Ray A. Bressan

Ion Homeostasis in NaCl Stress Environments

Homeostasis can be defined as the tendency of a cell or an organism to maintain internal steady state, even in response to any environmental perturbation or stimulus tending to disturb normality, because of the coordinate responses of its constituent components. Typically, ions constantly flux in and out of cells in a controlled fashion with net flux adjusted to accommodate cellular requirements, thus creating an ionic homeostasis. When plant cells are exposed to salinity, mediated by high NaCl concentrations, kinetic steady states of ion transport for Na+ and Cland other ions, such as K+ and Ca2+, are disturbed (Binzel et al., 1988). High apoplastic levels of Na+ and Clalter aqueous and ionic thermodynamic equilibria, resulting in hyperosmotic stress, ionic imbalance, and toxicity. Thus, it is vital for the plant to re-establish cellular ion homeostasis for metabolic functioning and growth, that is, to adapt to the saline environment. Comparisons of what have been interpreted to be adaptive responses among various species lead to the conclusion that some salt-tolerant plants have evolved specialized complex mechanisms that allow adaptation to saline stress conditions. In fact, these unique mechanisms, such as salt glands, exist in few plant species and cannot be presumed to be ubiquitously functional for salt adaptation of all plants. However, intrinsically cellular-based mechanisms appear to be common to all genotypes and are a requisite for salt tolerance. Of paramount importance are those mechanisms that function to regulate ion homeostasis while mediating osmotic adjustment through the accumulation and intracellular compartmentation of ions that are predominant in the external environment. In this update we will focus principally on Na+ homeostasis in sodic environments; however, we also include discussions of H+, K+, Ca2+, and Clbecause of the interrelationship of these ions with Na+ homeostasis. Ion transport processes across the plasma membrane and the tonoplast will be emphasized because these are presumed to be most essential for the control of intracellular Na+ uptake and vacuolar compartmentation.

Plant Defense Genes Are Synergistically Induced by Ethylene and Methyl Jasmonate.

Combinations of ethylene and methyl jasmonate (E/MeJA) synergistically induced members of both groups 1 and 5 of the pathogenesis-related (PR) superfamily of defense genes. E/MeJA caused a synergistic induction of PR-1b and osmotin (PR-5) mRNA accumulation in tobacco seedlings. E/MeJA also synergistically activated the osmotin promoter fused to a [beta]-glucuronidase marker gene in a tissue-specific manner. The E/MeJA responsiveness of the osmotin promoter was localized on a -248 to +45 fragment that exhibited responsiveness to several other inducers. E/MeJA induction also resulted in osmotin protein accumulation to levels similar to those induced by osmotic stress. Of the several known inducers of the osmotin gene, including salicylic acid (SA), fungal infection is the only other condition known to cause substantial osmotin protein accumulation in Wisconsin 38, a tobacco cultivar that does not respond hypersensitively to tobacco mosaic virus. Based on the ability of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine to block ethylene induction of PR-1b mRNA accumulation and its inability to block osmotin mRNA induction by ethylene, these two PR gene groups appeared to have at least partially separate signal transduction pathways. Stimulation of osmotin mRNA accumulation by okadaic acid indicated that another protein kinase system is involved in regulation of the osmotin gene. SA, which is known to induce pathogen resistance in tobacco, could not induce the osmotin gene as much as E/MeJA and neither could it induce PR-1b as much as SA and MeJA combined.

SIZ1-Mediated Sumoylation of ICE1 Controls CBF3/DREB1A Expression and Freezing Tolerance in Arabidopsis

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.

Regulation of protease inhibitors and plant defense

Protease inhibitors are an important element of the plant defense response to insect predation, and may also act to restrict infection by some nematodes. Production of these inhibitors is highly regulated by a signal transduction pathway that is initiated by predation and transduced as a wound response. Local and systemic extracellular inducers of the signal pathway are released by injury. Current evidence suggests that the production of the inhibitors occurs via the octadecanoid pathway, which catalyzes the breakdown of linolenic acid and the formation of jasmonic acid to induce protease inhibitor gene expression.

AtHKT1 is a salt tolerance determinant that controls Na^+ entry into plant roots

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.

Dissection of floral induction pathways using global expression analysis.

Flowering of the reference plant Arabidopsis thaliana is controlled by several signaling pathways, which converge on a small set of genes that function as pathway integrators. We have analyzed the genomic response to one type of floral inductive signal, photoperiod, to dissect the function of several genes transducing this stimulus, including CONSTANS, thought to be the major output of the photoperiod pathway. Comparing the effects of CONSTANS with those of FLOWERING LOCUS T, which integrates inputs from CONSTANS and other floral inductive pathways, we find that expression profiles of shoot apices from plants with mutations in either gene are very similar. In contrast, a mutation in LEAFY, which also acts downstream of CONSTANS, has much more limited effects. Another pathway integrator, SUPPRESSOR OF OVEREXPRESSION OF CO 1, is responsive to acute induction by photoperiod even in the presence of the floral repressor encoded by FLOWERING LOCUS C. We have discovered a large group of potential floral repressors that are down-regulated upon photoperiodic induction. These include two AP2 domain-encoding genes that can repress flowering. The two paralogous genes, SCHLAFMÜTZE and SCHNARCHZAPFEN, share a signature with partial complementarity to the miR172 microRNA, whose precursor we show to be induced upon flowering. These and related findings on SPL genes suggest that microRNAs play an important role in the regulation of flowering.

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.

The tomato gene Pti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the hypersensitive response

The Pto gene encodes a serine/threonine kinase that confers resistance to bacterial speck disease in tomato. Using the yeast two-hybrid system, we identified a second serine/threonine kinase, Pto-interacting 1 (Pti1), that physically interacts with Pto. Cross-phosphorylation assays revealed that Pto specifically phosphorylates Pti1 and that Pti1 does not phosphorylate Pto. Fen, another serine/threonine kinase from tomato that is closely related to Pto, was unable to phosphorylate Pti1 and was not phosphorylated by Pti1. Expression of a Pti1 transgene in tobacco plants enhanced the hypersensitive response to a P. syringae pv. tabaci strain carrying the avirulence gene avrPto. These findings indicate that Pti1 is involved in a Pto-mediated signaling pathway, probably by acting as a component downstream of Pto in a phosphorylation cascade.

Molecular Aspects of Osmotic Stress in Plants

Abstract Plant molecular responses to osmotic stress are complex as evidenced by the isolation of numerous OR (osmotic stress-regulated) genes. Although functions including osmolyte biosynthesis, membrane transport, signal transduction, and cellular protection have been predicted for OR genes, few of them have been established. Current efforts toward isolating and analyzing the expression of individual OR genes should be replaced by systematic approaches to analyze all OR genes simultaneously in selected plant species. Both transcriptional and posttranscriptional regulation of OR genes have been described. Cis-elements that respond to osmotic stress through abscisic acid (ABA)-dependent as well as ABA-independent pathways have been identified. Functional genetic approaches using yeast and plant model systems are expected to complement current molecular analysis of overexpression of OR genes in transgenic plants. These systems will help to establish functions of OR genes, to dissect osmotic stress-signalin...

AtHKT1 Facilitates Na+ Homeostasis and K+ Nutrition in Planta

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.