Assessment of leaf/stem ratio in wheat straw feedstock and impact on enzymatic conversion

The composition of wheat straw leaf and stem fractions were characterized using traditional strong acid hydrolysis, and monoclonal antibodies using comprehensive microarray polymer profiling (CoMPP). These results are then related to high throughput lignocellulose pretreatment and saccharification screening data. Pure leaf fraction of wheat straw was the least recalcitrant compared to pure stem and easily digested by commercial cellulases after moderate hydrothermal pretreatment; 63% and 31% (w/w) of glucan, 88% and 61% of xylan were released from the leaf and stem fractions, respectively. By preparing samples of various leaf‐to‐stem (L/S) ratios, we found shifting conversion behavior as processing parameters were modified. Increasing the enzyme dosage, pretreatment temperature and pretreatment time all significantly improved conversion rates in samples with more than 50% leaf content, whereas less impact was observed on samples with less than 50% leaf content. Enzyme affinity, desorption and readsorption with leaf and stem fractions may affect the sugar yield in wheat straw saccharification. The data suggest that the L/S ratio is an important parameter when adjusting or optimizing conversion processes and additionally in feedstock breeding. Furthermore, this highlights the need for rapid techniques for determining L/S ratio in wheat straw harvests. The CoMPP data on specific carbohydrates and leaf pectin highlight carbohydrate epitopes that may be useful as markers in the development of novel screening techniques; especially pectin or arabinogalactan proteins related epitopes are promising.     

Solid-phase chemical tools for glycobiology

Techniques involving solid supports have played crucial roles in the development of genomics, proteomics, and in molecular biology in general. Similarly, methods for immobilization or attachment to surfaces and resins have become ubiquitous in sequencing, synthesis, analysis, and screening of oligonucleotides, peptides, and proteins. However, solid-phase tools have been employed to a much lesser extent in glycobiology and glycomics. This review provides a comprehensive overview of solid-phase chemical tools for glycobiology including methodologies and applications. We provide a broad perspective of different approaches, including some well-established ones, such as immobilization in microtiter plates and to cross-linked polymers. Emerging areas such as glycan microarrays and glycan sequencing, quantum dots, and gold nanoparticles for nanobioscience applications are also discussed. The applications reviewed here include enzymology, immunology, elucidation of biosynthesis, and systems biology, as well as first steps toward solid-supported sequencing. From these methods and applications emerge a general vision for the use of solid-phase chemical tools in glycobiology.     

Stable transformation and reverse genetic analysis of Penium margaritaceum: a platform for studies of charophyte green algae,the immediate ancestors of land plants

The charophyte green algae (CGA, Streptophyta, Viridiplantae) occupy a key phylogenetic position as the immediate ancestors of land plants but, paradoxically, are less well‐studied than the other major plant lineages. This is particularly true in the context of functional genomic studies, where the lack of an efficient protocol for their stable genetic transformation has been a major obstacle. Observations of extant CGA species suggest the existence of some of the evolutionary adaptations that had to occur for land colonization; however, to date, there has been no robust experimental platform to address this genetically. We present a protocol for high‐throughput Agrobacterium tumefaciens‐mediated transformation of Penium margaritaceum, a unicellular CGA species. The versatility of Penium as a model for studying various aspects of plant cell biology and development was illustrated through non‐invasive visualization of protein localization and dynamics in living cells. In addition, the utility of RNA interference (RNAi) for reverse genetic studies was demonstrated by targeting genes associated with cell wall modification (pectin methylesterase) and biosynthesis (cellulose synthase). This provided evidence supporting current models of cell wall assembly and inter‐polymer interactions that were based on studies of land plants, but in this case using direct observation in vivo. This new functional genomics platform has broad potential applications, including studies of plant organismal biology and the evolutionary innovations required for transition from aquatic to terrestrial habitats.