CA2371659A1 - Method for enhancing cellulose and modifying lignin biosynthesis in plants - Google Patents

Abstract

This invention relates to polynucleotide molecules encoding cellulose synthase, promoters of cellulose synthase and cellulose synthase polypeptides, methods for genetically altering cellulose and lignin biosynthesis, and methods for improving strength properties of juvenile wood and fiber in trees.
The invention further relates to methods for identifying regulatory elements in a cellulose synthase promoter and transcription factors that bind to such regulatory elements, and to methods for augmenting expression of polynucleotides operably linked to a cellulose synthase promoter.

Description

METHOD FOR ENHANCING CELLULOSE AND
MODIFYING LIGNIN BIOSYNTHESIS IN PLANTS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/135,280 filed 21 May, 1999.
STATEMENT REGARDING FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
Not Applicable FIELD OF THE INVENTION
This invention relates to polynucleotide molecules encoding cellulose synthase, promoters of cellulose synthase and cellulose synthase polypeptides, methods for genetically altering cellulose and lignin biosynthesis, and methods for improving strength properties of juvenile wood and fiber in trees. The invention further relates to methods for identifying regulatory elements in a cellulose synthase promoter and transcription factors that bind to such regulatory elements, and to methods for augmenting expression of polynucleotides operably linked to a cellulose synthase promoter.
BACKGROUND OF THE INVENTION
Lignin and cellulose are the two major building blocks of plant cell walls that provide mechanical strength and rigidity. In plants, and especially in trees, these two organic materials exist in a dynamic equilibrium conferring mechanical strength, water transporting ability and protection from biotic and abiotic environmental stresses.
Normally, oven-dry wood contains 30 to 50% cellulose, 20 to 30% lignin and 20 to 30%
hemicellulose (Higuchi, 1997).
Proportions of lignin and cellulose are known to change with variation in the natural environment. For example, during the development of compression wood in conifers, the percentage of lignin increases from 30 to 40 %, and cellulose content proportionally decreases from 40 to 30% (Timmell, 1986). Conversely, in angiosperm tension wood the percentage of cellulose increases from 30 to 40%, while lignin content decreases from 30 to 20% (Timmell, 1986).
It was recently discovered that the genetic down-regulation of a key tissue-specific enzyme from the lignin biosynthesis pathway, 4CL, results in reduction of lignin

-2-content by up to 45% in transgenic aspen trees (Hu et al., 1999). This down-regulation is also associated with a 15% increase in the cellulose content. If the converse were true, i.e., that increasing cellulose content by genetic up-regulation of cellulose biosynthesis results in reduction of lignin content, then the pulp yield could be increased. This would allow tremendous savings in chemical and energy costs during pulping because, for example, lignin must be degraded and removed during the pulping process.
Cellulose is a linear glucan consisting of ~i-D-1,4-linked glucose residues.
It is formed by a cellulose synthase enzyme which catalyzes assembly of UDP-glucose units in plasma membrane complexes known as "particle rosettes" (Delmer and Amor, 1995). Cellulose synthase is thought to be anchored to the membrane by eight transmembrane binding domains to form the basis of the cellulose biosynthesis machinery in the plant cell wall (Pear et al., 1996).
In higher plants, the glucan chains in cellulose microfibrils of primary and secondary cell walls are different in their degree of polymerization (Brown et al., 1996).
For example, secondary cell walls are known to contain cellulose having a high degree of polymerization, while in primary cell walls the degree of polymerization is lower. In another example, woody cell walls suffering from tension stress produce tension wood on the upper side of a bent angiosperm tree in response to the stress. In these cells, there are elevated quantities of cellulose which have very high crystallinity. The formation of highly crystalline cellulose is important to obtain a higher tensile strength of the wood fiber. Woody cell walls located at the under side of the same stem experience a compression stress, but do not produce highly crystalline cellulose. Such variation in the degree of polymerization in cell walls during development is believed to be due to different types of cellulose synthases for organizing glucose units into different paracrystalline arrays (Haigler and Blanton, 1996). Therefore, it would be advantageous to determine the molecular basis for the synthesis of highly crystalline cellulose so that higher yields of wood pulp having superior strength properties can be obtained from transgenic trees. Production of highly crystalline cellulose in transgenic trees would also markedly improve the mechanical strength properties of juvenile wood formed in normal trees. This would be a great benefit to the industry because juvenile wood is generally undesirable for solid wood applications because it has inferior mechanical properties.
Since the deposition of cellulose and lignin in trees is regulated in a compensatory fashion, genetic augmentation of cellulose biosynthesis might have a repressive effect on lignin deposition. Since the degree of polymerization and crystallinity may depend upon the type of cellulose synthase incorporated in the cellulose biosynthesis machinery, the expression of heterologous cellulose synthase or a UDP-glucose binding region thereof (e.g., sweetgum protein expression in loblolly pine), could increase the quality of cellulose in transgenic plants. Over-expression of a heterologous cellulose dV0 00/71670 PCT/US00/13637

-3-synthase may also increase cellulose quantity in transgenic plants. Thus, genetic engineering of cellulose biosynthesis can provide a strategy to augment cellulose quality and quantity, while reducing lignin content in transgenic plants.
A better understanding of the biochemical processes that lead to wood formation would enable the pulp and paper industries to more effectively use genetic engineering as a tool to meet the increasing demands for wood from a decreasing production area. With this objective, many xylem-specific genes, including most lignin biosynthesis genes, have been isolated from developing xylem tissues of various plants including tree species (Ye and Varner, 1993; Fukuda, 1996; Whetten et al., 1998). Genes regulating cellulose biosynthesis in crop plants (Pear et al., 1996 and Arioli et al., 1998), versus in trees, have also been isolated. However, isolation of tree genes which are directly involved in cellulose biosynthesis has remained a great challenge.
For more than 30 years, no gene encoding higher plant cellulose synthase (CeIA) was identified. Recently, Pear et al. (1996) isolated the first putative higher plant CeIA cDNA, GhCeIA (GenBank No. GHU58283), by searching for UDP-glucose binding sequences in a cDNA library prepared from cotton fibers having active secondary wall cellulose synthesis. GhCeIA was considered to encode a cellulose synthase catalytic subunit because it is highly expressed in cotton fibers, actively synthesizes secondary wall cellulose, contains eight transmembrane domains, binds UDP-glucose, and contains two other domains unique to plants.
Recently, Arioli et al. (1998) cloned a CeIA homolog, RSWI (radial swelling) (GenBank No. AF027172), from Arabidopsis by chromosome walking to a defective locus of a temperature sensitive cellulose-deficient mutant.
Complementation of the rswl mutant with a wild type full-length genomic RSWI clone restored the normal phenotype. This complementation provided the first genetic proof that a plant CeIA gene encodes a catalytic subunit of cellulose synthase and functions in the biosynthesis of cellulose microfibrils. The full-length Arabidopsis RSWl represents the only known, currently available cellulose synthase cDNA available for further elucidating cellulose biosynthesis in transgenic systems (Wu et al., 1998).
The discovery of the RSWl gene substantiated the belief that the assembly of a cellulose synthase into the plasma membrane is required for functional cellulose biosynthetic machinery and for manufacturing crystalline cellulose microfibrils in plant cell walls. Most significantly, a single CeIA gene, e.g. RSWl, is sufficient for the biosynthesis of cellulose microfibrils in plants, e.g. Arabidopsis. Thus, RSWI
is a prime target for engineering augmented cellulose formation in transgenic plants.
Since many of society's fiber, chemical and energy demands are met through the industrial-scale production of cellulose from wood, genetic engineering of the cellulose biosynthesis machinery in trees could produce higher pulp yields.
This would

-4-allow greater returns on investment by pulp and paper industries. Therefore, it would be advantageous to isolate and characterize genes from trees that are involved in cellulose biosynthesis in order to improve the properties of wood.
SUMMARY OF THE INVENTION
The present invention relates to polynucleotides comprising a nucleotide sequence that encodes a cellulose synthase, regulatory sequences, including a stress-inducible promoter, of the cellulose s5mthase, a cellulose synthase protein or a functional domain thereof and methods for augmenting cellulose biosynthesis in plants.
Thus, in one aspect, the invention provides a polynucleotide comprising a sequence that encodes a cellulose synthase, or a polynucleotide fragment thereof, the fragment encoding a functional domain of cellulose synthase, such as a UDP-glucose binding domain. The invention also provides a cellulose synthase or a functional domain or fragment thereof, including a UDP-glucose binding domain and at least one of eight transmembrane domains. The invention further provides a cellulose synthase promoter, or a functional fragment thereof, which fragment contains one or more mechanical stress response elements (MSRE).
In another aspect, the present invention is directed to a method of improving the quality of wood by altering the quantity of cellulose in plant cells, and optionally decreasing the content of lignin ~ in the cell. The invention also relates to a method of altering the growth or the cellulose content of a plant by expressing an exogenous polynucleotide encoding a cellulose synthase or a UDP-glucose binding domain thereof in the plant. The invention further provides a method for causing a stress induced gene expression in a plant cell by expressing a polynucleotide of choice using a stress-inducible cellulose synthase promoter.
In yet another aspect, the invention relates to a method for determining a mechanical stress responsive element (MSRE) in a cellulose synthase promoters and a method for identifying transcription factors that binds to the MSRE.
In a further aspect, the invention provides a method for altering (increasing or decreasing) i.e., regulating, the expression of a cellulose synthase in a plant by expressing an exogenous polynucleotide encoding a transcription factor having the property of binding a positive MSRE of a cellulose synthase promoter or by expressing an antisense polynucleotide encoding a transcription factor having the property of binding a negative MSRE to block the expression of the transcription factor.
Other aspects of the invention will be appreciated by a consideration of the detailed description of the invention drawings and appended claims.

-5-DESCRIPTION OF THE DRAWINGS
Fig. 1 represents a nucleic acid sequence encoding a cellulose synthase from Populus trenauloides [SEQ ID NO: 1] and the protein sequence thereof [SEQ
ID
NO: 2].
Fig. 2 a-c (collectively referred to as Fig. 2) represent a Southern blot analysis of aspen genomic DNA probed with a fragment of the aspen cDNA
represented in Fig. 1 under low (panel a) and high stringency conditions (panel b), and a northern blot analysis of the total aspen RNA from stem internodes using the same probe (panel c).
Fig. 3 a-d (collectively referred to as Fig. 3) represent in situ localization of the cellulose synthase gene transcripts as shown in the transverse sections from second (panel a), fourth (panel b), sixth (panel c) and fifth (panel d) internode.
Fig. 4 represents a nucleic acid sequence of the 5' region of aspen cellulose synthase gene including the promoter region and the 5' portion of the coding sequence [SEQ ID NO: 3].
Fig. 5 a-f (collectively referred to as Fig. 5) represents a histochemical analysis (panels a-d and f) and fluorescence microscopy (panel e) of transgenic tobacco for GUS gene expression driven by a cellulose synthase promoter of the invention.
Fig. 6 a-d (collectively referred to as Fig. 6) represents a histochemical analysis of GUS gene expression driven by aspen cellulose synthase promoter of the invention; tangential and longitudinal sections were harvested before bending (panel a), and 4 (panel b), 20 (panel c) and 40 (panel d) hours after bending and stained for GUS
expression.
Fig. 7 represents a cDNA encoding cellulose synthase isolated from Arabidopsis [SEQ ID N0:4].
Fig. 8 represents an Arabidopsis cellulose synthase [SEQ ID NO:S]
encoded by the cDNA represented in Fig. 7.
DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications and references cited in this specification are hereby incorporated herein by reference in their entirety. In case of any inconsistency, the present disclosure governs.
Definitions The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the person of skill in the art in describing the compositions and methods of the invention and how to make and use them. It will be appreciated that the

-6-same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term.
Likewise, the invention is not limited to the preferred embodiments.
The term "plant" includes whole plants and portions of plants, including plant organs (e.g. roots, stems, leaves, etc.).
The term "angiosperm" refers to plants which produce seeds encased in an ovary. A specific example of an angiosperm is Liquidambar styraciflaca (L.)[sweetgum].
The term "gymnosperm" refers to plants which produce naked seeds, that is, seeds which are not encased in an ovary. Specific examples of a gymnosperm include Pinus taeda (L.)[loblolly pine].
The term "polynucleotide" or "nucleic acid molecule" is intended to include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense strands together or individually (although only sense or anti-sense stand may be represented herein). This includes single-and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.
An "isolated" nucleic acid molecule or polynucleotide refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurnng). An isolated nucleic acid or polypeptide may contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated. A
polynucleotide amplified using PCR so that it is sufficiently and easily distinguishable (on a gel, for example) from the rest of the cellular components is considered "isolated". The polynucleotides and polypeptides of the invention may be "substantially pure,"
i.e., having the highest degree of purity that can be achieved using purification techniques known in the art.
The term "hybridization" refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing. Polynucleotides are "hybridizable" to each other when at least one strand of one polynucleotide can anneal to a strand of another polynucleotide under defined stringency conditions.
Hybridization requires that the two polynucleotides contain substantially complementary sequences;

_7_ depending on the stringency of hybridization, however, mismatches may be tolerated.
Typically, hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of O.SX SSC at 65°C) requires that the sequences exhibit some high degree of complementarily over their entire sequence. Conditions of intermediate stringency (such as, for example, an aqueous solution of 2X SSC at 65°C) and low stringency (such as, for example, an aqueous solution of 2X SSC at 55°C), require correspondingly less overall complementarily between the hybridizing sequences. (1X
SSC is 0.15 M NaCI, 0.015 M Na citrate.) As used herein, the above solutions and temperatures refer to the probe-washing stage of the hybridization procedure.
The term "a polynucleotide that hybridizes under stringent (low, intermediate) conditions"
is intended to encompass both single and double-stranded polynucleotides although only one strand will hybridize to the complementary strand of another polynucleotide.
A "sequence-conservative variant" is a polynucleotide that contains a change of one or more nucleotides in a given codon position, as compared with another polynucleotide, but the change does not result in any alteration in the amino acid encoded at that position.
A "function-conservative variant" is a polypeptide (or a polynucleotide encoding the polypeptide) having a given amino acid residue that has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like).
Amino acids with have similar physico-chemical properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Sequence- and function-conservative variants are discussed in greater detail below with respect to degeneracy of the genetic code.
A "functional domain" or a "functional fragment" refers to any region or portion of a protein or polypeptide or polynucleotide which is a region or portion of a larger protein or polynucleotide, the region or portion having the specific activity or specific function attributable to the larger protein or polynucleotide, e.g., a functional domain of cellulose synthase is the UDP-glucose binding domain.
The term "% identity" refers to the percentage of the nucleotides/amino acids of one polynucleotide/polypeptide that are identical to the nucleotides/amino acids of another sequence of polynucleotide/polypeptide as identified by program GAP
from Genetics Computer Group Wisconsin (GCG) package (version 9.0) (Madison, WI).
GAP
uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. When parameters required to run the above algorithm are _g_ not specified, the default values offered by the program are contemplated. The following parameters are used by the GCG program GAP as default values (for polynucleotides): gap creation penalty:50; gap extension penalty:3; scoring matrix: nwsgapdna.cpm (local data file).
The "% similarity" or "~/o homology" between two polypeptide sequences is a function of the number of similar positions shared by two sequences on the basis of the scoring matrix used divided by the number of positions compared and then multiplied by 100. This comparison is made when two sequences are aligned (by introducing gaps if needed) to determine maximum homology. PowerBlast program, implemented by the National Center for Biotechnology Information, can be used to compute optimal, gapped alignments. GAP program from Genetics Computer Group Wisconsin package (version 9.0) (Madison, WI) can also be used. GAP uses the algorithm of Needleman and Wunsch (J Mol Biol 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. When parameters required to run the above algorithm are not specified, the default values offered by the program are contemplated. The following parameters are used by the GCG program GAP
as default values (for polypeptides): gap creation penalty:l2; gap extension penalty:4;
scoring matrix:Blosum62.cpm (local data file).
The term "oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of CeIA, or to detect the presence of nucleic acids encoding CeIA. In a further embodiment, an oligonucleotide of the invention can form a triple helix with a CeIA DNA
molecule. In still another embodiment, a library of oligonucleotides arranged on a solid support, such as a silicon wafer or chip, can be used to detect various polymorphisms of interest. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurnng phosphoester analog bonds, such as thioester bonds, etc.
The term "coding sequence" refers to that portion of the gene that contains the information for encoding a polypeptide. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
A "promoter" is a polynucleotide containing elements (e.g., a TATA box) which are capable of binding RNA polymerise in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerise. Examples of promoters that can be used in the present invention include PtCeIAP, 4CL-1 and 35S.
The term "constitutive promoter" refers to a promoter which typically, does not require positive regulatory proteins to activate expression of an associated coding sequence, i.e., a constitutive promoter maintains some basal level of expression. A
constitutive promoter is commonly used in creation of an expression cassette.
An example of a constitutive promoter are 35S CaMV (Cauliflower Mosaic Virus), available from Clonetech, Palo Alto, CA.
The term "inducible promoter" refers to the promoter which requires a positive regulation to activate expression of an associated coding sequence.
An example of such a promoter is a stress-inducible cellulose synthase promoter from aspen described herein.
A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerise transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
A "vector" is a recombinant nucleic acid construct, such as plasmid, phage genome, virus genome, cosmid, or artificial chromosome to which a polynucleotide of the invention may be attached. In a specific embodiment, the vector may bring about the replication of the attached segment, e.g., in the case of a cloning vector.
The term "expression cassette" refers to a polynucleotide which contains both a promoter and a protein coding sequence such that expression of a given protein is achieved upon insertion of the expression cassette into a cell.
A cell has been "transfected" by exogenous or heterologous polynucleotide when such polynucleotide has been introduced inside the cell. A cell has been "transformed" by exogenous or heterologous polynucleotide when the transfected polynucleotide effects a phenotypic change. Preferably, the transforming polynucleotide should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
"Exogenous" refers to biological material, such as a polynucleotide or protein, that has been isolated from a cell and is then introduced into the same or a different cell. For example, a polynucleotide encoding a cellulose synthase of the invention can be cloned from xylem cells of a particular species of tree, inserted into a plasmid and reintroduced into xylem cells of the same or different species.
The species thus contains an exogenous cellulose synthase polynucleotide.
"Heterologous polynucleotide" refers to an exogenous polynucleotide not naturally occurnng in the cell into which it is introduced.
"Homologous polynucleotide" refers to an exogenous polynucleotide that naturally exists in the cells into which it is introduced.
The present invention relates to isolation and characterization of polynucleotides encoding cellulose synthases from plants, especially trees, including full length or naturally occurring forms of cellulose synthases, functional domains, promoters and regulatory elements. Therefore, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA
techniques within the skill of the art. Such techniques are explained fully in the literature.
See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I
and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984);
Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Tra~zscription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Arcinzal Cell Culture [R.I.
Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B.
Perbal, A
Practical Guide To Molecular Clorzifzg (1984); F.M. Ausubel et al. (eds.), Curre~zt Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
The present invention relates to a novel, full-length cellulose synthase gene (CeIA), a novel stress inducible promoter of cellulose synthases (CeIAP), and cellulose synthase proteins from trees, including UDP-glucose catalytic domains thereof.
The invention enables the development of transgenic tree varieties having increased cellulose content, decreased lignin content and, therefore, improved wood fiber characteristics.
Production of increased cellulose quantity and quality in multiple varieties of commercially relevant, transgenic forest tree species in operational production scenarios are further contemplated. The invention further provides a new experimental system for study of CeIA gene expression and function in trees.

Polynucleotides encoding cellulose synthase and fragments thereof The present invention relates to polynucleotides which comprise the nucleotide sequence that encodes cellulose synthase of the invention or a functional fragment thereof. In a preferred embodiment, the polynucleotide comprises the sequence encoding a tree cellulose synthase and most preferrably, the sequence encoding a cellulose synthase from aspen. In one embodiment, a polynucleotide of the invention includes the entire cellulose synthase coding region, e.g., nucleotides 69 to 3,005 of SEQ
>D NO: 1. In another aspect of the invention, the polynucleotide encoding an Arabidopsis cellulose synthase is provided (see SEQ B7 N0:4 and the translated protein of SEQ ID
NO:S).
Also within the scope of the invention are fragments of the polynucleotides encoding cellulose synthase of the invention, which fragments encode at least one transmembrane domain and/or a LAP-glucose binding domain. For example, a polynucleotide comprising the nucleotides encoding a UDP-glucose binding domain of aspen cellulose synthase (e.g., nucleotides 660 to 2250 of SEQ ID NO:1) or corresponding nucleotides of SEQ 117 N0:4 are within the scope of the invention. The nucleotides encoding the UDP-glucose binding domain can be determined by, for example, alignment of protein sequences as described below.
The invention further relates to sequence conservative variants of the coding portion of SEQ ID NOS: 1 and 4.
Polynucleotides that hybridize under conditions of low, medium, and high stringency to SEQ >D NOS: 1 and 4, and their respective coding portions are also within the scope of the invention. Preferably, the polynucleotide that hybridizes to any of SEQ
ID NOS: 1 and 4, or their respective coding portions, is about the same length as that sequence, for example, not more than about 10 to about 20 nucleotides longer or shorter.
In another embodiment of the invention, the hybridizable polynucleotide is at least 1500 nucleotides long, preferably at least 2500 nucleotides long and most preferably at least 3000 nucleotides long. In yet another embodiment, the hybridizable polynucleotide comprises the UDP-glucose binding domain as found in SEQ ID NO:1 or 4, or at least the conserved region QVLRW. Most preferably, the hybridizable polynucleotide has a UDP
glucose binding activity.
The polynucleotides that occur originally in nature may be isolated from the organisms that contain them using methods described herein or well known in the art. The non-naturally occurnng polynucleotides may be prepared using various manipulations known in the field of recombinant DNA. For example, the cloned CeIA
polynucleotide can be modified according to methods described by Sambrook et al., 1989. The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated i~2 vitro. In the production of the modified polynucleotides, for example, care should be taken to ensure that the modified polynucleotide remains within the appropriate translational reading frame (if to be expressed) or uninterrupted by translational stop signals. As a further example, a CeIA-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further irz vitro modification. Preferably, such mutations enhance the functional activity of the mutated CeIA polynucleotide. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA
3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc.
Natl. Acad.
Sci. U.S.A. 83:710), use of TAB linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR
Technology: Principles and Applicatiorzs for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).
The polynucleotides of the present invention may be introduced into various vectors adapted for plant or non-plant replication. These are well known in the art, thus, choice; construction and use of such vectors is well within the skill of a person skilled in the art. Possible vectors include, but are not limited to, plasmids or modified viruses of plants, but the vector system must be compatible with the host cell used. An example of a suitable vector is Ti plasmid. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA
molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. An expression cassette containing cellulose synthase or recombinant molecules thereof can be introduced into host cells via silicon carbide whiskers, transformed protoplasts, transformation, e.g., Agrobacteriurn vectors (discussed below), electroporation, infection, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2m plasmid.
Transgenic plants containing the polynucleotides described herein are also within the scope of the invention. Methods for introducing exogenous polynucleotides into plant cells and regenerating transgenic plants are well known. Some are provided below.
In one embodiment, to introduce a plasmid containing a CeIA coding sequence or promoter of the invention into a plant, a 1:1 mixture of plasmid DNA
S containing a selectable marker expression cassette and plasmid DNA
containing a cellulose synthase expression cassette is precipitated with gold to form microprojectiles.
The microprojectiles are rinsed in absolute ethanol and aliquots are dried onto a suitable macrocarrier such as the macrocarrier available from BioRad in Hercules, CA.
Prior to bombardment, embryogenic tissue is preferably desiccated under a sterile laminar-flow hood. The desiccated tissue is transferred to semi-solid proliferation medium.
The prepared microprojectiles are accelerated from the macrocarner into the desiccated target cells using a suitable apparatus such as a BioRad PDS-1000/I~ particle gun. In a preferred method, each plate is bombarded once, rotated 180 degrees, and bombarded a second time. Preferred bombardment parameters are 1350 psi rupture disc pressure, 6 mm distance from the rupture disc to macrocarrier (gap distance), 1 cm macrocarrier travel distance, and 10 cm distance from macrocarrier stopping screen to culture plate (microcarrier travel distance). Tissue is then transferred to semi-solid proliferation medium containing a selection agent, such as hygromycin B, for two days after bombardment.
Cellulose synthase protein and fragment thereof A cellulose synthase of the invention is a plant protein that contains a catalytic subunit which has UDP-glucose binding activity for the synthesis of glucan from glucose, and eight transmembrane domains for localizing the cellulose synthase to the cell membrane. The cellulose synthase of the invention has eight transmembrane binding domains; two at the amino terminal and six at the carboxyl terminal. The UDP-glucose binding domain is located between transmembrane domains two and three.
Examples of this protein structure are seen in the aspen cellulose synthase as well as in those of RSWI
and GhCeIA. The location of the transmembrane domain may be identified as described below and as exemplified in the Example. Preferably, the cellulose synthase of the invention has an amino acid sequence of a tree cellulose synthase.
In one embodiment, the cellulose synthase protein of the invention is isolated from aspen. Aspen cellulose synthase contains about 978 amino acids and has a molecular weight of about 110 KDa and a pI of about 6.58. In one embodiment, the aspen cellulose synthase has the amino acid sequence of SEQ >D N0:2 as represented in Fig. 1.
In another aspect, the invention relates to cellulose synthase of SEQ ID NO:
5.
The invention further relates to fragments of plant cellulose synthases, such as fragments containing at least one transmembrane region and/or a UDP-glucose binding domain. The transmembrane regions may be identified as described in the Example by using the method of Hoffman and Stoffel (1993).
The cellulose synthase fragment containing the UDP-glucose binding domain is functional without the presence of the rest of the protein. This separable activity is as shown in the Example. This result was surprising and unexpected because previously identified UDP-glucose binding domains were not known to be functional when isolated from other portions of the protein. Thus, a fragment of any cellulose synthase (such as PtCeIA, RSWI, GhCeIA and SEQ )D NO:S) that contains a UDP-glucose binding domain and is independently functional is within the scope of the invention. The function of the UDP-glucose binding domain may be determined using the assay described in the Example. The UDP-glucose binding domain of the invention is located between the second and third transmembrane region of the cellulose synthase and has conserved amino acid sequences for UDP-glucose binding, such as the sequence QVLRW and conserved D
residues. The UDP-glucose binding domain and the conserved regions therein may be located in a cellulose synthase using the guidance of the present specification and the general knowledge in the art, for example Brown, 1996. In one embodiment, the UDP-glucose binding domain and the conserved regions therein may be identified by comparing the amino acid sequence of cellulose synthase of interest with the amino acid sequence of aspen cellulose synthase using the algorithms described in the specification or generally known in the art. For example, the UDP-glucose binding domain of SEQ ID N0:2 is in the position amino acids 220 to 749. The conserved QVLRW sequence is located at positions 715-719 of SEQ ID N0:2.
Polypeptides having at least 75%, preferably at least 85% and most preferably at least 95% similarity to the amino acid sequence of SEQ >D NO: 2, amino acids 220-749 of SEQ 1D N0:2, SEQ )D NO:S or its UDP-glucose binding domain using Power Blast or GAP algorithm described above. In a preferred embodiment, these polypeptides are of about the same length as the polypeptide of SEQ ID NO: 2 or amino acids 220-749 of SEQ ID N0:2. For example, the polypeptide may be from about 2-3 to about 5-7 and to about 10-15 amino acids longer or shorter. In another embodiment, the polypeptides described in this paragraph are not originally found (i.e., naturally occurring) in Arabidopsis or cotton. These polypeptides may be prepared by, for example, altering the nucleic acid sequence of a cloned polynucleotide encoding the protein of SEQ ID
NO:2 or SEQ >D NO:S using the methods well known in the art.
Function conservative variants of cellulose synthase are also within the scope of the invention and can be prepared by altering the sequence of a cloned polynucleotide encoding cellulose synthase or fragments thereof. Conventional methods used in the art can be used to make substitutions, additions or deletions in one or more amino acids, to provide functionally equivalent molecules. For example, a function conservative variant that has substitutions, deletions and/or additions in the amino and/or carboxyl terminus of the protein, outside of the UDP-glucose binding domain is within the scope of the invention. Preferably, variants are made that have enhanced or increased functional activity relative to native cellulose synthase. Methods of directed evolution can be used for this purpose.
The invention also includes function conservative variants which include altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point. Particularly preferred substitutions are: (i) Lys for Arg and vice versa such that a positive charge may be maintained; (ii) Glu for Asp and vice versa such that a negative charge may be maintained;
(iii) Ser for Thr such that a free -OH can be maintained; and (iv) Gln for Asn such that a free CONHZ can be maintained. Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces b-turns in the protein's structure.
The cellulose synthase of the invention can be isolated by expressing a cloned polynucleotide encoding the cellulose synthase as well as using direct protein purification techniques. These methods will be apparent to those of skill in the art.
Polmucleotides containing cellulose synthase promoter The present invention further relates to a cellulose synthase promoter. The promoter is a stress-inducible promoter and may be used to synthesize greater quantities of high crystalline cellulose in plant, and preferably in trees. This permits an increase in the proportion of cellulose in transgenic plants, greater strength of juvenile wood and fiber, and acceleration of overall growth rate.
In one embodiment, the promoter of the invention is from aspen and is represented in Figure 4. The promoter sequence is located within the region of nucleotides 1-840 of SEQ >D N0:3. A person of skill in the art will appreciate that not the entire sequence is required for the promoter function and can easily identify the critical regions by looking for conserves boxes and doing routine deletion analysis. Thus, functional fragments of SEQ ID NO:1 are within the scope of the invention.
Polynucleotides that hybridize under conditions of low, medium, and high stringency to SEQ ID N0:3, and its non-coding portion are also within the scope of the invention. The hybridizable polynucleotide may be about the same length as the sequence to which it hybridizes, for example, not more than about 10 to about 20 nucleotides longer or shorter. In another embodiment, the hybridizable polynucleotide is at least about 200 nucleotides long, preferably at least about 400 nucleotides long and most preferably at least 500 nucleotides long. In yet another embodiment, the hybridizable polynucleotide comprises at least one MSRE element identified according to the method described below.
A cellulose synthase promoter of the invention typically provides tissue specific gene regulation in xylem, but also permits up-regulation of gene expression in other tissues as well, e.g., phloem under tension stress. Furthermore, expression of cellulose synthase is localized to an area of the plant under stress.
This stress-inducible phenomenon is regulated by positive and negative mechanical stress response elements (MSREs). These MSREs upregulate (positive) or downregulate (negative) the expression of a cellulose synthase polynucleotide under stress conditions through binding of transcription factors. MSRE-regulated expression of cellulose synthase permits synthesis of cellulose with high crystallinity.
The MSREs of cellulose synthase can be modified or employed otherwise in methods to regulate expression of a polynucleotide, including a cellulose synthase, operatively linked to a promoter containing an MSRE in response to mechanical stress (e.g., tension or compression) to a transgenic plant.
Negative MSREs of a cellulose synthase promoter can be modified, removed or blocked to improve expression of a cellulose synthase, and thereby increase cellulose production and improve wood quality. Alternatively, positive MSREs can be removed or blocked to decrease expression of a cellulose synthase, which decreases cellulose production and increases lignin deposition. This is useful for increasing the fuel value of wood because lignin has a higher BTU value than cellulose. Moreover, a modified cellulose synthase promoter can be operatively linked to a polynucleotide of interest to control its expression upon mechanical stress to a plant harboring it.

The location of MSRE elements in the SEQ ID N0:3 may be identified, for example, using promoter deletion analysis, DNAse Foot Print Analysis, and Southwestern screening of an expression library for an MSRE. In one embodiment, cellulose synthase promoter that has one or more portions deleted, and is operatively linked to a reporter sequence, is introduced into a plant or a plant cell. A positive MSRE is detected by observing no relative change or increase in the amount of reporter in a transgenic plant or tissue, e.g., phloem after inducing a stress to the plant, and a negative MSRE
is detected by observing increases in the amount of reporter in the plant in the absence of any stress to the plant. A positive element is detected when by removing it, GUS expression goes down and by adding it kept at the same level or more. The negative element does not support, or suppreses, expression of GUS and by removing it, normal or enhanced GUS
expression is observed as compared to when negative element is present.
Manipulation of a MSRE binding sites and/or providing transcription factors that bind thereto, provides a mechanism to continuously produce high crystalline cellulose in woody plant cell walls of transgenic plants. For example, one having ordinary skill in the art can delete or block negative MSRE elements, or provide cDNA
encoding proteins) that bind the positive MSREs, to enable constitutive expression of a cellulose synthase without the requirement of a mechanical stress. The increased cellulose synthase, and therefore, increased cellulose content, can improve the strength properties of juvenile wood and fiber. It is also contemplated that the positive MSREs can be deleted or blocked, or cDNA in an antisense direction, which in the sense direction encodes a protein that binds a positive MSRE, can be provided, to reduce cellulose synthase activity and decrease cellulose production.
Method of Isolating Polynucleotides Encoding Cellulose Synthase The invention further relates to identifying and isolating polynucleotides encoding cellulose synthase in plants, e.g., trees, (in addition to those polynucleotides provided in the Example and represented in Fig. 1 and Fig. 7). These polynucleotides may be used to manipulate expression of cellulose synthase with an objective to improve the cellulose content and properties of wood.
The method comprises identifying a nucleic acid fragment containing a sequence encoding cellulose synthase or a portion thereof by using a fragment of SEQ ID
NOS:l or 4 as a probe or a primer. Once identified, the nucleic acid fragment containing a sequence encoding cellulose synthase or a portion thereof is isolated.
Polynucleotides encoding cellulose synthases of the invention, whether genomic DNA, cDNA, or fragments thereof, can be isolated from many sources, particularly from cDNA or genomic libraries from plants, preferably trees (e.g. aspen, sweetgum, loblolly pine, eucalyptus, and other angiosperms and gymnosperms).

Molecular biology methods for obtaining polynucleotides encoding a cellulose synthase are well known in the art, as described above (see, e.g., Sambrook et al., 1989, supra).
Accordingly, cells from any species of plant can potentially serve as a nucleic acid source for the molecular cloning of a polynucleotide encoding a cellulose S synthase of the invention. The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library"), and preferably is obtained from a cDNA
library prepared from tissues with high level expression of a cellulose synthase (e.g., xylem tissue, since cells in this tissue evidence very high levels of expression of CeIA), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purifred from a desired cell (see, for example, Sambrook et al., 1989, supra;
Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II). Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, a polynucleotide should be molecularly cloned into a suitable vector for its propagation.
In another embodiment for the molecular cloning of a polynucleotide encoding a cellulose synthase of the invention from genomic DNA, DNA fragments are generated from a genome of interest, such as from a plant, or more particularly a tree genome, part of which will correspond to a desired polynucleotide. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
Once the DNA fragments are generated, identification of the specific DNA
fragment containing a desired CeIA sequence may be accomplished in a number of ways.
For example, if an amount of a portion of a CeIA sequence or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA
fragments may be screened by nucleic acid hybridization to a labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad.
Sci.
U.S.A. 72:3961). For example, a set of oligonucleotides corresponding to the partial amino acid sequence information obtained for a CeIA protein from trees can be prepared and used as probes for DNA encoding cellulose synthase, or as primers for cDNA
or mRNA (e.g., in combination with a poly-T primer for RT-PCR). Preferably, a fragment is selected that is highly unique to a cellulose synthase of the invention, such as the UDP-glucose binding regions. Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used. In a specific embodiment, stringency hybridization conditions can be used to identify homologous CeIA sequences from trees or other plants.
Thus, in one embodiment, a labeled cellulose synthase cDNA from, e.g., Populus tremuloides (PtCeIA), can be used to probe a library of genes or DNA
fragments from various species of plants, especially angiosperm and gymnosperm, to determine whether any bind to a CeIA of the invention. Once genes or fragments are identified, they can be amplified using standard PCR techniques, cloned into a vector, e.g., pBluescript vector (StrataGene of LaJolla, CA), and transformed into a bacteria, e.g., DHSa E. coli strain (Gibco BRL of Gaithersburg, MD). Bacterial colonies are typically tested to determine whether any contains a cellulose synthase-encoding nucleic acid.
Once a positive clone is identified through binding, it is sequenced from an end, preferably the 3' end.
cDNA libraries can be constructed in various hosts, such as lambda ZAPII, available from Stratagene, LaJolla, CA, using poly(A) +RNA isolated from aspen xylem, according to the methods described by Bugos et al. (Biotechniques 19:734-737, 1995 ).
The above mentioned probes are used to assay the aspen cDNA library to locate cDNA
which codes for enzymes involved in production of cellulose synthases. Once a cellulose synthase sequence is located, it is then cloned and sequenced according to known methods in the art.
Further selection can be carned out on the basis of the properties of the gene, e.g., if the gene encodes a protein product having the isoelectric, electrophoretic, hydropathy plot, amino acid composition, or partial amino acid sequence of a cellulose synthase protein of the invention, as described herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones or DNA clones which hybrid-select the proper mRNAs can be used to produce a protein that has similar properties known for cellulose synthases of the invention. Such properties may include, for example, similar or identical electrophoretic migration patterns, isoelectric focusing or non-equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, hydropathy plots, or functional properties (such as isolated, functional UDP-glucose binding domains).
A cellulose synthase polynucleotide of the invention can also be identified by mRNA selection, i.e., by nucleic acid hybridization followed by in vitro translation. In this procedure, nucleotide fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified CeIA DNA, or may be synthetic oligonucleotides designed from the partial amino acid sequence information.
Functional assays (e.g., UDP-glucose activity) of the in vitro translation products of the products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments, that contain the desired sequences.

A radiolabeled CeIA cDNA can be synthesized using a selected mRNA as a template. The radiolabeled mRNA or cDNA may then be used as a probe to identify homologous CeIA DNA fragments from amongst other genomic DNA fragments.
It will be appreciated that other polynucleotides, in addition to a CeIA of the invention can be operatively linked to a CeIA promoter to control expression of the polynucleotide upon application of a mechanical stress.
Expression of CeIA Polypeptides The nucleotide sequence coding for CeIA, or a functional fragment, derivative or analog thereof, including chimeric proteins, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
Preferably, an expression vector includes an origin of replication. The elements are collectively termed herein a "promoter." Thus, a nucleic acid encoding CeIA of the invention can be operatively associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. The necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding CeIA and/or its flanking regions.
In addition to a CeIAP, expression of cellulose synthase can be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control CeIA polynucleotide expression include, constitutive, development-specific and tissue-specific. Examples of these promoters include 35S Cauliflower Mosaic Virus, terminal flower and 4CL-1. Thus, there are various ways to alter the growth of a plant using different promoters, depending on the needs of the practitioner.
The nucleotide sequence may be inserted in a sense or antisense direction depending on the needs of the practitioner. For example, if augmentation of cellulose biosynthesis is desired then polynucleotides encoding, e.g., cellulose synthase, can be inserted into the expression vector in the sense direction to increase cellulose synthase production and thus cellulose biosynthesis. Alternatively, if it is desired that cellulose biosynthesis is reduced or lignin content is increased, then polynucleotides encoding, e.g., cellulose synthase ,can be inserted in the antisense direction so that upon transcription the antisense mRNA hybridizes to other complementary transcripts in the sense orientation to prevent translation. In other embodiments, the polynucleotide encodes a UDP-glucose binding domain and is used in a similar manner as described.
A recombinant CeIA protein of the invention, or functional fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems for plants may be used to achieve high levels of stable gene expression, as discussed above. Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).
Expression vectors containing a nucleic acid encoding a CeIA of the invention can be identified by four general approaches: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection marker gene functions, (d) analyses with appropriate restriction endonucleases, and (e) expression of inserted sequences. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g., ~i-glucuronidase activity, resistance to antibiotics, transformation phenotype, etc.) caused by the insertion of foreign genes in the vector. In another example, if the nucleic acid encoding CeIA is inserted within the "selection marker" gene sequence of the vector, recombinants containing the CeIA insert can be identified by the absence of the CeIA gene function. In the fourth approach, recombinant expression vectors are identified by digestion with appropriate restriction enzymes. In the fifth approach, recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant, provided that the expressed protein assumes a functionally active conformation.
After a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to those vectors or their derivatives described above.
Vectors are introduced into the desired host cells by methods known in the art, e.g., Agrobacteriunz-mediated transformation (described in greater detail below), transfection, electroporation, microinjection, transduction, cell fusion, DEAE
dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).
The cell into which the recombinant vector comprising the nucleic acid encoding CeIA is cultured in an appropriate cell culture medium under conditions that provide for expression of CeIA by the cell. In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific fashion desired. Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (such as glycosylation, cleavage, e.g., of a signal sequence) of proteins.
Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
AQrobacterium-mediated transformation and inducing somatic embryos The culture media used in the invention, and for transforming Agrobacterium, contain an effective amount of each of the medium components (e.g. basal medium, growth regulator, carbon source) described above. As used in describing the present invention, an "effective amount" of a given medium component is the amount necessary to cause a recited effect. For example, an effective amount of a growth hormone in the primary callus growth medium is the amount of the growth hormone that induces callus formation when combined with other medium components. Other compounds known to be useful for tissue culture media, such as vitamins and gelling agents, may also be used as optional components of the culture media of the invention.
Transformation of cells from plants, e.g., trees, and the subsequent production of transgenic plants using Agrobacteriunz-mediated transformation procedures known in the art, and further described herein, is one example of a method for introducing a foreign gene into trees. Transgenic plants may be produced by various methods, such as by the following steps: (i) culturing Agrobacterium in low-pH induction medium at low temperature and preconditioning, i.e., coculturing bacteria with wounded tobacco leaf extract in order to induce a high level of expression of the Agrobacterium vir genes whose products are involved in the T-DNA transfer; (ii) coculturing a desired plant tissue explants, including zygotic and/or somatic embryo tissues derived from cultured explants, with the incited Agrobacterium; (iii) selecting transformed callus tissue on a medium containing antibiotics; and (v) and converting the embryos into plantlets.
Any non-tumorigenic A. tumefaciens strain harboring a disarmed Ti plasmid may be used in the method of the invention. Any Agrobacterium system may be used. For example, Ti plasmid/binary vector system or a cointegrative vector system with one Ti plasmid may be used. Also, any marker gene or polynucleotide conferring the ability to select transformed cells, callus, embryos or plants and any other gene, such as, W~ 00/71670 CA 02371659 2001-11-19 PCT/iJS00/13637 for example ,a gene conferring resistance to a disease, or one improving cellulose content, may also be used. Any promoter desired can be used, such as, for example, a PtCeIAP of the invention, and those promoters as described above. A person of ordinary skill in the art can determine which markers and genes are used depending on particular needs.
For purposes of the present invention, "transformed" or "transgenic" means that at least one marker gene or polynucleotide confernng selectable marker properties is introduced into the DNA of a plant cell, callus, embryo or plant.
Additionally, any gene may also be introduced.
To increase the infectivity of the bacteria, Agrobacterium is cultured in low-pH induction medium, i.e., any bacterium culture media with a pH value adjusted to from 4.5 to 6.0, most preferably about 5.2, and at low temperature such as for example about 19-30°C, preferably about 21-26°C. The conditions of low-pH and low temperature are among the well-defined critical factors for inducing virulence activity in Agrobacterium (e.g., Altmorbe et al., Mol. Plant-Microbe. Interac. 2: 301, 1989; Fullner et al., Science 273: 1107, 1996; Fullner and Nester, J. Bacteriol. 178: 1498, 1996).
The bacteria is preconditioned by coculturing with wounded tobacco leaf extract (prepared according to methods known generally known in the art) to induce a high level of expression of the Agrobacteriacm vir genes. Prior to inoculation of plant somatic embryos, Agrobacterium cells can be treated with a tobacco extract prepared from wounded leaf tissues of tobacco plants grown in vitro. To achieve optimal stimulation of the expression of Agrobacterium vir genes by wound-induced metabolites and other cellular factors, tobacco leaves can be wounded and pre-cultured overnight.
Culturing of bacteria in low pH medium and at low temperature can be used to further enhance the bacteria vir gene expression and infectivity. Preconditioning with tobacco extract and the vir genes involved in the T-DNA transfer process are generally known in the art.
Agrobacterium treated as described above is then cocultured with a plant tissue explant, such as for example zygotic and/or somatic embryo tissue. Non-zygotic (i.e., somatic) or zygotic tissues can be used. Any plant tissue may be used as a source of explants. For example, cotyledons from seeds, young leaf tissue, root tissues, parts of stems including nodal explants, and tissues from primary somatic embryos such as the root axis may be used. Generally, young tissues are a preferred source of explants.
The invention also relates to methods of altering the growth of a plant by expressing the polynucleotide of the invention, which as a result alters the growth of the plant. The polynucleotide used in the method may be a homologous polynucleotide or a heterologous polynucleotide and are described in detail above. For example, both full-length and UDP-glucose binding region containing fragments may be expressed.
Additionally, depending on the aim of the method, the polynucleotide may be introduced into the plant in the sense or in the antisense orientation. Any suitable promoter may be used to provide expression. The promoter or a functional fragment thereof is operatively linked to the polynucleotide. The promoter may be a constitutive promoter, a tissue-specific promoter or a development-specific plant promoter. Examples of suitable promoters are Cauliflower Mosaic Virus 35S, 4CL, cellulose synthase promoter, PtCeIAP
and terminal flower promoter.
The invention further relates to a method of altering the cellulose content in a plant by expressing the polynucleotide of the invention as described above.
The method may be used to increased the ratio of cellulose to lignin in the plant that have an exogenous polynucleotide of the invention introduced therein.
The invention further relates to a method for altering expression of a cellulose synthase in a plant cell by introducing into the cell a vector comprising a polynucleotide of the invention and expressing the polynucleotide. The polynucleotides and promoters described above may be used.
A method for causing stress-induced gene expression in a plant cell is also within the scope of the invention. The method comprises (i) introducing into the plant or a plant cell an expression cassette comprising a cellulose synthase promoter or a functional fragment thereof or providing a plant or a plant cell that comprises the expression cassette (The promoter of the cassette is operatively linked to a coding sequence of choice.); and (ii) applying mechanical stress to the plant to induce expression of the desired coding sequence.
A method for determining a positive mechanical stress responsive element (MSRE) in a cellulose synthase promoter is also within the scope of the invention and comprises (i) making serial deletions in the cellulose synthase promoter, such as for example, SEQ >D N0:3; (ii) introducing the deletion linked to a polynucleotide encoding a reporter sequence into a plant cell, and (iii) detecting a decrease in the amount of reporter in the plant after inducing a stress to the plant. Similarly, a method for determining a negative MSRE in a cellulose synthase promoter is provided. It comprises (i) making serial deletions in the cellulose synthase promoter, such as for example, SEQ
>D N0:3; (ii) introducing the deletion linked to a polynucleotide encoding a reporter sequence into a plant cell, and (iii) detecting an increase in the amount of reporter in the plant after inducing a stress to the plant.
The following methods are also within the scope of the invention: a method for expressing cellulose synthase in a tissue-specific manner comprising transforming a plant with a tissue specific promoter operatively linked to a polynucleotide encoding a cellulose synthase; a method for inducing expression of a cellulose synthase in a plant comprising introducing into a plant a cDNA encoding a protein that binds to a positive MSRE of a cellulose synthase promoter, thereby resulting in increased expression of cellulose in the plant, wherein the binding to the positive MSRE results in expression of a cellulose synthase; a method for reducing expression of a cellulose synthase comprising introducing into a plant a cDNA in an antisense orientation, wherein the cDNA
in a sense orientation encodes a protein that binds to a positive MSRE and results in expression of a cellulose synthase; a method for increasing cellulose biosynthesis in a plant comprising introducing into a plant a cDNA encoding a protein that binds to a positive MSRE of a cellulose synthase promoter, whereby binding of the protein to the positive MSRE results in expression of a cellulose synthase, and A method for reducing cellulose biosynthesis in a plant comprising introducing into a plant a cDNA in an antisense orientation, wherein the cDNA in a sense orientation encodes a protein that binds to a positive MSRE of a cellulose synthase promoter.
EXAMPLE
Molecular cloning of cellulose s nt This Example describes the first tree cellulose synthase cDNA (PtCeIA, GenBank No. AF072131) cloned from developing secondary xylem of aspen trees using RSWI cDNA.
Prior to the present invention, only partial clones of cellulose synthases from crop species and cotton GhCeIA have been discovered, which have significant homology to each other. The present inventors have discovered and cloned a new full-length cellulose synthase cDNA, AraxCelA (GenBank No. AF062485) (Fig. 7, [SEQ
ID
NO: 4]), from an Arabidopsis primary library. AraxCelA is a new member of cellulose synthase and shows 63-85% identity and 72-90% similarity in amino acid sequence with other Arabidopsis CeIA members.
Another cellulose synthase was cloned in aspen using a 32P-labeled 1651-by long EcoRI fragment of Arabidopsis CeIA cDNA, which encodes a centrally located UDP-glucose binding domain, was used as a probe to screen about 500,000 pfu of a developing xylem cDNA library from aspen (Populus tremuloides) (Ge and Chiang, 1996).
Four positive clones were obtained after three rounds of plaque purification.
Sequencing the 3' ends of these four cDNAs showed that they were identical clones. The longest cDNA
clone was fully sequenced and determined to be a full-length cDNA having a 3232 by nucleotide sequence (Fig. 1) [SEQ ID NO: 1], which encodes a protein of 978 amino acids [SEQ ID NO: 2].
Characterization of a cellulose synthase from as en The first AUG codon of PtCeIA was in the optimum context for initiation of transcription on the basis of optimal context sequence described by Joshi (1987a) and Joshi et al. (1997). A putative polyadenylation signal (AATACA) was found 16 by upstream of a polyadenylated tail of 28 bp, which is similar to the proposed plant structure (Joshi, 1987b). The 5' untranslated leader was determined to have 68 by and the 3' untranslated traitor was 227 bp. Both of these regions have a typical length observed in many plant genes (Joshi, 1987a and Joshi, 1987b). This cDNA clone exhibited 90%
amino acid sequence similarity with cellulose synthase from cotton (GhCeIA,) and 71°70 with cellulose synthase from Arabidopsis (RSWl ), suggesting that this particular tree homolog also encodes a cellulose synthase.
The full length cDNA was designated PtCeIA, and encodes a 110,278 Da polypeptide having an isoelectric point (pI) of 6.58 and 8 charged molecules.
The hydropathy curve indicated that this particular cellulose synthase has eight transmembrane binding domains; two at the amino terminal and six at the carboxyl terminal, using the method of Hoffman and Stoffel (1993). This protein structure is analogous to those of RSW1 and GhCeIA. All of the conserved domains for UDP-glucose binding, such as QVLRW and conserved D residues, are also present in a cellulose synthase of the invention, e.g., PtCeIA (Brown et al., 1996). Thus, based on sequence and molecular analyses, it was concluded that PtCeIA encodes a catalytic subunit which, like RSWI in Arabidopsis, is essential for the cellulose biosynthesis machinery in aspen.
ha situ localization of PtCeIA mRNA transcripts along the developmental gradient defined by stem primary and secondary growth demonstrated that cellulose synthase expression is confined exclusively to developing xylem cells undergoing secondary wall thickening. This cell-type-specific nature of PtCeIA gene expression was also consistent with xylem-specific activity of cellulose synthase promoter (PtCeIAP) based on heterologous promoter-13-glucuronidase (GUS) fusion analysis.
Overall, the results provide several lines of evidence that cellulose synthase is the gene primarily responsible for cellulose biosynthesis during secondary wall formation in woody xylem of trees, such as aspen. Previous results by the inventors (Hu et al., 1999) showed that cellulose and lignin are deposited in a compensatory fashion in wood. The discovery of a cellulose synthase in trees, such as aspen, permits the up-regulation of the protein to elevate cellulose production. Surprisingly, expression of CeIA in trees suppressed lignin biosynthesis to further improve wood properties of trees.
Preparation of transgenic plants The UDP-glucose binding sequence was subcloned into pBI121, which was used to prepare transgenic tobacco plants (Hu et al., 1998). The expression of a heterologous UDP-glucose binding sequence resulted in a remarkable growth-accelerating effect. This was surprising because current knowledge of the function of plant cellulose synthases teaches that a UDP-glucose sequence must remain intact with other functional domains in CeIA, e.g., the transmembrane domains, in order for cellulose synthase to initiate cellulose biosynthesis. The remarkable growth and tremendous increase in plant biomass observed in transgenic tobacco was due likely to an augmented deposition of cellulose, indicating that the UDP-glucose domain alone is sufficient for genetic augmentation of cellulose biosynthesis in plants.
Genome organization and expression of a novel cellulose synthase To confirm that the cDNA clone of Fig. 1 [SEQ >D NO: 1] was a cellulose synthase, genomic Southern blot analysis was performed under both high and low stringency conditions using the cDNA. Genomic DNA from aspen was digested with PstI
(lane P), HindIII (lane H) and EcoRI (lane E), and probed using a lkb 32P-labeled fragment from the 5' end of a cellulose synthase of Fig. 1. The Southern blot suggested the presence of a small family of cellulose synthase genes in aspen genome (Fig. 2, panels a and b). Repeated screening of the aspen xylem cDNA library with various plant CeIA
gene-related probes always resulted in the isolation of the same cellulose synthase cDNA
clone. This suggested that the cellulose synthase cDNA cloned (Fig. 1) [SEQ >D
NO: 1], represents the primary and most abundant cellulose synthase-encoding gene in developing xylem of trees, such as aspen, where active cellulose deposition takes place.
It also indicates that manipulation of cellulose synthase gene expression can have a profound influence on cellulose biosynthesis in trees.
In situ hybridization Northern blot analysis of total RNA from the internodes of aspen seedling stems (Fig. 2, panel c) using the labeled probe (as described above) revealed the near absence of cellulose synthase transcripts in tissues undergoing primary growth (internodes 1 to 4), and that the presence of cellulose synthase transcripts occurs during the secondary growth of stem tissues (internodes 5 to 11). However, weak northern signals in primary growth may only suggest that cellulose synthase gene expression is specific to xylem, of which there is little in primary growth tissue.
Xylogenesis in higher plants offers a unique model that involves sequential execution of cambium cell division, commitment to xylem cell differentiation, and culmination in xylem cell death (Fukuda, 1996). Although primary and secondary xylem cells originate from different types of cambia, namely procambium and inter/intrafasicular cambium, both exhibit conspicuous secondary wall development with massive cellulose and lignin deposition (Esau, 1965). To further investigate spatial and temporal cellulose synthase gene expression patterns at the cellular level, in situ hybridization was used to localize cellulose synthase mRNA along the developmental gradient defined by stem primary and secondary growth.
Localization of cellulose synthase gene transcripts (RNA) in stem at various growth stages was also observed. Fig. 3 shows transverse sections from 2°d, 4th and 6'h internodes hybridized with digoxygenin (DIG)-labeled cellulose synthase antisense or sense (control) RNA probes, as described.

PtCeIA transcripts were detected in young aspen stem sections by in sitar hybridization with transcripts of highly variable 5' region of PtCeIA cDNA (a 771 by long fragment generated from PstI and SacI). This region was first subcloned in the plasmid vector, pGEM,-3Zf (+) (Promega) for the production of digoxygenin (DIG)-labeled transcripts using T7 (for antisense transcripts) and SP6 (for sense transcripts) RNA
polymerase (DIG system: Boehringer Mannheim). Probes were subjected to mild alkaline hydrolysis by incubation in 100 m'VI NaHC03, pH 10.2 at 60 °C, which produced approximately 200 by fragments.
Aspen young stems were prepared for sectioning by fixation in 4% (w/v) paraformaldehyde in 100 mM phosphate buffer (pH 7.0) at 4 °C overnight, dehydrated through an ethanol series on ice, and embedded in Paraplast medium (Sigma).
Ten ~.m sections were mounted on Superfrost/plus (Fisher) slides at 42 °C
overnight, dewaxed and then rehydrated through a descending ethanol series. The sections were incubated with proteinase K (10 ~,g/ml in 100 mM Tris-HCI, 50 mM EDTA, pH 7.5) for 30 min and were post-fixed with FAA. The sections were acetylated with 0.33% (v/v) acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0) prior to hybridization. The sections were then incubated in a hybridization mixture (approximately 2 ~,g/ml DIG-labeled probes, 50%
(v/v) formamide, 2 X SSPE, 10% (w/v) dextran sulfate, 125 ~,g/ml tRNA, pH 7.5) at 45 °C
for 12-16 hrs. Nonhybridized single-stranded RNA probe was removed by treatment with 20 ~.g/ml RNase A in TE buffer with 500 mM NaCI. The sections were washed at 50 °C.
Hybridized DIG-labelled probe was detected on sections using anti-digoxygenin antiserum at a 1:1500 dilution, as described in the manufacturer's instruction (DIG
system:
Boehringer Mannheim). Sections were examined by Eclipse 400 light microscope (Nikon) and photographed.
During the primary growth stage (Fig. 3, panels a and b), strong expression of cellulose synthase was found localized exclusively to primary xylem (PX) cells. At this stage, young internodes are elongating, resulting in thickening of primary xylem cells through formation of secondary walls (Esau, 1968). The concurrence of shoot elongation with high expression of cellulose synthase strongly suggests the association of cellulose synthase protein with secondary cell wall cellulose synthesis. Later stages of primary growth (Fig. 3, panel b) are characterized by the appearance of an orderly alignment of primary xylem cells. Active cellulose biosynthesis accompanies cell elongation-induced wall thickening, as indicated by the strong expression of cellulose synthase in these primary xylem cells.
At the beginning of secondary growth in older internodes, it was observed that expression of cellulose synthase is also exclusively localized to xylem cells (Fig. 3, panel c). Instead of elongation in internodes distal to the meristematic activity, growth at this stage is mainly radial due to thickening in secondary cell walls of secondary xylem.

At the same time, expression of PtCeIA gene becomes localized to the secondary developing xylem cells (SX in Fig. 3, panel c), which is again consistent with the idea that PtCeIA encodes a secondary cell wall cellulose synthase. At this stage, secondary xylem cells cover the elongated and differentiated primary xylem cells in which PtCeIA gene expression is no longer detectable (Fig. 3, panel c). These results demonstrate that expression of PtCeIA gene is xylem-specific and the cellulose synthase of Fig.
1 [SEQ ID
NO: 1] encodes a cellulose synthase associated with cellulose biosynthesis in secondary walls of xylem cells. To further confirm xylem-specific expression of cellulose synthase, a cellulose synthase gene promoter sequence was cloned and characterized for regulatory activities.
Characterization of expression regulated by cellulose synthase promoter A 5' 1,200 by cDNA fragment of a cellulose synthase of Fig. 1 [SEQ >D
NO: 1] was used as a probe to screen an aspen genomic library for 5' regulatory sequences of a novel cellulose synthase gene, PtCeIA. The library was constructed by cloning aspen genomic DNA fragments, generated from an Sau3AI partial-digest and sucrose gradient-selected, into the BamHI site of a Lambda DASH II vector (Stratagene, La Jolla, CA).
Five positive clones were obtained from about 150,000 pfu and Lambda DNA was purified. One clone having about a 20 kb DNA insert size was selected for restriction mapping and partial sequencing. This resulted in the identification of a 5' flanking region of PtCeIA gene of approximately 1 kb. This genomic fragment, designated PtCeIAP (Fig.
4) [SEQ ID NO: 3], contained about 800 by of promoter sequence, 68 by of 5' end untranslated region and 160 by of coding sequence. To investigate regulation of tissue-specific cellulose synthase expression at the cellular level, promoter activity was analyzed in transgenic tobacco plants by histochemical staining of a GUS protein. A
PtCeIAP-GUS
fusion binary vector was constructed in pBIl21 with the 35S promoter replaced with PtCeIAP [SEQ ID NO: 3] and introduced into tobacco (Nicotiaoa tabacum) as per Hu et al. (1998).
Eleven independent transgenic lines harboring a CeIAP-GUS fusion were generated. Fig. 5 shows a histochemical analysis of GUS expression driven by a cellulose synthase promoter of the invention in transgenic tobacco plants. Transverse sections from the 3rd (panel a), 5th (panel b), 7th (panel c), and 8th (panels d and f) internodes were stained from GUS activity, and fluorescence microscopy was used to visualize expression under UV radiation.
GUS staining was detected exclusively in xylem tissue of stems, roots and petioles. In stems, strong GUS activity was found localized to xylem cells undergoing primary (Fig. 5, panel a) and secondary growth (Fig. 5 panels b-d and f). GUS
expression was confined to xylem cells in the primary growth stage and became more localized in developing secondary xylem cells during secondary growth. An entire section from the 8th internode stained for GUS activity (Fig. 5, panel f). These results are consistent with the in vivo expression patterns of cellulose synthase in aspen stems. Lignin autofluorescence was visualized after UV radiation. Phloem fibers, which are also active in cellulose and lignin biosynthesis (Fig. 5, panels d and e), did not show GUS activity, suggesting that cellulose synthase gene expression is not associated with cellulose biosynthesis in cell types other than xylem. Examination of GUS activity in roots, stems, leaves, anthers and fruit also showed GUS expression in xylem tissue of all these organs suggesting that cellulose synthases of the invention are xylem-specific cellulose and expressed in all plant organs.
Characterization of promoter activity and cellular expression of a cellulose synthase of the invention from one particular source (aspen) indicated hat expression produces a protein that encodes a secondary cell wall-specific cellulose synthase and is specifically compartmentalized in developing xylem cells. Characterization of the cellulose synthase gene promoter sequence not only confirms cell type-specific expression of cellulose synthase, but also provides a method for over-expressing cellulose synthase in a tissue-specific manner to augment cellulose production in xylem.
Expression of cellulose synthase under tension stress As described earlier, a cellulose synthase promoter of the invention is involved in a novel gene regulatory phenomenon of cellulose synthase. To further characterize a cellulose synthase of the invention, GUS expression driven by an aspen cellulose synthase promoter (PtCeIAP) was observed in transgenic tobacco plants without or under tension stress. The stress was induced by bending and affixing the plants to maintain the bent position (e.g., tying) over a 40 hour period. Tangential and longitudinal sections were taken before bending, and 4 hrs, 20 hrs and 40 hrs after bending (panels a-d, respectively).
The cellulose synthase promoter-GUS fusion binary constructs showed exclusive xylem-specific expression of GUS without any tension stress (Fig. 6, panel a).
However, under tension stress conditions endured by angiosperms in nature, the transgenic tobacco plants induced xylem and phloem-specific expression on the upper side of the stem within the first four hours of stress (Fig. 6, panel b).
This observation was surprising because during tension wood development fibers produce highly crystalline cellulose in order to provide essential mechanical strength to a bending stem. The present observation was the first showing of transcriptional up-regulation of a cellulose synthase, mediated through a cellulose synthase promoter that is directly responsible for development of highly crystalline cellulose in trees.
Furthermore, after 20 hrs of tension stress, both xylem and phloem exhibited GUS
expression, but only on the upper side of the stem that was under tensile stress, i.e., GUS
expression on the lower side was inhibited (Fig. 6, panel c). With extended stress (up to 40 hrs), GUS

expression was restricted to only one small region on the upper side of the stem where maximum tension stress was present (Fig. 6, panel d). Based on the observation of GUS
signal in woody cells upon tension stress and the absence of GUS under compression or no stress, it was concluded that a cellulose synthase promoter of the invention has mechanical stress responsive elements (MSREs) that turn cellulose synthase genes on and off depending on the presence and type of stress to the stem.
The results indicate that positive MSREs exist in a cellulose synthase promoter of the invention to bind transcription factors in response to tension stress for regulating the expression of cellulose synthase and increasing biosynthesis of higher crystalline cellulose. This is evident based on the expression of GUS in xylem and phloem tissue at the upper side of the stem subjected to tension stress, but not when tissue on the lower side was subjected to compression or no stress. Furthermore, the tissue at the lower side of the stem, which was subjected to compression stress, showed no GUS
expression, i.e., expression was turned off. This indicated the presence of negative MSREs, which bind transcription factors to turn off expression of cellulose synthase at the lower side of the stem. Negative MSREs likely suppress development of highly crystalline cellulose in normal wood.
These results provide a mechanism for genetically engineering synthesis of highly crystalline cellulose in juvenile wood for enhancing strength properties, and for synthesizing a higher percentage of cellulose in reaction wood. The positive MSREs and their cognate transcription factors are important in the synthesis of highly crystalline cellulose of high tensile strength, as are the negative MSREs and inhibition of cognate transcription factors thereto. The present invention thus provides a starting point for cloning cDNAs for the transcription factors that bind to positive and negative MSREs according to methods known in the art. Constitutive expression of cDNAs for positive MSRE transcription factors allows the continuous production of highly crystalline cellulose in transgenic trees, while expression of antisense cDNAs for negative MSRE
transcription factors inhibits those transcription factors so that cellulose synthase cannot turn off. This combination will assure continuous production of highly crystalline cellulose in trees.
Genetic en ing~ Bering of cellulose synthase in trans eg nic plants As discussed above, the nucleotide sequence of a cellulose synthase of the invention, e.g., PtCeIA cDNA from aspen, shows significant homology with other polynucleotides encoding cellulose synthase proteins that have been suggested as authentic cellulose synthase clones. To further characterize the activity of a cellulose synthase, four constructs were prepared in a PBI121 plasmid.
1) A constitutive plant promoter Cauliflower mosaic Virus 35S was operatively linked to PtCeIA (35SP-PtCeIA-s) and overexpressed in transgenic plants.

This causes excess production of cellulose, resulting in a reduction in lignin content.
Tobacco and aspen have been transformed with this construct.
2) Cauliflower mosaic Virus 35S was operatively linked to antisense RNA from PtCeIA (35S-PtCeIA-a) and constitutively expressed to reduce production of cellulose and increase lignin content in transgenic plants. This negative control construct may not result in healthy plants since cellulose is essential for plant growth and development. Aspen plants have been transformed with this construct.
3) Aspen 4CL-1 promoter (Hu et al., 1998) was operatively linked to PtCeIA (Pt4CLP-PtCeIA) (the 35S promoter of PBI121 was removed in this construct) and expressed in a tissue-specific manner in developing secondary xylem of transgenic aspen.
This expression augments the native cellulose production and reduces lignin content of angiosperm tissues. Tobacco and aspen have transformed with this construct.
4) The cytoplasmic domain of PtCeIA which contains three conserved regions thought to be involved in UDP-glucose binding during cellulose biosynthesis, was linked to a 35S promoter to produce binary constructs (35S-PtCeIA UDP-glucose).
Expression by this promoter permits constitutive expression of a UDP glucose binding domain of PtCeIA in transgenic plants. Tobacco and aspen have been transformed with this construct.
35S-GUS constructs (pBI121, ClonTech, CA) were used as controls for each experiment with the constructs. Transgenic tobacco plants were transformed with the constructs. The following table shows the general growth measurements of the TO tobacco plants. Plants carrying a PtCeIA construct grew much faster than control plants carrying a pBI121 (control) construct. In comparing developmental 4CL and constitutive promoter control of PtCeIA expression, the 35S was more effective, permitting faster growth of transgenic tobacco plants. The fastest growth was seen in transgenic plants carrying a 35S promoter driven UDP-G domain from PtCeIA.
It is noted that TO generation plants can have carry over effects from their tissue culture treatments. Therefore, seeds were collected for testing this growth phenomenon in T1 generations. The transgenic tobacco plants were analyzed for presence of the transferred genes and all tested positive for the respective gene constructs.

TABLE
Transgenic tobacco plant measurements after transfer in soil for about 1.5 months (N = 2) Construct Height Diameter Internode lengthNo. of Longest leaves leaf 35S-GUS 17 0.5 1 11 17 35S-PtCeIA 77 1.0 6 13 37 35S-UDPG 83 1.0 6 13 37 4CLP-PtCeIA 41 0.8 5 10 29 Note: All values were measured in centimeters, excluding cumber of leaves.
It will be appreciated by persons of ordinary skill in the art that the examples and preferred embodiments herein are illustrative, and that the invention may be practiced in a variety of embodiments which share the same inventive concept.

BIBLIOGRAPHY
Hu et al., 1999, Nature Biotechnology, In Press Whetten et al., 1998, Ann Rev Pl Physiol Pl Mol Biol, 49: 585-609 Arioli et al., 1998, Science, 279: 717-720 Wu et al., 1998, PI Physiol, 117: 1125 Hu et al., 1998, PNAS, 95: 5407-5412 Joshi et al., 1997, PMB, 35: 993-1001 Fukuda, 1996, Ann Rev Pl Physiol Pl Mol Biol, 47: 299-325 Pear et al., 1996, PNAS, 93: 12637-12642 Haigler and Blanton, 1996, PNAS, 93: 12082-12085 Ge and Chiang, 1996, Pl Physiol, 112: 861 Brown et al., 1996, Trends Pl Sci., l: 149-156 Delmer and Amor, 1995, PI Cell, 7: 987-1000 Hoffman and Stoffel, 1993, Biol Chem, Hoppe-Seyler 374: 166 Joshi, 1987, NAR, 15: 6643-6653 Joshi, 1987, NAR, 15: 9627-9640 Timmell, 1986, Compression Wood in Gymnopserms, Springer Verlag Esau, 1967, Plant Anatomy, Wiley and sons, NY
Higuchi, 1997, Biochemistry and Molecular Biology of Wood, Springer Verlag INTERNATIONAL SEARCH REPORT International application No.

Bo: I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This international report has not been established in respect of certain claims under Article 17(2xa) for the following reasons:

1. ~ Claims Nos.:

because they relate to subject matter not requmed to be searched by this Authority, namely:

2. ~ Claims Nos.:

because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be cartied out, specifically:

3. ~ Claims Nos.:

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Boz II Observations where unity of invention is lacking (Continuation of item 't of first sheet) This International Searching Authority found multiple inventions in this international application, as follows:

Please See Extra Sheet.

1. ~ As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims.

2. Q As all searehable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee.

3. ~ As only some of the required additional searcl. fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.:

4. ~ No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

1-4, 9-16, 25-31, 35, 40, 42-43, 45-46 Remark on Protest ~ The additional search fees were accompanied by the applicant's protest.

No protest accompanied the payment of additional search fees.

Form PCT/ISA/210 (continuation of first sheet(1)) (July 1998)*

INTERNATIONAL SEARCH REPORT ~ International application No.

A. CLASSIFICATION OF SUBJECT MATTER:
US CL
800/278, 286, 287, 295, 298; 435/G9.1, 320.1, 419; 536/23.2, 23.6, 24.1, 24.5 BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING
This ISA found multiple inventions as follows:
This application contains the following inventions or groups of inventions which are not so linked as to form a singe inventive concept under PCT Rule 13.1. In order for all inventions to be searched, the appropriate additional search fees must be paid.
Group I, claims) 1-4, 9-16, 25-31, 35, 40, 42-43, 45-46, drawn to polynucleotide having specific nucleic acid sequence or a fragment thereof encoding a functional domain of a cellulose synthase, methods of altering cellulose content of a transgenic plant.
Group II , claim(s)5-7, 32, 36-39, 41 44, drawn to cellulose synthase promoters and methods of their use.
Group III, claims) 8, drawn to cellulose synthase polypeptides Group IV , claim(s) 17, 19-24 , drawn to a polynucleotide encoding UDP-glucose binding domain, and transgenic plants expressing it.
Group V , claim(s)18, drawn to UDP-glucose polypeptide .
Group VI, claims) 33-34 , drawn to a method of identifying regulatory elements in a cellulose synthase promoter.
The inventions listed as Groups 1-VI do not relate to a single inventive concept under PCT Rule 13.1 because, under PCT Rule 13.2, they lack the same or corresponding special technical features for the following reasons:
The claimed polynucleotide sequences or a fragment thereof encoding a functional domain of a cellulose synthase is anticipated by Stalker et al (WO 98/18949) who teach plant cDNAs encoding functional units of cellulose synthase, and so do not constitute a single special technical feature which would be an advance over the prior art.
The invention of Group I, drawn to a polynucleotide encoding cellulose synthase, reguires a polynucleotide with speci5c sequence and transgenic plants expressing it, which are not reguired by any of the other groups.
The invention of Group 11, drawn to cellulose synthase promoter and a mechanical stress to a plant which are not reguired by any of the other groups.
The invention of Group III reguires isolated cellulose synthase polypeptides which are not reguired by any of the other groups.
The invention of Group IV reguires polynucleotides encoding UDP-glucose binding domain which are not reguired by any of the other groups.
The invention of Group V reguires UDP-glucose polypeptide which is not reguired by any of the other groups.
The invention of Group VI reguires methods for identifying regulatory elements which are not reguired by any of the other groups.
Form PCT/ISA/210 (extra sheet) (July 1998)*

SEQUENCE LISTING
<110> Board of Control of Michigan Technological Univers <120> METHOD FOR ENHANCING CELLULOSE AND MODIFYING LIGNIN
BIOSYNTHESIS IN PLANTS
<130> 66040/9675 <140>
<141>
<150> 60/135,280 <151> 1999-05-21 <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 3232 <212> DNA
<213> Populus tremuloides <220>
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aac 1598 Gln Arg Phe Asp Gly Ile Asp Lys Ser Asp Arg Tyr Ala Asn Arg Asn gta gtt ttc ttt gat gtt aac atg aaa ggg ttg gat ggc att caa gga 1696 Val Val Phe Phe Asp Val Asn Met Lys Gly Leu Asp Gly Ile Gln Gly cca gta tac gta gga act ggt tgt gtt ttc aac agg caa gca ctt tac 1699 Pro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Gln Ala Leu Tyr ggc tac ggg cct cct tct atg ccc agc tta cgc aag aga aag gat tct 1792 Gly Tyr Gly Pro Pro Ser Met Pro Ser Leu Arg Lys Arg Lys Asp Ser SUBSTITUTE SHEET (RULE 26) WO 00/71670 PCT/i1S00/13637 tcatcctgcttc tcatgttgc tgcccctca aagaagaag cctgetcaa 1790 SerSerCysPhe SerCysCys CysProSer LysLysLys ProAlaGln gatccagetgag gtatacaga gatgcaaaa agagaggat ctcaatget 1838 AspProAlaGlu ValTyrArg AspAlaLys ArgGluAsp LeuAsnAla gccatatttaat cttacagag attgataat tatgacgag catgaaagg 1886 AlaIlePheAsn LeuThrGlu IleAspAsn TyrAspGlu HisGluArg tcaatgctgatc tcccagttg agctttgag aaaactttt ggcttatct 1934 SerMetLeuIle SerGlnLeu SerPheGlu LysThrPhe GlyLeuSer tctgtcttcatt gagtctaca ctaatggag aatggagga gtacccgag 1982 SerValPheIle GluSerThr LeuMetGlu AsnGlyGly ValProGlu tctgccaactca ccaccattc atcaaggaa gcgattcaa gtcatcggc 2030 SerAlaAsnSer ProProPhe IleLysGlu AlaIleGln ValIleGly tgtggctatgaa gagaagact gaatgggga aaacagatt ggttggata 2078 CysGlyTyrGlu GluLysThr GluTrpGly LysGlnIle GlyTrpIle tatgggtcagtc actgaggat atcttaagt ggcttcaag atgcactgc 2126 TyrGlySerVal ThrGluAsp IleLeuSer GlyPheLys MetHisCys cgaggatggaga tcaatttac tgcatgccc gtaaggcct gcattcaaa 2174 ArgGlyTrpArg SerIleTyr CysMetPro ValArgPro AlaPheLys ggatctgcaccc atcaacctg tctgataga ttgcaccag gtcctccga 2222 GlySerAlaPro IleAsnLeu SerAspArg LeuHisGln ValLeuArg tgggetcttggt tctgtggaa attttcttt agcagacac tgtcccctc 2270 TrpAlaLeuGly SerValGlu IlePhePhe SerArgHis CysProLeu tggtacgggttt ggaggaggc cgtcttaaa tggctccaa aggcttgcg 2318 TrpTyrGlyPhe GlyGlyGly ArgLeuLys TrpLeuGln ArgLeuAla tatataaacacc attgtgtac ccatttaca tccctccct ctcattgcc 2366 TyrIleAsnThr IleValTyr ProPheThr SerLeuPro LeuIleAla tattgcacaatt cctgcagtt tgtctgctc accggaaaa ttcatcata 2414 TyrCysThrIle ProAlaVal CysLeuLeu ThrGlyLys PheIleIle cca acg ctc tca aac ctg gca agc atg ctg ttt ctt ggc ctc ttt atc 2462 SUBSTITUTE SHEET (RULE 26) ProThrLeuSer AsnLeuAlaSer MetLeu PheLeuGly LeuPheIle tccatcattgta actgcggtgctt gagcta agatggagc ggtgtcagc 2510 SerIleIleVal ThrAlaValLeu GluLeu ArgTrpSer GlyValSer attgaagattta tggcgtaatgaa caattc tgggtgatc ggaggtgtt 2558 IleGluAspLeu TrpArgAsnGlu GlnPhe TrpValIle GlyGlyVal tcagcccatctc tttgcggtcttc caggga ttcttaaaa atgttgget 2606 SerAlaHisLeu PheAlaValPhe GlnGly PheLeuLys MetLeuAla ggcatcgatacg aacttcactgtc acagca aaagcagcc gaagatgca 2654 GlyIleAspThr AsnPheThrVal ThrAla LysAlaAla GluAspAla gaatttggggag ctatatatggtc aagtgg acaacactt ttgattcct 2702 GluPheGlyGlu LeuTyrMetVal LysTrp ThrThrLeu LeuIlePro ccaaccacactt ctcattatcaat atgtcg ggttgtget ggattctct 2750 ProThrThrLeu LeuIleIleAsn MetSer GlyCysAla GlyPheSer gatgcactcaac aaaggatatgaa gcatgg gggcctctc tttggcaag 2798 AspAlaLeuAsn LysGlyTyrGlu AlaTrp GlyProLeu PheGlyLys gtgttctttget ttctgggtgatt cttcat ctctatcca ttccttaaa 2846 ValPhePheAla PheTrpValIle LeuHis LeuTyrPro PheLeuLys ggtctaatgggt cgccaaaaccta acacca accattgtt gttctctgg 2894 GlyLeuMetGly ArgGlnAsnLeu ThrPro ThrIleVal ValLeuTrp tcagtgctgttg gcctctgtcttc tctctc gtttgggtc aagatcaat 2942 SerValLeuLeu AlaSerValPhe SerLeu ValTrpVal LysIleAsn ccattcgttaac aaagttgataac accttg gttgcggag acctgcatt 2990 ProPheValAsn LysValAspAsn ThrLeu ValAlaGlu ThrCysIle tccattgattgc tgagctacct ccaataagtc 3042 tctcccagta ttttggggtt SerIleAspCys acaaaacctt tgggaattgg aatatgatcc tcgttgtagt ttccctcaag aaagcacata 3102 tcgctgtcag tatttaaatg aactgcaaga tgattgttct ctatgaagtt ttgaacagtt 3162 tgaaatgata ttatgttaaa atacaggttt tgattgtgtt gaaaaaaaaa aagaaaaaaa 3222 SUBSTITUTE SHEET (RULE 26) aaaaaaaaaa <210> 2 <211> 978 <212> PRT
<213> Populus tremuloides <400> 2 Met Met Glu Ser Gly Ala Pro Ile Cys His Thr Cys Gly Glv _ Gly His Asp Ala Asn Gly Glu Leu Phe Val Ala Cys His G1~
20 25 3' Tyr Pro Met Cys Lys Ser Cys Phe Glu Phe Glu Ile Asn Glv Lys Val Cys Leu Arg Cys Gly Ser Pro Tyr Asp Glu Asn Lei. _ Asp Val Glu Lys Lys Gly Ser Gly Asn Gln Ser Thr Met Al~

Leu Asn Asp Ser Gln Asp Val Gly Ile His Ala Arg His Ile _ Val Ser Thr Val Asp Ser Glu Met Asn Asp Glu Tyr Gly As:. _ Trp Lys Asn Arg Val Lys Ser Cys Lys Asp Lys Glu Asn Lye _ Lys Arg Ser Pro Lys Ala Glu Thr Glu Pro Ala Gln Val Prc _ Gln Gln Met Glu Glu Lys Pro Ser Ala Glu Ala Ser Glu Prc -Ile Val Tyr Pro Ile Pro Arg Asn Lys Leu Thr Pro Tyr Arc_ __ Ile Ile Met Arg Leu Val Ile Leu Gly Leu Phe Phe His Phe -180 185 19~_ Thr Asn Pro Val Asp Ser Ala Phe Gly Leu Trp Leu Thr Ser -Cys Glu Ile Trp Phe Ala Phe Ser Trp Val Leu Asp Gln Ph=

Trp Asn Pro Val Asn Arg Glu Thr Tyr Ile Glu Arg Leu Ser _ 225 230 ~ 235 Tyr Glu Arg Glu Gly Glu Pro Ser Gln Leu Ala Gly Val Asp -SUBSTITUTE SHEET (RULE 26) Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Ser Phe Glu Ser Leu Val Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys Phe Ser Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ser Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Gln Pro Ser Phe Val Lys Glu Arg Arg Ala Met Lys Arg Asp Tyr Glu Glu Tyr Lys Val Arg Val Asn Ala Leu Val Ala Lys Ala Gln Lys Thr Pro Glu Glu Gly Trp Thr Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly His Asp Ser Gly Leu Pro Trp Glu Ile Leu Gly Ala Arg Asp Ile Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr Gln His His Lys Lys Ala Gly Ala Glu Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala Pro Tyr Ile Leu Asn Val Asp Cys Asp His Tyr Val Asn Asn Ser Lys Ala Val Arg Glu Ala Met Cys Ile Leu Met Asp Pro Gln Val Gly Arg Asp Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Lys Ser Asp Arg Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Val Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Gly Pro Pro Ser Met Pro Ser Leu Arg Lys Arg Lys Asp Ser Ser Ser

7 SUBSTITUTE SHEET (RULE 26) Cys Phe Ser Cys Cys Cys Pro Ser Lys Lys Lys Pro Ala Gln Asp Pro Ala Glu Val Tyr Arg Asp Ala Lys Arg Glu Asp Leu Asn Ala Ala Ile Phe Asn Leu Thr Glu Ile Asp Asn Tyr Asp Glu His Glu Arg Ser Met Leu Ile Ser Gln Leu Ser Phe Glu Lys Thr Phe Gly Leu Ser Ser Val Phe Ile Glu Ser Thr Leu Met Glu Asn Gly Gly Val Pro Glu Ser Ala Asn Ser Pro Pro Phe Ile Lys Glu Ala Ile Gln Val Ile Gly Cys Gly Tyr Glu Glu Lys Thr Glu Trp Gly Lys Gln Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Ser Gly Phe Lys Met His Cys Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Val Arg Pro Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Phe Phe Ser Arg His Cys Pro Leu Trp Tyr Gly Phe Gly Gly Gly Arg Leu Lys Trp Leu Gln Arg Leu Ala Tyr Ile Asn Thr Ile Val Tyr Pro Phe Thr Ser Leu Pro Leu Ile Ala Tyr Cys Thr Ile Pro Ala Val Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Thr Leu Ser Asn Leu Ala Ser Met Leu Phe Leu Gly Leu Phe Ile Ser Ile Ile Val Thr Ala Val Leu Glu Leu Arg Trp Ser Gly Val Ser Ile Glu Asp Leu Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe Ala Val Phe Gln Gly Phe Leu Lys Met Leu Ala Gly Ile Asp Thr Asn Phe Thr Val Thr Ala Lys Ala Ala Glu Asp Ala Glu Phe

8 SUBSTITUTE SHEET (RULE 26) Gly Glu Leu Tyr Met Val Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu Leu Ile Ile Asn Met Ser Gly Cys Ala Gly Phe Ser Asp Ala Leu Asn Lys Gly Tyr Glu Ala Trp Gly Pro Leu Phe Gly Lys Val Phe Phe Ala Phe Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Leu Thr Pro Thr Ile Val Val Leu Trp Ser Val Leu Leu Ala Ser Val Phe Ser Leu Val Trp Val Lys Ile Asn Pro Phe Val Asn Lys Val Asp Asn Thr Leu Val Ala Glu Thr Cys Ile Ser Ile Asp Cys <210> 3 <211> 1010 <212> DNA
<213> Populus tremuloides <220>
<221> CDS
<222> (841)..(1008) <220>
<223> 5' flanking region of PtCelA coding sequence <400> 3 gaattcgccc ttttgaattc aggagacgat agtttccggt tcgttgaatg gctttgttca 60 cttctggtct agcaatttgc aaaagaagtt acaaaacaaa tgcatattat gtaaatttaa 120 caagagatgg gttctatggt cacttattta tgcccatcat ttgttctggg gttactcttt 180 atagtctgat tcgaagttgc aaactgccgt ttctggtatt gcaattatgt agccataaac 240 tgttaatcct gtagctatta gcggaccaac aaccagatat acgggatcag cgtcgtaaaa 300 gagatctcca ttctacgttt ctttctaatt tttccgtttc agtgagagaa ttaccctgat 360 acattgacat gatgattgat gattatggga accattccga tgttagacac gagaccatct 420 ggatcctgcc agttttcagt tcacatggca tctcagccca agatcatgtg tttatacgcc 480 taatgacttg tattgaaagt ttggtaagtt gaagatgtgc tctgcccaac agaaaccttc 540

9 SUBSTITUTE SHEET (RULE 26) cttaaatttc cagcaaatct ttcaaacttg gccttacacc ccgaaaatag acgtgcttct 600 acttgggttc ttggaaacca tgcaccaacc gccatacccc accaacccac caccctcaac 660 cttctcttcg ccattacaaa aatgtcagta ccaccctctg aaagacacca acacacccta 720 gctttggtta gggtatttga tataaaaaca aggccaaaac aaaagattgg aaggaagcag 780 aggaagaccc tcttgaaaga attgaagttg taaagagctg gtaaagtggt aataagcaag 840 atg atg gaa tct ggg get cct ata tgc cat acc tgt ggt gaa cag gtg 888 Met Met Glu Ser Gly Ala Pro Ile Cys His Thr Cys Gly Glu Gln Val ggg cat gat gca aat ggg gag cta ttt gtg get tgc cat gag tgt agc 936 Gly His Asp Ala Asn Gly Glu Leu Phe Val Ala Cys His Glu Cys Ser tat ccc atg tgc aag tct tgt ttc gag ttt gaa atc aaa gag ggc cgg 984 Tyr Pro Met Cys Lys Ser Cys Phe Glu Phe Glu Ile Lys Glu Gly Arg aaa gtt tgc ttg cgg tgt ggc tcg ag 1010 Lys Val Cys Leu Arg Cys Gly Ser <210> 4 <211> 56 <212> PRT
<213> Populus tremuloides <223> 5' flanking region of PtCelA coding sequence <400> 4 Met Met Glu Ser Gly Ala Pro Ile Cys His Thr Cys Gly Glu Gln Val Gly His Asp Ala Asn Gly Glu Leu Phe Val Ala Cys His Glu Cys Ser Tyr Pro Met Cys Lys Ser Cys Phe Glu Phe Glu Ile Lys Glu Gly Arg Lys Val Cys Leu Arg Cys Gly Ser <210> 5 <211> 3444 <212> DNA
<213> Arabidopsis thaliana <220>
<223> cellulose synthase mRNA
<400> 5 SUBSTITUTE SHEET (RULE 26) gcggccgcgg ttaatcgccg gttctcacaa caggaatgag tttgtcctca ttaatgccga 60 tgagaatgcc cgaataagat cagtccaaga gctgagtgga cagacatgtc aaatctgcag 120 agatgagatc gaattgactg ttgatggaga accgtttgtg gcatgtaacg aatgtgcatt 180 ccctgtgtgt agaccttgct atgagtacga aagacgagaa ggcaatcaag cttgtccaca 240 gtgcaaaacc cgtttcaaac gtcttaaagg aagtccaaga gttgaaggtg atgaagagga 300 agatgacatt gatgatttag acaatgagtt tgagtatgga aataatggga ttggatttga 360 tcaggtttct gaaggtatgt caatctctcg tcgcaactcc ggtttcccac aatctgattt 420 ggattcagct ccacctggct ctcagattcc attgctgact tacggcgacg aggacgttga 480 gatttcttct gatagacatg ctcttattgt tcctccttca cttggtggtc atggcaatag 540 agttcatcct gtttctcttt ctgacccgac cgtggctgca catcgaaggc tgatggtacc 600 tcagaaagat cttgcggttt atggttatgg aagtgtcgct tggaaagatc ggatggagga 660 atggaagaga aagcagaatg agaaacttca ggttgttagg catgaaggag atcctgattt 720 tgaagatggt gatgatgctg attttccaat gatggatgag ggaaggcagc cattgtctat 780 gaagatacca atcaaatcga gcaagataaa tccttaccgg atgttaattg tgctacgtct 840 tgtgattctt ggtctcttct ttcactaccg tattcttcac cccgtcaaag atgcatatgc 900 tttgtggctt atttctgtta tatgtgagat atggtttgct gtttcatggg ttcttgatca 960 gttccctaaa tggtacccta tcgagcgaga aacgtacttg~gaccgactct cattaagata 1020 tgagaaagaa gggaaaccgt cgggactatc ccctgtggat gtatttgtta gtacagtgga 1080 tccattgaaa gagcctccgc ttattactgc aaatactgtc ttgtctattc ttgctgttga 1140 ttatcctgtc gataaggttg cttgttacgt atctgatgat ggtgctgcta tgcttacttt 1200 cgaagctctt tctgagaccg ctgaattcgc aaggaaatgg gttcctttct gcaagaaata 1260 ttgtattgag cctcgtgctc ccgaatggta tttctgccat aaaatggact acttgaagaa 1320 taaagttcat cccgcatttg ttagggagcg gcgagccatg aagagagatt atgaagaatt 1380 caaagtaaag atcaatgctt tagtagcaac agcacagaaa gtgcctgagg atggttggac 1440 tatgcaagac ggtacacctt ggcccggtaa tagtgtgcga gatcatcctg gcatgattca 1500 ggtcttcctt ggaagtgacg gtgttcgtga tgtcgaaaac aacgagttgc ctcgattagt 1560 ttacgtttct cgtgagaaga gacccggatt tgatcaccat aagaaggctg gagctatgaa 1620 ttccctgata cgagtctctg gggttctatc aaatgctcct taccttctga atgtcgattg 1680 tgatcactac atcaacaata gcaaagctct tagagaagca atgtgtttca tgatggatcc 1740 tcagtcagga aagaaaatct gttatgttca gttccctcaa aggttcgatg ggattgatag 1800 gcacgatcga tactcaaatc gcaatgttgt gttctttgat atcaatatga aaggtttgga 1860 tgggctacaa gggcctatat acgtcggtac aggttgtgtt ttcaggaggc aagcgcttta 1920 cggatttgat gcaccgaaga agaagaaggg cccacgtaag acatgcaatt gctggccaaa 1980 atggtgtctc ctatgttttg gttcaagaaa gaatcgtaaa gcaaagacag tggctgcgga 2040 taagaagaag aagaataggg aagcgtcaaa gcagatccac gcattagaaa atatcgaaga 2100 gggccgcggt cataaagttc ttaacgtaga acagtcaacc gaggcaatgc aaatgaagtt 2160 gcagaagaaa tatgggcagt ctcctgtatt tgttgcatct gcgcgtctgg agaatggtgg 2220 gatggctaga aacgcaagcc cggcttgtct gcttaaagaa gccatccaag tcattagtcg 2280 cggatatgaa gataaaactg aatggggaaa agagattggg tggatctatg gttctgttac 2340 cgaagatatt cttacgggtt ctaagatgca ttctcatggt tggagacatg tttattgtac 2400 accaaagtta gcggctttca aaggatcagc tccaatcaat ctttcggatc gtctccatca 2460 agttcttcga tgggcgcttg ggtcggttga gattttcttg agtaggcatt gtcctatttg 2520 gtatggttat ggaggtgggt tgaaatggct tgagcggttg tcctacatta actctgtggt 2580 ttacccgtgg acctctctac cgctcatcgt ttactgttct ctccctgcca tctgtcttct 2640 cactggaaaa ttcatcgttc ccgagattag caactatgcg agtatcctct tcatggcgct 2700 cttctcgtcg attgcaataa cgggtattct cgagatgcaa tggggcaaag ttgggatcga 2760 tgattggtgg agaaacgaac agttttgggt cattggaggt gtttctgcgc atctgtttgc 2820 tctcttccaa ggtctcctca aggttcttgc tggtgtcgac actaacttca cagtcacatc 2880 aaaagcagct gatgatggag agttctctga cctttacctc ttcaaatgga cttcacttct 2940 catccctcca atgactctac tcatcataaa cgtcattgga gtcatagtcg gagtctctga 3000 tgccatcagc aatggatacg actcgtgggg accgcttttc ggaagactgt tctttgcact 3060 ttgggtcatc attcatcttt acccgttcct taaaggtttg cttgggaaac aagatagaat 3120 gccaaccatt attgtcgtct ggtccatcct cctggcctcg attcttacac ttctttgggt 3180 ccgggttaat ccgtttgtgg cgaaaggcgg tcctattctc gagatctgtg gtttagactg 3240 cttgtgattc gattgaccgg tggatgggtt ggtgaaaaag gtttaattcc cacggatcaa 3300 agagaggtaa gagagatatt gttttacctc taaaagactc cttcattgtg ttcattagat 3360 gatgaaaaat gaaaagaaaa agaagattta attttgttac gagaattgtt atttttgcaa 3420 SUBSTITUTE SHEET (RULE 26) gaatgtgttg tagatagcgg ccgc 3444 <210> 6 <211> 1080 <212> PRT
<213> Arabidopsis thaliana <220>
<223> cellulose synthase <400> 6 Arg Pro Arg Leu Ile Ala Gly Ser His Asn Arg Asn Glu Phe Val Leu Ile Asn Ala Asp Glu Asn Ala Arg Ile Arg Ser Val Gln Glu Leu Ser Gly Gln Thr Cys Gln Ile Cys Arg Asp Glu Ile Glu Leu Thr Val Asp Gly Glu Pro Phe Val Ala Cys Asn Glu Cys Ala Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Asn Gln Ala Cys Pro Gln Cys Lys Thr Arg Phe Lys Arg Leu Lys Gly Ser Pro Arg Val Glu Gly Asp Glu Glu Glu Asp Asp Ile Asp Asp Leu Asp Asn Glu Phe Glu Tyr Gly Asn Asn Gly Ile Gly Phe Asp Gln Val Ser Glu Gly Met Ser Ile Ser Arg Arg Asn Ser Gly Phe Pro Gln Ser Asp Leu Asp Ser Ala Pro Pro Gly Ser Gln Ile Pro Leu Leu Thr Tyr Gly Asp Glu Asp Val Glu Ile Ser Ser Asp Arg His Ala Leu Ile Val Pro Pro Ser Leu Gly Gly His Gly Asn Arg Val His Pro Val Ser Leu Ser Asp Pro Thr Val Ala Ala His Arg Arg Leu Met Val Pro Gln Lys Asp Leu Ala Val Tyr Gly Tyr Gly Ser Val Ala Trp Lys Asp Arg Met Glu Glu Trp Lys Arg Lys Gln Asn Glu Lys Leu Gln Val Val Arg His Glu Gly Asp Pro Asp Phe SUBSTITUTE S~IEET (RULE 26) Glu Asp Gly Asp Asp Ala Asp Phe Pro Met Met Asp Glu Gly Arg Gln Pro Leu Ser Met Lys Ile Pro Ile Lys Ser Ser Lys Ile Asn Pro Tyr Arg Met Leu Ile Val Leu Arg Leu Val Ile Leu Gly Leu Phe Phe His Tyr Arg Ile Leu His Pro Val Lys Asp Ala Tyr Ala Leu Trp Leu Ile Ser Val Ile Cys Glu Ile Trp Phe Ala Val Ser Trp Val Leu Asp Gln Phe Pro Lys Trp Tyr Pro Ile Glu Arg Glu Thr Tyr Leu Asp Arg Leu Ser Leu Arg Tyr Glu Lys Glu Gly Lys Pro Ser Gly Leu Ser Pro Val Asp Val Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ala Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr Phe Glu Ala Leu Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys Tyr Cys Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe Cys His Lys Met Asp Tyr Leu Lys Asn Lys Val His Pro Ala Phe Val Arg Glu Arg Arg Ala Met Lys Arg Asp Tyr Glu Glu Phe Lys Val Lys Ile Asn Ala Leu Val Ala Thr Ala Gln Lys Val Pro Glu Asp Gly Trp Thr Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Ser Val Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly Ser Asp Gly Val Arg Asp Val Glu Asn Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Asp His His Lys Lys Ala Gly Ala Met Asn Ser Leu Ile Arg SUBSTITUTE SHEET (RULE 26) Val Ser Gly Val Leu Ser Asn Ala Pro Tyr Leu Leu Asn Val Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met Asp Pro Gln Ser Gly Lys Lys Ile Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg His Asp Arg Tyr Ser Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Leu Gln Gly Pro Ile Tyr Val Thr Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr Gly Phe Asp Ala Pro Lys Lys Lys Lys Gly Pro Arg Lys Thr Cys Asn Cys Trp Pro Lys Trp Cys Leu Leu Cys Phe Gly Ser Arg Lys Asn Arg Lys Ala Lys Thr Val Ala Ala Asp Lys Lys Lys Lys Asn Arg Glu Ala Ser Lys Gln Ile His Ala Leu Glu Asn Ile Glu Glu Gly Arg Gly His Lys Val Leu Asn Val Glu Gln Ser Thr Glu Ala Met Gln Met Lys Leu Gln Lys Lys Tyr Gly Gln Ser Pro Val Phe Val Ala Ser Ala Arg Leu Glu Asn Gly Gly Met Ala Arg Asn Ala Ser Pro Ala Cys Leu Leu Lys Glu Ala Ile Gln Val Ile Ser Arg Gly Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Ser Lys Met His Ser His Gly Trp Arg His Val Tyr Cys Thr Pro Lys Leu Ala Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Phe Leu Ser Arg His Cys Pro Ile Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Trp SUBSTITUTE SFtEET (RULE 26,j Leu Glu Arg Leu Ser Tyr Ile Asn Ser Val Val Tyr Pro Trp Thr Ser Leu Pro Leu Ile Val Tyr Cys Ser Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Val Pro Glu Ile Ser Asn Tyr Ala Ser Ile Leu Phe Met Ala Leu Phe Ser Ser Ile Ala Ile Thr Gly Ile Leu Glu Met Gln Trp Gly Lys Val Gly Ile Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe Ala Leu Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Val Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ala Asp Asp Gly Glu Phe Ser Asp Leu Tyr Leu Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro Met Thr Leu Leu Ile Ile Asn Val Ile Gly Val Ile Val Gly Val Ser Asp Ala Ile Ser Asn Gly Tyr Asp Ser Trp Gly Pro Leu Phe Gly Arg Leu Phe Phe Ala Leu Trp Val Ile Ile His Leu Tyr Pro Phe Leu Lys Gly Leu Leu Gly Lys Gln Asp Arg Met Pro Thr Ile Ile Val Val Trp Ser Ile Leu Leu Ala Ser Ile Leu Thr Leu Leu Trp Val Arg Val Asn Pro Phe Val Ala Lys Gly Gly Pro Ile Leu Glu Ile Cys Gly Leu Asp Cys Leu SUBSTITUTE SHEET (RULE 26)

Claims (46)

-35-.
WHAT IS CLAIMED IS:

1. A polynucleotide which is (a) a polynucleotide having a nucleotide sequence of SEQ ID NO:1; (b) a polynucleotide having a nucleotide sequence of SEQ ID
NO: 4; or (c) a polynucleotide fragment of (a) or (b) encoding a functional domain of a cellulose synthase.

2. The polynucleotide of claim 1 wherein the polynucleotide is operably linked to a polynucleotide of SEQ ID NO: 3, or a functional fragment thereof.

3. A vector comprising a polynucleotide of claim 1.

4. A transgenic plant comprising a polynucleotide of claim 1.

5. A cellulose synthase promoter, or a functional fragment thereof which binds a transcription factor in a plant cell.

6. A vector comprising a promoter or a fragment of claim 5.

7. A transgenic plant comprising a promoter or a fragment of claim 5.

8. A polypeptide having an amino acid sequence of SEQ ID NO: 2, an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence sequence which a functional domain of cellulose synthase.

9. A method of altering the growth of a plant, comprising expressing in cells of the plant an exogenous polynucleotide encoding a cellulose synthase wherein the polynucleotide is expressed in an amount effective to alter the growth of the plant.

10. A method according to claim 9, wherein the polynucleotide comprises a homologous polynucleotide.

11. A method according to claim 9, wherein the polynucleotide comprises a heterologous polynucleotide.

12. A method according to claim 9, wherein the polynucleotide is in a sense orientation.

13. A method according to claim 9, wherein the polynucleotide is in an antisense orientation.

14. A method according to claim 9, wherein a plant promoter, or a transcription factor binding domain thereof, is operatively linked to the polynucleotide.

15. A method according to claim 14, wherein the promoter is selected from constitutive promoters, tissue-specific promoters and developmental-specific plant promoters.

16. A method according to claim 15, wherein the promoter is Cauliflower Mosaic Virus 35S, 4CL, cellulose synthase promoter, PtCelAP or terminal flower promoter.

17. A polynucleotide encoding a UDP-glucose binding domain of a cellulose synthase.

18. A polypeptide comprising a UDP-glucose catalytic subunit of cellulose synthase wherein the UDP-glucose catalytic subunit catalyzes the biosynthesis of cellulose.

19. A method of altering the growth of a plant, comprising incorporating into a plant genome a polynucleotide encoding a UDP-glucose catalytic subunit wherein expression of the polynucleotide alters the growth of the plant.

20. A method according to claim 19, wherein the polynucleotide comprises a homologous polynucleotide.

21. A method according to claim 19, wherein the polynucleotide comprises a heterologous polynucleotide.

22. A method according to claim 19, wherein the polynucleotide is in a sense orientation.

23. A method according to claim 19, wherein the polynucleotide is in a antisense orientation.

24. A method of altering the cellulose content in a plant comprising expressing an exogenous polynucleotide encoding a UDP-glucose binding domain in a plant genome to alter the cellulose content of the plant.

25. A transgenic plant having an increased ratio of cellulose to lignin in cells of the plant comprising an exogenous polynucleotide encoding a cellulose synthase operably linked to a promoter so that the polynucleotide is expressed in an amount effective to increase the cellulose content of the plant.

26. A transgenic plant having a decreased ratio of lignin to cellulose, the plant comprising an exogenous polynucleotide encoding a cellulose synthase.

27. A method of altering expression of a cellulose synthase in a plant cell comprising delivering into the cell a vector comprising a polynucleotide encoding a cellulose synthase.

28. The method according to claim 27, wherein the polynucleotide comprises a homologous polynucleotide.

29. The method according to claim 27, wherein the polynucleotide comprises a heterologous polynucleotide.

30. The method according to claim 27, wherein the polynucleotide is in a sense orientation.

31. The method according to claim 27, wherein the polynucleotide is in a antisense orientation.

32. A method of causing stress-induced gene expression in a plant cell comprising delivering into the cell a vector comprising a cellulose synthase promoter operatively linked to a gene, wherein the gene is expressed upon a mechanical stress to the plant.

33. A method of determining a positive mechanical stress responsive element (MSRE) in a cellulose synthase promoter comprising:
(i) introducing into a plant a cellulose synthase promoter that has a portion deleted, the cellulose synthase promoter operatively linked to a polynucleotide encoding a reporter, and (ii) detecting a decrease in the amount of reporter in the plant after inducing a stress to the plant.

34. A method of determining a negative MSRE in a cellulose synthase promoter comprising:
(i) introducing into a plant a cellulose synthase promoter that has a portion deleted, the cellulose synthase promoter operatively linked to a reporter gene, and (ii) detecting an increase in the amount of reporter in the plant after inducing a stress to the plant.

35. A method of expressing cellulose synthase in a plant in a tissue-specific manner comprising transforming the plant with a tissue-specific promoter operatively linked to a polynucleotide encoding a cellulose synthase.

36. A method of increasing expression of a cellulose synthase in a plant comprising delivering into the plant a cDNA encoding a protein that binds to a positive MSRE of a cellulose synthase promoter wherein the binding to the positive MSRE
results in expression of a cellulose synthase, resulting in increased expression of cellulose in the plant.

37. A method of reducing expression of a cellulose synthase in a plant comprising delivering into the plant a cDNA in an antisense orientation, the cDNA in a sense orientation encoding protein that binds to a positive MSRE and results in expression of a cellulose synthase.

38. A method of increasing cellulose biosynthesis in a plant comprising delivering into the plant a cDNA encoding a protein that binds to a positive MSRE of a cellulose synthase promoter, wherein binding of the protein to the positive MSRE results in expression of a cellulose synthase.

39. A method of reducing cellulose biosynthesis in a plant comprising delivering into the plant a cDNA in an antisense orientation, the cDNA in a sense orientation encoding protein that binds to a positive MSRE of a cellulose synthase promoter.

40. A transgenic plant containing a polynucleotide comprising a promoter and encoding a cellulose synthase, the polynucleotide expressed such that the growth of the plant is altered relative to a similar control plant that does not contain the polynucleotide.

41. A vector comprising a promoter functional in a plant cell, and a coding sequence for cellulose synthase, the coding sequence operably linked to the promoter, the promoter having a nucleotide sequence encoding a positive MSRE of cellulose synthase.

42. A method of altering a characteristic of a plant comprising genetically upregulating cellulose synthase in the plant, wherein the characteristic is accelerated growth, increased cellulose content or decreased lignin content.

43. The method of claim 42 wherein the plant is genetically upregulated through incorporation into the genome of the plant a cDNA having a nucleotide sequence encoding a cellulose synthase.

44. A method of regulating cellulose synthase expression in a plant comprising delivery in a plant (a) a cDNA encoding a polypeptide which is a positive MSRE
of a cellulose synthase promoter; or (b) a cDNA in an anti sense orientation of the cDNA of (a), in amount and under conditions effective to allow at least a portion of the plant's cells to take up the cDNA.

45. A method of altering cellulose content in a plant comprising:
(a) delivery into cells of the plant an expression cassette comprising a cDNA encoding a cellulose synthase operably linked to a promoter functional in a plan cell; and (b) expressing the cDNA in the cells of the plant in an amount effective to alter the cellulose content in the cells of the plant.

46. A DNA encoding a protein having cellulose synthase activity and comprising the amino acid sequence in SEQ ID NO:2, SEQ ID NO:5 or an amino acid sequence which is a functional domain of cellulose synthase.