Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-09-01
2003-05-06
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S243000, C435S252300, C435S320100, C435S325000, C435S410000, C536S023600, C800S295000
Reexamination Certificate
active
06558922
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to the field of floral scent production.
Floral scent is a key modulating factor in plant-insect interactions and plays a central role in successful pollination, and thus in fruit development, of many crop species. Flower fragrances vary widely among species in terms of the number, identity, and relative amounts of constituent volatile compounds (Knudsen and Tollsten, 1993, Knudsen et al., 1993). Closely related plant species, which rely on different insects for pollination, produce different odors (Henderson, 1986; Raguso and Pichersky,1995). Often, characteristic floral odors are correlated with the type of pollinators. Species pollinated by bees and flies tend to have scents that are defined (by humans) as “sweet,” whereas those pollinated by beetles have “musty,” “spicy,” or “fruity” odors (Dobson,1994).
Some volatile compounds found in floral scent also have important functions in vegetative processes. They may function as attractants to the natural predators of herbivores (Rose et al., 1996; Pare and Tumlinson, 1997) or as airborne signals that activate disease resistance via the expression of defense-related genes in the healthy tissues of infected plants and in neighboring plants (Farmer and Rayan, 1990; Shulaev et al., 1997; Seskar et al., 1998). They may also serve as repellents against herbivores (Levin, 1973; Rodriguez and Levin, 1976; Pellmyr et al, 1987; Gershenzon and Croteau, 1991).
Many volatile components of flowers have been identified; however, the mechanism of flower fragrance formation is not well understood. Recent investigations of floral scent production in
Clarkia breweri
are the first example of the isolation of enzymes and genes responsible for the biosynthesis of scent volatiles. The enzymes S-linalool synthase, S-adenosyl-L-methionine (SAM):(iso)eugenol O-methyl transferase, acetyl-coenzyme A:benzyl alcohol acetyltransferase, and S-adenosyl-L-methionine:salicylic acid carboxyl methyl transferase, which catalyze the formation of linalool, methyl(iso)eugenol, benzylacetate, and methyl salicylate, respectively, and their corresponding genes have been isolated and characterized (Dudareva et al., 1996; 1998a, b; Pichersky et al., 1994; 1995; Wang et al., 1997; Wang and Pichersky, 1998; Ross et al., 1999; reviewed in Dudareva and Pichersky, 2000). It has been shown that in
C. breweri
, flowers synthesize their scent compounds de novo in the tissues from which they are emitted, and that their emission levels, corresponding enzyme activities, and mRNA levels are all spatially and temporally correlated. In general, the expression of these genes is highest in petals just before anthesis and is restricted to the epidermal cell layer of floral tissues.
Although production of volatile scent compounds appears to be widespread in the plant kingdom, information about their de novo biosynthesis (as distinct from their possible release from glucosides; see Oka et al., 1999) and regulation of the genes involved is limited and based to date on the analysis of a single model system—moth-pollinated
C. breweri
. Whether similar molecular mechanisms are involved in regulation of floral scent production in other plant species is currently unclear. Several genes encoding flower pigment biosynthetic enzymes and also genes controlling flower development have been isolated from snapdragon (Coen et al., 1986; Sommer and Saedler, 1986; Coen and Meyerowitz, 1991; Irish and Yamamoto, 1995), but there is no information about enzymes and genes involved in the synthesis of flower scent compounds. There is thus a need for a better understanding of floral scent production, especially in snapdragon flowers which represent a very good model system. The present invention addresses this need.
SUMMARY OF THE INVENTION
A novel protein, S-adenosyl-L-methionine:benzoic acid carboxymethyltransferase, BAMT, that functions as an enzyme in the production of floral scent compounds, has been discovered. Accordingly, in a first aspect of the invention, purified BAMT proteins are provided.
In a second aspect of the invention, isolated nucleic acid molecules that encode BAMT proteins are provided. The nucleic acid molecules may be incorporated into a vector to form a recombinant nucleic acid molecule. Moreover, such recombinant nucleic acid molecules may be introduced into a host cell. Host cells, and transgenic plants, having the introduced nucleic acid nucleic acid molecules encoding a protein as described herein are specifically provided.
In a third aspect of the invention, a method of transforming a host cell is provided that includes introducing into a host cell a nucleic acid molecule encoding a protein described herein.
In fourth aspect of the invention, methods of expressing BAMT proteins are provided. The methods include transforming a host cell with a nucleotide sequence encoding a protein that functions in production of floral scent compounds as provided herein, and culturing the transformed host cells under conditions effective in achieving expression of BAMT proteins. The proteins may then be purified by conventional techniques.
It is an object of the invention to provide purified, functional BAMT proteins.
It is a further object of the invention to provide nucleotide sequences encoding functional BAMT proteins.
It is a further object of the invention to provide recombinant vectors that include nucleotide sequences encoding functional BAMT proteins.
It is yet another object of the invention to provide host cells containing introduced nucleotide sequences encoding functional BAMT proteins.
It is a further object of the invention to provide transgenic plants containing introduced nucleotide sequences encoding functional BAMT proteins.
These and other objects and advantages of the present invention will be apparent from the descriptions herein.
REFERENCES:
patent: 5849526 (1998-12-01), Pichersky
Wang et al.,Floral Scent Production in Clarkia breweri(Onagraceae), Plant Physiol.(1997) 114:213-221.
Bushue et al.,Floral Scent Production in Antirrhinum majus.Published in Jul. 1999 Meeting Abstract Booklet of the Phytochemical Society of North America, Evolution of Metabolic Pathways Conference, Concordia University, Montreal, Canada p. 80, Poster 289, (Jul. 1999).
Joshi, Chandrashekhar P. and Chiang, Vincent L.,Conserved Sequence Motifs in Plant S-adenosyl-L-methionine-dependent Methyltransferases, Plant Molecular Biology, (1998) 37: 663-674.
Bushue, et al., New Class of Plant Methyltransferases:Benzoic Acid Carboxyl Methyltransferase from Snapdragon Flowers, Published in Jul. 1999 Meeting Abstract Booklet of the American Society of Plant Physiology, Baltimore Maryland, p. 32.
Ibrahim, et al.,Plant O-methyltransferasess: Molecular Analysis, Common Signature and Classification, Plant Molecular Biology(1998) 36: 1-10.
Dudareva, et al.,Floral Scent Production in Clarkia breweri, Plant Physiol.(1998) 116: 599-604.
Knudsen, et al.,Flora Scents—A Checklist of Volatile Compounds Isolated by Head-Space Techniques, Phytochemistry, (1993) vol. 33, No. 2, 253-280.
Dudareva, et al.,Developmental Regulation of Methyl Benzoate Biosynthesis and Emission in Snapdragon Flowers, The Plant Cell(2000), vol. 12, 949-961.
Ross, et al.,S-Adenosyl-L-Methionine: Salicylic Acid Carboxyl Methyltransferase, an Enzyme Involved in Floral Scent Production and Plant Defense, Represents a New Class of Plant Methyltransferases, Archives of Biochemistry and Biophysics(1999) vol. 367, No. 1, 9-16.
Dudareva, N. and Pichersky, E., (2000) “Biochemical and Molecular Genetic Aspects of Floral Scents”Plant Physiology122:627-633.
Doudareva Natalia
Murfitt Lisa M.
Greenlee Winner & Sullivan PC
McKelvey Terry
Purdue Research Foundation
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