Hamilton Eric S, Haswell Elizabeth S
Department of Biology, Box 1137, Washington University in St. Louis, One Brookings Drive, Saint Louis, MO 63130, USA.
Plant Cell Physiol. 2017 Jul 1;58(7):1222-1237. doi: 10.1093/pcp/pcw230.
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 an 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 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.
所有细胞都会对渗透挑战作出反应，包括在正常生长和发育过程中所面临的挑战。机械敏感（MS）离子通道提供了一种保守机制，可通过在膜张力增加时传导离子来调节渗透力。我们之前证明，MS离子通道类MscS 8（MSL8）是花粉在受精正常过程中应对多种渗透挑战所必需的，并且它可以抑制花粉萌发。然而，这些生理功能是否需要通过MSL8提供的机械门控通道进行离子通量仍不清楚。我们在MSL8预测的孔衬里结构域中引入了两个点突变，以不同方式破坏了正常通道功能。Ile711Ser突变提高了MSL8通道的张力阈值，同时电导率不变，而Phe720Leu突变严重破坏了MSL8通道。当以内源水平表达时，这两种突变均损害了MSL8在水合过程中保持花粉活力以及维持花粉管完整性的能力。当在msl8 - 4缺失背景中过表达时，MSL8I711S可以部分挽救功能丧失表型，而MSL8F720L则不能。当在野生型Ler背景中过表达时，MSL8I711S抑制花粉萌发，类似于野生型MSL8。相比之下，MSL8F720L未能抑制花粉萌发并增加了花粉破裂，从而模拟了msl8 - 4突变体的表型。因此，完整的MSL8通道是花粉在水合和萌发过程中正常功能所必需的。这些数据确立了MSL8作为第一个满足先前确定的作为机械转导器标准的植物MS通道。
