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To manufacture composites with required properties, it is extremely important to select an appropriate filler. Carbon-based nano materials such as carbon black, graphene and carbon nanotube (CNT) have been extensively investigated as reinforcing and conducting fillers. Because of their high aspect ratio coupled with superior physical properties, 1-dimensional CNTs are ideal as filler materials to impart electrical conductivity to insulating polymers, while enhancing mechanical strength. In this study, we investigated the piezo-resistive and mechanical properties of composites consisting of two types of CNT classified as multi-walled CNT (MWNT) or single-walled CNT (SWNT) depending on the number of walls. Since MWNT and SWNT have different physical properties such as specific surface area and aspect ratio, this can affect the composite’s performance. To more effectively evaluate the effect of MWNTs and SWNTs in composites, we used thermoplastic polyurethane (TPU) as a matrix, which is an insulating stretchable elastomer. Morphological and mechanical/electrical characterizations were conducted to determine differences in the MWNT and SWNT composites. In addition, we conducted dynamic strain sensing tests on each type of CNT composites to compare the sensitivity as a strain sensor. Differences in piezo-resistive behaviors were attributed to the loss of electrical contact points during stretching. These results can serve as a useful design guideline for the wider use of CNT composites.

Border-like cells (BLCs) are sheets of cells that are continuously sloughed off and replenished at the Arabidopsis root cap surface.ROOT CAP POLYGALACTURONASE (RCPG)encodes a putative pectinase involved in BLC shedding. Xylogalacturonan (XGA) is a pectic polysaccharide whose synthesis is associated with cell detachment and secreted separately from other cell wall polysaccharides.BEARSKIN1 (BRN1)andBRN2areArabidopsisNAC family transcription factors, andRCPGexpression is inhibited inbrn1/2. To explore the link between XGA and RCPG, we examined XGA synthesis inArabidopsislines with altered RCPG levels. We found that RCPG was contained in XGA-carrying vesicles budding from thetrans-Golgi, but XGA synthesis was not affected in thercpgmutant. XGA was absent in BLCs ofbrn2, but not ofbrn1, indicating thatBRN2is necessary for XGA synthesis. Overexpression of functional RCPG-GFP (oeRCPG-GFP) caused upregulation ofBRN2, ectopic XGA synthesis, overaccumulation of endogenous RCPG, and accelerated BLC turnover, suggesting a positive regulatory loop between RCPG and BRN2. Inactivation ofBRN2inoeRCPG-GFPsuppressed RCPG-GFP expression, excess RCPG, and XGA synthesis. Our data provide evidence that XGA and RCPG are secreted together and that BRN2 controls XGA synthesis, which facilitates RCPG export and BLC separation.

In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

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.

Bacterial cell morphology might result from natural selection to gain a competitive advantage under environmentally stressful conditions such as nutrient limitation. A bacterial strain YC6860T isolated from the rhizosphere of rice (Oryza sativa L.) showed pleomorphic behavior with smooth cell morphology and wrinkled surface rods depending upon nutritional conditions. Based on scanning and transmission electron microscopy studies, we hypothesized that the surface-to-volume ratio of cells increases with decreasing nutrient concentrations. The transition from smooth to wrinkled cell surface morphology could be one of the adaptation strategies by which YC6860T maximizes its ability to access available nutrients. To characterize the properties of the wrinkled strain, we performed taxonomic and phylogenetic analyses. 16S rRNA gene sequencing results showed that the strain represented a novel, deep-rooting lineage within the order Rhizobiales with the highest similarity of 94.2% to Pseudorhodoplanes sinuspersici RIPI 110T. Whole genome sequencing was also performed to characterize its genetic features. The strain YC6860T might belong to a new genus, named Rugositalea, and a new species, named Rugositalea oryzae, In addition, taxonomic analysis showed that YC6860T is Gram-negative, aerobic, and rod-shaped with large regular wrinkles resembling a delicate twist of fusilli, measuring 0.5-0.6 µm in width and 1.5-1.6 µm in length under nutrient-limiting conditions. This unique cell structure with regular rugosity could be the first finding that has not been reported in the existing bacterial morphology.

The root cap is a multi-layered tissue covering the tip of a plant root that directs root growth through its unique functions such as gravity-sensing and rhizosphere interaction. To prevent damages from the soil environment, cells in the root cap continuously turn over through balanced cell division and cell detachment at the inner and the outer cell layers, respectively. Upon displacement toward the outermost layer, columella cells at the central root cap domain functionally transition from gravity-sensing cells to secretory cells, but the mechanisms underlying this drastic cell fate transition are largely unknown. By using live-cell tracking microscopy, we here show that organelles in the outermost cell layer undergo dramatic rearrangements, and at least a part of this rearrangement depends on spatiotemporally regulated activation of autophagy. Notably, this root cap autophagy does not lead to immediate cell death, but rather is necessary for organized separation of living root cap cells, highlighting a previously undescribed role of developmentally regulated autophagy in plants.Summary statementTime-lapse microscope imaging revealed spatiotemporal dynamics of intracellular reorganization associated with functional transition and cell separation in the Arabidopsis root cap and the roles of autophagy in this process.

The para-crystalline structure of prolamellar bodies (PLBs) and light-induced etioplasts-to-chloroplasts transformation have been investigated with electron microscopy methods. However, these studies suffer from chemical fixation artifacts and limited volumes of three-dimensional reconstruction. We have examined Arabidopsis thaliana cotyledon cells with electron tomography (ET) to visualize etioplasts and their conversion into chloroplasts. We employed the scanning mode of ET for imaging 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 hours after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form thylakoid sheets, a.k.a. pre-granal thylakoids. These pre-granal thylakoids in PLB’s vicinity folded and overlapped with each other to create grana stacks. Since the nascent pre-granal thylakoids had curved membranes in their tips, we examined the expression and localization of CURT1 proteins. CURT1A transcript was most abundant in de-etiolating cotyledon samples, and CURT1A concentrated to the PLB periphery. In curt1a etioplasts, PLB-associated thylakoids were swollen and failed to form grana stacks. By 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 the cubic crystal growth under darkness.

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, mechanisms of autophagosome maturation, i.e., delivery and fusion with the vacuole, remain largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1’s function is evolutionarily conserved in plants as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by connecting autophagosomes with the ESCRT-I component VPS23, leading to the formation of amphisomes. Disrupting the VPS23-CFS1 interaction affects autophagic flux and renders plants sensitive to starvation stress. Altogether, our results reveal a deeply conserved mechanism of vacuolar delivery in plants that is mediated by amphisomes.

The crystalline structure of prolamellar bodies (PLBs) and light-induced etioplasts-to-chloroplasts transformation have been investigated with electron microscopy methods. However, these studies suffer from chemical fixation artifacts and limited volumes of tomographic reconstruction. We have examined Arabidopsis thaliana cotyledon samples preserved by high-pressure freezing with scanning transmission electron tomography to visualize larger volumes in etioplasts and their conversion into chloroplasts. PLB tubules were arranged in a zinc blende-type lattice like carbon atoms in diamonds. Within 2 hours after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form fenestrated sheets that eventually matured into lamellar thylakoids. These planar thylakoids emerging from PLBs overlapped or folded into grana stacks in PLBs’ vicinity. Since the nascent lamellae had curved membrane at their tips, we examined the localization of CURT1 proteins. CURT1A transcript was most abundant in de-etiolating cotyledon samples, and CURT1A concentrated at the peripheral PLB. In curt1a mutant etioplasts, thylakoid sheets were swollen and failed to develop stacks. In curt1c mutant, however, PLBs had cracks in their lattices, indicating that CURT1C contributes to cubic crystal growth under darkness. Our data provide evidence that CURT1A and CURT1C play distinct roles in the etioplast and chloroplast biogenesis.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD–RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories., Metabolic enzymes often form supramolecular complexes to improve product yield. Here the authors use short peptide tags to create scaffold-free assemblies and synthetic metabolic nodes.

Exogenous metabolites from microbial and dietary origins have profound effects on host metabolism. Here, we report that a sub-population of lipid droplets (LDs), which are conserved organelles for fat storage, is defined by metabolite-modulated targeting of the C. elegans seipin ortholog, SEIP-1. Loss of SEIP-1 function reduces the size of a subset of LDs while over-expression of SEIP-1 has the opposite effect. Ultrastructural analysis reveals SEIP-1 enrichment in an endoplasmic reticulum (ER) subdomain, which co-purifies with LDs. Analyses of C. elegans and bacterial genetic mutants indicate a requirement of polyunsaturated fatty acids (PUFAs) and microbial cyclopropane fatty acids (CFAs) for SEIP-1 enrichment, as confirmed by dietary supplementation experiments. In mammalian cells, heterologously expressed SEIP-1 engages nascent lipid droplets and promotes their subsequent expansion in a conserved manner. Our results suggest that microbial and polyunsaturated fatty acids serve unexpected roles in regulating cellular fat storage by promoting LD diversity., Lipid droplets (LDs) are fat storage organelles that are initiated and expanded by seipins at ER contact sites. Here the authors show that the C. elegans seipin ortholog SEIP-1 is recruited to these sites by certain dietary fatty acids to support the expansion of a subset of LDs.

Structural analyses of organelles and localization of proteins in their confines are essential to investigate inner workings of eukaryotic cells. Electron tomography (ET) is a three-dimensional electron microscopy method with which we can extract sliced views of organelles from any direction and quantify their structural parameters at nanometer-level resolution. This advanced electron microscopy tool is suited for characterization of convoluted membrane compartments and of cellular constituents of dimensions smaller than 100 nm. ET studies of plant cells fixed by rapid freezing have expanded our understanding of the biogenesis and functions of plant organelles. Here we describe how the molecular imaging capacity of correlative light and electron microscopy can be integrated with ET in studies of plant organelles.

Plant vacuoles are dynamic organelles that play essential roles in regulating growth and development. Two distinct models of vacuole biogenesis have been proposed: separate vacuoles are formed by the fusion of endosomes, or the single interconnected vacuole is derived from the endoplasmic reticulum. These two models are based on studies of two-dimensional (2D) transmission electron microscopy and 3D confocal imaging, respectively. Here, we performed 3D electron tomography at nanometre resolution to illustrate vacuole biogenesis in Arabidopsis root cells. The whole-cell electron tomography analysis first identified unique small vacuoles (SVs; 400–1,000 nm in diameter) as nascent vacuoles in early developmental cortical cells. These SVs contained intraluminal vesicles and were mainly derived/matured from multivesicular body (MVB) fusion. The whole-cell vacuole models and statistical analysis on wild-type root cells of different vacuole developmental stages demonstrated that central vacuoles were derived from MVB-to-SV transition and subsequent fusions of SVs. Further electron tomography analysis on mutants defective in MVB formation/maturation or vacuole fusion demonstrated that central vacuole formation required functional MVBs and membrane fusion machineries. The plant vacuole plays important roles in regulating growth and development. Now, the vacuole in Arabidopsis root cells is presented at nanometre resolution by 3D whole-cell tomography, suggesting a cellular mechanism for plant vacuole formation.

Chloroplast development is a complex process that is critical to the growth and development of plants. However, the detailed mechanism of chloroplast development in woody plants remains unclear. In this study, we showed that chloroplasts with elaborate thylakoids could develop from proplastids in the cells of calli derived from leaf tissues of Populus tomentosa upon exposure to light. Chloroplast development was confirmed at the molecular and cellular levels. Transcriptome analysis revealed that genes related to photoreceptors and photosynthesis were significantly up-regulated during chloroplast development in a time-dependent manner. In light-induced chloroplast development, a key process was the removal of hydrogen peroxide, in which thylakoid-localized PtotAPX played a major role; light-induced chloroplast development was enhanced in PtotAPX-overexpressing transgenic P. tomentosa callus with lower levels of hydrogen peroxide, but was suppressed in PtotAPX antisense transgenic callus with higher levels of hydrogen peroxide. Moreover, the suppression of light-induced chloroplast development in PtotAPX antisense transgenic callus was relieved by the exogenous reactive oxygen species scavenging agent N,N′-dimethylthiourea (DMTU). Based on these results, we propose that PtotAPX-mediated removal of reactive oxygen species plays a key role in chloroplast development from proplastids upon exposure to light in P. tomentosa.

Plasmodesmata are intercellular channels that facilitate molecular diffusion between neighboring plant cells. The development and functions of plasmodesmata are controlled by multiple intra- and intercellular signaling pathways. Plasmodesmata are critical for dual-cell C4 photosynthesis in maize because plasmodesmata at the mesophyll and bundle sheath interface mediate exchange of CO2-carrying organic acids. We examined developmental profiles of plasmodesmata and chloroplasts in the maize leaf from young cells in the base to mature cell in the tip using microscopy approaches. Young mesophyll and bundle sheath cells in the leaf base had proplastids, and their plasmodesmata were simple, devoid of cytoplasmic sleeves. In maturing cells where Kranz anatomy and dimorphic chloroplasts were evident, we observed extensive remodeling of plasmodesmata that included acquisition of an electron-dense ring on the mesophyll side and cytoplasmic sleeves on the bundle sheath side. Interestingly, the changes in plasmodesmata involved a drop in symplastic dye mobility and suberin accumulation in the cell wall, implying a more stringent mesophyll-bundle sheath transport. We compared kinetics of the plasmodesmata and the cell wall modification in wildtype leaves with leaves from ppdk and dct2 mutants with defective C4 pathways. Plasmodesmata development, symplastic transport inhibition, and cell wall suberization were accelerated in the mutant lines, probably due to the aberrant C4 cycle. Transcriptomic analyses of the mutants confirmed the expedited changes in the cell wall. Our results suggest that a regulatory machinery at the mesophyll-bundle sheath boundary suppresses erroneous flux of C4 metabolites in the maize leaf.Significance StatementPlasmodesmata in the maize Kranz anatomy mediate the exchange of organic acids between mesophyll and bundle sheath. Since solute diffusion through plasmodesmata is governed by solute concentration gradients, a balanced distribution of C4 metabolites is critical for concentration of CO2 in the bundle sheath. Plasmodesmata bridging the mesophyll and bundle sheath cytoplasm have a cylindrical cavity, which can facilitate molecular movements, and a valve-like attachment. Construction of the sophisticated plasmodesmata was linked to C4 photosynthesis, and plasmodesmata assembly finished more rapidly in maize mutants with defective C4 pathways than in wild-type plants. These results suggest that the specialized plasmodesmata contribute to controlled transport of C4 metabolites.

Plants adapt to heatviathermotolerance pathways in which the activation of protein folding chaperones is essential. In eukaryotes, protein disulfide isomerases (PDIs) facilitate the folding of nascent and misfolded proteins in the secretory pathway by catalyzing the formation and isomerization of disulfide bonds and serving as molecular chaperones. In Arabidopsis, several members of the PDI family are upregulated in response to chemical inducers of the unfolded protein response (UPR), including both members of the non-classical PDI-M subfamily, PDI9 and PDI10. Unlike classical PDIs, which have two catalytic thioredoxin (TRX) domains separated by two non-catalytic TRX-fold domains, PDI-M isoforms are orthologs of mammalian P5/PDIA6 and possess two tandem catalytic domains. Here, PDI9 accumulation was found to be upregulated in pollen in response to heat stress. Histochemical staining of plants harboring thePDI9andPDI10promoters fused to thegusAgene indicated they were actively expressed in the anthers of flowers, specifically in the pollen and tapetum. Immunoelectron microscopy revealed that PDI9 localized to the endoplasmic reticulum in root and pollen cells. transfer DNA (T-DNA) insertional mutations in thePDI9gene disrupted pollen viability and development in plants exposed to heat stress. In particular, the pollen grains ofpdi9mutants exhibited disruptions in the reticulated pattern of the exine and an increased adhesion of pollen grains. Pollen in thepdi10single mutant did not display similar heat-associated defects, butpdi9 pdi10double mutants (DMs) completely lost exine reticulation. Interestingly, overexpression ofPDI9partially led to heat-associated defects in the exine. We conclude that PDI9 plays an important role in pollen thermotolerance and exine biogenesis. Its role fits the mechanistic theory of proteostasis in which an ideal balance of PDI isoforms is required in the endoplasmic reticulum (ER) for normal exine formation in plants subjected to heat stress.

International audience; Lipid droplets (LDs) are intracellular organelles found in a wide range of organisms and play important roles in stress tolerance. During nitrogen (N) starvation, Chlamydomonas reinhardtii stores large amounts of triacylglycerols (TAGs) inside LDs. When N is resupplied, the LDs disappear and the TAGs are degraded, presumably providing carbon and energy for regrowth. The mechanism by which cells degrade LDs is poorly understood. Here, we isolated a mutant ( dth1-1 , Delayed in TAG Hydrolysis 1) in which TAG degradation during recovery from N starvation was compromised. Consequently, the dth1-1 mutant grew poorly compared to its parental line during N recovery. Two additional independent loss-of-function mutants ( dth1-2 and dth1-3 ) also exhibited delayed TAG remobilization. DTH1 transcript levels increased sevenfold upon N resupply, and DTH1 protein was localized to LDs. DTH1 contains a putative lipid-binding domain (DTH1$^{LBD}$) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A–I. Recombinant DTH1$^{LBD}$ bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1$^{LBD}$ in Chlamydomonas phenocopied the dth1 mutant’s defective TAG degradation, suggesting that the function of DTH1 depends on its ability to bind PE. Together, our results demonstrate that the lipid-binding DTH1 plays an essential role in LD degradation and provide insight into the molecular mechanism of protein anchorage to LDs at the LD surface in photosynthetic cells.

Lipid droplets (LDs) are intracellular organelles found in a wide range of organisms and play important roles in stress tolerance. During nitrogen (N) starvation, Chlamydomonas reinhardtii stores large amounts of triacylglycerols (TAGs) inside LDs. When N is resupplied, the LDs disappear and the TAGs are degraded, presumably providing carbon and energy for regrowth. The mechanism by which cells degrade LDs is poorly understood. Here, we isolated a mutant (dth1-1, Delayed in TAG Hydrolysis 1) in which TAG degradation during recovery from N starvation was compromised. Consequently, the dth1-1 mutant grew poorly compared to its parental line during N recovery. Two additional independent loss-of-function mutants (dth1-2 and dth1-3) also exhibited delayed TAG remobilization. DTH1 transcript levels increased sevenfold upon N resupply, and DTH1 protein was localized to LDs. DTH1 contains a putative lipid-binding domain (DTH1(LBD)) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A–I. Recombinant DTH1(LBD) bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1(LBD) in Chlamydomonas phenocopied the dth1 mutant’s defective TAG degradation, suggesting that the function of DTH1 depends on its ability to bind PE. Together, our results demonstrate that the lipid-binding DTH1 plays an essential role in LD degradation and provide insight into the molecular mechanism of protein anchorage to LDs at the LD surface in photosynthetic cells.

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 ATG5 dependent and labelled by ATG8 proteins. Friendly, a member of the Clustered Mitochondria protein family, is recruited to the damaged mitochondria to mediate mitophagy. In addition to stress, mitophagy is also induced during de-etiolation, a major cellular transformation during photomorphogenesis that involves chloroplast biogenesis. De-etiolation triggered mitophagy regulates cotyledon greening, pointing towards an inter-organellar cross-talk mechanism. Altogether our results demonstrate how plants employ mitophagy to recycle damaged mitochondria during stress and development.

The plant trans-Golgi network (TGN) is a multifunctional organelle derived from the Golgi. It consists of tubulovesicular compartments scattered in the cytosol. They produce secretory vesicles delivering proteins and polysaccharides to the cell wall. They also serve as early endosomal compartments, receiving endocytic cargos from the plasma membrane. This versatility is thought to originate from functional variations among individual TGN compartments. Correlative light and electron microscopy (CLEM) combines the imaging capability of light microscopy and electron microscopy (EM) to determine the location of macromolecules in EM images in the cellular context. It is possible to identify organelles associated with specific fluorescent markers and examine their membrane architectures at nanometer-level resolutions using CLEM. In this chapter, we will explain the CLEM method that our lab uses to investigate functional and structural heterogeneity among individual TGN compartments in plant cells.

C4 plants enhance photosynthesis efficiency by concentrating CO2 to the site of Rubisco action. Chloroplasts in C4 plants exhibit structural dimorphism because thylakoid architectures vary depending on energy requirements. Advances in electron microscopy imaging capacity and sample preparation technologies allowed characterization of thylakoid structures and their macromolecular arrangements with unprecedented precision mostly in C3 plants. The thylakoid is assembled during chloroplast biogenesis through collaboration between the plastid and nuclear genomes. Recently, the membrane dynamics involved in the assembly process has been investigated with 3D electron microscopy, and molecular factors required for thylakoid construction have been characterized. The two classes of chloroplasts in C4 plants arise from common precursors, but little is known about how a single type of chloroplasts grow, divide, and differentiate to mature into distinct chloroplasts. Here, we outline the thylakoid structure and its assembly processes in C3 plants to discuss ultrastructural analyses of dimorphic chloroplast biogenesis in C4 plant species. Future directions for electron microscopy research of C4 photosynthetic systems are also proposed.

Germination requires sufficient water absorption by seeds, but excessive water in the soil inhibits plant growth. We therefore hypothesized that tolerance mechanisms exist that help young seedlings survive and develop in waterlogged conditions. Many ATP-BINDING CASSETTE TRANSPORTER subfamily G (ABCG) proteins protect terrestrial plants from harsh environmental conditions. To establish whether any of these proteins facilitate plant development under waterlogged conditions, we observed the early seedling growth of many ABCG transporter mutants under waterlogged conditions. abcg5 seedlings exhibited severe developmental problems under waterlogged conditions: the shoot apical meristem was small, and the seedling failed to develop true leaves. The seedlings had a high water content and reduced buoyancy on water, suggesting that they were unable to retain air spaces on and inside the plant. Supporting this possibility, abcg5 cotyledons had increased cuticle permeability, reduced cuticular wax contents, and a much less dense cuticle layer than the wild-type. These results indicate that proper development of plants under waterlogged conditions requires the dense cuticle layer formed by ABCG5 activity.

Chromatin is the main carrier of genetic information and is non-randomly distributed within the nucleus. Next-generation sequence-based chromatin conformation capture technologies have enabled us to directly examine its three-dimensional organization at an unprecedented scale and resolution. In the best-studied mammalian models, chromatin folding can be broken down into three hierarchical levels, compartment, domains, and loops, which play important roles in transcriptional regulation. Although similar structures have now been identified in plants, they might not possess exactly the same functions as the mammalian ones. Here, we review recent Hi-C studies in plants, compare plant chromatin structures with their mammalian counterparts, and discuss the differences between plants with different genome sizes.

The plant trans-Golgi network (TGN) is a multifunctional organelle derived from the Golgi. It consists of tubulovesicular compartments scattered in the cytosol. They produce secretory vesicles delivering proteins and polysaccharides to the cell wall. They also serve as early endosomal compartments, receiving endocytic cargos from the plasma membrane. This versatility is thought to originate from functional variations among individual TGN compartments. Correlative light and electron microscopy (CLEM) combines the imaging capability of light microscopy and electron microscopy (EM) to determine the location of macromolecules in EM images in the cellular context. It is possible to identify organelles associated with specific fluorescent markers and examine their membrane architectures at nanometer-level resolutions using CLEM. In this chapter, we will explain the CLEM method that our lab uses to investigate functional and structural heterogeneity among individual TGN compartments in plant cells.

Bienertia sinuspersici is a single-cell C4 plant species of which chlorenchyma cells have two distinct groups of chloroplasts spatially segregated in the cytoplasm. The central vacuole encloses most chloroplasts at the cell center and confines the rest of the chloroplasts near the plasma membrane. Young chlorenchyma cells, however, do not have large vacuoles and their chloroplasts are homogenous. Therefore, maturing Bienertia chlorenchyma cells provide a unique opportunity to investigate chloroplast proliferation in the central cluster and the remodeling of chloroplasts that have been displaced by the vacuole to the cell periphery. Chloroplast numbers and sizes increased, more notably, during later stages of maturation than the early stages. Electron tomography analyses indicated that chloroplast enlargement is sustained by thylakoid growth and that invaginations from the inner envelope membrane contributed to thylakoid assembly. Grana stacks acquired more layers, differentiating them from stroma thylakoids as central chloroplasts matured. In peripheral chloroplasts, however, grana stacks stretched out to a degree that the distinction between grana stacks and stroma thylakoids was obscured. In central chloroplasts undergoing division, thylakoids inside the cleavage furrow were kinked and severed. Grana stacks in the division zone were disrupted, and large complexes in their membranes were dislocated, suggesting the existence of a thylakoid fission machinery.

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.

Chloroplasts are multifunctional organelles whose morphology is affected by environmental stresses. Although the three-dimensional (3D) architecture of thylakoid membranes has been reported previously, a 3D visualization of chloroplast under stress has not been explored. In this work, we used a positive-strand RNA ((+)RNA) virus, barley stripe mosaic virus (BSMV) to observe chloroplast structural changes during infection by electron tomography. The analyses revealed remodeling of the chloroplast membranes, characterized by the clustering of outer membrane-invaginated spherules in inner membrane-derived packets. Diverse morphologies of cytoplasmic invaginations (CIs) were evident with spherules at the periphery and different sized openings connecting the CIs to the cytoplasm. Immunoelectron microscopy of these viral components verified that the aberrant membrane structures were sites for BSMV replication. The BSMV αa replication protein localized at the surface of the chloroplasts and played a prominent role in eliciting chloroplast membrane rearrangements. In sum, our results have revealed the 3D structure of the chloroplasts induced by BSMV infection. These findings contribute to our understanding of chloroplast morphological changes under stress conditions and during assembly of plant (+)RNA virus replication complexes.

The maize endosperm consists of three major compartmentalized cell types: the starchy endosperm (SE), the basal endosperm transfer cell layer (BETL), and the aleurone cell layer (AL). Differential genetic programs are activated in each cell type to construct functionally and structurally distinct cells. To compare gene expression patterns involved in maize endosperm cell differentiation, we isolated transcripts from cryo-dissected endosperm specimens enriched with BETL, AL, or SE at 8, 12, and 16 days after pollination (DAP). We performed transcriptome profiling of coding and long noncoding transcripts in the three cell types during differentiation and identified clusters of the transcripts exhibiting spatio-temporal specificities. Our analysis uncovered that the BETL at 12 DAP undergoes the most dynamic transcriptional regulation for both coding and long noncoding transcripts. In addition, our transcriptome analysis revealed spatio-temporal regulatory networks of transcription factors, imprinted genes, and loci marked with histone H3 trimethylated at lysine 27. Our study suggests that various regulatory mechanisms contribute to the genetic networks specific to the functions and structures of the cell types of the endosperm.