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Grain size is one of the most important traits that influence grain yield in rice. Here we report the isolation and characterization of the long grain1 (lin1) mutant, which exhibited long grains and increased grain weight. A field trial conducted over 2 years indicated that the grain weight increased 3.0–6.6% in the lin1 mutant compared with that of the wild-type Nipponbare. Whole-genome sequencing and genetic linkage analysis indicated that a 1-bp deletion within the coding sequence of Os06g0675200, which showed no homology to previously characterized genes, is responsible for the lin1 phenotype. Quantitative real-time PCR indicated that LIN1 transcripts were more abundant in young panicles than at advanced inflorescence developmental stages, which indicated that LIN1 controls grain length mainly at early stages of grain development. Sequence polymorphism analysis of LIN1 showed that all 15 temperate japonica cultivars tested as well as six out of the nine indica cultivars tested possessed the Nipponbare-type LIN1 allele, whereas the remaining three indica cultivars and one aus cultivar tested harbored an identical missense mutation in LIN1. These results revealed that the mutant allele of LIN1 has not been widely utilized in breeding temperate japonica cultivars currently in cultivation. Our findings indicate that the lin1 mutation may be useful to further improve grain length and presumably grain yield in temperate japonica and indica rice cultivars that harbor the Nipponbare-type LIN1 allele.

We isolated a cold sensitive virescent1 (csv1) mutant from a rice (Oryza sativa L.) population mutagenized by carbon ion irradiation. The mutant exhibited chlorotic leaves during the early growth stages, and produced normal green leaves as it grew. The growth of csv1 plants displayed sensitivity to low temperatures. In addition, the mutant plants that were transferred to low temperatures at the fifth leaf stage produced chlorotic leaves subsequently. Genetic and molecular analyses revealed translocation of a 13-kb genomic fragment that disrupted the causative gene (CSV1; LOC_Os05g34040). CSV1 encodes a plastid-targeted oxidoreductase-like protein conserved among land plants, green algae, and cyanobacteria. Furthermore, CSV1 transcripts were more abundant in immature than in mature leaves, and they did not markedly increase or decrease with temperature. Taken together, our results indicate that CSV1 supports chloroplast development under cold stress conditions, in both the early growth and tillering stages in rice. We isolated and characterized the rice virescent mutant csv1. The mutant showed low temperature sensitivity not only early growth stages but also the tillering stage.

Ion beams are recognized as alternative mutagens for plant and microbe breeding because they are thought to cause mutations by distinct mechanism from chemical mutagens or gamma rays. Valuable mutants have been successfully generated by ion beam irradiation not only in ornamental plants but also in crops such as rice. The characteristics of the ion-beam-induced mutations had been analyzed using specific marker genes; however, little is known about how many and what type of mutations are induced by ion beams in genomic level. Here, we conducted exome analysis of DNA samples of mutants identified in a carbon ion beam-irradiated rice population to understand the properties of mutations caused by ion beams at a genomic level. Rice seeds (Nipponbare) were irradiated with carbon ion beams (320 MeV 12C6+ at 40 Gy) accelerated by the cyclotron in QST Takasaki. Five mutant lines (2 dwarfs and 3 early heading date mutants) were selected and subjected to the exome analysis. As a result, we identified a total of 56 mutations including 24 single nucleotide variations and 28 insertions/deletions in the 5 lines. A large deletion (33.6k bp) and an inversion (534K bp) were also detected. In addition, we identified 6 mutations that potentially have high impact on protein function (frameshift or exon loss variants). Strong candidate genes that possibly link to the mutant phenotype were found in 4 lines out of 5 lines, suggesting that the combination of ion beam irradiation and genome analysis is helpful to narrow down a candidate gene responsible for a mutant phenotype., 16th International Symposium on Rice Functional Genomics

The biosynthetic pathway of lutein, the most abundant carotenoid in leaves, has been extensively studied, while its degradation pathway during senescence is poorly understood. We found that a novel carotenoid derivative, lutein 3-acetate, accumulates in senescent leaves of rice (Oryza sativa L.). The change in contents of lutein and lutein 3-acetate suggests that lutein is converted to lutein 3-acetate during senescence. Analysis of mutants involved in light-harvesting complex II (LHCII) accumulation and degradation suggests that the converting activity is induced during senescence and that only free lutein, not lutein bound to LHCII, is converted into lutein 3-acetate.

In order to analyze mutations induced by gamma irradiation in higher plants, we irradiated rice with gamma rays and screened for mutations expressing phenotypes of glutinous endosperm (wx), chlorophyll b deficiency, endosperm protein deficiency, gibberellin-related dwarfism, and shortened plastochron-in order to clarify types of mutations. Nucleotide sequence analysis showed that the most frequent mutation induced by gamma rays was deletion, particularly small deletion. Of the 24 mutations, 15 were small deletions (1-16 bp), four were large deletions (9.4-129.7 kbp), three were single-base substitutions, and two were inversions. Deletions 100 bp-8 kbp in length were not found, suggesting that gamma irradiation is unlikely to induce deletions of 100 bp to 8 kbp but is more likely to induce deletions between 1 and several ten bp or those of around 10 kbp or more. Based on the results, reverse genetics applications may be possible for gamma irradiation-induced deletions in rice by mismatch cleavage analysis used in Targeting Induced Local Lesions IN Genomes (TILLING) to detect small deletions and base substitutions or by using array comparative genomic hybridization (aCGH) to detect large deletions.

Mutants that retain greenness of leaves during senescence are known as “stay-green” mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR . Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a ) and chlorophyll b (Chl b ) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl–protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1 , which encode the first enzymes in the degrading pathways of Chl a and Chl b , respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes.

Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)–bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence.

Sequence rearrangement of rice waxy (wx) mutant N26 induced by thermal neutron irradiation was analyzed by PCR amplification and subsequent DNA sequencing. We found that the genome region of Norin 8, including the Wx locus, had been deleted, corresponding to 39 867 nucleotides for the japonica cultivar Nipponbare. Sequence analysis suggested that a short homologous sequence was used for rejoining without any addition of nucleotides. This is the first mutant in which a complete loss of the Wx locus was induced by thermal neutron irradiation in rice.

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll - protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4-1, a stay-green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4-1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation-related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity-dependent manner. Surprisingly, the Fv /Fm value remained high in nyc4-1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4-1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild-type and other non-functional stay-green mutants, including sgr-2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll - protein complexes during leaf senescence is discussed.

Yellowing, which is related to the degradation of chlorophyll and chlorophyll-protein complexes, is a notable phenomenon during leaf senescence. NON-YELLOW COLORING 1 (NYC1) in rice encodes a membrane-localized short-chain dehydrogenase/reductase (SDR) that is thought to represent a chlorophyll b reductase necessary for catalyzing the first step of chlorophyll b degradation. Analysis of the nyc1 mutant, which shows the stay-green phenotype, revealed that chlorophyll b degradation is required for the degradation of light-harvesting complex II and thylakoid grana in leaf senescence. Phylogenetic analysis further revealed the existence of NYC1-LIKE (NOL) as the most closely related protein to NYC1. In the present paper, the nol mutant in rice was also found to show a stay-green phenotype very similar to that of the nyc1 mutant, i.e. the degradation of chlorophyll b was severely inhibited and light-harvesting complex II was selectively retained during senescence, resulting in the retention of thylakoid grana even at a late stage of senescence. The nyc1 nol double mutant did not show prominent enhancement of inhibition of chlorophyll degradation. NOL was localized on the stromal side of the thylakoid membrane despite the lack of a transmembrane domain. Immunoprecipitation analysis revealed that NOL and NYC1 interact physically in vitro. These observations suggest that NOL and NYC1 are co-localized in the thylakoid membrane and act in the form of a complex as a chlorophyll b reductase in rice.

In order to suppress the somatic excision of the Ds element and increase the independent transposition events of the Ac/Ds transposon tagging system in rice, we employed promoters of two meiosis-specific genes of lily, LIM10 and LIM18. The LIM10 promoter directed GUS expression specifically in anthers, with the LIM18 promoter doing the same in the anthers and somatic tissue. Both promoters induced independent germinal transposition with the frequency of approximately 1%. The LIM10 promoter, lacking induction of somatic transposition, is considered to be useful for improving transposon-tagging efficiencies in rice.