SWEET11b transports both sugar and cytokinin in developing barley grains.

Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin. Barley plants harboring a loss-of-function mutation of HvSWEET11b could not set viable grains, while the distribution of sucrose and cytokinin was altered in developing grains of plants in which the gene was knocked down. Sucrose allocation within transgenic grains was disrupted, which is consistent with the changes to the cytokinin gradient across grains, as visualized by magnetic resonance imaging and Fourier transform infrared spectroscopy microimaging. Decreasing HvSWEET11b expression in developing grains reduced overall grain size, sink strength, the number of endopolyploid endosperm cells, and the contents of starch and protein. The control exerted by HvSWEET11b over sugars and cytokinins likely predetermines their synergy, resulting in adjustments to the grain's biochemistry and transcriptome.

Hello darkness, my old friend: 3-KETOACYL-COENZYME A SYNTHASE4 is a branch point in the regulation of triacylglycerol synthesis in Arabidopsis thaliana.

Plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness or extended darkness to a panel of ∼300 Arabidopsis (Arabidopsis thaliana) accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding an enzyme involved in very long chain fatty acid (VLCFA) synthesis, underlies the differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression of KCS4 in yeast and plants proved that KCS4 is a functional enzyme localized in the endoplasmic reticulum with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and leaf wax coverage, puTAG accumulation, and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlates with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation.

Integrated multi-omics analyses and genome-wide association studies reveal prime candidate genes of metabolic and vegetative growth variation in canola.

Genome-wide association studies (GWAS) identified thousands of genetic loci associated with complex plant traits, including many traits of agronomical importance. However, functional interpretation of GWAS results remains challenging because of large candidate regions due to linkage disequilibrium. High-throughput omics technologies, such as genomics, transcriptomics, proteomics and metabolomics open new avenues for integrative systems biological analyses and help to nominate systems information supported (prime) candidate genes. In the present study, we capitalise on a diverse canola population with 477 spring-type lines which was previously analysed by high-throughput phenotyping of growth-related traits and by RNA sequencing and metabolite profiling for multi-omics-based hybrid performance prediction. We deepened the phenotypic data analysis, now providing 123 time-resolved image-based traits, to gain insight into the complex relations during early vegetative growth and reanalysed the transcriptome data based on the latest Darmor-bzh v10 genome assembly. Genome-wide association testing revealed 61 298 robust quantitative trait loci (QTL) including 187 metabolite QTL, 56814 expression QTL and 4297 phenotypic QTL, many clustered in pronounced hotspots. Combining information about QTL colocalisation across omics layers and correlations between omics features allowed us to discover prime candidate genes for metabolic and vegetative growth variation. Prioritised candidate genes for early biomass accumulation include A06p05760.1_BnaDAR (PIAL1), A10p16280.1_BnaDAR, C07p48260.1_BnaDAR (PRL1) and C07p48510.1_BnaDAR (CLPR4). Moreover, we observed unequal effects of the Brassica A and C subgenomes on early biomass production.

From data to knowledge - big data needs stewardship, a plant phenomics perspective.

The research data life cycle from project planning to data publishing is an integral part of current research. Until the last decade, researchers were responsible for all associated phases in addition to the actual research and were assisted only at certain points by IT or bioinformaticians. Starting with advances in sequencing, the automation of analytical methods in all life science fields, including in plant phenotyping, has led to ever-increasing amounts of ever more complex data. The tasks associated with these challenges now often exceed the expertise of and infrastructure available to scientists, leading to an increased risk of data loss over time. The IPK Gatersleben has one of the world's largest germplasm collections and two decades of experience in crop plant research data management. In this article we show how challenges in modern, data-driven research can be addressed by data stewards. Based on concrete use cases, data management processes and best practices from plant phenotyping, we describe which expertise and skills are required and how data stewards as an integral actor can enhance the quality of a necessary digital transformation in progressive research.

Rhizosphere microbiome mediates systemic root metabolite exudation by root-to-root signaling.

Microbial communities associated with roots confer specific functions to their hosts, thereby modulating plant growth, health, and productivity. Yet, seminal questions remain largely unaddressed including whether and how the rhizosphere microbiome modulates root metabolism and exudation and, consequently, how plants fine tune this complex belowground web of interactions. Here we show that, through a process termed systemically induced root exudation of metabolites (SIREM), different microbial communities induce specific systemic changes in tomato root exudation. For instance, systemic exudation of acylsugars secondary metabolites is triggered by local colonization of bacteria affiliated with the genus Bacillus Moreover, both leaf and systemic root metabolomes and transcriptomes change according to the rhizosphere microbial community structure. Analysis of the systemic root metabolome points to glycosylated azelaic acid as a potential microbiome-induced signaling molecule that is subsequently exuded as free azelaic acid. Our results demonstrate that rhizosphere microbiome assembly drives the SIREM process at the molecular and chemical levels. It highlights a thus-far unexplored long-distance signaling phenomenon that may regulate soil conditioning.

Steroidal alkaloids defence metabolism and plant growth are modulated by the joint action of gibberellin and jasmonate signalling.

Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear. We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism. We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein-protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism. These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.

Method to analyze effects of low-level laser therapy on biological cells with a digital holographic microscope.

Low-level laser therapy (LLLT) is a therapeutic tool that uses the photobiochemical interaction between light and tissue. Its effectiveness is controversial due to a strong dependence on dosimetric parameters. In this work, we demonstrate that digital holographic microscopy is an effective label-free imaging technique to analyze the effects of LLLT on biological cells, and we propose the full methodology to create correct synthetic aperture phase maps for further extensive, highly accurate statistical analysis. The proposed methodology has been designed to provide a basis for many other biological experiments using quantitative phase imaging. We use SHSY-5Y and HaCaT cells irradiated with different doses of red light for the experiment. The analysis shows quantitative changes in cell dry mass density and the projected cell surface in response to different radiation doses.

Mitochondrial Network and Biogenesis in Response to Short and Long-Term Exposure of Human BEAS-2B Cells to Aerosol Extracts from the Tobacco Heating System 2.2.

BACKGROUND/AIMS: Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2). METHODS: Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot. RESULTS: In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process. CONCLUSION: Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.

A Multilevel Study of Melon Fruit Reticulation Provides Insight into Skin Ligno-Suberization Hallmarks.

The skin of fleshy fruit is typically covered by a thick cuticle. Some fruit species develop different forms of layers directly above their skin. Reticulation, for example, is a specialized suberin-based coating that ornaments some commercially important melon (Cucumis melo) fruit and is an important quality trait. Despite its importance, the structural, molecular, and biochemical features associated with reticulation are not fully understood. Here, we performed a multilevel investigation of structural attributes, chemical composition, and gene expression profiles on a set of reticulated and smooth skin melons. High-resolution microscopy, surface profiling, and histochemical staining assays show that reticulation comprises cells with heavily suberized walls accumulating large amounts of typical suberin monomers, as well as lignified cells localized underneath the specialized suberized cell layer. Reticulated skin was characterized by induced expression of biosynthetic genes acting in the core phenylpropanoid, suberin, lignin, and lignan pathways. Transcripts of genes associated with lipid polymer assembly, cell wall organization, and loosening were highly enriched in reticulated skin tissue. These signatures were exclusive to reticulated structures and absent in both the smooth surfaces observed in between reticulated regions and in the skin of smooth fruit. Our data provide important insights into the molecular and metabolic bases of reticulation and its tight association with skin ligno-suberization during melon fruit development. Moreover, these insights are likely to contribute to melon breeding programs aimed at improving postharvest qualities associated with fleshy fruit surface layers.

Distinction of sporadic and familial forms of ALS based on mitochondrial characteristics.

Bioenergetic failure, oxidative stress, and changes in mitochondrial morphology are common pathologic hallmarks of amyotrophic lateral sclerosis (ALS) in several cellular and animal models. Disturbed mitochondrial physiology has serious consequences for proper functioning of the cell, leading to the chronic mitochondrial stress. Mitochondria, being in the center of cellular metabolism, play a pivotal role in adaptation to stress conditions. We found that mitochondrial dysfunction and adaptation processes differ in primary fibroblasts derived from patients diagnosed with either sporadic or familial forms of ALS. The evaluation of mitochondrial parameters such as the mitochondrial membrane potential, the oxygen consumption rate, the activity and levels of respiratory chain complexes, and the levels of ATP, reactive oxygen species, and Ca2+ show that the bioenergetic properties of mitochondria are different in sporadic ALS, familial ALS, and control groups. Comparative statistical analysis of the data set (with use of principal component analysis and support vector machine) identifies and distinguishes 3 separate groups despite the small number of investigated cell lines and high variability in measured parameters. These findings could be a first step in development of a new tool for predicting sporadic and familial forms of ALS and could contribute to knowledge of its pathophysiology.-Walczak, J., Debska-Vielhaber, G., Vielhaber, S., Szymanski, J., Charzynska, A., Duszynski, J., Szczepanowska, J. Distinction of sporadic and familial forms of ALS based on mitochondrial characteristics.

Involvement of a gut-retina axis in protection against dietary glycemia-induced age-related macular degeneration.

Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf. LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut-retina axis to protect against diet- and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD.

Mitochondria as a possible target for nicotine action.

Mitochondria are multifunctional and dynamic organelles deeply integrated into cellular physiology and metabolism. Disturbances in mitochondrial function are involved in several disorders such as neurodegeneration, cardiovascular diseases, metabolic diseases, and also in the aging process. Nicotine is a natural alkaloid present in the tobacco plant which has been well studied as a constituent of cigarette smoke. It has also been reported to influence mitochondrial function both in vitro and in vivo. This review presents a comprehensive overview of the present knowledge of nicotine action on mitochondrial function. Observed effects of nicotine exposure on the mitochondrial respiratory chain, oxidative stress, calcium homeostasis, mitochondrial dynamics, biogenesis, and mitophagy are discussed, considering the context of the experimental design. The potential action of nicotine on cellular adaptation and cell survival is also examined through its interaction with mitochondria. Although a large number of studies have demonstrated the impact of nicotine on various mitochondrial activities, elucidating its mechanism of action requires further investigation.

Label-free deep shotgun proteomics reveals protein dynamics during tomato fruit tissues development.

Current innovations in mass-spectrometry-based technologies allow deep coverage of protein expression. Despite its immense value and in contrast to transcriptomics, only a handful of studies in crop plants engaged with global proteome assays. Here, we present large-scale shotgun proteomics profiling of tomato fruit across two key tissues and five developmental stages. A total of 7738 individual protein groups were identified and reliably measured at least in one of the analyzed tissues or stages. The depth of our assay enabled identification of 61 differentially expressed transcription factors, including renowned ripening-related regulators and elements of ethylene signaling. Significantly, we measured proteins involved in 83% of all predicted enzymatic reactions in the tomato metabolic network. Hence, proteins representing almost the complete set of reactions in major metabolic pathways were identified, including the cytosolic and plastidic isoprenoid and the phenylpropanoid pathways. Furthermore, the data allowed us to discern between protein isoforms according to expression patterns, which is most significant in light of the weak transcript-protein expression correspondence. Finally, visualization of changes in protein abundance associated with a particular process provided us with a unique view of skin and flesh tissues in developing fruit. This study adds a new dimension to the existing genomic, transcriptomic and metabolomic resources. It is therefore likely to promote translational and post-translational research in tomato and additional species, which is presently focused on transcription.

Assimilation of 'omics' strategies to study the cuticle layer and suberin lamellae in plants.

The assembly of the lipophilic cuticle layer and suberin lamellae, approximately 450 million years ago, was a major evolutionary development that enabled plants to colonize terrestrial habitats. The cuticle layer is composed of cutin polyester and embedded cuticular waxes, whereas the suberin lamellae consist of very long chain fatty acid derivatives, glycerol, and phenolics cross-linked with alkyl ferulate-embedded waxes. Due to their substantial biological roles in plant life, the mechanisms underlying the assembly of these structures have been extensively investigated. In the last decade, the introduction of 'omics' approaches, including genomics, transcriptomics, proteomics, and metabolomics, have been key in the identification of novel genetic and chemical elements involved in the formation and function of the cuticle layer and suberin lamellae. This review summarizes contemporary studies that utilized various large-scale, 'omics' strategies in combination with novel technologies to unravel how building blocks and polymers of these lipophilic barriers are made, and moreover linking structure to function along developmental programs and stress responses. We anticipate that the studies discussed here will inspire scientists studying lipophilic barriers to integrate complementary 'omics' approaches in their efforts to tackle as yet unresolved questions and engage the main challenges of the field to date.

Linking gene expression and membrane lipid composition of Arabidopsis.

Glycerolipid metabolism of plants responds dynamically to changes in light intensity and temperature, leading to the modification of membrane lipid composition to ensure optimal biochemical and physical properties in the new environment. Although multiple posttranscriptional regulatory mechanisms have been reported to be involved in the process, the contribution of transcriptional regulation remains largely unknown. Here, we present an integrative analysis of transcriptomic and lipidomic data, revealing large-scale coordination between gene expression and changes in glycerolipid levels during the Arabidopsis thaliana response to light and temperature stimuli. Using a multivariate regression technique called O2PLS, we show that the gene expression response is strictly coordinated at the biochemical pathway level and occurs in parallel with changes of specific glycerolipid pools. Five interesting candidate genes were chosen for further analysis from a larger set of candidates identified based on their close association with various groups of glycerolipids. Lipidomic analysis of knockout mutant lines of these five genes showed a significant relationship between the coordination of transcripts and glycerolipid levels in a changing environment and the effects of single gene perturbations.

Interaction of Mitochondria with the Endoplasmic Reticulum and Plasma Membrane in Calcium Homeostasis, Lipid Trafficking and Mitochondrial Structure.

Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them that contribute to the control and regulation of various cellular processes, such as calcium and lipid exchange or structural reorganization of the mitochondrial network. In recent years, many studies focused their attention on the structure and function of contacts between mitochondria and other organelles. From these studies, findings emerged showing that these contacts are involved in various processes, such as lipid synthesis and trafficking, modulation of mitochondrial morphology, endoplasmic reticulum (ER) stress, apoptosis, autophagy, inflammation and Ca 2 + handling. In this review, we focused on the physical interactions of mitochondria with the endoplasmic reticulum and plasma membrane and summarized present knowledge regarding the role of mitochondria-associated membranes in calcium homeostasis and lipid metabolism.

[Mechanism of mitochondrial fission - structure and function of Drp1 protein]. / Mechanizm fragmentacji mitochondriów - struktura i funkcja bialka Drp1.

In the cell mitochondria constitute a dynamic network undergoing continuous reshaping by fusion and fission. Mitochondrial fission is involved in several crucial cellular processes such as mitosis, apoptosis and mitophagy. Main mediator of mitochondrial fission is Dynamin related protein 1 (Drp1). This protein is able to assemble into higher order oligomers, what enables the formation of Drp1 spiral structures on the surface of mitochondrial network. These spirals constrict thanks to the energy gained from GTP hydrolysis, what results in mitochondrial fission. Mitochondrial fission process is precisely regulated by different mechanisms, especially by controlling Drp1 activity. This article presents our current understanding of mitochondrial fission with a particular focus on the role of Drp1 in this process and mechanisms that regulate activity of this protein.

Salt stress and senescence: identification of cross-talk regulatory components.

Leaf senescence is an active process with a pivotal impact on plant productivity. It results from extensive signalling cross-talk coordinating environmental factors with intrinsic age-related mechanisms. Although many studies have shown that leaf senescence is affected by a range of external parameters, knowledge about the regulatory systems that govern the interplay between developmental programmes and environmental stress is still vague. Salinity is one of the most important environmental stresses that promote leaf senescence and thus affect crop yield. Improving salt tolerance by avoiding or delaying senescence under stress will therefore play an important role in maintaining high agricultural productivity. Experimental evidence suggests that hydrogen peroxide (H2O2) functions as a common signalling molecule in both developmental and salt-induced leaf senescence. In this study, microarray-based gene expression profiling on Arabidopsis thaliana plants subjected to long-term salinity stress to induce leaf senescence was performed, together with co-expression network analysis for H2O2-responsive genes that are mutually up-regulated by salt induced- and developmental leaf senescence. Promoter analysis of tightly co-expressed genes led to the identification of seven cis-regulatory motifs, three of which were known previously, namely CACGTGT and AAGTCAA, which are associated with reactive oxygen species (ROS)-responsive genes, and CCGCGT, described as a stress-responsive regulatory motif, while the others, namely ACGCGGT, AGCMGNC, GMCACGT, and TCSTYGACG were not characterized previously. These motifs are proposed to be novel elements involved in the H2O2-mediated control of gene expression during salinity stress-triggered and developmental senescence, acting through upstream transcription factors that bind to these sites.

Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy.

The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.

Nuclear Rac1 controls nuclear architecture and cell migration of glioma cells.

Rac1 (Ras-related C3 botulinum toxin substrate 1) protein has been found in the cell nucleus many years ago, however, its nuclear functions are still poorly characterized but some data suggest its nuclear accumulation in cancers. We investigated nuclear Rac1 in glioma cancer cells nuclei and compared its levels and activity to normal astrocytes, and also characterized the studied cells on various nuclear properties and cell migration patterns. Nuclear Rac1 indeed was found accumulated in glioma cells, but only a small percentage of the protein was in active, GTP-bound state in comparison to healthy control. Altering the nuclear activity of Rac1 influenced chromatin architecture and cell motility in GTP-dependent and independent manner. This suggests that the landscape of Rac1 nuclear interactions might be as complicated and wide as its well-known, non-nuclear signaling.