# Patent

## Title
Plants having altered expression and activity of yield-related proteins

## Abstract

Transgenic plants that have enhanced yield-related traits, such as increased seed oil production, are produced by genetically engineering the plants to down-regulate the expression of at least one BPM protein. Such transgenic plants can, for example, be cultivated and yield higher seed oil production than control plants which have not been genetically engineered for down regulation of a BPM protein.

## Background
This invention was made with government support under MCB1020673 awarded by the National Science Foundation. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Field of the Invention
Embodiments of the invention generally relate to enhanced yield-related traits in plants. In particular, the invention provides plants that have been genetically engineered to down-regulate expression or reduce the activity of BPM proteins, resulting in enhanced yield-related traits including without limitation enhanced seed oil production, as well as products made by or from the plants.
Background of the Invention
Under field conditions, plant performance, for example in terms of growth, development, biomass accumulation and seed generation, depends on a plant's tolerance and acclimation ability to numerous environmental conditions, changes and stresses. There has always been a need for improving plant traits in crop cultivation. Breeding strategies foster crop properties to withstand biotic and abiotic stresses, to improve nutrient use efficiency and to alter other intrinsic crop specific yield parameters.
Plants are sessile organisms and consequently need to cope with various environmental stresses. Biotic stresses such as plant pests and pathogens on the one hand, and abiotic environmental stresses on the other hand are major limiting factors for plant growth and productivity (Boyer, 1982; Bohnert et al., 1995), thereby limiting plant cultivation and geographical distribution. Plants exposed to different stresses typically have low yields of plant material, like seeds, fruit or other produces. Crop losses and crop yield losses caused by abiotic and biotic stresses represent a significant economic and political factor and contribute to food shortages, particularly in many underdeveloped countries.
Conventional means for crop and horticultural improvements today utilize selective breeding techniques to identify plants with desirable characteristics. Advances in molecular biology have allowed for the production of transgenic plants with enhanced yield-related traits. Various yield-related traits in plants are important to many industries worldwide. In particular, plant seed oils are an important source of calories for human nutrition, as feedstocks for non-food uses such as soaps and polymers, and can serve as a high-energy biofuel. World production from oilseed crops in 2011 reached a value near US$120 billion with plant oil consumption expected to double by 2040 (Bates et al., 2013). As a result, methods for increasing seed oil biosynthesis have been an important research topic. However, previous attempts to modulate the transcription levels of factors critical for seed oil biosynthesis, such as WRINKLED1 (WRI1), resulted in relatively low increases in seed oil content (Liu et al., 2010; Shen et al., 2010; Pouvreau et al., 2011).
Effective regulatory mechanisms to time and control developmental and physiological processes in response to environmental cues are of utmost importance to plants due to their sessile life style. A mechanism that allows plants to quickly and flexibly respond is the ubiquitin (UBQ) proteasome pathway (Hua and Vierstra, 2011). It is highly conserved among eukaryotes and requires the concerted activities of an E1 UBQ activating enzyme, a UBQ conjugating enzyme E2, and an E3 UBQ ligase. While E1 and E2 activate the UBQ to modify target substrates, the E3 ligase binds the E2 and a substrate protein to facilitate transfer of the UBQ moiety. Upon building up a UBQ chain on the substrate, the ubiquitylated protein is marked for degradation via the 26S proteasome (Hua and Vierstra, 2011).
CUL3-based RING E3 ligases (CRL3) have been described only recently and mainly with respect to their basic architecture (Figueroa et al., 2005; Gingerich et al., 2005; Weber et al., 2005; Gingerich et al., 2007). They are composed of a cullin 3 protein, as the scaffolding subunit, that binds in its C-terminal region the RING-finger protein RBX1, while its N-terminal part is recognized by proteins containing a BTB/POZ (Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) fold (Figueroa et al., 2005; Weber et al., 2005). BTB/POZ proteins comprise a diverse group of proteins within Arabidopsis and rice, containing 80 and 149 members, respectively (Gingerich et al., 2007). They have been divided into 12 subgroups based on their secondary domains (Gingerich et al., 2007). While the BTB/POZ fold is required for assembly with the cullin and to interact with other BTB/POZ proteins, the secondary domain may function as an adaptor to allow binding of a substrate and delivery to the CRL3 core for ubiquitylation.
Based on its role as the central scaffolding subunit that assembles with potentially many BTB/POZ proteins, it is not surprising that the loss of CUL3 causes an embryo lethal phenotype. Reduced amounts of functional cullin 3 protein affects red light and ethylene signaling and impacts plant development (Dieterle et al., 2005; Thomann et al., 2009).
One BTB/POZ subfamily is the BPM (BTB/POZ-MATH) family that contains a BTB/POZ fold in their C-terminal region, and a MATH (Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog) domain located within the first 200 amino acids of their N-terminal region. BPM proteins are known in the art and may also be referred to as MATH-BTB/POZ proteins. The family comprises six members in Arabidopsis, all of which have molecular weights between 40-50 kDa (Weber et al., 2005). A recent study of Brassica rapa provided a phylogenetic analysis of select BPM proteins, but there has yet to be any functional characterization of these genes in the Brassica species (Zhao et al., 2013). In Zea mays, it was found that the loss of a BPM protein resulted in defects in female gametophyte development (Juranić et al., 2012). However, there has been no study linking the downregulation of BPM proteins to enhanced yield-related traits.
SUMMARY OF THE INVENTION
Based on the surprising finding that BPM proteins assemble widely with ERF/AP2 transcription factors, and as demonstrated with a selected member of this family, WRI1, that the interaction is a requirement to destabilize WRI1 in plants, embodiments of the invention provide genetically engineered plants having increased yield-related traits, in particular and without limitation, seed oil production as compared to non-transgenic plants or other control plants which have not been genetically engineered as described herein, wherein at least one BPM protein is down-regulated or its activity reduced, and methods of making the same. Aspects of the invention also relate to methods of producing and recovering seed oil in plants. Although increased expression of polypeptides containing BTB/POZ domains has been implicated in modifying plant yield-related traits (U.S. patent application Ser. No. 13/818,858), no study has suggested or shown that down-regulating the expression of such polypeptides comprising a MATH domain can enhance yield-related traits.
An embodiment the invention provides transgenic plants, wherein the plants are genetically engineered so as to down-regulate expression or reduce the activity of at least one BPM (BTB/POZ-MATH) protein as compared to a control plant such as a non-transgenic plant or a plant in which the expression or activity of a BPM protein has not been reduced through the genetic engineering described herein. In preferred embodiments, the transgenic plant is of the Brassicaceae family, in particular, Arabidopsis thaliana. In some embodiments, the transgenic plant exhibits enhanced yield-related traits as compared to a control plant. In exemplary embodiments, the transgenic plant of the claimed invention exhibits increased seed oil production as compared to a control plant.
Another aspect of the invention provides a method for recovering seed oil from a transgenic plant comprising cultivating said transgenic plant under conditions promoting plant growth and development, wherein said transgenic plant is genetically engineered to down-regulate expression or reduce the activity of at least one BPM protein as compared to a control plant; and recovering seed oil from the transgenic plant. In preferred embodiments, the transgenic plant is of the Brassicaceae family, in particular, Arabidopsis thaliana. In some embodiments, said step of genetically engineering comprises introducing artificial microRNA (amiRNA) to down-regulate said BPM protein. In still other embodiments, said step of genetically engineering comprises the expression of an exogenous MATH domain to compete with BPM protein.
Additional aspects of the invention provide a method for enhancing yield-related traits in a plant comprising genetically engineering the plant so as to down-regulate expression or reduce the activity of at least one BPM protein as compared to a control plant. In some embodiments, the method of the invention results in increased seed oil production in the transgenic plant as compared to a control plant. In preferred embodiments, the transgenic plant is of the Brassicaceae family, in particular, Arabidopsis thaliana. In some embodiments, the step of genetically engineering comprises introducing artificial microRNA (amiRNA) to down-regulate the BPM protein. In still other embodiments, the step of genetically engineering comprises the expression of an exogenous MATH domain to compete with BPM protein.
Further embodiments of the invention relate to a product produced by or from a transgenic plant which is genetically engineered to down-regulate expression or reduce the activity of at least one BPM protein as compared to a control plant.
Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-E: Verification of α-CUL3 and α-WRI1 antibodies. (A) Upper sequence: partial alignment of Arabidopsis CUL3a and CUL3b sequences (SEQ ID NO:65 and SEQ ID NO:66, respectively). Lower sequence: Arabidopsis WRI1 (SEQ ID NO:67). The peptide sequences used for antibody generation are highlighted with arrows. (B) Western blot analysis in WT, cul3a, cul3b and cul3hyp backgrounds with α-CUL3. (C) The specificity of α-CUL3 was confirmed by Western blot analysis in transient expression assays with a GFP:CUL3a construct in tobacco. (D) PCR identification of homozygous T-DNA mutant wri1-3. A WRI1 gene-specific product was amplified in WT but not in wri1-3, while one T-DNA specific product was amplified in wri1-3 but not in WT. (E) WRI1 is detectable in WT but not in wri1-3.
FIG. 2A-H: Interaction studies of WRI1 with BPM and CUL3 proteins. (A) BPM1 interacts with ERF4, RAV1 and DREB1a, but only poorly with ERF1 in Y2H assays. SDII, medium for transformation selection; SDIV, medium for test of interaction. Pictures of single spots were taken seven days after transformation. (B) WRI1 can assemble with itself in Y2H assay, as well as with representative members of the BPM family, BPM1, BPM3, BPM4, and BPM5. (C) In vitro translated and [355]-methionine labeled BPM1 protein was used in pull-down assay with E. coli expressed and GST and GST:WRI1. (D) Pulldown experiments with in E. coli expressed GST: WRI1 results in the precipitation of WRI1 and CUL3, while GST alone was ineffective. Asterisks indicate GST:WRI1 band, while the band below is plant WRI1. Pulldowns were first tested with α-WRI1, and then with α-CUL3 after the membrane had been stripped. (E) Silver-stained SDS-PAGE gel to illustrate the IPTG-induced expression of purified GST and GST: WRI1 proteins from E. coli. (F) Pulldown experiments with purified proteins show that His:WRI1 can precipitate GST:CUL3a if GST:BPM1 is present in the assay (left blot), while this is not the case when GST alone is used (right blot). Blots were probed first with a α-GST antibody, before stripped and subsequently probed with α-CUL3 and α-WRI1. PD, pulldown. (G) IP experiments with α-WRI1 antibody shows co-precipitation of CUL3 with WRI1 from Arabidopsis wild type (WT) protein extract. If not otherwise stated in this and subsequent figures 30 μg of total protein extract were loaded as input, and experiments were done with 14-days old seedlings. (H) WRI1 is present in the plant extract used for the IP shown in (G).
FIG. 3A-B: FPLC analysis and subcellular localization of WRI1. (A) Western blot analysis of FPLC fractions from two-week-old Arabidopsis WT plantlets tested with either α-WRI1 or α-CUL3, showed co-migration of the two proteins in fractions between 70 and 150 kDa. WRI1 is also present in fractions corresponding to proteins of around 40-50 kDa, and in fractions higher than 150 kDa indicating that the protein assembles also in complexes distinct from CUL3. (B) Transient expression analysis of GFP:WRI1 in tobacco shows that the fusion protein is located in the nucleus (green fluorescence derives from GFP; blue fluorescence from DAPI staining).
FIG. 4A-C: CHX treatment does not reduce the protein level of WRI1 but it induces its transcription level. (A) WRI1 protein levels do not decline 6 h after CHX treatment (both with 50 μM or 100 μM). (B) CHX but not MG132 treatment up-regulates WRI1 expression (p<0.01). (C) IAA5 (auxin-responsive protein IAA5) was used as positive control for CHX treatment. The expression of IAA5 was strongly induced by CHX treatment (p<0.01). In all cases two-week-old seedlings were used. Error bars illustrate standard error.
FIG. 5A-D: Stability and expression level of WRI1 in WT and cul3hyp. (A) Treatment of WT plants with ActD2 (6 h) and CHX (3 h) inhibitors shows instability of WRI1. This is blocked by co-treatment with MG132 (6 h). (B) WRI1 protein accumulates in cul3hyp double mutants in comparison to WT. (C) Expression of WRI1 is comparable to WT in cul3hyp plants. (D) WRI1 protein is more stable in cul3hyp than in WT. Error bars in this and all subsequent figures represent standard deviation.
FIG. 6: Predicted target sites for artificial microRNA (squares) on the different BPM genes. Numbers indicate base pairs.
FIG. 7A-D: Generation of 6×amiBPM and MATH-overexpressing lines and their impact on WRI1 protein levels. (A, B) qRT-PCR analysis show significantly reduced expression levels for six BPMs is in two representative 6×amiBPM lines compared to WT. (C) Expression levels of BPM1MATH show significant increases in two BPM1MATH lines, and two BPM1MATH:NLS lines compared to WT. (D) WRI1 protein content is strongly reduced in MATH-overexpressing lines when compared to WT, while both 6×amiBPM lines display increased WRI1 levels. All asterisks in this and subsequent figures indicate the statistical significant difference of at least p<0.05 (One-way ANOVA) to WT.
FIG. 8A-C: Sub-cellular localization of GFP:BPM1MATH and GFP:BPM1MATH:NLS. (A) Transient expression analysis of GFP:BPM1MATH in tobacco demonstrates that the fusion protein is present in the cytosol and nucleus (bar=5 μm). (B) GFP:BPM1MATH:NLS fusion protein is strictly localized to the nucleus (bar=10 μm). Arrows indicate nuclei. (C) The expression level of WRI1 in WT and two individual lines of GFP:BPM1MATH, GFP:BPM1MATH:NLS.
FIG. 9A-G: Phenotype analysis of WT, two 6×amiBPM lines, and MATH-overexpressing lines. (A) Primary root lengths of 2-weeks old Arabidopsis seedlings is strongly reduced in all transgenic lines when compared to WT (n=45). (B) Specifically 6×amiBPM plants have reduced numbers of lateral roots development (2-weeks old seedlings; n=45). (C) Root phenotype of WT and two 6×amiBPM lines. Picture was taken 14-days post-germination. (D) Overview of rosette phenotypes from WT and transgenic lines. Picture was taken 33 days after germination. (E) All transgenic lines are late flowering. Data were taken for 33-day-old plants (n=30). (F) Rosette leaf number at time of flowering (n=10). (G) Rosette area of 25-day-old Arabidopsis plants for each genetic background (n=30).
FIG. 10: Rosette leaf phenotype on the time of primary inflorescence. 6×amiBPM and MATH-overexpressing plants develop less and shorter leaves then WT. Leaves of transgenic plants frequently developed wider blades (scale=1 cm).
FIG. 11A-G: Studies of WRI1's protein level, WRI1's transcriptional activity, and complex assembly at the DNA level. (A) WRI1 protein is stabilized in MATH overexpressing and 6×amiBPM lines. (B) CUL3 could be precipitated with α-WRI1 from WT plant extracts but not from BPM1MATH (left half) or 6×amiBPM (right half) extracts. Input was tested for presence of WRI1 and CUL3 proteins using the respective antibodies. (C) Protein levels of WRI1, and expression of two WRI1 target genes, AtGLB1 and BCCP1, in WT and the different transgenic backgrounds. (D-G) ChIP-qPCR analysis on WT, two 6×amiBPM lines, and wri1-3 shows enrichment of the two WRI1 target promoters proBCCP1 and proAtGLB1 in WT (D) but not in a wri1-3 mutant (G) after IP with either α-WRI1 or α-CUL3 antibodies. While no enrichment was detectable in samples derived from α-CUL3 ChIPs in two 6×amiBPM lines (E, F), significant enrichment was detectable in α-WRI1 ChIP samples when compared to WT (E, F). ChIP-qPCR experiments were repeated at least three times independently. Error bar indicates the value of standard error. Asterisk indicates a statistical significant difference (One-Way ANOVA, p<0.05) compared to individual control.
FIG. 12A-B: Stability assays of WRI1 in WT, 6×amiBPM, and MATH overexpressing lines. In comparison to WT, WRI1 protein is stabilized in both (A) 6×amiBPM and (B) MATH overexpressing plants.
FIG. 13A-D: Seed weight, size, and fatty acid content in WT, wri1-3, and 6×amiBPM plants. (A, B) In comparison to WT, seed weight and size is significantly reduced in wri1-3, while it is increased in both 6×amiBPM lines, and to a greater extend in 6×amiBPM#1 then it is in #2 (data in (A) represent average of n=5 measurements of 20 seeds. Scale bar in (B) represents 1 mm. (C) Seeds in 6×amiBPM lines contain higher WRI1 levels which correlate with expression of the WRI1 target genes BCCP1 and AtGLB1. (D) Differences in total fatty acid contents for WT, wri1-3, and the two 6×amiBPM lines correlated with seed weights and sizes. Data represent average of n=5 measurements on 30 seeds. The asterisk shows the statistical significant difference to WT (p<0.05, T-test).
FIG. 14: Fatty acid profile and metabolic profile in seeds of WT, wri1-3, and two 6×amiBPM lines. All extractions and measurements were done from mature and desiccated seeds from WT, wri1-3, and 6×amiBPM lines. In the graph for fatty acids, numbers indicate the type of fatty acid (carbon chain length and number of double bonds). All values are based on five independent samples. Error bar indicates the value of standard deviation.
FIG. 15A-E: Seed phenotype analyzes for 6×amiBPM #3. (A) WRI1 protein is elevated in the mutant line in comparison to WT, which (B,C) correlates with seed size and weight, and (D) increased expression of BCCP1 and AtGLB1. (E) Fatty acid levels are significantly increased in 6×amiBPM #3 in comparison to WT (p<0.05, T-test).
FIGS. 16A-C: Sequence alignment of full length BPM proteins from 27 different plant species (SEQ ID NOs. 1 to 27) and a consensus sequence (SEQ ID NO. 28). Amino acid residues with black background color are fully conserved, those with a dark gray background are highly conserved. Arrows at the top of the alignment indicate start and end of either the predicted MATH (black solid line) or BTB/POZ (black dotted line) domains in the Arabidopsis thaliana BPM3 protein. (A) shows the predicted MATH domain and surrounding sequence. (B) shows the predicted BTB/POZ domain and surrounding sequence. (C) shows the c-terminal sequence of the BPM proteins. For example, in Arabidopsis the predicted MATH domain comprises residues 29-141 and the BTB/POZ domain comprises residues 194-303. The following are the Latin names of the different species listed as well as the accession numbers (AccNo) of the corresponding BPM proteins: Arabidopsis: Arabidopsis thaliana; AccNo BAH19418 (SEQ ID NO:1); Polish Canola: Brassica rapa; AccNo XP_009141835 (SEQ ID NO:2). Barbados Nut: Jatropha curcas; AccNo KDP44889.1 (SEQ ID NO:3). California poplar: Populus trichocarpa; AccNo XP_002311186 (SEQ ID NO:4). Cacao tree: Theobroma cacao AccNo XP_007009287 (SEQ ID NO:5). Clementine: Citrus clementina; AccNo XP_006435614 (SEQ ID NO:6). Castor oil plant: Ricinus communis; AccNo XP_002524218 (SEQ ID NO:7). Eucalyptus: Eucalyptus grandis; AccNo KCW65573 (SEQ ID NO:8). Grape vine: Vitis vinifera; AccNo XP_002282536 (SEQ ID NO:9). Peach: Prunus persica; AccNo XP_007218050 (SEQ ID NO:10). String bean: Phaseolus vulgaris; AccNo XP_007163464 (SEQ ID NO:11). Soybean: Glycine max AccNo XP_003552772 (SEQ ID NO:12). Date palm: Phoenix dactylifera; AccNo XP_008785535 (SEQ ID NO:13). Strawberry: Fragaria vesca subsp. Vesca; AccNo XP_004307466 (SEQ ID NO:14). Apple: Malus domestica; AccNo XP_008372026 (SEQ ID NO:15). Tomato: Solanum lycopersicum; AccNo XP_004239913 (SEQ ID NO:16). Potato: Solanum tuberosum; AccNo XP_006355691 (SEQ ID NO:17). Oryza: Oryza brachyantha; AccNo XP_006657384 (SEQ ID NO:18). Brachypodium: Brachypodium distachyon; AccNo XP_003557713 (SEQ ID NO:19). Rice: Oryza sativa Japonica Group; AccNo NP 001058677 (SEQ ID NO:20). Barley: Hordeum vulgare subsp. vulgare; AccNo BAJ94248 (SEQ ID NO:21). Spikemoss: Selaginella moellendorffii; AccNo XP_002961582 (SEQ ID NO:22). Barrel Clover: Medicago truncatula; AccNo KEH23724 (SEQ ID NO:23). Robusta coffee: Coffea canephora; AccNo CDP03595 (SEQ ID NO:24). Corn: Zea mays; AccNo NP_001142069 (SEQ ID NO:25). Sorghum: Sorghum bicolor; AccNo XP_002461292 (SEQ ID NO:26). Muskmelon: Cucumis melo; AccNo XP_008458543 (SEQ ID NO:27). A consensus sequence is also shown (SEQ ID NO:28).
FIG. 17: Comparison of protein identities and similarities of BPM proteins from 27 different plant species. Arab: Arabidopsis. Cano: Polish Canola. Barb: Barbados Nut. Popl: California poplar. Caca: Cacao tree. Clem: Clementine. Cast: Castor oil plant. Euca: Eucalyptus. Vine: Grape vine. Peac: Peach. Stri: String bean. Soy: Soybean. Palm: Date palm. Stra: Strawberry. Appl: Apple. Toma: Tomato. Pota: Potato. Oryz: Oryza. Brac: Brachypodium. Rice: Rice. Barl: Barley. Moss: Spikemoss. Cloy: Barrel Clover. Coff: Robusta coffee. Corn: Corn. Sorg: Sorghum. Musk: Muskmelon. For specific latin names and accession numbers, see FIG. 16.
FIGS. 18A-B: Salt stress assay. (A) For salt stress tolerance assays, wild type (WT) and bpm mutants (6×amiBPM and BPMMATH:NLS) were plated on solid minimal culture medium, and grown vertically for five days. Afterwards, they were carefully transferred to plates that were supplemented with 150 mM NaCl. The root length was measured for six days by tracking root tips. n=30 (B) Wild type root elongation growth at day six was significantly more inhibited under salt stress conditions than in bpm mutant plants. Asterisks indicate the statistical significant difference of at least P<0.05 (one-way analysis of variance) of WT plants in comparison to mutants.
FIG. 19: Photosynthetic parameters of dark adapted plants exposed to drought stress. Plants were grown for three weeks in soil under standard growth conditions (long day (16 h light: 8 h dark)). Drought stress was applied by withholding water over a period of 5 days which is indicated on the X-axis. At day four significant changes were observed between wild type and 6×amiBPM plants (n=12). The changes indicate increased sensitivity of the mutant towards drought stress. qL; Estimates the fraction of open PSII centers on the basis of the lake model for PSII. NPQ; Non-photochemical quenching. Monitors the apparent rate constant for heat loss from PSII. Fv/Fm Maximum quantum efficiency of PSII photochemistry. Maximum efficiency at which light absorbed by PSII is used for reduction of Qa. Healthy plants are usually between 0.8 and 0.85. Phi II; PSII operating efficiency. Estimates the efficiency at which light absorbed by PSII is used for Qa reduction, and provides an estimate of linear electron flux through PSII.
FIGS. 20A-B: Flowering phenotype analysis of WT, and 6×amiBPM mutants. (A) Expression level of Flowering Locus T (FT), a key regulator of the flowering time point, in rosette leaves at the end of the third, fourth, fifth, and sixth week after germination in wild type (WT) and 6×amiBPM plants. FT expression is significantly down regulated in 6×amiBPM plants when compared to WT which is in agreement with the late flowering phenotype of the mutants. (B) Schematic drawing of six different FT promoter regions analyzed via qPCR after α-CUL3 ChIP experiments. “0” indicates location of the start codon. (C) Significant enrichments were detectable in regions 1, 5 and 6 in WT, but not in a 6×amiBPM#1 control, indicating that CRL3BPM E3 ligases are directly involved in controlling FT expression. “NC” indicates negative control primer set afterwards of FT gene's ATG code. Asterisks indicate significant differences of mutant plants to WT (one-way ANOVA; P<0.05).
FIGS. 21A-E: Inducible 6×amiBPM constructs allow controlled increase in seed size. A, treatment of plants with estradiol over a time period of 24 hours leads to a significant down-regulation of all six BPM genes. B, pMDC7:6×amiBPM plants that carry an estradiol (E) inducible construct are indistinguishable from wild type (WT) when not treated with estradiol. Bar represents 5 cm. C-E, Plants carrying an estradiol (E) inducible 6×amiBPM construct were sprayed daily for around two weeks, starting at the onset of flowering with 10 mM estradiol (+E). The plants were indistinguishable in development from WT, except that seeds in E-treated 6×amiBPM plants were significantly larger then WT seeds (C, E), and heavier (D; data shown represent the average weight of 30 seeds). Bar represents 1 mm.
FIGS. 22A-J: Nucleotide sequence of full length BPM proteins from 27 different plant species (SEQ ID NOs. 68 to 94). Nucleotide sequences corresponding to the amino acid sequences listed in FIG. 16A-C.



DETAILED DESCRIPTION
Embodiments of the invention provide transgenic plants, wherein the expression of at least one BPM protein has been down-regulated or its activity reduced, resulting in enhanced yield-related traits, in particular but without limitation, increased seed oil production, as compared to a control plant. Control plants of the invention may be non-transgenic plants or plants wherein the expression or activity of a BPM protein has not been reduced or decreased by genetic engineering. Further embodiments of the invention provide methods for enhancing yield-related traits, in particular producing and recovering the seed oil from transgenic plants, wherein the expression of at least one BPM (BTB/POZ-MATH) protein is down-regulated or its activity reduced.
The following definitions are used throughout:
The terms “protein”, “polypeptide” and “peptide” refer to contiguous chains of amino acids that are covalently bonded (linked) to each other by peptide (amide) bonds. In general, a peptide contains up to about 50 amino acids and a polypeptide contains about 50 or more amino acids. Proteins may contain one or more than one polypeptide. Those of skill in the art will recognize that these definitions are considered somewhat arbitrary, and these terms may be used interchangeably herein. The terms encompass amino acid polymers that are synthesized (transcribed and translated) in vivo and amino acid polymers that are chemically synthesized using procedures well known to those skilled in the art.
As used herein, the terms “nucleic acid” or “polynucleotide” or “nucleic acid molecule” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Exemplary nucleic acids include DNA (including cDNA), RNA (e.g. mRNA, tRNA, rRNA, microRNA, amiRNA, antisense RNA, RNAi, etc.), and hybrids thereof.
The term “gene” means a segment of DNA that encodes a biologically active RNA, which may be further translated into a polypeptide chain. The term may or may not include regions preceding and following the coding region as well as intervening sequences (introns) between individual coding segments (exons). As used herein, a gene may be a recombinant or genetically engineered DNA sequence that encodes a polypeptide of interest from which introns have been eliminated.
The term “consensus sequence” or “motif” refers to a short conserved region in the sequence of evolutionary related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of a conserved domain.
As used herein, the term “transformation” refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell. As used herein, the term “genetic transformation” refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell. The term “transformant” refers to a cell, tissue or organism that has undergone transformation.
As used herein, the term “transgenic” refers to cells, cell cultures, organisms (e.g., plants), and progeny which comprise a modified or foreign (heterologous) gene, wherein the modified or foreign gene is not originally present in the host organism. The term “transgenic” also refers to modification of an endogenous gene through the introduction of a transgene that modifies the endogenous gene in a plant. Transgenic organisms may receive the transgene by one of the various methods of transformation, but may also receive the transgene via conventional breeding techniques whereby at least one of the parent organisms comprises such a transgene.
“Recombinant” refers to a product of genetic engineering, e.g. a nucleic acid such as recombinant DNA, a protein that results from the expression of recombinant DNA, and recombinant cells or organisms that are transformed with recombinant DNA.
As used herein, the terms “plant” and “plant tissue” refer to any part of a plant. Examples of plant organs include, but are not limited to the leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, and leaf sheath. Plants also include vegetables and fruit plants. “Lower plants” is a collective term for three main groups of plants (mosses, liverworts and lichens) which do not have roots and produce spores to reproduce, rather than flowers. “Higher plants” refers to plants that have vascular tissue (as known as tracheophytes). “Seed producing plants” is a term referring to those plants that produce seed (Spermatophytes) and includes “Flowering plants”, which refers to seed-producing plants, also known as Angiospermae or Magnoliophyta, as well as the Gymnospermae. Plants may be grown (e.g. in a field or a greenhouse) for production of food, fuel or fiber or other uses (e.g. wood, ornamentals). All such plants are encompassed by the present invention.
Exemplary plants or plant cells that may be utilized in the practice of the invention include but are not limited to: oil seed plants, canola, safflower, camelina, soybean, corn, sunflower, peanut, sesame, cotton rice, wheat, etc. Generally, oil seed plants (which may be trees) are cultivated so that oil, especially edible oil, can be produced from the seeds, nuts, tubers, etc. of the plants. Exemplary oil seed plants include but are not limited to: coconut, corn, cotton, olive, palm, peanut (ground nut), various rapeseed plants including canola, safflower, sesame, flax, soybean, sunflower, and the like. Various plant species that produce nuts from which oils are extracted may also be employed, including those that produce hazelnuts (e.g. from the common hazel), almond, beech (e.g. which produce Fagus sylvatica nuts), cashew macadamia, mongongo (or manketti, seeds of the Schinziophyton rautanenii tree), pecan, pine, pistachio, walnut, etc. Various citrus plants and trees produce seeds which are used to prepare edible oils, e.g. lemon, orange oil, grapefruit, sea-buckthorn, etc. Various melons and gourds may be utilized, e.g. watermelon (e.g. Citrullus vulgaris), members of the Cucurbitaceae family including gourds, melons, pumpkins, and squashes; the bitter gourd (Momordica charantia), bottle gourd (e.g. Lagenaria siceraria), buffalo gourd (Cucurbita foetidissima), butternut squash (e.g. Cucurbita moschata), egusi (Cucumeropsis mannii naudin, pumpkin, etc. Other plants and/or trees that may be utilized include borage (e.g. Borago officinalis), blackcurrant, evening primrose (e.g. Oenothera biennis), açai (e.g. any of several species of the Açai palm (Euterpe), black seed (e.g. from Nigella sativa), blackcurrant (e.g. Ribes nigrum), flax (linseed, e.g. Linum usitatissimum), carob, amaranth (e.g. from Amaranthus cruentus and Amaranthus hypochondriacus), apricot, apple, argan (e.g. from Argania spinosa), avocado, babassu r.g. Attalea speciosa), the seeds of Moringa oleifera, from which “ben” oil is extracted, species of genus Shorea, cape chestnut, the cacao plant, cocklebur (e.g. species of genus Xanthium), poppy, the Attalea cohune (cohune palm), coriander, date, Irvingia gabonensis, Camelina sativa, grape, hemp, Ceiba pentandra, Hibiscus cannabinus, Lallemantia iberica, Trichilia emetica, Sclerocarya birrea, meadowfoam, mustard, nutmeg (e.g. from cogeners of genus Myristica), okra (e.g. Abelmoschus esculentus), papaya, perilla, persimmon (e.g. Diospyros virginiana), Caryocar brasiliense, pili nut (e.g. Canarium ovatum), pomegranate (e.g. Punica granatum), prune quinoa, ramtil (e.g. several species of genus Guizotia abyssinica (Niger pea), rice, Prinsepia utilis, shea, Sacha inchi, sapote (e.g. Jessenia bataua), arugula (e.g. Eruca sativa), tea (Camellia), thistle (e.g. Silybum marianum), Cyperus esculentus, tobacco (e.g. Nicotiana tabacum and other Nicotiana species), tomato, and wheat, among others.
In some aspects, embodiments of the invention provide products produced by plants or from plants or parts of plants, for example, oils produced from the seeds or nuts of the transgenic plants. Exemplary oils of the invention include but are not limited to: Coconut oil, Corn oil, Cottonseed oil, Olive oil, Palm oil, Peanut oil (Ground nut oil), Rapeseed oil (including Canola oil) Safflower oil, Sesame oil, Soybean oil, and Sunflower oil. Various nut oils are also contemplated, including but not limited to: Almond oil, Beech nut oil, Cashew oil, Hazelnut oil, Macadamia oil, Mongongo nut oil (or manketti oil), Pecan oil, Pine nut oil, Pistachio oil, and Walnut oil. Various Citrus oils are also contemplated, including but not limited to: Grapefruit seed oil, Lemon oil, Orange oil, and sea-buckthorn oil. Oils from melon and gourd seeds are also contemplated, including but not limited to: Cucurbitaceae oils from e.g. gourds, melons, pumpkins, and squashes such as Watermelon seed oil, Bitter gourd oil, Bottle gourd oil, Buffalo gourd oil, Butternut squash seed oil, Egusi seed oil, and Pumpkin seed oil, Various other plant-derived oils are also encompassed by the invention, including but not limited to: Açai oil, Arabidopsis oil, Black seed oil, Blackcurrant seed oil, Borage seed oil, Evening primrose oil, Flaxseed oil (linseed oil), Carob seed pods, Apricot oil, Apple seed oil, Argan oil, Avocado oil, Babassu oil, Ben oil, Borneo tallow nut oil, Cape chestnut oil, Carob pod oil (Algaroba oil), Cocoa butter, Cocklebur oil, Cohune oil, Coriander seed oil Date seed oil, Dika oil, False flax oil Grape seed oil, Hemp oil, Kapok seed oil, Kenaf seed oil, Lallemantia oil, Mafura oil, Manila oil, Meadowfoam seed oil, Mustard oil (pressed), Poppyseed oil, Nutmeg butter, Okra seed oil, Papaya seed oil, Perilla seed oil, Persimmon seed oil, Pequi oil, Pili nut oil, Pomegranate seed oil, Prune kernel oil, Quinoa oil, Ramtil oil, Rice bran oil Royle oil, Sacha inchi oil, Sapote oil, Seje oil, Shea butter, Taramira oil, Tea seed oil (Camellia oil), Thistle oil, Tigernut oil (or nut-sedge oil) Tobacco seed oil, Tomato seed oil, and Wheat germ oil, etc.
It is an object of the invention to provide transgenic plants having enhanced yield-related traits as compared to a non-transgenic plant or other control plant which has not been similarly genetically engineered according to the teachings provided herein. Yield-related traits include, but are not limited to, seed oil production, flowering, stress-tolerance, and increased growth rate. In exemplary embodiments, the transgenic plants have increased seed oil production as compared to control plants (e.g., non-transgenic plants or plants not genetically engineered as described herein).
The plants of the present invention have been genetically engineered using molecular biology techniques to down-regulate the expression or reduce the activity of at least one BPM protein. Methods of producing transgenic plants are well known to those of ordinary skill in the art. Transgenic plants can be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment, e.g. using needle-like crystals (“whiskers”) of silicon carbide; viral-mediated transformation; Agrobacterium-, Rhizobium-, Mesorhizobium- and Sinorffizobium-mediated transformation. See, for example, U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318; 5,641,664; 5,736,369; 5,736369; and US patent applications 2005/0289672 and 2005/0289667; each of which is expressly incorporated herein by reference in entirety. Progeny of the transgenic plants of the invention are also encompassed.
BPM proteins are found in almost all plants and the exemplary amino acid sequences of BPM proteins from 27 different species and a consensus sequence are shown in FIGS. 16A-C. The corresponding nucleotide sequences are shown in FIGS. 22A-J. The species listed in FIGS. 16A-C contain members of the Eudicots as well as monocot groups and one spikemoss. The spikemoss is an ancient plant with members of this family existing before Angio- and Gymnosperms were present. The alignment as shown in FIG. 16 indicates that BPMs are functionally conserved. FIG. 17 compares the full length BPM proteins shown in FIGS. 16A-C for identity and similarity.
Aspects of the invention related to the down-regulation of at least one BPM protein in a transgenic plant. The term “down-regulation” refers to a decrease in endogenous gene expression and/or polypeptide levels as compared to a control, wherein the expression levels in the control have not been modulated. The reduction may be at least about 10% or more reduced compared to that of a non-transgenic control plant, preferably at least 40% and more preferably at least 60%. Methods of decreasing expression of proteins in plants are well known in the art and include, but are not limited to artificial microRNA (amiRNA), antisense RNA, RNAi or co-suppression, and T-DNA insertion. In exemplary embodiments, amiRNA is introduced into the plant to down-regulate expression of at least one BPM protein. Other methods for down-regulating expression of proteins, for example the use of CRISPR-Cas9 nucleases, can be used in the practice of this invention and the invention encompasses each of these methods (Hsu et al., 2014). One of ordinary skill in the art would be able to adapt the various known biological methods for silencing so as to reduce the expression of a gene in a plant or in parts thereof.
Additional aspects of the invention relate to the reduced activity of at least one BPM protein. The term “reduced activity” refers to the functional aspects of the BPM protein. For example, “reduced activity” is construed to mean that the ability of the BPM protein to assemble with cullin, to interact with other BTB/POZ proteins, and/or to function as an adaptor to allow binding of a substrate and delivery to the CRL3 core for ubiquitylation is fully or partially inhibited. Methods of altering the activity of a protein are known in the art and include, but are not limited to reducing expression (for example, amiRNA), substrate competition (for example, MATH domain overexpression), targeted mutation of amino acid residues required for binding to substrates, and targeted mutation of amino acid residues in substrate proteins required for recognition (by the MATH domain for example). In exemplary embodiments, an exogenous BPM domain, preferably the MATH domain, is expressed in the plant to compete with at least one BPM protein for binding with a substrate.
Exemplary amino acid sequences are provided, but those of skill in the art will recognize that various other modified forms (variants or derivatives) of the amino acid sequences disclosed herein may be made, and the invention encompasses all such variants/derivatives, as long as the resulting molecule retains a desired level of activity as described herein. Exemplary encoding nucleotide sequences are also provided, but those of skill in the art will recognize that, due to the redundancy of the genetic code, other nucleotide sequences may also encode the same protein/polypeptide.
The nucleic acid molecules described herein may be modified, for example, by codon optimization to facilitate expression in heterologous cells. This type of modification changes or alters the nucleotide sequence that encodes a protein of interest to use, throughout the sequence, codons that are more-commonly used in the transgenic expression host cell. In addition, changes may be made to the nucleotide sequence that encodes the protein to adjust the relative concentration of A/T and G/C base pairs to ratios that are more similar to those of the expression host.
In addition, nucleotide sequences encoding MATH domains of the invention may be further modified to encode other sequences such as those described above as being beneficial or desirable for inclusion in the plants of the invention, e.g. sequences which target or direct the polypeptide to a particular location or locations within the expression host cell, etc.
The invention also encompasses vectors that comprise the nucleic acid sequences described herein. “Vector” refers broadly to any plasmid or virus encoding an exogenous nucleic acid. (However, the term may also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like.) The vector may be a viral vector that is suitable as a delivery vehicle for delivery of the nucleic acid, or mutant thereof, to a cell, or the vector may be a non-viral vector which is suitable for the same purpose. Examples of viral and non-viral vectors for delivery of DNA to cells and tissues are well known in the art. Examples of viral vectors include, but are not limited to recombinant vaccinia, adeno-, retro-, adeno-associated, avian pox and other viral vectors. Examples of non-viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.
Embodiments of the invention provide transgenic plants with enhanced yield-related traits as compared to non-transgenic plants and methods for producing the same. In preferred embodiments, the transgenic plants have increased seed oil production. The amount of seed oil that can be recovered from plants of the present invention is more than about 1% in comparison to the oil recovered from non-transgenic plants, preferably more than about 25%, and more preferably more than about 50%. The more severe the down-regulation of BPMs or the reduction of the activity of BPMs, the higher the amount of seed oil that may be recovered from the plant. Methods of recovery of oil from a plant are known in the art and can be performed substantially as described in Focks and Benning, 1998. Methods of cultivating plants under conditions promoting plant growth and development are also known in the art.
Embodiments of the invention provide novel biotechnological approaches to improve yield-related traits in plants, in particular seed oil production, with beneficial impacts for biofuel or food-related products. Embodiments of the invention have many applications including, but not limited to, producing food, feed, or an industrial product comprising obtaining a plant or a part thereof, as herein described, including plants wherein the expression of at least one BPM protein is down-regulated or its activity reduced and preparing the food, feed or industrial product from the plant or part thereof. The food or feed may be oil, meal, grain, starch, flour or protein; or the industrial product may be biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical.
Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
EXAMPLES
Example 1. Arabidopsis BPM Proteins Function as Substrate Adaptors to a CUL3-Based E3 Ligase Affecting Fatty Acid Metabolism in Plants
Summary
Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity control because they play a critical role as key mediators assuring specific expression of target genes. In this example, it is shown that Cul3-based E3 ligases can interact with a broad range of ERF/AP2 transcription factors, mediated by MATH-BTB/POZ proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes changed fatty acid contents in mutant seeds. Overall the work provides a novel link between fatty acid metabolism and E3 ligase activities in plants, and establishes Cul3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.
Materials and Methods
Plant Materials and Growth Conditions
Arabidopsis thaliana wild-type ecotype Columbia plants and plants of the different genetic backgrounds were grown either on Arabidopsis thaliana (AT) medium without supplement of Suc (Estelle and Somerville, 1987) in a growth chamber at 22° C. with 120 μmol/m2/s light intensity or in soil in a greenhouse at 20° C. under long-day conditions (16 hours light/8 hours dark).
Clone Constructions
The cDNAs of BPM1MATH, BPM1MTH:NLS, CUL3s, ERF/AP2s, and BPMs were cloned into pDONR221 (Invitrogen). For Y2H studies, the corresponding cDNAs were shuffled into destination vectors pACT2 (prey) and pBTM116-D9 (bait) by Gateway technology (Invitrogen) as described (Weber et al., 2005). pDEST15 (Invitrogen) and pET-58-DEST (Merck) were used to express and purify GST- and His-tagged proteins in Escherichia coli, respectively; where necessary, elution of GST proteins from glutathione-agarose beads was done following standard procedures. pMDC43 was used for expression in plants and for subcellular localization studies as described (Curtis and Grossniklaus, 2003). The NLS sequence was adopted from Howard et al. (1992), extended, and attached to the MATH domain in a four-step-based PCR process. To generate artificial microRNAs, a protocol from the WMD2 microRNA designer Web page; see FIG. 6) was followed using pRS300 as starting vector. For expression in plants, ami constructs were first cloned into pDONR221 before being shuffled into the binary vector pGBW14. For primers used see Table 1 below.







TABLE 1







Primers used for different PCR-based approaches.








Name of Primer
Sequence of Primer (5′-3′)










Cloning of constructs








T7BPM1MATHFW 
TAATACGACTCACTATAGGGAGAATGTTC


(SEQ ID NO: 29)
AAGATCTGTGGGTAC


 


BPM1MATHRW 
CTACATTTCTAGACTGGACCTCCTG


(SEQ ID NO: 30)



 


BPM1-MATH-RW-NLS
TCGTCCTCAGTGGACGCTTAGAGAGCACT


(SEQ ID NO: 31)
TCTAGACTGGACCTCCTG


 


NLS-1RW 
ACGCTTACGCTCAGATGGCTCACCGTCGT


(SEQ ID NO: 32)
CGTCCTCACGTGGACGCTTAG


 


NLS-RW2 
CACGACCGTCCTTAGAACGCTCGTCACGC


(SEQ ID NO: 33)
TCACGCTTACGCTCAGATGGC


 


Attb2stop-NLS
AGAAAGCTGGGTCACGACGGTTACCACCA


(SEQ ID NO: 34)
CGACCGTCC


 


WRI1-FW
ATGAAGAAGCGCTTAACCAC


(SEQ ID NO: 35)



 


WRI-RW
TCAGACCAAATAGTTACAAG


(SEQ ID NO: 36)



 







qRT-PCR








ACTIN2-qRT FW
CCTGCCATGTATGTTGCCATT


(SEQ ID NO: 37)



 


ACTIN2-qRT RW 
AATCGAGCACAATACCGGTTGT


(SEQ ID NO: 38)



 


BPM1-qRT-FW
ATTGGCGTCTACTCTTGT


(SEQ ID NO: 39)



 


BPM1-qRT-RW
AATGATGCTGCTCTGCTA


(SEQ ID NO: 40)



 


BPM2-qRT-FW
TAATCGGCACAGACTTGA


(SEQ ID NO: 41)



 


BPM2-qRT-RW
ACTCGCATATTGTTCTAAGC


(SEQ ID NO: 42)



 


BPM3-qRT-FW
CACCAGTTCACGATTCAAG


(SEQ ID NO: 43)



 


BPM3-qRT-RW
CCACCAACGGAGAAGATAT


(SEQ ID NO: 44)



 


BPM4-qRT-FW
TCCTGATGGCAAGAATCC


(SEQ ID NO: 45)



 


BPM4-qRT-RW
CGAAGTGGCTATGAACCT


(SEQ ID NO: 46)



 


BPM5-qRT-FW
TTAGGCTCAGGTTGTTGT


(SEQ ID NO: 47)



 


BPM5-qRT-RW
TCATCCTTCATCTGTTGGTA


(SEQ ID NO: 48)



 


BPM6-qRT-FW
GCATAAGGTTCATAGCCATT


(SEQ ID NO: 49)



 


BPM6-qRT-RW
AGATGTCTCAAGCAAGGA


(SEQ ID NO: 50)



 


WRI1-qRT-FW
GAGCAACAAGAAGCAGAG


(SEQ ID NO: 51)



 


WRI1-qRT-RW 
CCACAACGATCCATTTCC


(SEQ ID NO: 52)



 


BCCP1-qRT-FW
CAGCCAAATCGTCACT


(SEQ ID NO: 53)



 


BCCP1-qRT-RW
GTTCCGGTATGGTCAG


(SEQ ID NO: 54)



 


AtGLB1-qRT-FW
CTTTCACCGTCTTAGGAACAAACAG


(SEQ ID NO: 55)



 


AtGLB1-qRT-RW
TAGGAACAGAGTTTCGATGTCTGAGAAC


(SEQ ID NO: 56)



 


BPM1-MATH-qRTFW
CGGAGGATAACTCGTCTT


(SEQ ID NO: 57)



 


BPM1-MATH-qRTRW
AATGGCTATGAACCTTATGC


(SEQ ID NO: 58)



 







ChIP-qPCR








EF1-qRT-FW
CTGGAGGTTTTGAGGCTGGTTA


(SEQ ID NO: 59)



 


EF1-qRT-RW
CCAAGGGTGAAAGCAAGAAGA


(SEQ ID NO: 60)



 


ProBCCP1-qRT-FW
AAGTGAACTGTTGTTGTT


(SEQ ID NO: 61)



 


ProBCCP1-qRT-RW
CGTCTTCTTATTGTTATTGG


(SEQ ID NO: 62)



 


Pro-GLB1-qRT-FW
TTCCAATAATTACCTCCTT


(SEQ ID NO: 63)



 


Pro-GLB1-qRT-RW
TTTAACACAACTTTCAAAG


(SEQ ID NO: 64)









Subcellular Localization Studies

The fluorescent fusion proteins GFP:WRI1, GFP:BPM1MATH, and BPM1MATH:NLS were transiently expressed in Nicotiana benthamiana epidermal cells following the method described by Sparkes et al. (2006). GFP expression was detected and documented with a Zeiss LSM 510 Meta confocal microscope.
Interaction and Complex Assembly Studies
Y2H studies were followed as described by Weber et al. (2005). SDII selection medium supplemented with uracil and His was used as a transformation control, while for interaction studies, SDIV minimal medium was chosen without uracil and His supplements. Photos were taken from single spots 7 days after plating. FPLC was performed with 2 mg protein injections and a flow rate of 50 μL/min using an AKTA FPLC system (GE Healthcare Science) and as described by Leuendorf et al. (2010). Pull-down analysis and IP studies were followed as described before (Hellmann et al., 2003; Bernhardt et al., 2006). Extraction and washing buffers contained at all times 1 mM PMSF (Sigma-Aldrich) and 10 μM MG132 (Sigma-Aldrich) to prevent proteolytic and proteasomal activities, respectively. For pull-down analysis with GST- and His-tagged proteins, GST-containing proteins were first eluted from glutathione-agarose beads before incubated with His:WRI1 that remained attached to tetradentated-chelated nickel resin. In general, proteins were incubated at least 1.5 h at 4° C. under shaking conditions before being centrifuged. Precipitates were washed no less than three times to remove unspecific bindings, before they were taken up in Laemmli buffer (Laemmli, 1970) and boiled (10 min, 95° C.). IPs were followed as described by Bernhardt et al. (2006). In brief, 1 mg of fresh protein extracts from 2-week-old seedlings were precleaned with 30 μL protein-A-agarose beads (Santa Cruz Biotechnology; 1.5 h, 4° C.). The beads were centrifuged, and the supernatant was transferred into a fresh tube and incubated first with α-WRI1 (1.5 h, 4° C.) before 30 μL protein-A-agarose beads were added (1.5 h, 4° C.). After brief centrifugation, four washing steps followed, after which precipitates were taken up in Laemmli buffer and boiled as described above. For pull-down and IP studies, precipitates were further analyzed by SDS-PAGE and protein gel blotting using standard procedures. Where applicable, membranes were stained with Ponceau S to detect transferred proteins. For immunodetection, custom-made (α-CUL3 [rabbit] and α-WRI1 [rabbit]; GeneScript) or commercially available antibodies (GST, secondary α-rabbit IgG-horseradish peroxidase; Santa Cruz) in combination with an ECL Plus Western Blotting Detection Kit (GE Healthcare Life Science) were used.
Stability Assays
For stability assays, Arabidopsis seedlings were cultured on solid AT medium for 2 weeks before being transferred to 5 mL AT liquid medium. Plants were incubated for 3 h with the transcriptional inhibitor ActD2 (Sigma-Aldrich; final concentration 10 μg/mL) and/or the proteasomal inhibitor MG132 (Sigma-Aldrich; 20 μM/mL, 6 h) before CHX (Sigma-Aldrich; 100 μM/mL, 3 h) was added to inhibit translation. DMSO was used as mock control and as a dissolvent for all inhibitors. Protein gel blot analysis and protein detection were conducted using standard procedures. The antibodies against WRI1 and CUL3 were designed and produced by GeneScript and used in a 1:1000 dilution. Protein detection was followed as described in the ECL Plus Western Blotting Detection Kit manual (GE Healthcare Life Science).
RNA Isolation and Expression Analysis
Total Arabidopsis RNA was extracted following the protocol of the Isolate RNA kit from Bioline; reverse transcription was done according to the manual for the high-capacity cDNA reverse transcription kit (Applied Biosystems). qRT-PCR reactions (95° C., 7 min; 95° C., 15 s; 60° C., 1 min; 40 cycles) were performed using the SYBR green method on a 7500 Fast Real-Time PCR system (Applied Biosystems). Relative gene expression analyses were calculated by the full quantification method with ACTIN2 as the internal control gene. Fourteen 2-week-old seedlings were pooled for each replicate. At least three biological replicates were performed for each individual experiment. Primers used for qRT-PCR are shown in Table 1 above.
ChIP Assays
For ChIP assays, an established protocol was followed (Morohashi et al., 2009). In brief, 60 mg (fresh weight) of 15-day-old seedlings were harvested for each ChIP experiment and cross-linked for 10 min under vacuum in cross-link buffer containing 1% formaldehyde as described by Morohashi et al. (2009). Cross-linked samples were incubated in 100 mM Gly for 5 min under a vacuum, thoroughly washed in double-distilled water, and snap frozen in liquid nitrogen. Frozen tissues were ground into fine powder and dissolved in nuclear isolation buffer (Morohashi et al., 2009) supplemented with 1× protease inhibitor cocktail (Sigma-Aldrich). After filtering through single-layered Miracloth (Merck), the samples were centrifuged (10 min, 1200 g, 4° C.). Pellets were resuspended in nuclear isolation buffer supplemented with 0.3% Triton X-100 and centrifuged again (10 min, 10,000 g, 4° C.). After resuspension in lysis buffer, the purified nuclei were then sonicated (three times, 20 s, 9 W; Fisher Scientific Model 100 dismembrator) to yield chromatin fragments of 300 to 500 bps. Sonicated chromatin fragments (2 mg) were first precleared with protein-A-agarose beads (Sigma-Aldrich) (1.5 h, 4° C.) before being incubated with specific antibodies (1 mg/mL; 1.5 h, 4° C.), followed by a fresh batch of protein-A-agarose beads (30 μL; 1.5 h, 4° C.) to IP protein-DNA complexes. After IP, cross-linking was reversed by incubating samples overnight at 65° C. in elution buffer (1% SDS, 0.1 M NaHCO3, and 0.25 mg/mL proteinase K; Morohashi et al., 2009), after which RNaseA (1 mg/mL) was added (30 min; room temperature). As input control for data normalization, a portion of sonicated, cross-linked, and precleared DNA was treated accordingly except for undergoing an IP. Samples were further cleaned up using a DNA purification kit (NuCleoSpin Extraction II; Macherey-Nagel) and quantified to use equal amounts of template (50 ng/reaction) for qRT-PCR analysis. To amplify promoter sequences that contain an AW-box recognized by WRI1 (Maeo et al., 2009), specific primers were designed (Table 1). EF1 was selected as a reference gene for internal control. qRT-PCR reactions (95° C., 10 min; 95° C., 15 s; 60° C., 1 min; 50 cycles) were done as described above and repeated with at least three independent biological replicates.
Metabolic Analysis
Metabolic profiling of 2-week-old seedlings grown on ATS plate (100 mg fresh weight) and seed samples (50 mg dry weight) of all backgrounds used in this study were analyzed according to earlier described protocols (Roessner-Tunali et al., 2003). Seed fatty acids were extracted exactly as described before (Focks and Benning, 1998). For quantification with gas chromatography, pentadecanoic acid was used as an internal standard (Browse et al., 1985).
Accession Numbers
Sequence data from this Example can be found in the GenBank/EMBL data libraries under the following accession numbers: ACTIN2, At3g18780; IAA5, At1g15580; BPM1, At5g19000; BPM2, At3g06190; BPM3, At2g39760; BPM4, At3g03740; BPM5, At5g21010; BPM6, At3g43700; DREB1a, At4g25480; ERF1, At3g23240; ERF4, At3g15210; RAV1, At1g13260; WRI1, At3g54320; BCCP1, At5g16390; and GLB1, At2g16060.
Results
BPM Proteins Interact Broadly with ERF/AP2 Transcription Factors
We have earlier described that BPM proteins assemble with several, but not all members, of the A6 group of ERF/AP2 transcription factors (Weber and Hellmann, 2009). According to Sakuma and co-workers, the ERF/AP2 superfamily can be divided into five subgroups: AP-2, RAV, DREB, ERF, and others (Sakuma et al., 2002). The A6 group belongs to the ERF subfamily. To investigate how broadly BPM proteins assemble with ERF/AP2 transcription factors, we also tested in yeast-2-hybrid (Y2H) assays additional members outside the A6 group using BPM1 as prey (FIG. 2A). The ERF subfamily member ERF1 (At3g23240) showed weak interaction, while the ERF-subfamily members WRI1 (At3g54320) and ERF4 (At3g15210), showed a strong interaction with BPM1 in the Y2H assay. WRI1 also tested positively for self-assembly in the yeast assay (FIG. 2B). Finally, DREB1a (At4g25480), which belongs to the DREB subfamily, and also RAV1 (At1g13260), a member of the RAV-subfamily, strongly interacted with BPM1 (FIG. 2A). Since BPMs interact also with CUL3 proteins, we tested interaction of the different ERF/AP2 transcription factors with CUL3a, and did not observe any in the yeast system (data not shown). We therefore concluded that a large number of ERF/AP2 transcription factors is recognized by BPM proteins in Arabidopsis. 
CUL3 and BPM Proteins Assemble in Planta with WRI1
To identify and demonstrate basic principles of CRL3BPM complex assembly with substrates, we focused on a single well-described protein, WRI1, which is a key player in fatty acid and carbohydrate metabolism (Cernac and Benning, 2004; Baud et al., 2009).
In agreement with findings from the Y2H assays, pulldown experiments using a GST:WRI1 fusion protein can co-precipitate in vitro translated BPM1 from rabbit reticulolysates (FIG. 2C). For further investigation of in planta complex assembly, specific peptide-based antibodies were raised against CUL3 and WRI1 (FIG. 1). The antibody against CUL3 does not distinguish between CUL3a and b (FIG. 1 A,C); however transient expression experiments in tobacco clearly demonstrated specificity of the α-CUL3 antibody. We only observed a single band of around 85 kDa appearing on Western blots with wild type (WT) or cul3 mutant plant extracts (FIG. 1B). Likewise, only a single band was detectable when the α-WRI1 antibody was used on total plant extracts from WT plants, and which was missing in a wri1-3 mutant when the α-WRI1 antibody was used on total plant extracts (FIG. 1E).
Pulldown experiments using GST:WRI1 against WT plant extract showed that the fusion protein precipitates both endogenous WRI1 and CUL3, however, this was not the case with GST alone (FIG. 2D). In addition, pulldown analysis with GST- and His-tag proteins expressed in and purified from E. coli demonstrated that BPM proteins are necessary to bridge assembly between WRI1 and CUL3 proteins. Here His:WRI1 is only capable of co-precipitating GST:CUL3a, if GST:BPM1 protein is present in the assay but not with GST alone (FIG. 2F). Also, immunoprecipitation (IP) studies using the α-WRI1 antibody successfully precipitated CUL3 from plant extracts in WT background (FIG. 2G). We also observed in FPLC studies co-migration of WRI1 with CUL3 (FIG. 3A), and detected GFP:WRI1 localized to the nucleus, as has been earlier shown for most BPM proteins and CUL3a (FIG. 3B; (Weber and Hellmann, 2009)). Overall these studies demonstrate that WRI1 assembles with CUL3 proteins in planta, and support the working hypothesis that the assembly is mediated by BPM proteins.
WRI1 is a CUL3-Dependent Target of the 26S Proteasome
One outcome of BPM assembly with CUL3 proteins is the proteolytic degradation of their substrates. Consequently, stability assays were performed using the translational inhibitor cycloheximide (CHX) and the proteasomal inhibitor MG132. In initial experiments, CHX treatments did not point to instability of the WRI1 protein (FIG. 4A), although accumulation of WRI1 was observed when plants were treated with MG132 (FIG. 4A).
To further characterize this phenomenon, WRI1 expression was tested in plants treated with CHX or with the proteasomal inhibitor. While plants incubated with MG132 did not show any change in WRI1 expression, CHX caused a strong up-regulation of the WRI1 gene (FIG. 4B). It was therefore decided to pre-treat plants with the transcriptional inhibitor actinomycin D2 (ActD2) before CHX was given. Under these conditions WRI1 protein was completely gone after 6 h treatment, and its disappearance was blocked by co-incubation with MG132 (FIG. 5A), demonstrating that WRI1 is unstable in a 26S proteasome-dependent manner. Notably, the accumulation of WRI1 protein in samples treated with all three inhibitors is most likely due to pre-treatment of plants with ActD2 and MG132 for three hours before CHX was supplemented.
A cul3hyp double mutant was previously described that is knocked-out for CUL3b and partially functional for CUL3a (Thomann et al., 2009). We took advantage of this mutant to investigate whether WRI1 is stabilized in this genetic background and to prove that the instability is mediated by a CUL3-based complex. Western-blot analysis on WT and cul3hyp plant extracts showed that WRI1 was present in the mutant in higher amounts than in WT (FIG. 5B). This was not based on increased transcriptional activities in the mutant since no significant difference in WRI1 expression was detectable in either plant (FIG. 5C). Stability assays with ActD2 and CHX showed no considerable change in protein content over six hour treatments in the mutant, while WRI1 was not detectable in WT extracts (FIG. 5D), revealing that WRI1 is instable in a 26S proteasome- and CUL3-dependent manner.
BPM Proteins Bridge the Assembly Between WRI1 and CUL3 and are Broadly Important for Development.
The results show that BPM proteins assemble with a broad range of ERF/AP2 transcription factors and that, if ERF/AP2 proteins are in complex with CUL3s, the BPMs likely function as their bridging substrate receptors. Since WRI1 is unstable, and because complex formation requires presence of a functional CUL3 protein in the plant, it was necessary to investigate whether loss of BPM proteins is also stabilizing WRI1.
Two strategies were followed to support the hypothesis that BPM proteins function as substrate receptors and are required for mediating WRI1 instability. First, because of a lack of T-DNA insertion mutants for nearly all BPM genes, a 35S artificial microRNA (amiRNA) construct was designed to down-regulate expression of all six members, based on predictions from the WMD 2—Web MicroRNA Designer; FIG. 6) (the lines are further denoted as 6×amiBPM). Second, the MATH domain from BPM1 was cloned under the control of a 35S promoter and behind a GFP reporter, and with (further denoted as BPM1MATH:NLS) or without (BPM1MATH) a nuclear localization signal attached to the end of the domain to affect subcellular localization. The MATH construct was generated to impose a competition in the plant where endogenous BPM proteins have reduced access to WRI1 and thus, hypothetically cause its stabilization.
Several independent plant lines were successfully generated, and two independent lines of the T4 generation were chosen for each construct for further analysis. In both 6×amiBPM lines a significant down-regulation in gene expression of all BPMs was measurable (FIG. 7A,B), while the BPM1MATH and BPM1MATH:NLS lines showed strong expression of the transgene (FIG. 7C). In addition, based on the GFP reporter, BPM1MATH constructs were detectable throughout the cell, including the nucleus (FIG. 8A), while BPM1MATH:NLS was exclusively present in the nucleus (FIG. 8B). Analysis of T4-generation plants showed that the 6×amiBPM lines consistently had higher WRI1 protein levels comparable to cul3hyp mutants, while surprisingly all MATH-overexpression lines had significantly less WRI1 protein (FIG. 7D). Although the absolute degree of WRI1 reduction in MATH-overexpression lines varied among tested plants, we never observed any levels that equaled or exceeded those in WT. Also of note is that this is not based on reduced WRI1 expression, since the gene is actually up-regulated in BPM1MATH and BPM1MATH:NLS lines (FIG. 8C).
The different plant lines were broadly affected in development. Primary root growth was significantly delayed in all six lines, and most strongly in the two 6×amiBPM lines (FIG. 9A). While lateral roots emerged at a lower frequency in the BPM1MATH lines, no significant changes were detectable in plants expressing the BPM1MATH:NLS construct (FIG. 9B). The 6×amiBPM plants developed very low numbers of lateral roots (FIG. 9B,C), and all transgenic lines were affected in shoot development. In addition all were late flowering, most strongly pronounced in BPM1MATH and 6×amiBPM lines (FIG. 9D,E), with less leaves present at the beginning of flowering (FIG. 4F), and a reduced rosette size (FIG. 9D,G). Besides being smaller and present in fewer numbers, the leaves of transgenic plants also had a tendency to develop wider blades then WT (FIG. 10).
To characterize the extent WRI1 protein stability is affected in the different lines, stability assays were performed on selected plants (FIG. 11 and FIG. 12). The assays consistently showed that in either the 6×amiBPM or MATH-overexpressing backgrounds, WRI1 was highly stable in comparison to WT (FIG. 11A; FIG. 12A, B).
IP experiments were carried out on two MATH-overexpressing and two 6×amiBPM lines. As shown in FIG. 11B, CUL3 protein was precipitated from WT plant extracts, while no precipitated CUL3 was detectable in either the MATH-overexpressing or the 6×amiBPM lines. These findings together with stability assays demonstrate that the BPM proteins are required (i) for assembly of WRI1 into a complex with CUL3, and (ii) for mediation of the transcription factor's degradation.
WRI1 Activity is Affected by CRL3BPM 
We showed stabilization of WRI1 and an effect on its protein content in the plant in three different genetic backgrounds. In both cul3hyp double mutants and 6×amiBPM lines, WRI1 levels are increased, while in MATH-lines WRI1 amounts are decreased. In 6×amiBPM lines BPM expression is reduced, and in the cul3hyp and MATH-backgrounds BPM protein levels are likely unchanged. However, based on each genetic background, the assembly of WRI1 into a CUL3-based complex is differently affected by either reduced CUL3 availability and/or functionality (cul3hyp), reduced BPM content (6×amiBPM), or reduced accessibility of BPMs to WRI1 (MATH-overexpressing lines). To show how these situations differently affect WRI1 transcriptional activities, expression of two confirmed WRI1 targets, BCCP1 and AtGLB1, were tested (Baud et al., 2009; Maeo et al., 2009). BCCP1 (At5 g16390), which encodes for a biotin carboxyl carrier protein, and AtGLB1 (At4g01900), which encodes for a PII protein, are both critical players in fatty acid biosynthesis, but also participate in carbon and nitrogen metabolism (Tissot et al., 1998; Chen et al., 2006).
qRT-PCR analysis showed loss of BCCP1 and AtGLB1 expression in the wri1-3 null mutant compared to WT (FIG. 11C). Expression of both genes in MATH-overexpressing lines was similarly reduced. In contrast, both genes were strongly up-regulated in 6×amiBPM lines correlating with changes in WRI1 protein content. Interestingly, no change in BCCP1 and AtGLB1 expression in comparison to WT was noticeable in the cul3hyp line, despite the fact that WRI1 protein levels were elevated comparable to 6×amiBPM lines. These findings indicate, based on the presumed presence (cul3hyp) or absence (6×amiBPM), that BPM proteins also negatively affect transcriptional activity of their target proteins.
CUL3 Assembles with WRI1 at the DNA Level
To show whether CUL3 forms a complex with WRI1 at the DNA level we performed chromosomal immunoprecipitation experiments (ChIP) (Morohashi et al. 2009). In WT plants, α-WRI1 and α-CUL3 based ChIP experiments resulted in a two to three-fold enrichment of WRI1 binding sites (proBCCP1 and proAtGLB1), respectively (FIG. 11D), while no enrichment was detectable in the wri1-3 null mutant, which served as a negative control (FIG. 11G). Interestingly, α-WRI1 ChIP in the two 6×amiBPM lines yielded higher levels of proAtGLB1 and proBCCP1 sites which was in agreement with higher WRI1 protein levels in these plants (FIG. 11E, F), as well as increased transcription of the corresponding genes (FIG. 11C). Finally, ChIP using the α-CUL3 antibody in 6×amiBPM lines did not lead to any enrichment of proAtGLB1 and proBCCP1 sites (FIG. 11E, F), which corroborates the finding that loss of BPMs disrupt the ability of CUL3 to assemble into a complex with WRI1 (FIG. 11B). Overall these results show that CUL3 proteins form a complex with WRI1 at the DNA level, and that this assembly requires BPM proteins.
Reduced BPM Content Affects Fatty Acid Metabolism in Seeds
The finding that a reduced BPM expression in 6×amiBPM lines increases both WRI1 protein content and expression of WRI1 target genes was intriguing as it opened up the possibility that seeds of 6×amiBPM lines may also contain elevated levels of fatty acids due to augmented levels of active WRI1 (Baud et al., 2009). In agreement with this idea, both 6×amiBPM lines showed significant increases in seed weights and size when compared to WT seeds (FIG. 13A,B). However, changes were much more pronounced in 6×amiBPM #1 than in 6×amiBPM #2 plants, which may be due to different activities of the 35S promoter in seeds of the two lines. They also showed increased WRI1 content in seeds and elevated expression of the two target genes BCCP1 and AtGLB1 (FIG. 13C). Similar to increases in weight, both lines also showed altered total fatty acid contents (FIG. 13D); and while changes in 6×amiBPM #2 plants were only very mild and furthermore non-significant ( ̃96 μg/30 seeds in average versus  ̃93 μg/30 seeds in WT), the total fatty acid content in 6×amiBPM #1 seeds was increased by around 50% ( ̃140 μg/30 seeds) when compared to WT. The wri1-3 line was used in these experiments as a control, and showed a significant reduction in both seed weight and total fatty acid contents ( ̃79 μg/30 seeds) when compared to WT and the two 6×amiBPM lines. While changes were observable for total fatty acid content measurements of individual fatty acids did not detect any significant changes (FIG. 14). In addition, no significant changes were observed in a general metabolic profile (amino and organic acids as well as soluble sugar) for wri1-3 or the two amiBPM lines when compared to WT (FIG. 14), indicating that the changes in seed size and weight for the mutants are primarily based on aberrant fatty acid contents. Overall these data further underscore that BPM proteins are critical regulators of WRI1 activity, and that their loss positively affects both WRI1 stability as well as its actions.
Because seeds of the two 6×amiBPM lines varied significantly in their weight and fatty acid content, we included a third 6×amiBPM line in our analysis to ensure that loss of BPMs is leading in a reproducible manner to increases in fatty acid content and seed size. As observed for the other two lines, 6×amiBPM #3 seeds are also increased in size (FIG. 15B) as well as in dry weight (FIG. 15C), and this correlated with elevated WRI1 content (FIG. 15A), as well as up-regulated expression of BCCP1 and AtGLB1 (FIG. 15D). Likewise, we also observed a significant increase in fatty acid content in these seeds in comparison to WT (FIG. 15E), substantiating findings for 6×amiBPM #1 plants that reduced BPM activity likely result in higher fatty acid levels in seeds.
Discussion
This example shows that BPM proteins have the ability to interact with a broad-range of ERF/AP2 proteins. Y2H studies indicate that many ERF/AP2 proteins are targeted in Arabidopsis by a CRL3BPM complex. IP and pull down studies in this work underscore that WRI1 assembles in vitro and in the plant into a complex with CUL3, and the missing CUL3-WRI1 assembly in 6×amiBPM and MATH-overexpressing backgrounds emphasizes that BPM proteins are required for this step. The studies further show that the interaction of BPMs with WRI1 results in the destabilization of their substrate. This is supported by the finding that WRI1 is stabilized in a cul3hyp background, as well as in MATH overexpression and 6×amiBPM plants. Moreover, the ChIP data strongly supports our conclusion that WRI1-CRL3BPM assembly occurs at the DNA level. Consequently, BPM proteins can be considered as negative regulators of WRI1 activities by mediating assembly with the CRL3 core, and ultimately causing its degradation via the 26S proteasome. This is also supported by the finding that fatty acid levels are significantly increased in seeds of 6×amiBPM #1 and #3 plants. These changes resemble earlier descriptions for plants overexpressing WRI1 (Cernac and Benning. 2004). However, it is significant to note that similar changes can be accomplished in Arabidopsis without ectopically expressing a transgenic WRI1 in seeds. The fact that overall metabolic changes were mostly restricted to total fatty acid contents also indicates that the function of BPM proteins in seeds is strongly connected with WRI1 activity. In this context, it is of note that WRI1 has very recently been described as part of a small gene family with a total of four members in Arabidopsis (To et al. 2012). Although WRI1 is the primary member that controls fatty acid biosynthesis in seeds, the other members also contribute to this pathway but in other tissues (To et al. 2012). The current findings also support the earlier suggestion that instability of RAP2.4, another BPM interacting protein, is mediated by a CRL3BPM ligase (Weber and Hellmann, 2009). Overall, it is likely that a general consequence of BPM interaction with ERF/AP2 transcription factors is degradation of the latter.
In this context, it is also important to note that the BPM family members have very recently been established as regulators of an ABA response by targeting the Homeodomain-Leucine Zipper transcription factor AtHB6 for degradation (Lechner et al., 2011). Consequently, plants with reduced levels of BPM1, 4, 5, and 6 (amiR-bpm) display aberrant responses in stomatal opening (Lechner et al., 2011); however, germinating amiR-bpm seedlings only display increased ABA resistance when combined with an AtHB6 overexpression background. The finding that a member of another transcription factor family is a substrate of BPM proteins, further increases the number of potential substrate proteins that are targeted by CRL3BPM for degradation. Furthermore, many members of the ERF/AP2 family have been described in context with stress tolerance including the ones tested in this study. For example DREB1a is a classical regulator of drought and cold tolerance responses in plants (Sakuma et al., 2002; Miura et al., 2007). ERF1 is known to play a role in biotic stress (Lorenzo et al., 2003; Zhang et al., 2011), and RAV1 has been described in context with senescence and different abiotic stress conditions (Sohn et al., 2006; Woo et al., 2010; Yun et al., 2010). It is therefore likely that both MATH overexpression and 6×amiBPM plants display different sensitivities towards stress such as cold or drought, and treatments with phytohormones such as ethylene or jasmonic acids, in addition to ABA.
It is also noteworthy that degradation of WRI1 appears to occur continuously rather than being stimulated by a specific signal, and this also holds true for RAP2.4 and AtHB6 (Weber and Hellmann, 2009; Lechner et al., 2011). It is unlikely that the cell is degrading these proteins always to the same amount since this seems to be a quite inefficient and uneconomical approach to control protein amounts. Rather one would expect that specific signals are in place that slow down turnover of CRL3BPM substrates similar to ethylene signal transduction, where ethylene disrupts proteasomal degradation of E1N3 mediated by the F-box proteins EBF1 and EBF2 (Guo and Ecker, 2003; Potuschak et al. 2003). In fact, ABA treatment has a stabilizing impact on AtHB6, but the kind of signal that may have a similar impact on WRI1 or RAP2.4 remains unclear.
The wide-ranging developmental changes in both 6×amiBPM, as well as MATH overexpression lines, emphasizes that the BPM family is widely required for plant development. amiR-BPM plants showed a reduced shoot growth (Lechner et al., 2011), which we also observed for 6×amiBPM lines. Interestingly, we could not detect any problems in fertility as observed by Lechner and co-workers for amiR-bpm plants. In addition, 6×amiBPM plants had a strongly reduced root development and fewer leaves, and it remains open whether these changes were also seen by Lechner et al (2011). Moreover, changes in root development were not apparent in MATH overexpression lines, indicating that reduced BPM expression and binding competition approaches differently affected the developmental program of the root in the corresponding plants.
The different approaches followed in this work to affect CRL3BPM-WRI1 interplay revealed two additional interesting aspects about the function of BPM proteins besides being substrate receptors to a CRL3BPM ligase. First, comparing cul3hyp and 6×amiBPM plants clearly demonstrated that in both genetic backgrounds WRI1 protein content is increased due to greater stability of the transcription factor. However, the transcriptional activity of WRI1 was only elevated in 6×amiBPM plants but not in cul3hyp, as indicated by the changed versus unchanged transcriptional levels of AtGLB1 and BCCP1. Given that in the cul3hyp mutant BPM protein levels are likely normal, these findings indicate that BPM proteins negatively interfere with WRI1 activity, most likely by binding to the transcription factor, while more active WRI1 is available in 6×amiBPM plants. Secondly, the reduced WRI1 amount was quite surprising and unexpected. Because WRI1 expression was up-regulated in MATH overexpressors, one may suggest that the reduced WRI1 content was sensed by the cell, and that changes on the transcriptional level represent a feedback-loop response. In addition, these data also clearly indicate that the MATH domain also interfered with post-transcriptional processes, and thus point out that BPM proteins may have even further diverse roles in addition to targeting ERF/AP2 or AtHB6 transcription factors for ubiquitylation and proteasomal degradation.
Finally, the ChIP data strongly indicate that CRL3BPM E3 ligase assembles with WRI1 at the DNA level while the transcription factor is bound to its target sites. In summary, the current work reveals a new link between fatty acid metabolism and CUL3-based E3 ligase activities. The work also confirms that BPM proteins function in planta as substrate receptor proteins to a CRL3BPM ligase with the purpose to destabilize bound substrates. These findings further indicate that a large number of ERF/AP2 proteins are targets of BPM proteins, and that this complex plays a major role in plant development and stress tolerance by broadly regulating transcriptional, and potentially post-transcriptional, processes in the plant.
Example 2. Modulation of BPM Expression or Activity Enhances Several Yield-Related Traits
The transgenic plants (6×amiBPM and BPMMATH:NLS) as described in Example 1 were tested under varying conditions to assess the effects on several yield-related traits. For salt stress tolerance assays, wild type (WT) and transgenic plants (6×amiBPM and BPMMATH:NLS) were plated on solid minimal culture medium, and grown vertically for five days. Afterwards, they were carefully transferred to plates that were supplemented with 150 mM NaCl. The transgenic plants had significantly increased root growth from day 3 to 6 after the addition of salt as compared to the WT plants (FIG. 18A). Wild type root elongation growth at day six was significantly more inhibited under salt stress conditions than in transgenic plants (FIG. 18B).
The 6×amiBPM plants were then tested under drought conditions. After withholding water for four days, significant changes were observed between WT and 6×amiBPM plants which indicate increased sensitivity of the transgenic plant towards drought stress (FIG. 19).
The 6×amiBPM plants were also found to affect the flowering phenotype as compared to WT plants. Expression of Flowering Locus T (FT), a key regulator of the flowering time point, is significantly down regulated in 6×amiBPM plants when compared to WT which is in agreement with the late flowering phenotype of the transgenic plants (FIG. 20A). FIG. 20B shows a schematic drawing of six different FT promoter regions analyzed via qPCR after α-CUL3 ChIP experiment. Significant enrichments were detectable in regions 1, 5 and 6 in WT, but not in a 6×amiBPM#1 control, indicating that CRL3BPM E3 ligases are directly involved in controlling FT expression (FIG. 20C).
Inducible 6×amiBPM constructs were generated and shown to allow for controlled increase in seed size (FIG. 21). Treatment of plants with estradiol over a time period of 24 hours leads to a significant down-regulation of all six BPM genes (FIG. 21A). pMDC7:6×amiBPM plants that carry an estradiol inducible construct are indistinguishable from wild type plants when not treated with estradiol (FIG. 21B). When the transgenic plants were sprayed with estradiol for about 2 weeks, the seeds in estradiol-treated 6×amiBPM plants were significantly larger (FIGS. 21C and E), and heavier (FIG. 21D) than WT seeds.
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While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

## Claims

1. A transgenic plant, wherein said plant is genetically engineered to express a nucleic acid so as to down-regulate expression or reduce the activity of all of the members of the BPM (BTB/POZ-MATH) protein family as compared to a control plant, wherein said transgenic plant exhibits enhanced yield-related traits as compared to said control plant, and wherein said enhanced yield-related traits are selected from the group consisting of increased seed oil production, increased salt stress-tolerance, and increased drought stress-tolerance as compared to said control plant.

2. The transgenic plant of claim 1, wherein said plant is of the Brassicaceae family.

3. The transgenic plant of claim 2, wherein said plant is Arabidopsis Thaliana.

4. The transgenic plant of claim 1, wherein said transgenic plant exhibits increased seed oil production as compared to said control plant.

5. A method for increasing seed oil production in a transgenic plant as compared to a control plant and recovering said seed oil, said method comprising: cultivating a transgenic plant under conditions promoting plant growth and development, wherein said transgenic plant is genetically engineered to express a nucleic acid so as to exhibit increased seed oil production as compared to a control plant by down-regulating expression or reducing the activity of all of the members of the BPM protein family as compared to a control plant; and recovering said seed oil from said transgenic plant.

6. The method of claim 5, wherein said plant is of the Brassicaceae family.

7. The method of claim 6, wherein said plant is Arabidopsis Thaliana.

8. A method for enhancing yield-related traits in a plant as compared to a control plant, said method comprising: genetically engineering said plant to express a nucleic acid so as to down-regulate expression or reduce the activity of all of the members of the BPM protein family as compared to a control plant, wherein said yield-related traits are selected from the group consisting of increased seed oil production, increased salt stress-tolerance, and increased drought stress-tolerance as compared to said control plant, and testing the genetically engineered plant to assess a yield-related trait selected from the group consisting of seed oil production, salt stress-tolerance, and drought stress-tolerance.

9. The method of claim 8, wherein said plant exhibits increased seed oil production as compared to a control plant.

10. The method of claim 8, wherein said plant is of the Brassicaceae family.

11. The method of claim 10, wherein said plant is Arabidopsis Thaliana.

12. The method of claim 8, wherein said step of genetically engineering comprises introducing artificial microRNA (amiRNA) to down-regulate all of the members of the BPM protein family.

13. A method for enhancing yield-related traits in a plant as compared to a control plant, said method comprising: genetically engineering said plant to express a nucleic acid so as to reduce the activity of at least one BPM protein as compared to a control plant, wherein said yield-related traits are selected from the group consisting of increased seed oil production, increased salt stress-tolerance, and increased drought stress-tolerance as compared to said control plant, wherein said step of genetically engineering comprises the expression of at least one exogenous MATH domain to compete with said BPM protein, and testing the genetically engineered plant to assess a yield-related trait selected from the group consisting of seed oil production, salt stress-tolerance, and drought stress-tolerance.

14. A product produced by or from a transgenic plant which is genetically engineered to express a nucleic acid so as to enhance yield-related traits by down-regulating expression or reducing the activity of all of the members of the BPM protein family as compared to a control plant, wherein said enhanced yield-related traits are selected from the group consisting of increased seed oil production, increased salt stress-tolerance, and increased drought stress-tolerance as compared to said control plant, and wherein the product comprises the expressed nucleic acid.