A high‐confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution

Abstract

Summary Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large‐scale systematic localization, defining a high‐confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD‐localized. Callose‐degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium‐like proteins, which also diversified into PD‐localized and non‐PD‐localized members on distinct phylogenetic clades. Our high‐confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large‐scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.

Comparative phyloproteomics identifies conserved plasmodesmal proteins

“…While this might arise from our fractionation methods being suboptimal for their extraction, or from usage of non-quantitative MS methods, we found that if we reduced the stringency of protein identification in both our Arabidopsis leaf and moss plasmodesmal fractions, allowing an identification probability of >50% threshold for peptide and protein identification and a minimum of one sample, we identify an additional 12 orthogroups present in at least four of five proteomes, one of which represents callose synthases ( Supplementary Tables S2 , S3 , S7 ). This low stringency analysis also reveals orthogroups containing heavy metal-associated isoprenylated proteins (HIPPs), which have been localized to plasmodesmata in Arabidopsis ( Guo et al , 2021 ) and N. benthamiana ( Cowan et al , 2018 ), and xyloglucan endotransglucosylase/hydrolase proteins, which have been confirmed as plasmodesmal proteins in a concomitant proteomic study ( Gombos et al , 2023 ). In essence, by requiring the identification of a protein in multiple independent proteomes, we are increasing the a priori likelihood of protein identification within a sample.…”

Plasmodesmata act as unconventional membrane contact sites regulating inter-cellular molecular exchange in plants

“…They consist of a C-terminal ER-anchor and three to four C2 lipid binding domains. In Arabidopsis, the MCTP family comprises 16 members, among which MCTP3, MCTP4, and MCTP6 were identified in plasmodesmata fractions in a systematic manner (Fernandez-Calvino et al, 2011; Kraner et al, 2017; Brault et al, 2019; Kirk et al, 2022; Miras et al, 2022a; Gombos et al, 2023; Johnston et al, 2023). Given that nearly all plant cells are interconnected by these communication bridges, it is anticipated that a primary component would be widely expressed and consistently associated with plasmodesmata, irrespective of tissue type or developmental stage.…”

CsEXL3 regulate mechanical harvest-related droopy leaves under the transcriptional activation of CsBES1.2 in tea plant

“…This different function might be related to tissue localization according to proteome in Arabidopsis and Physcomitrium patens . PsEXO1 was localized in plasmodesmata of P. patens and divided into Clade III of the phylogenetic tree and Clade III also contained AtEXO, AtEXL1, and AtEXL2 from Arabidopsis [ 23 ]. AtEXL3, AtEXL5, and AtEXL7 were divided into Clade II [ 23 ] and CsEXL3 was a homologous gene of AtEXL3 ( Fig.…”

“…PsEXO1 was localized in plasmodesmata of P. patens and divided into Clade III of the phylogenetic tree and Clade III also contained AtEXO, AtEXL1, and AtEXL2 from Arabidopsis [ 23 ]. AtEXL3, AtEXL5, and AtEXL7 were divided into Clade II [ 23 ] and CsEXL3 was a homologous gene of AtEXL3 ( Fig. S7 , see online supplementary material).…”