RESUMEN

Soil salinity is detrimental to plant growth and remains a major threat to crop productivity of the world. Plants employ various physiological and molecular mechanisms to maintain growth under salt stress. Identification of genes and genetic loci underlying plant salt tolerance holds the key to breeding salt tolerant crops. CIPK-CBL pathways regulate adaptive responses of plants (especially ion transport) to abiotic stresses via fine-tuned Ca2+ signal transduction. In this study, we showed that over-expression of OsCIPK17 in Arabidopsis enhanced primary root elongation under salt stress, which is in a Ca2+ dependent manner. Further investigation revealed that, under salt stress, OsCIPK17 transcript level was significantly induced and its protein moved from the cytosol to the tonoplast. Using both Y2H and BiFC, tonoplast-localised OsCBL2 and OsCBL3 were shown to interact with OsCIPK17. Interestingly, over-expressing salt-induced OsCBL2 or OsCBL3 in Arabidopsis led to enhanced primary root elongation under salt stress. In this process, OsCIPK17 was shown recruited to the tonoplast (similar to the effect of salt stress). Furthermore, transgenic Arabidopsis lines individually over-expressing OsCIPK17, OsCBL2 and OsCBL3 all demonstrated larger biomass and less Na + accumulation in the shoot under salt stress. All data combined suggest that OsCIPK17- OsCBL2/3 module is a major component of shoot Na+ exclusion and therefore plant salt tolerance, which is through enhanced Na + compartmentation into the vacuole in the root. OsCIPK17 and OsCBL2/3 are therefore potential genetic targets that can be used for delivering salt tolerant rice cultivars.

Asunto(s)

RESUMEN

In the course of past two decade anthropogenic activities have reinforced, begetting soil and water defilement. A plethora of heavy metals alters and limits plant growth and yield, with opposing effect on agricultural productivity. Silicon often perceived as plant alimentary 'nonentity'. A suite of determinants associated with silicon have been lately discerned, concerning plant physiology, chemistry, gene regulation/expression and interaction with different organisms. Exogenous supplementation of silicon renders resistance against heavy-metal stress. Predominantly, plants having significant amount of silicon in root and shoot thus are barely prone to pest onset and manifest greater endurance against abiotic stresses including heavy-metal toxicity. Silicon-mediated stress management involves abatement of metal ions within soil, co-precipitation of metal ions, gene modulation associated with metal transport, chelation, activation of antioxidants (enzymatic and non-enzymatic), metal ion compartmentation and structural metamorphosis in plants. Silicon supplementation also stimulates expression of stress-resistant genes under heavy-metal toxicity to provide plant tolerance under stress conditions. Ergo, to boost metal tolerance within crops, immanent genetic potential for silicon assimilation should be enhanced. Current study, addresses the potential role and mechanistic interpretation of silicon induced mitigation of heavy-metal stress in plants.

RESUMEN

Three plant pathways for the synthesis of putrescine have been described to date. These are the synthesis of putrescine from ornithine, by ornithine decarboxylase (ODC); the synthesis of putrescine from arginine by arginine decarboxylase, agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (NLP1); and arginine decarboxylase and agmatinase. To address how these pathways are organized in plants, we have used transient expression analysis of these genes in the leaves of Nicotiana benthamiana. Brassicas do not have ODC, but the single ODC gene from rice and one of the soybean genes, were localized to the ER. Transient expression of the rice agmatinase gene showed that it was localized to the mitochondria. In A. thaliana there are five isoforms of AIH and three isoforms of NLP1. Stable GFP-tagged transformants of the longest isoforms of AIH and NLP1 showed that both proteins were localized to the ER, but in tissues with chloroplasts, the localization was concentrated to lamellae adjacent to chloroplasts. Transient expression analyses showed that four of the isoforms of AIH and all of the isoforms of NLP1 were localized to the ER. However, AIH.4 was localized to the chloroplast. Combining these results with other published data, reveal that putrescine synthesis is excluded from the cytoplasm and is spatially localized to the chloroplast, ER, and likely the mitochondria. Synthesis of putrescine in the ER may facilitate cell to cell transport via plasmodesmata, or secretion via vesicles. Differential expression of these pathways may enable putrescine-mediated activation of hormone-responsive genes.

RESUMEN

The phenomenon of multicellular compartmentation in biosynthetic pathways has been documented for only a limited subset of specialized metabolites, despite its hypothesized significance in facilitating plant survival and adaptation to environmental stress. Transporters that shuttle metabolic intermediates between cells are hypothesized to be integral components enabling compartmentalized biosynthesis. Nevertheless, our understanding of the multicellular compartmentation of plant specialized metabolism and the associated intermediate transporters remains incomplete. The emergence of single-cell and spatial multiomics techniques holds promise for shedding light on unresolved questions in this field, such as the prevalence of multicellular compartmentation across the plant kingdom and the specific types of specialized metabolites whose biosynthetic pathways are prone to compartmentation. Advancing our understanding of the mechanisms underlying multicellular compartmentation will contribute to improving the production of specialized target metabolites through metabolic engineering or synthetic biology.

Asunto(s)

RESUMEN

As a novel type of catalytic material, hollow nanoreactors are expected to bring new development opportunities in the field of persulfate-based advanced oxidation processes due to their peculiar void-confinement, spatial compartmentation, and size-sieving effects. For such materials, however, further clarification on basic concepts and construction strategies, as well as a discussion of the inherent correlation between structure and catalytic activity are still required. In this context, this review aims to provide a state-of-the-art overview of hollow nanoreactors for activating persulfate. Initially, hollow nanoreactors are classified according to the constituent components of the shell structure and their dimensionality. Subsequently, the different construction strategies of hollow nanoreactors are described in detail, while common synthesis methods for these construction strategies are outlined. Furthermore, the most representative advantages of hollow nanoreactors are summarized, and their intrinsic connections to the nanoreactor structure are elucidated. Finally, the challenges and future prospects of hollow nanoreactors are presented.

RESUMEN

While the intracellular-extracellular distribution of lactate has been suggested to play a critical role in the healthy and diseased brain, tools are lacking to noninvasively probe lactate in intracellular and extracellular spaces. Here, we show that, by measuring the diffusion of lactate with diffusion-weighted magnetic resonance (MR) spectroscopy in vivo and comparing it to the diffusion of purely intracellular metabolites, noninvasive quantification of extracellular and intracellular lactate fractions becomes possible. More specifically, we detect alterations of lactate diffusion in the APP/PS1 mouse model of Alzheimer's disease. Data modeling allows quantifying decreased extracellular lactate fraction in APP/PS1 mice as compared to controls, which is quantitatively confirmed with implanted enzyme-microelectrodes. The capability of diffusion-weighted MR spectroscopy to quantify extracellular-intracellular lactate fractions opens a window into brain metabolism, including in Alzheimer's disease.

Asunto(s)

RESUMEN

White and brown adipocytes are central mediators of lipid metabolism and thermogenesis, respectively. Their function is tightly regulated by all three ß-adrenergic receptor (ß-AR) subtypes which are coupled to the production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). While known for decades in other cell types, compartmentation of adipocyte ß-AR/cAMP signaling by spatial organization of the pathway and by cAMP degrading phosphodiesterases (PDEs) as well as its role in the regulation of lipolysis is only beginning to emerge. Here, we provide a short overview of recent findings which shed light on compartmentalized signaling using live cell imaging of cAMP in adipocytes and discuss possible future directions of research which could open up new avenues for the treatment of metabolic disorders.

RESUMEN

BACKGROUND AND PURPOSE: In human airway smooth muscle (hASM) cells, not all receptors stimulating cAMP production elicit the same effects. This can only be explained if cAMP movement throughout the cell is restricted, yet the mechanisms involved are not fully understood. Phosphodiesterases (PDEs) contribute to compartmentation of many cAMP responses, but PDE activity alone is predicted to be insufficient if cAMP is otherwise freely diffusible. We tested the hypothesis that buffering of cAMP by protein kinase A (PKA) associated with A kinase anchoring proteins (AKAPs) slows cAMP diffusion and that this contributes to receptor-mediated, compartmentalized responses. EXPERIMENTAL APPROACH: Raster image correlation spectroscopy (RICS) was used to measure intracellular cAMP diffusion coefficients and evaluate the contribution of PKA-AKAP interactions. Western blotting and immunocytochemistry were used to identify the AKAPs involved. RNA interference was used to down-regulate AKAP expression and determine its effects on cAMP diffusion. Compartmentalized cAMP responses were measured using fluorescence resonance energy transfer (FRET) based biosensors. KEY RESULTS: Cyclic AMP movement was significantly slower than that of free-diffusion in hASM cells, and disrupting PKA-AKAP interactions significantly increased the diffusion coefficient. PKA associated with the outer mitochondrial membrane appears to play a prominent role in this effect. Consistent with this idea, knocking down expression of D-AKAP2, the primary mitochondrial AKAP, increased cAMP diffusion and disrupted compartmentation of receptor-mediated responses. CONCLUSION AND IMPLICATIONS: Our results confirm that AKAP-anchored PKA contributes to the buffering of cAMP and is consequential in the compartmentation of cAMP responses in hASM cells.

Asunto(s)

RESUMEN

Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

Asunto(s)

RESUMEN

Dysregulated macrophage responses and changes in tissue metabolism are hallmarks of chronic inflammation in the skin. However, the metabolic cues that direct and support macrophage functions in the skin are poorly understood. Here, we show that during sterile skin inflammation, the epidermis and macrophages uniquely depend on glycolysis and the TCA cycle, respectively. This compartmentalisation is initiated by ROS-induced HIF-1α stabilization leading to enhanced glycolysis in the epidermis. The end-product of glycolysis, lactate, is then exported by epithelial cells and utilized by the dermal macrophages to induce their M2-like fates through NF-κB pathway activation. In addition, we show that psoriatic skin disorder is also driven by such lactate metabolite-mediated crosstalk between the epidermis and macrophages. Notably, small-molecule inhibitors of lactate transport in this setting attenuate sterile inflammation and psoriasis disease burden, and suppress M2-like fate acquisition in dermal macrophages. Our study identifies an essential role for the metabolite lactate in regulating macrophage responses to inflammation, which may be effectively targeted to treat inflammatory skin disorders such as psoriasis.

Asunto(s)

RESUMEN

MAIN CONCLUSION: Auto-fluorescent condensed tannins specifically accumulated in mesophyll cells of non-salt secretor mangroves are involved in the compartmentation of Na+ and osmotic regulation, contributing to their salt tolerance. Salinity is a major abiotic stress affecting the distribution and growth of mangrove plants. The salt exclusion mechanism from salt secretor mangrove leaves is quite known; however, salt management strategies in non-salt secretor leaves remain unclear. In this study, we reported the auto-fluorescent inclusions (AFIs) specifically accumulated in mesophyll cells (MCs) of four non-salt secretor mangroves but absent in three salt secretors. The AFIs increased with the leaf development under natural condition, and applied NaCl concentrations applied in the lab. The AFIs in MCs were isolated and identified as condensed tannin accretions (CTAs) using the dye dimethyl-amino-cinnamaldehyde (DMACA), specific for condensed tannin (CT), both in situ leaf cross sections and in the purified AFIs. Fluorescence microscopy and transmission electron microscope (TEM) analysis indicated that the CTAs originated from the inflated chloroplasts. The CTAs had an obvious membrane and could induce changes in shape and fluorescence intensity in hypotonic and hypertonic NaCl solutions, suggesting CTAs might have osmotic regulation ability and play an important role in the osmotic regulation in MCs. The purified CTAs were labeled by the fluorescent sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM), confirming they were involved in the compartmentation of excess Na+ in MCs. This study provided a new view on the salt resistance-associated strategies in mangroves.

Asunto(s)

RESUMEN

Mitochondria undergo structural changes reflective of functional statuses. Ultrastructural characterizing of mitochondria is valuable for understanding mitochondrial dysfunction in various pathological conditions. PINK1, a Parkinson's disease (PD) associated gene, plays key roles in maintaining mitochondrial function and integrity. In Drosophila melanogaster, deficiency of PINK1 results in PD-like pathologies due to mitochondrial abnormalities. Here, we report the existence of a new type of mitochondrial-membrane deformity, mitochondrial spherical compartmentation (MSC), caused by PINK1 deficiency in Drosophila. The MSC is a three-dimensional spheroid-like mitochondrial membrane structure encompassing nonselective contents. Upregulation of dDrp1, downregulation of dMarf, and upregulation of dArgK1-A-all resulting in mitochondrial fragmentation-were able to suppress the formation of MSC. Furthermore, arginine kinase, only when localizing to the vicinity of mitochondria, induced mitochondrial fragmentation and reversed the MSC phenotype. In summary, this study demonstrates that loss of dPINK1 leads to the formation of mitochondrial-membrane deformity MSC, which responds to mitochondrial dynamics. In addition, our data suggest a new perspective of how phosphagen energy-buffer system might regulate mitochondrial dynamics.

RESUMEN

G protein-coupled receptors (GPCRs) are ligand-activated cell membrane proteins and represent the most important class of drug targets. GPCRs adopt several active conformations that stimulate different intracellular G proteins (and other transducers) and thereby modulate second messenger levels, eventually resulting in receptor-specific cell responses. It is increasingly accepted that not only the type of active signaling protein but also the duration of its stimulation and the subcellular location from where receptors signal distinctly contribute to the overall cell response. However, the molecular principles governing such spatiotemporal GPCR signaling and their role in disease are incompletely understood. Genetically encoded, fluorescent biosensors-in particular for the GPCR/cAMP signaling axis-have been pivotal to the discovery and molecular understanding of novel concepts in spatiotemporal GPCR signaling. These include GPCR priming, location bias, and receptor-associated independent cAMP nanodomains. Here, we review such technologies that we believe will illuminate the spatiotemporal organization of other GPCR signaling pathways that define the complex signaling architecture of the cell.

Asunto(s)

RESUMEN

The application of high throughput synthesis methodologies in the generation of active pharmaceutical ingredients (APIs) currently requires the use of automated and easily scalable systems, easy dispensing of supported reagents in solution phase organic synthesis (SPOS), and elimination of purification and extraction steps. The recyclability and recoverability of supported reagents and/or catalysts in a rapid and individualized manner is a challenge in the pharmaceutical industry. This objective can be achieved through a suitable compartmentalization of these pulverulent reagents in suitable devices for it. This work deals with the use of customized polypropylene permeable-capsule devices manufactured by 3D printing, using the fused deposition modeling (FDM) technique, adaptable to any type of flask or reactor. The capsules fabricated in this work were easily loaded "in one step" with polymeric reagents for use as scavengers of isocyanides in the work-up process of Ugi multicomponent reactions or as compartmentalized and reusable catalysts in copper-catalyzed cycloadditions (CuAAC) or Heck palladium catalyzed cross-coupling reactions (PCCCRs). The reaction products are different series of diversely substituted isatins, which were tested in cancerous cervical HeLa and murine 3T3 Balb fibroblast cells, obtaining potent antiproliferative activity. This work demonstrates the applicability of 3D printing in chemical processes to obtain anticancer APIs.

Asunto(s)

RESUMEN

The ATP-sensitive K+ (KATP) channel is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATP channel activity in pancreatic ß cells and the lesser-studied α cells, from both humans and mice, we provide evidence that a glycolytic metabolon locally controls KATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further show the presence of a plasma membrane-associated NAD+/NADH cycle whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.

Asunto(s)

RESUMEN

Enhanced detoxification is a prominent mechanism protecting plants from toxic xenobiotics and endows resistance to diverse herbicide chemistries in grass weeds such as blackgrass (Alopecurus myosuroides). The roles of enzyme families which impart enhanced metabolic resistance (EMR) to herbicides through hydroxylation (phase 1 metabolism) and/or conjugation with glutathione or sugars (phase 2) have been well established. However, the functional importance of herbicide metabolite compartmentalisation into the vacuole as promoted by active transport (phase 3), has received little attention as an EMR mechanism. ATP-binding cassette (ABC) transporters are known to be important in drug detoxification in fungi and mammals. In this study, we identified three distinct C-class ABCCs transporters namely AmABCC1, AmABCC2 and AmABCC3 in populations of blackgrass exhibiting EMR and resistance to multiple herbicides. Uptake studies with monochlorobimane in root cells, showed that the EMR blackgrass had an enhanced capacity to compartmentalize fluorescent glutathione-bimane conjugated metabolites in an energy-dependent manner. Subcellular localisation analysis using transient expression of GFP-tagged AmABCC2 assays in Nicotiana demonstrated that the transporter was a membrane bound protein associated with the tonoplast. At the transcript level, as compared with herbicide sensitive plants, AmABCC1 and AmABCC2 were positively correlated with EMR in herbicide resistant blackgrass being co-expressed with AmGSTU2a, a glutathione transferase (GST) involved in herbicide detoxification linked to resistance. As the glutathione conjugates generated by GSTs are classic ligands for ABC proteins, this co-expression suggested AmGSTU2a and the two ABCC transporters delivered the coupled rapid phase 2/3 detoxification observed in EMR. A role for the transporters in resistance was further confirmed in transgenic yeast by demonstrating that the expression of either AmABCC1 or AmABCC2, promoted enhanced tolerance to the sulfonylurea herbicide, mesosulfuron-methyl. Our results link the expression of ABCC transporters to enhanced metabolic resistance in blackgrass through their ability to transport herbicides, and their metabolites, into the vacuole.

RESUMEN

Autism is characterized by atypical social communication and stereotyped behaviors. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1-2% of patients with autism and intellectual disability, but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterized the behavior of Shank3 Δ11/Δ11 mice from 3 to 12 months of age. We observed decreased locomotor activity, increased stereotyped self-grooming and modification of socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEGs). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g., Grm2, Dlgap1), G-protein-signaling pathways (e.g., Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g., Gad2). Downregulated and upregulated genes were enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN), respectively. Several DEGs (Cnr1, Gnal, Gad2, and Drd4) were reported as striosome markers. By studying the distribution of the glutamate decarboxylase GAD65, encoded by Gad2, we showed that the striosome compartment of Shank3 Δ11/Δ11 mice was enlarged and displayed much higher expression of GAD65 compared to wild-type mice. Altogether, these results indicate altered gene expression in the striatum of Shank3-deficient mice and strongly suggest, for the first time, that the excessive self-grooming of these mice is related to an imbalance in the striatal striosome and matrix compartments.

RESUMEN

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

Asunto(s)

RESUMEN

Lipid droplets, also known as oil bodies or lipid bodies, are plant organelles that compartmentalize neutral lipids as a hydrophobic matrix covered by proteins embedded in a phospholipid monolayer. Some of these proteins have been known for decades, such as oleosins, caleosins, and steroleosins, whereas a host of others have been discovered more recently with various levels of abundance on lipid droplets, depending on the tissue and developmental stage. In addition to a growing inventory of lipid droplet proteins, the subcellular machinery that contributes to the biogenesis and degradation of lipid droplets is being identified and attention is turning to more mechanistic questions regarding lipid droplet dynamics. While lipid droplets are mostly regarded as storage deposits for carbon and energy in lipid-rich plant tissues such as seeds, these organelles are present in essentially all plant cells, where they display additional functions in signaling, membrane remodeling, and the compartmentalization of a variety of hydrophobic components. Remarkable metabolic engineering efforts have demonstrated the plasticity of vegetative tissues such as leaves to synthesize and package large amounts of storage lipids, which enable future applications in bioenergy and the engineering of high-value lipophilic compounds. Here, we review the growing body of knowledge about lipid droplets in plant cells, describe the evolutionary similarity and divergence in their associated subcellular machinery, and point to gaps that deserve future attention.

Asunto(s)