Qing-Tian Li

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.

An S-domain receptor-like kinase OsSIK2 confers abiotic stress tolerance and delays dark-induced leaf senescence in rice

A receptor-like kinase, part of a family of proteins contributing to plant development and defense, is involved in abiotic stress and the senescence process, integrating stress signals into a developmental program for adaptive growth. Receptor-like kinases play important roles in plant development and defense responses; however, their functions in other processes remain unclear. Here, we report that OsSIK2, an S-domain receptor-like kinase from rice (Oryza sativa), is involved in abiotic stress and the senescence process. OsSIK2 is a plasma membrane-localized protein with kinase activity in the presence of Mn2+. OsSIK2 is expressed mainly in rice leaf and sheath and can be induced by NaCl, drought, cold, dark, and abscisic acid treatment. Transgenic plants overexpressing OsSIK2 and mutant sik2 exhibit enhanced and reduced tolerance to salt and drought stress, respectively, compared with the controls. Interestingly, a truncated version of OsSIK2 without most of the extracellular region confers higher salt tolerance than the full-length OsSIK2, likely through the activation of different sets of downstream genes. Moreover, seedlings of OsSIK2-overexpressing transgenic plants exhibit early leaf development and a delayed dark-induced senescence phenotype, while mutant sik2 shows the opposite phenotype. The downstream PR-related genes specifically up-regulated by full-length OsSIK2 or the DREB-like genes solely enhanced by truncated OsSIK2 are all induced by salt, drought, and dark treatments. These results indicate that OsSIK2 may integrate stress signals into a developmental program for better adaptive growth under unfavorable conditions. Manipulation of OsSIK2 should facilitate the improvement of production in rice and other crops.

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.

Three SAUR proteins SAUR76, SAUR77 and SAUR78 promote plant growth in Arabidopsis

Ethylene perceived by a family of five receptors regulates many developmental processes in Arabidopsis. Here we conducted the yeast two-hybrid assay to screen for additional unidentified proteins that interact with subfamily II ethylene receptor ETR2. Three SAUR proteins, named SAUR76, 77 and 78, were identified to associate with both ETR2 and EIN4 in different assays. Interaction of SAUR76 and SAUR78 with ETR2 was further verified by co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays. Expressions of SAUR76-78 are induced by auxin and ethylene treatments. Compared with wild type, SAUR-overexpressing plants exhibit reduced ethylene sensitivity, while SAUR-RNAi lines exhibit enhanced ethylene sensitivity. Overexpressing the three SAURs partially complements the phenotype of subfamily II ethylene receptor loss-of-function double mutant etr2-3ein4-4, which has increased ethylene response and small cotyledon and rosette. saur76 mutation partially suppresses the reduced ethylene sensitivity of etr2-2. SAUR76/78 proteins are regulated by 26S proteasome system and larger tag increases their protein stability. These findings suggest that SAUR76-78 may affect ethylene receptor signaling and promote plant growth in Arabidopsis.

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.

Trihelix transcription factor GT - 4 mediates salt tolerance via interaction with TEM2 in Arabidopsis

BackgroundTrihelix transcription factor family is plant-specific and plays important roles in developmental processes. However, their function in abiotic stress response is largely unclear.ResultsWe studied one member GT-4 from Arabidopsis in relation to salt stress response. GT-4 expression is induced by salt stress and GT-4 protein is localized in nucleus and cytoplasm. GT-4 acts as a transcriptional activator and its C-terminal end is the activation domain. The protein can bind to the cis-elements GT-3 box, GT-3b box and MRE4. GT-4 confers enhanced salt tolerance in Arabidopsis likely through direct binding to the promoter and activation of Cor15A, in addition to possible regulation of other relevant genes. The gt-4 mutant shows salt sensitivity. TEM2, a member of AP2/ERF family was identified to interact with GT-4 in yeast two-hybrid, BiFC and Co-IP assays. Loss-of-function of TEM2 exerts no significant difference on salt tolerance or Cor15A expression in Arabidopsis. However, double mutant gt-4/tem2 shows greater sensitivity to salt stress and lower transcript level of Cor15A than gt-4 single mutant. GT-4 plus TEM2 can synergistically increase the promoter activity of Cor15A.ConclusionsGT-4 interacts with TEM2 and then co-regulates the salt responsive gene Cor15A to improve salt stress tolerance.

Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation

Translationally-controlled tumor protein binds with a class of ethylene receptors at the endoplasmic reticulum and affects protein degradation. Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation.

The Alfin‐like homeodomain finger protein AL5 suppresses multiple negative factors to confer abiotic stress tolerance in Arabidopsis

Plant homeodomain (PHD) finger proteins affect processes of growth and development by changing transcription and reading epigenetic histone modifications, but their functions in abiotic stress responses remain largely unclear. Here we characterized seven Arabidopsis thaliana Alfin1-like PHD finger proteins (ALs) in terms of the responses to abiotic stresses. ALs localized to the nucleus and repressed transcription. Except AL6, all the ALs bound to G-rich elements. Mutations of the amino acids at positions 34 and 35 in AL6 caused loss of ability to bind to G-rich elements. Expression of the AL genes responded differentially to osmotic stress, salt, cold and abscisic acid treatments. AL5-over-expressing plants showed higher tolerance to salt, drought and freezing stress than Col-0. Consistently, al5 mutants showed reduced stress tolerance. We used ChIP-Seq assays to identify eight direct targets of AL5, and found that AL5 binds to the promoter regions of these genes. Knockout mutants of five of these target genes exhibited varying tolerances to stresses. These results indicate that AL5 inhibits multiple signaling pathways to confer stress tolerance. Our study sheds light on mechanisms of AL5-mediated signaling in abiotic stress responses, and provides tools for improvement of stress tolerance in crop plants.

The transcriptomic signature of developing soybean seeds reveals the genetic basis of seed trait adaptation during domestication

Cultivated soybean has undergone many transformations during domestication. In this paper we report a comprehensive assessment of the evolution of gene co-expression networks based on the analysis of 40 transcriptomes from developing soybean seeds in cultivated and wild soybean accessions. We identified 2680 genes that are differentially expressed during seed maturation and established two cultivar-specific gene co-expression networks. Through analysis of the two networks and integration with quantitative trait locus data we identified two potential key drivers for seed trait formation, GA20OX and NFYA. GA20OX encodes an enzyme in a rate-limiting step of gibberellin biosynthesis, and NFYA encodes a transcription factor. Overexpression of GA20OX and NFYA enhanced seed size/weight and oil content, respectively, in seeds of transgenic plants. The two genes showed significantly higher expression in cultivated than in wild soybean, and the increases in expression were associated with genetic variations in the promoter region of each gene. Moreover, the expression of GA20OX and NFYA in seeds of soybean accessions correlated with seed weight and oil content, respectively. Our study reveals transcriptional adaptation during soybean domestication and may identify a mechanism of selection by expression for seed trait formation, providing strategies for future breeding practice.

Soybean GmDREBL Increases Lipid Content in Seeds of Transgenic Arabidopsis.

A DREB-type transcription factor gene GmDREBL has been characterized for its functions in oil accumulation in seeds. The gene is specifically expressed in soybean seeds. The GmDREBL is localized in nucleus and has transcriptional activation ability. Overexpression of GmDREBL increased the fatty acid content in the seeds of transgenic Arabidopsis plants. GmDREBL can bind to the promoter region of WRI1 to activate its expression. Several other genes in the fatty acid biosynthesis pathway were also enhanced in the GmDREBL-transgenic plants. The GmDREBL can be up-regulated by GmABI3 and GmABI5. Additionally, overexpression of GmDREBL significantly promoted seed size in transgenic plants compared to that of WT plants. Expression of the DREBL is at higher level on the average in cultivated soybeans than that in wild soybeans. The promoter of the DREBL may have been subjected to selection during soybean domestication. Our results demonstrate that GmDREBL participates in the regulation of fatty acid accumulation by controlling the expression of WRI1 and its downstream genes, and manipulation of the gene may increase the oil contents in soybean plants. Our study provides novel insights into the function of DREB-type transcription factors in oil accumulation in addition to their roles in stress response.