Analysis of rotational grazing management for sheep in mixed grassland.

Sown mixed grassland is rarely used for livestock raising and grazing; however, different forages can provide various nutrients for livestock, which may be beneficial to animal health and welfare. We established a sown mixed grassland and adopted a rotational grazing system, monitored the changes in aboveground biomass and sheep weights during the summer grazing period, measured the nutrients of forage by near-infrared spectroscopy, tested the contents of medium- and long-chain fatty acids by gas chromatography, and explored an efficient sheep fattening system that is suitable for agro-pastoral interlacing areas. The results showed that the maximum forage supply in a single grazing paddock was 4.6 kg DM/d, the highest dry matter intake (DMI) was 1.80 kg DM/ewe/d, the average daily weight gain (ADG) was 193.3 g, the DMI and ADG were significantly correlated (P < 0.05), and the average feed weight gain ratio (F/G) reached 8.02. The average crude protein and metabolizable energy intake by sheep were 286 g/ewe/d and 18.5 MJ/ewe/d respectively, and the n-6/n-3 ratio of polyunsaturated fatty acids in mutton was 2.84. The results indicated that the sheep fattening system had high feed conversion efficiency, could improve the yield and quality of sheep, and could be promoted in suitable regions.

Integrated mRNA and microRNA expression analysis of root response to phosphate deficiency in Medicago sativa.

The deficiency of available phosphate significantly limits plant growth and development. This study sought to investigate how alfalfa (Medicago sativa), a high-yielding and high-quality forage widely cultivated worldwide, responds to phosphate deficiency stress by integrating transcriptional and post-transcriptional data. In this study, 6,041 differentially expressed genes (DEGs) were identified in alfalfa roots under phosphate deficiency conditions. Furthermore, psRNATarget, RNAhybrid, and TargetFinder were used to predict the target genes of 137 differentially expressed miRNAs (DEMs) in the root. In total, 3,912 DEGs were predicted as target genes. Pearson correlation analysis revealed 423 pairs of miRNA-mRNA regulatory relationships. MiRNA negatively regulates mRNA involved in regulatory pathways of phosphate deficiency responses in alfalfa. miR156e targeted squamosa promoter-binding-like protein 13A (SPL13), miR160c targeted auxin response factor 18 (ARF18), and miR2587a controlled glycolysis and citrate cycle via Phosphoenolpyruvate carboxykinase (ATP) (PCKA). Novel-miR27 regulated SPX domain-containing protein that controls phosphate transport in alfalfa root, novel-miR3-targeted sulfoquinovosyl transferase SQD2 controlled sulfolipid synthesis and glutathione S-transferase (GST; mediated by miR169j/k and novel-miR159) regulated glutathione metabolism. miR399l regulated auxin-responsive protein SAUR72 involved in IAA signal transduction, while abscisic acid receptor PYL4 (regulated by novel-miR205 and novel-miR83) participated in ABA signal transduction. Combined miRNA-mRNA enrichment analysis showed that most miRNAs regulate the phosphate starvation response of alfalfa by modulating target genes involved in carbohydrate metabolism, sulfolipid metabolism, glutathione metabolism, and hormone signal transduction. Therefore, this study provides new insights into the post-transcriptional regulation mechanism of phosphate deficiency responses and new perspectives on phosphate assimilation pathways in alfalfa and other legumes.

Metabolomic changes in crown of alfalfa (Medicago sativa L.) during de-acclimation.

Alfalfa is a high-quality forage legume species that is widely cultivated at high latitudes worldwide. However, a decrease in cold tolerance in early spring seriously affects regrowth and persistence of alfalfa. There has been limited research on the metabolomic changes that occur during de-acclimation. In this study, a liquid chromatography-mass spectrometry system was used to compare the metabolites in two alfalfa cultivars during a simulated overwintering treatment. In four pairwise comparisons, 367 differential metabolites were identified, of which 31 were annotated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Many of these metabolites were peptides, carbohydrates, and lipids. At the subclass level, 17 major pathways were revealed to be significantly enriched (P < 0.05). The main differential metabolites included amino acids, peptides and analogs, carbohydrates, and glycerol phosphocholines. A metabolomic analysis showed that the up-regulation of unsaturated fatty acids and amino acids as well as the enhancement of the related metabolic pathways might be an effective strategy for increasing alfalfa cold tolerance. Furthermore, glycerophospholipid metabolism affects alfalfa cold tolerance in early spring. Study results provide new insights about the changes in alfalfa metabolites that occur during de-acclimation, with potential implications for the selection and breeding of cold-tolerant cultivars.

Plastic response of Medicago sativa L. root system traits and cold resistance to simulated rainfall events.

Climate change (rainfall events and global warming) affects the survival of alfalfa (Medicago sativa L.) in winter. Appropriate water management can quickly reduce the mortality of alfalfa during winter. To determine how changes in water affect the cold resistance of alfalfa, we explored the root system traits under different rainfall events and the effects on cold resistance in three alfalfa cultivars. These were exposed to three simulated rainfall events (SRE) × two phases in a randomized complete block design with six replications. The three cultivars were WL168, WL353 and WL440, and the three SRE were irrigation once every second day (D2), every four days (D4) and every eight days (D8). There were two phases: before cold acclimation and after cold acclimation. Our results demonstrated that a period of exposure to low temperature was required for alfalfa to achieve maximum cold resistance. The root system tended toward herringbone branching under D8, compared with D2 and D4, and demonstrated greater root biomass, crown diameter, root volume, average link length and topological index. Nevertheless, D8 had less lateral root length, root surface area, specific root length, root forks and fractal dimensions. Greater root biomass and topological index were beneficial to cold resistance in alfalfa, while more lateral roots and root forks inhibited its ability to survive winter. Alfalfa roots had higher proline, soluble sugar and starch content in D8 than in D2 and D4. In contrast, there was lower malondialdehyde in D8, indicating that alfalfa had better cold resistance following a longer irrigation interval before winter. After examining root biomass, root system traits and physiological indexes we concluded that WL168 exhibited stronger cold resistance. Our results contribute to greater understanding of root and cold stress, consequently providing references for selection of cultivars and field water management to improve cold resistance of alfalfa in the context of changes in rainfall patterns.

Metabolomic analyses reveal substances that contribute to the increased freezing tolerance of alfalfa (Medicago sativa L.) after continuous water deficit.

BACKGROUND: Alfalfa is a high-quality forage cultivated widely in northern China. Recently, the failure of alfalfa plants to survive the winter has caused substantial economic losses. Water management has attracted considerable attention as a method for the potential improvement of winter survival. The aim of this study was to determine whether and how changes in the water regime affect the freezing tolerance of alfalfa. RESULTS: The alfalfa variety WL353LH was cultivated under water regimes of 80 and 25% of water-holding capacity, and all the plants were subjected to low temperatures at 4/0 °C (light/dark) and then - 2/- 6 °C (light/dark). The semi-lethal temperatures were lower for water-stressed than well-watered alfalfa. The pool sizes of total soluble sugars, total amino acids, and proline changed substantially under water-deficit and low-temperature conditions. Metabolomics analyses revealed 72 subclasses of differential metabolites, among which lipid and lipid-like molecules (e.g., fatty acids, unsaturated fatty acids, and glycerophospholipids) and amino acids, peptides, and analogues (e.g., proline betaine) were upregulated under water-deficit conditions. Some carbohydrates (e.g., D-maltose and raffinose) and flavonoids were also upregulated at low temperatures. Finally, Kyoto Encyclopedia of Genes and Genomes analyses revealed 18 significantly enriched pathways involved in the biosynthesis and metabolism of carbohydrates, unsaturated fatty acids, amino acids, and glycerophospholipids. CONCLUSIONS: Water deficit significantly enhanced the alfalfa' freezing tolerance, and this was correlated with increased soluble sugar, amino acid, and lipid and lipid-like molecule contents. These substances are involved in osmotic regulation, cryoprotection, and the synthesis, fluidity, and stability of the cellular membrane. Our study provides a reference for improving alfalfa' winter survival through water management.

Retraction Note: Doubled haploid production in alfalfa (Medicago sativa L.) through isolated microspore culture.

This paper has been retracted.

Doubled haploid production in alfalfa (Medicago sativa L.) through isolated microspore culture.

Rapid production of doubled haploids (DHs) through isolated microspore culture is an important and promising method for genetic study of alfalfa. To induce embryogenesis in alfalfa, isolated microspores were submitted to abiotic stresses during their initial culture, in order to stimulate them to form embryos and plantlets. 'Baoding' and 'Zhongmu No 1' alfalfa cultivars supported reproducible and reliable proliferation response irrespective of any stress treatment of microspores. The microspore developmental stage for isolated microspore culture was studied and we found that uninucleate microspores were best to initiate culture. Exposure of microspores to appropriate low temperature or heat shock stresses were able to increase the efficiency of embryogenesis. The most effective low-temperature treatment was 4 °C for 24 h and the frequency of plantlets induction was 20.0%. The most effective heat shock treatment was 32 °C for 2 d and the frequency of plantlets induction was 14.17%. The analysis of ploidy level performed by flow cytometer revealed that the majority of 278 regenerated plantlets were haploid (65.83%) or doubled haploid (33.81%). This is the first report of haploid production in alfalfa through isolated microspore culture.

Analysis of physiological and miRNA responses to Pi deficiency in alfalfa (Medicago sativa L.).

KEY MESSAGE: The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake. Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.

Identification of Drought-Responsive MicroRNAs from Roots and Leaves of Alfalfa by High-Throughput Sequencing.

Alfalfa, an important forage legume, is an ideal crop for sustainable agriculture and a potential crop for bioenergy resources. Drought, one of the most common environmental stresses, substantially affects plant growth, development, and productivity. MicroRNAs (miRNAs) are newly discovered gene expression regulators that have been linked to several plant stress responses. To elucidate the role of miRNAs in drought stress regulation of alfalfa, a high-throughput sequencing approach was used to analyze 12 small RNA libraries comprising of four samples, each with three biological replicates. From the 12 libraries, we identified 348 known miRNAs belonging to 80 miRNA families, and 281 novel miRNAs, using Mireap software. Eighteen known miRNAs in roots and 12 known miRNAs in leaves were screened as drought-responsive miRNAs. With the exception of miR319d and miR157a which were upregulated under drought stress, the expression pattern of drought-responsive miRNAs was different between roots and leaves in alfalfa. This is the first study that has identified miR3512, miR3630, miR5213, miR5294, miR5368 and miR6173 as drought-responsive miRNAs. Target transcripts of drought-responsive miRNAs were computationally predicted. All 447 target genes for the known miRNAs were predicted using an online tool. This study provides a significant insight on understanding drought-responsive mechanisms of alfalfa.

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.

Co-downregulation of the hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase and coumarate 3-hydroxylase significantly increases cellulose content in transgenic alfalfa (Medicago sativa L.).

Lignin is a component of the cell wall that is essential for growth, development, structure and pathogen resistance in plants, but high lignin is an obstacle to the conversion of cellulose to ethanol for biofuel. Genetically modifying lignin and cellulose contents can be a good approach to overcoming that obstacle. Alfalfa (Medicago sativa L.) is rich in lignocellulose biomass and used as a model plant for the genetic modification of lignin in this study. Two key enzymes in the lignin biosynthesis pathway-hydroxycinnamoyl -CoA:shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3H)-were co-downregulated. Compared to wild-type plants, the lignin content in the modified strain was reduced by 38%, cellulose was increased by 86.1%, enzyme saccharification efficiency was increased by 10.9%, and cell wall digestibility was increased by 13.0%. The modified alfalfa exhibited a dwarf phenotype, but normal above ground biomass. This approach provides a new strategy for reducing lignin and increasing cellulose contents and creates a new genetically modified crop with enhanced value for biofuel.

Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa 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 AK and APR genes, modified AK, 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.