As a unique tree with significant ecological and economic value, understanding the molecular mechanisms behind its drought tolerance is essential for the conservation and cultivation of Ginkgo biloba in the face of climate change. Despite notable variation in drought tolerance among different ginkgo populations, the genetic and molecular mechanisms remain largely unexplored. In this study, we assessed 21 ginkgo families subjected to progressive drought stress over 0, 20, and 60 days. We analyzed a range of phenotypic, physiological, and biochemical traits related to photosynthesis, osmotic regulation, antioxidant activity, and pigment levels. Using principal component analysis (PCA) and a weighted membership function approach, we identified the SD115 family as drought-tolerant and the GS6-A family as drought-sensitive. Comparative transcriptomic analysis revealed that differentially expressed genes (DEGs) increased over time and were primarily enriched in pathways related to phenylpropanoid and sucrose metabolism, plant hormone signaling, and phenylalanine metabolism. Among these DEGs, we isolated GbWRKY57, a drought-responsive transcription factor located in the nucleus. Functional studies showed that overexpression of GbWRKY57 reduced drought tolerance in ginkgo calli and transgenic tobacco, whereas virus-induced gene silencing enhanced drought resistance. Dual-luciferase assays further demonstrated that GbWRKY57 functions as a transcriptional repressor of GbRD19 and GbPR1. Our findings not only provide candidate germplasm for drought-resistant ginkgo breeding but also uncover a negative regulatory mechanism mediated by GbWRKY57, offering new candidate molecular targets and theoretical insights for improving drought resilience in woody plants.

Codon usage bias (CUB), referring to the unequal preference for synonymous codons in genetic coding, is shaped by both mutation pressure and natural selection. However, a comprehensive understanding of codon usage trends and the evolutionary mechanisms influencing the chloroplast, mitochondrial, and nuclear genomes of yellowhorn (Xanthoceras sorbifolium Bunge), a tree species native to China with high ecological and economic value, remains lacking. Analysis of nucleotide composition revealed a marked bias toward A/T bases, especially at the third codon position, across all three genomic types. Notably, nuclear genes had a relatively higher GC content compared to organellar counterparts. Analyses using ENC–GC3s plots, PR2-bias assessments, and neutrality plots consistently indicated that natural selection exerted a stronger influence than mutation in determining codon usage patterns. Additionally, correspondence analysis showed that only a limited portion of codon usage variation could be attributed to relative synonymous codon usage, implying the involvement of other evolutionary forces. We identified 11, 13, and 23 optimal codons in the chloroplast, mitochondrial, and nuclear genomes, respectively. Among these, CUU (Leu) and GCA (Ala) were consistently preferred across all compartments. Overall, our findings highlight genome-specific codon preferences and evolutionary pressures, underscoring distinct functional and evolutionary characteristics across the nuclear and organellar genomes.

Understanding the genetic and regulatory mechanisms underlying wood traits and secondary cell wall (SCW) development in Ginkgo biloba is crucial for improving wood quality. We identified key genes related to wood traits and SCW development through integrated genome-wide association studies (GWAS), transcriptome-wide association studies (TWAS), and weighted gene co-expression network analysis (WGCNA). Cellulose biosynthesis in the SCW is catalyzed by the CesA4-CesA7-CesA8 complex encoded by GbCesA4, GbCesA7, and GbCesA8A/8B. These CesA genes form a co-expression network with TUBA/TUBB and EG, indicating coordination among cellulose synthesis, cytoskeletal guidance, and cell wall remodeling. Additionally, loss of function of GbCesA8B caused only a slight reduction in cellulose content, supporting potential functional redundancy between GbCesA8A and GbCesA8B. For hemicellulose biosynthesis, GbCSLA9A/9B and IRX9/IRX14 were major contributors to mannan/glucomannan and xylan synthesis, respectively, and formed a co-expression network with UXS, UXE, IRX7, GXMT, and URGT, spanning nucleotide sugar supply, transport, and polymer elongation and modification. Moreover, MYB46 may regulate mannan/glucomannan biosynthesis in the SCW by activating CSLA9 transcription. For lignin biosynthesis, TWAS identified multiple genes involved in phenylalanine biosynthesis, phenylpropanoid metabolism, and lignin monomer polymerization, including ADT/PDT, PAL, and PER, as well as MYB91 and several bHLH genes that may positively regulate lignin accumulation. Furthermore, several transcription factors potentially involved in SCW development were identified, including GATA9 as a putative positive regulator, WRKY12 and HB15 as potential negative regulators, and ELF6, which may facilitate tracheid expansion. Our findings provide valuable insights into the genetic regulation of wood traits and SCW development in Ginkgo.

Sophora moorcroftiana (Benth.) Baker is an ecologically important shrub dominating the dry valleys of the Yarlung Tsangpo River on the central Qinghai–Tibet Plateau. However, its population-level genomic variation and adaptive signatures remain poorly characterized. Here we used whole-genome resequencing (WGRS) to investigate genetic diversity and population structure in 180 individuals from nine natural populations in Xizang, China. Sequencing at 15× depth yielded 28.27 million high-quality SNPs. Within-population genetic diversity was moderate overall, with observed heterozygosity (Ho) ranging from 0.2178 to 0.3002, nucleotide diversity (π) from 0.2435 × 10⁻³ to 0.2789 × 10⁻³, and uniformly positive Tajima’s D values (1.0782–1.2805). Pairwise genetic differentiation was also moderate (mean FST = 0.066; range 0.0375–0.0896). ADMIXTURE, PCA, and a SNP-based phylogenetic tree consistently resolved four genetic clusters that corresponded primarily to major river-basin groupings rather than elevation. TreeMix analysis inferred a directional gene-flow event from the Cheshire population into Rinpung, while linkage disequilibrium decay varied markedly among populations, from rapid decay in Taktse to slow decay in Gonggar, indicating heterogeneous demographic histories. A selective-sweep comparison between two climatically contrasting but elevationally similar sites (Sangzhuzi vs. Sangri) identified 180 candidate genes, with 99 and 81 genes under putative selection in each population, respectively. These genes were significantly enriched in pathways related to flavonoid biosynthesis, terpene metabolism, oxidoreductase activity, and other secondary-metabolism and one-carbon processes associated with UV protection, drought tolerance, and oxidative-stress regulation. Together, our results provide the first genome-wide portrait of genetic diversity and fine-scale population structure in S. moorcroftiana, and highlight how microclimatic heterogeneity and valley-bounded dispersal shape local adaptation in a key dry-valley shrub of the Qinghai–Tibet Plateau.

The primary poisonous components in Ginkgo biloba seeds are ginkgotoxin (4’-O-methylpyridoxine, MPN) and its 5’-glucoside (MPN-5’-glucoside, MPNG). When Ginkgo biloba kernels are consumed in excess at one time, the total amount of MPN and MPNG (TMPN) can reach toxic levels. We investigated the effects of four storage methods (room temperature storage, sand storage, 4 °C storage, -20 °C storage) on MPN and MPNG levels and their distribution in embryos and endosperms of different Ginkgo biloba cultivars. Results showed that room temperature and sand storage promoted embryonic post-maturation of Ginkgo biloba seeds, whereas 4 °C and − 20 °C storage inhibited embryo development. During all storage treatments, the total TMPN mass in the whole kernel and its proportional distribution between embryo and endosperm remained stable, while the content concentrations (µg/g) of MPN, MPNG, and TMPN in the two tissues exhibited dynamic fluctuations driven by the interconversion of MPN and MPNG. Notably, over 93

Dihydroflavonol 4-reductase (DFR) plays a pivotal role in regulating flavonoid and anthocyanin biosynthesis, governing the accumulation of plant secondary metabolites. This study aimed to characterize the DFR gene family in Ginkgo biloba and elucidate the function of the predominant gene GbDFR2 in the flavonoid metabolic network. Through transcriptome analysis, three differentially expressed GbDFR genes were identified. Bioinformatic analysis revealed that all three GbDFR proteins are hydrophilic and acidic and belong to the NADB_Rossmann superfamily. RT-qPCR analysis of different tissues of ginkgo revealed that all three GbDFR genes exhibited the highest expression levels in the leaves. An overexpression vector of GbDFR2 was constructed and stably transformed into Nicotiana benthamiana. Metabolomic and qPCR analyses showed that heterologous GbDFR2 expression significantly remodeled the flavonoid profile, upregulating sakuranetin and 3,7-Di-O-methylquercetin while downregulating narcissin and naringenin chalcone. Additionally, it upregulated endogenous NbCHI and NbDFR, and suppressed the transcription factors NbMYL2b and NbERF4a. These findings suggest that GbDFR2 can act as a regulator of flavonol biosynthesis and provide a candidate gene for the metabolic engineering of flavonoids in woody plants.

Ginkgo biloba is a valuable timber tree species, particularly suitable for high-end furniture manufacturing. Understanding the variations in its wood properties and their relationship with environmental factors is crucial for the selection and cultivation of superior timber germplasm. In this study, the analysis of 13 wood property indicators from 289 Ginkgo biloba across 20 regions revealed a relatively high variation coefficient (7.076% to 37.719%). With the exception of hemicellulose content, significant differences in other properties were observed among various regions. Correlation analysis demonstrated relationships among multiple traits, particularly indicating that an increase in tracheid wall thickness and the width-to-lumen ratio, along with a decrease in tracheid lumen diameter, could result in higher wood density. The ginkgo wood properties across multiple regions were assessed using principal component analysis and membership function method. The HC and LA regions were identified as sources of high-quality ginkgo wood, and several exceptional individual trees suitable for high-end furniture manufacturing were selected. Further investigation into the influence of environmental factors on ginkgo wood properties revealed that temperature, precipitation, and their fluctuations significantly affect wood density, tracheid morphology, and chemical composition. Regions with higher temperatures and precipitation, along with smaller fluctuations, exhibit greater wood density and larger tracheid, making them more suitable for furniture manufacturing.

Artificial defoliation serves not only as a simulation of natural abiotic stress but also as a practical silvicultural measure. To investigate the effects of defoliation on growth and physiology in Ginkgo biloba L., and to examine how the timing and intensity of leaf harvesting influence flavonoid yield, two-year-old ginkgo seedlings were subjected to treatments in which either one-third (mild defoliation) or two-thirds (severe defoliation) of the leaves were removed in June, July, or August. Growth, carbon synthesis, allocation and storage traits, as well as oxidative defense indicators, were measured following defoliation. The results indicated that defoliation-induced growth inhibition was time-dependent, with significant reductions in seedling height and ground diameter increment. The net photosynthetic rate per unit leaf area decreased in remaining leaves, while soluble sugar and starch concentrations increased significantly in leaves but decreased in mature stems and roots. Severe defoliation resulted in a delayed recovery of carbon reserves in roots. Additionally, superoxide dismutase activity in leaf tissues increased progressively over time after defoliation, whereas soluble protein content was significantly elevated only in October. Mild defoliation led to a declining trend in flavonoid content over time, yet the total flavonoid yield across six treatment groups exceeded that of the control by 10.8

GbF3H acts as a hierarchical switch that suppresses NtFLS1 but activates NtFLS2, thereby diverting flavonoid flux away from flavonols and toward isoflavonoids in transgenic tobacco. Flavanone 3-hydroxylase (F3H) is a key branch-point enzyme in the flavonoid biosynthesis, catalyzing the hydroxylation of naringenin to dihydrokaempferol and thereby directing metabolic flux into distinct flavonoid branches. In this study, we employed an integrated approach combining bioinformatic, molecular characterization, and transgenic techniques to elucidate the function and regulatory role of F3H in Ginkgo biloba. The cloned GbF3H encodes a cytoplasm-localized hydrophobic acidic protein of 369 amino acids, containing the conserved DIOX_N and 2OG-FeII_Oxy domains characteristic of 2-oxoglutarate-dependent dioxygenases. Expression profiling revealed that GbF3H transcripts were most abundant in immature fruits, ovulate strobili, and juvenile leaves, and were significantly modulated by different nitrogen forms and abscisic acid (ABA). Heterologous overexpression of GbF3H in tobacco (Nicotiana tabacum cv. K326) substantially reshaped the flavonoid metabolome, characterized by the pronounced upregulation in isoflavonoids and concomitant downregulation in flavonols. Concurrent transcriptomic analysis demonstrated that GbF3H overexpression suppressed key flavonoid biosynthetic genes (Nt4CL, NtFLS1, and NtUFGT), while differentially regulating NtFLS2, indicating a hierarchical regulatory mechanism governing the flavonoid metabolic network. Collectively, these results establish GbF3H as a central switch of flavonoid metabolic flux partitioning in G. biloba, providing crucial mechanistic insights for understanding the regulation of flavonoid biosynthesis and providing a basis for future metabolic engineering strategies.

Ginkgo biloba L., a relict tree species with strong ecological resilience, shows high adaptability to precipitation variation, yet the mechanisms linking its rhizosphere metabolites and microbial communities to natural precipitation gradients remain unclear. This study investigated five sites along an eastern China precipitation gradient (651.7-1719 mm; Jinan, Pizhou, Taixing, Hangzhou, Ningbo) using integrated metabolomics and microbiomics. Results revealed that precipitation significantly shaped rhizosphere metabolite profiles: organic acids increased (22.7%-25.9%) with higher precipitation, while lipids increased (38.9%-39.2%) under reduced and increased rainfall. The main drivers of bacterial community assembly shifted from conventional nutrients (total potassium, available phosphorus, available potassium) under baseline conditions to metabolites (organic acids, lipids) under altered precipitation. Two bacterial orders, Bacillales and Rhizobiales, responded significantly to precipitation changes, with Bacillales enriched under extreme conditions as key growth-promoting taxa enhancing stress tolerance in ginkgo. Overall, precipitation indirectly influenced bacterial communities by reshaping metabolite composition. Under extreme moisture conditions, metabolites replaced soil nutrients as dominant drivers, indicating that ginkgo adapts by modulating root exudates to recruit beneficial microbes, providing potential targets for rhizosphere microbial engineering.

Ginkgo (Ginkgo biloba L.), a living relict of gymnosperms, possesses remarkable ecological, medicinal, and economic importance. Sustaining its conservation and advancing genetic improvement require a comprehensive understanding of genomic variation. However, progress in breeding has been constrained by the scarcity of informative molecular markers and limited knowledge of its genetic diversity. In this study, a genome-wide survey of simple sequence repeats (SSRs) was conducted with evaluation of the genetic diversity of 205 accessions collected from diverse geographic regions of China. A total of 2,374,726 SSR loci were identified across the 12 chromosomes, predominantly di- and tri-nucleotide repeats. Functional enrichment analysis showed that SSR-containing coding regions were associated with biological processes such as gibberellin signaling, RNA metabolism, and plant morphogenesis, suggesting potential regulatory roles in key developmental and metabolic pathways. To validate the in silico predictions, 20 polymorphic SSR markers were experimentally confirmed, providing deeper insights into the genetic variation of ginkgo germplasm. Across all SSR markers, the mean observed heterozygosity and polymorphism information content were 0.525 and 0.534, respectively. Population structure, phylogenetic, and principal component analyses revealed two well-defined genetic clusters largely corresponding to geographic origin. Furthermore, a core set of 32 accessions (15.61

Soil organic matter (SOM) molecular composition governs its stability and ecological functions in forest ecosystems. Nevertheless, how land-use changes (LUCs) regulate the SOM molecular composition remains poorly understood, particularly the underlying mechanisms mediated by soil properties. This study investigated the effects of LUCs on SOM molecular composition in a subtropical coastal region and examined the driving roles of soil nutrient availability and enzyme activities. The research was conducted in Huanghai National Forest Park, Jiangsu Province, China, focusing on four land-use types converted from historical wheat cropland (W, as control): monoculture plantations of Ginkgo biloba (G) and Metasequoia glyptostroboides (M), a ginkgo-metasequoia mixed forest (GM), and a ginkgo-wheat agroforestry system (GW). Soil samples were collected from 0 to 20 cm and 20-40 cm layers and analyzed for SOM molecular compositions using solid-state C-13 nuclear magnetic resonance (NMR) spectroscopy. Soil chemical properties and enzyme activity activities were also determined, with redundancy analysis (RDA) and correlation analysis applied to identify key influencing factors. Results demonstrated that LUCs significantly altered SOM molecular composition. The GW system exhibited the highest proportion of labile O-alkyl carbon (42.65%), while the M plantation accumulated greatest levels of stable aromatic carbon (up to 49.25%). During the initial decades following afforestation, soil nutrient availability and enzyme activities were confirmed as pivotal drivers of SOM molecular variation. Specifically, available potassium (AK), ammonium nitrogen (AN), and the carbon/phosphorus (C/P) ratio were significantly correlated with specific SOM components (p < 0.05). The elevated O-alkyl carbon proportion in GW was closely associated with its higher invertase activity. Notably, vertical differentiation in SOM stability was observed across land-use types, with the agroforestry system achieving the highest carbon pool management index in surface soil but showing a weakened capacity for subsoil C stabilization. RDA further confirmed that AK and AN were dominant factors shaping SOM molecular composition. In conclusion, LUCs modulate SOM chemical composition and stability primarily through altering soil nutrient availability and associated enzyme activities. Agroforestry system facilitates labile C accumulation in surface soil, whereas monoculture plantations are more conducive to stable C sequestration, especially in subsoil layers. These findings provide novel mechanistic insights into SOM dynamics following LUCs and offer a theoretical basis for formulating tailored management strategies to enhance C sequestration efficiency in subtropical coastal ecosystems.

Flavonoids are widely distributed in plants, including Ginkgo biloba, a traditional medicinal and edible species. Methylated flavonoids often exhibit enhanced biological activities compared with their unmethylated forms, and flavonoid O-methyltransferases (FOMTs) responsible for this modification have been identified in several plants but remain poorly characterized in ginkgo. In this study, transcriptomic analysis of ginkgo leaves at different developmental stages identified two S-adenosylmethionine-dependent FOMTs, GbFOMT3 and GbFOMT12. Enzymatic assays with seven flavonoid substrates showed that both enzymes methylate quercetin, eriodictyol, and luteolin to produce isorhamnetin, hesperetin, and chrysoeriol, respectively, through methylation at the 3'-OH and 4'-OH positions of the B ring. These findings expand the repertoire of ginkgo flavonoid O-methyltransferases and provide insights into flavonoid methylation in ginkgo.

Ginkgo (Ginkgo biloba L.), a unique gymnosperm with significant medicinal, edible, and ornamental value, has been underutilised in breeding programs due to limited understanding of its genetic diversity. This study evaluated 202 ginkgo germplasms using two physiological traits (flavonoid and soluble sugar content) and 10 start codon targeted (SCoT) markers to assess genetic diversity and construct a core germplasm collection. Flavonoid content showed marked variation and a significant negative correlation with soluble sugar content (P < 0.01). The SCoT markers revealed moderate genetic diversity, with an average Shannon’s diversity index (I = 0.452), expected heterozygosity (He = 0.298), and polymorphism information content (PIC = 0.357). Population structure, neighbour-joining phylogeny, and principal coordinates analyses clustered the germplasms into two major groups, reflecting geographical origins. A core collection of 64 accessions (31.68

Nitrogen nutrition monitoring is crucial in agriculture and forestry. With the development of Unmanned Aerial Vehicle (UAV) imaging technology, its application in nitrogen nutrition monitoring has gained attention. Traditional regression methods often struggle to accurately capture the nonlinear relationships between image features and nitrogen nutrition parameters. This study introduces Gaussian regression models to better model the relationship between UAV image features and nitrogen nutrition in Ginkgo. UAV RGB imagery of three-year-old Ginkgo biloba L. seedlings was used to extract nitrogen-related image features. Gaussian regression models were employed to select and model these features, creating regression models for nitrogen accumulation and nitrogen content in Ginkgo. The accuracy of the models was validated. Results indicated that the optimal canopy type for monitoring nitrogen accumulation in Ginkgo was the shadowed canopy, with the color feature BMR being the most important feature. For monitoring nitrogen content, sunlight and shadow canopy types were suitable, with BMR and b* being the key features. Gaussian regression demonstrated superior accuracy and robustness compared to traditional regression models. This study emphasizes the potential of Gaussian regression models to improve nitrogen monitoring through UAV imagery, offering valuable applications in precision agriculture and forestry management, particularly in supporting nitrogen fertilization and nutrition management for Ginkgo.

In vitro regeneration presents significant challenges for the propagation and genetic improvement of most woody plants, particularly gymnosperms. The WUSCHEL-related homeobox (WOX) genes are known to play vital roles as growth regulators in tissue culture regeneration in several plant species. However, the specific functions of WOX genes in the regeneration processes of gymnosperms had not been previously elucidated. This study aims to systematically identify and analyze the WOX gene family in Ginkgo biloba to understand its potential role in tissue culture regeneration. Thirteen WOX genes from Ginkgo biloba, designated as GbWUS and GbWOXs, were systematically identified. Phylogenetic analysis revealed the presence of nine genes in the WUSCHEL (WUS) clade, one in the intermediate clade, and three in the ancient clade. Transcriptome analysis indicated tissue-specific expression of seven GbWOXs, with two gymnosperm-specific GbWOXs characterized by extra-long introns exhibiting constitutive expression. Further investigation through Ginkgo tissue culture indicated that GbWOX2 was specifically expressed in embryos and facilitated callus induction, while GbWOX3A showed preferential expression during the early stages of embryo and callus development. Co-expression and Gene Ontology (GO) enrichment analyses suggested interactions and functional roles among GbWOXs. Three genes (GbWOX1, GbWOX2, and GbWOX3A) were then cloned and transformed into poplar and/or tobacco. Overexpression of GbWOX2 resulted in larger and denser callus formation, whereas GbWOX3A effectively enhanced shoot regeneration and noticeably increased the rate of adventitious shoot induction. This study provides the first comprehensive analysis of the WOX gene family in Ginkgo biloba and highlights its significant role in tissue culture regeneration. The findings suggest that specific GbWOXs are critical for embryo development and callus regeneration, which provides the foundation for the establishment of effective tissue culture systems in Ginkgo. Moreover, this research contributes valuable insights that could be beneficial for improving propagation techniques and genetic studies in other forest trees, especially within the gymnosperm group.

Sophora moorcroftiana (Benth.) Baker is a drought- and sand-resistant endemic shrub species in the family Fabaceae, native to the Tibetan Plateau along the Yarlung Tsangpo River (elevation: 2800–4400 m). This study offers a comprehensive review of the latest research on S. moorcroftiana, with a focus on its ecological functions, medicinal potential, pest and disease management, and germplasm conservation. By synthesizing existing studies, the review sheds light on the mechanisms that enable this species to thrive in extreme environments, highlights its unique secondary metabolites, and explores its critical role in biodiversity maintenance. Additionally, the article examines the current conservation status of S. moorcroftiana, identifies the key threats to its survival, and suggests future research directions and strategies for sustainable utilization. The goal of this review is to fill existing knowledge gaps by providing a theoretical foundation and practical guidance for future scientific research, applied uses, and conservation initiatives related to S. moorcroftiana.

WRKY transcription factors, as evolutionarily conserved regulators in higher plants, orchestrate transcriptional reprogramming that maintains metabolic homeostasis and facilitates adaptive responses under salinity stress. However, a comprehensive identification, characterisation, and functional analysis of XsWRKY transcription factors (TFs) in response to salt stress remains lacking in yellowhorn (Xanthoceras sorbifolium), a perennial woody oilseed species renowned for its remarkable salt tolerance. We employed a genome-wide mining approach to investigate the expression profiles and biological functions of XsWRKY TFs in response to salt stress. This analysis identified 43 XsWRKY TFs, all possessing a highly conserved domain. Phylogenetic analysis classified these TFs into three distinct subgroups, with genes within the same subgroup displaying similar motif compositions. Collinearity analysis revealed that some XsWRKY genes originated from segmental duplications, highlighting the conservation of their domains. Promoter cis-element analysis of XsWRKY genes revealed regulatory elements associated with hormonal signalling, abiotic stress responses, and developmental processes. Transcriptomic profiling under salt stress conditions highlighted distinct expression patterns, with XsWRKY30 emerging as a key regulator in the salt stress response. Experimental validation confirmed that XsWRKY30 exhibited transcriptional activation activity and was localised to the nucleus. Transient expression of XsWRKY30 in tobacco significantly reduced malondialdehyde (MDA) accumulation, while stable overexpression in Arabidopsis seedlings and yellowhorn calli enhanced antioxidant enzyme activities and proline levels, accompanied by a marked decrease in MDA content compared to wild-type controls. In addition, XsWRKY30-overexpressing Arabidopsis seedlings and yellowhorn calli exhibited a reduced Na*/K* ratio, reflecting improved ion homeostasis under salt stress. Several salt stress-responsive genes were also upregulated in the transgenic lines and calli, suggesting that XsWRKY30 played a positive regulatory role in salt tolerance. Overall, these findings demonstrated that XsWRKY30 overexpression alleviated oxidative damage and Na* toxicity under osmotic stress, providing new insights into the evolutionary conservation and functional significance of XsWRKY TFs in yellowhorn salt tolerance.

Woody plants have garnered significant attention in recent years for their essential ecological and economic contributions. Protoplasts, isolated from plant cells, have exhibited remarkable totipotency and offered immense potential in a broad array of biological and biotechnological fields. These include, but are not limited to, protein gene expression regulation, functional gene analysis, subcellular localization, interaction studies, gene editing and single-cell sequencing. This review offers a comprehensive overview of protoplast isolation methods, key influencing factors, purification techniques and viability assessment. It further explores the use of protoplast transient expression systems for gene function characterization, while highlighting the diverse applications of protoplast-based technologies, such as fusion, regeneration, genome editing and single-cell sequencing. With technological advancements, future breakthroughs in these areas will be poised to create new avenues for research, genetic improvement and biotechnological innovations in woody plants.

Globally, the increasing frequency of human activities such as fossil fuel combustion, pesticide and fertilizer use has led to a significant increase in atmospheric reactive nitrogen (N) emissions and continuously increasing N deposition to forests. This can significantly disrupt the elemental balance among components of the ecosystem and primary productivity. We reviewed the stoichiometry of the “plant-litter-soil” system in forests under elevated N deposition, specifically the stoichiometric characteristics of leaf, litter, and soil carbon (C), N, and phosphorus (P). This review also examines the effects of microbe-mediated and forest adaptation mechanisms on the stoichiometry of the “plant-litter-soil” system under N deposition. Findings demonstrated that (i) N deposition generally increased the plant leaf N and N: P ratio, especially under N-limited conditions, while the C: P ratio remained unchanged, suggesting a potential trend of increasing P limitation in forest ecosystems, (ii) the C: N ratio of litter tended to decrease, and N addition may accelerate its decomposition rate, (iii) N deposition increased the soil organic carbon (SOC) and N: P ratio, decreased the C: N ratio, negatively impacted microbial biomass C, N, P, and its effects on extracellular enzyme activities (EEAs) showed considerable variability, (iv) the response of forest stoichiometric characteristics was primarily influenced by the intensity and duration of N addition, while the forest’s own N-P resource availability also largely determined the outcome of stoichiometric response, (v) plants adapt to increased N availability by adjusting nutrient allocation, biomass distribution across organs, and nutrient resorption, (vi) changes in the composition and function of microbial communities were key mechanisms by which N deposition affects soil-microbe stoichiometry. This research helps to understand the dynamics of elemental cycling and ecological effects in forests under climate change, which is crucial for the protection of forest ecosystem stability and sustainable management.