Patent Application
20120017335
The embryonic stem or hypocotyl is an excellent model for studying both internal and external factors controlling growth in plants (Nozue, K. et al., Nature, 448:358-361 (2007)). Genetic screens in common laboratory accessions have yielded direct molecular insight into how light and hormone dependent signaling pathways interact with the circadian clock to regulate the final length of the hypocotyl (Nozue, K. et al., Nature, 448:358-361 (2007)). The power of the hypocotyl assay is its simplicity, as well as its obvious meaningfulness. When germinating seeds are exposed to low levels of light, such as those caused by a covering layer of debris, the hypocotyl has to grow for a while. Only after the surface has been broken by the tip of the hypocotyls can the embryonic leaves, the cotyledons, unfold. Conversely, if a seed has fallen on open ground, there is no need for the hypocotyls to be particularly long. Because of the ease and reproducibility with which hypocotyl length can be measured in thousands of individuals, it has also been a powerful model in mapping genes with more subtle effects on light and hormone regulated growth, using methods of quantitative genetics (Borevitz, J. O. et al., Genetics, 160:683-696 (2002)). Multiple light signaling genes controlling hypocotyl length have been characterized in quantitative trait locus (QTL) studies (Aukerman, M. J. et al., Plant Cell, 9:1317-1326 (1997); Balasubramanian, S. et al., Nat Genet, 38:711-715 (2006); Filiault, D. L. et al., Proc Natl Acad Sci USA, 105:3157-3162 (2008); Maloof, J. N. et al., Nat Genet, 29:441-446 (2001)).
The present invention provides for plants comprising a heterologous expression cassette comprising a promoter operably linked to a polynucleotide encoding a TZP polypeptide. In some embodiments, the promoter is heterologous to the polynucleotide. In some embodiments, the plant is characterized by increased size, speed of growth, elongation of organs or delay or extension of time of seed and/or fruit production compared to a plant lacking the heterologous expression cassette. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the TZP polypeptide is at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%) identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In addition to the above described plants, the present invention also provides for processed plant material, including but not limited to processed plant parts (e.g., seeds, fruit, nuts, tubers, leaves, etc.). The present invention further provides a method of making processed plant material, the method comprising processing a plant or plant part of the present invention, thereby generating processed plant material.
The present invention also provides for isolated nucleic acids comprising a promoter operably linked to a polynucleotide encoding a TZP polypeptide wherein the promoter is heterologous to the polynucleotide. In some embodiments, the TZP polypeptide is at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%) identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
The present invention also provides for methods for increasing size, speed of growth or biomass in a plant. In some embodiments, the method comprises, introducing an expression cassette into one or more plants, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a TZP polypeptide, and selecting a plant that has increased size, speed of growth, elongation of organs or delay or extension of time of seed and/or fruit production compared to a plant lacking the heterologous expression cassette. In some embodiments, the TZP polypeptide is at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, 98%, 99%) identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
The term “promoter” or “regulatory element” refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of plant origin, for example, promoters derived from plant viruses, such as the CaMV35S promoter, can be used in the present invention.
The term “plant” includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
A polynucleotide sequence is “heterologous to” a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from naturally occurring allelic variants.
An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. Antisense constructs or sense constructs that are not or cannot be translated are expressly included by this definition.
An “TZP polypeptide” is a polypeptide substantially identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18 or as otherwise described herein. TZP polypeptides comprise a tandem (e.g., two) zinc knuckle domains (CX2CX4HX4C; SEQ ID NO:13) as well as a PLUS3 domain. Exemplary TZP genomic and coding sequences are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively.
Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needle man and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.
“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The term “substantial identity” of polypeptide sequences means that a polypeptide comprises a sequence that has at least 25% sequence identity. Alternatively, percent identity can be any integer from 25% to 100%. Exemplary embodiments include at least: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. Accordingly, TZP sequences of the invention include nucleic acid sequences encoding a polypeptide that has substantial identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. TZP sequences of the invention also include polypeptide sequences having substantial identify to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Polypeptides which are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or a third nucleic acid, under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60° C.
The term “isolated”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.
(B) Phylogenetic tree of the PLUS3 domain across species and with other PLUS3 domains of Arabidopsis thaliana. The TZP PLUS3 domain is more closely related to the PLUS3 domain from TZP orthologs in other species, than other PLUS3 domains in Arabidopsis thaliana. Arabidopsis lyrata (Al), Carica papaya (Cp), Oryza sativa (Os), Ricinus communis (Rc), Populus trichocarpa (Pt), Sorghum bicolor (Sb), Brachypodium distachyon (Bd), Glycine max (Gm), Vitis vinifera (Vv) and Selaginella moellendorffii (Sm).
(F) SWI/PLUS3 (At2g16480)
The present invention provides for methods of improving plant traits including but not limited to increasing plant organ elongation, increasing plant height, delaying seed or fruit set, increasing the time of fruit or seed set, and/or increasing plant size. In some embodiments of the invention, a TZP polypeptide is ectopically or otherwise overexpressed. In some embodiments, for example, the invention provides for transgenic plants comprising a heterologous expression cassette for expressing a TZP polypeptide in the plant.
Alternatively, the present invention provides for plants with shortened stature. In these embodiments, endogenous TZP expression is reduced, e.g., by use of siRNA, antisense, or other gene expression reduction technology.
Isolated nucleic acid sequences encoding all or part of a TZP polypeptide can be used to prepare expression cassettes that enhance, or increase endogenous, TZP gene expression. Where overexpression of a gene is desired, the desired gene from a different species may be used to decrease potential sense suppression effects.
Any of a number of means well known in the art can be used to increase TZP activity in plants. Any organ can be targeted, such as shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit. Alternatively, one or several TZP genes can be expressed constitutively (e.g., using the CaMV 35S promoter).
One of skill will recognize that the polypeptides encoded by the genes of the invention, like other proteins, have different domains which perform different functions. Thus, the gene sequences need not be full length, so long as the desired functional domain of the protein is expressed.
To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for the desired polypeptide, for example a cDNA sequence encoding a full length 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 transformed plant.
For example, for overexpression, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill.
Alternatively, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, or flowers. 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 vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention can optionally comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.
In some embodiments, TZP nucleic acid sequences of the invention are expressed recombinantly in plant cells to enhance and increase levels of TZP polypeptides. Alternatively, antisense or other TZP constructs are used to suppress TZP levels of expression. A variety of different expression constructs, such as expression cassettes and vectors suitable for transformation of plant cells can be prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A TZP sequence coding for a TZP polypeptide, e.g., a cDNA sequence encoding a full length protein, can be combined with cis-acting (promoter) and trans-acting (enhancer) transcriptional regulatory sequences to direct the timing, tissue type and levels of transcription in the intended tissues of the transformed plant. Translational control elements can also be used.
The invention provides an TZP nucleic acid operably linked to a promoter that, in some embodiments, is capable of driving the transcription of the TZP coding sequence in plants. The promoter can be, e.g., derived from plant or viral sources. The promoter can be, e.g., constitutively active, inducible, or tissue specific. In construction of recombinant expression cassettes, vectors, transgenics, of the invention, a different promoters can be chosen and employed to differentially direct gene expression, e.g., in some or all tissues of a plant or animal. Typically, as discussed above, desired promoters are identified by analyzing the 5′ sequences of a genomic clone corresponding to the TZP genes described here.
A. Constitutive Promoters
A promoter fragment can be employed that will direct expression of TZP nucleic acid in all transformed cells or tissues, e.g. as those of a regenerated plant. The term “constitutive regulatory element” means a regulatory element that confers a level of expression upon an operatively linked nucleic molecule that is relatively independent of the cell or tissue type in which the constitutive regulatory element is expressed. A constitutive regulatory element that is expressed in a plant generally is widely expressed in a large number of cell and tissue types. Promoters that drive expression continuously under physiological conditions are referred to as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation.
A variety of constitutive regulatory elements useful for ectopic expression in a transgenic plant are well known in the art. The cauliflower mosaic virus 35S (CaMV 35S) promoter, for example, is a well-characterized constitutive regulatory element that produces a high level of expression in all plant tissues (Odell et al., Nature 313:810-812 (1985)). The CaMV 35S promoter can be particularly useful due to its activity in numerous diverse plant species (Benfey and Chua, Science 250:959-966 (1990); Futterer et al., Physiol. Plant 79:154 (1990); Odell et al., supra, 1985). A tandem 35S promoter, in which the intrinsic promoter element has been duplicated, confers higher expression levels in comparison to the unmodified 35S promoter (Kay et al., Science 236:1299 (1987)). Other useful constitutive regulatory elements include, for example, the cauliflower mosaic virus 19S promoter; the Figwort mosaic virus promoter; and the nopaline synthase (nos) gene promoter (Singer et al., Plant Mol. Biol. 14:433 (1990); An, Plant Physiol. 81:86 (1986)).
Additional constitutive regulatory elements including those for efficient expression in monocots also are known in the art, for example, the pEmu promoter and promoters based on the rice Actin-1 5′ region (Last et al., Theor. Appl. Genet. 81:581 (1991); Mcelroy et al., Mol. Gen. Genet. 231:150 (1991); Mcelroy et al., Plant Cell 2:163 (1990)). Chimeric regulatory elements, which combine elements from different genes, also can be useful for ectopically expressing a nucleic acid molecule encoding an TZP polynucleotide (Comai et al., Plant Mol. Biol. 15:373 (1990)).
Other examples of constitutive promoters include the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens (see, e.g., Mengiste (1997) supra; O'Grady (1995) Plant Mol. Biol. 29:99-108); actin promoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang (1997) Plant Mol. Biol. 1997 33:125-139); alcohol dehydrogenase (Adh) gene promoters (see, e.g., Millar (1996) Plant Mol. Biol. 31:897-904); ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565 (1989)), Gpc2 from maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)), other transcription initiation regions from various plant genes known to those of skill. See also Holtorf Plant Mol. Biol. 29:637-646 (1995).
B. Inducible Promoters
Alternatively, a promoter may direct expression of the TZP nucleic acid of the invention under the influence of changing environmental conditions or developmental conditions. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. Such promoters are referred to herein as “inducible” promoters. For example, the invention incorporates the drought-inducible promoter of maize (Busk (1997) supra); the cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant Mol. Biol. 33:897-909).
Alternatively, plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids of the invention. For example, the invention can use the auxin-response elements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) Mol. Plant. Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900-1902).
Promoters that are inducible upon exposure to chemicals reagents applied to the plant, such as herbicides or antibiotics, can also be used to express the nucleic acids of the invention. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. TZP coding sequence can also be under the control of, e.g., a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324; Uknes et al., Plant Cell 5:159-169 (1993); Bi et al., Plant J. 8:235-245 (1995)).
Other inducible regulatory elements include but are not limited to copper-inducible regulatory elements (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717 (1988)); tetracycline and chlor-tetracycline-inducible regulatory elements (Gatz et al., Plant J. 2:397-404 (1992); Roder et al., Mol. Gen. Genet. 243:32-38 (1994); Gatz, Meth. Cell Biol. 50:411-424 (1995)); ecdysone inducible regulatory elements (Christopherson et al., Proc. Natl. Acad. Sci. USA 89:6314-6318 (1992); Kreutzweiser et al., Ecotoxicol. Environ. Safety 28:14-24 (1994)); heat shock inducible regulatory elements (Takahashi et al., Plant Physiol. 99:383-390 (1992); Yabe et al., Plant Cell Physiol. 35:1207-1219 (1994); Ueda et al., Mol. Gen. Genet. 250:533-539 (1996)); and lac operon elements, which are used in combination with a constitutively expressed lac repressor to confer, for example, IPTG-inducible expression (Wilde et al., EMBO J. 11:1251-1259 (1992)). An inducible regulatory element useful in the transgenic plants of the invention also can be, for example, a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al., Plant Mol. Biol. 17:9 (1991)) or a light-inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et al., Mol. Gen. Genet. 226:449 (1991); Lam and Chua, Science 248:471 (1990)).
C. Tissue-Specific Promoters
Alternatively, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters). Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues.
Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue. Reproductive tissue-specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof.
Other tissue-specific promoters include seed promoters. Suitable seed-specific promoters are derived from the following genes: MAC1 from maize (Sheridan (1996) Genetics 142:1009-1020); Cat3 from maize (GenBank No. L05934, Abler (1993) Plant Mol. Biol. 22:10131-1038); vivparous-1 from Arabidopsis (Genbank No. U93215); atmyc1 from Arabidopsis (Urao (1996) Plant Mol. Biol. 32:571-57; Conceicao (1994) Plant 5:493-505); napA from Brassica napus (GenBank No. J02798, Josefsson (1987) JBL 26:12196-1301); and the napin gene family from Brassica napus (Sjodahl (1995) Planta 197:264-271).
A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can also be used to express the TZP nucleic acids of the invention. For example, promoters controlling patatin, the major storage protein of the potato tuber, can be used, see, e.g., Kim (1994) Plant Mol. Biol. 26:603-615; Martin (1997) Plant J. 11:53-62. The ORF13 promoter from Agrobacterium rhizogenes which exhibits high activity in roots can also be used (Hansen (1997) Mol. Gen. Genet. 254:337-343. Other useful vegetative tissue-specific promoters include: the tarin promoter of the gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra (1995) Plant Mol. Biol. 28:137-144); the curculin promoter active during taro corm development (de Castro (1992) Plant Cell 4:1549-1559) and the promoter for the tobacco root-specific gene TobRB7, whose expression is localized to root meristem and immature central cylinder regions (Yamamoto (1991) Plant Cell 3:371-382).
Leaf-specific promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters can be used. For example, the tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light-grown seedlings, only RBCS1 and RBCS2 are expressed in developing tomato fruits (Meier (1997) FEBS Lett. 415:91-95). A ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, described by Matsuoka (1994) Plant J. 6:311-319, can be used. Another leaf-specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter, see, e.g., Shiina (1997) Plant Physiol. 115:477-483; Casal (1998) Plant Physiol. 116:1533-1538. The Arabidopsis thaliana myb-related gene promoter (Atmyb5) described by L1 (1996) FEBS Lett. 379:117-121, is leaf-specific. The Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage. A leaf promoter identified in maize by Busk (1997) Plant J. 11:1285-1295, can also be used.
Another class of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters. For example, the “SHOOTMERISTEMLESS” and “SCARECROW” promoters, which are active in the developing shoot or root apical meristems and are described by Di Laurenzio (1996) Cell 86:423-433; and, Long (1996) Nature 379:66-69, can be used. Another promoter is the 3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene promoter, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto (1995) Plant Cell. 7:517-527). Additional promoter examples include the kn1-related gene promoters from maize and other species that show meristem-specific expression, see, e.g., Granger (1996) Plant Mol. Biol. 31:373-378; Kerstetter (1994) Plant Cell 6:1877-1887; Hake (1995) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51. One such example is the Arabidopsis thaliana KNAT1 promoter. In the shoot apex, KNAT1 transcript is localized primarily to the shoot apical meristem; the expression of KNAT1 in the shoot meristem decreases during the floral transition and is restricted to the cortex of the inflorescence stem (see, e.g., Lincoln (1994) Plant Cell 6:1859-1876).
One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.
In another embodiment, a TZP nucleic acid is expressed through a transposable element. This allows for constitutive, yet periodic and infrequent expression of the constitutively active polypeptide. The invention also provides for use of tissue-specific promoters derived from viruses which can include, e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong phloem-specific reporter gene expression; the cassaya vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol. 31:1129-1139).
DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. 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.
Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. EMBO J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).
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. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).
Transformed plant cells that are derived from any transformation technique can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, optionally relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan 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, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).
The nucleic acids of the invention can be used to confer desired traits on essentially any plant. Thus, the invention has use over a broad range of plants, including species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.
The following examples are offered to illustrate, but not to limit the claimed invention.
In this example, we utilize the hypocotyl assay to identify QTL controlling growth in two light and two temperature conditions. We identified a recessive large effect QTL on chromosome five controlling 40% of the growth variation segregating in Recombinant Inbred Lines (RILs) derived from the Bay-0 and Shandara accessions of A. thaliana. The QTL was fine-mapped and the causal factor shown to be a mutation affecting a tandem zinc knuckle/PLUS3 protein (At5g43630; TZP), which is encoded by a single-copy gene in all completely sequenced plant genomes. We show that TZP acts downstream of the circadian clock and light signaling, directly regulating blue light-dependent, morning (dawn) specific growth, during seedling development and beyond. Based on its nuclear localization and its novel domain structure, we argue that TZP functions at the transcriptional level to control growth-promoting pathways. TZP represents a new component of the growth pathway that was not previously identified using traditional genetic screens.
Intraspecific variation provides a fertile source of genetic combinations that can be utilized to map new genes (or new alleles) involved in complex traits such as growth. To investigate natural variation for hypocotyl elongation response to light and temperature, we phenotyped a core set of 164 RILs from the Bay-0×Shandara cross in four different environments combining two white-light (17 mmol/m−2s−1 μL1] and 10 μmol/m−2s−1 [L2]) and two temperature (22° C. and 26° C.) conditions (supporting information [SI]
The genetic architecture of variation in hypocotyl elongation under these environmental conditions is presented in SI
Identifying the Quantitative Trait Gene (QTG) underlying a QTL is a challenging task that requires several independent lines of proof that a gene is linked to a trait of interest and that variation in this gene explains the trait (Weigel, D. and Nordborg, M., Plant Physiol, 138:567-568 (2005)). The most general approach is fine-mapping to a very small physical candidate interval, which in the best case allows immediate identification of candidate polymorphisms (Quantitative Trait Nucleotide, QTN) within the causal gene or regulatory regions (Clark, R. M. et al., Science, 317:338-342 (2007)). For most QTL and situations in A. thaliana, phenotyping for a QTL effect remains much more limiting than genotyping many individuals. Therefore, an efficient strategy for fine-mapping is to first isolate recombinants within a segregating nearly-isogenic line based on genotype alone and then to phenotypically interrogate only the informative ones (in successive rounds) to reduce the candidate interval to the gene level (Peleman, J. D. et al., Genetics, 171:1341-1352 (2005)).
We followed the HIF strategy to build nearly-isogenic lines from a RIL (RIL350) that was segregating solely for the LIGHT5 region. Comparing plants homozygous for the Sha allele with plants homozygous for the Bay allele at the QTL region (in an otherwise identical genetic background) confirmed the phenotypic impact of LIGHT5 on hypocotyl elongation (
Screening for recombinants by genotyping 600 plants descended from the initial RIL350 individual (heterozygous over the whole QTL region) allowed us to identify 80 recombinants (recombined HIF, or rHIF) over the ˜1.9 Mb heterozygous region. Twenty-six of these rHIF were individually interrogated in successive rounds of progeny testing to score for the presence (or absence) of the QTL effect caused by the remaining interval. This first screen allowed us to narrow the candidate region to less than 300 kb between markers at 17.405 and 17.692 Mb (
Sequencing the 7 kb interval in Bay-0 and Shandara revealed dozens of SNPs and several indels, many of which resulted in non-silent changes in the coding regions of the three candidate genes. Eight, one and five SNPs caused amino-acid changed in At5g43630, At5g43640 and At5g43650, respectively. Two single amino-acid deletions and one larger deletion were discovered in At5g43630. Finally, an 8 by insertion in Bay-0 caused a frameshift and a premature stop within the coding region for the predicted PLUS3 domain of At5g43630 (
The three genes in the LIGHT5 interval are annotated as encoding a tandem zinc knuckle/PLUS3 (TZP, At5g43630), a 40S ribosomal protein (RPS15E, At5g43640), and a basic helix-loop-helix protein (bHLH092, At5g43650). TZP has no close homologs, but does share similarity with other Arabidopsis proteins that only have the zinc knuckle or PLUS3 domains. bHLH092 is part of the large bHLH family of transcription factors (Toledo-Ortiz, G. et al., Plant Cell, 15:1749-1770 (2003)), and a close homolog is TRANSPARENT TESTA 8 (TT8; At4g09820). There are four closely related RPS15E genes (At1g04270, At5g09500, At5g09510, and At5g09490) (Barakat, A. et al., Plant Physiol, 127:398-415 (2001)). The transcript/expression of all three annotated genes in the Columbia background was confirmed by tiling array data and full length cDNAs. Quantitative RT-PCR (qPCR) revealed that all three genes are expressed in Bay-0 and Shandara (data not shown).
T-DNA-insertions in RPS15E or bHLH092 did not result in hypocotyl elongation phenotypes (data not shown). No insertion mutants were available for TZP (an insertion GABI-KAT line could not be recovered). To determine the association of the stop codon in TZP with the hypocotyl phenotype, we sequenced the PLUS3 domain in other A. thaliana accessions. Although we identified 10 synonymous changes and 13 non-synonymous changes (including two changes affecting residues at least partially conserved in other species), we could not detect the Bay-0 premature stop codon polymorphism in a panel of ˜300 additional accessions (data not shown). We discovered that a Bay-0 single seed descent (SSD) line (Bay-0[41AV]), which was derived independently of the parent for the RIL set, did not have the 8 bp-insertion in TZP. Sequencing the entire 7 kb-candidate region revealed that this was the only sequence difference between Bay-0 and Bay-0[41AV] at LIGHT5. Genotyping additional markers in Bay-0[41AV] (as well as a careful phenotypic observation of the lines) showed that it is from the same genetic background as Bay-0 (data not shown). We crossed the two lines, with and without the TZP stop codon, to determine whether the mutation was responsible for the LIGHT5 phenotype. As shown in SI
Additionally, we employed a transgenic approach to confirm the role of TZP in hypocotyl growth. The Sha alleles of all three genes were overexpressed in the rHIF containing the Bay allele (rHIF138-8). Overexpression of TZP (TZP-OX) caused plants to have very long hypocotyls (
TZP is a single copy gene in A. thaliana. TZP orthologs are also single copy in the fully sequenced plant genomes (SI
The zinc knuckle (znkn; CX2CX4HX4C; SEQ ID NO:13) domain has been shown to be important for protein-protein interactions, as well as for binding single-stranded DNA (Vo, L. T. et al., Mol Cell Biol, 21:8346-8356 (2001)). There are at least 24 znkn-containing proteins in Arabidopsis, five of which are closely related to each other, but not to TZP (table S2 from our website data). Mutations in one of them, SWELLMAP 1 (SMP1), result in small plants with small cells because of a dysfunction in the commitment to the cell cycle (Clay, N. K. and Nelson, T., Plant Cell, 17: 1994-2008 (2005)). Another znkn protein, RSZ33, regulates interactions between splicing factors (Kalyna, M. et al., Mol Biol Cell, 14:3565-3577 (2003); Lopato, S. et al., Plant Mol Biol, 39:761-773 (1999)). In yeast, the znkn of MPE1 has been found to control 3′ end processing of pre-mRNA, and its human ortholog RBBP6 has been shown to interact with tumor suppressor pRB1 (Vo, L. T. et al., Mol Cell Biol, 21:8346-8356 (2001)). Znkn domains are also found in retroviral gag proteins (nucleocapsid), including that of HIV (De Guzman, R. N. et al., Science, 279:384-388 (1998)).
The PLUS3 domain is thought to play a role in nucleic acid binding through three conserved positive amino acids (de Jong, R. N. et al., Structure, 16:149-159 (2008)). There are five proteins in Arabidopsis with the PLUS3 domain (SI
Our initial QTL study suggested that LIGHT5 is involved in light-regulated hypocotyl growth. To determine whether the growth defects in LIGHT5 are specific to certain light environments, we measured hypocotyl lengths for both the rHIF and TZP-OX lines under different fluences of monochromatic red, blue and far-red light, and in continuous dark. We found that both loss and gain of TZP activity had a significant effect on hypocotyl length under a range of blue or white fluences, but not in red or far-red light, or in the dark (
We measured transcript abundance in Shandara, Bay-0, rHIF138-8, rHIF138-13, arHIF47-2, arHIF47-5, and TZP-OX line #3 seedlings grown in constant blue light (15 μmol/m2s) for five days and then harvested at subjective dawn (relative to the time when they were moved from stratification to light). The extent of growth paralleled TZP expression levels (SI
It is well established that hypocotyl elongation is controlled by the circadian clock (Nozue, K. et al., Nature, 448:358-361 (2007); Dowson-Day, M. J. and Millar, A. J., Plant J, 17:63-71 (1999)). Therefore, we tested whether TZP is clock regulated and if the circadian clock is altered in TZP-OX plants and the rHIF carrying the Bay-0 allele. We found that TZP cycles under both diurnal and circadian conditions with peak expression at dawn (transition from dark to light;
Since TZP is regulated by light/dark cycles and the circadian clock, we asked if the genes disrupted by TZP-OX were time-of-day specific. We used the PHASER time-of-day analysis tool (http://phaser.cgrb.oregonstate.edu/), which determines if there is a pattern of time-of-day co-expression in a given gene list compared to a background model. The peak expression of genes that were disrupted in TZP-OX, and differentially expressed in rHIF138-8 vs. rHIF138-13 is biased towards dawn (
In an effort to capture the entire effect of TZP-OX, we carried out a time course under light/dark cycles (12 hrs white light/12 hrs dark) in seven-day-old seedlings, sampling every four hours over one day in the same genotypes as above. We validated the overexpression of TZP using qPCR, which revealed that TZP continued to cycle despite overexpression (
Similar to the results in blue light, the genes that were upregulated in TZP-OX were dawn-specific (SI
Auxin-response genes, including WES™ [GH3.5; (Park, J. E. et al., Plant Cell Physiol, 48:1236-1241 (2007))], DFL1 [GH3.6; (Nakazawa, M. et al., Plant J, 25:213-221 (2001))] (SI
A similar number of genes were found to cycle across all three genotypes as reported for this condition previously [(Michael, T. P. et al., PLoS Genet, 4:e14 (2008)); ˜7,700 genes], and there was no significant difference between the number of genes cycling in rHIF lines and TZP-OX, although there were genes specific to each genotype. However, the peak transcript abundance of 362 genes was shifted by six hours or more in TZP-OX compared to rHIF138-8 (only genes that cycled in both genotypes were considered). Phytohormone-related (SIR1, ETO1, GH3.3, RGA1, ABF4), homeobox-leucine zipper (HAT2, HAT3), chromatin remodeling (HD2B, HDT4, SUVH9, CHR4, TAF1), leaf polarity (KAN3, AS1) and ribosomal genes were misphased in TZP-OX. PHYTOCHROME D (PHYD) was also phased eight hours earlier in TZP-OX (SI
Consistent with TZP controlling blue light dependent growth, LONG HYPOCOTYL IN FARRED 1 (HFR1) is overexpressed at dawn more than 100 fold with the same pattern as IRX1 (SI
Conclusions: Despite extensive forward genetics screens in A. thaliana, natural variation has recently made important contributions to the identification of genes not previously known to impact several different traits [e.g., (Bentsink, L. et al., Proc Natl Acad Sci USA, 103:17042-17047 (2006); Mouchel, C. F. et al., Genes Dev, 18:700-714 (2004); Macquet, A. et al., Plant Cell, 19:3990-4006 (2007); Baxter, I. et al., PLoS Genetics, 4:e100000438-41 (2008))]. Apart from being able to exploit allelic variation (in multiple genetic backgrounds) that cannot be generated by conventional mutagenesis, the success of these studies has often been due to the use of quantitative phenotyping, as opposed to the qualitative gauges employed in typical mutant screens. We have demonstrated here the power of QTL analysis to reveal a new component of the hypocotyl growth pathway in A. thaliana, TZP, a unique, tandem zinc knuckle/PLUS3 domain protein encoded by a single copy gene in the vascular plant lineage. TZP provides a direct link between light signaling and the pathways that control growth in an environmentally independent fashion.
A detailed and referenced version of this section is available online (SI Text).
Plant material and phenotyping. The Core-Population of 164 RILs from the Bay-0×Shandara set (http://dbsgap.versailles.inra.fr/vnat/) was phenotyped in four different light and temperature environments to map QTL affecting hypocotyl elongation. Complete phenotypic data from RILs is available in table S14 from our website data at www.inra.fr/vast/Files/Loudet_PNAS_SITables.xls. HIF350 was developed from an F7 line (RIL350) that still segregated for a single and limited genomic region around LIGHT5 locus. Plants still heterozygous for the QTL region were screened with adequate markers to isolate recombinants (rHIF) used in the fine-mapping process. Advanced rHIF crosses were generated from two different rHIFs recombined immediately to the north or immediately to the south of the LIGHT5 interval giving rise to lines arHIF47. Distinct Bay-0 lines from the stock center were used to find variants at LIGHT5. rHIF138-8[Bay] was complemented by over-expressing each of the three positional candidate genes cloned from rHIF138-13[Sha].
QTL mapping. Analyses used hypocotyl length mean values of an average of 16 seedlings (from two distinct experiments) per genotype per environment. QTL analyses were performed using QTL Cartographer, with classical parameters for interval mapping and composite interval mapping.
Microarray analysis. Microarray experiments were carried out per Affymetrix protocols (ATH1 GeneChip), on seven-day-old tissue harvested under either continuous blue at subjective dawn, or every four hours (starting at dawn) under 12 hrs white light/12 hrs dark cycles over one day (six time points). Hybridization intensities from all microarrays were normalized together using gcRMA implemented in the R statistical package. The blue dataset was then separated and differentially expressed genes were identified using linear modeling with the limma bioconductor package in R.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
Arabidopsis LIGHT5 genomic sequence
The present application claims benefit of priority to U.S. Provisional Patent Application No. 61/195,819, filed Oct. 10, 2008, which is incorporated by reference for all purposes.
This invention was made with government support under (identify the contract) awarded by the National Institutes of Health (NIH Grant No. GM62932). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/60232 | 10/9/2009 | WO | 00 | 10/4/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61195819 | Oct 2008 | US |
