Hybrid Rice Breeding Research Articles

Engineering synthetic apomixis in different hybrid rice varieties using the Fix strategy

Hybrid rice breeding has significantly increased the yield and stability of rice production. However, due to trait segregation in F2 progeny seeds, the F1 hybrid seeds must be renewed annually. Apomixis is a form of reproduction leading to clonal seeds, which has a revolutionary potential in fixing heterosis and realizing selfretention of hybrid rice seeds. Previously, we successfully engineered synthetic apomixis in rice by combining Mitosis instead of Meiosis (MiMe) with haploid induction (mutation of MATRILINEAL), a strategy known as Fix for Fixation of hybrids. However, only one hybrid rice variety has been tested for the Fix synthetic apomixis system. In this study, we expanded the application of the Fix strategy and achieved synthetic apomixis in multiple hybrid rice varieties. We observed significant variations in seed setting rate across diverse genetic backgrounds and witnessed a remarkable tenfold increase in clonal seed efficiency. Our findings will provide valuable insights into the application of the Fix strategy for synthetic apomixis in hybrid rice.

An epi-allele of SMS causes Sanming dominant genic male sterility in rice.

Male sterility is an important trait in rice for hybrid rice (Oryza sativa) breeding. However, the factors involved in dominant male sterility are largely unknown. Here, we identified a gene from Sanming dominant genic male sterile rice, named Sanming dominant male sterility (SMS), and reported that an epi-allele of this locus contributes to male sterility. Segregation analysis attributed dominant male sterility to a single locus, SMS, which we characterized using a male-sterile near isogenic line (NIL) of rice cultivar 93-11. The SMS locus was heterozygous in the male-sterile 93-11 NIL, containing an epi-allele identical to that in 93-11, and an epi-allele identical to that in rice cultivar Nipponbare, which we refer to as SMS9 and SMSN, respectively. SMS9 is silent and hyper-methylated, whereas SMSN is expressed and hypo-methylated in the 93-11 NIL. Overexpressing SMSN led to male sterility. Mutations in SMS rescued the male sterility of the 93-11 NIL. Interestingly, we observed the duplication of SMSN in Nipponbare, but did not observe the duplication of SMS9 in 93-11. Together, these findings suggest that the reduced methylation and enhanced expression of the SMSN epi-allele in the 93-11 NIL is responsible for its role in conferring dominant male sterility.

Genome-Wide Dissection of Quan 9311A Breeding Process and Application Advantages

Germplasm resource innovation is a crucial factor for cultivar development, particularly within the context of hybrid rice breeding based on the three-line system. Quan 9311A, a cytoplasmic male sterile (CMS) line, has been successfully cultivated using rice restoration materials and extensively empolyed as a female parent in hybrid breeding program in China. This line was developed by crossing the CMS line Zhong 9A with a two-line restorer line 93-11, with the intention of eliminating the restoring ability of 93-11 while retaining the sterility gene WA352c from Zhong 9A. Quan 9311A effectively amalgamates the most favorable agronomic traits from both parental lines. In this study, the relationship between phenotypic characteristics and the known functional genes of Quan 9311A were analyzed using the rice genome navigation analysis technology based on whole-genome sequencing. The findings revealed that Quan 9311A harbors multiple superior alleles from both 93-11 and Zhong 9A, providing exceptional agronomic traits that were unavailable in earlier CMS lines. Despite the removal of the fertility restorer gene Rf3 from 93-11, numerous chromosomal segments from 93-11 persist in the Quan 9311A genome. Furthermore, the hybrid rice Quanyousimiao (QYSM) and the restorer line Wushansimiao (WSSM) were used as examples to illustrate the important role of Quan 9311A as the female parent in heterosis. It was found that QYSM carries a great number of superior alleles, which accounts for its high grain yield and wide adaptability. These insights not only advanced the utilization of hybrid rice pairing groups but also provided guidance for future breeding endeavors. The study introduced innovative concepts to further integrate genomics with traditional breeding techniques. Ultimately, Quan 9311A signifies a significant milestone in rice breeding technology, opening up novel avenues for hybrid rice development.

Molecular Breeding of Zheyou810, an Indica–Japonica Hybrid Rice Variety with Superior Quality and High Yield

Rice heterosis has played a significant role in improving rice yields in China since the 1970s. The utilization of heterosis between the indica and japonica subspecies of rice is especially important in this regard. However, inter-subspecies hybridization between indica and japonica rice is hampered by the inadequate seed-setting rate and inferior rice quality of hybrid offspring, which has limited its development. The discovery and cloning of wide-compatibility gene S5-n and the granular starch synthase gene Wx have provided a solution to this problem, paving the way for the breeding of inter-subspecies hybrid rice. Here we demonstrated the breeding process of Zheyou810 and its performances in regional trials. Employing marker-assisted selection through the utilization of KASP (Kompetitive Allele-Specific PCR), a high-yielding and superior-quality indica–japonica hybrid rice variety named Zheyou810 was developed. Zheyou810 achieved an average yield of 10,737.75 kg/ha (12.58% higher than control Jiayou5) in the national regional trials across 2019 and 2020, and 11,777.25 kg/ha (13.36% higher than Jiayou5) in the production trial in 2020, respectively. The quality of rice has also been improved, reaching the standards set by the Ministry of Agriculture of the People’s Republic of China for Grade 3 premium rice (NY/T593). In 2021, Zheyou810 was approved by the national examination showing broad application prospects.

Identification of Good Restorer Lines through Molecular Confirmation of Rf3 and Rf4 Genes in Rice Hybrids

Pollen fertility can be restored by nuclear-encoded genes known as fertility restorer (Rf) genes. The initial stage in creating high-yielding heterotic hybrids involves the identification of restorers capable of effectively restoring the fertility of CMS lines. Hybrid rice breeding programs have proven effective in achieving these goals by utilizing cytoplasmic male sterility (CMS) systems in combination with restorer genes. Among these, Rf3 and Rf4 genes play critical roles in restoring male fertility and are essential components of successful hybrid rice breeding. In this study, we aimed to investigate the molecular conformation of Rf3 and Rf4 genes in a set of restorer lines and hybrids in rice. DNA was isolated from 10 restorer lines and 30 rice hybrids, two checks and KMR3R were used for comparison for positive alleles. Two functional markers, RM SF21-5 for Rf3 and RMS-PRR-9-1 for Rf4, were employed to identify the allelic status of the fertility restorer genes. Results revealed that among the 42 rice entries screened, five rice hybrids lacked both Rf3 and Rf4 genes and Four rice restorer lines and four hybrids were found to possess both Rf3 and Rf4 genes, making them valuable for hybrid breeding programs. Conversely, five hybrids lacked restorer genes and were unsuitable for such breeding programs. These findings establish Rf3and Rf4 as major fertility restoring genes in rice, consistently contributing to complete fertility restoration. The genotyping results provide valuable insights for selecting appropriate parental lines and restorers in hybrid rice breeding programs. The presence or absence of these fertility-restoring genes plays a crucial role in developing high-yielding and productive rice varieties through effective breeding strategies.

Combining Ability Analysis of Yield-Related Traits of Two Elite Rice Restorer Lines in Chinese Hybrid Rice.

Hybrid rice breeding is an important strategy for enhancing grain yield. Breeding high-performance parental lines and identifying combining abilities is a top priority for hybrid breeding. Yuenongsimiao (YNSM) and its derivative variety Yuehesimiao (YHSM) are elite restorer lines with a high ability of fertility restoration, from which 67 derived hybrid combinations have been authorized to different degrees in more than 110 instances in China. In this study, we found that YNSM and YHSM contained three candidate restorer-of-fertility (Rf) genes, Rf3, Rf4, and Rf5/Rf1a, that might confer their restoration ability. Subsequently, we investigated heterosis and combining ability of YNSM and YHSM using 50 F1 hybrids from a 5 × 10 incomplete diallelic mating design. Our results indicated that hybrid combinations exhibited significant genetic differences, and the additive effects of the parental genes played a preponderant role in the inheritance of observed traits. The metrics of plant height (PH), 1000-grain weight (TGW), panicle length (PL), and the number of spikelets per panicle (NSP) were mainly affected by genetic inheritance with higher heritability. Notably, the general combining ability (GCA) of YHSM exhibited the largest positive effect on the number of grains per panicle (NGP), NSP, PL, and TGW. Thus, YHSM had the largest GCA effect on yield per plant (YPP). In addition, the GCA of YNSM exhibited a positive impact on YPP, mainly due to the critical contribution of seed setting percentage (SSP). Moreover, YNSM and YHSM exhibited negative GCA effects on PH, implying that YNSM and YHSM could effectively enhance plant lodging resistance by reducing the plant height of the derived hybrids. Remarkably, among the hybrids, Yuanxiang A/YNSM (YXA/YNSM), Shen 08S/Yuemeizhan (S08S/YMZ), and Quan 9311A/YHSM (Q9311A/YHSM) represent promising new combinations with a higher specific combining ability (SCA) effect value on YPP with a value more than 3.50. Our research thus highlights the promising application for the rational utilization of YNSM and YHSM in hybrid rice breeding.

Two complementary genes in a presence-absence variation contribute to indica-japonica reproductive isolation in rice

Understanding the evolutionary forces in speciation is a central goal in evolutionary biology. Asian cultivated rice has two subspecies, indica and japonica, but the underlying mechanism of the partial reproductive isolation between them remains obscure. Here we show a presence-absence variation (PAV) at the Se locus functions as an indica-japonica reproductive barrier by causing hybrid sterility (HS) in indica-japonica crosses. The locus comprises two adjacent genes: ORF3 encodes a sporophytic pollen killer, whereas ORF4 protects pollen in a gametophytic manner. In F1 of indica-japonica crosses, pollen with the japonica haplotype, which lacks the sequence containing the protective ORF4, is aborted due to the pollen-killing effect of ORF3 from indica. Evolutionary analysis suggests ORF3 is a gene associated with the Asian cultivated rice species complex, and the PAV has contributed to the reproductive isolation between the two subspecies of Asian cultivated rice. Our analyses provide perspectives on rice inter-subspecies post-zygotic isolation, and will promote efforts to overcome reproductive barriers in indica-japonica hybrid rice breeding.

A natural gene drive system confers reproductive isolation in rice

Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.

Correlation and Path Coefficient Analysis for Yield and Yield Attributing Traits in Rice (Oryza sativa L.)

This study aimed to investigate the correlations among yield and yield component traits and estimate the direct and indirect effects of these traits on grain yield in rice. The experiment was conducted during kharif 2022. Correlation coefficients and path coefficients were analyzed to determine the relationships between these traits and seed yield. The results showed significant positive correlations between seed yield and traits such as effective tillers per plant, total tillers per plant, number of filled grains, spikelet fertility, biological yield, harvest index, and 100 seed weight. The genotypic correlations were slightly higher than the corresponding phenotypic correlations. Path coefficient analysis revealed that biological yield had the maximum direct effect on seed yield, followed by harvest index, panicle length, and number of filled grains per panicle, plant height, and effective tillers per plant, 100 seed weight, and days to 50% flowering. These findings provide valuable information for selecting superior cross combinations in hybrid rice breeding programs to improve grain yield.

Advances on the Study of Diurnal Flower-Opening Times of Rice

The principal goal of rice (Oryza sativa L.) breeding is to increase the yield. In the past, hybrid rice was mainly indica intra-subspecies hybrids, but its yield has been difficult to improve. The hybridization between the indica and japonica subspecies has stronger heterosis; the utilization of inter-subspecies heterosis is important for long-term improvement of rice yields. However, the different diurnal flower-opening times (DFOTs) between the indica and japonica subspecies seriously reduce the efficiency of cross-pollination and yield and increase the cost of indica–japonica hybrid rice seeds, which has become one of the main constraints for the development of indica–japonica hybrid rice breeding. The DFOT of plants is adapted to their growing environment and is also closely related to species stability and evolution. Herein, we review the structure and physiological basis of rice flower opening, the factors that affect DFOT, and the progress of cloning and characterization of DFOT genes in rice. We also analyze the problems in the study of DFOT and provide corresponding suggestions.

Transcriptomic and Physiological Analyses of Two Rice Restorer Lines under Different Nitrogen Supplies Provide Novel Insights into Hybrid Rice Breeding.

Improving plant nitrogen-use efficiency (NUE) has great significance for various crops, particularly in hybrid breeding. Reducing nitrogen inputs is key to achieving sustainable rice production and mitigating environmental problems. In this study, we analyzed the transcriptomic and physiological changes in two indica restorer lines (Nanhui511 [NH511] and Minghui23 [MH23]) under high nitrogen (HN) and low nitrogen (LN) conditions. Compared to MH23, NH511 was more sensitive to different nitrogen supplies and exhibited higher nitrogen uptake and NUE under HN conditions by increasing lateral root and tiller numbers in the seedling and maturation stages, respectively. NH511 also exhibited a lower survival rate than MH23 when planted in a chlorate-containing hydroponic solution, indicating its HN uptake ability under different nitrogen-supply conditions. Transcriptomic analysis showed that NH511 has 2456 differentially expressed genes, whereas MH23 had only 266. Furthermore, these genes related to nitrogen utilization showed differential expression in NH511 under HN conditions, while the opposite was observed in MH23. Our findings revealed that NH511 could be regarded as elite rice and used for breeding high-NUE restorer lines by regulating and integrating nitrogen-utilization genes, which provides novel insights for the cultivation of high-NUE hybrid rice.

Genome-wide identification of nitrate-responsive microRNAs by small RNA sequencing in the rice restorer cultivar Nanhui 511.

Rice productivity relies heavily on nitrogen fertilization, and improving nitrogen use efficiency (NUE) is important for hybrid rice breeding. Reducing nitrogen inputs is the key to achieving sustainable rice production and reducing environmental problems. Here, we analyzed the genome-wide transcriptomic changes in microRNAs (miRNAs) in the indica rice restorer cultivar Nanhui 511 (NH511) under high (HN) and low nitrogen (LN) conditions. The results showed that NH511 is sensitive to nitrogen supplies and HN conditions promoted the growth its lateral roots at the seedling stage. Furthermore, we identified 483 known miRNAs and 128 novel miRNAs by small RNA sequencing in response to nitrogen in NH511. We also detected 100 differentially expressed genes (DEGs), including 75 upregulated and 25 downregulated DEGs, under HN conditions. Among these DEGs, 43 miRNAs that exhibited a 2-fold change in their expression were identified in response to HN conditions, including 28 upregulated and 15 downregulated genes. Additionally, some differentially expressed miRNAs were further validated by qPCR analysis, which showed that miR443, miR1861b, and miR166k-3p were upregulated, whereas miR395v and miR444b.1 were downregulated under HN conditions. Moreover, the degradomes of possible target genes for miR166k-3p and miR444b.1 and expression variations were analyzed by qPCR at different time points under HN conditions. Our findings revealed comprehensive expression profiles of miRNAs responsive to HN treatments in an indica rice restorer cultivar, which advances our understanding of the regulation of nitrogen signaling mediated by miRNAs and provides novel data for high-NUE hybrid rice cultivation.

Genetic Analysis of S5 Regulating the Hybrid Sterility between Indica and Japonica Subspecies in Rice

Hybrid sterility is the major obstacle to the utilization of inter-subspecific heterosis in hybrid rice breeding. The S5 locus, composed of three adjacent genes ORF3, ORF4, and ORF5, plays a crucial role in regulating indica/japonica hybrids’ female sterility. Through a series of crosses involving 38 parents, three alleles of S5, ORF3+ORF4−ORF5n, ORF3+ORF4+ORF5n, and ORF3−/ORF4−/ORF5n, all could be regarded as wide-compatibility alleles, and when crossed with indica or japonica rice, they all showed significantly high fertility. Then, in order to explore the genes’ function, we further knocked out genes by using CRISPR/Cas9-based genome editing. Our results demonstrate that the ORF3+ was not just the protector in the killer-protector system, and knocking out ORF3 of the indica allele seriously affected the rice’s normal development. We observed the concrete enhancing hybrid spikelet fertility from the crosses between the ORF4+ knockout japonica materials with indica varieties. By conducting the comparative RNA-Seq analysis of young spikelets, we found that the ORF4+/ORF4− could modulate the hybrid fertility by affecting the expressions of genes related to the function of the Golgi apparatus. This study indicated that knocking out the ORF4+ of the japonica allele or using the alleles carrying ORF5n would provide effective approaches to overcome indica/japonica hybrid female sterility in rice breeding.

EVALUATION OF TEST CROSSES A WAY FORWARD TOWARDS THE IDENTIFICATION OF POTENTIAL RESTORERS & MAINTAINERS FOR THE DEVELOPMENT OF RICE HYBRIDS

Identification of desirable maintainers and potential restorers in a hybrid rice breeding program is the dire need for the development of potential rice hybrids. Seventeen (17) cytoplasmic male sterile (CMS) lines (15 imported from IRRI and 02 locally developed by RRI, KSK) of rice were crossed with one hundred and sixty (160) testers to identify their genetic nature to behave as restorer or maintainer in the year 2017 and 2018. A total of one hundred and sixty (160) rice test crosses were subjected to pollen and spikelet fertility when crossed with CMS lines. Most of the testers show differential fertility/sterility responses with CMS lines. From these evaluated test crosses thirty-three (33) restorers and thirty-four (34) maintainers (16 Basmati and 18 non-basmati lines) were recognized for further use to develop potential rice hybrids. Four basmati and twenty-nine coarse lines were identified as restorers from the tested genotypes. Most of the genotypes found to be partial restorer (PR) and partial maintainers (PM). Moreover, four promising heterotic combinations/potential restorers were also identified. Six heterotic hybrid combinations had more than 80% spikelet fertility. Potential restorers and maintainers identified in the present exploration could be used to develop high-yielding promising rice hybrids adaptable to the diversified climatic conditions of Punjab, Pakistan.

Yuan Longping, a great world hunger fighter

Yuan Longping, a great world hunger fighter

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice.

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC1F1 generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC1F1 generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC1F3 generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC1F5 generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC1F5 generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.

OsLDDT1, encoding a transmembrane structural DUF726 family protein, is essential for tapetum degradation and pollen formation in rice

Formation of the pollen wall, which is mainly composed of lipid substances secreted by tapetal cells, is important to ensure pollen development in rice. Although several regulatory factors related to lipid biosynthesis during pollen wall formation have been identified in rice, the molecular mechanisms controlling lipid biosynthesis are unclear. In this study, we isolated the male-sterile rice mutant oslddt1 (leaked and delayed degraded tapetum 1). oslddt1 plants show complete pollen abortion resulting from delayed degradation of the tapetum and blocked formation of Ubisch bodies and pollen walls. OsLDDT1 (LOC_Os03g02170) encodes a DUF726 containing protein of unknown function with highly conserved transmembrane and α/β Hydrolase domains. OsLDDT1 localizes to the endoplasmic reticulum and the gene is highly expressed in rice panicles. Genes involved in regulating fatty acid synthesis and formation of sporopollenin and pollen exine during anther development showed significantly different expression patterns in oslddt1 plants. Interestingly, the wax and cutin contents in mature oslddt1–1 anthers were decreased by 74.07 % and 72.22 % compared to WT, indicating that OsLDDT1 is involved in fatty acid synthesis and affects formation of the anther epidermis. Our results provide as deeper understanding of the role of OsLDDT1 in regulating male sterility and also provide materials for hybrid rice breeding.

Breeding of the Long-Grain Restorer of Indica-Japonica Hybrid Rice by Using the Genetic Effects of Grain Shape QTLs

Grain shape improvement, which determines grain yield, quality traits and commercial value, is an extremely important aspect of rice breeding. Grain size is controlled by multiple genes, and Maker Assistant Selection (MAS) breeding is effective for breeders in developing stable and efficient markers to aggregate these genes in order to speed up the selection of new lines with desirable traits during the breeding process. In this study, functional markers were developed based on the sequence differences of five grain-shaped genes (GL7, GW6a, GS6, GW5 and TGW6) between the long-grain japonica rice variety Zhendao and the indica-japonica restorer R2027. We then constructed a population of recombinant inbred lines (RILs) based on their cross. The newly designed functional markers were used to genotype grain-size genes, and a genetic effect analysis was conducted to screen high-quality long-grain restorers. Our results reveal diverse effects of different genes on grain size, and the five genotypes were distributed in the 36 selected BC1F8 lines. Specifically, gw5 positively regulates grain width and 1000-grain weight, gl7 and gs6 positively regulate grain length but negatively regulate grain width and 1000-grain weight, tgw6 positively regulates grain length and gw6a positively regulates 1000-grain weight. The most outstanding outcome is that 5 of the 36 lines achieved in this study showing an excellent performance of long grain and yield characters are ideal materials not only for studying the interaction and genetic effects between polygenes but also as restorers or donors for dominant genes in indica-japonica hybrid rice breeding.

Large-scale genomic and transcriptomic profiles of rice hybrids reveal a core mechanism underlying heterosis

BackgroundHeterosis is widely used in agriculture. However, its molecular mechanisms are still unclear in plants. Here, we develop, sequence, and record the phenotypes of 418 hybrids from crosses between two testers and 265 rice varieties from a mini-core collection.ResultsPhenotypic analysis shows that heterosis is dependent on genetic backgrounds and environments. By genome-wide association study of 418 hybrids and their parents, we find that nonadditive QTLs are the main genetic contributors to heterosis. We show that nonadditive QTLs are more sensitive to the genetic background and environment than additive ones. Further simulations and experimental analysis support a novel mechanism, homo-insufficiency under insufficient background (HoIIB), underlying heterosis. We propose heterosis in most cases is not due to heterozygote advantage but homozygote disadvantage under the insufficient genetic background.ConclusionThe HoIIB model elucidates that genetic background insufficiency is the intrinsic mechanism of background dependence, and also the core mechanism of nonadditive effects and heterosis. This model can explain most known hypotheses and phenomena about heterosis, and thus provides a novel theory for hybrid rice breeding in future.

Optimizing Agronomy Improves Super Hybrid Rice Yield and Nitrogen Use Efficiency through Enhanced Post-Heading Carbon and Nitrogen Metabolism

The super hybrid rice breeding program in China has raised genetic yield ceilings through morphological improvements and inter-subspecific heterosis. Despite this, little information on the physiological basis underlying this yield transformation exists, and less so on the genotype x environment x management conditions enabling consistent yield gains. Here, we assess grain yield, photosynthetic physiology, and leaf carbon and nitrogen (N) metabolic properties of super rice (Y-liangyou900) under four management practices (i.e., zero-fertilizer control, CK; farmers’ practice, FP; high-yield and high-efficiency management, OPT1; and super-high-yield management, OPT2) using a field experiment conducted over five years. Grain yield and agronomic N use efficiency (AEN) of OPT2 were 15% and 10% higher than OPT1, and 30% and 78% higher than FP, respectively. The superior yields of OPT2 were attributed to higher source production capacity, that is, higher leaf photosynthetic rate, carbon metabolic enzyme activity (i.e., AGP and SPS), nitrogen metabolic enzyme activity (i.e., NR, GS, and GOGAT), soluble protein and sugar content, and delayed leaf senescence (the latter due to elevated activity of protective enzyme systems) during grain filling. The higher AEN of OPT2 was associated with higher activity of leaf carbon metabolic enzyme (i.e., AGP and SPS), nitrogen metabolic enzyme (i.e., NR, GS, GDH, and GOGAT) and protective enzyme (POD) after heading, and lower C/N ratio in grains. We conclude that optimized management (optimized water and fertilizer management with appropriate dense planting) improved grain yield and N use efficiency simultaneously by enhancing post-heading leaf carbon and N metabolism and delayed leaf senescence.