Low mutation rate of spontaneous mutants enables detection of causative genes by comparing whole genome sequences.

In the early 1900s, mutation breeding to select varieties with desirable traits using spontaneous mutation was actively conducted around the world, including Japan. In rice, the number of fixed mutations per generation was estimated to be 1.38-2.25. Although this low mutation rate was a major problem for breeding in those days, in the modern era with the development of next-generation sequencing (NGS) technology, it was conversely considered to be an advantage for efficient gene identification. In this paper, we proposed an in silico approach using NGS to compare the whole genome sequence of a spontaneous mutant with that of a closely related strain with a nearly identical genome, to find polymorphisms that differ between them, and to identify the causal gene by predicting the functional variation of the gene caused by the polymorphism. Using this approach, we found four causal genes for the dwarf mutation, the round shape grain mutation and the awnless mutation. Three of these genes were the same as those previously reported, but one was a novel gene involved in awn formation. The novel gene was isolated from Bozu-Aikoku, a mutant of Aikoku with the awnless trait, in which nine polymorphisms were predicted to alter gene function by their whole-genome comparison. Based on the information on gene function and tissue-specific expression patterns of these candidate genes, Os03g0115700/LOC_Os03g02460, annotated as a short-chain dehydrogenase/reductase SDR family protein, is most likely to be involved in the awnless mutation. Indeed, complementation tests by transformation showed that it is involved in awn formation. Thus, this method is an effective way to accelerate genome breeding of various crop species by enabling the identification of useful genes that can be used for crop breeding with minimal effort for NGS analysis.

Effective use of legacy data in a genome-wide association studies improves the credibility of quantitative trait loci detection in rice.

Genome-wide association studies (GWASs) are used to detect quantitative trait loci (QTL) using genomic and phenotypic data as inputs. While genomic data are obtained with high throughput and low cost, obtaining phenotypic data requires a large amount of effort and time. In past breeding programs, researchers and breeders have conducted a large number of phenotypic surveys and accumulated results as legacy data. In this study, we conducted a GWAS using phenotypic data of temperate japonica rice (Oryza sativa) varieties from a public database. The GWAS using the legacy data detected several known agriculturally important genes, indicating reliability of the legacy data for GWAS. By comparing the GWAS using legacy data (L-GWAS) and a GWAS using phenotypic data that we measured (M-GWAS), we detected reliable QTL for agronomically important traits. These results suggest that an L-GWAS is a strong alternative to replicate tests to confirm the reproducibility of QTL detected by an M-GWAS. In addition, because legacy data have often been accumulated for many traits, it is possible to evaluate the pleiotropic effect of the QTL identified for the specific trait that we focused on with respect to various other traits. This study demonstrates the effectiveness of using legacy data for GWASs and proposes the use of legacy data to accelerate genomic breeding.

The gs3 allele from a large-grain rice cultivar, Akita 63, increases yield and improves nitrogen-use efficiency.

The Green Revolution allowed a large amount of nitrogen (N) fertilization to increase crop yield but has led to severe environmental pollution. Therefore, increasing the crop grain yield must be achieved without such considerable input of N fertilization. A large-grain japonica rice cultivar, Akita 63, significantly increased grain yield and improved N-use efficiency (NUE) for yield per amount of N absorbed by plants. This study found that the nonsense mutated GS3 gene, the gs3 allele of Akita 63, has a superior yield production with enlarged grain size. The gs3 allele increased the yield with improvements in harvest index and NUE for yields per plant N content by analyzing the near-isogenic line of rice plants with a large grain (LG-Notohikari), which was developed by introducing the gs3 allele of Akita 63 into normal-grain japonica cultivar, Notohikari. Thus, the gs3 allele would be promising for further yield increase without additional large input of N fertilization in non-gs3-allele rice varieties.

Expression of flavodiiron protein rescues defects in electron transport around PSI resulting from overproduction of Rubisco activase in rice.

Fragility of photosystem I has been observed in transgenic rice plants that overproduce Rubisco activase (RCA). In this study, we examined the effects of RCA overproduction on the sensitivity of PSI to photoinhibition in three lines of plants overexpressing RCA (RCA-ox). In all the RCA-ox plants the quantum yield of PSI [Y(I)] decreased whilst in contrast the quantum yield of acceptor-side limitation of PSI [Y(NA)] increased, especially under low light conditions. In the transgenic line with the highest RCA content (RCA-ox 1), the quantum yield of PSII [Y(II)] and CO2 assimilation also decreased under low light. When leaves were exposed to high light (2000 µmol photon m-2 s-1) for 60 min, the maximal activity of PSI (Pm) drastically decreased in RCA-ox 1. These results suggested that overproduction of RCA disturbs PSI electron transport control, thus increasing the susceptibility of PSI to photoinhibition. When flavodiiron protein (FLV), which functions as a large electron sink downstream of PSI, was expressed in the RCA-ox 1 background (RCA-FLV), PSI and PSII parameters, and CO2 assimilation were recovered to wild-type levels. Thus, expression of FLV restored the robustness of PSI in RCA-ox plants.

Suppression of chloroplast triose phosphate isomerase evokes inorganic phosphate-limited photosynthesis in rice.

The availability of inorganic phosphate (Pi) for ATP synthesis is thought to limit photosynthesis at elevated [CO2] when Pi regeneration via sucrose or starch synthesis is limited. We report here another mechanism for the occurrence of Pi-limited photosynthesis caused by insufficient capacity of chloroplast triose phosphate isomerase (cpTPI). In cpTPI-antisense transgenic rice (Oryza sativa) plants with 55%-86% reductions in cpTPI content, CO2 sensitivity of the rate of CO2 assimilation (A) decreased and even reversed at elevated [CO2]. The pool sizes of the Calvin-Benson cycle metabolites from pentose phosphates to 3-phosphoglycerate increased at elevated [CO2], whereas those of ATP decreased. These phenomena are similar to the typical symptoms of Pi-limited photosynthesis, suggesting sufficient capacity of cpTPI is necessary to prevent the occurrence of Pi-limited photosynthesis and that cpTPI content moderately affects photosynthetic capacity at elevated [CO2]. As there tended to be slight variations in the amounts of total leaf-N depending on the genotypes, relationships between A and the amounts of cpTPI were examined after these parameters were expressed per unit amount of total leaf-N (A/N and cpTPI/N, respectively). A/N at elevated [CO2] decreased linearly as cpTPI/N decreased before A/N sharply decreased, owing to further decreases in cpTPI/N. Within this linear range, decreases in cpTPI/N by 80% led to decreases up to 27% in A/N at elevated [CO2]. Thus, cpTPI function is crucial for photosynthesis at elevated [CO2].

Manganese toxicity disrupts indole acetic acid homeostasis and suppresses the CO2 assimilation reaction in rice leaves.

Despite the essentiality of Mn in terrestrial plants, its excessive accumulation in plant tissues can cause growth defects, known as Mn toxicity. Mn toxicity can be classified into apoplastic and symplastic types depending on its onset. Symplastic Mn toxicity is hypothesised to be more critical for growth defects. However, details of the relationship between growth defects and symplastic Mn toxicity remain elusive. In this study, we aimed to elucidate the molecular mechanisms underlying symplastic Mn toxicity in rice plants. We found that under excess Mn conditions, CO2 assimilation was inhibited by stomatal closure, and both carbon anabolic and catabolic activities were decreased. In addition to stomatal dysfunction, stomatal and leaf anatomical development were also altered by excess Mn accumulation. Furthermore, indole acetic acid (IAA) concentration was decreased, and auxin-responsive gene expression analyses showed IAA-deficient symptoms in leaves due to excess Mn accumulation. These results suggest that excessive Mn accumulation causes IAA deficiency, and low IAA concentrations suppress plant growth by suppressing stomatal opening and leaf anatomical development for efficient CO2 assimilation in leaves.

Co-overproducing Rubisco and Rubisco activase enhances photosynthesis in the optimal temperature range in rice.

Rubisco limits C3 photosynthesis under some conditions and is therefore a potential target for improving photosynthetic efficiency. The overproduction of Rubisco is often accompanied by a decline in Rubisco activation, and the protein ratio of Rubisco activase (RCA) to Rubisco (RCA/Rubisco) greatly decreases in Rubisco-overproducing plants (RBCS-ox). Here, we produced transgenic rice (Oryza sativa) plants co-overproducing both Rubisco and RCA (RBCS-RCA-ox). Rubisco content in RBCS-RCA-ox plants increased by 23%-44%, and RCA/Rubisco levels were similar or higher than those of wild-type plants. However, although the activation state of Rubisco in RBCS-RCA-ox plants was enhanced, the rates of CO2 assimilation at 25°C in RBCS-RCA-ox plants did not differ from that of wild-type plants. Alternatively, at a moderately high temperature (optimal range of 32°C-36°C), the rates of CO2 assimilation in RBCS-ox and RBCS-RCA-ox plants were higher than in wild-type plants under conditions equal to or lower than current atmospheric CO2 levels. The activation state of Rubisco in RBCS-RCA-ox remained higher than that of RBCS-ox plants, and activated Rubisco content in RCA overproducing, RBCS-ox, RBCS-RCA-ox, and wild-type plants was highly correlated with the initial slope of CO2 assimilation against intercellular CO2 pressures (A:Ci) at 36°C. Thus, a simultaneous increase in Rubisco and RCA contents leads to enhanced photosynthesis within the optimal temperature range.

Overproduction of Chloroplast Glyceraldehyde-3-Phosphate Dehydrogenase Improves Photosynthesis Slightly under Elevated [CO2] Conditions in Rice.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) limits the regeneration of ribulose 1,5-bisphosphate (RuBP) in the Calvin-Benson cycle. However, it does not always limit the rate of CO2 assimilation. In the present study, the effects of overproduction of GAPDH on the rate of CO2 assimilation under elevated [CO2] conditions, where the capacity for RuBP regeneration limits photosynthesis, were examined in transgenic rice (Oryza sativa). GAPDH activity was increased to 3.2- and 4.5-fold of the wild-type levels by co-overexpression of the GAPDH genes, GAPA and GAPB, respectively. In the transgenic rice plants, the rate of CO2 assimilation under elevated [CO2] conditions increased by approximately 10%, whereas that under normal and low [CO2] conditions was not affected. These results indicate that overproduction of GAPDH is effective in improving photosynthesis under elevated [CO2] conditions, although its magnitude is relatively small. By contrast, biomass production of the transgenic rice plants was not greater than that of wild-type plants under elevated [CO2] conditions, although starch content tended to increase marginally.

Phosphorus toxicity disrupts Rubisco activation and reactive oxygen species defence systems by phytic acid accumulation in leaves.

Phosphorus (P) is an essential mineral nutrient for plants. Nevertheless, excessive P accumulation in leaf mesophyll cells causes necrotic symptoms in land plants; this phenomenon is termed P toxicity. However, the detailed mechanisms underlying P toxicity in plants have not yet been elucidated. This study aimed to investigate the molecular mechanism of P toxicity in rice. We found that under excessive inorganic P (Pi) application, Rubisco activation decreased and photosynthesis was inhibited, leading to lipid peroxidation. Although the defence systems against reactive oxygen species accumulation were activated under excessive Pi application conditions, the Cu/Zn-type superoxide dismutase activities were inhibited. A metabolic analysis revealed that excessive Pi application led to an increase in the cytosolic sugar phosphate concentration and the activation of phytic acid synthesis. These conditions induced mRNA expression of genes that are activated under metal-deficient conditions, although metals did accumulate. These results suggest that P toxicity is triggered by the attenuation of both photosynthesis and metal availability within cells mediated by phytic acid accumulation. Here, we discuss the whole phenomenon of P toxicity, beginning from the accumulation of Pi within cells to death in land plants.

Effects of Overproduction of Rubisco Activase on Rubisco Content in Transgenic Rice Grown at Different N Levels.

It has been reported that overproduction of Rubisco activase (RCA) in rice (Oryza sativa L.) decreased Rubisco content, resulting in declining photosynthesis. We examined the effects of RCA levels on Rubisco content using transgenic rice with overexpressed or suppressed RCA under the control of different promoters of the RCA and Rubisco small subunit (RBCS) genes. All plants were grown hydroponically with different N concentrations (0.5, 2.0 and 8.0 mM-N). In RCA overproduced plants with > 2-fold RCA content (RCA-HI lines), a 10%-20% decrease in Rubisco content was observed at 0.5 and 2.0 mM-N. In contrast, at 8.0 mM-N, Rubisco content did not change in RCA-HI lines. Conversely, in plants with 50%-60% increased RCA content (RCA-MI lines), Rubisco levels remained unchanged, regardless of N concentration. Such effects on Rubisco content were independent of the promoter that was used. In plants with RCA suppression to < 10% of the wild-type RCA content, Rubisco levels were increased at 0.5 mM-N, but were unchanged at 2.0 and 8.0 mM-N. Thus, the effects of the changes in RCA levels on Rubisco content depended on N supply. Moreover, RCA overproduction was feasible without a decrease in Rubisco content, depending on the degree of RCA production.

Transgenic rice overproducing Rubisco exhibits increased yields with improved nitrogen-use efficiency in an experimental paddy field.

The green revolution's breeding of semi-dwarf rice cultivars in the 1960s improved crop yields, with large increases in the use of nitrogen (N) fertilizer. However, excess N application has caused serious environmental problems, including acid rain and the eutrophication of rivers and oceans. To use N to improve crop yields, while minimizing the associated environmental costs, there is a need to produce crops with higher N-use efficiency and higher yield components. Here we show that transgenic rice overproducing ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco)-the key enzyme of photosynthesis-exhibits increased yields with improved N-use efficiency for increasing biomass production when receiving sufficient N fertilization in an experimental paddy field. This field experiment demonstrates an improvement in photosynthesis linked to yield increase due to a higher N-use efficiency in a major crop.