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Plants maintain large repertoires of carbohydrate-active enzymes (CAZymes)-comprising between 3% and 10% of their genomes-to synthesize, modify, and degrade the polysaccharide components of the cell wall. We recently identified a unique group of plant endo-glucanases from Glycoside Hydrolase Family 16, viz. EG16 orthologs, which constitute a sister clade to the well-known XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products. Biochemical analysis of EG16 orthologs from poplar (Populus trichocarpa), grapevine (Vitis vinifera), and spreading earthmoss (Physcomitrium patens) has demonstrated that these endo-glucanases are distinctly active on cell wall matrix glycans, mixed-linkage β(1,3);β(1,4)-glucan and xyloglucan (XyG), and that enzyme structure and specificity is highly conserved across diverse plant lineages. However, the physiological role of EG16 orthologs in any species is presently unknown. To shed light on EG16 function in vivo, here we performed reverse genetics and protein localization analyses of the single EG16 ortholog in the model moss P. patens, where this gene is highly expressed in young, expanding tissues, particularly in protonema. Surprisingly, deletion of the PpEG16 gene by homologous recombination led to an increase in growth, as well as accelerated senescence. Notably, the PpEG16 protein was shown to co-localize with XyG in the cell wall of protonema tissue, specifically at cell tips, despite lacking a secretion signal peptide. Although the precise biological role of EG16 orthologs remains elusive, our results implicate these highly conserved glycoside hydrolases in cell wall polysaccharide remodeling and recycling. We anticipate that these foundational results will inform future studies on EG16 function across plant lineages.

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 β-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 EG16 enzymes.

Carbohydrate-active enzymes (CAZymes) are central to the biosynthesis and modification of the plant cell wall. An ancient clade of bifunctional plant endo-glucanases (EG16 members) was recently revealed and proposed to represent a transitional group uniting plant xyloglucan endo-transglycosylase/hydrolase (XTH) gene products and bacterial mixed-linkage endo-glucanases in the phylogeny of glycoside hydrolase family 16 (GH16). To gain broader insights into the distribution and frequency of EG16 and other GH16 members in plants, the PHYTOZOME, PLAZA, NCBI and 1000 PLANTS databases were mined to build a comprehensive census among 1289 species, spanning the broad phylogenetic diversity of multiple algae through recent plant lineages. EG16, newly identified EG16-2 and XTH members appeared first in the green algae. Extant EG16 members represent the early adoption of the β-jellyroll protein scaffold from a bacterial or early-lineage eukaryotic GH16 gene, which is characterized by loop deletion and extension of the N terminus (in EG16-2 members) or C terminus (in XTH members). Maximum-likelihood phylogenetic analysis of EG16 and EG16-2 sequences are directly concordant with contemporary estimates of plant evolution. The lack of expansion of EG16 members into multi-gene families across green plants may point to a core metabolic role under tight control, in contrast to XTH genes that have undergone the extensive duplications typical of cell-wall CAZymes. The present census will underpin future studies to elucidate the physiological role of EG16 members across plant species, and serve as roadmap for delineating the closely related EG16 and XTH gene products in bioinformatic analyses of emerging genomes and transcriptomes.

The colonization of land by plants was a pivotal event in the history of the biosphere, and yet the underlying evolutionary features and innovations of the first land plant ancestors are not well understood. Here we present the genome sequence of the unicellular algaPenium margaritaceum, a member of the Zygnematophyceae, the sister lineage to land plants. TheP. margaritaceumgenome has a high proportion of repeat sequences, which are associated with massive segmental gene duplications, likely facilitating neofunctionalization. Compared with earlier diverging plant lineages,P. margaritaceumhas uniquely expanded repertoires of gene families, signaling networks and adaptive responses, supporting its phylogenetic placement and highlighting the evolutionary trajectory towards terrestrialization. These encompass a broad range of physiological processes and cellular structures, such as large families of extracellular polymer biosynthetic and modifying enzymes involved in cell wall assembly and remodeling. Transcriptome profiling of cells exposed to conditions that are common in terrestrial habitats, namely high light and desiccation, further elucidated key adaptations to the semi-aquatic ecosystems that are home to the Zygnematophyceae. Such habitats, in which a simpler body plan would be advantageous, likely provided the evolutionary crucible in which selective pressures shaped the transition to land. Earlier diverging charophyte lineages that are characterized by more complex land plant-like anatomies have either remained exclusively aquatic, or developed alternative life styles that allow periods of desiccation.

The phytochemical indole-3-carbinol is produced in Cruciferous plants upon tissue rapture and deters herbivores. We recently showed that indole-3-carbinol modulates auxin signaling in root tips. Here we present transcript profiling experiments which further reveal the influence of indole-3-carbinol on auxin signaling in root tips, and also show that I3C affects auxin transporters. Brief treatment with indole-3-carbinol led to a reduction in the amount of PIN1 and to mislocalization of PIN2.