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Endosperm, the major storage organ in cereal grains, determines the grain yield and quality. Mitochondria provide the energy for dry matter accumulation, in the endosperm development. Although mitochondrial single-stranded DNA-binding proteins (mtSSBs) play a canonical role in the maintenance of single-stranded mitochondrial DNA, their molecular functions in RNA processing and endosperm development remain obscure. Here, we report a defective rice endosperm mutant, floury endosperm26 (flo26), which develops abnormal starch grains in the endosperm. Map-based cloning and complementation experiments showed that FLO26 allele encodes a mitochondrial single-stranded DNA-binding protein, named as mtSSB1.1. Loss of function of mtSSB1.1 affects the transcriptional level of many mitochondrially-encoded genes and RNA splicing of nad1, a core component of respiratory chain complex I in mitochondria. As a result, dysfunctional mature nad1 led to dramatically decreased complex I activity, thereby reducing ATP production. Our results reveal that mtSSB1.1 plays an important role in the maintenance of mitochondrial function and endosperm development by stabilizing the splicing of mitochondrial RNA in rice., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm., Competing Interests: Conflict of interest statement. The authors declare no conflict of interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Subcellular mRNA localization is an evolutionarily conserved mechanism to spatially and temporally drive local translation and, in turn, protein targeting. Hence, this mechanism achieves precise control of gene expression and establishes functional and structural networks during cell growth and development as well as during stimuli response. Since its discovery in ascidian eggs, mRNA localization has been extensively studied in animal and yeast cells. Although our knowledge of subcellular mRNA localization in plant cells lags considerably behind other biological systems, mRNA localization to the endoplasmic reticulum (ER) has also been well established since its discovery in cereal endosperm cells in the early 1990s. Storage protein mRNA targeting to distinct subdomains of the ER determines efficient accumulation of the corresponding proteins in different endosomal storage sites and, in turn, underlies storage organelle biogenesis in cereal grains. The targeting process requires the presence of RNA localization elements, also called zipcodes, and specific RNA-binding proteins that recognize and bind these zipcodes and recruit other factors to mediate active transport. Here, we review the current knowledge of the mechanisms and functions of mRNA localization to the ER in plant cells and address directions for future research.

Key Message: FLO15encodes a plastidic glyoxalase I protein, OsGLYI7, which affects compound starch granule formation and starch synthesis in rice endosperm. Starch synthesis in rice (Oryza sativa) endosperm is a sophisticated process, and its underlying molecular machinery still remains to be elucidated. Here, we identified and characterized two allelic rice floury endosperm 15 (flo15) mutants, both with a white-core endosperm. The flo15 grains were characterized by defects in compound starch granule development, along with decreased starch content. Map-based cloning of the flo15 mutants identified mutations in OsGLYI7, which encodes a glyoxalase I (GLYI) involved in methylglyoxal (MG) detoxification. The mutations of FLO15/OsGLYI7 resulted in increased MG content in flo15 developing endosperms. FLO15/OsGLYI7 localizes to the plastids, and the in vitro GLYI activity derived from flo15 was significantly decreased relative to the wild type. Moreover, the expression of starch synthesis-related genes was obviously altered in the flo15 mutants. These findings suggest that FLO15 plays an important role in compound starch granule formation and starch synthesis in rice endosperm.

Pyruvate kinase (PK) is a key enzyme in glycolysis and carbon metabolism. Here, we isolated a rice (Oryza sativa) mutant, w59, with a white-core floury endosperm. Map-based cloning of w59 identified a mutation in OsPKpα1, which encodes a plastidic isoform of PK (PKp). OsPKpα1 localizes to the amyloplast stroma in the developing endosperm, and the mutation of OsPKpα1 in w59 decreases the plastidic PK activity, resulting in dramatic changes to the lipid biosynthesis in seeds. The w59 grains were also characterized by a marked decrease in starch content. Consistent with a decrease in number and size of the w59 amyloplasts, large empty spaces were observed in the central region of the w59 endosperm, at the early grain-filling stage. Moreover, a phylogenetic analysis revealed four potential rice isoforms of OsPKp. We validated the in vitro PK activity of these OsPKps through reconstituting active PKp complexes derived from inactive individual OsPKps, revealing the heteromeric structure of rice PKps, which was further confirmed using a protein-protein interaction analysis. These findings suggest a functional connection between lipid and starch synthesis in rice endosperm amyloplasts., (© 2018 Institute of Botany, Chinese Academy of Sciences.)

In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury endosperm7 (flo7) mutant, which develops a floury-white endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral endosperm. Notably, flo7 peripheral endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral endosperm and provide new insights into the spatial regulation of endosperm development in rice., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Summary: Amylose content is a crucial physicochemical property responsible for the eating and cooking quality of rice (Oryza sativa L.) grain and is mainly controlled by the Waxy (Wx) gene. Previous studies have identified several Dull genes that modulate the expression of the Wxb allele in japonica rice by affecting the splicing efficiency of the Wxb pre‐mRNA. Here, we uncover dual roles for a novel Dull gene in pre‐mRNA splicing and microRNA processing. We isolated the dull mutant, du13, with a dull endosperm and low amylose content. Map‐based cloning showed that Du13 encodes a C2H2 zinc‐finger protein. Du13 coordinates with the nuclear cap‐binding complex to regulate the splicing of Wxb transcripts in rice endosperm. Moreover, Du13 also regulates alternative splicing of other protein‐coding transcripts and affects the biogenesis of a subset of microRNAs. Our results reveal an evolutionarily conserved link between pre‐mRNA splicing and microRNA biogenesis in rice endosperm. Our findings also provide new insights into the functions of Dull genes in rice and expand our knowledge of microRNA biogenesis in monocots. [ABSTRACT FROM AUTHOR]

Summary: Starch accounts for over 80% of the total dry weight in cereal endosperm and determines the kernel texture and nutritional quality. Amyloplasts, terminally differentiated plastids, are responsible for starch biosynthesis and storage. We screened a series of rice mutants with floury endosperm to clarify the mechanism underlying amyloplast development and starch synthesis. We identified the floury endosperm19 (flo19) mutant which shows opaque of the interior endosperm. Abnormal compound starch grains (SGs) were present in the endosperm cells of the mutant. Molecular cloning revealed that the FLO19 allele encodes a plastid‐localized pyruvate dehydrogenase complex E1 component subunit α1 (ptPDC‐E1‐α1) that is expressed in all rice tissues. In vivo enzyme assays demonstrated that the flo19 mutant showed decreased activity of the plastidic pyruvate dehydrogenase complex. In addition, the amounts of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were much lower in the developing flo19 mutant endosperm, suggesting that FLO19 participates in fatty acid supply for galactolipid biosynthesis in amyloplasts. FLO19 overexpression significantly increased seed size and weight, but did not affect other important agronomic traits, such as panicle length, tiller number and seed setting rate. An analysis of single nucleotide polymorphism data from a panel of rice accessions identified that the pFLO19L haplotype was positively associated with grain length, implying a potential application in rice breeding. In summary, our study demonstrates that FLO19 is involved in galactolipid biosynthesis which is essential for amyloplast development and starch biosynthesis in rice. [ABSTRACT FROM AUTHOR]

Background: The sequences of several important mitochondrion-encoded genes involved in respiration in higher plants are interrupted by introns. Many nuclear-encoded factors are involved in splicing these introns, but the mechanisms underlying this splicing remain unknown. Results: We isolated and characterized a rice mutant named floury shrunken endosperm 5 (fse5). In addition to having floury shrunken endosperm, the fse5 seeds either failed to germinate or produced seedlings which grew slowly and died ultimately. Fse5 encodes a putative plant organelle RNA recognition (PORR) protein targeted to mitochondria. Mutation of Fse5 hindered the splicing of the first intron of nad4, which encodes an essential subunit of mitochondrial NADH dehydrogenase complex I. The assembly and NADH dehydrogenase activity of complex I were subsequently disrupted by this mutation, and the structure of the mitochondria was abnormal in the fse5 mutant. The FSE5 protein was shown to interact with mitochondrial intron splicing factor 68 (MISF68), which is also a splicing factor for nad4 intron 1 identified previously via yeast two-hybrid (Y2H) assays. Conclusion: Fse5 which encodes a PORR domain-containing protein, is essential for the splicing of nad4 intron 1, and loss of Fse5 function affects seed development and seedling growth. [ABSTRACT FROM AUTHOR]

Chalkiness of rice grain is an important quality component of rice, as it has a profound influence on eating and milling qualities. We has determined the inheritance of percentage of grain with chalkiness (PGWC) using a set of chromosome segment substitution lines, made from a cross between cv. PA64s and cv. 9311. Two loci controlling PGWC, designated as qPGWC-6 and qPGWC-7, were located on, respectively, chromosomes 6 and 7. Comparisons were made between C-51 (a CSSL harbouring qPGWC-7 and having a chalky endosperm) and the recurrent parent 9311 (translucent endosperm) to characterize the physical and chemical differences between translucent and chalky endosperm. Unlike the translucent endosperm, the chalky endosperm contains loosely packed starch granules, and there were significant difference between C-51 and 9311 for amylopectin structure and degree of crystallinity, but not for either amylose content or starch viscosity. Segregation analysis of the F2 population from the cross between C-51 and 9311 showed PGWC is a semi-dominant trait, controlled by single nuclear gene. A large F2 population was constructed from the cross C51 × 9311, and used for the fine mapping of qPGWC-7, which was located to a 44-kb DNA fragment, containing thirteen predicted genes. This result provides a springboard for the map-based cloning of qPGWC-7 and allowed for marker-assisted selection for endosperm texture. [ABSTRACT FROM AUTHOR]

Starch accounts for about 80–85 % of the dry weight of grains and determines yield by impact on grain weight. And, the content and composition of starch also determine appearance, eating, cooking and nutritional quality of rice. By coordinating crucial reactions of the primary carbohydrate metabolism in all eukaryotes, fructose-2,6-bisphosphate (Fru-2,6-P 2) is a traffic signal in metabolism. However, the metabolic regulation of starch in plant sink tissues by Fru-2,6-P 2 remains unclear. Here we isolated rice mutant floury endosperm23 (flo23) which has opaque endosperm and anomalous compound starch grains (SGs). flo23 mutant grains had reduced contents of starch, lipids and proteins. Map-based cloning and genetic complementation experiments showed that FLO23 encodes a cytoplasmic Fructose-6-phosphate-2-kinase/Fructose-2,6-bisphosphatase (F2KP). Mutation of OsF2KP2 decreased Fru-2,6-P 2 content in endosperm cells, leading to drastically reduced phosphoenolpyruvate (PEP) and pyruvate contents and disordered glycolysis and energy metabolism. The results imply that OsF2KP2 participates in the glycolytic pathway by providing precursors and energy for synthesis of grain storage compounds. • flo23 displayed floury endospem consisted of anomalous starch granules. • FLO23 encoded a fructose-6-phosphate-2-kinase/fructose-2, 6-bisphosphatase. • OsF2KP2 functions through homo and heterocomplexes. • Glycolysis and storage product accumulation were defective in the flo23 mutant. [ABSTRACT FROM AUTHOR]