Effect of Biogas Slurry on the Soil Properties and Microbial Composition in an Annual Ryegrass-Silage Maize Rotation System over a Five-Year Period.

Soil health is seriously threatened by the overuse of chemical fertilizers in agricultural management. Biogas slurry is often seen as an organic fertilizer resource that is rich in nutrients, and its use has the goal of lowering the amount of chemical fertilizers used while preserving crop yields and soil health. However, the application of continuous biogas slurry has not yet been studied for its long-term impact on soil nutrients and microbial communities in a rotation system of annual ryegrass-silage maize (Zea mays). This study aimed to investigate the impacts on the chemical properties and microbial community of farmland soils to which chemical fertilizer (NPK) (225 kg ha-1), biogas slurry (150 t ha-1), and a combination (49.5 t ha-1 biogas slurry + 150 kg ha-1 chemical fertilizer) were applied for five years. The results indicated that compared to the control group, the long-term application of biogas slurry significantly increased the SOC, TN, AP, and AK values by 45.93%, 39.52%, 174.73%, and 161.54%, respectively; it neutralized acidic soil and increased the soil pH. TN, SOC, pH, and AP are all important environmental factors that influence the structural composition of the soil's bacterial and fungal communities. Chemical fertilizer application significantly increased the diversity of the bacterial community. Variation was observed in the composition of soil bacterial and fungal communities among the different treatments. The structure and diversity of soil microbes are affected by different methods of fertilization; the application of biogas slurry not only increases the contents of soil nutrients but also regulates the soil's bacterial and fungal community structures. Therefore, biogas slurry can serve as a sustainable management measure and offers an alternative to the application of chemical fertilizers for sustainable intensification.

The first two whole mitochondrial genomes for the genus Dactylis species: assembly and comparative genomics analysis.

BACKGROUND: Orchardgrass (Dactylis glomerata L.), a perennial forage, has the advantages of rich leaves, high yield, and good quality and is one of the most significant forage for grassland animal husbandry and ecological management in southwest China. Mitochondrial (mt) genome is one of the major genetic systems in plants. Studying the mt genome of the genus Dactylis could provide more genetic information in addition to the nuclear genome project of the genus. RESULTS: In this study, we sequenced and assembled two mitochondrial genomes of Dactylis species of D. glomerata (597, 281 bp) and D. aschersoniana (613, 769 bp), based on a combination of PacBio and Illumina. The gene content in the mitochondrial genome of D. aschersoniana is almost identical to the mitochondrial genome of D. glomerata, which contains 22-23 protein-coding genes (PCGs), 8 ribosomal RNAs (rRNAs) and 30 transfer RNAs (tRNAs), while D. glomerata lacks the gene encoding the Ribosomal protein (rps1) and D. aschersoniana contains one pseudo gene (atp8). Twenty-three introns were found among eight of the 30 protein-coding genes, and introns of three genes (nad 1, nad2, and nad5) were trans-spliced in Dactylis aschersoniana. Further, our mitochondrial genome characteristics investigation of the genus Dactylis included codon usage, sequences repeats, RNA editing and selective pressure. The results showed that a large number of short repetitive sequences existed in the mitochondrial genome of D. aschersoniana, the size variation of two mitochondrial genomes is due largely to the presence of a large number of short repetitive sequences. We also identified 52-53 large fragments that were transferred from the chloroplast genome to the mitochondrial genome, and found that the similarity was more than 70%. ML and BI methods used in phylogenetic analysis revealed that the evolutionary status of the genus Dactylis. CONCLUSIONS: Thus, this study reveals the significant rearrangements in the mt genomes of Pooideae species. The sequenced Dactylis mt genome can provide more genetic information and improve our evolutionary understanding of the mt genomes of gramineous plants.

Transcriptome Analysis Reveals the Potential Molecular Mechanisms of Tiller Bud Development in Orchardgrass.

Tillering is a special type of branching and one of the important contributors to the yield of cereal crops. Strigolactone and sucrose play a vital role in controlling tiller formation, but their mechanism has not been elucidated completely in most crops. Orchardgrass (Dactylis glomerata L.) is an important perennial forage with prominent tillering ability among crops. To date, the mechanism of tillering in orchardgrass is still largely unknown. Therefore, we performed a transcriptome and miRNA analysis to reveal the potential RNA mechanism of tiller formation under strigolactone and sucrose treatment in orchardgrass. Our results found that D3, COL5, NCED1, HXK7, miRNA4393-z, and miRNA531-z could be key factors to control tiller bud development in orchardgrass. In addition, strigolactones might affect the ABA biosynthesis pathway to regulate the tiller bud development of orchardgrass, which may be related to the expression changes in miRNA4393-z, NCED1, and D10. miRNA531-z could be involved in the interaction of strigolactones and sucrose in regulating tillering. These results will be further used to clarify the potential mechanism of tillering for breeding new high-tillering and high-production orchardgrass varieties and beneficial to improving the production and reproduction of crops.

Transcriptome Profiling Provides Insights into the Early Development of Tiller Buds in High- and Low-Tillering Orchardgrass Genotypes.

Orchardgrass (Dactylis glomerata L.) is among the most economically important perennial cool-season grasses, and is considered an excellent hay, pasture, and silage crop in temperate regions worldwide. Tillering is a vital feature that dominates orchardgrass regeneration and biomass yield. However, transcriptional dynamics underlying early-stage bud development in high- and low-tillering orchardgrass genotypes are unclear. Thus, this study assessed the photosynthetic parameters, the partially essential intermediate biomolecular substances, and the transcriptome to elaborate the early-stage profiles of tiller development. Photosynthetic efficiency and morphological development significantly differed between high- (AKZ-NRGR667) and low-tillering genotypes (D20170203) at the early stage after tiller formation. The 206.41 Gb of high-quality reads revealed stage-specific differentially expressed genes (DEGs), demonstrating that signal transduction and energy-related metabolism pathways, especially photosynthetic-related processes, influence tiller induction and development. Moreover, weighted correlation network analysis (WGCNA) and functional enrichment identified distinctively co-expressed gene clusters and four main regulatory pathways, including chlorophyll, lutein, nitrogen, and gibberellic acid (GA) metabolism pathways. Therefore, photosynthesis, carbohydrate synthesis, nitrogen efficient utilization, and phytohormone signaling pathways are closely and intrinsically linked at the transcriptional level. These findings enhance our understanding of tillering in orchardgrass and perennial grasses, providing a new breeding strategy for improving forage biomass yield.

Milletdb: a multi-omics database to accelerate the research of functional genomics and molecular breeding of millets.

Millets are a class of nutrient-rich coarse cereals with high resistance to abiotic stress; thus, they guarantee food security for people living in areas with extreme climatic conditions and provide stress-related genetic resources for other crops. However, no platform is available to provide a comprehensive and systematic multi-omics analysis for millets, which seriously hinders the mining of stress-related genes and the molecular breeding of millets. Here, a free, web-accessible, user-friendly millets multi-omics database platform (Milletdb, http://milletdb.novogene.com) has been developed. The Milletdb contains six millets and their one related species genomes, graph-based pan-genomics of pearl millet, and stress-related multi-omics data, which enable Milletdb to be the most complete millets multi-omics database available. We stored GWAS (genome-wide association study) results of 20 yield-related trait data obtained under three environmental conditions [field (no stress), early drought and late drought] for 2 years in the database, allowing users to identify stress-related genes that support yield improvement. Milletdb can simplify the functional genomics analysis of millets by providing users with 20 different tools (e.g., 'Gene mapping', 'Co-expression', 'KEGG/GO Enrichment' analysis, etc.). On the Milletdb platform, a gene PMA1G03779.1 was identified through 'GWAS', which has the potential to modulate yield and respond to different environmental stresses. Using the tools provided by Milletdb, we found that the stress-related PLATZs TFs (transcription factors) family expands in 87.5% of millet accessions and contributes to vegetative growth and abiotic stress responses. Milletdb can effectively serve researchers in the mining of key genes, genome editing and molecular breeding of millets.

Proteomic and physiological responses of contrasting two different heat-resistant orchardgrass genotypes to heat stress.

As an important forage crop worldwide, the growth and productivity of orchardgrass are greatly impacted by high temperatures. However, little information is known about how orchardgrass proteomic changes under heat conditions. Therefore, the present study investigated the proteomics and physiological changes in 667 [AKZ-NRGR667 (heat-tolerant)] and 7602 [PI237602 (heat-sensitive)] under heat stress (40/35 °C). In addition, the responses of translational regulating of heat stress in orchardgrass were analyzed through proteomic changes using the tandem mass tags (TMT) technique. Together, 410 differentially expressed proteins (DEPs) were identified from two orchardgrass genotypes under heat at 24 h. Proteomics analyses indicated that proteins related to substance metabolism, photosynthesis, and heat shock proteins (HSPs) were differentially expressed under heat stress and control conditions. Moreover, a large proportion of HSPs were expressed in the heat-tolerant genotype as compared to the heat-sensitive genotype. In conclusion, genotype 667 has higher adaptability and repairing capability due to stronger heat tolerance capacity that can make it more suited to sustaining its survival and growth than genotype 7602. These findings can provide the basis for genetic improvements in orchardgrass and other crops facing high-temperature stress or heat environment that may lead to heat resistance or tolerance.

Genome-Wide Identification, Characterization, and Expression of TCP Genes Family in Orchardgrass.

Plant-specific TCP transcription factors regulate several plant growth and development processes. Nevertheless, little information is available about the TCP family in orchardgrass (Dactylis glomerata L.). This study identified 22 DgTCP transcription factors in orchardgrass and determined their structure, phylogeny, and expression in different tissues and developmental stages. The phylogenetic tree classified the DgTCP gene family into two main subfamilies, including class I and II supported by the exon-intron structure and conserved motifs. The DgTCP promoter regions contained various cis-elements associated with hormones, growth and development, and stress responses, including MBS (drought inducibility), circadian (circadian rhythms), and TCA-element (salicylic acid responsiveness). Moreover, DgTCP9 possibly regulates tillering and flowering time. Additionally, several stress treatments upregulated DgTCP1, DgTCP2, DgTCP6, DgTCP12, and DgTCP17, indicting their potential effects regarding regulating responses to the respective stress. This research offers a valuable basis for further studies of the TCP gene family in other Gramineae and reveals new ideas for increasing gene utilization.

Pangenomic analysis identifies structural variation associated with heat tolerance in pearl millet.

Pearl millet is an important cereal crop worldwide and shows superior heat tolerance. Here, we developed a graph-based pan-genome by assembling ten chromosomal genomes with one existing assembly adapted to different climates worldwide and captured 424,085 genomic structural variations (SVs). Comparative genomics and transcriptomics analyses revealed the expansion of the RWP-RK transcription factor family and the involvement of endoplasmic reticulum (ER)-related genes in heat tolerance. The overexpression of one RWP-RK gene led to enhanced plant heat tolerance and transactivated ER-related genes quickly, supporting the important roles of RWP-RK transcription factors and ER system in heat tolerance. Furthermore, we found that some SVs affected the gene expression associated with heat tolerance and SVs surrounding ER-related genes shaped adaptation to heat tolerance during domestication in the population. Our study provides a comprehensive genomic resource revealing insights into heat tolerance and laying a foundation for generating more robust crops under the changing climate.

Genome-wide identification of MADS-box gene family in orchardgrass and the positive role of DgMADS114 and DgMADS115 under different abiotic stress.

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.

Metabolic Regulation and Lipidomic Remodeling in Relation to Spermidine-induced Stress Tolerance to High Temperature in Plants.

Beneficial effects of spermidine (Spd) on alleviating abiotic stress damage have been explored in plants for hundreds of years, but limited information is available about its roles in regulating lipids signaling and metabolism during heat stress. White clover (Trifolium repens) plants were pretreated with 70 µM Spd and then subjected to high temperature (38/33 °C) stress for 20 days. To further investigate the effect of Spd on heat tolerance, transgenic Arabidopsisthaliana overexpressing a TrSAMS encoding a key enzyme involved in Spd biosynthesis was exposed to high temperature (38/33 °C) stress for 10 days. A significant increase in endogenous Spd content in white clover by exogenous application of Spd or the TrSAMS overexpression in Arabidopsisthaliana could effectively mitigate heat-induced growth retardation, oxidative damage to lipids, and declines in photochemical efficiency and cell membrane stability. Based on the analysis of metabolomics, the amino acids and vitamins metabolism, biosynthesis of secondary metabolites, and lipids metabolism were main metabolic pathways regulated by the Spd in cool-season white clover under heat stress. Further analysis of lipidomics found the TrSAMS-transgenic plants maintained relatively higher accumulations of total lipids, eight phospholipids (PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; CL, cardiolipin; LPA, lysophosphatidic acid; LPC, lyso phosphatidylcholine; LPG, lyso phosphatidylglycerol; and LPI, lyso phosphatidylinositol), one glycoglycerolipid (DGDG, digalactosyl diacylglycerol), and four sphingolipids (Cer, ceramide; CerG2GNAc1, dihexosyl N-acetylhexosyl ceramide; Hex1Cer, hexosyl ceramide; and ST, sulfatide), higher ratio of DGDG: monogalactosyl diacylglycerol (MGDG), and lower unsaturation level than wild-type Arabidopsisthaliana in response to heat stress. Spd-induced lipids accumulation and remodeling could contribute to better maintenance of membrane stability, integrity, and functionality when plants underwent a long period of heat stress. In addition, the Spd significantly up-regulated PIP2 and PA signaling pathways, which was beneficial to signal perception and transduction for stress defense. Current findings provide a novel insight into the function of Spd against heat stress through regulating lipids signaling and reprograming in plants.

Complete Chloroplast Genomes of 14 Subspecies of D. glomerata: Phylogenetic and Comparative Genomic Analyses.

Orchardgrass (Dactylis glomerata L.) is a species in the Gramineae family that is highly important economically and valued for its role in ecology. However, the phylogeny and taxonomy of D. glomerata are still controversial based on current morphological and molecular evidence. The study of chloroplast (cp) genomes has developed into a powerful tool to develop molecular markers for related species and reveal the relationships between plant evolution and phylogenetics. In this study, we conducted comparative genomic analyses and phylogenetic inferences on 14 cp genomes of D. glomerata originating from the Mediterranean and Eurasia. The genome size ranged from 134,375 bp to 134,993 bp and exhibited synteny of gene organization and order. A total of 129-131 genes were identified, including 85-87 protein coding genes, 38 tRNA genes and 8 rRNA genes. The cp sequences were highly conserved, and key sequence variations were detected at the junctions of inverted repeats (IRs)/small single-copy (SSC) regions. Moreover, nine highly variable regions were identified among the subspecies based on a sequence divergence analysis. A total of 285 RNA editing sites were detected that were relevant to 52 genes, where rpoB exhibited the most abundant RNA editing sites. The phylogenetic analysis revealed that all Dactylis subspecies clustered into a monophyletic group and most branches provided a high support bootstrap. The main divergence time of D. glomerata was dated to the Miocene era, and this could have been due to changes in the climate. These findings will provide useful insights for further studies on phylogeny, the identification of subspecies and the development of hypotheses for the evolutionary history of the genus Dactylis and of the Gramineae family.

Genome-wide identification and characterization of bHLH family genes from orchardgrass and the functional characterization of DgbHLH46 and DgbHLH128 in drought and salt tolerance.

Basic helix-loop-helix (bHLH) is the second largest family of transcription factors that widely exist in plants and animals, and plays a key role in a variety of biological processes. As an important forage crop worldwide, little information is available about the bHLH family in orchardgrass (Dactylis glomerata L.), although a huge number of bHLH family have been identified and characterized in plants. In this study, we performed genome-wide analysis of bHLH transcription factor family of orchardgrass and identified 132 DgbHLH genes. The phylogenetic tree was constructed by using bHLH proteins of orchardgrass, with Arabidopsis thaliana and Oryza sativa bHLH proteins, to elucidate their homology and classify them into 22 subfamilies. The results of conserved motifs and gene structure support the classification of DgbHLH family. In addition, chromosomal location and gene duplication events of DgbHLH genes were further studied. Transcriptome data exhibited that DgbHLH genes were differentially expressed in different tissues of orchardgrass. We analyzed the gene expression level of 12 DgbHLH genes in orchardgrass under three types of abiotic stresses (heat, salt, and drought). Finally, heterologous expression assays in yeast indicated that DgbHLH46 and DgbHLH128 may enhance the resistance to drought and salt stress. Furthermore, DgbHLH128 may also be involved in abiotic stress by binding to the MYC element. Our study provides a comprehensive assessment of DgbHLH family of orchardgrass, revealing new insights for enhancing gene utilization and improving forage performance.

Spermine-mediated metabolic homeostasis improves growth and stress tolerance in creeping bentgrass (Agrostis stolonifera) under water or high-temperature stress.

Plants have developed diverse defense strategies to reduce the detrimental effects of a wide range of environmental stresses. The objectives of this study were to explore the function of spermine (Spm) on mediating growth and physiological changes in water homeostasis, photosynthetic performance, and oxidative damage and to further examine the regulatory mechanism of Spm on global metabolites reprogramming and associated metabolic pathways in horticultural creeping bentgrass (Agrostis stolonifera) under water and heat stresses. The 21-days-old plants were pretreated with or without 100 µM Spm for 3 days and then subjected to water stress (17% polyethylene glycol 6000), high-temperature stress (40/35°C, day/night), or normal condition (control without water stress and heat stress) for 18 days. Results demonstrated that exogenous application of Spm could significantly increase endogenous polyamine (PAs), putrescine (Put), spermidine (Spd), and Spm contents, followed by effective alleviation of growth retardant, water imbalance, photoinhibition, and oxidative damage induced by water and heat stress. Metabolites' profiling showed that a total of 61 metabolites were differentially or commonly regulated by Spm in leaves. Spm upregulated the accumulation of mannose, maltose, galactose, and urea in relation to enhanced osmotic adjustment (OA), antioxidant capacity, and nitrogen metabolism for growth maintenance under water and heat stress. Under water stress, Spm mainly induced the accumulation of sugars (glucose-1-phosphate, sucrose-6-phosphate, fructose, kestose, maltotriose, and xylose), amino acids (glutamic acid, methionine, serine, and threonine), and organic acids (pyruvic acid, aconitic acid, and ketoglutaric acid) involved in the respiratory pathway and myo-inositol associated with energy production, the ROS-scavenging system, and signal transduction. In response to heat stress, the accumulation of alanine, glycine, gallic acid, malic acid, or nicotinic acid was specifically enhanced by Spm contributing to improvements in antioxidant potency and metabolic homeostasis. This study provides novel evidence of Spm-induced,tolerance to water and heat stresses associated with global metabolites reprogramming in favor of growth maintenance and physiological responses in horticultural plants.

DNA hypermethylation promotes the flowering of orchardgrass during vernalization.

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.

Enhanced Adaptability to Limited Water Supply Regulated by Diethyl Aminoethyl Hexanoate (DA-6) Associated With Lipidomic Reprogramming in Two White Clover Genotypes.

Membrane lipid reprogramming is one of the most important adaptive strategies in plant species under unfavorable environmental circumstances. Therefore, the present experiment was conducted to elucidate the effect of diethyl aminoethyl hexanoate (DA-6), a novel synthetic plant growth regulator, on oxidative damage, photosynthetic performance, changes in lipidomic profile, and unsaturation index of lipids in two white clover (Trifolium repens) cultivars (drought-sensitive "Ladino" and drought-resistant "Riverdel") under PEG-6000-induced water-deficit stress. Results revealed that water-deficit stress significantly enhanced oxidative damage and decreased photosynthetic functions in both cultivars. However, the damage was less in Riverdel. In addition, water-deficit stress significantly decreased the relative content of monogalactocyl-diacylglycerols (MGDG), sulfoquinovosyl-diacylglycerols (SQDG), phosphatidic acisd (PA), phosphatidyl-ethanolamines (PE), phosphatidyl-glycerols (PG), phosphatidyl-serines (PS), ceramides (Cer), hexosylmonoceramides (Hex1Cer), sphingomyelins (SM), and sphingosines (Sph) in both cultivars, but a more pronounced decline was observed in Ladino. Exogenous application of DA-6 significantly increased the relative content of digalactocyl-diacylglycerols (DGDG), monogalactocyl-diacylglycerolsabstra (MGDG), sulfoquinovosyl-diacylglycerols (SQDG), phosphatidic acids (PA), phosphatidyl-ethanolamines (PE), phosphatidyl-glycerols (PG), phosphatidyl-inositols (PI), phosphatidyl-serines (PS), ceramides (Cer), hexosylmonoceramides (Hex1Cer), neutral glycosphingolipids (CerG2GNAc1), and sphingosines (Sph) in the two cultivars under water-deficit stress. DA-6-treated Riverdel exhibited a significantly higher DGDG:MGDG ratio and relative content of sphingomyelins (SM) than untreated plants in response to water deficiency. Furthermore, the DA-6-pretreated plants increased the unsaturation index of phosphatidic acids (PA) and phosphatidylinositols (PI) in Ladino, ceramides (Cer) and hexosylmonoceramides (Hex1Cer) in Riverdel, and sulfoquinovosyl-diacylglycerols (SQDG) in both cultivars under water stress. These results suggested that DA-6 regulated drought resistance in white clover could be associated with increased lipid content and reprogramming, higher DGDG:MGDG ratio, and improved unsaturation index of lipids, contributing to enhanced membrane stability, integrity, fluidity, and downstream signaling transduction.

Global Metabolites Reprogramming Induced by Spermine Contributing to Salt Tolerance in Creeping Bentgrass.

Soil salinization has become a serious challenge to modern agriculture worldwide. The purpose of the study was to reveal salt tolerance induced by spermine (Spm) associated with alterations in water and redox homeostasis, photosynthetic performance, and global metabolites reprogramming based on analyses of physiological responses and metabolomics in creeping bentgrass (Agrostis stolonifera). Plants pretreated with or without 0.5 mM Spm were subjected to salt stress induced by NaCl for 25 days in controlled growth chambers. Results showed that a prolonged period of salt stress caused a great deal of sodium (Na) accumulation, water loss, photoinhibition, and oxidative damage to plants. However, exogenous application of Spm significantly improved endogenous spermidine (Spd) and Spm contents, followed by significant enhancement of osmotic adjustment (OA), photosynthesis, and antioxidant capacity in leaves under salt stress. The Spm inhibited salt-induced Na accumulation but did not affect potassium (K) content. The analysis of metabolomics demonstrated that the Spm increased intermediate metabolites of γ-aminobutyric acid (GABA) shunt (GABA, glutamic acid, and alanine) and tricarboxylic acid (TCA) cycle (aconitic acid) under salt stress. In addition, the Spm also up-regulated the accumulation of multiple amino acids (glutamine, valine, isoleucine, methionine, serine, lysine, tyrosine, phenylalanine, and tryptophan), sugars (mannose, fructose, sucrose-6-phosphate, tagatose, and cellobiose), organic acid (gallic acid), and other metabolites (glycerol) in response to salt stress. These metabolites played important roles in OA, energy metabolism, signal transduction, and antioxidant defense under salt stress. More importantly, the Spm enhanced GABA shunt and the TCA cycle for energy supply in leaves. Current findings provide new evidence about the regulatory roles of the Spm in alleviating salt damage to plants associated with global metabolites reprogramming and metabolic homeostasis.

R2R3-MYB gene family: Genome-wide identification provides insight to improve the content of proanthocyanidins in Trifolium repens.

The R2R3-MYB family is one of largest transcription factor families in plants playing significant roles in regulating anthocyanin and proanthocyanidin biosynthesis. Proanthocyanidins are one of major objectives to improve the quality of white clover (Trifolium repens L.), which have a beneficial effect on ruminant to prevent the lethal pasture bloat. A total of 133 TrR2R3-MYB genes were identified and distributed on all 16 chromosomes based on the whole genome information of white clover. Also, by exploring the gene structure, motifs and duplication events of TrR2R3-MYBs, as well as the evolutionary relationship with TrR2R3-MYB genes of other species, 10 TrR2R3-MYB genes with the potential to regulate the anthocyanins and proanthocyanidins biosynthesis were screened. These TrR2R3-MYB genes responded significantly to low temperature in white clover. In addition, they have different expression patterns in leaves, petioles and inflorescences of white clover. Importantly, TrMYB116 and TrMYB118 may positively regulate anthocyanin accumulation and low temperature response in white clover. TrMYB118 may also be associated with anthocyanin pigmentation pattern in Purple leaves. This study provides a basis for verifying the function of TrR2R3-MYB and breeding white clover cultivars with high proanthocyanidins.

Metabolites Reprogramming and Na+/K+ Transportation Associated With Putrescine-Regulated White Clover Seed Germination and Seedling Tolerance to Salt Toxicity.

Soil salinization is a serious challenge to many countries worldwide. Putrescine (Put) is related to the improvement of seed germination under salt stress, but molecular and metabolic mechanisms are still not fully understood. The objectives of this study were to determine the effect of seed soaking with Put on germination characteristics under salt stress induced by 100 mm sodium chloride (NaCl) and to further analyze subsequent stress tolerance associated with amylolysis, oxidative damage, sodium (Na+)/ potassium (K+) accumulation and transportation, and metabolic homeostasis in white clover (Trifolium repens cv. Haifa) seedlings. The results showed that seed soaking with Put significantly alleviated salt-induced decreases in the endogenous Put content, germination rate, germination vigor, germination index, Rl/SL, and fresh/dry weight of seedlings. Put application also significantly promoted starch metabolism through activating α-amylase and ß-amylase activities under salt stress. The metabolomic analysis showed that seed soaking with Put significantly increased the accumulation of polyamines (Put and spermidine), amino acids (γ-aminobutyric acid, glutamate, alanine, proline, citrulline, etc.), organic acids (ketopentanic acid, malonic acid, malic acid, ketopentanic acid, cis-sinapinic acid, etc.), lipids and fatty acids (glycerol, stearic acid, linoleic acid, palmitic acid, etc.), sugars (levoglucosan, fucose, and anhydro-D-galactose), alcohols (myo-inositol, allo-inositol, hexadecanol, and threitol), and other metabolites (thymine, xanthine, adenine, guanine, and glycerol 1-phosphate, etc.) associated with enhanced tricarboxylic acid (TCA) cycle and γ-aminobutyric acid (GABA) shunt contributing to better osmotic adjustment, cell membrane stability, energy supply, and metabolic homeostasis when seeds germinated under salt stress. In addition, Put significantly up-regulated the AsSOS1, NHX6, SKOR, HKT1, and HKT8 expression levels which played critical roles in Na+ rejection and K+ retention resulting in higher K+/Na+ ratio during seed germination under salt stress. The Put-induced up-regulation of HAL2 transcription level could reduce the toxicity of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in cells. Current findings will provide an integrative understanding of Put-induced salt tolerance associated with amylolysis, metabolic regulation, and ionic homeostasis during seed germination.

Adaptive Responses of Common and Hybrid Bermudagrasses to Shade Stress Associated With Changes in Morphology, Photosynthesis, and Secondary Metabolites.

Alteration of ploidy in one particular plant species often influences their environmental adaptation. Warm-season bermudagrass is widely used as forage, turfgrass, and ground-cover plant for ecological remediation, but exhibits low shade tolerance. Adaptive responses to shade stress between triploid hybrid bermudagrass cultivars ["Tifdwarf" (TD), "Tifsport" (TS), and "Tifway" (TW)] and tetraploid common bermudagrass cultivar "Chuanxi" (CX) were studied based on changes in phenotype, photosynthesis, and secondary metabolites in leaves and stems. Shade stress (250 luminance, 30 days) significantly decreased stem diameter and stem internode length, but did not affect the leaf width of four cultivars. Leaf length of CX, TD, or TW showed no change in response to shade stress, whereas shade stress significantly elongated the leaf length of TS. The CX and the TS exhibited significantly higher total chlorophyll (Chl), Chl a, carotenoid contents, photosynthetic parameters [PSII photochemical efficiency (Fv/Fm), transpiration rate, and stomatal conductance] in leaves than the TW and the TD under shade stress. The CX also showed a significantly higher performance index on absorption basis (PIABS) in leaf and net photosynthetic rate (Pn) in leaf and stem than the other three cultivars under shade stress. In addition, the TS maintained higher proantho cyanidims content than the TW and the TD after 30 days of shade stress. Current results showed that tetraploid CX exhibited significantly higher shade tolerance than triploid TD, TS, and TW mainly by maintaining higher effective photosynthetic leaf area, photosynthetic performance of PSI and PSII (Pn and Fv/Fm), and photosynthetic pigments as well as lower Chl a/b ratio for absorption, transformation, and efficient use of light energy under shade stress. For differential responses to shade stress among three triploid cultivars, an increase in leaf length and maintenance of higher Fv/Fm, gas exchange, water use efficiency, carotenoid, and proanthocyanidin contents in leaves could be better morphological and physiological adaptations of TS to shade than other hybrid cultivars (TD and TW).

Comprehensive Transcriptome Analysis Uncovers Distinct Expression Patterns Associated with Early Salinity Stress in Annual Ryegrass (Lolium Multiflorum L.).

Soil salination is likely to reduce crop production worldwide. Annual ryegrass (Lolium multiflorum L.) is one of the most important forages cultivated in temperate and subtropical regions. We performed a time-course comparative transcriptome for salinity-sensitive (SS) and salinity-insensitive (SI) genotypes of the annual ryegrass at six intervals post-stress to describe the transcriptional changes and identify the core genes involved in the early responses to salt stress. Our study generated 215.18 Gb of clean data and identified 7642 DEGs in six pairwise comparisons between the SS and SI genotypes of annual ryegrass. Function enrichment of the DEGs indicated that the differences in lipid, vitamins, and carbohydrate metabolism are responsible for variation in salt tolerance of the SS and SI genotypes. Stage-specific profiles revealed novel regulation mechanisms in salinity stress sensing, phytohormones signaling transduction, and transcriptional regulation of the early salinity responses. High-affinity K+ (HAKs) and high-affinity K1 transporter (HKT1) play different roles in the ionic homeostasis of the two genotypes. Moreover, our results also revealed that transcription factors (TFs), such as WRKYs, ERFs, and MYBs, may have different functions during the early signaling sensing of salt stress, such as WRKYs, ERFs, and MYBs. Generally, our study provides insights into the mechanisms of the early salinity response in the annual ryegrass and accelerates the breeding of salt-tolerant forage.