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.

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.

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.

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.