Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1997-10-29
2001-08-07
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S320100, C435S419000, C536S023200, C536S023600
Reexamination Certificate
active
06271443
ABSTRACT:
INTRODUCTION
1. Technical Field
This invention relates to plant cellulose synthase cDNA encoding sequences, and their use in modifying plant phenotypes. Methods are provided whereby the sequences can be used to control or limit the expression of endogenous cellulose synthase.
This invention also relates to methods of using in vitro constructed DNA transcription or expression cassettes capable of directing fiber-tissue transcription of a DNA sequence of interest in plants to produce fiber cells having an altered phenotype, and to methods of providing for or modifying various characteristics of cotton fiber. The invention is exemplified by methods of using cotton fiber promoters for altering the phenotype of cotton fiber, and cotton fibers produced by the method.
2. Background
In spite of much effort, no one has succeeded in isolating and characterizing the enzyme(s) responsible for synthesis of the major cell wall polymer of plants, cellulose.
Numerous efforts have been directed toward the study of synthesis of cellulose (1,4-&bgr;-D-glucan) in higher plants. However, hampered by low rates of activity in vitro, the cellulose synthase of plants has resisted purification and detailed characterization (for reviews, see 1,2). Aided by the discovery of cyclic-di-GMP as a specific activator, the cellulose synthase of the bacterium
Acetobacter xylinum
can be easily assayed in vitro, has been purified to homogeneity, and a catalytic subunit identified (for reviews, see 2,3). Furthermore, an operon of four genes involved in cellulose synthesis in
A. xylinum
has been cloned (4-7).
Characterization of these genes indicates that the first gene, termed either BcsA (7) or AcsAB (6) codes for the 83 kD subunit of the cellulose synthase that binds the substrate UDP-glc and presumably catalyzes the polymerization of glucose residues to 1,4-&bgr;-D-glucan (8). The second gene (B) of the operon is believed to function as a regulatory subunit binding cyclic-di-GMP (9) while recent evidence suggests that the C and D genes may code for proteins that form a pore allowing secretion of the polymer and control the pattern of crystallization of the resulting microfibrils (6).
Recent studies with another gram-negative bacterium,
Agrobacterium tumefaciens
, have also led to cloning of genes involved in cellulose synthesis (10,11), although the proposed pathway of synthesis differs in some respects from that of
A. xylinum.
In
A. tumefaciens
, a CelA gene showing significant homology to the BcsA/AcsAB gene of
A. xylinum
, is proposed to transfer glc from UDP-glc to a lipid acceptor; other gene products may then build up a lipid oligosaccharide that is finally polymerized to cellulose by the action of an endo-glucanase functioning in a synthetic mode. In addition, homologs of the CelA, B, and C genes have been identified in
E. coli
, but, as this organism is not known to synthesize cellulose in vivo, the function of these genes is not clear (2).
These successes in bacterial systems opened the possibility that homologs of the bacterial genes might be identified in higher plants. However, experments in a number of laboratories utilizing the
A. xylinum
genes as probes for screening plant cDNA libraries have failed to identify similar plant genes. Such lack of success suggests that, if plants do contain homologs of the bacterial genes, their overall sequence homology is not very high. Recent studies analyzing the conserved motifs common to glycosyltransferases using either UDP-glc or UDP-GlcNAc as substrate suggest that there are specific conserved regions that might be expected to be found in any plant homolog of the catalytic subunit (referred to hereafter as CelA). In one of these studies, Delmer and Amor (2) identifed a motif common to many such glycosyltransferases including the bacterial CelA proteins. An independent analysis (6) also concluded that this motif was highly conserved in a group of similar glycosyltransferases.
Extending these studies further, Saxena et al. (12) presented an elegant model for the mechanism of catalysis for enzymes such as cellulose synthase that have the unique problem of synthesizing consecutive residues that are rotated approximately 180° with respect to each other. The model invokes independent UDP-glc binding sites and, based upon hydrophobic cluster analysis of these enzymes, the authors concluded that 3 critical regions in all such processive glycosyltransferases each contain a conserved aspartate (D) residue, while a fourth region contained a conserved QXXRW motif. The first D residue resides in the motif as previously analyzed (2,6).
In general, genetic engineering techniques have been directed to modifying the phenotype of individual prokaryotic and eukaryotic cells, especially in culture. Plant cells have proven more intransigent than other eukaryotic cells, due not only to a lack of suitable vector systems but also as a result of the different goals involved. For many applications, it is desirable to be able to control gene expression at a particular stage in the growth of a plant or in a particular plant part. For this purpose, regulatory sequences are required which afford the desired initiation of transcription in the appropriate cell types and/or at the appropriate time in the plant's development without having serious detrimental effects on plant development and productivity. It is therefore of interest to be able to isolate sequences which can be used to provide the desired regulation of transcription in a plant cell during the growing cycle of the host plant.
One aspect of this interest is the ability to change the phenotype of particular cell types, such as differentiated epidermal cells that originate in fiber tissue, i.e. cotton fiber cells, so as to provide for altered or improved aspects of the mature cell type. Cotton is a plant of great commercial significance. In addition to the use of cotton fiber in the production of textiles, other uses of cotton include food preparation with cotton seed oil and animal feed derived from cotton seed husks.
A related goal involving the control of cell wall and characteristics would be to affect valuable secondary tree characteristics of wood for paper forestry products. For instance, by altering the balance of cellulose and lignin, the quality of wood for paper production may be improved.
Finally, despite the importance of cotton as a crop, the breeding and genetic engineering of cotton fiber phenotypes has taken place at a relatively slow rate because of the absence of reliable promoters for use in selectively effecting changes in the phenotype of the fiber. In order to effect the desired phenotypic changes, transcription initiation regions capable of initiating transcription in fiber cells during development are desired. Thus, an important goal of cotton bioengineering research is the acquisition of a reliable promoter which would permit expression of a protein selectively in cotton fiber to affect such qualities as fiber strength, length, color and dyability.
Relevant Literature
Cotton fiber-specific promoters are discussed in PCT publications WO 94/12014 and WO 95/08914, and John and Crow, Proc. Natl. Acad. Sci. USA, 89:5769-5773, 1992. cDNA clones that are preferentially expressed in cotton fiber have been isolated. One of the clones isolated corresponds to mRNA and protein that are highest during the late primary cell wall and early secondary cell wall synthesis stages. John and Crow, supra.
In plants, control of cytoskeletal organization is poorly understood in spite of its importance for the regulation of patterns of cell division, expansion, and subsequent deposition of secondary cell wall polymers. The cotton fiber represents an excellent system for studying cytoskeletal organization. Cotton fibers are single cells in which cell elongation and secondary wall deposition can be studied as distinct events. These fibers develop synchronously within the boll following anthesis, and each fiber cell elongates for about 3 weeks, depositing a thin primary wall (Meinert and Delmer, (1984) Plant Physi
Delmer Deborah
Pear Julie R.
Stalker David M.
Calgene LLC
Nelson Amy J.
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