Global analysis of seed transcriptomes reveals a novel PLATZ regulator for seed size and weight control in soybean.

Seed size and weight are important factors that influence soybean yield. Combining the weighted gene co-expression network analysis (WGCNA) of 45 soybean accessions and gene dynamic changes in seeds at seven developmental stages, we identified candidate genes that may control the seed size/weight. Among these, a PLATZ-type regulator overlapping with 10 seed weight QTLs was further investigated. This zinc-finger transcriptional regulator, named as GmPLATZ, is required for the promotion of seed size and weight in soybean. The GmPLATZ may exert its functions through direct binding to the promoters and activation of the expression of cyclin genes and GmGA20OX for cell proliferation. Overexpression of the GmGA20OX enhanced seed size/weight in soybean. We further found that the GmPLATZ binds to a 32-bp sequence containing a core palindromic element AATGCGCATT. Spacing of the flanking sequences beyond the core element facilitated GmPLATZ binding. An elite haplotype Hap3 was also identified to have higher promoter activity and correlated with higher gene expression and higher seed weight. Orthologues of the GmPLATZ from rice and Arabidopsis play similar roles in seeds. Our study reveals a novel module of GmPLATZ-GmGA20OX/cyclins in regulating seed size and weight and provides valuable targets for breeding of crops with desirable agronomic traits.

Isolation of a Novel QTL, qSCM4, Associated with Strong Culm Affects Lodging Resistance and Panicle Branch Number in Rice.

Rice breeders are now developing new varieties with semi-high or even high plant height to further increase the grain yield, and the problem of lodging has re-appeared. We identified a major quantitative trait locus (QTL), qSCM4, for resistance to lodging by using an F2 segregant population and a recombinant self-incompatible line population from the cross between Shennong265 (SN265) and Lijiangxintuanheigu (LTH) after multiple years and multiple environments. Then, the residual heterozygous derived segregant population which consisted of 1781 individual plants, and the BC3F2 segregant population which consisted of 3216 individual plants, were used to shorten the physical interval of qSCM4 to 58.5 kb including 11 genes. DNA sequencing revealed the most likely candidate gene for qSCM4 was Os04g0615000, which encoded a functional protein with structural domains of serine and cysteine. There were 13 DNA sequence changes in LTH compared to SN265 in this gene, including a fragment deletion, two base changes in the 3' UTR region, six base changes in the exons, and four base changes in the introns. A near-isogenic line carrying qSCM4 showed that it improved the lodging resistance through increasing stem thickness by 25.3% and increasing stem folding resistance by 20.3%. Furthermore, it was also discovered that qSCM4 enhanced the primary branch per panicle by 16.7%, secondary branch by per panicle 9.9%, and grain number per panicle by 14.7%. All the above results will give us a valuable genetic resource for concurrently boosting culm strength and lodging resistance, and they will also provide a basis for further research on the lodging resistance mechanism of rice.

Zinc-finger protein GmZF351 improves both salt and drought stress tolerance in soybean.

Abiotic stress is one of the most important factors reducing soybean yield. It is essential to identify regulatory factors contributing to stress responses. A previous study found that the tandem CCCH zinc-finger protein GmZF351 is an oil level regulator. In this study, we discovered that the GmZF351 gene is induced by stress and that the overexpression of GmZF351 confers stress tolerance to transgenic soybean. GmZF351 directly regulates the expression of GmCIPK9 and GmSnRK, leading to stomata closing, by binding to their promoter regions, which carry two CT(G/C)(T/A)AA elements. Stress induction of GmZF351 is mediated through reduction in the H3K27me3 level at the GmZF351 locus. Two JMJ30-demethylase-like genes, GmJMJ30-1 and GmJMJ30-2, are involved in this demethylation process. Overexpression of GmJMJ30-1/2 in transgenic hairy roots enhances GmZF351 expression mediated by histone demethylation and confers stress tolerance to soybean. Yield-related agronomic traits were evaluated in stable GmZF351-transgenic plants under mild drought stress conditions. Our study reveals a new mode of GmJMJ30-GmZF351 action in stress tolerance, in addition to that of GmZF351 in oil accumulation. Manipulation of the components in this pathway is expected to improve soybean traits and adaptation under unfavorable environments.

GmJAZ3 interacts with GmRR18a and GmMYC2a to regulate seed traits in soybean.

Seed weight is usually associated with seed size and is one of the important agronomic traits that determine yield. Understanding of seed weight control is limited, especially in soybean plants. Here we show that Glycine max JASMONATE-ZIM DOMAIN 3 (GmJAZ3), a gene identified through gene co-expression network analysis, regulates seed-related traits in soybean. Overexpression of GmJAZ3 promotes seed size/weight and other organ sizes in stable transgenic soybean plants likely by increasing cell proliferation. GmJAZ3 interacted with both G. max RESPONSE REGULATOR 18a (GmRR18a) and GmMYC2a to inhibit their transcriptional activation of cytokinin oxidase gene G. max CYTOKININ OXIDASE 3-4 (GmCKX3-4), which usually affects seed traits. Meanwhile, the GmRR18a binds to the promoter of GmMYC2a and activates GmMYC2a gene expression. In GmJAZ3-overexpressing soybean seeds, the protein contents were increased while the fatty acid contents were reduced compared to those in the control seeds, indicating that the GmJAZ3 affects seed size/weight and compositions. Natural variation in JAZ3 promoter region was further analyzed and Hap3 promoter correlates with higher promoter activity, higher gene expression and higher seed weight. The Hap3 promoter may be selected and fixed during soybean domestication. JAZ3 orthologs from other plants/crops may also control seed size and weight. Taken together, our study reveals a novel molecular module GmJAZ3-GmRR18a/GmMYC2a-GmCKXs for seed size and weight control, providing promising targets during soybean molecular breeding for better seed traits.

Genome-wide association study uncovers major genetic loci associated with flowering time in response to active accumulated temperature in wild soybean population.

Flowering time and active accumulated temperature (AAT) are two key factors that limit the expanded production especially for soybean across different regions. Wild soybean provides an important germplasm for functional genomics study in cultivar soybean. However, the studies on genetic basis underlying flowering time in response to AAT especially in wild soybean were rarely reported. In this study, we used 294 wild soybean accessions derived from major soybean production region characterized by different AAT in Northeast of China. Based on genome-wide association study (GWAS), we identified 96 SNPs corresponded to 342 candidate genes that significantly associated with flowering time recorded in two-year experiments. Gene Ontology enrichment analysis suggests that the pathways of photosynthesis light reaction and actin filament binding were significantly enriched. We found three lead SNPs with -log10(p-value) > 32 across the two-year experiments, i.e., Chr02:9490318, Chr04:8545910 and Chr09:49553555. Linkage disequilibrium block analysis shows 28 candidate genes within the genomic region centered on the lead SNPs. Among them, expression levels of three genes (aspartic peptidase 1, serine/threonine-protein kinase and protein SCAR2-like) were significantly differed between two subgroups possessing contrasting flowering time distributed at chromosome 2, 4 and 9, respectively. There are 6, 7 and 3 haplotypes classified on the coding regions of the three genes, respectively. Collectively, accessions with late flowering time phenotype are typically derived from AAT zone 1, which is associated with the haplotypic distribution and expression levels of the three genes. This study provides an insight into a potential mechanism responsible for flowering time in response to AAT in wild soybean, which could promote the understanding of genetic basis for other major crops.

Genome-Wide Association Study of Partial Resistance to P. sojae in Wild Soybeans from Heilongjiang Province, China.

Phytophthora root rot (PRR) is a destructive disease of soybeans (Glycine max (L.) Merr) caused by Phytophthora sojae (P. sojae). The most effective way to prevent the disease is growing resistant or tolerant varieties. Partial resistance provides a more durable resistance against the pathogen compared to complete resistance. Wild soybean (Glycine soja Sieb. & Zucc.) seems to be an extraordinarily important gene pool for soybean improvement due to its high level of genetic variation. In this study, 242 wild soybean germplasms originating from different regions of Heilongjiang province were used to identify resistance genes to P. sojae race 1 using a genome-wide association study (GWAS). A total of nine significant SNPs were detected, repeatedly associated with P. sojae resistance and located on chromosomes 1, 10, 12, 15, 17, 19 and 20. Among them, seven favorable allelic variations associated with P. sojae resistance were evaluated by a t-test. Eight candidate genes were predicted to explore the mechanistic hypotheses of partial resistance, including Glysoja.19G051583, which encodes an LRR receptor-like serine/threonine protein kinase protein, Glysoja.19G051581, which encodes a receptor-like cytosolic serine/threonine protein kinase protein. These findings will provide additional insights into the genetic architecture of P. sojae resistance in a large sample of wild soybeans and P. sojae-resistant breeding through marker-assisted selection.

A transcriptional regulatory module controls lipid accumulation in soybean.

Soybean (Glycine max) is one of the most important oilseed crops. However, the regulatory mechanism that governs the process of oil accumulation in soybean remains poorly understood. In this study, GmZF392, a tandem CCCH zinc finger (TZF) protein which was identified in our previous RNA-seq analysis of seed-preferred transcription factors, was found to function as a positive regulator of lipid production. GmZF392 promotes seed oil accumulation in both transgenic Arabidopsis and stable transgenic soybean plants by binding to a bipartite cis-element, containing TG- and TA-rich sequences, in promoter regions, activating the expression of genes in the lipid biosynthesis pathway. GmZF392 physically interacts with GmZF351, our previously identified transcriptional regulator of lipid biosynthesis, to synergistically promote downstream gene expression. Both GmZF392 and GmZF351 are further upregulated by GmNFYA, another transcription factor involved in lipid biosynthesis, directly (in the former case) and indirectly (in the latter case). Promoter sequence diversity analysis showed that the GmZF392 promoter may have been selected at the origin of the Glycine genus and further mildly selected during domestication from wild soybeans to cultivated soybeans. Our study reveals a regulatory module containing three transcription factors in the lipid biosynthesis pathway, and manipulation of the module may improve oil production in soybean and other oilseed crops.

A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis.

Soybean (Glycine max) production is severely affected in unfavorable environments. Identification of the regulatory factors conferring stress tolerance would facilitate soybean breeding. In this study, through coexpression network analysis of salt-tolerant wild soybeans, together with molecular and genetic approaches, we revealed a previously unidentified function of a class B heat shock factor, HSFB2b, in soybean salt stress response. We showed that HSFB2b improves salt tolerance through the promotion of flavonoid accumulation by activating one subset of flavonoid biosynthesis-related genes and by inhibiting the repressor gene GmNAC2 to release another subset of genes in the flavonoid biosynthesis pathway. Moreover, four promoter haplotypes of HSFB2b were identified from wild and cultivated soybeans. Promoter haplotype II from salt-tolerant wild soybean Y20, with high promoter activity under salt stress, is probably selected for during domestication. Another promoter haplotype, III, from salt-tolerant wild soybean Y55, had the highest promoter activity under salt stress, had a low distribution frequency and may be subjected to the next wave of selection. Together, our results revealed the mechanism of HSFB2b in soybean salt stress tolerance. Its promoter variations were identified, and the haplotype with high activity may be adopted for breeding better soybean cultivars that are adapted to stress conditions.

An optimized double T-DNA binary vector system for improved production of marker-free transgenic tobacco plants.

OBJECTIVES: In the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB-LB-RB-LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB-RB-RB-LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI. RESULTS: We separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%). CONCLUSION: Our results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.

Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication.

Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops.

Transcriptomic comparison reveals genetic variation potentially underlying seed developmental evolution of soybeans.

Soybean (Glycine max) was domesticated from its wild relative Glycine soja. However, the genetic variations underlying soybean domestication are not well known. Comparative transcriptomics revealed that a small portion of the orthologous genes might have been fast evolving. In contrast, three gene expression clusters were identified as divergent by their expression patterns, which occupied 37.44% of the total genes, hinting at an essential role for gene expression alteration in soybean domestication. Moreover, the most divergent stage in gene expression between wild and cultivated soybeans occurred during seed development around the cotyledon stage (15 d after fertilization, G15). A module in which the co-expressed genes were significantly down-regulated at G15 of wild soybeans was identified. The divergent clusters and modules included substantial differentially expressed genes (DEGs) between wild and cultivated soybeans related to cell division, storage compound accumulation, hormone response, and seed maturation processes. Chromosomal-linked DEGs, quantitative trait loci controlling seed weight and oil content, and selection sweeps revealed candidate DEGs at G15 in the fruit-related divergence of G. max and G. soja. Our work establishes a transcriptomic selection mechanism for altering gene expression during soybean domestication, thus shedding light on the molecular networks underlying soybean seed development and breeding strategy.

Water regime-nitrogen fertilizer incorporation interaction: Field study on methane and nitrous oxide emissions from a rice agroecosystem in Harbin, China.

Water regime and nitrogen (N) fertilizer are two important factors impacting greenhouse gases (GHG) emission from paddy field, whereas their effects have not been well studied in cold region. In this study, we conducted a two-year field experiment to study the impacts of water regime and N fertilizer on rice yields and GHG emissions in Harbin, China, a cold region located in high latitudes. Our results showed that intermittent irrigation significantly decreased methane (CH4) emission compared with continuous flooding, however, the decrement was far lower than the global average level. The N2O emissions were very small when flooded but peaked at the beginning of the disappearance of floodwater. The N fertilizer treatments increased CH4 emissions at low level (75kgN/ha). But both CH4 and N2O emissions were uninfluenced at the levels of 150kgN/ha and 225kgN/ha. Rice yields increased under intermittent irrigation and were highest at the level of 150kgN/ha. From our results, we recommended that the intermittent irrigation and 150kgN/ha as the ideal water regime-nitrogen fertilizer incorporation for this area to achieve low GHG emissions without impacting rice yields.

The transcriptomic signature of developing soybean seeds reveals the genetic basis of seed trait adaptation during domestication.

Cultivated soybean has undergone many transformations during domestication. In this paper we report a comprehensive assessment of the evolution of gene co-expression networks based on the analysis of 40 transcriptomes from developing soybean seeds in cultivated and wild soybean accessions. We identified 2680 genes that are differentially expressed during seed maturation and established two cultivar-specific gene co-expression networks. Through analysis of the two networks and integration with quantitative trait locus data we identified two potential key drivers for seed trait formation, GA20OX and NFYA. GA20OX encodes an enzyme in a rate-limiting step of gibberellin biosynthesis, and NFYA encodes a transcription factor. Overexpression of GA20OX and NFYA enhanced seed size/weight and oil content, respectively, in seeds of transgenic plants. The two genes showed significantly higher expression in cultivated than in wild soybean, and the increases in expression were associated with genetic variations in the promoter region of each gene. Moreover, the expression of GA20OX and NFYA in seeds of soybean accessions correlated with seed weight and oil content, respectively. Our study reveals transcriptional adaptation during soybean domestication and may identify a mechanism of selection by expression for seed trait formation, providing strategies for future breeding practice.

Differential expression of a WRKY gene between wild and cultivated soybeans correlates to seed size.

Soybean (Glycine max) probably originated from the wild soybean (Glycine soja). Glycine max has a significantly larger seed size, but the underlying genomic changes are largely unknown. Candidate regulatory genes were preliminarily proposed by data co-localizing RNA sequencing with the quantitative loci (QTLs) for seed size. The soybean gene locus SoyWRKY15a and its orthologous genes from G. max (GmWRKY15a) and G. soja (GsWRKY15a) were analyzed in detail. The coding sequences were nearly identical between the two orthologs, but GmWRKY15a was significantly more highly expressed than GsWRKY15a. Four haplotypes (H1-H4) were found and they varied in the size of a CT-core microsatellite locus in the 5'-untranslated region of this gene. H1 (with six CT-repeats) was the only allelic version found in G. max, while H3 (with five CT-repeats) was the dominant G. soja allele. Differential expression of this gene in soybean pods was correlated with CT-repeat variation, and manipulation of the CT copy number altered the reporter gene expression, suggesting a regulatory role for the simple sequence repeats. Seed weight of wild soybeans harboring H1 was significantly greater than that of soybeans having haplotypes H2, H3, or H4, and seed weight was correlated with gene expression, suggesting the influence of GsWRKY15a in controlling seed size. However, the seed size might be refractory to increased SoyWRKY15a expression in cultivated soybeans. The evolutionary significance of SoyWRKY15a variation in soybean seed domestication is discussed.

GmWRKY27 interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants.

Soybean (Glycine max) is an important crop for oil and protein resources worldwide. The molecular mechanism of the abiotic stress response in soybean is largely unclear. We previously identified multiple stress-responsive WRKY genes from soybean. Here, we further characterized the roles of one of these genes, GmWRKY27, in abiotic stress tolerance using a transgenic hairy root assay. GmWRKY27 expression was increased by various abiotic stresses. Over-expression and RNAi analysis demonstrated that GmWRKY27 improves salt and drought tolerance in transgenic soybean hairy roots. Measurement of physiological parameters, including reactive oxygen species and proline contents, supported this conclusion. GmWRKY27 inhibits expression of a downstream gene GmNAC29 by binding to the W-boxes in its promoter region. The GmNAC29 is a negative factor of stress tolerance as indicated by the performance of transgenic hairy roots under stress. GmWRKY27 interacts with GmMYB174, which also suppresses GmNAC29 expression and enhances drought stress tolerance. The GmWRKY27 and GmMYB174 may have evolved to bind to neighbouring cis elements in the GmNAC29 promoter to co-reduce promoter activity and gene expression. Our study discloses a valuable mechanism in soybean for regulation of the stress response by two associated transcription factors. Manipulation of these genes should facilitate improvements in stress tolerance in soybean and other crops.

Effects of plant density on alfalfa (Medicago sativa L.) seed yield in western Heilongjiang areas.

Alfalfa (Medicago sativa L.) is known as the "king of forages". The aim of the current study is to determine the optimum planting density as the key cultivation technique for high yield of alfalfa seed. Alfalfa variety (Longmu 801) was planted in experimental fields from 2014 to 2017. In the planting density test, the row spacing was 65, 80, and 95 cm, and the plant spacing was 30, 45, 60, 75, and 90 cm. The seed yield and yield components in the row spacing and plant spacing tests were measured. On the basis of 3 years average of the experimental data, the highest seed yield of 225.49 kg ha-1 was obtained with row spacing vs plant spacing of 65 and 60 cm, respectively. Correlation analysis showed a significant positive correlation between the racemes per stem, pods per raceme, pods per stem, seeds per pod, and the seed yield. These results suggested that Longmu 801 should be cultivated with 65 cm row spacing and 60 cm plant spacing to maximize seed yields in western Heilongjiang areas.

OsBBX11 on qSTS4 links to salt tolerance at the seeding stage in Oryza sativa L. ssp. Japonica.

Rice has been reported to be highly sensitive to salt stress at the seedling stage. However, the lack of target genes that can be used for improving salt tolerance has resulted in several saline soils unsuitable for cultivation and planting. To characterize new salt-tolerant genes, we used 1,002 F2:3 populations derived from Teng-Xi144 and Long-Dao19 crosses as the phenotypic source to systematically characterize seedlings' survival days and ion concentration under salt stress. Utilizing QTL-seq resequencing technology and a high-density linkage map based on 4,326 SNP markers, we identified qSTS4 as a major QTL influencing seedling salt tolerance, which accounted for 33.14% of the phenotypic variation. Through functional annotation, variation detection and qRT-PCR analysis of genes within 46.9 Kb of qSTS4, it was revealed that there was one SNP in the promoter region of OsBBX11, which resulted in a significant response difference between the two parents to salt stress. Transgenic plants using knockout-based technology and demonstrated that Na+ and K+ in the roots of the functional-loss-type OsBBX11 were translocated largely to the leaves under 120 mmol/L NaCl compared with the wild-type, causing osbbx11 leaves to die after 12 days of salt stress due to an imbalance in osmotic pressure. In conclusion, this study identified OsBBX11 as a salt-tolerance gene, and one SNPs in the OsBBX11 promoter region can be used to identify its interacting transcription factors. This provides a theoretical basis for finding the molecular mechanism of OsBBX11 upstream and downstream regulation of salt tolerance and molecular design breeding in the future.

The genetic basis of high-latitude adaptation in wild soybean.

In many plants, flowering time is influenced by daylength as an adaptive response. In soybean (Glycine max) cultivars, however, photoperiodic flowering reduces crop yield and quality in high-latitude regions. Understanding the genetic basis of wild soybean (Glycine soja) adaptation to high latitudes could aid breeding of improved cultivars. Here, we identify the Tof4 (Time of flowering 4) locus, which encodes by an E1-like protein, E1La, that represses flowering and enhances adaptation to high latitudes in wild soybean. Moreover, we found that Tof4 physically associates with the promoters of two important FLOWERING LOCUS T (FT2a and FT5a) and with Tof5 to inhibit their transcription under long photoperiods. The effect of Tof4 on flowering and maturity is mediated by FT2a and FT5a proteins. Intriguingly, Tof4 and the key flowering repressor E1 independently but additively regulate flowering time, maturity, and grain yield in soybean. We determined that weak alleles of Tof4 have undergone natural selection, facilitating adaptation to high latitudes in wild soybean. Notably, over 71.5% of wild soybean accessions harbor the mutated alleles of Tof4 or a previously reported gain-of-function allele Tof5H2, suggesting that these two loci are the genetic basis of wild soybean adaptation to high latitudes. Almost no cultivated soybean carries the mutated tof4 allele. Introgression of the tof4-1 and Tof5H2 alleles into modern soybean or editing E1 family genes thus represents promising avenues to obtain early-maturity soybean, thereby improving productivity in high latitudes.

Combination of Water-Saving Irrigation and Nitrogen Fertilization Regulates Greenhouse Gas Emissions and Increases Rice Yields in High-Cold Regions, Northeast China.

Increased rice production, which benefitted from cropping areas expansion and continuous N applications, resulted in severe increases in greenhouse gases (GHG) emissions from 1983 to 2019 in Heilongjiang Province, China. Therefore, field trials were performed in the high-cold Harbin region, Northeast China, to determine the efficiency of incorporating water regimes with N fertilization in minimizing the impact of rice production on GHG emissions. Two water-saving irrigation strategies, intermittent irrigation (W1) and control irrigation (W2), were used relative to continuous flooding (W0), and we combined them with six fertilized treatments. Our results demonstrated that W1 and W2 significantly decreased seasonal CH4 emissions by 19.7-30.0% and 11.4-29.9%, enhanced seasonal N2O emissions by 77.0-127.0% and 16.2-42.4%, and increased significantly yields by 5.9-12.7% and 0-4.7%, respectively, compared with W0. Although trade-offs occurred between CH4 and N2O emissions, W1 and W2 resulted in significant reductions in global warming potential (GWP). Moreover, low N rates (<120 kg N ha-1) performed better in GWP than high N rates. N fertilization and irrigation regimes had remarkable effects on rice yields and GWP. In conclusion, the incorporation of W1 and a N application under 120 kg N ha-1 could simultaneously mitigate GWP while enhancing production in black soils in high-cold Northeast China.