Plant peroxisomes:Small organelles with versatility and complexity

Peroxisomes are small,highly dynamic,and multifunctional organelles in eukaryotes.Essential to plant survival,peroxisomes house various crucial metabolic activities,such as degradation of hydrogen peroxide(H2O2),lipid metabolism,photorespiration,and hormone biosynthesis and catabolism,and remodel their proteome in response to developmental and environmental changes(Hu et al.2012;Pan and Hu 2018).The four reviews and three research articles in this special issue on plant peroxisomes provide new insights into the diverse roles and dynamics of these structurally simple but functionally complicated organelles,raising exciting new questions for future investigations.

RNA editing machinery in plant organelles

RNA editing is a type of post-transcriptional modification that includes nucleotide insertion/deletion or conversion. Different categories of RNA editing have been widely observed in distinct RNAs from divergent organisms. In flowering plants, RNA editing usually alters cytidine to uridine in plastids and mitochondria, playing important roles in various plant developmental processes, including organelle biogenesis, adaptation to environmental changes, and signal transduction. Numerous studies have demonstrated that a number of factors are involved in plant RNA editing, such as pentatricopeptide repeat(PPR) proteins, multiple organelle RNA editing factors(MORF, also known as RIP), organelle RNA recognition motif(ORRM) containing proteins,protoporphyrinogen IX oxidase 1(PPO1) and organelle zinc finger 1(OZ1). These factors play diverse roles in plant RNA editing due to their distinct characteristics. In this review, we discuss the functional roles of the individual editing factors and their associations in plant RNA editing.

Compartmentalized regulation of organelle integrity in neurodegenerative diseases:lessons from the Drosophila motor neuron

Neurons are highly polarized,morphologically asymmetric,and functionally compartmentalized cells that contain long axons extending from the cell body.For this reason,their maintenance relies on spatiotemporal regulation of organelle distribution between the somatodendritic and axonal domains.Although some organelles,such as mitochondria and smooth endoplasmic reticulum,are widely distributed throughout the neuron,others are segregated to either the somatodendritic or axonal compartment.For example,Golgi outposts and acidified lysosomes are predominantly present in the somatodendritic domain and rarely distributed along the axon,whereas newly formed autophagosomes and synaptic vesicles are mainly distributed in the distal axon(Britt et al.,2016).

Recent Advances of AIEgens for Targeted Imaging of Subcellular Organelles

Fluorescence imaging based on luminogens with aggregation-induced emission(AIE)effect has drawn great attention in recent two decades,due to their superior advantages to overcome the technical difficulties.Thus,the AIE-active bioprobes with targeted ability at the subcellular level have been widely investigated to visualize the subcellular structures and monitor the biological processes.Considering the very rapid developments and the significance of selective imaging of subcellular structures,we summarize the recent two-year achievements about the AIEgens for targeted imaging of subcellular organelles including nuclei,membranes,lipid droplets(LDs),endoplasmic reticulum(ER),lysosomes,mitochondria and cytoplasm.The designed protocols and advantages of AIEgens,their mechanisms for targeted staining at organelles and the imaging performance are discussed.These AIE bioprobes exhibit great potentials for early diagnosis and therapeutics of diseases that related to subcellular organelles.Finally,the perspectives about AIEgens for these applications are also discussed.

Phase-separated bienzyme compartmentalization as artificial intracellular membraneless organelles for cell repair

Implanting artificial organelles in living cells is capable of correcting cellular dysfunctionalities for cell repair and biomedical applications. In this work, phase-separated bienzyme-loaded coacervate microdroplets are established as a model of artificial membraneless organelles in endothelial dysfunctional cells for the cascade enzymatic production of nitric oxide(NO) with a purpose of correcting cellular NO deficiency. We prepared the coacervate microdroplets via liquid-liquid phase separation of oppositely charged polyelectrolytes, in which glucose oxidase/horseradish peroxidase-mediated cascade reaction was compartmented. After the coacervate microdroplets were implanted in NO-deficient dysfunctional cells, the compartments maintained a phase-separated liquid droplet structure, which facilitated a significant enhancement of NO production in the dysfunctional cells. The recovery of NO production was further exploited to inhibit clot formation in blood plasma located in the cell suspension. This demonstrated a proof-of-concept design of artificial organelles in dysfunctional cells for cell repair and anticoagulation-related medical applications. Our results demonstrate an approach for the construction of coacervate droplets through phase separation for the generation of artificial membraneless organelles, which can be designed to provide an array of functionalities in living organisms that have the potential to be used in the field of cell engineering and medical therapy.

Zbed3 participates in the subcortical maternal complex and regulates the distribution of organelles

We previously identified a subcortical maternal complex （SCMC） that is essential for early embryogenesis and female fertility in mice. However, the molecular mechanism by which the SCMC affects female fertility remains largely uncharacterized. Here, we report that a novel maternal protein, zinc finger BED-type containing 3 （Zbed3）, participates in the SCMC. Depletion of maternal Zbed3 results in reduced fecundity of females, because of the impaired and delayed development in a proportion of mutant embryos. The loss of maternal Zbed3 results in asymmetric zygotic division and abnormal distributions of organeUes in the affected oocytes and zygotes, similar to the phenotypes observed in females with disrupted core SCMC genes. Further investiga- tion revealed that these phenotypes are associated with disrupted dynamics of microtubules and/or formation of cytoplasmic lat- tices （CPLs）. The stability and localization of Zbed3 depend on, but are not required for, the formation of the SCMC. Thus, our data suggest Zbed3 as one of downstream proteins mediating SCMC functions and provide further insights into the roles of the SCMC and CPLs in female fertility.

Role of plant myosins in motile organelles: Is a direct interaction required?

Plant organelles are highly motile, with speed values of 3-71μm/s in cells of land plants and about 20-60 μm/s in characean algal cells. This movement is believed to be important for rapid distribution of materials around the cell, for the plant＇s ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organelles within plant cells is based on the actin cytoskeleton and its motor proteins the myosins.Most plants express multiple members of two main classes： myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in all kingdoms. Paradoxically, while it was found that myosins from class XI regulate most organelle movement, it is not quite clear how or even if these motor proteins attach to the organelles whose movement they regulate.

Group-specific functional patterns of mitochondrion-related organelles shed light on their multiple transitions from mitochondria in ciliated protists

Adaptations of ciliates to hypoxic environments have arisen independently several times.Studies on mitochondrion-related organelle(MRO)metabolisms from distinct anaerobic ciliate groups provide evidence for understanding the transitions from mitochondria to MROs within eukaryotes.To deepen our knowledge about the evolutionary patterns of ciliate anaerobiosis,mass-culture and single-cell transcriptomes of two anaerobic species,Metopus laminarius(class Armophorea)and Plagiopyla cf.narasimhamurtii(class Plagiopylea),were sequenced and their MRO metabolic maps were compared.In addition,we carried out comparisons using publicly available predicted MRO proteomes from other ciliate classes(i.e.,Armophorea,Litostomatea,Muranotrichea,Oligohymenophorea,Parablepharismea and Plagiopylea).We found that single-cell transcriptomes were similarly comparable to their mass-culture counterparts in predicting MRO metabolic pathways of ciliates.The patterns of the components of the MRO metabolic pathways might be divergent among anaerobic ciliates,even among closely related species.Notably,our findings indicate the existence of group-specific functional relics of electron transport chains(ETCs).Detailed group-specific ETC functional patterns are as follows:full oxidative phosphorylation in Oligohymenophorea and Muranotrichea;only electron-transfer machinery in Armophorea;either of these functional types in Parablepharismea;and ETC functional absence in Litostomatea and Plagiopylea.These findings suggest that adaptation of ciliates to anaerobic conditions is group-specific and has occurred multiple times.Our results also show the potential and the limitations of detecting ciliate MRO proteins using single-cell transcriptomes and improve the understanding of the multiple transitions from mitochondria to MROs within ciliates.

Cryopreserved Fibroblast and Mesenchymal Stem Cells (MSCs) Being Alternative Mitochondrial Donors for Mitochondrial Organelle Transplantation (MOT)

Mitochondrial organelle transplantation (MOT) is an innovative strategy for the treatment of mitochondrial dysfunction such as cardiac ischemic reperfusion injuries, traumatic brain and spinal cord injuries, cerebral stroke, and neurodegenerative diseases. The earlier MOT results in better efficacy in animal models of urgent diseases such as ischemic stroke, and traumatic brain and spinal cord injuries. There is no long-term method to preserve mitochondria. Routine MOT procedure from cell growth to mitochondrial injection often takes serval weeks and is not satisfactory for urgent use cases. Hypothesis: Cryopreserved cells might be mitochondrial donors for MOT. Methods: We isolated mitochondria from cryopreserved human fibroblasts and mesenchymal stem cells (MSCs) in cell banks and compared the mitochondrial viability and transplantation with the mitochondria from fresh cells. Key findings: We found that mitochondria from fresh and cryopreserved cells are comparable in mitochondrial viability and transplantation. We also obtained data showing that mitochondria of fibroblasts and MSCs had similar membrane potential and transfer ability, but MSC’s mitochondria had higher ATP content than fibroblast’s mitochondria. In addition, oxygen consumption rates (OCRs) were higher in MSC’s mitochondria compared to fibroblast’s mitochondria and did not change between fresh and frozen cells. Conclusion: Cryopreserved fibroblasts and MSCs are alternative mitochondrial donors for MOT to fresh cells. MSCs could provide higher ATP-produced mitochondria than fibroblasts.

The BLOC Interactomes Form a Network in Endosomal Transport

With the identification of more than a dozen novel Hermansky-Pudlak Syndrome （HPS） proteins in vesicle trafficking in higher eukaryotes, a new class of trafficking pathways has been described. It mainly consists of three newly-defined protein com- plexes, BLOC-l, -2, and -3. Compelling evidence indicates that these complexes together with two other well-known complexes, AP3 and HOPS, play important roles in endosomal transport. The interactions between these complexes form a network in protein trafficking via endosomes and cytoskeleton. Each node of this network has intra-complex and extra-complex interactions. These complexes are connected by direct interactions between the subunits from different complexes or by indirect interactions through coupling nodes that interact with two or more subunits from different complexes. The dissection of this network facilitates the understanding of a dynamic but elaborate transport machinery in protein/membrane trafficking. The disruption of this network may lead to abnormal trafficking or defective organellar development as described in patients with Hermansky-Pudlak syndrome.

Ultrastructural Study of Secondary Wall Formation in the Stem Fiber of Phyllostachys pubescens

Ultrastructural changes in secondary wall formation of Phyllostachys pubescens Mazel fiber were investigated with transmission electron microscopy. Fiber developed initially with the elongation of cells containing ribosomes, mitochondria and Golgi bodies in the dense cytoplasm. During the wall thickening, the number of rough endoplasmic reticulum and Golgi bodies increased apparently. There were two kinds of Golgi vesicles, together with the ones from endoplasmic reticulum formed transport vesicles. Many microtubules were arranged parallel to the long axis of the cell adjacent to the plasmalemma. Along with the further development of fiber, polylamellate structure of the secondary wall appeared, with concurrent agglutination of chromatin in the nucleus, swelling and disintegration of organelles, while cortical microtubules were still arranged neatly against the inner side of plasmalemma. Lomasomes could be observed between the wall and plasmalemma. The results indicated that the organelles, such as Golgi bodies together with small vesicles, rough endoplasmic reticulum and lomasomes, played the key role in the thickening and lignification of the secondary wall of bamboo fiber, though cortical microtubules were correlative with the process as well.

Biological Effects of Stevia rebaudianum Induced by Carbon Ion Implantation

The biological effects during seed germination were investigated after the dry seeds of Stevia rebaudianum Bertoni were implanted with carbon ion beam of 75 keV and 10 14 ions/cm 2. The results showed that the germination rate of carbon ion implanted seeds was slightly higher than that of the control, but the survival rate of the treated seedlings, on the contrary, was lower than that of the control (P<0.02), while the height of the treated seedlings was significantly higher than that of the control (P<0.01). On the 4th day after germination, the leaf cell wall in the treated group was thick, some high electron_dense substance deposited in the enlarged plasmodesma; Cell membrane creased with high electron_dense granules deposited on it. The plasma membrane protruded towards cell wall, and the granules shifted via plasmodesma or deposited onto cell wall. These phenomena may be related to the conveyance of implanted ions across cell wall, or be related to the accumulation of callose. In addition, the implantation of carbon ions could increase the lamellae of the chloroplast and cause high development of the chloroplast which sometimes contained two plastid centers in an individual chloroplast. Also, the highly developed cristae, abundant mitochondria and typical crystalloid structure in microbody could be found. All these results indicated that the anabolic and catabolic activities in the seedlings implanted with carbon ions before germination were obviously more active than those in the controls.

Analysis on the Relationship between Selective Pressure and Nucleotide Substitution Rate in Nucleus and Organelle Genes of Sorghum and Maize

[Objective]The aim was to research the relationship between nucleotide substitutions rate and selective pressure.[Method]Synonymous and nonsynonymous substitutions and their ratios for some sorghum and maize genes in nucleus and organelle genomes were analyzed by statistical method,and comparative analysis of related functional genes were carried out.[Result]The pure selective pressures of the related functional genes were similar between nucleus and chloroplast genomes,but was lower in mitochondrial genome.The significant differences of nucleotide substitution rate between sorghum and maize orthologous genes in nucleus genome,and among different functional genes in nucleus genome were mainly due to the nonsynonymous substitution difference.[Conclusion]The molecular evolutional rate of different functional genes and different lineages were influenced by selective pressure.The differences of molecular evolutional rate among nucleus,chloroplast and mitochondria genomes had no direct relationship with selective pressure.

Subcellular Distribution of Calcium, Magnesium and Iron in Brassica napus Leaves during Cold Acclimatization

[Objective] The aim of the present study was to investigate the characteristics of calcium （Ca）, magnesium （Mg） and iron （Fe） subcellular distributions in oilseed rape （Brassica napus L.） leaves during cold acclimatization. [Method] A field experiment was conducted at two contrasting environments with three replicates and included a freezing sensitive variety （cv. Zhongshuang No. 11, ZS） and a freezing tolerant variety （cv. Ganyouza No. 1, GY）. [Result] The concentrations of Ca, Mg and Fe were significantly increased in plants roots, and the concentrations of Ca and Mg were significantly decreased but the Fe concentration was significantly increased in plants shoots. In leaves, the Ca concentration stored in soluble fraction was significantly increased and the Ca concentration of organelle-contained fraction and cell wall-associated fraction were significantly decreased with experiment time. The Mg concentrations stored in soluble fraction, organelle-contained fraction and cell wall-associated fraction were decreased with time. In contrary, the Fe concentration stored in soluble fraction, organelle-contained fraction and cell wall associated-fraction was significantly increased with time. Meanwhile, the changes of these measures of ZS were much higher than those of GY during cold acclimatization, which might be due to the GY is a freezing tolerant variety but ZS is not. [Conclusion] These results suggested that increasing the concentrations of Ca, Mg and Fe in plants shoots might increase the cold resistance of oilseed rape.

Phosphatidylglycerol-containing ER-transport vesicles built and restore outer mitochondrial membrane and deliver nuclear DNA translation products to generate cardiolipin in the inner mitochondrial membrane

Phosphatidylglycerol (PG) an important membrane phospholipid required for the synthesis of diphos-phatidylglycerol (DPG) commonly known as cardiolipin (CL) was identified in the fraction of endo-plasmic reticulum (ER)-derived transport vesicles which had no affinity for Golgi. The vesicles were produced in the presence of Brefeldin A (BFA), the agent known to inhibit ER-Golgi transport, and found to display affinity to mitochondria. The analysis revealed that their cargo was not containing proteins that are transported to Golgi, and that their membrane was free of phosphatidylinositol (PI) and ceramides (Cer). The incubation of PG-containing transport vesicles with mitochondria afforded incorporation of their membrane into the Outer Mito-chondrial Membrane (OMM) and formation of lyso-phosphatidylglycerol (LPG). In turn, upon further incubation with fresh transport active cytosol, the mitochondrial LPG was converted to PG. The results of analysis of the OMM, Inner Mitochondrial Mem-brane (IMM) and Inner Mitochondrial Space Components (IMSC) strongly suggest that PG-containing transport vesicles deliver nuclear DNA translation products to the IMSC and thus facilitate CL synthesis in the IMM. In summary, our studies provide evidence that ER-generated PG-enriched transport vesicles represent the general pathway for restitution of mitochondrial membranes and the delivery of nuclear DNA translation products that generate CL, and thus sustain the mitochondrial matrix CL-dependent metabolic reactions.

Label-free Super-resolution Microscopy for Long-term Monitoring the Dynamic Interactions of Cellular Organelles

The ideal method for imaging living cells is one that allows for long-term,label-free observation in real-time with super-resolution capabilities.Such a method would overcome the drawbacks of phototoxicity and photobleaching associated with fluorescence labeling microscopy.Fourier ptychography is a promising label-free imaging technique that surpasses the diffraction limit of conventional microscopy while avoiding issues related to fluorescent labeling.However,previous Fourier ptychography microscopy (FPM) systems lacked high spatiotemporal resolution,preventing real-time observation of subcellular organelle structures in living cells.To address this limitation,we have developed a high-speed super-resolution microscope based hemispherical digital illumination (HDI) device.This system utilizes 61 light-emitting diodes (LEDs) to provide high-angle illumination with a numerical aperture (NA) of 0.98.By employing a 40×/0.6 NA objective lens,we have achieved lateral resolutions of around 150 nm,enabling us to capture images at a speed of over 1 Hz with the field of view measuring 118×118 μm2.HDI-FPM allows for the monitoring of various cellular processes,such as vesicular transportation,mitochondrial fusion and division,as well as cell-cell fusion over a duration of 4 h.The development of HDI-FPM represents a significant advancement in label-free imaging of living cells.It offers a comprehensive understanding of the mechanisms underlying cellular activities.

Reconstruction of liver organoid using a bioreactor

AIM： To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver. METHODS： We previously reported that a radial-flow bioreactor （RFB） could provide a three-dimensional highdensity culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line （FLC-5） as hepatocytes together with mouse immortalized sinusoidal endothelial cell （SEC） line M1 and mouse immortalized hepatic stellate cell （HSC） line A7 as non parenchymal cells in the RFB. Two × 10^7 FLC-5 cells were incubated in the RFB. After 5 d, 2 × 10^7 A7 cells were added in a similar manner followed by another addition of 10^7 M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy （TEM） and scanning electron microscopy （SEM）. To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB. RESULTS： By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles （WO） and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased. CONCLUSION： Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.

Glyphosate exposure deteriorates oocyte meiotic maturation via induction of organelle dysfunctions in pigs

Background:Recently,defects in mammalian oocytes maturation induced by environmental pollution results in the decreasing animal reproduction.Animal exposed to glyphosate is largely unavoidable because glyphosate is one of the most widely used herbicide worldwide due to its high-efficiency and broad-spectrum effects,which causes glyphosate an environmental contaminant found in soil,water and food.During the last few years,the growing and wider use of glyphosate has raised great concerns about its effects of reproductive toxicity.In this study,using porcine models,we investigated effects of glyphosate on organelle functions during oocyte meiosis.Results:The results showed glyphosate exposure disrupted porcine oocyte maturation.Expression levels of cumulus expansion-related genes were interfered,further indicating the meiotic defects.The damaging effects were mediated by destruction of mitochondrial distribution and functions,which induced ROS accumulation and oxidative stress,also indicated by the decreased mRNA expression of related antioxidant enzyme genes.We also found an interference of endoplasmic reticulum(ER)distribution,disturbance of Ca^(2+)homeostasis,as well as fluctuation of ER stress,showing with the reduced ER stress-related mRNA or protein expression,which could indicate the dysfunction of ER for protein processing and signal transduction in glyphosate-exposed oocytes.Moreover,glyphosate exposure induced the disruption of lysosome function for autophagy,showing with the decrease of LAMP2 expression and autophagy-related genes mRNA expression.Additionally,our data showed the distribution of Golgi apparatus and the functions of ribosome were disturbed after glyphosate exposure,which might affect protein synthesis and transport.Conclusions:Collectively,our study showed that exposed to glyphosate could affect animal reproduction by compromising the quality of oocytes through its wide toxic effects on organelle functions.

Lipid-Protein Microinclusions in the Morphological Structures of Organelle Membranes Studied by Fluorescent Confocal Microscopy

Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.

Protective Actions of Blumea Flavanones on Primary Cultured Hepatocytes and Liver Subcellular Organelle against Lipid Peroxidation

To search for the protective actions of blumea flavanones (BFs) on hepatocytes and hepatic subcellular organelle against lipid peroxidation, monkey′s hepatocytes were isolated and cultured with or without blumea flavanones, then damaged by FeSO 4 cysteine or CCl 4. The lipid peroxidation (malondialdehyde production) and alteration in hepatocyte membrane (leakage of GPT) were estimated. Hepatic subcellular organelles were also isolated and incubated with or without blumea flavanones, then injured by FeSO 4 ascorbate. The generation of malondialdehyde(MDA) was measured. It was found that BFs 10 and 100 μmol·L 1 inhibited the MDA generation and GPT (glutamic pyruvic transaminase) leakage out of hepatocytes that were induced by CCl 4 or FeSO 4 cysteine. BFs could prevent lipid peroxidation initiated by FeSO 4 ascorbate in subcellular organelle suspension. Among BFs, BF 2 possessed the strongest activity. Conclusion: Blumea flavanones possess antioxidation activities that protect monkey′s hepatocytes and hepatic subcellular organelle against injuries induced by FeSO4 or CCl 4.