Chloroplast Envelopes Play a Role in the Formation of Autophagy-Related Structures in Plants.

Autophagy is a degradation process of cytoplasmic components that is conserved in eukaryotes. One of the hallmark features of autophagy is the formation of double-membrane structures known as autophagosomes, which enclose cytoplasmic content destined for degradation. Although the membrane source for the formation of autophagosomes remains to be determined, recent studies indicate the involvement of various organelles in autophagosome biogenesis. In this study, we examined the autophagy process in Bienertia sinuspersici: one of four terrestrial plants capable of performing C4 photosynthesis in a single cell (single-cell C4 species). We demonstrated that narrow tubules (stromule-like structures) 30-50 nm in diameter appear to extend from chloroplasts to form the membrane-bound structures (autophagosomes or autophagy-related structures) in chlorenchyma cells of B. sinuspersici during senescence and under oxidative stress. Immunoelectron microscopic analysis revealed the localization of stromal proteins to the stromule-like structures, sequestering portions of the cytoplasm in chlorenchyma cells of oxidative stress-treated leaves of B. sinuspersici and Arabidopsis thaliana. Moreover, the fluorescent marker for autophagosomes GFP-ATG8, colocalized with the autophagic vacuole maker neutral red in punctate structures in close proximity to the chloroplasts of cells under oxidative stress conditions. Together our results implicate a role for chloroplast envelopes in the autophagy process induced during senescence or under certain stress conditions in plants.

Protoplast Isolation and Transfection in the Single-Cell C4 Species Bienertia sinuspersici.

We have developed an optimized protocol for isolating protoplasts from chlorenchyma cells of the single-cell C4 species Bienertia sinuspersici. The isolated protoplasts maintained the integrity of the unique single-cell C4 intracellular compartmentation of organelles as observed in chlorenchyma cells after cell wall digestion. Approximately over 80% of isolated protoplasts expressed the fusion reporter gene following the polyethylene glycol-mediated transfection procedures. Overall, fluorescent protein fusion tagged with various intraorganellular sorting signals validated the potential use of the transient gene expression system in subcellular localization and organelle dynamics studies.

Tandem C2 domains mediate dynamic organelle targeting of a DOCK family guanine nucleotide exchange factor.

Multicellular organisms use dedicator of cytokinesis (DOCK) family guanine nucleotide exchange factors (GEFs) to activate Rac/Rho-of-plants small GTPases and coordinate cell shape change. In developing tissues, DOCK signals integrate cell-cell interactions with cytoskeleton remodeling, and the GEFs cluster reversibly at specific organelle surfaces to orchestrate cytoskeletal reorganization. The domain organizations among DOCK orthologs are diverse, and the mechanisms of localization control are poorly understood. Here, we use combinations of transgene complementation and live-cell imaging assays to uncover an evolutionarily conserved and essential localization determinant in the DOCK-GEF named SPIKE1. The SPIKE1-DHR3 domain is sufficient for organelle association in vivo, and displays a complicated lipid-binding selectivity for both phospholipid head groups and fatty acid chain saturation. SPIKE1-DHR3 is predicted to adopt a C2-domain structure and functions as part of a tandem C2 array that enables reversible clustering at the cell apex. This work provides mechanistic insight into how DOCK GEFs sense compositional and biophysical membrane properties at the interface of two organelle systems.

Development of C4 Biochemistry and Change in Expression of Markers for Photosystems I and II in the Single-Cell C4 Species, Bienertia sinuspersici

Bienertia sinuspersici is one of four identified terrestrial plants that perform C4 photosynthesis within a single chlorenchyma cell via the compartmentation of organelles and photosynthetic enzymes. The patterns of accumulation of key photosynthetic enzymes and transcripts in developing leaves were examined using immunolocalization and in situ hybridization. The polypeptides of Rubisco large subunit (RbcL) and pyruvate Pi dikinase (PPDK) accumulated equally in all chloroplasts before the formation of two intracellular cytoplasmic compartments: the central (CCC) and peripheral (PCC) cytoplasmic compartments. The differential accumulation of these enzymes was not completed until the leaf had reached maturity, indicating that the transition from C3 to C4 photosynthesis occurred during leaf maturation. In mature chlorenchyma cells, RbcL accumulated 20-fold higher in the CCC than in the PCC, while PPDK exhibited a concentration gradient that was the lowest in the chloroplasts in the central region of the CCC and the highest in PCC chloroplasts. The pattern of rbcL transcript accumulation followed that of its polypeptides in developing leaves, suggesting that the expression of this gene was likely controlled by transcriptional and/or post-transcriptional processes. Immunocytochemical results examining the distribution of photosystems I and II in the chloroplasts of chlorenchyma cells from mature leaves showed that PSII is more abundant in chloroplasts of the central compartment, whereas PSI is higher in those of the peripheral compartment. The quantitative real-time PCR results of rbcL, psbA, and psaB transcripts from the isolated chloroplasts of each compartment further supported this observation. Our results suggest that multiple levels of regulation play a role in controlling the differential accumulation of photosynthetic gene expression in the dimorphic chloroplasts of single-cell C4 species during leaf development.

Arabidopsis vascular complexity and connectivity controls PIN-FORMED1 dynamics and lateral vein patterning during embryogenesis.

Arabidopsis VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC) is a plant-specific transmembrane protein that controls the development of veins in cotyledons. Here, we show that the expression and localization of the auxin efflux carrier PIN-FORMED1 (PIN1) is altered in vcc developing cotyledons and that overexpression of PIN1-GFP partially rescues vascular defects of vcc in a dosage-dependent manner. Genetic analyses suggest that VCC and PINOID (PID), a kinase that regulates PIN1 polarity, are both required for PIN1-mediated control of vasculature development. VCC expression is upregulated by auxin, likely as part of a positive feedback loop for the progression of vascular development. VCC and PIN1 localized to the plasma membrane in pre-procambial cells but were actively redirected to vacuoles in procambial cells for degradation. In the vcc mutant, PIN1 failed to properly polarize in pre-procambial cells during the formation of basal strands, and instead, it was prematurely degraded in vacuoles. VCC plays a role in the localization and stability of PIN1, which is crucial for the transition of pre-procambial cells into procambial cells that are involved in the formation of basal lateral strands in embryonic cotyledons.

Real-time conversion of tissue-scale mechanical forces into an interdigitated growth pattern.

The leaf epidermis is a dynamic biomechanical shell that integrates growth across spatial scales to influence organ morphology. Pavement cells, the fundamental unit of this tissue, morph irreversibly into highly lobed cells that drive planar leaf expansion. Here, we define how tissue-scale cell wall tensile forces and the microtubule-cellulose synthase systems dictate the patterns of interdigitated growth in real time. A morphologically potent subset of cortical microtubules span the periclinal and anticlinal cell faces to pattern cellulose fibres that generate a patch of anisotropic wall. The subsequent local polarized growth is mechanically coupled to the adjacent cell via a pectin-rich middle lamella, and this drives lobe formation. Finite element pavement cell models revealed cell wall tensile stress as an upstream patterning element that links cell- and tissue-scale biomechanical parameters to interdigitated growth. Cell lobing in leaves is evolutionarily conserved, occurs in multiple cell types and is associated with important agronomic traits. Our general mechanistic models of lobe formation provide a foundation to analyse the cellular basis of leaf morphology and function.

Microtubule-Dependent Confinement of a Cell Signaling and Actin Polymerization Control Module Regulates Polarized Cell Growth.

Cell types with wildly varying shapes use many of the same signaling and cytoskeletal proteins to dynamically pattern their geometry [1-3]. Plant cells are encased in a tough outer cell wall, and growth patterns are indirectly controlled by the cytoskeleton and its ability to locally specify the material properties of the wall [4, 5]. Broad and non-overlapping domains of actin and microtubules are predicted to create sharp cell-wall boundaries with distinct mechanical properties [6] that are often proposed to direct growth patterns and cell shape [1, 6, 7]. However, mechanisms by which the cytoskeleton is patterned at the spatial and temporal scales that dictate cell morphology are not known. Here, we used combinations of live-cell imaging probes and unique morphology mutants in Arabidopsis to discover how the microtubule and actin systems are spatially coordinated to pattern polarized growth in leaf epidermal cells. The DOCK family guanine nucleotide exchange factor (GEF) SPIKE1 [8, 9] clusters and activates conserved heteromeric WAVE/SCAR and ARP2/3 complexes at the cell apex to generate organized actin networks that define general cytoplasmic flow patterns. Cortical microtubules corral punctate SPIKE1 signaling nodules and restrict actin polymerization within a broad microtubule-depletion zone at the cell apex. Our data provide a useful model for cell-shape control, in which a GEF, actin filament nucleation complexes, microtubules, and the cell wall function as interacting systems that dynamically pattern polarized growth.

Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter.

Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission.

Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.

The plant actin cytoskeleton is an unstable network of filaments that influences polarized growth through poorly understood mechanisms. Here, we used a combination of live cell imaging and finite element computational modelling of Arabidopsis trichome morphogenesis to determine how the actin and microtubule cytoskeletons cooperate to pattern the cell wall and growth. The actin-related protein (ARP)2/3 complex generates an actin meshwork that operates within a tip-localized, microtubule-depleted zone to modulate cell wall anisotropy locally. The actin meshwork also positions an actin bundle network that organizes organelle flow patterns. This activity is required to maintain cell wall thickness gradients that enable tip-biased diffuse growth. These newly discovered couplings between cytoskeletal patterns and wall textures provide important insights into the cellular mechanism of growth control in plants.

ARP2/3-dependent growth in the plant kingdom: SCARs for life.

In the human experience SCARs (suppressor of cAMP receptors) are permanent reminders of past events, not always based on bad decisions, but always those in which an interplay of opposing forces leaves behind a clear record in the form of some permanent watery mark. During plant morphogenesis, SCARs are important proteins that reflect an unusual evolutionary outcome, in which the plant kingdom relies heavily on this single class of actin-related protein (ARP) 2/3 complex activator to dictate the time and place of actin filament nucleation. This unusually simple arrangement may serve as a permanent reminder that cell shape control in plants is fundamentally different from that of crawling cells in mammals that use the power of actin polymerization to define and maintain cell shape. In plant cells, actin filaments indirectly affect cell shape by determining the transport properties of organelles and cargo molecules that modulate the mechanical properties of the wall. It is becoming increasingly clear that polarized bundles of actin filaments operate at whole cell spatial scales to organize the cytoplasm and dictate the patterns of long-distance intracellular transport and secretion. The number of actin-binding proteins and actin filament nucleators that are known to participate in the process of actin network formation are rapidly increasing. In plants, formins and ARP2/3 are two important actin filament nucleators. This review will focus on ARP2/3, and the apparent reliance of most plant species on the SCAR/WAVE (WASP family verprolin homologous) regulatory complex as the sole pathway for ARP2/3 activation.

Lewis X-carrying N-glycans regulate the proliferation of mouse embryonic neural stem cells via the Notch signaling pathway.

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells. Several signaling pathways have been shown to be involved in the fate determination process of NSCs, but the molecular mechanisms underlying the maintenance of neural cell stemness remain largely unknown. Our previous study showed that human natural killer carbohydrate epitopes expressed specifically by mouse NSCs modulate the Ras-MAPK pathway, raising the possibility of regulatory roles of glycoprotein glycans in the specific signaling pathways involved in NSC fate determination. To address this issue, we performed comparative N-glycosylation profiling of NSCs before and after differentiation in a comprehensive and quantitative manner. We found that Lewis X-carrying N-glycans were specifically displayed on undifferentiated cells, whereas pauci-mannose-type N-glycans were predominantly expressed on differentiated cells. Furthermore, by knocking down a fucosyltransferase 9 with short interfering RNA, we demonstrated that the Lewis X-carrying N-glycans were actively involved in the proliferation of NSCs via modulation of the expression level of Musashi-1, which is an activator of the Notch signaling pathway. Our findings suggest that Lewis X carbohydrates, which have so far been characterized as undifferentiation markers, actually operate as activators of the Notch signaling pathway for the maintenance of NSC stemness during brain development.

Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici.

Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis.

Structures, biosynthesis, and functions of gangliosides--an overview.

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nervous system. Heterogeneity and diversity of the structures in their carbohydrate chains are characteristic hallmarks of these lipids; so far, 188 gangliosides with different carbohydrate structures have been identified in vertebrates. The molecular structural complexity increases manifold if one considers heterogeneity in the lipophilic components. The expression levels and patterns of brain gangliosides are known to change drastically during development. In cells, gangliosides are primarily, but not exclusively, localized in the outer leaflets of plasma membranes and are integral components of cell surface microdomains with sphingomyelin and cholesterol from which they participate in cell-cell recognition, adhesion, and signal transduction. In this brief review, we discuss the structures, metabolism and functions of gangliosides.

Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells.

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.

Histone acetylation-mediated glycosyltransferase gene regulation in mouse brain during development.

Gangliosides are sialic acid-containing glycosphingolipids abundant in the central nervous tissues. The quantity and expression pattern of gangliosides in brain change drastically during early development and are mainly regulated through stage-specific expression of glycosyltransferase (ganglioside synthase) genes. It is still unclear, however, how the transcriptional activation of glycosyltransferase genes is regulated during development. In this study, we investigated the epigenetic regulation of two key glycosyltransferases, N-acetylgalactosaminyltransferase I (GA2/GM2/GD2/GT2-synthase) and sialyltransferase II (GD3-synthase), in embryonic, postnatal, and adult mouse brains. Combined bisulfite restriction analysis assay showed that DNA methylation in the 5' regions of these glycosyltransferase genes was not associated with their expression patterns. On the other hand, chromatin immunoprecipitation assay of both glycosyltransferase genes showed that their histone H3 acetylation was highly correlated to their mRNA expression levels during development. In fact, we confirmed that the expression patterns of gangliosides and glycosyltransferases in neuroepithelial cells were changed after treatment with a histone deacetylase inhibitor, sodium butyrate. Our studies provide the first evidence that efficient histone acetylation of the glycosyltransferase genes in mouse brain contributes to the developmental alteration of ganglioside expression.

Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici.

Although transient gene expression using reporters such as green fluorescent protein is a versatile tool for examining gene functions and intracellular protein trafficking, the establishment of a highly efficient gene manipulation method remains a challenge in many plant species. A reliable transformation protocol has not yet been established for the three single-cell C(4) species, despite their potential of serving as model systems for their extraordinary C(4) photosynthetic metabolism. We report the first protocol optimized for isolating a large-scale and homogenous population of protoplasts from chlorenchyma cells of the single-cell C(4) species Bienertia sinuspersici. Cytochemical staining confirmed the preservation of the unusual subcellular compartmentation of organelles in chlorenchyma cells after cell wall digestion. Approximately 84% of isolated protoplasts expressed the reporter fluorescent protein following our optimized polyethylene glycol-mediated transfection procedures. Fluorescent fusion protein tagged with various intracellular sorting signals demonstrated potential use of the transient gene expression system in subcellular protein localization and organelle dynamics studies. Further applications of the current protoplast isolation and transfection techniques in understanding the novel single-cell C(4) photosynthetic mechanism are discussed.

Stem cell glycolipids.

Glycolipids are compounds containing one or more monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety. Because of their expression patterns and the intracellular localization patterns, glycolipids, including stage-specific embryonic antigens (SSEA-3, SSEA-4, and possibly SSEA-1) and gangliosides (e.g., GD3, GD2, and A2B5 antigens), have been used as marker molecules of stem cells. In this review, I will introduce glycolipids expressed in pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells, very small embryonic-like stem cells, amniotic stem cells, and multilineage-differentiating stress enduring cells), multipotent stem cells (neural stem cells, mesenchymal stem cells, fetal liver multipotent progenitor cells, and hematopoietic stem cells), and cancer stem cells (brain cancer stem cells and breast cancer stem cells), and discuss their availability as biomarkers for identifying and isolating stem cells.

Ganglioside metabolism in a transgenic mouse model of Alzheimer's disease: expression of Chol-1α antigens in the brain.

The accumulation of Aß (amyloid ß-protein) is one of the major pathological hallmarks in AD (Alzheimer's disease). Gangliosides, sialic acid-containing glycosphingolipids enriched in the nervous system and frequently used as biomarkers associated with the biochemical pathology of neurological disorders, have been suggested to be involved in the initial aggregation of Aß. In the present study, we have examined ganglioside metabolism in the brain of a double-Tg (transgenic) mouse model of AD that co-expresses mouse/human chimaeric APP (amyloid precursor protein) with the Swedish mutation and human presenilin-1 with a deletion of exon 9. Although accumulation of Aß was confirmed in the double-Tg mouse brains and sera, no statistically significant change was detected in the concentration and composition of major ganglio-N-tetraosyl-series gangliosides in the double-Tg brain. Most interestingly, Chol-1α antigens (cholinergic neuron-specific gangliosides), such as GT1aα and GQ1bα, which are minor species in the brain, were found to be increased in the double-Tg mouse brain. We interpret that the occurrence of these gangliosides may represent evidence for generation of cholinergic neurons in the AD brain, as a result of compensatory neurogenesis activated by the presence of Aß.

HNK-1 epitope-carrying tenascin-C spliced variant regulates the proliferation of mouse embryonic neural stem cells.

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development.