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The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature-sensitive floury endosperm11-2 (flo11-2) mutant was isolated from ion beam-irradiated rice of 1116 lines. The flo11-2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole-exome sequencing of the flo11-2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid-localized 70-kDa heat shock protein 2 (cpHSP70-2). The cpHSP70-2 of the flo11-2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11-2 mutants that express the wild-type cpHSP70-2 showed significantly lower chalkiness than the flo11-2 mutant. Moreover, the accumulation level of cpHSP70-2 was negatively correlated with the chalky ratio, indicating that cpHSP70-2 is a causal gene for the chalkiness of the flo11-2 mutant. The intrinsic ATPase activity of recombinant cpHSP70-2 was lower by 23% at Vmax for the flo11-2 mutant than for the wild type. The growth of DnaK-defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild-type DnaK. The results indicate that the lowered cpHSP70-2 function is involved with the chalkiness of the flo11-2 mutant.

Summary Heavy‐ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost‐efficient whole‐exome sequencing procedure in rice, and its application to characterize an unselected population of heavy‐ion beam‐induced mutations. The bioinformatics pipeline identified single‐nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy‐ion beams at the population level. In total, 165 individual M2 lines derived from six irradiation conditions as well as eight pools from non‐irradiated ‘Nipponbare’ controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon‐ion beam‐induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M2 lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing‐based determination of the optimal irradiation condition for induction of mutations., Significance Statement We developed a multiplexed and cost‐efficient whole‐exome sequencing procedure and bioinformatics filtering pipeline for rice, and demonstrated the rapid identification of authentic mutations from a large dataset of candidate mutations. We applied this technique for the characterization of mutation spectra by heavy‐ion beams in the absence of the mutant selection, and showed that 9.06 ± 0.37 (average ± SE) mutations were induced within the target exon regions by 150 Gy irradiation of dry seeds in rice.

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.

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.

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.