Phenylalanine ammonia-lyase (PAL) serves as a pivotal regulatory enzyme at the initial branching point of the phenylpropanoid pathway, exerting a profound influence on downstream reactions essential for flavonoid biosynthesis. Glycyrrhiza species are important medicinal plants and provide plenty of roots as raw materials for further utilization, with the components of glycyrrhizic acid and flavonoids as two major active ingredients. However, functional studies of the PAL genes in the medicinal Glycyrrhiza species remain limited. In this study, we identified seven PAL family genes from each of the three medicinal Glycyrrhiza species, Glycyrrhiza uralensis Fisch., G. inflata Bat., and G. glabra L., and comprehensively analyzed their phylogenetic relationships, gene structures, motif distributions, and promoter cis-elements. Gene expression profiling revealed that PAL1 is highly expressed in roots and significantly induced by drought and salt stresses. We further selected G. uralensis GuPAL1 for functional investigation in Arabidopsis. GuPAL1-overexpression lines (GuPAL1-OE) demonstrated significant enhancements in plant growth, flavonoid accumulation, and hormone levels in Arabidopsis thaliana. Conversely, the Atpal1 mutant plants displayed marked reductions in these traits, while the transgenic lines of GuPAL1-OE in the Atpal1 mutant (Atpal1/GuPAL1) recovered to the normal phenotypes similar to wild type (WT). Transcriptomic analysis of the GuPAL1-OE plants compared to WT demonstrated that several key genes in the phenylpropanoid and flavonoid metabolic pathways (4CL, CCoAOMT, CAD, POD, F3H, FLS) were significantly enriched, suggesting that GuPAL1 may promote plant growth and flavonoid biosynthesis by regulating diverse cellular functions, metabolic pathways, and associated gene expressions. These findings highlight the functional importance of GuPAL1 in flavonoid biosynthesis, and provide valuable insights into the molecular mechanisms underlying the medicinal properties of Glycyrrhiza species.

Basic Pentacysteine (BPC) represents a class of plant-exclusive transcription factors, serving pivotal roles in orchestrating developmental processes and mediating responses to both biotic and abiotic stressors. However, the genome-wide characteristics and low-temperature response mechanism of the BPC gene family in cotton remain unclear. Employing a genome-wide screening approach, this study characterized 60 distinct BPC transcription factor genes across ten Gossypium species. Conserved structural analysis showed that all BPC members carried a highly conserved GAGA-binding domain. Concurrently, the exploration of cis-acting elements within promoter regions demonstrated the potential involvement of these BPC transcription factors in modulating developmental processes, hormone signaling cascades, and abiotic stress adaptation mechanisms. Genomic collinearity analysis shows that segmental duplication is the core mechanism for the expansion of this gene family. Expression analysis indicated that the transcription level of GhBPC4 was significantly increased under low-temperature stress. Genetic function studies displayed that VIGS-mediated GhBPC4 silencing reduced cotton cold tolerance. This study systematically analyzed the genomic characteristics of the cotton BPC transcription factor family and functionally validated the molecular mechanism of GhBPC4-mediated cryogenic response. These findings establish an important foundation for subsequent analysis of multidimensional regulatory networks and the breeding of cold-resistant cotton germplasms.

Isoflavone synthase (IFS) is the key enzyme in isoflavonoid biosynthesis and has been functionally characterized in numerous plant species. Glycyrrhiza species, valued for their medicinal properties, accumulate flavonoids with significant physiological activities. Among these, isoflavones play crucial roles in plant growth, development and stress responses. However, the IFS gene family in Glycyrrhiza remains poorly understood. In this study, we identified 10, 9 and 9 IFS genes in G. uralensis, G. inflata and G. glabra, respectively. Phylogenetic analysis classified these genes into four distinct clades (Clade A–D). Further characterization included chromosomal localization, gene structure, conserved motifs, cis-acting elements and synteny analysis. Using yeast one-hybrid (Y1H) screening, dual-luciferase assays and an electrophoretic mobility shift assay (EMSA), these results revealed that auxin response factor 4 (GgARF4) directly binds to the isoflavone synthase 9 (GgIFS9) promoter and activates its expression. Following indole-3-acetic acid (IAA) treatment, RNA-seq revealed that in the differentially expressed genes (DEGs), the genes involved in isoflavonoid and flavonoid biosynthesis pathways were significantly enriched. The result of quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that GgIFS9 was strongly induced by IAA. β-Glucuronidase (GUS) assays confirmed that IAA activates the expression of the GgIFS9 promoter in Nicotiana tabacum. Our findings reveal that, through GgARF4 and its downstream-activated gene GgIFS9, IAA may promote flavonoid synthesis in G. glabra. This study provides novel insights into the auxin-mediated regulation of secondary metabolism in Glycyrrhiza species.

Histidine triad (HIT) family proteins contain a conserved histidine triad motif and play key roles in fungal metabolism and pathogenicity. This study focused on VD9136, a member of the HIT family in Verticillium dahliae, aiming to elucidate its biological function and mechanism underlying its role in cotton pathogenesis. A systematic investigation of the VD9136 gene in V. dahliae was conducted using bioinformatics analysis, gene knockout, genetic complementation, and pathogenicity assays. The results showed that VD9136 protein consists of 136 amino acids and is a stable, neutral, and weakly hydrophilic protein that lacks transmembrane domains and signal peptides; it is localized to the extracellular space via a non-classical secretion pathway. Its secondary structure is predominantly composed of α-helices and random coils. Phylogenetic analysis revealed that VD9136 is closely related to VliHIT, a homologous protein from V. longisporum, the pathogen responsible for Verticillium wilt in rapeseed. The promoter region of VD9136 contains multiple cis-acting elements, including light-responsive, hormone-responsive, and stress-responsive elements, indicating that its transcription may be regulated by multiple signaling pathways. VD9136 was significantly upregulated during the early stage of cotton infection (6–24 h post-inoculation). Pathogenicity assays demonstrated that V. dahliae knockout mutants lacking VD9136 exhibited a significant reduction in virulence, as evidenced by a lower disease index, decreased fungal biomass within plant tissues, and attenuated vascular browning in cotton plants. The pathogenic phenotype was successfully restored in genetic complementation strains. This study identified VD9136 as a key regulatory factor in the pathogenic process of V. dahliae, and its loss of function reduces the pathogenicity of V. dahliae. The findings provide a theoretical basis for elucidating the pathogenic mechanism of cotton Verticillium wilt and for developing corresponding prevention and control strategies.