phaseolus vulgaris leaves in the light can be converted specifically to CA1P in the dark. 3 Rubisco activity is also regulated in response to changes in irradiance by another tight-binding inhibitor (daytime inhibitor) which is related to hamamelose-2,5- bisphosphate (HBP). HBP is synthesised from fructose-1,6- bisphosphate. Thus hamamelose is the precursor of metabolites which collectively regulate Rubisco activity. Hamamelose is ubiquitous among plant species and hamamelose phosphates have been detected in plants. The overall importance of tight binding inhibitors in the regulation of Rubisco will be investigated using extraction methods which do, or do not, clear the catalytic site of the enzyme. The daytime inhibitor will be identified; its contribution to regulating photosynthetic rate will be determined in different species in various environments. The potential for utilizing branched chain sugars to control photosynthetic rate and growth can only be determined through understanding the metabolic pathways. 4 Substrates for the pathway (identified and generated in project 011491) will be used to determine the catalytic steps and in the purification of the biosynthetic enzymes. Genetic manipulation of key enzymes (e.g CA1P- phosphatase) in transformed plants will not only enable their contribution to the regulation of Rubisco to be determined, but also demonstrate the potential for chemical or genetic manipulation of agronomic advantage. To identify the gene for CA1P phosphatase from a Phaseolus vulgaris leaf cDNA library. Oligonucleotide primers (based on peptide sequences) will be used for PCR amplification of a specific probe to screen the library. To purify sufficient daytime inhibitor to identify it chemically. The transfer of radiolabel from hamamelose and hamamelose phosphate (identified in project 011491 as precursors of CA1P) to CA1P and to the daytime inhibitor will be investigated. Assays for enzymes involved in the conversion of such precursors to CA1P will be developed. Embark on purification of enzymes involved in CA1P biosynthesis. Examine the physiological relevance of CA1P in Rubisco regulation. Determine further peptide sequences of CA1P phosphatase. To identify and isolate a full length cDNA for CA1P phosphatase from the Phaseolus vulgaris leaf cDNA library. Express the cDNA for P vulgaris Rubisco activase in E. coli. Investigate coordinate roles of Rubisco activase and CA1P phosphatase in the regulation of P. vulgaris Rubisco. Study of the metabolism of CA1P and its precursors in leaves. Purify the enzymes involved in these processes.

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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