Liqin Cheng

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.

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.

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.

LcWRKY5 : an unknown function gene from sheepgrass improves drought tolerance in transgenic Arabidopsis

Key messageThe expression ofLcWRKY5was induced significantly by salinity, mannitol and cutting treatments.Arabidopsis-overexpressingLcWRKY5greatly increased dehydration tolerance by regulating the expression of multiple stress-responsive genes.AbstractBased on the data of sheepgrass 454 high-throughout sequencing and expression analysis results, a drought-induced gene LcWRKY5 was isolated and cloned, and the biological role of the gene has not been reported until now. Bioinformatics analysis showed that LcWRKY5 contains one conserved WD domain and belongs to the group II WRKY protein family. LcWRKY5 shows high sequence identity with predicted or putative protein products of Hordeum vulgare, Aegilops tauschii, Triticum aestivum, Brachypodium distachyon, Oryza sativa, but it has low homology with WRKYs from dicotyledonous plants. Several drought-inducibility, fungal elicitor, MeJA-responsiveness, endosperm, light, anoxic specific inducibility, and circadian control elements were found in the promoter region of LcWRKY5. Tissue-specific expression patterns showed that LcWRKY5 is expressed in roots and leaves, without expression in other tissues. The expression of LcWRKY5 was induced significantly under salinity and mannitol stresses but was not notably changed under cold and Abscisic acid stress. The LcWRKY5 protein exhibits transcription activation activity in the yeast one-hybrid system. Overexpressing LcWRKY5 exhibited increased rates of cotyledon greening and plant survival in transgenic Arabidopsis compared with wild-type plants under drought stress, and the expression levels of DREB2A and RD29A in transgenic plants were enhanced under drought stress. These results indicated that LcWRKY5 may play an important role in drought-response networks through regulation of the DREB2A pathway. LcWRKY5 can be a candidate gene for engineering drought tolerance in other crops.

New Insights on Drought Stress Response by Global Investigation of Gene Expression Changes in Sheepgrass (Leymus chinensis)

Water is a critical environmental factor that restricts the geographic distribution of plants. Sheepgrass [Leymus chinensis, (Trin.) Tzvel] is an important forage grass in the Eurasia Steppe and a close germplasm for wheat and barley. This native grass adapts well to adverse environments such as cold, salinity, alkalinity and drought, and it can survive when the soil moisture may be less than 6% in dry seasons. However, little is known about how sheepgrass tolerates water stress at the molecular level. Here, drought stress experiment and RNA-sequencing (RNA-seq) was performed in three pools of RNA samples (control, drought stress, and rewatering). We found that sheepgrass seedlings could still survive when the soil water content (SWC) was reduced to 14.09%. Differentially expressed genes (DEGs) analysis showed that 7320 genes exhibited significant responses to drought stress. Of these DEGs, 2671 presented opposite expression trends before and after rewatering. Furthermore, ~680 putative sheepgrass-specific water responsive genes were revealed that can be studied deeply. Gene ontology (GO) annotation revealed that stress-associated genes were activated extensively by drought treatment. Interestingly, cold stress-related genes were up-regulated greatly after drought stress. The DEGs of MAPK and calcium signal pathways, plant hormone ABA, jasmonate, ethylene, brassinosteroid signal pathways, cold response CBF pathway participated coordinatively in sheepgrass drought stress response. In addition, we identified 288 putative transcription factors (TFs) involved in drought response, among them, the WRKY, NAC, AP2/ERF, bHLH, bZIP, and MYB families were enriched, and might play crucial and significant roles in drought stress response of sheepgrass. Our research provided new and valuable information for understanding the mechanism of drought tolerance in sheepgrass. Moreover, the identification of genes involved in drought response can facilitate the genetic improvement of crops by molecular breeding.

Overexpression of a novel cold-responsive transcript factor LcFIN1 from sheepgrass enhances tolerance to low temperature stress in transgenic plants.

As a perennial forage crop broadly distributed in eastern Eurasia, sheepgrass (Leymus chinensis (Trin.) Tzvel) is highly tolerant to low-temperature stress. Previous report indicates that sheepgrass is able to endure as low as -47.5 °C,allowing it to survive through the cold winter season. However, due to the lack of sufficient studies, the underlying mechanism towards the extraordinary low-temperature tolerance is unclear. Although the transcription profiling has provided insight into the transcriptome response to cold stress, more detailed studies are required to dissect the molecular mechanism regarding the excellent abiotic stress tolerance. In this work, we report a novel transcript factor LcFIN1 (L. chinensis freezing-induced 1) from sheepgrass. LcFIN1 showed no homology with other known genes and was rapidly and highly induced by cold stress, suggesting that LcFIN1 participates in the early response to cold stress. Consistently, ectopic expression of LcFIN1 significantly increased cold stress tolerance in the transgenic plants, as indicated by the higher survival rate, fresh weight and other stress-related indexes after a freezing treatment. Transcriptome analysis showed that numerous stress-related genes were differentially expressed in LcFIN1-overexpressing plants, suggesting that LcFIN1 may enhance plant abiotic stress tolerance by transcriptional regulation. Electrophoretic mobility shift assays and CHIP-qPCR showed that LcCBF1 can bind to the CRT/DRE cis-element located in the promoter region of LcFIN1, suggesting that LcFIN1 is directly regulated by LcCBF1. Taken together, our results suggest that LcFIN1 positively regulates plant adaptation response to cold stress and is a promising candidate gene to improve crop cold tolerance.