Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Reexamination Certificate
2000-04-28
2004-09-07
Fredman, Jeffrey (Department: 1634)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
C800S278000, C435S468000
Reexamination Certificate
active
06787683
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
BACKGROUND OF THE INVENTION
The chloroplasts of higher plants contain and elaborate many unique, interconnected biochemical pathways that produce an array of compounds that not only perform vital plastid functions but are also important from agricultural and nutritional perspectives. One class of lipid soluble, chloroplastically synthesized compounds are the prenyllipids, plastoquinone and tocopherols. Plastoquinone is a fundamentally important component of the chloroplast photosynthetic electron transport chain and accounts for up to 50% of the total prenyllipid pool in green tissues. Tocopherols collectively account for up to 40% of the total prenyllipids pool in green plastids and have a well documented role in mammals as an antioxidant [Liebler, 1993] and a similar, though less well understood antioxidant role in plants [Hess, 1993]. The essential nutritional value of tocopherols has been known for over 70 years [Mason, 1980]. Despite the well studied, wide-spread importance of these chloroplastic compounds to human nutrition, agriculture and biochemical processes within plant cells, much remains to be learned at the molecular level about their biosynthesis.
Plastoquinone and tocopherols are the most abundant prenyllipids in the plastid and are synthesized by the common pathway reviewed in Hess, 1993 and Soll, 1987. The head group for both compounds, homogentisic acid, is produced from p-hydroxyphenylpyruvic acid by the enzyme p-hydroxyphenylpyruvic acid dioxygenase in a reaction that catalyzes both an oxidation and decarboxylation. Homogentisic acid is subject to phytylation/prenylation (phytyl and solanyl, C20 and C45, respectively) coupled to a simultaneous decarboxylation to form the first true tocopherol and plastoquinone intermediates, 2-demethyl-phytylplastoquinol and 2-demethylplastoquinol-9, respectively. A single ring methylation occurs on 2-demethylplastoquinol to yield plastoquinol-9 that is then oxidized to plastoquinone-9. The preferred route in spinach for &agr;-tocopherol formation is thought to be 1) ring methylation of 2-demethylphytylplastoquinol, to yield phytylplastoquinol, 2) cyclization to yield gamma-tocopherol and, finally, 3) a second ring methylation to yield &agr;-tocopherol. The first ring methylation in both tocopherol and plastoquinone synthesis is thought to be carried out by a single enzyme that is specific for the sight of methylation on the ring but has broad substrate specificity and accommodates both classes of compounds. The final methylation enzyme (gamma tocopherol methyl transferase) is the only enzyme of the pathway that has been purified from plants to date (dHarlingue and Camara, 1985). All other enzymatic activities of tocopherol/plastoquinone synthesis have been localized to the inner chloroplast envelope by fractionation studies except p-hydroxyphenylpyruvic acid dioxygenase and the tocopherol cyclase enzyme. Difficulties with cell fractionation methods, low activities for some of the enzymes, substrate stability and availability and assay problems make studying the pathway biochemically extremely challenging.
The fact that tocopherol and plastoquinone levels, ratios and total amounts vary by orders of magnitude in different plant tissues and developmental stages indicates the pathway is both highly regulated and highly flexible and has potential for quantitative and qualitative manipulation. However, while biochemical analysis has been useful in deciphering the biosynthetic pathway such studies have provided almost no insight into how bulk carbon flow through the pathway is regulated or how differing amounts of tocopherols or plastoquinone are synthesized.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide nucleic acids and polypeptides relating to the biosynthesis of tocopherol and plastiquinone.
It is another object of the present invention to provide nucleic acids and polypeptides that can be used to identify proteins involved in tocopherol and plastiquinone biosynthesis.
It is another object of the present invention to provide antgenic fragments of the polypeptides of the present invention.
It is another object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention.
It is another object of the present invention to provide methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
It is another object of the present invention to provide a method for modulating the level of tocopherols and plastiquinone in a plant.
Other aspects of the present invention include expression cassettes comprising the nucleic acid operably linked to a promoter, host cells transfected with the expression cassette, and transgenic plants and seeds comprising the expression cassette.
In a further aspect, the present invention relates to a method of modulating expression of the nucleic acids in a plant, comprising the steps of
(a) transforming a plant cell with an expression cassette comprising a nucleic acid of the present invention operably linked to a promoter in sense or antisense orientation;
(b) growing the plant cell under plant growing conditions to produce a regenerated plant capable of expressing the nucleic acid for a time sufficient to modulate expression of the nucleic acids in the plant compared to a corresponding non-transformed plant.
Expression of the nucleic acids encoding the proteins of the present invention can be increased or decreased relative to a non-transformed control plant.
DETAILED DESCRIPTION OF THE INVENTION
Tocopherols are synthesized in the inner plastid membrane. The first committed step in the pathway is the condensation of the homogentisate head group with the phytyl tail catalyzed by an integral membrane protein: homogentisate: phytyl transferase. The present polypeptides catalyze the condensation of homogentisic acid with phytyldiphosphate or geranylgeranyl pyrophosphate to produce the first intermediates in tocopherol or tocotrienol synthesis, respectively.
The phytylation/prenylation of homogentisic acid is a likely key regulatory step for “tail” synthesis and in determining the relative amounts of tocopherols, tocotrienols and plastoquinone produced as it is the branchpoint for the tocopherol and plastoquinone arms of the pathway.
One purpose of this invention is to modulate a prenyllipid biosynthetic pathway, such as the plastoquinone and tocopherol pathways. The modulation of the pathway may be an up regulation or down regulation of the amount or activity of a prenyllipid (ie. plastoquinone or tocopherol), or of an intermediate in a pathway (ie. 2-demethyl-phytylplastoquinol or 2-demethylplastoquinol-9).
Definitions
The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.
The terms polypeptide, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms “polypeptide”, “peptide” and “protein” are also inclusive of modifications including, but not limited
Collakova Eva
Coughlan Sean J.
Helentjaris Timothy G.
Penna Dean Della
Callistein Steven J.
Fredman Jeffrey
Lappegart Kathryn K.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred Intl.
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