How do plants, lacking a central nervous system, translate environmental stimuli into physiological actions within milliseconds? Vesicular trafficking acts as a cellular core signal and material transport hub that facilitates this rapid adaptation, yet its dynamic nature has long remained a "black box". Traditional imaging approaches have struggled not only with optical resolution (the "unseen"), but critically with a lack of quantitative precision (the "immeasurable") and the inability to track molecular history (the "unknown age"). This review synthesizes a new paradigm that unlocks this black box by integrating advanced chemical biology with deep learning computational analysis. We detail how multimodal strategies combining pH-sensitive probes (e.g., pHluorin), covalent tags (HaloTag), and fluorescent timers visualize molecular events with unprecedented fidelity. Furthermore, we explore how integrating next generation FRAP/FCS variants (DeepFRAP, FCSNet) with deep learning allows for the rigorous mathematical modeling of vesicle kinetics. By resolving long-standing controversies such as endocytic stoichiometry and secretory sorting logic, this quantitative framework maps nanoscale membrane dynamics to organismal phenotypes, ultimately refining our understanding of plant stress resilience and signal transduction.

The mediator complex is a central hub in transcriptional regulation in eukaryotes, yet its biological function in woody plants remains largely unexplored. Here, we systematically identified and characterized the Mediator (MED) gene family in the forest model tree Populus alba × P. glandulosa and focused on PagMED20, a conserved subunit of the head module. Genome-wide analysis revealed significant diversity in the evolutionary history, protein structures, and cis-regulatory elements of the PagMED family. Among these subunits, PagMED20 was selected for further study based on its preferential expression in leaves and nuclear localization, suggesting a potential role in leaf-associated processes. Overexpression of PagMED20 in poplar resulted in enhanced vegetative growth, including increases in plant height, stem diameter, and biomass accumulation. Physiological analyses showed that these growth changes were accompanied by increased chlorophyll content, net photosynthetic rate, and stomatal conductance. Cytological observations further revealed increased stomatal density and enhanced secondary xylem development in the overexpression lines. At the molecular level, PagMED20 overexpression was associated with coordinated changes in the expression of genes related to photosystem components, carbon assimilation, and stomatal development. In addition, chlorophyll fluorescence analysis indicated increased electron transport rates and reduced non-photochemical quenching under the tested conditions, suggesting altered photosystem II energy utilization. Together, these results indicate that PagMED20 overexpression is associated with coordinated changes in growth, photosynthetic performance, and stomatal traits in poplar. This study provides a foundation for understanding the potential roles of Mediator subunits in woody plant growth and physiology and identifies PagMED20 as a candidate gene for improving biomass production in forest trees.

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.

The innovative HaloTag is a labeling technology that plays a crucial role in advanced fluorescence imaging. However, due to the complexity of plant materials, the application of HaloTag technology in the field of plants is still in its infancy. To expand the application of HaloTag technology in plant cells, we constructed six eukaryotic gene expression vectors with different localizations carrying HaloTag labels, then transformed Arabidopsis thaliana and Nicotiana benthamiana, and finally stained the living cells using HaloTag ligands. Live-cell imaging showed that HaloTag did not affect the localization pattern and biological functions of the target protein, and by improving the experimental conditions, the optimized HaloTag technology could be more effectively applied to plants. Remarkably, we first displayed that the optimized HaloTag technology exhibits superior labeling performance compared to conventional fluorescent proteins, including photostability, monomer properties, multicolor imaging and spatio-temporal differentiated labeling. More importantly, the combination of HaloTag technology with single-particle tracking reveals the specific dynamics of membrane proteins and the changes in the rearrangement of ER-PM connectivity, providing a powerful tool for analyzing protein dynamics. Taken together, this promising HaloTag technology enriches the labeling toolkit for studying spatiotemporal dynamics and various biological processes.

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.

The "salt tree", Rhus chinensis, holds significant economic and medicinal value due to its ability to produce Galla chinensis (Chinese gall/gallnut), a plant-derived medicinal material used in both traditional Chinese and modern medicine that is rich in tannins and flavonoids. It is also renowned for its remarkable stress tolerance. However, the genetic basis underlying its tannin and flavonoid biosynthesis and stress adaptation remains largely unexplored. Here, we assembled a chromosome-level genome of R. chinensis with a size of 357.62 Mb. A significant expansion of defence-related genes, particularly those involved in chitin catabolism and flavonoid biosynthesis, explains the tree's extensive environmental adaptability. We identified key genes involved in tannin biosynthesis and hydrolysis, with RcTA1 playing a central role in gallic acid accumulation, a precursor of hydrolyzable tannins. Notably, RcDIV1 promotes tannin hydrolysis by directly activating RcTA1 transcription. Additionally, we uncovered that well-developed multicellular glandular trichomes, regulated by RcGL2, along with an expanded array of transporters (e.g., ABCGs) and an enhanced ABA response, play critical roles in mediating salt tolerance. These factors collectively drive the production of salt-like secretions, including phenolic and organic acids, which coat the fruit surface. Our study provides profound insights into the genetic mechanisms governing abundant tannin accumulation, flavonoid biosynthesis, glandular trichome development, and stress resilience, offering valuable genetic resources for improving the medicinal and ecological traits of this species.

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

Bu et al. reveal that PagSNAT4‐mediated regulation of endogenous melatonin biosynthesis enhances drought and heat stress tolerance in Populus. This pathway is dually regulated through antagonistic transcriptional control by PagWRKY75 and PagWRKY46, which oppositely modulate PagSNAT4 expression.

Arabidopsis thaliana H+-ATPases (AHAs) are key plasma membrane enzymes that drive nutrient uptake and ion transport across the membrane. RAPID ALKALINIZATION FACTOR 1 (RALF1) induces phosphorylation of AHA2 and inhibits its activity. While the phosphorylation of AHA2 induced by RALF1 has been well described, the ultimate fate of AHA2 following phosphorylation remains unclear. Here, we analyzed the diffusion dynamics of GFP-AHA2 in Arabidopsis thaliana using single-particle tracking combined with variable-angle total internal reflection fluorescence microscopy. Treatment with RALF1, which causes extracellular alkalinization, markedly inhibited AHA2 activity and reduced the velocity of GFP-AHA2. RALF1 promotes the internalization and degradation of GFP-AHA2 through both clathrin-mediated and clathrin-independent endocytosis. In addition, single-particle tracking showed that phosphorylation influences the spatiotemporal dynamics of AHA2. These findings reveal a previously unreported role for RALF1 in promoting AHA2 internalization and degradation via synergistic endocytosis, offering new insights into plant signaling, environmental responses, and protein endocytosis.

>Dear Editor,Plant cell walls are complex and dynamic structures that are mainly composed of polysaccharide polymers such as cellulose,hemicellulose,and pectin,in addition to substantial amounts of the non-saccharide polymer lignin(McFarlane et al.,2014;German et al.,2023).

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.

In plants, cytoskeletal proteins assemble into dynamic polymers that play numerous roles in diverse fundamental cellular processes, including endocytosis, vesicle trafficking, and the spatial distribution of organelles and protein complexes. Plant elicitor peptides (Peps) are damage/danger-associated molecular patterns (DAMPs) that are perceived by the receptor-like kinases PEP RECEPTOR 1 (PEPR1) and PEPR2 to enhance innate immunity and inhibit root growth in Arabidopsis (Arabidopsis thaliana). To date, however, there is little evidence that the actin cytoskeleton of the host cell participates in DAMP-induced innate immunity. Here, we demonstrated that the actin cytoskeleton alters the Pep1-triggered immune response. In addition, dual-color total internal reflection fluorescence-structured illumination microscopy (TIRF-SIM) showed that PEPR1 diffusion on the plasma membrane is closely related to the actin cytoskeleton. We performed single-particle tracking to quantify individual protein particles and found that the actin cytoskeleton notably regulates PEPR1 mobility and cluster size. More importantly, we demonstrated that actin filament reconfiguration is sufficient to inhibit Pep1-induced internalization, which alters the immune response. Taken together, these findings suggest that the actin cytoskeleton functions as an integration node for Pep1 signaling and PEPR1 endocytosis.

Ginkgo (Ginkgo biloba L.) is one of the earliest extant species in seed plant phylogeny. Embryo development patterns can provide fundamental evidence for the origin, evolution, and adaptation of seeds. However, the architectural and morphological dynamics during embryogenesis in G. biloba remain elusive. Herein, we obtained over 2,200 visual slices from 3 stages of embryo development using micro-computed tomography imaging with improved staining methods. Based on 3-dimensional (3D) spatiotemporal pattern analysis, we found that a shoot apical meristem with 7 highly differentiated leaf primordia, including apical and axillary leaf buds, is present in mature Ginkgo embryos. 3D rendering from the front, top, and side views showed 2 separate transport systems of tracheids located in the hypocotyl and cotyledon, representing a unique pattern of embryogenesis. Furthermore, the morphological dynamic analysis of secretory cavities indicated their strong association with cotyledons during development. In addition, we identified genes GbLBD25a (lateral organ boundaries domain 25a), GbCESA2a (cellulose synthase 2a), GbMYB74c (myeloblastosis 74c), GbPIN2 (PIN-FORMED 2) associated with vascular development regulation, and GbWRKY1 (WRKYGOK 1), GbbHLH12a (basic helix-loop-helix 12a), and GbJAZ4 (jasmonate zim-domain 4) potentially involved in the formation of secretory cavities. Moreover, we found that flavonoid accumulation in mature embryos could enhance postgerminative growth and seedling establishment in harsh environments. Our 3D spatial reconstruction technique combined with multiomics analysis opens avenues for investigating developmental architecture and molecular mechanisms during embryogenesis and lays the foundation for evolutionary studies of embryo development and maturation. Three-dimensional spatiotemporal pattern analysis reveals a unique pattern of morphogenesis and molecular dynamics of embryonic development states in Ginkgo biloba.

Proteins are large molecules of specific conformations,and their functions vary depending on their structure.As the key components involved in life processes,proteins often assemble into large complexes called oligomers to execute important activities.Oligomers are of two types:Homologous and heterologous.The homologous oligomers contain subunits of the same polypeptide chain,whereas the heterologous ones consist of two or more polypeptide chains with different amino acids or different biopolymers,such as nucleic acids.These oligomer complexes play significant roles in regulating enzyme activity,signal transduction,and cell adhesion.Oligomerization can increase protein concentration in local space to provide higher stability and enhance the specificity in a molecular recognition process.Oligomerization is influenced by factors such as the types of ligands,proteins,and temperature.For example,receptor proteins are often assembled into dimeric or higher-order oligomers mediated by their corresponding ligands,which promote downstream signaling cascades to transmit signals.The formation mechanisms of oligomeric proteins can be categorized into the following four types:Domain exchange,ligand-induced oligomerization,interfacial point mutation,and post-translational modification.Domain exchange allows functional conversion between protein monomers and oligomers.Ligands can induce oligomerization to activate protein activity,as is typically observed in receptor proteins.Interfacial point mutation involves changes in the amino acid sequence,insertion,or deletion of residues in an interfacial region,which distinguishes different oligomerization states.Post-translational modifications are essential for functional protein formation,although errors in modifications can result in the formation and accumulation of abnormal oligomers,which potentially contribute to disease development.Therefore,analyzing protein oligomerization is crucial for understanding biological processes such as protein-protein/ligand interactions,signal transduction,and disease-related mechanisms.Researchers have optimized the study of protein oligomerization from different perspectives,including predicting and identifying interacting proteins,labeling target proteins,and observing them through imaging.The conventional techniques such as co-immunoprecipita-tion(Co-IP)and yeast two-hybrid precipitation have some limitations,such as the inability to investigate directly in living cells as well as the possibility of giving false-positive or false-negative results.In recent years,however,the emergence of new technologies has enabled real-time,dynamic,and in vivo observation of protein-protein interactions,which has significantly contributed to the advancements in this field.For instance,the use of more stable and photobleaching-resistant fluorescent probes,combined with high-speed imaging techniques,has improved the temporal and spatial resolution in observing the dynamic behavior and interactions of proteins.In addition,the combination of emerging technologies and algorithms has expanded the scope of protein explored at both temporal and spatial scales,which has deepened our understanding of the relation between protein structure and function.This review summarizes the classification and formation mechanisms of oligomeric proteins,as well as highlights several major technologies for analyzing protein complex oligomerization,and provides an overview of the recent research progress in protein labeling and in vivo detection technologies.Finally,it offers a prospective outlook on the development of research in protein oligomerization,intending to provide theoretical references for researchers in selecting appropriate analytical techniques.

The geometric shape and arrangement of individual cells play a role in shaping organ functions. However, analyzing multicellular features and exploring their connectomes in centimeter-scale plant organs remain challenging. Here, we established a set of frameworks named Large-Volume Fully Automated Cell Reconstruction (LVACR), enabling the exploration of three-dimensional (3D) cytological features and cellular connectivity in plant tissues. Through benchmark testing, our framework demonstrated superior efficiency in cell segmentation and aggregation, successfully addressing the inherent challenges posed by light sheet fluorescence microscopy (LSFM) imaging. Using LVACR, we successfully established a cell atlas of different plant tissues. Cellular morphology analysis revealed differences of cell clusters and shapes in between different poplar (P. simonii Carr. and P. canadensis Moench.) seeds, whereas topological analysis revealed that they maintained conserved cellular connectivity. Furthermore, LVACR spatiotemporally demonstrated an initial burst of cell proliferation, accompanied by morphological transformations at an early stage in developing the shoot apical meristem. During subsequent development, cell differentiation produced anisotropic features, thereby resulting in various cell shapes. Overall, our findings provided valuable insights into the precise spatial arrangement and cellular behavior of multicellular organisms, thus enhancing our understanding of the complex processes underlying plant growth and differentiation.