Yaguang Zhan

The basic helix–loop–helix (bHLH) transcription factor family regulates plant secondary metabolism, development and stress responses. Although triterpenoids such as betulinic acid (BA), betulin (BT) and oleanolic acid (OA) from Betula platyphylla Suk. are of pharmacological importance, the methyl jasmonate (MeJA)‐induced regulation of IVa bHLHs remains unclear. A total of 131 BpbHLH genes were identified, and functional characterisation was performed for two IVa members, BpbHLH42 and BpbHLH44 , in yeast, tobacco, birch cells and transgenic plants. Both enhanced triterpenoid biosynthesis but exhibited distinct specificities: BpbHLH42 primarily promoted OA accumulation by activating BpHMGR , BpSE1/3 , BpW and BpY6 , while BpbHLH44 favoured BT production through BpW , BpY6 and BpY11 . Both indirectly regulated BpY9 via BpMYB21 , forming a bHLH–MYB complex, and the MeJA repressor BpJAZ2 disrupted the BpbHLH42–MYB21 interaction. Transgenic birch lines further revealed divergent phenotypes: BpbHLH42 overexpression enlarged and smoothed leaves and increased OA levels, whereas BpbHLH44 enhanced alkali stress tolerance by elevating secondary metabolite accumulation. These results uncover the differential regulatory roles of BpbHLH42 and BpbHLH44 in triterpenoid biosynthesis and demonstrate their potential for metabolic engineering and genetic improvement in woody plants.

Fraxinus mandshurica exhibits high tolerance to ash dieback and superior wood mechanical properties, yet the genomic basis of these dual traits remains incompletely understood. Here, we present a chromosome-scale genome assembly of the elite clone 'M8' and integrate comparative genomics with temporal transcriptomics to investigate its adaptive architecture. We show that recent tandem and proximal duplications have contributed to the diversification and candidate positive selection of the NLR immune repertoire. Transcriptomic profiling reveals a stage-associated deployment pattern, in which expressed NLR genes are enriched during the Transition Stage, consistent with reinforced immune surveillance during rapid xylem expansion. As development progresses, the Latewood Stage shifts toward phenylpropanoid metabolism, lignification-related programs, and structural reinforcement. We identify and experimentally validate a latewood-recruited FmNAC104–FmPRX1 candidate regulatory module, in which FmNAC104 binds to and activates the FmPRX1 promoter and promotes endogenous FmPRX1 expression. Formal NAC/PRX gene family analysis further supports an enlarged NAC/PRX repertoire relative to P. trichocarpa and clarifies the orthogroup status of the FmNAC104–FmPRX1 module among related ash species. Collectively, our findings support a 'fortified defense' working model in which F. mandshurica temporally coordinates immune-receptor diversification with latewood-associated structural reinforcement. This study provides a genomic resource and candidate targets for breeding programs aimed at improving wood quality and disease resilience in Fraxinus.

Drought is a major abiotic stressor that severely threatens forest tree growth and survival worldwide. The SQUAMOSA promoter binding protein-like (SPL) family is a group of plant-specific transcription factors that play important roles in plant growth, development, and environmental adaptation. Our study showed that overexpression of Fraxinus mandshurica SPL2 (FmSPL2) promotes lignin biosynthesis and enhances drought tolerance. Phenotypic analysis indicated that the stomatal aperture of FmSPL2-OE1 and FmSPL2-OE2 was reduced by 12.05% and 19.23%, respectively, compared to the wild type (WT). Additionally, the xylem width of the sixth internode in these lines increased by 26.07% and 25.93% relative to WT. Correspondingly, lignin content was elevated by 19.87% and 18.48% compared with WT, respectively. Under drought stress, the lignin content in FmSPL2-OE plants remained elevated compared to WT. Furthermore, the overexpression lines exhibited increased SOD and POD activities, accompanied by reduced levels of MDA, H₂O₂, and superoxide anion. Transcriptome analysis indicated significant upregulation of multiple genes involved in phenylpropanoid biosynthesis in FmSPL2-OE plants, including phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and 4-coumarate-CoA ligase (4CL). Moreover, the yeast one-hybrid and dual-luciferase reporter assays demonstrated that FmSPL2 directly binds to the promoter regions of phenylpropanoid-related genes like PAL, CAD, and PRX, thereby activating the expression of these genes and enhancing lignin biosynthesis. These results elucidate a novel function of FmSPL2 in enhancing drought tolerance through the regulation of phenylpropanoid pathway-mediated lignin accumulation.

Fraxinus mandshurica Rupr. is a key forest species in Northeast Asia, primarily distributed in semi-humid regions. Given its wide range of provenances, F. mandshurica exhibits substantial genetic and phenotypic variations. To identify drought–tolerant lines, we evaluated physiological, biochemical, and growth traits of two-year-old seedlings grown in a semi-arid environment across spring, summar and autumn. Principal component analysis (PCA) of 24 trait variables yielded 10 principal components per season, accounting for approximately 85% of the cumulative variation. We developed a comprehensive drought evaluation metric (D value) for each line by calculating weighted averages of PCA scores. Cluster analysis base on seasonal D values categorized 69 F. mandshurica lines into three groups: drought-tolerant, weak drought-sensitive, and drought-sensitive. Using two growth traits, these lines were further classified into high-, medium-, and low-growth groups. From this, we identified five elite lines combining drought tolerance and rapid growth. Stepwise regression revealed that F. mandshurica employs distinct drought-tolerance strategies across seasons. We developled robust drought-tolerance evaluation models for F. mandshurica and selected five high-performing lines with dual traits of drought tolerance and fast growth. Our results provided a basis for afforestation efforts of F. mandshurica in semi-arid regions.

CRISPR/Cas9-mediated genome editing has revolutionized tree improvement by enabling precise trait modification, accelerating breeding cycles, and enhancing forestry sustainability. Fraxinus mandshurica, valued for its desirable traits and adaptability, serves as a strategic focus for the National Reserve Forest Project (NRFP) and forestry germplasm resource breeding and quality improvement in China. Developing a species-specific genome editing system is crucial for valuable yet recalcitrant species like F. mandshurica. In this study, the development of a species-specific CRISPR/Cas9 platform is presented for F. mandshurica, which incorporates endogenous promoter engineering, sgRNA optimization, light quality modulation, and temperature control protocols to enhance genome editing efficiency. Truncated endogenous FmU6 promoter variants (FmU6-6-4 and FmU6-7-4) drove sgRNA expression at levels 3.36 and 3.11 times higher than that of the AtU6-26 promoter. The expression of Cas9 was controlled by the endogenous constitutive FmECP3 promoter, exhibiting an activity 5.48 times greater than the positive control. A highly active sgRNA4 targeting FmPDS1/2 was identified, demonstrating a cleavage efficiency of 36.10%. Heat treatment at 37 °C effectively increased the Cas9 cleavage efficiency to 7.77 times that observed at 22 °C. Chimeric albino mutants with an editing efficiency of 18.2% were obtained through transient and stable transformations, combined with light quality optimization and heat treatment during different regeneration stages. The mutation types included nucleotide insertions, deletions, and substitutions, leading to early termination codons and truncated FmPDS1/2 protein. Additionally, mutations in FmPDS1/2 resulted in albino phenotypes and a reduction in chlorophyll content to 46.44%−58.88%. This optimized system provides a robust platform for functional genomics studies and trait improvement in F. mandshurica, with potential applications in forestry biotechnology.

Fraxinus mandshurica Rupr. (F. mandshurica) is a dioecious tree species with important ecological and application values. To delve deeper into the regulatory pathways and genes responsible for male and female flowers in F. mandshurica, we conducted transcriptome sequencing on male and female flowers at four distinct stages. The analysis revealed that the female database generated 38,319,967 reads while the male database generated 43,320,907 reads, resulting in 2930 differentially expressed genes with 1441 were up-regulated and 1489 down-regulated in males compared to females. Following an analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), four distinct pathways (hormone signal transduction, energy metabolism, flavonoid biosynthesis, and photoperiod) linked to female and male flowers were identified. Subsequently, qRT-PCR verification revealed that FmAUX/IAA, FmEIN3, and FmA-ARR genes in hormone signal transduction pathway are related to female flower development. Meanwhile, FmABF genes in hormone signal transduction pathway, FmGS and FmGDH genes in energy metabolism pathway, FmFLS genes in flavonoid biosynthesis pathway, and FmCaM, FmCRY, and FmPKA genes in photoperiod pathway are related to male flower development. This study was the first to analyze the transcriptome of male and female flowers of F. mandshurica, providing a reference for the developmental pathways and gene expression levels of male and female plants.

To elucidate the function of the cold-resistance regulatory gene FmNAC1 from Fraxinus mandshurica Rupr., this study identified the role that overexpression of the FmNAC1 gene plays in tobacco growth and cold-stress regulation. The cloned FmNAC1 gene from F. mandshurica is 891 bp in length and encodes 296 amino acids. Our subcellular localization analysis confirmed that FmNAC1 is primarily located in the nucleus and functions as a transcription factor. FmNAC1 is responsive to cold and NaCl stress, as well as to the induction of IAA, GA, and ABA hormone signals. To further elucidate its function in cold resistance, four transgenic tobacco lines expressing FmNAC1 (FmNAC1-OE) were generated through tissue culture after the Agrobacterium-mediated transformation of wild-type (WT) Nicotiana tabacum L. These FmNAC1-OE plants exhibited accelerated growth after transplantation. When exposed to low-temperature conditions at −5 °C for 24 h, the rates of wilting and yellowing of the FmNAC1-OE plants were significantly lower than those of the WT tobacco plants. Additionally, the membrane integrity, osmotic regulation, and reactive oxygen species (ROS)-scavenging abilities of the FmNAC1-OE tobacco lines were better than those of the WT plants, indicating the potential of the FmNAC1 gene to improve plant cold resistance. The gene expression results further revealed that the FmNAC1 transcription factor exhibits regulatory interactions with growth-related genes such as IAA and AUX1; cold-resistance-related genes such as ICE, DREB, and CBF1; and genes involved in the clearance of reactive oxygen species (ROS), such as CAT and SOD. All of this evidence shows that the FmNAC1 transcription factor from F. mandshurica plays a key role in contributing to the enhancement of growth, cold resistance, and ROS clearance in transgenic tobacco plants.

To elucidate the function of the cold resistance regulatory gene FmNAC1 from Fraxinus mandshurica, this study identified the role that the overexpression of the FmNAC1 gene plays in tobacco growth and cold stress regulation. The cloned FmNAC1 gene from F. mandshurica is 891 bp in length and encodes 296 amino acids. Our subcellular localization analysis confirmed that FmNAC1 is primarily located in the nucleus and functions as a transcription factor. FmNAC1 is responsive to cold and NaCl stress, as well as to the induction of IAA, GA, and ABA hormone signals. To further elucidate its function in cold resistance, four transgenic tobacco lines of FmNAC1 overexpression（FmNAC1-OE）were generated through tissue culture after the agrobacterium-mediated transformation of wild-type (WT) tobacco. These FmNAC1-OE plants exhibited accelerated growth after transplantation. When exposed to low-temperature conditions at -5°C for 24 hours, the rates of wilting and yellowing of the FmNAC1-OE plants were significantly lower than those of the WT tobacco plants. Additionally, the membrane integrity, osmotic regulation, and reactive oxygen species (ROS)-scavenging abilities of the FmNAC1-OE tobacco lines were better than those of the WT plants, indicating the potential of the FmNAC1 gene to improve plant cold resistance. The gene expression results further revealed that the FmNAC1 transcription factor exhibits regulatory interactions with growth-related genes such as IAA and AUX1; cold resistance-related genes such as ICE, DREB, and CBF1; and genes involved in the clearance of reactive oxygen species (ROS), such as CAT and SOD. All of this evidence shows that the FmNAC1 transcription factor from F. mandshurica plays a key role in contributing to the enhancement of growth, cold resistance, and ROS clearance in transgenic tobacco plants.