Elsevier BV Acta Medica Okayama 0960-9822 36 5 2026 Compound starch granule formation in grass seeds is associated with distinct temporal patterns of gene expression 1142 1155.e6 EN Alexander Watson-Lazowski John Innes Centre, Norwich Research Park Doreen Feike John Innes Centre, Norwich Research Park Qi Yang Ngai John Innes Centre, Norwich Research Park Lara Esch John Innes Centre, Norwich Research Park Naoko Fujita Department of Biological Production, Akita Prefectural University Ryo Matsushima Institute of Plant Science and Resources, Okayama University Alison M. Smith John Innes Centre, Norwich Research Park David Seung John Innes Centre, Norwich Research Park There is extensive interspecies variation in starch granule morphology in the endosperm of grass species, but the factors underpinning this variation are poorly understood. The two major granule morphology types among species are simple and compound granules, where simple granules arise from a single initiation per amyloplast, and compound granules form from multiple granule initiations. Here, we carried out an extensive survey of seed starch morphology, examining specimens from 105 species within the Pooideae using electron microscopy. This not only expanded our current knowledge of the diversity of starch granule types but also confirmed the homoplasy of simple and compound starch granules. We then selected species representing independent origins of simple (Brachyelytrum japonicum, Phaenosperma globosa, and Brachypodium distachyon) and compound granules (Nardus stricta, Melica altissima, and Calamagrostis brachytricha) for analysis of starch structure and gene expression. There were no clear patterns in starch content, amylopectin structure, or amylose content that could distinguish the two granule types. However, comparative transcriptomics over seed development revealed gene expression patterns associated with granule type, most notably increasing expression of STARCH SYNTHASE 3a (SS3a), STARCH BRANCHING ENZYME 1 (SBE1), and limit dextrinases (LDAs) between 3 and 9 days post anthesis in species with compound granules. Mutation of these genes in rice, which natively produces compound granules, resulted in altered granule shape and/or size. Our work highlights differences in gene expression that could contribute to natural variation in granule morphology within the Pooideae while providing important new starch phenotype and gene expression datasets that can be widely used to study endosperm development. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1467-7644 2026 Rice EMF3 Alleles Adjust Flower Opening Time to Enhance the Seed Setting Rate Under High Temperature Stress EN Takuma Ishizaki Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences (JIRCAS) Yoichi Hashida Faculty of Agriculture, Takasaki University of Health and Welfare Hideyuki Hirabayashi Institute of Crop Science, National Agriculture and Food Research Organization (NARO) Kazuhiro Sasaki Biological Resources and Post-Harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS) Hiroki Tokunaga Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences (JIRCAS) Eliza Vie M. Simon‐Ada Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute (IRRI) Masataka Wakayama Institute for Advanced Biosciences, Keio University Toshiyuki Takai Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute (IRRI) Hiroki Saito Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences (JIRCAS) Atsushi J. Nagano Institute for Advanced Biosciences, Keio University Hitoshi Sakakibara Graduate School of Bioagricultural Sciences, Nagoya University Mikiko Kojima RIKEN Center for Sustainable Resource Science Yumiko Takebayashi RIKEN Center for Sustainable Resource Science Sung‐Ryul Kim Rice Breeding Innovations Department, International Rice Research Institute (IRRI) Ryo Matsushima Institute of Plant Science and Resources, Okayama University Michael J. Thomson Plant Breeding, Genetics, and Biotechnology Division International Rice Research Institute (IRRI) Metro Manila Philippines Kazuhiko Sugimoto Institute of Crop Science, National Agriculture and Food Research Organization (NARO) Ken‐Ichiro Hibara 18Graduate School of Agricultural Regional Vitalization, Kibi International University Tsutomu Ishimaru Biological Resources and Post-Harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS) To safeguard global food security against rapid population growth and a warming world, the effective genetic improvement of cereals is imperative. Flower opening time (FOT) critically affects the seed setting rate. In this study, we identified a gene, EARLY-MORNING FLOWERING 3 (EMF3), in which single-nucleotide substitutions strongly modulate FOT in rice in a semi-dominant manner, resulting in wide variation in FOT from earlier to later FOT than the wild-type. EMF3 knock-out mutants showed significantly reduced FOT synchrony and disrupted anther dehiscence, leading to fertilisation failure. EMF3 encodes a plasma membrane-localised polypeptide of 723 amino acids with an armadillo repeat fold and four transmembrane segments. Furthermore, EMF3 is specifically expressed in the anthers starting from nighttime on the day of flowering, with substantial impacts on the transcriptomes of both anther and lodicule, which suggested an exclusive role of EMF3 in flowering events. Modifying EMF3 alleles of O. sativa enabled the adjustment of FOT among Oryza species and subspecies, potentially facilitating cross-fertilisation by overcoming one of the major challenges of inter-specific hybridisation to exploit heterosis. Introducing the EMF3 alleles with the earlier FOT into popular rice cultivars resulted in flowering at an earlier time of day when the temperature was cooler, efficiently increasing seed setting rate under heat stress. This discovery unveils the novel mechanism of anther control of flower opening time through the EMF3 gene, while also enabling the use of EMF3 alleles in breeding strategies for efficient fertilisation for increasing hybrid rice seed production and mitigating future heat-stress damage at flowering. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0028-646X 2026 Starch Synthase 3 isoforms are essential for normal starch granule initiation in wheat endosperm EN Jinjin Ding John Innes Centre, Norwich Research Park Brendan Fahy John Innes Centre, Norwich Research Park Ryo Matsushima Institute of Plant Science and Resources, Okayama University Qiantao Jiang State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Triticeae Research Institute, Sichuan Agricultural University David Seung John Innes Centre, Norwich Research Park No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0040-5752 137 9 2024 Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley 212 EN Ryo Matsushima Institute of Plant Science and Resources, Okayama University Hiroshi Hisano Institute of Plant Science and Resources, Okayama University June-Sik Kim Institute of Plant Science and Resources, Okayama University Rose McNelly John Innes Centre, Norwich Research Park Naoko F. Oitome Department of Biological Production, Akita Prefectural University David Seung John Innes Centre, Norwich Research Park Naoko Fujita Department of Biological Production, Akita Prefectural University Kazuhiro Sato Institute of Plant Science and Resources, Okayama University The genetic interactions among starch biosynthesis genes can be exploited to alter starch properties, but they remain poorly understood due to the various combinations of mutations to be tested. Here, we isolated two novel barley mutants defective in starch BRANCHING ENZYME 2a (hvbe2a-1 and hvbe2a-2) based on the starch granule (SG) morphology. Both hvbe2a mutants showed elongated SGs in the endosperm and increased resistant starch content. hvbe2a-1 had a base change in HvBE2a gene, substituting the amino acid essential for its enzyme activity, while hvbe2a-2 is completely missing HvBE2a due to a chromosomal deletion. Further genetic crosses with barley isoamylase1 mutants (hvisa1) revealed that both hvbe2a mutations could suppress defects in endosperm caused by hvisa1, such as reduction in starch, increase in phytoglycogen, and changes in the glucan chain length distribution. Remarkably, hvbe2a mutations also transformed the endosperm SG morphology from the compound SG caused by hvisa1 to bimodal simple SGs, resembling that of wild-type barley. The suppressive impact was in competition with floury endosperm 6 mutation (hvflo6), which could enhance the phenotype of hvisa1 in the endosperm. In contrast, the compound SG formation induced by the hvflo6 hvisa1 mutation in pollen was not suppressed by hvbe2a mutations. Our findings provide new insights into genetic interactions in the starch biosynthetic pathway, demonstrating how specific genetic alterations can influence starch properties and SG morphology, with potential applications in cereal breeding for desired starch properties. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0040-5752 136 4 2023 FLOURY ENDOSPERM 6 mutations enhance the sugary phenotype caused by the loss of ISOAMYLASE1 in barley 94 EN Ryo Matsushima Institute of Plant Science and Resources, Okayama University Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Ivan Galis Institute of Plant Science and Resources, Okayama University Satoko Miura Department of Biological Production, Akita Prefectural University Naoko Crofts Department of Biological Production, Akita Prefectural University Yuto Takenaka College of Life Sciences, Ritsumeikan University Naoko F. Oitome Department of Biological Production, Akita Prefectural University Takeshi Ishimizu College of Life Sciences, Ritsumeikan University Naoko Fujita Department of Biological Production, Akita Prefectural University Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Starch is a biologically and commercially important glucose polymer synthesized by plants as semicrystalline starch granules (SGs). Because SG morphology affects starch properties, mutants with altered SG morphology may be useful in breeding crops with desirable starch properties, including potentially novel properties. In this study, we employed a simple screen for mutants with altered SG morphology in barley (Hordeum vulgare). We isolated mutants that formed compound SGs together with the normal simple SGs in the endosperm and found that they were allelic mutants of the starch biosynthesis genes ISOAMYLASE1 (HvISA1) and FLOURY ENDOSPERM 6 (HvFLO6), encoding starch debranching enzyme and CARBOHYDRATE-BINDING MODULE 48-containing protein, respectively. We generated the hvflo6 hvisa1 double mutant and showed that it had significantly reduced starch biosynthesis and developed shrunken grains. In contrast to starch, soluble α-glucan, phytoglycogen, and sugars accumulated to higher levels in the double mutant than in the single mutants. In addition, the double mutants showed defects in SG morphology in the endosperm and in the pollen. This novel genetic interaction suggests that hvflo6 acts as an enhancer of the sugary phenotype caused by hvisa1 mutation. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 9 2019 Imaging Amyloplasts in the Developing Endosperm of Barley and Rice 3745 EN Ryo Matsushima Institute of Plant Science and Resources, Okayama University Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Amyloplasts are plant-specific organelles responsible for starch biosynthesis and storage. Inside amyloplasts, starch forms insoluble particles, referred to as starch grains (SGs). SG morphology differs between species and SG morphology is particularly diverse in the endosperm of Poaceae plants, such as rice (Oryza sativa) and barley (Hordeum vulgare), which form compound SGs and simple SGs, respectively. SG morphology has been extensively imaged, but the comparative imaging of amyloplast morphology has been limited. In this study, SG-containing amyloplasts in the developing endosperm were visualized using stable transgenic barley and rice lines expressing amyloplast stroma-targeted green fluorescent protein fused to the transit peptide (TP) of granule-bound starch synthase I (TP-GFP). The TP-GFP barley and rice plants had elongated amyloplasts containing multiple SGs, with constrictions between the SGs. In barley, some amyloplasts were connected by narrow protrusions extending from their surfaces. Transgenic rice lines producing amyloplast membrane-localized SUBSTANDARD STARCH GRAIN6 (SSG6)-GFP were used to demonstrate that the developing amyloplasts contained multiple compound SGs. TP-GFP barley can be used to visualize the chloroplasts in leaves and other plastids in pollen and root in addition to the endosperm, therefore it provides as a useful tool to observe diverse plastids. No potential conflict of interest relevant to this article was reported.