BRITTLE STALK 2 GENE FAMILY AND RELATED METHODS AND USES

Abstract

This invention relates to isolated polynucleotides encoding BRITTLE STALK 2-like (Bk2L) family polypeptides. The invention also relates to the construction of a chimeric gene encoding all or a portion of a Bk2L polypeptide, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the Bk2L polypeptide in a transformed host cell.

Description

FIELD OF THE INVENTION

The field of invention relates to plant molecular biology, and in particular, to BRITTLE STALK 2-like genes, BRITTLE STALK 2-like polypeptides, and uses thereof.

BACKGROUND OF THE INVENTION

Plant primary growth is mainly driven by an enlargement of the cells, which occurs through the irreversible yielding of the primary cell wall to turgor pressure inside the cell. Although cell division is required to produce new cells, the growth results from the expansion of these cells, not simply from their division. Cellulose microfibrils, which are embedded in a matrix of hemicellulose and lignin in the wall, are the main determinants of tensile strength (Appenzeller et al., Cellulose 11:287-299 (2004)). A cell usually expands along the axis that is perpendicular to the orientation of the microfibrils. For example, radial deposition of microfibrils favors cell expansion along the longitudinal axis.

Secondary wall differs from primary wall in that it is richer in cellulose and lignin and its deposition commences toward the end of cell expansion. Modulation of primary cell wall synthesis has applications in altering growth rate and size (stature) of a plant whereas that of secondary wall can be useful in improving biomass accumulation and tissue strength (Appenzeller et al., Cellulose 11:287-299 (2004)).

Cellulose in general is the major wall constituent in mature plant cells forming vegetative tissues. The paracrystalline structure of cellulose that results from energy minimization by the formation of inter- and intra-chain hydrogen bonds makes it mechanically one of the strongest organic molecule known on density basis. It is natural then that cellulose is the primary determinant of strength in structural tissues.

Plant mechanical strength is one of the most important agronomic traits. Plant mutants that are defective in stem strength have been isolated and characterized. Barley brittle culm (bc) mutants were first described based on the physical properties of the culms, which have an 80% reduction in the amount of cellulose and a twofold decrease in breaking strength compared with those of wildtype plants (Kokubo et al., Plant Physiol. 97:509-514 (1991)). Rice brittle culm1 (bc1) mutants show a reduction in cell wall thickness and cellulose content (Qian et al., Chi. Sci. Bull. 46:2082-2085 (2001)). Li et al. described the identification of rice BRITTLE CULM1 (BC1), a gene that encodes a COBRA-like protein (The Plant Cell 15(9):2020-2031 (2003)). Their findings indicated that BC1 functions in regulating the biosynthesis of secondary cell walls to provide the main mechanical strength for rice plants.

The stalks of maize brittle stalk 2 (bk2) mutant exhibit a dramatically reduced mechanical strength compared to their wild type counterparts (Langham, MNL 14:21-22 (1940)). Maize bk2 mutants have stalk and leaves that are very brittle and break easily. The main chemical constituent deficient in the mutant stalk is cellulose. Therefore, stalk mechanical strength appears to be dependent primarily on the amount of cellulose in a unit length of the stalk below the ear.

Furthermore, genes encoding cellulose synthase catalytic subunits (CesA) have been implicated in cell wall synthesis and are represented by a large family in plants. Ten genes were identified in Arabidopsis after complete genome sequencing and twelve genes have been isolated from maize by EST sequencing (U.S. Pat. Nos. 6,803,498 and 6,930,225). Three of the CesA genes from each Arabidopsis and maize have been reported to make secondary wall whereas the rest apparently make primary wall (Taylor et al., Proc. Natl. Acad. Sci. U.S.A. 100:1450-1455 (2003)). Mutations in three of the CesA genes from Arabidopsis resulted in collapsed xylem and reduced mechanical strength of the stem-like peduncle. When related CesA genes from rice were mutated the culms became brittle, indicating the role of these genes in secondary wall formation. In each case, reduced mechanical strength was correlated with diminished cellulose content.

In general, mutations in the CesA genes involved in primary wall formation cause severe phenotypic alterations whereas those in secondary wall-forming genes do not alter the visual phenotype as much as they affect mechanical strength (Appenzeller et al., Cellulose 11:287-299 (2004)).

As insufficient stalk strength is a major problem in corn breeding, it is desirable to provide compositions and methods for manipulating cellulose concentration in the cell wall and thereby alter plant stalk strength and/or quality for improved standability or silage quality.

SUMMARY OF THE INVENTION

The present invention includes:

In one embodiment, an isolated polynucleotide comprising (a) a nucleotide sequence encoding a polypeptide associated with stalk mechanical strength, wherein said polypeptide has an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any integer in between 80% and 100%, based on the Clustal V method of alignment, when compared to SEQ ID NOs:16 or 18, or (b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.

In another embodiment, a method of altering (preferably increasing) stalk mechanical strength in a plant comprising (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a)(i); and (b) regenerating a transgenic plant from said transformed plant cell, wherein said transgenic plant comprises in its genome said recombinant DNA construct and wherein said transgenic plant exhibits an alteration (preferably an increase) in stalk mechanical strength, when compared to a control plant not comprising said recombinant DNA construct. The method may further comprise (c) obtaining a progeny plant derived from said transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct.

In another embodiment, a method of evaluating stalk mechanical strength in a plant comprising (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a)(i); (b) regenerating a transgenic plant from said transformed plant cell; and (c) evaluating said transgenic plant for stalk mechanical strength. The method may further comprise (d) obtaining a progeny plant derived from said transgenic plant; and (e) evaluating said progeny plant for stalk mechanical strength.

In another embodiment, a method of evaluating stalk mechanical strength in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a)(i); (b) regenerating a transgenic plant from said transformed plant cell; (c) obtaining a progeny plant derived from said transgenic plant; and (d) evaluating said progeny plant for stalk mechanical strength.

The present invention also includes:

In one embodiment, a plant comprising in its genome: (a) a first recombinant DNA construct comprising at least one promoter that is functional in a plant operably linked to at least one of a first isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) a second recombinant DNA construct comprising at least one promoter that is functional in a plant operably linked to at least one of a second isolated polynucleotide selected from the group consisting of (iv) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; (v) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and (vi) a full-length complement of the polynucleotide of (b)(iv) or (b)(v).

In another embodiment, a plant comprising in its genome at least one regulatory sequence operably linked to: (a) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii), and wherein said plant exhibits increased cell wall cellulose content or enhanced growth rate when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).

In another embodiment, a plant comprising in its genome a suppression DNA construct comprising a promoter functional in a plant operably linked to (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, or any integer from 51% up to and including 100% sequence identity, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a polypeptide selected from the group consisting of Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9, and wherein said plant exhibits reduced stalk mechanical strength when compared to a control plant not comprising said suppression DNA construct.

In another embodiment, a plant comprising in its genome a suppression DNA construct comprising a promoter functional in a plant operably linked to (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L3 polypeptide, and wherein said plant exhibits reduced plant height and/or reduced organ size when compared to a control plant not comprising said suppression DNA construct.

In another embodiment, a plant comprising in its genome a suppression DNA construct comprising a promoter functional in a plant operably linked to (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:10, or (ii) a full complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L5 polypeptide, and wherein said plant exhibits male sterility when compared to a control plant not comprising said suppression DNA construct.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application.

FIGS. 1A-1F show a Clustal V alignment, using default parameters, of the amino acid sequences of the Bk2 and Bk2-like proteins set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16 and 18.

FIG. 2 shows a chart setting forth a comparison of the percent identity (and percent divergence in the lower half triangle), using the Clustal V alignment method, between the nine amino acid sequences shown in FIGS. 1A-1F.

FIG. 3 shows Solexa MPSS™ gene expression analysis of gene Bk2.

FIG. 4 shows the correlation of expression patterns of the Bk2 gene with members of the CesA gene family.

FIGS. 5A-5B show the correlation among the expression level of all the different Bk2 and CesA genes from maize as studied from Solexa MPSS™.

FIG. 6 shows the phylogenetic analysis of the Bk2L proteins from maize, BC1L proteins from rice, and COBL proteins from Arabidopsis (NCBI Accession Nos. are in parenthesis). The numbers along the branches are the bootstrap values obtained from a heuristic search over 5,000 replications. The bootstrap values for only the monophyletic groups that were supported >50% of the time are shown. The branch lengths are proportional to the inferred amino acid differences.

SEQ ID NO:1 is the 1784 bp nucleotide sequence containing the open reading frame (ORF) (nucleotides 89-1438) of the BRITTLE STALK 2 (Bk2) gene from maize flanked by additional untranslated regions (UTR) 5′ (nucleotides 1-88) and 3′ (nucleotides 1439-1784) to this ORF region.

SEQ ID NO:2 is the deduced amino acid sequence of a maize BRITTLE STALK 2 (Bk2) polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:1.

SEQ ID NO:3 is the 3152 bp nucleotide sequence containing the ORF (nucleotides 586-2586) of the BRITTLE STALK 2-Like1 (Bk2L1) gene from maize flanked by additional UTR regions 5′ (nucleotides 1-585) and 3′ (nucleotides 2587-3152) to this ORF region.

SEQ ID NO:4 is the deduced amino acid sequence of a maize BRITTLE STALK 2-Like1 (Bk2L1) polypeptide derived from ORF of the nucleotide sequence set forth in SEQ ID NO:3.

SEQ ID NO:5 is the 2094 bp nucleotide sequence containing the ORF (nucleotides 281-1624) of the BRITTLE STALK 2-Like3 (Bk2L3) gene from maize flanked by additional UTR regions 5′ (nucleotides 1-280) and 3′ (nucleotides 1625-2094) to this ORF region.

SEQ ID NO:6 is the deduced amino acid sequence of a maize BRITTLE STALK 2-Like3 (Bk2L3) polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:5.

SEQ ID NO:7 is the 2102 bp nucleotide sequence containing the ORF (nucleotides 326-1672) of the BRITTLE STALK 2-Like4 (Bk2L4) gene from maize flanked by additional UTR regions 5′ (nucleotides 1-325) and 3′ (nucleotides 1673-2102) to this ORF region.

SEQ ID NO:8 is the deduced amino acid sequence of a maize BRITTLE STALK 2-Like4 (Bk2L4) polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:7.

SEQ ID NO:9 is the 2422 bp nucleotide sequence containing the ORF (nucleotides 216-2249) of the BRITTLE STALK 2-Like5 (Bk2L5) gene from maize flanked by additional UTR regions 5′ (nucleotides 1-215) and 3′ (nucleotides 2250-2422) to this ORF region.

SEQ ID NO:10 is the deduced amino acid sequence of a maize BRITTLE STALK 2-Like5 (Bk2L5) polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:9.

SEQ ID NO:11 is the 1845 bp nucleotide sequence containing the ORF (nucleotides 184-1563) of the BRITTLE STALK 2-Like6 (Bk2L6) gene from maize flanked by additional UTR regions 5′ (nucleotides 1-183) and 3′ (nucleotides 1564-1845) to this ORF region.

SEQ ID NO:12 is the deduced amino acid sequence of a maize BRITTLE STALK 2-Like6 (Bk2L6) polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:11.

SEQ ID NO:13 is the 1644 bp nucleotide sequence containing the ORF (nucleotides 85-1425) of the BRITTLE STALK 2-Like7 (Bk2L7) gene from maize flanked by additional UTR regions 5′ (nucleotides 1-84) and 3′ (nucleotides 1426-1644) to this ORF region.

SEQ ID NO:14 is the deduced amino acid sequence of a maize BRITTLE STALK 2-Like7 (Bk2L7) polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:13.

SEQ ID NO:15 is the 2108 bp nucleotide sequence containing the ORF (nucleotides 144-2105) of the BRITTLE STALK 2-Like8 (Bk2L8) gene from maize flanked by additional UTR regions 5′ (nucleotides 1-143) and 3′ (nucleotides 2106-2108) to this ORF region.

SEQ ID NO:16 is the deduced amino acid sequence of a maize BRITTLE STALK 2-Like8 (Bk2L8) polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:15.

SEQ ID NO:17 is the 1335 bp nucleotide sequence containing the ORF (nucleotides 1-1332) of the BRITTLE STALK 2-Like9 (Bk2L9) gene from maize flanked by additional UTR regions 5′ (nucleotides 0) and 3′ (nucleotides 1963-1965) to this ORF region.

SEQ ID NO:18 is the deduced amino acid sequence of a maize BRITTLE STALK 2-Like9 (Bk2L9) polypeptide derived from the nucleotide sequence set forth in SEQ ID NO:17.

SEQ ID NO:19 is the 3780 bp nucleotide sequence containing the ORF (nucleotides 201-3428) of the CesA1 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-200) and 3′ (nucleotides 3429-3780) to this ORF region.

SEQ ID NO:20 is the deduced amino acid sequence of a maize CesA1 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:19.

SEQ ID NO:21 is the 3725 bp nucleotide sequence containing the ORF (nucleotides 179-3403) of the CesA2 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-178) and 3′ (nucleotides 3404-3725) to this ORF region.

SEQ ID NO:22 is the deduced amino acid sequence of a maize CesA2 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:21.

SEQ ID NO:23 is the 2830 bp nucleotide sequence containing the ORF (nucleotides 3-2468) of the CesA3 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-2) and 3′ (nucleotides 2469-2830) to this ORF region.

SEQ ID NO:24 is the deduced amino acid sequence of a maize CesA3 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:23.

SEQ ID NO:25 is the 3773 bp nucleotide sequence containing the ORF (nucleotides 338-3571) of the CesA4 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-337) and 3′ (nucleotides 3572-3773) to this ORF region.

SEQ ID NO:26 is the deduced amino acid sequence of a maize CesA4 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:25.

SEQ ID NO:27 is the 3704 bp nucleotide sequence containing the ORF (nucleotides 272-3502) of the CesA5 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-271) and 3′ (nucleotides 3503-3704) to this ORF region.

SEQ ID NO:28 is the deduced amino acid sequence of a maize CesA5 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:27.

SEQ ID NO:29 is the 3568 bp nucleotide sequence containing the ORF (nucleotides 63-3242) of the CesA6 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-62) and 3′ (nucleotides 3243-3568) to this ORF region.

SEQ ID NO:30 is the deduced amino acid sequence of a maize CesA6 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:29.

SEQ ID NO:31 is the 3969 bp nucleotide sequence containing the ORF (nucleotides 144-3404) of the CesA7 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-143) and 3′ (nucleotides 3405) to this ORF region.

SEQ ID NO:32 is the deduced amino acid sequence of a maize CesA7 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:31.

SEQ ID NO:33 is the 3813 bp nucleotide sequence containing the ORF (nucleotides 215-3499) of the CesA8 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-214) and 3′ (nucleotides 3500-3813) to this ORF region.

SEQ ID NO:34 is the deduced amino acid sequence of a maize CesA8 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:33.

SEQ ID NO:35 is the 3799 bp nucleotide sequence containing the ORF (nucleotides 238-3477) of the CesA9 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-237) and 3′ (nucleotides 3478-3799) to this ORF region.

SEQ ID NO:36 is the deduced amino acid sequence of a maize CesA9 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:35.

SEQ ID NO:37 is the 3470 bp nucleotide sequence containing the ORF (nucleotides 29-3265) of the CesA10 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-28) and 3′ (nucleotides 3266-3470) to this ORF region.

SEQ ID NO:38 is the deduced amino acid sequence of a maize CesA10 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:37.

SEQ ID NO:39 is the 3231 bp nucleotide sequence containing the ORF (nucleotides 21-3044) of the CesA11 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-20) and 3′ (nucleotides 3045-3231) to this ORF region.

SEQ ID NO:40 is the deduced amino acid sequence of a maize CesA11 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:39.

SEQ ID NO:41 is the 3028 bp nucleotide sequence containing the ORF (nucleotides 52-2835) of the CesA12 gene from maize flanked by additional UTR regions 5′ (nucleotides 1-51) and 3′ (nucleotides 2836-3028) to this ORF region.

SEQ ID NO:42 is the deduced amino acid sequence of a maize CesA12 polypeptide derived from the ORF of the nucleotide sequence set forth in SEQ ID NO:41.

The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and publications cited throughout the application are hereby incorporated by reference in their entirety.

As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants, reference to “a cell” includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.

In the context of this disclosure, a number of terms shall be utilized.

“Transgenic” includes any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

“Genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises within its genome a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.

“Heterologous” with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid fragment” are used interchangeably and is a polymer of RNA or DNA that is single or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.

“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product have been removed.

“Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.

“Isolated” refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. “Recombinant” also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.

“Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controlling transcription in plant cells whether or not its origin is from a plant cell.

“Operably linked” refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.

“Expression” refers to the production of a functional product. For example, expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation and transient transformation.

“Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.

“Transient transformation” refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.

“Allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. Different alleles of a gene differ in their DNA sequence. When the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant are the same that plant is homozygous at that locus. If the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant differ that plant is heterozygous at that locus. If a transgene is present on one of a pair of homologous chromosomes in a diploid plant that plant is hemizygous at that locus.

“Contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to a nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of the nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

The term “amplified” means the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.

The term “chromosomal location” includes reference to a length of a chromosome which may be measured by reference to the linear segment of DNA which it comprises. The chromosomal location can be defined by reference to two unique DNA sequences, i.e., markers.

The term “marker” includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A “polymorphic marker” includes reference to a marker which appears in multiple forms (alleles) such that different forms of the marker, when they are present in a homologous pair, allow transmission of each of the chromosomes in that pair to be followed. A genotype may be defined by use of one or a plurality of markers.

Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.

Unless otherwise stated, “BLAST” sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=⁻4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

As used herein, “any integer from 51% up to and including 100%” means 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%”.

As used herein, “any integer from 61% up to and including 100%” means 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%”.

As used herein, “any integer from 81% up to and including 100%” means 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%”.

As used herein, “80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer in between 80% and 100%” means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).

Turning now to preferred embodiments:

Preferred embodiments include isolated polynucleotides and polypeptides, recombinant DNA constructs, compositions (such as plants or seeds) comprising these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs.

Preferred Isolated Polynucleotides and Polypeptides

The present invention includes the following preferred isolated polynucleotides and polypeptides:

In one preferred embodiment, an isolated polynucleotide comprises (a) a nucleotide sequence encoding a polypeptide associated with stalk mechanical strength, wherein said polypeptide has an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer in between 80% and 100%, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16 or 18; or (b) a complement of the nucleotide sequence of (a), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary (i.e., a full-length complement of the nucleotide sequence of (a)). Preferably, the polypeptide is associated with maize stalk mechanical strength, and the amino acid sequence of the polypeptide is compared to SEQ ID NOs:16 or 18.

In another preferred embodiment, an isolated polynucleotide comprises (a) a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17, or (b) a full-length complement of said nucleic acid sequence of (a).

In another preferred embodiment, an isolated polypeptide associated with stalk mechanical strength comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer in between 80% and 100%, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16 or 18.

Several methods may be used to measure the stalk mechanical strength of plants. Preferably, the mechanical strength may be measured with an electromechanical test system. In the case of maize stalk mechanical strength, in a preferred method, the internodes below the ear may be subjected to a three-point bend test using an Instron, Model 4411 (Instron Corporation, 100 Royall Street, Canton, Mass. 02021), with a span-width of 200 mm between the anchoring points and a speed of 200 mm/minute of the third point attached to a load cell; the load needed to break the internode can be used as a measure of mechanical strength (hereinafter “the three-point bend test”). Internodal breaking strength has been shown to be highly correlated with the amount of cellulose per unit length of the maize stalk (see U.S. Patent Application No. 2004068767 A1, herein incorporated by reference).

A polypeptide is “associated with stalk mechanical strength” in that the absence of the polypeptide in a plant results in a reduction of stalk mechanical strength of the plant when compared to a control plant that expresses the polypeptide.

A polypeptide is “associated with maize stalk mechanical strength” in that the absence of the polypeptide in a maize plant results in a reduction of stalk mechanical strength of the maize plant when compared to a control maize plant that expresses the polypeptide.

It is understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences. Alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

Preferred Recombinant DNA Constructs and Suppression DNA Constructs

The present invention also includes a recombinant DNA construct comprising at least one polynucleotide operably linked to at least one regulatory sequence (e.g., preferably, a promoter that is functional in said plant), wherein said polynucleotide comprises any isolated polynucleotide of the present invention.

In one preferred embodiment, a recombinant DNA construct comprises a promoter that is functional in a plant operably linked to (a) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a).

In another preferred embodiment, a recombinant DNA construct comprises a promoter that is functional in a plant operably linked to (a) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17, or (b) a full-length complement of said polynucleotide of (a).

The present invention also includes a suppression DNA construct.

In one preferred embodiment, a suppression DNA construct comprises a promoter functional in a plant operably linked to (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a polypeptide selected from the group consisting of Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9.

“Suppression DNA construct” is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in “silencing” of a target gene in the plant. The target gene may be endogenous or transgenic to the plant. “Silencing,” as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality. The term “suppression” includes lower, reduce, decline, decrease, inhibit, eliminate and prevent. “Silencing” or “gene silencing” does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending upon the approach to be utilized, the region may be 100% identical or less than 100% identical (e.g., at least 50% or any integer between 51% and 100% identical) to all or part of the sense strand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.

“Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcripts capable of suppressing the expression of the target protein. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al. (1998) Plant J. 16:651-659; and Gura (2000) Nature 404:804-808).

Another variation describes the use of plant viral sequences to direct the suppression of proximal mRNA encoding sequences (PCT Publication WO 98/36083 published on Aug. 20, 1998).

Recent work has described the use of “hairpin” structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential “stem-loop” structure for the expressed RNA (PCT Publication WO 99/53050 published on Oct. 21, 1999). In this case the stem is formed by polynucleotides corresponding to the gene of interest inserted in either sense or anti-sense orientation with respect to the promoter and the loop is formed by some polynucleotides of the gene of interest, which do not have a complement in the construct. This increases the frequency of cosuppression or silencing in the recovered transgenic plants. For review of hairpin suppression see Wesley, S. V. et al. (2003) Methods in Molecular Biology, Plant Functional Genomics: Methods and Protocols 236:273-286.

A construct where the stem is formed by at least 30 nucleotides from a gene to be suppressed and the loop is formed by a random nucleotide sequence has also effectively been used for suppression (WO 99/61632 published on Dec. 2, 1999).

The use of poly-T and poly-A sequences to generate the stem in the stem-loop structure has also been described (WO 02/00894 published Jan. 3, 2002).

Yet another variation includes using synthetic repeats to promote formation of a stem in the stem-loop structure. Transgenic organisms prepared with such recombinant DNA fragments have been shown to have reduced levels of the protein encoded by the nucleotide fragment forming the loop as described in PCT Publication WO 02/00904, published 3 Jan. 2002.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., Nature 391:806 1998). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing (PTGS) or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., Trends Genet. 15:358 1999). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., Nature 409:363 (2001)). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir et al., Genes Dev. 15:188 (2001)). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., Science 293:834 (2001)). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementarity to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., Genes Dev. 15:188 (2001)). In addition, RNA interference can also involve small RNA (e.g., miRNA) mediated gene silencing, presumably through cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see, e.g., Allshire, Science 297:1818-1819 (2002); Volpe et al., Science 297:1833-1837 (2002); Jenuwein, Science 297:2215-2218 (2002); and Hall et al., Science 297:2232-2237 (2002)). As such, miRNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al. (Nature 391:806 (1998)) were the first to observe RNAi in C. elegans. Wianny and Goetz (Nature Cell Biol. 2:70 (1999)) describe RNAi mediated by dsRNA in mouse embryos. Hammond et al. (Nature 404:293 (2000)) describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., (Nature 411:494 (2001)) describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells.

Small RNAs play an important role in controlling gene expression. Regulation of many developmental processes, including flowering, is controlled by small RNAs. It is now possible to engineer changes in gene expression of plant genes by using transgenic constructs which produce small RNAs in the plant.

Small RNAs appear to function by base-pairing to complementary RNA or DNA target sequences. When bound to RNA, small RNAs trigger either RNA cleavage or translational inhibition of the target sequence. When bound to DNA target sequences, it is thought that small RNAs can mediate DNA methylation of the target sequence. The consequence of these events, regardless of the specific mechanism, is that gene expression is inhibited.

It is thought that sequence complementarity between small RNAs and their RNA targets helps to determine which mechanism, RNA cleavage or translational inhibition, is employed. It is believed that siRNAs, which are perfectly complementary with their targets, work by RNA cleavage. Some miRNAs have perfect or near-perfect complementarity with their targets, and RNA cleavage has been demonstrated for at least a few of these miRNAs. Other miRNAs have several mismatches with their targets, and apparently inhibit their targets at the translational level. Again, without being held to a particular theory on the mechanism of action, a general rule is emerging that perfect or near-perfect complementarity causes RNA cleavage, whereas translational inhibition is favored when the miRNA/target duplex contains many mismatches. The apparent exception to this is microRNA 172 (miR172) in plants. One of the targets of miR172 is APETALA2 (AP2), and although miR172 shares near-perfect complementarity with AP2 it appears to cause translational inhibition of AP2 rather than RNA cleavage.

MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24 nucleotides (nt) in length that have been identified in both animals and plants (Lagos-Quintana et al., Science 294:853-858 (2001), Lagos-Quintana et al., Curr. Biol. 12:735-739 (2002); Lau et al., Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001); Llave et al., Plant Cell 14:1605-1619 (2002); Mourelatos et al., Genes. Dev. 16:720-728 (2002); Park et al., Curr. Biol. 12:1484-1495 (2002); Reinhart et al., Genes. Dev. 16:1616-1626 (2002)). They are processed from longer precursor transcripts that range in size from approximately 70 to 200 nt, and these precursor transcripts have the ability to form stable hairpin structures. In animals, the enzyme involved in processing miRNA precursors is called Dicer, an RNAse III-like protein (Grishok et al., Cell 106:23-34 2001; Hutvagner et al., Science 293:834-838 (2001); Ketting et al., Genes. Dev. 15:2654-2659 (2001)). Plants also have a Dicer-like enzyme, DCL1 (previously named CARPEL FACTORY/SHORT INTEGUMENTS1/SUSPENSOR1), and recent evidence indicates that it, like Dicer, is involved in processing the hairpin precursors to generate mature miRNAs (Park et al., Curr. Biol. 12:1484-1495 (2002); Reinhart et al., Genes. Dev. 16:1616-1626 (2002)). Furthermore, it is becoming clear from recent work that at least some miRNA hairpin precursors originate as longer polyadenylated transcripts, and several different miRNAs and associated hairpins can be present in a single transcript (Lagos-Quintana et al., Science 294:853-858 (2001); Lee et al., EMBO J 21:4663-4670 2002). Recent work has also examined the selection of the miRNA strand from the dsRNA product arising from processing of the hairpin by DICER (Schwartz, et al. Cell 115:199-208 (2003)). It appears that the stability (i.e. G:C vs. A:U content, and/or mismatches) of the two ends of the processed dsRNA affects the strand selection, with the low stability end being easier to unwind by a helicase activity. The 5′ end strand at the low stability end is incorporated into the RISC complex, while the other strand is degraded.

MicroRNAs appear to regulate target genes by binding to complementary sequences located in the transcripts produced by these genes. In the case of 11n-4 and let-7, the target sites are located in the 3′ UTRs of the target mRNAs (Lee et al., Cell 75:843-854 (1993); Wightman et al., Cell 75:855-862 (1993); Reinhart et al., Nature 403:901-906 (2000); Slack et al., Mol. Cell 5:659-669 (2000)), and there are several mismatches between the lin-4 and let-7 miRNAs and their target sites. Binding of the lin-4 or let-7 miRNA appears to cause downregulation of steady-state levels of the protein encoded by the target mRNA without affecting the transcript itself (Olsen and Ambros, Dev. Biol. 216:671-680 (1999)). On the other hand, recent evidence suggests that miRNAs can in some cases cause specific RNA cleavage of the target transcript within the target site, and this cleavage step appears to require 100% complementarity between the miRNA and the target transcript (Hutvagner and Zamore, Science 297:2056-2060 (2002); Llave et al., Plant Cell 14:1605-1619 (2002)). It seems likely that miRNAs can enter at least two pathways of target gene regulation: Protein downregulation when target complementarity is <100%, and RNA cleavage when target complementarity is 100%. MicroRNAs entering the RNA cleavage pathway are analogous to the 21-25 nt short interfering RNAs (siRNAs) generated during RNA interference (RNAi) in animals and posttranscriptional gene silencing (PTGS) in plants (Hamilton and Baulcombe (1999); Hammond et al., (2000); Zamore et al., (2000); Elbashir et al., (2001)), and likely are incorporated into an RNA-induced silencing complex (RISC) that is similar or identical to that seen for RNAi.

Identifying the targets of miRNAs with bioinformatics has not been successful in animals, and this is probably due to the fact that animal miRNAs have a low degree of complementarity with their targets. On the other hand, bioinformatic approaches have been successfully used to predict targets for plant miRNAs (Llave et al., Plant Cell 14:1605-1619 (2002); Park et al., Curr. Biol. 12:1484-1495 (2002); Rhoades et al., Cell 110:513-520 (2002)), and thus it appears that plant miRNAs have higher overall complementarity with their putative targets than do animal miRNAs. Most of these predicted target transcripts of plant miRNAs encode members of transcription factor families implicated in plant developmental patterning or cell differentiation.

Preferred regulatory elements of recombinant DNA constructs and suppression DNA constructs

A number of promoters can be used in recombinant DNA constructs and suppression DNA constructs of the present invention. The promoters can be selected based on the desired outcome, and may include constitutive, tissue-specific, inducible, or other promoters for expression in the host organism.

High level, constitutive expression of the candidate gene under control of the 35S promoter may have pleiotropic affects. However, tissue specific and/or stress-specific expression may eliminate undesirable affects but retain the ability to enhance drought tolerance. This affect has been observed in Arabidopsis (Kasuga et al., Nature Biotechnol. 17:287-91 (1999)). As such, candidate gene efficacy may be tested when driven by different promoters.

Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

In choosing a promoter to use in the methods of the invention, it may be desirable to use a tissue-specific or developmentally regulated promoter. A tissue-specific or developmentally regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in the cells/tissues of a plant critical to tassel development, seed set, or both, and limits the expression of such a DNA sequence to the period of tassel development or seed maturation in the plant. Any identifiable promoter may be used in the methods of the present invention which causes the desired temporal and spatial expression.

A preferred stalk-specific promoter is the alfalfa stalk-specific S2A gene (Abrahams et al., Plant Mol. Biol. 27:513-528 (1995))

Promoters which are seed or embryo specific and may be useful in the invention include soybean Kunitz trysin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1:1079-1093 (1989)), patatin (potato tubers) (Rocha-Sosa et al., EMBO J. 8:23-29 (1989)), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W. G., et al. Mol. Gen. Genet. 259:149-157 (1991); Newbigin, E. J., et al., Planta 180:461-470 (1990); Higgins, T. J. V., et al., Plant. Mol. Biol. 11:683-695 (1988)), zein (maize endosperm) (Schemthaner, J. P., et al., EMBO J. 7:1249-1255 (1988)), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)), phytohemagglutinin (bean cotyledon) (Voelker, T. et al., EMBO J. 6:3571-3577 (1987)), B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, et al., EMBO J. 7:297-302 (1988)), glutelin (rice endosperm), hordein (barley endosperm) (Marris, C., et al., Plant Mol. Biol. 10:359-366 (1988)), glutenin and gliadin (wheat endosperm) (Colot, V., et al., EMBO J. 6:3559-3564 (1987)), and sporamin (sweet potato tuberous root) (Hattori, T., et al., Plant Mol. Biol. 14:595-604 (1990)). Promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructions maintain their temporal and spatial expression pattern in transgenic plants. Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds (Vanderkerckhove et al., Bio/Technology 7:L929-932 (1989)), bean lectin and bean beta-phaseolin promoters to express luciferase (Riggs et al., Plant Sci. 63:47-57 (1989)), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al., EMBO J. 6:3559-3564 (1987)).

Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.

Promoters which are timed to stress include the following: 1) the RD29A promoter (Kasuga et al., Nature Biotechnol. 17:287-291 (1991)); 2) barley promoter, B22E; expression of B22E is specific to the pedicel in developing maize kernels (“Primary Structure of a Novel Barley Gene Differentially Expressed in Immature Aleurone Layers”. Klemsdae, S. S. et al., Mol. Gen. Genet. 228(1/2):9-16 (1991)); and 3) maize promoter, Zag2 (“Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS”, Schmidt, R. J. et al., Plant Cell 5(7):729-737 (1993)). Zag2 transcripts can be detected 5 days prior to pollination to 7 to 8 DAP, and directs expression in the carpel of developing female inflorescences and Ciml which is specific to the nucleus of developing maize kernels. Ciml transcript is detected 4 to 5 days before pollination to 6 to 8 DAP. Other useful promoters include any promoter which can be derived from a gene whose expression is maternally associated with developing female florets.

Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B., Biochemistry of Plants 15:1-82 (1989).

Particularly preferred promoters may include: alfalfa stalk-specific S2A gene promoter, RIP2, mLIP15, ZmCOR1, Rab17, CaMV 35S, RD29A, SAM synthetase, ubiquitin, CaMV 19S, nos, Adh, sucrose synthase, R-allele, or root cell promoter. Other preferred promoters include any of the CesA10, CesA11, and CesA12 promoters disclosed in United States Patent Publication 200510086712A1, which is hereby incorporated by reference in its entirety.

Recombinant DNA constructs and suppression DNA constructs of the present invention may also include other regulatory sequences, including but not limited to, translation leader sequences, introns, and polyadenylation recognition sequences. In another preferred embodiment of the present invention, a recombinant DNA construct of the present invention further comprises an enhancer or silencer.

An intron sequence can be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).

If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.

A translation leader sequence is a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D., Molecular Biotechnology 3:225 (1995)).

Any plant can be selected for the identification of regulatory sequences and genes to be used in creating recombinant DNA constructs and suppression DNA constructs of the present invention. Examples of suitable plant targets for the isolation of genes and regulatory sequences would include but are not limited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radiscchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat, yams, and zucchini. Particularly preferred plants for the identification of regulatory sequences are Arabidopsis, corn, wheat, soybean, and cotton.

In another preferred embodiment of the present invention, a recombinant DNA construct of the present invention further comprises an enhancer.

Preferred Compositions

A preferred composition of the present invention is a plant comprising in its genome any of the recombinant DNA constructs of the present invention (such as those preferred constructs discussed above).

Another preferred composition is a plant whose genome comprises a disruption (e.g., an insertion, such as a transposable element, or sequence mutation) of at least one gene (which may be heterologous or endogenous to the genome) selected from the group consisting of Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9.

Still another preferred composition is a plant whose genome comprises other recombinant DNA constructs as discussed below (e.g., constructs involving nucleic acid sequences and amino acid sequences relating to SEQ ID NOs:20-42).

Preferred compositions also include any progeny of the plant, and any seed obtained from the plant or its progeny. Progeny includes subsequent generations obtained by self-pollination or out-crossing of a plant. Progeny also includes hybrids and inbreds.

Preferably, in hybrid seed propagated crops, mature transgenic plants can be self-crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced recombinant DNA construct. These seeds can be grown to produce plants that would contain the recombinant DNA construct in its genome and exhibit the associated phenotype(s) as described herein, or used in a breeding program to produce hybrid seed, which can be grown to produce plants that would contain the recombinant DNA construct and exhibit the associated phenotype(s) as described herein. Preferably, the seeds are maize.

Preferably, the plant is a monocotyledonous or dicotyledonous plant, more preferably, a maize or soybean plant, even more preferably a maize plant, such as a maize hybrid plant or a maize inbred plant. The plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley or millet.

Preferably, any recombinant DNA construct is stably integrated into the genome of the plant.

Particularly preferred embodiments include:

1. A plant (preferably maize) comprising in its genome a recombinant DNA construct comprising at least one regulatory element operably linked to (a) a nucleotide sequence encoding a polypeptide associated with stalk mechanical strength, wherein said polypeptide has an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer in between 80% and 100%, based on the Clustal V method of alignment, when compared to SEQ ID NOs:16 or 18; or (b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary (i.e., a full length complement of the nucleotide sequence of (a)). Preferably, the at least one regulatory element is a promoter that is functional in a plant.

2. A plant (preferably maize) comprising in its genome (a) a first recombinant DNA construct comprising at least one promoter that is functional in a plant operably linked to at least one of a first isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) a second recombinant DNA construct comprising at least one promoter that is functional in a plant operably linked to at least one of a second isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii). Preferably, the plant exhibits increased cell wall cellulose content and/or enhanced growth rate when compared to a control plant not comprising said first recombinant DNA construct and said second recombinant DNA construct.

3. A plant (preferably maize) comprising in its genome at least one regulatory sequence operably linked to (a) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii). Preferably, the plant exhibits increased cell wall cellulose content and/or enhanced growth rate when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).

4. A plant (preferably maize) comprising in its genome at least one regulatory sequence operably linked to (a) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:2; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:1; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:38, 40, and 42; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:37, 39, and 41; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii). Preferably, the plant exhibits increased cell wall cellulose content when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).

5. A plant (preferably maize) comprising in its genome at least one regulatory sequence operably linked to (a) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:5; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 32, and 34; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:19, 31, and 33; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii). Preferably, the plant exhibits enhanced growth rate when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).

6. A plant (preferably maize) comprising in its genome at least one regulatory sequence operably linked to at least two isolated polynucleotides selected from the group consisting of (a) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (b) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (c) a full-length complement of the polynucleotide of (a) or (b).

7. A plant (preferably maize) comprising in its genome a suppression DNA construct comprising a promoter functional in a plant operably linked to (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or (ii) a full complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a polypeptide selected from the group consisting of BK2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9. Preferably, the plant exhibits reduced stalk mechanical strength when compared to a control plant not comprising said suppression DNA construct. Preferably, the suppression DNA construct comprises a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double-stranded RNA-producing construct, RNAi construct, or small RNA construct (e.g., an siRNA construct or an miRNA construct).

8. A plant (preferably maize) whose genome comprises a disruption of at least one gene encoding a polypeptide selected from the group consisting of BK2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9. Preferably, the disruption results in said plant exhibiting reduced stalk mechanical strength when compared to a control plant not comprising said disruption. Preferably, the disruption comprises an insertion, such as a transposable element or sequence mutation.

9. A plant (preferably maize) comprising in its genome a suppression DNA construct comprising a promoter functional in a plant operably linked to (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6, or (ii) a full complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L3 polypeptide. Preferably, the plant exhibits reduced plant height and/or reduced organ size when compared to a control plant not comprising said suppression DNA construct. Preferably, the suppression DNA construct comprises a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double-stranded RNA-producing construct, RNAi construct, or small RNA construct (e.g., an siRNA construct or an miRNA construct).

10. A plant (preferably maize) whose genome comprises a disruption of at least one gene encoding a Bk2L3 polypeptide. Preferably, said disruption results in said plant exhibiting reduced plant height and/or reduced organ size when compared to a control plant not comprising said disruption. Preferably, the disruption comprises an insertion, such as a transposable element or sequence mutation.

11. A plant (preferably maize) comprising in its genome a suppression DNA construct comprising a promoter functional in a plant operably linked to (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:10, or (ii) a full complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L5 polypeptide. Preferably, the plant exhibits male sterility when compared to a control plant not comprising said suppression DNA construct. Preferably, the suppression DNA construct comprises a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double-stranded RNA-producing construct, RNAi construct, or small RNA construct (e.g., an siRNA construct or an miRNA construct).

12. A plant (preferably maize) whose genome comprises a disruption of at least one gene encoding a BKL5 polypeptide. Preferably, the disruption results in said plant exhibiting male sterility when compared to a control plant not comprising said disruption. Preferably, the disruption comprises an insertion, such as a transposable element or sequence mutation.

13. Any progeny of the above plants, any seeds of the above plants, any seeds of progeny of the above plants, and cells from any of the above plants and progeny.

One of ordinary skill in the art would readily recognize a suitable control or reference plant for use in comparing or measuring relative to a plant comprising within its genome a recombinant DNA construct (or suppression DNA construct). For example, by way of non-limiting illustrations:

• • Progeny of a transformed plant which is hemizygous with respect to a recombinant DNA construct (or suppression DNA construct), such that the progeny are segregating into plants either comprising or not comprising the recombinant DNA construct (or suppression DNA construct): progeny comprising the recombinant DNA construct (or suppression DNA construct) would be typically measured relative to the progeny not comprising the recombinant DNA construct (or suppression DNA construct).
  • Introgression of a recombinant DNA construct (or suppression DNA construct) into an inbred line, such as in corn, or into a variety, such as in soybean: the introgressed line would typically be measured relative to the parent inbred or variety line.
  • Two hybrid lines, where the first hybrid line is produced from two parent inbred lines, and the second hybrid line is produced from the same two parent inbred lines except that one of the parent inbred lines contains a recombinant DNA construct (or suppression DNA construct): the second hybrid line would typically be measured relative to the first hybrid line.
  • A plant comprising a recombinant DNA construct (or suppression DNA construct) in its genome (or a plant comprising a disruption of a gene in its genome): the plant may be measured relative to a control plant not comprising the recombinant DNA construct (or suppression DNA construct) in its genome (or to a control plant not comprising the disruption) but otherwise having a comparable genetic background to the plant (e.g., sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of nuclear genetic material compared to the plant comprising the recombinant DNA construct or suppression DNA construct or disruption). There are many laboratory-based techniques available for the analysis, comparison and characterization of plant genetic backgrounds; among these are Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats (SSRs) which are also referred to as Microsatellites.

The introduction of recombinant DNA constructs of the present invention into plants may be carried out by any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector mediated DNA transfer, bombardment, or Agrobacterium mediated transformation. Where multiple or stacked recombinant DNA constructs or isolated polynucleotides are desired to be integrated into the genome (e.g., to effect co-expression of two or more isolated polynucleotides), the individual isolated polynucleotides may be introduced into parent lines and crossed through traditional breeding techniques to provide the desired combination or stack in subsequent progeny plants.

Preferred techniques are set forth below in Example 3 for transformation of maize plant cells and in Example 8 for transformation of soybean plant cells.

Other preferred methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants include those published for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011, McCabe et. al., Bio/Technology 6:923 (1988), Christou et al., Plant Physiol. 87:671 674 (1988)); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653 657 (1996), McKently et al., Plant Cell Rep. 14:699 703 (1995)); papaya; and pea (Grant et al., Plant Cell Rep. 15:254 258, (1995)).

Transformation of monocotyledons using electroporation, particle bombardment, and Agrobacterium have also been reported and are included as preferred methods, for example, transformation and plant regeneration as achieved in asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux, Plant Physiol 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204 (1988), Gordon-Kamm et al., Plant Cell 2:603 618 (1990), Fromm et al., Bio/Technology 8:833 (1990), Koziel et al., Bio/Technology 11: 194, (1993), Armstrong et al., Crop Science 35:550 557 (1995)); oat (Somers et al., BiolTechnology 10: 15 89 (1992)); orchard grass (Horn et al., Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppl. Genet. 205:34, (1986); Part et al., Plant Mol. Biol. 32:1135 1148, (1996); Abedinia et al., Aust. J. Plant Physiol. 24:133 141 (1997); Zhang and Wu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant Cell Rep. 7:379, (1988); Battraw and Hall, Plant Sci. 86:191 202 (1992); Christou et al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature 325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992)); tall fescue (Wang et al., Bio/Technology 10:691 (1992)), and wheat (Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated.

The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif., (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign, exogenous isolated nucleic acid fragment that encodes a protein of interest is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art. Assays to detect proteins may be performed by SDS-polyacrylamide gel electrophoresis or immunological assays. Assays to detect levels of substrates or products of enzymes may be performed using gas chromatography or liquid chromatography for separation and UV or visible spectrometry or mass spectrometry for detection, or the like. Determining the levels of mRNA of the enzyme of interest may be accomplished using northern-blotting or RT-PCR techniques. Once plants have been regenerated, and progeny plants homozygous for the transgene have been obtained, plants will have a stable phenotype that will be observed in similar seeds in later generations.

Preferred Methods

The present invention also includes methods for altering stalk mechanical strength in a plant; methods for evaluating stalk mechanical strength in a plant; methods for evaluating cellulose content in plant; methods for altering cell wall cellulose content and/or growth rate in a plant, methods for conferring male sterility in a plant, and methods for reducing plant height and/or organ size in a plant. Preferably, the plant is a monocotyledonous or dicotyledonous plant, more preferably, a maize or soybean plant, even more preferably a maize plant. The plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley or millet.

A preferred method for altering (preferably increasing) stalk mechanical strength of a plant comprises (a) introducing a recombinant DNA construct into a regenerable plant cell to produce a transformed plant cell, the recombinant DNA construct comprising at least one regulatory element (preferably, a promoter that is functional in a plant) operably linked to (i) a nucleotide sequence encoding a polypeptide associated with stalk mechanical strength, wherein said polypeptide has an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer in between 80% and 100%, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16 or 18, or (ii) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary; and (b) regenerating a transgenic plant from said transformed plant cell, wherein said transgenic plant comprises in its genome said recombinant DNA construct and wherein said transgenic plant exhibits an alteration (preferably an increase) in stalk mechanical strength, when compared to a control plant not comprising said recombinant DNA construct.

A preferred method of evaluating stalk mechanical strength in a plant comprises (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (ii) a full-length complement of said polynucleotide of (a)(i); (b) regenerating a transgenic plant from said transformed plant cell; and (c) evaluating said transgenic plant for stalk mechanical strength. This method may further comprise (d) obtaining a progeny plant derived from said transgenic plant; and (e) evaluating said progeny plant for stalk mechanical strength.

Another preferred method of evaluating stalk mechanical strength in a plant comprises (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a)(i); (b) regenerating a transgenic plant from said transformed plant cell; (c) obtaining a progeny plant derived from said transgenic plant; and (d) evaluating said progeny plant for stalk mechanical strength.

A preferred method of evaluating cellulose content in a plant, comprises (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a polynucleotide operably linked to a promoter that is functional in a plant, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18; (b) regenerating a transgenic plant from said transformed plant cell; and (c) evaluating said transgenic plant for cellulose content. This method may further comprise (d) obtaining a progeny plant derived from said transgenic plant; and (e) evaluating said progeny plant for cellulose content.

Another preferred method of evaluating cellulose content in a plant comprises (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a polynucleotide operably linked to a promoter that is functional in a plant, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18; (b) regenerating a transgenic plant from said transformed plant cell; (c) obtaining a progeny plant derived from said transgenic plant; and (d) evaluating said progeny plant for cellulose content.

A preferred method for selecting a plant with altered cellulose content comprises (a) obtaining any plant of the present invention (such as any of the preferred embodiments discussed above); (b) evaluating the plant obtained in step (a) for cellulose content; and (c) selecting the evaluated plant of step (b) when its cellulose content is altered when compared to a control plant. Preferably, the evaluated plant is selected when its cellulose content is increased, even more preferably, when the cellulose content is at least 35%, 40%, 45%, 50%, 55%, or 60% and/or when the cellulose dry matter content is at least 100 mg/cm, 200 mg/cm, 300 mg/cm, 400 mg/cm, or 500 mg/cm. Preferred methods for measuring cellose content are set forth herein in Example 10.

A preferred method for altering (preferably increasing) cell wall cellulose content and/or for altering (preferably enhancing) growth rate in a plant comprises integrating (e.g., through transgenic techniques or a combination of transgenic techniques and traditional breeding) into the genome of a plant one or more recombinant DNA constructs such that the co-expression is obtained of (a) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 81% sequence identity, or any integer up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; (ii) a polynucleotide having a nucleic acid sequence of at least 61% sequence identity, or any integer up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii).

Preferably, a method for increasing cell wall cellulose content in a plant comprises integrating into the genome of a plant one or more recombinant DNA constructs such that the co-expression is obtained of (a) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:2; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:1; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:38, 40, and 42; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:37, 39, and 41; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii).

Preferably, a method for enhancing plant growth rate comprises integrating into the genome of a plant one or more recombinant DNA constructs such that the co-expression is obtained of (a) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:5; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and (b) at least one isolated polynucleotide selected from the group consisting of (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, or any integer from 81% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 32, and 34; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, or any integer from 61% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:19, 31, and 33; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii).

A preferred method of conferring male sterility in a plant comprises: (a) introducing into a regenerable plant cell a suppression DNA construct comprising a promoter functional in a plant operably linked to (i) all or part of (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:10, or (B) a full complement of the nucleic acid sequence of (i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L5 polypeptide; and (b) regenerating a transgenic plant from said transformed plant cell, wherein said transgenic plant comprises in its genome said suppression DNA construct and wherein said transgenic plant exhibits reduced plant height and/or reduced organ size when compared to a control plant not comprising said suppression DNA construct. The method may further comprise: (c) obtaining a progeny plant derived from said transgenic plant, wherein said progeny plant comprises in its genome the suppression DNA construct.

A preferred method of reducing plant height and/or reducing organ size in a plant comprises: (a) introducing into a regenerable plant cell a suppression DNA construct comprising a promoter functional in a plant operably linked to (i) all or part of (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6, or (B) a full complement of the nucleic acid sequence of (i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, or any integer from 51% up to and including 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L3 polypeptide; and (b) regenerating a transgenic plant from said transformed plant cell, wherein said transgenic plant comprises in its genome said suppression DNA construct and wherein said transgenic plant exhibits reduced plant height and/or reduced organ size when compared to a control plant not comprising said suppression DNA construct. The method may further comprise: (c) obtaining a progeny plant derived from said transgenic plant, wherein said progeny plant comprises in its genome the suppression DNA construct.

The isolated nucleic acids and proteins and any embodiments of the present invention can be used over a broad range of plant types, particularly monocots such as the species of the Family Graminiae including Sorghum bicolor and Zea mays. The isolated nucleic acid and proteins of the present invention can also be used in species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Triticum, Bambusa, Dendrocalamus, and Melocanna.

EXAMPLES

The present invention is further illustrated in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1

Characterization of Maize cDNA Encoding Bk2-like Proteins

The maize brittle stalk 2 (bk2) phenotype was first reported in 1940 (Langham, MNL 14:21-22 (1940)), and was mapped by phenotype to chr9L between the markers umc95 and bnl7.13 around the 100 centiMorgan region (Howell et al., MNL 65:52-53 (1991)). Previously, clone csc1c.pk005.k4:fis (SEQ ID NO:1) was shown to encode a BRITTLE STALK 2 polypeptide (SEQ ID NO:2) (International Application No. PCT/US2005/035450 which claims the benefit of U.S. Provisional Application No. 60/615,868, filed Oct. 6, 2004, the entire contents of which are herein incorporated by reference). Also disclosed were two other members of the Bk2 gene family (SEQ ID NOs:7 and 8 and SEQ ID NOs:13 and 14). In the instant disclosure these genes have been named as Bk2-like (Bk2L).

Search for additional maize cDNA sequences homologous at the nucleic acid and amino acid level to the maize BRITTLE STALK 2 (Bk2) sequence (SEQ ID NO:1) was conducted using BLASTN or TBLASTN algorithm provided by the National Center for Biotechnology Information (NCBI) against several databases, including, but not limited to, DuPont's internal proprietary database (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)) and publicly available Maize Genomic Survey Sequences (GSS) and TIGR Maize genomic assemblies (The TIGR Gene Index Databases, The Institute for Genomic Research, Rockville, Md. 20850; Quackenbush et al., J. Nucleic Acids Res. 28(1):141-145 (2000)). Six new members of the Bk2 gene family were isolated (Bk2L1, Bk2L3, Bk2L5, Bk2L6, Bk2L8 and Bk2L9). Table 1 lists all the Bk2-like proteins disclosed in the instant specification, in addition to Bk2 itself.

TABLE 1
Brittle Stalk 2-like Proteins
	SEQ ID NO:
	Protein	Nucleotide	Amino Acid
	Bk2	1	2
	Bk2L1	3	4
	Bk2L3	5	6
	Bk2L4	7	8
	Bk2L5	9	10
	Bk2L6	11	12
	Bk2L7	13	14
	Bk2L8	15	16
	Bk2L9	17	18

FIGS. 1A-1F show a Clustal V alignment, using default parameters, of the amino acid sequences reported in Table 1. FIG. 2 is a chart setting forth a comparison of the percent identity (and percent divergence in the lower half triangle), using the Clustal V alignment method, between the nine amino acid sequences shown in FIGS. 1A-1F.

The possible function of the polypeptide encoded by each cDNA was further identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)) searches of the ESTs against public databases. The searches were conducted for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The sequences were analyzed for similarity using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish, W. and States, D. J., Nature Genetics 3:266-272 (1993)) provided by the NCBI. Shown in Table 2 are the “Score” results obtained for the amino acid sequences of the entire Bk2-like proteins encoded by the entire cDNA inserts comprising the indicated cDNA clones. The data in Table 2 also presents the results obtained for the calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16 and 18, with the sequences identified in the NCBI General Identifier No. column.

TABLE 2
BLAST Results for Sequences Encoding Polypeptides
Homologous to Bk2-like Proteins
	NCBI General
Gene	Identifier No.		Percent
(SEQ ID NO:)	(Accession No.)	Score (bits)	Identity
Bk2L1	NCBI GI 34733385	1266	100%
(SEQ ID NO: 3)	(AAQ81633.1)
Bk2L3	NCBI GI 30090026	868	95%
(SEQ ID NO: 5)	(AAO17706.1)
Bk2L4	NCBI GI 30090026	922	97%
(SEQ ID NO: 7)	(AAO17706.1)
Bk2L5	NCBI GI 52076665	1079	79%
(SEQ ID NO: 9)	(BAD45565.1)
Bk2L6	NCBI GI 50939113	742	81%
(SEQ ID NO: 11)	(XP_479084.1)
Bk2L7	NCBI GI 50939113	751	82%
(SEQ ID NO: 13)	(XP_479085.1)
Bk2L8	NCBI GI 34898176	838	65%
(SEQ ID NO: 15)	(NP_910434.1)
Bk2L9	NCBI GI 50927043	597	63%
(SEQ ID NO: 17)	(XP_473354.1)

FIG. 6 shows the phylogenetic analysis of the Bk2L proteins from maize, BC1L proteins from rice, and COBL proteins from Arabidopsis (NCBI Accession Nos. are in parenthesis). The numbers along the branches are the bootstrap values obtained from a heuristic search over 5,000 replications. The bootstrap values for only the monophyletic groups that were supported >50% of the time are shown. The branch lengths are proportional to the inferred amino acid differences.

Example 2

Gene Expression Analysis of Bk2-Like Proteins

The tissue specificity of expression of the Bk2-like gene family disclosed in Table 1 was examined using Solexa's Massively Parallel Signature Sequencing (MPSS™) technology (see Table 3) (Brenner et al., Nat. Biotechnol. 18:630-634 (2000); Brenner et al., Proc. Natl. Acad. Sci. U.S.A. 97:1665-1670 (2000)). MPSS™ involves the generation of seventeen base signature tags from mRNA samples that have been reverse transcribed. The tags are simultaneously sequenced and assigned to genes or ESTs. The abundance of these tags is given a number value that is normalized to parts per million (PPM) which then allows the tag expression, or tag abundance, to be compared across different tissues. Thus, the MPSS™ platform can be used to determine the expression pattern of a particular gene and its expression level in different tissues. The numbers are averages over multiple libraries for each tissue listed in the second column.

TABLE 3
Expression in PPM of the Bk2 Gene Family in Maize
Tissue	Lib. #	Bk2	Bk2L1	Bk2L3	Bk2L4	Bk2L5	Bk2L6	Bk2L7	Bk2L8	Bk2L9
anther	3	1	73	49	51	0	0	0	9	1
ear	17	0	48	17	30	0	0	0	0	0
embryo	10	0	18	61	18	0	0	0	0	3
endosperm	26	0	18	48	23	0	4	1	3	0
husk	1	75	68	490	16	0	0	39	0	0
kernel	5	2	86	103	51	0	1	1	15	1
leaf	46	17	20	87	10	0	0	0	32	0
meristem	20	2	60	81	19	0	0	0	2	0
pericarp	6	4	16	290	54	0	0	0	2	0
pollen	2	0	6	2	13	794	0	0	0	0
root	43	52	69	263	14	0	0	2	8	0
seedling	7	8	16	72	21	0	0	0	8	0
silk	9	0	36	69	47	29	0	2	0	0
spikelet	12	17	86	205	111	0	0	12	0	0
stalk	15	172	48	474	15	0	0	8	14	0
tassel	2	4	72	53	62	0	0	16	0	0
vascular	2	182	56	117	11	0	0	0	7	0
bundles
whorl	7	152	9	126	33	0	0	0	2	2

Bk2 (Table 3, column 3 and FIG. 3) is expressed in husk, leaf, root, stalk and isolated vascular bundles, but not in the kernel, meristem, pollen or silk tissues. This expression pattern is consistent with the role of the Bk2 gene in secondary wall formation as all the tissues it is expressed in contain at least some lignified cells. The correlation coefficient analysis of the expression level of Bk2 with the expression levels of the twelve maize CesA genes is shown in FIG. 4 (also see FIG. 5A, column 2). The expression pattern of the Bk2 gene is very similar to that of the previously disclosed secondary wall-forming CesA genes, CesA10, 11 and 12 (see FIG. 5 of U.S. Pat. No. 6,930,225, granted Aug. 16, 2005, the entire contents of which are herein incorporated by reference). More specifically, Bk2 shows a higher correlation coefficient, approximately >0.8, with each of the maize CesA10, 11, and 12 genes than with any other gene in this class. Since the three CesA genes are also co-expressed, it is likely that their corresponding proteins form a functional complex along with the Bk2 protein. Table 4 lists all the primary and secondary wall-forming CesA proteins known to date (U.S. Pat. No. 6,930,225, supra; U.S. Pat. No. 6,803,498, granted Oct. 12, 2004, the entire contents of which are herein incorporated by reference). The maize CesA10, 11, and 12 genes and their orthologs from Arabidopsis and rice have been implicated in secondary wall formation (Tanaka et al., Plant Physiol. 133:73-83 (2003); Taylor et al., Proc. Natl. Acad. Sci. U.S.A. 100:1450-1455 (2003); Appenzeller et al., Cellulose 11:287-299 (2004)). The co-expression of the Bk2 and secondary wall-forming CesA genes supports a role for Bk2 in secondary wall formation in maize.

TABLE 4
Primary and Secondary Wall-forming CesA Proteins
	SEQ ID NO:
	Protein	(Nucleotide)	(Amino Acid)
	CesA1	19	20
	CesA2	21	22
	CesA3	23	24
	CesA4	25	26
	CesA5	27	28
	CesA6	29	30
	CesA7	31	32
	CesA8	33	34
	CesA9	35	36
	CesA10	37	38
	CesA11	39	40
	CesA12	41	42

Another Bk2L gene that shows correlated expression with CesA genes is Bk2L3. The expression pattern of Bk2L3, is very similar to the CesA genes that were reported previously to be involved in primary wall formation (Holland et al., Plant Physiol. 123:1313-1323 (2000); Dhugga, Curr. Opin. Plant Biol. 4:488-493 (2001); Appenzeller et al., Cellulose 11:287-299 (2004)). Three genes in particular, CesA1, 7 and 8 appear to likely form a functional cellulose synthase complex for primary wall formation. The expression of the Bk2L3 gene is highly correlated with these three CesA genes that it appears that, analogous to the secondary wall cellulose synthase complex consisting of three CesA proteins and a Bk2 protein, these four proteins may form a functional cellulose synthase complex for primary wall formation.

Bk2L5 is expressed only in pollen. Some expression in silk most likely results from the pollen tube growing through it. Bk2L8 appears like is leaf-preferred and Bk2L6 is endosperm-specific.

Correlation among the expression level of all the different Bk2 and CesA genes from maize as studied from Solexa MPSS™ is shown in FIGS. 5A and 5B.

Example 3

Prophetic Example

Engineering Increased Stalk Strength by Overexpression of Maize Bk2-Like Genes Under a Strong, Stalk-Specific Promoter

A chimeric transgene is constructed to directly overexpress the Bk2 gene/polypeptide in a tissue specific manner. The transgene construct comprises a maize cDNA encoding Bk2L3 and/or Bk2L6 (e.g., SEQ ID NO:5 or SEQ ID NO:11) operably linked to the promoter from the alfalfa stalk-specific S2A gene (Abrahams et al., Plant Mol. Biol. 27:513-528 (1995)). The DNA containing the Bk2L3 or Bk2L6 ORF is then fused to the S2A promoter on the 5′ end and pinII terminator on the 3′ end to produce an expression cassette as illustrated in FIG. 3. The construct is then linked to a selectable marker cassette containing a bar gene driven by CaMV 35S promoter and a pinII terminator. It is appreciated that one skilled in the art could employ different promoters, 5′ end sequences and/or 3′ end sequences to achieve comparable expression results. Transgenic maize plants are produced by transforming immature maize embryos with this expression cassette using the Agrobacterium-based transformation method by Zhao (U.S. Pat. No. 5,981,840, issued Nov. 9, 1999; the contents of which are hereby incorporated by reference). While the method below is described for the transformation of maize plants with the S2A promoter-Bk2L3 (or Bk2L6) expression cassette, those of ordinary skill in the art recognize that this method can be used to produce transformed maize plants with any nucleotide construct or expression cassette that comprises a promoter linked to maize Bk2L3 (or Bk2L6) gene for expression in a plant.

Immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the S2A promoter-Bk2L3 (or Bk2L6) expression cassette (illustrated above) to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step, the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step is included. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed calli are recovered (step 4: the selection step). Preferably, the immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells.

The resulting calli are then regenerated into plants by culturing the calli on solid, selective medium (step 5: the regeneration step).

Example 4

Prophetic Example

Engineering Increased Stalk Strength by Transgenic Expression of Maize Bk2-Like Genes with an Enhancer Element in the Promoter Region Under a Strong, Stalk-Specific Promoter

The expression of the Bk2L3 (or Bk2L6) gene is increased by placing a heterologous enhancer element in the promoter region of the native Bk2L3 (or Bk2L6) gene. An expression cassette is constructed comprising an enhancer element such as CaMV 35S fused to the native promoter of Bk2L3 (or Bk2L6) and the full-length cDNA. Transgenic maize plants can then be produced by transforming immature maize embryos with this expression cassette as described in Example #3.

Example 5

Prophetic Example

Engineering Increased Stalk Strength by Overexpression of Maize Bk2-like and CesA Genes

Whereas the secondary wall-forming genes mainly affect the mechanical strength of the plant tissues and not the morphological phenotype, the primary wall-forming genes can affect plant growth rate and thus their modulation can be employed to increase the rate of growth. The maize genes CesA1, 7, and 8 were previously shown to be co-expressed across multiple tissues, suggesting that they might form a functional enzyme complex. Bk2L3 is co-expressed with these three CesA genes, strongly suggesting that the protein products of all of these four genes form a functional enzyme complex. Simultaneous over-expression of these four genes as a single multi-gene construct or as separate constructs containing different combinations of these genes in maize driven by different promoters, preferably by the promoters of genes whole expression is associated with cell elongation, can be employed to produce transgenic plants with enhanced growth rate. Any of the other Bk2L genes can also be used in combination with the mentioned three CesA genes as described above to produce transgenic plants with enhanced growth rate.

Example 6

Prophetic Example

Engineering Increased Stalk Strength by Overexpression of Maize Bk2-Like and CesA Genes

Aside from contributing to mechanical strength, secondary wall accounts for a majority of the biomass in plants. Whereas mechanical strength has applications in reducing in crop lodging, quality and amount biomass are important for many other applications, including ethanol production. The Bk2 gene along with the maize CesA10, 11, and 12 genes offers an avenue to increase the ratio of cellulose in the cell wall. The efficiency of ethanol production is directly related to the amount of cellulose in the biomass. Replacement of lignin with cellulose will also be useful in silage digestibility.

The Bk2 gene can be co-expressed with the CesA10, 11, and 12 genes as described in Example 5 for the primary wall-forming genes but under the control of secondary wall-specific promoters to produce transgenic plants with improved stalk strength and biomass quality.

Example 7

Prophetic Example

Engineering Down-Regulation of Maize Bk2-Like Genes

Since primary wall forming CesA genes contribute to cell expansion, their limited down-regulation can be employed to reduce plant height or organ size. In particular, the expression of the Bk2L3 gene is highly correlated with the primary wall-forming CesA genes. Whereas the overexpression of all the members of a functional enzyme complex may be required to increased enzyme activity, down-regulation of only one member may be sufficient to reduce activity. The down-regulation of Bk2L3, for example (and/or Bk2L5 for male sterility), can be accomplished by any of the technologies of co-suppression, RNAi, antisense RNA, or micro RNA resulting in dwarf transgenic plants. Height reduction has applications in some crop plants where harvest index is low and needs to be increased. Modern wheat and rice varieties, for example, are considerably shorter than their older counterparts. The ability to reduce plant height was mainly the cause of green revolution in each of these crops.

Example 8

Prophetic Example

Expression of Recombinant DNA Constructs in Dicot Cells Under a Strong, Stalk-Specific Promoter

An expression cassette composed of the promoter from the alfalfa stalk-specific S2A gene (Abrahams et al., Plant Mol. Biol. 27:513-528 (1995)) 5-prime to the cDNA fragment can be constructed and be used for expression of the instant polypeptides in transformed soybean. The pinII terminator can be placed 3-prime to the cDNA fragment. Such construct may be used to overexpress the Bk2-like genes. It is realized that one skilled in the art could employ different promoters and/or 3-prime end sequences to achieve comparable expression results.

The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.

Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

Soybean embryogenic suspension cultures can be maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5′ region, the fragment encoding the instant polypeptides and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

To 504 of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/4), 204 spermidine (0.1 M), and 504 CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 404 of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 9

Prophetic Example

Expression of Recombinant DNA Constructs in Microbial Cells Under a Strong, Stalk-Specific Promoter

The cDNAs (SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15 or 17) encoding the instant BRITTLE STALK 2-like polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 is constructed by first destroying the EcoRI and HindIII sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites is inserted at the BamHI site of pET-3a. This creates pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the NdeI site at the position of translation initiation is converted to an NcoI site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, is converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% low melting agarose gel. Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies, Madison, Wis.) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25° C. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

Example 10

Characteristics of the Stalk Tissue of the Wildtype (Bk2) and of the Brittle Stalk (bk2-ref) Mutant of Maize

The maize stock containing the reference allele of bk2 (bk2-ref) was obtained from the Maize Genetics COOP Stock Center (USDA/ARS & Crop Sciences/UIUC, S-123 Turner Hall, 1102 S. Goodwin Avenue, Urbana, Ill. 61801-4798). Three greenhouse-grown plants each of the bk2-ref/bk2-ref and its wildtype sibling, Bk2/bk2-ref, both derived from seeds obtained from the same selfed ear, were evaluated for different traits approximately two weeks after flowering. Three internodes below the ear (internodes 3, 4, and 5, numbered from the ear node) were subjected to a three-point flexural test using a model 4411 Instron electromechanical testing device (Instron Corp., Canton, Mass.). The span width between the anchor points was 20 cm. The anvil was vertically driven at a constant speed of 20 cm/min against the internodal zone ˜3 cm above the node on a horizontally placed stalk until it collapsed or snapped. The maximum load to break was used as a measure of strength to differentiate the internodes and stalks.

Total dry matter was measured in the stalk portion below the ear node. Structural dry matter and cellulose contents were determined in duplicates on each of the three plants from the third and fourth internodes below the ear node by boiling the powdered stalk material twice with buffer (25 mM MOPS, pH 7) for 30 minutes. The remaining material was suspended in methanol/chloroform (3/1, v/v) for 1 hour, dried and weighed. Crystalline cellulose was determined by the Updegraff method (Updegraff, Anal. Biochem. 32:120-124 (1969)). Briefly, ground stalk material was place in a boiling water bath in an 8:2:1 mixture of acetic acid:water:nitric acid for 1 hour, the crystalline material washed three times with water and then with 95% ethanol followed by drying in a Speedvac. Klason lignin was determined by incubating the ground stalk material with 72% (w/w) sulfuric acid for 1 hour, washing twice with a 1:20 dilution of 72% sulfuric acid in water, heating at 65° C. for 30 minutes, washing once with water, and drying the residue at 80° C. overnight. Sugar composition was determined as described in (Appenzeller et al., Cellulose 11:287-299 (2004)).

In summary, reduction in mechanical strength in the stalk tissue was highly correlated with a reduction in the amount of cellulose and an uneven deposition of secondary cell wall material in the subepidermal and perivascular sclerenchyma fibers. Lower amount of cellulose and thinner walls of the mutant were reflected in reduced dry matter content per unit length of the stalk.

TABLE 5
Measurement of Stalk Composition and Mechanical Strength
	Trait	Wildtype	bk2-ref
	ear height (cm)	102.00 ± 8.8	106.33 ± 11.8
	stalk diameter (mm)	23.84 ± 0.27	23.40 ± 0.46
	stalk dry mass (g)	89.43 ± 3.39	62.08 ± 8.46
	moisture (%)	79.20 ± 0.21	84.87 ± 1.04
	dry matter (g/cm)	0.68 ± 0.04	0.43 ± 0.07
	displacement to break (mm)	11.83 ± 0.46	6.51 ± 1.10
	load to break (kg)	23.68 ± 2.25	9.04 ± 2.66
	insoluble dry matter (%)	51.57 ± 1.00	45.20 ± 1.69
	cellulose (%)	33.30 ± 0.56	23.76 ± 0.68
	lignin (%)	9.07 ± 0.21	10.28 ± 0.63
	remainder cell wall (%)	9.20 ± 1.68	11.16 ± 2.03
	cellulose (g/cm)	0.24 ± 0.114	0.11 ± 0.019

Claims

1. An isolated polynucleotide comprising:

(a) a nucleotide sequence encoding a polypeptide associated with stalk mechanical strength, wherein said polypeptide has an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer in between 80% and 100%, based on the Clustal V method of alignment, when compared to SEQ ID NOs:16 or 18; or

(b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.

2. A recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to a promoter that is functional in a plant.

3. A method for altering stalk mechanical strength of a plant, comprising:

(a) introducing into a regenerable plant cell the recombinant DNA construct of claim 2 to produce a transformed plant cell; and

(b) regenerating a transgenic plant from said transformed plant cell, wherein said transgenic plant comprises in its genome said recombinant DNA construct and wherein said transgenic plant exhibits an alteration in stalk mechanical strength, when compared to a control plant not comprising said recombinant DNA construct.

4. The method of claim 3, further comprising (c) obtaining a progeny plant derived from said transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct.

5. The method of claim 3, wherein the transgenic plant exhibits an increase in stalk mechanical strength.

6. A plant comprising in its genome the recombinant DNA construct of claim 2.

7. The isolated polynucleotide of claim 1, wherein said stalk mechanical strength is measured by the three-point bend test.

8. A method of evaluating stalk mechanical strength in a plant, comprising:

(a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a)(i);

(b) regenerating a transgenic plant from said transformed plant cell; and

(c) evaluating said transgenic plant for stalk mechanical strength.

9. The method of claim 8, further comprising:

(d) obtaining a progeny plant derived from said transgenic plant; and

(e) evaluating said progeny plant for stalk mechanical strength.

10. A method of evaluating stalk mechanical strength in a plant, comprising:

(a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a)(i);

(b) regenerating a transgenic plant from said transformed plant cell;

(c) obtaining a progeny plant derived from said transgenic plant; and

(d) evaluating said progeny plant for stalk mechanical strength.

11. A plant comprising in its genome:

(a) a first recombinant DNA construct comprising at least one promoter that is functional in a plant operably linked to at least one of a first isolated polynucleotide selected from the group consisting of: (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and

(b) a second recombinant DNA construct comprising at least one promoter that is functional in a plant operably linked to at least one of a second isolated polynucleotide selected from the group consisting of: (iv) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; (v) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and (vi) a full-length complement of the polynucleotide of (b)(iv) or (b)(v)

and wherein said plant exhibits increased cell wall cellulose content or enhanced growth rate when compared to a control plant not comprising said first recombinant DNA construct and said second recombinant DNA construct.

12. A plant comprising in its genome at least one regulatory sequence operably linked to:

(a) at least one isolated polynucleotide selected from the group consisting of: (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and

(b) at least one isolated polynucleotide selected from the group consisting of: (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii),

and wherein said plant exhibits increased cell wall cellulose content or enhanced growth rate when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).

13. The plant of claim 12, wherein:

(a) said at least one polynucleotide is selected from the group consisting of: (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:2; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:1; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and

(b) said at least one isolated polynucleotide is selected from the group consisting of: (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:38, 40, and 42; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:37, 39, and 41; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii),

and wherein said plant exhibits increased cell wall cellulose content when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).

14. The plant of claim 12, wherein:

(a) said at least one polynucleotide is selected from the group consisting of: (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:5; and (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and

(b) said at least one isolated polynucleotide is selected from the group consisting of: (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 32, and 34; (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:19, 31, and 33; and (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii),

and wherein said plant exhibits enhanced growth rate when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).

15. A plant comprising in its genome at least one regulatory sequence operably linked to at least two isolated polynucleotides selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18;

(b) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and

(c) a full-length complement of the polynucleotide of (a) or (b).

16. A plant comprising in its genome:

a suppression DNA construct comprising a promoter functional in a plant operably linked to: (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a polypeptide selected from the group consisting of Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9,

and wherein said plant exhibits reduced stalk mechanical strength when compared to a control plant not comprising said suppression DNA construct.

17. The plant of claim 16, wherein said suppression DNA construct comprises a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double-stranded RNA-producing construct, RNAi construct, or small RNA construct.

18. A plant comprising in its genome:

a suppression DNA construct comprising a promoter functional in a plant operably linked to: (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L3 polypeptide,

and wherein said plant exhibits reduced plant height and/or reduced organ size when compared to a control plant not comprising said suppression DNA construct.

19. A plant comprising in its genome:

a suppression DNA construct comprising a promoter functional in a plant operably linked to: (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:10, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L5 polypeptide,

and wherein said plant exhibits male sterility when compared to a control plant not comprising said suppression DNA construct.