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Alfalfa (Medicago sativa L.) is the most widely cultivated perennial leguminous forage crop woldwide. MicroRNA156 (miR156) precursor genes from dicotyledonous species are reportedly useful for improving alfalfa plant architecture and abiotic stress resistance. However, there has been no report on whether a miR156 precursor gene from a monocotyledonous species functions in alfalfa. We introduced two tandem precursor genes of miR156, rice Osa-MIR156b and Osa-MIR156c (Osa-MIR156bc), into alfalfa. The expression of miR156 in the transgenic (TG) alfalfa was significantly elevated. Compared to wild-type plants, the TG plants overexpressing miR156 had more branches and leaves and showed improved salt and drought tolerance. Overexpression of miR156 slightly reduced plant height, but the biomass yield of TG plants grown in flowerpots was still increased. Forage quality of TG plants was markedly improved by reduction of acid detergent lignin (ADL) content and increase in crude protein content. The expression of the putative miR156 target genes MsSPL6, MsSPL12, and MsSPL13 in TG plants was repressed by miR156 overexpression, and that of all tested MsSPLs would be sharply increased under drought or salt stress. RNA sequencing revealed that overexpression of miR156 affected the expression of genes associated with abiotic stress resistance and plant development in multiple pathways. This first report of overexpression of monocot miR156 precursors in alfalfa sheds light on the function of miRNA156 precursors from the monocot species rice that could be used for genetic improvement of the dicot forage crop alfalfa.

Overexpression of OsDST-SRDX chimeric gene in switchgrass promotes plant growth and improves the salt tolerance of transgenic switchgrass by improving its antioxidative ability. Switchgrass (Panicum virgatum L.) is a forage and model feedstock plant. To avoid competing with crops in arable land utilization, improving salt tolerance of switchgrass is required to use marginal saline land for switchgrass production. To improve salt tolerance of switchgrass, a chimeric DROUGHT AND SALT TOLERANCE (DST) gene OsDST-SRDX was constructed using the Chimeric REpressor gene-Silencing Technology (CRES-T), and introduced into switchgrass genome by Agrobacterium-mediated transformation. Compared to wild-type (WT) plants, OsDST-SRDX transgenic (TG) switchgrass plants showed wider leaves and thicker stems. They performed better under salt stress, had higher relative leaf water content, lower electrolyte leakage and lower malondialdehyde (MDA) content, and accumulated less Na+ and more K+ than WT controls. The transgenic plants had also higher activities of antioxidant enzymes associated with suppressed expressing of genes in H2O2 homeostasis, including glutathione S-transferase (GST2, GST6), cytochrome P450, peroxidase 24 precursor, and induced expressing of CAT and SOD under salt stress to eliminate excess H2O2. Our results indicate that overexpression of the chimeric gene OsDST-SRDX improves salt tolerance of switchgrass, a C4 biofuel crop.

Transgenic switchgrass overexpressing Lolium perenne L. delta1-pyrroline 5-carboxylate synthase (LpP5CS) in group I (TG4 and TG6 line) and group II (TG1 and TG2 line) had significant P5CS and ProDH enzyme activities, with group I plants (TG4 and TG6) having higher P5CS and lower ProDH enzyme activity, while group II plants had higher ProDH and lower P5CS enzyme activity. We found group II transgenic plants showed stunted growth, and the changed proline content in overexpressing transgenic plants may influence the growth and development in switchgrass. RNA-seq analysis showed that KEGG enrichment included phenylpropanoid biosynthesis pathway among group I, group II and WT plants, and the expression levels of genes related to lignin biosynthesis were significantly up-regulated in group II. We also found that lignin content in group II transgenic plants was higher than that in group I and WT plants, suggesting that increased lignin content may suppress switchgrass growth and development. This study uncover that proline can appropriately reduce lignin biosynthesis to improve switchgrass growth and development. Therefore, appropriate reduction in lignin content and increase in biomass are important for bioenergy crop to lower processing costs for biomass fermentation-derived fuels.

Intercropping can effectively control some plant soil-borne diseases. However, few studies on intercropping have focused on forage grass as companion plants. In this experiment, Festuca arundinacea (tall fescue) was selected as the intercropping forage to explore whether it could control tomato stem rot. We found that: (1) tomato intercropped with tall fescue had a significantly lower disease incidence and disease index of tomato stem rot than sole tomato; (2) the antifungal activities of the root exudates of tomato and tall fescue in intercropping system were significantly higher than those of sole tomato or tall fescue. Meanwhile, it was inferred that the main allelochemicals might be cyclohexane-1, 2-diol and putrescine based on the GC-MS analysis of root exudates of tall fescue. (3) RNA-seq suggested that intercropping with tall fescue significantly upregulated the expression of genes related to pathogenesis-related proteins and hormone metabolism of tomato compared to those in sole tomato.

PvArf regulate proline biosynthesis by physically interacting with PvP5CS1 to improve salt tolerance in switchgrass. The genetic factors that contribute to stress resiliency are yet to be determined. Here, we identified three ADP-ribosylation factors, PvArf1, PvArf-B1C, and PvArf-related, which contribute to salinity tolerance in transgenic switchgrass (Panicum virgatum L.). Switchgrass overexpressing each of these genes produced approximately twofold more biomass than wild type (WT) under normal growth conditions. Transgenic plants accumulated modestly higher levels of proline under normal conditions, but this level was significantly increased under salt stress providing better protection to transgenic plants compared to WT. We found that PvArf genes induce proline biosynthesis genes under salt stress to positively regulate proline accumulation, and further demonstrated that PvArf physically interact with PvP5CS1. Moreover, the transcript levels of two key ROS-scavenging enzyme genes were significantly increased in the transgenic plants compared to WT, leading to reduced H2O2 accumulation under salt stress conditions. PvArf genes also protect cells against stress-induced changes in Na+ and K+ ion concentrations. Our findings uncover that ADP-ribosylation factors are key determinants of biomass yield in switchgrass, and play pivotal roles in salinity tolerance by regulating genes involved in proline biosynthesis.

Melatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic signaling molecule that plays important roles in plant growth, development and stress responses. Alfalfa (Medicago sativa L.) is an important and widely cultivated leguminous forage crop with high biomass yield and rich nutritional value. The effects of exogenous melatonin content regulation on alfalfa stress tolerance have been investigated in recent years. Here, we isolated and introduced the MsASMT1 (N-acetylserotonin methyltransferase) gene into alfalfa, which significantly improved the endogenous melatonin content. Compared with wild-type (WT) plants, MsASMT1 overexpression (OE-MsASMT1) plants exhibited a series of phenotypic changes, including vigorous growth, increased plant height, enlarged leaves and robust stems with increased cell sizes, cell numbers and vascular bundles, as well as more branches. We also found that the flavonoid content and lignin composition of syringyl to guaiacyl ratio (S/G) the lignin monomer S/G ratio was were decreased and the cellulose content was increased in OE-MsASMT1 transgenic alfalfa. Further transcriptomic and metabolomic exploration revealed that a large group of genes in phenylalanine pathway related to flavonoids and lignin biosynthesis were significantly altered, accompanied by significantly reduced concentrations of the glycosides of quercetin, kaempferol, formononetin and biochanin in OE-MsASMT1 transgenic alfalfa. Our study first uncovers the effects of endogenous melatonin on alfalfa growth and metabolism. This report provides insights into the regulation regulatory effects and mechanisms of melatonin on manipulating plant growth and phenylalanine metabolism pathway, especially flavonoids and lignin biosynthesis.

Figure S1. Total mapped and unmapped RNA-seq clean reads for transgenic lines and WT plants. (PDF 119 kb)

Figure S4 Expression analysis of selected RNA-seq genes by qRT-PCR. FPKM (fragments per kilobase of exon per million fragments mapped) values obtained with RNA-seq and qPCR values in the analysis of selected genes. Error bars represent the standard error for three independent experimental replicates. (PDF 146 kb)

Figure S3 Differentially expressed transcription factors in transgenic switchgrass. Blue bar represents down-regulated DEGs; red bar indicates up-regulated DEGs. (PDF 133 kb)

Figure S2. Enriched gene ontologies in differentially expressed genes of transgenic switchgrass. Each box shows the GO term number, the p-value in parenthesis, and GO term. Box colors indicates levels of statistical significance: yellow = 0.05; orange = e-05; and red = e-09. (PDF 269 kb)

Table S1. Significantly upregulated genes involved in cell division in transgenic compared to WT plants. (DOCX 15 kb)

Table S4. Number of genes identified from KEGG pathways. (DOCX 16 kb)

Table S3. Significant upregulated transporters in transgenic compared to WT plants. (DOCX 16 kb)

Background Intracellular Na+ (K+)/H+ antiporters (NHXs) have pivotal functions in regulating plant growth, development, and resistance to a range of stresses. To gain insight into the molecular events underlying their actions in switchgrass (Panicum virgatum L.), we analyzed transcriptomic changes between PvNHX1-overexpression transgenic lines and wild-type (WT) plants using RNA sequencing (RNA-seq) technology. Results The comparison of transcriptomic data from the WT and transgenic plants revealed a large number of differentially expressed genes (DEGs) in the latter. Gene ontology (GO) and KEGG pathway analyses showed that these DEGs were associated with a wide range of functions, and participated in many biological processes. For example, we found that PvNHX1 had an important role in plant growth through its regulation of photosynthetic activity and cell expansion. In addition, PvNHX1 regulated K+ homeostasis, cell expansion and pollen development, indicating that it has unique and specific roles in flower development. We also found that transgenic switchgrass exhibited a higher level of transcription of defense-related genes, especially those involved in disease resistance. Conclusion We showed that PvNHX1 had an important role in plant growth and development through its regulation of photosynthetic activity, cell expansion, K+ homeostasis, and pollen development. Additionally, PvNHX1 overexpression activated a complex signal transduction network in response to various biotic and abiotic stresses. In relation to plant growth, development, and defense responses, PvNHX1 also had a vital regulatory role in the formation of a series of plant hormones and transcription factors (TFs). The reliability of the RNA-seq data was confirmed by quantitative real-time PCR. Our data provide a valuable foundation for further research into the molecular mechanisms and physiological roles of NHXs in plants. Electronic supplementary material The online version of this article (10.1186/s12870-018-1278-5) contains supplementary material, which is available to authorized users.

Understanding and enhancing switchgrass (Panicum virgatum L.) photosynthesis will help to improve yield and quality for bio-industrial applications on cellulosic biofuel production. In the present study, leaf anatomical traits and physiological characteristics related to photosynthetic capacity of both lowland and upland switchgrass were recorded from four varieties across the vegetative, elongation and reproductive growth stages. Compared with the upland varieties, the lowland switchgrass showed 37–59, 22–64 and 27–73% higher performance on height, stem and leaf over all three growth stages. Leaf anatomical traits indicated that the leaves of lowland varieties provided more space for carbon assimilation and transportation caused by enhanced cell proliferation with more bundles sheath cells and larger contact areas between the bundle sheath and mesophyll cells (CAMB), which lead to the 32–72% higher photosynthetic capacity found in the lowland varieties during vegetative and elongation growth. However, photosynthetic capacity became 22–51% higher in the upland varieties during the reproductive stage, which is attributed to more photosynthetic pigment. In conclusion, lowland varieties gain a photosynthetic advantage with enhanced bundle sheath cell proliferation, while the upland varieties preserved more photosynthetic pigments. Our study provides new insights for improving the yield in crops by enhancing photosynthesis with anatomical and physiological strategies.

Table S5. Primer sequences used in the experiments. (DOCX 16Â kb)

Figure S1. Total mapped and unmapped RNA-seq clean reads for transgenic lines and WT plants. (PDF 119Â kb)

In plants, Δ1-pyrroline- 5-carboxylate synthase (P5CS) is the rate-limiting enzyme in proline biosynthesis. In this study, we introduced the LpP5CS (Lolium perenne L.) gene into switchgrass by Agrobacterium-mediated transformation. The transgenic lines (TG) were classified into two groups based on their phenotypes and proline levels. The group I lines (TG4 and TG6) had relatively high proline levels and improved biomass yield. The group II lines (TG1 and TG2) showed low proline levels, severely delayed flowering, stunted growth and reduced biomass yield. Additionally, we used RNA-seq analysis to detect the most significant molecular changes, and we analyzed differentially expressed genes, such as flowering-related and CYP450 family genes. Moreover, the biomass yield, physiological parameters, and expression levels of reactive oxygen species scavenger-related genes under salt stress all indicated that the group I plants exhibited significantly increased salt tolerance compared with that of the control plants, in contrast to the group II plants. Thus, genetic improvement of switchgrass by overexpressing LpP5CS to increase proline levels is feasible for increasing plant stress tolerance.

The Drought and Salt Tolerance gene (DST) encodes a C2H2 zinc finger transcription factor, which negatively regulates salt tolerance in rice (Oryza sativa). Phylogenetic analysis of six homologues of DST genes in different plant species revealed that DST genes were conserved evolutionarily. Here, the rice DST gene was linked to an SRDX domain for gene expression repression based on the Chimeric REpressor gene-Silencing Technology (CRES-T) to make a chimeric gene (OsDST-SRDX) construct and introduced into perennial ryegrass by Agrobacterium-mediated transformation. Integration and expression of the OsDST-SRDX in transgenic plants were tested by PCR and RT-PCR, respectively. Transgenic lines overexpressing the OsDST-SRDX fusion gene showed obvious phenotypic differences and clear resistance to salt-shock and to continuous salt stresses compared to non-transgenic plants. Physiological analyses including relative leaf water content, electrolyte leakage, proline content, malondialdehyde (MDA) content, H2O2 content and sodium and potassium accumulation indicated that the OsDST-SRDX fusion gene enhanced salt tolerance in transgenic perennial ryegrass by altering a wide range of physiological responses. To our best knowledge this study is the first report of utilizing Chimeric Repressor gene-Silencing Technology (CRES-T) in turfgrass and forage species for salt-tolerance improvement.