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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.

The extracellular matrix of plants can contain the hydrophobic biopolymers lignin, suberin and/or cutin, which provide mechanical strength and limit water loss and pathogen invasion. Due to their remarkable chemical resistance, these polymers have a high potential in various biotechnological applications and can replace petrol-based resources, for example, in the packing industry. However, despite the importance of these polymers, the regulation of their precursor biosynthesis is far from being fully understood. This is particularly true for suberin and cutin, which hinders efforts to engineer their formation in plants and produce customised biopolymers. This review brings attention to knowledge gaps in the current research and highlights some of the most recent findings on transcription factors that regulate lignin, suberin and cutin precursor biosynthesis. Finally, we also briefly discuss how some of the remaining knowledge gaps can be closed.

Seaweed farming: Biomass production from algae Algae are a high-potential yet underexplored source of renewable biomass. Here Kaus Herburger explores whether seaweed farming be the future for biomass production? The increasing demand for energy required for transport, heating and electricity is exhausting the world's natural resources and results into habitat loss, pollution and an aggravation of anthropogenic climate change. In order to meet future energy needs and comply with mandated CO2 reductions, it will be vital to utilise a wide range of sources for renewable materials and energy. A high-potential yet underexplored source of renewable biomass are algae, which are dominating the oceans as primary producers and can help overcoming shortcomings of classical land-based biomass production by serving as a pillar of the blue economy.

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 and3000 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.

SUMMARY Certain transglucanases can covalently graft cellulose and mixed‐linkage β‐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‐β‐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., Significance Statement The enzyme HTG can graft segments of cellulose molecules onto xyloglucan (a hemicellulose), thereby re‐structuring the cell wall via hetero‐transglycosylation. In native walls, two endogenous hemicelluloses competed with cellulose (and with each other) as substrate. HTG more readily selected cellulose as substrate if an expansin‐enriched preparation was added. Hetero‐transglycosylation was inhibited by cellobiose – a potential tool for exploring HTG’s biological functions. Interestingly, cellobiose retarded Equisetum stem elongation, suggesting a role for HTG in growth.

The Equisetum enzyme hetero-trans-β-glucanase (HTG) covalently grafts native plant cellulose (donor-substrate) to xyloglucan (acceptor-substrate), potentially offering a novel ‘green’ method of cellulose functionalisation. However, the range of cellulosic and non-cellulosic donor substrates that can be utilised by HTG is unknown, limiting our insight into its biotechnological potential. Here we show that HTG binds all celluloses tested (papers, tissues, hydrogels, bacterial cellulose) to radioactively- or fluorescently-labelled xyloglucan-heptasaccharide (XXXGol; acceptor-substrate). Glycol-chitin, glycol-chitosan and chitosan also acted as donor substrates but less effectively than cellulose. Cellulose-XXXGol conjugates were formed throughout the volume of a block of hydrogel, demonstrating penetration. Plant-derived celluloses (cellulose Iβ) became more effective donor-substrates after ‘mercerisation’ in ≥3 M NaOH; the opposite was true for bacterial cellulose Iα. Cellulose-XXXGol bonds resisted boiling 6 M NaOH, demonstrating strong glycosidic bonding. In conclusion, HTG stably grafts native and processed celluloses to xyloglucan-oligosaccharides, which may carry valuable ‘cargoes’, exemplified by sulphorhodamine. We thus demonstrate HTG's biotechnological potential to modify various cellulose-based substrates such as textiles, pulps, papers, packaging, sanitary products and hydrogels., We thank Ms. Amy Wallace (The University of Edinburgh) for help in producing xyloglucan–[3H]XXXGol, MLG–[3H]XXXGol and cellulose–[3H]XXXGol, Dr. Tom Simmons (The University of Edinburgh) for heterologous production of EfHTG in Pichia, Ms. Lucile Druel (CEMEF, Mines ParisTech) for the preparation of cellulose hydrogels, and Ms. Anna Roig (ICMAB, Bellaterra) for help in preparing bacterial cellulose. We acknowledge The Royal Society, London, for organising a meeting (‘Cellulose: prospects and challenges’, 15–16 March 2017) which initiated part of this collaboration. This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/N002458/1]; and the BBSRC Impact Acceleration Account (IAA) project [grant number PIII057].

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.

The multi-scale structural dynamics of amylopectin-only barley starch (APBS), normal barley starch (NBS), and amylose-only barley starch (AMBS) digested with amylolytic enzymes for 120 min were investigated in this study. For all starches, 0–20 min was identified as a rapid digestion stage, followed by a 20–120 min slow digestion. For APBS and NBS, the content of short chains (DP ≤ 12) and the thickness of crystalline and amorphous nano-lamellae increased, whereas the crystallinity decreased, and pores were generated on the granular surface during the rapid digestion stage. At the following slow digestion stage, the branching degree and relative amount of double helices increased, the crystalline and lamellar structures were lost, and the hydrolyzed starch segments aggregated. For AMBS, the rapid digestion stage was characterized by an increased content of short chains (DP ≤ 8) and B-type crystals and decreased amounts of V-type crystals. Furthermore, the lamellar and granular structures were lost, and digestion residues aggregated during the first stage of AMBS, which were formed in the second digestion stage of APBS and NBS. The second slow digestion stage of AMBS was characterized by an increased branching degree and a decreased relative content of single helices. It is suggested that the aggregated digestion residues at the initial digestion stage are the main reason for the overall low digestibility of AMBS.

BackgroundEnzymatic modification of foods is an important tool for enhancing starch functionality due to its safety, high efficiency, and specificity. Enzymatic modification of gelatinized starch systems is well reported in literature. However, to our knowledge, a review describing and comparing the effects of granular vs. gelatinized systems and enzyme-assisted modification of starch is both timely and lacking.Scope and approachWe summarize, discuss and point at future aspects of the consequences of using various techniques of enzymatic treatment of granular and gelatinized starch for modifying the structure and physicochemical properties, including the crystalline and granular structure, and functional properties. For gelatinized starch systems, we generally discuss catalytic mechanism, substrate specificity and product molecular structures for different types of starch-acting enzymes with emphasis on the resulting molecular structures. We critically discuss the problems of enzyme attack on granular starch in relation to two common strategies for starch granular pretreatment, namely, the preparation of porous starch and cold water-swelling of granular starch aimed at increasing the enzyme catalytic efficiency. Finally, we point out research directions to expand the applications of enzymatic treatments in starch modifications.Key findings and conclusionThe catalytic efficiency of starch-acting enzymes is higher on amorphous gelatinized starch than on semi-crystalline granular starch. To enhance the catalytic efficacy on granular starch, granules can be engineered to allow enzyme binding more efficiently onto granular surfaces. Additionally, we in-depth describe efforts to increase the specific surface area of the starch granular entities by I) generating pores and channels at the granular surface via treatment with hydrolases or by II) swelling granules via e.g., thermal pretreatment in the presence of ethanol. It is generally found that strategy I reduces the starch yield due to the hydrolytic effect, decreasing the pasting properties. Strategy II keeps a high yield of the starch product and generates granular starch with high pasting stability.

Octenyl succinate anhydride (OSA) modified high amylose maize starch (HAMS) showed limited emulsion stability due to the OSA groups were located on the surface of starch granules. However, amylose is enriched in the internal region of HAMS and provides the reaction site for OSA groups. In this study, five types of starches with different amylose content were prepared using optimized reaction conditions. Our data showed that amylose content (AC) was the most important parameter controlling the degree of substitution (DS), in comparison with temperature, pH, starch concentration and time. DS reached a maximum (2.1%) at AC of 58%. Importantly, OSA groups were mainly located in the internal regions of HAMS under the optimized reaction conditions. This was supported by (1) the decrease of the crystallinity, (2) the weaker fluorescence intensity of the starch fluorophore probe 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) in the internal regions of HAMS, and (3) unchanged signals from Fourier transform infrared spectroscopy which characterized the granular surface structure ordering. This new OSA reaction pattern permits HAMS to be used in stabilizing Pickering emulsions This is the first report that OSA groups located in the internal regions of HAMS granules.

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.

Supplemental irrigation (SI) is an important strategy to improve the water-use efficiency (WUE) of crops without compromising the yield. However, such strategy can influence the starch and grain quality. Hence, the effects of SI on winter wheat starch structure and functionality were studied on ridge-furrow (RF) and flat tillage (FT) treated fields. Flat irrigation was set as control. RF + SI significantly increased the grain yield throughout the study period (2016–2018). SI decreased the amylose content and the content of amylopectin chains with DP 13–24 but increased the proportions of amylopectin chains with DP 6–12 and 25–36. The starch granule relative crystallinity decreased, and more B-type granules were produced by SI treatment. SI significantly increased the resistant starch content in both raw and cooked starch systems. Flat tillage enhanced the effect of SI on granule specific surface area (SSA) and viscosity, which increased starch paste viscosity, while SI + RF showed the opposite effects. Our study demonstrates important combined effects of SI and tillage on wheat starch quality.

The agile product innovation method Lean Startup Approach is used by startups to quickly adapt to the fast changing market conditions and customer needs. The authors carried out a preliminary study which showed that global challenges such as digital transformation are urging companies to become more dynamic and flexible, and that this method is already used by large companies to meet the needs. Based on the preliminary study, the authors conducted in-depth interviews with experts to examine the procedure for introducing the Lean Startup Approach in large manufacturing companies, and examined the feasibility of applying the method to SMEs..

Mougeotia spp. collected from field samples were investigated for their conjugation morphology by light-, fluorescence-, scanning- and transmission electron microscopy. During a scalarifom conjugation, the extragametangial zygospores were initially surrounded by a thin cell wall that developed into a multi-layered zygospore wall. Maturing zygospores turned dark brown and were filled with storage compounds such as lipids and starch. While M. parvula had a smooth surface, M. disjuncta had a punctated surface structure and a prominent suture. The zygospore wall consisted of a polysaccharide rich endospore, followed by a thin layer with a lipid-like appaerance, a massive electron dense mesospore and a very thin exospore composed of polysaccharides. Glycan microarray analysis of zygospores of different developmental stages revealed the occurrence of pectins and hemicelluloses, mostly composed of homogalacturonan (HG), xyloglucans, xylans, arabino-galactan proteins and extensins. In situ localization by the probe OG7-13AF 488 labelled HG in young zygospore walls, vegetative filaments and most prominently in conjugation tubes and cross walls. Raman imaging showed the distribution of proteins, lipids, carbohydrates and aromatic components of the mature zygospore with a spatial resolution of ~ 250 nm. The carbohydrate nature of the endo- and exospore was confirmed and in-between an enrichment of lipids and aromatic components, probably algaenan or a sporopollenin-like material. Taken together, these results indicate that during zygospore formation, reorganizations of the cell walls occured, leading to a resistant and protective structure.

The plant apoplast contains the four hydrophobic polymer, lignin, suberin, cutin, and cutan, that are crucial for stress resistance, controlling solute diffusion, and strengthening the cell wall. Some of these polymers are widely used in industry and daily life products, such as all wood-containing goods (lignin) and wine cork (suberin). Despite the importance of these polymers, several aspects of their formation remain unknown. This mini review highlights technical bottlenecks in the current research and summarizes recent insights into the precursor transmembrane transport, an essential step in the polymer formation. We also briefly discuss how some of the remaining knowledge gaps can be closed and how a better understanding of these biopolymers will benefit other research fields.

A method for efficient functional modification of starch granules by thermal ethanol pre-treatment and subsequent maltogenic α-amylase (MA) and branching enzyme (BE) post-treatments is described. Ethanol pre-treatment significantly increased the swelling power of starch granules thereby increasing the MA and BE susceptibility. Ethanol pre-treated granules became shrunk and twisted after incubating in buffer. Sequential MA post-treatments remarkably increased the α-1,6 to α-1,4 ratio and the content of amylopectin short chains (DP 1–10), contributing to the low retrogradation rate. BE post-treatments significantly decreased the product yield, increased the relative crystallinity of starch granules, suggesting BE had intramolecular transglucosylation activity which altered the branch position and reduced the molecular size by forming cyclic structures. Moreover, BE post-treatments showed an α-1,6 to α-1,4 transglucosylation activity by decreasing the α-1,6 to α-1,4 ratio, especially during simultaneous MA and BE catalysis. However, in the simultaneous MA and BE post-catalysis, MA dosage was predominant by noticeably hydrolyzing amylopectin and amylose molecules and increasing the α-1,6 to α-1,4 ratio, thereby leading to the lowest digestibility and retrogradation.

Due to the semi-crystalline structure of native starch granules, enzymatic modification of these solid, raw, entities by branching enzyme (BE) is limited. Here, we describe a method to efficiently modify starch by BE after maltogenic α-amylases pre-treatment. This pre-treatment produced pores at the starch granule surface, which decreased the granular yield, but increased the branching degree in starch molecules. BE post-treatments recovered the yield, increased the content of long amylose chains, and the starch crystallinity. WAXS analysis showed that BE transformed the unresolved doublet peak at 2θ 17° and 18° to a strong peak at 2θ 17°, i.e. transformed the granules from the A-type to a mixed A-, B-type allomorph. Syneresis of starch gels increased with increasing BE concentrations and increased the content of slowly digested starch in retrograded starch preparations. Rheology data demonstrated that low and medium BE concentrations produced starch gels with higher G′ and G″ after storage for 1day, whereas high BE concentrations reduced both G′ and G’’. Our data demonstrate the potential of clean, enzyme-based protocols using sequential addition of starch active enzymes for post-harvest modification of raw starch granules to obtain clean and functional starch.

Many alpine streams inhabit conspicuous epilithic biofilms on pebbles and rocks that are formed by members of the cyanobacterial genus Chamaesiphon (Synechococcales). In the Austrian Alps, some Chamaesiphon species can even overgrow up to 70% of the surface of river rocks, and hence they must play an important but still unstudied ecological role in the organic matter flux. Since photo-biological traits have not been investigated so far, photosynthetic features, pigments, and UV-sunscreen compounds were studied in three Chamaesiphon morphospecies (C. geitleri, C. polonicus, C. starmachii). These species form conspicuously differently colored spots on cobbles and boulders in the alpine streams. While C. polonicus typically forms red crusts on flat pebble conglomerate, C. geitleri and C. starmachii are characterized by dark brown and black biofilms in the field, respectively. Photosynthesis-irradiance (PE) curves indicate that all three Chamaesiphon species have different light requirements for photosynthesis, with C. starmachii and C. polonicus preferring high and low photon fluence rates, respectively, while C. geitleri takes a position in between. This low-light requirement of C. polonicus is also reflected in ca. ten-times lower chlorophyll a, zeaxanthin, and s-carotene concentrations, as well as in a lack of the UV-sunscreen scytonemin. All Chamaesiphon morphospecies exhibit the mycosporine-like amino acid porphyra-334. The physiological and biochemical data indicate strong intraspecific differences in photosynthetic activity and pigment patterns, which explain well the distinct preferences of the three studied Chamaesiphon morphospecies for sun-exposed or shaded habitats.

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 soil and freshwater habitats. 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. This indicates 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.

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglucan or mixed-linkage β-d-glucan (MLG). However, Equisetum hetero-trans-β-glucanase (HTG) grafts cellulose onto xyloglucan oligosaccharides (XGOs) – and, we now show, xyloglucan polysaccharide – in vitro, thus exhibiting CXE (cellulose:xyloglucan endotransglucosylase) activity. In addition, HTG also catalyzes MLG-to-XGO bonding (MXE activity). In this study, we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds. To quantify and visualize CXE and MXE action, we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics. We demonstrated covalent cellulose–xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15% and 60% of total transglucanase action, respectively, and peaked in aging, strengthening tissues: CXE in xylem and cells bordering intercellular canals and MXE in sclerenchyma. Recombinant bacterial expansin (EXLX1) strongly augmented CXE activity in vitro. CXE and MXE action in living Equisetum structural tissues potentially strengthens stems, while expansin might augment the HTG-catalyzed CXE reaction, thereby allowing efficient CXE action in muro. Our methods will enable surveys for comparable reactions throughout the plant kingdom. Furthermore, engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance., Studying a hetero-transglucanase showed that cellulose and mixed-linkage glucan can be covalently linked to the hemicellulose xyloglucan in native plant cell walls. These reactions increase with tissue age and predominate in structural tissues and in cells surrounding cavities, suggesting a role in tissue strengthening. Expansin strongly augments cellulose hetero-transglucosylation but has no effect on mixed-linkage glucan.

Global competition, rapid market changes and the need for more and faster innovations lead to a new risk situation for manufacturing companies. As dynamics increase due to the Digital Transformation, the reliability of success forecasts decreases and many of the product design innovations launched on the market fail. Manufacturing companies, which design products down to the last detail and then develop them over a long period of time and with high investments are highly likely to find that their product will not be accepted on the market. To reduce this risk, agile processes and iterative product design is useful. This approach is based on multiple, iterative tests with customers and the possibility of implementing the knowledge gained. It is important to constantly check, whether the products meet the demand of the market and to design the product in such a variable way, that changes can be taken up immediately and learning effects can be implemented. The Lean-Startup approach is based on these principles. It has been established as a way of thinking for startups and young High-Tech companies who want to optimize their initial market approach through iterative testing of prototypes and continuous improvement through learning. However, the Lean-Startup approach is also suitable for larger companies, which want to make their product development more agile and flexible. This enables companies to react more flexibel to external influences, such as market competition, technologies and politics and to integrate customer needs are more effectively into the product design process. This constant monitoring of the product market fit reduces the risk of developing past the market. This paper introduces an approach for migrating the Lean-Startup approach to large manufacturing companies.

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.

Many alpine streams inhabit conspicuous epilithic biofilms on pebbles and rocks that are formed by members of the cyanobacterial genus Chamaesiphon (Synechococcales). In the Austrian Alps, some Chamaesiphon species can even overgrow up to 70% of the surface of river rocks, and hence they must play an important but still unstudied ecological role in the organic matter flux. Since photo-biological traits have not been investigated so far, photosynthetic features, pigments, and UV-sunscreen compounds were studied in three Chamaesiphon morphospecies (C. geitleri, C. polonicus, C. starmachii). These species form conspicuously differently colored spots on cobbles and boulders in the alpine streams. While C. polonicus typically forms red crusts on flat pebble conglomerate, C. geitleri and C. starmachii are characterized by dark brown and black biofilms in the field, respectively. Photosynthesis-irradiance (PE) curves indicate that all three Chamaesiphon species have different light requirements for photosynthesis, with C. starmachii and C. polonicus preferring high and low photon fluence rates, respectively, while C. geitleri takes a position in between. This low-light requirement of C. polonicus is also reflected in ca. ten-times lower chlorophyll a, zeaxanthin, and ß-carotene concentrations, as well as in a lack of the UV-sunscreen scytonemin. All Chamaesiphon morphospecies exhibit the mycosporine-like amino acid porphyra-334. The physiological and biochemical data indicate strong intraspecific differences in photosynthetic activity and pigment patterns, which explain well the distinct preferences of the three studied Chamaesiphon morphospecies for sun-exposed or shaded habitats.

Highlights ► We investigate the osmotic potential of four polar green algal strains. ► We examine effects of osmotic stress on ultrastructure and photosynthesis. ► One investigated strain belongs to a different phylogenetic lineage. ► Arctic strains show higher osmotic values (more negative osmotic potentials). ► Physiological performance and ultrastructure are affected by osmotic stress., The osmotic potential and effects of plasmolysis on photosynthetic oxygen evolution and chlorophyll fluorescence were studied in two Arctic Zygnema sp. (strain B, strain G) and two Antarctic Zygnema sp. (strain E, strain D). Antarctic strain D was newly characterized by rbcL sequence analysis in the present study. The two Antarctic strains, D and E, are most closely related and may represent different isolates of the same species, in contrast, strain B and G are separate lineages. Incipient plasmolysis in the cells was determined by light microscopy after incubating cells in sorbitol solutions ranging between 200 mM and 1000 mM sorbitol for 3, 6 and 24 h. In Zygnema strain B and G incipient plasmolysis occurred at ∼600 mM sorbitol solution (720 mOsmol kg−1, ψ = −1.67 MPa) and in strains D and E at ∼300 mM (318 mOsmol kg−1, ψ = −0.8 MPa) sorbitol solution. Hechtian strands were visualized in all plasmolysed cells, which is particularly interesting, as these cells lack pores or plasmodesmata. Ultrastructural changes upon osmotic stress were a retraction of the condensed cytoplasm from the cell walls, damages to chloroplast and mitochondrial membranes, increasing numbers of plastoglobules in the chloroplasts and membrane enclosed particles in the extraplasmatic space. Maximum photosynthetic rates (Pmax) in light saturated range were between 145.5 μmol O2 h−1 mg−1 Chl a in Zygnema G and 752.9 μmol O2 h−1 mg−1 Chl a in Zygnema E. After incubation in 800 mM sorbitol for 3 h Pmax decreased to the following percentage of the initial values: B: 16.3%, D: 16.8%, E: 26.1% and G: 35.0%. Osmotic stress (800 mM sorbitol) decreased maximum photochemical quantum yield of photosystem II (Fv/Fm) when compared to controls. Maximum values of relative electron transport rates of photosystem II (rETRmax) decreased after incubation in 400 mM sorbitol in Zygnema D and E, while they decreased in Zygnema B and G only after incubation in 800 mM sorbitol. The kinetics of the rETR curves were similar for the Arctic strains Zygnema B and G, but distinct from the Antarctic strains Zygnema D and E, which were similar when compared with each other. This suggests that the investigated Arctic Zygnema sp. strains might be better adapted to tolerate osmotic water stress than the investigated strains from the Antarctic.

The phylogenetic placement of Prasiola calophylla, from an anthropogenic habitat previously shown to contain a novel UV sunscreen compound, was confirmed by analysis of its rbcL gene. This alga has the capacity to tolerate strong water-limiting conditions. The photosynthetic performance and ultrastructural changes under desiccation and osmotic stress were investigated. Freshly harvested thalli showed an effective quantum yield of PSII [Y(II)] of 0.52 ± 0.06 that decreased to ∼60% of the initial value at 3000 mM sorbitol, and 4000 mM sorbitol led to a complete loss of Y(II). The Y(II) of thalli exposed to controlled desiccating conditions at 60% relative humidity (RH) ceased within 240 min, whereas zero values were reached after 120 min at 20% RH. All investigated samples completely recovered Y(II) within ∼100 min after rehydration. Relative electron transport rates (rETR) were temperature dependent, increasing from 5, 10, to 25 °C but strongly declining at 45 °C. Transmission electron microscopy of samples desiccated for 2.5 h showed an electron dense appearance of the entire cytoplasm when compared to control samples. Thylakoid membranes were still visible in desiccated cells, corroborating the ability to recover. Control and desiccated cells contained numerous storage lipids and starch grains, providing reserves. Overall, P. calophylla showed a high capacity to cope with water-limiting conditions on a physiological and structural basis. A lipophilic outer layer of the cell walls might contribute to reduce water evaporation in this poikilohydric organism.

Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.

Green algae of the genus Klebsormidium (Klebsormidiales, Streptophyta) are typical members of biological soil crusts (BSCs) worldwide. The phylogeny and ecophysiology of Klebsormidium has been intensively studied in recent years, and a new lineage called superclade G, which was isolated from BSCs in arid southern Africa and comprising undescribed species, was reported. Three different African strains, that have previously been isolated from hot-desert BSCs and molecular-taxonomically characterized, were comparatively investigated. In addition, Klebsormidium subtilissimum from a cold-desert habitat (Alaska, USA, superclade E) was included in the study as well. Photosynthetic performance was measured under different controlled abiotic conditions, including dehydration and rehydration, as well as under a light and temperature gradient. All Klebsormidium strains exhibited optimum photosynthetic oxygen production at low photon fluence rates, but with no indication of photoinhibition under high light conditions pointing to flexible acclimation mechanisms of the photosynthetic apparatus. Respiration under lower temperatures was generally much less effective than photosynthesis, while the opposite was true for higher temperatures. The Klebsormidium strains tested showed a decrease and inhibition of the effective quantum yield during desiccation, however with different kinetics. While the single celled and small filamentous strains exhibited relatively fast inhibition, the uniserate filament forming isolates desiccated slower. Except one, all other strains fully recovered effective quantum yield after rehydration. The presented data provide an explanation for the regular occurrence of Klebsormidium strains or species in hot and cold deserts, which are characterized by low water availability and other stressful conditions.

The green-algal class Klebsormidiophyceae (Streptophyta), which occurs worldwide, comprises the genera Klebsormidium, Interfilum, Entransia, and Hormidiella. Ecophysiological research has so far focused on the first two genera because they are abundant in biological soil crust communities. The present study investigated the photosynthetic performances of Hormidiella attenuata and two strains of Entransia fimbriata under light, temperature, and desiccation stress. Their ultrastructure was compared using transmission electron microscopy. The two Entransia strains showed similar physiological responses. They used light more efficiently than Hormidiella, as indicated by higher oxygen production and relative electron transport rate under low light conditions, lower light saturation and compensation points, and higher maximum oxygen production during light saturation. Their requirement for low light levels explains the restriction of Entransia to dim limnetic habitats. In contrast, Hormidiella, which prefers drier soil habitats, responded to light gradients similarly to other aero-terrestrial green algae. Compared to Entransia, Hormidiella was less affected by short-term desiccation, and rehydration allowed full recovery of the photosynthetic performance. Nevertheless, both strains of Entransia coped with low water availability better than other freshwater algae. Photosynthetic oxygen production in relation to respiratory consumption was higher in low temperatures (Entransia: 5 °C, Hormidiella: 10 °C) and the ratio decreased with increasing temperatures. Hormidiella exhibited conspicuous triangular spaces in the cell wall corners, which were filled either with undulating cell wall material or with various inclusions. These structures are commonly seen in various members of Klebsormidiophyceae. The data revealed significant differences between Hormidiella and Entransia, but appropriate adaptations to their respective habitats. Electronic supplementary material The online version of this article (doi:10.1007/s00709-015-0889-z) contains supplementary material, which is available to authorized users.

Zygogonium ericetorum, the type species of the genus, was studied from a natural population collected in Mt. Schönwieskopf, Tyrol, Austria. Generic concepts of Zygogonium and Zygnema were tested with atpB, psbC, and rbcL gene sequence analysis, which showed a sister relationship between Z. ericetorum and Mesotaenium, in an early branching clade sister to a grouping of Zygnema and several other filamentous and unicellular zygnematalean taxa. A variety of light, confocal, transmission electron microscopy, and cytochemical techniques provided new data on the variable chloroplast shape of Z. ericetorum, and its aplanospore structure and development, which has been previously considered taxonomically important but has been ambiguously interpreted. Zygogonium can be distinguished from other zygnematophytes (particularly Zygnema), based on the combination of two characters: (i) irregular, compressed plate-like chloroplasts and (ii) residual cytoplasmic content left in sporangia outside of the fully developed aplanospores or zygospores. The presence of a sporangial wall that separates the spores from the parent cell should be excluded from the definition of Zygogonium, because it is also observed in Zygnema. Similarly, the ecological characterization of Zygogonium as acidophilic is not unique to the genus. The names of 18 species currently belonging to Zygogonium are here changed to Zygnema, because of incompatibility with this new proposed Zygogonium concept. In the species transferred to Zygnema, chloroplasts are typically stellate in three-dimensions, and the entire content of fertile cells is transformed into the spore, so there is no cytoplasmic residue.