Shuangyan Chen

Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana

Tocopherol cyclase (VTE1, encoded by VTE1 gene) catalyzes the penultimate step of tocopherol synthesis. Transgenic tobacco plants overexpressing VTE1 from Arabidopsis were exposed to drought conditions during which transgenic lines had decreased lipid peroxidation, electrolyte leakage and H2O2 content, but had increased chlorophyll compared with the wild type. Thus VTE1 can be used to increase vitamin E content of plants and also to enhance tolerance to environmental stresses.

Cloning two P5CS genes from bioenergy sorghum and their expression profiles under abiotic stresses and MeJA treatment.

Sweet sorghum (Sorghum bicolor (Linn.) Moench) has promise as a bioenergy feedstock in China and other countries for its use in the production of ethanol as the result of its high fermentable sugar accumulation in stems. To boost biofuel production and extend its range, we seek to improve its stress tolerance. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS (Δ1-pyrroline-5-carboxylate synthetase) is a key regulatory enzyme that plays a crucial role in proline biosynthesis. We isolated two closely related P5CS genes from sweet sorghum, designated SbP5CS1 (GenBank accession number: GQ377719) and SbP5CS2 (GenBank accession number: GQ377720), which are located on chromosome 3 and 9 and encode 729 and 716 amino acid polypeptides, respectively. The homology between the two sweet sorghum P5CS genes was 76%. Promoter analysis of the two P5CS genes revealed that both sequences not only contained the expected cis regulatory regions such as TATA and CAAT boxes, but also had many stress response elements. Expression analysis revealed that SbP5CS1 and SbP5CS2 transcripts were up-regulated after treatment of 10-day-old seedlings of sweet sorghum with drought, salt (250mM NaCl) and MeJA (10μM). The expression levels of the both SbP5CS genes were significantly increased after 3-day drought stress. Under high salt treatment, peak SbP5CS1 expression was detected at 4h and 8h for SbP5CS2 in roots, while the trends of expression were nearly identical in leaves. In contrast, under drought and high salt stress, the up-regulated expression of SbP5CS1 was higher than that of SbP5CS2. When the seedlings were exposed to MeJA, rapid transcript induction of SbP5CS1 was detected at 2h in leaves, and the SbP5CS2 expression level increase was detected at 4h post-treatment. SbP5CS1 and SbP5CS2 also show different temporal and spatial expression patterns. SbP5CS2 gene was ubiquitously expressed whereas SbP5CS1 was mainly expressed in mature vegetative and reproductive organs. Proline concentration increased after stress application and was correlated with SbP5CS expression. Our results suggest that the SbP5CS1 and SbP5CS2 are stress inducible genes but might play non-redundant roles in plant development. The two genes could have the potential to be used in improving stress tolerance of sweet sorghum and other bioenergy feedstocks.

Major Energy Plants and Their Potential for Bioenergy Development in China

China is rich in energy plant resources. In this article, 64 plant species are identified as potential energy plants in China. The energy plant species include 38 oilseed crops, 5 starch-producing crops, 3 sugar-producing crops and 18 species for lignocellulosic biomass. The species were evaluated on the basis of their production capacity and their resistance to salt, drought, and/or low temperature stress. Ten plant species have high production and/or stress resistance and can be potentially developed as the candidate energy plants. Of these, four species could be the primary energy plants in China: Barbados nut (Jatropha curcas L.), Jerusalem artichoke (Helianthus tuberosus L.), sweet sorghum (Sorghum bicolor L.) and Chinese silvergrass (Miscanthus sinensis Anderss.). We discuss the use of biotechnological techniques such as genome sequencing, molecular markers, and genetic transformation to improve energy plants. These techniques are being used to develop new cultivars and to analyze and manipulate genetic variation to improve attributes of energy plants in China.

The transcriptional factor LcDREB2 cooperates with LcSAMDC2 to contribute to salt tolerance in Leymus chinensis

S-Adenosyl-methionine decarboxylase (SAMDC) and dehydration responsive element-binding proteins (DREBs) can improve plant resistance to abiotic stresses. These proteins have been extensively studied, but the mechanism for transcriptional regulation of SAMDC remains unclear. In this paper, the LcSAMDC2 gene and its promoter were isolated from Leymus chinensis. Two DRE cis-elements were identified from the promoter of LcSAMDC2 and shown to bind with LcDREB2. Subcellular localization and yeast one-hybrid assay revealed that LcDREB2 is a transcription factor. An electrophoretic mobility shift assay (EMSA) showed that LcDREB2 can bind to the LcSAMDC2 promoter probe containing a DRE element. Over-expression of LcDREB2 in L. chinensis callus increased expression of LcSAMDC2. Co-expression of LcDREB2 and the promoter of LcSAMDC2 fused with GUS in tobacco activated GUS activity. These results indicate that LcSAMDC2 is the downstream gene of LcDREB2. In addition, transgenic expression of LcDREB2 and LcSAMDC2 in Arabidopsis can improve the salt stress tolerance of transgenic lines. These results indicate that LcDREB2 cooperating with LcSAMDC2 contributes to resistance to abiotic stress.

Overexpression of sheepgrass R1-MYB transcription factor LcMYB1 confers salt tolerance in transgenic Arabidopsis.

Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is a dominant, rhizomatous grass that has extensive plasticity in adapting to various harsh environments. Based on data from 454 high-throughput sequencing (GS FLX) exposure to salt stress, an unknown functional MYB-related gene LcMYB1 was identified from sheepgrass. Tissue specific expression profiles showed that the LcMYB1 gene was expressed ubiquitously in different tissues, with higher expression levels observed in the rhizome and panicle. The expression of LcMYB1 was induced obviously by high salt, drought and abscisic acid and was induced slightly by cold. A fusion protein of LcMYB1 with green fluorescent protein (GFP) was localized to the nucleus, and yeast one-hybrid analysis indicated that LcMYB1 was an activator of transcriptional activity. LcMYB1-overexpressing plants were more tolerant to salt stress than WT plants. The amounts of proline and soluble sugars were higher in transgenic Arabidopsis than in WT plants under salt stress conditions. The overexpression of LcMYB1 enhanced the expression levels of P5CS1 and inhibited other salt stress response gene markers. These findings demonstrate that LcMYB1 influences the intricate salt stress response signaling networks by promoting different pathways than the classical DREB1A- and MYB2-mediated signaling pathway. Additionally, LcMYB1 is a promising gene resource for improving salinity tolerance in crops.

Transcriptome Analysis in Sheepgrass (Leymus chinensis): A Dominant Perennial Grass of the Eurasian Steppe

Background Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development. Results The transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat. Conclusions This research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species.

Proteomics analysis of rice seedling responses to ovine saliva

Grazing is accompanied by a multitude of processes including wounding, saliva deposition, and defoliation. Previous studies have focused on the effects of the grazing or clipping intensity on plant regrowth, survival, and composition in the grassland. However, the impact of saliva deposition on plants is poorly understood. In this study, rice was used as a model plant to study the differentially expressed proteins after ovine saliva treatment. The shoots of 2-week-old seedlings were crosscut and the lower parts were daubed with ovine saliva at the cut surface. After 2, 6, 12 and 24h, proteomics analysis was performed using proteins extracted from the saliva-treated shoots. The results showed that proteins involved in multiple pathways were differentially expressed in response to ovine saliva, including catalase (CAT), peroxiredoxin (Prx), ATP synthase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Moreover, real-time quantitative reverse-transcription-PCR (RT-PCR) data showed that most of the genes were also regulated at the transcript level. Our results indicate the ovine saliva induces an early response in the rice seedling by stress-related pathways. This study provides information about the response of rice seedlings to ovine saliva at the protein level.

LcSAIN1, a Novel Salt-Induced Gene from SheepGrass, Confers Salt Stress Tolerance in Transgenic Arabidopsis and Rice

Previously, we identified >1,500 genes that were induced by high salt stress in sheepgrass (Leymus chinensis, Gramineae: Triticeae) when comparing the changes in their transcription levels in response to high salt stress by next-generation sequencing. Among the identified genes, a gene of unknown function (designated as Leymus chinensis salt-induced 1, LcSAIN1) showed a high sequence identity to its homologs from wheat, Hordeum vulgare and Oryza sativa, but LcSAIN1 and its homologs produce hypothetical proteins with no conserved functional domains. Transcription of the LcSAIN1 gene was up-regulated by various stresses. The overexpression of LcSAIN1 in Arabidopsis and rice increased the greening rate of cotyledons, the fresh weight, root elongation, plant height and the plant survival rate when compared with control plants and conferred a tolerance against salt stress. Subcellular localization analysis indicated that LcSAIN1 is localized predominantly in the nucleus. Our results show that the LcSAIN1 gene might play an important positive modulation role in increasing the expression of transcription factors (MYB2 and DREB2A) and functional genes (P5CS and RAB18) in transgenic plants under salt stress and that it augments stress tolerance through the accumulation of compatible solutes (proline and soluble sugar) and the alleviation of changes in reactive oxygen species. The LcSAIN1 gene could be a potential resource for engineering salinity tolerance in important crop species.

Transcriptome analysis reveals common and distinct mechanisms for sheepgrass (Leymus chinensis) responses to defoliation compared to mechanical wounding.

Background Herbivore grazing is a multiple-component process that includes wounding, defoliation, and saliva deposition. Despite the extensive published research on mechanical wounding and defoliation, no analysis to identify the genes that specify defoliation and mechanical wounding has been performed. Moreover, the influence of the expression of these genes on plant regrowth after defoliation remains poorly understood. Results Seven cDNA libraries for RNA samples collected from stubble tissues that had been mechanically wounded or defoliated at 2, 6 and 24 h along with the control were sequenced using the Illumina/Solexa platform. A comparative transcriptomic analysis of the sequencing data was conducted. In total, 1,836 and 3,238 genes were detected with significant differential expression levels after wounding and defoliation, respectively, during one day. GO, KOG and pathway-based enrichment analyses were performed to determine and further understand the biological functions of those differentially expressed genes (DEGs). The results demonstrated that both wounding and defoliation activated the systemic synthesis of jasmonate (JA). However, defoliation specifically reduced the expression levels of ribosomal protein genes, cell division or cell expansion-related genes, and lignin biosynthesis genes and may have negatively affected plant growth. Further analysis revealed that the regrowth of elongating leaves was significantly retarded after defoliation at 6 h through the following 7 days of measurement, suggesting that the gene expression pattern and phenotype are consistent. Fifteen genes were selected, and their expression levels were confirmed by quantitative RT-PCR (qRT-PCR). Thirteen of them exhibited expression patterns consistent with the digital gene expression (DGE) data. Conclusions These sequencing datasets allowed us to elucidate the common and distinct mechanisms of plant responses to defoliation and wounding. Additionally, the distinct DEGs represent a valuable resource for novel gene discovery that may improve plant resistance to defoliation from various processes.

A novel salt-induced gene from sheepgrass, LcSAIN2, enhances salt tolerance in transgenic Arabidopsis

Salt stress affects plant growth and development, and limits the productivity of crops. Sheepgrass can grow well under various environmental and soil conditions and is a good wild resource in Triticeae. Using 454 high throughout sequencing technique, a large number of salt stress responsive genes have been picked out from sheepgrass. In this study, a novel salt-induced gene and its promoter were cloned and the gene was designated as LcSAIN2 (Leymus chinensissalt-induced 2). Bioinformatics analysis predicted that LcSAIN2 has one transmembrane helix and is localized in nucleus. Experiments of subcellular localization in tobacco leaf cells also indicated that it was mainly localized in nucleus. Several stress responsive elements were found in the promoter region of the LcSAIN2 gene. The expression analysis confirmed that LcSAIN2 was induced by salinity, PEG, ABA, and cold stresses, especially by high salinity. Overexpression of LcSAIN2 in Arabidopsis enhanced salt tolerance of transgenic plants by accumulating osmolytes, such as soluble sugars and free proline, and improving the expression levels of some stress-responsive transcription factors and key genes. Our results suggest that LcSAIN2 might play an important positive modulation role in salt stress tolerance and be a candidate gene utilized for enhancing stress tolerance in wheat and other crops.