Functional Analysis of Conserved Motifs in the Mechanosensitive Channel Homolog MscS-Like2 from Arabidopsis thaliana

Abstract

The Mechanosensitive channel of Small conductance (MscS) of Escherichia coli has become an excellent model system for the structural, biophysical, and functional study of mechanosensitive ion channels. MscS, a complex channel with multiple states, contributes to protection against lysis upon osmotic downshock. MscS homologs are widely and abundantly dispersed among the bacterial and plant lineages, but are not found in animals. Investigation into the eukaryotic branch of the MscS family is in the beginning stages, and it remains unclear how much MscS homologs from eukaryotes resemble E. coli MscS with respect to structure, function, and regulation. Here we test the effect of mutating three conserved motifs on the function of MscS-Like (MSL)2, a MscS homolog localized to the plastids of Arabidopsis thaliana. We show that 1) a motif at the top of the cytoplasmic domain, referred to here as the PN(X)9N motif, is essential for MSL2 function and for its proper intraplastidic localization; 2) substituting polar residues for two large hydrophobic residues located in the predicted pore-lining transmembrane helix of MSL2 produces a likely gain-of-function allele, as previously shown for MscS; and 3) mis-expression of this allele causes severe defects in leaf growth, loss of chloroplast integrity, and abnormal starch accumulation. Thus, two of the three conserved motifs we analyzed are critical for MSL2 function, consistent with the conservation of structure and function between MscS family members in bacteria and plants. These results underscore the importance of plastidic mechanosensitive channels in the maintenance of normal plastid and leaf morphology.

Plastid osmotic stress influences cell differentiation at the plant shoot apex

“…1E, second row from top). As previously documented (Haswell and Meyerowitz, 2006;Jensen and Haswell, 2012;Wilson et al, 2014), msl2 msl3 leaves were small and malformed. To facilitate comparison, the same leaf from each genotype is marked with an asterisk in the time course in Fig.…”

Plastid Osmotic Stress Activates Cellular Stress Responses in Arabidopsis

“…Chloroplasts in the msl2 msl3 mutant are greatly enlarged and exhibit defective division site selection (Haswell and Meyerowitz, 2006;Wilson et al, 2011) but do not appear round, and we speculate that they have a second pathway for osmoregulation in addition to mechanosensitive ion channels. Loss of MSL2 and/or MSL3 function also results in whole-plant phenotypes, including leaves with variegation, notching, and rumpled surfaces and dwarfing of the adult plant (Haswell and Meyerowitz, 2006;Jensen and Haswell, 2012). These data suggest that organellar osmotic stress responses are integrated into a larger cellular context and that msl2 msl3 mutants might provide a powerful tool to study the cellular response to plastid hypoosmotic stress.…”

“…We have previously documented the presence of enlarged and abnormally round leaf epidermal plastids in the msl2-1 msl3-1 mutant background (Haswell and Meyerowitz, 2006;Veley et al, 2012) and the msl2-3 mutant background (Jensen and Haswell, 2012). Although msl2-1 and msl3-1 are likely to be intermediate loss-of-function alleles, msl2-3 is a null allele (Wilson et al, 2011).…”

Arabidopsis MSL10 Has a Regulated Cell Death Signaling Activity That Is Separable from Its Mechanosensitive Ion Channel Activity

“…The MSL10 cDNA was cloned previously into pENTR/D-TOPO (Haswell et al, 2008). This clone was used as a template for making all MSL10 variants by the introduction of point mutations via site-directed mutagenesis as described (Jensen and Haswell, 2012). These pENTR constructs were then used in recombination reactions with pK7FWG2 (Karimi et al, 2002) to create C-terminal GFP fusion overexpression constructs (P35S:MSL10-GFP).…”