Huazhong Shi

The Putative Plasma Membrane Na+/H+ Antiporter SOS1 Controls Long-Distance Na+ Transport in Plants

The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na+/H+ antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na+ transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na+ transporters, SOS1 was able to reduce Na+ accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1–green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter–β-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na+ than did the wild-type shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na+ than did the wild type. There also was greater Na+ content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na+ transport from root to shoot. We present a model in which SOS1 functions in retrieving Na+ from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na+ into the xylem.

Overexpression of a plasma membrane Na + /H + antiporter gene improves salt tolerance in Arabidopsis thaliana

High concentrations of Na+ in saline soils inhibit plant growth and reduce agricultural productivity. We report here that CaMV 35S promoter driven overexpression of the Arabidopsis thaliana SOS1 gene, which encodes a plasma membrane Na+/H+ antiporter, improves plant salt tolerance in A. thaliana. Transgenic plants showed substantial upregulation of SOS1 transcript levels upon NaCl treatment, suggesting post-transcriptional control of SOS1 transcript accumulation. In response to NaCl treatment, transgenic plants overexpressing SOS1 accumulated less Na+ in the xylem transpirational stream and in the shoot. Undifferentiated callus cultures regenerated from the transgenic plants were also more tolerant of salt stress, which was correlated with reduced Na+ content in the transgenic cells. These results show that improved salt tolerance could be achieved by limiting Na+ accumulation in plant cells.

Salt Cress. A Halophyte and Cryophyte Arabidopsis Relative Model System and Its Applicability to Molecular Genetic Analyses of Growth and Development of Extremophiles

Salt cress (Thellungiella halophila) is a small winter annual crucifer with a short life cycle. It has a small genome (about 2 × Arabidopsis) with high sequence identity (average 92%) with Arabidopsis, and can be genetically transformed by the simple floral dip procedure. It is capable of copious seed production. Salt cress is an extremophile native to harsh environments and can reproduce after exposure to extreme salinity (500 mm NaCl) or cold to −15°C. It is a typical halophyte that accumulates NaCl at controlled rates and also dramatic levels of Pro (>150 mm) during exposure to high salinity. Stomata of salt cress are distributed on the leaf surface at higher density, but are less open than the stomata of Arabidopsis and respond to salt stress by closing more tightly. Leaves of salt cress are more succulent-like, have a second layer of palisade mesophyll cells, and are frequently shed during extreme salt stress. Roots of salt cress develop both an extra endodermis and cortex cell layer compared to Arabidopsis. Salt cress, although salt and cold tolerant, is not exceptionally tolerant of soil desiccation. We have isolated several ethyl methanesulfonate mutants of salt cress that have reduced salinity tolerance, which provide evidence that salt tolerance in this halophyte can be significantly affected by individual genetic loci. Analysis of salt cress expressed sequence tags provides evidence for the presence of paralogs, missing in the Arabidopsis genome, and for genes with abiotic stress-relevant functions. Hybridizations of salt cress RNA targets to an Arabidopsis whole-genome oligonucleotide array indicate that commonly stress-associated transcripts are expressed at a noticeably higher level in unstressed salt cress plants and are induced rapidly under stress. Efficient transformation of salt cress allows for simple gene exchange between Arabidopsis and salt cress. In addition, the generation of T-DNA-tagged mutant collections of salt cress, already in progress, will open the door to a new era of forward and reverse genetic studies of extremophile plant biology.

Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na+ homeostasis

The Arabidopsis thaliana SOS1 protein is a putative Na+/H+ antiporter that functions in Na+ extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca2+ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na+ tolerance of yeast mutants lacking endogenous Na+ transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1-dependent Na+ tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2/SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2/SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K+ uptake at low external concentrations, neither the unmodified nor the SOS2/SOS3-activated SOS1 protein showed K+ transport capacity in vivo, suggesting that the role of SOS1 on K+ uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca2+ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na+ transporter SOS1.

The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf.

Soil Bacteria Confer Plant Salt Tolerance by Tissue-Specific Regulation of the Sodium Transporter HKT1

Elevated sodium (Na(+)) decreases plant growth and, thereby, agricultural productivity. The ion transporter high-affinity K(+) transporter (HKT)1 controls Na(+) import in roots, yet dysfunction or overexpression of HKT1 fails to increase salt tolerance, raising questions as to HKT1s role in regulating Na(+) homeostasis. Here, we report that tissue-specific regulation of HKT1 by the soil bacterium Bacillus subtilis GB03 confers salt tolerance in Arabidopsis thaliana. Under salt stress (100 mM NaCl), GB03 concurrently down- and upregulates HKT1 expression in roots and shoots, respectively, resulting in lower Na(+) accumulation throughout the plant compared with controls. Consistent with HKT1 participation in GB03-induced salt tolerance, GB03 fails to rescue salt-stressed athkt1 mutants from stunted foliar growth and elevated total Na(+) whereas salt-stressed Na(+) export mutants sos3 show GB03-induced salt tolerance with enhanced shoot and root growth as well as reduced total Na(+). These results demonstrate that tissue-specific regulation of HKT1 is critical for managing Na(+) homeostasis in salt-stressed plants, as well as underscore the breadth and sophistication of plant-microbe interactions.

Involvement of Arabidopsis HOS15 in histone deacetylation and cold tolerance

Histone modification in chromatin is one of the key control points in gene regulation in eukaryotic cells. Protein complexes composed of histone acetyltransferase or deacetylase, WD40 repeat protein, and many other components have been implicated in this process. Here, we report the identification and functional characterization of HOS15, a WD40-repeat protein crucial for repression of genes associated with abiotic stress tolerance through histone deacetylation in Arabidopsis. HOS15 shares high sequence similarity with human transducin-beta like protein (TBL), a component of a repressor protein complex involved in histone deacetylation. Mutation of the HOS15 gene renders mutant plants hypersensitive to freezing temperatures. HOS15 is localized in the nucleus and specifically interacts with histone H4. The level of acetylated histone H4 is higher in the hos15 mutant than in WT plants. Moreover, the stress inducible RD29A promoter is hyperinduced and associated with a substantially higher level of acetylated histone H4 in the hos15 mutant under cold stress conditions. Our results suggest a critical role for gene activation/repression by histone acetylation/deacetylation in plant acclimation and tolerance to cold stress.

Regulation of expression of the vacuolar Na + /H + antiporter gene AtNHX1 by salt stress and abscisic acid

The Arabidopsis thalianaAtNHX1 gene encodes a vacuolar Na+/H+ antiporter that is important in salt tolerance. We report here the tissue distribution and regulation of AtNHX1 expression by salt stress and abscisic acid (ABA). The steady-state level of AtNHX1 transcript was up-regulated by treatment with NaCl, KCl or ABA. AtNHX1 promoter-GUS analysis in transgenic Arabidopsis showed that AtNHX1 was expressed in all tissues except the root tip. Strong GUS expression was detected in guard cells, suggesting that AtNHX1 may play a role in pH regulation and/or K+ homeostasis in the specialized cells. AtNHX1 promoter activity was substantially up-regulated by NaCl, KCl or ABA, demonstrating that salt and ABA regulation of AtNHX1 expression occurs at the transcriptional level. Strong induction of GUS activity in root hair cells was observed, which suggests a role of AtNHX1 in storing Na+ in the enlarged vacuoles in root hair cells. The up-regulation of AtNHX1 transcript levels by NaCl was reduced in abi1-1, aba2-1 and aba3-1, but not in abi2-1, sos1, sos2 or sos3 mutants. ABA-induced AtNHX1 expression was also decreased in abi1-1 but not in abi2-1. These results suggest that salt stress up-regulates AtNHX1 expression transcriptionally and the up-regulation is partially dependent on ABA biosynthesis and ABA signaling through ABI1.

Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity

We conducted an analysis of the topology of AtNHX1, an Arabidopsis thaliana vacuolar Na+/H+ antiporter. Several hydrophilic regions of the antiporter were tagged with a hemagglutinin epitope, and protease protection assays were conducted to determine the membrane topology of the antiporter by using yeast as a heterologous expression system. The overall structure of AtNHX1 is distinct from the human Na+/H+ antiporter NHE1 or any known Na+/H+ antiporter. It is comprised of nine transmembrane domains and a hydrophilic C-terminal domain. Three hydrophobic regions do not appear to span the tonoplast membrane, yet appear to be membrane associated. Our results also indicate that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region resides in the vacuolar lumen. Deletion of the hydrophilic C terminus resulted in a dramatic increase in the relative rate of Na+/H+ transport. The ratio of Na+/K+ transport was twice that of the unmodified AtNHX1. This altered ratio resulted from a relatively small decrease in K+/H+ transport with a large increase in Na+/H+ transport. The vacuolar localization of the C terminus of the AtNHX1, taken together with the regulation of the antiporter selectivity by its C terminus, demonstrates the existence of luminal vacuolar regulatory mechanisms of the antiporter activity.

An Enhancer Mutant of Arabidopsis salt overly sensitive 3 Mediates both Ion Homeostasis and the Oxidative Stress Response

ABSTRACT The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3.