Dennison K L, Robertson W R, Lewis B D, Hirsch R E, Sussman M R, Spalding E P
Biotechnology Center, University of Wisconsin, 425 Henry Mall, Madison, WI 53706, USA.
Plant Physiol. 2001 Nov;127(3):1012-9.
A reverse genetic strategy was used to isolate Arabidopsis plants containing "knockout" mutations in AKT1 and AKT2, two members of a K+ channel gene family. Comparative studies of growth and membrane properties in wild-type and mutant seedlings were performed to investigate the physiological functions of these two related channels. The growth rates of plants supplied with rate-limiting concentrations of K+ depended on the presence of AKT1 but not AKT2 channels. This result indicates that AKT1 but not AKT2 mediates growth-sustaining uptake of K+ into roots, consistent with the expression patterns of these two genes. K+ -induced membrane depolarizations were measured with microelectrodes to assess the contribution each channel makes to the K+ permeability of the plasma membrane in three different organs. In apical root cells, AKT1 but not AKT2 contributed to the K+ permeability of the plasma membrane. In cotyledons, AKT1 was also the principal contributor to the K+ permeability. However, in the mesophyll cells of leaves, AKT2 accounted for approximately 50% of the K+ permeability, whereas AKT1 unexpectedly accounted for the remainder. The approximately equal contributions of AKT1 and AKT2 in leaves detected by the in vivo functional assay employed here are not in agreement with previous RNA blots and promoter activity studies, which showed AKT2 expression to be much higher than AKT1 expression in leaves. This work demonstrates that comparative functional studies of specific mutants can quantify the relative contributions of particular members of a gene family, and that expression studies alone may not reliably map out distribution of gene functions.
采用反向遗传学策略分离拟南芥植株,这些植株在K⁺通道基因家族的两个成员AKT1和AKT2中含有“敲除”突变。对野生型和突变型幼苗的生长和膜特性进行了比较研究,以探究这两个相关通道的生理功能。供应有限浓度K⁺的植株的生长速率取决于AKT1通道的存在,而不依赖于AKT2通道。这一结果表明,是AKT1而非AKT2介导了根系中维持生长所需的K⁺吸收,这与这两个基因的表达模式一致。用微电极测量K⁺诱导的膜去极化,以评估每个通道对三种不同器官中质膜K⁺通透性的贡献。在根尖细胞中,对质膜K⁺通透性有贡献的是AKT1而非AKT2。在子叶中,AKT1也是K⁺通透性的主要贡献者。然而,在叶片的叶肉细胞中,AKT2约占K⁺通透性的50%,而AKT1意外地占其余部分。此处采用的体内功能测定法检测到AKT1和AKT2在叶片中的贡献大致相等,这与之前的RNA印迹和启动子活性研究不一致,后者表明叶片中AKT2的表达远高于AKT1的表达。这项工作表明,对特定突变体的比较功能研究可以量化基因家族特定成员的相对贡献,而且仅靠表达研究可能无法可靠地描绘出基因功能的分布情况。