De novo transcriptome analysis of Medicago falcata reveals novel insights about the mechanisms underlying abiotic stress-responsive pathway

BACKGROUND: The entire world is facing a deteriorating environment. Understanding the mechanisms underlying plant responses to external abiotic stresses is important for breeding stress-tolerant crops and herbages. Phytohormones play critical regulatory roles in plants in the response to external and internal cues to regulate growth and development. Medicago falcata is one of the stress-tolerant candidate leguminous species and is able to fix atmospheric nitrogen. This ability allows leguminous plants to grow in nitrogen deficient soils. METHODS: We performed Illumina sequencing of cDNA prepared from abiotic stress treated M. falcata. Sequencedreads were assembled to provide a transcriptome resource. Transcripts were annotated using BLASTsearches against the NCBI non-redundant database and gene ontology definitions were assigned. Acomparison among the three abiotic stress treated samples was carried out. The expression of transcriptswas confirmed with qRT-PCR. RESULTS: We present an abiotic stress-responsive M. falcata transcriptome using next-generation sequencing data from samples grown under standard, dehydration, high salinity, and cold conditions. We combined reads from all samples and de novo assembled 98,515 transcripts to build the M. falcata gene index. A comprehensive analysis of the transcriptome revealed abiotic stress-responsive mechanisms underlying the metabolism and core signalling components of major phytohormones. We identified nod factor signalling pathways during early symbiotic nodulation that are modified by abiotic stresses. Additionally, a global comparison of homology between the M. falcata and M. truncatula transcriptomes, along with five other leguminous species, revealed a high level of global sequence conservation within the family. CONCLUSIONS: M. falcata is shown to be a model candidate for studying abiotic stress-responsive mechanisms in legumes. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to abiotic stresses. Our data provides many gene candidates that might be used for herbage and crop breeding. Additionally, FalcataBase (http://bioinformatics.cau.edu.cn/falcata/) was built for storing these data. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-2019-x) contains supplementary material, which is available to authorized users

Response of Alfalfa (Medicago sativa L.) to Abrupt Chilling as Reflected by Changes in Freezing Tolerance and Soluble Sugars

Abrupt-chilling events threaten the survival of alfalfa plants, the ability to cope with such condition should be considered during cultivar selection in the production. To assess biochemical and molecular responses of alfalfa to abrupt chilling, the cultivars “WL440HQ” (WL) and “ZhaoDong” (ZD) were subjected to a five-phase experimental regime that included two abrupt-chilling events. The freezing tolerance of the crown was determined as the semi-lethal temperature (LT50) calculated from electrolyte leakage. Soluble sugar concentrations were quantified by ion chromatography. The mRNA transcript levels of four genes encoding enzymes (β-amylase, sucrose phosphate synthase, galactinol synthase, and stachyose synthase) involved in sugar metabolism and two cold-regulated genes (Cas15A and K3-dehydrin) were quantified using quantitative real-time PCR analysis. During the abrupt-chilling events, the LT50 decreased significantly in ZD but not in WL. The rapid response of ZD to abrupt chilling may have been due to the large increases in raffinose and stachyose concentrations, which were consistent with increased transcript levels of the galactinol synthase and stachyose synthase genes. Transcript levels of the cold-regulated genes Cas15A and K3-dehydrin were correlated with increased freezing tolerance under abrupt chilling. The results provide a reference for selection of appropriate cultivars to reduce the risk of crop damage in production areas where early autumn or late spring frosts are likely

The Effect of Fertilizers on Biomass and Biodiversity on a Semi-Arid Grassland of Northern China

Semi-arid grassland in northern China faces degradation and desertification problems, with fertilizer application appearing to be a potential solution by improving soil fertility and plant fields or biodiversity. In this study, mineral and organic fertilizers were used in a semi-arid natural grassland in Hebei Province for three years. The plant characteristics, biomass and species diversity index were assessed and analyzed. In the years 2016 and 2017, mineral fertilizers (RC) significantly increased the total aboveground biomass and the aboveground biomass, natural height, density and coverage of Leymus chinensis compared with organic fertilizers (RO), especially at a moderate application rate (RC2). Leymus chinensis was first divided into its own group and then separated into four groups via cluster tree analysis. The importance values of Leymus chinensis showed continuous increases in mineral fertilizer treatments, but not for organic fertilizers. Margalef’s species richness indexes increased significantly (to 2.09) in the organic treatment (RO1) when compared with RC2. Thus, it was concluded that mineral fertilizers could enhance the position of Leymus chinensis in the natural grassland, while organic fertilizers could promote species biodiversity. This study also provides recommendations regarding the use of fertilizers for the purposes of increasing plant biomass and biodiversity in semi-arid grasslands of northern China

Effects on Soil Bacterial Organisms in an Alfalfa Monocropping System after Corn Insertion and Nitrogen Fertilization

Alfalfa (Medicago sativa L.) and corn (Zea mays L.) are common forage plants for feeding livestock, and their effects on soil bacterial organisms have been extensively studied. However, there is little comprehensive research on soil bacterial organisms and their function in a long-term alfalfa monocropping system after corn insertion and fertilization. The effects of alfalfa–corn rotation (AF: alfalfa monocropping, RA: alfalfa and corn rotation) and nitrogen fertilization (RA0 and RA15) were investigated in a field experiment. The results showed that fertilization significantly increased the aboveground biomass (AGB) and soil nitrate nitrogen, and corn insertion significantly decreased the nitrate nitrogen (NO3−-N) (p p NO3−-N and pH were the main environmental factors affecting the bacterial community structure in the RA system

Organic fertilizer and hydrothermal conditions change the distribution of Medicago sativa L. productivity and soil bacterial diversity in coastal saline soil

Abstract Background Alfalfa (Medicago sativa L.) is a perennial leguminous forage that can improve the properties of saline soil. In addition, the supplementation with fertilizer to promote soil bacterial activity is critical to improve the productivity of coastal saline soils. However, the association between fertilizer application, bacterial community characteristics, and alfalfa yield in saline soil remains unclear. Aim To understand the interaction of different fertilizer and environmental factors on soil bacterial diversity and alfalfa yield in coastal saline soil. Results A 4-year field study was conducted to examine the interaction of different fertilizer treatments and environmental factors on soil bacterial diversity and alfalfa yield in coastal saline soil in China. Three organic fertilizer treatments (fulvic acid potassium, fulvic acid potassium + wood vinegar, and fulvic acid potassium + Bacillus), one biochar treatment (bio-charcoal), one inorganic fertilizer treatment (diammonium phosphate), and a control (no fertilizer) were included. The effects of the  six treatments and 15 cutting times on alfalfa yield and soil bacterial community diversity were assessed. The productivity in fulvic acid potassium + Bacillus and fulvic acid potassium treatments was 68.37 and 67.90 t·hm−2 in 4 years, which was significantly higher than that in the bio-charcoal treatment and control. Hydrothermal conditions and timing of alfalfa harvest had significant effects on the soil bacterial community. Proteobacteria, Acidobacteria, and Actinobacteria were the principal bacterial phyla detected in the soil samples, collectively accounting for approximately 60% of the total bacterial abundance. The relative abundance of Bacteroidetes, Firmicutes, and Nitrospirae was significantly correlated with alfalfa yield, and the abundance of these phyla was also affected by the maximum temperature and precipitation. Fertilizer supplementation in coastal saline soil can effectively increase the yield of alfalfa. Among the fertilizers tested, fulvic acid potassium had the strongest effect, whereas bio-charcoal fertilizer had little effect on alfalfa yield. Graphical Abstrac

Co-Expression of Bacterial Aspartate Kinase and Adenylylsulfate Reductase Genes Substantially Increases Sulfur Amino Acid Levels in Transgenic Alfalfa (<i>Medicago sativa</i> L.)

<div><p>Alfalfa (<i>Medicago sativa</i> L.) is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR) catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial <i>AK</i> and <i>APR</i> genes, modified <i>AK</i>, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs), such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.</p></div

PCR and Southern blot analysis of transgenic alfalfa plants.

<p>A. PCR analysis of <i>AK</i> and <i>APR</i> genes in the transgenic alfalfa plants. Lane WT: wild-type line; Lane1-6: Line1-6 transgenic alfalfa lines; Lane 7: water; Lane 8: pDESAK-APR vector; Lane M: GeneRuler TM 1kb DNA ladder (MBI Fermentas, Maryland, USA). Arrows on the left indicate the standard marker bands: 800 bp, 1,000 bp and 1,500 bp. B. Southern blot analysis of <i>AK</i> and <i>APR</i> genes in the transgenic alfalfa plants. Lane L2-1,L2-2,L6-1,L6-2,L8-1,L8-2: two replicates of each transgenic line (Line 2, Line 6, Line 8), Lane WT: wild-type line. M: M: GeneRuler TM 1 kb DNA ladder. Arrows on the right indicate the standard marker bands: 3,000 bp, 5,000 bp, 6,000 bp, 8,000 and 10,000 bp.</p

Analysis of total amino acids and forage products in wild-type and transgenic plants.

<p>A. Total amino acid content in leaves of transgenic and wild-type plants. m/m, % represented mass ratio of total amino acids from dry weight; WT: wild-type plant; 1–12: 12 lines of transgenic alfalfa plants; the bar represented ±SE. B. Weight of up-ground part and leaves products and height of wild-type and transgenic plants. WT : wild-type plant; OE: the average number of 12 lines of transgenic alfalfa plants, the bar represented ±SE.</p

Relative expression levels of SAT, CGS and MS in wild-type and transgenic plants.

<p>Lane WT: wild-type line; Lane 2,6,8: Line 2,6,8 transgenic alfalfa lines. SAT: serine acetyltransferase; CGS: cystathionine γ-synthase; MS: methionine synthase. * represents statistically significant differences (P<0.05). ** represents statistically significant differences (P<0.01).</p

Amino acid contents in the leaves of wild-type and transgenic alfalfa plants over-expressing <i>AK</i> and <i>APR.</i>

<p>The data are presented as the means ±SE obtained from three independent measurements.</p><p>* represents statistically significant differences (P<0.05). ** represents statistically significant differences (P<0.01). The amounts of amino acids were calculated from dry weight of samples as detected and are given inµmol/g of dry weight (DW) of leaves samples. The total amino acid content did not differ significantly between wild-type and transgenic lines. Three plantlets of each type were analyzed.</p