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Symbiotic nitrogen fixation boosts legume growth and production in nitrogen-poor soils. It has long been assumed that fixed nitrogen increases reproductive success, but until now, the regulatory mechanism was unknown. Here, we report a symbiotic flowering pathway that couples symbiotic and nutrient signals to the flowering induction pathway in legumes. We show that the symbiotic microRNA-microRNA172c (miR172c) and fixed nitrogen systemically and synergistically convey symbiotic and nutritional cues from roots to leaves to promote soybean ( Glycine max ) flowering. The combinations of symbiotic miR172c and local miR172c elicited by fixed nitrogen and development in leaves activate florigen-encoding FLOWERING LOCUS T ( FT ) homologs ( GmFT2a/5a ) by repressing TARGET OF EAT1-like 4a ( GmTOE4a ). Thus, FTs trigger reproductive development, which allows legumes to survive and reproduce under low-nitrogen conditions.

Soybean is an important model crop for photoperiodic response studies in plants and contributes significantly to the study of plant development and physiology in the past century. Because soybean plant is much bigger in size and longer in life cycle than Arabidopsis , it needs much more space for growth and time for investigation, which significantly hamper the efficiency of research. In the current study, we tested the photoperiodic response of a distinctive artificially-made cotyledon-only plant (COP) using a photoperiod-sensitive soybean variety Zigongdongdou (ZGDD) and other varieties with diverse sensitivity to photoperiod. ZGDD COPs flowered 39.4 ± 2.5 d after emergence under short-day conditions but maintained vegetative growth under long-day and night break conditions, which is similar to the case in the intact ZGDD plants. The COPs of early-maturing and medium-maturing soybean varieties also grew and flowered normally under natural day-length conditions. At the molecular level, the key genes in the photoperiodic pathway such as E1 , GmFT1a , GmFT2a , and GmFT5a in the COPs also showed the same photoperiod sensitivity as in the intact plants. In addition, a simpler material of COP with only one cotyledon and root was generated and found to be sensitive to photoperiod as well. Notably, the COPs are only one-fifth the height of intact plants and one-third the maximum diameter of the intact plants grown in chambers 30 d after emergence. Based on COPs, we established a novel experimental system characterized by an entire photoperiodic response and longer longevity of cotyledons in addition to small plant size, ensuring the consistency, reliability, and stability of plant materials. COPs have the potential to be a novel model material for studies of the developmental biology of soybean and other dicots., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Xu, Chen, Yuan, Wu, Jiang, Sapey, Wu, Sun, Guo and Han.)

Increasing soybean production and ensuring greater access to soybean protein and lipids is critical for global food security and human health. Seed size (i.e., seed weight) is one of the most important agronomic traits of soybean, which not only determines the seed yield, but can also affect the yield of protein and lipids. In China, farmers favor soybean cultivars with large seeds, which they believe produce more protein and lipids; however, experimental evidence supporting this belief is lacking. Therefore, we conducted field experiments from 2017 to 2020 at 35 locations across the Huang-Huai-Hai region (HHH) of China with 64 soybean cultivars. The seed yield, seed protein content, and seed lipids content of soybean, and their relationship with seed size were investigated. The highest seed yield (i.e., seed weight per unit area) was 2996.5 kg ha−1 in the north of HHH. However, the highest seed protein content was found in the south of HHH (42.5%) for the higher temperature, which was significantly higher than that of the middle (41.7%) and north of HHH (40.2%). In contrast, the highest seed lipids content was 20.7% in the north of HHH. Temperature, which had a path coefficient on seed yield of 0.519, can promote soybean seed yield. The correlation analysis indicated that the selection of the large seed size cultivar did not increase seed yield, and even led to a reduction of seed yield under high-yield environmental conditions. The seed protein content of soybean was not increased in the cultivars with large seed sizes. In addition, under different levels of seed lipids content (<20.30% or >20.30%), a significantly negative relationship was found between seed lipids content and hundred seed weight. Therefore, it is recommended that farmers choose to plant cultivars with smaller soybean seed sizes, so as to ensure high and stable soybean seed yield and obtain more vegetable protein and lipids per unit area.

Key Message: The genetic basis of soybean root system architecture (RSA) and the genetic relationship between shoot and RSA were revealed by integrating data from recombinant inbred population grafting and QTL mapping. Variations in root system architecture (RSA) affect the functions of roots and thus play vital roles in plant adaptations and agricultural productivity. The aim of this study was to unravel the genetic relationship between RSA traits and shoot-related traits in soybean. This study characterized RSA variability at seedling stage in a recombinant inbred population, derived from a cross between cultivated soybean C08 and wild soybean W05, and performed high-resolution quantitative trait locus (QTL) mapping. In total, 34 and 41 QTLs were detected for RSA-related and shoot-related traits, respectively, constituting eight QTL clusters. Significant QTL correspondence was found between shoot biomass and RSA-related traits, consistent with significant correlations between these phenotypes. RSA-related QTLs also overlapped with selection regions in the genome, suggesting the cultivar RSA could be a partial consequence of domestication. Using reciprocal grafting, we confirmed that shoot-derived signals affected root development and the effects were controlled by multiple loci. Meanwhile, RSA-related QTLs were found to co-localize with four soybean flowering-time loci. Consistent with the phenotypes of the parental lines of our RI population, diminishing the function of flowering controlling E1 family through RNA interference (RNAi) led to reduced root growth. This implies that the flowering time-related genes within the RSA-related QTLs are actually contributing to RSA. To conclude, this study identified the QTLs that determine RSA through controlling root growth indirectly via regulating shoot functions, and discovered superior alleles from wild soybean that could be used to improve the root structure in existing soybean cultivars., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a revolutionary genome editing technology that has been used to achieve site-specific gene knock-out, large fragment deletion, or base editing in many plant species including soybean ( Glycine max ). The Streptococcus pyogenes Cas9 (SpCas9) is widely used in plants at present, although there are some reports describing the application of CRISPR/Cpf1 in soybean. Therefore, the selection range of PAM (protospacer adjacent motif) sequences for soybean is currently limited to 5'-NGG-3' (SpCas9) or 5'-TTTN-3' (Cpf1), which in turn limits the number of genes that can be mutated. Another Cas9 enzyme from Staphylococcus aureus (SaCas9) recognizes the PAM sequence 5'-NNGRRT-3' (where R represents A or G), which can provide a wider range of potential target sequences. In this study, we developed a CRISPR/SaCas9 system and used this tool to specifically induce targeted mutations at five target sites in the GmFT2a (Glyma.16G150700) and GmFT5a (Glyma.16G044100) genes in soybean hairy roots. We demonstrated that this tool can recognize the PAM sequences 5'-AAGGGT-3', 5'-GGGGAT-3', 5'-TTGAAT-3', and 5'-TAGGGT-3' in soybean, and it achieved mutation rates ranging from 34.5% to 73.3%. Our results show that we have established a highly efficient CRISPR/SaCas9 tool that is as suitable as SpCas9 for genome editing in soybean, and it will be useful for expanding the range of target sequences for genome editing.

Pseudo-response regulator (PRR) family members serve as key components of the core clock of the circadian clock, and play important roles in photoperiodic flowering, stress tolerance, growth, and the development of plants. In this study, 14 soybean PRR genes were identified, and classified into three groups according to phylogenetic analysis and structural characteristics. Real-time quantitative PCR analysis revealed that 13 GmPRRs exhibited obvious rhythmic expression under long-day (LD) and short-day (SD) conditions, and the expression of 12 GmPRRs was higher under LD in leaves. To evaluate the effects of natural variations in GmPRR alleles on soybean adaptation, we examined the sequences of GmPRRs among 207 varieties collected across China and the US, investigated the flowering phenotypes in six environments, and analyzed the geographical distributions of the major haplotypes. The results showed that a majority of non-synonymous mutations in the coding region were associated with flowering time, and we found that the nonsense mutations resulting in deletion of the CCT domain were related to early flowering. Haplotype analysis demonstrated that the haplotypes associated with early flowering were mostly distributed in Northeast China, while the haplotypes associated with late flowering were mostly cultivated in the lower latitudes of China. Our study of PRR family genes in soybean provides not only an important guide for characterizing the circadian clock-controlled flowering pathway but also a theoretical basis and opportunities to breed varieties with adaptation to specific regions and farming systems.

Onset of flowering of plants is precisely controlled by extensive environmental factors and internal molecular networks, in which FLOWERING LOCUS T ( FT ) is a key flowering integrator. In soybean, a typical short-day plant, 11 FT homologues are found in its genome, of which several homologues are functionally diversified in flowering pathways and the others including GmFT3a are yet unknown. In the current study, we characterized GmFT3a , which is located on the same chromosome as the flowering promoters GmFT2a and GmFT5a . Overexpression of GmFT3a significantly promoted flowering of Arabidopsis under the inductive long-day (LD) photoperiod. GmFT3a over-expressed soybean also flowered earlier than the control under LD, but they were not significantly different under inductive short-day (SD) conditions, indicating that GmFT3a acts as a flowering promoter in the non-inductive photoperiod in soybean. Compared with other GmFT homologues, GmFT3a exhibited a slighter effect in flowering promotion than GmFT2a , GmFT5a and GmFT2b under LD conditions. GmFT3a promoted flowering by regulating the expression of downstream flowering-related genes and also affected the expression of other GmFTs . According to the re-sequencing data, the regional distributions of two major haplotypes in 176 soybean varieties were analyzed. The varieties with GmFT3a -Hap2 haplotype matured relatively early, and relative higher expression of GmFT3a was detected in early maturing varieties, implying that Hap2 variation may contribute to the adaptation of soybean to higher latitude regions by increasing expression level of genes in metabolism and signaling pathways. The early flowering germplasm generated by overexpression of GmFT3a has potential to be planted at higher latitudes where non-inductive long day is dominant in the growing season, and GmFT3a can be used to fine-tune soybean flowering and maturity time and improve the geographical adaptation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yuan, Wang, Wang, Zhang, Zhang, Jiang, Wu, Chen, Xu, Cai, Sun, Chen, Song, Wu, Hou, Yu and Han.)

Preserving viable pollen is of great interest to breeders to maintain desirable germplasm for future inbreeding. Ultra-low temperature preservation of pollen is an effective and safe way for long-term storage of plant germplasm resources. In this study, we improved methods for the preservation of soybean pollen at ultra-low temperature. Soybean flowers at the initially-open stage were collected at 6-10 a.m. during the fully-bloom stage of soybean plants and were dehydrated for 10 h and then frozen and stored at -196 or -80°C. In vitro culture experiments showed that the viability of preserved pollen remained as high as about 90%. The off-season (local site Heihe) and off-site (Beijing, after long-distance express delivery from Heihe) hybridization verification was conducted, and no significant difference in true hybrid rate was founded between the preserved pollen and the fresh pollen. The ultra-low temperature preservation technology for soybean pollen could break the spatiotemporal limit of soybean hybridization and facilitate the development of engineered soybean breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jia, Liang, Zhang, Zhang, Sapey, Liu, Sun, Sun, Yan, Lu and Han.)

CONSTANS ( CO ) plays a critical role in the photoperiodic flowering pathway. However, the function of soybean CO orthologs and the molecular mechanisms in regulating flowering remain largely unknown. This study characterized the natural variations in CO family genes and their association with flowering time and maturity in soybeans. A total of 21 soybean CO family genes ( GmCOL s) were cloned and sequenced in 128 varieties covering 14 known maturity groups (MG 0000-MG X from earliest to latest maturity). Regarding the whole genomic region involving these genes, GmCOL1 , GmCOL3 , GmCOL8 , GmCOL9 , GmCOL10 , and GmCOL13 were conserved, and the remaining 15 genes showed genetic variation that was brought about by mutation, namely, all single-nucleotide polymorphisms (SNPs) and insertions-deletions (InDels). In addition, a few genes showed some strong linkage disequilibrium. Point mutations were found in 15 GmCOL genes, which can lead to changes in the potential protein structure. Early flowering and maturation were related to eight genes ( GmCOL1 / 3 / 4 / 8 / 13 / 15 / 16 / 19 ). For flowering and maturation, 11 genes ( GmCOL2 / 5 / 6 / 14 / 20 / 22 / 23 / 24 / 25 / 26 / 28 ) expressed divergent physiognomy. Haplotype analysis indicated that the haplotypes of GmCOL5-Hap2 , GmCOL13-Hap2/3 , and GmCOL28-Hap2 were associated with flowering dates and soybean maturity. This study helps address the role of GmCOL family genes in adapting to diverse environments, particularly when it is necessary to regulate soybean flowering dates and maturity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Awal Khan, Zhang, Emon, Chen, Song, Wu, Yuan, Wu, Hou, Sun, Fu, Jiang and Han.)

Key Message: GmFULa improved soybean yield by enhancing carbon assimilation. Meanwhile, different from known yield-related genes, it did not alter flowering time or maturity. Soybean (Glycine max (L.) Merr.) is highly demanded by a continuously growing human population. However, increasing soybean yield is a major challenge. FRUITFULL (FUL), a MADS-box transcription factor, plays important roles in multiple developmental processes, especially fruit and pod development, which are crucial for soybean yield formation. However, the functions of its homologs in soybean are not clear. Here, through haplotype analysis, we found that one haplotype of the soybean homolog GmFULa (GmFULa-H02) is dominant in cultivated soybeans, suggesting that GmFULa-H02 was highly selected during domestication and varietal improvement of soybean. Interestingly, transgenic overexpression of GmFULa enhanced vegetative growth with more biomass accumulated and ultimately increased the yield but without affecting the plant height or changing the flowering time and maturity, indicating that it enhances the efficiency of dry matter accumulation. It also promoted the yield factors like branch number, pod number and 100-seed weight, which ultimately increased the yield. It increased the palisade tissue cell number and the chlorophyll content to promote photosynthesis and increase the soluble sugar content in leaves and fresh seeds. Furthermore, GmFULa were found to be sublocalized in the nucleus and positively regulate sucrose synthases (SUSs) and sucrose transporters (SUTs) by binding with the conserved CArG boxes in their promoters. Overall, these results showed GmFULa promotes the capacity of assimilation and the transport of the resultant assimilates to increase yield, and provided insights into the link between GmFULa and sucrose synthesis with transport-related molecular pathways that control seed yield., (© 2021. The Author(s).)

Background: In soybean, some circadian clock genes have been identified as loci for maturity traits. However, the effects of these genes on soybean circadian rhythmicity and their impacts on maturity are unclear., Results: We used two geographically, phenotypically and genetically distinct cultivars, conventional juvenile Zhonghuang 24 (with functional J/GmELF3a, a homolog of the circadian clock indispensable component EARLY FLOWERING 3) and long juvenile Huaxia 3 (with dysfunctional j/Gmelf3a) to dissect the soybean circadian clock with time-series transcriptomal RNA-Seq analysis of unifoliate leaves on a day scale. The results showed that several known circadian clock components, including RVE1, GI, LUX and TOC1, phase differently in soybean than in Arabidopsis, demonstrating that the soybean circadian clock is obviously different from the canonical model in Arabidopsis. In contrast to the observation that ELF3 dysfunction results in clock arrhythmia in Arabidopsis, the circadian clock is conserved in soybean regardless of the functional status of J/GmELF3a. Soybean exhibits a circadian rhythmicity in both gene expression and alternative splicing. Genes can be grouped into six clusters, C1-C6, with different expression profiles. Many more genes are grouped into the night clusters (C4-C6) than in the day cluster (C2), showing that night is essential for gene expression and regulation. Moreover, soybean chromosomes are activated with a circadian rhythmicity, indicating that high-order chromosome structure might impact circadian rhythmicity. Interestingly, night time points were clustered in one group, while day time points were separated into two groups, morning and afternoon, demonstrating that morning and afternoon are representative of different environments for soybean growth and development. However, no genes were consistently differentially expressed over different time-points, indicating that it is necessary to perform a circadian rhythmicity analysis to more thoroughly dissect the function of a gene. Moreover, the analysis of the circadian rhythmicity of the GmFT family showed that GmELF3a might phase- and amplitude-modulate the GmFT family to regulate the juvenility and maturity traits of soybean., Conclusions: These results and the resultant RNA-seq data should be helpful in understanding the soybean circadian clock and elucidating the connection between the circadian clock and soybean maturity., (© 2021. The Author(s).)

It has been commonly accepted that soybean domestication originated in East Asia. Although East Asia has the historical merit in soybean production, the USA has become the top soybean producer in the world since 1950s. Following that, Brazil and Argentina have been the major soybean producers since 1970s and 1990s, respectively. China has once been the exporter of soybean to Japan before 1990s, yet she became a net soybean importer as Japan and the Republic of Korea do. Furthermore, the soybean yield per unit area in East Asia has stagnated during the past decade. To improve soybean production and enhance food security in these East Asian countries, much investment has been made, especially in the breeding of better performing soybean germplasms. As a result, China, Japan, and the Republic of Korea have become three important centers for soybean genomic research. With new technologies, the rate and precision of the identification of important genomic loci associated with desired traits from germplasm collections or mutants have increased significantly. Genome editing on soybean is also becoming more established. The year 2019 marked a new era for crop genome editing in the commercialization of the first genome-edited plant product, which is a high-oleic-acid soybean oil. In this review, we have summarized the latest developments in soybean breeding technologies and the remarkable progress in soybean breeding-related research in China, Japan, and the Republic of Korea.

Aluminium (Al) toxicity restrains water and nutrient uptake and is toxic to plant roots, ultimately inhibiting crop production. Here, we isolated and characterized a soybean glycine-rich protein-like gene (GmGRPL) that is mainly expressed in the root and that is regulated by Al treatment. Overexpression of GmGRPL can alleviate Al-induced root growth inhibition in Arabidopsis. The levels of IAA and ethylene in GmGRPL-overexpressing hairy roots were lower than those in control and RNA interference-exposed GmGRPL hairy roots with or without Al stress, which were mainly regulated by TAA1 and ACO, respectively. In transgenic soybean hairy roots, the MDA, H 2 O 2 and O 2 -· content in GmGRPL-overexpressing hairy roots were less than that in control and RNA interference-exposed GmGRPL hairy roots under Al stress. In addition, IAA and ACC can enhance the expression level of the GmGRPL promoter with or without Al stress. These results indicated that GmGRPL can alleviate Al-induced root growth inhibition by regulating the level of IAA and ethylene and improving antioxidant activity.