# Patent

## Title
Engineering lipids in vegetative tissues of plants

## Abstract

The present invention discloses gene targets and methods for the genetic control of lipid accumulation in vegetative (non-seed) portions of plants. Enhanced lipid, e.g. triacylglycerol (TAG), accumulation in vegetative portions of plants may be obtained by down-regulation of activity of At4g24160 or a homolog thereof. Plants, plant parts, seeds comprising down-regulated AT4G24160 activity, or activity of a homolog thereof, are also provided, as well as products prepared therefrom.

## Background
This application claims the priority of U.S. Provisional Appl. Ser. No. 61/148,952, filed Jan. 31, 2009, the entire disclosure of which is incorporated herein by reference.


GOVERNMENT INTEREST
This invention was made with government support under Grant HL 20948 and Grant GM 52016 awarded by the National Institute of Health. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the engineering of plants to alter lipid content in vegetative portions of the plant.
2. Description of Related Art
Plants synthesize and store oil primarily in cytosolic lipid droplets, and much of the Earth's fossil fuel reserves are the consequence of this process. In domesticated oilseeds, these stored triacylglycerols (TAGs) represent a major source of calories for human and animal nutrition, an excellent feedstock for diesel fuels, and a reservoir for the deposition of industrial fatty acids for chemical feedstocks. A number of acyltransferases are involved in the biosynthesis of TAGs, including acyl-CoA:cholesterol acyltransferases (ACATs), diacylglycerol acyltransferases (i.e., DGAT1s and DGAT2s), lecithin:cholesterol acyltransferases (LCATs), phospholipid:diacylglycerol acyltransferases (PDATs), glycerol-3-phosphate acyltransferases (GPATs) and acyl-CoA lysophosphatidic acid acyltransferases (LPAATs). In plants, TAG is the primary component of vegetable oil that is used by the seed as a stored form of energy to be used during seed germination.
Higher plants are believed to synthesize oils via a metabolic pathway commonly referred to as the Kennedy pathway (Kennedy et al., 1956; Finnlayson et al., 1980). Fatty acids are made in plastids from acetyl-CoA through a series of reactions catalyzed by enzymes known collectively as Fatty Acid Synthase (FAS). The fatty acids produced in plastids are exported to the cytosolic compartment of the cell, and are esterified to coenzyme A. These acyl-CoAs are the substrates for glycerolipid synthesis on the endoplasmic reticulum (ER). Glycerolipid synthesis itself is a series of reactions leading first to phosphatidic acid (PA) and 1,2-diacylglycerol (DAG). Either of these metabolic intermediates may be directed to membrane phospholipids such as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), or phosphatidylcholine (PC), or they may be directed on to form neutral triacylglycerol (TAG). DAG is synthesized from glycerol-3-phosphate and fatty acyl-CoAs in two steps catalyzed sequentially by glycerol-3-phosphate acyltransferase (G3PAT), and lysophosphatidic acid acyltransferase (LPAAT) to make PA, and then an additional hydrolytic step catalyzed by phosphatidic acid phosphatase (PAP) to make DAG. In most cells, DAG is used to make membrane phospholipids, the first step being the synthesis of PC catalyzed by CTP-phosphocholine cytidylyltransferase. In cells producing storage oils, DAG is acylated with a third fatty acid in a reaction catalyzed by DAGAT.
Factors governing the oil content of a vegetative plant part are not well known, and the role of a CGI58 homolog in lipid accumulation in vegetative tissues of plants has not been previously described. As such, materials and methods for increasing the neutral lipid (e.g. oil content, including TAG) content in cells of plant vegetative tissues by use of CGI58-related sequences have not been previously described.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: shows partial amino acid sequence alignments of Arabidopsis At4g24160 (CGI58-homolog) splice variants (Arabidopsis 1, 2), and homologs from grape, rice, human, mouse, C. elegans, zebrafish, and moss (Physcomitrella) (SEQ ID NOs: 9-18). Motif analysis by MEME/MAST or visual analysis reveals four distinct domains common to all proteins (shaded boxes). A classical GXSXG (SEQ ID NO:1) esterase/lipase sequence (boxed and annotated above the sequence) is present in the plant and nematode sequences, and the acyltransferase motif (H(X)4D (SEQ ID NO:17; also boxed and annotated) is present in all but the truncated Arabidopsis splice variant. Consensus sequences of each motif are shown at the bottom right (SEQ ID NOs: 2-5). Conserved residues are noted with an asterisk, strongly similar amino acids with a colon, and weakly similar amino acids with a period. Two positions in the Arabidopsis amino acid sequence marked 176 and 315 correspond to residues, that when mutated in the human sequence, interfere with lipid body binding and cause disease. “Arabidopsis.1” represents aligned portions of SEQ ID NO:7; “Arabidopsis.2” (SEQ ID NO:9) represents aligned portions of the shorter polypeptide (SEQ ID NO:38) produced by the At4g24160 splice variant, also indicating the differences with the longer polypeptide encoded at the same locus.
FIG. 2: (A) the relative gene exon/intron structure of the At4g24160 locus, which gives rise to two transcripts; and (B) Detection of both transcripts by RT-PCR in wild-type seedlings; neither is detected in seedlings of T-DNA insert mutant lines. Nucleotide sequence of full length transcript (mRNA) is given in SEQ ID NO:6. The corresponding amino acid sequence is given in SEQ ID NO:7. The nucleotide sequence of the second transcript is given in SEQ ID NO:37, and the corresponding amino acid sequence is given in SEQ ID NO:38. Ubiquitin transcripts expressed in seedlings of the same wild-type and mutant lines are shown as a control. cDNAs corresponding to the protein coding sequences of both transcripts were amplified from seedling mRNA.
FIG. 3: Representative confocal microscope images of WT (Col 0), and two T-DNA mutant lines stained with Nile red to reveal lipid bodies (left) in 21-day old seedlings (petiole region of true leaves)(left). Bar=20 microns. TLC separation of neutral lipids isolated from 14-day old seedlings (450 mg FW each) of WT (lane 1), and both T-DNA mutants (lanes 2 and 3)(upper right). Standards are sterylesters (StE), triacylglycerols (TAG) (arrow), free fatty acids (FFA), diacylglycerols (DAG), and monoacylglycerols (MAG). Representative epifluorescence images of purified lipid bodies from 14-day old wild-type and T-DNA mutant seedlings (stained with BODIPY 493). Bar=20 microns(lower right).
FIG. 4: Nanospray ionization and mass spectroscopy of triacylglycerol molecular species in Arabidopsis Col 0 wild-type seedlings. Full scan, positive ion nanospray-MS species were identified as ammonium adducts (M+NH4]+ using a ProXeon nanospray ionization source (ProXeon Biosystems, Odense, Denmark) and analyzed with a LCQ Deca XP Plus quadrupole ion trap (Thermo Fisher Scientific, Waltham, Mass.). A 50× dilution of reconstituted TAG extract in chloroform was prepared with 1:1 (v/v) chloroform:methanol plus 10 mM ammonium acetate. Typical scanning conditions were carried out in positive ion mode with a 0.8-1.0 kV spray voltage, scan range of 700-1000 m/z, 275° C. capillary temperature, 3 microscans per second with a maximum injection time of 200 msec.
FIG. 5: Same as for FIG. 4, but carried out on At4g24160 T-DNA exon insertional line SALK—0136871.
FIG. 6: Nanospray ionization and mass spectroscopy of triacylglycerol molecular species in Arabidopsis; tandem scans were typically carried out with an isolation width of 2.0 m/z, 35% normalized collision energy, 0.200 activation Q, and 30 msec activation time with a scan range from 200 to 1000 m/z. Representative tandem scans performed on the wild-type TAG extracts (FIG. 6) show the typical diacylglycerol fragmentation products as notated by the TAG plus initial adduct [LnLnLn+NH4] minus a free fatty acid as underlined [LnLnLn+NH4-LnCOOH]+ and compared to known masses (Byrdwell, 2005).
FIG. 7: shows representative confocal fluorescence images of leaves of 40 day old Arabidopsis plants (wild-type and T-DNA insertional mutant line Salk—136871). Leaves were fixed in paraformaldehyde and stained with BODIPY 493/520 (Invitrogen), a neutral lipid-specific stain. BODIPY-fluorescence and chlorophyll autofluorescence were imaged together. Excitation of both chlorophyll and BODIPY stain were at 493 nm. Emission wavelength for chlorophyll was 692 nm, exposed for 0.4 seconds. Emission wavelengths for BODIPY-stained lipid bodies was 520 nm, exposed for 10 seconds. Scale bar=10 microns. Images were acquired with a Zeiss 200M optical microscope fitted with a CSU-10 Yokogawa confocal scanner (McBain Instruments, Simi Valley, CA) and captured with a digital camera (Hamamatsu, Phoenix, AZ).
FIG. 8: shows a three-dimensional projection of lipid droplets in SALK—136871 (cgi58 mutant) mesophyll cells of mature Arabidopsis leaves, about 40 days after emergence. The projection was prepared from a Z-stack of thirteen optical sections imaged by confocal fluorescence micrographs acquired from the T-DNA insertional mutant line. Leaves were prepared and imaged as in FIG. 7. Each image is a 1 micron section (Scale bar=20 microns). Lipid bodies are abundant throughout the cytoplasm of these mutants. Lipid droplets are distinctly separated from the chloroplasts in these cells.
FIG. 9: shows a Clustal alignment of the sequence of Arabidopsis TIGR cDNA clone TC287358 (SEQ ID NO:8), containing the At4g24160 gene (CGI58 homolog), with cDNA sequences of other organisms: B. napus, poplar, rice, wheat, potato, onion, pine, spruce, sorghum, sugarcane, maize, barley, tobacco, cotton, grape, Lotus japonicus, soybean, and alfalfa (SEQ ID NOs:18-36). The gene is located on Arabidopsis chromosome 4, spanning bases 12,539,796-12,542,321, according to the Arabidopsis genome TC alignment (version 5), available at the Gene Index Project (compbio.dfci.harvard.edu/tgi; Quackenbush et al., 2001).
FIG. 10: Electrospray ionization mass spectrometry (MS) of TAG molecular species in wild-type and At4g24160 T-DNA knockout plants. Positive ion MS analysis of neutral lipids of wildtype (A) and CGI-58-exon disruption (B) identified TAG species as ammonium adducts [M+NH4]+ using a LCQ Deca XP Plus quadrupole ion trap. The neutral lipid fractions from combined leaf tissues of mature plants (about 45 d old) were dissolved in 1:1 (v/v) chloroform:methanol plus 10 mM ammonium acetate. Peaks are labeled according to the three fatty acids present in each TAG molecular species, and low abundance TAG species with the same molecular mass are indicated in parentheses. Positions of fatty acids on the glycerol backbone were not determined. Fatty acid abbreviations are: He, 16:3-hexadecatrienoic acid; L, 18:2-linoleic acid; Ln, α-18:3-linolenic acid; P, 16:0-palmitic acid; Po, 16:1-palmitoleic acid; S, 18:0-stearic acid. (C) Regions of several relevant, representative MS/MS scans showing the diagnostic diacylglycerol fragmentation ions minus a free fatty acid compared to known masses (e.g. Byrdwell, 2005). Molecular ions were in some cases a combination of isomers as revealed in MS/MS (e.g., first two panels, each with three combinations of DAG species derived from same parent ion, but with a different fatty acid loss).
FIG. 11: shows that lipid droplets are abundant in leaves of cgi58 mutants. A) Representative confocal fluorescence micrograph of mesophyll tissues of mature wild-type leaves (about 40 d after emergence), showing chloroplasts and a few lipid droplets (arrows) stained with BODIPY 493. The boxed region in the left panel is shown at higher magnification on the right. (B) Confocal fluorescence micrograph of mesophyll tissues of same-age leaves of cgi-58 T-DNA knockouts showing similar chloroplast distribution, but with considerably more lipid droplets accumulated in the cytosol outside of chloroplasts. (C) Z-stack of thirteen optical sections (taken at 1 micron increments) of the cgi-58 T-DNA knockout mutant (Salk—136871), reconstructed to reveal the cellular organization of lipid droplets and their relationship to chloroplasts, as also seen in FIG. 8. D) The percent area of lipid droplets and chloroplasts in mesophyll cells was quantified by morphometric analysis as the percent of image area fluorescing from either the lipid droplets or chloroplasts channels. Averages and standard deviations are plotted for ten digital images of 25,000 microns2 each taken from several leaves at each stage. There were significant differences between the amounts of lipid droplets in mutants and wild-types in leaves at 40 d (p<0.0001) and at 65 d (p<0.002), but not at 15 d (p>0.63). Abundance was most dramatic in mature, 40 d leaves. No statistical differences were observed in the relative percentage of chloroplasts between mutant and wild-type, suggesting the mutation only affected lipid accumulation in cytosolic droplets. Scale Bar=20 microns.
FIG. 12: shows temporal differences in lipid droplet accumulation in wild-type and cgi-58-disrupted Arabidopsis plant cells. Representative confocal fluorescence images of Arabidopsis leaves at 15 days after emergence, 40 d, or 65 d (WT and T-DNA mutant, Salk—136871). Lipid droplets are green and chloroplasts are red; imaging acquisition conditions were the same as for FIG. 11. Bar=20 microns.
FIG. 13: shows expression of At4g24160 isoforms in different tissues and different developmental stages, as determined by semi-quantitative RT-PCR. Lane legends are as follows: “L”=full-length transcript; “S”=alternatively spliced shorter transcript for a smaller CGI-58 protein which lacks the acyltransferase domain; “U”=ubiquitin control. “WT”=RNA isolated from wild-type Arabidopsis; “M”=RNA isolated from cgi58 mutant Arabidopsis. 
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a plant comprising increased lipid accumulation in vegetative tissues relative to a wild type plant of the same species and wherein activity of an At4g24160 gene product or a homolog thereof has been down-regulated in the plant. In one embodiment, down-regulation of an At4g24160 gene product or a homolog thereof comprises at least one method selected from the group consisting of: a) expressing in the plant a RNA molecule complementary to all or a portion of an mRNA expressed from a gene comprising a sequence selected from the group consisting of SEQ ID NOs:18-36, wherein the RNA molecule inhibits the function of the At4g24160 gene product or homolog thereof in said plant; b) mutagenizing said At4g24160 gene, or homolog thereof, so that the function thereof is down-regulated relative to wild type At4g24160 or a homolog thereof, in a plant of the same species; and c) co-suppression.
In certain embodiments, the plant is a transgenic plant; in other embodiments, the plant is non-transgenic. The plant may further be defined as a fertile R0 transgenic plant, or as a progeny plant of any generation of a fertile R0 transgenic plant. A part of such a plant is an embodiment of the invention. In certain embodiments, the plant part is selected from the group consisting of a cell (including a cell (e.g. tissue) culture), a leaf, a stem, a petiole, pollen, a tuber, and root tissue.
Another aspect of the invention relates to a seed of a plant comprising increased lipid accumulation in vegetative tissues relative to a wild type plant of the same species and wherein activity of an At4g24160 gene product or a homolog thereof has been down-regulated in the plant, further wherein the seed is of a plant that comprises increased lipid accumulation in vegetative tissues, relative to a wild type plant of the same species.
Another aspect of the invention relates to a method of altering lipid content in a plant (including a plant cell or plant cell culture) comprising down-regulating the function of an At4g24160 gene product or homolog thereof. In certain embodiments, the sequence of the gene encoding the homolog, or of the homolog, comprises a sequence selected from the group consisting of: SEQ ID NOs:18-36; a sequence at least about 70% identical to a any of SEQ ID NOs:6 or 18-36; and a sequence at least about 75% identical to any of SEQ ID NOs:10-16.
In some embodiments, down-regulating the function of an At4g24160 gene product or homolog thereof comprises eliminating the function of said an At4g24160 gene product or homolog thereof. In certain embodiments, down-regulating the function of an At4g24160 gene product or homolog thereof comprises expressing in the plant a RNA molecule complementary to all or a portion of an mRNA expressed from a gene comprising a sequence selected from the group consisting of SEQ ID NOs:18-36, wherein the RNA molecule inhibits the function or expression of an At4g24160 gene product or homolog thereof in said plant. In particular embodiments the RNA molecule is a single stranded RNA molecule. In yet other embodiments, the RNA molecule is a double stranded RNA molecule. In still yet other embodiments, down-regulating the function of an At4g24160 gene product or homolog thereof comprises mutagenizing said At4g24160 gene, or homolog thereof, so that the function thereof is down-regulated relative to the wild type At4g24160 or homolog thereof in a plant of the same species. In further embodiments, down-regulating the function of an At4g24160 gene product or homolog thereof comprises co-suppression.
In certain embodiments, the plant is of a species selected from the group consisting of corn, sugarcane, sorghum, millet, rice, wheat, barley, soybean, Glycine sp., grape, canola, Arabidopsis, Brassica sp., cotton, tobacco, sugar beet, sunflower, bamboo, switchgrass (Panicum virgatum), giant reed (Arundo donax), reed canarygrass (Phalaris arundinacea), Miscanthus×giganteus, Miscanthus sp., Sericea lespedeza (Lespedeza cuneata), ryegrass (Lolium multiflorum, Lolium sp.), timothy, kochia (Kochia scoparia), forage soybeans, alfalfa, clover, turf grass, sunn hemp, kenaf, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, indiangrass, fescue (Festuca sp.) including tall fescue, Dactylis sp., Brachypodium distachyon, smooth bromegrass, orchardgrass, kentucky bluegrass, yellow nutsedge (Cyperus esculentus), pine, poplar (Populus sp.), willow, and eucalyptus.
Another aspect of the invention comprises a method of producing lipids in a plant comprising: (a) obtaining a plant comprising increased lipid accumulation in vegetative tissues relative to a wild type plant of the same species and wherein activity of an At4g24160 gene product or a homolog thereof has been down-regulated in the plant; and (b) isolating lipid from said plant. In certain embodiments, the fatty acid content of lipids accumulated in vegetative tissue comprise a rationally defined fatty acid profile. In other embodiments, the neutral lipids accumulated in vegetative tissue are leaf-specific fatty acids. In particular embodiments the fatty acids are rich in omega-3 fatty acids.
Yet another aspect of the invention relates to a method of plant breeding comprising: identifying a plant comprising a reduced level of function of an At4g24160 gene product or a homolog thereof relative to that found in an otherwise isogenic plant that displays a wild-type level of function of an At4g24160 gene product or homolog thereof; and selecting the plant for crossing with a second plant. In certain embodiments, the method may further be defined as comprising a step wherein identifying comprises at least one method selected from the group consisting of: PCR, single strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, cleavage fragment length polymorphism analysis and/or DNA sequencing. In certain embodiments the method comprises: a) identifying at least a first polymorphism at the At4g24160 locus, or a locus encoding a homolog thereof, in a crop plant that confers increased neutral lipid content in vegetative tissues of the plant; b) assaying a crop plant for the presence of the polymorphism; and c) selecting at least a first crop plant comprising the polymorphism. The method further comprises, in certain embodiments, crossing the first crop plant with a second crop plant of the same species or variety to produce a progeny plant comprising the polymorphism. In some embodiments the polymorphism comprises an insertion, a deletion, or at least one single nucleotide polymorphism (SNP) at a position in the At4g24160 locus, or a locus encoding a homolog thereof, that reduces the function of At4g24160, or corresponding homolog thereof, relative to that found in an otherwise isogenic plant displaying a wild-type level of function of an At4g24160 gene product, or homolog thereof.
Another aspect of the invention relates to a method of producing food, feed, or oil comprising: (a) obtaining a plant or progeny thereof, according to the method of plant breeding comprising: identifying a plant comprising a reduced level of function of an At4g24160 gene product or a homolog thereof relative to that found in an otherwise isogenic plant that displays a wild-type level of function of an At4g24160 gene product or homolog thereof; and selecting the plant for crossing with a second plant; (b) cultivating said plant to produce a plant product; and (c) preparing food, feed, or oil from said plant or plant product. In certain embodiments, the oil comprises TAG. In other embodiments, the plant product comprises leaves, stems, shoots, tubers, or roots.
In certain embodiments, the neutral lipids isolated from vegetative tissues of a plant comprise a rationally designed fatty acid profile. In particular embodiments, the neutral lipids may comprise hydroxyl, epoxy, cyclic, acetylenic, saturated, polyunsaturated, short-chain fatty acids, long-chain fatty acids, TAGs, wax-esters, or steryl-esters. In other embodiments, the neutral lipids isolated from vegetative tissues of the plant comprise a leaf-specific fatty acid profile. Thus, in particular embodiments, the neutral lipids isolated from vegetative tissues of the plant may comprise hexadecatrienoic and octadecatetraenoic fatty acids, or do not contain eicosaenoic fatty acid.



DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description of the invention provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention.
The present invention relates to altering the lipid composition of a plant, and vegetative production of lipids in particular, by affecting the expression of certain plant genes, such as a At4g24160 gene of Arabidopsis or plant homologs thereof. This specifically includes, but is not limited to, plants such as corn, sugarcane, sorghum, millet, rice, wheat, barley, soybean, Glycine sp., grape, canola, Arabidopsis, Brassica sp., cotton, tobacco, bamboo, sugar beet, sunflower, willow, switchgrass (Panicum virgatum), giant reed (Arundo donax), reed canarygrass (Phalaris arundinacea), Miscanthus×giganteus, Miscanthus sp., Sericea lespedeza (Lespedeza cuneata), ryegrass (Lolium multiflorum, lolium sp.), timothy, kochia (Kochia scoparia), forage soybeans, alfalfa, clover, turf grass, sunn hemp, kenaf, bahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem, indiangrass, fescue (Festuca sp.) including tall fescue, Dactylis sp., Brachypodium distachyon, smooth bromegrass, orchardgrass, kentucky bluegrass, yellow nutsedge, pine, poplar (Populus sp.), and eucalyptus, among others. The present invention thus provides methods for controlling levels of lipids in plants.
Chanarin-Dorfman Syndrome is a neutral-lipid storage disorder (Lefevre et al., 2001; Bruno et al., 2008). CGI58, also known as ABHD5, associates with lipid droplets in human cells and participates in storage lipid hydrolysis. A mutation in this protein results in hyperaccumulation of lipid droplets in cells and the pathology associated with this syndrome. The CGI58 protein sequence includes a so-called “alpha/beta hydrolase fold” that is shared by members of the esterase/lipase/thioesterase family, suggesting that it might be a TAG lipase. Recent analyses of its functional properties have indicated that the mammalian polypeptide stimulates the activity of a lipase called ATGL (Adipose Triglyceride Lipase), which is the major lipase responsible for catalyzing the initial step of TAG breakdown in both adipose and non-lipid storing cell types (e.g. Lass et al., 2006; Yen & Farese, 2006; Schweiger et al., 2006; Yamaguchi et al., 2007). Interestingly, CGI58 also possesses lysophosphatidic acid acyltransferase (LPAAT) activity in vitro, suggesting that, in addition to its role in stimulating lipase activity, it may play a role in recycling of fatty acids into membrane phospholipids (Ghosh et al., 2008).
At4g24160 has been identified by the inventors as a putative homolog of human CGI58, in Arabidopsis thaliana. The gene in Arabidopsis is apparently expressed as two alternative transcripts (two distinct cDNAs corresponding to the same gene have been identified) and the predicted protein products share domain architecture with other lipases/esterases and acyltransferases (FIG. 1). Surprisingly, Arabidopsis mutant lines lacking the function of the CGI58 homolog (i.e. At4g24160) contained vegetative (i.e. non-seed) tissues with metabolic machinery capable of synthesizing and storing oil as TAG, demonstrating that there are mechanisms in place to regulate this process in non-seed tissues. It is thus provided for the first time that this mechanism can be exploited for the synthesis and accumulation in substantial quantities of desired neutral lipid compounds (i.e. oils) in above-ground biomass of plants, as well as in plant cell cultures.
Triacylglycerols can be synthesized in non-seed tissues (Murphy, 2001); however, their abundance is low and these storage lipids are presumed to be metabolized rapidly, perhaps for the recycling of fatty acids for energy or the synthesis of membrane lipids. Thus the regulation and transient accumulation of storage oils in non-seed tissues is not well understood.
In certain embodiments, “controlling levels of lipids” as used herein refers to control of lipid levels in vegetative portions of the plants. Likewise, “controlling fatty acid composition” refers to altering the molecular composition of fatty acids in neutral lipids produced in vegetative tissues of plants with decreased level of At4g24160 function, to obtain lipids displaying a particular profile of fatty acids, such as a leaf-specific fatty acid profile. Thus, the fatty acids may include hydroxyl, epoxy, cyclic, acetylenic, saturated, polyunsaturated, short-chain or long-chain fatty acids that are incorporated into neutral lipids that can be compartmentalized in lipid droplets, including TAGs, wax-esters, and steryl-esters.
“Down-regulating” as used herein refers to reducing the expression or function of the At4g24160 gene or homolog thereof. Such reduction may be a 25%, 50%, 75%, or up to 90%, or more, reduction in gene expression, or in function of a At4g24160 polypeptide or homolog thereof. For instance, this may be measured by comparing the level of mRNA transcript(s) derived from the At4g24160 locus, or homolog thereof, in a given plant cell with the level of such transcript(s) found in an otherwise isogenic plant cell, but differing in function or regulation of the At4g24160 locus, or homolog thereof, including functional activity of a polypeptide encoded at this locus, under comparable conditions. Such “down-regulated” function may also be measured by assaying the enzymatic activity of a polypeptide, such as ATGL (Adipose Triglyceride Lipase) or homolog, that is regulated by a polypeptide encoded at the At4g24160 locus or a locus homologous thereto.
“Homolog,” as used herein, refers to genes related to each other by descent from a common ancestral DNA sequence, and such genes, as understood herein, may share about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or greater sequence similarity at the nucleotide level with any of the nucleotide sequences given in SEQ ID NOs:6, 8, and 18-37. In some embodiments, homologs comprise the domain structure shown in FIG. 1, for instance one or more, two or more, or three or more of the four domains indicated by shading in FIG. 1, and/or one or more of the esterase/lipase and acyltransferase sequence motifs described in FIG. 1. Homologs may also be defined, in certain embodiments, as polypeptides comprising at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, sequence similarity, at the corresponding amino acid sequences, with one or more of the polypeptide sequences given in SEQ ID NOs:7, 10-16, and 38.
By “vegetative portions” is meant non-seed portions, including leaves, stems, shoots, buds, tubers, and roots, among others. Thus, in specific embodiments, levels of neutral lipids such as triacylglycerols (TAGs), diacylglycerols (DAGs, e.g. 1,2-diacylglycerol, or 1,3-diacylglycerol), and monoacylglycerols in vegetative portions of a plant are increased relative to the levels typically seen in such portions of a given plant (e.g. in a similar and otherwise isogenic but non cgi58-mutant plant). This increase may result in a vegetative tissue neutral lipid content of, for example, about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or greater, on a w/w basis. Thus, the amount of TAG in vegetative tissues of mature cgi58 mutant plants, such as cgi58 mutant Arabidopsis, may be measured by electrospray MS against a tripentadecanoyl standard (or by another method known in the art) and be about 50% higher than in otherwise similar wild-type plants. The level of neutral lipids in vegetative portions of a plant may be measured at various stages of vegetative tissue growth, for instance when true leaves are expanding, when they are fully expanded but not senescing, or when they are beginning to senesce (e.g., corresponding to developmental stages of about 15, 40, and 65 days after emergence as seen for Arabidopsis plants). In some embodiments, TAG content in a vegetative portion of a plant that displays decreased function of At4g24160 or a homolog thereof, may increase 2-fold, or 3-fold, 5-fold, 10-fold, or more, relative to that found in an otherwise isogenic plant of the same variety or species that differs by displaying a wild-type level of At4g24160 homolog function, such as the activity of a polypeptide encoded at the At4g24160 locus, or a locus homologous thereto.
Additionally, the fatty acid profile of neutral lipids synthesized in vegetative portions of a plant that displays decreased function of At4g24160 or a homolog thereof may show a molecular composition similar to that found in wild-type leaves, for instance demonstrating an increase in omega-3 fatty acid content, or an increase in the ratio of omega-3 to omega-6 fatty acids, relative to this level or ratio of neutral lipids as typically found in seed tissues. Thus, such TAGs, DAGs, and/or monoacylglycerols being produced in vegetative portions of a plant that displays decreased function of At4g24160 or a homolog thereof may, for instance, comprise levels of 16:3 and 18:3 hexadecatrienoic and octadecatrienoic fatty acids, as is typically found in leaf tissue, while, for instance, not containing the level of 20:1 eicosaenoic fatty acids typically found in seed oil bodies such as those of Arabidopsis. Thus, the fatty acid composition (as well as the level of neutral lipids) may be manipulated in vegetative tissues of plants with altered (e.g. decreased) level of At4g24160 function, to yield particular useful lipid species, including ones that are not found, or found at such levels, in seed oil. This further allows for the production of plants and tissue cultures which display rationally designed fatty acid profiles in the neutral lipids produced in their vegetative tissues, such as neutral lipids from vegetative tissues which are enriched in omega-3 fatty acid content, relative to the omega-3 fatty acid content found in neutral lipids of seed oils. For instance, one or more enzymes specific for production of a particular fatty acid of interest, or which are known to alter the fatty acid profile of lipids in an organism, as is known in the art, may be introduced into a cgi58 mutant plant, or other plant displaying decreased At4g24160 function, in order to produce a plant, or plant-derived oil, comprising neutral lipids (e.g. vegetatively-produced neutral lipids) displaying a specific non-naturally occurring fatty acid profile, or which contain one or more specific fatty acid(s) of interest. This may, for instance, include any fatty acid for industrial purposes (e.g. for production of food, feed, or fuel), for which an enzymatic step or steps can be introduced or modified in plants, including hydroxyl, epoxy, cyclic, acetylenic, saturated, polyunsaturated, short-chain or long-chain fatty acids that are incorporated into neutral lipids that can be compartmentalized in lipid droplets, including TAGs, wax-esters, and steryl-esters.
The substantial elevation of what is almost entirely TAG in cgi58 homolog T-DNA knockouts (e.g. see FIG. 3 upper right) indicates that multiple molecular strategies can be used for oil engineering in vegetative tissues in accordance with the invention. Because this biomass would not necessarily compete with food crops directly, this strategy could allow better utilization of GM and non-GM strategies. Extraction and recovery of this oil from biomass will also not be a considerable issue, since extrusion technologies and/or solvent-based approaches may be adapted to recovery of oil before other uses. It should be recognized that even a minor percentage of oil in biomass represents a revolutionary change in oil production. For example, a conservative estimate of TAG content at 3% on a dry weight basis would be comparable to total yields from oilseed crops. Yields of biomass from switchgrass are in the range of 5 tons of dry matter per acre (Fuentes and Taliaferro, 2002). A 3% yield of oil from this amount of biomass would represent 300 lbs of oil/acre, which is comparable to the current soybean oil yield (335 lbs/acre). A goal of 10% oil yield from plant vegetative tissues would be realistic in terms of causing minimal physiological consequences and representing a relatively minor impact on overall carbon allocation, especially if this is coordinated with senescence (e.g. expression of a sequence designed for suppression of expression of a At4g24160 polypeptide or homolog is placed under the control of a senescence-specific promoter) or chemical treatment. This amount of oil from biomass (1000 lbs/acre) would rival the highest oilseed crop yields in the temperate regions of the world. Canola is currently the highest yielder at about 850 lbs/acre, thus making biofuel production more economical. The success in Arabidopsis supports a rapid translation to crop systems. This strategy could be readily adapted for use in food crops, where oils could be recovered from vegetative crop residues (e.g., corn stover), or for use with dedicated energy crops where oil could be recovered from, for instance, stems and leaves prior to carbohydrate utilization.
In particular embodiments, the neutral lipid content of the vegetative portions of such plants, with a decreased activity of an AT4G24160 gene product or homolog thereof, is increased. An mRNA sequence of the At4g24160 gene is found in SEQ ID NO:6. The amino acid sequence of the full length polypeptide encoded at the At4g24160 locus is given in SEQ ID NO:7, and is also found at GenBank Accession B029749. Differences between the two polypeptides encoded by the At4g24160 locus are shown in FIG. 1. Altered plants with a reduced activity (partial or complete reduction in function) of a At4g24160 homolog, as compared to a wild type, for example, have dramatically increased levels of TAG in their vegetative tissues. The neutral lipid content of such plant vegetative parts may further be altered by cultural methods, for instance by exposing a plant to low temperature or to an increased concentration of ozone (i.e. above ambient).
In another aspect, the invention provides methods for screening crop plants, including cultivars, accessions, and breeding lines and materials of the crop plants listed above, for the presence of a mutation in a At4g24160 gene, or homolog thereof present in their genomes. Such screening may, in certain embodiments, be phenotypic (e.g. by chemically, visually, or microscopically assessing the oil content, neutral lipid content, or TAG content of plant vegetative tissues), or genetic (e.g. by detection based on nucleic acid probes or primers). In certain embodiments, use of an immuno-technique may be employed. In other embodiments, a method may employ marker-assisted breeding to identify plants, including cultivars or breeding lines, displaying a trait of interest, such as elevated levels of neutral lipids in vegetative portions of plant biomass.
At4g24160 Homolog Isolation
Isolation of additional At4g24160 homologs from other plant species may be accomplished by laboratory procedures well known and commonly used in the art. Standard techniques are used for identification, cloning, isolation, amplification, and purification of nucleic acid sequences and polypeptides. These techniques and various others are generally performed as described for instance in Sambrook et al., 1989. Genome walking techniques may be performed according to manufacturer's specifications (CLONTECH Laboratories, Inc., Palo Alto, Calif.).
One such technique for isolation of At4g24160-homologs is the use of oligonucleotide probes based on sequences disclosed in this specification to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by digestion with restriction endonucleases and then ligating the resultant segments with vector DNA to form concatemers that can be packaged into an appropriate vector. To prepare a cDNA library, mRNA is isolated from the desired organ, such as seed tissue, and a cDNA library is prepared from the mRNA.
A cDNA or genomic DNA library can be screened using a probe based upon the sequence of a cloned At4g24160 gene (e.g. SEQ ID NO:6), or the sequence encoding another identified homolog (e.g. SEQ ID NOs:18-36). Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Usefully employed such probes include, without limitation, 5′ UTRs which, may function as promoters. Alternatively, antibodies raised against an At4g24160 polypeptide, or homolog thereof, can be used to screen an mRNA expression library to isolate sequences of interest. At4g24160 homologs may also be identified in silico, for instance by similarity-based database searches as described below.
Nucleic acid sequences can be screened for the presence of protein encoding sequence that is homologous to genes of other organisms with known protein encoding sequence using any of a variety of search algorithms. Such search algorithms can be homology-based or predictive-based. Similarity-based searches (e.g., GAP2, BLASTX supplemented by NAP and TBLASTX) can detect conserved sequences during comparison of DNA sequences or hypothetically translated protein sequences to public and/or proprietary DNA and protein databases. Existence of a gene is inferred if significant sequence similarity extends over the majority of the target gene. Since such methods may overlook genes unique to the source organism, for which homologous nucleic acid molecules have not yet been identified in databases, gene prediction programs may also be used. Gene prediction programs generally use “signals” in the sequences, such as splice sites or “content” statistics, such as codon bias, to predict gene structures (Stormo, 2000).
Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For example, polymerase chain reaction technology can be used to amplify the sequences of a At4g24160 gene or At4g24160 gene homolog directly from genomic DNA, from cDNA, from genomic libraries, and cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, in cloning nucleic acids sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
Appropriate primers and probes for identifying At4g24160 and homolog sequences from plant tissues are generated from comparisons of the sequences provided herein. For a general overview of PCR, see, Innis, et al., eds., 1990.
As noted above, the nucleic acids used in the context of the present invention are characterized by the presence of sequence encoding an AT4G24160 homolog polypeptide. Primers that specifically amplify At4g24160 coding regions of the exemplified genes may be useful for identification of particular AT4G24160 homologs from different crop species. Primers suitable for this purpose are designed based on the sequence of At4g24160 homolog genes disclosed herein (e.g. SEQ ID NOs:18-36), using well known methods.
In certain embodiments of the invention, down-regulation of the activity of a polypeptide encoded by a gene comprising any of SEQ ID NO:18-36 may be accomplished using antisense-mediated-, or dsRNA-mediated-, or other forms of RNA-mediated-interference (RNAi), as is well known in the art. Methods for identification of candidate nucleotide sequences for RNA-mediated gene suppression, and design of oligonucleotides and constructs to achieve RNA-mediated gene suppression, are well known (e.g. Reynolds et al., 2004; Lu and Mathews, 2008). DNA sequences of genes encoding At4g24160 homologs may also be mutagenized, such as by T-DNA insertion, transposon insertion, or homologous recombination, to create plants comprising down-regulated activity of a At4g24160 homolog.
PCR or other primers may be used under standard PCR conditions, preferably using nucleic acid sequences as identified in EST libraries or other GenBank accessions as a template. The PCR products generated by any of the reactions can then be used to identify nucleic acids useful in the context of the present invention by their ability to hybridize to known At4g24160 homolog genes found in GenBank and other databases, as well as their ability to mediate down-regulation of activity of a At4g24160 homolog such as those given in FIG. 1, FIG. 9, and SEQ ID NOs:18-36. Alternatively, primers that specifically hybridize to highly conserved regions in a At4g24160 gene or At4g24160 gene homolog can be used to amplify sequences from widely divergent plant species such as switchgrass, canola, soybean, and tobacco, for example.
Polynucleotides may be synthesized by well-known techniques, as described in the technical literature. See, e.g., Carruthers et al., 1982, and Adams et al., 1983. 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 a suitable primer sequence.
In a particular embodiment, the expression of a At4g24160 gene or gene homolog is reduced relative to the level of expression found in an otherwise isogenic wild-type plant. This reduction in expression may be partial or complete, relative to the expression found in cells of an otherwise isogenic wild-type plant. Reduction in activity or function of the polypeptide encoded by At4g24160, or homolog thereof, may be accomplished by methods well known in the art including through co-suppression, and RNAi-mediated approaches, including via dsRNA and siRNA, among others.
DsRNA or siRNA nucleotide sequences comprise double strands of polymerized ribonucleotide and may include modifications to either the phosphate-sugar backbone or the nucleoside. Modifications in RNA structure may be tailored to allow specific genetic inhibition. In one embodiment, the dsRNA molecules may be modified through an enzymatic process so that siRNA molecules may be generated. Alternatively, a construct may be engineered to express a nucleotide segment for use in an miRNA- or siRNA-mediated resistance approach. The siRNA can efficiently mediate the down-regulation effect for target genes. This enzymatic process may be accomplished by utilizing an RNAse III enzyme or a DICER enzyme of the RNAi pathway (Elbashir et al., 2001; Hamilton and Baulcombe, 1999).
Use of variants of At4g24160 sequences may be employed, for instance, by preparing mutant At4g24160 homolog alleles. In vitro mutagenesis and selection, site-directed mutagenesis, or other means can be employed to obtain mutations of naturally-occurring At4g24160 homolog sequences. Such mutagenesis may, for instance, comprise use of transposon or T-DNA insertions
Plant Transformation
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., 1988; and Sambrook et al., 1989. Methods of plant cell culture are well known in the art. 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 that will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.
Vectors used for plant transformation may include, for example, plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), plant artificial chromosomes (PACs), or any suitable cloning system. It is contemplated the utilization of cloning systems with large insert capacities will allow introduction of large DNA sequences comprising more than one selected gene. Introduction of such sequences may be facilitated by use of BACs or YACs, or even PACs. For example the use of BACs for Agrobacterium-mediated transformation was disclosed by Hamilton et al., 1999.
Particularly useful for transformation are expression cassettes that have been isolated from such vectors. DNA segments used for transforming plant cells will, of course, generally comprise the cDNA, gene or genes that one desires to introduce into and have expressed in the host cells. These DNA segments can further include structures such as promoter, enhancers, 3′ untranslated regions (such as polyadenylation sites), polylinkers, or even regulatory genes as desired. The DNA segment or gene chosen for cellular introduction may encode a protein that will be expressed in the resultant recombinant cells resulting in a screenable or selectable trait and/or will impart an improved phenotype to the resulting transgenic plant. However, this may not always be the case, and the present invention also encompasses transgenic plants incorporating non-expressed transgenes. Preferred components are described below.
A number of promoters that are active in plant cells have been described in the literature, and are preferred elements included in the context of the present invention. Such promoters would include but are not limited to those isolated from the following genes: nopaline synthase (NOS; Ebert et al., 1987) and octopine synthase (OCS): cauliflower mosaic virus (CaMV) 19S (Lawton et al. 1987) and 35S (Odell et al., 1985), as well as the enhanced CaMV 35S promoter (e35S; described by Kay et al., 1987); figwort mosaic virus (FMV) 35S; the small subunit of ribulose bisphosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide); napin (Kridl et al., 1991); Adh (Walker et al., 1987); sucrose synthase (Yang et al., 1990); tubulin; actin (Wang et al., 1992); cab (Sullivan et al., 1989); PEPCase (Hudspeth et al., 1989); 7S-alpha'-conglycinin (Beachy et al., 1985); R gene complex promoters (Chandler et al. 1989); tomato E8; patatin; ubiquitin; mannopine synthase (mas); soybean seed protein glycinin (Gly); soybean vegetative storage protein (vsp); waxy; Brittle; Shrunken 2; Branching enzymes I and II; starch synthases; debranching enzymes; oleosins; glutelins; globulin 1; BETL1; and Arabidopsis banyuls promoter. The rice actin 1 promoter, the AGL11 promoter, the BETL1 promoter, and the e35S promoter may find use in the practice of the present invention. All of these promoters have been used to create various types of DNA constructs that have been expressed in plants (see, for example, Rogers et al., WO 84/02913).
Promoter hybrids can also be constructed to enhance transcriptional activity (Hoffman, U.S. Pat. No. 5,106,739, herein incorporated by reference), or to combine desired transcriptional activity, inducibility, and tissue or developmental specificity. Promoters that function in plants include but are not limited to promoters that are classified as, among others, inducible, viral, synthetic, constitutive, tissue-specific, developmentally-regulated, chemically or environmentally inducible, or senescence-related, for instance as described (Odell et al., 1985). Promoters that are tissue specific, tissue-enhanced, or developmentally regulated are also known in the art and envisioned to have utility in the practice of this present invention. For instance, a tissue specific promoter, such as the ST-LS1 promoter (e.g. Stockhaus et al., 1989), that is functional in plant vegetative tissues such as leaves, stems, and/or roots, may be of use. Such a promoter may also be expressed to at least some degree in seed or embryo tissues. In certain embodiments, the promoter to be utilized may be expressed preferentially in green parts of a plant such as leaves or stems. A senescence-related promoter (e.g. from SAG12) may also be utilized.
The promoters used in the present invention may be modified to affect their control characteristic. Promoters can be derived by means of ligation with operator regions, random or controlled mutagenesis, or other means well known in the art. Furthermore the promoter regions can be altered to contain multiple enhancer sequences to assist in elevating gene expression. Examples of such enhancer sequences have been reported (Kay et al., 1987).
Where an enhancer is used in conjunction with a promoter for the expression of a selected protein, it is believed that it will be preferred to place the enhancer between the promoter and the start codon of the selected coding region. However, one could also use a different arrangement of the enhancer relative to other sequences and still realize the beneficial properties conferred by the enhancer. For example, the enhancer could be placed 5′ of the promoter region, within the promoter region, within the coding sequence, or 3′ of the coding region. The placement and choice of sequences used as enhancers is known to those of skill in the art in light of the present disclosure. Transformation constructs prepared in accordance with the current invention will typically include a 3′ untranslated region (3′ UTR), and typically contains a polyadenylation sequence. One type of 3′ UTR that may be used is a 3′ UTR from the nopaline synthase gene of Agrobacterium tumefaciens (NOS 3′-end; Bevan et al., 1983). Other 3′ UTR sequences can be used and are commonly known to those of skill in the art.
A number of selectable marker genes are known in the art and can be used in the present invention (Wilmink and Dons, 1993). By employing a selectable or screenable marker gene in addition to the gene of interest, one can provide or enhance the ability to identify transformants. Useful selectable marker genes for use in the present invention would include genes that confer resistance to compounds such as antibiotics like kanamycin and herbicides like glyphosate or dicamba. Other selectable markers known in the art may also be used and would fall within the scope of the present invention.
DNA constructs of the present invention may be introduced into the genome of the desired plant host by a variety of techniques that are well known in the art. 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 DNA particle bombardment.
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., 1984. Electroporation techniques are described in Fromm et al., 1985. Ballistic transformation techniques are described in Klein et al., 1987.
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 direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch, 1984; and Fraley, 1983.
After transformation by any of the above transformation techniques, the transformed plant cells or tissues may be grown in an appropriate medium to promote cell proliferation and regeneration. Plant regeneration from cultured protoplasts is described in Evans et al., 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21 73, CRC Press, Boca Raton, 1985. For gene gun transformation of wheat and maize, see, U.S. Pat. Nos. 6,153,812 and 6,160,208. See also, Christou, 1996. See, also, U.S. Pat. Nos. 5,416,011; 5,463,174; and 5,959,179 for Agrobacterium-mediated transformation of soy; U.S. Pat. Nos. 5,591,616 and 5,731,179 for Agrobacterium-mediated transformation of monocots such as maize; and U.S. Pat. No. 6,037,527 for Agrobacterium-mediated transformation of cotton. Other Rhizobiaceae may be used for plant cell transformation as well (e.g. Broothaerts et al., 2007).
Methods for Identifying and Breeding Plant Germplasm
In still yet another aspect, the invention provides a method of obtaining crop plant germplasm, comprising the steps of: a) identifying at least a first polymorphism in a crop plant genomic region conferring reduced function or expression of a plant At4g24160 homolog; b) assaying other crop plants of the same order, family, genus, or species for the presence of the polymorphism; and c) selecting at least a first crop plant comprising the polymorphism. In certain embodiments the polymorphism results in a loss of function phenotype. In other embodiments, the polymorphism may comprise a full or partial deletion or a point mutation in the gene sequence of the At4g24160 homolog. Detecting the polymorphism may be carried out by any method, for instance PCR, single strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, cleavage fragment length polymorphism analysis and/or DNA sequencing. Since the cgi58 mutant phenotype of interest is a loss-of-function mutation, many of the commonly used techniques for screening mutants (e.g., TILLING; McCallum et al., 2000) in commercial crop species may be adapted to identify cgi58 mutant alleles.
In still yet another aspect, the invention provides a method of plant breeding comprising the steps of: a) identifying in a crop plant a genomic region conferring reduced function or expression of a plant At4g24160-homolog allele; b) selecting at least a first crop plant comprising the genomic region; and c) crossing the first crop plant to a second crop plant to produce progeny plants comprising the genomic region. In certain embodiments the genomic region comprises a sequence polymorphism relative to the nucleotide sequence of the corresponding genomic region of a wild-type plant. The polymorphism may comprise, for instance, an insertion, a deletion, or a single nucleotide polymorphism (SNP) at the locus encoding At4g24160, or homolog thereof. Identifying the polymorphism may be carried out by any method, including PCR, single strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, cleavage fragment length polymorphism analysis and/or DNA sequencing, among others. The method may further comprise the step of: d) selecting a progeny plant comprising the polymorphism and crossing the progeny plant with a third crop plant to produce additional progeny plants. In the method the second and third plants may be of the same variety. In certain embodiments, the method further comprises repeating step d) about 2-10 times.
Uses For Vegetative Plant Tissues With Enhanced Lipid content
Common uses for oils comprising neutral lipids include the preparation of food for human consumption, feed for non-human animal consumption and industrial uses such as for preparation of biofuels. As used herein, “industrial use” or “industrial usage” refers to non-food and non-feed uses for products prepared from plant parts prepared according to the present invention. As used herein, “biofuel” refers to a fuel combusted to provide power, heat, or energy, e.g. for an internal combustion engine, comprising at least 1%, 5%, 10%, 20% or more, by weight, of an oil, or product thereof, produced from a plant of the present invention, or by a method of the present invention. Also included in this invention are plants, plant cell cultures, and plant parts thereof, including seeds, containing one or more various transgene construct(s) or mutagenized loci utilized for decreasing expression of At4g24160 or a homolog thereof, oil obtained from the vegetative tissues of such plants and cells and progeny thereof, animal feed derived from the processing of such tissues, the use of the foregoing oil in food, animal feed, biofuels, cooking oil or industrial applications, and products made from the hydrogenation, fractionation, interesterification or hydrolysis of such oil.
The following examples are included to demonstrate specific embodiments of the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the present invention, and thus can be considered to constitute exemplary modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present invention.
EXAMPLES
Example 1
Oil bodies Accumulate in Tissues of Arabidopsis CGI58 Gene Knockout Lines
Two Arabidopsis lines with T-DNA disruptions in the first exon or first intron of the At4g24160 locus (SALK—127083 and SALK—0136871; Arabidopsis Biological Resource Center, Ohio State University, Columbus, Ohio) were obtained and characterized. There appeared to be no obvious growth differences between the mutant and wildtype Arabidopsis plants. The corresponding non-segregating homozygous mutant lines and the location and orientation of the T-DNA insert in the 0136871 were verified by DNA sequencing. The T-DNA insertion found in SALK—0136871 is annotated in the first exon of the At4g24160 locus, while the insertion found in SALK—127083 is found near the first exon of the At4g24160 locus. As annotated by the Arabidopsis information site (www.arabidopsis.org) it is located in the first intron.
The Arabidopsis cgi58-homolog T-DNA mutants were stained with either Nile red or BODIPY 493 (e.g. Invitrogen, or Molecular Probes, Inc., Eugene, OR), two selective neutral lipid stains, and found to show neutral lipid particles. This lipid droplet phenotype was found in petioles, roots and leaves of Arabidopsis plants imaged at 14-21 days after germination, and in leaves of 28-35 day old plants (e.g. see FIGS. 7-8). Representative images of petioles from leaves from 14-day old wild-type [Col 0] and both T-DNA mutant lines are shown (FIG. 3 left). To further support the identity of these stained particles as lipid bodies, subcellular fractions were isolated from homogenates of 14-day old wild-type seedlings and from seedlings of both T-DNA lines (by flotation 3× through sucrose medium according to Chapman and Trelease, 1991). Isolated lipid bodies were stained with BODIPY 493 (and Nile red, not shown), and were substantially more numerous in purified fractions from T-DNA mutants than from corresponding wild-type (Col 0; FIG. 3 lower right). In addition to subcellular fractionation, an increase in neutral lipid content was confirmed in both T-DNA mutants, most of which comprised TAGs (FIG. 3 upper right). Taken together, these results indicated that a disruption in the At4g24160 locus, encoding a putative homolog of human CGI58, lead to an accumulation of TAG in vegetative tissues of plants, and that this TAG is compartmentalized into lipid droplets in the cytoplasm of most all vegetative cells. Because only a small number of lipid bodies are normally found in vegetative tissues, this is evidently due to an elaboration of an otherwise normal cellular process of transient neutral lipid storage and recycling.
Example 2
Arabidopsis CGI58 is Alternatively Spliced
The At24160 gene gives rise to two alternative transcripts (FIG. 1A): one transcript, At4g24160.1, (SEQ ID NO:6; see GenBank Accession NM—118548) predicted to encode the full-length protein (SEQ ID NO:7), while the other transcript, At4g24160.2 (SEQ ID NO:37), encodes a shorter protein (SEQ ID NO:38) that lacks the last two exons but possesses a longer 3′ untranslated region (UTR). RT-PCR confirms the occurrence of both transcripts in Arabidopsis tissues (and lack thereof in mutants) (FIG. 1) and both cDNAs have been isolated (FIG. 1). The nucleotide sequences of the splice variants were confirmed by DNA sequencing.
Expression of At4g24160 isoforms in different tissues and different developmental stages was followed, to further characterize the temporal profile and location of neutral lipid accumulation in vegetative tissues of cgi58 mutant plants. Transcript abundance was estimated by RT-PCR using a one-Step RT-PCR system from Takara Bio (Shiga, Japan). The following transcript-specific primers were used for At4G24160:








5′-ATGAACTTGAGCCGTTTTGCTTCGAGA-3′;
(SEQ ID NO: 39)


 


5′-AACCAATCGTAGACCATCTAGGAG-3′;
(SEQ ID NO: 40)


and


 


5′-GCAATGTTTTTGGTGGACATACCT-3′.
(SEQ ID NO: 41)






Both long (R1) and short (R2) transcripts were amplified with the same forward primer (SEQ ID NO:39) but different reverse primers (SEQ ID NOs:40-41, respectively). RT-PCR reactions were performed with 0.2 μg total RNA and the following RT-PCR conditions: 42° C. for 15 min, followed by 35 cycles of 95° C. for 2 min, 94° C. for 10 s, 56° C. for 25 s, 72° C. for 1 min 30 s. Amplification of ubiquitin transcripts was used as a control for comparisons. Amplimers were separated by agarose gel (1%) electrophoresis and visualized by ethidium bromide staining.
Thus, cDNAs corresponding to the two alternatively spliced At4g24160 (At4g24160.1 and At4g24160.2) transcripts (see FIG. 1) were amplified from seedling mRNA and used to verify sequences predicted in FIG. 1. Analysis of mRNA abundance using semi-quantitative RT-PCR revealed that the longer splice variant (1,340 bp; see FIG. 13, lanes labeled “L” in each sample) was expressed in both leaves and roots of wild-type (WT) plants cultivated under a variety of conditions. The shorter splice variant (1,121 bp; FIG. 13 lanes labeled “S”), however, was only detected at very low levels in leaves of 40 day old plants, and seedlings grown on liquid or solid media. Notably, no transcripts for either the longer or shorter form of At4g24160 were detected in the T-DNA mutant plants (M). The level of ubiquitin transcripts expressed in the same wild-type and mutant plants (lanes labeled “U”) are shown as a control.
Using semi-quantitative RT-PCR, it was found that the longer (full-length) transcript was expressed in all wild-type tissues examined, whereas the shorter, truncated transcript was expressed, albeit at low levels, only in leaves of wild-type mature plants and in seedlings cultured in liquid or solid media (FIG. 13). The longer (full-length) transcript was also the major form detected in vegetative tissues of soil-grown, wild-type plants (FIG. 13). Neither transcript was detected in any tissues from mutants, confirming the lack of CGI-58 expression in these plants. Based on these results, it is likely that the larger CGI-58 protein product is responsible for regulating neutral lipid accumulation in vegetative tissues. The physiological relevance of the alternatively-spliced smaller CGI-58 protein, which lacks the acyltransferase domain, is presently unclear.
Example 3
The Arabidopsis At4g24160 Protein Products are CGI58 Homologs
An alignment of CGI58 proteins from various species indicates the presence of several conserved domains as well as specific amino acid motifs that are known to be important for certain enzyme activities. For instance, Domain 2, which starts at about position 178 in FIG. 1, contains a GXSXG (SEQ ID NO:1) motif (boxed in FIG. 1) where X is any amino acid) that is associated with esterase/lipase/thioesterase function (Schrag and Cygler, 1997), and in mammalian CGI58 the critical serine residue in the middle of this motif has been substituted with an asparagine (Lefèvre et al., 2001; see FIG. 1). While this substitution is known to disrupt the esterase/lipase function, the plant homologs do have a serine at this position, suggesting that the plant (and C. elegans) proteins might have lipase activities themselves (rather than stimulate a lipase, like the mammalian proteins). A second motif (HX4D) is present towards the C-terminal end of the proteins (also boxed in FIG. 1), and this motif has been implicated in acyltransferase activity (Heath and Rock, 1998). Whether this motif is important for the LPAAT activity of mammalian (or Arabidopsis) CGI58 polypeptides, and homologs thereof, is unknown, However, this motif is absent in the short form of the Arabidopsis protein (FIG. 2A; due to alternative splicing).
Example 4
Enhancement of Oil Content in Vegetative Tissues of Plants
Lipid bodies may be found in plant tissues other than seeds (e.g. see wild-type stems in FIG. 3 left). However, the elaboration of this compartment to the level seen in the cgi58 T-DNA knock out lines (FIG. 3) represents an unexpected result, and allows for use of non-seed, food or non-food vegetative biomass for plant oil production. Rough estimates of TAG content in the cgi58 T-DNA mutants suggests that the level of TAGs has increased by at least two to three times that of corresponding wild-type (FIG. 3 upper right). TAGs were identified and analyzed by nanospray ionization and mass spectrometry (see FIGS. 4-6).
Example 5
Further Enhancement of Oil Content in Plants Displaying a cgi58 Mutant Phenotype
Factors that might be combined with At4g24160 homolog loss-of-function mutations, or in other plants wherein At4g24160 is down-regulated, to increase the amount of oil accumulated in vegetative tissues such as leaf tissues may include the following: 1) ectopic expression of embryo-associated transcription factors that have been shown to result in oil accumulation in leaves (e.g, WR11 or LEC; Cernac and Benning, 2003); 2) chemical or natural induction of senescence to stimulate lipid turnover (Guo and Gan, 2005) and boost accumulation of neutral lipid droplets in At4g24160-homolog mutant plants prior to harvest; 3) ectopic expression of oleosin proteins (Murphy, 1993; Capuano et al., 2003; Abell et al, 2004) to stabilize lipid droplets and prevent their turnover; or 4) combination of At4g24160 down-regulation with alterations in endoplasmic reticulum (ER) machinery to stimulate the abnormal accumulation of lipid droplets (Chapman et al., 2008).
Example 6
Identification of At4g24160 Homologs
Plant homologs of the Arabidopsis At4g24160 locus were initially found via in silico analysis of genomic and cDNA (e.g. EST) databases. Searches were conducted with the sequence of At4g24160 (SEQ ID NO:6 or SEQ ID NO:8), and multiple putative homologs were identified. FIG. 9 provides a nucleotide alignment of sequences encoding some homologs, sequences of which are given in SEQ ID NOs:18-36.
Example 7
Triacylglycerol Composition of Neutral Lipids Produced in Vegetative Tissues
The neutral lipid fractions from combined leaf tissues of mature plants (about 45 day old) were dissolved in 1:1 (v/v) chloroform:methanol with 10 mM ammonium acetate. TAG molecular species were identified by neutral loss fragmentation spectra in tandem. Typical scanning conditions were carried out in positive ion mode with a 4-4.5 kV spray voltage, scan range of 650 m/z to 950 m/z, 275° C. capillary temperature, 3 microscans per full scan with a maximum injection time of 200 msec. Tandem scans (MS/MS) were performed with an isolation width of 5.0 m/z, 35% normalized collision energy, and 30 msec activation time with a scan range from 200 m/z to 1000 m/z. Tripentadecanoyl glycerol (tri 15:0) was added at the time of extraction and used as a quantitative standard. All solvents were optima grade from Thermo-Fisher Scientific.
Previously, ectopic over-expression of seed transcription factors was shown to increase TAG content in Arabidopsis seedling tissues, and this appeared to be caused by an up-regulation of a seed-specific program (e.g. Cernac & Benning, 2004) because the TAG profiles were more similar to those found in seeds than in leaves. For instance, the TAGs in certain overexpression mutants were rich in 20:1/eicosaenoic fatty acid typically found in Arabidopsis seed oil bodies (Graham, 2008). In contrast, however, analysis of TAGs in above-ground vegetative tissues of cgi58 mutant plants by electrospray ionization and tandem mass (MS) unexpectedly showed that their molecular composition was similar to that found in wild-type leaves (see FIG. 10). That is, these TAGs were composed of typical leaf tissue fatty acids such as 16:3 and 18:3/hexadecatrienoic and octadecatrienoic fatty acids and did not contain 20:1 fatty acid. Moreover, the TAG profiles in the cgi58 mutants were reminiscent of the TAG composition generated in leaf tissues of a Arabidopsis beta-oxidation mutant (e.g. Yang & Ohlrogge, 2009). Thus, also in combination with genes encoding other fatty acid synthesis-related genes which are known in the art, the fatty acid composition (i.e. “profile”), as well as the level of neutral lipids, may be controlled in vegetative tissues of plants displaying a decreased level of At4g24160 function, to yield particular useful lipid species and profiles, including ones that are not otherwise found, or found at such levels, in seed oils.
Example 8
Quantification of Lipids in Vegetative Tissues
In addition to the studies described for instance in Example 4, additional imaging with a second neutral-lipid-specific stain (such as 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY®493); available from Molecular Probes-Invitrogen; Eugene, Oreg.), which has improved and more selective spectral characteristics (Tavian & Colombo, 2007) confirmed that there are more lipid droplets in mesophyll cells of mutant leaves compared to wild-type leaves (FIGS. 11A and B). Imaging both chloroplasts (red chlorophyll autofluorescence) and lipid droplets (green BODIPY 493 fluorescence, arrows) together in the mutants also showed that lipid droplets were extraplastidial and that there appeared to be a dramatic elaboration of lipid droplets in mutants compared to wild-type leaf cells (FIGS. 11A and B). Moreover, three-dimensional reconstructions of multiple Z-stacked confocal images of mutants specifically revealed that the lipid droplets accumulated in the cytosol and not inside chloroplasts (FIG. 11C), which is unlike the lipid-rich plastoglobuli that tend to accumulate in the chloroplast stroma of stressed or senescing tissues (Munne-Bosch, 2005). Morphometric analysis comparing wild-type and mutant mesophyll tissues revealed a  ̃50-fold increase in area occupied by lipid droplets in mutant cells (quantified as % area in the green channel) compared to wild-type cells (3.71%±1.17 vs. 0.072%±0.040; p<0.0001), whereas the areas occupied by chloroplasts in these same groups of cells was approximately equivalent (quantified as % area in the red channel) (FIG. 11D). The amount of TAG in mature Arabidopsis cgi58 mutant plants quantified by direct electrospray MS against a tripentadecanoyl glycerol standard was 50% higher than in wild-type plants (e.g. 699±74 mg/g dw; vs 466±98 mg/g dw; n=9, p<0.0001).
Example 9
Developmental Aspects of Cytosolic Lipid Droplet Accumulation
A difference in cytosolic lipid droplet abundance was seen, depending upon the developmental stage of the leaves. That is, there were significantly more lipid droplets in mature, fully expanded leaves of the mutants (e.g., 40-day old), than either in younger leaves (15 days after emergence) or older, senescing leaves (65 days after emergence) of the mutants. Representative confocal fluorescence images of Arabidopsis leaves emerged at 15 d, 40 d, or 65 d (WT and T-DNA mutant, Salk—136871) are shown in FIG. 12. Lipid droplets are green and chloroplasts are red; imaging acquisition conditions were the same as for FIG. 11. The percent area of lipid droplets and chloroplasts (graphs in lower panel) were quantified as the percent of image area fluorescing from either the green or red channels, respectively. Averages and standard deviations are plotted from ten digital images of 25,000 microns each over several leaves at each stage. There were significant differences between the amounts of lipid droplets in mutants and wild-type leaves at 40 days (p<0.0001) and at 65 days (p<0.002), but not at 15 days (p>0.63). Abundant droplets were most evident in mature, 40 day-old leaves of mutants. No statistical differences were observed in the relative percentage of chloroplasts between mutant and wild-type leaves, suggesting the mutation affected lipid accumulation in cytosolic droplets, not lipid biosynthesis which primarily occurs in chloroplasts.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of the foregoing illustrative embodiments, it will be apparent to those of skill in the art that variations, changes, modifications, and alterations may be applied to the composition, methods, and in the steps or in the sequence of steps of the methods described herein, without departing from the true concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.
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## Claims

1. An Arabidopsis or Brassica plant comprising increased lipid accumulation in vegetative tissues relative to a wild type plant of the same species, wherein activity of an At4g24160 gene product or homolog thereof is down-regulated in the plant, wherein said down-regulation comprises:
expressing in the plant a RNA molecule complementary to all or a portion of an mRNA expressed from a gene comprising a sequence selected from the group consisting of SEQ ID NOs: 6, and 37, wherein the RNA molecule inhibits the function of the At4g24160 gene product or homolog thereof in said plant.

2. The plant of claim 1, wherein the plant is a transgenic plant.

3. The plant of claim 2, further defined as a fertile R0 transgenic plant.

4. The plant of claim 2, further defined as a progeny plant of any generation of a fertile R0 transgenic plant.

5. A seed of the plant of claim 1, wherein the seed is of a plant that comprises increased lipid accumulation in vegetative tissues relative to a wild type plant of the same species and wherein an At4g24160 gene product is down-regulated in the plant, wherein said seed comprises said RNA molecule.

6. A plant part of the plant of claim 1, wherein said plant part comprises said RNA molecule.

7. The plant part of claim 6, selected from the group consisting of a cell, a leaf, stem, a petiole, pollen, a tuber, and root tissue.

8. A method of altering lipid content in an Arabidopsis or Brassica plant or plant cell comprising down-regulating the function of an At4g24160 gene product or homolog thereof, wherein said down-regulating comprises expressing in the plant a RNA molecule complementary to all or a portion of an mRNA expressed from a gene comprising a sequence selected from the group consisting of SEQ ID NOs: 6, and 37, wherein the RNA molecule inhibits the function of an At4g24160 gene product or homolog thereof in said plant or plant cell.

9. The method of claim 8, wherein the RNA molecule is a single stranded RNA molecule.

10. The method of claim 8, wherein the RNA molecule is a double stranded RNA molecule.

11. A method of altering lipid content in an Arabidopsis or Brassica plant or plant cell comprising down-regulating the function of an At4g24160 gene product or homolog thereof by co-suppression, wherein said down-regulating comprises expressing in the plant a cDNA molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 6, and 37, wherein the cDNA molecule inhibits the function of an At4g24160 gene product or homolog thereof in said plant or plant cell.

12. A method of producing lipids in a plant comprising:
(a) obtaining the Arabidopsis or Brassica plant according to claim 1; and
(b) isolating lipid from said plant.

13. A method of producing food, feed, or oil comprising:
(a) obtaining the plant or progeny thereof expressing said RNA molecule according to claim 1;
(b) cultivating said plant to obtain a plant product; and (c) preparing food, feed, or oil from said plant or plant product.

14. The method of claim 13, wherein the oil comprises triacylglycerol (TAGI.

15. The method of claim 13, wherein the neutral lipids isolated from vegetative tissues of the plant comprise a rationally designed fatty acid profile.

16. The method of claim 15, wherein the neutral lipids comprise hydroxyl, epoxy, cyclic, acetylenic, saturated, polyunsaturated, short-chain fatty acids, long-chain fatty acids, TAGs, wax-esters, or steryl-esters.

17. The method of claim 13, wherein the neutral lipids isolated from vegetative tissues of the plant comprise a leaf-specific fatty acid profile.

18. The method of claim 17, wherein the neutral lipids isolated from vegetative tissues of the plant comprise hexadecatrienoic and octadecatetraenoic fatty acids, or do not contain eicosaenoic fatty acid