Observation of Vacuoles in Arabidopsis Developing Pollen Grains with Whole-Cell Electron Tomography.

Arabidopsis thaliana developing pollen grains serve as an excellent system for studying vacuole dynamics. Here, we present a methodological approach that utilizes the serial tomography package in Etomo software from IMOD to generate whole-cell tomograms on A. thaliana developing pollens for visualizing vacuoles on the whole-cell scale. In order to understand the vacuole dynamics along with the pollen maturation, we also introduce a sampling method aimed at harvesting the pollen grains at various stages, marked by the vegetative nucleus or generative cell. The cryo-fixation/freeze-substitution technique can then be applied to preserve the fine structures of the pollen grains and facilitate detailed ultrastructure examination. Through this method, large-volume whole-cell electron tomograms regarding vacuolar morphologies and ultrastructural changes during pollen development and maturation have been obtained. Overall, the method presented here provides valuable insights into the dynamic nature of vacuoles in Arabidopsis developing pollen.

Correlation Analysis of IL-17, IL-21, IL-23 with Non-Alcoholic Liver Fibrosis and Cirrhosis.

Objective: This research aimed to explore the involvement of interleukins (IL) - IL-6, IL-17, IL-21, and IL-23 - in the evolution and diagnosis of non-alcoholic liver fibrosis and cirrhosis. Methods: The study subjects were selected from the patients who visited the Department of Hepatology of X Hospital in Y City from August 2021 to April 2023. Peripheral blood samples were collected. All participants were divided into liver fibrosis, cirrhosis, hepatitis, and healthy subjects four groups. IL-21, IL-17, IL23, IL-6 were detected by double antibody sandwich. Results: The results showed that there was a significant difference in the levels of IL-17, IL-21, and IL-23 among the 4 groups (P<0.0001). ROC curve analysis showed that the AUC values of IL-17, IL-21 and liver fiber 4 items were >0.70, suggesting that the diagnostic efficacy of IL-17, IL-21 was similar to that of liver fiber 4 items. Spearman correlation analysis showed that IL-17 had a positive correlation with collagen type III N-peptide, type IV collagen, and Laminin (P < 0.05), and no correlation with Hyaluronic acid (P > 0.05). Conclusion: IL-17, IL-21, and IL-23 play a pivotal role in the inflammatory pathways associated with liver injuries, establishing themselves as potent auxiliary diagnostic markers in identifying liver fibrosis and cirrhosis.

CRISPR RNA-Guided Transposases Facilitate Dispensable Gene Study in Phage.

Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn6677, encoding a nuclease-deficient type I-F CRISPR-Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.

Targeting the pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase (PDC/PDK) axis to discover potent PDK inhibitors through structure-based virtual screening and pharmacological evaluation.

Proliferating cancer cells are characterized by the Warburg effect, a metabolic alteration in which ATP is generated from cytoplasmic glycolysis instead of oxidative phosphorylation. The pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase (PDC/PDK) axis plays a crucial role in this effect and has been identified as a potential target for anticancer drug development. Herein, we present the discovery and pharmacological evaluation of potent PDK inhibitors targeting the PDK/PDC axis. We successfully identified 6 compounds from a small molecule library through a structure-based virtual screening campaign and evaluated their enzymatic inhibitory potencies for PDK1-4. Our results indicated that compound 1 exhibited submicromolar inhibitory activities against PDK1-3 (IC50 = 109.3, 135.8, and 458.7 nM, respectively), but is insensitive to PDK4 (IC50 = 8.67 µM). Furthermore, compound 1 inhibited the proliferation of A549 cells with an EC50 value of 10.7 µM. In addition, compound 1 induced cell apoptosis, arrested the cell cycle at the S phase, and reduced cell invasion and migration, while showing low in vivo toxicity at a high dose. Based on these observations, it can be concluded that compound 1 is a promising anti-PDK1-3 lead that merits further investigation.

Recent advances in plant endomembrane research and new microscopical techniques.

The endomembrane system consists of various membrane-bound organelles including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, and the lysosome/vacuole. Membrane trafficking between distinct compartments is mainly achieved by vesicular transport. As the endomembrane compartments and the machineries regulating the membrane trafficking are largely conserved across all eukaryotes, our current knowledge on organelle biogenesis and endomembrane trafficking in plants has mainly been shaped by corresponding studies in mammals and yeast. However, unique perspectives have emerged from plant cell biology research through the characterization of plant-specific regulators as well as the development and application of the state-of-the-art microscopical techniques. In this review, we summarize our current knowledge on the plant endomembrane system, with a focus on several distinct pathways: ER-to-Golgi transport, protein sorting at the TGN, endosomal sorting on multivesicular bodies, vacuolar trafficking/vacuole biogenesis, and the autophagy pathway. We also give an update on advanced imaging techniques for the plant cell biology research.

Embryo sac development relies on symplastic signals from ovular integuments in Arabidopsis.

Ovules are female reproductive organs of angiosperms, consisting of sporophytic integuments surrounding female gametophytes, that is, embryo sacs. Synchronization between integument growth and embryo sac development requires intracellular communication. However, signaling routes through which cells of the two generations communicate are unclear. We report that symplastic signals through plasmodesmata (PDs) of integuments are critical for the development of female gametophytes. Genetic interferences of PD biogenesis either by functional loss of CHOLINE TRANSPORTER-LIKE1 (CTL1) or by integument-specific expression of a mutated CALLOSE SYNTHASE 3 (cals3m) compromised PD formation in integuments and reduced fertility. Close examination of pINO:cals3m or ctl1 ovules indicated that female gametophytic development was either arrested at various stages after the formation of functional megaspores. In both cases, defective ovules could not attract pollen tubes, leading to the failure of fertilization. Results presented here demonstrate a key role of the symplastic route in sporophytic control of female gametophytic development.

Arabidopsis ORP2A mediates ER-autophagosomal membrane contact sites and regulates PI3P in plant autophagy.

Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein-related protein 2A (ORP2A) in mediating ER-autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) in vivo. Taken together, ORP2A mediates ER-autophagosomal MCSs and regulates autophagy through PI3P redistribution.

Electron tomography of prolamellar bodies and their transformation into grana thylakoids in cryofixed Arabidopsis cotyledons.

The para-crystalline structures of prolamellar bodies (PLBs) and light-induced etioplast-to-chloroplast transformation have been investigated via electron microscopy. However, such studies suffer from chemical fixation artifacts and limited volumes of 3D reconstruction. Here, we examined Arabidopsis thaliana cotyledon cells by electron tomography (ET) to visualize etioplasts and their conversion into chloroplasts. We employed scanning transmission ET to image large volumes and high-pressure freezing to improve sample preservation. PLB tubules were arranged in a zinc blende-type lattice-like carbon atoms in diamonds. Within 2 h after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form thylakoid sheets (pre-granal thylakoids), which folded and overlapped with each other to create grana stacks. Since the nascent pre-granal thylakoids contained curved membranes in their tips, we examined the expression and localization of CURT1 (CURVATURE THYLAKOID1) proteins. CURT1A transcripts were most abundant in de-etiolating cotyledon samples, and CURT1A was concentrated at the PLB periphery. In curt1a etioplasts, PLB-associated thylakoids were swollen and failed to form grana stacks. In contrast, PLBs had cracks in their lattices in curt1c etioplasts. Our data provide evidence that CURT1A is required for pre-granal thylakoid assembly from PLB tubules during de-etiolation, while CURT1C contributes to cubic crystal growth in the dark.

ADP-ribosylation factor D1 modulates Golgi morphology, cell plate formation, and plant growth in Arabidopsis.

ADP-ribosylation factor (ARF) family proteins, one type of small guanine-nucleotide-binding (G) proteins, play a central role in regulating vesicular traffic and organelle structures in eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains more than 21 ARF proteins, but relatively little is known about the functional heterogeneity of ARF homologs in plants. Here, we characterized the function of a unique ARF protein, ARFD1B, in Arabidopsis. ARFD1B exhibited both cytosol and punctate localization patterns, colocalizing with a Golgi marker in protoplasts and transgenic plants. Distinct from other ARF1 homologs, overexpression of a dominant-negative mutant form of ARFD1B did not alter the localization of the Golgi marker mannosidase I (ManI)-RFP in Arabidopsis cells. Interestingly, the ARFD1 artificial microRNA knockdown mutant arfd1 displayed a deleterious growth phenotype, while this phenotype was restored in complemented plants. Further, confocal imaging and transmission electron microscopy analyses of the arfd1 mutant revealed defective cell plate formation and abnormal Golgi morphology. Pull-down and liquid chromatography-tandem mass spectrometry analyses identified Coat Protein I (COPI) components as interacting partners of ARFD1B, and subsequent bimolecular fluorescence complementation, yeast (Saccharomyces cerevisiae) two-hybrid, and co-immunoprecipitation assays further confirmed these interactions. These results demonstrate that ARFD1 is required for cell plate formation, maintenance of Golgi morphology, and plant growth in Arabidopsis.

Friendly mediates membrane depolarization-induced mitophagy in Arabidopsis.

The oxidative environment within the mitochondria makes them particularly vulnerable to proteotoxic stress. To maintain a healthy mitochondrial network, eukaryotes have evolved multi-tiered quality control pathways. If the stress cannot be alleviated, defective mitochondria are selectively removed by autophagy via a process termed mitophagy. Despite significant advances in metazoans and yeast, in plants, the molecular underpinnings of mitophagy are largely unknown. Here, using time-lapse imaging, electron tomography, and biochemical assays, we show that uncoupler treatments cause loss of mitochondrial membrane potential and induce autophagy in Arabidopsis. The damaged mitochondria are selectively engulfed by autophagosomes that are labeled by ATG8 proteins in an ATG5-dependent manner. Friendly, a member of the clustered mitochondria protein family, is recruited to the damaged mitochondria to mediate mitophagy. In addition to the stress, mitophagy is also induced during de-etiolation, a major cellular transformation during photomorphogenesis that involves chloroplast biogenesis. De-etiolation-triggered mitophagy is involved in cotyledon greening, pointing toward an inter-organellar crosstalk mechanism. Altogether, our results demonstrate how plants employ mitophagy to recycle damaged mitochondria during stress and development.