Plant Metabolism.

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Louise Weston

Plant Metabolism

To what extent has genetic manipulation of the Calvin cycle forced the reappraisal of our understanding of the control of metabolic pathways in plants? What do studies of transgenic plants reveal about the integration of metabolism?

The Calvin cycle or the reductive pentose phosphate pathway is quantitatively the most important metabolic pathway in biology, which involves 13 reactions catalysed by 11 enzymes in the stroma of the chloroplast. All photosynthetic eukaryotes reduce CO2 to carbohydrate by this same mechanism. The cycle involves CO2 and water from the environment being enzymatically combined with a five carbon accepter molecule; ribulose-1,5-bisphospahte where a total of 3 molecules of ATP and 2 molecules of NADPH produced from the light reactions are consumed for each CO2 molecule converted into carbohydrate. The Calvin cycle proceeds in three stages;

  • Carboxylation of the CO2 acceptor, forming two molecules of 3-phosphoglycerate, the first stable intermediate of the Calvin cycle.
  • Reduction of 3-phosphoglycerate, forming glyceraldehydes-3-phosphate.
  • Regeneration of the CO2 acceptor ribulose-1,5-bisphosphate from glyceraldehydes-3-phosphate.

From the fixation of three molecules of CO2, six molecules of phosphoglycerate are formed and are converted to six molecules of glyceraldehydes-3-phosphate (triose phosphate.) Of these only one molecule of triose phosphate is gain, which is provided to the cell for various biosynthetic processes; it is a central intermediate in glycolysis, much of the triose phosphate produced is exported to the cytosol where is can be converted to fructose 6-phosphate and glucose 1-phosphate by the reversal of glycolytic reactions. These are precursors for sucrose synthesis. Most of the triose phosphate that remains in the chloroplast is converted into starch in the stroma.

The diagram below summarises the various reactions of the Calvin cycle;

As shown there are four irreversible steps in the cycle; carboxylation, hydrolysis of fructose, bisphosphate and sedoheptulose bisphosphate, and phosphorylation of ribulose 5-phosphate.  Traditionally the enzymes that catalyse irreversible reactions were thought to be the points of control in a pathway. These are generally the enzymes under ‘fine’ regulation. That is regulation that involves perturbation of the activity of the pre-existing protein activity by changes in the levels of substrates, inhibitors or activators and by post-translational modification. It is the traditional view that control of pathways must reside in a relatively few such enzymes whose in vitro properties suggest that they could be controlling flux in vivo e.g displacement of reaction from equilibrium, irreversibility, response to effectors, cooperative kinetics etc. They are often referred to as ‘rate limiting steps.’ These are qualitative ideas that have been hard to test experimentally. This essay will examine how transgenic plants have since been used to examine various enzymes within the above cycle and to predict the extent of their control. This has resulted in a reappraisal of these traditional ideas of the control of metabolic pathways in plants.

Many molecular tools have been used to manipulate the metabolic processes of plants. Directed changes require a transformation system, a suitable gene, suitable promoter sequences for cell- and tissue-specific expression, and effective targeting signals to direct protein to its final destinations within the cell. Metabolic design can involve antisense inhibition or heterologous overexpression of plant-related or foreign genes. There are various plant transformation systems, for example transfer without a vector e.g direct uptake into protoplasts (PEG/electroporation), liposome mediated fusion into protoplasts, microinjection into cells/inflorescence, and use of a biolistic gun. Additionally there is transfer with a vector using RNA viruses and DNA viruses and there is agrobacterium-mediated transfer. Agrobacterium tumerfaciens is the causative agent of crown gall disease. Phenolic substance released from a wounded area in a plant signal virulence genes in the tumor inducing plasmid of the agrobacterium, these genes encode virulence proteins which enable the transfer of tumor-inducing genes contained in T DNA into the plant genome. This system has been manipulated by modifying Ti plasmids and introducing binary vectors to integrate foreign genes in their functional state into the plant genome.

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This is the normal biological system of DNA transfer and has recently been exploited in antisense inhibition (and sense suppression) of endogenous plant genes. The discovery of these kind of transformations and the development of metabolic control analysis in the 1970’s has meant that the alteration in the expression of endogenous plant genes allows investigations of their contribution to plant growth and thus a much better understanding of the control of metabolic processes. The approach involves the production of a set of plants in which the expression of one enzyme is progressively decreased, in as specific a manner as possible. ...

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