MutL homolog 1 participates in interference-sensitive meiotic crossover formation in soybean.

MutL homolog 1 (MLH1), a member of the MutL-homolog family, is required for normal recombination in most organisms. However, its role in soybean (Glycine max) remains unclear to date. Here, we characterized the Glycine max female and male sterility 1 (Gmfms1) mutation that reduces pollen grain viability and increases embryo sac abortion in soybean. Map-based cloning revealed that the causal gene of Gmfms1 is Glycine max MutL homolog 1 (GmMLH1), and CRISPR/Cas9 knockout approach further validated that disruption of GmMLH1 confers the female-male sterility phenotype in soybean. Loss of GmMLH1 function disrupted bivalent formation, leading to univalent mis-segregation during meiosis and ultimately to female-male sterility. The Gmmlh1 mutant showed about a 78.16% decrease in meiotic crossover frequency compared to the wild type. The residual chiasmata followed a Poisson distribution, suggesting that interference-sensitive crossover formation was affected in the Gmmlh1 mutant. Furthermore, GmMLH1 could interact with GmMLH3A and GmMLH3B both in vivo and in vitro. Overall, our work demonstrates that GmMLH1 participates in interference-sensitive crossover formation in soybean, and provides additional information about the conserved functions of MLH1 across plant species.

Understanding the Molecular Regulatory Networks of Seed Size in Soybean.

Soybean being a major cash crop provides half of the vegetable oil and a quarter of the plant proteins to the global population. Seed size traits are the most important agronomic traits determining the soybean yield. These are complex traits governed by polygenes with low heritability as well as are highly influenced by the environment as well as by genotype x environment interactions. Although, extensive efforts have been made to unravel the genetic basis and molecular mechanism of seed size in soybean. But most of these efforts were majorly limited to QTL identification, and only a few genes for seed size were isolated and their molecular mechanism was elucidated. Hence, elucidating the detailed molecular regulatory networks controlling seed size in soybeans has been an important area of research in soybeans from the past decades. This paper describes the current progress of genetic architecture, molecular mechanisms, and regulatory networks for seed sizes of soybeans. Additionally, the main problems and bottlenecks/challenges soybean researchers currently face in seed size research are also discussed. This review summarizes the comprehensive and systematic information to the soybean researchers regarding the molecular understanding of seed size in soybeans and will help future research work on seed size in soybeans.

Disruption of CHORISMATE SYNTHASE1 leads to yellow-green variegation in soybean leaves.

Yellow-green variegation leaf phenotype adds more value to ornamental plants, but it is regarded as an undesirable trait in crop plants, affecting their yields. Until recently, the underlying mechanism regulating the yellow-green variegation phenotype has remained largely unexplored in soybean. In the present study, we indentified four Glycine max leaf yellow/green variegation mutants, Gmvar1, Gmvar2, Gmvar3, and Gmvar4, from artificial mutagenesis populations. Map-based cloning, together with the allelic identification test and CRISPR-based gene knockout, proved that mutated GmCS1 controls yellow-green variegation phenotype of the Gmvar mutants. GmCS1 encodes a chorismate synthase in soybean. The content of Phe, Tyr, and Trp were dramatically decreased in Gmcs1 mutants. Exogenous supply of three aromatic amino acid mixtures, or only Phe to Gmvar mutants, leads to recovery of the mutant phenotype. The various biological processes and signalling pathways related to metabolism and biosynthesis were altered in Gmvar mutants. Collectively, our findings provide new insights about the molecular regulatory network of yellow-green variegation leaf phenotype in soybean.

miR319-Regulated TCP3 Modulates Silique Development Associated with Seed Shattering in Brassicaceae.

Seed shattering is an undesirable trait that leads to crop yield loss. Improving silique resistance to shattering is critical for grain and oil crops. In this study, we found that miR319-targeted TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCPs) inhibited the process of post-fertilized fruits (silique) elongation and dehiscence via regulation of FRUITFULL (FUL) expression in Arabidopsis thaliana and Brassica napus. AtMIR319a activation resulted in a longer silique with thickened and lignified replum, whereas overexpression of an miR319a-resistant version of AtTCP3 (mTCP3) led to a short silique with narrow and less lignified replum. Further genetic and expressional analysis suggested that FUL acted downstream of TCP3 to negatively regulate silique development. Moreover, hyper-activation of BnTCP3.A8, a B. napus homolog of AtTCP3, in rapeseed resulted in an enhanced silique resistance to shattering due to attenuated replum development. Taken together, our findings advance our knowledge of TCP-regulated silique development and provide a potential target for genetic manipulation to reduce silique shattering in Brassica crops.

Soybean GmHY2a encodes a phytochromobilin synthase that regulates internode length and flowering time.

Plant height and flowering time are important agronomic traits that directly affect soybean [Glycine max (L.) Merr.] adaptability and yield. Here, the Glycine max long internode 1 (Gmlin1) mutant was selected from an ethyl methyl sulfonate (EMS)-mutated Williams 82 population due to its long internodes and early flowering. Using bulked segregant analysis (BSA), the Gmlin1 locus was mapped to Glyma.02G304700, a homologue of the Arabidopsis HY2 gene, which encodes a phytochromobilin (PΦB) synthase involved in phytochrome chromophore synthesis. Mutation of GmHY2a results in failure of the de-etiolation response under both red and far-red light. The Gmlin1 mutant exhibits a constitutive shade avoidance response under normal light, and the mutations influence the auxin and gibberellin pathways to promote internode elongation. The Gmlin1 mutant also exhibits decreased photoperiod sensitivity. In addition, the soybean photoperiod repressor gene E1 is down-regulated in the Gmlin1 mutant, resulting in accelerated flowering. The nuclear import of phytochrome A (GmphyA) and GmphyB following light treatment is decreased in Gmlin1 protoplasts, indicating that the weak light response of the Gmlin1 mutant is caused by a decrease in functional phytochrome. Together, these results indicate that GmHY2a plays an important role in soybean phytochrome biosynthesis and provide insights into the adaptability of the soybean plant.

Genetic Mapping of the Gmpgl3 Mutant Reveals the Function of GmTic110a in Soybean Chloroplast Development.

The generation of oxygen and organic matter in plants mainly depends on photosynthesis, which directly affects plant growth and development. The chloroplast is the main organelle in which photosynthesis occurs. In this study, a Glycine max pale green leaf 3-1 (Gmpgl3-1) mutant was isolated from the soybean mutagenized population. The Gmpgl3-1 mutant presented with decreased chlorophyll contents, reduced chloroplast stroma thylakoids, reduced yields, and decreased numbers of pods per plant. Bulked segregant analysis (BSA) together with map-based cloning revealed a single-nucleotide non-synonymous mutation at the 341st nucleotide of the first exon of the chloroplast development-related GmTic110a gene. The phenotype of the knockout plants was the same as that of the mutant. The GmTic110a gene was highly expressed in the leaves at various developmental stages, and its protein was localized to the inner chloroplast membrane. Split luciferase complementation assays and coimmunoprecipitation (co-IP) experiments revealed that GmTic110a interacted with GmTic20, GmTic40a, and GmTic40b in tobacco leaves. These results indicated that the GmTic110a gene plays an important role in chloroplast development.

The miR166 mediated regulatory module controls plant height by regulating gibberellic acid biosynthesis and catabolism in soybean.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play critical roles in regulating plant growth and development. Here, we used Short Tandem Target Mimic (STTM) technology to generate soybean (Glycine max (L.) Merr.) miRNA knockdown lines and identify miRNAs that regulate plant height, a key agronomic trait that affects yield. STTM166 successfully silenced miR166 in soybean and upregulated the expression of miR166 target genes, such as ATHB14-LIKE. The miR166 knockdown lines (GmSTTM166) displayed a reduced plant height phenotype. Moreover, GmSTTM166 plants contained lower levels of bioactive gibberellic acid (GA3) than wild-type plants, and application of exogenous GA partially rescued the dwarf phenotype of GmSTTM166. Knockdown of miR166 altered the expression of genes involved in GA biosynthesis and catabolism. Further analysis revealed that ATHB14-LIKE directly represses transcription of the GA biosynthesis genes GmGA1 and GmGA2, while activating transcription of the GA catabolic gene GIBBERLLIN 2 OXIDASE 2 (GmGA2ox2). Collectively, these results reveal a pivotal role for miR166 in the genetic control of plant height in soybean, thereby providing invaluable insights for molecular breeding to improve soybean yield.

GmPGL2, Encoding a Pentatricopeptide Repeat Protein, Is Essential for Chloroplast RNA Editing and Biogenesis in Soybean.

Chloroplast biogenesis and development are highly complex processes requiring interactions between plastids and nuclear genomic products. Pentatricopeptide repeat (PPR) proteins play an essential role in the development of chloroplasts; however, it remains unclear how RNA editing factors influence soybean development. In this study, a Glycine max pale green leaf 2 mutant (Gmpgl2) was identified with decreased chlorophyll contents. Genetic mapping revealed that a single-nucleotide deletion at position 1949 bp in the Glyma.05g132700 gene in the Gmpgl2 mutant, resulting in a truncated GmPGL2 protein. The nuclear-encoded GmPGL2 is a PLS-type PPR protein that localizes to the chloroplasts. The C-to-U editing efficiencies of rps16, rps18, ndhB, ndhD, ndhE, and ndhF were reduced in the Gmpgl2 mutant. RNA electrophoresis mobility shift assay (REMSA) analysis further revealed that GmPGL2 binds to the immediate upstream sequences at RNA editing sites of rps16 and ndhB in vitro, respectively. In addition, GmPGL2 was found to interact with GmMORF8, GmMORF9, and GmORRM6. These results suggest that GmPGL2 participates in C-to-U RNA editing via the formation of a complex RNA editosome in soybean chloroplasts.

A Robust and Rapid Candidate Gene Mapping Pipeline Based on M2 Populations.

The whole-genome sequencing-based bulked segregant analysis (WGS-BSA) has facilitated the mapping candidate causal variations for cloning target plant genes. Here, we report an improved WGS-BSA method termed as M2-seq to expedite the mapping candidate mutant loci by studying just M2 generation. It is an efficient mutant gene mapping tool, rapid, and comparable to the previously reported approaches, such as Mutmap and Mutmap+ that require studying M3 or advanced selfed generations. In M2-seq, background variations among the M2 populations can be removed efficiently without knowledge of the variations of the wild-type progenitor plant. Furthermore, the use of absolute delta single-nucleotide polymorphism (SNP) index values can effectively remove the background variation caused by repulsion phase linkages of adjacent mutant alleles; and thereby facilitating the identification of the causal mutation in target genes. Here, we demonstrated the application of M2-seq in successfully mapping the genomic regions harboring causal mutations for mutant phenotypes among 10 independent M2 populations of soybean. The mapping candidate mutant genes just in M2 generation with the aid of the M2-seq method should be particularly useful in expediting gene cloning especially among the plant species with long generation time.

GmCCD4 controls carotenoid content in soybeans.

To better understand the mechanisms regulating plant carotenoid metabolism in staple crop, we report the map-based cloning and functional characterization of the Glycine max carotenoid cleavage dioxygenase 4 (GmCCD4) gene, which encodes a carotenoid cleavage dioxygenase enzyme involved in metabolizing carotenoids into volatile ß-ionone. Loss of GmCCD4 protein function in four Glycine max increased carotenoid content (gmicc) mutants resulted in yellow flowers due to excessive accumulation of carotenoids in flower petals. The carotenoid contents also increase three times in gmicc1 seeds. A genome-wide association study indicated that the GmCCD4 locus was one major locus associated with carotenoid content in natural population. Further analysis indicated that the haplotype-1 of GmCCD4 gene was positively associated with higher carotenoid levels in soybean cultivars and accumulated more ß-carotene in engineered E. coli with ectopic expression of different GmCCD4 haplotypes. These observations uncovered that GmCCD4 was a negative regulator of carotenoid content in soybean, and its various haplotypes provide useful resources for future soybean breeding practice.

The genetic control of leaf and petal allometric variations in Arabidopsis thaliana.

BACKGROUND: Organ shape and size covariation (allometry) factors are essential concepts for the study of evolution and development. Although ample research has been conducted on organ shape and size, little research has considered the correlated variation of these two traits and quantitatively measured the variation in a common framework. The genetic basis of allometry variation in a single organ or among different organs is also relatively unknown. RESULTS: A principal component analysis (PCA) of organ landmarks and outlines was conducted and used to quantitatively capture shape and size variation in leaves and petals of multiparent advanced generation intercross (MAGIC) populations of Arabidopsis thaliana. The PCA indicated that size variation was a major component of allometry variation and revealed negatively correlated changes in leaf and petal size. After quantitative trait loci (QTL) mapping, five QTLs for the fourth leaf, 11 QTLs for the seventh leaf, and 12 QTLs for petal size and shape were identified. These QTLs were not identical to those previously identified, with the exception of the ER locus. The allometry model was also used to measure the leaf and petal allometry covariation to investigate the evolution and genetic coordination between homologous organs. In total, 12 QTLs were identified in association with the fourth leaf and petal allometry covariation, and eight QTLs were identified to be associated with the seventh leaf and petal allometry covariation. In these QTL confidence regions, there were important genes associated with cell proliferation and expansion with alleles unique to the maximal effects accession. In addition, the QTLs associated with life-history traits, such as days to bolting, stem length, and rosette leaf number, which were highly coordinated with climate change and local adaption, were QTL mapped and showed an overlap with leaf and petal allometry, which explained the genetic basis for their correlation. CONCLUSIONS: This study explored the genetic basis for leaf and petal allometry and their interaction, which may provide important information for investigating the correlated variation and evolution of organ shape and size in Arabidopsis.

A malectin-like receptor kinase regulates cell death and pattern-triggered immunity in soybean.

Plant cells can sense conserved molecular patterns through pattern recognition receptors (PRRs) and initiate pattern-triggered immunity (PTI). Details of the PTI signaling network are starting to be uncovered in Arabidopsis, but are still poorly understood in other species, including soybean (Glycine max). In this study, we perform a forward genetic screen for autoimmunity-related lesion mimic mutants (lmms) in soybean and identify two allelic mutants, which carry mutations in Glyma.13G054400, encoding a malectin-like receptor kinase (RK). The mutants exhibit enhanced resistance to both bacterial and oomycete pathogens, as well as elevated ROS production upon treatment with the bacterial pattern flg22. Overexpression of GmLMM1 gene in Nicotiana benthamiana severely suppresses flg22-triggered ROS production and oomycete pattern XEG1-induced cell death. We further show that GmLMM1 interacts with the flg22 receptor FLS2 and its co-receptor BAK1 to negatively regulate flg22-induced complex formation between them. Our study identifies an important component in PTI regulation and reveals that GmLMM1 acts as a molecular switch to control an appropriate immune activation, which may also be adapted to other PRR-mediated immune signaling in soybean.

Corrigendum: Genetic Mapping of a Light-Dependent Lesion Mimic Mutant Reveals the Function of Coproporphyrinogen III Oxidase Homolog in Soybean.

[This corrects the article DOI: 10.3389/fpls.2020.00557.].

GmNAP1 is essential for trichome and leaf epidermal cell development in soybean.

KEY MESSAGE: Map-based cloning revealed that two novel soybean distorted trichome mutants were due to loss function of GmNAP1 gene, which affected the trichome morphology and pavement cell ploidy by regulating actin filament assembly. Trichomes increase both biotic and abiotic stress resistance in soybean. In this study, Gmdtm1-1 and Gmdtm1-2 mutants with shorter trichomes and bigger epidermal pavement cells were isolated from an ethyl methylsulfonate mutagenized population. Both of them had reduced plant height and smaller seeds. Map-based cloning and bulked segregant analysis identified that a G-A transition at the 3' boundary of the sixth intron of Glyma.20G019300 in the Gmdtm1-1 mutant and another G-A transition mutation at the 5' boundary of the fourteenth intron of Glyma.20G019300 in Gmdtm1-2; these mutations disrupted spliceosome recognition sites creating truncated proteins. Glyma.20G019300 encodes a Glycine max NCK-associated protein 1 homolog (GmNAP1) in soybean. Further analysis revealed that the GmNAP1 involved in actin filament assembling and genetic information processing pathways during trichome and pavement cell development. This study shows that GmNAP1 plays an important role in soybean growth and development and agronomic traits.

Genetic Mapping of a Light-Dependent Lesion Mimic Mutant Reveals the Function of Coproporphyrinogen III Oxidase Homolog in Soybean.

Lesion mimic mutants provide ideal genetic materials for elucidating the molecular mechanism of cell death and disease resistance. Here, we isolated a Glycine max lesion mimic mutant 2-1 (Gmlmm2-1), which displayed a light-dependent cell death phenotype. Map-based cloning revealed that GmLMM2 encods a coproporphyrinogen III oxidase and participates in tetrapyrrole biosynthesis. Knockout of GmLMM2 led to necrotic spots on developing leaves of CRISPR/Cas9 induced mutants. The GmLMM2 defect decreased the chlorophyll content by disrupting tetrapyrrole biosynthesis and enhanced resistance to Phytophthora sojae. These results suggested that GmLMM2 gene played an important role in the biosynthesis of tetrapyrrole and light-dependent defense in soybeans.

GmPGL1, a Thiamine Thiazole Synthase, Is Required for the Biosynthesis of Thiamine in Soybean.

Thiamine is an essential cofactor in several enzymatic reactions for all living organisms. Animals cannot synthesize thiamine and depend on their diet. Enhancing the content of thiamine is one of the most important goals of plant breeding to solve the thiamine deficiency associated with the low-thiamin staple crops. In this study, a Glycine max pale green leaf 1 (Gmpgl1) mutant was isolated from the EMS mutagenized population of soybean cultivar, Williams 82. Map-based cloning of the GmPGL1 locus revealed a single nucleotide deletion at the 292th nucleotide residue of the first exon of Glyma.10g251500 gene in Gmpgl1 mutant plant, encoding a thiamine thiazole synthase. Total thiamine contents decreased in both seedlings and seeds of the Gmpgl1 mutant. Exogenous application of thiazole restored the pale green leaf phenotype of the mutant. The deficiency of thiamine in Gmpgl1 mutant led to reduced activities of the pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC), and decreased contents of six amino acids as compared to that in the wild type plants. These results revealed that GmPGL1 played an essential role in thiamine thiazole biosynthesis.

The Soybean Laccase Gene Family: Evolution and Possible Roles in Plant Defense and Stem Strength Selection.

Laccase is a widely used industrial oxidase for food processing, dye synthesis, paper making, and pollution remediation. At present, laccases used by industries come mainly from fungi. Plants contain numerous genes encoding laccase enzymes that show properties which are distinct from that of the fungal laccases. These plant-specific laccases may have better potential for industrial purposes. The aim of this work was to conduct a genome-wide search for the soybean laccase genes and analyze their characteristics and specific functions. A total of 93 putative laccase genes (GmLac) were identified from the soybean genome. All 93 GmLac enzymes contain three typical Cu-oxidase domains, and they were classified into five groups based on phylogenetic analysis. Although adjacent members on the tree showed highly similar exon/intron organization and motif composition, there were differences among the members within a class for both conserved and differentiated functions. Based on the expression patterns, some members of laccase were expressed in specific tissues/organs, while some exhibited a constitutive expression pattern. Analysis of the transcriptome revealed that some laccase genes might be involved in providing resistance to oomycetes. Analysis of the selective pressures acting on the laccase gene family in the process of soybean domestication revealed that 10 genes could have been under artificial selection during the domestication process. Four of these genes may have contributed to the transition of the soft and thin stem of wild soybean species into strong, thick, and erect stems of the cultivated soybean species. Our study provides a foundation for future functional studies of the soybean laccase gene family.

Evolution and Expression Divergence of the CYP78A Subfamily Genes in Soybean.

Gene expression divergence is an important evolutionary driving force for the retention of duplicate genes. In this study, we identified three CYP78A subfamily genes in soybean, GmCYP78A70, GmCYP78A57 and GmCYP78A72, which experienced different duplication events. GmCYP78A70 was mainly expressed in leaf tissue and the vegetative phase, whereas GmCYP78A57 was mainly expressed in floral tissue and seed, i.e., the reproductive phase. Expression of GmCYP78A72 could be detected in all the tissues and phases mentioned above. The expression levels of GmCYP78A70 and GmCYP78A57 in different soybean cultivars showed positive correlations with leaf size and 100-seed weight, respectively. The population genetics analysis indicated that the three genes had experienced different selective pressures during domestication and improved breeding of soybean. Deciphering the function of this subfamily of genes may well prove useful to breeders for improving soybean's agronomic traits.

Identification of ZHOUPI Orthologs in Rice Involved in Endosperm Development and Cuticle Formation.

The endosperm occupies most of the available space within mature rice seeds, contains abundant nutrients, and directly influences both the quality and quantity of rice production. Initial reports noted that AtZHOUPI (AtZOU) coordinates endosperm breakdown and the concomitant separation of the embryo from this structure in Arabidopsis. The results of this study show that rice genomes contain two most closely related homologs of AtZOU, OsZOU-1 and OsZOU-2; of these, OsZOU-1 expression is limited to within the endosperm where it can be detected throughout this structure 5 days after pollination (DAP). Its expression gradually decreases from seven DAP to nine DAP. The second of the two most closely related homologs, OsZOU-2, is highly expressed in leaves and stem, but is not detected in developing seeds. Heterologous expression of OsZOU-1 and OsZOU-2 in Atzou-4 mutants also revealed that OsZOU-1 partially complements the seed phenotypes of these individuals, while its counterpart, OsZOU-2, was unable to recover these phenotypes. The over-expression of OsZOU-1 severely disrupts both seed development and plant growth in transgenic rice lines, as plants in which this gene has been knocked down failed in the separation of endosperm from embryo and cuticle formation during seed development. The results of this study therefore suggest that OsZOU-1 is orthologous to the AtZOU, and regulates both endosperm development and cuticle formation in rice.

GmILPA1, Encoding an APC8-like Protein, Controls Leaf Petiole Angle in Soybean.

Leaf petiole angle (LPA) is an important plant architectural trait that affects canopy coverage, photosynthetic efficiency, and ultimately productivity in many legume crops. However, the genetic basis underlying this trait remains unclear. Here, we report the identification, isolation, and functional characterization of Glycine max Increased Leaf Petiole Angle1 (GmILPA1), a gene encoding an APC8-like protein, which is a subunit of the anaphase-promoting complex/cyclosome in soybean (Glycine max). A gamma ray-induced deletion of a fragment involving the fourth exon of GmILPA1 and its flanking sequences led to extension of the third exon and formation of, to our knowledge, a novel 3'UTR from intronic and intergenic sequences. Such changes are responsible for enlarged LPAs that are associated with reduced motor cell proliferation in the Gmilpa1 mutant. GmILPA1 is mainly expressed in the basal cells of leaf primordia and appears to function by promoting cell growth and division of the pulvinus that is critical for its establishment. GmILPA1 directly interacts with GmAPC13a as part of the putative anaphase-promoting complex. GmILPA1 exhibits variable expression levels among varieties with different degrees of LPAs, and expression levels are correlated with the degrees of the LPAs. Together, these observations revealed a genetic mechanism modulating the plant petiole angle that could pave the way for modifying soybean plant architecture with optimized petiole angles for enhanced yield potential.