The Tension-sensitive Ion Transport Activity of MSL8 is Critical for its Function in Pollen Hydration and Germination

Abstract

All cells respond to osmotic challenges, including those imposed during normal growth and development. Mechanosensitive (MS) ion channels provide a conserved mechanism for regulating osmotic forces by conducting ions in response to increased membrane tension. We previously demonstrated that the MS ion channel MscS-Like 8 (MSL8) is required for pollen to survive multiple osmotic challenges that occur during the normal process of fertilization, and that it can inhibit pollen germination. However, it remained unclear whether these physiological functions required ion flux through a mechanically gated channel provided by MSL8. We introduced two point mutations into the predicted pore-lining domain of MSL8 that disrupted normal channel function in different ways. The Ile711Ser mutation increased the tension threshold of the MSL8 channel while leaving conductance unchanged, and the Phe720Leu mutation severely disrupted the MSL8 channel. Both of these mutations impaired the ability of MSL8 to preserve pollen viability during hydration and to maintain the integrity of the pollen tube when expressed at endogenous levels. When overexpressed in a msl8-4 null background, MSL8I711S could partially rescue loss-of-function phenotypes, while MSL8F720L could not. When overexpressed in the wild type Ler background, MSL8I711S suppressed pollen germination, similar to wild type MSL8. In contrast, MSL8F720L failed to suppress pollen germination and increased pollen bursting, thereby phenocopying the msl8-4 mutant. Thus, an intact MSL8 channel is required to for normal pollen function during hydration and germination. These data establish MSL8 as the first plant MS channel to fulfill previously established criteria for assignment as a mechanotransducer.

Nonpolar residues in the presumptive pore‐lining helix of mechanosensitive channel MSL10 influence channel behavior and establish a nonconducting function

“…MSL10 has six Phe residues scattered through the pore‐lining domain. MSL8 has six Phe residues in its TM6; five of these are conserved with MSL10 (indicated in yellow, Figure c), and F720 is essential for channel function (Hamilton & Haswell, ). To determine whether Phe residues in TM6 are also critical for MSL10 channel function, we used site‐directed mutagenesis to change F544, F553, and F563 to smaller nonpolar residues, and to introduce a series of increasingly disruptive changes at F553.…”

“…MSL1 is localized to the mitochondrial inner membrane (Lee et al., ), while MSL8 and MSL10 are primarily localized to the plasma membrane (Hamilton et al., ; Haswell et al., ). The unitary conductances of MSL8 and MSL10 expressed in Xenopus oocytes are approximately 60 pS and 105 pS, respectively (compare to 340 pS for Ec MscS expressed in oocytes) (Hamilton & Haswell, ; Hamilton et al., ; Maksaev & Haswell, , ). MSL8 and MSL10 have a slightly higher preference for anions (P Cl :P Na = 5.6–6.3) than Ec MscS.…”

“…MSL8 is primarily localized to the plasma membrane of pollen grains, where it is required for full survival of rehydration, germination, and tube growth. Two mutations in the presumptive pore‐lining helix of MSL8, I711S and F720L, alter channel behavior and fail to complement these mutant phenotypes (Hamilton & Haswell, ). These observations link ion flux through the MSL8 channel to protection from osmotic stresses during pollen development.…”

Calcium transport across plant membranes: mechanisms and functions

“…The fifth transmembrane domain plays the role of a pore region, while the region encoding a voltage sensor is not found in MSLs. Group 3 MSLs (MSL8, MSL9 and MSL10) are localized to the PM and have six transmembrane domains, assembled as tetramers (or multimers), with the sixth transmembrane domain playing the role of the pore region (Hamilton & Haswell, ). Compared with other Ca 2+ ‐permeable channels, MSLs demonstrate higher unitary conductances, from 45 to 140 pS (Haswell et al ., ) and potentially higher preference to anions (Haswell et al ., ; Maksaev & Haswell, ).…”

“…Functions of MSLs include regulation of programmed cell death (MSL10; Veley et al ., ), water and ion balance in pollen tubes (MSL8; Hamilton & Haswell, ), regulation of electrical potential difference across the inner membrane of mitochondria (MSL1; Lee et al ., ), mechanical sensing in roots (MSL9 and MSL10; Peyronnet et al ., ) and chloroplast volume under normal and stress conditions (MSL2 and MSL3; Veley et al ., ), along with other possible roles (Hamilton et al ., ) (Table ).…”