AU778221B2 - Expression of phytase in plants as a method of modifying plant productivity - Google Patents

Description

WO 01/22806 PCT/AU00/01183 -1- METHOD OF MODIFYING PLANT PRODUCTIVITY FIELD OF THE INVENTION The present invention relates generally to a method of modifying plant productivity comprising expressing in a plant cell, tissue or organ one or more genes capable of facilitating a plant's ability to utilise soil phosphorus. More particularly, the present invention provides a method of increasing plant productivity comprising expressing in the root of a plant an isolated nucleic acid molecule encoding a phytase polypeptide for a time and under conditions sufficient for said phytase to be secreted from the root, and preferably, further comprising modifying the chemistry of the soil around the root using an organic acid. The present invention extends to novel phytase-encoding genes; genetic constructs which are useful for performing the inventive method; and to transgenic plants produced therewith having improved productivity compared to their otherwise isogenic counterparts.

GENERAL

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

WO 01/22806 PCT/AU00/01183 -2- Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

Reference herein to prior art, including any one or more prior art documents, is not to be taken as an acknowledgment, or suggestion, that said prior art is common general knowledge in Australia or forms a part of the common general knowledge in Australia.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.

This specification contains nucleotide and amino acid sequence information prepared using the programme Patentln Version 2.0, presented herein after the claims. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier <210>1, <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively.

Nucleotide and amino acid sequences referred to in the specification are defined by descriptor "SEQ ID NO:" followed by the numeric identifier. For example, SEQ ID NO: 1 refers to the information provided in the numeric indicator field designated <400> 1, etc.

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide WO 01/22806 PCT/AUOO/01183 -3other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

The designation of amino acid residues referred to herein are also those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein three-letter and one-letter abbreviations for naturally-occurring amino acids are listed in Table 1. In addition to the abbreviations listed in Table 1, the three-letter symbol Asx, or the one-letter symbol B, denotes Asp or Asn; and the three-letter symbol GIx, or the one-letter symbol Z, denotes glutamic acid or glutamine or a substance, such as, for example, 4-carboxyglutamic acid (Gla) or 5-oxoproline (GIp) that yields glutamic acid upon the acid hydrolysis of a peptide.

The designation of plasmid pBS389 herein shall be taken to mean the plasmid depicted in Figure 3, which is also known by those skilled in the art as plasmid pPLEX502, and includes the SCSV promoter and terminator sequences taught in Intemational Patent Application No. PCT/AU95/00552.

Amino acid designations referred to herein are listed in Table 1.

WO 01/22806 WO 0122806PCT/AUOO/01183 -4- TABLE I Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala

A

Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gin a Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ilie I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Praline Pro P Serine Ser S Th reonine Thr

T

Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid as above Xaa X WO 01/22806 PCT/AUOO/01183 BACKGROUND TO THE INVENTION In light of the dwindling supply of land available for agriculture, intensive agriculture production is an imperative for the purposes of feeding the increasing worldwide population. To achieve this end, it is necessary to increase the productivity of agriculture plants. High productivity is of great agricultural and horticultural value, because increased growth reduce times-to-harvest and yield of crop plants. This improvement is of considerable value in the case of both forage and grain crops.

It is well known that phosphorus may boost or even optimise plant productivity. Soil phosphorus may originate from the deposition of organic material in the soil, which form can account for at least 50-85% of total soil phosphorus. However, organic forms of soil phosphorus, such as, for example, inositol phosphorus (soil phytate), may also account for a substantial component of total soil phosphorus. For example, in Australian soils, phytate accounts for up to 38% of total organic phosphorus, and organic phosphorus may account for 50-85% of total soil phosphorus.

Present methods for boosting plant productivity include the application of phosphate-based fertilisers to the soil. High costs of intensive agriculture, particularly in respect of producing agronomically-important crops, are incurred by the requirement to apply phosphate-based fertilisers to the soil. This is especially evident in regions where the soils are deficient in forms of phosphorus that are readily utilisable by plants. Additionally, there is a considerable environmental cost associated with the use of phosphate fertilisers in particular, due to run-off entering the water catchment and resulting in algal blooms under appropriate conditions.

In spite of the benefits to be derived from providing phosphorus to plants in terms of increased productivity, the use of phosphate-based fertilisers has declined recently, in part due to the high economic costs associated therewith and in part due to the high environmental costs. This decline in phosphate-based fertiliser usage has occurred in regions where soils are deficient in forms of phosphate that are WO 01/22806 PCT/AU00/01183 -6available to plants. This has meant a reduction in plant productivity, particularly in those regions having phosphorus-deficient soils.

Notwithstanding the high proportion of total phosphorus present in the soil in the form of soil phytate, plants almost exclusively derive their phosphorus requirement from soluble phosphate anions, and possess a very limited capacity to directly obtain phosphorus from soil phytate, because phytate is not absorbed by plant roots and further, because phytate is inefficiently hydrolysed to inositol and phosphorus in the soil.

STATEMENT OF THE PRIOR ART Microorganisms and fungi in the soil are known to contain phytase enzymes that catalyse the conversion of phytate to inositol and inorganic phosphate and phytaseencoding genes of Aspergillus niger have been described previously in United States Patent No. 5, 436, 156 issued on 25 July, 1995 (hereinafter 'Van Gorcom et al., 1995").

Additionally, Van Ooijen et al. (United States Patent No. 5, 593, 963 issued on 14 January, 1997) expressed the Aspergillus ssp. phytase gene in the cells of plants, in particular the seeds, with a view to increasing the level of available phosphorus in feedstock containing the transgenic plants. However, the transgenic plants produced by Van Ooijen et al. do not possess improved phosphorus nutrition by virtue of an ability to utilise soil phytate.

Hayes et al (1999) measured the phytase and acid phosphatase activities in root extracts of burr medic, phalaris, white clover, and subterranean clover. These authors concluded that phytase activity was less than 5% of the total acid phosphatase activity in extracts of roots of these plant species. The authors also characterised the biochemical properties of the phytase enzyme in root extracts of a phosphorus-deficient subterranean clover plant, and showed that the enzyme was inhibited by cobalt, zinc, or arsenate, and suggested that cysteine and EDTA may be effective in the chelation of heavy metals that interfere with phytase activity.

WO 01/22806 PCT/AU00/01183 -7- Hayes et al (1999) concluded that a number of pasture plants have limited ability to use phytate-derived phosphorus as a substrate for growth, consistent with the earlier conclusions of Hubel and Beck (1996) that phytase was unlikely to play a role in the phosphorus nutrition of Zea mays, notwithstanding that phytase enzyme and phytate were measurable in the root of that species.

SUMMARY OF THE INVENTION In work leading up to the present invention, the present inventors sought to improve the phosphorus nutrition and yield of plants without the extensive application of phosphate-based fertilisers, by increasing or improving the ability of a plant to utilise phytate, in particular soil phytate. Surprisingly, the present inventors have found that by ectopically expressing phytase enzyme in the roots, and secreting the phytase into the extracellular environment outside the root, the ability of the plant to utilise phytate as a source of phosphorus is markedly improved. Plants produced according to the inventive method provide considerable benefits to the agriculture sector, in the form of reduced economic and environmental costs, and improved plant productivity relative to their otherwise isogenic counterparts. These benefits are further enhanced if the inventive method is coupled with the step of modifying the chemistry of the soil around the root using an organic acid.

Accordingly, one aspect of the invention provides a method of enhancing the phosphorus nutrition of a plant comprising ectopically expressing in the root of a plant an isolated nucleic acid molecule encoding a phytase polypeptide for a time and under conditions sufficient for said phytase to be secreted from the root.

In a preferred embodiment of the invention, the phytase enzyme is secreted from root cells in the region of the root tip and/or the zone of elongation, that divide more rapidly than those cells that are more distal to the root tip.

Preferably, the secretion of phytase from the root provides a high local concentration of active phytase enzyme in the vicinity surrounding those root cells that are involved in active phosphorus uptake. In work leading up to the present WO 01/22806 PCT/AU00/01183 -8invention the inventors found that mere cell damage or sloughing that occurs during the movement of the root through the soil fails to provide sufficient phytase activity in the region surrounding those root cells involved in phosphorus uptake, and that an active secretion mechanism is important to achieve the improved phosphorus nutrition of the invention, Accordingly, a preferred embodiment of the present invention provides for the phytase enzyme to be produced as a fusion polypeptide with a secretory signal sequence that is active in plant cells and capable of achieving protein transport not merely outside of a root cell, but outside the root surface. In a particularly preferred embodiment, the phytase enzyme is produced as a fusion polypeptide with the leader sequence of the carrot extensin polypeptide to facilitate extracellular targeting of phytase outside the root surface.

In an alternative embodiment, the present invention provides a method of enhancing the phosphorus nutrition of a plant comprising: ectopically expressing in the root of a plant an isolated nucleic acid molecule encoding a phytase polypeptide for a time and under conditions sufficient for said phytase to be secreted from the root; and (ii) modifying the chemistry of the soil around the root or other growth medium around the root using an organic acid, preferably for a time and under conditions sufficient to solubilise phosphorus produced by the action of said phytase enzyme on phytate, or preferably for a time and under conditions sufficient to make the phytate accessible to the phytase enzyme.

The application of the inventive method results in the production of plants having higher biomass production and/or increased phosphorus content compared to otherwise isogenic counterparts, including higher rates of hypocotyl and epicotyl production, leading to a greater accumulation of biomass and larger plants, without the need for extensive application of phosphate-based fertilisers in soils that comprise phytate or to which phytate has accumulated as a result of past agricultural practice. By virtue of its relative insolubility compared to phosphate anions in soil, phytate-based fertilizers may also provide environmental advantages WO 01/22806 PCT/AUOO/01183 -9relative to super-phosphate based fertilizers. It is also known that phytate is abundant in the excreta of animals, particularly monogastric animals, such as, for example, pigs and poultry, wherein animal excreta represent a significant source of phosphorus contamination into the environment. The inventive method described herein provides a significant solution to the problems of using phytate as a fertilizer for plants, from both the perspective of unlocking those phytate reserves in soils and animal excreta, and from the perspective of reducing environmental contamination associated with the reliance upon superphosphates. Accordingly, the present invention clearly provides for the production and use of "phytate-based fertilisers" in conjunction with the inventive method.

Accordingly, a further aspect of the invention contemplates a plant fertiliser comprising phytate and/or a fertiliser composition comprising phytate and a suitable carrier for application to plants and/or the soil.

The present invention further extends to the plants produced by the performance of the inventive method.

A further aspect of the present invention provides an isolated nucleic acid molecule encoding a phytase polypeptide and having more than about 92% nucleotide sequence identity to SEQ ID NO: 1 and/or which is capable of hybridising to SEQ ID NO: 1 or a complementary nucleotide sequence thereto under high stringency hybridisation conditions. Preferably, the isolated nucleic acid molecule of the invention is derived from a microbial source such as, for example, the filamentous fungi Aspergillus ssp.

Alternatively, the Isolated nucleic acid molecule comprises a nucleotide sequence that encodes an amino acid sequence having more than about 95% identity to the sequence set forth in SEQ ID NO: 2 or an enzymically-active fragment thereof.

This aspect of the invention does not extend to the publicly available PhyA-1 gene of Aspergillus niger (GenBank Accession No. M94550; SEQ ID NO: 3), WO 01/22806 PCT/AUOO/01183 notwithstanding that the invention clearly extends to the use of the PhyA-1 gene or the PhyA-2 gene in performing the inventive method described herein.

In one embodiment, the isolated nucleic acid molecule encoding phytase is obtainable by the method of: a) hybridising under at least low stringency conditions plant genomic DNA, RNA or cDNA derived therefrom with one or more nucleic acid probes or primers of at least 10 nucleotides in length for a period of time and under conditions sufficient to form a double-stranded nucleic acid molecule, wherein said probes or primers comprise a nucleotide sequence obtainable from SEQ ID NO: 1 or a nucleotide sequence that is complementary thereto; b) detecting the hybridised nucleic acid molecule; and c) isolating said hybridised nucleic acid molecule comprising said genetic sequence.

In a particularly preferred embodiment, the isolated nucleic acid molecule of the invention comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1 or a fragment thereof encoding an active phytase enzyme. Altematively, the isolated nucleic acid molecule comprises or consists of a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO: 2 or an enzymicallyactive fragment thereof.

A further aspect of the present invention extends to gene constructs comprising a phytase-encoding nucleotide sequence connected in-frame to a secretory signalencoding nucleotide sequence, and placed operably in connection with a promoter sequence that is operable in the root cells of a plant. Preferably, the phytaseencoding nucleotide sequence comprises or consists of the Aspergillus niger PhyA- 2 nucleotide sequence set forth in SEQ ID NO: 1 or a homologue or derivative thereof as described herein.

Preferably, the secretory-signal-encoding nucleotide sequence is the carrot extensin secretory signal or equivalent.

WO 01/22806 PCT/AUOO/01183 -11- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a copy of a representation of a nucleotide sequence alignment between the open reading frames of the chimeric genes produced between the 99 bp nucleotide sequence encoding the carrot extensin leader sequence (bold type) and either the Aspergillus niger PhyA-1 gene (PhyA-1.seq; GenBank Accession No.

M94550; SEQ ID NO: 3) or the A. niger PhyA-2 gene (PhyA-2.seq; SEQ ID NO: 1) obtained by the present inventors. The alignment was produced using the CLUSTAL W algorithm of Thompson et al (1994). Numbering refers to the nucleotide positions from the start of the chimeric ext::PhyA genes. Bars between the sequences represent identical nucleotide residues.

Figure 2 is a copy of a representation of an amino acid sequence alignment between two fusion polypeptides comprising the carrot extensin leader sequence (bold type) fused to either the Aspergillus niger PhyA-1 polypeptide (PhyA-1.pro; GenBank Accession No. M94550; SEQ ID NO: 4) or the A. niger PhyA-2 polypeptide (PhyA-2.pro; SEQ ID NO: 2) obtained by the present inventors. The alignment was produced using the CLUSTAL W algorithm of Thompson et al (1994). Numbering refers to the amino acid positions from the start of the chimeric polypeptides. Bars between the sequences represent identical amino acid residues.

Figure 3 is a copy of a representation of the pPLEX vector plasmid designated pBS389. This plasmid contains the sub-clover stunt virus (SCSV) region 1 promoter Sc1 Pr; International Patent Application No. PCT/AU95/00552) and SCSV region 3 terminator (Sc3 International Patent Application No. PCT/AU95/00552) operably connected to a kanamycin-resistance gene (nptll) for expression in plants, flanked by the Agrobacterium tumefaclens left-border (LB) and right-border (RB) integration sequences; a bacterial-operable spectinomycin/streptomycin resistance gene (Sp- R/St-R); an Agrobacterium origin of replication (oriVRK2) and E.coli origin of WO 01/22806 PCT/AU00/01183 -12replication (oricolEl) and intergenic spacer (IS1/oriT). Positions of restriction sites are indicated.

Figure 4 is a copy of a representation of the plasmid pART 7. Plasmid pART7 is a vector containing bacterial two origins of replication (fl ori and ori), an ampicillin resistance gene for bacterial selection (AmpR). Plasmid pART7 also contains two Notl restriction sites flanking a CaMV 35S promoter-multiple cloning site(MCS)-NOS 3' cassette, wherein the MCS permits cloning of structural genes in operable connection with said promoter and terminator sequences.

Figure 5 is a copy of a representation of the plasmid pAER02, containing the carrot extensin leader-encoding sequence (ext) in-frame with the A. niger PhyA-1 gene (PhyA-1; SEQ ID NO: 3) and placed operably in connection with the CaMV promoter sequence and OCS terminator sequence. This vector is based upon plasmid pBS389 (Figure 3).

Figure 6 is a copy of a representation of the plasmid pAER04, containing the carrot extensin leader-encoding sequence (ext) in-frame with the A. niger PhyA-2 gene (PhyA-2; SEQ ID NO: 1) and placed operably in connection with the CaMV promoter sequence and OCS terminator sequence. This vector is based upon plasmid pBS389 (Figure 3).

Figure 7 is a photographic representation showing the growth of transgenic Arabidopsis thaliana (C24 ecotype) plants (T1 generation) containing a binary vector selected from the group consisting of: plasmid pBS389 (row marked (ii) a plasmid containing the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence; row marked and (iii) plasmid pAER04 (Figure 6; row marked Transgenic plants were grown for 42 days, at a density of three plants per tube, in sterile agar media supplemented with 0.8 mM Na 2

HPO

4 (column marked "Phosphate"), or phytate, equivalent to 0.8 mM phosphate (column marked "Phytate"), or without added phosphorus (column marked "No Duplicate results are indicated for each condition stated herein.

WO 01/22806 PCT/AU00/01183 13- Figure 8 is a photographic representation showing growth on phytate-containing agar media, of transgenic A. thaliana (C24 ecotype) plants containing a binary vector selected from the group consisting of: plasmid pBS389 a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); plasmid pAER02 (Figure a plasmid containing the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence); and plasmid pAER04 (Figure Transgenic plants were grown for 42 days, at a density of three plants per tube, in sterile agar media supplemented with phytate, equivalent to 0.8 mM phosphate. Duplicate results are indicated for each condition stated herein.

Figure 9 is a photographic representation showing growth on media lacking added phosphorus (panel or on phytate-containing agar media having a phytate concentration equivalent to 0.8 mM phosphate (panels B, C, of transgenic A.

thaliana (C24 ecotype) plants containing a binary vector selected from the group consisting of: plasmid pAER02 (Figure a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); plasmid pAER04 (Figure and plasmid pAER02 (Figure Plants were grown for days at a density of about thirty plants per agar plate.

Figure 10 is a photographic representation showing growth on sterile agar media plates supplemented with phytate at a concentration equivalent to 0.8 mM phosphate (panels A, or 0.8 mM Na 2

HPO

4 (panel of transgenic A. thaliana (C24 ecotype) plants containing a binary vector selected from the group consisting of: a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); plasmid pAER02 (Figure and plasmid pAER02 (Figure Plants were grown for 22 days and plates were stained with 0.03% FeCI 3 to determine regions where phytate was absent from the medium.

Regions of darker shading in the plates, in particular the heavier regions along the margins of the roots in Panel C show the absence of phytate from the medium, which phytate has been utilised by the plants.

WO 01/22806 PCT/AU00/01183 -14- Figure 11 is a photographic representation of a northern blot experiment showing ectopic expression of phytase genes in transgenic A. thaliana (C24 ecotype) plants.

The upper panel is an ethidium bromide-stained agarose gel containing 10 Lg of total RNA isolated from transgenic shoots. The lower panel shows an autoradiograph of the same mRNAs following hybridization under stringent conditions at 65°C, in a buffer comprising 0.5xSSC and 0.1% SDS] with the PhyA-1 sequence (SEQ ID NO: The mRNA samples indicated were derived from lines of transformed plants containing a plasmid selected from the group consisting of: plasmid pBS389 a plasmid containing the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence); plasmid pAER04 (Figure 6); a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); and plasmid pAER02 (Figure Duplicate lines are indicated for each plasmid. For RNA isolation, the plants were grown for 36 days on sterile agar supplied with 0.8 mM phosphorus.

Figure 12 is a photographic representation of a Southern blot of total DNA from transgenic A. thaliana (C24 ecotype) plants produced using a plasmid selected from the group consisting of: plasmid pBS389 a plasmid containing the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence); plasmid pAER04 (Figure a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); and plasmid pAER02 (Figure DNA samples gg digested with EcoRI) were hybridized under stringent conditions at 65*C, in a buffer comprising 0.5xSSC and 0.5% SDS] with the PhyA-1 sequence (SEQ ID NO: Multiple lines are indicated for C, D, and E. For DNA isolation, the plants were grown for 36 days on sterile agar supplied with 0.8 mM phosphorus. Size markers (kb) are indicated at the left of the Figure. The lane marked is a control lane containing DNA from non-transgenic A. thaliana.

Figure 13 is a photographic representation of a northern blot experiment showing ectopic expression of phytase genes in transgenic Trifolium subterraneum (subterranean clover, cultivar Dalkeith) plants (To generation). The upper panel is an ethidium bromide-stained agarose gel containing 10 lpg of total RNA isolated WO 01/22806 PCT/AU00/01183 from transgenic shoots. The centre panel shows an autoradiograph of the same mRNAs following hybridization under stringent conditions at 65°C, in a buffer comprising O.5xSSC and 0.1% SDS] with the PhyA-1 sequence (SEQ ID NO: The lower panel indicates the number of transgene inserts as determined by Southern blot analysis (not shown). The mRNA samples indicated were derived from 10 lines of transformed plants, of which five were independent lines (lines i, ii, iii, iv and produced by transformation of explants with the plasmid pAER02 (Figure The lane marked is a positive control containing RNA from transgenic A. thaliana plants produced using the vector pAER02 (see Figure 12E). For RNA isolation, tissue culture-derived explant material was grown to maturity under glasshouse conditions.

Figure 14 is a graphical representation showing the release of phosphorus from soil by extraction of air-dried soil in 50 mM citric acid. Panel (a):The hydrolysis of organic phosphorus in soil extracts was measured for up to 8 hr in the presence of either no added phytase enzyme or alternatively, in the presence of different concentrations of commercially-available phytase enzyme, as follows: 0.005 nkat phytase g" 1 soil 0.05 nkat phytase g-1 soil or 0.50 nkat phytase g'1 soil Panel (b):The hydrolysis of organic phosphorus in soil extracts was measured for up to 8 hr in the presence of either no added phytase enzyme or alternatively, in the presence of different concentrations of purified phytase enzyme, as follows: 0.23 nkat phytase g' 1 soil or 2.28 nkat phytase g-1 soil Soil was from Rutherglen Research Institute, Victoria, Australia, and had been fertilised with superphosphate phosphorus) at a rate of 125 kg ha- 1 each alternate year since 1914. Time (hr) is indicated on the abscissa, and phosphorus (pg phosphorus g' 1 soil) is indicated on the ordinate. Each point represents the mean of three observations. Bars indicate standard error and, where not shown, were smaller than the symbol.

Figure 15 is a graphical representation showing the effect of citric acid on the hydrolysis of phytate in air-dried soil, as measured by the release of phytase-labile organic phosphorus. The hydrolysis of organic phosphorus in soil extracts was WO 01/22806 PCT/AUOO/01183 -16measured for 6 hr in the presence of either 0.50 nkat commercial phytase soil or 2.28 nkat purified phytase g-1 soil and various concentrations of citric acid indicated on the abscissa. Phytase-labile organic phosphorus (pg phosphorus g-1 soil) is indicated on the ordinate. Total extractable organic phosphorus (pg phosphorus g-1 soil) is also shown in the figure Soil was from Rutherglen Research Institute, Victoria, Australia, and had been fertilised with superphosphate phosphorus) at a rate of 125 kg ha' 1 each alternate year since 1914. Each point represents the mean of three observations. Bars indicate standard error and, where not shown, were smaller than the symbol. Data show a significant positive correlation between the hydrolysis of phytate and the concentration of citric acid in the soil extract.

Figure 16 is a graphical representation showing the effect of pH on the hydrolysis of phytate in air-dried soil, as measured by the release of phytase-labile organic phosphorus. The hydrolysis of organic phosphorus in soil extracts was measured for 6 hr in the presence of either 0.50 nkat commercial phytase g- 1 soil or 2.28 nkat purified phytase g-1 soil in the presence of 50 mM citric acid at the pH values indicated on the abscissa. Phytase-labile organic phosphorus (pg phosphorus g-1 soil) is indicated on the ordinate. Total extractable organic phosphorus (pg phosphorus g-1 soil) is also shown in the figure Soil was from Rutherglen Research Institute, Victoria, Australia, and had been fertilised with superphosphate phosphorus) at a rate of 125 kg ha' 1 each alternate year since 1914. Each point represents the mean of three observations. Bars indicate standard error and, where not shown, were smaller than the symbol. Data show that, whilst extractable phosphorus increased with pH, the hydrolysis of phytate by phytase was similar across the range of pH values tested.

Figure 17 is a graphical representation showing the organic phosphorus (filled bars) or inorganic phosphorus (open bars) contents of soils from two sites in Australia that are extractable by water (panels a, 50 mM citric acid (panels c, or 500 mM sodium bicarbonate (pH 8.5; panels e, and the organic phosphorus that was hydrolysed by phytase in soil extracts for 6 hr in the presence of either 0.50 nkat WO 01/22806 PCT/AUOO/01183 -17commercial phytase soil (open-hatched bars), or 2.28 nkat purified phytase g- 1 soil (densely-hatched bars), in each of the above conditions. One soil sample was from Rutherglen Research Institute, Victoria, Australia, that had been either unfertilised or fertilised with superphosphate phosphorus) at a rate of 125 kg ha 1 each alternate year since 1914 or fertilised as for FR soils and also topdressed with nine applications of lime at the rate of 1.25 tonnes ha- 1 in the period 1914-1948 (F+LR) The other soil sample was from Ginninderra Experiment Station (Wallaroo 3 paddock) that had been either unfertilised or received three autumn applications of triple superphosphate (20.7% phosphorus) at a rate of either 416 kg ha (F1G) or 675 kg ha- 1 (F2G). Extractable phosphorus (pg phosphorus g-1 soil) is indicated on the ordinate. Each bar represents the mean of three observations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One aspect of the invention provides a method of improving the phosphorus nutrition of a plant comprising ectopically expressing in the root of a plant an isolated nucleic acid molecule encoding a phytase polypeptide for a time and under conditions sufficient for said phytase to be secreted from the root.

As used herein, the term "phosphorus nutrition" shall be taken to refer to the utilisation by a plant of an external source of phosphorus in any form, including organic phosphorus and/or phosphate anion and/or phytate and/or phosphate and/or orthophosphate and/or pyrophosphate, amongst others. By "external source of phosphorus" is meant phosphorus that is taken up by the plant from the external environment.

An "improved phosphorus nutrition" refers to a greater ability of the plant to utilise an existing phosphorus source in the soil or growth medium in which said plant grows.

WO 01/22806 PCT/AUOO/01183 -18- Accordingly, the present invention is directed to a method of improving the ability of a plant to utilise an extemal source of phosphorus. In a particularly preferred embodiment, the external source of phosphorus is phytate.

The term "phytate" shall be taken to refer to any storage phosphorus source comprising inositol phosphate, including aggregates and polymers thereof, and phytin, a generic term applied to complex salts of phytic acid (for a review see Graf, 1986).

The term "phytase polypeptide" refers to any amino acid sequence, peptide, oligopeptide, polypeptide, or protein molecule, with or without additional non-amino acid substituents or non-naturally-occurring amino acid substituents, that is capable of catalysing the removing a phosphate-containing moiety from phytate as hereinbefore defined. Preferably, a phytase polypeptide will further be capable of catalysing the conversion of phytate to inositol and phosphate, which may be in any form, such as, for example, an anion, or metal complex, a transition metal complex, or a weak acid, amongst others. Those skilled in the art will be aware of those forms of soil phosphate that are readily utilisable by plants, and the present invention clearly extends to phytase enzymes capable of converting phytate to any such form of soil phosphate.

Whilst the present invention is not limited by the source of phytase, the phytase polypeptide of the invention is preferably derived from a plant, microorganism, or animal cell. Those skilled in the art will be aware that phytases are widely-occurring enzymes in nature, derivable from bacteria, such as, for example, Bacillus subtilis (Paver and Jagannathan, 1982), Pseudomonas (Cosgrove, 1970); yeasts, such as, for example, Saccharomyces cerevisiae (Nayini and Markakis, 1984); fungi, such, for example, Aspergillus fumigatus (Wyss et al., 1999), Aspergillus terreus (Yamada et al., 1986), A. niger (Mullaney et al., 1991; van Hartingsveldt et al., 1993); plants (Loewus, 1990) such as, for example, maize (Laboure et al., 1993; Hubel and Beck, 1996; Maugenest et al., 1997), potato (Gellady and Lefebvre, 1990), and soybean (Gibson et al., 1988), amongst others. Conveniently, the phytase enzyme WO 01/22806 PCT/AUOO/01183 -19employed in the performance of the present invention possesses high specific activity and/or high Vmax in a soil environment and/or low Km for phytate in a soil environment.

In a particularly preferred embodiment, the phytase enzyme employed in the performance of the present invention is derived from a fungus, more preferably Aspergillus spp., and even more preferably from A. niger. As exemplified herein, the A. niger phytase enzymes comprising the amino acid sequences set forth in SEQ ID NO: 2 or SEQ ID NO: 4 provides high biomass production and/or increased phosphorus content when expressed in the roots of transgenic plants and secreted therefrom into the surrounding growth medium.

For the purposes of nomenclature, the amino acid sequence set forth in SEQ ID NO: 2 relates to the A. niger PhyA-2 phytase polypeptide, which has been produced by expression of the A. niger PhyA-2 gene (SEQ ID NO: The present inventors have modified the naturally-occurring gene to be more suitable for expression in plants.

The amino acid sequence set forth in SEQ ID NO: 4 relates to a variant of the A.

niger PhyA-1 polypeptide (Mullaney et al., 1991; Van Hartingsveldt et al., 1993; GenBank Accession No. M94550), having the leader sequence removed and a different translation start site inserted relative to the naturally-occurring PhyA-1 polypeptide. To express the PhyA-1 polypeptide in plants, the present inventors modified the corresponding PhyA-1 gene sequence to remove the endogenous A.

niger leader sequence-encoding nucleotide sequence and intron sequence, and introduced a new translation start site immediately prior to and in-frame with, the nucleotide sequence encoding the mature PhyA-1 polypeptide.

As used herein, the terms "in-frame" and "in the same reading frame" refer to one or more codons of a nucleotide sequence being in the same open reading frame as one or more other codons of said nucleotide sequence. Similarly, the term "in-frame fusion" refers to the linkage between two or more heterologous nucleotide WO 01/22806 PCT/AUOO/01183 sequences such that the amino acid sequences encoded thereby are expressed in the same reading frame and, as a consequence, as a single polypeptide molecule.

The phytase polypeptide may be expressed throughout the length of the root, or alternatively, in a localised region of the root, preferably in the zone of elongation and/or the root tip. Conveniently, expression occurs in the epidermal cells and/or the cortex, to facilitate the secretion of the phytase to the root surface, either by reducing the number of cell layers through which the phytase must be transported or alternatively, by facilitating transport of the phytase to the epidermal cells.

Preferably, secretion of the phytase polypeptide from the root cells in which it is expressed is achieved by expressing the phytase as an in-frame fusion polypeptide with a secretory signal sequence capable of directing transport of the phytase to the root surface. According to this embodiment, the secretory signal sequence may be placed at the N-terminal and/or C-terminal end of the phytase polypeptide. Those skilled in the art will be aware that such signal sequences have been demonstrated to be operable in either configuration. Alternatively, or in addition, a secretory signal sequence may be embedded in the phytase polypeptide, the only requirement being that the embedding of the secretory signal sequence in the phytase does not inactivate the phytase enzymic activity in the soil or growth medium. The present invention further encompasses the use of a cryptic secretory signal sequence, either as an in-frame fusion with phytase or alternatively, by mutation of a region of the phytase polypeptide to produce an amino acid variant phytase polypeptide, such as a substitutional variant or insertional variant or deletional variant, that comprises a cryptic secretory signal sequence therein.

Substitutional variants are those in which at least one residue in the phytase amino acid sequence has been removed and a different residue inserted in its place.

Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues. and deletions will range WO 01/22806 PCT/AU00/01183 -21from about 1-20 residues. Preferably, amino acid substitutions will comprise conservative amino acid substitutions, such as those described supra.

Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the phytase protein.

Insertions can comprise amino- terminal and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues.

Deletional variants are characterised by the removal of one or more amino acids from the phytase sequence.

Phytase amino acid variants may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art. For example, techniques for making substitution mutations at.predetermined sites in DNA having known sequence are well known to those skilled in the art, such as by M13 mutagenesis or other site-directed mutagenesis protocol.

Preferred secretory signal sequences according to this embodiment of the invention are derived from plants, fungi, yeasts, bacteria or animal cells, the only requirement being that they function in the root of a plant. Such function can be readily determined without undue experimentation, by determining the level of phytase transported to the cell surface or root surface following its ectopic expression in the root as an in-frame fusion with the secretory signal sequence. Alternatively, or in addition, the efficacy of the signal sequence can be tested by determining the ability of a plant ectopically expressing the phytase as an in-frame fusion with the signal sequence to grow on phytate as a source of phosphorus. As exemplified herein, and as shown in Figures 7, 8, and 9, plants that ectopically express phytase as an WO 01/22806 PCT/AUOO/01183 -22in-frame fusion with the secretory signal sequence derived from the carrot extensin protein, exhibit improved growth on phytate relative to otherwise isogenic nontransformed plants. Using such tests, those skilled in the art can readily determine the optimum secretory signal sequence for performing the present invention, and the optimum placement of said secretory signal sequence relative to the phytase polypeptide.

The secretory signal sequence is conveniently derived from the full-length potato patatin polypeptide (Iturriaga et a,1989; Li et al., 1997), the tobacco PR-S polypeptide (Comelissen et al. 1986; Pen et al., 1993), the lupin acid phosphatase (LASAP 1; Wasaki et al., 1999) polypeptide or the carrot extensin polypeptide (Chen and Vamer, 1985).

In a particularly preferred embodiment, the secretory signal sequence is derived from the carrot extensin polypeptide and placed at the N-terminus of the phytase polypeptide.

Nucleotide sequences of the secretory signal-encoding nucleotide sequences of the carrot extensin and lupin acid phosphatase genes are set forth herein, as SEQ ID NOs: 5 and 7, respectively. The amino acid sequences of the carrot extensin and lupin acid phosphatase secretory signal peptides are also set forth herein, as SEQ ID NOs: 6 and 8, respectively.

Additionally, the nucleotide sequences of chimeric genes encoding the carrot extensin leader sequence fused to the PhyA-2 or PhyA-1 gene, are shown in Figure 1 and SEQ ID Nos: 9 and 11, respectively. The amino acid sequences of fusion polypeptides comprising the carrot extensin leader sequence fused to the modified PhyA-2 or PhyA-1 polypeptides, are shown in Figure 2 and SEQ ID Nos: 10 and 12, respectively. These chimeric genes have been expressd by the inventors in several plant species by the methods described herein to enhance the phosphorus nutrition of those plants.

WO 01/22806 PCT/AU00/01183 -23- The word "express" or variations such as "expressing" and "expression" as used herein shall be taken in their broadest context to refer to the transcription of a particular genetic sequence to produce sense or antisense mRNA or the translation of a sense mRNA molecule to produce a peptide, polypeptide, oligopeptide, protein or enzyme molecule. In the case of expression comprising the production of a sense mRNA transcript, the word "express" or variations such as "expressing" and "expression" may also be construed to indicate the combination of transcription and translation processes, with or without subsequent post-translational events which modify the biological activity, cellular or sub-cellular localisation, tumover or steadystate level of the peptide, polypeptide, oligopeptide, protein or enzyme molecule.

Without being bound by any theory or mode of action, the expression of phytase in the root cell and its subsequent secretion to the root surface provides a localised high concentration of active phytase enzyme the is capable of diffusing into the soil to catalyse the conversion of phytate into inositol and phosphate, such that the phosphate is then able to be absorbed or actively taken up by the root.

The term "ectopic expression" refers to the de novo and/or increased expression of a peptide, oligopeptide, polypeptide or protein from an introduced nucleic acid molecule, such as, for example, by means of transfection or transformation of a cell, tissue or organ with nucleic acid encoding the peptide, oligopeptide, polypeptide or protein. Ectopic expression can also be achieved by the infection or transformation of a cell, tissue or organ with a foreign organism containing nucleic acid encoding the peptide, oligopeptide, polypeptide or protein.

Accordingly, to ectopically-express a phytase polypeptide comprising an in-frame fusion with a secretory signal sequence, it is necessary to produce a corresponding nucleic acid molecule encoding both the secretory signal sequence and the phytase polypeptide in the same reading frame. This may be achieved by those skilled in the art without undue experimentation.

WO 01/22806 PCT/AUOO/01183 -24- For the ectopic expression of a peptide, oligopeptide, polypeptide or protein in a plant cell, tissue or organ, the nucleic acid molecule encoding said peptide, oligopeptide, polypeptide or protein in a plant-expressible format, such as, for example, in an appropriate gene construct comprising a promoter sequence operably connected to said nucleic acid molecule, and optionally a transcription termination sequence comprising a polyadenylation signal, amongst others.

By "expressible format" is meant that the isolated nucleic acid molecule is in a form suitable for being transcribed into mRNA and/or translated to produce a protein, either constitutively or following induction by an intracellular or extracellular signal, such as an environmental stimulus or stress (anoxia, hypoxia, temperature, salt, light, dehydration, low phosphate, low P, etc) or a chemical compound such as an antibiotic (tetracycline, ampicillin, rifampicin, kanamycin) hormone (eg. gibberellin, auxin, cytokinin, glucocorticoid, etc), hormone analogue (iodoacetic acid (IAA), 2,4- D, etc) metal (zinc, copper, iron, etc), or dexamethasone, amongst others. As will be known to those skilled in the art, expression of a functional protein may also require one or more post-translational modifications, such as glycosylation, phosphorylation, dephosphorylation, or one or more protein-protein interactions, amongst others. All such processes are included within the scope of the term "expressible format".

The term "plant-expressible format' refers to an expressible format that pertains to expression of proteins in plant cells, tissues or organs.

Preferably, the ectopic expression of phytase is effected by introducing an isolated nucleic acid molecule encoding phytase, such as a cDNA molecule, genomic gene, synthetic oligonucleotide molecule, mRNA molecule or open reading frame, to a plant cell, tissue or organ, operably in connection with a promoter sequence that is capable of conferring expression in a plant root cell, albeit not necessarily exclusively in the root cell.

WO 01/22806 PCT/AU00/01183 Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences derived from a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or are capable of conferring expression on a structural gene sequence the protein-coding region of a gene) in a tissue-specific manner, conveniently in the roots.

In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a plant cell, tissue or organ.

Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably connected. For example, copper-responsive, glucocorticoid-responsive or dexamethasone-responsive regulatory elements may be placed adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule to confer copper inducible, glucocorticoid-inducible, or dexamethasone-inducible expression respectively, on said nucleic acid molecule.

Included within the scope of the present invention is the use of strong constitutive promoter sequences, cell-specific promoter sequences, inducible promoter sequence, tissue-specific promoter sequences, organ-specific promoter sequences, and constitutive promoter sequences that have been modified to confer expression in a particular part of the plant at any one time, such as by integration of said constitutive promoter within a transposable genetic element (Ac, Ds, Spm, En, or other transposon).

The term "constitutive" will be known by those skilled in the art to indicate that expression is observed predominantly throughout the plant, albeit not necessarily in WO 01/22806 PCT/AU00/01183 -26every cell, tissue or organ under all conditions. In the present context, a preferred strong constitutive promoter is one which confers a high level of ectopic expression on a phytase structural gene to which it is operably connected, predominantly throughout the plant and at least in the root, albeit not necessarily in every cell, tissue or organ under all conditions.

The term "cell-specific" shall be taken to indicate that expression is predominantly in a particular plant cell or plant cell-type, albeit not necessarily exclusively in that plant cell or plant cell-type. In the present context, a preferred cell-specific promoter will confer expression on a phytase gene in at least one cell type of the root, preferably, a root epidermal cell or root cortical cell.

Similarly, the term "tissue-specific" shall be taken to indicate that expression is predominantly in a particular plant tissue or plant tissue-type, albeit not necessarily exclusively in that plant tissue or plant tissue-type. In the present context, a preferred tissue-specific promoter will confer expression on one or more tissues of the root, such as, for example, in the zone of elongation, the root vasculature, or the root tip.

Similarly, the term "organ-specific" shall be taken to indicate that expression is predominantly in a particular plant organ albeit not necessarily exclusively in that plant organ. In the present context, a preferred organ-specific promoter will confer expression on a phytase gene throughout the root.

Those skilled in the art will be aware that an "inducible promoter" is a promoter the transcriptional activity of which is increased or induced in response to a developmental, chemical or physical stimulus.

Preferably, the promoter is a root-specific, phosphate-regulated promoter derived from a phosphate transport gene, such as, for example, the phosphate transport genes of A. thaliana or barley plants, which are induced under conditions of WO 01/22806 PCT/AUOO/01183 -27phosphate deficiency in the plant. These promoters are obtainable from CSIRO Tropical Agriculture, Queensland, Australia.

The present invention is not to be limited by the choice of promoter sequence and those skilled in the art will readily be capable of selecting appropriate promoter sequences for use in regulating appropriate expression of phytase or modified phytase from publicly-available or readily-available sources, without undue experimentation.

Placing a phytase-encoding nucleic acid molecule under the regulatory control of a promoter sequence, or in operable connection with a promoter sequence, means positioning said nucleic acid molecule such that expression is controlled by the promoter sequence.

A promoter is usually, but not necessarily, positioned upstream, or at the and within 2 kb of the start site of transcription, of the nucleic acid molecule which it regulates.

In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting the gene from which the promoter is derived). As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting the gene from which it is derived). Again, as is known in the art, some variation in this distance can also occur.

Examples of strong constitutive promoters and root-specific promoters that are suitable for use in expressing phytase in the roots of plants include those listed in Table 2, amongst others. The promoters listed in Table 2 are provided for the WO 01/22806 PCT/AU00/01183 -28purposes of exemplification only and the present invention is not to be limited by the list provided therein. Those skilled in the art will readily be in a position to provide additional promoters that are useful in performing the present invention.

Preferred tissue-specific inducible promoter sequences include the anoxia-inducible and hypoxia-inducible maize Adhl gene promoter (Howard et al., 1987; Walker et al., 1987). Such environmentally-inducible promoters are reviewed in detail by Kuhlemeier et al. (1987).

Preferred chemically-inducible promoters include the 3-P- indoylacrylic acidinducible Tip promoter; IPTG-inducible lac promoter; phosphate-inducible promoter; L-arabinose-inducible araB promoter, heavy metal-inducible metallothionine gene promoter; dexamethasone-inducible promoter; glucocorticoid-inducible promoter; ethanol-inducible promoter (Zeneca); the N,N-diallyl-2,2-dichloroacetamideinducible glutathione-S-transferase gene promoter (Wiegand et al., 1986); or any one or more of the chemically-inducible promoters described by Gatz et al.

(1996;1998), amongst others.

Preferred wound-inducible or pathogen-inducible promoters include the phenylalanine ammonia lyase (PAL) gene promoter (Ebel et al., 1984), chalcone synthase gene promoter (Ebel et al., 1984) or the potato wound-inducible promoter (Cleveland et al., 1987), amongst others.

In the case of constitutive promoters or promoters that induce expression throughout the entire plant, such sequences may be modified by the addition of nucleotide sequences derived from one or more of the root-specific promoters listed in Table 2, and optionally, additional nucleotide sequences derived from one or more inducible promoters, to confer inducible tissue-specificity thereon. For example, the CaMV 35S promoter may be modified by the addition of maize Adhl promoter sequence, to confer anaerobically-regulated root-specific expression thereon, as described previously (Ellis et al., 1987). Such modifications can be achieved by routine experimentation by those skilled in the art.

WO 01/22806 PCT/AU00/01183 -29- In a particularly preferred embodiment of the present invention, the phytase is ectopically expressed under control of the CaMV 35S promoter sequence.

In each of the preceding embodiments of the present invention, the phytase protein or a homologue, analogue, or derivative thereof, in particular the A. niger phytase protein PhyA-1 or PhyA-2, is expressed under the operable control of a promoter sequence operable in the root. As will be known those skilled in the art, this is generally achieved by introducing a gene construct or vector into plant cells by transformation or transfection means. The nucleic acid molecule or a gene construct comprising same may be introduced into a cell using any known method for the transfection or transformation of said cell. Wherein a cell is transformed by the gene construct of the invention, a whole organism may be regenerated from a single transformed cell, using methods known to those skilled in the art.

By "transfect" is meant that the gene construct or vector or an active fragment thereof comprising the PhyA-1 or PhyA-2 gene or a homologue, analogue or derivative thereof, operably under the control of the promoter sequence is introduced into said cell without integration into the cell's genome.

By "transform" is meant that the gene construct or vector or an active fragment thereof comprising the PhyA-1 or PhyA-2 gene or a homologue, analogue or derivative thereof, operably under the control of the plant-expressible promoter sequence is stably integrated into the genome of the cell.

In an alternative embodiment, the present invention provides a method of improving the phosphorus nutrition of a plant comprising: ectopically expressing in the root of a plant an isolated nucleic acid molecule encoding a phytase polypeptide for a time and under conditions sufficient for said phytase to be secreted from the root; and (ii) modifying the chemistry of the soil around the root or other growth medium around the root.

WO 01/22806 PCT/AU00/01183 As used herein, the term "modifying the chemistry of the soil around the root or other growth medium around the root using an organic acid" or similar shall be taken to include any effect of an organic acid on increasing the ability of a plant to utilise phytase-labile phosphorus and/or total organic phosphorus, such as, for example, acidification, and/or a chelating effect that facilitates the solubilisation of phosphorus and/or phosphorus uptake, or to make the phytate accessible to the phytase enzyme, amongst others. The present invention is not to be limited by the mode of action of the organic acid in improving phosphorus nutrition, the only requirement being that the amount of organic acid in the vicinity of the root is increased, such as by direct application, extracellular secretion or active transport, amongst others.

The present invention particularly extends to the use of any agent known to those skilled in the art to modify the chemistry of the soil by chelation, preferably by chelation of a metal, such as, for example a transition metal, to facilitate phosphate uptake by a plant. In this regard, phosphate in the soil which is released by the breakdown of phytate may form complexes with various metals in the soil, such as, for example, aluminium. Without being bound by any theory or mode of action, the presence of an organic acid in the vicinity of the root where phytase acts in accordance with the inventive method may increase access of phytase enzyme to the phytrate substrate by chelation of metal ions, such as, for example, aluminium, iron or calcium, that are known to associate with phytate in the soil. This hypothesis is consistent with data provided as Example 3 herein which show that organic acids increase the availability of organic P substrates and makes them more amendable to dephosphorylation by phytase.

According to this embodiment, the chemistry of the soil around the root may be altered in accordance with the present invention by any means known to those skilled in the art, including the application of organic acids to the soil or growth medium, or the addition of agents known to those skilled in the art that chelate metals but not phosphorus.

WO 01/22806 PCT/AUOO/01183 -31 However, a particularly preferred means comprises expressing an organic acid biosynthetic enzyme in the roots of the plant so as to increase the intracellular level of organic acids, and the subsequent efflux of organic acids from the root.

Preferably, the expression of the organic acid biosynthetic enzyme is targeted to the same cells in which the phytase gene is expressed, to optimise the local extracellular concentrations of available phosphorus in that region of the root which is involved in phosphate uptake.

P

referably, the organic acid biosynthesis enzyme is citrate synthase. Expression of citrate synthase may be increased in the region around the root by ectopicallyexpressing a citrate synthase-encoding nucleic acid molecule in the root under the control of a root-specific promoter sequence or constitutive promoter sequence as described herein, and preferably, targeting the citrate synthase polypeptide product of such expression to the root surface.

As will be apparent to those skilled in the art, the ectopic expression of citrate synthase and targeting of the citrate synthase polypeptide to the root surface may be performed in a similar manner to the expression and secretion of the phytase polypeptide, in accordance with the description provided herein. Preferably, in the performance of this embodiment of the invention, the nucleic acid molecules encoding phytase and the organic acid biosynthesis enzyme are placed operably in connection with different promoter sequences, to minimise competition therebetween for nuclear transcription factors, which competition may reduce expression of one or other structural gene.

Alternatively or in addition, the chemistry of the soil or other growth medium is increased by expressing an organic acid transporter polypeptide in the roots of the plant for a time and under conditions sufficient for the rate or amount of organic acid outside the root to increase.

WO 01/22806 PCT/AU00/01183 -32- A further aspect of the present invention clearly provides a gene construct or vector to facilitate the ectopic expression and/or maintenance of the phytase proteinencoding sequence and promoter in a plant cell, tissue or organ.

It will be apparent from the preceding statements that the gene construct of the invention will at least comprise a phytase protein-encoding sequence, optionally further comprising nucleotide sequences encoding a secretory signal sequence operable in plant cells, and a promoter sequence operable in the root cells of a plant operably connected thereto.

The present invention clearly encompasses genetic constructs that further comprise a nucleotide sequence that encodes an organic acid biosynthesis enzyme, in particular citrate synthase, placed operably under the control of a further rootoperable or constitutive promoter sequence.

Additionally, the gene construct of the present invention may further comprise one or more terminator sequences.

The term "terminator"' refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.

TABLE 2 EXEMPLARY PROMOTERS FOR USE IN THE PERFORMANCE OF THE PRESENT INVENTION GENE SOURCE EXPRESSION PATTERN REFERENCE root-expressible genes roots Tingey et a. (1987); An et al. (1988); tobacco auxin-inducible gene root tip Van der ZaaI et a. (1991 P-tubulin root Oppenheimer et a. (1988) tobacco root-specific genes root Conkling et (1990) B. napus G1 -3b gene root United States Patent No. 5, 401, 836 SbPRP1 roots Suzuki et a. (1993) AtPRP1; AtPRP3 roots: root hairs htto://salusmedium.edu/mm/tierne/html RD2 gene root cortex http://www2.cnsu.edu/ncsu/research TobRB37 gene root vasculature http://www2 .cnsu .edu/ncsu/research Actin constitutive CaMV 35S constitutive Odell et al (1985) CaMV 19S constitutive Octopine synthase (OCS) constitutive Koncz et al., (1984) Nopaline Synthase (NOS) constitutive Depicker et (1982) gos2 constitutive de Pater eta!. (1992) UBQ1 constitutive Callis et al., (1990) INDUCIBLE PROMOTERS INDUCTANT REFERENCE (delta(1)-pyrroline-5- salt, water Zhang etal (1997) carboxylate syntase) cold Hajela et al (1990) cold Wilhelm et a(1993) (-305 to +78 nt) cold, drought Baker et al (1994) rd29 salt, drought, cold Kasuga et al (1999) heat shock proteins, including heat Barros et al (1992); Marrs etal (1993); Schoffl et a (1989) artificial promoters containing the heat shock element (HSE) smHSP (small heat shock heat Waters et al. (1989) proteins) wcs120 cold Oullet et a (1998) ci7 cold Kirch et a (1997) Adh cold, drought, hypoxia Dolferus et al(1994) pwsi18 water: salt and drought Joshee et al (1998) ci21A cold Schneider et al (1997) Trg-31 drought Chaudhury et al (1996) osmotin osmotic stress Raghothama et a (1993) WO 01/22806 PCT/AUOO/01183 \Examples of terminators particularly suitable for use in the gene constructs of the present invention include the Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator, the Agrobacterium tumefaciens octopine synthase (OCS) gene terminator sequence, the Cauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryza sativa ADP-glucose pyrophosphorylase terminator sequence (t3'Bt2), the Zea mays zein gene terminator sequence, the rbcs-1A gene terminator, and the rbcs-3A gene terminator sequences, amongst others.

Those skilled in the art will be aware of additional promoter sequences and terminator sequences which may be suitable for use in performing the invention.

Such sequences may readily be used without any undue experimentation.

The gene constructs of the invention may further include an origin of replication sequence which is required for maintenance and/or replication in a specific cell type, for example a bacterial cell, when said gene construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.

Preferred origins of replication include, but are not limited to, the fl-ori and colE1 origins of replication.

The gene construct may further comprise a selectable marker gene or genes that are functional in a cell into which said gene construct is introduced.

As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a gene construct of the invention or a derivative thereof.

Suitable selectable marker genes contemplated herein include the ampicillin resistance tetracycline resistance gene (Tcr), bacterial kanamycin resistance gene phosphinothricin resistance gene, neomycin phosphotransferase gene (nptll), hygromycin resistance gene, -glucuronidase WO 01/22806 PCT/AU00/01183 -36- (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), and luciferase gene, amongst others.

In fact, the phytase protein-encoding sequence may also be used as a selectable marker gene as defined herein, by virtue of the improved phosphorus nutrition of plants secreting phytase from their roots in accordance with the inventive method, including their ability to grow on phytate-containing media. Accordingly, the present invention clearly encompasses the selection of transformed plants by their ability to regenerate on media having phytate as the source of phosphorus.

In a further preferred embodiment, the present invention provides a method of modifying the phosphorus nutrition of a plant comprising: introducing to a plant cell, tissue or organ a gene construct or vector comprising a nucleotide sequence that encodes phytase in a secretable form, operably in connection with a promoter sequence capable of conferring expression in the roots of a plant; (ii) regenerating a whole plant therefrom; and (ii) expressing said phytase in the root or one or more of said cells or tissues thereof such that it is secretable to the surface of the root.

A further aspect of the invention provides a transformed plant ectopically-expressing phytase in secretable form, preferably as an in-frame fusion with a secretable signal sequence.

Means for introducing recombinant DNA into bacterial cells, yeast cells, or plant, insect, fungal (including mould), avian or mammalian tissue or cells include, but are not limited to, transformation using CaCI, and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990), electroporation (Fromm et al., 1985), microinjection of DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or cells (Christou et al, WO 01/22806 PCT/AU00/01183 -37- 1988; Sanford et al., 1987; Finer and McMullen, 1990; Finer et al., 1992; Sanford et al., 1993; Karunaratne et al., 1996; and Abedinia et al., 1997), vacuum-infiltration of tissue with nucleic acid, or T-DNA-mediated transfer from Agrobacterium to the plant tissue (An et al.1985; Herrera-Estrella et al., 1983a; 1983b; 1985).

For example, the transformed plants can be produced by the method of in planta transformation method using Agrobacterium tumefaciens (Bechtold et al.,1993; Clough et al.,1998), wherein A. tumefaciens is applied to the outside of the developing flower bud and the binary vector DNA is then introduced to the developing microspore and/or macrospore and/or the developing seed, so as to produce a transformed seed. Those skilled in the art will be aware that the selection of tissue for use in such a procedure may vary, however it is preferable generally to use plant material at the zygote formation stage for in planta transformation procedures.

Alternatively, microparticle bombardment of cells or tissues may be used, particularly in cases where plant cells are not amenable to transformation mediated by A.

tumefaciens. In such procedures, microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. Patent No. 5,122.466) and Sanford and Wolf Patent No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to 5 /m gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

A whole plant may be regenerated from the transformed or transfected cell, in accordance with procedures well known in the art. Plant tissue capable of WO 01/22806 PCT/AU00/01183 -38subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a gene construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue apical meristem, axillary buds, and root meristems), and induced meristem tissue cotyledon meristem and hypocotyl meristem).

The term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centres.

The term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformant, and the T2 plants further propagated through classical breeding techniques.

The generated transformed organisms contemplated herein may take a variety of forms. For example, they may be chimeras of transformed cells and nontransformed cells; clonal transformants all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues in plants, a transformed root stock grafted to an untransformed scion The present invention is applicable to any plant, in a particular monocotyledonous plants or dicotyledonous plant including fodder or forage legume, companion plant, food crop, tree, shrub, or ornamental selected from the list comprising Acacia spp., WO 01/22806 WO 0122806PCT/AUOO/01 183 -39- Acer spp., Actinidia spp.,Aesculus spp., Agathis australis, Albizia amara, Also phila tricolor, Andrapogon spp., A. thaliana, Arachis spp, Areca, catechu, Astelia fragrans, Astragalus cicer, Baikiaea plunjuga, Betula spp., Brassica, spp., Brvguiera gymnorrhiza, Burkea afficana, Butea frondosa, Cadaba faninosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp.,Cinnamomum cassia, Coffea arabica, Cola phospermum ma pane, Coronilia varia, Cotaneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetania, Davallia divaricata, Desmodiumn spp., Dicksonia squarosa, Diheteropogan amplectens, Dioclea spp, Dolichos spp., Dot ycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Era grestis spp., Erythnina spp., Eucalyptus spp., Euclee schimpen, Eulalia villosa, Fagopyrumn spp., Feqoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksi, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Glinicidia spp, Gassypium hirsutum, Gre villea spp., Guibourtia coleosperma, Hedysarumn spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericumn erectum, Hyperthelia dissoluta, Indigo incamnata, Iris spp., Leptarrhena pyralifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesi Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Qnobtychis spp., Omnithopus spp., Oryza spp., Peltopho rum afticanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormiumn cookianum, Photinia spp., Picea glauca, Pin us spp., Pisumn sativumn, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cinerania, Pseudotsuga, menziesi, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossulania, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus ala pecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium, distichum, Themeda WO 01/22806 PCT/AU00/01183 triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp.Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, rice, straw, amaranth, onion, asparagus, sugar cane, soybean, sugarbeet, sunflower, carrot, celery, cabbage, canola, tomato, potato, lentil, flax, broccoli, oilseed rape, cauliflower, brussel sprout, artichoke, okra, squash, kale, collard greens, and tea, amongst others, or the seeds of any plant specifically named above or a tissue, cell or organ culture of any of the above species.

Preferably, the plant is a plant that is capable of being transfected or transformed with a genetic sequence, or which is amenable to the introduction of a protein by any art-recognised means, such as microprojectile bombardment, microinjection, Agrobacterium-mediated transformation, protoplast fusion, protoplast transformation, in planta transformation, or electroporation, amongst others.

In a particularly preferred embodiment, the transformed plant is A. thaliana or subterranean clover. As will be known to those skilled in the art, the provision of both transformed species is sufficient to enable the enhancement of phosphorus nutrition in any plant species using the inventive method described herein.

This aspect of the invention further extends to plant cells, tissues, organs and plants parts, propagules and progeny plants of the primary transformed or transfected cells, tissues, organs or whole plants that also comprise the introduced isolated nucleic acid molecule or gene construct comprising same, and, as a consequence, exhibit similar phenotypes to the primary transformants/transfectants or at least are useful for the purpose of replicating or reproducing said primary transformants/transfectants.

A further aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a phytase peptide, oligopeptide, polypeptide, protein or enzyme having at least about 93% nucleotide sequence identity to the WO 01/22806 PCT/AU00/01183 -41- Aspergillus oiger PhyA-2 gene sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 9 or a complementary nucleotide sequence thereto.

Preferably, the percentage identity to SEQ ID NO: 1 or SEQ ID NO: 9 is at least about 95%, more preferably at least about 97%, and still more preferably at least about 99%. In a particularly preferred embodiment, the isolated nucleic acid molecule of the invention comprises or contains the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 9 or a fragment thereof that encodes an enzymicallyfunctional phytase peptide, oligopeptide or polypeptide.

In determining whether or not two nucleotide or amino acid sequences fall within defined percentage identity or similarity limits referred to herein, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison. In such comparisons or alignments, differences will arise in the positioning of non-identical residues depending upon the algorithm used to perform the alignment. In the present context, references to percentage identities between two or more sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard algorithm known to those skilled in the art. In particular, nucleotide and/or amino acid identities can be calculated using the GAP programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, 1984), which utilizes the algorithm of Needleman and Wunsch (1970) or alternatively, the CLUSTAL W algorithm of Thompson et al (1994) for multiple alignments, to maximise the number of identical/similar residues and to minimise the number and/or length of sequence gaps in the alignment.

Alternatively or in addition, the present invention encompasses those phytaseencoding nucleotide sequences that are capable of hybridising under high stringency hybridisation conditions to SEQ ID NO: 1 or SEQ ID NO: 9 or a complementary nucleotide sequence thereto, but not including the PhyA-1 gene sequence.

WO 01/22806 PCT/AU00/01183 -42- For the purposes of defining the level of stringency, those skilled in the art will be aware that several different hybridisation conditions may be employed. As used herein, a high stringency may comprise a standard reaction buffer used in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to template DNA at temperatures higher than 42 0 C, or altematively, a standard DNA/DNA hybridisation and/or wash carried out in 0.1xSSC buffer, 0.1% SDS at a temperature of at least 65"C, or equivalent annealing/hybridisation conditions.

As will be known to those skilled in the art, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS in a standard hybridisation, and/or increasing the temperature of the annealing/hybridisation of PCR or a standard hybridisation, and/or increasing the temperature of the wash in a standard hybridisation. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification (to parameters affecting hybridisation between nucleic acid molecules), reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.

As will be known to those skilled in the art, the specificity of PCR may also be increased by reducing the number of cycles, or the time per cycle, or by the use of specific PCR formats, such as, for example, a nested PCR, a format that is wellknown to those skilled in the art. For the purposes of clarification of the parameters affecting the specificity of PCR, reference is made herein to McPherson et al. (1991) which is incorporated by way of reference.

Particularly preferred variants of the A. niger PhyA-2 gene exemplified herein comprise degenerate nucleotide sequences homologues) that encode the amino acid sequence set forth in SEQ ID NO: 2.

WO 01/22806 PCT/AUOO/01183 -43- Preferably, the isolated nucleic acid molecule encodes a phytase polypeptide, protein or enzyme as an in-frame fusion with a secretory signal peptide, in particular the carrot extensin signal peptide.

Homologues, analogues and derivatives of the phytase-encoding nucleotide sequence of the present invention may be obtained by any standard procedure known to those skilled in the art, such as by nucleic acid hybridization (Ausubel et al, 1987), polymerase chain reaction (McPherson et al, 1991) screening of expression libraries using antibody probes (Huynh et al, 1985), and the invention encompasses all such homologues, analogues and derivatives falling within the above-mentioned sequence identity and/or hybridisation limitations.

In nucleic acid hybridizations, genomic DNA, mRNA or cDNA or a part of fragment thereof, in isolated form or contained within a suitable cloning vector such as a plasmid or bacteriophage or cosmid molecule, is contacted with a hybridizationeffective amount of a nucleic acid probe derived from SEQ ID NO: 1 for a time and under conditions sufficient for hybridization to occur and the hybridized nucleic acid is then detected using a detecting means.

Detection is performed preferably by labelling the probe with a reporter molecule capable of producing an identifiable signal, prior to hybridization. Preferred reporter molecules include radioactively-labelled nucleotide triphosphates and biotinylated molecules.

Preferably, variants of the A. niger PhyA-2 gene exemplified herein, including genomic equivalents, are isolated by hybridisation under high stringency conditions, to the probe.

In the polymerase chain reaction (PCR), a nucleic acid primer molecule comprising at least about 14 nucleotides in length derived from the A. niger PhyA-2 gene is hybridized to a nucleic acid template molecule and specific nucleic acid molecule WO 01/22806 PCT/AU00/01183 -44copies of the template are amplified enzymatically as described in McPherson et al, (1991), which is incorporated herein by reference.

In expression screening of cDNA libraries or genomic libraries, protein- or peptideencoding regions are placed operably under the control of a suitable promoter sequence in the sense orientation, expressed in a prokaryotic cell or eukaryotic cell in which said promoter is operable to produce a peptide or polypeptide. screened with a monoclonal or polyclonal antibody molecule or a derivative thereof against one or more epitopes of a phytase polypeptide and the bound antibody is then detected using a detecting means, essentially as described by Huynh et al (1985) which is incorporated herein by reference. Suitable detecting means according to this embodiment include 125 1-labelled antibodies or enzyme-labelled antibodies capable of binding to the first-mentioned antibody, amongst others.

A still further aspect of the present invention provides an isolated or recombinant phytase polypeptide selected from the group consisting of: a phytase polypeptide having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; (ii) a phytase polypeptide encoded by a nucleotide sequence having at least 93% identity to SEQ ID NO: 1; (iii) fragments of or (ii) that possess phytase enzyme activity; (iv) in-frame fusion polypeptides comprising and/or (ii) and/or (iii) linked to a secretory signal peptide, in particular the carrot extensin secretory signal peptide.

It will be apparent from the preceding description that a recombinant phytase polypeptide or an in-frame fusion polypeptide comprising same may be produced by standard means by expressing a phytase-encoding nucleotide sequence operably under the control of a suitable promoter sequence in a host cell for a time and under conditions sufficient for translation to occur. Such expression may be carried out in a prokaryotic cell, such as, for example, a bacterial cell. Alternatively, such expression WO 01/22806 PCT/AUOO/01183 may be performed in a eukaryotic cell such as an insect cell, mammalian cell, plant cell, fungal cell, or yeast cell, amongst others. In any case, unless the sense molecule is expressed under the control of a strong universal promoter, it is important to select a promoter sequence which is capable of regulating expression in the cell comprising the said nucleic acid molecule in an expressible format. Persons skilled in the art will be in a position to select appropriate promoter sequences for expression of the sense molecule without undue experimentation.

Examples of promoters useful in performing this embodiment include the CaMV promoter, NOS promoter, octopine synthase (OCS) promoter, A. thaliana SSU gene promoter, napin seed-specific promoter, P 32 promoter, BK5-T imm promoter, lac promoter, tac promoter, phage lambda X or k promoters, CMV promoter (U.S.

Patent No. 5,168,062), T7 promoter, lacUV5 promoter, SV40 early promoter (U.S.

Patent No. 5,118,627), SV40 late promoter Patent No. 5,118,627), adenovirus promoter, baculovirus P10 or polyhedrin promoter Patent Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051 and 5,169,784), and the like. In addition to the specific promoters identified herein, cellular promoters for so-called housekeeping genes are useful.

In a preferred embodiment, the recombinant phytase polypeptide is provided in a sequencably-pure format or a pure format substantially free of conspecific proteins.

By "sequencably pure" is meant that the subject polypeptide or a homologue, analogue, derivative or epitope thereof is purified sufficiently to facilitate amino acid sequence determination.

Preferably, said' polypeptide or a homologue, analogue, derivative or epitope is at least about 20% pure, more preferably at least about 40% pure, even more preferably at least about 60% pure and even more preferably at least about pure or 95% pure on a weight basis.

WO 01/22806 PCT/AU00/01183 -46- For the purposes of describing the present invention in more detail, a plasmid comprising A. niger PhyA-2 gene as an in-frame fusion with the carrot extensin secretion signal-encoding nucleotide sequence, and operably in connection with the CaMV 35S promoter, was deposited on 23 September, 1999, with the Australian Govemment Analytical Laboratories (AGAL) at 1, Suakin Street Pymble, New South Wales 2073, Australia, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and accorded AGAL Accession No. NM99/06795. Accordingly, the presently-described invention clearly extends to the use of the deposited plasmid and/or the phytase-encoding portion thereof or variants thereof, with or without the secretory signal-encoding portion of said plasmid, in accordance with the scope of each and every embodiment described herein.

The present invention is further described with reference to the following non-limiting Examples and to the drawings.

EXAMPLE 1 Ectopic expression of phytase under control of the CaMV 35S promoter I. The PhyA-2 gene PCR primers PHYF2 and PHYR3 were used to amplify a derivative of the PhyA-1 gene from genomic DNA isolated from Aspergillus niger, strain ATCC9029. The sequence of the PCR primers (which also contained cloning sites for EcoRI and Clal, respectively; underlined lower case) were as follows: Forward(PHYF2): cgcgaattcATGCTGGCAGTCCCCGCCTCG (SEQ ID NO: 13); and Reverse(PHYR3): ggcatcgatCTAAGCAAAACACTCCGC (SEQ ID NO: 14) The amplified PhyA-2 gene is a modified version of the PhyA-1 gene that is functional in plants, and contains no leader sequence or first intron; a "new" ATG translation start in the open reading frame, immediately prior to, and in frame with, the nucleotide sequence encoding the mature phytase polypeptide. The sequence WO 01/22806 PCT/AU00/01183 -47of the reverse primer resulted in the TAG translation stop codon being identical to that in the published A. niger PhyA-1 gene. The amplified gene has been designated "PhyA-2".

The PhyA-2 gene has approximately 92% DNA sequence identity to the PhyA-1 gene. Additionally, there is about 95% amino acid sequence identity between PhyA-1 and PhyA-2 polypeptides (Figures 1 and 2).

The nucleotide sequence of the PhyA-2 gene is set forth in SEQ ID NO: 1, and the derived amino acid sequence encoded by the PhyA-2 gene is set forth in SEQ ID NO: 2.

II. The PhyA-1 gene The PhyA-1 gene was originally obtained from Dr Mullaney (United States Department of Agriculture) as a 7.016 kb plasmid (pMD4.21), containing a 2.7 kb Sphl clone of the Aspergillus ficuum strain NRRL3135 (now termed A. niger) PhyA-1 gene, cloned into the plasmid vector pBR322.

The 2.7 kb insert contained a genomic clone of the PhyA-1 gene with 5' and 3' flanking sequences, including an Aspergillus 5' leader sequence and an intron (102 bp) upstream of the coding region for the mature protein (Genbank Accession No.

M94550; Van Hartingsveldt et al.,1993).

Modifications were made to the PhyA-1 gene, to delete the leader sequence and the intron, using PCR. These modifications introduced a "new" ATG translation start in the open reading frame, immediately prior to, and in frame with, the nucleotide sequence for the mature peptide. The sequence of the reverse primer resulted in the TAG translation stop codon being identical to that in the published A. niger PhyA-1 gene.

WO 01/22806 PCT/AUOO/01183 -48- The sequence of the PCR primers (which also contained cloning sites for EcoRI and Clal, respectively; underlined lower case) to obtain a modified version of the gene that is functional in plants were as follows: Forward(PHYF2): cgcgaattcATGCTGGCAGTCCCCGCCTCG (SEQ ID NO: 13); and Reverse(PHYR3): ggcatcgatCTAAGCAAAACACTCCGC (SEQ ID NO: 14) The nucleotide sequence of the modified PhyA-1 gene is set forth in SEQ ID NO: 3, and the derived amino acid sequence encoded by this PhyA-2 gene is set forth in SEQ ID NO: 4.

III. The Carrot extensin secretory signal sequence.

Nucleotide sequences encoding the secretory signal sequence of the carrot extensin gene was amplified using PCR, from plasmid pSEGON, obtained from D. Llewellyn, Commonwealth Scientific and Industrial Research Organisation, Canberra, Australia, which plasmid contains nucleotide sequences encoding the extensin signal peptide upstream of a glucose oxidase-encoding gene.

The PCR primers used to amplify the extensin secretory signal sequence were as follows, wherein cloning sites in the primers are underlined and in lower case: Forward: gcgtctagagaattcATGGGAAGAATTGCTAG (SEQ ID NO: 15); and Reverse: cgcggatccgcggccgcAGCTGTGGTTTCGGAAGC (SEQ ID NO: 16).

The amplified product was first subcloned as a XbaVBamHI fragment into plasmid pBSIIKS (Stratagene, Palo Alto, Califomia, USA). The amplified fragment was 99 bp in length and codes for 33 amino acids. The sequence of the leader is set forth in SEQ ID NO: IV. ExtensinlPhytase Gene Fusion WO 01/22806 PCT/AU00/01183 -49- In-frame" fusions between the extensin secretory signal-encoding sequence and both the PhyA-1 and PhyA-2 genes were generated by ligation between the Notl site located at the 3' end of the extension secretory signal-encoding sequence and the EcoRI site at the 5' end of the PhyA-1 and PhyA-2 genes, respectively. This was achieved by generating blunt flushed) ends of the restriction enzymes sites prior to ligation.

The junction sequence generated from the extensin/phytase fusion comprised the nucleotide sequence 5'-ACGCTGCCATGCTGGCA-3' (SEQ ID NO: 17), encoding the junction amino acid sequence Thr-Ala-Ala-Met-Leu-Ala (SEQ ID NO: 18), which amino acid sequence comprises two codons derived from extensin, an inserted alanine (encoded by the Noti-EcoRI fusion), and three codons derived from the phytase genes (PhyA-1 or PhyA-2 as appropriate).

Without being bound by any theory or mode of action, the Alanine residue at the extensin::phytase junction provides a suitable site for protease cleavage of the precursor polypeptide to remove the extensin secretory signal sequence.

The extensin/phytase in-frame fusions were generated and verified in plasmid pBSIIKS.

The nucleotide sequence of the extensin::PhyA-2 chimeric gene is set forth in SEQ ID NO: 9, and the amino acid sequence encoded therefor is set forth in SEQ ID NO: The nucleotide sequence of the extensin::PhyA-1 chimeric gene is set forth in SEQ ID NO: 11, and the amino acid sequence encoded therefor is set fourth in SEQ ID NO: 12.

V. Construction of vectors for plant transformation Four gene expression cassettes were produced, comprising the PhyA-1 gene, chimeric extensin::PhyA-1 gene, PhyA-2 gene, and chimeric extensin::PhyA-2 gene, by subcloning these genes, as EcoRI-Kpnl fragments, from their plasmid pBSIIKS WO 01/22806 PCT/AU00/01183 vector (see above) into plasmid pART7 (Figure which contains a CaMV promoter and ocs terminator.

Accordingly, in the resultant plasmid constructs, the phytase genes were placed in operable connection with the CaMV 35S promoter sequence, to produce the plasmids: 1. 35S+PhyA-1+ocs; 2. 35S+extensin::PhyA-1 +ocs; 3. 35S+PhyA-2+ocs; and 4. 35S+extensin::PhyA-2+ocs.

The four phytase transgenes were then subcloned as Nofl fragments into the binary vector pBS389 (Figure This vector is suitable for plant transformation and contains the selectable marker nptll under control of the SCSV Sc promoter and Sc3 terminator sequences. The phytase transgenes were cloned such that their orientation (determining the direction of transcription) was the same as the orientation as the selectable marker.

WO 01/22806 PCT/AU00/01183 -51 VI. Plant transformation The pBS389 vectors containing the various phytase gene constructs were transferred to Agrobactenum tumefaciens strain AGL1 using standard, tri-parental mating techniques. These strains were used to transform tobacco, A. thaliana and subterranean clover using published protocols. Transformed plants of all three species were generated and have been verified to contain the various phytase transgenes by selection on Kanamycin, PCR and by Southern blot analysis using the PhyA-1 gene as a hybridisation probe.

Figure 12 shows a representative Southern blot hybridisation to the PhyA-1 nucleotide sequence under high stringency conditions, of DNAs derived from several lines of A. thaliana (C24 ecotype) plants generated using plasmid pBS389, or the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence), or plasmid pAER04 (Figure or plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); or plasmid pAER02 (Figure Data indicate the presence of the PhyA-1-hybridising signal only in plants produced using the PhyA-1-containing or PhyA-2-containing gene constructs.

VII. Utilization of organic phosphorus substrates by non-transformed plants When grown under sterile conditions, wild-type non-transformed) A. thaliana plants were able to access phosphorus from a range of organic phosphorus substrates, and their growth and phosphorus nutrition were comparable to that for plants supplied with inorganic phosphate. By contrast, wild-type plants were unable to acquire P from phytate. When supplied with phytate, a lower shoot dry weight was observed, and the total phosphorus content of the shoots was only 9.3% of the total phosphorus content of plants supplied with inorganic phosphate as Na 2

HPO

4 and not significantly different (P<0.05) from plants grown in the absence of external phosphorus.

Similarly, shoot phosphorus concentrations of non-transformed plants supplied with phytate, or alternatively, grown without an external phosphorus source were WO 01/22806 PCT/AU00/01183 -52significantly lower and these plants exhibited markedly lower shoot to root ratios than plants grown on inorganic phosphate as Na 2

HPO

4 In general, no major differences in root dry weights were observed across the various phosphorus treatments, although lower root mass was evident for plants grown in the absence of phosphorus.

The observed phenotype of plants grown on phytate are indicative of a phosphorus deficiency.

Total extracellular acid phosphomonoesterase activities of between 13.6 and 25.5 mU mg-" dry weight were measured for roots of A. thaliana plants, with no significant differences (P<0.05) occurring between phosphorus-deficient plant grown on phytate or in the absence of phosphorus, and phosphorus-sufficient plants grown on inorganic phosphate as Na 2

HPO

4 Similarly, no differences were observed in total acid phosphomonoesterase in crude extracts prepared from whole roots. In these extracts, phytase activity constituted less than 1% of the total phosphomonoesterase activity, while in separate experiments we were unable to detect extracellular phytase activity.

VIII. Analysis of transformed plants A) Production of phytase-encoding mRNA Northem blot hybridisations have been conducted to confirm ectopic expression of the introduced phytase genes transgenic tobacco, A. thaliana and subterranean clover plants.

Figure 11 shows a representative northern blot hybridisation to the PhyA-1 nucleotide sequence, of several lines of transgenic A. thaliana (C24 ecotype) plants carrying the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence), or plasmid pAER04 (Figure or plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); or plasmid pAER02 (Figure Data WO 01/22806 PCT/AU00/01183 -53obtained indicated that, in most cases, highest levels of phytase-encoding mRNA were present in plants that contain the phytase genes (PhyA-1 or PhyA-2) fused to the extensin secretory signal-encoding nucleotide sequence, suggesting that these sequences may also increase mRNA stability.

Figure 13 shows a northern blot experiment showing ectopic expression of phytase genes in five independent lines of transgenic Trifolium subterraneum (subterranean clover, cultivar Dalkeith; To generation), containing plasmid pAERO2 (Figure Data indicate a positive correlation between copy number of the introduced transgene and the level of expression of the chimeric extensin::PhyA-2 transgene.

b) Phytase enzyme activity Transformed tobacco plants Phytase enzyme assays were performed using leaf material derived from transformed tobacco plants.

The phytase activity in leaf extracts of control plants was 0.24 nkat phytase g fresh wt (0,04 nkat phytase mg protein), compared to 30.4 nkat g fresh wt (6.2 nkat phytase mg protein) for transformed plants containing the extensin::PhyA-1 gene construct, representing a 130-fold increase in enzyme activity. Similar results are obtained for phytase assays of roots.

(ii) Transformed subterranean clover plants Phytase enzyme activity increased in the leaves and roots of transgenic subterranean clover that contain the introduced phytase gene constructs. In particular, the phytase activity in the shoots of primary transformants of subterranean clover generated from tissue culture explant material transformed with plasmid pAER02 was approximately 24-fold that observed in the shoots of control plants transformed with the vector pBS389 (Table In contrast, the phytase activity of transformed plants carrying the PhyA-1 gene without the extensin signal peptide, WO 01/22806 PCT/AUOO/01183 -54was not significantly different from that of the control plants transformed with pBS 3 8 9 TABLE 3 Phytase activity of transgenic subterranean clover plants: TO generation Construct Number of plants Phytase activity (mU phytase fresh wt) mean range control* 3 100.1 35.5 190.5 PhyA-1 9 76.8 38.7 96.9 ext::PhyA-1 20 2443.5 413.3 -4346.0 Control plants were transformed with the binary vector pBS389. The phytase containing lines were transformed with derivatives of pBS389 containing PhyA-1 either without (PhyA-1) or with (ext::PhyA-1) the extracellular-targeting sequence from the carrot extensin gene.

The high phytase activity of the TO plant lines expressing the extensin::PhyA-1 fusion polypeptide was transmissable to the T1 generation, and, for the line designated "iv" there was a 3:1 segregation ratio of high phytase:low phytase, consistent with a single gene insertion in the primary transformant (Table For the line designated there was a 15:1 segregation ratio of high phytase:low phytase, consistent with a double gene insertion in the primary transformant giving rise to that line(Table 4).

The number of insertions of the ex::phyA gene was confirmed by Southern blot analyses.

WO 01/22806 PCT/AU00/01183 TABLE 4 Phytase activity of transgenic subterranean clover plants: T1 population LINE IV LINE I plant phytase activity plant phytase activity number (mU phytase g-1 FW) number (mU phytase g- 1

FW)

1 34.3 1 1220.9 2 1253.2 2 1840.5 3 110.9 3 1517.6 4 544.9 4 1995.9 1327.9 5 1247.2 6 165.5 6 837.5 7 813.3 7 1279.5 8 998.9 8 994.9 9 990.9 9 991.0 1545.9 10 577.2 11 238.2 11 861.7 12 94.8 12 1961.6 13 879.9 13 974.7 14 1247.2 14 226.1 161.5 15 2036.2 16 1693.2 16 244.2 17 938.4 17 1461.1 18 1495.4 18 750.7 19 871.8 19 379.4 1245.2 20 1428.8 control-1i 106.9 control-1 197.7 control-2 50.5 control-2 60.5 Control plants were transformed with the binary vector pBS389. The phytase containing lines were transformed with PhyA-1 containing the extracellular-targeting sequence (ext::phyA-1) from the carrot extensin gene.

WO 01/22806 PCT/AU00/01183 -56- (iii) Transformed A. thaliana plants We determined the phytase enzyme activity in root extracts for two independently (a and b) transformed lines of A. thaliana (C24 ecotype) for each transgene referred to herein above. The plants were grown for 36 days on sterile agar (~120 plants per replicate sample) containing 0.8 mM inorganic phosphate as For P determination, the plants were supplied with an equivalent concentration of P as phytate and were grown for 42 days (3 plants tube 1 Figure 3a-e).

High levels of phytase activity were observed in soluble root extracts prepared from transgenic lines of A. thaliana that ectopically-expressed phytase from either the chimeric ext::PhyA-1 or ext::PhyA-2 genes (Table Relative to control plants, the phytase activity of roots from lines generated by transformation with the ex::PhyA-2 and ex::PhyA-1 chimeric genes were increased by approximately 20- to 25-fold, and by up to 1500-fold, respectively.

In contrast, no difference in phytase activitity was observed between control plants, and transformed plants produced by transformation with the PhyA-1 and PhyA-2 genes without the signal transport peptide-encoding gene sequence attached; Table Notwithstanding the absence of a detectable difference in phytase enzyme activity, we could confirm the expression of mRNA encoding phytase in Northern blot experiments (Figure 11).

WO 01/22806 PCT/AU00/01183 -57- TABLE Phytase activity and P nutrition of transgenic A. thaliana plants Root phytase Total shoot Shoot phosphorus Transgenic line* (mU mg' protein) P content t (pg mg-' dry wt) (pg P) Control* a 11.4 a§ 6.3 2.6 a b 9.3 a 7.3a 1.9 PhyA-2 a 3.9 a 6.7 a 2.4 a b 6.6 6.9 a 2.3 ext::phyA-2 a 224.2b 9 7 .4 b 11.2 b b 432.6 b 80.7 b 10.2 b PhyA-1 a 14.3 a 6.4 a 2.0 0 b 11.7 a 5.9 a 3.0 a ext::phyA-1 a 992.5 C 99.1 b 10.4 b b 14, 9 8 0 .4 d 81.9 11.7 b Control plants were transformed with pBS389.

tAcross all transgenic lines, the mean phosphorus content of shoots from plants that were grown under identical conditions, but were supplied with inorganic phosphate or alternatively, that were grown with no added phosphorus, was 79.9 28.7 and 1.1 0.6 pg, respectively.

:Across all transgenic lines, the mean shoot phosphorus content for control plants supplied with P1, or alternatively, were grown without added phosphorus (as above), were 10.1± 1.0 and 1.5 0.6 pg mg- 1 dry weight, respectively.

Within each column, values followed by different superscripts are significantly different Phytase activities were log o-transformed prior to analysis.

Seedlings of the selected lines were grown in sterile nutrient solution and phytase activity was determined between days 10 and 12 of growth. Extracellular phytase activity was only detected in transgenic A. thaliana that contained gene constructs carrying the leader sequence. Under the assay conditions used, we determined that about 10% of the total soluble root phytase activity in these lines was secreted per day as an extracellular enzyme into the growth medium (Table In contrast, WO 01/22806 PCT/AU00/01183 -58measurable phytase activity was not secreted from roots of the control plants carrying plasmid pBS389 alone, or from the roots of plants that expressed phytase without the leader sequence attached (Table Accordingly, significant zones of phytate depletion were only observed around the roots of those transgenic lines contianing the chimeric ext::PhyA-1 or ext::PhyA-2 genes (Figure TABLE 6 Extracellular phytase activity of transgenic A. thaliana (C24 ecotype) plants.

Secreted phytase activity (mU) Transgenic Activity per Activity per Activity plant line plant per day gram f. wt per ug per day protein Control a 0.

0 1 t 1.5 0.1 PhyA-2 a 0.01 1.5 0.1 Ext::PhyA-2 a 0.19 0.04 26.0 4.8 4.5 0.9 PhyA-1 b 0 1 1.5 0.1 79.1 5.1 ext::PhyA-1 b 10.07 1.04 1538.0 171.5 0 The control line was transformed with pBS389.

t Each observation is the mean of 4 replicates and is shown 1 standard deviations. Where shown with the value was below the indicated limit for detection.

WO 01/22806 PCT/AUOO/01183 -59c) Plant Growth on phytate-containing media The growth responses of transgenic plants in media supplemented with phytate were determined for transformed A. thaliana plants.

We compared the growth of transformed A. thaliana plants in media containing either no added phosphate, or using 0.8 mM Na 2

HPO

4 or phytate (equivalent to 0.8 mM phosphate), as a source of phosphorus. Whilst plants transformed with the pBS389 control plasmid, or expressing the PhyA-2 gene without the extensin leader sequence, could only grow efficiently in the presence of 0.8 mM Na 2

HPO

4 plants transformed with the gene construct pAER04 (Figure 6) and expressing the extensin::PhyA-2 fusion protein grew efficiently on phytate as well soluble phosphate as a source of phosphorus. Accordingly, data shown in Figure 7 show that plants ectopically-expressing the extensin-phytase fusion polypeptide grow more rapidly on media containing phytate as a sole source of phosphorus.

We also compared the enhancement of growth, using phytate as a sole source of phosphorus, that was obtainable using the PhyA-1 or PhyA-2 genes. As shown in Figure 8, transformed A. thaliana plants carrying either the PhyA-1 or PhyA-2 genes fused to a nucleotide sequence encoding the carrot extensin leader sequence grew efficiently in media having phytate as the sole source of phosphorus (see panels C, E of Figure In contrast, plants expressing the phytase genes without the leader sequence (see panels B, D of Figure 8) attached grew poorly on phytate, as did plants not expressing any phytase gene (panel A of Figure Accordingly, both modified phytase genes work equally-well to improve the phosphorus nutrition of plants, however significantly more plant growth was obtained for plants expressing the extensin-phytase fusion polypeptide compared to plants expressing phytase but unable to target the phytase to the extracellular space.

To test the requirement for the extensin leader sequence, we also compared the growth of plants expressing the extensin::PhyA-1 fusion polypeptide on media lacking added phosphorus (Figure 9, panel to the growth of plants expressing the WO 01/22806 PCT/AUOO/01183 PhyA-1 polypeptide without the extensin leader sequence on phytate (Figure 9, panel or plants expressing the PhyA-2 polypeptide on phytate (Figure 9, panel or expressing the extensin::PhyA-2 fusion polypeptide on phytate (Figure 9, panel After 40 days growth, at a density of about thirty plants per agar plate, significantly enhanced growth was observed on phytate, even for lines wherein the introduced phytase-encoding gene lacked nucleotide sequences encoding the carrot extensin leader sequence, compared to plants grown in the absence of a phosphorus source. However, optimum growth was clearly observed where the extensin leader sequence was present.

To demonstrate that the enhanced growth on phytate is due to enhanced utilisation of phytate, we also stained media plates with 0.03% FeCI 3 to determine regions where phytate was absent from the medium after 22 days of growth. As shown in Figure 10, staining was observed along the margins of the roots in Panel C, showing the absence of phytate from the medium, which phytate has been utilised by plants expressing the extensin::PhyA-1 fusion protein and grown on phytate, but not soluble phosphorus (0.8 mM Na 2

HPO

4 Accordingly, the enhanced nutrition on phytate-containing media is correlated with enhanced phytate utilisation by the transgenic plants.

EXAMPLE 2 Phytase activities of plant roots and localisation of activity The phytase activities in the roots of a wide range of agriculturally important legume and grass species, including subterranean clover, burr medic, white clover, lucere, tobacco, A. thaliana, wheat, phalaris, ryegrass and danthonia, have been determined.

In summary, phytase activities in extracts prepared from roots of these species ranged between 0.1 and 1.7 nkat g -1 root fresh wt (equivalent to 6.0 mU g root fresh wt and 102.0 mU g -1 root fresh wt respectively), or alternatively, 0.2 to nkat mg -1 total protein (equivalent to 12.0 mU mg protein and 90.0 mU mg WO 01/22806 PCT/AU00/01183 -61protein, respectively), with levels of activity increased by up to 3.3-fold (9.8-fold on a total root protein basis) when seedlings were grown in conditions of phosphorus deficiency.

In contrast, acid phosphatase activity measured in the same extracts ranged between 20 and 60 nkat g -1 root fresh wt (equivalent to 1200 mU g root fresh wt and 3600 mU g root fresh wt, respectively). Phytase activity was a small component only (less than 5% for the range of species investigated) of the total acid phosphatase activity of plant roots, irrespective of the level of phosphorus nutrition.

The extracellular component of root phytase activity is a minor proportion of the total phytase activity measurable in the roots of naturally-occurring plants. For example, less than 0.042 nkat phytase activity g root fresh wt less than 4.7% of the activity measured in root extracts) could be eluted from roots of subterranean clover.

The estimated extracellular root phytase activity of intact roots of subterranean clover seedlings is as low as 0.03 nkat g root fresh wt 3% or less of the activity measured in soluble root extracts).

However, supplementation of subterranean clover roots with phytase, at a rate that is equivalent to 0.13 nkat g root fresh wt, resulted in a significant enhancement of the ability of the plants to acquire phosphorus from phytate.

EXAMPLE 3 Effect of organic acids on the phytase-labile component of soil phosphorus We have also demonstrated that the efficacy of organic acids in improving the extractability of phosphorus from the soil, and to show that a significant component of the organic phosphorus in soil actually extracted by citrate is amenable to dephosphorylation by phytase.

Phytase-labile organic phosphorus was determined in various extracts prepared from two Australian pasture soils with contrasting fertiliser histories.

WO 01/22806 PCT/AUOO/01183 -62- Materials and Methods Soils Soil samples were collected to a depth of 10 cm, under permanent pastures (containing perennial grass and annual legume components) located at two sites: (i) Rutherglen Research Institute, Victoria; and (ii) Ginninderra Experiment Station, Canberra, ACT. Individual cores (2.5 cm diameter) of soil (-30 samples) from each treatment at each site were bulked as a composite sample which was air-dried, passed through a 2 mm sieve to remove large, particulate matter and stored at room temperature.

Samples from the Rutherglen site were collected in 1993 from treatments of a nonreplicated fertiliser trial, the details for which are published in Ridley et al. (1990).

Briefly, the trial was established in 1914 on three 1.5 ha fields. Two fields (FR and F+LR) received approximately 125 kg ha of single superphosphate (9% phosphorus) each alternate year from 1914 to 1986, while the third field (UR) was unfertilised. One of the fertilised fields (F+LR) was also top-dressed with lime from 1914 to 1948, receiving nine applications of 1.25 t ha 1 Samples from Ginninderra Experiment Station (Wallaroo 3 Paddock) were collected in October, 1996. Three phosphate fertiliser treatments were imposed across the trial on its establishment in 1994. Plots were either unfertilised (UG) or received three autumn applications of triple superphosphate (20.7% phosphate), totalling 416 kg ha- 1 (F1G) or 675 kg ha 1 (F2G).

Properties of the two soils are presented in Table 7. Total inorganic phosphorus and organic phosphorus were determined by the ignition-extraction (0.5 M H2SO 4 procedure (Olsen and Sommers 1982) and Colwell phosphorus by a 16 h extraction with 0.5 M NaHCO 3 (Colwell 1963), followed by determination of inorganic phosphorus using the molybdate-blue colour reaction (Murphy and Riley 1962).

Organic carbon was determined by the Modified Mebius procedure (Nelson and Sommers 1982) and soil pH was measured in CaCl 2 WO 01/22806 PCT/AU00/01183 -63- Substrate specificities of phytase preparations The substrate specificities of commercial preparations of wheat germ acid phosphatase (EC 3.1.3.2; Sigma Chemical Aspergillus niger phytase (EC 3.1.3.8; Sigma) and a purified preparation of the A. niger NRRL 3135 phytase (kindly provided by Dr Markus Wyss; F. Hoffmann-La Roche, Switzerland) were determined using a range of organic phosphorus compounds. The specific activities of the three enzyme preparations were tested at 27 OC, in 50 mM MES buffer (pH 5.5) containing 1 mM EDTA, against the following substrates: myo-inositol hexaphosphoric acid (dodecasodium salt; IHP), a-D-glucose 1-phosphate (disodium salt; G1P), ribonucleic acid (type VI; RNA), adenosine-5'-triphosphate (ATP), D(-)3-phosphoglyceric acid (trisodium salt; PGA), p-nitrophenyl phosphate (disodium salt; pNPP), and bis(pnitrophenyl) phosphate (sodium salt; bis-pNPP). With the exception of ATP (Boehringer Mannheim), all substrates were obtained from Sigma Chemical Co.

Assays were performed in 1 ml volumes, using 50 pg mr 1 acid phosphatase; 1.14 pg ml" commercial phytase; or 0.45 pg ml 1 purified phytase. These amounts of enzyme were chosen on the basis of the reported specific activities of the preparations, against either IHP or pNPP. Enzyme assays were conducted over 30 min at a range of substrate concentrations between 2.4 mM and 4.8 mM phosphorus, with three replicates. Activities were measured against p-nitrophenol standards (Bessey et al.

1946) for the pNPP and bis-pNPP samples, and for all other samples by the release of inorganic phosphorus as determined using the malachite-green reaction (Irving and McLaughlin 1990).

Table 7 Properties of two pasture soils (0 to 10 cm depth) from Ginninderra and Rutherglen Site Soil Type Fertiliser treatmenta phosphorus (mg kg' soil; pH Organic C Colwell ignition/extraction method) phosphorus (Stace et al. Total Inorganic Organic (CaCI 2 (mg kg'' soil) 1968) phosphorus phosphorus phosphorus Ginninderra Yellow podzolic UG (Unfertilised) 203 53 150 4.67 1.88 11.3 Experiment F1G (86 kg P 242 79 163 4.57 1.91 25.0 Station F2G (140 kg P ha 254 97 157 4.57 1.95 43.7 Rutherglen Grey-brown to UR (Unfertilised) 154 30 123 4.60 1.99 7.8 Research yellow podzolics FR (-450 kg P ha 312 113 199 4.21 3.07 38.8 Institute F+LR (-450 kg P ha' 287 95 192 4.55 2.57 26.4 'and 11.25 tonnes lime ha"') "Total amount of fertiliser applied since Inception of trial WO 01/22806 PCT/AU00/01183 Measures of enzyme-labile soil Soil extraction and total, organic and inorganic phosphorus determinations Amounts of air-dried soil of between 2 and 10 g were extracted in 50 ml polypropylene tubes using two volumes of sterile extractant solution, usually deionised water, 50 mM citric acid (pH or 0.5 M Na-bicarbonate (pH Other extractants included 0.025 and 0.1 M HCI, and 0.01 M CaCI 2 (pH The soils were extracted at 22 2 OC for 30 min on a reciprocal shaker (300 rpm), followed by centrifugation (10, 300 g) for 15 min. Soil extracts were decanted from the pelleted material and stored at 4 oC prior to enzyme analyses.

The phosphorus contents of soil extracts were determined on 1 ml sub-samples of each solution. Inorganic phosphorus was determined by measuring the inorganic phosphorus content of solutions with malachite-green reagent (Irving and McLaughlin 1990). In order to determine total phosphorus, the samples were autoclaved (120 kPa/ 121 OC; 40 min) in the presence of 0.6 M H 2 S0 4 and 3.3% ammonium persulphate (Schoenau and Huang 1991), and were similarly analysed for inorganic phosphorus content. Corrections for volume loss during autoclaving were made as necessary, based on gravimetric analyses. Organic phosphorus in the extracts was calculated by deduction of inorganic from total phosphorus.

Incubation of soil extracts with enzyme Standard assays involved incubation of 1 ml of soil extract in the presence of excess enzyme: either 0.25 nkat 0.50 nkat g-1 soil) of commercial phytase, as determined against IHP substrate for the incubation conditions specified herein, or 1.14 nkat (2.28 nkat g-1 soil) of purified phytase. These amounts of enzyme equated to 1.56 (3.12 nkat g' 1 soil) and 0.03 nkat (0.05 nkat g' 1 soil) of acid phosphatase activity as determined against pNPP, for the commercial and purified preparations, respectively. Samples were adjusted to pH -5.5 with either diluted HCI or NaOH, 300 pl of 250 mM MES buffer (pH 5.5; containing 5 mM EDTA) was added, and the solutions were made up to a final volume of 1.5 ml with deionised WO 01/22806 PCT/AU00/01183 -66water. Immediately on addition of the enzyme to each reaction, an aliquot was removed and mixed with a one-fifth volume of 25% trichloro-acetic acid (TCA).

Remaining solutions were incubated at 27 OC for 6 h, after which TCA was added to terminate the reaction. The TCA-treated samples were centrifuged in an Eppendorf microfuge (12,000 rpm, 10 min) prior to analysis for inorganic phosphorus using malachite-green reagent. Control treatments were routinely included, whereby either soil extract or enzyme were omitted.

Statistics Enzyme assays and other soil measures were replicated three times and the data were analysed to investigate variation associated with soil extraction and incubation procedures, using one- and two-way analyses of variance (ANOVAs). Where Fratios were significant treatment means were compared by least significant difference (LSD).

Results Substrate specificities and specific activities of phytase preparations The specific activities of the three enzymes for IHP ranged from 1.0 nkat mg 1 protein (wheat bran acid phosphatase) to 560.9 nkat mg 1 (purified A. niger phytase; Table The purified A. niger phytase preparation had a 12-fold higher specific activity for IHP than the commercial phytase. Moreover, purified phytase showed a narrow substrate specificity, with specific activities for a range of organic phosphorus substrates that were 10% or less than the specific activity for IHP. The commercial A. niger phytase preparation was less substrate-specific, with highest activity observed against pNPP. The activity profile of the commercial phytase preparation was similar to that of wheat bran acid phosphatase.

It is likely that the commercial Aspergillus phytase preparation contained a considerable amount of phosphatase activity that was not specific for phytate. This was removed from the more purified preparation. The wheat bran acid WO 01/22806 PCT/AUOO/01183 -67phosphatase was not used in subsequent experiments because of its similarity to commercial phytase.

TABLE 8 Substrate specificities of various phytase enzyme preparations Specific Activity (nkat mg-1 protein) Substrate Acid Sigma phytase Purified phosphatase phytase myo-inositol 1.0 46.9 560.9 hexakisphosphate Glucose 1-phosphate 0.4 84.8 0.1 Ribonucleic acid 0.4 38.5 0.3 Adenosine triphosphate 4.2 55.2 59.4 Phosphoglyceric acid 3.6 78.7 54.5 p-nitrophenyl phosphate 7.0 293.0 13.1 bis(p-nitrophenyl) 0.2 11.5 35.5 phosphate LSD (P=0.05) 0.3 14.5 51.7 Inorganic and organic phosphorus contents of extracts from unfertilised Ginninderra soil Unfertilised soil from Ginninderra Experiment Station (UG) was selected initially for phosphorus measurements because of its low phosphorus status (Table There was a requirement for soil extracts to contain sufficient levels of organic phosphorus, so that any change upon incubation with enzyme would be detectable.

Various extracts of field-moist UG soil were prepared, in which recoverable organic phosphorus was determined. Water, 0.01 M CaCI 2 0.025 M HCI and 0.1 M HCI extracted less than 1.0 pg organic phosphorus g-1 soil. By contrast, up to 22.5 pg phosphorus g' soil was extracted with citric acid and 0.5 M Na-bicarbonate. Na- WO 01/22806 PCT/AUOO/01183 -68bicarbonate, citric acid and water were thus selected as extractants for further analysis. A solution-to-soil ratio of 2:1 extracted higher concentrations of organic phosphorus (as compared to a 5:1 ratio) and was adopted for the subsequent incubation experiments.

Enzyme-labile phosphorus in extracts from fertilised Rutherglen soil.

Fertilised Rutherglen soil (FR) was used to determine the appropriate conditions to be employed for enzyme incubation studies with all soil-extractant combinations.

The hydrolysis of organic phosphorus from extracts of soil FR was measured for up to 8 h in the presence of three concentrations of commercial or purified phytase, or in the absence of enzyme.

The release of inorganic phosphorus from incubated citric acid soil extracts is shown in Figure 14. Additions of 0.50 nkat commercial phytase or 2.28 nkat purified phytase g-1 soil were required for reactions to approach completion within 8 h. With lower amounts of enzyme, reactions proceeded more slowly and did not reach completion during the incubation period. For all subsequent measures of labile phosphorus, including various soils containing lower organic phosphorus concentrations, soil extracts were incubated for 6 h in the presence of 0.50 or 2.28 nkat phytase g' 1 soil, using commercial or purified preparations, respectively.

To determine the contribution of soil microbe-derived enzyme activities to the observed rates of hydrolysis in amended soil extracts, duplicate samples were passed through 0.45 pm filters (Millex-HA, Millipore). There were no differences in either the rate or total quantity of inorganic phosphorus liberated from citric acid, water or Na-bicarbonate extracts of soil FR. between filtered and unfiltered samples (data not shown). Therefore, over the 6 h incubation period, microbial enzyme activity did not contribute to the observed rates of hydrolysis. A low, and insignificant, level of hydrolysis was measured when no enzyme was added to the extracts (Figure 14).

WO 01/22806 PCT/AU00/01183 -69- Effects of citric acid concentration and pH on phytase-labile phosphorus from fertilised Rutherglen soil Citric acid concentration Extractable organic phosphorus from soil FR increased significantly with increasing concentrations of citric acid to 50 mM (P<0.05; Figure 15). The amounts of organic phosphorus that were hydrolysed by the commercial and purified phytases, and the proportion of the total organic phosphorus which was enzyme-labile, also increased with citric acid concentration. For the commercial phytase, labile phosphorus represented 13.5% and 82% of the organic phosphorus in water and 50 mM citric acid soil extracts, respectively. Purified phytase-labile organic phosphorus was approximately half of that hydrolysed by the commercial enzyme preparation, representing between 1.8% and 44.7% of the total organic phosphorus across the same range of citric acid concentrations.

Effect of pH of citric acid Soil extractions were prepared using 50 mM citric acid solutions adjusted to between pH 2.3 and pH 6.0. Extractable organic phosphorus increased with pH, from 7.5 phosphrous g- 1 soil at pH 2.3 to 25.3 pg phosphorus g-1 soil at pH (Figure 16). However, enzyme-labile phosphorus was similar across the entire pH range, with -7.8 pg phosphorus g' soil and -4.5 pg phosphorus g-1 soil hydrolysed by commercial phytase and purified phytase, respectively (Figure 16).

Comparison between citric and hydrochloric acids as extractants Total organic phosphorus, and commercial and purified phytase-labile organic phosphorus in water, citric acid and hydrochloric acid extracts from soil FR are presented in Table 9.

WO 01/22806 PCT/AUOO/01183 70 TABLE 9 Extractable organic phosphorus and enzyme-labile phosphorus (pg g-1 soil) using two sources of phytase from A. niger, in water, citric acid and HCI extracts of the fertilised Rutherglen soil (FR).

n.d. not detectable.

Organic Enzyme-labile Po (pg g'1 soil)

P

Extractant (pg g-1 Commercial phytase Purified phytase soil) of Po) of Po) Water 2.10 0.26 (12.4) 0.07 (3.3) mM citric acid 7.65 6.15 (80.4) 3.35 (43.8) (pH 2.3) mM HCI (pH 1.01 0.09 n.d.

1.45) mM HCI, pH 2.3 2.09 0.17 0.07 (3.3) LSD (P=0.05) 0.54 0.30 0.57 Two HCI extractants were used: one at the same molar concentration as citric acid mM) and the other at the same pH (pH 2.3, at -5 mM). When used at an equivalent molar concentration, HCI extracted only 13% of the quantity of organic phosphorus extracted with citric acid, and the enzyme-labile phosphorus component was negligible relative to that of citric acid extracts. When the concentration of HCI was adjusted for a solution of pH 2.3, the amount of extractable organic phosphorus was increased, but was still only 27% of that extracted by citric acid. Likewise, the enzyme-labile organic phosphorus component was less than 3% compared to citric acid. While commercial and purified phytase-labile organic phosphorus in 50 mM citric acid extracts represented 80% and 44% of the extractable organic WO 01/22806 PCT/AU00/01183 -71 phosphorus, respectively, less than 13% and 4% of organic phosphorus in the other extracts were hydrolysed by either of the enzyme preparations (Table 9).

Extractable and phytase-labile organic phosphorus from soils with different fertiliser histories The total and organic phosphorus contents of water, 50 mM citric acid (pH and 0.5 M Na-bicarbonate (pH 8.5) extracts of soil from phosphorus fertiliser trials at two sites are illustrated in Figure 17, along with the fractions of extractable organic phosphorus that were hydrolysed by commercial and purified phytase during separate incubations.

While Na-bicarbonate extracted more organic phosphorus than the other solutions (between 12 and 30 pg g- 1 soil; Figure 17, panels e, citric acid extracted the most enzyme-labile organic phosphorus (up to 5.7 pg phosphorus soil; Figure 17c, d), with between 56% and 79% of the organic phosphorus in citric acid extracts being hydrolysed by the commercial phytase preparation. In contrast, only 7 to 17% and 2 to 9% of the organic phosphorus in water and Na-bicarbonate extracts, respectively, was hydrolysed by the commercial phytase (Figure 17a, b; Figure 17e, A smaller component of the organic phosphorus was hydrolysed by purified phytase. In citric acid extracts, 28 to 40% of the organic phosphorus was purified phytase-labile (Figure 17c, while only 3 to 8% and 1 to 2% of the organic phosphorus in water and Na-bicarbonate extracts, respectively, was hydrolysed (Figure 17a, b; Figure 17e, I).

Organic, inorganic and enzyme-labile phosphorus extracted by citric acid was significantly higher (P<0.05) in the fertilised Rutherglen soils (FR and F+LR) than the unfertilised soil (UR; Figure 17c). In addition, both total extractable organic phosphorus and the component which was phytase-labile, were greater for the fertilised soil (FR) than soil which had received both fertiliser and lime While organic and inorganic phosphorus extracted by Na-bicarbonate were significantly greater (P<0.05) for both fertilised Rutherglen soils, only the treatment which had WO 01/22806 PCT/AUOO/01183 -72received fertiliser alone contained a higher level of extractable, commercial phytaselabile phosphorus than the unfertilised control (Figure 17e). No differences in fertiliser treatment were evident for purified phytase-labile phosphorus.

For the soil from Ginninderra, which had received only recent applications of phosphorus fertiliser, there were no differences in either extractable organic phosphorus or enzyme-labile phosphorus across fertiliser treatments (Figure 17).

However, the extractable inorganic phosphorus component increased significantly (P<0.05) with phosphorus fertility, in both citric acid and Na-bicarbonate extracts (Figure 17d, f).

Discussion Characterising phytase-labile organic phosphorus in extracts of soil Two A. niger phytase preparations with markedly different substrate specificities were used to measure the amounts of enzyme-labile organic phosphorus present in extracts of soil. A commercial preparation of A. niger phytase (from Sigma Chemical Co.) showed activity against a range of organic phosphorus substrates, with highest activity for pNPP. In accordance with previous reports (Wyss et al.

1999), a purified form of the A. niger phytase was highly specific for phytate. The contrasting substrate specificities of the two phytase preparations were exploited to characterise the enzyme lability of organic phosphorus extracted from soils.

Material hydrolysed by the purified phytase was considered likely to be indicative of the phytate content of the extracts, whereas hydrolysis by the commercial "phytase" preparation reflected a more general acid phosphatase activity. However, it should be noted that the activities of the two preparations were not entirely mutually exclusive, as shown by their activities against a range of organic phosphorus substrates (Table 8).

In water extracts from soils collected from Ginninderra Experiment Station and Rutherglen Research Institute, less than 8% of the organic phosphorus present was hydrolysed by the purified phytase and only 7 to 17% was hydrolysed by the WO 01/22806 PCT/AU00/01183 -73commercial preparation (Figure 17). Previous measures of enzyme-labile organic phosphorus in water extracts of soils have not been made using phytase with a narrow specificity for IHP.

The present work suggests that low levels of phytate occur in soil solution (less than 0.22 pg phosphorus g- soil), and also that organic phosphorus esters accessible to a more general phosphatase (ie. commercial phytase) activity are present in only small quantities. In contrast, higher amounts of organic phosphorus in water extracts (between 42 and 70%; Pant et al. 1994) and soil solution (48 to 62%; Shand and Smith 1997) from Scottish soils, were hydrolysed by a phytase (from wheat bran) which was not specific for phytate.

Several extractants were used to assess enzyme-labile organic phosphorus.

Sodium-bicarbonate has previously been considered to extract a component of soil organic phosphorus which is readily mineralisable (Bowman and Cole, 1978). On this basis, sequential soil organic phosphorus extraction procedures have been developed to include bicarbonate-extractable organic phosphorus as a labile fraction (Hedley et al. 1982; Sharpley 1985). However, the present experiments do not support the supposition that bicarbonate-extractable organic phosphorus is labile. While Na-bicarbonate extracted the largest quantities of organic phosphorus relative to other soil extractants (up to 30 pg phosphorus g-1 soil), only a small proportion (between 1 and was enzyme-labile, even when using phytase with activity against a wide range of phosphate esters (Figure 17). Otani and Ae (1999) have similarly shown that negligible amounts of organic phosphorus were enzymelabile in Na-bicarbonate extracts from a range of soils.

In contrast, 50 mM citric acid extracted intermediate amounts of organic phosphorus (3.8 to 7.7 pg phosphorus g- 1 soil), of which up to 79% could be hydrolysed by the commercial phytase preparation and up to 40% was hydrolysed by the purified preparation. Otani and Ae (1999) also used a range of extractants and found that only citrate extracted organic phosphorus that was readily accessible to either acid WO 01/22806 PCT/AU00/01183 -74phosphatase or broad-specificity phytase. From the present work, it is evident that citric acid and Na-bicarbonate extracts contain different components of the total organic phosphorus pool.

Up to 40% of citrate-extractable soil organic phosphorus was hydrolysed by the purified phytase preparation (Table 9; Figure 17), indicating that a considerable amount may occur as phytate. Citrate is an effective chelator of trivalent metal ions such as Fe 3 and A1 3 (Jones and Darrah 1994). In soils, citrate can release inorganic phosphorus into solution either by anion exchange with Fe- and Alassociated phosphates on soil adsorption surfaces (Gerke 1992), or by chelation of precipitates to form soluble compounds (eg. Gardner et al. 1983). Phytate undergoes similar adsorption and precipitation reactions in soils to produce inorganic phosphorus (Ognalaga et al. 1994). It is conceivable that soil phytate is released into solution in the presence of citric acid via similar mechanisms.

Citrate also extracted organic phosphorus that was readily accessible to a phytase preparation possessing general acid phosphatase activity (Figure 17). The purified and commercial phytase preparations showed markedly different specific activities against a range of phosphate esters (Table However, it was evident that they also hydrolysed a common component of the citrate-extractable organic phosphorus; when combined, the amounts of extractable organic phosphorus hydrolysed by the two enzymes often exceeded the total quantity of extractable soil organic phosphorus (Figure 17). Enzyme-labile organic phosphorus in citric acid extracts may represent a component of soil phosphorus that can potentially be used by plants. In previous work, we have observed that plants grown in sterile culture were able to use organic phosphorus substrates, such as glucose 1-phosphate and P-glycerophosphate, essentially as equivalent phosphorus sources to inorganic phosphorus for growth, while phytate was a relatively poor source of phosphorus (JE Hayes et al. submitted to Plant and Soil, AE Richardson, PA Hadobas and JE Hayes submitted to Plant, Cell and Environment).

WO 01/22806 PCT/AU00/01183 Higher concentrations of citric acid extracted more soil organic phosphorus and also increased the proportion of organic phosphorus that was enzyme-labile. By contrast, HCI (used at the same molarity as citric acid) extracted minimal phytaselabile organic phosphorus. Consequently, the chelating properties of citric acid, rather than acidification effects, were considered to be largely responsible for the extraction of enzyme-labile organic phosphorus. The amount of extractable organic phosphorus also increased with the pH of the citric acid extractant. It is likely that this was due to the greater chelation ability of the citrate 2 /citrate 3 species, which would predominate over citrate/citrate' species at the higher pH. Gerke and Meyer (1995) found that citrate adsorption to a humic podzol was higher at pH compared to pH 5.5. Concurrently, they observed greater mobilisation of phosphorus. In the present work, the amount of enzyme-labile organic phosphorus did not increase with higher citric acid pH.

Effect of fertiliser history on extractable, phytase-labile soil organic phosphorus Measures of phosphorus in extracts of soils with contrasting fertiliser histories indicated that there were marked differences in the amounts of both total organic phosphorus and enzyme-labile organic phosphorus. Similar amounts of organic and hydrolysed phosphorus were observed in fertilised and unfertilised soils from plots at a recently established field site (Ginninderra). Only the inorganic phosphorus content of the soil at this site had been increased by the relatively recent phosphorus fertiliser applications. By contrast, clear distinctions could be made between fertiliser treatments for soil with a long history of fertiliser application, but which had received no fertiliser for seven seasons prior to sampling (Rutherglen). Differences between phosphorus treatments were especially evident in citric acid extracts, where the extracts from the fertilised soil contained approximately 3-fold greater amounts of enzyme-labile organic phosphorus than extracts from unfertilised soil. Extracts of soil which had received both fertiliser and lime similarly contained higher levels of total organic phosphorus and enzyme-labile organic phosphorus. These results may indicate that, with extended periods of WO 01/22806 PCT/AUOO/01183 -76fertiliser application, a greater proportion of applied phosphorus accumulates in soil as labile organic phosphorus. This component of labile organic phosphorus may make an important contribution to the phosphorus nutrition of pastures.

Mineralisable fractions of soil organic phosphorus have been reported to be important for phosphorus cycling within permanent pasture systems (McLaughlin et al. 1990).

Conclusions We have shown that a only small component of the organic phosphorus in water and Na-bicarbonate extracts of soil can be hydrolysed by phytase and general acid phosphatase activities. This may indicate that only low levels of potentially plantavailable organic phosphorus occur in soil solution. By contrast, greater amounts of enzyme-labile organic phosphorus were extracted using citrate. Citrate is exuded into the rhizosphere by roots of a number of plant species and, in many instances, exudation is enhanced under conditions of phosphorus deficiency (eg. Lipton et al.

1987; Hoffland et al. 1989; Grierson 1992; Keerthisinghe et al. 1998). Citrate exudation by plant roots is considered to be an important mechanism for increasing the acquisition of soil inorganic phosphorus. Our results imply that citrate also increases the availability to plants of soil organic phosphorus, by solubilising a fraction that can be hydrolysed by enzymes.

Enzyme-labile soil organic phosphorus was also influenced by soil fertiliser history.

While inorganic phosphorus was the dominant labile fraction in soils which had received recent applications of fertiliser, in soils of low fertility (eg. soil UG) or with a long history of fertiliser application (Rutherglen soil), the enzyme-labile component of citrate-extractable organic phosphorus was equivalent to, or exceeded the quantity of extractable inorganic phosphorus.

C,

WO 01/22806 WO 0122806PCT/AUOO/O1 183 77

REFERENCES

1. Abedinia et al., (1997) J. Plant Physiol. 24: 133-14 1.

2. An et EMBO J 4:277-284, 1985.

3. An, et Plant Physiol. 88& 547, 1988 4. Armstrong, et Plant Cell Reports 9: 335-339, 1990.

Ausubel, F. et al. (1987). In: Current Protocols in Molecular Biology.

Wiley I nterscience (ISBN 047150338).

6. Baker etat., Plant Mo! Bil. 24(5):701-713, 1994.

7. Barros et Plant Mol Biol, 19(4).665-675,1992.

8. Bechtold, et C.R. Acad. Sci. (Pans, Sciences de la vie! Life Sciences) 316: 1194-1199, 1993.

9. Bessey OA, et a. J. Biol. Chem. 164:,321-329, 1946.

Bowman RA, Cole CV Sodl Sci. 125:49-54, 1978.

11. Callis et al. J Biol. Chem. 265(21): 12486-12493, 1990.

12. Chaudhury etat, Plant Mol Biol, 30(6):1247-57, 1996.

13. Chen, J. and Varner J.E. (1985) The EMBO Journal 4:2145-2151.

14. Christou, et Plant Physiol 87: 671-674, 1988.

Cleveland, T. E. et Plant Mo!. BioL 8.199-208, 1987.

16. Clough et a! Plant J 16: 735-743, 1998.

17. Colwell JD Aust. J Exp. Agric. 3:190-198, 1963.

18. Comelissen eta!. (1986) Nature 321:531-532.

19. Conkling, et at, Plant Physiol. 93:1203, 1990.

Cosgrove, D. J. (1970) Aust. J Biol. Sd. 23: 1207.

21. Crossway et Mo!. Gen. Genet. 202:1 7PhyA-285, 1986.

22. de Pater et Plant J. 2: 837-44. 1992 23. Depicker. et J. Mo!. App!. Genet. 561-573, 1982.

24. Devereux, eta!. (1984) NucI. Acids Res. 12: 387-395.

Dolferus et aL Plant Physiol. 105(4):1075- 1087, 1994.

26. Ebel, et Arch. Biochem. Biophys. 232:240-248, 1984.

WO 01/22806 WO 0122806PCT/AUOO/01183 78 27. Ellis et at., EMBO Journal 6.11-16, 1987.

28. Finer and McMullen (1990) Plant Cell Rep. 8: 586-589.

29. Finer et at (1992) Plant Cell Rep. 11: 323-328.

Fromm et at Proc. Natl. Acad. Sci. (USA) 82:5824-5828, 1985.

31. Gardner WK, etal. Plant Soil 70:107-124, 1983.

32. Gatz et at Trends in Plant Science 3: 352-352. 1998.

33. Gatz et Cuff. Opinion Biotech. 7: 168-172, 1996.

34. Gellady, and Lefebvre, 0.0. (1990) Plant Physiol. supp. 93: Abstract 562.

35. Gerke J. Z Pflanzenemahr. Bodenk. 155:339-343, 1992.

36. Gerke J, Meyer U J. Plant Nutr. 18:2409-2429, 1995.

37. Gibson et at (1988) J. Cell. Biochem. 12C: Abstract L407.

38. Graf, F. (1986) Phytic acid: Chemistry and Applications, Pilatus Press, Minneapolis, Minn., USA (whole of text).

39. Gnierson PF Plant Soil 144:259-265, 1992.

Hajela et aL Plant Physiol. 93:1246-1252, 1990.

41. Hanahan, 0. J. Mol.Biol. 166, 557-560, 1983.

42. Haseloff, et at, Proc. NalAcad. Sci. USA 94: 2122-2127, 1997.

43. Hayes, et at, Aust, J. Plant Physiol. 26, 801-809, 1999.

44. Hedley MJ, et at Soil Sci. Soc. Am. J. 46:970-976, 1982.

Herrera-Estella et at., Nature 303: 209-213, 1 983a.

46. Herrera-Estella et at. EMBO J. 2: 987-995, 1 983b.

47. Herrera-Estella et at In: Plant Genetic Engineering, Cambridge University Press, pp 63-93, 1985.

48. Hoffland E, et at Plant Soil 113:161-165, 1989.

49. HOWe F, Beck E Plant Soil 157:1-9, 1993.

Hubei and Beck (1996) Plant Physiol. 112: l42PhyA-2436.

51. Huynh, et aL(1985) In: DNA Cloning Vol. 1: A Practical Approach (D.M.

Glover, ed) IRL Press Limited, Oxford. pp49-78.

52. Howard et at., Planta 17a.535-540, 1987.

53. Irving GCJ, McLaughlin MJ Commun. Soil Sci. Plant Anal. 21:2245-2255, 1990.

54. Iturriaga et at (1989) Plant Cell 1: 381-390.

Jones DL, Darrah PR Plant Soil 166:247-257, 1994.

WO 01/22806 PCT/AUOO/01183 -79- 56. Joshee et al., Plant Cell Physiol. 39(1):64-72, 1998.

57. Karunaratne et al. (1996) Aust. J. Plant Physiol. 23: 429-435.

58. Kasuga et al, Nature Biotech. 18: 287-291, 1999.

59. Keerthisinghe G, et al. Plant Cell Environ. 21:467-478, 1998.

60. Kirch et al., Plant Mol Biol. 33(5):897-909,1997.

61. Koncz, et al., EMBO J. 1029-1037, 1984.

62. Krens, et al., Nature 296: 72-74, 1982.

63. Kuhlemeier et al., Ann. Rev. Plant Physiol., 38:221-257, 1987.

64. Laboure et al. (1993) Biochem. J. 295: 413-419.

65. Li et al., (1997) Plant Physiol. 114:1103-1111.

66. Lipton DS, et al. Plant Physiol. 85:315-317, 1987.

67. Loewus, F. A. (1990) In: Plant Biology, Vol. 9 "Inositol metabolism in plants" (eds. D. J. Morre et page 13.

68. McLaughlin MJ, et al. Aust. J. Soil Res. 28:371-385, 1990.

69. McPherson, et al. (1991) PCR: A Practical Approach. (series eds, D.

Rickwood and B.D. Hames) IRL Press Limited, Oxford. ppl-253.

Marrs et al., Dev Genet., 14(1): 27-41, 1993.

71. Maugenest, et al. (1997) Biochem J. 322: 511-517.

72. Mullaney et al., (1991)App. Microbiol. and Biotech. 35: 611-614.

73. Murphy J, Riley JP Anal. Chim. Acta 27:31-36, 1962.

74. Nayini, N. and Markakis, P. (1984) Lebens. Wissenschaft Technol. 17: 24.

Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453.

76. Nelson DW, Sommers LE (1982) In: Page AL, Miller RH, Keeney DR (Eds) Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties, vol. 2. ASA-SSSA, Madison, pp 561-573.

77. Odell et al., Nature 313:810-812, 1985 78. Ognalaga M, et al. Soil Sci. Soc. Am. J. 58332-337, 1994.

79. Olsen SR, Sommers LE (1982) In: Page AL, Miller RH, Keeney DR (Eds) Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties, vol. 2. ASA-SSSA, Madison, pp 403-430.

Oppenheimer, et al., Gene 63: 87, 1988.

81. Otani T, Ae N Soil Sci. Plant Nutr. 45:151-161, 1999.

82. Ouellet et al., FEBS Lett. 423: 324-328,1998.

WO 01/22806 WO 0122806PCT/AUOO/01 183 80 83. Pant HK, et at. Bil. Fertit. Soils 17:196-200, 1994.

84. Paver, V. and Jagannathan, V. J. (1982) L. Bacteniot. 151: 1102.

Paszkowski et at., EMBO J. 3:2717-2722, 1984.

86. Pen et at., (1993) Blooechnotogy 11: 811-814.

87. Raghothama et at., Ptant Mot Bilt. 23(6):1 117-1128, 1993.

88. Ridley AM, et at. Aust. J. Exp. Agric. 30:529-537, 1990.

89. Sanford, J. et at., Particulate Science and Technotogy 5: 27-37, 1987.

Sanford, et at. (1993) Methods Enzymot. 21 7: 483-509.

91. Schneider et at. Ptant Physio. 1 13(2):335-345, 1997 92. Schoenau JJ, Huang WZ Commun. Soit Sc. Ptant Anal. 22:465-492, 1991.

93. Schoffi et at., Mot Gen. Genetics 217.246-253, 1989.

94. Shand CA, Smith S Biol. Ferti. Soils 24:183-1 87, 1997.

Sharpley AN Soit Sd. Soc. Am. J. 49:905-911, 1995.

96. Stace HCT, et at (1968) A Handbook of Australian Soils Rellim Technical Publications, Glenside, South Australia.

97. Suzuki et at., Ptant Mot Biot. 21: 1lOPhyA-219, 1993.

98. Thompson, et at. (1994) Nucl. Acids Res. 22: 4673-4680.

99. Tingey, et at., EMBO J. 6: 1, 1987.

100. Van der Zaal, et at, Plant Mot. Bil. 16, 983, 199 1.

101. Van Hartingsveldt et at. (1993) Gene 127: 87-94.

102. Walker et at., Proc. NaU. Acad. Sdi. (USA) 84:6624-6628, 1987.

103. Wasaki, et at., Soil Sc. Plant Nutr. 45, 439-449, 1999.

104. Waters et at J Experimental Bot. 47.:296-53, 1989.

105. Wiegand, R. et at, Plant Mot Bil. 7: 235-243, 1986.

106. Wilhelm et al, Plant Mot Bilt. 23(5):1073-1077, 1993.

107. Wyss et at (1999) App. Environ. Microbiot. 65: 367-373.

108. Yamada, et at (1986) Agric. Bilt Chem. 32: 1275.

109. Zhang et al, Plant Science. 129(1):81-89, 1997.

EDITORIAL NOTE APPLICATION NUMBER 78882/00 The following sequence listing pages 1 26 are part of the description. The claims pages follow on pages 81 86.

WO 01/22806 WO 0122806PCT/AUOO/01 183 -1I- SEQUENCE LISTING <110> Commonwealth Scientific and Industrial Research Organisation AND Australian Wool Research and Promotion Organisation <120> Method of modifying plant productivity <130> p:\oper\mro\phytase.pct <140> PCT/AUOO/XXXXX <141> 2000-09-23 <150> AU PQ3049 <151> 1999-09-24 <160> 18 <170> Patentln Ver. <210> <211> <212> <213> <220> <221> <222> 1 1350

DNA

Aspergillus niger

CDS

.(1347) <400> 1 atg ctg gca gtc ccc gcc tcg Met Leu Ala Val Pro Ala Ser 1 5 gat cag ggg tat caa tgc ttc Asp Gin Gly Tyr Gin Cys Phe tac gcg ccc ttc ttt tct ctg Tyr Ala Pro Phe Phe Ser Leu aga aat caa tcc act tgc gat acg gtc 48 Arg Asn Gin Ser Thr Cys Asp Thr Val 10 tcg gag act tcg cat ctt tgg ggc caa 96 Ser Glu Thr Ser His Leu Trp Gly Gin 25 gca aac aaa tcg gcc atc tcc cct gat 144 Ala Asn Lys Ser Ala Ile Ser Pro Asp WO 01/22806 WO 0122806PCT/AUOO/01 183 gtt cct gcc gga tgc cat gtc act ttc gcc cag Val Pro Ala Giy Cys His Val Thr Phe Ala Gin ctc tcc cgc cat Leu Ser Arg His gga gca cgg tat ccg Gly Ala Arg Tyr Pro gac tcc aag ggc Asp Ser Lys Gly aaa tac tcc gct Lys Tyr Ser Ala atc gag gag atc cag cag aac gcg aca Ilie Giu Giu Ilie Gin Gin Asn Ala Thr ttc gag ggg aaa Phe Giu Gly Lys tat gcc Tyr Ala 28 ttc ctg aag Phe Leu Lys tac aac tac agc Tyr Asn Tyr Ser ggc gcg gat gat Gly Ala Asp Asp ctg act ccc Leu Thr Pro 110 tac cag cga Tyr Gin Arg ttc gga gag cag gag ctg gtc Phe Gly Giu Gin Giu Leu Vai 115 tcc ggc gtc aag Ser Gly Val Lys tac gaa Tyr Giu 130 tcg ctc aca aga Ser Leu Thr Arg aac att gtc ccg ttc atc cga tcc tca ggc Asn Ile Val Pro Phe Ile Arg Ser Ser Gly 135 140 tct ggc aat aaa ttc atc gag ggc ttc cag Ser Gly Asn Lys Phe Ile Giu Gly Phe Gin 155 160 tcc Ser 145 aac cgc gtg att Asn Arg Val Ile agc act aag ctg Ser Thr Lys Leu gat cct cgt gcc Asp Pro Arg Ala ccc ggc caa tcg Pro Gly Gin Ser tcg ccc Ser Pro 175 aag atc gac Lys Ile Asp gat ccg ggc Asp Pro Gly 195 gtc att tca gag gcc agc aca tcc aac Val Ile Ser Giu Ala Ser Thr Ser Asn 185 aac act ctc Asn Thr Leu 190 acc tgc acc gtt Thr Cys Thr Val gaa gat agc gaa Giu Asp Ser Giu ttg gcc gat gac Leu Ala Asp Asp atc gaa gcc aat ttc acc gcc acg ttc gtc ccc tcc att cgt caa cgt Ile Glu Ala Asn Phe Thr Ala Thr Phe Val Pro Ser Ile Arg Gin Arg WO 01/22806 WO 0122806PCT/AUOO/01 183 ct g Leu 225 gag aat gac ttg Glu Asn Asp Leu ggc gtg tct ctC Gly Val Ser Leu acg gac Thr Asp 235 aca gaa gtg Thr Glu Val tac ctc atg gac Tyr Leu Met Asp tgc tcc ttc gac Cys Ser Phe Asp atc tcc acc agc Ile Ser Thr Ser acc gtc Thr Val 255 gac acc aag Asp Thr Lys atc aac tac Ile Asn Tyr 275 gca ggt aac Ala Gly Asn 290 tcc ccc ttc tgt Ser Pro Phe Cys ctg ttc acc cat Leu Phe Thr His gaa gaa tgg Glu Giu Trp 270 ggc cat ggc Gly His Gly gac tac ctc cag Asp Tyr Leu Gin ccg aac aaa tac Pro Asn Lys Tyr ccg ctc ggc Pro Leu Gly acc cag ggc gtc Thr Gin Gly Val tac gct aac gag Tyr Ala Asn Giu ctc Leu 305 atc gcc cgt ctc Ile Ala Arg Leu cac tcg cct gtc His Ser Pro Val gat gac acc agc Asp Asp Thr Ser 960 1008 aac cac aca ttg Asn His Thr Leu tcc aac ccg gct Ser Asn Pro Aia ttc ccg ctc aac Phe Pro Leu Asn tcc act Ser Thr 335 ctc tat gcg Leu Tyr Ala gct ttg ggt Ala Leu Gly 355 ttt tcg cat gat aac ggc atc atc tct Phe Ser His Asp Asn Gly Ile Ile Ser 345 atc ctc ttt Ile Leu Phe 350 acg acc gcg Thr Thr Ala 1056 ctg tac aac ggc acc Leu Tyr Asn Gly Thr 360 aag ccg ctg Lys Pro Leu tct tcc Ser Ser 365 1104 gag aat atc acc cag acc gat ggg ttc tca tct Glu Asn Ile Thr 370 Gin Thr Asp Giy Phe Ser Ser gcc tgg acg gtt cct Ala Trp Thr Vai Pro 380 1152 WO 01/22806 WO 0122806PCT/AUOO/01 183 gcg tcg cgc atg Ala Ser Arg Met gtc gag atg atg caa tgc cag tcc gag Val Giu Met Met Gin Cys Gin Ser Glu 395 1200 1248 gag cct ttg gtc cgt gtc ttg gtt aat gat cgt gtt gtt ccg Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro ggc tgt ccg Gly Cys Pro gtt gat Val Asp 420 gct ttg gga aga tgt acg cgg gat Ala Leu Gly Arg Cys Thr Arg Asp 425 agc ttc gtg Ser Phe Val 430 gag tgt ttt Glu Cys Phe 1296 aag ggg ttg Lys Gly Leu 435 agc ttt gcc aga Ser Phe Ala Arg ggc ggt gat tgg Gly Gly Asp Trp 1344 1350 gct tag Al a <210> 2 <211> 449 <212> PRT <213> Aspergillus niger <400> 2 Met Leu Ala Val Pro Ala Ser Arg Asn Gin Ser Thr Cys Asp Thr Val Asp Gin Gly Tyr Ala Pro Gin Cys Phe Ser Thr Ser His Leu Trp Gly Gin Phe Phe Ser Leu Asn Lys Ser Ala Ile Ser Pro Asp Val Pro Ala Gly Cys His Val Thr Phe Ala Gin Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Asp Ser Lys Gly Lys Lys Tyr Ser Ala Ile Giu Giu Ile Gin Gin Asn Ala Thr Thr Phe Giu Gly Lys Tyr Ala WO 01/22806 WO 0122806PCT/AUOO/01 183 Phe Leu Lys Phe Gly Glu 115 Tyr Asn Tyr Ser Leu Giy Ala Asp Asp 105 Leu Thr Pro 110 Tyr Gin Arg Gin Giu Leu Val Ser Gly Val Lys Tyr Giu 130 Ser Leu Thr Arg Asn Ile Val Pro Phe 135 Arg Ser Ser Gly Asn Arg Val Ile Ser Gly Asn Lys Phe Ile Giu Gly Phe 155 Ser Thr Lys Leu Asp Pro Arg Ala Pro Gly Gin Ser Ser Pro 175 Lys Ilie Asp Asp Pro Giy 195 Val Vai 180 Ile Ser Giu Ser Thr Ser Asn Asn Thr Leu 190 Ala Asp Asp Thr Cys Thr Vai Giu Asp Ser Giu Ile Giu 210 Ala Asn Phe Thr Thr Phe Val Pro Ile Arg Gin Arg Giu Asn Asp Leu Gly Val Ser Leu Asp Thr Giu Val Tyr Leu Met Asp Thr Lys Ile Asn Tyr 275 Asp Met 245 Leu Ser 260 Cys Ser Phe Asp Ile Ser Thr Ser Thr Vai 255 Pro Phe Cys Leu Phe Thr His Giu Giu Trp 270 Giy His Gly Asp Tyr Leu Gin Pro Asn Lys Tyr Ala Gly 290 Asn Pro Leu Giy Thr Gin Gly Vai Tyr Ala Asn Giu Leu Ile Ala Arg Leu Thr His Ser Pro Val His Asp Asp Thr Ser Ser WO 01/22806 WO 0122806PCT/AUOO/01 183 -6- ~305 Asn His Thr Leu Ser Asn Pro Ala Phe Pro Leu Asn Ser Thr 335 Leu Tyr Ala Ala Leu Gly 355 Phe Ser His ASP Gly Ile Ile Ser Ile Leu Phe 350 Thr Thr Ala Leu Tyr Asn Gly Lys Pro Leu Ser Glu Asn 370 Ile Thr Gin Thr Gly Phe Ser Ser Trp Thr Val Pro Phe 385 Ala Ser Arg Met Val Giu Met Met Cys Gin Ser Glu Giu Pro Leu Val Val Leu Val Asn Arg Val Vai Pro Leu His 415 Giy Cys Pro Lys Gly Leu 435 Asp Ala Leu Giy Cys Thr Arg Asp Ser Phe Vai 430 Giu Cys Phe Ser Phe Ala Arg Gly Gly Asp Trp <210> <211> <212> <213> <220> (221> <222> 3 1350

DNA

Aspergilius niger

CDS

(1347) (400> 3 atg ctg gca gtc ccc gcc tcg aga aat caa tcc agt tgc gat acg gtc 48 Met Leu Ala Val Pro Ala Ser Arg Asn Gin Ser Ser Cys Asp Thr Val WO 01/22806 PCT/AUOO/01 183 -7gat cag ggg Asp Gin Gly tac gca ccg Tyr Ala Pro caa tgc ttc tcc Gin Cys Phe Ser act tcg cat ctt Thr Ser His Leu tgg ggt caa Trp Giy Gin tcc cct gag Ser Pro Glu ttc ttc tct ctg Phe Phe Ser Leu aac gaa tcg gtc Asn Git' Ser Val gtg ccc Val Pro gcc gga tgc aga Ala Gly Cys Arg act ttc gct cag Thr Phe Ala Gin ctc tcc cgt cat Let' Ser Arg His gga gcg cgg tat ccg Gly Ala Arg Tyr Pro gac tcc aag ggc Asp Ser Lys Gly aaa tac tcc gct Lys Tyr Ser Ala att gag gag atc cag cag aac gcg acc Ile Giu Gi' Ile Gin Gin Asn Ala Thr ttt gac gga aaa Phe Asp Gly Lys tat gcc Tyr Ala ttc ctg aag Phe Let' Lys tac aac tac agc ttg ggt gca gat gac Tyr Asn Tyr Ser Leu Gly Ala Asp Asp 105 ctg act ccc Let' Thr Pro 110 tac cag cgg Tyr Gin Arg ttc gga gaa cag gag cta. gtc Phe Gly Giu Gin Giu Leu Val 115 tcc ggc atc aag Ser Gly Ile Lys tac gaa Tyr Giu 130 tcg ctc aca agg Ser Let' Thr Arg atc gti cca ttc Ile Val Pro Phe cga tcc tct ggc Arg Ser Ser Gly tcc agc cgc gtg atc gcc tcc ggc aag aaa Ser Ser Arg Vai Ile Ala Ser Gly Lys Lys atc gag ggc ttc Ile Glu Gly Phe agc acc aag ctg Ser Thr Lys Leu gat cct cgt gcc cag ccc ggc caa tcg ASP Pro Arg Aia Gin Pro Gly Gin Ser 170 tcg ccc Ser Pro 175 WO 01/22806 WO 0122806PCT/AUOO/01 183 -8aag atc gac Lys Ile Asp gac cca ggc Asp Pro Gly 195 gtc gaa gcc Val Glu Ala 210 gtc att tcc gag Val Ile Ser Glu agc tca tcc aac Ser Ser Ser Asn aac act ctC Asn Thr Leu 190 gcc gat acc Ala Asp Thr acc tgc act gtc Thr Cys Thr Val gaa gac agc gaa Giu Asp Ser Glu aat ttc acc Asn Phe Thr acg ttc gtc ccc Thr Phe Val Pro ait cgt caa cgt Ile Arg Gin Arg ctg Leo 225 gag aac gac ctg Giu Asn Asp Leu ggt gtg act ctc Gly Val Thr Leu gac aca gaa gtg Asp Thr Glu Val tac ctc atg gac Tyr Leo Met Asp tgc tcc ttc gac Cys Ser Phe Asp atc tcc acc agc Ile Ser Thr Ser acc gtc Thr Val 255 gac acc aag Asp Thr Lys atc aac tac Ile Asn Tyr 275 tcc ccc ttc tgt Ser Pro Phe Cys ctg ttc acc cat Leo Phe Thr His gac gaa tgg Asp Glu Trp 270 ggc cat ggt Giy His Giy gac tac ctc cag Asp Tyr Leo Gin ttg aaa aag tat Leu Lys Lys Tyr gca ggt aac Ala Gly Asn 290 ccg ctc ggc Pro Leo Gly acc cag ggc gtc Thr Gin Gly Val tac gct aac gag Tyr Ala Asn Glu ctc Leo 305 atc gcc cgt ctg Ile Ala Arg Leo acc cac Thr His 310 tcg cct gtc Ser Pro Val gat gac acc agt Asp Asp Thr Ser 960 1008 aac cac act ttg Asn His Thr Leu tcg agc ccg gct Ser Ser Pro Ala acc ttt Thr Phe 330 ccg ctc aac tct act Pro Leu Asn Ser Thr 335 ctc tac gcg gac ttt tcg cat gac aac ggc atc atc tcc att ctc ttt Leu Tyr Ala Asp Phe Ser His Asp Asn Giy Ile Ile Ser Ile Leo Phe 1056 WO 01/22806 WO 0122806PCT/AUOO/0 1183 gct tta ggt Ala Len Gly 355 ctg tac aac ggc Len Tyr Asn Gly aag ccg cta tct Lys Pro Leu Ser acg acc gtg Thr Thr Val 1104 gag aat Glu Asn 370 atc acc cag aca Ile Thr Gin Thr gga ttc tcg tct Gly Phe Ser Ser gct tgg acg gtt ccg Ala Trp Thr Val Pro 380 tgt cag gcg gag cag Cys Gin Ala Gin Gin 1152 gct tcg cgt ttg Ala Ser Arg Leu gtc gag atg atg Val Glu Met Met 1200 gag ccg ctg gtc cgt gtc ttg gtt aat gat cgc gtt gtc ccg Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro 1248 ggg tgt ccg Gly Cys Pro gat gct ttg ggg Asp Ala Len Gly aga tgt Arg Cys 425 acc cgg gat Thr Arg Asp agc ttt gtg Ser Phe Val 430 gag tgt ttt Glu Cys Phe 1296 1344 agg ggg ttg Arg Gly Len 435 agc ttt gct aga Ser Phe Ala Arg ggg ggt gat Gly Gly Asp tgg gcg Trp Ala 445 gct tag Ala 1350 <210> 4 <211> 449 <212> PRT <213> Aspergillus niger <400> 4 Met Len Ala Val Pro Ala Ser Arg Asn Gin Ser Ser Cys Asp Thr Val Asp Gin Gly Tyr Gin Cys Phe Ser Gin Thr Ser His Len Trp Gly Gin 25 WO 01/22806 WO 0122806PCT/AUOO/01 183 Tyr Ala Pro Phe Phe Ser Leu Ala Asfl Giu Ser Val Ile Ser Pro Glu Val Pro Gly Ala Ala Gly Cys Arg Thr Phe Ala Gin Leu Ser Arg His Arg Tyr Pro Thr Asp Ser Lys Gly Lys Lys Tyr Ser Ala Leu Tyr Ala Ilie Giu Giu Ilie Gin Gin Asn Ala Thr Thr Phe Asp Gly Lys Phe Leu Lys Thr Tyr Asn Tyr Ser Leu Gly Ala Asp Asp Leu Thr Pro 100 105 110 Phe Gly Giu Gin Giu Leu Vai 115 Ser Gly Ile Lys Tyr Gin Arg Tyr Giu 130 Ser Leu Thr Arg Asn Ile Val Pro Phe 135 Arg Ser Ser Giy Ser Arg Val Ilie Ala Ser Gly Lys Lys Phe Ile Giu Gly Phe Ser Thr Lys Leu Lys Asp Pro Arg Ala 165 Pro Gly Gln Ser Ser Pro 175 Lys Ile Asp Val Val Ile Ser Giu Ala Ser Ser Ser Asn Asn Thr Leu 190 Ala Asp Thr Asp Pro Gly 195 Thr Cys Thr Val Phe Glu 200 Asp Ser Giu Val Glu 210 Ala Asn Phe Thr Ala Thr Phe Val Pro 215 Ile Arg Gin Arg Glu Asn Asp Leu Gly Val Thr Leu Thr Asp Thr Glu Val Thr 235 240 Tyr Leu Met Asp Cys Ser Phe Asp Ile Ser Thr Ser Thr Val 255 WO 01/22806 WO 0122806PCT/AUOO/01 183 -1I1I- Asp Thr Lys Ser Pro Phe Cys Leu Phe Thr His Asp Giu Trp 270 Gly His Giy Ile Asn Tyr Asp Tyr Leu Gin 275 Leu Lys Lys Tyr Ala Gly 290 Asn Pro Leu Gly Thr Gin Gly Val Tyr Ala Asn Giu Leu 305 Ile Ala Arg Leu His Ser Pro Val Asp Asp Thr Ser Asn His Thr Leu Ser Ser Pro Ala Phe Pro Leu Asn Ser Thr 335 Leu Tyr Ala Ala Leu Gly 355 Phe Ser His Asp Asn Gly Ile Ile Ser 345 Ile Leu Phe 350 Thr Thr Val Leu Tyr Asn Giy Lys Pro Leu Ser Glu Asn 370 Ilie Thr Gin Thr Asp Gly Phe Ser Ser 375 Trp Thr Val Pro Phe Ala Ser Arg Leu Val Giu Met Met Cys Gin Ala Glu Giu Pro Leu Val Val Leu Val Asn Arg Vai Val Pro Leu His 415 Gly Cys Pro Asp Ala Leu Gly Cys Thr Arg Asp Ser Phe Val 430 Ala Giu Cys Phe 445 Arg Gly Leu Ser 435 Phe Ala Arg Ser Gly Gly Asp Trp 440 <210> <211> 96 WO 01/22806 WO 0122806PCT/AUOO/01 183 -12- <212> DNA <213> Daucus carota <220> <221> CDS <222> <400> atg gga aga Met Gly Arg 1 ttg ctt gta.

Leu Leu Val att gct aga ggc tca aaa Ile Ala Arg Gly Ser Lys 5 agt tct ctc Ser Ser Leu att gtg tct 48 Ile Val Ser acc aca gct 96 Thr Thr Ala ttg gtg tca ctc Leu Val Ser Leu ttg gct tcc gaa.

Leu Ala Ser Glu <210> 6 <211> 32 <212> PRT <213> Daucus carota <400> 6 Met Gly Arg Ile Ala Arg Gly Ser 15 Lys Met Ser Ser Leu Ile Val Ser 10 Leu Leu Val Val Leu Val Ser Leu Asn Leu Ala Ser Glu Thr Thr Ala 25 <210> 7 <211> 93 <212> DNA <213> Lupinus luteus <220> <221> CDS <222> (93) <400> 7 atg ggt tat tat tca att tat tgt ttg ata gtg tta gtg aat gta ttg 48 Met Gly Tyr Tyr Ser Ile Tyr Cys Leu Ile Val Leu Val Asn Val Leu WO 01/22806 WO 0122806PCT/AUOO/01 183 13 gtg ttt tgc gat gga ggg aag acg agt agt ttt gtt agg gaa tct Val Phe Cys Asp Gly Gly Lys Thr Ser Ser Phe Val Arg Glu Ser 25 <210> 8 <211> 31 <212> PRT <213> Lupinus luteus <400> 8 Met Gly Tyr Tyr Ser Ilie Tyr Cys 1 5 Leu Ile Val Leu Val 10 Asn Val Leu Val Phe Cys Asp Gly Gly Lys Thr Ser Ser Phe Val Arg Glu Ser 25 <210> 9 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:chimeric ext::phyA-2 gene <220> <221> <222>

CDS

(1)..(1446) <400> 9 atg gga aga att gct aga Met Gly Arg Ilie Ala Arg 1 5 ttg ctt gta gta ttg gtg Leu Leu Val Val Leu Val ggc tca aaa Gly Ser Lys agt tct ctc att Ser Ser Leu Ile gtg tct Val Ser tca ctc aat ttg Ser Leu Asn Leu gct tcc gaa Ala Ser Glu acc aca gct Thr Thr Ala WO 01/22806 WO 0122806PCT/AUOO/01 183 14 gcc atg ctg gca gtc ccc gcc Ala Met Leu Ala Val Pro Ala aga aat caa tcc act tgc gat acg Arg Asn Gin Ser Thr Cys Asp Thr gtc gat Val Asp cag ggg tat caa Gin Gly Tyr Gin ttc tcg gag act Phe Ser Giu Thr cat ctt tgg ggc His Leu Trp Gly caa Gin tac gcg ccc ttc Tyr Ala Pro Phe tct ctg gca aac Ser Leu Ala Asn tcg gcc atc tcc Ser Ala Ile Ser gat gtt cct gcc gga tgc cat gtc act ttc gcc cag gtt ctc Asp Val Pro Ala Gly Cys His Val Thr Phe Ala Gin Val Leu tcc cgc Ser Arg cat gga gca His Gly Ala tat ccg acc gac Tyr Pro Thr Asp aag ggc aag aaa Lys Gly Lys Lys tac tcc gct Tyr Ser Ala 110 ggg aaa tat Gly Lys Tyr ctc atc gag gag atc cag cag Leu Ilie Giu Giu Ilie Gin Gin 115 gcc ttc ctg aag aca tac a Ala Phe Leu Lys Thr Tyr Asn 130 135 gcg aca acc ttc Ala Thr Thr Phe tac agc ctg ggc Tyr Ser Leu Gly gat gat ctg act Asp Asp Leu Thr ccc ttc gga gag cag gag ctg gtc aac tcc Pro Phe Gly Giu Gin Giu Leu Val Asn Ser gtc aag ttc tac Val Lys Phe Tyr cga tac gaa tcg ctc aca aga aac att gtc ccg ttc atc cga Arg Tyr Giu Ser Leu Thr Arg Asn Ile Val Pro Phe Ile Arg tcc tca Ser Ser 175 ggc tcc aac cgc Gly Ser Asn Arg 180 cag agc act aag Gin Ser Thr Lys gtg att gcc tct Val Ile Ala Ser aat aaa ttc Asn Lys Phe atc gag ggc ttc Ile Giu Gly Phe 190 ctg aag gat cct cgt gcc cag ccc ggc caa tcg tcg Leu Lys Asp Pro Arg Ala Gin Pro Gly Gin Ser Ser WO 01/22806 WO 0122806PCT/AUOO/01 183 15 200 ccc aag Pro Lys 210 atc gac gtg gtc Ile Asp Val Val att tca Ile Ser 215 gag gcc agc aca.

GiU Ala Ser Thr tcc aac aac act Ser Asn Asn Thr gat ccg ggc acc Asp Pro Gly Thr acc gtt ttc gaa Thr Val Phe Glu agc gaa ttg gcc Ser Giu Leu Ala 720 gac atc gaa gcc aat ttc acc gcc acg Asp Ile Glu Ala Asn Phe Thr Ala Thr 245 gtc ccc tcc att Val Pro Ser Ile cgt caa Arg Gin 255 768 cgt ctg Arg Leu gag aat gac ttg tct ggc Giu Asn Asp Leu Ser Gly 260 tct ctc acg gac Ser Leu Thr Asp aca gaa gtg Thr Giu Vai 270 acc agc acc Thr Ser Thr acc tac cic Thr Tyr Leu 275 gtc gac acc Val Asp Thr 290 atg gac atg tgc Met Asp Met Cys ttc gac acc atc Phe Asp Thr Ilie aag ctg tcc Lys Leu Ser ttc tgt gac ctg Phe Cys Asp Leu acc cat gaa gaa Thr His Giu Glu atc aac tac gac Ile Asn Tyr Asp ctc cag tcc ccg aac aaa. tac tac ggc Leu Gin Ser Pro Asn Lys Tyr Tyr Giy 315 ggc gca ggt aac Giy Ala Giy Asn ctc ggc ccg acc Leu Gly Pro Thr gqc gtc ggc tac Gly Val Giy Tyr gct aac Ala Asn 335 1008 gag ctc atc gcc cgt ctc acc cac Giu Leu Ile Ala Arg Leu Thr His 340 cct gtc cac gat Pro Val His Asp gac acc agc Asp Thr Ser 350 ctc aac tcc Leu Asn Ser 1056 tcc aac cac Ser Asn His 355 aca ttg gac tcc Thr Leu Asp Ser aac ccg Asn Pro 360 gct act ttc Ala Thr Phe 1104 WO 01/22806 WO 0122806PCT/AUOO/0 1183 16 act ctc Thr Leu 370 tat gcg gac Tyr Ala Asp ttt tcg Phe Ser 375 cat gat aac ggc His Asp Asn Gly atc tct atc ctc Ile Ser Ile Leu ttt Phe 385 gct ttg ggt ctg Ala Leu Gly Leu aac ggc acc aag Asn Gly Thr Lys ctg tct tcc acg Leu Ser Ser Thr 1152 1200 1248 1296 gcg gag aat atc Ala Glu Asn Ilie acc cag acc gat ggg Thr Gin Thr Asp Gly 405 tca ict gcc tgg Ser Ser Ala Trp cct ttc gcg Pro Phe Ala cgc aig tac gtc gag atg atg caa tgc Arg Met Tyr Val Giu Met Met Gin Cys 425 cag tcc gag Gin Ser Glu 430 cag gag cct Gin Giu Pro 435 ttg gtc cgt gtc ttg gtt aat gat cgt gtt gtt ccg ctg Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro Leu 1344 cat ggc His Gly 450 tgt ccg gtt gat Cys Pro Val Asp ttg gga aga tgt Leu Gly Arg Cys cgg gat agc ttc Arg Asp Ser Phe 1392 1440 aag ggg ttg agc Lys Gly Leu Ser gcc aga tct ggc Ala Arg Ser Gly gat tgg gcg gag Asp Trp Ala Glu 1449 ttt gct tag Phe Ala <210> <211> 482 <212> PRT <213> Artificial Sequence <400> met Gly Arg Ile Ala Arg Gly Ser Lys Met Ser Ser Leu Ile Val Ser 1 5 10 Leu Leu Val Val Leu Val Ser Leu Asn Leu Ala Ser Glu Thr Thr Ala WO 01/22806 WO 0122806PCT/AUOO/01 183 -17- Ala Met Leu Val Asp Gin Gin Tyr Ala Val Pro Ala Asn Gin Ser Cys Asp Thr Leu Trp Gly Gly Tyr Gin Cys Ser Giu Thr Pro Phe Ser Leu Ala 65 Asp Asn Lys Phe Ala Ala Ile Ser Val Pro Ala Gly Cys His Val Thr Gin Val Leu Ser Arg His Gly Ala Arg 100 Leu Ile Giu Glu 115 Ala Phe Leu Lys Tyr Pro Thr Asp Lys Gly Lys Lys Ile Gin Gin Thr Tyr Asn 135 Gin Giu Leu Thr Thr Phe Tyr Ser Aia 110 Giy Lys Tyr Asp Leu Thr Ser Leu Gly Pro 145 Arg Gly Giu Vai Asn Ser Gly 155 Lys Phe Tyr Tyr Giu Ser Arg Asn Ile Val Pro Phe 170 Ile Arg Giy Ser Asn Gln Ser Thr 195 Pro Lvs Ile Ile Ala Ser Asn Ala Leu Lys Asp Lys Phe Ile Giu Gly Phe 190 Gin Pro Gly Gin Ser Ser 205 Ser Thr Ser Asn Asn Thr Asp Val Val Glu Ala Leu 225 Asp Pro Gly Thr Val Phe Giu Giu Leu Ala Ile Glu Ala Asn lie Gu Al Asn Thr Ala Thr Phe PrSe leAg Pro Ser Ile Arg WO 01/22806 WO 0122806PCTAUOO/01 183 18- Arg Leu Glu Thr Tyr Leu 275 Asp Leu Ser Giy Ser Leu Thr Asp Thr Glu Val 270 Thr Ser Thr Met Asp Met Cys Phe Asp Thr Ile Val Asp 290 Thr Lys Leu Ser Phe Cys Asp Leu Thr His Giu Glu Trp Ile Asn Tyr 305 Asp Tyr 310 Pro Leu 325 Leu Gin Ser Pro Asn Lys Tyr Tyr Gly 315 Gly Ala Gly Asn Gly Pro Thr Gly Val Gly Tyr Ala Asn 335 Giu Leu Ile Ser Asn His 355 Arg Leu Thr His Pro Val His Asp Asp Thr Ser ~350 Leu Asn Ser Thr Leu Asp Ser Pro Ala Thr Phe Thr Leu 370 Tyr Ala Asp Phe His Asp Asn Gly Ile Ser Ile Leu Ala Leu Giy Leu Tyr 390 Asn Gly Thr Lys Leu Ser Ser Thr Ala Giu Asn Ile Gin Thr Asp Gly Ser Ser Ala Trp Thr Val 415 Pro Phe Ala Gin Giu Pro 435 Arg Met Tyr Val Met Met Gin Cys Gin Ser Giu 430 Val Pro Leu Leu Val Arg Val Val Asn Asp Arg His Gly 450 Cys Pro Val Asp Ala 455 Leu Giy Arg Cys Thr Arg Asp Ser Phe 460 Val Lys Gly Leu Ser Phe Ala Arg Ser Gly Gly Asp Trp Ala Giu Cys WO 01/22806 WO 0122806PCT/AUOO/01 183 Phe Ala <210> <211> <212> <213> 11 1449

DNA

Artificial Sequence <220> <223> <220> <221> <222> Description of Artificial Sequence:chimeric ext::phyA-l gene

CDS

(1)..(1446) <400> 11 atg gga aga att gct aga ggc tca aaa Met Gly Arg Ile Ala Arg Gly Ser Lys agt tct ctc att Ser Ser Leu Ile gtg tct Val Ser ttg ctt gta Leu Leu Val ttg gtg tca ctc aat ttg gct tcc gaa Leu Val Ser Leu Asn Leu Ala Ser Glu acc aca gct Thr Thr Ala gcc atg ctg Ala Met Leu.

gca gtc ccc gcc Ala Val Pro Ala tcg aga aat Ser Arg Asn caa tcc agt tgc gat acg Gln Ser Ser Cys Asp Thr gtc gat Val Asp cag ggg tat caa tgc ttc tcc gag act Gln Gly Tyr Gln Cys Phe Ser Giu Thr cat ctt tgg ggt His Leu Trp Gly caa tac gca ccg ttc ttc tct ctg gca aac gaa tcg gtc atc tcc cct Gln Tyr Ala Pro Phe Phe Ser Leu Ala Asn Glu Ser Val Ile Ser Pro 70 75 gag gtg ccc gcc gga tgc aga gtc act ttc gct cag gtc ctc tcc cgt Glu Vai Pro Ala Gly Cys Arg Val Thr Phe Ala Gin Val Leu Ser Arg WO 01/22806 WO 0122806PCT/AUOO/01 183 20 cat gga gcg His Gly Ala ctc att gag Leu Ile Glu 115 tat ccg acc gac Tyr Pro Thr Asp aag ggc aag aaa tac tcc gct Lys Gly Lys Lys Tyr Ser Ala 110 gag atc cag cag Glu Ile Gin Gin gcg acc acc ttt Ala Thr Thr Phe gga aaa tat Gly Lys Tyr 384 gcc ttc Ala Phe 130 ctg aag aca tac Leu Lys Thr Tyr aac tac agc ttg ggt gca gat gac ctg act Asn Tyr Ser Leu Gly Ala Asp Asp Leu Thr 135 140 cta gtc aac tcc ggc atc aag ttc tac cag Leu Val Asn Ser Gly Ilie Lys Phe Tyr Gin 155 160 432 ccc Pro 145 ttc gga gaa cag Phe Gly Glu Gin cgg tac gaa tcg ctc aca agg aac atc Arg Tyr Giu Ser Leu Thr Arg Asn Ile 165 cca ttc atc cga tcc tct Pro Phe Ile Arg Ser Ser 175 ggc tcc agc Gly Ser Ser cag agc acc Gin Ser Thr 195 gtg atc gcc tcc Val Ile Ala Ser aag aaa ttc atc Lys Lys Phe Ile gag ggc ttc Giu Gly Phe 190 caa tcg tcg Gin Ser Ser aag ctg aag gat Lys Leu Lys Asp cgt gcc cag ccc Arg Ala Gin Pro ccc aag Pro Lys 210 atc gac gtg gtc Ile Asp Val Val tcc gag gcc agc Ser Giu Ala Ser tcc aac aac act Ser Asn Asn Thr ctc gac cca ggc acc Leu Asp Pro Gly Thr act gtc ttc gaa Thr Val Phe Giu agc gaa ttg gcc Ser Glu Leu Ala acc gtc gaa gcc Thr Val GiU Ala aat ttc acc Asn Phe Thr 245 gcc acg ttc Ala Thr Phe 250 gtc ccc tcc att Val Pro Ser Ile cgt caa Arg Gin 255 WO 01/22806 PCT/AUOO/01183 -21 cgt ctg gag Arg Leu Glu acc tac ctc Thr Tyr Leu 275 gac ctg tcc ggt Asp Leu Ser Gly act ctc aca Thr Leu Thr gac aca gaa gtg Asp Thr Glu Val 270 atg gac atg tgc Met Asp Met Cys ttc gac acc atc Phe Asp Thr Ile acc age acc Thr Ser Thr gtc gac Val Asp 290 acc aag ctg tcc Thr Lys Leu Ser ttc tgt gac ctg ttc acc cat gac gaa Phe Cys Asp Leu Phe Thr His Asp Glu 300 tgg Trp 305 atc aac tac gac Ile Asn Tyr Asp ctc cag tcc ttg Leu Gin Ser Leu aag tat tac ggc Lys Tyr Tyr Gly 960 1008 ggt gca ggt aac Gly Ala Gly Asn ctc ggc ccg acc Leu Gly Pro Thr ggc gtc ggc tac Gly Val Gly Tyr gag ctc atc Glu Leu Ile cgt ctg acc cac Arg Leu Thr His cct gtc cac gat Pro Val His Asp gac acc agt Asp Thr Ser 350 ctc aac tct Leu Asn Ser tcc aac cac act Ser Asn His Thr 355 ttg gac tcg Leu Asp Ser ccg gct acc ttt Pro Ala Thr Phe 1056 1104 1152 act ctc Thr Leu 370 tac gcg gac ttt Tyr Ala Asp Phe cat gac aac ggc His Asp Asn Gly atc tcc att ctc Ile Ser Ile Leu ttt Phe 385 gct tta ggt ctg Ala Leu Gly Leu gag aat atc acc Glu Asn Ile Thr 405 tac aac ggc act aag Tyr Asn Gly Thr Lys 390 cag aca gat gga ttc Gin Thr Asp Gly Phe 410 cta tct acc acg Leu Ser Thr Thr 1200 1248 gtg Val tcg tct gct tgg Ser Ser Ala Trp acg gtt Thr Val 415 ccg ttt get tcg cgt ttg tac gtc gag atg atg cag tgt cag gcg gag Pro Phe Ala Ser Arg Leu Tyr Val Glu Met Met Gin Cys Gln Ala Glu 1296 WO 01/22806 WO 0122806PCT/AUOO/01 183 22 cag gag ccg Gin Glu Pro 435 ctg gtc cgt gtc Leu Val Arg Val gtt aat gat cgc Val Asn Asp Arg gtc ccg ctg Val Pro Leu 1344 cat ggg His Gly 450 tgt ccg gtt gat Cys Pro Val Asp ttg ggg aga tgt Leu Gly Arg Cys cgg gat agc ttt Arg Asp Ser Phe 1392 agg ggg ttg agc Arg Gly Leu Ser gct aga tct ggg Ala Arg Ser Gly gat tgg gcg gag Asp Trp Ala Glu 1440 ttt gct tag Phe Ala <210> 12 <211> 482 <212> PRT <213> Artificial Sequence 1449 <400> 12 Met Gly Arg Ile Ala Arg Gly Ser Lys 1 5 Ser Ser Leu Ile Val Ser Leu Leu Vai Val Leu Val Ser Leu Asn 25 Leu Ala Ser Giu Thr Thr Ala Cys Asp Thr Ala Met Leu Ala Val Pro Ala Ser Arg Asn Gin Ser Val ASP Gln Gly Tyr Gln Cys Phe Ser Giu Thr Ser His Leu Trp Gly Gln Tyr Ala Pro Phe Phe Ser Leu Ala Asn Giu Ser Val Ile 70 75 Glu Val Pro Ala Gly Cys Arg Val Thr Phe Ala Gln Val Leu Ser Pro Ser Arg WO 01/22806 WO 0122806PCT/AUOO/01 183 23 His Gly Ala Tyr Pro Thr Asp Ser Lys Gly Lys 105 Lys Tyr Ser Ala 110 Leu Ile Giu Giu Ile Gin Gin Asn Ala Thr Thr Phe Asp Gly Lys Tyr Ala Phe 130 Leu Lys Thr Tyr Tyr Ser Leu Gly Ala Asp Asp Leu Thr 140 Pro Phe Gly Glu Gin 145 Leu Val Asn Ser Ile Lys Phe'Tyr Arg Tyr Glu Ser Leu Thr Arg Asn Ile 165 Pro Phe Ile Arg Ser Ser 175 Gly Ser Ser Gin Ser Thr 195 Val Ile Ala Ser Lys Lys Phe Ile Giu Gly Phe 190 Lys Leu Lys Asp Arg Ala Gin Pro Gly Gin Ser Ser 205 Pro Lys 210 Ile Asp Val Val Ser Giu Ala Ser Ser Asn Asn Thr Leu 225 Asp Pro Gly Thr Thr Val Phe Giu Ser Giu Leu Ala Thr Val Giu Ala Phe Thr Ala Thr Val Pro Ser Ile Arg Gin 255 Arg Leu Giu Asp Leu Ser Gly Thr Leu Thr Asp Thr Giu Val 270 Thr Ser Thr Thr Tyr Leu Met 275 Asp Met Cys Phe Asp Thr Ile Val Asp Thr Lys Leu Ser Pro Phe 290 295 Cys Asp Leu Thr His Asp Giu Ile Asn Tyr Asp Leu Gin Ser Leu Lys 315 Lys Tyr Tyr Gly WO 01/22806 WO 0122806PCT/AUOO/01 183 24 Gly Ala Gly Asfl Leu Gly Pro Thr Gly Val Gly Tyr Ala Asn 335 Glu Leu Ile Arg Leu Thr His Ser Pro Val His Asp 345 Asp Thr Ser 350 Leu Asn Ser Ser Asn His 355 Thr Len Asp Ser Pro Ala Thr Phe Thr Len 370 Tyr Ala Asp Phe His Asp Asn Gly Ile Ser Ile Leu Ala Leu Gly Leu Tyr Asn Gly Thr Lys 390 Leu Ser Thr Thr Val Glu Asn Ile Gin Thr Asp Gly Phe 410 Ser Ser Ala Trp Thr Val 415 Pro Phe Ala Arg Leu Tyr Val Glu Met Met Gin Cys 425 Gin Ala Glu 430 Val Pro Leu Gin Glu Pro 435 Leu Val Arg Val Val Asn Asp Arg His GiY 450 Cys Pro Val Asp Leu Gly Arg Cys Thr Arg Asp Ser Phe 460 Arg Gly Leu Ser Ala Arg Ser Gly Asp Trp Ala Glu Cys 480 Phe Ala <210> 13 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer WO 01/22806 WO 0122806PCT/AUOO/O1 183 <400> 13 cgcgaattca tgctggcagt ccccgcctcg <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 14 ggcatcgatc taagcaaaac actccgc <210> <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> gcgtctagag aattcatggg aagaattgct ag <210> 16 <211> <212> DNA <213> Artificial Sequence <220> <223> Description Of Artificial Sequence:primer <400> 16 cgcggatccg cgqccgcagc tgtggtttcg gaagc <210> 17 <211> 17 WO 01/22806 WO 0122806PCT/AUOO/01183 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:ext: :phytase junction <400> 17 acgctgccat gctggca <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:ext::phytase junction <400> 18 Thr Ala Ala Met Leu Ala

Claims (35)

1. A method of enhancing the phosphorus nutrition of a plant comprising ectopically expressing in the root of a plant an isolated nucleic acid molecule encoding a phytase polypeptide for a time and under conditions sufficient for said phytase to be secreted from the root.

2. The method according to claim 1 wherein the secretion of phytase from the root is achieved by ectopically expressing the phytase as a fusion protein with a secretory signal peptide.

3. The method according to claim 2 wherein the secretory signal peptide is selected from the group consisting of the carrot extensin signal peptide and the lupin acid phosphatase signal peptide.

4. The method according to any one of claims 1 to 3 wherein the phytase polypeptide is from Aspergillus niger. The method according to any one of claims 1 to 4 wherein the phytase polypeptide has at least about 93% identity to SEQ ID NO: 2.

6. The method according to claim 5 wherein the phytase polypeptide is selected from the group consisting of SEQ ID Nos: 2 and 4.

7. The method according to claim 5 wherein the phytase polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID Nos: 1, 3 and degenerate nucleotide sequence thereto.

8. The method according to claim 5 wherein the phytase polypeptide is encoded by a nucleotide sequence contained within the plasmid assigned AGAL Accession No. NM99/06795 WO 01/22806 PCT/AUOO/01183 -82-

9. A method of enhancing the phosphorus nutrition of a plant comprising ectopically expressing in the root of a plant an isolated nucleic acid molecule encoding a fusion polypeptide between a secretory signal peptide and a phytase polypeptide for a time and under conditions sufficient for said fusion polypeptide to be secreted from the root, wherein said isolated nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 9, 11, the phytase-encoding nucleotide sequence contained in the plasmid assigned AGAL Accession No. NM99/06795, and degenerate nucleotide sequences thereto. The method according to claim 9 wherein the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: and 12.

11. A method comprising: ectopically expressing in the root of a plant an isolated nucleic acid molecule encoding a phytase polypeptide for a time and under conditions sufficient for said phytase to be secreted from the root; and (ii) modifying the chemistry of the soil around the root or other growth medium around the root using an organic acid.

12. The method according to claim 11 wherein the organic acid is citric acid.

13. The method according to claims 11 or 12 wherein the secretion of phytase from the root is achieved by ectopically expressing the phytase as a fusion protein with a secretory signal peptide.

14. The method according to claim 13 wherein the secretory signal peptide is selected from the group consisting of the carrot extensin signal peptide and the lupin acid phosphatase signal peptide. WO 01/22806 PCT/AUOO/01183 -83 The method according to any one of claims 11 to 14 wherein the phytase polypeptide is from Aspergillus niger.

16. The method according to any one of claims 11 to 15 wherein the phytase polypeptide has at least about 93% identity to SEQ ID NO: 2.

17. The method according to claim 16 wherein the phytase polypeptide is selected from the group consisting of SEQ ID Nos: 2 and 4.

18. The method according to claim 17 wherein the phytase polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID Nos: 1, 3 and degenerate nucleotide sequence thereto.

19. The method according to claim 17 wherein the phytase polypeptide is encoded by a nucleotide sequence contained within the plasmid assigned AGAL Accession No. NM99/06795. A method comprising: ectopically expressing in the root of a plant an isolated nucleic acid molecule encoding a fusion polypeptide between a secretory signal peptide and a phytase polypeptide for a time and under conditions sufficient for said fusion polypeptide to be secreted from the root, wherein said isolated nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 9, 11, a phytase- encoding nucleotide sequence contained within the plasmid assigned AGAL Accession No. NM99/06795, and degenerate nucleotide sequences thereto and (ii) modifying the chemistry of the soil around the root or other growth medium around the root using an organic acid.

21. The method according to claim 20 wherein the organic acid is citric acid. P.AOPER\ nmsSpecificaionsl782a00 doc.29D9O 4 -84-

22. The method according to claims 20 or 21 wherein the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 10 and 12.

23. The method according to any one of claims 11 to 22 when used to enhance the phosphorus nutrition of a plant or the growth of a plant on a phosphorus source comprising phytate and/or increase the phosphorus content of a plant.

24. The method according to any one of claims 11 to 22 when used to enhance the biomass produced by a plant. The method according to any one of claims 11 to 22 when used to enhance the rate of hypocotyl production or the rate of epicotyl production.

26. A transformed plant that ectopically expresses a secretable phytase polypeptide in its roots wherein said phytase polypeptide is secreted from the roots of said plant, wherein said plant is produced by a process comprising performing the method according to any one of claims 1 to

27. The progeny of the transformed plant of claim 26 wherein said progeny ectopically expresses a secretable phytase polypeptide in its roots wherein said phytase polypeptide is secreted from the roots of said plant. a. 25 28. The transformed plant of claim 26 wherein said plant grows on a i phosphorus source comprising phytate more efficiently than an isogenic plant that does not ectopically express the phytase enzyme.

29. The progeny of claim 27 wherein said progeny grows on a phosphorus 30 source comprising phytate more efficiently than an isogenic plant that does not ectopically express the phytase enzyme. 0 a WO 01/22806 PCT/AU00/01183 The transformed plant of claims 26 or 28 wherein said plant exhibits a larger biomass than an isogenic plant that does not ectopically express the phytase enzyme when grown on a phosphorus source comprising phytate.

31. The progeny of claims 27 or 29 wherein said progeny exhibits a larger biomass than an isogenic plant that does not ectopically express the phytase enzyme when grown on a phosphorus source comprising phytate.

32. The transformed plant of claims 26 or 28 wherein said plant exhibits an enhanced rate of epicotyl or hypocotyl production than an isogenic plant that does not ectopically express the phytase enzyme when grown on a phosphorus source comprising phytate.

33. The progeny of claims 27 or 29 wherein said progeny exhibits an enhanced rate of epicotyl or hypocotyl production than an isogenic plant that does not ectopically express the phytase enzyme when grown on a phosphorus source comprising phytate.

34. A process comprising producing a plant that ectopically expresses phytase in secretable form in its roots or a progeny of said plant that expresses said phytase in secretable form and growing said plant or progeny in a plant growth medium comprising phytate and a suitable carrier for application to plants and/or the growth medium.

35. The process according to claim 34 further comprising modifying the chemistry around the root of the plant or progeny using an organic acid.

36. The process according to claim 35 wherein the organic acid is citric acid. PAPERSpcciaf sk88U2.0 dc.29MM9A4 -86-

37. The process according to any one of claims 34 to 36 wherein the secretion of phytase from the root is achieved by ectopically expressing the phytase as a fusion protein with a secretory signal peptide.

38. The process according to claim 37 wherein the secretory signal peptide is selected from the group consisting of the carrot extensin signal peptide and the lupin acid phosphatase signal peptide.

39. The process according to any one of claims 34 to 38 wherein the phytase polypeptide is from Aspergillus niger. The process according to any one of claims 34 to 39 wherein the phytase polypeptide has at least about 93% identity to SEQ ID NO: 2.

41. The process according to claim 40 wherein the phytase polypeptide is selected from the group consisting of SEQ ID Nos: 2 and 4.

42. The process according to claim 40 wherein the phytase polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID Nos: 1, 3 and degenerate nucleotide sequences thereto.

43. The process according to claim 5 wherein the phytase polypeptide is encoded by a nucleotide sequence contained within the plasmid assigned AGAL Accession No. NM99/06795. Dated this 2 9 t h day of September 2004. Commonwealth Scientific and Industrial Research Organisation AND Australian Wool Innovation Limited 30 By their Patent Attorneys Davies Collison Cave