EP1221830A1 - Expression de phytase dans des plantes, procede en modifiant le rendement - Google Patents

Expression de phytase dans des plantes, procede en modifiant le rendement

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Publication number
EP1221830A1
EP1221830A1 EP00969056A EP00969056A EP1221830A1 EP 1221830 A1 EP1221830 A1 EP 1221830A1 EP 00969056 A EP00969056 A EP 00969056A EP 00969056 A EP00969056 A EP 00969056A EP 1221830 A1 EP1221830 A1 EP 1221830A1
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EP
European Patent Office
Prior art keywords
phytase
plant
seq
polypeptide
phosphorus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00969056A
Other languages
German (de)
English (en)
Other versions
EP1221830A4 (fr
Inventor
Alan Edward Richardson
Julie Ellen Hayes
Richard Joseph Simpson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Australian Wool Innovation Ltd
Original Assignee
Commonwealth Scientific and Industrial Research Organization CSIRO
Australian Wool Research and Promotion Organization AWRAP
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Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO, Australian Wool Research and Promotion Organization AWRAP filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Publication of EP1221830A1 publication Critical patent/EP1221830A1/fr
Publication of EP1221830A4 publication Critical patent/EP1221830A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • 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.
  • 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.
  • 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 (e.g. ⁇ 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.
  • SEQ ID NO: 1 refers to the information provided in the numeric indicator field designated ⁇ 400> 1 , etc.
  • 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 other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
  • amino acid residues referred to herein are also those recommended by the lUPAC-IUB Biochemical Nomenclature Commission, wherein three-letter and one-letter abbreviations for naturally-occurring amino acids are listed in Table 1.
  • the three-letter symbol Asx, or the one-letter symbol B denotes Asp or Asn
  • the three-letter symbol Glx, 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 (Glp) that yields glutamic acid upon the acid hydrolysis of a peptide.
  • 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 International Patent Application No. PCT/AU95/00552.
  • Soil 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.
  • organic forms of soil phosphorus such as, for example, inositol phosphorus (soil phytate)
  • inositol phosphorus soil phytate
  • 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.
  • Microorganisms and fungi in the soil are known to contain phytase enzymes that catalyse the conversion of phytate to inositol and inorganic phosphate and phytase- encoding 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 a/., 1995").
  • 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.
  • 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) 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 H ⁇ bel 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • active phytase enzyme in work leading up to the present invention 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.
  • 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.
  • 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
  • the present invention provides a method of enhancing the phosphorus nutrition of a plant comprising: (i) 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.
  • phytate-based fertilizers may also provide environmental advantages relative to super-phosphate based fertilizers.
  • 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.
  • 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.
  • the isolated nucleic acid molecule of the invention is derived from a microbial source such as, for example, the filamentous fungi Aspergillus ssp.
  • 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.
  • 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.
  • the isolated nucleic acid molecule of the invention comprises or consists of the nucleotide sequence set forth in SEQ ID NO:
  • 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 enzymically- active 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 signal- encoding nucleotide sequence, and placed operably in connection with a promoter sequence that is operable in the root cells of a plant.
  • the phytase- encoding nucleotide sequence comprises or consists of the Aspergillus niger PhyA-
  • the secretory-signal-encoding nucleotide sequence is the carrot extensin secretory signal or equivalent.
  • 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 extr.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
  • 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.
  • FIG. 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 3'; International Patent Application No.
  • SCSV sub-clover stunt virus
  • PCT/AU95/00552 operably connected to a kanamycin-resistance gene (nptll) for expression in plants, flanked by the Agrobactetium tumefaciens left-border (LB) and right-border (RB) integration sequences; a bacterial-operable spectinomycin/streptomycin resistance gene (Sp- R/St-R); an Agrobactetium origin of replication (oriVRK2) and E.coli origin of replication (oricolEI) and intergenic spacer (IS1/oriT). Positions of restriction sites are indicated.
  • FIG 4 is a copy of a representation of the plasmid pART 7.
  • Plasmid pART7 is a vector containing bacterial two origins of replication (f1 oh and ori), an ampicillin resistance gene for bacterial selection (Amp R ).
  • 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.
  • MCS CaMV 35S promoter-multiple cloning site
  • 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 35S 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 35S 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: (i) plasmid pBS389 (row marked “A”); (ii) a plasmid containing the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence; row marked “B”); and (iii) plasmid pAER04 ( Figure 6; row marked "C”).
  • 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 P”). Duplicate results are indicated for each condition stated herein.
  • Figure 8 is a photographic representation showing growth on phytate-containing agar media, of transgenic A.
  • 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 A), or on phytate-containing agar media having a phytate concentration equivalent to 0.8 mM phosphate (panels B, C, D), of transgenic A. thaliana (C24 ecotype) plants containing a binary vector selected from the group consisting of: (A) plasmid pAER02 ( Figure 5); (B) a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); (C) plasmid pAER04 ( Figure 6); and (D) plasmid pAER02 ( Figure 5). Plants were grown for 40 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, C), or 0.8 mM Na 2 HPO (panel B), of transgenic A. thaliana (C24 ecotype) plants containing a binary vector selected from the group consisting of: (A) a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); (B) plasmid pAER02 ( Figure 5); and (C) plasmid pAER02 ( Figure 5).
  • FIG. 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 ⁇ g of total RNA isolated from transgenic shoots.
  • the lower panel shows an autoradiograph of the same mRNAs following hybridization under stringent conditions [e.g.
  • RNA samples indicated were derived from lines of transformed plants containing a plasmid selected from the group consisting of: (A) plasmid pBS389 ; (B) a plasmid containing the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence); (C) plasmid pAER04 ( Figure 6); (D) a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); and (E) plasmid pAER02 ( Figure 5). 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: (A) plasmid pBS389 ; (B) a plasmid containing the PhyA-2 gene (SEQ ID NO: 1) in plasmid pBS389 (no leader sequence); (C) plasmid pAER04 ( Figure 6); (D) a plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); and (E) plasmid pAER02 ( Figure 5).
  • DNA samples ( ⁇ 6 ⁇ g digested with EcoRI) were hybridized under stringent conditions [e.g. at 65°C, in a buffer comprising O. ⁇ xSSC and 0.5% (w/v) SDS] with the PhyA-1 sequence (SEQ ID NO: 3). 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 Thfolium subterraneum
  • the upper panel is an ethidium bromide-stained agarose gel containing 10 ⁇ g of total RNA isolated from transgenic shoots.
  • the centre panel shows an autoradiograph of the same mRNAs following hybridization under stringent conditions [e.g. at 65°C, in a buffer comprising O. ⁇ xSSC and 0.1% (w/v) SDS] with the PhyA-1 sequence (SEQ ID NO: 3).
  • 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 v), produced by transformation of explants with the plasmid pAER02 ( Figure 5).
  • the lane marked # is a positive control containing RNA from transgenic A. thaliana plants produced using the vector pAER02 (see Figure 12E).
  • 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.
  • 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 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 (D), and various concentrations of citric acid indicated on the abscissa.
  • Phytase-labile organic phosphorus ⁇ g phosphorus (P) g "1 soil is indicated on the ordinate.
  • 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 (D), in the presence of 50 mM citric acid at the pH values indicated on the abscissa.
  • Phytase-labile organic phosphorus ⁇ g phosphorus (P) g "1 soil is indicated on the ordinate.
  • 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, b), 50 mM citric acid (panels c, d), or 500 mM sodium bicarbonate (pH 8.5; panels e, f); and the organic phosphorus that was hydrolysed by phytase in soil extracts for 6 hr in the presence of either 0.50 nkat commercial phytase g "1 soil (open-hatched bars), or 2.28 nkat purified phytase g "1 soil (densely-hatched bars), in each of the above conditions.
  • 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.
  • 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.
  • 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. Accordingly, the present invention is directed to a method of improving the ability of a plant to utilise an external source of phosphorus.
  • the external source of phosphorus is phytate.
  • 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).
  • 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.
  • 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.
  • the phytase polypeptide of the invention is preferably derived from a plant, microorganism, or animal cell.
  • 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.
  • the phytase enzyme employed 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.
  • 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.
  • 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.
  • 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: 1).
  • 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.
  • 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.
  • 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.
  • in-frame fusion refers to the linkage between two or more heterologous nucleotide 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.
  • 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.
  • the secretory signal sequence may be placed at the N-terminal and/or C-terminal end of the phytase polypeptide.
  • 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 from about 1-20 residues.
  • 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.
  • 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.
  • plants that ectopically express phytase as an in-frame fusion with the secretory signal sequence derived from the carrot extensin protein exhibit improved growth on phytate relative to otherwise isogenic non- transformed plants.
  • 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 al, 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 Varner, 1985).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, turnover or steady- state level of the peptide, polypeptide, oligopeptide, protein or enzyme molecule.
  • 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.
  • 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.
  • 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.
  • “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.
  • an environmental stimulus or stress anoxia, hypoxia, temperature, salt, light, dehydration, low phosphate, low P, etc
  • an antibiotic tetracycline, ampicillin, rifampicin, kanamycin
  • 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".
  • plant-expressible format refers to an expressible format that pertains to expression of proteins in plant cells, tissues or organs.
  • 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.
  • an isolated nucleic acid molecule encoding phytase such as a cDNA molecule, genomic gene, synthetic oligonucleotide molecule, mRNA molecule or open reading frame
  • promoter 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 (i.e. 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 (i.e. the protein-coding region of a gene) in a tissue-specific manner, conveniently in the roots.
  • TATA box which is required for accurate transcription initiation
  • CCAAT box sequence i.e. upstream activating sequences, enhancers and silencers
  • additional regulatory elements i.e. upstream activating sequences, enhancers and silencers
  • 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.
  • 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.
  • constitutive promoter sequences include 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).
  • a transposable genetic element Ac, Ds, Spm, En, or other transposon
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • organ-specific shall be taken to indicate that expression is predominantly in a particular plant organ albeit not necessarily exclusively in that plant organ.
  • a preferred organ-specific promoter will confer expression on a phytase gene throughout the root.
  • an "inducible promoter” is a promoter the transcriptional activity of which is increased or induced in response to a developmental, chemical or physical stimulus.
  • 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 phosphate deficiency in the plant.
  • a phosphate transport gene such as, for example, the phosphate transport genes of A. thaliana or barley plants, which are induced under conditions of phosphate deficiency in the plant.
  • 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 5'-end, and within 2 kb of the start site of transcription, of the nucleic acid molecule which it regulates.
  • 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 (i.e., 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.
  • 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 (i.e., 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 purposes 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.
  • tissue-specific inducible promoter sequences include the anoxia-inducible and hypoxia-inducible maize Adh1 gene promoter (Howard et al, 1987; Walker et al., 1987). Such environmentally-inducible promoters are reviewed in detail by Kuhlemeier er a/. (1987).
  • Preferred chemically-inducible promoters include the 3- ⁇ - indoylacrylic acid- inducible 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-dichloroacetamide- inducible 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.
  • PAL phenylalanine ammonia lyase
  • 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.
  • the CaMV 35S promoter may be modified by the addition of maize Adh1 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.
  • the phytase is ectopically expressed under control of the CaMV 35S promoter sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides a method of improving the phosphorus nutrition of a plant comprising:
  • modifying the chemistry of the soil around the root or other growth medium around the root 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.
  • a metal such as, for example a transition metal
  • 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.
  • 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.
  • metal ions such as, for example, aluminium, iron or calcium
  • 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.
  • 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.
  • 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.
  • the organic acid biosynthesis enzyme is citrate synthase.
  • Expression of citrate synthase may be increased in the region around the root by ectopically- expressing 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.
  • 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.
  • 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.
  • 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.
  • 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 protein- encoding sequence and promoter in a plant cell, tissue or organ.
  • 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 root- operable or constitutive promoter sequence.
  • gene construct of the present invention may further comprise one or more terminator sequences.
  • 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
  • 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.
  • NOS nopaline synthase
  • OCS Agrobacterium tumefaciens octopine synthase
  • CaMV Cauliflower mosaic virus
  • t3'Bt2 Oryza sativa ADP-glucose pyrophosphorylase terminator sequence
  • Zea may
  • 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.
  • 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 f1-o ⁇ and co/E1 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.
  • 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 (Amp r ), tetracycline resistance gene (Tc r ), bacterial kanamycin resistance gene (Kan r ), phosphinothricin resistance gene, neomycin phosphotransferase gene (tiprll), hygromycin resistance gene, ⁇ -glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), and luciferase gene, amongst others.
  • 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.
  • the present invention provides a method of modifying the phosphorus nutrition of a plant comprising:
  • 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 2 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, 1988; Sanford ef 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
  • 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.
  • Agrobacterium tumefaciens Bactatumefaciens
  • 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.
  • 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. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050).
  • the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
  • 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 subsequent clonal propagation 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.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • existing meristematic tissue e.g., apical meristem, axillary buds, and root meristems
  • induced meristem tissue e.g., cotyledon meristem and hypocotyl meristem.
  • organogenesis means a process by which shoots and roots are developed sequentially from meristematic centres.
  • embryogenesis 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.
  • 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 non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., 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., Acer spp., Actinidia spp.,Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., A.
  • Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, lea 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.
  • 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.
  • any art-recognised means such as microprojectile bombardment, microinjection, Agrobacterium-mediated transformation, protoplast fusion, protoplast transformation, in planta transformation, or electroporation, amongst others.
  • the transformed plant is A. thaliana or subterranean clover.
  • 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 Aspergillus niger PhyA-2 gene sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 9 or a complementary nucleotide sequence thereto.
  • 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%.
  • 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 enzymically- functional phytase peptide, oligopeptide or polypeptide.
  • 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 ef al, 1984), which utilizes the algorithm of Needleman and Wunsch (1970) or alternatively, the CLUSTAL W algorithm of Thompson ef 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.
  • the present invention encompasses those phytase- encoding 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.
  • SEQ ID NO: 1 or SEQ ID NO: 9 or a complementary nucleotide sequence thereto but not including the PhyA-1 gene sequence.
  • 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°C, or alternatively, a standard DNA/DNA hybridisation and/or wash carried out in O.lxSSC buffer, 0.1% (w/v) SDS at a temperature of at least 65°C, or equivalent annealing/hybridisation conditions.
  • PCR polymerase chain reaction
  • 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.
  • reference is found in pages 2.10.8 to 2.10.16. of Ausubel ef al. (1987), which is herein incorporated by reference.
  • 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 well- known to those skilled in the art.
  • specific PCR formats such as, for example, a nested PCR, a format that is well- known to those skilled in the art.
  • Particularly preferred variants of the A. niger PhyA-2 gene exemplified herein comprise degenerate nucleotide sequences (i.e. homologues) that encode the amino acid sequence set forth in SEQ ID NO: 2.
  • 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.
  • a suitable cloning vector such as a plasmid or bacteriophage or cosmid molecule
  • Detection is performed preferably by labelling the probe with a reporter molecule capable of producing an identifiable signal, prior to hybridization.
  • reporter molecules include radioactively-labelled nucleotide triphosphates and biotinylated molecules.
  • variants of the A. niger PhyA-2 gene exemplified herein, including genomic equivalents are isolated by hybridisation under high stringency conditions, to the probe.
  • 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 copies of the template are amplified enzymatically as described in McPherson et al, (1991), which is incorporated herein by reference.
  • protein- or peptide- encoding 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 include 125 l-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 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.
  • a eukaryotic cell such as an insect cell, mammalian cell, plant cell, fungal cell, or yeast cell, amongst others.
  • 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.
  • 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.
  • promoters useful in performing this embodiment include the CaMV 35S 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 ⁇ L or ⁇ R promoters, CMV promoter (U.S.
  • Patent No. 5,168,062 T7 promoter, lacUV ⁇ promoter, SV40 early promoter (U.S.
  • Patent No. 5,118,627 SV40 late promoter (U.S. Patent No. 5,118,627), adenovirus promoter, baculovirus P10 or polyhedrin promoter (U.S. Patent Nos. 5,243,041 ,
  • the recombinant phytase polypeptide is provided in a sequencably-pure format or a pure format substantially free of conspecific proteins.
  • polypeptide or a homologue, analogue, derivative or epitope thereof is purified sufficiently to facilitate amino acid sequence determination.
  • 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 80% pure or 95% pure on a weight basis.
  • 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 Government 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.
  • AGAL Australian Government Analytical Laboratories
  • 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.
  • 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 C/al, 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 of 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
  • the derived amino acid sequence encoded by the PhyA-2 gene is set forth in SEQ ID NO: 2.
  • 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 Sph ⁇ 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).
  • 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.
  • 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.
  • 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 Xoal BamHI fragment into plasmid pBSIIKS (Stratagene, Palo Alto, California, 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: 5.
  • Extensin/Phytase Gene Fusion 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 (ie., 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 ⁇ /ofl-EcoRI fusion), and three codons derived from the phytase genes (PhyA-1 or PhyA-2 as appropriate).
  • 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 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: 10.
  • 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.
  • the phytase genes were placed in operable connection with the CaMV 35S promoter sequence, to produce the plasmids:
  • the four phytase transgenes were then subcloned as ⁇ / fl fragments into the binary vector pBS389 ( Figure 3).
  • This vector is suitable for plant transformation and contains the selectable marker npt ⁇ under control of the SCSV Sc1 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.
  • the pBS389 vectors containing the various phytase gene constructs were transferred to Agrobacterium 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 6), or plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); or plasmid pAER02 ( Figure 5).
  • Data indicate the presence of the signal only in plants produced using the P/7yA-7-containing or Pfjy/A-2-containing gene constructs.
  • shoot phosphorus concentrations of non-transformed plants supplied with phytate, or alternatively, grown without an external phosphorus source were significantly lower and these plants exhibited markedly lower shoot to root ratios than plants grown on inorganic phosphate as Na 2 HPO 4 .
  • Figure 1 1 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 6), or plasmid containing the PhyA-1 gene (SEQ ID NO: 3) in plasmid pBS389 (no leader sequence); or plasmid pAER02 ( Figure 5).
  • 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; T 0 generation), containing plasmid pAER02 ( Figure 5). Data indicate a positive correlation between copy number of the introduced transgene and the level of expression of the chimeric extensin::P/7y>4-2 transgene.
  • 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 "1 fresh wt (0,04 nkat phytase mg "1 protein), compared to 30.4 nkat g ⁇ 1 fresh wt (6.2 nkat phytase mg '1 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.
  • Phytase enzyme activity increased in the leaves and roots of transgenic subterranean clover that contain the introduced phytase gene constructs.
  • 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 3).
  • the phytase activity of transformed plants carrying the PhyA-1 gene without the extensin signal peptide was not significantly different from that of the control plants transformed with pBS389.
  • 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 4). For the line designated "i” 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 exr.phyA gene was confirmed by Southern blot analyses. TABLE 4 Phytase activity of transgenic subterranean clover plants: T1 population
  • control-2 20 1245.2 20 1428.8 control-1* 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 (iii) Transformed A. thaliana plants
  • the growth responses of transgenic plants in media supplemented with phytate were determined for transformed A. thaliana plants.
  • 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.
  • the phytase activities in the roots of a wide range of agriculturally important legume and grass species including subterranean clover, burr medic, white clover, lucerne, tobacco, A. thaliana, wheat, phalaris, ryegrass and danthonia, have been determined.
  • 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 "1 root fresh wt and 102.0 mU g "1 root fresh wt , respectively), or alternatively, 0.2 to 1.5 nkat mg _1 total protein (equivalent to 12.0 mU mg " protein and 90.0 mU mg "1 protein, 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.
  • acid phosphatase activity measured in the same extracts ranged between 20 and 60 nkat g "1 root fresh wt (equivalent to 1200 mU g "1 root fresh wt and 3600 mU g '1 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 "1 root fresh wt (ie., 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 "1 root fresh wt (ie., 3% or less of the activity measured in soluble root extracts).
  • 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.
  • the substrate specificities of commercial preparations of wheat germ acid phosphatase (EC 3.1.3.2; Sigma Chemical Co.), 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 °C, in 50 mM MES buffer (pH 5.5) containing 1 mM EDTA, against the following substrates: myo-inositol hexaphosphoric acid (dodecasodium salt; IHP), ⁇ -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(p- nitrophenyl) phosphate (sodium salt; b/ ' s-pNPP).
  • myo-inositol hexaphosphoric acid diodecasodium salt; IHP
  • ⁇ -D-glucose 1-phosphate diisodium salt
  • G1P
  • 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 (-1.0%) citric acid (pH ⁇ 2.3) or 0.5 M Na-bicarbonate (pH 8.5). Other extractants included 0.025 and 0.1 M HCl, and 0.01 M CaCI 2 (pH 5.5).
  • the soils were extracted at 22 ⁇ 2 °C 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 °C 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 °C; 40 min) in the presence of 0.6 M H 2 SO 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.
  • Standard assays involved incubation of 1 ml of soil extract in the presence of excess enzyme: either 0.25 nkat (ie., 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.
  • 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.
  • 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 8).
  • the purified A. niger phytase preparation had a 12-fold higher specific activity for IHP than the commercial phytase.
  • 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.
  • Citric acid concentration Extractable organic phosphorus from soil F R increased significantly with increasing concentrations of citric acid to 50 M (P ⁇ 0.05; Figure 15).
  • 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.
  • 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 ⁇ g phosphorus g "1 soil at pH 6.0 ( Figure 16). However, enzyme-labile phosphorus was similar across the entire pH range, with ⁇ 7.8 ⁇ g phosphorus g '1 soil and ⁇ 4.5 ⁇ g phosphorus g "1 soil hydrolysed by commercial phytase and purified phytase, respectively ( Figure 16).
  • Extractable organic phosphorus P 0
  • enzyme-labile phosphorus ⁇ g g "1 soil
  • Extractant ⁇ g g "1 Commercial phytase Purified phytase soil) (% Of Po) (% Of Po)
  • HCl extractants Two HCl extractants were used: one at the same molar concentration as citric acid (50 mM) and the other at the same pH (pH 2.3, at ⁇ 5 M). When used at an equivalent molar concentration, HCl 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 HCl 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.
  • 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) was used to measure the amounts of enzyme-labile organic phosphorus present in extracts of soil.
  • citric acid extracted intermediate amounts of organic phosphorus (3.8 to 7.7 ⁇ g 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 phosphatase 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.
  • citrate is an effective chelator of trivalent metal ions such as Fe 3+ and Al 3+ (Jones and Darrah 1994).
  • citrate can release inorganic phosphorus into solution either by anion exchange with Fe- and Al- associated 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 8). 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.
  • 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.

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Abstract

L'invention porte sur un procédé modifiant le rendement de plantes consistant à faire exprimer dans une cellule, un tissu ou un organe de plante un ou plusieurs gènes capables d'en faciliter la capacité à utiliser le phosphore du sol, et en particulier un gène de phytase exprimant un polypeptide de phytase sous forme sécrétable. L'invention porte plus particulièrement sur un procédé d'accroissement de la productivité de plantes consistant à faire exprimer dans la racine de la plante une molécule isolée d'acide nucléique codant pour un polypeptide de phytase pendant un temps et sous des conditions suffisants pour que ladite phytase soit sécrétée par la plante. Dans l'exécution préférée la chimie du sol autour de la racine est modifiée par application d'un acide organique. L'invention porte également sur de nouveaux gènes codant pour ladite phytase, sur des produits d'assemblage géniques servant à mettre en oeuvre le procédé de l'invention, et sur des plantes transgéniques produites par son intermédiaire présentant des rendements améliorés par rapport à leurs pendants isogéniques.
EP00969056A 1999-09-24 2000-09-22 Expression de phytase dans des plantes, procede en modifiant le rendement Withdrawn EP1221830A4 (fr)

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US8114443B2 (en) 2002-03-13 2012-02-14 Academia Sinica Phytase-expressing transgenic plants
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WO1997038096A1 (fr) * 1996-04-05 1997-10-16 Kyowa Hakko Kogyo Co., Ltd. Nouvelle phytase et gene codant pour ladite phytase
WO1998005785A1 (fr) * 1996-08-01 1998-02-12 Biocem Phytases de plantes et applications biotechnologiques

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