Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin

• Published in: The Plant journal : for cell and molecular biology Vol. 104; no. 3; pp. 752 - 767
• Main Authors: Stratilová, Barbora, Šesták, Sergej, Mravec, Jozef, Garajová, Soňa, Pakanová, Zuzana, Vadinová, Kristína, Kučerová, Danica, Kozmon, Stanislav, Schwerdt, Julian G., Shirley, Neil, Stratilová, Eva, Hrmova, Maria
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.11.2020
• Subjects: Anions - metabolism; Barley; Catalytic Domain; Cell Wall - metabolism; Cell walls; clustered heatmap of expression profiles; evolution; Fluoresceins - chemistry; Gene expression; Glucans - metabolism; Glycosidases; Glycoside hydrolase; glycoside hydrolase 16 family; Glycosylation; Glycosyltransferases - chemistry; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; homo‐ and hetero‐transglycosylation; Hordeum - cytology; Hordeum - genetics; Hordeum - metabolism; Hordeum vulgare; Hydrogen-Ion Concentration; Hydrolase; Isoenzymes; Isoforms; Models, Molecular; Molecular modelling; Multigene Family; mutants; Oligosaccharides; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Pectin; pectin fragment; Pectins - metabolism; Phylogeny; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Roots - cytology; Plant Roots - metabolism; Polymerase chain reaction; RT‐qPCR; Substrate Specificity; Substrates; Sulfonic Acids - chemistry; Three dimensional models; Xylans - metabolism; Xyloglucan; pectin fragment; homo- and hetero-transglycosylation; mutants; clustered heatmap of expression profiles; evolution; RT-qPCR; glycoside hydrolase 16 family
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.14964

SUMMARY We report on the homo‐ and hetero‐transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488‐labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta‐galacturonide [α(1‐4)GalAp]5 acceptor – the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1‐4)GalAp]5 acceptor substrates at the −4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed‐localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero‐transglycosylation reaction with the xyloglucan/[α(1‐4)GalAp]5 substrate pair, while wild‐type TmXET6.3 lacked this activity. Expression data obtained by real‐time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co‐expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero‐transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1‐4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan‐pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re‐modelling, and affect the structural features of walls. Significance Statement The substrate specificity definition of xyloglucan xyloglucosyl transferases (XETs) is needed to understand the roles of XETs in cell wall formation, assembly and re‐modelling. Here, we defined the substrate specificity of barley XET3, XET4 and XET6 isoforms, and discovered that they catalyse hetero‐transglycosylation reactions with the xyloglucan/pectin fragment substrate pair. We visualised this activity in barley roots, defined expression profiles and the evolutionary origin of XETs, and discussed their significance in wall structure and function.