Modifications of plant traits using cyclin A

This application claims priority to U.S. provisional application Ser. No. 60/533,036, filed on Dec. 30, 2003, which is specifically incorporated in its entirety herein by reference.

The instant application contains a “lengthy” Sequence Listing which has been submitted via CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. Said CD-R, recorded on May 12, 2005, are labeled “CRF”, “Copy 1” and “Copy 2”, respectively, and each contains only one identical 1.63 MB file (44463360.APP).

The present invention relates generally to the field of plant cell cycle genes and polypeptides encoded by such genes, and the use of such polynucleotide and polypeptide sequences for regulating a plant cell cycle. The invention specifically provides cell cycle polynucleotide and polypeptide sequences isolated from Eucalyptus and Pinus and sequences related thereto.

Cell growth and division are controlled by the temporal expression of different sets of genes, allowing the dividing cell to progress through the different phases of the cell cycle. Continued growth and organogesis in plants requires precise function of the cell cycle machinery. Plant development, which is directly affected by cell division rates and patterns, also is influenced by environmental factors, such as temperature, nutrient availability, light, etc. See Gastal and Nelon, Plant Physiol. 105:191-7 (1994), Ben-Haj-Sahal and Tardieu, Plant Physiol. 109:861-7 (1995), and Sacks et al., Plant Physiol. 114:519-27 (1997). Plant development and phenotype are connected with the cell cycle, and altering expression of the genes involved in the cell cycle can be a useful method of modifying plant development and altering plant phenotype.

The ability to alter expression of cell cycle genes is extremely powerful because the cell cycle drives plant development, including growth rates, responses to environmental cues, and resulting plant phenotype. Control of the plant cell cycle and phenotypes associated with alteration of cell cycle gene expression, in the vascular cambium, in particular, has applications for, inter alia, alteration of wood properties and, in particular, lumber and wood pulp properties. For example, improvements to wood pulp that can be effected by altering cell cycle gene expression include increased or decreased lignin and cellulose content, and altered length, diameter, and lumen diameter of cells. Manipulating the plant cell cycle, and in particular the cambium cell cycle (i.e. the rate and angle of cell division), can also engineer better lumber having increased dimensional stability, increased tensile strength, increased shear strength, increased compression strength, increased shock resistance, increased stiffness, increased or decreased hardness, decreased spirality, decreased shrinkage, and desirable characteristics with respect to weight, density, and specific gravity.

A. Cell Cycle Genes and Proteins

1. Cyclin Dependent Protein Kinase

Progression through the cell cycle is regulated primarily by cyclin-dependent kinases (CDKs). CDKs are a conserved family of eukaryotic serine/threonine protein kinases, which require heterodimer formation with a cyclin subunit for activity. For review see, e.g. Joubes et al., Plant Mol. Biol. 43: 607-20 (2000), Stals and Inze, Trends Plant Sci. 6:359-64 (2001), and John et al., Protoplasma 216: 119-42 (2001).

The are five subclasses of CDK's, each having a different cyclin binding consensus sequence. In CDK type A the cyclin binding consensus sequence is PSTAIRE (SEQ ID NO: 778). Id. The cyclin binding consensus sequence in CDK types B-1, B-2, and C are PPTTLRE (SEQ ID NO: 779), PPTALRE (SEQ ID NO: 780), and PITAIRE (SEQ ID NO: 781), respectively. Joubes et al, Plant Physiol, 126: 1403-15 (2001).

Cell cycle progression is directed, in part, by changes in CDK activity. CDK activity is modulated by a number of different cell cycle protein components, such as changes in the abundance of individual cyclins due to changing rates of biosynthesis and proteolysis. Fluctuations in cyclin concentrations result in commensurate fluctuations in CDK activity. Cyclin accumulation is especially important in terminating the G1 phase of the cell cycle because DNA replication is initiated by an increase in CDK activity.

Activation of CDK also requires phosphorylation of a threonine residue within the T-loop of CDK by a CDK-activating kinase (CAK). Umeda et al., Proc. Nat'l Acad. Sci. U.S.A. 97: 13396-400 (2000). It was suggested by Yamaguchi et al., Plant J. 24: 11-20 (2000), that cyclin H is a regulatory subunit of CAK. CDK activity is further regulated by interaction with a CDK regulatory subunit, a small (70-100 AA) protein involved in cell cycle regulation.

A cell must exit the cell cycle in order to commit to differentiation, senescence or apoptosis. This process involves the down-regulation of CDK activities. CDK inhibitors (CKI) are low molecular weight proteins, which are important for cell cycle regulation and development. CKIs bind stoichiometrically to CDK and down-regulate the activity of CDKs.

Many biochemical properties of ICK1, the first plant CKI to be identified from Arabidopsis thaliana , are known. Wang et al., Nature 386:451-2 (1997) Wang et al., Plant J. 24: 613-23 (2000). ICK1 is expressed at low levels in many tissue types, and there can be a threshold level of ICK1 that must be overcome before a cell can enter the cell cycle. Wang et al., Plant J. 24: 613-23 (2000). ICK1 is induced by the plant growth regulator abscisic acid (ABA), which inhibits cell division by blocking DNA replication. When the expression of ICK 1 increases, there is a corresponding decrease in Cdc2-like H1 histone activity. ICK1 has been shown to bind in vitro with the cyclins C2c2a and CycD3, and deletion experiments have identified different domain regions for these two interactions.

Altering the expression of CDK regulatory protein or a subunit thereof is known to cause changes in plant phenotype. Overexpression of the Arabidopsis CDK regulatory subunit, CKS1At, resulted in a reduction of leaf size, root growth rates and meristem size. Additionally, overexpression of CKS1At resulted in inhibition of cell-cycle progression, with an extension in the duration of the G1 and G2 phases of the cell cycle.

2. Cyclins

Cyclins are positive regulatory subunits of cyclin-dependent kinase (CDK) enzymes and are required for CDK activity. Fowler et al., Mol. Biotech. 10, 123, 126. Cyclins and CDK complexes provide temporal regulation of transition through the cell cycle. Evidence also suggests that cyclins provide spatial regulation of specific CDK activity, differentially targeting the cytoskeleton, spindle, phragmoplast, nuclear envelope, and chromosomes.

Plant cyclins are classified into five major groups: A, B, C, D, and H. Renaudin et al., Plant Mol. Biol. 32: 1003-18 (1996) and Yamaguchi et al., (supra 2000). Cyclins can be divided into mitotic cyclins (A and B) and G 1 cyclins.

The mitotic cyclins possess a consensus sequence (R-x-x-L-x-x-I-x-N. SEQ ID NO: 782) located at the N-terminal region, termed a destruction box, adjacent to a lysine-rich region. The destruction box and lysine-rich region target the mitotic cyclins for ubiquitin-dependent proteolysis during mitosis. Stals, supra at 361, and Fowler, supra at 126. The destruction box in A versus B cyclins differs slightly and this difference is thought to result in slightly different timing of degradation of A versus B cyclins. Fowler, supra at 126. A-type cyclins accumulate during the S, G2, and early M phase of the cell cycle, whereas B-type cyclins accumulate during the late G2 and early M phase. Mironov et al., Plant Cell 11: 509-22 (1999). Three subgroups of A-type cyclins are known in plants, but only one is known in animals. Cyclin A1 (cycA1;zm;1 from Zea cans ) is most concentrated during cytokinesis at the microtubule-containing phragmoplast. Expression of cyclin A2 is upregulated by auxins in roots, and by cytokinins in the shoot apex. Abrahams et al., Biochim. Biophys. Acta 28: 1-2 (2001).

D-type cyclins, of which five subgroups are known, are thought to control the progression through the G1 phase in response to growth factors and nutrients. Riou-Khamlichi et al., Mol. Cell Biol. 20: 4513-21 (2000). For example, the expression of D-type cyclins is upregulated by sucrose as shown by an increase in cycD2 mRNA 30 minutes after sucrose exposure, and an increase in cycD3 four hours after sucrose exposure. This timing corresponds to early G1-phase and late G1-phase, respectively. Cockcroft et al., Nature 405: 575-9 (2000). Furthermore, in Arabidopsis , a D3 cyclin was shown to be upregulated by the brassinosteroid, epi-brassinolide.

Cyclin D2 proteins bind with CDKA to produce an active complex, which binds to and phosphorylates retinoblastoma-related protein (Rb). This process is found in actively proliferating tissue, suggesting it plays an important function during late G1- and early S-phase. Three different D3-type cyclins are active during tomato fruit development. These proteins all contain a retinoblastoma binding motif and a PEST-destruction motif. There are differences in the spatial and temporal expression of these D3 cyclins, inferring different roles during fruit development.

Overexpression of cyclin D was shown to increase overall growth rate. Over-expression of cyclin D2 in tobacco increases causes shortening the G1-phase which producing a faster rate of cell cycling.

C- and H-type cyclins were characterized in poplar ( Populus tremula ×tremuloides ) and rice ( Oryza sativa ) but their exact function is still unclear. Putative cyclins with a lesser degree of peptide sequence conservation have also been identified. For example, Arabidopsis CycJ18 has only 20% identity with homologues over the cyclin box domain. CycJ18 is expressed predominantly in young seedlings. Arabidopsis F3O9.13 protein also has similarity to the cyclin family.

3. Histone Acetyltransferase/Deacetyltransferase

Histone acetyltransferase (HA) and histone deacetyltransferase (HAD) control the net level of acetylation of histones. Histone acetylation and deacetylation are thought to exert their regulatory effects on gene expression by altering the accessibility of nucleosomal DNA to DNA-binding transcriptional activators, other chromatin-modifying enzymes or multi-subunit chromatin remodeling complexes capable of displacing nucleosomes. Lusser et al., Nucleic Acids Res. 27: 4427-35 (1999). Therefore, in general, the HDAs are involved in the repression of gene expression, while HAs are correlated with gene activation.

HA effects acetylation at the ε-amino group of conserved lysine residues clustered near the amino terminus of core histones which up-regulates gene expression.

HDAs remove acetyl groups from the core histones of the nucleosome. There are numerous family members in the HDA group, many of which are conserved throughout evolution. Lechner et al., Biochim Biophys Acta 5:181-8 (1996). HDAs fimction as part of multi-protein complexes facilitating chromatin condensation.

HDAs and HAs recognize highly distinct acetylation patterns on the nucleosome. It is thought that different types of HDAs interact with specific regions of the genome, to influence gene silencing.

Schultz et al., Genes Dev. 15: 428-43 (2001), demonstrated that the superfamily of Kruppel-associated-box zinc finger proteins (KRAB-ZFPs) are linked to the nucleosome remodelling and histone deacetylation complex via the PHD (plant homeodomain) and bromodomains of co-repressor KAP-1, to form a cooperative unit that is required for transcriptional repression. A maize HDAC (HD2) has been identified that has no sequence homology to other eukaryotic HDACs, but instead contains sequence similarity to peptidyl-prolyl cis-trans isomerases (PPIases).

The effects of interfering with histone deacetylation are discussed in e.g. Tian and Chen, Proc. Nat'l Acad. Sci. USA 98: 200-5 (2001).

4. Peptidyl Prolyl Cis-Trans Isomerase

Peptidylprolyl isomerases (e.g., peptidylprolyl cis-trans isomerase, peptidyl-prolyl cis-trans isomerase, PPIase, rotamase, cyclophilin) catalyze the interconversion of peptide bonds between the cis and trans conformations at proline residues. Sheldon and Venis, Biochem J. 315: 965-70 (1996). This interconversion is thought to be the rate limiting step of protein folding. PPIases belong to a conserved family of proteins that are present in animals, fungi, bacteria and plants. PPIases are implicated in a number of responses including the response to environmental stress, calcium signals, transcriptional repression, cell cycle control, etc. Viaud, et al., Plant Cell 14: 917-30 (2002).

5. Retinoblastoma-Related Protein

Retinoblastoma (Rb)-related protein putatively regulates progression of the cell cycle through the G1 phase and into S phase. Xie et al., EMBO J. 15: 4900-8 (1996) and Ach et al., Mol. Cell Biol. 17: 5077-86 (1997).

Although Rb is well-characterized in mammalian systems, the role of Rb-related proteins in regulation of G1 phase progression and S phase entry is not well characterized in plants. It is known, however, that RB-related protein functions through its association with various other cellular proteins involved in cell cycle regulation, such as the cyclins, WD40 proteins, Soni et al., Plant. Cell. 7:85-103 (1995); Grafi et al., Proc. Natl. Acad. Sci. U.S.A. 93:8962 (1996); Ach et al., Plant Cell 9:1595-606 (1997); Umen and Goodenough, Genes Dev. 15:1652-61 (2001); Mariconti et al., J. Biol. Chem. 277:9911-9 (2002).

6. WD40 Repeat Protein

WD40 is a common repeating motif involved in many different protein-protein interactions. The WD40 domain is found in proteins having a wide variety of functions including adaptor/regulatory modules in signal transduction, pre-mRNA processing and cytoskeleton assembly. Goh et al., Eur. J. Biochem. 267: 434-49 (2000).

The WD40 domain, which is 40 residues long, typically contains a GH dipeptide 11-24 residues from the N-terminus and the WD dipeptide at the C-terminus. Id. Between the GH dipeptide and the WD dipeptide lies a conserved core which serves as a stable platform where proteins can bind either stably or reversibly. The core forms a propeller-like structure with several blades. Each blade is composed of a four-stranded anti-parallel β-sheet. Each WD40 sequence repeat forms the first three strands of one blade and the last strand in the next blade. The last C-terminal WD40 repeat completes the blade structure of the first WD40 repeat to create the closed ring propeller-structure. The residues on the top and bottom surface of the propeller are proposed to coordinate interactions with other proteins and/or small ligands.

Studies in yeast demonstrated that Cdc20, which contains the WD40 motif, is required for the proteolysis of mitotic cyclins. This process is mediated by an ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome. Following ubiquitination and proteolysis by the 26S proteasome, the cell can segregate chromosomes, and exit from mitosis. Cdc20 also contains a destruction-box domain.

7. WEE1-Like Protein

WEE1 controls the activity of cyclin-dependent kinases. WEE1 itself is a serine/threonine kinase. Sorrell et al., Planta 215: 518-22 (2002). The enzymatic activity of these protein kinases is controlled by phosphorylation of specific residues in the activation segment of the catalytic domain, sometimes combined with reversible conformational changes in the C-terminal autoregulatory tail. This process is conserved among eukaryotes, from fungi to animals and plants. Similarly, there is a high degree of homology between WEE1 proteins from various organisms. For example, there is 50% identity between the protein kinase domains of the human and maize WEE1 proteins.

Expression of WEE1 is shown to occur only in actively dividing tissues and is believed to inhibit cell division by acting as a negative regulator of mitosis. WEE1 is believed to prevent entry from G2 to M by protecting the nucleus from cytoplasmically-activated cyclin B1-complexed CDC2 before the onset of mitosis. For example, over-expression of AtWEE1 (from Arabidopsis ) and ZmWEE1 (from Zea cans ) in fission yeast inhibits cell division which results in elongated cells. Sun et al., Proc. Nat'l Acad. Sci . USA 96: 4180-5 (1999).

B. Expression Profiling and Microarray Analysis in Plant Development

The multigenic control of plant phenotype presents difficulties in determining the genes responsible for phenotypic determination. One major obstacle to identifying genes and gene expression differences that contribute to phenotype in plants is the difficulty with which the expression of more than a handful of genes can be studied concurrently. Another difficulty in identifying and understanding gene expression and the interrelationship of the genes that contribute to plant phenotype is the high degree of sensitivity to environmental factors that plants demonstrate.

There have been recent advances using genome-wide expression profiling. In particular, the use of DNA microarrays has been useful to examine the expression of a large number of genes in a single experiment. Several studies of plant gene responses to developmental and environmental stimuli have been conducted using expression profiling. For example, microarray analysis was employed to study gene expression during fruit ripening in strawberry, Aharoni et al., Plant Physiol. 129:1019-1031 (2002), wound response in Arabodopsis , Cheong et al., Plant Physiol. 129:661-7 (2002), pathogen response in Arabodopsis , Schenk et al., Proc. Nat'l Acad. Sci. 97:11655-60 (2000), and auxin response in soybean, Thibaud-Nissen et al., Plant Physiol. 132:118. Whetten et al., Plant Mol. Biol. 47:275-91 (2001) discloses expression profiling of cell wall biosynthetic genes in Pinus taeda L . using cDNA probes. Whetten et al. examined genes which were differentially expressed between differentiating juvenile and mature secondary xylem. Additionally, to determine the effect of certain environmental stimuli on gene expression, gene expression in compression wood was compared to normal wood. 156 of the 2300 elements examined showed differential expression. Whetten, supra at 285. Comparison of juvenile wood to mature wood showed 188 elements as differentially expressed. Id. at 286.

Although expression profiling and, in particular, DNA microarrays provide a convenient tool for genome-wide expression analysis, their use has been limited to organisms for which the complete genome sequence or a large cDNA collection is available. See Hertzberg et al., Proc. Nat'l Acad. Sci. 98:14732-7 (2001a), Hertzberg et al., Plant J, 25:585 (2001b). For example, Whetten, supra, states, “A more complete analysis of this interesting question awaits the completion of a larger set of both pine and poplar ESTs.” Whetten et al. at 286. Furthermore, microarrays comprising cDNA or EST probes may not be able to distinguish genes of the same family because of sequence similarities among the genes. That is, cDNAs or ESTs, when used as microarray probes, may bind to more than one gene of the same family.

Methods of manipulating gene expression to yield a plant with a more desirable phenotype would be facilitated by a better understanding of cell cycle gene expression in various types of plant tissue, at different stages of plant development, and upon stimulation by different environmental cues. The ability to control plant architecture and agronomically important traits would be improved by a better understanding of how cell cycle gene expression effects formation of plant tissues, how cell cycle gene expression causes plant cells to enter or exit cell division, and how plant growth and the cell cycle are connected. Among the large number of genes, the expression of which can change during development of a plant, only a fraction are likely to effect phenotypic changes during any given stage of the plant development.

Accordingly, there is a need for tools and methods useful in determining the changes in the expression of cell cycle genes that occur during the plant cell cycle. There is also a need for polynucleotides useful in such methods. There is a further need for methods which can correlate changes in cell cycle gene expression to phenotype or stage of plant development. There is a further need for methods of identifying cell cycle genes and gene products that impact plant phenotype, and that can be manipulated to obtain a desired phenotype.

In one aspect, the present invention provides an isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof.

In another aspect, the present invention provides a DNA construct comprising at least one polynucleotide having the sequence of any one of SEQ ID NOs: 1-260 and conservative variants thereof.

Another aspect of the invention is a plant cell transformed with a DNA construct of comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof.

A further aspect of the invention is a transgenic plant comprising a plant cell transformed with a DNA construct comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof.

Another aspect of the invention is an isolated polynucleotide comprising a sequence encoding the catalytic or substrate-binding domain of a polypeptide selected from of any one of SEQ ID NOs: 261-520, wherein the polynucleotide encodes a polypeptide having the activity of said polypeptide selected from any one of SEQ ID NOs: 261-520.

A further aspect of the invention is a method of making a transformed plant comprising transforming a plant cell with a DNA construct comprising at least one polynucleotide having the sequence of any of SEQ ID NOs: 1-260; and culturing the transformed plant cell under conditions that promote growth of a plant.

In another aspect, the invention provides a wood obtained from a transgenic tree.

In a further aspect, the invention provides a wood pulp obtained from a transgenic tree which has been transformed with the DNA construct of the invention.

Another aspect of the invention is a method of making wood, comprising transforming a plant with a DNA construct comprising a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof; culturing the transformed plant under conditions that promote growth of a plant; and obtaining wood from the plant.

The invention further provides a method of making wood pulp, comprising transforming a plant with a DNA construct comprising a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof; culturing the transformed plant under conditions that promote growth of a plant; and obtaining wood pulp from the plant.

In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence encoded by the isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof.

The invention also provides, an isolated polypeptide comprising an amino acid sequence selected from the group consisting of 261-520.

The invention further provides a method of altering a plant phenotype of a plant, comprising altering expression in the plant of a polypeptide encoded by any one of SEQ ID NOs: 1-260.

In another aspect, the invention provides a polynucleotide comprising a nucleic acid selected from the group comprising of SEQ ID NOs: 521-772.

An aspect of the invention is a method of correlating gene expression in two different samples, comprising detecting a level of expression of one or more genes encoding a product encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof in a first sample; detecting a level of expression of the one or more genes in a second sample; comparing the level of expression of the one or more genes in the first sample to the level of expression of the one or more genes in the second sample; and correlating a difference in expression level of the one or more genes between the first and second samples.

A further aspect of the invention is a method of correlating the possession of a plant phenotype to the level of gene expression in the plant of one or more genes comprising detecting a level of expression of one or more genes encoding a product encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof in a first plant possessing a phenotype; detecting a level of expression of the one or more genes in a second plant lacking the phenotype; comparing the level of expression of the one or more genes in the first plant to the level of expression of the one or more genes in the second plant; and correlating a difference in expression level of the one or more genes between the first and second plants to possession of the phenotype.

In a further aspect, the invention provides a method of correlating gene expression to a stage of the cell cycle, comprising detecting a level of expression of one or more genes encoding a product encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 and conservative variants thereof in a first plant cell in a first stage of the cell cycle; detecting a level of expression of the one or more genes in a second plant cell in a second, different stage of the cell cycle; comparing the level of the expression of the one or more genes in the first plant cells to the level of expression of the one or more genes in the second plants cells; and correlating a difference in expression level of the one or more genes between the first and second samples to the first or second stage of the cell cycle.

An aspect of the invention is a combination for detecting expression of one or more genes, comprising two or more oligonucleotides, wherein each oligonucleotide is capable of hybridizing to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260.

Another aspect of the invention is a combination for detecting expression of one or more genes, comprising two or more oligonucleotides, wherein each oligonucleotide is capable of hybridizing to a nucleic acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260.

The invention further provides a microarray comprising a combination for detecting expression of one or more genes, comprising two or more oligonucleotides, wherein each oligonucleotide is capable of hybridizing to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 or wherein each oligonucleotide is capable of hybridizing to a nucleic acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260, wherein each of said two or more oligonucleotides occupies a unique location on said solid support.

In another aspect, the invention provides a method for detecting one or more genes in a sample, comprising contacting the sample with two or more oligonucleotides, wherein each oligonucleotide is capable of hybridizing to a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 under standard hybridization conditions; and detecting the one or more genes of interest which are hybridized to the one or more oligonucleotides.

The invention also provides a method for detecting one or more nucleic acid sequences encoded by one or more genes in a sample, comprising contacting the sample with two or more oligonucleotides, wherein each oligonucleotide is capable of hybridizing to a nucleic acid sequence encoded by a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-260 under standard hybridization conditions; and detecting the one or more nucleic acid sequences which are hybridized to the one or more oligonucleotides.

The invention further provides a kit for detecting gene expression comprising the microarray of the invention together with one or more buffers or reagents for a nucleotide hybridization reaction.

Other features, objects, and advantages of the present invention are apparent from the detailed description that follows. It should be understood, however, that the detailed description, while indicating preferred embodiments of the invention, are given by way of illustration only, not limitation. Various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from the detailed description.

FIG. 1 : Exemplary microarray sampling parameters.

FIG. 2 : Plasmid map for pWVK202.

FIG. 3 : Plasmid map for pGrowth14.

FIG. 4 : Plasmid map for pGrowth15.

FIG. 5 : Plasmid map for pGrowth16.

FIG. 6 : Plasmid map for pGrowth18.

FIG. 7 : Plasmid map for pGrowth19.

FIG. 8 : Plasmid map for pGrowth20.

Table 1: shows genes having greater than doubled signal with any one sample as compared to the mean signal of the other three samples.

Table 2: identifies plasmid(s), genes, and Genesis ID numbers for constructs described in Example 17.

Table 3: Rooting medium for Populus deltoids.

Table 4: pGrowth information.

Table 5: shows genes having greater than doubled signal with any one sample as compared to the mean signal of the other three samples.

Table 6: Differentially expressed cDNAs.

Table 7: Consensus ID information.

Table 8: pGrowth information.

Table 9 : Eucalyptus grandis cell cycle genes and proteins.

Table 10 : Pinus radiata cell cycle genes and proteins.

Table 11: Annotated peptide sequences of the present invention.

Table 12 : Eucalyptus in silico data.

Table 13: Pine in silico data.

Table 14: Oligo table.

Table 15: Peptide table.

Table 16: BLAST sequence alignment table.

The inventors have discovered novel isolated cell cycle genes and polynucleotides useful for identifying the multigenic factors that contribute to a phenotype and for manipulating gene expression to affect a plant phenotype. These genes, which are derived from plants of commercially important forestry genera, pine and eucalyptus, are involved in the plant cell cycle and are, at least in part, responsible for expression of phenotypic characteristics important in commercial wood, such as stiffness, strength, density, fiber dimensions, coarseness, cellulose and lignin content, and extractives content. Generally speaking, the genes and polynucleotides encode a protein which can be a cyclin, cyclin dependent kinase, cyclin dependent kinase inhibitor, histone acetyltransferase, histone deacetylase, peptidyl-prolyl cis-trans isomerase, retinoblastoma-related protein, WEE1-like protein, or WD40 repeat protein, or a catalytic domain thereof, or a polypeptide having the same function, and the invention further includes such proteins and polypeptides.

The methods of the present invention for selecting cell cycle gene sequences to target for manipulation will permit better design and control of transgenic plants with more highly engineered phenotypes. The ability to control plant architecture and agronomically important traits in commercially important forestry species will be improved by the information obtained from the methods, such as which genes affect which phenotypes, which genes affect entry into which stage of the cell cycle, which genes are active in which stage of plant development, and which genes are expressed in which tissue at a given point in the cell cycle or plant development.

Unless indicated otherwise, all technical and scientific terms are used herein in a manner that conforms to common technical usage. Generally, the nomenclature of this description and the described laboratory procedures, including cell culture, molecular genetics, and nucleic acid chemistry and hybridization, respectively, are well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, oligonucleotide synthesis, cell culture, tissue culture, transformation, transfection, transduction, analytical chemistry, organic synthetic chemistry, chemical syntheses, chemical analysis, and pharmaceutical formulation and delivery. Generally, enzymatic reactions and purification and/or isolation steps are performed according to the manufacturers' specifications. Absent an indication to the contrary, the techniques and procedures in question are performed according to conventional methodology disclosed, for example, in Sambrook et al., M OLECULAR C LONING A L ABORATORY M ANUAL, 2d ed. (Cold Spring Harbor Laboratory Press, 1989), and C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY , John Wiley & Sons, 1989). Specific scientific methods relevant to the present invention are discussed in more detail below. However, this discussion is provided as an example only, and does not limit the manner in which the methods of the invention can be carried out.

A. Plant Cell Cycle Genes and Proteins

1. Cell Cycle Genes, Polynucleotide and Polypeptide Sequences

One aspect of the present invention relates to novel plant cell cycle genes and polypeptides encoded by such genes. As used herein, the term “plant cell cycle genes” refers to genes encoding proteins that function during the plant cell cycle, and the term “plant cell cycle proteins” refers to proteins that function during the plant cell cycle. There are several known families of plant cell cycle proteins, including cyclin, cyclin dependent kinase, cyclin dependent kinase inhibitor, histone acetyltransferase, histone deacetylase, peptidyl-prolyl cis-trans isomerase, retinoblastoma-related protein, WEE1-like protein, and WD40 repeat protein. Although there is significant sequence homology within each gene and protein family, each member of each family can display different biochemical properties and altering the expression of at least one of these genes can result in a different plant phenotype.

The present invention provides novel plant cell cycle genes and polynucleotides and novel cell cycle proteins and polypeptides. In accordance with one embodiment of the invention, the novel plant cell cycle genes are the same as those expressed in a wild-type plant of a species of Pinus or Eucalyptus . Exemplary novel plant cell cycle gene sequences of the invention are set forth in Tables 9 and 10, which depict Eucalyptus grandis sequences and Pinus radiata sequences, respectively. Corresponding gene products, i.e., oligonucleotides and polypeptides, are also listed in Tables 14, 15, and 16. The Sequence Listing in APPENDIX 1 provides the sequences of these aspects of the invention.

The sequences of the invention have cell cycle activity and encode proteins that are active in the cell cycle, such as proteins of the cell cycle families discussed above. As discussed in more detail below, manipulation of the expression of the cell cycle genes and polynucleotides, or manipulation of the activity of the encoded proteins and polypeptides, can result in a transgenic plant with a desired phenotype that differs from the phenotype of a wild-type plant of the same species.

Throughout this description, reference is made to cell cycle gene products. As used herein, a “cell cycle gene product” is a product encoded by a cell cycle gene, and includes both nucleotide products, such as RNA, and amino acid products, such as proteins and polypeptides. Examples of specific cell cycle genes of the invention include SEQ ID NOs: 1-260. Examples of specific cell cycle gene products of the invention include products encoded by any one of SEQ ID NOs: 1-260. Reference also is made herein to cell cycle proteins and cell cycle polypeptides. Examples of specific cell cycle proteins and polypeptides of the invention include polypeptides encoded by any of SEQ ID NOs: 1-260 or polypeptides comprising the amino acid sequence of any of SEQ ID NOs: 261-520. One aspect of the invention is directed to a subset of these cell cycle genes and cell cycle gene products, namely SEQ ID NOs: 1-12, 14-58, 60-62, 64-70, 72-75, 77-83, 85-86, 88-91, 93-119, 121-130, 132-148, 150-156, 158-191, 193-207, 209-218, 220-221, 223-231, 233-237, their respective conservative variants (as that term is defined below), and the nucleotide and amino acid products encoded thereby. Another aspect of the invention is directed to a subset of the cell cycle genes and cell cycle gene products, namely SEQ ID NOs: 1-12, 14, 16-26, 30-37, 40-41, 43-76, 78-103, 106, 108-113, 116-121, 124-125, 128-147, 150-152, 154-155, 161-162, 164-172, 174, 177-183, 185-191, 193-197, 200-204, 208-213, 215-218, 220-221, 223-231, and 233-234 their respective conservative variants, and the nucleotide and amino acid products encoded thereby. A further aspect of the invention is directed to a subset of the cell cycle genes and cell cycle gene products, namely SEQ ID NOs: 1-12, 14, 16-26, 30-37, 40-41, 43-58, 60-62, 64-70, 72-75, 78-83, 85-86, 88-91, 93-103, 106, 108-113, 116-119, 121, 124-125, 128-130, 132-147, 150-152, 154-155, 161-162, 164-172, 174, 177-183, 185-191, 193-197, 200-204, 209-213, 215-218, 220-221, 223-231, and 233-234 their respective conservative variants, and the nucleotide and amino acid products encoded thereby.

The present invention also includes sequences that are complements, reverse sequences, or reverse complements to the nucleotide sequences disclosed herein.

The present invention also includes conservative variants of the sequences disclosed herein. The term “variant,” as used herein, refers to a nucleotide or amino acid sequence that differs in one or more nucleotide bases or amino acid residues from the reference sequence of which it is a variant.

Thus, in one aspect, the invention includes conservative variant polynucleotides. As used herein, the term “conservative variant polynucleotide” refers to a polynucleotide that hybridizes under stringent conditions to an oligonucleotide probe that, under comparable conditions, binds to the reference gene the conservative variant is a variant of. Thus, for example, a conservative variant of SEQ ID NO: 1 hybridizes under stringent conditions to an oligonucleotide probe that, under comparable conditions, binds to SEQ ID NO: 1. One aspect of the invention provides conservative variant polynucleotides that exhibit at least about 75% sequence identity to their respective reference sequences.

“Sequence identity” has an art-recognized meaning and can be calculated using published techniques. See C OMPUTATIONAL M OLECULAR B IOLOGY , Lesk, ed. (Oxford University Press, 1988), B IOCOMPUTING : I NFORMATICS A ND G ENOME P ROJECTS , Smith, ed. (Academic Press, 1993), C OMPUTER A NALYSIS O F S EQUENCE D ATA , P ART I, Griffin & Griffin, eds., (Humana Press, 1994), S EQUENCE A NALYSIS I N M OLECULAR B IOLOGY , Von Heinje ed., Academic Press (1987), S EQUENCE A NALYSIS P RIMER , Gribskov & Devereux, eds. (Macmillan Stockton Press, 1991), and Carillo & Lipton, SIAM J. Applied Math. 48: 1073 (1988). Methods commonly employed to determine identity or similarity between two sequences include but are not limited to those disclosed in G UIDE T O H UGE C OMPUTERS , Bishop, ed, (Academic Press, 1994) and Carillo & Lipton, supra. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include but are not limited to the GCG program package (Devereux et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Mol. Biol. 215: 403 (1990)), and FASTDB (Brutlag et al., Comp. App. Biosci. 6: 237 (1990)).

The invention includes conservative variant polynucleotides having a sequence identity that is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% to any one of SEQ ID NOs: 1 to 237. In such variants, differences between the variant and the reference sequence can occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

Additional conservative variant polynucleotides contemplated by and encompassed within the present invention include polynucleotides comprising sequences that differ from the polynucleotide sequences of SEQ ID NO: 1-237, or complements, reverse complements or reverse sequences thereof, as a result of deletions and/or insertions totaling less than 10% of the total sequence length.

The invention also includes conservative variant polynucleotides that, in addition to sharing a high degree of similarity in their primary structure (sequence) to SEQ ID NOs: 1 to 260, have at least one of the following features: (i) they contain an open reading frame or partial open reading frame encoding a polypeptide having substantially the same functional properties in the cell cycle as the polypeptide encoded by the reference polynucleotide, or (ii) they have nucleotide domains or encoded protein domains in common. The invention includes conservative variants of SEQ ID NOs: 1-260 that encode proteins having the enzyme or biological activity or binding properties of the protein encoded by the reference polynucleotide. Such conservative variants are functional variants, in that they have the enzymatic or binding activity of the protein encoded by the reference polynucleotide.

In accordance with the invention, polynucleotide variants can include a “shuffled gene” such as those described in e.g. U.S. Pat. Nos. 6,500,639, 6,500,617 6,436,675, 6,379,964, 6,352,859 6,335,198 6,326,204, and 6,287,862. A variant of a nucleotide sequence of the present invention also can be a polynucleotide modified as disclosed in U.S. Pat. No. 6,132,970, which is incorporated herein by reference.

In accordance with one embodiment, the invention provides a polynucleotide that encodes a cell cycle protein from one of the following families: cyclin, cyclin dependent kinase, cyclin dependent kinase inhibitor, histone acetyltransferase, histone deacetylase, peptidyl-prolyl cis-trans isomerase, retinoblastoma-related protein, WEE1-like protein, or WD40 repeat protein. SEQ ID NOs: 1-260 provide examples of such polynucleotides.

In accordance with another embodiment, a polynucelotide of the invention encodes the catalytic or protein binding domain of a polypeptide encoded by any of SEQ ID NOs: 1-260 or of a polypeptide comprising any of SEQ ID NOs: 261-520. The catalytic and protein binding domains of the cell cycle proteins of the invention are known in the art. The conserved sequences of these proteins are shown in Entries 1-248 as underlined, bold, and/or italicized text.

The invention also encompasses as conservative variants polynucleotides that differ from the sequences discussed above but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide which is the same as that encoded by a polynucleotide of the present invention. The invention also includes as conservative variants polynucleotides comprising sequences that differ from the polynucleotide sequences discussed above as a result of substitutions that do not affect the amino acid sequence of the encoded polypeptide sequence, or that result in conservative substitutions in the encoded polypeptide sequence.

The present invention also includes an isolated polypeptide encoded by a polynucleotide comprising any of SEQ ID NOs: 1-260 or any of the conservative variants thereof discussed above. The invention also includes polypeptides comprising SEQ ID NOs: 261-520 and conservative variants of these polypeptides. Another aspect of the invention include polypeptides comprising SEQ ID NOs: 261-272, 274-318, 320-322, 324-330, 332-335, 337-343, 345-346, 348-351, 353-379, 381-390, 392-408, 410-416, 418-451, 453-467, 469-478, 480-481, 483-491, and 493-494 and conservative variants thereof. A further aspect of the invention includes polypeptides comprising SEQ ID NOs: 261-272, 274, 276-286, 289, 290-297, 300-301, 303-345, 347-363, 366, 368-373, 376-381, 384-385, 388-407, 410-412, 414-415, 420-422, 424-432, 434, 437-443, 445-451, 453-457, 460-464, 468-473, and 475-494 and conservative variants thereof. Another aspect of the invention includes polypeptides comprising SEQ ID NOs: 261-272, 274, 276-286, 290-297, 300-301, 303-318, 320-322, 324-330, 332-335, 337-343, 345, 348-351, 353-363, 366, 368-373, 376-381, 384-385, 388-390, 392-407, 410-412, 414-415, 421-422, 424-432, 434, 437-443, 445-451, 453-457, 460-464, 469-473, 475-478, 480-481, 483-491, and 493-494 and conservative variants thereof.

In accordance with the invention, a variant polypeptide or protein refers to an amino acid sequence that is altered by the addition, deletion or substitution of one or more amino acids.

The invention includes conservative variant polypeptides. As used herein, the term “conservative variant polypeptide” refers to a polypeptide that has similar structural, chemical or biological properties to the protein it is a conservative variant of. Guidance in determining which amino acid residues can be substituted, inserted, or deleted can be found using computer programs well known in the art such as Vector NTI Suite (InforMax, MD) software. In one embodiment of the invention, conservative variant polypeptides that exhibit at least about 75% sequence identity to their respective reference sequences.

Conservative variant protein includes an “isoform” or “analog” of the polypeptide. Polypeptide isoforms and analogs refers to proteins having the same physical and physiological properties and the same biological function, but whose amino acid sequences differs by one or more amino acids or whose sequence includes a non-natural amino acid.

Polypeptides comprising sequences that differ from the polypeptide sequences of SEQ ID NO: 261-520 as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by and encompassed within the present invention.

One aspect of the invention provides conservative variant polypeptides that have the same function in the cell cycle as the proteins of which they are variants, as determined by one or more appropriate assays, such as those described below. The invention includes variant polypeptides that function as cell cycle proteins, such as those having the biological activity of cyclin, cyclin dependent kinase, cyclin dependent kinase inhibitor, histone acetyltransferase, histone deacetylase, peptidyl-prolyl cis-trans isomerase, retinoblastoma-related protein, WEE1-like protein, and WD40 repeat protein, and are thus capable of modulating the cell cycle in a plant. As discussed above, the invention includes variant polynucleotides that encode polypeptides that function as cell cycle proteins.

The activities and physical properties of cell cycle proteins can be examined using any method known in the art. The following examples of assay methods are not exhaustive and are included to provide some guidance in examining the activity and distinguishing protein characteristics of cell cycle protein variants.

CDK activity can be assessed using roscovitine as described in Yamaguchi et al., Proc. Natl. Acad. Sci. U.S.A. 100:8019 (2003). CDK histone kinase activity can be assayed using autoradiography to detect histone H1 phosphorylation by CDK as described in Joubes et al., Plant Physiol. 121:857 (1999).

CKI activity can be assayed using a variation of the method described in Zhou et al., Planta. 6:604 (2003). The modified method can employ co-transformation or subsequent transformations to identify the interaction of CKI and cyclins in vivo. For example, in the first transformation pine tissue can be transformed using the method described in U.S. Patent Application Publication No. 2002/0100083 using geneticin selection to obtain transgenic plants possessing cycD3 and cdc2a homologs. The second transformation can be performed using alpha-methyltryptophan as a selectable marker to obtain transformants having an ICK1 homologue as described in U.S. Provisional Application No. 60/476,189. Tissue capable of growing on both on geneticin and on alpha-methyltryptophan contains the ICK1 homologue and the cycD3 and cdc2a homologues. The CKI activity is determined by comparison of the phenotype of transformants having the cycD3 and cdc2a homologues to the transformants having ICK1 homologue and the cycD3 and cdc2a homologs.

Histone deacetylase activity can be assessed by complementation of the Arabidopsis mutants described in Tian et al., Genetics 165:399 (2003). Histone acetyltransferase activity can be assayed using anacardic acid as described in Balasubramanyam et al., J. Biol. Chem. 278:19134 (2003). Histone acetyltransferase also can be assayed using trichostatin A-treated plant lines as is described in Bhat et al., Plant J. 33:455 (2003). The plant lines described in Bhat et al., supra, also can be used to assay retinoblastoma-related proteins using the co-precipitation method described in Rossi et al., Plant Mol. Biol. 51:401 (2003).

Peptidyl-prolyl isomerase can be assayed as described in Edvardsson et al., FEBS Lett. 542:137 (2003). WD40 proteins can be evaluated based on the possession of the WD40 motif as well as their ability to interact with cdc2. WEE-1 can be assayed using any kinase activity assay known in the art.

2. Methods of Using Cell Cycle Genes, Polynucleotide and Polypeptide Sequences

The present invention provides methods of using plant cell cycle genes and conservative variants thereof. The invention includes methods and constructs for altering expression of plant cell cycle genes and/or gene products for purposes including, but not limited to (i) investigating function during the cell cycle and ultimate effect on plant phenotype and (ii) to effect a change in plant phenotype. For example, the invention includes methods and tools for modifying wood quality, fiber development, cell wall polysaccharide content, fruit ripening, and plant growth and yield by altering expression of one or more plant cell cycle genes.

The invention comprises methods of altering the expression of any of the cell cycle genes and variants discussed above. Thus, for example, the invention comprises altering expression of a cell cycle gene present in the genome of a wild-type plant of a species of Eucalyptus or Pinus . In one embodiment, the cell cycle gene comprises a nucleotide sequence selected from SEQ ID NOs: 1-260, from the subset thereof comprising SEQ ID NOs: 1-12, 14-58, 60-62, 64-70, 72-75, 77-83, 85-86, 88-91, 93-119, 121-130, 132-148, 150-156, 158-191, 193-207, 209-218, 220-221, 223-231, and 233 -237, from the subset thereof comprising SEQ ID NOs: 1-12, 14, 16-26, 30-37, 40-41, 43-76, 78-103, 106, 108-113, 116-121, 124-125, 128-147, 150-152, 154-155, 161-162, 164-172, 174, 177-183, 185-191, 193-197, 200-204, 208-213, and 215-234, from the subset thereof comprising SEQ ID NOs: 1-12, 14, 16-26, 30-37, 40-41, 43-58, 60-62, 64-70, 72-75, 78-83, 85-86, 88-91, 93-103, 106, 108-113, 116-119, 121, 124-125, 128-130, 132-147, 150-152, 154-155, 161-162, 164-172, 174, 177-183, 185-191, 193-197, 200-204, 209-213, 215-218, 220-221, 223-231, and 233-234, or the conservative variants thereof, as discussed above.

Techniques which can be employed in accordance with the present invention to alter gene expression, include, but are not limited to: (i) over-expressing a gene product, (ii) disrupting a gene's transcript, such as disrupting a gene's mRNA transcript; (iii) disrupting the function of a polypeptide encoded by a gene, or (iv) disrupting the gene itself. Over-expression of a gene product, the use of antisense RNAs, ribozymes, and the use of double-stranded RNA interference (dsRNAi) are valuable techniques for discovering the functional effects of a gene and for generating plants with a phenotype that is different from a wild-type plant of the same species.

Over-expression of a target gene often is accomplished by cloning the gene or cDNA into an expression vector and introducing the vector into recipient cells. Alternatively, over-expression can be accomplished by introducing exogenous promoters into cells to drive expression of genes residing in the genome. The effect of over-expression of a given gene on cell function, biochemical and/or physiological properties can then be evaluated by comparing plants transformed to over-express the gene to plants that have not been transformed to over-express the gene.

Antisense RNA, ribozyme, and dsRNAi technologies typically target RNA transcripts of genes, usually mRNA. Antisense RNA technology involves expressing in, or introducing into, a cell an RNA molecule (or RNA derivative) that is complementary to, or antisense to, sequences found in a particular mRNA in a cell. By associating with the mRNA, the antisense RNA can inhibit translation of the encoded gene product. The use of antisense technology to reduce or inhibit the expression of specific plant genes has been described, for example in European Patent Publication No. 271988, Smith et al., Nature, 334:724-726 (1988); Smith et. al., Plant Mol. Biol., 14:369-379 (1990)).

A ribozyme is an RNA that has both a catalytic domain and a sequence that is complementary to a particular mRNA. The ribozyme functions by associating with the mRNA (through the complementary domain of the ribozyme) and then cleaving (degrading) the message using the catalytic domain.

RNA interference (RNAi) involves a post-transcriptional gene silencing (PTGS) regulatory process:, in which the steady-state level of a specific mRNA is reduced by sequence-specific degradation of the transcribed, usually fully processed mRNA without an alteration in the rate of de novo transcription of the target gene itself. The RNAi technique is discussed, for example, in Elibashir, et al., Methods Enzymol. 26: 199 (2002); McManus & Sharp, Nature Rev. Genetics 3: 737 (2002); PCT application WO 01/75164; Martinez et al., Cell 110: 563 (2002); Elbashir et al., supra; Lagos-Quintana et al., Curr. Biol. 12: 735 (2002); Tuschl et al., Nat. Biotechnol. 20:446 (2002); Tuschl, Chembiochem. 2: 239 (2001); Harborth et al., J. Cell Sci. 114: 4557 (2001); et al, EMBO J. 20:6877 (2001); Lagos-Quintana et al., Science. 294: 8538 (2001); Hutvagner et al., loc cit, 834; Elbashir et al., Nature. 411: 494 (2001).

The present invention provides a DNA construct comprising at least one polynucleotide of SEQ ID NOs: 1-260 or conservative variants thereof, such as the conservative variants discussed above. Any method known in the art can be used to generate the DNA constructs of the present invention. See, e.g. Sambrook et al., supra.

The invention includes DNA constructs that optionally comprise a promoter. Any suitable promoter known in the art can be used. A promoter is a nucleic acid, preferably DNA, that binds RNA polymerase and/or other transcription regulatory elements. As with any promoter, the promoters of the invention facilitate or control the transcription of DNA or RNA to generate an mRNA molecule from a nucleic acid molecule that is operably linked to the promoter. The RNA can encode a protein or polypeptide or can encode an antisense RNA molecule or a molecule useful in RNAi. Promoters useful in the invention include constitutive promoters, inducible promoters, temporally regulated promoters and tissue-preferred promoters.

Examples of useful constitutive plant promoters include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (Odel et al. Nature 313:810(1985)); the nopaline synthase promoter (An et al. Plant Physiol. 88:547 (1988)); and the octopine synthase promoter (Fromm et al., Plant Cell 1: 977 (1989)). It should be noted that, although the CaMV 35S promoter is commonly referred to as a constitutive promoter, some tissue preference can be seen. The use of CaMV 35S is envisioned by the present invention, regardless of any tissue preference which may be exhibited during use in the present invention.

Inducible promoters regulate gene expression in response to environmental, hormonal, or chemical signals. Examples of hormone inducible promoters include auxin-inducible promoters (Baumann et al. Plant Cell 11:323-334(1999)), cytokinin-inducible promoters (Guevara-Garcia, Plant Mol. Biol. 38:743-753(1998)), and gibberellin-responsive promoters (Shi et al. Plant Mol. Biol. 38:1053-1060(1998)). Additionally, promoters responsive to heat, light, wounding, pathogen resistance, and chemicals such as methyl jasmonate or salicylic acid, can be used in the DNA constructs and methods of the present invention.

Tissue-preferred promoters allow for preferred expression of polynucleotides of the invention in certain plant tissue. Tissue-preferred promoters are also useful for directing the expression of antisense RNA or siRNA in certain plant tissues, which can be useful for inhibiting or completely blocking the expression of targeted genes as discussed above. As used herein, vascular plant tissue refers to xylem, phloem or vascular cambium tissue. Other preferred tissue includes apical meristem, root, seed, and flower. In one aspect, the tissue-preferred promoters of the invention are either “xylem-preferred,” “cambium-preferred” or “phloem-preferred,” and preferentially direct expression of an operably linked nucleic acid sequence in the xylem, cambium or phloem, respectively. In another aspect, the DNA constructs of the invention comprise promoters that are tissue-specific for xylem, cambium or phloem, wherein the promoters are only active in the xylem, cambium or phloem.

A vascular-preferred promoter is preferentially active in any of the xylem, phloem or cambium tissues, or in at least two of the three tissue types. A vascular-specific promoter is specifically active in any of the xylem, phloem or cambium, or in at least two of the three. In other words, the promoters are only active in the xylem, cambium or phloem tissue of plants. Note, however, that because of solute transport in plants, a product that is specifically or preferentially expressed in a tissue may be found elsewhere in the plant after expression has occurred.

In another embodiment, the promoter is under temporal regulation, wherein the ability of the promoter to initiate expression is linked to factors such as the stage of the cell cycle or the stage of plant development. For example, the promoter of a cyclin D2 gene may be expressed only during the G1 and early S-phase, and the promoters of particular cyclin genes may be expressed only within the primary vascular poles of the developing seedling.

Additionally, the promoters of particular cell cycle genes may be expressed only within the cambium in developing secondary vasculature. Within the cambium, particular cell cycle gene promoters may be expressed exclusively in the stem or in the root. Moreover, the cell cycle promoters may be expressed only in the spring (for early wood formation) or only in the summer.

A promoter may be operably linked to the polynucleotide. As used in this context, operably linked refers to linking a polynucleotide encoding a structural gene to a promoter such that the promoter controls transcription of the structural gene. If the desired polynucleotide comprises a sequence encoding a protein product, the coding region can be operably linked to regulatory elements, such as to a promoter and a terminator, that bring about expression of an associated messenger RNA transcript and/or a protein product encoded by the desired polynucleotide. In this instance, the polynucleotide is operably linked in the 5′- to 3′-orientation to a promoter and, optionally, a terminator sequence.

Alternatively, the invention provides DNA constructs comprising a polynucleotide in an “antisense” orientation, the transcription of which produces nucleic acids that can form secondary structures that affect expression of an endogenous cell cycle gene in the plant cell. In another variation, the DNA construct may comprise a polynucleotide that yields a double-stranded RNA product upon transcription that initiates RNA interference of a cell cycle gene with which the polynucleotide is associated. A polynucleotide of the present invention can be positioned within a t-DNA, such that the left and right t-DNA border sequences flank or are on either side of the polynucleotide.

It should be understood that the invention includes DNA constructs comprising one or more of any of the polynucleotides discussed above. Thus, for example, a construct may comprise a t-DNA comprising one, two, three, four, five, six, seven, eight, nine, ten, or more polynucleotides.

The invention also includes DNA constructs comprising a promoter that includes one or more regulatory elements. Alternatively, the invention includes DNA constructs comprising a regulatory element that is separate from a promoter. Regulatory elements confer a number of important characteristics upon a promoter region. Some elements bind transcription factors that enhance the rate of transcription of the operably linked nucleic acid. Other elements bind repressors that inhibit transcription activity. The effect of transcription factors on promoter activity can determine whether the promoter activity is high or low, i.e. whether the promoter is “strong” or “weak.”

A DNA construct of the invention can include a nucleotide sequence that serves as a selectable marker useful in identifying and selecting transformed plant cells or plants. Examples of such markers include, but are not limited to, a neomycin phosphotransferase (nptII) gene (Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985)), which confers kanamycin resistance. Cells expressing the nptII gene can be selected using an appropriate antibiotic such as kanamycin or G418. Other commonly used selectable markers include a mutant EPSP synthase gene (Hinchee et al., Bio/Technology 6:915-922 (1988)), which confers glyphosate resistance; and a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulphonylurea resistance (European Patent Application 154,204, 1985).

The present invention also includes vectors comprising the DNA constructs discussed above. The vectors can include an origin of replication (replicons) for a particular host cell. Various prokaryotic replicons are known to those skilled in the art, and function to direct autonomous replication and maintenance of a recombinant molecule in a prokaryotic host cell.

In one embodiment, the present invention utilizes a pWVR8 vector as described in U.S. Application No. 60/476,222, filed Jun. 6, 2003, or pART27 as described in Gleave, Plant Mol. Biol, 20:1203-27 (1992).

The invention also provides host cells which are transformed with the DNA constructs of the invention. As used herein, a host cell refers to the cell in which a polynucleotide of the invention is expressed. Accordingly, a host cell can be an individual cell, a cell culture or cells that are part of an organism. The host cell can also be a portion of an embryo, endosperm, sperm or egg cell, or a fertilized egg. In one embodiment, the host cell is a plant cell.

The present invention further provides transgenic plants comprising the DNA constructs of the invention. The invention includes transgenic plants that are angiosperms or gymnosperms. The DNA constructs of the present invention can be used to transform a variety of plants, both monocotyledonous (e.g grasses, corn, grains, oat, wheat and barley), dicotyledonous (e.g., Arabidopsis , tobacco, legumes, alfalfa, oaks, eucalyptus , maple), and Gymnosperms (e.g., Scots pine; see Aronen, Finnish Forest Res. Papers , Vol. 595, 1996), white spruce (Ellis et al., Biotechnology 11:84-89, 1993), and larch (Huang et al., In Vitro Cell 27:201-207, 1991).

The plants also include turfgrass, wheat, maize, rice, sugar beet, potato, tomato, lettuce, carrot, strawberry, cassava, sweet potato, geranium, soybean, and various types of woody plants. Woody plants include trees such as palm oak, pine, maple, fir, apple, fig, plum and acacia. Woody plants also include rose and grape vines.

In one embodiment, the DNA constructs of the invention are used to transform woody plants, i.e., trees or shrubs whose stems live for a number of years and increase in diameter each year by the addition of woody tissue. The invention includes methods of transforming plants including eucalyptus and pine species of significance in the commercial forestry industry such as plants selected from the group consisting of Eucalyptus grandis and its hybrids, and Pinus taeda , as well as the transformed plants and wood and wood pulp derived therefrom. Other examples of suitable plants include those selected from the group consisting of Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus contorta, Pinus coulteri, Pinus echinata, Pinus eldarica, Pinus ellioti, Pinusjeffreyi, Pinus lambertiana, Pinus massoniana, Pinus monticola, Pinus nigra, Pinus palustris, Pinus pinaster, Pinus ponderosa, Pinus radiata, Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, Pinus virginiana, Abies amabilis, Abies balsamea, Abies concolor, Abies grandis, Abies lasiocarpa, Abies magnifica, Abies procera, Chamaecyparis lawsoniona, Chamaecyparis nootkatensis, Chamaecyparis thyoides, Juniperus virginiana, Larix decidua, Larix laricina, Larix leptolepis, Larix occidentalis, Larix siberica, Libocedrus decurrens, Picea abies, Picea engelmanni, Picea glauca, Picea mariana, Picea pungens, Picea rubens, Picea sitchensis, Pseudotsuga menziesii, Sequoia gigantea, Sequoia sempervirens, Taxodium distichum, Tsuga canadensis, Tsuga heterophylla, Tsuga mertensiana, Thuja occidentalis, Thuja plicata, Eucalyptus alba, Eucalyptus bancroflii, Eucalyptus botryoides, Eucalyptus bridgesiana, Eucalyptus calophylla, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus coccifera, Eucalyptus curtisii, Eucalyptus dalrympleana, Eucalyptus deglupta, Eucalyptus delagatensis, Eucalyptus diversicolor, Eucalyptus dunnii, Eucalyptus ficifolia, Eucalyptus globulus, Eucalyptus gomphocephala, Eucalyptus gunnii, Eucalyptus henryi, Eucalyptus laevopinea, Eucalyptus macarthurii, Eucalyptus macrorhyncha, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus megacarpa, Eucalyptus melliodora, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nova-angelica, Eucalyptus obliqua, Eucalyptus occidentalis, Eucalyptus obtusiflora, Eucalyptus oreades, Eucalyptus pauciflora, Eucalyptus polybractea, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus robusta, Eucalyptus rudis, Eucalyptus saligna, Eucalyptus sideroxylon, Eucalyptus stuartiana, Eucalyptus tereticornis, Eucalyptus torelliana, Eucalyptus urnigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, and Eucalyptus youmanni.

As used herein, the term “plant” also is intended to include the fruit, seeds, flower, strobilus, etc. of the plant. A transformed plant of the current invention can be a direct transfectant, meaning that the DNA construct was introduced directly into the plant, such as through Agrobacterium , or the plant can be the progeny of a transfected plant. The second or subsequent generation plant can be produced by sexual reproduction, i.e., fertilization. Furthermore, the plant can be a gametophyte (haploid stage) or a sporophyte (diploid stage).

As used herein, the term “plant tissue” encompasses any portion of a plant, including plant cells. Plant cells include suspension cultures, callus, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, seeds and microspores. Plant tissues can be grown in liquid or solid culture, or in soil or suitable media in pots, greenhouses or fields. As used herein, “plant tissue” also refers to a clone of a plant, seed, progeny, or propagule, whether generated sexually or asexually, and descendents of any of these, such as cuttings or seeds.

In accordance with one aspect of the invention, a transgenic plant that has been transformed with a DNA construct of the invention has a phenotype that is different from a plant that has not been transformed with the DNA construct.

As used herein, “phenotype” refers to a distinguishing feature or characteristic of a plant which can be altered according to the present invention by integrating one or more DNA constructs of the invention into the genome of at least one plant cell of a plant. The DNA construct can confer a change in the phenotype of a transformed plant by modifying any one or more of a number of genetic, molecular, biochemical, physiological, morphological, or agronomic characteristics or properties of the transformed plant cell or plant as a whole.

In one embodiment, transformation of a plant with a DNA construct of the present invention can yield a phenotype including, but not limited to any one or more of increased drought tolerance, herbicide resistance, reduced or increased height, reduced or increased branching, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced resistance of the wood to decay, enhanced resistance to fingal diseases, altered attractiveness to insect pests, enhanced heavy met al tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, improved flower longevity, production of novel resins, and production of novel proteins or peptides.

In another embodiment, the affected phenotype includes one or more of the following traits: propensity to form reaction wood, a reduced period of juvenility, an increased period of juvenility, self-abscising branches, accelerated reproductive development or delayed reproductive development, as compared to a plant of the same species that has not been transformed with the DNA construct.

In a further embodiment, the phenotype that is different in the transgenic plant includes one or more of the following: lignin quality, lignin structure, wood composition, wood appearance, wood density, wood strength, wood stiffness, cellulose polymerization, fiber dimensions, lumen size, other plant components, plant cell division, plant cell development, number of cells per unit area, cell size, cell shape, cell wall composition, rate of wood formation, aesthetic appearance of wood, formation of stem defects, average microfibril angle, width of the S2 cell wall layer, rate of growth, rate of root formation ratio of root to branch vegetative development, leaf area index, and leaf shape.

Phenotype can be assessed by any suitable means. The plants can be evaluated based on their general morphology. Transgenic plants can be observed with the naked eye, can be weighed and their height measured. The plant can be examined by isolating individual layers of plant tissue, namely phloem and cambium, which is further sectioned into meristematic cells, early expansion, late expansion, secondary wall formation, and late cell maturation. See, e.g., Hertzberg, supra. The plants also can be assessed using microscopic analysis or chemical analysis.

Microscopic analysis includes examining cell types, stage of development, and stain uptake by tissues and cells. Fiber morphology, such as fiber wall thickness and microfibril angle of wood pulp fibers can be observed using, for example, microscopic transmission ellipsometry. See Ye and Sundström, Tappi J., 80:181 (1997). Wood strength, density, and grain slope in wet wood and standing trees can be determined by measuring the visible and near infrared spectral data in conjunction with multivariate analysis. See, U.S. Patent Application Publication Nos. 2002/0107644 and 2002/0113212. Lumen size can be measured using scanning electron microscopy. Lignin structure and chemical properties can be observed using nuclear magnetic resonance spectroscopy as described in Marita et al., J. Chem. Soc., Perkin Trans. I 2939 (2001).

The biochemical characteristic of lignin, cellulose, carbohydrates and other plant extracts can be evaluated by any standard analytical method known including spectrophotometry, fluorescence spectroscopy, HPLC, mass spectroscopy, and tissue staining methods.

As used herein, “transformation” refers to a process by which a nucleic acid is inserted into the genome of a plant cell. Such insertion encompasses stable introduction into the plant cell and transmission to progeny. Transformation also refers to transient insertion of a nucleic acid, wherein the resulting transformant transiently expresses the nucleic acid. Transformation can occur under natural or artificial conditions using various methods well known in the art. Transformation can be achieved by any known method for the insertion of nucleic acid sequences into a prokaryotic or eukaryotic host cell, including Agrobacterium -mediated transformation protocols, viral infection, whiskers, electroporation, microinjection, polyethylene glycol-treatment, heat shock, lipofection, and particle bombardment. Transformation can also be accomplished using chloroplast transformation as described in e.g. Svab et al., Proc. Natl Acad. Sci. 87:8526-30 (1990).

In accordance with one embodiment of the invention, transformation in Eucalyptus is performed as described in U.S. Patent Application No. 60/476,222 (supra) which is incorporated herein by reference in its entirety. In accordance with another embodiment, transformation of Pinus is accomplished using the methods described in U.S. Patent Application Publication No. 2002/0100083.

Another aspect of the invention provides methods of obtaining wood and/or making wood pulp from a plant transformed with a DNA construct of the invention. Methods of producing a transgenic plant are provided above and are known in the art. A transformed plant can be cultured or grown under any suitable conditions. For example, pine can be cultured and grown as described in U.S. Patent Application Publication No. 2002/0100083 . Eucalyptus can be cultured and grown as in, for example, Rydelius, et al., G ROWING E UCALYPTUS FOR P ULP AND E NERGY , presented at the Mechanization in Short Rotation, Intensive Culture Forestry Conference, Mobile, Ala., 1994. Wood and wood pulp can be obtained from the plant by any means known in the art.

As noted above, the wood or wood pulp obtained in accordance with this invention may demonstrate improved characteristics including, but not limited to any one or more of lignin composition, lignin structure, wood composition, cellulose polymerization, fiber dimensions, ratio of fibers to other plant components, plant cell division, plant cell development, number of cells per unit area, cell size, cell shape, cell wall composition, rate of wood formation, aesthetic appearance of wood, formation of stem defects, rate of growth, rate of root formation ratio of root to branch vegetative development, leaf area index, and leaf shape include increased or decreased lignin content, increased accessibility of lignin to chemical treatments, improved reactivity of lignin, increased or decreased cellulose content increased dimensional stability, increased tensile strength, increased shear strength, increased compression strength, increased shock resistance, increased stiffness, increased or decreased hardness, decreased spirality, decreased shrinkage, and differences in weight, density, and specific gravity.

B. Expression Profiling of Cell Cycle Genes

The present invention also provides methods and tools for performing expression profiling of cell cycle genes. Expression profiling is useful in determining whether genes are transcribed or translated, comparing transcript levels for particular genes in different tissues, genotyping, estimating DNA copy number, determining identity of descent, measuring mRNA decay rates, identifying protein binding sites, determining subcellular localization of gene products, correlating gene expression to a phenotype or other phenomenon, and determining the effect on other genes of the manipulation of a particular gene. Expression profiling is particularly useful for identifying gene expression in complex, multigenic events. For this reason, expression profiling is useful in correlating gene expression to plant phenotype and formation of plant tissues and the interconnection thereof to the cell cycle.

Only a small fraction of the genes of a plant's genome are expressed at a given time in a given tissue sample, and all of the expressed genes may not affect the plant phenotype. To identify genes capable of affecting a phenotype of interest, the present invention provides methods and tools for determining, for example, a gene expression profile at a given point in the cell cycle, a gene expression profile at a given point in plant development, and a gene expression profile a given tissue sample. The invention also provides methods and tools for identifying cell cycle genes whose expression can be manipulated to alter plant phenotype or to alter the biological activity of cell cycle gene products. In support of these methods, the invention also provides methods and tools that distinguish expression of different genes of the same family.

As used herein, “gene expression” refers to the process of transcription of a DNA sequence into an RNA sequence, followed by translation of the RNA into a protein, which may or may not undergo post-translational processing. Thus, the relationship between cell cycle stage and/or developmental stage and gene expression can be observed by detecting, quantitatively or qualitatively, changes in the level of an RNA or a protein. As used herein, the term “biological activity” includes, but is not limited to, the activity of a protein gene product, including enzyme activity.

The present invention provides oligonucleotides that are useful in these expression profiling methods. Each oligonucleotide is capable of hybridizing under a given set of conditions to a cell cycle gene or gene product. In one aspect of the invention, a plurality of oligonucleotides is provided, wherein each oligonucleotide hybridizes under a given set of conditions to a different cell cycle gene product. Examples of oligonucleotides of the present invention include SEQ ID NOs: 521-772. Each of the oligos of SEQ ID NOs 521-772 hybridizes under standard conditions to a different gene product of one of SEQ ID NOs: 1-260. The oligonucleotides of the invention are useful in determining the expression of one or more cell cycle genes in any of the above-described methods.

1. Cell, Tissue, Nucleic Acid, and Protein Samples

Samples for use in methods of the present invention may be derived from plant tissue. Suitable plant tissues include, but are not limited to, somatic embryos, pollen, leaves, stems, calli, stolons, microtubers, shoots, xylem, male strolbili, pollen cones, vascular tissue, apical meristem, vascular cambium, xylem, root, flower, and seed.

According to the present invention “plant tissue” is used as described previously herein. Plant tissue can be obtained from any of the plants types or species described supra.

In accordance with one aspect of the invention, samples are obtained from plant tissue at different stages of the cell cycle, from plant tissue at different developmental stages, from plant tissue at various times of the year (e.g. spring versus summer), from plant tissues subject to different environmental conditions (e.g. variations in light and temperature) and/or from different types of plant tissue and cells. In accordance with one embodiment, plant tissue is obtained during various stages of maturity and during different seasons of the year. For example, plant tissue can be collected from stem dividing cells, differentiating xylem, early developing wood cells, differentiated spring wood cells, and differentiated summer wood cells. As another example, gene expression in a sample obtained from a plant with developing wood can be compared to gene expression in a sample obtained from a plant which does not have developing wood.

Differentiating xylem includes samples obtained from compression wood, side-wood, and normal vertical xylem. Methods of obtaining samples for expression profiling from pine and eucalyptus are known. See, e.g., Allona et al., Proc. Nat'l Acad. Sci. 95:9693-8 (1998) and Whetton et al., Plant Mol. Biol. 47:275-91, and Kirst et al., I NT'L U NION OF F ORESTRY R ESEARCH O RGANIZATIONS B IENNIAL C ONFERENCE , S6.8 (June 2003, Umea, Sweden).

In one embodiment of the invention, gene expression in one type of tissue is compared to gene expression in, a different type of tissue or to gene expression in the same type of tissue in a difference stage of development. Gene expression can also be compared in one type of tissue which is sampled at various times during the year (different seasons). For example, gene expression in juvenile secondary xylem can be compared to gene expression in mature secondary xylem. Similarly, gene expression in cambium can be compared to gene expression in xylem. Furthermore, gene expression in apical meristems can be compared to gene expression in cambium.

In an alternative embodiment, differences in gene expression are determined as cells from different tissues advance during the cell cycle. In this method, the cells from the different tissues are synchronized and their gene expression is profiled. Methods of synchronizing the stage of cell cycle in a sample are known. These methods include, e.g., cold acclimation, photoperiod, and aphidicoline. See, e.g., Nagata et al., Int. Rev. Cytol. 132:1-30 (1992), Breyne and Zabeau, Curr. Opin. Plant Biol. 4:136-42, 140 (2001). A sample is obtained during a specific stage of the cell cycle and gene expression in that sample is compared to a sample obtained during a different stage of the cell cycle. For example, tissue can be examined in any of the phases of the cell cycle, such as mitosis, G1, G1, S, and G2. In particular, one can examine the changes in gene expression at the G1, G2, and metaphase checkpoints.

In another embodiment of the invention, a sample is obtained from a plant having a specific phenotype and gene expression in that sample is compared to a sample obtained from a plant of the same species that does not have that phenotype. For example, a sample can be obtained from a plant exhibiting a fast rate of growth and gene expression can be compared with that of a sample obtained from a plant exhibiting a normal or slow rate of growth. Differentially expressed genes identified from such a comparison can be correlated with growth rate and, therefore, useful for manipulating growth rate.

In a further embodiment, a sample is obtained from clonally propagated plants. In one embodiment the clonally propagated plants are of the species Pinus or Eucalyptus . Individual ramets from the same genotype can be sacrificed at different times of year. Thus, for any genotype there can be at least two genetically identical trees sacrificed, early in the season and late in the season. Each of these trees can be divided into juvenile (top) to mature (bottom) samples. Further, tissue samples can be divided into, for example, phloem to xylem, in at least 5 layers of peeling. Each of these samples can be evaluated for phenotype and gene expression. See Entry 196.

Where cellular components may interfere with an analytical technique, such as a hybridization assay, enzyme assay, a ligand binding assay, or a biological activity assay, it may be desirable to isolate the gene products from such cellular components. Gene products, including nucleic acid and amino acid gene products, can be isolated from cell fragments or lysates by any method known in the art.

Nucleic acids used in accordance with the invention can be prepared by any available method or process, or by other processes as they become known in the art. Conventional techniques for isolating nucleic acids are detailed, for example, in Tijssen, L ABORATORY T ECHNIQUES IN B IOCHEMISTRY AND M OLECULAR B IOLOGY : H YBRIDIZATION W ITH N UCLEIC A CID P ROBES , chapter 3 (Elsevier Press, 1993), Berger and Kimmel, Methods Enzymol. 152:1 (1987), and G IBCO BRL & L IFE T ECHNOLOGIES T RIZOL RNA I SOLATION P ROTOCOL , Form No. 3786 (2000). Techniques for preparing nucleic acid samples, and sequencing polynucleotides from pine and eucalyptus are known. See, e.g., Allona et al., supra and Whetton et al., supra, and U.S. Application No. 60/476,222.

A suitable nucleic acid sample can contain any type of nucleic acid derived from the transcript of a cell cycle gene, i.e., RNA or a subsequence thereof or a nucleic acid for which an mRNA transcribed from a cell cycle gene served as a template. Suitable nucleic acids include cDNA reverse-transcribed from a transcript, RNA transcribed from that cDNA, DNA amplified from the cDNA, and RNA transcribed from the amplified DNA. Detection of such products or derived products is indicative of the presence and/or abundance of the transcript in the sample. Thus, suitable samples include, but are not limited to, transcripts of the gene or genes, cDNA reverse-transcribed from the transcript, cRNA transcribed from the cDNA, DNA amplified from the genes, and RNA transcribed from amplified DNA. As used herein, the category of “transcripts” includes but is not limited to pre-mRNA nascent transcripts, transcript processing intermediates, and mature mRNAs and degradation products thereof.

It is not necessary to monitor all types of transcripts to practice the invention. For example, the expression profiling methods of the invention can be conducted by detecting only one type of transcript, such as mature mRNA levels only.

In one aspect of the invention, a chromosomal DNA or cDNA library (comprising, for example, fluorescently labeled cDNA synthesized from total cell mRNA) is prepared for use in hybridization methods according to recognized methods in the art. See Sambrook et al., supra.

In another aspect of the invention, mRNA is amplified using, e.g., the MessageAmp kit (Ambion). In a further aspect, the mRNA is labeled with a detectable label. For example, mRNA can be labeled with a fluorescent chromophore, such as CyDye (Amersham Biosciences).

In some applications, it may be desirable to inhibit or destroy RNase that often is present in homogenates or lysates, before use in hybridization techniques. Methods of inhibiting or destroying nucleases are well known. In one embodiment of the invention, cells or tissues are homogenized in the presence of chaotropic agents to inhibit nuclease. In another embodiment, RNase is inhibited or destroyed by heat treatment, followed by proteinase treatment.

Protein samples can be obtained by any means known in the art. Protein samples useful in the methods of the invention include crude cell lysates and crude tissue homogenates. Alternatively, protein samples can be purified. Various methods of protein purification well known in the art can be found in Marshak et al., S TRATEGIES FOR P ROTEIN P URIFICATION AND C HARACTERIZATION : A L ABORATORY C OURSE M ANUAL (Cold Spring Harbor Laboratory Press 1996).

2. Detecting Level of Gene Expression

For methods of the invention that comprise detecting a level of gene expression, any method for observing gene expression can be used, without limitation. Such methods include traditional nucleic acid hybridization techniques, polymerase chain reaction (PCR) based methods, and protein determination. The invention includes detection methods that use solid support-based assay formats as well as those that use solution-based assay formats.

Absolute measurements of the expression levels need not be made, although they can be made. The invention includes methods comprising comparisons of differences in expression levels between samples. Comparison of expression levels can be done visually or manually, or can be automated and done by a machine, using for example optical detection means. Subrahmanyam et al., Blood. 97: 2457 (2001); Prashar et al., Methods Enzymol. 303: 258 (1999). Hardware and software for analyzing differential expression of genes are available, and can be used in practicing the present invention. See, e.g., GenStat Software and GeneExpress® GX Explorer™ Training Manual, supra; Baxevanis & Francis-Ouellette, supra.

In accordance with one embodiment of the invention, nucleic acid hybridization techniques are used to observe gene expression. Exemplary hybridization techniques include Northern blotting, Southern blotting, solution hybridization, and S1 nuclease protection assays.

Nucleic acid hybridization typically involves contacting an oligonucleotide probe and a sample comprising nucleic acids under conditions where the probe can form stable hybrid duplexes with its complementary nucleic acid through complementary base pairing. For example, see PCT application WO 99/32660; Berger & Kimmel, Methods Enzymol. 152: 1 (1987). The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. The detectable label can be present on the probe, or on the nucleic acid sample. In one embodiment, the nucleic acids of the sample are detectably labeled polynucleotides representing the mRNA transcripts present in a plant tissue (e.g., a cDNA library). Detectable labels are commonly radioactive or fluorescent labels, but any label capable of detection can be used. Labels can be incorporated by several approached described, for instance, in WO 99/32660, supra. In one aspect RNA can be amplified using the MessageAmp kit (Ambion) with the addition of aminoallyl-UTP as well as free UTP. The aminoallyl groups incorporated into the amplified RNA can be reacted with a fluorescent chromophore, such as CyDye (Amersham Biosciences)

Duplexes of nucleic acids are destabilized by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under low stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA:DNA, RNA:RNA or RNA:DNA) will form even where the annealed sequences are not perfectly complementary. Thus, specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature and/or lower salt and/or in the presence of destabilizing reagents) hybridization tolerates fewer mismatches.

Typically, stringent conditions for short probes (e.g., 10 to 50 nucleotide bases) will be those in which the salt concentration is at least about 0.01 to 1.0 M at pH 7.0 to 8.3 and the temperature is at least about 30° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.

Under some circumstances, it can be desirable to perform hybridization at conditions of low stringency, e.g., 6×SSPE-T (0.9 M NaCl, 60 mM NaH 2 PO 4 , pH 7.6, 6 mM EDTA, 0.005% Triton) at 37° C, to ensure hybridization. Subsequent washes can then be performed at higher stringency (e.g., 1×SSPE-T at 37° C.) to eliminate mismatched hybrid duplexes. Successive washes can be performed at increasingly higher stringency (e.g., down to as low as 0.25×SSPE-T at 37° C. to 50° C.) until a desired level of hybridization specificity is obtained.

In general, standard conditions for hybridization is a compromise between stringency (hybridization specificity) and signal intensity. Thus, in one embodiment of the invention, the hybridized nucleic acids are washed at successively higher stringency conditions and read between each wash. Analysis of the data sets produced in this manner will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular oligonucleotide probes of interest. For example, the final wash may be selected as that of the highest stringency that produces consistent results and that provides a signal intensity greater than approximately 10% of the background intensity.

a. Oligonucleotide Probes

Oligonucleotide probes useful in nucleic acid hybridization techniques employed in the present invention are capable of binding to a nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing via hydrogen bond formation. A probe can include natural bases (i.e., A, G, U, C or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the nucleotide bases in the probes can be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, probes can be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.

Oligonucleotide probes can be prepared by any means known in the art. Probes useful in the present invention are capable of hybridizing to a nucleotide product of cell cycle genes, such as one of SEQ ID NOs: 1-260. Probes useful in the invention can be generated using the nucleotide sequences disclosed in SEQ ID NOs: 1-260. The invention includes oligonucleotide probes having at least a 2, 10,15, 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 100 nucleotide fragment of a corresponding contiguous sequence of any one of SEQ liD NOs: 1-260. The invention includes oligonucleotides of less than 2, 1, 0.5, 0.1, or 0.05 kb in length. In one embodiment, the oligonucleotide is 60 nucleotides in length.

Oligonucleotide probes can be designed by any means known in the art. See, e.g., Li and Stormo, Bioinformatics 17: 1067-76 (2001). Oligonucleotide probe design can be effected using software. Exemplary software includes ArrayDesigner, GeneScan, and ProbeSelect. Probes complementary to a defined nucleic acid sequence can be synthesized chemically, generated from longer nucleotides using restriction enzymes, or can be obtained using techniques such as polymerase chain reaction (PCR). PCR methods are well known and are described, for example, in Innis et al. eds., PCR P ROTOCOLS : A G UIDE TO M ETHODS AND A PPLICATIONS , Academic Press Inc. San Diego, Calif. (1990). The probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag. Optimally, the nucleic acids in the sample are labeled and the probes are not labeled. Oligonucleotide probes generated by the above methods can be used in solution or solid support-based methods.

The invention includes oligonucleotide probes that hybridize to a product of the coding region or a 3′ untranslated region (3′ UTR) of a cell cycle gene. In one embodiment, the oligonucleotide probe hybridizes to the 3′ UTR of any one of SEQ ID NOs: 1-260. The 3′ UTR is generally a unique region of the gene, even among members of the same family. Therefore, the probes capable of hybridizing to a product of the 3′ UTR can be useful for differentiating the expression of individual genes within a family where the coding region of the genes likely are highly homologous. This allows for the design of oligonucleotide probes to be used as members of a plurality of oligonucleotides, each capable of uniquely binding to a single gene. In another embodiment, the oligonucleotide probe comprises any one of SEQ ID NOs: 521-772. In another embodiment, the oligonucleotide probe consists of any one of SEQ ID NOs: 521-772.

b. Oligonucleotide Array Methods

One embodiment of the invention employs two or more oligonucleotide probes in combination to detect a level of expression of one or more cell cycle genes, such as the genes of SEQ ID NOs: 1-260. In one aspect of this embodiment, the level of expression of two or more different genes is detected. The two or more genes may be from the same or different cell cycle gene families discussed above. Each of the two or more oligonucleotides may hybridize to a different one of the genes.

One embodiment of the invention employs two or more oligonucleotide probes, each of which specifically hybridize to a polynucleotide derived from the transcript of a gene provided by SEQ ID NOs: 1-260. Another embodiment employs two or more oligonucleotide probes, at least one of which comprises a nucleic acid sequence of SEQ ID NOs: 521-772. Another embodiment employs two or more oligonucleotide probes, at least one of which consists of SEQ ID NOs: 521-772.

The oligonucleotide probes may comprise from about 5 to about 60, or from about 5 to about 500, nucleotide bases, such as from about 60 to about 100 nucleotide bases, including from about 15 to about 60 nucleotide bases.

One embodiment of the invention uses solid support-based oligonucleotide hybridization methods to detect gene expression. Solid support-based methods suitable for practicing the present invention are widely known and are described, for example, in PCT application WO 95/11755; Huber et al., Anal. Biochem. 299: 24 (2001); Meiyanto et al., Biotechniques. 31: 406 (2001); Relogio et al., Nucleic Acids Res. 30:e51 (2002). Any solid surface to which oligonucleotides can be bound, covalently or non-covalently, can be used. Such solid supports include filters, polyvinyl chloride dishes, silicon or glass based chips, etc.

One embodiment uses oligonucleotide arrays, i.e. microarrays, which can be used to simultaneously observe the expression of a number of genes or gene products. Oligonucleotide arrays comprise two or more oligonucleotide probes provided on a solid support, wherein each probe occupies a unique location on the support. The location of each probe may be predetermined, such that detection of a detectable signal at a given location is indicative of hybridization to an oligonucleotide probe of a known identity. Each predetermined location can contain more than one molecule of a probe, but each molecule within the predetermined location has an identical sequence. Such predetermined locations are termed features. There can be, for example, from 2, 10, 100, 1,000, 2,000 or 5,000 or more of such features on a single solid support. In one embodiment, each oligonucleotide is located at a unique position on an array at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 times.

Oligonucleotide probe arrays for detecting gene expression can be made and used according to conventional techniques described, for example, in Lockhart et al., Nat'l Biotech. 14: 1675 (1996), McGall et al., Proc. Nat'l Acad Sci. USA 93: 13555 (1996), and Hughes et al., Nature Biotechnol. 19:342 (2001). A variety of oligonucleotide array designs is suitable for the practice of this invention.

In one embodiment the one or more oligonucleotides include a plurality of oligonucleotides that each hybridize to a different gene expressed in a particular tissue type. For example, the tissue can be developing wood.

In one embodiment, a nucleic acid sample obtained from a plant can be amplified and, optionally labeled with a detectable label. Any method of nucleic acid amplification and any detectable label suitable for such purpose can be used. For example, amplification reactions can be performed using, e.g. Ambion's MessageAmp, which creates “antisense” RNA or “aRNA” (complementary in nucleic acid sequence to the RNA extracted from the sample tissue). The RNA can optionally be labeled using CyDye fluorescent labels. During the amplification step, aaUTP is incorporated into the resulting aRNA. The CyDye fluorescent labels are coupled to the aaUTPs in a non-enzymatic reaction. Subsequent to the amplification and labeling steps, labeled amplified antisense RNAs are precipitated and washed with appropriate buffer, and then assayed for purity. For example, purity can be assay using a NanoDrop spectrophotometer. The nucleic acid sample is then contacted with an oligonucleotide array having, attached to a solid substrate (a “microarray slide”), oligonucleotide sample probes capable of hybridizing to nucleic acids of interest which may be present in the sample. The step of contacting is performed under conditions where hybridization can occur between the nucleic acids of interest and the oligonucleotide probes present on the array. The array is then washed to remove non-specifically bound nucleic acids and the signals from the labeled molecules that remain hybridized to oligonucleotide probes on the solid substrate are detected. The step of detection can be accomplished using any method appropriate to the type of label used. For example, the step of detecting can accomplished using a laser scanner and detector. For example, on can use and Axon scanner which optionally uses GenePix Pro software to analyze the position of the signal on the microarray slide.

Data from one or more microarray slides can analyzed by any appropriate method known in the art.

Oligonucleotide probes used in the methods of the present invention, including microarray techniques, can be generated using PCR. PCR primers used in generating the probes are chosen, for example, based on the sequences of SEQ ID NOs: 1-260, to result in amplification of unique fragments of the cell cycle genes (i.e., fragments that hybridize to only one polynucleotide of any one of SEQ ID NOs: 1-260 under standard hybridization conditions). Computer programs are useful in the design of primers with the required specificity and optimal hybridization properties. For example, Li and Stormo, supra at 1075, discuss a method of probe selection using ProbeSelect which selects an optimum oligonucleotide probe based on the entire gene sequence as well as other gene sequences to be probed at the same time.

In one embodiment, oligonucleotide control probes also are used. Exemplary control probes can fall into at least one of three categories referred to herein as (1) normalization controls, (2) expression level controls and (3) negative controls. In microarray methods, one or more of these control probes may be provided on the array with the inventive cell cycle gene-related oligonucleotides.

Normalization controls correct for dye biases, tissue biases, dust, slide irregularities, malformed slide spots, etc. Normalization controls are oligonucleotide or other nucleic acid probes that are complementary to labeled reference oligonucleotides or other nucleic acid sequences that are added to the nucleic acid sample to be screened. The signals obtained from the normalization controls, after hybridization, provide a control for variations in hybridization conditions, label intensity, reading efficiency and other factors that can cause the signal of a perfect hybridization to vary between arrays. In one embodiment, signals (e.g., fluorescence intensity or radioactivity) read from all other probes used in the method are divided by the signal from the control probes, thereby normalizing the measurements.

Virtually any probe can serve as a normalization control. Hybridization efficiency varies, however, with base composition and probe length. Preferred normalization probes are selected to reflect the average length of the other probes being used, but they also can be selected to cover a range of lengths. Further, the normalization control(s) can be selected to reflect the average base composition of the other probes being used. In one embodiment, only one or a few normalization probes are used, and they are selected such that they hybridize well (i.e., without forming secondary structures) and do not match any test probes. In one embodiment, the normalization controls are mammalian genes.

Expression level controls probes hybridize specifically with constitutively expressed genes present in the biological sample. Virtually any constitutively expressed gene provides a suitable target for expression level control probes. Typically, expression level control probes have sequences complementary to subsequences of constitutively expressed “housekeeping genes” including, but not limited to certain photosynthesis genes.

“Negative control” probes are not complementary to any of the test oligonucleotides (i.e., the inventive cell cycle gene-related oligonucleotides), normalization controls, or expression controls. In one embodiment, the negative control is a mammalian gene which is not complementary to any other sequence in the sample.

The terms “background” and “background signal intensity” refer to hybridization signals resulting from non-specific binding or other interactions between the labeled target nucleic acids (i.e., mRNA present in the biological sample) and components of the oligonucleotide array. Background signals also can be produced by intrinsic fluorescence of the array components themselves.

A single background signal can be calculated for the entire array, or a different background signal can be calculated for each target nucleic acid. In a one embodiment, background is calculated as the average hybridization signal intensity for the lowest 5 to 10 percent of the oligonucleotide probes being used, or, where a different background signal is calculated for each target gene, for the lowest 5 to 10 percent of the probes for each gene. Where the oligonucleotide probes corresponding to a particular cell cycle gene hybridize well and, hence, appear to bind specifically to a target sequence, they should not be used in a background signal calculation. Alternatively, background can be calculated as the average hybridization signal intensity produced by hybridization to probes that are not complementary to any sequence found in the sample (e.g., probes directed to nucleic acids of the opposite sense or to genes not found in the sample). In microarray methods, background can be calculated as the average signal intensity produced by regions of the array that lack any oligonucleotides probes at all.

c. PCR-Based Methods

In another embodiment, PCR-based methods are used to detect gene expression. These methods include reverse-transcriptase-mediated polymerase chain reaction (RT-PCR) including real-time and endpoint quantitative reverse-transcriptase-mediated polymerase chain reaction (Q-RTPCR). These methods are well known in the art. For example, methods of quantitative PCR can be carried out using kits and methods that are commercially available from, for example, Applied BioSystems and Stratagene®. See also Kochanowski, Q UANTITATIVE PCR P ROTOCOLS (Humana Press, 1999); Innis et al., supra.; Vandesompele et al., Genome Biol. 3: RESEARCH0034 (2002); Stein, Cell Mol. Life Sci. 59: 1235 (2002).

Gene expression can also be observed in solution using Q-RTPCR. Q-RTPCR relies on detection of a fluorescent signal produced proportionally during amplification of a PCR product. See Innis et al., supra. Like the traditional PCR method, this technique employs PCR oligonucleotide primers, typically 15-30 bases long, that hybridize to opposite strands and regions flanking the DNA region of interest. Additionally, a probe (e.g., TaqMan®, Applied Biosystems) is designed to hybridize to the target sequence between the forward and reverse primers traditionally used in the PCR technique. The probe is labeled at the 5′ end with a reporter fluorophore, such as 6-carboxyfluorescein (6-FAM) and a quencher fluorophore like 6-carboxy-tetramethyl-rhodamine (TAMRA). As long as the probe is intact, fluorescent energy transfer occurs which results in the absorbance of the fluorescence emission of the reporter fluorophore by the quenching fluorophore. As Taq polymerase extends the primer, however, the intrinsic 5′ to 3′ nuclease activity of Taq degrades the probe, releasing the reporter fluorophore. The increase in the fluorescence signal detected during the amplification cycle is proportional to the amount of product generated in each cycle.

The forward and reverse amplification primers and internal hybridization probe is designed to hybridize specifically and uniquely with one nucleotide derived from the transcript of a target gene. In one embodiment, the selection criteria for primer and probe sequences incorporates constraints regarding nucleotide content and size to accommodate TaqMan® requirements.

SYBR Green® can be used as a probe-less Q-RTPCR alternative to the Taqman®-type assay, discussed above. ABI P RISM® 7900 S EQUENCE D ETECTION S YSTEM U SER G UIDE A PPLIED B IOSYSTEMS , chap. 1-8, App. A-F. (2002).

A device measures changes in fluorescence emission intensity during PCR amplification. The measurement is done in “real time,” that is, as the amplification product accumulates in the reaction. Other methods can be used to measure changes in fluorescence resulting from probe digestion. For example, fluorescence polarization can distinguish between large and small molecules based on molecular tumbling (see U.S. Pat. No. 5,593,867).

d. Protein Detection Methods

Proteins can be observed by any means known in the art, including immunological methods, enzyme assays and protein array/proteomics techniques.

Measurement of the translational state can be performed according to several protein methods. For example, whole genome monitoring of protein—the “proteome”—can be carried out by constructing a microarray in which binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of proteins having an amino acid sequence of any of SEQ ID NOs: 261-520 or proteins encoded by the genes of SEQ ID NOs: 1-260 or conservative variants thereof. See Wildt et al., Nature Biotechnol. 18: 989 (2000). Methods for making polyclonal and monoclonal antibodies are well known, as described, for instance, in Harlow & Lane, A NTIBODIES : A L ABORATORY M ANUAL (Cold Spring Harbor Laboratory Press, 1988).

Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well-known in the art and typically involves isoelectric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. See, e.g., Hames et al, , G EL E LECTROPHORESIS OF P ROTEINS : A P RACTICAL A PPROACH (IRL Press, 1990). The resulting electropherograms can be analyzed by numerous techniques, including mass spectrometric techniques, western blotting and immunoblot analysis using polyclonal and monoclonal antibodies, and internal and N-terminal micro-sequencing.

3. Correlating Gene Expression to Phenotype and Tissue Development

As discussed above, the invention provides methods and tools to correlate gene expression to plant phenotype. Gene expression may be examined in a plant having a phenotype of interest and compared to a plant that does not have the phenotype or has a different phenotype. Such a phenotype includes, but is not limited to, increased drought tolerance, herbicide resistance, reduced or increased height, reduced or increased branching, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced resistance of the wood to decay, enhanced resistance to fungal diseases, altered attractiveness to insect pests, enhanced heavy met al tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, improved flower longevity, production of novel resins, and production of novel proteins or peptides.

In another embodiment, the phenotype includes one or more of the following traits: propensity to form reaction wood, a reduced period of juvenility, an increased period of juvenility, self-abscising branches, accelerated reproductive development or delayed reproductive development.

In a further embodiment, the phenotype that is differs in the plants compares includes one or more of the following: lignin quality, lignin structure, wood composition, wood appearance, wood density, wood strength, wood stiffness, cellulose polymerization, fiber dimensions, lumen size, other plant components, plant cell division, plant cell development, number of cells per unit area, cell size, cell shape, cell wall composition, rate of wood formation, aesthetic appearance of wood, formation of stem defects, average microfibril angle, width of the S2 cell wall layer, rate of growth, rate of root formation ratio of root to branch vegetative development, leaf area index, and leaf shape.

Phenotype can be assessed by any suitable means as discussed above.

In a further embodiment, gene expression can be correlated to a given point in the cell cycle, a given point in plant development, and in a given tissue sample. Plant tissue can be examined at different stages of the cell cycle, from plant tissue at different developmental stages, from plant tissue at various times of the year (e.g. spring versus summer), from plant tissues subject to different environmental conditions (e.g. variations in light and temperature) and/or from different types of plant tissue and cells. In accordance with one embodiment, plant tissue is obtained during various stages of maturity and during different seasons of the year. For example, plant tissue can be collected from stem dividing cells, differentiating xylem, early developing wood cells, differentiated spring wood cells, differentiated summer wood cells.

It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. Throughout the specification, any and all references to a publicly available document, including a U.S. patent, are specifically incorporated by reference.