A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

The Fly Brain Atlas.

The brain's synaptic networks endow an animal with powerfully adaptive biological behavior. Maps of such synaptic circuits densely reconstructed in those model brains that can be examined and manipulated by genetic means offer the best prospect for understanding the underlying biological bases of behavior. That prospect is now technologically feasible and a scientifically enabling possibility in neurobiology, much as genomics has been in molecular biology and genetics. In Drosophila, two major advances are in electron microscopic technology, using focused ion beam-scanning electron microscopy (FIB-SEM) milling to capture and align digital images, and in computer-aided reconstruction of neuron morphologies. The last decade has witnessed enormous progress in detailed knowledge of the actual synaptic circuits formed by real neurons. Advances in various brain regions that heralded identification of the motion-sensing circuits in the optic lobe are now extending to other brain regions, with the prospect of encompassing the fly's entire nervous system, both brain and ventral nerve cord.

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

Immuno-scanning electron microscopy of islet primary cilia.

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.

Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and 3D Focused Ion Beam-Scanning Electron Microscopy, we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or break-down revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

Precise Fabrication and Manipulation of Individual Polymer Nanofibers.

Polymer nanofibers hold promise in a wide range of applications owing to their diverse properties, flexibility, and cost effectiveness. In this study, we introduce a polymer nanofiber drawing process in a scanning electron microscope and focused ion beam (SEM/FIB) instrument with in situ observation. We employed a nanometer-sharp tungsten needle and prepolymer microcapsules to enable nanofiber drawing in a vacuum environment. This method produces individual polymer nanofibers with diameters as small as ∼500 nm and lengths extending to millimeters, yielding nanofibers with an aspect ratio of 2000:1. The attachment to the tungsten manipulator ensures accurate transfer of the polymer nanofiber to diverse substrate types as well as fabrication of assembled structures. Our findings provide valuable insights into ultrafine polymer fiber drawing, paving the way for high-precision manipulation and assembly of polymer nanofibers.

Large and Pressure-Dependent c-Axis Piezoresistivity of Highly Oriented Pyrolytic Graphite near Zero Pressure.

The c-axis piezoresistivity is a fundamental and important parameter of graphite, but its value near zero pressure has not been well determined. Herein, a new method for studying the c-axis piezoresistivity of van der Waals materials near zero pressure is developed on the basis of in situ scanning electron microscopy and finite element simulation. The c-axis piezoresistivity of microscale highly oriented pyrolytic graphite (HOPG) is found to show a large value of 5.68 × 10-5 kPa-1 near zero pressure and decreases by 2 orders of magnitude to the established value of ∼10-7 kPa-1 when the pressure increases to 200 MPa. By modulating the serial tunneling barrier model on the basis of the stacking faults, we describe the c-axis electrical transport of HOPG under compression. The large c-axis piezoresistivity near zero pressure and its large decrease in magnitude with pressure are attributed to the rapid stiffening of the electromechanical properties under compression.

Chemomechanical Origins of the Dynamic Evolution of Isolated Li Filaments in Inorganic Solid-State Electrolytes.

The penetrating growth of Li into the inorganic solid-state electrolyte (SSE) is one key factor limiting its practical application. Research to understand the underlying mechanism of Li penetration has been ongoing for years and is continuing. Here, we report an in situ scanning electron microscopy methodology to investigate the dynamic behaviors of isolated Li filaments in the garnet SSE under practical cycling conditions. We find that the filaments tend to grow in the SSE, while surprisingly, those filaments can self-dissolve with a decrease in the current density without a reversal of the current direction. We further build a coupled electro-chemo-mechanical model to assess the interplay between electrochemistry and mechanics during the dynamic evolution of filaments. We reveal that filament growth is strongly regulated by the competition between the electrochemical driving force and mechanical resistive force. The numerical results provide rational guidance for the design of solid-state batteries with excellent properties.

Stability of Plasmonic Mg-MgO Core-Shell Nanoparticles in Gas-Phase Oxidative Environments.

Magnesium is a recent addition to the plasmonic toolbox: nanomaterials that efficiently utilize photons' energy due to their ability to sustain localized surface plasmon resonances. Magnesium nanoparticles protected by a native oxide shell can efficiently absorb light across the solar spectrum, making them a promising photocatalytic material. However, their inherent reactivity toward oxidation may limit the number of reactions in which Mg-MgO can be used. Here, we investigate the stability of plasmonic Mg-MgO core-shell nanoplates under oxidative conditions. We demonstrate that the MgO shell stabilizes the metallic Mg core against oxidation in air at up to 400 °C. Furthermore, we show that the reactivity of Mg-MgO nanoplates with water vapor (3.5 vol % in N2) decreases with temperature, with no oxidation of the Mg core detected from 200 to 400 °C. This work unravels the potential of Mg-MgO nanoparticles for a broad range of catalytic transformations occurring in oxidative environments.

A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

Focused ion beam-SEM 3D study of osteodentin in the teeth of the Atlantic wolfish Anarhichas lupus.

The palette of mineralized tissues in fish is wide, and this is particularly apparent in fish dentin. While the teeth of all vertebrates except fish contain a single dentinal tissue type, called orthodentin, dentin in the teeth of fish can be one of several different tissue types. The most common dentin type in fish is orthodentin. Orthodentin is characterized by several key structural features that are fundamentally different from those of bone and from those of osteodentin. Osteodentin, the second-most common dentin type in fish (based on the tiny fraction of fish species out of ∼30,000 extant fish species in which tooth structure was so far studied), is found in most Selachians (sharks and rays) as well as in several teleost species, and is structurally different from orthodentin. Here we examine the hypothesis that osteodentin is similar to anosteocytic bone tissue in terms of its micro- and nano-structure. We use Focused Ion Beam-Scanning Electron Microscopy (FIB/SEM), as well as several other high-resolution imaging techniques, to characterize the 3D architecture of the three main components of osteodentin (denteons, inter-denteonal matrix, and the transition zone between them). We show that the matrix of osteodentin, although acellular, is extremely similar to mammalian osteonal bone matrix, both in general morphology and in the three-dimensional nano-arrangement of its mineralized collagen fibrils. We also document the presence of a complex network of nano-channels, similar to such networks recently described in bone. Finally, we document the presence of strings of hyper-mineralized small 'pearls' which surround the denteonal canals, and characterize their structure.

Melanin granules morphology and distribution in human black hair investigated by focused ion beam scanning electron microscopy: Differences between Asian and Caucasian hair.

Melanin granules (melanosomes) in Asian and Caucasian black hairs were investigated by focused ion beam scanning electron microscopy (FIB-SEM). This technique facilitates a direct evaluation of the three-dimensional distribution and morphology of melanin granules without requiring their isolation from hair. Three-dimensional reconstructed images of melanin granule distribution in hair samples were obtained using serial SEM images observed by FIB-SEM. Melanin granules in black hair tended to be three-dimensionally dense in the outer periphery of the cortex. The morphometric parameters of melanin granules were calculated using the reconstructed three-dimensional images. The results confirmed that melanin granules in Caucasian black hair were much smaller those in Asian black hair. Moreover, it was indicated that the relative frequency distribution of the volume of melanin granules was significantly different between Asians and Caucasians.

Cell-induced collagen alignment in a 3D in vitro culture during extracellular matrix production.

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Inoculation of heavy metal resistant bacteria alleviated heavy metal-induced oxidative stress biomarkers in spinach (Spinacia oleracea L.).

Most vegetable crops are severely affected by the uptake of heavy metals from the soil. Heavy metals in vegetable bodies generate reactive oxygen species (ROS) that unbalance the antioxidant defense system. This study was initiated to determine the physiological and biochemical characteristics of spinach plants grown on soil contaminated with heavy metals and responding to Bacillus cereus and Bacillus aerius were isolated from soil contaminated with heavy metals. Heavy metal contamination led to a significant reduction in seed germination, seedling biomass, protein, and total nitrogen content of spinach plants grown in contaminated soils compared to control soils. In contrast, a significant increase in the content of metallothioneins and antioxidant enzymes was observed. Plants inoculated with B. cereus and B. aerius significantly reduced the oxidative stress induced by heavy metals by improving seed germination (%), seedling growth, nitrogen, and protein content. The content of metallothioneins and the activities of antioxidant enzymes were reduced in spinach plants grown from seeds inoculated with bacterial strains. In addition, plants inoculated with, B. cereus and B. aerius showed greater stomata opening than plants grown on soil contaminated with heavy metals, whose stomata were almost closed. These results suggested that both bacterial strains enhanced plant growth by reducing oxidative stress caused by metals.

Utilizing bio-synthesis of nanomaterials as biological agents for controlling soil-borne diseases in pepper plants: root-knot nematodes and root rot fungus.

The utilization of Trichoderma longibrachiatum filtrate as a safe biocontrol method for producing zinc nanoparticles is a promising approach for managing pests and diseases in agricultural crops. The identification of Trichoderma sp. was achieved through PCR amplification and sequencing of 18s as ON203115, while the synthesis of ZnO-NPs was accomplished by employing Trichoderma filtration. The presence of ZnO-NPs was confirmed by observing a color change to dark green, along with the use of visible and UV spectrophotometers, and the formation and chemical structure of ZnO-NPs were examined. Direct exposure to ZnO-NPs exhibited a significant inhibitory effect on the growth of Fusarium oxysporum at 80.73% compared with control. Also, the percent mortality of Meloidogyne incognita second juveniles stage (J2s) results showed 11.82%, 37.63%, 40.86%, and 89.65% after 6, 12, 24, and 72 h, respectively in vitro. Disease resistance was assessed in the greenhouse against M. incognita and F. oxysporum using the drench application of ZnO-NPs. The application of ZnO-NPs significantly reduced the disease severity of F. oxysporum and improved the quality and quantity of sweet pepper yield. In addition, the application of ZnO-NPs to M. incognita resulted in a significant reduction in the number of nematode galls, egg masses per root, eggs/egg mass, and females by 98%, 99%, 99.9%, and 95.5% respectively.Furthermore, it was observed that the application of ZnO-NPs to pepper plants not only inhibited the growth of F. oxysporum and M. incognita, but also promoted the recovery of pepper plants as indicated by improvements in stem length by 106%, root length 102%, fresh weight 112%, root fresh weight 107%, and leaf area 118% compared to healthy control plants. Additionally, real-time PCR application and DD-PCR technique revealed that the application of ZnO-NPs stimulated the secretion of certain enzymes. These findings suggest that the biosynthesized ZnO-NPs possess anti-nematode and antifungal properties, making them effective for protecting plants against M. incognita and F. oxysporum invasion in soil. This study significantly contributes to our understanding of the nematicidal and fungicidal activities of ZnO-NPs in suppressing soil-borne diseases.

Potential fungicidal and antiaflatoxigenic effects of cinnamon essential oils on Aspergillus flavus inhabiting the stored wheat grains.

Wheat is one of the essential crops for the human and animal nutrition, however, contamination with aflatoxigenic fungi, due to the improper storage conditions and high humidity, was the main global threats. So, preventing the growth of aflatoxigenic fungi in stored wheat grains, by using different essential oils was the main objective of this work. Aspergillus flavus EFBL-MU12 PP087400, EFBL-MU23 PP087401 and EFBL-MU36 PP087403 isolates were the most potent aflatoxins producers inhabiting wheat grains. The effect of storage conditions of wheat grains "humidity, temperature, incubation period, and pH" on growth of A. flavus, was assessed by the response surface methodology using Plackett-Burman design and FCCD. The highest yield of aflatoxins EFBL-MU12 B1 and B2 by A. flavus grown on wheat grains were 145.3 and 7.6 µg/kg, respectively, at incubation temperature 35°C, 16% moisture contents, initial pH 5.0, and incubated for 14 days. The tested oils had a powerful antifungal activity for the growth and aflatoxins production by A. flavus in a concentration-dependent manner. Among these oils, cinnamon oil had the highest fungicidal activity for A. flavus at 0.125%, with about 85-90 % reduction to the aflatoxins B1 and B2, conidial pigmentation and chitin contents on wheat grains. From the SEM analysis, cinnamon oils had the most deleterious effect on A. flavus with morphological aberrations to the conidial heads, vegetative mycelia, alteration in conidiophores identity, hyphae shrank, and winding. To emphasize the effect of the essential oils on the aflatoxins producing potency of A. flavus, the molecular expression of the aflatoxins biosynthetic genes was estimated by RT-qPCR. The molecular expression of nor-1, afLR, pKsA and afLJ genes was suppressed by 94-96%, due to cinnamon oil at 0.062% compared to the control. Conclusively, from the results, cinnamon oils followed by the peppermint oils displayed the most fungicidal activity for the growth and aflatoxins production by A. flavus grown on wheat grains.

Volatile compositions and glandular trichomes of Zataria multiflora in different phenological stages under normal and drought stress conditions.

BACKGROUND: Zataria multiflora Boiss. is a medicinal and aromatic plant from the Lamiaceae family. It is extensively used in Iranian traditional medicine, mostly as a replacement for Thyme species. This study was focused on the analysis of chemical composition and the distribution and types of trichomes of Z. multiflora grown under different conditions. Equilibrium headspace analysis in combination with GC-FID-MS was used to identify volatile compounds released by aerial parts of Z. multiflora in development stages of 50 and 100% flowering under normal and drought-stress conditions. RESULTS: The main constituents were p-cymene (20.06-27.40%), γ-terpinene (12.44-16.93%), and α-pinene (6.91-16.58%) and thymol (8.52-9.99%). The highest content of p-cymene (27.40%) and thymol (9.99%) was observed in the 50% flowering stage at the 90% field capacity, while the maximum γ-terpinene (16.93%) content was recorded in the 100% flowering stage under normal conditions. Using the SEM method, it was found that peltate glandular and non-glandular trichomes are distributed on the surface of the leaf, stem, and outer side of the calyx. However, capitate trichomes only are detected on the stem and calyx in the 100% flowering and beginning of blooming stages, respectively. The type and structure of trichomes do not vary in different development stages, but they differ in density. The highest number of leaf peltate glandular trichomes was observed in the vegetative and beginning of blooming stages at 50% and 90% field capacity, respectively. Non-glandular trichomes of the stem were observed with high density in both normal and stress conditions, which are more densely in 90% field capacity. CONCLUSIONS: Since this plant has strong potential to be used in the food and pharmacological industries, this study provides valuable information for its cultivation and harvesting at specific phenological stages, depending on desired compounds and their concentrations.

Differences and similarities in biophysical and biological characteristics between U87 MG glioblastoma and astrocyte cells.

Current cancer studies focus on molecular-targeting diagnostics and interactions with surroundings; however, there are still gaps in characterization based on topological differences and elemental composition. Glioblastoma (GBM cells; GBMCs) is an astrocytic aggressive brain tumor. At the molecular level, GBMCs and astrocytes may differ, and cell elemental/topological analysis is critical for identifying potential new cancer targets. Here, we used U87 MG cells for GBMCS. U87 MG cell lines, which are frequently used in glioblastoma research, are an important tool for studying the various features and underlying mechanisms of this aggressive brain tumor. For the first time, atomic force microscopy (AFM), scanning electron microscopy (SEM) accompanied by energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS) are used to report the topology and chemistry of cancer (U87 MG) and healthy (SVG p12) cells. In addition, F-actin staining and cytoskeleton-based gene expression analyses were performed. The degree of gene expression for genes related to the cytoskeleton was similar; however, the intensity of F-actin, anisotropy values, and invasion-related genes were different. Morphologically, GBMCs were longer and narrower while astrocytes were shorter and more disseminated based on AFM. Furthermore, the roughness values of these cells differed slightly between the two call types. In contrast to the rougher astrocyte surfaces in the lamellipodial area, SEM-EDS analysis showed that elongated GBMCs displayed filopodial protrusions. Our investigation provides considerable further insight into rapid cancer cell characterization in terms of a combinatorial spectroscopic and microscopic approach.

Salt tolerance in mungbean is associated with controlling Na and Cl transport across roots, regulating Na and Cl accumulation in chloroplasts and maintaining high K in root and leaf mesophyll cells.

Salinity tolerance requires coordinated responses encompassing salt exclusion in roots and tissue/cellular compartmentation of salt in leaves. We investigated the possible control points for salt ions transport in roots and tissue tolerance to Na+ and Cl- in leaves of two contrasting mungbean genotypes, salt-tolerant Jade AU and salt-sensitive BARI Mung-6, grown in nonsaline and saline (75 mM NaCl) soil. Cryo-SEM X-ray microanalysis was used to determine concentrations of Na, Cl, K, Ca, Mg, P, and S in various cell types in roots related to the development of apoplastic barriers, and in leaves related to photosynthetic performance. Jade AU exhibited superior salt exclusion by accumulating higher [Na] in the inner cortex, endodermis, and pericycle with reduced [Na] in xylem vessels and accumulating [Cl] in cortical cell vacuoles compared to BARI Mung-6. Jade AU maintained higher [K] in root cells than BARI Mung-6. In leaves, Jade AU maintained lower [Na] and [Cl] in chloroplasts and preferentially accumulated [K] in mesophyll cells than BARI Mung-6, resulting in higher photosynthetic efficiency. Salinity tolerance in Jade AU was associated with shoot Na and Cl exclusion, effective regulation of Na and Cl accumulation in chloroplasts, and maintenance of high K in root and leaf mesophyll cells.