Nucleic acid molecules from plants encoding enzymes which...

Details Nucleic acid molecules from plants encoding enzymes which... Nucleic acid molecules from plants encoding enzymes which...

2000-08-11

2003-07-08

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters carbohydrate production in the plant

C800S278000, C800S286000, C800S320100, C435S069100, C435S101000, C435S320100, C435S419000, C435S468000, C536S023600

Reexamination Certificate

active

06590141

ABSTRACT:

FIELD OF THE INVENTION
The invention furthermore relates to vectors and to host cells transformed with the described nucleic acid molecules or vectors, in particular plant cells and plants which can be regenerated from these.
There are also described methods for the generation of transgenic plant cells and plants which, owing to the introduction of DNA molecules encoding a starch synthase, synthesize a starch whose properties are altered. The present invention also relates to the starch which can be obtained from the plant cells and plants according to the invention, and to processes for the production of this starch.
Bearing in mind the recently increasing importance of plant constituents as renewable raw materials, it is one of the tasks of biotechnology research to attempt to adapt these plant raw materials to the demands of the processing industry. Thus, to make possible the use of renewable raw materials in as many fields of application as possible, it is necessary to make available a great variety of materials. Not only oils, fats and proteins, but also polysaccharides, constitute important renewable raw materials from plants. A pivotable position in the polysaccharides is taken up not only by cellulose, but also by starch, which is one of the most important storage materials in higher plants. In this context, maize is one of the most interesting plants, since it is the most important crop plant worldwide for starch production.
The polysaccharide starch is a polymer of chemically uniform base units, the glucose molecules. However, it is a highly complex mixture of different forms of molecules which differ with regard to their degree of polymerization and the occurrence of branchings in the glucose chains. Starch is therefore no uniform raw material. In particular, we differentiate between amylose starch, an essentially unbranched polymer of &agr;-1,4-glycosidically linked glucose molecules, and amylopectin starch, which, in turn, constitutes a complex mixture of differently branched glucose chains. Other branchings are generated by the occurrence of additional &agr;-1,6-glycosidic linkages. In typical plants used for starch production such as, for example, maize or potatoes, the starch synthesized consists of approx. 20%-25% of amylose starch and approx. 75%-80% of amylopectin starch.
To allow as broad an application of starch as possible, it appears desirable to provide plants which are capable of synthesizing modified starch which is particularly suitable for various purposes. One possibility of providing such plants is—besides plant-breeding measures—the targeted genetic alteration of the starch metabolism of the starch-producing plants by recombinant methods. However, a prerequisite for this is the identification and characterization of the enzymes which participate in starch synthesis and/or starch modification, and the isolation of the relevant DNA molecules which encode these enzymes.
The biochemical synthetic pathways which lead to the synthesis of starch are essentially known. In plant cells, starch synthesis takes place in the plastids. In photosynthetically active tissues, these are the chloroplasts, in photosynthetically inactive, starch-storing tissues, the amyloplasts.
The most important enzymes which participate in starch synthesis are the starch synthases and the branching enzymes. Amongst the starch synthases, various isoforms have been described, all of which catalyze a polymerization reaction by transferring a glucosyl residue from ADP-glucose to &agr;-1,4-glucans. Branching enzymes catalyze the introduction of &agr;-1,6-branchings into linear &agr;-1,4-glucans. Two classes of starch synthases can be distinguished: the granule-bound starch synthases (GBSS) and the soluble starch synthases (SS). However, this distinction is not clear-cut in each individual case, since some of the starch synthases are present both in granule-bound form and in soluble form (Denyer et al., Plant J. 4 (1993), 191-198; Mu et al., Plant J. 6 (1994), 151-159).
Besides the class of the granule-bound starch synthases, GBSSI, at least three different isoforms have been described in maize plants within the class of the soluble starch synthases, based on cDNA and amino acid sequence comparisons. Isoform I of starch synthase (SSI) includes genes, which, in maize, encode an approx. 76 kDa protein zSSI (Mu et al., Plant J 6, (1994), 151-159) and which have as yet only been described for monocotyledonous plants such as, for example, for rice (Baba et al., Plant Physiol. 103, (1993), 565-573), are expressed mainly in the endosperm. As a rule, these proteins are stimulated by citrate and are independent of so-called primer molecules.
In contrast, isoform II starch synthases (=SSII) are, as a rule, dependent on primer molecules and show the highest sequence homology with the SSII Isoforms—some of which used to be termed GBSSII—from pea (Dry et al., Plant J. 2, (1992), 193-202) and potato (Edwards et al., Plant J 8, (1995), 283-294).
When considering the maize SSII, a distinction must be made between the genes, or cDNAs, which are termed zSSIIa and zSSIIb in the literature (Ham et al., Plant Mol. Biol. 37, (1998), 639-649; Imparl-Radosevich, Arch. Biochem. Biophys. 362, (1999), 131-138), and the so-called SSII protein, an approx. 180 kDa protein (molecular weight determined by means of gel filtration (Mu et al., Plant J. 6, (1994), 151-159)) from maize endosperm, whose name is based on earlier biochemical studies (Boyer and Preiss, Plant Physiol. 67, (1981), 1141-1145; Mu et al., Plant J. 6, (1994), 151-159). The question of which gene actually corresponds to these 180 kDa proteins is currently not conclusively answered (Imparl-Radosevich, Arch. Biochem. Biophys. 362, (1999), 131-138). Cao et al. (Plant Physiol. 120, (1999), 205-215) propose the so-called du1 gene as the gene which corresponds to the 180 kDa protein.
The third class of starch synthase genes which has been described to date, termed SSIII, encode, in potatoes, an 139 kDa protein (Abel et al., Plant J. 10, (1996), 981-991; Marshall et al., Plant Cell 8, (1996), 1121-1135), which amount to 80% of the total starch synthase activity in potato tubers. Since certain sequence regions of the C-terminus are highly conserved in comparison with the potato SSIII amino acid sequence, it was proposed to rename the maize gene originally termed du1 gene “zSSIII” (Cao et al., Plant Physiol. 120, (1999), 205-215), the prefix “z” denoting the organism of origin
Zea mays.
The detailed function in starch synthesis has as yet only been determined for the isoform GBSS I. Plants in which this enzyme activity is greatly or fully reduced synthesize an amylase-free “waxy” starch (Shure et al., Cell 35 (1983), 225-233; Visser et al., Mol. Gen. Genet. 225 (1991), 289-296; WO9211376A1), so that an important role in amylose starch synthesis is attributed to this enzyme. This phenomenon is likewise observed in cells of the green algae
Chlamydomonas reinhardtii
(Delrue et al., J. Bacteriol. 174 (1992), 3612-3620). In Chlamydomonas, it was additionally possible to demonstrate that GBSS I not only participates in amylose synthesis, but also affects amylopectin synthesis. Mutants which have no GBSSI activity lack a particular fraction of the usually synthesized amylopectin, which contains longer-chain glucans.
The functions of the isoforms of the soluble starch synthases remain unclear, it is assumed that the soluble starch synthases together with branching enzymes participate in amylopectin synthesis (see, for example, Ponstein et al., Plant Physiol. 92 (1990), 234-241) and that they play an important role in the regulation of the starch synthesis rate.
Besides maize, soluble starch synthases were also identified in a series of other plant species. For example, soluble starch synthases have been isolated until homogeneous from pea (Denyer and Smith, Planta 186 (1992), 609-617) and potato (Edwards et al., Plant J. 8 (1995), 283-294). It emerged in these cases that the isoform of the soluble starch synthase which is identified as SS II is identical

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