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Oil bodies have multiple functions: oleosin-mediated freezing tolerance of seeds, direct interaction with glyoxysomes for lipid degradation in seedlings, and antifungal compound production in leaves.

Eukaryotic cells acquired novel organelles during evolution through mechanisms that remain largely obscure. The existence of the unique oil body compartment is a synapomorphy of liverworts that represents lineage-specific acquisition of this organelle during evolution, although its origin, biogenesis, and physiological function are yet unknown. We find that two paralogous syntaxin-1 homologs in the liverwort Marchantia polymorpha are distinctly targeted to forming cell plates and the oil body, suggesting that these structures share some developmental similarity. Oil body formation is regulated by an ERF/AP2-type transcription factor and loss of the oil body increases M . polymorpha herbivory. These findings highlight a common strategy for the acquisition of organelles with distinct functions in plants, via periodical redirection of the secretory pathway depending on cellular phase transition. Liverworts have a unique oil body organelle unrelated to lipid stores found in other eukaryotes. Here the authors show that oil body formation is analogous to that of cell plates, relying on periodic redirection of the secretory pathway and a syntaxin-1 homolog, and that oil bodies contribute to defence against herbivory.

Lipid droplets (LDs) are lipid storage organelles in plant leaves and seeds. Seed LD proteins are well known, and their functions in lipid metabolism have been characterized; however, many leaf LD proteins remain to be identified. We therefore isolated LDs from leaves of the leaf LD–overaccumulating mutant high sterol ester 1 ( hise1 ) of Arabidopsis thaliana by centrifugation or co-immunoprecipitation. We then performed LD proteomics by mass spectrometry and identified 3,206 candidate leaf LD proteins. In this study, we selected 31 candidate proteins for transient expression assays using a construct encoding the candidate protein fused with green fluorescent protein (GFP). Fluorescence microscopy showed that MYOSIN BINDING PROTEIN14 (MYOB14) and two uncharacterized proteins localized to LDs labeled with the LD marker. Subcellular localization analysis of MYOB family members revealed that MYOB1, MYOB2, MYOB3, and MYOB5 localized to LDs. LDs moved along actin filaments together with the endoplasmic reticulum. Co-immunoprecipitation of myosin XIK with MYOB2-GFP or MYOB14-GFP suggested that LD-localized MYOBs are involved in association with the myosin XIK–LDs. The two uncharacterized proteins were highly similar to enzymes for furan fatty acid biosynthesis in the photosynthetic bacterium Cereibacter sphaeroides , suggesting a relationship between LDs and furan fatty acid biosynthesis. Our findings thus reveal potential molecular functions of LDs and provide a valuable resource for further studies of the leaf LD proteome.

To increase food production under the challenges presented by global climate change, the concept of de novo domestication—utilizing stress-tolerant wild species as new crops—has recently gained considerable attention. We had previously identified mutants with desired domestication traits in a mutagenized population of the legume Vigna stipulacea Kuntze (minni payaru) as a pilot for de novo domestication. Given that there are multiple stress-tolerant wild legume species, it is important to establish efficient domestication processes using reverse genetics and identify the genes responsible for domestication traits. In this study, we identified VsPSAT1 as the candidate gene responsible for decreased hard-seededness, using a Vigna stipulacea isi2 mutant that takes up water from the lens groove. Scanning electron microscopy and computed tomography revealed that the isi2 mutant has lesser honeycomb-like wax sealing the lens groove than the wild-type, and takes up water from the lens groove. We also identified the pleiotropic effects of the isi2 mutant: accelerating leaf senescence, increasing seed size, and decreasing numbers of seeds per pod. While doing so, we produced a V. stipulacea whole-genome assembly of 441 Mbp in 11 chromosomes and 30,963 annotated protein-coding sequences. This study highlights the importance of wild legumes, especially those of the genus Vigna with pre-existing tolerance to biotic and abiotic stresses, for global food security during climate change.

Sterols are important lipid constituents of cellular membranes in plants and other organisms. Sterol homeostasis is under strict regulation in plants because excess sterols negatively impact plant growth. HIGH STEROL ESTER 1 (HISE1) functions as a negative regulator of sterol accumulation. If sterol production exceeds a certain threshold, excess sterols are detoxified via conversion to sterol esters by PHOSPHOLIPID STEROL ACYL TRANSFERASE 1 (PSAT1). We previously reported that the Arabidopsis thaliana double mutant hise1-3 psat1-2 shows 1.5-fold higher sterol content than the wild type and consequently a severe growth defect. However, the specific defects caused by excess sterol accumulation in plants remain unknown. In this study, we investigated the effects of excess sterols on plants by analyzing the phenotypes and transcriptomes of the hise1-3 psat1-2 double mutant. Transcriptomic analysis revealed that 435 genes were up-regulated in hise1-3 psat1-2 leaves compared with wild-type leaves. Gene ontology (GO) enrichment analysis revealed that abiotic and biotic stress-responsive genes including RESPONSIVE TO DESICCATION 29B/LOW-TEMPERATURE-INDUCED 65 (RD29B/LTI65) and COLD-REGULATED 15A (COR15A) were up-regulated in hise1-3 psat1-2 leaves compared with wild-type leaves. Expression levels of senescence-related genes were also much higher in hise1-3 psat1-2 leaves than in wild-type leaves. hise1-3 psat1-2 leaves showed early senescence, suggesting that excess sterols induce senescence of leaves. In the absence of sucrose, hise1-3 psat1-2 exhibited defects in seedling growth and root elongation. Together, our data suggest that excess sterol accumulation disrupts cellular activities of vegetative organs including leaves and roots, resulting in multiple damages to plants.

Endoplasmic reticulum structures facilitate the increased secretion of proteins during the plant immune response.

Oil bodies are intracellular structures present in the seed and leaf cells of many land plants. Seed oil bodies are known to function as storage compartments for lipids. However, the physiological function of leaf oil bodies is unknown. Here, we show that leaf oil bodies function as subcellular factories for the production of a stable phytoalexin in response to fungal infection and senescence. Proteomic analysis of oil bodies prepared from Arabidopsis (Ambidopsis thaliana) leaves identified caleosin (CLO3) and a-dioxygenase (α-DOX1). Both CLO3 and α-DOX1 were localized on the surface of oil bodies. Infection with the pathogenic fungus Colletotrichum higginsianum promoted the formation of CLO3-and α-DOX1-positive oil bodies in perilesional areas surrounding the site of infection. α-DOX1 catalyzes the reaction from α-linolenic acid (a major fatty acid component of oil bodies) to an unstable compound, 2-hydroperoxyoctadecatrienoic acid (2-HPOT). Intriguingly, a combination of α-DOX1 and CLO3 produced a stable compound, 2-hydroxyoctadecatrienoic acid (2-HOT), from α-linolenic acid. This suggests that the colocalization of α-DOX1 and CLO3 on oil bodies might prevent the degradation of unstable 2-HPOT by efficiently converting 2-HPOT into the stable compound 2-HOT. We found that 2-HOT had antifungal activity against members of the genus Colletotrichum and that infection with C. higginsianum induced 2-HOT production. These results defined 2-HOT as an Arabidopsis phytoalexin. This study provides, to our knowledge, the first evidence that leaf oil bodies produce a phytoalexin under a pathological condition, which suggests a new mechanism of plant defense.

Plants strictly regulate the levels of sterol in their cells, as high sterol levels are toxic. However, how plants achieve sterol homeostasis is not fully understood. We isolated an Arabidopsis thaliana mutant that abundantly accumulated sterol esters in structures of about 1 µm in diameter in leaf cells. We designated the mutant high sterol ester 1 ( hise1 ) and called the structures sterol ester bodies. Here, we show that HISE1, the gene product that is altered in this mutant, functions as a key factor in plant sterol homeostasis on the endoplasmic reticulum (ER) and participates in a fail-safe regulatory system comprising two processes. First, HISE1 downregulates the protein levels of the β-hydroxy β-methylglutaryl-CoA reductases HMGR1 and HMGR2, which are rate-limiting enzymes in the sterol synthesis pathway, resulting in suppression of sterol overproduction. Second, if the first process is not successful, excess sterols are converted to sterol esters by phospholipid sterol acyltransferase1 (PSAT1) on ER microdomains and then segregated in SE bodies. Sterols are essential structural components of cell membranes and have functions in many different physiological processes. For example, brassinosteroids are a class of sterol phytohormones that are important in regulating plant growth and development. This study describes the identification of a protein factor that regulates sterol homeostasis in Arabidopsis .

Eukaryotic cells acquired novel organelles during evolution through mechanisms that remain largely obscure. The existence of the unique oil body compartment is a synapomorphy of liverworts that represents lineage-specific acquisition of this organelle during evolution, although its origin, biogenesis, and physiological function are yet unknown. We found that two Syntaxin 1 paralogs in the liverwort, Marchantia polymorpha, are distinctly targeted to forming cell plates and the oil body, suggesting these structures share some developmental similarity. Oil body formation is under the regulation of an ERF/AP2-type transcription factor and loss of the oil body increased M. polymorpha herbivory. These findings highlight a common strategy for the acquisition of organelles with distinct functions in plants, via periodical switching in secretion direction depending on cellular phase transition. Competing Interest Statement The authors have declared no competing interest.

Abstract Soybean is an important crop plant for food and biofuel production, and there have been considerable efforts to develop transgenic soybean lines with higher seed oil contents and/or seed yields. However, the process of screening transgenic lines is laborious and requires a large amount of space. Here, we describe a powerful screening method, Glycine max Fluorescence-Accumulating Seed Technology (GmFAST), which is based on a seed-specific fluorescent marker. The marker is composed of a soybean seed-specific promoter coupled to the OLE1-GFP gene, which encodes GFP fused to the oil-body membrane protein OLEOSIN1 of Arabidopsis thaliana. We introduced the marker gene into cotyledonary nodes of G. max Kariyutaka via Agrobacterium-mediated transformation and regenerated heterozygous transgenic plants. OLE1-GFP-expressing soybean seeds can be selected nondestructively using a fluorescence stereomicroscope. Among T2 seeds, the most strongly fluorescent seeds were homozygous. GmFAST uses one-tenth of the growing space required for the conventional method. This space-saving method will contribute to facilitating transformation of soybean. OLE1-GFP was localized specifically to oil bodies in the cotyledon cells of seeds, but it did not affect oil content per seed, the size and density of the oil bodies, or oil composition. One of the homozygous lines (line #8) showed a 44% increase in the seed pod number, which resulted in 41% and 30% increases in seed yield and total oil production, respectively, compared with the wild type. In line #8, OLE1-GFP was inserted into the intron of Glyma13g30950, causing its overexpression. An increase in seed pod number was confirmed in Arabidopsis thaliana plants that overexpressed the Arabidopsis ortholog of Glyma13g30950, E6L1. These results suggest that line #8 is a valuable resource for agricultural and industrial applications. Taken together, GmFAST provides a space-saving visual and non-destructive screening method for soybean transformation, thereby increasing the chance of developing useful soybean lines.