WO1996037614A1 - Gene and protein for modification of the gravitropic response - Google Patents

Abstract

Nucleotide sequences encoding a profilin-like polypeptide, as well as the polypeptide itself, and methods for modifying the gravitropic response of a plant, are provided.

Description

GENE AND PROTEIN FOR MODIFICATION OF THE GRAVITROPIC RESPONSE

FIELD OF THE INVENTION The present invention relates to the field of plant molecular biology. More specifically, the present invention relates to isolated lazy genes and the profilin- like protein encoded thereby, and to plants having a modified gravitropic response.

BACKGROUND Gravitropism is the mechanism by which plants change the orientation of growth in response to gravity. A theory to explain this phenomenon must involve both the perception of gravity and the subsequent reorientation of growth. For the past decades two models of gravitropism have been tested. The starch- statholith theory is based on heavy starch granules in amlyopiasts that sediment in response to gravity. In some way sedimentation 'tells' the cell where it is in relation to the force of gravity. However, mutants of Arabidopsis that do not store starch yet still respond, albeit more slowly, to a change in orientation, show that although statholiths may be involved, they do not adequately explain the entire phenomenon (Casper and Prickard, 1989). The Cholodeny-Went theory explains gravitropic bending due to asymmetric accumulation of auxin. Thus when reoriented with respect to gravity, auxin is transported laterally and accumulates in the basipetal regions of the stem. Auxin, in turn, enhances cell division resulting in bending and the resumption of negative gravitropic growth.

The lazy (lal) locus of maize is a recessive gravitropic mutant first described in 1923 (Jenkins and Gerhardt, 1931 ). Homozygous lazy plants grow up out of the soil, but approximately one month prior to flowering, begin to bend just above the nodes and grow toward gravity (positive gravitropic growth) until they are prostrate. Male and female flowers differentiate and mature normally. If lazy plants are staked at this bending stage, positive gravitropic growth continues.

The lazy phenotype can be observed much earlier in seedlings by changing the direction in which the plants are oriented. Whereas non-mutant plants bend away from gravity to restore negative gravitropic growth, homozygous lazy plants do not bend. Van Overbeek measured the auxin, indoleacetic acid (IAA) in lazy and normal coleoptiles after the direction of the gravity vector was altered to right angles (Van Overbeek, 194-). Normal plants had higher IAA in the basal longitudinal section, whereas lazy seedlings had higher IAA in the apical region. His conclusion was that the lazy gene interferes with the auxin distribution in the stem and thus the shoot bends on the side the concentration of IAA is highest. By some mechanism, the lazy seedlings have the opposite IAA levels compared to their normal siblings. Profilin is an actin binding protein. It is believed to bind both actin and phosphoinositides and to provide storage capabilities for actin. Its role in signal transduction has been postulated: an external signal reacts with a ligand on the cell membrane's surface; a kinase is activated which causes dephosphorylation of profilin; once dephosphorylated, profilin has a higher affinity for actin, and causes rapid depolymerization of actin microfiliments and the subsequent collapse of the cytoskeleton.

Positive gravitropic growth is a desirable growth habit in the ornamental industry, where it is only rarely found in naturally occurring variants. In addition, an "opposite" logical extension of this thinking leads to the conclusion that it might in some instances be advantageous to make organs which normally grow down grown up instead.

In the past, plant breeders have had to rely on the lucky, serendipitous or fortuitous discovery of rare gravitropic mutants, since there has been no known method for direct breeding of a modified gravitropic response. Many ornamentals are not amenable to breeding and are propagated vegetatively, and this fact further reduces the opportunity to find or exploit gravitropic mutants. The mimicking of mutants has been accomplished by training woody species with strong wires, and this sort of training has been practiced extensively in the orient. However, a molecular genetic method for the alteration or modification of a plant's gravitropic response is highly desirable. SUMMARY

It is therefore an object of the present invention to provide a nucleotide sequence encoding a plant profilin-like polypeptide (the sequence of which is shown in FIGURE 1 ), and the plant profilin-like polypeptide encoded thereby (the sequence of which is shown in FIGURE 1 ). It is a further object of this invention to provide expression cassettes, useful in the transformation of plant cells, that contain the nucleotide sequences encoding the profilin-like proteins of the invention or their functional equivalent.

It is also an object of the present invention to provide a method for modifying the gravitropic response of a plant. This can be accomplished either by selection of non-transgenic mutants using Mutator transposon methodology, or by generation of transgenic plants comprising the lazy gene of the invention, in either the sense or the antisense orientation, such that the transgenic plant displays the opposite gravitropic growth response than that of non-transgenic, non-mutated (wild type) plants of the same species. In addition, the present invention also provides a method of modifying the gravitropic response in plants of appropriate taxa by selecting plants from sexually compatible taxa that have been transformed with DNA encoding the profilin-like polypeptide of the invention and sexually crossing such plants with plants from a taxon in which the gravitropic response is to be altered. Reproductive material is recovered from the progeny of the cross, and insect resistant plants are grown up from that reproductive material. To modify the gravitropic response in taxa consisting of substantially homozygous plants the method further comprises repetitive backcrossing of gravitropically modified progeny with substantially homozygous non-modified plants of the taxon, and selecting for expression of the profilin-like polypeptide along with any other desired characteristics of the non-modified plants from among the progeny of the backcross, until the desired percentage of the characteristics of the originally non-modified taxon are present in the progeny, along with the modified gravitropic response. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the double stranded nucleotide sequence of the lazy gene of the invention, and also shows the amino acid sequence encoded by the nucleotide sequence.

DETAILED DESCRIPTION The present invention provides a novel nucleotide encoding a profilin-like protein, the profilin-like polypeptide encoded for by that gene, and methods for modifying the gravitropic response of a plant.

Transposon tagging has been highly successful for cloning plant genes. See, for example, (Johal and Briggs, Reductase Activity Encoded by the Hm1 Disease Resistance Gene in Maize, Science 258:985-987, 1992). Briefly, mutations in the gene of interest are identified in families harboring transposabie elements. DNA restriction fragments are identified that hybridize to a transposon probe and are present in plants with the phenotypic mutation. These restriction fragments are then cloned and tested for identity as the gene of interest. This methodology was used by the inventors to isolate the lazy gene, as described in detail below in Example!

The present invention includes the lazyl gene, its DNA sequence as set forth in FIGURE 1 , the polypeptide encoded for by that sequence and having the amino acid sequence set forth in FIGURE 1 , and structural and functional equivalents thereof. The present invention also includes expression cassettes including the DNA or RNA sequences encoding the profilin-like polypeptide, and transformed plant cells and plants comprising such expression cassettes. In addition, the invention provides a method for the modification of a plant's gravitropic response. By "transgenic plant" is meant any plant or plant cell that has become transformed by the introduction, stable and heritable incorporation, into the subject plant or plant cell, of either native DNA that is under the control of a promoter other than the promoter that typically drives expression of that DNA in a wild-type plant, and that has been introduced back into its host plant, or foreign

DNA, i.e. DNA encoding for a protein not normally found within that plant species. "Plantlet" refers to a plant sufficiently developed to have a shoot and a root that is asexually reproduced by cell culture.

"Explant" refers to a section or piece of tissue from any part of a plant for culturing.

The term "callus" and its plural "calli", refer to an unorganized group of cells formed in response to cutting, severing, or other injury inflicted on plant tissue. Excised pieces of plant tissue and isolated cells can be induced to form callus under the appropriate culture conditions. Callus can be maintained in culture for a considerable time by transferring or subculturing parts of the callus to fresh medium at regular intervals. The transfer of callus to liquid medium leads to dispersion of the tissue and the formation of a plant cell suspension culture. Callus can be induced to undergo organized development to form shoots and roots.

"Embryoid" refers to a structure similar in appearance to a plant zygotic embryo. By the term "taxon" herein is meant a unit of botanical classification of genes or lower. It thus includes genes, species, cultivar, variety, variant, and other minor taxonomic groups that lack a consistent nomenclature.

"Somatic hybrid" and "somatic hybridization" refers generally to stable combination of cellular material, be it protoplast/protoplast or protoplast/cytoplast combinations, and includes cybrids and cybridization.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.

As used herein, the term "nucleotide sequence" means a DNA or RNA sequence, and can include a cDNA, or genomic DNA, or synthetic DNA sequence, a structural gene or a fragment thereof, or an mRNA sequence, that encodes an active unfunctional polypeptide. A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A DNA "coding sequence" is a DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

A "promoter sequence" or a "promoter" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bound at its 3' terminus by the translation start codon (ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eucaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Procaryotic promoters can contain Shine- Dalgarno sequences.

DNA "control sequences" refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell.

A coding sequence is "operably linked to" or "under the control of control sequences in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence. A "host cell" is a cell which has been transformed, or is capable of undergoing transformation, by an exogenous DNA sequence.

A cell has been "transformed" by endogenous or exogenous DNA when such DNA has been introduced inside the cell membrane. The DNA may or may not be integrated into (covalently linked to) chromosomal DNA making up the genome of the transformed cell. In procaryotes and yeasts, for example, the DNA may be maintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the DNA.

A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA, RNA or polypeptide sequences are "substantially homologous" or "structurally equivalent" when at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982; Brown, T. A., Gene Cloning: An Introduction, (2nd Ed.) Chapman & Hall, London, 1990.

A "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a bacterial gene, the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacterium. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). "Heterologous" DNA also refers to DNA not found within the host cell in nature. Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as these terms are used herein. "Native", "autologous" or "endogenous" DNA, as used herein, refer to DNA that is typically present in the host in nature. The term "polypeptide" as used herein is used in its broadest sense, i.e., any polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the term "polypeptide" includes proteins, oligopeptides, protein fragments, analogues, muteins, fusion proteins and the like. The term also encompasses amino acid polymers as described above that include additional non-amino acid moieties. Thus, the term "polypeptide" includes glycoproteins, lipoproteins, phosphoproteins, metalloproteins, nucleoproteins, as well as other conjugated proteins. The term "polypeptide" contemplates polypeptides as defined above that are recombinantly produced, isolated from an appropriate source, or synthesized. In carrying out this invention, it will be appreciated that numerous plant expression cassettes and vectors are well known in the art. By the term "expression cassette" is meant a complete set of control sequences including initiation, promoter and termination sequences which function in a plant cell when they flank a structural gene in the proper reading frame. Expression cassettes frequently and preferably contain an assortment of restriction sites suitable for cleavage and insertion of any desired structural gene. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence. In addition, the plant expression cassette preferably includes a strong promoter sequence at one end to causes the gene to be transcribed at a higher frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA. An example of a preferred (empty) expression cassette into which the DNA sequence of the present invention can be inserted is the pPHI414 plasmid developed by Beach et al. of Pioneer Hi-Bred International, Inc., Johnston, IA. Preferred plant expression cassettes can be designed to include one or more selectable marker genes, including, for example, kanamycin resistance or herbicide tolerance genes. By the term "vector" herein is meant a DNA sequence which is able to replicate and express a foreign gene in a host cell. Typically, the vector has one or more endonuclease recognition sites which may be cut in a predictable fashion by use of the appropriate enzyme. Such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic or herbicide resistance, which then serve as selectable markers to identify and separate transformed cells. Preferred selection agents include, for example, kanamycin, chlorosulfuron, phosphonothricin, hygromycin and methotrexate, and preferred markers are genes conferring resistance to these compounds. A cell in which the foreign genetic material in a vector is functionally expressed has been "transformed" by the vector and is referred to as a "transformant". A "foreign" gene refers to any gene introduced into a plant by transformation techniques.

A particularly preferred vector is a plasmid, by which is meant a circular double-stranded DNA molecule that is not a part of the chromosomes of the cell. As mentioned above, genomic, synthetic, and/or cDNA representing the nucleotide sequence of the lazy gene and encoding the profilin-like polypeptide may be used in this invention. The vector of interest may also be constructed partially from a cDNA clone, partially from a synthetic sequence and partially from a genomic clone. When the lazy nucleotide sequence is in hand, genetic constructs are made which contain the necessary regulatory sequences to provide for efficient expression of the gene in the host cell. According to this invention, the genetic construct will contain a genetic sequence coding for the profilin-like polypeptide and one or more regulatory sequences operably linked on either side of the nucleotide sequence. The nucleotide sequence encoding the profilin-like polypeptide may be incorporated into the genome of a plant in either the sense or antisense direction. The use of antisense is described generally in Van der Krol et al., (1990) Mol. Gen. Genet. 220:204-212. The use of sense DNA sequences is described in various references, including Napoli et al. (1990) Plant Cell, 2:279-289, and Van der Krol et al. (1990) Plant Cell, 2:291- 299. Alternative down-regulatory methods are also known in the molecular biology arts, including, for example, TranSwitch™ technology, as described in U.S. Patents 5,034,323 and 5,231 ,020 (both Jorgensen et al.), and can be applied in the practice of the present invention. Typically, the regulatory sequences will be selected from the group comprising of promoters and terminators. The regulatory sequences may be from autologous or heterologous sources.

Promoters that may be used in the genetic sequence include, for example, nos, ocs, phaseolin, caMV, FMV, ubiquitin, and other promoters isolated from the DNA of plants or other sources, both natural and synthetic. A tissue-specific promoter can be used in instances where it may be desirable to localize production of the lazy expression product to a particular tissue type.

An efficient plant promoter that may be used is an overproducing plant promoter. Overproducing plant promoters that may be used in this invention include the promoter of the small sub-unit (ss) of the ribulose-1 , 5-bisphosphate carboxylase from soybean (Berry-Lowe et al., J. Molecular and App. Gen.. 1 :483- 498 (1982)), and the promoter of the chlorophyll a-b binding protein. These two promoters are known to be light-included in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, An Agricultural Perspective. Cashmore, Pelham, New York, 1983, pp. 29-38, G. Coruzzi et al., J. Biol. Chem., 258:1399 (1983), and P. Dunsmuir, et al., J. Molecular and App. Gen., 2:285 (1983)) and may be particularly desirable in the practice of the present invention. An especially preferred constitutive promoter is the 35S promoter from Cauliflower Mosaic Virus.

Root-specific promoters are also well known and can be selected from the many available from the literature or isolated de novo from various compatible species. For example, Hirel, B., Marsolier, M.C., Hoarau, A., Hoarau, J., Brangeon, J., Schafer, R., and Verma, D.P.S., Plant Molecular Biology. Oct. 1992. v. 20 (2), pp. 207-218, describe a root-specific glutamine synthetase gene from soybean. Keller, B. and Baumgartner, C, The Plant Cell, Oct. 1991 , v. 3 (10), pp. 1051-1061 , describe a root-specific control element in the GRP 1.8 gene of French bean. Sanger, M., Daubert, S., and Goodman, R.M., Plant Molecular Biology, Mar. 1990, v. 14 (3), pp. 433-443, discuss the root-specific promoter of the Mannopine Synthase (MAS) gene of Agrobacterium tumefaciens. Miao, G.H., Hirel, B., Marsolier, M.C., Ridge, R.W., and Verma, D.P.S., The Plant Cell, Jan. 1991 , v. 3 (1 ), pp. 11-22, describe a full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean. Bogusz, D., Llewellyn, D.J., Craig, S., Dennis, E.S., Appleby, C , and Peacock, W.J., The Plant Cell, July 1990, v. 2(7), pp. 633- 641 , discusses two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing noniegume Parasponia andersonii and the related non- nitrogen-fixing noniegume Trema tomentosa. The promoters of these genes were linked to a beta-glucuronidase reporter gene and introduced into both the noniegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach, F. and Aoyagi, K., Plant Science (Limerick) 1991 , 79 (1 ):69-76, describe their analysis of the promoters of the highly expressed ro1 C and ro1 D root-inducing genes of Agrobacterium rhizogenes. They concluded that enhancer and tissue-specific DNA determinants are dissociated in those promoters. Teeri, T.H., Lehvaslaiho, H., Franck, M., Uotila, J., Heino, P., Palva, E.T., Montagu, M. van, and Herrera- Estrella, L, EMBO Journal, 1989, 8 (2):343-350, used gene fusions to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene was root specific in the instant plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene. The TR1' gene, fused to NPTII, (neomycin phosphotransferase II) showed similar characteristics.

Various promoters particularly useful in plants are disclosed and discussed in U.S. Patent No. 5,234,834 (Fischer et al., Aug. 10, 1993). Fischer et al. also discuss expression cassette and vector construction useful for plant genetic applications. The expression cassette comprising the structural gene for profilin operably linked to the desired control sequences can be ligated into a suitable cloning vector. In general, plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used. The cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotyic selection markers in transformed host cells. Typically, genes conferring resistance to antibiotics or selected herbicides are used. After the genetic material is introduced into the target cells, successfully transformed cells and/or colonies of cells can be isolated by selection on the basis of these markers.

Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells. Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as E. coli, S. typhimurium, and S. marcescens. Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention. The isolated cloning vector will then be introduced into the plant cell using any convenient technique, including electroporation, retroviruses, microparticle bombardment, and microinjection into cells from any plant species, including monocotyledonous or dicotyledonous plants, and particularly including ornamental plant species, in cell or tissue culture, or in whole plants where applicable, to provide transformed plant cells or plants containing as foreign DNA at least one copy of the DNA sequence of the plant expression cassette. Using known techniques, protoplasts can be regenerated and cell or tissue culture can be regenerated to form whole fertile plants which carry and express the desired nucleotide sequence for the selected polypeptide(s). See generally Chapters 6, 7 and 9 in Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology. CRC Press, Boca Raton, 1993.

It will also be appreciated by those of ordinary skill that the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens, which can then be used to transfer the vector into susceptible plant cells. Thus, this invention provides a method for altering a plant's gravitropic response in Agrobacterium tumefaciens-s scepX\b\e plants in which the expression cassette is introduced into the cells by infecting the cells with Agrobacterium tumefaciens, a plasmid of which has been modified to include a plant expression cassette which expresses the profilin-like polypeptide in the manner of this invention. Additionally, this can be accomplished, in appropriate plant taxa, by culturing regenerable cells or tissues from at least one plant of the taxon, introducing into the cells of the cell or tissue culture at least one copy of an expression cassette comprising the nucleotide sequence encoding the profilin- like lazy polypeptide of the invention, the nucleotide sequence being operably linked to plant regulatory sequences that cause expression of peptides in the cells, and regenerating whole plants with an altered gravitropic response from the cell or tissue culture. Once whole plants have been obtained they can be sexually or clonally reproduced in such a manner that at least one copy of the sequence provided by the expression cassette is present in the cells of progeny of the reproduction.

Alternatively, once a single transformed plant has been obtained by the foregoing method, conventional plant breeding methods can be used, in amendable taxa, to transfer the structural gene for the profilin-like polypeptide of the invention and regulatory sequences via crossing and backcrossing. Such a method would further comprise sexually crossing the transformed plant with a plant the gravitropic response of which is to be altered, recovering reproductive material from the progeny of the cross, and growing gravitropically altered plants from the reproductive material. Where desirable or necessary, particularly desirable traits or characteristics of the gravitropically altered plant can be substantially preserved by expanding this method to further include repetitively backcrossing the transformed, altered gravitropic response progeny with unaltered plants, and selecting for altered gravitropic response among the progeny of the backcross, until the desired percentage of the characteristics of the unaltered plants are present in the progeny along with the gene for altered gravitropic response.

The following description further exemplifies the compositions of this invention and the methods of making and using them. However, it will be understood that other methods, known by those of ordinary skill in the art to be equivalent, can also be employed.

EXAMPLE 1 in a screen of selfed progeny derived from parents with Mutator (Mu) transposons, a family segregating the lazy mutation was identified. A test cross of mutant progeny to the reference Ia1 allele from the Maize Genetic Stock Center verified the mutation was an allele of the previously described lazy gene. The new allele is designated Ia1-m1. Southern hybridization analysis identified a 7.6 kb Sacl DNA restriction fragment which hybridized to the Mu1 probe, MuED4 (420 bp BstEII-Notl fragment) and was present in all of the Ia1-m1 mutant progeny. Subsequent probing of forty additional Ia-m1 plants with Mu1 confirmed the presence of the 7.6 kb Sacl fragment. This restriction fragment was cloned using lambda zap (Stratagene). To verify the clone was indeed the fragment identified with the Mu1 probe, a subfragment of pSKSacl 7.6, BI1 II 0.5 kb, was hybridized to the same blot. The same fragment that hybridized to the Mu1 probe and was present in all Ia-m1 individuals was identified by the Bg1 II 0.5 kb probe. To confirm pSKSacl 7.6 was indeed part of the lazy locus, the Bgl ll 0 probe was used to detect novel polymorphisms between progenitors and derived mutant alleles of the lazy gene. Several lazy alleles have been identified from maize lines carrying active transposons. These alleles include, Ia1-m2 (from Activator-Dissociation line, P-vv, r-scm-3), 1 (from Suppressor-mutator c2-m1 ), and Ia1-m4 (from Mutator Mu-DR, a1 -m). The Bgl ll 0.5 gene specific probe detects a novel insertion in 1a1-m2 individuals that is not present in the progenitors. This novel insert had 4.3 kb and includes a Sacl and EcoRI restriction site. The detection of a novel insertion in Ia1-m2 by the Bg1 II 0.5 kb probe verified that it is the lazy gene. A cDNA for the lazy gene was cloned from a lambda gt10 library made from shoot apical meristems of the maize inbred, W22. Using the Sacl 7.6 kb genomic clone as the probe, two cDNA clones were recovered from 600,000 plaques. The EcoRI inserts were cloned into pSK- (Stratagene) and the largest 1.0 kb cDNA (cLa14) was sequenced. Genomic mapping of the cDNA localized the coding region adjacent to the Mu1 insertion. Using a primer to the end of Mutator elements (Benson et al., 1995), the genomic clone pSKSac7.6 was sequenced. Sequence matching the cDNA was encountered at position 247 with the initial ATG at position of the genomic sequence. Thus, the Mu1 insertion is 326 nucleotides 5-prime of the start of transcription in the genomic clone of Ia1- ml .

To determine in which tissues the lazy gene is expressed, Northern hybridizations were done using the cDNA, cLa14, as a probe to total RNA isolated from a variety of maize tissues. The cDNA hybridized to a 1 kb RNA species in wild type and mutant leaves and immature ears but not roots, tassels or pollen.

The nucleotide sequence was compared for homology to other sequences in Genbank using BLAST (Altschul et al., 1990). The sequence of cl-A14 is similar to other profilin sequences, and there is a high degree of homology to profilin sequences identified from many species of plants.

Profilins in plants are more similar to each other than to profilins in other eukaryotes (Staiger et al., 1993). In fact, only 9 amino acids are conserved in eukaryotes (Staiger et al., 1993). Yet, functionally, microinjected birch pollen profilin terminates cytoplasmic steaming by depolymerization of the microfiliments (Staiger et al., 1994). The greatest homology to cLa14 is with a maize cDNA, Zea mays 5C (Shen et al., 19 ). It is intriguing that cLa14 is more similar to profilin sequences from Arabidopsis, rice, wheat, timothy grass, and Brassica than to other profilins isolated from maize (Zea mays PR01-3; Figure 4). Without intending to be limited by theory, the outgrowth of Zea mays PR01-3 suggests a functional divergence of profilin sequences. Although the function of Z. mays PR01-3 is not known for certain, it may be postulated to be involved in the direction of pollen tube growth by orienting microfiliments. By analogy, the function of the lazy profilins in gravitropism may be to provide a rapid way to change the orientation microfiliments and thus the cytoskeleton during a change in direction gravity.

EXAMPLE 2 Transgenic plants can be produced using any of several art-recognized methods. See, generally, Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, 1993. For example, ornamental species have been transformed by inoculating stem and leaf fragments with Agrobacterium rhizogenes. Pellegrineschi et al., BioTechnology 12:64-68, 1994. Such plants can be transformed with a cDNA or genomic DNA or synthetic DNA sequence (i.e., a nucleotide sequence) coding for the profilin- like polypeptide of the invention or a structural and functional equivalent, operably linked to an over-producing promoter that drives expression of the polypeptide, either constituatively or in a tissue specific manner. Such plants can then be tested for expression of the polypeptide and/or monitored for altered or modified gravitropic response using art-recognized techniques.

All references cited herein are hereby expressly incorporated herein by reference.

Claims

WHAT IS CLAIMED:

1. A profilin-like polypeptide having the amino acid sequence shown in FIGURE 1.

2. A DNA molecule having the nucleotide sequence of FIGURE 1.

3. A nucleotide sequence encoding the amino acid sequence of the profilin- like polypeptide having the amino acid sequence shown in FIGURE 1 or a structural and functional equivalent thereto.

4. An RNA sequence according to Claim 3.

5. A DNA sequence according to Claim 3.

6. An expression cassette containing the DNA sequence of Claims 2 or 5, oriented in the sense or antisense direction, operably linked to one or more regulatory sequences which cause the expression of the DNA sequence in plant cells.

7. Transformed plant cells containing at least one copy of the expression cassette of Claim 6.

8. Transformed cells according to Claim 7, further characterized in being cells of an ornamental species.

9. A transformed plant comprising transformed plant cells containing at least one copy of the expression cassette of Claim 6.

10. A method for modification of the gravitropic response of a plant, the method comprising growing a transformed plant comprising the expression cassette of Claim 6.

11. The method of Claim 10 wherein the plant is an ornamental plant.

12. A plant having a gravitropic response that has been modified using the method of Claim 10.

13. A method for modifying the gravitropic response of a plant comprising inserting into the genome of the plant a nucleotide sequence that codes for the profilin-like polypeptide of Claim 1 , in proper reading frame relative to transcription initiator and promoter sequences active in plants, to cause expression of the nucleotide sequence at levels that provide an amount of the polypeptide effective in modifying the gravitropic response of the plant.

14. The method of Claim 13 wherein the plant is an ornamental plant.

15. The method of Claim 13, further comprising culturing cells or tissues from the plant, introducing into the cells of the cell or tissue culture by a transformation method at least one copy of an expression cassette comprising a nucleotide sequence encoding the profilin-like polypeptide of

Claim 1 or a structural and functional equivalent, and regenerating whole plants from the cell or tissue culture.

16. The method of Claim 15, further comprising sexually or clonally reproducing the whole plant so that at least one copy of the nucleotide sequence provided by the expression cassette is present in the cells of progeny of the reproduced plant.

17. A method of modifying the gravitropic response of a plant, the method comprising selecting a fertile gravitropically modified plant prepared by the method of Claim 16 from a sexually compatible taxon, sexually crossing the modified plant with a plant of the taxon to be modified, recovering reproductive material from the progeny of the cross, and growing gravitropically modified plants from the reproductive material.

18. The method of Claim 17 for modifying the gravitropic response in a non- modified taxon consisting of substantially homozygous plants, the method further comprising repetitively backcrossing the insect resistant progeny with substantially homozygous, non-modified plants from the taxon, and selecting for expression of modified gravitropic response along with any other desired characteristics of the originally non-modified taxon from among the progeny of the backcross, until the desired percentage of the characteristics of the originally non-modified taxon are present in the progeny along with the modified gravitropic response.