Patent Publication Number: US-10308950-B2

Title: Methods for increasing oil content in plant tissues by suppressing hydrophobic lipid droplet protein

Description:
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/350,843, entitled “Methods for Increasing Oil content in Plant Tissues by Suppressing Hydrophobic Lipid Droplet Protein,” filed on Jun. 16, 2016, the entire contents of which are hereby incorporated by reference. 
    
    
     BACKGROUND 
     This disclosure pertains to regulation of oil content in plant tissues through manipulation of proteins. 
     Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles found in a wide range of unicellular and multicellular organisms. Uniquely delineated by a single phospholipid monolayer and coated with a diverse set of proteins, LDs function primarily in the storage of energy-rich neutral lipids, such as triacylglycerols. In plants, LDs have been studied mostly in oilseeds, although all plant cell types, including vegetative tissues, have the machinery required for accumulating and storing lipids in LDs. 
     A number of high value oils are produced by plants, and increasing the oil content in the cells of these oil-producing plants would be highly advantageous to the supply of these oils. 
     SUMMARY 
     The present disclosure relates generally to increasing oil content in plant cells. In particular, this disclosure pertains to manipulation of proteins in order to affect regulation of cytoplasmic lipid droplets (LDs) in plant cells. 
     A class of proteins in  Arabidopsis thaliana  ( Arabidopsis ) has been identified and named LD-associated proteins (LDAPs). These proteins are abundant components of LDs in non-seed cell types and are required for their proper compartmentation. In an effort to identify other novel LD proteins, the LDAPs were used as ‘bait’ proteins in yeast two-hybrid screens against an  Arabidopsis  cDNA ‘prey’ library. One LDAP3 interactor, encoded by gene At5g16550 (based on the genome sequence annotation for  Arabidopsis  at TAIR [The  Arabidopsis  Information Resource]), is a protein annotated to be of unknown function, but possesses a unique mycobacterial membrane protein large (MMPL) domain found in members of the  M. tuberculosis  protein family involved in lipid transport. Similar to the LDAPs, At5g16550 is expressed in  Arabidopsis  in a variety of tissues and at various developmental stages, and is targeted to the LD surface with high specificity in leaf cells. Furthermore, analyses of an  Arabidopsis  at5g16550 mutant revealed conspicuously enlarged LDs and increased neutral lipid content in both leaves and mature seeds, demonstrating that At5g16550 plays a role in regulating LD size and neutral lipid levels in plant cells. 
     The present disclosure relates generally to reduction or elimination of gene expression of At5g16550 or its homolog in plants in order to increase oil content. Preferred embodiments include transgenic plants or seeds having suppressed gene expression of AT5G16550 or its homologs and which produce increased oil content through the inclusion of substantially larger LDs. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  shows a phylogenic tree of  Arabidopsis  At5g16550 (**At1) and its predicted protein homologs in plants, including other angiosperms (dicots and monocots), as well as gymnosperms and mosses, generated using TreeView with sequence alignment generated using BioEdit. 
         FIG. 1B . shows the genomic sequence of the  Arabidopsis  At5g16550 gene, with sequences corresponding to the three exons in the At5g16550 gene in bold type and 5′ and 3′ untranslated regions (UTRs) italicized (SEQ ID NO:2). 
         FIG. 2  shows a hydropathy profile of  Arabidopsis  At5g16550 (based on the Center of Biological Sequence Analysis [CBS; cbs.dtu.dk]). 
         FIG. 3A  shows confocal laser-scanning microscopy (CLSM) images of transiently-expressed Cherry-tagged full-length  Arabidopsis  At5g16550 localized to BODIPY 493/503-stained cytoplasmic lipid droplets (LDs) in a tobacco leaf cell, with the corresponding merged image. 
         FIG. 3B  shows CSLM images of transiently-expressed Cherry alone and BODIPY 493/503-stained cytoplasmic LDs in a tobacco leaf cell. 
         FIG. 4  shows At5g16550 expression in different  Arabidopsis  tissues, organs, and developmental stages. 
         FIG. 5  shows the results of yeast two-hybrid assays for At5g16550 and LDAP3, and the LDAP3 mutant LDAP3 C100  along with empty vector controls. 
         FIG. 6A  shows CLSM images of the bimolecular fluorescence complementation (BiFC) of (co)transiently-expressed cCFP-tagged At5g16550 and nVenus-tagged LDAP3 or the LDAP3 C100  mutant along with an expressed Cherry-PTS1 transformation marker protein in tobacco leaf cells. 
         FIG. 6B  shows the relative amounts of BiFC signal for cotransiently-expressed cCFP-At5g16550 and either nVenus-tagged LDAP3 or LDAP3 C100  in tobacco leaf cells. 
         FIG. 6C  shows CLSM images of the cCFP-At5g16550 and nVenus-LDAP3 BiFC fluorescence signal colocalized with MDH-stained cytoplasmic LDs in a transiently-transformed tobacco leaf cell. 
         FIG. 7A  shows T-DNA locations, zygosity, and At5g16550 transcript levels in the  Arabidopsis  at5g16550 knockdown (SALK_084555) mutant line. 
         FIG. 7B  shows T-DNA locations, zygosity, and At5g16550 transcript levels in the  Arabidopsis  at5g16550 knockout (SAIL_335_H11) mutant line. 
         FIG. 8A  shows CLSM micrographs of LDs stained with BODIPY 493/503 along with chlorophyll autofluorescence of 15 day-old leaves from WT and at5g16550 suppression mutant lines (SALK_084555 [knockdown] and SAIL_335_H11 [knockout]) at the end of the night, when LD abundance is high in WT, with arrowheads highlighting examples of enlarged lipid droplets in the knockout mutant leaves. 
         FIG. 8B  shows quantification of number and sizing of LDs in 15 day-old leaves from wild-type (WT) and the at5g16550 knockdown (SALK_084555) and knockout (SAIL_335_H11) at the end of day or the end of the night. 
         FIG. 9A  shows quantification of neutral lipid content in 15 day-old leaves from WT and at5g16550 suppression mutant lines (SALK_084555 [knockdown] and SAIL_335_H11 [knockout]) at the end of the day and end of the night as indicated. 
         FIG. 9B  shows quantification of fatty acid composition in 15 day-old leaves from WT and at5g16550 suppression mutant lines (SALK_084555 [knockdown] and SAIL_335_H11 [knockout]) at the end of the day and end of the night. 
         FIG. 10A  shows CLSM images of BODIPY-stained LDs in wild-type (WT), and at5g16550 SAIL_335_H11 (knockout) and SALK_084555 (knockdown) mature, dry  Arabidopsis  seeds, with arrowheads highlighting examples of enlarged LDs in both knockdown and knockout mutant seeds. 
         FIG. 10B  shows total fatty acid content (left) and content of different fatty acids (right) in WT and at5g16550 SAIL_335_H11 (knockout) and SALK_084555 (knockdown) mature, dry  Arabidopsis  seeds. 
         FIG. 11A  shows CLSM images of BODIPY-stained LDs in WT and at5g16550 SAIL_335_H11 (knockout) and SALK_084555 (knockdown)  Arabidopsis  seedlings 1, 2 and 4 days after germination (DAG), shown in top, middle and bottom row of micrographs, respectively. 
         FIG. 11B  shows total fatty acid content in WT and at5g16550 SAIL_335_H11 (knockout) and SALK_084555 (knockdown)  Arabidopsis  seedlings 1, 2 and 4 days after germination. 
         FIG. 11C  shows content of different fatty acids in WT and at5g16550 SAIL_335_H11 (knockout) and SALK_084555 (knockdown)  Arabidopsis  seedlings 1, 2 and 4 days after germination, shown in top and bottom left and right graphs, respectively. 
         FIG. 12  shows a CLUSTALW amino acid alignment of  Arabidopsis  At5g16550 (SEQ ID NO:1) with potential orthologous proteins from major oilseed crops, including palm (Genbank accession XP_010905612.1) (SEQ ID NO:3), soybean (XP_003538382.1) (SEQ ID NO:4), rapeseed (XP_013649840.1) (SEQ ID NO:5), camelina (XP_010492567.1) (SEQ ID NO:6), sunflower (OTF96048.1) (SEQ ID NO:7), and peanut (PeanutBase accession Aradu.C5TRQ) (SEQ ID NO:8), with identical and similar amino acid residues indicated with asterisks and colons or periods, respectively. 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     Generally, the present disclosure relates to increasing the occurrence and size of cytoplasmic lipid droplets (LDs) in seeds and leaves through reduction or elimination of expression of certain proteins, namely a protein encoded by gene At5g16550 in  Arabidopsis thaliana , or its homolog in related plant species. Preferred embodiments include methods for producing plants having increased size of LDs or oil content by introducing mutations in At5g16550 protein-coding genes or their homologs to reduce or eliminate expression of these proteins. Additional preferred embodiments include plants, plant cells, or plant seeds having mutations in At5g16550 like proteins, which produce LDs with increased size or oil content in the resulting plants. Further, overexpression of At5g16550 or its homologs in plant cells will yield an increase in LD number. 
     The examples described herein are carried out with particular attention to  Arabidopsis  plants and the  Arabidopsis  protein encoded by gene At5G16550 (gene locus identifier based on the genome sequence annotation for  Arabidopsis  at TAIR). However,  Arabidopsis  is frequently used as a representative plant system (Koornneef and Meinke, 2010; Provart et al., 2016), including for oilseed crops (Wallis and Brown, 2010; Li-Beisson et al., 2013) and, accordingly, it is expected that similar results would be obtained for any plant through similar manipulation of homologs of At5g16550 proteins. Homologs of  Arabidopsis  of At5g16550 are broadly conserved in higher to lower plant species and the majority of plant species contain one or, in those plants whose genomes underwent whole-genome duplications, two At5g16550-like proteins. 
     EXAMPLE 1 
     Sequence and Hydropathy Profile 
     The deduced amino acid sequence of  Arabidopsis  At5g16550 is shown below: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 1) 
                   
               
            
           
           
               
               
               
            
               
                 MAQDHDETEN KTFADVVGGD DVGEIVNGGT KNGYRKPDSV EKEDDEDLKS 
                  50 
                   
               
               
                   
               
               
                 LYSLICLTIG SILFPDSKTG DASSFLERVR NSVAENGPKL REASERTGRE 
                 100 
               
               
                   
               
               
                 ILLWTRRGSS LRALLVITMG TIVLLTTMAL VVFTLFFVAA TANAIIISLL 
                 150 
               
               
                   
               
               
                 ISLAVAGGFL ALFFLCLTGV YIGALSVAAF VISTATVSAV VSVLIASGWI 
                 200 
               
               
                   
               
               
                 GFFYAVWLGT RGSLRLAKQS VSVVGSAISG NTISRHQHQD REVNIESTN* 
                 249 
               
            
           
         
       
     
     This protein was identified in a yeast two-hybrid screen of an  Arabidopsis  cDNA library using the  Arabidopsis  lipid droplet-associated protein isoform 3 (LDAP3) as ‘bait.’ At5g16550 is the only protein in  Arabidopsis  annotated (based on previous annotations at TAIR (The  Arabidopsis  Information Resource) and the National Centre for Biotechnology Information [NCBI; ncbi.nlm.nih.gov]) to contain an MMPL domain, a membrane transport protein domain. The N-terminal sequence upstream of the MMPL domain in  Arabidopsis  At5g16550 is found at residues 1-106, the C-terminal sequence downstream of the MMPL domain is found at residues 212-249, and the MMPL domain sequence is found at residues 107-211. MMPL domain-containing proteins are present also in the mycobacterial membrane protein large (MMPL) family of proteins in  M. tuberculosis , wherein they serve as cell membrane proteins involved in transport of various lipids across the cell membrane to the cell wall (Tekaia et al., 1999; Pacheco et al., 2013; Viljoen et al., 2017 and references therein). Similar to the plant-specific LDAPs (Gidda et al., 2012; Horn et al., 2013; Divi et al., 2016), homologs of  Arabidopsis  At5g16550 are broadly conserved in all plant species, including mosses, gymnosperms, and angiosperms (with reference to the phylogenetic tree shown in  FIG. 1A ), and other than the abovementioned MMPL-domain-containing proteins in  M. tuberculosis , they appear to be specific to the plant kingdom. Further, while many plant species contain only one copy of an At5g16550-like gene, others, such as grasses, Brassiceae and legumes, possess two copies, consistent with their past whole-genome duplications.  FIG. 1B  shows the full sequence of the At5g16550 gene (SEQ ID NO:2), based on TAIR (The  Arabidopsis  Information Resource). In  FIG. 1A , sequences were obtained from Phytozome and abbreviations are based on the genus and species and gene copy number, e.g., in  Arabidopsis thaliana  (At1) At5g16550 is present as a single copy; two At5g16550-like genes exist in  Glycine max  (Gm1 and Gm2). 
       FIG. 2  shows a hydropathy profile of  Arabidopsis  At5g16550 (based on the Center of Biological Sequence Analysis [CBS; cbs.dtu.dk]), which reveals an extensive hydrophobic region corresponding to the protein&#39;s MMPL domain-like sequence (residues 107-211). Without wanting to be bound by theory, based on the characterization of members of the  M. tuberculosis  MMPL family (Converse et al., 2003; Varela et al., 2012; Pacheco et al., 2013), the MMPL-domain-like sequence in the  Arabidopsis  At5g16550 protein may serve as a membrane anchor and/or a channel for transporting lipids. 
     EXAMPLE 2 
     Intracellular Localization, Expression, and Interaction with LDAP3 
       FIG. 3A  shows representative confocal laser-scanning microscopy (CLSM) images of Cherry-tagged full-length  Arabidopsis  At5g16550 localized to cytoplasmic lipid droplets (LDs) in tobacco leaf epidermal (pavement) cells. Leaves of 4-week-old tobacco ( Nicotiana benthamiana ) plants were co-infiltrated with  Agrobacterium tumefaciens  carrying binary vectors encoding Cherry-At5g16550 (pMDC32/Cherry-At5g16550 driven by the 35S constitutive promoter) or the tomato bushy stunt virus P19 to enhance transgene expression according to the methods described elsewhere (McCartney et al., 2005; Petrie et al., 2010; Cai et al., 2015). LDs were stained with the fluorescent neutral lipid dye BODIPY 493/503. Microscopic images were acquired using a Leica DM RBE microscope with a Leica TCS SP2 scanning head, and the Leica TCS NT software package (Leica). Based on the merged image, Cherry-At5g16550 localizes to LDs in tobacco leaf cells, similar to the intracellular localization reported for its putative interacting protein partner, LDAP3 (Horn et al., 2012; Gidda et al., 2016). Further, compared to the CLSM images of BODIPY-stained LDs in tobacco leaf cells transiently expressing the Cherry fluorescent protein alone ( FIG. 3B ), ectopic expression of Cherry-At5g16550 appears to result in a relative increase in LD numbers in tobacco leaf cells (compare BODIPY staining in  FIGS. 3A and 3B ). 
       FIG. 4  shows At5g16550 expression in  Arabidopsis . RT-PCR analysis revealed that At5g16550 transcripts are detectable in a variety of  Arabidopsis  tissues/organs and developmental stages (as indicated by labels), including in the mature (dry) seed and germinating seed (i.e., 24 h post-imbibition), germinated seedlings, and in various vegetative tissue of 30-day-old plants, suggesting that the protein is a constitutive component of the LD proteome throughout the plant lifecycle. Endogenous tubulin was used as a reference gene in a parallel set of RT-PCRs. All RT-PCRs were performed using cDNA derived from isolated total RNA. 
       FIGS. 5 and 6  show the interaction of At5g16550 and LDAP3 in yeast and plant cells. Yeast strains were co-transformed with indicated pairs of GAL4-activation domain (AD) and GAL4-binding domain (BD) fusion proteins or the corresponding empty BD or AD control plasmids and then spotted (as serial dilutions) onto agar plates containing either low-stringency (SD-Leu,Trp) or high-stringency media (SD-Leu,Trp,His,Ade), where cell growth is dependent on two-hybrid protein interaction. As shown in  FIG. 5A , co-expression of AD-At5g16550 and BD-LDAP3, but not a mutant version of BD-LDAP3 (BD-LDAP3 C100 ), whereby the C-terminal 100 amino acid residues of the protein known to be necessary for targeting to LDs were removed (Gidda et al., 2016), resulted in significant yeast growth on high selection media, indicating that At5g16550 and LDAP3 interact. Consistent with these data,  FIG. 6  shows the results of in vivo bimolecular fluorescence complementation (BiFC) assays indicating that At5g16550 and LDAP3 interact at LDs in plant cells. Tobacco leaves were co-infiltrated with binary vectors encoding the C-terminal half of the cyan fluorescent protein (cCFP) appended to At5g16550 and the N-terminal half of the Venus fluorescent protein (nVenus) appended to either full-length LDAP3 or LDAP3 C100 . In addition, cells were transformed with a peroxisomal marker protein (consisting of Cherry fused to a type 1 peroxisomal targeting signal [Cherry-PTS1] (Ching et al., 2012), which was used to identify transformed cells and also served as an internal normalization for reconstituted BiFC, minimizing differences due to cell-to-cell variability in expression levels. Representative CLSM images ( FIG. 6A ) and quantifications of BiFC signal based on &gt;25 leaf areas expressing Cherry-PTS1 from at least two separate infiltrations ( FIG. 6B ) revealed that cells co-transformed with cCFP-At5g16550 and nVenus-LDAP3 displayed significantly more BiFC (punctate) signal than cells co-transformed with LDAP3 C100 . Further, higher magnified CLSM imaging of the cCFP-At5g16550 and nVenus-LDAP3 BiFC punctate (as seen in  FIG. 6A ), along with staining of LDs with the blue-fluorescent neutral lipid dye monodansylpentane (MDH), confirmed that the two proteins were interacting at LDs in plant cells ( FIG. 6C ). 
     EXAMPLE 3 
     Transgenic  Arabidopsis  at5g16550 Suppression Mutant Lines 
     Shown in  FIG. 7  are the T-DNA locations, zygosity, and AT5G16550 transcript levels in the  Arabidopsis  at5g16550 knockdown (SALK_084555) ( FIG. 7A ) and knockout (SAIL_335_H11) ( FIG. 7B ) mutant lines obtained from the  Arabidopsis  Biological Resource Centre (ABRC). The schematic diagrams show the organization of the AT5G16550 gene, which is annotated by TAIR to contain three exons (represented by black bars), the second of which contains the T-DNA insert in the knockout line, and two introns, the first of which contains the T-DNA insert in the knockdown line. The relative positions of At5g16550-specific primer pairs are also shown in each diagram as black arrows. Two independent lines for each mutant (i.e., SALK #3 and #5; SAIL #1 and #4) were confirmed to be homozygous, as evidenced by the PCR screening results (see ‘Zygosity check’). Ethidium bromide-stained agarose gels revealed that PCR amplicons were not generated when using gene-specific primers CF-FP and CF-RP, which flank the location of the T-DNA insert. Conversely, using wild-type (WT) DNA, the same primers generated an amplicon of a specific size due the absence of a T-DNA. The location of the T-DNA was also confirmed in the two plants using a T-DNA-specific primer (LBb or SAIL-RP) and the At5g16550 gene-specific primer CF-FP. As shown under ‘RT-PCR’, a reduction or apparent lack of full-length AT5G16550 transcript levels was observed in both sets of lines (i.e., SALK #3 and #5; SAIL #1 and #4) compared to wild-type levels using primers RT MMPL-FP and RT MMPL-RP. Primers designed for the amplification of endogenous α-tubulin transcripts were used as a control. Taken together, these results confirm that SALK_084555 and SAIL_335_H11 are bona fide at5g16550 knockdown and knockout  Arabidopsis  mutant lines, respectively. 
     EXAMPLE 4 
     Increased LD Size Heterogeneity and Neutral Lipid Content in Leaves in AT5G16650 Suppression Mutant Lines 
     As shown in  FIG. 8 , LD size and abundance in leaves are affected in transgenic  Arabidopsis  At5g16550 suppression mutant lines. Leaves of 15-day-old WT, At5g16550 suppression mutant (SALK_084555), and At5g16550 knockout mutant (SAIL_335_H11) plants were formaldehyde fixed at the end of the dark cycle, when LD abundance is highest in WT plants (Gidda et al., 2016), then LDs were stained with BODIPY 493/503 and imaged (along with chlorophyll autofluorescence), using CLSM, as described elsewhere (Cai et al., 2015; Gidda et al., 2016). Representative micrographs are shown in  FIG. 8A , and LD numbers and sizes for each line (n=9) at both end of the night and end of the day, when LD abundance is highest and lowest in WT plants, respectively (Gidda et al., 2016), were quantified using ImageJ according to Cai et al. (2015) ( FIG. 8B ). Arrowheads in the representative micrograph of the knockout mutant ( FIG. 8A , right panel) highlight the presence of several ‘supersized’ LDs, which were not observed in leaves of WT or the knockdown mutant. Consistent with these observations, quantification of LD size and abundance (see corresponding graphs in  FIG. 8B ) revealed that while the total number of LDs in the knockdown and knockout mutants at both the end of day and end of the night was significantly lower than that of the WT (i.e., ˜28 and 23 versus 48, and ˜340 and 210 versus 510; compare y-axes), the number of LDs with diameters greater than the average LD diameter in WT was significantly greater in the knockout mutant. That is, the sizes of LDs in the knockout mutant ranged from the typical 0.2 μm seen in WT (and knockdown plants) at the end of the day and end of the night to over 1.6 μm. 
       FIG. 9  shows an analysis of neutral lipid and fatty acid content in 15-day old WT and at5g16550 suppression mutant lines (i.e., SALK_084555 [knockdown] and SAIL_335_H11 [knockout]) at the end of the night and end of the day, when as already mentioned, LD abundance is highest and lowest, respectively in WT plants (Gidda et al., 2016). Briefly, five biological replicates of leaf material were weighed and flash frozen, then total lipids were extracted from the tissue using a hexane/isopropanol method. Neutral lipids were subsequently isolated by solid-phase extraction and analyzed by gas chromatograpy and flame ionization detection. Values represent average and standard deviation; *p&lt;0.05. Note the relative increase in neutral lipid content in both the knockdown and knockout at5g16550 mutant lines compared to WT at both the end of the day and end of the night ( FIG. 9A ), due primarily to increases in polyunsaturated (i.e., 18:2 and 18:3) fatty acids ( FIG. 9B ). Taken together and with those presented above, these data indicate that At5g16550 plays an important role in LD biogenesis (i.e., number and size) and also the accumulation of total neutral lipids in leaves. 
     EXAMPLE 5 
     Increased LD Size and Seed Oil Content in at5g16650 Suppression Mutant Lines 
     Suppression of At5g16550 gene expression results in the presence of abnormally large LDs in seeds and an increase in total seed oil content.  FIG. 10A  shows representative, CLSM images of BODIPY-stained LDs in WT, SAIL_335_H11 (knockout) and SALK_084555 (knockdown) mature, dry  Arabidopsis  seeds. Briefly, seeds were imbibed in deionized water for 20 minutes and the embryos were rolled out of the seed coats with a cover slip and imaged via CLSM. Note the presence of grossly enlarged, ‘supersized’ LDs in both the knockout and knockdown mutant lines (refer to arrowheads). LDs of this size are not seen in WT seeds. These data reveal a novel role for At5g16550 in the biogenesis and/or stability of LDs in seeds. 
       FIG. 10B  shows an analysis of seed oil content and composition. Five biological replicates of seeds were weighed, then fatty acid methyl esters were prepared using methanolic-HCl, then the content and composition of seed oil was determined using GC-FID and tri-heptadecanoin as an internal standard. Values represent average and standard deviation; *p&lt;0.05. Note the increase in total seed oil content in both knockout and knockdown lines, with increases in stoichiometric amounts of nearly all fatty acid components of seed oil. Taken together and with those presented above, these data indicate that At5g16550 plays an important role not only in determining the cellular properties of LD in seeds, but also the accumulation of total oil within developing seeds. 
       FIG. 11  shows that seed oil breakdown is moderately affected in At5g16550 mutant seeds and correlates with the presence of abnormally large LDs during post-germinative growth.  FIG. 11A  shows representative, CLSM images of BODIPY-stained LDs in WT, SAIL_335_H11 (knockout) and SALK_084555 (knockdown) in  Arabidopsis  seedlings 1, 2 and 4 days after germination (shown in top, middle and bottom row of micrographs, respectively). Note the persistence of ‘supersized’ LDs in the knockdown mutant line at 2 and 4 days after germination (middle and bottom rows of micrographs, respectively).  FIGS. 11B and 11C  show an analysis of seed oil content and composition. Five biological replicates of seedlings were weighed, then fatty acid methyl esters were prepared using methanolic-HCl, then the content and composition of seed oil was determined using GC-FID and tri-heptadecanoin as an internal standard. Values represent average and standard deviation; *p&lt;0.05 (n=3). In  FIG. 11B  the increase in total seed oil content in both knockout and knockdown lines in mature seeds and seedlings occurred one day after germination, but only in the knockdown line 2 and 4 days after germination. Similarly, there were increases in stoichiometric amounts of nearly all fatty acid components of seed oil in both the knockdown and knockout seedlings 1, 2 and 4 days after germination. 
     The results discussed above show that  Arabidopsis  At5g16550 is a plant-specific protein ( FIG. 1 ), containing an MMPL domain ( FIG. 2 ), that was identified as an interacting protein with the LDAP isoform 3 ( FIGS. 5 and 6 ), and localizes to LDs of plant cells ( FIG. 3 ). The At5g16550 gene was expressed in all  Arabidopsis thaliana  (plant) tissues examined ( FIG. 4 ). Two mutants were identified with reduced or no expression ( FIG. 7 ), and these mutants had fewer but substantially larger lipid droplets in plant leaves ( FIG. 8 ) and increased neutral lipid content ( FIG. 9 ), suggesting that the At5g16550 protein may function to regulate LD size, abundance, and neutral lipid content in plants. Likewise, in dry seed cells of these mutants, there were also larger lipid droplets visible, and there was significantly more total lipid in these mutant seeds ( FIG. 10 ), as well as at several stages of post-germinative seedling growth ( FIG. 11 ). Given that genes similar to At5g16550 are easily identified in several commercially-important oilseed crops ( FIG. 12 ), and that  Arabidopsis thaliana  has been used as, and is accepted as, a representative plant system for oilseed crops (Wallis and Brown, 2010; Li-Beisson et al, 2013), this demonstrates that loss-of At5g16550-like gene function should increase the oil content in tissues of oil crops, like canola, Camelina, soybean, sunflower, safflower, cotton, palm, coconut, peanut, and others. 
     REFERENCES CITED 
     The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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