WRKY proteins are a superfamily of transcription factors (TFs) that play multiple roles in plants’ growth, development, and environmental stress response. In this study, a novel WRKY gene called GsWRKY23 that is specifically upregulated in salt-tolerant Glycine soja accession BB52 seedlings was identified by transcriptomic analysis under salt stress. How the physiological functions and mechanisms of the GsWRKY23 gene affect salt tolerance was investigated using transformations of soybean hairy roots and Arabidopsis, including wild-type (WT) and atwrky23-mutant plants. The results showed that GsWRKY23 in the roots, stems, and leaves of BB52, along with its promoter in the cotyledons and root tips of GsWRKY23pro::GUS Arabidopsis seedlings, displayed enhanced induction under salt stress. GsWRKY23 localises to the nucleus and shows transcriptional activation ability in yeast cells. Compared to GsWRKY23-RNAi wild soybean hairy-root composite plants under salt stress, obvious improvements, such as superior growth appearance, plant height and fresh weight (FW), and leaf chlorophyll and relative water content (RWC), were displayed by GsWRKY23-overexpressing (OE) composite plants. Moreover, their relative electrolytic leakage (REL) values and malondialdehyde (MDA) contents in the roots and leaves declined significantly. Most of the contents of Na⁺ and Cl⁻ in the roots, stems, and leaves of GsWRKY23-OE plants decreased significantly, while the content of K⁺ in the roots increased, and the content of NO₃⁻ displayed no obvious change. Ultimately, the Na⁺/K⁺ ratios of roots, stems, and leaves, along with the Cl⁻/NO₃⁻ ratios of roots and stems, decreased significantly. In the transgenic WT-GsWRKY23 and atwrky23-GsWRKY23 Arabidopsis seedlings, the salt-induced reduction in seed germination rate and seedling growth was markedly ameliorated; plant FW, leaf chlorophyll content, and RWC increased, and the REL value and MDA content in shoots decreased significantly. In addition, the accumulation of Na⁺ and Cl⁻ decreased, and the K⁺ and NO₃⁻ levels increased markedly to maintain lower Na⁺/K⁺ and Cl⁻/NO₃⁻ ratios in the roots and shoots. Taken together, these results highlight the role of GsWRKY23 in regulating ionic homeostasis in NaCl-stressed overexpressed soybean composite plants and Arabidopsis seedlings to maintain lower Na⁺/K⁺ and Cl⁻/NO₃⁻ ratios in the roots and shoots, thus conferring improved salt tolerance.

The growth of Flammulina filiformis is strongly dependent on low-temperature cues for the initiation of primordia formation. To obtain a comprehensive understanding of the molecular mechanisms that govern the mycelial response to cold stress, de novo genome sequencing of the F. filiformis monokaryon and multi-omics data (transcriptome and metabolome) analyses of the mycelia, primordia, and fruiting bodies were conducted in the present study. Genome sequencing based on PacBio HiFi and Hi-C resulted in a 36.3 Mb genome sequence that mapped to 12 chromosomes, comprising 11,886 protein-coding genes. A total of 25 cold-responsive (COR) genes and 520 cold-adapted enzymes were identified in the genome. Multi-omics analyses showed that the pathways related to carbohydrate metabolism in the mycelia under low temperature (10 °C) were significantly enriched. Further examination of the expression profiles of carbohydrate-active enzymes (CAZymes) involved in carbohydrate metabolism revealed that out of 515 CAZyme genes in F. filiformis, 58 were specifically upregulated in mycelia under low-temperature conditions. By contrast, the expression levels of these genes in primordia and fruiting bodies reverted to those prior to low-temperature exposure. These indicate that CAZyme genes are important for the low-temperature adaptation of F. filiformis. This research contributes to the targeted breeding of F. filiformis.

Although gene editing technologies have advanced considerably, research on gene editing in Flammulina filiformis remains in its infancy, and a systematic evaluation of how ssODN donor properties affect HDR efficiency in this species is still lacking. To enable precise genome editing in the edible mushroom F. filiformis, we established an LbCas12a ribonucleoprotein (RNP)-mediated homology-directed repair (HDR) platform in protoplasts and systematically evaluated key ssODN donor design parameters: PAM-site mutation, homology arm distance, RAD51-preferred sequences (RPSs), truncated Cas target sequences (tCTSs), the RS-1 enhancer, and thermodynamic stability (ΔG). A PAM-disrupting mutation greatly increased HDR efficiency, up to 58.5%. Extending the left homology arm 174 bp upstream of the break still yielded 20.2% HDR, expanding the tolerable distance. RPSs showed context-dependent effects: enhancing HDR up to 287.8% in some designs but reducing it by 61.9% in another. Both tCTS and RS-1 consistently suppressed HDR. No strong linear correlation between ΔG and HDR efficiency was observed. Collectively, from experiments conducted at a single genomic locus, our results not only establish a versatile precision-editing platform for F. filiformis through optimized ssODN design, but also offer tentative and transferable insights into the HDR-mediated genome editing of other heterokaryotic basidiomycetes facing similar genetic operation challenges.

Flammulina filiformis is a widely cultivated edible mushroom valued for its taste and nutrition. However, its stipe often develops a fibrous and stringy texture that unpleasantly lodges between teeth during chewing. Texture analysis confirmed a distinct toughness gradient, with the upper stipe being more brittle and less tough than the lower part. UHPLC-MS/MS-based metabolomics of these regions identified 953 metabolites, predominantly spanning lipids and lipid-like molecules, organic acids and derivatives, and nucleosides, nucleotides, and analogues. Comparative analysis revealed that the tender upper stipe was characterized by a widespread downregulation of primary metabolites, including severe depletion of key signaling molecules (cAMP, cGMP) and amino acids such as L-tryptophan. In contrast, the tough lower stipe was enriched with metabolites indicative of an oxidative environment, notably a broad spectrum of oxidized lipids and phenolic compounds. KEGG pathway analysis attributed this dichotomy to distinct metabolic programs. While the upper stipe exhibited downregulation in tryptophan and purine metabolism, the lower stipe was enriched for pathways associated with redox homeostasis and lipid peroxidation, including glutathione metabolism and lipid peroxidation. The co-accumulation of oxidized lipids and phenolics suggests a potential mechanism for oxidation-driven tissue fortification. This study reveals a spatially programmed metabolic basis for the textural differentiation in F. filiformis stipes, providing a framework for understanding tissue development and highlighting potential regulatory targets for breeding varieties with improved eating quality.