Rice Endosperm Research Articles

Seeing the unseen in characterizing RNA editome during rice endosperm development

Rice (Oryza sativa L.) endosperm is essential to provide nutrients for seed germination and determine grain yield. RNA editing, a post-transcriptional modification essential for plant development, unfortunately, is not fully characterized during rice endosperm development. Here, we perform systematic analyses to characterize RNA editome during rice endosperm development. We find that most editing sites are C-to-U CDS-recoding in mitochondria, leading to increased hydrophobic amino acids and changed structures of mitochondrial proteins. Comparative analysis of RNA editome reveals that CDS-recoding sites present higher editing frequencies with lower variabilities and their resultant recoded amino acids tend to exhibit stronger evolutionary conservation across many land plants. Furthermore, we classify mitochondrial genes into three groups, presenting distinct patterns in terms of CDS-recoding events. Besides, we conduct genome-wide screening to detect pentatricopeptide repeat (PPR) proteins and construct PPR-RNA binding profiles, yielding candidate PPR editing factors related to rice endosperm development. Taken together, our findings provide valuable insights for deciphering fundamental mechanisms of rice endosperm development underlying RNA editing machinery.

Functional Diversification of Sec13 Isoforms for Storage Protein Trafficking in Rice Endosperm Cells.

Coat protein complex II (COPII) vesicles play crucial roles in mediating the endoplasmic reticulum (ER) exit of newly synthesized proteins to the Golgi in eukaryotic cells. However, the molecular functions of COPII components and their functional diversifications in plant seeds remain obscure. Here, we showed that the rice (Oryza sativa) glutelin precursor accumulation12 (gpa12) mutant is defective in storage protein export from the ER, resulting in the formation of aggregated protein bodies. Map-based cloning revealed that GPA12 encodes a COPII outer layer protein, Sec13a, that mainly localizes to endoplasmic reticulum exit sites (ERES) and partially localizes to the Golgi. Biochemical experiments verified that Sec13a physically interacts with Sec31 and Sec16, and mutation in Sec13 compromises its interaction with Sec31 and Sec16, thereby affecting the membrane association of the inner complex components Sar1b and Sec23c. Apart from Sec13a, the rice genome encodes two other Sec13 isoforms, Sec13b and Sec13c. Notably, we observed an abnormal accumulation of globular ER structures in the sec13bc double mutant but not in the single mutants, suggesting a functional redundancy of Sec13b and Sec13c in modulating ER morphology. Taken together, our results substantiated that Sec13a plays an important role in regulating storage protein export from the ER, while Sec13b and Sec13c are required for maintaining ER morphology in rice endosperm cells. Our findings provide insights into the functional diversification of COPII components in plants.

Phenotypic Analysis and Gene Cloning of Rice Floury Endosperm Mutant wcr (White-Core Rice).

The composition and distribution of storage substances in rice endosperm directly affect grain quality. A floury endosperm mutant, wcr (white-core rice), was identified, exhibiting a loose arrangement of starch granules with a floury opaque appearance in the inner layer of mature grains, resulting in reduced grain weight. The total starch and amylose content remained unchanged, but the levels of the four component proteins in the mutant brown rice significantly decreased. Additionally, the milled rice (inner endosperm) showed a significant decrease in total starch and amylose content, accompanied by a nearly threefold increase in albumin content. The swelling capacity of mutant starch was reduced, and its chain length distribution was altered. The target gene was mapped on chromosome 5 within a 65 kb region. A frameshift mutation occurred due to an insertion of an extra C base in the second exon of the cyOsPPDKB gene, which encodes pyruvate phosphate dikinase. Expression analysis revealed that wcr not only affected genes involved in starch metabolism but also downregulated expression levels of genes associated with storage protein synthesis. Overall, wcr plays a crucial role as a regulator factor influencing protein synthesis and starch metabolism in rice grains.

Multi-omics analyses reveal mechanism for high resistant starch formation in an indica rice SSIIIa mutant

Soluble starch synthase IIIa (SSIIIa) is a key enzyme involved in amylopectin biosynthesis in rice, and deficiency of SSIIIa results in high content of resistant starch, which is benefit to human health. However, little is known about metabolic differences and carbon re-allocation in the seeds of the indica rice ss3a mutant. We found that SSIIIa deficiency impaired the storage of starch, but increased the soluble sugars, free amino acids and lipids. By multi-omic analyses, we found inactivation of SSIIIa triggered carbon repartitioning by downregulating sucrose synthase, grain incomplete filling 1, fructokinase and hexokinase (HK), and promoted the accumulation of soluble sugars. Meanwhile, the downregulation of HK and upregulation of plastidic phosphoglucomutase reduced the carbon flow through glycolysis and promoted glycogenesis. The downregulation of OsbZIP58 and the deleterious effect on ribosome formation might result in the reduction of storage protein synthesis and increased free amino acids content in ss3a. The higher levels of amylose and lipids could form more amylose-lipid complexes (starch phospholipids), resulting in a higher resistant starch content. Taken together, our study unraveled a functional cross talk between starch, protein and lipids in rice endosperm during seed development of ss3a, providing new insights for formation of high resistant starch in rice.

Upstream regulator of genomic imprinting in rice endosperm is a small RNA-associated chromatin remodeler

Genomic imprinting is observed in endosperm, a placenta-like seed tissue, where transposable elements (TEs) and repeat-derived small RNAs (sRNAs) mediate epigenetic changes in plants. In imprinting, uniparental gene expression arises due to parent-specific epigenetic marks on one allele but not on the other. The importance of sRNAs and their regulation in endosperm development or in imprinting is poorly understood in crops. Here we show that a previously uncharacterized CLASSY (CLSY)-family chromatin remodeler named OsCLSY3 is essential for rice endosperm development and imprinting, acting as an upstream player in the sRNA pathway. Comparative transcriptome and genetic analysis indicated its endosperm-preferred expression and its likely paternal imprinted nature. These important features are modulated by RNA-directed DNA methylation (RdDM) of tandemly arranged TEs in its promoter. Upon perturbation of OsCLSY3 in transgenic lines, we observe defects in endosperm development and a loss of around 70% of all sRNAs. Interestingly, well-conserved endosperm-specific sRNAs (siren) that are vital for reproductive fitness in angiosperms are also dependent on OsCLSY3. We observed that many imprinted genes and seed development-associated genes are under the control of OsCLSY3. These results support an essential role of OsCLSY3 in rice endosperm development and imprinting, and propose similar regulatory strategies involving CLSY3 homologs among other cereals.

Lysine 98 in NAC20/NAC26 transcription factors: a key regulator of starch and protein synthesis in rice endosperm

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.

Molecular protocol to develop β-carotene-biofortified rice events via molecular optimization

Providing food with nutrition and functionality is crucial for sustaining human life. Rice (Oryza sativa L.) is a representative staple crop with high carbohydrate content but low amounts of essential amino acids, micronutrients, and carotenoids such as provitamin A. To improve the nutritional quality, rice endosperm was biofortified to accumulate carotenoids such as β-carotene through genetic engineering (i.e., using synthetic carotenoid biosynthetic genes, a nonmammalian viral polycistronic sequence, and an optimized promoter and transit peptide) and high-throughput rice transformation (approximately 300 transgenic plants per construct). To facilitate the safety assessment of genetically modified food, molecular characterization was performed to select elite lines equipped with a single intergenic insertion of T-DNA, high transgene expression, in this case leading to high carotenoid content, and with phenotypic and compositional substantial equivalence. In this study, we present β-carotene-biofortified rice event candidate lines eligible for commercial use and a disclosed molecular protocol for the development of biotech rice crops.

Multilayered epigenetic control of persistent and stage-specific imprinted genes in rice endosperm.

In angiosperms, epigenetic profiles for genomic imprinting are established before fertilization. However, the causal relationships between epigenetic modifications and imprinted expression are not fully understood. In this study, we classified 'persistent' and 'stage-specific' imprinted genes on the basis of time-course transcriptome analysis in rice (Oryza sativa) endosperm and compared them to epigenetic modifications at a single time point. While the levels of epigenetic modifications are relatively low in stage-specific imprinted genes, they are considerably higher in persistent imprinted genes. Overall trends revealed that the maternal alleles of maternally expressed imprinted genes are activated by DNA demethylation, while the maternal alleles of paternally expressed imprinted genes with gene body methylation (gbM) are silenced by DNA demethylation and H3K27me3 deposition, and these regions are associated with an enriched motif related to Tc/Mar-Stowaway. Our findings provide insight into the stability of genomic imprinting and the potential variations associated with endosperm development, different cell types and parental genotypes.

Relationships between the appearance quality and starch structure of soft rice under different nitrogen levels

AbstractThis study aims to explore the formation mechanism of starch structure and the relationships between the appearance quality and starch structure of soft rice under different nitrogen levels. We comprehensively investigated the physiological aspects, starch structure variations, and appearance quality of soft rice in response to different nitrogen applications. The results showed that under the moderate nitrogen application (270 N), the soft rice exhibited the highest AGPase activity, the highest large‐starch granule content, and the lowest chalkiness. Under the highest nitrogen application (360 N), the soft rice exhibited the highest GBSS and DBE activity and the lowest SBE activity, the highest content of long‐branched amylopectin, the lowest relative crystallinity, the fewest ordered structures, the most amorphous structures, the largest semi‐crystalline lamellar thicknesses, and the highest transparency of chalk‐free rice. In conclusion, moderate nitrogen fertilization (270 N) improved the AGPase activity, which leaded to fuller starch granules and more compact endosperm in soft rice. Thus, the grain chalkiness of soft rice decreased. Continuous nitrogen application (0‐360 N) constantly increased the GBSS and DBE activity and reduced the SBE activity in soft rice, leading a lower content of short‐branched amylopectin and a higher content of long‐branched amylopectin in soft rice. Thus, the relative crystallinity and ordered structures of soft rice were reduced. These structures improved the transparency phenotype of soft rice.

Involvement of a rice mutation in storage protein biogenesis in endosperm and its genomic location.

A mutation was first found to cause the great generation of glutelin precursors (proglutelins) in rice (Oryza sativa L.) endosperm, and thus referred to as GPGG1. The GPGG1 was involved in synthesis and compartmentation of storage proteins. The PPR-like gene in GPGG1-mapped region was determined as its candidate gene. In the wild type rice, glutelins and prolamins are synthesized on respective subdomains of rough endoplasmic reticulum (ER) and intracellularly compartmentalized into different storage protein bodies. In this study, a storage protein mutant was obtained and characterized by the great generation of proglutelins combining with the lacking of 13 kD prolamins. A dominant genic-mutation, referred to as GPGG1, was clarified to result in the proteinous alteration. Novel saccular composite-ER was shown to act in the synthesis of proglutelins and 14 kD prolamins in the mutant. Additionally, a series of organelles including newly occurring several compartments were shown to function in the transfer, trans-plasmalemmal transport, delivery, deposition and degradation of storage proteins in the mutant. The GPGG1 gene was mapped to a 67.256kb region of chromosome 12, the pentatricopeptide repeat (PPR)-like gene in this region was detected to contain mutational sites.

Rice floury endosperm26 encoding a mitochondrial single-stranded DNA-binding protein is essential for RNA-splicing of mitochondrial genes and endosperm development

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.

The cake fortified with rice bran and market available one: An evaluation of texture, colour and nutritional qualities

Rice bran is the outer pericarp of the rice endosperm. It is the underutilized milling bi-product of rice, which is generally used for cattle feeds and extraction of oil. However, the whole bran is packed with many essential bioactive molecules and fibers, which have many health benefits. In this study, the whole bran was utilized for making the cake. The textural profile analysis reveals that cakes fortified with rice bran (CRB) showed lower hardness (661.10-1429.90 g) but higher adhesiveness (-3.5 to -19.6 g-sec) and cohesiveness (72.6 to 80.6%) as compared to market available cakes (MAC). With respect to colour parameters, CRB was generally darker (L* = 33.96 to 39.25) than MAC (L* = 37.39 to 37.92) except market available chocolate cake (L*=17.34), which was darkest one. However, CRB was superior to MAC for different nutritional parameters. The minerals (Fe, Zn) content of CRB (Fe: 22.25 to 28.30 ppm and Zn: 14.68 to 16.14 ppm) was higher than MAC (Fe: 11.25 to 17.03 ppm and Zn: 6.24 to 7.61 ppm). There was higher level of phenolics content, flavonoids content(0.43 to 0.76 mg CE/g) and protein content (12.94 to 18.76%) in case of all the CRBs as compared to MACs. The total soluble sugar content was lower but antioxidant activity was higher in CRBs as compared to MACs. Therefore, CRB is one of the healthy alternatives for proper utilization of rice milling bi-product, rice bran.

Activation of the native PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis-acting elements induces carotenoid biosynthesis in embryogenic rice callus

Key messageModification of silent latent endosperm-enabled promoters (SLEEPERs) allows the ectopic activation of non-expressed metabolic genes in rice callusMetabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

Analysis of the global transcriptome and miRNAome associated with seed dormancy during seed maturation in rice (Oryza sativa L. cv. Nipponbare)

BackgroundSeed dormancy is a biological mechanism that prevents germination until favorable conditions for the subsequent generation of plants are encountered. Therefore, this mechanism must be effectively established during seed maturation. Studies investigating the transcriptome and miRNAome of rice embryos and endosperms at various maturation stages to evaluate seed dormancy are limited. This study aimed to compare the transcriptome and miRNAome of rice seeds during seed maturation.ResultsOryza sativa L. cv. Nipponbare seeds were sampled for embryos and endosperms at three maturation stages: 30, 45, and 60 days after heading (DAH). The pre-harvest sprouting (PHS) assay was conducted to assess the level of dormancy in the seeds at each maturation stage. At 60 DAH, the PHS rate was significantly increased compared to those at 30 and 45 DAH, indicating that the dormancy is broken during the later maturation stage (45 DAH to 60 DAH). However, the largest number of differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs) were identified between 30 and 60 DAH in the embryo and endosperm, implying that the gradual changes in genes and miRNAs from 30 to 60 DAH may play a significant role in breaking seed dormancy. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses confirmed that DEGs related to plant hormones were most abundant in the embryo during 45 DAH to 60 DAH and 30 DAH to 60 DAH transitions. Alternatively, most of the DEGs in the endosperm were related to energy and abiotic stress. MapMan analysis and quantitative real-time polymerase chain reaction identified four newly profiled auxin-related genes (OsSAUR6/12/23/25) and one ethylene-related gene (OsERF087), which may be involved in seed dormancy during maturation. Additionally, miRNA target prediction (psRNATarget) and degradome dataset (TarDB) indicated a potential association between osa-miR531b and ethylene biosynthesis gene (OsACO4), along with osa-miR390-5p and the abscisic acid (ABA) exporter-related gene (OsMATE19) as factors involved in seed dormancy.ConclusionsAnalysis of the transcriptome and miRNAome of rice embryos and endosperms during seed maturation provided new insights into seed dormancy, particularly its relationship with plant hormones such as ABA, auxin, and ethylene.

Exploring the structural assembly of rice ADP-glucose pyrophosphorylase subunits using MD simulation

ADP-glucose pyrophosphorylase plays a pivotal role as an allosteric enzyme, essential for starch biosynthesis in plants. The higher plant AGPase comparises of a pair of large and a pair of small subunits to form a heterotetrameric complex. Growing evidence indicates that each subunit plays a distinct role in regulating the underlying mechanism of starch biosynthesis. In the rice genome, there are four large subunit genes (OsL1-L4) and three small subunit genes (OsS1, OsS2a, and OsS2b). While the structural assembly of cytosolic rice AGPase subunits (OsL2:OsS2b) has been elucidated, there is currently no such documented research available for plastidial rice AGPases (OsL1:OsS1). In this study, we employed protein modeling and MD simulation approaches to gain insights into the structural association of plastidial rice AGPase subunits. Our results demonstrate that the heterotetrameric association of OsL1:OsS1 is very similar to that of cytosolic OsL2:OsS2b and potato AGPase heterotetramer (StLS:StSS). Moreover, the yeast-two-hybrid results on OsL1:OsS1, which resemble StLS:StSS, suggest a differential protein assembly for OsL2:OsS2b. Thus, the regulatory and catalytic mechanisms for plastidial AGPases (OsL1:OsS1) could be different in rice culm and developing endosperm compared to those of OsL2:OsS2b, which are predominantly found in rice endosperm.

Regulatory loops between rice transcription factors OsNAC25 and OsNAC20/26 balance starch synthesis.

Several starch synthesis regulators have been identified, but these regulators are situated in the terminus of the regulatory network. Their upstream regulators and the complex regulatory network formed between these regulators remain largely unknown. A previous study demonstrated that NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20 and OsNAC26 (OsNAC20/26), redundantly and positively regulate the accumulation of storage material in rice (Oryza sativa) endosperm. In this study, we detected OsNAC25 as an upstream regulator and interacting protein of OsNAC20/26. Both OsNAC25 mutation and OE resulted in a chalky seed phenotype, decreased starch content, and reduced expression of starch synthesis-related genes, but the mechanisms were different. In the osnac25 mutant, decreased expression of OsNAC20/26 resulted in reduced starch synthesis; however, in OsNAC25-overexpressing plants, the OsNAC25-OsNAC20/26 complex inhibited OsNAC20/26 binding to the promoter of starch synthesis-related genes. In addition, OsNAC20/26 positively regulated OsNAC25. Therefore, the mutual regulation between OsNAC25 and OsNAC20/26 forms a positive regulatory loop to stimulate the expression of starch synthesis-related genes and meet the great demand for starch accumulation in the grain filling stage. Simultaneously, a negative regulatory loop forms among the 3 proteins to avoid the excessive expression of starch synthesis-related genes. Collectively, our findings demonstrate that both promotion and inhibition mechanisms between OsNAC25 and OsNAC20/26 are essential for maintaining stable expression of starch synthesis-related genes and normal starch accumulation.

Characterization of rice endosperm-derived antidepressant-like peptide (REAP): An orally active novel tridecapeptide derived from rice protein

It is ideal to ingest bioactive substances from daily foods to stay healthy. Rice is the staple food for almost half of the human population. We found that an orally administered enzymatic digest of rice endosperm protein exhibits antidepressant-like effects in the tail suspension test (TST) using mice. A comprehensive peptide analysis of the digest using liquid chromatography-tandem mass spectrometry was performed, and a tridecapeptide QQFLPEGQSQSQK, detected in the digest, was chemosynthesized. Oral administration of the tridecapeptide exhibited antidepressant-like effects at a low dose comparable to classical antidepressant in the TST. This also exhibited anti-depressant-like effect in the forced swim test. We named it rice endosperm-derived antidepressant-like peptide (REAP). Intriguingly, intraperitoneal administration had no effect. Orally administered REAP(8−13) but not REAP(1−7) exhibited antidepressant-like activity, suggesting that the C-terminal structure is important for the antidepressant-like effect. We confirmed the presence of REAP, corresponding to rice glutelin type B4(130−142) and B5(130−142), in the digest. The effects of REAP were blocked by both dopamine D1 and D2 antagonists. These results suggest that it exerts its antidepressant-like activity through activation of the dopamine system.Taken together, oral administration of a novel tridecapeptide exhibited antidepressant-like effects via the dopamine system. This is the first report of a rice-derived peptide that exhibits antidepressant-like effects.

Selenium in rice: Impact on protein content and distribution for enhanced food and feed security in agroclimatic challenges

Countries face exasperating and inclement climate worldwide. Food and feed security could be their paramount life objective. The study aimed to investigate the impact of selenium on the protein content and distribution in different parts of rice. For this purpose, advanced selenium biofortified breeding material developed after generations of breeding efforts was investigated at the field area, rice research institute, Chengdu, China during cropping season 2021–22. The accumulation and distribution of selenium and protein contents were observed in various fractions of selenium-enriched rice (Z3057B) and positive control (727). The correlation studies for selenium and protein quantification leads to the optimization of the breeding material and relevance in virtue. The rice fractions indicated rice embryo retains highest selenium contents, which gradually decreases in succession (other rice parts). The difference in protein content between the embryo and endosperm of Se-enriched rice is significant, while that between embryo and aleurone layer is not obvious. The selenium protein was found with molecular weight of 13.6–122.6 kDa. The protein of each molecular weight is found to bind with selenium, but the binding strength of selenium is negatively correlated with the molecular weight of protein. The 67.5% of the total selenium sticks with protein having molecular weight less than 38.8 kDa. In summary, protein with low molecular weight (13.4 kDa) binds maximum selenium and accounts for highest total protein content (40.76%).

Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

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.