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The current hypothesis is that land plants originated from a charophycean green alga and that a prominent feature for adaptation to land was their development of alternating life cycles. Our work on cell wall evolution and morphological and physiological observations in the charophycean green algae challenged us to reassess how land plants became terrestrial. Our hypothesis is simple in that the charophycean green algae ancestors were already living on land and had been doing so for some time before the emergence of land plants. The evolution of alternate life cycles merely made the ancestral land plants evolutionary successful and had nothing to do with terrestrialization per se . [ABSTRACT FROM AUTHOR]

• Preparation of an Alcohol Insoluble Residue (AIR) is an important first step in the analysis of cell wall carbohydrates. • Variation in the protocols used for AIR preparation impacts the downstream extractability and detection of cell wall components. • Carbohydrate microarrays were used to analyse carbohydrate extractability. • 70-CHCl3/MeOH L protocol can be considered a reasonable starting point for optimisation of carbohydrate extraction. Almost all plant cells are surrounded by a wall constructed of co-extensive networks of polysaccharides and proteoglycans. The capability to analyse cell wall components is essential for both understanding their complex biology and to fully exploit their numerous practical applications. Several biochemical and immunological techniques are used to analyse cell walls and in almost all cases the first step is the preparation of an alcohol insoluble residue (AIR). There is significant variation in the protocols used for AIR preparation, which can have a notable impact on the downstream extractability and detection of cell wall components. To explore these effects, we have formally compared ten AIR preparation methods and analysed polysaccharides subsequently extracted using high-performance anion exchange chromatography (HPAEC-PAD) and Micro Array Polymer Profiling (MAPP). Our results reveal the impact that AIR preparation has on downstream detection of cell wall components and the need for optimisation and consistency when preparing AIR. [ABSTRACT FROM AUTHOR]

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface--and wall--associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgilocalized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana. GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-β-1,3-galactosidase activity of GH43 enzymes is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall--extracted glycoproteins or AGP-rich gumarabic. These results indicate that the lack of exoβ-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract: Potato pulp is rich in dietary fibres and is an underutilised material produced in large quantities by the potato starch factories. Potato fibres are especially rich in rhamnogalacturonan I (RG I). RG I is a pectic polysaccharide with a high degree of branching and until now undegraded RG I has only been extracted in small amounts limiting the application possibilities for RG I. The present paper describes a large-scale extraction process providing large quantities of undegraded RG I readily available. The extraction process includes enzymatic starch removal using purified Termamyl, enzymatic RG I solubilisation using a highly purified polygalacturonase, and finally purification using depth filtration and ultrafiltration. The extracted RG I has a high molecular weight and a monosaccharide composition comparable to RG I extracted by analytical extraction procedures. The large amount of RG I available by the presented method allows for thorough structure–function analyses and tailoring of RG I to specific functionalities. [Copyright &y& Elsevier]

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in Gal- NAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designedO-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts. [ABSTRACT FROM AUTHOR]

Abstract: Marama bean (Tylosema esculentum) is an important component of the diet around the Kalahari Desert in Southern Africa where this drought resistant plant can grow. The marama bean contains roughly 1/3 proteins, 1/3 lipids and 1/3 carbohydrates, but despite its potential as dietary supplement little is known about the carbohydrate fraction. In this study the carbohydrate fraction of “immature” and “mature” marama seeds are characterised. The study shows that the marama bean contains negligible amounts of starch and soluble sugars, both far less than 1%. The cell wall is characterised by a high arabinose content and a high resistance to extraction as even a 6M NaOH extraction was insufficient to extract considerable amounts of the arabinose. The arabinose fraction was characterised by arabinan-like linkages and recognised by the arabinan antibody LM6 and LM13 indicating that it is pectic arabinan. Two pools of pectin could be detected; a regular CDTA (1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid) or enzymatically extractable pectin fraction and a recalcitrant pectin fraction containing the majority of the arabinans, of which about 40% was unextractable using 6M NaOH. Additionally, a high content of mannose was observed, possibly from mannosylated storage proteins. [Copyright &y& Elsevier]

Abstract: Potato (Solanum tuberosum cv. Posmo) was transformed with a fungal endo-α-1,5-l-arabinanase gene (E.C. 3.2.1.99) and the enzyme secreted to the apoplast. Using promoters that are known to be sucrose inducible in potato—the granule bound starch synthase promoter and the promoter of the patatin storage protein gene—strong expression of the arabinanase was observed in developing sinks, notably the meristems giving rise to tubers, stolons and side shoots. Expression of the arabinanase interfered with normal meristem function and depending on the promoter used, different phenotypes were obtained devoid of tubers or both tubers, stolons and side shoots. The altered ratio of source to sink tissues in the transformants was used to study the regulation of photoassimilate allocation to different tissues. Tuberless transformants were unable to fully balance supply with demand and excess photoassimilates were deposited as starch granules with the structure of tuber starch in above ground tissues, notably in the stem pith. Transcripts normally associated with tuber development were detected in the starch accumulating tissues and the tuberlike transcript profile was not limited to transcripts that are directly sucrose inducible. These observations are discussed in relation to organ identity and source–sink regulation in potato. [Copyright &y& Elsevier]

• Engineered amylose and waste CNF generate good bioplastics. • CNF enhanced crystallinity, mechanics and permeability. • Cellulose nanofibers and amylose showed domain phase separation. Thermoplastic, polysaccharide-based plastics are environmentally friendly. However, typical shortcomings include lack of water resistance and poor mechanical properties. Nanocomposite manufacturing using pure, highly linear, polysaccharides can overcome such limitations. Cast nanocomposites were fabricated with plant engineered pure amylose (AM), produced in bulk quantity in transgenic barley grain, and cellulose nanofibers (CNF), extracted from agrowaste sugar beet pulp. Morphology, crystallinity, chemical heterogeneity, mechanics, dynamic mechanical, gas and water permeability, and contact angle of the films were investigated. Blending CNF into the AM matrix significantly enhanced the crystallinity, mechanical properties and permeability, whereas glycerol increased elongation at break, mainly by plasticizing the AM. There was significant phase separation between AM and CNF. Dynamic plasticizing and anti-plasticizing effects of both CNF and glycerol were demonstrated by NMR demonstrating high molecular order, but also non-crystalline, and evenly distributed 20 nm-sized glycerol domains. This study demonstrates a new lead in functional polysaccharide-based bioplastic systems. [ABSTRACT FROM AUTHOR]