Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.

High-amylose starch: Structure, functionality and applications.

Starch with a high amylose (AM) content (high AM starch, HAS) has attracted increasing research attention due to its industrial application potential, such as functional foods and biodegradable packaging. In the past two decades, HAS structure, functionality, and applications have been the research hotspots. However, a review that comprehensively summarizes these areas is lacking, making it difficult for interested readers to keep track of past and recent advances. In this review, we highlight studies that benefited from rapidly developing techniques, and systematically review the structure, functionality, and applications of HAS. We particularly emphasize the relationships between HAS molecular structure and physicochemical properties.

3D-reconstructions of zygospores in Zygnema vaginatum (Charophyta) reveal details of cell wall formation, suggesting adaptations to extreme habitats.

The streptophyte green algal class Zygnematophyceae is the immediate sister lineage to land plants. Their special form of sexual reproduction via conjugation might have played a key role during terrestrialization. Thus, studying Zygnematophyceae and conjugation is crucial for understanding the conquest of land. Moreover, sexual reproduction features are important for species determination. We present a phylogenetic analysis of a field-sampled Zygnema strain and analyze its conjugation process and zygospore morphology, both at the micro- and nanoscale, including 3D-reconstructions of the zygospore architecture. Vegetative filament size (26.18 ± 1.07 µm) and reproductive features allowed morphological determination of Zygnema vaginatum, which was combined with molecular analyses based on rbcL sequencing. Transmission electron microscopy (TEM) depicted a thin cell wall in young zygospores, while mature cells exhibited a tripartite wall, including a massive and sculptured mesospore. During development, cytological reorganizations were visualized by focused ion beam scanning electron microscopy (FIB-SEM). Pyrenoids were reorganized, and the gyroid cubic central thylakoid membranes, as well as the surrounding starch granules, degraded (starch granule volume: 3.58 ± 2.35 µm3 in young cells; 0.68 ± 0.74 µm3 at an intermediate stage of zygospore maturation). Additionally, lipid droplets (LDs) changed drastically in shape and abundance during zygospore maturation (LD/cell volume: 11.77% in young cells; 8.79% in intermediate cells, 19.45% in old cells). In summary, we provide the first TEM images and 3D-reconstructions of Zygnema zygospores, giving insights into the physiological processes involved in their maturation. These observations help to understand mechanisms that facilitated the transition from water to land in Zygnematophyceae.

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation.

Certain transglucanases can covalently graft cellulose and mixed-linkage ß-glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero-transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero-transglucosylation, we studied Equisetum fluviatile hetero-trans-ß-glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo-transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia-produced EfHTG was up to approximately 300% increased by addition of methanol-boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol-stable factor is proposed to be expansin-like, a suggestion supported by observations of pH dependence. Screening numerous low-molecular-weight compounds for hetero-transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero-transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero-transglucosylation in planta by identifying factors that govern this reaction.

Localisation and substrate specificities of transglycanases in charophyte algae relate to development and morphology.

Cell wall-modifying enzymes have been previously investigated in charophyte green algae (CGA) in cultures of uniform age, giving limited insight into their roles. Therefore, we investigated the in situ localisation and specificity of enzymes acting on hemicelluloses in CGA genera of different morphologies and developmental stages. In vivo transglycosylation between xyloglucan and an endogenous donor in filamentous Klebsormidium and Zygnema was observed in longitudinal cell walls of young (1 month) but not old cells (1 year), suggesting that it has a role in cell growth. By contrast, in parenchymatous Chara, transglycanase action occurred in all cell planes. In Klebsormidium and Zygnema, the location of enzyme action mainly occurred in regions where xyloglucans and mannans, and to a lesser extent mixed-linkage ß-glucan (MLG), were present, indicating predominantly xyloglucan:xyloglucan endotransglucosylase (XET) activity. Novel transglycosylation activities between xyloglucan and xylan, and xyloglucan and galactomannan were identified in vitro in both genera. Our results show that several cell wall-modifying enzymes are present in CGA, and that differences in morphology and cell age are related to enzyme localisation and specificity. This indicates an evolutionary significance of cell wall modifications, as similar changes are known in their immediate descendants, the land plants. This article has an associated First Person interview with the first author of the paper.

A Trihelix Family Transcription Factor Is Associated with Key Genes in Mixed-Linkage Glucan Accumulation.

Mixed-linkage glucan (MLG) is a polysaccharide that is highly abundant in grass endosperm cell walls and present at lower amounts in other tissues. Cellulose synthase-like F (CSLF) and cellulose synthase-like H genes synthesize MLG, but it is unknown if other genes participate in the production and restructuring of MLG. Using Brachypodium distachyon transcriptional profiling data, we identified a B distachyon trihelix family transcription factor (BdTHX1) that is highly coexpressed with the B distachyon CSLF6 gene (BdCSLF6), which suggests that BdTHX1 is involved in the regulation of MLG biosynthesis. To determine the genes regulated by this transcription factor, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) experiments using immature B distachyon seeds and an anti-BdTHX1 polyclonal antibody. The ChIP-seq experiment identified the second intron of BdCSLF6 as one of the most enriched sequences. The binding of BdTHX1 to the BdCSLF6 intron sequence was confirmed using electrophoretic mobility shift assays (EMSA). ChIP-seq also showed that a gene encoding a grass-specific glycoside hydrolase family 16 endotransglucosylase/hydrolase (BdXTH8) is bound by BdTHX1, and the binding was confirmed by EMSA. Radiochemical transglucanase assays showed that BdXTH8 exhibits predominantly MLG:xyloglucan endotransglucosylase activity, a hetero-transglycosylation reaction, and can thus produce MLG-xyloglucan covalent bonds; it also has a lower xyloglucan:xyloglucan endotransglucosylase activity. B distachyon shoots regenerated from transformed calli overexpressing BdTHX1 showed an abnormal arrangement of vascular tissue and seedling-lethal phenotypes. These results indicate that the transcription factor BdTHX1 likely plays an important role in MLG biosynthesis and restructuring by regulating the expression of BdCSLF6 and BdXTH8.

Photosynthetic plasticity in the green algal species Klebsormidium flaccidum (Streptophyta) from a terrestrial and a freshwater habitat.

The genus Klebsormidium (Klebsormidiales, Streptophyta) has a worldwide distribution in terrestrial habitats. In the present study, we focused on two strains of Klebsormidium flaccidum, the type species of the genus. The isolates used in this study were isolated from a soil and freshwater habitat. Photosynthetic activity was evaluated under different controlled gradients of light, temperature and desiccation. The data clearly indicate that both isolates of K. flaccidum exhibit conspicuously different photosynthetic response patterns to photon fluence rate, temperature and desiccation, and thus can be related to their different habitats. Although both strains represent the same species, their physiological response patterns to abiotic gradients, as well as their morphology differed to some extent, indicating high phenotypic plasticity of K. flaccidum, which was maintained even after long-term culture and thus can be explained by the formation of physiologically distinct ecotypes.

Conjugation morphology of Zygogonium ericetorum (Zygnematophyceae, Charophyta) from a high alpine habitat.

Reproductive characteristics are important for defining taxonomic groups of filamentous Zygnematophyceae, but they have not been fully observed in the genus Zygogonium. Specimens of Z. ericetorum previously studied and used to clarify the generic concept lacked fertile material, which was obtained recently. This study illustrates for the first time, using color light microscopic and fluorescence images, a consequent conjugation stage in Z. ericetorum, including completely developed zygospores and purple cytoplasmic residue content left outside the zygospores, similar to aplanospore formation. Structures confirmed earlier reports and provided new observation informative regarding phylogenetically relevant reproductive characters of Z. ericetorum.

Localization and Quantification of Callose in the Streptophyte Green Algae Zygnema and Klebsormidium: Correlation with Desiccation Tolerance.

Freshwater green algae started to colonize terrestrial habitats about 460 million years ago, giving rise to the evolution of land plants. Today, several streptophyte green algae occur in aero-terrestrial habitats with unpredictable fluctuations in water availability, serving as ideal models for investigating desiccation tolerance. We tested the hypothesis that callose, a ß-d-1,3-glucan, is incorporated specifically in strained areas of the cell wall due to cellular water loss, implicating a contribution to desiccation tolerance. In the early diverging genus Klebsormidium, callose was drastically increased already after 30 min of desiccation stress. Localization studies demonstrated an increase in callose in the undulating cross cell walls during cellular water loss, allowing a regulated shrinkage and expansion after rehydration. This correlates with a high desiccation tolerance demonstrated by a full recovery of the photosynthetic yield visualized at the subcellular level by Imaging-PAM. Furthermore, abundant callose in terminal cell walls might facilitate cell detachment to release dispersal units. In contrast, in the late diverging Zygnema, the callose content did not change upon desiccation for up to 3.5 h and was primarily localized in the corners between individual cells and at terminal cells. While these callose deposits still imply reduction of mechanical damage, the photosynthetic yield did not recover fully in the investigated young cultures of Zygnema upon rehydration. The abundance and specific localization of callose correlates with the higher desiccation tolerance in Klebsormidium when compared with Zygnema.

Desiccation tolerance in the chlorophyte green alga Ulva compressa: does cell wall architecture contribute to ecological success?

MAIN CONCLUSION: Desiccation leads to structural changes of the inner pectic cell wall layers in Ulva compressa. This contributes to protection against mechanical damage due to desiccation-rehydration cycles. Ulva compressa, characterized by rbcL phylogeny, is a common species in the Mediterranean Sea. Ulva as an intertidal species tolerates repeated desiccation-rehydration cycles in nature; the physiological and structural basis were investigated under experimental conditions here. Desiccation to 73% relative water content (RWC) led to a significant decrease of the maximum quantum yield of photosystem II (F v/F m) to about half of the initial value. A reduction to 48 or 27% RWC caused a more drastic effect and thalli were only able to recover fully from desiccation to 73% RWC. Relative electron transport rates were stimulated at 73% RWC, but decreased significantly at 48 and 27% RWC, respectively. Imaging-PAM analysis demonstrated a homogenous desiccation process within individual thallus discs. The different cell wall layers of U. compressa were characterized by standard staining procedures, i.e. calcofluor white and aniline blue for structural components (cellulose, callose), ruthenium red for pectins and toluidine blue for acidic polysaccharides. Already a reduction to 73% RWC caused severe changes of the cell walls. The inner pectin-rich layers followed the shrinkage process of the cytoplasm, while the outer denser fibrillar layers maintained their shape. In this way, the thalli were not plasmolyzed during water loss, and upon recovery not negatively influenced by any mechanical damage. Transmission electron microscopy corroborated the arrangement of the different layers clearly distinguishable by their texture and electron density. We suggest the flexibility of the pectin-rich cell wall layers as a major contribution to desiccation tolerance in Ulva.

Dehydration, temperature, and light tolerance in members of the aeroterrestrial green algal genus interfilum (streptophyta) from biogeographically different temperate soils.

Unicellular green algae of the genus Interfilum (Klebsormidiales, Streptophyta) are typical components of biological soil crusts. Four different aeroterrestrial Interfilum strains that have previously been molecular-taxonomically characterized and isolated from temperate soils in Belgium, Czech Republic, New Zealand, and Ukraine were investigated. Photosynthetic performance was evaluated under different controlled abiotic conditions, including dehydration, as well as under a light and temperature gradient. For standardized desiccation experiments, a new methodological approach with silica gel filled polystyrol boxes and effective quantum yield measurements from the outside were successfully applied. All Interfilum isolates showed a decrease and inhibition of the effective quantum yield under this treatment, however with different kinetics. While the single cell strains exhibited relatively fast inhibition, the cell packet forming isolates dried slower. Most strains fully recovered effective quantum yield after rehydration. All Interfilum isolates exhibited optimum photosynthesis at low photon fluence rates, but with no indication of photoinhibition under high light conditions suggesting flexible acclimation mechanisms of the photosynthetic machinery. Photosynthesis under lower temperatures was generally more active than respiration, while the opposite was true for higher temperatures. The presented data provide an explanation for the regular occurrence of Interfilum species in soil habitats where environmental factors can be particularly harsh.

Phylogenetic position of Zygogonium ericetorum (zygnematophyceae, charophyta) from a high alpine habitat and ultrastructural characterization of unusual aplanospores.

Characeae (Charophyceae, Charophyta) contains two tribes with six genera: tribe Chareae with four genera and tribe Nitelleae, which includes Tolypella and Nitella. This paper uses molecular and morphological data to elucidate the phylogeny of Tolypella species in North America. In the most comprehensive taxonomic treatment of Characeae, 16 Tolypella species worldwide were subsumed into two species, T. intricata and T. nidifica, in two sections, Rothia and Tolypella respectively. It was further suggested that Tolypella might be a derived group within Nitella. In this investigation into species diversity and relationships in North American Tolypella, sequence data from the plastid genes atpB, psbC, and rbcL were assembled for a broad range of charophycean and land plant taxa. Molecular data were used in conjunction with morphology to test monophyly of the genus and species within it. Phylogenetic analyses of the sequence data showed that Characeae is monophyletic but that Nitelleae is paraphyletic with Tolypella sister to a monophyletic Nitella + Chareae. The results also supported the monophyly of Tolypella and the sections Rothia and Tolypella. Morphologically defined species were supported as clades with little or no DNA sequence differences. In addition, molecular data revealed several lineages and a new species (T. ramosissima sp. nov.), which suggests greater species diversity in Tolypella than previously recognized.

Antarctic benthic diatoms after 10 months of dark exposure: consequences for photosynthesis and cellular integrity.

Antarctic algae are exposed to prolonged periods of extreme darkness due to polar night, and coverage by ice and snow can extend such dark conditions to up to 10 months. A major group of microalgae in benthic habitats of Antarctica are diatoms, which are key primary producers in these regions. However, the effects of extremely prolonged dark exposure on their photosynthesis, cellular ultrastructure, and cell integrity remain unknown. Here we show that five strains of Antarctic benthic diatoms exhibit an active photosynthetic apparatus despite 10 months of dark-exposure. This was shown by a steady effective quantum yield of photosystem II (Y[II]) upon light exposure for up to 2.5 months, suggesting that Antarctic diatoms do not rely on metabolically inactive resting cells to survive prolonged darkness. While limnic strains performed better than their marine counterparts, Y(II) recovery to values commonly observed in diatoms occurred after 4-5 months of light exposure in all strains, suggesting long recovering times. Dark exposure for 10 months dramatically reduced the chloroplast ultrastructure, thylakoid stacking, and led to a higher proportion of cells with compromised membranes than in light-adapted cells. However, photosynthetic oxygen production was readily measurable after darkness and strong photoinhibition only occurred at high light levels (>800 µmol photons m-2 s-1). Our data suggest that Antarctic benthic diatoms are well adapted to long dark periods. However, prolonged darkness for several months followed by only few months of light and another dark period may prevent them to regain their full photosynthetic potential due to long recovery times, which might compromise long-term population survival.

Structural evolution of maize starches with different amylose content during pasting and gelation as evidenced by Rapid Visco Analyser.

This study examined multi-scale structural alterations of maize starches varying in amylose content during pasting and gelation, using Rapid Visco Analyser (RVA). At 50 °C, starch granules maintained their morphology with low viscosity. As the temperature increased to 95 °C, helical and crystal structures were destroyed, leading to granule swelling, distortion and porosity, as identified by Wide Angle X-ray Scattering and Fourier Transforms Infrared measurements at 90% moisture. This resulted in increased viscosity and the formation of a loose gel network structure. Subsequently, maintaining the temperature at 95 °C caused a decrease in viscosity as most granules disappeared, forming a reorganized flaky gel structure with larger pores. As the temperature decreased, gel porosity reduced. In high amylose content starch, the viscosity remained low and granules were partially gelatinized since the heating temperature was below the gelatinization temperature. This study is the first to detail starch multilevel structural dynamics during RVA gelatinization.

Optimizing planting density enhances the multi-scale structural characteristics and in vitro digestibility of maize starch via modulating the size distribution of granules.

The modification of starch through agricultural practices is becoming increasingly significant for producing healthy foodstuffs and raw materials for industrial applications, consequently gaining momentum in academic research. This study examined how three different planting densities influenced the distribution of granule sizes, multi-scale structural characteristics, and in vitro digestibility of maize starch. The results showed that planting density significantly enhanced grain yield and relative crystallinity, and significant increases were also observed in the contents of both rapidly and slowly digestible starch. The surface- and volume-weighted mean diameter of granules significantly increased under the medium level (6.75 × 104 plants ha-1), and then decreased under high planting density level. As planting density level increased, the amylose content, peak viscosity, and hardness varied from 23.3 to 26.4 %, from 1962 to 2659 mPa·s, and from 129.3 to 307.6 g, respectively. However, no change was found in crystalline structure of maize starch. These results indicated that optimizing planting density could effectively improve grain yield and starch characteristics of maize, with the best effect under the level of 6.75 × 104 plants ha-1.

The effects of different types of high-amylose maize starches on viscosity and digestion of acidified milk gels.

High-amylose maize starch (HAMS) can provide dietary fiber to foods. In this study, we investigated the effects of three HAMSs (Gelose 50, Hylon VII, and NAFU50) on the functionality of casein (CA) and/or whey protein (WP) networks in acidified milk gels using normal maize starch (NMS) as a control thickener. When compared with NMS, HAMSs performed better in increasing the resistant starch content (RS), storage modulus, loss modulus, and complex viscosity of all the milk gels. The results are attributed to the retention of the starch granular integrity of HAMSs during the preparation of the milk gels, as observed by microscopy. HylonVII exhibited the highest RS and viscosity in all milk gel systems, especially in WP gels (71.8 % RS and >3000 Pa.s at 1 Hz viscosity). Our data provide support for the potential of using HAMS to develop healthier yogurt products using functional thickeners from natural sources.

Strategies for starch customization: Agricultural modification.

Raw starch is commonly modified to enhance its functionality for industrial applications. There is increasing demand for 'green' modified starches from both end-consumers and producers. It is well known that environmental conditions are key factors that determine plant growth and yield. An increasing number of studies suggest growth conditions can expand affect starch structure and functionality. In this review, we summarized how water, heat, high nitrogen, salinity, shading, CO2 stress affect starch biosynthesis and physicochemical properties. We define these treatments as a fifth type of starch modification method - agricultural modification - in addition to chemical, physical, enzymatic and genetic methods. In general, water stress decreases peak viscosity and gelatinization enthalpy of starch, and high temperature stress increases starch gelatinization enthalpy and temperature. High nitrogen increases total starch content and regulates starch viscosity. Salinity stress mainly regulates starch and amylose content, both of which are genotype-dependent. Shading stress and CO2 stress can both increase starch granule size, but these have different effects on amylose content and amylopectin structure. Compared with other modification methods, agricultural modification has the advantage of operating at a large scale and a low cost and can help meet the ever-rising market of clean-label foods and ingredients.

Bricks out of the wall: polysaccharide extramural functions.

Plant polysaccharides are components of plant cell walls and/or store energy. However, this oversimplified classification neglects the fact that some cell wall polysaccharides and glycoproteins can localize outside the relatively sharp boundaries of the apoplastic moiety, where they adopt functions not directly related to the cell wall. Such polysaccharide multifunctionality (or 'moonlighting') is overlooked in current research, and in most cases the underlying mechanisms that give rise to unconventional ex muro trafficking, targeting, and functions of polysaccharides and glycoproteins remain elusive. This review highlights major examples of the extramural occurrence of various glycan cell wall components, discusses the possible significance and implications of these phenomena for plant physiology, and lists exciting open questions to be addressed by future research.

Shank-localized cell wall growth contributes to Arabidopsis root hair elongation.

Root hairs are highly elongated tubular extensions of root epidermal cells with a plethora of physiological functions, particularly in establishing the root-rhizosphere interface. Anisotropic expansion of root hairs is generally thought to be exclusively mediated by tip growth-a highly controlled apically localized secretion of cell wall material-enriched vesicles that drives the extension of the apical dome. Here we show that tip growth is not the only mode of root hair elongation. We identified events of substantial shank-localized cell wall expansion along the polar growth axis of Arabidopsis root hairs using morphometric analysis with quantum dots. These regions expanded after in vivo immunolocalization using cell wall-directed antibodies and appeared as distinct bands that were devoid of cell wall labelling. Application of a novel click chemistry-enabled galactose analogue for pulse chase and real-time imaging allowed us to label xyloglucan, a major root hair glycan, and demonstrate its de novo deposition and enzymatic remodelling in these shank regions. Our data reveal a previously unknown aspect of root hair growth in which both tip- and shank-localized dynamic cell wall deposition and remodelling contribute to root hair elongation.

The effects of drought treatments on biosynthesis and structure of maize starches with different amylose content.

We investigated the effects of drought stress (DS) on maize varieties with different amylose content (AC). In starches with AC of 33 %, DS increased the contents of amylopectin (AP) chains with a degree of polymerization (DP) > 36 and decreased the AP chains with DP ≤ 36, while the AC was unchanged. DS decreased the crystallinity, the thickness of both amorphous and crystalline lamellae, and average granular size. In contrast, the digestibility increased. For starches with AC of 45 %, DS increased the content of AP chains with DP > 24 and AC, while the contents of AP chains with DP ≤ 24 decreased. DS produced starch with thinner crystalline lamellae, thicker amorphous lamellae, more elongated and larger granules. The digestibility of the starches decreased. In starches with AC of 53 %, moderate DS led to similar structural and functional changes as found for starches with AC of 45 %. Finally, severe DS resulted in the decrease of AC.