Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3-1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1 sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3-1 hkt1-1 and sos3-1 hkt1-2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1-1 and hkt1-2 mutations can suppress to an equivalent extent the Na ؉ sensitivity of sos3-1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3-1 hkt1-1 and sos3-1 hkt1-2 seedlings are able to maintain [K ؉ ]int in medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K ؉ ͞Na ؉ than the sos3-1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3-1 mutant that is caused by K ؉ deficiency on culture medium with low Ca 2؉ (0.15 mM) and <200 M K ؉ . Interestingly, the capacity of hkt1 mutations to suppress the Na ؉ hypersensitivity of the sos3-1 mutant is reduced substantially when seedlings are grown in medium with low Ca 2؉ (0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na ؉ entry and high affinity K ؉ uptake. The hkt1 mutations have revealed the existence of another Na ؉ influx system(s) whose activity is reduced by high [Ca 2؉ ]ext. H igh [NaCl] ext disturbs intracellular ion homeostasis of plants, which leads to membrane dysfunction, attenuation of metabolic activity, and secondary effects that cause growth inhibition and lead ultimately to cell death (1). Both glycophytes and halophytes use a similar strategy that involves regulation of net Na ϩ flux across the plasma membrane and vacuolar compartmentalization of the internalized cation to mediate intracellular Na ϩ homeostasis. This strategy requires the coordinated function of numerous ion transport determinants and effectively partitions the toxic ion away from critical cytosolic and organellar machinery. Under conditions of high [Na ϩ ] ext , the functioning of these determinants also facilitates the use of Na ϩ as an osmolyte to mediate osmotic adjustment that is necessary for cell expansion (1-3). Because vacuolar expansion is the primary mechanism of plant cell enlargement, this strategy is likely to be an essential adaptation to saline environments.Recently, putative plasma membrane and tonoplast localized Na ϩ ͞H ϩ transporters were identified in plants that are presumed to mediate energized transport of Na ϩ outward from the cytosol to the apoplast or into the vacuole (4-7). These transporters are apparently the molecular effectors of Na ϩ ͞H ϩ antiporter activities associated with plasma membrane and tonoplast vesicles that were described more than a decade ago (1,3,8,9). The plasma membrane Na ϩ ͞H ϩ

SummaryProgrammed cell death (PCD) is a fundamental cellular process conserved in metazoans, plants and yeast. Evidence is presented that salt induces PCD in yeast and plants because of an ionic, rather than osmotic, etiology. In yeast, NaCl inhibited growth and caused a time-dependent reduction in viability that was preceded by DNA fragmentation. NaCl also induced the cytological hallmarks of lysigenoustype PCD, including nuclear fragmentation, vacuolation and lysis. The human anti-apoptotic protein Bcl-2 increased salt tolerance of wild-type yeast strain and calcineurin-de®cient yeast mutant (cnb1D) that is defective for ion homeostasis, but had no effect on the NaCl or sorbitol sensitivity of the osmotic hypersensitive hog1D mutant ± results that further link PCD in the response to the ion disequilibrium under salt stress. Bcl-2 suppression of cnb1D salt sensitivity was ENA1 (P-type ATPase gene)-dependent, due in part to transcriptional activation. Salt-induced PCD (TUNEL staining and DNA laddering) in primary roots of both Arabidopsis thaliana wild type (Col-1 gl1) and sos1 (salt overly sensitive) mutant seedlings correlated positively with treatment lethality. Wild-type plants survived salt stress levels that were lethal to sos1 plants because secondary roots were produced from the shoot/root transition zone. PCD-mediated elimination of the primary root in response to salt shock appears to be an adaptive mechanism that facilitates the production of roots more able to cope with a saline environment. Both salt-sensitive mutants of yeast (cnb1D) and Arabidopsis (sos1) exhibit substantially more profound PCD symptoms, indicating that salt-induced PCD is mediated by ion disequilibrium.

Dynamic cytoplasmic streaming, organelle positioning, and nuclear migration use molecular tracks generated from actin filaments arrayed into higher-order structures like actin cables and bundles. How these arrays are formed and stabilized against cellular depolymerizing forces remains an open question. Villin and fimbrin are the best characterized actin-filament bundling or cross-linking proteins in plants and each is encoded by a multigene family of five members in Arabidopsis thaliana. The related villins and gelsolins are conserved proteins that are constructed from a core of six homologous gelsolin domains. Gelsolin is a calcium-regulated actin filament severing, nucleating and barbed end capping factor. Villin has a seventh domain at its C terminus, the villin headpiece, which can bind to an actin filament, conferring the ability to crosslink or bundle actin filaments. Many, but not all, villins retain the ability to sever, nucleate, and cap filaments. Here we have identified a putative calcium-insensitive villin isoform through comparison of sequence alignments between human gelsolin and plant villins with x-ray crystallography data for vertebrate gelsolin. VILLIN1 (VLN1) has the least well-conserved type 1 and type 2 calcium binding sites among the Arabidopsis VILLIN isoforms. Recombinant VLN1 binds to actin filaments with high affinity (K d ;1 mM) and generates bundled filament networks; both properties are independent of the free Ca 2þ concentration. Unlike human plasma gelsolin, VLN1 does not nucleate the assembly of filaments from monomer, does not block the polymerization of profilin-actin onto barbed ends, and does not stimulate depolymerization or sever preexisting filaments. In kinetic assays with ADF/cofilin, villin appears to bind first to growing filaments and protects filaments against ADF-mediated depolymerization. We propose that VLN1 is a major regulator of the formation and stability of actin filament bundles in plant cells and that it functions to maintain the cable network even in the presence of stimuli that result in depolymerization of other actin arrays.