Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.Peer reviewe

Xylan biosynthesis and modification

Wood is an important renewable material used by humans for a variety of downstream applications. The basic subcellular structure in wood is the cell wall, mainly consisting of the cross-linked polymers cellulose, hemicellulose, and lignin. Xylan is the main hemicellulose found in angiosperm wood, and its biosynthesis and effects on wood properties in hybrid aspen (Populus tremula x tremuloides) were the object of the present study. We identified a glycoside hydrolase in developing wood, PtxtXyn10A—a previously suggested xylanase—and found that it rather has xylan transglycosylase activity. This xylan transglycosylase probably rearranges xylan chains during secondary wall deposition. Interestingly, it affected the orientation of cellulose microfibrils, giving new insights into the interaction of cell wall polymers and their control during deposition into the wall. To study genes involved in secondary wall formation and to modify wood properties using transgenic approaches, it is essential to efficiently target transgene expression to the secondary wall forming cells. We isolated and tested a promoter, pGT43B, which proved to be very effective in altering gene expression and in generating intended chemical modifications in woody cells. We applied this GT43B promoter to modify xylan acetylation and polymerisation in hybrid aspen wood. Furthermore, it enabled the identification of the sucrose transporter SUT3, elucidating how sucrose is transported from ray to developing fiber and vessel cells. Glycosyltransferases (GTs) are enzymes involved in polysaccharide biosynthesis. We characterised the xylan biosynthetic GT43 gene family in hybrid aspen and found that reduced GT43 expression levels positively affected overall plant growth, wood mechanical strength, and saccharification efficiency, indicating potential applications for engineering bioenergy feedstocks. We conclude that cell specific modification of xylan properties in hybrid aspen can alter cellulose deposition and result in activation of cambial proliferation which has a positive impact on overall biomass yield and properties for downstream woodutilisation

Bra fråga! En studie om hur gymnasielärare använder frågandet i naturkunskapsundervisningen

Genom klassrumsobservationer och lärarintervjuer undersöker denna studie vilka frågandetekniker lärare använder för att stimulera elevers tänkande och ge produktiva diskussioner i klassrummet. För att analysera dialogen mellan lärare och elever utvecklades en metod som visualiserar händelser i förloppet av en lektion på ett översiktligt sätt. Visualiseringsmetoden gjorde det möjligt att enkelt identifiera både trender i klassrumsdialogen och enskilda moment i samtalet som verkade intressanta att undersöka vidare. Undersökningens fokus var att analysera vilka produktiva frågandetekniker gymnasielärare i naturkunskapsundervisningen använder och hur lärare själva uppfattar att de använder frågor i klassrummet. Jag observerade flera exempel av produktiva frågandetekniker i klassrummen, till exempel ”verbal jigsaw” och ”framing”. Med hjälp av visualiseringsmetoden av klassrumsdialogen identifierade jag dessutom undervisningssituationer där elever var muntligt engagerade. Studien visade i enlighet med tidigare forskning att elever ställer flest frågor när detta uttryckligen efterfrågas av läraren, när de personligen kan relatera till innehållet och när de arbetar i små grupper. Lärarna som deltog i studien var alla positivt inställda till elevernas egna frågor och försökte uppmuntra elevengagemang

PURA-Related Developmental and Epileptic Encephalopathy: Phenotypic and Genotypic Spectrum

Background and ObjectivesPurine-rich element-binding protein A (PURA) gene encodes Pur-alpha, a conserved protein essential for normal postnatal brain development. Recently, a PURA syndrome characterized by intellectual disability, hypotonia, epilepsy, and dysmorphic features was suggested. The aim of this study was to define and expand the phenotypic spectrum of PURA syndrome by collecting data, including EEG, from a large cohort of affected patients.MethodsData on unpublished and published cases were collected through the PURA Syndrome Foundation and the literature. Data on clinical, genetic, neuroimaging, and neurophysiologic features were obtained.ResultsA cohort of 142 patients was included. Characteristics of the PURA syndrome included neonatal hypotonia, feeding difficulties, and respiratory distress. Sixty percent of the patients developed epilepsy with myoclonic, generalized tonic-clonic, focal seizures, and/or epileptic spasms. EEG showed generalized, multifocal, or focal epileptic abnormalities. Lennox-Gastaut was the most common epilepsy syndrome. Drug refractoriness was common: 33.3% achieved seizure freedom. We found 97 pathogenic variants in PURA without any clear genotype-phenotype associations.DiscussionThe PURA syndrome presents with a developmental and epileptic encephalopathy with characteristics recognizable from neonatal age, which should prompt genetic screening. Sixty percent have drug-resistant epilepsy with focal or generalized seizures. We collected more than 90 pathogenic variants without observing overt genotype-phenotype associations