MiRiQ Database: A Platform for In Silico Rice Mutant Screening.

Genetic studies using mutant resources have significantly contributed to elucidating plant gene function. Massive mutant libraries sequenced by next-generation sequencing technology facilitate mutant identification and functional analysis of genes of interest. Here, we report the creation and release of an open-access database (https://miriq.agr.kyushu-u.ac.jp/index.php), called Mutation-induced Rice in Kyushu University (MiRiQ), designed for in silico mutant screening based on a whole-genome-sequenced mutant library. This database allows any user to easily find mutants of interest without laborious efforts such as large-scale screening by PCR. The initial version of the MiRiQ database (version 1.0) harbors a total of 1.6 million single-nucleotide variants (SNVs) and InDels of 721 M1 plants that were mutagenized by N-methyl-N-nitrosourea treatment of the rice cultivar Nipponbare (Oryza sativa ssp. japonica). The SNVs were distributed among 87% of all 35,630 annotated protein-coding genes of the Nipponbare genome and were predicted to induce missense and nonsense mutations. The MiRiQ database provides built-in tools, such as a search tool by keywords and JBrowse for mutation searches. Users can request mutant seeds in the M2 or M3 generations from a request form linked to this database. We believe that the availability of a wide range of gene mutations in this database will benefit the plant science community and breeders worldwide by accelerating functional genomic research and crop improvement.

Collection, preservation and distribution of Oryza genetic resources by the National Bioresource Project RICE (NBRP-RICE).

Biological resources are the basic infrastructure of bioscience research. Rice (Oryza sativa L.) is a good experimental model for research in cereal crops and monocots and includes important genetic materials used in breeding. The availability of genetic materials, including mutants, is important for rice research. In addition, Oryza species are attractive to researchers for both finding useful genes for breeding and for understanding the mechanism of genome evolution that enables wild plants to adapt to their own habitats. NBRP-RICE contributes to rice research by promoting the usage of genetic materials, especially wild Oryza accessions and mutant lines. Our activity includes collection, preservation and distribution of those materials and the provision of basic information on them, such as morphological and physiological traits and genomic information. In this review paper, we introduce the activities of NBRP-RICE and our database, Oryzabase, which facilitates the access to NBRP-RICE resources and their genomic sequences as well as the current situation of wild Oryza genome sequencing efforts by NBRP-RICE and other institutes.

BELL1-like homeobox genes regulate inflorescence architecture and meristem maintenance in rice.

Inflorescence architecture is diverse in angiosperms, and is mainly determined by the arrangement of the branches and flowers, known as phyllotaxy. In rice (Oryza sativa), the main inflorescence axis, called the rachis, generates primary branches in a spiral phyllotaxy, and flowers (spikelets) are formed on these branches. Here, we have studied a classical mutant, named verticillate rachis (ri), which produces branches in a partially whorled phyllotaxy. Gene isolation revealed that RI encodes a BELL1-type homeodomain transcription factor, similar to Arabidopsis PENNYWISE/BELLRINGER/REPLUMLESS, and is expressed in the specific regions within the inflorescence and branch meristems where their descendant meristems would soon initiate. Genetic combination of an ri homozygote and a mutant allele of RI-LIKE1 (RIL1) (designated ri ril1/+ plant), a close paralog of RI, enhanced the ri inflorescence phenotype, including the abnormalities in branch phyllotaxy and rachis internode patterning. During early inflorescence development, the timing and arrangement of primary branch meristem (pBM) initiation were disturbed in both ri and ri ril1/+ plants. These findings suggest that RI and RIL1 were involved in regulating the phyllotactic pattern of the pBMs to form normal inflorescences. In addition, both RI and RIL1 seem to be involved in meristem maintenance, because the ri ril1 double-mutant failed to establish or maintain the shoot apical meristem during embryogenesis.

Axillary Meristem Formation in Rice Requires the WUSCHEL Ortholog TILLERS ABSENT1.

Axillary shoot formation is a key determinant of plant architecture. Formation of the axillary shoot is regulated by initiation of the axillary meristem or outgrowth of the axillary bud. Here, we show that rice (Oryza sativa) TILLERS ABSENT1 (TAB1; also known as Os WUS), an ortholog of Arabidopsis thaliana WUS, is required to initiate axillary meristem development. We found that formation of the axillary meristem in rice proceeds via a transient state, which we term the premeristem, characterized by the expression of OSH1, a marker of indeterminate cells in the shoot apical meristem. In the tab1-1 (wus-1) mutant, however, formation of the axillary meristem is arrested at various stages of the premeristem zone, and OSH1 expression is highly reduced. TAB1/WUS is expressed in the premeristem zone, where it shows a partially overlapping pattern with OSH1. It is likely, therefore, that TAB1 plays an important role in maintaining the premeristem zone and in promoting the formation of the axillary meristem by promoting OSH1 expression. Temporal expression patterns of WUSCHEL-RELATED HOMEOBOX4 (WOX4) indicate that WOX4 is likely to regulate meristem maintenance instead of TAB1 after establishment of the axillary meristem. Lastly, we show that the prophyll, the first leaf in the secondary axis, is formed from the premeristem zone and not from the axillary meristem.

Analysis of the Rice ADP-Glucose Transporter (OsBT1) Indicates the Presence of Regulatory Processes in the Amyloplast Stroma That Control ADP-Glucose Flux into Starch.

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[(14)C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.

Development and evaluation of rice giant embryo mutants for high oil content originated from a high-yielding cultivar 'Mizuhochikara'.

Rice bran oil is a byproduct of the milling of rice (Oryza sativa L.). It offers various health benefits and has a beneficial fatty acid composition. To increase the amount of rice bran as a sink for triacylglycerol (TAG), we developed and characterized new breeding materials with giant embryos. To induce mutants, we treated fertilized egg cells of the high-yielding cultivar 'Mizuhochikara' with N-methyl-N-nitrosourea (MNU). By screening M2 seeds, we isolated four giant embryo mutant lines. Genetic analysis revealed that the causative loci in lines MGE12 and MGE13 were allelic to giant embryo (ge) on chromosome 7, and had base changes in the causal gene Os07g0603700. On the other hand, the causative loci in lines MGE8 and MGE14 were not allelic to ge, and both were newly mapped on chromosome 3. The TAG contents of all four mutant lines increased relative to their wild type, 'Mizuhochikara'. MGE13 was agronomically similar to 'Mizuhochikara' and would be useful for breeding for improved oil content.

The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme.

Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme.

Influence of single-nucleotide polymorphisms in the gene encoding granule-bound starch synthase I on amylose content in Vietnamese rice cultivars.

Amylose content is one of the most important factors influencing the physical and chemical properties of starch in rice. Analysis of 352 Vietnamese rice cultivars revealed a wide range of variation in apparent amylose content and the expression level of granule-bound starch synthase. On the basis of single-nucleotide polymorphisms (SNP) at the splicing donor site of the first intron and in the coding region of the granule-bound starch synthase I gene, Waxy gene, alleles can be classified into seven groups that reflect differences in apparent amylose content. The very low and low apparent amylose content levels were tightly associated with a G to T in the first intron whereas intermediate and high amylose was associated with a T genotype at SNP in exon 10. The correlation between the combination of T genotype at SNP in the first intron, C in exon 6, or C in exon 10 was predominant among low amylose rice varieties. Our analysis confirmed the existence of Wx (op) allele in Vietnamese rice germplasm. The results of this study suggest that the low amylose properties of Vietnamese local rice germplasm are attributable to spontaneous mutations at exons, and not at the splicing donor site.

Whole-Genome Sequencing of Rice Mutant Library Members Induced by N-Methyl-N-Nitrosourea Mutagenesis of Fertilized Egg Cells.

Although targeted genome editing technology has become a powerful reverse genetic approach for accelerating functional genomics, conventional mutant libraries induced by chemical mutagens remain valuable for plant studies. Plants containing chemically induced mutations are simple yet effective genetic tools that can be grown without regard for biosafety issues. Whole-genome sequencing of mutant individuals reduces the effort required for mutant screening, thereby increasing their utility. In this study, we sequenced members of a mutant library of Oryza sativa cv. Nipponbare derived from treating single fertilized egg cells with N-methyl-N-nitrosourea (MNU). By whole-genome sequencing 266 M1 plants in this mutant library, we identified a total of 0.66 million induced point mutations. This result represented one mutation in every 146-kb of genome sequence in the 373 Mb assembled rice genome. These point mutations were uniformly distributed throughout the rice genome, and over 70,000 point mutations were located within coding sequences. Although this mutant library was a small population, nonsynonymous mutations were found in nearly 61% of all annotated rice genes, and 8.6% (3248 genes) had point mutations with large effects on gene function, such as gaining a stop codon or losing a start codon. WGS showed MNU-mutagenesis using rice fertilized egg cells induces mutations efficiently and is suitable for constructing mutant libraries for an in silico mutant screening system. Expanding this mutant library and its database will provide a useful in silico screening tool that facilitates functional genomics studies with a special emphasis on rice.

A role for the cysteine-rich 10 kDa prolamin in protein body I formation in rice.

The rice prolamins consist of cysteine-rich 10 kDa (CysR10), 14 kDa (CysR14) and 16 kDa (CysR16) molecular species and a cysteine-poor 13 kDa (CysP13) polypeptide. These storage proteins form protein bodies (PBs) composed of single spherical intracisternal inclusions assembled within the lumen of the rough endoplasmic reticulum. Immunofluorescence and immunoelectron microscopy demonstrated that CysR10 and CysP13 were asymmetrically distributed within the PBs, with the former concentrated at the electron-dense center core region and the latter distributed mainly to the electron-lucent peripheral region. These results together with temporal expression data showed that the formation of prolamin-containing PB-I in the wild-type endosperm was initiated by the accumulation of CysR10 to form the center core. In mutants deficient for cysteine-rich prolamins, the typical PB-I structures containing the electron-dense center core were not observed, and instead were replaced by irregularly shaped, electron-lucent, hypertrophied PBs. Similar, deformed PBs were observed in a CysR10 RNA interference plant line. These results suggest that CysR10, through its formation of the central core and its possible interaction with other cysteine-rich prolamins, is required for tight packaging of the proteins into a compact spherical structure.

Serine hydroxymethyltransferase participates in the synthesis of cysteine-rich storage proteins in rice seed.

The low level of cysteine-rich proteins (lcrp) mutation indicates a decrease in cysteine-rich (CysR) prolamines, α-globulin, and glutelin. To identify the causing factor of lcrp mutation, to elucidate its function, and to elucidate the role of CysR proteins in the formation of protein bodies (PBs), lcrp mutant was analyzed. A linkage map of the LCRP gene was constructed and genomic DNA sequencing of a predicted gene within the mapped region demonstrated that LCRP encodes a serine hydroxymethyltransferase, which participates in glycine-serine interconversion of one-carbon metabolism in the sulfur assimilation pathway. The levels of l-Ser, Gly, and Met in the sulfur assimilation pathway in the lcrp seeds increased significantly compared to that in the wildtype (WT). As the lcrp mutation influences the growth of shoot and root, the effects of the addition to the medium of amino acids and other compounds on the sulfur assimilation pathway were studied. Electron-lucent PBs surrounded by ribosome-attached membranes were observed accumulating cysteine-poor prolamines in the lcrp seeds. Additionally, glutelin-containing PBs were smaller and distorted in the lcrp seeds compared to those in the WT. These analyses of PBs in the lcrp seeds suggest that cysteine-rich proteins play an important role in the formation of PBs in rice.

Genetic analysis of cysteine-poor prolamin polypeptides reduced in the endosperm of the rice esp1 mutant.

The esp1 mutant CM21 specifically exhibits reduced levels of cysteine-poor (CysP) prolamin bands with pIs of 6.65, 6.95, 7.10, and 7.35 in rice seed. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis demonstrated that the bands with pIs 6.65, 6.95, and 7.35 are encoded by different structural genes. These results suggest that the Esp1 locus encodes a regulatory factor involved in the synthesis and/or accumulation of CysP prolamin molecules. Isoelectric focusing (IEF) analysis of CysP prolamins in chromosome substitution lines showed that structural genes for bands with pI values of 6.95, 7.10, and 7.35, which are reduced in esp1 mutant lines, are located as a gene cluster in the 44.2 cM region on chromosome 5.

MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice.

Mutant populations are indispensable genetic resources for functional genomics in all organisms. However, suitable rice mutant populations, induced either by chemicals or irradiation still have been rarely developed to date. To produce mutant pools and to launch a search system for rice gene mutations, we developed mutant populations of Oryza sativa japonica cv. Taichung 65, by treating single zygotic cells with N-methyl-N-nitrosourea (MNU). Mutagenesis in single zygotes can create mutations at a high frequency and rarely forms chimeric plants. A modified TILLING system using non-labeled primers and fast capillary gel electrophoresis was applied for high-throughput detection of single nucleotide substitution mutations. The mutation rate of an M(2) mutant population was calculated as 7.4 x 10(-6) per nucleotide representing one mutation in every 135 kb genome sequence. One can expect 7.4 single nucleotide substitution mutations in every 1 kb of gene region when using 1,000 M(2) mutant lines. The mutations were very evenly distributed over the regions examined. These results indicate that our rice mutant population generated by MNU-mutagenesis could be a promising resource for identifying mutations in any gene of rice. The modified TILLING method also proved very efficient and convenient in screening the mutant population.