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Secondary growth in woody plants generates new cells and tissues via the activity of the vascular cambium and drives the radial expansion of stems and roots. It is regulated by a series of endogenous factors, especially transcription factors. Here, we cloned the basic helix–loop–helix (bHLH) transcription factor gene UNFERTILIZED EMBRYO SAC12 (UNE12) from poplar (Populus alba × Populus glandulosa Uyeki) and used biochemical, molecular, and cytological assays to investigate the biological functions and regulatory mechanism of PagUNE12. PagUNE12 mainly localized in the nucleus and possessed transcriptional activation activity. It was widely expressed in vascular tissues, including primary phloem and xylem and secondary phloem and xylem. Poplar plants overexpressing PagUNE12 showed significantly reduced plant height, shorter internodes, and curled leaves compared with wild-type plants. Optical microscopy and transmission electron microscopy revealed that overexpressing PagUNE12 promoted secondary xylem development, with thicker secondary cell walls than wild-type poplar. Fourier transform infrared spectroscopy, confocal Raman microscopy, and 2D Heteronuclear Single Quantum Correlation analysis indicated that these plants also had increased lignin contents, with a lower relative abundance of syringyl lignin units and a higher relative abundance of guaiacyl lignin units. Therefore, overexpressing PagUNE12 promoted secondary xylem development and increased the lignin contents of secondary xylem in poplar, suggesting that this gene could be used to improve wood quality in the future.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, Nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of Lonicera maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in chlorogenic acid biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

Biochemical and genetic approaches have been extensively used to study transcription factor (TF) functions, but their dynamic behaviors and the complex ways in which they regulate transcription in plant cells remain unexplored, particularly behaviors such as translocation and binding to DNA. Recent developments in labeling and imaging techniques provide the necessary sensitivity and resolution to study these behaviors in living cells. In this review, we present an up-to-date portrait of the dynamics and regulation of TFs under physiologically relevant conditions and then summarize recent advances in fluorescent labeling strategies and imaging techniques. We then discuss future prospects and challenges associated with the application of these techniques to examine TFs’ intricate dance in living plants.

Aquaporins such as the plasma membrane intrinsic proteins (PIPs) allow water to move through cell membranes and are vital for stomatal movement in plants. Despite their importance, the dynamic changes in aquaporins during water efflux and influx have not been directly observed in real time in vivo. Here, to determine which factors regulate these changes during the bidirectional translocation of water, we examined aquaporin dynamics during the stomatal immune response to the bacterial flagellin-derived peptide flg22. The Arabidopsis (Arabidopsis thaliana) aquaporin mutant pip2;1 showed defects in the flg22-induced stomatal response. Variable-angle total internal reflection fluorescence microscopy revealed that the movement dynamics and dwell times of AQ6]GFP-AtPIP2;1 in guard cells and subsidiary cells exhibited cell type-specific dependencies on flg22. The cytoskeleton, rather than the cell wall, was the major factor regulating AtPIP2;1 dynamics, although both the cytoskeleton and cell wall might form bounded domains that restrict the diffusion of AtPIP2;1 in guard cells and subsidiary cells. Finally, our analysis revealed the different roles of cortical actin and microtubules in regulating AtPIP2;1 dynamics in guard cells, as well as subsidiary cells, under various conditions. Our observations shed light on the heterogeneous mechanisms that regulate membrane protein dynamics in plants in response to pathogens.

Ca2+-calmodulin (Ca2+-CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca2+-CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca2+-CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca2+-CaM signaling rapidly induced an increase in extracellular Ca2+ influx, resulting in dramatically increased cytosolic Ca2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca2+-CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca2+-CaM in the regulation of tip growth machinery.

The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner, while hardly similar effects were detected when cysteine-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanate-phalloidin and β-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.

We assessed the effects of brefeldin A (BFA) on pollen tube development in Picea meyeri using fluorescent marker FM4-64 as a membrane-inserted endocytic/recycling marker, together with ultrastructural studies and Fourier transform infrared analysis of cell walls. BFA inhibited pollen germination and pollen tube growth, causing morphological changes in a dose-dependent manner, and pollen tube tip growth recovered after transferring into BFA-free medium. FM4-64 labeling showed typical bright apical staining in normally growing P. meyeri pollen tubes; this apical staining pattern differed from the V-formation pattern found in angiosperm pollen tubes. Confocal microscopy revealed that exocytosis was greatly inhibited in the presence of BFA. In contrast, the overall uptake of FM4-64 dye was about 2-fold that in the control after BFA (5 μg mL−1) treatment, revealing that BFA stimulated endocytosis in a manner opposite to the induced changes in exocytosis. Transmission electron microscopic observation showed that the number of secretory vesicles at the apical zone dramatically decreased, together with the disappearance of paramural bodies, while the number of vacuoles and other larger organelles increased. An acid phosphatase assay confirmed that the addition of BFA significantly inhibited secretory pathways. Importantly, Fourier transform infrared microspectroscopy documented significant changes in the cell wall composition of pollen tubes growing in the presence of BFA. These results suggest that enhanced endocytosis, together with inhibited secretion, is responsible for the retarded growth of pollen tubes induced by BFA.

MicroRNAs (miRNAs) are endogenous small RNAs that repress target gene expression posttranscriptionally, and are critically involved in various developmental processes and responses to environmental stresses in eukaryotes. MiRNA857 is not widely distributed in plants and is encoded by a single gene, AtMIR857, in Arabidopsis (Arabidopsis thaliana). The functions of miR857 and its mechanisms in regulating plant growth and development are still unclear. Here, by means of genetic analysis coupled with cytological studies, we investigated the expression pattern and regulation mechanism of miR857 and its biological functions in Arabidopsis development. We found that miR857 regulates its target gene, Arabidopsis LACCASE7, at the transcriptional level, thereby reducing laccase activity. Using stimulated Raman scattering and x-ray microtomography three-dimensional analyses, we showed that miR857 was involved in the regulation of lignin content and consequently morphogenesis of the secondary xylem. In addition, miR857 was activated by SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 in response to low copper conditions. Collectively, these findings demonstrate the role of miR857 in the regulation of secondary growth of vascular tissues in Arabidopsis and reveal a unique control mechanism for secondary growth based on the miR857 expression in response to copper deficiency.

Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.