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Starch and proteins are main storage product to determine the appearance, cooking, texture, and nutritional quality of rice (Oryza sativa L.). OsNAC20 and OsNAC26, as pivotal transcription factors, redundantly regulate the expression of genes responsible for starch and protein synthesis in the rice endosperm. Any knockout of OsNAC20 or OsNAC26 did not result in visible endosperm defects. In this study, we had isolated and characterized a mutant named as floury endosperm25 (flo25). The caryopsis of the flo25 mutant exhibits a floury endosperm, accompanied by reductions in both the 1000-grain weight and grain length, as well as diminished levels of total starch and protein. Through map-based cloning, it was determined that FLO25 encodes a NAM, ATAF, and CUC (NAC) transcription factors, namely OsNAC26, with a lysine to asparagine substitution at position 98 in the flo25 mutant. Remarkably, lysine 98 is conserved across plants species, and this mutation does not alter the subcellular localization of OsNAC26 but significantly attenuates its transcriptional activity and its ability to activate downstream target genes. Furthermore, the mutant protein encoded by OsNAC26 -flo25 could interact with OsNAC20, disrupting the native interaction between OsNAC20 proteins. Additionally, when lysine 98 is substituted with asparagine in OsNAC20, the resulting mutant protein, OsNAC20(K98N), similarly disrupts the interaction between OsNAC26 proteins. Collectively, these findings underscore the pivotal role of Lysine 98 (K) in modulating the transcriptional activity of NAC20/NAC26 within the rice endosperm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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.)

Cereal crops accumulate large amounts of starch which is synthesized and stored in amyloplasts in the form of starch grains (SGs). Despite significant progress in deciphering starch biosynthesis, our understanding of amyloplast development in rice (Oryza sativa) endosperm remains largely unknown. Here, we report a novel rice floury mutant named enlarged starch grain1 (esg1). The mutant has decreased starch content, altered starch physicochemical properties, slower grain-filling rate and reduced 1000-grain weight. A distinctive feature in esg1 endosperm is that SGs are much larger, mainly due to an increased number of starch granules per SG. Spherical and loosely assembled granules, together with those weakly stained SGs may account for decreased starch content in esg1. Map-based cloning revealed that ESG1 encodes a putative permease subunit of a bacterial-type ABC (ATP-binding cassette) lipid transporter. ESG1 is constitutively expressed in various tissues. It encodes a protein localized to the chloroplast and amyloplast membranes. Mutation of ESG1 causes defective galactolipid synthesis. The overall study indicates that ESG1 is a newly identified protein affecting SG development and subsequent starch biosynthesis, which provides novel insights into amyloplast development in rice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rice varieties with suitable flour-making qualities are required to promote the rice processed-food industry and to boost rice consumption. A rice mutation, Namil(SA)-flo1, produces grains with floury endosperm. Overall, grains with low grain hardness, low starch damage, and fine particle size are more suitable for use in flour processing grains with waxy, dull endosperm with normal grain hardness and a high amylose content. In this study, fine mapping found a C to T single nucleotide polymorphism (SNP) in exon 2 of the gene encoding cytosolic pyruvate phosphate dikinase (cy OsPPDK ). The SNP resulted in a change of serine to phenylalanine acid at amino acid position 101. The gene was named FLOURY ENDOSPERM 4-5 ( FLO4-5 ). Co-segregation analysis with the developed cleaved amplified polymorphic sequence (CAPS) markers revealed co-segregation between the floury phenotype and the flo4-5 . This CAPS marker could be applied directly for marker-assisted selection. Real-time RT-PCR experiments revealed that PPDK was expressed at considerably higher levels in the flo4-5 mutant than in the wild type during the grain filling stage. Plastid ADP-glucose pyrophosphorylase small subunit (AGPS2a and AGPS2b) and soluble starch synthase ( SSIIb and SSIIc) also exhibited enhanced expression in the flo4-5 mutant.

Pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP1) reversibly converts fructose 6-phosphate and pyrophosphate to fructose 1, 6-bisphosphate and orthophosphate during glycolysis, and has diverse functions in plants. However, mechanisms underlying the regulation of starch metabolism by PFP1 remain elusive. This study addressed the function of PFP1 in rice floury endosperm and defective grain filling. Compared with the wild type, pfp1-3 exhibited remarkably low grain weight and starch content, significantly increased protein and lipid content, and altered starch physicochemical properties and changes in embryo development. Map-based cloning revealed that pfp1-3 is a novel allele and encodes the regulatory β-subunit of PFP1 (PFP1β). Measurement of nicotinamide adenine dinucleotide (NAD+) showed that mutation of PFP1β markedly decreased its enzyme activity. PFP1β and three of four putative catalytic α-subunits of PFP1, PFP1α1, PFP1α2, and PFP1α4, interacted with each other to form a heterotetramer. Additionally, PFP1β, PFP1α1 and PFP1α2 also formed homodimers. Furthermore, transcriptome analysis revealed that mutation of PFP1β significantly altered expression of many essential enzymes in starch biosynthesis pathways. Concentrations of multiple lipid and glycolytic intermediates and trehalose metabolites were elevated in pfp1-3 endosperm, indicating that PFP1 modulates endosperm metabolism, potentially through reversible adjustments to metabolic fluxes. Taken together, these findings provide new insights into seed endosperm development and starch biosynthesis and will help in the breeding of rice cultivars with higher grain yield and quality., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants. Recent studies revealed the functions of PPR proteins in organellar RNA metabolism and plant development, but the functions of most PPR proteins, especially PPRs localized in the nucleus, remain largely unknown. Here, we report the isolation and characterization of a rice mutant named floury and growth retardation1 (fgr1). fgr1 showed floury endosperm with loosely arranged starch grains, decreased starch and amylose contents, and retarded seedling growth. Map-based cloning showed that the mutant phenotype was caused by a single nucleotide substitution in the coding region of Os08g0290000. This gene encodes a nuclear-localized PPR protein, which we named OsNPPR1, that affected mitochondrial function. In vitro SELEX and RNA-EMSAs showed that OsNPPR1 was an RNA protein that bound to the CUCAC motif. Moreover, a number of retained intron (RI) events were detected in fgr1. Thus, OsNPPR1 was involved in regulation of mitochondrial development and/or functions that are important for endosperm development. Our results provide novel insights into coordinated interaction between nuclear-localized PPR proteins and mitochondrial function., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Mutations of stromal Hsp70 cause chloroplast developmental abnormalities and knockout mutants of stromal Hsp70 usually exhibit protein import deficiencies. However, their effects have not been studied in amyloplast development. Here, we identified an amyloplast abnormal development mutant, floury endosperm11 (flo11) that exhibited an opaque phenotype in the inner core and the periphery of grains. Semi-thin section revealed defective amyloplast development in the flo11 endosperm. Map-based cloning and subsequent complementation test demonstrated that FLO11 encoded a plastid-localized heat shock protein 70 (OsHsp70cp-2). OsHsp70cp-2 was abundantly expressed in developing endosperm, whereas its paralogous gene OsHsp70cp-1 was mainly expressed in photosynthetic tissues. Ectopic expression of OsHsp70cp-1 under the control of OsHsp70cp-2 promoter rescued the mutant phenotype of flo11. Moreover, simultaneous knockdown of both OsHsp70cp genes resulted in white stripe leaves and opaque endosperm. BiFC and Co-IP assays revealed that OsHsp70cp-2 was associated with Tic complex. Taken together, OsHsp70cp-2 may regulate protein import into amyloplasts, which is essential for amyloplast development in rice., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Pyruvate orthophosphate dikinase (PPDK) is a component of glycolysis to mediate endosperm energy charge by adjusting the ratio of ATP to ADP and AMP that proposed to balance the flow of carbon into starch, protein, fatty acid and amino acid biosynthesis. However, these were inconsistent with the first report of a T-DNA insertional knockout mutant of the rice PPDK gene (flo4) showed that rice with inactivated PPDK gene failed to produce a opaque seeds. Therefore, the PPDK might have multifaceted functions in grain filling stage, which in some ways might depend on the direction of the reversible catalysis. Suweon 542 is a rice (Oryza sativa L.) mutant developed from Oryza sativa ssp. japonica cv. Namil. Suweon 542 has a milky-white floury endosperm suitable for dry filling, with low starch damage, low grain hardness, and fine flour particle size. The mutant locus on chromosome 5 controls the floury endosperm phenotype of Suweon 542. Fine mapping of this locus is required for efficient breeding of rice germplasm suitable for dry milling. In this study, whole genome of Suweon 542 and Milyang 23 were re-sequenced using Illumina HiSeq 2500. Co-segregation analysis of F 3:4 family populations derived from Suweon 542/Milyang 23 was performed using eight CAPS markers and phenotypic evaluation of the endosperm. The target region was mapped to a 33 kb region and identified to encode cytosolic pyruvate orthophosphate dikinase protein (cyOsPPDK). A G→A SNP in exon 8 of cyOsPPDK resulting in a missense mutation from Gly to Asp at amino acid position 404 was responsible for the floury endosperm of Suweon 542. qRT-PCR experiments revealed that FLO4-4 was expressed to a considerably higher level in Suweon 542 than in Namil during the grain filling stage. Overall, fine mapping of FLO4-4 and candidate gene analysis provided further insight into the floury endosperm of rice, and reveal a novel SNP in cyOsPPDK gene can affect the floury endosperm phenotype through active PPDK gene during grain filling stage.

Starch, as a main energy storage substance, plays an important role in plant growth and human life. Despite the fact that several enzymes and regulators involved in starch biosynthesis have been identified, the regulating mechanism of starch synthesis is still unclear. In this study, we isolated a rice floury endosperm mutant M14 from a mutant pool induced by 60 Co. Both total starch content and amylose content in M14 seeds significantly decreased, and starch thermal and pasting properties changed. Compound starch granules were defected in the floury endosperm of M14 seeds. Map-based cloning and a complementation test showed that the floury endosperm phenotype was determined by a gene of OsPPDKB , which encodes pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1). Subcellular localization analysis demonstrated that PPDK was localized in chloroplast and cytoplasm, the chOsPPDKB highly expressed in leaf and leaf sheath, and the cyOsPPDKB constitutively expressed with a high expression in developing endosperm. Moreover, the expression of starch synthesis-related genes was also obviously altered in M14 developing endosperm. The above results indicated that PPDK played an important role in starch metabolism and structure in rice endosperm.

Key Message: Decreased PFPase activity in rice perturbs the equilibration of carbon metabolism during grain filling but has no visible phenotypic effects during the vegetative and reproductive growth stages. Starch is a primary energy reserve for various metabolic processes in plant. Despite much advance has been achieved in pathways involved in starch biosynthesis, information was still lacked for precise regulation related to carbon metabolism during seed filling in rice (Oryza sativa). The objective of this study was to identify and characterize new gene associated with carbon metabolism during grain filling. By screening our chemical mutant pool, two allelic mutants exhibiting floury endosperm were isolated. No visible phenotypic defects were observed during both the vegetative and reproductive growth stages, except for the floury-like endosperm of grains with significantly reduced kernel thickness, 1000-grain weight and total starch content. Map-based cloning revealed that the mutant phenotypes were controlled by a gene encoding pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) β subunit (PFPβ), which catalyzes reversible interconversion between fructose-6-phosphate and fructose-1, 6-bisphosphate. The identity of PFP β was further confirmed by a genetic complementation test. Subcellular analysis demonstrated that PFPβ was localized in cytoplasm. Quantitative PCR and histochemical staining indicated PFP β was ubiquitously expressed in various tissues. Furthermore, we found PFP β could express in both the early and late phases of starch accumulation during grain filling and decreased activity of PFP β in pfp mutants resulted in compromised carbon metabolism with increased soluble sugar contents and unfavorable starch biosynthesis. Our results highlight PFPβ functions in modulating carbon metabolism during grain filling stage.

Alteration of storage substances, in particular the major storage form starch, leads to floury endosperm. Because floury mutants have physical attributes for milling processes, identification and characterization of those mutants are valuable. In this study we identified a floury endosperm mutant caused by a T-DNA insertion in Oryza sativa alanine-aminotransferase1 (OsAlaAT1). OsAlaAT1 is localized in the cytosol and has aminotransferase enzyme activity. The osalaat1 mutant has less amylose and its amylopectin is structurally altered. OsAlaAT1 is predominantly expressed in developing seeds during active starch synthesis. AlaAT catalyzes the interconversion of pyruvate to alanine, and this pathway is activated under low-oxygen conditions. Consistently, OsAlaAT1 is induced by such conditions. Expression of the starch synthesis genes AGPases, OsSSI, OsSSIIa, and OsPPDKB is decreased in the mutant. Thus, our observations suggest that OsAlaAT1 plays an essential role in starch synthesis in developing seeds that are exposed to low concentrations of oxygen., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Storage lipid is a vital component for maintaining structure of seed storage substances and valuable for rice quality and food texture. However, the knowledge of lipid transporting related genes and their function in seed development have not been well elucidated yet. In this study, we identified OsLTPL36, a homolog of putative lipid transport protein, and showed specific expression in rice developing seed. Transcriptional profiling and in situ hybridization analysis confirmed that OsLTPL36 was exclusively expressed in developing seed coat and endosperm aleurone cells. Down-regulated expression of OsLTPL36 led to decreased seed setting rate and 1000-grain weight in transgenic plants. Further studies showed that suppressed expression of OsLTPL36 caused chalky endosperm and resulted in reduced fat acid content in RNAi lines as compared with wild type (WT). Histological analysis showed that the embryo development was delayed after down regulation of OsLTPL36. Moreover, impeded seed germination and puny seedling were also observed in the OsLTPL36 RNAi lines. The data demonstrated that OsLTPL36, a lipid transporter, was critical important not only for seed quality but also for seed development and germination in rice., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)