Conservation of endo-glucanase 16 (EG16) activity across highly divergent plant lineages

Abstract

Plant cell walls are highly dynamic structures that are composed predominately of polysaccharides. As such, endogenous carbohydrate active-enzymes (CAZymes) are central to the synthesis and subsequent modification of plant cells during morphogenesis. The endo-glucanase 16 (EG16) members constitute a distinct group of plant CAZymes, angiosperm orthologs of which were recently shown to have dual beta-glucan/xyloglucan hydrolase activity. Molecular phylogeny indicates that EG16 members comprise a sister clade with a deep evolutionary relationship to the widely studied apoplastic xyloglucan endo-transglycosylases/hydrolases (XTH). A cross-genome survey indicated that EG16 members occur as a single ortholog across species and are widespread in early-diverging plants, including the non-vascular bryophytes, for which functional data were previously lacking. Remarkably, enzymological characterization of an EG16 ortholog from the model moss Physcomitrella patens (PpEG16) revealed that EG16 activity and sequence/structure are highly conserved across 500 million years of plant evolution, vis-à-vis orthologs from grapevine and poplar. Ex vivo biomechanical assays demonstrated that the application of EG16 gene products caused abrupt breakage of etiolated hypocotyls rather than slow extension, thereby indicating a mode-of-action distinct from endogenous expansins and microbial endo-glucanases. The biochemical data presented here will inform future genomic, genetic, and physiological studies of these enzymes.

Physcomitrium (Physcomitrella) patens endo‐glucanase 16 is involved in the cell wall development of young tissue

“…Pp EG16 also exhibits significant activity on XyG (Behar et al, 2021), which has been reported in mosses (Roberts et al, 2012). Immunofluorescence of XyG using the LM15 antibody, which targets XXXG‐type non‐fucosylated XyGs (Marcus et al, 2008; Vincken et al, 1997), revealed wall‐specific labeling of wild‐type and ΔPpEG16‐1 protonema.…”

“…Despite extensive biochemical and structural characterization that has defined polysaccharide specificity in divergent examples (Behar et al, 2021; Eklöf et al, 2013; McGregor et al, 2017), the biological function of EG16 orthologs is presently unknown. Since EG16 genes are strongly represented in non‐angiosperm genomes (Behar et al, 2018), we selected the moss P. patens as a well‐established model organism (Cove, 2005; Quatrano et al, 2007; Rensing et al, 2020) to explore the role of an exemplar EG16 in vivo.…”

“…As such, the Pp EG16 sequence lacks the characteristic C‐terminal extension of the XTHs (Baumann et al, 2007; Eklöf et al, 2013; Johansson et al, 2004; McGregor et al, 2017) and contains distinguishing sequence motifs (Behar et al, 2018). Like other EG16 orthologs, Pp EG16 has predominant hydrolytic activity on MLG and secondary activity on XyG and soluble cellulosic substrates (Behar et al, 2021). This specificity for MLG is particularly enigmatic because although P. patens cell walls contain the matrix polysaccharide XyG, MLG is absent (Popper et al, 2011).…”

“…P. patens encodes a single EG16 ortholog, PpEG16 , which was initially identified as an XTH ( Pp XTH32) by sequence similarity in an early genomic survey (Yokoyama et al, 2010). Subsequently, refined phylogenetic and biochemical analysis revealed that Pp EG16 is a bona fide EG16 ortholog (Behar et al, 2018; 2021). As such, the Pp EG16 sequence lacks the characteristic C‐terminal extension of the XTHs (Baumann et al, 2007; Eklöf et al, 2013; Johansson et al, 2004; McGregor et al, 2017) and contains distinguishing sequence motifs (Behar et al, 2018).…”

Building an extensible cell wall

“…In my view no plant enzyme has been documented to possess cell wall creep activity, even though many enzymes have at times been asserted to have such activity, for example, PME, pectinase, pectate lyase, xyloglucanase, cellulase, and others. Some bifunctional microbial endoglucanases (with xyloglucanase and cellulase activity) can induce plant cell wall creep ( Park and Cosgrove, 2012b ), but no plant equivalent has been discovered ( Behar et al, 2021 ). The results of Zhang et al (2019) showed that various wall lytic enzymes may soften cell walls (reduce their resistance to applied forces) yet not loosen the walls (not induce cell wall creep).…”