DWARF TILLER1 regulates apical-basal pattern formation and proper orientation of rice embryos.

Body axis establishment is one of the earliest patterning events in plant embryogenesis. Asymmetric zygote division is critical for apical-basal axis formation in Arabidopsis (Arabidopsis thaliana). However, how the orientation of the cell division plane is regulated and its relation to apical-basal axis establishment and proper position of embryos in grasses remain poorly understood. By characterizing mutants of 3 rice (Oryza sativa) WUSCHEL HOMEOBOX9 (WOX9) genes, whose paralogs in Arabidopsis play essential roles in zygotic asymmetric cell division and cell fate determination, we found 2 kinds of independent embryonic defects: topsy-turvy embryos, in which apical-basal axis twists from being parallel to the longitudinal axis of the seed to being perpendicular; and organ-less embryos. In contrast to their Arabidopsis orthologs, OsWOX9s displayed dynamic distribution during embryo development. Both DWT1/OsWOX9A and DWL2/WOX9C play major roles in the apical-basal axis formation and initiation of stem cells. In addition, DWT1 has a distinct function in regulating the first few embryonic cell divisions to ensure the correct orientation of the embryo in the ovary. In summary, DWT1 acts in 2 steps during rice embryo pattern formation: the initial zygotic division, and with DWL2 to establish the main body axes and stem cell fate 2 to 3 d after pollination.

A multiomic study uncovers a bZIP23-PER1A-mediated detoxification pathway to enhance seed vigor in rice.

Seed vigor in crops is important in terms of improving grain quality and germplasm conservation; however, little is known about its regulatory mechanisms through the encoded proteome and gene network. Comparative analyses of transcriptome (RNA sequencing [RNA-seq]) and broadly targeted metabolic profiling of two subspecific rice cultivars with distinct seed vigor during accelerated aging revealed various biological pathways and metabolic processes as key influences explaining trait differences. RNA-seq coexpression regulatory network analyses identified several transcription factors, including bZIP23 and bZIP42, that act as nodes in the gene network. Importantly, transgenic seeds of overexpression of bZIP23 enhanced seed vigor, whereas its gene knockout reduced seed vigor, suggesting that the protein it encodes functions as a positive regulator. Similarly, overexpression and knockout of PER1A that encodes a key player in the detoxification pathway enhanced and decreased seed vigor, respectively. We further demonstrated a direct interaction of the PER1A promoter with bZIP23 in seeds, which activates the expression of PER1A, and the genetic evidence suggested that bZIP23 most likely functions in a common pathway with and acts upstream of PER1A to modulate seed vigor. In addition, the control of seed vigor by the bZIP23-PER1A module was connected with that of the abscisic acid signaling pathway. Collectively, we revealed the genetic architecture of variation in seed vigor and uncovered the bZIP23-PER1A-mediated detoxification pathway that enhances the trait in rice.

Comparison of transcriptomic adjustments to availability of sugar, cellular energy, and oxygen in germinating rice embryos.

During germination, the availability of sugars, oxygen, or cellular energy fluctuates under dynamic environmental conditions, likely affecting the global RNA profile of rice genes. Most genes that exhibit sugar-regulation in rice embryos under aerobic conditions are responsive to low energy and anaerobic conditions, indicating that sugar regulation is strongly associated with energy and anaerobic signaling. The interference pattern of sugar regulation by either anaerobic or low energy conditions indicates that induction is likely the more prevalent regulatory mechanism than repression for altering the expression of sugar-regulated genes. Among the aerobically sugar-regulated genes, limited genes exhibit sugar regulation under anaerobic conditions, indicating that anaerobic conditions strongly influence sugar regulated gene expression. Anaerobically responsive genes substantially overlap with low energy responsive genes. In particular, the expression levels of anaerobically downregulated genes are consistent with those provoked by low energy conditions, suggesting that anaerobic downregulation results from the prevention of aerobic respiration due to the absence of the final electron acceptor, i.e., molecular oxygen. It has been noted that abscisic acid (ABA) responsive genes are over representative of genes upregulated under low energy conditions, in contrast to downregulated genes. This suggests that either ABA itself or upstream signaling components of the ABA signaling pathway are likely to be involved in the signaling pathways activated by low energy conditions.

EMBRYO SAC DEVELOPMENT 1 affects seed setting rate in rice by controlling embryo sac development.

Seed setting rate is one of the critical factors that determine rice yield. Grain formation is a complex biological process, whose molecular mechanism is yet to be improved. Here we investigated the function of an OVATE family protein, Embryo Sac Development 1 (ESD1), in the regulation of seed setting rate in rice (Oryza sativa) by examining its loss-of-function mutants generated via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) technology. ESD1 was predominantly expressed at Stage 6 of panicle development, especially in the ovules. esd1 mutants displayed reduced seed setting rates with normal stamen development and pollen tube growth but abnormal pistil group. Investigation of embryo sacs revealed that during the mitosis of functional megaspores, some egg cells degraded during differentiation in esd1 mutants, thereby hindering subsequent fertilization process and reducing seed setting rate. In addition, the transcriptional level of O. sativa anaphase-promoting complex 6, a reported embryo sac developing gene, was significantly reduced in esd1 mutants. These results support that ESD1 is an important modulator of ESD and seed setting rate in rice. Together, this finding demonstrates that ESD1 positively regulates the seed setting rate by controlling ESD in rice and has implications for the improvement of rice yield.

Oral Administration of Germinated, Pigmented, Giant Embryo Rice (Oryza sativa L. cv. Keunnunjami) Extract Improves the Lipid and Glucose Metabolisms in High-Fat Diet-Fed Mice.

Obesity is a significant risk factor for chronic diseases. The effect of ethanol extract from germinated Keunnunjami, blackish-purple rice with a giant embryo, compare to ordinary brown rice, on the body weight and lipid and glucose metabolism in high-fat diet-fed mice was analyzed. Mice were fed with a high-fat diet-fed for 3 weeks and then orally administered with either distilled water (HF) or extract (0.25%, w/w) from brown, germinated brown, Keunnunjami, and germinated Keunnunjami rice for 4 weeks. Control mice were fed with a normal diet and orally administered with distilled water. The HF group showed markedly higher body weight and triglyceride, cholesterol, fatty acid, glucose, and insulin levels than the control group. However, the oral administration of rice extracts ameliorated this high-fat diet-induced obesity, hyperlipidemia, and hypoglycemia through the modulation of adipokine production, lipogenic and glucose-regulating enzyme activities, and mRNA expression of genes associated with lipid and glucose metabolism. The germinated Keunnunjami extract exhibited greater hypolipidemic, hypoglycemic, and body weight-lowering effects than the other rice extracts. The results demonstrated that germination could further enhance the physiological properties of rice and that germinated Keunnunjami extract has a strong therapeutic potential against high-fat diet-induced obesity, hyperlipidemia, and hyperglycemia.

Overexpression of the ascorbate peroxidase through enhancer-trapped pSB111 bar vector for alleviating drought stress in rice.

Rice (Oryza sativa L.) plant growth and productivity is adversely affected by various stress factors. Overexpression of drought tolerance-related genes is one of the best approaches for developing drought-resistant transgenics. Agrobacterium tumefaciens has been widely used in generating transgenic plants through plasmid vector to obtain desired characteristics and to know the specific expression profiles of genes in the plant. The enhancer trap method was developed to know the specific expression of genes at different stages of growth by entrapping the genes of an organism. In the present study, we designed a vector molecule with a feature of promoting the expression of a specific gene more than four times than its normal expression and it is useful for efficient transformation to higher plants by utilizing the trans configuration of vir genes of the plasmid A. tumefaciens, to transfer right and left sequence bordered of transferred DNA (T-DNA) into the nuclear genome of plants. We developed a binary vector consisting of 1.8-kb green fluorescent protein (GFP) cassette as a reporter gene and 1.4-kb tetramer of CaMv35S enhancer (4XEn) were cloned at HindIII site of pSB11 bar intermediate vector to tag and know the genes and their expression profiles, then mobilized into A. tumefaciens to produce a super-binary vector pSB111-bar-4XEn-GFP. The resultant construct was confirmed by polymerase chain reaction and restriction digestion methods. Finally, we discuss the role of overexpressed ascorbate peroxidase in drought stress.

Conserved Imprinted Genes between Intra-Subspecies and Inter-Subspecies Are Involved in Energy Metabolism and Seed Development in Rice.

Genomic imprinting is an epigenetic phenomenon in which a subset of genes express dependent on the origin of their parents. In plants, it is unclear whether imprinted genes are conserved between subspecies in rice. Here we identified imprinted genes from embryo and endosperm 5-7 days after pollination from three pairs of reciprocal hybrids, including inter-subspecies, japonica intra-subspecies, and indica intra-subspecies reciprocal hybrids. A total of 914 imprinted genes, including 546 in inter-subspecies hybrids, 211 in japonica intra-subspecies hybrids, and 286 in indica intra-subspecies hybrids. In general, the number of maternally expressed genes (MEGs) is more than paternally expressed genes (PEGs). Moreover, imprinted genes tend to be in mini clusters. The number of shared genes by R9N (reciprocal crosses between 9311 and Nipponbare) and R9Z (reciprocal crosses between 9311 and Zhenshan 97), R9N and RZN (reciprocal crosses between Zhonghua11 and Nipponbare), R9Z and RZN was 72, 46, and 16. These genes frequently involved in energy metabolism and seed development. Five imprinted genes (Os01g0151700, Os07g0103100, Os10g0340600, Os11g0679700, and Os12g0632800) are commonly detected in all three pairs of reciprocal hybrids and were validated by RT-PCR sequencing. Gene editing of two imprinted genes revealed that both genes conferred grain filling. Moreover, 15 and 27 imprinted genes with diverse functions in rice were shared with Arabidopsis and maize, respectively. This study provided valuable resources for identification of imprinting genes in rice or even in cereals.

A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.

21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells.

In grasses, phased small interfering RNAs (phasiRNAs), 21- or 24-nucleotide (nt) in length, are predominantly expressed in anthers and play a role in regulating male fertility. However, their targets and mode of action on the targets remain unknown. Here we profile phasiRNA expression in premeiotic and meiotic spikelets as well as in purified male meiocytes at early prophase I, tetrads and microspores in rice. We show that 21-nt phasiRNAs are most abundant in meiocytes at early prophase I while 24-nt phasiRNAs are more abundant in tetrads and microspores. By performing highly sensitive degradome sequencing, we find that 21-nt phasiRNAs direct target mRNA cleavage in male germ cells, especially in meiocytes at early prophase I. These targets include 435 protein-coding genes and 71 transposons that show an enrichment for carbohydrate biosynthetic and metabolic pathways. Our study provides strong evidence that 21-nt phasiRNAs act in a target-cleavage mode and may facilitate the progression of meiosis by fine-tuning carbohydrate biosynthesis and metabolism in male germ cells.

Mechanism of delayed seed germination caused by high temperature during grain filling in rice (Oryza sativa L.).

High temperature during grain filling considerably reduces yield and quality in rice (Oryza sativa L.); however, how high temperature affects seed germination of the next generation is not yet well understood. Here, we report that seeds from plants exposed to high temperature during the grain filling stage germinated significantly later than seeds from unstressed plants. This delay remained even after dormancy release treatments, suggesting that it was not due to primary seed dormancy determined during grain filling. In imbibed embryos of heat-stressed seeds, expression of abscisic acid (ABA) biosynthesis genes (OsNCEDs) was higher than in those of control seeds, whereas that of ABA catabolism genes (OsABA8'OHs) was lower. In the aleurone layer, despite no change in GA signaling as evidenced by no effect of heat stress on OsGAMYB gene expression, the transcripts of α-amylase genes OsAmy1C, OsAmy3B, and OsAmy3E were significantly down-regulated in heat-stressed seeds in comparison with controls. Changes in promoter methylation levels were consistent with transcriptional changes of ABA catabolism-related and α-amylase genes. These data suggest that high temperature during grain filling results in DNA methylation of ABA catabolism-related and α-amylase gene promoters, delaying germination of heat-stressed seeds.

OsLFR is essential for early endosperm and embryo development by interacting with SWI/SNF complex members in Oryza sativa.

Rice (Oryza sativa L.) endosperm provides the developing embryo with nutrients and provides human beings with a staple food. The embryo eventually develops into a new sporophyte generation. Despite their important roles, the molecular mechanisms underlying early-stage endosperm and embryo development remain elusive. Here, we established the fundamental functions of rice OsLFR, an ortholog of the Arabidopsis SWI/SNF chromatin-remodeling complex (CRC) component LFR. OsLFR was expressed primarily in the rice spikelets and seeds, and the OsLFR protein was localized to the nucleus. We conducted genetic, cellular and molecular analyses of loss-of-function mutants and transgenic rescue lines. OsLFR depletion resulted in homozygous lethality in the early seed stage through endosperm and embryo defects, which could be successfully recovered by the OsLFR genomic sequence. Cytological observations revealed that the oslfr endosperm had relatively fewer free nuclei, had abnormal and arrested cellularization, and demonstrated premature programed cell death: the embryo was reduced in size and failed to differentiate. Transcriptome profiling showed that many genes, involved in DNA replication, cell cycle, cell wall assembly and cell death, were differentially expressed in a knockout mutant of OsLFR (oslfr-1), which was consistent with the observed seed defects. Protein-protein interaction analysis showed that OsLFR physically interacts with several putative rice SWI/SNF CRC components. Our findings demonstrate that OsLFR, possibly as one component of the SWI/SNF CRC, is an essential regulator of rice seed development, and provide further insights into the regulatory mechanism of early-stage rice endosperm and embryo development.

Biolistic Transformation of Japonica Rice Varieties.

Genetic improvement of rice is crucial to achieve global food security as rice is an important staple crop for more than half of the global population. One of the methodologies for genetic improvement is biolistic delivery of genetic components into plant cells. In this chapter, we describe steps involved in introducing plasmid DNA carrying gene of interest into rice mature embryos using Biolistic® PDS-1000/He particle delivery system. We also provide information required for recovery of transformed plants and production of transgenic seed for next generation analysis. Using this protocol, typical 50-70 putative independent transgenic callus lines can be generated from 100 bombarded embryos. Transgenic rice plantlets can be produced within 2 months after the initiation of seed germination for transformation.

CHR721, interacting with OsRPA1a, is essential for both male and female reproductive development in rice.

KEY MESSAGE: CHR721 functions as a chromatin remodeler and interacts with a known single-stranded binding protein, OsRPA1a, to regulate both male and female reproductive development in rice. Reproductive development and fertility are important for seed production in rice. Here, we identified a sterile rice mutant, chr721, that exhibited defects in both male and female reproductive development. Approximately 5% of the observed defects in chr721, such as asynchronous dyad division, occurred during anaphase II of meiosis. During the mitotic stage, approximately 80% of uninucleate microspores failed to develop into tricellular pollen, leading to abnormal development. In addition, defects in megaspore development were detected after functional megaspore formation. CHR721, which encodes a nuclear protein belonging to the SNF2 subfamily SMARCAL1, was identified by map-based cloning. CHR721 was expressed in various tissues, especially in spikelets. CHR721 was found to interact with replication protein A (OsRPA1a), which is involved in DNA repair. The expressions of genes involved in DNA repair and cell-cycle checkpoints were consistently upregulated in chr721. Although numerous genes involved in male and female development have been identified, the mode of participation of chromatin-remodeling factors in reproductive development is still not well understood. Our results suggest that CHR721, a novel gene cloned from rice, plays a vital role in both male and female reproductive development.

In Vitro Production of Zygotes by Electrofusion of Rice Gametes.

In angiosperms, fertilization and embryogenesis occur in the embryo sac, which is deeply embedded in ovular tissue. In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect postfertilization events in angiosperms, such as egg activation, zygotic development, and early embryogenesis. In addition, using IVF systems, interspecific zygotes and polyploid zygotes have been artificially produced, and their developmental profiles/mechanisms have been analyzed. Taken together, the IVF system can be considered a powerful technique for investigating the fertilization-induced developmental sequences in zygotes and generating new cultivars with desirable characteristics. Here, we describe the procedures for the isolation of rice gametes, electrofusion of gametes, and the culture of the produced zygotes and embryo.

RADICLELESS 1 (RL1)-mediated nad4 intron 1 splicing is crucial for embryo and endosperm development in rice (Oryza sativa L.).

Pentatricopeptide repeat (PPR) proteins are one of the largest protein families in land plants. PPR proteins exhibit sequence-specific RNA-binding activity and are implicated in plant growth and development related processes. In this study, we report that the radicleless 1 (rl1) mutant in rice (Oryza sativa L.) exhibited defective radicle emergence in embryos and compromised grain filling in endosperms. Gene cloning and confirmation via genetic complementation analyses showed that RL1 encodes a P-type PPR protein, which is localized to mitochondria. The RL1 protein was specifically involved in the splicing of intron 1 of the mitochondrial nad4 transcript, which encodes a subunit of the mitochondrial NADH dehydrogenase complex. Consistent with this observation, the rl1 mutant exhibited altered mitochondrial morphology and lower ATP accumulation compared with the wild type. Thus, our findings suggest that RL1-mediated nad4 splicing is crucial for embryo and endosperm development in rice.

Quantitative morphological phenomics of rice G protein mutants portend autoimmunity.

The heterotrimeric G protein complex, composed of Gα, Gß, and Gγ subunits, plays some role in structural development in plants but this role could be indirect because loss-of-function mutations do not alter the body plan and post-embryonic organs differ only morphologically and not in their identity. This uncertainty has been compounded by the fact that loss of the Gß subunit in cereals, but not Arabidopsis, is seedling lethal and that loss of maize Gα subunit confers prolificacy of a reproductive organ. In this study, we comprehensively profiled the root and shoot structural traits of rice Gα-null and viable Gß-RNAi "knockdown" mutants, and found anomalous morphologies caused by Gß-RNAi that are distinct from the Arabidopsis orthologue. The rice Gß-RNAi mutant exhibited reduced radial growth of aerial parts as well as a more compact root architecture, among which smaller root mass seems mainly due to increased necrosis when grown on soil. In addition, three dimensional analyses of rice root system architecture revealed that the smaller root architecture of Gß-RNAi plant is also due to both reduced root elongation and adventitious root formation. This contrasts to the Arabidopsis Gß-null mutation that promotes cell proliferation. There is elevated cell senescence activity both visualized by Evans Blue staining and inferred from an expression analysis of cell-death marker genes. We propose that the morphological phenotypes of rice Gß-RNAi plants are predominantly associated with the mediation of various stresses and cell senescence, consistent with an indirect role for Arabidopsis Gß in development where the orthologous gene ablation mainly confers altered cell proliferation. We also elaborate our speculative working hypothesis that cell division is a type of stress and as such due to impairment in responding to stress in the G protein mutants, manifests as altered morphology and architecture but not an altered body plan or organ identities.

Characterization of AtWRI1 in fatty acids and starch synthesis in rice.

WRINKLED1 (WRI1) belongs to AP2/EREBP transcription factor. Its function in dicots for fatty acids synthesis has been deeply studied, but its role in monocot, especially in rice, is still poorly understood. Here, with the overexpression of AtWRI1 in rice, we found its overexpression increased fatty acids content in vegetative organs and seed coat including aleurone layer (SCAL) but decreased fatty acids content in endosperm. Meanwhile, the overexpression of AtWRI1 increased starch content in endosperm. These results provide a new insight into the function of AtWRI1in monocot and make a previous basement for the study of the connection of fatty acids and starch synthesis in rice.

Specification of basal region identity after asymmetric zygotic division requires mitogen-activated protein kinase 6 in rice.

Asymmetric cell division is a key step in cellular differentiation in multicellular organisms. In plants, asymmetric zygotic division produces the apical and basal cells. The mitogen-activated protein kinase (MPK) cascade in Arabidopsis acts in asymmetric divisions such as zygotic division and stomatal development, but whether the effect on cellular differentiation of this cascade is direct or indirect following asymmetric division is not clear. Here, we report the analysis of a rice mutant, globular embryo 4 (gle4). In two- and four-cell-stage embryos, asymmetric zygotic division and subsequent cell division patterns were indistinguishable between the wild type and gle4 mutants. However, marker gene expression and transcriptome analyses showed that specification of the basal region was compromised in gle4 We found that GLE4 encodes MPK6 and that GLE4/MPK6 is essential in cellular differentiation rather than in asymmetric zygotic division. Our findings provide a new insight into the role of MPK in plant development. We propose that the regulation of asymmetric zygotic division is separate from the regulation of cellular differentiation that leads to apical-basal polarity.

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.

Photosynthetic Performance of Rice Seedlings Originated from Seeds Exposed to Spaceflight Conditions.

The mechanism of the regulation on photosynthesis after spaceflight has not been fully understood. To learn more information about this, we conducted a series of experiments of photosystem, including photosynthetic physiological characteristics (fluorescence parameters, pigment contents), gene expression and proteomic change. We want to examine the response of rice (Oryza sativaDN416), whose seeds were placed in Bio-Radiation Box on the ShiJian-10(SJ-10) recoverable satellite. Our results demonstrated that the photosynthesis capacity of plants after spaceflight declined, compared to ground control plants. Specifically, Fv/Fm is significantly reduced for 7.5%. Chlorophyll content decreased in the three growth stages of rice, trefoil, tillering and mature stages. To further analyze changes under spaceflight environment, quantitative real-time PCR technology and isobaric tags for relative and absolute quantization (iTRAQ) labeling technology were deployed. We found that the gene expression of important subunits of key enzymes and important structures had been decreased after spaceflight. As for the results of changes in proteins, we discovered that the content of proteins related to electron transport and photosynthesis key enzyme declined. Our experiments can provide reference for further research to learn more about the effects of spaceflight on photosynthesis.