Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na+ homeostasis in planta and by this function modulates K+ nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na+ content in the shoot and lower content of the ion in the root, reducing the Na+ imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K+ to Na+ ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K+ to Na+ ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na+ homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K+-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K+ deficiency of sos3-1 or wildtype seedlings. Together, these results indicate that AtHKT1 controls Na+ homeostasis in planta and through this function regulates K+ nutrient status.

Cold, hyperosmolarity, and abscisic acid (ABA) signaling induce RD29A expression, which is an indicator of the plant stress adaptation response. Two nonallelic Arabidopsis thaliana(ecotype C24) T-DNA insertional mutations, cpl1 and cpl3, were identified based on hyperinduction of RD29A expression that was monitored by using the luciferase (LUC) reporter gene (RD29A::LUC) imaging system. Genetic linkage analysis and complementation data established that the recessive cpl1 and cpl3 mutations are caused by T-DNA insertions in AtCPL1 (Arabidopsis C-terminal domain phosphatase-like) and AtCPL3, respectively. Gel assays using recombinant AtCPL1 and AtCPL3 detected innate phosphatase activity like other members of the phylogenetically conserved family that dephosphorylate the C-terminal domain of RNA polymerase II (RNAP II). cpl1 mutation causes RD29A::LUC hyperexpression and transcript accumulation in response to cold, ABA, and NaCl treatments, whereas the cpl3 mutation mediates hyperresponsiveness only to ABA. Northern analysis confirmed that LUC transcript accumulation also occurs in response to these stimuli. cpl1 plants accumulate biomass more rapidly and exhibit delayed flowering relative to wild type whereas cpl3 plants grow more slowly and flower earlier than wild-type plants. Hence AtCPL1 and AtCPL3 are negative regulators of stress responsive gene transcription and modulators of growth and development. These results suggest that C-terminal domain phosphatase regulation of RNAP II phosphorylation status is a focal control point of complex processes like plant stress responses and development. AtCPL family members apparently have both unique and overlapping transcriptional regulatory functions that differentiate the signal output that determines the plant response.

To study low-temperature signaling in plants, we previously screened for cold stress response mutants using bioluminescent Arabidopsis plants that express the firefly luciferase reporter gene driven by the stress-responsive RD29A promoter. Here, we report on the characterization and cloning of one mutant, frostbite1 (fro1), which shows reduced luminescence induction by cold. fro1 plants display reduced cold induction of stress-responsive genes such as RD29A, KIN1, COR15A, and COR47. fro1 leaves have a reduced capacity for cold acclimation, appear water-soaked, leak electrolytes, and accumulate reactive oxygen species constitutively. FRO1 was isolated through positional cloning and found to encode a protein with high similarity to the 18-kD Fe-S subunit of complex I (NADH dehydrogenase, EC 1.6.5.3) in the mitochondrial electron transfer chain. Confocal imaging shows that the FRO1:green fluorescent protein fusion protein is localized in mitochondria. These results suggest that cold induction of nuclear gene expression is modulated by mitochondrial function.

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 Ca2+ (0.15 mM) and <200 μM 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 Ca2+ (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 [Ca2+]ext.