▪ Abstract Plant responses to salinity stress are reviewed with emphasis on molecular mechanisms of signal transduction and on the physiological consequences of altered gene expression that affect biochemical reactions downstream of stress sensing. We make extensive use of comparisons with model organisms, halophytic plants, and yeast, which provide a paradigm for many responses to salinity exhibited by stress-sensitive plants. Among biochemical responses, we emphasize osmolyte biosynthesis and function, water flux control, and membrane transport of ions for maintenance and re-establishment of homeostasis. The advances in understanding the effectiveness of stress responses, and distinctions between pathology and adaptive advantage, are increasingly based on transgenic plant and mutant analyses, in particular the analysis of Arabidopsis mutants defective in elements of stress signal transduction pathways. We summarize evidence for plant stress signaling systems, some of which have components analogous to those that regulate osmotic stress responses of yeast. There is evidence also of signaling cascades that are not known to exist in the unicellular eukaryote, some that presumably function in intercellular coordination or regulation of effector genes in a cell-/tissue-specific context required for tolerance of plants. A complex set of stress-responsive transcription factors is emerging. The imminent availability of genomic DNA sequences and global and cell-specific transcript expression data, combined with determinant identification based on gain- and loss-of-function molecular genetics, will provide the infrastructure for functional physiological dissection of salt tolerance determinants in an organismal context. Furthermore, protein interaction analysis and evaluation of allelism, additivity, and epistasis allow determination of ordered relationships between stress signaling components. Finally, genetic activation and suppression screens will lead inevitably to an understanding of the interrelationships of the multiple signaling systems that control stress-adaptive responses in plants.

SIZ1 is a SUMO E3 ligase that facilitates conjugation of SUMO to protein substrates. siz1-2 and siz1-3 T-DNA insertion alleles that caused freezing and chilling sensitivities were complemented genetically by expressing SIZ1, indicating that the SIZ1 is a controller of low temperature adaptation in plants. Cold-induced expression of CBF/DREB1, particularly of CBF3/DREB1A, and of the regulon genes was repressed by siz1. siz1 did not affect expression of ICE1, which encodes a MYC transcription factor that is a controller of CBF3/DREB1A. A K393R substitution in ICE1 [ICE1(K393R)] blocked SIZ1-mediated sumoylation in vitro and in protoplasts identifying the K393 residue as the principal site of SUMO conjugation. SIZ1-dependent sumoylation of ICE1 in protoplasts was moderately induced by cold. Sumoylation of recombinant ICE1 reduced polyubiquitination of the protein in vitro. ICE1(K393R) expression in wild-type plants repressed cold-induced CBF3/DREB1A expression and increased freezing sensitivity. Furthermore, expression of ICE1(K393R) induced transcript accumulation of MYB15, which encodes a MYB transcription factor that is a negative regulator of CBF/DREB1. SIZ1-dependent sumoylation of ICE1 may activate and/or stabilize the protein, facilitating expression of CBF3/DREB1A and repression of MYB15, leading to low temperature tolerance.

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 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 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 .

SUMO (small ubiquitin-related modifier) conjugation (i.e., sumoylation) to protein substrates is a reversible posttranslational modification that regulates signaling by modulating transcription factor activity. This paper presents evidence that the SUMO E3 ligase SIZ1 negatively regulates abscisic acid (ABA) signaling, which is dependent on the bZIP transcripton factor ABI5. Loss-of-function T-DNA insertion siz1–2 and siz1–3 mutations caused ABA hypersensitivity for seed germination arrest and seedling primary root growth inhibition. Furthermore, expression of genes that are ABA-responsive through ABI5-dependent signaling (e.g., RD29A , Rd29B , AtEm6 , RAB18 , ADH1 ) was hyperinduced by the hormone in siz1 seedlings. abi5–4 suppressed ABA hypersensitivity caused by siz1 ( siz1–2 abi5–4 ), demonstrating an epistatic genetic interaction between SIZ1 and ABI5 . A K391R substitution in ABI5 [ABI5(K391R)] blocked SIZ1-mediated sumoylation of the transcription factor in vitro and in Arabidopsis protoplasts, indicating that ABI5 is sumoylated through SIZ1 and that K391 is the principal site for SUMO conjugation. In abi5–4 plants, ABI5(K391R ) expression caused greater ABA hypersensitivity (gene expression, seed germination arrest and primary root growth inhibition) compared with ABI5 expression. Together, these results establish that SIZ1-dependent sumoylation of ABI5 attenuates ABA signaling. The double mutant siz1–2 afp-1 exhibited even greater ABA sensitivity than the single mutant siz1 , suggesting that SIZ1 represses ABI5 signaling function independent of AFP1.

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 wild-type seedlings. Together, these results indicate that AtHKT1 controls Na⁺ homeostasis in planta and through this function regulates K⁺ nutrient status.

Summary: Programmed 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 lysigenous‐type PCD, including nuclear fragmentation, vacuolation and lysis. The human anti‐apoptotic protein Bcl‐2 increased salt tolerance of wild‐type yeast strain and calcineurin‐deficient yeast mutant (cnb1Δ) that is defective for ion homeostasis, but had no effect on the NaCl or sorbitol sensitivity of the osmotic hypersensitive hog1Δ mutant – results that further link PCD in the response to the ion disequilibrium under salt stress. Bcl‐2 suppression of cnb1Δ 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 (cnb1Δ) and Arabidopsis (sos1) exhibit substantially more profound PCD symptoms, indicating that salt‐induced PCD is mediated by ion disequilibrium.