Morphology and chemical composition of Taiwan oil millet (Eccoilopus formosanus) epicuticular wax.

MAIN CONCLUSION: Taiwan oil millet has two types of epicuticular wax: platelet wax composed primarily of octacosanol and filament wax constituted essentially by the singular compound of octacosanoic acid. Taiwan oil millet (TOM-Eccoilopus formosanus) is an orphan crop cultivated by the Taiwan indigenous people. It has conspicuous white powder covering its leaf sheath indicating abundant epicuticular waxes, that may contribute to its resilience. Here, we characterized the epicuticular wax secretion in TOM leaf blade and leaf sheath using various microscopy techniques, as well as gas chromatography to determine its composition. Two kinds of waxes, platelet and filaments, were secreted in both the leaf blades and sheaths. The platelet wax is secreted ubiquitously by epidermal cells, whereas the filament wax is secreted by a specific cell called epidermal cork cells. The newly developed filament waxes were markedly re-synthesized by the epidermal cork cells through papillae protrusions on the external periclinal cell wall. Ultrastructural images of cork cell revealed the presence of cortical endoplasmic reticulum (ER) tubules along the periphery of plasma membrane (PM) and ER-PM contact sites (EPCS). The predominant wax component was a C28 primary alcohol in leaf blade, and a C28 free fatty acid in the leaf sheath, pseudopetiole and midrib. The wax morphology present in distinct plant organs corresponds to the specific chemical composition: platelet wax composed of alcohols exists mainly in the leaf blade, whereas filament wax constituted mainly by the singular compound C28 free fatty acids is present abundantly in leaf sheath. Our study clarifies the filament wax composition in relation to a previous study in sorghum. Both platelet and filament waxes comprise a protection barrier for TOM.

Similar but different: Characterization of dddD gene-mediated DMSP metabolism among coral-associated Endozoicomonas.

Endozoicomonas are often predominant bacteria and prominently important in coral health. Their role in dimethylsulfoniopropionate (DMSP) degradation has been a subject of discussion for over a decade. A previous study found that Endozoicomonas degraded DMSP through the dddD pathway. This process releases dimethyl sulfide, which is vital for corals coping with thermal stress. However, little is known about the related gene regulation and metabolic abilities of DMSP metabolism in Endozoicomonadaceae. In this study, we isolated a novel Endozoicomonas DMSP degrader and observed a distinct DMSP metabolic trend in two phylogenetically close dddD-harboring Endozoicomonas species, confirmed genetically by comparative transcriptomic profiling and visualization of the change of DMSP stable isotopes in bacterial cells using nanoscale secondary ion spectrometry. Furthermore, we found that DMSP cleavage enzymes are ubiquitous in coral Endozoicomonas with a preference for having DddD lyase. We speculate that harboring DMSP degrading genes enables Endozoicomonas to successfully colonize various coral species across the globe.

Enhancing polyhydroxyalkanoate production in Cupriavidus sp. L7L through wcaJ gene deletion.

Cupriavidus sp. L7L synthesizes a high content of ductile polyhydroxyalkanoate. However, during fermentation, the medium's viscosity gradually increases, eventually reaching a level similar to 93 % glycerol, leading to fermentation termination and difficulties in cell harvest. A non-mucoid variant was isolated from a mini-Tn5 mutant library with the transposon inserted at the promoter sequence upstream of the wcaJ gene. Deletion of wcaJ eliminated the mucoid-colony appearance. The complementation experiment confirmed the association between wcaJ gene expression and mucoid-colony formation. Additionally, the wild-type strain exhibited a faster specific growth rate than the deletion strain using levulinate (Lev) as a carbon source. In fed-batch fermentation, Cupriavidus sp. L7L∆wcaJ showed similar PHA content and monomer composition to the wild-type strain. However, the extended fermentation time resulted in a 42 % increase in PHA concentration. After fed-batch fermentation, the deletion strain's medium had only 8.75 % of the wild-type strain's extracellular polymeric substance content. Moreover, the deletion strain's medium had a much lower viscosity (1.04 mPa·s) than the wild-type strain (194.7 mPa·s), making bacterial cell collection easier through centrifugation. In summary, Cupriavidus sp. L7L∆wcaJ effectively addressed difficulties in cell harvest, increased PHA production, and Lev-to-PHA conversion efficiency, making these characteristics advantageous for industrial-scale PHA production.

The use of immobilised bacteria cross-linked within magnetic alginate beads enhances the treatment of benzophenone-type UV filter chemicals by the SBR system.

Benzophenone-n compounds (BPs) are applied in a broad spectrum of commercial products, one of which is sunscreen. These chemicals are frequently detected in a variety of environmental matrices worldwide, especially water bodies. BPs are defined as emerging contaminants as well as endocrine-disrupting contaminants; thus, it has become necessary to develop aggressive and green treatments to remove BPs. In this study, we used immobilised BP-biodegrading bacteria linked to reusable magnetic alginate beads (MABs). The MABs were added to a sequencing batch reactor (SBR) system to enhance the removal of 2,4-dihydroxybenzophenone (BP-1) and oxybenzone (BP-3) from sewage. The BP-1 and BP-3 biodegrading bacteria in the MABs consisted of strains from up to three genera to allow for efficient biodegradation. The strains used were Pseudomonas spp., Gordonia sp., and Rhodococcus sp. The optimal composition of the MABs consisted of 3% (w/v) alginate and 10% (w/v) magnetite. The MABs resulted in 60.8%-81.7% recovery by weight after 28 days, and there was a continuous release of bacteria. Moreover, the biological treatment of the BPs sewage improved after adding 100 g of BP1-MABs (1:27) and also 100 g BP3-MABs (1:27) into the SBR system at a hydraulic retention time (HRT) of 8 h. Compared with the SBR system without MABs, the removal rates of BP-1 and BP-3 increased from 64.2% to 71.5% and from 78.1% to 84.1%, respectively. Furthermore, the COD removal increased from 36.1% to 42.1%, and total nitrogen increased from 30.5% to 33.2%. Total phosphorus remained constant at 29%. The bacterial community analysis showed that the Pseudomonas population was <2% before the MAB addition, but increased to 56.1% by day 14. In contrast, the Gordonia sp. And Rhodococcus sp. Populations (<2%) remained unchanged throughout the 14-day treatment period.

Synthesis of cross-linked magnetic chitosan beads immobilised with bacteria for aerobic biodegrading benzophenone-type UV filter.

Bacterial immobilisation is a technique by which bacteria are embedded into or adsorbed onto a carrier material thereby increasing bacterial tolerance to harsh environments. This technique can be used to enhance bacterial activity and to degrade pollutants. Immobilised bacterial beads that contain nanomagnetic particles allow bead recycling and reuse. In this study, our objective was to produce cross-linked nanomagnetic chitosan beads (MCBs) for the biodegradation of benzophenone-type UV filter chemicals such as 2,4-dihydroxybenzophenone (BP-1) and oxybenzone (BP-3). We found that the optimal concentration for creating these MCBs to be 1.2% by weight chitosan and 10% by weight nano-magnetite. We selected and isolated six benzophenone-n (BPs)-biodegrading bacteria identified to be various Pseudomonas spp., a Gordonia sp., and Rhodococcus zopfii; these were used to create MCBs that were able to effectively biodegrade BP-1 or BP-3 as a sole carbon source. Both BPs were effectively biodegraded and mineralised over 8 days in the presence of the selected MCB-immobilised bacterial strains. The highest pseudo-first-order constant rates for BP biodegradation were 8.7 × 10-3 h-1 for BP-1 (strain BP1-D) and 1.02 × 10-3 h-1 for BP-3 (strain BP3-1). The mechanical strength of the MCBs was measured to be above 90% based on recovered weight. The MCBs released their bacteria at rates in the range of 104-105 CFU/day. We also determined the pathway through which the BPs were being aerobically biodegraded based on the GC/MS profiles of the intermediates. Our findings provide a novel strategy for treating BPs via the use of reusable and recyclable MCBs that are cheap, easy and fast to synthesise.

TFEB- and TFE3-dependent autophagy activation supports cancer proliferation in the absence of centrosomes.

Centrosome amplification is a phenomenon frequently observed in human cancers, so centrosome depletion has been proposed as a therapeutic strategy. However, despite being afflicted with a lack of centrosomes, many cancer cells can still proliferate, implying there are impediments to adopting centrosome depletion as a treatment strategy. Here, we show that TFEB- and TFE3-dependent autophagy activation contributes to acentrosomal cancer proliferation. Our biochemical analyses uncover that both TFEB and TFE3 are novel PLK4 (polo like kinase 4) substrates. Centrosome depletion inactivates PLK4, resulting in TFEB and TFE3 dephosphorylation and subsequent promotion of TFEB and TFE3 nuclear translocation and transcriptional activation of autophagy- and lysosome-related genes. A combination of centrosome depletion and inhibition of the TFEB-TFE3 autophagy-lysosome pathway induced strongly anti-proliferative effects in cancer cells. Thus, our findings point to a new strategy for combating cancer.Abbreviations: AdCre: adenoviral Cre recombinase; AdLuc: adenoviral luciferase; ATG5: autophagy related 5; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DKO: double knockout; GFP: green fluorescent protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LTR: LysoTracker Red; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MITF: melanocyte inducing transcription factor; PLK4: polo like kinase 4; RFP: red fluorescent protein; SASS6: SAS-6 centriolar assembly protein; STIL: STIL centriolar assembly protein; TFEB: transcription factor EB; TFEBΔNLS: TFEB lacking a nuclear localization signal; TFE3: transcription factor binding to IGHM enhancer 3; TP53/p53: tumor protein p53.

Degradation of Decabromodiphenyl Ether in an Aerobic Clay Slurry Microcosm Using a Novel Immobilization Technique.

A novel chitosan immobilization technique that entraps photocatalyst and microbes was developed and applied to decompose decabromodiphenyl ether (BDE-209) in a clay slurry microcosm. The optimized conditions for immobilization were obtained by mixing 1.2% (w/v) chitosan dissolved in 1% (v/v) acetic acid with nano-TiO2 particles and the BDE-209-degrading bacterial mixed culture. This aqueous mixture was injected into 1% (w/v) water solution containing sodium tripolyphosphate to form spherical immobilized beads. The surface of the immobilized beads was reinforced by 0.25% (v/v) glutaraldehyde cross-linking. These beads had enough mechanical strength during BDE-209 degradation to maintain their shape in the system at a stirring rate of 200-rpm, while undergoing continuous 365 nm UVA irradiation. This novel TiO2-Yi-Li immobilized chitosan beads system allowed a successful simultaneous integration of photolysis, photocatalysis and biodegradation to remove BDE-209. The remaining percentage of BDE-209 was 41% after 70 days of degradation using this system. The dominant bacteria in the BDE-209-degrading bacterial mixed culture during remediation were Chitinophaga spp., Methyloversatilis spp., Terrimonas spp. and Pseudomonas spp. These bacteria tolerated the long-term UVA irradiation and high-level free radicals present, while utilizing BDE-209 as their primary carbon resource. This new method has great potential for the treatment of a range of pollutants.

Maize Golden2-like transcription factors boost rice chloroplast development, photosynthesis, and grain yield.

Chloroplasts are the sites for photosynthesis, and two Golden2-like factors act as transcriptional activators of chloroplast development in rice (Oryza sativa L.) and maize (Zea mays L.). Rice OsGLK1 and OsGLK2 are orthologous to maize ZmGLK1 (ZmG1) and ZmGLK2 (ZmG2), respectively. However, while rice OsGLK1 and OsGLK2 act redundantly to regulate chloroplast development in mesophyll cells, maize ZmG1 and ZmG2 are functionally specialized and expressed in different cell-specific manners. To boost rice chloroplast development and photosynthesis, we generated transgenic rice plants overexpressing ZmG1 and ZmG2, individually or simultaneously, with constitutive promoters (pZmUbi::ZmG1 and p35S::ZmG2) or maize promoters (pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2). Both ZmG1 and ZmG2 genes were highly expressed in transgenic rice leaves. Moreover, ZmG1 and ZmG2 showed coordinated expression in pZmG1::ZmG1/pZmG2::ZmG2 plants. All Golden2-like (GLK) transgenic plants had higher chlorophyll and protein contents, Rubisco activities and photosynthetic rates per unit leaf area in flag leaves. However, the highest grain yields occurred when maize promoters were used; pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2 transgenic plants showed increases in grain yield by 51%, 47%, and 70%, respectively. In contrast, the pZmUbi::ZmG1 plant produced smaller seeds without yield increases. Transcriptome analysis indicated that maize GLKs act as master regulators promoting the expression of both photosynthesis-related and stress-responsive regulatory genes in both rice shoot and root. Thus, by promoting these important functions under the control of their own promoters, maize GLK1 and GLK2 genes together dramatically improved rice photosynthetic performance and productivity. A similar approach can potentially improve the productivity of many other crops.

Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages.

Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs.

Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis.

X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.

Studies of a rice sterile mutant sstl from the TRIM collection.

BACKGROUND: Rice (Oryza sativa) is one of the main crops in the world, and more than 3.9 billion people will consume rice by 2025. Sterility significantly affects rice production and leads to yield defects. The undeveloped anthers or abnormal pollen represent serious defects in rice male sterility. Therefore, understanding the mechanism of male sterility is an important task. Here, we investigated a rice sterile mutant according to its developmental morphology and transcriptional profiles. RESULTS: An untagged T-DNA insertional mutant showed defective pollen and abnormal anthers as compared with its semi-sterile mutant (sstl) progeny segregates. Transcriptomic analysis of sterile sstl-s revealed several biosynthesis pathways, such as downregulated cell wall, lipids, secondary metabolism, and starch synthesis. This downregulation is consistent with the morphological characterization of sstl-s anthers with irregular exine, absence of intine, no starch accumulation in pollen grains and no accumulated flavonoids in anthers. Moreover, defective microsporangia development led to abnormal anther locule and aborted microspores. The downregulated lipids, starch, and cell wall synthesis-related genes resulted in loss of fertility. CONCLUSIONS: We illustrate the importance of microsporangia in the development of anthers and functional microspores. Abnormal development of pollen grains, pollen wall, anther locule, etc. result in severe yield reduction.

3D Imaging of Tapetal Mitochondria Suggests the Importance of Mitochondrial Fission in Pollen Growth.

Mitochondrial fission occurs frequently in plant cells, but its biological significance is poorly understood because mutants specifically impaired in mitochondrial fission do not show obvious defects in vegetative growth. Here, we revealed that the production of viable pollen was reduced in mutants lacking one of the three main proteins involved in mitochondrial fission in Arabidopsis (Arabidopsis thaliana), DYNAMIN-RELATED PROTEIN3A (DRP3A)/Arabidopsis DYNAMIN-LIKE PROTEIN2A, DRP3B, and ELONGATED MITOCHONDRIA1 (ELM1). In drp3b and elm1, young microspores contained an abnormal number of nuclei, and mature pollen had aberrant accumulation of lipids in their coat and an irregular pollen outer wall. Because the formation of the pollen wall and coat is mainly associated with tapetal function, we used 3D imaging to quantify geometric and textural features of cells and mitochondria in the tapetum at different stages, using isolated single tapetal cells in which the in vivo morphology and volume of cells and mitochondria were preserved. Tapetal cells and their mitochondria changed in the volume and morphology at different developmental stages. Defective mitochondrial fission in the elm1 and drp3b mutants caused changes in mitochondrial status, including mitochondrial elongation, abnormal mitochondrial ultrastructure, a decrease in cross-sectional area, and a slight alteration of mitochondrial distribution, as well as a large reduction in mitochondrial density. Our studies suggest that mitochondrial fission is required for proper mitochondrial status in the tapetum and possibly in pollen as well and therefore plays an important role for the production of viable pollen.

Metagenomic, phylogenetic, and functional characterization of predominant endolithic green sulfur bacteria in the coral Isopora palifera.

BACKGROUND: Endolithic microbes in coral skeletons are known to be a nutrient source for the coral host. In addition to aerobic endolithic algae and Cyanobacteria, which are usually described in the various corals and form a green layer beneath coral tissues, the anaerobic photoautotrophic green sulfur bacteria (GSB) Prosthecochloris is dominant in the skeleton of Isopora palifera. However, due to inherent challenges in studying anaerobic microbes in coral skeleton, the reason for its niche preference and function are largely unknown. RESULTS: This study characterized a diverse and dynamic community of endolithic microbes shaped by the availability of light and oxygen. In addition, anaerobic bacteria isolated from the coral skeleton were cultured for the first time to experimentally clarify the role of these GSB. This characterization includes GSB's abundance, genetic and genomic profiles, organelle structure, and specific metabolic functions and activity. Our results explain the advantages endolithic GSB receive from living in coral skeletons, the potential metabolic role of a clade of coral-associated Prosthecochloris (CAP) in the skeleton, and the nitrogen fixation ability of CAP. CONCLUSION: We suggest that the endolithic microbial community in coral skeletons is diverse and dynamic and that light and oxygen are two crucial factors for shaping it. This study is the first to demonstrate the ability of nitrogen uptake by specific coral-associated endolithic bacteria and shed light on the role of endolithic bacteria in coral skeletons.

PeERF1, a SHINE-Like Transcription Factor, Is Involved in Nanoridge Development on Lip Epidermis of Phalaenopsis Flowers.

Phalaenopsis orchids have a spectacular floral morphology with a highly evolved lip that offers a landing platform for pollinators. The typical morphological orchid lip features are essential for the special pollination mechanism of Phalaenopsis flowers. Previously, we found that in the lip, a member of the AP2/EREBP protein family was highly expressed. Here, we further confirmed its high expression and characterized its function during lip development. Phylogenetic analysis showed that AP2/EREBP belongs to the Va2 subgroup of ERF transcription factors. We named it PeERF1. We found that PeERF1 was only expressed at stage 5, as flowers opened. This coincided with both thickening of the cuticle and development of nanoridges. We performed knockdown expression of PeERF1 using CymMV-based virus-induced gene silencing in either the AP2 conserved domain, producing PeERF1_AP2-silenced plants, or the SHN specific domain, producing PeERF1_SHN-silenced plants. Using cryo-SEM, we found that the number of nanoridges was reduced only in the PeERF1_AP2-silenced group. This change was found on both the abaxial and adaxial surfaces of the central lip lobe. Expression of PeERF1 was reduced significantly in PeERF1_AP2-silenced plants. In cutin biosynthesis genes, expression of both PeCYP86A2 and PeDCR was significantly decreased in both groups. The expression of PeCYP77A4 was reduced significantly only in the PeERF1_AP2-silenced plants. Although PeGPAT expression was reduced in both silenced plants, but to a lesser degree. The expression of PeERF1 was significantly reduced in the petal-like lip of a big-lip variant. PeCYP77A4 and PeGPAT in the lip were also reduced, but PeDCR was not. Furthermore, heterologous overexpression of PeERF1 in the genus Arabidopsis produced leaves that were shiny on the adaxial surface. Taken together, our results show that in Phalaenopsis orchids PeERF1 plays an important role in formation of nanoridges during lip epidermis development.

Cultivation temperature modulated the monomer composition and polymer properties of polyhydroxyalkanoate synthesized by Cupriavidus sp. L7L from levulinate as sole carbon source.

Cupriavidus sp. L7L, a newly isolated wild-type soil bacterium, was found to synthesize polyhydroxyalkanoate (PHA) terpolymers from levulinic acid (LA), a top bio-based platform chemical, as the sole carbon source. NMR spectra showed that the terpolymers consisted mainly of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and a small amount (2.1-4.8 mol%) of 4-hydroxyvalerate (4HV). The combination of monomers together with high molecular weights improved markedly the thermal and mechanical properties of the terpolymers. Increasing cultivation temperature of Cupriavidus sp. L7L increased the proportions of 3HB and decreased the proportions of 3HV. A fed-batch fermentation using LA as the sole carbon source without pH control produced a biomass of 15.8 dry weight g L-1 that contained 81 dry weight% of a terpolymer P(3HB-co-33.7 mol% 3HV-co-2.1 mol% 4HV), equaling a productivity of 0.213 g PHA L-1 h-1. The terpolymer showed a melting point of 92 °C and elongation at break of 630%, compared to 84.6 °C and 462%, respectively, for the copolymer P(3HB-co-35.9 mol% 3HV) that contained no 4HV. This study showed that Cupriavidus sp. L7L exhibited a great potential for producing PHA polymers with excellent mechanical property that could be modulated by cultivation temperature when cultivated exclusively in LA.

Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components.

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.

Equilibrative Nucleoside Transporter 3 Regulates T Cell Homeostasis by Coordinating Lysosomal Function with Nucleoside Availability.

T cells are a versatile immune cell population responding to challenges by differentiation and proliferation followed by contraction and memory formation. Dynamic metabolic reprogramming is essential for T cells to meet the biosynthetic needs and the reutilization of biomolecules, processes that require active participation of metabolite transporters. Here, we show that equilibrative nucleoside transporter 3 (ENT3) is highly expressed in peripheral T cells and has a key role in maintaining T cell homeostasis by supporting the proliferation and survival of T cells. ENT3 deficiency leads to an enlarged and disturbed lysosomal compartment, resulting in accumulation of surplus mitochondria, elevation of intracellular reactive oxygen species, and DNA damage in T cells. Our results identify ENT3 as a vital metabolite transporter that supports T cell homeostasis and activation by regulating lysosomal integrity and the availability of nucleosides. Moreover, we uncovered that T cell lysosomes are an important source of salvaged metabolites for survival and proliferation.

Characterization of Root Epidermal Cell Patterning and Differentiation in Arabidopsis.

The root epidermis of Arabidopsis thaliana has been established as a model system for elucidating the mechanisms which govern the spatial patterningAbstract and morphogenesis of plant cells. Investigations into root hairs focus on various aspects of the biology of epidermal cells, using methods specifically developed to dissect the biological question under study. Despite the large number of studies related to epidermal cell differentiation, a survey of methods to analyze the phenotypic readout resulting from environmental conditions or the genetic background of the plant has not been provided so far. This protocol describes how to analyze the spatial arrangement and morphologic characteristics of cells in the root epidermis based on whole mount roots or cross sections, using confocal, scanning electron and light microscopy. This comparison of methods aids in selecting the most suitable strategy to examine the differentiation of root epidermal cells at different developmental stages.