Qingxin Song

Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing.

BackgroundMicroRNAs (miRNAs) regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max) is limited, and global identification of the related miRNA targets has not been reported in previous research.ResultsIn our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3) was detected as a target of the newly identified miRNA Soy_25, suggesting the presence of feedback control of miRNA biogenesis.ConclusionsWe have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development.

Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants

NAC transcription factors play important roles in plant growth, development and stress responses. Previously, we identified multiple NAC genes in soybean (Glycine max). Here, we identify the roles of two genes, GmNAC11 and GmNAC20, in stress responses and other processes. The two genes were differentially induced by multiple abiotic stresses and plant hormones, and their transcripts were abundant in roots and cotyledons. Both genes encoded proteins that localized to the nucleus and bound to the core DNA sequence CGT[G/A]. In the protoplast assay system, GmNAC11 acts as a transcriptional activator, whereas GmNAC20 functions as a mild repressor; however, the C-terminal end of GmANC20 has transcriptional activation activity. Over-expression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 over-expression only improves salt tolerance. Over-expression of GmNAC20 also promotes lateral root formation. GmNAC20 may regulate stress tolerance through activation of the DREB/CBF-COR pathway, and may control lateral root development by altering auxin signaling-related genes. GmNAC11 probably regulates DREB1A and other stress-related genes. The roles of the two GmNAC genes in stress tolerance were further analyzed in soybean transgenic hairy roots. These results provide a basis for genetic manipulation to improve the agronomic traits of important crops.

Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation.

Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.

Genome-Wide Analysis of DNA Methylation in Soybean

Cytosine methylation is an important mechanism for dynamical regulation of gene expression and transposable element (TE) mobility during plant developmental processes. Here, we identified the transcription start sites of genes using high-throughput sequencing and then analyzed the DNA methylation status in soybean roots, stems, leaves, and cotyledons of developing seeds at single-base resolution. Profiling of DNA methylation in different organs revealed 2162 differentially methylated regions among organs, and a portion of hypomethylated regions were correlated with high expression of neighboring genes. Because of the different distribution of class I TEs (retrotransposons) and class II TEs (DNA transposons), the promoters of the lowest-expressed genes showed higher levels of CG and CHG methylation but a lower level of CHH methylation. We further found that the CHH methylation level of class II TEs was higher than class I TEs, possibly due to the presence of more smRNAs in class II TEs. In cotyledons of developing seeds, smRNA abundance was roughly positively correlated with hypermethylated regions but negatively related to hypomethylated regions. These studies provide significant insights into the complicated interplays among DNA methylation, smRNA abundance, TE distribution, and gene expression in soybean.

Epigenetic and developmental regulation in plant polyploids

Polyploidy or whole-genome duplication occurs in some animals and many flowering plants, including many important crops such as wheat, cotton and oilseed rape. The prevalence of polyploidy in the plant kingdom suggests it as an important evolutionary feature for plant speciation and crop domestication. Studies of natural and synthetic polyploids have revealed rapid and dynamic changes in genomic structure and gene expression after polyploid formation. Growing evidence suggests that epigenetic modifications can alter homoeologous gene expression and reprogram gene expression networks, which allows polyploids to establish new cytotypes, grow vigorously and promote adaptation in local environments. Sequence and gene expression changes in polyploids have been well documented and reviewed elsewhere. This review is focused on developmental regulation and epigenetic changes including DNA methylation and histone modifications in polyploids.

Identification of Rice Ethylene-Response Mutants and Characterization of MHZ7/OsEIN2 in Distinct Ethylene Response and Yield Trait Regulation

Ethylene plays essential roles in adaptive growth of rice plants in water-saturating environment; however, ethylene signaling pathway in rice is largely unclear. In this study, we report identification and characterization of ethylene-response mutants based on the specific ethylene-response phenotypes of etiolated rice seedlings, including ethylene-inhibited root growth and ethylene-promoted coleoptile elongation, which is different from the ethylene triple-response phenotype in Arabidopsis. We establish an efficient system for screening and a set of rice mutants have been identified. Genetic analysis reveals that these mutants form eight complementation groups. All the mutants show insensitivity or reduced sensitivity to ethylene in root growth but exhibit differential responses in coleoptile growth. One mutant group mhz7 has insensitivity to ethylene in both root and coleoptile growth. We identified the corresponding gene by a map-based cloning method. MHZ7 encodes a membrane protein homologous to EIN2, a central component of ethylene signaling in Arabidopsis. Upon ethylene treatment, etiolated MHZ7-overexpressing seedlings exhibit enhanced coleoptile elongation, increased mesocotyl growth and extremely twisted short roots, featuring enhanced ethylene-response phenotypes in rice. Grain length was promoted in MHZ7-transgenic plants and 1000-grain weight was reduced in mhz7 mutants. Leaf senescent process was also affected by MHZ7 expression. Manipulation of ethylene signaling may improve adaptive growth and yield-related traits in rice.

Soybean GmMYB73 promotes lipid accumulation in transgenic plants

BackgroundSoybean is one of the most important oil crops. The regulatory genes involved in oil accumulation are largely unclear. We initiated studies to identify genes that regulate this process.ResultsOne MYB-type gene GmMYB73 was found to display differential expression in soybean seeds of different developing stages by microarray analysis and was further investigated for its functions in lipid accumulation. GmMYB73 is a small protein with single MYB repeat and has similarity to CPC-like MYB proteins from Arabidopsis. GmMYB73 interacted with GL3 and EGL3, and then suppressed GL2, a negative regulator of oil accumulation. GmMYB73 overexpression enhanced lipid contents in both seeds and leaves of transgenic Arabidopsis plants. Seed length and thousand-seed weight were also promoted. GmMYB73 introduction into the Arabidopsis try cpc double mutant rescued the total lipids, seed size and thousand-seed weight. GmMYB73 also elevated lipid levels in seeds and leaves of transgenic Lotus, and in transgenic hairy roots of soybean plants. GmMYB73 promoted PLDα1 expression, whose promoter can be bound and inhibited by GL2. PLDα1 mutation reduced triacylglycerol levels mildly in seeds but significantly in leaves of Arabidopsis plants.ConclusionsGmMYB73 may reduce GL2, and then release GL2-inhibited PLDα1 expression for lipid accumulation. Manipulation of GmMYB73 may potentially improve oil production in legume crop plants.

Soybean GmbZIP123 gene enhances lipid content in the seeds of transgenic Arabidopsis plants

Soybean is one of most important oil crops and a significant increase in lipid content in soybean seeds would facilitate vegetable oil production in the world. Although the pathways for lipid biosynthesis in higher plants have been uncovered, our understanding of regulatory mechanism controlling lipid accumulation is still limited. In this study, we identified 87 transcription factor genes with a higher abundance at the stage of lipid accumulation in soybean seeds. One of these genes, GmbZIP123, was selected to further study its function in regulation of lipid accumulation. Overexpression of GmbZIP123 enhanced lipid content in the seeds of transgenic Arabidopsis thaliana plants. The GmbZIP123 transgene promoted expression of two sucrose transporter genes (SUC1 and SUC5) and three cell-wall invertase genes (cwINV1, cwINV3, and cwINV6) by binding directly to the promoters of these genes. Consistently, the cell-wall invertase activity and sugar translocation were all enhanced in siliques of GmbZIP123 transgenic plants. Higher levels of glucose, fructose, and sucrose were also found in seeds of GmbZIP123 transgenic plants. These results suggest that GmbZIP123 may participate in regulation of lipid accumulation in soybean seeds by controlling sugar transport into seeds from photoautotrophic tissues. This study provides novel insights into the regulatory mechanism for lipid accumulation in seeds and may facilitate improvements in oil production in soybean and other oil crops through genetic manipulation of the GmbZIP123 gene.

Polyploidy and small RNA regulation of cotton fiber development

Cotton is not only the most important source of renewal textile fibers, but also an excellent model for studying cell fate determination and polyploidy effects on gene expression and evolution of domestication traits. The combination of A and D-progenitor genomes into allotetraploid cotton induces intergenomic interactions and epigenetic effects, leading to the unequal expression of homoeologous genes. Small RNAs regulate the expression of transcription and signaling factors related to cellular growth, development and adaptation. An example is miRNA-mediated preferential degradation of homoeologous mRNAs encoding MYB-domain transcription factors that are required for the initiation of leaf trichomes in Arabidopsis and of seed fibers in cotton. This example of coevolution between small RNAs and their homoeologous targets could shape morphological traits such as fibers during the selection and domestication of polyploid crops.

The transcription factor AtDOF4.2 regulates shoot branching and seed coat formation in Arabidopsis

Plant-specific DOF (DNA-binding with one finger)-type transcription factors regulate various biological processes. In the present study we characterized a silique-abundant gene AtDOF (Arabidopsis thaliana DOF) 4.2 for its functions in Arabidopsis. AtDOF4.2 is localized in the nuclear region and has transcriptional activation activity in both yeast and plant protoplast assays. The T-M-D motif in AtDOF4.2 is essential for its activation. AtDOF4.2-overexpressing plants exhibit an increased branching phenotype and mutation of the T-M-D motif in AtDOF4.2 significantly reduces branching in transgenic plants. AtDOF4.2 may achieve this function through the up-regulation of three branching-related genes, AtSTM (A. thaliana SHOOT MERISTEMLESS), AtTFL1 (A. thaliana TERMINAL FLOWER1) and AtCYP83B1 (A. thaliana CYTOCHROME P450 83B1). The seeds of an AtDOF4.2-overexpressing plant show a collapse-like morphology in the epidermal cells of the seed coat. The mucilage contents and the concentration and composition of mucilage monosaccharides are significantly changed in the seed coat of transgenic plants. AtDOF4.2 may exert its effects on the seed epidermis through the direct binding and activation of the cell wall loosening-related gene AtEXPA9 (A. thaliana EXPANSIN-A9). The dof4.2 mutant did not exhibit changes in branching or its seed coat; however, the silique length and seed yield were increased. AtDOF4.4, which is a close homologue of AtDOF4.2, also promotes shoot branching and affects silique size and seed yield. Manipulation of these genes should have a practical use in the improvement of agronomic traits in important crops.