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Abiotic stress, a major factor limit growth and productivity of major crops. Orchardgrass is one of the most important cool-season forage grasses in the world, and it is highly tolerant to abiotic stress. The MADS-box transcription factor family is one of the largest families in plants, and it plays vital roles in multiple biological processes. However, MADS-box transcription factors in orchardgrass, especially those involved in abiotic stress, have not yet been elucidated. Here, 123 DgMADS-box members were identified in orchardgrass and a detailed overview has been presented. Syntenic analysis indicated that the expansion of the DgMADS-box genes in orchardgrass is mainly dependent on tandem duplication events. Some DgMADS-box genes were induced by multiple abiotic stresses, indicating that these genes may play critical regulatory roles in orchardgrass response to various abiotic stresses. Heterologous expression showed that DgMADS114 and DgMADS115 could enhance stress tolerance of transgenic Arabidopsis, as revealed by longer root length or higher survival rates under PEG, NaCl, ABA, and heat stress. The results of this study provide a scientific basis for clarifying the functional characterization of MADS-box genes in orchardgrass in response to environmental stress can be further used to improve forages and crops via breeding programs.

Vernalization, influenced by environmental factors, is an essential process associated with the productivity of temperate crops, during which epigenetic regulation of gene expression plays an important role. Although DNA methylation is one of the major epigenetic mechanisms associated with the control of gene expression, global changes in DNA methylation in the regulation of gene expression during vernalization-induced flowering of temperate plants remain largely undetermined. To characterize vernalization-associated DNA methylation dynamics, we performed whole-genome bisulfite-treated sequencing and transcriptome sequencing in orchardgrass (Dactylis glomerata) during vernalization. The results revealed that increased levels of genome DNA methylation during the early vernalization of orchardgrass were associated with transcriptional changes in DNA methyltransferase and demethylase genes. Upregulated expression of vernalization-related genes during early vernalization was attributable to an increase in mCHH in the promoter regions of these genes. Application of an exogenous DNA methylation accelerator or overexpression of orchardgrass NUCLEAR POLY(A) POLYMERASE (DgPAPS4) promoted earlier flowering, indicating that DNA hypermethylation plays an important role in vernalization-induced flowering. Collectively, our findings revealed that vernalization-induced hypermethylation is responsible for floral primordium initiation and development. These observations provide a theoretical foundation for further studies on the molecular mechanisms underlying the control of vernalization in temperate grasses.

The C2H2-type zinc finger protein (ZFP) family is one of the largest transcription factor families in the plant kingdom and its members are involved in plant growth, development, and stress responses. As an economically valuable perennial graminaceous forage crop, orchardgrass (Dactylis glomerata) is an important feedstuff resource owing to its high yield and quality. In this study, 125 C2H2-type ZFPs in orchardgrass (Dg-ZFPs) were identified and further classified by phylogenetic analysis. The members with similar gene structures were generally clustered into the same groups, with proteins containing the conserved QALGGH motif being concentrated in groups VIII and IX. Gene ontology and miRNA target analyses indicated that Dg-ZFPs likely perform diverse biological functions through their gene interactions. The RNA-seq data revealed differentially expressed genes across tissues and development phases, suggesting that some Dg-ZFPs might participate in growth and development regulation. Abiotic stress responses of Dg-ZFP genes were verified by qPCR and Saccharomyces cerevisiae transformation, revealing that Dg-ZFP125 could enhance the tolerance of yeasts to osmotic and salt stresses. Our study performed a novel systematic analysis of Dg-ZFPs in orchardgrass, providing a reference for this gene family in other grasses and revealing new insights for enhancing gene utilization.

Miscanthus sinensis is an ornamental grass, non-food bioenergy crop, and forage with high feeding value. It can adapt to many kinds of soil conditions due to its high level of resistance to various abiotic stresses. However, a low level of seed germination restricts the utilization and application of M. sinensis. It is reported that the Homeodomain-leucine zipper (HD-Zip) gene family participates in plant growth and development and ability to cope with outside environment stresses, which may potentially regulate seed germination and stress resistance in M. sinensis. In this study, a complete overview of M. sinensis HD-Zip genes was conducted, including gene structure, conserved motifs, chromosomal distribution, and gene duplication patterns. A total of 169 members were identified, and the HD-Zip proteins were divided into four subgroups. Inter-chromosomal evolutionary analysis revealed that four pairs of tandem duplicate genes and 72 segmental duplications were detected, suggesting the possible role of gene replication events in the amplification of the M. sinensis HD-Zip gene family. There was an uneven distribution of HD-Zip genes on 19 chromosomes of M. sinensis. Also, evolutionary analysis showed that M. sinensis HD-Zip gene family members had more collinearity with monocotyledons and less with dicotyledons. The gene structure analysis showed that there were 93.5% of proteins with motif 1 and motif 4, while motif 10 was only found in group IV. Based on the cis-elements analysis, it appeared that most of the genes were related to plant growth and development, various hormones, and abiotic stress. Furthermore, qRT-PCR analysis showed that Misin06G303300.1 was significantly expressed in seed germination and Misin05G030000.1 and Misin06G303300.1 were highly expressed under chromium, salt, and drought stress. Results in this study will provide a basis for further exploring the potential role of HD-Zip genes in stress responses and genetic improvement of M. sinensis seed germination.

Leaves are the primary and critical feed for herbivores. They directly determine the yield and quality of legume forage. Trifolium repens (T. repens) is an indispensable legume species, widely cultivated in temperate pastures due to its nutritional value and nitrogen fixation. Although the leaves of T. repens are typical trifoliate, they have unusual patterns to adapt to herbivore feeding. The number of leaflets in T. repens affects its production and utilization. The KNOX gene family encodes transcriptional regulators that are vital in regulating and developing leaves. Identification and characterization of TrKNOX gene family as an active regulator of leaf development in T. repens were studied. A total of 21 TrKNOX genes were identified from the T. repens genome database and classified into three subgroups (Class I, Class II, and Class M) based on phylogenetic analysis. Nineteen of the genes identified had four conserved domains, except for KNOX5 and KNOX9, which belong to Class M. Varying expression levels of TrKNOX genes were observed at different developmental stages and complexities of leaves. KNOX9 was observed to upregulate the leaf complexity of T. repens. Research on TrKNOX genes could be novel and further assist in exploring their functions and cultivating high-quality T. repens varieties.

Leaves are the primary and critical feed for herbivores. They directly determine the yield and quality of legume forage.lt;igt;Trifolium repenslt;/igt; (lt;igt;T. repenslt;/igt;) is an indispensable legume species, widely cultivated in temperate pastures due to its nutritional value and nitrogen fixation. Although the leaves oflt;igt;T. repenslt;/igt; are typical trifoliate, they have unusual patterns to adapt to herbivore feeding. The number of leaflets inlt;igt;T. repenslt;/igt; affects its production and utilization. Thelt;igt;KNOXlt;/igt; gene family encodes transcriptional regulators that are vital in regulating and developing leaves. Identification and characterization oflt;igt;TrKNOXlt;/igt; gene family as an active regulator of leaf development inlt;igt;T. repenslt;/igt; were studied. A total of 21lt;igt;TrKNOXlt;/igt; genes were identified from the T. repens genome database and classified into three subgroups (Class I, Class II, and Class M) based on phylogenetic analysis. Nineteen of the genes identified had four conserved domains, except forlt;igt;KNOX5lt;/igt; andlt;igt;KNOX9lt;/igt;, which belong to Class M. Varying expression levels of TrKNOX genes were observed at different developmental stages and complexities of leaves.lt;igt;KNOX9lt;/igt; was observed to upregulate the leaf complexity of T. repens. Research onlt;igt;TrKNOXlt;/igt; genes could be novel and further assist in exploring their functions and cultivating high-qualitylt;igt;T. repenslt;/igt; varieties.

Free-standing flexible supercapacitive electrodes have practical application for wearable energy storage devices. In this paper, graphene paper (GP), a flexible electrode substrate, was prepared by one-step reduction of graphene oxide (GO) using HI solution. GP can be used independently as a flexible electrode with specific capacitance of 227 F/g. In order to make up for the shortage of GP specific capacitance storage, polyaniline (PANI) with high specific capacitance and good electrical conductivity was selected to composite with GP by electrochemical polymerization approach. This method to fabricate electrode material by direct electrochemical polymerization avoids the use of conductive binder and organic solvent. Owing to the specific capacitance contribution of PANI and GP, the PANI/GP composites exhibit higher specific capacitance when the polymerization time is 30 s and the polymerization voltage is 0.8 V. At 1 A/g current density, the specific capacitance of composite is up to 759 F/g, which is 3.34 times of neat GP.

Orchardgrass (Dactylis glomerata L.) is an economically important perennial forage grass with a high biomass, abundant carbohydrates, shade tolerance, and broad adaptability. The time of flowering is a crucial agronomic trait that is strongly relevant to the quality and yield of orchardgrass. However, there has been little information provided about the genes expressed during the critical flower development stages. In this study, we performed a comparative transcriptome analysis at different stages in which we compared the early- and late-flowering phenotypes of orchardgrass to identify the regulators and pathways that are involved in timing of flowering. Based on our results, 2561 and 3885 differentially expressed genes (DEGs) were identified between the late- and early-flowering phenotypes of orchardgrass in vernalization stage (VE) and before heading (BH) stages, respectively. Gene ontology analysis indicated that the DEGs in VE stage were primarily related to plant hormones, development processes, and environmental stimulation in response to vernalization induction. At the BH stage, DEGs were related to plant development processes and organ formation and differentiation. A large number of heat shock factors (HSFs) were detected in cooperation with plant hormone regulators in the heading (HT) stage. Ultimately, 50 flowering genes discovered were studied as DEGs between different phenotypes in flowering time, and 25 of those flowering DEGs were involved in the photoperiod pathway. These 50 DEGs revealed gene transcripts that corresponded to flowering-associated differences, and they provide novel insights into the molecular mechanisms that underlie regulation of flowering in early-flowering and late-flowering phenotypes of orchardgrass.

Background Tillering is an important agronomic trait underlying the yields and reproduction of orchardgrass (Dactylis glomerata), an important perennial forage grass. Although some genes affecting tiller initiation have been identified, the tillering regulatory network is still largely unknown, especially in perennial forage grasses. Thus, unraveling the regulatory mechanisms of tillering in orchardgrass could be helpful in developing selective strategies for high-yield perennial grasses. In this study, we generated high-throughput RNA-sequencing data from multiple tissues of tillering stage plants to identify differentially expressed genes (DEGs) between high- and low-tillering orchardgrass genotypes. Gene Ontology and pathway enrichment analyses connecting the DEGs to tillering number diversity were conducted. Results In the present study, approximately 26,282 DEGs were identified between two orchardgrass genotypes, AKZ-NRGR667 (a high-tillering genotype) and D20170203 (a low-tillering genotype), which significantly differed in tiller number. Pathway enrichment analysis indicated that DEGs related to the biosynthesis of three classes of phytohormones, i.e., strigolactones (SLs), abscisic acid (ABA), and gibberellic acid (GA), as well as nitrogen metabolism dominated such differences between the high- and low-tillering genotypes. We also confirmed that under phosphorus deficiency, the expression level of the major SL biosynthesis genes encoding DWARF27 (D27), 9-cis-beta-carotene 9′,10′-cleaving dioxygenase (CCD7), carlactone synthase (CCD8), and more axillary branching1 (MAX1) proteins in the high-tillering orchardgrass genotype increased more slowly relative to the low-tillering genotype. Conclusions Here, we used transcriptomic data to study the tillering mechanism of perennial forage grasses. We demonstrated that differential expression patterns of genes involved in SL, ABA, and GA biosynthesis may differentiate high- and low-tillering orchardgrass genotypes at the tillering stage. Furthermore, the core SL biosynthesis-associated genes in high-tillering orchardgrass were more insensitive than the low-tillering genotype to phosphorus deficiency which can lead to increases in SL biosynthesis, raising the possibility that there may be distinct SL biosynthesis way in tillering regulation in orchardgrass. Our research has revealed some candidate genes involved in the regulation of tillering in perennial grasses that is available for establishment of new breeding resources for high-yield perennial grasses and will serve as a new resource for future studies into molecular mechanism of tillering regulation in orchardgrass.

Trifolium repens is the most widely cultivated perennial legume forage in temperate region around the world. It has rich nutritional value and good palatability, seasonal complementarity with grasses, and can improve the feed intake and digestibility of livestock. However, flowering time and inflorescence development directly affects the quality and yield of T. repens, as well as seed production. Squa promoter binding protein–like (SPL) gene family is a plant specific transcription factor family, which has been proved to play a critical role in regulating plant formation time and development of flowers. In this study, a total of 37 TrSPL genes were identified from the whole genome of T. repens and were divided into 9 clades based on phylogenetic tree. The conserved motif of squamosa promoter binding protein (SBP) contains two zinc finger structures and one NLS structure. Gene structure analysis showed that all TrSPL genes contained SBP domain, while ankyrin repeat region was just distributed in part of genes. 37 TrSPL genes were relatively dispersedly distributed on 16 chromosomes, and 5 pairs of segmental repeat genes were found, which indicated that segmental duplication was the main way of gene expansion. Furthermore, the gene expression profiling showed that TrSPL11, TrSPL13, TrSPL22 and TrSPL26 were highly expressed only in the early stage of inflorescence development, while TrSPL1 and TrSPL6 are highly expressed only in the mature inflorescence. Significantly, the expression of TrSPL4 and TrSPL12 increased gradually with the development of inflorescences. The results of this study will provide valuable clues for candidate gene selection and elucidating the molecular mechanism of T. repens flowering regulation.

The heat shock protein 90 (Hsp90) is a protein produced in plants in response to stress. This study identified and analyzed Hsp90 gene family members in the perennial ryegrass genome. From the results, eight Hsp90 proteins were obtained and their MW, pI and number of amino acid bases varied. The amino acid bases ranged from 526 to 862. The CDS also ranged from 20 (LpHsp0-4) to 1 (LpHsp90-5). The least number of CDS regions was 1 (LpHsp90-5) with 528 kb amino acids, while the highest was 20 (LpHsp90-4) with 862 kb amino acids, which showed diversity among the protein sequences. The phylogenetic tree revealed that Hsp90 genes in Lolium perenne, Arabidopsis thaliana, Oryza sativa and Brachypodium distachyon could be divided into two groups with five paralogous gene pairs and three orthologous gene pairs. The expression analysis after perennial ryegrass was subjected to heat, salt, chromium (Cr), cadmium (Cd), polyethylene glycol (PEG) and abscisic acid (ABA) revealed that LpHsp90 genes were generally highly expressed under heat stress, but only two LpHsp90 proteins were expressed under Cr stresses. Additionally, the expression of the LpHsp90 proteins differed at each time point in all treatments. This study provides the basis for an understanding of the functions of LpHsp90 proteins in abiotic stress studies and in plant breeding.

The NAC transcription factor family is deemed to be a large plant-specific gene family that plays important roles in plant development and stress response. Miscanthus sinensis is commonly planted in vast areas of marginal lang as forage, ornamental grass, or bioenergy crop which demand a relatively high resistance to abiotic stresses. The recent release of a draft chromosome-scale assembly genome of M. sinensis provided a basic platform for the genome-wide investigation of NAC proteins. In this study, a total of 261 M. sinensis NAC genes were identified and a complete overview of the gene family was presented, including gene structure, conserved motif compositions, chromosomal distribution, and gene duplications. Results showed that gene length, molecular weights (MW), and theoretical isoelectric points (pI) of NAC family were varied, while gene structure and motifs were relatively conserved. Chromosomal mapping analysis found that the M. sinensis NAC genes were unevenly distributed on 19 M. sinensis chromosomes, and the interchromosomal evolutionary analysis showed that nine pairs of tandem duplicate genes and 121 segmental duplications were identified, suggesting that gene duplication, especially segmental duplication, is possibly associated with the amplification of M. sinensis NAC gene family. The expression patterns of 14 genes from M. sinensis SNAC subgroup were analyzed under high salinity, PEG, and heavy metals, and multiple NAC genes could be induced by the treatment. These results will provide a very useful reference for follow-up study of the functional characteristics of NAC genes in the mechanism of stress-response and potential roles in the development of M. sinensis.

Orchardgrass (Dactylis glomerata L.) is an important forage grass for cultivating livestock worldwide. Here, we report an similar to 1.84-Gb chromosome-scale diploid genome assembly of orchardgrass, with a contig N50 of 0.93 Mb, a scaffold N50 of 6.08 Mb and a super-scaffold N50 of 252.52 Mb, which is the first chromosome-scale assembled genome of a cool-season forage grass. The genome includes 40 088 protein-coding genes, and 69% of the assembled sequences are transposable elements, with long terminal repeats (LTRs) being the most abundant. The LTRretrotransposons may have been activated and expanded in the grass genome in response to environmental changes during the Pleistocene between 0 and 1 million years ago. Phylogenetic analysis reveals that orchardgrass diverged after rice but before three Triticeae species, and evolutionarily conserved chromosomes were detected by analysing ancient chromosome rearrangements in these grass species. We also resequenced the whole genome of 76 orchardgrass accessions and found that germplasm from Northern Europe and East Asia clustered together, likely due to the exchange of plants along the 'Silk Road' or other ancient trade routes connecting the East and West. Last, a combined transcriptome, quantitative genetic and bulk segregant analysis provided insights into the genetic network regulating flowering time in orchardgrass and revealed four main candidate genes controlling this trait. This chromosome-scale genome and the online database of orchardgrass developed here will facilitate the discovery of genes controlling agronomically important traits, stimulate genetic improvement of and functional genetic research on orchardgrass and provide comparative genetic resources for other forage grasses. National Basic Research Program (973 Program) in ChinaNational Basic Research Program of China [2014CB138705]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China [NSFC 31872997]; Earmarked Fund for Modern Agro-industry Technology Research System [CARS-34]; National Project on Sci-Tec Foundation Resources Survey [2017FY100602] This research work was funded by the National Basic Research Program (973 Program) in China (No. 2014CB138705), the National Natural Science Foundation of China (NSFC 31872997), the Earmarked Fund for Modern Agro-industry Technology Research System (No. CARS-34) and the National Project on Sci-Tec Foundation Resources Survey (2017FY100602). Public domain – authored by a U.S. government employee

SPL (SQUAMOSA promoter binding protein-like) is a plant-specific transcription factor family that contains the conserved SBP domain, which plays a vital role in the vegetative-to-reproductive phase transition, flowering development and regulation, tillering/branching, and stress responses. Although the SPL family has been identified and characterized in various plant species, limited information about it has been obtained in orchardgrass, which is a critical forage crop worldwide. In this study, 17 putative DgSPL genes were identified among seven chromosomes, and seven groups that share similar gene structures and conserved motifs were determined by phylogenetic analysis. Of these, eight genes have potential target sites for miR156. cis-Element and gene ontology annotation analysis indicated DgSPLs may be involved in regulating development and abiotic stress responses. The expression patterns of eight DgSPL genes at five developmental stages, in five tissues, and under three stress conditions were determined by RNA-seq and qRT-PCR. These assays indicated DgSPLs are involved in vegetative-to-reproductive phase transition, floral development, and stress responses. The transient expression analysis in tobacco and heterologous expression assays in yeast indicated that miR156-targeted DG1G01828.1 and DG0G01071.1 are nucleus-localized proteins, that may respond to drought, salt, and heat stress. Our study represents the first systematic analysis of the SPL family in orchardgrass. This research provides a comprehensive assessment of the DgSPL family, which lays the foundation for further examination of the role of miR156/DgSPL in regulating development and stress responses in forages grasses.

Background Orchardgrass (Dactylis glomerata L.) is one of the most important cool-season perennial forage grasses that is widely cultivated in the world and is highly tolerant to stressful conditions. However, little is known about the mechanisms underlying this tolerance. The NAC (NAM, ATAF1/2, and CUC2) transcription factor family is a large plant-specific gene family that actively participates in plant growth, development, and response to abiotic stress. At present, owing to the absence of genomic information, NAC genes have not been systematically studied in orchardgrass. The recent release of the complete genome sequence of orchardgrass provided a basic platform for the investigation of DgNAC proteins. Results Using the recently released orchardgrass genome database, a total of 108 NAC (DgNAC) genes were identified in the orchardgrass genome database and named based on their chromosomal location. Phylogenetic analysis showed that the DgNAC proteins were distributed in 14 subgroups based on homology with NAC proteins in Arabidopsis, including the orchardgrass-specific subgroup Dg_NAC. Gene structure analysis suggested that the number of exons varied from 1 to 15, and multitudinous DgNAC genes contained three exons. Chromosomal mapping analysis found that the DgNAC genes were unevenly distributed on seven orchardgrass chromosomes. For the gene expression analysis, the expression levels of DgNAC genes in different tissues and floral bud developmental stages were quite different. Quantitative real-time PCR analysis showed distinct expression patterns of 12 DgNAC genes in response to different abiotic stresses. The results from the RNA-seq data revealed that orchardgrass-specific NAC exhibited expression preference or specificity in diverse abiotic stress responses, and the results indicated that these genes may play an important role in the adaptation of orchardgrass under different environments. Conclusions In the current study, a comprehensive and systematic genome-wide analysis of the NAC gene family in orchardgrass was first performed. A total of 108 NAC genes were identified in orchardgrass, and the expression of NAC genes during plant growth and floral bud development and response to various abiotic stresses were investigated. These results will be helpful for further functional characteristic descriptions of DgNAC genes and the improvement of orchardgrass in breeding programs.

The AP2/ERF transcription factor (TF) family is of great importance in developmental regulation and responses to stress and pathogenic stimuli. Orchardgrass (Dactylis glomerata), a perennial cold-season forage of high quality in the world's temperate zones, contributes to grazing land through mixed sowing with alfalfa (Medicago sativa) and white clover (Trifolium repens). However, little is known about AP2/ERF TFs in orchardgrass. In this study, 193 AP2/ERF genes were classified into five subfamilies and 13 subgroups through phylogenetic analysis. Chromosome structure analysis showed that AP2/ERF family genes in orchardgrass were distributed on seven chromosomes and specific conservative sequences were found in each subgroup. Exon-intron structure and motifs in the same subgroup were almost identical, and the unique motifs contributed to the classification and functional annotation of DgERFs. Expression analysis showed tissue-specific expression of DgERFs in roots and flowers, with most DgERFs widely expressed in roots. The expression levels of each subgroup (subgroups Vc, VIIa, VIIIb, IXa, and XIa) were high at the before-heading and heading stages (BH_DON and H_DON). In addition, 12 DgERFs in various tissues and five DgERFs associated with abiotic stresses were selected for qRT-PCR analysis showed that four dehydration-responsive element binding (DREB) genes and one ERF subfamily gene in orchardgrass were regulated with PEG, heat and salt stresses. DgERF056 belonged to ERF subfamily was involved in the processes of flowering and development stage. This study systematic explored the DgERFs at the genome level for the first time, which lays a foundation for a better understanding of AP2/ERF gene function in Dactylis glomerata and other types of forage.

NAC is one of the largest family of plant-specific transcription factors, and it plays important roles in plant development and stress responses. The study identified 72 LpNACs genes from the perennial ryegrass genome database. Gene length, MW and pI of NAC family transcription factors varied, but the gene structure and motifs were relatively conserved in bioinformatics analysis. Phylogenetic analyses of perennial ryegrass, rice and Arabidopsis were performed to study the evolutionary and functional relationships in various species. The expression of LpNAC genes that respond to various abiotic stresses including high salinity, ABA, high temperature, polyethylene glycol (PEG) and heavy metal was comprehensively analyzed. The present study provides a basic understanding of the NAC gene family in perennial ryegrass for further abiotic stress studies and improvements in breeding.

Additional file 9 : Supplemental Figure S9 The results of protein sequence alignment for genes involved in SL biosynthesis in orchardgrass. D27, DWARF27; CCD7, 9-cis-beta-carotene 9′,10′-cleaving dioxygenase; CCD8, carlactone synthase; MAX1, more axillary branching1.

Background Annual ryegrass (Lolium multiflorum L.) is a commercially important, widely distributed forage crop that is used in the production of hay and silage worldwide. Drought has been a severe environmental constraint in its production. Nevertheless, only a handful of studies have examined the impact of short-term drought stress on annual ryegrass. The aim of this study was to explore how stress-induced core metabolic processes enhance drought tolerance, or adaptation to drought, in annual ryegrass. Results We profiled the transcriptomes, proteomes, and metabolomes of two annual ryegrass genotypes: the drought-resistant genotype “Abundant 10” and drought-susceptible genotype “Adrenalin 11.” We identified differentially expressed metabolites and their corresponding proteins and transcripts that are involved in 23 core metabolic processes, in response to short-term drought stress. Protein–gene–metabolite correlation networks were built to reveal the relationships between the expression of transcripts, proteins, and metabolites in drought-resistant annual ryegrass. Furthermore, integrated metabolic pathways were used to observe changes in enzymes corresponding with levels of amino acids, lipids, carbohydrate conjugates, nucleosides, alkaloids and their derivatives, and pyridines and their derivatives. The resulting omics data underscored the significance of 23 core metabolic processes on the enhancement of drought tolerance or adaptation to drought in annual ryegrass. Conclusions The regulatory networks were inferred using MCoA and correlation analysis to reveal the relationships among the expression of transcripts, proteins, and metabolites that highlight the corresponding elements of these core metabolic pathways. Our results provide valuable insight into the molecular mechanisms of drought resistance, and represent a promising strategy toward the improvement of drought tolerance in annual ryegrass. Electronic supplementary material The online version of this article (10.1186/s12870-018-1239-z) contains supplementary material, which is available to authorized users.

The GRAS gene family is a family of transcription factors that regulates plant growth and development. Despite being well-studied in many plant species, little is known about this gene family in orchardgrass (Dactylis glomerata L.), one of the top four economically important perennial forage grasses cultivated worldwide. We identified 46 GRAS genes in orchardgrass and analyzed their characteristics by phylogenetic, gene structural, motifs and expression patterns analysis. The phylogenetic analysis of eight species revealed that DgGRAS family had the evolutional conservation and closer homology relationship with the GRAS family of rice, barley and Brachypodium distachyon. Moreover, 46 DgGRAS proteins were divided into eight subfamilies based on the tree topology and rice or Arabidopsis classification, and LISCL subfamily was the largest one. Besides, we found that the motif 15 may be unique to the orchardgrass LISCL subfamily, and the motif 6 and motif 17 had indispensable functions in the orchardgrass LISCL subfamily. We further analyzed the expression profiles of DgGRAS genes at mature and seeding stage. And we found that DgGRAS17 played an important role in the growth and development no matter what stage it was at. DgGRAS5, DgGRAS28, DgGRAS31, DgGRAS42 and DgGRAS44 got involved in processes of the growth and development at seeding stage instead of mature stage. These results indicated that the major expression patterns and detailed functions of the DgGRAS genes varied with developmental stages. Taken together, this is the first systematic analysis of the GRAS gene family in the orchardgrass genome and the results provide insights into the potential functions of GRAS genes.

King grass, a hybrid grass between pearl millet and elephant grass, has many excellent characteristics such as high biomass yield, great stress tolerance, and enormous economic and ecological value, which makes it ideal for development of phytoremediation. At present, the physiological and molecular response of king grass to cadmium (Cd) stress is poorly understood. Transcriptome analysis of early response (3 h and 24 h) of king grass leaves and roots to high level Cd (100 &micro, M) has been investigated and has shed light on the molecular mechanism underlying Cd stress response in this hybrid grass. Our comparative transcriptome analysis demonstrated that in combat with Cd stress, king grass roots have activated the glutathione metabolism pathway by up-regulating glutathione S-transferases (GSTs) which are a multifunctional family of phase II enzymes that detoxify a variety of environmental chemicals, reactive intermediates, and secondary products of oxidative damages. In roots, early inductions of phenylpropanoid biosynthesis and phenylalanine metabolism pathways were observed to be enriched in differentially expressed genes (DEGs). Meanwhile, oxidoreductase activities were significantly enriched in the first 3 h to bestow the plant cells with resistance to oxidative stress. We also found that transporter activities and jasmonic acid (JA)-signaling might be activated by Cd in king grass. Our study provided the first-hand information on genome-wide transcriptome profiling of king grass and novel insights on phytoremediation.

King grass, a hybrid grass between pearl millet and elephant grass, has many excellent characteristics such as high biomass yield, great stress tolerance, and enormous economic and ecological value, which makes it ideal for development of phytoremediation. At present, the physiological and molecular response of king grass to cadmium (Cd) stress is poorly understood. Transcriptome analysis of early response (3 h and 24 h) of king grass leaves and roots to high level Cd (100 µM) has been investigated and has shed light on the molecular mechanism underlying Cd stress response in this hybrid grass. Our comparative transcriptome analysis demonstrated that in combat with Cd stress, king grass roots have activated the glutathione metabolism pathway by up-regulating glutathione S-transferases (GSTs) which are a multifunctional family of phase II enzymes that detoxify a variety of environmental chemicals, reactive intermediates, and secondary products of oxidative damages. In roots, early inductions of phenylpropanoid biosynthesis and phenylalanine metabolism pathways were observed to be enriched in differentially expressed genes (DEGs). Meanwhile, oxidoreductase activities were significantly enriched in the first 3 h to bestow the plant cells with resistance to oxidative stress. We also found that transporter activities and jasmonic acid (JA)-signaling might be activated by Cd in king grass. Our study provided the first-hand information on genome-wide transcriptome profiling of king grass and novel insights on phytoremediation.

Background Flowering is a critical reproductive process in higher plants. Timing of optimal flowering depends upon the coordination among seasonal environmental cues. For cool season grasses, such as Dactylis glomerata, vernalization induced by low temperature provides competence to initiate flowering after prolonged cold. We combined analyses of the transcriptome and microRNAs (miRNAs) to generate a comprehensive resource for regulatory miRNAs and their target circuits during vernalization and heading stages. Results A total of 3,846 differentially expressed genes (DEGs) and 69 differentially expressed miRNAs were identified across five flowering stages. The expression of miR395, miR530, miR167, miR396, miR528, novel_42, novel_72, novel_107, and novel_123 demonstrated significant variations during vernalization. These miRNA targeted genes were involved in phytohormones, transmembrane transport, and plant morphogenesis in response to vernalization. The expression patterns of DEGs related to plant hormones, stress responses, energy metabolism, and signal transduction changed significantly in the transition from vegetative to reproductive phases. Conclusions Five hub genes, c136110_g1 (BRI1), c131375_g1 (BZR1), c133350_g1 (VRN1), c139830_g1 (VIN3), and c125792_g2 (FT), might play central roles in vernalization response. Our comprehensive analyses have provided a useful platform for investigating consecutive transcriptional and post-transcriptional regulation of critical phases in D. glomerata and provided insights into the genetic engineering of flowering-control in cereal crops. Electronic supplementary material The online version of this article (10.1186/s12864-018-5104-0) contains supplementary material, which is available to authorized users.

Simple sequence repeat (SSR) markers are considered very useful molecular markers for genetic and molecular breeding, specifically for non-model species. To develop a set of whole genome-wide distributed SSR marker for annual ryegrass (Lolium multiflorum L.), we obtained the first transcriptome dataset of annual ryegrass subjected to drought stress and identified a total of 11,254 EST-SSR. Fifty EST-SSR markers were selected to test the amplification and polymorphism rates in 23 accessions of Lolium and Festuca diploid species. The results showed that all of the 50 functionally relevant markers were polymorphic, with allele number and polymorphism content averaging 7.4 and 0.839, respectively, and fully transferable across 23 accessions of Lolium and Festuca species. Sequencing of PCR products showed that the nucleotide sequences of Lolium and Festuca species had a high level of conservation. Different types of tri-repeats as abundant SSR motifs were observed in repeat sequence regions. A number of EST-SSRs were mapped to genome sequences of Lolium perenne and chromosomes of Brachypodium distachyon. Among them, 973 and 1375 Lolium SSR markers were located on the five chromosomes of B. distachyon and L. perenne scaffolds, respectively. In addition, we confirmed that EST-SSRs as well as SRAP markers could clearly identify varieties of Lolium and Festuca species, and the two sets of molecular markers had a significant correlation (r = 0.74). This set of EST-SSR markers are valuable genetic tools for genetic diversity, evolutionary, and association mapping studies in Lolium, Festuca, and many other grass species.

Detailed information of genes used for real-time-PCR analysis (DOCX 15 kb)

The photoluminescent and persistent luminescence properties of BaMg2(PO4)2:Eu2+, Ln3+ (Ln=Ce, Pr, Tb and Lu) were investigated. These phosphors were prepared by a solid-state reaction at high temperature. The persistent phosphors were investigated by XRD, photoluminescence, persistent luminescence and thermoluminescence glow curves. Radiative/nonradiative energy transfer from Eu2+ to Pr3+/Tb3+ were observed due to the overlap between the Eu2+ emission and Pr3+/Tb3+ absorption spectra. The relevant transitions of Pr3+ and Tb3+ are respectively spin allowed and forbidden, so that the energy transfer occurs in different manners. After irradiated by ultraviolet light, all these codoped phosphors show a broad-band persistent luminescence located at ~435 nm at room temperature. Codoping with Tb3+ considerably enhances the afterglow intensity while Pr3+ codoped sample only shows weak afterglow. These properties can also be confirmed by their thermoluminescence glow curves. The emissions for both photoluminescence and persistent luminescence are due to the 5d→4f transition of Eu2+. The influence of auxiliary codopants is discussed in terms of ionization energy and ionic radius.

A series of Ce 3+ or Tb 3+ doped and Ce 3+ /Tb 3+ co-doped CaMgP 2 O 7 phosphors were prepared by the solid state reaction method. The fluorescence and energy transfer property of samples were studied in details. CaMgP 2 O 7 :Ce 3+ , Tb 3+ shows both an ultraviolet (UV) emitting (300–450 nm) from Ce 3+ and a yellowish-green emitting (543 nm) from Tb 3+ with considerable intensity under UV excitation (278 nm). The energy transfer mechanism from Ce 3+ to Tb 3+ ions was proved to be dipole–dipole interaction. The Ce 3+ and Tb 3+ co-doped CaMgP 2 O 7 phosphors are potential UV-convertible candidates with green light emitting in UV-LEDs for the high efficient energy transfer from Ce 3+ to Tb 3+ ions.

Italian ryegrass (Lolium multiflorum) is an important cool-season, annual forage crop for the grassland rotation system in Southern China. The primary aim of breeding programs is always to seek to improve forage quality in the animal productivity system; however, it is time- and labor-consuming when analyzed excessive large number of samples. The main objectives of this study were to construct near-infrared reflectance spectroscopy (NIRS) models to predict the forage chemistry quality of Italian ryegrass including the concentrations of crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), and water soluble carbohydrate (WSC). The results showed that a broader range of CP, NDF, ADF and WSC contents (%DM) were obtained (4.45–30.60, 21.29–60.47, 11.66–36.17 and 3.95–51.52, respectively) from the samples selected for developing NIRS models. In addition, the critical wavelengths identified in this study to construct optimal NIRS models were located in 4,247–6,102 and 4,247–5,450 cm-1 for CP and NDF content, and both wavelengths 5,446–6,102 and 4,247–4,602 cm-1 could for ADF and WSC. Finally, the optimal models were developed based on the laboratory data and the spectral information by partial least squares (PLS) regression, with relatively high coefficients of determination (R2CV, CP = 0.99, NDF = 0.94, ADF = 0.92, WSC = 0.88), ratio of prediction to devitation (RPD, CP = 8.58, NDF = 4.25, ADF = 3.64, WSC = 3.10). The further statistics of prediction errors relative to laboratory (PRL) and the range error ratio (RER) give excellent assessments of the models with the PRL ratios lower than 2 and the RER values greater than 10. The NIRS models were validated using a completely independent set of samples and have coefficients of determination (R2V, CP = 0.99, NDF = 0.91, ADF = 0.95, WSC = 0.91) and ratio of prediction to deviation (RPD, CP = 9.37, NDF = 3.44, ADF = 4.40, WSC = 3.39). The result suggested that routine screening for forage quality parameters with large numbers of samples is available with the NIRS model in Italian ryegrass breeding programs, as well as facilitating graziers to monitor the forage development stage for improving grazing efficiency.

Drought is a major abiotic stress that impairs growth and productivity of Italian ryegrass. Comparative analysis of drought responsive proteins will provide insight into molecular mechanism in Lolium multiflorum drought tolerance. Using the iTRAQ-based approach, proteomic changes in tolerant and susceptible lines were examined in response to drought condition. A total of 950 differentially accumulated proteins was found to be involved in carbohydrate metabolism, amino acid metabolism, biosynthesis of secondary metabolites, and signal transduction pathway, such as β-D-xylosidase, β-D-glucan glucohydrolase, glycerate dehydrogenase, Cobalamin-independent methionine synthase, glutamine synthetase 1a, Farnesyl pyrophosphate synthase, diacylglycerol, and inositol 1, 4, 5-trisphosphate, which might contributed to enhance drought tolerance or adaption in Lolium multiflorum. Interestingly, the two specific metabolic pathways, arachidonic acid and inositol phosphate metabolism including differentially accumulated proteins, were observed only in the tolerant lines. Cysteine protease cathepsin B, Cysteine proteinase, lipid transfer protein and Aquaporin were observed as drought-regulated proteins participating in hydrolysis and transmembrane transport. The activities of phospholipid hydroperoxide glutathione peroxidase, peroxiredoxin, dehydroascorbate reductase, peroxisomal ascorbate peroxidase and monodehydroascorbate reductase associated with alleviating the accumulation of reactive oxygen species in stress inducing environments. Our results showed that drought-responsive proteins were closely related to metabolic processes including signal transduction, antioxidant defenses, hydrolysis, and transmembrane transport.

A series of Ce 3+ or Tb 3+ doped and Ce 3+ /Tb 3+ co-doped Ba 2 ZnSi 2 O 7 phosphors were prepared via the conventional high temperature solid state reaction method. The photoluminescence and energy transfer properties of samples were studied in detail. The optimal proportion of Ce 3+ single doping is 2 mol% with maximal fluorescence intensity. Ba 1.98− x ZnSi 2 O 7 :0.02Ce 3+ , x Tb 3+ shows both a blue emission (428 nm) from Ce 3+ and a yellowish-green emission (542 nm) from Tb 3+ with considerable intensity under ultraviolet (UV) excitation (352 nm). The emission chromaticity coordinates can be adjusted from blue to green region by tuning the concentration of Tb 3+ ions from 0.00 to 0.06 through an energy transfer process. The energy transfer mechanism from Ce 3+ to Tb 3+ ions was proved to be dipole–dipole interaction. The Ce 3+ and Tb 3+ co-doped Ba 2 ZnSi 2 O 7 phosphors are potential UV-convertible candidates with green light emitting in UV-LEDs for the high efficient energy transfer from Ce 3+ to Tb 3+ ions.

Stoichiometric phosphors LiGd 1− x Eu x (PO 3 ) 4 ( x =0, 0.2, 0.4, 0.6, 0.8, 1.0) were synthesized via traditional solid state reactions. The X-ray powder diffraction measurements show that all prepared samples are isostructural with LiNd(PO 3 ) 4 . Eu 3+ doped phosphors can emit intense reddish orange light under the excitation of near ultraviolet light from 370 to 410 nm. The strongest two at 591 and 613 nm can be attributed to the transitions from excited state 5 D 0 to ground states 7 F 1 and 7 F 2 , respectively. The typical chromaticity coordinates ( x =0.620, y =0.368) of Eu 3+ doped phosphors are in red area. The recorded absorbance spectra indicate that there is effective absorbance in the near UV region for all Eu 3+ doped samples. Present research indicates that LiGd 1– x Eu x (PO 3 ) 4 is a promising phosphor for white light-emitting diodes.

Un-doped and Sm3+ doped Y2WO6 were synthesized with solid state reactions. X-ray diffraction measurements show that all the samples present single-phase Y2WO6. The combination of blue emission in a broad band from the host and several groups of emission lines in yellow, orange, and red spectral region results in a desired white light. The chromaticity coordinates and color temperature can be tuned by changing the doping concentration of Sm3+. The mechanisms of energy transfer were analyzed. The present results prove Sm3+ doped Y2WO6 is a promising single-phase, color-tunable, broadband-excited white light-emitting phosphor based on AlGaN-based ultraviolet light-emitting diodes.

Water deficit adversely affects the growth and productivity of annual ryegrass (Lolium multiflorum Lam.). The exogenous application of chitosan (CTS) has gained extensive interests due to its effect on improving drought resistance. This research aimed to determine the role of exogenous CTS on annual ryegrass in response to water stress. Here, we investigated the impact of exogenous CTS on the physiological responses and transcriptome changes of annual ryegrass variety &ldquo, Tetragold&rdquo, under osmotic stress induced by exposing them to 20% polyethylene glycol (PEG)-6000. Our experimental results demonstrated that 50 mg/L exogenous CTS had the optimal effect on promoting seed germination under osmotic stress. Pre-treatment of annual ryegrass seedlings with 500 mg/L CTS solution reduced the level of electrolyte leakage (EL) as well as the contents of malondialdehyde (MDA) and proline and enhanced the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbic acid peroxidase (APX) under osmotic stress. In addition, CTS increased soluble sugars and chlorophyll (Chl) content, net photosynthetic rate (A), stomatal conductance (gs), water use efficiency (WUE), and transpiration rate (E) in annual ryegrass seedlings in response to three and six days of osmotic stress. Transcriptome analysis further provided a comprehensive understanding of underlying molecular mechanisms of CTS impact. To be more specific, in contrast of non-treated seedlings, the distinct changes of gene expressions of CTS-treated seedlings were shown to be tightly related to carbon metabolism, photosynthesis, and plant hormone. Altogether, exogenous CTS could elicit drought-related genes in annual ryegrass, leading to resistance to osmotic stress via producing antioxidant enzymes and maintaining intact cell membranes and photosynthetic rates. This robust evidence supports the potential of the application of exogenous CTS, which will be helpful for determining the suitability and productivity of agricultural crops.

The long afterglow phosphors Ba 3.94 (Si 3 O 8 ) 2 :0.02Eu 2+ , 0.04Re 3+ (Re:Dy,Ho) were prepared by the high-temperature solid state reaction. Ba 3.94 (Si 3 O 8 ) 2 :0.02Eu 2+ , 0.04Ho 3+ exhibited a long-lasting phosphorescence whose duration was more than 4 h visible to the naked eyes, and the initial phosphorescence intensity was over 3 cd/m 2 ; Ba 3.94 (Si 3 O 8 ) 2 :0.02Eu 2+ , 0.04Dy 3+ exhibited a weak phosphorescence (the initial intensity: 0.3 cd/m 2 ) lasting for half hour. The crystal structure, excitation spectra, emission spectra, the fluorescence lifetime, long afterglow decaying curves and thermoluminescence (TL) curves of the two sample were obtained in our work, reflecting that the phosphorescence mechanism was different: the TL curve half width of Ba 3.94 (Si 3 O 8 ) 2 :0.02Eu 2+ , 0.04Dy 3+ was constant with different waiting time and the depth of the traps was about 0.748 eV, indicating that the process of releasing carriers was apt to the first order kinetics. However, the TL curve half width of Ba 3.94 (Si 3 O 8 ) 2 :0.02Eu 2+ , 0.04Ho 3+ decreased gradually indicating that the inner traps energy was not a single level but a energy band ranging from 0.608 eV to 1.156 eV.

Drought is a major environmental stress that limits growth and development of cool-season annual grasses. Drought transcriptional profiles of resistant and susceptible lines were studied to understand the molecular mechanisms of drought tolerance in annual ryegrass (Lolium multiflorum L.). A total of 4,718 genes exhibited significantly differential expression in two L. multiflorum lines. Additionally, up-regulated genes associated with drought response in the resistant lines were compared with susceptible lines. Gene ontology enrichment and pathway analyses revealed that genes partially encoding drought-responsive proteins as key regulators were significantly involved in carbon metabolism, lipid metabolism, and signal transduction. Comparable gene expression was used to identify the genes that contribute to the high drought tolerance in resistant lines of annual ryegrass. Moreover, we proposed the hypothesis that short-term drought have a beneficial effect on oxidation stress, which may be ascribed to a direct effect on the drought tolerance of annual ryegrass. Evidence suggests that some of the genes encoding antioxidants (HPTs, GGT, AP, 6-PGD, and G6PDH) function as antioxidant in lipid metabolism and signal transduction pathways, which have non-independent and promoting roles in drought resistance. This study provides the first transcriptome data on the induction of drought-related gene expression in annual ryegrass, especially via modulation of metabolic homeostasis, signal transduction, and antioxidant defenses to improve drought tolerance response to short-term drought stress.

Praseodymium (Pr3+) with rich f energy levels can generate various visible emissions, but the luminescence of Pr3+ in a CaWO4 matrix has not yet been studied. In this article, the phosphors of Pr3+-doped CaWO4 were synthesized via a high temperature solid-state reaction, and the photoluminescence (PL) and afterglow properties of Pr3+ in the CaWO4 matrix were investigated. The phase identifications of the samples show that an impure monoclinic phase appears when the Pr3+ concentration is high. The results of PL exhibit the usual Pr3+ characteristic emissions as well as an emission that is produced by the charge transfer from 5d of W6+ to 2p of O2− under a shortwave ultraviolet excitation. An intermediate energy transfer occurs during the charge transfer which induces the afterglow. The quenching point of Pr3+ in CaWO4 is 2 mol%, corresponding to the critical energy transfer distance of ~20 A. The quenching mechanism in the Pr3+-doped CaWO4 is the dipole-dipole interaction. The Ca2+ vacancies (VCa) are the defects that induce the traps and capture holes from O2−. The thermal energy of holes liberation from VCa to O2− results in the charge transfer accompanied by an energy transfer to Pr3+, and consequently generates the afterglow. The experimental results also show that the afterglow characteristics of Pr3+ are prolonged by incorporating Pr3+ in the CaWO4 matrix.

The Ca 2 Al 2 SiO 7 samples doped with Ce 3+ and Eu 2+ are synthesized via a high temperature solid-state reaction. Ca 2 Al 2 SiO 7 : Ce 3+ emits a strong UV–violet emission while Ca 2 Al 2 SiO 7 : Eu 2+ emits a blue–green emission. The Stokes shift of the latter is greater due to a stronger crystal repulsion from ligands to Eu 2+ ions. Ca 2 Al 2 SiO 7 : Ce 3+ exhibits a stronger initial intensity and longer duration of afterglow due to the higher liberated probability of the trapped carriers. The thermoluminescence curves reveal that at least three traps exist in the phosphors. Ca 2+ vacancies may enhance the electron trapping and then lead to a stronger afterglow. A possible explanation will be provided.

A series of Eu3+ activated Li6Y1−xEux(BO3)3 (0.05 ⩽ x ⩽ 1) phosphors were synthesized by solid-state reaction method. The structures and photoluminescent properties of the phosphors were investigated at room temperature. The results of XRD patterns indicate that these phosphors are isotypic to the monoclinic Li6Gd(BO3)3. The excitation spectra indicate that these phosphors can be effectively excited by near UV (370–410 nm) light. The red emission from transition 5D0→7F2 is dominant. The emission spectra exhibit strong red performance (CIE chromaticity coordinates: x = 0.65, y = 0.35), which is due to the 5D0−7FJ transitions of Eu3+ ions. The relationship between the structure and the photoluminescent properties of the phosphors was studied. The concentration quenching occurs at x ≈ 0.85 under near UV excitation. Li6Y(BO3)3:Eu3+ has potential application as a phosphor for white light-emitting diodes.

The luminescence and energy transfer of cerium and terbium codoped Ba2Mg(PO4)2 were investigated. The phosphor was prepared by a solid-state reaction at high temperature. The photoluminescence properties were investigated under UV excitation. From a powder X-ray diffraction analysis, the formation of single-phased Ba2Mg(PO4)2 with a monoclinic structure was confirmed. The photoluminescence excitation spectrum of Ba2Mg(PO4)2:Ce3+ shows that the excitation band from 220 to 350 nm and the broad-band UV-blue emission are attributed to the allowed 4f ↔ 5d transitions of Ce3+. Upon excitation of Ce3+ band, Ba2Mg(PO4)2:Ce3+,Tb3+ phosphors exhibit both Ce3+ emission band the characteristic Tb3+ emission lines. Also, efficient energy transfer from Ce3+ to Tb3+ in Ba2Mg(PO4)2 was verified by observing the excitation spectra of Ba2Mg(PO4)2:Ce3+,Tb3+. An energy level scheme is constructed to depict the energy transfer process.