Analysis of Plant-Specific ANTH Domain-Containing Protein in Marchantia polymorpha.

Membrane trafficking is a fundamental mechanism for protein and lipid transport in eukaryotic cells and exhibits marked diversity among eukaryotic lineages with distinctive body plans and lifestyles. Diversification of the membrane trafficking system is associated with the expansion and secondary loss of key machinery components, including RAB GTPases, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and adaptor proteins, during plant evolution. The number of AP180 N-terminal homology (ANTH) proteins, an adaptor family that regulates vesicle formation and cargo sorting during clathrin-mediated endocytosis, increases during plant evolution. In the genome of Arabidopsis thaliana, 18 genes for ANTH proteins have been identified, a higher number than that in yeast and animals, suggesting a distinctive diversification of ANTH proteins. Conversely, the liverwort Marchantia polymorpha possesses a simpler repertoire; only two genes encoding canonical ANTH proteins have been identified in its genome. Intriguingly, a non-canonical ANTH protein is encoded in the genome of M. polymorpha, which also harbors a putative kinase domain. Similar proteins have been detected in sporadic lineages of plants, suggesting their ancient origin and multiple secondary losses during evolution. We named this unique ANTH group phosphatidylinositol-binding clathrin assembly protein-K (PICALM-K) and characterized it in M. polymorpha using genetic, cell biology-based and artificial intelligence (AI)-based approaches. Our results indicate a flagella-related function of MpPICALM-K in spermatozoids, which is distinct from that of canonical ANTH proteins. Therefore, ANTH proteins have undergone significant functional diversification during evolution, and PICALM-K represents a plant-unique ANTH protein that is delivered by neofunctionalization through exon shuffling.

Wound-inducible WUSCHEL-RELATED HOMEOBOX 13 is required for callus growth and organ reconnection.

Highly efficient tissue repair is pivotal for surviving damage-associated stress. Plants generate callus upon injury to heal wound sites, yet regulatory mechanisms of tissue repair remain elusive. Here, we identified WUSCHEL-RELATED HOMEOBOX 13 (WOX13) as a key regulator of callus formation and organ adhesion in Arabidopsis (Arabidopsis thaliana). WOX13 belongs to an ancient subclade of the WOX family, and a previous study shows that WOX13 orthologs in the moss Physcomitrium patens (PpWOX13L) are involved in cellular reprogramming at wound sites. We found that the Arabidopsis wox13 mutant is totally defective in establishing organ reconnection upon grafting, suggesting that WOX13 is crucial for tissue repair in seed plants. WOX13 expression rapidly induced upon wounding, which was partly dependent on the activity of an AP2/ERF transcription factor, WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1). WOX13 in turn directly upregulated WIND2 and WIND3 to further promote cellular reprogramming and organ regeneration. We also found that WOX13 orchestrates the transcriptional induction of cell wall-modifying enzyme genes, such as GLYCOSYL HYDROLASE 9Bs, PECTATE LYASE LIKEs and EXPANSINs. Furthermore, the chemical composition of cell wall monosaccharides was markedly different in the wox13 mutant. These data together suggest that WOX13 modifies cell wall properties, which may facilitate efficient callus formation and organ reconnection. Furthermore, we found that PpWOX13L complements the Arabidopsis wox13 mutant, suggesting that the molecular function of WOX13 is partly conserved between mosses and seed plants. This study provides key insights into the conservation and functional diversification of the WOX gene family during land plant evolution.