WO2006084342A2

WO2006084342A2 - Isolated nucleic acid molecules encoding plant transcription factors in the knox family

Info

Publication number: WO2006084342A2
Application number: PCT/BR2006/000018
Authority: WO
Inventors: Fabio Papes; Isabel Rodrigues Gerhardt; Paulo Arruda
Original assignee: Alellyx SA
Current assignee: Monsanto do Brasil Ltda
Priority date: 2005-02-10
Filing date: 2006-02-08
Publication date: 2006-08-17
Anticipated expiration: 2007-08-10
Also published as: WO2006084342A3

Abstract

Polynucleotides, DNA constructs and methods are disclosed for the modification of size, architecture of aerial and/or non-aerial portions of a plant as well as modification of the structure and/or chemical composition of the xylem of a plant. Plants are transformed with a gene encoding an active transcription factor belonging to the KNOX family. The over-expression of this gene in the plant's vascular system leads to increased overall size and architecture both in the shoot and root systems of the transgenic plant.

Description

ISOLATED NUCLEIC ACID MOLECULES ENCODING PLANT TRANSCRIPTION

FACTORS IN THE KNOX FAMILY

RELATED APPLICATION

[0001] This application claims priority of U. S. Provisional Application Serial No. 60/651,826, filed on February 10, 2005, the disclosure of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to genetic engineering as applied to plants. More specifically, it relates to the isolation of certain nucleic acid molecules which are transcription factors and which are useful in producing genetically engineered, recombinant plants. The resulting recombinant plants have increased or altered root number, root size, shoot number, or shoot position. The resulting plants also exhibit altered xylem structure, chemical composition, or both. Also a part of the invention are processes for making the recombinant plants.

BACKGROUND AND PRIOR ART

[0003] Every plant species exhibits a wide discrepancy in the growth of different individuals in a given population, in which maximal growth potential of individual plants is usually not met under most environmental conditions.

[0004] The growth rate of an individual plant is influenced, directly or indirectly, by many factors. These include, for example, the availability and accessibility of mineral nutrients in the soil, to which shoots and roots respond differently.

[0005] While there is a direct relationship between nutrient availability and growth rate, this direct relationship is not maintained over a wide nutrient concentration range. Both the availability of plant nutrients, required for cell growth, as well as developmental signalling pathways limit the growth of a plant. Genetic engineering provides an avenue for modifying a plant's "architecture" (explained infra) or its ability to absorb nutrients from the soil or other growing media.

[0006] It is known that plants rarely have optimal conditions in which to grow. The availability of water and nutrients in the soil, weather conditions and other factors shortening the growth season impact the ability of a plant to flourish and grow. Many plant species exhibit drought tolerant genotypes, and these genotypes usually correlate with the ability of the plant's roots to penetrate the growth medium or soil.

[0007] The manipulation of gene expression in plants provides a general methodology for introducing new characteristics, such as an increase in the size of a root system, which in turn leads to increased nutrient assimilation. Moreover, increasing shoot growth enhances the plant's ability to use solar energy. It is therefore of interest to be able to control the growth of entire plants, or specific organs of the plant which are targeted for specific applications.

[0008] The "HD" family of transcription factors is characterized by a conserved domain of 30-60 amino acids, referred to as "homeodomain" (also known as "homeobox domain"). Many of the proteins in this family are recognized as sequence-specific DNA- binding transcription factors.

[0009] The present invention involves the isolation and cloning of nucleic acid molecules which serve as transcription factors. The group of nucleic acid molecules and proteins of the invention will be referred to as the KN molecules. Genetic alternation of plants via these nucleic acid molecules results in transgenic plants that differ from their non- transgenic counterparts in terms of larger and more complex aerial shoot systems, and altered xylem structure or chemical composition.

[0010] How this is achieved will be seen from the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 shows the domain organization of the polypeptides of the invention.

[0012] Figure 2 shows a generic plant expression vector which delineates the various parts of the vector. Variations are described in the specification.

[0013] Figures 3A and 3B present results obtained following genetic transformation and growth of Nicotiana benthamiana using expression vectors in accordance with the invention. The transformants in each figure are on the left, while the non-transformed control plants are on the right.

[0014] Figure 3A shows differences in root architecture, while 3B, especially with the position indicated by the arrow, shows the increased vigor, numbers of vein branches, and the darker color of leaves in the transgenic plant as compared to the control plant. [0015] Figure 4 shows the general aspect of plants in the segregating Tl population. These plants were obtained from seeds collected from primary transformant number 30 (described, infra, at Example 5) harboring a construct comprising the KN nucleic acid from Populus deltoides under the control of a xylem-preferred alpha-tubulin promoter. Note the stunted phenotype (Figure 4A) and increased number of primary and secondary veins in leaves (Figure 4B) of AA and A0 plants when compared to non-transgenic siblings (00).

[0016] Figure 5 shows free-hand (A) and microtome (B) histological sections of Tl plants obtained from planting seeds of primary transgenic event 30, as described infra. Note the smaller width of secondary xylem in AA and A0 plants as compared with control sibling 00 plants from the same segregating progeny.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] All technical terms used herein are terms commonly used in biochemistry, molecular biology and agriculture, and are readily understood by one of ordinary skill in the art. See, e.g., Molecular Cloning: A Laboratory Manual, 3^rd ed., vol. 1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001; Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing Associates and Wiley- Interscience, New York (1988); Short Protocols in Molecular Biology: A compendium of Methods from Current Protocols in Molecular Biology, 5^th ed., vol. 1-2, ed. Ausubel et al., John Wiley & Sons, Inc. (2002); Genome Analysis: A Laboratory Manual, vol. 1-2, ed. Green et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1997). Methods involving plant biology techniques are described herein and are described in detail in works such as Methods in Plant Molecular Biology: A Laboratory Course Manual, ed. Maliga et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995. Various techniques using PCR are described, e.g., in Iπnis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, 1990 and in Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003. PCR-primer pairs can be derived from known sequences by known techniques such as using computer programs intended for that purpose (e.g., Primer, Version 0.5 (1991), Whitehead Institute for Biomedical Research, Cambridge, MA). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Caruthers, Terra. Letts., 22:1859-1862 (1981) and Matteucci and Caruthers, J. Am. Chem. Soc, 103:3185 (1981). [0018] Restriction enzyme digestions, phosphorylations, ligations and transformations were done as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press. All reagents and materials used for the growth and maintenance of bacterial cells are widely available commercially.

EXAMPLE 1

[0019] Expressed Sequence Tags (ESTs) from Populus sp. were clustered using the CAP3 program (Huang and Madan, Genome Res., 9:868-877 (1999)), incorporated by reference. The 150,000 ESTs were obtained from libraries representing the following tissues: apical shoot, bark, cambium, seed, xylem, leaf and root. The set of clusters thus generated was searched for clusters composed of at least 90% of EST reads from libraries representing Populus cambium and xylem tissues. Additionally, the set of clusters was searched for clusters composed of at least three EST reads from cambium and xylem tissues and preferably less than two reads from other libraries.

[0020] Clusters selected using these parameters were then aligned using the Blast-X algorithm with a cutoff e-value <= le-5 (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997)) to sequences from a curated Arabidopsis thaliana transcription factor database composed of sequences obtained from the Arabidopsis Gene Regulator Information Server (AGRIS) AtTFDB database. The comparison results were stored in a local database of Populus transcription factors and browsed via a web-based interface to filter specific transcription factor cluster sequences of genes expressed specifically or preferably in the xylem and cambium tissues of Populus sp. Clusters coding for HD transcription factors were retrieved using this procedure as xylem/cambium-preferred HD transcription factors. One cluster thus selected, composed of 7 EST reads from xylem and cambium cDNA libraries and only one read from another library (apical shoot), represents a KNOX-subfamily member. The sequence of the longest read in this cluster is set forth herein as SEQ. ID. NO: 1, which codes for the polypeptide disclosed herein under SEQ. ID. NO: 3.

EXAMPLE 2

[0021] (a) Preparation of RNA from Populus deltoides cambium/xylem and cDNA synthesis.

[0022] Bark was removed from stem cuttings of one-year-old Populus deltoides trees. The inner part of the stem, containing cambium, xylem and pith, was cut in small pieces, frozen in liquid nitrogen and used for RNA extraction using the cetyltrimethyl-ammonium bromide (CTAB) extraction method (Aldrich and Cullis, Plant MoI. Biol. Report., 11:128-141 (1993)). A cDNA pool was used in RT-PCR experiments in which the isolated total RNA was used as template, and Superscript II reverse transcriptase (Invitrogen) and oligo(dT) primer were used to synthesize the first-strand cDNA. Double-stranded cDNA was obtained by the subsequent polymerase reaction, using gene-specific primers, as described below.

[0023] (b) Design of PCR Primers and RT-PCR reaction.

[0024] Oligomers, based on SEQ. ID. NO: 1 were synthesized as primers for PCR, including either the region around the first ATG codon or around the termination codon of the main ORF encoding the polypeptide.

[0025] Primers were designed to amplify the entire coding region of the main ORF, i.e., from the ATG through the translation stop codon. The sequences of the primers:

[0026] SEQ. ID. NO: 5

[0027] ATCCATATGGAGGACTACAATCAAATGAGTG

[0028] SEQ. ID. NO: 6

[0029] ATCTCTAGATCATGGACCCAGCCGATAATGAC

[0030] The cDNA pool obtained in (a) was used as the template in a PCR reaction with the primers of SEQ. ID. NO: 5 and 6. The PCR involved 40 cycles of 1 minute at 94°C, 1 minute at 51⁰C, and 2 minutes at 72°C followed by an extra step of elongation at 72°C for 7 minutes. The PCR products were isolated by gel electrophoresis on 1.0% agarose followed by ethidium bromide staining of the electrophoresed gel and detection of amplified bands on a UV transilluminator. The detected amplified band was verified and cut out of the agarose gel with a razor. The pieces of gel were transferred to 1.5mL microtubes, and the DNA fragments were isolated and purified using a GFX PCR clean-up and gel band purification kit. The recovered DNA fragments were subcloned in a commercially available cloning vector, transformed into E. coli, and then used to prepare plasmid DNA, which was then sequenced by the dideoxy method (Messing, Methods in Enzymol. 101, 20-78 (1983)) using standard methods. The nucleotide sequence SEQ. ID. NO: 2, which codes for the polypeptide disclosed herein under SEQ. ID. NO: 4, ensued.

EXAMPLE 3

[0031] The nucleic acid molecule from Populus deltoides obtained in Example 2 above was introduced into a plant host to produce transgenic tobacco plants. [0032] The nucleic acid molecule isolated from Populus deltoides and obtained in Example 2 was cloned into the aforementioned expression vector downstream of a xylem- preferred alpha-tubulin gene (TUB) promoter. The resulting expression construct was amplified in E. coli, and then transformed by chemical transformation into Agrobacterium tumefaciens LBA4404 strain.

[0033] Agrobacterium-mediated transformation of Nicotiαnα benthαmiαnα was accomplished using the leaf disk method of Horsch et al. (Science 227, 1229, (1985)). In short, LBA4404 Agrobacterium strain was grown overnight until it reached mid-log phase growth. The cultures were diluted 1:10 in sterile water and co-cultivated for 20 min with leaf disks from sterile grown young Nicotiana benthamiana plants. These disks were incubated on Murashige-Skoog medium in the dark. After 48 hours, leaf disks were placed upside down on fresh plates of the same growth medium supplemented with 0.4 mg/L of indoleacetic acid (IAA), 2 mg/L benzyl-aminopurine (hOBAP), lmg/L phosphinothricin and 500 mg/L carbenicillin. When shoots formed, they were removed from the leaf disk and placed on fresh medium supplemented with just 1 mg/L phosphinothricin. Shoots of primary transformants of Nicotiana benthamiana, heterozygous for the transgene, were allowed to root on Murashige and Skoog medium, and subsequently transferred to soil and grown in the greenhouse. The conditions (~50 pM/m2/sec of white light, 27°C) were sufficient to identify those transgenic plants which exhibited altered size and/or architecture of the aerial parts, or altered size and/or architecture of the non-aerial parts, or altered xylem structure and/or xylem chemical composition, or a combination of these effects, according to the descriptions provided herein.

EXAMPLE 4

[0034] PCR was used to verify the integration of the gene construct in the genome of the transgenic plants. A pair of primers was synthesized to amplify a 400bp DNA sequence from the selectable marker gene Bar. Additionally, another pair was synthesized to amplify the endogenous Nicotiana benthamiana chalcone synthase (CHS) gene:

[0035] SEQ. ID. NO: 7

[0036] Bar 35 Length: 20

[0037] TCTACCATGAGCCCAGAACG

[0038] SEQ. ID. NO: 8 [0039] Bar 36 Length: 23

[0040] AATTCGGGGGATCTGGATTTTAG

[0041] SEQ. ID. NO: 9

[0042] CHS 150 Length: 24

[0043] GCCAGCCCAAATCCAAGATTACTC

[0044] SEQ. ID. NO: 10

[0045] CHS 151 Length: 23

[0046] AATGTTAGCCCAACTTCACGGAG

[0047] These primers were used to PCR-amplify part of the T-DNA portion of the expression construct containing a nucleic acid molecule of the invention, i.e., from genomic DNA of Nicotiana benthamiana transformants.

[0048] The PCR reaction mixture contained 100 ng genomic DNA of transformed plant, prepared using the cetyltrimethyl-ammonium bromide (CTAB) extraction method (Aldrich and Cullis, Plant MoI. Biol. Report., 11:128-141 (1993)), 0.2 μM of each primer for the Bar gene, 0.2 μM of each primer for the endogenous CHS control gene, 100 μM of each deoxyribonucleotide triphosphate, IxPCR buffer and 2.5 Units of AmpliTaq DNA polymerase (Applied Biosystems) in a total volume of 50 μL. The cycling parameters were as follows: 94°C. for 1 minute, 57°C. for 1 minute and 72°C for 1 minute, for 40 cycles, with 5 minutes at 72⁰C. extension. The PCR products were electrophoresed on a 1% agarose gel.

EXAMPLE 5

[0049] Several independent heterozygous primary transformants were self-pollinated to yield the Tl generation. One of these primary transformants (event 30) and its Tl progeny are described here, but other transgenic events had similar phenotypes. Thirty Tl plants from transgenic event 30 were allowed to grow in the greenhouse under conditions sufficient to identify those Tl plants which exhibited altered size and/or architecture of the aerial parts, or altered size and/or architecture of the non-aerial parts, or altered xylem structure and/or xylem chemical composition, or a combination of these effects, according to the descriptions provided herein. Seeds from each plant in this segregating progeny were collected and plated onto MS-agar plates containing 4.5 mg/L phospliinothricin to allow for rapid genotyping of the Tl plants.

[0050] AA and A0 Tl plants exhibited a stunted phenotype (FIG. 4A), curled smaller leaves with extra primary and secondary veins (FIG. 4B) and more lateral branches when compared to 00 Tl plants from the same progeny.

[0051] Hand-sectioned stem cuts taken from the midpoint of the stem from representative plants possessing each of the three possible Tl genotypes (AA, A0 and 00) were stained with the lignin-specific dye phloroglucinol and observed under either a Leica stereomicroscope (FIG. 5A) or a Leica visible light transmission microscope (FIG. 5B). AA and A0 plants exhibited a marked reduction in secondary xylem width (FIGs. 5A and 5B) when compared with 00 plants from the same segregating progeny, a phenotype consistent with the notion (discussed infra) that KNOX transcription factors regulate stem cell identity and maintenance in plants. When over-expressed under the control of the constitutive CaMV 35S promoter, a KNOX transcription factor from Arabidopsis was shown to induce the formation of curled and knotted leaves due to the appearance of ectopic meristematic cell aggregates in the leaves, along with inflorescence stem showing abnormal impairment in the lignification of extraxylary and xylary fibres (MeIe et al., Genes and Development, 17: 2088- 2093 (2003)).

[0052] The main stems of 5 months-old AA, A0 and 00 Tl plants were collected, air-dried, milled and separately subjected to Klason lignin assay and liquid chromatography to determine cellulose and hemicellulose contents. AA and A0 plants showed a statistically significant reduction in insoluble lignin percentual content (9.4% ± 0.28% and 9.57% ± 0.05%, respectively, as opposed to 15.5% ± 0.05% in 00 plants; P<0.1, t-test). These results may reflect the lower amount of lignified cells in xylem in Tl plants harboring the transgene, but may also be the result of direct negative regulation on genes coding for enzymes in the lignin biosynthetic pathway, as has been shown for KNOX transcription factors in Arabidopsis (MeIe et al., Genes and Development, 17: 2088-2093 (2003)).

[0053] This marked reduction in lignin content was also accompanied by an increase in cellulose content in AA and A0 Tl plants (59.26% ± 0.06% and 59.5% ± 0.08%, respectively, as compared to 51.27% ± 0.35% in 00 plants; PO.l, t-test). On the other hand, the content of xylans and xyloglucans was higher in 00 plants (15.36% ± 0.00%) than in AA and A0 plants (9.0% ± 0.35% and 9.42% ± 0.2%, respectively; PO.l, t-test). Other types of hemicelluloses did not show significant alterations. [0054] The foregoing disclosure and examples describe various features of the invention, which essentially involve the isolation and cloning of nucleic acid molecules which encode transcription factors and are useful in the production of genetically engineered plants. Recombinant plants which have been transformed or transfected with such isolated nucleic acid molecules exhibit improvement in overall plant architecture and/or chemical composition, via one or more of improved aerial shoot systems, improved xylem structure, and improved xylem chemical composition. Examples of altered plant architecture include, but are not limited to, alteration in the shape or number of stem branches, leaves or root branches, and abnormal development of the leaf, stem or root. Examples of altered xylem structure or chemical composition include, but are not limited to, alteration in the number, position or size of xylem cells, and quantitative alteration in lignin or cellulose content and/or composition.

[0055] These isolated nucleic acid molecules encode transcription factors which are members of the "homeodomain-containing," or "HD," family of transcription factors. Members of this family share a conserved DNA-binding domain of 30-60 amino acids, which was first identified in Drosophila homeotic and segmentation proteins, and which binds to DNA as described supra, i.e., through a "helix turn helix" structure.

[0056] These HD transcription factors are divided into three groups, of which the "KNOTTED - like homeobox family," abbreviated as "KNOX," is relevant here. The members of this family share a common structural organization, consisting of six regions. These include conserved KNOX and ELK domains, as well as the HD domain referred to supra. Within this family, KNOX domains are further divided into KNOX-I and KNOX-II classes. While no specific function has been attributed to KNOX-II, most plant KNOX-I proteins have in fact been implicated in the development of and cell fate determination events in the shoot apical meristem. The isolated nucleic acid molecules of the invention encode HD transcription factors in the KNOX-I subfamily.

[0057] In species with simple leaves, class I members of the KNOX family are expressed in shoot meristems and stems, but not leaves or roots (Nishimura et al., Plant J., 18: 337-347 (1999)). Loss-of-function phenotypes have been described for a few KNOX genes, and demonstrate a role in meristem maintenance (Long et al., Nature, 379: 66-69 (1996); Vollbrecht et al., Development, 127: 3161-3172 (2000)). Mutations in the KNOX gene Brevipedicellus (BP) results in plants with shorter internodes, downward pointing siliques and increased lignification in the inflorescence stem (Venglat et al., Proc. Natl. Acad. ScL USA, 99: 4730-4735 (2002); MeIe et al., Genes and Development, 17: 2088-2093 (2003)). BP overexpression results in changes in leaf shape, increased cytokinin levels in several species (Lincoln et al., Plant Cell, 6: 1859-1876 (1994); Chuck et al., Plant Cell, 8: 1277-1289 (1996); Ori et al., Development, 127: 5523-5532 (2000); Frugis et al., Plant Physiol, 126: 1370-1380 (2001)) along with impaired lignin deposition in the inflorescence stem (MeIe et al., Genes and Development, 17: 2088-2093 (2003)). This latter phenotype was further attributed to the fact that the BP protein seems to directly regulate several genes in the lignin biosynthetic pathway at the transcriptional level (MeIe et al., Genes and Development, 17: 2088-2093 (2003)), which further stresses the use of KNOX transcription factor genes to produce transgenic plants with altered lignification for biotechnological applications.

[0058] What distinguishes the nucleic acid molecules and proteins of the invention is their preferred expression pattern in secondary xylem and cambium tissues.

[0059] The isolated nucleic acid molecules of the invention are represented by, but are not limited to, those defined as comprising the nucleotide sequence of SEQ. ID. NO: 1 or 2, as well as those which exhibit 65% or more homology therewith, as is discussed infra.

[0060] The isolated nucleic acid molecules of the invention are capable of hybridizing, under stringent conditions, to one or both of SEQ. ID. NOS: 1 and 2. "Stringent conditions" as defined herein refers to conditions at least as stringent as those where hybridization is carried out in 3.5x SSC, Ix Denhardt's solution, 25mM sodium phosphate buffer (pH 7.0), 0.5% SDS, and 2mM EDTA for 18 hours, at 65⁰C, followed by four washes at 65⁰C for 20 minutes per wash, in 2x SSC, 0.1% SDS, and a final wash for up to 20 minutes, in 0.5x SSC, 0.1% SDS, or lower concentrations of SSC, e.g., 0.3x SSC, 0.1% SDS or O.lx SSC, 0.1% SDS, for even greater stringency. These conditions will serve as "stringent conditions" throughout this disclosure. Also a part of the invention are those nucleic acid molecules which encode the same protein as the nucleic acid molecules described supra, but differ in nucleotide sequence as a result of degeneracy of the genetic code, as well as those nucleic acid molecules which encode proteins that have at least about 60%, preferably at least about 70%, more preferably at least about 80% and most preferably at least 90% of the activity of SEQ. ID. NOS: 3 and 4.

[0061] The nucleic acid molecules of the invention include, e.g., allelic variants. These are nucleic acid molecules which vary in nucleotide sequence from the reference sequences supra, and lead to changes in amino acid translation, but are subject to no more than about 5% variation in the nucleotide sequence.

[0062] Also included are analogs of the sequences, where the analog has the same function as the sequence, but evolved in a different organism. Orthologs and paralogs are included herein, as these terms are known to the art.

[0063] In one preferred embodiment of the invention, the nucleic acid molecules of the invention contain regions which encode conserved domains KNOXA and KNOXB. With respect to the amino acid sequence listings herein, SEQ. ID. NOS: 3 and 4 present KNOXA domains at amino acids 106-150, and KNOXB domains at amino acids 157-209.

[0064] In addition to this, the invention encompasses those nucleic acid molecules which encode proteins that include amino acid sequences that are at least 75%, preferably at least 80%, more preferably 85%, even more preferably at least 90% and most preferably at least 95% identical to the KNOXA and/or KNOXB domains described supra, and which have at least 60% of the biological activity of the proteins encoded by SEQ. ID. NO: 1 or 2. "Biological activity" refers to the properties described in the examples, supra.

[0065] The isolated nucleic acid molecules of the invention may be incorporated into expression vectors, such that they are in operable linkage with a promoter. Preferably, the promoter is one known to operate in plant cells, and more preferably to be operable in cells of specific plant organs or tissues, such as roots, shoots, leaves, xylem, etc. The nucleic acid molecules of the invention may be placed in operable linkage with constitutive or inducible promoters. Alternatively, the nucleic acid molecules of the invention may be placed in operable linkage with promoters which direct the expression of the downstream gene preferably or specifically to an organ or tissue of the plant, such as xylem and cambium.

[0066] The expression vectors of the invention may also contain termination sequences, which are positioned downstream of the nucleic acid molecules of the invention, such that transcription of mRNA is terminated, and polyA sequences added. Exemplary of such terminators are the cauliflower mosaic virus (CaMV) 35S terminator and the nopaline synthase gene (Tnos) terminator.

[0067] The expression vectors of the invention may, in addition to tissue-specific promoters, include a developmentally regulated promoter, an organelle specific promoter, a seed specific promoter, a plastid specific promoter, and so forth. Coding sequences for targeting, transit, or secretion protein encoding molecules may also be included. Enhancers, transcription terminators, start codons, splicing signal sequences, and so forth, may also be included. For example, if a transcription initiation region is included, it may be, e.g., any of the various opine initiation regions, such as octopine, mannopine, nopaline and the like that are found in the Ti plasmids of Agrobacterium tumefaciens. Alternatively, plant viral promoters can also be used, such as the cauliflower mosaic virus 35S promoter, to control gene expression in a plant. Bi addition, plant promoters such as leaf-specific promoters, root- specific promoters, seed-specific promoters, etc. can also be used. The most preferred promoters' as explained, supra, are tissue-preferred promoters which are active in, e.g., the cambium and/or xylem, particularly xylem fiber cells.

[0068] Included within the meaning of promoter is a sequence sufficient to direct transcription, such as promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, environmentally- or developmentally-regulated, or expression that is inducible by external signals or agents. Such elements may be located in the 5' and/or 3' regions of the construct. Both constitutive and inducible promoters are included (see e.g., Bitter et al., Methods in EnzymoL, 153:516-544 (1987), incorporated by reference).

[0069] The expression of genes employed in the present invention may be driven by more than one promoter in different circumstances. Although the endogenous promoter of a structural gene of interest, for example that of a gene of the present invention, maybe utilized for transcriptional regulation of the gene, the promoter may also be a foreign regulatory sequence. For plant expression vectors, suitable viral promoters include the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., Nature, 310:51-61 (1984); Odell et al., Nature, 313:810 (1985)); the enhanced CaMV 35S promoter; the Figwort Mosaic Virus (FMV) promoter (Gowda et al., J. Cell Biochem., 13D:301 (1989); Richins et al., Nucleic Acids Res., 15(20): 8451-66 (1987)) and the coat protein promoter of TMV (Takamatsu et al., EMBO J., 6:307 (1987)), leading to the over-expression of the nucleic acid molecules of the invention in most tissues, in turn leading to changes in xylem structure and/or xylem composition and/or plant size and/or plant architecture.

[0070] Tissue specific promoters may also be utilized in the present invention. As used herein, the term "tissue-specific promoter" means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, effecting expression of said selected DNA sequence in specific cells, e.g., in the root or in the shoot of a plant. The term also covers so-called "leaky" promoters, which regulate expression of a selected DNA primarily in one tissue, but may cause minor expression in other tissues as well. Such promoters may also include additional DNA sequences that are necessary for expression, such as introns and enhancer sequences. These additional sequences may be either endogenous to the organism from which the promoter is derived, endogenous to the plant which is being transformed, or may be exogenous to both. Examples of tissue-specific promoters active in floral meristems are the promoters of the Apetala3 and Apetalal genes which are described in Jack et al., Cell, 76:703 (1994) and Hempel et al., Development, 124:3845 (1997).

[0071] In addition, vascular system-specific, xylem-specific or xylem preferred promoters may be useful to promote expression of the nucleic acid molecules of the invention specifically in vascular tissues, especially xylem tissue. The use of a constitutive promoter, in general, affects protein levels and functions in all parts of the plant, while use of a tissue- preferred promoter permits targeting of the modified gene expression to specific plant parts, leading to more controllable phenotypes. Thus, in applying the invention, it may be found convenient to use a promoter that will give expression during xylem development, whereby the proteins of the invention would only be overproduced in the organ(s) or tissue(s) or cell type(s) in which its action is required for the uses disclosed herein. Vascular tissue-specific, xylem-specific promoters, vascular tissue-preferred and xylem-preferred promoters that could be used include, but are not limited to, the xylem-preferred tubulin (TUB) gene promoter, the xylem-preferred lipid transfer protein (LTP) gene promoter and the xylem-preferred coumarate-4-hydroxylase (C4H) gene promoter. Other suitable xylem-preferred promoters are disclosed in International Patent Application PCT/BR2005/000041, filed on March 28, 2005, Publication No. WO2005/096805, which is incorporated here by reference.

[0072] Promoters useful in the invention include both natural constitutive and inducible promoters as well as genetically engineered promoters. To be most useful, an inducible promoter should 1) provide low expression in the absence of the inducer; 2) provide high expression in the presence of the inducer; 3) use an induction scheme that does not interfere with the normal physiology of the plant; and 4) have no effect on the expression of other genes. Examples of inducible promoters useful in plants include those induced by chemical means, such as the yeast metallothionein promoter, which is activated by copper ions (Mett et al., Proc. Natl. Acad. Sci. U.S.A., 90:4567 (1993)); In2-1 and hi2-2 regulator sequences, which are activated by substituted benzenesulfonamides, e.g., herbicide safeners (Hershey et al., Plant MoI. Biol., 17:679 (1991)); the GRE regulatory sequences, which are induced by glucocorticoids (Schena et al., Proc. Natl. Acad. Sci., U.S.A., 88:10421 (199100; and ethanol-inducible promoters (Caddick et al., Nature Biotech., 16:177 (1998)). Other promoters, both constitutive and inducible, and enhancers will be known to the skilled artisan and will not be discussed here.

[0073] The particular promoter selected should be capable of causing sufficient expression to result in the over-expression of the protein of the invention, to modify the size of the aerial portion of a plant, to modify the size of the non-aerial portion of a plant or to modify the structure and chemical composition of the xylem of a plant or yet a combination of these effects.

[0074] In practice, a constitutive promoter (such as the CaMV promoter discussed supra) or a tissue-specific promoter, preferably a xylem preferred promoter as described supra, is typically ligated to the protein encoding region using standard techniques known in the art. The promoter may be operably linked to the nucleic acid coding region in any manner known to one of skill in the art. The expression unit may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer elements.

[0075] Transcription terminators or termination regions downstream of the structural gene provide for efficient transcription termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene. If the mRNA encoded by the structural gene is to be efficiently processed, DNA sequences which direct polyadenylation sequences including, but not being limited to, the Agrobacterium octopine synthase signal (Gielen et al., EMBO J., 3:835-846 (1984)) or the nopaline synthase signal (Depicker et al., MoI. And Appl. Genet., 1:561-573 (1982)), are also used.

[0076] When inserted into plants or plant cells, the expression vectors of the invention will typically contain, in addition to the protein coding nucleotide sequence, a plant promoter region, a transcription initiation site and a transcription termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the expression unit may also be included to allow for easy insertion into a preexisting vector.

[0077] An additional embodiment of the invention is a recombinant expression vector comprising a promoter, said promoter being functional in a plant cell, and a nucleic acid molecule in accordance with the invention, operably linked to said promoter, wherein said nucleic acid molecule comprises a nucleotide sequence of at least 21 contiguous nucleotides from a sequence chosen from SEQ DD. NOS: 1 or 2, in antisense orientation. More preferably, the sequence contains 21 - 300 contiguous nucleotides from a sequence chosen from SEQ ID. NOS: 1 or 2, in antisense orientation. Such constructs are useful for inhibiting and/or decreasing expression of the relevant genes. In another embodiment, the nucleotide sequence comprises a nucleic acid molecule selected from the group consisting of: (a) SEQ. ID. NOS: 1 or 2, or a part thereof, or a complement thereof; (b) a nucleotide sequence that hybridizes to said nucleotide sequence of (a) under a wash stringency equivalent to 0.1X SSC to LOX SSC, 0.1 % SDS, at 50-65°C, and which encodes a polypeptide having at least 60% of that of the polypeptide of Populus deltoides disclosed herein; (c) a nucleotide sequence encoding a protein having the same amino acid sequence as is encoded by the nucleotide sequence of (a), but which is degenerate in accordance with the degeneracy of the genetic code; and (d) a nucleotide sequence encoding the same amino acid sequence as said nucleotide sequence of (b), but which is degenerate in accordance with the degeneracy of the genetic code.

[0078] Another embodiment of the invention is a recombinant expression vector comprising a promoter functional in a plant cell, a nucleic acid molecule which is a homologue of the nucleotide sequences described supra, and which encodes a polypeptide whose amino acid sequence contains regions that are at least 70% identical to the KNOXA and/or KNOXB domains of SEQ ID. NOS: 3 and 4, as described supra, and which exhibits biological activity equivalent to that of the polypeptides shown in SEQ ID. NOS: 3 or 4.

[0079] More preferably, the nucleic acid molecule encodes a polypeptide whose amino acid sequence contains regions that are at least 75% or even 80% identical to the KNOXA and/or KNOXB sequences supra.

[0080] Expression vectors of the invention may also contain a selection marker by which transformed plant cells can be identified in culture. The marker may be associated with the heterologous nucleic acid molecule, i.e., the gene operably linked to a promoter. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype that permits the selection of, or the screening for, a plant or plant cell containing the marker. Usually, the marker gene will encode antibiotic resistance. This allows for selection of transformed cells from among cells that are not transformed or transfected. Alternatively the marker gene may confer herbicide resistance.

[0081] Examples of suitable selectable markers include adenosine deaminase, dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidne kinase, xanthine- guanine phospho-ribosyltransferase, glyphosate and glufosinate resistance and aminoglycoside 3'-O-phosphotranserase (kanamycin, neomycin and G418 resistance). These markers include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin. The construct may also contain the selectable marker gene Bar that confers resistance to herbicidal phosphinothricin analogs like ammonium gluphosinate (Thompson et al., EMBO J. 9:2519- 2523 (1987)) Other suitable selection markers will be known to those of skill in the art.

[0082] Replication sequences, of bacterial or viral origin, may also be included to allow the vector to be cloned in a bacterial or phage host. Preferably, a broad host range prokaryotic origin of replication is used. A selectable marker for bacteria may be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such a kanamycin or tetracycline.

[0083] Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For instance, when Agrobacterium is the host, T-DNA sequences may be included to facilitate the subsequent transfer to and incorporation into plant chromosomes.

[0084] A further feature of the invention are proteins and polypeptides encoded by the nucleic acid molecules of the invention, exemplified by, but not being limited to, the polypeptides which have the amino acid sequences of SEQ ID. NOS: 3 and 4. Preferably, the polypeptides of the invention have amino acid sequences which contain regions that are at least 70% identical to the conserved KNOXA and/or KNOXB domains referred to supra. Identity greater than 75% is preferred, while identity greater than 80%, 85%, 90% or even 95% with respect to the KNOX domains is preferred, hi all cases, the polypeptide has at least 60% of the biological activity of a polypeptide from the group shown in SEQ ID. NOS: 3 or 4.

[0085] As with any protein or polypeptide, antibodies that are specific thereto can be generated, and these antibodies, which may be polyclonal, monoclonal, chimeric, or produced via recombinant means in some way, manner or form are all covered by the invention as are the means of producing these, including, e.g,. hybridomas, recombinant cells containing nucleic acid molecules that produce the antibodies, and the antibody-producing nucleic acid molecules themselves.

[0086] The presence of the proteins, polypeptides, and/or nucleic acid molecules in a particular cell can be measured to determine if, e.g., a cell has been successfully transformed or transfected. The ability to carry out such assay is well known and need not be reiterated here.

[0087] The nucleic acid molecules of the invention may be used "neat," or preferably in expression vector constructs, to transform or transfect cells, such as plant cells. Standard molecular biological techniques, well known to the skilled artisan, may be used. [0088] Both dicot and monocot cells may be so transformed or transfected. Cells can be so treated before differentiating into plant organs, and can also be treated so as to transform or transfect particular plant organs, such as seeds, fruit, leaves, stems, wood, flowers and so forth. Exemplary of such plants are those which have economic value such as, but not being limited to, turf grasses, leafy plants such as tobacco, spinach, and lettuce, maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes, solanaceous plants such as, potato, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, forage crops, seed- bearing plant, such as rice, corn, soy and bean, and woody trees, such as poplar, eucalyptus, pine, spruce, fir, etc.

[0089] The methods of the present invention rely on altering plants using molecular techniques. AU technical terms used herein are terms commonly used in biochemistry, molecular biology and agriculture, and can be understood by one of ordinary skill in the art to which this invention belongs. Those technical terms can be found in: Molecular Cloning: A Laboratory Manual, 3^rd ed., vol. 1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing Associates and Wiley-Interscience, New York (1988, with periodic updates); Short Protocols in Molecular Biology: A compendium of Methods from Current Protocols in Molecular Biology, 5^th ed., vol. 1-2, ed. Ausubel et al., Jon Wiley & Sons, Inc. (2002); Genome Analysis: A Laboratory Manual, vol. 1-2, ed. Green et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1997). Methods involving plant biology techniques are described herein and are described in detail in methodology treatises such as Methods in Plant Molecular Biology: A Laboratory Course Manual, ed. Maligna et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1995).

[0090] Use of these methods in combination with the nucleic acid molecules of the invention, results in the transgenic plants and transgenic plant cells discussed supra. Agrobacterium such as A. tumefaciens or A. rhizogenes can be used, for example, in accordance with Nagel, et al. Microbiol Lett 67: 325 (1990). In brief, the method teaches that Agrobacterium may be transformed with a plant expression vector via, e.g., electroporation, after which the Agrobacterium is introduced to plant cells via, e.g., the well known leaf-disk method. Additional methods for accomplishing this include, but are not limited to, electroporation, particle gun bombardment, calcium phosphate precipitation, and polyethylene glycol fusion, transfer into germinating pollen grains, direct transformation (Lorz, et al., MoI. Genet. 199: 179-182 (1985), and other methods known to the art. If a selection marker, such as kanamycin resistance, is employed, it makes it easier to determine which cells have been successfully transformed.

[0091] It is to be noted that the Agrobacterium transformation methods discussed supra have been known as being useful for transforming dicots; recently, however, de Ia Pena, et al., Nature 325: 274-276 (1987), Rhodes, et al., Science 240: 204-207 (1988), and Shimamato, et al., Nature 328: 274-276 (1989), all of which are incorporated by reference, have transformed cereal monocots using Agrobacterium. Also see Bechtold, et al., CR. Acad. Sci. Paris 316 (1994), showing the use of vacuum infiltration for Agrobacterium-mediatQd transformation.

[0092] Of course, it is to be understood that additional methods for transforming and/or transfecting plants with the nucleic acid molecules of the invention will be known to the skilled artisan, and need not be reiterated here.

Claims

1. A method for producing a transgenic plant or a transgenic plant part which, has (i) altered size and/or architecture of its aerial portion, (ii) modified architecture and/or size of its non-aerial portion, (iii) altered xylem structure or chemical composition, or (iv) a combination thereof, comprising introducing into a plant cell a nucleic acid molecule which encodes a polypeptide with transcription factor activity, wherein said nucleic acid molecule hybridizes to at least one of the nucleotide molecules set forth in SEQ. ID. NOS: 1 and 2 under stringent conditions, and cultivating said plant cell to obtain said transgenic plant or plant part.

2. The method of claim 1, wherein said nucleic acid molecule encodes a polypeptide of SEQ. ID. NO: 3 or SEQ. ID. NO: 4, or an active portion thereof.

3. The method of claim 1, wherein said nucleic acid molecule has the nucleotide sequence of SEQ. ID. NO: 1 or 2.

4. The method of claim 1, wherein said nucleic acid molecule encodes an amino acid sequence comprising regions that are 70% or more identical to the regions composed of amino acid residues 106-150, 157-209, or 273-332 of SEQ. ID. NO: 3 or SEQ. ID. NO: 4.

5. The method of claim 1, wherein said nucleic acid molecule is from a non-Arabidopsis dicot, a monocot, or a gymnosperm.

6. The method of claim 1, comprising introducing said nucleic acid molecule in the form of a construct in which it is operably linked to a promoter.

7. The method of claim 6, wherein said construct further comprises a selection marker.

8. A transgenic plant or plant part produced by the method of claim 1.

9. The transgenic plant of claim 8, wherein said plant is a dicot, monocot, or gymnosperm.

10. The transgenic plant of claim 9, wherein said plant is a woody tree.

11. The transgenic plant of claim 10, wherein said woody tree is Eucalyptus, Populus, or Pinus.

12. The transgenic plant part of claim 8, wherein said plant part is a leaf, a stem, a flower, an ovary, a fruit, a seed, a callus, or wood.

13. An isolated nucleic acid molecule which comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleotide sequence set forth in SEQ. ID. NO: 1 or 2, and which encodes a plant transcription factor.

14. The isolated nucleic acid molecule of claim 13, comprising SEQ. ID. NO: 1 or 2.

15. The isolated nucleic acid molecule of claim 13, which encodes a protein having the amino acid sequence of SEQ. BD. NO: 3 or 4, or an active portion thereof.

16. The isolated nucleic acid molecule of claim 13, which encodes an amino acid sequence that comprises regions that are 70% or more identical to the regions composed of amino acid residues 106-150, 157-209, or 273-332 of SEQ. ID. NO: 3 or 4.

17. Expression vector comprising the isolated nucleic acid molecule of claim 13, operably linked to a promoter.

18. The expression vector of claim 17, further comprising a selection marker.

19. Recombinant cell, transformed or transfected with the expression vector of claim 17.