Genome-wide association study of leaf photosynthesis using a high-throughput gas exchange system in rice.

Enhancing leaf photosynthetic capacity is essential for improving the yield of rice (Oryza sativa L.). Although the exploitation of natural genetic resources is considered a promising approach to enhance photosynthetic capacity, genomic factors related to the genetic diversity of leaf photosynthetic capacity have yet to be fully elucidated due to the limitation of measurement efficiency. In this study, we aimed to identify novel genomic regions for the net CO2 assimilation rate (A) by combining genome-wide association study (GWAS) and the newly developed rapid closed gas exchange system MIC-100. Using three MIC-100 systems in the field at the vegetative stage, we measured A of 168 temperate japonica rice varieties with six replicates for three years. We found that the modern varieties exhibited higher A than the landraces, while there was no significant relationship between the release year and A among the modern varieties. Our GWAS scan revealed two major peaks located on chromosomes 4 and 8, which were repeatedly detected in the different experiments and in the generalized linear modelling approach. We suggest that high-throughput gas exchange measurements combined with GWAS is a reliable approach for understanding the genetic mechanisms underlying photosynthetic diversities in crop species.

Metabolomic analysis of rice brittle culm mutants reveals each mutant- specific metabolic pattern in each organ.

INTRODUCTION: Plant cell walls play an important role in providing physical strength and defence against abiotic stress. Rice brittle culm (bc) mutants are a strength-decreased mutant because of abnormal cell walls, and it has been reported that the causative genes of bc mutants affect cell wall composition. However, the metabolic alterations in each organ of bc mutants have remained unknown. OBJECTIVES: To evaluate the metabolic changes in rice bc mutants, comparative analysis of the primary metabolites was conducted. METHODS: The primary metabolites in leaves, internodes, and nodes of rice bc mutants and wild-type control were measured using CE- and LC-MS/MS. Multivariate analyses using metabolomic data was performed. RESULTS: We found that mutations in each bc mutant had different effects on metabolism. For example, higher oxalate content was observed in bc3 and bc1 bc3 mutants, suggesting that surplus carbon that was not used for cell wall components might be used for oxalate synthesis. In addition, common metabolic alterations such as a decrease of sugar nucleotides in nodes were found in bc1 and Bc6, in which the causative genes are involved in cellulose accumulation. CONCLUSION: These results suggest that metabolic analysis of the bc mutants could elucidate the functions of causative gene and improve the cell wall components for livestock feed or bioethanol production.

Genetic architecture of leaf photosynthesis in rice revealed by different types of reciprocal mapping populations.

The improvement of leaf net photosynthetic rate (An) is a major challenge in enhancing crop productivity. However, the genetic control of An among natural genetic accessions is still poorly understood. The high-yielding indica cultivar Takanari has the highest An of all rice cultivars, 20-30% higher than that of the high-quality japonica cultivar Koshihikari. By using reciprocal backcross inbred lines and chromosome segment substitution lines derived from a cross between Takanari and Koshihikari, we identified three quantitative trait loci (QTLs) where the Takanari alleles enhanced An in plants with a Koshihikari genetic background and five QTLs where the Koshihikari alleles enhanced An in plants with a Takanari genetic background. Two QTLs were expressed in plants with both backgrounds (type I QTL). The expression of other QTLs depended strongly on genetic background (type II QTL). These beneficial alleles increased stomatal conductance, the initial slope of An versus intercellular CO2 concentration, or An at CO2 saturation. Pyramiding of these alleles consistently increased An. Some alleles positively affected biomass production and grain yield. These alleles associated with photosynthesis and yield can be a valuable tool in rice breeding programs via DNA marker-assisted selection.

QTLs maintaining grain fertility under salt stress detected by exome QTL-seq and interval mapping in barley.

Enhancing salt stress tolerance is a key strategy for increasing global food production. We previously found that long-term salinity stress significantly reduced grain fertility in the salt-sensitive barley (Hordeum vulgare) accession, 'OUC613', but not in the salt-tolerant accession, 'OUE812', resulting in large differences in grain yield. Here, we examined the underlying causes of the difference in grain fertility between these accessions under long-term treatment with 150 or 200 mM NaCl from the seedling stage to harvest and identified quantitative trait loci (QTLs) for maintaining grain fertility. In an artificial pollination experiment of the two accessions, grain fertility was significantly reduced only in OUC613 plants produced using pollen from plants grown under NaCl stress, suggesting that the low grain fertility of OUC613 was mainly due to reduced pollen fertility. Using QTL-seq combined with exome-capture sequencing and composite interval mapping of recombinant inbred lines derived from a cross between OUE812 and OUC613, we identified a QTL (qRP-2Hb) for grain fertility on chromosome 2H. The QTL region includes two genes encoding an F-box protein and a TIFY protein that are associated with male sterility, highlighting the importance of this region for maintaining grain fertility under salt stress.

Leaf photosynthetic rate and mesophyll cell anatomy changes during ontogenesis in backcrossed indica × japonica rice inbred lines.

The high-yielding indica rice variety, 'Takanari', has the high rate of leaf photosynthesis compared with the commercial japonica varieties. Among backcrossed inbred lines from a cross between 'Takanari' and a japonica variety, 'Koshihikari', two lines, BTK-a and BTK-b, showed approximately 20% higher photosynthetic rate than that of 'Takanari' for a flag leaf at full heading. This is a highest recorded rate of rice leaf photosynthesis. Here, the timing and cause of the increased leaf photosynthesis in the BTK lines were investigated by examining the photosynthesis and related parameters, as well as mesophyll cell anatomy during ontogenesis. Their photosynthetic rate was greater than that of 'Takanari' in the 13th leaf, as well as the flag leaf, but there were no differences in the 7th and 10th leaves. There were no consistent differences in the stomatal conductance, or the leaf nitrogen and Rubisco contents in the 13th and flag leaves. The total surface area of mesophyll cells per leaf area (TAmes) in the 13th and flag leaves increased significantly in the BTK lines due to the increased number and developed lobes of mesophyll cells compared with in 'Takanari'. The mesophyll conductance (g m) became greater in the BTK lines compared with 'Takanari' in the flag leaves but not in the 10th leaves. A close correlation was observed between TAmes and g m. We concluded that the increased mesophyll conductance through the development of mesophyll cells during the reproductive period is a probable cause of the greater photosynthetic rate in the BTK lines.

Introgression of two chromosome regions for leaf photosynthesis from an indica rice into the genetic background of a japonica rice.

Increases in rates of individual leaf photosynthesis (P n) are critical for future increases of rice yields. A previous study, using introgression lines derived from a cross between indica cultivar Habataki, with one of the highest recorded values of P n, and the Japanese elite cultivar Koshihikari, identified four QTLs (qCAR4, qCAR5, qCAR8, and qCAR11) that affect P n. The present study examined the combined effect of qCAR4 and qCAR8 on P n in the genetic background of Koshihikari. The pyramided near-isogenic line NIL(qCAR4+qCAR8) showed higher P n than both NIL(qCAR4) and NIL(qCAR8), equivalent to that of Habataki despite being due to only two out of the four QTLs. The high P n of NIL(qCAR4+qCAR8) may be attributable to the high leaf nitrogen content, which may have been inherited from NIL(qCAR4), to the large hydraulic conductance due to the large root surface area from NIL(qCAR4), and to the high hydraulic conductivity from NIL(qCAR8). It might be also attributable to high mesophyll conductance, which may have been inherited from NIL(qCAR4). The induction of mesophyll conductance and the high leaf nitrogen content and high hydraulic conductivity could not be explained in isolation from the Koshihikari background. These results suggest that QTL pyramiding is a useful approach in rice breeding aimed at increasing P n.

The mesophyll anatomy enhancing CO2 diffusion is a key trait for improving rice photosynthesis.

Increases in rates of individual leaf photosynthesis (P(n)) are critical for future increases in yields of rice plants. Although many efforts have been made to improve rice P(n) with transgenic technology, the desired increases in P(n) have not yet been achieved. Two rice lines with extremely high values of P(n) were identified among the backcrossed inbred lines derived from the indica variety Takanari, one of the most productive varieties in Japan, and the elite japonica variety Koshihikari (Koshihikari/Takanari//Takanari). The P(n) values of the two lines at an ambient CO(2) concentration of 370µmol mol(-1) as well as at a saturating concentration of CO(2) were 20-50% higher than those of the parental varieties. Compared with Takanari, these lines had neither a higher content nor a higher activity of ribulose 1,5-bisphosphate carboxylase/oxygenase when the leaf nitrogen contents were similar, but they did have high mesophyll conductance with respect to CO(2) flux due to their higher density and more highly developed lobes of mesophyll cells. These lines also had higher electron transport rates. The plant growth rates of these lines were higher than that of Takanari. The findings show that it is possible to increase P(n) significantly, both at the current atmospheric concentration of CO(2) and at the increased concentration of CO(2) expected in the future, using appropriate combinations of genetic resources that are available at present.

Identification of Novel Quantitative Trait Loci for Culm Thickness of Rice Derived from Strong-Culm Landrace in Japan, Omachi.

Increasing the lodging resistance of rice through genetic improvement has been an important target in breeding. To further enhance the lodging resistance of high-yielding rice varieties amidst climate change, it is necessary to not only shorten culms but strengthen them as well. A landrace rice variety, Omachi, which was established more than 100 years ago, has the largest culm diameter and bending moment at breaking in the basal internodes among 135 temperate japonica accessions. Using unused alleles in such a landrace is an effective way to strengthen the culm. In this study, we performed quantitative trait locus (QTL) analysis to identify the genetic factors of culm strength of Omachi using recombinant inbred lines (RILs) derived from a cross between Omachi and Koshihikari, a standard variety in Japan. We identified three QTLs for the culm diameter of the 5th internode on chromosomes 3 (qCD3) and 7 (qCD7-1, qCD7-2). Among them, qCD7-2 was verified by QTL analysis using the F2 population derived from a cross between one of the RILs and Koshihikari. RNA-seq analysis of shoot apex raised 10 candidate genes underlying the region of qCD7-2. The increase in culm strength by accumulating Omachi alleles of qCD3, qCD7-1 and qCD7-2 was 25.0% in 2020. These QTLs for culm diameter pleiotropically increased spikelet number per panicle but did not affect days to heading or culm length. These results suggest that the Omachi alleles of qCD3, qCD7-1 and qCD7-2 are useful for breeding to increase lodging resistance and yield.

Identification of Genomic Regions for Deep-Water Resistance in Rice for Efficient Weed Control with Reduced Herbicide Use.

Deep-water (DW) management in rice fields is a promising technique for efficient control of paddy weeds with reduced herbicide use. Maintaining a water depth of 10-20 cm for several weeks can largely suppress the weed growth, though it also inhibits rice growth because the DW management is usually initiated immediately after transplanting. Improving the DW resistance of rice during the initial growth stage is essential to avoid suppressing growth. In this study, we demonstrate a large genetic variation in the above-ground biomass (AGB) after the end of DW management among 165 temperate japonica varieties developed in Japan. Because the AGB closely correlated with plant length (PL) and tiller number (TN) at the early growth stage, we analyzed genomic regions associated with PL and TN by conducting a genome-wide association study. For PL, a major peak was detected on chromosome 3 (qPL3), which includes a gene encoding gibberellin biosynthesis, OsGA20ox1. The rice varieties with increased PL had a higher expression level of OsGA20ox1 as reported previously. For TN, a major peak was detected on chromosome 4 (qTN4), which includes NAL1 gene associated with leaf morphological development and panicle number. Although there was less difference in the expression level of NAL1 between genotypes, our findings suggest that an amino acid substitution in the exon region is responsible for the phenotypic changes. We also found that the rice varieties having alternative alleles of qPL3 and qTN4 showed significantly higher AGB than the varieties with the reference alleles. Our results suggest that OsGA20ox1 and NAL1 are promising genes for improving DW resistance in rice.

Pyramiding of multiple strong-culm genes originating from indica and tropical japonica to the temperate japonica rice.

Severe lodging has recurrently occurred at strong typhoon's hitting in recent climate change. The identification of quantitative trait loci and their responsible genes associated with a strong culm and their pyramiding are important for developing high-yielding varieties with a superior lodging resistance. To evaluate the effects of four strong-culm genes on lodging resistance, the temperate japonica near isogenic line (NIL) with the introgressed SCM1 or SCM2 locus of the indica variety, Habataki and the other NIL with the introgeressed SCM3 or SCM4 locus of the tropical japonica variety, Chugoku 117 were developed. Then, we developed the pyramiding lines with double,triple and quadruple combinations derived from step-by-step crosses among NIL-SCM1-NIL-SCM4. Quadruple pyramiding line (NIL-SCM1 + 2 + 3 + 4) showed the largest culm diameter and the highest culm strength among the combinations and increased spikelet number due to the pleiotropic effects of these genes. Pyramiding of strong culm genes resulted in much increased culm thickness, culm strength and spikelet number due to their additive effect. SCM1 mainly contributed to enhance their pyramiding effect. These results in this study suggest the importance of identifying the combinations of superior alleles of strong culm genes among natural variation and pyramiding these genes for improving high-yielding varieties with a superior lodging resistance.

Hydraulic conductance as well as nitrogen accumulation plays a role in the higher rate of leaf photosynthesis of the most productive variety of rice in Japan.

An indica variety Takanari is known as one of the most productive rice varieties in Japan and consistently produces 20-30% heavier dry matter during ripening than Japanese commercial varieties in the field. The higher rate of photosynthesis of individual leaves during ripening has been recognized in Takanari. By using pot-grown plants under conditions of minimal mutual shading, it was confirmed that the higher rate of leaf photosynthesis is responsible for the higher dry matter production after heading in Takanari as compared with a japonica variety, Koshihikari. The rate of leaf photosynthesis and shoot dry weight became larger in Takanari after the panicle formation and heading stages, respectively, than in Koshihikari. Roots grew rapidly in the panicle formation stage until heading in Takanari compared with Koshihikari. The higher rate of leaf photosynthesis in Takanari resulted not only from the higher content of leaf nitrogen, which was caused by its elevated capacity for nitrogen accumulation, but also from higher stomatal conductance. When measured under light-saturated conditions, stomatal conductance was already decreased due to the reduction in leaf water potential in Koshihikari even under conditions of a relatively small difference in leaf-air vapour pressure difference. In contrast, the higher stomatal conductance was supported by the maintenance of higher leaf water potential through the higher hydraulic conductance in Takanari with the larger area of root surface. However, no increase in root hydraulic conductivity was expected in Takanari. The larger root surface area of Takanari might be a target trait in future rice breeding for increasing dry matter production.

Identification and characterization of genomic regions on chromosomes 4 and 8 that control the rate of photosynthesis in rice leaves.

DNA marker-assisted selection appears to be a promising strategy for improving rates of leaf photosynthesis in rice. The rate of leaf photosynthesis was significantly higher in a high-yielding indica variety, Habataki, than in the most popular Japanese variety, Koshihikari, at the full heading stage as a result of the higher level of leaf nitrogen at the same rate of application of nitrogen and the higher stomatal conductance even when the respective levels of leaf nitrogen were the same. The higher leaf nitrogen content of Habataki was caused by the greater accumulation of nitrogen by plants. The higher stomatal conductance of Habataki was caused by the higher hydraulic conductance. Using progeny populations and selected lines derived from a cross between Koshihikari and Habataki, it was possible to identify the genomic regions responsible for the rate of photosynthesis within a 2.1 Mb region between RM17459 and RM17552 and within a 1.2 Mb region between RM6999 and RM22529 on the long arm of chromosome 4 and on the short arm of chromosome 8, respectively. The designated region on chromosome 4 of Habataki was responsible for both the increase in the nitrogen content of leaves and hydraulic conductance in the plant by increasing the root surface area. The designated region on chromosome 8 of Habataki was responsible for the increase in hydraulic conductance by increasing the root hydraulic conductivity. The results suggest that it may be possible to improve photosynthesis in rice leaves by marker-assisted selection that focuses on these regions of chromosomes 4 and 8.

Maintaining higher leaf photosynthesis after heading stage could promote biomass accumulation in rice.

Leaf photosynthetic rate changes across the growing season as crop plants age. Most studies of leaf photosynthesis focus on a specific growth stage, leaving the question of which pattern of photosynthetic dynamics maximizes crop productivity unanswered. Here we obtained high-frequency data of canopy leaf CO2 assimilation rate (A) of two elite rice (Oryza sativa) cultivars and 76 inbred lines across the whole growing season. The integrated A value after heading was positively associated with crop growth rate (CGR) from heading to harvest, but that before heading was not. A curve-smoothing analysis of A after heading showed that accumulated A at > 80% of its maximum (A80) was positively correlated with CGR in analyses of all lines mixed and of lines grouped by genetic background, while the maximum A and accumulated A at ≤ 80% were less strongly correlated with CGR. We also found a genomic region (~ 12.2 Mb) that may enhance both A80 and aboveground biomass at harvest. We propose that maintaining a high A after heading, rather than having high maximum A, is a potential target for enhancing rice biomass accumulation.

Potential of rice landraces with strong culms as genetic resources for improving lodging resistance against super typhoons.

It is generally believed that rice landraces with long culms are susceptible to lodging, and have not been utilized for breeding to improve lodging resistance. However, little is known about the structural culm strength of landraces and their beneficial genetic loci. Therefore, in this study, genome-wide association studies (GWAS) were performed using a rice population panel including Japanese rice landraces to identify beneficial loci associated with strong culms. As a result, the landraces were found to have higher structural culm strength and greater diversity than the breeding varieties. Genetic loci associated with strong culms were identified, and it was demonstrated that haplotypes with positive effects of those loci were present in a high proportion of these landraces. These results indicated that the utilization of the strong culm-associated loci present in Japanese rice landraces may further improve the lodging resistance of modern breeding varieties that have relied on semi-dwarfism.

Characterization of a rice variety with high hydraulic conductance and identification of the chromosome region responsible using chromosome segment substitution lines.

BACKGROUND AND AIMS: The rate of photosynthesis in paddy rice often decreases at noon on sunny days because of water stress, even under submerged conditions. Maintenance of higher rates of photosynthesis during the day might improve both yield and dry matter production in paddy rice. A high-yielding indica variety, 'Habataki', maintains a high rate of leaf photosynthesis during the daytime because of the higher hydraulic conductance from roots to leaves than in the standard japonica variety 'Sasanishiki'. This research was conducted to characterize the trait responsible for the higher hydraulic conductance in 'Habataki' and identified a chromosome region for the high hydraulic conductance. METHODS: Hydraulic conductance to passive water transport and to osmotic water transport was determined for plants under intense transpiration and for plants without transpiration, respectively. The varietal difference in hydraulic conductance was examined with respect to root surface area and hydraulic conductivity (hydraulic conductance per root surface area, L(p)). To identify the chromosome region responsible for higher hydraulic conductance, chromosome segment substitution lines (CSSLs) derived from a cross between 'Sasanishiki' and 'Habataki' were used. KEY RESULTS: The significantly higher hydraulic conductance resulted from the larger root surface area not from L(p) in 'Habataki'. A chromosome region associated with the elevated hydraulic conductance was detected between RM3916 and RM2431 on the long arm of chromosome 4. The CSSL, in which this region was substituted with the 'Habataki' chromosome segment in the 'Sasanishiki' background, had a larger root mass than 'Sasanishiki'. CONCLUSIONS: The trait for increasing plant hydraulic conductance and, therefore, maintaining the higher rate of leaf photosynthesis under the conditions of intense transpiration in 'Habataki' was identified, and it was estimated that there is at least one chromosome region for the trait located on chromosome 4.

Landraces of temperate japonica rice have superior alleles for improving culm strength associated with lodging resistance.

Lodging can reduce grain yield and quality in cereal crops including rice (Oryza sativa L.). To achieve both high biomass production and lodging resistance, the breeding of new cultivars with strong culms is a promising strategy. However, little is known about the diversity of culm strength in temperate japonica rice and underlying genetic factors. Here, we report a wide variation of culm strength among 135 temperate japonica cultivars, and some landraces having the strongest culms among these cultivars. The genome-wide association study (GWAS) identified 55 quantitative trait loci for culm strength and morphological traits, and revealed several candidate genes. The superior allele of candidate gene for culm thickness, OsRLCK191, was found in many landraces but had not inherited to the modern improved cultivars. Our results suggest that landraces of temperate japonica rice have unutilized superior alleles for contributing future improvements of culm strength and lodging resistance.

Influence of rice-husk biochar and Bacillus pumilus strain TUAT-1 on yield, biomass production, and nutrient uptake in two forage rice genotypes.

Biochar is widely used as a soil amendment to increase crop yields. However, the impact of the interaction between the biochar and microbial inoculants (e.g., biofertilizer) on plant nutrient uptake and yield in forage rice is not fully understood. A greenhouse study was conducted to evaluate the synergistic effects of rice-husk biochar and Bacillus pumilus strain TUAT-1 biofertilizer application on growth, yield, and nutrient uptake in two forage rice genotypes; Fukuhibiki and the newly bred line, LTAT-29. Positive effects of biochar and biofertilizer, alone or in a combination, on growth traits, nutrient uptake, and yield components were dependent on the rice genotypes. Biochar and TUAT-1 biofertilizer influenced the overall growth of plants positively and increased straw and above-ground biomass in both genotypes. However, although biochar application significantly increased grain yield in LTAT-29, this was not the case in Fukuhibiki. Biochar and TUAT-1 biofertilizer, either alone or combined, significantly affected plant nutrient uptake but the effect largely depended on rice genotype. Results of this study indicate that biochar amendment and TUAT-1 biofertilizer can enhance forage rice productivity depending on genotypes, and therefore, there is a need to consider plant genetic composition when evaluating the potential for crop response to these soil amendments before application on a commercial scale.

Next generation long-culm rice with superior lodging resistance and high grain yield, Monster Rice 1.

During late 1960s Green Revolution, researchers utilized semidwarf 1 (sd1) to improve the yield and lodging resistance in rice (Oryza sativa L.). However, sd1 has a negative effect to culm strength and biomass production. To increase yield dramatically in 21th century, development of next generation long-culm rice for non-lodging and high grain yield independent of sd1 has been needed. The present study developed Monster Rice 1, a long-culm and heavy-panicle type of rice line and compared it with Takanari, a high-yielding semidwarf rice variety about yield and lodging resistance associated traits. Brown rice yield and bending moment at breaking of the basal elongated internode were higher in Monster Rice 1 than those in Takanari due to a large number of spikelets per panicle and thicker culm. Furthermore, to identify QTLs with superior alleles for these traits, QTL and haplotype analyses were performed using F2 population and recombinant inbred lines derived from a cross between Monster Rice 1 and Takanari. The results from this study suggest that long-culm and heavy-panicle type of rice with a superior lodging resistance by culm strength can perform its high yield potential by using these identified QTLs contributing yield and lodging resistance.

Finding the superior allele of japonica-type for increasing stem lodging resistance in indica rice varieties using chromosome segment substitution lines.

BACKGROUND: In cereal crops, stem lodging can be classified into two types: stem-breaking type and stem-bending type. To improve stem-lodging resistance, the strong culm traits of superior lodging-resistant varieties must be characterized. The identification of quantitative trait loci (QTLs) and the corresponding genes associated with the parameters for bending moment at breaking (M) and flexural rigidity (FR) is expected to enable the efficient development of lodging-resistant varieties. A set of Chromosome Segment Substitution Lines (CSSLs) derived from the cross between Takanari and Koshihikari were used in this study to identify QTLs associated with lodging resistance. RESULTS: The indica variety Takanari possesses large M due to its large section modulus (SM) despite its small bending stress (BS), whereas Takanari also has large FR due to its large secondary moment of inertia (SMI) and Young's modulus (YM). The QTLs for BS were assigned to chromosomes 3, 5, 6, 8, 9, 10, 11, and 12. Koshihikari alleles increased BS in these QTLs. The YM was increased by substitution of the Koshihikari chromosomal segments on chromosomes 2, 10, and 11. Other QTLs mapped to chromosomes 7 and 12, such that the Koshihikari alleles contributed to the decrease of YM. QTLs for cellulose density were assigned to chromosomes 1, 3, and 5, which were replaced by substitutions of Koshihikari segments. The QTLs for hemicellulose, cellulose, and holocellulose densities identified on chromosome 5 overlapped with those for BS, indicating the positive effect of the Koshihikari segment on increasing BS. CONCLUSIONS: These results suggested that the QTLs for the densities of cell wall materials in japonica varieties contributed to increased BS and might be utilized for improving lodging resistance in indica varieties of rice.

Fine Mapping of Carbon Assimilation Rate 8, a Quantitative Trait Locus for Flag Leaf Nitrogen Content, Stomatal Conductance and Photosynthesis in Rice.

Increasing the rate of leaf photosynthesis is one important approach for increasing grain yield in rice (Oryza sativa). Exploiting the natural variation in CO2 assimilation rate (A) between rice cultivars using quantitative genetics is one promising means to identify genes contributing to higher photosynthesis. In this study, we determined precise location of Carbon Assimilation Rate 8 (CAR8) by crossing a high-yielding indica cultivar with a Japanese commercial cultivar. Fine mapping suggested that CAR8 encodes a putative Heme Activator Protein 3 (OsHAP3) subunit of a CCAAT-box-binding transcription factor called OsHAP3H. Sequencing analysis revealed that the indica allele of CAR8 has a 1-bp deletion at 322 bp from the start codon, resulting in a truncated protein of 125 amino acids. In addition, CAR8 is identical to DTH8/Ghd8/LHD1, which was reported to control rice flowering date. The increase of A is largely due to an increase of RuBP regeneration rate via increased leaf nitrogen content, and partially explained by reduced stomatal limitation via increased stomatal conductance relative to A. This allele also increases hydraulic conductivity, which would promote higher stomatal conductance. This indicates that CAR8 affects multiple physiological aspects relating to photosynthesis. The detailed analysis of molecular functions of CAR8 would help to understand the association between photosynthesis and flowering and demonstrate specific genetic mechanisms that can be exploited to improve photosynthesis in rice and potentially other crops.