US20050223431A1

US20050223431A1 - Methods of modulating glucosinolate production in plants

Info

Publication number: US20050223431A1
Application number: US10/969,808
Authority: US
Inventors: Soren Bak; Rene Feyereisen
Original assignee: Arizona State University ASU
Current assignee: Arizona State University ASU
Priority date: 2000-12-18
Filing date: 2004-10-20
Publication date: 2005-10-06
Also published as: US20080222754A1; US7009088B1

Abstract

The present invention relates to methods for modulating glucosinolate production in plants, specifically by modulating CYP83A1 expression. The present invention also relates to transgenic plants that overexpress and underexpress CYP83A1.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e)(1) of provisional patent application Ser. No. 60/256,693, filed Dec. 18, 2000, and provisional patent application Ser. No. 60/317,374, filed Sep. 4, 2001, which applications are incorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention relates to novel methods of regulating plant phenotypes. In particular, the invention relates to methods of modulating glucosinolate production by overexpressing or underexpressing CYP83A1.

BACKGROUND

Indole-3-acetic acid (IAA) is the primary plant auxin. The biosynthetic routes resulting in IAA production and the mechanism securing an optimal IAA concentration at the cellular level are poorly understood. Several biosynthetic pathways have been proposed. Mutant studies have provided some knowledge of IAA and indole metabolism, and have led to a current picture of a metabolic grid consisting of several redundant pathways operating at different developmental stages (Normanly and Bartel (1999) Curr. Opin. Plant Biol. 2:207-213). Tryptophan dependent as well as independent pathways have been proposed to occur in Arabidopsis thaliana seedlings based on the ability of the tryptophan auxotrophic mutants trp3-1 and trp2-1 to accumulate increased levels of IAA-conjugates in spite of reduced tryptophan synthesis (Normanly et al. (1993) Proc. Natl. Acad. Sci. USA 90:10355-10359). However, pleiotropic effects caused by these mutants renders it difficult to draw conclusions with respect to IAA synthesis under normal growth conditions. Thus mature trp3-1 plants accumulate high levels of indole-3-glycerophosphate (IGP) and increased levels of tryptophan-derived indole glucosinolates and indole-3-acetonitrile (IAN) whereas the level of free IAA is normal (Müller and Weiler (2000) Planta. 211:855-863). The latter observations question the operation of the proposed tryptophan-independent IAA pathway, because IGP is non-enzymatically converted to IAA under the alkaline conditions used to hydrolyze IAA conjugates (Müller and Weiler (2000) Planta. 211:855-863). Superroot2 (sur2) was described in 1998 as an auxin mutant that accumulated elevated levels of free IAA and less conjugated IAA (Delarue et al. (1998) Plant J. 14:603-611). Based on these observations the sur2 gene was predicted to encode a protein involved in homeostasis of IAA by controlling auxin conjugation. It has recently been shown that sur2, which is allelic to rnt1-1, encodes a cytochrome P450, CYP83B1 (see, e.g., copending, commonly owned application entitled “Methods of Modulating Auxin Production in Plants” filed on even date herewith (attorney docket no. 2225-0029), which application is incorporated herein by reference in its entirety; Barlier et al. (2000) Proc Natl Acad. Sci. USA 97:14819-14824; Bak et al. (2001) Plant Cell. 13:101-111) involved in the conversion of indole-3-acetaldoxime to S-alkylthiohydroxymates in the biosynthesis of indole glucosinolates (Bak et al. (2001) Plant Cell. 13:101-111).
Cytochromes P450 are monooxygenases catalyzing key steps in numerous metabolic pathways (Kahn and Durst (2000) In Recent advances in phytochemistry. Evolution of metabolic pathways. Elsevier Science Ltd, Amsterdam, pp 151-190). CYP83B1/RNT1/SUR2 catalyzes the initial conversion of indole-3-acetaldoxime, a proposed intermediate in IAA biosynthesis, to the corresponding S-alkylthiohydroxymate. This is the first committed step in the biosynthesis of indole glucosinolates e.g. glucobrassicin (Bak et al. (2001) Plant Cell. 13:101-111). Indole-3-acetaldoxime thus constitutes a metabolic branch point in IAA and indole glucosinolate biosynthesis and the level of IAA can be regulated by the flux of indole-3-acetaldoxime through CYP83B1. IAN has generally been assumed to be a product of indole-3-acetaldoxime metabolism in IAA biosynthesis (e.g. Normanly et al. (1995) Plant Physiol. 107:323-329; Bartel B. (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:51-66; Normanly and Bartel (1999) Curr. Opin. Plant Biol. 2:207-213; Hull et al. (2000) Proc. Natl. Acad. Sci. USA 97:2379-2384). However, the nit1-1 mutation which renders Arabidopsis seedlings insensitive to the IAA effects of exogenously applied IAN (Normanly et al. (1997) Plant Cell 9:1781-1790), is unable to mitigate the auxin phenotype of rnt1-1 in double mutants (Bak et al. (2001) Plant Cell. 13:101-111). This evidence argues against a role for IAN as a direct metabolite of indole-3-acetaldoxime (Bak et al. (2001) Plant Cell. 13:101-111). Instead, IAN may be regarded as a degradation product derived from turnover of indole glucosinolates that are hydrolyzed by a nitrilase belonging to the NITF3 group (Andersen and Muir (1966) Plant Physiol 19:1038-1048; Ludwig-Müller et al. (1999) Planta 208:409-419; Bak et al. (2001) Plant Cell. 13:101-111, Vorwerk et al. (2001) Planta. 212:508-516).
The post-oxime metabolizing enzymes in IAA biosynthesis in A. thaliana still await identification. The closest homologue to CYP83B 1 in the A. thaliana genome is CYP83A1 showing 63% sequence identity and 78% sequence similarity at the amino acid level (Paquette et al. (2000) DNA Cell Biol. 19:307-317). Both CYP83B1 and CYP83A1 transcripts are expressed in roots, leaves, stems, flowers and siliques (Mizutani et al. (1998) Plant Mol. Biol. 37:39-52; Xu et al. (2001) Gene. In Press). However, while CYP83B1 is preferentially expressed in roots and induced by wounding or by dehydration, CYP83A1 is preferentially expressed in leaves and wounding reduces its expression (Mizutani et al. (1998) Plant Mol. Biol. 37:39-52; Reymond et al. (2000) The Plant Cell 1′:707-719). CYP83B1 transcription was shown recently to be induced by IAA as well (Barlier et al. (2000) Proc. Nat. Acad. Sci. USA 97:14819-14824), strengthening the connection between indole glucosinolate and IAA synthesis.
Thus, there remains a need for the identification and characterization of enzymes that regulate auxin and glucosinolate synthesis.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that CYP83A1 is a regulator of glucosinolate production in Arabidopsis and functions in the metabolic grid of IAA and indole glucosinolate biosynthesis. Indeed, overexpression of CYP83A1 compensates for the total lack of CYP83B1. However, the expression patterns of the two genes are different and the two enzymes operate on different substrates in vivo thereby serving different purposes. Thus the CYP83A1 and CYP83B1 genes are not redundant.
Accordingly, in one aspect, the present invention includes a transgenic plant that displays an altered phenotype relative to the wild-type plant. In another embodiment, the transgenic plant has altered CYP83A1 expression.
In another aspect, the invention includes a method of producing a transgenic plant with altered CYP83A1 expression relative to the wild-type plant. The method comprises the steps of (a) introducing an expression construct described herein into a plant cell to produce a transformed plant cell, wherein the expression construct comprises a polynucleotide encoding a CYP83A1 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide; and (b) producing a transgenic plant from the transformed plant cell with altered CYP83A1 expression. In certain embodiments, at least one polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter or a constitutive promoter. The polypeptide can be overexpressed, underexpressed, or it can inhibit expression of CYP83A1. In a still further embodiment, at least two polynucleotides are introduced into the plant cell. Each polynucleotide is operably linked to a different tissue-specific promoter such that one polynucleotide is overexpressed while the other inhibits expression of CYP83A1.
In another embodiment, the invention relates to a method of producing a transgenic plant with altered CYP83A1 expression relative to the wild-type plant. The method comprises: (a) introducing a polynucleotide that inhibits expression of a CYP83A1 polynucleotide into a plant cell to produce a transformed plant cell; and (b) producing a transgenic plant from the transformed plant cell with altered CYP83A1 expression.
The altered phenotype due to CYP83A1 over- or underexpression includes altered morphological appearance and altered biochemical activity, for example, altered (reduced or increased) cell length in any cell or tissue, altered (extended or decreased) periods of flowering, altered (increased or decreased) branching, altered (increased or decreased) seed production, altered (increased or decreased) leaf size, altered (elongated or shortened) hypocotyls, altered (increased or decreased) plant height, altered cytochrome P450 activity, altered heme-thiolate enzyme activity, altered CYP83A1 expression (under- or overexpressed), regulation of glucosinolate and auxin synthesis and altered resistance to plant pathogens.
In yet another aspect, the invention includes a method for altering the biochemical activity of a cell comprising the following steps: introducing an expression construct described herein into a plant cell to produce a transformed plant cell, wherein the expression construct comprises a polynucleotide encoding a CYP83A1 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide; and growing the cell under conditions such that the biochemical activity of the cell is altered. Biochemical activity includes, for example, altered CYP83A1 enzyme activity and regulation of glucosinolates. In certain embodiments, the expression construct is introduced ex vivo. In other embodiments, the expression construct is provided to the cell in vivo. In still other embodiments, more than one expression construct is provided to the cell.
In yet another, aspect, the invention includes a method for regulating the cell cycle of a plant cell comprising the following steps: providing a polynucleotide as described herein to a plant cell; and expressing the polynucleotide to provide the encoded polypeptide, wherein the polypeptide is provided in amounts such that cell cycling is regulated. In certain embodiments, the plant cell is provided in vitro and is cultured under conditions suitable for providing the polypeptide. In still other embodiments, the polynucleotide is provided in vivo.
In another aspect, the invention provides a method of producing a transgenic plant with altered expression of a cytochrome P450 that catalyzes the conversion of aldoxime to glucosinolate, the method comprising introducing an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide into a plant cell to produce a transformed plant cell, and producing a transgenic plant from the transformed plant cell with altered cytochrome P450 expression. In certain embodiments, the cytochrome P450 is CYP83A1, and in other embodiments, the polynucleotide encoding a cytochrome P450 polypeptide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter. The glucosinolate can be aliphatic, aromatic or indolic, anc can be obtained from a corresponding aldoxime. The aldoxime is obtained from the conversion of an aliphatic amino acid or a chain-elongated form thereof, an aromatic amino acid, or tryptophan to the corresponding N-hydroxy amino acid, and the conversion of the N-hydroxyamino acid to the aldoxime. The aliphatic amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine and chain-elongated forms thereof, and the aromatic amino acid is phenylalanine or tyrosine.
In another aspect, the invention provides a method of producing a cytochrome P450 that catalyzes the conversion of aldoxime to the corresponding aci-nitro and the conversion of the aci-nitro to the corresponding S-alkyl-thiohydroximate and the conversion of the S-alkyl-thiohydroximate to glucosinolate, the method comprising introducing an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide into a host cell to produce a transformed host cell, expressing the cytochrome P450 in the host cell, and isolating the expressed cytochrome P450. In certain embodiments, the cytochrome P450 is CYP83A1, and in other embodiments, the polynucleotide encoding the cytochrome P450 polypeptide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter. The aldoxime is obtained from the conversion of an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, tyrosine, and phenylalanine to the corresponding N-hydroxy amino acid, and the conversion of N-hydroxyamino acid to the aldoxime.
In yet another aspect, the invention includes a method for producing a glucosinolate, the method comprising contacting a cytochrome P450 with an aldoxime, and isolating the glucosinolate. In certain embodiments, the cytochrome P450 is CYP83A1. In certain other embodiments, the method uses a transformed host cell overexpressing a cytochrome P450, where the transformed host cell comprises an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing the polypeptide, wherein the promoter is selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter.
Any of the polynucleotides or polypeptides described herein can be used in diagnostic assays; to generate antibodies. Further, the antibodies and fragments thereof can also be used in diagnostic assays, to produce immunogenic compositions or the like.
These and other objects, asp-ects, embodiments and advantages of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the genomic sequence of native CYP83A1.
FIG. 2 depicts the amino acid sequence of native CYP83A1.
FIG. 3 shows that ectopic expression of CYP83A1 cDNA in rnt1-1 complements the indole glucosinolate deficiency in the CYP83B1 knock out. Indole glucosinolates were measured colorimetrically as thiocyanite (SCN⁻). Data are represented as mean±SE calculated pr mg fresh weight, n=10 seedlings. The corresponding mean indole glucosinolate level pr individual seedling are: wild type (1.46±0.05 nmol) rnt1-1 (0.62±0.03 nmol) 2.8.6 (1.48±0.15 nmol), 2.9.5 (1.60±0.07 nmol), and 2.24.3 (1.15±0.10 nmol).
FIG. 4 shows the products of CYP83B1 metabolism of M [5⁻3H]indole-3-acetaldoxime in the presence and absence of nucleophiles. Reaction mixtures were analyzed by thin-layer chromatography. The components applied at the origin were focused by pre-electrophoresis (2 cm) in 100% methanol before development in chloroform/methanol/water (85:14:1) (v/v/v). FIG. 4A shows that in the absence (−) of a nucleophile CYP83B1 catalysis is inhibited, and the radioactivity accumulates as an aggregate at the origin of application. In the presence (+) of β-mercaptoethanol an adduct is formed (←). Samples were analyzed after 0 and 15 min. incubation in MOPS buffer. FIG. 4B shows various structurally different nuceophiles form adducts with similar turnover. 1) β-mercaptoethanol, 2) ethanthiol, 3) 1-thio-β-D-glucose, 4) L-cysteine, 5) reduced glutathione. Samples were incubated for 15 min in the absence (−) or presence (+) of NADPH in tris buffer. ← shows the position of the adduct. Due to the volatility and immiscibility of ethanthiol in aqueous solutions adducts were identified at both the origin (●) as well as with the buffer tris (*).
FIG. 5 shows that CYP83 μl and CYP83B1 metabolize indole-3-acetaldoxime with different affinity. Kinetics with indole-3-acetaldoxime as substrate and using cysteine as thiol donor were compared for both CYP83A1(●) and CYP83B1 (▪). Computed regression curves as well as the experimental data points are shown. The correlation coefficients (R²) for CYP83B1 and CYP83A1 regression analyses are 0.985 and 0.999, respectively.
FIG. 6 is a spectral characterization of CYP83A1 and CYP83B1. Type II spectra were recorded with 0.15 μM of CYP83A1 or 0.44 μM of CYP83B1 using 200 μM ligands. 1) tryptamine, 2) β-phenylethylamine, 3) tyramine, 4) n-octylamine, 5) 5-OH-tryptamine, 6) 3-OH-tyramine, B) baseline.
FIG. 7 shows that CYP83A1 and CYP83B1 have different affinity for tryptamine and β-phenyletylamine. 0.15 μM of CYP83A1 or 0.44 μM of CYP83B1 were incubated with increasing amounts of either tryptamine (●) or β-phenylethylamine (▪) and the difference in amplitude of the type II difference spectra were plotted as a function of concentration of ligand. To compensate for ligand absorbance, the experimental data were fitted to a hyperbolic curve using the equation A=A_max*X/(K_s+X)+C*X, where A is the amplitude of the spectra, X the concentration of ligand, and C the contribution from ligand absorbance. The computed regression curve is shown as well as the experimental data points. Correlation coefficients (R²) for CYP83B1 interaction with tryptamine and β-phenylethylamine are 0.983 and 0.987, respectively, and for CYP83A1 interaction with tryptamine and β-phenylethylamine 0.933 and 0.989, respectively.
FIG. 8 shows that CYP83A1 and CYP83A1 are not redundant enzymes CYP83B1 is primarily involved in biosynthesis of indole glucosinolates whereas CYP83A1 is involved in glucosinolates not derived from indole-3-acetaldoxime. The use of a separate CYP83 for indole glucosinolate biosynthesis ensures a tight control of the flux of the shared tryptophan derived intermediate, indole-3-acetaldoxime, for IAA and indole glucosinolate biosynthesis.

DETAILED DESCRIPTION

The present inventors have shown that CYP83A1 is a cytochrome P450 that regulates glucosinolate production in Arabidopsis. As shown in the examples, although expression of CYP83A1 under control of its endogenous promoter in the rnt1-1 background does not prevent the auxin excess and indole glucosinolate deficit phenotype caused by the lack of the CYP83B 1 gene, ectopic overexpression of CYP83A1 using a ³⁵S promoter rescues the rnt1-1 phenotype. CYP83A1 and CYP83B1 heterologously expressed in yeast cells show marked differences in their substrate specificity. Both enzymes convert indole-3-acetaldoxime to a thiohydroximate adduct in the presence of NADPH and a nucleophilic thiol donor. However, indole-3-acetaldoxime has a 50-fold higher affinity towards CYP83B1 than towards CYP83A1. Both enzymes also metabolize the phenylalanine- and tyrosine-derived aldoximes. Enzyme kinetic comparisons of CYP83 μl and CYP83B1 show that indole-3-acetaldoxime is the physiological substrate for CYP83B1 but not for CYP83A1. Instead, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. The two closely related CYP83 subfamily members are therefore not redundant. The presence of putative auxin responsive cis-acting elements, AuxRes, in the CYP83B1 promoter, but not in the CYP83A1 promoter evidences that CYP83B1 has evolved to selectively metabolize a tryptophan-derived aldoxime intermediate shared with the pathway of auxin biosynthesis in A. thaliana. (See, e.g., copending, commonly owned application entitled “Methods of Modulating Auxin Production in Plants” filed on even date herewith (attorney docket no. 2225-0029) incorporated herein by reference in its entirety, for a detailed discussion of CYP83B1.) Accordingly, the present invention represents an important discovery in understanding and regulating plant cell growth.
Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified molecules or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. In addition, the practice of the present invention will employ, unless otherwise indicated, conventional methods of plant biology, virology, microbiology, molecular biology, recombinant DNA techniques and immunology all of which are within the ordinary skill of the art. Such techniques are explained fully in the literature. See, e.g., Evans, et al., Handbook of Plant Cell Culture (1983, Macmillan Publishing Co.); Binding, Regeneration of Plants, Plant Protoplasts (1985, CRC Press); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); A Practical Guide to Molecular Cloning (1984); and Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes a mixture of two or more polypeptides, and the like.

The following amino acid abbreviations are used throughout the text:



	Alanine: Ala (A)	Arginine: Arg (R)
	Asparagine: Asn (N)	Aspartic acid: Asp (D)
	Cysteine: Cys (C)	Glutamine: Gln (Q)
	Glutamic acid: Glu (E)	Glycine: Gly (G)
	Histidine: His (H)	Isoleucine: Ile (I)
	Leucine: Leu (L)	Lysine: Lys (K)
	Methionine: Met (M)	Phenylalanine: Phe (F)
	Proline: Pro (P)	Serine: Ser (S)
	Threonine: Thr (T)	Tryptophan: Trp (W)
	Tyrosine: Tyr (Y)	Valine: Val (V)

Definitions
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. This term refers only to the primary structure of the molecule and thus includes double- and single-stranded DNA and RNA. It also includes known types of modifications, for example, labels which are known in the art, methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelates (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Nonlimiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
Techniques for determining nucleic acid and amino acid “sequence identity” are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure. M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research, Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR.
Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 43%-60%, preferably 60-70%, more preferably 70%-85%, more preferably at least about 85%-90%, more preferably at least about 90%-95%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, or any percentage between the above-specified ranges, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule) such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence “selectively hybridize,” or bind, to each other to form a hybrid molecule: A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under “moderately stringent” typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
A “gene” as used in the context of the present invention is a sequence of nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.) with which a genetic function is associated. A gene is a hereditary unit, for example of an organism, comprising a polynucleotide sequence that occupies a specific physical location (a “gene locus” or “genetic locus”) within the genome of an organism. A gene can encode an expressed product, such as a polypeptide or a polynucleotide (e.g., tRNA). Alternatively, a gene may define a genomic location for a particular event/function, such as the binding of proteins and/or nucleic acids, wherein the gene does not encode an expressed product. Typically, a gene includes coding sequences, such as, polypeptide encoding sequences, and non-coding sequences, such as, promoter sequences, polyadenlyation sequences, transcriptional regulatory sequences (e.g., enhancer sequences). Many eucaryotic genes have “exons” (coding sequences) interrupted by “introns” (non-coding sequences). In certain cases, a gene may share sequences with another gene(s) (e.g., overlapping genes).
A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are typically determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence. Other “control elements” may also be associated with a coding sequence. A DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence. “Encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide encoded by the sequence.
Typical “control elements”, include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), translation enhancing sequences, and translation termination sequences. Transcription promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), tissue-specific promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced only in selected tissue), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
A control element, such as a promoter, “directs the transcription” of a coding sequence in a cell when RNA polymerase will bind the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
“Expression enhancing sequences” typically refer to control elements that improve transcription or translation of a polynucleotide relative to the expression level in the absence of such control elements (for example, promoters, promoter enhancers, enhancer elements, and translational enhancers (e.g., Shine and Delagarno sequences).
“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered “operably linked” to the coding sequence.
A “heterologous sequence” as used herein typically refers to a nucleic acid sequence that is not normally found in the cell or organism of interest. For example, a DNA sequence encoding a polypeptide can be obtained from a plant cell and introduced into a bacterial cell. In this case the plant DNA sequence is “heterologous” to the native DNA of the bacterial cell.
The “native sequence” or “wild-type sequence” of a gene is the polynucleotide sequence that comprises the genetic locus corresponding to the gene, e.g., all regulatory and open-reading frame coding sequences required for expression of a completely functional gene product as they are present in the wild-type genome of an organism. The native sequence of a gene can include, for example, transcriptional promoter sequences, translation enhancing sequences, introns, exons, and poly-A processing signal sites. It is noted that in the general population, wild-type genes may include multiple prevalent versions that contain alterations in sequence relative to each other and yet do not cause a discernible pathological effect. These variations are designated “polymorphisms” or “allelic variations.”
“Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
By “vector” is meant any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus etc., which is capable of transferring gene sequences to target cells. Generally, a vector is capable of replication when associated with the proper control elements. Thus, the term includes cloning and expression vehicles, as well as viral vectors and integrating vectors.
As used herein, the term “expression cassette” refers to a molecule comprising at least one coding sequence operably linked to a control sequence which includes all nucleotide sequences required for the transcription of cloned copies of the coding sequence and the translation of the mRNAs in an appropriate host cell. Such expression cassettes can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue-green algae, plant cells, yeast cells, insect cells and animal cells, either in vivo or in vitro. Under the invention, expression cassettes can include, but are not limited to, cloning vectors, specifically designed plasmids, viruses or virus particles. The cassettes may further include an origin of replication for autonomous replication in host cells, selectable markers, various restriction sites, a potential for high copy number and strong promoters.
A cell has been “transformed” by an exogenous polynucleotide when the polynucleotide has been introduced inside the cell. The exogenous polynucleotide may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
“Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
The term “CYP83A1 polynucleotide” refers to a polynucleotide derived from the gene encoding the CYP83A1 polypeptide that encodes a polynucleotide that retains CYP83A1 enzymatic activity. CYP83A1 is a cytochrome P450 that is a regulator of glucosinolate production in Arabidopsis. The inventors herein have shown that CYP83A1 converts indole-3-acetaldoxime to a thiohydroximate adduct in the presence of NADPH and a nucleophilic thiol donor. CYP83A1 also metabolizes the phenylalanine- and tyrosine-derived aldoximes. In particular, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. The CYP83A1 polynucleotide sequence and corresponding amino acid sequence are shown in FIGS. 1 and 2, respectively. The term as used herein encompasses a polynucleotide including a native sequence depicted in FIG. 1, as well as modifications and fragments thereof.
Thus, the term encompasses alterations to the polynucleotide sequence. Generally, such alteration will result in a molecule displaying at least one CYP83A1 biochemical activity, as described above. The activity displayed by such mutant molecules need not be at the same level as the native molecule. In some cases, it may be desirable to completely destroy CYP83A1 activity. CYP83A1 activity can be assessed using the methods described herein. Such modifications typically include deletions, additions and substitutions, to the native CYP83A1 sequence. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of plants which express the polynucleotide or errors due to PCR amplification. The term encompasses expressed allelic variants of the wild-type sequence which may occur by normal genetic variation or are produced by genetic engineering methods.
The term “phenotype” as used herein refers to any microscopic or macroscopic change in structure or morphology of a plant, such as a transgenic plant, as well as biochemical differences, which are characteristic of a plant which overproduces or underproduces glucosinolate or auxin, compared to a progenitor, wild-type plant cultivated under the same conditions. Generally, such morphological differences include loss or increase of apical dominance, reduced or increased hypocotyl length, reduced or increased number of inflorescences, reduced or increased height, a bushy appearance due to extensive branching and reduced seed set, epinastic cotyledons, exfoliation of the hypocotyl, adventitious root formation from the hypocotyl, enhanced secondary root and root hair formation and, eventually, callus formation and increasing disintegration of the seedling. Additional phenotypic morphological attributes of the auxin phenotype are summarized in the Examples.
A “polypeptide” is used in it broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or by other bonds, for example ester, ether, etc. As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is typically called a polypeptide or a protein. Full-length proteins, analogs, mutants and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, as ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide may be obtained as an acidic or basic salt, or in neutral form. A polypeptide may be obtained directly from the source organism, or may be recombinantly or synthetically produced (see further below).
A “CYP83A1” polypeptide is a polypeptide as defined above, which is derived from the CYP83A1 polypeptide and that retains CYP83A1 enzymatic activity. As explained above, this enzyme is a cytochrome P450 and converts indole-3-acetaldoxime to a thiohydroximate adduct in the presence of NADPH and a nucleophilic thiol donor.
Moreover, CYP83A1 metabolizes phenylalanine- and tyrosine-derived aldoximes. In particular, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. The CYP83A1 amino acid sequence is shown in FIG. 2. The term encompasses mutants and fragments of the native sequence so long as the protein functions for its intended purpose.
The term “CYP83A1 analog” refers to derivatives of CYP83A1, or fragments of such derivatives, that retain desired function, e.g., as measured in assays as described further below. In general, the term “analog” refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy desired activity. Preferably, the analog has at least the same activity as the native molecule. Methods for making polypeptide analogs are known in the art and are described further below.
Particularly preferred analogs include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. It is to be understood that the terms include the various sequence polymorphisms that exist, wherein amino acid substitutions in the protein sequence do not affect the essential functions of the protein.
By “purified” and “isolated” is meant, when referring to a polypeptide or polynucleotide, that the molecule is separate and discrete from the whole organism with which the molecule is found in nature; or devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith. It is to be understood that the term “isolated” with reference to a polynucleotide intends that the polynucleotide is separate and discrete from the chromosome from which the polynucleotide may derive. The term “purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present. An “isolated polynucleotide which encodes a particular polypeptide” refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
By “fragment” is intended a polypeptide or polynucleotide consisting of only a part of the intact sequence and structure of the reference polypeptide or polynucleotide, respectively. The fragment can include a 3′ or C-terminal deletion or a 5′ or N-terminal deletion, or even an internal deletion, of the native molecule. A polynucleotide fragment of a CYP83A1 sequence will generally include at least about 15 contiguous bases of the molecule in question, more preferably 18-25 contiguous bases, even more preferably 30-50 or more contiguous bases of the CYP83A1 molecule, or any integer between 15 bases and the full-length sequence of the molecule. Fragments which provide at least one CYP83A1 phenotype as defined above are useful in the production of transgenic plants. Fragments are also useful as oligonucleotide probes, to find additional CYP83A1 sequences, e.g., in different plant species.
Similarly, a polypeptide fragment of a CYP83A1 molecule will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, anti most preferably at least about 20-50 or more contiguous amino acid residues of the full-length CYP83A1 molecule, or any integer between 10 amino acids and the full-length sequence of the molecule. Such fragments are useful for the production of antibodies and the like.
By “transgenic plant” is meant a plant into which one or more exogenous polynucleotides have been introduced. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, and the like. In the context of the present invention, the transgenic plant contains a CYP83A1 polynucleotide which is either over- or underexpressed and which confers at least one phenotypic trait to the plant, as defined above. The transgenic plant therefore exhibits altered structure, morphology or biochemistry as compared with a progenitor plant which does not contain the transgene, when the transgenic plant and the progenitor plant are cultivated under similar or equivalent growth conditions. A transgenic plant may also over- or underexpress glucosinolates. Such a plant containing the exogenous polynucleotide is referred to here as an R₁generation transgenic plant. Transgenic plants may also arise from sexual cross or by selfing of transgenic plants into which exogenous polynucleotides have been introduced. Such a plant containing the exogenous nucleic acid is also referred to here as an R₁generation transgenic plant. Transgenic plants which arise from a sexual cross with another parent line or by selfing are “descendants or the progeny” of a R₁plant and are generally called F_nplants or S_nplants, respectively, n meaning the number of generations.
General Overview
The inventors herein have discovered that CYP83A1, a cytochrome P450, regulates glucosinolate production in Arabidopsis. In particular, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. Plants which overexpress or underexpress this enzyme, therefore, have altered phenotypes, as described above. Thus, plant growth, nutritional values and plant pathogens can be affected by modulating levels of expression of this enzyme.
The molecules of the present invention are therefore useful in the production of transgenic plants which display at least one altered phenotype, so that the resulting plants have altered structure or morphology. The present invention particularly provides for altered structure or morphology such as reduced cell length, extended flowering periods, increased size of leaves or fruit, increased branching, and increased seed production relative wild-type plants. The CYP83A1 polypeptides can be expressed to engineer a plant with desirable properties. The engineering is accomplished by transforming plants with nucleic acid constructs described herein which may also comprise promoters and secretion signal peptides. The transformed plants or their progenies are screened for plants that express the desired polypeptide.
Engineered plants exhibiting the desired altered structure or morphology can be used in plant breeding or directly in agricultural production or industrial applications. Plants having the altered phenotypes can be crossed with other altered plants engineered with alterations in other growth modulation enzymes, proteins or polypeptides to produce lines with even further enhanced altered structural morphology characteristics compared to the parents or progenitor plants.
The present invention also pertains to methods of producing glucosinolates and indole-3-acetic acid. Glucosinolates are hydrophilic, non-volatile thioglycosides found within several orders of dicotyledoneous angiosperms (Cronquist, The Evolution and Classification of Flowering Plants, New York Botanical Garden, Bronx, 1988). The greatest economic significance of glucosinolates is their presence in all members of the Brassicaceae (order of Capparales) that are a source of condiments, relishes, salad crops and vegetables as well as fodders and forage crops. Additionally, these compounds are pharmaceutically significant and may find use as anti-cancer agents. More recently, rape (especially Brassica napus and Brassica campestris) has emerged as a major oil seed of commerce.
About 100 different glucosinolates are known which possess the same general chemical structure but differ in the nature of the side chain. Generally, glucosinolates are grouped into three different classes: aliphatic, aromatic and indole, depending on whether they are derived from aliphatic amino acids, aromatic amino acids, or tryptophan. The amino acids can be converted into glucosinolates either directly or after the side chains on the amino acids have been modified, for example, by chain-elongation. Initially, the amino acids or chain-elongated amino acids are converted to the labile aldoximes by cytochrome P450s, the aldoximes are hydroxylated by another cytochrome P450 of the CYP83 family and eventually metabolized to form a glucosinolate.
The glucosinolates are derived from seven protein amino acids, namely alanine, valine, leucine, isoleucine, tyrosine, tryptophan, and phenylalanine, chain-elongated forms thereof, as well as homophenylalanine and several chain-elongated homologues of methionine. In vivo biosynthetic studies have shown that N-hydroxyamino acids, nitro compounds, aldoximes, thiohydroximates, and desulfoglucosinolates are precursors of glucosinolates.
The first step in the biosynthesis of glucosinolate and indole glucosinolates is catalyzed by cyctochromes P450 of the CYP79 subfamily. CYP79 catalyzes the conversion of amino acids to their corresponding aldoximes via N-hydroxyamino intermediates. The aldoximes are then acted on by another subfamily of cytochromes P450, CYP83A1 and CYP83B 1, which convert aldoximes to glucosinolates and indole glucosinolates, respectively. The cytochromes are thought to act by adding a hydroxyl group at the nitrogen atom of the oxime function which generates a highly reactive aci-nitro compound. The α-carbon atom of the aci-nitro compound is a target for a nucleophilic attack from a sulfhydryl group, resulting in the formation of the corresponding S-alkylthiohydroximate or indole-3-S-alkylthiohydroximate. The S-alkylthiohydroximate can be cleaved presumably by a C-S lyase to generate thiohydroximates. It is well established that thiohydroximates are glucosylated by a soluble UDPG:thiohydroximate glucosyltransferase to form desulfoglucosinolates that are subsequently sulfated. Thus, for the biosynthesis of glucosinolates, aliphatic or aromatic amino acids are catalyzed by CYP79B2 or CYP79B3 to acetaldoximes. CYP83 μl catalyzes the conversion of acetaldoximes to the corresponding aci-nitro compounds which converts to S-alkyl-thiohydroximate which in turn converts to glucosinolate.
Isolation of Nucleic Acid Sequences from Plants
The isolation of CYP83A1 polynucleotides may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed herein can be used to identify the desired gene in a cDNA or genomic DNA library from a desired plant species. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a library of tissue-specific cDNAs, mRNA is isolated from tissues and a cDNA library which contains the gene transcripts is prepared from the mRNA.
The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned gene such as the polynucleotides disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR.RTM and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
Appropriate primers and probs for identifying CYP83A1-specific genes from plant tissues are generated from comparisons of the sequences provided herein. For a general overview of PCR see Innis et al. eds, PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990). Appropriate primers for this invention include, for instance, primers derived from the CYP83A1 polynucleotide sequence depicted in FIG. 1 herein. Suitable amplifications conditions may be readily determined by one of skill in the art in view of the teachings herein, for example, including reaction components and amplification conditions as follows: 10 mM Tris-HCl, pH 8.3, 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.001% gelatin, 200 μM DATP, 200 μM dCTP, 200 μM dGTP, 200 μM dTTP, 0.4 μM primers, and 100 units per mL Taq polymerase; 96° C. for 3 min., 30 cycles of 96° C. for 45 seconds, 50° C. for 60 seconds, 72° C. for 60 seconds, followed by 72° C. for 5 min.
Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers, et al. (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418, and Adams, et al. (1983) J. Am. Chem. Soc. 105:661. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
The polynucleotides of the present invention may also be used to isolate or create other mutant cell gene alleles. Mutagenesis consists primarily of site-directed mutagenesis followed by phenotypic testing of the altered gene product. Some of the more commonly employed site-directed mutagenesis protocols take advantage of vectors that can provide single stranded as well as double stranded DNA, as needed. Generally, the mutagenesis protocol with such vectors is as follows. A mutagenic primer, i.e., a primer complementary to the sequence to be changed, but consisting of one or a small number of altered, added, or deleted bases, is synthesized. The primer is extended in vitro by a DNA polymerase and, after some additional manipulations, the now double-stranded DNA is transfected into bacterial cells. Next, by a variety of methods, the desired mutated DNA is identified, and the desired protein is purified from clones containing the mutated sequence. For longer sequences, additional cloning steps are often required because long inserts (longer than 2 kilobases) are unstable in those vectors. Protocols are known to one skilled in the art and kits for site-directed mutagenesis are widely available from biotechnology supply companies, for example from Amersham Life Science, Inc. (Arlington Heights, Ill.) and Stratagene Cloning Systems (La Jolla, Calif.).
Control Elements
Regulatory regions can be isolated from the CYP83A1 gene and used in recombinant constructs for modulating the expression of the gene or a heterologous gene in vitro and/or in vivo. This region may be used in its entirety or fragments of the region may be isolated which provide the ability to direct expression of a coding sequence linked thereto.
Thus, promoters can be identified by analyzing the 5′ sequences of a genomic clone including the CYP83A1 gene and sequences characteristic of promoter sequences can be used to identify the promoter. Sequences controlling eukaryotic gene expression have been extensively studied. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. In most instances the TATA box is required for accurate transcription initiation. In plants, further upstream from the TATA box, at positions −80 to −100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T) N G. (See, J. Messing et al., in Genetic Engineering in Plants, pp. 221-227 (Kosage, Meredith and Hollaender, eds. (1983)). Methods for identifying and characterizing promoter regions in plant genomic DNA are described, for example, in Jordano et al. (1989) Plant Cell 1:855-866; Bustos et al (1989) Plant Cell 1:839-854; Green et al. (1988) EMBO J. 7:4035-4044; Meier et al. (1991) Plant Cell 3:309-316; and Zhang et al (1996) Plant Physiology 110:1069-1079).
Additionally, the promoter region may include nucleotide substitutions, insertions or deletions that do not substantially affect the binding of relevant DNA binding proteins and hence the promoter function. It may, at times, be desirable to decrease the binding of relevant DNA binding proteins to “silence” or “down-regulate” a promoter, or conversely to increase the binding of relevant A binding proteins to “enhance” or “up-regulate” a promoter. In such instances, the nucleotide sequence of the promoter region may be modified by, e.g., inserting additional nucleotides, changing the identity of relevant nucleotides, including use of chemically-modified bases, or by deleting one or more nucleotides.
Promoter function can be assayed by methods known in the art, preferably by measuring activity of a reporter gene operatively linked to the sequence being tested for promoter function. Examples of reporter genes include those encoding luciferase, green fluorescent protein, GUS, neo, cat and bar.
Polynucleotides comprising untranslated (UTR) sequences and intron/exon junctions may also be identified. UTR sequences include introns and 5′ or 3′ untranslated regions (5′ UTRs or 3′ UTRs). These portions of the gene, especially UTRs, can have regulatory functions related to, for example, translation rate and mRNA stability. Thus, these portions of the gene can be isolated for use as elements of gene constructs for expression of polynucleotides encoding desired polypeptides.
Introns of genomic DNA segments may also have regulatory functions. Sometimes promoter elements, especially transcription enhancer or suppressor elements, are found within introns. Also, elements related to stability of heteronuclear RNA and efficiency of transport to the cytoplasm for translation can be found in intron elements. Thus, these segments can also find use as elements of expression vectors intended for use to transform plants.
The introns, UTR sequences and intron/exon junctions can vary from the native sequence. Such changes from those sequences preferably will not affect the regulatory activity of the UTRs or intron or intron/exon junction sequences on expression, transcription, or translation. However, in some instances, down-regulation of such activity may be desired to modulate traits or phenotypic or in vitro activity.
Use of Nucleic Acids of the Invention to Inhibit Gene Expression
The isolated sequences prepared as described herein, can be used to prepare expression cassettes useful in a number of techniques. For example, expression cassettes of the invention can be used to suppress (underexpress) endogenous CYP83A1 gene expression. Inhibiting expression can be useful, for instance, in producing an glucosinolate phenotype, as described above. Further, the inhibitory polynucleotides of the present invention can also be used in combination with overexpressing constructs described below, for example, using suitable tissue-specific promoters linked to polynucleotides described herein. In this way, the polynucleotides can be used to modulate glucosinolate phenotypes in selected tissue and, at the same time, modulate glucosinolate phenotypes in different tissue(s).
A number of methods can be used to inhibit gene expression in plants. For instance, antisense technology can be conveniently used. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA is produced. In plant cells, antisense RNA may inhibit gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al (1988) Proc. Nat. Acad. Sci. USA 85:8805-8809, and Hiatt et al., U.S. Pat. No. 4,801,340.
The nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous gene or genes to be repressed. The sequence, however, need not be perfectly identical to inhibit expression. The vectors of the present invention can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the target gene.
For antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full length nucleotides should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 500 nucleotides is especially preferred. It is to be understood that any integer between the above-recited ranges is intended to be captured herein.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression of CYP83A1 genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs which are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in Haseloff et al (1988) Nature 334:585-591.
Another method of suppression is sense suppression. Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al (1990) The Plant Cell 2:279-289 and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.
Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 50%-65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% absolute identity would be most preferred. It is to be understood that any integer between the above-recited ranges is intended to be captured herein. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.
For sense suppression, the introduced sequence in the expression cassette, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants which are overexpressers. A higher identity in a shorter than full length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern and identity of non-coding segments will be equally effective. Normally, a sequence of the size ranges noted above for antisense regulation is used.
Use of Nucleic Acids of the Invention to Enhance Gene Expression
The present invention may also be used to overexpress CYP83A1. For example, by operably linking the CYP83A1 coding sequence to a promoter which allows for overexpression of the gene. (See the discussion regarding promoters below.) The exogenous CYP83A1 polynucleotides do not have to code for exact copies of the endogenous CYP83A1 proteins. Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described for instance, in Sambrook et al., supra. Hydroxylamine can also be used to introduce single base mutations into the coding region of the gene (Sikorski et al (1991) Meth. Enzymol. 194: 302-318). For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
It will be apparent that the polynucleotides described herein can be used in a variety of combinations. For example, the polynucleotides can be used to produce different phenotypes in the same organism, for instance by using tissue-specific promoters to overexpress a CYP83A1 polynucleotide in certain tissues (e.g., leaf tissue) while at the same time using tissue-specific promoters to inhibit expression of in other tissues. In addition, fusion proteins of the polynucleotides described herein with other known polynucleotides (e.g., polynucleotides encoding products involved in the brassinosteroid pathway) can be constructed and employed to obtain desired phenotypes.
Any of the polynucleotides described herein can also be used in standard diagnostic assays, for example, in assays for mRNA levels (see, Sambrook et al, supra); as hybridization probes, e.g., in combination with appropriate means, such as a label, for detecting hybridization (see, Sambrook et al., supra); as primers, e.g., for PCR (see, Sambrook et al., supra); attached to solid phase supports and the like.
Preparation of Recombinant Vectors
To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described further below as well as in the technical and scientific literature. See, for example, Weising et al (1988) Ann. Rev. Genet. 22:421-477. A DNA sequence coding for the desired polypeptide, for example a cDNA sequence encoding the full-length CYP83A1 protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transgenic plant.
Such regulatory elements include but are not limited to the promoters derived from the genome of plant cells (e.g., heat shock promoters such as soybean hsp17.5-E or hsp17.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565); the promoter for the small subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie et al (1984) Science 224:838-843); the promoter for the chlorophyll a/b binding protein) or from plant viruses viral promoters such as the ³⁵S RNA and 19S RNA promoters of CaMV (Brisson et al. (1984) Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al. (1987) EMBO J. 6:307-311), cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, heat shock promoters (e.g., as described above) and the promoters of the yeast alpha-mating factors.
In construction of recombinant expression cassettes of the invention, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) ³⁵S transcription initiation region, the T-DNA mannopine synthetase promoter (e.g., the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens), and other transcription initiation regions from various plant genes known to those of skill.
Alternatively, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, or flowers such as tissue- or developmental-specific promoter, such as, but not limited to the CHS promoter, the PATATIN promoter, etc. The tissue specific E8 promoter from tomato is particularly useful for directing gene expression so that a desired gene product is located in fruits.
Other suitable promoters include those from genes encoding embryonic storage proteins. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light. If proper polypeptide expression is desired, a polyadenylation region at the 3′-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. In addition, the promoter itself can be derived from the CYP83A1 gene, as described above.
The vector comprising the sequences (e.g., promoters or coding regions) from CYP83A1 will typically comprise a marker gene which confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.
Production of Transgenic Plants
DNA constructs may be introduced into the genome of the desired plant host by a variety of conventional techniques. For reviews of such techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al (1987) Nature 327:70-73). Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al (1984) Science 233:496-498, and Fraley et al (1983) Proc. Nat'l Acad. Sci USA 80:4803. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivation procedure (Horsch et al (1985) Science 227:1229-1231). Generally, the Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al (1982) Ann. Rev. Genet 16:357-384; Rogers et al (1986) Methods Enzymol. 118:627-641). The Agrobacterium transformation system may also be used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells. (see Hemalsteen et al (1984) EMBO J3:3039-3041; Hooykass-Van Slogteren et al (1984) Nature 311:763-764; Grimsley et al (1987) Nature 325:1677-179; Boulton et al (1989) Plant Mol. Biol. 12:31-40; and Gould et al (1991) Plant Physiol. 95:426-434).
Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation through calcium, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984) EMBO J. 3:2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell 4:1495-1505). Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618).
Transformed plant cells which are produced by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans, et al., “Protoplasts Isolation and Culture” in Handbook of Plant Cell Culture, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al (1987) Ann. Rev. of Plant Phys. 38:467-486.
The nucleic acids of the invention can be used to confer desired traits on essentially any plant. A wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present invention and the various transformation methods mentioned above. In preferred embodiments, target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis). Thus, the invention has use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.
One of skill in the art will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
A transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the β-glucuronidase, luciferase, B or C1 genes) that may be present on the recombinant nucleic acid constructs of the present invention. Such selection and screening methodologies are well known to those skilled in the art.
Physical and biochemical methods also may be used to identify plant or plant cell transformants containing the gene constructs of the present invention. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S1 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art.
Effects of gene manipulation using the methods of this invention can be observed by, for example, northern blots of the RNA (e.g., mRNA) isolated from the tissues of interest. Typically, if the amount of mRNA has increased, it can be assumed that the endogenous CYP83A1 gene is being expressed at a greater rate than before. Other methods of measuring CYP83A1 activity can be used. For example, cell length can be measured at specific times. Because CYP83A1 affects the glucosinolate biosynthetic pathway, an assay that measures the amount of glucosinolate can also be used, as well as assays that measure the direct step where CYP83A1 is involved. Such assays are known in the art. Different types of enzymatic assays can be used, depending on the substrate used and the method of detecting the increase or decrease of a reaction product or by-product. In addition, the levels of CYP83A1 protein expressed can be measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based assays well known to those of skill in the art, by electrophoretic detection assays (either with staining or western blotting), and glucosinolate detection assays.
The transgene may be selectively expressed in some tissues of the plant or at some developmental stages, or the transgene may be expressed in substantially all plant tissues, substantially along its entire life cycle. However, any combinatorial expression mode is also applicable.
The present invention also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct. The present invention further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
Polypeptides
The present invention also includes CYP83A1 polypeptides, including such polypeptides as a fusion, or chimeric protein product (comprising the protein, fragment, analogue, mutant or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
As noted above, the phenotype due to over or underexpression of CYP83A1 includes any macroscopic, microscopic or biochemical changes which are characteristic of over or underexpression of glucosinolate or auxin. Thus, the phenotype (e.g., activities) can include any activity that is exhibited by the native CYP83A1 polypeptide including, for example, in vitro, in vivo, biological, enzymatic, immunological, substrate binding activities, etc. Non-limiting examples of such activities include:

- (a) activities displayed by other heme-thiolate enzymes;
- (b) characteristic Soret absorption peak at 450 nm when the substrate-bound reduced form is exposed to the lights (see, e.g., Jefcoate et al., infra);
- (c) oxidation, dealkylation, deaminoation, dehalogenation, and sulfoxide formation that are involved in a variety of biological events in plants and animals (e.g., catabolism, anabolism, and xenobiotic activities);
- (d) activity on indole-3-acetaldoxime;
- (e) glucosinolate phenotypic activities such as modulation of cell length, periods of flowering, branching, seed production and leaf size;
- (f) regulation of glucosinolates and auxins; and
- (g) induce resistance to plant pathogens (see, e.g., U.S. Pat. No. 5,952,545).

A CYP83A1 analog, whether a derivative, fragment or fusion of native CYP83A1 polypeptides, is capable of at least one CYP83A1 activity. Preferably, the analogs exhibit at least 60% of the activity of the native protein, more preferably at least 70% and even more preferably at least 80%, 85%, 90% or 95% of at least one activity of the native protein.
Further, such analogs exhibit some sequence identity to the native CYP83A1 polypeptide sequence. Preferably, the variants will exhibit at least 35%, more preferably at least 59%, even more preferably 75% or 80% sequence identity, even more preferably 85% sequence identity, even more preferably, at least 90% sequence identity; more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity.
CYP83A1 analogs can include derivatives with increased or decreased activities as compared to the native CYP83A1 polypeptides. Such derivatives can include changes within the domains, motifs and/or consensus regions of the native CYP83A1 polypeptide.
One class of analogs is those polypeptide sequences that differ from the native CYP83A1 polypeptide by changes, insertions, deletions, or substitution; at positions flanking the domain and/or conserved residues. For example, an analog can comprise (1) the domains of a CYP83A1 polypeptide and/or (2) at conserved or nonconserved residues. For example, an analog can comprise residues conserved between the CYP83A1 polypeptide and other cytochrome P450 proteins with other regions of the molecule changed.
Another class of analogs includes those that comprise a CYP83A1 polypeptide sequence that differs from the native sequence in the domain of interest or conserved residues by a conservative substitution.
Yet another class of analogs includes those that lack one of the in vitro activities or structural features of the native CYP83A1 polypeptides, for example, dominant negative mutants or analogs that comprise a heme-binding domain but other inactivated domains.
CYP83A1 polypeptide fragments can comprise sequences from the native or analog sequences, for example fragments comprising one or more of the following P450 domains or regions: A, B, C, D, anchor binding, and proline rich. Such domains and regions are known.
Fusion polypeptides comprising CYP83A1 polypeptides (e.g., native, analogs, or fragments thereof) can also be constructed. Non-limiting examples of other polypeptides that can be used in fusion proteins include chimeras of CYP83A1 polypeptides and fragments thereof; and other known P450 polypeptides or fragments thereof.
In addition, CYP83A1 polypeptides, derivatives (including fragments and chimeric proteins), mutants and analogues can be chemically synthesized. See, e.g., Clark-Lewis et al. (1991) Biochem. 30:3128-3135 and Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156. For example, CYP83A1, derivatives, mutants and analogues can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 50-60). CYP83A1, derivatives and analogues that are proteins can also be synthesized by use of a peptide synthesizer. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 198′, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 34-49).
Further, the polynucleotides and polypeptides described herein can be used to generate antibodies that specifically recognize and bind to the protein products of the CYP83A1 polynucleotides. (See, Harlow and Lane, eds. (1988) “Antibodies: A Laboratory Manual”). The polypeptides and antibodies thereto can also be used in standard diagnostic assays, for example, radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), “sandwich” immunoassays, immunoradiometric assays, in situ immunoassay, western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.
Applications
The present invention finds use in various applications, for example, including but not limited to those listed above. In particular, the present invention contemplates production of transgenic plants that over or underexpress CYP83A1, thereby producing any of the various phenotypes specified above. Thus, the CYP83A1 polynucleotides may be placed in recombinant vectors which may be inserted into host cells to express the CYP83A1 protein, under the control of promoters that either enhance or decrease CYP83A1 expression.
The nucleic acid molecules may be used to design plant CYP83A1 antisense molecules, useful, for example, in plant CYP83A1 gene regulation or as antisense primers in amplification reactions of plant gene nucleic acid sequences. With respect to plant gene regulation, such techniques can be used to regulate, for example, plant growth, development or gene expression. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for gene regulation.
Thus, the molecules of the present invention can be used to provide plants with increased seed and/fruit production, extended flowering periods and increased branching, by altering the glucosinolate composition of a plant. A still further utility of the molecules of the present invention is to provide a tool for studying the biosynthesis of glucosinolates, both in vitro and in vivo.
The Arabidopsis CYP83A1 protein can be used in any biochemical applications (experimental or industrial), for example, but not limited to, regulation of glucosinolate synthesis, modification of elongating plant structures, and experimental or industrial biochemical applications known to those skilled in the art.

EXPERIMENTAL

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Materials and Methods
Plants.
Plants were grown at a photosynthetic flux of 100-120 μmol photons m⁻²s⁻¹and 70% humidity, 22° C. for a 12 h photoperiod. For morphometric analyses, seedlings were grown vertically on Murashige and Skoog (MS) agar plates without addition of antibiotics and grown for a 16 h photoperiod. Morphometric analyses are shown with their standard error of the mean.
The molecularly complemented rnt1-1 line used in this study was line 3.25.11 (Bak et al. (2001) Plant Cell. 13:101-111). For functional complementation of rnt1-1, overexpression constructs comprising the CYP83A1 cDNA under control of a cauliflower mosaic virus ³⁵S promoter and polyadenylation site were made in pPZP221 (Hajdukiewicz et al. (1994) Plant Mol Biol 25:989-994). Primary transformants were selected on MS plates supplemented with 2% sucrose, 0.9% Bacto agar, 50 μg/ml kanamycin and 200 μg/ml gentamycin. Lines homozygous for the T-DNA insertion in CYP83B1 and homozygous for the introduced ³⁵S::CYP83A1 construct were identified by co-segregation analysis on selective MS agar plates.
Indole glucosinolate content in 10-day-old seedlings grown as described for the morphometric analyses were quantified colorimetrically as the degradation product thiocyanite (SCN) as previously described (Bak et al. (2001) Plant Cell. 13:101-111).
Analysis of Recombinant CYP83A1 and CYP83B1 Enzyme.
Microsomes from yeast WAT11 cells expressing the CYP83A1 and CYP83B1 cDNA using the pYeDP60 vector were isolated and the amount of functional enzyme quantified essentially according to Pompon et al. (1996) Methods Enzymol. 272:51-64. Indole-3-acetaldoxime and radiolabelled indole-3-acetaldoxime were prepared as described in Bak et al. (2001) Plant Cell. 13:101-111 and references therein. V_maxand K_mwere determined as previously described using 2.2 nM of CYP83A1 or CYP83B1 and 50 mM L-cysteine as thiol donor (Bak et al. (2001) Plant Cell. 13:101-111). Type II spectra were recorded using 0.44 μM CYP83B1 or 0.15 μM CYP83A1 and in the presence of 0.200 μM ligand and using a Lambda19 spectrophotometer (Perkin Elmer). V_max, K_m, and K_swere calculated using SigmaPlot 5.0 (SPSS Inc.). For analysis of CYP83B1 activity in the presence or absence of thiol donors, recombinant CYP83B1 was reconstituted and analyzed as previously described (Bak et al. (2001) Plant Cell. 13:101-111).
Identification and Quantification of Substrates and Products by GC-MS.
For structural analysis of the products of CYP83A1 and CYP83B1 catalysis, 0.5 μM recombinant enzyme was reconstituted using 1 mM of either indole-3-acetaldoxime, p-hydroxyphenylacetaldoxime or phenylacetaldoxime and incubated for 20 min at 28° C. and analyzed using GC-MS essentially as previously described (Bak et al. (2001) Plant Cell. 13:101-111). Turnover numbers were calculated based on the relative areas under the substrate and product peaks. Silylated substrates and products were identified by their fragmentation pattern in both electron impact mode and chemical ionization mode.
Fragments identified by chemical ionization mode: Silylated indole-3-acetaldoxime (15.447 min): [M+H]⁺m/z 319, major fragmentation ion m/z 202. Silylated S-mercaptoyl-indole-3-acetaldoxime (21.032 min): [M+H]+m/z 467, major fragmentation ions m/z 229, and m/z 202. Silylated (E+Z)-p-hydroxyphenylacetaldoxime (11.860 and 11.942 min): [M+H]⁺m/z 296 major fragmentation ion m/z 179. Silylated S-mercaptoyl-p-hydroxyphenylacetaldoxime (17.433 min): [M+H]⁺m/z 444, major fragmentation ions m/z 206 and m/z 179. Silylated phenylethylacetaldoxime (11.066 min): [M+H]⁺m/z 209. Silylated S-mercaptoyl-phenylacetaldoxime (14.666 min): [M+H]⁺m/z 356, major fragmentation ions m/z 226, m/z 206, m/z 118 and m/z 91.

Example 1

CYP83A1 Functionally Complements CYP83B1 in rnt1-1

To determine whether CYP83A1 is a functional homolog of CYP83B1, CYP83A1 cDNA was ectopically expressed in rnt1-1 under control of the ubiquitous ³⁵S cauliflower mosaic virus promoter (CaMV). Plants heterozygous for knockout of CYP83B1 (rnt1-1/RNT1) were used for transformation because the homozygous plant is not optimal for transformation due to its severe phenotype (Bak et al. (2001) Plant Cell. 13:101-111). Out of 26 primary transformants, 15 were viable. These 15 primary transformants were selfed, and the seeds were germinated on double selection plates to select for lines containing both the ³⁵S:: CYP83A1 construct and the T-DNA insertion in CYP83B 1. Out of these 15 original viable lines, 5 lines did not display the characteristic rnt1-1 seedling phenotype in a rnt1-1/rnt1-1 background.
Lines complemented by CYP83A1 under control of the ³⁵S CaMV promoter displayed significantly shorter hypocotyls and non-epinastic cotyledons as compared to one-week-old rnt1-1 seedlings. When compared to wild type seedlings, the hypocotyls of the CYP83A1-coinplemented lines were shorter. This had also been observed in rnt1-1 seedlings complemented with a genomic clone comprising the CYP83B1 gene (Bak et al. (2001) Plant Cell. 13:101-111). The appearance of primary roots of one-week-old rnt1-1, wild type or complemented seedlings did not differ. However, the characteristic extensive proliferation of root hairs and secondary roots from the primary root as well as the development of secondary roots from the vascular tissue in the hypocotyl in two-week-old rnt1-1 seedlings (Delarue et al. (1998) Plant J. 14:603-611; Bak et al. (2001) Plant Cell. 13: 101-111) were abolished in the complemented lines.
Whereas the visual phenotypes of the complemented seedlings were very similar, changes were observed in mature plants. Some of the complemented lines appeared slightly bigger than wild type (lines 2.8.6 and 2.9.5), whereas other lines such as 2.24.3 were characterized by being shorter and bushier compared to e.g. the lines 2.8.6 and 2.9.5. In the latter line, up to 20 inflorescences could be observed. In addition, this line exhibited flower abnormalities and many of the siliques contained none or only a few seeds.
Indole-3-acetaldoxime is the metabolic branch point in tryptophan-dependent IAA and indole glucosinolate biosynthesis. Molecular complementation of rnt1-1 using a 5.5 kb genomic fragment comprising the CYP83B 1 gene has previously been shown (Bak et al. (2001) Plant Cell. 13:101-111). In accordance with the hypothesis that indole-3-acetaldoxime is the metabolic branch point, the functionally complemented rnt1-1 lines ectopically expressing CYP83A1 cDNA complemented both the high IAA phenotype and the deficiency in indole glucosinolates (see, FIG. 3).
In A. thaliana, indole-3-acetaldoxime is a metabolic branch point in IAA and indole glucosinolate biosynthesis and the level of IAA can be regulated by the flux of indole-3-acetaldoxime through CYP83B1 (FIG. 8) (Bak et al. (2001) Plant Cell. 13:101-111). The above study demonstrates that ectopic expression of CYP83A1 cDNA can functionally complement CYP83B 1 by suppressing the high IAA phenotype and deficiency in indole glucosinolate of rnt1-1.
Knock-out of CYP83B 1 results in plants characterized by increased apical dominance and elongated hypocotyls (Barlier et al. (2000) Proc Natl Acad. Sci. USA 97:14819-14824; Bak et al. (2001) Plant Cell. 13:101-111) due to an increase of free IAA (Delarue et al. (1998) Plant J. 14:603-611; Barlier et al. (2000) Proc Natl Acad. Sci. USA 97:14819-14824). Ectopic overexpression of CYP83B1 cDNA using the ³⁵S promoter in wild type Arabidopsis also showed a bushier phenotype in 3 out of 13 transformants. Similarly, bushy phenotypes were seen in 2 out of 18 rnt1-1 lines molecularly complemented with a genomic fragment comprising the CYP83B1 gene (Bak et al. (2001) Plant Cell. 13:101-111). Multiple insertions as well as position effects may result in lines that phenotypically resemble overexpression lines. The phenotype of plants like 2.24.3 is similar to the phenotype of strong alleles of axr1, characterized by decreased apical dominance, reduced hypocotyl length and fertility as a result of reduced sensing of auxin (Estelle and Sommerville (1987) Mol. Gen. Genet. 206:200-206; Lincoln et al. (1990) Plant Cell 2:1071-1080; Leyser et al. (1993) Nature 364:161-164; Collett et al. (2000) Plant Physiol. 124:553-561). Arabidopsis seedlings overexpressing the bacterial enzyme tryptophan monooxygenase (iaaM) have up to 4-fold higher IAA levels than wild type and are characterized by having elongated hypocotyls (Romano et al. (1995) Plant Mol. Biol. 27:1071-1083). Conversely, plants that overexpress iaaL have reduced levels of free IAA and shorter hypocotyls due to increased conjugation of IAA to Lys (Romano et al. (1991) Genes Dev. 5:438-446; Jensen et al. (1998) Plant Physiol. 116:455-462).
Although functional complementation of CYP83B1 in rnt1-1 by overexpression of CYP83A1 under the control of the ³⁵S promoter was demonstrated, the CYP83A1 gene is not redundant compared to CYP83B1, because CYP83A1 cannot prevent the rnt1-1 phenotype when expressed under the control of its native promoter in the rnt1-1 background.

Example 2

CYP83A1 and CYP83B1 Metabolize indole-3-acetaldoxime with Different Affinity

It has previously been shown that CYP83B1, when co-expressed in yeast with A. thaliana NADPH cytochrome P450 reductase, metabolizes indole-3-acetaldoxime in the presence of thiol compounds to S-alkyl-thiohydroxymates (Bak et al. (2001) Plant Cell. 13: 101-111). The nature of the initially monooxygenated product of CYP83B1 catalysis is not formally known, but it has been proposed to be an aci-nitro compound, 1-aci-nitro-2-indolyl-ethane originating from N-hydroxylation of indole-3-acetaldoxime (Ettlinger and Kjaer (1968) Rec. Adv. Phytochem. 1:49-144; Bak et al. (2001) Plant Cell. 13:101-111). This proposed aci-nitro compound is a strong electrophile that non-enzymatically reacts preferentially with thiol compounds to form S-alkylthiohydroximate adducts (FIG. 4). In the absence of β-mercaptoethanol, the enzymatic reaction is inhibited: less indole-3-acetaldoxime is metabolized (FIG. 4A). As the conjugate formed in the absence of a nucleophile does not migrate on thin-layer chromatography (FIG. 4A), it most likely represents the conjugate formed by the electrophilic product of the enzymatic reaction with the nucleophilic sites of the enzyme, thereby leading to the inactivation of the enzyme (FIG. 4).
To determine if CYP83A1 metabolizes indole-3-acetaldoxime in a similar manner to CYP83B1, CYP83A1 was produced in yeast cells. Reconstitution experiments using yeast microsomes in the presence of thiol compounds showed that yeast microsomes containing CYP83A1 also metabolize indole-3-acetaldoxime leading to thiohydroximate adducts. Kinetics with indole-3-acetaldoxime as substrate and using cysteine as thiol donor were compared for both enzymes (FIG. 5). CYP83B1 had a K_mof 3.1±0.4 μM and a V_maxof 52±2 m⁻¹(Bak et al. (2001) Plant Cell. 13:101-111), whereas the corresponding values for CYP83A1 were 150±15 μM and 140±10 min⁻¹respectively. Based on these apparent enzyme parameters, CYP83B1 exhibits a 50 fold lower K_sand a 20 fold higher catalytic efficiency (V_max/K_m) compared to CYP83A1.
Thus, in accordance with the in planta complementation results described in Example 1, indole-3-acetaldoxime was identified as a substrate for recombinant CYP83A1. Oximes are generally unstable and considered toxic compounds that do not accumulate in the cell. To optimize and control catalytic activities most biosynthetic enzymes have K_m's in the range of the concentration of their substrate. The in vivo concentration of indole-3-acetaldoxime in A. thaliana is not known. However, in the related cruciferous plant Chinese Cabbage, Brassica campestris, the indole-3-acetaldoxime concentration has been reported to be less than 50 pmol/g fresh weight (Helminger et al. (1985) Phytochemistry 24:2497-2502). Indole-3-acetaldoxime constitutes a metabolic branch point between IAA and indole glucosinolate biosynthesis, and it has previously been determined that enzymes in indole glucosinolate and IAA biosynthesis utilize the same indole-3-acetaldoxime pool (Bak et al. (2001) Plant Cell. 13:101-111). This implies that an enzyme working in such a branch point must have a K_min the same range as CYP83B 1 in order to efficiently compete for the substrate. The 50 fold higher K_mof CYP83A1 relative to CYP83B1 thus argues that indole-3-acetaldoxime is not a substrate for CYP83A1 under normal conditions.
As shown in Example 1, overexpression of CYP83A1 cDNA in the rnt1-1 background did not result in elevated indole glucosinolate levels as compared to wild type seedlings (FIG. 3). This is in contrast to overexpression of CYP83B 1 cDNA (Bak et al. (2001) Plant Cell. 13:101-111) which resulted in increased levels of indole glucosinolates. These data imply that CYP83A1 does not to the same extent as CYP83B 1 compete with an indole-3-acetaldoxime metabolizing enzyme in IAA biosynthesis. Low levels of indole glucosinolates are present in rnt1-1 seedlings (Bak et al. (2001) Plant Cell. 13:101-111; FIG. 3). Thus, some of the indole glucosinolates present in the seedlings may not originate from de novo synthesis, but by translocation of indole glucosinolates from the seed.
Without being bound by a particular theory, there are two reasonable explanations for the ability of the CYP83 μl cDNA to functionally complement rnt1-1: (1) in rnt1-1, indole-3-acetaldoxime accumulates to levels that makes it available to CYP83A1; or (2) ectopic expression of CYP83A1 restores the channeling of indole glucosinolate biosynthesis by restoring a supra molecular enzymatic complex with e.g. CYP79B. The latter explanation satisfies the observation that increased levels of indole glucosinolates were not seen in the functionally complemented lines (FIG. 3).
The catalytic mechanism by which CYP83B1 and CYP83 μl convert aldoximes is not known. However, the oxygen atom of the oxime function may lodge between the heme iron and the P450 I-helix thereby replacing a water molecule as sixth ligand to the heme iron. This replacement of wafer by the oxime may explain the absence of a strong type I binding spectrum. Subsequent introduction of an additional hydroxyl group at the nitrogen atom of the oxime function generates a highly reactive aci-nitro compound. The α-carbon atom of the aci-nitro compound is a target for a nucleophilic attack from a sufhydryl group resulting in the formation of indole-3-S-alkylthiohydroxymate with a dehydration reaction taking place either before or after adduct formation. An aci-nitro compound has previously been proposed as an intermediate in glucosinolate biosynthesis (Ettlinger and Kjaer (1968) Rec. Adv. Phytochem. 1:49-144). Liver microsomes have in a similar manner been suggested to catalyze the conversion of n-butyraldoxime to nitrobutane via an aci-nitro compound (DeMaster et al. (1992) J. Org. Chem. 57:5674-5075). The observed ability to form S-alkylthiohydroximate adducts with a wide range of structurally very different thiol compounds in vitro suggests that formation of the adduct proceeds non-enzymatically outside the active site (FIG. 4). In accordance with this proposed mechanism, indole-3-acetaldoxime metabolism in the absence of a nucleophile eventually inactivates the enzyme (FIG. 4A).
CYP83B1 has recently been shown to be induced by IAA. Accordingly, we analyzed in silico 2.5 Kb upstream of the start codon of CYP83B 1 for cis-acting elements (Higo et al. (1999) Nucleic Acids Research 27:297-300), and identified four putative AuxREs (auxin-responsive cis-acting elements; Guilfoyle et al. (1998) Plant Physiol. 118:341-347; Ulmasov et al. (1999) Plant J. 19:309-319). It has previously been shown that a 5.5 Kb genomic fragment comprising this putative CYP83B1 promoter is sufficient to achieve molecular complementation of rnt1-1 (Bak et al. (2001) Plant Cell. 13:101-111). Conversely, no AuxREs could be identified 2.5 Kb region upstream of CYP83A1. Accordingly, cDNA micro array data show that in rnt1-1 seedlings CYP83A1 transcripts are not induced but down regulated 3.5 fold. This suggests that CYP83B1, but not CYP83A1, is under the regulation of auxin.

Example 3

Interaction with Ligands

To characterize the topology of the active sites of CYP83 μl and CYP83B 1, we have taken advantage of the ability of nitrogen-containing ligands like primary amines to produce type II spectra with cytochrome P450 enzymes by binding to the active site bringing the electron lone pairs of the amine group in close vicinity to the heme iron (Jefcoate C. R. (1978) Methods Enzymol 27:258-279). This gives rise to a characteristic spectrum with a trough around 390 nm and a peak around 425 nm. It has previously been reported that tryptamine is a ligand that binds to the active site and inhibits metabolism of indole-3-acetaldoxime by CYP83B1 (Bak et al. (2001) Plant Cell. 13:101-111). Similar results were obtained with CYP83A1. Likewise, type II spectra were observed for CYP83B1 and CYP83A1 with n-octylamine and the amines corresponding to phenylalanine (P-phenylethylamine), and tyrosine (tyramine) (FIG. 6). Indole-3-acetonitrile (IAN) did not produce a type II spectrum, showing that the nitrogen atom of the indole ring system does not contribute. Introduction of a hydroxyl group at the 5 position of tryptamine (5-OH-tryptamine/serotonin) abolished binding. Similarly, tyramine produced a weak type II spectrum, whereas 3-OH-tyramine (i.e. dopamine) and histamine did not. This indicates that introduction of hydroxyl groups or an electronegative group in the aromatic ring causes significant reduction of ligand binding to the active site. Based on the sizes of the amplitudes of the type II spectra recorded using 200 μM ligand, the relative affinity for ligand binding to CYP83B1 is tryptamine >>β-phenylethylanine>n-octylamine>tyramine. CYP83A1 shows a different affinity for the same amines: n-octylamine>>α-phenylethylamine=tryptarine>tyramine. The observed difference in affinity for the amines tested argues that although the same ligands bind to CYP83B 1 and CYP83A1, the topology of their active site differ.
By titrating the amplitude of the type II difference spectra with increasing concentrations of ligand, Ks values were determined for tryptamine and β-phenylethylamine (FIG. 7). K_svalues of 18±5 μM and 240±180 μM were calculated for tryptamine for CYP83B 1 and CYP83A1 respectively. K, values of 540±180 μM and 390±70 μM were estimated for P-phenylethylamine binding to CYP83B1 and CYP83A1 respectively. Accordingly, CYP83B1 binds tryptamine 13-fold stronger compared to CYP83A1. Compared to P-phenylethylamine, tryptamine is a 30 fold stronger ligand for CYP83B 1. In contrast, CYP83A1 displays similar high binding constants for tryptamine and β-phenylethylamine. Due to high absorbance and low amplitude of the type II spectra, K_svalues could not be determined for tyramine.

Example 4

Interaction with Oximes

Indole-3-acetaldoxime is a substrate for CYP83B1 and CYP83A1 as shown by heterologous expression studies and by the ability of CYP83A1 to functionally complement CYP83B1 in rnt1-1. Substrates for cytochromes P450 often give rise to the formation of a type I or reverse type I spectrum upon binding, depending on the spin state of the heme iron (Jefcoate C. R. (1978) Methods Enzymol 27:258-279). Besides CYP83A1 and CYP83B 1, the only other plant cytochrome P450 known to metabolize an aldoxime is CYP71E1 from sorghum. CYP71E1 is involved in the biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin and catalyzes the conversion of p-hydroxyphenylacetaldoxime top-hydroxymandelonitrile (Kahn et al. (1997) Plant Physiol. 115:1661-1670; Bak et al. (1998) Plant Mol. Biol. 36:393-405; Kahn (1999) Arch. Biochem. Biophys. 363:9-18). The substrate binding spectra obtained using p-hydroxyphenylacetaldoxime as a substrate for sorghum CYP71E1 were not trivial and prone to peculiar artifacts (Kahn et al. (1997) Plant Physiol. 115:1661-1670; Kahn (1999) Arch. Biochem. Biophys. 363:9-18). Similarly, spectral analysis of a cytochrome P450 in rat liver microsomes displayed peculiar binding spectra with aryl and alkyl aldoximes (Boucher et al. (1994) Biochmeistry 33:7811-7818). Only a weak reverse type I spectrum was recorded upon indole-3-acetaldoxime binding to CYP83B1 (Bak et al. (2001) Plant Cell. 13:101-111). Accordingly, a K_svalue of 0.2 μM for indole-3-acetaldoxime binding to CYP83B1 was determined by exploiting the ability of indole-3-acetaldoxime to displace the ligand tryptamine from the active site of CYP83B1 (Bak et al. (2001) Plant Cell. 13:101-111). In that approach, CYP83B1 was first saturated with 100 μM tryptamine. Tryptamine was subsequently displaced from the active site by titration with increasing amounts of indole-3-acetaldoxime, causing a gradual appearance of a reverse type II spectrum. Unfortunately, a similar approach could not be used for CYP83A1 because (1) much higher levels of tryptamine (1000 μM) are required to saturate CYP83A1 giving rise to interfering levels of ligand absorbance (FIG. 6); (2) the amplitude of the type II spectra produced by tryptamine binding to CYP83A1 is much weaker than for CYP83B 1 (FIG. 6); and (3) indole-3-acetaldoxime absorbance interferes significantly at concentrations higher than 1 μM.

Reconstitution experiments were also conducted to compare the ability of CYP83A1 and CYP83B1 to metabolize other oximes. The putative substrates tested were p-hydroxyphenylacetaldoxime derived from tyrosine and phenylacetaldoxime derived from phenylalanine. In all studies, β-mercaptoethanol was the thiol donor. After incubation in the presence or absence of NADPH, reaction mixtures were extracted with ethyl acetate, and the ethyl acetate phase containing both substrate and product was lyophilized, silylated and analysed by GC-MS (gas chromatography—mass spectrometry) as previously described (Bak et al. (2001) Plant Cell. 13:101-111). As expected, the turnover of indole-3-acetaldoxime was lower using CYP83A1 compared to CYP83B1 under the experimental conditions applied (Table 1). Conversely, the turnover of p-hydroxyphenylacetaldoxime was higher using CYP83A1 compared to CYP83B 1. Phenylacetaldoxime was identified as a substrate for CYP83A1 as well as for CYP83B1 but the turnover numbers were low (Table 1).

TABLE 1


CYP83A1 and CYP83B1 have overlapping substrate - and ligand affinity in vitro.

		Turnover p-	Turnover
K_mindole-3-	Turnover, indole-	hydroxyphenyl-	phenylethyl-	K_sfor	K_sfor β-
acetaldoxime	3-acetaldoxime	acetaldoxime	acetaldoxime	tryptamine	phenylethylamine

CYP83B1	3.1 μM	26 min⁻¹	9.8 min ⁻¹	15 min⁻¹	18 μM	540 μM
CYP83A1	150 μM	10 min⁻¹	25 min⁻¹	7.2 min⁻¹	240 μM	390 μM

Based on the above studies, the inventors herein have shown that CYP83A1 is a regulator of glucosinolate production in Arabidopsis and that CYP83A1 and CYP83B 1 are not redundant enzymes. Indole-3-acetaldoxime, phenylacetaldoxime and p-hydroxyphenylacetaldoxime are all substrates for CYP83A1 and CYP83B1 in vitro. Based on the turnover numbers using high substrate concentrations (1 mM), p-hydroxyphenylacetaldoxime is the preferred substrate for CYP83A1 as compared to CYP83B1. Arabidopsis contains at least 24 glucosinolates derived from tryptophan and chain elongated homologs of phenylalanine and methionine (Hogge et al. (1988) Chromog. Sci. 26:551-556; Petersen et al. (2001) Planta in press). In Arabidopsis, seven functional CYP79 homologs and six CYP79 pseudogenes have been identified. These CYP79s appear to catalyze the conversion of amino acids and chain-elongated amino acids to their corresponding aldoximes (Bak et al. (1998b) Plant Mol. Biol. 38:725-734), as has been documented for CYP79A2, CYP79B2, CYP79B3 and CYP79F1 (Hull et al. (2000) Proc. Natl. Acad. Sci. USA 97:2379-2384; Mikkelsen et al. (2000) J. Biol. Chem. 275:33712-33717; Wittstock and Halkier (2000) J. Biol. Chem. 275:14659-14666; Hansen et al. (2001) J. Biol. Chem. 276:11078-11085; Reintanz et al. (2001) Plant Cell. 13:351-367). These CYP79 homologs are highly substrate-specific and are thought to determine the substrate specificity of glucosinolate biosynthesis. In contrast, only two CYP83 homologs are present in the Arabidopsis genome.
Based on the foregoing, CYP83B 1 appears to be primarily involved in the biosynthesis of indole glucosinolates whereas CYP83A1 is involved in biosynthesis of those glucosinolates that are not derived from tryptophan (FIG. 8). Use of a separate CYP83 for indole glucosinolate biosynthesis insures tight control of the flux of the shared intermediate, indole-3-acetaldoxime, for indole glucosinolate and IAA biosynthesis as is also indicated by the presence of putative AuxREs in the CYP83B 1 but not in the CYP83A1 promoter. The evidence demonstrates that CYP83s and other post-oxime enzymes have a low substrate specificity.
Thus, novel methods for modulating glucosinolate synthesis are disclosed. Although preferred embodiments 6f the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined herein.

Claims

1. A transgenic plant with altered CYP83A1 expression relative to the corresponding wild-type plant.

2. The transgenic plant of claim 1, wherein CYP83A1 is overexpressed.

3. The transgenic plant of claim 1, wherein CYP83A1 is underexpressed.

4. A method of producing a transgenic plant with altered CYP83A1 expression relative to the wild-type plant, said method comprising:

(a) introducing an expression construct that comprises a polynucleotide encoding a CYP83A1 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide into a plant cell to produce a transformed plant cell; and

(b) producing a transgenic plant from the transformed plant cell with altered CYP83A1 expression.

5. The method of claim 4, wherein CYP83A1 is overexpressed.

6. The method of claim 4, wherein CYP83A1 is underexpressed.

7. The method of claim 4, wherein the polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter.

8. A method of producing a transgenic plant with altered CYP83A1 expression relative to the wild-type plant, said method comprising:

(a) introducing a polynucleotide that inhibits expression of a CYP83 μl polynucleotide into a plant cell to produce a transformed plant cell; and

9. A method for altering the biochemical activity of a cell, said method comprising:

(b) growing the cell under conditions such that the biochemical activity of the cell is altered.

10. The method of claim 9, wherein the expression construct is introduced into the cell ex vivo.

11. The method of claim 9, wherein the expression construct is introduced into the cell in vivo.

12. A method for altering the biochemical activity of a cell, said method comprising:

(a) introducing a polynucleotide that inhibits expression of a CYP83A1 polynucleotide into a plant cell to produce a transformed plant cell; and

13. The method of claim 12, wherein the polynucleotide is introduced into the cell ex vivo.

14. The method of claim 12, wherein the polynucleotide is introduced into the cell in vivo.

15. A method of producing a transgenic plant with altered expression of a cytochrome P450 that catalyzes the conversion of an aldoxime to a glucosinolate, the method comprising:

(a) introducing an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide, into a plant cell to produce a transformed plant cell; and

(b) producing a transgenic plant from the transformed plant cell with altered cytochrome P450 expression.

16. The method of claim 15, wherein the cytochrome P450 is CYP83A1.

17. The method of claim 16, wherein CYP83A1 is overexpressed.

18. The method of claim 16, wherein CYP83A1 is underexpressed.

19. The method of claim 15, wherein the polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter.

20. The method of claim 15, wherein the glucosinolate is aliphatic, aromatic or indolic.

21. The method of claim 20, wherein the glucosinolate is aliphatic.

22. The method of claim 21, wherein the aliphatic glucosinolate is obtained from a corresponding aldoxime.

23. The method of claim 22, wherein the aldoxime is obtained from the conversion of an aliphatic amino acid or a chain-elongated form thereof to the corresponding N-hydroxy amino acid, and the conversion of the N-hydroxyamino acid to the aldoxime.

24. The method of claim 23, wherein the aliphatic amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine and chain-elongated forms thereof.

25. The method of claim 20, wherein the glucosinolate is aromatic.

26. The method of claim 25, wherein the aromatic glucosinolate is obtained from the corresponding aldoxime.

27. The method of claim 26, wherein the aldoxime is obtained from the conversion of an aromatic amino acid to the corresponding N-hydroxy amino acid, and the conversion of the N-hydroxyamino acid to the aldoxime.

28. The method of claim 27, wherein the aromatic amino acid is phenylalanine or tyrosine.

29. A method of producing a cytochrome P450 that catalyzes the conversion of aldoxime to a corresponding aci-nitro and the conversion of the aci-nitro to a corresponding S-alkyl-thiohydroximate and the conversion of the S-alkyl-thiohydroximate to glucosinolate, the method comprising:

(a) introducing an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing or underexpressing the polypeptide, into a host cell to produce a transformed host cell;

(b) expressing the cytochrome P450 in the host cell; and

(c) isolating the expressed the cytochrome P450.

30. The method of claim 29, wherein the cytochrome P450 is CYP83A1.

31. The method of claim 30, wherein CYP83 μl is overexpressed.

32. The method of claim 29, wherein the polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter.

33. The method of claim 29, wherein the aldoxime is obtained from the conversion of an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, tyrosine, and phenylalanine to a corresponding N-hydroxy amino acid, and the conversion of N-hydroxyamino acid to the aldoxime.

34. A method for producing a glucosinolate, the method comprising contacting an acetaldoxime with a cytochrome P450, and isolating the glucosinolate.

35. The method of claim 34, wherein the cytochrome P450 is CYP83A1.

36. The method of claim 34, wherein the cytochrome P450 is overexpressed in a transformed host cell.

37. The method of claim 36, wherein the transformed host cell comprises an expression construct that comprises a polynucleotide encoding a cytochrome P450 polypeptide operably linked to a promoter which is capable of overexpressing the polypeptide.

38. The method of claim 37, wherein the polynucleotide is operably linked to a promoter selected from the group consisting of a tissue-specific promoter, an inducible promoter and a constitutive promoter.