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Background: Soy isoflavones have been reported to exhibit anti-tumor effects. We hypothesize that genetic variants in soy isoflavone metabolism-related genes are associated with the risk of lung cancer., Methods: A two-stage case-control study design was conducted in this study. The discovery stage included 300 lung cancer cases and 600 healthy controls to evaluate the association of candidate genetic variants with lung cancer risk. The validation stage involved 1200 cases and 1200 controls to validate the associations found. Furthermore, qPCR was performed to assess the mRNA expression levels of different genotypes of the SNP. ELISA was used to explore the association between genotype and soy isoflavone levels, as well as the association between soy isoflavone levels and lung cancer risk., Results: A nonlinear association was observed between plasma soy isoflavone levels and lung cancer risk, with higher soy isoflavone levels associated with lower lung cancer risk (P < 0.001). The two-stage case-control study identified that UGT1A1 rs3755319 A > C was associated with decreased lung cancer risk (Recessive model: adjusted OR = 0.69, 95 %CI = 0.57-0.84, P < 0.001). Moreover, eQTL analysis showed that the expression level of UGT1A1 in the rs3755319 CC genotype was lower than in the AA + AC genotype (P < 0.05). The plasma concentration of soy isoflavones in the rs3755319 CC genotype was higher than in the AA + AC genotype (P = 0.008)., Conclusions: We identified a potentially functional SNP, UGT1A1 rs3755319 A > C, as being associated with decreased lung cancer risk. Further experiments will be needed to explore the mechanisms underlying the observed associations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Vegetative soybean (maodou or edamame) serves as a nutrient-rich food source with significant potential for mitigating global nutritional deficiencies. This study undertook a thorough examination of the nutritional profiles and transcriptomic landscapes of six soybean cultivars, including three common cultivars (Heinong551, Heinong562, and Heinong63) and three fresh maodou cultivars (Heinong527, HeinongXS4, and HeinongXS5). Nutrient analysis of the seeds disclosed notable differences in the levels of protein, fat, soluble sugars, vitamin E, calcium, potassium, magnesium, manganese, iron, and zinc across the cultivars. Through comparative transcriptome profiling and RNA sequencing, distinct variations in differentially expressed genes (DEGs) were identified between fresh and traditional maodou cultivars. Functional enrichment analyses underscored the involvement of DEGs in critical biological processes, such as nutrient biosynthesis, seed development, and stress responses. Additionally, association studies demonstrated robust correlations between specific DEG expression patterns and seed nutrient compositions across the different cultivars. Sankey diagrams illustrated that DEGs are strongly linked with seed quality traits, revealing potential molecular determinants that govern variations in nutritional content. The identified DEGs and their relationships with nutritional profiles offer valuable insights for breeding programs focused on developing cultivars with improved nutritional quality, tailored to specific dietary needs or industrial applications., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Soybean seed-protein content and composition is important because it contributes over half the value of this $61 billion crop. Historic negative correlation between seed-protein content and oil have been reported. Similarly, negative correlation between seed-protein content and yield have been reported but may be at least in part mitigated by increasing the genetic diversity of the elite soybean germplasm. Improvements in amino acid composition of seed-protein would increase the value of soybean meal and protein for animal and human consumption. We have identified a genetic resource in wild soybean germplasm, PI424025B, with elevated seed-protein, elevated cysteine in the seed and increased sulfur content in the seed. We have developed a population of Recombinant Inbred Lines derived from a cross between NC-Raleigh and PI424025B. We evaluated the carbon, nitrogen and sulfur (C, N and S) content of seeds from the progeny, the parents and other high-seed protein soybean lines. N content and C/N (ratio of C to N) were well correlated with protein content measured by NIR. PI424025B had a N content comparable to high-protein soybean lines and a superior N/S. These traits were inherited by some of the progeny. Quantitative Trait Loci (QTL) associated with high-seed protein were identified on chr2, chr20 and chr15 and colocalizes with well characterized loci on chr 15 and chr 20 known to affect seed-protein content and quality. A potentially unique QTL was identified on Chr15. Unlike the chr20 QTL, the chr15 QTL improved S content relative to N content and was superior to other high seed-protein phenotypes tested. These data indicate that PI424025B is a valuable resource to diversify the genetics of soybean while improving soybean seed-protein content and composition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Objectives: Wild soybean (Glycine soja), the ancestor of domesticated soybean, retains a higher level of genetic diversity and adaptability to harsh environments, making it highly valuable for breeding. Here, we re-sequenced 69 wild soybean individuals collected by the Shandong Academy of Agricultural Sciences and identified 1,613,162 high-quality SNPs which not only enriches our understanding of the genetic structure of wild soybean, but also provides valuable resources for further genomic research and genetic improvement of soybean., Data Description: In this study, we collected 69 wild soybean accessions from Shandong Province, China, and performed re-sequencing on the DNBSEQ platform, followed by SNPs identification. We then integrated ADMIXTURE, neighbor-joining tree, and principal component analysis to illustrate population characteristics. The results showed that these wild soybean accessions could be divided into three distinct subpopulations, exhibiting significant genetic differences., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Key Message: Two novel and one known genomic regions associated with R-gene resistance to Phytophthora sojae were identified by genome-wide association analysis and linkage analysis in soybean. Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is a severe disease that causes substantial economic losses in soybean [Glycine max (L.) Merr.]. The primary approach for successful disease management of PRR is using R-gene-mediated resistance. Based on the phenotypic evaluation of 376 cultivated soybean accessions for the R-gene type resistance to P. sojae (isolate 2457), a genome-wide association analysis identified two regions on chromosomes 3 and 8. The most significant genomic region (20.7-21.3 Mbp) on chromosome 8 was a novel resistance locus where no Rps gene was previously reported. Instead, multiple copies of the UDP-glycosyltransferase superfamily protein-coding gene, associated with disease resistance, were annotated in this new locus. Another genomic region on chromosome 3 was a well-known Rps cluster. Using the Daepung × Ilpumgeomjeong RIL population, a linkage analysis confirmed these two resistance loci and identified a resistance locus on chromosome 2. A unique feature of the resistance in Ilpumgeomjeong was discovered when phenotypic distribution was projected upon eight groups of RILs carrying different combinations of resistance alleles for the three loci. Interestingly, the seven groups carrying at least one resistance allele statistically differed from the other with none, regardless of the number of resistance alleles. This suggests that the respective three different resistance genes can confer resistance to P. sojae isolate 2457. Deployment of the three regions via marker-assisted selection will facilitate effectively improving resistance to particular P. sojae isolates in soybean breeding programs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: The QTLs and candidate genes governing the multifoliolate phenotype were identified by combining linkage mapping with BSR-seq, revealing a possible interplay between genetics and the environment in soybean leaf development. Soybean, as a legume, is typified by trifoliolate leaves. Although multifoliolate leaves (compound leaves with more than three leaflets each) have been reported in soybean, including sporadic appearances in the first compound leaves in a recombinant inbred line (RIL) population from a cross between cultivated soybean C08 and wild soybean W05 from this study, the genetic basis of this phenomenon is still unclear. Here, we integrated quantitative trait locus (QTL) mapping with bulked segregant RNA sequencing (BSR-seq) to identify the genetic loci associated with the multifoliolate phenotype in soybean. Using linkage mapping, ten QTLs related to the multifoliolate trait were identified. Among these, a significant and major QTL, qMF-2-1 on chromosome 2 and consistently detected across biological replicates, explained more than 10% of the phenotypic variation. Together with BSR-seq analyses, which analyzed the RILs with the highest multifoliolate frequencies and those with the lowest frequencies as two distinct bulks, two candidate genes were identified: Glyma.06G204300 encoding the transcription factor TCP5, and Glyma.06G204400 encoding LONGIFOLIA 2 (LNG2). Transcriptome analyses revealed that stress-responsive genes were significantly differentially expressed between high-multifoliolate occurrence lines and low occurrence ones, indicating environmental factors probably influence the appearance of multifoliolate leaves in soybean through stress-responsive genes. Hence, this study offers new insights into the genetic mechanism behind the multifoliolate phenotype in soybean., (© 2024. The Author(s).)

The enzyme serine hydroxymethyltransferase (SHMT) plays a key role in folate metabolism and is conserved in all kingdoms of life. SHMT is a pyridoxal 5'-phosphate (PLP) - dependent enzyme that catalyzes the conversion of L-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. Crystal structures of multiple members of the SHMT family have shown that the enzyme has a single conserved cis proline, which is located near the active site. Here, we have characterized a Pro to Ser amino acid variant (P285S) that affects this conserved cis proline in soybean SHMT8. P285S was identified as one of a set of mutations that affect the resistance of soybean to the agricultural pathogen soybean cyst nematode. We find that replacement of Pro285 by serine eliminates PLP-mediated catalytic activity of SHMT8, reduces folate binding, decreases enzyme stability, and affects the dimer-tetramer ratio of the enzyme in solution. Crystal structures at 1.9-2.2 Å resolution reveal a local reordering of the polypeptide chain that extends an α-helix and shifts a turn region into the active site. This results in a dramatically perturbed PLP-binding pose, where the ring of the cofactor is flipped by ∼180° with concomitant loss of conserved enzyme-PLP interactions. A nearby region of the polypeptide becomes disordered, evidenced by missing electron density for ∼10 residues. These structural perturbations are consistent with the loss of enzyme activity and folate binding and underscore the important role of the Pro285 cis-peptide in SHMT structure and function., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The precise control of flowering time is of utmost importance for crop adaptation to varying environmental conditions and consequently determines grain yield and plant fitness. Soybean E2 , the homolog of Arabidopsis GIGANTEA , is a major locus contributing to high-latitude adaptation and is involved in photoperiod sensitivity. However, due to major effects of E2 , additional genetic loci controlling soybean flowering and adaptation have historically been masked and difficult to identify. Here, by eliminating the effect of E2 , we identified a Tof9 locus controlling flowering in which ZEITLUPE 2 ( ZTL2 ) is the causal gene. ZTL2 encodes an F-box E3 ubiquitin ligase with homology to Arabidopsis ZEITLUPE and is shown to play a key role in the soybean photoperiodic flowering pathway. ZTL2 physically interacts with E2 to mediate its degradation. Intriguingly, ZTL2 and FKF1, both belong to the F-box-type E3 ubiquitin-ligase family, exhibit opposite roles in regulating soybean flowering. ZTL2 degrades E2, leading to early flowering, while FKF1 stabilizes E2, resulting in delayed flowering. The balance between ZTL2-mediated degradation and FKF1-mediated stabilization enables soybeans to finetune flowering time and maximize grain yield. Field-grown ztl2 mutants are taller, flower late, and have increased yield parameters. ZTL2 and FKF1b bear contrasting artificial-selection patterns to adapt to different latitudes. This antagonistic regulation is crucial for soybean adaptation to diverse ecological settings and allows plants to fine-tune their flowering time in response to photoperiod and latitudinal changes., Competing Interests: Competing interests statement:The authors declare no competing interest.

Understanding the regulatory mechanisms controlling storage lipid accumulation will inform strategies to enhance seed oil quality and quantity in crop plants. The WRINKLED1 transcription factor (WRI1 TF) is a central regulator of lipid biosynthesis. We characterized the genome-wide binding profile of soybean (Gm)WRI1 and show that the TF directly regulates genes encoding numerous enzymes and proteins in the fatty acid and triacylglycerol biosynthetic pathways. GmWRI1 binds primarily to regions downstream of target gene transcription start sites. We showed that GmWRI1-bound regions are enriched for the canonical WRI1 DNA binding element, the ACTIVATOR of Spomin::LUC1/WRI1 (AW) Box (CNTNGNNNNNNNCG), and another DNA motif, the CNC Box (CNCCNCC). Functional assays showed that both DNA elements mediate transcriptional activation by GmWRI1. We also show that GmWRI1 works in concert with other TFs to establish a regulatory state that promotes fatty acid and triacylglycerol biosynthesis. In particular, comparison of genes targeted directly by GmWRI1 and by GmLEC1, a central regulator of the maturation phase of seed development, reveals that the two TFs act in a positive feedback subcircuit to control fatty acid and triacylglycerol biosynthesis. Together, our results provide unique insights into the genetic circuitry in which GmWRI1 participates to regulate storage lipid accumulation during seed development., Competing Interests: Competing interests statement:The authors declare no competing interest.

Aluminum (Al) toxicity adversely impacts soybean (Glycine max) growth in acidic soil. Reversible protein phosphorylation plays an important role in adapting to adverse environmental conditions by regulating multiple physiological processes including signal transduction, energy coupling and metabolism adjustment in higher plant. This study aimed to reveal the Al-responsive phosphoproteins to understand their putative function and involvement in the regulation of Al resistance in soybean root. We used immobilized metal affinity chromatography to enrich the key phosphoproteins from soybean root apices at 0, 4, or 24 h Al exposure. These phosphoproteins were detected using liquid chromatography-tandem mass spectrometry measurement, verified by parallel reaction monitoring (PRM), and functionally characterized via overexpression in soybean hairy roots. A total of 638 and 686 phosphoproteins were identified as differentially enriched between the 4-h and 0-h, and the 24-h and 0-h Al treatment comparison groups, respectively. Typically, the phosphoproteins involved in biological processes including cell wall modification, and RNA and protein metabolic regulation displayed patterns of decreasing enrichment (clusters 3, 5 and 6), however, the phosphoproteins involved in the transport and metabolic processes of various substrates, and signal transduction pathways showed increased enrichment after 24 h of Al treatment. The enrichment of phosphoproteins in organelle organization bottomed after 4 h of Al treatment (cluster 1). Next, we selected 26 phosphoproteins from the phosphoproteomic profiles, assessed their enrichment status using PRM, and detected enrichment patterns similar to those observed via phosphoproteomic analysis. Among them, 15 phosphoproteins were found to reduce the accumulation of Al and callose in Al-stressed soybean root apices when their corresponding genes were individually overexpressed in soybean hairy roots. In summary, the findings of this study facilitated a comprehensive understanding of the protein phosphorylation events involved in Al resistance responses and revealed some critical phosphoproteins that enhance Al resistance in soybean roots., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The WRKY transcription factor gene family in soybean [Glycine max (L.) Merr.] (GmWRKY) is critical for the plant's development and stress responses. This study examines the evolutionary dynamics of the GmWRKY gene family, focusing on its synonymous codon usage bias (CUB) in a comprehensive set of 179 coding sequences. CUB was analyzed using various indices, revealing a preference for A/T-ending codons and relatively low codon bias. Codon adaptation index (CAI) analysis suggested that these genes are optimized for efficient translation despite relatively low bias, reflecting a balance between codon diversity and translation efficiency. Neutrality and NC plots indicated that selective forces dominate over mutational forces in shaping codon usage, while selection signature analysis showed purifying selection being prevalent across the gene family. However, episodic positive selection was also detected in certain clades, highlighting potential adaptive diversification in response to environmental stress. Additionally, promoter binding site analysis uncovered correlations between codon usage and transcriptional regulation, indicating a context-dependent relationship between CUB and gene expression. Phylogenetic analysis identified 11 well-supported clades in the modern GmWRKY gene family and ancestral sequence reconstruction revealed more relaxed codon preferences and reduced selection constraints in modern GmWRKY genes, potentially linked to neofunctionalization and adaptation to environmental changes. These findings provide a framework for optimizing gene expression in transgenic soybean crops with resilience. Further functional validation of positively selected genes is recommended to elucidate their role in stress responses., (© 2024. The Author(s).)

Main Conclusion: The redox homeostasis and photosynthetic pigments changes vary with Tetranychus ludeni infestation, with longer-cycle genotypes showing greater tolerance and efficiency in antioxidant defense. Infestations of Tetranychus ludeni Zacher (Tetranychidae) have been frequently observed in soybean plants. In this context, understanding the oscillation of redox homeostasis is crucial for detecting and assessing the stress levels caused in the plants by these organisms. The impacts of these infestations on redox metabolism and photosynthetic pigments are currently unknown. Therefore, we examined the hypothesis that T. ludeni infestations in soybean plants can influence redox homeostasis and photosynthetic pigments in a spatiotemporal manner, varying between different infestation times, modules and genotypes. For this purpose, soybean plants of the genotypes Monsoy, maturity group 5.7, and Brasmax, maturity group 6.3, grown in a controlled environment, were exposed to infestation and evaluated at two periods: 14 and 24 days. A variation in the distribution of T. ludeni within the infested plants over time increased the activity of ascorbate peroxidase and catalase, especially in Monsoy, reducing the content of hydrogen peroxide and superoxide, which prevented lipid peroxidation in the apical region in both genotypes. In the basal region, low chlorophyll indices corroborated by the yellow coloration of trifoliate leaves, high levels of membrane stability loss, and accumulation of hydrogen peroxide and superoxide characterized senescent trifoliate leaves in Brasmax, 24 days post infestation. Thus, the infestation of T. ludeni has a complex and significant impact on the redox metabolism of soybean plants, especially in shorter-cycle genotypes such as Brasmax. Furthermore, the oscillation of homeostasis can be considered as a good biochemical marker for selecting more suitable genotypes that are less sensitive and prone to infestations., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We aimed to investigate the regulatory function of the soybean transcription factor R2R3-MYB (GmMYB68) in isoflavone biosynthesis. Through comprehensive subcellular and chromosomal localization analyses, we found that GmMYB68 was predominantly localized to the nucleus and mapped to chromosome Gm04. Notably, SSR markers near this gene significantly correlated with seed isoflavone content. GmMYB68 overexpression markedly increased isoflavone contents, confirming its positive role in regulating isoflavone synthesis. GmMYB68 also played a crucial role in the response of soybean to abiotic stress. Using RNA-seq and yeast one-hybrid techniques, we discovered an intricate interaction between GmMYB68 and key isoflavone biosynthesis genes GmCHS7 and GmCHS8. These findings provide novel insights into the mechanisms underlying isoflavone biosynthesis. Furthermore, using yeast two-hybrid experiments, we identified proteins interacting with GmMYB68, suggesting roles in the synthesis of physiologically active compounds and abiotic stress response. We not only elucidated the regulatory mechanisms of GmMYB68 in isoflavone biosynthesis and abiotic stress response but also constructed a molecular network encompassing GmMYB68, GmCHS7, and GmCHS8. This network provides a theoretical basis for a better understanding of and strategies for improving soybean isoflavone biosynthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Rhizobia are common symbiotic microorganisms in the root system of leguminous plants that can usually provide nitrogen to the host through nitrogen fixation. Studies have shown that rhizobium-preinoculated soybean plants usually exhibit improved salt tolerance, but the underlying mechanism is not fully understood. In this paper, transcriptome sequencing (RNA-seq) revealed that preinoculation with rhizobia affected polyamine (PA) metabolism in soybean roots. The assay of PA contents showed that preinoculation with rhizobia significantly increased the putrescine (Put) content in roots and leaves during short-term salt treatment (0-5 d). Long-term salt treatment (5-7 d) resulted in a high Put content and significantly increased Spm and Spd contents, resulting in a rapid increase in the Put/(Spd + Spm) ratio (0-5 d) and subsequent decrease. Moreover, rhizobium preinoculation of soybean plants resulted in increased contents of conjugated and bound PAs under salt stress. Further transcriptome sequencing, PA contents, PA synthase expression and activity analysis revealed that GmADC may be a key gene related to salt tolerance in rhizobium-preinoculated soybean plants, and the GmADC-overexpressing soybean hairy-root composite plants exhibited less ROS damage, lower Cl - /NO 3 - ratios and Na + /K + ratios, and stabilized ion homeostasis. Taken together, preinoculation with rhizobia increased the expression level and enzyme activity of arginine decarboxylase (ADC) in soybean roots, increased the content of Put in roots and leaves, and increased the content of conjugated and bound PAs in soybean plants, thereby alleviating the oxidative and ionic injuries of soybean plants and enhancing the salt tolerance., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Masson SAS.)

Soybean is an important plant source of protein worldwide. Increasing demands for soybean can be met by improving the quality of its seed protein. In this study, GmCG-1, which encodes the β-conglycinin α' subunit, was identified via combined genome-wide association study and transcriptome analysis. We subsequently knocked down GmCG-1 and its paralogues GmCG-2 and GmCG-3 with CRISPR-Cas9 technology and generated two stable multigene knockdown mutants. As a result, the β-conglycinin content decreased, whereas the 11S/7S ratio, total protein content and sulfur-containing amino acid content significantly increased. Surprisingly, the globulin mutant exhibited salt tolerance in both the germination and seedling stages. Little is known about the relationship between seed protein composition and the salt stress response in soybean. Metabonomics and RNA-seq analysis indicated that compared with the WT, the mutant was formed through a pathway that was more similar to that of active salicylic acid biosynthesis; however, the synthesis of cytokinin exhibited greater defects, which could lead to increased expression of plant dehydrin-related salt tolerance proteins and cell membrane ion transporters. Population evolution analysis suggested that GmCG-1, GmCG-2, and GmCG-3 were selected during soybean domestication. The soybean accessions harboring GmCG-1 Hap1 presented relatively high 11S/7S ratios and relatively high salt tolerance. In conclusion, knockdown of the β-conglycinin α and α' subunits can improve the nutritional quality of soybean seeds and increase the salt tolerance of soybean plants, providing a strategy for designing soybean varieties with high nutritional value and high salt tolerance., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cultivar Williams 82 has served as the reference genome for the soybean research community since 2008, but is known to have areas of genomic heterogeneity among different sub-lines. This work provides an updated assembly (version Wm82.a6) derived from a specific sub-line known as Wm82-ISU-01 (seeds available under USDA accession PI 704477). The genome was assembled using Pacific BioSciences HiFi reads and integrated into chromosomes using HiC. The 20 soybean chromosomes assembled into a genome of 1.01Gb, consisting of 36 contigs. The genome annotation identified 48 387 gene models, named in accordance with previous assembly versions Wm82.a2 and Wm82.a4. Comparisons of Wm82.a6 with other near-gapless assemblies of Williams 82 reveal large regions of genomic heterogeneity, including regions of differential introgression from the cultivar Kingwa within approximately 30 Mb and 25 Mb segments on chromosomes 03 and 07, respectively. Additionally, our analysis revealed a previously unknown large (>20 Mb) heterogeneous region in the pericentromeric region of chromosome 12, where Wm82.a6 matches the 'Williams' haplotype while the other two near-gapless assemblies do not match the haplotype of either parent of Williams 82. In addition to the Wm82.a6 assembly, we also assembled the genome of 'Fiskeby III,' a rich resource for abiotic stress resistance genes. A genome comparison of Wm82.a6 with Fiskeby III revealed the nucleotide and structural polymorphisms between the two genomes within a QTL region for iron deficiency chlorosis resistance. The Wm82.a6 and Fiskeby III genomes described here will enhance comparative and functional genomics capacities and applications in the soybean community., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The transport, compartmentation and allocation of sugar are critical for plant growth and development, as well as for stress resistance, but sugar transporter genes have not been comprehensively characterized in soybean. Here, we performed a genome-wide identification and expression analyses of sugar transporter genes in soybean in order to reveal their putative functions. A total of 122 genes encoding sucrose transporters (SUTs) and monosaccharide transporters (MSTs) were identified in soybean. They were classified into 8 subfamilies according to their phylogenetic relationships and their conserved motifs. Comparative genomics analysis indicated that whole genome duplication/segmental duplication and tandem duplication contributed to the expansion of sugar transporter genes in soybean. Expression analysis by retrieving transcriptome datasets suggested that most of these sugar transporter genes were expressed in various tissues, and a number of genes exhibited tissue-specific expression patterns. Several genes including GmSTP21, GmSFP8, and GmPLT5/6/7/8/9 were predominantly expressed in nodules, and GmPLT8 was significantly induced by rhizobia inoculation in root hairs. Transcript profiling and qRT-PCR analyses suggested that half of these sugar transporter genes were significantly induced or repressed under stresses like salt, drought, and cold. In addition, GmSTP22 was found to be localized in the plasma membrane, and its overexpression promoted plant growth and salt tolerance in transgenic Arabidopsis under the supplement with glucose or sucrose. This study provides insights into the evolutionary expansion, expression pattern and functional divergence of sugar transporter gene family, and will enable further understanding of their biological functions in the regulation of growth, yield formation and stress resistance of soybean., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Houqing Zeng reports financial support was provided by Hangzhou Normal University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Phosphorus (P) is an essential macronutrient for plant growth and development. Rapid alkalisation factors (RALFs) play crucial roles in plant responses to nutrient stress. However, the functions of Glycine max RALFs (GmRALFs) under low P (LP) stress remain elusive. In this study, we first identified 27 GmRALFs in soybean and then revealed that, under LP conditions, GmRALF10, GmRALF11, and GmRALF22 were induced in both roots and leaves, whereas GmRALF5, GmRALF6, and GmRALF25 were upregulated in leaves. Furthermore, GmRALF22 was found to be the target gene of the transcription factor GmPHR1, which binds to the P1BS cis-element in the promoter of GmRALF22 via electrophoretic mobility shift assay and dual-luciferase experiments. Colonisation with Bacillus subtilis which delivers GmRALF22, increases the expression of the high-affinity phosphate (Pi) transporter genes GmPT2, GmPT11, GmPT13, and GmPT14, thus increasing the total amount of dry matter and soluble Pi in soybeans. RNA sequencing revealed that GmRALF22 alleviates LP stress by regulating the expression of jasmonic acid- (JA-), salicylic acid- (SA-), and immune-related genes. Finally, we verified that GmRALF22 was dependent on FERONIA (FER) to promote Arabidopsis primary root growth under LP conditions. In summary, the GmPHR1-GmRALF22 module positively regulates soybean tolerance to LP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As a non-collinear expression form of genetic information, chimeric RNAs increase the complexity of transcriptome in diverse organisms. Although chimeric RNAs have been identified in plants, few common features have been revealed. Here, we systemically explored the landscape of chimeric RNAs across multi-accession and multi-tissue using pan-genome and transcriptome data of four plants: rice, maize, soybean, and Arabidopsis. Among the four species, conserved characteristics of breakpoints and parental genes were discovered. In each species, chimeric RNAs displayed a high level of diversity among accessions, and the clustering of accessions using chimeric events was generally concordant with clustering based on genomic variants, implying a general relationship between genetic variations and chimeric RNAs. Through mass spectrometry, we confirmed a fusion protein OsNDC1-OsGID1L2 and observed its subcellular localization, which differed from the original proteins. Phenotypic cues in transgenic rice suggest the potential functions of OsNDC1-OsGID1L2. Moreover, an intriguing chimeric event Os01g0216500-Os01g0216900, generated by a large deletion in basmati rice, also exists in another accession without the deletion, demonstrating its convergence in evolution. Our results illuminate the characteristics and hint at the evolutionary implications of plant chimeric RNAs, which serve as a supplement to genetic variations, thus expanding our understanding of genetic diversity., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Leaves are important sites for photosynthesis and can convert inorganic substances into organic matter. Photosynthetic performance is an important factor affecting crop yield. Leaf colour is closely related to photosynthesis, and leaf colour mutants are considered an ideal material for studying photosynthesis., Results: We obtained a yellow-green leaf mutant jym165, using ethyl methane sulfonate (EMS) mutagenesis. Physiological and biochemical analyses indicated that the contents of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll in the jym165 mutant decreased significantly compared with those in Jiyu47 (JY47). The abnormal chloroplast development of jym165 led to a decrease in net photosynthetic rate and starch content compared with that of JY47. However, quality traits analysis showed that the sum of oil and protein contents in jym165 was higher than that in JY47. In addition, the regional yield (seed spacing: 5 cm) of jym165 increased by 2.42% compared with that of JY47 under high planting density. Comparative transcriptome analysis showed that the yellow-green leaf phenotype was closely related to photosynthesis and starch and sugar metabolism pathways. Genetic analysis suggests that the yellow-green leaf phenotype is controlled by a single recessive nuclear gene. Using Mutmap sequencing, the candidate regions related of leaf colour was narrowed to 3.44 Mb on Chr 10., Conclusions: Abnormal chloroplast development in yellow-green mutants leads to a decrease in the photosynthetic pigment content and net photosynthetic rate, which affects the soybean photosynthesis pathway and starch and sugar metabolism pathways. Moreover, it has the potentiality to increase soybean yield under dense planting conditions. This study provides a useful reference for studying the molecular mechanisms underlying photosynthesis in soybean., (© 2024. The Author(s).)

Genes involved in iron deficiency responses have been well characterized in Arabidopsis thaliana , but their roles in crop species have not been well explored. Reliance on model species may fail to identify novel iron stress mechanisms present within crop species, likely selected by hundreds of years of selection. Fiskeby III (PI 438471) is a soybean line from Sweden that demonstrates high levels of resilience to numerous stresses. Earlier Fiskeby III studies have identified a suite of genes responding to iron deficiency stress in Fiskeby III that are also associated with Arabidopsis iron deficiency responses. We were interested in determining how canonical iron genes function in Fiskeby III under normal and iron stress conditions. To investigate this, we used virus-induced gene silencing to knock down gene expression of three iron deficiency response genes (FER-like iron deficiency induced transcription factor (FIT), elongated hypocotyl 5 (HY5) and popeye (PYE)) in Fiskeby III. Analyses of RNAseq data generated from silenced plants in iron-sufficient and -deficient conditions found silencing FIT and HY5 altered general stress responses but did not impact iron deficiency tolerance, confirming Fiskeby III utilizes novel mechanisms to tolerate iron deficiency stress.

Inositol polyphosphate-5-phosphatase (5PTase) is a key enzyme in the inositol signaling pathway. It hydrolyzes the 5-phosphate on the inositol ring of inositol phosphate (IP) or phosphatidylinositol phosphate (PIP). However, there is limited reports on the homologous genes in soybean. This study cloned the salt tolerant gene Gs5PTase8 from wild soybean ( Glycine soja S. & Z.) and explored its substrate. Gs5PTase8 encodes 493 amino acid residues. The sequence alignment and phylogenetic tree showed that this gene was conserved in plants. RT-qPCR was employed to determine the expression of Gs5PTase8 in different tissues of soybean and the results showed that Gs5PTase8 was mainly expressed in soybean roots. To investigate the hydrolytic substrates, we constructed pET28a- Gs5PTase8 and pGEX4T1- Gs5PTase8 for the Escherichia coli expression system and only obtained the recombinant protein GST-Gs5PTase8. The induction conditions for the protein expression including the isopropyl beta-d-thiogalactopyranoside (IPTG) concentration and temperature (16 ℃, 30 ℃, and 37 ℃) were optimized. The expression level was highest when the expression was induced overnight with 0.2 mmol/L IPTG at 16 ℃. The SDS-PAGE results showed that the recombinant protein had a relative molecular weight of 75 kDa and presented a single band after purification, with the purity reaching over 95%. The yield of the recombinant protein determined by the BCA method was 4.9 mg/L LB. The hydrolytic substrates of this enzyme in vitro included IP 3 [inositol(1, 4, 5)trisphosphate], IP 4 [inositol(1, 3, 4, 5)tetrakisphosphate], PI(4, 5)P 2 [phosphatidylinositol(4, 5) bisphosphate] and PI(3, 4, 5)P 3 [phosphatidylinositol(3, 4, 5)trisphosphate]. This study provides a scientific basis for further research on the molecular mechanism of Gs5PTase8 involved in salt tolerance.

The WRKY transcription factor gene family is a plant-specific transcription factor that plays important roles defense responses. Studies in model plant Arabidopsis demonstrated that WRKYs function downstream of mitogen activated-protein kinase (MAPK) signaling cascade and participate in defense responses through activating the expression of defense-related genes. However, the roles of WRKYs in defense responses have not been previously investigated in paleopolyploidy soybean. Bioinfomatic analysis revealed that there are three pair of GmWRKY33 genes in the soybean genome. The identity of first two pair of GmWRKY33 genes is greater than 84% (named as GmWRKY33A ). The identity of genes within the same pair is greater than 95%. A 300 bp fragment highly homologous to these four GmWRKY33A was chosen to clone into bean pod mosaic virus (BPMV)-based silencing vector (BPMV-VIGS) to achieve the goal of silencing four GmWRKY33A genes simultaneously. In this study, we simultaneously silenced four homologous genes of GmWRKY33A using a bean pod mottle virus (BPMV) vector carrying a single fragment of GmWRKY33A . Comparing the silenced plants with the vector control plants, no evident morphological phenotypes were observed. However, the GmWRKY33A- silenced plants exhibited significantly reduced resistance to Pseudomonas syringae pv. glycinea ( Psg ), Xanthomonas axonopodis pv. glycine ( Xag ), as well as to soybean mosaic virus (SMV). Furthermore, we demonstrated that silencing these GmWRKY33A genes significantly inhibited the activation of Gm MPK3/ Gm MPK6 induced by Psg infection. Collectively, our results suggest that Gm WRKY33As are involved in soybean immunity through regulating the transcription of GmMPK3/6 genes or activating the kinase activities of Gm MPK3/6. Taken together, our results demonstrated that Gm WRKY33As are positive regulators of soybean immune responses.

Roots play a crucial role in plant development, serving to absorb water and nutrients from the soil while also providing structural stability. However, the impacts of global warming can impede root growth by altering soil conditions that hinder overall plant growth. To address this challenge, there is a need to screen and identify plant genotypes with superior Root System Architecture traits (RSA), that can be used for future breeding efforts in enhancing their resilience to these environmental changes. In this project, 500 mid to late-maturity soybean accessions were grown on blue blotting papers hydroponically with six replicates and assessed seven RSA traits. Genome-Wide Association Studies (GWAS) were carried out with root phenotypic data and SNP data from the SoySNP50K iSelect SNP BeadChip, using both the TASSEL 5.0 and FarmCPU techniques. A total of 26 significant SNP-trait correlations were discovered, with 11 SNPs on chromosome 13. After SNP selection, we identified 14 candidate genes within 100-kb regions flanking the SNPs, which are related to root architecture. Notably, Glyma.17G258700, which exhibited substantial differential expression in root tips and its Arabidopsis homolog, AT4G24190 (GRP94) is involved in the regulation of meristem size and organization. Other candidate genes includes Glyma.03G023000 and Glyma.13G273500 that are also play a key role in lateral root initiation and root meristem growth, respectively. These findings significantly contribute to the discovery of key genes associated with root system architecture, facilitating the breeding of resilient cultivars adaptable to changing climates., (© 2024. The Author(s).)

Key Message: The soybean Rxp gene, encoding a bHLH transcription factor and an ACT-like domain, has an rxp allele producing a truncated protein that confers resistance to pustule-causing Xanthomonas axonopodis pv. glycines. In soybean, bacterial pustules caused by Xanthomonas axonopodis pv. glycines lead to premature defoliation and decreased yield in warm, wet climates. In the USA, approximately 70 years ago, bacterial pustules were eliminated by introducing a recessive resistance allele, rxp, of the Rxp gene, representing the first example of successful soybean breeding for durable disease resistance in North America. In this study, we isolated this historical Rxp gene from resistant soybean varieties using positional cloning. The 1.06 Mb region where Rxp was reported to reside was narrowed down to an 11.1 kb region containing a single gene, Glyma.17g090500. The resistance allele, rxp, contains a T insertion. A complementation test of the Rxp allele in resistant plants confirmed the identification of the Rxp gene. The product of the susceptible wild-type allele, Rxp, is presumed to be a basic helix-loop-helix (bHLH) transcription factor with an aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain. This gene was mainly expressed in extended leaves, and its homologs were identified to be distributed in angiosperms. A total of six alleles were obtained: four from spontaneous variation, including the wild-type and three mutant alleles that encoded truncated proteins, and two from ethyl methanesulfonate mutants, including an allele that encoded a truncated protein and a missense allele. By evaluating the resistance of these six alleles, we found that the loss of function of RXP decreased the bacterial pustule lesions. This study provides important insights into the soybean rxp allele, which confers durable resistance to bacterial pustules., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Soybean cyst nematode disease represents the major soil-borne disease of soybean. Identifying disease-resistant genes in soybean has a substantial impact on breeding of disease-resistant crops and genetic improvement. The present work created the F 2 population with the disease-resistant line H-10 and disease-susceptible line Chidou4. 30 respective F 2 disease-resistant and disease-susceptible individuals for forming two DNA pools for whole-genome re-sequencing were selected. As a result, a total of 11,522,230 single nucleotide polymorphism (SNPs) markers from these two parental lines and two mixed pools were obtained. Accordng to SNP-index based association analysis, there were altogether 741 genes out of 99% confidence interval, which were mainly enriched into regions of 38,524,128∼39,849,988 bp with a total length of 1.33 Mb contain 111 genes on chromosome 2, 27,821,012∼29,612,574 bp with a total length of 1.79 Mb contain 92 genes on chromosome 3, 308∼348,214 bp with a total of length 0.35 Mb contain 34 genes on chromosome 10, and 53,867,581∼58,017, 852 bp with a total length of 4.15 Mb contain 504 genes on chromosome 18. Bulk segregant analysis in F 2 generations (BSA-seq) was correlated with a disease resistance interval containing 15 genes. Then, using bioinformatics analysis and differential expression analysis, five candidate genes were identified: Glyma.02G211400 , Glyma.18G252800 , Glyma.18G285800, Glyma.18G287400 and Glyma.18G298200 . Our results provides a key basis for analyzing the soybean resistance mechanism against soybean cyst nematode and cloning soybean resistance genes., Competing Interests: The authors declare there are no competing interests., (©2024 Hu et al.)

Sulfur is a critical element for plant growth and development, serving as a component of amino acids (cysteine and methionine), iron-sulfur clusters, proteins, glutathione, coenzymes, and auxin precursors. Deficiency or low concentrations of sulfur in the soil can lead to significant growth retardation in plants. The objective of our study was to examine the effects of sulfur (S) deficiency and excess on morphological symptoms, sulfur and nitrogen (N) metabolism, as well as antioxidant activity in soybean. We found that S starvation decreased the fine root length, biomass, and activity, and the chlorophyll content was reduced, while excess sulfur promotes lateral root growth. In contrast to sulfur excess, sulfur deficiency inhibits N and S metabolism levels in both subsurface and above-ground parts, and induced the expression of some sulfur transporters (SULTRs). In this study, we created soybean hairy root lines overexpressing the SULTR gene ( GmSULTR2;1a ) to observe metabolic changes following sulfur deficiency treatment. The results showed that GmSULTR2;1a saved the sulfur-deficient phenotype, and the antioxidant enzyme activity was much higher than that of the wildtype in the absence of sulfur. Our study revealed the important role of sulfur element in soybean growth and development and the regulation of sulfur deficiency by GmSULTR2;1a .

The major crop, soybean, forms root nodules with symbiotic rhizobia, providing energy and carbon to the bacteria in exchange for bioavailable nitrogen. The relationship is host-specific and highly host-regulated to maximize energy efficiency. Symbiotic nitrogen fixation (SNF) is greener than synthetic fertilizer for replenishing soil fertility, contributing to yield increase. Nodulation Outer Protein P (NopP) and NopI of the type 3 secretion system (T3SS) of the rhizobium determine host specificity. Sinorhizobium fredii CCBAU25509 (R2) and CCBAU45436 (R4) have different NopP and NopI variants, affecting their respective symbiotic compatibilities with the cultivated soybean C08 and the wild soybean W05. Swapping the NopP variants between R2 and R4 has been shown to switch their compatibility with C08 with the rj2/Rfg1 genotype. To understand the effects of Nops on host compatibility, analyses on the transcriptomic data of W05 roots and nodules inoculated with S. fredii strains containing Nop variants uncovered many differentially expressed genes related to nodulation and nodule functions, providing important information on the effects of Nops on hosts and nodules., (© 2024. The Author(s).)

Background: Drought stress is a significant abiotic stressor that hinders growth, development, and crop yield in soybeans. Strigolactones (SLs) positively regulate plant resistance to drought stress. However, the impact of foliar application of SLs having different concentrations on soybean growth and metabolic pathways related to osmoregulation remains unknown. Therefore, to clarify the impact of SLs on soybean root growth and cellular osmoregulation under drought stress, we initially identified optimal concentrations and assessed key leaf and root indices. Furthermore, we conducted transcriptomic and metabolic analyses to identify differential metabolites and up-regulated genes., Results: The results demonstrated that drought stress had a significant impact on soybean biomass, root length, root surface area, water content and photosynthetic parameters. However, when SLs were applied through foliar application at appropriate concentrations, the accumulation of ABA and soluble protein increased, which enhanced drought tolerance of soybean seedlings by regulating osmotic balance, protecting membrane integrity, photosynthesis and activating ROS scavenging system. This also led to an increase in soybean root length, lateral root number and root surface area. Furthermore, the effects of different concentrations of SLs on soybean leaves and roots were found to be time-sensitive. However, the application of 0.5 µM SLs had the greatest beneficial impact on soybean growth and root morphogenesis under drought stress. A total of 368 differential metabolites were screened in drought and drought plus SLs treatments. The up-regulated genes were mainly involved in nitrogen compound utilization, and the down-regulated metabolic pathways were mainly involved in maintaining cellular osmoregulation and antioxidant defenses., Conclusions: SLs enhance osmoregulation in soybean plants under drought stress by regulating key metabolic pathways including Arachidonic acid metabolism, Glycerophospholipid metabolism, Linoleic acid metabolism, and Flavone and flavonol biosynthesis. This study contributes to the theoretical understanding of improving soybean adaptability and survival in response to drought stress., (© 2024. The Author(s).)

Key Message: Desiccation-stage transcription factors perform similar functions, with early ones focused on desiccation tolerance and later ones on development. Gene networks governing late embryo development diverge between soybean and Arabidopsis. To understand gene activities programming seed embryo development, we profiled the soybean embryo transcriptome across embryonic morphogenesis through post-germination. Transcriptomic landscapes across embryo development feature highly prevalent transcripts, categorized into early and late groups, with shared and distinct functions. During the mid-storage reserve accumulation stage, the upregulated genes are enriched with regulatory tasks at both the transcriptional and chromatin levels, including DNA methylation and chromatin remodeling. The epigenetic-related functions also dominate in the upregulated genes during germination, involving core histone variants and histone chaperones. Gene network analysis reveals both stage-specific modules and modules active across multiple stages. The desiccation-associated gene module integrates diverse transcription factors (TFs) that are sequentially active during different desiccation stages, transitioning from abiotic stress functions early on to developmental functions later. Two TFs, active during the early and mid-desiccation stages were functionally assessed in Arabidopsis overexpression lines to uncover their potential roles in desiccation processes. Interestingly, nearly half of the Arabidopsis orthologs of soybean TFs active in the desiccation-associated module are inactive during Arabidopsis desiccation. Our results reveal that chromatin and transcriptional regulation coordinate during mid-storage reserve accumulation, while distinct epigenetic mechanisms drive germination. Additionally, gene modules either perform stage-specific functions or are required across multiple stages, and gene networks during late embryogenesis diverge between soybean and Arabidopsis. Our studies provide new information on the biological processes and gene networks underlying development from embryonic morphogenesis to post-germination., (© 2024. The Author(s).)

Serine/arginine-rich (SR) proteins mostly function as splicing factors for pre-mRNA splicing in spliceosomes and play critical roles in plant development and adaptation to environments. However, detailed study about SR proteins in legume plants is still lacking. In this report, we performed a genome-wide investigation of SR protein genes in wild soybean ( Glycine soja ) and identified a total of 31 GsSR genes from the wild soybean genome. The analyses of chromosome location and synteny show that the GsSR s are unevenly distributed on 15 chromosomes and are mainly under the purifying selection. The GsSR proteins can be phylogenetically classified into six sub-families and are conserved in evolution. Prediction of protein phosphorylation sites indicates that GsSR proteins are highly phosphorylated proteins. The protein-protein interaction network implies that there exist numerous interactions between GsSR proteins. We experimentally confirmed their physical interactions with the representative SR proteins of spliceosome-associated components such as U1-70K or U2AF35 by yeast two-hybrid assays. In addition, we identified various stress-/hormone-responsive cis -acting elements in the promoter regions of these GsSR genes and verified their expression patterns by RT-qPCR analyses. The results show most GsSR genes are highly expressed in root and stem tissues and are responsive to salt and alkali stresses. Splicing analysis showed that the splicing patterns of GsSR s were in a tissue- and stress-dependent manner. Overall, these results will help us to further investigate the biological functions of leguminous plant SR proteins and shed new light on uncovering the regulatory mechanisms of plant SR proteins in growth, development, and stress responses.

Symbiotic nitrogen fixation (SNF) in legume-rhizobia serves as a sustainable source of nitrogen (N) in agriculture. However, the addition of inorganic N fertilizers significantly inhibits SNF, and the underlying mechanisms remain not-well understood. Here, we report that inorganic N disrupts iron (Fe) homeostasis in soybean nodules, leading to a decrease in SNF efficiency. This disruption is attributed to the inhibition of the Fe transporter genes Natural Resistance-Associated Macrophage Protein 2a and 2b (GmNRAMP2a&2b) by inorganic N. GmNRAMP2a&2b are predominantly localized at the tonoplast of uninfected nodule tissues, affecting Fe transfer to infected cells and consequently, modulating SNF efficiency. In addition, we identified a pair of N-signal regulators, nitrogen-regulated GARP-type transcription factors 1a and 1b (GmNIGT1a&1b), that negatively regulate the expression of GmNRAMP2a&2b, which establishes a link between N signaling and Fe homeostasis in nodules. Our findings reveal a plausible mechanism by which soybean adjusts SNF efficiency through Fe allocation in response to fluctuating inorganic N conditions, offering valuable insights for optimizing N and Fe management in legume-based agricultural systems., (© 2024. The Author(s).)

Spatially resolved transcriptomics (SRT) combines gene expression profiles with the physical locations of cells in their native states but suffers from unpredictable spatial noise due to cell damage during cryosectioning and exposure to reagents for staining and mRNA release. To address this noise, we developed SpotGF, an algorithm for denoising SRT data using optimal transport-based gene filtering. SpotGF quantifies diffusion patterns numerically, distinguishing widespread expression genes from aggregated expression genes and filtering out the former as noise. Unlike conventional denoising methods, SpotGF preserves raw sequencing data, thereby avoiding false positives that can arise from imputation. Additionally, SpotGF demonstrates superior performance in cell clustering, identifying potential marker genes, and annotating cell types. Overall, SpotGF has the potential to become a crucial preprocessing step in the downstream analysis of SRT data. The SpotGF software is freely available at GitHub. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A genome-wide association study (GWAS) identifies trait-associated loci, but identifying the causal genes can be a bottleneck, due in part to slow decay of linkage disequilibrium (LD). A transcriptome-wide association study (TWAS) addresses this issue by identifying gene expression-phenotype associations or integrating gene expression quantitative trait loci with GWAS results. Here, we used self-pollinated soybean (Glycine max [L.] Merr.) as a model to evaluate the application of TWAS to the genetic dissection of traits in plant species with slow LD decay. We generated RNA sequencing data for a soybean diversity panel and identified the genetic expression regulation of 29 286 soybean genes. Different TWAS solutions were less affected by LD and were robust to the source of expression, identifing known genes related to traits from different tissues and developmental stages. The novel pod-color gene L2 was identified via TWAS and functionally validated by genome editing. By introducing a new exon proportion feature, we significantly improved the detection of expression variations that resulted from structural variations and alternative splicing. As a result, the genes identified through our TWAS approach exhibited a diverse range of causal variations, including SNPs, insertions or deletions, gene fusion, copy number variations, and alternative splicing. Using this approach, we identified genes associated with flowering time, including both previously known genes and novel genes that had not previously been linked to this trait, providing insights complementary to those from GWAS. In summary, this study supports the application of TWAS for candidate gene identification in species with low rates of LD decay., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Egyptian-farmed soybeans have a wide range of genetic diversity which is most important in plant improvement programs in order to develop new higher yielding soybean genotypes. The present study is designed to determine the genetic variability among seventeen genotypes of cultivated soybean ( Glycine max L.) by examining the phenotypic level at the seedling stage, field performance over two years 2022/2023 and genetically using Start Codon Targeted (SCoT) markers. Results indicated that the SCoT markers, 100 seed weight, and tip angle (TA) traits were positively correlated with H2L12, DR 101, H15L5, and H117 genotypes. In addition, the number of branches per plant and plant height were associated with H113, H32, Crowford, H129, and D7512035. Furthermore, the length of the first internode (LFI), root width (RW), root length (RL), and shoot length (SL) were more associated with Giza 111, NC105, and Hutcheson. The hierarchical cluster analysis (HCA) and its associated heatmap explored the differences among the genotypes. It showed that all examined parameters were clustered into four distinct clusters. The obtained results showed that genotypes NC105, H30, D75&#95;12035, and H2L12 have promising phenological and morphological traits besides tracking the inheritance of nearby genes surrounding the ATG translation start codon since they are in a monoclades. The obtained results will help the breeder plan appropriate selection strategies for improving seed yield in soybeans through hybridization from divergent clusters., Competing Interests: The authors declare there are no competing interests., (©2024 Abdel-Sattar et al.)

Phytophthora sansomeana is an emerging oomycete pathogen causing root rot in many agricultural species including soybean. However, as of now, only one potential resistance gene has been identified in soybean, and our understanding of how genetic and epigenetic regulation in soybean contributes to responses against this pathogen remains largely unknown. In this study, we performed whole genome bisulfite sequencing (WGBS) on two soybean lines, Colfax (resistant) and Williams 82 (susceptible), in response to P. sansomeana at two time points: 4 and 16 hours post-inoculation to compare their methylation changes. Our findings revealed that there were no significant changes in genome-wide CG, CHG (H = A, T, or C), and CHH methylation. However, we observed local methylation changes, specially an increase in CHH methylation around genes and transposable elements (TEs) after inoculation, which occurred earlier in the susceptible line and later in the resistant line. After inoculation, we identified differentially methylated regions (DMRs) in both Colfax and Williams 82, with a predominant presence in TEs. Notably, our data also indicated that more TEs exhibited changes in their methylomes in the susceptible line compared to the resistant line. Furthermore, we discovered 837 DMRs within or flanking 772 differentially expressed genes (DEGs) in Colfax and 166 DMRs within or flanking 138 DEGs in Williams 82. These DEGs had diverse functions, with Colfax primarily showing involvement in metabolic process, defense response, plant and pathogen interaction, anion and nucleotide binding, and catalytic activity, while Williams 82 exhibited a significant association with photosynthesis. These findings suggest distinct molecular responses to P. sansomeana infection in the resistant and susceptible soybean lines., Competing Interests: Conflicts of interest The authors declare no conflicts of interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Background: Vegetable soybean is rich in nutrients and has a unique flavor. It is highly preferred by people because of its pharmacological activities, including those that regulate the intestines and lower blood pressure. The pod color of vegetable soybeans is an important quality that indicates their freshness and has a significant impact on their commercialization., Results: In this study, pod color was evaluated in 301 vegetable soybean accessions collected from various regions. Genome-wide association analysis was carried out using the Mixed linear model (MLM), a total of 18 quantitative trait loci including 117 SNPs were detected. Two significant QTLs located on chromosomes 6 (qGPCL4 /qGPCa1/qGPCb2) and 18 (qGPCL10/qGPCb3) were consistently detected across different variables. Based on gene functional annotation, 30 candidate genes were identified in these two candidate intervals. Combined with transcriptome analysis, Glyma.18g241700 has been identified as a candidate gene for regulating pod color in vegetable soybeans. Glyma.18g241700 encodes a chlorophyll photosystem I subunit XI. which localizes to the chloroplast named GmPsaL, qRT-PCR analysis showed that GmPsaL was specifically highly expressed in developing pods. Furthermore, overexpression of GmPsaL in transgenetic Arabidopsis plants produced dark green pods., Conclusions: These findings may be useful for clarifying the genetic basis of the pod color of vegetable soybeans. The identified candidate genes may be useful for the genetic improvement of the appearance quality of vegetable soybeans., (© 2024. The Author(s).)

New selection methods, using trait-specific markers (marker-assisted selection (MAS)) and/or genome-wide markers (genomic selection (GS)), are becoming increasingly widespread in breeding programs. This new era requires innovative and cost-efficient solutions for genotyping. Reduction in sequencing cost has enhanced the use of high-throughput low-cost genotyping methods such as genotyping-by-sequencing (GBS) for genome-wide single-nucleotide polymorphism (SNP) profiling in large breeding populations. However, the major weakness of GBS methodologies is their inability to genotype targeted markers. Conversely, targeted methods, such as amplicon sequencing (AmpSeq), often face cost constraints, hindering genome-wide genotyping across a large cohort. Although GBS and AmpSeq data can be generated from the same sample, an efficient method to achieve this is lacking. In this study, we present the Genome-wide & Targeted Amplicon (GTA) genotyping platform, an innovative way to integrate multiplex targeted amplicons into the GBS library preparation to provide an all-in-one cost-effective genotyping solution to breeders and research communities. Custom primers were designed to target 23 and 36 high-value markers associated with key agronomical traits in soybean and barley, respectively. The resulting multiplex amplicons were compatible with the GBS library preparation enabling both GBS and targeted genotyping data to be produced efficiently and cost-effectively. To facilitate data analysis, we have introduced Fast-GBS.v3, a user-friendly bioinformatic pipeline that generates comprehensive outputs from data obtained following sequencing of GTA libraries. This high-throughput low-cost approach will greatly facilitate the application of DNA markers as it provides required markers for both MAS and GS in a single assay., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

High-affinity potassium transporters (HKTs) play an important role in plants responding to salt stress, but the transport properties of the soybean HKT transporters at the molecular level are still unclear. Here, using Xenopus oocyte as a heterologous expression system and two-electrode voltage-clamp technique, we identified four HKT transporters, GmHKT1;1, GmHKT1;2, GmHKT1;3 and GmHKT1;4, all of which belong to type I subfamily, but have distinct ion transport properties. While GmHKT1;1, GmHKT1;2 and GmHKT1;3 function as Na+ transporters, GmHKT1;1 is less selective against K+ than the two other transporters. Astonishingly, GmHKT1;4, which lacks transmembrane segments and has no ion permeability, is significantly expressed, and its gene expression pattern is different from the other three GmHKTs under salt stress. Interestingly, GmHKT1;4 reduced the Na+/K+ currents mediated by GmHKT1;1. Further study showed that the transport ability of GmHKT1;1 regulated by GmHKT1;4 was related to the structural differences in the first intracellular domain and the fourth repeat domain. Overall, we have identified one unique GmHKT member, GmHKT1;4, which modulates the Na+ and K+ transport ability of GmHKT1;1 via direct interaction. Thus, we have revealed a new type of HKT interaction model for altering their ion transport properties., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Soybean cyst nematode (SCN, Heterodera glycines ) is a sedentary endoparasite nematode that results in severe economic losses in soybean crops. miRNAs play crucial roles in plant responses to nematode. However, the role of miR2119 responding to SCN stress in soybean. Here, we demonstrated that the transcript levels of polycistronic precursors containing miR2119 and miR398a were significantly reduced in soybean upon nematode infection. Promoter of the miR2119-398a precursor analysis was conducted containing a GUS reporter gene. GUS activity assays demonstrated a decrease in miR2119-398a promoter during SCN infection. Overexpression of polycistronic precursor miR2119-398a (OE-premiR2119-398a) and miR2119 precursor (OE-premiR2119) rendered soybean more susceptible to SCN. Conversely, silencing miR2119 (STTM2119) increased soybean resistance against SCN. Furthermore, RNA-seq analysis revealed that miR2119 is involved in many defense signaling pathways. GUS reporter gene assays demonstrated that miR2119 targets GmADH1.1a and GmADH1.1b . Functional analysis indicated that ADHs act as a major role in responding to H. glycines by modulating reactive oxygen species (ROS) levels. Together, the findings reveal a novel mechanism by which the polycistronic precursor miR2119-398a coordinately regulates in response to H. glycines . Additionally, miR2119 becomes an essential element contributing to H. glycines by modulating ADH activity and ROS homeostasis in soybean.

This study reports the use of the Arabidopsis KASII promoter ( AtKASII ) to develop an efficient CRISPR/Cas9 system for soybean genome editing. When this promoter was paired with Arabidopsis U6 promoters to drive Cas9 and single guide RNA expression, respectively, simultaneous editing of the three fatty acid desaturase genes GmFAD2-1A , GmFAD2-1B , and GmFAD3A occurred in more than 60% of transgenic soybean lines at T 2 generation, and all the triple mutants possessed desirable high-oleic traits. In sharp contrast, not a single line underwent simultaneous editing of the three target genes when AtKASII was replaced by the widely used AtEC1.2 promoter. Furthermore, our study showed that the stable and inheritable mutations in the high-oleic lines did not alter the overall contents of oil and protein or amino acid composition while increasing the oleic acid content up to 87.6% from approximately 23.8% for wild-type seeds, concomitant with 34.4- and 3.7-fold reductions in linoleic and linolenic acid, respectively. Collectively, this study demonstrates that the AtKASII promoter is highly promising for optimization of the CRISPR/Cas9 system for genome editing in soybean and possibly beyond.

Wild soybean Glycine soja is the progenitor of cultivated soybean Glycine max Information on soybean functional centromeres is limited despite extensive genome analysis. These species are an ideal model for studying centromere dynamics for domestication and breeding. We performed a detailed chromatin immunoprecipitation analysis using centromere-specific histone H3 protein to delineate two distinct centromeric DNA sequences with unusual repeating units with monomer sizes of 90-92 bp (CentGm-1) and 413-bp (CentGm-4) shorter and longer than standard nucleosomes. These two unrelated DNA sequences with no sequence similarity are part of functional centromeres in both species. Our results provide a comparison of centromere properties between a cultivated and a wild species under the effect of the same kinetochore protein. Possible sequence homogenization specific to each chromosome could highlight the mechanism for evolutionary conservation of centromeric properties independent of domestication and breeding. Moreover, a unique barcode system to track each chromosome is developed using CentGm-4 units. Our results with a unifying centromere composition model using CentGm-1 and CentGm-4 superfamilies could have far-reaching implications for comparative and evolutionary genome research., (© 2024 Tek et al.)

Soybean provides protein, oil and multiple health-related compounds. Understanding the effects of structural variations (SVs) on economic traits in modern breeding is important for soybean improvement. Here we assembled the high-quality genome of modern cultivar Nongdadou2 (NDD2) and identified 25,814 SV-gene pairs compared to 29 reported genomes, with 13 NDD2-private SVs validated in 547 deep-resequencing (average = 18.05-fold) accessions, which advances our understanding of genomic variation biology. We found some insertions/deletions involved in seed protein and weight formation, an inversion related to adaptation to drought and a large intertranslocation implicated in a key divergence event in soybean. Of 749,714 SVs from 547 accessions, 6,013 were significantly associated with 22 yield-related and seed-quality-related traits determined in ten location × year environments. We uncovered 1,761 associated SVs that hit genes or regulatory regions, with 12 in GmMQT influencing oil and isoflavone contents. Our work provides resources and insights into SV roles in soybean improvement., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Although much interest has been focused on the role of selenium (Se) in plant nutrition over the last 20 years, the influences of organic selenium (selenomethionine; Se-Met) and inorganic selenium (potassium selenite; Se-K) on the growth and physiological characters of cadmium (Cd)-stressed Glycine max L.) seedlings have not yet been studied. In this study, the impacts of Se-Met or Se-K on the growth, water physiological parameters (gaseous exchange and leaf water content), photosynthetic and antioxidant capacities, and hormonal balance of G. max seedlings grown under 1.0 mM Cd stress were studied. The results showed that 30 μM Se-K up-regulates water physiological parameters, photosynthetic indices, antioxidant systems, enzymatic gene expression, total antioxidant activity (TAA), and hormonal balance. In addition, it down-regulates levels of reactive oxygen species (ROS; superoxide free radicals and hydrogen peroxide), oxidative damage (malondialdehyde content as an indicator of lipid peroxidation and electrolyte leakage), Cd translocation factor, and Cd content of Cd-stressed G. max seedlings. These positive findings were in favor of seedling growth and development under Cd stress. However, 50 μM Se-Met was more efficient than 30 μM Se-K in promoting the above-mentioned parameters of Cd-stressed G. max seedlings. From the current results, we conclude Se-Met could represent a promising strategy to contribute to the development and sustainability of crop production on soils contaminated with Cd at a concentration of up to 1.0 mM. However, further work is warranted to better understand the precise mechanisms of Se-Met action under Cd stress conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Background: ADP-glucose pyrophosphorylase (AGPase) is the key rate-limiting enzyme in starch biosynthesis pathway, and has been identified as a potential target for manipulation strategies aimed at improving crop yield and quality., Objective: To identify the AGPase gene family members in soybean, and explore the potential implications of GmAGPS2 in drought stress tolerance., Methods: The genome-wide identification and sequence analysis of soybean AGPase gene family was carried out by bioinformatics methods. The GmAGP gene expression was analyzed using transcriptome data and quantitative real-time PCR (qRT-PCR). Furthermore, transgenic yeast strains overexpressing GmAGPS2 were generated, and their growth was observed under drought stress., Results: In this study, we searched for AGPase genes (GmAGP) in the soybean genome and identified a total of 14 GmAGP genes. The GmAGP proteins had a unique conserved NTP&#95;transferase domain and were mainly located in the chloroplast and cytosol. Evolutionarily, the GmAGP proteins can be clustered into two distinct subgroups; within the same subgroup, they displayed a similar distribution pattern of conserved motifs. The GmAGP genes exhibited an uneven distribution on 10 chromosomes, and segmental duplication contributed to AGPase gene family expansion in soybean. The GmAGP genes presented different tissue expression pattern, in which GmAGPL6, GmAGPL9, and GmAGPL10 mainly exhibited tissue-specific expression pattern. The promoter of GmAGP genes had multiple cis-acting elements related to phytohormones and stress responses, and 8 GmAGP genes contained drought-responsive cis-acting elements. qRT‒PCR analysis demonstrated a significant upregulation expression of GmAGPL6, GmAGPL10, and GmAGPS2 in response to drought stress. Further functional analysis indicated that GmAGPS2 gene could improve yeast growth under drought stress conditions and enhance the drought tolerance of yeast., Conclusion: These results will contribute to further elucidation of the function of GmAGP genes, and offer important candidate genes for the genetic improvement of starch and yield-related traits and the breeding of high drought stress tolerance varieties in soybean., (© 2024. The Author(s) under exclusive licence to The Genetics Society of Korea.)

Soybean cyst nematode (SCN, Heterodera glycines ) is most effectively managed through planting resistant soybean cultivars, but the repeated use of the same resistance sources has led to a widespread emergence of virulent SCN populations that can overcome soybean resistance. Resistance to SCN HG type 0 (Race 3) in soybean cultivar Forrest is mediated by an epistatic interaction between the soybean resistance genes rhg1-a and Rhg4 . We previously developed two SCN inbred populations by mass-selecting SCN HG type 0 (Race 3) on susceptible and resistant recombinant inbred lines, derived from a cross between Forrest and the SCN-susceptible cultivar Essex, which differ for Rhg4 . To identify SCN genes potentially involved in overcoming rhg1-a / Rhg4 -mediated resistance, we conducted RNA sequencing on early parasitic juveniles of these two SCN inbred populations infecting their respective hosts, only to discover a handful of differentially expressed genes (DEGs). However, in a comparison with early parasitic juveniles of an avirulent SCN inbred population infecting a resistant host, we discovered 59 and 171 DEGs uniquely up- or downregulated in virulent parasitic juveniles adapted on the resistant host. Interestingly, the proteins coded by these 59 DEGs included vitamin B-associated proteins (reduced folate carrier, biotin synthase, and thiamine transporter) and nematode effectors known to play roles in plant defense suppression, suggesting that virulent SCN may exert a heightened transcriptional response to cope with enhanced plant defenses and an altered nutritional status of a resistant soybean host. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

The final phase in root nodule development is nodule senescence. The mechanism underlying the initiation of nodule senescence requires further elucidation. In this study, we investigate the intrinsic signals governing soybean (Glycine max L. Merr.) nodule senescence, uncovering ethylene as a key signal in this intricate mechanism. Two AP2/ethylene response factor (ERF) transcription factor (TF) genes, GmENS1 and GmENS2 (Ethylene-responsive transcription factors required for Nodule Senescence), exhibit heightened expression levels in both aged nodules and nodules treated with ethylene. An overexpression of either GmENS1 or GmENS2 accelerates senescence in soybean nodules, whereas the knockout or knockdown of both genes delays senescence and enhances nitrogenase activity. Furthermore, our findings indicate that GmENS1 and GmENS2 directly bind to the promoters of GmNAC039, GmNAC018, and GmNAC030, encoding 3 NAC (NAM, ATAF1/2, and CUC2) TFs essential for activating soybean nodule senescence. Notably, the nodule senescence process mediated by GmENS1 or GmENS2 overexpression is suppressed in the soybean nac039/018/030 triple mutant compared with the wild-type control. These data indicate GmENS1 and GmENS2 as pivotal TFs mediating ethylene-induced nodule senescence through the direct activation of GmNAC039/GmNAC018/GmNAC030 expression in soybean., Competing Interests: Conflict of interest statement. Authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The wild soybean Glycine soja Sieb. et Zucc. is an ancestor of the cultivated soybean Glycine max (L.) Merr. and a source of many valuable genes missing in the G. max genome, including genes that determine stress resistance to adverse environmental factors. Biochemical parameters (protein, oil, ascorbic acid, carotene, higher fatty acids, and specific activities and multiple forms of enzymes of the oxidoreductase and hydrolase classes) were studied in five G. soja accessions from the collection of the All-Russian Institute of Soybean (КА-1413, КА-342, КBl-29, КBl-24, and Kеl-72). The accessions provide unique natural gene banks. Wild seeds were collected in three districts (Arkharinskii, Blagoveshchensk, and Belogorskii) of Amur Oblast. Based on superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), ribonuclease (RNase), acid phosphatase, esterase, and amylase (AML) activities and biochemical parameters of seeds, the G. soja accession KA-1413 was found to have higher contents of protein, oleic acid, and linolenic acid; a lower polyphenol oxidase specific activity; and higher activities of SODs, esterases, and RNases. The accession KA-1413 was therefore recommended to use as a source of dominant genes in breeding to increase the adaptive potential of new soybean varieties. A higher heterogeneity of multiple forms was observed for SOD, AML, RNase, and esterase, which can provide markers of adaptation to environmental conditions., (© 2024. Pleiades Publishing, Ltd.)

Soybean is one of the most important crops for producing high quality oil and protein. Mineral nutrient deficiencies are frequently observed in soybeans. However, there are few studies to understand the absorption process of mineral nutrients in soybeans. Here, we investigated the functions of soybean (Glycine max L.) IRT1.1 (IRON-REGULATED TRANSPORTER 1.1) in the transportation of mineral elements. Heterologous expression of GmIRT1.1 in yeast mutants revealed that GmIRT1.1 compensated for the growth defects of Δfet3fet4 and Δsmf1 mutants under iron (Fe) and manganese (Mn) deficiency conditions, respectively, and enhanced the sensitivity of the Δycf1 mutant to cadmium (Cd) toxicity. Expression analysis revealed that GmIRT1.1 was only significantly induced by Fe deficiency and was primarily expressed in roots. Furthermore, the GmIRT1.1 overexpression lines enhanced Arabidopsis tolerance to Fe deficiency, leading to increased accumulation of Fe in the roots and shoots. Additionally, the transgenic lines increased the sensitivity to Mn and Cd toxicity. Subcellular localization analysis revealed that GmIRT1.1 was localized on the plasma membrane. Moreover, the results obtained from the soybean hairy roots system indicated that the localization of GmIRT1.1 was dependent on the regulation of Fe homeostasis in plant. Consequently, these results suggested that GmIRT1.1 was responsible for the transportation of Fe, Mn and Cd., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Expanded agriculture production is required to support the world's population but can impose substantial environmental and climate change costs, particularly with intensifying animal production and protein demand. Shifting from an animal- to a plant-based protein diet has numerous health benefits. Soybean (Glycine max [L.] Merr.) is a major source of protein for human food and animal feed; improved soybean protein content and amino acid composition could provide high-quality soymeal for animal feed, healthier human foods, and a reduced carbon footprint. Nonetheless, during the soybean genome evolution, a balance was established between the amount of seed protein, oil, and carbohydrate content, burdening the development of soybean cultivars with high proteins (HPs). We isolated 2 high-seed protein soybean mutants, HP1 and HP2, with improved seed amino acid composition and stachyose content, pointing to their involvement in controlling seed rebalancing phenomenon. HP1 encodes β-conglycinin (GmCG-1) and HP2 encodes sucrose-binding protein (GmSBP-1), which are both highly expressed in soybean seeds. Mutations in GmSBP-1, GmCG-1, and the paralog GmCG-2 resulted in increased protein levels, confirming their role as general regulators of seed protein content, amino acid seed composition, and seed vigor. Biodiversity analysis of GmCG and GmSBP across 108 soybean accessions revealed haplotypes correlated with protein and seed carbohydrate content. Furthermore, our data revealed an unprecedented role of GmCG and GmSBP proteins in improving seed vigor, crude protein, and amino acid digestibility. Since GmSBP and GmCG are present in most seed plants analyzed, these genes could be targeted to improve multiple seed traits., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)