The Chinese pine genome and methylome unveil key features of conifer evolution.

Conifers dominate the world's forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers' adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development.

3D reconstruction and multi-omics analysis reveal a unique pattern of embryogenesis in Ginkgo biloba.

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 Ginkgo biloba (G. biloba) remain elusive. Herein, we obtained over 2200 visual slices from three stages of embryo development using micro-computed tomography imaging with improved staining methods. Based on 3D spatio-temporal pattern analysis, we found that a shoot apical meristem with seven 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 two 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), GbJAZ4 (jasmonate zim-domain 4) potentially involved in the formation of secretory cavities. Moreover, we found that flavonoid accumulation in mature embryos could enhance post-germinative growth and seedling establishment in harsh environments. Our 3D spatial reconstruction technique combined with multi-omics analysis opens avenues for investigating developmental architecture and molecular mechanisms during embryogenesis and lays the foundation for evolutionary studies of embryo development and maturation.

PagARGOS promotes low-lignin wood formation in poplar.

Wood formation, which occurs mainly through secondary xylem development, is important not only for supplying raw material for the 'ligno-chemical' industry but also for driving the storage of carbon. However, the complex mechanisms underlying the promotion of xylem formation remain to be elucidated. Here, we found that overexpression of Auxin-Regulated Gene involved in Organ Size (ARGOS) in hybrid poplar 84 K (Populus alba × Populus tremula var. glandulosa) enlarged organ size. In particular, PagARGOS promoted secondary growth of stems with increased xylem formation. To gain further insight into how PagARGOS regulates xylem development, we further carried out yeast two-hybrid screening and identified that the auxin transporter WALLS ARE THIN1 (WAT1) interacts with PagARGOS. Overexpression of PagARGOS up-regulated WAT1, activating a downstream auxin response promoting cambial cell division and xylem differentiation for wood formation. Moreover, overexpressing PagARGOS caused not only higher wood yield but also lower lignin content compared with wild-type controls. PagARGOS is therefore a potential candidate gene for engineering fast-growing and low-lignin trees with improved biomass production.

Suberin deficiency and its effect on the transport physiology of young poplar roots.

The precise functions of suberized apoplastic barriers in root water and nutrient transport physiology have not fully been elucidated. While lots of research has been performed with mutants of Arabidopsis, little to no data are available for mutants of agricultural crop or tree species. By employing a combined set of physiological, histochemical, analytical, and transport physiological methods as well as RNA-sequencing, this study investigated the implications of remarkable CRISPR/Cas9-induced suberization defects in young roots of the economically important gray poplar. While barely affecting overall plant development, contrary to literature-based expectations significant root suberin reductions of up to 80-95% in four independent mutants were shown to not evidently affect the root hydraulic conductivity during non-stress conditions. In addition, subliminal iron deficiency symptoms and increased translocation of a photosynthesis inhibitor as well as NaCl highlight the involvement of suberin in nutrient transport physiology. The multifaceted nature of the root hydraulic conductivity does not allow drawing simplified conclusions such as that the suberin amount must always be correlated with the water transport properties of roots. However, the decreased masking of plasma membrane surface area could facilitate the uptake but also leakage of beneficial and harmful solutes.

Membrane microdomains: Structural and signaling platforms for establishing membrane polarity.

Cell polarity results from the asymmetric distribution of cellular structures, molecules, and functions. Polarity is a fundamental cellular trait that can determine the orientation of cell division, the formation of particular cell shapes, and ultimately the development of a multicellular body. To maintain the distinct asymmetric distribution of proteins and lipids in cellular membranes, plant cells have developed complex trafficking and regulatory mechanisms. Major advances have been made in our understanding of how membrane microdomains influence the asymmetric distribution of proteins and lipids. In this review, we first give an overview of cell polarity. Next, we discuss current knowledge concerning membrane microdomains and their roles as structural and signaling platforms to establish and maintain membrane polarity, with a special focus on the asymmetric distribution of proteins and lipids, and advanced microscopy techniques to observe and characterize membrane microdomains. Finally, we review recent advances regarding membrane trafficking in cell polarity establishment and how the balance between exocytosis and endocytosis affects membrane polarity.

PagUNE12 encodes a basic helix-loop-helix transcription factor that regulates the development of secondary vascular tissue in poplar.

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.

High-resolution genome mapping and functional dissection of chlorogenic acid production in Lonicera maackii.

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 L. 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 CGA 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.

Cross-talk between clathrin-dependent post-Golgi trafficking and clathrin-mediated endocytosis in Arabidopsis root cells.

Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.

Synaptotagmins at the endoplasmic reticulum-plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress.

Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

Spatial regulation of RBOHD via AtECA4-mediated recycling and clathrin-mediated endocytosis contributes to ROS accumulation during salt stress response but not flg22-induced immune response.

Various environmental stresses can induce production of reactive oxygen species (ROS) to turn on signaling for proper responses to those stresses. Plasma membrane (PM)-localized respiratory burst oxidase homologs (RBOHs), in particular RBOHD, produce ROS via the post-translational activation upon abiotic and biotic stresses. Although the mechanisms of RBOHD activation upon biotic stress have been elucidated in detail, it remains elusive how salinity stress activates RBOHD. Here, we present evidence that trafficking of PM-localized RBOHD to endosomes and then its recycling back to the PM is critical for ROS accumulation upon salinity stress. ateca4 plants that were defective in recycling of proteins from endosomes to the PM and clc2-1 and chc2-1 plants that were defective in endocytosis showed a defect in salinity stress-induced ROS production. In addition, ateca4 plants showed a defect in transient accumulation of GFP:RBOHD to the PM at the early stage of salinity stress. By contrast, ateca4 plants showed no defect in the increase in the ROS level and accumulation of RBOHD to the PM upon flg22 treatment as wild-type plants. Based on these observations, we propose that factors involved in the trafficking machinery such as AtECA4 and clathrin are important players in salt stress-induced, but not flg22-induced, ROS accumulation.

The RALF1-FERONIA interaction modulates endocytosis to mediate control of root growth in Arabidopsis.

The interaction between the receptor-like kinase (RLK) FERONIA (FER) and the secreted peptide RAPID ALKALINIZATION FACTOR1 (RALF1) is vital for development and stress responses in Arabidopsis Ligand-induced membrane dynamics affect the function of several RLKs, but the effects of the RALF1-FER interaction on the dynamics of FER and the ensuing effects on its functionality are poorly understood. Here, we show that RALF1 modulated the dynamics and partitioning of FER-GFP at the plasma membrane (PM). Moreover, FER was internalized by both clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) under steady-state conditions. After RALF1 treatment, FER-GFP internalization was primarily enhanced via the CME pathway, raising FER-GFP levels in the vacuole. RALF1 treatment also modulated trafficking of other PM proteins, such as PIN2-GFP and BRI1-GFP, increasing their vacuolar levels by enhancing their internalization. Importantly, blocking CME attenuated RALF1-mediated root growth inhibition independently of RALF1-induced early signaling, suggesting that the RALF1 can also exert its effects via the CME pathway. These findings reveal that the RALF1-FER interaction modulates plant growth and development, and this might also involve endocytosis of PM proteins.

Multiscale microscopy to decipher plant cell structure and dynamics.

New imaging methodologies with high contrast and molecular specificity allow researchers to analyze dynamic processes in plant cells at multiple scales, from single protein and RNA molecules to organelles and cells, to whole organs and tissues. These techniques produce informative images and quantitative data on molecular dynamics to address questions that cannot be answered by conventional biochemical assays. Here, we review selected microscopy techniques, focusing on their basic principles and applications in plant science, discussing the pros and cons of each technique, and introducing methods for quantitative analysis. This review thus provides guidance for plant scientists in selecting the most appropriate techniques to decipher structures and dynamic processes at different levels, from protein dynamics to morphogenesis.

Transcription factor dynamics in plants: Insights and technologies for in vivo imaging.

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.

Endoplasmic reticulum membrane contact sites: cross-talk between membrane-bound organelles in plant cells.

Eukaryotic cells contain organelles surrounded by monolayer or bilayer membranes. Organelles take part in highly dynamic and organized interactions at membrane contact sites, which play vital roles during development and response to stress. The endoplasmic reticulum extends throughout the cell and acts as an architectural scaffold to maintain the spatial distribution of other membrane-bound organelles. In this review, we highlight the structural organization, dynamics, and physiological functions of membrane contact sites between the endoplasmic reticulum and various membrane-bound organelles, especially recent advances in plants. We briefly introduce how the combined use of dynamic and static imaging techniques can enable monitoring of the cross-talk between organelles via membrane contact sites. Finally, we discuss future directions for research fields related to membrane contact.

Plasmodesmal endoplasmic reticulum proteins regulate intercellular trafficking of cucumber mosaic virus in Arabidopsis.

Plasmodesmata (PD) are plasma membrane-lined cytoplasmic nanochannels that mediate cell-to-cell communication across the cell wall. A range of proteins are embedded in the PD plasma membrane and endoplasmic reticulum (ER), and function in regulating PD-mediated symplasmic trafficking. However, knowledge of the nature and function of the ER-embedded proteins in the intercellular movement of non-cell-autonomous proteins is limited. Here, we report the functional characterization of two ER luminal proteins, AtBiP1/2, and two ER integral membrane proteins, AtERdj2A/B, which are located within the PD. These PD proteins were identified as interacting proteins with cucumber mosaic virus (CMV) movement protein (MP) in co-immunoprecipitation studies using an Arabidopsis-derived plasmodesmal-enriched cell wall protein preparation (PECP). The AtBiP1/2 PD location was confirmed by TEM-based immunolocalization, and their AtBiP1/2 signal peptides (SPs) function in PD targeting. In vitro/in vivo pull-down assays revealed the association between AtBiP1/2 and CMV MP, mediated by AtERdj2A, through the formation of an AtBiP1/2-AtERdj2-CMV MP complex within PD. The role of this complex in CMV infection was established, as systemic infection was retarded in bip1/bip2w and erdj2b mutants. Our findings provide a model for a mechanism by which the CMV MP mediates cell-to-cell trafficking of its viral ribonucleoprotein complex.

Roles of the wound hormone jasmonate in plant regeneration.

Plants have remarkable abilities to regenerate in response to wounding. How wounding triggers rapid signal transduction to induce a cellular response is a key topic for understanding the molecular mechanism of plant regeneration. An increasing body of evidence indicates that jasmonate, a hormone that is produced rapidly in response to wounding, plays multiple roles in different plant regeneration processes. In this review, we summarize recent advances on the roles of jasmonate in tissue repair, the formation of wound-induced callus, de novo organ regeneration, and somatic embryogenesis. Physiological and molecular analyses indicate that jasmonate can regulate stem cell activities, cell proliferation, cell fate transition, and auxin production, thereby contributing to plant regeneration. In addition, jasmonate is strictly controlled in plant cells via restriction of the jasmonate concentration and its signalling pathway in a spatial and temporal manner during regeneration. Overall, jasmonate acts as the hormone linking wounding to distinct types of regeneration in plants.

Multifeature analyses of vascular cambial cells reveal longevity mechanisms in old Ginkgo biloba trees.

Aging is a universal property of multicellular organisms. Although some tree species can live for centuries or millennia, the molecular and metabolic mechanisms underlying their longevity are unclear. To address this, we investigated age-related changes in the vascular cambium from 15- to 667-y-old Ginkgo biloba trees. The ring width decreased sharply during the first 100 to 200 y, with only a slight change after 200 y of age, accompanied by decreasing numbers of cambial cell layers. In contrast, average basal area increment (BAI) continuously increased with aging, showing that the lateral meristem can retain indeterminacy in old trees. The indole-3-acetic acid (IAA) concentration in cambial cells decreased with age, whereas the content of abscisic acid (ABA) increased significantly. In addition, cell division-, cell expansion-, and differentiation-related genes exhibited significantly lower expression in old trees, especially miR166 and HD-ZIP III interaction networks involved in cambial activity. Disease resistance-associated genes retained high expression in old trees, along with genes associated with synthesis of preformed protective secondary metabolites. Comprehensive evaluation of the expression of genes related to autophagy, senescence, and age-related miRNAs, together with analysis of leaf photosynthetic efficiencies and seed germination rates, demonstrated that the old trees are still in a healthy, mature state, and senescence is not manifested at the whole-plant level. Taken together, our results reveal that long-lived trees have evolved compensatory mechanisms to maintain a balance between growth and aging processes. This involves continued cambial divisions, high expression of resistance-associated genes, and continued synthetic capacity of preformed protective secondary metabolites.

SNARE proteins VAMP721 and VAMP722 mediate the post-Golgi trafficking required for auxin-mediated development in Arabidopsis.

The plant hormone auxin controls many aspects of plant development. Membrane trafficking processes, such as secretion, endocytosis and recycling, regulate the polar localization of auxin transporters in order to establish an auxin concentration gradient. Here, we investigate the function of the Arabidopsis thaliana R-SNAREs VESICLE-ASSOCIATED MEMBRANE PROTEIN 721 (VAMP721) and VAMP722 in the post-Golgi trafficking required for proper auxin distribution and seedling growth. We show that multiple growth phenotypes, such as cotyledon development, vein patterning and lateral root growth, were defective in the double homozygous vamp721 vamp722 mutant. Abnormal auxin distribution and root patterning were also observed in the mutant seedlings. Fluorescence imaging revealed that three auxin transporters, PIN-FORMED 1 (PIN1), PIN2 and AUXIN RESISTANT 1 (AUX1), aberrantly accumulate within the cytoplasm of the double mutant, impairing the polar localization at the plasma membrane (PM). Analysis of intracellular trafficking demonstrated the involvement of VAMP721 and VAMP722 in the endocytosis of FM4-64 and the secretion and recycling of the PIN2 transporter protein to the PM, but not its trafficking to the vacuole. Furthermore, vamp721 vamp722 mutant roots display enlarged trans-Golgi network (TGN) structures, as indicated by the subcellular localization of a variety of marker proteins and the ultrastructure observed using transmission electron microscopy. Thus, our results suggest that the R-SNAREs VAMP721 and VAMP722 mediate the post-Golgi trafficking of auxin transporters to the PM from the TGN subdomains, substantially contributing to plant growth.

In vivo single-particle tracking of the aquaporin AtPIP2;1 in stomata reveals cell type-specific dynamics.

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.

MADS-box transcription factors MADS11 and DAL1 interact to mediate the vegetative-to-reproductive transition in pine.

The reproductive transition is an important event that is crucial for plant survival and reproduction. Relative to the thorough understanding of the vegetative phase transition in angiosperms, a little is known about this process in perennial conifers. To gain insight into the molecular basis of the regulatory mechanism in conifers, we used temporal dynamic transcriptome analysis with samples from seven different ages of Pinus tabuliformis to identify a gene module substantially associated with aging. The results first demonstrated that the phase change in P. tabuliformis occurred as an unexpectedly rapid transition rather than a slow, gradual progression. The age-related gene module contains 33 transcription factors and was enriched in genes that belong to the MADS (MCMl, AGAMOUS, DEFICIENS, SRF)-box family, including six SOC1-like genes and DAL1 and DAL10. Expression analysis in P. tabuliformis and a late-cone-setting P. bungeana mutant showed a tight association between PtMADS11 and reproductive competence. We then confirmed that MADS11 and DAL1 coordinate the aging pathway through physical interaction. Overexpression of PtMADS11 and PtDAL1 partially rescued the flowering of 35S::miR156A and spl1,2,3,4,5,6 mutants in Arabidopsis (Arabidopsis thaliana), but only PtMADS11 could rescue the flowering of the ft-10 mutant, suggesting PtMADS11 and PtDAL1 play different roles in flowering regulatory networks in Arabidopsis. The PtMADS11 could not alter the flowering phenotype of soc1-1-2, indicating it may function differently from AtSOC1 in Arabidopsis. In this study, we identified the MADS11 gene in pine as a regulatory mediator of the juvenile-to-adult transition with functions differentiated from the angiosperm SOC1.