Investigating the molecular role of bryophyte-specific protein SHORT-LEAF (SHLF) in gametophore development of moss (Physcomitrium patens)

Abstract

Plant development is mediated by the intricate interplay of genes and phytohormones. Though there is a common repertoire of genes amongst all plant lineages, many of them lack functional conservation across different clades. Lineage-specific genes (LSGs) represent the genetic novelty of each plant group, contributing to unique mechanisms necessary to thrive in their specific-ecological niche. Previously, we reported a unique bryophyte-specific gene family - SHORT-LEAF, which regulates gametophore development and auxin distribution pattern in moss. The disruption of this gene by Tnt1 insertion resulted in the short-leaf (shlf) mutant, defective in gametophore development and auxin response. SHLF encodes an N-terminal signal peptide, longest reported four TDRs (Tandem Direct Repeats) and a C-terminal tail, but has no known conserved protein domain. The unique nature of SHLF makes it intriguing to study its nature, molecular role and processing. As SHLF traffics to ER and undergoes cleavage, we carried out mass spectrometric analysis of wild type secretome to validate the secretory role of signal peptide and identified several SHLF peptides. Overexpression of a construct lacking signal peptide failed to recover the shlf phenotypes and SHLF peptides were absent in this secretome, indicating that secretion is necessary for SHLF function. Supplementation of both WT secretome to shlf exhibited complete recovery, proving that secretory SHLF is functional. We report a minimal functional protein (miniSHLF) comprising of the N-TDR1-2. Here, we also showed that 2nd TDR in essential for SHLF function and undergoes cleavage to form secretory peptides. Supplementation of 2nd TDR specific synthetic peptide, SHLFpep3 rescued all the shlf phenotypes. RNA-seq analysis and metabolomic profiling of shlf showed the up-regulation of several stress responsive genes and metabolites, which are recovered upon SHLFpep3 supplementation. Co-immunoprecipitation experiments using SHLF specific antibody resulted in several proteins involved in mediating plant development and stress response further implicating the dual role of SHLF in moss. In summary, we have demonstrated that SHLF behaves as a modular protein and that the conserved SHLF TDRs forms functional secretory peptides. SHLFpep3 derived from conserved TDRs affects auxin distribution pattern, gametophore development and stress response of moss. Future studies using SHLF may shed light on the evolutionary significance of lineage specific protein-hormone cross-talk in land plants.