Inhibition Of A Novel Gene Involved In Phosphorylating Starch In TransgenicPotato Tubers Leads To The Repression Of Cold Induced Sweetening

May 14, 1998

Scientists at the Max Planck Institute of Molecular Plant Physiology in Golm, Germany, have isolated a novel gene that is responsible for the phosphorylation of starch. The effects which occur if the expression of this gene is inhibited are described in the May issue of Nature Biotechnology.

Starch is composed of amylose and amylopectin, which are slightly or more highly branched glucose polymers. In addition, amylopectin can, depending on the plant organ where it is manufactured, contain different levels of phosphate monoesters. The phosphate content in potato tubers is exceptionally high as compared with other plant storage organs. Starch biosynthesis is accomplished by different forms of starch synthase which polymerize the glucose monomers using ADP-glucose, and isoforms of branching enzyme which introduce the branch points. Other enzymes are needed to determine the final starch structure, e.g. the presence of a debranching enzyme is a prerequisite to synthesize the semi-crystalline starch granules in contrast to non-crystalline glycogen, which accumulates in eukaryotes other than plants, in bacteria, or in mutants of different plant species lacking this enzyme.

Even though many starch biosynthetic enzymes are known, it has never been possible to synthesize semi-crystalline glucans in vitro, nor has it been explained how the phosphate monoesters are incorporated into starch. Furthermore, it is not clear at the moment which enzymes are responsible for the breakdown of starch in vegetative plant organs. In order to address these open questions in starch metabolism, researchers working in Jens Kossmann¹s in the department headed by Lothar Willmitzer at the Max Planck Institute of Molecular Plant Physiology in Golm-Potsdam have isolated proteins bound to potato starch granules, raised antisera to these proteins and used the antisera to screen cDNA libraries for corresponding clones. One of the resulting clones, designated R1, is encoding a 160 kDa protein that is partially localized on starch granules.

Lorberth et al. describe in Nature Biotechnology (Vol.16 (5) - May 98) the effects on starch metabolism exerted if the expression of this gene is inhibited in transgenic potato plants using antisense technology. A major observation made is the reduction of the phosphate monoester content of the starch synthesized in the transgenic lines down to 10% as compared with wild-type plants. This indicates that the R1 protein is responsible for the phosphorylation of starch, which is also supported by the fact that the expression of the protein in Escherichia coli leads to elevated phosphate contents of the synthesized glycogen. The existance of the R1 protein is not confined to the Solanaceous species; corresponding sequences are also found in Arabidopsis and rice, but not in bacteria, mammals and yeasts, indicating that it is a general but unique component of starch biosynthesis.

The biochemical mechanism by which the R1 protein phosphorylates amylopectin remains to be elucidated. It is not known which substrate acts as a phosphoryl donor, nor which type of glucans are phosphate acceptors. Evidence that phosphoenolpyruvate may act as a phosphoryl donor comes from slight sequence homology of the R1 protein to the PEP synthases from different bacteria. The question of a phosphoryl acceptor is far more difficult to address, as the different intermediates between ADP-glucose and amylopectin are plentifold and not easily obtained.

A further phenotype associated with the reduction of phosphate in the starch is linked to its degradability in different organs of the potato. Initially it was observed that the transgenic potatoes were unable to degrade their starch in leaves even after extremely prolonged (7 days) periods of darkness. The same was later measured in cold-stored potato tubers. The phenomenon of starch degradation and concomittant accumulation of sugars in plant organs induced by low temperatures (cold-induced sweetening) was first described by Mueller-Thurgau, a famous German grapevine breeder, in 1888. Many attempts have been undertaken to circumvent this phenomenon in potato tubers; however, most of them failed. The researchers interpret this observation as a side-effect of the altered starch properties on the starch-degradative machinery present in potato plants. It is hypothesized that the altered starch is not degradable by the enzymes present in potato tubers.

Both of the phenomena observed, the influence on starch phosphorylation and degradation, have a significant impact on plant biotechnology. The high phosphate content of potato starch is an attribute which makes potato starch superior to other commercially available starches. If it was possible to transfer this characteristic to graminaceous and other species, it would open a wide range of novel applications of starch as a renewable resource. In addition, cold sweetening is a severe problem in the potato processing industry as a high sugar content leads to the occurence of the Maillard reaction during frying, which is manifested by the undesired dark staining of potato chips and french fries. Since potato tubers are stored in the cold in order to prevent sprouting, the reduction of cold sweetening will improve potato processing.
-end-


Max-Planck-Gesellschaft

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