WO2000047715A2 - Dwf4 polynucleotides, polypeptides and uses thereof - Google Patents
Dwf4 polynucleotides, polypeptides and uses thereof Download PDFInfo
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- WO2000047715A2 WO2000047715A2 PCT/US2000/003820 US0003820W WO0047715A2 WO 2000047715 A2 WO2000047715 A2 WO 2000047715A2 US 0003820 W US0003820 W US 0003820W WO 0047715 A2 WO0047715 A2 WO 0047715A2
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- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12N15/8291—Hormone-influenced development
- C12N15/8298—Brassinosteroids
Definitions
- the present invention relates to novel polynucleotides isolated from dwarf plants.
- the dwf4 polynucleotides encode all, or a portion of, a DWF4 polypeptide, a cytochrome P450 enzyme that mediates multiple steps in synthesis of brassinosteroids.
- the present invention also relates to isolated polynucleotides that encode regulatory regions of dwf4. Uses of the dwf4 polypeptides and polynucleotides are also disclosed.
- BACKGROUND Plant growth is accomplished by orderly cell division and tightly regulated cell expansion. In plants, the contribution of cell expansion to growth is of much greater significance than in most other organisms; all plant organs owe their final size to a period of significant cell elongation, which usually follows active cell division. Further, the sessile nature of plants requires that they make fine but responsive adjustments in growth to survive harsh environmental conditions and to optimize their use of limited resources (Trewavas (1986) "Resource allocation under poor growth conditions: A major role for growth substances in developmental plasticity" In Plasticity in Plants, D.H. Jennings and A.J. Trewavas, eds (Cambridge, UK: Company of Biologists Ltd.), pp. 31-76).
- the internal components of plant signaling are generally mediated by chemical growth regulators (phytohormones; reviewed in Klee, H., and Estelle, M. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:529-551 ).
- plant growth in response to environmental factors is modulated by plant hormones acting alone or in concert (Evans "Functions of hormones at the cellular level of organization” In Hormonal Regulation of Plant Physiology, T.K. Scott, ed (Berlin: Springer- Verlag), pp. 23-79), and growth depends on regulated cellular events, such as division, elongation, and differentiation.
- GA Gibberellic acid
- cytokinins promote flowering; in addition, GA stimulates stem elongation, whereas cytokinins have the opposite effect, reducing apical dominance by stimulating increased axillary shoot formation.
- auxins promote apical dominance and stimulate elongation by a process postulated to require acidification of the cell wall by a K " -dependent H + -pumping ATPase (Rayle, D.L., and Cleland, R.E. (1977) Curr. Top. Dev. Biol. 11 :187-214).
- BRs brassinosteroids
- brassinolide (BL; 2 , 3 ⁇ , 22(R), 23(R)-tetrahydroxy-24(S)- methyl-B-homo-7-oxa-5 ⁇ -cholestan-6-one) has been shown to be the most biologically active (reviewed in Mandava (1988) Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:23-52 ).
- BRs stimulate longitudinal growth of young tissues via cell elongation and cell division (reviewed in Clouse (1996), supra; Fujioka and Sakurai (1997a) Nat. Prod. Rep. 14:1-10).
- BRs are synthesized via multiple parallel pathways (Fujioka et al. (1996) Plant Cell Physiol. 37:1201-1203; Choi et al. (1997), supra).
- campesterol campesterol
- the BR intermediates undergo a series of hydroxylations, reductions, an epimerization, and a Baeyer-Villiger ⁇ type oxidation leading to the most oxidized form, BL (Fujioka and Sakurai (1997b) Physiol. Plant. 100:710-715; Figure 1).
- Castasterone (CS) oxidation the last step in BR biosynthesis, is not found in some species, such as mung bean. In that case, CS plays a role as the major BR rather than BL (Yokota et al. (1991) "Metabolism and biosynthesis of brassinosteroids" in Brassinosteroids: Chemistry, Bioactivity, and Application, H.G.
- GA biosynthetic mutants may also have no or defective flower development and are marked by an absence of viable pollen. Reduced levels of endogenous gibberellins are also a characteristic (Barendse et al.(1986) Physiol. Plant. 67:315-319; Talon et al. (1990) Proc. Natl. Acad. Sci. USA 87:7983-7987), and their phenotype can be nearly restored to that of the wild type by the addition of exogenous GA. (Koornneef and Van der Veen (1980) Theor. Appl. Genet. 58:257-263).
- auxin resistant2 results in plants with a dwarf phenotype both in the light and in darkness as well as increased resistance to high levels of auxin, ethylene, and abscisic acid (Timpte et al. (1992) Planta 188:271-278).
- CAB chlorophyll a/b binding protein gene
- DET2 was shown to encode a putative steroid 5 -reductase, mediating an early step in BR biosynthesis (Li et al. (1996), supra , Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554-3559; Fujioka et al. (1997) Plant Cell 9:1951-1962; Figure 1).
- detl and det2 have a decreased requirement for cytokinins in tissue culture and appear to be saturated for a cytokinin-dependent delay in senescence (Chory et al. (1994) Plant Physiol. 104:339-347).
- CPD has been proposed to be a novel cytochrome P450 (CYP90A1; Szekeres et al. (1996), supra), encoding a putative 23 -hydroxylase that acts in BR biosynthesis.
- CYP90A1 Szekeres et al. (1996), supra
- dwarfs that are insensitive to one of these hormones, such as bri (brassinosteroid insensitive; Clouse et al. (1996) Plant Physiol. I l l :671-678; Li and Chory (1997) Cell 90:929-938), gai (gibberellic acid insensitive; Koornneef et al. (1985) Physiol. Plant. 65:33-39), and axr2 (auxin resistant2; Timpte et al. (1994) Genetics 138:1239-1249). Clouse et al.
- the invention includes an isolated dwf4 polynucleotide comprising an open reading frame that encodes a polypeptide comprising (i) a sequence having greater than 43% identity to the amino acid sequence of SEQUENCE LD NO:2; (ii) a sequence comprising at least about 10 contiguous amino acids that have greater than 43% identity to 10 contiguous amino acids of SEQUENCE ID NO:2, or a complement or reverse complement of said polynucleotide.
- the polynucleotide will have at least 70% identity to the DWF4 polypeptide-coding region of SEQ ID NO:l or to complements and reverse complements of this region.
- the isolated dwf4 polynucleotide comprises the nucleotide sequence of SEQ ID NO:l, complements and reverse complements thereof.
- the polynucleotide may also comprise at least 30 consecutive nucleotides of SEQ ID NO:l.
- the invention includes an isolated dwf4 polynucleotide comprising (i) a sequence having at least 50% identity to SEQ ID NO:l, complements and reverse complements thereof or (ii) a sequence comprising at least about 15 contiguous nucleotides that has at least 50% identity to SEQ ID NO: 1 , complements and reverse complements thereof.
- the isolated dwf4 polynucleotide has at least 50% identity to the DWF4 polypeptide-coding region of SEQ ID NO:l, complements and reverse complements thereof.
- the isolated dwfl polynucleotides described herein comprise the nucleotide sequence of SEQ ID NO: 1 , complements and reverse complements thereof or nucleotide sequences comprising at least 30 consecutive nucleotides of SEQ ID NO: 1.
- Any of the dwf4 polynucleotides described herein may be genomic DNA and may include introns.
- the dwf4 polynucleotide includes a dwf4 control control element comprising a polynucleotide selected from the group consisting of (i) a sequence having at least 50% identity to nucleotides 1 to 3202 of SEQ ID NO:l; (ii) a fragment of (i) which includes a dwf4 control element; and (iii) complements and reverse complements of (i) or (ii).
- the polynucleotide includes a dwfl control element comprising a polynucleotide selected from the group consisting of (i) a sequence having at least 50% identity to nucleotides 6111 to 6468 corresponding to the 3' UTR of SEQ ID NO:l; (ii) a fragment of (i) which includes a dwfl 3' UTR; and (iii) complements and reverse complements of (i) or (ii).
- the polynucleotide includes a dwfl polynucleotide selected from the group consisting of (i) a sequence having at least 50% identity to the sequences corresponding to the introns of SEQ ID NO:l; (ii) a fragment of (i) which includes a dwf4 intro; and (iii) complements and reverse complements of (i) and (ii).
- Introns are found, for example, in the following regions: nucleotides 3424 to 3503 of SEQ ID NO:l; nucleotides 3829 to 3913 of SEQ ID NO:l; nucleotides 4067 to 4164 of SEQ ID NO:l; nucleotides 4480 to 4531 of SEQ ID NO:l; nucleotides 4725 to 4815 of SEQ ID NO: 1; nucleotides 4895 to 5000 of SEQ ID NO:l; and nucleotides 5111 to 5864 of SEQ ID NO:l. 54.
- any of the polynucleotides described herein can operably linked to a nucleic acid molecule encoding a heterologous polypeptide (e.g., a cytochrome P450 polypeptide), for example, as a chimeric polynucleotide.
- a heterologous polypeptide e.g., a cytochrome P450 polypeptide
- the invention includes recombinant vectors comprising (i) one or more of the polynucleotides described above; and (ii) control elements operably linked to the one or more polynucleotides, whereby a coding sequence within said polynucleotide can be transcribed and translated in a host cell.
- the recombinant vector comprises (a) any of the polynucleotides which include a dwf4 control element described above (e.g., promoter or intron); and (b) a nucleic acid molecule comprising a coding sequence operably linked to the dwfl control element.
- Host cells comprising and/or transformed with any of the recombinant vectors described herein are also provided.
- the host cells are cultured ex vivo while in other embodiments, the dwf4 polynucleotide is provided the host cell in vivo.
- the DWF4 polypeptide is provided in amounts such that a plant is regenerated.
- the present invention includes a method of modulating a
- DWF4 polypeptide comprising the following steps: (a) providing a host cell as described herein; and (b) culturing said host cell under conditions whereby the dwf4 polynucleotide included in the host cell is transcribed. In certain embodiments, the dwfl polynucleotide is overexpressed. Alternatively, in other embodiments, the polynucleotide included in the host cell inhibits expression of dwfl.
- the present invention includes a transgenic plant comprising any of the recombinant vectors described herein.
- the invention includes a method of producing a recombinant polypeptide comprising the following steps: (a) providing a host cell as described herein; and (b) culturing said host cell under conditions whereby the recombinant polypeptide encoded by the coding sequence present in said recombinant vector is expressed.
- the invention includes a method of producing a transgenic plant comprising the steps of (a) introducing a polynucleotide described herein into a plant cell to produce a transformed plant cell; and (b) producing a transgenic plant from the transformed plant cell.
- Methods for producing a transgenic plant having an altered phenotype relative to the wild-type plant comprising the following steps: introducing at least one polynucleotide described herein into a plant cell; and producing a transgenic plant from the plant cell, said transgenic plant having an altered phenotype relative to the wild-type plant are also included in the present invention.
- the altered phenotype 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 heme-thiolate enzyme activity, altered monooxygenase activity, altered 22 ⁇ -hydroxylase activity, regulation of brassinosteriod synthesis, regulation of gibberellic acid, regulation of cytokinins, regulation of auxins, altered resistance to plant pathogens, altered growth at low temperatures, altered growth in dark conditions and altered sterol composition.
- the 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 polynucleotide can be overexpressed or it can inhibit expression of dwf4.
- 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 dwf4.
- the invention includes a method for altering the biochemical activity of a cell comprising the following steps: introducing at least one polynucleotide described herein; and culturing the cell under conditions such that the biochemical activity of the cell is altered.
- Biochemical activity includes, for example, altered heme-thiolate enzyme activity, altered monooxygenase activity, altered 22 ⁇ - hydroxylase activity, regulation of gibberellic acid, regulation of cytokinins, regulation of auxins, and altered sterol composition.
- the cell is cultured ex vivo.
- the dwfl polynucleotide is provided to the cell in vivo.
- the invention includes a method for regulating the cell cycle of a plant cell comprising the following steps providing a dwfl polynucleotide to a plant cell; and expressing the dwff polynucleotide to provide a DWF4 polypeptide, wherein the DWF4 polypeptide is provided in amounts such that cell cycling is regulated.
- the plant cell is provided in vitro and is cultured under conditions suitable for providing the DWF4 polypeptide.
- the dwf4 polynucleotide is provided in vivo.
- the invention includes an isolated DWF4 polypeptide comprising (i) a sequence having greater than 43% identity to SEQ ID NO:2 or (ii) fragments of (i) that confer a DWF4 phenotype when expressed in a host organism.
- the isolated DWF4 polypeptide comprises the amino acid sequence of SEQ ID NO:2.
- the invention includes a chimeric polypeptide comprising a DWF4 polypeptide as described herein and a heterologous polypeptide, for example a cytochrome P450 polypeptide.
- 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.
- Figure 1 depicts a proposed biosynthetic pathway for BL.
- CR goes through at least two different pathways, referred to as the early C-6 oxidation (right column) and late C-6 oxidation (left column) pathways.
- Steps mediated by DWF4, CPD (Szerkeres et al. (1996), supra), DET2 (Fujioka and Skaurai (1997a), infra; Li et al. (1997), supra) and LKB (Yokota et al. (1997), infra) are indicated.
- Figures 2 A and B depict schematic representations of the DWF4 gene and protein.
- Figure 2 A depicts the DWF4 coding sequence (1542 bp) and shows that the coding sequence contains eight exons and seven introns. The exons and introns range in length from 93 to 604 and 84 to 754 bp, respectively. All of the introns are bordered by typical consensus splice junctions, 5'-GU and AG-3'. Closed rectangles indicate exons.
- the T-DNA position in dwff-1 is marked with an arrow.
- Figure 2B shows the relative positions of the major domains in DWF4 cytochrome P450. All of the major domains found in the cytochrome P450 superfamily are conserved in DWF4.
- the estimated molecular mass and isoelectric point of the DWF4 protein were 58 kD and 7.28, respectively.
- Hydropathy plotting and protein localization prediction by the PSORT software package suggested that the protein may reside in a membrane of the endoplasmic reticulum as an integral protein. Mutations identified in the other dwfl alleles are indicated.
- Figure 3 depicts alignment of cytochrome P450 proteins that exhibited the most similarity to DWF4 in BLAST searches.
- GenBank accession numbers are AF044216 (DWF4; CYP90B), X87368 (CPD; CYP90A), U54770 (tomato; CYP85), D64003 (cyanobacteria; CYP120), U32579 (maize; CYP88), U68234 (zebrafish; CYP26), and M13785 (human; CYP3A3X).
- Dashes indicate gaps introduced to maximize alignment. Domains indicated in Figure 2B are highlighted in a box.
- Amino acid residues that are conserved >50% between the compared sequences are highlighted by a reverse font, and identical residues between DWF4 and CPD are boxed and italicized. Open triangles are placed under the 100%) conserved residues. Closed triangles locate functionally important amino acid residues, for example, threonine (T ) at 369, which is thought to bind molecular oxygen, and cysteine (C) at 516, which links to a heme prosthetic group by a thiolate bond. X's indicate mutated residues in dwfl alleles. Multiple sequence alignment was performed using PILEUP in the Genetics Computer Group package, and box shading was made possible by the ALSCRIPT package (Barton (1993) Protein Eng. 6:37-40).
- Figure 4 depicts the phylogenetic Relationship between DWF4 and Selected Cytochrome P450s.
- DWF4 did not cluster with the group A plant cytochrome P450s that are known to mediate plant-specific reactions (Durst and Nelson 1995).
- CYP90A, CYP85, and DWF4 which are thought to be involved in BR metabolism, branched from CYP88, which mediates GA biosynthesis.
- GenBank accession numbers for the group A cytochrome P450s are M32885 (avocado; CYP71 Al), P48421 (Arabidopsis; CYP83), P48418 (petunia; CYP75A1), and X71658 (eggplant; CYP76A1).
- Figure 5 depicts a comparison of wild-type and dwfl hypocotyl growth rates. Circles indicate wild-type and square indicate dwfl. Each data point represents the average of 10 seedlings.
- Figure 6 depicts responses to cell elongation signals.
- BL measurements were performed with dwft-3 and the corresponding wild-type control, Enkheim. Open bars indicate the wild type. Filled bars indicate dwfl. Lines above the bars represent one standard deviation.
- light refers to light-grown controls;
- dark refers to dark-grown controls;
- hy2 refers to DWF4 and dwff plants in a hy2 background;
- GA refers to plants grown in 10 "5 M GA;
- 2,4-D refers to plants grown in 10 '8 M 2,4-D;
- -BR refers to liquid-grown controls; and
- +BR refers to liquid-grown controls with 10 "8 M BL.
- Figure 7 depicts pedicel elongation of dwff mature plants in response to exogenous application of BR. Measurements were performed with the BR-fed plants. dwft-1 plants were more sensitive to intermediates belonging to the late C-6 oxidation pathway (10 "6 M 6-deoxoCT and 10 "6 M 6-deoxoTE) compared with compounds in the early C-6 pathway (10 "5 M CT and 10 "5 M TE). BL (10 '7 M) induced almost the same amount of elongation with one-tenth the concentration of its precursors.
- Figure 9 depicts the increase in seed production of three transformants which overexpress dwf4 as compared to wild type (Ws-2). Seeds were harvested from individual plants of each genotype (n>5). Seeds from each plant were weighed and a mean value calculated. The Figure shows percent increase over wild type.
- Figures 10(A)- 10(G) depict the nucleotide sequence of wild-type dwfl (SEQ ID NO: 1 , see, also, GenBank Accession Number AF044216).
- the dwf4 polynucleotide includes a coding region between nucleotides 3203 and 6110, inclusive.
- the coding region includes the following eight exons: nucleotides 3203 to 3423, inclusive; nucleotides 3504 to 3828, inclusive; nucleotides 3914 to 4066, inclusive; nucleotides 4165 to 4479, inclusive; nucleotides 4632 to 4724, inclusive; nucleotides 4816 to 4894, inclusive; nucleotides 5001 to 5110, inclusive and nucleotides 5865 to 6110, inclusive.
- the exons are indicated by a bar beneath the nucleotide sequence.
- a 5' control region (e.g., promoter) extends from nucleotides 1 to 3202.
- a 3' untranslated region corresponds to the region extending from nucleotide to 6011 to approximately nucleotide 6468 of Figure 10 (SEQ ID NO:l) and a TATA signal extending approximately from nucleotides 3060 to 3125.
- mutant alleles of dwff have also been characterized.
- dwf4-l contains an approximately 20 kb insert between nucleotides 5202 and 5203.
- dwfl-2 has a 9 base pair deletion corresponding to amino acids 324-326.
- the polypeptide is 513 amino acids in length.
- Figure 12 depicts seedling phenotypes of twelve-day-old dwf4-l, wild type, epi-BL-treated wild type, and AOD4 lines grown in the light and dark, particularly quantification of hypocotyl and root growth.
- the average lengths of 16 seedlings are displayed with the standard deviation.
- Increased BR concentration supplied exogenously or endogenously resulted in both elongated hypocotyls and shortened roots.
- the novel dwff polynucleotides and DWF4 polypeptides described herein are important molecules in regulating cell growth and sterol synthesis.
- the present inventors have shown that dwff encodes a cytochrome P450 monooxygenase having 43%o sequence identity to the protein termed Constitutive Phoromorphogenesis and Dwfarism (CPD).
- CPD Constitutive Phoromorphogenesis and Dwfarism
- both CPD and DWF4 polypeptides appear to regulate biosynthesis of brassinosteriods, for example brassinolide (BL).
- DWF4 appears to act as a "gatekeeper" in these biosynthetic pathways in that its substrates (e.g., 6-Oxo campestanol and 6 ⁇ -Hydroxy campestanol) are approximately 500 times more prevalent than the downstream molecules.
- substrates e.g., 6-Oxo campestanol and 6 ⁇ -Hydroxy campestanol
- the present invention represents an important discovery in understanding and regulating cell growth.
- Cysteine Cys (C) Glutamine: Gin (Q)
- Threonine Thr (T) Tryptophan: Tip (W) Tyrosine: Tyr (Y) Valine: Val (V) Definitions
- 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.
- 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, intemucleotide 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., alkylators, those with
- 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 (rnRNA), 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).
- A adenine
- C cytosine
- G guanine
- T thymine
- U uracil
- T thymine
- T uracil
- T thymine
- 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.
- such techniques include determining the nucleotide sequence of the rnRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence.
- 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 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 Davhoff, Atlas of Protein Sequences and Structure. M.O. Dayhoff ed., 5 suppl.
- 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.
- 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.
- a nucleic acid probe 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 10% 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, DC; IRL Press).
- 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).
- 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.
- a gene typically 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).
- 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).
- 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.
- 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.
- 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
- 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.
- 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.
- vector any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus etc., which is capable of transferring gene sequences to target cells.
- a vector is capable of replication when associated with the proper control elements.
- 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.
- 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.
- 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.
- the exogenous DNA may be maintained on an episomal element, such as a plasmid.
- 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 mo ⁇ hology 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.
- dwfl polynucleotide refers to a polynucleotide derived from the dwff gene.
- the gene encodes the protein referred to herein as DWF4.
- DWF4 is a cytochrome P450 cytochrome P450 that mediates multiple 22 ⁇ -hydroxylation steps in brassinosteroid biosynthesis (see, Figure 1).
- the dwf4 polynucleotide sequence and corresponding amino acid sequence are shown in Figures 10 and 11 (SEQ LD NO:l, SEQ ED NO:2 and GenBank accession No. AF044216).
- the dwff coding sequence spans the region from nucleotide positions 3203 to 6110 and the upstream 5' UTR, including the promoter region, spans nucleotide positions 1 to 3202.
- a functional 1.1 kb control element is also described in the Examples.
- a 3' UTR spans nucleotide positions 6111 to approximately 6468 of SEQ ID NO: 1.
- the term as used herein encompasses a polynucleotide including a native sequence depicted in Figure 10, as well as modifications and fragments thereof.
- the term encompasses alterations to the polynucleotide sequence, so long as the alteration results in a plant displaying one or more dwf4 phenotypic traits (described below) when the polynucleotide is expressed in a plant.
- modifications typically include deletions, additions and substitutions, to the native dwff sequence, so long as the mutation results in a plant displaying a dwff phenotype as defined below.
- These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of plants which express the dwff polynucleotide or errors due to PCR amplification.
- the term encompasses expressed allelic variants of the wild-type dwff sequence which may occur by normal genetic variation or are produced by genetic engineering methods and which result in a detectable change in the wild-type dwff phenotype.
- dwff phenotype refers to any microscopic or macroscopic change in structure or mo ⁇ hology of a plant, such as a transgenic plant, as well as biochemical differences, which are characteristic of a dwf4 plant, compared to a progenitor, wild-type plant cultivated under the same conditions.
- mo ⁇ hological differences include multiple short stems, short rounded leaves, loss of fertility due to reduced stamen length, and delayed development. Dark-grown dwff seedlings possess short hypocotyls, open cotyledons, and developing leaves.
- dwff hypocotyls are converted to wild-type length with the application of BL.
- 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.
- 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.
- 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 "DWF4" polypeptide is a polypeptide as defined above, which is derived from a 22 ⁇ -hydroxylase that functions in the brassinolide (BL) biosynthetic pathway (see, Figure 1).
- the native sequence of full-length DWF4 is shown in Figure 11
- DWF4 analog refers to derivatives of DWF4, or fragments of such derivatives, that retain desired function, e.g., as measured in assays as described further below.
- 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.
- 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.
- 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.
- 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.
- the terms include the various sequence polymo ⁇ hisms that exist, wherein amino acid substitutions in the protein sequence do not affect the essential functions of the protein.
- 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.
- 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.
- 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 dwf4 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 dwff molecule, or any integer between 15 bases and the full-length sequence of the molecule.
- Fragments which provide at least one dwff phenotype as defined above are useful in the production of transgenic plants. Fragments are also useful as oligonucleotide probes, to find additional dwff sequences.
- a polypeptide fragment of a DWF4 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, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length DWF4 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.
- 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.
- the transgenic plant contains a polynucleotide which is not normally present in the corresponding wild-type plant and which confers at least one dwff phenotypic trait to the plant.
- the transgenic plant therefore exhibits altered structure, mo ⁇ hology 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 plant containing the exogenous polynucleotide is referred to here as an R, generation 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 R, generation transgenic plant.
- Transgenic plants which arise from a sexual cross with another parent line or by selfing are "descendants or the progeny" of a R, plant and are generally called F n plants or S n plants, respectively, n meaning the number of generations.
- the molecules of the present invention are therefore useful in the production of transgenic plants which display at least one dwff phenotype, so that the resulting plants have altered structure or mo ⁇ hology.
- the present invention particularly provides for altered structure or mo ⁇ hology such as reduced cell length, extended flowering periods, increased size of leaves or fruit, increased branching, increased seed production and altered sterol composition relative wild-type plants.
- the DWF4 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 mo ⁇ hology can be used in plant breeding or directly in agricultural production or industrial applications. Plants having the altered polypeptide 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 mo ⁇ hology characteristics compared to the parents or progenitor plants.
- oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library from a desired plant species.
- 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.
- 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.
- 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.
- 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 pu ⁇ oses.
- Appropriate primers and probes for identifying c ⁇ v/ ⁇ -specific genes from plant tissues are generated from comparisons of the sequences provided herein.
- Appropriate primers for this invention include, for instance, those primers described in the Examples and Sequence Listings, as well as other primers derived from the dwff sequences disclosed 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 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 200 ⁇ M dTTP, 0.4 ⁇ M 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.
- the desired mutated DNA is identified, and the desired protein is purified from clones containing the mutated sequence.
- 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, 111.) and Stratagene Cloning Systems (La Jolla, Calif).
- Regulatory regions can be isolated from the dwff gene and used in recombinant constructs for modulating the expression of the dwff gene or a heterologous gene in vitro and/or in vivo.
- the coding region of the dwff gene begins at nucleotide position 1133.
- the region of the gene spanning nucleotide positions 990-1132 of Figure 10 includes the dwff promoter. 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.
- promoters can be identified by analyzing the 5' sequences of a genomic clone corresponding to the Jw/ ⁇ -specific genes described here. 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.
- 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 DNA binding proteins to "enhance” or "up-regulate” a promoter.
- 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.
- reporter genes include those encoding luciferase, green fluorescent protein, GUS, neo, cat and bar.
- UTR sequences include introns and 5' or 3' untranslated regions ( 5' UTRs or 3' UTRs).
- the dwff gene sequence includes eight exons and seven introns. These portions of the dwff 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.
- the 5' control element region of dwf4 extends from nucleotides 1 through 3202 of SEQ ID NO: 1. Further, as described in Example 11 , a 1.1 kb portion of this region that is directly upstream of the translation initiation site contains elements necessary for transcriptional control of dwff. In contrast, a 280 bp fragment of the dwff control element region that includes the TATA-like region does not appear to contain all of the necessary transcriptional control elements (see, Example 11).
- 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.
- expression cassettes of the invention can be used to suppress (underexpress) endogenous dwff gene expression. Inhibiting expression can be useful, for instance, in suppressing the phenotype (e.g., dwarf appearance, 22 ⁇ -hydroxylase activity) exhibited by dwff plants.
- 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.
- the polynucleotides can be used to promote dwf4 phenotypes (e.g., activity) in selected tissue and, at the same time, inhibit dwf4 phenotypes (e.g., activity) 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.
- antisense RNA inhibits 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. Patent Number 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.
- 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.
- RNA molecules or ribozymes can also be used to inhibit expression of dwff 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.
- RNAs 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) N ⁇ t ⁇ re 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.
- this method to modulate expression of endogenous genes see, ⁇ apoli et al (1990) The Plant Cell 2:279-289 and U.S. Patent Numbers 5,034,323, 5,231,020, and 5,283,184.
- 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% to 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.
- the effect should apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.
- 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.
- 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.
- the polynucleotides of the invention can be used to increase certain features such as extending flowering, producing larger leaves or fruit, producing increased branching and increasing seed production. This can be accomplished by the overexpression of dwff polynucleotides.
- Modified DWF4 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).
- 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.
- the polynucleotides described herein can be used in a variety of combinations.
- the polynucleotides can be used to produce different phenotypes in the same organism, for instance by using tissue-specific promoters to overexpress a dwf4 polynucleotide in certain tissues (e.g., leaf tissue) while at the same time using tissue-specific promoters to inhibit expression of dwff in other tissues.
- tissue-specific promoters to overexpress a dwf4 polynucleotide in certain tissues (e.g., leaf tissue) while at the same time using tissue-specific promoters to inhibit expression of dwff in other tissues.
- fusion proteins of the polynucleotides described herein with other known polynucleotides e.g., polynucleotides encoding products involved in the BR pathway
- any of the dwff polynucleotides described herein can also be used in standard diagnostic assays, for example, in assays 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.
- 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 DWF4 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 hspl7.5-E or hspl7.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565); the promoter for the small subunit of RUBISCO (Coruzzi et al. (1984) EMBOJ. 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.
- promoters derived from the genome of plant cells e.g., heat shock promoters such as soybean hspl7.5-E or hspl7.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565
- TMV coat protein promoter of TMV
- cytomegalovirus hCMV immediate early gene the early or late promoters of SV40 adenovirus
- the lac system the tip system
- the TAC 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.
- 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.
- constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the T-D ⁇ A 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.
- 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).
- 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 dwff promoter, the CHS promoter, the PATATIN promoter, etc.
- tissue specific E8 promoter from tomato is particularly useful for directing gene expression so that a desired gene product is located in fruits.
- 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.
- the promoter itself can be derived from the dwff gene, as described above.
- the vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene which confers a selectable phenotype on plant cells.
- 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.
- DNA constructs of the invention 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.
- 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).
- 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.
- 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 Hernalsteen et al (1984) E ROJ3:3039-3041; Hooykass-Van Slogteren et al
- 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.
- PEG polyethylene glycol
- 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.
- 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 pu ⁇ oses (e.g., Arabidopsis).
- crops including grain crops (e.g., wheat, maize, rice, millet, barley
- 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.
- 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 ⁇ -glucuronidase, luciferase, B or Cl 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.
- any visible marker genes e.g., the ⁇ -glucuronidase, luciferase, B or Cl genes
- 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, S 1 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.
- RNA e.g., mRNA
- mRNA RNA isolated from the tissues of interest.
- dwff dwf4 gene
- cell length can be measured at specific times.
- an assay that measures the amount of BL can also be used. 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.
- the levels of DWF4 protein expressed can be measured immunochemically, i.e., ELISA, RIA, ELA 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 sterol (BL) 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.
- the present invention also includes DWF4 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)).
- 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.
- DWF4 phenotype includes any macroscopic, microscopic or biochemical changes which are characteristic of over- or under-expression of dwff.
- DWF4 polypeptide phenotype e.g., activities
- DWF4 activities can include any activity that is exhibited by the native DWF4 polypeptide including, for example, in vitro, in vivo, biological, enzymatic, immunological, substrate binding activities, etc.
- Non-limiting examples of DWF4 activities include:
- campestanol CN
- 6- deoxocastasterone 6-deoxoCS
- a DWF4 analog whether a derivative, fragment or fusion of native DWF4 polypeptides, is capable of at least one DWF4 activity.
- 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 DWF4 polypeptide sequence.
- 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.
- DWF4 analogs can include derivatives with increased or decreased activities as compared to the native DWF4 polypeptides. Such derivatives can include changes within the domains, motifs and/or consensus regions of the native DWF4 polypeptide, which are described in detail in Example 3.
- an analog can comprise (1) the domains of a DWF4 polypeptide and/or (2) residues conserved between the DWF4 polypeptide and other cytochrome P450 proteins, for example as shown in Figure 3 and described in Example 3.
- Another class of analogs includes those that comprise a DWF4 polypeptide sequence that differs from the native sequence in the domain of interest or conserved residues by a conservative substitution.
- an analog that exhibits increased sterol binding can have optimized sterol binding domain sequences that differ from the native sequence.
- Yet another class of analogs includes those that lack one of the in vitro activities or structural features of the native DWF4 polypeptides, for example, dominant negative mutants or analogs that comprise a heme-binding domain but contain an inactivated steroid binding domain.
- DWF4 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 shown in Figures 2B, 3 and described in Example 3.
- Fusion polypeptides comprising DWF4 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 DWF4 polypeptides and fragments thereof; and P450 polypeptides or fragments thereof, such as those shown in Figure 3.
- DWF4 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.
- DWF4, 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).
- DWF4 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, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 34-49).
- the dwf4 polynucleotides and DWF4 polypeptides described herein can be used to generate antibodies that specifically recognize and bind to the protein products of the dwf4 polynucleotides.
- the DWF4 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, immunoprecipitationassays, immuno fluorescent assays and PAGE-SDS.
- the present invention finds use in various applications, for example, including but not limited to those listed above.
- the polynucleotide sequences may additionally be used to isolate mutant dwf4 gene alleles. Such mutant alleles may be isolated from plant species either known or proposed to have a genotype which contributes to altered plant mo ⁇ hology. Additionally, such plant dwff gene sequences can be used to detect plant dwff gene regulatory (e.g., promoter or promoter/enhancer) defects which can affect plant growth.
- the molecules of the present invention can be used to provide plants with increased seed and/fruit production, extended flowering periods and increased branching.
- the molecules described herein can be used to alter the sterol composition of a plant, thereby increasing or reducing cholesterol content in the plant.
- a still further utility of the molecules of the present invention is to provide a tool for studying the biosynthesis of brassinosteriods, both in vitro and in vivo.
- the dwf4 gene of the invention also has utility as a transgene encoding a cytochrome P450 protein that mediates multiple 22 hydroxylation steps in brassinosteriod biosynthesis which results in a transgenic plant to alter plant structure or mo ⁇ hology.
- the dwff gene also has utility for encoding the DWF4 protein in recombinant vectors which may be inserted into host cells to express the DWF4 protein.
- the dwf4 polynucleotides of the invention may be utilized (1) as nucleic acid probes to screen nucleic acid libraries to identify other enzymatic genes or mutants; (2) as nucleic acid sequences to be mutated or, modified to produce DWF4 protein variants or derivatives; (3) as nucleic acids encoding 22 ⁇ -hydroxylase in molecular biology techniques or industrial applications commonly known to those skilled in the art.
- the dwff nucleic acid molecules may be used to design plant dwff antisense molecules, useful, for example, in plant dwff gene regulation or as antisense primers in amplification reactions of plant dwff gene nucleic acid sequences.
- dwff 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 dwff gene regulation.
- the dwff control element (e.g., promoter) of the present invention may be utilized as a plant promoter to express any protein, polypeptide or peptide of interest in a transgenic plant. In particular, the dwff promoter may be used to express a protein involved in brassinosteriod biosynthesis.
- the Arabidopsis DWF4 protein of the invention can be used in any biochemical applications (experimental or industrial) where 22 ⁇ -hydroxylase activity is desired, for example, but not limited to, regulation of BL synthesis, regulation of other sterol synthesis, modification of elongating plant structures, and experimental or industrial biochemical applications known to those skilled in the art.
- Plant Growth Conditions The conditions used for plant growth were essentially as described previously
- agar-solidified medium contained 0.5% sucrose. Seedlings up to 2 weeks of age (6 weeks of age for dark-growth experiments) were grown on 0.8%) agar-solidified medium containing 1 x Murashige and Skoog 1962 salts (Murashige, T., and Skoog, F. (1962) Physiol. Plant.
- the pots were covered with plastic wrap and cold treated (4°C) for two days before transfer to a growth chamber (16:8, light [240 ⁇ mol m "2 sec " ']:dark; 22 and 21 °C, respectively, and 75 to 90% humidity).
- the plastic wrap was removed 5 days after germination, and the pots were subirrigated in distilled water as required. Germination of seeds for dark growth experiments was induced by overnight exposure of the seeds to light immediately after removing the plates from incubation at 4°C.
- the dwff-1 and dwff-2 mutations were in the Arabidopsis thaliana ecotype Wassilewskija (Ws-2) background; the dwff-3 and dwff-4 mutations were in the Enkheim (En-2) background.
- Green tissue was weighed, frozen in liquid nitrogen, and extracted in dim light with 80% acetone in the presence of a mixture of equal parts sand, NaHCO 3 , and Na-,SO 4 . After brief centrifugation, the supernatant was collected and the extraction was repeated twice, pooling the supematants from each sample. Chlorophylls a and b were measured spectrophotometrically, as described in Chory et al. (1991), supra.
- Brassinolide (BL) response was determined in liquid culture, as described by Clouse et al. (1993), supra, except that three or four seedlings were grown in each well of a 24-well culture plate for 7 days. Measurements were taken for 10 to 20 seedlings for each genotype and condition, under a dissection microscope fitted with an ocular micrometer.
- the tissues were treated after fixation with 1% tannic acid in buffer for 30 min, washed three times, and postfixed in 1% OsO 4 in buffer for 2 hr, followed by five washes and dehydration through an ethanol series.
- Samples for transmission electron microscopy were embedded in Spurr's resin. Sections (90 nm) were stained with saturated uranyl acetate followed by Reynolds's lead citrate (Reynolds (1963) J. Cell Biol. 17:208-212) and examined in a JEOL (Tokyo, Japan) 100-CX instrument.
- samples were transferred to freon 113, critical point dried, and sputter-coated with 30 to 50 nm of gold.
- Electron microscopy was performed at the Electron Microscope Facility, Division of Biotechnology, Arizona Research Laboratories, University of Arizona.
- the dwff-1 mutation was identified in a screen of 14,000 transformants of Arabidopsis, resulting in a dwarfed phenotype similar to dwfl (Feldmann and Marks (1987) Mol. Gen. Genet. 208:1-9; Feldmann et al. (1989) Science 243:1351-1354; referred to as diminuto in Takahashi et al. (1995) Genes Dev. 9:97-107 and Szekeres et al., supra) and det2 (Azpiroz et al. (1998), supra). Two independent lines were found that segregated for a similar phenotype: both were shorter than dwfl, but their rosette diameter was comparable to that mutant.
- dwff-1 The dwff mutation was subsequently shown to be inherited as a monogenic, recessive Mendelian trait that, in dwff-1, cosegregates with the dominant kanamycin resistance marker contained in the T-DNA, suggesting that the mutation in this line may be a dismpted, tagged allele.
- dwf4-2 also contains a single kanamycin resistance marker, but it failed to cosegregate with the dwarf phenotype.
- Two additional alleles (dwff-3 and dwff-4) were identified among dwarf mutants obtained from the Nottingham Arabidopsis Resource Centre (Nottingham, UK; N365 and N374). Unless otherwise indicated, all experiments presented below were performed with dwff-1.
- the resulting colonies were screened on ampicillin.
- Five colonies from the left border transformation contained plant DNA flanking the insertion site.
- the restriction pattern displayed two different types of plant DNA. Three contained a 5.6-kb insert, whereas the other two contained a 1.1 -kb insert. This result suggested that the T-DNA insert in dwff-1 was flanked by two left border sequences. The existence of two left border sequences was confirmed by gel blot analysis with genomic DNA, using the putative plant flanking DNAs as probes. A single wild-type EcoRI fragment was split into two fragments in dwff-1.
- Wild-type genomic clones were isolated from a library made from Ws-2 DNA by using the 5.6-kb fragment as a probe.
- the library was constructed using ⁇ DASH-II arms (Stratagene, La Jolla, CA). Approximately 10,000 primary plaques were screened. Duplicate-filter screening resulted in 12 positives. Restriction mapping of the secondary clones revealed that some contained part of the DWF4 locus. In fact, one of the clones, D4G12-1, contained an intact 13-kb DNA spanning the T-DNA insertion site. The 13-kb insert in D4G12-1 was subcloned into pBluescript SK- (Stratagene).
- Superscript II reverse transcriptase (BRL, Gaithersburg, MD) was used for the cDNA synthesis, according to the manufacturer's protocol. Briefly, 7 ⁇ g of total RNA was mixed with the reverse primer, D4R3. To the heat-denatured RNA-primer mix, the RT mixture was added and incubated for 1 hr at 43 °C. Two microliters of RT product was used for PCR amplification by using different primers sets intended to cover all of the putative coding region.
- RT-PCR products were fractionated on an 0.8% agarose gel (Sambrook et al. 1989); the expected bands were purified using a Geneclean kit (BIO 101, Inc., Vista, CA), further amplified, and sequenced to determine the coding region.
- D4OVERF l-ATGTTCGAAACAGAGCATCATACT-24 (SEQ ID NO:3); D4PRM, (-l)-CCTCGATCAAAGAGAGAGA-(-21) (SEQ ID NO:4); D4RTF, 143-TTCTTGGTGAAACCATCGGTTATCTTAAA-171 (SEQ ID NO:5); D4RTR, 853-TATGATAAGCAGTTCCTGGTAGATTT-828 (SEQ ID NO:6); D4F1, (-242)-CGAGGCAAC-AAAAGTAATGAA-(-222) (SEQ ID NO:7); D4R1, 689-GTTAGAAACTCTAAAGATTCA-669 (SEQ ID NO: 8); D4F2, 576-GATTCTTGGCAACAAAACTCTAT-598 (SEQ ID NO:9); D4R2, 1685-CCGAACATCTTTGAGTGCTT-1666 (SEQ ID NO: 10); D4F3,
- Genomic DNA isolated from the mutants was subjected to PCR, using these primer sets.
- the amplified DNA fragments were fractionated on 0.8%o TAE agarose gel (Sambrook et al. 1989), purified using Geneclean (BIO 101, Inc.) or QiaquickTM columns (Qiagen Inc.,
- TAT AT is found in the putative promoter region between nucleotides -143 to -78
- AATAA polyadenylation signal sequences
- RNA from both light-grown and dark-grown seedlings yielded the expected RT-PCR products
- RNA from dark-grown seedlings generated significantly more.
- the bands were gel purified and sequenced. Alignment of the genomic and cDNA sequences indicated that the DWF4 gene was composed of eight exons and seven introns (Figure 2A; Figure 10). Sequence analysis of the dwf4-l allele revealed that the T-DNA was inserted in the 5' end of intron 7 ( Figure 2A). In addition, sequence analysis of the left border plant junctions indicated that at one junction (5'), 75 bp of unknown DNA was inserted, whereas at the other junction (3'), 24 bp of left border and 19 bp of plant DNA were deleted.
- dwff-2 contained a deletion of three conserved amino acids (324 to 326) caused by a 9-bp deletion
- dwf4-3 contained a premature stop codon (289) caused by changing a tryptophan codon (UGG) to a nonsense codon (UGA). Due to a premature stop codon, translation is predicted to be terminated before the heme binding domain, which is essential for cytochrome P450 function (Poulos et al. (1985) J. Biol. Chem. 260:16122-16130). Because T-DNA-generated alleles dwf4-l and dwf4-2 and an additional mutant allele all possess loss-of-function mutations affecting the same protein, we conclude that we have cloned the DWF4 gene.
- Example 3 The DWF4 Gene Encodes a Cytochrome P450
- Cytochrome P450s are heme-thiolate enzymes. They display a characteristic Soret abso ⁇ tion peak at 450 nm when the substrate-bound, reduced form is exposed to the light (Jefcoate (1978) "Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy" in Methods in Enzymology, Vol. 52, S.
- microsomal cytochrome P450s hydroxylate various substrates via their monooxygenase activity, which utilizes molecular oxygen and reducing equivalents from NAD(P)H.
- cytochrome P450 enzymes In addition to the hydroxylation, other activities of cytochrome P450 enzymes, such as oxidation, dealkylation, deamination, dehalogenation, and sulfoxide formation, are involved in a variety of biological events in catabolism, anabolism, and xenobiotic metabolism in plants as well as animals (reviewed in West (1980) "Hydroxylases, monooxygenases, and cytochrome P-450" in The Biochemistry of Plants: A Comprehensive Treatise, Vol. 2, Metabolism and Respiration, D.D. Davies, ed (New York: Academic Press), pp. 317-365; Nebert and Gonzalez (1987), supra; Guengerich (1990) Crit. Rev. Biochem. Mol. Biol.
- cytochrome P450 Biochemical Characteristics, Genetic Engineering and Practical Implications, K. Ruckpaul and H. Rein, eds (London: Taylor and Francis), pp. 191-232; Bolwell et al. (1994) Phytochemistry 37:1491-1506; Durst and Nelson (1995), supra; Schuler (1996) CRC Crit. Rev. Plant Sci. 15:235-284). Evolutionarily, cytochrome P450s have been found in a broad spectrum of living organisms, and they share significant homology at the amino acid sequence level.
- cytochrome P450s were derived from a common ancestor (Nelson and Strobel (1987) Mol. Biol. Evol. 4:572-593).
- Typical cytochrome P450s contain four characteristic domains as defined by
- microsomal cytochrome P450 enzymes can be identified by their characteristic signature sequences, including the heme binding domain, domain A (also referred to as dioxygen binding), domain B (steroid binding), and domain C (Nebert and Gonzalez (1987) Annu. Rev. Biochem. 56:945-993; Kalb and Loper (1988) Proc. Natl. Acad. Sci. USA 85:7221-7225). All of these signature sequences were found in DWF4; the relative positions of the domains are indicated in Figure 2B.
- cytochrome P450s into two distinct groups based on their clustering nature in a phylogenetic tree. All of the group A families cluster and are assumed to originate from a common plant P450 ancestor.
- the group A cytochrome P450s conform to the characteristic consensus sequences (A/G)GX(D/E)T(T/S) in domain A (also called helix I) and PFG(A/S/V)GRRXC(P/A/V)G of the heme binding domain (D) with only a few exceptions.
- Group A cytochrome P450s appear to catalyze plant-specific reactions such as lignin biosynthesis ( Figure 6; GenBank accession number P48421).
- P450s that do not belong to group A are scattered in the phylogenetic tree. They share more amino acid identity/similarity with P450s found in animals, microbes, and fungi than with those found in plants.
- the non-A P450s possess functions, such as steroid metabolism, that are not limited to plants.
- non-A P450s have limited homology with known domains described for group A.
- DWF4 represents a second member of the CYP90 family and is designated CYP90B 1.
- cytochrome P450 sequences with the greatest homology to DWF4 CYP90A1, CYP85, CYP88 (Winkler and Helentjaris (1995) Plant Cell 7:1307-1317; GenBank accession number U32579), cyanobacteria CYP120 (Kaneko et al. (1996) DNA Res. 3:109-136; GenBank accession number D64003), human CYP3A3X (Molowa et al. (1986) Proc. Natl. Acad. Sci. USA 83:5311-5315; GenBank accession number M13785), and zebrafish CYP26 (White et al. White (1996) J. Biol. Chem.
- domain A of DWF4 is a monooxygenase, similar to P450s of group A, that utilizes molecular oxygen as a source of the hydroxyl group, but it mediates some reaction(s) that are not necessarily specific for plants, for instance, steroid hormone biosynthesis, which is a critical event for animals.
- the similarity of DWF4 to the rat testosterone 6 ⁇ -hydroxylase (34%; GenBank accession number 631895) or glucocorticoid-inducible hydroxylase (31%; Molowa et al. 1986; GenBank accession number M13785) supports this idea.
- a plant with a dwarf phenotype is one that has a short, robust stem and short, dark green leaves, dwff mutants are significantly smaller than the wild type and are dark green in color. They have short, rounded leaves.
- the dwff phenotype is reminiscent of the light-regulatory mutant det2 (Chory et al., supra); however, complementation analysis has shown that the two mutations are not allelic, with the dwff mutation mapping to the lower arm of chromosome 3 and det2 mapping to chromosome 2 (Chory et al, supra).
- Table 1 The results presented in Table 1 show that soil-grown dwff plants attained a height of ⁇ 3 cm at 5 weeks, whereas wild-type plants grew to >25 cm. Moreover, individual organs, such as leaves, were invariably shorter in dwarf plants, dwff siliques were also markedly shorter than those of the wild type and were infertile. The loss of fertility of dwff was due to the reduced length of the stamen filaments relative to the gynoecium, which resulted in mature pollen deposition on the ovary wall rather than on the stigmatic surface. Hand pollination of dwf4 flowers with either mutant or wild-type pollen resulted in good seed set without significantly changing the size of the siliques.
- dwff plants Another feature of dwff plants is a reduction in apical dominance, as was evident by the threefold increase in the number of inflorescences at 5 weeks of age (Table 1). Mutants also had twice the number of rosette leaves, which may be explained by a prolonged vegetative phase in the dwf4 plants. Development of flowers on the primary inflorescence was delayed by ⁇ 4 days in dwf4, but the flowering phase was significantly longer in the mutant, with senescence of the last flower occurring at ⁇ 98 days compared with -57 days for the wild type. One result of this delay in senescence was that dwff plants contained almost three times the number of siliques as did the wild type (Table 1).
- the reduced stature observed in soil-grown dwff was also observed in hypocotyls of agar-grown seedlings. Measurements of hypocotyl length over time indicated that not only were dwf4 seedlings shorter than wild-type seedlings immediately after germination but also that the rate of growth was retarded in the mutants (Figure 5). In addition, dwff hypocotyls reached their terminal length in ⁇ 5 days, whereas wild-type seedlings continued to grow. In sum, the dwff phenotype can be described as being due to both primary and secondary effects of reduced cell elongation. The primary effect is simply a reduction in the length of individual organs exclusively along their normal growth axis; that is, organ width is not reduced (Table 1).
- the secondary effects of reduced cell elongation are themselves due to the reduction in organ length.
- the dark green color of the leaves may be due exclusively to the existence of a wild-type number of chlorop lasts in a significantly smaller cell.
- the sterility of mutants is a consequence of the shortness of the stamens, which fail to deposit their pollen on the stigmatic surface.
- mutants display delayed development, the first sign of which occurs at flowering (Table 1). Because rosette leaves are produced continuously during vegetative development, delayed flowering results in dwff rosettes having almost twice the number of leaves observed in the wild type.
- Example 5 The Growth Defect of dwff Is Due to a Reduction in Cell Length
- Both the short stature and the reduced growth rate of dwff could be due to a defect in cell division or cell elongation or both.
- Table 2 the average cell size in dwff is significantly smaller than in wild-type plants, whereas no differences were detected in the number of cells along the length of either organ between the wild type and dwff. Therefore, the short stature and reduced organ length of dwff are largely or exclusively due to a failure of individual cells to elongate. No differences were observed in the number of cell layers contained in the wild type and dwff.
- the small size of dwff cells offers a possible explanation for the dark green color of the mutant plants. Chlorophyll measurements were taken, and leaf mesophyll protoplasts were prepared, stained, and measured to visualize and count chlorop lasts, as described in Methods. Although there were no significant differences in total chlorophyll content, the chlorophyll a/b ratio, or the abso ⁇ tion spectra between wild-type plants and mutants, the mean plane area (the apparent two-dimensional surface area of mounted cells) of dwff leaf mesophyll protoplasts was 376 mm 2 , whereas that of wild-type protoplasts was 599 mm 2 . The two-dimensional comparison of plane area represents a dramatic reduction in volume for dwff cells.
- the rate of growth was significantly reduced in agar-grown dwff seedlings, which ceased to grow when their hypocotyl length was ⁇ 20%> of the final wild-type length. Because all of the cells in a hypocotyl before the initiation of leaf development are present in the embryo, the initial growth of seedlings is due exclusively to cell expansion, which therefore must be reduced in dwff. A similar situation applies to soil-grown plants. Five weeks after germination, well after plants had bolted, dwf4 plants were shorter than wild-type plants (Table 1). Although the mutants continued growing for several weeks more than did the wild type, they remained shorter through senescence.
- Organ growth by cell elongation in plants occurs as part of normal development in response to a variety of input signals. Mutants that are defective in these signaling pathways invariably fail to elongate normally in response to the appropriate stimuli. A mutant with a block at a step that is common to several individual pathways would therefore be expected to have defective responses to all of the corresponding signals, dwff appears to be such a mutant.
- Figure 6 shows that elongation induced by the hy2 mutation is blocked in a dwff hy2 double mutant. Not su ⁇ risingly, in view of this result, dwff also failed to display hypocotyl elongation as a response to growth in complete darkness.
- dwff was capable of perceiving GA, but its response was severely compromised. This mutant could also respond to the inhibitory effects of auxin but was incapable of auxin-stimulated elongation. It was only exogenous BL that fully restored wild-type length to dwff hypocotyls (Choe et al. (1998), supra).
- the reduced length of cells in dwff hypocotyls and inflorescence stems is indicative of a failure of these cells to elongate during development.
- a variety of endogenous and environmental signals is responsible for stimulating elongation in plants; therefore, a series of experiments was performed to determine whether dwf4 is affected in a specific signaling pathway or is blocked in elongation as a response to various signals.
- auxin Higher concentrations of auxin were inhibitory for both wild-type and dwff seedlings, and lower concentrations had no effect. In view of the inhibition of root growth, it is clear that dwff is not auxin resistant; rather, its elongation response is compromised.
- hy mutants share the common phenotype of an elongated hypocotyl that mimics part of the etiolation response in the light.
- hy2 is deficient in active phytochrome because chromophore biosynthesis does not take place (Chory et al. (1989a) Plant Cell 1:867-880).
- Figure 6 shows that dwff hy2 double mutants displayed a dwarfed phenotype indistinguishable from that of dwf4 HY2 (light-grown control); therefore, the elongation block due to the dwff mutation is epistatic to a defect in phytochrome activity.
- BL is required for cell elongation as a response to darkness as well as GA and auxin.
- previous studies Karlmann et al. (1996), supra; Li et al. (1996), supra; Szekeres et al. (1996), supra) and the work described herein show that BR can compensate for the cell elongation defect of mutants as diverse as det2, cpd, dwff, detl, copl, and dwfl. This places BRs downstream of all the cellular functions affected in these mutants.
- at least one of the BR biosynthetic genes has been shown to be modulated by light, cytokinins, and the carbon source (Szekeres et al. (1996), supra).
- axr2 mutants are resistant to auxin, ethylene, and abscisic acid and have defective root and shoot gravitropism.
- the dwarf phenotype in axr2 mutants has been shown to be due to reduced cell elongation and is rescued by BL (Szekeres et al. (1996), supra). This suggests that at least one of the multiple hormone signaling pathways affected in axr2 involves a BR-dependent step.
- the BR-deficient mutant det2 was originally identified as defective in regulation by light (Chory et al. (1991), supra). Given the similarity of det2 and dwff phenotypes and functions and in view of the observation that dwff is epistatic to hy2, one can predict that the etiolation response, which includes significant hypocotyl elongation, would not be normal in dwff. To assess to what extent the etiolation response is affected by BR-dependent cell elongation, we grew dwff and wild-type plants on agar under continuous light or in complete darkness, as described above in Example 1.
- Example 8 Abnormal Skoto morphogenesis as a Consequence of the Dwarf Growth Habitat When dwf4 is grown in the light, its mo ⁇ hology is similar to that of various cop and det mutants, with multiple short stems, short rounded leaves, loss of fertility due to reduced stamen length, and delayed development (Figure 6). Dark-grown dwff seedlings possess short hypocotyls, open cotyledons, and developing leaves. Therefore, it is plausible to speculate that this mutant may be defective in the control of light-regulated processes.
- dwff sterility in dwff is hypothesized to be mechanical, which means that the filaments are shorter than the ca ⁇ els such that the pollen is shed onto the ovary walls rather than onto the stigmatic surface.
- dwf4 plants are hand pollinated using dwff pollen, fertility increases.
- Fujioka and Sakurai (1997b), supra have demonstrated that there are at least two branched biochemical pathways to the end product BL ( Figure 1; Fujioka and Sakurai (1997a), supra, Fujioka and Sakurai (1997b), supra; Sakurai and Fujioka (1997), supra).
- Figure 1 Fujioka and Sakurai (1997a), supra, Fujioka and Sakurai (1997b), supra; Sakurai and Fujioka (1997), supra.
- they are referred to as the early or late C-6 oxidation pathways.
- the C-6 is oxidized to a ketone at campestanol (CN), whereas in the late pathway it is oxidized at 6-deoxocastasterone (6-deoxoCS). Otherwise, the two pathways share equivalent reactions.
- the C-6 hydroxylated BRs for example, 6-OHCT, 6-hydroxyteasterone, and so on, may be possible intermediates in this network. If so, the intermediates in this pathway may play a role as bridging molecules between the early and late C-6 oxidation pathways. Alternatively, it might be possible that 22-OHCR merges into one of the two pathways to be metabolized.
- the late C-6 oxidation pathway is the best candidate; our unpublished data show that 22-OHCR is more effective in the light in rescuing the dwff phenotype, which is true for all of the intermediates in the late C-6 oxidation pathway.
- each intermediate may have nascent bioactivity.
- the in vivo ratio or composition of BRs at different oxidation states may result in different responses. Noticeably distinctive phenotypes for the various BR dwarfs, defective in different biosynthetic steps, support this idea.
- the biosynthetic rate of each pathway toward production of the end product may differ. In this case, the biosynthetic rate could be modulated by controlling the level of gene expression or the activity of participating enzymes. Certain signals, requiring different rates of BR biosynthesis, may induce one of the subpathways, which would then affect the concentration of the intermediates in one pathway relative to the other.
- the 22 -hydroxylation reaction has been suggested to be the rate-limiting step (Fujioka et al.
- promoter constructs Two promoter constructs were used for the DWF4-promoter: :GUS (D4G) analysis.
- D4G polymerase chain reaction
- PCR polymerase chain reaction
- pD4GL a promoterless GUS vector pBIl 01; this 1.1 kb promoter: :GUS construct was named pD4GL.
- pD4GS construct pD4GL was digested with Hindlll, the small restriction fragment was removed, and the remaining vector with the partial promoter was self-ligated. The constructs were introduced into Agrobacterium strain GV3101 through electroporation.
- PCR products were made by using D4OVERFA (5'-GAATTCTAGAATGTTCGAAACAGAGCATCATA-3') (SEQ ID NO: 18) and D4R2 (5'-CCGAACATCTTTGAGTGCTT-3') (SEQ ID NO: 10) primers and Wassilewskija-2 (Ws-2) genomic DNA.
- the PCR products were cut with Xbal and Hindlll, and inserted into the same restriction sites of genomic clone SCH25 containing a 2.5 kb Hindlll fragment of the DWF4 DNA corresponding to the 3' half of the gene.
- the resulting recombinant DNA clone pD4CDS containing the whole coding sequence from the translation initiation site to 694 bp downstream of the stop codon, was cut with Xbal and transferred to an overexpression vector pART27 (Gleave (1992), Plant Molec. Bio. 20:1203-1207).
- the resulting binary construct was named pOD4. This construct was introduced into Agrobacterium through electroporation.
- dwff-4 plants were used without decapitation.
- a single colony selected on 20 ⁇ g/ml kanamycin in Luria-Bertani (LB) medium (10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl per liter, pH 7) was inoculated into 100 ml liquid LB media, and grown for 3 days.
- LB Luria-Bertani
- the Agrobacterium suspension was sprayed onto plants on the third day after decapitation. To avoid physical contact with possibly hazardous Silwet vapor, protective glasses were used and the spraying was done in a fume hood. To test the efficiency of repeated spraying, plants were sprayed every third day (3x). Sprayed plants were grown to maturity and seeds harvested.
- T2 seeds were collected from individual transformants (Tl), and plated again on the selection media to determine segregation ratios for drug-resistant versus sensitive plants.
- Arabidopsis transformants were named Arabidopsis Overexpressor of DWF4 (AOD4) when harboring an overexpression construct pOD4, and DWF4 -promoter:: GUS (D4G) for transformants containing a GUS fusion gene. Homozygosity for the transgene was determined when no sensitive T4 seedlings segregated from >500 T3 individuals. Mo ⁇ hometric analysis of AOD4 lines and GUS histochemical analysis of D4GL plants was performed using plants homozygous for the transgene.
- Transgenic tobacco plants harboring the pOD4 constructs were produced in the plant tissue culture laboratory at the University of Arizona. Protocols for the regeneration of transgenic plants from lead discs of Nicotiana tabacum var Samsun will be provided on request. Fifteen independent transformants for both the control and OD4 constructs were grown for seeds. Mo ⁇ hological analysis of the TOD4 lines was performed using T2 plants in the course of growth for 4 months in the green house (30°C). Methods for Arabidopsis growth and RNA gel blot analysis were previously described herein. Briefly, seeds of wild type and the two AOD4 lines were germinated on M&S agar media.
- RNA gel blot analysis with total RNA isolated from nine different tissues of three- week old plants was performed.
- the DWF4 transcript was barely detectable in shoot tips, roots, dark-grown seedlings, callus and axilary buds, but the levels were below the detectable limit in the other tissues examined, including stems, siliques, pedicels, and rosette leaves.
- the expression of the GUS reporter gene controlled by the DWF4 promoter was examined.
- GUS staining patterns in T2 plants homozygous for D4GL revealed that GUS activity was present in tissues with actively dividing or elongating cells. These include shoot apical meristems, leaf primordia, collet (the junction between hypocotyl and root), and root tips, including lateral root primordia, as shown in 6-day old light-grown seedlings. Interestingly, dark-grown seedlings displayed GUS activity in cotyledons whereas the staining was not detectable in the cotyledons of light-grown seedlings.
- GUS activity was detected in floral primordia, ca ⁇ els, and the basal end of the filaments of unopened flowers, whereas GUS activity in sepals, petals, and mature pedicels was not detected.
- the shoot tips, bases of emerging branches, and primordia of axilary inflorescences were GUS positive, whereas elongated intemodes were negative.
- Embryos in the seeds of the fully elongated siliques were weakly positive for GUS staining, suggesting a role for BRs in embryo development.
- Leaf primordia, young leaves, expanding leaf margins, and the base of petioles displayed GUS activity, but old leaf blades were negative for GUS staining.
- the tissues positive for GUS staining confirmed the expression pattern examined by northern analysis with the tissue-specific RNA.
- DWF4 is proposed to be a key enzyme in the BR biosynthetic pathway
- DWF4 transcription could be regulated by an end-product feedback mechanism.
- D4GL was expressed in different genetic backgrounds including two BR deficient mutants, dwf/-l and dw ⁇ -1, and a BR-enriched line, AOD4.
- GUS activity was increased in dwf7-l and dw ⁇ -1 but decreased in AOD4 lines.
- DWF7 is a C-5 desaturase that acts in the sterol specific part of the pathway.
- D4GL activity in dwf7-l was found in the same tissues as wild type but. dw ⁇ -1 is defective in a BR biosynthetic step downstream of CPD.
- dw ⁇ -1 the intensity of the D4GL activity was noticeably stronger as compared to wild type but the expression patterns were relatively diffuse, dw ⁇ -1 was also found to express GUS at nascent sites as compared to wild type.
- D4GL expression in the cotyledons of light-grown seedlings was not detected, but dw ⁇ -1 displayed considerable D4GL activity in the cotyledons.
- GUS activity was detected throughout the hypocotyls of dw ⁇ -1 light-grown seedlings, suggesting that D4GL transcription is upregulated in dw ⁇ -1 in a more general manner. Conversely, GUS activity was greatly reduced in AOD4-4 plants.
- D. DWF4 overexpression results in elongated hypocotyls in Arabidopsis and tobacco seedlings
- a DWF4 overexpression construct (pOD4) was made by placing the DWF4 genomic DNA under the control of the CaMV 35S promoter.
- RNA gel blot analysis with total RNA isolated from the transgenic lines containing the overexpression construct, showed that DWF4 transcripts were greatly increased in both Arabidopsis and tobacco, whereas the level was not readily detectable in either wild type or in dwf4-l plants. Similar to increased mRNA transcripts, the 80 independent AOD4 transgenic plants had longer hypocotyls and inflorescences.
- dwft-1 displayed greatly reduced hypocotyl length both in the light and dark as compared to wild type. Wild-type roots are shortened when grown in the dark, but dwf4-l root length was not significantly reduced in the dark compared with the reduction in hypocotyl length.
- AOD4 plants tend to fall over earlier than the Ws-2 wild type.
- comparison of rosette leaf size between wild type and AOD4 indicates that leaves, both rosette and cauline, are larger, especially in adult plants.
- TOD4 plants also possessed leaves that were larger, and had longer petioles relative to the control.
- additional secondary branches were found both in Arabidopsis and tobacco overexpression lines. In AOD4 plants, this additional branching was associated with >2 times increased number of siliques per plant, leading to a 33 and 59% increase in seed production (Table 3).
- the increased seed production in the AOD4 lines was mainly due to the increased number of seeds per plant than increase in the seed size, because the size was not significantly increased (Table 3).
- the length of silique as well as the length of an intemode between the first silique in a main inflorescence and the base of plant was increased (Table 3).
- Figure 8 shows that stem growth is increased more than 20% compared to wild type in DWF4 overexpression lines and Figure 9 shows that seed production is increased significantly over wild type in the DWF4-overexpressed lines.
- Figure 12 depicts hypocotyl length and root length in light and dark. Further, the height of AOD4 lines was greater than wild type over the days examined. In addition, although wild type plants ceased growth around five weeks after germination, AOD4 plants continued to grow up to seven weeks.
- the DWF4 locus is defined by at least four mutant alleles.
- One of these is the result of a T-DNA insertion. Plant DNA flanking the insertion site was cloned and used as a probe to isolate the entire DWF4 gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450 monooxygenase with 43 % identity to the putative Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE
- PHOTOMORPHOGENESIS AND DWARFISM Sequence analysis of two other mutant alleles revealed deletions or a premature stop codon, confirming that DWF4 had been cloned. This sequence similarity suggests that DWF4 functions in specific hydroxylation steps during BR biosynthesis.
- the dwarf phenotype can be rescued with exogenously supplied brassinolide.
- dwf4 mutants display features of light-regulatory mutants, but the dwarfed phenotype is entirely and specifically brassinosteroid dependent; no other hormone can rescue dwf4 to a wild-type phenotype.
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EP1389904A2 (en) * | 2001-05-16 | 2004-02-25 | The Arizona Board of Regents on behalf of The University of Arizona | Dwf12 and mutants thereof |
WO2005111216A2 (en) * | 2004-04-23 | 2005-11-24 | Ceres Inc. | Methods for modifying plant characteristics |
US7253336B2 (en) | 1999-02-11 | 2007-08-07 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | DWF4 polynucleotides, polypeptides and uses thereof |
WO2007064724A3 (en) * | 2005-12-01 | 2007-09-13 | Cropdesign Nv | Plants having improved growth characteristics and methods for making the same |
CN1926235B (en) * | 2003-10-16 | 2012-07-18 | 美国无烟烟草有限责任公司 | Cloning of cytochrome P450 genes from nicotiana |
EP2710128A4 (en) * | 2011-05-20 | 2015-05-06 | Frontier Agri Science Inc | Plants having enhanced abiotic stress resistance |
CN110628737A (en) * | 2019-10-14 | 2019-12-31 | 南京农业大学 | Related gene for regulating cucumber dwarfing character and application thereof |
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CN109609541B (en) * | 2018-11-27 | 2021-10-19 | 杭州瑞丰生物科技有限公司 | Method for improving crop traits |
CN109913465A (en) * | 2019-03-12 | 2019-06-21 | 天津大学 | The sedum lineare gene of resistance to Ni SlDWF4 and its application |
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US7253336B2 (en) | 1999-02-11 | 2007-08-07 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | DWF4 polynucleotides, polypeptides and uses thereof |
US7935532B2 (en) | 1999-02-11 | 2011-05-03 | Arizona Board Of Regents For And On Behalf Of Arizona State University | DWF4 polynucleotides, polypeptides and uses thereof |
US7589255B2 (en) | 1999-02-11 | 2009-09-15 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | DWF4 polynucleotides, polypeptides and uses thereof |
US7304205B2 (en) | 2001-05-16 | 2007-12-04 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | DWF12 and mutants thereof |
EP1389904A2 (en) * | 2001-05-16 | 2004-02-25 | The Arizona Board of Regents on behalf of The University of Arizona | Dwf12 and mutants thereof |
EP1389904A4 (en) * | 2001-05-16 | 2005-04-27 | Univ Arizona | Dwf12 and mutants thereof |
CN1926235B (en) * | 2003-10-16 | 2012-07-18 | 美国无烟烟草有限责任公司 | Cloning of cytochrome P450 genes from nicotiana |
WO2005111216A3 (en) * | 2004-04-23 | 2006-06-01 | Ceres Inc | Methods for modifying plant characteristics |
WO2005111216A2 (en) * | 2004-04-23 | 2005-11-24 | Ceres Inc. | Methods for modifying plant characteristics |
US7897839B2 (en) | 2004-04-23 | 2011-03-01 | Ceres, Inc. | Methods for modifying plant characteristics |
WO2007064724A3 (en) * | 2005-12-01 | 2007-09-13 | Cropdesign Nv | Plants having improved growth characteristics and methods for making the same |
US8487160B2 (en) | 2005-12-01 | 2013-07-16 | Cropdesign N.V. | Plants having improved growth characteristics and methods for making the same |
EP2710128A4 (en) * | 2011-05-20 | 2015-05-06 | Frontier Agri Science Inc | Plants having enhanced abiotic stress resistance |
CN110628737A (en) * | 2019-10-14 | 2019-12-31 | 南京农业大学 | Related gene for regulating cucumber dwarfing character and application thereof |
CN110628737B (en) * | 2019-10-14 | 2022-06-07 | 南京农业大学 | Related gene for regulating cucumber dwarfing character and application thereof |
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