The Lesion Simulating Disease (LSD) gene family as a variable in soybean response to Phakopsora pachyrhizi infection and dehydration

The Lesion Simulating Disease (LSD) genes encode a family of zinc finger proteins that are reported to play an important role in the hypersensitive response and programmed cell death (PCD) that are caused by biotic and abiotic stresses. In the present study, 117 putative LSD family members were identified in Viridiplantae. Genes with one, two, or three conserved LSD domains were identified. Proteins with three LSD domains were highly represented in the species analyzed and were present in basal organisms. Proteins with two LSD domains were identified only in the Embryophyte clade, and proteins possessing one LSD domain were highly represented in grass species. Expression analyses of Glycine max LSD (GmLSD) genes were performed by real-time quantitative polymerase chain reaction. The results indicated that GmLSD genes are not ubiquitously expressed in soybean organs and that their expression patterns are instead organ-dependent. The expression of the majority of GmLSD genes is modulated in soybean during Phakopsora pachyrhizi infection. In addition, the expression of some GmLSD genes is modulated in plants under dehydration stress. These results suggest the involvement of GmLSD genes in the response of soybean to both biotic and abiotic stresses.     

Genome-wide analysis of the PHB gene family in <Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.

Prohibitins (PHBs) have one SPFH domain in common and present in species ranging from prokaryotes to eukaryotes. Although a number of researches on PHBs were performed in different plant species, a systematic analysis of the PHB family in soybean is still remains uncharacterized. In the present study, 24 putative PHB genes have been first systemically identified in soybean. According to phylogenetic analysis, these GmPHBs could be classified into four groups. Gene structures and motif patterns showed high levels of conservation within the phylogenetic subgroups. Several members of this family have undergone purifying selection based on Ka/Ks analysis on duplicated PHB genes in soybean. We performed microsynteny analysis across four legume species based on the comparisons among the specific regions contained in PHB genes. As a result, numerous microsyntenic gene pairs among soybean, Medicago, Lotus and Phaseolus were identified. Most soybean PHB genes exhibited different expression levels in various tissues and developmental stages through expression analysis using publicly available RNA-seq datasets. The 11 GmPHB genes from III_B subgroup were examined by qPCR for their expression in two soybean cultivar after infection by Phytophthora sojae. Besides three GmPHB genes previous reported by us, here other four genes also were rapidly induced by P. sojae infection in the resistant genotype, while induction was very weak in the susceptible genotype. The comprehensive overview of the PHB gene family in soybean genome will provide useful information for further functional analysis of the PHB gene family in soybean.     

A Comprehensive Analysis of the Cupin Gene Family in Soybean (Glycine max)

Cupin superfamily of proteins, including germin and germin-like proteins (GLPs) from higher plants, is known to play crucial roles in plant development and defense. To date, no systematic analysis has been conducted in soybean (Glycine max) incorporating genome organization, gene structure, expression compendium. In this study, 69 putative Cupin genes were identified from the whole-genome of soybean, which were non-randomly distributed on 17 of the 20 chromosomes. These Gmcupin proteins were phylogenetically clustered into ten distinct subgroups among which the gene structures were highly conserved. Eighteen pairs (52.2%) of duplicate paralogous genes were preferentially retained in duplicated regions of the soybean genome. The distributions of GmCupin genes implied that long segmental duplications contributed significantly to the expansion of the GmCupin gene family. According to the RNA-seq data analysis, most of the Gmcupins were differentially expressed in tissue-specific expression pattern and the expression of some duplicate genes were partially redundant while others showed functional diversity, suggesting the Gmcupins have been retained by substantial subfunctionalization during soybean evolutionary processes. Selective analysis based on single nucleotide polymorphisms (SNPs) in cultivated and wild soybeans revealed sixteen Gmcupins had selected site(s), with all SNPs in Gmcupin10.3 and Gmcupin07.2 genes were selected sites, which implied these genes may have undergone strong selection effects during soybean domestication. Taken together, our results contribute to the functional characterization of Gmcupin genes in soybean.     

Expression of soybean plant hemoglobin gene family under abiotic stresses

Many abiotic stresses induce the generation of nitric oxide (NO) in plant tissues, where it functions as a signal molecule in stress responses. Plants modulate NO by oxidizing it to NO₃⁻ with plant hemoglobin (GLB), because excess NO is toxic to cells. At least eight genes encoding GLB have been identified in soybean, in three clades: GLB1, GLB2, and GLB3. However, it is still unclear which GLB genes are responsible for NO regulation under abiotic stress in soybean. We exposed soybean roots to flooding, salt, and two NO donors—sodium pentacyanonitrosylferrate (III) dihydrate (SNP) and S-nitroso-N-acetyl-d,l-penicillamine (SNAP)—and analyzed expression of GLB genes. GmGLB1, one of two GLB1 genes of soybean, significantly responded to both SNP and SNAP, and its induction was almost completely repressed by a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. GmGLB1 responded to flooding but not to salt, suggesting that it is responsible for NO regulation under NO-inducing abiotic stresses such as flooding. GmGLB3, one of two GLB3 genes of soybean, did not respond to NO donors at all but did respond to flooding, at a lower level than GmGLB1. These results suggest that flooding induces not only NO but also unknown factor(s) that induce GmGLB3 gene in soybean.     

Genomic,molecular evolution,and expression analysis of NOX genes in soybean (Glycine max)

Reactive oxygen species (ROS) are versatile signaling molecules in sensing stresses and play critical roles in signaling and development. Plasma membrane NADPH oxidases (NOXs) are key producers of ROS, and play important roles in the regulation of plant-pathogen interactions. Here, we performed a comprehensive analysis of the NOX gene family in the soybean genome (Glycine max) and 17 NOX (GmNOX) genes were identified. Structural analysis revealed that the GmNOX proteins in soybean were as conserved as those in other plants. 8 duplicated gene pairs were formed by a Glycine-specific whole-genome duplication (WGD) event approximately 13 million years ago (Mya). The Ka/Ks ratios of GmNOX genes ranged from 0.04 to 0.28, suggesting that the GmNOX family had undergone purifying selection in soybean. Gene expression patterns showed different expression of these duplicate genes, suggesting that the GmNOXs were retained by substantial subfunctionalization during the soybean evolutionary processes. Subsequently, the expression of GmNOXs in response to drought and phytohormones were characterized via qPCR. Importantly, four GmNOXs showed strong expression in nodules, pointing to their probable involvement in nodulation. Thus, our results shed light on the evolutionary history of this family in soybean and contribute to the functional characterization of GmNOX genes in soybean.     

The IQD Gene Family in Soybean: Structure,Phylogeny, Evolution and Expression

Members of the plant-specific IQ67-domain (IQD) protein family are involved in plant development and the basal defense response. Although systematic characterization of this family has been carried out in Arabidopsis, tomato (Solanum lycopersicum), Brachypodium distachyon and rice (Oryza sativa), systematic analysis and expression profiling of this gene family in soybean (Glycine max) have not previously been reported. In this study, we identified and structurally characterized IQD genes in the soybean genome. A complete set of 67 soybean IQD genes (GmIQD1–67) was identified using Blast search tools, and the genes were clustered into four subfamilies (IQD I–IV) based on phylogeny. These soybean IQD genes are distributed unevenly across all 20 chromosomes, with 30 segmental duplication events, suggesting that segmental duplication has played a major role in the expansion of the soybean IQD gene family. Analysis of the Ka/Ks ratios showed that the duplicated genes of the GmIQD family primarily underwent purifying selection. Microsynteny was detected in most pairs: genes in clade 1–3 might be present in genome regions that were inverted, expanded or contracted after the divergence; most gene pairs in clade 4 showed high conservation with little rearrangement among these gene-residing regions. Of the soybean IQD genes examined, six were most highly expressed in young leaves, six in flowers, one in roots and two in nodules. Our qRT-PCR analysis of 24 soybean IQD III genes confirmed that these genes are regulated by MeJA stress. Our findings present a comprehensive overview of the soybean IQD gene family and provide insights into the evolution of this family. In addition, this work lays a solid foundation for further experiments aimed at determining the biological functions of soybean IQD genes in growth and development.     

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots

As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.