How do the carbon and nitrogen sources affect the synthesis of ß-(1,3/1,6)-glucan, its structure and the susceptibility of Candida utilis yeast cells to immunolabelling with ß-(1,3)-glucan monoclonal antibodies?

BACKGROUND: The need to limit antibiotic therapy due to the spreading resistance of pathogenic microorganisms to these medicinal substances stimulates research on new therapeutic agents, including the treatment and prevention of animal diseases. This is one of the goals of the European Green Deal and the Farm-To-Fork strategy. Yeast biomass with an appropriate composition and exposure of cell wall polysaccharides could constitute a functional feed additive in precision animal nutrition, naturally stimulating the immune system to fight infections. RESULTS: The results of the research carried out in this study showed that the composition of Candida utilis ATCC 9950 yeast biomass differed depending on growth medium, considering especially the content of ß-(1,3/1,6)-glucan, α-glucan, and trehalose. The highest ß-(1,3/1,6)-glucan content was observed after cultivation in deproteinated potato juice water (DPJW) as a nitrogen source and glycerol as a carbon source. Isolation of the polysaccharide from yeast biomass confirmed the highest yield of ß-(1,3/1,6)-glucan after cultivation in indicated medium. The differences in the susceptibility of ß-(1,3)-glucan localized in cells to interaction with specific ß-(1,3)-glucan antibody was noted depending on the culture conditions. The polymer in cells from the DPJW supplemented with glycerol and galactose were labelled with monoclonal antibodies with highest intensity, interestingly being less susceptible to such an interaction after cell multiplication in medium with glycerol as carbon source and yeast extract plus peptone as a nitrogen source. CONCLUSIONS: Obtained results confirmed differences in the structure of the ß-(1,3/1,6)-glucan polymers considering side-chain length and branching frequency, as well as in quantity of ß-(1,3)- and ß-(1,6)-chains, however, no visible relationship was observed between the structural characteristics of the isolated polymers and its susceptibility to immunolabeling in whole cells. Presumably, other outer surface components and molecules can mask, shield, protect, or hide epitopes from antibodies. ß-(1,3)-Glucan was more intensely recognized by monoclonal antibody in cells with lower trehalose and glycogen content. This suggests the need to cultivate yeast biomass under appropriate conditions to fulfil possible therapeutic functions. However, our in vitro findings should be confirmed in further studies using tissue or animal models.

The distal central pair segment is structurally specialised and contributes to IFT turnaround and assembly of the tip capping structures in Chlamydomonas flagella.

BACKGROUND INFORMATION: Cilia and flagella are dynamic organelles whose assembly and maintenance depend on an activetrafficking process known as the IntraFlagellar Transport (IFT), during which trains of IFT protein particles are moved by specific motors and carry flagellar precursors and turnover products along the axoneme. IFT consists of an anterograde (from base to tip) and a retrograde (from tip to base) phase. During IFT turnaround at the flagellar tip, anterograde trains release their cargoes and remodel to form the retrograde trains. Thus, turnaround is crucial for correct IFT. However, current knowledge of its mechanisms is limited. RESULTS: We show here that in Chlamydomonas flagella the distal ∼200 nm central pair (CP) segment is structurally differentiated for the presence of a ladder-like structure (LLS). During IFT turnaround, the IFT172 subunit dissociates from the IFT- B protein complex and binds to the LLS-containing CP segment, while the IFT-B complex participates in the assembly of the CP capping structures. The IFT scaffolding function played by the LLS-containing CP segment relies on anchoring components other than the CP microtubules, since IFT turnaround occurs also in the CP-devoid pf18 mutant flagella. CONCLUSIONS: During IFT turnaround in Chlamydomonas flagella, i) the LLS and the CP terminal plates act as anchoring platforms for IFT172 and the IFT-B complex, respectively, and ii) during its remodeling, the IFT-B complex contributes to the assembly of the CP capping structures. SIGNIFICANCE: Our results indicate that in full length Chlamydomonas flagella IFT remodeling occurs by a specialized mechanism that involves flagellar tip structures and is distinct from the previously proposed model in which the capability to reverse motility would be intrinsic of IFT train and independent by any other flagellar structure.

Gradients of cell wall nano-mechanical properties along and across elongating primary roots of maize.

To test the hypothesis that particular tissues can control root growth, we analysed the mechanical properties of cell walls belonging to different tissues of the apical part of the maize root using atomic force microscopy. The dynamics of properties during elongation growth were characterized in four consecutive zones of the root. Extensive immunochemical characterization and quantification were used to establish the polysaccharide motif(s) related to changes in cell wall mechanics. Cell transition from division to elongation was coupled to the decrease in the elastic modulus in all root tissues. Low values of moduli were retained in the elongation zone and increased in the late elongation zone. No relationship between the immunolabelling pattern and mechanical properties of the cell walls was revealed. When measured values of elastic moduli and turgor pressure were used in the computational simulation, this resulted in an elastic response of the modelled root and the distribution of stress and strain similar to those observed in vivo. In all analysed root zones, cell walls of the inner cortex displayed moduli of elasticity that were maximal or comparable with the maximal values among all tissues. Thus, we propose that the inner cortex serves as a growth-limiting tissue in maize roots.

The Escherichia coli Outer Membrane ß-Barrel Assembly Machinery (BAM) Crosstalks with the Divisome.

The BAM is a macromolecular machine responsible for the folding and the insertion of integral proteins into the outer membrane of diderm Gram-negative bacteria. In Escherichia coli, it consists of a transmembrane ß-barrel subunit, BamA, and four outer membrane lipoproteins (BamB-E). Using BAM-specific antibodies, in E. coli cells, the complex is shown to localize in the lateral wall in foci. The machinery was shown to be enriched at midcell with specific cell cycle timing. The inhibition of septation by aztreonam did not alter the BAM midcell localization substantially. Furthermore, the absence of late cell division proteins at midcell did not impact BAM timing or localization. These results imply that the BAM enrichment at the site of constriction does not require an active cell division machinery. Expression of the Tre1 toxin, which impairs the FtsZ filamentation and therefore midcell localization, resulted in the complete loss of BAM midcell enrichment. A similar effect was observed for YidC, which is involved in the membrane insertion of cell division proteins in the inner membrane. The presence of the Z-ring is needed for preseptal peptidoglycan (PG) synthesis. As BAM was shown to be embedded in the PG layer, it is possible that BAM is inserted preferentially simultaneously with de novo PG synthesis to facilitate the insertion of OMPs in the newly synthesized outer membrane.

The Escherichia coli Outer Membrane ß-Barrel Assembly Machinery (BAM) Anchors the Peptidoglycan Layer by Spanning It with All Subunits.

Gram-negative bacteria possess a three-layered envelope composed of an inner membrane, surrounded by a peptidoglycan (PG) layer, enclosed by an outer membrane. The envelope ensures protection against diverse hostile milieus and offers an effective barrier against antibiotics. The layers are connected to each other through many protein interactions. Bacteria evolved sophisticated machineries that maintain the integrity and the functionality of each layer. The ß-barrel assembly machinery (BAM), for example, is responsible for the insertion of the outer membrane integral proteins including the lipopolysaccharide transport machinery protein LptD. Labelling bacterial cells with BAM-specific fluorescent antibodies revealed the spatial arrangement between the machinery and the PG layer. The antibody detection of each BAM subunit required the enzymatic digestion of the PG layer. Enhancing the spacing between the outer membrane and PG does not abolish this prerequisite. This suggests that BAM locally sets the distance between OM and the PG layer. Our results shed new light on the local organization of the envelope.

Aging results in accumulation of M1 and M2 hepatic macrophages and a differential response to gadolinium chloride.

Macrophages have vital roles in innate immunity by modulating the inflammatory response via their ability to alter their phenotype from pro-inflammatory (M1) to anti-inflammatory (M2). Aging increases activation of the innate immune system, and macrophage numbers increase in the aged liver. Since macrophages also produce free radical molecules, they are a potential source of age-related oxidative injury in the liver. This study evaluated macrophage phenotype in the aged liver and whether the increase in the number of macrophages with aging is associated with enhanced hepatic oxidative stress. Hepatic macrophage phenotype and oxidative stress were evaluated 2 days after a single intraperitoneal injection of saline or gadolinium chloride (GdCl3, 10 mg/kg) in young (6 months) and aged (24 months) Fischer 344 rats. GdCl3 has been shown to decrease the expression of macrophage-specific markers and impair macrophage phagocytosis in the liver. Saline-treated aged rats demonstrated greater numbers of both M1 (HO-1+/iNOS+) and M2 (HO-1+/CD163+) macrophages, without evidence of a phenotypic shift. GdCl3 did not alter levels of dihydroethidium fluorescence or malondialdehyde, suggesting that macrophages are not a major contributor to steady-state levels of oxidative stress. However, GdCl3 decreased M1 and M2 macrophage markers in both age groups, an effect that was attenuated in aged rats. In old animals, GdCl3 decreased iNOS expression to a greater extent than HO-1 or CD163. These results suggest a novel effect of aging on macrophage biology and that GdCl3 shifts hepatic macrophage polarization to the M2 phenotype in aged animals.

Synthesis and ultrastructural observation of arbutoid mycorrhizae of black truffles (Tuber melanosporum and T. aestivum).

Arbutus unedo (the strawberry tree) is a Mediterranean shrub which forms arbutoid mycorrhizae with a variety of Asco- and Basidiomycetes. After the discovery of the mycorrhizal symbiosis between A. unedo and Tuber borchii, in this study, arbutoid mycorrhizae were synthetized in greenhouse with Tuber aestivum and Tuber melanosporum. Six months after inoculation, both species colonized the roots of all inoculated A. unedo seedlings, but mature mycorrhizae were only observed after 12 months. Ultrastructure analysis of Tuber arbutoid mycorrhizae was described for the first time, showing, as observed in typical endosymbiosis, a rearrangement of host cells and the creation of an interface compartment with both truffle species. Immunolabelling experiments suggested that pectins are not present in the interface matrix surrounding the intracellular hyphae. Thus, the ability to establish symbiosis with A. unedo seems to be a common feature in the genus Tuber, opening up the possibility to use this plant for mycorrhization with valuable truffles. This could represent an important economic opportunity in Mediterranean areas by combining the production of truffles, edible fruits and valued honey.

Protein localisation by electron microscopy reveals the architecture of the yeast spliceosomal B complex.

The spliceosome assembles on a pre-mRNA intron by binding of five snRNPs and numerous proteins, leading to the formation of the pre-catalytic B complex. While the general morphology of the B complex is known, the spatial arrangement of proteins and snRNP subunits within it remain to be elucidated. To shed light on the architecture of the yeast B complex, we immuno-labelled selected proteins and located them by negative-stain electron microscopy. The B complex exhibited a triangular shape with main body, head and neck domains. We located the U5 snRNP components Brr2 at the top and Prp8 and Snu114 in the centre of the main body. We found several U2 SF3a (Prp9 and Prp11) and SF3b (Hsh155 and Cus1) proteins in the head domain and two U4/U6 snRNP proteins (Prp3 and Lsm4) in the neck domain that connects the main body with the head. Thus, we could assign distinct domains of the B complex to the respective snRNPs and provide the first detailed picture of the subunit architecture and protein arrangements of the B complex.

Ultrastructural localization of 5-methylcytosine on DNA and RNA.

DNA methylation is the major epigenetic modification and it is involved in the negative regulation of gene expression. Its alteration can lead to neoplastic transformation. Several biomolecular approaches are nowadays used to study this modification on DNA, but also on RNA molecules, which are known to play a role in different biological processes. RNA methylation is one of the most common RNA modifications and 5-methylcytosine presence has recently been suggested in mRNA. However, an analysis of nucleic acid methylation at electron microscope is still lacking. Therefore, we visualized DNA methylation status and RNA methylation sites in the interphase nucleus of HeLa cells and rat hepatocytes by ultrastructural immunocytochemistry and cytochemical staining. This approach represents an efficient alternative to study nucleic acid methylation. In particular, this ultrastructural method makes the visualization of this epigenetic modification on a single RNA molecule possible, thus overcoming the technical limitations for a (pre-)mRNA methylation analysis.

Neuroprotective effect of Moringa oleifera leaf extract on aluminium-induced temporal cortical degeneration.

Aluminium (Al), one of the metals implicated in neurodegeneration easily gain access to the nervous system through its presence in many manufactured foods, medicines and drinking water, and causes neurotoxicity utilizing the reactive oxygen specie pathway. The need to curtail these effects on the nervous system motivated the use of the plant Moringa oleifera (MO). This study thus, investigated the neuroprotective effects of MO leaf extract on aluminium-induced temporal cortical degeneration in rats. 24 male albino Wistar rats were grouped (n = 6) into control (1 ml/kg distilled water), l00 mg/kg aluminium chloride (AlCl3), 300 mg/kg MO, and 100 mg/kg AlCl3 and 300 mg/kg MO groups. The administration lasted for 28 days and the rats were sacrificed on day 29 by perfusion-fixation after blood was obtained for serum Al estimation. The brain tissues were then routinely processed for some histological and immunnolabelling studies. There was no significant difference in serum Al in the test groups. Histological results showed atrophied and karyorrhetic cells with loss of Nissl substance in the temporal cortex of the AlCl3 group, while no adverse effect was observed in the cytoarchitecture of the temporal cortex and Nissl substance of the MO group. However, groups which were administered AlCl3 simultaneously with MO extract showed less degenerative features in the cyto-architecture of the temporal cortex with normal Nissl substance staining. There was increased neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expressions in the AlCl3 group, while the MO group also showed increased NSE but decreased GFAP expression. However, the group which were administered AlCl3 simultaneously with MO extract showed less expression of NSE and GFAP. In conclusion, MO protects against Al-induced neurotoxicity of the temporal cortex of rats.

Exploring the Functions of 9-Lipoxygenase (DkLOX3) in Ultrastructural Changes and Hormonal Stress Response during Persimmon Fruit Storage.

Lipoxygenase (LOX) initiates the hydroperoxidation of polyunsaturated fatty acids and is involved in multiple physiological processes. In this study, investigation of various microscopic techniques showed that the fruit peel cellular microstructure of the two persimmon cultivars differed after 12 days of storage, resulting in fruit weight loss and an increased number and depth of microcracks. Analysis of subcellular localization revealed that greater amounts of DkLOX3-immunolabelled gold particles accumulated in "Fupingjianshi" than in "Ganmaokui" during storage. In addition, the expression of DkLOX3 was positively up-regulated by abscisic acid (ABA), concomitant with the promotion of ethylene synthesis and loss of firmness, and was suppressed by salicylic acid (SA), concomitant with the maintenance of fruit firmness, inhibition of ethylene production and weight loss. In particular, the expression of DkLOX3 differed from the ethylene trajectory after methyl jasmonate (MeJA) treatment. Furthermore, we isolated a 1105 bp 5' flanking region of DkLOX3 and the activity of promoter deletion derivatives was induced through various hormonal treatments. Promoter sequence cis-regulatory elements were analysed, and two conserved hormone-responsive elements were found to be essential for responsiveness to hormonal stress. Overall, these results will provide us with new clues for exploring the functions of DkLOX3 in fruit ripening and hormonal stress response.

Biological effect of LOXL1 coding variants associated with pseudoexfoliation syndrome.

Pseudoexfoliation (PEX) syndrome is a systemic disease involving the extracellular matrix. It increases the risk of glaucoma, an irreversible cause of blindness, and susceptibility to heart disease, stroke and hearing loss. Single nucleotide polymorphisms (SNPs) in the LOXL1 (Lysyl oxidase-like 1) gene are the major known genetic risk factor for PEX syndrome. Two coding SNPs, rs1048861 (G > T; Arg141Leu) and rs3825942 (G > A; Gly153Asp), in the LOXL1 gene are strongly associated with the disease risk in multiple populations worldwide. In the present study, we investigated functional effects of these SNPs on the LOXL1 protein. We show through molecular modelling that positions 141 and 153 are likely surface residues and hence possible recognition sites for protein-protein interactions; the Arg141Leu and Gly153Asp substitutions cause charge changes that would lead to local differences in protein electrostatic potential and in turn the potential to modify protein-protein interactions. In RFL-6 rat fetal lung fibroblast cells ectopically expressing the LOXL1 protein variants related to PEX (Arg141_Gly153, Arg141_Asp153 or Leu141_Gly153), immunoprecipitation of the secreted variants showed differences in their processing by endogenous proteins, possibly Bone morphogenetic protein-1 (BMP-1) that cleaves and leads to enzymatic activation of LOXL1. Immunofluorescence labelling of the ectopically expressed protein variants in RFL-6 cells showed no significant difference in their extracellular accumulation tendency. In conclusion, this is the first report of a biological effect of the coding SNPs in the LOXL1 gene associated with PEX syndrome, on the LOXL1 protein. The findings indicate that the disease associated coding variants themselves may be involved in the manifestation of PEX syndrome.

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.

Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula × alba.

BACKGROUND AND AIMS: The efficiency and safety functions of xylem hydraulics are strongly dependent on the pits that connect the xylem vessels. However, little is known about their biochemical composition and thus about their hydraulic properties. In this study, the distribution of the epitopes of different wall components (cellulose, hemicelluloses, pectins and lignins) was analysed in intervessel pits of hybrid poplar (Populus tremula × alba). METHODS: Immunogold labelling with transmission electron microscopy was carried out with a set of antibodies raised against different epitopes for each wall polysaccharide type and for lignins. Analyses were performed on both immature and mature vessels. The effect of sap ionic strength on xylem conductance was also tested. KEY RESULTS: In mature vessels, the pit membrane (PM) was composed of crystalline cellulose and lignins. None of the hemicellulose epitopes were found in the PM. Pectin epitopes in mature vessels were highly concentrated in the annulus, a restricted area of the PM, whereas they were initially found in the whole PM in immature vessels. The pit border also showed a specific labelling pattern, with higher cellulose labelling compared with the secondary wall of the vessel. Ion-mediated variation of 24 % was found for hydraulic conductance. CONCLUSIONS: Cellulose microfibrils, lignins and annulus-restricted pectins have different physicochemical properties (rigidity, hydrophobicity, porosity) that have different effects on the hydraulic functions of the PM, and these influence both the hydraulic efficiency and vulnerability to cavitation of the pits, including ion-mediated control of hydraulic conductance. Impregnation of the cellulose microfibrils of the PM with lignins, which have low wettability, may result in lower cavitation pressure for a given pore size and thus help to explain the vulnerability of this species to cavitation.

Pollen tube cell walls of wild and domesticated tomatoes contain arabinosylated and fucosylated xyloglucan.

BACKGROUND AND AIMS: In flowering plants, fertilization relies on the delivery of the sperm cells carried by the pollen tube to the ovule. During the tip growth of the pollen tube, proper assembly of the cell wall polymers is required to maintain the mechanical properties of the cell wall. Xyloglucan (XyG) is a cell wall polymer known for maintaining the wall integrity and thus allowing cell expansion. In most angiosperms, the XyG of somatic cells is fucosylated, except in the Asterid clade (including the Solanaceae), where the fucosyl residues are replaced by arabinose, presumably due to an adaptive and/or selective diversification. However, it has been shown recently that XyG of Nicotiana alata pollen tubes is mostly fucosylated. The objective of the present work was to determine whether such structural differences between somatic and gametophytic cells are a common feature of Nicotiana and Solanum (more precisely tomato) genera. METHODS: XyGs of pollen tubes of domesticated (Solanum lycopersicum var. cerasiforme and var. Saint-Pierre) and wild (S. pimpinellifolium and S. peruvianum) tomatoes and tobacco (Nicotiana tabacum) were analysed by immunolabelling, oligosaccharide mass profiling and GC-MS analyses. KEY RESULTS: Pollen tubes from all the species were labelled with the mAb CCRC-M1, a monoclonal antibody that recognizes epitopes associated with fucosylated XyG motifs. Analyses of the cell wall did not highlight major structural differences between previously studied N. alata and N. tabacum XyG. In contrast, XyG of tomato pollen tubes contained fucosylated and arabinosylated motifs. The highest levels of fucosylated XyG were found in pollen tubes from the wild species. CONCLUSIONS: The results clearly indicate that the male gametophyte (pollen tube) and the sporophyte have structurally different XyG. This suggests that fucosylated XyG may have an important role in the tip growth of pollen tubes, and that they must have a specific set of functional XyG fucosyltransferases, which are yet to be characterized.

Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata.

BACKGROUND AND AIMS: In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata. METHODS: Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples. KEY RESULTS: Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly. CONCLUSIONS: This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.

Changes in hemicellulose metabolism in banana peel during fruit development and ripening.

Hemicellulose is key in determining the fate of plant cell wall in almost all growth and developmental stages. Nevertheless, there is limited knowledge regarding its involvement in the development and ripening of banana fruit. This study investigated changes in the temporal-spatial distribution of various hemicellulose components, hemicellulose content, activities of the main hydrolysis enzymes, and transcription level of the main hemicellulose-related gene families in banana peels. Both hemicellulose and xylan contents were positively correlated to the fruit firmness observed in our previous study. On the contrary, the xylanase activity was negatively correlated to xylan content and the fruit firmness. The vascular bundle cells, phloem, and cortex of bananas are abundant in xyloglucan, xylan, and mannan contents. Interestingly, the changes in the signal intensity of the CCRC-M104 antibody recognizing non-XXXG type xyloglucan are positively correlated to hemicellulose content. According to RNA-Seq analysis, xyloglucan and xylan-related genes were highly active in the early stages of growth, and the expression of MaMANs and MaXYNs increased as the fruit ripened. The abundance of plant hormonal and growth-responsive cis-acting elements was detected in the 2 kb upstream region of hemicellulose-related gene families. Interaction between hemicellulose and cell wall-specific proteins and MaKCBP1/2, MaCKG1, and MaHKL1 was found. The findings shed light on cell wall hemicellulose's role in banana fruit development and ripening, which could improve nutrition, flavor, and reduce postharvest fruit losses.

An advanced fast method for the evaluation of multiple immunolabelling using gold nanoparticles based on low-energy STEM.

We present a powerful method for the simultaneous detection of Au nanoparticles located on both sides of ultrathin sections. The method employs a high-resolution scanning electron microscope (HRSEM) operating in scanning transmission electron microscopy (STEM) mode in combination with the detection of backscattered electrons (BSE). The images are recorded simultaneously during STEM and BSE imaging at the precisely selected accelerating voltage. Under proper imaging conditions, the positions of Au nanoparticles on the top or bottom sides can be clearly differentiated, hence showing this method to be suitable for multiple immunolabelling using Au nanoparticles (NPs) as markers. The difference between the upper and lower Au NPs is so large that it is possible to apply common software tools (such as ImageJ) to enable their automatic differentiation. The effects of the section thickness, detector settings and accelerating voltage on the resulting image are shown. Our experimental results correspond to the results modelled in silico by Monte Carlo (MC) simulations.

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

BACKGROUND AND AIMS: The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. METHODS: A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. KEY RESULTS: Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. CONCLUSIONS: The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

Rapid in-solution preparation of somatic and meiotic plant cell nuclei for high-quality 3D immunoFISH and immunoFISH-GISH.

BACKGROUND: Though multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situ detection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity. The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH). RESULTS: The mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations. CONCLUSION: A simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise.