Thin-walled Cells Research Articles

Comparative Study on Pigment Components (Flavonols and Anthocyanins) and Antioxidant Capacity in Different Colored Double-petal Lotus

Lotus, an edible flower with high ornamental values, takes forms in many sizes, shapes, and colors. Among which, double-petal lotus not only has higher ornamental values but also gives a yield advantage in exploitation due to the large number of petals. Flower color is an important ornamental indicator, and flavonoids are the key basis for color presentation as well as important active components in lotus. This research used petals of four different colored double-petal lotus cultivars to identify their petal color, pigment localization, and pigment components (flavonols and anthocyanins), as well as the antioxidant activity of the petal extraction solution using the ABTS method. It was discovered that the white YMQ was the brightest but had the lowest Chroma and the third-highest Hue angle, while the dark red WFMH was the darkest but had both the highest Chroma and Hue angle. Most of the colored pigments were located in all the thin-walled cells immediately adjacent to both the upper and lower epidermis. The yellow MLQS had the highest amount of Quercetin derivatives in flavonols but the lowest antioxidant activity, the dark red WFMH had the highest amount of anthocyanin, and the pink MDZ had the highest antioxidant activity. This research offers insights into the potential medical usage of certain double-petal lotus cultivars like MLQS and WFMH, hopefully providing a reference for the identification of raw materials for extracting pigments to improve sleep patterns and prevent Alzheimer’s Disease.

Exploring the medicinal potential of Dendrobium: Uncovering the spatial distribution of flavonoids and alkaloids in 15 species of Dendrobium using MALDI-MSI

Dendrobium, the second largest genus in the Orchidaceae family, has been regarded as a valuable medicinal plant throughout Chinese history. Secondary metabolites present in the stem of Dendrobium, including alkaloids and flavonoids, confer significant pharmacological effects such as anti-inflammatory, antibacterial, immunomodulatory, antitumor, and antioxidant properties. To our knowledge, this study is the first to apply matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technology to comprehensively identify the spatial distribution of secondary metabolites, particularly flavonoids and alkaloids, in the mature stems of 15 common Dendrobium species. By analyzing the matching scores and relative concentrations of compounds, 20 flavonoids and 19 alkaloids with high identification credibility were identified, along with 4 sesquiterpenoid compounds. Their spatial distributions and compositions were analyzed, and the results revealed that despite the similar structural basis of the stems in the 15 Dendrobium species, significant differences existed in the distribution of secondary metabolites among different species. Flavonoids and alkaloids with different medicinal effects exhibited certain specific distribution patterns; for instance, flavonoids with antioxidant effects were mostly concentrated in the outer circle of thin-walled cells of Dendrobium stems, whereas those with antitumor effects were found in higher concentrations in the central areas of some Dendrobium species than in the periphery. Alkaloids with antitumor properties typically exhibited a higher concentration in peripheral thin-walled cells than in central regions. Furthermore, statistical analysis was conducted on the content of secondary metabolites with different effects among the 15 Dendrobium species to identify species with greater medicinal potential in terms of antioxidant and antitumor effects. The findings of this study not only enhance our understanding of the distribution patterns of flavonoids and alkaloids within Dendrobium stems but also offer valuable insights for the medicinal development and quality assessment of Dendrobium.

A preliminary investigation of banana pseudo-stem (Musa cavendish) for pulp and paper production: morphology, chemical composition, FTIR, XRD and thermogravimetric analysis

Abstract In today’s world, the use of paper and cardboard increasing but the availability of raw materials and the environmental impact on the paper industry is a big concern. To address these concerns, researchers are exploring the potential of agricultural waste products as raw materials for pulp production. This study uses morphological, chemical composition, FTIR, XRD, and thermogravimetric analysis to examine the potential of banana pseudo-stem as a raw material for paper pulp to address environmental concerns and raw material shortages in the paper industry. The study reveals favorable characteristics for papermaking, including long fiber length (1750 μm), thin cell wall thickness (9.7 μm), and large lumen diameter (22.15 μm). The chemical composition of banana pseudo-stem contains cellulose (44.93 %), hemicellulose (23.7 %), and Klason lignin (11.1) showing its suitability for pulp production. FTIR analysis highlights the functional groups present on the banana pseudo-stem. The XRD analysis shows that it has a similar cellulosic peak and crystallinity index with common raw materials used in pulp production. The thermogravimetric analysis shows that the banana pseudo-stem has high thermal stability. The findings demonstrate that banana pseudo-stem, both by itself and in combination with other raw materials, might be a potential raw material for the pulp production.

Towards a better understanding of xylem: Helical thickenings in vessels of woody angiosperms are associated with thinner cell walls and lower vessel diameter

Towards a better understanding of xylem: Helical thickenings in vessels of woody angiosperms are associated with thinner cell walls and lower vessel diameter

Putative APSES family transcription factor mbp1 plays an essential role in regulating cell wall synthesis in the agaricomycete Pleurotus ostreatus

The clade A APSES family transcription factors (Mbp1, Swi4, and Swi6) contribute to cell wall synthesis regulation in fungi. Herein, evolutionary relationships among these proteins were clarified by phylogenetic analysis using various ascomycetes and basidiomycetes, and then the detailed function of Mbp1 in cell wall synthesis regulation was analyzed in Pleurotus ostreatus. Our phylogenetic analysis revealed that Mbp1 and Swi6 are widely conserved among various fungi, whereas Swi4 is a protein specific for Saccharomycotina. In P. ostreatus, two putative clade A APSES family transcription factors, protein ID 83192 and 134090, were found and identified as Mbp1 and Swi6, respectively. The mbp1 gene was then disrupted through homologous recombination using P. ostreatus 20b strain (Δku80) as a host to obtain mbp1 disruption strains (Δmbp1). Disruption of mbp1 significantly decreased the growth rate and shortened aerial hyphae, suggesting that Mbp1 is involved in mycelial growth, especially aerial hyphal growth. Furthermore, thinner cell walls, decreased relative percentage of β-glucan, and downregulation of all β-glucan synthase genes were observed in Δmbp1 strains. Therefore, Mbp1 plays an essential role in β-glucan synthesis regulation in P. ostreatus. Disruption of mbp1 also impacted the expression profiles of chitin synthase genes, septum formation, and sensitivity to a chitin synthesis inhibitor, suggesting that Mbp1 also regulates chitin synthesis. In conclusion, Mbp1 is responsible for normal mycelial growth and regulates β-glucan and chitin synthesis in P. ostreatus. To the best of our knowledge, this is the first report on the detailed function of Mbp1 in cell wall synthesis regulation in fungi.

Eco-friendly drugs induce cellular changes in colistin-resistant bacteria

Abstract Background and objective As a last option, multidrug-resistant Gram-negative infections (caused by Enterobacteriaceae) are treated with the antibiotic colistin, also known as polymyxin E. Colistin-resistant superbugs predispose people to untreatable infections, possibly leading to a high mortality rate. This project aims to study the effect of Acacia nilotica aqueous extract and zinc oxide nanoparticles (ZnO-NPs) on colistin-resistant Klebsiella pneumonia (CRKP). Materials and methods ZnO-NPs were synthesized using the green method and characterized by UV-vis, Fourier transform infrared spectroscopy, and X-ray diffraction. The zone of inhibition (ZI) was measured using the agar-well diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration were estimated to determine the antimicrobial activity of the tested compound. Scanning electron microscopy (SEM) and transmission electronic microscopy (TEM) were used to investigate the alterations in bacterial cells that were treated with the tested drugs. Results The synthesized ZnO-NPs presented good chemical and physical properties, and the plant extract and ZnO-NPs displayed a large ZI. ZnO-NPs had the lowest MIC (0.2 mg·mL−1). SEM and TEM observations revealed various morphological modifications in CRKP cells, including cell shrinkage, cell damage, cytoplasm loss, cell wall thinning, and cell death. Conclusion A. nilotica aqueous extract and ZnO-NPs could be used as alternative natural products to produce antibacterial drugs and to prevent CRKP infection.

Zygospore formation in Zygnematophyceae predates several land plant traits.

Recent research on a special type of sexual reproduction and zygospore formation in Zygnematophyceae, the sister group of land plants, is summarized. Within this group, gamete fusion occurs by conjugation. Zygospore development in Mougeotia, Spirogyra and Zygnema is highlighted, which has recently been studied using Raman spectroscopy, allowing chemical imaging and detection of changes in starch and lipid accumulation. Three-dimensional reconstructions after serial block-face scanning electron microscopy (SBF-SEM) or focused ion beam SEM (FIB-SEM) made it possible to visualize and quantify cell wall and organelle changes during zygospore development. The zygospore walls undergo strong modifications starting from uniform thin cell walls to a multilayered structure. The mature cell wall is composed of a cellulosic endospore and exospore and a central mesospore built up by aromatic compounds. In Spirogyra, the exospore and endospore consist of thick layers of helicoidally arranged cellulose fibrils, which are otherwise only known from stone cells of land plants. While starch is degraded during maturation, providing building blocks for cell wall formation, lipid droplets accumulate and fill large parts of the ripe zygospores, similar to spores and seeds of land plants. Overall, data show similarities between streptophyte algae and embryophytes, suggesting that the genetic toolkit for many land plant traits already existed in their shared algal ancestor. This article is part of the theme issue 'The evolution of plant metabolism'.

GhCASPL1 regulates secondary cell wall thickening in cotton fibers by stabilizing the cellulose synthase complex on the plasma membrane.

Cotton (Gossypium hirsutum) fibers are elongated single cells that rapidly accumulate cellulose during secondary cell wall (SCW) thickening, which requires cellulose synthase complex (CSC) activity. Here, we describe the CSC-interacting factor CASPARIAN STRIP MEMBRANE DOMAIN-LIKE1 (GhCASPL1), which contributes to SCW thickening by influencing CSC stability on the plasma membrane. GhCASPL1 is preferentially expressed in fiber cells during SCW biosynthesis and encodes a MARVEL domain protein. The ghcaspl1 ghcaspl2 mutant exhibited reduced plant height and produced mature fibers with fewer natural twists, lower tensile strength, and a thinner SCW compared to the wild type. Similarly, the Arabidopsis (Arabidopsis thaliana) caspl1 caspl2 double mutant showed a lower cellulose content and thinner cell walls in the stem vasculature than the wild type but normal plant morphology. Introducing the cotton gene GhCASPL1 successfully restored the reduced cellulose content of the Arabidopsis caspl1 caspl2 mutant. Detergent treatments, ultracentrifugation assays, and enzymatic assays showed that the CSC in the ghcaspl1 ghcaspl2 double mutant showed reduced membrane binding and decreased enzyme activity compared to the wild type. GhCASPL1 binds strongly to phosphatidic acid (PA), which is present in much higher amounts in thickening fiber cells compared to ovules and leaves. Mutating the PA-binding site in GhCASPL1 resulted in the loss of its colocalization with GhCesA8, and it failed to localize to the plasma membrane. PA may alter membrane structure to facilitate protein-protein interactions, suggesting that GhCASPL1 and PA collaboratively stabilize the CSC. Our findings shed light on CASPL functions and the molecular machinery behind SCW biosynthesis in cotton fibers.

Identification of BpEXP family genes and functional characterization of the BpEXPA1 gene in the stems development of Betula platyphylla

Expansins (EXPs) are unique plant cell wall proteins with the ability to induce cell wall expansion and play potential roles in xylem development. In the present study, a total of 25 BpEXP genes were identified in Betula platyphylla. Results of bioinformatics analysis described that BpEXP gene family was highly conserved in the process of evolution. All these genes were clustered into four groups, EXPA (Expansin A), EXPB (Expansin B), EXLA (Expansin-like A) and EXLB (Expansin-like B), according to phylogenetic analysis and BpEXPA1 was highly homologous to PttEXP1 and PttEXP2. The results of RT-qPCR showed that BpEXPA1 was expressed higher in stems and preferentially expressed in the first internodes, followed by apical buds and the third internodes, promoter expression analysis with GUS assay demonstrated that it was expressed in developing xylem, suggesting that BpEXPA1 might be involved in the development of the primary stems of birch. Overexpression of BpEXPA1 can promote cortex cell expansion and then enlarge the cortex cell area and layer, however inhibit the secondary cell wall deposition and result in the thinner cell wall and larger lumens of xylem fiber in transgenic plants. This study will provide information for investigating the regulation mechanism of BpEXP family genes and gene resources for birch genetics improvement.

The DUF579 proteins GhIRX15s regulate cotton fiber development by interacting with proteins involved in xylan synthesis

Cotton provides the most abundant natural fiber for the textile industry. The mature cotton fiber largely consists of secondary cell walls with the highest proportion of cellulose and a small amount of hemicellulose and lignin. To dissect the roles of hemicellulosic polysaccharides during fiber development, four IRREGULAR XYLEM 15 (IRX15) genes, GhIRX15-1/-2/-3/-4, were functionally characterized in cotton. These genes encode DUF579 domain-containing proteins, which are homologs of AtIRX15 involved in xylan biosynthesis. The four GhIRX15 genes were predominantly expressed during fiber secondary wall thickening, and the encoded proteins were localized to the Golgi apparatus. Each GhIRX15 gene could restore the xylan deficient phenotype in the Arabidopsis irx15irx15l double mutant. Silencing of GhIRX15s in cotton resulted in shorter mature fibers with a thinner cell wall and reduced cellulose content as compared to the wild type. Intriguingly, GhIRX15-2 and GhIRX15-4 formed homodimers and heterodimers. In addition, the GhIRX15s showed physical interaction with glycosyltransferases GhGT43C, GhGT47A and GhGT47B, which are responsible for synthesis of the xylan backbone and reducing end sequence. Moreover, the GhIRX15s can form heterocomplexes with enzymes involved in xylan modification and side chain synthesis, such as GhGUX1/2, GhGXM1/2 and GhTBL1. These findings suggest that GhIRX15s participate in fiber xylan biosynthesis and modulate fiber development via forming large multiprotein complexes.

Fruit anatomy and histogenesis in Mediterranean species of Arbutus (Ericaceae: Arbutoideae): ecological and morphogenetic aspects

Abstract Details of fruit anatomy and pericarp histogenesis were studied in Mediterranean species of Arbutus with the aim to determine the morphogenetic fruit type, to reveal the functional and ecological significance of fruit structure, and to suggest the most probable scenarios of fruit structural transformations in Arbutoideae (Ericaceae). The pericarp of the coenocarpous berries of Arbutus is differentiated into: one-layered exocarp, multilayered parenchymatous mesocarp with scattered solitary and groups of sclereids, and one-layered endocarp composed of thin-walled cells. The warts covering the fruit surface are formed by parenchymatous cells of the mesocarp and the exocarp. The process of pericarp development in Arbutus is divided into four periods, which correlate with the phenology and climatic factors of the Mediterranean region. The origin of the dormancy period in the middle part of fruit development in Arbutus is interpreted as an important adaptation to the arid Mediterranean climate. The data obtained suggest that both the berry of Arbutus and the pyrenariums of Arctostaphylos and Arctous are highly specialized fruit types that could not have derived from each other. The capsule of Hamamelis type of Enkianthoideae is recognized as the original fruit type of the berries and pyrenariums of Arbutoideae.

Roles of α-1,3-glucosyltransferase in growth and polysaccharides biosynthesis of Ganoderma lucidum

Ganoderma lucidum polysaccharides are valuable natural compounds possessing significant biological activity, with glycosyltransferases playing a crucial role in their biosynthesis. Although the function of β-1,3-glucosyltransferase in polysaccharides production is well understood, the role of α-1,3-glucosyltransferase in edible fungi remains unclear. In this study, over-expression of the α-1,3-glucosyltransferase gene in G. lucidum (glagt) was found to suppress the growth, with the maximum biomass and mycelial growth rate decreasing by 21.78 % and 79.61 %, respectively, a behavior distinct from β-1,3-glucosyltransferase. The fungal pellet diameter decreased by 38 % and the cell-wall thickness by 32.44 %, whereas intracellular and extracellular polysaccharides production increased by 27.58 % and 66.08 %, respectively. In the transcription level, overexpressing the glagt gene i) downregulated the citrate synthase and isocitrate dehydrogenase gene in the TCA cycle, disrupting energy metabolism and fungal growth; ii) upregulated key enzymes involved in UDP-glucose synthesis and glycosyltransferases (gl24465, gl24971, and gl22535); and iii) universally increased the transcriptional level of glucosidases gl21451, gl30087, and gl24581 by 22 %–397 %, contributing to cell-wall thinning to facilitate polysaccharides export. Conversely, the glagt gene downregulation promoted G. lucidum growth and decreased polysaccharides production. The results elucidate the roles of GLAGT and are expected to inspire in-depth exploration of polysaccharides biosynthesis pathways.

Controlling the crystal morphology of high-hardness TPU through two pre-crystallization processes and its impact on physical foaming behavior

Clarifying the relationship between crystal evolution and the physical foaming behavior of high-hardness thermoplastic polyurethane (HD-TPU) is essential for preparing HD-TPU foams with controllable cellular morphology. In this study, two thermal treatment methods, isothermal annealing and isothermal crystallization, were utilized to control the crystal structure of HD-TPU. The effect of crystals on the physical foaming behavior of HD-TPU foams was examined. The annealing process produced mainly lamellae and Form Ⅰ structure, while isothermal crystallization primarily led to Form Ⅱ spherulites. Foams based on original and pre-treated HD-TPU were prepared by pressure quench foaming. The evolution of foam density and cell morphology was discussed. At low foaming temperatures, crystals induced the heterogeneous nucleation. Realizing a uniform cell structure at high foaming temperatures could be challenging when the spherulite size was large. When the size of the spherulite was small but loosely distributed, larger cells with thinner cell walls could be obtained. However, if the spherulite distribution was dense, the spherulites hindered cell growth, resulting in unevenly distributed and deformed cells.

The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors

Inactivation of flbA in Aspergillus niger results in thinner cell walls, increased cell lysis, abolished sporulation, and an increased secretome complexity. A total of 36 transcription factor (TF) genes are differentially expressed in ΔflbA. Here, seven of these genes (abaA, aslA, aslB, azf1, htfA, nosA, and srbA) were inactivated. Inactivation of each of these genes affected sporulation and, with the exception of abaA, cell wall integrity and protein secretion. The impact on secretion was strongest in the case of ΔaslA and ΔaslB that showed increased pepsin, cellulase, and amylase activity. Biomass was reduced of agar cultures of ΔabaA, ΔaslA, ΔnosA, and ΔsrbA, while biomass was higher in liquid shaken cultures of ΔaslA and ΔaslB. The ΔaslA and ΔhtfA strains showed increased resistance to H2O2, while ΔaslB was more sensitive to this reactive oxygen species. Together, inactivation of the seven TF genes impacted biomass formation, sporulation, protein secretion, and stress resistance, and thereby these genes explain at least part of the pleiotropic phenotype of ΔflbA of A. niger.

Functional analysis of basidiomycete specific chitin synthase genes in the agaricomycete fungus Pleurotus ostreatus

Chitin is an essential structural component of fungal cell walls composed of transmembrane proteins called chitin synthases (CHSs), which have a large range of reported effects in ascomycetes; however, are poorly understood in agaricomycetes. In this study, evolutionary and molecular genetic analyses of chs genes were conducted using genomic information from nine ascomycete and six basidiomycete species. The results support the existence of seven previously classified chs clades and the discovery of three novel basidiomycete-specific clades (BI–BIII). The agaricomycete fungus Pleurotus ostreatus was observed to have nine putative chs genes, four of which were basidiomycete-specific. Three of these basidiomycete specific genes were disrupted in the P. ostreatus 20b strain (ku80 disruptant) through homologous recombination and transformants were obtained (Δchsb2, Δchsb3, and Δchsb4). Despite numerous transformations Δchsb1 was unobtainable, suggesting disruption of this gene causes a crucial negative effect in P. ostreatus. Disruption of these chsb2–4 genes caused sparser mycelia with rougher surfaces and shorter aerial hyphae. They also caused increased sensitivity to cell wall and membrane stress, thinner cell walls, and overexpression of other chitin and glucan synthases. These genes have distinct roles in the structural formation of aerial hyphae and cell walls, which are important for understanding basidiomycete evolution in filamentous fungi.

Effects of microwave-assisted softening treatment on dynamic mechanical properties and chemical composition of bamboo

In response to the global plastic pollution crisis, a sustainable manufacturing strategy for replacing plastic with bamboo is proposed. Bamboo softening treatment is important for bamboo processing. In this study, the effects of microwave-assisted alkali/boiling treatment on the microstructure, chemical composition, glass transition temperature, compression ratio, and surface wettability of the bamboo species Phyllostachys edulis and Dendrocalamus sinicus were studied. Microwave-assisted alkali (0.1 % NaOH)/boiling (2 h) treatment increased the flexibility and processability of both species. Dynamic mechanical analysis showed that the storage modulus of P.edulis slices was reduced from 6429 MPa to 5128 MPa, loss modulus was reduced from 134.1 MPa to 100.6 MPa, and glass transition temperature was reduced from 204.25 ℃ to 100.39 ℃ indicating that bamboo stiffness was reduced and elasticity was enhanced. With the temperature increase, the alkali solution destroyed the dense fiber structure and broke the bonds between lignin, cellulose, and hemicellulose. The glass transition temperature decreased in P.edulis by 103.86℃, which was 1.14-times higher than that of D. sinicus, and the compression ratio increased form 10.52–37.58 %, which indicated better thermoplasticity compared with D.sinicus. The vascular bundles and thin-walled cells of the specimens showed different degrees of shrinkage and deformation, and the hydrophobicity of bamboo increased resulting in improved thermal stability and optimal process conditions, providing an effective solution for the realization of replacing plastic with bamboo. This lays the theoretical foundation for subsequent furniture-bending components and molded products.

Crushing behavior of closed-cell metallic foams: Anisotropy and temperature effects

Due to their lightweight features and special energy absorption properties, metal foams (MFs) are gradually replacing fully-dense solid materials in top industries. Having a porous structure with thin cell-walls, MFs are quite sensitive to various operating conditions such as temperature (TT) and loading direction (LD), aspects that should not be neglected. However, the dual TT-LD impact on the mechanical behavior of anisotropic MFs has not yet been addressed. In this regard, the temperature range 25–550 °C (in steps of 75 °C) and the three orthogonal directions (X, Y and Z) were used to experimentally investigate the compressive crushing responses of MFs under the combined temperature-anisotropy effect. The studied MFs had closed cells and were manufactured from Al alloys (AlSi12Mg0.6) through powder metallurgy route. The main strength properties and energy absorption performance of the investigated foams were found to be strongly dependent on the LD and the TT. The best mechanical characteristics are highlighted by the Z-LD, followed in order by the Y and X directions. Thus, improvements in properties were obtained by over 45% (compressive modulus), 71% (compressive strength), 28% (densification strain) and 53% (energy absorption) for Z-LD compared to the other two LDs. Finally, the failure mechanisms differ considerably depending on the imposed test condition (TT or LD).

Summer heat induced the decline of Pinus taiwanensis forests at its southern limit in humid Subtropical China

Warming-induced aridity has caused forest decline and mortality for many sites with water-limiting conditions. However, equatorward rear-edge Pinus taiwanensis trees at the Daiyun Mountains in humid subtropical China are also suffering die-backs and decline, but the roles played by heat or drought stress still remain unclear. Here, we compared the tree-ring radial width, anatomical features, stable carbon isotope (δ13C) and intrinsic water use efficiency (iWUE) between die-back and healthy trees to elucidate potential causes driving the decline. Die-back trees showed sustained growth reductions and produced tracheids with thinner cell walls over the recent decade, indicative of reduced carbon assimilation. The climate response pattern and Vaganov-Shashkin (V-S) model indicated the critical role of summer (June-August) temperature in recent growth decline. Long-term higher wood δ13C and iWUE within die-back trees indicated that actual growth decline already started several decades earlier. This conservative growth strategy was at the cost of low efficiency of photosynthesis due to chronic stomatal closure. When the lethal heatwaves arrived, these weakened trees were not able to access sufficient carbonhydrates to maintain metabolism, causing a distinct decline and mortality. We concluded that recent decline in Pinus taiwanensis trees was mainly caused by long-term carbon starvation.

The role of the Flb protein family in the life cycle of Aspergillus niger

Genes flbA-E are involved in sporulation and vegetative growth in Aspergillus nidulans. Inactivation of either of these genes results in a fluffy phenotype with delayed or even abolished sporulation. Previously, a non-sporulating phenotype was obtained by inactivating flbA in Aspergillus niger, which was accompanied by lysis, thinner cell walls, and an increased secretome complexity. Here, we further studied the role of the flb genes of A. niger. Strains ΔflbA, ΔflbB and ΔflbE showed increased biomass formation, while inactivation of flbA-D reduced, or even abolished, formation of conidia. Strain ΔflbA was more sensitive to H2O2, DTT, and the cell wall integrity stress compounds SDS and Congo Red (CR). Also, ΔflbC was more sensitive to SDS, while ΔflbB, ΔflbD, and ΔflbE were more sensitive to CR. On the other hand, inactivation of flbE increased resistance to H2O2. Enzyme secretion was impacted when the Δflb strains were grown on xylose. Strain ΔflbE showed reduced xylanase, cellulase and amylase secretion. On the other hand, amylase secretion at the periphery of the ΔflbA colony was reduced but not in its center, while secretion of this enzyme was increased in the center of the ΔflbB colony but not at its periphery. Inactivation of flbC and flbD also impacted zonal cellulase and amylase activity. Together, the Flb protein family of A. niger function in biomass formation, sporulation, stress response, and protein secretion.

Zygospore development of Spirogyra (Charophyta) investigated by serial block-face scanning electron microscopy and 3D reconstructions.

Sexual reproduction of Zygnematophyceae by conjugation is a less investigated topic due to the difficulties of the induction of this process and zygospore ripening under laboratory conditions. For this study, we collected field sampled zygospores of Spirogyra mirabilis and three additional Spirogyra strains in Austria and Greece. Serial block-face scanning electron microscopy was performed on high pressure frozen and freeze substituted zygospores and 3D reconstructions were generated, allowing a comprehensive insight into the process of zygospore maturation, involving storage compound and organelle rearrangements. Chloroplasts are drastically changed, while young stages contain both parental chloroplasts, the male chloroplasts are aborted and reorganised as 'secondary vacuoles' which initially contain plastoglobules and remnants of thylakoid membranes. The originally large pyrenoids and the volume of starch granules is significantly reduced during maturation (young: 8 ± 5 µm³, mature: 0.2 ± 0.2 µm³). In contrast, lipid droplets (LDs) increase significantly in number upon zygospore maturation, while simultaneously getting smaller (young: 21 ± 18 µm³, mature: 0.1 ± 0.2 and 0.5 ± 0.9 µm³). Only in S. mirabilis the LD volume increases (34 ± 29 µm³), occupying ~50% of the zygospore volume. Mature zygospores contain barite crystals as confirmed by Raman spectroscopy with a size of 0.02 - 0.05 µm³. The initially thin zygospore cell wall (~0.5 µm endospore, ~0.8 µm exospore) increases in thickness and develops a distinct, electron dense mesospore, which has a reticulate appearance (~1.4 µm) in Spirogyra sp. from Greece. The exo- and endospore show cellulose microfibrils in a helicoidal pattern. In the denser endospore, pitch angles of the microfibril layers were calculated: ~18 ± 3° in S. mirabilis, ~20 ± 3° in Spirogyra sp. from Austria and ~38 ± 8° in Spirogyra sp. from Greece. Overall this study gives new insights into Spirogyra sp. zygospore development, crucial for survival during dry periods and dispersal of this genus.