WO1998004701A1 - Methods and compositions for improving a plant's ability to take in phosphate from soil - Google Patents

Abstract

The present invention relates to methods and materials in the field of molecular biology and the regulation of protein synthesis through plant genetic engineering. More particularly, the invention relates to newly-isolated nucleotide sequences, nucleotide sequences having substantial identity thereto and proteins encoded thereby. The invention also involves the introduction of foreign nucleotide sequences into a plant genome, wherein the introduction of the nucleotide sequence effects an increase in the plant's ability to transport phosphate. Plant transformants harboring a DNA construct comprising a promoter operably linked to an inventive nucleotide sequence demonstrate increased levels of phosphate transporter proteins, rendering the plant better able to sustain growth in a phosphate-deficient substrate.

Description

METHODS AND COMPOSITIONS FOR IMPROVING A PLANT'S ABILITY TO TAKE IN PHOSPHATE FROM SOIL

This invention was made with government support under the following grant: number 94-37100-0834 awarded by the USDA. The government has certain rights in the invention.

REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.

60/022.391, filed July 29, 1996. which is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to methods and materials in the field of molecular biology and the regulation of protein synthesis through plant genetic engineering. More particularly, the invention relates to newly-isolated nucleotide sequences, nucleotide sequences having substantial identity thereto and proteins encoded thereby. The invention also involves plant nutrition, specifically, the introduction of foreign nucleotide sequences into a plant genome, wherein the introduction of the nucleotide sequence effects an increase in the plant^'s ability to take in phosphate (Pi) from the soil. Plant transformants harboring an inventive DNA construct comprising a promoter operably linked to an inventive nucleotide coding sequence demonstrate increased levels of phosphate transporter protein production, rendering the plant better able to withstand a phosphate deficiency.

Discussion of Related Art

Phosphorus is one of the essential and major plant nutrients, and phosphorus availability is considered as one of the major growth limiting factors for plants in many natural ecosystems. Most commercial farming worldwide depends upon phosphatic fertilizers as a source of phosphorus. The availability of rock phosphate, a non-renewable source of phosphorus, is predicted to last only for the next 60-90 years. In view of the importance of phosphorus as a nutrient and the non-renewable nature of the raw material, there is a neet to enhance the efficiency of its uptake by plant roots. Plants have developed adaptive mechanisms to overcome Pi stress. Changes in the root growth and architecture, increased production of phosphatases and RNAases. and altered activity of several enzymes of the glycolytic pathway are among the well characterized responses to Pi deficiency in plants. In addition, an increase in phosphate uptake rate of roots and cell cultures following phosphate starvation has been observed in several plant species.

Phosphate is acquired by plants in an energy mediated cotransport process, driven by a proton gradient generated by plasma membrane H^τ-ATPases. Phosphate absorption is accompanied by H+ influx with a stoichiometry of 2 to 4 H7H_:PO₄ ^" transported. A dual-mechanism model for uptake of ions, including phosphate, has been proposed. This is characterized by a high-affinity transport system operating at low (μM) concentration and a low-affinity system functioning at high concentration (mM) of ions. Measurements of phosphate in saturation extracts of a large number of soils showed that phosphate concentration in the majority of samples ranged between 0.65 to 2.5 μM. Under these conditions, the high-affinity transport system is considered to be the major mechanism for phosphate acquisition. Additionally, the kinetic characterization of the Pi uptake system by whole plants and cultured cells indicates a high-affinity transport activity operating at low concentration (μM range).

As stated above, an increase in phosphate uptake rate of roots and cultured cells following phosphate starvation has been observed in several plant species. The enhanced uptake appears to be, in part, due to an increased synthesis of a carrier system in response to phosphate deprivation or starvation. Studies with cultured cells of Catharanthus roseus has lead to the conclusion that the low affinity system is apparently expressed constitutively. while the high affinity system is regulated by the availability of phosphorus. When cells grown in Pi rich medium were transferred to Pi depleted medium, the high-affinity uptake activity increased significantly within 2 days. Phosphorus stress in microorganisms is known to result in transcriptional activation of high-affinity Pi transporters and phosphatases. Based upon high-affinity phosphate transporter genes from fungi which have been cloned and characterized. Pi transporter proteins are predicted to have a structure containing 12 membrane spanning domains separated into two groups of six by a charged hydrophilic region. Although the control of phosphate uptake in plants is well-documented at the physiological level, information regarding the molecular structure of transport proteins and their genetic regulation is incomplete. The present inventors have discovered, isolated and characterized four nucleotide sequences which encode phosphate transporters, two having been isolated from Arabidopsis thaliana and two having been isolated from Lycopersicon esculentum (tomato). The inventors have further discovered that these genes are induced in a tissue specific manner in response to phosphate starvation. The present invention, therefore, provides materials and methods for producing transgenic plants which over-express phosphate transporter proteins, preferably in their roots, these transgenic plants being more efficient in absorbing phosphate from the soil. The regulated expression of efficient forms of phosphate transporters should lead to greater agricultural productivity and reduced fertilizer costs to growers. Incorporation of phosphate uptake-efficiency traits in breeding programs will be an added benefit to biotechnology and seed companies trading with tropical countries, where soils are naturally deficient in phosphorus.

SUMMARY OF THE INVENTION

The present invention relates to the isolation, purification and use of nucieotide sequences and proteins encoded thereby. Inventive nucleotide sequences are advantageously integrated into a DNA construct which comprises a nucleotide coding sequence which encodes a phosphate transporter protein and a promoter capable of eliciting expression of the nucleotide coding sequence in a transformed plant. In a preferred aspect of the invention, the nucleotide sequence encoding a phosphate transporter has substantial identity to the sequence set forth in SEQ ID NO: 1 ; SEQ ID NO:2; SEQ ID NO:3; or SEQ ID NO:4. In one preferred aspect of the invention, the promoter is a tissue specific promoter. In another preferred aspect of the invention, the promoter is an inducible promoter which elicits expression of the nucleotide sequence when a plant experiences a phosphate deficiency. In one preferred aspect of the invention, the inventive DNA construct comprises a promoter having substantial identity to a native high-affinity phosphate transporter promoter such as, for example, AtPTl. AtPT2, LePTl or LePT2. In another preferred aspect of the invention the promoter has substantial identity to the nucleotide sequence set forth in SEQ ID NO:9. As discussed in the Background above, a phosphate transporter protein selected in accordance with the invention is an essential part of the high- affinity phosphate uptake mechanism, and over-expression thereof in the proper plant tissues results in an increased ability by the plant to survive and flourish in a low phosphate soil environment.

It is presently shown that inventive DNA constructs may advantageously be used according to the invention to transform a plant, thereby providing an inventive transformed plant which over-expresses a phosphate transporter protein. The term ''expresses", as used herein, refers to the production of the protein product encoded by a nucleotide coding sequence. "Over-expresses" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non- transformed organisms. Thus, a plant transformed in accordance with the invention produces an amount of a phosphate transporter protein that is greater than that of a non-transformed plant of the same species. The present invention thus provides methods for genetically engineering plants to provide inventive transformed plants which have an increased ability to withstand phosphate deprivation by increasing the plant^'s ability to bring phosphate into the plant^'s roots. This increased ability results from an increase in the rate of expression of phosphate transporter coding sequences. The invention features DNA constructs comprising a promoter sequence and a coding sequence as set forth herein, as well as DNA constructs comprising nucleotide sequences having substantial identity thereto and having similar levels of functionality. Inventive constructs may be inserted into an expression vector to produce a recombinant DNA expression vector which is also an aspect of the invention.

In a preferred aspect of the invention, there is provided an isolated nucleic-acid construct comprising a promoter and a nucleotide sequence having substantial identity to the sequence set forth in SEQ ID NO: l (AtPTl cDNA); SEQ ID NO:2 (AtPT2 cDNA); SEQ ID NO:3 (LePTl cDNA) or SEQ ID NO:4 (LePT2 cDNA). In a preferred aspect of the invention, the protein encoded thereby preferably has an amino acid sequence having substantial identity to the sequence set forth in SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7 or SEQ ID NO:8, wherein the amino acid sequence may include amino acid substitutions, additions and deletions that do not alter the function of the phosphate transporter protein.

It is an object of the present invention to provide an isolated DNA construct which comprises a tissue-specific promoter and a nucleotide sequence encoding a phosphate transporter protein, the construct finding advantageous use when incorporated into a vector or plasmid as a transformant for a plant. Additionally, it is an object of the invention to provide transformed plants which produce one or more phosphate transporter proteins at a rate greater than a native plant of the same species, thereby providing to the transformed plant the improved ability to survive and flourish in low-phosphate soil without the need for phosphorus- containing fertilizers. Further objects, advantages and features of the present invention will be apparent from the detailed description herein.

BRIEF DESCRIPTION OF THE FIGURES

Although the characteristic features of this invention will be particularly pointed out in the claims, the invention itself, and the manner in which it may be made and used, may be better understood by referring to the following description taken in connection with the accompanying figures forming a part hereof.

Figure 1 A illustrates the alignment of the deduced amino acid sequence of LePTl . LePT2. AtPTl and AtPT2 with that of potato (STPT1 and STPT2) and Catharanthus roseus (PIT1) phosphate transporters. Identical amino acids are indicated by asterisks (*) and conserved substitutions are indicated by dots (.). The membrane spanning domains of LePTl and LePT2 as predicted by TopPred are underlined and their numbering is indicated by roman numerals (I-XII). The shaded sequences are consensus sites for N-linked glycosylation; boxed sequences are consensus sites for phosphorylation by casein kinase II; and boxed and shaded sequences are consensus sites for phosphorylation by protein kinase C. Figure IB is a summary of percent amino acid identity between tomato and other plant phosphate transporters.

Figure 2 sets forth the results of a Northern blot analysis of the expression of phosphate transporter genes in Arabidopsis. Total R A from roots or leaves of Arabidopsis plants grown hydroponically in a solution containing 250 μm (+) or no (-) phosphate was hybridized with labeled probe from either AtPTl , AtPT2 or rDNA. The blots were exposed to x-ray film for 2 days (AtPTl), 5 days (AtPT2) or 5 hr (rDNA).

Figure 3 sets forth the results of a Northern blot analysis of the expression of tomato phosphate transporter genes. Total RNA from roots (R) or leaves (L) of tomato plants grown aeroponically and misted with a solution containing 250 μM (+) or no (-) phosphate was hybridized with labeled probe from either LePTl or LePT2. Ethidium bromide-stained gel picture indicating uniform loading and integrity of RNA samples is shown at the bottom.

Figure 4 sets forth the results of a Southern blot analysis of Arabidopsis genomic DNA digested with BamHI (B), EcoRl (E). or H dIII (H). The blots were hybridized with labeled cDNA inserts from AtPTl or AtPT2.

Figure 5 sets forth the results of a Southern blot analysis of tomato genomic DNA digested with Pstl (P), Hindlll (H). or EcoRI (E). Blots were hybridized with labeled cDNA inserts from LePTl and LePT2.

Figure 6 provides the results of the procedure set forth in Example 6 herein relating to the in situ localization of tomato phosphate transporter transcripts in roots and leaves. Tissue localization of the message was done using DIG labeled LePTl and LePT2 sense (A and C), and antisense (B and D) RNA probes. Sections are from roots and leaves of plants grown with (A and B) and without (C and D) phosphorus for 5 days. Abbreviations are as follows: ep = epidermis; cp = cortical parenchyma; cc = central cylinder; pp = palisade parenchyma and sp = spongy parenchyma.

Figure 7 provides the results of the procedure set forth in Example 7 herein relating to the complementation of yeast high-affinity phosphate transporter mutant NS219. Figure 7(a) sets forth acid phosphatase activity in NS219 transformants containing either the vector (pYES2) or the vector containing AtPTl (pYES2 + AtPTl )/AtPT2 (pYES2 + AtPT2) coding regions. The cells were grown on SD medium— high-phosphate (1 ImM) plates containing 3% glycerol and .5% galactose for 4 days before staining for acid phosphatase activity. The red color indicates presence of acid phophatase activity. Three independent transformants (A-C) for each construct are shown. Figure 7(b) sets forth growth of NS219 transformants in SD medium — low phosphate (1 10 μm) medium containing 2% galactose and 0.5% sucrose. Values are the averages from two experiments using three independent transformants for each construct. Error bars indicate the standard deviation. DETAILED DESCRIPTION OF THE INVENTION

For purposes of promoting an understanding of the principles of the invention, reference will now be made to particular embodiments of the invention and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the invention, and such further applications of the principles of the invention as described herein being contemplated as would normally occur to one skilled in the art to which the invention pertains.

The present invention relates to DNA constructs that may be integrated into a plant to provide an inventive transformed plant which over-expresses a phosphate transporter protein. Over-expression of a phosphate transporter protein in the proper plant tissues results in an increased uptake of phosphate from the soil in which the plant is grown. In soil environments where there is a phosphate deficiency, this results in an increased probability that the plant will survive and flourish without the addition of phosphorus-containing fertilizers. The present inventors have discovered, isolated and characterized four nucleotide sequences which encode phosphate transporters, two having been isolated from Arabidopsis thaliana and two having been isolated from Lycopersicon esculentum (tomato). The inventors have further discovered that these sequences regulated by inducible promoters which are induced in a root specific manner in response to a phosphate deficiency.

Therefore, the present invention relates to the nucleotide sequences set forth in SEQ ID NO: 1 ; SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4, which encode phosphate transporter proteins, and sequences having substantial identity thereto. Also, a nucleotide sequence selected in accordance with the invention may be operably linked to a promoter to provide a novel DNA construct which may be used to transform a plant to provide a transformed plant having the ability to withstand a phosphate deficiency. When heightened expression of a phosphate transporter protein is achieved in a transformed plant in accordance with the present invention, the transformed plant is able to accumulate an adequate amount of phosphate, and thereby is more readily able to survive and/or grow in phosphate deficient soil. As such, advantageous features of the present invention include the transformation of a wide variety of plants of various agriculturally and/or commercially valuable plant species to provide transformed plants having the above-discussed advantageous properties. A nucleotide sequence selected for use in accordance with the present invention is one that effectively expresses a functional phosphate transporter protein in tissues that are involved in taking up phosphate from the soil. Preferably, the nucleotide sequence encodes a protein selected from the group consisting of AtPTl , AtPT2. LePTl. LePT2 and proteins having substantial identity to these. It is not intended, however, that this list be limiting, but only provide examples of nucleotide sequences which may be advantageously used in accordance with the present invention to provide over-expression of a functional phosphate transporter protein in cells involved with the phosphate uptake.

It is well known that plants of a wide variety of species commonly express and utilize analogous enzymes and/or proteins which have varying degrees of degeneracy, and yet which effectively provide the same or a similar function. In this manner, sequences encoding phosphate transporters commonly differ to some degree between species; however, it is understood that the present invention is intended to encompass genes which encode phosphate transporter proteins in a wide variety of plant species. While nucleotide sequences encoding two phosphate transporters of the species

Arabidopsis thaliana are set forth in SEQ ID NO: 1 and SEQ ID NO:2 herein (AtPTl and AtPT2, respectively), and nucleotide sequences encoding two phosphate transporters of the species Lycopersicon esculentum are set forth in SEQ ID NO:3 and SEQ ID NO:4 herein (LePTl and LePT2. respectively), it is not intended that the present invention be limited to these exemplary sequences, but include sequences having substantial identity thereto and sequences from different plant species that encode phosphate transporter proteins of that species.

The term "nucleotide sequence," as used herein, is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, and derivatives thereof. The terms "encoding" and "coding" refer to the process by which a gene, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional protein, such as. for example, an active enzyme. It is understood that the process of encoding a specific amino acid sequence may involve DNA sequences having one or more base changes (i.e.. insertions, deletions, substitutions) that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. A preferred DNA construct selected or prepared in accordance with the invention expresses a phosphate transporter protein, or a protein having substantial identity thereto and having a level of activity suitable to achieve the advantageous result of the invention. A preferred amino acid sequence encoded by an inventive DNA construct is an amino acid sequence set forth in SEQ ID NO:5; SEQ ID NO:6 SEQ ID NO:7; SEQ ID NO:8 or a sequence having substantial identity thereto. The terms "protein" and "amino acid sequence" are used interchangeably herein to designate a plurality of amino acids linked in a serial array. Skilled artisans will recognize that through the process of mutation and/or evolution, proteins of different lengths and having differing constituents, e.g., with amino acid insertions, substitutions, deletions, and the like, may arise that are related to the proteins of the present invention by virtue of (a) amino acid sequence homology; and (b) good functionality with respect to phosphate transport activity. For example, a phosphate transporter protein isolated from one species and/or the nucleotide sequence encoding it. may differ to a certain degree from the sequences set forth herein, and yet have excellent functionality in accordance with the invention. Such a protein and/or nucleotide sequence falls directly within the scope of the present invention. While many deletions, insertions, and, especially, substitutions, are not expected to produce radical changes in the characteristics of the protein, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect may be evaluated by routine screening assays.

In addition to the exemplary amino acid sequences set forth in SEQ ID NOS:5-8. therefore, the present invention also contemplates proteins having substantial identity thereto. The term "substantial identity." as used herein with respect to an amino acid sequence, is intended to mean sufficiently similar to have suitable functionality when expressed in a plant transformed in accordance with the invention to achieve the advantageous result of the invention. In one preferred aspect of the present invention, variants having such potential modifications as those mentioned above, which have at least about 50% identity to an amino acid sequence set forth in SEQ ID NOS:5-8, are considered to have "'substantial identity" thereto. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents. More preferably, inventive proteins have at least about 75% identity to those set forth in SEQ ID NOS:5-8, more preferably at least about 85% identity and most preferably at least about 95% identity. It is believed that the identity required to maintain proper functionality is related to maintenance of the tertiary structure of the protein such that specific interactive sequences will be properly located and will have the desired activity. As such, it is believed that there are discrete domains and motifs within the amino acid sequence which must be present for the protein to retain its advantageous functionality and specificity. While it is not intended that the present invention be limited by any theory by which it achieves its advantageous result, it is contemplated that a protein including these discrete domains and motifs in proper spatial context will retain good enzymatic activity. It is therefore understood that the invention also encompasses more than the specific exemplary nucleotide sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce '"silent" changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alterations in the nucleotide sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, 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 biologically equivalent product.

Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity in the encoded products. As a related matter, it is understood that similar base changes may be present in a promoter sequence without substantially affecting its valuable functionality. Such variations to a promoter sequence are also within the purview of the invention.

In a preferred aspect, therefore, the present invention contemplates nucleotide sequences having substantial identity to those set forth in SEQ ID NOS. 1, 2, 3 and 4. The term "substantial identity^" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence has a sequence sufficiently similar to one of those explicitly set forth herein that it will hybridize therewith under moderately stringent conditions, this method of determining identity being well known in the art to which the invention pertains. Briefly, moderately stringent conditions are defined in Sambrook et al.. Molecular Cloning: a Laboratory Manual, 2ed. Vol. 1, pp. 101- 104. Cold Spring Harbor Laboratory Press (1989) as including the use of a prewashing solution of 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization and washing conditions of about 55 C, 5 x SSC. A further requirement of the term "substantial identity^" as it relates to an inventive nucleotide coding sequence in accordance with this embodiment is that it must encode a protein having substantially similar functionality to a phosphate transporter protein set forth in SEQ ID NOS:5-8. i.e.. one which is capable of effecting an increase in a plant^'s ability to withstand phosphate deficiency when over-expressed in the plant's tissues involved in the uptake of phosphate from the soil.

Suitable DNA sequences selected for use according to the invention may be obtained, for example, by cloning techniques using cDNA libraries corresponding to a wide variety of plant species, these techniques being well known in the relevant art, or may be made by chemical synthesis techniques which are also well known in the art. Suitable nucleotide sequences may be isolated from DNA libraries obtained from a wide variety of species by means of nucleic acid hybridization or PCR. using as hybridization probes or primers nucleotide sequences selected in accordance with the invention, such as those set forth in SEQ ID NOS: 1, 2, 3 and 4; nucleotide sequences having substantial identity thereto; or portions thereof. As was stated above, a nucleotide sequence which encodes a phosphate transporter, or a protein having substantial identity thereto and having suitable activity with respect to phosphate uptake, may be isolated and/or amplified from a wide variety of plant species. Nucleotide sequences specifically set forth herein or selected in accordance with the invention may be advantageously used in a wide variety of plant species, including but not limited to the species from which it is isolated.

Inventive DNA sequences can be incorporated into the genome of a plant using conventional recombinant DNA technology, thereby making a transformed plant better capable of withstanding phosphorus deprivation. In this regard, the term "genome" as used herein is intended to refer to DNA which is present in a plant and which is heritable by progeny during propagation of the plant. As such, an inventive transgenic plant may alternatively be produced by breeding a transgenic plant made according to the invention with a second plant or selfing an inventive transgenic plant to form an FI or higher generation plant. Transformed plants and progeny thereof are all contemplated by the invention and are all intended to fall directly within the meaning of the term "transgenic plant."

Generally, transformation of a plant involves inserting a DNA sequence into an expression vector in proper orientation and correct reading frame. The vector contains the necessary elements for the transcription of the inserted protein-encoding sequence. A wide variety of vector systems known in the art can be advantageously used in accordance with the invention, such as plasmids, bacteriophage viruses or other modified viruses. Suitable vectors include, but are not limited to the following viral vectors: lambda vector system gtl 1, gtlO, Charon 4. and plasmid vectors such as pBI121. pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18. pUC19, pLG339, pRK290, pKC37, pKClOl, pCDNAII, and other similar systems. The DNA sequences are cloned into the vector using standard cloning procedures in the art, for example, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), which is hereby incorporated by reference. The plasmid pBI121 is available from Clontech Laboratories, Palo Alto, California. It is understood that related techniques may be advantageously used according to the invention to transform microorganisms such as, for example, Agrobacterium sp., yeast, E.coli and Pseudomonas sp.

In order to obtain satisfactory expression of a nucleotide sequence which encodes an inventive phosphate transporter in a plant, a promoter must be present in the expression vector. Promoters selected for use in accordance with one preferred aspect of the present invention effectively target phosphate transporter expression to those tissues that are involved with phosphorus uptake (i.e., in most plants, the roots). In another preferred aspect of the invention, the promoter is inducible by phosphorus deficiency in a plant. Preferably, the promoter is one isolated from a native gene which encodes a phosphate transporter protein. For example, over-expression of a phosphate transporter may preferably be obtained in target plant tissues using one of the following promoters: AtPTl , AtPT2. LePTl , LePT2. It is not intended, however, that this list be limiting, but only provide examples of promoters which may be advantageously used in accordance with the present invention to provide over- expression of phosphate transporter proteins in cells involved in phosphorus uptake. In one preferred aspect of the invention, the promoter is the AtPT2 promoter, which is set forth in SEQ ID NO:9 herein. Although promoters for certain classes of genes commonly differ between species, it is understood that the present invention includes promoters which regulate expression of phosphate transporters in a wide variety of plant species.

An expression vector according to the invention may be either naturally or artificially produced from parts derived from heterologous sources, which parts may be naturally occurring or chemically synthesized, and wherein the parts have been joined by ligation or other means known in the art. The introduced coding sequence is under control of the promoter and thus will be generally downstream from the promoter. Stated alternatively, the promoter sequence will be generally upstream (i.e., at the 5' end) of the coding sequence. The phrase "under control of contemplates the presence of such other elements as may be necessary to achieve transcription of the introduced sequence. As such, in one representative example, enhanced production of a phosphate transporter protein may be achieved by inserting an inventive nucleotide sequence in a vector downstream from and operably linked to a promoter sequence capable of driving over-expression in a host cell. Two DNA sequences (such as a promoter region sequence and a phosphate transporter-encoding nucleotide sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter region sequence.

RNA polymerase normally binds to the promoter and initiates transcription of a DNA sequence or a group of linked DNA sequences and regulatory elements (operon). A transgene, such as a nucleotide sequence selected in accordance with the present invention, is expressed in a transformed plant to produce in the cell a protein encoded thereby. Briefly, transcription of the DNA sequence is initiated by the binding of RNA polymerase to the DNA sequence's promoter region. During transcription, movement of the RNA polymerase along the DNA sequence forms messenger RNA ("mRNA") and, as a result, the DNA sequence is transcribed into a corresponding mRNA. This mRNA then moves to the ribosomes of the cytoplasm or rough endoplasmic reticulum which, with transfer RNA ("tRNA"), translates the mRNA into the protein encoded thereby. Proteins of the present invention thus produced in a transformed host then perform an important function in the plant's ability to take in phosphate. It is well known that there may or may not be other regulatory elements (e.g., enhancer sequences) which cooperate with the promoter and a transcriptional start site to achieve transcription of the introduced (i.e., foreign) coding sequence. Also, the recombinant DNA will preferably include a transcriptional termination sequence downstream from the introduced sequence. Once the DNA construct of the present invention has been cloned into an expression vector, it is ready to be transformed into a host plant cell. Transformation may be achieved using one of a wide variety of techniques. One technique of transforming plants with a DNA construct in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising the DNA construct. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for about 48 to about 72 hours on regeneration medium without antibiotics at about 25- 28°C. Bacteria from the genus Agrobacterium may be advantageously utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants. Another technique which may advantageously be used is vacuum-infiltration of flower buds using Agrobacterium-based vectors.

Another approach to transforming plant cells with a DNA sequence selected in accordance with the present invention involves propelling inert or biologically active particles at plant tissues or cells. This technique is disclosed in U.S. Patent Nos. 4.945.050. 5,036.006 and 5,100,792. all to Sanford et al.. which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector . Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA material sought to be introduced) can also be propelled into plant cells. It is not intended, however, that the present invention be limited by the choice of vector or host cell. It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same vector expression system. However, one of skill in the art may make a selection among vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of this invention.

Once the recombinant DNA is introduced into the plant tissue, successful transformants can be screened using standard techniques such as the use of marker genes, e.g., genes encoding resistance to antibiotics. Additionally, the level of expression of the foreign DNA may be measured at the transcriptional level, as protein synthesized or by assaying to determine the level of phosphorus uptake by the plant. An isolated DNA construct selected in accordance with the present invention may be utilized in an expression vector to increase phosphorus uptake capabilities in a wide variety of plants, including gymnosperms. monocots and dicots. Inventive DNA constructs are particularly useful in plant species which are commonly grown in low phosphorus soil. For example, tropical soils are known to have low phosphorus content. Therefore, application of the invention to plants that are to be grown in tropical soils would be extremely advantageous. The invention finds advantageous use. for example, in transforming the following plants: gymnosperms. rice, wheat, barley, rye. corn, potato, carrot, sweet potato, bean, pea. chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. Those skilled in the art will recognize the commercial and agricultural advantages inherent in plants transformed to have increased or selectively increased expression of phosphate transporter proteins and/or of proteins having substantial identity thereto. Such plants are expected to have substantially improved ability to survive and flourish in low-phosphate soil environments and, therefore, are expected to be more agriculturally valuable compared to a corresponding non-transformed plant. Additionally, the invention will reduce or eliminate the need to include phosphorus in materials used to fertilize such plants, this being an excellent feature of the invention in view of the non-renewable nature of rock phosphate resources.

The invention will be further described with reference to the following specific Examples. It will be understood that these Examples are illustrative and not restrictive in nature.

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. The meaning of abbreviations is as follows: "h" means hour(s), "min" means minute(s), "sec" means second(s), "d" means day(s), "μL" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "g" means gram(s), "mg" means milligram(s), "μg" means microgram(s), "nm" means nanometer(s), "m" means meter(s), "E" means Einstein(s). EXAMPLE ONE

Plant Material Arabidopsis thaliana var. Columbia seeds were germinated on a 300-μm mesh nylon screen placed on a petri plate containing solidified agar supplemented with 1/10 Murashige-Skoog salts as described by Poirier et al. One week after germination , the nylon filter was removed along with the intact root system and transferred to a sterile floating membrane raft (Lift raft, Sigma). The floating device carrying seedlings was placed in a GA-7 (Sigma) tissue culture box containing 100 ml of half- strength Hoagland II nutrient solution. After 1 week, the plants were transferred to fresh half-strength Hoagland II nutrient solution containing either 250 μM phosphate or no phosphate (KH_:SO₄). The roots and leaves were harvested from these plants after 5 days following the initiation of phosphate starvation.

Tomato {Lycopersicon esculentum) plants were grown in an aeroponic growth facility similar to the one described in Liu et al., 1997. Tomato seeds var. OS4 were germinated in seedling trays filled with Scotts ready earth plug mix (Scotts Co.. Marysville. OH). When plants reached the 4 leaf stage (20 days after sowing) they were removed from the growing media, roots were washed free of media and transferred to aerophonics. In aeroponic culture, roots were sprayed with a fine mist of half strength Hoagland's solution for 3 seconds every 10 minutes. Phosphorus starvation treatments were initiated one week after the plants were transferred to aeroponics. For divided root system studies, tomato plants were grown in aeroponics for one week as described above. Three aeroponic plants were transferred to an aerated hydroponic solution containing either 250 μM or 0 μM Pi. Leaves and roots from P+ (250 μM Pi), P- (0 μM Pi) and divided root system plants were harvested separately, frozen in liquid nitrogen and stored at -70°C.

EXAMPLE TWO

RNA Isolation Total RNA was isolated from the roots and leaves of Arabidopsis plants and of tomato plants by hot phenol extraction and lithium chloride precipitation. Poly(A)^" RNA was isolated by the oligo(dT) cellulose batch binding method.

EXAMPLE THREE Northern Analysis

Total RNA (lOμg) was electrophoretically separated on 1% denaturing formaldehyde agarose gels and blotted onto BA-S (Schleicher & Schuell) nitrocellulose membrane. The nitrocellulose filters were hybridized overnight with ³²P-labeled DNA probe (10⁶ cpm/ml) in a solution containing 50% formamide, 5x Denhardt's solution. 0.1% SDS, 6x SSPE, and 100 μg/ml denatured salmon sperm

DNA at 42°C. Filters were washed twice in 2x SSC and 0.2 SDS at room temperature for 10 min, twice in lx SSC and 0.2% SDS at 50°C for 15 min. and twice in 0. lx SSC and 0.2% SDS at 62°C for 20 min before autoradiography.

The expression of LePTl and LePT2 in tomato plants grown either in the presence of 250 μM phosphate or no phosphate was compared by Northern blot analysis of total RNA isolated from different tissues. Both probes hybridized to about 2.0 kb transcripts. Their expression was markedly increased in plants grown under Pi limiting conditions. LePTl is primarily expressed in roots, with a small amount of the message also detectable in leaves, stems and petioles of tomato plants subject to Pi starvation. LePT2 is expressed only in the roots.

Expression of AtPTl and AtPT2 transcripts was compared by Northern blot analysis of total RNA isolated from roots and leaves of Arabidopsis plants grown either in the presence of 250 μM phosphate or no phosphate. A 1.9-kb transcript was detected for both of these genes in roots. Their expression was markedly increased in the roots of plants subjected to phosphate starvation. The AtPTl message was more abundant compared with that of AtPT2 n phosphate-starved roots. There was no detectable message for either of the genes in leaves even under phosphate starvation. Northern blots are set forth in Figures 2 and 3. -II

EXAMPLE FOUR

Southern Analysis High molecular weight genomic DNA was isolated from young leaves of Arabidopsis and from young leaves of tomato as described by Dellaporta et al. Genomic DNA (10 μg) was digested with restriction enzymes, electrophoretically separated through 0.8% agarose gels, denatured, and transferred to a supported nitrocellulose membrane. The hybridization and washing conditions were the same as those described above for Northern blots. Results are set forth in Figures 4 and 5 herein.

EXAMPLE FIVE cDNA Library Construction and Screening— Arabidopsis A cDNA library representing the mR A isolated from Arabidopsis roots starved for phosphate for 7 days was constructed in the EcoRl-X ol site of the Uni- ZAP XR vector according to manufacturer's instructions (Stratagene). The Arabidopsis expressed sequence tag clones (stock nos. 134M11T7, 178H14T7. And ATTS2854) showing similarity to yeast PHO84 were obtained from the Arabidopsis Biological Resource Center (Columbus, OH). The inserts from these clones were radio-labeled by random priming (DECAprime II. Ambion. Austin. TX) and used for screening the cDNA library according to standard procedures. Final washing of the filters was done with 0.1X SSC and 0.2% SDS at 62°C for 40 min. Two sets of cDNA clones were obtained from this screening based on their hybridization with either 178H14T7 insert or a mixture of ATTS2854 and 134M1 1T7 inserts. Based on the insert size and restriction mapping, one full-length representative from each of these sets was used for further analysis. The sequences of these two clones (AtPTl and AtPT2) were determined on both strands by the dideoxy method using Sequenase (United States Biochemical). The Genetics Computer Group (Madison, WI) software package was used for sequence analysis and data base searches. AtPTl is 1754 bp long and contains an open reading frame encoding a 524 amino acid polypeptide (molecular mass of 57.6 kDa), whereas AtPT2 is 1897 bp long and encodes a 534 amino acid polypeptide (molecular mass of 58.6 kDa). The open reading frames of AtPTl and AtPT2 are flanked by 47 and 151 bp of untranslated sequence at the 5' end, and by 135 and 144 bp of untranslated sequence including the poly(A) tail at the 3' end, respectively. These two clones are 70% similar in ther nucleotide sequence within the coding region. The two polypeptides are 78% identical in their amino acid sequence.

cDNA Library Construction and

Screening— Tomato A cDNA library representing the mRNA isolated from tomato roots starved for phosphate for five days was constructed in the XhoI-EcoRI site of the Uni-ZAP™- XR vector according to manufacturer instructions (Stratagene. CA). The two Arabidopsis thaliana cDNA clones (AtPTl and AtPT2) encoding the phosphate transporters were radiolabeled by random priming (DECAprime™ II, Ambion, TX) and used for screening the tomato cDNA library according to standard procedures (Sambrook et al., 1989). Hybridizations for screening were carried out in a solution containing 50% (v/v) formamide at 38°C. Final washing of the filters was done with 1 X SSC, 0.2% (w/v) SDS at 60°C for 30 min. Two sets of cDNA clones were obtained from this screening. Based on the insert size, and restriction mapping, one full length representative from each of these sets was used for further analysis. The sequences of these two clones (LePTl and LePT2) were determined on both strands by the dideoxy method using Sequenase (USB, OH). The Genetics Computers Group (Madison. WI) software package was used for sequence analysis and database searches.

LePTl is 2023 bp long and contains an open reading frame encoding a 538 aa polypeptide (molecular mass of 58.7 kDa), whereas LePT2 is 1826 bp long and encodes a 528 aa polypeptide (molecular mass 57.8 kDa). The open reading frames of LePTl and LePT2 are flanked by 151 and 37 bp of untranslated sequence at the 5^' end and by 258 and 205 bp of untranslated sequence including the poly(A) tail at the 3^" end. The LePT and LePT2 polypeptides are 80% identical in their amino acid sequence. The two polypeptides share substantial similarity with the phosphate transporters from potato. Arabidopsis and Catharanthits roseus. Based upon the amino acid sequence identity, LePTl is more similar to potato STPT1 and Arabidopsis AtPT2, whereas LePT2 is more similar to STPT2 and AtPTl . Phylogenetically, the phosphate transporters from plants and fungi belong to a closely related family, even though the similarity between the plant transporters is significantly higher than that between plants and fungal transporters.

Hydropathy plots of the deduced polypeptides suggest that both tomato transporters are integral membrane proteins consisting of 12 membrane spanning regions, a common feature shared by proteins responsible for transport of substrates as diverse as sugars, ions, antibiotics and amino acids. The position and spacing of these membrane-spanning regions in tomato transporters are very similar to those in other Pi-transporters, as is shown in Figure 1. Based upon secondary structure analysis, both N and C termini of the polypeptides are predicted to be on the cytoplasmic side of the plasma membrane. The amino acid domains for protein kinase C and casein kinase II-mediated phosporylation. as well as N-linked glycosylation are present in similar conserved regions as seen with Arabidopsis Pi transporters.

EXAMPLE SIX

In situ Localization of Tomato Phosphate Transporter Transcripts

Roots of tomato plants grown in aeroponics were sprayed with nutrient solutions containing Pi (250 μM), or without Pi for 5 days. Root and leaf samples were harvested and fixed in a solution containing 3.7% (v/v) formaldehyde. 5% (v/v) acetic acid and 50% (v/v ethanol (Niu et al., 1996). Fixed tissue samples were dehydrated in an ethanoi dilution series and embedded in wax (Paraplast from Fisher Scientific Co. IL). Ten μm sections cut with a microtome were transferred to Super- Frost plus slides (Fisher Scientific Co. IL). and incubated at 42°C overnight. Sense and antisense probes representing LePTl and LePT2 were transcribed by T3 or T7 RNA polymerase (Ambion, CA) from linearized pBluescript-SK-containing the cDNA. The probes were labeled with digozigenin (DIG) following the procedure described by the manufacture (Boehringer Mannheim, IN). Tissue section pretreatment and in situ hybridization were performed as described by Niu et al., (1996). Successive sections from roots obtained from three plants were used for hybridizing with sense and anti-sense probes. After color development for 16 to 24 hr. sections were photographed using a Nikon Optiphot microscope.

In tomato plants grown under phosphorus deficient conditions, a significant amount of chromogenic product signal for LePTl and LePT2 transcripts was observed in the root epidermis. Low levels of LePTl transcripts in other cell types including the central cylinder was also noticed in Pi starved roots. In addition, accumulation of LePTl message was also detected in palisade parenchyma and phloem cells of leaves under phosphate starvation. The significance of the presence of higher transcript levels in palisade parenchyma in leaves is not clear. Higher demand for phosphorus by the actively photosynthesizing palisade parenchyma cells may be one of the reasons for increased expression of phosphate transporters. These transporters may be involved in active transport of Pi from neighboring tissues, or Pi released in the apoplastic space, into the palisade parenchyma cells. Expression of LePT2 was not detected in leaf tissue even under phosphate starvation. In situ localization of phosphate transporter message agree with Northern analysis of RNA. Results are set forth in Figure 6 herein. EXAMPLE SEVEN

Cloning and Expression in Saccharomyces cerevisiae.

The yeast strain NS219 contains a mutation in the PHO84 gene and thereby lacks the high-affinity phosphate transport system. As a result of this mutation, NS219 cells exhibit reduced rates of phosphate uptake and growth in low-phosphate medium. In addition, the mutant cells continue to produce an acid phosphatase when grown in high-phosphate medium, whereas this activity is repressed in wild-type cells under these conditions. To test the ability of AtPTl - and AtPT2 -encoded polypeptides to complement this mutation, the coding regions of the cDNAs were ligated into a yeast expression vector pYES2 and transformed into NS219.

The yeast high-affinity phosphate transporter mutant NS219 was provided by Satoshi Harashima (Osaka University, Japan). The coding regions of AtPTl and AtPT2 cDNAs were subcloned in yeast expression vector pYES2 (Invitrogen) downstream of the GAL 1 promoter. NS219 was transformed by a Licl/PEG method. The procedures and synthetic defined (SD) medium used for growth and selection of transformants were similar to those described earlier. The acid phosphatase activity of the NS219 transformants was detected by a staining method based on the diazo-coupling reaction. For growth assays, single colonies of transformants were grown in high phosphate (1 1 mM) SD medium containing 2% galactose and 0.5% sucrose to an OD₆₀₀ of 1.0. The cells were collected by centrifugation, washed twice with SD medium containing no phosphate, and then resuspended in low phosphate (1 10 μM) SD medium containing 2% galactose and 0.5% sucrose to an OD₆₀₀ of 0,05. At regular time intervals, aliquots of the culture were removed for OD₆₀₀ measurements.

The cells transformed with only the pYES2 vector exhibited the acid phosphatase activity on high-phosphate medium as seen from their red color after staining. The NS219 transformants expressing either AtPTl or AtPT2 mimicked wild-type cells, showing no acid phosphatase activity. NS219 transformants expressing either AtPTl or AtPT2 were also able to grow much faster in low- phosphate ( 1 lOμM) medium than the cells containing pYES2. The average generation time of these cells was about 2.5 times higher than the control cells during the initial phases of their growth in low-phosphate medium. Results are set forth in Figure 7 herein.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: Raghothama, K. G.

(i) APPLICANT: Muchhal , Umes S.

( ii ) TITLE OF INVENTION : METHODS AND COMPOS ITIONS FOR IMPROVING A PLANT'S ABILITY TO TAKE IN PHOSPHATE FROM

SOIL

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS

(A) ADDRESSEE: Thomas Q. Henry

Woodard, Emhardt, Naughton, Moriarty & McNett

(B) STREET: 111 Monument Circle, Suite 3700

(C) CITY: Indianapolis (D) STATE: Indiana

<E) COUNTRY: USA

(F) POSTAL CODE (ZIP) : 46204-5137

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.5:, 1.44Mb

(B) COMPUTER: Hewlett Packard

(C) OPERATING SYSTEM: MSDOS

(D) SOFTWARE: ASCII

(v) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Unknown

(B) FILING DATE: 29-JUL-1997

(C) CLASSIFICATION: unknown

(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 60/022,391

(B) FILING DATE: 29-JUL-1996

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Henry, Thomas Q.

(B) REGISTRATION NO. : 28-309

(C) REFERENCE/DOCKET NUMBER: 7024-257

(ix) TELECOMMUNICATION INFORMATION (A) TELEPHONE: (317) 634-3456

(B) TELEFAX: (317) 637-7561

(2) INFORMATION FOR SEQ ID NO : 1 ;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : cDNA

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 1 (AtPTl ) :

GAGAAGTTCT TAATTTCTCC TGCCAAGCTG ATTAAGAGCT CTAGGAAATG GCCGAACAAC 60

AACTAGGAGT GCTAAAGGCA CTCGATGTTG CGAAGACGCA ACTTTATCAT TTCACGGCGA 120

TTGTCATCGC CGGTATGGGT TTCTTTACCG ATGCCTACGA TCTTTTTTGC GTGTCCTTGG 180

TGACGAAACT CCTTGGCCGC ATCTACTATT TCAATCCGGA GTCAGCGAAG CCTGGCTCAC 240

TTCCCCCTCA TGTTGCGGCC GCGGTCAACG GTGTGGCCCT TTGTGGAACC CTTTCTGGTC 300

AACTCTTCTT CGGTTGGCTC GGTGACAAAC TCGGACGGAA AAAAGTGTAC GGTCTCACTT 360

TGGTAATGAT GATCTTGTGC TCTGTCGCTT CTGGTCTCTC TTTTGGCCAC GAAGCCAAGG 420

GTGTCATGAC CACCCTTTGC TTCTTCAGGT TTTGGTTGGG ATTTGGTATT GGAGGTGACT 480

ACCCACTTTC TGCCACCATC ATGTCTGAAT ACGCAAACAA GAAGACCCGT GGGGCTTTCA 540

TCGCAGCTGT CTTCGCCATG CAAGGTGTCG GTATCTTGGC TGGAGGTTTC GTGGCACTCG 600

CAGTATCTTC TATATTCGAC AAAAAGTTCC CAGCTCCAAC ATATGCAGTA AACAGGGCCC 660

TCTCAACGCC TCCTCAAGTT GACTACATTT GGCGAATCAT CGTCATGTTT GGTGCTTTAC 720

CCGCAGCTTT GACTTACTAC TGGCGTATGA AGATGCCTGA AACTGCCCGT TACACCGCTT 780 TGGTTGCCAA GAACATCAAA CAAGCCACAG CCGACATGTC CAAGGTCTTA CAAACAGATA 840

TCGAGCTTGA GGAAAGGGTG GAGGATGACG TCAAAGACCC CAAACAAAAC TATGGCTTGT 900

TCTCCAAGGA ATTCCTTAGA CGCCATGGGC TTCATCTCCT TGGAACTACC TCCACATGGT 960

TTTTGCTTGA CATTGCCTTC TACAGCCAAA ACTTGTTCCA GAAGGATATT TTCTCGGCCA 1020

TCGGATGGAT CCCAAAGGCA GCCACCATGA ACGCCACCCA TGAGGTTTTC AGGATTGCTA 1080

GGGCTCAGAC TCTTATCGCC CTTTGCAGTA CAGTCCCAGG CTACTGGTTC ACAGTTGCGT 1140

TTATTGATAC CATTGGAAGG TTTAAGATCC AACTAAATGG ATTTTTCATG ATGACCGTGT 1200

TTATGTTTGC CATTGCCTTC CCTTACAACC ACTGGATCAA ACCAGAAAAC CGTATCGGAT 1260

TTGTGGTTAT GTACTCTCTT ACTTTCTTCT TCGCCAATTT TGGTCCAAAT GCAACCACTT 1320

TTATTGTCCC TGCTGAGATA TTCCCGGCCA GGCTAAGGTC TACATGTCAT GGAATATCAG 1380

CCGCGGCTGG TAAGGCTGGA GCCATTGTTG GAGCCTTTGG GTTCCTATAT GCGGCTCAAT 1440

CACAAGACAA GGCCAAGGTA GACGCAGGAT ACCCACCAGG CATCGGAGTT AAGAACTCAT 1500

TGATCATGCT TGGTGTTCTT AACTTTATCG GTATGCTCTT CACCTTCCTT GTCCCAGAGC 1560

CCAAAGGCAA GTCCCTTGAA GAACTCTCTG GTGAGGCTGA GGTTAGCCAT GACGAGAAAT 1620

AATTATGTAT GTTTATTTTG TTATTTGGAG TGCGATGTTT GGTTTTGTTT TCATTTTATT 1680

GGCTCGTTGA CCTTAAGTTA TGATGTTATA AGAATATTTA TGATATCATT TAAATCTAAA 1740

AAAAAAAAAA AAAA 1 54

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1897 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2(AtPT2):

CGATCATTCC ACTTCCTTCT TCCTCTCTCT CAACATTTTC CCCTGAAAAT AAGGAAACTA 60

AAGATTCTTC CTCTCTCTTT CTACACTCTT CTGACAATAC TAAAACACTT TATCAGATCA 120

GATCCCACAT AAACTTTCTA GGAGAAGAAG AATGGCAAGG GAACAATTAC AAGTGTTGAA 180

TGCACTTGAC GTGGCCAAGA CGCAATGGTA CCATTTCACG GCGATCATAA TCGCCGGAAT 240

GGGATTCTTC ACTGATGCTT ACGATCTCTT TTGCATCTCT CTCGTAACGA AGCTCCTCGG 300

TCGTATTTAT TACCACGTGG AAGGCGCACA AAAGCCTGGG ACTCTCCCTC CCAACGTCGC 360 AGCCGCCGTC AATGGCGTTG CCTTCTGTGG GACTCTCGCC GGTCAGCTCT TTTTCGGGTG 420

GCTTGGTGAT AAGCTCGGGA GGAAGAAAGT TTACGGTATG ACGTTGATGG TCATGGTCCT 480

TTGTTCAATA GCCTCTGGTC TCTCTTTCGG ACATGAGCCA AAAGCTGTGA TGGCCACGCT 540

CTGTTTTTTT CGGTTTTGGC TTGGATTTGG CATCGGTGGT GACTACCCTT TATCCGCAAC 600

CATCATGTCT GAATATGCGA ACAAGAAGAC TCGCGGAGCC TTTGTCTCTG CGGTTTTTGC 660

TATGCAAGGG TTCGGAATCA TGGCTGGTGG TATTTTCGCT ATTATAATTT CCTCTGCTTT 720

TGAAGCTAAG TTTCCATCCC CGGCCTATGC GGATGATGCC TTGGGATCCA CGATTCCTCA 780

AGCTGATTTG GTATGGCGGA TAATCCTGAT GGCTGGTGCT ATCCCTGCGG CTATGACGTA 840

TTACTCAAGG TCGAAGATGC CTGAGACCGC AAGGTACACG GCTTTGGTTG CTAAGGACGC 900

AAAGCAGGCA GCTTCGGACA TGTCTAAGGT TCTGCAAGTG GAGATAGAGC CAGAACAACA 960

GAAATTGGAA GAGATCTCAA AGGAGAAGTC CAAGGCCTTT GGATTGTTCT CAAAGGAATT 1020

CATGAGTCGC CATGGGCTTC ATTTGCTAGG CACTACATCG ACATGGTTCC TTCTCGACAT 1080

TGCTTTCTAC AGTCAAAACC TTTTCCAAAA GGATATTTTC AGCGCGATCG GATGGATTCC 1140

TCCCGCGCAA AGCATGAACG CAATTCAAGA GGTTTTCAAG ATTGCCCGTG CGCAAACGCT 1200

AATCGCCTTG TGTAGCACGG TACCTGGTTA CTGGTTCACA GTTGCGTTCA TCGACGTCAT 1260

TGGAAGATTT GCGATTCAGA TGATGGGTTT CTTTTTCATG ACGGTCTTTA TGTTTGCTCT 1320

GGCTATTCCT TACAACCACT GGACTCACAA GGAGAACCGA ATCGGATTTG TTATCATGTA 1380

CTCGTTAACA TTCTTTTTCσ CCAACTTTGG ACCCAATGCT ACAACCTTCG TTGTGCCGGC 1440

CGAAATCTTC CCAGCCAGGT TCAGATCAAC CTGCCACGGT ATCTCTGCAG CATCAGGAAA 1500

ATTAGGAGCA ATGGTTGGTG CGTTCGGGTT CTTGTACTTG GCTCAGAACC CAGACAAGGA 1560

CAAGACCGAC GCAGGATACC CTCCAGGGAT TGGGGTCAGG AACTCGCTTA TTGTGTTGGG 1620

TGTAGTTAAC TTCTTAGGTA TCCTCTTCAC TTTCTTGGTA CCTGAATCTA AAGGTAAGTC 1680

ACTCGAGGAA ATGTCCGGTG AAAATGAAGA CAATGAGAAT AGCAACAATG ATAGTAGAAC 1740

GGTCCCAATA GTTTAGGTGA TATAATACGC CTTTTGTAAT AATTTTCGTT _{ττττcτττcτ 180}o

CCTTGTCTCT AGCAACTCAA GTTGTTCTTT GTGTAATCCA TTGATACCTA ATTAATGCTA 1860

GAGAAATCAA AATTTTCAAA AAAAAAAAAA AAAAAAA 1897

(2) INFORMATION FOR SEQ ID NO: 3.

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 2023 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE. cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 (LePTl) TAAAAATAAA AATGCAAAAT AATCCAAAGT TTTGATTTTT AATTTTGGGT ATTTTTGTTG 60

GAGTTCTTGA ACGTAAAAAA AAGACCAAAT CTTGAAATTT GGTTTTTTTT TTGGTGGAAA 120

TTGAAGATTT ACTCTTTAAG GAAGTTTAGT CATGGCGAAC GATTTGCAAG TGCTAAATGC 180

ACTAGATGTC GCGAAGACAC AACTGTATCA CTTCACAGCG ATTGTGATTG CTGGCATGGG 240

TTTTTTTACT GATGCTTATG ACCTTTTCTG CATTTCTATG GTCACTAAAT TGCTTGGTCG 300

TCTTTACTAC CATCATGACG GTGCATTGAA ACCTGGCTCT CTGCCCCCTA ATGTTTCAGC 360

AGCTGTTAAT GGAGTCGCCT TCTGTGGCAC CCTTGCTGGA CAGTTGTTCT TCGGGTGGCT 420

TGGAGATAAA ATGGGAAGGA AGAAAGTCTA TGGAATGACC CTTATGATTA TGGTCATTTG 480

TTCAATTGCC TCGGGGCTTT CATTTGGCCA TACACCAAAA GGTGTTATGA CTACGCTTTG 540

TTTCTTCAGA TTCTGGCTAG GATTTGGCAT TGGTGGTGAT TATCCCCTTT CTGCCACCAT 600

CATGTCTGAG TATGCTAACA AAAAGACCCG TGGAGCGTTC ATTGCTGCTG TGTTTGCTAT 660

GCAAGGTTTC GGAATTCTGG CTGGTGGAAT GGTGGCAATC ATTGTTTCTG CAGCATTCAA 720

GGGCGCATTC CCTGCACCAG CATATGAGGT TGATGCTATT GGTTCAACAG TCCCTCAGGC 780

TGATTTCGTG TGGCGTATAA TTCTCATGTT TGGTGCAATC CCTGCTGGAC TTACTTATTA 840

CTGGCGTATG AAGATGCCTG AAACTGCCCG TTACACTGCC TTGGTCGCCA AGAACTTGAA 900

ACAGGCAGCT AACGACATGT CCAAGGTGTT GCAAGTCGAA ATTGAAGCAG AGCCAGAGAA 960

AGTTACAGCT ATTTCTGAAG CAAAAGGAGC CAATGACTTT GGTTTGTTCA CTAAGGAGTT 1020

CCTCCGTCGC CATGGACTTC ACTTGCTTGG AACTGCTAGC ACATGGTTCT TGTTGGACAT 1080

TGCTTTCTAC AGTCAAAACC TTTTCCAGAA GGACATTTTC AGTGCAATTG GATGGATTCC 1140

ACCAGCACAA ACCATGAACG CGTTGGAAGA AGTTTACAAG ATTGCAAGGG CACAAACACT 1200

TATTGCTCTT TGTAGTACTG TTCCTGGTTA CTGGTTCACA GTTGCATTCA TCGATAAGAT 1260

TGGTCGATTT GCAATTCAGT TGATGGGATT CTTCTTCATG ACAGTCTTCA TGTTTGCCTT 1320

AGCCATTCCA TACCATCACT GGACTCTCAA GGATCACAGA ATTGGCTTCG TGGTCATGTA 1380

CTCATTCACC TTTTTCTTCG CCAATTTTGG TCCAAACGCC ACAACATTCG TCGTCCCTGC 1440

TGAGATTTTC CCAGCCAGGC TTAGGTCCAC ATGCCATGGA ATATCAGCAG CAGCAGGTAA 1500

AGCAGGAGCT ATGGTTGGTG CATTTGGATT CTTATACGCT GCTCAGCCCA CGGATCCAAC 1560

AAAGACTGAC GCCGGTTACC CTCCTGGCAT TGGTGTGAGG AACTCGTTGA TCGTCCTTGG 1620

TTGTGTTAAC TTCCTCGGTA TGCTGTTCAC ATTCTTGGTT CCAGAATCCA ATGGGAAGTC 1680

ATTGGAAGAT TTGTCGAGGG AAAACGAAGG GGAAGAGGAA ACTGTAGCTG AAATAAGAGC 1740

AACAAGTGGA AGGACAGTTC CTGTGTGAGT TTTAGACAAG TTATCAGTTA GTATACACTA 1800

CAATGCAGTT TGAGTTAATT TGTGGTATTT GGGATTAGAA AGAGATTGTT TGTTGGTTTG 1860

TTATAAGAAG ATGGAATAAG CTCTTATCTT TTTGTTTGTT TGTTTGGGTA ATTAAACATT 1920

ATTACCTTAC TTCTGCAAAT CTCAGAAATT CTGAGATTAT ATAAAGTAAC CAAAGGAGGT 1980

TCTTTGGTTG TCTATCTCTT TTATAAAAAA AAAAAAAAAA AAA 2023

(2) INFORMATION FOR SEQ ID NO : 4 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1826 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4(LePT2>:

AGAAAGTGCA CAATTTTTTG AACCAATTTT AGGCATTATG GCTGTGGGGG ATAATGATAA 60

TAATAATTTA CAAGTGCTTA ATGCACTTGA TTTGGCTAAA ACTCAACTTT ACCATTTTAC 120

AGCTATTGTT ATTGCTGGAA TGGGATTTTT TACTGATGCT TATGATCTGT TTAGTATTTC 180

TCTTGTCACT AAATTGCTTG GGCGTTTATA CTATACCAAA CCGGATCTTT TGAAACCCGG 240

AACACTCCCT CCGGCCGTAT CAGCCTCGGT CACGGGTGTT GCCCTTGTTG GCACCCTAGC 300

CGGCCAGCTA TTCTTTGGCT GGCTAGGTGA CAAAATGGGA CGAAAAAAGG TGTATGGTAT 360

GACTTTAGTT CTCATGGTTG TGTGTTCTGT TGCTTCAGGG CTCTCCTTTG GTAGTACACC 420

TAAGGGTGTT ATGACTACAT TGTGTTTTTT TAGGTTTTGG CTTGGATTTG GTATTGGTGG 480

TGATTACCCT TTGTCCGCTA CCATCATGTC TGAATACGCT AACAAAAAGA CACGTGGTGC 540

ATTCATAGCG GCGGTATTCG CTATGCAAGG TTTTGGAATT TTGTTTAGTG GGATAGTTGC 600

ACTTATCACA GCAGCTGGAT TCGATCACGC GTATAGATCC CCAACTTTCG AGGAAAATGC 660

TGCTCTATCT ACTGTACCAC AATCGGACTA CATTTGGCGT ATCATTCTCA TGTTTGGATC 720

ATTGCCAGCT GCCCTCACTT ACTACTGGCG CATGAAAATG CCCGAAACAG CACGTTACAC 7B0

GGCTCTAGTC GCGAAAGACG CGAAGAGAGC CGCGCAGGAC ATGGGTAAGG TACTACAGGT 840

GGAAATTGAA AGCGAGGAAO CCAAAATCGA ACAAATTTCA AGGAATGAAA CTAATCAATT 900

CGGTCTGTTT TCGTGGGAAT TTGTTCGACG GCACGGATTG CATTTATTCG GAACGTGTTC 960

CACGTGGTTT TTATTAGATA TTGCTTTCTA CAGTCAGAAT TTGTTTCAGA AGGATGTGTT 1020

CTCCGCCGTC GGATGGATCC CTAAAGCTCC AACAATGAAC GCTGTTCAGG AAGTTTACAA 1080

AATTGCGAGA GCGCAAACAT TAATTGCGCT CTGTAGCACG GTGCCAGGGT ACTGGTTCAC 1140

CGTGGCGTTT ATTGACATTA TTGGAAGGTT CGCTATCCAA TTGATGGGAT TCTTCTTCAT 1200

GACAGTGTTT ATGTTTGCGA TTGCCATCCC TTACCATCAT TGGACACTGG AGGCTAACCG 1260

CATTGGATTC ATTGTAATGT ATTCGCTTAC ATTTTTCTTT GCGAATTTTG GGCCTAACGC 1320

TACTACATTT GTGGTCCCGG CGGAGATTTT CCCAGCGAGG CTTCGATCAA CATGTCATGG 1380

AATTTCAGCG GCTGCAGGTA AGGCTGGAGC TATAGTGGGA GCGTATGGGT TCTTATACGC 1440

TGCACAAAGT AAAGATCCAA ACAAAACAGA TGCAGGATAC CCAGCGGGAA TTGGAATAAA 1500

AAACTCGCTA ATTGTTCTAG GCTGTATCAA TGCACTTGGA ATGTTATGTA CATTTTGTGT 1560

GCCAGAGCCA AAAGGAAAAT CTCTAGAGGA AGCATCACAA GAAACTATAA CTGGAGAAGC 1620

ATGAGATCAT TAAATGATTG GAAAATCACT TTTTTTTTTA ATGTTTTTGT TGGTTTAATT 1680

TCAGTTTTCT TTGTATTGTG TAAGCTTGTT TGTTGCAAGT TTTTATTGTA TTATTTTTTT 1740 GTGGCCAAGT GTGTTATTGT AAAATGGTTT TGCTATTTAT TTATATTATA AATAAAAAAA 1800 TTGTTTGTAA AAAAAAAAAA AAAAAA 1826

(2) INFORMATION FOR SEQ ID NO : 5 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 534 amino acids

(B) TYPE: ammo acid

(C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown

( ii ) MOLECULE TYPE : protein

( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 5 ( AtPTl ) :

Met Ala Glu Gin Gin Leu Gly Val Leu Lys Ala Leu Asp Val Ala Lys

1 5 10 15

Thr Gin Leu Tyr His Phe Thr Ala He Val He Ala Gly Met Gly Phe 20 25 30

Phe Thr Asp Ala Tyr Asp Leu Phe Cys Val Ser Leu Val Thr Lys Leu 35 40 45

Leu Gly Arg He Tyr Tyr Phe Asn Pro Glu Ser Ala Lys Pro Gly Ser 50 55 60

Leu Pro Pro His Val Ala Ala Ala Val Asn Gly Val Ala Leu Cys Gly 65 70 75 80

Thr Leu Ser Gly Gin Leu Phe Phe Gly Trp Leu Gly Asp Lys Leu Gly

85 90 95

Arg Lys Lys Val Tyr Gly Leu Thr Leu Val Met Met He Leu Cys Ser 100 105 110

Val Ala Ser Gly Leu Ser Phe Gly His Glu Ala Lys Gly Val Met Thr 115 120 125 Thr Leu Cys Phe Phe Arg Glu Trp Leu Gly Phe Gly He Gly Gly Asp 130 135 140

Tyr Pro Leu Ser Ala Thr He Met Ser Glu Tyr Ala Asn Lys Lys Thr 145 150 155 160

Arg Gly Ala Phe He Ala Ala Val Phe Ala Met Gin Gly Val Gly He

165 l o 175

Leu Ala Gly Gly Phe Val Ala Leu Ala Val Ser Ser He Phe Asp Lys

180 185 190

Lys Phe Pro Ala Pro Thr Tyr Ala Val Asn Arg Ala Leu Ser Thr Pro 195 200 205

Pro Gin Val Asp Tyr He Trp Arg He He Val Met Phe Gly Ala Leu 210 215 220

Pro Ala Ala Leu Thr Tyr Tyr Trp Arg Met Lys Met Pro Glu Thr Ala 225 230 235 240

Arg Tyr Thr Ala Leu Val Ala Lys Asn He Lys Gin Ala Thr Ala Asp 245 250 255

Met Ser Lys Val Leu Gin Thr Asp He Glu Leu Glu Glu Arg Val Glu

260 265 270

Asp Asp Val Lys Asp Pro Lys Gin Asn Tyr Gly Leu Phe Ser Lys Glu 275 280 285

Phe Leu Arg Arg His Gly Leu His Leu Leu Gly Thr Thr Ser Thr Trp 290 295 300

Phe Leu Leu Asp He Ala Phe Tyr Ser Gin Asn Leu Phe Gin Lys Asp 305 310 315 320

He Phe Ser Ala He Gly Trp He Pro Lys Ala Ala Thr Met Asn Ala 325 330 335 Thr His Glu Val Phe Arg He Ala Arg Ala Gin Thr Leu He Ala Leu

340 345 350

Cys Ser Thr Val Pro Gly Tyr Trp Phe Thr Val Ala Phe He Asp Thr

355 360 365

He Gly Arg Phe Lys He Gin Leu Asn Gly Phe Phe Met Met Thr Val

370 375 380

Phe Met Phe Ala He Ala Phe Pro Tyr Asn His Trp He Lys Pro Glu

385 390 395 400

Asn Arg He Gly Phe Val Val Met Tyr Ser Leu Thr Phe Phe Phe Ala

405 410 415

Asn Phe Gly Pro Asn Ala Thr Thr Phe He Val Pro Ala Glu He Phe

420 425 430

Pro Ala Arg Leu Arg Ser Thr Cys His Gly He Ser Ala Ala Ala Gly 435 440 445

Lys Ala Gly Ala He Val Gly Ala Phe Gly Phe Leu Tyr Ala Ala Gin 450 455 460

Ser Gin Asp Lys Ala Lys Val Asp Ala Gly Tyr Pro Pro Gly He Gly 465 470 475 480

Val Lys Asn Ser Leu He Met Leu Gly Val Leu Asn Phe He Gly Met 485 490 495

Leu Phe Thr Phe Leu Val Pro Glu Pro Lys Gly Lys Ser Leu Glu Glu 500 505 510

Leu Ser Gly Glu Ala Glu Val Ser His Asp Glu Lys 515 520 524

(2) INFORMATION FOR SEQ I Asp NO : 6 : (ι) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 534 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : unknown (D) TOPOLOGY: unknown

(li) MOLECULE TYPE: protein

ixi) SEQUENCE DESCRIPTION: SEQ ID NO:6(AtPT2) :

Met Ala Arg Glu Gin Leu Gin Val Leu Asn Ala Leu Asp Val Ala Lys

1 5 10 15

Thr Gin Trp Tyr His Phe Thr Ala He He He Ala Gly Met Gly Phe 20 25 30

Phe Thr Asp Ala Tyr Asp Leu Phe Cys He Ser Leu Val Thr Lys Leu 35 40 45

Leu Gly Arg He Tyr Tyr His Val Glu Gly Ala Gin Lys Pro Gly Thr 50 55 60

Leu Pro Pro Asn Val Ala Ala Ala Val Asn Gly Val Ala Phe Cys Gly 65 70 75 80

Thr Leu Ala Gly Gin Leu Phe Phe Gly Trp Leu Gly Asp Lys Leu Gly 85 90 95

Arg Lys Lys Val Tyr Gly Met Thr Leu Met Val Met Val Leu Cys Ser 100 105 110

He Ala Ser Gly Leu Ser Phe Gly His Glu Pro Lys Ala Val Met Ala 115 120 125

Thr Leu Cys Phe Phe Arg Phe Trp Leu Gly Phe Gly He Gly Gly Asp 130 135 140 Tyr Pro Leu Ser Ala Thr He Met Ser Glu Tyr Ala Asn Lys Lys Thr

145 150 155 160

Arg Gly Ala Phe Val Ser Ala Val Phe Ala Met Gin Gly Phe Gly He

165 1"70 175

Met Ala Gly Gly He Phe Ala He He He Ser Ser Ala Phe Glu Ala

180 185 190

Lys Phe Pro Ser Pro Ala Tyr Ala Asp Asp Ala Leu Gly Ser Thr He 195 200 205

Pro Gin Ala Asp Leu Val Trp Arg He He Leu Met Ala Gly Ala He 210 215 220

Pro Ala Ala Met Thr Tyr Tyr Ser Arg Ser Lys Met Pro Glu Thr Ala

225 230 235 240

Arg Tyr Thr Ala Leu Val Ala Lys Asp Ala Lys Gin Ala Ala Ser Asp

245 250 255

Met Ser Lys Val Leu Gin Val Glu He Glu Pro Glu Gin Gin Lys Leu

260 265 270

Glu Glu He Ser Lys Glu Lys Ser Lys Ala Phe Gly Leu Phe Ser Lys

275 280 285

Glu Glu Met Ser Arg His Gly Leu His Leu Leu Gly Thr Thr Ser Thr 290 295 300

Trp Phe Leu Leu Asp He Ala Phe Tyr Ser Gin Asn Leu Phe Gin Lys

305 310 315 320

Asp He Phe Ser Ala He Gly Trp He Pro Pro Ala Gin Ser Met Asn

325 330 335

Ala He Gin Glu Val Phe Lys He Ala Arg Ala Gin Thr Leu He Ala 340 345 350 Leu Cys Ser Thr Val Pro Gly Tyr Trp Phe Thr Val Ala Phe He Asp 355 360 365

Val He Gly Arg Phe Ala He Gin Met Met Gly Phe Phe Phe Met Thr 370 375 380

Val Phe Met Phe Ala Leu Ala He Pro Tyr Asn His Trp Thr His Lys 385 390 395 400

Glu Asn Arg He Gly Phe Val He Met Tyr Ser Leu Thr Phe Phe Phe 405 410 415

Ala Asn Phe Gly Pro Asn Ala Thr Thr Phe Val Val Pro Ala Glu He 420 425 430

Phe Pro Ala Arg Glu Arg Ser Thr Cys His Gly He Ser Ala Ala Ser 435 440 445

Gly Lys Leu Gly Ala Met Val Gly Ala Phe Gly Phe Leu Tyr Leu Ala 450 455 460

Gin Asn Pro Asp Lys Asp Lys Thr Asp Ala Gly Tyr Pro Pro Gly He 465 470 475 480

Gly Val Arg Asn Ser Leu He Val Leu Gly Val Val Asn Phe Leu Gly 485 490 495

He Leu Phe Thr Phe Leu Val Pro Glu Ser Lys Gly Lys Ser Leu Glu 500 505 510

Glu Met Ser Gly Glu Asn Glu Asp Asn Glu Asn Ser Asn Asn Asp Ser 515 520 525

Arg Thr Val Pro He Val 530 534

(2) INFORMATION FOR SEQ ID NO : 7 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 538 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7(LePTl) :

Met Ala Asn Asp Leu Gin Val Leu Asn Ala Leu Asp Val Ala Lys Thr 1 5 10 15

Gin Leu Tyr His Phe Thr Ala He Val He Ala Gly Met Gly Phe Phe

20 25 30

Thr Asp Ala Tyr Asp Leu Phe Cys He Ser Met Val Thr Lys Leu Leu 35 40 45

Gly Arg Leu Tyr Tyr His His Asp Gly Ala Leu Lys Pro Gly Ser Leu 50 55 60

Pro Pro Asn Val Ser Ala Ala Val Asn Gly Val Ala Phe Cys Gly Thr 65 70 5 80

Leu Ala Gly Gin Leu Phe Phe Gly Trp Leu Gly Asp Lys Met Gly Arg

85 90 95

Lys Lys Val Tyr Gly Met Thr Leu Met He Met Val He Cys Ser He 100 105 110

Ala Ser Gly Leu Ser Phe Gly His Thr Pro Lys Gly Val Met Thr Thr

115 120 125

Leu Cys Phe Phe Arg Phe Trp Leu Gly Phe Gly He Gly Gly Asp Tyr 130 135 140

Pro Leu Ser Ala Thr He Met Ser G u Tyr Ala Asn Lys Lys Thr Arg 145 150 155 160

Gly Ala Phe He Ala Ala Val Phe Ala Met Gin Gly Phe Gly He Leu 165 170 175

Ala Gly Gly Met Val Ala He He Val Ser Ala Ala Phe Lys Gly Ala 180 185 190

Phe Pro Ala Pro Ala Tyr Glu Val Asp Ala He Gly Ser Thr Val Pro 195 200 205

Gin Ala Asp Phe Val Trp Arg He He Leu Met Phe Gly Ala He Pro 210 215 220

Ala Gly Leu Thr Tyr Tyr Trp Arg Met Lys Met Pro Glu Thr Ala Arg 225 230 235 240

Tyr Thr Ala Leu Val Ala Lys Asn Leu Lys Gin Ala Ala Asn Asp Met 245 250 255

Ser Lys Val Leu Gin Val Glu He Glu Ala Glu Pro Glu Lys Val Thr 260 265 270 Ala He Ser Glu Ala Lys Gly Ala Asn Asp Phe Gly Leu Phe Thr Lys 275 280 285

Glu Phe Leu Arg Arg His Gly Leu His Leu Leu Gly Thr Ala Ser Thr 290 295 300

Trp Phe Leu Leu Asp He Ala Phe Tyr Ser Gin Asn Leu Phe Gin Lys 305 310 315 320

Asp He Phe Ser Ala He Gly Trp He Pro Pro Ala Gin Thr Met Asn

325 330 335

Ala Leu Glu Glu Val Tyr Lys He Ala Arg Ala Gin Thr Leu He Ala 340 345 350

Leu Cys Ser Thr Val Pro Gly Tyr Trp Phe Thr Val Ala Phe He Asp 355 360 365

Lys He Gly Arg Phe Ala He Gin Leu Met Gly Phe Phe Phe Met Thr 370 375 380

Val Phe Met Phe Ala Leu Ala He Pro Tyr His His Trp Thr Leu Lys 385 390 395 400

Asp His Arg He Gly Phe Val Val Met Tyr Ser Phe Thr Phe Phe Phe

405 410 415

Ala Asn Phe Gly Pro Asn Ala Thr Thr Phe Val Val Pro Ala Glu He 420 425 430

Phe Pro Ala Arg Leu Arg Ser Thr Cys His Gly He Ser Ala Ala Ala 435 440 445

Gly Lys Ala Gly Ala Met Val Gly Ala Phe Gly Phe Leu Tyr Ala Ala 450 455 460

Gin Pro Thr Asp Pro Thr Lys Thr Asp Ala Gly Tyr Pro Pro Gly He 465 470 475 480 Gly Val Arg Asn Ser Leu He Val Leu Gly Cys Val Asn Phe Leu Gly

485 490 495

Met Leu Phe Thr Phe Leu Val Pro Glu Ser Asn Gly Lys Ser Leu Glu 500 505 510

Asp Leu Ser Arg Glu Asn Glu Gly Glu Glu Glu Thr Val Ala Glu He

515 520 525

Arg Ala Thr Ser Gly Arg Thr Val Pro Val 530 535 538

(2) INFORMATION FOR SEQ ID NO .8

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 528 ammo acids

(B) TYPE, amino acid

(C) STRANDEDNESS unknown (D) TOPOLOGY unknown

(ii) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION. SEQ ID NO 8(LePT2)

Met Ala Val Gly Asp Asn Asp Asn Asn Asn Leu Gin Val Leu Asn Ala

1 5 10 15

Leu Asp Leu Ala Lys Thr Gin Leu Tyr His Phe Thr Ala He Val He 20 25 30

Ala Gly Met Gly Phe Phe Thr Asp Ala Tyr Asp Leu Glu Ser He Ser

35 40 45

Leu Val Thr Lys Leu Leu Gly Arg Leu Tyr Tyr Thr Lys Pro Asp Leu

50 55 60 Leu Lys Pro Gly Thr Leu Pro Pro Ala Val Ser Ala Ser Val Thr Gly 65 70 75 80

Val Ala Leu Val Gly Thr Leu Ala Gly Gin Leu Phe Phe Gly Trp Leu

85 90 95

Gly Asp Lys Met Gly Arg Lys Lys Val Tyr Gly Met Thr Leu Val Leu 100 105 11C

Met Val Val Cys Ser Val Ala Ser Gly Leu Ser Phe Gly Ser Thr Pro 115 120 125

Lys Gly Val Met Thr Thr Leu Cys Phe Phe Arg Phe Trp Leu Gly Phe 130 135 140

Gly He Gly Gly Asp Tyr Pro Leu Ser Ala Thr He Met Ser Glu Tyr 145 150 155 160

Ala Asn Lys Lys Thr Arg Gly Ala Phe He Ala Ala Val Phe Ala Met

165 170 175

Gin Gly Phe Gly He Leu Phe Ser Gly He Val Ala Leu He Thr Ala 180 185 190

Ala Gly Phe Asp His Ala Tyr Arg Ser Pro Thr Phe Glu Glu Asn Ala 195 200 205

Ala Leu Ser Thr Val Pro Gin Ser Asp Tyr He Trp Arg He He Leu 210 215 220

Met Phe Gly Ser Leu Pro Ala Ala Leu Thr Tyr Tyr Trp Arg Met Lys 225 230 235 240

Met Pro Glu Thr Ala Arg Tyr Thr Ala Leu Val Ala Lys Asp Ala Lys

245 250 255

Arg Ala Ala Gin Asp Met Gly Lys Val Leu Gin Val Glu He Glu Ser 260 265 270 Glu Glu Ala Lys He Glu Gin He Ser Arg Asn Glu Thr Asn Gin Phe 275 280 285

Gly Leu Phe Ser Trp Glu Phe Val Arg Arg His Gly Leu His Leu Phe 290 295 300

Gly Thr Cys Ser Thr Trp Phe Leu Leu Asp He Ala Phe Tyr Ser Gin 305 310 315 320

Asn Leu Phe Gin Lys Asp Val Phe Ser Ala Val Gly Trp He Pro Lys

325 330 335

Ala Pro Thr Met Asn Ala Val Gin Glu Val Tyr Lys He Ala Arg Ala 340 345 350

Gin Thr Leu He Ala Leu Cys Ser Thr Val Pro Cys Tyr Trp Phe Thr 355 360 365

Val Ala Phe He Asp He He Gly Arg Phe Ala He Gin Leu Met Gly 370 375 380

Phe Phe Phe Met Thr Val Phe Met Phe Ala He Ala He Pro Tyr His 385 390 395 400

His Trp Thr Leu Glu Ala Asn Arg He Gly Phe He Val Met Tyr Ser

405 410 415

Leu Thr Phe Phe Phe Ala Asn Phe Gly Pro Asn Ala Thr Thr Phe Val 420 425 430

Val Pro Ala Glu He Phe Pro Ala Arg Leu Arg Ser Thr Cys His Gly 435 440 445

He Ser Ala Ala Ala Gly Lys Ala Gly Ala He Val Gly Ala Tyr Gly 450 455 460 Phe Leu Tyr Ala Ala Gin Ser Lys Asp Pro Asn Lys Thr Asp Ala Gly

465 470 475 480

Tyr Pro Ala Gly He Gly He Lys Asn Ser Leu He Val Leu Gly Cys

485 490 495

He Asn Ala Leu Gly Met Leu Cys Thr Phe Cys Val Pro Glu Pro Lys

500 505 510

Gly Lys Ser Leu Glu Glu Ala Ser Gin Glu Thr He Thr Gly Glu Ala 515 520 525 528

(2) INFORMATION FOR SEQ ID NO : 9 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1272 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n ) MOLECULE TYPE: genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9(AtPT2 promoter):

GTCGACTCGA TCATTTTAAT GCAACAACTA TATGTTAAAG GGAAATTGGT CTAGAAGCGG 60

CTATTTCTTG GTCTTGAAAT CATATTGTTC TTCTATAGTG TAGTGACATT TCCTATAATT 120

AATTTGAAAA AAGGAAGAAA TTGTGTTGGC AATGAAAACA TCATATGTAT GGTGTGAAGT 180 ATATACAAAA AAAAAAATCC CATTCGTGAA TGAAAACTAC GGTGTATATA TGTGAAAGAC 240

ATATATGGAG CCTTCACTAT ACGGTGTAGT TCATTTACAT AAGAATGGTT GGAAATGGAG 300

ATGC ATATT TTTTTTTTTT TCCACAATGG AGATGCCATA TCTMTAAAAA AAGAAAAGAG 360

GTTGAACTAG TTGGGTCGGC GCGACRAAAA GAGAAAATAC AACTTGCTGG GCTAAATCTA 420

GAAATTTCCA TTTCTGTAAA TGCCTTAAAT TAATGGCTCT TATTTATCAA ATACGGAACA 430 AACCCTCTTT ACACCTTACA ACTTACGGGT ATAGGGTGTT TATTCTCCCG TACCCGTTCA 540

AACTACACTA TATAATAAAC CATTGACATT GTTAGACCTA TTACACATCC TGCAGTTATT 600

GGCTTATTGC GATCTTTATT AAATCCAAAG AT CATACTA TATCGAAGAA ACAAAAAGTC 660

AAGAAATAAT AAAACGAAAA TAA TGAAGG CATCAATAAA AGCTTACCGC TCACATGTTT 720

ATTTTCTAAT AACTAATTTT TATTTAAAAA GCAGTTTATA CATCTACCAA ATTTATTTCT 780 TAGCATAAAT ATATATTTGG GTTTTGACTT TTAAGTTCTT TCTGACTTCT GAGTGATAAT 840

CACCAGTTTG CAACTTATAT TTGCCTAAAC CGCATGCCAA TTGTCATGTA TCGTATCTAG 900 TAAT3GT.-.TT AATGACC-ACG ATCCCAAAAT TTAAATTCCA CTTTCCAAGC ATTGAGCTCT 96C

TTAAACAATT CATGGTCAA TTAATTACAA GGAAAAAAAA AGAACTTATT GTTATAGTGG 102C

AACAGCTATT TTTTTGGATA TTAAAAGAAT AATAACAGCA AAACAGAATT ATCGTGTCCT L03C

AATAATA T AAGGTCCTAA ACGAAGCAAA AAAGTTGGTA AATAAGGAAG AGAAAACCTA 1140

CAAGATATTA AAACGGTGTC GTTGTTCGGA AGAATATACC GAAGTAGCAA AAGGAATATC 1200

TCATTAGAGA GTCCCTTATA AATGACCGTT TTAATACACT TCAACTCTGT CCTTGTTCAT 1260

AGGCAGCTTC AA 1272

Claims

What is claimed is:

1 . An isolated DNA segment comprising a nucleotide sequence having substantial identity to the sequence set forth in SEQ ID NO: l ; SEQ ID NO:2: SEQ ID NO:3: or SEQ ID NO:4.

2. A DNA construct comprising a promoter operably linked to a DNA segment which may be expressed in a host cell to produce a phosphate transporter protein: wherein the promoter regulates expression of the nucleotide sequence in the host cell; wherein the host cell expresses the nucleotide sequence; and wherein the phosphate transporter protein has substantial identity to an amino acid sequence set forth in SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; or SEQ ID NO:8.

3. The DNA construct according to claim 2, wherein the DNA segment has substantial identity to the sequence set forth in SEQ ID NO: l ; SEQ ID NO:2: SEQ ID NO:3: or SEQ ID NO:4.

4. The DNA construct according to claim 2. wherein the promoter is a plant promoter.

5. The DNA construct according to claim 2. wherein the promoter is a tissue-specific plant promoter.

6. The DNA construct according to claim 2. wherein the promoter is an inducible plant promoter.

7. The DNA construct according to claim 2, wherein the promoter has substantial identity to a native phosphate transporter promoter.

8. The DNA construct according to claim 2. wherein the protein has an amino acid sequence having substantial identity to the amino acid sequence set forth in SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; or SEQ ID NO:8.

9. The DNA construct according to claim 2, wherein the promoter is selected from the group consisting of an AtPTl promoter, an AtPT2 promoter, an LePTl promoter and an LePT2 promoter..

10. The DNA construct according to claim 2, wherein the promoter is an AtPT2 promoter or a LePT2 promoter.

11. A vector useful for transforming a cell, said vector comprising the DNA construct according to claim 2.

12. A plant transformed with the vector of claim 12, or progeny thereof, the plant being capable of expressing the nucleotide sequence.

13. The plant according to claim 12, the plant being selected from the group consisting of gymnosperms, rice, wheat, barley, rye, corn, potato, carrot, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, zucchini, cucumber. apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

14. The plant according to claim 12. the plant being selected from the group consisting of gymnosperms, rice, wheat, barley, rye, corn, potato, carrot, sweet potato, bean. pea. cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, eggplant, pepper, celery, cucumber, apple, pear, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

15. A cell having incorporated therein a foreign nucleotide sequence comprising a promoter operably linked to a DNA sequence having substantial identity to SEQ ID NO: 1 ; SEQ ID NO:2; SEQ ID NO:3; or SEQ ID NO:4.

16. The cell according to claim 15, wherein the cell is a microorganism.

17. The cell according to claim 15. wherein the cell is a plant cell.

18. A plant having incorporated into its genome a foreign DNA construct comprising a promoter operably linked to a DNA sequence having substantial identity to SEQ ID NO: 1 ; SEQ ID NO:2; SEQ ID NO:3; or SEQ ID NO:4.

19. A method for improving a plant's ability to grow in phosphate-deficient soil, comprising: incorporating into the plant's genome an DNA construct comprising a promoter operably linked to a DNA sequence having substantial identity to SEQ ID NO: 1 ; SEQ ID NO:2; SEQ ID NO:3; or SEQ ID NO:4 to provide a transformed plant; wherein the transformed plant is capable of over-expressing phosphate transporter proteins.

20. A method for improving a plant's ability to grow in phosphate-deficient soil, comprising: providing a vector comprising a promoter operably linked to a nucleotide sequence encoding a phosphate transporter protein; wherein the promoter regulates expression of the nucleotide sequence in a host plant cell: and transforming the target plant with the vector to provide a transformed plant, the transformed plant being capable of expressing the nucleotide sequence.

21. The method according to claim 20, wherein the protein comprises an amino acid sequence having substantial identity to the sequence set forth in SEQ ID NO: 5; SEQ ID NO: 6: SEQ ID NO: 7; or SEQ ID NO: 8.

22. The method according to claim 20, wherein the transformed plant produces phosphate transporter proteins at a rate substantially greater than the rate at which phosphate transporter proteins are produced by a non-transformed plant of the same species.

23. The method according to claim 20, wherein the target plant is selected from the group consisting of gymnosperms, rice, wheat, barley, rye, corn, potato, carrot, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

24. The method according to claim 20, wherein the nucleotide sequence has substantial identity to the nucleotide sequence of SEQ ID NO: 1 ; SEQ ID NO:2 SEQ ID

NO:3 or SEQ ID NO:4.

25. A transgenic plant obtained according to the method of claim 20 or progeny thereof.