Conifers exhibit distinct age-related transitions in wood development, yet the molecular mechanisms governing these changes remain poorly characterized. Here, we integrate multi-omics and functional analyses to unravel a transcriptional regulatory network underlying age-related wood formation in Chinese pine (Pinus tabuliformis). Cross-sectional and Raman microspectroscopy analyses revealed age-dependent enhancement of lignification and carbohydrate deposition in secondary xylem, coinciding with increased pith proportion. Temporal transcriptome profiling identified PtARN (AGE-RELATED NAC TRANSCRIPTION FACTOR) as a key age-correlated regulator, whose expression is directly activated by the conserved age timer PtDAL1 through promoter binding. We conducted a genome-wide identification of secondary cell wall biosynthesis pathway genes in P. tabuliformis, and genome-wide DNA affinity purification analysis uncovered that PtARN direct binding to promoters of 189 secondary cell wall biosynthesis genes. Strikingly, PtARN represses PtMYB162, a lignin biosynthesis suppressor, forming a regulatory cascade to fine-tune lignification. Collectively, our work establishes PtARN as a master regulator orchestrating age-dependent secondary cell wall deposition and lignification in the secondary xylem of Chinese pine, and delineates a previously uncharacterized PtDAL1-PtARN-mediated molecular regulatory network in P. tabuliformis. These findings significantly advance our understanding of the molecular regulatory network underlying wood formation in conifers, and bridging developmental timing with cell wall remodelling, and provide a robust framework for conifer-specific wood formation studies.

The age-regulation mechanism plays a crucial role throughout the entire life cycle of plants, including vegetative growth, growth phase transition, reproductive development, senescence, and regeneration. While this mechanism has been elucidated in angiosperms, it remains poorly understood in gymnosperms. DEFICIENS AGAMOUS-LIKE 1 (DAL1) is a conserved age biomarker in gymnosperms that integrates the aging pathway and reproductive development. However, the molecular regulatory mechanisms governing DAL1 expression patterns are not well understood. In this study, we revealed that DAL1 can activate its own expression through a positive feedback loop by directly binding to its own promoter region. Furthermore, the jasmonic acid (JA)-responsive transcriptional regulator TIFY25 acts as a 'brake' by directly interacting with DAL1 via protein-protein interactions. This interaction inhibits the self-activation of DAL1, ensuring that its expression increases gradually and steadily with age, thereby preventing premature or excessive activation. These findings provide insights into the age-regulation mechanism and integrate JA into the age pathway in P. tabuliformis.

Conifers, as long-lived gymnosperms, employ age-regulatory mechanisms distinct from the miR156-miR172 module dominant in angiosperms. The MADS-box gene DAL1 serves as a conserved age marker in conifers, yet the hormonal modulation of its activity remains elusive. Here, we reveal that gibberellin (GA) and jasmonic acid (JA) exert antagonistic control over DAL1 protein abundance through mechanistically distinct pathways in Pinus tabuliformis. GA promotes DAL1 accumulation via the conserved GA-GID1-DELLA cascade. Conversely, JA suppresses DAL1 primarily through the transcription factor MYC2, which directly interacts with DAL1 to mediate its degradation. Strikingly, we uncovered a compensatory mechanism wherein JA-induced TIFY25, a JAZ family protein, stabilizes DAL1 by competitively disrupting the DAL1-DPL1 interaction, thereby counterbalancing the negative regulation by MYC2. This dual regulatory architecture enables conifers to maintain age-dependent DAL1 expression while integrating growth-promoting (GA) and defense-related (JA) signals. Our findings establish a novel GA-JA crosstalk mechanism in conifers and provide fundamental insights into how long-lived trees coordinate developmental timing with phytohormone-mediated environmental adaptation.

Conifers serve as the cornerstone of global forest ecosystems, yet their genetic transformation faces notorious challenges. To overcome the intrinsic resistance of conifer adventitious roots to Agrobacterium/Rhizobium-mediated transformation, we systematically and iteratively engineered the binary vector by introducing chimeric Ri plasmid derived T-DNA borders, a phosphomimetic VirG mutant (VirGN54D) and a hyperactive replication origin mutant (pVS1 R106H). This optimised system enabled efficient, cross-species genetic transformation in diverse plants, including the recalcitrant gymnosperm Ginkgo biloba, thus establishing a broadly applicable genetic toolkit for plant research. Leveraging this system, we established a non-aseptic efficient root transformation system for Pinus tabuliformis. Given that conifers are evolutionarily ancient gymnosperms, this readily accessible system provides an unprecedented window to probe fundamental questions in functional genomics and evolutionary mechanisms, such as the dynamics of long-distance macromolecular trafficking. Transgenic deployment of Arabidopsis florigen FT in Chinese pine roots uncovered conserved protein level long-distance mobility, despite lacking functional FT orthologs in conifer genomes. Conversely, the conifer age biomarker DAL1 exhibited no detectable mobility even when engineered fusion with tRNA-like sequence (TLS) tag, which has been shown to facilitate long-distance mRNA transport in angiosperms, exposing lineage-specific constraints on mobile mRNA. Our work provides a versatile genetic tool for challenging plant species and offers new insights into long-distance signalling evolution. The streamlined protocols and universal vector system address critical bottlenecks in conifer biotechnology and open avenues for functional genomics in other non-model plants.

The Response Regulator (RR) gene family functions as a molecular “switch” in plant signal transduction, with TOC1 (PRR1) frequently acting as a transcriptional repressor that integrates both internal and external signals. While RR homologs in gymnosperms differ from those in angiosperms, research on their function remains sparse. This study presents a genome-wide analysis of the RR gene family in Pinus tabuliformis, identifying 35 RR genes that are grouped into three subfamilies: A-type ARRs (17 genes), B-type ARRs (16 genes), and pseudo-RRs (2 genes). Except for PtPRR43, these genes are distributed across various chromosomes, with 16 displaying broad expression in multiple organs. Our findings revealed that PtTOC indirectly promotes flowering by suppressing PtTFL2, a floral repressor, suggesting its role in balancing circadian rhythms and environmental signals. Additionally, PtTOC overexpression reduces cold tolerance, likely through the downregulation of cold-responsive pathways, suggesting a trade-off between promoting flowering and stress resilience. Subcellular localization analysis suggests PtTOC undergoes phosphorylation-regulated phase transitions, highlighting its dynamic role in cellular signaling and adaptation. Collectively, these results position PtTOC as a central integrator of environmental signals and circadian regulation, offering insights into strategies for enhancing reproductive performance and resilience in gymnosperms.

Extrachromosomal circular DNA (eccDNA) has been reported to play important roles in regulating genome replication, immune response and cellular communications in humans and animals. Recently, the presence of eccDNA has also recently been discovered in Arabidopsis, Amaranthus palmeri and Oryza sativa. Nevertheless, whether eccDNA exists and has roles in woody plants remains enigmatic. Here, we conducted a comprehensive analysis of morphological imaging, transcriptome and eccDNA expression profiles during different development stages of vascular cambium in Chinese pine (Pinus tabuliformis (P. tabuliformis)). It was found that eccDNA existed in the different development stages of vascular cambium and derived from each chromosome of P. tabuliformis. Further analysis revealed that eccDNA was not entirely random but rather exhibited a certain preference in exon regions. We also identified a high frequency of AA/AT/TT/TA dinucleotide repeats at the junctions of eccDNA and found that the length distribution of eccDNA was clustered between 158 and 316 bp. Notably, integration analysis revealed that differentially expressed eccDNAs and their annotated genes exhibited more significant dynamic changes in the dormant stage as compared with other stages. Taken together, our results provide new insights into the important mechanisms by which eccDNA influences vascular cambium development, enhancing our understanding of its role in tree plasticity.

Age-dependent reproductive development is pivotal for plant adaptability and for species perpetuation. While substantial progress has been made in elucidating age-related regulatory mechanisms in angiosperms, the molecular basis of how aging pathway interacts with reproductive development in gymnosperms, particularly conifers, remains elusive. Here, we demonstrate that DEFICIENS-AGAMOUS-LIKE 1 (DAL1), an age marker protein in conifers, binds to and activates two LEAFY (LFY) homologs, PtLEAFY (PtLFY) and PtNEEDLY (PtNLY), in Pinus tabuliformis. Furthermore, PtLFY and PtNLY directly regulate class B and C MADS-box genes, which are essential for specifying reproductive organ identity. Notably, PtLFY exerts a stronger regulatory effect on downstream class B and C genes compared to PtNLY, suggesting potential functional divergence between these two paralogous genes. This study reveals a PtDAL1-mediated regulatory cascade that links aging to reproductive development in conifers. These findings provide insights into the evolutionary conservation and divergence of the reproduction-associated regulatory networks across plant lineages, broadening our understanding of the genetic and molecular mechanisms underlying the reproductive development of gymnosperms.

The Pinus tabuliformis (Chinese pine), a keystone conifer species native to northern China with extended distributions into central and southern regions (e.g., Henan), plays a critical role in regional vegetation dynamics. Unraveling the molecular mechanisms underlying its seed dormancy and germination is vital for guiding effective ecological conservation and reforestation efforts. In order to elucidate the germination mechanism of Chinese pine seeds, we performed the transcriptome analysis of dormant seeds (S1), non-dormant seeds (S2), and germinating seeds (S3). We obtained high-quality transcriptome data from seeds at three developmental stages using the Illumina sequencing platform and conducted time-series trend analysis. The results revealed four gene modules significantly associated with the germination of Pinus tabuliformis seeds, alongside 857 DEGs (differentially expressed genes). WGCNA (Weighted Gene Co-expression Network Analysis) further pinpointed a key module comprising 153 genes strongly correlated with germination, of which 24 were prioritized as putative regulators. Expression profiling of 12 representative candidates across developmental stages revealed that at least 7 genes exhibited marked expression shifts during the dormancy-to-germination transition. Notably, PtbZIP25 (Pt4G12300) a homolog of Arabidopsis thaliana bZIP transcription factors, was functionally validated as a negative regulator of germination via overexpression and mutant assays. This gene modulate the expression of dormancy-related markers (DOG1, CYP707A2), indicating its potential role in ABA signaling. Our findings provide novel insights into the molecular basis of conifer seed germination and offer potential targets for optimizing afforestation practices.

As perennial evergreen non-deciduous plants, conifer face severe challenges from cold and drought stresses during autumn and winter. Understanding how conifers balance growth and stress responses is crucial for forest conservation and tree genetic improvement under climate change. While TFL2 has been identified as a core regulatory factor mediating dormancy with distinct seasonal rhythmic expression in conifers, its downstream regulatory mechanisms remain largely unknown. Here, we systematically screened the TFL2 interactome via Y2H-seq at the whole-transcriptome scale, identifying 448 non-redundant interacting proteins, including 76 transcription factors (TFs) and 16 transcriptional regulators (TRs). Among these, PtbZIP43 and PtERF21 were confirmed as direct interactors of PtTFL2, displaying temporal expression patterns highly correlated with PtTFL2, peaking sequentially in autumn. This hierarchy forms a PtTFL2-PtbZIP43-PtERF21 cascade module that binds to downstream gene promoters to regulate seasonal growth transitions. Overexpression of the PtTFL2, PtbZIP43 and PtERF21 significantly delayed flowering time while enhancing resistance to cold and drought stresses of transgenic plants. These findings demonstrate that the PtTFL2-PtbZIP43-PtERF21 module enhancing conifer tolerance to autumn/winter stresses by coordinates growth and stress responses. This study elucidates a hierarchical transcriptional network integrating seasonal cues with stress adaptation in conifers, offering targets for genetic improvement of forest tree resilience.

This study reveals the molecular mechanisms of seed dormancy and germination in Chinese pine, highlighting the key roles of GA and ABA pathways and specific genes in regulating germination, growth, and stress resistance. Chinese pine (Pinus tabuliformis) is an ecologically and economically vital conifer species in China, serving critical functions in forest regeneration, soil conservation, and carbon sequestration. Despite its ecological importance, the molecular mechanisms governing seed dormancy release and germination in this species remain poorly understood. Using an integrative approach that combines advanced microscopy, hormone profiling, metabolomics, and whole transcriptome sequencing, we uncovered novel molecular insights into these crucial developmental processes. Optical and transmission electron microscopy revealed that protein bodies aggregated in aleurone cells during dormancy release. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis revealed distinct hormonal antagonism: gibberellin (GA) levels peaked during dormancy release (S2), with GA3 increased by 8.1-fold (from 41.37 ± 8.68 ng/g to 336.97 ± 106.14 ng/g), whereas abscisic acid (ABA) decreased by 63.6

Conifers have developed intricate dormancy strategies to cope with extreme climates in winter. Among various environmental cues, shortened photoperiods in the fall induce conifers to enter dormancy, leading to growth cessation, bud set, and enhanced freezing tolerance. However, the molecular mechanisms of short-day-induced dormancy are not fully understood. In this study, we treated Pinus tabuliformis seedlings with different photoperiods and confirmed that short days rapidly induced and promoted dormancy. Transcriptomic analysis of photoperiod and annual cycle conditions revealed that the expression of TFL1-like (PtTFL2) was highly associated with various biological processes associated with dormancy. Heterologous overexpression studies showed that PtTFL2 promoted dormancy in poplar and delayed vegetative growth and enhanced freezing tolerance in both poplar and Arabidopsis thaliana. The transcription factor co-expression network centered on PtTFL2 identified for the co-transcription factors PtNF-YC18 and PtNAC67. PtTFL2 physically interacted with PtNF-YC18 to synergistically regulate both vegetative growth and growth cessation during dormancy. Additionally, PtTFL2 interacted with PtNAC67 to directly activate PtDHN2 expression, enhancing freezing tolerance during dormancy. Our findings highlight the role of PtTFL2 in conifer dormancy rapidly induced by short days and outline a regulatory network centered on PtTFL2 wherein 2 modules (PtTFL2-PtNF-YC18 and PtTFL2-PtNAC67) trigger growth-defense tradeoffs in the onset of winter dormancy.

Chinese sweetgum (Liquidambar formosana) is valued as a source of resin and timber and is an important ornamental tree due to its showy fall foliage. Here, we report the chromosome-level assembly of the Chinese sweetgum genome. Phylogenomic analyses showed the basal phylogenetic position of Chinese sweetgum in core eudicots. Comparative genomic analyses revealed that the well-known gamma event in the common ancestors of core eudicots is evident in the Chinese sweetgum genome, and ancestral triplicated blocks resulting from that event are more intact in Chinese sweetgum than in grapevine (Vitis vinifera). Because of its conserved genome structure, very slow rate of evolution, and basal phylogenetic position, the Chinese sweetgum genome is a good reference for comparative genome studies. Further, we reconstructed the entire metabolic pathway for anthocyanins and potential regulatory networks of autumn leaf coloration of this species via metabolomics and transcriptomics. The transcription factors LfMYB69, basic helix-loop-helix (LfbHLH4), and WD40-repeat (LfWDR1) may collectively regulate the transcription of anthocyanin biosynthetic genes. The regulation of chalcone synthase genes (LfCHS1-3) and dihydroflavonol 4-reductase genes (LfDFR1-2) by the LfMYB69-LfbHLH4-LfWDR1 complex was confirmed by luciferase assays. Epigenomic analyses revealed that 5 structural genes, including LfCHS1, and 2 regulatory LfMYBs are epigenetically regulated. This study expands our understanding of autumn leaf coloration and provides valuable genomic resources for comparative biology, breeding, and biotechnology.

The age biomarker gene PtDAL1 in conifers undergoes expression resetting during pollen maturation, but its regulatory mechanisms remain unclear. This study identifies PtABI3, a B3 transcription factor (TF) in Pinus tabuliformis, as a key repressor of PtDAL1. PtABI3 exhibits strict spatiotemporal complementarity with PtDAL1. Phylogenetic and structural studies revealed PtABI3 as a gymnosperm homolog of angiosperm ABI3, retaining conserved B3 domains critical for DNA binding and transcriptional repression. Ectopic expression of PtABI3 in Arabidopsis recapitulated the classic ABI3 late-flowering phenotype. Mechanistically, PtABI3 directly binds the RY motif (CATGCA) within the PtDAL1 promoter via its B3 domain, as demonstrated by dual-luciferase assays, yeast one-hybrid assays, and electrophoretic mobility shift assays (EMSA). These results revealed that PtABI3 is an evolutionary conserved transcriptional repressor that silences PtDAL1 during pollen maturation, providing the first evidence of B3 TFs mediating age timer gene resetting in conifers. This work bridges gymnosperm and angiosperm regulatory paradigms, highlighting ancient mechanisms for gene expression resetting in plants.

Age-dependent reproductive development is pivotal for plant adaptability and for species perpetuation. While substantial progress has been made in elucidating age-related regulatory mechanisms in angiosperms, the molecular basis of how aging pathways interact with reproductive development in gymnosperms, particularly conifers, remains elusive. Here, we demonstrate that DEFICIENS-AGAMOUS-LIKE 1 (DAL1), an age marker protein in conifers, binds to and activates 2 LEAFY (LFY) homologs, PtLEAFY (PtLFY) and PtNEEDLY (PtNLY), in Pinus tabuliformis. Furthermore, PtLFY and PtNLY directly regulate class B and C MADS-box genes, which are essential for specifying reproductive organ identity. Notably, PtLFY exerts a stronger regulatory effect on downstream class B and C genes compared with PtNLY, suggesting potential functional divergence between these 2 paralogous genes. This study reveals a PtDAL1-mediated regulatory cascade that links aging to reproductive development in conifers. These findings provide insights into the evolutionary conservation and divergence of the reproduction-associated regulatory networks across plant lineages, broadening our understanding of the genetic and molecular mechanisms underlying the reproductive development of gymnosperms.

Plasma membranes (PMs) are highly dynamic structures where lipids and proteins can theoretically diffuse freely. However, reports indicate that PM proteins do not freely diffuse within their planes but are constrained by cytoskeleton networks, though the mechanisms for how the cytoskeleton restricts lateral diffusion of plant PM proteins are unclear. Through single-molecule tracking, we investigated the dynamics of 6 Arabidopsis (Arabidopsis thaliana) PM proteins with diverse structures and found distinctions in sizes and dynamics among these proteins. Moreover, we showed that the cytoskeleton, particularly microtubules, limits the diffusion of PM proteins, including transmembrane and membrane-anchoring proteins. Interestingly, the microfilament skeleton regulates intracellular transport of endocytic cargo. Therefore, these findings indicate that the cytoskeleton controls signal transduction by limiting diffusion of PM proteins in specific membrane compartments and participating in transport of internalized cargo vesicles, thus actively regulating plant signal transduction.

The geographical variation and domestication of tree species are an important part of the theory of forest introduction, and the tracing of the germplasm is the theoretical basis for the establishment of high-quality plantations. Chinese pine (Pinus tabuliformis Carr.) is an important native timber tree species widely distributed in northern China, but it is unclear exactly where germplasm of the main Chinese pine plantation populations originated. Here, using two mtDNA markers, we analyzed 796 individuals representing 35 populations (matR marker), and 873 individuals representing 38 populations (nad5-1 marker) of the major natural and artificial populations in northern China, respectively (Shanxi, Hebei and Liaoning provinces). The results confirmed that the core position of natural SX* populations ("*" means natural population) in the Chinese pine populations of northern China, the genetic diversity of HB and LN plantations was higher than that of natural SX* populations, and there was a large difference in genetic background within the groups of SX* and LN, HB showed the opposite. More importantly, we completed the "point by point" tracing of the HB and LN plantings. The results indicated that almost all HB populations originated from SX* (GDS*, ZTS*, GCS*, and THS*), which resulted in homogeneity of the genetic background of HB populations. Most of germplasm of the LN plantations originated from LN* (ZJS* and WF*), and the other part originated from GDS* (SX*), resulting in the large differences in the genetic background within the LN group. Our results provided a reliable theoretical basis for the scientific allocation, management, and utilization of Chinese pine populations in northern China, and for promoting the high-quality establishment of Chinese pine plantations.

Reversal of plant developmental status from the mature to the juvenile phase, thus leading to the restoration of the developmental potential, is referred to as plant rejuvenation. It involves multilayer regulation, including resetting gene expression patterns, chromatin remodeling, and histone modifications, eventually resulting in the restoration of juvenile characteristics. Although plants can be successfully rejuvenated using some forestry practices to restore juvenile morphology, physiology, and reproductive capabilities, studies on the epigenetic mechanisms underlying this process are in the nascent stage. This review provides an overview of the plant rejuvenation process and discusses the key epigenetic mechanisms involved in DNA methylation, histone modification, and chromatin remodeling in the process of rejuvenation, as well as the roles of small RNAs in this process. Additionally, we present new inquiries regarding the epigenetic regulation of plant rejuvenation, aiming to advance our understanding of rejuvenation in sexually and asexually propagated plants. Overall, we highlight the importance of epigenetic mechanisms in the regulation of plant rejuvenation, providing valuable insights into the complexity of this process.

Genetic transformation has been a cornerstone in plant molecular biology research and molecular design breeding, facilitating innovative approaches for the genetic improvement of trees with long breeding cycles. Despite the profound ecological and economic significance of conifers in global forestry, the application of genetic transformation in this group has been fraught with challenges. Nevertheless, genetic transformation has achieved notable advances in certain conifer species, while these advances are confined to specific genotypes, they offer valuable insights for technological breakthroughs in other species. This review offers an in-depth examination of the progress achieved in the genetic transformation of conifers. This discussion encompasses various factors, including expression vector construction, gene-delivery methods, and regeneration systems. Additionally, the hurdles encountered in the pursuit of a universal model for conifer transformation are discussed, along with the proposal of potential strategies for future developments. This comprehensive overview seeks to stimulate further research and innovation in this crucial field of forest biotechnology.