Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2002-07-23
2004-11-30
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S440000, C435S419000, C435S320100, C435S252300, C800S281000
Reexamination Certificate
active
06825039
ABSTRACT:
TECHNICAL FIELD
This invention relates to plant genes useful for the genetic manipulation of plant characteristics. More specifically, the invention relates to the identification, isolation and introduction of genes useful, for example, for altering the seed oil content, seed size, flowering and/or generation time, or vegetative growth of commercial or crop plants.
BACKGROUND
Through a coordination of the light and dark reactions of photosynthesis, plants assimilate CO
2
in the formation of sugars. Via the catabolic and anabolic reactions of metabolism, these sugars are the basis of plant growth, and ultimately plant productivity. In the process of plant growth, respiration, which involves the consumption of O
2
and catabolism of sugar or other substrates to produce CO
2
, plays a central role in providing a source of energy, reducing equivalents and an array of intermediates (carbon skeletons) as the building blocks for many essential biosynthesic processes. It is known that any two plants with equal photosynthetic rates often differ in both total biomass production and harvestable product. Therefore, the relationship between rate of respiration and crop productivity has been one of the most intensively studied topics in plant physiology. In a biochemical sense, respiration can be taken to be composed of glycolysis, the oxidative pentose phosphate pathway, the Kreb's (tricarboxylic acid, TCA) cycle and the mitochondrial electron transport system. The intermediate products of respiration are necessary for growth in meristematic tissues, maintenance of existing phytomass, uptake of nutrients, and intra- and inter-cellular transport of organic and inorganic materials. In soybean there is evidence that an increase in respiration rate by the pod can lead to an increase in seed growth (Sinclair et al., 1987), while decreased respiration can result in decreased reproductive growth (Gale, 1974). Respiration is therefore important to both anabolic and catabolic phases of metabolism.
Although the pathways of carbon metabolism in plant cells are quite well known, control of the flux of carbon through these pathways in vivo is poorly understood at present. The mitochondrial pyruvate dehydrogenase complex (mtPDC), which catalyzes the oxidative decarboxylation of pyruvate to give acetyl CoA, is the primary entry point of carbohydrates into the Krebs cycle. The mtPDC complex links glycolytic carbon metabolism with the Krebs cycle, and, because of the irreversible nature of this reaction, the pyruvate dehydrogenase complex (PDC) is a particularly important site for regulation.
Mitochondrial PDC has been studied intensively in mammalian systems, and available knowledge about the molecular structure of plant mtPDC is largely based on studies of the mammalian mtPDC. The mtPDC contains the enzymes E1 (EC 1.2.4.1), E2 (EC 2.3.1.12) and E3 (EC 1.8.1.4) and their associated prosthetic groups, thiamine PPi, lipoic acid, and FAD, respectively. The E1 and E3 components are arranged around a core of E2. The E2 and E3 components are single polypeptide chains. In contrast, the E1 enzyme consists of two subunits, E1&agr; and E1&bgr;. Their precise roles remain unclear. Another subunit, the E3-binding protein, is thought to play a role in attaching E3 to the E2 core. The E1 kinase and phosphatase are associated regulatory subunits (Grof et al., 1995).
Plants are unique in having PDH complexes in two isoforms, one located in the mitochondrial matrix as in other eukaryotic cells, and another located in the chloroplast or plastid stroma (Randall et al., 1989). Although both plastidial and mitochondrial PDH complex isoforms are very sensitive to product feedback regulation, only the mitochondrial PDH complex is regulated through inactivation/reactivation by reversible phosphorylation/dephosphorylation (Miernyk and Randall, 1987; Gemel and Randall, 1992; Grof et al., 1995). More specifically, the activity of mitochondrial PDC (mtPDC) is regulated through product feedback inhibition (NADH and acetyl-CoA) and the phosphorylation state of mtPDC is determined by the combined action of reversible phosphorylation of the E1&agr; subunit by PDC kinase (PDCK) and its dephosphorylation by PDC phosphatase. PDCK phosphorylates and inactives PDC, while PDC phosphatase dephosphorylates and reactivates the complex. Maximum PDC activity also appears to vary developmentally, with the highest catalytic activity observed during seed germination and early seedling development (e.g., in post-germinative cotyledons, Hill et al., 1992; Grof et al., 1995).
Acetyl-CoA, the product of PDC, is also the primary substrate for fatty acid synthesis. While it is known that plant fatty acid biosynthesis occurs in plastids, the origin of the acetyl-CoA used for the synthesis of fatty acids in plastids has been the subject of much speculation. It remains a major question which has not been resolved. Because of the central role of acetyl-CoA in many metabolic pathways, it is likely that more than one pathway could contribute to maintaining the acetyl-CoA pool (Ohlrogge and Browse, 1995).
One school of thought takes the view that carbon for fatty acid synthesis is derived directly from the products of photosynthesis. In this scenario, 3-phosphoglycerate (3-PGA) would give rise to pyruvate, which would be converted to acetyl-CoA by pyruvate dehydrogenase in plastids (Liedvogel, 1986). This hypothesis has many appealing aspects, but also several unaddressed questions: (1) fatty acid synthesis occurs in photosynthetic (chloroplasts) and non-photosynthetic plastids (in root, developing embryo cotyledons, endosperm leucoplasts); (2) some plastids may lack 3-phosphoglycerate mutase (Kleinig and Liedvogel, 1980), an essential enzyme for converting 3-PGA, the immediate product of CO
2
fixation, to pyruvate. (3) Acetate is the preferred substrate for fatty acid synthesis using isolated intact plastids, and there is evidence that a multienzyme system including acetyl-CoA synthetase and acetyl-CoA carboxylase, exists in plastids, which channels acetate into lipids (Roughan and Ohlrogge, 1996). It is almost certain that at least some of the acetyl-CoA in plastids is formed by plastidic pyruvate dehydrogenase, using pyruvate imported from the cytosol or produced locally by plastidial glycolysis.
A further possibility, especially in non-photosynthetic tissues (e.g., roots and developing embryos), is that acetyl-CoA, generated in the mitochondria, is an alternate means to provide acetate moieties for fatty acid synthesis (Ohlrogge and Browse, 1995). Mitochondrially-generated acetyl-CoA could be hydrolysized to yield free acetate, which could move into the plastid for conversion to acetyl-CoA via plastidial acetyl-CoA synthetase, an enzyme with 5- to 15-fold higher activity than the in vivo rate of fatty acid synthesis (Roughan and Ohlrogge, 1994). Alternatively, the mitochondrial acetyl-CoA could be converted to acetylcarnitine and transported directly into the plastid. Hence, in theory, the mitochondrial pyruvate dehydrogenase complex has an important role to play in fatty acid biosynthesis (see
FIG. 1
of the accompanying drawings). The proof of this hypothesis has been hindered by the difficulties of directly measuring the existence of acetate in the cytosol.
The mitochondrial PDC (mtPDC) is a tightly regulated mutiple subunit complex. As mentioned previously, one of the key regulatory components of this complex is PDH_kinase (PDHK). PDHK functions as a negative regulator by inactivating PDH via phosphorylation. By modulating the PDCK, the activity of PDC can be genetically engineered.
Various attempts have been made to increase or channel additional carbon towards fatty acid biosynthesis. Targets have included genetically modifying acetyl-CoA carboxylase and pyruvate kinase gene expression through over-expression and antisense mRNA techniques with limited or no success.
However, there are many examples of successful modifications to plant metabolism that have been achieved by genetic engineering to transfer new genes or to alter the expression of e
Taylor David C.
Zou Jitao
Katcheves Konstantina
Ketter James
TraskBritt
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