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Cyperus esculentus is widely representing one of the important oil crops around the world, which provides valuable resources of edible tubers called tiger nut. The chemical composition and high ability to produce fats emphasize the role of tiger nut in promoting oil crop productivity. However, the underlying molecular mechanism of the production and accumulation of lipids in tiger nut development still remains unclear. Here, we conducted comprehensive transcriptomics and lipidomics analyses at different developmental stages of tuber in Cyperus esculentus. Lipidomic analyses confirmed that the accumulation of lipids including glycolipids, phospholipids, and glycerides were significantly enriched during tuber development from early to mature stage. The proportion of phosphatidylcholines (PC) declined during all stages and phosphatidyl ethanolamine (PE) was significantly declined in early and middle stages. These findings implied that PC is actively involved in triacylglycerol (TAG) biosynthesis during the tubers development, whereas PE may participate in TAG metabolism during early and middle stages. Comparative transcriptomics analyses indicated several genomic and metabolic pathways associated with lipid metabolism during tuber development in tiger nut. The Pearson correlation analysis showed that TAG synthesis in different developmental stages was attributed to 37 candidate transcripts including CePAH1. The up-regulation of diacylglycerol (DAG) and oil content in yeast, resulted from the inducible expression of exogenous CePAH1 confirmed the central role of this candidate gene in lipid metabolism. Our results demonstrated the foundation of an integrative metabolic model for understanding the molecular mechanism of tuber development in tiger nut, in which lipid biosynthesis plays a central role.

GmGASA is the GASA gibberellin regulated cysteine-rich protein family. The expression of GmGASA is up-regulated by gibberellin, which is the longest plant hormone in plants playing vital roles in plant development. However, very few reports explaining the direct regulation of downstream genes by GASA gene are available. In the current study, the GmGASA32, a member of the GASA family affecting soybean height was identified. In the early stage, preliminary verification of the response of GmGASA32 to gibberellin through phenotypic experiment was done. The promoter activity analysis confirmed that GmGASA32 was induced by gibberellin. Subcellular localization showed that GmGASA32-GFP fusion protein enriched in the nucleus after gibberellin treatment. In order to confirm the function of GmGASA32 in the nucleus, we confirmed that the GASA domain in the C terminal of GmGASA32 can interact with GmCDC25 (cell cycle-associated protein) through the bimolecular fluorescence complementation (BiFC) assay.

Background: Oil crop varieties are currently subjected to an increasing worldwile demand and the tiger nut (Cyperus esculentus L.) attracts significant attention because of its huge capacity of lipids production. In eukaryotic cells, the balance between the accumulation of lipids and the distribution of certain pivotal molecules is fundamental for the regulation of many complex transcriptional regulatory networks. However, many studies have struggled to understand the dynamic of lipids and the transcriptomic mechanisms governing their biosynthesis and accumulation process during plant development. Results: Our results displayed dynamic patterns for key lipids like glycolipid, phospholipid, and glyceride during the development of tiger nut tubers. Lipidomic analysis showed molecular species distribution of lipid class during developing stages. Here, we also characterrized transcription profiles of key transcripts that determined biosynthesis and distribution of natural lipids in tiger nuts tuber. The expression of FAD2 exhibited a significant influence on the molecular composition of phosphatidylcholines (PC) and phosphatidyl ethanolamine (PE) in tiger nuts. Moreover, during lipids accumulation, the expression pattern of three candidate transcripts of oleosin genes (OLE9, OLE10 and OLE11) also displayed significant leverage on the size of lipid drops. Conclusion: We described the significant alterations in the composition of lipids class during developing stages of tiger nut tuber, we also revealed transcriptional profiles of genes invloed in lipid biosynthesis and accumulation. These results provided new landscapes for research on lipid composition, synthesis and accumulation during different developmental stages of plant tubers.

Calcineurin B-like protein-interacting protein kinases (CIPKs) are key elements of plant abiotic stress signaling pathways. CIPKs are SOS2 (Salt Overly Sensitive 2)-like proteins (protein kinase S [PKS] proteins) which all contain a putative FISL motif. It seems that the FISL motif is found only in the SOS2 subfamily of protein kinases. In this study, the full-length cDNA of a soybean CIPK gene (GmPKS4) was isolated and was revealed to have an important role in abiotic stress responses. A qRT-PCR analysis indicated that GmPKS4 expression is upregulated under saline conditions or when exposed to alkali, salt-alkali, drought, or abscisic acid (ABA). A subcellular localization assay revealed the presence of GmPKS4 in the nucleus and cytoplasm. Further studies on the GmPKS4 promoter suggested it affects soybean resistance to various stresses. Transgenic Arabidopsis thaliana and soybean hairy roots overexpressing GmPKS4 had increased proline content as well as high antioxidant enzyme activities but decreased malondialdehyde levels following salt and salt-alkali stress treatments. Additionally, GmPKS4 overexpression activated reactive oxygen species scavenging systems, thereby minimizing damages due to oxidative and osmotic stresses. Moreover, upregulated stress-related gene expression levels were detected in lines overexpressing GmPKS4 under stress conditions. In conclusion, GmPKS4 improves soybean tolerance to salt and salt-alkali stresses. The overexpression of GmPKS4 enhances the scavenging of reactive oxygen species, osmolyte synthesis, and the transcriptional regulation of stress-related genes.

Following an in-depth transcriptomics-based approach, we first screened out and analyzed (in silico) cis motifs in a group of 63 drought-inducible genes (in soybean). Six novel synthetic promoters (SynP14-SynP19) were designed by concatenating 11 cis motifs, ABF, ABRE, ABRE-Like, CBF, E2F-VARIANT, G-box, GCC-Box, MYB1, MYB4, RAV1-A, and RAV1-B (in multiple copies and various combination) with a minimal 35s core promoter and a 222 bp synthetic intron sequence. In order to validate their drought-inducibility and root-specificity, the designed synthetic assemblies were transformed in soybean hairy roots to drive GUS gene using pCAMBIA3301. Through GUS histochemical assay (after a 72 h 6% PEG6000 treatment), we noticed higher glucuronidase activity in transgenic hairy roots harboring SynP15, SynP16, and SynP18. Further screening through GUS fluorometric assay flaunted SynP16 as the most appropriate combination of efficient drought-responsive cis motifs. Afterwards, we stably transformed SynP15, SynP16, and SynP18 in Arabidopsis and carried out GUS staining as well as fluorometric assays of the transgenic plants treated with simulated drought stress. Consistently, SynP16 retained higher transcriptional activity in Arabidopsis roots in response to drought. Thus the root-specific drought-inducible synthetic promoters designed using stimulus-specific cis motifs in a definite fashion could be exploited in developing drought tolerance in soybean and other crops as well. Moreover, the rationale of design extends our knowledge of trial-and-error based cis engineering to construct synthetic promoters for transcriptional upgradation against other stresses.

Following an in-depth transcriptomics-based approach, we first screened out and analyzed (in silico) cis motifs in a group of 63 drought-inducible genes (in soybean). Six novel synthetic promoters (SynP14-SynP19) were designed by concatenating 11 cis motifs, ABF, ABRE, ABRE-Like, CBF, E2F-VARIANT, G-box, GCC-Box, MYB1, MYB4, RAV1-A, and RAV1-B (in multiple copies and various combination) with a minimal 35s core promoter and a 222 bp synthetic intron sequence. In order to validate their drought-inducibility and root-specificity, the designed synthetic assemblies were transformed in soybean hairy roots to drive GUS gene using pCAMBIA3301. Through GUS histochemical assay (after a 72 h 6% PEG6000 treatment), we noticed higher glucuronidase activity in transgenic hairy roots harboring SynP15, SynP16, and SynP18. Further screening through GUS fluorometric assay flaunted SynP16 as the most appropriate combination of efficient drought-responsive cis motifs. Afterwards, we stably transformed SynP15, SynP16, and SynP18 in Arabidopsis and carried out GUS staining as well as fluorometric assays of the transgenic plants treated with simulated drought stress. Consistently, SynP16 retained higher transcriptional activity in Arabidopsis roots in response to drought. Thus the root-specific drought-inducible synthetic promoters designed using stimulus-specific cis motifs in a definite fashion could be exploited in developing drought tolerance in soybean and other crops as well. Moreover, the rationale of design extends our knowledge of trial-and-error based cis engineering to construct synthetic promoters for transcriptional upgradation against other stresses.

GmGASA is the GASA gibberellin regulated cysteine-rich protein family. The expression of GmGASA is up-regulated by gibberellin, which is the longest plant hormone in plants playing vital roles in plant development. However, very few reports explaining the direct regulation of downstream genes by GASA gene are available. In the current study, the GmGASA32, a member of the GASA family affecting soybean height was identified. In the early stage, preliminary verification of the response of GmGASA32 to gibberellin through phenotypic experiment was done. The promoter activity analysis confirmed that GmGASA32 was induced by gibberellin. Subcellular localization showed that GmGASA32-GFP fusion protein enriched in the nucleus after gibberellin treatment. In order to confirm the function of GmGASA32 in the nucleus, we confirmed that the GASA domain in the C terminal of GmGASA32 can interact with GmCDC25 (cell cycle-associated protein) through the bimolecular fluorescence complementation (BiFC) assay.

Additional file 16: Table S7. Details regarding the primers used in this study.

Vacuolar Na+/H+ antiporters play central roles in plant saline (NaCl) stress response. However, the functional characterizations of antiporters involved in plant saline-alkali (Na2CO3/NaHCO3) tolerance are limited. In this study, SucNHX1, a Na+/H+ antiporter gene was isolated from Suaeda corniculata, a halophyte native to the saline-alkali soil, and further studied in transgenic Arabidopsis thaliana. The SucNHX1 gene contains 2123 nucleotides with an open reading frame of 1665 nucleotides (GenBank accession number: DQ512716), encoding a polypeptide of 554 amino acids with a predicted molecular mass of 61.2 kDa. A northern blot analysis indicated that the enhanced mRNA accumulation of SucNHX1 was induced by sodium carbonate (Na2CO3) rather than by NaCl, polyethylene glycol or abscisic acid in S. corniculata seedlings. SucNHX1 was localized in membrane, nucleus and cytoplasm when it was transiently expressed in tobacco mesophyll cells. The expression of SucNHX1 in salt sensitive yeast mutant ΔenaΔnhx1 which lost of yeast vacuolar Na+/H+ antiporter ScNHX1 showed functional complementation. The transgenic Arabidopsis overexpressing SucNHX1 had higher Na2CO3 and NaCl tolerance levels than the wild-type plants. The expression patern of SucNHX1 under Na2CO3 and high NaCl stress in transgenic plants showed that SucNHX1 higher expressed under stress condition than normal control. The analysis of ion contents indicated that under Na2CO3 and NaCl conditions, there were more Na+ in the roots and higher K+/Na+ ratio in the leaves in the transgenic plants than in the wild-type plants. Thus, SucNHX1 may play a significant role in plant saline-alkali tolerance.

Designing the expression cassettes with desired properties remains the most important consideration of gene engineering technology. One of the challenges for predictive gene expression is the modeling of synthetic gene switches to regulate one or more target genes which would directly respond to specific chemical, environmental, and physiological stimuli. Assessment of natural promoter, high-throughput sequencing, and modern biotech inventory aided in deciphering the structure of cis elements and molding the native cis elements into desired synthetic promoter. Synthetic promoters which are molded by rearrangement of cis motifs can greatly benefit plant biotechnology applications. This review gives a glimpse of the manual in vivo gene regulation through synthetic promoters. It summarizes the integrative design strategy of synthetic promoters and enumerates five approaches for constructing synthetic promoters. Insights into the pattern of cis regulatory elements in the pursuit of desirable "gene switches" to date has also been reevaluated. Joint strategies of bioinformatics modeling and randomized biochemical synthesis are addressed in an effort to construct synthetic promoters for intricate gene regulation.

Potassium is a major cationic nutrient involved in numerous physiological processes in plants. The uptake of K+ is mediated by K+ channels and transporters, and the Shaker K+ channel gene family plays an essential role in K+ uptake and stress resistance in plants. However, little is known regarding this family in soybean. In this study, 14 members of the Shaker K+ channel gene family were identified in soybean and were classified into five groups. Protein domain analysis revealed that Shaker K+ channel gene members have an ion transport domain (ion trans), a cyclic nucleotide-binding domain, ankyrin repeat domains, and a dimerization domain in the potassium ion channel. Quantitative real-time polymerase chain reaction analysis indicated that the expression of eight genes (notably GmAKT1) in soybean leaves and roots was significantly increased in response to salt and drought stress. Furthermore, the overexpression of GmAKT1 in Arabidopsis enhanced root length, K+ concentration, and fresh/dry weight ratio compared with wild-type plants subjected to salt and drought stress; this suggests that GmAKT1 improves the tolerance of soybean to abiotic stress. Our results provide important insight into the characterization of Shaker K+ channel gene family members in soybean and highlight the function of GmAKT1 in soybean plants under salt and drought stress.

Fifteen transcription factors in the CAMTA (calmodulin binding transcription activator) family of soybean were reported to differentially regulate in multiple stresses, however, their functional analyses had not yet been attempted. To characterize their role in stresses, we first comprehensively analyzed the GmCAMTA family in silico and thereafter determined their expression pattern under drought. The bioinformatics analysis revealed multiple stress-related cis-regulatory elements including ABRE, SARE, G-box and W-box, 10 unique miRNA (microRNA) targets in GmCAMTA transcripts and 48 proteins in GmCAMTAs&rsquo, interaction network. We then cloned the 2769 bp CDS (coding sequence) of GmCAMTA12 in an expression vector and overexpressed in soybean and Arabidopsis through Agrobacterium-mediated transformation. The T3 (Transgenic generation 3) stably transformed homozygous lines of Arabidopsis exhibited enhanced tolerance to drought in soil as well as on MS (Murashige and Skoog) media containing mannitol. In their drought assay, the average survival rate of transgenic Arabidopsis lines OE5 and OE12 (Overexpression Line 5 and Line 12) was 83.66% and 87.87%, respectively, which was ~30% higher than that of wild type. In addition, the germination and root length assays as well as physiological indexes such as proline and malondialdehyde contents, catalase activity and leakage of electrolytes affirmed the better performance of OE lines. Similarly, GmCAMTA12 overexpression in soybean promoted drought-efficient hairy roots in OE chimeric plants as compare to that of VC (Vector control). In parallel, the improved growth performance of OE in Hoagland-PEG (polyethylene glycol) and on MS-mannitol was revealed by their phenotypic, physiological and molecular measures. Furthermore, with the overexpression of GmCAMTA12, the downstream genes including AtAnnexin5, AtCaMHSP, At2G433110 and AtWRKY14 were upregulated in Arabidopsis. Likewise, in soybean hairy roots, GmELO, GmNAB and GmPLA1-IId were significantly upregulated as a result of GmCAMTA12 overexpression and majority of these upregulated genes in both plants possess CAMTA binding CGCG/CGTG motif in their promoters. Taken together, we report that GmCAMTA12 plays substantial role in tolerance of soybean against drought stress and could prove to be a novel candidate for engineering soybean and other plants against drought stress. Some research gaps were also identified for future studies to extend our comprehension of Ca-CaM-CAMTA-mediated stress regulatory mechanisms.

Flavonoid is one of the widespread groups of plant secondary metabolites that provide several health benefits. However, the explicit mechanism of flavonoid biosynthesis in plants largely remains unclear. Chalcone isomerase an important class of enzyme presents crucial role during flavonoid metabolism in many plants. Here, we isolated the full-length cDNA (1161 bp) of a novel Chalcone Isomerase from safflower encoding 217 amino acid polypeptide using oligos from 5′ and 3′ ends. The result of Sanger sequencing and phylogenetic analysis revealed that CtCHI is highly homologous to other plants, including typical polyadenylation signals AATAA and Poly A tail. The transient expression in tobacco mesophyll cells using Green Fluorescent Protein tagging determined the subcellular localization of CtCHI in cell membrane and nucleus. The CtCHI ectopic expression in different safflower varieties at different flowering stages showed that CtCHI were found in abundance at the bud stage of Jihong No. 1. Further correlation analysis between CtCHI expression and flavonoid accumulation at various flowering phases suggested that CtCHI might play a potential role during flavonoid biosynthesis in safflower. In addition, the overexpression of pBASTA-CtCHI in transgenic Arabidopsis infiltrated with floral dip transformation showed relatively higher expression level and increased flavonoid accumulation than wild type. Moreover, the in vitro enzymatic activity and HPLC analysis of transgenic Arabidopsis confirmed the de novo biosynthesis of Rutin. Taken together, our findings laid the foundation of identifying an important gene that might influence flavonoid metabolism in safflower. Electronic supplementary material The online version of this article (10.1186/s13568-019-0854-x) contains supplementary material, which is available to authorized users.

Diacylglycerol kinase (DGK) is an enzyme that plays a pivotal role in abiotic and biotic stress responses in plants by transforming the diacylglycerol into phosphatidic acid. However, there is no report on the characterization of soybean DGK genes in spite of the availability of the soybean genome sequence. In this study, we performed genome-wide analysis and expression profiling of the DGK gene family in the soybean genome. We identified 12 DGK genes (namely GmDGK1-12) which all contained conserved catalytic domains with protein lengths and molecular weights ranging from 436 to 727 amino acids (aa) and 48.62 to 80.93 kDa, respectively. Phylogenetic analyses grouped GmDGK genes into three clusters&mdash, cluster I, cluster II, and cluster III&mdash, which had three, four, and five genes, respectively. The qRT-PCR analysis revealed significant GmDGK gene expression levels in both leaves and roots coping with polyethylene glycol (PEG), salt, alkali, and salt/alkali treatments. This work provides the first characterization of the DGK gene family in soybean and suggests their importance in soybean response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of this gene family.

Additional file 1: Table S1. List of primers. Fig. S1. Plant over expression vector. Fig. S2. Detection of transgenic plants. Fig. S3. Strongest transgenic Arabidopsis lines obtained in our study. Fig. S4. Other putative CHIs in safflower. Fig. S5. Graphical representation of pCAMBIA1302-GFP-35S vector.

GmGASA is the GASA gibberellin regulated cysteine-rich protein family. The expression of GmGASA is up-regulated by gibberellin, which is the longest plant hormone in plants playing vital roles in plant development. However, very few reports explaining the direct regulation of downstream genes by GASA gene are available. In the current study, the GmGASA32, a member of the GASA family affecting soybean height was identified. In the early stage, preliminary verification of the response of GmGASA32 to gibberellin through phenotypic experiment was done. The promoter activity analysis confirmed that GmGASA32 was induced by gibberellin. Subcellular localization showed that GmGASA32-GFP fusion protein enriched in the nucleus after gibberellin treatment. In order to confirm the function of GmGASA32 in the nucleus, we confirmed that the GASA domain in the C terminal of GmGASA32 can interact with GmCDC25 (cell cycle-associated protein) through the bimolecular fluorescence complementation (BiFC) assay.

Human rotavirus (HRV) is the primary cause of severe gastroenteritis in children. However, there is currently no protective virus for rotavirus available. In the present study, an HRVVP7-cholera toxin B subunit (CTB) fusion protein was expressed in Arabidopsis thaliana. To determine the adjuvant effect of HRVVP7-CTB, HRVVP7 without CTB was expressed in the same manner. HRVVP7-CTB accounted for 0.39% of the total soluble protein (TSP) in the transgenic seeds and 52.65 µg/g of HRVVP7 protein was expressed in these seeds. Mice were immunized with TSP from the transformed seeds and produced serum immunoglobulin G (IgG) and mucosal IgA specifically directed against HRVVP7. Antibody titers were highest in mice orally immunized with the plant-expressed HRVVP7-CTB protein, whereas HRVVP7-CTB-specific IgG neutralized the rotavirus. Suckling pups born from dams immunized with the HRVVP7-CTB fusion protein were protected against challenge with virulent rotavirus. The results of the present study suggest that the HRVVP7-CTB fusion protein produced in A. thaliana may be a rotaviral-specific candidate subunit vaccine.

Previously, it was reported that miR396s interact with growth-regulating factors (GRFs) to modulate plant growth, development, and stress resistance. In soybean, 11 gma-miR396 precursors (Pre-miR396a–k) were found, and 24 GmGRFs were predicted as targets of seven mature gma-miR396s (gma-miR396a/b/c/e/h/i/k). To explore the roles of the miR396–GRF module in low water availability response of soybean, we analyzed the expression of Pre-miR396a–k, and found that Pre-miR396a/i/bdgk/e/h were up-regulated in leaves and down-regulated in roots; on the contrary, GmGRF5/6/7/8/15/17/21 were down-regulated in leaves and GmGRF1/2/17/18/19/20/21/22/23/24 were up-regulated in roots of low water potential stressed soybean. Any one of gma-miR396a/b/c/e/h/i/k was able to interact with 20 GmGRFs (GmGRF1/2/6–11/13–24), confirming that this module represents a multi-to-multi network interaction. We generated Arabidopsis plants over-expressing each of the 11 gma-miR396 precursors (Pre-miR396a–k), and seven of them (miR396a/b/c/e/h/i/k-OE transgenic Arabidopsis) showed altered development. The low water availability of miR396a/b/c/e/h/i/k-OE was enhanced in leaves but reduced in seeds and roots. Contrary to previous reports, miR396a/b/c/i-OE seedlings showed lower survival rate than WT when recovering after rewatering under soil drying. In general, we believe our findings are valuable to understand the role of gma-miR396 family in coordinating development and low water availability responses, and can provide potential strategies and directions for soybean breeding programs to improve seed yield and plant drought tolerance.

Plant microRNAs are small non-coding, endogenic RNA molecule (containing 20-24 nucleotides) produced from miRNA precursors (pri-miRNA and pre-miRNA). Evidence suggests that up and down regulation of the miRNA targets the mRNA genes involved in resistance against biotic and abiotic stresses. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful technique to analyze variations in mRNA levels. Normalizing the data using reference genes is essential for the analysis of reliable RT-qPCR data. In this study, two groups of candidate reference mRNAs and miRNAs in soybean leaves and roots treated with various abiotic stresses (PEG-simulated drought, salinity, alkalinity, salinity+alkalinity, and abscisic acid) were analyzed by RT-qPCR. We analyzed the most appropriate reference mRNA/miRNAs using the geNorm, NormFinder, and BestKeeper algorithms. According to the results, Act and EF1b were the most suitable reference mRNAs in leaf and root samples, for mRNA and miRNA precursor data normalization. The most suitable reference miRNAs found in leaf and root samples were 166a and 167a for mature miRNA data normalization. Hence the best combinations of reference mRNAs for mRNA and miRNA precursor data normalization were EF1a + Act or EF1b + Act in leaf samples, and EF1a + EF1b or 60s + EF1b in root samples. For mature miRNA data normalization, the most suitable combinations of reference miRNAs were 166a + 167d in leaf samples, and 171a + 156a or 167a + 171a in root samples. We identified potential reference mRNA/miRNAs for accurate RT-qPCR data normalization for mature miRNA, miRNA precursors, and their targeted mRNAs. Our results promote miRNA-based studies on soybean plants exposed to abiotic stress conditions.

Salt, saline-alkali and drought stresses are major environmental constraints for the production and yield of soybean worldwide. To identify genes responsible for stress tolerance, the transcriptional profiles of genes in leaves and roots of seedlings (two-leaf stage) of the soybean inbred line HJ-1 were examined after 48 h under various stress conditions; salt (120 mM NaCl), saline-alkali (70 mM NaCl and 50mM NaHCO(3)) and drought (2% PEG 8000). Gene expression at the transcriptional level was investigated using high-throughput Illumina sequencing technology and bioinformatics tools. Under salt, saline-alkali and drought stress, 874, 1897, and 535 genes, respectively, were up-regulated in leaves, and 1822, 1731 and 1690 genes, respectively, were up-regulated in roots, compared with expression in the corresponding organ in control plants. Comparisons among salt, saline-alkali and drought stress yielded similar results in terms of the percentage of genes classified into each GO category. Moreover, 69 genes differentially expressed in both organs with similar expression patterns clustered together in the taxonomic tree across all conditions. Furthermore, comparison of gene expression among salt, saline-alkali and drought treated plants revealed that genes associated with calcium-signaling and nucleic acid pathways were up-regulated in the responses to all three stresses, indicating a degree of cross-talk among these pathways. These results could provide new insights into the stress tolerance mechanisms of soybean.

Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the Poaceae (also called Gramineae) family, which adapts well to drought, saline and alkaline conditions. However, little is known about the stress tolerance of L. chinensis at the molecular level. microRNAs (miRNAs) are known to play critical roles in nutrient homeostasis, developmental processes, pathogen responses, and abiotic stress in plants. In this study, we used Solexa sequencing technology to generate high-quality small RNA data from three L. chinensis groups: a control group, a saline-alkaline stress group (100 mM NaCl and 200 mM NaHCO3), and a drought stress group (20% polyethylene glycol 2000). From these data we identified 132 known miRNAs and 16 novel miRNAs candidates. For these miRNAs we also identified target genes that encode a broad range of proteins that may be correlated with abiotic stress regulation. This is the first study to demonstrate differentially expressed miRNAs in L. chinensis under saline-alkali and drought stress. These findings may help explain the saline-alkaline and drought stress responses in L. chinensis.

BackgroundLeymus chinensis (Trin.) Tzvel. is a high saline-alkaline tolerant forage grass genus of the tribe Gramineae family, which also plays an important role in protection of natural environment. To date, little is known about the saline-alkaline tolerance of L. chinensis on the molecular level. To better understand the molecular mechanism of saline-alkaline tolerance in L. chinensis, 454 pyrosequencing was used for the transcriptome study.ResultsWe used Roche-454 massive parallel pyrosequencing technology to sequence two different cDNA libraries that were built from the two samples of control and under saline-alkaline treatment (optimal stress concentration-Hoagland solution with 100 mM NaCl and 200 mM NaHCO(3)). A total of 363,734 reads in control group and 526,267 reads in treatment group with an average length of 489 bp and 493 bp were obtained, respectively. The reads were assembled into 104,105 unigenes with MIRA sequence assemable software, among which, 73,665 unigenes were in control group, 88,016 unigenes in treatment group and 57,576 unigenes in both groups. According to the comparative expression analysis between the two groups with the threshold of "log2 Ratio ≥1", there were 36,497 up-regulated unegenes and 18,218 down-regulated unigenes predicted to be the differentially expressed genes. After gene annotation and pathway enrichment analysis, most of them were involved in stress and tolerant function, signal transduction, energy production and conversion, and inorganic ion transport. Furthermore, 16 of these differentially expressed genes were selected for real-time PCR validation, and they were successfully confirmed with the results of 454 pyrosequencing.ConclusionsThis work is the first time to study the transcriptome of L. chinensis under saline-alkaline treatment based on the 454-FLX massively parallel DNA sequencing platform. It also deepened studies on molecular mechanisms of saline-alkaline in L. chinensis, and constituted a database for future studies.