Complete genome sequence of Bacillus velezensis strain Ag109, a biocontrol agent against plant-parasitic nematodes and Sclerotinia sclerotiorum.

Soybean is the main oilseed cultivated worldwide. Even though Brazil is the world's largest producer and exporter of soybean, its production is severely limited by biotic factors. Soil borne diseases are the most damaging biotic stressors since they significantly reduce yield and are challenging to manage. In this context, the present study aimed to evaluate the potential of a bacterial strain (Ag109) as a biocontrol agent for different soil pathogens (nematodes and fungi) of soybean. In addition, the genome of Ag109 was wholly sequenced and genes related to secondary metabolite production and plant growth promotion were mined. Ag109 showed nematode control in soybean and controlled 69 and 45% of the populations of Meloidogyne javanica and Pratylenchus brachyurus, respectively. Regarding antifungal activity, these strains showed activity against Macrophomia phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. For S. sclerotiorum, this strain increased the number of healthy plants and root dry mass compared to the control (with inoculation). Based on the average nucleotide identity and digital DNA-DNA hybridization, this strain was identified as Bacillus velezensis. Diverse clusters of specific genes related to secondary metabolite biosynthesis and root growth promotion were identified, highlighting the potential of this strain to be used as a multifunctional microbial inoculant that acts as a biological control agent while promoting plant growth in soybean.

Bowman-Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities.

Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman-Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities.

Identification of Gene-Allele System Conferring Alkali-Tolerance at Seedling Stage in Northeast China Soybean Germplasm.

Salinization of cultivated soils may result in either high salt levels or alkaline conditions, both of which stress crops and reduce performance. We sampled genotypes included in the Northeast China soybean germplasm population (NECSGP) to identify possible genes that affect tolerance to alkaline soil conditions. In this study, 361 soybean accessions collected in Northeast China were tested under 220 mM NaHCO3:Na2CO3 = 9:1 (pH = 9.8) to evaluate the alkali-tolerance (ATI) at the seedling stage in Mudanjiang, Heilongjiang, China. The restricted two-stage multi-locus model genome-wide association study (RTM-GWAS) with gene-allele sequences as markers (6503 GASMs) based on simplified genome resequencing (RAD-sequencing) was accomplished. From this analysis, 132 main effect candidate genes with 359 alleles and 35 Gene × Environment genes with 103 alleles were identified, explaining 90.93% and 2.80% of the seedling alkali-tolerance phenotypic variation, respectively. Genetic variability of ATI in NECSGP was observed primarily within subpopulations, especially in ecoregion B, from which 80% of ATI-tolerant accessions were screened out. The biological functions of 132 candidate genes were classified into eight functional categories (defense response, substance transport, regulation, metabolism-related, substance synthesis, biological process, plant development, and unknown function). From the ATI gene-allele system, six key genes-alleles were identified as starting points for further study on understanding the ATI gene network.

Mapping Locus RSC11K and predicting candidate gene resistant to Soybean mosaic virus strain SC11 through linkage analysis combined with genome resequencing of the parents in soybean.

Soybean mosaic virus (SMV) strain SC11 was prevalent in middle China. Its resistance was controlled by a Mendelian single dominant gene RSC11K in soybean Kefeng-1. This study aimed at mapping RSC11K and identifying its candidate gene. RSC11K locus was mapped ~217 kb interval between two SNP-linkage-disequilibrium-blocks (Gm02_BLOCK_11273955_11464884 and Gm02_BLOCK_11486875_11491354) in W82.a1.v1 genome using recombinant inbred lines population derived from Kefeng-1 (Resistant) × NN1138-2 (Susceptible), but inserted with a ~245 kb segment in W82.a2.v1 genome. In the entire 462 kb RSC11K region, 429 SNPs, 142 InDels and 34 putative genes were identified with more SNPs/InDels distributed in non-functional regions. Thereinto, ten genes contained SNP/InDel variants with high and moderate functional impacts on proteins, among which Glyma.02G119700 encoded a typical innate immune receptor-like kinase involving in virus disease process and responded to SMV inoculation, therefore was recognized as RSC11K's candidate gene. The novel RSC11K locus and candidate genes may help developing SMV resistance germplasm.

The Identification of a Quantative Trait Loci-Allele System of Antixenosis against the Common Cutworm (Spodoptera litura Fabricius) at the Seedling Stage in the Chinese Soybean Landrace Population.

Common cutworm (CCW) is an omnivorous insect causing severe yield losses in soybean crops. The seedling-stage mini-tray identification system with the damaged leaf percentage (DLP) as an indicator was used to evaluate antixenosis against CCW in the Chinese soybean landrace population (CSLRP) under three environments. Using the innovative restricted two-stage multi-locus genome-wide association study procedure (RTM-GWAS), 86 DLP QTLs with 243 alleles (2-11/QTL) were identified, including 66 main-effect loci with 203 alleles and 57 QTL-environment interaction loci with 172 alleles. Among the main-effect loci, 12 large-contribution loci (R2 ≥ 1%) explained 25.45% of the phenotypic variation (PV), and 54 small-contribution loci (R2 < 1%) explained 16.55% of the PV. This indicates that the CSLRP can be characterized with a DLP QTL-allele system complex that has not been found before, except for a few individual QTLs without alleles involved. From the DLP QTL-allele matrix, the recombination potentials expressed in the 25th percentile of the DLP of all possible crosses were predicted to be reduced by 41.5% as the maximum improvement and 14.2% as the maximum transgression, indicating great breeding potential in the antixenosis of the CSLRP. From the QTLs, 62 candidate genes were annotated, which were involved in eight biological function categories as a gene network of the DLP. Changing from susceptible to moderate plus resistant varieties in the CSLRP, 26 QTLs had 32 alleles involved, in which 19 genes were annotated from 25 QTL-alleles, including eight increased negative alleles on seven loci and 11 decreased positive alleles on 11 loci, showing the major genetic constitution changes for the antixenosis enhancement at the seedling stage in the CSLRP.

An Improved Genome-Wide Association Procedure Explores Gene-Allele Constitutions and Evolutionary Drives of Growth Period Traits in the Global Soybean Germplasm Population.

In soybeans (Glycine max (L.) Merr.), their growth periods, DSF (days of sowing-to-flowering), and DFM (days of flowering-to-maturity) are determined by their required accumulative day-length (ADL) and active temperature (AAT). A sample of 354 soybean varieties from five world eco-regions was tested in four seasons in Nanjing, China. The ADL and AAT of DSF and DFM were calculated from daily day-lengths and temperatures provided by the Nanjing Meteorological Bureau. The improved restricted two-stage multi-locus genome-wide association study using gene-allele sequences as markers (coded GASM-RTM-GWAS) was performed. (i) For DSF and its related ADLDSF and AATDSF, 130-141 genes with 384-406 alleles were explored, and for DFM and its related ADLDFM and AATDFM, 124-135 genes with 362-384 alleles were explored, in a total of six gene-allele systems. DSF shared more ADL and AAT contributions than DFM. (ii) Comparisons between the eco-region gene-allele submatrices indicated that the genetic adaptation from the origin to the geographic sub-regions was characterized by allele emergence (mutation), while genetic expansion from primary maturity group (MG)-sets to early/late MG-sets featured allele exclusion (selection) without allele emergence in addition to inheritance (migration). (iii) Optimal crosses with transgressive segregations in both directions were predicted and recommended for breeding purposes, indicating that allele recombination in soybean is an important evolutionary drive. (iv) Genes of the six traits were mostly trait-specific involved in four categories of 10 groups of biological functions. GASM-RTM-GWAS showed potential in detecting directly causal genes with their alleles, identifying differential trait evolutionary drives, predicting recombination breeding potentials, and revealing population gene networks.

Nitrogen allocation for CO2 fixation promotes nitrogen use in the photosynthetic compensation of soybean under heterogeneous light after a pre-shading.

The spatial and temporal distribution of sunlight around plants is constantly changing in natural and farmland environments. Previous studies showed that the photosynthesis of crops responds significantly to heterogeneous light conditions in fields. However, the underlying mechanisms remain unclear. In the present study, soybean plants were treated by heterogeneous light after a pre-shading (SH-HL) to simulate the light condition in relay strip intercropping. Gas exchange and nitrogen (N) of leaves were measured to evaluate the photosynthetic performance, as well as photosynthetic N- and water-use efficiency (PNUE and PWUE). Chlorophylls (Chl) and Rubisco were analyzed as representative photosynthetic N components. Results suggest that SH-HL treated soybean exhibited evident photosynthetic compensation as the net photosynthetic rate (Pn) increased significantly in unshaded leaves, from which the export of photosynthates was enhanced. Under SH-HL, leaf N concentration remained relatively stable in unshaded leaves. While Chl concentration decreased but Rubisco concentration increased in unshaded leaves, indicating preferential allocation of leaf N for CO2 fixation. Results also showed that PNUE increased and PWUE decreased in unshaded leaves under SH-HL. Therefore, we suggest that within-leaf N allocation for CO2 fixation in unshaded leaves rather than within-plant N distribution to unshaded leaves drives the photosynthetic compensation under heterogeneous light after a pre-shading. However, enhanced water loss from unshaded leaves is a cost for efficient N-use under these conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01392-8.

A ß-lactamase gene of Fusarium oxysporum alters the rhizosphere microbiota of soybean.

The rhizosphere is a multitrophic environment, and for soilborne pathogens such as Fusarium oxysporum, microbial competition in the rhizosphere is inevitable before reaching and infecting roots. This study established a tritrophic interaction among the plant growth-promoting rhizobacterium Burkholderia ambifaria, F. oxysporum and Glycine max (soybean) to study the effects of F. oxysporum genes on shaping the soybean microbiota. Although B. ambifaria inhibited mycelial growth and increased bacterial propagation in the presence of F. oxysporum, F. oxysporum still managed to infect soybean in the presence of B. ambifaria. RNA-Seq identified a putative F. oxysporum secretory ß-lactamase-coding gene, FOXG_18438 (abbreviated as Fo18438), that is upregulated during soybean infection in the presence of B. ambifaria. The ∆Fo18438 mutants displayed reduced mycelial growth towards B. ambifaria, and the complementation of full Fo18438 and the Fo18438 ß-lactamase domain restored mycelial growth. Using the F. oxysporum wild type, ∆Fo18438 mutants and complemented strains with full Fo18438, Fo18438 ß-lactamase domain or Fo18438 RTA1-like domain for soil inoculation, 16S rRNA amplicon sequencing revealed that the abundance of a Burkholderia operational taxonomic unit (OTU) was increased in the rhizosphere microbiota infested by the strains with Fo18438 ß-lactamase domain. Non-metric multidimensional scaling and PICRUSt2 functional analysis revealed differential abundance for the bacterial ß-lactam-related functions when contrasting the genotypes of F. oxysporum. These results indicated that the Fo18438 ß-lactamase domain provides F. oxysporum with the advantage of growing into the soybean rhizosphere, where ß-lactam antibiosis is involved in microbial competition. Accordingly, this study highlights the capability of an F. oxysporum gene for altering the soybean rhizosphere and taproot microbiota.

Nuclear transport factor GmNTF2B-1 enhances soybean drought tolerance by interacting with oxidoreductase GmOXR17 to reduce reactive oxygen species content.

Drought is a critical abiotic stressor that modulates soybean yield. Drought stress drastically enhances reactive oxygen species (ROS) formation, and maintaining ROS content above a cytostatic level but below a cytotoxic level is essential for normal biology processes in plants. At present, most of the known ROS-scavenging systems are in the cytoplasm, and the mechanism of ROS regulation in the nucleus remains unclear. GmNTF2B-1 is a member of the IV subgroup in the nucleus transporter family. Its expression is localized to the roots and is stimulated by drought stress. In this study, the overexpression of GmNTF2B-1 was found to improve the drought tolerance of transgenic soybean by influencing the ROS content in plants. An oxidoreductase, GmOXR17, was identified to interact with GmNTF2B-1 in the nucleus through the yeast two-hybrid, coimmunoprecipitation and bimolecular fluorescence complementation assays. The drought tolerance of GmOXR17 transgenic soybean was similar to that of GmNTF2B-1. GmNTF2B-1 was expressed in both cytoplasm and nucleus, and GmOXR17 transferred from the cytoplasm to the nucleus under drought stress. The overexpression of GmNTF2B-1 enhanced the nuclear entry of GmOXR17, and the overexpression of GmNTF2B-1 or GmOXR17 could decrease the H2 O2 content and oxidation level in the nucleus. In conclusion, the interaction between GmNTF2B-1 and GmOXR17 may enhance the nuclear entry of GmOXR17, thereby enhancing nuclear ROS scavenging to improve the drought resistance of soybean.

Growth period QTL-allele constitution of global soybeans and its differential evolution changes in geographic adaptation versus maturity group extension.

Soybean (Glycine max (L.) Merr.) has been disseminated globally as a photoperiod/temperature-sensitive crop with extremely diverse days to flowering (DTF) and days to maturity (DTM) values. A population with 371 global varieties covering 13 geographic regions and 13 maturity groups (MGs) was analyzed for its DTF and DTM QTL-allele constitution using restricted two-stage multi-locus genome-wide association study (RTM-GWAS). Genotypes with 20 701 genome-wide SNPLDBs (single-nucleotide polymorphism linkage disequilibrium blocks) containing 55 404 haplotypes were observed, and 52 DTF QTLs and 59 DTM QTLs (including 29 and 21 new ones) with 241 and 246 alleles (two to 13 per locus) were detected, explaining 84.8% and 74.4% of the phenotypic variance, respectively. The QTL-allele matrix characterized with all QTL-allele information of each variety in the global population was established and subsequently separated into geographic and MG set submatrices. Direct comparisons among them revealed that the genetic adaptation from the origin to geographic subpopulations was characterized by new allele/new locus emergence (mutation) but little allele exclusion (selection), while that from the primary MG set to emerged early and late MG sets was characterized by allele exclusion without allele emergence. The evolutionary changes involved mainly 72 DTF and 71 DTM alleles on 28 respective loci, 10-12 loci each with three to six alleles being most active. Further recombination potential for faster maturation (12-21 days) or slower maturation (14-56 days) supported allele convergence (recombination) as a constant genetic factor in addition to migration (inheritance). From the QTLs, 44 DTF and 36 DTM candidate genes were annotated and grouped respectively into nine biological processes, indicating multi-functional DTF/DTM genes are involved in a complex gene network. In summary, we identified QTL-alleles relatively thoroughly using RTM-GWAS for direct matrix comparisons and subsequent analysis.

Key Soybean Seedlings Drought-Responsive Genes and Pathways Revealed by Comparative Transcriptome Analyses of Two Cultivars.

Seedling drought stress is one of the most important constraints affecting soybean yield and quality. To unravel the molecular mechanisms under soybean drought tolerance, we conducted comprehensive comparative transcriptome analyses of drought-tolerant genotype Jindou 21 (JD) and drought-sensitive genotype Tianlong No.1 (N1) seedlings that had been exposed to drought treatment. A total of 6038 and 4112 differentially expressed genes (DEGs) were identified in drought-tolerant JD and drought-sensitive N1, respectively. Subsequent KEGG pathway analyses showed that numerous DEGs in JD are predominately involved in signal transduction pathways, including plant hormone signaling pathway, calcium signaling pathway, and MAPK signaling pathway. Interestingly, JA and BR plant hormone signal transduction pathways were found specifically participating in drought-tolerant JD. Meanwhile, the differentially expressed CPKs, CIPKs, MAPKs, and MAP3Ks of calcium and MAPK signaling pathway were only identified in JD. The number of DEGs involved in transcription factors (TFs) is larger in JD than that of in N1. Moreover, some differently expressed transcriptional factor genes were only identified in drought-tolerant JD, including FAR1, RAV, LSD1, EIL, and HB-PHD. In addition, this study suggested that JD could respond to drought stress by regulating the cell wall remodeling and stress-related protein genes such as EXPs, CALSs, CBPs, BBXs, and RD22s. JD is more drought tolerant than N1 owing to more DEGs being involved in multiple signal transduction pathways (JA, BR, calcium, MAPK signaling pathway), stress-related TFs, and proteins. The above valuable genes and pathways will deepen the understanding of the molecular mechanisms under drought stress in soybean.

Transcriptome Analysis Reveals the Genes Related to Pollen Abortion in a Cytoplasmic Male-Sterile Soybean (Glycine max (L.) Merr.).

Cytoplasmic male sterility (CMS) lays a foundation for the utilization of heterosis in soybean. The soybean CMS line SXCMS5A is an excellent CMS line exhibiting 100% male sterility. Cytological analysis revealed that in SXCMS5A compared to its maintainer SXCMS5B, its tapetum was vacuolated and abnormally developed. To identify the genes and metabolic pathways involving in pollen abortion of SXCMS5A, a comparative transcriptome analysis was conducted between SXCMS5A and SXCMS5B using flower buds. A total of 372,973,796 high quality clean reads were obtained from 6 samples (3 replicates for each material), and 840 differentially expressed genes (DEGs) were identified, including 658 downregulated and 182 upregulated ones in SXCMS5A compared to SXCMS5B. Among them, 13 DEGs, i.e., 12 open reading frames (ORFs) and 1 COX2, were mitochondrial genome genes in which ORF178 and ORF103c were upregulated in CMS lines and had transmembrane domain(s), therefore, identified as CMS candidate mitochondrial genes of SXCMS5A. Furthermore, numerous DEGs were associated with pollen wall development, carbohydrate metabolism, sugar transport, reactive oxygen species (ROS) metabolism and transcription factor. Some of them were further confirmed by quantitative real time PCR analysis between CMS lines with the same cytoplasmic source as SXCMS5A and their respective maintainer lines. The amount of soluble sugar and adenosine triphosphate and the activity of catalase and ascorbic acid oxidase showed that energy supply and ROS scavenging decreased in SXCMS5A compared to SXCMS5B. These findings provide valuable information for further understanding the molecular mechanism regulating the pollen abortion of soybean CMS.

Confirmation of GmPPR576 as a fertility restorer gene of cytoplasmic male sterility in soybean.

In soybean, heterosis achieved through the three-line system has been gradually applied in breeding to increase yield, but the underlying molecular mechanism remains unknown. We conducted a genetic analysis using the pollen fertility of offspring of the cross NJCMS1A×NJCMS1C. All the pollen of F1 plants was semi-sterile; in F2, the ratio of pollen-fertile plants to pollen-semi-sterile plants was 208:189. This result indicates that NJCMS1A is gametophyte sterile, and the fertility restoration of NJCMS1C to NJCMS1A is a quality trait controlled by a single gene locus. Using bulked segregant analysis, the fertility restorer gene Rf in NJCMS1C was located on chromosome 16 between the markers BARCSOYSSR_16_1067 and BARCSOYSSR_16_1078. Sequence analysis of genes in that region showed that GmPPR576 was non-functional in rf cultivars. GmPPR576 has one functional allele in Rf cultivars but three non-functional alleles in rf cultivars. Phylogenetic analysis showed that the GmPPR576 locus evolved rapidly with the presence of male-sterile cytoplasm. GmPPR576 belongs to the RFL fertility restorer gene family and is targeted to the mitochondria. GmPPR576 was knocked out in soybean N8855 using CRISPR/Cas9. The T1 plants showed sterile pollen, and T2 plants produced few pods at maturity. The results indicate that GmPPR576 is the fertility restorer gene of NJCMS1A.

GmFULa improves soybean yield by enhancing carbon assimilation without altering flowering time or maturity.

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.

Two TGA Transcription Factor Members from Hyper-Susceptible Soybean Exhibiting Significant Basal Resistance to Soybean mosaic virus.

TGA transcription factors (TFs) exhibit basal resistance in Arabidopsis, but susceptibility to a pathogen attack in tomatoes; however, their roles in soybean (Glycine max) to Soybean mosaic virus (SMV) are unknown. In this study, 27 TGA genes were isolated from a SMV hyper-susceptible soybean NN1138-2, designated GmTGA1~GmTGA27, which were clustered into seven phylogenetic groups. The expression profiles of GmTGAs showed that the highly expressed genes were mainly in Groups I, II, and VII under non-induction conditions, while out of the 27 GmTGAs, 19 responded to SMV-induction. Interestingly, in further transient N. benthamiana-SMV pathosystem assay, all the 19 GmTGAs overexpressed did not promote SMV infection in inoculated leaves, but they exhibited basal resistance except one without function. Among the 18 functional ones, GmTGA8 and GmTGA19, with similar motif distribution, nuclear localization sequence and interaction proteins, showed a rapid response to SMV infection and performed better than the others in inhibiting SMV multiplication. This finding suggested that GmTGA TFs may support basal resistance to SMV even from a hyper-susceptible source. What the mechanism of the genes (GmTGA8, GmTGA19, etc.) with basal resistance to SMV is and what their potential for the future improvement of resistance to SMV in soybeans is, are to be explored.

Heat-Responsive miRNAs Participate in the Regulation of Male Fertility Stability in Soybean CMS-Based F1 under High Temperature Stress.

MicroRNAs (miRNAs), a class of noncoding small RNAs (sRNAs), are widely involved in the response to high temperature (HT) stress at both the seedling and flowering stages. To dissect the roles of miRNAs in regulating male fertility in soybean cytoplasmic male sterility (CMS)-based F1 under HT, sRNA sequencing was performed using flower buds from HT-tolerant and HT-sensitive CMS-based F1 combinations (NF1 and YF1, respectively). A total of 554 known miRNAs, 59 new members of known miRNAs, 712 novel miRNAs, and 1145 target genes of 580 differentially expressed miRNAs (DEMs) were identified under normal temperature and HT conditions. Further integrated analysis of sRNA and transcriptome sequencing found that 21 DEMs and 15 differentially expressed target genes, such as gma-miR397a/Laccase 2, gma-miR399a/Inorganic phosphate transporter 1-4, and gma-miR4413a/PPR proteins, mitochondrial-like, were negatively regulated under HT stress. Furthermore, all members of the gma-miR156 family were suppressed by HT stress in both NF1 and YF1, but were highly expressed in YF1 under HT condition. The negative correlation between gma-miR156b and its target gene squamosa promoter-binding protein-like 2b was confirmed by expression analysis, and overexpression of gma-miR156b in Arabidopsis led to male sterility under HT stress. With these results, we proposed that miRNAs play an important role in the regulation of male fertility stability in soybean CMS-based F1 under HT stress.

Changes in photosynthetic traits and their responses to increasing fertilization rates in soybean (Glycine max (L.) Merr.) during decades of genetic improvement.

BACKROUND: Changes in photosynthetic traits (PTs) during the long-term genetic improvement of soybean (Glycine max (L.) Merr.) yield have been studied, but detailed information on whether PT responses to environmental stress have improved, and their correlations with seed yield, are still unknown. Our objectives were to describe the changes in soybean PTs - leaf area index (LAI), leaf chlorophyll content (Chl), net photosynthetic rate (PN ), stomatal conductance (gs ), and transpiration rate (E) - during decades of genetic improvement, and to detect whether the responses to increasing fertilizer application rates (FRs) of the PTs of 13 different soybean cultivars released in various decades differed. RESULTS: All of the soybean PTs increased significantly along with the year in which each cultivar was released, under different FR treatments, indicating that PTs have improved during decades of genetic breeding. Medium FR (nitrogen) treatment (150 kg ha -1 ) increased PT values, to different extents, at all the investigated growth stages. Leaf area index, Chl, and PN of the old and middle cultivar groups at the full bloom (R2), full seed (R6), and beginning maturity (R7) stages decreased significantly under high FR treatment (300 kg ha-1 ) compared with the medium FR treatment. The former had no effect on any of the PTs of new cultivar group, or had promotive effects. Thus, the photosynthetic capacities of the new cultivars are more tolerant to high FR-related stress than older cultivars. CONCLUSIONS: The photosynthetic capacities, and tolerance to high FR-related stress, of soybean cultivars that were released in different years improved after long-term genetic breeding. © 2021 Society of Chemical Industry.

Double mutation of two homologous genes YL1 and YL2 results in a leaf yellowing phenotype in soybean [Glycine max (L.) Merr].

KEY MESSAGE: Two homologous, chloroplast located CAAX proteases were identified to be functional redundancy in determining soybean leaf color, and they probably play essential roles in regulating light harvesting and absorption during photosynthesis process. Leaf color mutants are ideal materials for studying photosynthesis and chlorophyll metabolism. The soybean [Glycine max (L.) Merr.] yellowing leaf (yl) variation is a recombinant mutant characterized by yellow foliage, which derived from the specific cross between green seed-coated and yellow seed-coated soybean varieties. Molecular cloning and subsequent gene silencing revealed that the yellow leaf trait of yl was controlled by two recessive nuclear genes, glyma11g04660 and glyma01g40650, named as YL1 and YL2 respectively, and the latter was confirmed to be same as the earlier reported green seed-coat gene G. Both YL1 and YL2 belonged to chloroplast-located proteases possessing Abi domain, and these genes were expressed in various tissues, especially in young leaves. In yl, the expression of YL1 and YL2 were suppressed in most tissues, and the young leaf of yl presented an increased maximal photochemical efficiency (Fv/Fm) as well as enhanced net photosynthesis activity (Pn), indicating that YL1 and YL2 are involved in light absorption regulation during photosynthesis process. Collectively, the identification and description of YL1 and YL2 in our study provides insights for the regulatory mechanism of photosynthesis process, and these findings will further assist to clarify the close relationship between photosynthesis and chlorophyll metabolism.

One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation.

BACKGROUND: Agrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions. RESULTS: Here, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A. rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1, which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato. CONCLUSIONS: We established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.

Natural variation and CRISPR/Cas9-mediated mutation in GmPRR37 affect photoperiodic flowering and contribute to regional adaptation of soybean.

Flowering time is a critical determinant of the geographic distribution and regional adaptability of soybean (Glycine max) and is strongly regulated by photoperiod and temperature. In this study, quantitative trait locus (QTL) mapping and subsequent candidate gene analysis revealed that GmPRR37, encoding a pseudo-response regulator protein, is responsible for the major QTL qFT12-2, which was identified from a population of 308 recombinant inbred lines (RILs) derived from a cross between a very late-flowering soybean cultivar, 'Zigongdongdou (ZGDD)', and an extremely early-flowering cultivar, 'Heihe27 (HH27)', in multiple environments. Comparative analysis of parental sequencing data confirmed that HH27 contains a non-sense mutation that causes the loss of the CCT domain in the GmPRR37 protein. CRISPR/Cas9-induced Gmprr37-ZGDD mutants in soybean exhibited early flowering under natural long-day (NLD) conditions. Overexpression of GmPRR37 significantly delayed the flowering of transgenic soybean plants compared with wild-type under long photoperiod conditions. In addition, both the knockout and overexpression of GmPRR37 in soybean showed no significant phenotypic alterations in flowering time under short-day (SD) conditions. Furthermore, GmPRR37 down-regulated the expression of the flowering-promoting FT homologues GmFT2a and GmFT5a, and up-regulated flowering-inhibiting FT homologue GmFT1a expression under long-day (LD) conditions. We analysed haplotypes of GmPRR37 among 180 cultivars collected across China and found natural Gmprr37 mutants flower earlier and enable soybean to be cultivated at higher latitudes. This study demonstrates that GmPRR37 controls soybean photoperiodic flowering and provides opportunities to breed optimized cultivars with adaptation to specific regions and farming systems.