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  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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  • C12N15/09—Recombinant DNA-technology
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  • 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
  • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
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  • 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
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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  • 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
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2445—Beta-glucosidase (3.2.1.21)
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  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01021—Beta-glucosidase (3.2.1.21)
• • 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

• Phosphorus is one of the most important nutrients for plants. It is essential for their growth and is a structural component of nucleic acids, phospholipids, intermediary metabolites and numerous other biological molecules .
• plants Because they are sessile organisms, plants must deal biochemically with environmental stresses such as temperature extremes, nutrient deficiency and drought. This is also true for other photosynthetic organisms which are either sessile or limited in movement. Plants and other photosynthetic organisms, therefore, require signal transduction pathways in order to trigger cellular responses to adverse environmental stimuli.
• ADP and ATP which are the currency of cellular energy
• phosphorus is critical to bioenergetics .
• covalent addition or removal of a phosphate group to or from a biological substrate phosphorylation and dephosphorylation, respectively
• phosphorylation and dephosphorylation are key to various signal transduction pathways, including pathways of plant hormones such as ethylene (Kieber et al . , 1993) and abscisic acid (Anderberg and Walker-Simmons, 1992) .
• Self- incompatibility with respect to pollination and fertilization also involves the activity of protein kinases encoded by S-locus genes (Tantikanjana et al . , 1993; Zhang and Walker, 1993) .
• This invention provides the means to modify phosphorus metabolism in plants and other photosynthetic organisms by altering the expression and/or activity of one or more proteins involved in the response of plants or other photosynthetic organisms to phosphorus deprivation.
• This invention further provides means for more efficient metabolic utilization of phosphorus by plants and other photosynthetic organisms.
• the compounds of this invention provide the means to change plant morphology by altering phosphorus metabolism. In some applications, the modification will be restricted to seeds, where it can lower the amount of phytate, an anti-nutritive phosphorus storage compound.
• This invention relates to isolated DNA (genes) encoding proteins involved in phosphorus uptake and metabolism of plants and other photosynthetic organisms inducible by phosphate deficiency (psr proteins) , as well as DNA complementary to these genes, and recombinant DNA constructs and vectors containing DNA encoding such proteins or such complementary DNA, in whole or portions thereof .
• the present invention provides DNA (genes) encoding protein kinases, ⁇ -glucosidases, and phosphate transporters of Arabidopsi s thaliana and Brassica nigra , whose transcription is inducible by phosphate starvation, and further provides the RNA so transcribed.
• the nucleic acids (both DNA and RNA) of this invention encode proteins which differ from other protein kinases in having a unique portion of their amino acid sequence which is different from any other known protein kinase.
• the ⁇ -glucosidases and phosphate transporters of this invention differ from other known ⁇ -glucosidases and phosphate transporters in sequence and because their level of expression is specifically dependent on phosphate deprivation.
• nucleic acids of the invention include nucleic acids with sequences complementary to the nucleic acid sequences of Arabidopsis thaliana and Brassica nigra, or portions thereof; nucleic acids with sequences related to, but distinct from, the nucleic acid sequences of Arabidopsis thaliana and Brassica nigra and inducible under conditions of phosphate deficiency; and nucleic acid sequences that differ from the nucleic acid sequences of Arabidopsis thaliana and Brassica nigra , such as modified analogs, due to alteration of the sequence through mutation, substitution, deletion and the like.
• nucleic acid of the invention is an antisense oligonucleotide, a triple helix-forming oligonucleotide, or other oligonucleotide that can be used to inhibit the expression of the psr proteins encoded by the nucleic acids described herein.
• oligonucleotides can block the expression of any of these proteins in a number of ways; for example, preventing transcription of a psr protein-encoding gene by triple helix formation, or by binding to the mRNA transcribed by the gene ' in any manner that prevents a functional protein from being assembled.
• the oligonucleotides of the invention comprise a specific sequence of about 20 to about 200 or more nucleotides which are identical or complementary to a specific sequence of nucleotides of the psr protein-encoding gene or transcribed mRNA.
• the invention further provides nucleic acids of the invention operatively linked to a regulatory sequence, and plasmids or recombinant expression vectors for producing the nucleic acids encompassed by this invention.
• a recombinant expression vector comprising the nucleic acids operatively linked to a regulatory sequence is adapted for transformation of a plant cell .
• the invention also provides transformed or transgenic cells expressing one or more of the psr proteins of the invention.
• the transgenic cells are plant cells.
• the invention includes transgenic plants produced with nucleic acids or vectors of the invention which express psr proteins provided by the invention.
• the invention further includes transgenic plant parts, including seeds, as well as tissue culture or protoplasts produced with nucleic acids or vectors of the invention.
• the invention also provides a recombinant expression vector adapted for transformation of a plant cell, comprising a DNA molecule operatively linked to a regulatory sequence to allow expression of an RNA molecule that is antisense to a nucleic acid sequence having substantial sequence homology with any of the nucleotide sequences represented by SEQ ID NO : 1 , SEQ ID NO: 3, SEQ ID NO : 5 , SEQ ID NO : 7 , SEQ ID NO : 9 , SEQ ID NO: 11 through SEQ ID NO: 17, and SEQ ID NO: 19 through SEQ ID NO: 27.
• the invention further provides a method of preparing a psr protein having psr protein activity using the nucleic acids of the invention.
• the method comprises culturing a transformant or transgenic cell including a recombinant expression vector comprising a nucleic acid of the invention and a regulatory sequence operatively linked to the nucleic acid in a suitable medium until the psr protein is expressed, and then isolating the psr protein.
• the invention also provides an isolated psr protein or polypeptide having psr protein activity and substantial sequence homology with either or both of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO : 4 , SEQ ID NO : 6 , SEQ ID NO : 8 , SEQ ID NO: 10, SEQ ID NO: 18 or a portion thereof.
• Antibodies and antibody fragments which bind to the novel psr proteins described herein (or to portions of these sequences) are also included in this invention.
• the antibody is a monoclonal antibody.
• the invention further provides a method for reducing expression of a psr protein of a photosynthetic organism, preferably a plant, comprising the step of incorporating into the organism an isolated nucleic acid which is antisense to a nucleic acid having substantial sequence homology with the nucleotide sequence of a gene encoding a psr protein, especially SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO : 5 , SEQ ID NO : 7 , SEQ ID NO : 9 , SEQ ID NO:
• the methods of this invention can also be used to override dominant alleles thereby producing lowered expression rates or, at an extreme, the "null allele" phenotype .
• the antisense transcripts can cause a reduction m steady- state sense mRNA levels, perhaps because of increased turnover, or specific duplex attack by double-stranded RNases (Murray and Crockett (1992)).
• antisense genes must take into consideration that expression levels have to be sufficiently high and be temporally coincident with target gene expression.
• sequence specificity must be assured. This can be achieved by selecting fragments of the nucleic acid sequences encoding psr proteins from translated or from untranslated regions.
• the invention further provides a method for reducing expression of a psr protein of a plant, comprising the step of incorporating into a plant, an isolated nucleic acid which causes co-suppression of genes which are identical to or which have substantial sequence homology to the nucleic acid sequences of psr proteins .
• the invention further provides a method for lowering or increasing the activity of a psr protein of a plant, comprising the step of incorporating into the plant an isolated nucleic acid which causes the production of an altered psr protein such that it is either more active, or is dysfunctional and interferes with the native (naturally-occurring) functional psr protein in any way that its activity is reduced.
• this invention provides means for regulating the response of a photosynthetic organism to varying levels of phosphate in its environment as well as a mechanism for modifying the phosphate metabolism of such organisms.
• This approach to modifying the phosphate pathways of plants has several advantages over traditional plant breeding methods, most importantly, the modifications can be made quickly and specific traits can be modified, even introducing a new trait which is not part of the plant genome.
• Figure 2 is a densitometric scan of an autoradiogra of an SDS-polyacrylamide gel of 3S S- labelled in vi tro translation products of poly(A) + RNA extracted from B . nigra suspension cells grown for 7 days in MS media containing either no P 1 (dotted line) , 1.25 mM P 1 (dashed line) or 10 mM P x (solid line).
• Panel A shows the high molecular weight region of the gel;
• panel B the medium molecular weight region; and
• panel C the low molecular weight region.
• Arrows indicate peaks corresponding to induced polypeptides. Estimated molecular weights are presented on the x-axis.
• Figure 3 is a histogram showing the relative amounts of differentially expressed mRNA species in B . nigra suspension cells cultured in various concentrations of P 2 . The length of each bar represents the area under the corresponding peak and, therefore, the relative abundance of the mRNA species. Peak designations are as in Figure 2. Estimated molecular weights are presented on the y-axis.
• Figure 4 shows northern blots of total RNA extracted from 7-day old (a) minus P, -treated, (b) 1.25 mM P j -fed, and (c) 10 mM Pi-fed B. nigra suspension cells.
• Tub A is the -tubulin gene that was used as a standard. Values given to the left of each panel are size estimates of mRNA species corresponding to the respective psr clones. Each lane contained 30 ⁇ g total RNA.
• Figure 5 shows the DNA sequence (SEQ ID NO:l) of phosphate starvation- induced protein kinase psrPK (psrl ) from Arabidopsis thaliana and the encoded amino acid sequence of the protein kinase (SEQ ID NO: 2) .
• Figure 6 is a comparison of the cDNA sequence (SEQ ID NO:l) encoding the phosphate starvation- induced protein kinase psrPK ⁇ psrl) from Arabidopsis thaliana with the cDNA sequence (SEQ ID NO: 3) encoding a homologous protein kinase from Brassi ca nigra . Boxed residues indicate conserved nucleotides between the two sequences .
• Figure 7 shows the DNA sequence (SEQ ID NO:l) encoding the phosphate starvation-induced psrPK from Arabidopsi s thaliana with its unique 3' terminal sequence capitalized and underlined.
• Figure 8 is a comparison of the amino acid sequences of Arabidopsis thaliana psrPK (psrl ) (SEQ ID NO: 2) and B . nigra psrl (SEQ ID NO:4) with the amino acid sequences of other protein kinases.
• Figure 9 shows a computer analysis of Arabidopsis thaliana psrPK protein deduced amino acid sequence.
• Figure 10 shows the results of a nuclear runoff experiment described in Example 7.
• Figure 11 is a comparison of the 3 ' end of the cDNA sequences of Arabidopsis thaliana psrPK (psrl) and B . nigra psrl with the 3 ' end of the DNA sequences of other protein kinases.
• Figures 12A - 12D depict schematic representations of sense and antisense psrPK constructs in which a constitutive (CaMV-35S) or a seed-specific (Arabin-pro) promoter is fused with the sense (psrl) or antisense ( - psrl ) psrPK genes .
• Figure 13 is a map of the Arabidopsis thaliana clone (determined by Southern blotting of the restriction enzyme-digested DNA probed with the B . nigra psr3 . 1 cDNA) with the location of the psr3.2 indicated by an arrow.
• Figures 14A and 14B show the DNA sequence (SEQ ID NO: 5) of phosphate starvation- induced ⁇ -glucosidase (psr3.2) from Arabidopsis thaliana and its deduced amino acid sequence (SEQ ID NO: 6)
• Figure 15 is the nucleotide (SEQ ID NO: 7) and deduced amino acid sequence (SEQ ID NO: 8) of Brassica nigra psr3 . 1 cDNA clone (psr3.1B) .
• Figure 16 is the nucleotide (SEQ ID NO: 9) and deduced amino acid sequence (SEQ ID NO: 10) of Arabidopsi s thaliana psr3.1 cDNA clone (psr3.1A) .
• Figures 17A and 17B show a comparison of the amino acid sequences of Arabidopsis thaliana psr3 . 2 , psr3.1A, and B . nigra psr3 . 1B with the amino acid sequences of other plant ⁇ -glucosidases.
• Figure 18 is the partial DNA sequence of psr2 (SEQ ID NO: 11 and SEQ ID NO: 12) from Brassi ca nigra .
• Figure 19 is the partial DNA sequence of psr4 (SEQ ID NO: 13 and SEQ ID NO: 14) from Brassica nigra .
• Figure 20 is the partial DNA sequence of psr5 (SEQ ID NO: 15 and SEQ ID NO: 16) from Brassica nigra .
• Figure 21 is the DNA sequence (SEQ ID NO: 17) and encoded amino acid sequence (SEQ ID NO: 18) of psr6 from Brassica nigra .
• Figure 22 is the partial DNA sequence of psr7 (SEQ ID NO: 19 and SEQ ID NO:20) from Brassica nigra .
• Figure 23 is the partial DNA sequence of psr8 (SEQ ID NO: 21) from Brassica nigra .
• Figure 24 is the partial DNA sequence of psr9 (SEQ ID NO: 22 and SEQ ID NO: 23) from Brassica nigra .
• Figure 25 is the partial DNA sequence of psrlO (SEQ ID NO:24 and SEQ ID NO:25) from Brassica nigra .
• Figure 26 is the partial DNA sequence of psrll (SEQ ID NO: 26 and SEQ ID NO: 27) from Brassica nigra .
• Figures 27A-27B show the Southern Blot analysis of Arabidopsis genomic DNA.
• Figure 28 is a diagram of Arabidopsis thaliana transformation and production of subsequent generations.
• the present invention provides novel methods for producing photosynthetic organisms, especially plants, the organisms so produced and methods of their use.
• This invention is based, in part, on the discovery that the transcription and expression of several proteins are induced in phosphate-starved cells of photosynthetic organisms.
• this invention provides isolated DNA encoding at least a functional portion of a protein (psr protein) of a photosynthetic organism in which transcription of the DNA is induced by phosphate deficiency.
• the genes encoding three classes of psr proteins, ser/thr (serine/threonine) protein kinases, ⁇ -glucosidases, and phosphate transporters have been isolated and sequenced .
• all nucleic acids which encode psr polypeptides, and homologues of these psr nucleic acids are encompassed by this invention.
• B . nigra suspension cells were grown in medium containing 1.25 mM P, for 7 days, so that all cells would be in the same metabolic state. The cells were then subcultured into media with various initial concentrations of P x . Growth conditions for the next 7 days were either severe P. deprivation (0 P , mild P 1 deprivation (1.25 mM P.
• a cDNA library was constructed from mRNA isolated from the severely deprived B. nigra cells. Screening by differential hybridization was performed on this cDNA library using cDNA probes prepared from minus P - reated and 10 mM P x -fed (well-fed) B. nigra cells. A number of clones representing mRNA species preferentially transcribed under P, -deficiency were identified. These phosphate-starvation responsive ⁇ psr) clones (121 clones) were placed into eleven different homology groups as determined by cross-hybridization.
• Northern blots showed that the expression of each of the eleven distinct groups of genes is controlled at the level of transcription (Malboobi and Lefebrve, 1995) .
• the Northern blots showed that corresponding genes are inducible in both mild and severe P.-starvation conditions; that is, possible side effects of extremely stressful conditions leading to cell death on the induction of these genes can be ruled out.
• the induced Arabidopsis thaliana gene encodes a polypeptide designated psrPK (or psrl) which, along with the B . nigra polypeptide, has regions of high homology to other protein kinases (see Example 9 and Figure 8, infra) , and possesses serine/threonine (ser/thr) protein kinase activity.
• psrPK or psrl
• a thaliana psrPK and its B . nigra homologue are different from previously described protein kinases because they have a unique C-terminal region of the protein kinase. This unique region could be involved m P, concentration detection or in receiving or delivering signals, or more than one of these functions.
• the protein kinases' substrates could be other components of the phosphate-starvation response pathway or enzymes involved in the response itself. These proteins have no apparent N-termmal signal peptide, organellar targeting sequence or membrane spanning regions, which indicates they probably function in the cytoplasm of the cell.
• Protein kinases catalyze phosphorylation of protein substrates and are found in all living organisms. They are known to be involved in regulatory processes, wherein phosphorylation/dephosphorylation functions as a type of switch for the activation/deactivation (or vice versa) of the substrate protein. Certain types of protein kinases are involved in the phosphate starvation response of fungi and bacteria; however, this is the first time that a plant protein kinase has been shown to be inducible to high levels under P. starvation. Because psrPK is particularly active during periods when phosphate is unavailable, it is probable that it has a switch-like role in the control of the plant response to phosphate deprivation.
• the psrPK protein is homologous to SNF1, which is expressed in carbon-starved bacteria and has been shown to be involved in governing metabolic reactions under such conditions.
• modulation of the expression of the psrPK kinase could alter the expression of whole pathways involved in phosphate metabolism, thereby producing valuable phenotypes.
• a genomic library of A . thaliana was screened at high stringency to isolate the corresponding genomic clone.
• the resultant clone was designated psr3.2 (SEQ ID NO: 5) because of its sequence divergence from isolated psr3.1 cDNA clones.
• Northern blotting with probes derived from the coding region of the genomic clone showed that this gene is expressed at high levels in P.-starved roots and enhancement occurs within two days of growth in medium lacking P x .
• this gene is repressed by heat shock and anaerobic conditions, and it is not significantly induced by high salinity, or by nitrogen or sulphur deprivation.
• Sequence analysis of the genomic clone revealed the existence of thirteen exons interrupted by twelve AT-rich introns and shows high homology with the B . nigra psr3.1B, as well as various other ⁇ -glucosidase genes from other species. Sequence similarity and divergence percentages between the deduced amino acid sequences of the psr3 clones and other ⁇ -glucosidases suggests that these genes should be included along with two other Brassicaceae genes in a distinct subfamily of the BGA glycosidase gene family. The presence of an endoplasmic reticulum retention signal at the carboxy terminus indicates that this is the cellular location of psr3.2. The possible metabolic and regulatory roles of this enzyme during the Pj- starvation response are described infra .
• the DNA sequence of psr4 from Brassica nigra most closely resembles DNA encoding an envelope protein
• the DNA sequence of psr5 from Brassica nigra most closely resembles DNA encoding an aspartate kinase ( Figure 20) .
• This sequence shows some homology to DNA encoding disintegrin, another aspartate kinase, which inhibits the signal transduction pathway for the cell cycle .
• the DNA sequence of psr7 from Brassica nigra most closely resembles DNA encoding a histidine kinase at the T3 portion of the sequence and a skeletal muscle calcium release channel protein at the T7 portion of the sequence ( Figure 22) .
• the DNA sequence of psrlO from Brassica nigra most closely resembles DNA encoding a calcium channel protein (G-protein) ( Figure 25) .
• the DNA sequence of psrll from Brassica nigra most closely resembles DNA encoding a phosphatidylinositol kinase at the T3 portion of the sequence and a tripeptidyl peptidase protein at the T7 portion of the sequence ( Figure 26) .
• nucleic acid includes DNA and RNA, as well as single-stranded and double-stranded species.
• DNA or nucleic acids referred to herein as “isolated” are DNA or nucleic acids separated away from the nucleic acids of the genomic DNA or cellular RNA of their source or origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library) , and may have undergone further processing.
• isolated DNA or nucleic acids include DNA or nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure DNA or nucleic acids, DNA or nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids which are isolated.
• Nucleic acids referred to herein as "recombinant” are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial recombination, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes.
• “Recombinant” nucleic acids are also those that result from recombination events that occur through the natural mechanisms of cells, but are selected for after the introduction to the cells of nucleic acids designed to allow and make probable a desired recombination event.
• the isolated DNA can comprise: (a) SEQ ID NO:l, SEQ ID NO: 3, or a truncated nucleic acid sequence which encodes a functional portion of the protein encoded by SEQ ID NO:l or SEQ ID NO : 3 or (b) a nucleic acid sequence having at least 80% homology to SEQ ID NO : 1 , SEQ ID NO : 3 , or a truncated nucleic acid sequence which encodes a functional portion of the protein encoded by a nucleic acid sequence having at least 80% homology to
• a "functional portion” means a portion of a psr protein which when expressed will affect the phosphate uptake and/or metabolism of the native (naturally- occurring) photosynthetic organism in which the endogenous psr protein is produced.
• a preferred embodiment is a truncated DNA sequence comprising isolated DNA consisting of nucleotide residues 677 to 1020 of SEQ ID NO : 1 , encoding the unique region of the protein kinase.
• DNA or RNA having 50% homology, preferably 80% homology, or more preferably 90% homology, or which hybridizes under moderately stringent conditions to the DNA of Claim 3.
• Truncated nucleic acid sequences of the above-described DNA or nucleic acids which consist of 10-20 or more contiguous nucleotides are also provided and can find use as probes and primers.
• One cDNA of this invention is shown in Figure 5 (SEQ ID NO:l) and comprises a 1020 nucleotide open reading frame, bounded by ATG start and TAG stop codons, encoding 339 amino acids.
• the cDNA further comprises 89 bp 5' untranslated nucleotides and 141 bp 3' untranslated nucleotides, including a 3 1 polyA tail for a functional mRNA.
• the protein encoded by this cDNA is discussed in detail below.
• the isolated DNA further comprises: (a) SEQ ID NO:l) and comprises a 1020 nucleotide open reading frame, bounded by ATG start and TAG stop codons, encoding 339 amino acids.
• the cDNA further comprises 89 bp 5' untranslated nucleotides and 141 bp 3' untranslated nucleotides, including a 3 1 polyA tail for a functional mRNA.
• the protein encoded by this cDNA
• nucleic acids Polypeptides encoded by these nucleic acids are also encompassed by this invention.
• an isolated nucleic acid encoding a protein having ⁇ - glucosidase activity and an amino acid sequence with at least 80% sequence homology with SEQ ID NO: 5 or 50% homology with SEQ ID NO : 6 is also provided, as is a nucleic acid encoding a protein having phosphate transporter activity and an amino acid sequence with at least 80% sequence homology with SEQ ID NO 17 or 50% homology with SEQ ID NO: 18.
• Truncated nucleic acid sequences of the above-described DNA or nucleic acids which consist of 10-20 or more contiguous nucleotides are also provided and can find use as probes and primers .
• homology does not refer to common evolutionary origin, but rather to similarity between sequences.
• the degree of homology between two sequences can be determined by optimally aligning the sequences for comparison, as is commonly known in the art, and comparing a position in the first sequence with a corresponding position in the second sequence. When the compared positions are occupied by the same nucleotide or amino acid, as the case may be, the two sequences are homologous at that position.
• the degree of homology between two sequences is expressed as a percentage representing the ratio of the number of matching or homologous positions in the two sequences to the total number of positions compared.
• sequence in question has slight or insignificant sequence variations from, for example, the sequences of SEQ ID N0:1, SEQ ID NO : 3 , SEQ ID NO: 2 or SEQ ID NO : 4. That is, a nucleic acid sequence "having substantial sequence homology" to SEQ ID NO:l or SEQ ID NO : 3 encodes substantially the same protein product as the actual sequence; i.e., a protein kinase having a regulatory function in phosphate metabolism. It is expected that certain substitutions or other alterations will be able to be made in various portions of SEQ ID NO : 1 or SEQ ID NO : 3 which do not significantly affect protein function. The sequence variations may derive from mutation.
• a protein having a homologous sequence to that of SEQ ID NO: 2 or SEQ ID NO: 4 would have a similar catalytic activity to that of the protein whose sequence is shown in SEQ ID NO: 2 or SEQ ID NO : 4.
• isoforms of the protein of SEQ ID NO: 2 or SEQ ID NO : 4 that have protein kinase activity could exist.
• a sequence having substantial homology to that of SEQ ID NO: 2 can be a homologue from another plant variety or species, as is SEQ ID NO: 4.
• Such isoforms and homologous proteins may be immunologically cross-reactive.
• nucleic acid encoding a protein comprising an amino acid sequence having about 80% homology with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or about 50% homology with the amino acid residues 190 to 340 of SEQ ID NO: 2, will produce a functional protein kinase, and the invention provides such a nucleic acid.
• Proteins comprising an amino acid sequence that is about 60%, 70%, 80% or 90% homologous with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:4, or greater, are also expected to have protein kinase activity.
• the invention encompasses isolated nucleic acids encoding a protein having protein kinase activity, and having a sequence that differs from the nucleotide sequence of SEQ ID NO:l or SEQ ID NO : 3 due to degeneracy in the genetic code.
• “Degeneracy” is understood to mean that each of several different amino acids is designated by more than one nucleotide triplet or codon .
• AAA and AAG each code for lysine. This is an example of a "silent mutation" occurring in the third (or "wobble") nucleotide of a codon wherein the amino acid encoded remains the same.
• the invention also encompasses mutations that are not silent or other alterations wherein at least 80% amino acid homology with SEQ ID NO: 2 or SEQ ID NO : 4 , or at least 50% homology with amino acid residues 190 to 340 of SEQ ID NO:2, is maintained.
• nucleic acid and amino acid sequences having substantial sequence homology to any of SEQ ID NO: 5 through SEQ ID NO: 27 are also encompassed by this invention .
• the invention includes different forms of the nucleic acids of the invention arising from alternative splicing of an mRNA corresponding to a cDNA of the invention.
• the invention further provides a nucleic acid that hybridizes under high or moderate stringency conditions to a nucleic acid encoding at least a portion of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or other psr proteins or polypeptides.
• Stringency conditions for hybridization is a term of art which refers to the conditions of temperature and buffer concentration which permit hybridization of a particular nucleic acid to another nucleic acid in which the first nucleic acid may be perfectly complementary to the second, or the first and second may share some degree of complementarity which is less than perfect. See, sections 2 and 6 in Current Protocols in Molecular Biology (Ausubel, F.M. et al . , eds . , Vol. 1, Supplement 29, 1995) . Hybridization conditions are described generally in Mamatis et al . , 1982 and Sambrook et al . , 1989.
• High stringency hybridization procedures can (1) employ low ionic strength and high temperature for washing, such as 0.015 M NaCL/0.0015 M sodium citrate, pH 7.0 (O.lx SSC) with 0.1% sodium dodecyl sulfate (SDS) at 50°C; (2) employ during hybridization, 50% (vol/vol) formamide with 5x Denhardt ' s solution (0.1% weight/volume highly purified bovine serum album ⁇ n/0.1% wt/vol F ⁇ coll/0.1% wt/vol polyvmylpyrrolidone) , 50 mM sodium phosphate buffer at pH 6.5 and 5x SSC at 42°C; or (3) employ hybridization with 50% formamide, 5x SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate , 5x Denhardt ' s solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°
• This invention also provides nucleic acids and polypeptides with structures that have been altered by different means, including but not limited to, alterations using transposons, site-specific and random mutagenesis, and engineered nucleotide substitution, deletion, or addition.
• This invention also relates to psr proteins or psr polypeptides, for example the proteins encoded by SEQ ID N0:1, SEQ ID NO : 3 , SEQ ID NO : 5 , SEQ ID NO : 7 , SEQ ID
• proteins and polypeptides of the present invention can be isolated and/or recombinant .
• Proteins or polypeptides referred to herein as "isolated” are proteins or polypeptides purified to a state beyond the naturally-occurring state in which they exist endogenously in cells.
• a preferred embodiment is an essentially pure protein or polypeptide free of other proteins or polypeptides.
• isolated proteins or polypeptides include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, including essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis, or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated.
• Proteins or polypeptides referred to herein as "recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids. In their native state, the transcription of these proteins is induced under conditions of phosphate deficiency and the proteins have varying activities which affect levels and kinds of phosphate content in cells, such as protein kinase, ⁇ -glucosidase, and phosphate transporter activity.
• amino acid sequence of these proteins shows at least 80% sequence homology with SEQ ID NO : 2 , SEQ ID NO : 4 , SEQ ID NO: 6, SEQ ID NO: 18 or 50% homology with amino acid residues 190 to 340 of SEQ ID NO:2.
• Nucleic acids or portions thereof provided by this invention can be used to isolate homologous nucleic acids from cells of other species of photosynthetic organisms which contain genes encoding one or more psr proteins similar in function to the nucleic acids of this invention.
• psr protein means a protein whose expression is induced to higher levels under conditions of phosphate deficiency than under conditions of phosphate sufficiency. The protein may not be expressed at all if the cell has adequate levels of available phosphate. Further such a protein is not associated with mechanisms which bring about cell death.
• the inventors have isolated nucleic acids of the invention using B . nigra suspension cells as starting material.
• the inventors created a cDNA library from B . nigra suspension cells that had been starved for phosphate for seven days.
• the library was screened using two sets of cDNA probes generated from B . nigra suspension cells that had been grown under conditions of no phosphate or 5 mM phosphate, respectively. Clones that hybridized strongly to the first set of probes, but not the second set, were isolated.
• the cDNA inserts of these clones were subjected to dideoxy nucleotide sequencing (Sanger, 1981) to determine nucleotide sequence, and amino acid sequence was predicted therefrom.
• the psrPK gene was recognized to encode a novel protein kinase.
• Other differential hybridization, cloning and sequencing methods are known to those skilled in the art, and can be employed to obtain the protein kinase genes isolated by the inventors, other psr genes, or homologues thereof .
• a nucleic acid of the invention can be isolated in the following manner.
• a nucleic acid probe comprising at least a portion of the sequence of SEQ ID NO:l (or another psr sequence) , or a homologue is chemically synthesized or prepared using recombinant DNA techniques.
• the probe is radiolabelled and used to screen a cDNA or genomic DNA library according to standard techniques. It can be prepared from B. nigra , a different organism or another source having a homologue or transgene of the invention that could be identified under appropriate hybridization conditions.
• the DNA identified by screening the library is then cloned and sequenced using standard techniques.
• a third alternative method for isolating a nucleic acid of the invention is to isolate or chemically synthesize a peptide of the protein kinase (or another psr gene) and use this peptide to produce an antibody to the encoded psr protein in an animal.
• the antibody is then used according to standard techniques to screen a cDNA library, from B . nigra or another source, for lmmunoreactive clones. DNA from such clones are then sequenced as is known in the art.
• a fourth alternative method for isolating a nucleic acid of the invention is to selectively amplify such a nucleic acid using polymerase chain reaction (PCR)
• RNA DNA or RNA as a template.
• total mRNA may be isolated from cells using one of the methods common in the art, described in Maniatis et al . , 1982.
• the retroviral enzyme reverse transcriptase is then used to synthesize cDNA complementary to the mRNA.
• Appropriate oligonucleotide primers for the amplification of the chosen nucleic acid are designed and synthesized, and PCR performed on a mixture of the primers and cDNA using standard technology (Innis et al . , 1990).
• the PCR protocol can additionally include 5' or 3 ' RACE (rapid amplification of cDNA ends) methodology (Innis et al . , 1990) .
• the amplified DNA fragment produced is cloned into an appropriate vector.
• An RNA molecule of the invention can also be constructed by cloning an appropriate cDNA or an amplified DNA molecule as described above into one of the commonly available transcription vectors.
• the DNA would usually be cloned downstream of a promoter, for example, the SP6 promoter of the vector pGEM 3Z
• RNA polymerase m this example, SP6 polymerase
• transcription reactions performed according to the manufacturer's specifications.
• Other useful promoters carried by commonly used transcription vectors are the bacteriophage T7 and T3 promoters .
• Another well-known method of producing a nucleic acid or oligonucleotide of the invention is chemical synthesis.
• Various machines for DNA synthesis are well- known in the art, such as, for example, those sold by Applied Biosystems, Inc. of Foster City, CA and by Millipore Corp. of Bedford, MA, and can be used for such syntheses.
• recombinant nucleic acids are constructed in which psrPK or another psr protein is operatively linked to regulatory sequences, such as promoters, that control the level, timing or tissue-specificity of gene expression.
• Standard recombinant DNA techniques can be employed to engineer a recombinant expression vector including a nucleic acid of the invention that allows expression of at least a portion of a protein kinase or other psr protein of the invention. See, e.g., Sambrook, et al . , 1989.
• the engineered vector would also include a regulatory sequence "operatively linked" to the nucleic acid of the invention to allow such expression.
• regulatory sequence includes promoters, enhancers and other sequences that control expression or message stability, as are well-known in the art. Examples of known promoters suitable for these purposes are given infra . Those of skill in the art can recognize that these examples are not limiting and other promoters can be adapted for particular purposes of modulating the phosphate uptake and metabolism of photosynthetic organisms .
• the regulatory element can provide tissue-specific expression.
• the two-part term "operatively linked” means both that the regulatory sequence contains sufficient element (s) to allow expression of the nucleic acid in question and that the nucleic acid is linked to the regulatory sequence appropriately.
• the nucleic acid of the invention is in the appropriate orientation and in phase with an initiation codon.
• promoter or “promoter region” refers to a sequence of DNA, usually upstream (5 1 ) to the coding sequence of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and/or other factors required for transcription to start at the correct site.
• constitutive and inducible promoters There are generally two types of promoters, constitutive and inducible promoters.
• constitutive does not necessarily indicate that a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in abundance is often detected.
• An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically a protein factor (or factors) , that binds specifically to an inducible promoter to activate transcription, is present in an inactive form which is then directly or indirectly converted to an active form by the inducer.
• the inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus.
• the inducer can also be an illumination agent such as light, darkness and light's various aspects which include wavelength, intensity, fluence, direction and duration.
• a cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods. If it is desirable to activate the expression of a gene at a particular time during plant development, the inducer can be so applied at that time.
• inducible promoters include heat shock promoters, such as the inducible hsp70 heat shock promoter of Drosphilia melanogaster (Freeling, et al . , 1985; a cold inducible promoter, such as the cold inducible promoter from B . napus (White, et al . , 1994,); and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, et al . , 1986).
• sequences known to be useful in providing for constitutive gene expression are regulatory regions associated with Agrobacterium genes, such as nopaline synthase (Nos) , mannopine synthase (Mas) or octopine synthase (Ocs) , as well as regions regulating the expression of viral genes such as the 35S and 19S regions of cauliflower mosaic virus (CaMV) (Brisson, et al . 1984), or the coat promoter of TMV (Takamatsu, et al . , 1987) .
• Other useful plant promoters include promoters which are highly expressed in phloem and vascular tissue of plants such as the glutamine synthase promoter (Edwards, et al .
• the maize sucrose synthetase 1 promoter (Yang et al . , 1990), the promoter from the Rol- C gene of the TLDNA of Ri plasmid (Sagaya, et al . , 1989) , and the phloem-specific region of the pRVC-S-3A promoter (Aoyagi , et al . , 1988) .
• plant promoters such as the small subunit of Rubisco (Rbcs) promoter (Coruzzi, et al . , 1984; Broglie, et al . , 1984), or heat shock promoters, e.g., soybean HPS17.5-E or HPS17.3-B (Gurley, et al . , 1986) can be used.
• promoters which can be used according to the present invention include, but are not limited to:
• root and shoot promoters such as the ACS1 promoter (Rodrigues-Pousada et al . , 1993);
• seed-specific promoters such as the 22 kDa zein protein from maize (Unger et al . , 1993) the psl lectin promoter from pea (de Pater et al . , 1993), the phaseolin promoter from Phaseolus vulgaris (Frisch et al . , 1995) ;
• late embryo-abundant promoters such as the lea promoter (T.L. Thomas, 1993);
• fruit-specific promoters such as the E8 gene promoter from tomato, (Cordes et al . , 1989);
• PCNA promoter (Kosugi et al . , 1995);
• pollen-specific promoters such as the NTP303 promoter (Weterings et al . , 1995);
• late embryogenesis stage-specific promoters such as the OSEM promoter (Hattori et al . , 1995);
• ADP-glucose pyrophosphorylase tissue-specific promoters for guard cells and tuber parenchyma cells such as the ADP GP from potato (Muller-Rober et al . , 1994) ;
• conductive tissue-specific promoters such as the Myb promoter from barley (Wissenbach et al . , 1993); and
• Plastocyanin promoters in young green tissues such as the plastocyanin promoter from Arabidopsis (Vorst et al . , 1993) .
• a plant transformed with a recombinant nucleic acid of this invention would over- or under-express a psr protein, either in chosen plant parts or throughout the plant, and/or at different times in the life history of the plant. Changes in plant size, relative sizes of different plant parts, time of flowering, level of phytate, starch and oil accumulated in seeds, or other phenotypic characteristics can thus be engineered.
• a recombinant expression vector can be engineered for expression of a psr protein, such as psrPK, in prokaryotic cells, for example, Escherichia coli , or in eukaryotic cells, for example, Saccharomyces cerevisiae (yeast) and Arabidopsi s thaliana , tobacco or canola.
• the recombinant expression vector can be a plasmid, a bacteriophage or a virus.
• Plant gene constructs of the present invention can be introduced using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and the like, as described, supra .
• Common expression vectors often include a marker gene that permits easy screening for transformed cells.
• Some common vectors also include a sequence encoding at least a portion of another functional protein, such as firefly luciferase or bacterial ⁇ -galactosidase .
• a nucleic acid of the invention would be linked in frame to this coding sequence such that a fusion protein would be produced comprising at least a portion of the protein kinase of the invention and the other functional protein.
• Cells transformed with the engineered vector can be screened for expression of the luciferase, ⁇ - galactosidase or other fused protein.
• the other protein fused to the psr protein may not be useful for screening, but can instead provide a useful property, such as increased solubility, or can be exploited in a protein purification scheme or in industrial applications such as the addition of purified enzyme to a reaction.
• the vectors of this invention can be constructed containing nucleic acids encoding a psr protein with which to transform a wide variety of crop and horticultural plants, including monocots, dicots and gymnosperms . Modification can be targeted to the whole plant, or to a specific tissue, organ or plant part, such as a seed. Further, expression of the gene can be limited to particular developmental stages or environmental conditions.
• the gene delivery systems used to incorporate the constructs will vary depending on the target plant species; however, those of skill in the art can recognize that present molecular techniques can be applied to successfully modify particularly useful crop plants, such as rice, wheat, barley, rye, corn, soybeans, canola, sunflower, oranges, grapefruit, lemons, limes, potato, carrots, sweet potato, beans, peas, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, pepper, carrots, pumpkins, cucumber, apples, pears, melons, plum, cherry, peaches, nectarines, apricot, strawberry, grape, raspberry, pineapple, tobacco, bananas, sorghum, sugarcane, and the like.
• particularly useful crop plants such as rice, wheat, barley, rye, corn, soybeans, canola, sunflower, oranges, grapefruit, lemons, limes, potato, carrots, sweet potato, beans, peas, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, onion
• ⁇ kinase protein kinase
• canola canola
• tobacco and Arabidopsis have been genetically engineered to include vectors designed both to lower and to increase the amount of protein kinase within the entire plant or in the developing seeds.
• eight different constructs were incorporated into Arabidopsis cells with either constitutive (CaMV 35S) or seed-specific (Arabin) promoters. These constructs are described in general in Table 1 below.
• 35S promoter CaMV 35S C7 full length anti-sense psrl in pBI121 with CaMV 35S promoter Dl full length sense psrl in pBI121 with seed specific arabm promoter D4 full length anti-sense psrl in pBI121 with seed specific arabm promoter No .2 full length psrl with a mutated ATP binding site in pBI121 with
• CaMV 35S promoter (Lys changed to Glu at ammo acid 33) No.26 full length psrl with deleted Lys (ammo acid 33) in ATP binding site n pBI121 with CaMV 35S promoter No.6 full length psrl with mutated kinase active site in pBI121 with CaMV 35S promoter (Asp changed to Glu at amino acid 123) .
• psrl full length psrl in pBI121 with CaMV 35S promoter (identical to Cl).
• Transformation 170 plants / construct/ transformation experiment trans ormation experiments: A, B, ...
• the invention provides host cells transformed with a recombinant expression vector of the invention.
• the terms “transformed with”, “transformant “ , “transformation”, “transfect with”, “transfectant “ and “transfection” all refer to the introduction of a nucleic acid into a cell by one of the numerous methods known to persons skilled in the art. Transformation of prokaryotic cells, for example, is commonly achieved by treating the cells with calcium chloride so as to render them "competent” to take up exogenous DNA, and then mixing such DNA with the competent cells. Prokaryotic cells can also be infected with a recombinant bacteriophage vector. Nucleic acids can be introduced into cells of higher organisms by viral infection, bacteria-mediated transfer ( e .
• nucleic acid constructs of this invention can also be incorporated into specific plant parts such as those described infra through the transformation and transfection techniques described herein.
• the constructs of this invention are further manipulated to include genes coding for plant selectable markers.
• Useful selectable markers include enzymes which provide for resistance to an antibiotic such as gentamycin, hygromycin, kanamycin, or the like.
• the NOS/NPTII kanamycin-resistant gene is used to detect transfected plant cells.
• enzymes providing for production of a compound identifiable by color change such as GUS ( ⁇ -glucuronidase) , or by luminescence, such as luciferase, are useful.
• the invention provides transformant cells in which the introduced DNA has integrated into the genome, and transformant cells in which the introduced DNA exists as an extrachromosomal element. In the latter case, maintenance of an extrachromosomal element can be easily obtained by including a selectable marker in the recombinant expression vector and then, after introduction of the vector, growing the cells under conditions where expression of the marker gene is required.
• cells transformed with a recombinant expression vector of the invention are screened using protein kinase activity as a selectable marker. Such transformed cells can be screened, for example, under conditions of phosphate starvation.
• T2 generation seed from initially transformed plants has been obtained from constructs Nos . 2, 6, and 26, and from construct psrl.
• T3 generation seed from 10, 11, 18 and 4 kanamycin- resistant plants has been obtained from constructs Cl , C7, Dl, and D4 , respectively.
• Plants and other photosynthetic organisms containing the nucleic acid constructs described herein are further provided by this invention.
• photosynthetic organism is meant to include the members of the kingdom Planta, including vascular and nonvascular plants (angiosperms, gyrnnosperms , ferns, mosses, bryophytes, etc.), the algae, photosynthetic protists (single-celled eukaryotes) , the Cyanophyta (blue-green algae or cyanobacteria) and the photosynthetic bacteria.
• Suitable plants include both monocotyledons and dicotyledons. Examples of preferred monocotyledons are commercially-important crop plants such as rice, corn, wheat, rye, sugarcane and sorghum.
• Examples of preferred dicotyledons are canola, sunflower, citrus, tomato, broccoli, and lettuce.
• Algae can be used as a hosts for the constructs described herein. Examples of such algae are Chlamydomonas reinhardtii , Chlamydomonas moewusii , Euglena gracilis, Porphyra purpurea , Cryptomonas sp . , and Ochromonas sinensis .
• Prokaryotes can also provide suitable host cells. Specific examples include Anacystis nidulans, Synechococcus sp . , Rhodobacter sphaeroides, Rhodobacter capsulatus, Chloroflexus aurantiacus , and Heliobacterium chlorum.
• constructs of the present invention can be introduced into plants, plant parts, or other cells of photosynthetic organisms using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro- injection, electroporation, etc.
• Ti plasmids Ri plasmids
• plant virus vectors direct DNA transformation, micro- injection, electroporation, etc.
• transformed plant cells are cultured in an appropriate medium, which can contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells.
• Selected transformed plant cells can be induced to form callus tissue. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants .
• transformants or descendants of transgenic plants so produced can be evaluated with respect to growth and productivity.
• Transgenic plants can also be assessed for flowering characteristics, root and shoot size ratios, and seed phytate, starch and oil content.
• the transformants would be used directly or could be subjected to further modifications by genetic engineering or classical techniques.
• Transgenic plants containing the constructs of this invention can also be regenerated from plant tissues, plant parts, or protoplasts by methods known to those of skill in the art and as shown in Figure 28.
• Plant part is meant to include any portion of a plant capable of producing a regenerated plant.
• this invention encompasses cells, tissue (especially meristematic and/or embryonic tissue), protoplasts, epicotyls, hypocotyls, cotyledons, cotyledonary nodes, pollen, ovules, stems, roots, leaves, and the like. Plants can also be regenerated from explants. Methods will vary according to the plant species.
• Seed can be obtained from the regenerated plant or from a cross between the regenerated plant and a suitable plant of the same species.
• the plant can be vegetatively propagated by culturing plant parts under conditions suitable for the regeneration of such plant parts.
• the plants can then be used to establish repetitive generations containing an altered genotype with respect to phosphate- inducible psr protein activity, either from seeds or using vegetative propagation techniques (see, e . g. , Figure 28).
• the invention further encompasses homology-dependent gene silencing including silencing for the nucleic acid of the invention mediated by DNA-DNA pairing and by RNA.
• DNA-DNA pairing DNA-DNA pairing
• RNA-mediated silencing includes antisense technologies in which a nucleic acid or oligonucleotide that is antisense to a nucleic acid of the invention is introduced into cells.
• Such an antisense molecule is capable of base-pairing (hydrogen bonding) with the nucleic acid of the invention in an anti-parallel manner, according to the standard pair rules, i.e., G pairs with C, and A pairs with T or U.
• the antisense molecule can be complementary to a coding or non-coding region of a nucleic acid of the invention, including a non-coding regulatory region, or to portions of both (Gogarten et al . , 1992; Shotkoski and Fallon, 1994).
• the region of complementarity can precede or span the first codon of SEQ ID N0:1.
• An antisense molecule according to the invention can include a region complementary to a regulatory sequence, for example a non-coding regulatory sequence that is operatively linked to a gene of the invention in an expression construct.
• catalytic antisense RNA directed at the psr protein gene transcript (a ribozyme) can be employed to reduce gene expression (Heinrich et al . , 1993; de Feyter et al . , 1996).
• the antisense molecule can be produced by chemical synthesis, PCR or an expression vector, using certain of the techniques discussed in the previous section, or by other standard techniques. (Meyer, P. and Saedler, H., 1996) .
• Antisense constructs include, but may not be limited to, the following: 1. A whole psr gene placed in reverse orientation in respect to a promoter; 2. An antisense sequence complementary to the unique 3' region (nucleotides 930 through 1272) of Figure 7 ;
• An antisense sequence complementary to the 3'- untranslated region up to the polyA tail (nucleotides 1008-1240) ; or
• Sense or antisense nucleic acid according to the invention can be delivered to cells using any of a variety of methods known to persons skilled in the art.
• Xs.c_lated Proteins This invention provides an isolated protein having protein kinase, ⁇ -glucosidase, phosphate transporter, or other psr protein activity and, in particular, an amino acid sequence with substantial sequence homology with the amino acid sequence shown as SEQ ID NO: 2, SEQ ID N0:4, SEQ ID NO:6, SEQ ID N0:8, SEQ ID NO:10, or SEQ ID NO: 18.
• an isolated protein of the invention can be encoded by a nucleic acid that hybridizes under low or high stringency conditions to the nucleotide sequence of SEQ ID NO : 1 , SEQ ID NO : 3 , SEQ ID NO: 5, SEQ ID NO : 7 , SEQ ID NO : 9 , SEQ ID NO: 11 through SEQ ID NO: 17, SEQ ID NO: 19 through SEQ ID NO: 27, or a protein encoded by any psr gene. It should be understood that the invention includes protein fragments demonstrating such homology and having psr protein activity (i.e., functional portions).
• the mature, unmodified protein having the amino acid sequence shown as SEQ ID NO : 2 is predicted to have a molecular weight of 39,040 kDa. Prosite searches were used to determine the following characteristics of the PSRPK protein. It contains ser/thr protein kinase active site between amino acid positions 119-131, and an ATP-binding site between positions 9-33. An hydrophobicity plot of the protein does not indicate any long regions of membrane associated protein and an antigenicity plot of the protein indicates several areas that would be appropriate for employment as peptides for antibody production against the protein. These include but are not restricted to the last 150 amino acids at the C-terminus.
• N-terminal signal peptide or organellar targeting sequences the protein appears to be cytoplasmically localized.
• potential phosphorylation sites which include, but may not be restricted to, positions that are indicated by motifs for PKC, CK2 , and tyrosine kinase phosphorylation sites as shown in Figure 9. These may or may not be autophosphorylation sites.
• a psr protein of the invention can be purified from cells or from culture medium into which it has been secreted. Alternatively, it can be chemically synthesized, as is well-known in the art. As discussed above, for the purposes of this disclosure, the term “isolated” means that the protein is substantially free of other cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when produced by chemical synthesis .
• the invention further provides a method of preparing a protein having protein kinase activity, ⁇ - glucosidase activity, phosphate transporter activity, or other psr protein activity, or a fragment of such a protein, which includes the following steps: (i) transforming cells with a recombinant expression vector including a nucleic acid of the invention and a regulatory sequence operatively linked to the nucleic acid, (ii) culturing the transformant cells in a suitable medium until the psr protein is formed, and (iii) isolating the psr protein.
• a person skilled in the art would be able to devise a scheme for the isolation of the protein from other cellular material and culture medium using conventional techniques (Scopes, 1982) . These include chromatographic methods such as gel filtration, ion-exchange chromatography and affinity chromatography, as well as batch methods employing ion-exchange or affinity resins. Precipitation with ammonium sulfate, followed by resuspension and dialysis is another common purification and concentration protocol.
• a fusion protein comprising at least a functional portion of the psr protein of the invention can be prepared by the method whose steps are detailed above.
• the psr protein or functional portion thereof can be fused to a signal sequence which directs secretion of the fusion protein from the transformant cells.
• the secreted protein can be isolated using standard techniques.
• the invention encompasses an antibody that is specific for a psr protein of this invention.
• the invention includes both polyclonal and monoclonal antibodies.
• the term "antibody” includes antibody fragments that are specific for a psr protein, such as a protein kinase, ⁇ - glucosidase, or phosphate transporter as described herein. Such fragments include F ab fragments generated by proteolysis of an antibody.
• an antibody can be directed to an epitope unique to the psr protein of the invention. If the protein is a protein kinase of this invention, for example, these can include the protein kinase active site and the ATP-binding site as well as the unique C-terminus sequence of 150 amino acids .
• Intact psr proteins or an i munogenic fragment thereof can be used to prepare antibodies.
• the protein or fragment chosen as the antigen can be injected into an animal (e.g., rabbit, hamster, goat, mouse), causing the animal to produce antibodies specific to the injected antigen.
• the antigen is often combined with an adjuvant, such as Freund's adjuvant.
• an adjuvant such as Freund's adjuvant.
• the antigen is conjugated to a hapten, or carrier molecule.
• a person skilled in the art would be aware of appropriate antigen dosages for the size and species of animal, how to design a schedule of repeated injections if required, and how to titer and purify the antibody raised in the animal's serum.
• lymphocytes raised against the antigen are first harvested, and then fused with myeloma cells using standard procedures (Harlow and Lane, 1988) .
• the immortalized hybridoma cells so produced are screened for psr protein-specific antibodies using conventional immunoassay methods such as ELISA (enzyme-linked immunosorbent assay) .
• the antibodies can then be purified as is known in the art.
• An antibody of the invention can be physically coupled to any of a number of detectable substances that are known in the art. These include: a radioisotope, a fluorescent molecule, and an enzyme capable of catalyzing a colorimetric reaction. Examples of such an enzyme include alkaline phosphatase and horseradish peroxidase, which are commonly used in laboratory assays.
• this invention provides a method of detecting the expression of a phosphate-starvation inducible protein, such as a protein kinase, a ⁇ -glucosidase, or a phosphate transporter in a plant or plant part or other photosynthetic organism comprising: (a) inducing protein expression by depriving the plant, plant part, or other photosynthetic organism of sufficient levels of available phosphorus; (b) contacting a portion of a plant, plant part, or other photosynthetic organism with antibody to the protein so that an antibody : antigen complex is formed by the binding of the antibody to an epitope of the protein; and (c) detecting the antibody : antigen complex; wherein the detection of the antibody : antigen complex is indicative of the expression of the protein.
• a phosphate-starvation inducible protein such as a protein kinase, a ⁇ -glucosidase, or a phosphate transporter in a plant or plant part or other photosy
• a more modern approach to modifying the characteristics of plants (and other photosynthetic organisms) is to subject plants to mutagenesis by radiation or chemical treatmen . Such exposure randomly generates mutations in the DNA molecules comprising the plant genome which sometimes produces the desired traits.
• the mutagenized plants are screened for the traits and subsequently bred. While mutagenesis has the advantage of producing variations in plant DNA much faster than natural selection, it is not possible to select and generate preferred traits; the process is random. Further, exposing plants to mutagenic agents can induce additional, undesirable mutations to the plant genome. Some of these may not be immediately apparent and, further, may not be able to be "bred out" of a plant carrying a useful mutation.
• the nucleic acids and vectors of the invention can also be used to produce transgenic plants and other photosynthetic organisms which express the protein of the invention.
• the genome of a transgenic organism includes an integrated DNA transgene that was introduced either into that particular organism or into its ancestor.
• the introduced DNA including the transgene can comprise regulatory element (s) appropriate for the type of organism or tissue being transfected.
• a nucleic acid of the invention when introduced as a transgene, can be operatively linked to a tissue-specific DNA regulatory sequence such that protein kinase is specifically produced in the target tissue.
• a seed- specific promoter would permit expression of the protein kinase only in seeds.
• a suitable 3' region such as the 3 ' region containing a polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes can be included.
• other suitable 3' sequences derived from any characterized gene from plants as well as from other organisms such as animals, if they are deemed appropriately functional m the environment of a transgenic plant cell can be used.
• the constructs of this invention can be further manipulated to include genes coding for plant selectable markers.
• genetic engineering provides a method of producing a transgenic plant or other photosynthetic organism having altered growth, reproduction, or metabolic content by introducing into a cell or tissue of a plant or other photosynthetic organism, an exogenous nucleic acid which encodes a psr protein, such as a ⁇ - glucosidase, protein kinase or phosphate transporter, which is transcribed under conditions of phosphate deprivation in naturally-occurring species in which it occurs, and whose presence in the transgenic plant or photosynthetic organism results in altered growth, reproduction, or metabolic content, and by maintaining the cell or tissue containing the exogenous nucleic acid under conditions appropriate for growth of the cell or tissue, whereby a transgenic plant or other photosynthetic organism having an altered growth, reproduction, or metabolic content can be produced.
• a psr protein such as a ⁇ - glucosidase, protein kinase or phosphate transporter
• nucleic acids comprising the psrPK, psr3.2 , psr3.1A, psr3.
• IB genes and homologues can be used in this method to produce a transgenic plant from a species which belongs to the vascular plants including angiosperms, gymnosperms, monocots, and dicots, the nonvascular plants, and the algae.
• plants and other photosynthetic organisms are provided wherein the naturally-occurring phosphate-starvation induced protein kinase or ⁇ -glucosidase activity is increased. Because the introduced gene is stably integrated into the genome, seed of a transgenic plant and, therefore future generations of descendants, with this alteration, are also provided by this invention.
• Photosynthetic organisms have evolved a number of adaptive strategies to cope with growth-limiting amounts of exogenous inorganic phosphate. These strategies include enhancing the availability of endogenous phosphate (Lefebvre et al . , 1990; Sachay et al . , 1991), and using it efficiently in order to maintain essential metabolic pathways (Duff et al . , 1994), as well as, in times of plenty, storing excess phosphate in vacuoles (Lee et al . , 1990; Mimura et al . , 1990; Tu et al . , 1990) so that it can later be used to replenish the cytoplasmic pool as required (Rebeille et al . , 1983).
• root systems secrete acids and phosphatases that increase phosphate availability by releasing P, from rock phosphate and phosphate esters, respectively (Lefebvre et al . , 1990; Sachay et al . , 1991).
• the mechanisms of these relief strategies involve changes in either protein synthesis and degradation (Duff et al . , 1991), or secretion of pre-existing proteins, including phosphatases (Goldstein, 1992) . They invoke changes in phosphate-dependent reactions of photosynthesis (Rao et al . , 1990; Usuda and Shimogawara, 1993), respiration (Duff et al . , 1989b; Duff et al .
• ribonucleases and phosphatases act in tandem to cleave and dephosphorylate RNA molecules in a P, recycling process (Goldstein, 1992).
• One extracellular (Glund and Goldstein, 1993; Nurnberger et al . , 1990) and four intracellular ribonucleases were reported to be induced in P 1 -starved tomato (Loffler et al . , 1992).
• the activities of phosphatase enzymes are known to increase in plants experiencing P 1 deficiency (Duff et al . , 1989a; Duff et al . , 1991; Duff et al . , 1994; Goldstein et al .
• a P ⁇ starvation inducible ⁇ -glucosidase could be involved in the deglycosylation and, hence regulation of, certain enzymes during P, stress (Ballou and Fisher, 1986; Gellatly et al . , 1994).
• the nucleic acids, constructs, and methods of this invention can be applied to regulate aspects of the phosphate-starvation response of photosynthetic organisms, as well as phosphate metabolism of such organisms in general.
• a change in regulation of the phosphate metabolism pathways can be effected.
• phosphate metabolism efficiency could be improved, allowing growth of organisms in phosphate-poor soils without addition of phosphate fertilizers, or improved growth even m phosphate-sufficient environments. Reducing the amount of phosphate fertilizer required for crop plants and/or improvement in yields, would have significant and desirable economic and environmental ramifications .
• the timing of certain plant phenomena that depend on phosphate metabolism can be altered.
• the temporal and quantitative aspects of flowering can be modified in photosynthetic organisms in which reproductive evocation is responsive to the ratio of nitrogen to phosphorus in their environments. Earlier flowering would shorten the growing season for crops and reduce the seed- to- flowering time for bedding plants. Conversely, delay in flowering is desirable in crops harvested for their biomass, such as lettuce and spinach. Alteration of phosphate metabolism of plants can also result in an altered biomass ratio between root and shoot, and be used to produce a commercial benefit in root crops, such as carrots. Additionally, the ability to induce larger root systems on plants early in the growing season would contribute to drought tolerance later during drier months.
• a plant is used to produce a protein in quantity for subsequent purification, it can be differentially expressed in the roots of the plant, and the larger roots in plants of this invention can be especially useful to increase the root biomass and the resulting yield of harvested protein.
• Alte ⁇ ng cellular Pi levels can also affect the response of a photosynthetic organism to cold and/or frost .
• Plants in the field show improved growth in the cold if the plants are phosphate limited. Further, inorganic pyrophosphatase has been shown to increase sucrose levels in the leaves of plants, which may, m part by decreasing Pi levels at the same time, improve cold acclimation and frost tolerance. Thus, one method of increasing cold and frost tolerance in photosynthetic organisms could comprise inhibition or other alteration of psr protein expression.
• Another embodiment of this invention is the increase in expression of a phosphate transporter protein, such as psr ⁇ or a functional portion thereof in a photosynthetic organism to increase absorption of phosphate from the environment .
• a phosphate transporter protein such as psr ⁇ or a functional portion thereof in a photosynthetic organism to increase absorption of phosphate from the environment .
• These proteins can also be used in phytoremediation applications.
• Pi deficiency can be stimulated with regulatory protein genes which make the plant or other photosynthetic organism absorb and store more Pi because of the inefficiency of use of Pi.
• photosynthetic organisms can be modified to increase the nutritive value of vegetative or reproductive organs.
• seed plants such as canola, soybean and corn, store phosphate m the form of phytate (the salt of 1, 2, 3, 4, 5, 6- cyclohexanehexolphosphoric acid) .
• phytate the salt of 1, 2, 3, 4, 5, 6- cyclohexanehexolphosphoric acid
• the presence of phytate is a problem where the seed is made into meal and used as feed for animals.
• Monogastric animals cannot metabolize phytate and utilize its phosphate.
• phytate binds to essential minerals, such as calcium, manganese and zinc, making them relatively unavailable to the animal.
• General constitutive promoters can be employed to increase expression of psrPK and similar genes throughout the transformed organism to increase its ability to utilize phosphate, thereby growing better under conditions of phosphate limitation and imparting the ability of the plant or other organism to grow better under any regime of phosphate nutrition.
• Tissue-specific expression in the roots can have the effect of increasing root size as well as increasing the root's ability to acquire phosphate from the environment and to use it more efficiently in their root metabolism.
• Shoot-specific expression is anticipated to alter the temporal aspects and magnitude of flowering, as well as increasing the efficiency of phosphate utilization in the shoot .
• Seed-specific expression can alter the efficiency of phosphate utilization in the seed thereby causing more seeds to set and larger seeds to be formed. This strategy can also reduce phytate storage pools in the seed. This can also increase the starch and oil storage capacity of the seed.
• Phosphate-deprived B . nigra suspension cells store more fixed carbon than phosphate- sufficient cells (Lefebvre et al .
• tissue-specific expression sites can include root hairs for increased phosphate uptake, and other tissues where excessive or inadequate expression can be deleterious to cells and can cause cell death. This can be employed in the production of male sterile lines for hybridization purposes, among other applications.
• the invention encompasses methods of reducing expression of a nucleic acid or a protein of the invention.
• a transgenic organism can be produced which expresses a molecule that binds directly to an endogenous psrPK protein and reduces its protein kinase activity or its ability to bind a substrate or cofactor.
• expression of a molecule which binds to a cis-acting regulatory element or a trans-acting regulatory factor so as to interfere with their function can decrease protein kinase expression.
• a third method can employ the modification of a nucleic acid or a protein such that it is dysfunctional and interferes with the native (naturally-occurring) functional protein in any way so that its protein kinase activity is reduced or eliminated.
• a fourth method can employ an antisense molecule as described in section II above.
• the inventors contemplate that, when an antisense molecule of the invention is delivered to target plant cells, it will hydrogen bond with endogenous nucleic acid molecules encoding the protein kinase, thereby reducing gene expression of the protein kinase.
• the antisense molecule can be designed such that its region of complementarity with an endogenous protein kinase-encoding nucleic acid molecule includes the initiation codon of the sense strand.
• the antisense molecule can include regions complementary to coding or noncoding regions of the sense strand, or both.
• a fifth method can employ transformation such that the introduced nucleic acid comprising part or all of the nucleic acid of the invention in the sense orientation reduces protein kinases expression by any means including gene co-suppression (Meyer and Saedler, 1996) .
• Non-photosynthetic, rapidly growing Brassica nigra cell suspensions were cultured as described (Lefebvre et al . , 1990) in MS medium (Murashige and Skoog, 1962) containing 6% sucrose (17.5 mM) , and 2 mg/1 2,4- dichlorophenoxyacetic acid (2,4-D) at 24°C with 130 r.p.m. rotary shaking. To assure the same nutritional state for all cultured cells, 6 ml of 7-day old cultures containing 3 ml of cells (packed cell volume) were inoculated into 44 ml of fresh MS medium containing 1.25 mM Pi .
• Packed cell volume was determined by allowing the cells to settle from the culture medium in sterile graduated cylinders for 45 min. After 7 days of culture, the same quantity of cells was subcultured into 44 ml of fresh MS medium containing either 10 mM, 1.25 mM, or no phosphate and incubated for an additional 7 days.
• filter-sterilized KH ? PO well adjusted to pH 5.8 with KOH was added to the 1.25 mM P 1 medium.
• an equal molarity of KC1 replaced the KH 2 P0 4 .
• the patterns of growth and endogenous phosphate concentrations of these cultured cells are shown in Figure 1.
• Example 2 ⁇ Extraction of total RNA and ⁇ roRN Total RNA from the harvested B. nigra cells of
• Example 1 was isolated as described by Chirgwin (Chirgwin et al . , 1979) .
• the cells were homogenized at ice-cold temperature using a homogenization buffer containing 4 M guanidinium isothio ⁇ yanate (GIBCO/BRL, Burlington, Canada).
• Poly (A) + RNA (mRNA) was purified by using the mRNA purification kit and recommended protocol of Pharmacia (Uppsala, Sweden and Piscataway, NJ) .
• RNA and mRNA isolated from minus P,- treated, 1.25 mM P, - fed and 10 mM P.- fed cells were quantified spectrophotometrically and examined on formaldehyde/agarose gels (Sambrook et al . , 1989).
• SDS-PAGE denaturing SDS-polyacrylamide gel electrophoresis
• LKB 2010 Macrophor electrophoresis apparatus Pharmacia Biotech, Inc., Baie d'Urfe, Canada
• Laemmli Laemmli, 1970
• Molecular weight standards electrophoresed in parallel were 14 C-labelled ⁇ -lactalbumin, carbonic anhydrase, glyceraldehyde- 3 -phosphate dehydrogenase, chicken egg albumin and bovine serum albumin having molecular weights of 14, 29, 36, 45 and 66 kDa, respectively (Sigma, St.
• the gels were 0.4 mm thick slabs containing 1% SDS.
• the acrylamide monomer concentrations were 5% (w/v) for the stacking gel and 10% for the separating gel.
• the separating gel was 35 cm long, to maximize resolution of protein species. Electrophoresis was performed at 25°C for 5 h at a constant current of 30 mA.
• the gel Upon completion of electrophoresis, the gel, bound to a bind-silane-treated (Pharmacia) glass plate, was incubated in 600 ml of fixing solution containing 20% trichloroacetic acid (TCA) and 10% methanol for 20 min. The gel was then washed three times in 600 ml of post- fixing wash solution containing 10% ethanol and 5% acetic acid, and dried to the glass plate overnight in a fume hood at room temperature. The dried gel was exposed to X-ray film using a Cronex intensifying screen (DuPont, Wilmington, DE) at -70°C and the film was developed using Kodak developer and fixer solutions.
• TCA trichloroacetic acid
• the autoradiogram was inspected visually, and scanned using an LKB enhanced UltraScan XL Laser Densitometer (Pharmacia) .
• the densitometric scan was analyzed with GelScan XL Software, Version 2 (Pharmacia) .
• FIG. 3 is a summary of the standardized data of the expression differences between polypeptides produced from 10 mM P ⁇ fed, 1.25 mM P,-fed and P ⁇ deprived cells. Based on these analyses, P, deprivation caused the copy number of mRNAs to increase for ten polypeptides, whereas six others decreased. Of translatable RNAs showing altered expression, four species corresponding to proteins with estimated molecular weights of 31.7, 32.3, 52.5, and 64.8 kDa were only detected in the P,-starved treatment.
• polypeptides By comparison with the other polypeptides, these were expressed at relatively high levels during P 1 deprivation. The repression of a 43.5 kDa polypeptide was also noted in minus P L -treated cells.
• Example.4 Construction of_ cDNA library .from minua. P. - treated_.Brag.sica iiigua__cells
• RNA purified from cells deprived of P, for 7 days was used to synthesize a double-stranded cDNA library using the c-CLONE II cDNA Synthesis kit (Clontech, Palo Alto, CA) according to manufacturer's instructions.
• the oligo-d (T) -tailed cDNA products produced were fractionated on a Chroma Spin-400 column (Bio/Can Scientific, Mississauga, Canada) to obtain cDNA molecules greater than 400 bp in length. The size range of such molecules was estimated to be from about 0.5 kb to about 3 kb in length.
• the size-selected cDNA molecules were ligated into EcoRI predigested/phosphatase-treated ⁇ ZAPII bacteriophage arms as recommended by the manufacturer (Stratagene, La Jolla, CA) .
• Recombinant phages were packaged with Stratagene ' s Gigapack II Gold and titered on a lawn of E. coli XL1- Blue bacteria grown on LB agar plates according to the manufacturer's protocol.
• the ligation efficiency at an optimal ratio of cDNA: vector (44 ng : 500 ng) was 7.5 x 10 6 plaque forming units (pfu) / ⁇ g cDNA.
• the cDNA library from minus P 1 -treated B . nigra cells described above was differentially screened by hybridizing to cDNA probes from both minus P 3 -treated and 10 mM P. -fed B . nigra cells, in order to identify genes responsible for the differences in protein and mRNA profiles between these cells.
• Single-stranded cDNA probes were synthesized from mRNAs isolated from the Pj-deprived and 10 mM P L -fed cells to make -P and +P probes, respectively. This was done as previously described (Sambrook et al .
• the isolated clones were digested with EcoRI , PstI and TagI restriction enzymes and the resulting DNA fragments were separated on 1% agarose gels and transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) in 10X SSC overnight (Sambrook et al . , 1989).
• 32 P-labelled single-stranded probes were generated from the liberated B . nigra inserts of individual clones using PCR (Konat et al . , 1991), employing T 3 and T 7 primers in the first round and either T 3 or T 7 primers in the second round of reactions.
• Membranes to which DNA from the isolated clones was bound were first baked for 30 min. at 80 °C, then prehybridized for 5 min. at 65°C in 0.25 M NaH 2 P0 4 (pH 7.2), 7% SDS, 1 mM EDTA. Next, radiolabelled probe was added and hybridization was allowed to proceed for 2 hr . The membranes were then washed twice in 40 mM NaH 2 P0 4 (pH 7.2), 5% SDS, 1 mM EDTA and twice in 40 mM NaH 2 P0 4 (pH 7.2), 1% SDS, 1 mM EDTA, each time for 30-60 min. at 65 °C. Autoradiography was as described above.
• the membranes were stripped of bound probe by washing twice in 0.1X SSC and 0.5% SDS at 95°C for 20 min.
• the results of the cross-hybridization analysis permitted the majority of the P,-inducible clones to be placed into eleven different homology groups, which were designated plant "P j -starvation responsive" (psr) groups 1 to 11. Because these genes are particularly active during periods where phosphate is unavailable, they are assumed to be involved in the plant response to phosphate starvation.
• Example_j___ RNA analysis c_f_psr_cDNA._clones_nsing northern blots The induction and relative abundance of psr mRNAs under the different phosphate growth conditions were further assessed by northern blotting, results of which are shown in Figure 4.
• RNA extracted from each of the minus P.-treated, 1.25 mM P,-fed and 10 mM P x -fed cells were electrophoresed on a 2.2 M formaldehyde/ 1% agarose gel (Sambrook et al . , 1989) and transferred to Nytran Plus membrane (Schleicher & Schuell) according to the manufacturer's protocol.
• bound probes were removed by washing in 0.1% SDS for 5 min, followed by equilibration in 5X SSC, 0.1% SDS at room temperature for 20 min. and stripping in the same solution for 2 min. at 95°C.
• Plasmid DNA carrying the B . nigra psrl cDNA or A . thaliana ⁇ -tubulin inserts was alkaline denatured and applied to Nytran-Plus membranes (Schleicher and Schuell, Guelph, Canada) using Bio-Dot Microfiltration apparatus (Bio-Rad) at five ⁇ g per dot.
• Bio-Rad Bio-Dot Microfiltration apparatus
• transcriptionally active nuclei were isolated from 5-6 g of 5 mM P x -fed and P ⁇ -starved root tissues as described (Willimizer and Wagner, 1981) .
• RNAs were purified as described by Somssich et al . (1989) . Approximately 10 fi cpm of RNA probe was used for hybridization with the dot blots as described (Malboobi and Lefebvre, 1995) . After the last wash, the blot was treated with 20 ⁇ g/ml of ribonuclease A in 2X SSC for 30 min at room temperature. The blot was then washed with 2X SSC, 0.5% SDS, twice and 2X SSC, twice. Dot blots were exposed to X-ray film for 7 or more days. The results are shown in Figure 10.
• Arabidopsis thaliana (var. Columbia) seeds were surface sterilized for ten minutes in 30% bleach (Javex) , 0.03% Triton X-100 (Sigma) and washed with sterilized water six times. Seeds were transferred onto a 1 cm 2 piece of steel mesh placed on solid MS (Murashige and Skooge) plates containing 0.5% agar and 2% sucrose. After 11 days, plantlets grown and rooted through the mesh were transferred to 15 ml of half strength liquid MS medium containing 1.25 mM P, and 1% sucrose in 125 ml Erlenmeyer flasks at 24°C, 540 lux light with 80 r.p.m. rotary shaking.
• the psr cDNA inserts were excised in vivo from selected recombinant ⁇ ZAPII phages into BluescriptTM plasmid vectors in the presence of R408 helper phage according to supplier's instructions (Stratagene).
• Plasmid DNA was prepared by CsCl gradient centrifugation (Sambrook et al . , 1989).
• Clone psrl was identified as a gene coding for a phosphate-starvation inducible protein kinase. It has therefore been given the additional name psrPK.
• the psrPK sequence possesses high homology to protein kinases isolated from other plants, such as Glycine max protein kinase 2 (SPK2, GB accession #L19360, 63% homology), Arabidopsis thaliana protein kinase 2 (ASK2 , GB Accession # Z12120, 70% homology) (Park et al .
• An A . thaliana APRL2 cDNA library (CD4-7) was obtained from the Ohio State Arabidopsis Biological Resource Center. It was screened at high stringency with a probe consisting of the entire JS. nigra psrPK cDNA insert. The B . nigra psrPK cDNA insert was used for random priming probe synthesis and subsequent hybridization in non-radioactive Du Pont RenaissanceTM kit according to the manufacturer's instructions (Du Pont NEN, Boston, MA) . A strongly hybridizing plaque (designated p ⁇ rl -1) was purified and its bacteriophage DNA isolated. The A .
• FIG. 10 shows the cDNA sequence of B . nigra psrPK, aligned with the cDNA sequence of the corresponding protein kinase from Arabidopsis thaliana .
• Example 10 Genomic clone .isolation
• a genomic library of A . thaliana (Var. Colombia) in EMBL3 was screened at high stringency with a probe consisting of the unique 3' sequence of the A . thaliana psrPK cDNA insert. This insert was used for random priming probe synthesis and subsequent hybridization in non-radioactive Du Pont RenaissanceTM kit according to the manufacturer's instructions (DuPont NEN, Boston). Positive clones were then rescreened until homogenous and subjected to Southern blot analysis using the same probe as above. Inserts from positive clones were excised and digested with appropriate restriction enzymes and subcloned into plasmids. The inserts were then sequenced by dideoxy nucleotide sequencing of both strands.
• Arabidopsis thaliana was transformed in planta with eight different constructs employing constitutive and tissue-specific promoters attached to sense and antisense nucleic acids of the entire and partial sequences of psrPK from A . thaliana . See Figures 12A-12D, Table 2 and Figure 28.
• Figure 12A or antisense ( ⁇ -psrl) ( Figure 12B) direction to produce a sense or antisense construct under the control of a constitutive (CaMV-35S) promoter.
• the CaMV- 35S promoter (cauliflower mosaic virus 35S promoter) of pBI121 is fused upstream of GUS gene.
• the GUS gene was removed from this vector by Smal and EcoICRI digestion.
• a cDNA encoding psrPK was cloned in pZLl vector (isolated from Arabidopsis Resource Center cDNA library) . This clone was digested by S al and Ba HI enzymes . The BamHI site was subsequently filled in by Klenow fragment of DNA polymerase I .
• Competent E. coli DH 5- ⁇ was transformed by the ligation products and plated on LB/kan. The growing colonies were picked and mini-prepped. Digestion with Sail and EcoRI enzymes distinguished the sense and antisense constructs by appearance of 1.6 Kb and 0.3 Kb fragments, respectively.
• Agrobacterium tumefaciens strain GV3101 bearing the helper nopaline plasmid MP90 and a binary vector containing the psrPK gene construct and a plant selectable marker was grown overnight. Wound sites of excised primary and secondary inflorescence shoots were exposed to cultures of the transformed Agrobacterium cells three times to inoculate the plant tissues. The treated plants were grown to maturity, and the seeds harvested and screened for transformants on selective medium ( Figure 28) . Confirmation of transformation was made by determining if the plants contain the transferred genes through Southern blots or polymerase chain reaction techniques using the psrPK and associated sequences.
• the psrPK protein was expressed in the E. coli expression vector pGEX (Promega) .
• the insert was amplified out of the Bluescript plasmid containing the Arabidopsis psrPK such that a fragment was produced which contained the translational start and which stopped one codon short of the stop codon (i.e. excluding the stop codon) .
• This fragment was cloned into a modified pGEX plasmid containing six extra codons for histidine at the 3' end of the inserted psrPK cDNA.
• the protein was then expressed in the bacteria and the psrPK protein was purified from crude extract using columns which exploit the affinity properties of the added histidine residues.
• Protein kinase activity was determined by three different experiments using the purified psrPK protein. Manser, et al . (1994); Manser, et al . , (1992); Manser, et al . , (1995) .
• the first two experiments involve activity determinations on proteins obtained from plants grown minus phosphate. Between 1-5 g of plant tissue was homogenized in a buffer containing 15 mM Hepes/KOH pH7.6, 40 mM KC1, 5mM MgCl 2 , 1 mM dithiothreitol (DTT) , 0.1 mM phenylmethysulfonyl fluoride (PMSF) (Sigma) .
• DTT dithiothreitol
• PMSF phenylmethysulfonyl fluoride
• the homogenate was kept on ice for 30 min. before centrifuging at 13000 xg for 15 min, twice.
• the protein concentration was determined by using Bio-Rad Protein Assay Reagent Concentrate as described by the manufacturer (Bio-Rad Lab. , Richmond, CA) . Fifty ⁇ g of each protein extract was loaded onto a denaturing SDS- PAGE or a native gel (Laemmli, 1970) .
• the final concentration of acrylamide monomer in the 0.75 mm thick mini gel was 4% for the stacking gel and 10% for the separating gel.
• the SDS-PAGE was run at 200V at room temperature and stained with Coomassie R-250 (Sigma) .
• the final concentration of acrylamide monomer was 3% for the stacking gel and 7% for the separating gel.
• the gel was pre-run at 4°C at 200V for 1 hr prior to loading samples and then run at 200V for 45 min. at 4°C.
• the separated proteins were blotted onto Immobilon P PVDF membrane (Millipore, Mississauga, Ontario) and, in the case of the SDS-denatured samples, the blots were exposed to denaturing steps of 6M Guanidine HC1 in MES buffer, diluted 50% with MES buffer, five cycles, followed by a renaturing step of PBS buffer for 3 hours.
• the blots were immediately exposed to 25 mM MES, pH 6.5, containing either purified protein and 25 ⁇ Ci ⁇ 32 P-ATP or 25 ⁇ Ci ⁇ 32 P-ATP alone for 5 min. at 22°C, then 10 min. at 4°C. These blots were also washed, dried and exposed to X-ray film for detection purposes.
• the third activity experiment used casein or histones as artificial substrates for psrPK (Uesono, et al . , (1992)) .
• the psrPK protein expressed and purified from bacteria was dialyzed into 20 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 , and 1 mM ⁇ -mercaptoethanol .
• An equal volume of this mixture was added to 20 ⁇ M ATP, 2 ⁇ M/ml dephosphorylated casein (or histone) and 25 ⁇ Ci ⁇ 32 P-ATP.
• the sample was run on an SDS-Page gel as described above and the gel dried and exposed to X-ray film for detection purposes.
• the psrPK protein phosphorylated both casein and histone.
• Example 13 Plant culture for psr ⁇ -glucosidase expression analysis under other stresses
• Arabidopsis thaliana (var. Columbia) seeds were germinated and grown essentially as described in Example 8.
• the concentration of P j was kept at 5 mM when investigating responses to other environmental stresses.
• sterilized NaCl solution was added to a final concentration of 100 mM, a sublethal concentration (Saleki, et al . , (1993).
• KN0 3 and NH 4 N0 3 were replaced by an equal molarity of KC1.
• MgS0 4 was replaced by an equal molarity of MgCl 2 .
• Heat shock was performed by incubating 14 day-old plants at 39°C for 2 hours.
• Anaerobic conditions were created by blowing argon gas into the flasks containing 13 day-old plants through sterilized tubes plugged with cotton for 24 hours. In all cases, the culture medium was removed and replaced with fresh medium every 4 days to ensure that there was no depletion of supplied nutrients. Treated plants were harvested after 14 days except for plants grown without nitrogen that were harvested on day 11 due to onset of severe deprivation symptoms, and plants starved for P 1 for 14 days and resupplied with 5 mM P, that were harvested after a further 1 or 3 days. Only P x deprivation caused significant increases in mRNA levels for ⁇ -glucosidase.
• Example 14 Southern blots and genomic library screening Genomic DNA was extracted from A . thaliana plant material in a CTAB extraction buffer according to Saghai-Maroof , et al . (1984). About 10 ⁇ g of genomic DNA was digested with either SamHl , EcoRI , Sail, or BamHI/ Sail restriction enzymes overnight at 37°c. After separating the digestion products on a 0.8% agarose gel, DNA fragments were transferred to Nytran-Plus membrane as described by the manufacturer (Schleicher and Schuell, Guelph, Canada). The Brassica nigra psr3 .
• a genomic library of A . thaliana ecotype Columbia cloned into EMBL3 was provided by Kenton Ko, Queen's University, Kingston, Canada. About 200,000 plaque forming units (pfu) were screened with probe derived from the B. nigra p ⁇ r3 . 1 cDNA clone under high stringency conditions (Malboobi and Lefebvre, (1995)). Positive clones were carried through secondary and tertiary screening. Probes derived from the isolated genomic DNA inserts were used in Southern blotting to determine which genomic clones corresponded to the B . nigra psr3 . 1 . A genomic clone with a similar blotting pattern to that of B . nigra psr3 . 1 cDNA clone was chosen for restriction enzyme site mapping and subcloning by standard methods (Sambrook, et al . , (1989)).
• the DNA fragments resulting from Ecoi.1 , and EcoRl / Sail digestions of the psr3 genomic clone were prepared and inserted into the pBluescript KS-vector (Stratagene, La Jolla, CA) , and these constructs were used to transform competent E. coli strain DH5- ⁇ (GIBCO BRL, Burlington, Canada) through standard cloning techniques (Sambrook, et al . , 1989) . Plasmid DNA was prepared with the WizardTM Megapreps DNA Purification System (Promega, Madison, WI) .
• Transcriptional start sites for ⁇ -glucosidase were determined by primer extension analysis.
• RNA extracted from P,- starved roots then subjected to primer extension by AMV reverse transcriptase (Pharmacia, Baie D'Urfe, Canada) as described in Ausubel, et al . , (1995) .
• AMV reverse transcriptase Pharmacia, Baie D'Urfe, Canada
• the homogenate was kept on ice for 30 min. before centrifuging at 13000xgr for 15 min. The supernatant was removed and centrifuged for a further 15 min.
• the protein concentration of this final supernatant was determined using Bio-Rad Protein Assay Dye Reagent Concentrate as described by the manufacturer (Bio-Rad, Richmond, CA) . Fifty ⁇ g of total protein were electrophoresed in each lane in either denaturing SDS-PAGE or native PAGE gels (Laemmli, (1970)). For the SDS-PAGE, the final concentration of acrylamide monomer in the 0.75 mm thick mini-gel (Bio-Rad) was 4% and 10% in the stacking and separating gels, respectively. The gel was run at 200V at room temperature and stained with Coomassie R-250
• the native gel SDS was eliminated from all reagents and the final concentrations of acrylamide monomer were 3% and 7% for the stacking and separating gels, respectively.
• the native gel was prerun at 4°c and 100 V for 30 min prior to sample loading and then run at 200V for 3 h at 4°C.
• the native gel was equilibrated in a 100 mM sodium acetate, pH 6.5 (Sigma) buffer containing 20 mM CaCl 2 for 15 min. This was followed by incubation for 3 hours in the same solution plus 0.02%, (w/v) Fast Garnet GBC salt

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