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.

Elongation of Siliques Without Pollination 3 Regulates Nutrient Flow Necessary for Embryogenesis.

Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce the endosperm. The endosperm is essential for embryogenesis, and its development is suppressed until fertilization. We show that the expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the flowering Wageningen homeodomain transcription factor containing a StAR-related lipid transfer domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar to those in fertilized seeds, while those related to autophagy were downregulated similar to those in fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization.

Multi-omic characterization of the maize GPI synthesis mutant gwt1 with defects in kernel development.

BACKGROUND: Glycosylphosphatidylinositol (GPI) and GPI-anchored proteins (GAPs) are important for cell wall formation and reproductive development in Arabidopsis. However, monocot counterparts that function in kernel endosperm development have yet to be discovered. Here, we performed a multi-omic analysis to explore the function of GPI related genes on kernel development in maize. RESULTS: In maize, 48 counterparts of human GPI synthesis and lipid remodeling genes were identified, in which null mutation of the glucosaminyl-phosphatidylinositol O-acyltransferase1 gene, ZmGWT1, caused a kernel mutant (named gwt1) with defects in the basal endosperm transport layer (BETL). We performed plasma membrane (PM) proteomics to characterize the potential GAPs involved in kernel development. In total, 4,981 proteins were successfully identified in 10-DAP gwt1 kernels of mutant and wild-type (WT), including 1,638 membrane-anchored proteins with different posttranslational modifications. Forty-seven of the 256 predicted GAPs were differentially accumulated between gwt1 and WT. Two predicted BETL-specific GAPs (Zm00001d018837 and Zm00001d049834), which kept similar abundance at general proteome but with significantly decreased abundance at membrane proteome in gwt1 were highlighted. CONCLUSIONS: Our results show the importance of GPI and GAPs for endosperm development and provide candidate genes for further investigation of the regulatory network in which ZmGWT1 participates.

The phenomenon of autonomous endosperm in sexual and apomictic plants.

Endosperm is a key nutritive tissue that supports the developing embryo or seedling, and serves as a major nutritional source for human and livestock feed. In sexually-reproducing flowering plants, it generally develops after fertilization. However, autonomous endosperm (AE) formation (i.e. independent of fertilization) is also possible. Recent findings of AE loci/ genes and aberrant imprinting in native apomicts, together with a successful initiation of parthenogenesis in rice and lettuce, have enhanced our understanding of the mechanisms bridging sexual and apomictic seed formation. However, the mechanisms driving AE development are not well understood. This review presents novel aspects related to AE development in sexual and asexual plants underlying stress conditions as the primary trigger for AE. Both application of hormones to unfertilized ovules and mutations that impair epigenetic regulation lead to AE development in sexual Arabidopsis thaliana, which may point to a common pathway for both phenomena. Apomictic-like AE development under experimental conditions can take place due to auxin-dependent gene expression and/or DNA methylation.

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development.

Most of the reported P-type pentatricopeptide repeat (PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22 (flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein. Mutation of FLO22 resulting in defective trans-splicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22 mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly up-regulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

Introduction of glucan synthase into the cytosol in wheat endosperm causes massive maltose accumulation and represses starch synthesis.

We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic ß-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of ß-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan.

Identification and characterization of miRNAs and PHAS loci related to the early development of the embryo and endosperm in Fragaria × ananassa.

BACKGROUND: The strawberry fleshy fruit is actually enlarged receptacle tissue, and the successful development of the embryo and endosperm is essential for receptacle fruit set. MicroRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) play indispensable regulatory roles in plant growth and development. However, miRNAs and phasiRNAs participating in the regulation of strawberry embryo and endosperm development have yet to be explored. RESULTS: Here, we performed genome-wide identification of miRNA and phasiRNA-producing loci (PHAS) in strawberry seeds with a focus on those involved in the development of the early embryo and endosperm. We found that embryos and endosperm have different levels of small RNAs. After bioinformatics analysis, the results showed that a total of 404 miRNAs (352 known and 52 novel) and 156 PHAS genes (81 21-nt and 75 24-nt genes) could be found in strawberry seed-related tissues, of which four and nine conserved miRNA families displayed conserved expression in the endosperm and embryo, respectively. Based on refined putative annotation of PHAS loci, some auxin signal-related genes, such as CM3, TAR2, AFB2, ASA1, NAC and TAS3, were found, which demonstrates that IAA biosynthesis is important for endosperm and embryo development during early fruit growth. Additionally, some auxin signal-related conserved (miR390-TAS3) and novel (miR156-ASA1) trigger-PHAS pairs were identified. CONCLUSIONS: Taken together, these results expand our understanding of sRNAs in strawberry embryo and endosperm development and provide a genomic resource for early-stage fruit development.

The catalytic core of DEMETER guides active DNA demethylation in Arabidopsis.

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.

Natural alleles of a uridine 5'-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice.

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5'-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1YZN compared to EDR1YD1 , resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1YD1 and EDR1YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1YD1 and EDR1YZN varieties indicated that rice varieties harboring EDR1YZN and EDR1YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.

Functional divergence of two duplicated Fertilization Independent Endosperm genes in rice with respect to seed development.

Fertilization Independent Endosperm (FIE) is an essential member of Polycomb Repressive Complex 2 (PRC2) that plays important roles in the developmental regulation of plants. OsFIE1 and OsFIE2 are two FIE homologs in the rice genome. Here, we showed that OsFIE1 probably duplicated from OsFIE2 after the origin of the tribe Oryzeae, but has a specific expression pattern and methylation landscape. During evolution, OsFIE1 underwent a less intensive purifying selection than did OsFIE2. The mutant osfie1 produced smaller seeds and displayed reduced dormancy, indicating that OsFIE1 predominantly functions in late seed development. Ectopic expression of OsFIE1, but not OsFIE2, was deleterious to vegetative growth in a dose-dependent manner. The newly evolved N-terminal tail of OsFIE1 was probably not the cause of the adverse effects on vegetative growth. The CRISPR/Cas9-derived mutant osfie2 exhibited impaired cellularization of the endosperm, which suggested that OsFIE2 is indispensable for early seed development as a positive regulator of cellularization. Autonomous endosperm was observed in both OsFIE2+- and osfie1/OsFIE2+- but at a very low frequency. Although OsFIE1-PRC2 exhibited H3K27me3 methyltransferase ability in plants, OsFIE1-PRC2 is likely to be less important for development in rice than is OsFIE2-PRC2. Our findings revealed the functional divergence of OsFIE1 and OsFIE2 and shed light on their distinct evolution following duplication.

The co-expression of genes involved in seed coat and endosperm development promotes seed abortion in grapevine.

MAIN CONCLUSION: The seed coat gene VviAGL11 coordinates with endosperm development genes FIS2, PHERESE1 and IKU2 and functions as the key regulator in seed development and abortion processes in grapevine. Seed development is essential for the reproduction of flowering plants. Seed abortion is a specific characteristic that produces seedless berries and is often observed in cultivated grapevines. Although seedlessness is an important trait for table and dried grapevine production, the mechanism of seed abortion remains poorly understood. This research aimed to analyze the co-expression of the seed coat development gene VviAGL11 and the endosperm development genes FERTILIZATION INDEPENDENT SEED2 (FIS2), PHERESE1 and HAIKU2 (IKU2) that regulate seedless fruit development in grapevine. The transcript levels of VviAGL11, FIS2, PHERESE1 and IKU2 all decreased during seed abortion in the seedless grape 'Thompson Seedless' plants, compared to those of the seeded grape 'Pinot Noir'. The transcript levels of the salicylic acid (SA)-dependent defense response genes EDS1, NPR1, NDR1 and SID2 were higher in 'Thompson Seedless' than 'Pinot Noir' during seed development. Also, WRKY3, WRKY6 and WRKY52, which participate in the SA pathway, were higher expressed in 'Thompson Seedless' than in 'Pinot Noir', indicating that SA-dependent defense responses may regulate seed abortion. The genes related to synthesis and metabolism of gibberellic acid (GA) and abscisic acid (ABA) also showed differential expression between 'Thompson Seedless' and 'Pinot Noir'. Exogenous applications of plant growth regulators (PGRs) to inflorescences of three stenospermocarpy grapevines before flowering showed that GA3 was critical prominently in seed development. Therefore, the co-expression of seed coat and endosperm development-related genes, SA pathway genes, and genes for the synthesis and metabolism of GA3 together enhance seed abortion in seedless grapes.

Expansion and Functional Diversification of TFIIB-Like Factors in Plants.

As sessile organisms, plants have evolved unique patterns of growth and development, elaborate metabolism and special perception and signaling mechanisms to environmental cues. Likewise, plants have complex and highly special programs for transcriptional control of gene expression. A case study for the special transcription control in plants is the expansion of general transcription factors, particularly the family of Transcription Factor IIB (TFIIB)-like factors with 15 members in Arabidopsis. For more than a decade, molecular and genetic analysis has revealed important functions of these TFIIB-like factors in specific biological processes including gametogenesis, pollen tube growth guidance, embryogenesis, endosperm development, and plant-microbe interactions. The redundant, specialized, and diversified roles of these TFIIB-like factors challenge the traditional definition of general transcription factors established in other eukaryotes. In this review, we discuss general transcription factors in plants with a focus on the expansion and functional analysis of plant TFIIB-like proteins to highlight unique aspects of plant transcription programs that can be highly valuable for understanding the molecular basis of plant growth, development and responses to stress conditions.

Maize endosperm development.

Recent breakthroughs in transcriptome analysis and gene characterization have provided valuable resources and information about the maize endosperm developmental program. The high temporal-resolution transcriptome analysis has yielded unprecedented access to information about the genetic control of seed development. Detailed spatial transcriptome analysis using laser-capture microdissection has revealed the expression patterns of specific populations of genes in the four major endosperm compartments: the basal endosperm transfer layer (BETL), aleurone layer (AL), starchy endosperm (SE), and embryo-surrounding region (ESR). Although the overall picture of the transcriptional regulatory network of endosperm development remains fragmentary, there have been some exciting advances, such as the identification of OPAQUE11 (O11) as a central hub of the maize endosperm regulatory network connecting endosperm development, nutrient metabolism, and stress responses, and the discovery that the endosperm adjacent to scutellum (EAS) serves as a dynamic interface for endosperm-embryo crosstalk. In addition, several genes that function in BETL development, AL differentiation, and the endosperm cell cycle have been identified, such as ZmSWEET4c, Thk1, and Dek15, respectively. Here, we focus on current advances in understanding the molecular factors involved in BETL, AL, SE, ESR, and EAS development, including the specific transcriptional regulatory networks that function in each compartment during endosperm development.

Rice FLOURY ENDOSPERM 18 encodes a pentatricopeptide repeat protein required for 5' processing of mitochondrial nad5 messenger RNA and endosperm development.

Pentatricopeptide repeat (PPR) proteins, composing one of the largest protein families in plants, are involved in RNA binding and regulation of organelle RNA metabolism at the post-transcriptional level. Although several PPR proteins have been implicated in endosperm development in rice (Oryza sativa), the molecular functions of many PPRs remain obscure. Here, we identified a rice endosperm mutant named floury endosperm 18 (flo18) with pleiotropic defects in both reproductive and vegetative development. Map-based cloning and complementation tests showed that FLO18 encodes a mitochondrion-targeted P-type PPR protein with 15 PPR motifs. Mitochondrial function was disrupted in the flo18 mutant, as evidenced by decreased assembly of Complex I in the mitochondrial electron transport chain and altered mitochondrial morphology. Loss of FLO18 function resulted in defective 5'-end processing of mitochondrial nad5 transcripts encoding subunit 5 of nicotinamide adenine dinucleotide hydrogenase. These results suggested that FLO18 is involved in 5'-end processing of nad5 messenger RNA and plays an important role in mitochondrial function and endosperm development.

Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation.

Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

Autonomous and non-autonomous functions of the maize Shohai1 gene, encoding a RWP-RK putative transcription factor, in regulation of embryo and endosperm development.

In plants, establishment of the basic body plan during embryogenesis involves complex processes of axis formation, cell fate specification and organ differentiation. While molecular mechanisms of embryogenesis have been well studied in the eudicot Arabidopsis, only a small number of genes regulating embryogenesis has been identified in grass species. Here, we show that a RKD-type RWP-RK transcription factor encoded by Shohai1 (Shai1) is indispensable for embryo and endosperm development in maize. Loss of Shai1 function causes variable morphological defects in the embryo including small scutellum, shoot axis bifurcation and arrest during early organogenesis. Analysis of molecular markers in mutant embryos reveals disturbed patterning of gene expression and altered polar auxin transport. In contrast with typical embryo-defective (emb) mutants that expose a vacant embryo pocket in the endosperm, the endosperm of shai1 kernels conforms to the varied size and shape of the embryo. Furthermore, genetic analysis confirms that Shai1 is required for autonomous formation of the embryo pocket in endosperm of emb mutants. Analyses of genetic mosaic kernels generated by B-A translocation revealed that expression of Shai1 in the endosperm could partially rescue a shai1 mutant embryo and suggested that Shai1 is involved in non-cell autonomous signaling from endosperm that supports normal embryo growth. Taken together, we propose that the Shai1 gene functions in regulating embryonic patterning during grass embryogenesis partly by endosperm-to-embryo interaction.

Differential expression networks and inheritance patterns of long non-coding RNAs in castor bean seeds.

Long non-coding RNAs (lncRNAs) serve as versatile regulators of plant growth and development. The potential functions and inheritance patterns of lncRNAs, as well as the epigenetic regulation of lncRNA itself, remain largely uncharacterized in plant seeds, especially in the persistent endosperm of the dicotyledons. In this study, we investigated diverse RNA-seq data and catalogued 5356 lncRNAs in castor bean seeds. A small fraction of lncRNAs were transcribed from the same direction as the promoters of protein-coding genes (PCgenes) and exhibited strongly coordinated expression with the nearby PCgene. Co-expression analysis with weighted gene co-expression network analysis (WGCNA) showed these lncRNAs to be involved in differential transcription networks between the embryo and endosperm in the early developing seed. Genomic DNA methylation analyses revealed that the expression level of lncRNAs was tightly linked to DNA methylation and that endosperm hypomethylation could promote the expression of linked lncRNAs. Intriguingly, upon hybridization, most lncRNAs with divergent genome sequences between two parents could be reconciled and were expressed according to their parental genome contribution; however, some deviation in the expression of allelic lncRNAs was observed and found to be partially dependent on parental effects. In triploid endosperm, the expression of most lncRNAs was not dosage sensitive, as only 20 lncRNAs had balanced dosage. Our findings not only demonstrate that lncRNAs play potential roles in regulating the development of castor bean endosperm and embryo, but also provide novel insights into the parental effects, allelic expression and epigenetic regulation of lncRNAs in dicotyledonous seeds.

Rice FLOURY ENDOSPERM10 encodes a pentatricopeptide repeat protein that is essential for the trans-splicing of mitochondrial nad1 intron 1 and endosperm development.

Endosperm, the major storage organ in cereal grains, determines grain yield and quality. Despite the fact that a role for P-type pentatricopeptide repeat (PPR) proteins in the regulation of endosperm development has emerged, molecular functions of many P-type PPR proteins remain obscure. Here, we report a rice endosperm defective mutant, floury endosperm10 (flo10), which developed smaller starch grains in starchy endosperm and abnormal cells in the aleurone layer. Map-based cloning and rescued experiments showed that FLO10 encodes a P-type PPR protein with 26 PPR motifs, which is localized to mitochondria. Loss of function of FLO10 affected the trans-splicing of the mitochondrial nad1 intron 1, which was accompanied by the increased accumulation of the nad1 exon 1 and exons 2-5 precursors. The failed formation of mature nad1 led to a dramatically decreased assembly and activity of complex I, reduced ATP production, and changed mitochondrial morphology. In addition, loss of function of FLO10 significantly induced an alternative respiratory pathway involving alternative oxidase. These results reveal that FLO10 plays an important role in the maintenance of mitochondrial function and endosperm development through its effect on the trans-splicing of the mitochondrial nad1 intron 1 in rice.

Instability of endosperm development in amphiploids and their parental species in the genus Avena L.

KEY MESSAGE: The development of oat endosperm is modified by chromatin and nuclei elimination, intrusive growth of cell walls, and polyploidisation of cell clones. The last event is correlated with somatic crossing-over. Grass endosperm is a variable tissue in terms of its cytogenetics and development. Free-nuclear syncytium and starchy and aleurone endosperm were the main focus of the research. These were studied in oat amphiploids (4x, 6x, and 8x) and parental species (2x, 4x, and 6x). What the levels of cytogenetic disorders and developmental anomalies in species versus hybrids are, and, what the factors are determining phenotypes of both tissue components, are open questions for oats. Chromosome bridges and micronuclei are the main cytogenetic disorders showing the elimination of parts of genomes. Bridges are formed by the AT-heterochromatin-rich and -free ends of chromosomes. In the starchy tissue, various sectors are separated structurally due to the elongation or intrusive growth of aleurone cells. The development of the aleurone layer is highly disturbed locally due to the amplification of aleurone cell divisions. Changes related to their structure and metabolism occur in the aleurone cells, for example, clones of small versus large aleurone cells. Somatic crossing-over (SCO) is expressed in clones of large polyploidised cells (r = 0.80***), giving rise to new aleurone phenotypes. The multivariate description of the endosperm instability showed that endospermal disorders were more frequent in amphiploids than in the oat species. Avena strigosa and the amphiploid A. fatua × A. sterilis appeared to be extreme units in an ordination space. Nuclear DNA elimination, periclinal and multidirectional cytokineses, polyploidisation, intrusive growth, and SCO appeared to be important factors determining oat endospermal variations.

WB1, a Regulator of Endosperm Development in Rice, Is Identified by a Modified MutMap Method.

Abnormally developed endosperm strongly affects rice (Oryza sativa) appearance quality and grain weight. Endosperm formation is a complex process, and although many enzymes and related regulators have been identified, many other related factors remain largely unknown. Here, we report the isolation and characterization of a recessive mutation of White Belly 1 (WB1), which regulates rice endosperm development, using a modified MutMap method in the rice mutant wb1. The wb1 mutant develops a white-belly endosperm and abnormal starch granules in the inner portion of white grains. Representative of the white-belly phenotype, grains of wb1 showed a higher grain chalkiness rate and degree and a lower 1000-grain weight (decreased by ~34%), in comparison with that of Wild Type (WT). The contents of amylose and amylopectin in wb1 significantly decreased, and its physical properties were also altered. We adopted the modified MutMap method to identify 2.52 Mb candidate regions with a high specificity, where we detected 275 SNPs in chromosome 4. Finally, we identified 19 SNPs at 12 candidate genes. Transcript levels analysis of all candidate genes showed that WB1 (Os04t0413500), encoding a cell-wall invertase, was the most probable cause of white-belly endosperm phenotype. Switching off WB1 with the CRISPR/cas9 system in Japonica cv. Nipponbare demonstrates that WB1 regulates endosperm development and that different mutations of WB1 disrupt its biological function. All of these results taken together suggest that the wb1 mutant is controlled by the mutation of WB1, and that the modified MutMap method is feasible to identify mutant genes, and could promote genetic improvement in rice.