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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Omata, Sato, Mishiro-Sato, Kano, Ueda, Hara-Nishimura and Shimada.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Takahashi, Sakai, Ariga, Teramoto, Shimada, Eun, Muto, Naito and Tomooka.)