Sodium (Na + ) is toxic to most plants, but the molecular mechanisms of plant Na + uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na + transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na + levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na + levels, and athkt1 and athkt1/ sos3 shoots are Na + -hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na + distribution and counteracts salt stress in leaves by reducing leaf Na + accumulation. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The plasma membrane proton pump (H ϩ -ATPase) found in plants and fungi is a P-type ATPase with a polypeptide sequence, structure, and in vivo function similar to the mammalian sodium pump (Na ϩ , K ϩ -ATPase). Despite its hypothetical importance for generating and maintaining the proton motive force that energizes the carriers and channels that underlie plant nutrition, genetic evidence for such a central function has not yet been reported. Using a reverse genetic approach for investigating each of the 11 isoforms in the Arabidopsis H ϩ -ATPase (AHA) gene family, we found that one member, AHA3, is essential for pollen formation. A causative role for AHA3 in male gametogenesis was proven by complementation with a normal transgenic gene and rescue of the mutant phenotype back to wild type. We also investigated the requirement for phosphorylation of the penultimate threonine, which is found in most members of the AHA family and is thought to be involved in regulating catalytic activity. We demonstrated that a T948D mutant form of the AHA3 gene rescues the mutant phenotype in knockout AHA3 plants, but T948A does not, providing the first in planta evidence in support of the model in which phosphorylation of this amino acid is essential. 1995). Its role in the phloem is presumed to be the conwhich is used directly by most secondary transporters trol of sugar loading for long-distance sucrose transport, to mediate the movement of solutes into and out of the a process that is critical for plant nutrition. ization studies were useful in determining the expresIn Arabidopsis, the electrochemical potential has been sion pattern of AHA3, we wanted to more closely and recorded at very negative levels, such as Ϫ230 mV, while directly examine the in planta role of this gene. To this the corresponding protein in animal cells, the Na ϩ ,K ϩ -purpose, we took a reverse genetic approach and characATPase, typically generates membrane potentials of only terized the effect of the absence of functional AHA3 ‫ف‬Ϫ100 mV (Hirsch et al. 1998). This potential, together on the plant. Through transmission studies, microscopic with a chemical gradient of protons, is thought to be analysis, and functional complementation, we have identiessential for diverse cellular processes, including nutrified an essential role for AHA3 in pollen development. ent transport, cellular expansion, and osmoregulation.Knockout plants are useful for structure/function exThus, the proton ATPase has been hypothesized to play periments, which are designed to test the roles of spea crucial role in many important physiological processes. cific domains. Results of heterologous experiments with The family of genes encoding plasma membrane yeast suggested that T948, at the extreme C terminus of H ϩ -ATPases in Arabidopsis has 11 members (Palmgren AHA3, is essential for activation of the pump. To test the 2001). Expression data have implicated differing roles hypothesis that this residue is important for functions of for family members in numerous tissues, includi...

A reverse genetic strategy was used to isolate Arabidopsis plants containing "knockout" mutations in AKT1 and AKT2, two members of a K ϩ 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 ϩ depended on the presence of AKT1 but not AKT2 channels. This result indicates that AKT1 but not AKT2 mediates growth-sustaining uptake of K ϩ into roots, consistent with the expression patterns of these two genes. K ϩ -induced membrane depolarizations were measured with microelectrodes to assess the contribution each channel makes to the K ϩ permeability of the plasma membrane in three different organs. In apical root cells, AKT1 but not AKT2 contributed to the K ϩ permeability of the plasma membrane. In cotyledons, AKT1 was also the principal contributor to the K ϩ permeability. However, in the mesophyll cells of leaves, AKT2 accounted for approximately 50% of the K ϩ 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.The most abundant inorganic solute in plant cells is K ϩ . The transport in and out of cells of this essential element is a highly regulated process mediated by specific transporters within the plasma membrane (Maathuis et al., 1997; Chrispeels et al., 1999). Present at concentrations on the order of 100 mm, K ϩ serves as an osmoticum important to turgor pressure, and may act as an essential cofactor for certain enzymes. Its abundance contributes to the electrolyte character of cytoplasm and affects electrostatic interactions between charged entities such as proteins and other biopolymers. The transport of K ϩ helps set the electric potential difference across the plasma membrane, which powers the transport of other substances. Because K ϩ serves such fundamental functions throughout the plant, understanding the molecular mechanisms of its uptake and redistribution is an important goal. Progress in this regard may also spawn novel strategies for improving plant mineral nutrition and fertilizer application in the field.The first isolation of Arabidopsis genes encoding plasma membrane K ϩ channels by complementation of yeast K ϩ uptake mutants marked a major step toward this goal (Anderson et al., 1992;Sentenac et al., 1992). The transport properties displayed by AKT1 and KAT1 channels expressed in heterologous systems (Schachtman et al., 1992; Bertl et al., 1995 ...